Science 168 (2005) 105–111 www.elsevier.com/locate/plantsci

Low-molecular weight substances in the poikilohydric plant serbica during dehydration and rehydration

Tamara Zˇ ivkovic´a, Mike Frank Quartaccib, Branka Stevanovic´a, Franca Marinonec, Flavia Navari-Izzob,*

aInstitute of , University of Belgrade, Takovska 43, 11000 Belgrade, 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 to 4% in the desiccated ones, plants regaining the initial RWC upon complete rehydration. The decrease in the osmotic potential at full turgor from 0.7 MPa in the control plants to 2.2 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 Cl. 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+,Cl) 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 dry weight)/ and to control the rate of water loss. It is considered a (saturated weight 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 2 fully watered until the beginning of the experiment. One set detector was used for the determination of SO4 H2PO4 , 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 proline contents

Free amino acids and proline contents in leaves and in the cell sap of detached turgid leaves were determined by the ninhydrin method. Briefly, free amino acids and proline were extracted from leaves with 5 ml of 85% ethanol for 30 min. An aliquot (0.2 ml) of the filtered samples or cell sap was added to 1 ml of 0.2 M citrate buffer (pH 5.0) containing 0.4 N NaOH, 0.72 mM SnCl22H2O and 2% ninhydrin in ethylene glycol. The mixture was incubated in boiling water for 20 min. Samples were cooled and diluted with 5 ml of water:propanol (1:1, v/v). After 15 min, the absorbance of the samples at 570 and 440 nm was read with a Varian Cary 1 spectrophotometer for the determination of free amino acids and proline, respectively. Leucine and proline were used for calibration curves.

2.6. Soluble sugar content

Soluble sugar content in the cell sap of detached turgid leaves was determined by the H2SO4/phenol method of Fig. 1. Relative water content of Ramonda serbica leaves subjected to Roughan and Batt [21]. For sugar determination in leaves, dehydration and rehydration. The arrow indicates the onset of rehydration. 5 g of leaves were homogenised with 100 ml of 70% Results are the means of ten repetitions of three independent experiments methanol and extracted for 6 h under continuous stirring. S.E. (n = 30). The suspension was left at 4 8C overnight, filtered (What- man No. 40) and vacuum dried at 40 8C. The residue was taken up with 60 ml of distilled water and sonicated to 3. Results facilitate dissolution. Lipophilic substances were removed from soluble sugars and polar compounds by ethyl Throughout the dehydration-rehydration cycle, leaves of acetate extractions (4 15 ml). After phase separation, control fully hydrated plants did not show significant the organic fraction was discarded whereas the aqueous one differences in the RWC and in the biochemical parameters. was evaporated at reduced pressure and used for gas For this reason the data relative to control plants represent chromatographic analyses. Samples (about 30 mg) of the the mean value of the control plants at each harvest time. aqueous extracts, added with 2 mg of the internal standard During the 13 days of dehydration the RWC of R. serbica phenyl-b-D-glucopyranoside, were derivatised with a sily- leaves continuously decreased from 98% in the fully lating reagent made up of pyridine:hexamethyldisilazane hydrated plants (C) to 4% in the desiccated ones (D4) (HMDS):trimethylchlorosilane (TMS) (2:1:1, v/v/v). The (Fig. 1). RWC showed a remarkable decline especially in the mixture was heated at 70 8C for 30 min and vacuum dried. first 6 days decreasing to 67 (D1), 45 (D2) and 23% (D3) The residue was solubilised with iso-octane and filtered after 4, 5 and 6 days from dehydration, respectively. Its using a silica:celite (1:1, w/w) column under a nitrogen flux. further reduction to 4% (D4) occurred in 1 week. Upon Soluble sugar analysis was performed by FID-GC (Fisons rehydration, plants showed a rapid recovery of their water 8180) using a EASY SEP-54 non-polar capillary column content regaining 23, 50 and 95% RWC after 6, 24 and 72 h (Analitycal Technology), 25 m length, 0.32 mm i.d. and from rewatering, respectively (Fig. 1). 0.15 mm film-thickness. Column temperature was in a range The decrease in leaf water content caused a dramatic from 100 to 340 8C (100–280 8C, 10 8C/min; 280–340 8C, reduction of the cs value, which decreased from 0.7 MPa 15 8C/min; 340 8C for 15 min), detector temperature was at in the control leaves to 56.2 MPa in the desiccated ones 2 360 8C and the gas pressure (H2) was at 0.3 kg/cm . (Table 1). Upon rehydration, the cs was rapidly recovered,

