Plant Science 168 (2005) 105–111 www.elsevier.com/locate/plantsci Low-molecular weight substances in the poikilohydric plant Ramonda serbica during dehydration and rehydration Tamara Zˇ ivkovic´a, Mike Frank Quartaccib, Branka Stevanovic´a, Franca Marinonec, Flavia Navari-Izzob,* aInstitute of Botany, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia and Montenegro bDipartimento di Chimica e Biotecnologie Agrarie, Universita` di Pisa, Via del Borghetto 80, 56124 Pisa, Italy cDipartimento di Chimica Organica, Universita` di Pavia, Via Taramelli 10, 27100 Pavia, Italy Received 30 January 2004; accepted 20 July 2004 Available online 13 August 2004 Abstract The desiccation-tolerant plant Ramonda serbica Panc. was subjected to dehydration by withholding water for 13 days and then rehydrated by rewatering for 72 h. Dehydration reduced the relative water content (RWC) from 97% in the fully hydrated plants to 4% in the desiccated ones, plants regaining the initial RWC upon complete rehydration. The decrease in the osmotic potential at full turgor from À0.7 MPa in the control plants to À2.2 MPa in the desiccated leaves indicates that an osmotic adjustment came into play. The osmotic adjustment in the dried leaves was due primarily to the high concentration of inorganic ions (71% of total solutes), especially K+ and ClÀ. Other detected compounds such as soluble sugars and free amino acids gave a rather low contribution to the osmotic potential at full turgor of the desiccated leaves. Generally, upon rehydration all the osmotically active substances almost returned to their initial concentrations. The presence of increased amounts of sucrose detected during desiccation is discussed in relation to its role in membrane stabilisation. # 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Desiccation tolerance; Osmoregulation; Osmotic potential; Ramonda serbica; Resurrection plants; Solutes; Sugars 1. Introduction to injury and need to be maintained or quickly repaired as soon as water enters again the cells [2–4]. The ability of vascular flowering plants to tolerate Osmotic adjustment, the lowering of osmotic potential by dehydration is very rare. An exceptional adaptation among the net increase in intracellular solutes, is generally higher plants occurs in the poikilohydric resurrection plants, recognised as an adaptative mechanism to water stress whose fully differentiated tissues tolerate prolonged con- and is considered as a major component of drought-tolerant ditions of almost total protoplast desiccation and revive upon mechanisms allowing continued influx of water. Inorganic rehydration. Desiccation tolerance may depend on different ions (especially K+,Na+,ClÀ) have been shown to have a physiological and biochemical strategies carried out by the key role in turgor regulation and in turgor regulatory systems plants in order to survive and to regain normal metabolic such as the movement of guard cells [5]. Under water deficit processes upon rehydration [1,2]. Of crucial importance in conditions, also organic solutes increase, including sugars, desiccation-tolerant plants are the physical and chemical amino acids, mainly proline, quaternary ammonium properties of membranes, which are very sensitive and liable compounds and organic acids [5–8]. Membrane-compatible solutes, in contrast to perturbing solutes such as chaotropic ions, may stabilise folded protein structures [9],so protecting enzyme activities against the increase in ion Abbreviations: RWC, relative water content; cs, osmotic potential; 100 concentration following drought. It is worth to mention that s , osmotic potential at full turgor * Corresponding author. Tel.: +39 050 971921; fax: +39 050 598614. these compounds operate both in osmoregulation and in E-mail address: [email protected] (F. Navari-Izzo). preservation and/or stabilisation of cell structures [2,10]. 0168-9452/$ – see front matter # 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2004.07.018 106 T. Zˇivkovic´ et al. / Plant Science 168 (2005) 105–111 Contribution of carbohydrates, particularly disacchar- Samples were harvested from fully hydrated leaves (C), and ides, as protectants against the dry-induced damage of cell then after 4 (D1), 5 (D2), 6 (D3) and 13 (D4) days after the membranes is a common feature of all living beings able to beginning of dehydration. Rehydration was started by cope with complete dehydration [11]. In seeds of higher spraying the plants with water to simulate rainfall and plants, a correlation between the accumulation of soluble keeping the soil damp. Leaves were collected upon sugars and the acquisition of desiccation tolerance has been rewatering of whole plants after 6, 24 and 72 h (R1, R2 observed [12]. Sucrose, raffinose and trehalose are