Plant Physiol. (1978) 61, 639-643
Phase Behavior of Chloroplast and Microsomal Membranes
during Leaf Senescence'
Received for publication July 11, 1977 and in revised form November 21, 1977
BRYAN D. MCKERSIE AND JOHN E. THOMPSON
Department of Biology, University of Waterloo, Ontario, Canada
ABSTRACT
Wide angle x-ray diffraction of chloroplast and microsomal membranes
from primary leaves of Phaseolus vulgars has revealed that for both types
of membrane, portions of the lipid become crystalline as the tissue senesces.
For young leaves the transition temperature Is about 23 C for microsomes
and below -30 C for chloroplast membranes, Indicating that at physiological temperature the lipid is entirely liquid-crystalline. Between 2 and 3
weeks after planting the transition temperature rises to 38 C for microsomes, but for chloroplasts this Increase to a point above physiological
temperature does not occur until between 3 and 4 weeks. Thereafter the
transition temperature continues to rise for both types of membrane with
advancing senescence, although the rate of increase is greater for chloro-
plasts than for microsomes. Tbe appearance at physiological temperature
of gel phase lipid in the microsomes coincides temporally with the initiation
of a decline in total protein in the tissue, and the incidence of crystalinity
in chloroplasts coincides with loss of chlorophyll. This change in phase
behavior cannot be attrbuted to an alteration in fatty acid composition,
but for both membrane systems it correlates with an increase of about 4fold In the sterol to phospholipid ratio.
Foliar senescence is known to entail conspicuous changes in
metabolism and cell structure. Photosynthesis, e.g. declines before
the leaf is visibly senescent, and chloroplasts are the first organelles
to show morphological symptoms of deterioration. They decrease
in size coincident with loss of stroma, and the thylakoid membranes break down (1). The lipid framework of the thylakoids
appears to be dismantled into globules that accumulate in the
chloroplast interior, occupying 70 to 80% of its volume by late
senescence (1). Breakdown of macromolecules has also been observed in the senescing leaf. Chl, RNA, and protein levels decline
dramatically, and this appears to reflect loss of synthetic capability
rather than increased hydrolytic activity, since digestive enzyme
activity also decreases with advancing senescence (21).
Leaf senescence is not fundamentally different from cotyledon
senescence. In bean, e.g. metabolic activity is high in the young
cotyledons, but then declines during the later stages of germination
as senescence intensifies (17). The organelles of the storage cells
are initially intact, but with advancing senescence there is a general
deterioration of cytoplasmic structure (18). Loss of membrane
structure and function features prominently in this deterioration.
The activities of several membrane-bound enzymes decline (12)
and, perhaps of greater significance, portions of the membrane
lipid become crystalline as the tissue senesces (13, 14). The physical
state of membrane lipids is an important factor in determining
such key membrane functions as permeability and enzyme activity
(3, 19), and it is conceivable that the changing physical state of
the lipid in senescing membranes from cotyledons contributes to
their loss of function. In view of the similarities between foliar
and cotyledon senescence, we have examined the phase behavior
of chloroplast and microsomal membranes from senescing primary
leaves of Phaseolus vulgaris to determine whether lipid crystallization in membranes might be a feature of senescence in systems
other than the cotyledon.
MATERIALS AND METHODS
Growth Conditions and Fractionation. Seeds of P. vulgaris (var.
Kinghorn) were germinated in a mixture of sand, peat moss, and
soil (1:1:2). The plants were grown under greenhouse conditions
with a 16-hr photoperiod. The mean growth temperature was 26 C.
Primary leaves were harvested at various stages of senescence;
10-g samples were used for dry wt determinations, and for isolation
of chloroplasts and microsomes 100 g of tissue was homogenized
with a Sorvall Omni-Mixer in 50 ml of cold 0.3 M sucrose-0.05 M
NaHCO3 (pH 7). The homogenate was filtered through cheesecloth and centrifuged at 200g for 10 min. The supernatant was
centrifuged at 2,000g for 10 min and the pellet was resuspended
in the same volume of homogenizing buffer and centrifuged again
at 2,000g for 10 min to yield a crude chloroplast fraction. A
portion of this crude chloroplast fraction was retained for x-ray
diffraction and lipid analysis. The remainder was centrifuged
through a 10 to 18% continuous gradient of Ficoll in 0.5 M sucrose0.05 M NaHCO3 (pH 7) at 20,000g for 30 min as described by
Brandt and Benveniste (2) in order to purify the chloroplasts. The
lower layer on the gradient, which comprised the purified chloroplast fraction, was removed, diluted with homogenizing buffer,
and pelleted by centrifugation.
