Temperature and Aging Effects on Leaf Membranes of a Cold Hardy Perennial, Fragaria virginiana.

Abstract

The lipid composition of leaves of wild strawberry (Fragaria virginiana Duchesne) was analyzed throughout an annual growth cycle in the field. Cellular hardiness to temperature stress was assessed concomitantly by a solute leakage technique. Leaves were shown to be very sensitive to an applied temperature of -5 degrees C during the summer months but insensitive to a 35 degrees C treatment. This general pattern was also seen in young overwintering leaves but was reversed after a period of low-temperature hardening of these same leaves. Associated with cold hardening of the overwintering leaves was a twofold increase in the phospholipid content of the leaf membranes with a proportionately smaller increase in free sterols. The large increase in phospholipids presumably is due primarily to the proliferation of a sterol-poor membrane fraction, probably the endoplasmic reticulum. These quantitative changes in membrane material may be important in increasing freezing tolerance in the overwintering leaf cells by enhancing the overall capacity of the cell for plasma membrane and tonoplast extension through vesicle fusion using components from this endomembrane pool. Analysis of electron micrographs of hardened leaf cells showed an increase in vesiculated smooth endoplasmic reticulum and tonoplast membrane over nonhardened leaf cells, the latter resulting in an enhanced tonoplast surface area to vacuolar volume ratio. During this same period, no changes in the fatty acid or free sterol composition were detectable, suggesting that regulation of membrane fluidity via these components is not required for cold acclimation in this species. During aging and senescence of both the overwintering and the summer leaves, the cellular membranes remained functionally intact but became progressively more vulnerable to temperature stress. Free sterol content increased during this time. This feature may be related to the inability of the older leaves to withstand environmental stress. Increasing sensitivity of the cellular membranes to stress may, in turn, be causally related to the actual onset of senescence in these leaves, thus explaining why only the older leaves senesce when the plant is challenged by periodic environmental stress.

Full text

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PlantPhysiol.(1981) 68, 1409-1415
0032-0889/81/68/1409/07/$00.50/0
Temperature and Aging Effects on Leaf Membranes of a Cold
Hardy Perennial, Fragaria virginiana'
Received for publication January 23, 1981 and in revised form July 24, 1981
SHARMAN D. O'NEILL2, DAVID A. PRIESTLEY3, AND BRIAN F. CHABOT
Section ofEcology and Systematics, Cornell University, Ithaca, New York 14850 (S. D. O., B. F. C.); and
Boyce Thompson Institutefor Plant Research, Ithaca, New York 14853 (D.A.P.)
ABSTRACT
The lipid composition of leaves of wild strawberry (Fragaria virpgaa
Duchesne) was analyzed throughout an annual growth cycle in the field.
CeLlular hardiness to temperature stress was assessed concomitantly by a
solute leakage technique. Leaves were shown to be very sensitive to an
applied temperature of -5°C during the summer months but insensitive to
a 35C treatment. This general pattern was also seen in young overwinter-
ing leaves but was reversed after a period oflow-temperautre hardening of
these same leaves. Associated with cold hardening of the overwintering
leaves was a twofold increase in the phospholipid content of the leaf
membranes with a proportionately smaller increase in free sterols. The
large increase in phospholipids presumably is due primarily to the prolif-
eration of a sterol-poor membrane fraction, probably the endoplasmic
reticulum. These quantitative changes in membrane material may be
important in increasing freezing tolerance in the overwintering leafcells by
enhancing the overall capacity of the cell for plasma membrane and
tonoplast extension through vesicle fusion using components from this
endomembrane pool. Analysis of electron micrographs of hardened leaf
cells showed an increase in vesiculated smooth endoplasmic reticulum and
tonoplast membrane over nonhardened leaf cells, the latter resulting in an
enhanced tonoplast surface area to vacuolar volume ratio. During this
same period, no changes in the fatty acid or free sterol composition were
detectable, suggesting that regulation of membrane fluidity via these
components is not required for cold acclimation in this species. During
aging and senescence of both the overwintering and the summer leaves,
the cellular membranes remained functionally intact but became progres-
sively more vulnerable to temperature stress. Free sterol content increased
during this time. This feature may be related to the inability of the older
leaves to withstand envirommental stress. Increasing sensitivity of the
cellular membranes to stress may, in turn, be causally related to the actual
onset of senescence in these leaves, thus explaining why only the older
leaves senesce when the plant is challenged by periodic environmental
stress.
