Photosynthesis Research 67: 79–88, 2001. © 2001 Kluwer Academic Publishers. Printed in the Netherlands.
79
Regular paper
Photosystem II photochemical efficiency, zeaxanthin and antioxidant contents in the poikilohydric Ramonda serbica during dehydration and rehydration A. Augusti1, A. Scartazza1 , F. Navari-Izzo2 , C.L.M. Sgherri2 , B. Stevanovic3 & E. Brugnoli1,∗ 1 CNR,
Istituto per l’Agroselvicoltura, Via Marconi 2, 05010 Porano (TR), Italy; 2 Dipartimento di Chimica e Biotecnologie Agrarie, Universit`a degli Studi di Pisa, 56124 Pisa, Italy; 3 Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Yugoslavia; ∗ Author for correspondence (e-mail:
[email protected]; fax: +39-0763-374330)
Received 9 April 2000; Accepted in revised form 16 June 2000
Key words: antioxidants, dehydration, non-photochemical quenching, poikilohydric plants, zeaxanthin
Abstract Changes in photochemical efficiency, non-radiative energy dissipation (NRD), de-epoxidation state of xanthophyll cycle components (DPS) and contents of the antioxidants ascorbic acid and glutathione were studied in leaves of the poikilohydric Ramonda serbica Panc. (Gesneriaceae) during cycles of dehydration and subsequent rehydration. In drying leaves, the intrinsic efficiency of PS II photochemistry and the photon yield of PS II electron transport showed strong progressive decreases. Simultaneously, the fraction of excitation energy dissipated as heat in the PS II antenna increased markedly. The energy-dependent component of non-photochemical quenching (NPQ) showed an increase in dehydrating leaves down to relative water contents (RWC) values near 30%. Further decreases in RWC below these values caused a decrease in NPQ. Accordingly, DPS showed a similar behaviour, with a sharp increase and a subsequent decrease at very low RWC, although the maximum DPS was reached at slightly lower RWC than that for the maximum NPQ. The pools of reduced ascorbate and glutathione increased strongly when the RWC values fell below 40% and remained high in fully dehydrated leaves. When plants were re-watered photosynthetic efficiency, NRD, DPS and antioxidant contents recovered their initial control values. However, during rehydration, the zeaxanthin content showed a transient increase, as did NPQ, indicating an increasing demand for non-radiative dissipation. On the other hand, the contents of reduced ascorbate and reduced glutathione decreased but were still relatively high in the initial phase of rehydration, when the rate of photosynthetic electron transport, proton pumping and NRD were still relatively low. These results indicate that several photoprotective mechanisms are operating in R. serbica. Protection from photo-oxidation and photoinhibition appears to be achieved by coordinated contributions by ascorbate, glutathione and zeaxanthin-mediated NPQ. This variety of photoprotective mechanisms may be essential for conferring desiccation-tolerance. Abbreviations: A – antherazanthin; AsA – reduced ascorbate content; DPS – de-epoxidation state of xanthophyll cycle components; Fm – maximum fluorescence in the dark after relaxation of fast relaxing components of quenching; F m – maximum fluorescence under illumination; Fv – variable fluorescence in the dark; F v – variable fluorescence under illumination; GSH – reduced glutathione content; NPQ – fast-relaxing component of non-photochemical quenching; NRD – non-radiative energy dissipation; V – violaxanthin; RWC – relative water content; Z – zeaxanthin
80 Introduction In nature, terrestrial plants are exposed to a variety of adverse environmental conditions that threaten growth and survival. Under field conditions, plants are subjected to multiple stresses, such as drought and unfavourable temperature concomitant with high irradiance. Any stress that reduces the rate of photosynthetic electron transport and the light-saturated rate of photosynthesis, either directly or mediated by stomatal closure, enhances the amount of excess excitation energy (Björkman 1989). Exposure to excessive energy, arising either from high irradiance incident on the leaf or as a consequence of drought stress, is potentially harmful and can induce photoinhibition of PS II reaction centers and enhanced production of reactive oxygen species. However, it