Microsomes were isolated as previously described from a
10,000g, 20-min supernatant on a discontinuous sucrose gradient
designed to separate the membranes from free ribosomes (13). For
this purpose 9 ml of the supernatant were layered over 3 ml of 1.8
M sucrose and centrifuged at 150,000g for 2 hr. The microsomal
membranes which collected at the interface were removed, diluted
with 3 volumes of homogenizing buffer, and pelleted by centrifugation at 165,000g for I hr.
X-Ray Diffraction. Samples of the microsomal and chloroplast
fractions were prepared for x-ray diffraction as previously described (13, 14). Wide angle diffraction patterns were recorded
using CuKa radiation from a point-focused x-ray tube (type PW
2103/01) on a Philips (type 1030) camera under conditions in
which the samples retain 50 to 75% moisture with respect to final
dried wt (13). For each sample patterns were recorded at room
temperature (approximately 22 C), and the lipid phase transition
temperature was determined to within I C as the highest temperature at which gel phase lipid could be detected.
Lipid Analysis. Lipids were extracted from the isolated fractions
and washed according to the method of Nichols (16). Fatty acids
were obtained by suspending an aliquot of the lipid extract in I
ml of 0.1 N methanolic NaOH and heating at 95 C for 2 hr under
I
Supported by a grant-in-aid from the National Research Council of
Canada.
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639
Copyright © 1978 American Society
of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 61, 1978
McKERSIE AND THOMPSON
640
N2. One ml of water was added and the sample was washed three
times with 2 ml of pentane. The free fatty acids were recovered by
acidifying the methanolic layer with HCI and extracting with 2 ml
of pentane. Methyl esters of the fatty acids were prepared by
adding 1 ml of 14% BF3 in methanol and heating the mixture to
95 C for 10 min in a N2 atmosphere (15). The esters were extracted
with 2 ml of pentane and 1 ml of water and their purity confirmed
by TLC. The methyl esters were separated and identified by flame
ionization gas chromatography using a stainless steel column
(182.9 x 0.64 cm) packed with 10%1o EGSS-X in 100/120 Supelcoport and maintained at 170 C. Peak areas were determined with
a mechanical disc integrator.
Sterol levels were determined by procedures described by Grunwald (8). A portion of the lipid extract was taken up in 4 ml of
acetone-ethanol (1:1) and the free sterols were precipitated by
adding 2 ml of 0.5% digitonin (Sigma) and leaving the mixture
overnight at room temperature. The precipitate was washed once
with acetone-diethyl ether (1:1) and twice more with diethyl ether.
The digitonin-sterol complex was then broken down by adding 2
ml of pyridine containing cholestane, which was used as an
internal standard, and heating to 60 C for 1 hr. After storing
overnight at room temperature the mixture was diluted with 25
ml of diethyl ether and the digitonin was removed by centrifugation at 10,000g for 30 min. The supernatant was evaporated to
dryness and trimethylsilyl derivatives of the sterols were prepared
using equal volumes of acetonitrile and BSTFA (Chromatographic Specialties). The derivatized sterols were analyzed by
GLC on a glass column (182.9 x 0.64 cm) packed with 3% OV-17
Chromosorb W 100/120 mesh at 260 C. The sterols were
identified on the basis of their retention times relative to known
standards. Relative weight responses to cholestane were determined for cholesterol, campesterol, stigmasterol and f-sitosterol
using authentic standards obtained from Sigma and Applied
Science. The relative weight responses for the other sterols for
which standards could not be obtained was assumed to be 1.
