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Phycomyces

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Phycomyces helical growth and turgor pressure behavior during death by asphyxiation
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Olson, Jessica Elyn
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English
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164 leaves : illustrations ; 28 cm

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Subjects / Keywords:
Phycomyces ( lcsh )
Asphyxia ( lcsh )
Death -- Causes ( lcsh )
Turgor ( lcsh )
Asphyxia ( fast )
Death -- Causes ( fast )
Phycomyces ( fast )
Turgor ( fast )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Bibliography:
Includes bibliographical references (leaves 161-164).
General Note:
Department of Mechanical Engineering
Statement of Responsibility:
by Jessica Elyn Olson.

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|University of Colorado Denver
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ocm45536158
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LD1190.E55 2000m .O57 ( lcc )

Full Text
PHYCOMYCES: HELICAL GROWTH AND TURGOR PRESSURE
BEHAVIOR
DURING DEATH BY ASPHYXIATION
by
Jessica Elyn Cunning Olson
B.S., University of Colorado at Denver, 1998
A thesis submitted to the
University of Colorado at Denver
in partial fulfillment
of the requirements for the degree of
Master of Science
Mechanical Engineering
2000


This thesis for the Master of Science
Degree by
Jessica Elyn Cunning Olson
Has been approved
By
Cs> /% 1 /OC
Date


Olson, Jessica Elyn Cunning (M.S., Mechanical Engineering Department,
University of Colorado at Denver)
Phycomyces: Helical Growth and Turgor Pressure Behavior During Death by
Asphyxiation
Thesis directed by Professor Dr. Joseph K.E. Ortega
ABSTRACT
The helical growth components (elongation and rotation growth) and
turgor pressure behavior of stage IVb sporangiophores of Phycomyces
blakesleeanus are studied during death by asphyxiation. In the first group of
experiments that studied elongation growth, two phases are observed during
death by asphyxiation. The first phase is a slowing phase, where the elongation
growth gradually stops. The second phase is a contraction phase, where the
elongation growth is negative and ends in the collapse of the sporangiophore.
In related experiments, the rotation growth is measured together with
elongation growth. The results demonstrate that the rotation growth generally
stops soon after the elongation growth stops. In other experiments, markers are
used to measure longitudinal displacement at different locations along the stalk.


The results demonstrate that the entire length of the stalk is contracting during
asphyxiation.
Interestingly, many of the sporangiophores display a sudden increase in
length, a jump in elongation during the contraction phase. Importantly it is
found that there is no change in rotation during these jumps in elongation. The
markers on the stalk show a qualitatively similar jump in elongation. This
finding indicates that the jump in elongation occurs throughout the length of the
stalk.
The second group of experiments studies the turgor pressure (measured
with the pressure probe) of the sporangiophore during asphyxiation. The results
demonstrate that an initial small decrease in turgor pressure occurs when the
oxygen concentration is decreased to less than 1%. This initial decrease is
followed by a linear decay and then an exponential decay in turgor pressure. The
turgor pressure decays to a value near zero, before the sporangiophore collapses.
Furthermore, it is shown that the elongation growth rate decreases (often times to
zero) at the same time as the initial small decrease in turgor pressure.
Interestingly and importantly, jumps in elongation are accompanied by
simultaneous jumps in turgor pressure.
IV


This abstract accurately represents the content of the candidates thesis. I
recommend its publication.
Signed
Joseph K.E. Ortega


DEDICATION
This thesis is dedicated to my husband Jacob Olson, to who has supported me in
my endeavors to discover and fulfill my dreams. In addition Id like to dedicate
this work to my life long learning companion, Melissa Fearing.


ACKNOWLEDGMENT
A special thanks is extended to Elena Ortega for all the time that was spent in
doing the experiments for this thesis. Also I want to thank Melissa Fearing for
her expertise and advice. And most importantly I want to thank my advisor, Dr.
Joseph K.E. Ortega, for his wisdom and direction to achieve an excellent
education.


CONTENTS
Figures............................................................x
Chapter
1. Introduction...................................................1
1.1 General Introduction............................................1
1.2 Fungi...........................................................4
1.3 The Sporangiophore of Phycomyces blakesleeanus..................5
1.4 Developmental Stages of the Sporangiophores.....................6
1.5 Growth Mechanisms in Sporangiophores...........................11
1.6 Turgor Pressure in Sporangiophores.............................14
1.7 Preview of Thesis Content......................................27
2. Materials and Methods..........................................29
2.1 Plant Material.................................................29
2.2 Elongation Growth Measurements................................29
2.3 Growth Nitrogen Chamber.......................................34
2.4 Rotation Growth Measurements..................................36
2.5 Pressure Probe................................................36
2.6 Turgor Pressure Measurements..................................41
2.7 Pressure Probe Nitrogen Chamber...............................46
viii


3. Results..........................................................51
3.1 Elongation Growth Behavior During Death..........................51
3.2 Elongation and Rotation Growth Behavior During Death.............70
3.3 Markers, Elongation and Rotation Growth Behavior During Death....99
3.4 Turgor Pressure Behavior During Death...........................109
4. Elongation Jumps.................................................127
4.1 Elongation Jumps During Death...................................127
4.2 Rotation Growth During Elongation Jumps.........................127
4.3 Markers, Rotation Growth During Elongation Jumps................136
4.4 Turgor Pressure During Elongation Jumps.........................141
5. Discussion.......................................................150
5.1 Elongation Growth Behavior During Death.........................150
5.2 Elongation and Rotation Growth Behavior During Death............155
5.3 Turgor Pressure Behavior During Death...........................156
5.4 Elongation Jumps During Death...................................158
Appendix
A. Pressure Probe Nitrogen Chamber Drawing...........................160
References ........................................161
IX


FIGURES
Figure
1.1 Developmental Stages of the Sporangiophore......................7
1.2 Sporangiophore Modeled As a Spring and Dashpot.................17
1.3 Pressure Trace of a Pressure Relaxation Experiment.............22
1.4 Volumetric Elastic Modulus and Turgor Pressure Relationship....25
2.1 Experimental Set Up For Growth Readings........................32
2.2 Pressure Probe Schematic......................................38
2.3 Experimental Set Up For Turgor Pressure Readings..............44
2.4 Back Support and Microcapillary Tip...........................47
3.1 First Pattern Elongation Growth Experiments....................54
3.2 Collapse of the Sporangiophore............................... 59
3.3 Second Pattern Elongation Growth Experiments..................61
3.4 Third Pattern Elongation Growth Experiments...................66
3.5 First Pattern Elongation and Rotation Growth Experiments......72
3.6 Second Pattern Elongation and Rotation Growth Experiments.....77
3.7 Third Pattern Elongation and Rotation Growth Experiments......83
3.8 Reduced Rotation Pattern......................................88
3.9 Temporary Reversal in Rotation Pattern........................91


3.10 Reversal Rotation Pattern.......................................95
3.11 First Pattern Elongation and Rotation Growth With Markers......100
3.12 Second Pattern Elongation and Rotation Growth With Markers.....103
3.13 Third Pattern Elongation and Rotation Growth With Markers......105
3.14 First Pattern Elongation Growth With Turgor Pressure...........110
3.15 Third Pattern Elongation Growth With Turgor Pressure...........112
4.1 Elongation Jumps During Death....................................128
4.2 Rotation Growth During Elongation Jumps.........................132
4.3 Markers, Rotation Growth During Elongation Jumps................137
4.4 Turgor Pressure During Elongation Jumps.........................142
5.1 Drawings of a Collapsing Sporangiophore.........................151
XI


