Cold- and light-induced changes of metabolite and antioxidant levels in two high mountain plant species Soldanella alpina and Ranunculus glacialis and a lowland species Pisum sativum

Leaves of the two cold-acclimated alpine plant species Ranunculus glacialis and Soldanella alpina and, for comparison, of the non-acclimated lowland species Pisum sativum were illuminated with high light intensity at low temperature. The light- and cold-induced changes of antioxidants and of the major carbon and phosphate metabolites were analysed to examine which metabolic pathways might be limiting in non-acclimated pea leaves and whether alpine plants are able to circumvent such limitation. During illumination at low temperature pea leaves accumulated high quantities of sucrose, glucose-6-phosphate, fructose-6-phosphate, mannose-6-phosphate and phosphoglycerate (PGA) whereas ATP/ADP-ratios decreased. Although the PGA content also increased in leaves of R. glacialis the other metabolites did not accumulate and ATP/ADP-ratios remained fairly constant in either alpine species. These data indicate a inorganic phosphate (Pi)-limitation in the chloroplasts of pea leaves but not in the alpine species. However, the total phosphate pool and the percentage of free Pi were highest in pea and did not change during illumination in cold. In contrast, free Pi contents declined markedly in R. glacialis leaves, suggesting that Pi is available for metabolism in this species. In S. alpina leaves contents of ascorbate and glutathione doubled in light and cold, while the contents of sugars did not increase. Obviously, S. alpina leaves can use assimilated carbon for ascorbate synthesis, rather than for the synthesis of sugars. A high capacity for ascorbate synthesis might prevent the accumulation of mannose-6-phosphate and Pi-limitation.     

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

Two high mountain plants Soldanella alpina (L.) and Ranunculus glacialis (L.) were transferred from their natural environment to two different growth conditions (22 degrees C and 6 degrees 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 degrees 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 degrees 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.     

Resistance to photoinhibition of photosystem II and catalase and antioxidative protection in high mountain plants

In leaves of three alpine high mountain plants, Homogyne alpina, Ranunculus glacialis and Soldanella alpina, both photosystem II (PSII) and the enzyme catalase appeared to he highly resistant to photoinactivation under natural field conditions. While the Dl protein of PSII and catalase have a rapid turnover in light and require continuous new protein synthesis in non-adapted plants, little apparent photoinactivation of PSII or catalase was induced in the alpine plants by translation inhibitors or at low temperature, suggesting that turnover of the Dl protein and catalase was slow in these leaves. In vitro PSII was rapidly inactivated in light in isolated thylakoids from H. alpina and R. glacialis. In isolated intact chloroplasts from R. glacialis, photoinactivation of PSII was slower than in thylakoids. Partially purified catalase from R. glacialis and S. alpina was as sensitive to photoinactivation in vitro as catalases from other sources. Catalase from H. alpina had, however, a 10-fold higher stability in light. The levels of xanthophyll cycle carotenoids, of the antioxidants ascorbate and glulathione, and of the activities of catalase, superoxide dismutase and glutathione reductase were very high in S. alpina, intermediate in H. alpina, but very low in R. glacialis. However, isolated chloroplasts from all three alpine species contained much higher concentrations of ascorbate and glutathione than chloroplasts from lowland plants.     

High Temperature Effects on Light Sensitivity in the Two High Mountain Plant Species Soldanella alpina (L.) and Rannunculus glacialis (L.)

Abstract: The susceptibility to high temperature‐induced photoinhibition was investigated in leaves of two high mountain plant species, S. alpina and R. glacialis. In both species, PSII was similarly photoinactivated at 38 °C in the light. However, recovery from damage was much faster in S. alpina and depended on protein synthesis. In contrast, recovery was independent from protein synthesis in R. glacialis. Heat‐induced photoinactivation in both species was accompanied by: (1) a decrease in relative photosynthetic electron transport rates, (2) an increase in non‐photochemical chlorophyll fluorescence quenching, (3) a strong accumulation of zeaxanthin, (4) a marked decrease in soluble carbon metabolites and (5) an increase in lipid metabolism products, which was more pronounced in R. glacialis than in S. alpina. These results indicate that carbon assimilation was inhibited and that membranes were affected. Lipid peroxidation and possible membrane disintegration might limit the repair of damaged PSII in R. glacialis, while S. alpina appears to be protected by carotenoids and antioxidants. A marked decrease in α‐tocopherol content and an increase in reduced ascorbate indicated lipid peroxide scavenging activity in S. alpina. When zeaxanthin synthesis was impaired by DTT, photoinhibition increased and α‐tocopherol accumulated in R. glacialis. The increased susceptibility of R. glacialis leaves to light‐induced photoinhibition after growth at moderate temperature (Streb et al., 2003a) and the inability to repair heat‐induced damage might limit the distribution of R. glacialis to lower altitudes in the Alps.     

