California State University, Northridge
CHANGES IN LIPID COMPOSITION DURING
MICROCONIDIAL DIFFERENTIATION IN A MUTANT
OF NEUROSPORA
A thesis submitted in partial satisfaction of the
requirements for the degree of Master of Science in
Biology
by
Leslie R. Ballou
January, 1978
._
The 1rhesis of Leslie R. Ballou is approved:
California State University, Northridge
- ii
ACKNOWLEDGEMENTS
Iwould like to express my sincere appreciation
to Dr. Donald Bianchi for his guidance and help in
the preparation of this paper.
I am also grateful to Dr. Kenneth A. Wilson and
Dr. Kenneth E. Jones for their assistance in the
preparation of this thesis.
_iii
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS
iii
•
TABLE OF CONTENTS
TABLES
iv
v
•
FIGURES
• •
ABSTRACT
vi
vii
INTRODUCTION
1
Iv"..ATERIALS AND METHODS
4
RESULTS
DISCUSSION
12
53
•
SUMMARY
69
REFERENCES
70
iv
TABLES
Table
1.
2.
3.
4.
5.
6.
Page
Relative percentage of each lipid class
with respect to the total lipids present
after 2, 3, and 4 days of growth
respectively • • • • • • • -. • • • • • •
Changes in fatty acid methyl ester
composition of the total lipid extract
with respect to time • • • • • • • • •
..
20
...
25
Changes in fatty acid methyl ester
composition of the phosopholipid fraction
of the total lipid extract with respect
to time . . . . . . . . . . . . . . . . .
Changes in fatty acid methyl ester
composition of the triglyceride fraction
of the total lipid extract with respect
to time . . . . . . . . . . . . . . . .
.
..
30
34
Changes in the fatty acid methyl ester
composition of the monoglyceride and
diglyceride fractions of the total
lipid extract with respect to time • •
40
Changes in th~ fatty acid methyl ester
composition of the free fatty acid
fraction of the total lipid extract with
respect to time
• • • • • • • • • • • •
44
v
FIGURES
Page
1.
2.
3.
4.
Change in milligram dry weight of
mycelium for both liquid and dialysis
tubing cultures grown on Vogels (M)
medium at 2soc. • • • • • • • • • • •
...
-
16
•.. .
19
Quantities of the five major lipid
classes detected in the peach, fluffy,
cot mutant of Neurospora crassa
expressed in microgram/culture.
Cultures were grown using the dialysis
tubing technique with measurements
taken after 2, 3, and 4 days of growth
Changes in fatty acid methyl ester
composition of the total lipid extract
with respect to time ~ • • • • • • • •
Changes in the fatty methyl ester acid
composition of the phospholipid fraction
of the total lipid extract with respect
to time ..
e
e
•
•
•
• .. •
•
•
•
•
•
•
•
..
32
Changes in the fatty acid methyl ester
composition of the triglyceride fraction
of the total lipid extract with respect
to time . . . . . . . . . . . . . . . .
..
36
Changes in the fatty acid methyl ester
composition of the monoglyceride and
diglyceride fractions of the total lipid
extract with respect to time
• • • • •
39
Change in fatty acid methyl ester
composition of the free fatty acid
fraction of the total lipid extract
with respect to time
• • • • • • • • •
43
Composition of the mycelial fatty acid
methyl esters compared to the composition
of the microconidial fatty acid methyl
esters in 4 day old cultures • • •
48
Percent germination of microconidia
with respect to time
•••••••
52
0
5.
6.
7.
8.
9.
27
vi
....
ABSTRACT
CHANGES IN LIPID COMPOSITION DURING
MICROCONIDIAL DIFFERENTIATION IN MUTANT
OF NEUROSPORA
by
Leslie R. Ballou
Master of Science in Biology
December, 1977
The peach-fluffy-cot mutant of Neurospora is unable
to produce macroconidia but is able to differentiate
great numbers of microconidia after a predictable
-
length of time of vegetative growth.
The relationship
of lipid metabolism·to the differentiation of microconidia was studied by determining changes in the
composition of the major lipid classes and the fatty
acid composition of these lipid classes during the
transition from vegetative growth to reproductive growth:
The evidence strongly suggests a relationship between
the synthesis of sterols during vegetative growth and
the initiation of the microcbnidial differentiation
process.
The selective localization of sterols and
sterol esters in the microconidia themselves and the
vii
rapid degradation of these microconidial lipids after
differentiation is complete is further evidence of an
inductive role associated with the sterols.
It is not
clear whether the changes in fatty acid composition
affect the morphogenesis of Neurospora, or if the changes
are simply part of a NADPH-deficiency common to many
mutants of Neurospora.
viii-
1
INTRODUCTION
The growth cycle of N. crassa has been studied
extensively (Turian and Bianchi, 1971, 1972)
1965),
~
(Weiss,
(Zalokar, 1959a and 1959b) and there are three
distinct phases in this organism's growth cycle.
The
first phase of growth is begun with the germination of
a spore which produces a germ tube or single vegetative
hypha.
The second phase is a period during which
individual hyphae elongate and branch, each branch
giving rise to further successive branches.
The result
of the second phase is the formation of a dense mycelial
mat composed of many multi-branched and intertwined
hyphae.
The third stage of growth is the reproductive
phase during which spores differentiate.
The peach-fluffy-cot mutant of Neurospora crassa
differentiates many microconidia and is therefore an
excellent organism for studying the metabolic changes
that occur during the shift from vegetative to reproductive growth.
Microconidia, even though they can
germinate like macroconidia are structurally and
functionally considered as spermatia (Backus, 1939).
Conidiation in both mutant and wild type strains
of Neurospora is essentially an aerobic process (Kobe,
2
1965,
(Turian and Bianchi, 1971 and 1972).
Under
favorable conditions complex metabolic changes such
as alterations in enzyme systems and the redistribution·
of organells in the pre-differentiating vegetative
hyphae occur within the vegetative hyphae preceeding
the differention of conidia.
Until recently little information was available
regarding the importance of lipid metabolism relative
to morphological differentiation in fungi.
Currently,
many researchers have found lipids to exert profound
effect~
1970),
on fungal metabolism (Weete, 1973),
(Rattray, 1975),
(Hendrix,
(Smith, 1969a and 1969b), bdt
few studies have been made that correlate quantitative
changes in lipid composition with morphological changes
in fungi, particularly in the differentiation of reproductive structures.
According to Bianchi and Turian
(1967) quantitative changes occur in lipid composition
at the time of differentiation of macroconidia in wild
type N. crassa.
Also, morphological mutants of Neurospora
with low levels of reduced pyridine nucleotides have
altered saturated ?nd unsaturated fatty acid composition
compared with wild type (Brody and Nyc, 1970).
In this study, the role of lipid metabolism in the
differentiation of microconidia was investigated.
Quantitative and qualitative changes in the composition
of each lipid class_was measured as was fatty acid methyl
ester composition of each lipid ·class prior to and following microconidial differentiation.
The lipid content
of the microconidia was also determined.
The information
obtained from these experiments was examined.to determine
whether or not changes in lipid composition could be
correlated with microconidial differentiation.
4
MATERIALS
;~D
METHODS
In this study a peach, fluffy, cot mutant of
Neurospora crassa was used (48743m;L;Cl02(t)).
