Cross tolerance to heavy-metal and cold-induced photoinhibiton in leaves of Pisum sativum acclimated to low temperature.

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.

Molecular identification, heterologous expression and properties of light-insensitive plant catalases.

Most catalases are inactivated by light in a heme-sensitized and O2-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.

Mode of translational activation of the catalase (cat1) mRNA of rye leaves (Secale cereale L.) and its control through blue light and reactive oxygen.

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 (H(2)O(2), tertiary butyl hydroperoxide) and conditions enforcing an H(2)O(2) 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 Ca(2+) 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 Ca(2+) by EGTA the cat1 mRNA was rapidly degraded.

Changes in gene expression during dehardening of cold-hardened winter rye (Secale cereale L.) leaves and potential role of a peptide methionine sulfoxide reductase in cold-acclimation.

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 degrees C of cold-hardened 4 degrees 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 degrees 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 degrees C in light and was not affected by exposure to 4 degrees C in darkness. However, the ScPMSR mRNA was also induced by prolonged exposure (48 h) to high light at 22 degrees C, or by treatment with 2 muM 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.

Increased capacity for synthesis of the D1 protein and of catalase at low temperature in leaves of cold-hardened winter rye (Secale cereale L.).

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 degrees C, both the uptake of L-[(35)S]methionine into leaf sections and its incorporation into proteins were reduced, relative to 25 degrees C. However, much lower reductions were observed in CHL than in NHL. In particular, the proportion of the L-[(35)S]methionine uptake incorporated into membrane proteins at 4 degrees C was considerably higher in CHL than in NHL. At 25 degrees C, the incorporation of L-[(35)S]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 degrees 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 degrees C, while the amounts of the catalase transcript declined under these conditions. However, during a recovery period at 22 degrees C, subsequent to an exposure of NHL to 4 degrees C and high light, transient increases of the D1 and catalase mRNAs were observed.

Malate metabolism and reactions of oxidoreduction in cold-hardened winter rye (Secale cereale L.) leaves.

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.

Post-transcriptional mechanisms control catalase synthesis during its light-induced turnover in rye leaves through the availability of the hemin cofactor and reversible changes of the translation efficiency of mRNA.

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).

Preferential photoinactivation of catalase and photoinhibition of photosystem II are common early symptoms under various osmotic and chemical stress conditions.

Activity of catalase (EC 1.11.1.6) and variable fluorescence (F) were measured in sections of rye leaves (Secale cereale L. cv. Halo) that were exposed for 24 h to moderately high irradiance under osmotic or chemical stress conditions (paraquat, DCMU, mannitol, NaCl, CdCl2 , CuSO4 , Pb(NO3 )2 , KNO2 , or K2 SO3 ). Changes of the chlorophyll content and of enzyme activities related to peroxide metabolism, such as glycolate oxidase, glutathione reductase, and peroxidase, were assayed for comparison. In the presence of the herbicides paraquat and low DCMU concentrations that exert only partial inhibition of photosynthesis, as well as after most treatments with osmotic or chemical stress factors, catalase markedly declined due to a preferential photoinactivation. At higher DCMU levels catalase did not decline. At low KNO2 concentrations catalase activity was preferentially increased. In general, photoinactivation of catalase was accompanied by a decline of the F/Fm ratio, indicating photoinhibition of photosystem II, while other parameters were much more stable. Inasmuch as both catalase and the D1 reaction center protein of photosystem II have a rapid turnover in light, their steady state levels appear to decline whenever stress effects either excessively enhance deleterious oxidative conditions and degradation (e. g. Paraquat, low DCMU), or inhibit repair synthesis. Photoinactivation of catalase and of photosystem II represent specific and widely occurring early symptoms of incipient photodamage indicating stress conditions where the repair capacity is not sufficient. During prolonged exposures, e. g. to NaCl and CuSO4 , chlorophyll was bleached in light and the rate of its photodegradation increased in proportion as the catalase level had declined. The results suggest that the enhanced susceptibility of leaf tissues to photooxidative damage which is widely observed in stressed plants is related to the early loss of catalase.