thought and R3 stages, respectively). During the dehydration- to be involved in glass formation and/or to interact rehydration cycle harvested detached leaves were rewatered protectively with membrane phospholipids [13,14]. Further- to full turgor before chemical analyses. The experiment was more, carbohydrate changes which occur following drought run in triplicate and for each experiment, at each sampling are of particular importance due to their direct relationship date, three samples were collected. All measurements were with physiological processes such as photosynthesis, carried out on mature and fully expanded leaves comparable respiration, nutrient uptake and transport. in size and collected from the middle of the rosettes. According to Oliver [15], Ramonda serbica belongs to a small group of poikilohydric angiosperms of the northern 2.2. Relative water content hemisphere. It is a perennial herbaceous shade-adapted plant belonging to the group of resurrection plants which are able The RWC of leaves, collected at regular intervals during to withstand desiccation, even for months, using both the dehydration-rehydration cycle, was calculated according morphological and physiological mechanisms to slow down to the formula: 100  [(fresh weight À dry weight)/ and to control the rate of water loss. It is considered a (saturated weight À dry weight)] and expressed as the mean homoiochlorophyllous plant, since during desiccation value of ten replicates for each treatment. Saturated weight preserves more than 80% of its chlorophyll content [16]. was determined after incubation of the leaves in water for A particular feature of this resurrection plant is the 24 h at room temperature. Dry weight was measured recovery of its full photosynthetic activity following water following oven drying at 105 8C until constant weight. supply [17]. This species has also the ability to maintain cell membrane integrity preserving its plasma membrane lipid 2.3. Leaf osmotic potential composition and semipermeability during dehydration [4,18] as well as activating protective mechanisms such as higher At each harvest time mature leaves were harvested, put in levels of zeaxanthin and reduced ascorbate and glutathione 1 ml plastic vials and immediately frozen in liquid nitrogen. [17]. The observed increase in phenolic acids during For extraction of the cell sap, the frozen leaves were thawed dehydration might also play a role in protecting Ramonda and centrifuged at 21,000  g for 5 min, and the sap membranes from desiccation-induced damages [19]. collected from the disrupted tissues. The osmotic potential Our major aim was to follow the changes of low- (cs) of the cell sap was measured by a Roebling molecular weight components of leaf cell sap during a cycle microosmometer and quantified by van’t Hoff equation of dehydration and rehydration, to investigate the contribute X of these solutes to osmotic adjustment and to define, when c ¼ RT s cj possible, their role in the resurrection phenomenon. Due to P their involvement in membrane stabilisation, composition where cj represents the sum of the molal concentration of and content of sugars were investigated in leaves as well. all solutes [20]. Measurement of the osmotic potential at full turgor was performed on detached leaves using the same procedure described above after incubation of leaves in 2. Material and methods water for 24 h at room temperature to regain full turgor and freezing them in liquid nitrogen. Contribution of indi- 100 2.1. Plant material vidual solutes to cs was calculated by the van’t Hoff equation reported above. Plants of the desiccation-tolerant species R. serbica Panc., comparable in size and appearance, were collected 2.4. Determination of inorganic ions from their natural habitat in the south-east region of Serbia (Sicevo gorge) together with the layer of soil on which they Inorganic ions were determined in the cell sap extracted grew. Plants were transferred to the greenhouse of the for cs evaluation of detached turgid leaves. An ion Dipartimento di Chimica e Biotecnologie Agrarie of the chromatograph (Dionex DX-100) equipped with an IonPac University of Pisa and acclimated for 6 weeks keeping them AS4A column (4 mm  250 mm) and a conductivity 2À À fully watered until the beginning of the experiment. One set detector was used for the determination of SO4 H2PO4 , À À 2+ 2+ + + of plants was dehydrated by withholding water at room NO3 and Cl anions. Ca ,Mg ,Na and K cations were temperature and ambient photoperiod, whereas another set determined by a flame atomic absorption spectophotometer was watered daily during the whole experimental period. (Perkin-Elmer 373). T. Zˇivkovic´ et al. / Plant Science 168 (2005) 105–111 107 2.5. Free amino acid and