1. Introduction
1.1 General Introduction
Every living organism dies, therefore death is an important bio-
mechanism to understand. Insights to death maybe obtained by studying the
death process. Generally, the breakdown of tissues and cells are observed during
the death process.
Typically, cell death is divided into three categories: necrosis, apoptosis,
and cell deletion. Necrosis is death by means of either trauma or disease and
apoptosis and cell deletion are genetically programmed deaths.
Cellular swelling, rupture of plasma membranes and internal membranes,
and leakage of cellular contents are characteristic features of necrosis (Bowen
and Lockshin, 1981). This form of cellular death most often results from
external stimulus. The early signs of necrosis are cytoplasmic swelling,
enlargement of the endoplasmic reticulum, mitochondrial swelling, and the
breakdown of polysomes structures. In general, all of these changes are
reversible. However, if the cell is dying, these symptoms continue and become
irreversible. The mitochondria continue to swell, and extensive cytoplasmic
swelling continues until disintegration of cytoplasmic organelles and rupture of
1


the plasma membranes. Generally, the loss of control of the cell volume results
in cell death.
Apoptosis and programmed cell deletion are believed to be under genetic
control. Also they are a biomechanism used for change from one stage of
development to another (Bowen, 1984). For example, the loss of a tadpole tail is
apoptosis cell death. Unlike necrosis, in apoptosis cell death, the cells shrink and
contract. Apoptosis in cells generally results in cell fragmentation. Here cell
death is necessary for existence and survival. An example of programmed cell
deletion is the continual renewal of dying cells from the skin and other organs.
The easiest means of studying cell death is by observing and measuring
tissues and cells (Bowen, 1984). This kind of measurement relies upon the idea
that control of homeostasis is an important mechanism for a living tissue or cell.
Therefore data on changes in volume, length, weight and other characteristics are
correlated with death. In general, dying cells either through apoptosis or
necrosis, result in cell fragmentation and debris of the original cell. These types
of measurements exploit the idea that dead cells are significantly smaller,
deformed and unable to maintain membrane integrity.
Information concerning cell death can also be obtained through observing
changing tissue and morphology during death (Bowen, 1984). This type of study
2


entails not only measurements of volume and weight but include measuring
properties of a cell. These properties such as respiration, reproduction and waste
production define living organisms.
Other means of studying cell death include studying biochemical changes
in tissues and cells during death (Bowen, 1984). This type of experimentation
uses stains and dyes to enhance and demonstrate vital cellular functions. For
example, a dye would stain a vital organelle such as the mitochondria in a dead
cell but not in a living cell. In another example, the nuclei of dying cells that are
stained show clumping and fragmentation of chromatin. In general, these types
of studies involve stains and chemicals to observe changes in tissues and cells in
death.
Some of the previous methods used to study dead tissue provide
descriptive information concerning biological changes of tissue. However, they
do not provide insight to the biophysical and biomechanical changes that occur
during cellular death. These descriptive studies involve tissue, which is a more
complex system than a single cell.
An advantage to using a single-celled organism is that a better
interpretation of the experimental results maybe obtained. The study of a single-
3


celled organism can be conducted with a more rigorous methodology and may
provide a deeper understanding to the process of cellular death.
Phycomyces blakesleeanus is a single-celled organism that has been used
as a model system for biophysical studies (Ortega, 1976, 1990), and for sensory
and growth studies (Cerda-Olmedo and Lispson, 1987). In this thesis,
Phycomyces blakesleeanus is used to study the biophysics and biomechanics of
death.
1.2 Fungi
Fungi are characterized by several distinct differences that separate them
from plants; a few include a mycelium, hyphae, and spores (Ainsworth and
Sussman, 1965). The mycelium is a filamentous, multinucleated vegetative
structure. The mycelium consists of a network of hyphae, which is a complex
system of walled cylindrical tubes that contain protoplasm. The hyphae
continually branches out and extents by apical growth and lateral branching. The
spores are a means of reproduction, which occurs in fungi after a period of
growth. These spores are uni- or multicellular bodies that can separate (by
contact) from the parent organism and start a new generation. The main
difference from fungal and plant cells is that fungal cells do not photosynthesize.
4


Plant and fungal cells are very similar in anatomy. Typically, only the
building material of the cell wall of the plant and fungal cell is different. The
fungal cell wall is composed of chitin and chitosan where the plant cell wall is
composed of cellulose and hemicellulose. The fungal cell itself consists of an
outer firm cell wall and inner protoplasm (Campbell, 1990). A cell membrane
encloses the protoplasm and contains the cell organelles.
1.3 The Sporangiophore of Phycomyces blakesleeanus
Phycomyces have two life cycles, the vegetative or asexual cycle and the
sexual life cycle (Cerda-Olmedo and Lipson, 1987). In this thesis, the vegetative
cycle is used because it allows for rapid reproduction and growth of many
sporangiophores. The vegetative cycle relies upon vegetative spores to
reproduce. The spores can be kept in dormancy in a water medium in a
refrigerator or kept in a dry stock. In order to grow sporangiophores the spores
are heat shocked, by placing the spores in a heated water bath. The spores can
then be inoculated on a sterile growth medium where they grow and expand.
A mycelium (collection of germ tubes) forms within five hours after
inoculation (Ortega, 1976). The individual hyphae of the mycelium only grow at
the tips and produce branches that form lateral tubes creating a webbed structure.
The hyphal tips grow at a rate of 0.3 (im/s and grow in thickness by several
5


millimeters. The mycelium will produce a thick hyphal pad. The whole
mycelium is considered a single collective cell. The hyphal growth changes to
produce the sporangiophores. The sporangiophores are essentially aerial hyphae,
but are larger and arent branched and webbed like the hyphae. The
sporangiophore grows straight up and varies in length, but can reach up to more
than 100 mm.
The sporangiophore goes through five stages of development. A
sporangium is formed during the second and third stage, which houses the spores
for the vegetative reproduction cycle (Cerda-Olmedo and Lipson, 1987). In
these experiments, the sporangiophore is the large type or a macrophore. The
sporangium is about 500 pm in diameter and can contain up to 10A5 spores. In
previous research the macrophores are more commonly used because they are
bigger and more durable for laboratory conditions (Cerda-Olmedo and Lipson,
1987).
1.4 Developmental Stages of the Sporangiophores
There are five main developmental stages to the macrophore
sporangiophores (See Figure 1.1) (Cerda-Olmedo and Lipson, 1987). The
sporangiophore grows by means of elongation and rotation. The rotation of the
sporangiophore is about the longitudinal axis of the stalk of the sporangiophore,
6


Figure 1.1
This figure demonstrates the five developmental stages of the
sporangiophores (Ortega, 1971).
7


8


when viewed from above. The elongation and rotation growth occurs in the
growing zone of the sporangiophore, which is in different locations during the
stages of development.
In stage I, the tip of the sporangiophore rotates in a clockwise (CW)
direction and elongates at a rate of 0.3-0.6 |im/s. All the growth occurs in a short
region, the growing zone, which is approximately 1 mm in length and located at
the apical tip. In this stage the sporangiophore can reach a total length of
approximately 15 mm in about 10 hours.
In stage 13 there is no rotation or elongation of the sporangiophore
(Cerda-Olmedo and Lipson, 1987). This stage is primarily concerned with the
formation of the sporangium. To start the process, the growing zone of the
sporangiophore swells to a bulb and forms a spherical sporangium. It takes
approximately three hours for the sporangium to develop to a diameter of about
0.5 mm, which signifies the end of the stage.
Stage HI is thought to be a state of rest, because there is no rotation or
elongation of the sporangiophore (Cerda-Olmedo and Lipson, 1987). However,
this stage seems to be linked with the formation of spores. The anatomy of the
sporangium also changes in this stage with the formation of the spores. A wall
9


called the columella is developed. It divides the sporangium, which separates the
sporangiophore from the spores. This stage is a few hours long.
Stage IV is divided into several stages. However, the main characteristic
of this stage is the formation of a new growing zone (Cerda-Olmedo and Lipson,
1987). The growing zone is a cylindrical area that starts 500 |im under the
sporangium. Here the cell wall is thin to allow growth, intercalary growth. The
growing zone has been defined with the use of several markers along the stalk of
the sporangiophore in different locations (Ortega, 1976). If a marker has less
than 20 |im displacement over a 10-20 min interval, then this marker is
considered out of the growing zone. A marker that is in the growing zone will be
displaced depending on the stage of the sporangiophore.
The substage IVa has a counterclockwise (CCW) rotation of the
sporangium and the growing zone (Cerda-Olmedo and Lipson, 1987). This
substage lasts for approximately an hour and also includes the darkening of the
sporangium as the growth accelerates. The rotation changes from substage IVas
CCW rotation to CW in substage IVb. Substage IVb is the most common
experimental stage, because the elongation and rotation growth rates are
relatively constant, at 1 (im/s and 0.27s. Lastly, this substage has a black
sporangium that contains mature spores. The last substage IVc has another
10


