Journal of Experimental Botany, Vol. 54, No. 381, pp. 405-418,
January 2, 2003
© 2003 Oxford University Press
Reversibility of cold- and light-stress tolerance and accompanying changes of metabolite and antioxidant levels in the two high mountain plant species Soldanella alpina and Ranunculus glacialis
Received 1 July 2002; Accepted 15 September 2002
Station Alpine du Lautaret et Laboratoire de Physiologie Cellulaire Végétale, Unité Mixte de Recherche 5019 (Commissariat à l’Energie Atomique, Centre National de la Recherche Scientifique, Université Joseph Fourier), Département de Biologie Moléculaire et Structurale, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble cedex 9, France
1 Present address and to whom correspondence should be sent: Laboratoire d‘Ecophysiologie Végétale, Bâtiment 362, UFR Scientifique d‘Orsay Université Paris XI, 91405 Orsay Cedex, France. Fax: +33 (0)1 69 15 72 38. E-mail: peter.streb{at}eco.u-psud.fr
Abbreviations: qN, non-photochemical quenching of chlorophyll fluorescence; VAZ, violaxanthin+antheraxanthin+zeaxanthin.
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Abstract |
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Two high mountain plants Soldanella alpina (L.) and Ranunculus glacialis (L.) were transferred from their natural environment to two different growth conditions (22 °C and 6 °C) at low elevation in order to investigate the possibility of de-acclimation to light and cold and the importance of antioxidants and metabolite levels. The results were compared with the lowland crop plant Pisum sativum (L.) as a control. Leaves of R. glacialis grown for 3 weeks at 22 °C were more sensitive to light-stress (defined as damage to photosynthesis, reduction of catalase activity (EC 1.11.1.6) and bleaching of chlorophyll) than leaves collected in high mountains or grown at 6 °C. Light-stress tolerance of S. alpina leaves was not markedly changed. Therefore, acclimation is reversible in R. glacialis leaves, but constitutive or long-lasting in S. alpina leaves. The different growth conditions induced significant changes in non-photochemical fluorescence quenching (qN) and the contents of antioxidants and xanthophyll cycle pigments. These changes did not correlate with light-stress tolerance, questioning their role for light- and cold-acclimation of both alpine species. However, ascorbate contents remained very high in leaves of S. alpina under all growth conditions (12–19% of total soluble carbon). In cold-acclimated leaves of R. glacialis, malate represented one of the most abundant compounds of total soluble carbon (22%). Malate contents declined significantly in de-acclimated leaves, suggesting a possible involvement of malate, or malate metabolism, in light-stress tolerance. Leaves of the lowland plant P. sativum were more sensitive to light-stress than the alpine species, and contained only low amounts of malate and ascorbate.
Key words: Antioxidants, cold-acclimation, malate, NMR-spectroscopy, photoinhibition, xanthophyll cycle.
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Introduction |
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Plants growing at high altitude in the Alps are frequently exposed to high light intensities at low temperature. It was shown previously that the two high mountain plant species S. alpina and R. glacialis are highly resistant to such environmental conditions (Streb et al., 1997; Körner, 1999). Clearly, alpine plants are acclimated to low-temperature at high irradiance and are probably not stressed under these conditions (Körner, 1999) because they have efficient protective mechanisms. Nevertheless, non-acclimated lowland plants suffer from severe photo-oxidative damage under such conditions (Streb et al., 1997). Even though light intensities of 1000 µmol m–2 s–1 PAR at low temperature might be non-stressful for alpine plants, these conditions are defined here as light-stress with respect to the majority of non-acclimated lowland plants.
The light-inactivation of the reaction centre protein D1 of PSII and of the peroxisomal enzyme catalase are among the early and widespread symptoms of photodamage at low temperature in many plant species (Feierabend et al., 1992; Huner et al., 1993). Hence, these parameters were often used as indicators of light-stress (Streb et al., 1997, 1998, 1999). As expected, the turnover of these light-sensitive proteins in intact leaves of S. alpina and R. glacialis was low, while the isolated proteins were similarly inactivated as proteins isolated from lowland plants (Streb et al., 1997; Shang and Feierabend, 1998). Some lowland species can be acclimated to low temperature at high light intensity (Huner et al., 1993; Streb et al., 1999). However, the tolerance to light-stress achieved in cold-acclimated lowland plants was less than that observed in high mountain plants under comparable conditions (Streb et al. 1998, 1999), suggesting that acclimation in alpine plants is characterized by unique metabolic pathways and protective mechanisms. The possible contribution of metabolites and of protective mechanisms to light-stress tolerance was investigated further in this study.