Fragaria virginiana Duchesne, the wild strawberry, is a perennial
species that retains one or two leaves throughout the winter
months. During the growing season, each individual plant main-
tains a more or less constant number of leaves by sequential
senescence of the older leaves and production of new leaves.
'Supported in part by National Science Foundation Grant No. DEB
77-08432.
2Present address: Division of Natural Sciences, Thimann Labs, Uni-
versity of California, Santa Cruz, CA 95064.
3Present address: Afdeling Plantenphysiologie, Botanisch Lab, Arbor-
etumlaan 4, Wageningen, Netherlands.
Temperature extremes in Ithaca, NY, range from -25°C in the
winter to 35°C in the summer; thus, the wild strawberry must
adapt to a broad range ofenvironmental conditions. This situation
offers an unusual opportunity to investigate tolerance to both high
and low temperatures, as well as age-dependent changes in the
same plant.
Cellular membranes have been implicated as the site ofprimary
lesions in both chilling and freezing injury (8, 26). Interruption of
membrane-associatedmetabolic
phase transitions is thought to be a principal effect of chilling
damage at temperatures above freezing (8). Freezing injury, how-
ever, is more closely associated with cellular dehydration occurring
as the result of extracellular ice formation (10). Steponkus and
Wiest (25) have presented evidence suggesting that membrane
lesions occur as the result of a freeze-induced reduction in cell
membrane surface area during volumetric contraction, which
renders the protoplast incapable of rapidly regaining its original
size upon thawing. In contrast, Palta (15) has concluded that
freeze-induced lesions involve alterations in membrane semi-
permeability, mediated by effects on specific membrane proteins
involved in transport processes, and that general membrane dis-
ruption is not the critical lesion.
The cellular membranes have been the focus of considerable
attention in freezing injury. Several studies have looked at changes
in lipid composition under various temperature regimes and in
several different plants (18). Most investigations of plants in their
natural environment have investigated changes in cambial tissue,
roots, or evergreen needles during cold acclimation. In this inves-
tigation, we have selected an overwintering perennial species, F.
virginiana Duchesne, in order to study changes in overwintering
leaves. This has been an ideal system for contrasting the cold,
hardy with the nonhardy condition in leaves that have the same
genetic potential for acclimation and for investigating the effects
of temperature acclimation on lifespan, since cold temperature
apparently interrupts the aging process.
Solute leakage has been used to assess membrane integrity in
response to environmental stresses such as chilling (7), freezing
(16), and dehydration (4). In this study, it has been useful in
assessing the sensitivity of strawberry leaf tissue to low and high
temperatures. Since the first step in the cold acclimation ofstraw-
berry leaves involves low but not freezing temperatures, we began
our investigation by examining the regulation ofmembrane fluid-
ity during early cold hardening. Typical mechanisms of fluidity
regulation might involve changes in fatty acid unsaturation and
sterol composition ofthe cellular membranes. Homeoviscous reg-
ulation ofmembrane fluidity is well documented and is considered
to be an important aspect of temperature acclimation in many
animal and bacterial systems (11, 20, 23). The situation in plants
is not so clear. We also investigated quantitative changes in the
principal membrane lipids throughout an annual cycle. Overall,
the research reported here was undertaken to determine to what
1409
activity accompanying
lipid

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Plant Physiol. Vol. 68, 1981
extent the lipid composition of membranes is altered by environ-
mental stress, aging, and senescence and, further, to assess the
functional and structural changes that result from such membrane
changes.
