Molecular cloning,characterization and expression analysis of two catalase isozyme genes in barley

Clones representing two distinct barley catalase genes, Cat1 and Cat2, were found in a cDNA library prepared from seedling polysomal mRNA. Both clones were sequenced, and their deduced amino acid sequences were found to have high homology with maize and rice catalase genes. Cat1 had a 91% deduced amino acid sequence identity to CAT-1 of maize and 92% to CAT B of rice. Cat2 had 72 and 79% amino acid sequence identities to maize CAT-2 and-3 and 89% to CAT A of rice. Barley, maize or rice isozymes could be divided into two distinct groups by amino acid homologies, with one group homologous to the mitochondria-associated CAT-3 of maize and the other homologous to the maize peroxisomal/glyoxysomal CAT-1. Both barley CATs contained possible peroxisomal targeting signals, but neither had favorable mitochondrial targeting sequences. Cat1 mRNA occurred in whole endosperms (aleurones plus starchy endosperm), in isolated aleurones and in developing seeds, but Cat2 mRNA was virtually absent. Both mRNAs displayed different developmental expression patterns in scutella of germinating seeds. Cat2 mRNA predominated in etiolated seedling shoots and leaf blades. Barley genomic DNA contained two genes for Cat1 and one gene for Cat2. The Cat2 gene was mapped to the long arm of chromosome 4, 2.9 cM in telomeric orientation from the mlo locus conferring resistance to the powdery mildew fungus (Erysiphe graminis f.sp. hordei).     

Catalase activity and hydrogen peroxide levels are inversely correlated in maize scutella during seed germination

Temporal patterns of hydrogen peroxide (H2O2) levels and total catalase activity are presented for post-imbibition scutella from six maize inbred lines expressing variable catalase activity. In all lines examined, H2O2 levels were highest during the initial days post-imbibition (1-2 dpi) and decreased thereafter, while total catalase activity was lowest during early dpi (1-2 dpi) and reached maximal activity at 4-6 dpi. In three of the six lines tested, a simple inverse correlation between catalase activity and H2O2 level was significant by Spearman's rank (P < 0.01). In addition to the general decline in H2O2 level throughout the dpi period, a reproducible increase in H2O2 level was observed at 4-5 dpi in five of six lines examined. Mutant lines lacking CAT-3 activity demonstrated a temporal shift in the occurrence of this increase. The role of total catalase (and individual isozymes) in controlling H2O2 levels during germination and the role of H2O2 as a potential regulator of catalase expression during germination are discussed.     

Catalase activity and hydrogen peroxide levels are inversely correlated in maize scutella during seed germination

Abstract

Temporal patterns of hydrogen peroxide (H₂O₂) levels and total catalase activity are presented for post-imbibition scutella from six maize inbred lines expressing variable catalase activity. In all lines examined, H₂O₂ levels were highest during the initial days post-imbibition (1–2 dpi) and decreased thereafter, while total catalase activity was lowest during early dpi (1–2 dpi) and reached maximal activity at 4–6 dpi. In three of the six lines tested, a simple inverse correlation between catalase activity and H₂O₂ level was significant by Spearman's rank (P <0.01). In addition to the generaldecline in H₂O₂level throughout the dpi period, a reproducible increase in H₂O₂ level was observed at 4–5 dpi in five of six lines examined. Mutant lines lacking CAT-3 activity demonstrated a temporal shift in the occurrence of this increase. The role of total catalase (and individual isozymes) in controlling H₂O₂ levels during germination and the role of H₂O₂ as a potential regulator of catalase expression during germination are discussed.     

Effect of chlorophyll reduction in Arabidopsis thaliana by methyl jasmonate or norflurazon on antioxidant systems.

Methyl jasmonate (MeJA) and norflurazon (NF) treatments resulted in a substantial decrease in photosynthetic activities and chlorophylls (Chls) in Arabidopsis thaliana plants, causing a senescence-like yellowing and a bleaching in MeJA- and NF-treated plants, respectively. Non-radiative energy dissipation through q(N) and non-photochemical quenching increased greatly in NF-treated plants in concomitance with an increase in photoprotectants antheraxanthin and zeaxanthin from interconversion of violaxanthin, although they were not changed in MeJA-treated plants. A significant accumulation of anthocyanin was observed only in MeJA-treated plants, not in NF-treated plants. Total activities of catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7), superoxide dismutase (EC 1.15.1.1) and glutathione reductase (EC 1.6.4.2) increased greatly in response to MeJA, particularly a 100-fold increase in POD activity 7 days after MeJA treatment. NF application to plants exhibited less increase in antioxidant enzymes than MeJA-treated plants. NF-treated young leaves had a greater decline in Chls and CAT activity, and less zeaxanthin accumulation compared to NF-treated mature leaves, indicating that NF-treated young leaves are more susceptible to excess light exposure and a possible photooxidative stress. Both MeJA- and NF-treated Arabidopsis plants suffered destruction of Chls, however, they developed differential antioxidant responses during the stress, in large part by an increased anthocyanin level in the epidermis and enzymatic antioxidants in MeJA-treated plants and by accumulation of antheraxanthin and zeaxanthin, and enhanced energy dissipation in NF-treated plants.     

