PHOSPHOLIPID SYNTHESIS AND FATTY ACID CONTENT, h
, _- 0F PHOSPHOLIPIDS IN GERMINATING SEEDS AND
SEEDLINGS OFVCHILLII‘I'G RESISTANT AND CHILLING : =
SENSITIVE VEGETABLE SPECIES ’

Dissertation for the Degree of Ph. D.

MICHIGAN STATE UNIVERSITY '

‘CONSTANTINOS CH‘RISTOS DOGRAS
1975

 

LIBRARY

a b. Micmgan mate
Univcmty

 

This is to certify that the

thesis entitled

Phospholipid Synthesis and Fatty Acid Content of
Phospholipids in Germinating Seeds and Seedlings
of Chilling Resistant and Chilling Sensitive

Vegetable Species

presented by

Constantinos Christos Dogras

hummews fulfillment
of the requirements for

13119 Ph.D. deg-33in Horticulture

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ABSTRACT
PHOSPHOLIPID SYNTHESIS AND FATTY ACID CONTENT OF
PHOSPHOLIPIDS IN GERMINATING SEEDS AND SEEDLINGS

OF CHILLING RESISTANT AND CHILLING SENSITIVE
VEGETABLE SPECIES

BY

Constantinos Christos Dogras

Chilling sensitive plant species are injured by
chilling temperatures, below about 12°C but above 0°C,
while chilling resistant ones are not. Chilling injury has
been related by some workers to a phase change in the lipid
portion of cellular membranes and this has been correlated
with the degree of saturation of those lipids.

In this study, the incorporation of 14

C-glycerol
into ph03pholipids in germinating seeds and seedlings of
chilling sensitive and resistant species, at 25°C and 10°C,
has been investigated. In addition, the fatty acid content
of the major phosPholipids was determined.

Broad beans and peas, chilling resistant species,
germinate at 10°C while the chilling sensitive lima beans
do not. During seed imbibition at 10°C, a higher percentage
phosphatidyl choline (PC) and a lower percentage phosphatidyl
ethanolamine (PE) and phosphatidyl glycerol (PG) were

observed in both axes and cotyledons of broad beans and peas

the
at
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10°
of
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whi

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Sehs

Constantinos Christos Dogras

than in lima beans. Furthermore, lima bean seeds, imbibed
at 10°C, exhibited a lower percentage PC and a higher per-
centage PE and PG than at 25°C. The chilling resistant
species, broad beans and peas, had about the same PC and
PE at both temperatures.

Apparently, in the chilling sensitive lima beans at
10°C, there is a shift in metabolism towards the synthesis
of more PG and PE largely at the expense of PC. A higher
PC/PE ration, observed in broad beans and peas, may allow
membranes to remain fluid and funCtional at low temperature
(since PC remains liquid at lower temperatures than PE)
while a lower ratio, observed in lima beans, may result in
a phase change of the membrane lipids which would cause
chilling injury and lack of seed germination.

In the seedling stage, there were no consistent
differences between chilling resistant species (broad beans,
peas, cabbage, beets, lettuce) and chilling sensitive ones
(lima beans, watermelon, cucumber) in the relative percen—
tages of individual phospholipids labeled.

In general, exposure of plants of chilling resistant
species to 10°C continuously or shifting plants of chilling
resistant and sensitive species from 25°C to 10°C caused an
increase in percentage PC and a decrease in percentage PE
in comparison to plants grown continuously at 25°C. These
results suggest, that in the seedling stage of chilling

sensitive species, a shift in metabolism occurs which

18

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Constantinos Christos Dogras

produces more PC and less PE, similar to chilling resistant
species,which allows them to survive exposure to chilling
temperatures.

The fatty acid content of the three major phospho-
lipids (phosphatidyl choline, phosphatidyl ethanolamine
and phosphatidyl inositol) in germinating seeds of broad
beans, peas, and lima beans revealed that the percentage
18:1 was higher and the percentage 18:3 was lower in the
chilling resistant species than in lima beans. Also, over
a 6-day germination period at 10°C in broad beans and peas,
the percentage 18:1 decreased and the percentage 18:3
increased from the initial levels during imbibition. The
fatty acid content of lima beans did not change during this
6-day period. This indicates that in chilling resistant
plants, there is a shift in metabolism of fatty acids
towards a more highly unsaturated state while in lima beans
no shift occurs. This lack of a shift in fatty acid meta-
bolism in lima beans may reflect the fact that these seeds
were injured by the chilling temperature and no lipid
metabolism occurred.

In the seedling stage, the chilling resistant plants
had a higher percentage 18:2 and a lower percentage 18:3
than the chilling sensitive plants. In chilling resistant
and sensitive species, with few exceptions, the percentage
18:3 increased after chilling hardening. The degree of
unsaturation of fatty acids, as is reflected in the tradi-

tionally used indexes (double bond index and unsaturated

Constantinos Christos Dogras

fatty acids/saturated fatty acids ratio), was not always
positively correlated to the degree of chilling resistance
of the species studied. The results of this study suggest
that the chilling sensitivity of the species examined is
related to the nature of cellular phospholipids, as charac-
terized by the polar group of the molecule; furthermore,
it is apparent that the fatty acid composition of phospho-
lipids in chilling sensitive and resistant species is
different, in terms of relative quantities of individual

fatty acids.

PHOSPHOLIPID SYNTHESIS AND FATTY ACID CONTENT OF
PHOSPHOLIPIDS IN GERMINATING SEEDS AND SEEDLINGS
OF CHILLING RESISTANT AND CHILLING SENSITIVE

VEGETABLE SPECIES

BY

Constantinos Christos Dogras

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Horticulture

1975

ACKNOWLEDGMENTS

I wish to express my sincere thanks and deep
appreciation to Dr. R. C. Herner for his constant support,
assistance, counsel and encouragement during the course of
my graduate studies.

I am most grateful to Dr. D. R. Dilley for assis~
tance and counsel during the course of this work and for
serving on my guidance committee.

I would like to especially thank Drs. L. O.
Copeland, C. J. Pollard, and J. E. Motes for counsel and
service on my guidance committee.

I would like to extend my sincere thanks to
Ms. Julie Chamberlain and Mr. Christopher J. Rajzer for
assistance in the laboratory during the course of this
work.

My greatest debt is to my wife, Catherine, and to
the rest of my family for their understanding and

encouragement throughout my graduate studies.

ii

TABLE OF CONTENTS

Page
LIST OF TABLES O O O O O O O O O O O O O I

INTRODUCTION . . . . . . . . . . . . . . . 1

Literature Review . . . . . . . . 2
Lipids in Cell Membranes . . . . . . 2
Physical Properties of PhOSpholipids

in Relation to Temperature . . z . . 4
Lipids and Cold Sensitivity in Organisms . 6

Patterns of Phospholipid Synthesis in

Relation to Temperature of Growth

and Cold Resistance . . . . . . . 8
Effect of the Physical State of Membrane

Lipids on the Activity of Enzymes

Associated with Them . . . . . . . 9
Summary . . . . . . . . . . . . 10

Thesis Objectives . . . . . . . . . . 11

SECTION I PHOSPHOLIPID SYNTHESIS IN GERMINATING SEEDS
OF SOME VEGETABLE SPECIES IN RELATION TO

CHILLING RESISTANCE . . . . . . . . . 13
Introduction . . . . . . . . . . 13
Materials and Methods . . . . . . . . . 16
Results . . . . . . . . . . . . . . 19

Discussion . . . . . . . . . . . . . 29

SECTION II PHOSPHOLIPID SYNTHESIS IN SEEDLINGS OF
SOME VEGETABLE SPECIES IN RELATION TO
CHILLING RESISTANCE . . . . . . . . 35

Introduction . . . . . . . . . . . 35
Materials and Methods . . . . . . . . . 36
Results . . . . . . . . . . . . . . 39
Discussion . . . . . . . . . . . . . 46

iii

Page

SECTION III FATTY ACID CONTENT OF PHOSPHOLIPIDS IN
GERMINATING SEEDS OF SOME CHILLING
RESISTANT AND CHILLING SENSITIVE
VEGETABLE SPECIES . . . . . . . . . 52

Introduction . . . . . . . . . . . . 52
Materials and Methods . . . . . . . . . 54
Results . . . . . . . . . . . . . . 56
Discussion . . . . . . . . . . . . . 75

SECTION IV FATTY ACID CONTENT OF PHOSPHOLIPIDS IN
SEEDLINGS OF SOME VEGETABLE SPECIES IN

RELATION TO CHILLING RESISTANCE . . . . 79
Introduction . . . . . . . . . . . . 79
Materials and Methods . . . . . . . . . 80
Results . . . . . . . . . . . . . . 80
Discussion . . . . . . . . . . . . . 86

SUMMARY AND CONCLUS IONS . . . . . . . . . . . 89
MPENDIX O O O O O O O O O O O O O O O O 9 5

BIBLIOGRAPHY . . . . . . . . . . . . . . .. 100

iv

LIST OF TABLES

Table Page
Section I

1. Distribution of radioactivity (percentages)
among phospholipids of seed axis tissue
after a 24 hour imbibition with l4C-glycerol
at 10°C or 25°C . . . . . . . . . . 21

2. Distribution of radioactivity (percentages)
among phospholipids of cotyledonary tissue
after a 24 hour imbibition with 14C-glycerol
at 10°C or 25°C . . . . . . . . . . 22

3. Distribution of radioactivity (percentages)
among seed phospholipids after a 24 hour
imbibition with 14C-glycerol at 10°C or
25°C. Comparison of temperatures and
seed parts . . . . . . . . . . . . 23

4. Comparison of radioactivity distributed (per-
centages) in phosphatidyl choline vs.
phosphatidyl ethanolamine and in total
phospholipids vs. total lipids after a
24 hour imbibition with 14C-glycerol at
10°C or 25°C . . . . . . . . . . . 27

Section II

1. Characteristics of seedlings incubated with
14C-glycerol . . . . . . . . . . . 38

2. Distribution of radioactivity (percentages)
among phOSpholipids of seedlings after a
24 hour incubation with l4C-glycerol at
10°C or 25°C. Comparison of individual
phospholipids . . . . . . . . . . . 41

Table

Section

1.

Distribution of radioactivity (percentages)
among phospholipids of seedlings after
a 24 hour incubation with 14C-glycerol
at 10°C or 25°C. Comparison of tempera-
tures and species . . . . . . . .

Comparison of radioactivity distributed
(percentages) in phosphatidyl choline vs.
phosphatidyl ethanolamine, in total phos-
pholipids vs. total lipids, and in
phosphatidic acid vs. total lipids, after
a 24 hour incubation with 1 C-glycerol at
10°C or 25°C . . . . . . . . .

III

Phosphatidyl choline: Percentages of fatty
acids after 24 hours of germination at
10°C or 25°C. Comparison of species .

Phosphatidyl choline: Percentages of fatty
acids after 24 hours of germination at
10°C or 25°C. Comparison of temperatures
and seed parts . . . . . . . . .

Percentage of fatty acids in phospholipids
(PC, PE, PI) after 6 days of germination
at 10°C. Comparison of species . . .

Percentages of fatty acids in phospholipids
(PC, PE, PI) after 6 days of germination
at 10°C. Comparison of seed parts . .

PhOSphatidyl choline: Percentage of fatty
acids after 24 hours or 6 days of germi-
nation at 10°C . . . . . . . . .

Phosphatidyl ethanolamine: Percentages of
fatty acids after 24 hours of germination
at 10°C or 25°C. Comparison of species

Phosphatidyl ethanolamine: Percentages of
fatty acids after 24 hours of germination
at 10°C or 25°C. Comparison of tempera-
tures and seed parts . . . . . . .

Phosphatidyl ethanolamine: Percentages of

fatty acids after 24 hours or 6 days of
germination at 10°C . . . . . . .

vi

Page

42

45

58

59

60

62

64

66

67

70

Table Page

9. Phosphatidyl inositol: Percentages of fatty

acids after 24 hours of germination at

10°C or 25°C. Comparison of species . . . 71
10. Phosphatidyl inositol: Percentages of fatty

acids after 24 hours of germination at
10°C or 25°C. Comparison of temperatures
and seed parts . . . . . . . . . . . 72

ll. Phosphatidyl inositol: Percentages of fatty
acids after 24 hours or 6 days of germi-
nation at 10°C . . . . . . . . . . . 73

Section IV

1. Phosphatidyl choline: Percentages of fatty
acids in seedlings of chilling sensitive
and chilling resistant species grown at
10°C, 25°C or after transfer from 25°C to
10°C . . . . . . . . . . . . . . 82

2. Phosphatidyl ethanolamine: Percentages of
fatty acids in seedlings of chilling
sensitive and chilling resistant species
grown at 10°C, 25°C or after transfer from
25°C to 10°C . . . . . . . . . . . 83

3. Phosphatidyl inositol: Percentages of fatty
acids in seedlings of chilling sensitive
and chilling resistant species grown at
10°C, 25°C or after transfer from 25°C to
10°C . . . . . . . . . . . . . . 84

Appendix
A-l. Chemical formulas of the major plant
phospholipids . . . . . . . . . . . 95
A-2. Melting point of some fatty acids . . . . . 96
A-3. Approximate transition temperature (Tc), from
liquid-crystalline to crystalline state, of
some phospholipids . . . . . . . . . 97

vii

Table

A-4.

Page

Distribution of radioactivity in lipids of seeds
imbibed for 24 hours with 4C-glycerol . . . 98

Distribution of radioactivity among lipids of

seedlings after a 24 hour incubation with
14C-glycerol . . . . . . . . . . . 99

viii

INTRODUCTION

INTRODUCTI ON

Many plants, usually those originating in tropical
or subtropical regions, are injured or fail to germinate
and grow when they are exposed to chilling temperatures:
that is, below about 12°C but above 0°C (Levitt, 1972;
Lyons, 1973).

The damage caused by chilling temperatures is called
chilling injury and is a time—temperature response, with the
injury becoming more severe at lower temperatures or with
longer exposure to the chilling temperatures (Lyons, 1973).
Chilling resistance is determined by genetic factors but it
is possible to partially condition or harden some chilling
sensitive plants against chilling injury by exposing them,
for a relatively short period of time, to temperatures
slightly higher than the ones causing chilling injury (Wilson
and Crawford, 1974; Wheaton and Morris, 1967). Investiga-
tions made so far support the theory of Lyons (1973) that
upon chilling, a phase transition occurs in the membrane
lipids of the chilling sensitive plant tissue, with con-

comitant changes in the physical properties of the membranes

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resulting in disruption of cell metabolism, ultimately
leading to the observed chilling injury symptoms. Chilling
resistant plants do not show the described phase change in
membranes in the chilling temperature range. This suggests
that membrane lipids of chilling resistant plants may be
different from the ones of chilling sensitive plants and
indeed some differences have been shown.

