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Dimensional analyses to quantify cell wall deformation and stress relaxation in stiff mutants of phycomyces blanesleeanus : experimental investigations

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Title:
Dimensional analyses to quantify cell wall deformation and stress relaxation in stiff mutants of phycomyces blanesleeanus : experimental investigations
Creator:
Munoz, Cindy M.
Place of Publication:
Denver, CO
Publisher:
University of Colorado Denver
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Language:
English

Thesis/Dissertation Information

Degree:
Doctorate ( Doctor of philosophy)
Degree Grantor:
University of Colorado Denver
Degree Divisions:
College of Engineering and Applied Sciences, CU Denver
Degree Disciplines:
Engineering and applied science
Committee Chair:
Welch, Samuel
Committee Members:
Ortega, Joseph K. E.
Yakacki, Christopher M.
Carpenter, R. Dana
Roane, Timberley

Notes

Abstract:
Stiff mutant sporangiophores of Phycomyces blakesleeanus exhibit diminished tropic (bending) responses when compared to wild type sporangiophores. Insight into sporangiophore growth of two stiff mutants is obtained by quantifying the biophysical processes that produce expansive growth. The overall objective of this investigation is to learn if the cell wall is affected by the altered genes to cause the diminished tropic responses. In the first part of this study the dimensionless parameters Πpe, Πpv, and Πev obtained from the Ortega Growth Equations are used to quantitate changes in wall deformation rates and stress relaxation rates. The results provide insight into changes in wall loosening chemistry, wall architecture, and wall composition of the stiff mutants of P. blakesleeanus. In vivo turgor pressure step-up experiments are conducted to measure the longitudinal volumetric modulus, εL, that is needed to determine the magnitudes of Πpe and Πev. The dimensionless parameters Πpe, Πpv, and Πev are compared for wild type and stiff mutants. The results demonstrate that the altered genes reduce the magnitudes of Πpv (plastic deformation rates) and Πpe (stress relaxation rates), indicating a significant change in wall loosening chemistry in stiff mutants. Also shown is that the magnitudes of Πev (elastic deformation rates) are similar in wild type and stiff mutants. This indicates that the altered genes did not substantially change the general wall composition and architecture in stiff mutants. In the second part of this study, experiments are conducted to obtain insight into whether the cell wall architecture (microfibril orientation) and the wall dynamics (microfibril reorientation) of the stiff mutants are changed by the altered genes. Sporangiophores of P. blakesleeanus exhibit helical growth (simultaneous elongation and rotation of the wall along the longitudinal axis). Experiments are conducted in which the sporangiophore’s rotation and elongation are measured and used to determine the ratio of rotation rate and elongation rate (R) as a function of elongation rate (dL/dt). Stiff mutant curves are compared to the previously published wild type curve demonstrating the behavior to be similar. This finding suggests that the mutant genes did not substantially alter the fibrils orientation (cell wall architecture) or the fibril reorientation (wall dynamics). The quantitative methods presented here are relevant to cells with walls in the plant, algae, and fungi kingdoms. This quantitative method that employs dimensionless numbers can be used to obtain insight into changes in underlying biochemical processes, wall structure, and wall dynamics. The use of such methods can help optimize and tailor cells walls for the industrial and pharmaceutical sectors.
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University of Colorado Denver
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Auraria Library
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Full Text
DIMENSIONAL ANALYSES TO QUANTIFY CELL WALL DEFORMATION AND
STRESS RELAXATION IN STIFF MUTANTS OF PHYCOMYCES BLAKESLEEANUS:
EXPERIMENTAL INVESTIGATIONS
by
CINDY M. MUNOZ
B.S., University of Colorado Denver, 2010 M.S., University of Colorado Denver, 2014
A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy
Engineering and Applied Science Program
2018


©2018
CINDY M. MUNOZ
ALL RIGHTS RESERVED


This thesis for the Doctor of Philosophy degree by
Cindy M. Munoz has been approved for the Engineering and Applied Science Program by
Samuel Welch, Chair Joseph K.E. Ortega, Advisor Christopher M. Yakacki R. Dana Carpenter Timberley Roane
Date: December 15, 2018


Munoz, Cindy M. (PhD, Engineering and Applied Science Program)
Dimensional Analyses to Quantify Cell Wall Deformation and Stress Relaxation in Stiff Mutants of Phycomyces blakesleeanus: Experimental Investigations Thesis directed by Professor Emeritus Joseph K.E. Ortega
ABSTRACT
Stiff mutant sporangiophores of Phycomyces blakesleeanus exhibit diminished tropic (bending) responses when compared to wild type sporangiophores. Insight into sporangiophore growth of two stiff mutants is obtained by quantifying the biophysical processes that produce expansive growth. The overall objective of this investigation is to learn if the cell wall is affected by the altered genes to cause the diminished tropic responses.
In the first part of this study the dimensionless parameters npe, npv, and nev obtained from the Ortega Growth Equations are used to quantitate changes in wall deformation rates and stress relaxation rates. The results provide insight into changes in wall loosening chemistry, wall architecture, and wall composition of the stiff mutants of P. blakesleeanus. In vivo turgor pressure step-up experiments are conducted to measure the longitudinal volumetric modulus, a_, that is needed to determine the magnitudes of npe and nev. The dimensionless parameters npe, npv, and nev are compared for wild type and stiff mutants. The results demonstrate that the altered genes reduce the magnitudes of npv (plastic deformation rates) and npe (stress relaxation rates), indicating a significant change in wall loosening chemistry in stiff mutants. Also shown is that the magnitudes of flev (elastic deformation rates) are similar in wild type
IV


and stiff mutants. This indicates that the altered genes did not substantially change the general wall composition and architecture in stiff mutants.
In the second part of this study, experiments are conducted to obtain insight into whether the cell wall architecture (microfibril orientation) and the wall dynamics (microfibril reorientation) of the stiff mutants are changed by the altered genes. Sporangiophores of P. blakesleeanus exhibit helical growth (simultaneous elongation and rotation of the wall along the longitudinal axis). Experiments are conducted in which the sporangiophore’s rotation and elongation are measured and used to determine the ratio of rotation rate and elongation rate (R) as a function of elongation rate (dL/df). Stiff mutant curves are compared to the previously published wild type curve demonstrating the behavior to be similar. This finding suggests that the mutant genes did not substantially alter the fibrils orientation (cell wall architecture) or the fibril reorientation (wall dynamics).
The quantitative methods presented here are relevant to cells with walls in the plant, algae, and fungi kingdoms. This quantitative method that employs dimensionless numbers can be used to obtain insight into changes in underlying biochemical processes, wall structure, and wall dynamics. The use of such methods can help optimize and tailor cells walls for the industrial and pharmaceutical sectors.
The form and content of this abstract are approved. I recommend its publication.
Approved: Joseph K.E. Ortega
v


DEDICATION
I would like to dedicate this work to my family, their unconditional love has motivated me to persevere. To my mother, Mrs. Juanita Munoz, and my father, Mr. Ricardo Munoz, who have given up so much to support their children’s dreams. Without their loving upbringing and nurturing I would not have been where I am today and what I am today. Had it not been for my mother’s support, my dreams of excelling in education would have remained mere dreams. I thank my mother with all my heart and will forever be grateful. I thank my father for teaching me work ethic and humbleness. To my sister, Norma Munoz, and brother, Jesus Munoz, whom I’ve become a role model to. You have pushed me to go beyond my expectations. This work is also dedicated to a special person, Anahi Rubio, who has accompanied me in the struggles of this journey and who has not once stopped believing in me.
VI


ACKNOWLEDGEMENTS
Without you, GOD, I wouldn’t be able to accomplish the most difficult challenge in my educational career. Thank you for giving me the strength, knowledge, ability and opportunity to undertake this research and to persevere.
I would like to express my gratitude to Dr. Samuel Welch for providing me with the financial means to continue the pursuit of a Doctorate degree. Even more so, thank you for giving me the opportunity to explore the world of teaching. Thank you for your time when I needed support and guidance. Without your help, this thesis would not have been possible.
I would also like to express my gratitude to Dr. Christopher Yakacki for providing materials needed for research. Thank you for being involved in my research struggles and provided support by listening and offering genuine advice to help me complete this work.
In my journey towards this degree, I have found a teacher, an inspiration, and a role model, Dr. Timberley Roane. She has provided her heartfelt support and guidance at all times and has given me invaluable guidance, inspiration, and suggestions in my quest for knowledge. Without her guidance, this thesis would not have been possible and I shall eternally be grateful to her for her assistance.
I would also like to express my gratitude to Dr. Kenneth Ortega for encouraging independent thought and helping me grow as a researcher. Without him, I would have not been introduced to the wonderful world of research. Thank you for providing me with the opportunity to learn interdisciplinary research and unconventional thinking.
vii


It would be inappropriate if I omit to mention the names of people that helped me with technical problems throughout this research. Thank you to Jac Corless for helping me with machining the pressure probe. Thank you to Beatriz Bermudez for helping me with the design and 3D printing of microscope stands. Their help contributed to the completion of this thesis.
VIII


TABLE OF CONTENTS
CHAPTER
INTRODUCTION......................................................1
Expansive Growth of Cells with Walls..............................2
Turgor Pressure, Water Uptake and Cell Wall Stress Relaxation..3
Mechanical Behavior of the Cell Wall and Relevant Biochemical
Behaviors......................................................4
Principles of Expansive Growth.................................5
Quantitative Modeling of Expansive growth of Cells with Walls.....7
Lockhart Growth Equations......................................7
Ortega Growth Equations.......................................10
Dimensionless Ortega Growth Equations.........................12
Phycomyces Blakesleenaus.........................................16
Sporangiophore Development I24 49 52!.............17
Sporangiophore Cell Wall......................................20
Sensory Responses.............................................21
Light Response..................................................22
Avoidance Response..............................................22
Geotropic Response..............................................23
Stiff Mutants.................................................23
IX


PREVIOUS RELEVANT STUDIES IN EXPANSIVE GROWTH OF
PHYCOMYCES BLAKESLEEANUS.............................................24
In vivo creep to determine biophysical variables Pc, cpi, and mg.26
Effects on Elongation Growth Rate with Turgor, P, Changes........26
Comparison of Cell Wall Mechanical Properties of Wild Type and Stiff
Mutant Sporangiophores...........................................30
Cell Wall loosening in the Wild Type Sporangiohore of Phycomyces
blakesleeanus....................................................34
Anoxia Experiments..................................................34
Low pH Frozen-Thawed Experiments....................................35
III. RESEARCH OBJECTIVES..................................................39
IV. DIMENSIONLESS CHARACTERIZATION OF CELL WALL DEFORMATION RATES AND STRESS RELAXATION RATES TO HELP IDENTIFY CHANGES IN WALL LOOSENING CHEMISTRY, AND WALL COMPOSITION AND ARCHITECTURE IN STIFF MUTANTS OF
PHYCOMYCES BLAKESLEEANUS.............................................41
Introduction.........................................................41
Materials and Methods................................................42
Biological Material..............................................42
Elongation.......................................................42
Turgor Pressure..................................................45
In-vivo Step-up Experiments......................................47
x


Protocol for in-vivo step-up experiments.........................48
Dimensionless Parameters.......................................50
Experimental Results..............................................51
Longitudinal Volumetric Elastic Modulus, el....................51
Dimensionless Parameters npe, npvand nev.......................55
Discussion........................................................59
Limitations.......................................................62
V. CELL WALL ARCHITECTURE AND DYNAMICS OF STIFF MUTANT
SPORANGIOPHORES OF PHYCOMYCES BLAKESLEEANUS.......................63
Introduction......................................................63
Fibril-Slippage-Reorientation Hypothesis for Helical Growth in
Phycomyces.....................................................63
Materials and Methods.............................................65
Biological Material............................................65
Elongation rate................................................66
Rotation rate..................................................66
Results...........................................................67
Discussion........................................................70
Limitations.......................................................71
VI. CONCLUSIONS AND FUTURE WORK.......................................72
Conclusions.......................................................72
XI


Suggestions for Future Work
74
REFERENCES................................................................76
APPENDIX
A. Mathematics for Chapter IV......................................86
B. Innacurate Data.................................................87
C. Supplemental Results for In-Vivo Turgor Pressure Step-Up Experiments.... 91
D. Dimensionless Variables used by Ortega l48l to make the Ortega growth
equations dimensionless ............................................108
E. Computation of fl Dimensionless Parameters......................109
xii


LIST OF TABLES
TABLE
Table 1. Biophysical Variables for WT and Stiff mutants.....................31
Table 2. A comparison of relevant biophysical variables used in computing magnitudes of the ripe, nPv, and nev dimensionless parameters: a_, m, Pc, and dL/dffor stage IVb sporangiophores for WT, C216, and C149 strains....................55
XIII


LIST OF FIGURES
FIGURE
Figure 1. Expansive growth process for cells with walls......................6
Figure 2. An illustration of a cylindrical cell depicting the stresses caused by turgor
pressure..........................................................7
Figure 3. Comparison of the npe values for different cell species............15
Figure 4. Developmental stages of Phycomyces blakesleeanus...................20
Figure 5. Growth modeled by a dashpot and spring in series...................25
Figure 6. In vivo creep experiment with four small pressure step-ups (P = 0.0077 MPa) .............................................................................28
Figure 7. In vivo creep experiment with one large turgor pressure step-up (P = 0.031
MPa).............................................................29
Figure 8. In vivo creep experiment with one large turgor pressure step-up (P = 0.046
MPa).............................................................30
Figure 9. Normalized values for dL/df, mg, and P-Pc for WT and stiff mutant
sporangiopgores..................................................32
Figure 10. Normalized values for Lg, mg, and cpg for wild type and stiff mutant
sporangiophores..................................................33
Figure 11. Stiff mutant and wild type depiction of tropic response dependent on the
magnitude of the growth zone.....................................33
XIV


Figure 12. Turgor pressure and elongation behavior as a function of time for a stage
IVb anoxia experiment............................................35
Figure 13. Average extension behavior of fifteen FT and eight FTB walls of stage IV
sporangiophores..................................................37
Figure 14. Snapshot of a sporangiophore depicting the method of calibration in ImageJ for elongation measurements using the sporangium as a scale reference. .............................................................................44
Figure 15. Snapshot of the sporangiophore as it is elongated by the addition of oil using
a microcapillary attached to a pressure probe.......................45
Figure 16. Manual pressure probe with a USB pressure transducer..................46
Figure 17. Snapshot of a sporangiophore being impaled by the microcapillary tip..47
Figure 18. Experimental set-up for an in vivo pressure step-up experiment........49
Figure 19. Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus behavior for a single stage IVb C149 sporangiophore during an in vivo turgor pressure step-up experiment..........................................53
Figure 20. Comparison of average a_for stage III (non-growing) sporangiophores
between WT and stiff mutants (C216 and C149)....................54
Figure 21. Comparison of average a for stage IV (growing) sporangiophores between WT and stiff mutants (C216 and C149)........................................54
Figure 22. A comparison of the npe values calculated for growing sporangiophores of P.
blakesleeanus (Stage IVb).......................................57
xv


Figure 23. A comparison of the npv values calculated for growing sporangiophores of P.
blakesleeanus (Stage IVb)........................................58
Figure 24. A comparison of the nev values calculated for growing sporangiophores of P.
blakesleeanus (Stage IV).........................................59
Figure 25. Fibril reorientation and fibril slippage mechanism postulated by Ortega et al [62] for a fast growing with relatively long growing zone sporangiophore.. 65
Figure 26 (A) Snapshots showing a thin piece of hair attached to the sporangium used
as a reference point to determine rotation rate..................67
Figure 27. Results from a typical rotation experiment on a single stage IVb C149
sporangiophore...................................................69
Figure 28. Average Ravg values versus elongation rates for stage IVb sporangiophores
for WT and stiff mutant strains
70


CHAPTER I
INTRODUCTION
Expansive growth of cells with walls and its regulation is central to the life and development of these cells. Regulation of expansive growth and morphogenesis is determined by controlling the cell wall mechanical properties which are altered by biochemical changes. Learning how these cells regulate their mechanical properties to control growth and growth responses to environmental stresses and environmental stimuli can help with the development of novel uses for cells with walls.
The study of cell wall mechanics in the plant, algae, and fungi kingdoms has gained increasing interest due to the applicability in the industrial and pharmaceutical sectors. In these sectors, the end product is highly dependent on optimal cell wall composition. For example, in the paper industry, the removal of lignin in wood increases the cost of production, such that a lower lignin content in processed wood could help lower production costs. Wood is a type of plant with a cell wall that has a high lignin content, therefore, modification of the cell wall composition can reduce processing costs. However, lower lignin content in wood comes at a cost because it is important for tree stability and cannot be reduced arbitrarily without knowing how to compensate for this reduction hi. In biofuel production, it is necessary to effectively degrade cell walls into fermentable sugars for ethanol production l2l In agriculture, plant pathogens significantly reduce the production and quality of the crops Pathogens directly penetrate the cell wall to access water and nutrients of the plant protoplast Thus a rigid cell wall can help increase the crop quality and crop survival rates l4l Furthermore,
1


learning how the cell wall is remodeled in response to environmental stresses can help design stress-tolerant crops. In the pharmaceutical sector, the fungal cell wall is an attractive target in anti-fungal agents because it has been shown that the death of the fungus can result from inhibition of cell wall polysaccharide synthases Therefore knowledge and advancement in the area of cell wall mechanics allow for the optimization of cell walls for the industrial and pharmaceutical sectors.
Expansive Growth of Cells with Walls
The cells of plants, algae, and fungi have walls, a distinguishable characteristic that separates these cells from mammalian cells. The cell wall is a complex extracellular matrix that surrounds all cells in the organism. Cell walls are crucially important for processes in cell expansive growth, morphogenesis and reproduction. The cell wall provides structural and mechanical support, helps maintain and determine cell shape, control rate and direction of growth, protect against pathogens and environmental factors, and it’s the site for cell signaling and cell to cell interaction [6]. A main prerequisite of cell walls is to resist high internal turgor pressures while being able to deform during growth and elastic expansion of the cell. Cell walls vary in shape, chemical composition and structure depending on cell type and developmental stage. Primary walls surround growing and dividing cells while being thin and extensible, but strong [7]. In this thesis, experimental work and reference will be based on primary cell walls that are able to undertake expansive growth.
2


Turgor Pressure. Water Uptake and Cell Wall Stress Relaxation
Turgor pressure is the hydrostatic pressure in excess of atmospheric pressure that builds up in cells with walls (plants, algae, and fungi)[8]. It is produced by an osmotic pressure difference that drives water into the cells from its surroundings across a selectively permeable membrane [8]. Cells with walls use turgor pressure to produce the necessary cell wall stresses for wall deformation and expansion. The dependence of turgor pressure in cell wall growth is a consequence of the facts that 1) stress is needed for wall stress relaxation (to ensure water uptake) and 2) the stress relaxation rate is a function of the wall stress relaxation [9]. Thus, turgor pressure is recognized as the driving force in cell wall expansion and expansive growth.
A central process in expansive growth of cells with walls is stress relaxation [9'12]. Research has shown that wall stress relaxation creates the reduced water potential needed to drive water uptake and thus cell wall expansion [9_11’ 13'15]. In growing cells, wall stress relaxation is necessary for irreversible deformation (expansive growth) to occur. Breaking bonds between load-bearing polymers in the cell wall (cell wall loosening) result in stress relaxation [10]. Stress relaxation reduces turgor pressure in the wall initiating water uptake and cell wall expansion and the restoration of cell wall stress [9] and is a consequence of biochemical wall loosening (Figure 1). The rate of stress relaxation serves as a measure of wall chemistry necessary for wall loosening. The process of relaxation and expansion occur simultaneously and equal in measure so that the cell wall is not damaged [9].
3


Mechanical Behavior of the Cell Wall and Relevant Biochemical Behaviors
The shape and deformation of a material depend on the force exerted on the material and the mechanical properties of the material. Similarly in cells with walls, the control of their shape and expansive growth is regulated by the turgor pressure and the cell wall mechanical properties. The wall deformation behavior depends on the wall’s composition, structure, architecture and chemical reactions in the wall, thus the mechanical properties are biochemically mediated [8]. Research has shown that the magnitude of expansive growth is sometimes regulated by the magnitude of turgor pressure I16-19!. The direction and magnitude of expansive growth observed during morphogenesis and tropic responses are controlled by regulating the mechanical properties of the wall I20-26!.
Experimental evidence demonstrates that wall mechanical properties and wall behavior are modified by biochemical agents that loosen the wall[13’24’ 27'36]. Cell wall loosening is essential for irreversible deformation to occur in that it provides the necessary stress relaxation for water uptake and expansion of the cell volume [9’28]. For higher plants (stems, roots, and leaves) pH-dependent proteins have been shown to produce wall loosening and controlled polymer creep (time-dependent deformation under an applied load) I27 29-31], it is hypothesized that algal cells loosen their walls by breaking calcium bridges and reforming them between pectin polymers I33-35!. It is unknown which biochemical agents regulate cell wall loosening in fungi, but some evidence exists that the mechanism might be similar to higher plants in that it is low pH-mediated [24].
4


Principles of Expansive Growth
Expansive growth and its regulation are central to the life and development of cells with walls. Expansive growth is the irreversible increase in cell volume and surface area. This complex phenomenon features three interrelated and simultaneous processes: cell membrane hydraulics, cell wall deformation, and cell wall chemorheology (chemically mediated flow and deformation of polymeric material). Cell membrane hydraulics consists of water uptake and its regulation. Water uptake produces cell turgor pressure which in turn produce the cell wall stresses necessary for cell wall expansion and growth. Cell wall deformation is produced by cell wall stresses producing both irreversible and reversible deformations necessary for cell wall expansion and growth (Figure 2). Irreversible deformation produces permanent increases in the cell wall and is generally regulated by biochemical wall loosening [8]. Reversible deformation provides the cell wall stresses and the resistance strength necessary to sustain high turgor pressures. The elastic deformation and elastic properties are generally a function of cell wall structure and architecture [8]. Cell wall chemorheology involves the molecular modification of the wall network (loosening and stiffening of bonds) by biochemical reactions. The cell wall maintains its integrity by simultaneously synthetizing new cell wall material. The process of expansive growth is summarized by Ortega and Welch [37] as follows:
5


“The cell transports or produces active solutes inside the plasma membrane and absorbs water from its surroundings through the process of osmosis. The absorption of water produces turgor pressure that stresses the wall. The wall is biochemically loosened, reducing both wall stresses (stress relaxation) and turgor pressure (turgor pressure relaxation). More water is absorbed in response to the decrease in turgor pressure, extending (deforming) the loosened wall. The wall deformation produces an increase in wall surface area (expansion) and a thinner wall. New wall polymers and other wall materials are added to maintain a nearly constant wall thickness. The series of events (i.e. wall loosening, wall stress relaxation, turgor pressure relaxation, water uptake, wall expansion, an increase in turgor pressure, an increase in wall stresses, and wall loosening again) is repeated continually during expansive growth.”
(1)
1 i/ i
i i Turgor
pressure
j/ \
'k i J
*— wall stress—►
â–º
s
(2)

Reduced wall stress and turgor
J
A wall loosening
l
stress relaxation
restoring turgor and wall stress
Figure 1. Expansive growth process for cells with walls. The illustration shows the concept of stress relaxation and how it is related to wall loosening and growth. (1) Cell reaches osmotic-equilibrium with wall stresses counterbalancing turgor pressure and induced water flows (for a non-growing well hydrated cell). (2) The wall is biochemically loosened resulting in turgor pressure and wall stress relaxation (for a growing cell). (3) Water uptake takes place in consequense of stress relaxation and the cell wall expands. In consequense the cell wall restores its wall stresses. This figure was taken from Cosgrove l9l
6


Figure 2. An illustration of a cylindrical cell depicting the stresses caused by turgor pressure. The blue arrows depict the turgor pressure inside the cell and the red arrows show the cell wall stresses, both radial and longitudinal, caused by the turgor pressure. Figure taken from Ortega et al t8T
Quantitative Modeling of Expansive growth of Cells with Walls
The cell wall mechanical properties regulate the expansive growth of cells with walls and biochemical processes alter these properties to give controlled expansive growth and morphogenesis. Quantitative models allow the investigation of the interplay between the mechanical properties of the cell wall and the underlying biochemical processes.
Lockhart Growth Equations
For over 50 years researchers have studied different types of cells with walls in the plant, algae, and fungi kingdoms to decipher the mechanisms associated with
7


expansive growth. The physical processes associated with expansive growth gave rise to quantitative modeling of this phenomenon. Lockhart l18l was the first to develop the foundation for a quantitative model for the expansive growth of cells with walls. The biophysical equations model two underlying physical processes that are necessary for expansive growth: water uptake and cell wall irreversible deformation. Equation (1.1) describes the relationship between the relative rate of change in water volume within the cell, (d\/w/dt)/V, and the relative rate of water uptake, L(An-P).
Equation (1.1) is written relative to the volume, V (the terms are divided by the volume); t is the time, L is the relative membrane hydraulic conductance (L = LPA IV), Lp is the membrane hydraulic conductivity, A is the membrane area, An is the osmotic pressure difference across the membrane (where the solute reflection coefficient is assumed to be unity and omitted from these equations), and P is the turgor pressure. Equation (1.2) describes the relative rate of change of the volume of the cell wall chamber, (dV/d t)cwc/Vcwc, as equal to the relative irreversible (plastic) deformation rate of the wall. The biophysical variable, (1.1)
(1.2)
8


The rates of change in volume of the cell wall chamber and water uptake are approximately equal, {6Vcwcldt)IV ~ (d\/w/dt)/\/), and an equation for the equilibrium or steady state turgor pressure, Peq, can be obtained,
LAn + eq ~ cp + L
Equations (1.1), (1.2) and (1.3) are the Lockhart Growth Equations which are all interrelated and coupled by P. Equation (1.1) was derived from the physical laws of the deformation of viscoplastic materials specifically that of a Bingham fluid. Although it is a simple model, much insightful information was obtained through these equations. For example, through experiments [25’38’391 and Equation (1.2) it was found that cp and Pcare not static mechanical properties. Insight into how the cell wall responds to changes in turgor pressure and how it affects growth has also been provided by Lockhart’s equations I16’ 21.38,40]
Lockhart’s equations helped demonstrate that it is imperative to understand the biophysical parameters in expansive growth to fully understand growth regulation and morphogenesis. It is important to note that the versatility in these equations comes with the fact that the same physical processes are exhibited by diverse cells with walls (plant, fungi, and algae). This biophysical approach assumes that the biophysical parameters help control and regulate biological and biochemical processes. The growth model developed by Lockhart is a quantitative relationship between the rate of cell expansion and the biophysical parameters that could be measured in vivo. This is very important because the validation of the model through experimentation demonstrates its applicability.
9


While Lockhart’s equations set the foundation for additional expansive growth models, the equations were lacking some important features of the expansive growth of cells with walls. The force acting on the cell was assumed to be constant, which implied the cell wall stress, o, and turgor pressure, P, are constant. This eliminated the elastic deformation component in the total cell wall deformation equation. Lockhart also assumed that the cell behaves as a linear viscoplastic model with the irreversible deformation being a linear function of turgor pressure in which the material can only flow and permanently deform when the turgor pressure, P, exceeds the critical turgor pressure, Pc. These assumptions within the derivation gave rise to the irreversible cell wall deformation equation as presented in Equation (1.2). When deriving the relationship between the increase in water volume and the net rate of water uptake the model assumed no transpiration, 7=0, this gave rise to Equation (1.1). Lockhart’s equations are able to model steady-state growth in cells surrounded by free water where transpiration is nonexistent, tissue tensions are negligible, and the stresses can be assumed to be proportional to turgor pressure and where the elastic changes are negligible, such as the growth of Nitella internode cells (algal cells) I16 21.38] M0st importantly, these growth equations cannot model periodic stress and stress relaxation exhibited by living growing cells I7’14’15’17’37’ 41-43l It has been shown that elastic deformation is required for stress relaxation I11’15’ 42l and needed to accurately describe the instantaneous changes in wall deformation after turgor pressure changes I44’ 45l
Ortega Growth Equations
Ortega [42 461 augmented the Lockhart Growth Equations to address the limits in the model. Equation (1.1) was augmented with a transpiration rate, 7, term producing
10