Table 1 100 Osmotic potential (cs) and osmotic potential at full turgor (cs ) of leaves of Ramonda serbica subjected to dehydration and rehydration C D4R1R2R3 cs (MPa) 0.7 0.1 56.2 2.9 5.8 0.3 1.8 0.2 0.8 0.1 100 cs (MPa) 0.7 0.2 2.2 0.3 1.6 0.2 1.0 0.2 0.8 0.1 C, fully hydrated plants (98% RWC); D4, desiccated plants (4% RWC); R1, R2, R3, rehydrated plants (23, 50 and 95% RWC, respectively). Results are the means of three independent experiments each analysed twice S.E. (n = 6). 108 T. Zˇivkovic´ et al. / Plant Science 168 (2005) 105–111 reaching in the fully rehydrated plants the same value Table 3 detected in control leaves. The c100 showed the same trend, Contribution (%) of low molecular weight solutes to osmoregulation in the s cell sap of Ramonda serbica detached turgid leaves during dehydration and desiccated plants undergoing a decrease of about three-fold rehydration in comparison with the control. Rewatering resulted in a 100 CD4R1R2R3 complete regain of the initial cs value (Table 1). As regards solute concentration, the most remarkable Inorganic cations 34.8 39.5 44.9 42.3 34.1 Inorganic anions 22.3 31.6 25.1 20.8 14.3 changes during the dehydration–rehydration cycle were Organic acids 12.5 8.0 19.8 21.5 19.8 found in the amount of inorganic ions (Table 2). With the Soluble sugars 16.4 3.4 7.3 11.1 14.9 exception of H2PO4 , which remained almost constant Free amino acids 0.3 0.1 0.1 0.1 0.2 during the whole cycle, they accumulated upon desiccation, Others 13.7 17.4 2.8 4.2 16.7 thus actively participating in osmotic adjustment. Among C, fully hydrated plants (98% RWC); D4, desiccated plants (4% RWC); R1, inorganic ions, K+ was the one present in far higher R2, R3, rehydrated plants (23, 50 and 95% RWC, respectively). Results are concentration. In contrast to all the other ions, which showed the means of three independent experiments each analysed twice (n = 6). the highest concentration in the cell sap of desiccated leaves (Table 2), K+ reached the highest amount after 6 h from 34.8% of total solutes in the cell sap of control plants up to rewatering (227 mM at R1 stage). By further rehydration, K+ 44.9% in the leaves rewatered for 6 h (R1), returning to the concentration decreased and returned to almost the same initial value at the end of rehydration. Anions showed the 100 value detected in control plants. As regards the other highest contribution to cs in the desiccated plants inorganic ions, following a significant accumulation during (31.6%), decreasing afterwards during water supply. water shortage their concentrations in the cell sap gradually Organic anions were calculated from the difference of lowered during rehydration. In comparison with the control, cations–anions, which have been shown to relate stoichio- organic acid concentration doubled at the end of desiccation metrically to the amounts of carboxylates [22]. Organic and was four-fold higher after 6 h of rewatering, then acids gave a significant contribution to osmotic adjustment, decreasing during further rehydration. Differently from ions with the lowest values detected in the desiccated leaves and organic acids, concentration of total soluble sugars in (Table 3). Compared to control plants, contribution of sugars 100 the cell sap did not increase during dehydration showing, on to the cs showed a five-fold decrease in the dehydrated the contrary, a 34% reduction (Table 2). Sugars regained the plants and a regain of the initial value at the end of the initial value as soon as water was supplied. Free amino acids, rehydration period. Free amino acids gave a negligible detected in very small amounts, did not undergo any contribution to osmoregulation. significant change throughout the whole cycle. Soluble sugars detected in leaves were glucose, fructose, Taking into account all the osmotically active substances sucrose, raffinose and the galactosyl inositole galactinol detected, results show that during the dehydration-rehydra- (Fig. 2). The amount of raffinose as well as those of the tion cycle inorganic ions gave the greatest contribution to the 100 cs of leaves (Table 3). Cation contribution increased from

