Under high light intensity, low temperatures as well as heavy metals induce photoinhibition of PSII and oxidative stress in leaves. Since cold acclimation of leaves ameliorates their capacity of antioxidative defence, cross tolerance between cold-induced and heavy metal-induced photoinhibition was investigated in pea leaves grown at either 22 °C or 6 °C. The experimental conditions were chosen to induce a uniform level of short-term photoinhibition at low temperature or in the presence of CuSO4 or CdCl2 in leaves grown at 22 °C. Under all conditions photoinhibition of PSII was lower in cold-acclimated (6°C-grown) than in non-acclimated (22°C-grown) pea leaves. In darkness PSII was not affected by all treatments. Other parameters like catalase activity, chlorophyll content and metabolite contents were most sensitive to CuSO4, but less affected by CdCl2 and low temperature treatments. Strong oxidation of ascorbate and concomitant loss of catalase activity showed the enhanced oxidative stress in CuSO4-treated leaves. Generally, all measured parameters were less affected in cold-acclimated leaves than in non-acclimated leaves under all experimental conditions. Cold-acclimated pea leaves contained higher levels of ascorbate and particularly of glutathione and a higher capacity to keep the primary electron acceptor of PSII more oxidised. Incubation with heavy metals caused a nearly complete loss of reduced glutathione. It is suggested that reduced glutathione served as a source for phytochelatin synthesis. The extraordinarily high glutathione content in cold-acclimated pea leaves might therefore increase their ability to chelate heavy metals and thus to protect leaves from heavy-metal induced damage.

Most catalases are inactivated by light in a heme-sensitized and O-2-dependent reaction. In leaves of the alpine plant Homogyne alpina and in the peroxisomal cores of Helianthus annuus, light-insensitive catalases were observed. For the catalases Hacat1 of H. alpina and HnncatA3 of H. annuus, cDNA clones were obtained. Expression of recombinant active enzymes in insect cells confirmed that they coded for light-insensitive catalases. Kinetic and catalytic properties of light-sensitive or light-insensitive catalases did not differ substantially. However, the specific activity of the latter was markedly lower. The light-insensitive catalase HaCAT-1 was not resistant against inactivation by superoxide. Amino acid sequences of the light-insensitive catalases HaCAT-1 and HNNCATA3 were highly identical. They showed only a few exceptional amino acid substitutions at positions that are highly conserved in other catalases. These appeared to be localized mainly in a surface cavity at the entrance of a minor channel leading to the central heme, suggesting that this region played some, though yet undefined, role for light sensitivity. While the replacement of a highly conserved His by Thr225 was the most unique substitution, a single exchange of His225 by Thr in the light-sensitive catalase SaCAT-1 by mutagenesis was not sufficient to reduce its sensitivity to photoinactivation.

The enzyme catalase (EC 1.11.1.6) is inactivated by light and must be continuously replaced by new synthesis in order to maintain a constant enzyme activity in leaves. In winter rye leaves (Secale cereale L.) posttranscriptional mechanisms determine the rate of new catalase synthesis, including a light-controlled reversible modification of the catalase cat1 mRNA by methylation which greatly enhanced its translation efficiency. The specificity and regulation of this mRNA activation were further investigated. The translation efficiency of the rye cat1 mRNA was much more enhanced by N-7 methylation of the cap than that of an lhcb transcript. Investigations with truncated rye cat1 mRNAs indicated that the translational enhancement resulting from N-7 cap methylation did not require the presence of specific sequences of cat1 5′- and 3′-untranslated regions. Translational activation of the cat1 mRNA in rye leaves was independent of photosynthesis and most effectively induced by blue light. Peroxides (H2O2, tertiary butyl hydroperoxide) and conditions enforcing an H2O2 accumulation in the leaves (aminotriazole, paraquat) also caused an activation of the cat1 mRNA. A search for further signalling systems controlling the replenishment of inactivated catalase in light suggested that an inositol-1,4,5-triphosphate-mediated liberation of Ca2+ from internal stores and a protein phosphatase played some role. However, these signalling systems did not affect the activation of the cat1 mRNA. After removal of Ca2+ by EGTA the cat1 mRNA was rapidly degraded.