is now widely accepted that plants are protected by several mechanisms capable of preventing potential damage. An enormous contribution to the knowledge of photoprotection in higher plants derives from the extensive work developed in Olle Björkman’s laboratory. Björkman and co-workers contributed with pioneering experiments to the understanding of photoinhibition and photoprotection. In a report about the interaction of water stress and high light, Björkman and Powles (1982), anticipating by several years more recent results, wrote: ...tolerance of the photosynthetic system to injury under water stress may in large part depend on the plant’s ability to minimize photoinhibition. Hence, any mechanism that minimize light interception under conditions of water stress, such as paraheliotropic leaf movements, increased leaf reflectance, or any mechanism that permits an increased dissipation of excessive excitation energy should alleviate water stress-induced damage to the photosynthetic system. Subsequent studies in his laboratory stimulated a large number of investigations and the role of zeaxanthin in the dissipation of excessive energy under various stress conditions has emerged (Demmig-Adams 1990). Carotenoids play relevant roles in photoprotection. They protect from photo-oxidative damage by quenching triplet chlorophylls, hence, preventing singlet oxygen formation. Additionally, carotenoids can quench singlet oxygen directly providing further protection from oxidative stress. Furthermore, it is now universally recognised that the xanthophyll zeaxanthin is involved, directly or indirectly, in non-radiative energy dissipation (NRD) in the light-harvesting antenna
of PS II (Demmig-Adams and Adams 1992; Horton et al. 1996). NRD effectively protects PS II by diverting energy away from reaction centers, and contributing to minimising the reduction state of PS II traps (Björkman and Demmig-Adams 1994). From a series of studies, it is known that NRD, measured as non-photochemical fluorescence quenching (NPQ), requires a trans-thylakoid pH, the presence of zeaxanthin and other complex changes in the lightharvesting antenna evident as a light-induced conformational change (reviewed by Yamamoto and Bassi 1996; Horton et al. 1996; Gilmore 1997). These different factors can be easily resolved in chloroplasts by differential effects of inhibitors (Yamamoto and Bassi 1996; Horton et al. 1996). Recent experiments with Arabidopsis specific mutants for NPQ have allowed the genetic dissection of the complex of mechanisms contributing to NPQ (Niyogi et al. 1998; Björkman and Niyogi 1998; see review by Niyogi 1999). Hence, it has been unequivocally demonstrated that Z is essential for NPQ development, and the specific polypeptide required for the conformational change linked to NPQ has been identified (Li et al. 2000). In addition to carotenoids, other antioxidants as ascorbate and glutathione may be important in protection from photo-oxidation and oxidative stress in general. These components, among others, protect different cellular compartments and the thiol status of proteins against free radicals and reactive oxygen species (Asada and Takahashi 1987; Gilbert et al. 1990). Increased glutathione content was demonstrated in the resurrection plant Boea hygroscopica during dehydration (Navari-Izzo et al. 1997). Glutathione and ascorbate are also relevant in metabolising hydrogen peroxide in the ascorbate/glutathione cycle, or via the glutathione peroxidase (Foyer and Halliwell 1976; Asada 1999). Ascorbate is also linked to the xanthophyll cycle and to NPQ since it is required for violaxanthin de-epoxidation (see review by Yamamoto and Bassi 1996). Poikilohydric plants, also called ‘resurrection plants’, because of their desiccation-tolerance, require a very efficient photoprotection system to withstand severe drought and excessive excitation energy. In fact, these plants are able to survive, maintaining metabolic functions, in an almost completely dehydrated state and, subsequently, are able to recover their activity readily upon rehydration (e.g. Gaff 1977; Bewley and Krochko 1982; Schwab et al. 1989). Ramonda serbica Panc. (Gesneriaceae) belongs to a small group of poikilohydric angiosperms of the northern hemisphere.