Equation (1.4). Where T= (d VtI dt)/\/ is the relative rate of change in water volume lost
via transpiration (relative transpiration rate).
(1.4)
Equation (1.2) was augmented with an elastic component Me (dP/dt) which now describes changes in turgor pressure and reversible cell wall deformation producing Equation (1.5). Where e is the volumetric elastic modulus which measures how the cell wall changes volume in response to pressure. The underlying constitutive equation is now of a viscoelastic material with a yield stress (Maxwell-Bingham viscoelastic model) which now models deformation at any applied stress.
Similarly, when obtaining Equation (1.3) an equation for the turgor pressure is also derived, Equation (1.6). Note that this equation now addresses changes in turgor pressure in time and it is no longer constant as assumed in Lockhart’s equations.
With the addition of the transpiration rate, cells exposed to air where the effect of transpiration cannot be neglected can be modeled more accurately. An example of such case is in the fungal cell P. blakesleeanus, where water uptake takes place at the base and transpiration takes place throughout the long cylindrical stalk exposed to the air. When modeling stress relaxation water uptake is eliminated, and transpiration is suppressed, the Ortega Growth Equations (also known as Augmented Growth Equations) are reduced to Equations (1.7) and (1.8). Equation (1.8) describes the
(1.5)
— = e{L(An - P) - (1.6)
11


pressure decay from an initial pressure, Pi, at time, t= 0, with a stress relaxation time constant tc={€(p)A. For an ideal stress relaxation, e is constant and known throughout the pressure decay range l17l. This is the pressure decay observed experimentally for plant and fungal cells I14’15’ 17T
dP
— = -£(p(P - P^
(1.7)
P(0 = (.Pi - Pc) exp(~e The utility of the Ortega Growth Equations is that they were derived based on physical principles to model physical processes that are relevant for plant, algal, and fungal cells. Water uptake and cell deformation are the same for these cells with walls. The differences lie in the chemorheological (wall loosening chemistry) aspects of each cell, cell structure and synthesis of wall material. These differences are embedded in the biophysical variables of these equations which can be extracted from specifically designed experiments.
Dimensionless Ortega Growth Equations
Dimensionless parameters are frequently used in the physical sciences to better understand complex phenomena. For example, in the area of heat transfer and fluid mechanics, the Reynolds number (which is interpreted to be the ratio of inertia and viscous forces) is used to study different flows, in particular, if a flow is laminar or turbulent l47T Some of the reasons equations are made dimensionless is to reduce the number of experiments that need to be conducted, reduce the number of times one might need to solve equations, to obtain insight into what parameters might be small
12


and ignored or approximated, to separate relevant processes, and to scale up solutions by model similarity. The Ortega Growth Equations were made dimensionless and used to separate biophysical variables with their relevant processes. Ortega l12l used constant reference parameters, vs (steady relative volumetric growth rate), vst (steady relative volumetric transpiration rate), and Pc (critical turgor pressure) to make the equations dimensionless. Equations (1.9), (1.10), and (1.11) are the dimensionless equations corresponding to Equations (1.4), (1.5) and (1.6). See Appendix B for dimensionless variable definitions.
The ratios in parenthesis are dimensionless and are interpreted into ratios of relevant processes:
(1.9)
(1.10)
(1.11)
relative volumetric plastic deformation rate of the wall
)
relative volumetric growth rate
13


relative volumetric elastic deformation rate of the wall
)
relative volumetric growth rate
= © = (
relative volumetric water uptake rate
)
relative volumetric elastic deformation rate of the wall
relative volumetric transpiration rate
)
relative volumetric elastic rate of the wall
relative volumetric plastic deformation rate of the wall
)
relative volumetric elastic deformation rate of the wall
The ripe dimensionless parameter was shown to be central to stress relaxation [12] a necessary process for the expansive growth of cells with walls. This parameter directly relates cell wall mechanical properties, cell wall deformation rates and cell wall loosening chemistry. Wall loosening chemistry regulates wall loosening and alters mechanical properties to produce the wall deformation necessary for irreversible deformation (expansive growth). This dimensionless parameter can be computed through in vivo creep and in vivo stress relaxation tests I12’ 48l
Where T1/2, is the halftime of the exponential decay of turgor pressure in an in vivo stress relaxation experiment.
Ortega l12l computed npe values for three different species (plant, algal, and fungal cells) and compared the values (Figure 3). The npe values for different species are dramatically different from each other. The difference in magnitudes of the npe value
s
Ilpe (in vivo stress relaxation) =
14


for each cell species suggests a different cell wall loosening chemistry. There is evidence that shows that wall loosening chemistry is different between plants and algal cells l26’27’29’31 ’ 33'36l. A different wall chemistry would produce different ratios of plastic and elastic deformation rates of the cell wall, similar to what is seen in the npe values for different cell species (Figure 3). Most importantly different wall loosening chemistry would produce different wall stress relaxation rates.
The ripe parameter can be further decomposed into dimensionless reversible and irreversible wall deformation rates through the npv and nev parameters. These help to identify direct changes in biochemical wall loosening (npv) and wall architecture and composition (n ev).
Figure 3. Comparison of the npe values for different cell species. The npe values are different by several orders of magnitudes. Data to produce the figure was taken from Ortega [12].
15


Phycomyces Blakesleenaus
Phycomyces blakesleeanus is a single-celled fungus that has been used as a model organism in sensory transduction and cell wall mechanics research [23’ 49'51]. It is a filamentous fungus that belongs to the class Zygomycetes. The sporangiophores of P. blakesleeanus are long aerial cylindrical stalks that carry a sporangium filled with spores at the top that can reach a length of 10 cm or more. The sporangium is approximately 500 pm in diameter and can hold up to 105 spores for the vegetative reproductive cycle I49’ 52l The spores can be kept in dormancy in a water medium or as dry stock in the refrigerator. Vegetative spores are heat shocked and inoculated on an adequate growth medium such of potato dextrose agar to grow sporangiophores. The sporangiophore grows by simultaneously elongating and rotating along its longitudinal axis exhibiting helical growth. The elongation and rotation occur in the growing zone of the sporangiophore which is in different locations depending on the stage of development.
The sporangiophores exhibit different tropic (bending) responses to external stimuli. They detect and respond to light intensity, gravity, mechanical stretch, solid objects and air currents l49l. These stimuli control the growth rate, producing time-dependent changes in growth rate as well as tropisms l49l. The output (growth rate) has been the focal point of sensory transduction studies because of information gained in the response of various external stimuli l49l. The external stimuli must react with internal signals to control the growth rate. The objective of sensory transduction studies has been to gain information about the steps in this transduction process, from external stimuli, receptors, organelles, and chemical reactions in a controlled manner I49’52'54l.
16


Growth is limited by the cell wall making it apparent that expansive growth is dependent on cell wall formation and the growth responses being exhibited may reflect changes in the cell wall structure and cell wall chemistry. Studies have focused on cell wall mechanics to characterize cell wall mechanical properties and structure and relate this to growth I23 55-63!. These studies provided information about how cell wall mechanical properties regulate growth, and how external stimuli regulated mechanical properties.
Phycomyces has especially served as a model organism in understanding the expansive growth of cells with walls l23l. It is similar in morphology and growth behavior to higher cells that the study and understanding of its expansive growth provide useful knowledge in understanding expansive growth of higher cells.
Sporanqiophore Development t24’49’ 52l
Phycomyces has particularly served as a model organism because it is responsive to different external stimuli and because of its distinct developmental stages. The sporangiophore develops through five main developmental stages (Figure 4).
• In stage I, the sporangiophore appears as a pointed tube elongating at the tip at 0.3-0.6 pm/sec and rotating clockwise (seen from above) at approximately 30 °h"1. During this stage, growth occurs between the apical tip and 1.0 mm below it (Figure I-4).
• In stage II, no elongation or rotation occurs. During this stage the formation of sporangium begins. This stage lasts for approximately 3
17


hours in which a yellow sporangium is fully formed of approximately 0.5 mm in diameter.
• Stage III is a period of approximately 2 hours of rest with no elongation or rotation. This stage is hypothesized to be linked with active spore formation.
Stage IV is divided into three different stages, IVa, IVb, and IVc. The main characteristic of this stage is the formation of a new growing zone and intercalary growth occurring in this region.
• Stage IVa begins with elongation and counter-clockwise rotation at the short growing zone located approximately 0.6 mm below the base of the sporangium (Figure 1-4). Counter-clockwise rotation continues for approximately 1 hour before the rotation rate gradually decreases to zero and clockwise rotation. During this stage, the sporangium gradually darkens.
• Stage IVb begins with clockwise rotation. Elongation and rotation are maintained relatively constant (typical values are 1 pm/sec and 0.2°s'1) for many hours. This stage exhibits a dark sporangium containing mature spores. Most biophysical work is conducted with stage IVb because it exhibits nearly constant elongation, rotation and growing zone length for 2-4 hours. Elongation rates are between 35 and 60 pm min'1. The growing zone extends from 0.1 mm to approximately 2.5 mm below the sporangium. The cell wall in this region is extensible and deforms
18


plastically when stretched longitudinally l64l. The cell wall in the nongrowing region deforms elastically when subjected to a longitudinal load
[49, 56]
• Stage IVc exhibits another twist reversal to counter-clockwise rotation (I-4). During this stage, the sporangiohores are long (>10 cm).
• Stage V is the final stage characteristic of old sporangiophores and not exhibiting growth. During this stage, the sporangium can easily burst releasing spores that easily stick to most surfaces.
The rotation and elongation behavior has been measured and used as an indirect method to gain insight on cell wall architecture (microfibril orientation) and wall dynamics (microfibril reorientation)[61_63’ 65'67].
19


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O
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111,1,1,1,11
8 9 10
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TIME (hr)
28
30
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J_l
E
E
23 ^
LU
22
21
20
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18
GROWTH ZONE C CLOCKWISE ROTATION O COUNTER CLOCKWISE ROTATION
Figure 4. Developmental stages of Phycomyces blakesleeanus. The sporangiophores exhibit helical growth (simultaneous elongating and rotating about its longitudinal axis). The cell wall elongates in a region termed the growing zone (depicted in light green). Stage IVb is used in biophysical studies due to its nearly constant elongation and rotation. See section “Sporangiophore Development” for detailed information on each stage. Figure was taken from Ortega et al. l24l
Sporangiophore Cell Wall
The fungal cell wall consists of two types of components: 1) a matrix made of glycoproteins, amorphous polysaccharides, and lipids 2) microfibrils of skeletal
20


polysaccharides of chitin and B-glucans [49]. Phycomyces belongs to the class Zygomycetes which are characterized by having chitin and chitosan in their cell walls I68! The majn components in sporangiophore cell walls are chitin, chitosan, and polyuronides plus the neutral sugars, mannose, glucose, galactose, and flucose I49’ 52l The cell wall of Phycomyces is about 600 nm thick with chitin microfibrils of 15-20 nm in diameter and several micrometers long l52l.
Ruiz-Herrera l69l found that there exist microvesicles (named chitosomes) which contain chitin synthase in the cytoplasm of the yeast M. rouxii. In Phycomyces chitosomes have also been reported to be present in the cytoplasmic material of the tip of sporangiophores l49l The chitosomes (40-70 nm in diameter) move from the cytoplasm to areas where expansive growth occurs in cells with a growth zone l49l Chitin synthase in the chitosomes can be activated by protease and incubation with UDPGIcNAc substrate to synthetize chitin microfibrils l49l. Ruiz-Herrera ^calculated that 80% of chitin content in the cell is in these chitosomes.
Sensory Responses
Sporangiophores of Phycomyces respond to different sensory stimuli by changes in growth rate and/or direction of growth. The stimuli and responses are linked by sensory pathways that process information [49]. Each sensory pathway acquires information from one or more sensors, evaluates and transfers it, and finally governs a response mechanism [49]. The sensory transducers (elements in sensory transduction pathway) must be either proteins made under the direct command of genes or other chemicals manufactured with the participation of gene products [49]. In most cases, a
21


stimulus applied symmetrically to the longitudinal axis of the sporangiophore produces a change in elongation growth only while an asymmetrical stimulus produces differential growth on opposite sides of the stalk. The differential growth results in the sporangiophore troping (bending). There are different variations of responses that can be elicited through various symmetric and asymmetric stimuli and combinations of. Three main responses (light response, avoidance response, and geotropic response) are reviewed below.
Light Response
The sporangiophore responds to a spatially symmetric increase or decrease in light intensity by exhibiting a transient increase (positive response) or decrease (negative response) in growth rate respectively. The response is only elicited when the level of light intensity is higher or lower than the light intensity to which the sporangiophore was adapted to [49].
A phototropic response is elicited in the sporangiophore when being subjected to spatially asymmetric light distribution. The sporangiophore grows towards the region of maximum light intensity [49]. The sporangiophore has a lens-like property in the growing zone which allows unilateral light to be focused at the distal side of the sporangiophore [49]. This results in asymmetric growth rate with respect to the sporangiophore’s cross-section and troping towards the unilateral light source.
Avoidance Response
The sporangiophore detects objects in close proximity (< 5 mm) to the growing zone. It responds to this stimuli by growing away from the object with a response
22


latency of 2-3 min. The maximum growth rate may increase up to 20-60% higher than the basal rate. During the increase in growth rate, the trope rate is relatively constant at a rate of 3° min-1. The sporangiophore usually stops troping within 20-30 min after the barrier is placed. A maximum of 40° to 50° troping angle has been observed [49].
Geotropic Response
The sporangiophore detects a gravitational field and responds by growing away from the gravitational source, named the negatively geotropic response. When a sporangiophore is placed horizontally, it responds to gravity by growing towards a vertical axis until completely vertical. The latency towards the vertical axis is 30-180 minutes after being placed horizontally with and average troping rate of 0.3° min'1 I49 52!.
Stiff Mutants
Mutants with altered growth behavior have been classified into phenotypic classes based on their sensory responses to light, gravity, and avoidance l71Ho identify the genes, proteins, and enzymes affecting the operation of each step of the sensory pathways [49]. The mad mutants have been classified into three groups: classl .1, class 1.2 and class 2. Class 2 are said to be defective in the output side (growth rate) I49’ 71l. Class 2 mutants display a reduced sensitivity to visible light. Mutants in class 2 with defects in the genes mad D, E, F, G, and J (termed stiff mutants) have diminished or non-existent tropic responses I71-73!. In this research two stiff mutants, C216 and C149 with altered genes mad D, E, F, G, and J are studied to further elucidate on the expansive growth of P. blakesleeanus and to learn if the altered genes affect the cell wall properties thus affecting the tropic responses.
23


CHAPTER II
PREVIOUS RELEVANT STUDIES IN EXPANSIVE GROWTH OF PHYCOMYCES
The sporangiophores of P. blakesleeanus grow predominately in length, L. The radial growth (cross-sectional growth rate in area) is small compared to the elongation growth rate dL/df, therefore the radial growth rate can be neglected in expansive growth considering only elongation growth. Sporangiophores of stages I and IV exhibit intercalary growth meaning they only grow within a growth zone that is shorter than the rest of the cell’s length (Figure 5B). The equation for the elongation growth rate is modeled by Equation (2.1) l23l. Equation (2.1) represents the growth within the growing stalk region, Lg, and the non-growing stalk, Ls. Where mg is the longitudinal irreversible cell wall extensibility of the growing stalk, and £i_g and els are the longitudinal volumetric elastic modulus within the growing stalk and the longitudinal volumetric elastic modulus within the non-growing stalk, respectively. Within the growing region, both irreversible and reversible deformation occurs, in contrast to the non-growing region where only reversible deformation is exhibited (Figure 5B).
BLAKESLEEANUS
(2.1)
24


Cell Wall In Tension (Diffuse Growth) A
Cell Wall In Tension (Tip Growth) B
Figure 5. A Diffuse growth modeled by a dashpot and spring in series. This model represents growth for cells that elongate throughout the entire stalk (plants and algal cells). B Tip growth modeled by the two separate regions (growing and non-growing). The growing region has a dashpot and spring in series attached (in series) to another spring which represents the non-growing region. Figure was taken from Ortega [23].
When turgor pressure, P, is constant, the elastic deformation rate is eliminated from Equation (2.1) and the elongation growth rate is simply equal to the irreversible
deformation rate of the growing region, Equation (2.3).
dL
Tt = m^p ~ Pc)
(2.3)
Equation (2.3) can be written relative to the growing zone length, Lg\
1 dL
Lq dt
ma
-r(p-pc) Lg
(2.4)
25


Where cpg=mgILg is defined as the relative irreversible cell wall extensibility for the growth
zone.
In vivo creep to determine biophysical variables Pc, An in vivo creep experiment is used to determine biophysical variables Pc, cpi, and mg. This experiment requires an instantaneous increase in turgor pressure (turgor pressure step-up) imposed on the cell ^7\ The growth rate behavior after the turgor pressure step-up is used to determine Pcand mg. During an in vivo creep experiment Pc and /r?g are assumed to be constant l17l. Using finite differences Equation (2.4) becomes:
The average growth rate is usually obtained from the 10-min interval before and after the pressure step-up. The critical turgor pressure, Pc, can be determined from Equation
(2.3) using mg, P, and dL/df before the pressure step-up since they are known and constant.
Effects on Elongation Growth Rate with Turgor, P, Changes
Ortega et al. l17l demonstrated that the elongation growth rate in sporangiophores of P. blakesleeanus responds differently to changes in turgor pressure. The
(2.5)
Solving for mg\
m,
(2.6)
A P
26


experiments described in l17l demonstrate the changes in elongation growth behavior exhibited by sporangiophores when subjected to different magnitudes of pressure step-ups. It was demonstrated that the growth behavior elicited was related to the magnitude of the pressure step-up. Turgor pressure step-ups less than 0.02 MPa elicited an increase in growth rate (Figure 6). This response is predicted by the Ortega Growth Equation (Equation 2.3). Also shown was that turgor pressure step-ups larger than 0.02 MPa elicit a decrease in growth rate (Figure 7). Larger magnitudes of pressure step-ups produce larger decreases in growth for longer periods (Figure 8). The decrease in growth rate is related to the magnitude of the pressure step-up. Ortega et al. l17l attributed this effect as strain-hardening of the wall after a large (P> 0.02 MPa) pressure step-up.
27


P(MPa)
Figure 6. In vivo creep experiment with four small pressure step-ups (P = 0.0077 MPa). Turgor pressure (upper graph) and natural logarithm of the length, In L, (lower graph) are plotted as a function of time. The fat short arrow indicates when the cell was impaled and the long thin arrows indicate when the turgor pressure step-ups were imposed on the cell. Figure was obtained from Ortega et al. [17].
28


Figure 7. In vivo creep experiment with one large turgor pressure step-up (P = 0.031 MPa). Turgor pressure (upper graph) and natural logarith of the length, In L, (lower graph) are plotted as a function of time. The fat short arrow indicates when the cell was impaled and the long thin arrow when the turgor pressure step-up was imposed on the cell. Figure was obtained from Ortega et al.[17].
29


P (m Pa)
Figure 8. In vivo creep experiment with one large turgor pressure step-up (P = 0.046 MPa). Turgor pressure (upper graph) and natural logarith of the length, In L, (lower graph) are plotted as a function of time. The fat short arrow indicates when the cell was impaled and the long thin arrow when the a the turgor pressure step-up was imposed on the cell. Figure was obtained from Ortega et al. ^7\
Comparison of Cell Wall Mechanical Properties of Wild Type and Stiff Mutant Sporanqiophores
Sensory responses in stage IVb sporangiophores of P. blakesleeanus are produced by regulating the magnitude of elongation growth rate and differential elongation growth rate. Stiff mutant sporangiophores are deficient in the madD, E, F, G, and J genes I71-73!. The stiff mutant sporangiophores exhibit significantly reduced tropic responses to light, gravity, and the presence of solid surfaces I71-73!. Ortega et al. [24]
30


investigated whether the defective genes affect the biophysical variables that regulate growth (mg, P, and Pc). The length of the growth zone, Lg, was also determined and used to offer an explanation on why the stiff mutants exhibit diminished tropic responses and how the variables studied affect each other. Table 1 are the results from this study comparing biophysical variables between wildtype (WT) and stiff mutants.
Table 1. Biophysical Variables for WT and Stiff mutants. Table was taken from Ortega et al. I24l.
Variable (units) Wild type Mean±SEM (n) C216 Mean ±SEM (n) C149 Mean ± SEM (n)
dUdt lum 34 ±3 (20) 34±3 (181 29 ±6 (8)
min-’)
mg Ipm 997 ±164 120) 222 ± 40 (18) 170 ±30 181
min-1 MPa-1)
P IMPal 0.32 ± 0.01 (20) 0.40 ± 0.01 (18) 0.41 ±0.02 (8)
Pc (MPa) 0.26 ± 0.01 (20) 0.13 ±0.05 (18) 0.18 ±0.08 (8)
P-Pc IMPal 0.05 ± 0.01 (20) 0.27 ± 0.05(18) 0.23 ±0.06 (8)
Lg (|im) 2072 ± 81 (26) 1634 ± 89 (22) 1125 ± 31 (21)
i'g (MPa-1 **0.48 ^0.14 *»0.15
min-1)
Ortega et al. [24] found that the magnitudes of mg exhibited in stiff mutants were significantly smaller compared to the WT (Figure 9). This result indicated a diminished ability to irreversibly deform in the longitudinal direction in stiff mutants. Also found was that the magnitude of P-Pc significantly increased in stiff mutants (Figure 9). This indicated an increase in the magnitude of the effective stress in the wall responsible for irreversible deformation. The lower magnitudes of mg in stiff mutants produced less irreversible cell wall deformation and should produce a lower growth rate, however, the
31


magnitudes of P and P-Pc increased producing higher wall stresses necessary for irreversible deformation. Essentially the higher stresses in the wall were compensated for the lower longitudinal irreversible cell wall extensibility, mg, to produce similar growth rates exhibited by the WT (Figure 9). The decrease in the magnitude of mg (mg = cpg Lg) exhibited by the stiff mutants was shown to be produced by the decrease in Lg and cpg (Figure 10). The decrease in Lg and cpg was postulated to affect the ability to produce differential elongation growth because bending rates are dependent on mg in a similar fashion as growth rates are dependent on this variable. If a sporangiophore produces a constant bend rate per unit length then a shorter growing zone, Lg, would produce a lower bending rate because there is less length to bend (Figure 11).
1.2
1.0
a>
-5 0 8
| 06 ra
I 04
Z
02
0
Figure 9. Normalized values for dL/df, mg, and P-Pc for WT and stiff mutant sporangiopgores. The lower mg values in stiff mutants were compensated by the higher P-Pc values. There was no significant difference in dL/df between the strains. Figure was taken from Ortega et al. l24l
â–¡ dL/dt arrig bP-F^
WT C216 C149
32


1.2
a>
5
TJ
0>
N
15
E
â–¡ Lg DmB U Figure 10. Normalized values for Lg, mg, and cpg for wild type and stiff mutant sporangiophores. The values were significantly smaller in the stiff mutants compared to the wild type. Figure was taken from Ortega et al. l24l.
Stiff Mutant Wild Type
GROWTH ZONE | |
Figure 11. Stiff mutant and wild type depiction of tropic response dependent on the magnitude of the growth zone. Growth zone is depicted as a light green region. The stiff mutant exhibits a lower tropic response because of the decrease in magnitude of the growth zone length. Figure was taken from Ortega et al. [24].
33


Although it was found that the altered genes significantly reduced the irreversible extensibility of the cell wall, cp, the study did not provide evidence to learn whether the defective genes altered wall deformation rates and stress relaxation rates. Chapter IV focuses on investigating these questions and makes use of the results of Ortega et al. l24l to predict plastic deformation rates for the stiff mutants.
Cell Wall loosening in the Wild Type Sporanqiohore of Phycomyces blakesleeanus
Cell wall loosening is necessary for irreversible deformation in cells with walls. Many studies in cell wall loosening have been conducted on plant and algal cells to elucidate on chemorheological aspect of expansive growth ^ 27â– 29- s'134-36], however, no studies had been conducted on fungal cells. Ortega et al. l26l studied the cell wall loosening mechanism in P. blakesneeanus. The study provides evidence of the existence of chemistry in the fungal cell wall of P. blakesleeanus responsible for wall loosening, irreversible deformation, and elongation growth.
Anoxia Experiments
Ortega et al. [26] designed experiments to isolate the wall from new cell wall material addition reducing metabolic influences to gain insight on the existence of cell wall chemistry by subjecting the sporangiophore to an anoxic (without oxygen) environment. Results demonstrated that elongation growth continues on average for more than 10 minutes after an anoxic environment when small turgor pressure step-ups (P < 0.02 MPa) were imposed on the cell. This demonstrates that wall chemistry continues the chemorheogical process to deform the cell wall irreversibly during anoxia (Figure 12). The wall deformation during anoxia is irreversible because the turgor
34


pressure step-ups are held constant and dP/df=0, with no elastic deformation occurring after the first minute after the step-up (Equation 1.13). Ortega et al. l26l hypothesized that the wall material added to the cell wall before anoxia is used to continue the cell wall loosening process and enable irreversible deformation until the cell wall material is depleted.
0.70
0.60
0.50
0.40 *
a.
?
0.30 iC 0.20 0.10 0.00
0 10 20 30 40 50 60
Time (min)
Figure 12. Turgor pressure and elongation behavior as a function of time for a stage IVb anoxia experiment. Times when the cell was impaled and oxygen level was lower than 1% are represented in the graph by short thick and long thin arrows, respectively. Approximately 5 minutes after anoxia, turgor pressure step-ups ( P = 0.021 MPa) were imposed on the cell. Elongation growth rate and irreversible deformation continue for approximately 23 minutes. Graph was taken from Ortega et al. l26l.
Low pH Frozen-Thawed Experiments
Other experiments conducted by Ortega et al. l26l are similar to the low pH
frozen-thawed similar to those conducted by Cosgrove l74l on cucumber hypocotyls.
These experiments further confirmed the existence of wall chemistry and a better insight
into the cell wall loosening of P. blakesleeanus.
35


Ortega et al. l26l conducted constant-tension extension experiments on frozen-thawed (FT) and frozen-thawed boiled (FTB) sporangiophore walls to identify whether a decrease in pH increased cell wall extension and whether a wall-loosening protein may be involved in fungal cell wall deformation. Freezing the cell wall deactivates enzymatic activity but retains the wall enzymes. A relevant treatment that affects the cell wall chemistry would activate the enzymes within the wall producing cell wall extension. Boiling of the cell wall after freezing and thawing serves as a denaturing treatment. Boiling destroys cell wall proteins necessary for cell wall extension. If a treatment has an effect on FTB walls then cell wall extension is not enzymatic.
Results from Ortega et al. l26l demonstrated that when FT walls were subjected to a low pH environment under a constant load, the walls immediately extended and continued to extend (creep) for minutes after (Figure 13). Interestingly the FTB walls showed a smaller initial extension immediately when the pH was lowered and did not continue to extend and no creep was observed (Figure 13). FT walls exhibited an average creep time of five minutes. The results demonstrated that lowering the pH produces a chemorheological reaction, irreversible extension, and creep. In contrast to the FTB walls which exhibited a lower initial irreversible extension with no creep. The inhibition of creep in the FTB walls suggested that a wall protein may be involved in wall loosening and creep in P. blakesleeanus. The results proved to be similar qualitatively to those of cucumber hypocotyls ^7A\ but different quantitatively. The creep times were over 24 hrs in cucumber hypocotyls while on average creep time in the fungal cell P. blakesleeanus was 5 min. Ortega et al. [26] hypothesized that different cell wall material, structure and cell size could affect creep behavior and account for this difference. Also
36


hypothesized was that wall chemistry exists in walls of P. blakesleeanus to make and break load-bearing bonds between microfibrils producing the chemorheology necessary to regulate mechanical wall properties and wall deformation behavior.
300
250
200
E
a 150
100
50
0
0 2 4 6 8 10 12 14 16 18
Time (min)
4.6
Figure 13. Average extension behavior of fifteen FT and eight FTB walls of stage IV sporangiophores. The curves show the extension behavior before and after the pH of the bathing water’s solution was lowered to 4.6. The FT walls show increased initial extension and creep while the FTB wall shows reduced initial extension and no creep. Figure was taken from Ortega et al. l26l.
Many studies have been conducted on sporangiophores of P. blakesleeanus to gain insight on expansive growth and cell wall deformation. Ortega et al. [17] demonstrated that the elongation growth rate behavior of growing sporangiophores is different when the magnitude of the turgor pressure step-ups is changed. In this thesis, the results from large turgor pressure step-ups are used to develop an experimental protocol to determine the longitudinal volumetric elastic modulus, a, of stage IVb (growing) stiff mutant sporangiophores. Results from Ortega et al.[24] demonstrated that
37


the elongation growth rate is dependent on the magnitudes of cpg, Lg and P-Pc. Also shown is that stiff mutant genes madD, E, F, G, and J decreased the magnitudes of mg, (pg, and Lg. The smaller

38


CHAPTER III
RESEARCH OBJECTIVES
A general objective of this research is to demonstrate that the quantitative measures used in the studies outlined in the following chapters can be used to detect changes in the mechanical and rheological behavior of cell walls. Traditional Mechanical Engineering methods and experimental methods are adapted to suit a living organism and extract relevant cell wall material properties and cell wall behavior. The underlying biophysical principles in cell wall expansion and expansive growth allows for a quantitative approach that has been validated throughout the years. Changes in wall deformation rates and stress relaxation rates are translated into biochemical changes which can be used to identify molecular changes. The information provided from these methods along with biochemical methods can provide a better understanding of expansive growth and wall deformation to ultimately help tailor cell walls for industrial and pharmaceutical sectors. It is envisioned that quantitative studies such as these can help guide future biochemical studies. The model organism Phycomyces blakesleeanus is used to demonstrate how these methods can be used to predict biochemical changes associated with the cell wall.
The objective of Chapter IV is to quantify cell wall deformation rates and stress relaxation rates that can provide insight to changes in wall loosening chemistry, and wall composition and architecture in two stiff mutants of P. blakesleeanus. The dimensionless parameters npe, npv, and nev that were obtained from the Ortega Growth Equations can be used to achieve this objective. The magnitudes of npe and npv are substantially reduced in stiff mutants compared to the WT. In contrast, the magnitudes
39


of llevare similar in stiff mutants and WT. These results demonstrate that mutant (altered) genes reduced the irreversible (plastic) deformation rates of the wall and stress relaxation rates. This implies a significant alteration to the cell wall loosening chemistry in the stiff mutant walls. The similar nev values in stiff mutants and WT demonstrates that the altered genes did not substantially change the elastic deformation rate of the wall, implying that the cell wall architecture and composition are not significantly changed.
The objective of Chapter V is to obtain experimental evidence that can help determine whether the altered genes in stiff mutants significantly change wall architecture and wall dynamics. This is accomplished by using an experimental approach in which changes in microfibril orientation and reorientation is indirectly determined by measuring the sporangiophore’s rotation and elongation rates. Results demonstrate that the curves of R (the ratio of rotation rate to elongation rate) vs. elongation rate (dL/df) in the stiff mutants are essentially the same as those obtained from WT. This suggests that the altered genes did not substantially change the microfibril orientation and reorientation. It is concluded that the altered genes only significantly affect the cell wall plastic deformation rates and wall loosening chemistry.
40