rotation change from CW to CCW. This stage is also characteristic of long
sporangiophore of over 100 mm long.
Finally the last stage, stage V, is of old sporangiophores that dont rotate
or elongate (Cerda-Olmedo and Lipson, 1987). In this stage the sporangium can
easily be burst by contact. The sticky spores can then be spread upon a sterile
agar plate for another generation of sporangiophores.
1.5 Growth Mechanisms in Sporangiophores
In the process of extension and growth the cell wall is continually being
replenished with new material from the cytoplasm. This is needed for the cell to
be in homeostasis (Wold, 1985). In a stage IVb sporangiophore the cell wall
extension occurs in the growing zone (Ortega et al, 1974). There are three
subsections of the stalk of a stage IVb sporangiophore. Subzone I extends from
the sporangium to about 100 jam below. In this subzone there is no extension or
rotation. Although, this subzone is shown to have the thickest call wall, it is also
known to be the softest region of the growing zone (Gamow and Bottger, 1980).
Subzone II is where the majority of growth occurs in both elongation and
rotation. The rotation growth in this subzone is entirely CW, and this zone
extends to approximately 2000 p.m below the sporangium (Cohen and Delbruck,
1958; Ortega, et al, 1974; Ortega, 1976). Subzone III is out of the growing zone
11


but compromises the rest of the stalk of the sporangiophore. In this subzone
there is no elongation or rotation growth. It has also been shown that this
subzone is extremely rigid with very little mechanical ability for extension.
There are two concurring models of cell wall growth in Phycomyces
called the fibril reorientation model and the fibril slippage model (Ortega and
Gamow, 1974; Ortega, 1976). Both of the models begin with the microfibrils of
the cell wall oriented in an oblique CCW direction. These microfibrils passively
reorient themselves parallel to the longitudinal axis of the sporangiophore
because of the cell wall extension in the longitudinal direction. The reorientation
of the microfibrils will cause CW rotation of the cell wall during the elongation
growth.
It is postulated that the fibril slippage model only occurs during the short
period of stage IVa (Ortega and Gamow, 1974; Ortega, 1976). Fibril slippage is
postulated to occur when the non-growing rigid cell wall of stage III is in
transition to a growing cell wall of stage IVa, where the bonds between the
microfibrils are weakened and broke by enzymatic action. This allows the
microfibrils to slide over one another. The fibril slippage will cause cell wall
extension as well as produce CCW rotation. There have been several
12


experiments done to support this model that involve strain hardening of the cell
wall and determining its direction of rotation (Gamow and Bottger, 1980).
The studies involving the mechanical behavior of the sporangiophore cell
wall in the growing zone area include the loading of a sporangiophore at a
loading rate of more than five times its regular growth rate (Ahlquist and
Gamow, 1973). This has been done to show that the growing zone of stage IVb
sporangiophores can deform non-elastically, or undergo plastic extension. The
follow on experiment includes strain hardening the cell followed by a saturating
light stimulus (Ortega, Ahlquist and Gamow, 1975). This light stimulus causes
the sporangiophore to release cell wall-loosening enzymes, which will allow the
microfibrils to undergo fibril slippage for more plastic extension of the cell wall
during the period of the light growth response.
In fast growing stage IVb sporangiophores it is postulated that both the
fibril reorientation and the fibril slippage are occurring together (Ortega, 1976).
There is some evidence that the two models are occurring together throughout
the growing cell wall.
Through the two methods of extension, the cell wall thins unless there is
a continual replenishing of the microfibrils and other cell wall materials. These
microfibrils in the sporangiophore together with an amorphous matrix form
13


stress bearing walls to provide structure and support for the cell (Wold, 1985). If
there is not a continual replenishment of the microfibrils and other cell wall
materials, the cell wall becomes thinner and thinner until finally the cell wall can
no longer support the cell and it loses its ability to maintain homeostasis (Cerda-
Olmedo and Lipson, 1987).
1.6 Turgor Pressure in Sporangiophores
In the sporangiophore, a rigid cell wall surrounds the cytoplasm and
provides structure to the sporangiophore (Wold, 1985). This structure is similar
to a thin wall pressure vessel. The osmotic pressure of the cytoplasm provides
the driving force for water uptake into the cell. The uptake of water into the cell
increase the internal pressure of the sporangiophore; the turgor pressure.
There are several things that occur in order for plant or fungal cell
growth. They include biophysical processes and physical processes. The
biophysical processes include biochemical, and metabolic processes. The
physical processes are expressed through biophysical and biomechanical terms.
Governing equations were derived to describe the physical processes in terms of
biophysical and biomechanical parameters (Ortega, 1990, 1994).
Fungal cell enlargement is dependent upon two simultaneous and
interdependent physical processes, the net rate of water uptake and the rate of
14


cell wall extension (Ortega, 1990, 1993, 1994). The turgor pressure is an
important biophysical parameter that is implicit in both water uptake and cell
wall extension. The pressure probe has been used to measure the turgor pressure
of the cell (Cosgrove et al, 1987). Pressure probe methods have been developed
to determine important biomechanical and biophysical parameters (Ortega et al,
1992). In order to determine the magnitude of these parameters it is necessary to
have a known relationship between the turgor pressure and these biophysical and
biomechanical parameters of the cell. These relationships are described by
equations termed the Augmented Growth Equations (Ortega, 1990, 1994).
The first Augmented Growth Equation derived by Ortega (1985)
accounts for both irreversible and reversible extension of the cell wall. This
equation equates the sum of the relative rate of irreversible (plastic) extension
and relative rate of reversible (elastic) extension to the relative rate of change in
volume of the cell wall, vc (Ortega, 1985, 1990).
vc = (dVc/dt)/Vc= Where the parameters include Vr the volume of the cell wall chamber, t time, (j)
the relative irreversible wall extensibility, P the turgor pressure, Y the yield
15


threshold and £ the volumetric elastic modulus. The term the relative rate of the plastic extension. The last term (dP/dt)/ e describes
relative rate of the elastic extension. Equation 1.1 can be interpreted as a
mechanical system of a spring and dashpot in series (See Figure 1.2).
This schematic shows that a sporangiophore is analogous to a mechanical
system (Ortega, 1994). This system relates the extension behavior of the cell
wall (plastic and elastic extension) to a dashpot and spring.
This figure also demonstrates the second Augmented Growth Equation
using the conservation of water. The figure shows that the net rate of water
uptake available for growth is the difference between the rate of water uptake
and the rate of transpiration of the cell (Ortega, 1990, 1994). The mathematical
expression of the conservation of water mass leads to the second Augmented
Growth Equation (Ortega et al, 1988, Ortega, 1990).
v, = (dVw /dt)/Vw = L(oAn P)-T (1.2)
The parameters include vM. the relative rate of change in volume of the cell
contents, VM, the volume of the cell contents, L the relative hydraulic
conductance of the cell membrane, o the solute reflection coefficient of the cell
16


Figure 1.2
This figure demonstrates that a sporangiophore is analogous to a
mechanical system (Ortega, 1994). This system relates the extension
behavior of the cell wall (plastic and elastic extension) to a dashpot and
spring. This figure also demonstrates the second Augmented Growth
Equation using the conservation of water. The figure shows that the net
rate of water uptake available for growth is the difference between the
rate of water uptake and the rate of transpiration of the cell (Ortega et al,
1988; Ortega, 1990)
17