MATERIALS AND METHODS
Plant Material. The plant material used in this study consisted
of overwintering leaves and summer leaves of wild strawberry (F.
virginiana Duchesne) plants growing in an old field near Ithaca,
NY. Overwintering leaves are initiated from late September until
mid October, usually two or more per plant. These leaves do not
senesce until late April or early May, coincident with new leaf
expansion and flowering. Thus, overwintering leaves are relatively
long-lived (210 to 240 days) as compared to leaves initiated in the
summer (60 to 90 days). Leaf age was determined by growth
analysis of 25 field plants over the annual cycle (S. D. O'Neill,
unpublished data). During this study, leaf order was determined,
and samples represented a cohort of leaves produced at approxi-
mately the same time. Summer leaf material consisted of the
fourth leafproduced during the growth season. Overwintering leaf
material consisted of the last two leaves produced during the
growth season.
The sampling procedure used in this study consisted of single
collections of leaf material from three sites at various intervals
throughout the annual cycle (dates listed below). Fifty to 70 leaves
were randomly selected for each lipid analysis or leakage experi-
ment. Replicate determinations represent analyses of subsamples
taken from these field collections. Replicate field samples were
not taken due to the large amount of plant material required for
analysis over the entire season from a limited population of plants.
We feel that this experimental design reflects the actual seasonal
changes occurring in these leaves because of the large pooled
sample size used and the homogeneity of the plant population
sampled.
Leakage. The permeability of cellular membranes during tem-
perature stress and aging was assessed by a solute leakage tech-
nique modified from the procedure of Martineau el al. (9). Solute
leakage experiments were performed on the following dates: No-
vember 25, 1979; and January 14, March 8, April 20, June 7,
August 11, and October 4, 1980. Leaves were collected from the
field as nearly as possible to the time of lipid extraction (see
below) and cut into leaf discs with a 1-cm cork borer. Leaf discs
were placed in vials (10-20 discs/vial), washed three times in
deionized H20 to remove surface electrolytes, drained, and sub-
jected to temperature treatment (-5, 0, 25, and 35°C) for 1, 4, 8,
or 12 h in sealed vials. Following temperature treatment, a mea-
sured amount of deionized H20 was added to each vial, and the
tissue was allowed to incubate in the dark at 15 or 25°C, for
overwintering and summer leaves, respectively. After 12 h, elec-
trical conductance ofthe leachate was measured using a Beckman
Model RC 16B2 conductivity bridge. The relative amount of
leakage was expressed as a percentage of the maximum conduct-
ance, measured after autoclaving of the sample.
Lipid Analysis. Fully expanded leaves of wild strawberry were
collected from the field immediately prior to solvent extraction of
total leaf lipids on the following dates: October 3, 1979; and
January 8, March 5, April 11, June 2, June 11, June 22, and July
5, 1980. Leaves were weighed, washed, and rehydrated to full
turgor in distilled H20 and reweighed to ensure uniformity of
fresh weight determinations between extractions. Relative water
content was calculated and found to be similar throughout the
growing season.
Leaves were blended with boiling isopropanol for 2 min in a
Waring Blendor, and the homogenate was vacuum-filtered
through Whatman GF/A glass microfiber paper. The filter residue
was reextracted for 2 min with chloroform:isopropanol (1:1, v/v).
The combined filtrates were dried in vacuo and resolubilized in
chloroform:methanol (2:1, v/v). The solvent extract was parti-
tioned against 0.2 volumes of 1% (w/v) NaCl and then against 0.5
volumes of methanol:1% (w/v) NaCl (1:1, v/v). The solution was
subsequently evaporated to dryness in vacuo, and the lipid residue
was redissolved in chloroform:methanol (2:1, v/v). The final lipid
extract was stored under argon in the dark at -20°C.
Total lipid samples were separated for qualitative analysis of
phospholipids by two-dimensional TLC, using precoated Silica
Gel G plates (Applied Science Labs) with a layer thickness of 250
jim. The plates were developed in the first dimension using
chloroform:methanol:7 M NH40H (65:30:4, v/v/v) and in the
second dimension using chloroform:methanol:acetic acid:water
(170:25:25:4, v/v/v/v). Phospholipids were selectively visualized
using spray reagents, as described by Kates (6), and identified
with reference to authentic standards (Sigma, Supelco, and Ap-
plied Science). TLC was also performed in one dimension for the
identification and separation of total galactolipids (MGDG4 and
DGDG) from the total lipid extract, using chloroform:methanol:
acetic acid:water (85:15:10:3, v/v/v/v) as solvent.