81 It is a perennial herbaceous shade-adapted plant and is considered a homoiochlorophyllous poikilohydric plant, since it preserves more than 80% of the chlorophyll content during dehydration (Markovska et al. 1994). For this reason, and for the potential excitation pressure during dehydration, R. serbica is an interesting system to study photoprotection and NRD. In addition, previous studies on photoprotection with resurrection vascular plants have been confined to ferns (Casper et al. 1993; Eickmeier et al. 1993). The objectives of the present work were to study the role of zeaxanthin in non-radiative energy dissipation during dehydration and rehydration, and to investigate the importance of ascorbate and glutathione systems in photoprotection under conditions of very low relative water contents.
Materials and methods Plant material Plants of the poikilohydric angiosperm Ramonda serbica Panˇc. (Gesneriaceae) were collected in the southeast regions of Serbia (Stevanovic et al. 1998) during fall 1998. Plants were allowed to acclimate inside a greenhouse at CNR, Porano. The growth PFD level was around 100 µmol photons m−2 s−1 and plants were irrigated to maintain an optimal plant water status. Subsequently, plants were subjected to dehydration using two different protocols. In a set of experiments, plants were subjected to drought by withholding irrigation, leaving the soil drying in pot. In a second set of experiments, dehydration was accelerated by removing most of the soil and leaving the plants and most of the roots in air. The rate of dehydration was variable because of different methods used and differences in leaf area of individual plants. Dehydration was conducted in the laboratory. Air temperature was about 22 ◦ C, RH was around 80% and the PFD was around 100–150 µmol m−2 s−1 of diffuse natural light. Measurements were started under optimal water status and, subsequently, under the effect of dehydration, fluorescence, pigment and antioxidants contents were measured. After reaching the minimum relative water content (RWC, Figure 1), plants were re-watered and the kinetics of recovery of photosynthesis and pigment, ascorbate and glutathione contents were analysed until the fully hydrated conditions were reached.
Chlorophyll fluorescence measurements Chlorophyll fluorescence experiments were conducted using the set-up described by Brugnoli and Björkman (1992) as modified by Brugnoli et al. (1994). Fluorescence was detected using a PAM 101 modulated fluorometer (Walz, Effeltrich, Germany). Actinic light and saturating flashes were provided by two KL-1500 light sources. Actinic PFD was varied interposing neutral density filters. The PFD of saturating flashes was around 8000 µmol m−2 s−1 , with a flash duration of 1 s. Measurements were conducted during dehydration and following rehydration on fullyexpanded leaves. All leaves were pre-darkened in order to allow relaxation of fast relaxing components of non-photochemical quenching (NPQ), before fluorescence experiments; the PFD during measurement was 100 µmol m−2 s−1 . The intrinsic efficiency of PS II photochemistry was calculated as Fv /Fm , immediately after the pre-darkening period. The actual efficiency of PS II electron transport during illumination was estimated at steady state as PS II = (F m −F)/F m according to Genty et al. (1989), where F m is the maximum fluorescence under illumination. The fastrelaxing component of non-photochemical fluorescence quenching was estimated according to the SternVolmer equation as NPQ=Fm /F m −1. The efficiency of PS II in the presence of energy-dependent nonphotochemical quenching was estimated as F v /F m , and [1− (F v /F m )] is a measure of the relative proportion of the energy absorbed and dissipated as heat in the PS II antennae (Demmig-Adams et al. 1996). Fluorescence nomenclature is according to Van Kooten and Snel (1990). When values of fluorescence parameters reached the steady state level, the illuminated leaf disks were collected and used for pigment analysis and relative water content (RWC). In several samples, ascorbate and glutathione contents were also measured. Pigment analysis Leaf disks were punched using a cork borer of 0.85 cm2 . Disks were immediately frozen in liquid nitrogen and stored at −80 ◦ C until extraction and analysis. Chlorophyll and carotenoid contents were determined by HPLC as described by Brugnoli et al. (1994). The de-epoxidation state was measured as DPS = (Z + A)/(V + A + Z), where V, A and Z are the xanthophyll violaxanthin, antheraxanthin and zeaxanthin, respectively.