Dynamic acclimation to sunlight in an alpine plant,Soldanella alpina L.

In the French Alps, Soldanella alpina (S. alpina) grow under shade and sun conditions during the vegetation period. This species was investigated as a model for the dynamic acclimation of shade leaves to the sun under natural alpine conditions, in terms of photosynthesis and leaf anatomy. Photosynthetic activity in sun leaves was only slightly higher than in shade leaves. The leaf thickness, the stomatal density and the epidermal flavonoid content were markedly higher, and the chlorophyll/flavonoid ratio was significantly lower in sun than in shade leaves. Sun leaves also had a more oxidised plastoquinone pool, their PSII efficiency in light was higher and their non-photochemical quenching (NPQ) capacity was higher than that of shade leaves. Shade-sun transferred leaves increased their leaf thickness, stomatal density and epidermal flavonoid content, while their photosynthetic activity and chlorophyll/flavonoid ratio declined compared to shade leaves. Parameters indicating protection against high light and oxidative stress, such as NPQ and ascorbate peroxidase, increased in shade-sun transferred leaves and leaf mortality increased. We conclude that the dynamic acclimation of S. alpina leaves to high light under alpine conditions mainly concerns anatomical features and epidermal flavonoid acclimation, as well as an increase in antioxidative protection. However, this increase is not large enough to prevent damage under stress conditions and to replace damaged leaves.     

Divergent strategies of photoprotection in high-mountain plants

Leaves of high-mountain plants were highly resistant to photoinhibitory damage at low temperature. The roles of different photoprotective mechanisms were compared. Mainly, the alpine species Ranunculus glacialis (L.) and Soldanella alpina were investigated because they appeared to apply greatly divergent strategies of adaptation. The ratio of electron transport rates of photosystem II/photosystem I measured in thylakoids from R. glacialis did not indicate a specific acclimation to high irradiance. Low rates of a chloroplast-mediated inactivation of catalase (EC 1.11.1.6) in red light indicated, however, that less reactive oxygen was released by isolated chloroplasts from R. glacialis than by chloroplasts from lowland plants. Leaves of S. alpina and of Homogyne alpina (L.) Cass, but not those of R. glacialis, had a very high capacity for antioxidative protection, relative to lowland plants, as indicated by a much higher tolerance against paraquat-mediated photooxidative damage and a higher -tocopherol content. Accordingly, ascorbate and glutathione were strongly oxidized and already largely destroyed at low paraquat concentrations in leaves of R. glacialis, but were much less affected in leaves of  S. alpina. Non-radiative dissipation of excitation energy was essential for photoprotection of leaves of  S. alpina and depended on the operation of the xanthophyll cycle. Strong non-photochemical quenching of chlorophyll fluorescence occurred also in R. glacialis leaves at high irradiance, but was largely independent of the presence of zeaxanthin or antheraxanthin. For R. glacialis, photorespiration appeared to provide a strong electron sink and a most essential means of photoprotection, even at low temperature. Application of phosphinothricin, which interferes with photorespiration by inhibition of glutamine synthetase, caused a striking reduction of electron transport through photosystem II and induced marked photoinhibition at both ambient and low temperature in leaves of R. glacialis, while  S. alpina was less affected. Received: 18 March 1998 / Accepted: 7 August 1998     

Photoinhibition of photosystem II in tobacco plants overexpressing glutathione reductase and poplars overexpressing superoxide dismutase