A literature review is following on the subject of
the resistance of plants, and some other organisms, to

chilling temperatures.

Literature Review

 

Lipids in Cell Membranes

 

It is believed that cellular lipids are mostly bound
in membranes, with the exception of seed oils and leaf waxes
(Kates, 1970). In all models of membranes, lipids are con-
sidered to be combined with structural proteins and a basic
role in membrane structure and function is attributed to
phospholipids (Van Deenen, 1965; Bishop, 1971).

Some investigators have proposed that the majority
of the cell phospholipids are present in the membranes and
it is speculated that only biological membranes contain
phospholipids (Van Deenen, 1965; Chapman, 1967).

Subcellular organelles have been shown to be very
rich in phospholipids (Van Deenen, 1965). During cell

division an increased phospholipid synthesis is observed,

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apparently because phospholipids are necessary to build
new membranes in the newly formed cells (Van Deenen, 1965).

Phospholipids, in addition to their structural role
in membranes, are believed to be necessary for the function
of certain enzymes associated with cell membranes (Cronan
and Vagelos, 1972). The importance of the phospholipid
composition of membranes in determining their permeability
properties has been established in model and natural systems
(Demel et_al., 1972; Van Deenen, 1965; Williams and Chapman,
1970; Chapman and Leslie, 1970). There is a large variety
of phospholipid classes associated with cell membranes
(Chapman and Leslie, 1970; Kates, 1970).

In plants the major phOSpholipids are:* PI, PE, PC,
PG and the minor ones are: PA, PS and CL (Kates, 1970).

The need for several phospholipid classes in biolo-
gical membranes is not yet understood (Cronan and Vagelos,
1972), but it is suggested that the phospholipid variation
may contribute to the several membrane functions. This
idea is supported by the observation that a given biological
membrane has a constant and characteristic lipid composi-
tion, suggesting that the physical properties of membrane
lipids are essential for the function of membranes (Van

Deenen, 1965).

 

*PA: Phosphatidic Acid. PC: Phosphatidyl Choline.

PE: Phosphatidyl Ethanolamine. P57. Phosphatidyl Glycerol.
PI: Phosphatidyl Inositol. PS; —Phosphatidyl Serine.

EL: Cardiolipin. For chemical formulas see Appendix,

Table A-1.

Furthermore it has been suggested that the presence
of several phospholipid classes in biological membranes is
necessary in order to produce the correct liquid cyrstalline
phase organization required for membrane function (Williams
and Chapman, 1970).

Physical Properties of Phospholipids
in Relation to Temperature

 

 

Research with artificial membranes, prepared from
chemically pure phospholipids, established that for each
phospholipid a transition temperature (To) exists below
which the phospholipid occurs in a crystalline form and
above which it exists in a liquid-crystalline type of
organization (Williams and Chapman, 1970; Chapman, 1967;
Chapman and Leslie, 1970). The transition temperature is
observed many degrees below the capillary melting point and
depends on:

(1) The chain length of the hydrocarbons of the
phospholipid; i.e., the shorter the chain
length, the lower the Tc.

(2) The degree of unsaturation of the hydrocarbon
chains; i.e., the more unsaturated the chains,
the lower the Tc.

(3) The nature of the polar group of the phospho-
lipid; i.e., PC exhibits lower Tc than PE
when they have exactly the same kind of hydro-

carbon chains (Appendix, Table A-3).

Same

(4) The amount of water present in the membrane
system (the higher the water concentration, up
to a limit, the lower the Tc).

(5) The amount and type of water soluble materials
present.

(6) The presence of cholesterol.

It has been prOposed (Williams and Chapman, 1970;
Chapman, 1967), that the phase transition of phospholipids
may be important in relation to the behavior of phospho-
lipids in biological membranes at different temperatures.
Furthermore it has been suggested (Chapman, 1967 and 1968;
Cronan and Vagelos, 1972), that differing chain lengths and
degrees of unsaturation of the fatty acids in the lipids of
biological membranes allows the bio-membrane the correct
fluidity, at a particular environmental temperature, in
order to function properly.

Natural phospholipids extracted from mitochondria,
glyoxysomes and proplastids of castor bean endosperm (Wade
§£_21., 1974) exhibited a phase transition at about 10°C,
below which chilling injury occurs for this species. The
same phase transition, at the same temperature, was observed
to occur in the whole organelles and their isolated membranes.
A similar observation was made in mitochondrial lipids of
the chilling sensitive sweet potato, while no phase change
was observed in the chilling resistant potato at the chill-
ing temperature of 12°C (Raison et_§l,, 1971a). Phospho-

lipids of Mycgplasma laidlawii exhibit a phase transition at

 

n

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h.

b

low temperatures that is similar to the one observed

in chemically pure phospholipids (Reinert and Steim, 1970).
Egg yolk lecithin, a natural phosphatidyl choline, shows a
marked endothermic phase transition at a temperature of
about 20°C (Chapman, 1967).

The coincidence of a phase change occurring at low
temperatures in chemically pure phospholipids, in natural
phospholipids and in biological membranes collectively
supports the idea that chilling injury may be related to
physical changes occurring in the cell membranes.

Lipids and Cold Sensitivity
in Organigms

 

 

The permeability of biological membranes is believed
to be related to the fluidity of their lipids (Oldfield and
Chapman, 1971). The length and degree of unsaturation of
the hydrocarbon chains of the membrane lipids may determine
the fluidity of the membrane at low temperature (Chapman,
1968; Bishop, 1971). It has been found (Lyons and Asmundson,
1965) that a small alteration (e.g., 5%) in the unsaturation
of fatty acids in fatty acid mixtures, prepared from fatty
acids similar to the ones occurring in biological membranes,
results in a marked change of the freezing point of the
mixture.

Mitochondrial lipids of chilling resistant plant
species had more unsaturated fatty acids than the ones of

chilling sensitive species (Lyons et a1., 1964). The

mi tI

mitochondria of the chilling resistant species were more
flexible, a characteristic attributed to the higher degree
of unsaturation.

On the other hand, it has been shown (Raison et_al.,
197ld) that the lipid components of the membranes of mito-
chondria from a chilling sensitive plant (sweet potato)
undergo a thermal phase transition at about 12°C, where
chilling injury occurs in many chilling sensitive species.
No phase change was observed with mitochondria or the lipids
extracted from mitochondria of a chilling resistant plant
(potato) at that temperature. An increase in fatty acid
unsaturation, in all lipid classes, in plants grown at low
temperatures, has been shown in rye and wheat seedlings
(Redshaw and Zalik, 1968; De La Roche gt_al,, 1973) and
alfalfa plants (Gerloff §t_§1,, 1966).

Membrane lipids in E. ggli_grown at lower tempera-
tures were more unsaturated (Haest §£_al., 1969). Mito-
chondrial lipids of wheat seedlings (Miller §E_al., 1974)
exhibited a lower Tc at low temperatures than at high ones,
suggesting that some modification in the composition of
lipids takes place at low temperatures.

Furthermore, it has been reported that some
poikilothermic organisms at low temperatures, have more
unsaturated phospholipids than their counterparts at higher
temperatures (e.g., E, 9911, Cronan and Vagelos, 1972;

Candida lipolytica, Kates and Paradis, 1973; Bacilus

 

Nil

31

cereus, Kaneda, 1972; alfalfa, Grenier and Willemot, 1974;
wheat plants, De La Roche et al., 1972). Cold resistant
alfalfa varieties had more unsaturated phospholipids than
cold sensitive ones (Grenier and Willemot, 1974).

Patterns of Phospholipid Synthesis in

Ralation to Temperature of Growth and
Cold ReSistance

 

 

 

More phospholipids, in comparison to other lipid

classes, were synthesized in Populus euramericana, L.

 

(Yoshida and Sakai, 1973)and Robinia pseudoacacia

 

(Siminovitch §t_al., 1968) during the development of cold
hardiness.

In some cases, the variation of the temperature or
the development of cold hardiness resulted in the preferen-
tial accumulation of specific phospholipid classes. This
may be important for the elucidation of the mechanism of
cold resistance because different phospholipids show a
phase transition from a liquid-crystalline to a solid-
crystalline state at different temperatures as discussed
earlier. In alfalfa (Kuiper, 1970), more PG and PI accumu-
lated at high temperatures and more PC and PE at low ones.
Cold resistant alfalfa varieties had more PC and PE at low
temperatures than the cold sensitive ones. Also, in rye
seedlings (Thomson and Zalik, 1973) more PI was synthesized

at high temperatures than at low ones.

Smaller changes in phospholipids with temperature,
were observed in wheat Seedlings (Willemot, 1975) but
clearly more PC was synthesized at low temperatures,
especially in a cold resistant variety. Some workers, how-
ever, have shown no variation in the pattern of phospholipid
synthesis with temperature or cold resistance changes as
seen in E. 9911 (Haest gt_§l., 1969; Cronan and Vagelos,
1972) and wheat seedlings (De La Roche, et_§l,, 1973).

Other factors may be involved in the determination
of cold resistance in organisms, by altering the To of
cellular lipids. For example, it has been suggested that
cholesterol may affect the To of biological membranes
(Lyons, 1973). This idea is supported by the finding that
cholesterol lowers the Tc of phospholipids in model systems
(Ladbrooke gt_al,, 1968).

Effect of the Physical State of Membrane

Lipids on the Activity ofEngymes
Associated withCThem

 

Many of the enzymic reactions in cells take place on
cellular membranes (Bishop, 1971). It has been demonstrated
in chilling sensitive plants, that the activation energy
for some enzyme systems, e.g. electron transport systems of
both mitochondria and chloroplasts as well as protein
synthesis systems, increases suddenly at chilling temperatures.
This is suggestive of a conformational change in the membrane

bound enzymes, probably because of a phase change observed to

10

occur in the membrane lipids associated with the enzymes,
at the chilling temperatures (Towers §t_§l,, 1972; Towers
§E_§l., 1973; Yamaki and Uritani, 1974; Shneyour e£;313,
1973; Raison, 1973; Raison gt_al., 1971b; Phillips and
McWilliam, 1971; Lyons and Raison, 1970). In cold resis-
tant species a similar sudden increase in the activation
energy of the same membrane 'bound enzyme systems occurred
at much lower temperatures than in the cold sensitive
species. It is currently believed (Lyons, 1973) that at
chilling temperatures the membrane lipids of the chilling
sensitive species solidify and this causes an alteration of
membrane permeability as well as an increase in the activa-
tion energy of some membrane bound enzymes. Both changes
result in an imbalance of cell metabolism followed by the

appearance of chilling injury symptoms.

Summary

(1) Cell membranes and their lipids are probably
involved in the mechanims of chilling resistance in
organisms.

(2) Phospholipids, major membrane lipids, may be
important factors in chilling resistance. The hydrocarbon
chain and polar groups in the phospholipid molecule probably
contribute to the maintenance of a certain membrane fluidity
at a particular temperature, allowing the membrane to

function.

ll

(3) Exposure of biological membranes to low tempera-
tures presumably causes a physical change in their lipids
from a liquid-crystalline to a solid-crystalline state.
This results in some conformational changes of the enzymes
associated with the lipids with an increase in activation
energy and consequently an alteration in cell metabolism
that may end in failure of growth and chilling injury.

(4) Chilling resistant or chilling hardened plants,
apparently have (by inheritance or by modification in
response to environmental temperature) membrane lipids that
are subject to a phase change at lower temperatures than
those of chilling sensitive species. This allows them to
continue functioning normally at chilling temperatures

(0-12°C).

Thesis Objectives

 

The discussion in the preceding pages shows that:
(1) a specific relationship between temperature of growth
or chilling resistance of plants and certain phospholipid
classes, has not been established; (2) the knowledge on the
fatty acid content of phospholipids of chilling sensitive,
chilling resistant or chilling hardened plants is insuffi-
cient.

Because of the possible significance of phospho-
lipids in the mechanism of chilling resistance and chilling

injury investigations were initiated to determine: (1) the

C0
C1“;

V8

12

pattern of phospholipid synthesis, and (2) the fatty acid
content of the major phOSpholipids (PI, PC, PE), in some
chilling resistant, chilling sensitive and chilling hardened
vegetable species under several temperature treatments.

The results of those studies are presented in the

following four sections of this thesis.

SECTION I

PHOSPHOLIPID SYNTHESIS IN GERMINATING SEEDS
OF SOME VEGETABLE SPECIES IN RELATION

TO CHILLING RESISTANCE

PHOSPHOLIPID SYNTHESIS IN GERMINATING SEEDS
OF SOME VEGETABLE SPECIES IN RELATION

TO CHILLING RESISTANCE

Introduction

 

Chilling injury during the early stages of imbibition
in germinating seeds of chilling sensitive species has been
reported in many cases (Obendorf and Hobbs, 1970; Pollock
and Toole, 1966; Pollock, 1969; Woodstock and Pollock,
1965; Greencia and Bramlage, 1971; Christianson, 1968).
This injury results in inhibition of germination or in
development of abnormal seedlings.

It has been shown, for many chilling sensitive
plants exposed to chilling temperatures, that lipids of
their cellular membranes are subject to a phase transition
from a liquid to a solid gel state. This phase change has
been correlated with a sudden increase of the activation
energies of some enzyme systems associated with these mem—
branes, resulting in the disruption of cell metabolism that

is followed by the appearance of chilling injury symptoms

13

14

(Towers §£_§l,, 1972 and 1973, Shneyour et_gl,, 1973;
Raison g£_al., 1971b; Lyons and Raison, 1970; Phillips
and McWilliam, 1971).

PhOSpholipids are considered one of the most impor-
tant lipid groups in biological membranes (Van Deene, 1965;
Chapman, 1967; Bishop, 1971). It has been shown, that
synthesized and natural phospholipids (Williams and Chapman,
1970; Chapman, 1967; Wade §£_al., 1974) exhibit a phase
transition from a liquid-crystalline to a crystalline state
at temperatures determined by, among other factors, the
degree of unsaturation of the hydrocarbon groups and the
nature of the polar group (Williams and Chapman, 1970;
Chapman, 1967).

In some plant species, the pattern of phospholipid
synthesis was found to be affected by the temperature of
growth or the degree of cold resistance (Kuiper, 1970;
Willemot, 1975; Thomson and Zalik, 1973). This suggests
that a relationship may exist between specific classes of
phospholipids and cold resistance. However, a clear rela-
tionship has not yet been established.

Lipid synthesis in many species has been observed
to start during the early hours of seed imbibition (H6121
and Wagner, 1971; Katayama and Funahashi, 1969; Shewry
gt_§1,, 1973; Macher §E_213, 1975; Nawa and Asahi, 1971;

Kagawa et al., 1973).

