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 GlisÏic 2, Branka Stevanovic 2 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 Key words: Dehydration, lipids, plasma membrane, Ramonda serbica, resurrection plants. 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 isol- Introduction ated from leaves using a two-phase aqueous polymer partition system. Compared with well-hydrated (con- Flowering plants growing in hot and arid regions usually trol) leaves, dehydrated leaves suffered a reduction of survive the harsh environmental conditions either by about 75% in their plasma membrane lipid content, avoiding the stressful events or by very promptly activat- which returned to the control level following rewater- ing adaptative resistance mechanisms. Only a small ing. Also the lipid to protein ratio decreased after number of higher plants, mostly originating from the dehydration, almost regaining the initial value after southern hemisphere and called desiccation-tolerant or rehydration. Lipids extracted from the plasma mem- resurrection plants, are capable of surviving almost brane of fully-hydrated leaves were characterized by a complete dehydration for prolonged periods. Ramonda high level of free sterols and a much lower level of spp, as well as other species belonging to the family phospholipids. Smaller amounts of cerebrosides, acy- Gesneriaceae, are among the resurrection plants which lated steryl glycosides and steryl glycosides were grow in the northern hemisphere. Ramonda serbica is a also detected. The main phospholipids of control rare resurrection plant growing in the Balkan peninsula leaves were phosphatidylcholine and phosphatidy- (Gaff, 1981; StevanovicÂ, 1986). This species is capable of lethanolamine, whereas sitosterol was the free sterol surviving long dry periods between the wet periods, present in the highest amount. Following dehydration, passing quickly from anabiosis, which can last much leaf plasma membrane lipids showed a constant level longer than three months depending on water de®cit of free sterols and a reduction in phospholipids com- severity and temperatures, to the state of full biological pared with the well-hydrated leaves. Both phosphati- activity in less than 8±10 h if the favourable water balance dylcholine and phosphatidylethanolamine decreased in the soil re-establishes suddenly. following dehydration, their molar ratio remaining In spite of the fact that metabolic processes are almost unchanged. Among free sterols, the remarkably high stopped in resurrection plant dry leaves, the cell mem- cholesterol level present in the control leaves (about branes as well as most of the enzymatic systems are 14 mol%) increased 2-fold as a result of dehydration. protected in different ways (Bewley and Krochko, 1982; Dehydration caused a general decrease in the unsa- Oliver, 1996; Navari-Izzo and Rascio, 1999). It has been turation level of individual phospholipids and total suggested that the rapid and ef®cient recovery and full lipids as well. Upon rehydration the lipid composition reconstitution of membrane organization and functionality, of leaf plasma membranes restored very quickly as well as the presence of effective membrane defence approaching the levels of well-hydrated leaves. 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; Navari- Materials and methods Izzo et al., 1995; Navari-Izzo and Rascio, 1999; Sgherri Plant material et al., 2000). Specimens of the desiccation-tolerant plant Ramonda serbica PancÏ. According to Oliver (1996), Ramonda serbica seems to & Petrov. were collected from their natural habitat in the south-east belong to the group of resurrection plants which are able to region of Serbia, in a gorge near the town of Nis. There, the plants withstand desiccation using both morphological and grow on rocky slopes, exposed from north to north-east, on a thin physiological mechanisms to slow down and, for a while, layer of rich, mature, organo-mineral and dark mountain soil (pH 8.4) spread over limestone. During summer the habitat is to control the rate of water loss. The results of some recent characterized by high air temperatures and a remarkable decrease investigations indicate