Journal of Experimental Botany, Vol. 53, No. 378, pp. 2159±2166, November 2002
DOI: 10.1093/jxb/erf076
Plasma membrane lipids in the resurrection plant
Ramonda serbica following dehydration and rehydration
Mike F. Quartacci1, Olivera GlisÏicÂ2, Branka StevanovicÂ2 and Flavia Navari-Izzo1,3
1
Dipartimento di Chimica e Biotecnologie Agrarie, UniversitaÁ di Pisa, Via del Borghetto, 80, 56124 Pisa, Italy
2
Institute of Botany, University of Belgrade, Takovska 43, 11000 Belgrade, Yugoslavia
Received 10 December 2001; Accepted 3 July 2002
Abstract
Plants of Ramonda serbica were dehydrated to 3.6%
relative water content (RWC) by withholding water for
3 weeks, afterwards the plants were rehydrated for 1
week to 93.8% RWC. Plasma membranes were isolated from leaves using a two-phase aqueous polymer
partition system. Compared with well-hydrated (control) leaves, dehydrated leaves suffered a reduction of
about 75% in their plasma membrane lipid content,
which returned to the control level following rewatering. Also the lipid to protein ratio decreased after
dehydration, almost regaining the initial value after
rehydration. Lipids extracted from the plasma membrane of fully-hydrated leaves were characterized by a
high level of free sterols and a much lower level of
phospholipids. Smaller amounts of cerebrosides, acylated steryl glycosides and steryl glycosides were
also detected. The main phospholipids of control
leaves were phosphatidylcholine and phosphatidylethanolamine, whereas sitosterol was the free sterol
present in the highest amount. Following dehydration,
leaf plasma membrane lipids showed a constant level
of free sterols and a reduction in phospholipids compared with the well-hydrated leaves. Both phosphatidylcholine and phosphatidylethanolamine decreased
following dehydration, their molar ratio remaining
unchanged. Among free sterols, the remarkably high
cholesterol level present in the control leaves (about
14 mol%) increased 2-fold as a result of dehydration.
Dehydration caused a general decrease in the unsaturation level of individual phospholipids and total
lipids as well. Upon rehydration the lipid composition
of leaf plasma membranes restored very quickly
approaching the levels of well-hydrated leaves.
Key words: Dehydration, lipids, plasma
Ramonda serbica, resurrection plants.
membrane,
Introduction
Flowering plants growing in hot and arid regions usually
survive the harsh environmental conditions either by
avoiding the stressful events or by very promptly activating adaptative resistance mechanisms. Only a small
number of higher plants, mostly originating from the
southern hemisphere and called desiccation-tolerant or
resurrection plants, are capable of surviving almost
complete dehydration for prolonged periods. Ramonda
spp, as well as other species belonging to the family
Gesneriaceae, are among the resurrection plants which
grow in the northern hemisphere. Ramonda serbica is a
rare resurrection plant growing in the Balkan peninsula
(Gaff, 1981; StevanovicÂ, 1986). This species is capable of
surviving long dry periods between the wet periods,
passing quickly from anabiosis, which can last much
longer than three months depending on water de®cit
severity and temperatures, to the state of full biological
activity in less than 8±10 h if the favourable water balance
in the soil re-establishes suddenly.
In spite of the fact that metabolic processes are almost
stopped in resurrection plant dry leaves, the cell membranes as well as most of the enzymatic systems are
protected in different ways (Bewley and Krochko, 1982;
Oliver, 1996; Navari-Izzo and Rascio, 1999). It has been
suggested that the rapid and ef®cient recovery and full
reconstitution of membrane organization and functionality,
as well as the presence of effective membrane defence
mechanisms, are the most important prerequisites for
3
To whom correspondence should be addressed. Fax: +39 050 598614. E-mail: [email protected]
Abbreviations: ASG, acylated steryl glycosides; FS, free sterols; PA, phosphatidic acid; PL, phospholipids; PC, phosphatidylcholine; PE,
phosphatidylethanolamine; PI, phosphatidylinositol; PG, phosphatidylglycerol; PM, plasma membrane; RWC, relative water content; SG, steryl glycosides.
ã Society for Experimental Biology 2002
2160 Quartacci et al.
survival upon rehydration (Stevanovic et al., 1992; NavariIzzo et al., 1995; Navari-Izzo and Rascio, 1999; Sgherri
et al., 2000).