We studied photoinhibition in two cultivars of tobacco ( Nicotiana tabacum L.) expressing the bacterial gor gene in the cytosol and in four lines of poplar ( Populus tremula × P. alba ) expressing the FeSOD gene of Arabidopsis thaliana in the chloroplast. The respective total activities of glutathione reductase (EC 1.6.4.2) in leaves of gor tobaccos and superoxide dismutase (EC 1.15.1.1) in the FeSOD poplars were 5–8 times higher than in the respective untransformed control plants. Leaves of control and transformed plants were subjected to high-light stress at 20°C, and photoinhibition of photosystem II (PSII) was measured by oxygen evolution and chlorophyll fluorescence. The leaves were illuminated both in the presence and absence of lincomycin, which inhibits chloroplast protein synthesis. In both cases, the time course of loss of PSII activity was identical in plants overproducing superoxide dismutase (SOD) and in the untransformed controls, suggesting that the ability to convert superoxide to hydrogen peroxide is not a limiting factor in protection against photoinhibition, or in the repair of photoinhibitory damage or that the site of O₂⁻ production is not accessible to the transgene product. The rate constant of photoinhibition, measured in lincomycin-treated leaves, was smaller in glutathione reductase (GR) overproducing tobacco cv. Samsun than in the respective wild-type, but this difference was not seen in cv. Bel W3. The steady-state level of PSII activity measured when the PSII repair cycle was allowed to equilibrate with photoinhibitory damage under high light was not higher in the GR overproducing cv. Samsun, suggesting that the repair of photoinhibitory damage was not enhanced in plants overproducing GR in the cytosol.     

Effects of temperature and light on the formation of chloroplast protrusions in leaf mesophyll cells of high alpine plants

Chloroplasts of many alpine plants have the ability to form marked, stroma-filled protrusions that do not contain thylakoids. Effects of temperature and light intensity on the frequency of chloroplasts with such protrusions in leaf mesophyll cells of nine different alpine plant species (Carex curvula All., Leontodon helveticus Merat., Oxyria digyna (L.) Hill., Poa alpina L. ssp. vivipara, Polygonum viviparum L., Ranunculus glacialis L., Ranunculus alpestris L., Silene acaulis L. and Soldanella pusilla Baumg.) covering seven different families were studied. Leaves were exposed to either darkness and a stepwise increase in temperature (10-38 degrees C) or to different light intensities (500 and 2000 micromol photons m(-2) s(-1)) and a constant temperature of 10 or 30 degrees C in a special temperature-regulated chamber. A chloroplast protrusions index characterising the relative proportion of chloroplasts with protrusions was defined. Seven of the nine species showed a significant increase in chloroplast protrusions when temperature was elevated to over 20 degrees C. In contrast, the light level did not generally affect the abundance of chloroplasts with protrusions. Chloroplast protrusions lead to a dynamic enlargement of the chloroplast surface area. They do not appear to be directly connected to a distinct photosystem II (PSII) (F(v)/F(m)) status and thus seem to be involved in secondary, not primary, photosynthetic processes.     

Mechanism for photoinactivation of PSII by methyl viologen at two temperatures in the leaves of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

We studied the photooxidative effects of methyl viologen (MV) on PSII in rice (Oryza sativa L). Leaves were held at either room temperature (RT) or 4°C. In the presence of MV, the photochemical efficiency of PSII, or F_v/F_m, was more depressed at RT than at the low temperature (LT), but the loss of D1 protein that was detected at RT was not observed at LT. However, the decline in the content of functional PSII, 1/F_o - 1/F_m, was similar for MV-treated leaves at either temperature. These results suggest that, at LT, PSII is not protected from MV-induced photooxidation, although degradation of the D1 protein is delayed. The 1/F_o - 1/F_m decreased by MV treatment at RT was significantly recovered during dark incubation for 2 h. Recovery of a small portion of 1/F_o - 1/F_m was also possible, even for tissues treated with MV at LT. Therefore, we believe that MV-induced reversible photoinactivation may exist This possibility is further discussed in terms of changes in the de-epoxidation state and the rate of PSII-driven electron transport.     

Brassinosteroids accelerate recovery of photosynthetic apparatus from cold stress by balancing the electron partitioning,carboxylation and redox homeostasis in cucumber