Total phospholipids were determined by a colorimetric phos-
phate assay using the procedure of Raheja et al. (19). Total
MGDG and DGDG were estimated according to the procedure
ofRoughan and Batt (21). Neutral lipid diglyceridesandtriglyc-
erides were not quantified, since they were found to form avery
minor component when the total leaflipidfraction wasseparated
by TLC. Free fatty acids, although identifiable following TLC,
also representedaquantitativelyminorcomponent,as assessedby
their reaction with 12 and H2S04. Fatty acids were preparedfor
analysis by esterification in methanol:benzene:sulfuric acid(100:
5:5, v/v/v) for 2 h at 80°C. The fatty acid methylesters were
partitioned into ii-hexane and analyzed usinga Hewlett-Packard
5730 A gas chromatograph equipped with a flame ionization
detector and coupled to a Hewlett-Packard 3380 S integrator.
Separations were done on a 180-cmglass column (4mm internal
diameter) packed with 3% (w/w) Silar-5 CP on 100/120 mesh
Gas-chrom Q (Applied Science Labs) at 200°Cwith N2 as the
carrier gas. Heneicosanoic acid (C 21:0)was used as the internal
standard(Sigma).Free sterols wereprepared by precipitationwith
0.5%digitonin,as describedbyGrunwald(3),with 5a-cholestane
(Sigma) as the internal standard. Followingdissociation of the
sterol-digitonin complex, the sterol residue was derivatized in
acetonitril-bis(trimethylsilyl)trifluoroacetamide (1:1, v/v)for 1 h
at roomtemperature.Thetrimethylsilylderivatives wereanalyzed
by GLC. Separationswereperformedon a 180-cmglasscolumn
(4mm internaldiameter) packedwith 3%(w/w)OV-17 on 100/
120 mesh Chromsorb W-HP (Supelco)at255°Cwith N2 as the
carriergas.
ElectronMicroscopy.Leaf tissue was collected from the field at
0800 h. A 100-mm2 portion of the central leaflet was fixed in 2%
glutaraldehyde in 20 mol m-3 K-phosphate (pH 7.1)for 3 h,
followinga modification of theprocedureof Mohr andCocking
(13). Post-fixation was carried out in 2% OS04for 3 h at4°C (pH
7). Dehydration, infiltration, andembeddingwere doneusinga
graded ethanol to water series, followedby propyleneoxide and
Spurr medium (24). Polymerizationwasaccomplishedin astep-
wise manner for 2dayseach at 24, 37, and80°C.
Thin sections (pale gold to silver)were made on a Reicher
OMU2 microtomeequippedwith a diamond knife. The sections
were stained for 20 min with 2%aqueous uranylacetate and for
7 to 8 min with lead citrate. Electronmicrographswere taken on
aPhilipsEM 3000 electronmicroscope (KV 60).The leafmaterial
examined byelectronmicroscopyconsisted of tworepresentative
specimenseach from theoverwintering (January, 1980)and sum-
mer-leaftypes (June, 1980)and onespecimenfrom an unhardened
4Abbreviations: MGDG, monogalactosyl diglyceride;DGDG,digalac-
tosyl diglyceride.
1410
O'NEILL ET AL.

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COLD ACCLIMATION AND AGING OF MEMBRANES
overwintering leaf (October, 1980) for a follow-up comparison.
Other leaf material was examined by light microscopy as a sup-
plement to data obtained by electron microscopy. The palisade
mesophyll cells were used for all electron micrograph comparisons.
RESULTS AND DISCUSSION
Leakage. Solute leakage from overwintering leaves in October
indicates that these leaves were in a nonhardened state (Fig. IA).