15

In this study an investigation was conducted on
phospholipid synthesis during the imbibition stage of
seed germination, in chilling resistant and chilling sensi-

tive species, at chilling or non-chilling temperatures. The
14

incorporation of C—glycerol into phospholipids during the
first 24 hours of seed imbibition was determined. The
incorporation of l4C—glycerol into phospholipids of germi-
nating seeds, hazel and soybeans, has been demonstrated
earlier by others (Stobart and Pinfield, 1970; H6121 and
Wagner, 1971).

The incorporation studies in this work were con-

ducted in germinating seeds of the chilling resistant

species Vicia faba, L., and Pisum sativum, L. as well as

 

 

of the chilling sensitive Phaseolus lunatus, L. Germina-

 

tion studies with lima beans (Pollock and Toole, 1966;
Wbodstock and Pollock, 1965) have shown that this species
is subject to chilling injury during the imbibition period
when the temperature is below 15°C. The chilling resis—
tance of the other two Species examined has been well
established (Robinson, 1968; Knott, 1962; Kotowski, 1926).
Germination studies conducted with lima beans, broad beans
and peas, at 10°C for‘a period of 25 days, revealed that
lima beans did not germinate at that temperature while the
other two species germinated and develOped normal seedlings
even though their growth was much slower at 10°C than at

25°C.

 

 

{C

16

Materials and Methods

Materials

 

Seeds of lima beans (Phaseolus lunatus, L., cv.

 

Henderson bush) and peas (Pisum sativum, L., cv. Little

 

Marvel) were purchased from Northrup, King & Co. (Fresno,

California) seeds of broad beans (Vicia faba cv. Broad

 

Windsor) were purchased from Stokes Seed Inc. (Buffalo,
N.Y.). The seeds used were about one year old and of high
germinability. Glycerol [14C(U)] was purchased from New
England Nuclear (Boston, Mass.). Precoated TLC plates
(20x20 cm, 0.250 mm) were purchased from Analtech, Inc.,
(Newark, Delaware). Lipid standards were purchased from
Applied Science Laboratories, Inc. (State College,
Pennsylvania) and Supelco, Inc. (Bellefonte, Pennsylvania).
Cab-O-Sil was purchased from Research Products International
Corp. (Elk Grove Village, Illinois). All organic solvents
used were distilled in glass and contained the antioxidant

BHT (butylated hydroxytoluene) at 0.1mg/l (Turner and

Rouser, 1970).

Methods

Seeds of lima beans, broad beans and peas were
surface sterilized with 1% NaOCl and imbibed for 24 hours
in the dark at 10°C or 25°C in sterilized water containing

14C-glycerol (121 or 133.4 mCi/m mole at 0.2 uCi/ml). The

D)

K)

In

l7

moisture content of the seeds was 10-11% on fresh weight
basis. Three groups of seeds from each species were treated.
Each group consisted of 20 seeds (lima beans and peas) and

12 seeds of broad beans. At the end of the imbibition, the
seeds were frozen in liquid nitrogen and 1yophilized, to
avoid enzymatic degradation of lipids (Haelzl and Wagner,
1966; Thomson and Zalik, 1973). The lyOphilized seeds were

stored at -30°C until lipid extraction.

Lipid Extraction. The 1yophilized seeds were

 

separated into axes and cotyledons and ground to form a
powder, using a glass homogenizer. The lipids were extracted
from the ground tissue according to the method of Folch
§E_al. (1957),by shaking with chloroform: methanol (C/M)
(2:1 v/v) for 30-40 minutes at 30-35°C. The C/M superna-
tant was decanted and filtered through Whatman No. 41
filter paper. The residue was re—extracted two more times
with C/M 2:1; the extracts were combined and washed with
0.2 volumes of 0.9% NaCl (Dawson ep_§l., 1969) and shaken
vigorously for two minutes. The mixture was allowed to
settle and the upper phase was discarded while the lower
one, containing the lipids was washed two more times with
Folch's mixture (chloroform: methanol:water, 3:48:47 v/v)
also containing 0.9% NaCl, to prevent partitioning of
phospholipids to the upper phase wash. After the second

washing, the lower, chloroform phase containing the lipids

18

was evaporated to dryness under nitrogen, and the lipids
were redissolved in chloroform for application to thin

layer chromatography plates.

Thin Layer Chromatography (TLC): (1) Separation of
phospholipids: The lipids extracted from the seeds were
applied on precoated TLC plates (20x20 cm), coated with
0.250 mm Silica gel G (E. Merck, Darmstadt) layers, follow—
ing activation of the layers at 110°C for 60 minutes. The
phospholipids were separated from other lipids and from
each other by 2 dimensional thin layer chromatography at
about 25°C. The plates were developed in the first direc-
tion in chloroform/methanol/28% NH4OH (65/25/4 v/v/v) and
after drying for about 15-20 minutes, they were developed in
the second direction in chloroform/methanol/acetic acid/
water (85/15/10/3 v/v/v/v).

(2) Detection of phospholipids on the TLC plates:
The chromatographed lipids were separated into the follow-
ing phospholipids, among other lipid classes: (1) Major
ones, containing more than 95% of the radioactivity counted
in phospholipids: PI, PC, PE and PG, and (2) minor ones:
PA, PS, CL and lysophosphatidyl choline (LPC). The phos-
pholipids were identified by comparison of their migration
on the TLC plates with that of pure standards and by using
the following chromogenic reagents (Skipski and Barclay,

1969):

19

(a) Dragendorff's reagent for PC and LPC.
(b) Ninhydrin reagent for PE and PS.
(c) Periodate-Schiff reagent for PI and PG.
(d) Molybdenum blue reagent for all phospholipids.
PI migrated close to PS and their separation
was not complete but all the other phospho-
lipids were completely separated.
(3) Measurement of radioactivity of phospholipids:
The silica gel of the TLC plate, containing the labeled
phospholipids, was scraped off the plate into scintilation
vials, the scintilation liquid added, and the vials were
shaken, for 4-5 hours to insure good distribution of the
labeled phospholipids throughout the scintilation liquid.
The scintilation liquid was made by mixing toluene with
Triton-X-lOO (2:1 v/v) and adding 49 of PPO (2,5—
diphenyloxazole) and 0.19 of dimethyl POPOP (1,4—bis
[2-(4-methyl-5-pheny10xazole)l-benzene) per liter of
toluene (Patterson and Greene, 1965). Also included in the
mixture was 3% Cab-O-Sil. The radioactivity of the phos-
pholipids was measured in a Beckman LS-100 Liquid Scintila-
tion Counter. The counting efficiency was about 95%.

Corrections for quenching were made.

Results

14

In this experiment the label of C-glycerol was

incorporated primarily into PI, PC, PE and PG, representing

20

more than 95% of the label distributed among all phospho-
lipid, with the remaining label in PA, PS, CL and LPG
(Appendix, Table A-4). The results will be presented as
percentages of the CPM recorded for each phospholipid

in relation to the total CPM of the four major phOSpho-

CPM for PI
CPM for (PI+PC+PE+PG)

 

lipids, i.e., x 100 = percentage PI.

The results for each phOSpholipid will be discussed
with reference to species, seed parts (axes or cotyledons)

and temperature treatments in that order.

Phosphatidyl Choline (PC)

 

(1) Significant differences in the percentage PC
between all three species at both temperatures were found
in axes and cotyledons (Table 1, 2). Lima beans, with the
exception of axis tissue at 25°C, had a significantly
lower percentage PC than broad beans and peas (Tables 1
and 2). With the exception of lima bean cotyledons at
10°C, the incorporation of 14C-glycerol into PC was higher
than in any other phospholipid (Tables 1 and 2).

(2) When axes and cotyledons are compared at 10°C
(Table 3) the cotyledons of broad beans and peas had a
higher percentage PC, while in lima beans the axes had the
higher percentage. At 25°C, broad bean cotyledons showed
a higher percentage PC than axes, peas had the same
percentage PC in both axes and cotyledons, while in lima

beans, the axes had the higher percentage PC.

21

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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comm no mcfiofiaonomono wcowm Ammwwpcoonmov mpa>fiuomoaomn mo cospsnfinpmfio .H edema

 

22

 

 

 

 

 

 

 

 

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mm mm om Hm
ooa A cause Hmpmm capes Amo.ov oo Amcoeoflspoov
cdmfiaonemonm mm\om .Q.m.q manpwanSmB mmfiomom

 

 

 

 

momentoonmo ofioaaonemonm

 

 

 

 

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.m

anwsoomazpoo no mofiefiaonomono mcoem Am pcmonmev zufi>auom0Homn mo coauonfinpmfia

.m mHDMB

23

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

.m.z mm.s .m.z mo.m so.H .m.z Amo.oV.o.m.q
mm.z mm.m Hm.om :w.o NH.N mm.m mw.m :m.: mm.m mcoomahuoo 0m
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mm.H .m.z m~.m .m.z No.0 o:.m Amo.ov.Q.m.A
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coaanHbEH ado: am m sebum mcfiofiaocomoca comm mcoEm Amowwpcmonoov zufl>fipomofiomn ho soapsnflppmfio .m magma

24

(3) In cotyledons and axes of lima beans the per—
centage PC was higher at 25°C than 25 10°C, while in
cotyledons of broad beans and peas no differences were
observed with temperature (Table 3). However, in pea axes
a higher percentage PC was found at 10°C while in broad
bean axes a higher percentage PC was observed at 25°C
than at 10°C (Table 3). It should be especially noted that
the difference in percentage PC, between 25°C and 10°C, was
quite large in lima beans (both axes and cotyledon) compared
to differences in these two tissues of broad beans and

peas (Table 3).

Phosphatidyl Ethanolamine (PE)

 

(l) Lima bean axes and cotyledons, at 10°C and
25°C, always had a higher percentage PE than the cold
resistant species (Tables 1 and 2). In the axes of all
species, at 10°C or 25°C, PE was the 2nd ranking phospho-
lipid and the same was observed in cotyledons with the
exception of lima beans at 10°C, where PE was the lst
ranking phospholipid (Table l).

(2) No consistent differences in percentage PE
between axes and cotyledons were detected in all three
Species (Table 3).

(3) A higher percentage PE was observed at 10°C
than at 25°C in lima bean axes and cotyledons as well as

in broad bean axes while no difference was observed in

25

peas (Table 3). In broad bean cotyledons more PE was syn-

thesized at 25°C.

Phosphatidyl Inositol (PI)

 

(1) Significant differences observed between species
in percentage PI of axis tissue, at 10°C and 25°C. Lima
bean axes had a lower percentage PI at both temperatures
than did axes from peas and broad beans (Table 1). Con-
sistent differences between species were not noted in
cotyledonary tissue (Table 2).

(2) Cotyledonary tissue of lima beans had a higher
percentage PI at both temperatures than axis tissue. Peas
and broad beans had the same percentage PI in cotyledons
and axes (Table 3).

(3) In all three Species, a higher percentage PI
was observed at 25°C than at 10°C, with the exception of
broad bean cotyledons which exhibited about the same amount

of PI at both temperatures (Table 3).

PhOSphatidyl Glycerol (PG)

 

(1) When species were compared, a significantly
higher percentage PG was observed in lima beans at 10°C,
in both axes and cotyledons (Tables 1 and 2). At 25°C no
differences were observed between species in axis tissue
while in cotyledons the highest percentage PG was observed

in peas and the lowest one in broad beans (Tables 1 and 2).

26

(2) There were no consistent differences in percen—
tage PG, in axes or cotyledons, between chilling sensitive
lima beans and chilling resistant peas and broad beans
(Table 3) .

(3) In both axes and cotyledons of broad beans and
lima beans a higher percentage PG was found at 10°C
than at 25°C. No differences occurred with temperature in
pea axes. A higher percentage PG was observed in pea
cotyledons at 25°C than at 10°C (Table 3).

Phosphatidyl Choline_/ Phosphatidyl
Ethanolamine Ratio

Because the transition temperature (To) of phOSpho-
lipids is affected by the nature of the polar group (PC
exhibits lower Tc than PE) and the fact that chilling sensi-
tive lima bean axes and cotyledons at 10°C had a lower
percentage PC and a higher percentage PE than chilling
resistant peas and broad beans (Tables 1 and 2) the PC/PE
ratio was investigated.

(1) Lima beans exhibited the lowest PC/PE ratio,
at 10°C and 25°C, in both axes and cotyledons compared to
broad beans and peas (Tables 1 and 2).

(2) When axes and cotyledons are compared (Table 4)
the axes of lima beans, at both temperatures, had the
higher ratio. In broad beans and peas, cotyledonary tissue

had a higher or same ratio as the axis tissue.

27

  
 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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losononm
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28

(3) In both axes and cotyledons of lima beans the
PC/PE ratio was higher at 25°C than at 10°C (Table 4). In
cotyledons and axes of peas the PC/PE ratio did not change
with temperature. In broad bean axis the PC/PE was higher
at 25°C than at 10°C but in cotyledons the ratio was
higher at 10°C.

Phospholipids as Percent of
Total Lipids

 

 

Since the quantity of phospholipids synthesized,
in relation to other lipid classes, may reflect membrane
building activity and because the growth of some plant
Species at low temperatures resulted in accumulation of
phospholipids (Kuiper, 1970; De La Roche §£_al,, 1972) the
percentage of phospholipids in relation to total lipids was
calculated. In this study the following were observed:

(1) No significant differences were observed among
Species in the relative quantities of phospholipids synthe-
sized (Tables 1 and 2).

(2) At 10°C, more phospholipids were synthesized in
cotyledons than axes of the chilling resistant species
(Table 4) while in lima beans no significant difference
was observed between seed parts. At 25°C, in all Species,
no difference was observed between seed parts.

(3) Some differences were observed between tempera-

tures but they were not consistent (Table 4).

29

Discussion

 

The d3.ppyp_synthesis of phOSpholipids is essential
for the production of new and for the repairing of old mem-
branes (Van Deenen, 1965). During seed germination phospho-
lipids are synthesized, presumably for use in the building
of cellular and organellar membranes in the seedlings
(Ching, 1972). The increase in mitochondrial phospholipids
observed in pea cotyledons during imbibition (Nawa and
Asahi, 1971) indicates synthesis of new or repair of old
membranes. In this experiment, phospholipids were labeled
in both axes and cotyledons of all species studied and it
is assumed that de_ppyp_synthesis of phospholipids
occurred since glycerol, that was used as the labeling
source, is believed to be a precursor for synthesis of
new phospholipid molecules (Mazliak, 1973). The results
Show that some of the phospholipids may be related to the
chilling resistance of the studied Species or to the
temperature at which the seeds germinated.