Phospholipid levels were determined by measuring the Pi content of the lipid extracts after perchloric acid digestion according
to the method of Fiske and SubbaRow as described by Dittmer
and Wells (5).
Protein and Chi Analyses. Homogenate protein levels were
determined by the method of Lowry et al. (10). Chl content was
determined by extracting 0.1 ml of the homogenate with 10 ml of
acetone and reading the A at 665 nm as described by Phillips et al.
on
(21).
RESULTS
Profiles of protein and Chl attenuation during senescence of the
primary leaf of P. vulgaris are illustrated in Figure 1. Total protein
expressed relative to dry wt rose during the early stages of growth
to reach a peak about 2 weeks after planting, and thereafter
declined as senescence set in to reach a low level by 4 weeks. Chl
levels also rose during the early stages of seedling development to
reach a peak 3 weeks after planting, and then subsequently
declined through to 5 weeks. By this time the leaves were extensively senescent and beginning to abscise.
Wide angle x-ray diffraction patterns of crude and purified
chloroplast fractions as well as microsomal membranes were recorded throughout the senescence period. For 2-week-old leaf
tissue, the room temperature patterns of all three fractions featured
two broad reflections centered at Bragg spacings of 4.6 A and 10
A (Fig. 2, A, C and E). These patterns are typical of those obtained
ereviously for many different types of membranes (3, 11). The 10
A reflection is thought to derive from membrane protein, but is
not well characterized (6). The diffuse 4.6 A band is a lipid
reflection deriving from the fatty acid side chains of the membrane
lipid arranged in a liquid-crystalline (fluid) phase (3, 11).
Diffraction patterns recorded at room temperature for fractions
obtained from older leaf tissue were identical, except that a sharp
I
I
as0 c
4.0[
0
39
0'
3.
-c
150 oc
b0.' 3.0
.0
r-
4..
'00n
< 2.0
E.E
50
I.C
2
3
4
5
Age (weeks)
FIG. 1. Changes in protein (0) and Chl (0) content of the primary leaf
of P. vulgaris during senescence. Values are the means of three to six
determinations; standard errors of the means are shown. Dry weights were
determined in triplicate for each sample. Protein and Chl were determined
in duplicate from tissue homogenates.
band centered at a Bragg spacing of 4.15 A became increasingly
apparent as senescence intensified. This is illustrated in Figure 2,
B, D and F which show patterns for microsomal, crude chloroplast,
and purified chloroplast membranes from 5-week-old tissue. In
addition, a fainter ring was evident in the patterns for the crude
chloroplast fraction at a Bragg spacing of 3.75 A (Fig. 2D). The
sharp 4.15 A reflection has been well characterized, and its appearance in wide angle patterns from membranes indicates the
presence of a gel (crystalline) phase in which there is a rigid,
hexagonal packing of the hydrocarbon chains (11). The 3.75 A
band reflects an alternative crystalline state of the lipid in which
there is an orthorhombic rather than hexagonal packing of the
hydrocarbon chains (14). For microsomal membranes the gel
phase first appeared 3 weeks after planting, but in chloroplast
membranes it was not detectable until 4 weeks. The intensity of
the 4.15 A reflection increased in all fractions with age indicating
that as senescence advanced an increasing proportion of lipid in
both microsomal and chloroplast membranes was converted from
a liquid-crystalline to a gel phase. This intensity change was
greater for the crude chloroplast fraction than for the purified
chloroplasts (compare Fig. 2, D and F), suggesting that the more
senescent chloroplasts are excluded from the purified fraction.
However, even by the 5th week, the proportion of lipid in the gel
phase for all three fractions remained small in comparison to that
in the liquid-crystalline phase as indicated by the relative intensities of the 4.15 A and 4.6 A peaks (Fig. 2, B, D and F). Even
when diffraction patterns for 5-week-old chloroplat and microsomal membranes were recorded at -30 C (approximately 70 C
below the transition temperature) the relative intensities of the
4.15 A and 4.6 A bands did not change markedly, indicating that
only a small proportion of the lipid is able to form a gel phase.