Cellular leakage following the -5°C treatment showed that the
leaves were susceptible at this time to freezing injury but insensi-
tive to the high temperature treatment of 35°C. Leakage rates at
0°C were similar to those ofthe 25°C treatments in this and every
other experiment throughout the annual cycle, verifying that the
strawberry plant is not sensitive to cold (but above freezing)
temperatures even when in a nonhardy state. These data are not
presented in Figure 1 because of the overlap with the data for
250C. At the time ofthis experiment, weekly average temperature
had not fallen below 80C (Fig. 2).
By late November, some acclimation ofthe overwintering leaves
751
50[
0
-
0
w
(D
4c
od
Ul
J
25
overwintering leaves
A
B
0-5 C
025 C
635 C
1
DC
75k
50F
25k
4
8
12
4
8
12
HOURS OF TREATMENT
FIG.
1. Cellular leakage as percentage of total conductivity for over-
wintering leaves in October (A), November (B), January (C), and April
(D). Bars represent the SD ofthe mean for three replicates.
30
Ul
-
w
IL 30
w
> 10
ww
w
3:0O
C-10
w
10/79
1/80
4/80
7/80
MONTH
FIG. 2. Average weekly air temperature (OC) recorded in Ithaca, NY.
had occurred, as indicated by the decreased cellular leakage at
-5°C (Fig. 1B). The results ofthe freezing treatment (-5°C) were
interesting, in that the amount of subsequent leakage showed an
apparent decrease if the freezing stress was imposed for 8 h or
more. The pronounced decrease in leakage after 4 h in the
unhardened overwintering leaves is difficult to explain. We feel
that it is not anomalous, given the replication involved in these
experiments and its appearance in both October and November.
While a time-dependent membrane alteration is suggested by the
data, a metabolically based repair is unlikely to occur at -5°C.
Instead, the data indicate that an alteration in membrane perme-
ability occurred several h following the onset of freezing stress,
which would suggest that a molecular rearrangement in the plasma
membrane may eventually take place at subzero temperatures. It
has been suggested (25) that a thermodynamically favorable mo-
lecular rearrangement can occur during membrane contraction,
which may result in a decrease in membrane permeability.
By January, the overwintering leaves showed complete accli-
mation to freezing stress. Solute leakage at -5°C was well below
the level of that in both the October and November leaves and
was not significantly different from the leakage at 25°C (Fig. IC).
Both average weekly minimum and mean temperatures were
mostly below 0°C for 3 weeks prior to this experiment.
In April, cellular leakage results showed that the overwintering
leaves still exhibited considerable tolerance to -5°C treatment,
even while in the process of deacclimating (Fig. ID). Average
weekly mean temperatures had risen above 0°C, although mini-
mum weekly temperatures were still below freezing (Fig. 2).
Leakage rates at 35°C were well above the October leakage rates
at 350C (Fig. IA), although 25°C leakage rates were essentially
the same as they were in all previous experiments (Fig. 1, A-C).
This indicates a loss of the previously attained high temperature
tolerance but only slightly increased leakage at nonstressful tem-
peratures (0°C and 25°C) as these leaves aged.
Overwintering leaves illustrate the effects ofacclimation to low
temperature, in addition to the effects of aging. Indeed, the major
difference between summer and overwintering leaves lies in the
ability of the latter to undergo a pronounced acclimation, at the
cellular level, to low temperatures. Tolerance to-5°C temperature
treatment was not lost during aging. Total phospholipid levels
also remained higher than did the October prehardened levels, as
discussed in the following section. Since these functional changes
in leakage were associated with alterations in the phospholipid
fraction, a causal relationship is suggested.
The pattern of leakage in summer leaves was essentially the
reverse of that observed in the overwintering leaves, suggesting
that acclimation to either high or low temperature in wild straw-
berry involves different cellular mechanisms. Cellular leakage at
-50C was comparatively high in the summer leaves, reflecting
considerable membrane damage (Fig. 3A). This leakage at -5°C
was greater as the leaves aged (Fig. 3B). Conversely, at 35°C,
leakage was no higher than in the control in young summer leaves
(Fig. 3A). As in the overwintering leaves, sensitivity to high
temperature apparently did increase with age, as shown by higher
leakage at 35°C for the same group of summer leaves at a later
date.