According to Oliver (1996), Ramonda serbica seems to
belong to the group of resurrection plants which are able to
withstand desiccation using both morphological and
physiological mechanisms to slow down and, for a while,
to control the rate of water loss. The results of some recent
investigations indicate that Ramonda serbica has the
ability to maintain cell membrane integrity, i.e. to preserve
semipermeability during dehydration (Stevanovic et al.,
1998), as well as to activate protective mechanisms that
increase the level of zeaxanthin and the amounts of
reduced ascorbate and glutathione, which are crucial for
photoprotection during the dehydration/rehydration cycle
(Augusti et al., 2001). Furthermore, it has been found that
an increased amount of phenolic acids also protects
Ramonda membranes during desiccation (Sgherri et al.,
2000).
In the last decade, only a few studies on lipids extracted
from thylakoids or the whole plant have been undertaken
in order to explain desiccation tolerance in resurrection
plants (Stevanovic et al., 1992; Stefanov et al., 1992;
Navari-Izzo et al., 1995; Quartacci et al., 1997). The
investigations showed the general tendency of dehydrated
plants to adapt their membranes to the altered conditions,
and to recover quickly on rehydration both the lipid
composition and the order parameters of the well hydrated
plants. However, there is a complete lack of knowledge
about changes of lipids during dehydration and rehydration
for the plasma membrane (PM). The composition and
organization of PM lipids are crucial for intracellular
metabolism. Many vital activities of cells originate in the
membrane, the structure and function of which are
profoundly altered following water stress that leads to
destructive events such as phase transition, fusion and
increased permeability. The composition and physical
state of the lipid bilayer in¯uence lipid±protein and
protein±protein associations, membrane-bound enzyme
activities and the carrier-mediated transport capacity of
membranes (Navari-Izzo and Rascio, 1999; Leprince et al.,
2000; Navari-Izzo et al., 2000; Kerkeb et al., 2001).
Preserving membrane integrity, resurrection plants are
capable of surviving during anabiosis and returning
quickly to the complex and dynamic whole-organism
functionality upon rehydration (Quartacci et al., 1997;
Navari-Izzo et al., 1995, 2000).
The aim of the present study was to examine the
composition of lipids of plasma membranes isolated
from Ramonda serbica leaves, as well as to determine
changes during a dehydration/rehydration cycle in order
to explain, at least in part, the plant's ability to
reactivate its physiological functions so rapidly after
rewatering.
Materials and methods
Plant material
Specimens of the desiccation-tolerant plant Ramonda serbica PancÏ.
& Petrov. were collected from their natural habitat in the south-east
region of Serbia, in a gorge near the town of Nis. There, the plants
grow on rocky slopes, exposed from north to north-east, on a thin
layer of rich, mature, organo-mineral and dark mountain soil (pH
8.4) spread over limestone. During summer the habitat is
characterized by high air temperatures and a remarkable decrease
in air humidity and the plants pass to, and stay in, the anabiotic state,
although they never receive sunlight directly. Plants of the same age
were harvested together with the layer of soil on which they grew.
After collection, plants were acclimated for 2 weeks keeping them
fully watered until the beginning of the experiments. Plants were
dehydrated for 3 weeks by withholding water at room temperature
and ambient photoperiod. Rehydration was started by spraying the
plants with water to simulate rainfall and keeping the soil damp. The
rehydrated samples were collected after 1 week during which they
were watered daily.
Relative water content
At regular intervals during the dehydration/rehydration cycle
measurements of the relative water content (RWC) of the leaves
were carried out as previously reported (Sgherri et al., 1994). For the
analyses, mature and fully expanded leaves from the middle of
the rosettes and comparable in size were selected. The RWC of the
leaves was calculated according to the formula: 1003[(fresh
weight±dry weight)/(saturated weight±dry weight)] and expressed
as the mean value of ten replicates for each treatment.
Solute leakage
For solute leakage determination samples of the same weight were
obtained from leaves comparable in size. The plant material was
washed in double-distilled water to remove the contents of the cut
cells, soaked in 25 ml of double-distilled water, shaken at room
temperature for 24 h and aliquots for leachate measurements were
taken. Samples were then immersed for 5 min in liquid N2, placed
again in the same vial containing the leachate and shaken for an
additional hour prior to the measurement of the maximum conductivity (Metrohm 660 conductometer). The injury index was calculated according to the formula: injury index %=1±[(T±C)/T]3100,
where T and C represent the conductivity of the leachate after and
before liquid N2 treatment, respectively.