Determinations of the lipid phase transition temperatures confirmed that structural modifications had occurred in the lipid
component of both chloroplast and microsomal membranes during senescence. The transition temperature is defined as the highest
temperature at which gel phase lipid can be detected. At temper-
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Copyright © 1978 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 61, 1978
A
C
641
PHASE BEHAVIOR OF SENESCING MEMBRANES
atures above the transition only a liquid-crystalline phase is present; at temperatures below the transition, both liquid-crystalline
and gel phases are detectable. Gel phase lipid was not detectable
in either purified or crude chloroplast preparations from young
tissue at temperatures as low as -30 C (Table I). However, by
week 4 a liquid-crystalline to gel phase transition was initiated at
31 C for the purified chloroplasts and at 46 C for the crude
chloroplasts. By week 5, the transition temperatures had risen still
B1 | twwrr 8 further to 42 and 52 C for the purified and crude chloroplast
fractions, respectively (Table I).
The transition temperature for microsomal membranes from
young tissue proved to be much higher than that for the corresponding chloroplast membranes, but still remained below the
_N4^>:'
mean growth temperature (Table 1). However, by week 3 it had
risen to 38 C, a point well above the growth temperature, but
i _ e2e''5,tr:,
thereafter increased only slightly through to week 5 (Table 1).
Since fatty acids can influence the phase behavior of biological
membranes (3), the effect of senescence on the fatty acid composition of chloroplast and microsomal membranes was examined.
The total extracted lipid from both types of membrane contained
six fatty acids-the saturated fatty acids palmitic (16:0) and stearic
(18:0), and the unsaturated fatty acids palmitoleic (16:1), oleic
(18:1), linoleic (18:2), and linolenic (18:3). Crude and purified
chloroplast fractions from 9-day-old leaf tissue contained predominantly linolenic acid, which comprises 60% of the total fatty acid
complement, and palmitic acid, which comprises 20%Yo (Table II).
The microsomes had a similar fatty acid composition-47% linolenic and 24% palmitic-although the proportion of linoleic acid
was substantially higher (Table II).
This proportionality among the fatty acids changed as the leaf
aged (Table II). In the chloroplast membranes lnolenic acid
D
.
increased to over 70%o by 3 weeks, coincident with a corresponding
decline in palmitic acid. Subsequently, linolenic acid decreased,
reaching about 60% of the total by 5 weeks, and palmitic acid rose
,1-_..again to 20%. There was also a progressive decrease, especially
after 3 weeks, in the proportion of palmitoleic acid (Table II). For
microsomes the only conspicuous change during senescence was
a loss of linoleic acid (Table II).
,,
:
E
Changes in the unsaturated to saturated fatty acid ratio have
been graphed in Figure 3 to enable a better comparison with the
changes in phase behavior detected by x-ray diffraction. The
microsomal membranes show only a slight decrease in this ratio
during senescence. However, the ratio for the chloroplast membranes peaks at 3 weeks and subsequently declines to reach a level
by 5 weeks that is slightly below that of 9-day-old tissue (Fig. 3).
Analysis of free sterols in the microsomal and chloroplast
fractions revealed the presence of at least seven. However, /8sitosterol and stigmasterol collectively comprised about 85% of the
total in each membrane system. Others which were detectable
.
Fw
F
Table I. Changes in Lipid Phase Transition Temperatures of Chloroplast
and Microsomal Membranes from the Primary Leaf of Phaseolus vulgaris
during Senescence.
The values are means of 3-4 determinations and represent the highest
temperature at which gel phase lipid could be detected by wide angle
x-ray diffraction. Standard errors of the means are shown.