Suppression subtractive hybridization and differential display polymerase chain reactions were used to identify genes that were differentially expressed in cold-hardened and dehardened leaves of winter rye (Secale cereale L.). The transcripts of nine genes declined during dehardening at 22°C of cold-hardened 4°C-grown leaves, indicating some role in cold-acclimation. Among the genes that were strongly expressed in cold-hardened leaves were five genes of photosynthetic metabolism, the gene of the antioxidative enzyme peptide methionine sulfoxide reductase (PMSR) and three genes of RNA and protein metabolism. Four genes were identified that were more strongly expressed during dehardening of cold-hardened leaves at 22°C. A full-length cDNA for a presumed cytosolic PMSR (EC 1.8.4.6) of rye leaves was identified. After heterologous expression in Escherichia coli, an antiserum against the ScPMSR was produced. The content of the ScPMSR protein, visualized by immunoblotting, was much higher in cold-hardened than in non-hardened leaves and declined during dehardening. In non-hardened leaves the mRNA of ScPMSR increased only slowly during exposures to 4°C in light and was not affected by exposure to 4°C in darkness. However, the ScPMSR mRNA was also induced by prolonged exposure (48 h) to high light at 22°C, or by treatment with 2 μM paraquat. Consequently, the induction of cytosolic ScPMSR is a late response to prolonged photooxidative stress conditions, as expected during growth at low temperature in light. In cold-hardened leaves, PMSR may protect proteins from photodamage and thus prevent their degradation and the need for repair.

The effect of low temperature on protein synthesis, particularly the synthesis of the photolabile proteins D1 of photosystem II and catalase (EC 1.11.1.6), was compared in non-hardened leaves (NHL) and cold-hardened leaves (CHL) of winter rye (Secale cereale L.). At 4°C, both the uptake of L-[35S]methionine into leaf sections and its incorporation into proteins were reduced, relative to 25°C. However, much lower reductions were observed in CHL than in NHL. In particular, the proportion of the L-[35S]methionine uptake incorporated into membrane proteins at 4°C was considerably higher in CHL than in NHL. At 25°C, the incorporation of L-[35S]methionine into both the D1 protein and catalase was lower in CHL than in NHL, in accord with a slower light-induced turnover in CHL. At 4°C, the incorporation into the D1 protein and catalase was, however, much higher in CHL than in NHL, indicating that their de novo synthesis was less suppressed by the low temperature. The results indicate that cold-acclimated leaves had an improved ability to repair the photolabile proteins D1 and catalase at low temperature, relative to NHL. mRNAs for the D1 protein and for leaf catalase were not increased in CHL, relative to NHL. The superior capacity of CHL for repair at low temperature must result from posttranscriptional mechanisms. The translational efficiency of the catalase mRNA was similarly increased in both NHL and CHL during 7-h exposures to high light at 4°C, while the amounts of the catalase transcript declined under these conditions. However, during a recovery period at 22°C, subsequent to an exposure of NHL to 4°C and high light, transient increases of the D1 and catalase mRNAs were observed.

In cold-hardened leaves (CHL) of winter rye (Secale cereale L.) much higher levels of malate were detected by (13)C-NMR than in non-hardened leaves (NHL). As this was not observed previously, malate metabolism of CHL was studied in more detail by biochemical assays. The activities of several enzymes of malate metabolism, NADP-malate dehydrogenase, NAD-malate dehydrogenase, phosphoenolpyruvate carboxylase, and NADP-malic enzyme, were also increased in CHL. Short exposures to low temperature of 1-3 d did not induce increases in the malate content or in the activities of enzymes of malate metabolism in mature NHL. The malate content and the enzyme activities declined within 1-2 d after a transfer of CHL from their growing temperature of 4 degrees C to 22 degrees C. The malate content was further increased when CHL were exposed to a higher light intensity at 4 degrees C. In CO(2)-free air the malate content of CHL strongly declined at 4 degrees C. Malate may thus serve as an additional carbon sink and as a CO(2)-store in CHL. It may further function as a vacuolar osmolyte balancing increased concentrations of soluble sugars previously observed in the cytosol of CHL. Malate was not used as a source of reductants when CHL were exposed to photo-oxidative stress by treatment with paraquat. However, the activities of enzymes of the oxidative pentose phosphate pathway were markedly increased in CHL and may serve as non-photosynthetic sources of NADPH and thus contribute to the previously observed superior capacity of CHL of winter rye to maintain their antioxidants in a reduced state in the presence of paraquat.