This
mutant is capable of producing microconidia in great
numbers and is incapable of producing macroconidia or
ascospores.
The mutant is also temperature sensitive
and will grow as a colony at temperatures above 32oc.
Stock cultures were maintained either on Vogels (M)
medium slants and plates at 2soc in the dark, or by
embedding microconidia on silica gel (6-16 mesh) and
storing at soc.
Vogels (M) medium (SOX)
(Vogel et al.
19S6) of the following:
ADD:
Na3·Citrate.2H20
12Sgm
KH2P04 (Anhydrous)
2SOgm
NH4N03 (Anhydrous)
lOOgm
MgS04•?H20
lOgm
CaC1•2H20
Sgm
Distilled Water
1 liter
Biotin
2.Sml
Sucrose
2%
Trace elements
Sml
5
The trace elements solution contained the following:
Citric Acid
5gm
ZnS04
5gm
Fe(NH4(2(S04)2•6H20
lgm
CuS04
0.25gm
MnS04
0.05gm
H3B04
0.05gm
Na2Mo04•2H20
0.05gm
Distilled Water
lOOml
All experimental innoculations were made from cultures which had grown for several days at 25°C on petri
plates containing Vogels (M) medium.
After sufficient
growth had taken place a sterile blade was used to
section the agar into small blocks about lcm. square.
These blocks were then transferred to plates containing
Vogels (M) medium in which the surface of the medium was
covered with a circular sterile sheet of dialysis tubing.
This technique allowed nutrients to diffuse from the
medium to the fungus and yet did not allow the fungus to
grow directly into the agar itself.
This facilitated
easy separation of the fungus from the surface of the
medium.
Newly innoculated cultures were then incubated for
48, 72 and 96 hours respectively, at 250 in the dark.
6
The production of microconidia occurred after approximately 86 hours$
After incubation for the appropriate
length of time the cultures were harvested by_simply
scraping the mycelial mat from the sterile dialysis
tubing circles into a solution of chloroform-methanol
(2: 1 v/v) •
EXTRACTION PROCEDURE
Extraction of the total lipids was carried out by
soaking the mycelial mats in chloroform-methanol contained in a lOOml flask.
At least 17 ml of the solution
was used for each gram dry weight equivalent of mycelium.
After about 10 minutes the 2:1 solution was poured
into a clean flask.
Additional chloroform-methanol
solution was then added to the already extracted mycelial
suspension and was allowed to stand for another 10 minutes.
This solution was then poured into the flask which contained the previous extract.
After most of the liquid
had been poured off, the mycelial mats were washed from
the flask into a funnel lined with Whatman #1 filter
paper, previously washed with chloroform.
The mats were
then washed several times with chloroform-methanol to
insure a complete extraction.
After extraction was
complete the mycelial mats were placed into tared
weighing pans and dried to a constant weight.
7
The total lipid extract was then washed with 0.2
volumes of water and centrifuged for 5 minutes at 5,000
RPM at 5°C after which the bottom layer, containing the
lipid fraction, was drawn off and collected in test
tubes.
Before storage the solvent volume was reduced
to less than lOml, placed in a lOml volumetric flask
and brought up to exactly 10 ml with
chloroform~methanol.
This solution could then be stored in the freezer under
nitrogen to prevent oxidation, although in every case
experimental procedures \vere carried out as soon after
extraction as possible to minimize the affect of oxidation.
ISOLATION AND IDENTIFICATION OF LIPID EXTRACTS
Isolation and identification of the various lipid
classes was carried out by thin layer chromatography (TLC)
on silica gel G~
Plates were coated to a thickness of
250 microns {Woe.lm) •
Each plate was washed in chloroform
prior to its use and the adsorbent was activated by heating at ll0°C for at least 2 hours before use.
Lipid extract was spotted at about 1.5cm from the
bottom of the TLC plate and 30 microliter of the extract
was applied.
The right side of the plate was reserved
for the application of known standard solutions which
would be. compared to the unknovms on the left side of
the plate.
8
All neutral lipid runs were developed in a tank
saturated with a solvent system which contained pentane,
diethyl ether, and acetic acid in the proportions 80:20:1
(v/v/v) •
The solvent was allowed to rise about lScm
above the origin.
After separation was complete the
plate was removed from the TLC tank, allowed to air dry,
a.nd was then sprayed with 3% cupric acetate in orthophosphori.c acid.
The plate was then dried in an oven
for 40 minutes at 11ooc to char clearly the various
spots.
Essentially the same procedure was used in the analysis on the polar lipids except for the fact that a different solvent system was used.
In this case a mixture
of chloroform, methanol, acetic acid and water was used
(100:55 :16:8).
After development of all spots, identification of
the various spots was accomplished by comparison with
knovm compounds •
QUANTIFICATION OF LIPID EXTRACTS
Quantification was carried out by using a Photovolt
Densitometer to compare spot densities of experimental
extracts with densities of known quantities of lipid as
a function of peak area.
This procedure was used for
polar and nonpolar lipid analysis.
HYDROLYSIS AND METHYLATION OF TOTAL LIPID EXTRACT FOR
GAS CHROMATOGRAPHIC ANALYSIS OF FATTY ACIDS
The procedure followed fOr hydrolysis and methylation
-
is essentially that of Morrison and Smith (1969) and is
sumn1arized below.
1.
The lipid extract is evaporated to dryness under
nitrogen.
2.
BF3-methanol, redistilled benzene and redistilled
methanol were added in the proportions lml:lml:2ml.
This solution is then put into a screw top test tube
with a cap lined with teflon tape and heated in
boiling water for 10 minutes.
3.
'I'he solution is cooled to room temperature.
Pentane (8ml, chromatography quality) and 4ml of
de-ionized water are added and the solution was
shaken.
4.
The solution is transferred to centrifuge tubes
and centrifuged for 5 minutes in the cold.
5.
The top layer containing the methylated fatty
acids in pentane is drawn off for chromatographic
analysis.
Analysis-of the fatty acid methyl esters (FAME) was
carried out on a Beckman GC-72-5 Gas Chromatograph using
a Flame Ionization Detector and a Beckman Model 1005
recorder equipped with an integrator.
A six foot
10
·(
stainless steel column (O.D. 1/8 inch) packed with 20%
diethyl glycol succinate as the partition liquid and
Chromasorb WHP (mesh size 80/100) as the support material
was used in this analysis.
gas.
Helium was used as the carrier.
The gas flow and temperature settings are listed
below.
Column temperature
Detector temperature
Inlet temperature
21ooc
Line temperature
2300C
Air flow rate
300cc/minute
He flow rate
lOOcc/minute
He flow rates through
rotometers
20cc/minute
GAS CHROMATOGRAPHIC ANALYSIS OF FAME'S
5ml of 48, 72 and 96 hour lipid extracts were
evaporated to lml.
400 microliter of extract was then
spott.ed on_ a TLC plate and placed in a development chamber
until separation was complete.
Then, each lipid
fractio~
was scraped from the TLC plate into separate flasks and
re-extracted with chloroform-methanol three times.
separated lipid fractions were then methylated and
hydrolyzed for analysis by gas chromatography.
The
11
SEPA!~TION
OF MICROCONIDIA FROM THE MYCELIUM
Separation of microconidia from the mycelium_was
accomplished by simply shaking mycelial mats in distilled
water to shake the microconidia from the mycelial mats.