Numerous different protective mechanisms against light- and cold-stress are discussed in the literature and their relative importance appears to be species dependent (Huner et al., 1993; Falk et al., 1996; Nishida and Murata, 1996; Savitch et al., 2001). Both low temperature and high irradiance increase the production of reactive oxygen species and the risk of oxidative damage (Wise, 1995). Hence the enhanced capacity of cold-acclimated leaves to scavenge reactive oxygen species was regarded as an adaptive response (Schöner and Krause, 1990; Polle, 1997). Similarly, cold-acclimated leaves showed increased formation of the xanthophyll cycle pigment zeaxanthin, which is believed to mediate the non-radiative energy dissipation of excess excitation energy (Verhoeven et al., 1996; Thiele et al., 1996). High contents of antioxidants and carotenoids also correlated positively with the altitude where alpine plants were collected (Wildi and Lütz, 1996). However, the capacity of the antioxidative system and the xanthophyll cycle differed greatly in leaves of S. alpina and R. glacialis. The amounts of ascorbate, glutathione and 
-tocopherol and the activities of catalase, SOD and glutathione reductase were very high in S. alpina, but markedly lower in R. glacialis leaves (Wildi and Lütz, 1996; Streb et al., 1997, 1998). Furthermore, leaves of S. alpina were resistant to oxidative stress as generated by the herbicide paraquat and sensitive to the inhibition of zeaxanthin formation by dithiothreitol, suggesting the involvement of antioxidants and xanthophyll cycle activity in light-stress tolerance. In contrast, leaves of R. glacialis were very sensitive to paraquat and only slightly affected by dithiothreitol (Streb et al., 1998), suggesting that other, unidentified, mechanisms must be involved in acclimation.
In cereals, cold- and light-stress tolerance is correlated with an increased capacity to keep the photosynthetic electron transport chain in the oxidized state during illumination at low temperature (Öquist and Huner, 1993). Similar observations were made in R. glacialis leaves compared to leaves of S. alpina (Streb et al., 1998). Higher capacities of CO2-assimilation, higher rates of photorespiration and higher rates of sucrose synthesis in cold-acclimated plants all might explain this ability (Huner et al., 1993; Heber et al., 1996; Falk et al., 1996; Strand et al., 1999; Savitch et al., 2001).
Cold-acclimated rye leaves can be partially de-acclimated within 3 d at ambient temperature. This de-acclimation is not accompanied by a major loss in antioxidative protection or xanthophyll cycle activities, questioning their importance for the acclimation process (Streb et al., 1999). These results stimulated the present investigation. The possibility of de-acclimation in alpine species and the associated changes in antioxidant and carbon metabolism were investigated. Therefore, high mountain plants were transferred to low elevation at two different growth temperatures under weak light. A wide range of soluble carbon metabolites was screened by 13C-NMR-spectroscopy for a possible correlation with cold-acclimation in S. alpina and R. glacialis leaves. Any change could be related to processes involved in the resistance to high light and cold and triggered by the growth condition experienced in the natural environment. As a lowland plant reference, pea leaves were used because their response to cold and light is well characterized (Feierabend et al., 1992) and the NMR-spectra from pea are of high quality. The advantages of NMR-spectroscopy is that while other techniques are limited to the metabolites that are specifically chosen for study, NMR-spectroscopy offers the opportunity to reveal unexpected information (Bligny and Douce, 2001). Thus, it is possible that special metabolites that are of particular importance for cold-acclimation escape detection by other analytical methods.
The aims of the present investigation were (1) to determine whether cold- and light-stress tolerance is constitutive or can be reversed in alpine plant species and (2) whether changes of light- and stress-tolerance are accompanied by changes in metabolite or antioxidant contents which might be necessary for acclimation.