that Ramonda serbica has the in air humidity and the plants pass to, and stay in, the anabiotic state, ability to maintain cell membrane integrity, i.e. to preserve although they never receive sunlight directly. Plants of the same age semipermeability during dehydration (Stevanovic et al., were harvested together with the layer of soil on which they grew. 1998), as well as to activate protective mechanisms that After collection, plants were acclimated for 2 weeks keeping them fully watered until the beginning of the experiments. Plants were increase the level of zeaxanthin and the amounts of dehydrated for 3 weeks by withholding water at room temperature reduced ascorbate and glutathione, which are crucial for and ambient photoperiod. Rehydration was started by spraying the photoprotection during the dehydration/rehydration cycle plants with water to simulate rainfall and keeping the soil damp. The (Augusti et al., 2001). Furthermore, it has been found that rehydrated samples were collected after 1 week during which they were watered daily. an increased amount of phenolic acids also protects Ramonda membranes during desiccation (Sgherri et al., Relative water content 2000). At regular intervals during the dehydration/rehydration cycle In the last decade, only a few studies on lipids extracted measurements of the relative water content (RWC) of the leaves from thylakoids or the whole plant have been undertaken were carried out as previously reported (Sgherri et al., 1994). For the in order to explain desiccation tolerance in resurrection analyses, mature and fully expanded leaves from the middle of the rosettes and comparable in size were selected. The RWC of the plants (Stevanovic et al., 1992; Stefanov et al., 1992; leaves was calculated according to the formula: 1003[(fresh Navari-Izzo et al., 1995; Quartacci et al., 1997). The weight±dry weight)/(saturated weight±dry weight)] and expressed investigations showed the general tendency of dehydrated as the mean value of ten replicates for each treatment. plants to adapt their membranes to the altered conditions, and to recover quickly on rehydration both the lipid Solute leakage composition and the order parameters of the well hydrated For solute leakage determination samples of the same weight were obtained from leaves comparable in size. The plant material was plants. However, there is a complete lack of knowledge washed in double-distilled water to remove the contents of the cut about changes of lipids during dehydration and rehydration cells, soaked in 25 ml of double-distilled water, shaken at room for the plasma membrane (PM). The composition and temperature for 24 h and aliquots for leachate measurements were organization of PM lipids are crucial for intracellular taken. Samples were then immersed for 5 min in liquid N2, placed again in the same vial containing the leachate and shaken for an metabolism. Many vital activities of cells originate in the additional hour prior to the measurement of the maximum conduct- membrane, the structure and function of which are ivity (Metrohm 660 conductometer). The injury index was calcu- profoundly altered following water stress that leads to lated according to the formula: injury index %=1±[(T±C)/T]3100, destructive events such as phase transition, fusion and where T and C represent the conductivity of the leachate after and increased permeability. The composition and physical before liquid N2 treatment, respectively. state of the lipid bilayer in¯uence lipid±protein and Plasma membrane preparation protein±protein associations, membrane-bound enzyme Plasma membranes were prepared using a two-phase aqueous activities and the carrier-mediated transport capacity of polymer partition system. Leaves were cut into pieces and imme- membranes (Navari-Izzo and Rascio, 1999; Leprince et al., diately homogenized in the isolation medium consisting of 50 mM 2000; Navari-Izzo et al., 2000; Kerkeb et al., 2001). Tris-HCl, pH 7.5, 0.25 M sucrose, 3 mM Na2EDTA, 10 mM ascorbic acid, and 5 mM diethyldithiocarbamic acid. The homogenate was Preserving membrane integrity, resurrection plants are ®ltered through four layers of a nylon cloth and centrifuged at 10 