The distilled water was then poured through a layer of
glass wool which allowed·the microconidia to pass through
but held the mycelium.
This procedure was repeated
several times in insure complete separation.
After
separation the microconidia were removed from the distilled water by centrifugation.
MICROCONIDIAL VIABILITY STUDY
Microconidial viability was studied by making serial
dilutions of conidial suspensions of variously aged
microconidia _and allowing the microconidia to germinate
on a modified Vogels medium.
Modification of the medium
was achieved by replacing the 2% sucrose with 1.5% sucrose
and 0.5%
L-sorbose~
This modification allowed the micro-
conidia to· germinate but caused the growth of the mycelium
to be restricted to small colonies so that they could be
more easily counted.
RESULTS
This study consisted of several groups of interrelated experiments.
The first was to determine the
general growth pattern of the peach-fluffy-cot mutant
of Neurospora by rate of dry weight increase and the
time of initiation of microconidiation.
After obtaining information of the growth pattern,
the second step was to identify each of the lipid classes
present during vegetative growth and microconidiation.
The third step was to measure the relative composition of the fatty acid methyl
este~s
(FAME) of the total
lipid fraction with respect to time and to measure the
FAMEs of each major lipid class separately with respect
to time.
Based upon the information obtained from the measurement of the amount and type of lipids present and the
relative composition of the FAMEs of these lipids two
further groups of experiments were carried out.
These
experiments consisted of the separation of mycelia from
microconidia in 96 hour cultures and analyzing the lipid
content of each component separately as well as FAMEs of
each component.
Also analyzed were the lipids present
in 7 day old microconidia to determine changes in the
13
quantity of the various lipids that were stored in the
microconidium itself.
Finally, a microconidial viability
study was done to determine the affect of time on the
ability of the microconidium to germinate.
It is extremely important to mention at this point
that there were several possible ways in which to express
the results of the experiments done in this study.
Specifically, the purpose of this study was to measure
the changes in composition of the major lipid classes and
the changes in FAME composition with respect to time.
However, the organism increases in mass several fold
during the course of the experiments.
The quantities of
· lipid could then be expressed in terms of dry weight of
mycelia to reflect the increase in mass of the organism
-
or in absolute terms.
When for example, microgram lipid/
mg dry weight mycelium was used, this could be misleading
in terms of actual changes in lipid composition because a
decrease in microgram dry weight could cause an apparent
increase ih lipid even though no such increase may have
actually existed.
Decreasing dry weight does occur
during the later so called "staitionary" phases of growth.
For this reason all values were expressed in microgram
lipid/culture.
All FAME results were expressed in rela-
ti ve percent (peak area of a single FAME/total FMJ!..E peak
area).
14
GROWTH AND MICROCONIDIATION
Growth experiments were carried out using two
techniques.
First, cultures were grown in 125ml flasks
in liquid Vogels (M) medium and dry weights were taken
at 24 hour intervals.
be seen in Figure 1.
R~sults
of these experiments can
Liquid culture growth followed the
typical pattern for fungi.
Initially there was a lag
phase followed by a logarithmic phase and finally a
stationary phase.
Conidia are not produced in liquid
culture unless the liquid is drawn off and the mycelial
mats are allowed to dry.
Under these conditions induction
of microconidiation does not occur at regular time intervals.
Cultures grown on agar media covered with dialysis
tubing were found tq be most suitable for this study
since in every case microconidia were produced in about
86 hours.
The growth pattern for these cultures is also
shown in Figure 1 and followed the same general pattern
as the growth in liquid culture. The lag phase and
.
logarithmic phase were of about the same duration but
during the final stationary phase there was a distinct
decrease in dry weight by 9.64%.
During the logarithmic
phase dry weight increase by about 300% between 48 and
72 hours of growth.
15
Figure 1 ..
The change in milligram dry weight of mycelium
for both liquid and dialysis tubing cultures
grovm on Vogels (M) medium at 2soc.
2000
~.
0
..-!
.p
cj
..-!
1875
ru
liquid culture
..-!
~
0
1750
C)
0
H
C)
1625
I
·r-1
!Ol
1500
dialysis tubing technique
1375
1250 1125
.I
.p
.c
bD
...-!
1000
~
p
ro~
875
s
750 -
cj
H
bD
....-!
.-I
r-1
625
...-!
s
500
375
250
125
1
2
3
4
5
DA.YS
6
7
8
9
17
QUALITATIVE ANALYSIS OF TOTAL LIPIDS
Qualitative analysis of the total lipid extract
tv as accomplished by TLC and comparison with known lipid
standards.
Seven major lipid components were identified
and are listed in order of depreasing polarity.
1.
2.
3.
4.
5.
6.
7.
Phospholipids
Monoglycerides (trace)
Sterols
Diglycerides
(trace)
Free fatty acids
Triglycerides
Sterol esters
Each of these classes of compounds was found during
vegetative growth and microconidiation.
The phospholipid fraction was separated further by
TLC and two major polar lipid components were identified,
phosphatidyl-ethanolamine and phosphatidyl inositol.
QUANTITATIVE ANALYSIS OF TOTAL LIPIDS
Amounts of each lipid class were measured after 48,
72 and 96 hours respectively.
Figure 2 shows the results
of the lipid analyses and all results are expressed in
microgram lipid/culture.
Table 1 shows the relative
percentages of each lipid class relative to the total
lipids present •.
After 48 hours of growth the triglycerides were
found in the highest concentration and comprised 44.18%
18
Figure 2.
Quantities of the five major lipid classes
detected in the peach-fluffy-cot mutant of
Neurospora crassa expressed in microgram/
culture.
Cultures were grown using the
dialysis tubing technique with measurements
taken after 2, 3, and 4 days of growth.
19
0.8
0.6
T
0.5
<D
H
:::1
+=>
:::1
.--1
0.4
C)
s
ro
----·.-I
0,
-.-I
.--1
0.3
0.0
:i
0.2
Sterol Ester
Phospholipid
0.1
Free Fatty
Acid
2
3
DAYS
4
~u
2
3
Sterol
Ester
7.89%
5.86%
9.15%
Triglyceride
44.18%
37.22%
41.26%
Free
Fatty
Acid
6.57%
1.70%
2.19%
D:tys
4
-
-·
Sterol
27.30%
46.02%
40.43%
Phospholipid
14.06%
9.21%
6.96%
Table 1.
The relative percentage of each lipid class with
respect to the total lipids present after 2, 3,
and 4 days of growth respectively.
21
of the toal lipid and 0.61 microgram/culture.
Sterols
were 27.30% of the total lipid and 0.38 microgram/
culture.
Phospholipids equalled 0.19 microgram/culture
and comprised 14.06% of the total lipid.
Sterol esters
and free fatty acids were detected in small amounts and
equalled 0.11 micro/culture and 0.09 micro/culture
respectively corresponding to 7.89% and 6.57% of the
total lipid present after 48 hours of growth.
After 72 hours of growth the dry weight of the fungus
had about tripled and microconidiation was only a few
hours away.
At this point there was a dramatic increase
in the sterol fraction increasing from 0.38 microgram/
culture to 0.815 microgram/culture to become the most
concentrated of the fractions, at 46.02% of the total
lipid.
This was an increase of 116% over the 48 hour
level.
The triglyceride fraction increased slightly
from 0.61 microgram/culture after 48 hours to 0.66
microgram/culture after 72 hours, an increase of 7.7%.