The enzyme catalase is light-sensitive. In leaves, losses caused by photoinactivation are replaced by new enzyme and the rate of de novo synthesis must be rapidly and flexibly attuned to fluctuating light conditions. In mature rye leaves, post-transcriptional mechanisms were shown to control the rate of catalase synthesis. The amount of the leaf catalase (CAT-1) transcript did not increase with light intensity, but was even higher after dark exposure of light-grown leaves. Initiation was apparently not limiting translation in the dark, as the association of the Cat1 mRNA with polysomes did not change notably under different light conditions. By analysing the translation of catalase polypeptides in cell-free systems with poly(A)+ RNA from leaves or with mRNA transcribed from a Cat1-containing cDNA clone, two mechanisms of post-transcriptional control were identified. First, translation of catalase depended on the presence of hemin. In leaves, the availability of hemin may signal the extent of catalase degradation as the hemin of the inactivated enzyme is recycled. Second, the translation efficiency of the Cat1 transcripts was reversibly modulated in a dose-dependent manner by the light intensity to which leaves were exposed, prior to extraction. The Cat1 mRNA from light-exposed leaves was translated much more efficiently than mRNA from dark-exposed leaves. The increase of its translation activity in vivo was not blocked by cordycepin but was suppressed by methylation inhibitors, indicating a reversible modification of pre-existing mRNA by methylation. Translation of in vitro synthesized Cat1 mRNA required a methylated cap (m7GpppG), but was virtually below detection when it contained an unmethylated cap (GpppG).

The enzyme catalase, which detoxifies the H2O2 produced during photorespiration and thus represents an important step of photoprotection, is generally light-sensitive. In leaves catalase has a light-induced turnover. However, usually a constant level of activity is maintained because the loss by inactivation is continuously replaced by de novo synthesis. Mechanisms by which the rate of catalase synthesis is attuned to fluctuating light conditions, were analysed in mature rye leaves (Secale cereale L.). Light-modulated changes of the rate of synthesis were not related to changes in the amounts of mRNA but determined by posttranscriptional controls. Conditions for the translation of catalase mRNA were investigated in vitro with poly[A]+RNA from rye leaves in a cell-free wheat germ lysate. Incorporation into catalase was visualized by fluorography after immunoprecipitation and electrophoretic separation. The rate of catalase synthesis was determined by the availability of the heme cofactor. Furthermore, the translational activity of the catalase mRNA was reversibly changed and attuned to the light conditions to which the leaves were exposed, prior to RNA extraction. In darkness the translational activity of the catalase mRNA declined (half-life: 2h). Light induced dose-dependent increases. The light-induced increases were not prevented when the accumulation of new catalase mRNA was blocked by cordycepin. The change of the translational activity must be due to some reversible modification of the existing mRNA. The translational activity of the catalase mRNA depended on the methylation pattern of the cap structure.

A cDNA clone (ScRPS7) encoding the cytoplasmic ribosomal protein S7 was isolated from a rye leaf cDNA library and sequenced. The deduced protein of 192 amino acids with M(r) 22189 shows identity of 52% or 47%, respectively, relative to S7 proteins of human or yeast. A RPS7 mRNA accumulation is higher in the meristematic zone at the leaf base than in the non-meristematic middle and upper section of leaves. Short periods of cold stress sharply reduce the mRNA level while leaves of cold hardened plants contain normal levels of ScRPS7 transcripts.

Glycolate oxidase that was partially purified from pea leaves was inactivated in vitro by blue Light in the presence of FMN. Inactivation was greatly retarded in the absence of O-2. Under aerobic conditions H2O2 was formed. The presence of catalase, GSH or dithiothreitol protected glycolate oxidase against photoinactivation. Less efficient protection was provided by ascorbate, histidine, tryptophan or EDTA. The presence of superoxide dismutase or of hydroxyl radical scavengers had no, or only minor, effects. Glutathione suppressed H2O2 accumulation and was oxidized in the presence of glycolate oxidase in blue light. Glycolate oxidase was also inactivated in the presence of a superoxide-generating system or by H2O2 in darkness. In intact leaves photoinactivation of glycolate oxidase was not observed. However, when catalase was inactivated by the application of 3-amino-1,2,4-triazole or depleted by prolonged exposure to cycloheximide a strong photoinactivation of glycolate oxidase was also seen in leaves. In vivo blue and red light were similarly effective. Furthermore, glycolate oxidase was photoinactivated in leaves when the endogenous GSH was depleted by the application of buthionine sulfoximine. Both catalase and antioxidants, in particular GSH, appear to be essential for the protection of glycolate oxidase in the peroxisomes in vivo.