The aim of this study was to examine the role of brassinosteroids (BRs) in protecting the photosynthetic apparatus from cold‐induced damage in cucumber (Cucumis sativus) plants. Recovery at both high light (HL) and low light (LL) after a cooling at 10/7°C induced irreversible inhibition of CO₂ assimilation, photoinhibition at photosystem I (PSI) and inhibition of enzyme activities of Calvin cycle and ascorbate (AsA)‐reduced glutathione (GSH) cycle, followed by accumulation of H₂O₂ and malondialdehyde. However, cold‐induced photoinhibition at PSII was fully recovered at LL but not at HL. Meanwhile, recovery at HL increased electron flux to O₂‐dependent alternative pathway [Ja(O₂‐dependent)]. Foliar application of 24‐epibrassinolide (EBR) accelerated recovery from photoinhibition of PSII but not of PSI. EBR also significantly increased CO₂ assimilation, activity of Calvin cycle enzymes and electron flux to carbon reduction [Je(PCR)], with a concomitant decrease in Ja(O₂‐dependent); meanwhile EBR increased the activity of enzymes in AsA‐GSH cycle and cellular redox states. However, the positive effect of EBR on plant recovery was observed only at HL, but not LL. These results indicate that BR accelerates the recovery of photosynthetic apparatus at HL by activation of enzymes in Calvin cycle and increasing the antioxidant capacity, which in turn mitigate the photooxidative stress and the inhibition of plant growth during the recovery.     

Resistance of cold-hardened winter rye leaves (Secale cereale L.) to photo-oxidative stress

Catalase and photosystem II (PSII) were strongly inactivated during exposure to 4 °C and moderate light in 22 °C-grown non-hardened leaves (NHL) of winter rye (Secale cereale L.), but highly resistant to photo-inactivation at low temperature in 4 °C-grown cold-hardened leaves (CHL). Resistance of CHL to chilling-induced photo-inactivation of catalase and PSII depended partially on more efficient de novo synthesis at 4 °C and partially on improved protection. Lower rates of chloroplast-mediated inactivation of catalase in vitro indicated that less reactive oxygen was released by chloroplasts from CHL than by chloroplasts from NHL. The contents of xanthophyll cycle carotenoids, α-tocopherol, ascorbate, glutathione, the activities of superoxide dismutase and glutathione reductase, and the tolerance against paraquat-induced photo-oxidative damage were greatly increased in CHL, relative to NHL. Zeaxanthin-related thermal energy dissipation was only of minor importance for paraquat-tolerance and protection of catalase in CHL. When CHL were transferred to a higher temperature of 22 °C the increased resistance to photo-inactivation of catalase and PSII and the increased paraquat-tolerance were largely lost within 3 d, whereas most non-enzymic and enzymic antioxidants retained higher levels than in NHL. The decline of resistance to photodamage during dehardening was not related to concomitant changes of antioxidants or antioxidative enzymes.     

Photoinhibition and recovery of photosynthesis in intact barley leaves at 5 and 20°C

Photoinhibition of photosynthesis and its recovery were studied in intact barley ( Hordeum vuigare L. cv. Gunilla) leaves grown in a controlled environment by exposing them to two temperatures, 5 and 20°C, and a range of photon flux densities in excess of that during growth. Additionally, photoinhibtion was examined in the presence of chloramphenicol (CAP, an inhibitor of chloroplast protein synthesis) and of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Susceptibility to photoinhibition was much higher at 5 than at 20°C. Furthermore, at 20°C. CAP exacerbated photoinhibition strongly, whereas CAP had little additional effect (10%) at 5°C. These results support the model that net photoinhibition is the difference between the inactivation and repair of photosystem II (PSII); i.e. the degradation and synthesis of the reaction centre protein, Dl. Furthermore, the steady-state extent of photoinhibition was strongly dependent on temperature and the results indicated this was manifested through the effects of temperature on the repair process of PSII. We propose that the continuous repair of PS II at 20°C conferred at least some protection from photoinhibition. At 5°C the repair process was largely inhibited, with increased photoinhibition as a consequence. However, we suggest where repair is inhibited by low temperature, some protection is alternatively conferred by the photoinhibited reaction centres. Providing they are not degraded, such centres could still dissipate excitation energy non-radiatively, thereby conferring protection of remaining photochemically active centres under steady-state conditions.
A fraction of PS II centres were capable of resisting photoinhibition when the repair process was inhibited by CAP. This is discussed in relation to PS II heterogeneity. Furthermore, the repair process was not apparently activated within 3 h when barley leaves were transferred to photoinhibitory light conditions at 20°C.     