The triglycerides now comprised 37.22% of the total
lipids.
The phospholipid fraction decreased from 0.19
microgram/culture to 0.16 microgram/culture during this
period, a decrease of 16.41% to become 9.21% of the 72
hour total lipid.
Sterol esters showed vert little
change, decreasing 0.11 microgram/culture to 0.10
microgram/culture.
This was a drop of 4.59% to become
22
5.86% of the total lipid.
The free fatty acids decreased
from 0.91 microgram/culture to 0.30
microgram/cul~ure,
a
67.03% decrease to 1.70% of the total 72 hour lipids.
After the 72 hour measurement and before the 96 hour
measurement the production of microconidia occurred and
the values given in the next section represent an entire
lipid extract of both mycelia and microconidia.
The 96 hour lipid extract contained about equal
amounts of trigylceride and sterol, 0.82 microgram/
culture and 0.80 microgram/culture respectively, corresponding to 41.26% and 40.43% of the 96 hour total lipid.
This represented a minor decrease in sterols (1.47%) but
a rather significant increase of the triglycerides
(24.28%) over the 72 hour quantities.
The phospholipids
continued a steady decrease from 0.16 microgram/culture
to 0.14 microgram/culture, a drop of 15.34% to comprise
6.96% of the total 96 hour lipid.
Sterol esters showed
a significant increase, rising from 0.10 microgram/
culture to· 0.18 microgram/culture, an increase of 75.0%
to become 9.15% of the total lipid.
Free fatty acids
increased from 0.30 microgram/culture to 0.044 microgram/
culture for an increase of 46.67% to become 2.19% of the
total 96 hour lipid.
23
Overall, the total. lipids increased rather steadily
from 1.384 microgram/culture after 48 hours to 1.770
microgram/culture after 72 hours, an increase of 27.89%.
After 96 hours of growth the total lipids again increased
from 1.770 microgram/culture to 1.986 microgram/culture
which was an increase of-another 12.20% over the 72 hour
level.
It is interesting to note that sterols were most ·
responsible for the increase in total lipid between 48
and 72 hours, whereas triglycerides and sterol esters
were most responsible for the increase in total lipid
between 72 and 96 hours.
QUALITATIVE ANALYSIS OF THE FAMEs OF THE TOTAL
LIPID EXTRACT
·-
The FAMEs of the total lipid fraction were analyzed
by GLC (Gas-Liquid Chromatography) and compared with
known FAMEs.
In the unknown lipid extract six peaks
were identified as follows:
16:0
Palmitic Acid
16:1
Palmitoleic Acid (trace amounts)
18:0
Stearic Acid
18:1
Oleic Acid
18:2
Linoleic Acid
18:3
Linolenic Acid
24
All of these
F&~s
were present during each measurement
but in varying amounts.
QUANTITATIVE ANALYSIS OF FAMEs IN THE TOTAL
LIPID EXTRACT
Each FAME will be expressed as a percentage of the
total FAME; the peak area of one FAME divided by the
total peak area for all FAMEs.
Table 2 gives the
relative percentages of each FAME with respect to time
and Figure 3 is simply these percentages presented in
graphic form.
After 48 hours of growth the linoleic acid fraction
was at 49.14% of the total lipid extract FAMEs and made
up the larges·t component of the FAMEs.
Palmi tic acid
comprised 19.!54%, linolenic acid comprised 14.72%, oleic
acid was at 14.33% and stearic acid made up 2.27% of the
total lipid extract FAMEs.
Measurement of the total lipid extract FAMEs after
72 hours of growth revealed that the linoleic fraction
had increased from 49.14% of the total FiillEs to 52.65%
of the total.
The palmitic acid fraction also increased
from 19.54% to 24.68% of the total.
The linolenic acid
level decreased from 14.72% to 9.75%, oleic acid
25
2
3
4
Palmitic
Acid
19.54%
24.68%
21.85%
Stearic
Acid
2.27%
2.06%
1.05%
Oleic
Acid
14.33%
10.85%
7.14%
Days
Linoleic
Acid
49.14%
52.65%
63.51%
14.72%
9-75%
6.45%
--
Linolenic
Acid
.
Table 2.
Changes in fatty acid methyl ester composition
of the total lipid extract with respect to time.
26
Figure 3.
Changes in fatty acid methyl ester composition
of the total lipid extract with respect to time.
27
18:2
Linoleic
Acid
4o%
16:0
Palmitic
Acid
JJinolenic Acid
Oleic Acid
20%
10%
l
18:1
18:3
-""":ii!J----:---=4. .~.
~~~~ric ~------:
2
3
DAYS
4
18:0
28
decreased from 14.33% to 10.85% and stearic acid remained
relatively stable changing from 2.27% to 2.06% of the
total lipid extract FAMEs.
After 96 hours of growth the linoleic acid fraction
again increased significantly from 52.65% to 63.51%.
All other FAMEs decreased; pa·lmi tic acid dropped from
24.68% to 21.85%, oleic acid decreased from 10.85% to
7.14%, linolenic went from 9.75% to 6.45% and stearic
acid decreased from 2.06% to 1.05% of the total lipid
extract FAMEs.
From Figure 3 it can be easily seen that the linoleic
acid fraction of the FAMEs is the major FAME component
and is the only component to increase steadily with time.
The palmitic acid fraction was the next highest in
concentratio~ throughout vegetative growth and micro-
conidial production-and increased along with the linoleic
acid fraction prior to microconidial differentiation but
decreased following microconidiation.
The relative per-
centages OI oleic acid, linolenic acid and stearic acid
decreased with time and in general it could be said thateach FAME tended to remain in the same relative position
regardless of the stage of growth of the organism.
It is interesting to note that the linoleic acid
fraction was the only FAME fraction to increase relative
29
to the other FAMEs after microconidiation and that the
linoleic acid and palmitic acid fractions were the only
FAME fractions to increase relative to the other FAMEs
prior to microconidiation.
k~ALYSIS
OF THE FAMEs OF THE SEPARATED
LIPID COMPONENTS
Based upon the information obtained from the analysis of the total lipid extract FAMEs the next step in
this study was to separate the total lipid extract into
its various components and analyze the FAMEs of each
individual lipid class.
PHOSPHOLIPID
Figure
~
and Table 3 show the relative percentages
of each FAME found in the phospholipid fraction of the
total lipid extract with respect to time.
After 48
hours of growth the linoleic acid fraction was measured
as 51.48% of the total FAMEs.
The linoleic acid·fraction
decreased with time relative to the other FAMEs dropping
to 49.87% after 72 hours and to 48.72% after 96 hours of
growth.
The palmitic acid fraction was detected in higher
amounts in the phospholipid fraction than in the total
lipid extract FAME analysis.
After 48 hours the palmitic
30
2
3
4
Palmitic
Acid
35.24%
37.38%
4o.65%
Stearic
Acid
2.87%
2.62%
2.77%
Oleic
Acid
3.90%
5.02%
9.88%
fuys
1-·
-
:--
Linoleic
Acid
51.48%
49.87%
Linolenic
Acid
6.53%
5.18%
1.~8. 73%
o%
Table 3.
Changes in fatty acid methyl ester composition
of the phosopholipid fraction of the total lipid
extract with respect to time.
31
Figure 4.