CHAPTER IV
DIMENSIONLESS CHARACTERIZATION OF CELL WALL DEFORMATION RATES AND STRESS RELAXATION RATES TO HELP IDENTIFY CHANGES IN WALL LOOSENING CHEMISTRY, AND WALL COMPOSITION AND ARCHITECTURE IN STIFF MUTANTS OF PHYCOMYCES BLAKESLEEANUS
Introduction
The main objectives of this chapter are to quantitate cell wall deformation and stress relaxation rates in an attempt to gain insight into changes in wall loosening chemistry, and wall composition and architecture of two stiff mutants (C216 and C149) of the model organism Phycomyces blakesleeanus. The stiff mutants are defective in the genes madD, E, F, G, and J exhibiting diminished tropic (bending) responses compared to the wild type (WT) I71-73!. This has prompted this investigation to learn if the cell wall is affected by the altered genes to cause the smaller tropic responses. Prior experimental research shows that the irreversible cell wall extensibility, cp, for stiff mutants C216 and C149 is significantly smaller than that of the WT [24]. No evidence was provided to learn whether the wall deformation rates and stress relaxation rates were affected by the altered genes. In this study, the |""|pe, |"lpv, and [lev dimensionless parameters are used to quantify cell wall deformation rates and stress relaxation rates to gain insight in changes of cell wall loosening chemistry, wall composition and architecture of the two stiff mutants, C216 and C149, of P. blakesleeanus. Based on the smaller cell wall extensibility reported for stiff mutant cell walls it is predicted that the magnitude of flpv (irreversible wall deformation rate) for stiff mutants is smaller than the WT. However, because the magnitude of the longitudinal volumetric elastic modulus, a,
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is not known, the magnitudes of |""|pe (stress relaxation rate) and |""|ev (wall composition and architecture) cannot be predicted. Here, in vivo turgor pressure step-up experiments are used to measure a_, and compare measured values between stiff mutants and WT. Measured average values of el and published data from in vivo creep experiments are used to compute the magnitudes of [Ipe, flpv, and [lev for stiff mutants and WT. Ortega [12] published the magnitude of flpe of in vivo creep tests for WT sporangiophores of P. blakesleeanus. This value is used to compare flpe magnitudes computed for the stiff mutants C216 and C149.
Materials and Methods Biological Material
Vegetative spores of the stiff mutant gene strain C216 geo -(-) were originally obtained from Ishinomaki Senshu University, Miyagi, Japan. The C149 madD120(~) strain was obtained from ATCC: The Global Research Center, Virginia, USA. Sporangiophores were inoculated on sterile growth medium consisting of 4% (w/v) YM agar. After inoculating, the sporangiophores were incubated under diffuse incandescent light and constant temperature (22° C ± 2°). Stage IVb sporangiophores, 2-2.5 cm in length, were selected for experiments from the second to the seventh crop. Stage IVb sporangiophores are used because they exhibit a nearly constant growth rate and rotation I49’ 52l
Elongation
The change in elongation in the sporangiophore is determined by measuring the change in length, AL, of a reference point, in this case, the edge of the sporangium is
42


used. The length is measured by taking snapshots using a USB camera (720P HD, GUCEE HD92 Skype Web Camera) attached to a long focal length horizontal microscope (Gaertner;7011 Keyepiece and 32m/m EFL objective) mounted to a 3-D micromanipulator (Line ToolCo.;modelH-2, with digital micrometer heads). Snapshots were acquired using open source software Vividia Ablescope and were analyzed using open source software ImageJ. The diameter of the sporangium for each sporangiophore was measured before initiation of the experiment to use as a scale reference in ImageJ. Figures 14 and 15 show the snapshots of the sporangiophore obtained by the method described above.
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Figure 14. Snapshot of a sporangiophore depicting the method of calibration in ImageJ for elongation measurements using the sporangium as a scale reference. The sporangium’s diameter was measured with a micrometer attached to the microscope set-up.
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Figure 15. Snapshot of the sporangiophore as it is elongated by the addition of oil using a microcapillary attached to a pressure probe.
Turgor Pressure
The turgor pressure of the sporangiophore is measured continuously using a manual version of the pressure probe I17’ 25l Figure 16 shows the version of the pressure probe used. A USB gage pressure transducer is used in the pressure probe which was purchased from Ellison Sensors Inc, Boca Raton, Florida, USA (model GD4200 USB-100BAR) and tested for calibration inside the pressure probe with a Fleise Bourdon Tube Pressure Gauge (Dresser Industries, Newton, CT, USA; model CMM, 0-200 PSIG Range). The transducer’s output was recorded on the software package included with the USB transducer.
The pressure probe was mounted on a 3-D manipulator so that the microcapillary tip (10-15 pm in diameter) could be guided to impale the sporangiophore under visual observation using a High Resolution Digital Microscope (Jiusion 40 to 1000x
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Magnification Endoscope, 8 LED USB 2.0 Digital Microscope, AMAZON, Seattle, WA, USA). The microcapillary of the pressure probe was filled with inert silicon oil (Dow Corning Corp.; fluid 200, 1.5 centistoke viscosity). After the cell was impaled, the cell sap oil interface was pushed to the surface of the cell vacuole and maintained at the fixed location to measure the turgor pressure of the sporangiophore I17’ 25l (Figure 17). The higher turgor pressure was maintained by small injections of inert silicon oil and also ensuring silicon oil was flowing into the cell vacuole for immediate step-up in turgor pressure.
Figure 16. Manual pressure probe with a USB pressure transducer.
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Figure 17. Snapshot of a sporangiophore being impaled by the microcapillary tip. The cell sap interface flows out of the cell and needs to be brought to the surface of the cell in order to read the turgor pressure and ensure oil flows into the cell vacuole. This is accomplised by increasing the pressure with the pressure probe.
In-vivo Step-up Experiments
The in vivo step-up experiment requires constant short time interval (30 sec) increases in turgor pressure of 0.03-0.04 MPa (turgor pressure step-ups) produced to the sporangiophore and immediate measurement of the elongation. The biophysical variable, a_, can be determined by this method and through the Ortega Growth Equation representing the wall deformation (elongation rate for longitudinal extending cells)[23’42], Equation (4.1). When pressure step-ups are large (P > 0.03 MPa) and at short time intervals it ensures that the irreversible component in Equation (4.1) is minimized and can be neglected (see Appendix A for mathematical proof). The equation is then simplified and can be solved for el (Equation 4.2). Where AP is the magnitude of the
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pressure step-up and AL is the change in elongation after the pressure step-up. This is validated by experimental research in which large turgor pressure step-ups cause a transient decrease in elongation growth rate of sporangiophores of P. blakesleeanus[17’
25]
1 dL
1 dP
(4.1)
A P
(4.2)
Protocol for in-vivo step-up experiments
A stage III or IVb sporangiophore (typically 2-2.5 cm in length) in a glass shell vial is selected and adapted for 20 minutes to the room temperature of 21 -23°C (Figure 18), to room lights (cool-white fluorescent lamps hung from the ceiling), and to bilateral swan-neck light guides (from Schoelly Fiber-optic; the end of each light guide is positioned approximately 8-12 cm on either side of the sporangiophore at an angle of about 30° from the horizontal) from a fiber-optic illuminator (Flexilux 90; FILU Light Source 90/Wfrom Schoelly Fiber-optic, Denzlingen, FRG, which filtered out nearly all of the infrared light). The sporangium is measured and recorded using the micrometer attached to the USB camera microscope set-up. Following this adaptation period, the length of the sporangiophore is measured and the pressure transducer software is initiated. The pressure in the pressure probe is increased between 0.03-0.09 MPa before impaling the cell and the cell is immediately impaled by the micro capillary tip of the pressure probe to measure the turgor pressure. Once a cell-sap interface is located the Image acquisition software is also initiated to take snapshots of the sporangiophore
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at 30-second intervals with a 5-second delay from the pressure step-ups. The snapshots are later analyzed to measure elongation using ImageJ. For the first 30-60 seconds into the experiment, the turgor pressure is maintained, 30 seconds after, the turgor pressure is increased by 0.03-0.04 MPa and maintained for 30 seconds by injecting inert silicon oil into the cell vacuole (Figure 15). This same procedure (turgor pressure step-up) is followed until the cell ruptures by not sustaining any more oil into the vacuole and bursting or until a leak is present. Figure 18 shows the experimental set-up for an in vivo turgor pressure step-up experiment.
Horizontal
Figure 18. Experimental set-up for an in vivo pressure step-up experiment. A horizontal microscope with a USB camera is used to capture elongation of the sporangiophore. A USB miscroscope helps guide the microcapillary needle into the sporangiophore. A pressure probe is used to impale, measure, and control the cell’s turgor pressure.
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Dimensionless Parameters
The ripe, ripv, and |""| ev dimensionless parameters are used to quantitate cell wall deformation rates and stress relaxation rates to gain insight on wall loosening chemistry, and wall composition and architecture in the stiff mutants. The |"|pe parameter was shown to control stress relaxation in walled cells [12]. It was identified as the ratio of relative volumetric plastic and elastic deformation rates of the cell wall[48]. The |""|pe parameter reflects the wall loosening and hardening characteristics of the cell wall chemistry[12] which regulates wall mechanical properties and thus wall deformation and expansive growth of cells with walls.
£(o relative volumetric plastic deformation rate n - _L = ---------------------1--------1--------------
y vs relative volumetric elastic deformation rate
This dimensionless number can be used to compare different cell species or mutants from same cell species in terms of their wall deformation rates, stress relaxation rates, wall loosening chemistry, and expansive growth. Plastic and elastic deformation rates of the wall can independently be computed using the dimensionless parameters |""|pvand |""|ev[481. The |""|pv parameter reflects cell wall loosening chemistry and the |""|ev parameter reflects wall composition and architecture. These parameters can be used to gain further insight into the individual processes represented.
(pPc relative volumetric plastic deformation rate
Ylvv —
Vc
relative volumetric growth rate
Pc relative volumetric elastic deformation rate ^ev e relative volumetric growth rate
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Experimental Results
Longitudinal Volumetric Elastic Modulus, el
The magnitude of the longitudinal volumetric elastic modulus, el, is determined from in vivo turgor pressure step-up experiments conducted on stage III (non-growing) and stage IVb (growing) sporangiophores from stiff mutant strains, C216 geo- (-) and C149 madD120 (-). Average values of a for stage III (non-growing) sporangiophores were determined to get the range of values that could be exhibited by stage IVb (growing) sporangiophore a-values.
A typical in vivo turgor pressure step-up experiment for a stage IVb sporangiophores is shown in Figure 19 with 0.04 MPa pressure step-ups (See Appendix C for more similar experimental results). Turgor pressure step-ups larger or equal than 0.03 MPa at short time intervals ensures that the irreversible deformation rate (steady-state growth rate since P=constant for the time interval) is negligible and eliminated from Equation (4.1). For the growing sporangiophores the first pressure step-up is not considered to produce a purely elastic elongation and therefore is not considered as part of the a-values used to compute an average el. All other values that correspond to the pressure step-ups after the first are considered to produce purely elastic elongations. For stage III sporangiophores (non-growing) all pressure step-ups are considered to be part of the average a-value. Average values for each sporangiophore tested are used to determine the mean value for each stiff mutant and stage.
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B
Time (sec)
*ref (sec)
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Figure 19. Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus behavior for a single stage IVb C149 sporangiophore during an in vivo turgor pressure step-up experiment. A. Turgor pressure behavior. The cell is impaled at the second mark represented by the red arrow and the turgor pressure is increased until the cell-sap interface is brought to the surface of the cell. Turgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressure step-up is applied to the cell (green arrow). The new turgor pressure is maintained constant until 30 seconds later and the next pressure step-up is applied. tref=o indicates the time when elongation measurements began. A total of 6 pressure step-ups were applied to the sporangiophore which resulted in purely elastic elongation. B. Elongation as a function of time. Elongation was graphed for the corresponding pressure step-ups at 30-sec intervals. The time interval, W = 30-210 sec (elongations after the black dashed line), represents purely elastic elongations considered for an average a-value. The cell sustains a maximum pressure of« .75 MPa before a leak occurs. The leak is represented by a decrease in elongation due to the leakage of oil and cell material (purple arrow). The change in elongation, AL, and AP, are used to determine el for each pressure step-up. A total of 6 a-values are determined for this experiment and an average value is computed. C. Plot of el as a function of turgor pressure, P. Each a_-value is graphed with its corresponding pressure. The average a-value for this sporangiophore is represented as a red dashed line (a_average= 92 MPa). The longitudinal volumetric elastic modulus is approximately constant for the range of turgor pressures the cell was subjected to.
Figure 20 compares average a-values of stage III (non-growing) between WT and stiff mutants. Results from student f-tests revealed no difference for a-values between WT and stiff mutants, but the results were less significant (p=0.07 and 0.06). Figure 21 compares average a-values of stage IVb (growing sporangiophores) between WT and stiff mutants. No significant difference was found for a-values between WT and C149 (p=0.96). Furthermore, a-values between WT and C216 are not different, but the results are less significant (p= 0.054). Additionally, student f-tests results confirmed that no significant difference existed between stage III (non-growing) and IVb (growing) sporangiophore a-values.
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100 r
90 -
80 -
70 -
*â–  60 -Q.
§ 50 -* 40 -
30 -20 -10 -0 -
â–  C216
â–  C149
â–  WT
Stage
Figure 20. Comparison of average a_for stage III (non-growing) sporangiophores between WT and stiff mutants (C216 and C149). a-values for stiff mutants were measured using in vivo turgor pressure step-up experiments while a_-values for WT were obtained from Ortega [23l The vertical bars represent the standard error (SE) of the mean value and the horizontal bars enclose the area of statistical comparison for p-values.
Figure 21. Comparison of average a for stage IV (growing) sporangiophores between WT and stiff mutants (C216 and C149). a-values for stiff mutants were measured using in vivo turgor pressure step-up experimements while el-values for WT were obtained from Ortega [23]. The vertical bars represent the standard error (SE) of the mean value and the horizontal bars enclose the area of statistical comparison for p-values.
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Dimensionless Parameters nPe. nPVand flev
Table 2 shows the biophysical variables dL/df (elongation growth rate), m (longitudinal irreversible cell wall extensibility), el (longitudinal volumetric elastic modulus), and Pc (critical turgor pressure) for WT and stiff mutants C216 and C149. All data for WT sporangiophores was obtained from Ortega [23] and Ortega et al[24] published results. Stiff mutant data for variables m and Pc are obtained from Ortega et al l24l published results. Results from student t-tests revealed that m and Pcwere significantly different between WT and both mutants [24]. The values in Table IV-1 are used to compute the magnitudes of the npe, npv, and nev dimensionless parameters. The maximum and minimum values for each variable in Table 2 are used to compute maximum and minimum values for the dimensionless parameters (see Appendix D for analysis).
Table 2. A comparison of relevant biophysical variables used in computing magnitudes of the npe, npv, and nev dimensionless parameters: a_, m, Pc, and dL/df for stage IVb sporangiophores for WT, C216, and C149 strains. Values for dL/df, m, and PcforWT are obtained from Ortega et al[24]. Values for m and Pc for stiff mutants are obtained from Ortega et al l24\ el and dL/df values were measured in this study.
Variable Wild type C216 C149
units Mean ± SEM (n) Mean ± SEM (n) Mean ± SEM (n)
el (MPa) 60 ±5.1 (27) 49.2 ±2.6 (24) 60.5 ±5.3 (17)
m (pm min_1 MPa'1) 997 ±164 (20) 222 ±40 (18) 170 ±30 (8)
Pc (MPa) 0.26 ± 0.01 (20) 0.13 ±0.05 (18) 0.18 ±0.08 (8)
dL/df (pm min'1) 34 ±3.1 (20) 27.3 ±2.5 (59) 27.8 ±3.1 (59)
Figure 22 compares magnitudes of the npe parameter between WT and stiff mutants determined from in vivo creep and in vivo step-up experiments conducted on intact stage IVb sporangiophores (see Appendix E for calculations of fl magnitudes).
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The magnitudes of npeand npvforthe stiff mutants C216 and C149 are dramatically smaller than for WT (Figures 22 and 23). The difference in magnitudes between WT and stiff mutants of the npe and npv parameters provides a comparison of the stress relaxation rates and plastic deformation rates further providing direct evidence on whether cell wall loosening chemistry has changed. The magnitudes of the nev parameter were also computed and compared to gain insight on the magnitude of elastic deformation rates in the wall for WT and stiff mutants (Figure 24). The magnitudes of the nev parameter for stiff mutants were similar to the WT. Plastic deformation rates and stress relaxation rates were reduced in stiff mutants while the elastic deformation rates of the wall were not substantially affected (Figures 22, 23, and 24).
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3000
2500
2000
Q.
n. 1500 1000 500 0
â–  C216
â–  C149
â–  WT
Figure 22. A comparison of the npe values calculated for growing sporangiophores of P. blakesleeanus (Stage IVb). The maximum and minimum values of the npe parameter are represented by the confidence intervals that are calculated from the statistical data (standard deviation and standard error) presented in this research and in papers: [23’24].
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12
10 -
8 -
>
Q.
6 -
4 -
2 -
â–  C216
â–  C149
â–  WT
Figure 23. A comparison of the npv values calculated for growing sporangiophores of P. blakesleeanus (Stage IVb). The maximum and minimum values of the npv parameter are represented by the confidence intervals that are calculated from the statistical data (standard deviation and standard error) presented in the paper:[24].
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Figure 24. A comparison of the nev values calculated for growing sporangiophores of P. blakesleeanus (Stage IV). The maximum and minimum values of the nev parameter are represented by the confidence intervals that are calculated from the statistical data (standard deviation and standard error) presented in this research and papers: I23 24!.
Discussion
The dimensionless parameter,Pipe, is central to stress relaxation and expansive growth [12]. This dimensionless parameter is the ratio of relative volumetric plastic and elastic deformation rates of the cell wall which reflects the wall loosening and hardening characteristics of the cell wall[12]. The flpe parameter can be further decomposed into the dimensionless plastic deformation rate and elastic deformation rate of the wall by the ripvand [levparameters. The flpvparameter reflects cell wall loosening chemistry and the [V parameter reflects cell wall architecture and composition.
In this study, the [Ipe, f|pv, and [lev dimensionless parameters were used to quantitate cell wall deformation rates and stress relaxation rates in two stiff mutants of
59


P. blakesleeanus to gain insight on whether the mutant genes madD, E, F, G, and J genes affect these processes. The results provide insight into changes in wall loosening chemistry, wall architecture and wall composition of the stiff mutants. Prior experimental research showed that the magnitudes of the irreversible cell wall extensibility, cp, for stiff mutants C216 and C149 are significantly smaller than the WT l24\ but no further evidence was provided to learn whether the wall deformation rates and stress wall relaxation rates were affected by the altered genes.
In vivo turgor pressure step-up experiments were conducted to measure the longitudinal volumetric elastic modulus, a_, in stiff mutants to compute magnitudes for the ripe and [lev parameters. Previous data from in vivo creep experiments on stiff mutants and WT that measured the longitudinal irreversible cell wall extensibility, m, and critical turgor pressure were used to compute the magnitudes of the flpv parameter for stiff mutants and WT. Magnitudes of ripe, flpv, and flevwere compared between stiff mutants and WT. The magnitudes of [Ipe and flpv were substantially smaller in stiff mutants compared to the WT (Figures 22 and 23). The results demonstrate that the altered genes reduced stress relaxation rates and plastic deformation rates to significantly alter cell wall loosening chemistry. The magnitudes of the |“|ev parameter for stiff mutants and WT were also computed to learn if the cell wall composition and architecture was affected by the altered genes. The magnitudes of the |“|ev parameter were similar in stiff mutants and WT (Figure 24). These results indicate that the wall elastic deformation rate did not substantially change and suggests that the general wall composition and architecture has not significantly changed with the altered genes. The results from dimensionless numbers ripe, flpv, and \~\ev demonstrate that the defective
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genes madD, E, F, G and J substantially reduce irreversible deformation rates and stress relaxation rates in stiff mutants which reflects a significant change in wall loosening chemistry. Furthermore, the altered genes did not substantially change the reversible deformation rates in the wall of stiff mutants to significantly produce a change in cell wall composition and architecture. The cell wall is the expression site of tropisms in P. blakesleenus; thus a decrease in wall loosening chemistry would be consistent with the observed diminished tropic responses exhibited by the stiff mutants.
From the results in this study, it can be predicted that 1) there are less cell wall remodeling enzymes necessary to break load-bearing bonds in the stiff mutants and/or 2) the function of these cell wall remodeling enzymes was reduced in the stiff mutants. There is evidence to believe that chitinases (hydrolytic enzymes found in fungal cell walls) may be involved in breaking glycosidic bonds in chitin I75-77!. Flerrera-Estrella and Ruiz-Flerrera [78] showed that the cell wall of Phycomyces sporangiophores was softened by illuminating with white light dark adapted sporangiophores. The investigators labeled reducing ends in chitin which increased after the light stimulation. Each poly-GIcNAc chain in chitin contains only one reducing end, therefore, an increase in reducing ends indirectly measures breakage of chitin microfibrils. Most importantly, it was shown that madB and madD mutants did not show this increase in reducing ends. This evidence along with the results presented in this study point to chitinases as being involved in cell wall plastic deformation, wall stress relaxation and wall loosening of P. blakesleeanus.
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Limitations
In the past, pressure probe experiments with Phycomyces have been conducted with two people, one to operate the pressure probe, the other to measure elongation. In other areas of cell wall research, the cells tested are large enough to attach an extensometer to measure the elongation. Due to the size and fragility of Phycomyces this method cannot be used. Therefore experiments with Phycomyces have been a two-person job enabling higher accuracy results. In this study, a data acquisition system was designed to run the experiments with one person actively involved in the operation of the pressure probe and not in the elongation measurements. Elongation measurements were automated using a USB camera and image capturing software to take snapshots of the cell at specified time intervals. The snapshots were later analyzed with a cell measurement software. Throughout the different steps in this process, the accuracy of the elongation measurements could be reduced affecting the overall data. Care was taken to accurately acquire and measure snapshots, but this limitation did exist. In the future, a higher resolution camera could be used with better image acquisition software. All software used in this study is open source and easily operated which prompted its use in this study.
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CHAPTER V
CELL WALL ARCHITECTURE AND DYNAMICS OF STIFF MUTANT SPORANGIOPHORES OF PHYCOMYCES BLAKESLEEANUS
Introduction
In the previous chapter, it was demonstrated that the madD, E, F, G, and J genes substantially reduced cell wall plastic deformation rates and stress relaxation rates which reflected a significant alteration in wall loosening chemistry in the stiff mutants. The objective of this chapter is to investigate whether these altered genes affect cell wall architecture and wall dynamics. Measurements of the sporangiophore’s rotation and elongation indirectly provide evidence for microfibril orientation (wall architecture) and reorientation (wall dynamics).
Fibril-Slippaqe-Reorientation Hypothesis for Helical Growth in Phycomyces
Helical growth is observed in many plants, algal, and fungal cells. The model organism Phycomyces blakesleeanus undergoes helical growth (simultaneous elongation and rotation of the wall along the longitudinal axis) throughout its development. Helical growth in P. blakesleeanus has been investigated to elucidate the biophysical and molecular mechanisms associated with this phenomenon [60’66’67’ 79'81]. Early investigations were not able to assess whether the ratio of rotation rate and elongation rate (R) was independent of the magnitude of elongation rate and assumed R to be constant and independent of elongation rate I61'80’ 82l From this assumption, the fibril-reorientation and fibril-slippage mechanisms were founded [61’ 63'65]. Later Ortega et
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al[62] demonstrated that the ratio of rotation rate and elongation rate (R) decreases as the elongation rate (dL/df) increases for stage IVb WT sporangiophores. The authors proposed a modified fibril-reorientation mechanism to account for this new finding. The proposed mechanism postulates there is a fibril-reorientation subzone in which elongation and rotation occur and a fibril-slippage subzone in which elongation and no rotation occurs for fast-growing sporangiophores (Figure 25). In the fibril-slippage subzone, the fibrils are oriented in the longitudinal direction (direction of irreversible cell wall deformation) and can no longer reorient. The modified model predicts that in fastgrowing sporangiophores with a relatively long growth zone the region farthest away from the sporangium only produces elongation contributing to the elongation rate but no rotation is exhibited. In the slow-growing sporangiophores with relatively short growing zones there only exists the fibril-reorientation subzone producing a larger rotation.
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Figure 25. Fibril reorientation and fibril slippage mechanism postulated by Ortega et al l62l for a fast growing with relatively long growing zone sporangiophore. It was postulated that the large growth zone is divided into a fibril-reorientation (elongation with rotation occurs) subzone and a fibril-slippage subzone (elongation occurs with no rotation). Microfibril orientation closest to the sporangium lies transversely and those
farthest away from the sporangium lie longitudinally. Figure was taken from Ortega et al
[62]
Materials and Methods Biological Material
Vegetative spores of the stiff mutant gene strain C216 geo -(-) were originally obtained from Ishinomaki Senshu University, Miyagi, Japan. The C149 madD120(~) strain was obtained from ATCC: The Global Research Center, Virginia, USA. Sporangiophores were inoculated on sterile growth medium consisting of 4% (w/v) YM agar. After inoculating, the sporangiophopres were incubated under diffuse
65


incandescent light and constant temperature (22° C ± 2°). Stage IVb sporangiophores, 1.5-2.5 cm in length, were selected for experiments from the second to the seventh crop. Stage IVb sporangiophores are used because they exhibit constant growth rate and rotation I49’ 52l
Elongation rate
The change in elongation in the sporangiophore is determined by measuring the change in length, AL, of a reference point, in this case, the edge of the sporangium is used. The length is measured by taking snapshots at 1-minute intervals using a web camera (720P HD, GUCEE HD92 Skype Web Camera) attached to a long focal length horizontal microscope (Gaertner;7011 Keyepiece and 32m/m EFL objective) mounted to a 3-D micromanipulator (Line ToolCo.;modelH-2, with digital micrometer heads). Snapshots were acquired using open source software Vividia Ablescope and were analyzed using open source software ImageJ. The elongation rate was obtained by dividing the AL over the time interval, At, in which it was measured. The diameter of the sporangium for each sporangiophore was measured before initiation of the experiment to use as a scale reference in ImageJ.
Rotation rate
A thin piece of hair (7-10 mm long) was attached to the top of the sporangium of the stage IVb sporangiophore, perpendicular to the longitudinal axis of the cell. Sometimes a small amount of petroleum jelly was used to help the piece of hair adhere to the sporangium. When viewed from above the piece of hair rotates in the clockwise rotation for a stage IVb sporangiophore. The rotation rate was determined by measuring
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the rotation rate of the piece of hair using a USB microscope to acquire snapshots at 1-minute intervals and later analyzed using ImageJ software. Figure 26 shows snapshots for elongation rate and rotation rate measurements.
Figure 26 (A) Snapshots showing a thin piece of hair attached to the sporangium used as a reference point to determine rotation rate. (B) Snapshots used to determine the elongation rate for each sporangiophore. The same time interval was used to determine rotation and elongation rate.
Results
The elongation and rotation rates were measured concurrently for stage IVb sporangiophores of C216 and C149 (n=59 for each strain). In constant conditions, the elongation rate fluctuates around an average value with small fluctuations. The
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elongation and rotation rate was averaged over the same 10-minute interval in which the elongation rate was observed to be constant and the ratio of rotation rate and elongation rate, R, was calculated (Figure 27). R was calculated for each sporangiophore and averaged with values of R obtained from other sporangiophores growing within a specified range of elongation growth rates (i.e. 1-10.9, 11-20.9, 21-30.9, etc); see Figure 28. Each average R-value is plotted as a function of the elongation rate, all three strains WT, C216, and C149 are plotted in the same graph for comparison (Figure 28). The behavior exhibited by the stiff mutants is similar to the WT in which R decreases with increasing elongation rate. The large standard error bars of R for the slowest growing sporangiophores in stiff mutants suggest that this value lies in the same range of the slow-growing sporangiophores of WT and that the difference might not be significant.
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Time (min)
Time (min)
Figure 27. Results from a typical rotation experiment on a single stage IVb C149 sporangiophore . The elongation rate and rotation rate is measured to compute R. For this sporangiophore R= 0.15. The same time interval is used to average both elogantion and rotation rates.
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Figure 28. Average Ravg values versus elongation rates for stage IVb sporangiophores for WT and stiff mutant strains. Each Ravg-value represents the mean value of R for sporangiophores with elongation rates at specified elongation rates: 1-10.9, 11-20.9, 21-30.9, 31-40.9, 51-60.9, and 61-70.9. The vertical bars represent the standard error (SE) of the mean value (n=59 for stiff mutants and n=171 for WT). Data to reproduce WT behavior was obtained from Ortega et al l62l.
Discussion
It is found that there is a similar dependence of R (ratio of rotation rate to elongation rate) on elongation growth rate in the stiff mutants as in the WT {R decreases as dL/df increases) (Figure 28). The results presented indicate that the wall architecture (microfibril orientation) and wall dynamics (reorienting of microfibrils during elongation growth) of stiff mutants is similar to that of WT sporangiophores. The results indicate that the altered genes did not significantly change the wall architecture and wall dynamics. In the previous chapter, it was found that the dimensionless parameter that defines the reversible wall deformation rate (nev) was not substantially different in stiff mutants and WT. This indicated that the altered genes did not significantly change the wall composition and architecture. Thus similar flev values further support the
70