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Figure 1. Change in relative water content (RWC) during dehydration (left panel) and rehydration (right panel) in R. serbica leaves. Different symbols and lines indicate different plants: Circles indicate plant dehydrated slowly in pots while squares indicate plant dehydrated removing roots from the soil. The two curves give the maximum extent of variability in RWC changes during dehydration and rehydration.
Antioxidant determinations Fresh leaf tissue was homogenized in ice-cold 5% trichloroacetic acid (w/v), centrifuged at 12 000 g for 15 min. The supernatant was used for total and reduced ascorbate and total and reduced glutathione. Ascorbate content was determined according to Wang et al. (1991). Total ascorbate was determined by reduction of dehydroascorbate (DHA) to AsA by dithiothreitol. The content of DHA was evaluated subtracting AsA from total ascorbate. Total and reduced glutathione contents were determined by the 5,5 -dithio-bis-(2nitrobenzoic acid) reductase recycling procedure, as in Anderson et al. (1992). Oxidised glutathione was determined after removal of GSH in the extract by 2vinylpyridine derivatizations. Changes in absorbance of the reaction mixture were measured at 412 nm at 25 ◦ C. The content of total and oxidised glutathione were measured as described by Sgherri et al. (1994). Reduced glutathione, as oxidised glutathione equivalents, was determined by the difference between total and oxidised glutathione contents. Statistical treatment Experiments were repeated several times in order to test the statistical significance of results. As discussed above, because of variation in the kinetics of dehydration and, to a lesser extent, of re-hydration, the statistical treatment of results as a function of time was difficult. However, irrespective of the method
used (potted or bare-root plants) the responses of fluorescence, pigment and antioxidant contents were univocally dependent on the RWC. Hence, responses of different parameters were analysed as function of RWC and potted and bare-root plants were pooled and analysed together (Figures 2 and 3).
Results Effects of dehydration on photochemical activity and photoprotection Dehydration caused sharp decreases in RWC in Ramonda serbica (Figure 1). Different kinetics in RWC changes were mostly due to the different dehydration protocols used, either removing or not removing roots from the soil. Additionally, limited variation in the rate of water loss was also observed among plants dehydrated using the same protocol, because of individual variation in plant leaf area and stomatal conductance (data not shown). However, irrespective of the method used, variations in various fluorescence parameters, pigment and antioxidant contents were found to be dependent on RWC (Figures 2 and 3). Marked decreases in RWC from values around 80– 90% in fully irrigated control plants, to values of about 10% after dehydration were observed in all plants irrespective of the dehydration protocol used. Conversely, after re-irrigation, plants showed a fast recovery and,
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Figure 2. Relative water content (RWC) dependencies of intrinsic efficiency of PS II photochemistry (Fv /Fm , panel A), actual efficiency of PS II electron transport in the light (PS II , panel B), relative proportion of excitation energy dissipated as heat in the PS II antennae (1 − F v /F m , panel C) and fast relaxing component of non-photochemical fluorescence quenching (NPQ, panel D) in Ramonda serbica during dehydration. Data points represent the average of different measurements on potted and bare-root plants (n = 5–7). Error bars indicate S.E. The insets in each panel show the variations in Fv /Fm , PS II , 1−F v /F m , and NPQ as function of days of dehydration. Different symbols and lines in the insets indicate different plants dehydrated either with bare-roots (−−−) or in pots (—–).
in about 40 h (Figure 1), the RWC reached values close to those observed before dehydration. Figure 2 shows the relationships between fluorescence parameters and RWC during dehydration, while the kinetics of these parameters are shown in the insets. The Fv /Fm ratio (Figure 2, inset) was initially unaffected, with values around 0.8 until d 5 and d 9 for the fast and slow dehydration treatments, respectively. However, in both treatments, a strong decrease in Fv /Fm started at the threshold RWC of about 30–40% (Figure 2). Subsequently, with lower RWC values, Fv /Fm declined down to values as low as 0.2–0.1. Similarly, the actual efficiency of PS II electron transport (PS II ) showed a sharp decrease, but starting at RWC values near 50–60% (Figure 2), higher than the threshold found for Fv /Fm .