000 capable of surviving during anabiosis and returning g for 10 min. The supernatant was further centrifugated at 65 000 g quickly to the complex and dynamic whole-organism for 30 min to yield a microsomal pellet, which was resuspended in 2 functionality upon rehydration (Quartacci et al., 1997; ml of a resuspension buffer (5 mM K-phosphate, pH 7.8, 0.25 M Navari-Izzo et al., 1995, 2000). sucrose, and 3 mM KCl). Plasma membranes were isolated by loading the microsomal suspension (1.0 g) onto an aqueous two- The aim of the present study was to examine the phase polymer system to give a ®nal concentration of 6.6% (w/w) composition of lipids of plasma membranes isolated Dextran T500, 6.6% (w/w) polyethylenglycol, 5 mM K-phosphate from Ramonda serbica leaves, as well as to determine (pH 7.8), 0.25 M sucrose, and 3 mM KCl. The PM was further changes during a dehydration/rehydration cycle in order 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 to explain, at least in part, the plant's ability to sucrose, and centrifuged for 30 min at 100 000 g. The resultant PM reactivate its physiological functions so rapidly after pellet was resuspended in the same buffer containing 30% rewatering. ethylenglycol and stored at ±80 °C for lipid analyses. All steps of Plasma membrane lipids in Ramonda 2161 Table 1. Distribution of marker enzymes (mmol mg±1 protein min±1) 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 Well hydrated leaves Dehydrated leaves

UP LP UP LP ATPase 0.42160.021 0.26760.019 0.51460.022 0.31760.015 Vanadate-sensitive ATPase 0.06360.005 0.15560.014 0.03160.004 0.20160.012 ± NO3 -sensitive ATPase 0.37460.026 0.02560.004 0.51260.020 0.06360.004 NADH-cyt c reductase 0.00660.001 0.18760.010 0.03260.003 0.22760.011 Cyt c oxidase 0.00460.001 0.03060.003 0.00460.001 0.09860.009 Latent IDPase 0.04160.005 0.10660.009 0.02160.003 0.07260.006

the isolation were carried out at 4 °C. Plasma membrane pellets for glucose and KH2PO4 as standards, respectively. All procedures were enzyme activity determinations were used immediately. performed in the presence of silica gel from TLC. In order to check the purity of the PM, the activity of the vanadate- sensitive ATPase as a marker enzyme was determined (Table 1). Sterol analysis Cytochrome c oxidase, antimycin A-insensitive NADH cyt c ± Individual free sterol (FS) components were separated and reductase, NO3 -sensitive ATPase and latent IDPase activities quantiti®ed by GLC as underivatized residues. The sterol moieties were used as markers of mitochondria, endoplasmic reticulum, dissolved in ethyl acetate were analysed with a Perkin-Elmer Sigma tonoplast, and Golgi membranes, respectively (Navari-Izzo et al., 2B gas chromatograph using a ¯ame ionization detector and a 30 1993). Chlorophyll was not detected in the PM fraction. Tests with m30.32 mm SPB-5 fused silica capillary column (Supelco). The the markers showed that the partition behaviour of both PM and operating conditions were: column temperature 250 °C, injector and intracellular membranes may be in¯uenced by water contents since ±1 detector temperatures 280 °C, N2 was the carrier gas at 1 ml min the net charge density of membranes is also related to their polar (split ratio 1:70). Compound identi®cation was made on the basis of head group composition. The protein content was determined taking the retention time relative to known standards. Cholestane was the aliquots of the PM suspension. The analysis was performed internal standard, and corrections were made for differences in according to Bradford (1976) with bovine serum albumin as a detector response. standard.