In green or etiolated rye leaves catalase was most efficiently inactivated by blue light absorbed by its prosthetic heme. Red light was ineffective at low intensity but induced marked inactivation in green leaves at higher photon flux, while far-red light was ineffective. At identical intensities of photosynthetically active radiation, Photosystem II (PS II) was equally inactivated by both blue and red light. Since catalase was insensitive to red light and no sensitizer for red light was detected in isolated peroxisomes, the inactivation of catalase observed in leaves in red light must result from photooxidative reactions initiated in the chloroplasts. In a simplified model system the inactivation of isolated catalase was induced by the presence of a suspension of either intact or broken chloroplasts in red light. This chloroplast-mediated inactivation of catalase in vitro was O2-dependent. It was greatly retarded at low temperature, fully suppressed by the radic al scavenger Trolox, partially retarded by superoxide dismutase, but only little diminished by the singlet oxygen quencher histidine and not affected by dimethylsulfoxide, a hydroxyl radical scavenger. Chloroplast-mediated catalase inactivation in vitro was suppressed by suitable electron acceptors, in particular by methyl viologen. A comparison of the effects of inhibitors, donors, or acceptors for specific sites of the photosynthetic electron transport indicated that an overreduction of PS II and plastoquinone represented the major sources for the formation of O2ċ and some unidentified radical that appeared to mediate the inactivation of catalase outside of the chloroplasts. Chloroplast-mediated catalase inactivation provides a means for the detection of a redox signalling system of chloroplasts that was postulated to indicate overreduction of plastoquinones. Similarly as in the in vitro system, catalase inactivation in red light was also in leaves temperature-dependent and stimulated by DBMIB (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone). These results provide strong evidence that inactivation of catalase initiated by chloroplastic reactions in red light occurred also in leaves under identical conditions as in the model system in vitro.

ABSTRACTThe contents of ascorbate and glutathione and the activities of superoxide dismutase and glutathione reductase were increased to levels as high as those in cold‐hardened leaves (CHL) by incubating non‐hardened leaves (NHL) of winter rye (Secale cereale L.) with the precursor substrates L‐galactonic acid‐γ‐lactone and 2‐oxothiazolidine‐4‐carboxylate. Reduced glutathione was rapidly depleted from NHL after application of D, L‐buthionine sulfoximine, an inhibitor of its biosynthesis. In spite of greatly divergent antioxidant contents the rates of photo‐inactivation of photosystem II (PSII) and catalase observed in the presence of translation inhibitors did not differ greatly. The paraquat‐induced catalase inactivation and chlorophyll degradation in light were reduced in NHL with increased antioxidant levels. Paraquat‐induced photo‐inactivation of PSII was, however, not mitigated. The CHL had a higher capacity to prevent paraquat‐induced oxidation of ascorbate and glutathione than NHL with increased antioxidant contents. Increased antioxidant contents did not establish resistance to low temperature‐induced photo‐inactivation of PSII and catalase in NHL. The resistance of CHL to low temperature‐induced photo‐inactivation of PSII and catalase required repair at low temperature and active carbon assimilation but was only little affected when photorespiration was suppressed by phosphinothricin. Protection of PSII depended also on non‐photochemical quenching of excitation energy.

Plant Gene Register titles for PGR 99–174 to PGR 99–187 appear below. The sequences have beendeposited in GenBank and the articles listed online through the World Wide Web.To cite an electronic Plant Gene Register article as a bibliographic reference, follow the stylegiven below:Park S, Thornburg RW (1998) Characterization of UMP kinase cDNAs from rice (accession nos.AF187062 and AF187063) (PGR 99–174). Plant Physiol

Catalase and photosystem II (PSII) were strongly inactivated during exposure to 4 degrees C and moderate light in 22 degrees C grown non-hardened leaves (NHL) of winter rye (Secale cereale L.), but highly resistant to photo-inactivation at low temperature in 4 degrees 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 degrees 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, alpha-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 degrees C the increased resistance to photoinactivation 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.

Five varieties of Sorghum bicolor (L.) Moench., differing in their drought tolerance under field conditions have been used to study the effect of individual components of drought stress, namely high light intensity stress, heat stress and water stress, on their photosynthetic performance. Chlorophyll content, chlorophyll fluorescence, ribulose-1,5-bisphosphate carboxylase (Rubisco, EC 4.1.1.39) content, phosphoenolpyruvate carboxylase (PEPcase, EC 4.1.1.31) activity and photosynthetic oxygen evolution were used as key parameters to assess photosynthetic performance. The results indicated that photochemical efficiency of photosystem II (PSII) was severely reduced by all three stress components, whereas PEPcase activity was more specifically reduced by water stress. Degradation of Rubisco and chlorophyll loss occurred under high light and water stress conditions. Of the four drought-tolerant varieties, E 36-1 showed higher PEPcase activity, Rubisco content and photochemical efficiency of PSII, and was able to sustain a higher maximal rate of photosynthetic oxygen evolution under each stress condition as compared to the other varieties. A high stability to stress-induced damage, or acclimation of photosynthesis to the individual components of drought stress may contribute to the high yields of E 36-1 under drought conditions. In the E 36-1 variety markedly higher levels of the chloroplastic chaperonin 60 (cpn 60) were observed under all stress conditions than in the susceptible variety CSV 5.