The decrease in PS II was accompanied by a corresponding increase in (1−F v /F m ), which is indicative of an increased proportion of thermal energy dissipation in the antenna. Non-photochemical quenching showed two distinct components: A fast-relaxing and a slow-relaxing component. The fast-relaxing component of non-photochemical quenching, here reported as NPQ, showed an increase until RWC reached values near 30%; subsequently, further decreases in RWC caused a drop of NPQ (Figure 2). During dehydration, leaf pigment composition showed no significant changes. Chl content, chl a/b ratio and carotenoid contents showed limited changes, statistically not significant (data not shown). The xanthophyll cycle components were subjected to wide changes during dehydration. The deepoxidation state (DPS) of xanthophyll cycle, i.e. a measure of the con-
84 tents of zeaxanthin and antheraxanthin relative to the total pool of V + A + Z, showed a sharp increase during dehydration. The maximum level of zeaxanthin content was observed at RWC close to 20% (Figure 3). However, when RWC fell to lower values zeaxanthin content showed a significant decrease during dehydration, with a corresponding increase in violaxanthin. On the other hand, the levels of reduced ascorbate (AsA) and reduced glutathione (GSH) showed no change until the RWC reached values close to 40% and, subsequently, increased markedly and continuously until the leaves were dehydrated to the minimum RWC of about 10%. Recovery during rehydration After reaching the final level of dehydration (RWC around 10%), plants were re-watered to follow the recovery of photosynthesis and of the photoprotective mechanisms. Upon rehydration, Fv /Fm and PS II increased back to control values (Figure 4). Also, the antioxidant system and the xanthophyll cycle components returned to control values, but with different kinetics. In details, Fv /Fm increased and the control value, about 0.8, was reached after approx. 20–30 h from the start of rehydration. Concurrently, the actual efficiency of PS II increased up to values of about 0.6. In addition, the decrease in (1−F v /F m ), indicative of a decrease in NRD in the antenna, was associated with an increase in photosynthetic capacity. Rehydration caused an initial increase in DPS during the initial 10–15 h and a subsequent reduction in the late part of rehydration, corresponding to the recovery of full photochemical activity. This transient increase in DPS was similar to that observed during dehydration. Reduced ascorbate and glutathione contents during rehydration showed a fast recovery, reaching values slightly lower than those of initial controls in about 10 h. Discussion
Figure 3. Relative water content (RWC) dependencies of de-epoxidation state (DPS) of xanthophyll cycle components (panel A), reduced ascorbate (AsA, panel B) and reduced glutathione (GSH, panel C) in Ramonda serbica during dehydration. Data points represent the average of different measurements on potted and bare-root plants (n = 4–7). Error bars indicate S.E. The insets in each panel show the variations in DPS, AsA and GSH as function of days of dehydration. Different symbols and lines in the insets indicate different plants dehydrated either with bare-roots (−−−) or in pots (—–).