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Materials and methods |
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Plant material and growing conditions
P. sativum plants were grown under controlled conditions (14/10 h light/dark) in white light at an intensity of 100 µmol m–2 s–1 PAR in vermiculite moistened with tap water. Plants were either grown at a temperature of 22 °C (day) and 18 °C (night) for 10–12 d or at a constant temperature of 6 °C for 16 weeks. At this stage 22 °C-grown plants and 6 °C-grown plants had similar leaf numbers and chlorophyll contents on a fresh weight basis.
Leaves of S. alpina and R. glacialis were collected in the morning at the Col du Galibier in the French Alps at elevations between 2400–2700 m as described by (Streb et al., 1997). For de-acclimation, whole plants were taken from their natural site at the Col du Galibier, placed in pots together with soil from this site and were grown in Grenoble at a low elevation of 200 m for 3 weeks at 22 °C or at 6 °C under the same conditions as described for pea plants. In order to be sure that de-acclimation occurred, the de-acclimation period was extended to 3 weeks as compared to 3 d for rye leaves (Streb et al., 1999). However, since longer periods at low elevation might cause senescence the de-acclimation period was limited to 3 weeks.
Intact leaves were incubated on water in Petri dishes (10 ml for 0.5 g leaves) at 25 °C in the presence or absence of the protein synthesis inhibitors lincomycin (3 mM) and cycloheximide (35.5 µM) or in ice-cooled water at approximately 5–7 °C and illuminated with white light at 1000 µmol m–2 s–1 PAR (4x100 W low-voltage halogen lamps). Leaves illuminated in the presence of lincomycin were pre-treated for 1 h in darkness.
13C-NMR spectroscopy
For NMR-measurements, leaf material of 10 g fresh weight was frozen in liquid nitrogen and stored at –20 °C. The frozen material was ground to a fine powder in liquid nitrogen and extracted with perchloric acid as described (Aubert et al., 1998). The extracts were adjusted to pH 5.0, lyophilized, dissolved in 10% 2H2O, and neutralized to pH 7.5 with KOH (Aubert et al., 1998). Extracts were measured with a NMR-spectrometer (AMX 400, Bruker, Bilerica, MA) in a 10 mm multinuclear tube tuned at 100.6 MHz for 13C-NMR. The resonance signal of 2H2O was used as a lock signal. Data acquisition conditions were as described (Aubert et al., 1998), except that 13C-NMR-data were recorded with 225 scans.
The peaks in the NMR-spectra were identified by comparison with standard solutions of known compounds reported in previous publications (Aubert et al., 1998). In some cases, authentic compounds were added to the extracts. For the identification of the 13C-NMR-spectrum of ranunculin, a test version of the ACD/CNMR program (Sigma/Aldrich) was used.
For the estimation of metabolite contents the relative peak response on the same fresh weight basis was calculated. The concentration limit for the detection of metabolites in the spectra is approximately 1 mM. Since the quality of the NMR-spectra from different plants and different growing conditions varied, changes in metabolite levels were calculated as a percentage of the sum of the metabolites present in the spectra, assuming that the different compounds recovered similarly, as described previously (Aubert et al., 1998). Since, in all three plants, some peaks of the 13C-NMR-spectra could not be identified (see also Figs 4 and 6), their quantitative contribution to total soluble carbon was estimated. Therefore, the carbon number of unknown metabolites was estimated according to their similar relative peak response and appearance after a wide range of different incubation conditions (not shown).
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Fluorescence and oxygen evolution measurements
A Mini-PAM (Waltz, Germany) was used for the chlorophyll-fluorescence measurements. The ratio Fv/Fm in 30 min dark-adapted samples and the non-photochemical fluorescence quenching (qN) were measured and calculated as described previously (Streb et al., 1998). Light-dependent oxygen evolution was measured at 25 °C with a Hansatech (Kings Lynn, Norfolk, UK) leaf-disc electrode at saturating photon flux in the presence of saturating CO2 (Streb et al., 1997).
Enzyme measurements and analytical methods
For the estimation of enzyme activity and contents of antioxidants and pigments, approximately 0.4 g fresh weight of leaf material was used. Extraction and estimation of catalase activity, dry weight and protein content were as described previously (Streb et al., 1997). Total chlorophyll contents were determined from 80% (v/v) acetone extracts (Arnon, 1949), or after separation by HPLC (Streb et al., 1998). The antioxidants ascorbate, dehydroascorbate, reduced and oxidized glutathione were extracted in 1% metaphosphoric acid and assayed by HPLC as described (Streb et al., 1998). For analysis of pigment and -tocopherol content by HPLC, leaf material was frozen in liquid nitrogen. Frozen leaves were ground with a mortar and pestle in liquid nitrogen and extracted with 99% acetone essentially as described by Thayer and Björkman (1990). The separation and quantification of the different pigments and of -tocopherol was carried out as described previously (Streb et al., 1998). Contents of carotenoids and of -tocopherol were expressed on the basis of total chlorophyll content, as determined in the separation of the same samples.