conclusion that wall architecture was not significantly changed by the altered genes. Furthermore, the fibril-reorientation-slippage mechanism postulated by Ortega et al l62l is further validated by the stiff mutant rotation and elongation behavior.
Limitations
Statistical analysis was unable to be conducted on the results presented in this chapter due to the unavailability of the WT raw data. It is suggested that to help get a stronger conclusion the rotation and elongation behavior be measured for the WT sporangiophores to properly conduct statistical analysis.
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CHAPTER VI
CONCLUSIONS AND FUTURE WORK
The principal objective of this work was to quantify cell wall deformation rates and stress relaxation rates to gain insight into the wall loosening chemistry, and wall architecture and wall dynamics of two stiff mutants of Phycomyces blakesleeanus. Dimensionless parameters and biophysical experiments were used to reach this objective. The stiff mutants exhibit diminished tropic responses compared to the WT prompting an investigation to learn if the cell wall is affected by the altered genes to cause the deficient responses. The results presented demonstrate that the cell wall is indeed affected further affecting the sensory transduction pathway by the genes madD, E, F, G, and J. Specifically, the results demonstrate that the altered genes reduce the plastic deformation rates and stress relaxation rates to significantly affect the wall loosening chemistry. A general objective of this work was to demonstrate that wall deformation rates and stress relaxation rates can be quantified to gain insight on changes of wall loosening chemistry, and cell wall composition and architecture using the dimensionless parameters npe, npv, and nev.
Conclusions
In Chapter IV of this study, it was proposed that three dimensionless parameters obtained from the Ortega Growth Equations be used to quantitate cell wall deformation rates and stress relaxation rates to gain insight into changes in wall loosening chemistry and cell wall composition and architecture. It was found that the irreversible (plastic) deformation rates and stress relaxation rates in stiff mutants were substantially smaller
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compared to the wild type. This implied that the altered genes significantly changed the cell wall loosening chemistry responsible for irreversible deformation (expansive growth). Also found was that the reversible deformation rates of the wall were similar in the stiff mutants and WT. This indicated that the cell wall composition and architecture was not significantly changed by the altered genes. The results in this chapter demonstrated that the |"|pe, ripvand |"|ev dimensionless parameters can be used to predict changes at the biochemical level. Based on the results it was predicted that the stiff mutants have a lower amount of chitin cleaving enzymes known as chitinases and/or that the altered genes affected the chitinases enzymatic activity.
In Chapter V, the wall architecture and wall dynamics were investigated to gain further information on whether the altered genes affected these behaviors. It was found that the stiff mutants exhibit a similar dependence of R (ratio of rotation rate to elongation rate) vs. dL/dt (elongation rate) as in the WT. This indicates that stiff mutants have a similar wall architecture (microfibril orientation) and wall dynamics (microfibril reorientation) as the WT demonstrating that the altered genes did not significantly affect these behaviors. Furthermore, it was shown that the Fibril-slippage-reorientation mechanism postulated by Ortega et al l62l was further validated by the stiff mutant cell wall dynamics behavior.
Based on the results from the studies presented in Chapters IV and V it is concluded that the madD, E, F, G, and J genes only substantially alter cell wall loosening chemistry affecting irreversible deformation. The results demonstrated that the important processes in expansive growth were affected by the altered genes (stress relaxation and irreversible deformation). This indicates that the diminished tropic
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responses exhibited by the stiff mutants is due to an alteration in the cell wall loosening chemistry by the madD, E, F, G, and J genes.
Suggestions for Future Work
One of the predictions that arise from the results of Chapter IV can further be investigated to learn if the chitinase activity in stiff mutants is lowered or if it’s simply affected or both. The amount of chitinase activity can be indirectly accounted for by measuring the number of chitosomes in stiff mutants and WT. It has been shown that chitosomes carry 80% of chitin content in Phycomyces[70]. To learn if chitinase activity is lowered in stiff mutants the in vitro activation of chitosomes in stiff mutants can be investigated by similar methods used by Herrera-Estrella et al [83]and Martinez-Cadena and Ruiz-Herrera [84].
An important area of investigation is with chitin synthetase mutants. Fungal chitinases are diverse and are used in multiple functions in fungi I85 86!. These functions include 1) degradation of exogenous chitin present in fungal cell walls of dead hyphal fragments or in the skeletons of dead arthropods, and the use of degradation products as a nutrient source; 2) cell wall remodeling during the fungal life cycle, which includes roles in hyphal growth, branching, hyphal fusion, and autolysis; 3) competition and defense against other fungi and arthropods in the fungal habitat[87]. Some fungi use chitinases to attack other fungi, insects or nematodes [87]. Thus, not all class of chitinases would elicit changes in cell wall deformation and stress relaxation that reflect changes in cell wall loosening chemistry. In filamentous fungi, chitinases are classified into subgroup A (class V), B (class III) and subgroup C (other classes)[87]. It has been
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suggested that chitinases in subgroup B are responsible for cell wall remodeling in fungi [87-90] Qurrently, there is no evidence to report whether mutants in this subgroup are affected in cell wall deformation rates and stress relaxation rates. Investigating mutants in this subgroup and the other subgroups can help narrow down the number of chitinases responsible for cell wall remodeling, morphology and expansive growth in Phycomyces blakesleeanus and other fungi. Furthermore, these investigations can help with novel methods in the control of fungal diseases.
Another future investigation could consist of microscopically identifying wall architecture and composition in the WT and stiff mutant sporangiophores. The results from such a study can help validate the results presented in Chapters IV and V. It would be expected that the microfibril arrangement and composition is similar in mutants and wild type.
One can think of using the dimensionless parameters as a way to rule out genes, proteins and enzymes that do not produce the expected wall loosening chemistry, wall stress relaxation rates, and wall deformation rates observed in expansive growth. The dimensionless parameters can help gain better insight into other behaviors exhibited by cells with walls such as helical growth in Phycomyces blakesleeanus (Chapter V).
These methods can help with optimizing cell walls for the use by industrial and pharmaceutical sectors.
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REFERENCES
[1] H. Vogler, D. Felekis, B. Nelson, and U. Grossniklaus, "Measuring the
Mechanical Properties of Plant Cell Walls," Plants, vol. 4, no. 2, p. 167, 2015.
[2] S.-Y. Ding, Y.-S. Liu, Y. Zeng, M. E. Himmel, J. 0. Baker, and E. A. Bayer, "How
does plant cell wall nanoscale architecture correlate with enzymatic digestibility?," Science, vol. 338, no. 6110, pp. 1055-1060, 2012.
[3] R. N. Strange and P. R. Scott, "Plant disease: a threat to global food security," Annu. Rev. Phytopathol., vol. 43, pp. 83-116, 2005.
[4] J. G. Vallarino and S. Osorio, "Signaling role of oligogalacturonides derived
during cell wall degradation," Plant signaling & behavior, vol. 7, no. 11, pp. 1447-1449, 2012.
[5] J.-P. Latge, "The cell wall: a carbohydrate armour for the fungal cell," Molecular
Microbiology, vol. 66, no. 2, pp. 279-290, 2007.
[6] S. M. Bowman and S. J. Free, "The structure and synthesis of the fungal cell wall," Bioessays, vol. 28, no. 8, pp. 799-808, 2006.
[7] D. J. Cosgrove, "Growth of the plant cell wall," Nature reviews molecular cell biology, vol. 6, no. 11, p. 850, 2005.
[8] J. K. Ortega, J. T. Truong, C. M. Munoz, and E. L. Ortega, "Expansive Growth of Cells with Walls: Force Generation and Growth Regulation," Cells, Forces, and the Microenvironment, p. 357, 2015.
[9] D. J. Cosgrove, "Plant cell wall extensibility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall-modifying enzymes," Journal of Experimental Botany, vol. 67, no. 2, pp. 463-476, 2015.
[10] D. J. Cosgrove, "Water uptake by growing cells: an assessment of the controlling roles of wall relaxation, solute uptake, and hydraulic conductance," International Journal of Plant Sciences, vol. 154, no. 1, pp. 10-21, 1993.
76


[11] D. J. Cosgrove, "Wall relaxation and the driving forces for cell expansive growth," Plant physiology, vol. 84, no. 3, pp. 561-564, 1987.
[12] J. K. Ortega, "Dimensionless number is central to stress relaxation and
expansive growth of the cell wall," Scientific reports, vol. 7, no. 1, p. 3016, 2017.
[13] D. J. Cosgrove, "Wall extensibility: its nature, measurement and relationship to plant cell growth," New Phytologist, vol. 124, no. 1, pp. 1-23, 1993.
[14] D. J. Cosgrove, "Cell wall yield properties of growing tissue: evaluation by in vivo stress relaxation," Plant physiology, vol. 78, no. 2, pp. 347-356, 1985.
[15] D. J. Cosgrove, "Wall relaxation in growing stems: comparison of four species and assessment of measurement techniques," Planta, vol. 171, no. 2, pp. 266-278, 1987.
[16] P. B. Green, "Growth physics in Nitella: a method for continuous in vivo analysis of extensibility based on a micro-manometer technique for turgor pressure," Plant Physiology, vol. 43, no. 8, pp. 1169-1184, 1968.
[17] J. K. Ortega, E. G. Zehr, and R. G. Keanini, "In vivo creep and stress relaxation experiments to determine the wall extensibility and yield threshold for the sporangiophores of Phycomyces," Biophysical journal, vol. 56, no. 3, p. 465, 1989.
[18] J. A. Lockhart, "An analysis of irreversible plant cell elongation," Journal of theoretical biology, vol. 8, no. 2, pp. 264-275, 1965.
[19] J. H. Kroeger, R. Zerzour, and A. Geitmann, "Regulator or driving force? The role of turgor pressure in oscillatory plant cell growth," PloS one, vol. 6, no. 4, p. e18549, 2011.
[20] M. Probine, "Chemical control of plant cell wall structure and of cell shape," Proc. R. Soc. Lond. B, vol. 161, no. 985, pp. 526-537, 1965.
77


[21] L. Taiz, "Plant cell expansion: regulation of cell wall mechanical properties," Annual Review of Plant Physiology, vol. 35, no. 1, pp. 585-657, 1984.
[22] A. Geitmann and J. K. Ortega, "Mechanics and modeling of plant cell growth," Trends in plant science, vol. 14, no. 9, pp. 467-478, 2009.
[23] J. K. Ortega, "Growth rate regulation of cells with walls: The sporangiophores of Phycomyces blakesleeanus used as a model system," Rec. Res. Dev. Plant Physiol, vol. 5, pp. 1-19, 2012.
[24] J. K. Ortega, C. M. Munoz, S. E. Blakley, J. T. Truong, and E. L. Ortega, "Stiff mutant genes of Phycomyces affect turgor pressure and wall mechanical properties to regulate elongation growth rate," Frontiers in plant science, vol. 3, p. 99, 2012.
[25] J. K. Ortega, M. E. Smith, A. J. Erazo, M. A. Espinosa, S. A. Bell, and E. G. Zehr, "A comparison of cell-wall-yielding properties for two developmental stages of Phycomyces sporangiophores," Planta, vol. 183, no. 4, pp. 613-619, 1991.
[26] J. K. Ortega, J. T. Truong, C. M. Munoz, and D. G. Ramirez, "Cell wall loosening in the fungus, Phycomyces blakesleeanus," Plants, vol. 4, no. 1, pp. 63-84, 2015.
[27] D. J. Cosgrove, "Loosening of plant cell walls by expansins," Nature, vol. 407, no. 6802, p. 321,2000.
[28] D. J. Cosgrove, "Diffuse growth of plant cell walls," Plant physiology, vol. 176, no. 1, pp. 16-27, 2018.
[29] D. J. Cosgrove, "Enzymes and other agents that enhance cell wall extensibility," Annual review of plant biology, vol. 50, no. 1, pp. 391-417, 1999.
[30] D. J. Cosgrove, "Wall structure and wall loosening. A look backwards and forwards," Plant physiology, vol. 125, no. 1, pp. 131-134, 2001.
78


[31] S. McQueen-Mason, D. M. Durachko, and D. J. Cosgrove, "Two endogenous proteins that induce cell wall extension in plants," The Plant Cell, vol. 4, no. 11, pp. 1425-1433, 1992.
[32] H. G. Gerken, B. Donohoe, and E. P. Knoshaug, "Enzymatic cell wall
degradation of Chlorellavulgaris and other microalgae for biofuels production," Planta, vol. 237, no. 1, pp. 239-253, 2013.
[33] R. Palin and A. Geitmann, "The role of pectin in plant morphogenesis," Biosystems, vol. 109, no. 3, pp. 397-402, 2012.
[34] T. E. Proseus and J. S. Boyer, "Calcium deprivation disrupts enlargement of
Chara corallina cells: further evidence for the calcium pectate cycle," Journal of experimental botany, vol. 63, no. 10, pp. 3953-3958, 2012.
[35] T. E. Proseus and J. S. Boyer, "Pectate chemistry links cell expansion to wall deposition in Chara corallina," Plant signaling & behavior, vol. 7, no. 11, pp. 1490-1492, 2012.
[36] Y. B. Park and D. J. Cosgrove, "Changes in cell wall biomechanical properties in the xyloglucan-deficient xxt1/xxt2 mutant of Arabidopsis," Plant physiology, vol. 158, no. 1, pp. 465-475, 2012.
[37] J. Ortega and S. Welch, "Mathematical models for expansive growth of cells with walls," Mathematical Modelling of Natural Phenomena, vol. 8, no. 4, pp. 35-61, 2013.
[38] P. Green, R. Erickson, and J. Buggy, "Metabolic and physical control of cell elongation rate: in vivo studies in Nitella," Plant Physiology, vol. 47, no. 3, pp. 423-430, 1971.
[39] N. P. Money and F. M. Harold, "Extension growth of the water mold Achlya:
interplay of turgor and wall strength," Proceedings of the National Academy of Sciences, vol. 89, no. 10, pp. 4245-4249, 1992.
[40] J. Passioura and S. Fry, "Turgor and cell expansion: beyond the Lockhart equation," Functional Plant Biology, vol. 19, no. 5, pp. 565-576, 1992.
79


[41] A. Geitmann, MA. Geitmann and JKE Ortega, Trends Plant Sci. 14, 467 (2009)," Trends Plant Sci, vol. 14, p. 467, 2009.
[42] J. K. Ortega, "Augmented growth equation for cell wall expansion," Plant physiology, vol. 79, no. 1, pp. 318-320, 1985.
[43] J. S. Boyer, A. Cavalieri, and E.-D. Schulze, "Control of the rate of cell
enlargement: excision, wall relaxation, and growth-induced water potentials," Planta, vol. 163, no. 4, pp. 527-543, 1985.
[44] T. E. Proseus, J. K. Ortega, and J. S. Boyer, "Separating growth from elastic
deformation during cell enlargement," Plant physiology, vol. 119, no. 2, pp. 775-784, 1999.
[45] J. K. Ortega, "Plant cell growth in tissue," Plant physiology, p. pp. 110.162644, 2010.
[46] J. K. Ortega, R. G. Keanini, and K. J. Manica, "Pressure probe technique to study transpiration in Phycomyces sporangiophores," Plant physiology, vol. 87, no. 1, pp. 11-14, 1988.
[47] R. Fox, A. McDonald, P. Pritchard, and J. Leylegian, "Fluid Mechanics. 8th. 1," ed: New York: Wiley, 2011.
[48] J. Ortega, "Dimensional analysis of expansive growth of cells with walls," Res Rev: J Bot Sci, vol. 5, no. 3, pp. 17-24, 2016.
[49] E. C.-O. a. E. D. Lipson, Phycomyces. Cold Spring Flarbor, NY: Cold Spring Flarbor Laboratory Press, 1987.
[50] P. Galland, A. Palit, and E. Lipson, "Phycomyces: phototropism and light-growth response to pulse stimuli," Planta, vol. 165, no. 4, pp. 538-547, 1985.
[51] G. Loser and E. Schafer, "ARE THERE SEVERAL PHOTORECEPTORS
INVOLVED IN PHOTOTROPISM OF Phycomyces blakesleeunus? KINETIC
80


STUDIES OF DICHROMATIC IRRADIATION," Photochemistry and photobiology, vol. 43, no. 2, pp. 195-204, 1986.
[52] K. Bergman et ai, "Phycomyces," Bacteriological Reviews, vol. 33, no. 1, pp. 99-157, 1969.
[53] M. Delbruck, A. Katzir, and D. Presti, "Responses of Phycomyces indicating optical excitation of the lowest triplet state of riboflavin," Proceedings of the National Academy of Sciences, vol. 73, no. 6, pp. 1969-1973, 1976.
[54] M. Delbruck and W. Reichardt, "System analysis for the light growth reactions of Phycomyces," Cellular mechanisms in differentiation and growth, vol. 14, p. 3, 1956.
[55] C. N. Ahlquist and R. I. Gamow, "Phycomyces: Mechanical Behavior of Stage II and Stage IV 1," Plant physiology, vol. 51, no. 3, p. 586, 1973.
[56] D. S. Dennison and C. C. Roth, "Phycomyces sporangiophores: fungal stretch receptors," Science, vol. 156, no. 3780, pp. 1386-1388, 1967.
[57] J. K. E. Ortega and R. I. Gamow, "An Increase in Mechanical Extensibility during the Period of Light-stimulated Growth," Plant Physiology, vol. 57, no. 3, pp. 456-457, 1976.
[58] J. K. Ortega and R. I. Gamow, "Phycomyces: an increase in mechanical
extensibility during the avoidance growth response," Plant physiology, vol. 60, no. 5, pp. 805-806, 1977.
[59] J. K. Ortega, R. I. Gamow, and C. N. Ahlquist, "Phycomyces: a change in
mechanical properties after a light stimulus," Plant physiology, vol. 55, no. 2, pp. 333-337, 1975.
[60] M. J. Middlebrook and R. Preston, "Spiral growth and spiral structure: III. Wall structure in the growth zone of Phycomyces," Biochimica et biophysica acta, vol. 9, pp. 32-48, 1952.
81


[61] J. K. Ortega and R. I. Gamow, "The problem of handedness reversal during the spiral growth of Phycomyces," Journal of theoretical biology, vol. 47, no. 2, pp. 317-332, 1974.
[62] J. K. Ortega et al., "Helical growth of stage-IVb sporangiophores of Phycomyces blakesleeanus: the relationship between rotation and elongation growth rates," Planta, vol. 216, no. 4, pp. 716-722, 2003.
[63] K. Yoshida, T. Ootaki, and J. Ortega, "Spiral growth in the radially-expanding piloboloid mutants ofPhycomyces blakesleeanus," Planta, vol. 149, no. 4, pp. 370-375, 1980.
[64] J. K. E. Ortega, "Phycomyces: The Mechanical and Structural Dynamics of Cell Wall Growth," Doctor of Philosophy Aerospace Engineering, University of Colorado Boulder, UMI Dissertation Information Service, 1976.
[65] J. K. Ortega, J. F. Harris, and R. I. Gamow, "The analysis of spiral growth in
Phycomyces using a novel optical method," Plant physiology, vol. 53, no. 3, pp. 485-490, 1974.
[66] E. S. Castle, "Spiral growth and reversal of spiraling in Phycomyces, and their
bearing on primary wall structure," American Journal of Botany, vol. 29, no. 8, pp. 664-672, 1942.
[67] A. Oort, "The spiral-growth of Phycomyces," 1931: Koninklijke Akademie van Wetenschappen.
[68] S. Bartnicki-Garcia, "Cell wall chemistry, morphogenesis, and taxonomy of fungi," Annual Reviews in Microbiology, vol. 22, no. 1, pp. 87-108, 1968.
[69] J. Ruiz-Herrera, E. Lopez-Romero, and S. Bartnicki-Garcia, "Properties of chitin synthetase in isolated chitosomes from yeast cells of Mucor rouxii," Journal of Biological Chemistry, vol. 252, no. 10, pp. 3338-3343, 1977.
[70] J. Ruiz-Herrera, C. E. Bracker, and S. Bartnicki-Garcia, "Sedimentation
properties of chitosomes fromMucor rouxii," Protoplasma, vol. 122, no. 3, pp. 178-190, 1984.
82


[71] T. Ootaki and A. Miyazaki, "Genetic nomenclature and strain catalogue of Phycomyces," Sendai, Japan: Tohoku University, 1993.
[72] V. Campuzano, P. Galland, M. I. Alvarez, and A. P. Eslava, "Blue-Light Receptor Requirement for Gravitropism, Autochemotropism and Ethylene Response in Phycomyces*," Photochemistry and Photobiology, vol. 63, no. 5, pp. 686-694, 1996.
[73] F. Grolig, P. Eibel, C. Schimek, T. Schapat, D. S. Dennison, and P. A. Galland, "Interaction between Gravitropism and Phototropism in Sporangiophores of Phycomyces blakesleeanus," Plant Physiology, vol. 123, no. 2, pp. 765-776, 2000.
[74] D. J. Cosgrove, "Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls," Planta, vol. 177, no. 1, pp. 121-130, 1989.
[75] B. Cubero, J. Ruiz-Herrera, and E. Cerda-Olmedo, "Chitin synthetase mutants of Phycomyces blakesleeanus," Molecular and General Genetics MGG, vol. 240, no. 1, pp. 9-16, 1993.
[76] M. Atsushi, M. Momany, P. J. Szaniszlo, M. Jayaram, and O. Tamotsu, "Chitin synthase-encoding gene (s) of the Zygomycete fungus Phycomyces blakesleeanus," Gene, vol. 134, no. 1, pp. 129-134, 1993.
[77] Y. N. Jan, "Properties and cellular localization of chitin synthetase in
Phycomyces blakesleeanus," Journal of Biological Chemistry, vol. 249, no. 6, pp. 1973-1979, 1974.
[78] L. Herrera-Estrella and J. Ruiz-Herrera, "Light response inPhycomyces blakesleeanus: evidence for roles of chitin biosynthesis and breakdown," Experimental mycology, vol. 7, no. 4, pp. 362-369, 1983.
[79] A. Heyn, "Further investigations on the mechanism of cell elongation and the properties of the cell wall in connection with elongation," Protoplasma, vol. 25, no. 1, pp. 372-396, 1936.
83


[80] E. S. Castle, "The distribution of velocities of elongation and of twist in the growth zone of Phycomyces in relation to spiral growth," Journal of Cellular and Comparative Physiology, vol. 9, no. 3, pp. 477-489, 1937.
[81] M. J. Middlebrook and R. Preston, "Spiral growth and spiral structure: IV. Growth studies and mechanical constants in the cell wall," Biochimica et biophysica acta, vol. 9, pp. 115-126, 1952.
[82] R. Cohen and M. Delbruck, "Distribution of stretch and twist along the growing zone of the sporangiophore of Phycomyces and the distribution of response to a periodic illumination program," Journal of cellular and comparative physiology, vol. 52, no. 3, pp. 361-388, 1958.
[83] L. Herrera-Estrella, B. Chavez, and J. Ruiz-Herrera, "Presence of chitosomes in the cytoplasm ofPhycomyces blakesleeanus and the synthesis of chitin microfibrils," Experimental Mycology, vol. 6, no. 4, pp. 385-388, 1982.
[84] G. Martinez-Cadena and J. Ruiz-Herrera, "Activation of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin," Archives of Microbiology, journal article vol. 148, no. 4, pp. 280-285, October 01 1987.
[85] G. W. Gooday, "Physiology of microbial degradation of chitin and chitosan," in Biochemistry of microbial degradation: Springer, 1994, pp. 279-312.
[86] P. Jolles and R. A. Muzzarelli, "Chitin and chitinases," EXS(Basel), 1999.
[87] V. Seidl, "Chitinases of filamentous fungi: a large group of diverse proteins with multiple physiological functions," Fungal Biology Reviews, vol. 22, no. 1, pp. 36-42, 2008.
[88] D. J. Adams, "Fungal cell wall chitinases and glucanases," Microbiology, vol. 150, no. 7, pp. 2029-2035, 2004.
[89] R. Hurtado-Guerrero and D. M. van Aalten, "Structure of Saccharomyces cerevisiae chitinase 1 and screening-based discovery of potent inhibitors," Chemistry & biology, vol. 14, no. 5, pp. 589-599, 2007.
84


Full Text

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D IMENSIONAL ANALYSES TO QUANTIFY CELL WALL DEFORMATION AND STRESS RELAXATION IN STIFF MUTANTS OF PHYCOMYCES BLAKESLEEANUS : EXPERIMENTAL INVESTIGATIONS by CINDY M. MUNOZ B.S. , Uni versity of Colorado Denver, 2010 M.S. , University of Colorado Denver, 2014 A t hesis s ubmitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Engineering and Applied Science Program 2018

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ii © 2018 CINDY M. MUNOZ ALL RIGHTS RESERVED

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iii This thesis for the Doctor of Philosophy degree by Cindy M. Munoz has been approved for the Engineering and Applied Science Program by Samuel Welch, Chair Joseph K.E. Ortega, Advisor Christopher M. Yakacki R. Dana Carpenter Timb erley Roane Date: December 15, 2018

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iv Mun oz, Cindy M. ( PhD, Engineering and Applied Science Program) Dimensional Analyses to Quantify Cell Wall Deformation and Stress Relaxation in Stiff Mutants of Phycomyces blakesleeanus : Experimental Investigations The sis directed by Professor Emeritus Joseph K.E. Ortega A BSTRACT Stiff mutant sporangiophores of Phycomyces blakesleeanus exhibit diminished tropic (bending) responses when compared to wild type sporangiophores. Insight into sporangiophore growth of two stif f mutants is obtained by quantifying the biophysical processes that produce expansive growth. The overall objective of this investigation is to learn if the cell wall is affected by the altered genes to cause the diminished tropic responses. In the first part of this study the dimensionless parameters pe , pv , and ev obtained from the Ortega Growth Equations are used to quantitate changes in wall deformation rates and stress relaxation rates. The results provide insight into changes in wall loosening che mistry, wall architecture , and wall composition of the stiff mutants of P. blakesleeanus . In vivo turgor press ure step up experiments are conducted to measure the longitudinal volumetric modulus, L , that is needed to determine the magnitudes of pe e v . The dimensionless parameters pe pv , and ev are compared for wild type and stiff mutants. The result s demonstrate that the altered genes reduce the magnitudes of pv ( plastic deformation rates ) and pe ( stress relaxation rates ), indicating a signific ant change in wall loosening chemistry in stiff mutants. Also shown is that the magnitude s of ev (elastic deformation rates) are similar in wild type

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v and stiff mutants. This indicates that the altered genes did not substantially change the general wall co mposition and architecture in stiff mutants . In the second part of this study , experiments are conducted to obtain insight into whether the cell wall architecture ( micro fibril orientation ) and the wall dynamics (micro fibril reorientation) of the stiff muta nts are changed by the altered genes. Sporangiophores of P. blakesleeanus exhibit helical growth ( simultaneous elongation and rotation of the wall along the longitudinal axis). Experiments are conducted in which are measured and used to determine the ratio of rotation rate and elongation rate ( R ) as a function of elongation rate (d L /d t ) . Stiff mutant curves are compared to the previously published wild type curve demonstrating the behavior to be similar . This fin ding suggests that the mu tant genes did not substantially alter the fibrils orientation (cell wall architecture) or the fibril reorientation (wall dynamics) . The quantitative methods presented here are relevant to cells with walls in the plant, algae, and fungi kingdoms. This quantitative method that employs dimensionless numbers can be used to obtain insight into changes in underlying biochemical processes, wall structure, and wall dynamics. The use of such methods can help optimize and tailor cells walls for the industrial and pharmaceutical sectors . The form and content of this abstract are approved. I recommend its publication. Approved: Joseph K.E. Ortega

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vi D EDICATION I would like to dedicate this work to my family, their unconditional love has motivate d me to persevere. To my mother , Mrs. Juanita Munoz , and my father , Mr. Ricardo Munoz , dreams. Without their loving upbringing and nurturing I would not have been where I am today and what I am today. H would have remained mere dreams. I thank my mother with all my heart and will forever be grateful. I thank my father for teaching me work ethic and humbleness. To my sister, Norma Munoz , and brot her , Jesus Munoz , to . You have pushed me to go beyond my expectations. This work is also dedicated to a special person, Anahi Rubio, who has accompanied me in the struggles of this journey and who has not once s topped believing in me.

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vii A CKNOWLEDGEMENTS my educational career. Thank you for giving me the strength, knowledge, ability and opportunity to undertake this research an d to persevere. I would like to express my gratitude to Dr. Samuel Welch for providing me with the financial means to continue the pursuit of a Doctorate degree. Even more so, thank you for giving me the opportunity to explore the world of teaching. Th ank you for your time when I needed support and guidance. Without your hel p, this thesis would not have been possible. I would also like to express my gratitude to Dr. Christopher Yakacki for providing materials needed for research. Thank you for being involv ed in my research struggles and provided support by listening and offering genuine advice to help me complete this work . In my journey towards this degree, I have found a teacher, an inspiration, and a role model, Dr. Tim berley Roane. She has provided her heartfelt support and guidance at all times and has given me invaluable guidance, inspiration , and suggestions in my quest for knowledge. Without her guidance, this thesis would not have been possible and I shall eternally be grateful to her for her assis tance. I would also like to express my gratitude to Dr. Kenneth Ortega for encouraging independent thought and helping me grow as a researcher. Without him , I would have not been introduced to the wonderful world of research. Thank you for providing me wit h the opportunity to learn interdisciplinary research and unconventional thinking.

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viii It would be inappropriate if I omit to mention the names of people that helped me with technical problems throughout this research. Thank you to Jac Corless for helping me w ith machining the pressure probe. Thank you to Beatriz Bermudez for helping me with the design and 3D printing of microscope stands. Their help contributed to the completion of this thesis.