Changes in the fatty acid methyl ester
composition of the phospholipid fraction
of the total lipid extract with respect
to time.
32
0
0
.-!
><:
ro
OJ
70%
H
ro
.-"<:
ro
6o%
~
50%
OJ
0.
rl
18:2 e
e
---e Linoleic acid
ro
.j..)
0
.j..)
......._,
cd
OJ
16:0
H
ro
.-"<:
ro
30%
~
20%
OJ
0.
OJ
.-1
Palmitic acid
40%
gp
..-I
Ul
10%
Oleic acid
18:3
18:1
18:0
o Stearic acid
Linolenic acid
2
3
Days
4
33
acid Fl\.I"'!Es comprised 35.24% of the total FAMEs present
which then increased to 37.38% after 72 hours and
increased again after 96 hours to 40.65% of the total
FAMEs.
The linolenic acid fraction comprised 6.53% of the
total FAMEs after 48 hours, dropped to 5.18% after 72
hours and became completely undetectable after 96 hours.
Oleic acid FAMEs made up 3.90% of the total FAMEs
after 48 hours, 5.02% after 72 hours, and after 96 hours
of grow·th rose again to 9. 88% of the total FAMEs.
The stearic acid fraction showed little change with
time being at 2,87% after 48 hours, 2.62% after 72 hours
and 2.77% of the total after 96 hours.
TRIGLYCERIDE FAMEs
Relative triglyceride FAMEs can be seen in Table 4
and Figure 5.
In the triglyceride fraction the palmitic
acid FAMEs were predominant, in contrast to the linoleic
acid fraction, that predominated in the total lipid
extract and phospholipid extracts.
The palmitic acid
level increased from 36.31% after 48 hours to 39.08%
after 72 hours and increased further to 51.55% after
96 hours.
The stearic acid FAMEs were found to make up a very
high percentage of the FAMEs as compared \'lith the total
34
-
2
3
Palmitic
Acid
36.31%
39.08%
51.55%
Stearic
Acid
28.42%
35.40%
43.12%
Oleic
Acid·
12.91%
8.87%
o%
Linoleic
Acid
17.10%.
13.93%
5.39%
Linolenic
Acid
5.26%
2.71%
O%
Days
4
Table 4.
Changes in fatty acid methyl ester composition
of the triglyceride fraction of the total lipid
extract with respect to time.
35
Figure 5.
Changes in the fatty acid methyl ester
composition of the triglyceride fraction
of the total lipid extract with respect to
time.
36
100%
90%
0
~
X
ro(!)
SO%
H
ro
.!4
ro
(!)
70%
0.
~
60%
.-!
ro
-P
0
.p
ro
(!)
-----H
ro
.!4
ro(!)
Palmitic
Acid
Stearic
Acid
50%
4o%
0.
I
30%
(!)
.-!
bO
s::
·rl
til
20%
18:2
,a___ _ _ _ _
18:1-----Linoleic
Acid
2
3
DA.YS
4
37
lipid extract and the phospholipid extract.
In the
triglyceride fraction the stea.ric acid FAMEs comprised
..
28.42% of the total after 48 hours which increased to
35.40% after 72 hours and increased again after 96 hours
to 43.12% of the total FAMEs.
In general all of the unsaturated FAMEs decreased
with time and all the saturated FAMEs increased with
time.
MONOGLYCERIDE AND DIGLYCERIDE FAMEs
Even though monoglycerides and diglycerides were
only found in trace amounts and were not measured
quanti·t:atively, FAMEs. were found to be in measureable
amounts in these two fractions.
Figure 6 and Table 5 show that only three FAMEs
were detected.
The stearic acid FAMEs showed significant
variation with time being at 51.4% after 48 hours,
decreased to 39.2% after 72 hours and increased to 50.6%
of the total
Fh~s
after 96 hours.
The palmitic acid fraction changed very slightly
with time going from 45.6% after 48 hours to 49.5% after
72 hours and 49.4% after 96 hours.
Very small amounts of oleic acid were detected after
48 hours of growth and comprised only 2.4% of the total
38
Figure 6.
Changes in the fatty acid methyl ester
composition of the monoglyceride and
diglyceride fractions of the total lipid
extract with respect to time.
39
100%
70%
6o%
50%
18:0
4)
Stearic acid
Palmitic
Acid
16:0
40%
30%
10%
.18:1-=:
~
~----------~-----~~----~----·~~--------~~~.~O:le:l~·c~acid
2
3
DAYS
4
'!V
DAYB
2
3
4
Palmitic
Acid
45.6%
49.5%
49.4%
Stearic
Acid
51.4%
39.2%
50.6%
Oleic
Acid
2.4%
5-9%
O%
o%
o%
O%
O%
O%
o%
JJinoleic
Acid
Linolenic
L_
•rable 5.
Changes in the fatty acid methyl ester composition
of the monoglyceride and diglyceride fractions of
the total lipid extract with respect to time.
41
FA¥£s.
After 72 hours oleic acid made up 5.9% of the
total.
The oleic acid FAMEs were not detected after
96 hours of growth.
FREE FATTY ACID FAMEs
In the free fatty acid fraction only two FAMEs were
detected and those were the two saturated
acid and s·t:earic acid.
Fill~s,
palmitic
The relationship of these two
FAMEs can be seen in Figure 7 and Table 6.
Stearic acid was found to decrease with time with
the drop being more pronounced between the 48 and 72
hour measurements going from 58.2% to 52.7% during this
period.
After 96 hours the stearic acid fraction dropped
to 52.1% of the total FAMEs.
The palmitic acid fraction FAMEs tended to increase
with time.
After 48 hours there was 41.8% palmitic acid
going to 47.3% after 72 hours and after 96 hours increased
slightly again to 47.9% of the total FAMEs present.
STEROL ESTER AND STEROL FAMEs
No FAMEs were· detected in these two fractions.
This
is not unexpected since fatty acids are not associated
with the structure of these types of lipid.
42
Figure 7.
Changes in fatty acid methyl ester composition
of the free fatty acid fraction of the total
lipid extract with respect to time.
100%
90%
0
0
r-i
X
So%
a1
Q)
H
a1
.!4
a1
70%
Q)
P-i
~
~
60%
18:0
r-1
Stearic Acid
ro
+>
0
+>
.......__
50%
Palmitic
Acid
a1
Q)
H
a1
.!4
ro
4o%
16:0
Q)
P-i
~
30%
p:.j
(Jj
r-1
i??
.,...;
{))
20% .
10%
2
3
DAYS
4
44
2
3
4
Palmitic
Acid
41.8%
47.3%
47.9%
Stearic
Acid
58.2%
52.7%
52.1%
Oleic
Acid
----
----
----
DAYS
~~
Linoleic
Acid
----
----
----
Linolenic
Acid
---=-
----
----
Table 6.
Changes in the fatty acid methyl ester composition
of the free fatty acid fraction of the total lipid
extract with respect to time.
45
ANALYSIS OF 96 HOUR CULTURES WITH MYCELIA AND
MICROCONIDIA SEPARATED
Microconidia are produced after about 86 hours of
incubation.
In previous experiments data 96 hour cultures
represented a combination of mycelia and microconidia.
In the experiments carried out in this section of the
study 96 hour mycelia and 10 hour old microconidia were
separated from one another and analyzed separately.
In
the mycelial fraction the same lipid components were
detected as reported for the previous 96 hour cultures.