The role of electron transport in determining the temperature dependence of the photosynthetic rate in spinach leaves grown at contrasting temperatures

The temperature response of the uncoupled whole-chain electron transport rate (ETR) in thylakoid membranes differs depending on the growth temperature. However, the steps that limit whole-chain ETR are still unclear and the question of whether the temperature dependence of whole-chain ETR reflects that of the photosynthetic rate remains unresolved. Here, we determined the whole-chain, PSI and PSII ETR in thylakoid membranes isolated from spinach leaves grown at 30 degrees C [high temperature (HT)] and 15 degrees C [low temperature (LT)]. We measured temperature dependencies of the light-saturated photosynthetic rate at 360 microl l(-1) CO2 (A360) in HT and LT leaves. Both of the temperature dependences of whole-chain ETR and of A360 were different depending on the growth temperature. Whole-chain ETR was less than the rates of PSI ETR and PSII ETR in the broad temperature range, indicating that the process was limited by diffusion processes between the PSI and PSII. However, at high temperatures, whole-chain ETR appeared to be limited by not only the diffusion processes but also PSII ETR. The C3 photosynthesis model was used to evaluate the limitations of A360 by whole-chain ETR (Pr) and ribulose bisphosphate carboxylation (Pc). In HT leaves, A360 was co-limited by Pc and Pr at low temperatures, whereas at high temperatures, A360 was limited by Pc. On the other hand, in LT leaves, A360 was solely limited by Pc over the entire temperature range. The optimum temperature for A360 was determined by Pc in both HT and LT leaves. Thus, this study showed that, at low temperatures, the limiting step of A360 was different depending on the growth temperature, but was limited by Pc at high temperatures regardless of the growth temperatures.     

Effects of Rubisco kinetics and Rubisco activation state on the temperature dependence of the photosynthetic rate in spinach leaves from contrasting growth temperatures

Recently, several studies reported that the optimum temperature for the initial slope [IS(Ci)] of the light-saturated photosynthetic rate (A) versus intercellular CO2 concentration (Ci) curve changed, depending on the growth temperature. However, few studies compare IS(Ci) with ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) properties. Here, we assessed Rubisco activation state and in vitro Rubisco kinetics, the main determinants of IS(Ci), in spinach leaves grown at 30/25 [high temperature (HT)] and 15/10 degrees C [low temperature (LT)]. We measured Rubisco activation state and A at a CO2 concentration of 360 microL L(-1) (A360) at various temperatures. In both HT and LT leaves, the Rubisco activation state decreased with increasing temperatures above the optimum temperatures for A360, while the activation state remained high at lower temperatures. To compare Rubisco characteristics, temperature dependences of the maximum rate of ribulose 1,5-bisphosphate (RuBP) carboxylation (Vcmax), specificity factor (Sc/o) and thermal stability were examined. We also examined Vcmax, and thermal stability in the leaves that were transferred from HT to LT conditions and were subsequently kept under LT conditions for 2 weeks (HL). Rubisco purified from HT, LT and HL leaves are called HT, LT and HL Rubisco, respectively. Thermal stabilities of LT and HL Rubisco were similar and lower than that of HT Rubisco. Both Vcmax and Sc/o in LT Rubisco were higher than those of HT Rubisco at low temperatures, while these were lower at high temperatures. Vcmax in HL Rubisco were similar to those of LT Rubisco at low temperatures, and to those of HT Rubisco at high temperatures. The predicted photosynthetic rates, taking account of the Rubisco kinetics and the Rubisco activation state, agreed well with A360 in both HT and LT leaves. This study suggests that photosynthetic performance is largely determined by the Rubisco kinetics at low temperature and by Rubisco Kinetics and the Rubisco activation state at high temperature.     

Effect of zinc on antioxidant response in maize (Zea mays L.) leaves

Maize (Zea mays L. cv kanaujia) plants grown with Zn [10 (control), 0.1 (low) and 20 microM (high)], were investigated for concentration of antioxidants and activities of antioxidative enzymes in leaves. Young leaves of low Zn plants developed whitish-necrotic spots. Leaves of both low and high Zn plants showed decrease in chlorophyll concentration and accumulation of lipid peroxides, ascorbate and dehydroascorbate, associated with a decrease in the activity of ascorbate peroxidase and superoxide dismutase. Low and high Zn, however, showed diverse effect on glutathione reductase. While low Zn increased the activity of glutathione reductase, high Zn decreased its activity. Zinc effect on antioxidative constituents suggested Zn involvement in sustaining the antioxidative defense system in maize leaves.     