The experiments were repeated independently at least three times. Alpine plants were investigated within the vegetation period (July and August) of two consecutive years (1998 and 1999). Mean values with standard errors of the mean are indicated. For statistical analysis the Student’s t-test was applied.
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Results |
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Effect of growth conditions on leaf morphology and basic parameters
S. alpina and R. glacialis plants were collected at their natural site at the Col du Galibier (2400–2700 m elevation) in the French Alps and either studied directly or after 3 weeks growth under controlled conditions at 22 °C or at 6 °C at low elevation. Newly developed leaves of R. glacialis grown at 22 °C were smaller and lost succulence relative to leaves from the natural site. No changes of leaf morphology were observed in S. alpina grown under the different conditions.
Basic parameters (dry weight, protein and chlorophyll content) of the alpine species grown at 6 °C did not differ significantly from plants collected at the high mountain site, with the exception of a low protein content in 6 °C-grown R. glacialis leaves. By contrast, plants grown at 22 °C differed markedly from plants grown at the two other conditions (Table 1). In both species the dark-adapted Fv/Fm decreased slightly at 22 °C, whereas the chlorophyll content was higher. As a consequence, photosynthetic oxygen evolution was low on a chlorophyll basis in 22 °C-grown leaves, but higher on a leaf area basis than it was in 6 °C-grown leaves (Table 1).
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Catalase activity in leaves from both species declined significantly during the growth season and is, therefore, shown separately for the two months of investigation. In general, catalase activity was higher after growth at 22 °C and lower at 6 °C, as compared to leaves collected in the mountains, particularly leaves collected in July (Table 1).
Effect of growth conditions on susceptibility to light- and cold-stress
S. alpina leaves from all growth conditions were equally sensitive to high light at low temperature or in the presence of protein synthesis inhibitors at 25 °C. In contrast, the 22 °C-grown leaves of R. glacialis were significantly more light-sensitive than leaves collected at the Col du Galibier or grown at 6 °C. This is evident from a stronger reduction in Fv/Fm in the presence of lincomycin and on ice, from a lower rate of oxygen evolution after cold treatment and even from a reduction in chlorophyll content in the presence of cycloheximide. In addition, catalase was completely inactivated in the presence of cycloheximide and decreased strongly during illumination on ice (Figs 1B, 2). The light sensitivity of the measured parameters was not significantly different in 6 °C-grown leaves and leaves from the Col du Galibier (Figs 1B, 2).
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Photoinhibition of photosystem II in the non-alpine control plant P. sativum was markedly higher than in the two alpine plant species, irrespective of growing conditions (Fig. 1), indicating that pea leaves could not achieve a comparable resistance to cold-induced photodamage as the alpine species.. However, the reduction in Fv/Fm was slightly delayed in 6 °C-grown leaves, compared to 22 °C-grown leaves (Fig. 1).
Effect of growth conditions on antioxidants, carotenoids and fluorescence quenching
The ascorbate content of S. alpina leaves was markedly elevated at the 22 °C-growth temperature, while the glutathione content was highest at the 6 °C-growth temperature. The ascorbate and glutathione contents did not vary significantly in leaves of R. glacialis from the different conditions. Increased glutathione contents were observed in 6 °C-grown pea leaves as compared to the 22 °C control (Table 2). Contents of -tocopherol decreased markedly in S. alpina leaves grown at low elevation, but increased strongly in 22 °C-grown leaves of R. glacialis (Table 2).