Plasma membrane preparation
Plasma membranes were prepared using a two-phase aqueous
polymer partition system. Leaves were cut into pieces and immediately homogenized in the isolation medium consisting of 50 mM
Tris-HCl, pH 7.5, 0.25 M sucrose, 3 mM Na2EDTA, 10 mM ascorbic
acid, and 5 mM diethyldithiocarbamic acid. The homogenate was
®ltered through four layers of a nylon cloth and centrifuged at 10 000
g for 10 min. The supernatant was further centrifugated at 65 000 g
for 30 min to yield a microsomal pellet, which was resuspended in 2
ml of a resuspension buffer (5 mM K-phosphate, pH 7.8, 0.25 M
sucrose, and 3 mM KCl). Plasma membranes were isolated by
loading the microsomal suspension (1.0 g) onto an aqueous twophase polymer system to give a ®nal concentration of 6.6% (w/w)
Dextran T500, 6.6% (w/w) polyethylenglycol, 5 mM K-phosphate
(pH 7.8), 0.25 M sucrose, and 3 mM KCl. The PM was further
puri®ed using a two-step batch procedure. The resulting upper-phase
was diluted 4-fold with 50 mM Tris-HCl, pH 7.5, containg 0.25 M
sucrose, and centrifuged for 30 min at 100 000 g. The resultant PM
pellet was resuspended in the same buffer containing 30%
ethylenglycol and stored at ±80 °C for lipid analyses. All steps of
Plasma membrane lipids in Ramonda 2161
±1
±1
Table 1. Distribution of marker enzymes (mmol mg protein min ) in the upper and lower phases of the partition system used for
the isolation of plasma membrane from well hydrated and dehydrated leaves of Ramonda serbica
Results are the means of three independent experiments 6SE. UP, upper phase; LP, lower phase.
Marker enzymes
ATPase
Vanadate-sensitive ATPase
NO3±-sensitive ATPase
NADH-cyt c reductase
Cyt c oxidase
Latent IDPase
Well hydrated leaves
Dehydrated leaves
UP
LP
UP
LP
0.42160.021
0.06360.005
0.37460.026
0.00660.001
0.00460.001
0.04160.005
0.26760.019
0.15560.014
0.02560.004
0.18760.010
0.03060.003
0.10660.009
0.51460.022
0.03160.004
0.51260.020
0.03260.003
0.00460.001
0.02160.003
0.31760.015
0.20160.012
0.06360.004
0.22760.011
0.09860.009
0.07260.006
the isolation were carried out at 4 °C. Plasma membrane pellets for
enzyme activity determinations were used immediately.
In order to check the purity of the PM, the activity of the vanadatesensitive ATPase as a marker enzyme was determined (Table 1).
Cytochrome c oxidase, antimycin A-insensitive NADH cyt c
reductase, NO3±-sensitive ATPase and latent IDPase activities
were used as markers of mitochondria, endoplasmic reticulum,
tonoplast, and Golgi membranes, respectively (Navari-Izzo et al.,
1993). Chlorophyll was not detected in the PM fraction. Tests with
the markers showed that the partition behaviour of both PM and
intracellular membranes may be in¯uenced by water contents since
the net charge density of membranes is also related to their polar
head group composition. The protein content was determined taking
aliquots of the PM suspension. The analysis was performed
according to Bradford (1976) with bovine serum albumin as a
standard.
glucose and KH2PO4 as standards, respectively. All procedures were
performed in the presence of silica gel from TLC.
Lipid extraction and separation
Lipids were extracted from the PM suspension by the addition of
boiling isopropanol followed by chloroform:methanol (2:1 v/v)
containing butylhydroxytoluol (50 mg ml±1) as an antioxidant. The
solvent mixture was then washed with 0.88% KCl to separate the
chloroform phase. The upper water phase was re-extracted with
chloroform, the chloroform phases combined and dried under a
stream of N2. The lipid extracts were stored at ±20 °C and retained
for further separation. Lipids were fractionated into neutral lipid,
glycolipid and phospholipid (PL) fractions on Sep-Pak cartridges
(Waters) (Uemura and Steponkus, 1994). Lipid extracts dissolved in
chloroform:acetic acid (100:1 v/v) were transferred to the Sep-Pak
cartridges and sequentially eluted with 20 ml of chloroform:acetic
acid (100:1 v/v) for neutral lipids, 10 ml of acetone and 10 ml of
acetone:acetic acid (100:1 v/v) for glycolipids and 7.5 ml of
methanol:chloroform:water (100:1 v/v) for PL. Chloroform (2.25
ml) and water (3 ml) were added successively to the eluate
containing the PL to obtain a phase separation and to facilitate their
recovery. The separation of individual lipids was performed by TLC
(Silica Gel 60, 0.25 mm thickness; Merck) with the following
solvent mixture: petroleum ether:ethyl ether:acetic acid (80:35:1 by
vol.) for neutral lipids (free sterols and sterol esters); chloroform:methanol:water (65:25:4 by vol.) for glycolipids (steryl glycosides
and
cerebrosides);
chloroform:methanol:acetic
acid:water
(85:15:10:3.5 by vol.) for PL. After development, the bands were
located with iodine vapour or spraying the plates with 0.1%
Rhodamine 6G in ethanol. Individual lipids were identi®ed by cochromatography with authentic standards.