Table 2 Concentration (mM) of low molecular weight solutes in the cell sap of Ramonda serbica detached turgid leaves during dehydration and rehydra- tion CD4R1R2R3 K+ 74 6 144 9 227 18 145 17 96 3 Na+ 5 274 18 10 27 13 1 Ca2+ 10 287 844 329 26 1 Mg2+ 11 152 514 110 17 1 P cations 100 357 295 191 112

2 SO4 11 166 628 114 19 1 H2PO4 10 19 114 112 211 3 NO3 25 11 82 154 729 111 2 Cl 18 3 128 18 69 13 39 616 1 P anions 64 285 165 94 47 Organic acids 36 372 3 130 297 365 3 Soluble sugars 47 331 248 450 449 2 Free amino acids 0.8 0.2 0.5 0.1 0.4 0.1 0.5 0.1 0.5 0.1 C, fully hydrated plants (98% RWC); D4, desiccated plants (4% RWC); R1, R2, R3, rehydrated plants (23, 50 and 95% RWC, respectively). Results are Fig. 2. Soluble sugar content and composition of Ramonda serbica leaves the means of three independent experiments each analysed twice S.E. subjected to dehydration and rehydration. Results are the means of three (n = 6). independent experiments each analysed twice S.E. (n = 6). T. Zˇivkovic´ et al. / Plant Science 168 (2005) 105–111 109

