Plasma Membrane Lipids in the Resurrection Plant Ramonda Serbica Following Dehydration and Rehydration
Total Page：16
File Type：pdf, Size：1020Kb
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.