Fatty acid analysis
The fatty acid methyl ester derivates from individual and total PL
were obtained as previously described (Quartacci et al., 1997) and
separated by GLC on a Dani 86.10 HT gas chromatograph equipped
with a 60 m30.32 mm SP-2340 fused silica capillary column
(Supelco) coupled to a ¯ame ionization detector (column temperature 175 °C). Both the injector and detector were maintained at 250
°C. Nitrogen was used as the carrier gas at 0.9 ml min±1 with a split
injector system (split ratio 1:100).
Quanti®cation of lipids after TLC
Quantitative analyses of sterols, cerebrosides (CER) and PL were
performed as reported by Navari-Izzo et al. (1993) using cholesterol,
Sterol analysis
Individual free sterol (FS) components were separated and
quantiti®ed by GLC as underivatized residues. The sterol moieties
dissolved in ethyl acetate were analysed with a Perkin-Elmer Sigma
2B gas chromatograph using a ¯ame ionization detector and a 30
m30.32 mm SPB-5 fused silica capillary column (Supelco). The
operating conditions were: column temperature 250 °C, injector and
detector temperatures 280 °C, N2 was the carrier gas at 1 ml min±1
(split ratio 1:70). Compound identi®cation was made on the basis of
the retention time relative to known standards. Cholestane was the
internal standard, and corrections were made for differences in
detector response.
Statistical analysis
A completely random experimental design was run in triplicate. Data
from each experimental design determined in triplicate were
analysed by a one-way analysis of variance. The signi®cance of
differences was determined according to Tukey's test. P values
<0.05 are considered to be signi®cant.
Results
Following dehydration by withholding water for 3 weeks
the RWC decreased from 87.0% in the fully hydrated
plants to the value of 3.6% in the desiccated ones,
dehydrating very slowly especially in the ®rst 15 d. After
rewetting, the plants regained quickly their hydration state
reaching the RWC of 93.8% after a week (Fig. 1).
The injury index calculated from the solute leakage
measurements decreased from 13.0% in the well-watered
plants to 6.8% in the desiccated ones, regaining the value
of 13.2% in the rehydrated leaves (not shown).
2162 Quartacci et al.
Lipid composition (mmol mg±1 protein), PL to FS
molar ratio and lipid to protein mass ratio of plasma
membranes isolated from leaves of Ramonda serbica during
dehydration and rehydration
Table 2.
Lipid to protein ratio is expressed as mg mg±1. In brackets, lipid class
proportion (mol%) relative to total content. Results are the means of
three independent experiments. For comparisons among means an
analysis of variance was used. For each treatment means in rows
followed by different letters are signi®cantly different at P <0.05
level. tr, trace.
Fig. 1. Relative water content (RWC) of leaves of Ramonda serbica
following dehydration and rehydration. Results are the means 6SE of
ten measurements.
In PM isolated from fully hydrated Ramonda leaves a
total lipid content of 1.05 mmol mg±1 protein was detected
(Table 2). The amount of PM total lipids, as well as all the
individual components of the dried leaves suffered a
dramatic reduction and were reduced to one-quarter of the
hydrated leaves. Upon rehydration, the PM lipid content of
leaves was restored and approached the amount of the
hydrated leaves (0.93 mmol mg±1 protein). The lipid to
protein ratio of PM showed a reduction in the dehydrated
leaves from 3.5 to 2.1, but upon rehydration regained the
value of 3.1. The same trend was followed also by the PL
to FS molar ratio which decreased by 20% in the
dehydrated leaves (Table 2).