harvest time detached leaves were rewatered to attain full turgor before solute measurement, thus taking into account the mistakes in solute determination arising from the loss of water. In R. serbica the decrease in osmotic potential during dehydration was due not only to a water loss from the leaf tissues but also to an effective increase of cell solute concentration which allows an osmotic adjustment, as 100 shown by the decrease in cs (Table 1). Osmotic adjustment in R. serbica resulted mainly from an accumulation of inorganic ions and, to a lesser extent, from an increase in sucrose content (Table 2 and Fig. 2). Indeed, the osmotic adjustment accomplished by the accumulation of inorganic ions is considered an energetically cheaper way in comparison with the synthesis of organic osmolytes [23]. The fact that the uptake of the majority of inorganic ions was enhanced during dehydration due to an increased mass flow from the more concentrated soil solution into the root, is in agreement with the results of some other authors [5,20,24]. In the course of the dehydration-rehydration cycle, K+ ion concentration in leaves exceeded all the inorganic ions Fig. 3. Free amino acid and proline contents of Ramonda serbica leaves analysed (Table 2), confirming its important role in subjected to dehydration and rehydration. Results are the means of three osmoregulation. The further remarkable increase in K+ independent experiments each analysed twice S.E. (n = 6). concentration during the first few hours after rewatering, reducing sugars, glucose and fructose, decreased during when water balance was already partially recovered (RWC dehydration compared to control plants. Upon rehydration 23%), indicates its importance on stomata opening and their concentrations increased, but in rehydrated plants their consequent increase in CO2 uptake. This observation is contents were still about 50% lower than those detected in supported by a previous study on R. serbica which showed fully hydrated leaves. Sucrose content increased starting that in the same period the intrinsic PS II efficiency was from day 4 (D1 stage), being in desiccated plants 2.5-fold recovered by almost 30% [17]. Furthermore, it is likely that higher than in the control ones (Fig. 2). Even though an increased K+ content did not cause any negative effect on decreasing, at the end of rehydration sucrose amount cell structures as this ion is regarded as a compatible solute remained 60% higher compared to the control. In the [24]. The content of other inorganic ions, such as Na+ and desiccated plants galactinol resulted 5.5-fold lower than in Cl, were also significantly increased during dehydration the hydrated ones. Upon rewatering galactinol content did (Table 2). Their possible detrimental effect on the cellular not change. structures could have been avoided or limited by restoration Both total free amino acid and proline contents followed of ionic balance and/or by their vacuolar sequestration. The almost the same behaviour during the whole dehydration- excess of cationic charge, derived from the difference rehydration cycle. Water deficit caused a reduction in the between major cations and anions, indicates that organic total free amino acid and proline contents of leaves during anions are required to balance the excess of positive charges the first 6 days (Fig. 3), followed by an increase during the (Table 2). remaining period of dehydration. At the end of desiccation It is well known that in resurrection plants changes in the Ramonda leaves showed a 50% reduction in the total free content of soluble sugars during water stress play one of the amino acids and a much lower decrease in proline content, key roles in overcoming desiccation-induced damages to the latter representing 71% of the total free amino acid membranes [13,25–27]. The decrease in the amounts of content. Upon the first 6 h of rehydration, both free amino reducing sugars such as glucose and fructose detected in R. acid and proline amounts quickly decreased. By further serbica (Fig. 2) and previously in R. nathaliae and R. myconi rewatering, free amino acids increased without regaining the leaves [27] may have a positive effect on desiccation value detected in the fully hydrated leaves, whereas proline tolerance since it has been shown that their presence in high content did not change (Fig. 3). concentrations is detrimental, primarily because they alter protein structure, increase respiration and mitochondrial electron transport. These processes may impair cell 4. Discussion metabolism favouring energy production and provoking damages to cell membranes by active oxygen species In order to distinguish solute accumulation processes formation [2]. associated with osmoregulation from the simple concentra- In contrast, an increase in sucrose amount during drought tion of solutes which accompanies loss of water, at each (Fig. 2) is a characteristic response of resurrection species 110 T. Zˇivkovic´ et al. / Plant Science 168 (2005) 105–111 due to the fundamental role of this sugar in both of a delicate balance of some enzyme activities of either osmoregulation and membrane stabilisation [28,29]. It has biosynthetic or degradative pathways which are impaired been observed that sucrose was the predominant soluble under water deficit conditions [36–38]. As a consequence, carbohydrate in R. nathaliae and R. myconi and that during proline did not affect osmotic adjustment in this resurrection desiccation its level steadily increased in both intact and plant during desiccation even though its potential role in excised leaves [27], indicating that sucrose accumulation is antioxidative defence systems or in gene expression may not connected to desiccation tolerance in . Indeed, be disregarded as evidenced by studies on some other in water deficit conditions sucrose interacts with polar head species [37–39]. groups of membrane phospholipids, thus preserving the In conclusion, desiccation tolerance in R. serbica under membrane native structure and preventing leakage due to stressful water deficit conditions and its rapid recovery upon phase transition. Furthermore, sucrose interacts with rewatering are achieved through a complex of functional and proteins to form hydrogen bonds with specific polar groups structural adaptations, among which the osmotic adjustment on the protein surface, not allowing the formation of and sucrose accumulation play an important role. hydrogen bonds within the proteins themselves that would irreversibly change their tertiary structure [30]. In Craterostigma plantagineum, despite the increase in Acknowledgements sucrose upon dehydration, the overall sugar content was similar in hydrated and dried plants [31]. It is likely that This study was performed within the International Inter sugar composition, rather than just concentration, is related University Cooperation Convention between the University to desiccation tolerance. The capacity of sucrose to vitrify is of Pisa (promoter F. Navari-Izzo) and the University of facilitated by the presence of raffinose [27,32,33], the only Belgrade (promoter B. Stevanovic´). Travelling expenses of oligosaccharide detected in the leaves of R. serbica (Fig. 2). T.Z. were supported by Project 1505 of Ministry of Sciences, Its decrease during dehydration is in agreement with Technologies and Development of Serbia. previous results on the Gesneriaceae resurrection species R. nathaliae, R. myconi and Haberlea rhodopensis [27].We might hypothesise that raffinose content, despite its decrease, was sufficient for sucrose vitrification to occur. References As raffinose is formed by galactosylation of sucrose, it could [1] J.D. Bewley, J.E. 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