The observation that PC/PE ratio at 10°C, was lower
in the chilling sensitive lima beans than in the chilling
resistant broad beans and peas suggests that PC and PE may
be implicated in the determination of chilling resistance
or cold sensitivity in these species.

It is known (Chapman, 1967; Williams and Chapman,
1970) that PC changes phase from liquid-crystalline to

crystalline state at lower temperatures than PE and it has

30

been suggested that this may be important for the function
of biological membranes at different temperatures. Similar
information is not available for PI and PG, however their
capillary melting points are quite different: m.p. of PG
is 67°C and of PI is 136°C (Williams and Chapman, 1970).
The following are the most important conclusions from the
data of this study:

(1) A higher percentage of radioactivity was found
in PC in the chilling resistant species (broad beans and
peas) than in the chilling sensitive lima beans at 10°C,
especially in the cotyledonary tissue (Table 1). Further-
more, in lima beans, the percentage PC at 10°C was much
lower than at 25°C in both axes and cotyledons (Table 3).
This may suggest a shortage of available PC, at 10°C, for
the building of new membranes or the repair of old ones
leading to failure of germination and chilling injury. On
the other hand, no significant differences or small differ-
ences were obserfed in percentage PC between temperatures
in the chilling resistant Species (Table 3). The above
results suggest that PC may play an important role in the
expression of chilling resistance in the species studied.
The report concerning higher percentage PC in cold resis—
tant alfalfa varieties (Kuiper, 1970) agrees with the
results of this study of higher percent PC in chilling
resistant species. Furthermore, it has been reported
(Willemot, 1975) that more PC was synthesized, in wheat

seedlings, at low than at high temperatures.

31

(2) Lima beans at 10°C and 25°C, always had a
higher percentage PE than the chilling resistant species
(Tables 1 and 2). In addition lima beans had a higher per-
centage PE at 10°C than at 25°C, while no differences or
much smaller differences were observed with change in
temperature in the chilling resistant species (Table 3).
Combining the results of a higher percentage PE and a lower
percentage PC observed in lima beans at 10°C with the fact
that PC has a lower Tc than PE, it may be suggested that
this chilling sensitive Species cannot maintain a proper
PC/PE ratio at chilling temperatures. Consequently, its
cellular membranes cannot maintain the liquid crystalline
phase that is necessary for biological function at low
temperatures.

Our results do not agree with the results reported
for alfalfa (Kuiper, 1970) where cold resistant varieties
exhibited higher PE than cold sensitive ones at low tempera-
ture.

(3) A greater percentage PI was observed at 25°C
than at 10°C, in most of the cases, suggesting that
apparently more PI is synthesized at higher temperature.
These results agree with those reported for alfalfa plants
(Kuiper, 1970) and rye seedlings (Thomson and Zalik, 1973).

(4) Lima beans exhibit a very large percentage PG
at 10°C in both axes and cotyledons compared to the chill-

ing resistant broad beans and peas (Table 3). At 25°C,

32

there were no large differences between species. This
suggests that in the chilling sensitive Species (lima beans)
there is a shift of metabolism which results in a large
amount of PG at the expense, apparently, of PC at 10°C.
This large shift in percentage of individual thSpholipidS
do not appear to occur in the chilling resistant species
(broad beans and peas). The above results suggest that at
chilling temperatures the chilling resistant species are
able to maintain a balanced phospholipid metabolism
required for their growth and development while chilling
sensitive species are not able to do so and consequently
are subject to chilling injury. The higher percentage of
PG observed at 10°C than at 25°C apparently does not agree
with the report on alfalfa plants (Kuiper, 1970) where it
was shown that more PG was accumulated at high temperatures
than at low ones. The differences observed between axes
and cotyledons can be explained, at least partially, by the
fact that cotyledons represent an aging tissue while axes
represent an actively growing one. Differences in the
pattern of phospholipid synthesis between axes and cotyledons
have been reported in mung beans (Katayama and Funahashi,
1969) during the incorporation of 32P into lipids.

The possibility that the observed preferential
synthesis of some of the phospholipids in cold resistant
species or at different temperatures may simply reflect

faster turnover of some phospholipids over others at the

33

temperatures and the species studied, cannot be ignored. It
should be mentioned that when E. 99;; cells (Okuyama, 1969)
were transferred from 37°C to 10°C, a faster turnover for

PE was observed. The phospholipids exhibit various turnover
rates in different cellular fractions of the same plant
tissue (Mazliak, 1973). The measured "half-lives" in

plants varied from 3 to 32 days. Since the incubation
period, in this study, was 24 hours and the seeds were
analyzed at the end of the incubation period it is assumed
that the turnover effect on the data presented here was
minimal. The possibility of a differential effect of some
temperature treatments on the turnover rates of certain
phospholipids could cause changes in phospholipid content
without having a cause and effect on cold sensitivity in

the plants involved. Furthermore, the possibility of an
enzymatic breakdown of the phospholipids during the lipid
extraction in this experiment should not be ignored. It

has been reported (De La Roche §£_§E,, 1973, Quarles and
Dawson, 1969), that phospholipase D may cause artifacts
during lipid extraction from plant tissues containing water,
by breaking phospholipids, and especially PC, into PA and
the alcohol moiety of the molecule. By freezing with
liquid nitrogen and immediately lyophilizing the plant
tissues in this experiment, it is assumed, that the enzy-
matic breakdown of phospholipids was prevented or at least

was minimized to a great degree. In addition, the absence

34

of substantial quantities of PA in the lipid extracts may
narrow the possibility of artifacts because of enzymatic
breakdown.

Glycerol pool size differences, could contribute to
differences observed in labeling of lipids among different
species. Pool size differences Should be reflected in the
total amount of lipids labeled. However, if we assume that
the provided labeled glycerol molecules had the same chance
to enter phospholipid synthesis as the ones of the cell
pool, then, the distribution of the label among the newly
synthesized phospholipids (reflected in the percentages of
phospholipids discussed here) Should not be affected by

differences in pool size among the species.

SECTION II

PHOSPHOLIPID SYNTHESIS IN SEEDLINGS OF SOME
VEGETABLE SPECIES IN RELATION TO

CHILLING RESISTANCE

PHOSPHOLIPID SYNTHESIS IN SEEDLINGS OF SOME
VEGETABLE SPECIES IN RELATION TO

CHILLING RESISTANCE

Introduction

 

Chilling sensitive plants are injured by chilling
temperatures, 0-10°C (Lyons, 1973), but they can be
hardened, at least partially, against chilling injury by
conditioning them for relatively short periods of time at
temperatures slightly above the chilling temperatures
(Wheaton and Morris, 1967; Wilson and Crawford, 1974).

Studies, presented in Section I, indicated marked
differences in the pattern of phospholipid synthesis
according to chilling sensitivity of the species studied
and the temperature during seed germination.

In this study, plants of chilling resistant (broad
beans, peas, lettuce, beets and cabbage) and of chilling
sensitive (lima beans, cucumber and watermelon) species, in
the seedling stage, were employed to gain further information
regarding phospholipid synthesis in relation to chilling
sensitivity and the development of chilling resistance. The
degree of chilling resistance of the species used has been
well established (Robinson, 1968; Knott, 1962; Kotowski,

1926).

35

36

Materials and Methods

 

Materials

 

The seeds used were purchased from Joseph Harris
Seed Co., Rochester, N.Y. (beet, cabbage, watermelon,
cucumber, lettuce), Northrup, King & Co., Fresno, Califor-
nia (lima bean and pea) and Stokes Seed Co., Buffalo, N.Y.
(broad bean).

All the other materials used were the same as in
the germination experiments (Section I). The following
species and cultivars were used:

1. Beet (Beta vulgaris, L., cv. Early Wonder)

 

2. Broad bean (Vicia faba, L., cv. Broad Windsor)

 

3. Cabbage (B£E§Sica oleracea, L., var. capitata

 

cv. Market Price)

4. Cucumber (Cucumis sativus, L., cv. Wisconsin

 

SMR 8)

5. Lettuce (Lactuca sativa, L., cv. Ruby)

 

6. Lima bean (Phaseolus lunatus, L., cv.

 

Henderson Bush)

7. Peas (Pisug_sativum, L., cv. Little Marvel)

 

8. Watermelon [Citrulus lanaEus, (Thunberg)

 

Matsumura and Nakai, cv. Charleston Gray].
Methods

The seeds were germinated and the seedlings grown

in moist vermiculite, in the dark, at 10° (the chilling

37

resistant species) and at 25°C (both chilling resistant
and chilling sensitive Species) for a designated number of

days before they were incubated with 14

C-glycerol. Since
the chilling sensitive species do not germinate and grow at
10°C there were no treatments of continuous 10°C for these
species. For the temperature conditioning (cold hardening)
experiment the seedlings were grown for a number of days at
25°C and then transferred to 10°C where they remained for

3 days before they were incubated with l4C-glycerol. The
seedlings grown at 10°C and the seedlings of the tempera-
ture conditioning treatment were healthy and did not Show
any chilling injury symptom at the time of incubation with
l4C-glycerol.

Within each species the morphological stage of
plants treated at 10°C or 25°C or treated after cold harden-
ing was uniform. Each treatment was applied in 3 groups of
plants for each species. Details of the characteristics of
the treated plants as well as the period of time the
several temperature treatments were applied before incuba—
tion in glycerol are shown in Table l. The hypocotyls or
epicotyles of plants were cut near the radical and they
were soaked for 24 hours in sterilized water containing
14C-glycerol (7.4 mCi/m mole at 0.5 uCi/ml water. Cucumber,
especially, was treated with 14C-glycerol of 121 or 133.4
mCi/m mole). At the end of the 24 hour incubation period

the plants were frozen in liquid nitrogen and 1y0philized

to avoid enzymatic degradation of lipids (Hoelzl and

38

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

~H aHINH m » ll II II NH :HINH m coaoEHouoz
NH aalma m » ll ll ll NH zHINH HH monsooso
0H maloa m M II II II 0H maloa m mcwmm mafia
om mum m m om mlm Hm om mlm Ha mpmom
om mum m m om mlm Hm om mlm HH mwmnnwo
om mum m m om mum Hm om mum HH mosuumq
0H ml» m m 0H ml» ma 0H ml» m mwmm
0H :HINH m m II II II 0H zHlNH m mcwom cmonm

coauMO Eo ucoEpwoLp coca on cognac So pcospmohp coupmo So pcoauwohu

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pcwam ucmflm um ammo pa nzmm mucmam pcmam we when musmam ucwam pm when
o.OH+T0.mm mascoapfiocoo engagemQEme QOOH comm

.Honmomawl02H Spa: omumb50cH mwcflaoomm mo moapmfipmpomhmso .H magma

 

39

Wagner, 1966; Thomson and Zalik, 1973). The lyophylized
plants were stored at -30°C until lipid extraction.

The methods for lipid extraction, thin layer
chromatography and radioactivity counting were the same as

in Section I.
Results

In this study, lipids generally and phospholipids
especially were labeled heavily after a 24 hour incubation
in 14C-glycerol. The label was found primarily in PA, PI,
PC, PE and PG.* Very little label was detected in PS,

LPC and C1, adding up to about 2% of the radioactivity
counted in the labeled phospholipids (Appendix, Table A-S).
It has been reported (H6121 and Wagner, 1971; Stobart and

Pinfield, 1970) that 1‘

C-glycerol is incorporated, 1p.yiyp,
into several plant phospholipids.

The results are presented as percentages of the CPM
recorded for each phospholipid in relation to the total
CPM recorded for PI, PC, PE and PG (the major phOSpholipids
in these plants). Phosphatidic acid was not included in
the calculations of the percentages of the major phospho-

lipids because PA is considered to be a general intermediate

in glyceride synthesis (Van Deenen, 1965; Bishop, 1971;

 

*EE: Phosphatidic Acid; EE: PhOSphatidyl Choline;
PE: Phosphatidyl Ethanolamine; El; Phosphatidyl Inositol;
: Phosphatidyl Glycerol; EE: Phosphatidyl Serine;
LPC: Lyso-Phosphatidyl Choline; EE: Cardiolipin (Dipho-

sphatidyl Glycerol).

40

Kates, 1970; Mazliak, 1973; Macher gE_§E., 1975). The
results for each phospholipid will be discussed with
reference to species and temperature treatments.

The calculations of the percentage of each phospho-

lipid have been made as follows:

CPM for PI
CPM for (PI+PC+PE+PG)

 

e.g.: x 100 = percentage PI

Phosphatidyl Choline (PC)

 

(1) In the chilling resistant species, with some
exceptions, the percentage PC was the highest among the
percentages of other phospholipids at 10°C, and was ranked
first or second at 25°C and after plants were transferred
from 25°C to 10°C (Table 2).

(2) Continuous exposure to 10°C and transfer from
25°C to 10°C generally caused an increased in percentage PC
in chilling resistant Species compared to the continuous
25°C treatment (Table 3). In chilling sensitive species,
transfer from 25°C to 10°C caused an increase in percen-

tage PC compared to the 25°C treatment.

Phosphatidyl Ethanolamine (PE)

 

(1) In most cases, the percentage PE was the highest
or second highest among the percentages of phospholipids in
seedlings at 25°C. There was no correlation between percen-
tage PE and the degree of chilling resistance of species

(Table 2).

41

 

 

 

 

 

 

 

 

 

 

Table 2. Distribution of radioactivity (percentages) among
phospholipids of fieedlings after a 24 hour
incubation with 1 C-glycerol at 10°C or 25°C.
Comparison of individual phospholipids.

S 1 Tempera- Phospholipid Percentages L.S.D.
pec es ture °c PI PC PE PG (0.05)
Broad Beans 10°C -- -— _- -- __
25—a10°C 13.37 58.46 10.30 17.84 1.25
25°C 14.49 52.81 21.73 10.93 2.57
Peas 10°C 20.63 40.40 17.40 21.43 5.41
25-plO°C 21.22 45.07 12.83 20.86 3.04
25°C 17.11 43.98 23.75 13.79 1.74
Lettuce 10°C 12.66 39.09 26.19 22.03 3.38
25-910°C 10.48 45.30 24.20 19.98 1.86
25°C 23.81 18.84 42.85 14.47 7.28
Beets 10°C 10.87 23.42 41.59 24.08 5.79
25-910°C 14.58 32.13 31.02 22.24 2.78
25°C 13.21 30.65 39.60 16.50 4.13
Cabbage 10°C 10.65 34.80 29.15 25.36 2.30
25—S10°C 17.09 32.19 27.83 22.93 3.02
25°C 10.82 28.84 39.27 21.04 5.57
Lima Beans 10°C -- -- -- —- --
25—>10°C 24.01 49.71 14.40 11.85 2.36
25°C 21.80 38.07 25.62 14.47 2.66
Cucumber 10°C -- —— __ _- _-
25->10°C 22.27 42.48 19.57 15.65 1.75
25°C 6.37 29.13 53.70 10.77 2.69
Watermelon 10°C -- -- —_ __ _-
25-s10°C 20.92 40.27 24.61 14.18 4.79
25°C 20.51 33.82 34.69 10.96 3.40

 

 

42

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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»num II II II mH.mm mm.Hc 0H.0N 0:.»H ll 0H mm

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=N.0H II II II 0m.=m ~=.mm 00.0m 02.0: ll 0H 0m

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6.9a 2.33:8 9:320 95838 8:35 -8889 .2825
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coaumDSocH ado: :m m amuwm mwcfiacmmm mo mcfioaHOSQmoso wcoEm Antwancmonoov mufi>fipom0Homn mo coHpSManmHQ

.m manme

 

43

(2) Generally, a higher percentage PE was observed
at 25°C than at 10°C in all chilling resistant species,
except beets. Chilling sensitive species exhibited much
lower percentage PE when transferred from 25°C to 10°C

than at 25°C continuously (Table 3).