The main PM lipids of Ramonda serbica leaves were FS
which accounted for more than half of the total lipids in
both hydrated and desiccated plants (Table 2). Their
proportion did not change during the dehydration/rehydration cycle. The other PM lipids included a relatively large
amount of PL (which declined from 30.6 to 25.8 mol%
during dehydration and regained the control value upon
rehydration), and a smaller amount of CER (increasing
from 6.6 to 10.9 mol% following dehydration and
recovering the control value in rehydrayed leaves).
Acylated steryl glycosides (ASG) decreased during
desiccation from 5.1 to 2.0 mol% and were restored
upon rehydration. A small proportion of steryl glycosides
(SG) was also detected, which remained constant during
the dehydration/rehydration cycle (Table 2).
The predominant PM phospholipids of hydrated leaves
were phosphatidylcholine (PC) and phosphatidylethanolamine (PE) (Table 3). During dehydration the proportions
of these PLs declined by about 50%, regaining the control
levels when rehydrated. The other PLs were present in
smaller amounts and increased remarkably during dehydration, especially PA which almost doubled its level from
PL
FS
ASG
SG
CER
Total content
PL/FS
Lipid/protein
Hydrated
Dehydrated
Rehydrated
0.32 b (30.6)
0.59 b (56.2)
0.05 b (5.1)
0.02 a (1.5)
0.07 b (6.6)
1.05 b
0.54 b
3.5 b
0.06 a (25.8)
0.14 a (60.1)
0.01 a (2)
tr (1.2)
0.03 a (10.9)
0.24 a
0.43 a
2.1 a
0.29 b (31.3)
0.52 b (55.8)
0.05 b (5.8)
0.02 a (1.7)
0.05 a (5.4)
0.93 b
0.56 b
3.1 b
8.3 to 15 mol%. In the PM isolated from rehydrated leaves
the individual PLs approached the values of the wellhydrated leaves with the exception of PG which further
decreased. The changes in the PL composition due to water
shortage did not cause any variation in the PC to PE molar
ratio, which remained constant during the dehydration/
rehydration cycle (Table 3).
The most abundant PM free sterol was sitosterol
followed by campesterol and cholesterol with lesser
amounts of stigmasterol (Table 4). The sitosterol level
decreased constantly during the dehydration/rehydration
cycle (from 54.8 to 45.3 mol%), whereas stigmasterol
remained constant. Campesterol signi®cantly decreased in
the desiccated leaves (from 22.6 to 14.8 mol%) and
exceeded the control value in the rehydrated ones (33.8
mol%). The cholesterol level signi®cantly increased
during dehydration, being in desiccated leaves 2-fold
higher than in well-hydrated plants, and then regained the
amount of 13.5 mol% upon rehydration (Table 4). The
more planar (cholesterol+campesterol) to less planar
(sitosterol+stigmasterol) molar ratio continuously increased during dehydration and rehydration.
The main fatty acids of PM phospholipids were palmitic
(16:0) and linoleic (18:2) acids, followed by lower
amounts of oleic (18:1) and stearic (18:0) acids, and
much smaller proportions of linolenic (18:3), miristic
(14:0), and palmitoleic (16:1) fatty acids (Table 5).
Palmitic acid was the most abundant fatty acid in all the
individual PLs with the exception of PC, in which the main
fatty acid, linoleic acid, drastically decreased by 3-fold or
more in dried leaves. Almost all fatty acids, especially the
most abundant ones, i.e. 16:0 and 18:2, quickly restored
their levels in rehydrated leaves, returning almost to the
same value of the fresh leaves, with the exception of 18:0
Plasma membrane lipids in Ramonda 2163
Table 3.
Phospholipid composition (mol%) of plasma
membranes isolated from leaves of Ramonda serbica during
dehydration and rehydration
Table 4. Free sterol composition (mol%) and more planar to
less planar molar ratio of plasma membranes isolated from
leaves of Ramonda serbica during dehydration and rehydration
Results are the means of three independent experiments. For
comparisons among means an analysis of variance was used. The
signi®cance of the letters is the same as in Table 2.
Results are the means of three independent experiments. For
comparisons among means an analysis of variance was used. The
signi®cance of the letters is the same as in Table 2.