Lipid Analysis. The fatty acid profile for strawberry leaves (Fig.
4) is dominated by the unsaturated fatty acids, mostly linoleic acid
(18:2) and linolenic acid (18:3) which, together, constitute about
75% of the total fatty acid. Other fatty acids present in the total
lipid extract include palmitic (16:0), palmitoleic (16:1), stearic (18:
0), and oleic (18:1) acids. Fatty acid levels ofoverwintering leaves
remained essentially unchanged during both cold acclimation and
cold deacclimation, with no substantial increase in unsaturated
forms. Slight fluctuations occurred in the mol percent of linoleic
and linoleic acids during late winter and early spring, but, since
no consistent trend is indicated by the data, the physiological
1411
PlantPhysiol.Vol. 68, 1981
r
II

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Plant Physiol. Vol. 68, 1981
summer leaves
O- 0
0
IC
Lu
-J
75
50
25
4
8
12
4
8
12
HOURS OF TREATMENT
FIG. 3. Cellular leakage as percentage of total leakage for summer
leaves in June (A) and August (B). Bars represent the SD of the mean for
three replicates.
70
overwintering leaves
summer leaves
80
60 o
~
d)
18:3
50
16:0
<2
18>
1
:2]
10/79
1/80
4/80
7/80
MONTH
FIG. 4. Changes in fatty acid composition duringthe annual cycle, with
each point a single observation.
significance of these slight fluctuations is difficult to assess.
The fatty acid composition ofthe summerleaflipidwasvirtually
identical to that of the overwintering leaf lipid, which supports
the proposal that large shifts in the level of unsaturation of
membrane lipids are not required for cold acclimation in wild
strawberry. Changes in unsaturation are not necessarily expected
in this species, since the high levels of unsaturated forms in the
strawberry leaf membranes at ali times of the year suggest that
this plant exists at temperatureswell above theregionofthephase
transition of its membrane lipids. This is an interesting phenom-
enon in plants, in that organisms that do exhibit homeoviscous
adaptation via fatty acid, and sterol changes are metabolically
active at temperatures very close to the upper limits of the phase
transition of their membrane lipids (23). The only notable change
throughout the annual cycle in the fatty acid composition was
encountered in the summer leaves during the latter one-half of
their lifespan. This consisted of a decrease in the level of unsatu-
ration due to a proportional decline in both linoleic and linolenic
acids.
The results of the free sterol analysis are presented in Figures
5 and 6. The major free sterol in strawberry leaflipid is sitosterol
(about 95 mol
and stigmasterol (Fig. 5). As with fatty acid composition, free
%o), with lesser amounts ofcholesteroL campesterol,
100
985
E
-
0
lr
ul
ui
It.
90
10.
5
10/79
1/80
4/80
7/80
MONTH
FIG. 5. Changes in free sterol composition during the annual cycle.
Data points represent the mean of three observations, except where *
indicates a single observation.
-9
z
z
10/79
I/80
4/80
7/80
01
m
D
0
r0
a
MONTH
FIG. 6. Changes in total phospholipids (0), free sterols (@), and the
ratio of free sterols to phospholipids (A). Data points and bars represent
the mean and SE for phospholipids with n = 6 and for free sterols with n
= 3, except where*indicates a single observation. Ratio is based on mean
values, except at *.
sterol composition remained qualitatively stable throughout the
lifespans of both the overwintering and the summer leaves (Fig.
5). The most notable quantitative change was a steady gradual
increase in the average levels of free sterols (on a fresh-weight
basis) during the aging and senescence ofboth leaftypes (Fig. 6).
This increase in free sterols represents a nonspecific enhancement
of all the sterol classes, given that the proportion of individual
sterols remained constant throughout. In the overwintering leaves,
the gradual increase between the prehardened October levels and
the midspring levels amounts to about 80%o augmentation in total
free sterols, based on average values. The major portion of this
increase occurred after the time of the January lipid extraction.