Transition Temperature
(C°)
Tissue
Age
9
2
3
4
5
FIG. 2. Wide angle x-ray diffraction patterns recorded at room temperature (22 C) from A: 2-week-old microsomes; B: 5-week-old microsomes; C: 2-week-old crude chloroplasts; D: 5-week-old crude chloroplasts;
E: 2-week-old purified chloroplasts; and F: 5-week-old purified chloroplasts isolated from the primary leaf of P. vulgaris. A, C, and E show (from
day
week
week
week
week
Crude Chloroplasts
Below
Below
Below
46 ±
52 +
Purified Chloroplasts
-30
-30
-30
2
1
Below
Below
Below
31 ±
42 ±
-30
-30
-30
6
1
Microsomes
23 ± 2
22 + 3
38 + 2
43 ± 0.3
43 ± 1
outside to inside) diffuse bands at 4.6 A and about 10 A; B and F show
(from outside to inside) a sharp band at 4.15 A and diffuse bands at 4.6
A and about 10 A; D shows sharp bands at 3.75 A and 4.15 A, and diffuse
bands at 4.6 A and about 10 A.
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Copyright © 1978 American Society of Plant Biologists. All rights reserved.
642
Plant Physiol. Vol. 61, 1978
McKERSIE AND THOMPSON
.
Table II. Changes in the Fatty Acid Composition of the Total Extracted
Lipid from Chloroplast and Microsomal Fractions of the Primary Leaf of
Phaseolus vulgaris during Senescence.
. .
v
-
800
Values are the means of 3-6 determinations. Standard errors of the means
were routinely less than 1%, except for 16-0 and 18-3 which were always
less than 2.5%.
._
8
Fraction
Crude
Chloroplast
Purified
Chloroplast
Microsomes
1
Fatty
Acid
a
% of Total Fatty Acids
1
06
9 day
2 week
3 week
4 week
5 week
16:0
16:1
18:0
18:1
18:2
18:3
18.9
8.0
1.8
3.0
9.3
59.2
13.6
7.7
1.4
1.2
4.5
71.6
12.2
6.3
1.4
2.3
5.5
72.1
15.8
5.7
2.0
1.6
5.0
69.8
20.8
3.1
2.8
4.8
6.7
61.2
16:0
16:1
18:0
18:1
18:2
18:3
17.6
15.3
4.9
3.1
3.7
4.8
66.0
6.5
2.3
2.9
3.8
69.1
12.4
6.9
1.7
2.0
4.3
72.7
17.2
3.8
3.3
3.0
4.8
67.4
23.4
2.4
4.3
6.5
6.6
56.9
16:0
16:1
18:0
18:1
18:2
18:3
23.6
4.8
24.1
3.8
2.3
25.7
2.6
3.8
5.2
12.6
50.1
26.8
2.4
4.1
3.2
10.1
53.4
27.2
1.5
4.5
5.1
10.5
51.2
2.3
2.9
19.9
46.6
4.4
13.9
50.7
16:0, palmitic acid; 16:1, palmitoleic acid; 18:0, stearic acid; 18:1,
oleic acid; 18:2, linoleic acid; 18:3, linolenic acid.
600[
E
E
400
0
q8,
200
E
2
3
Age (weeks)
4
5
FIG. 4. Changes in total free sterol content of crude chloroplast (0),
purified chloroplast (0), and microsomal (A) fractions of the primary leaf
of P. vulgaris during senescence. Values are expressed relative to phospholipid content and are means of three to six determinations. All three
fractions show a significant increase with age at the 1% level as determined
by a one-way analysis of variance. Least significant differences at the 1%
level are: purifi'ed chloroplasts, 123; crude chloroplasts, 144; microsomes,
167.
7.0
(Fig. 4). The microsomal membranes consistently showed a higher
sterol to phospholipid ratio than did the chloroplast membranes,
and the ratio was also somewhat higher for the crude chloroplast
fraction than for the purified chloroplasts (Fig. 4).
6.0
5.0[
DISCUSSION
The use ofwide angle x-ray diffraction for monitoring the phase
0
behavior of polar lipids in biological membranes is well docu3.0[
mented (11). For most membranes the hydrocarbon side chains of
these lipids are in a liquid-crystalline (fluid) phase under physiological conditions. Lipid in this form registers as a broad reflection
2.0[
centered at a Bragg spacing of 4.6 A in wide angle diffraction
patterns and has been observed for liquid paraffins (11) as well as
1.0
both artificial and biological membranes (6, 11). The gel phase, in
which the fatty acid chains are packed hexagonally into a crystal4
line lattice, registers as a sharp reflection at a Bragg spacing of
3
5
2
4.15 A in diffraction patterns. This phase is found in long chain
Age (weeks)
FIG. 3. Changes in fatty acid unsaturation of the total extracted lipids paraffms but is not generally observed for biological membranes
from crude chloroplast (0), purified chloroplast (0), and microsomal (A) except at low temperatures (3) or in membranes from bacterial
fractions of the primary leaf of P. vulgaris during senescence. Values are fatty acid auxotrophs which have unusually high saturation levels
expressed as the ratio of the unsaturated fatty acids (16:1, 18:1, 18:2, and (23).