Lipid extraction and separation Fatty acid analysis Lipids were extracted from the PM suspension by the addition of The fatty acid methyl ester derivates from individual and total PL boiling isopropanol followed by chloroform:methanol (2:1 v/v) were obtained as previously described (Quartacci et al., 1997) and containing butylhydroxytoluol (50 mgml±1) as an antioxidant. The separated by GLC on a Dani 86.10 HT gas chromatograph equipped solvent mixture was then washed with 0.88% KCl to separate the with a 60 m30.32 mm SP-2340 fused silica capillary column chloroform phase. The upper water phase was re-extracted with (Supelco) coupled to a ¯ame ionization detector (column tempera- chloroform, the chloroform phases combined and dried under a ture 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 stream of N2. The lipid extracts were stored at ±20 °C and retained for further separation. Lipids were fractionated into neutral lipid, injector system (split ratio 1:100). glycolipid and phospholipid (PL) fractions on Sep-Pak cartridges (Waters) (Uemura and Steponkus, 1994). Lipid extracts dissolved in Statistical analysis chloroform:acetic acid (100:1 v/v) were transferred to the Sep-Pak A completely random experimental design was run in triplicate. Data cartridges and sequentially eluted with 20 ml of chloroform:acetic from each experimental design determined in triplicate were acid (100:1 v/v) for neutral lipids, 10 ml of acetone and 10 ml of analysed by a one-way analysis of variance. The signi®cance of acetone:acetic acid (100:1 v/v) for glycolipids and 7.5 ml of differences was determined according to Tukey's test. P values methanol:chloroform:water (100:1 v/v) for PL. Chloroform (2.25 <0.05 are considered to be signi®cant. 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 Results solvent mixture: petroleum ether:ethyl ether:acetic acid (80:35:1 by Following dehydration by withholding water for 3 weeks vol.) for neutral lipids (free sterols and sterol esters); chloroform:- methanol:water (65:25:4 by vol.) for glycolipids (steryl glycosides the RWC decreased from 87.0% in the fully hydrated and cerebrosides); chloroform:methanol:acetic acid:water plants to the value of 3.6% in the desiccated ones, (85:15:10:3.5 by vol.) for PL. After development, the bands were dehydrating very slowly especially in the ®rst 15 d. After located with iodine vapour or spraying the plates with 0.1% rewetting, the plants regained quickly their hydration state Rhodamine 6G in ethanol. Individual lipids were identi®ed by co- reaching the RWC of 93.8% after a week (Fig. 1). chromatography with authentic standards. The injury index calculated from the solute leakage Quanti®cation of lipids after TLC measurements decreased from 13.0% in the well-watered Quantitative analyses of sterols, cerebrosides (CER) and PL were plants to 6.8% in the desiccated ones, regaining the value performed as reported by Navari-Izzo et al. (1993) using cholesterol, of 13.2% in the rehydrated leaves (not shown). 2162 Quartacci et al. Table 2. 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 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. Hydrated Dehydrated Rehydrated PL 0.32 b (30.6) 0.06 a (25.8) 0.29 b (31.3) FS 0.59 b (56.2) 0.14 a (60.1) 0.52 b (55.8) ASG 0.05 b (5.1) 0.01 a (2) 0.05 b (5.8) SG 0.02 a (1.5) tr (1.2) 0.02 a (1.7) CER 0.07 b (6.6) 0.03 a (10.9) 0.05 a (5.4) Total content 1.05 b 0.24 a 0.93 b PL/FS 0.54 b 0.43 a 0.56 b Fig. 1. Relative water content (RWC) of leaves of Ramonda serbica Lipid/protein 3.5 b 2.1 a 3.1 b 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 8.3 to 15 mol%. In the PM isolated from rehydrated leaves (Table 2). The amount of PM total lipids, as well as all the the individual PLs approached the values of the well- individual components of the dried leaves suffered a hydrated leaves with the exception of PG which further dramatic reduction and were reduced to one-quarter of the decreased. The changes in the PL composition due to water hydrated leaves. Upon rehydration, the PM lipid content of shortage did not cause any variation in the PC to PE molar leaves was restored and approached the amount of the ratio, which remained constant during the dehydration/ hydrated leaves (0.93 mmol mg±1 protein). The lipid to rehydration cycle (Table 3). protein ratio of PM showed a reduction in the dehydrated The most abundant PM free sterol was sitosterol leaves from 3.5 to 2.1, but upon rehydration regained the followed by campesterol and cholesterol with lesser value of 3.1. The same trend was followed also by the PL amounts of stigmasterol (Table 4). The sitosterol level to FS molar ratio which decreased by 20% in the decreased constantly during the dehydration/rehydration dehydrated leaves (Table 2). cycle (from 54.8 to 45.3 mol%), whereas stigmasterol The main PM lipids of Ramonda serbica leaves were FS remained constant. Campesterol signi®cantly decreased in which accounted for more than half of the total lipids in the desiccated leaves (from 22.6 to 14.8 mol%) and both hydrated and desiccated plants (Table 2). Their exceeded the control value in the rehydrated ones (33.8 proportion did not change during the dehydration/rehydra- mol%). The cholesterol level signi®cantly increased tion cycle. The other PM lipids included a relatively large during dehydration, being in desiccated leaves 2-fold amount of PL (which declined from 30.6 to 25.8 mol% higher than in well-hydrated plants, and then regained the during dehydration and regained the control value upon amount of 13.5 mol% upon rehydration (Table 4). The rehydration), and a smaller amount of CER (increasing more planar (cholesterol+campesterol) to less planar from 6.6 to 10.9 mol% following dehydration and (sitosterol+stigmasterol) molar ratio continuously in- recovering the control value in rehydrayed leaves). creased during dehydration and rehydration. Acylated steryl glycosides (ASG) decreased during The main fatty acids of PM phospholipids were palmitic desiccation from 5.1 to 2.0 mol% and were restored (16:0) and linoleic (18:2) acids, followed by lower upon rehydration. A small proportion of steryl glycosides amounts of oleic (18:1) and stearic (18:0) acids, and (SG) was also detected, which remained constant during much smaller proportions of linolenic (18:3), miristic the dehydration/rehydration cycle (Table 2). (14:0), and palmitoleic (16:1) fatty acids (Table 5). The predominant PM phospholipids of hydrated leaves Palmitic acid was the most abundant fatty acid in all the were phosphatidylcholine (PC) and phosphatidylethanola- individual PLs with the exception of PC, in which the main mine (PE) (Table 3). During dehydration the proportions fatty acid, linoleic acid, drastically decreased by 3-fold or of these PLs declined by about 50%, regaining the control more in dried leaves. Almost all fatty acids, especially the levels when rehydrated. The other PLs were present in most abundant ones, i.e. 16:0 and 18:2, quickly restored smaller amounts and increased remarkably during dehy- their levels in rehydrated leaves, returning almost to the dration, especially PA which almost doubled its level from 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 Table 4. Free sterol composition (mol%) and more planar to membranes isolated from leaves of Ramonda serbica during less planar molar ratio of plasma membranes isolated from dehydration and rehydration leaves of Ramonda serbica during dehydration and rehydration Results are the means of three independent experiments. For Results are the means of three independent experiments. For comparisons among means an analysis of variance was used. The comparisons among means an analysis of variance was used. The signi®cance of the letters is the same as in Table 2. signi®cance of the letters is the same as in Table 2. Hydrated Dehydrated Rehydrated Hydrated Dehydrated Rehydrated PC 28.7 b 15.0 a 29.0 b Cholesterol 13.8 a 28.0 b 13.5 a PE 21.2 b 11.0 a 23.6 b Campesterol 22.6 b 14.8 a 33.8 c PG 12.8 b 18.6 c 6.5 a Stigmasterol 8.8 a 8.0 a 7.4 a PI 17.5 a 22.8 b 16.9 a Sitosterol 54.8 b 49.2 a 45.3 a PS 11.5 a 17.6 a 13.0 a More planar/less planar 0.57 a 0.75 b 0.90 c PA 8.3 a 15.0 b 11.0 a PC/PE 1.35 a 1.36 a 1.23 a

(Lynch and Steponkus, 1987; Palta et al., 1993; Rochester et al., 1987) and in the halophyte species Spartina patens which generally remained in smaller amounts in rehy- (Wu et al., 1998). drated leaves. As for the degree of unsaturation, dehydra- The lipid bilayer is the major barrier to free diffusion in tion induced a higher saturation in the PM fatty acids the selectively permeable membrane, and the permeability compared with well-hydrated or rehydrated leaves properties of the bilayer are greatly in¯uenced by its (Table 5). chemical composition and, in particular, by steryl lipids (Navari-Izzo et al., 1993). Under physiological conditions, Discussion FS act as the main lipid rigidi®er by increasing the The ability of R. serbica, as