In the summer leaves, the level of free sterol rose about 17%
between June 22 and July 5 (Fig. 6). As with the overwintering
leaves, this free sterol increase was coincident with a substantial
loss of Chl (Table 1). Comparable leaves in the field senesced
completely within several days of the July lipid extraction. Sum-
mer leaves used in this final (July) lipid extraction were in a more
advanced stage of senescence than were the overwintering leaves
at the time oftheir final (April) extraction, as evidenced by greater
Chl loss (Table I) and other qualitative observations on leaf
condition.
Increased sterol levels in plants have been linked to senescence-
o-o sitosterol
cholesterol
h-a campesterol
6-A stigmasterol
20l
100
P2ospolipid
15--l-7
RiO
.
-A/
I
|00
X //-
----
50
S
t
-
Sterol(Xto l
25
.
-1--&
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O'NEILL ET AL.

Page 5

COLD ACCLIMATION AND AGING OF MEMBRANES
Table 1.Total chlorophyll content and chlorophyll
a/b ratios during the annual cycle.
Total
Leaf type/
Chlorophyll
chlorophyll
extraction date
a/b ratio
(mg/g)
Overwintering
10/3/79
1.63
2.25
1/8/80
1.69
2.02
3/5/80
1.70
2.49
4/22/80
1.02
2.51
Summer
6/2/80
0.61
2.21
6/11/80
1.35
2.45
6/22/80
1.35
2.47
7/5/80
0.51
1.71
related changes occurring in cellular membranes leading to in-
creased membrane viscosity (12). Since free sterols also increased
during the aging of summer leaves, we believe that the sterol
increases observed in the overwintering leaves are also age-related,
rather than a response to temperature per se, and represent an
increase in the microviscosity ofthe cellular membranes.
The most dramatic change in the lipid composition of the leaf
membranes involved increases in total phospholipid during cold
acclimation. Between October, 1979, and January, 1980, the total
phospholipid content of the overwintering leaves increased more
than 100% (Fig. 6). During this same interval, the free sterols
showed a lesser increase based on average values, amounting to a
30% enhancement overall by midwinter. The ratio offree sterol to
phospholipid (Fig. 6) was at its lowest during acclimation and
when the leaves were in their most cold hardy state (October to
January). Deacclimation of overwintering leaves was marked by
a progressive loss of phospholipids relative to the January peak
level. Phospholipids decreased 25% by March and 45% by April.
Despite these decreases, the phospholipid level remained well
above that of the prehardened October level. For the summer
leaves, there were no notable changes in the phospholipid content,
even during advanced senescence.
Galactolipids were measured in all the leaves. The levels ex-
ceeded the standard curves run at the same time; thus, absolute
quantitative data cannot be reported. Qualitatively, galactolipid
levels in the overwintering leaves remained relatively stable
through midwinter but showed a considerable decline in both
MGDG and DGDG in early spring. In April, this marked loss of
galactolipids appeared to be reversed in part, when an increase in
total galactolipids was observed. This increase was coincident with
the resumption of growth metabolism. The galactolipid levels of
the summer leaves were similar to those of the overwintering
leaves. A decline in both MDGD and DGDG was observed
towards the latter part of the summer leaf lifespan with the onset
of senescence.
Electron Microscopy. Summer leaf cells in June (Fig. 7) had a
FIG. 7. Electron micrograph of a palisade parenchyma cell of a repre-
sentative summer leaf in June. (x 19,820).
FIG. 8. Electron micrograph of palisade parenchyma cells of a repre-
sentative overwintering leaf in early October. (x 11,149).
single large vacuole. As a result, the nucleus occupied a lateral
position within the cell, and all other cellular contents and organ-
elles were peripheral, surrounding the large central vacuole. No
vesiculated material was visible in the cytoplasm, although an
occasional dictyosome and RER were visible. The chloroplasts
were filled with starch, and no osmiophilic globules were observed.
Overwintering leaves in October (Fig. 8) had several large vacu-
oles. Cytoplasm was mainly peripheral, although transvacuolar
strands were observed. Both RER and cisternae-like SER were
1413
PlantPhysiol.Vol. 68, 1981