When the microconidia were analyzed for lipid content
only sterols and sterol esters were found in measureable
amounts.
In addition two new polar lipid compounds were
detected which chromatographed between phosopholipid and
sterols but were unidentified.
Traces of triglyceride
were detected and only a trace of phospholipid could be
seen.
No free fatty acids were detected.
In the mycelia alone the triglyceride fraction
contained 0.81 microgram/culture as opposed to 0.82
microgram/culture in the combined fraction.
The sterol
fraction contained 0.71 microgram/culture (0.80 microgram/
culture combined) and 0.10 microgram/culture was detected
in the sterol ester fraction (0.18 microgram/culture
combined).
In the phospholipid fraction 0.13 microgram/
46
culture was detected (0.14 microgram/culture combined)
and in the free fatty acid fraction there was 0.41
microgram/culture and in the combined extract there was
0.44 microgram/culture.
When the lipids were analyzed in the microconidia
alone, two lipid classes were measured quantitatively.
The sterol fraction contained 0.08 microgram/culture and
the sterol ester fraction contained 0.03 microgram/
culture.
Therefore, 9.5% of the total sterols present
in 96 hour cultures was restricted to the microconidia
and 24.26% of the sterol esters present in the 96 hour
cultures was restricted to the microconidia.
ANA_LYSIS OF LIPIDS PRESENT IN 7 DAY OLD MICROCONIDIA
Seven day old microconidia were analyzed for lipid
content to determine whether or not the lipids found in
young microconidia were degraded with time or were stored
for extended periods of time.
After chromatographic
analysis of these older microconidia no lipids were
detected in measureable amounts.
There was only a trace
of sterol.
FAME ANALYSIS OF MYCELIAL AND MICROCONIDIAL FRACTIONS
Figure 8 shows the relationship of mycelial and
microconidial FAMEs.
The palmitic acid FAMEs are very
47
Figure 8.
The composition of the mycelial fatty acid
methyl esters compared to the composition
of the microconidial fatty acid methyl esters
in 4 day old cultures.
48
..
-
-
60%
Q)
m
Q)
H H
;::l ;::l
+'+'
.-I.-I
;::l ;::l
C) C)
--...._--...._
~~
Iii iii
r-i .-1
40%
cO cO
•rl ..-1
§:'0
c
m
.-1 ro
Q) •rl
C) s:l
c
50%
30%
1'.)
~t:.~~
20%
-
c
me
m
m
I~
I
16:0
18:0
'
18:1
18:2
18:3
49
close at 31.15% (mycelial) and 32.84% (microconidial).
There was a more significant difference in the stearic
acid fraction with the mycelial fraction accounting for
3.83% of the total FAMEs while in the microconidial
fraction stearic acid accounted for 12.44% of the total.
The oleic acid fractions were very close to the stearic
acid fractions, making up 7.79% of the mycelial FAMEs
and 7.46% of the microconidial FAMEs.
The mycelial
fraction contained 53.01% linoleic acid while the
microconidial fraction contained 46.77% linoleic acid
FAME.
The linoleic acid FAMEs comprised 4.23% of the
mycelial fraction and only 0.49% of the microconidial
fraction FAMEs. _
In
gene~al,
the microconidial fraction contained
more saturate-d FAMEs while the mycelial fraction contained relatively more unsaturated FAMEs.
MICROCONIDIAL VIABILITY
r'ticroconidial viability was determined by a series
of experiments that compared the number of germinated
microconidia/plate to the total number of microconidia/
plate with respect to microconidial age.
Figure 9 shows percent germination of 2 day old
microconidia through 7 day old microconidia.
From this
50
graph it can easily be seen that microconidial viability
decreased very rapidly with time.
The percent germination
decreased from 71.24% after 2 days to 47.26% after 3 days.
Only 11.91% of the microconidia were viable after 4 days.
Microconidia more than 5 days old were inviable.
51
Figure 9.
Percent germination of microconidia with
respect to time.
52
100%
(!)
+'
~
60%
0.
----s:l
0
...-!
50%
+=>
~
...-!
e 4o%
(!)
QO
*
30%
7
Conidial Age (Days)
8
53
DISCUSSION
The results of this study indicate that the
differentiation of microconidia in peach-fluffy-cot
mutants of Neurospora crassa occurs after a consistantly
predictable period of vegetative growth.
The predicta-
bility of the time of initiation of the microconidial
differentiation process suggests that when a specific
set of metabolic requirements are met microconidia are
produced.
This st'udy investigated whether or not changes
·in lipid composition can be correlated to microconidial
differentiation.
Little is known about the role of lipid
metabolism in the differentiation of a microconidia.
However, the-results of this study strongly suggests
that basic
relation~hips
do exist between lipid metabolism
and microconidial differentiation in
peach-fluffy-~
mutants of N. crassa.
I.
-
Vegetative Growth and the Differentiation of
Microconidia
The peach-fluffy-cot mutant of N. crassa was cultured
in liquid media or on solid media covered with sterile
dialysis tubing.
In cultures grown in liquid medium the
54
growth kinetics of this organism consists of three phases.
Initially there is a lag phase during which time growth
is slow and barely visible.
Following the lag phase a .
logarithmic growth phase occurs during which time growth
is accelerated and, in this case, the dry weight of
mycelium increases by over 300%.
After the logarithmic
phase a stationary phase of growth begins during which
logarithmic growth ceases and a static level of mycelial
growth is maintained with no significant increases in
mycelial dry weight.
is common in fungi
'l'his triphasic pattern of growth
(Emerson, 1950),
(Cochrane, 1958) .
In liquid culture vegetative growth is vigorous but'
microconidia
ar~
not produced.
Gr9wth is vigorous
because filamentous fungi are able to elongate their
hyphal tips in low 02 tensions associated with liquid
culture (Kobr et al., 1965).
Vegetative hyphal metabolism,
providing energy for synthetic reactions, is predominantly
of an anaerobic, glycolytic type and thus, vigorous
vegetative· growth is possible (Turian, 1971).
Inversely,
aerobic or oxidative conditions favor conidial differentiation (Turian an9 Bianchi, 1971).
Oxidative conditions
trigger a shift in various enzyme systems from fermentative to oxidative metabolism and also the redistribution
of organelles to the site of conidial differentiation.
55
Until oxidative metabolism is achieved conidial
differentiation will not occur (Turian and Bianchi,
1971).
Aerobic conditions also favor lipid synthesis
(Prill, et al., 1935),
(Gurr, et al., 1971), (Brody,
et al., 1970) although, reduced lipid synthesis appears
to have little if any effect on the vegetative growth
potential of this organism.
Cultures grown in liquid
rneoia have the ability to microconidiate when prooxidative conditions are present.
Cultures grown using a surface culture technique
follmv the same triphasic growth pattern as observed in
the liquid cultures but in surface culture microconidia
are differentiated after 86 hours following innoculation.
In surface culture the organism begins growth with a lag
phase followed by a logarithmic phase very similar to the
liquid cultures.
During this time the mass of the myce-
lium increases significantly until microconidia are
differentiated.
After microconidial differentiation
the organism ente~s a period of decreasing mass rather
than a staitionary phase.
During and after differentiation of microconidia
there are changes in the properties of the cell membranes
which could allow much of the cellular contents to diffuse
out of the cell.