Phosphatidyl Inositol (PI)

 

(1) Although significant differences observed in
percentage PI between species at 25°C and when transferred
from 25°C to 10°C, no consistent differences occurred
between chilling sensitive and chilling resistant Species
(Table 3) .

(2) Among the temperature conditioned plants the
percentage PI was generally higher in chilling sensitive

species with the exception of peas (Table 3).

Phogphatidyljglyperol (PG)

 

(l) A higher percentage PG was observed in chilling
resistant species at 10°C than at 25°C with the exception
of cabbage (Table 3).

(2) The temperature conditioning treatment (transfer
from 25°C to 10°C) resulted in an increase of percentage PG
in comparison to 25°C in most of the Species (Table 3).
Among the chilling hardened plants, most chilling resistant
Species had higher percentage PG than the chilling sensi-
tive ones (Table 3) and PG was a higher ranking phospholipid

in chilling resistant species (Table 2).

44

Phosphatidyl Choline / Phosphatidyl
Ethanolamine Ratio

 

(1) In general, a higher PC/PE ratio was found at
10°C than at 25°C in the chilling resistant species
(Table 4).

(2) The temperature conditioning treatment (25°C+
10°C) resulted in an increase of the PC/PE ratio in com-
parison to the one at 25°C, in all chilling resistant
and sensitive species (Table 4).

Phospholipids as Percent of
Total Lipids*

 

 

It has been reported that during the development of
cold hardiness (Siminovitch §E_3E,, 1968; Yoshida and
Sakai, 1973) or growth at low temperatures (Kuiper, 1970;
De La Roche §E_El°' 1972), some plant species accumulate
increasing quantities of phospholipids. The radioactivity
incorporated in all phospholipids in relation to the radio-
activity incorporated in all lipids was calculated with the
following results:

(1) In chilling resistant species no significant or
consistent differences were observed between 10°C and 25°C
in total quantities of phospholipids synthesized in rela-

tion to total lipids (Table 4).

 

*PA was not included in the calculations.

45

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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as I I I :34 2.00 mg; Ems I 2 W1.
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0
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46

(2) The degree of chilling resistance in species
was not correlated to the total phospholipids synthesized
(Table 4).

(3) In most of the treated species the chilling
hardening caused few alterations in the total amount of
phospholipids synthesized in comparison to the 25°C
treatment.

Phosphatidic Acid as Percent
of Total Phospholipid

 

 

The phosphatidic acid labeled in comparison to the
labeled total lipids showed no consistent relationships to
the degree of chilling resistance of the Species, the
temperature of growth or the temperature conditioning

treatment (Table 4).

Discussion

 

The Eg_ppyg synthesis of phospholipids is believed
to be essential for the building of new cell and subcellular
membranes as well as for the maintenance of the existing
membranes (Van Deenen, 1965).

In our labeling studies, with a variety of chilling
sensitive and chilling resistant vegetable species, lipids
and especially phospholipids were labeled heavily over an
incubation period of 24 hours.

It has been reported (Chapman, 1967; Williams and '

Chapman, 1970) that phospholipids, natural or artificial,

ir
re
if

PC

re

Id‘

of
shi
to

in

Fla;
trdI

Cont

POSS
COHt

Sens

47

change phase from a liquid crystalline to a crystalline
state at temperatures much lower than their melting points.
This change may be of importance Since phospholipids are
major components of membranes. According to those studies
PC changes phase at a much lower temperature than PE
(Appendix, Table A-3). This difference could be important
in relation to the function of membranes of chilling
resistant and chilling sensitive species at low temperatures
if the membranes contain differing amounts or ratios of

PC and PE.

More PC has been found at low temperatures in cold
resistant alfalfa cultivars than in cold sensitive ones
(Kuiper, 1970) as well as in wheat seedlings grown at low
rather than high temperatures (Willemot, 1975).

The results of this study indicate that exposure
of chilling resistant plants to 10°C continuously, or
shifting chilling resistant and sensitive plants from 25°C
to 10°C generally causes an increase in the percentage PC
in comparison with the continuous 25°C treatment (Table 3).
A similar difference in percentage PG was also noted when
plants of chilling resistant species grown at 10°C or
transferred from 25°C to 10°C compared to plants grown
continuously at 25°C (Table 3).

These results suggest that an increase in PC and
possibly PG upon exposure to low temperatures could be
contributing to hardening of both chilling resistant and

sensitive Species. Presumably, the higher PC and PG

48

percentages would allow the membranes to maintain a liquid-
crystalline phase at 10°C and normal functioning could
occur. In contrast, the percentage PE in chilling resistant
species was lower when the seedlings were grown at 10°C

than at 25°C. In addition, conditioning plants, by trans-
ferring them from 25°C to 10°C, resulted in a decrease in
percentage PE in both chilling resistant and sensitive
Species compared to those plants grown at 25°C continuously.

PI was found to be a higher ranking phospholipid
in chilling sensitive Species than in chilling resistant
ones when temperature conditioned plants were compared
(Table 2). Chilling sensitive species had a higher percen-
tage of PI than the majority of chilling resistant ones
under lower temperature conditions (Table 3). PI may play
some role in chilling sensitivity.

The percentage PE appears to decrease and the
percentage PC increases when chilling sensitive and chill—
ing resistant plants are transferred from 25°C to 10°C.

This suggests that the low temperature causes a shift in
metabolism of phospholipids towards PC at the expense of

PE. This may contribute to the synthesis of membranes

which will remain flexible and will function at low tempera—
ture.

This Shift is reflected in the PC/PE ratio comparing
seedlings exposed to 25°C or grown at 25°C and transferred
to 10°C. Exposure to low temperature results in an

increase in the PC/PE ratio.

49

There appears to be a basic difference in how chill—
ing sensitive lima beans respond to low temperature treat-
ment (in terms of phospholipid synthesis) between the
initial imbibition stage of germination and the seedling
stage. It was shown earlier (Section I) that lima beans
synthesized less PC and more PE at 10°C than at 25°C during
the seed imbibition stage. Large changes in PC and PE
synthesis did not occur in broad beans or peas when imbibed
at 10°C or 25°C.

In addition, lima beans exhibited a significantly
lower percentage PC and a higher percentage PE at 10°C than
broad beans and peas. It is known that lima beans are
extremely sensitive to chilling injury during this early
imbibition stage (Pollock and Toole, 1966; Woodstock and
Pollock, 1965).

In the seedling Stage both the chilling resistant
species (broad beans and peas) and the chilling sensitive
one (lima beans) synthesized more PC and less PE after they
were transferred from 25°C to 10°C than at 25°C. Lima
beans are less sensitive to cold temperatures after the
initial stages of imbibition have passed (Pollock and
Toole, 1966).

This information suggests that lima beans in the
imbibition stage are not capable of shifting metabolism
to produce the type of phospholipids necessary at low
temperature for proper maintenance of cellular integrity,

and chilling injury and death of the seed occurs. Lima

50

beans (as well as cucumber and watermelon) in the seedling
stage appear to have the capability, at low temperatures,
of Shifting metabolism to higher PC and lower PE levels.
This is similar to the chilling resistant species. Some
degree of hardening apparently occurs that protects the
chilling sensitive plant from chilling injury, as occurs
in the cold resistant species. In this experiment, the
temperature conditioned chilling sensitive plants were
observed to stay alive, but with a small growth rate, for
about 30 days at 10°C. This may indicate that, in addition
to a proper phospholipid metabolism, some other factors
determine growth at chilling temperatures.

However the possibility that a 24 hour phospholipid
synthesis may not represent the phospholipid synthesis
mechanism in the studied plants at different temperatures
should not be ignored and therefore any conclusions should
be made cautiously.

The possibility also exists that our data have been
affected by a differential effect of temperature on the
turnover rates of the several phOSpholipid classes result-
ing in a picture that may have nothing to do with the
mechanism of chilling resistance (see discussion in Section
I). Furthermore, there is a possibility that an artifact,
because of enzymatic degradation of lipids during extrac—
tion, altered the constituents of the analyzed lipid

mixture. In fact it has been reported (De La Roche et al.,

51

1973; Quarles and Dawson, 1969) that phOSpholipase D can
cause artifacts during lipid extraction, in the presence
of water, by breaking phospholipids, especially PC, into
PA and the alcoholic moiety of the molecule.

It is assumed that the possibility of an enzymatic
degradation of phospholipids during the procedure of lipid
extraction from the tissues was minimized by the freezing
in liquid nitrogen and lyophilization of the plants (Hoelzl

and Wagner, 1966; Thomson and Zalik, 1973).

The fact that PA was heavily labeled in the extracted
lipids (Table 4) may support the idea of an artifact because
of an enzymatic breakdown of phospholipids by phospholipase
D. But since PA is a precursor in glyceride synthesis
(Kates, 1970; Mazliak, 1973; Macher §E_§l,, 1975) we suggest
that the labeled PA was synthesized §§_ppyp_from l4C—glycerol
as an intermediate step for lipid synthesis. A high PA
concentration has been reported in other cases (De La Roche
23.213! 1973; Wilson and Rinne, 1974) and it was explained
as synthesized §g_ppy9,

The comparison of the pattern of lipid synthesis of
different Species may be subject to an error because of
differences in pool Size of glycerol as it has been dis-

cussed earlier (Section I).

SECTION III

FATTY ACID CONTENT OF PHOSPHOLIPIDS IN GERMINATING
SEEDS OF SOME CHILLING RESISTANT AND CHILLING

SENSITIVE VEGETABLE SPECIES

FATTY ACID CONTENT OF PHOSPHOLIPIDS IN GERMINATING
SEEDS OF SOME CHILLING RESISTANT AND CHILLING

SENSITIVE VEGETABLE SPECIES

Introduction

 

Chilling injury during the early stages of seed
imbibition in chilling sensitive Species has been Shown to
cause failure of germination or abnormal seedlings (Obendorf
and Hobbs, 1970; Pollock and Toole, 1966; Pollock, 1969;
Woodstock and Pollock, 1965; Christianson, 1968). Chilling
sensitivity and chilling injury have been correlated to a
phase change occurring in lipids of cellular membranes of
chilling sensitive plants at chilling temperatures, 0—120c,
(Lyons and Raison, 1970; Raison g£_§l,, 1971b; Shneyour
EE_El'v 1973; Towers §E_§l,, 1972; Towers gE_§E,, 1973;
Yamaki and Uritani, 1974; Phillips and McWilliam, 1971).
Such a phase change of lipids occurs at much lower tempera-
tures in chilling resistant plants.

Membrane lipids of chilling sensitive plants have
been found to contain less unsaturated fatty acids than
those of chilling resistant plants (Lyons EE_§E., 1964).

In addition, membrane lipids of micro—organisms and plants

52

53

grown at high temperatures contain less unsaturated fatty
acids than those from organisms grown at low temperatures
(Haest EEE313, 1969; Miller §E_El3' 1974).

The higher the unsaturation of a fatty acid the
lower its melting point (Appendix, Table A-2). It has been
suggested that the correct fluidity of biological membranes,
for function at a particular temperature, is provided by
the distribution in the membrane lipids of fatty acids with
the proper chain length and degree of unsaturation (Chapman,
1967 and 1968; Cronan and Vagelos, 1972).

Phospholipids, which are important cell membrane
components (Bishop, 1971; Chapman, 1967; Van Deenen, 1965)
have been found to be more unsaturated when growth occurs
at low temperatures, in some micro-organisms (Kaneda, 1972;
Cronan and Vagelos, 1972; Kates and Paradis, 1973) alfalfa
(Grenier and Willemot, 1974) and wheat plants (De La Roche
§E_§E., 1972). Cold resistant alfalfa varieties also had
more unsaturated phospholipids than did sensitive ones
(Grenier and Willemot, 1974).

Since cellular lipids apparently are involved in the
cold sensitivity of plants, knowledge of the distribution of
the fatty acids in phospholipids of germinating seeds of
chilling sensitive and chilling resistant plant species may
contribute to an understanding of the chilling injury
mechanism. Chilling sensitive lima beans and chilling

resistant broad beans and peas were employed to investigate

54

the relationship of phospholipid fatty acid composition to

chilling sensitivity.

Materials and Methods

 

Materials

 

Seeds and other materials described earlier (Section
I) were also used for the study of fatty acids in phospho-

lipids.

Methods

Seeds of lima beans, broad beans and peas were sur-
face sterilized with 1 percent NaOCl and imbibed for 24
hours in distilled water at 10°C or 25°C in the dark. At
the end of the imbibition period the seeds were frozen in
liquid nitrogen and lyophilized before lipid extraction.
Other groups of seeds were maintained at 10°C, in moist
vermiculite in the dark, for 6 days before they were
analyzed for fatty acid content of phospholipids.

Methods for lipid extraction and separation of
phospholipids by thin layer chromatography were described
earlier (Section I).

The phospholipids were located on the thin layer
plates by spraying with 2',7'-dichloro fluorescein and

observation under UV light (Mackender and Leech, 1974).

55

Fatty Acid Analysis. The fatty acids of phosphatidyl

 

inositol (PI) phosphatidyl ethanolamine (PE) and phosphatidyl
choline (PC) were determined.

The phospholipid areas were scraped from the thin
layer plates and the lipids were eluted from the silica gel
using chloroform: methanol: water (10:10:0.5 v/v/v). The
methyl esters of the fatty acids of the phospholipids were
prepared using BF3—methanol according to Metcalfe §E_§l3,
l966;after saponification of the phosphalipids with 0.5N
NaOH in methanol the free fatty acids were methylated with
BF3-methanol (14% w/v). The methyl esters of the fatty

acids were removed from the reaction medium by partitioning

in hexane for analysis by gas liquid chromatography (GLC).