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The sum of the xanthophyll cycle pigments (VAZ) was significantly lowest in leaves of both alpine plants when grown at 22 °C. The contents of the other carotenoids were similar in all growth conditions (Table 2). The significance of the lower VAZ-contents on potential zeaxanthin formation was examined during illumination with high light on ice. In 22 °C-grown S. alpina leaves, zeaxanthin formation was significantly lower and in 6 °C-grown leaves slightly lower as compared to leaves collected at the alpine growing site. In R. glacialis zeaxanthin formation was slightly delayed in 22 °C-grown leaves (Fig. 3A). However, leaves of both alpine species under all growing conditions converted approximately the same percentage of xanthophyll cycle pigments into zeaxanthin (Fig. 3B), suggesting that zeaxanthin formation is limited by the low pool of xanthophyll cycle pigments (Table 2) and not by de-epoxidase activities.
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Chlorophyll fluorescence quenching was only analysed at 25 °C. At this temperature, light inactivation of PSII and catalase was only apparent in the presence of protein synthesis inhibitors (Figs 1B, 2). However, chlorophyll fluorescence quenching was markedly changed in both alpine species grown at different temperatures, but not significantly affected in pea leaves (Table 2). Non-photochemical fluorescence quenching (qN) was markedly higher in S. alpina leaves grown 22 °C and at 6 °C compared to leaves collected in the Alps. In leaves of R. glacialis qN increased only after growth at 6 °C (Table 2). However, although the contents of xanthophyll cycle pigments were low in 22 °C-grown leaves of both alpine species the magnitude of qN was not diminished compared to leaves with higher VAZ contents. (Table 2).
Effect of growth conditions on carbon-metabolite levels
Representative 13C-NMR-spectra of S. alpina leaves show that ascorbate represented the second most abundant soluble carbon compound after sucrose (Fig. 4). Ascorbate levels appear to be limited by low growth temperature because ascorbate content was highest in 22 °C-grown leaves, as measured by HPLC and NMR-spectroscopy (Fig. 5; Table 2).
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In 13C-NMR-spectra of R. glacialis leaves 11 major peaks were observed, which appeared to belong to a single metabolite (Fig. 6). This compound was identified as ranunculin by the ACD/CNMR-program, by comparing the calculated 13C-NMR-spectra of ranunculin with the spectra of the extracts. Furthermore, the peaks of ranunculin disappeared in the 13C-NMR-spectra after alkaline treatment of the extract, while the amount of glucose increased (not shown). Ranunculin is already known as a major compound in R. glacialis (Ruijgrok, 1963) consisting of a glucoside of the lactone of
-hydroxyvinylacrylic acid and is rapidly cleaved into protoanemonin and glucose in alkaline media (Hill and Van Heyningen, 1951). In leaves of R. glacialis collected at the Col du Galibier, ranunculin and malate were the major soluble carbon compounds (Figs 6, 7). In 22 °C-grown leaves malate was reduced by 50% relative to the other growth conditions (Fig. 7), and this decrease in malate content was accompanied by an increase in light sensitivity (Figs 1B, 2). In addition to malate the amount of citrate was lower and that of glucose higher in 22 °C-grown leaves, as compared to leaves from the mountains. However, similar changes in citrate and glucose levels were also observed in 6 °C-grown leaves where sensitivity to photodamage was not affected (Fig. 7). Leaves of R. glacialis from all growth conditions contained significant amounts of glycerate, a metabolite that was not observed in other plants (Figs 6, 7).
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In pea leaves, glucose is the most abundant soluble carbon compound. The slightly enhanced tolerance to photoinhibition in leaves grown at 6 °C (Fig. 1) was paralleled by particularly higher sucrose, fructose, inositol, and homoserine levels and a lower amount of asparagine than in 22 °C-grown leaves (Fig. 8). However, neither malate nor ascorbate contents differed significantly in pea leaves from the different growth conditions (Fig. 8).
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In all three plants from the 22°C-growth condition sucrose levels were the lowest and glucose levels were the highest. In contrast, in plants from the 6 °C-growth condition, the free amino acid levels were elevated, in particular in P. sativum (Figs 5, 7, 8).
Several peaks in the 13C-NMR-spectra of all three species could not be identified (Figs 4, 6). These unidentified peaks belonged to several unknown metabolites in every plant species and not to a single compound. This was concluded from the appearance and response of these peaks after a series of different incubation conditions (not shown). In leaves of S. alpina, unknown metabolites increased markedly in leaves from plants grown at low elevation, particularly in plants grown at 6 °C, compared with leaves collected in the Alps (Fig. 5). By contrast, unidentified carbon metabolites decreased in P. sativum leaves at 6 °C growth temperature (Fig. 8). The amount of unidentified carbon metabolites was low in R. glacialis leaves and did not vary markedly under the different growing conditions (Figs 6, 7).