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ix T ABLE OF CONTENTS CHAPTER I. INTRODUCTION ................................ ................................ ......................... 1 Expansive Growth of Cells with Walls ................................ .......................... 2 Turgor Pressure, Water Uptake and Cell Wall Stress Relaxation .......... 3 Mechanical Behavior of the Cell Wall and Relevant Biochemical Behaviors ................................ ................................ .............................. 4 Principles of Expansive Growth ................................ ............................. 5 Quantitative Modeling of Expansive growth of Cells with Walls ................... 7 Lockhart Growth Equations ................................ ................................ ... 7 Ortega Growth Equations ................................ ................................ .... 10 Dimensionless Ortega Growth Equations ................................ ............ 12 Phycomyces Blakesleenaus ................................ ................................ ...... 16 Sporangiophore Development [24, 49, 52] ................................ ................ 17 Sporangiophore Cell Wall ................................ ................................ .... 20 Sensory Responses ................................ ................................ ............ 21 Light Response ................................ ................................ ........................ 22 Avoidance Response ................................ ................................ ............... 22 Geotropic Response ................................ ................................ ................ 23 Stiff Mutants ................................ ................................ ......................... 23

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x II. PREVIOUS RELEVANT STUDIES IN EXPANSIVE GROWTH OF PHYCOMYCES BLAKESLEEANUS ................................ .......................... 24 In vivo creep to determine biophysical variables P c , L , and m g ........... 26 Effects on Elongation Growth Rate with Turgor, P , Changes .............. 26 Comparison of Cell Wall Mechanical Properties of Wild Type and Stiff Mutant Sporangiophores ................................ ................................ ..... 30 Cell Wall loosening in the Wild Type Sporangi ohore of Phycomyces blakesleeanus ................................ ................................ ...................... 34 Anoxia Experiments ................................ ................................ ................. 34 Low pH Frozen Thawed Experiments ................................ ...................... 35 III. RESEARCH OBJECTIVES ................................ ................................ ........ 39 IV. DIMENSIONLESS CHARACTERIZATION OF CELL WALL DEFORMATION RATES AND STRESS RELAXATION RATES TO HELP IDENTIFY CHANGES IN WALL LOOSENING CHEMI STRY, AND WALL COMPOSITION AND ARCHITECTURE IN STIFF MUTANTS OF PHYCOMYCES BLAKESLEEANUS ................................ .......................... 41 Introduction ................................ ................................ ................................ 41 Materials and Method s ................................ ................................ ............... 42 Biological Material ................................ ................................ ............... 42 Elongation ................................ ................................ ........................... 42 Turgor Pressure ................................ ................................ ................... 45 In vivo Step up Experiments ................................ ................................ 47

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xi Protocol for in vivo step up experiments ................................ .................. 48 Dimensionless Parameters ................................ ................................ .. 50 Experimental Results ................................ ................................ ................. 51 Longitudinal Volumetric Elastic Modulus, L ................................ ........ 51 pe pv ev ................................ ........ 55 Discussion ................................ ................................ ................................ .. 59 Limitations ................................ ................................ ................................ .. 62 V. CELL WALL ARCHITECTURE AND DYNAMICS OF STIFF MUTANT SPORANGIOPHORES OF PHYCOMYCES BLAKESLEEANUS .............. 63 Introduction ................................ ................................ ................................ 63 Fibril Slippage Reorientation Hypothesis for Helical Growth in Phycomyces ................................ ................................ ........................ 63 Materials and Methods ................................ ................................ ............... 65 Biological Material ................................ ................................ ............... 65 Elongation rate ................................ ................................ .................... 66 Rotation rate ................................ ................................ ........................ 66 Results ................................ ................................ ................................ ....... 67 Discussion ................................ ................................ ................................ .. 70 Limitations ................................ ................................ ................................ .. 71 VI. CONCLUSIONS AND FUTURE WORK ................................ ..................... 72 Conclusions ................................ ................................ ............................... 72

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xii Suggestions for Future Work ................................ ................................ ..... 74 RE FERENCES ................................ ................................ ................................ .............. 76 APPENDIX A . Mathematics for Chapter IV ................................ ................................ ........... 86 B . Innacurate Data ................................ ................................ ............................. 87 C . Supplemental Results for In Vivo Turgor Pressure Step Up Experiments .... 91 D . Dimensionless Variables used by Ortega [48] to make the Ortega growth equations dimensionless ................................ ................................ ................. 108 E . Computation of ................................ ............. 109

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xiii L IST OF TABLES TABLE Table 1. Biophysical Variables for WT and Stiff mutants . ................................ ............. 31 Table 2. A comparison of relevant biophysical variabl es used in computing magnitudes of the pe pv , ev dimensionless parameters: L , m , P c , and d L /d t for stage IVb sporangiophores for WT, C216, and C149 strains . .................. 55

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xiv L IST OF FIGURES FIGURE Fig ure 1. Expansive growth process for cells with walls ................................ ................. 6 Figure 2. An illustration of a cylindrical cell depicting the stresses caused by turgor pressure ................................ ................................ ................................ ..... 7 Figure 3. Comparison of the pe values for different cell species ................................ . 15 Figure 4. Developmental stages of Phycomyces blakesleeanus ................................ .. 20 Figure 5. G rowth modeled by a dashpot and spring in series ................................ ....... 25 Figure 6. In vi vo creep experiment with four small pre ssure step ups (P = 0.0077 MPa) ................................ ................................ ................................ ................. 28 Figure 7. In vivo creep experiment with one large turgor p ressure step up (P = 0.031 MPa) ................................ ................................ ................................ ........ 29 Figure 8. In vivo creep exper iment with one large turgor p ressure step up (P = 0.046 MPa) ................................ ................................ ................................ ........ 30 Figure 9. Normalized values for d L /d t , m g , and P P c for WT a nd stiff mutant sporangiopgores ................................ ................................ ...................... 32 Figure 10. Normalized values for L g , m g , and g for wild type a nd stiff mutant sporangiophores ................................ ................................ ...................... 33 Figure 11. Stiff mutant and wild type depiction of tropic response dependent on t he magnitude of the growth zone ................................ ................................ .. 33

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xv Figure 12. Turgor pressure and elongation behavior as a function of time fo r a stage IVb anoxia experiment ................................ ................................ ............. 35 Figure 13. Average extension behavior of fifteen FT and eight FTB wa lls of stage IV sporangiophores ................................ ................................ ...................... 37 Figure 14. Snapshot of a sporangiophore depicting the method of calibration in ImageJ for elongation measurements using the sp orangium as a scale reference. ................................ ................................ ................................ ................. 44 Figure 15. Snapshot of the sporangiophore as it is elongated by the addition of oil using a microcapillary attached to a pressure probe. ................................ ........ 45 Figure 16. Manual pressure probe with a USB pressure transducer. ........................... 46 Figure 17. Snapshot of a sporangiophore being im paled by the mic rocapillary tip ....... 47 Figure 18. Experimental set up for an in vivo pressure step up experiment ................. 49 Figure 19. Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus behavior for a si ngle stage IVb C149 sporangiophore during an in vivo turgor pressure step up experiment.. ................................ ....................... 53 Figure 20. Comparison of average L for stage III (non growing) sporangiophores between WT an d stiff mutants (C216 and C149) ................................ ..... 54 Figure 21. Comparison of average L for stage IV (growing) sporangiophores between WT an d stiff mutants (C216 and C149) ................................ .................... 54 Figure 22. A comparison of the pe values calculated for growing sporangiophores of P. blakesleeanus (Stage IVb) ................................ ................................ ....... 57

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xvi Figure 23. A comparison of the pv values calculated for growing sporangiophores of P. blakesleeanus (Stage IVb) ................................ ................................ ....... 58 Figure 24. A compar ison of the ev values calculated for growing sporangiophores of P. blakesleeanus (Stage IV) ................................ ................................ ......... 59 Figure 25. Fibril reorientation and fibril slippage mechanism postulated by Ortega et al [62] for a fast growing with relatively long growing zone sporang iophore .. 65 Figure 26 ( A ) Snapshots showing a thin piece of hair attached to the sporangium used as a reference poi nt to determine rotation rate ................................ ........ 67 Figure 27. Results from a typical rotati on experiment on a single stage IVb C149 sp orangiophore ................................ ................................ ........................ 69 Figure 28. Average R avg values versus elongation rates for stage IVb sporangiophores for WT and stiff mutant strains ................................ ................................ . 70

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1 CHAPTER I INTRODUCTION Expansive growth of cells with walls and its regulation is central to the life and development of these cells. Regulation of expansive growth and morphogenesis is determined by controlling the cell wall mechanical properties which ar e altered by biochemical changes. Learning how these cells regulate their mechanical properties to control growth and growth responses to environmental stresses and environmental stimuli can help with the development of novel uses for cells with walls. Th e study of cell wall mechanics in the plant, algae , and fungi kingdoms has gained increasing interest due to the applicability in the industrial and pharmaceutical sectors. In these sectors , the end product is highly dependent on optimal cell wall composit ion . For example , in the paper industry , the removal of lignin in wood increases the cost of production , such that a lower lignin content in proce ssed wood could help lower production costs. Wood is a type of plant with a cell wall that has a high lignin c ontent , therefore , modific ation of the cell wall composition can reduce processing costs. However, lower lignin content in wood comes at a cost because it is important for tree stability and cannot be reduced arbitrarily without knowing how to compensate f or this reduction [ 1 ] . In biofuel production , it is necessary to effectively degrade cell walls into fermentable sugars for ethanol production [ 2 ] . In a griculture , plant pathogens significantly reduce the production and quality of the crops [ 3 ] . Pathogens directly penetrate the cell wall to access water and nutrients of the plant protoplast [ 4 ] . Thus a rigid cell wall can help increase the crop quality and crop survival rates [ 4 ] . Furthermore ,

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2 learning how the cell wall is remodeled in resp onse to environmental stresses can help design stress tolerant crops. In the pharmaceutical sector , the fungal cell wall is an attractive target in anti fungal agents because it has been shown that the death of the fun gus can result from inhibition of cell wall polysaccharide synthases [ 5 ] . Therefore knowledge and advancement in the ar ea of cell wall mechanics allow for the optimization of cell walls for the industrial and pharmaceutical sectors. Expansive Growth of Cells with Walls The cells of p lants, a lgae, and fungi have walls, a distinguishable characteristic that separates these cells from mamma l i an cells. The cell wall is a complex extracellular matrix that surrounds all cells in the organism. Cell walls are crucially important for processes in cell expansive growth, morphoge nesis and reproduction. The cell wall provides structural and mechanical support, helps maintain and determine cell shape, con trol rate and direction of growth , protect against pathogens an d environmental the site for cell signalin g and c ell to cell interaction [ 6 ] . A main prerequisite of cell walls is to resist high internal turgor pressures while being able to deform during growth and elastic expansion of the cell. Cell walls vary in shape, chemical composition and structure depending on cell type and developmen tal stage. Primary walls surround growing and dividing cells while being thin and extensible, but strong [ 7 ] . In this thesis, experimental work and reference will be ba sed on primary cell walls that are able to undertake expansive growth .

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3 Turgor Pressure, Water Uptake and Cell Wall Stress Relaxation Turgor pressure is the hydrostatic pressure in excess of atmospheric pres sure that builds up in cells with walls (plants, algae, and fungi) [ 8 ] . It is produced by an osmotic pressure difference that drives water into the cells from its surroundings across a selectively pe rmeable membrane [ 8 ] . Cells with walls use turgor pressure to produce the necessary cell wall stresses for wall deformation and expansion . The dependence of t urgor pressure in cell wall growth is a consequence of th e facts that 1) stress is needed for wall stress relaxation ( to ensure water uptake) and 2) the stress relaxation rate is a function of the wall stress relaxation [ 9 ] . Thus, turgor pressure is recognized as the driving force in cell wall expansion and expansive growth. A central process in expansive growth of cells with walls is stress relaxation [ 9 12 ] . Research has shown that wall stress relaxation creates the reduced water potential needed to drive water uptake and thus cell wall expansion [ 9 11 , 13 15 ] . In growing cells , wall stress relaxation is necessary for irreversible deformation (expansive growth) to occur. Breaking bonds between loa d bearing polymers in the cell wall (cell wall loosening) result in st ress relaxation [ 10 ] . Stress relaxation reduces turgor pressure in the wall initiating water uptake and cell wall expansion and the restoration of c ell wall stress [ 9 ] and is a consequence of bioch emical wall loosening (Figure 1). The rate of stress relaxation serves as a measure of wall chemistry necessary for wall loosening. The process of relaxation and expansion occ ur simultaneously and equal in measure so that the cell wall is not damaged [ 9 ] .

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4 Mechanical Behavio r of the Cell Wall and Relevant Biochemical Behaviors T he shape and d eformation of a material depend on the force exerted on the material and the mechani cal properties of the material. Similarly in cells with walls , the control of their shape and expansive growth is regulated by the turgor pressure and the composition, structure, architecture and chemical reactions in the wall, thus the mechanical properties are biochemical ly mediated [ 8 ] . Research has shown that the magnitude of expansive growth is sometimes regu lated by the magnitude of turgor pressure [ 16 19 ] . The direction and magnitude of expansive growth observed during morph ogenesis and tropic responses are controlled by regulating the mechanical properties of the wall [ 20 26 ] . Experimental evidence demonstrates that wall mechanical properties and wall behavior are modified b y biochemical agents that loosen the wall [ 13 , 24 , 27 36 ] . Cell wall loosening is essential for irreversible deformation to occur in that it provides the necessary stress relaxation for water uptake and expansion of the cell volume [ 9 , 28 ] . For higher plants (stems, roots , and leaves) pH dependent proteins have been shown to produce wall loosening and controlled polymer creep ( time d ependent deformation under an applie d load) [ 27 , 29 31 ] . It is hypothesized that algal cells loosen their walls by breaking calcium bridges and reforming them between pectin polymers [ 33 35 ] . It is unknown which biochemical agents regulate cell wall loosening in fungi, but some evide nce exists that the mech anism might be similar to higher plants in that it is low pH mediated [ 24 ] .

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5 Principles of Expansive Growth Expansive growth and its regulation are central to the life and development of cells with walls. Expansive growth is the irreversible increase in cell volume and surface area. This complex phenomenon features three interrelated and simultaneous processes: cell membrane hydraulics, cell wall deformat ion, and cell wall chemorheology (chemically mediated flow and deformation of polymeric material) . Cell membrane hydraulics c onsists of water uptake and its regulation. Water uptake produces cell turgor pressure which in turn produce the cell wall stresses necessary for cell wall expansion and growth . Cell wall deformation is produced by cell wall stresses producing both irrever sible and reversible deformations necessary for cell wall expansion and growth (Figure 2) . Irreversible deformation produces permanent increases in the cell wall and is generally regulated by biochemical wall loosening [ 8 ] . R eversible deformation provides the cell wall stresses and the resistance strength necessary to sustain high turgor pressure s . The elastic deformation and elastic properties are generally a function of cell wall structure and architecture [ 8 ] . Cell wall chemorheology involves the molecular modification of the wall network (loosening and stiffening of bonds) by biochemical reactions . The cell wall maintains its integrity by simultaneously synt hetizing new cell wall mat erial. Th e process of expansive growth is summa rized by Ortega and Welch [ 37 ] as follows:

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6 s inside the plasma membrane and absorbs water from its surroundings through the process of osmosis. The absorption of water produces turgor pressure that stresses the wall. The wall is biochemically loosened, reducing both wall stresses (stress relaxation ) and turgor pressure (turgor pressure relaxation). More water is absorbed in response to the decrease in turgor pressure, extending (deforming) the loosened wall. The wall deformation produces an increase in wall surface area (expansion) and a thinner wal l. New wall polymers and other wall materials are added to maintain a nearly constant wall thickness. The series of events (i.e. wall loosening, wall stress relaxation, turgor pressure relaxation, water uptake, wall expansion, an increase in turgor pressur e, an increase in wall stresses, and wall loosening again) is Figure 1 . Expansive growth process for cells with walls . The illustration shows the concept of stress relaxation and how it is related to wall loosening and growth. (1) Cell reaches osmotic equilibrium with wall stresses counterbalancing turgor pressure and induced water flows (for a non growing well hydrated cell). (2) The wall is biochemically loosened resulting in turgor pressure and wall stress relaxation (for a growing cell) . (3) Water uptake takes place in consequense of stress relax ation and the cell wall expands. In consequense the cell wall restores its wall stresses. This figure was taken from Cosgrove [ 9 ] .

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7 Figure 2 . An illustration of a cylindrical cell depicting the stresses caus ed by turgor pressure. The blue arrows depict the turgor pressure inside the cell and the red arrows show the cell wall stresses, both radial and longitudinal, caused by the turgor pressure . Figure taken from Ortega et al [ 8 ] . Quantitative Modeling of Expansive growth of Cells with Walls The cell wall mechanical properties regulate the expansive growth of cells with walls and biochemical processes alter these properties to give controlled expansive growth and morphogenes is . Quantitative models allow the investigation of the interplay between the mechanical properties of the cell wall and the under lying biochemical processes. Lockhart Growth Equations For over 50 years researchers have studied different types of cells with walls in the plant, algae, and fungi kingdoms to decipher the mechanisms associated with

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8 expansive growth. The physical process es associated with expansive growth gave rise to quantitative modeling o f this phenomenon. Lockhart [ 18 ] was the first to develop the foundation for a quantitative model for the expansive growth of cells with walls. The biophysical equations model two underlying physical processes that are necessary for expansive growth: water uptake and ce ll wall irreversible deformation. Equation (1 .1) describes the relationship between the relative rate of change in water volume within the cell, ( d V w /dt)/V, and the relative rate of water uptake, L P ). (1 .1) Equation (1 .1) is written relative to the volume, V (the terms are divided by the volume); t is the time, L is the relative membrane hydraulic conductance ( L = L p A / V ), L p is the membrane hydraulic conductivity, A is the membrane area, is the osmotic pressure difference across the membrane (where the solute reflection coefficient is assumed to be unity and omitted from these equations), and P is the turgor pressure. Equation (1 .2) describes the relative rate of change of the v olume of the cell wall chamber, ( d V / d t ) cwc / V cwc , as equal to the relative irreversible (plastic) deformation rate of the wall. The bi ophysical variable, , is the relative irreversible extensibility of the wall (a measure of P c is the critical turgor pressure which is related to the yield threshold, Y (the value that must be exc eeded to produce irreversible deformation of the wall). (1 .2)

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9 The rates of change in volume of the cell wall chamber and water uptake are approximately equal, (d V cwc /dt)/ V (d V w /dt)/ V ), and an equatio n for the equilibrium or steady state turgor pressure, P eq , can be obtained, (1 .3) Equations (1.1), (1.2) and (1 .3) are the Lockhart Growth E quations which are all interrelated and coupled by P . Equation (1 .1) was deriv ed from the physical laws of the deformation of viscoplastic materials specifically that of a Bingham fluid. Al though it is a simple model, much insightful information was obtained through these equations . For example, through experiments [ 25 , 38 , 39 ] and Equation (1.2) it was found that and P c are not static mechanical properties. Insight into how the cell wall responds to changes in turgor pressure and how it affects growth has also be e quations [ 16 , 21 , 38 , 40 ] . quations helped demonstrate that it is imperat ive to understand the biophysical parameters in expansive growth to fully understand growth regulation and morphogenesis. It is important to note that the versatility in these equations comes with the fact that the same physical processes are exhibited by diverse cells with walls (plant, fungi , and algae). This biophysical approach assumes that the biophysical parameters help control and regulate biological and biochemical processes. The growth model developed by Lockhart is a quantitative relationship betw een the rate of cell expansion and the biophysical parameters that could be measured in vivo. This is very important because the validation of the model through experimentation demonstrates its applicability.

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10 While Loc quations set the foundation f or additional expansive growth models , the equations were lacking some important features of the expansive growth of cells with walls. T he force acting on the cell was assumed to be constant, which implied the cell wall stress, , and turgor pressure, P , a re constant. This eliminated the elastic deformation component in the total cell wall deformation equation . Lockhart also assumed that the cell behaves as a linear viscoplastic model with the irreversible deformation being a linear function of turgor press ure in which the material can only flow and permanently deform when the turgor pressure, P , exceeds the critical turgor pressure, P c . These assumptions within the derivation gave rise to the irreversible cell wall deformation equa tion as presented in Equat ion (1 .2). When deriving the relationship between the increase in water volume and the net rate of water uptake the model assumed no transpiration, T = 0, this gave rise to Equation (1 .1) e quations are able to model steady state growth in cells s urrounded by free water where transpiration is nonexistent, tissue tensions are negligible, and the stresses can be assumed to be proportional to turgor pressure and where the elastic changes are negligible, such as the growth of Nitella inte rnode cells (a lgal cells) [ 16 , 21 , 38 ] . Most importantly, these growth equation s cannot model periodic stress and stress relaxation exhibited by living growing cells [ 7 , 14 , 15 , 17 , 37 , 41 43 ] . It has been shown that elastic deformation i s required for stress relaxation [ 11 , 15 , 42 ] and needed t o accurately describe the instantaneous changes in wall deformation after turgor pressure changes [ 44 , 45 ] . Ortega Growth Equations Ortega [ 42 , 46 ] augmented the Lockhart Growth E quations to address the limits in the mod el. E quation (1 .1) was augmented with a transpiration rate, , term producing

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11 Equation (1 .4). Where = (d V T / dt)/ V is the relative rate of change in water volume lost via transpiration (relative transpiration rate). (1 .4) E quation (1 .2) was augmented with an elastic component 1/ (d P /dt) which now describes changes in turg or pressure and reversible cell wall d eformation producing Equation (1 .5). Where is the volumetric elastic modulus which measures how the cell wall changes volume in response to pressure . The under lying constitutive equatio n is now of a viscoelastic material with a yield stress (Maxwell Bingham viscoelastic model ) which now models deformation at any applied stress . (1 .5) Simil arly, w hen obtaining Equation (1 .3) an equation for the tu rgor pressure is also derived, Equation (1 .6). Note that this equation now addresses changes in turgor pressure in time and it is no longer constant a quations. (1 .6) With the addition of the transpiratio n rate, cells exposed to air where the effect of transpiration cannot be neglected can be modeled more accurately. An example of such case is in the fungal cell P. blakesleeanus , where water uptake takes place at the base and transpiration takes place throughout the long cylindrical stalk exposed to the air. When modeling stress relaxation water uptake is eliminated, and transpiration is su ppressed, the Ortega Growth E quat ions (also kno wn as Augmented Growth E quations) are reduced to Equations (1.7) and (1 .8). Equation (1 .8) describes the

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12 pressure decay from an initial pressure, P i , at time, t =0, with a stress relaxation time constant t c = ( ) 1 . For an ideal stress relaxation, is constant and known throughout the pressure decay range [ 17 ] . This is the pressure decay observed experimentally for plant and fungal cells [ 14 , 15 , 17 ] . (1 .7) (1 .8) The utility of the Ortega Growth E quations is that they were derived based on physical principles to model physical processes that are relevant for plant, algal, and fungal cells. Water uptake and cell deformation are the same for these cells with walls. The differences lie in the chemorheological (wall loosening chemistry) aspects of each cell, cell structure and synthesis of wall material. These differences are embedded in the biophysical variables of these equations which can be extracted from specifically designed experiments. Dimensionless Ortega Growth Equations Dime nsionless parameters are frequently us ed in the physical sciences to better understand complex phenomena. For example, in the area of heat transfer and fluid mechanics, the Reynolds number ( which is interpreted to be the ratio of inertia and viscous forces ) is used to study different flows, in particular , if a flow is laminar or turb ulent [ 47 ] . Some of the reasons equatio ns are made dimensionless is to reduce the number of experiments that need to be conducted, reduce the number of ti mes one might need to solve equations, to obtain insight into what parameters might be small

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13 and ignored or approximated, to separate relevant processes, and to scale up solutions by model similarity. The Ortega Growth E quations were made dimensionless and used to separate biophysical variables with their re levant processes. Ortega [ 12 ] used constant reference parameters, v s (steady relative volumetric growth rate), v sT (steady relative volumetric transpiration rate), and P C ( critical turgor pressure) to make the equat ions dimensionless. Equations (1.9), (1.10) , and (1 .11) are the dimensionless equatio ns corresponding to Equations (1.4), (1.5) and (1.6). See Appendix B for dimensionless variable definitions. (1 .9) (1 . 1 0) (1 .11) The ratios in paren thesis are dimensionless and are interpreted into ratios of relevant processes:

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14 The pe dimensionless parameter was shown to be central to stress relaxation [ 12 ] a necessary process for the expansive growth of cells with walls. This parameter directly relates cell wall mechanical properties, cell wall deformation rates and cell wall loosening chemi stry. Wall loosening chemistry regulates wall loosening and alters mechanical properties to produce the wall defor mation necessary for irreversible deformation ( expansive growth ) . This dimensionless parameter can be computed through in vivo creep and in vi vo stress relaxation tests [ 12 , 48 ] . Where T 1/2 , is the halftime of the exponential decay of turgor pressure in an in vivo stress relaxation experiment. Orte ga [ 12 ] computed pe values for three different species (plant, algal , and fungal cells) and compared the values (Figure 3) pe values for dif ferent species are dramatically different from each other. The difference in magnitudes of the pe value

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15 for each cell s pecies suggests a different cell wall loosening chemistry . There is evidence that shows that wall loosening chemistry is different betw een plants and algal cells [ 26 , 27 , 29 , 31 , 33 36 ] . A different wall chemist ry would produce different ratios of plastic and elastic defo rmation rates of the cell wall, similar to what is seen in the pe values for different cell species (Figure 3) . Most importantly different wall loosening chemistry would produce different wall s tress relaxation rates. The pe parameter can be further decomposed into dimensionless reversible and ir reversible wall deformation rates through the pv and e v paramet ers. These help to identify direct changes in biochemical wall loosening ( pv ) and wal l architecture and composition ( e v ) . Figure 3 . Comparison of the pe value s for different cell species. The pe value s are different by several orders of magnitudes. Data to produce the figure was taken from Ortega [ 12 ] . 0 500 1000 1500 2000 2500 pe Plant cells in pea stems Internode algal cells Fungal sporangiophores

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16 Phycomyces Blakesleenaus Phycomyces blakesleeanus is a single cell ed fungus that has been used as a model organism in sensory transduction and cell wall mechanics research [ 23 , 49 51 ] . It is a filamentous fungus that belongs to the class Z ygomycetes. The sporangiophores of P. blakesleeanus are long aerial cylindrical stalks that carry a sporangium filled with spores at the top that can reach a length of 10 cm or more. The s porangium is approximately 500 m in diameter and can hold up to 10 5 spores for the vegetative reproductive cycle [ 49 , 52 ] . The spores can be kept in dormancy in a water medium or as dry stock in the refrigerator. Vegetative spores are heat shocked and inoculated on an adequate growth medium s uch of potato dextrose agar to grow sporangiophores. The sporangiophore grows by simultaneously elongating and rotating along its longitudinal axis exhibiting helical growth . Th e elongation and rotation occur in the growing zone of the sporangiophore which is in different locations depending on th e stage of development. The sporangiophores exhibit different tropi c (bending) responses to external stimuli . They detect and respond to light intensity, gravity, mechanical stretch, solid objects and ai r currents [ 49 ] . These stimuli control the growth rate, producing time dependent changes in growth rate as well as tropisms [ 49 ] . The output (growth rate) has been the focal point of sensory transduction studies because of information gained in the response of various external stimuli [ 49 ] . The external stimuli must react with internal signals to control the growth rate. The objective of sensory transduction studies has been to gain information about the steps in this transduction process, from e xternal stimuli, receptors, organelles, and chemical reactions in a controlled manner [ 49 , 52 54 ] .

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17 Growth is l imited by the cell wall making it apparent that expansive growth is dependent on cell wall formation and the growth responses being exhibited may reflect changes in the cell wall structure and cell wall chemistry. S tudies have focused on cell wall mechanic s to characterize cell wall mechanical properties and structure and relate this to growth [ 23 , 55 63 ] . These s tudies provided information about how cell wall mechanical properties regulate growth, and how external stimuli regulated mechanical properties. Phycomyces has especially served as a model organism in understanding the expansive growth of cells with walls [ 23 ] . It is similar in mo rphology and growth behavior to higher cells that the study and understanding of its expansive growth provide useful knowledge in understanding exp ansive growth of higher cells. Sporangiophore Development [ 24 , 49 , 52 ] Phycomyces has particularly served as a model organism because it is responsive to different external stimuli and because of its distinct developmental stages. The sporangiophore develops through five main developmental stages (Figure 4 ). In st age I, the sporangiophore appears as a pointed tube el ongating at the tip at 0.3 0.6 m/sec and rotating clockwise (seen from above) at approximately 30 ° h 1 . During this stage , growth occurs between the apical tip and 1.0 mm below it (Figure I 4 ). In stage II, no elongation or rotation occurs. During this stage the formation of sporangiu m begins. This stage lasts for approximately 3

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18 hours in which a yellow sporangium is fully formed of approximately 0.5 mm in diameter. Stage III is a period of approximately 2 hours of rest with no elongation or rotation. This stage is hypothesized to be linked with active spore formation. Stage IV is divided into three different stages, IVa, IVb, and IVc. The main characteristic of this stage is the formation of a new growing zone and intercalary growth occurring in this region. Stage IVa begins with el ongation and counter clockwise rotation at the short growing zone located approximately 0.6 mm below the ba se of the sporangium (Figure I 4 ). Counter clockwise rotation continues for approximately 1 hour before the rotation rate gradually decreases to zero and clockwise rotation. During this stage , the sporangium gradually darkens. Stage IVb begins with clockwise rotation. Elongation and rotation are maintained relatively constant (typical values are 1 m/sec and 0.2 ° s 1 ) for many hours. This stage exhibits a dark sporangium containing mature spores. Most biophysical work is conducted with stage IVb because it exhibits nearly constant elongation, rotation and growing zone length for 2 4 hours . Elongati on rates are between 35 and 60 m min 1 . The growing zone extends from 0.1 mm to approximately 2.5 mm below the sporangium. The cell wall in this region is extensible and deforms

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19 plastically when stretched longitudinally [ 64 ] . The cell wall in the non growing region deforms elastically when subjected to a longitudinal load [ 49 , 56 ] . Stage IVc exhibits another twist reversal to counte r clockwise rotation (I 4 ). During this stage , the sp orangiohores are long (>10 cm). Stage V is the final stage characteristic of old sporangiophores and not exhibiting growth. During this stage , the sporangium can easily burst releasing spores that easily stick to most surfaces. The rotation and elongation behavior has been measured and used as an indirect method to gain insight on cell wall archite cture (micro fibril orientation) and wall dynamics (micro fibril reorientation) [ 61 63 , 65 67 ] .