PC
PE
PG
PI
PS
PA
PC/PE
Hydrated
Dehydrated
Rehydrated
28.7 b
21.2 b
12.8 b
17.5 a
11.5 a
8.3 a
1.35 a
15.0
11.0
18.6
22.8
17.6
15.0
1.36
29.0 b
23.6 b
6.5 a
16.9 a
13.0 a
11.0 a
1.23 a
a
a
c
b
a
b
a
which generally remained in smaller amounts in rehydrated leaves. As for the degree of unsaturation, dehydration induced a higher saturation in the PM fatty acids
compared with well-hydrated or rehydrated leaves
(Table 5).
Discussion
The ability of R. serbica, as well as of other poikilohydric
plants, to survive complete desiccation, i.e. to live in an
anabiotic state, is the result of adaptations that both
maintain the structure of membranes or allow it to be
regained during rewatering and also prevent the functional
impairment of cell metabolism during water loss and
subsequent rehydration. Indeed, in spite of the changes
observed in PM lipid composition, R. serbica was capable
of pursuing its normal metabolic activity upon rehydration,
rapidly recovering without accelerating physiological
ageing as shown by its persistence over time, i.e. viability
in the vegetative state, as well as by its capacity to ¯ower
and to enter again into anabiosis.
A decrease in the lipid content is a common response of
plants to water de®cit and, in general, to environmental
stresses (Navari-Izzo and Rascio, 1999). A similar
behaviour has already been found in Ramonda species
(Stevanovic et al., 1992) and in the resurrection plants
Boea hygroscopica and Sporobolus stap®anus following
severe dehydration (Navari-Izzo et al., 1995, 2000;
Quartacci et al., 1997). The reduction in lipids following
dehydration (Table 2) is generally interpreted as causing a
decrease in the total membrane area of the cells, and may
alter the speci®c interactions between lipids and membrane-intrinsic proteins, essential for the maintenance of
membrane integrity (HernandeÂz and Cooke, 1997).
The PM isolated from Ramonda leaves showed a
relatively high FS level in comparison with PL (Table 2).
High sterol contents are not a unique characteristic of the
PM of this resurrection plant. Similar levels were also
observed in other species such as rye, potato and barley
Cholesterol
Campesterol
Stigmasterol
Sitosterol
More planar/less planar
Hydrated
Dehydrated
Rehydrated
13.8 a
22.6 b
8.8 a
54.8 b
0.57 a
28.0 b
14.8 a
8.0 a
49.2 a
0.75 b
13.5 a
33.8 c
7.4 a
45.3 a
0.90 c
(Lynch and Steponkus, 1987; Palta et al., 1993; Rochester
et al., 1987) and in the halophyte species Spartina patens
(Wu et al., 1998).
The lipid bilayer is the major barrier to free diffusion in
the selectively permeable membrane, and the permeability
properties of the bilayer are greatly in¯uenced by its
chemical composition and, in particular, by steryl lipids
(Navari-Izzo et al., 1993). Under physiological conditions,
FS act as the main lipid rigidi®er by increasing the
ef®ciency of PL packing. Sterol enrichment of membranes
has been interpreted as a mechanism of adaptation based
on sterol-induced membrane rigidi®cation (Yoshida and
Uemura, 1990; Quartacci et al., 2001). The increase in the
FS to PL molar ratio during dehydration may be an
indication of reduced ¯uidity of the PM, as also suggested
indirectly by the lower lipid to protein ratio (Table 2) and
the injury index, and might have altered the physical
architecture and permeability of membranes. The increase
in FS upon dehydration, observed earlier in water-stressed
maize, soybean and sun¯ower (Navari-Izzo et al., 1988,
1989, 1990, 1993) may provide an advantage to plants
growing under water-de®cit conditions since it has been
shown that higher sterol amounts in the bilayer reduce the
rate of permeation by water (Schroeder, 1984). Besides the
FS level, their composition (Table 4) also alters membrane
status because of the speci®c effect of the individual sterol
involved (Navari-Izzo et al., 1993). It is worth mentioning
that PM from leaves of R. serbica, irrespective of their
hydration state, were characterized by a relatively high
amount of cholesterol (Table 4) as already found in leaves
of Boea hygroscopica (Navari-Izzo et al., 1995). Among
free sterols, cholesterol has been found to be more
effective in controlling membrane permeability and ¯uidity due to the more planar con®guration of the molecule
(Grunwald, 1974). The more planar to less planar sterol
molar ratio plays a fundamental role in allowing the plant
to tolerate stress, as the ratio is considered to be an index of
membrane permeability and functioning (Navari-Izzo
et al., 1989; Surjus and Durand, 1996). The higher molar
ratio value in the PM of dehydrated and, perhaps more
2164 Quartacci et al.
Table 5. Fatty acid composition (mol%) of individual and total phospholipids in plasma membranes isolated from leaves of
Ramonda serbica during dehydration and rehydration
Results are the means of three independent experiments. For comparisons among means an analysis of variance was used. For each treatment
means in columns followed by different letters are signi®cantly different at P <0.05 level. tr, trace.