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Discussion |
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Leaves of the two alpine species, S. alpina and R. glacialis, use different mechanisms to cope with high light at low temperature (Streb et al., 1998). They also differ in their ability to retain light-stress tolerance after growth at a low elevation under controlled conditions. As shown here, leaves of R. glacialis grown at 22 °C partially lost their light-stress tolerance when compared either to high altitude or to 6 °C growing conditions. This was concluded from (1) a larger decrease of the Fv/Fm-ratio both at low temperature and in the presence of lincomycin; (2) a larger decrease in the photosynthetic oxygen evolution capacity after cold treatment; (3) a greater inactivation of catalase in the cold and in the presence of cycloheximide, and (4) increased degradation of chlorophyll in the presence of cycloheximide (Figs 1B, 2). By contrast, light-stress tolerance did not change significantly in leaves of S. alpina. Therefore, acclimation to light and cold appears to be constitutive in leaves of S. alpina, but reversible in leaves of R. glacialis.
The de-acclimation of R. glacialis leaves occurred only at 22 °C, and it was independent of elevation or growth at low light intensity, because plants grown at 6 °C and low elevation and low light intensity showed the same resistance to high irradiance as plants taken from the high mountains. Similar to other crop plants grown at low temperature (Huner et al., 1993; Falk et al., 1996; Streb et al., 1999), pea leaves acquired a slightly higher tolerance to photoinhibition of PSII at low temperature. However, photoinhibition in cold-acclimated pea leaves was still much higher than in those of the two alpine species (Fig. 1).
The light- and cold-stress sensitivity of plants from different growing conditions was studied to assess the possible role of antioxidants, carotenoids and soluble carbon metabolites in acclimation.
Increased antioxidant contents were frequently related to enhanced stress tolerance in several cold-acclimated plant species (Schöner and Krause, 1990; Badiani et al., 1993; Wise, 1995; Kocsy et al., 2000). However, the contents of carotenoids and of -tocopherol increased in the de-acclimated leaves of R. glacialis, while ascorbate and glutathione contents remained unchanged. These results support previous observations that enhanced antioxidative protection is not related to the light-stress tolerance of R. glacialis leaves (Streb et al., 1997, 1998). In contrast, -tocopherol contents decreased significantly in S. alpina leaves grown at low elevation without any accompanying increase in light sensitivity. Obviously, -tocopherol contents were not limiting for cold-acclimation in S. alpina leaves. This does not, however, exclude a possible role of other antioxidants for acclimation of S. alpina leaves. In S. alpina leaves ascorbate contents were very high in plants grown under all growth regimes, representing the second most abundant soluble carbon metabolite (12–19% of total soluble carbon). Ascorbate contents in 22 °C-grown S. alpina leaves increased further, but this was not accompanied by higher stress tolerance. Obviously, the mere increase of leaf antioxidant contents does not protect from photoinhibitory damage at low temperature, as was recently shown for rye and maize leaves (Streb and Feierabend, 1999; Leipner et al., 2000). However, in pea leaves higher contents of glutathione correlated with a slightly enhanced light-stress tolerance of 6 °C-grown leaves as compared to the 22 °C-grown control.
Besides antioxidants, zeaxanthin-related non-photochemical quenching of chlorophyll fluorescence is supposed to protect photosystem II from excess light in cold-acclimated leaves (Verhoeven et al., 1996; Thiele et al., 1996). Accordingly, the contents of VAZ and the synthesis of zeaxanthin decreased in de-acclimated R. glacialis leaves. However, as shown previously, the complete inhibition of zeaxanthin synthesis by dithiothreitol did not enhance photoinhibition of photosystem II in R. glacialis leaves at low temperature, suggesting that zeaxanthin synthesis is only of minor importance for photoprotection in this species (Streb et al., 1998). Similarly, VAZ contents were also very low in 22 °C-grown leaves of S. alpina, and zeaxanthin synthesis was even more affected in this species (Table 2; Fig. 3). However, the magnitude of qN was not diminished in 22 °C-grown leaves of either species with low VAZ contents, and photoinhibitory damage did not increase in 22 °C-grown leaves of S. alpina. Therefore, the present results also exclude a direct correlation between high VAZ contents, zeaxanthin synthesis, qN and photoinhibitory damage in S. alpina leaves. The zeaxanthin contents in S. alpina leaves growing in the high mountains are obviously in excess of what is necessary for protection. The photoprotective effect of zeaxanthin might thus be limited to a rather small number of molecules (Noctor et al., 1991; Hurry et al., 1997; Bukhov et al., 2001) and the efficiency of zeaxanthin-related qN might be more dependent on the presence and function of the psbS protein (Peterson and Havir, 2001).
