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.

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.

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.

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

The peroxisomal enzyme catalase is inactivated by visible light in the presence of 02 both in vitro and in vivo. The photoinactivation is mediated by blue light absorbed by the prosthetic heme groups. In addition, at higher photon flux, in leaves catalase is photoinactivated in red light absorbed by chlorophylls, through activated oxygen products transmitted from the chloroplasts. In mature green leaves with constant catalase activity the catalase apoprotein undergoes a permanent degradation and turnover in light which increases with the photon flux, while the heme groups remain largely undamaged. The light-dependent changes in the rate of catalase apoprotein synthesis are not related to changes of its mRNA but they must be determined by translational control mechanisms. Under various stress conditions which enhance oxidative stress or impair translation (e.g. low temperature or heat- shock, salt) a rapid loss of catalase activity is observed as early symptom together with the photoinhibition of photosystem II. The decline of catalase in stress-exposed, e.g. NaCl-treated, leaves contributes to enhanced oxidative damage in light, which is indicated by the bleaching of chlorophyll as well as increases in the proportions of the oxidized forms of ascorbate and glutathione, but no H202 accumulation was detected. Plants have developed adaptive mechanisms to avoid photoinactivation of catalase in vivo. Leaves of cold- hardened rye or several alpine high-altitude plants are tolerant against catalase photoinactivation in spite of high light and low temperature.

The isolation of cDNAs is described which encode the complete sequence of a precursor protein for a HSP90 homologue consisting of an N-terminal transit peptide of 5850 Da and a mature protein (cpHSP82) of 82 260 Da, located in the plastids of rye leaves (Secale cereale). Hybridization analysis indicated the presence of a single gene in the DNA of rye and a transcript size of 2.8 kb. A phylogenetic tree constructured on the basis of sequence comparisons for HSP90 homologues from different species and compartments indicated that the plastidic HSP82 from rye was more closely related to an eubacterial protein than to HSP90 homologues of the cytosol or ER from both plants and animals. The results suggest that during chloroplast evolution the gene for cpHSP82 was transferred to the nucleus from a prokaryotic endosymbiont. Immunoblots with specific antibodies and Percoll gradient-purified organelles confirmed the location of cpHSP82 in chloroplasts or non-green plastids. In green rye leaves cpHSP82 was constitutively expressed and equally distributed among tissues of different age. The expression of cpHSP82 was enhanced within 2 h by exposure to 42 °C. The cpHSP82 transcript and protein were much more strongly expressed in non-green tissues, such as etiolated, 70S ribosome-deficient 32 °C-grown, or herbicide-bleached, than in normal green leaves. Also chromoplasts from the pericarp of tomato fruits contained high levels of a HSP90 polypeptide while a photosynthetic protein, the large subunit of the ribulose-1,5-bisphosphate carboxylase was largely degraded during ripening.

Two different cDNA clones (SCL12-1 and SCL12-2) encoding precursors of a chloroplast ribosomal protein with homology to L12 from Escherichia coli were isolated from rye leaf cDNA libraries and sequenced. The corresponding polypeptide of rye chloroplast ribosomes was identified. The sequences for the mature proteins of Mr 13447 and 13609 share 85% amino acid identity. The mature polypeptide of clone SCL12-1 has an amino acid identity of 71%, 72% or 44%, respectively, relative to L12 proteins from spinach, tobacco, or E. coli. Codon usage of the rye L12 cDNAs shows a high preference (97% and 82%) for G or C in the third base position.