14:0
16:0
16:1
18:0
18:1
18:2
18:3
Unsaturation
PC
Hydrated
Dehydrated
Rehydrated
tr
3
tr
23.2 a
43.6 b
25.8 a
1.4
tr
tr
10.4 b
20.4 c
3.7 a
16.7 a
20.9 a
12.3 a
43.2 b
8.7 a
54.9 c
5.1 a
3.4 a
3.3 a
66.4 b
33.0 a
70.5b
PE
Hydrated
Dehydrated
Rehydrated
1.3 a
tr
0.5 a
42.8 a
47.7 b
37.2 a
4.6
tr
tr
12.6 b
25.3 c
3.4 a
5.8 a
16.1 b
6.4 a
30.1 b
10.9 a
49.6 c
2.8 a
tr
2.9 a
43.3 b
27.0 a
58.9 c
PG
Hydrated
Dehydrated
Rehydrated
2.6 a
8.0 b
1.7 a
49.6 a
46.4 a
46.9 a
tr
tr
tr
19.0 b
22.7 b
14.2 a
10.3 a
11.5 a
14.1 a
18.0 b
8.8 a
20.5 b
0.5 a
2.6 b
2.6 b
28.8 a
22.9 a
37.2 b
PI
Hydrated
Dehydrated
Rehydrated
1.2 a
3.9 b
tr
44.6 a
42.5 a
41.1 a
4.9
tr
tr
13.1 b
15.5 b
3.9 a
6.7 a
26.5 b
11.3 a
24.5 b
7.0 a
37.3 c
5.0 a
4.6 a
6.4 a
41.1 a
38.1 a
55.0 b
PS
Hydrated
Dehydrated
Rehydrated
1.0 a
3.0 a
tr
39.2 a
47.5 b
32.9 a
7.5
tr
tr
17.9 a
17.1 a
23.8 a
9.9 a
21.4 b
22.7 b
23.3 b
7.6 a
20.6 b
1.2 a
3.4 a
tr
41.9 b
32.4 a
43.3 b
PA
Hydrated
Dehydrated
Rehydrated
5.2 a
7.4 a
tr
38.4 a
53.6 c
46.9 b
1.0
tr
tr
4.0 a
7.2 a
3.4 a
11.1 a
22.8 b
11.7 a
31.6 b
9.0 a
32.6 b
7.8 a
tr
5.4 a
51.5 b
31.8 a
49.7 b
Total
Hydrated
Dehydrated
Rehydrated
1.3 a
4.4 b
tr
33.5 a
46.4 b
35.6 a
3.2
tr
tr
12.7 b
19.4 c
6.9 a
10.5 a
20.2 b
12.1 a
35.0 b
7.0 a
41.9 b
3.8 a
2.6 a
3.3 a
52.5 b
29.8 a
57.3 b
importantly, of rehydrated leaves compared with the
control may have maintained membrane functionality
due to the higher effectiveness of the ¯at con®gurations of
cholesterol and campesterol in stabilizing the bilayer
architecture (Navari-Izzo et al., 1989; Stalleart and Geuns,
1994).
Changes in the ASG to SG ratio or an increase in the
proportion of ASG plus FS at the expense of SG could be
important in modulating the phase behaviour of PM during
dehydration, and could have a relevant impact on the
physical properties of the PM (Palta et al., 1993; Zhang
et al., 1997). The change in the relative proportions of
ASG and SG in the PM from dehydrated leaves (Table 2)
may indicate that alterations in sterol conjugation play a
role in R. serbica membrane functioning as previously
observed in the PM of wheat roots subjected to copper
stress (Quartacci et al., 2001).
Cerebrosides are characterized by an extensive hydrogen bonding ability and high gel to liquid crystalline phase
transition temperatures. This lipid class is, therefore,
generally considered to stabilize the PM physically and
to reduce ion permeability of the cells (Uemura and
Steponkus, 1994). The increase in CER level following
dehydration (Table 2) probably contributed to the overall
membrane response to unfavourable events during the
desiccation period. Indeed, it has been suggested that CER
stabilize the interaction of bulk lipids and proteins by
facilitating a tighter sealing of proteins into the lipid head
groups, thus regulating the retention of water (Wu et al.,
1998) and intracellular solutes. If this is the case, the
increase in CER in the dehydrated PM should play an
important role in stabilizing membrane structural integrity
when bulk lipid content is reduced and speci®c interactions
between lipids and membrane-intrinsic proteins are altered
(Table 2).