GLC Analysis. The fatty acid composition of the

 

methyl esters was determined using a Packard 7300 gas-
liquid chromatograph equipped with a flame ionization
detector. A 1.80M, 3mm I.D. glass column packed with 10%
SP-222 PS (brand name of Supelco, Inc. for diethylene
glycol succinate) was employed at a temperature of 190°C.
The temperature of the injector and the detector was 220°C
and the flow rate of the nitroqen carrier gas was 50 ml/min.
Authentic standards were used for the identifica-
tion of the methyl ester peaks in the chromatoqrams. The
major fatty acids (representing more than 90% of the total

fatty acids) were identified as 16:0, 18:0, 18:1, 18:2

56

and 18:3* (the lst number indicating the number of carbon
atoms and the second one the number of double bonds in the
molecule).

The relative concentration of each fatty acid was
calculated by triangulation of the peak areas on the chroma-
tograms (corrections were made for detector sensitivity) and

was expressed as percentage of the total peak area

. 16:0 _ .
e'9" l6:0+18:0+18:1+18:2+18:3 X 100 ” percentage 16°°

Other calculations made were:

 

 

 

(1) Unsaturated ratio = 18:l+l8:2+18:3
Saturated 16:0+l8:0
(2) Double Bond = Q of 18:1)x1+(% of 18:2)x2+(% of 18:3)x3
Index (D.B.I.) 100
Results

Content of Fatty Acids in
Phosphatidyl Choline

 

(1) Species Differences. Considerable differences
in percentage of individual fatty acids were observed between
species during both imbibition and after 6 days of germina-

tion.

 

*16:0: Palmitic; 18:0: Stearic; 18:1: Oleic; 18:2:
Linoleic; 18:3: Linolenic.

(a)

(b)

(C)

(d)

(e)

57

Lima beans exhibited a considerably higher percen-
tage 16:0 in both axes and cotyledons during the
imbibition stage (Table 1) and in cotyledons on
the 6th day of germination (Table 2) than did the
chilling resistant Species.

The percentage 18:1 was much higher in peas and
broad beans (cotyledons and axes) than in lima
beans during imbibition (Table 1). However the
percentage 18:1 on the 6th day was different from
the imbibition stage. On the 6th day the percen-
tage 18:1 in broad bean and pea axes was much lower
than in lima beans while the cotyledons of peas
and broad beans had the highest percentage 18:1
(Table 3).

The Situation for 18:2 of cotyledons and axes was
mixed, with lima beans showing a higher percentage
in cotyledons during imbibition (Table l) and a
lower percentage in axes on the 6th day (Table 3).
No other consistent relationship occurred.

Lima beans, in all cases, had a much higher percen-
tage 18:3 than the chilling resistant species
(Tables 1 and 3).

The double bond index (D.B.I.) of lima beans was
larger than the chilling resistant Species during
imbibition (Table 3)4at 10°C and 25°C. However,
on the 6th day, in cotyledons, lima beans had the

lowest D.B.I. (Table 3).

58

 

 

 

 

 

 

 

 

 

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60

 

 

 

 

 

 

 

 

Table 3. Percentages of fatty acids in phospholipids (PC, PE, PI)
after 6 days of germination at 10°C. Comparison of species.
Phospho— Species Seed Fatty Adds D3223: Unsat.
lipid pert 16:0 18:0 18:1 18:2 18:3 Index sat°
PC Broad Beans Axes 11.60 1.81 3.53 71.01 12.00 1.81 6.67
PC Peas Axes 16.48 3.04 4.93 61.48 14.04 1.70 4.12
PC Lima Beans Axes 12.69 3.17 19.13 43.41 21.54 1.70 5.42
L.S.D.(0.05) 2.83 N.S 1.38 4.84 5.30 0.09 1.60
PC Broad Beans Cotyl. 9.79 1.82 33.11 52.91 2.34 2.92 7.72
PC Peas Cotyl. 7.78 5.29 22.65 58.93 5.30 3.03 6.70
PC Lima Beans Cotyl. 22.57 3.20 7.26 53.32 10.02 1.44 2.81
L.S.D.(0.05) 3.85 1.22 6.81 N.S. 1.15 0.294 1.85
PE Broad Beans Axes 18.59 2.92 2.98 64.84 13.15 1.72 3.99
PE Peas Axes 22.43 3.07 3.33 55.74 15.16 1.61 2.97
PE Lima Beans Axes 25.12 23.3 15.81 40.53 16.04 1.45 2.63
L.S.D.(0.05) N.S. N.S 1.32 13.61 N.S. N.S. N.S.
PE Broad Beans Cotyl. 13.59 2.71 29.59 51.75 2.31 1.40 5.20
PE Peas Cotyl. 15.35 5.64 16.54 58.45 3.97 1.45 3.79
PE Lima Beans Cotyl. 27.90 2.23 5.78 55.22 8.83 1.42 2.34
L.S.D.(0.05) 4.93 0.99 5.87 N.S. 1.23 N.S. 1.17
PI Broad Beans Axes 37.68 3.45 2.93 46.69 9.20 1.24 1.44
PI Peas Axes 32.13 4.24 4.29 42.40 16.95 1.40 1.75
PI Lima Beans Axes 30.18 5.55 10.75 31.12 22.37 1.40 1.80
L.S.D.(0.05) N.S. 0.80 1.23 3.64 3.22 0.10 N.S.
PI Broad Beans Cotyl. 32.95 3.57 16.81 45.27 1.36 1.11 1.75
PI Peas Cotyl. 34.74 7.81 12.79 39.81 4.81 1.07 1.35
PI Lima Beans Cotyl. 36.31 5.86 4.91 42.13 10.74 1.21 1.42
L.S.D.(0.05) N.S. 1.36 3.13 N.S. 1.56 N.S. N.S.

 

61

(f) During imbibition in both axes and cotyledons, and
in cotyledons only on the 6th day, peas and broad
beans had a higher ratio of unsaturated to saturated

fatty acids than lima beans (Tables 1 and 3).

(2) Cotyledons vs. Axes. When cotyledons and axes
were compared there were differences in percentage of
individual fatty acids in the imbibition stage but no con-
sistent trends were evident in both chilling resistant and
sensitive species (Table 2). For example, the percentage
18:1 was higher in cotyledons than axes of broad beans and
lima beans while the opposite was true in peas.

After 6 days of germination at 10°C (Table 4) the
following were observed:

(a) The cotyledons of the chilling resistant species
had a much higher percentage 18:1 than the axes
while the opposite was true in lima beans.

(b) The percentage 18:3 was higher in the axes than
in cotyledons, in all species.

(c) The D.B.I. was larger in cotyledons in comparison
to axes of the chilling resistant plants but in
lima beans the opposite was true.

(d) The cotyledons of chilling resistant species
had the same or higher unsaturated/saturated
ratio than the corresponding axes while the axis
tissue of lima beans had a higher ratio than the

cotyledonary tissue.

62

 

 

 

 

 

 

 

 

 

 

 

 

Table 4. Percentages of fatty acids in phospholipids (PC, PE, PI)
after 6 days of germination at 10°C. Comparison of seed parts.
‘Phospho— Species Seed Fatty A°1ds ”Eggée Unsat.
lipid Part 16:0 18:0 18:1 18:2 18:3 Index sat“
Axes 11.60 1.81 3.53 71.01 12.00 1.81 6.66
PC Broad Beans Cotyl. 9.79 1.82 33.11 52.91 2.34 2.92 7.72
L.S.D.(0.05) N.S. N.S. 7.94 9.09 2.22 0.257 N.S
PC Pea Axes 16.48 3.04 4.93 61.48 14 04 1.70 4.12
S Cotyl. 7.78 5.29 22.65 58.93 5.30 3.03 6.70
L.S.D.(0.05) 3.08 1.650 5.37 N.S. 6.638 0.247 1.40
Axes 12.69 3.17 19.13 43.41 21.54 1.70 5.42
PC Lima Beans Cotyl. 22.57 3.20 7.26 53.32 10.02 1.44 2.81
L.S.D.(0.05) 5.55 N.S 1.19 N.S. 2.79 0.240 2.06
Axes 18.59 2.92 2.98 64.84 13.15 1.72 3.99
PE“ Broad Beans Cotyl. 13.59 2.71 29.59 51.75 2.31 1.40 5.20
L.S.D.(0.05) N.S. N.S 5.99 N.S. 1.41 N.S N.S
PE Peas Axes 22.43 3.07 3.33 55.74 15.16 1.61 2.97
Cotyl. 15.35 5.64 16.54 58.45 3.97 1.45 3.79
L.S.D.(0.05) 5.69 1.46 5.05 N.S. 4.29 0.08 N.S
Axes 25.12 2.48 15.81 40.53 16.04 1.45 2.63
PE— Lima Beans Cotyl. 27.90 2.43 5.78 55.22 8.83 1.42 2.34
L.S.D.(0.05) N.S. N.S 2.88 8.74 1.12 N.S N.S
Axes 37.68 3.45 2.93 46.69 9.20 1.24 1.44
PI Broad Beans Cotyl. 32.50 3.57 16.81 45 27 1.36 1.11 1.74
L.S.D.(0.05) N.S. N.S 4.36 N.S. 1.27 0.10 N.S
PI Peas Axes 32.13 4.24 4.29 42.40 16.95 1.40 1.75
Cotyl. 34.74 7.81 12.79 39.81 4.81 1.07 1.35
L.S.D.(0.05) 2.52 1.00 1.37 N.S. 2.39 0.04 0.148
Axes 30.18 5.55 10.75 31.12 22.37 1.40 1.80
PI Lima Beans Cotyl. 36.31 5.86 4.91 42.13 10.74 1.21 1.42
L.S.D.(0.05) N.S. N.S 0.97 N.S. 4.17 N.S. N.S

 

63

(3) 25°C vs. 10°C Imbibition. When imbibition at

 

10°C was compared with imbibition at 25°C no consistent dif-
ferences in the percentage of any fatty acids could be
detected in any of the species and no relationship was seen

between chilling resistant and sensitive species.

(4) Imbibition Stage vs. 6th Day of Germination at
19:9. There was a large change in percentage of individual
fatty acids between imbibition and the 6th day of germina-
tion at 10°C in broad beans and peas but not in lima beans
(Table 5), presumably because lima beans cannot germinate
at that temperature and consequently they do not have an
active lipid metabolism.

Specifically, in axis tissue of broad beans and peas,
there was an increase in percentage 16:0, 18:2 and 18:3 and
a decrease in percentage 18:1 when comparing fatty acid
content at the initial imbibition stage and after 6 days
germination of 10°C (Table 5).

In cotyledons, there was an increase in percentage
18:2 and a decrease in percentage 18:1 the 6th day of germi-
nation compared to the imbibition stage in broad beans and
peas (Table 5). There was little or no change in lima

beans.

64

 

 

 

 

 

 

 

 

 

 

 

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65

Content of Fatty Acids in

 

Phosphatidyl Ethanolamine

 

(a)

(b)

(C)

(d)

(e)

(1) Species Differences.

Lima beans had a considerably higher percentage
16:0 than the chilling resistant Species, except
in axis tissue at 10°C, both during imbibition
(Table 6) and the 6th day of germination at 10°C
(Table 3).

The chilling resistant species had a higher per-
centage 18:1 than lima beans in most cases (except
axis of lima beans on 6th day) (Tables 6 and 3).
In general, the percentage 18:3 was highest in
lima beans.

In most cases, the unsaturated/saturated ratio

was highest for the chilling resistant species and
lowest for lima beans (Tables 6 and 3).

The D.B.I. was not related to chilling resistance

or sensitivity.

(2) Axes vs. Cotyledons. During imbibition, the

main difference between axes and cotyledons was that broad

beans and peas had the same or higher percentage 18:1 in

cotyledons than in axis tissue while lima beans had a con-

siderably higher percentage 18:1 in axis tissue (Table 7).

On the 6th day, broad beans and peas had a higher

percentage 18:1 in cotyledons than in the axes. Lima beans

66

 

 

 

 

 

 

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68

had just the opposite (Table 4). All 3 species had a higher

percentage 18:3 in axis tissue (Table 4).

(3) 25°C vs. 10°C Imbibition. In general, no signi-
ficant or consistent differences were seen for any of the
fatty acids, for D.B.I. or unsaturated/saturated ratio during
imbibition at 25°C vs. 10°C (Table 7).

This may be explained by the fact that the 24 hour
imbibition at 25°C or 10°C was a short period of time for
large quantities of new phospholipids to be synthesized,
containing fatty acids characteristic of the particular
temperature. Consequently, the seeds analyzed contained
mostly the same fatty acids in their phospholipids as there
were at the time of seed maturity. In one of the few
exceptions observed, lima bean axes exhibited a higher
D.B.I. and unsaturated/saturated ratio at 10°C than at 25°C
(Table 7). Apparently the actively growing axis at 25°C
had sufficient time to produce quite large quantities of
new phospholipids with fatty acids characteristic of

25°C.

(4) Imbibition Stage vs. 6th Day of Germination.

 

No significant changes in the percentages of fatty acids
between the imbibition stage and the 6th day of germination,
at 10°C, were observed for broad bean and lima bean cotyle-
dons (Table lO). In peas, a decrease in percentage 18:1
and an increase in percentage 18:2 was observed during this

6 day period.

69

However, in the axes a lO-fold decrease in percen-
tage 18:1 and a S-fold increase in percentage 18:3 were
observed in the chilling resistant species while no signi-
ficant differences were observed in lima beans at the two
stages of germination (Table 8), apparently because of
chilling injury and lack of metabolism in this species at
10°C. There were few significant differences between the
D.B.I. and unsaturated/saturated ratio which could be
related to chilling resistance or sensitivity.

Content of Fatty Acids in
Phosphatidyl Inositol '—

 

 

(1) Species Differences.

 

(a) Large differences were observed in percentage 18:1
and 18:3 in most cases.‘ Chilling resistant Species
generally exhibited a higher percentage 18:1 and a
lower percentage 18:3 than lima beans after 24 hours
of imbibition (Tables 8 and 10).

(b) On the 6th day the chilling resistant species
maintained the highest percentage 18:1 in cotyledons,
while in axes, lima beans had the highest one (Table
3).

Lima beans maintained the highest percentage
18:3 among species after 6 days of germination and
they had a higher D.B.I. during the imbibition

period than the other species. No significant

 

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70

 

 

 

 

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71

 

 

 

 

 

 

 

 

 

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73

differences between chilling resistant and sensi-
tive species were observed on the 6th day (Tables

3, 4 and 9).

(2) Axes vs. Cotyledons. During imbibition, there

 

were few consistent differences among species in the con-
tent of fatty acids comparing axes and cotyledons. After

6 days germination, broad beans and peas had a higher per-
centage 18:1 in cotyledons than axis while the opposite was
true in lima beans (Table 4). All three species had a

higher percentage 18:3 in axis tissue (Table 4).