Water Uptake
18


membrane, An the difference in osmotic pressure between the cell sap, and T
the relative transpiration rate. The term hipAn P) describes the relative rate
of water uptake and the last term T represents the relative transpiration rate of
water out of the cell (Ortega, 1990, 1994).
The last Augmented Growth Equation derived by Ortega (1994)
describes the turgor pressure behavior and is the combination of the first and
second augmented equations. Because the volume of the cell wall chamber and
the volume of the cell contents are equal, the third Augmented Growth Equation
is formed with the elimination of the volume parameter to become:
dP/dt + e Many of the parameters in the Augmented Growth Equations are shown
to be dependent upon the turgor pressure P. For instance £ L and An all vary
with the turgor pressure. Therefore three more equations are included for the
parameters that are turgor pressure dependent.
£(P)=£- [(e. -£0)exP(-kp)] (1.4)
lp (p) = (Ko ~ lp~ )exp(- aP)+ lp- (1.5)
19


An (P)= n, (P)-ne = (n0-ne)-n0[P/£ (/>)]
(1.6)
These equations represent the foundation for a set of governing equations for
plant and fungal cell growth (Ortega, 1994).
Most of the pressure probe methods developed are either a pressure
relaxation method, where the pressure is allowed to decay, or a pressure clamp
method, where the pressure is fixed at a constant value (Ortega et al, 1991,
1992). These two methods are used to obtain values for all the biophysical and
biomechanical parameters, and to obtain different insights into the mechanical
behavior of the cell (Ortega, 1994).
A pressure probe method of particular interest is the transpiration-
pressure relaxation method (Ortega et al, 1988). Pressure relaxation occurs when
the water source is removed (Ortega, 1993). In this experiment, a
sporangiophore is removed from its mycelial base (plucked) and placed in a
water source. This allows the water source to be removed during the protocol of
the experiment. Then using a pressure probe with a microcapillary tip, the
sporangiophore is impaled to measure the turgor pressure. Once the
sporangiophore turgor pressure stabilizes the water is removed from the
sporangiophore. Because there is no water uptake, and transpiration continues,
20


the turgor pressure decays. The transpiration rate of the cell can be calculated
from the turgor pressure decay rate, using the third augmented growth equation.
Note that the term (P Y)= 0, because the cell is not growing (stage HI) and
the term L(oAn -P)=0, because there is no water source for the cell. This
leaves equation 1.7, which expresses that the relative transpiration rate is equal to
the turgor pressure decay rate divided by the volumetric elastic modulus.
T = ~{dp/dt)/£ (1.7)
This allows for the calculation of the transpiration rate, if the volumetric elastic
modulus is known and the turgor pressure decay rate is measured.
The pressure probe is connected to a strip chart recorder and can be used
to measure the turgor pressure decay (Ortega, 1993) (See Figure 1.3). From this
plot it can be seen that the turgor pressure decays with a function of time after
the water source is removed (indicated in the plot by the arrow). It can also be
seen from this plot that the early section of the trace is linear, followed by an
exponential curve. This linear section is indicative of high turgor pressures,
greater than 0.2 MPa. This graph also shows that the exponential curve is where
the turgor pressures are less than 0.2 MPa.
21


Figure 1.3
This figure demonstrates a pressure trace of a pressure relaxation
experiment, where the pressure decays in a linear and exponential curve
(Ortega, 1993). The large arrow represent where the water source is
removed.
22


P(MPa)


The volumetric elastic modulus £ however can be approximated by
knowing that the elastic modulus is a non-linear function of the turgor pressure
(Ortega, 1993). Therefore the volumetric elastic modulus can be approximated
by:
£(?)=£ -£0)exP(-^)]- (!-8)
Where £mis the maximum elastic modulus, £0 the elastic modulus at zero turgor
pressure, and k an experimental value (Ortega, 1993). By using the relationship
of the volumetric elastic modulus as a function of turgor pressure, indicated by
the following graph, gives a method to approximate the volumetric elastic
modulus for a range of turgor pressures (See Figure 1.4). It can be seen that at
low pressures a linear relationship exists, which can be mathematically
represented by the equation, £ = £0 + mP where m is the slope of the line.
Finally at high pressures the relationship becomes constant, therefore at high
pressures £=£,. Using these ranges of turgor pressure, equation 1.7 can be
used to obtain appropriate values for T.
24


Figure 1.4
This graph represents the relationship between the volumetric elastic
modulus and turgor pressure (Ortega, 1993).
25


t
(MPa)
PtMPa)
26


The turgor pressure of the sporangiophore is one of the necessary forces
that is utilized for the reorientation of the microfibrils to the longitudinal axis and
to drive cell wall extension (Ortega and Gamow, 1974). This is why the turgor
pressure of the sporangiophore is an important parameter that must be measured
and monitored.
1.7 Preview of Thesis Content
This thesis is the study of the growth and turgor pressure behavior during
asphyxiation of the sporangiophore Phycomyces blakesleeanus. Chapter three
reports the results of experiments that asphyxiate a sporangiophore. The first
group of experiments studies the two growth components of helical growth in the
asphyxiation process. These experiments are divided into three categories. The
first category studies elongation growth measurements during the asphyxiation
process. The second category studies elongation and rotation growth
measurements during the asphyxiation process. The third category studies the
helical growth components with markers to measure elongation growth along the
length of the sporangiophore. Finally the second group of experiments studies
the turgor pressure along with elongation growth during the asphyxiation
process.
27


Chapter four reports experiments of sporangiophores that demonstrated a
jump in elongation growth during the asphyxiation process. This chapter
includes the two components of helical growth as well as turgor pressure
measurements, which demonstrate a jump in elongation growth.
Chapter five is a discussion that attempts to provide insight to the
biophysical and biomechanical changes in the sporangiophore as it is
asphyxiated. This includes the analysis of the contraction and collapse of the
sporangiophore as it is asphyxiated.
28


2. Materials and Methods
2.1 Plant Material
The fungal spores of a wild type strain of Phycomyces blakesleeanus
NRRL1555(-) are used to inoculate a sterile growth medium. The sterile growth
medium consists of 4% (wt/vol) potato dextrose agar, 0.1% (vol/vol) Wesson oil,
and 0.006% (wt/vol) B-l Vitamin (Ortega et al, 1988). Glass shell vials hold the
growth medium and spores. After using an autoclave to sterilize the growth
medium and once the agar is inoculated with spores, the vials are stored in an
incubator. Here they receive continuous light, and are kept at high humidity and
constant temperature (201C). High humidity is achieved by storing the glass
vials upright in a bath of sterile water. Typically after three days, the
sporangiophores grow and are pluck daily for a fresh crop of sporangiophores for
the next day and experiment.
2.2 Elongation Growth Measurements
In order to monitor the change of elongation growth in a sporangiophore,
a growth-scope is used. The growth-scope consists of a long focal length
horizontal microscope (Gaertner Scientific, Chicago, IL; 701 IK eyepiece and 32
m/m EFL objective) mounted to a three dimensional micromanipulator (model
29


H-2; Line Tool Company, Allentown, PA, with large barrel micrometer heads or
digital micrometer heads, 2 (im error).
The elongation growth is monitored by using the growth-scope with an
ocular cross hair marked at the junction of the sporangium and the cylindrical
stalk of the sporangiophore (also called the neck marker). The stage of
sporangiophores used in these experiments is stage IVb, which is a stage of
sporangiophore that primarily just elongates rather than grows radially.
Therefore the growth-scope accurately measures the change in elongation growth
of the sporangiophore. By taking readings at regular time intervals of one
minute, the change in length per minute can be calculated. The minute time
intervals are measured using a stopwatch.
In order to see the sporangiophore clearly in the growth-scope for
measurements, lights are needed to illuminate the sporangiophore. Bilateral
lights from fiber-optic illuminator (Flexilux 90; HLU Light Source 90AV from
Schoelly Fiberoptic, Denzlingen, FRG) are used to reduce the heat of the lamp
on the sporangiophore and illuminate the sporangiophore with two swan-neck
\
light guides (Schoelly Fiberoptic).
In order for the sporangiophore to adapt to the light of the experimental
set up, a 30 minute adaptation time is given before starting the readings of the
30


elongation growth rate. After the adaptation period, 10 minutes of readings are
taken and are used to show that the sporangiophore is a normal healthy growing
sporangiophore. These 10 minutes of elongation growth readings are also to
show that the elongation growth rate of the sporangiophore is nearly constant.
Following the 10 minutes of elongation growth measurements the nitrogen gas
(N2) is turned on and the asphyxiation process is started. There are two methods
that are used in placing the chamber over the sporangiophore before the start of
the asphyxiation process (See Figure 2.1).
The first method places the chamber over the sporangiophore after the
adaptation time but before the 10 minutes of readings. However, placing the
chamber over the sporangiophore takes about 10 minutes to seal and set up. This
allows the sporangiophore to adapt to the chamber. The chamber however, is
large enough that it doesnt show a house response, a momentary increase in
elongation and rotation. Once the chamber is sealed and then after 10 minutes of
elongation growth readings, the room air is replaced with N2.
31