well as of other poikilohydric ef®ciency of PL packing. Sterol enrichment of membranes plants, to survive complete desiccation, i.e. to live in an has been interpreted as a mechanism of adaptation based anabiotic state, is the result of adaptations that both on sterol-induced membrane rigidi®cation (Yoshida and maintain the structure of membranes or allow it to be Uemura, 1990; Quartacci et al., 2001). The increase in the regained during rewatering and also prevent the functional FS to PL molar ratio during dehydration may be an impairment of cell metabolism during water loss and indication of reduced ¯uidity of the PM, as also suggested subsequent rehydration. Indeed, in spite of the changes indirectly by the lower lipid to protein ratio (Table 2) and observed in PM lipid composition, R. serbica was capable the injury index, and might have altered the physical of pursuing its normal metabolic activity upon rehydration, architecture and permeability of membranes. The increase rapidly recovering without accelerating physiological in FS upon dehydration, observed earlier in water-stressed ageing as shown by its persistence over time, i.e. viability maize, soybean and sun¯ower (Navari-Izzo et al., 1988, in the vegetative state, as well as by its capacity to ¯ower 1989, 1990, 1993) may provide an advantage to plants and to enter again into anabiosis. growing under water-de®cit conditions since it has been A decrease in the lipid content is a common response of shown that higher sterol amounts in the bilayer reduce the plants to water de®cit and, in general, to environmental rate of permeation by water (Schroeder, 1984). Besides the stresses (Navari-Izzo and Rascio, 1999). A similar FS level, their composition (Table 4) also alters membrane behaviour has already been found in Ramonda species status because of the speci®c effect of the individual sterol (Stevanovic et al., 1992) and in the resurrection plants involved (Navari-Izzo et al., 1993). It is worth mentioning Boea hygroscopica and Sporobolus stap®anus following that PM from leaves of R. serbica, irrespective of their severe dehydration (Navari-Izzo et al., 1995, 2000; hydration state, were characterized by a relatively high Quartacci et al., 1997). The reduction in lipids following amount of cholesterol (Table 4) as already found in leaves dehydration (Table 2) is generally interpreted as causing a of Boea hygroscopica (Navari-Izzo et al., 1995). Among decrease in the total membrane area of the cells, and may free sterols, cholesterol has been found to be more alter the speci®c interactions between lipids and mem- effective in controlling membrane permeability and ¯uid- brane-intrinsic proteins, essential for the maintenance of ity due to the more planar con®guration of the molecule membrane integrity (HernandeÂz and Cooke, 1997). (Grunwald, 1974). The more planar to less planar sterol The PM isolated from Ramonda leaves showed a molar ratio plays a fundamental role in allowing the plant relatively high FS level in comparison with PL (Table 2). to tolerate stress, as the ratio is considered to be an index of High sterol contents are not a unique characteristic of the membrane permeability and functioning (Navari-Izzo PM of this resurrection plant. Similar levels were also et al., 1989; Surjus and Durand, 1996). The higher molar observed in other species such as rye, potato and barley 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 tr 23.2 a 1.4 10.4 b 16.7 a 43.2 b 5.1 a 66.4 b Dehydrated 3 43.6 b tr 20.4 c 20.9 a 8.7 a 3.4 a 33.0 a Rehydrated tr 25.8 a tr 3.7 a 12.3 a 54.9 c 3.3 a 70.5b

PE Hydrated 1.3 a 42.8 a 4.6 12.6 b 5.8 a 30.1 b 2.8 a 43.3 b Dehydrated tr 47.7 b tr 25.3 c 16.1 b 10.9 a tr 27.0 a Rehydrated 0.5 a 37.2 a tr 3.4 a 6.4 a 49.6 c 2.9 a 58.9 c

PG Hydrated 2.6 a 49.6 a tr 19.0 b 10.3 a 18.0 b 0.5 a 28.8 a Dehydrated 8.0 b 46.4 a tr 22.7 b 11.5 a 8.8 a 2.6 b 22.9 a Rehydrated 1.7 a 46.9 a tr 14.2 a 14.1 a 20.5 b 2.6 b 37.2 b

PI Hydrated 1.2 a 44.6 a 4.9 13.1 b 6.7 a 24.5 b 5.0 a 41.1 a Dehydrated 3.9 b 42.5 a tr 15.5 b 26.5 b 7.0 a 4.6 a 38.1 a Rehydrated tr 41.1 a tr 3.9 a 11.3 a 37.3 c 6.4 a 55.0 b