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20 Figure 4 . Developmental stages of Phycomyces blakesleeanus . The sporangiophores exhibit helical growth ( simultaneous elongating and rotating about its l ongitudinal axis ) . The cell wall elongates in a region termed the growing zone (depicted in light green). Stage IVb is used in biophysical studies due to its nearly constant elongation and ormation on each stage. Figure was taken from Ortega et al . [ 24 ] . Sporangiophore Cell Wall The fungal cell wall consists of two types of components: 1) a matrix made of glycoproteins, amorphous polysaccharides , and lipids 2) micro fibril s of skeletal

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21 polysaccha rides of chitin an d B glucans [ 49 ] . Phycomyces belongs to the class Zygomycetes which are charac terized by having chitin and chitosan in their cell walls [ 68 ] . The main components in sporangiophore cell walls are chitin, chitosan, and polyuronides plus the neutral sugars, mannose, glucose, galactose, and flucose [ 49 , 52 ] . The cell wall of Phycomyces is about 600 nm thick with chitin microfibrils of 15 20 nm in diameter and several micrometers long [ 52 ] . R uiz Herrera [ 69 ] found that there exist m icro vesicles (named chi tosomes) which contain chitin synthas e in the cytoplasm of the yeast M. rouxii . In Phycomyces chitosomes have also been reported to be present in the cytoplasmic material of the tip of sporangiophores [ 49 ] . The chitosomes (40 70 nm in diameter) move from the cytoplasm to areas where expansive growth occurs in cells with a growth zon e [ 49 ] . C hitin synthase in the chitosomes can be activated by protease and inc ubation with UDPGlcNAc substrate to synthetize chitin mic ro fibrils [ 49 ] . Ruiz Herrera [ 70 ] calculated that 80% of chitin content in t he cell is in these chitosomes. Sensory Responses Sporangiophores of Phycomyces respond t o different sensory stimuli by changes in growth rate and/or direction of growth. The stimuli and responses are linked by sensory pathways that process information [ 49 ] . Each sensory pathway acquires information from one or more sensors, evaluates and transfers it, and finally governs a response mechanism [ 49 ] . The sensory transducers (elements in sensory transduction pathway) must be either proteins made und er the direct command of genes or other chemical s manufactured with the participation of gen e products [ 49 ] . In most cases, a

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22 stimulus applied symmetrically to the longitudinal axis of the sporangiophore produces a change in elongation growth only while an asymmetrical stimulus produces differential growth on opposite sides of the s talk. The differential growth results in the sporangiophore troping (bending) . There are different variations of responses that can be elicited through various symmetric and asymmetric s timuli and combinations of. Three main responses (light response, avoi dance response, and g eotropic response) are reviewed below. Light Response The sporangiophore responds to a spatially symmetric increase or decrease in light intensity by exhibiting a transient increase (positive response) or decrease (negative response) i n growth rate respectively. The response is only elicited when the level of light intensity is higher or lower than the light intensity to which the sporangiophore was adapted to [ 49 ] . A phototropic response is elicited in the sporangiophore when being subjected to spatially asymmetric light distribution. The sporangiophore grows t owards the region of maximum light intensity [ 49 ] . The sporangiophore has a le ns like property in the growing zone which allows unilateral light to be focused at the distal s ide of the sporangiophore [ 49 ] . This results in asymmetric growth rate with respec section and troping towards the unilateral light source. Avoidance Response The sporangiopho re detects objects in close pro ximity (< 5 mm) to the growing zone. It responds to this stimuli by growing away from the object with a response

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23 latency of 2 3 min. The maximum growth rate may increase up to 20 60% higher than the basal rate. During the increase in growth rate , the trope rate is relatively constant at a rate of 3 ° min 1 . The sporangiophore usually stops troping within 20 30 min after the barrier is placed. A maximum of 40 ° to 50 ° troping angle has bee n observed [ 49 ] . Geotropic Response The sporangiophore detects a gravitational field and responds by growing away from the gravitational source, name d the negatively geotropic response. When a sporangiophore is placed horizontally, it responds to gravity by growing towards a vertical axis until completely vertical. The latency towards the vertical axis is 30 180 minutes after being placed horizontally with and average troping rate of 0.3 ° min 1 [ 49 , 52 ] . Stiff Mutants Mutants with altered growth behavior have been classified into phenotypic classes based on their sensory responses to light, gravity , and avoidance [ 71 ] to identify the genes , prot eins , and enzymes affecting the operation of each step of the sensory pathways [ 49 ] . The mad mutants have been classified into three groups: class1.1, class 1.2 and class 2. Class 2 are said to be defective in the output side (growth rate) [ 49 , 71 ] . Class 2 mutants display a reduced sensitivity to visible light. Mutants in class 2 with defects in the genes mad D, E, F, G, and J (termed stiff mutants) have diminished or non existent tropic responses [ 71 73 ] . In this res earch two stiff mutants, C216 and C149 with altered genes mad D, E, F, G, and J are studied to further elucidate on the expansive growth of P. blakesle eanus and to learn if the altered genes affect the cell wall properties thus affecting the tropic responses.

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24 CHAPTER II PREVIOUS RELEVANT STUDIES IN EXPANSIVE GROWTH OF PHYCOMYCES BLAKESLEEANUS The sporangiophores of P. blakesleeanus grow predominately in length, L. The radial growth (cross sectional growth rate in area) is small compared to the elongation growth rate d L /d t , therefore the radial growth rate can be neglected in expansive growth considering only elongation growth . Sporangiophores of stages I and IV exh ibit intercalary growth meaning they only grow within a growth zone that is shorter than the rest B). The equation for the elongation growth r ate is modeled by Equation (2 .1) [ 23 ] . Equation (2.1) represents the growth within the growing stalk region, L g , and the non growing stalk, L s . Where m g is the longitudinal irreversible cell wall extensibility of the growing stalk, and Lg and Ls are the l ongitudinal volumetric elastic modulus within the growing stalk and the longitudinal volumetric elastic modulus within the non growing stalk, respectively. Within the growing region , both irreversible and reversible deformation occurs, in contrast to the n on growing region where only reversible defor mation is exhibited (Figure 5B ). (2 .1)

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25 Figure 5 . A Diffuse growth modeled by a dashpot and spring in series. This model represents growth for cells that elongate througho ut the entire stalk (plants and algal cells). B Tip growth modeled by the two separate regions (growing and non growing). The growing region has a dashpot and spring in series attached (in series) to another spring which represents the non growing region. Figure was taken from Ortega [ 23 ] . When turgor pressure, P , is constant , the ela stic deformation rate is eliminated from Equation (2.1 ) and the elongation growth rate is sim ply equal to the irreversible deformation rate of t he growing region, Equation (2. 3 ). (2. 3 ) Equation (2. 3 ) can be written relative to the growing zone length, L g : (2. 4 )

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26 Where g = m g / L g is defined as the relative irreversible cell wall extensibili ty f or the growth zone. In vivo creep to determine biophysical variables P c , L , and m g An in vivo creep experiment is used to determine biophysical variables P c , L , and m g . This experiment requires an instantaneous increase in turgor pressure (turgor pr essure step up) imposed on the cell [ 17 ] . The growth rate behavior after the turgor p ressure step up is used to determine P c and m g . During an in vivo creep experiment P c and m g are assumed to be constant [ 17 ] . Using finite differences Equation (2.4) becomes: (2. 5 ) Solving for m g : (2. 6 ) The average growth rate is usually obtained from the 10 min interval before and after the pressure step up. The critical turgor pressure, P c , can be determined from Equation (2.3) using m g , P , and d L /d t before the pressure step up since they are known and constant. Effects on Elongation Growth Rate with Turgor, P , Changes Ortega et al . [ 17 ] demonstrated that the elongation growth rate in sporangiophores of P. blake sleeanus responds differently to changes in turgor pressu re . The

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27 experiments described in [ 17 ] demonstrate the changes in elongation growth behavior exhibited by spor a ngiophores when subjected to different magnitudes of pressure step ups. It was demonstrated that the growth behavior elicited was related to the magnitude of the pressure ste p up. Turgor pressure step ups less than 0.02 MPa elicited an increase in growth rate (Figure 6 ) . This response is predicted by the Ortega Growth Equation (Equatio n 2.3 ). Also shown was that turgor pressure step ups larger than 0.02 MPa elicit a decrease i n growth rate (Figure 7 ) . Larger magnitudes of pressure step ups produce larger decreases in growth for longer periods (Figure 8 ) . The decrease in growth rate is related to the magnitude of the pressure step up. Ortega et al. [ 17 ] at tributed this effect as s train hardening of the wall after a large (P> 0.02 MPa) pressure step up.

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28 Figur e 6 . In vivo creep experiment with four small pressure step ups (P = 0.0077 MPa) . Turgor pressure (upper graph) and natural logarithm of the length, ln L , (lower graph) are plotted as a function of time. The fat short arrow indicat es when the cell was impaled and the long thin arrows indicate when the turgor pressure step ups were imposed on the cell. Figure was o btained from Ortega et al . [ 17 ] .

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29 Figure 7 . In vivo creep experiment with one large turgor pressure step up (P = 0 .031 MPa). Turgor pressure (upper graph) and natural logarith of t he length, ln L , (lower graph) are plotted as a function of time. The fat short arrow indicates when the cell was impaled and the long thin arrow when the turgor pressure step up was imposed on the cell. Figure was o btained from Ortega et al . [ 17 ] .

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30 Figure 8 . In vivo creep experiment with one large turgor press ure step up (P = 0 .046 MPa). Turgor pressure (upper graph) and natural logarith of the length, ln L , (lower graph) are plotted as a function of time. The fat short arrow indicates when the cell was impaled and the long thin arrow when the a the turgor pres sure step up was imposed on the cell. Figure was o btained from Ortega et al . [ 17 ] . Co mparison of Cell Wall Mechanical Properties of Wild Type and Stiff Mutant Sporangiophores Sensory responses in stage IV b sporangiophores of P. blakesleeanus are produced by regulating the magnitude of elongation growth rate and differential elongation grow th rate. Stiff mutant sporangiophores are deficient in the madD , E , F , G , and J genes [ 71 73 ] . The stiff m utant sporangiophores exhibit significantly reduced tropi c responses to light, gravity, and the presen ce of solid surfaces [ 71 73 ] . Ortega et al . [ 24 ]

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31 investigated whether the defective genes affect the biophysical variables that regulate gro wth ( m g , P , and P C ). The length of the growth zone, L g , was also determined and used to offer an explanation on why the stiff mutants exhibit diminished tropic responses and how the variables studied affect each other. Table 1 are the results from this stu dy comparing biophysical variables between wildtype (WT) and stiff mutants. Table 1 . Biophysical Variables for WT and Stiff mutants . Table was taken from Ortega et al. [ 24 ] . Ortega et al . [ 24 ] found that the magnitudes of m g exhibited in stiff mutants were significantly s maller compared to the WT (Figure 9 ). This result indicate d a diminished ability to irreversibly deform in the longi tudinal direction in stiff mutants. Also found was that the magnitude of P P c significantly increas ed in stiff mutants (Figure 9 ). This indi cate d an increase in the magnitude of the effective stress in the wall responsible for irreversible deformation. The lower magnitudes of m g in stiff mutants produce d less irreversible cell wall deformation and should produce a lower growth rate, however , t he

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32 magnitudes of P and P P c increased producing higher wall stresses necessary for irreversible deformation. Essentially the higher stresses in the wall were compensate d for the lower longitudinal irreversible cell wall extensibility, m g , to produce simila r growth rates exhibited by the WT (Figure 9 ). The decrease in the magnitude of m g ( m g = g L g ) e xhibited by the stiff mutants was shown to be produced by the decrease in L g and g (Figure 10 ). The decrease in L g and g was postulated to affect the abilit y to produce differential elongation growth because bending rates are dependent on m g in a similar fashion as growth rates are dependent on this variable. If a sporangiophore produces a constant bend rate per unit length then a shorter growing zone, L g , wo uld produce a lower bending rate because there is less length to bend (Figure 11 ). Figure 9 . Normalized values for d L /d t , m g , and P P c for WT and stiff mutant sporangiopgores. The lower m g values in stiff mutants were compensate d by the higher P P c values. There was no signi fi cant difference in d L /d t between the strains. Figure was taken from Ortega et al. [ 24 ] .

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33 Figure 10 . Normalized values for L g , m g , and g for wild type and stiff mutant sporangiophores. The values were significantly smaller in the stiff mutants compared to the wild type . Figure was taken from Ortega et al. [ 24 ] . Figure 11 . Stiff mutant and wild type depiction of tropic response dependent on the magnitude of the growth zone. Growth zone is depicted a s a light green region. The stiff mutant exhibits a lower tropic response because of the decrease in magnitude of the growth zone length. Figure was taken from Ortega et al. [ 24 ] .

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34 Although it was found that the altered genes significantly reduced the irreversible extensibility of the cell wall, , the study did not provide evidence to learn whether the defective genes altered wall deformat ion rates and stress relaxation rates . Chapter I V focus es on investigating these questions and makes use of the results of Ortega et al . [ 24 ] to predict plastic def ormation rates for the stiff mutants. Cell Wall loosening in the Wild Type Sporangiohore of Phycomyces blakesleeanus Cell wall loosening is necessary for irreversible deformation in cells with walls. Many studies in cell wall loosening have been conducted on plant and algal cells to elucidate on chemorheological aspect of expansive growth [ 7 , 27 , 29 , 31 , 34 36 ] , however , no studies had been conducted on fungal cells. Ortega et al . [ 26 ] studied the cell wall loosening mechanism in P. blakesneeanus . The study provides evidence of the existence of chemistry in the fungal cell wall of P. blakesleeanus responsib le for wall loosening, irreversible deformation , and elongation growth. Anoxia Experiments Ortega et al . [ 26 ] designed experiments to isolate the wall from new cell wall material addition reducing metabolic influences to gain insight on the existence of cell wall chemistry by subjecting the sporangiophore to an anoxic (without oxygen) environment. Results demonstrated that elongation growth continues on avera ge for more than 10 minutes after an anoxic environ ment when small turgor pressure step ups (P 0.02 MPa) were imposed on the cell. This demonstrates that wall chemistry continues the chemorheogical process to deform the cell wall irreversibly during anox ia (Figure 12 ) . The wall deformation during anoxia is irreversible because the turgor

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35 pressure step ups are held constant and d P /d t =0, with no elastic deformation occurring after the first minute after the step up (Equation 1.13 ). Ortega et al . [ 26 ] hypothesized that the wall material added to the cell wall before anoxia is used to continue the cell wall loosening process and enable irreversible deformation un til the cell wall material is depleted. Figure 12 . Turgor pressure and elongation behavior as a function of time for a stage IVb anoxia experiment. Times when the cell was impaled and oxygen level was lower than 1% are represent ed in the graph by short thick and long thin arrows, respectivel y. Approximately 5 minutes after anoxia, turgor pressure step ups ( P = 0.021MPa) were imposed on the cell. Elongation growth rate and irreversible deformation continue for approximately 23 mi nutes. Graph was taken from Ortega et al . [ 26 ] . Low pH Frozen Thawed Experiments Other experiments conducted by Ortega et al . [ 26 ] are similar to the low pH frozen thawed similar to those conducted by Cosgrove [ 74 ] on cucumber hypocotyls. These experiments further confirmed the existence of wall chemistry and a better insight into the cell wall loosening of P. blakesleeanus .

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36 Ortega et al . [ 26 ] cond ucted c onstant tension exte nsion experiments on frozen thawed (FT) and frozen thawed boiled (FTB) sporangiophore walls to identify whether a decrease in pH increased cell wall extension and whether a wall loosening protein may be involved in fungal cell wa ll deformation. Freezing the cell wall deactivates enzymatic activity but retains the wall enzymes. A relevant treatment that affects the cell wall chemistry would activate the enzymes within the wall producing cell wall extension. Boiling of the cell wall after freezing and thawing serves as a denaturing treatment. Boiling destroys cell wall proteins necessary for cell wall extension. If a treatment has an effect on FTB walls then cell wall extension is not enzymatic. Results from Ortega et al . [ 26 ] demonstrated that when FT walls were subjected to a low pH environment under a constant load, the walls immediately extended and continued to extend (cre ep) for mi nutes after (Figure 13 ). Interestingly the FTB walls showed a smaller initial extension immediately when the pH was lowered and did not continue to extend and no creep was observed (Figure 13 ). FT walls exhibited an average creep time of five minutes. The results demonstrated that lowering the pH produces a chemorheological reaction, irreversi ble extension , and creep. In contrast to the FTB walls which exhibited a lower initial irreversible extension with no creep. The inhibition of creep in the FTB walls s uggested that a wall protein may be involved in wall loosening and creep in P. blakesleeanus . The results proved to be similar qualitatively to those of cucumber hypocotyls [ 74 ] , but dif ferent quantitative ly . The creep times were over 24 hrs in cucumber hypocotyls while on average creep time in the fungal cell P. blakesleeanus was 5 min. Ortega et al . [ 26 ] hypothesized that different cell wall material, structure and cell size could affect creep behavior and account for this difference. Also

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37 hypothesized was that wall chemistry exists in walls of P. blakesleeanus to make and break load bearin g bonds between microfibrils producing the chemorheology necessary to regulate mechanical wall properties and wall deformation behavior. Figure 13 . Average extension behavior of fifteen FT and eight FTB walls of stage IV sporang iophores. The curves show the extension behavior before and after the pH of s show increased initial extension and creep while the FTB wall shows reduced initial extension and no creep. Figure was taken from Ortega et al . [ 26 ] . Many studies have been conducted on sporangiophores of P. blakesleeanus to gain insight on expansive growth and cell wall def ormation. Ortega et al . [ 17 ] demonstrated that the elongation growth rate behavior of growing sporangiophores is different when the magnitude of the turgor pressure step ups is changed. In this thesis , the results from l arge turgor pressure step ups are used to develop an experimental protocol to determine the longitudinal volumetric elast ic modulus , L , of stage IVb (growing) stiff mutant sporangiophores. Results from Ortega et al . [ 24 ] demonstrated that

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38 the elongation growth rate is dependent on the magnitudes of g , L g and P P c . Also shown is that stiff mutant genes madD , E , F , G , and J decreased the magnitudes of m g , g , and L g . The smaller values found in the stiff mutants provides the basis for further studying the cell wall of these mutants. This result is u sed to make a prediction on the magnitudes of plastic deformation rates in stiff mutants. Furthermore, results from Ortega et al . [ 26 ] demonstrated that ther e is cell wall chemistry necessary for cell wall loosening and irreversible deformation in P. blakesleeanus . Re sults from this study encourage the quantification of cell wall deformation rates and stress relaxation rates to learn about the changes in wall loosening chemistry and wall architecture and composition in the stiff mutants. The two studies presented in this thesis make use of the information presented in the studies above to gain a better and more quantitative understanding of cell wall deformatio n rates and stress relaxation rates to gain insight on cell wall loosening chemistry, wall composition, microfibril orientation (wall architecture) , and reorientation (wall dynamics) of this model organism .

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39 CHAPTER III RESEARCH OBJECTIVES A general objective of this research is to demonstrate that the quantitative measures used in the studies outlined in the following chapters can be used to detect changes in the me chanical and rheological behavio r of cell walls. T raditional Mechanical Engineering methods and exp erimental methods are adapted to suit a living organism and extract relevant cell wall material properties and cell wall behavior . The underlying bio physical principles in cell wall expansion and expansive growth allows for a quantitative approach that has been validated throughout the years. Changes in wall deformation rates and stress relaxation rates are translated into biochemical changes which can be used to identify molecular changes . The information provided from these methods along with biochemical methods can provide a better understanding of expansive growth and wall deformation to ultimately help tailor cell walls for industrial and pharmaceutical sectors. It is envisioned that quantitative studies such as these can help guide future biochemical s tudies. The model organism Phycomyces blakesleeanus is used to demonstrate how these methods can be used to predict biochemical changes associated with the cell wall . The objective of Chapter IV is to quanti fy cell wall deformation rates and stress relaxa tion rat es that can provide insight to changes in wall loosening chemistry , and wall composition and architecture in two stiff mutants of P. blakesleeanus . T he dimensionless parameters pe , pv , and ev that were obtained from the Ortega Growth Equations c an be used to achieve this objective . T he magnitudes of pe and pv are substantially reduced in stiff mutants compared to the WT . In contrast, the magnitudes

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40 of ev are similar in stiff mutants and WT. These results demonstrate that mutant (altered) genes reduced the irreversible (plastic) deformation rates of the wall and stress relaxation rates. This implies a significant alteration to the cell wall loosening chemistry in the stiff mutant walls. The similar ev values in stiff mutants and WT demonstrates that the altered genes did not substantially change the elastic deformation rate of the wall , implying that the cell wall architecture and composition are not significantly changed. The objective of Chapter V is to obtain experimental evidence that can h elp determine whether the altered genes in stiff mutants significantly change wall architecture and wall dynamics. This is accomplished by using an experimental approach in which changes in microfibril orientation and reorientation is indirectly determined demonstrate that the curves of R (the ratio of rotation rate to elongation rate) vs. elongation rate (d L /d t ) in the stiff mutants are essential ly the same as those obtained from WT . This suggests that the alt ered genes did not substantially change the microfibril orientation and reorientation. It is concluded that the altered genes only significantly affect the cell wall plastic deformation rates and wall loosening chemistry.

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41 CHAPTER IV D IMENSIONLESS CHARACTERIZATION OF C ELL WALL DEFORMATION RATES AND STRESS RELAXATION RATES TO HELP IDENTIFY CHANGES IN WALL LOOSENING CHEMISTRY , AND WALL COMPOSITION AND ARCHITECTURE IN STIFF MUTANTS OF PHYCOMYCES BLAKESLEEANUS Introducti on T he main objectiv es of this chapter are to quantitate cell wall defo rmation and stress relaxation rates in an attempt to gain insight into changes in wall loosening chemistry , and wall composition and architecture of two stiff mutants (C216 and C149) of the model organism Phycomyces blakesleeanus . The stiff mutants are defective in the genes madD , E , F , G , and J exhibiting diminished tropic (bending) respon ses compared to the wild type (WT) [ 71 73 ] . This has prompted this investigation to learn if the cell w all is affected by the altered genes to cause the smaller tropic responses. Prior experimental research shows that the irreversible cell wall extensibility, , for stiff mutants C216 and C149 is significa ntly sm aller than that of the WT [ 24 ] . No evidence was provided to learn whet her the wall deformation rates and stress relaxation r ates were affected by the altered genes. In this study , the pe , pv , and e v dimensionless parameters are used to quantify cell wall deformation rat es and stress relaxation rates to gain insight in changes of cell wall loosening chemistry , wall composition and architecture of the two stiff mutants, C216 and C149, of P. blakesleeanus . Based on the smaller cell wall extensibility reported for stiff mutant cell walls it is predicted that the magnitude of pv (irreversible wall deformation rate) for stiff mutan ts is smaller than the WT . However, because the magnitude o f the longitudinal volumetric elastic modulus, L ,

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42 is not known , the magnitudes of pe (stress relaxation rate) and e v (wall composition and architecture) cannot be predicted. Here, i n vivo turgor pressure step up experiments are used to measure L , and c ompare measured val ues bet ween stiff mutants and WT . Measured average values of L and published data from in vivo creep experiments are used to compute the magnitudes of pe , pv, and ev for stiff mutants and WT . Ortega [ 12 ] published the magnitude of pe of in vivo creep tests for WT sporangiophores of P. blakesleeanus . This value is used to compare pe magnitudes computed for the stiff mutants C216 and C149. Materials and Methods Biological Material Vegetative spores of the stiff mutant gene strain C 216 geo ( ) were originally obtained from Ishinomaki Senshu University, Miyagi, Japan . The C149 madD120( ) strain was obtained from ATCC: The G lobal Research Center, Virginia , USA. Sporangiophores were inoculated on sterile growth medium consisting of 4% (w/v) YM agar. After i noculating, the sporangiophores were incubated under diffuse incandescent light and constant temperature ( 22° C ± 2° ). Stage IVb sporangiophores, 2 2.5 cm in length, were selected for experiments from the second to the seventh crop. S tage IVb sporangiophores are used because they exhibit a nearly constant growth rate an d rotation [ 49 , 52 ] . Elongation The change in elongation in the sporangiophore is determined by measuring the change in length, L , of a reference point, in this case , the edge of the sporangium is

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43 used. The length is measured by taking snapshots using a USB camera ( 720P HD, GUCEE HD92 Skype Web Camera) attached to a long focal length horizontal microscope (Gaertner;7011Keyepiece a nd 32m/m EFL objective) mou nted to a 3 D micromanipulator (Line ToolCo.;modelH 2, with digital micrometer heads). Snapshots were acquired using open source software Vividia Ablescope and were analyzed using open source software ImageJ. The diameter of the sporangium for each sporangiophore was measured before initiation of the experiment to use as a scale reference in ImageJ. Figure s 14 and 15 show the s napshots of the sporangiophore obtained by the method described above.

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44 Figure 14 . Snapshot of a sporangiophore depicting the method of calibration in ImageJ for elongation measurements using the sporangium as a scale reference . The set up. D =515 m

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45 Figur e 15 . Snapshot of the spor angiophore as it is elongated by the addition of oil using a microcapillary attached to a pressure probe . Turgor Pressure The turgor pressure of the sporangiophore is measured continuously using a manual v ersion of the pressure probe [ 17 , 25 ] . Figure 16 shows the version of the pressure probe used. A USB gage pressure transducer is used in the pressure probe which was purchased from Ellison Sensors Inc, Boca Raton, Florida, USA (model GD4200 USB 100BAR) and tested for calibration inside the pressure probe with a Heise Bourdon Tube Pressure Gauge (Dresser Industries, Newton, CT, USA; model CMM, 0 200 PSIG Range). t was recorded on the software package included with the USB transducer. The pressure probe was mounted on a 3 D manipulator so that the micro capillary tip (10 15 m in diameter) could be guided to impale the sporangiophore under visual observation using a High Resolution Digital Microscope (J iusion 40 to 1000x

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46 Magnification Endoscope, 8 LED USB 2.0 Digital Microscope, AMAZON, Seattle, WA, USA). The microcapillary o f the pressure probe was filled with inert silicon oil (Dow Corning Corp.; fluid 200, 1.5 centistoke viscosity). After the cell was impaled, the cell sap oil interface was pushed to the surface of the cell vacuole and maintained at the fixed loc ation to me asure the turgor pressure of the sporangiophore [ 17 , 25 ] (Figure 17 ) . The higher turgor pressure was maintained by small injections of inert silicon oil and also ensuring silicon oi l was flowing into the cell vacuole for immediate step up in turgor pressure. Figure 16 . Manual pressure p robe with a USB pressure transd u c er .

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47 Figure 17 . Snapshot of a sporangiophore being impaled by the microcapillary tip. The cell sap interface flows out of the cell and needs to be brought to the surface of the cell in order to read the turgor pressure and ensure o il flows into the cell vacuole. This is accomplised by increasing the pressure with th e pressure probe. In vivo Step up Experiments The in vivo step up experiment requires constant short time interval (30 sec) increases in turgor pressure of 0.03 0.04 MPa (turgor pressure step ups) produced to the sporangiophore and immediate measurement o f the elongation. The biophysical variable, L , can be determined by this method and through the Ortega Growth E quation representing the wall deformation (elongation rate for longitudinal extending cells) [ 23 , 42 ] , Equation (4.1 ). When pressure step ups are large (P 0.03 MPa) and at short time intervals it ensures that the irreversible component in Equation ( 4. 1) is minimized and can be neglected (see Appendix A for mathematical proof) . The equation is then simplified and can be solved for L (Equation 4. 2) P is the magnitude of the

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48 pressure step L is the change in elongation after the pressure step up. This is validated by experimental research in which large turgor pressure step ups cau se a transient decrease in elongation growth rate of sporangiophores of P. blakesleeanus [ 17 , 25 ] . ( 4. 1 ) ( 4. 2 ) Protocol for in vivo step up experiments A stage III or I Vb sporangiophore (typically 2 2.5 cm in length) in a glass shell vial is selected and adapted f or 20 minutes to the room temperature of 21 23 ° C (Figure 18 ), to room lights (cool white fluorescent lamps hung from the ceiling), and to bilateral swan neck light guides (from Schoelly Fiber optic; the end of each light guide is positioned approximately 8 12 cm on either side of the sporangiophore at an angle of about 30 ° from the horizontal) from a fiber optic illuminator (Flexilux 90; HLU Light Source 90/Wfrom Schoelly Fiber optic, Denzlingen, FRG, which filtered out nearly all of the infrared light ). Th e sporangium is measured and recorded using th e micrometer attached to the USB camera microscope set up . Following this adaptation period, the length of the sporangiophore is measured and the pressure transducer software is initiated. The pressure in the pressure probe is increased between 0.03 0.09 MPa before impaling the cell and the cell is immediately impaled by the micro capillary tip of the pressure probe to measure the turgor pressure. Once a cell sap interface is located the Image acquisition softw are is also initiated to take snapshots of the sporangiophore

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49 at 30 second intervals with a 5 second delay from the pressure step ups. The snapshots are later analyzed to measure elongation using ImageJ. For the first 30 60 seconds into the experiment, the turgor pressure is maintained, 30 seconds after, the turgor pressure is increased by 0.03 0.04 MPa and maintained for 30 seconds by injecting inert silicon oil into the cell vacuole (Figure 15 ) . This same procedure (turgor pressure step up) is followed un til the cell ruptures by not sustaining any more oil into the vacuole and bursting or until a leak is present. Figure 18 shows the experimental set up for an in vivo turgor pressure step up experiment. Figure 18 . Experimental se t up for an in vivo pressure step up experi ment . A horizontal microscope with a USB camera is used to capture elongation of the sporangiophore. A USB miscroscope helps guide the microcapillary needle into the sporangiophore. A pressure probe is used to im pale, measure ,

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50 Dimensionless Parameters The pe , pv , and ev dimensionless parameters are used to quantitate cell wall deformation rate s and stress relaxation rates to gain insight on wall loosening chemistry , and wall composition and architecture in the stiff mutants. The pe parameter was shown to control stress relaxation in walled cells [ 12 ] . It was identified as the ratio of relative volumetric plastic and elastic deformation rates of the cell wall [ 48 ] . The pe parameter reflects the wall loosening and hardening characteristics of the ce ll wall chemistry [ 12 ] which regulates wall mechanical properties and thus wall deformation and expansive growth of cells with walls. This dimensionless number can be used to compare different cell species or mutants from same cell species in terms of their wall deformation rates, stress relaxation rates, wall loosening chemistry , and expansive growth. Plastic and elastic deformation rates of the wall can independently be computed using the dim ensionless parameters pv and e v [ 48 ] . The pv parameter reflects cell wall loosening chemistry and the e v parameter reflects wall composition and architecture. These parameters can be used to gain further insight into the individual processes represente d.