Figure 2.1
This is the experimental set up for the elongation and rotation growth
readings. This set up shows the acrylic square box with growth scope for
growth readings and the tubing for the N2.
32


33


A second method is also used to assure that the chamber is not affecting
the results. Several experiments are done that have the chamber on prior to the
30 min adaptation time. This is done to show that either method produces the
same results. The rest of the method is identical, followed by 10 minutes of
readings and then the N2 is turned on.
An oxygen sensor is used to monitor the change in % O2 in the chamber.
The oxygen sensor placed near the sporangiophore reads about 0.24%- 0% O2.
The sensor monitors this amount of O2 in the chamber while the N2 is being feed
into the chamber. This percentage continues approximately 20 minutes after the
N2 runs out. The elongation growth readings are continually monitored for the
duration of the experiment or until the asphyxiation process causes the
sporangiophore to collapse.
2.3 Growth Nitrogen Chamber
In order to asphyxiate a sporangiophore, a nitrogen chamber is
constructed to enclose the cell. The first chamber is constructed of four flat
pieces of clear acrylic with a smaller fifth piece acting as the top. In order to
create a sufficient seal, a large piece of gasket material somewhat larger than the
top of the chamber and about 5mm thick, is used to seal the bottom. An outline
of the four ends of the flat pieces of the chamber is carved into the gasket
34


material, such that the chamber fits snuggly into the impression. To create an
even better seal, once the sporangiophore is placed in the center of the chamber,
petroleum jelly is placed around the gasket and acrylic walls to keep the N2 from
escaping.
In order to keep the N2 from escaping and because N2 is atomically
lighter than air, an entrance hole for the N2 is placed at the side top of the
chamber. Here a tube from a cylinder of N2 is placed. There is also a hole at the
very bottom of the chamber acting as a feedback loop for the N2. Once the
chamber is filled with N2 it can exit through the bottom hole, where it enters a
tube that feeds back into the top hole of the chamber.
Because the chamber has to fit around the sporangiophore and not
interfere with the growth-scope, the chamber has to be small enough to fit within
the parameters of the growth-scope. Later the nitrogen chamber is re-designed to
fit the parameters of the pressure probe. The chamber has to provide a
transparent view through the sides of the walls for the growth-scope(s), as well
as a transparent view through the top for the rotation growth measurements. The
clear acrylic provides the optics needed for both the elongation growth readings
as well as the rotation growth readings.
35


2.4 Rotation Growth Measurements
The rotation growth measurements are made by a vertically mounted
EMZ-2TR trinocular zoom stereomicroscope (Meiji Labax Optical Limited,
Tokyo, Japan) with a micromanipulator (model H-2; Line Tool Company,
Allentown, PA, with large barrel micrometer heads or digital micrometer heads)
which manipulates the stereomicroscope above the sporangiophore. The
measurement of the rotation of the sporangium starts by placing a small fiber or
hair upon the sporangium, without damaging the sporangium. If the hair is place
in the center of the sporangium the hair will rotate at the same rate as the
sporangium. Using the stereomicroscope with an ocular protractor micrometer
disc can monitor the rotation.
The rotation of the sporangium is monitored just as the elongation growth
readings. The readings are taken every minute. Again a stopwatch is used to
regulate the readings.
2.5 Pressure Probe
The basic building materials of a pressure probe include a 1.5 inch thick
sheet of acrylic, round stock acrylic (1.25 inch diameter), a small diameter (1.3
mm) piece of piano wire, a micrometer and a pressure transducer. The sheet of
acrylic will hold the heart of the pressure probe, by connecting the pressure
36


transducer, piano wire and oil chamber. Both a CNC drill press and lathe are
used for all of the machining of the pressure probe.
Most of the machining is on the heart of the pressure probe, the block of
acrylic. The block of acrylic that makes up the pressure probe is approximately
2.5 inches by 3 inches. The oil chamber is drilled such that it runs the length of
the 3 inches of the acrylic block (See Figure 2.2). The port or hole for the
pressure transducer is then drilled perpendicular to the oil chamber. This hole is
then threaded to fit the pressure transducer.
After the transducer hole is threaded, a boss (a threaded protrusion) is
machined out of the acrylic block at one end of the oil chamber. The boss is
threaded and covered with a threaded cap made from the round stock acrylic.
The cap is machined out of 1.25 inch diameter round stock acrylic using a lathe.
A small diameter hole is drilled through the cap such that the oil chamber
extends through the acrylic block, boss and through the cap tip. A pulled
microcapillary tip is then placed in the cap and partially through the boss. It is
the pulled microcapillary tip that will actually have physical contact with the
sporangiophore. In order to hold pressure and prevent leaks from the boss and
the cap, a rubber gasket is used in between the boss and the cap. This gasket has
a small hole to accommodate the pulled microcapillary tip. When the cap is
37


Figure 2.2
This figure demonstrates the schematic of the pressure probe (Ortega,
1993). This schematic shows the relationship between the components
that make up the pressure probe and how it is used to impale a
sporangiophore.
38


Cell Wall
Pressure Transducer
39


tighten down it will cause the gasket to spread and seal the oil chamber and the
pulled microcapillary tip, such that it will hold pressure.
In order to control the pressure in the oil chamber a control rod is
constructed. At the opposite end of the boss, a control rod is used to manipulate
the oil inside the oil chamber, which can either retract cell sap or push cell sap
into the sporangiophore. In other words the control rod allows the manipulation
or control of the turgor pressure inside the sporangiophore. The control rod is
manufactured from piano wire that is attached to a micrometer through a coupler.
In order to seal the end of the acrylic block a large threaded bolt is used, which is
drilled with a hole down the center to accommodate the piano wire. A rubber
gasket is used at the junction of the oil chamber and the bolt to seal the oil
chamber and hold pressure. The pressure probe must hold pressure and not leak.
If it does leak then there is no way to accurately measure the turgor pressure of a
sporangiophore.
The pressure probe is then mounted onto a three dimensional
micropositioner, for fine movements of the microcapillary tip. This allows easy
control of the pressure probe to impale a sporangiophore.
40


2.6 Turgor Pressure Measurements
In order to continually monitor and measure the turgor pressure of a
sporangiophore a pressure probe is used. The pressure probe uses a gage type
pressure transducer, which measures the difference between the absolute
pressure and the local atmospheric pressure (Ortega et al, 1989). The continuous
readings of the pressure probe are transcribed onto a chart recorder, a Houston
omniscribe stripchart recorder (model D5217-2). This type of pressure
transducer can be obtained from Kulite Semiconductor Products Inc., Ridgefield,
NJ (model XT-190-300G).
To calibrate the pressure transducer a Heise Bourdon Tube pressure
gauge is used (Dresser Ind., Newton, CT, model CMM, 0-200 PSIG range)
(Ortega et al, 1989). The output of the pressure transducer used in the pressure
probe is linear over the range of turgor pressures used in these experiments
(4.61 mV = 0.1 MPa).
The pressure probe is then mounted to a 3-D micromanipulator such that
small micro adjustments can be made for the microcapillary tip to impale the
cell. To impale a cell, a horizontally mounted EMZ-2TR trinocular zoom
stereomicroscope (Meiji Labax Optical Limited, Tokyo, Japan) is used to view
the microcapillary tip and the cell. The microcapillary tip is made from
41