Traditionally, alterations in fatty acid unsaturation
degree are related to changes in bilayer thickness and
¯uidity, and it is known that a decrease in fatty acid
unsaturation results in a decrease in membrane ¯uidity
(Wu et al., 1998; Navari-Izzo et al., 2000), even though it
is unlikely that the ¯uidity of the bulk lipid phase has any
important effect on the function of membrane proteins
(Lee et al., 1989). The remarkable reduction of the acyl
chain unsaturation detected in dehydrated PM (Table 5)
may have contributed, together with the increase in FS and
CER, to render the bilayer tighter and more rigid, as
con®rmed indirectly by the reduced solute leakage. A
lower unsaturation compared with control plants has been
observed in PM isolated from roots of wheat grown in
excess copper (Quartacci et al., 2001), and the reduced PM
unsaturation was demonstrated to be linked to a lower
permeability to glucose and a tighter molecular packing
Plasma membrane lipids in Ramonda 2165
(Berglund et al., 2000). By contrast, PC and PE
unsaturation of the resurrection plants B. hygroscopica
and S. stap®anus increased following dehydration (NavariIzzo et al., 1995; Quartacci et al., 1997), indicating a
different defence and/or adaptation mechanism depending
on the species and the dehydration severity and time
course.
Lyotropic phase transitions and non-bilayer lipid structures have been reported in dehydrated cell membranes
and liposomes (Uemura et al., 1995). The removal of water
and the subsequent tightening of the lipid bilayers is
suggested to induce lipid±lipid demixing, which facilitates
the lamellar-to-hexagonal (HII) transition due to the
intrinsic curvature (bending energy) in membrane monolayers caused by dehydration-induced packing stress
(Uemura and Steponkus, 1994). Increased membrane
stability may be achieved by altering the lipid composition
so that demixing and/or phase transitions are precluded or
reduced. It has been shown that ASG are much more
effective than FS in increasing the propensity for dehydration-induced formation of the HII phase (Webb et al.,
1995), and that PE, especially polyunsaturated species of
PE, is the PM lipid most likely to form the HII structure
(Uemura and Steponkus, 1994). In this study, the lack of
changes in the PE level following desiccation, its low
content (2.8% of total PM lipids in the dehydrated leaves)
and the decrease in its unsaturation (Tables 3, 4), together
with ASG reduction (Table 2), may have limited the
tendency to form non-lamellar con®gurations which are
otherwise favoured by the altered hydration characteristics
of the membranes. Indeed, high proportions of highly
hydrated species (e.g. PL) and poorly hydrated species
(e.g. FS and CER) increase the tendency for dehydrationinduced lipid±lipid demixing and hence the tendency to
form HII con®gurations which, at low water contents
(<20%, w/w), minimize the lateral pressure in the bilayer
and thus the bending energy (Gruner, 1989). Nevertheless,
it is likely that the tendency for dehydration-induced
formation of the HII phase is in¯uenced by the collective
changes in the various lipid classes rather than in a speci®c
lipid class or species (Uemura et al., 1995). Among PLs,
the high PA amount present in the dehydrated PM
(Table 2), besides the result of PL degradation, may be
considered as a storage of PL precursors which can be
readily used as soon as stress conditions are released.
The full regaining of photosynthetic activity a few hours
after rehydration (Augusti et al., 2001) as well as the active
membrane defence systems against oxidative stress
(Sgherri et al., 2000; Augusti et al., 2001) indicates that
Ramonda leaves completely restored their thylakoid
membrane integrity and functionality. In addition, the
rapid recovery upon rehydration of the lipid PM composition indicates the importance of ef®cient mechanisms that
are necessary for repairing membranes after rewatering.
Lipid modulation, and ¯uidity as a consequence, seems to
play a major role in the adaptation to altered conditions and
in regenerating the original membrane structure and
functioning.
Acknowledgements
This study was performed by collaboration between the University
of Pisa (promoter F Navari-Izzo) and the University of Belgrade
(promoter B StevanovicÂ). This paper is dedicated to the memory of
O GlisÏicÂ.
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