PS Hydrated 1.0 a 39.2 a 7.5 17.9 a 9.9 a 23.3 b 1.2 a 41.9 b Dehydrated 3.0 a 47.5 b tr 17.1 a 21.4 b 7.6 a 3.4 a 32.4 a Rehydrated tr 32.9 a tr 23.8 a 22.7 b 20.6 b tr 43.3 b

PA Hydrated 5.2 a 38.4 a 1.0 4.0 a 11.1 a 31.6 b 7.8 a 51.5 b Dehydrated 7.4 a 53.6 c tr 7.2 a 22.8 b 9.0 a tr 31.8 a Rehydrated tr 46.9 b tr 3.4 a 11.7 a 32.6 b 5.4 a 49.7 b

Total Hydrated 1.3 a 33.5 a 3.2 12.7 b 10.5 a 35.0 b 3.8 a 52.5 b Dehydrated 4.4 b 46.4 b tr 19.4 c 20.2 b 7.0 a 2.6 a 29.8 a Rehydrated tr 35.6 a tr 6.9 a 12.1 a 41.9 b 3.3 a 57.3 b importantly, of rehydrated leaves compared with the stabilize the interaction of bulk lipids and proteins by control may have maintained membrane functionality facilitating a tighter sealing of proteins into the lipid head due to the higher effectiveness of the ¯at con®gurations of groups, thus regulating the retention of water (Wu et al., cholesterol and campesterol in stabilizing the bilayer 1998) and intracellular solutes. If this is the case, the architecture (Navari-Izzo et al., 1989; Stalleart and Geuns, increase in CER in the dehydrated PM should play an 1994). important role in stabilizing membrane structural integrity Changes in the ASG to SG ratio or an increase in the when bulk lipid content is reduced and speci®c interactions proportion of ASG plus FS at the expense of SG could be between lipids and membrane-intrinsic proteins are altered important in modulating the phase behaviour of PM during (Table 2). dehydration, and could have a relevant impact on the Traditionally, alterations in fatty acid unsaturation physical properties of the PM (Palta et al., 1993; Zhang degree are related to changes in bilayer thickness and et al., 1997). The change in the relative proportions of ¯uidity, and it is known that a decrease in fatty acid ASG and SG in the PM from dehydrated leaves (Table 2) unsaturation results in a decrease in membrane ¯uidity may indicate that alterations in sterol conjugation play a (Wu et al., 1998; Navari-Izzo et al., 2000), even though it role in R. serbica membrane functioning as previously is unlikely that the ¯uidity of the bulk lipid phase has any observed in the PM of wheat roots subjected to copper important effect on the function of membrane proteins stress (Quartacci et al., 2001). (Lee et al., 1989). The remarkable reduction of the acyl Cerebrosides are characterized by an extensive hydro- chain unsaturation detected in dehydrated PM (Table 5) gen bonding ability and high gel to liquid crystalline phase may have contributed, together with the increase in FS and transition temperatures. This lipid class is, therefore, CER, to render the bilayer tighter and more rigid, as generally considered to stabilize the PM physically and con®rmed indirectly by the reduced solute leakage. A to reduce ion permeability of the cells (Uemura and lower unsaturation compared with control plants has been Steponkus, 1994). The increase in CER level following observed in PM isolated from roots of wheat grown in dehydration (Table 2) probably contributed to the overall excess copper (Quartacci et al., 2001), and the reduced PM membrane response to unfavourable events during the unsaturation was demonstrated to be linked to a lower desiccation period. Indeed, it has been suggested that CER permeability to glucose and a tighter molecular packing Plasma membrane lipids in Ramonda 2165 (Berglund et al., 2000). By contrast, PC and PE play a major role in the adaptation to altered conditions and unsaturation of the resurrection plants B. hygroscopica in regenerating the original membrane structure and and S. stap®anus increased following dehydration (Navari- functioning. Izzo et al., 1995; Quartacci et al., 1997), indicating a different defence and/or adaptation mechanism depending Acknowledgements on the species and the dehydration severity and time course. This study was performed by collaboration between the University Lyotropic phase transitions and non-bilayer lipid struc- of Pisa (promoter F Navari-Izzo) and the University of Belgrade tures have been reported in dehydrated cell membranes (promoter B StevanovicÂ). This paper is dedicated to the memory of O GlisÏicÂ. and liposomes (Uemura et al., 1995). 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