cylindrical glass tubes (A-M Systems, Inc., Everett, WA, Glass, Standard 1.0
MM x .5MM, 6 inch) that are pulled using a microcapillary puller (David KOPF
Instuments, Tujunga, California, Vertical Pipette Puller, Model 720). These
pullers can make the outer diameter of the microcapillary tip be about 10 pm
(Ortega et al, 1992). The microcapillary of the pressure probe is filled with inert
silicon oil (Dow Coming Corp., Midland, MI, fluid 200, 1 centistoke viscosity)
(Ortega et al, 1989). The small diameter microcapillary tips are necessary to
impale the sporangiophore without damaging the cell wall and cell membrane of
the sporangiophore. If too large of a tip is used the cell wall and cell membrane
will leak the internal cell sap and the sporangiophore will lose turgor pressure.
In order to accurately measure the turgor pressure of the cell; the
interface method is used. This method is characterized by keeping the interface
of the cell sap and the inert silicon oil fixed in one location in the microcapillary
tip. Once the cells equilibrium turgor pressure is found the interface is fixed in
one location. If the impale process is successful, the sporangiophore will not be
affected and will continue with a normal growth rate. However, if it is
unsuccessful there will be a decrease in normal elongation growth rate and a
likelihood of cell sap leaking out around the microcapillary tip. When successful
the mesh of the cell wall and cell membrane will accommodate the tip size of the
42


microcapillary and even seal the tip with cell sap. This method is used to
measure the continuous turgor pressure of the sporangiophore.
The chamber used to measure the turgor pressure of the sporangiophore
in an environment of N2 is complex and cant be simply placed over the
sporangiophore due to the pressure probe (See Figure 2.3). Hence the extruded
acrylic piece is placed around the sporangiophore with all its attachments before
the adaptation period. Prior to the adaptation period the microcapillary tip is
broken to the size that is desirable and removed from the set up to allow the
sporangiophore to adapt to the chamber. After the adaptation period the
microcapillary tip is slowly maneuvered into a small hole near the
sporangiophore. At this point the 10 minutes of reading can begin.
After 10 minutes of measuring a constant elongation growth rate the
microcapillary tip is used to impale the cell. At this point it is necessary to make
sure that impaling didnt affect the sporangiophore. Making sure that the
elongation growth rate is still constant and that equilibrium pressure can be
obtained can prove this. After a period of 10 minutes of holding equilibrium
turgor pressure the chamber is flooded with N2. The N2 is continually feed into
the chamber such that the oxygen sensor reads approximately 0.24%- 0% oxygen
for the remainder of the experiment. The composition of air is comprised mainly
43


Figure 2.3
This is the experimental set up for turgor pressure readings. This figure
demonstrates the acrylic box that encloses the sporangiophore and allows
access for the pressure probe. The sporangiophore is located in the center
of the acrylic box.
44


45


of N2 (78.03%) and O2 (20.99%) (Kotz and Purcell, 1991). Continuous readings
of the change in turgor pressure, % O2, and the growth rate are measured until
the cell collapses due to asphyxiation.
2.7 Pressure Probe Nitrogen Chamber
Again this special environmental chamber is used to asphyxiate the
sporangiophore. However, this second chamber is created because the first
chamber couldnt accommodate the pressure probe. This chamber is made
smaller than the first such that the pressure probe could reach the sporangiophore
to impale the cell. A professional machined this chamber being much more
complicated than the original four sided acrylic box (Ramon Hoagland, R&R
Machining). It also went through several iterations before it was ideal for the
types of experiments that were desired.
The last chamber iteration design resulted in an extruded acrylic square,
which is 2.25 inch by 2.25 inch by 6.5 inch. This piece is also fitted with several
necessary items that allow the use of a pressure probe (See Figure 2.4). First
there are two small holes on opposite sides of the extruded acrylic square. The
holes are approximately 2 mm in diameter and at the same height. One hole will
fit a microcapillary tip coming from the pressure probe and the other a 1.5 mm
diameter metal back support (See Appendix) (Drawings done by Jacob Olson).
46


Figure 2.4
This figure demonstrates how the back support and the microcapillary tip
have access to the sporangiophore in the acrylic box. This also shows
how the back support holds the sporangiophore.
47


48


The back support is essentially a long cylindrical 1.5 mm diameter metal
rod that functions as a support for the sporangiophore. It is necessary because it
offers a hold to the sporangiophore when impaling the wall of the cell. The rod
is attached to a perpendicular rod that is mounted to a 3-D micromanipulator
(model H-2, Line Tool Company, Allentown, PA, with large barrel micrometer
heads). The back support holds the sporangiophore in view of the
stereomicroscope and growth-scope.
Because the extruded acrylic square is not very transparent when viewing
through it with a growth-scope, windows had to be cut into the square for
visibility. The two windows are on opposite sides of the square piece and are
therefore on the opposite sides of the two holes. The windows are made from
sheets of acrylic, which are very transparent. One of the windows allows the
growth-scope to monitor the elongation growth rate of the sporangiophore. The
other window is for the horizontally mounted EMZ-2TR trinocular zoom
stereomicroscope that is used to see for impaling the cell. The windows provide
an excellent view of the sporangiophore as it is asphyxiated.
In order to asphyxiate the sporangiophore the ends of the extruded acrylic
square are fitted into acrylic blocks with an airtight seal. The top block is fitted
with an access hole that is threaded for a valve to fit into. The top valve will fit
49


the tubing from the cylinder of N2. The bottom hole is for the over pressure
valve. The bottom valve is situated near the stand that holds the sporangiophore.
Because the two holes for the back support and the pressure probe limit
the location of the sporangiophore, special stands are designed to hold the
sporangiophores. So several round acrylic rods are built at varying lengths to
accommodate the change in lengths of the sporangiophores.
The next iteration to this chamber after the stands is the addition of an
oxygen sensor. The oxygen sensor is from maxtec Inc., called the MAX-250
Galvanic oxygen sensor (2425 South 900 West, Suite B, Salt Lake City, Utah
84119). The two main components of air are approximately 21 % 02 and 78%
N2 (Kotz and Purcell, 1991). The oxygen sensor is mainly used to monitor the
asphyxiation time and to monitor the % of 02 in the chamber. To mount the
oxygen sensor in the chamber a hole is drilled in the side above the back support
hole. Here the oxygen sensor is closest to the sporangiophore. This chamber is
the final design of the chamber used to monitor the turgor pressure of the
sporangiophore in an environment of N2.
50


3. Results
3.1 Elongation Growth Behavior During Death
The following experiments are conducted to gain insight into the
biophysical and biomechanical changes that occur during the death of a
sporangiophore. A transparent chamber filled with nitrogen gas (N2) is used to
asphyxiate the sporangiophore until it dies and or collapses. There are several
reasons why N2 is chosen to asphyxiate the sporangiophore. Nitrogen gas is a
natural component of air (78% of air is N2) and it is inert (Kotz and Purcell,
1991). It seems also that N2 will not affect the regular metabolic activities of the
sporangiophore as it has been used to asphyxiate sporangiophores before.
Gamow and Goodell (1969) used N2 to asphyxiate various locations of
the sporangiophore stalk, in order to study the changes in metabolic activities at
these locations. They found that when the sporangiophores were placed in N2
the growth rates dropped to 10% of normal growth rates within three to four
minutes and continued to drop for the next 15 minutes. When they returned the
sporangiophore to regular air, the growth rate increased to approximately normal
rates. Goodell (1971) also asphyxiated sporangiophores by placing part of a
sporangiophore in a capillary, where it consumed all the oxygen (O2) and then
51


was asphyxiated. Goodell did this to find the changes in O2 consumption of
different locations along the sporangiophore stalk.
The experiments presented in this thesis demonstrate that when the room
air in a chamber is replaced by N2, the elongation growth stops within a few
minutes. Subsequently, similar experiments are conducted to study rotation
growth rate and turgor pressure behavior during death by asphyxiation. These
experiments include measurements of the elongation growth rate. The purpose
of the initial experiments is to see how asphyxiating the sporangiophore affects
the elongation growth rate.
In constant conditions the elongation growth rate of a stage IVb
sporangiophore is nearly constant, ranging anywhere between 20 |J,m/min to 60
|im/min. All of the experiments begin by measuring the basal elongation growth
rate. Ten minutes of normal growth rate provides a control, to demonstrate that
the sporangiophore is growing at a steady rate. This shows that the
sporangiophore is in equilibrium with the environment. A 30 min adaptation
period prior to the 10 min control is used to adapt the sporangiophore to the
experimental set up.
After this 10 min period, the sporangiophore is asphyxiated, using a
special air tight, transparent chamber that surrounds the sporangiophore. The
52