Small light-harvesting antenna does not protect from photoinhibition

High-light-induced decrease in photosystem II (PSII) electron transfer activity was studied in high- and low-light-grown pumpkin (Cucurbita pepo L.) plants in vivo and in vitro. The PSII light-harvesting antenna of the low-light leaves was estimated to be twice as big as that of the high-light leaves. The low-light leaves were more susceptible to photoinhibition in vivo. However, thylakoids isolated from these two plant materials were equally sensitive to photoinhibition when illuminated in the absence of external electron acceptors. Only the intensity of the photoinhibitory light and the chlorophyll concentration of the sample, not the size of the light-harvesting antenna, determined the rate of PSII photoinhibition in vitro. Because excitation of the reaction center and not only the antenna chlorophylls is a prerequisite for photoinhibition of PSII activity, independence of photoinhibition on antenna size provides support for the hypothesis (Schatz EH, Brock H, Holzwarth AR [1988] Biophys J 54: 397-405) that the excitations of the antenna chlorophylls are in equilibrium with the excitations of the reaction centers. Better tolerance of the high-light leaves in vivo was due to a more active repair process and more powerful protective mechanisms, including photosynthesis. Apparently, some protective mechanism of the high-light-grown plants is at least partially active at low temperature. The protective mechanisms do not appear to function in vitro.     

Photoprotective function of betacyanin in leaves of <Emphasis Type="Italic">Amaranthus cruentus</Emphasis> L. under water stress

The photoprotective function of leaf betacyanin in water-stressed Amaranthus cruentus plants was examined by comparing leaves of two strains which differ significantly in the amount of betacyanin. At 0, 1, and 2 days after the imposed water stress, leaves were subjected to high-light (HL) treatment to assess their photosynthetic capacity and photoinhibition susceptibility. The water stress equally reduced leaf relative water content (RWC), gas-exchange rate and chlorophyll (Chl) contents in both leaves, indicating that the severity of water stress was comparable between the strains. Consequently, the extent of photoinhibition after the HL treatment increased in both strains as water stress developed; however, it was significantly greater in acyanic leaves than in betacyanic leaves, suggesting lower photoinhibition susceptibility in the betacyanic strain. The betacyanic leaves also exhibited approximately 30% higher values for photochemical quenching coefficient (q_P) during the period of water stress despite the nonphotochemical quenching coefficient (q_N) did not differ significantly between the strains. These results may be partially explained by the increased amount of leaf betacyanin under water stress. Moreover, a decrease in Chl content in betacyanic leaves might have enhanced light screening effect of betacyanin by increasing relative abundance of betacyanin to Chl molecule. In addition, reduced Chl content increased light penetrability of leaves. As a result, the extent of photoinhibition at the deeper tissue was exacerbated and the Chl fluorescence emitted from these tissues was more readily detected, facilitating assessment of photoinhibition at deeper tissues where the effect of betacyanic light screening is considered to be most apparent. Our results demonstrated that leaf betacyanin contributes to total photoprotective capacity of A. cruentus leaves by lowering excitation pressure on photosystem II (PSII) via attenuation of potentially harmful excess incident light under water stress.     

Reversible photoinhibition of unhardened and cold-acclimated spinach leaves at chilling temperatures

The photoinhibition of photosynthesis at chilling temperatures was investigated in cold-acclimated and unhardened (acclimated to +18° C) spinach (Spinacia oleracea L.) leaves. In unhardened leaves, reversible photoinhibition caused by exposure to moderate light at +4° C was based on reduced activity of photosystem (PS) II. This is shown by determination of quantum yield and capacity of electron transport in thylakoids isolated subsequent to photoinhibition and recovery treatments. The activity of PSII declined to approximately the same extent as the quantum yield of photosynthesis of photoinhibited leaves whereas PSI activity was only marginally affected. Leaves from plants acclimated to cold either in the field or in a growth chamber (+1° C), were considerably less susceptible to the light treatment. Only relatively high light levels led to photoinhibition, characterized by quenching of variable chlorophyll a fluorescence (F_V) and slight inhibition of PSII-driven electron transport. Fluorescence data obtained at 77 K indicated that the photoinhibition of cold-acclimated leaves (like that of the unhardened ones) was related to increased thermal energy dissipation. But in contrast to the unhardened leaves, 77 K fluorescence of cold-acclimated leaves did not reveal a relative increase of PSI excitation. High-light-treated, cold-acclimated leaves showed increased rates of dark respiration and a higher light compensation point. The photoinhibitory fluorescence quenching was fully reversible in low light levels both at +18° C and +4° C; the recovery was much faster than in unhardened leaves. Reversible photoinhibition is discussed as a protective mechanism against excess light based on transformation of PSII reaction centers to fluorescence quenchers.Abbreviations F_O initial fluorescence - F_M maximal fluorescence - F_V devariable fluorescence (f_m-f_o) - PFD photon flux density - PS photosystem - SD standard deviation The authors thank the Deutsche Forschungsgemeinschaft and the Academy of Finland for financial support.     