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51 Experimental Results Longitudinal Volumetric Elastic Modulus, L The magnitude of the longitudinal volumetric elastic modulus, L , is determined from in vi vo turgor pressure step up experiments conducted on stage III (non growing) and stage IVb (growing) sporangiophores from stiff mutant strains, C216 geo and C149 . Average values of L for stage III (non growing) sporangiophores were determi ned to get the range of values that could be exhibited by stage IVb (growing) sporangiophore L values. A typical in vivo turgor pressure step up experiment for a stage IVb sporangiophores is shown in Figure 19 with 0.04 MPa pressure step ups (See Appen di x C for more similar experimental results). Turgor p ressure step ups larger or equal than 0.03 MPa at short time intervals ensures that the irreversible deformation rate ( ste ady state growth rate since P =constant for the time interval) is negligible and el iminated from Equation ( 4. 1). For the growing sporangiophores t he first pressure step up is not considered to produce a purely elastic elongation and therefore is not considered as part of the L values used to compute an average L . All other values that correspond to the pressure step ups after the first are considered to produce purely elastic elongations. For stage III sporangiophores (non growing) all pressure step ups are considered to be part of the average L value. Average values for each sporangiophore tested a re used to determine the mean value for each stiff mutant and stage .

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52 A B C

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53 Figure 19 . Turgor pressure, Elongation and Longitudinal Volumetric Elastic M odulus behavior for a single stage IVb C149 sporangiophore during an in vivo turgor pressure step up experiment. A . Turgor pressure behavior. The cell is impaled at the second mark represented by the red arrow and the turgor pressure is increased until the cell sap interface is brought to the surface of the cell. Turgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressure step up is applied to the cell (green arrow). The new turgor pressure is maintained const ant until 30 seconds later and the next pressure st ep up is applied . t ref=0 indicates the time when elongation measurements began. A total of 6 pressure step ups were applied to the sporangiophore which resulted in purely elastic elongation. B . Elongation as a function of time. Elongation was graphed for the corres ponding pressure step ups at 30 sec intervals. The time interval, t ref = 30 210 sec (elongations after the black dashed line) , represent s pure ly elastic elongations considered for an average L value. The cell sust The leak is represented by a decrease in elongation due to the leakage of oi l and cell material (purple arrow ). The change in elongation, L , and P , are used to determine L for each pressure step up. A total of 6 L values are determined for this experiment and an average value is computed. C. Plot of L as a function of turgor pressure, P . Each L value is graphed with its corresponding pressure. The average L value for th is sporangiophore is represented as a red dashed line ( L average = 92 MPa) . The longitudinal volumetric elastic modulus is approximately constant for the range of turgor pressures the cell was subjected to. Figure 20 compares average L values of stage III (non growing) between WT and stiff mutants. Results from student t tests revealed n o difference for L values between WT and stiff mutants, but the results were less significant ( p=0.07 and 0.06 ). Figure 21 compares average L values of stage IVb (growing sporangiophores) between WT and stiff mutants. No significant difference was found for L values between WT and C149 ( p=0.96 ) . Furthermore, L values between WT and C216 are not different, but the results are less significant ( p= 0.054 ). Additionally, stu dent t tests results confirmed that no significant difference existed between stage III (non growing) and IVb (growing) sporangiophore L values .

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54 Figure 20 . Comparison of average L for stage III (non growing) sporangiophor es between WT and stiff mutants (C216 and C149). L values for stiff mutants were measured using in vivo turgor pressure step up experimen ts while L values for WT were ob tained from Ortega [ 23 ] . The vertical bars represent the standard error (SE) of the mean value and the horizontal bars enclose the area of statistical comparison for p values . Figure 21 . Comparison of average L for stage IV (growing) sporangiophores between WT and stiff mutants (C216 and C149). L v alues for stiff mutants were measured using in vivo turgor pressure step up experimements while L values for WT were ob tained from Ortega [ 23 ] . The vertical bars represent the standard error (SE) of the mean value and the horizontal bars enclose the area of statistical comparison for p values. 0 10 20 30 40 50 60 70 80 90 100 L (MPa) Stage III C216 C149 WT 0 10 20 30 40 50 60 70 80 L (MPa) Stage IV c216 C149 WT p=0.05 p=0.96 p =0.07 p=0.06 N=13 N=17 N=13 N=24 N=17 N=27

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55 Dimensionless Parameter s pe , pv and e v Table 2 shows the biophysical variables d L /d t (elongation growth rate), m ( longitudinal irreversible cell wall extensibility), L (longitudinal volumetric elastic modulus ) , and P c (critical turgor pressure) for WT and stiff mutants C216 an d C149. All data for WT sporangiophores was obtained from Ortega [ 23 ] and Ortega et al [ 24 ] published results. Stiff mutant data for variables m and P c are ob tained from Ortega et al [ 24 ] published results. R esults fr om student t tests revealed that m and P c were significantly different between WT and both mutants [ 24 ] . The values in T able IV 1 are used to compute the magnitudes of the pe pv , ev dimensionless parameters. The maximum and minim um values for each variable in T able 2 ar e used to compute maximum and minimum values for the dimensionless parameters (see Appendix D for analysis). Table 2 . A comparison of relevant biophysical variables used in computing magnitudes of the pe pv , ev dimensionless parameters: L , m , P c , and d L /d t for stage IVb sporangiophores for WT, C216 , and C149 strains. Values for d L /d t , m , and P c for WT are obtained from Ortega et al [ 24 ] . Values for m and P c for stiff mutants are obtained from Ortega et al [ 24 ] , L and d L /d t values were measured in this study. Variable Wild type C216 C149 units Mean ± SEM (n) Mean ± SEM (n) Mean ± SEM (n) L (MPa) 60 ± 5.1 (27) 49.2 ± 2.6 (24) 60.5 ± 5.3 (17) m ( m min 1 MPa 1 ) 997 ± 164 (20) 222 ± 40 (18) 170 ± 30 (8) P c (MP a ) 0.26 ± 0.01 (20) 0.13 ± 0.05 (18) 0.18 ± 0.08 (8) d L /d t ( m min 1 ) 34 ± 3.1 (20) 27.3 ± 2.5 (59) 27.8 ± 3.1 (59) Figure 22 compares magnitudes of the pe parameter between WT and stiff mutants determined from in vivo creep and in vivo step up experime nts conducted on inta ct stage IVb sporangiophores (see Appendix E for calculations of magnitudes ) .

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56 The magnitudes of pe and pv for the stiff mutants C216 and C149 are dramati cally smaller than for WT (Figures 22 and 23 ) . The difference in magnitude s be tween WT and stiff mutants of the pe and pv parameters provide s a comparison of the stress relaxation rates and plastic deformation rates further providing direct evidence on whether cell wall loosening chemistry has chan ged. The magnitudes of the ev pa rameter were also computed and compared to gain insight on the magnitude of elastic deformation rates in the wall fo r WT and stiff mutants (Figure 24 ). The magnitudes of the ev parameter for stiff mutants were similar to the WT. Plastic deformation rates and stress relaxation rates were reduced in stiff mutants while the elastic deformat ion rates of the wall were not substantially affected (Figures 22 , 23, and 24 ).

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57 Figure 22 . A comparison of the pe values calculated for growing sporangiophores of P. blakesleeanus (Stage IV b ). The maximum and minimum values of the pe parameter are represented by the confidence intervals that are calculated from the statistical data (standard deviation and standard error) presented in this resear ch and in papers: [ 23 , 24 ] . 0 500 1000 1500 2000 2500 3000 pe C216 C149 WT

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58 Figure 23 . A comparison of the pv values calculated for growing sporangiophores of P. blakesleeanus (Stage IV b ). The maximum and minimum values of the pv parameter are represented by the confidence intervals that are calculated from the statistical data (standard deviation and standar d error) presented in the paper: [ 24 ] . 0 2 4 6 8 10 12 pv C216 C149 WT

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59 Figure 24 . A comparison of the ev values calculated for growing sporangiophores of P. blakesleeanus (Stage IV). The maximum and minimum values of the e v parameter are represented by the confidence intervals t hat are calculated from the statistical data (standard deviation and standard error) presented in this research and papers : [ 23 , 24 ] . Discussion The dimensionless parameter, pe , is central to stress relaxation and expansive growth [ 12 ] . This dimensionless parameter is the ratio of relative volumetric plastic and elastic deformation rates of the cel l wall which reflects the wall loosening and hardening characteristics of the ce ll wall [ 12 ] . The pe parameter can be further decomposed into the dimensionless plastic deforma tion rate and elastic deformation rate of the wall by the pv and ev parameters. The pv parameter reflects cell wall loosening chemistry and the ev parameter reflects cell wall architecture and composition . In this study , the pe , pv , and e v dimensi onless parameters were used to quantitate cell wall deformation rates and stress relaxation rates in two stiff mutants of 0 0.001 0.002 0.003 0.004 0.005 0.006 0.007 ev C216 C149 WT

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60 P. blakesleeanus to gain insight on whether the mutant genes madD , E , F , G , and J genes affect these processes. The results provide in sight into changes in wall loosening chemistry, wall architecture and wall composition of the stiff mutants . Prior experimental research showed that the magnitudes of the irreversible cell wall extensibility, , for stiff mutants C216 and C149 are significantly smaller than the WT [ 24 ] , b ut no further evidence was provided to learn whether the wall deformation rates and st ress wall relaxation rates were affected by the altered genes. In vivo turgor pressu re step up experiments were conducted to measure the longitudinal volumetric elastic modulus, L , in stiff mutants to compute magnitudes for the pe and e v parameters. P revious data from in vivo creep experiments on stiff mutants and WT that measured the longitudinal irreversible c ell wall extensibility, m , and critical turgor pressure were used to compute the magnitudes of the pv parameter for stiff mutants and WT. Magnitudes of pe , pv , and ev were compared bet ween stiff mutants and WT . The magnitudes of pe and pv were subst antially smaller in stiff mutants compared to the WT (Figure s 22 and 23 ) . The results demonstrate t hat the altered genes reduce d stress relaxation rates and plastic deformation rates to significantly alter cell wall loosening chemistry. The magnitudes of t he ev parameter for stiff mutants and WT were also computed to learn if the cell wall composition and architecture was affected by the altered genes. The magnitudes of the ev parameter were simila r in stiff mutants and WT (Figure 24 ) . These results indicate that the w all elastic deformation rate did not substantially change and suggests that the general wall composition and architecture has not significantly changed with the altered genes . The results from dimensionless numbers pe , pv , and ev demonstrate that the defective

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61 genes madD , E , F , G and J substantially reduce irr evers ible deformation rates and stress relaxation rates in stiff mutants which reflects a significant change in wall loosening chemistry . Furthermore, the altered g enes did not substanti ally change the reversible deformation rates in the wall of stiff mutants to sig nificantly produce a change in cell w all composition and architecture . The cell wall is the expression site of tropisms in P. blakesleenus ; thus a decrease in wall loosening ch emistry would be consistent with the observed diminished tropic responses exhibited by the stiff mutants . From the results in this study , it can be predicted that 1) there are less cell wall remodeling enzymes necessary to break load bearing bonds in the stiff mutants and/or 2) the function of these cell wall remodeling enzymes was reduced in the stiff mutants. There is evidence to believe that chitinases (hydrolytic enzymes found in fungal cell walls) may be involved in breaking glycosidic bonds in chitin [ 75 77 ] . Herrera Estrella and Ruiz Herrera [ 78 ] showed that the cell wall of Phycomyces sporangiophores was softened by illuminating with white light dark adapted sporangio phores. The investigators label ed reducing ends in chitin which increased after the light stimul ation. Each poly GlcNAc chain in chitin contains only one reducing end , therefore , an increase in reducing ends indirectly measures breakage of chitin microfibrils. Most importantly , it was shown that madB and madD mutants did not show this increase in red ucing ends. This evidence along with the results presented in this study point to chitinases as being involved in cell wall plastic deformation , wall stress relaxation and wall loosening of P. blakesleeanus .

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62 Limitations In the past , pressure probe experi ments with Phycomyces have been conducted with two people, one to operate the pre ssure probe, the other to measure elongation . In other areas of cell wall research , the cells tested are large enough to attach an extensometer to measure the elonga tion . Due to the size and fragility of Phycomyces this method cannot be used. Therefore experiments with Phycomyces have been a two person job enabling higher accuracy results. In this study , a data acquisition system was designed to run the experiments with one per son actively involved in the operation of the pressure probe and not in the elongation measurements. Elongation measurements were automated using a USB camera and image capturing software to take snapshots of the cell at specified time intervals. The snaps hots were later analyzed with a cell measurement software. Throughout the different steps in this process , the accuracy of the elongation measurements could be reduced affecting the overall data. Care was taken to accurately acquire and measure snapshots, but this limitation d id exist. In the future , a higher resolution camera could be used with better image acquisition software. All software used in this study is open source and easily operated which prompted its use in this study.

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63 CHAPTER V CELL WALL ARCHITECTUR E AND DYNAMICS OF STIFF MUTANT SPORANGIOPHORES OF PHYCOMYCES BLAKESLEEANUS Introduction In the previous chapter , it was demonstrated that the madD , E , F , G , and J genes substantially reduced cell wall plastic deformation rates and stress relaxation rates which reflect ed a significant alteration in wall loosening chemistry in the stiff mutants. The objective of this chapter is to inv estigate whether these altered genes affect cell wall architecture and wall dynamics. Measurem ation and elongation indirectly provide evidence for microfi bril orientation (wall architecture) and re orientat ion ( wall dynamics ). Fibril Slippage Reorie ntation Hypothesis for Helical G rowth in Phycomyces Helical growth is observed in many plants, algal , and fungal cells. The model organism Phycomyces blakesleeanus undergoes helical growth ( simultaneous elongation and rotation of the wall along the longitudinal axis ) throughout its development. Helical growth in P. blakesleeanus has been investigated to e lucidate the biophysical and molecular mechanisms associated with this phenomenon [ 60 , 66 , 67 , 79 81 ] . Early investigations were not able to assess whether the ratio of rotation rate and elongation rate ( R ) was independent of the magnitude of elongation rate and as sumed R to be constant and independent of elongation rate [ 61 , 80 , 82 ] . From this assumption , the fibril reorientation and fibril slippage mechanisms were founded [ 61 , 63 65 ] . Later Ortega et

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64 al [ 62 ] demonstrated that the ratio of rotation rate and elongation rate ( R ) decreases as the elongation rate (d L /d t ) increases for stage IVb WT sporangiophores. The authors proposed a modi fied fibril reorientation mechanism to account for this new finding. The proposed mechanism postulates there is a fibril reorientation subzone in whic h elongation and rotation occur and a fibril slippage subzone in which elongation and no rotation occurs f or fast growing sporangiophores (Figure 25 ) . In the fibril slippage subzone , the fibrils are oriented in the longitudinal direction (direction of irreversible cell wall deformation) and can no longer reorient. The modified model predicts that in fast grow ing sporangiophores with a relatively long growth zone the region farthest away from the sporangium only produces elongation contributing to the elongation rate but no rotation is exhibited. In the slow growing sporangiophores with relatively short growing zones there only exists the fibril reorient at ion subzone producing a larger rotation.

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65 Figure 25 . Fibril reorientation and fibril slippage mechanism p ostulated by Ortega et al [ 62 ] for a fast growing with relatively long growing zone sporangiophore . It was postulated that the large growth zone is divided into a fibril reorientation (elongation with rotation occurs) subzone and a fibril slippage subzone (elongation occurs with no rotation). Microfibril orientation closes t to the sporangium lies transversely and those farthest away from the sporangium lie longitud inally. Figure was taken from Ortega et al [ 62 ] . Materials and Methods Biological Material Vegetative spores of the stiff mutant gene strain C216 geo ( ) were originally obtained from Ishinomaki Senshu University, Miyagi, Japan . The C149 madD120( ) strain was obtained from ATCC: The Global Researc h Center, Virginia , USA. Sp orangiophores were inoculated on sterile growth medium consisting of 4% (w/v) YM agar. After inoculating, the sporangiophopres were incubated under diffuse

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66 incandescent light and constant temperature ( 22° C ± 2° ). Stage IVb sporangiophores, 1.5 2.5 cm in l ength, were selected for experiments from the second to the seventh crop. Stage IVb sporangiophores are used because they exhibit constant growth rate an d rotation [ 49 , 52 ] . Elongation rate The change in elongation in the sporangiophore is determined by measuring the change in length, L , of a reference point, in this case , the edge of the sporangium is used. The length is measured by taking snapshots at 1 minute intervals using a web camera ( 720P HD, GUCEE HD92 Skype Web Camera) attached to a long focal length horizontal microscope (Ga ertner;7011Keyepiece and 32m/m EFL objective) mou nted to a 3 D micromanipulator (Line ToolCo.;modelH 2, with digital micrometer heads). Snapshots were acquired using open source software Vividia Ablescope and were analyzed using open source software ImageJ . The elongation rate was obtained by dividing the L over the time interval, t , in which it was measured. The diameter of the sporangium for each sporangiophore was measured before initiation of the experiment to use as a scal e reference in ImageJ. Rotation rate A thin piece of hair (7 10 mm long) was attached to the top of the sporangium of the stage IVb sporangiophore, perpendicular to the longitudinal axis of the cell. Sometimes a small amount of petroleum jelly was used to help the piece of hair adhere to the sporangium. When viewed from above the p iece of hair rotates in the clockwise rotation for a stage IVb sporangiophore. The rotation rate was determined by measuring

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67 the rotation rate of the piece of hair using a USB microscope to acquire snapshots at 1 minute intervals and later analyz ed using I mageJ software. Figure 26 shows snapshots for elongation rate and rotation rate measurements. Figure 26 ( A ) Snapshots showing a thin piece of hair attached to the sporangium used as a reference point to determine rotation rate . ( B ) S napshots used to determine the elongation rate for each sporangiophore. The same time interval was used to determine rotation and elongation rate. Results The elongation and rotation rates were measured concurrently for stage IVb sporangiophores of C 216 and C149 (n=59 for each strain). In constant conditions , the elongation rate fluctuates around an average value with small fluctuations. The

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68 elongation and rotation rate was averaged over the same 10 minute interval in which the elongation ra te was obs erved to be constant and the ratio of rotation rate and elongation rate, R , was calculated (Figure 27 ) . R was calculated for each sporangiophore and averaged with values of R obtained from other sporangiophores growing within a specified range of elongatio n growth rates (i.e. 1 10.9, 11 20.9, 21 30.9, etc) ; see Figure 28 . Each average R value is plotted as a function of the elongation rate, all three strains WT, C216, and C149 are plotted in the same graph for comparison (Figure 28 ). T he behavior exhibited by the stiff mutants is similar to the WT in which R decreases with increasing elongation rate. The large standard error bars of R for the slowest growing sporangiophores in stiff mutants suggest that this value lie s in the same range of the slow growing s porangiophores of WT and that the difference might not be significant .

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69 Figure 27 . Results from a typical rotation experiment on a single stage IVb C149 sporangiophore . The elongation rate and rotation rate is measured to comp u t e R . For this sporangiophore R = 0.15. The same time interval is used to average both elogantion and rotation rates.

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70 Figure 28 . Average R avg values versus elongation rates for stage IVb sporangiophores for WT and stiff mutant st rains. Each R avg value represents the mean value of R for sporangiophores with elongation rates at specified elongation rates: 1 10.9, 11 20.9, 21 30.9, 31 40.9, 51 60.9, and 61 70.9. The vertical bars represent the standard error (SE) of the mean value (n =59 for stiff mutants and n=171 for WT) . Data to reproduce WT behavior was obtained from Ortega et al [ 62 ] . Discussion It is found that there is a similar dependence of R (ratio of rotation rate to elongati on rate) on elongation growth rate in the stiff mutants as in the WT ( R decreases as d L /d t increases) (Figure 28 ). T he results presented indicate that the wall architecture (microfibril orientation ) and wall dynamics (reorienting of micro fibrils during elo ngation growth) of stiff mutants is similar to that of WT sporangiophores. The results indicate that the altered g enes did not significantly change the wall architecture and wall dynamics. In the previous chapter , it was found that the dimensionless parame ter that defines the reversible wall deformation rate ( ev ) was not substantially different in stiff mutants and WT. This indicated that the altered genes did not significantly change the wall composition and architecture. Thus similar ev values further s upport the 0 0.2 0.4 0.6 0.8 1 1.2 1.4 0 10 20 30 40 50 60 70 80 R avg (degrees/ m) d L /d t ( m/min) C149 C216 WT

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71 conclusion that wall architectu re was not significantly changed by the altered genes. Furthermore, the fibril reorientation slippage mechan ism postulated by Ortega et al [ 62 ] is further validated by the stiff mutant rotation and elongation behavior. Limitations Statistical analysis was unable to be conducted on the results presented in this chapter due to the unavailability of the WT raw data. It is suggested that to help get a stronger conclusion the rotation and elongation behav ior be measured for the WT sporangiophores to proper ly conduct statistical analysis.

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72 CHAPTER VI CONCLUSION S AND FUTURE WORK The principal objective of this work was to quantify cell wall deformation rat es and stress relaxation rate s to gain insight into the wall loosening chemistry , and w all architecture and wall dynamics of two stiff mutants of Phycomyces blakesleea nus . D imensionless parameters and biophysical experiments were used to reach this objective . The stiff mutants exhibit diminished tropi c responses compared to the WT prompting an investigation to learn if the cell wall is affected by the altered genes to cause the deficient responses. The results presented demonstrate that the cell wall is indeed affected further affectin g the sensory transduction pathway by the genes madD , E , F , G , and J . Specifically , the results demonstrate that t he altered genes reduce the plastic deformation rates and stress relaxat ion rates to significantly affect the wall loosening chemistry . A gene ral objective of this work was to demonstrate that wall deformation rates and stress relaxation rates can be quantified to gain insight o n changes of wall loosening chemistry , and cell wall composition and architecture using the dimensionless parameters p e , pv , and e v . Conclusions In C hapter IV of this study, it was proposed that three di mensionless parameters obtained from the Ortega Growth Equations be used to quantita te cell wall deformation rates and stress relaxation rates to gain insight into chang es in wall loosening chemistry and cell wall composition and architecture . It was found that the irreversible (plastic) deformation rate s and stress relaxation rate s in stiff mutants were substantially smaller

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73 compared to the wild type . This implied that t he altered genes significantly changed the cell wall loosening chemistry responsible for irreversible deformation (expansive growth). Also found was that the reversible deformation rates of the wa ll were similar in the stiff mutants and WT. This indicated that the cell wall composition and architecture was not signi ficantly changed by the altered genes. The results in this chapter demonstrated that the pe , pv and ev dimensionless parameters can be used to predict changes at the biochemical level. Based o n the results i t was predicted that the stiff mutants have a lower amount of chitin cleaving enzymes known as chitinases and/ or that the altered genes affected the chitinases enzy matic activity . In Chapter V , the wall architecture and wall dynamics were i nvestigated to gain further information on whether the altered genes affected these behaviors . I t was found t hat the stiff mutants exhibit a similar dependence of R (ratio of rotation rate to elongation rate) vs. dL/dt (elon gation rate) as in the WT . This indicates that stiff mutants have a similar wall architecture (microfibril orientation ) and wall dynamics (microfib ril reorientation) as the WT demonstrating that the a ltered genes did not significantly affect these behavior s . Furthermore, it was shown tha t the Fibril slippage reori e ntation mechanism postulated by Ortega et al [ 62 ] was further validated by the stiff mutant cell wall dynamics behavior. Based on the results from the s tudies presented in Chapt ers IV and V it is concluded that the madD , E , F , G , and J genes only substantially alter cell wall loosening chemistry affecting irreversible deformation. The results demonstrated that the important processes in expansive growth were affected by the alter ed genes (stress relaxation and irreversible deformation). This indicates that the diminished tropic

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74 responses exhibited by the stiff mutants is due to an alteration in the cell wall loosening chemistry by the madD , E , F , G , and J genes. Suggestions for Fu ture Work One of the predictions that aris e from the results of Chapter IV can further be investigated to learn if the chitinase activity in stiff mutants is lowered or if simply affected or both . The amount of c hitinase activity can be indirectly acc ounted for by measuring the number of ch itosome s in stiff mutants and WT . It has been shown that chitosomes carry 80% of chitin content in Phycomyces [ 70 ] . To learn if chitinase activity is lowered in stiff mutants the in vitro activation of chitosomes in stiff mutants can be investigated by sim ilar methods used by Herrera Estrella et al [ 83 ] and Martinez Cadena and Ruiz Herrera [ 84 ] . An important area of inv estigation is with chitin synthe tase mutants. Fungal chitinases are diverse and are used in multiple functions in fungi [ 85 , 86 ] . These functions include 1) degradation of exogenous chitin present in fungal cell walls of dead hyphal fragments or in the skeletons of dead arthropods, and the use of degradation products as a nutrient source; 2) cell wall remodeling during the fungal life cycle, which include s roles in hyphal growth, branching, hyphal fusion , and autolysis; 3) competition and defense against other fungi and arthropods in the fungal habitat [ 87 ] . Some fungi use chitinases to attack other fungi, insects or nematodes [ 87 ] . Thus, not all class of chitinases would elicit changes in cell wall deformation and stress relaxation that reflect changes in cell wall loosening chemistry. In filamentous fungi, chitinases are classified into subgro up A (class V) , B (class III) and subgroup C (other classes) [ 87 ] . It has been

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75 suggested that chitinases in subgroup B are responsible for cell wall remodeling in fungi [ 87 90 ] . Currently , there is no evidence to report whether mutants in this subgroup are aff ected in cell wall deformation rates and stress relaxation rate s . Investigating mutants in this subgroup and the other subgroups can help narrow down the number of chitinases responsible for cell wall rem odeling, morphology and expansive growth in Phycomyces blakesleeanus and other fungi . Furthermore, these investigat ions can help with novel methods in the control of fungal diseases. Another future investigation could consist of microscopically identifying wall architecture and composition in the WT and stiff mutant sporangiophores . The results from such a study can h elp validate the r esults presented in Chapters IV and V. It w ould be expected that the micro fibril arrangement and composition is similar in mutants and wild type. One can think of using the dimensionless parameters as a way to rule out genes , proteins a nd enzymes that do not produce the expected wall loosening chemistry, wall stress relaxation rates , and wall deformation rates observed in expansive growth. The dimensionless parameters can help gain better insight into other behaviors exhibited by cells w ith walls such as helical growth in Phy comyces blakesleeanus (Chapter V ). These methods can help with optimizing cell walls for the use by industr ial and pharmaceutical sectors.

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76 R EFERENCES [1] H. Vogler, D. Felekis, B. Nelson, and U. G rossniklaus, "Measuring the Mechanical Properties of Plant Cell Walls," Plants, vol. 4, no. 2, p. 167, 2015. [2] S. Y. Ding, Y. S. Liu, Y. Zeng, M. E. Himmel, J. O. Baker, and E. A. Bayer, "How does plant cell wall nanoscale architecture correlate with enz ymatic digestibility?," Science, vol. 338, no. 6110, pp. 1055 1060, 2012. [3] R. N. Strange and P. R. Scott, "Plant disease: a threat to global food security," Annu. Rev. Phytopathol., vol. 43, pp. 83 116, 2005. [4] J. G. Vallarino and S. Osorio, "Signalin g role of oligogalacturonides derived during cell wall degradation," Plant signaling & behavior, vol. 7, no. 11, pp. 1447 1449, 2012. [5] J. P. Latgé, "The cell wall: a carbohydrate armour for the fungal cell," Molecular Microbiology, vol. 66, no. 2, pp. 2 79 290, 2007. [6] S. M. Bowman and S. J. Free, "The structure and synthesis of the fungal cell wall," Bioessays, vol. 28, no. 8, pp. 799 808, 2006. [7] D. J. Cosgrove, "Growth of the plant cell wall," Nature reviews molecular cell biology, vol. 6, no. 11, p. 850, 2005. [8] J. K. Ortega, J. T. Truong, C. M. Munoz, and E. L. Ortega, "Expansive Growth of Cells with Walls: Force Generation and Growth Regulation," Cells, Forces, and the Microenvironment, p. 357, 2015. [9] D. J. Cosgrove, "Plant cell wall extensi bility: connecting plant cell growth with cell wall structure, mechanics, and the action of wall modifying enzymes," Journal of Experimental Botany, vol. 67, no. 2, pp. 463 476, 2015. [10] D. J. Cosgrove, "Water uptake by growing cells: an assessment of th e controlling roles of wall relaxation, solute uptake, and hydraulic conductance," International Journal of Plant Sciences, vol. 154, no. 1, pp. 10 21, 1993.

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77 [11] D. J. Cosgrove, "Wall relaxation and the driving forces for cell expansive growth," Plant phy siology, vol. 84, no. 3, pp. 561 564, 1987. [12] J. K. Ortega, "Dimensionless number is central to stress relaxation and expansive growth of the cell wall," Scientific reports, vol. 7, no. 1, p. 3016, 2017. [13] D. J. Cosgrove, "Wall extensibility: its nat ure, measurement and relationship to plant cell growth," New Phytologist, vol. 124, no. 1, pp. 1 23, 1993. [14] D. J. Cosgrove, "Cell wall yield properties of growing tissue: evaluation by in vivo stress relaxation," Plant physiology, vol. 78, no. 2, pp. 3 47 356, 1985. [15] D. J. Cosgrove, "Wall relaxation in growing stems: comparison of four species and assessment of measurement techniques," Planta, vol. 171, no. 2, pp. 266 278, 1987. [16] P. B. Green, "Growth physics in Nitella: a method for continuous in vivo analysis of extensibility based on a micro manometer technique for turgor pressure," Plant Physiology, vol. 43, no. 8, pp. 1169 1184, 1968. [17] J. K. Ortega, E. G. Zehr, and R. G. Keanini, "In vivo creep and stress relaxation experiments to determin e the wall extensibility and yield threshold for the sporangiophores of Phycomyces," Biophysical journal, vol. 56, no. 3, p. 465, 1989. [18] J. A. Lockhart, "An analysis of irreversible plant cell elongation," Journal of theoretical biology, vol. 8, no. 2, pp. 264 275, 1965. [19] J. H. Kroeger, R. Zerzour, and A. Geitmann, "Regulator or driving force? The role of turgor pressure in oscillatory plant cell growth," PloS one, vol. 6, no. 4, p. e18549, 2011. [20] M. Probine, "Chemical control of plant cell wall structure and of cell shape," Proc. R. Soc. Lond. B, vol. 161, no. 985, pp. 526 537, 1965.