(3) 25°C vs. 10°C Imbibition. There were only a
few significant differences in the percentages of fatty
acids in axes or cotyledons in all species, due to tempera-

ture during the imbibition stage (Table 10).

(4) Imbibition Stage vs. 6 Dangermination. In axis
tissue, there was a decrease in percentage 18:1 and an
increase in percentage 18:3 in all three species from the
imbibition stage to the 6th day of germination (Table 11).
In addition, the percentage 18:2 of broad beans and peas
did not change with time, but in lima beans it increased
lO-fold (Table 11).

Similar changes with time did not occur in cotyle-

donary tissue (Table 11).

74

 

 

 

 

 

 

 

 

 

 

 

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75

Discussion

 

There are many reports in the literature corre-
lating cold hardiness or chilling resistance with highly
unsaturated lipids as it was discussed earlier.

Results reported here show that there are large
differences in some fatty acids of phospholipids between
chilling resistant broad beans and peas and chilling
sensitive lima beans.

The fatty acids analyzed in this study were a
reflection of total fatty acid content of the total phos-
pholipid present after a 24 hour imbibition of seeds rather
than a representation of newly synthesized fatty acids.
When the fatty acids of three major phospholipids (PC, PE
and PI) were determined in the 3 species studied, there
were some very consistent differences.

In all three phospholipids, at 10°C and 25°C,
broad beans and peas had a higher percentage 18:1 and a
lower percentage 18:3 than lima beans. In PC, lima beans
generally had a higher percentage 16:0 than did peas and
broad beans in both cotyledon and axis tissue. In PE,
lima beans had a higher percentage 16:0 than the other two
species in cotyledons but not in axis tissue.

The melting point of 16:0 is 63°C and of 18:1 is
about 16°C. The high percentage 16:0 observed in lima

beans in PC, and partially in PE, may be related to the

76

transition temperature from liquid crystalline to crystal-
line state for those phospholipids in the cell membranes.
This may contribute to the cold sensitivity of lima beans.
On the other hand the fact that lima beans generally had

a higher percentage 18:3 (melting point -10°C) than the
chilling resistant species is inconsistent with the above
concept. Differences in the percentage of 18:0 and 18:2
were not consistently related to the degree of chilling
resistance.

The double bond index (D.B.I.) has been used to
indicate differences in unsaturation of fatty acids. A
high D.B.I. is usually related to cold resistance. The
results reported here are contrary to the above in that
lima beans had a higher D.B.I. than the chilling resistant
Species. On the other hand, the unsaturated to saturated
raio was higher,,especially in cotyledons of the chilling
resistant species. This discrepancy is, apparently,
because the unsaturated/saturated ratio does not give a
higher weight to 18:3 than to 18:1 or 18:2 even though
the 18:3 fatty acid is more highly unsaturated and may
contribute more to the lowering of the Tc of phospholipids.

The data presented here point out dramatic differ-
ences between lima beans, broad beans, and peas in
the fatty acid changes which occur over a 6 day germina-
tion period at 10°C. In general, in the two major phospho-

lipids (PC and PE), the percentage 18:1 in broad beans

77

and peas decreased from imbibition to 6 days later and the
percentage 18:3 increased. This is especially true in
axis tissue. This indicates that in these chilling resis-
tant plants, there is a shift in metabolism of fatty acids
toward a more highly unsaturated state even though this is
not reflected in the D.B.I. or unSaturated/saturated ratio.
This again is compatible with the idea that exposure to
low temperatures can cause shifts in lipid metabolism of
many organisms towards more unsaturated fatty acids. It
should be emphasized that even small changes in the overall
melting points of the membrane lipids may be crucial for
normal functioning at chilling temperatures. Lima beans,
which are very sensitive to chilling temperatures, did not
exhibit any change in fatty acid composition from imbibi-
tion to 6 days later. This probably indicates that the
seed was injured by the low temperature and no shift in
metabolism was possible because no growth was occurring.

The possibility exists, however, that the changes
observed to occur from imbibition to the 6th day at 10°C,
could also occur at 25°C. Therefore, definite conclusions
cannot be made at this time.

An increase in the desaturation of fatty acids at
low temperatures, in a number of plant tissues including
seeds (Harris and James, 1969a,b) has been attributed to a
higher solubility of oxygen at low temperatures; oxygen is

required for the desaturation reactions of fatty acids.

78

The observed increase in the percentage of 18:3, and some—
times of 18:2, from the imbibition stage to the 6th day
of germination (Tables 5, 8 and 11) may be explained accord-
ing to the above relationship. Accordingly, the majority
of fatty acids analyzed in the imbibed seeds apparently
were synthesized at the higher temperatures of the period
of seed development and consequently under relatively low
oxygen concentrations in the cellular environment. On the
contrary the majority of the fatty acids analyzed the 6th
day of germination, presumably, were synthesized during
the six days of germination at 10°C, apparently under
higher oxygen concentrations; therefore, they were more
unsaturated.

The regulation of the level of the unsaturation of
fatty acids by the available oxygen suggested by Harris
and James is not known to have any cause-effect relationship

with the development of cold hardiness in plants.

SECTION IV

FATTY ACID CONTENT OF PHOSPHOLIPIDS IN SEEDLINGS

OF SOME VEGETABLE SPECIES IN RELATION TO

CHILLING RESISTANCE

FATTY ACID CONTENT OF PHOSPHOLIPIDS IN SEEDLINGS
OF SOME VEGETABLE SPECIES IN RELATION TO

CHILLING RESISTANCE

Introduction

 

Chilling sensitive plants are injured by chilling
temperatures, below about 12°C but above 0°C (Lyons, 1973).
A partial hardening of those plants against injury from
chilling temperatures can be achieved by conditioning them
for relatively short periods of time, at temperatures
slightly above the chilling range (Wheaton and Morris,
1967; Wilson and Crawford, 1974).

For the reasons presented in the introduction of
Sections I and III, it was thought that the analysis of the
fatty acids of the major phospholipids (PI, PC and PE) in
seedlings of some chilling resistant and chilling sensitive
vegetable species, may contribute to the understanding of
the mechanism of chilling injury.

The species used to study phospholipid synthesis
(Section II) were also used for the analysis of fatty

acids of their major phospholipids.

79

80

Materials and Methods

 

Materials

 

The materials (seeds, chemicals, etc.) described in
Section II were employed to conduct the experiments pre-

sented in this section.

Methods

The plants analyzed for fatty acids were grown as
described in Section II; however they were one day older
than those described in Table l of that Section. The fatty
acid analysis of chilling hardened plants was performed in
plants that were maintained for 4 days at 10°C after trans-
fer from 25°C. The plants were frozen in liquid nitrogen,
lyophilized and their lipids were extracted and fractionated
in individual phospholipids using methods described in
Section I.

Methyl ester preparation and GLC analysis as well
as calculation of the data was made as described in Section
III. The fatty acids analyzed here represent the ones of
total PI, PE and PC occurring in the plants at the time of

analysis.

Results

(1) Comparison of Species

 

(a) No correlation between the degree of chilling

resistance of the species and the percentage 16:0

(b)

(C)

(d)

(e)

81

could be detected for any of the phospholipids at
25°C or following transfer from 25°C to 10°C
(Tables 1, 2 and 3).

In general, the chilling sensitive species had a
higher percentage 18:0 than the chilling resis-
tant ones at 25°C and after transfer from 25°C

to 10°C (Tables 1 and 2). However, there were
some exceptions; peas and cabbage exhibited about
the same percentage 18:0 as the chilling sensitive
species did. In many comparisons, the differences
were not significant.

There were no consistent differences in percentage
18:1, in all phospholipids, between chilling
resistant and chilling sensitive Species which
could be related to sensitivity to chilling injury
(Tables 1, 2 and 3).

Chilling resistant species had a higher percentage
18:2 than chilling sensitive ones had, in all
phOSpholipids, at 25°C or after transfer from

25°C to 10°C (Tables 1, 2 and 3).

Chilling sensitive species had a much higher
percentage 18:3 than the chilling resistant ones
had (with few exceptions), in all phospholipids,
at 25°C or after transfer from 25°C to 10°C

(Tables 1, 2 and 3).

Table 1. Phosphatidyl Choline:
and chilling resistant species grown at 10°C, 25°C or after transfer from 25°C to

10°C.

82

Percentages of fatty acids in seedlings of chilling sensitive

 

Chilling resistant species

Chilling sensitive species

 

 

 

 

 

 

 

 

 

 

 

Fatty Broad Lima Cucum- Hater- L.S.D.

Acids Temp. Beans Peas Lettuce Beets Cabbage Beans ber melon (0.05)

16:0 lO'C 23.20 “2.58 2u.0u 22.70 16.96 -- -- -- 6.7“
" 25-a10°c 29.56 “6.19 -- 25.05 16.8“ 35.10 19.69 23.75 “.60
" 25°C 2“.12 32.89 26.52 31.89 22.05 “0.95 2“.72 30.36 5.39

L.S.D.(0.05) 5.2“ 9.31 N.S. N.S. 3.71 N.S. 2.69

18:0 10°C 3.25 6.99 1.75 1.3“ 0.6“ -- -- -- 1.85
" 25-+10°C 3.7“ 8.19 -- 2.““ 1.62 7.5“ 5.27 8.38 1.26
" 25°C “.15 11.00 2.26 2.16 3.87 10.56 6.70 8.6“ 1.32

L.S.D.(0.05) N.S. 2.81 N.S. N.S 0.“38 N.S. 0.73 N.S

18:1 lo'C 1.29 1.“8 “.78 3.0“ 9.92 -— -- -— 1.01
” 25—910°C 2.6“ 3.20 -- 8.95 1“.33 7.77 0.70 1.65 “.30
" 25°C 2.7“ 5.27 18.63 6.19 17.2“ 9.58 1.62 1.65 3.37

L.S.D.(0.05) 0.85 1.50 5.61 N.S. N.S. N.S. 0.218 N.S

18:2 10°C 6“.16 “3.83 52.91 71.08 39.12 -- -- -- 7.19
" 25—910°C 58.71 “0.06 -- 63.03 “3.87 32.27 19.37 26.66 “.97
" 25°C 6“.89 “7.53 “6.66 58.18 39.37 27.35 19.96 30.89 6.3“

L.S.D.(0.05) N.S. N.S. N.S. N.S N.S. N.S. N.S. 2.66

18:3 10°C 8.05 5.06 16.“5 1.75 33.27 -- -- -- 2.61
" 25—010°C 5.29 2.28 -- 1.78 23.31 17.33 5“.93 39.““ 2.50
" 25°C “.06 3.26 5.91 1.5“ l7.““ 11.53 “7.02 29.“1 3.35

L.S.D.(0.05) 2.1“ 1.59 2.50 N.S “.02 N.S. “.68 3.33

10°C 2.816 1.020 2.876 3.“76 5.010 -- -- -- 1.50“

Unsat. 25-10°C 2.016 0.8“3 -- 2.730 “.“20 1.363 3.016 2.110 0.508
Sat. 25°C 2.553 1.316 2.“83 1.936 2.870 0.9“3 2.186 1.590 0.“o9

L.S.D.(0.05) N.S. N.S. 0.“02 N.S. 1.031 N.S. 0.““9 0.30“

Double 10°C 1.536 1.0“0 1.600 1.500 1.880 —- -- -- 0.1“8

Bond 25-¢10°C 1.360 0.900 -- 1.380 1.720 1.2“3 2.0“3 1.733 0.118

Index 25° 1.““6 1.100 1.293 1.273 1.“83 0.986 1.826 1.516 0.155

L.S.D.(0.05) 0.127 N.S. 0.119 N.S. 0.099 N.S. 0.087 0.090

 

Table 2. Phosphatidyl Ethanolamine:

813

 

 

Percentages of fatty acids in seedlings of chilling

sensitive and chilling resistant Species grown at 10°C, 25°C or after transfer
Itxxn 25°C to 10°C.

 

Chilling resistant species

Chilling sensitive species

 

 

 

 

 

 

 

 

 

 

 

Fatty Broad ' Lima Cucum— water- LuS.D.
Acids Temp. Beans Peas Lettuce Beets Cabbage Beans ber melon (0.05)
16:0 10°C 32.“9 “1.03 27.57 27.39 17.90 - - - 6.“1
'" 25-»10°c 38.93 “6.07 28.39 3“.10 25.28 “1.27 31.61 38.57 5.65
" 25°C 32.98 “8.9“ 28.29 “3.70 26.55 “2.2“ 3“.23 39.68 “.97

L.S.D.(0.05) N.S. N.S. N.S. 7.59 “.15 N.S. N.S. N.S.

18:0 10°C 2 08 5.26 1.20 1.22 1.2“ -— -- - 1.31
" 25-»10°c 2.16 “.81 0.83 2.82 3.12 3.09 2.55 3.05 1.“8
" 25°C 3 3“ 5.16 1.53 1.“6 2.86 7.03 2.83 3.37 0.76

L.S.D.(0.05) 0.72 N.S. N.S. N.S. N.S. 0.25 N.S. N.S.

18:1 10°C 0.3“ 0.89 2.“5 1.22 11.30 -— - —— 0.99
" 25-910°C 0.87 1.51 5.03 “.26 12.“5 3.78 0.““ 0.52 2.32
" 25°C 1.03 1.5“ 7.03 3.96 9.72 6.86 0.83 0.51 1.19

L.S.D.(0.05) 0.5“ N.S. 1.23 1.55 N.S. 2.39 N.S. N.S.

18:2 10°C 58.6“ “6.86 53.1“ 69.32 “0.12 —— -— —— 6.07
" 25—+1o°c 55.81 ““.32 58.13 56.93 “0.5“ 35.93 18.13 21.53 “.65
" 25° 58.“0 “2.12 57.59 50.““ “2.56 33.“8 18.83 25.72 “.“0

L.S.D.(0.05) N.S. N.S. 2.95 7.11 N.S. N.S. N.S. 2.19

18:3 10°C 6.“2 5.95 15.60 1.29 29.73 -— —— —— 2.07
" 25—910°c 3.“7 3.22 7.66 1.8“ 18.62 15.93 “7.19 36.26 2.25
" 25°C “.21 2.20 5.52 0.35 18.27 10.u0 “3.33 29.69 2.“6

L.S.D.(0.05) N.S. N.S. 1.37 1.09 2.08 “.37 N.S. 2.07

Unc t 10°C 1.916 1.213 2.500 2.596 “.2“6 —— -— -— 0.725

-§§%—L 25—>10°c 1.500 0.966 2.“13 1.730 2.523 1.286 1.926 1.“06 0.33“

' 25°C 1.780 0.860 2.356 1.213 2.“30 1.023 1.706 1.926 0.378

L.S.D.(0.05) N.S. 0.26“ N.S. 0.878 0.571 N.S. N.S. N.S.