The de-acclimation of R. glacialis leaves was accompanied by a decrease in leaf size and leaf thickness and paralleled by a large decrease in the amount of malate, which is the second-most abundant metabolite in this species. Increases in mesophyll cell size following cold acclimation have been shown for a number of plant species and this was related to higher leaf sucrose contents (Huner et al., 1993; Strand et al., 1999), which increase the osmotic potential and freezing tolerance. However, the amount of the soluble sugars fructose and glucose increased in R. glacialis leaves after growth at 22 °C and the amount of sucrose and ranunculin did not differ significantly from leaves grown at high elevation or at 6 °C. Because ranunculin and its cleavage products have a bitter taste (Ruijgrok, 1963) the high concentration of this metabolite in R. glacialis appears to protect this plant species from being eaten by animals. It was not determined whether mesophyll cell size is affected in the different species grown under different thermal regimes, but the succulence of cold-acclimated R. glacialis leaves was lost following growth at 22 °C. Hence, malate might replace sucrose as an osmolyte to increase leaf size and freezing tolerance in cold-acclimated leaves of this species. Moreover, since no significant correlation was observed between changes of other soluble carbon metabolites and the increase in light sensitivity, it is suggested that malate might play a more important role for high light tolerance in R. glacialis leaves.
Malate is involved in the C4 and the CAM pathway of photosynthesis, but both pathways rarely occur in alpine plants and are suggested to be insignificant at higher altitude (Körner, 1999). Microscopic preparations of R. glacialis leaves did not show any differentiation into mesophyll and bundle sheath cells, as in C4-plants (Lütz and Moser, 1977; Lütz, 1987). In addition, both during the course of the day and during high light treatment, malate content did not decrease in R. glacialis leaves as would be expected for the operation of the CAM pathway and phosphoenolpyruvate-carboxylase activity remains low (P Streb, unpublished results), suggesting that the high malate contents are not associated with the C4 or the CAM pathway in this plant. Malate can transport reducing equivalents via the malate shuttle from the chloroplast to the cytoplasm (Scheibe, 1987). Therefore, the reduction of oxaloacetate to malate in the chloroplast stroma could contribute to prevent over-reduction of the photosynthetic electron transport chain and protect from high light damage. The large capacity of R. glacialis leaves to keep the photosynthetic electron transport chain oxidized in cold and light (Streb et al., 1998) suggested the existence of an efficient sink for reducing equivalents. Accordingly, an elevated activity of NADP-malate dehydrogenase was recently reported for cold-acclimated wheat leaves (Savitch et al., 2000). However, the NADP-malate dehydrogenase activity of R. glacialis leaves appears to be insufficient to serve as a major sink of reducing equivalents in the chloroplast (P Streb, unpublished results). Recently, fumaric acid was described as a significant carbon sink and possible transport metabolite in leaves of several plants, accumulating to even higher levels than starch and soluble sugars (Chia et al., 2000). Fumaric acid was not present in the 13C-NMR-spectra of R. glacialis, but malate is directly synthesized from fumaric acid in the mitochondria and thus might replace the function of fumaric acid in this plant.
Pea leaves grown at 6 °C, which remained relatively sensitive to light-stress, had neither very high malate contents nor high ascorbate levels, supporting their possible involvement in the cold acclimation of alpine plants.
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Acknowledgements |
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We thank Anne-Marie Boisson, Michelle Quemin and Christel van Oijen for excellent technical assistance. We are grateful to Jürgen Feierabend, Gabriel Cornic and Vaughan Hurry for critical reading of the manuscript and valuable discussion. Financial support by a scholarship to PS by the DFG (Germany) is greatly appreciated.
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References |
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