Enhanced photochemical light utilization and decreased chilling-induced photoinhibition of photosystem II in cotton overexpressing genes encoding chloroplast-targeted antioxidant enzymes

The aim of this study was to determine whether increases in stromal superoxide dismutase (SOD; EC 1.15.1.1), ascorbate peroxidase (APX; EC 1.11.1.11) and glutathione reductase (GR; EC 1.6.4.2) via transformation could reduce photosystem (PS) II photoinhibition at low temperature for cotton (Gossypium hirsutum L.) plants and to determine by what mechanism this protection may be realized. During 3-h exposures of lincomycin-treated leaf discs to 10 degrees C and a photon flux density of 500 &mgr;mol m-2 s-1, all transgenic plants exhibited significantly greater PSII activity and O2 evolution than did wild-type plants. Also, the rate constant of PSII photoinactivation was significantly lower for all transgenic plants than for wild-type plants. No significant differences existed between genotypes in non-photochemical quenching of chlorophyll a fluorescence and the regulated component of the thermal dissipation of excitation energy. The relationship between changes in variable to maximum chlorophyll fluorescence (Fv/Fm) and the time-dependent averaged excessive light exposure was similar for all genotypes. This observation excluded the possibility that differences in PSII photodamage were due to improvements in the direct protection of PSII from active oxygen by antioxidant enzyme overproduction. Similar decreases in Fv/Fm during the stress treatment for all genotypes when leaves were pre-treated with 3-(3',4'-dichlorophenyl)-1,1-dimethylurea (DCMU) suggested that the effect of overproduction involved events downstream of PSII in the electron transfer pathway. Since all transgenic plants exhibited a significantly higher photochemical quenching of chlorophyll fluorescence during the chilling treatment, we concluded that, under the conditions used in this study, the enhancement of the protection of PSII from photodamage by increasing the stromal antioxidant enzyme activity in cotton leaves was due to the maintenance of a higher rate of electron transport and, consequently, a lower reduction state of QA.     

Cost and benefit of the repair of photodamaged photosystem II in spinach leaves: roles of acclimation to growth light

When visible light is excess, the photosynthetic machinery is photoinhibited. The extent of net photoinhibition of photosystem II (PSII) is determined by a balance between the rate of photodamage to D1 and some other PSII proteins and the rate of the turnover cycle of these proteins. It is widely believed that the protein turnover requires much energy cost. The aims of this study are to (1) evaluate the energy cost of PSII repair, (2) measure the benefit in terms of photosynthetic gain realized by the repairing of the photodamaged PSII, and (3) know whether acclimation of photosynthesis to growth light affects the rates of the photodamage and repair. We grew spinach in high-light (HL) and low-light (LL) and measured the rates of D1 photodamage and repair in these leaves. We determined the rate constants of photodamage (k (pi)) and repair (k (rec)) by the PAM fluorometry in the presence or in the absence of lincomycin, an inhibitor of 70S protein synthesis. HL leaves showed smaller k (pi) and greater k (rec) than LL leaves. The energy cost of the repairing of the photodamaged D1 protein was <0.5?% of ATP produced by photophosphorylation at PPFDs ranging from 400 to 1600?μmol?m(-2)?s(-1) and was greater in HL leaves than in LL leaves. The benefits brought about by the repair were more than from 35 to 270 times the cost at PPFDs ranging from 400 to 1600?μmol?m(-2)?s(-1). The benefits of HL leaves were greater than those of LL leaves because of the higher photosynthesis rates in HL leaves. Running a simple simulation of daily photosynthesis using the parameters obtained in this study, we discuss why the plants need to pay the cost of D1 protein turnover to repair the photodamaged PSII.