chamber is filled with N2 to asphyxiate the sporangiophore. Nitrogen gas
continually flows into the chamber at a slow rate for approximately 60 min to
ensure a low O2 environment for the sporangiophore. After approximately 60
min of N2 being fed into the system, the system is sealed to hold in the N2. This
continues to asphyxiate the sporangiophore for approximately another 20 min as
the O2 % slowly increases.
Generally graphs of the elongation growth verses time (L(t)) reveals two
phases once the sporangiophore is asphyxiated (See Figure 3.1). The first phase
is a slowing phase, where the elongation growth gradually slows. The second
phase is a contraction phase, where the elongation growth is negative resulting in
the collapse of the sporangiophore. The term contraction refers to the decrease
in length.
The figures in 3.1 demonstrate the two phases of slowing and contraction
of the asphyxiation process in the L(t) curve. Initially in these experiments the
10 min control is the constant elongation growth rate, this is followed by the
arrow indicating when the N2 is turned on. Note that the oxygen level decrease
from approximately 21% to approximately 1% when the N2 is turned on. When
the N2 is turned on, this signifies the beginning of the slowing phase followed by
the contraction phase.
53


Figure 3.1.1
Graph a, is a graph of the L(t) of a stage 4b sporangiophore. This graph
includes the change in % O2 throughout the experiment. The experiment
starts with 21% O2 in the chamber, as this is the approximate percentage
of O2 in the air. As can be seen in this graph once the N2 is turned on the
O2 % drops to less than 1%. The N2 is turned on after the 10 min control.
Graph b, is the L(t). In this graph the small arrow after the 10 min
control indicates when the N2 is turned on. The elongation growth is
measured from the junction of the sporangium and the stalk of the
sporangiophore, or the neck marker.
54


a
Time (min)
b
55


Figure 3.1.2 and 3.1.3
These are graphs of the L(t) of stage 4b sporangiophores. In these
graphs the small arrow after the 10 min control indicates when the N2 is
turned on. The elongation growth is measured from the junction of the
sporangium and the stalk of the sporangiophore, or the neck marker.
56


3.1.2
3.1.3
57


First Pattern. In the first pattern of the L(t) curve, it can be seen that
there is a relatively short period of time in the slowing phase. The L(t) curve is
mostly in the contraction phase. The slowing phase is approximately 5-10 min
where as the contraction phase consumes the duration of the experiment. The
contraction phase demonstrates a negative elongation growth, which continues
until the sporangiophore collapses (See Figure 3.2). Because of the relative
duration of the experiments and to accurately describe the duration of the
slowing phase and the contraction phase, a ratio of the slowing phase to the
contraction phase is created and defined as the s/c ratio. In the first pattern the
ratio of s/c is generally less than 0.1, s/c < 0.1.
Second Pattern. The second pattern of the L(t) curve, again has the two
phases of slowing and contraction of the asphyxiation process (See Figure 3.3).
In general, the second pattern has approximately equal duration slowing phase
and contraction phase. The ratio of the s/c is generally between 1.5 and 0.1. The
ratio is 1.5 > s/c >0.1.
The figures in 3.3 demonstrate the two phases of slowing and contraction
of the asphyxiation process in the L(t) curve. Initially in these experiments the
10 min control is the constant elongation growth rate, this is followed by the
arrow indicating when the N2 is turned on. Note that the oxygen level decreases
58


Figure 3.2
This figure demonstrates the collapse of the sporangiophore. Nearing the
end of the contraction phase the sporangiophore starts to buckle and bend
over in the growing zone (approximately -1000 pm down from the
sporangium). The stalk will continue to bend over until the sporangium
hits the stalk.
59


60


Figure 3.3.1
Graph a, is a graph of the L(t) of a stage 4b sporangiophore. This graph
includes the change in % O2 throughout the experiment. The experiment
starts with 21% O2 in the chamber, as this is the approximate percentage
of O2 in the air. As can be seen in this graph once the N2 is turned on the
O2 % drops to less than 1%. The N2 is turned on after the 10 min control.
Graph b, is the L(t). In this graph the small arrow after the 10 min
control indicates when the N2 is turned on. The elongation growth is
measured from the junction of the sporangium and the stalk of the
sporangiophore, or the neck marker.
61


62


Figure 3.3.2 and 3.3.3
These are graphs of the L(t) of stage 4b sporangiophores. In these
graphs the small arrow after the 10 min control indicates when the N2 is
turned on. The elongation growth is measured from the junction of the
sporangium and the stalk of the sporangiophore, or the neck marker.
63


0 20 40 60 80 100 120
Time (min)
64


from approximately 21% to approximately 1% when the N2 is turned on. When
the N2 is turned on, this signifies the beginning of the slowing phase followed by
the contraction phase.
Third Pattern. The third pattern of the L(t) curve has the two phases of
slowing and contraction of the asphyxiation process. However, in these
experiments the slowing phase is the longest duration (See Figure 3.4). This is
again followed by a contraction phase that demonstrates a negative elongation
growth that continues until the sporangiophore collapses. However, this
contraction phase is more abrupt than the other two phases. The ratio of s/c is
generally in these experiments greater than 1.5, s/c > 1.5.
The figures of 3.4 demonstrate the two phases of slowing and contraction
of the asphyxiation process in the L(t) curve. Initially in these experiments the
10 min control is the constant elongation growth rate, this is followed by the
arrow indicating when the N2 is turned on. Note that the oxygen level decreases
from approximately 21% to approximately 1% when the N2 is turned on. When
the N2 is turned on, this signifies the beginning of the slowing phase followed by
the contraction phase.
65


Figure 3.4.1
Graph a, is a graph of the L(t) of a stage 4b sporangiophore. This graph
includes the change in % O2 throughout the experiment. The experiment
starts with 21% O2 in the chamber, as this is the approximate percentage
of O2 in the air. As can be seen in this graph once the N2 is turned on the
O2 % drops to less than 1%. The N2 is turned on after the 10 min control.
Graph b, is the L(t). In this graph the small arrow after the 10 min
control indicates when the N2 is turned on. The elongation growth is
measured from the junction of the sporangium and the stalk of the
sporangiophore, or the neck marker.
66


a
67


Figure 3.4.2 and 3.4.3
These are graphs of the L(t) of stage 4b sporangiophores. In these
graphs the small arrow after the 10 min control indicates when the N2 is
turned on. The elongation growth is measured from the junction of the
sporangium and the stalk of the sporangiophore, or the neck marker.
68


3.4.2
69


The elongation growth experiments demonstrate three patterns in the L(t)
curve. These three patterns contain the two phases of the asphyxiation process,
the slowing phase and the contraction phase. However, the three patterns show a
different duration in the two phases. The first pattern has a short slowing phase
with a long contraction phase, where the ratio of s/c is less than 0.1, s/c < 0.1.
The second pattern has an almost equal duration between the two phases, 1.5 >
s/c >0.1. The third pattern has a long slowing phase with a short contraction
phase, where s/c >1.5. These patterns dont seem to be dependent upon the
change in availability of oxygen in the chamber, due to the fact that the C>2% is
always less than 1% once the N2 is turned on.
3.2 Elongation and Rotation Growth Behavior
During Death
As the sporangiophore elongates, the sporangium rotates (due to the
rotation of the growing zone) in both clockwise (CW) and counter-clockwise
(CCW) directions, depending on the stage of development. Experiments are
conducted on stage IVb sporangiophores, which rotate in the CW direction. It
has been shown in other studies that the rotation and elongation are not
70


completely coupled; they differ in magnitudes along the sporangiophore (Cohen
and Delbruck, 1958; Ortega et. al., 1974). The following experiments were
designed to study elongation and rotation growth when the sporangiophore is
being asphyxiated. The following graphs demonstrate the elongation growth
verses time, L(t), and rotation growth verse time, R(t) (See Figure 3.5).
In constant conditions the rotation growth rate of a stage IVb
sporangiophore is nearly constant, ranging anywhere between 10 7min to 15
7min. Ten minutes of normal rotation growth rate provides a control, to
demonstrate that the sporangiophore is growing at a steady rate.
The figures of 3.5 demonstrate the two phases of slowing and contraction
of the asphyxiation process in the L(t) curve. Initially in these experiments the
10 min control is the constant elongation and rotation growth rates, this is
followed by the arrow indicating when the N2 is turned on. Note that the oxygen
level decrease from approximately 21% to approximately 1% when the N2 is
turned on. When the N2 is turned on, this signifies the beginning of the slowing
phase followed by the contraction phase.
71