Chlorophyll fluorescence analysis indicated that Ramonda serbica leaves undergo a decrease in photochemical activity during dehydration, even at a light level corresponding to the prevailing growth light intensity. The decline in the intrinsic efficiency of PS II photochemistry, monitored as Fv /Fm , was matched by a corresponding decrease in the quantum yield of PS II electron transport (PS II ) under illumination (Figure 2). However, the decrease in variable fluorescence was mostly attributable to a strong decrease
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Figure 4. Effects of rehydration on: Intrinsic efficiency of PS II photochemistry (Fv /Fm , panel A), actual efficiency of PS II electron transport in the light (PS II , panel B), relative proportion of excitation energy dissipated as heat in the PS II antennae (1−F v /F m , panel C), de-epoxidation state of xanthophyll cycle components (DPS, panel D), reduced ascorbate (AsA, panel E) and reduced glutathione (GSH, panel F) contents in leaves of R. serbica. Error bars indicate S.E. Number of observations was 5–7 for fluorescence measurements and 3–4 for AsA and GSH determinations.
in Fm , accompanied by a moderate decrease in Fo (data not shown). Only at very low RWC values, Fo showed slight increases (data not shown), indicating limited photoinhibitory damage to PS II reaction centers (Björkman 1987).
Indeed, the decline in variable fluorescence observed during dehydration was mostly attributable to non-radiative energy dissipation, as indicated by the sharp increase in (1−F v /F m ) and by the general increase in non-photochemical fluorescence quenching (Figure 2). The increase in the fast-relaxing compon-
86 ent of NPQ was especially relevant in the early stage of dehydration, while it showed a decline when the RWC dropped to very low values (below 30%). Hence, it can be concluded that NRD has a major role in preventing photoinhibition in the early stage of dehydration, in agreement with previous reports with poikilohydric plants (Eickmeier et al. 1993; Casper et al. 1993). The relevance of NPQ appears to be maximum at intermediate RWC values, while at extremely low RWCs, other mechanisms may become important to avoid photodamage. In order to analyse in detail the mechanisms involved in photoprotection, we measured the content and composition in carotenoids, especially the xanthophyll cycle components, and two major components of the antioxidative defense system in leaves, namely, ascorbate and glutathione. During dehydration, the content of zeaxanthin (and antheraxanthin) increased sharply, to maximum values between 60 and 80% of the V + A + Z pool (Figure 3), and decrease to variable extent in the late stage of dehydration, when the RWC dropped to extremely low values. It is interesting to note that, although with a slight shift in the respective maximum, variation in DPS (Figure 3) and in NPQ (Figure 2) were remarkably similar, as expected on the basis of the knowledge that zeaxanthin is involved in the development of non-photochemical quenching (Demmig-Adams and Adams 1992; Björkman and Demmig-Adams 1994; Horton et al. 1996; Niyogi 1999). More recently, it has been demonstrated in Arabidopsis mutants that zeaxanthin is essential for pH-dependent NPQ, as is a specific gene product for the light-induced conformational change (Björkman and Niyogi 1998; Niyogi 1999). In agreement with the evidence that Z is a prerequisite for NPQ, the increase in Z content was associated with development and extent of the fast relaxing NPQ in Ramonda serbica leaves during dehydration. In addition, DPS was always negatively correlated with PS II (r = 0.87) demonstrating a role of zeaxanthin in the modulation of PS II electron transport. These results are in agreement with those obtained previously with other resurrection plants such as Selaginella lepidophylla (Eickmeier et al. 1993; Casper et al. 1993), showing a role for zeaxanthin in photoprotection during dehydration. The contents of AsA and GSH showed continuous increases during dehydration, indicating also a potential role of these antioxidants in preventing permanent damage to the photosynthetic system, especially under conditions of extremely low RWC.