The cellular contents then could diffuse
through the sterile dialysis tubing covering the surface
56
of the medium, and would be lost in the medium.
If so,
they would not be considered in the dry weight of the
mycelial mat.
Surface culture provides the aerobic conditions
necessary for microconidial differentiation.
However,
aerobic conditions alone are not responsible for the
predictable initiation of the differentiation process
86 hours after culture inoculation.
Microconidial
initiation after a specific period of vegetative growth
ls probably a result of a reduction in the carbon source
of the nutrient agar at some point prior to 86 hours of
grm.,-th (Enebo, et al., 1976),
(Gyllenber, et al., 1952).
(Borrow, et al., 1961),
Therefore, synchronization
of microconidiation in separate cultures is possible
since the nutrients from the medium are utilized at
similar rates and in each culture the carbon source is
exhausted in approximately the same time after inoculation.
II.
Qualitative Analysis of the Total Lipid Extract
The seven classes of lipid detected in the peach-
fluffy-cot mutant of N. crassa are common to many fungi
(Kanetsuna, et al., 1969), (Domer, 1971).
However,
investigators have found that the lipid components of
57
fungi may vary greatly between species and differ between
members of the same species grown under different
conditions (Kanetsuna, et al., 1971).
The published
evidence suggests that a great deal of variability is
present in the lipid components of fungi but the
significance of this variability is not well understood
since in many cases changes in lipid composition has
little correlation with fungal morphology.
In the phospholipid fraction of the total lipid
extract phosphatidyl ethanolamine and phosphatidyl
inositol were detected, both of which are considered
to be common components of the fungal phospholipid
fraction (Rattray, et al., 1975). -Many investigators
have found phosphatidyl choline to be one of the
predominate phospholipid fractions in ascomycetes
(Al-Doory, et al., 1962),
(Bard, et al., 1974),
(Bartnicki-Garcia, 1968),
(Barton, et al., 1972, 1973)
but I found no detectable phosphatidyl choline present
in the
peach~fluffy-cot
mutant phospholipid fraction.
This phosphatidyl choline deficiency may be due to the
inability of the organism to synthesize phosphatidyl
choline through the acylation of phosphatidic acid,
since phosphatidic acid is not present in the phospholipid fraction (Rattray, et al., 1975),
(Steiner, et
58
al., 1972).
It is also possible that the organism is
unable to methylate phosphatidyl ethanolamine sequentially (Waechter, et al., 1971).
III.
Quantitative Analysis of the Total Lipid Extract
In this organism the lipid content per culture
increases with time and is certainly a reflection of
the increasing mass of hyphae.
The increase in total
lipid is due to significant increases in one or two
of the lipid classes while other lipid classes either
remain constant or decrease slightly.
According to
Bovman the accumulation of lipids is very common in
fungi and not only do the amounts of lipid change with
time but also the relative composition of the lipid
classes.
Other investigators report an accumulation
of lipid during the lag phase growth followed by a
period of rapid utilization of lipid during the
staitionary growth phase and during reproduction-(Weete,
1973),
(Singh, 1955).
Since lipids are being accumulated
in the peach-fluffy-cot mutant at about the same rate
prior to and following microconidiation it appears that
lipid utilization has little to do with the change from
vegetative to reproductive growth.
The composition of the lipids found in this mutant
vary significantly with respect to time and the stage
of hyphal development.
After 48 hours of growth the
triglycerides were found to be the predominate lipid
component followed by the sterols, .phospholipids,
sterol esters, and free fatty acids.
The quantities
of lipid present after 48 hours of growth represent
the lipids synthesized and accumulated during the lag
phase of growth.
At this point there is an abundance
of nutrients in the medium to allow for maximum lipid
synthesis.
Between 48 and 72 hours, growth is entirely in the
loga:ci thmic phase during which time many nev.r cells
differentiate.
During this period the triglycerides
increased only slightly even though the mass of hyphae
increased rapidly.
This is probably because the
triglycerides were being used as an energy source as
quickly as they were being synthesized to provide energy
during logarithmic growth (Weete, 1971).
Also during
this period of rapid growth triglycerides probably were
incorporated into the many new cell walls that were
differentiating since triglycerides are considered to be
one of the primary lipid components of the cell wall
(Kanetsuna, et al., 1971) ,
(Domer, 1971) •
60
The most significant change in lipid composition
occurs during the lag phase of growth.
Between 48 and
72 hours of growth there is a tremendous increase in
free sterols.
This increase makes the free sterols
the predominate 72 hour lipid component.
The increase
in free sterols is significant since the major function
of sterols in fungi is thought to be one of structural
influences on the dynamic state of the cell membrane
(Proudluck, et al., 1968, 1969).
It is also known that
sterols influence phospholipid-protein interactions,
membrane bound enzyme activities, and membrane permeability (Papahadjopoulous, et al., 1973).
Based upon
this information, it is probable that the properties
of the cell membranes of this organism are different
after 72 hours of growth than they were after 48 hours
-
of growth because of shift in membrane composition.
This change in lipid composition is even more significant
when it is known that microconidia differentiate only a
few hours after this increase in sterols.
Weete (1971)
and Hendrix (19701 reported that in many fungal species
sterol production is required for the production of
conidia and for the induction of sexual reproductive
processes.
Unfortunately, the mechanism by which sterols
exert their influence is difficult to study.
However,
the evidence strongly suggests that sterols play some
sort of inductive role in the microconidiation process.
cultur~s
Post microconidial
provide
evid~nce
that
sterols act as an initiator of microconidial differentiation since sterol synthesis ceases following the
initiation of microconidiation.
This suggests that
some critical level of sterols is reached in order to
initiate
micro~onidial
differentiation.
After
rnicroconidiation no new sterols are synthesized.
Throughout the growth cycle of this organism it
appears that the phospholipids, sterol esters and free
fatty acids are all involved in a basic biochemical
function since these components remain relatively stable
regardless of the stage of growth (Weete,
1971)~
The
sterol ester fraction does, however, rise following the
differentiation of microconidia.
This may be the result
of ·the conversion of free sterols to sterol esters
(Rattray, et al., 1975),
IV.
(Gurr, 1971).
Total Lipid Extract FAMEs
The fatty acid methyl esters (FAMEs) detected in
the
~-fluf~-cot
mutant of Neurospora crassa are
directly comparable to other mutant strains of Neurospora
as analyzed by Brody et al.,
(1970).
The findings of
62
Brody indicate that certain morphological mutants of
!i·
crassa contain considerably less linolenic acid than
the wild type.
This restriction in the linolenic acid
fraction is a result of the inability of the organism
to convert linoleic acid (18:2) to linolenic acid (18:3)
because of a NADPH-deficiency.
Apparently this defi-
ciency also exists in peach-fluffy-cot mutants although
NADPH levels were never directly measured.
The FAME
composition of this mutant reflects an inability to
desaturate linoleic acid to linolenic acid.
The
relationship between linolenic acid content and observed
morphological changes cannot be evaluated since externally applied linolenic acid does not reverse the
morphology of the mutants studied (Brody, 1970).
According to Henry (197la, 197lb, 1973a, 1973b)
and Jones (1962), fa·tty acid composition appears to be
the most important variable in determining membrane
morphology which in turn may influence oxygen uptake
by the cell (Eletr and Keith, 1974).
The FAME composition of the peach-fluffy-cot mutant
does not change significantly with respect to time.