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78 [21] L. Taiz, "Plant cell expansion: regulation of cell wall mechanical properties," Annual Review of Plant Physiology, vol. 35, no. 1, pp. 585 657, 1984. [22] A. G eitmann and J. K. Ortega, "Mechanics and modeling of plant cell growth," Trends in plant science, vol. 14, no. 9, pp. 467 478, 2009. [23] J. K. Ortega, "Growth rate regulation of cells with walls: The sporangiophores of Phycomyces blakesleeanus used as a m odel system," Rec. Res. Dev. Plant Physiol, vol. 5, pp. 1 19, 2012. [24] J. K. Ortega, C. M. Munoz, S. E. Blakley, J. T. Truong, and E. L. Ortega, "Stiff mutant genes of Phycomyces affect turgor pressure and wall mechanical properties to regulate elongatio n growth rate," Frontiers in plant science, vol. 3, p. 99, 2012. [25] J. K. Ortega, M. E. Smith, A. J. Erazo, M. A. Espinosa, S. A. Bell, and E. G. Zehr, "A comparison of cell wall yielding properties for two developmental stages of Phycomyces sporangiopho res," Planta, vol. 183, no. 4, pp. 613 619, 1991. [26] J. K. Ortega, J. T. Truong, C. M. Munoz, and D. G. Ramirez, "Cell wall loosening in the fungus, Phycomyces blakesleeanus," Plants, vol. 4, no. 1, pp. 63 84, 2015. [27] D. J. Cosgrove, "Loosening of pla nt cell walls by expansins," Nature, vol. 407, no. 6802, p. 321, 2000. [28] D. J. Cosgrove, "Diffuse growth of plant cell walls," Plant physiology, vol. 176, no. 1, pp. 16 27, 2018. [29] D. J. Cosgrove, "Enzymes and other agents that enhance cell wall exte nsibility," Annual review of plant biology, vol. 50, no. 1, pp. 391 417, 1999. [30] D. J. Cosgrove, "Wall structure and wall loosening. A look backwards and forwards," Plant physiology, vol. 125, no. 1, pp. 131 134, 2001.

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7 9 [31] S. McQueen Mason, D. M. Durac hko, and D. J. Cosgrove, "Two endogenous proteins that induce cell wall extension in plants," The Plant Cell, vol. 4, no. 11, pp. 1425 1433, 1992. [32] H. G. Gerken, B. Donohoe, and E. P. Knoshaug, "Enzymatic cell wall degradation of Chlorellavulgaris and other microalgae for biofuels production," Planta, vol. 237, no. 1, pp. 239 253, 2013. [33] R. Palin and A. Geitmann, "The role of pectin in plant morphogenesis," Biosystems, vol. 109, no. 3, pp. 397 402, 2012. [34] T. E. Proseus and J. S. Boyer, "Calcium deprivation disrupts enlargement of Chara corallina cells: further evidence for the calcium pectate cycle," Journal of experimental botany, vol. 63, no. 10, pp. 3953 3958, 2012. [35] T. E. Proseus and J. S. Boyer, "Pectate chemistry links cell expansion to wall deposition in Chara corallina," Plant signaling & behavior, vol. 7, no. 11, pp. 1490 1492, 2012. [36] Y. B. Park and D. J. Cosgrove, "Changes in cell wall biomechanical properties in the xyloglucan deficient xxt1/xxt2 mutant of Arabidopsis," Plant ph ysiology, vol. 158, no. 1, pp. 465 475, 2012. [37] J. Ortega and S. Welch, "Mathematical models for expansive growth of cells with walls," Mathematical Modelling of Natural Phenomena, vol. 8, no. 4, pp. 35 61, 2013. [38] P. Green, R. Erickson, and J. Buggy , "Metabolic and physical control of cell elongation rate: in vivo studies in Nitella," Plant Physiology, vol. 47, no. 3, pp. 423 430, 1971. [39] N. P. Money and F. M. Harold, "Extension growth of the water mold Achlya: interplay of turgor and wall strengt h," Proceedings of the National Academy of Sciences, vol. 89, no. 10, pp. 4245 4249, 1992. [40] J. Passioura and S. Fry, "Turgor and cell expansion: beyond the Lockhart equation," Functional Plant Biology, vol. 19, no. 5, pp. 565 576, 1992.

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80 [41] A. Geitman n, "A. Geitmann and JKE Ortega, Trends Plant Sci. 14, 467 (2009)," Trends Plant Sci, vol. 14, p. 467, 2009. [42] J. K. Ortega, "Augmented growth equation for cell wall expansion," Plant physiology, vol. 79, no. 1, pp. 318 320, 1985. [43] J. S. Boyer, A. Ca valieri, and E. D. Schulze, "Control of the rate of cell enlargement: excision, wall relaxation, and growth induced water potentials," Planta, vol. 163, no. 4, pp. 527 543, 1985. [44] T. E. Proseus, J. K. Ortega, and J. S. Boyer, "Separating growth from el astic deformation during cell enlargement," Plant physiology, vol. 119, no. 2, pp. 775 784, 1999. [45] J. K. Ortega, "Plant cell growth in tissue," Plant physiology, p. pp. 110.162644, 2010. [46] J. K. Ortega, R. G. Keanini, and K. J. Manica, "Pressure pro be technique to study transpiration in Phycomyces sporangiophores," Plant physiology, vol. 87, no. 1, pp. 11 14, 1988. [47] R. Fox, A. McDonald, P. Pritchard, and J. Leylegian, "Fluid Mechanics. 8th. 1," ed: New York: Wiley, 2011. [48] J. Ortega, "Dimensio nal analysis of expansive growth of cells with walls," Res Rev: J Bot Sci, vol. 5, no. 3, pp. 17 24, 2016. [49] E. C. O. a. E. D. Lipson, Phycomyces . Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1987. [50] P. Galland, A. Palit, and E. Lipso n, "Phycomyces: phototropism and light growth response to pulse stimuli," Planta, vol. 165, no. 4, pp. 538 547, 1985. [51] G. Löser and E. Schäfer, "ARE THERE SEVERAL PHOTORECEPTORS INVOLVED IN PHOTOTROPISM OF Phycomyces blakesleeunus? KINETIC

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81 STUDIES OF D ICHROMATIC IRRADIATION," Photochemistry and photobiology, vol. 43, no. 2, pp. 195 204, 1986. [52] K. Bergman et al. , "Phycomyces," Bacteriological Reviews, vol. 33, no. 1, pp. 99 157, 1969. [53] M. Delbrück, A. Katzir, and D. Presti, "Responses of Phycomyc es indicating optical excitation of the lowest triplet state of riboflavin," Proceedings of the National Academy of Sciences, vol. 73, no. 6, pp. 1969 1973, 1976. [54] M. Delbrück and W. Reichardt, "System analysis for the light growth reactions of Phycomy ces," Cellular mechanisms in differentiation and growth, vol. 14, p. 3, 1956. [55] C. N. Ahlquist and R. I. Gamow, "Phycomyces: Mechanical Behavior of Stage II and Stage IV 1," Plant physiology, vol. 51, no. 3, p. 586, 1973. [56] D. S. Dennison and C. C. R oth, "Phycomyces sporangiophores: fungal stretch receptors," Science, vol. 156, no. 3780, pp. 1386 1388, 1967. [57] J. K. E. Ortega and R. I. Gamow, "An Increase in Mechanical Extensibility during the Period of Light stimulated Growth," Plant Physiology, v ol. 57, no. 3, pp. 456 457, 1976. [58] J. K. Ortega and R. I. Gamow, "Phycomyces: an increase in mechanical extensibility during the avoidance growth response," Plant physiology, vol. 60, no. 5, pp. 805 806, 1977. [59] J. K. Ortega, R. I. Gamow, and C. N. Ahlquist, "Phycomyces: a change in mechanical properties after a light stimulus," Plant physiology, vol. 55, no. 2, pp. 333 337, 1975. [60] M. J. Middlebrook and R. Preston, "Spiral growth and spiral structure: III . Wall structure in the growth zone of Phycomyces," Biochimica et biophysica acta, vol. 9, pp. 32 48, 1952.

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82 [61] J. K. Ortega and R. I. Gamow, "The problem of handedness reversal during the spiral growth of Phycomyces," Journal of theoretical biology, vol. 47, no. 2, pp. 317 332, 1974. [62] J. K. Ortega et al. , "Helical growth of stage IVb sporangiophores of Phycomyces blakesleeanus: the relationship between rotation and elongation growth rates," Planta, vol. 216, no. 4, pp. 716 722, 2003. [63] K. Yoshida, T. Ootaki, and J. Ortega, "Spiral growth in the radially expanding piloboloid mutants ofPhycomyces blakesleeanus," Planta, vol. 149, no. 4, pp. 370 375, 1980. [64] J. K. E. Ortega, "Phycomyces: The Mechanical and Structural Dynamics of Cell Wall Growth," D octor of Philosophy Aerospace Engineering, University of Colorado Boulder, UMI Dissertation Information Service, 1976. [65] J. K. Ortega, J. F. Harris, and R. I. Gamow, "The analysis of spiral growth in Phycomyces using a novel optical method," Plant physi ology, vol. 53, no. 3, pp. 485 490, 1974. [66] E. S. Castle, "Spiral growth and reversal of spiraling in Phycomyces, and their bearing on primary wall structure," American Journal of Botany, vol. 29, no. 8, pp. 664 672, 1942. [67] A. Oort, "The spiral grow th of Phycomyces," 1931: Koninklijke Akademie van Wetenschappen. [68] S. Bartnicki Garcia, "Cell wall chemistry, morphogenesis, and taxonomy of fungi," Annual Reviews in Microbiology, vol. 22, no. 1, pp. 87 108, 1968. [69] J. Ruiz Herrera, E. Lopez Romero, and S. Bartnicki Garcia, "Properties of chitin synthetase in isolated chitosomes from yeast cells of Mucor rouxii," Journal of Biological Chemistry, vol. 252, no. 10, pp. 3338 3343, 1977. [70] J. Ruiz Herrera, C. E. Bracker, and S. Bartnicki Garcia, "Sedi mentation properties of chitosomes fromMucor rouxii," Protoplasma, vol. 122, no. 3, pp. 178 190, 1984.

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83 [71] T. Ootaki and A. Miyazaki, "Genetic nomenclature and strain catalogue of Phycomyces," Sendai, Japan: Tohoku University, 1993. [72] V. Campuzano, P. Galland, M. I. Alvarez, and A. P. Eslava, "Blue Light Receptor Requirement for Gravitropism, Autochemotropism and Ethylene Response in Phycomyces*," Photochemistry and Photobiology, vol. 63, no. 5, pp. 686 694, 1996. [73] F. Grolig, P. Eibel, C. Schimek, T . Schapat, D. S. Dennison, and P. A. Galland, "Interaction between Gravitropism and Phototropism in Sporangiophores of Phycomyces blakesleeanus," Plant Physiology, vol. 123, no. 2, pp. 765 776, 2000. [74] D. J. Cosgrove, "Characterization of long term extension of isolated cell walls from growing cucumber hypocotyls," Planta, vol. 177, no. 1, pp. 121 130, 1989. [75] B. Cubero, J. Ruiz Herrera, and E. Cerdá Olmedo, "Chitin synthetase mutants of Phycomyces blakesleeanus," Molecular and General Geneti cs MGG, vol. 240, no. 1, pp. 9 16, 1993. [76] M. Atsushi, M. Momany, P. J. Szaniszlo, M. Jayaram, and O. Tamotsu, "Chitin synthase encoding gene (s) of the Zygomycete fungus Phycomyces blakesleeanus," Gene, vol. 134, no. 1, pp. 129 134, 1993. [77] Y. N. Ja n, "Properties and cellular localization of chitin synthetase in Phycomyces blakesleeanus," Journal of Biological Chemistry, vol. 249, no. 6, pp. 1973 1979, 1974. [78] L. Herrera Estrella and J. Ruiz Herrera, "Light response inPhycomyces blakesleeanus: evi dence for roles of chitin biosynthesis and breakdown," Experimental mycology, vol. 7, no. 4, pp. 362 369, 1983. [79] A. Heyn, "Further investigations on the mechanism of cell elongation and the properties of the cell wall in connection with elongation," Pr otoplasma, vol. 25, no. 1, pp. 372 396, 1936.

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84 [80] E. S. Castle, "The distribution of velocities of elongation and of twist in the growth zone of Phycomyces in relation to spiral growth," Journal of Cellular and Comparative Physiology, vol. 9, no. 3, pp. 4 77 489, 1937. [81] M. J. Middlebrook and R. Preston, "Spiral growth and spiral structure: IV. Growth studies and mechanical constants in the cell wall," Biochimica et biophysica acta, vol. 9, pp. 115 126, 1952. [82] R. Cohen and M. Delbrück, "Distribution of stretch and twist along the growing zone of the sporangiophore of Phycomyces and the distribution of response to a periodic illumination program," Journal of cellular and comparative physiology, vol. 52, no. 3, pp. 361 388, 1958. [83] L. Herrera Estrell a, B. Chavez, and J. Ruiz Herrera, "Presence of chitosomes in the cytoplasm ofPhycomyces blakesleeanus and the synthesis of chitin microfibrils," Experimental Mycology, vol. 6, no. 4, pp. 385 388, 1982. [84] G. Martinez Cadena and J. Ruiz Herrera, "Activat ion of chitin synthetase from Phycomyces blakesleeanus by calcium and calmodulin," Archives of Microbiology, journal article vol. 148, no. 4, pp. 280 285, October 01 1987. [85] G. W. Gooday, "Physiology of microbial degradation of chitin and chitosan," in Biochemistry of microbial degradation : Springer, 1994, pp. 279 312. [86] P. Jollès and R. A. Muzzarelli, "Chitin and chitinases," EXS(Basel), 1999. [87] V. Seidl, "Chitinases of filamentous fungi: a large group of diverse proteins with multiple physiologic al functions," Fungal Biology Reviews, vol. 22, no. 1, pp. 36 42, 2008. [88] D. J. Adams, "Fungal cell wall chitinases and glucanases," Microbiology, vol. 150, no. 7, pp. 2029 2035, 2004. [89] R. Hurtado Guerrero and D. M. van Aalten, "Structure of Sacchar omyces cerevisiae chitinase 1 and screening based discovery of potent inhibitors," Chemistry & biology, vol. 14, no. 5, pp. 589 599, 2007.

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85 [90] A. K. Jaques et al. , "Disruption of the gene encoding the ChiB1 chitinase of Aspergillus fumigatus and character ization of a recombinant gene product," Microbiology, vol. 149, no. 10, pp. 2931 2939, 2003.

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86 APPENDIX A Below are the m athematics to explain how the irrever sible deformation rate in Equation ( 4. 1) of Chapter IV is eliminated. First Equation ( 4.1) is r ewritten using finite differences: ( 4. 1 ) Multiplying Equation (4.1 ) by t and rearranging to solve for L : ( 4 .1a ) As the limit of t app roaches zero, ( 4.1b ) the term L ( P P c ) also approaches zero and becomes negligible in Equation ( 4. 1). Now L becomes Equation ( 4. 2) in Chapter IV: ( 4. 2 ) Furthermo re, because the pressure step ups are larger than 0.02 MPa, the steady state growth rate , ( L ( P P c ) = steady state growth rate when P =constant) is reduced at the first pressure step up for the growing spora ngiophores. This also makes the irreversible defo rm ation term smaller and therefore negligible .

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87 APPENDIX B Inaccurate Data Figure A 1 represents an experiment that results in inaccurate results obtained from the microcapillary tip being too large and causing leaks at impalement. Figure A 2 is a snapsho t captured through a USB microscope depicting a large (30 40 m) microcapillary tip size and the leak caused by this. A good tip size is the smallest possible without blocking oil flow and cell sap through the microcapillary (Figure A 3 ) . Too small of a tip causes the cell sap to clog the tip and read inaccurate tur gor pressure readings . Too large of a tip causes leaks at impalement site and causes inaccurate turgor pressure reading s as well. A good tip size for mutant sporangiophores was between 10 15 m .

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88

PAGE 105

89 Figure A 1 Example of an in vivo turgor pressure step up e xperi ment displaying innacurate results. (UPPER GRAPH) Turgor pressure behavior. The cell is impaled at the second mark r epresented by the red arrow. The cell sap interface was not located since there was a leak at impalement. The turgor pressure is innacu rate and results in a low measured value ( P 0.02 MPa) . The t urgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressure step up is applied . Due to the leak at impalement all other measuremnts are innacurate , L and L . Figure A 2 . Microcapillary t ip size is too large (30 40 m) causing a leak at i mpalement site. Cell material leaks out and an accurate turgor pressure measurement cannot be accomplished.

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90 Figure A 2 . Microcapillary tip is the correct size (10 15 m). Here a leak is not caused when impaling the cell, this can be observed because cell sap flows into the microcapillary enabling turgor pressure reading .

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91 APPENDIX C Supplemental Results for In V ivo Turgor Pressure Step Up Experiments The following two examples of in vivo pressure step up experiments are for stage III ( non growing ) sporangiophores. In stage I II sporangiophores the wall deformation is purely elastic thus all elongations elicited by the turgor pressure step ups are considered when computing an average L value .

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92 A C B

PAGE 109

93 Figure A 3 . Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus beha vior for a single stage III C 216 sporangiophore during an in vivo turgor pressure step up experiment . A . Turgor pressure behavior. The cell is impaled at the second mark represented by the red arrow and the turgor pressure is increased until the cell sap interface is brought to the surface of the cell. Turgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressur e step up is a pplied to the cell . The new turgor pressure is maintained constant until 30 seconds later and the next pressure st ep up is applied. t ref=0 indicates the time when elongation measurements began. A total of 5 pressure step ups were applied to the sporangiophore which resulted in purely elastic elongation. B . Elongation as a function of time. Elongation was graphed for the corresponding pressure step ups at 30 sec intervals. All elongations are purely elastic since the cell is non growing. The c ell sust The leak is represented by a decrease in elongation due to the leakage of oil and cell material (purple arrow ). The change in elongation, L , and P , are used to determine L for eac h pressure step up. A total of 5 L values are determined for this experiment and an average value is computed. C. Plot of L as a function of turgor pressure, P . Each L value is graphed with its corresponding pressure. The average L value for this sporangiophore is rep resented as a red dashed line ( L = 47 MPa) . The volumetric elastic modulus is approximately constant for the range of turgor pressures the cell was subjected to.

PAGE 110

94 A B C

PAGE 111

95 Figure A 4 . Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus beha vior for a single stage III C149 sporangiophore during an in vivo turgor pressure step up experiment . A . Turgor pressure behavior. The cell is impaled at the second mark represented by the red arrow and the turgor pressure is increased until the cell sap interface is brought to the surface of the cell. Turgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressure step up is a pplied to the cell . The new turgor pressure is maintained constant until 30 seconds later and the next pressure st ep up is applied. t ref=0 indicates the time when elongation measurements began. A total of 9 pressure step ups were applied to the sporangiophore which resulted in purely elastic elongation. B . Elongation as a function of ti me. Elongation was graphed for the corresponding pressure step ups at 30 sec intervals. All elongations are purely elastic since the cell is non growing. cell sust ains a The leak is represented by a decrease in elongation due to the leakage of oil and cell material (purple arrow ). The change in elongation, L , and P , are used to determine L for eac h pressure step up. A total of 9 L values are determined for this experiment and an average value is computed. C. Plot of L as a function of turgor pressure, P . Each L value is graphed with its corresponding pressure. The average L value for this sporangiophore is rep resented as a red dashed line ( L = 49 MPa) . The volumetric elastic modulus is approximately constant for the range of turgor pressures the cell was subjected to. T he following 6 results depict typical in vivo pre ssure step ups experiments conducted on sta ge IVb (growing) sporangiophores for C216 and C149 strains .

PAGE 112

96 A B C

PAGE 113

97 Figure A 5 . Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus behavior for a single stage IVb C216 sporangiophore during an in vivo turgor pressure step up experiment . A . Turgor pressure behavior. The cell is impaled at the second mark represented by the red arrow and the turgor pressure is increased until the cell sap interface is brought to the surface of the cell. Turgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressure step up is applied to the cell (green arrow). The new turgor pressure is maintained constant until 30 seconds later and the next pressure st ep up is applied. t ref=0 indicates the time when elongation m easurements began. A total of 6 pressure step ups were applied to the sporangiophore which resulted in purely elastic elongation. B . Elongation as a function of time. Elongation was graphed for the corresponding pressure step ups at 30 sec intervals. E long ations after the black dashed line are purely elast ic . The cell sust The leak is represented by a decrease in elongation due to the leakage of oil and cell material (purple arrow ). The change in elon gation, L , and P , are used to determine L for eac h pressure step up. A total of 6 L values are determined for this experiment and an average value is computed. C. Plot of L as a function of turgor pressure, P . Each L value is graphed with its corresp onding pressure. The average L value for this sporangiophore is represented as a red dashed line ( L = 50 MPa) . The volumetric elastic modulus is approximately constant for the range of turgor pressures the cell was subjected to.

PAGE 114

98 A C B

PAGE 115

99 Figure A 6 . Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus behavior for a single stage IVb C216 sporangiophore during an in vivo turgor pressure step up experiment . A . Turgor pressure behavior. The cell is impaled at the second mark represented by th e red arrow and the turgor pressure is increased until the cell sap interface is brought to the surface of the cell. Turgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressure step up is applied to the cell (green arrow). The new turgor pressure is maintained constant until 30 seconds later and the next pressure st ep up is applied. t ref=0 indicates the time when elongation measurements began. A total of 6 pressure step ups were applied to the sporangiophore which resulted in purely elastic elongation. B . Elongation as a function of time. Elongation was graphed for the corresponding pressure step ups at 30 sec intervals. Elongations after the black dashed line are purely elastic. The cell sust ains a maximum pr essure of P The leak is represented by a decrease in elongation due to the leakage of oil and cell material (purple arrow ). The change in elongation, L , and P , are used to determine L for eac h pressure step up. A total of 6 L values are determined for this experiment and an average value is computed. C. Plot of L as a function of turgor pressure, P . Each L value is graphed with its corresponding pressure. The average L value for this sporangiophore is represented as a r ed dashed line ( L = 126 MPa) . The volumetric elastic modulus is approximately constant for the range of turgor pressures the cell was subjected to.

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100 A C B

PAGE 117

101 Figure A 6 . Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus behavior for a sin gle stage IVb C216 sporangiophore during an in vivo turgor pressure step up experiment . A . Turgor pressure behavior. The cell is impaled at the second mark represented by the red arrow and the turgor pressure is increased until the cell sap interface is br ought to the surface of the cell. Turgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressure step up is applied to the cell (green arrow). The new turgor pressure is maintained constant until 30 seconds late r and the next pressure st ep up is applied. t ref=0 indicates the time when elongation measurements began. A total of 5 pressure step ups were applied to the sporangiophore which resulted in purely elastic elongation. B . Elongation as a function of time. E longation was graphed for the corresponding pressure step ups at 30 sec intervals. Elongations after the black dashed line are purely elastic. The cell sust ains a maximum pressure of P The leak is represented by a decrease in elongation due to the leakage of oil and cell material (purple arrow ). The change in elongation, L , and P , are used to determine L for eac h pressure step up. A total of 5 L values are determined for this experiment and an average value is computed. C. Plot of L as a function of turgor pressure, P . Each L value is graphed with its corresponding pressure. The average L value for this sporangiophore is represented as a red dashed li ne ( L = 37 MPa) . The volumetric elastic modulus is approximately constant for the range of turgor pressures the cell was subjected to.

PAGE 118

102 A C B

PAGE 119

103 Figure A 8 . Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus behavior for a single stage I Vb C149 sporangiophore during an in vivo turgor pressure step up experiment . A . Turgor pressure behavior. The cell is impaled at the second mark represented by the red arrow and the turgor pressure is increased until the cell sap interface is brought to th e surface of the cell. Turgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressure step up is applied to the cell (green arrow). The new turgor pressure is maintained constant until 30 seconds later and the n ext pressure st ep up is applied. t ref=0 indicates the time when elongation measurements began. A total of 8 pressure step ups were applied to the sporangiophore which resulted in purely elastic elongation. B . Elongation as a function of time. Elongation w as graphed for the corresponding pressure step ups at 30 sec intervals. Elongations after the black dashed line are purely elastic. The cell sust ains a maximum pressure of P The leak is represented by a decrease in elongation due to the leakage of oil and cell material (purple arrow ). The change in elongation, L , and P , are used to determine L for eac h pressure step up. A total of 8 L values are determined for this experiment and an average value is computed. C. Plot of L as a function of turgor pressure, P . Each L value is graphed with its corresponding pressure. The average L value for this sporangiophore is represented as a red dashed l ine ( L = 60 MPa) . The volumetric elastic modulus is approximately constant for the range of turgor pressures the cell was subjected to.

PAGE 120

104 A C B

PAGE 121

105 Figure A 9 . Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus behavior for a single stage IV b C 149 sporangiophore during an in vivo turgor pressure step up experiment . A . Turgor pressure behavior. The cell is impaled at the second mark represented by the red arrow and the turgor pressure is increased until the cell sap interface is brought to the surface of the cell. Turgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressure step up is applied to the cell (green arrow). The new turgor pressure is maintained constant until 30 seconds later and the ne xt pressure st ep up is applied. t ref=0 indicates the time when elongation measurements began. A total of 7 pressure step ups were applied to the sporangiophore which resulted in purely elastic elongation. B . Elongation as a function of time. Elongation wa s graphed for the corresponding pressure step ups at 30 sec intervals. Elongations after the black dashed line are purely elastic. The cell sust ains a maximum pressure of P The leak is represented by a decrease in elongation due to the leakage of oil and cell material (purple arrow ). The change in elongation, L , and P , are used to determine L for eac h pressure step up. A total of 7 L values are determined for this experiment and an average value is computed. C. Plot of L as a function of turgor pressure, P . Each L value is graphed with its corresponding pressure. The average L value for this sporangiophore is represented as a red dashed l ine ( L = 32 MPa) . The volumetric elastic modulus is approximately constant for the range of turgor pressures the cell was subjected to.

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106 A C B

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107 Figure A 10 . Turgor pressure, Elongation and Longitudinal Volumetric Elastic Modulus behavior for a single stage I Vb C149 sporangiophore during an in vivo turgor pressure step up experiment . A . Turgor pressure behavior. The cell is impaled at the second mark represented by the red arrow and the turgor pressure is increased until the cell sap interface is brought to th e surface of the cell. Turgor pressure is maintained constant (blue arrow) by injecting inert silicon oil until the first pressure step up is applied to the cell (green arrow). The new turgor pressure is maintained constant until 30 seconds later and the n ext pressure st ep up is applied. t ref=0 indicates the time when elongation measurements began. A total of 8 pressure step ups were applied to the sporangiophore which resulted in purely elastic elongation. B . Elongation as a function of time. Elongation w as graphed for the corresponding pressure step ups at 30 sec intervals. Elongations after the black dashed line are purely elastic. The cell sust ains a maximum pressure of P The leak is represented by a decrease in elongation due to the leakage of oil and cell material (purple arrow ). The change in elongation, L , and P , are used to determine L for eac h pressure step up. A total of 8 L values are determined for this experiment and an average value is computed. C. Plot of L as a function of turgor pressure, P . Each L value is graphed with its corresponding pressure. The average L value for this sporangiophore is represented as a red dashed l ine ( L = 57 MPa) . The volumetric elastic modulus is approximately constant for the range of turgor pressures the cell was subjected to.

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108 APPENDIX D Dimensionless Variables used by Ortega [ 48 ] to make the Ortega growth equatio ns dimensionless

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109 APPENDIX E Computation of Dimensionless Parameters The same proc ess was used as in Ortega [ 48 ] to compute stiff mutant values. The process consists of computing the largest value and the smallest value to obtain an average value. For reference , computation of the pe value for intact WT sporangiophores is presented. All other values for WT and stiff mutants are computed here as pa rt of the s tudy presented in Chapter IV . Wild Type values The following values were obtain ed for intact stage IVb WT sporangiophores of Phycomyces blakesleeanus . WT pe magnitude is from Ortega [ 12 ] . Values for variables L , d L /d t , m , and were obtained from [ 12 , 48 ] : Wild Type pe : L = 30 mm = 3.0 X 10 4 m d L /d t = 34 ± 3.1 (SE) m min 1 (n=20) m = 997 ± 160 (SE) m min 1 (n=20)

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110 = 60.9 ± 5.1 (SE) MPa (n=27) Wild Type pv : Wild Type ev :

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111 C216 values The following values were obtain ed for intact stage IVb C216 sporangiophores of Phycomyces blakesleeanus . Values for variables L , d L /d t , and L were determined in the study presente d in chapter II and values for m were obtained from [ 24 ] : C216 pe : L = 25 mm = 2.5 X 10 4 m d L /d t = 27.3 ± 2.5 (SE) m min 1 (n=59 ) m = 222 ± 4 0 (SE) m min 1 (n=18 )

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112 = 49.2 ± 2.6 (SE) MPa (n =24 ) C216 pv : C126 ev :

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113 C149 values The following values were obtain ed for intact stage IVb C149 sporangiophores of Phycomyces blakesleeanus. Values for variables L , d L /d t , and L were determined in the study presented in chapter II and values for m were obtained from [ 24 ] : C149 pe : L = 25 mm = 2.5 X 10 4 m d L /d t = 27.8 ± 3.1 (SE) m min 1 (n=59 ) m = 170 ± 30 (SE) m min 1 (n=8 ) L = 60.5 ± 5.3 (SE) MPa (n =17 )

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114 C149 pv : C149 ev :