It is clear from the diffraction data that the lipid of both
18:3) to the saturated fatty acids (16:0 and 18:0) as detailed in Table II.
Significance was determined by a one-way analysis of variance, and least chloroplast and microsomal membranes from young primary
significant differences (LSD) were calculated for each fraction during leaves of P. vulgaris is exclusively liquid-crystalline. However, the
senescence. LSD values are: crude chloroplasts, 1.6; purified chloroplasts, appearance of the sharp 4.15 A reflection in diffraction patterns
1.8; microsomes, not significant.
for older membranes indicates that as the tissue senesces portions
of the lipid matrix convert to the gel phase under physiological
included campesterol, isofucosterol, cholesterol, and two uniden- conditions. The intensity of the 4.15 A reflection was greater for
tified sterols with retention times greater than that for isofucos- the crude chloroplast fraction than for the purified chloroplast
terol, which collectively comprised less than 5% of the total free fraction. The most logical interpretation of this observation is that
sterol. The sterol compositions of the chloroplast and microsomal the more senescent chloroplasts, which would also have a greater
membranes were very similar and the relative proportions of the proportion of crystalline lipid, were excluded from the purified
sterols did not change appreciably with age apart from a slight fraction by reason of structural and morphological alterations
decrease in the amount of fi-sitosterol.
which changed their centrifugation properties. This is supported
The most striking change in lipid composition during senescence by the fact that the transition temperatures of these two fractions,
was a dramatic increase in the total free sterol to phospholipid which in effect reflect the lipid composition of the crystalline
ratio in both chloroplast and microsomal membranes (Fig. 4). regions, change in parallel during the senescence period. MoreThis increase was initiated between 2 and 3 weeks for both over, they do so in a manner that can be distinguished temporally
fractions, and continued almost
linearly throughout senescence from corresponding changes for microsomes. The transition temDownloaded from on July 28, 2017 - Published by www.plantphysiol.org
4.0 -
N
Copyright © 1978 American Society of Plant Biologists. All rights reserved.
Plant Physiol. Vol. 61, 1978
PHASE BEHAVIOR OF SENESCING MEMBRANES
peratures for chloroplast and microsomal membranes from young
tissue are quite distinct (below -30 C and approximately 22 C,
respectively), and the rise in transition temperature with age is
initiated earlier in the microsomal membranes.
The appearance of gel phase lipid during senescence can be
correlated with parameters of tissue autolysis. For example, the
initial decline in the protein content of the leaf between 2 and 3
weeks of age coincides temporally with the appearance of gel
phase lipid in the microsomal membranes. Similarly, the initial
loss of Chl between 3 and 4 weeks correlates temporally with
formation of the gel phase in the chloroplast membranes. It is not
clear from these observations whether the presence of the gel
phase contributes to the initiation of tissue autolysis or is merely
a result of it. It has been shown (19, 25) in studies with liposomes
that permeability to ions increases dramatically at the phase
transition when both liquid-crystalline and gel phases are present.
Gel phase lipid has also been detected in senescing membranes
from P. vulgaris cotyledons (13, 14). It is conceivable, therefore,
that the presence of gel phase lipid, which would give rise to
discontinuities in the bilayer where it interfaced with adjacent
liquid-crystalline lipid, contributes to loss of cellular compartmentalization during senescence.