The
linoleic acid fraction increases proportionally with
time but this is not unexpected since this mutant lacks
the ability to desaturate linoleic acid to linolenic
acid effectively.
63
The degree of fatty acid unsaturation changes
slightly with time but these changes many not be
directly related to morphological change.
according to several workers (Singh, 1955),
In-fact,
(Brody,
et al., 1970) culture temperature alone has a significant
affect on fatty acid unsaturation without affecting the
morphology of the organism.
v.
Separation and Analysis of the FAMEs of the
Individual Lipid Classes
PHOSPHOLIPIDS
The resul·ts obtained from the analysis of the FAME
composition of the phospholipid fraction of the peach_f;!-uf_:fy-cot mutant were similar to the results obtained
by Brody and Nyc.
However, the phospholipid FAME
composition showed some significant differences from
the total lipid extract FJI.ME composition.
The phospho-
lipid fraction contained almost 20% more palmitic acid
(16:0) tha~ the total lipid extract and the palmitic
acid fraction made up as much as 40.65% of the total
FAMEs at any given time.
The significance of this high
degree of saturation in the phospholipid fraction is not
clear but one possible explanation for this is that
saturated fatty acids are known to be more resistant
to oxidation than unsaturated fatty acids (Gurr, 1971).
Since phospholipids are thought to play a structural
role in the cell membrane (Jollow, et al., 1968) perhaps
the high degree of saturation adds to the stability of
the membrane or plays a role in the high fermentative
activity associated with microconidial mutants (Turian,
1966a and b).
VI.
Analysis of Triglyceride, Monoglyceride, Diglyceride,
and Free Fatty Acid Fraction FAMEs
'I'he information obtained from the anlysis of the
FN.ffis of the various lipid fractions
(other than the
total lipid extract and the phospholipid fraction) was
i.nconsistant because changes in FM1E composition were
occurring that could not be attributed to normal
metabolic activity.
The inconsistant results in FAME composition is
a result of auto-oxidation and chemical oxidation of
lipids (Gurr, 1971).
The time necessary to separate the
total lipid extract into its major components is long
enough for
signifi~ant
oxidation to take place.
Oxidation
has a profound effect on fatty acid composition, affecting
part.icularly the more unstable unsaturated fatty acids.
Oxidation causes unsaturated fatty acids to be broken
down into more saturated forms
(Gurr, 1971).
This
65
explains the very high levels of stearic acid and palmitic
acid detected in samples analyzed for fatty acid content
imn1ediately following separation by TLC.
The phospholipid
fraction of the total lipid extract was unaffected by the
oxidative process.
This is probably because the solvent
system used for separation of the total lipid extract had
a no apparent effect on the phospholipid fraction since
the phospholipid fraction had an Rf value of zero.
Furthermore, the phospholipids appeared to be unaffected
by oxidation since the data obtained in this study were
comparable to the results obtained by Brody et al.
VII.
(1970).
Analysis of Mycelial and Microconidial Lipid Content
This experiment was done to determine whether or not
there is a preferential transfer of specific lipid components to the microconidium during microconidiogenesis.
It was found that free and esterified sterols are
preferentially localized in the microconidium during
microconidiogenesis along with trace amounts of phospholipid and triglyceride.
Also detected were two polar
lipid fraction not- seen before which are probably
triglyceride precursors.
Transport of lipids to spores
through' the mycelium has been studied by
Bartnicki~Garcia
and Reyes (1968) using species of Mucor and they found
that preferential transp9rt of lipids does occur.
66
Bianchi and Turian (1967) found tha·t the lipid content
of the macroconidium was mostly phospholipid .(94%) with
sterol esters and free sterols only making up_about 4% of
the total lipids.
Triglycerides comprised only about 2%
of the total lipid.
These
res~lts
vary greatly from the
information obtained in this study.
The microconidium of
this mutant of Neurospor0:. contains only a small amount of
phospholipid and triglyceride (which made up 96% of the
macroconidial lipids).
The predominate lipid components
of the microconidium are free and esterified sterols.
Aout 10% of the sterols present in the vegetative mycelium
is localized in the microconidia.
During microconidiation
there is an increase in sterol ester content of the
mycelium and about 24% of the sterol ester content is
localized in the microconidia.
Turian et al. have stated that prior to conidial
differentiation there is a localization of specific
compounds that have controlling effects on many different
metabolic processes at the site which is about to become
differentiated,
i~e.,
the portion of the vegetative
hyphae about to give rise to conidia.
Therefore, it
is quite possible that sterols are localized at the
specific point in the vegetative hyphae prior to the
onset of microconidiation and are important in the
initiation of the differentiation process.
Localization
of sterols at the point of differentiation would also
67
facilitate the transport of these compounds to the
microconidium.
Sterol esters apparently have little
to do with the initiation of
m~croconidial
differentiation
since increases in sterol ester content occur after
differentiation has already begun.
However, sterol
esters are the predominate lipid component of the
microconidia and must therefore play an important role
in microconidial metabolism.
Bec~use
of the preferential storage of sterols and
sterol esters in the microconidium (not phospholipids
as in the macroconidium) the microconidia were tested
for their ability to germinate after being aged for
various lengths of time.
Probably due to a low percentage
of phospholipid and triglyceride storage in the microconidia, the ability of the microconidia to germina·te
decreased rather rapidly with age, losing their ability
to germinate within five days.
It was determined that
the lipids stored in the microconidium v1ere being utilized
in microcohidial metabolic processes.
Bianchi and Turian
(1967) noted that_lipids were consumed in the macroconidium and concluded that they were being consumed
as an energy source for.conidial metabolism.
Also, lipids
apparently have little to do with the actual germination
process since the sugar trehalose is thought to be the
important factor in conidial germination
(Bianc~i
and
68
Turian, 1967).
Even though trehalose is used in conidial
germination if the microconidia have exhausted the stored
lipid energy source before the proper conditions for
germination vmre met, the microconidium may have already
died.
Thus, the comparatively small amounts of phospho-
lipid and triglyceride which would be used as the energy
source is certainly directly related to the short life
span of the microconidium.
VIII.
FAME Analysis of Microconidial Extracts and
Post-Microconidial Mycelial Extracts
The FAME composition of the microconidial extracts
suggest,s that the FAMEs present in- the microconidiu.rn are
from the phospholipid fraction since the FAME composition
of the microconidia and phospholipid fraction are very
similar.
The major difference in the FAME composition of
the microconidia and the post-microconidial mycelial
extracts is that the micronidial FAMEs are more
unsaturated than the mycelial FAMEs.
This is probably
because a more acute NADPH-deficiency exists in the
microconidium than in the mycelium.
69
SUMMARY
The results of this study strongly suggest that
there is a direct relationship between changes in lipid
composition and the developmental events that lead to
the differentiation of microconidia.
The most important
aspect of lipid metabolism relative to microconidial
differentiation is the apparent inductive influence of
sterols during the transition from vegetative to
reproductive growth and the preferential storage of
sterols and sterol esters in the microconidia themselves.
These results are supported by evidence that many fungal
species are known to require sterols for the production
of sexual spores (Hendrix, 1970),
1967, 1968).
(Siestsma, et al.,
Further study is necessary to determine
the mechanisms by which sterols exert their influences
during the transition from vegetative growth to the
differentiation of sexual spores.
70
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