Antioxidative defense systems may be involved in different ways in preventing damage. For example, ascorbate is an essential specific electron donor for violaxanthin de-epoxidase (Yamamoto and Bassi 1996), the enzyme responsible of conversion of violaxanthin to zeaxanthin, via the intermediate antheraxanthin. Furthermore, ascorbate-dependent Mehler-peroxidase reaction can mediate a proton pumping activity sufficient to induce Z formation and NPQ (Neubauer and Yamamoto 1992) in the pseudocyclic electron transport activity. In general, AsA and GSH protect against oxidative stress, participate in metabolising hydrogen peroxide through the ascorbate-glutathione cycle (Drotar et al. 1985; Sgherri and Navari-Izzo 1995), can directly quench activated oxygen species and can control the redox state of the chloroplasts (Neubauer and Yamamoto 1994; Asada 1999). Nevertheless, the response of AsA and GSH contents in plants subjected to drought is not univocal and varies with species and conditions, and its regulation mechanism is still unclear (Asada 1999). While relatively low levels were reported in wheat and in the resurrection plant Sporobolus stapfianus under drought (Sgherri et al. 1994; Loggini et al. 1999), higher contents were found in other species (Buckland et al. 1991) and, especially, in the poikilohydric species Boea hygroscopica (NavariIzzo et al. 1997). From the present results with the poikilohydric R. serbica, it is clear that the massive increase in GSH and AsA contents plays a relevant role in photosynthetic regulation, together with the xanthophyll cycle components. In fact, AsA was negatively correlated with PS II (r = 0.93), while the correlation for GSH was not significant (data not shown). Indeed, the highest levels of AsA and GSH were reached at very low RWC values, when zeaxanthin, although present, was not at highest concentration. In addition, the total ascorbate and glutathione pools also increased, as did the proportion of reduced to total ascorbate and glutathione (data not shown). Hence, it may be hypothesised that AsA and GSH may be important in avoiding damages when photochemical activity and, consequently proton pumping, are decreasing to values close to zero. This is in agreement with previous reports showing that these antioxidants respond to the level of excess energy (Logan et al. 1998). These antioxidants were also maintained high in the inactive, completely dehydrated state. Hence, they may confer protection upon the onset of rehydration, when photosynthesis is still inactive and excitation pressure can be high. From the present results, it
87 is also evident that disassembly of PS II reactions centers, or their conversion to PS II-β, with low photochemical yield, occurred during dehydration. However, before reaction centers are disassembled zeaxanthin-mediated NRD and antioxidants are relevant in photoprotection against permanent damage. When leaves of R. serbica were allowed to take up water, they showed a fast recovery of photosynthetic activity. Most of the recovery of Fv /Fm and PS II was accomplished in about 20–25 h (Figure 4). As mentioned above, it is obvious that the early stage of rehydration is potentially harmful for PS II reaction centers, because of low RWC, low electron transport capacity and high excitation energy. During this stage, an increase in DPS was evident, and concurrently the level of NPQ was maintained high. On the other hand, the levels of GSH and AsA showed a fast decrease. Hence, it can be concluded that while GSH and AsA appear to be crucial at very low RWC, zeaxanthinmediated NPQ may be most relevant at intermediate values of RWC (20–40%), when there is electron transport activity and proton pumping present. It is also evident that zeaxanthin was always retained in dehydrated leaves, and this may be crucial for mediating NRD (Demmig-Adams et al. 1998) and preventing damage during dehydration and, perhaps more importantly, during rehydration, when inactive PS II centers are reconverted back to the fully competent form. This interpretation is also in agreement with the results reported by Casper et al. (1993). In conclusion, leaves of the resurrection Ramonda serbica possess different photoprotective mechanisms allowing them to withstand profound dehydration during drought periods, maintaining the integrity of metabolism. It is clear that zeaxanthin plays a major role in non-radiative energy dissipation, that protects against photoinhibition. While the maximum level of zeaxanthin was found at intermediate levels of RWC (20– 30%), some zeaxanthin was also maintained in the completely dehydrated inactive state, and this may be crucial for contributing energy dissipation during rehydration. On the other hand, our results show that reduced ascorbate and reduced glutathione play major roles in preventing photo-oxidation, especially when dehydrating leaves approach the lowest water content. The increase in the pools of these antioxidants may be relevant for conferring desiccation-tolerance. Evidently, zeaxanthin, ascorbate and glutathione contribute to the modulation of photosynthetic electron transport and appear to be crucial for photoprotection during dehydration and rehydration in poikilohydric plants.
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