The onset of lipid crystallization in membranes from senescing
tissue presumably reflects chemical changes, particularly in the
lipid. Increased levels of fatty acid saturation are known to induce
a transition to the gel phase (3), but it is clear that the incidence
of crystallinity in the membranes of senescing leaf cannot be
attributed solely to changes in the degree of hydrocarbon saturation. For microsomes the unsaturated to saturated fatty acid ratio
does not change appreciably during senescence, and for chloroplasts the ratio at 9 days of age when there is no gel phase lipid in
the membranes is comparable to that for 4-week-old chloroplasts
which do contain gel phase lipid. Small changes in the saturation
of individual polar lipids would not be detected by these measurements since total lipid extracts were used for the fatty acid
analyses. However, the large changes in saturation, which are
required for comparable shifts in lipid transition temperature of
bacterial membranes (23), are clearly not incurred. This is consistent with our previous observation that changes in the phase
behavior of membranes from senescing cotyledon tissue as well
are not due solely to changes in fatty acid saturation (13). The
microsomal and chloroplast membranes from leaf contain a high
proportion of linolenic acid which by reason of its high unsaturation would not readily pack into a hexagonal lattice. This may
well account for the fact that for both types of membrane only a
small proportion of the lipid became crystalline, even at temperatures 70 C below the phase transition temperature.
The appearance of crystalline lipid in senescent chloroplast and
microsomal membranes could reflect a lateral redistribution of the
polar lipids in the plane of the membrane, whereby localized
regions in the lipid matrix become disproportionately rich in
saturated fatty acids and form a separated gel phase. This is more
conceivable when it is realized that the various polar lipids of this
tissue have distinctly different degrees of fatty acid saturation (22).
A number of agents including divalent cations (3), protein (20),
and cholesterol (9, 24) are known to influence the mobility of
phospholipids. Cholesterol, for example, alters the phase transition
temperature of pure phospholipids (9, 24) and can interact selectively with specific classes of phospholipids (4). The effect of plant
sterols on the phase behavior of lipid bilayers has not been
established. Nevertheless, it may be significant that the sterol to
phospholipid ratio increases by about 4-fold in both microsomal
and chloroplast membranes during leaf senescence. Chloroplast
643
membranes are rich in galactolipids, but galactolipids and phospholipids change in parallel during senescence of this tissue (7),
and thus measurements of the sterol to phospholipid ratio still
provide a realistic indication of changes in the sterol to fatty acid
ratio. It is conceivable that the higher levels of sterols in the older
membranes reflect a more rapid loss of polar lipid than of sterols
during senescence. There may also be some conversion of sterol
esters and sterol glycosides to free sterols as senescence intensifies.
In any case, whatever the cause, it seems reasonable to propose
that the increase in free sterols together with the loss of Chl and
presumably protein help to bring about a redistribution of polar
lipids in the plane of the membrane that results in formation of
the gel phase.
LITERATURE CITED
1. BARTON R 1966 Fine structure of mesophyll cells in senescing leaves of Phaseolus. Planta 71:
314-325
2. BRANDT RD, P BENVENISTE 1972 Isolation and identification of sterols from subcellular
fractions of bean leaves (Phaseohs vulgans). Biochim Biophys Acta 282: 85-92
3. CHAPMAN D 1975 Fluidity and phase transitions of cell membranes In H Eisenbeig, E
Katchalski-Katzin, LA Manson, eds, Biomembranes, Vol 7. Plenum Press, New York, pp
1-9
4. DEMEL RA, JWCM JANSEN, PWM VAN DUCK, LLM VAN DEENEN 1977 Preferential inter
action of cholesterol with different classes of phosphollpids. Biochim Biophys Acta 465: 1-10
5. DIrrMER JC, MA WELLS 1969 Quantitative and qualitative analysis of lipids and lipid
components. Methods Enzymol 14: 482-530
6. ESFAHANI M, AR LIMBRICK, S KNu-roN, T OKA, SJ WAKIL 1971 The molecular organization
of lipids in the membranes of E. cobl: phase transitions. Proc Nat Acad Sci USA 68:
3180-3184
7. FoNac F, RL HEATH 1977 Age dependent changes in phospholipids and galactolipids in
primary bean leaves (Phasohu vulgars). Phytochemistry 16: 215-217
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