Double 10°C 1.390 1.123 1.553 1.“30 1.810 —— -- - 0.1“5
Bond 25—¥IO°C 1.233 1.000 1.“33 1.236 1.“90 1.233 1 780 1.520 ,0.132
Index 25°C 1.306 0.923 1.386 1.056 1.“93 1.0“6 1.683 1.“10 0.122

L.S.D.(0.05) N.S. N.S. 0.079 0.176 0.107 N.S. N.S. 0.06“

 

84

Table 3. Phosphatidyl Inositol: Percentages of fatty acids in seedlings of chilling sensi-
tive and chilling resistant species grown at 10°C, 25°C or after transfer fran
25 C to 10°C.

 

 

 

 

Chilling resistant species Chilling sensitive species]
Fatty Broad Lina Cucum- Water- L.S . D.
Acids Temp. Beans Peas Lettuce Beets Cabbage. Beans ber melon (0 . 05)

 

 

16:0 10°C “2.62 51.38 52.29 39.7“ “6.67 - -— -—
" 25—510°C 35.65 56.28 “0.6“ 37.95 50.30 “6.00 35.11 38.12' 5.“0
" 25°C 39.1“ 53.53 52.75 53.17 ““.28 “9.83 “1.06 ““.15 “.29

 

 

 

 

 

 

L.S.D.(0.05) N.S. N.S. 1.0“ 6.79 N.S. N.S. 5.“6 3.68
18:0 10°C 2.69 3.93 2.“6 “.32 3.28 —- - - N.S.
" 25—910°C 2.2“ 5.19 3.95 6.21 3.29 5.55 “.91 9.11 3.0“
" 25°C 3.38 6.00 2.90 5.33 “.27 7.95 “.01 7.02 1.77
L.S.D.(0.05) 0.“6 N.S. 0.90 N.S. N.S. N.S. N.S. N.S.
18:1 10°C 1.08 0.81 0.95 3.12 3.26 - -— -— 0.90
" 25-p10°C 1.87 5.68 “.70 7.51 “.23 3.98 1.09 3.93 N.S.
" 25° 1.76 2.88 2.22 2.53 7.32 “.56 1.07 1.29 1.69
L.S.D.(0.05) 0.6“ N.S. 1.08 2.32 1.7“ N.S. N.S. N.S.
18:2 10°C “5.79 35.18 “l.“3 “7.98 22.66 —- —- - 6.35
" 25—e10°C 53.58 29.89 “6.85 ““.83 25.88 25.29 11.29 15.02 3.“7
" 25° 51.67 3“.39 39.90 36.8“ 25.89 23.5“ 11.73 18.93 5.31
L.S.D.(0.05) N.S. N.S. 1.93 N.S. N.S. N.S. N.S. 2.79
18:3 10°C 7.8“ 8.70 2.8“ 5.05 2“.07 - - - 3.17
" 25—910°C 6.61 3.15 3.80 3.51 16.“3 19.17 “7.58 33.75 6.05
" 25° “.2“ 3.29 2.“3 3.0“ 18.1“ 1“.10 “2.16 28.80 1.7“
L.S.D.(0.05) 1.22 2.66 N.S. N.S. 2.3“ 3.96 “.““ N.S.
Unsat. 10°C 1.229 0.823 0.826 1.270 1.013 -- - -— 0.285
Sat. 25-)10°C 1.666 0.630 1.2“3 1.270 0.863 0.9“0 1.503 1.126 0.267
25° 1.363 0.683 0.798 0.713 1.070 0.730 1.223 0.960 0.1“3
L.S.D.(0.05) N.S. N.S. 0.03“ 0.216 N.S. 0.168 0.239 N.S.
Double 10°C 1.160 0.976 0.920 1.1“0 1.206 -- - -— 0.155
Bond 25—910°C 1.290 0.753 1.100 1.076 1.056 1.120 1.666 1.350 0.159
Index 25° 1.363 0.813 0.882 0.820 1.136 0.9“0 1.506 1.253 0.087

L.S.D.(0.05) N.S. 0.169 0.019 0.103 N.S. 0.120 0.111 N.S.

 

(f)

(g)

85

The ratio of unsaturated/saturated fatty acids,
in all phospholipids, did not correlate well with
the known differences in chilling sensitivity
between species (Tables 1, 2 and 3).

The chilling sensitive species exhibited a higher
D.B.I. than the chilling resistant ones in most

of the cases (Tables 1, 2 and 3).

(2) Temperature Comparisons

 

(a)

(b)

(C)

(d)

In most of the species no Significant change or

a decrease hipercentage 16:0 was observed when the
temperature was lowered from 25°C to 10°C. The same
was true in chilling resistant Species when the 25°C
and 10°C treatments were compared (Tables 1, 2 and
3).

The percentage of 18:0 either did not change with
temperature or it was lower in plants growing con—
tinuously at 10°C or transferred from 25°C to 10°C
(Tables 1, 2 and 3).

In general, the percentage 18:1 followed a Similar
pattern, when comparing temperature treatments
within species, as Shown for 16:0 and 18:0 (Tables
1, 2 and 3).

No consistent differences in percentage 18:2 due

to temperature treatment were detected in any of

the species (Tables 1, 2 and 3).

86

(e) In most of the cases the percentage 18:3 was higher
at 10°C than at 25°C, in both chilling resistant
and sensitive species (Tables 1, 2 and 3).

(f) In general, the unsaturated/saturated fatty acid
ratio remained the same or increased upon exposure
to low temperatures for all the species.

(9) The D.B.I. remained constant or increased in all

species upon exposure to low temperatures.

Discussion

 

More unsaturated phospholipids have been found in
micro-organisms (Cronan and Vagelos, 1972; Kates and
Paradis, 1973; Kaneda, 1972) and plants (Grenier and
Willemot, 1974; De La Roche §t_al., 1972) grown at lower
temperatures; in cold resistant varieties of alfalfa vs.
cold sensitive ones (Grenier and Willemot, 1974); in chill—

ing hardened plants of Phaseolus vulgaris and Gossypium

 

 

hirsutum VS. non-hardened ones (Wilson and Crawford, 1974).

On the other hand in alfalfa (Kuiper, 1970) the
unsaturation of the fatty acids of phospholipids was not
different in plants grown at low or high temperatures or
in varieties differing in cold hardiness.

It is generally believed that chilling or cold
resistance is associated with highly unsaturated membrane
lipids (Lyons et_31., 1964; Lyons, 1973).

The results of this study support, with some excep-

tions, the findings of others about the relationship of

87

fatty acid unsaturation and chilling resistance. In
general, the chilling resistant Species had a lower percen-
tage 18:0 and 18:3 and a higher percentage 18:2 than did the
chilling sensitive ones. The differences in 18:0 and 18:2
between sensitive and resistant species appears to be con-
sistent with the expected higher degree of unsaturation in
resistant Species. However, the high level of 18:3 in
chilling sensitive species was unexpected and difficult to
explain.

The unsaturated/saturated ratio did not correlate
well with chilling sensitivity and the D.B.I. was actually
higher in chilling sensitive Species, which is opposite of
what was expected.

Exposure of all species to low temperature resulted
in a decrease in 16:0, 18:0, 18:1 and in an increase in
18:3, unsaturated/saturated ratio and D.B.I. These results
support the findings of others which generally Show an
increase in unsaturation upon exposure to low temperatures.
Few differences in the fatty acid content, in the chilling
hardened plants, among chilling resistant and chilling
sensitive Species were observed. This was, probably, due
to the fact that chilling sensitive species in the seedling
stage are able to modify their fatty acid metabolism, in
response to low temperatures, towards forms Similar to the
ones observed in chilling resistant species; this modifi-

cation may contribute to the relative chilling resistance

88

of the lima bean seedling as opposed to the chilling
sensitivity of the germinating lima bean seed (Section III).
Furthermore, there is a possibility that the
observed higher percentage of 18:3 at 10°C or after
transfer from 25°C to 10°C in comparison to 25°C, can be
explained according to the findings of Harris and James

(1969a,b) as it has been discussed in Section III).

SUMMARY AND CONCLUS IONS

SUMMARY AND CONCLUSIONS

Significant differences in phospholipid synthesis
were observed between chilling resistant and chilling
sensitive Species as well as between low and high tempera—
tures. In general, PC and to a lesser extent PG, was shown
to correlate positively with chilling resistance or growth
at low temperatures. The opposite was Shown for PE and to
a lesser extent for PI.

(1) Specifically, the chilling resistant Species
(broad beans and peas) during the imbibition stage, at
10°C and 25°C, exhibited a much higher PC/PE ratio than the
chilling sensitive species lima beans. In general, chilling
resistant species, during imbibition, exhibited a higher
percentage PG and PC and a lower percentage PE and PI at
10°C than at 25°C. An apparent shift in phospholipid meta-
bolism towards PE and PG, largely at the expense of PC and
PI, was observed in imbibed lima bean seeds at 10°C in
comparison to 25°C.

(2) A higher percentage of PG, and in some cases of
PC, and a lower percentage of PE was observed at 10°C than

at 25°C in seedlings of chilling resistant Species.

89

90

Transferring chilling resistant and sensitive plants
from 25°C to 10°C generally caused an increase in percen-
tage PC and PG and a decrease in percentage PE. It is sug-
gested that lima beans, in the seedling stage, have the
ability to Shift phospholipid metabolism towards a preferen-
tial synthesis of PC at the expense of PE when plants are
transferred from 25 to 10°C and probably avoiding chilling
injury for that reason, while they are unable to do so at
the imbibition stage.

In general, a higher ratio PC/PE was observed at
lower temperatures than at higher ones. Generally, the
percentage PG was higher in chilling resistant Species vs.
chilling sensitive ones but other phOSpholipids could not
be related to the degree of chilling resistance of the
Species.

The observed differences in phospholipid synthesis
may be related to the degree of chilling resistance in the
Species studied since the physical prOpertieS of individual
phospholipids in relation to temperature are different.

Preferential synthesis of some phospholipid classes
by chilling resistant plants or by both chilling resistant
and sensitive plants when they are exposed to chilling
temperatures, may provide the plant cells with the ability
to build membranes which have a Specific physical state
suitable for normal functioning at low temperatures. On

the other hand, it is also possible that the differences

91

in phospholipid synthesis observed in this study have no
cause-effect relationship with cold resistance but may
represent genetic differences or temperature responses
related to other functions of plant metabolism.

Much more research is required to elucidate the
possible relationship of chilling or cold resistance to
phospholipids. Quantitative and qualitative phospholipid
analyses, coupled with physical measurements in yiyg and
in yitrg, in membrane systems (e.g. mitochondria, chloro-
plasts, plasmalemma, etc.) will be useful to study the
problem. The discovery of mutants, unable to synthesize
some of the phospholipid classes, may help to investigate
the importance of each phospholipid class in relation to
temperature.

(3) The analysis for fatty acid content in PI, PC
and PE in germinating chilling resistant and sensitive
Species revealed considerable differences. In general,
broad beans and peas had a higher percentage 18:1 and a
lower percentage 18:3 and 16:0 than lima beans. Consider-
ing the melting point of those fatty acids (presumably
this affects the temperatures at which the phospholipids
change phase from a liquid crystalline to a crystalline
state) it appears that the chilling sensitive lima beans
exhibited an unexpected high percentage 18:3 and D.B.I.
However, the unsaturated/saturated ratio was higher in the

chilling resistant species as was expected. During the

92

6 day germination period for chilling resistant Species, the
percentage 18:1 decreased and 18:3 increased indicating a
shift of fatty acid metabolism towards more unsaturated
forms. This Shift may be a response to low temperatures or
a characteristic of the chilling resistant species. How-
ever, it is possible that the Shift is also observed at
high temperature both in chilling resistant and sensitive
Species. In the chilling sensitive lima beans no changes
were observed in individual fatty acids between the imbibi-
tion stage and the 6th day of germination presumably
because of the absence of normal metabolism at 10°C. It
Should be pointed out, however, that the exposure of lima
beans to chilling temperatures did not result in a break
down of phospholipids Since they maintained about the same
fatty acid content during the six day chilling.

(4) In seedlings grown continuously at 25°C or
temperature conditioned at 10°C, the chilling resistant
Species generally had a higher percentage 18:2 and a lower
percentage 18:0 and 18:3 than chilling sensitive species.

Chilling sensitive Species, unexpectedly, had a
higher D.B.I. than chilling resistant ones, while the ratio
of unsaturated/saturated was not correlated with chilling
sensitivity of the species. In general, the percentage
18:3 was higher at 10°C than at 25°C or after transferring

from 25°C to 10°C, in chilling resistant species.

93

Exposure of all species to low temperatures gener-
ally resulted in a decrease in 16:0, 18:0, 18:1 and an
increase in 18:3, unsaturated/saturated ratio and D.B.I.
These results support the findings of other workers which
Show an increase in unsaturation upon exposure to low tem-
perature.

The lack of a consistently higher unsaturated/
saturated ratio and D.B.I. in chilling resistant species
vs. chilling sensitive Species may suggest that the degree
of unsaturation of cellular lipids is not the most impor-
tant factor determining the chilling resistance of plants.
The same suggestion which minimizes the importance of fatty
acid unsaturation in determining cold sensitivity has been
made earlier by Lyons (1973) but it cannot be proven on the
basis of the knowledge presently available. In E. 9211.
(Esfahani gt_al., 1971) and Sheep liver mitochondria (Lyons,
1973) the fatty acid composition of lipids determined the
transition temperature (Tc) of the lipid portion of the
respective membranes. In contrast to the above, it has
been found, that dietary modifications altered the fatty
acid composition of lipids in mitochondrial membranes of
rat liver but no considerable Shifting of Tc was observed
(Williams §t_al,, 1972); there is no Similar information
available for higher plants.

The results of Section I and II suggest that the

polar groups of cellular phospholipids are important in

94

determining the degree of chilling resistance of the Species

studied as well as the development of chilling hardiness.

APPENDIX

95

Table A-1. Chemical formulas of the major plant

 

phospholipids.
O
H
O Hzi—O-C-R
fl
GENERAL FORMULA OF PHOSPHOLIPIDS R-C-O-CH O
In
HZC-O-P-O-X
0
Phosphatidyl-Choline X = -CH2-CH2-N+ (CH3)3
Phosphatidyl-Ethanolamine X = -CH2-CH2-NH2
MHZ
Phosphatidyl—Serine X = -CH2-CH-COOH
Phosphatidyl-Glycerol X = -CH2-CH-CH2011
OH
OH OH
Phosphatidyl-Inositol X =
OH

OH

96

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