Figure 3.5.1
Graph a, is a graph of the L(t) of a stage 4b sporangiophore. This graph
includes the change in % O2 throughout the experiment. The experiment
starts with 21% O2 in the chamber, as this is the approximate percentage
of O2 in the air. As can be seen in this graph once the N2 is turned on the
O2 % drops to less than 1%. The N2 is turned on after the 10 min control.
Graph b, is the L(t) and the R(t). In this graph the small arrow after the
10 min control indicates when the N2 is turned on. The elongation
growth is measured from the junction of the sporangium and the stalk of
the sporangiophore, or the neck marker. The rotation growth is measured
using a hair on the sporangium and an ocular protractor.
72


b
Time (min)
73


Figure 3.5.2 and 3.5.3
These are graphs of the L(t) and R(t) of stage 4b sporangiophores. In
these graphs the small arrow after the 10 min control indicates when the
N2 is turned on. The elongation growth is measured from the junction of
the sporangium and the stalk of the sporangiophore, or the neck marker.
The rotation growth is measured using a hair on the sporangium and an
ocular protractor.
74


3.5.2
3.5.3
Time (min)
75


The elongation growth and the rotation growth experiments have similar
patterns to the L(t) curves that are shown in the elongation growth experiments,
shown previously. These patterns consist of different lengths in duration in the
slowing phase and the contraction phase.
First Pattern. The figure 3.5 demonstrates the short duration slowing
phase with a long duration contraction phase. The slowing to contraction ratio
s/c is less than 0.1, s/c < 0.1.
Second Pattern. The second pattern in the L(t) curve has the longer
slowing phase, which is followed by a contraction phase (See Figure 3.6). Again
in these experiments it seems that the slowing phase and the contraction phase
are similar in duration. However, the ratio of s/c is 1.5 > s/c >0.1.
Third Pattern. The third pattern is the long duration slowing phase
followed by an abrupt contraction phase (See Figure 3.7). The ratio of s/c is
greater than 1.5, s/c >1.5.
76


Figure 3.6.1
Graph a, is a graph of the L(t) of a stage 4b sporangiophore. This graph
includes the change in % O2 throughout the experiment. The experiment
starts with 21% O2 in the chamber, as this is the approximate percentage
of O2 in the air. As can be seen in this graph once the N2 is turned on the
O2 % drops to less than 1%. The N2 is turned on after the 10 min control.
Graph b, is the L(t) and the R(t). In this graph the small arrow after the
10 min control indicates when the N2 is turned on. The elongation
growth is measured from the junction of the sporangium and the stalk of
the sporangiophore, or the neck marker. The rotation growth is measured
using a hair on the sporangium and an ocular protractor.
77


b
180
160
140
120 f
O)
100
80 I
ra
60 o
EC
40
20
0
78


Figure 3.6.2 and 3.6.3
These are graphs of the L(t) and R(t) of stage 4b sporangiophores. In
these graphs the small arrow after the 10 min control indicates when the
N2 is turned on. The elongation growth is measured from the junction of
the sporangium and the stalk of the sporangiophore, or the neck marker.
The rotation growth is measured using a hair on the sporangium and an
ocular protractor.
79


3.6.2
250
200
150
100
50
0
u
0)
k_
o>
o
c
o
o
cc
Time (min)
Time (min)
80


Figure 3.6.4 and 3.6.5
These are graphs of the L(t) and R(t) of stage 4b sporangiophores. In
these graphs the small arrow after the 10 min control indicates when the
N2 is turned on. The elongation growth is measured from the junction of
the sporangium and the stalk of the sporangiophore, or the neck marker.
The rotation growth is measured using a hair on the sporangium and an
ocular protractor.
81


Figure 3.7.1
Graph a, is a graph of the L(t) of a stage 4b sporangiophore. This graph
includes the change in % O2 throughout the experiment. The experiment
starts with 21% O2 in the chamber, as this is the approximate percentage
of O2 in the air. As can be seen in this graph once the N2 is turned on the
O2 % drops to less than 1%. The N2 is turned on after the 10 min control.
Graph b, is the L(t) and the R(t). In this graph the small arrow after the
10 min control indicates when the N2 is turned on. The elongation
growth is measured from the junction of the sporangium and the stalk of
the sporangiophore, or the neck marker. The rotation growth is measured
using a hair on the sporangium and an ocular protractor.
83


b
Time (min)
84


Figure 3.7.2 and 3.7.3
These are graphs of the L(t) and R(t) of stage 4b sporangiophores. In
these graphs the small arrow after the 10 min control indicates when the
N2 is turned on. The elongation growth is measured from the junction of
the sporangium and the stalk of the sporangiophore, or the neck marker.
The rotation growth is measured using a hair on the sporangium and an
ocular protractor.
85


3.7.3
250
200
150
d>
a>
O)
0)
TJ
C
o
100 b
rj
+*
o
cc
50
86


Three different patterns of the rotation growth due to the asphyxiation
process emerge from these experiments. The three patterns demonstrate that
when the asphyxiation starts both the rotation growth as well as the elongation
growth have a slowing phase. In general, in the slowing phase, the rotation stops
once the asphyxiation process starts. However in some experiments the R(t)
curve demonstrates a change from the normal rotation rate of 10-15 7min to 1-5
7min. However, unlike the L(t) curve that has a slowing phase followed by a
contraction phase, the R(t) curve does not seem to contract. Rather it seems that
the R(t) curve remains in the slowing phase for the duration of the experiment or
until the collapse of the cell. However, there are three different patterns to the
rotation growth during the slowing phase.
Reduced Rotation Pattern. In the first pattern of the R(t) the rotation
continues to rotate CW for the duration of the experiment (See Figure 3.8). In
this pattern the rotation growth rate continues to rotate CW at approximately 1-5
7min after the N2 is turned on.
87


Figure 3.8
This is a graph of the L(t) and the R(t) of a stage 4b sporangiophore. In
this graph the small arrow after the 10 min control indicates when the N2
is turned on. The R(t) curve in this graph demonstrates the reduced
rotation pattern, where the rotation growth continues to rotate CW until
the collapse of the sporangiophore. The rotation growth rate is on
average of 1-5 /min. The elongation growth is measured from the
junction of the sporangium and the stalk of the sporangiophore, or the
neck marker. The rotation growth is measured using a hair on the
sporangium and an ocular protractor.
88


3.8
250
200
150
100 m
50
0
89


Temporary Reversal in Rotation Pattern. In the second pattern of the
R(t) curve the rotation growth rate slows from the normal 10-15/min to 1-5
/min in a CW direction, however at some point in the experiment the rotation
reverses to rotate CCW (See Figure 3.9). In a period of time the rotation
resumes the CW direction. In some experiments the direction of rotation often
goes back and forth between CW and CCW.
Reverse Rotation Pattern. In the third pattern of the R(t) curve the
rotation growth rate slows from the normal 10-15/min to 2-5/min in a CW
direction, however at some point in the experiment the rotation reverses to rotate
CCW (See Figure 3.10). Unlike the temporary reversal in rotation pattern these
experiments continue to rotate CCW for the duration of the experiment until the
collapse of the sporangiophore.
In comparing the different patterns of the R(t) experiments it seems that
the patterns of the R(t) are independent of the different patterns of the L(t)
experiments. This is demonstrated in the experiments, because in categorizing
the R(t) patterns, the L(t) patterns are varied among the reduced, temporary
reversal, and the reverse patterns of the rotation.
90