Structural organization, regulation, and expression of the chloroplastic superoxide dismutase Sod1 gene in maize.

A cDNA and genomic clone encoding maize chloroplastic Cu/Zn superoxide dismutase Sod1 were isolated. Southern blot analysis indicated little homology between the chloroplastic (Sod1) and the cytosolic (Sod2, Sod4, Sod4A) cDNAs. Sequence analysis of the genomic clone revealed a promoter, transit peptide, and partial coding sequence. The promoter contained several response elements (e.g., for light, cold temperature, xenobiotics) that may be involved in the regulation of the Sod1 gene. Sod1 expression during development and in response to physiological and chemical stressors such as temperature, xenobiotics (paraquat), and light were examined.     

Effect of the free radical-generating herbicide paraquat on the expression of the superoxide dismutase (Sod) genes in maize

10-day-old maize leaves were treated with the oxygen free radical-generating herbicide paraquat for 12 h. Paraquat treatments (10(-5) M) resulted in a 40% increase in superoxide dismutase activity and a smaller increase in catalase activity. The increase in total superoxide dismutase (SOD) activity correlates with higher levels of specific isozymes. The chloroplast (SOD-1) and cytosolic (SOD-2 and SOD-4) forms were increased significantly; however, the mitochondrial form (SOD-3) was increased only slightly. Higher levels of SOD-4 and SOD-3 after paraquat exposure were the result of increased synthesis of these proteins, as determined by labeling in vivo with [35S]methionine. Isolation and in vitro translation of polysomes from 10(-5) M paraquat-treated leaves indicated that paraquat increased the amount of polysomal mRNA which codes for SOD-4 and SOD-3. Superoxide dismutase induction does not appear to be a response that is specific to paraquat, since another superoxide-generating compound, juglone, caused a similar increase in total superoxide dismutase activity. Therefore, the effect of these compounds on the expression of the maize Sod genes is exerted via their ability to generate superoxide.     

Analysis of variants affecting the catalase developmental program in maize scutellum

Summary The catalase of maize scutella is coded for by two loci, Cat1 and Cat2, which are differentially expressed in this tissue during early seedling growth. Two variant lines have been previously identified in which the developmental program for the expression of the Cat2 structural gene in the scutellum has been altered. Line R6–67 exhibits higher than normal levels of CAT-2 catalase in this tissue after four days of postgerminative growth. This phenotype is controlled by a temporal regulatory gene designated Car1. Line A16 exhibits a CAT-2 null phenotype. Further analysis of Car1 verifies the initial indication that it is trans-acting and exhibits strict tissue (scutellum) specificity. A screen of other available inbred lines uncovered eight additional catalase high-activity lines. All eight lines exhibit significantly higher than normal levels of CAT-2 protein. Two of these lines have been shown to be regulated by Car1 as in R6–67. Another line (A338) uncovered during the screen exhibits a null phenotype for CAT-2 protein and resembles A16. Catalase activity levels are low in the scutellum and no CAT-2 CRM (cross-reacting material) is present in the tissues of this line. Also, unlike most maize lines, CAT-2 cannot be induced in the leaf tissue of A338 upon exposure to light. Finally, a single line (A337), demonstrating a novel catalase developmental program, was identified.     

Synthesis of Isozymes of Superoxide Dismutase in Maize Leaves in Response to O3, SO2 and Elevated O2

Matters, G. L. and Scandalios, J. G. 1987. Synthesis of isozymesof superoxide dismutase in maize leaves in response to O₃ SO₂and elevated O₂.—J. exp. Bot 38: 842–852. The activities of the enzymes superoxide dismutase (SOD) andcatalase were determined in maize leaves treated with O₃or SO₂for8 h, or with elevated levels of oxygen for up to 96 h. NeitherO₃nor SO₂significantly increased the levels of superoxide dismutaseor catalase activity. However, after 72 h in an atmosphere containing90% oxygen, superoxide dismutase activity was increased, butnot the activities of catalase, ascorbate pcroxidase, and malatedehydrogenase. Immunological analysis showed that amounts ofthe cytosolic superoxide dismutase isozymes, SOD-2 and SOD-4,were increased by the elevated oxygen but not the chroloplast(SOD-1) or mitochondrial (SOD-3) isozymes. Immunoprecipitationof translation products of leaf polysomes indicated that thehigher levels of SOD-2 and SOD-4 were due to increased amountsof polysome-bound mRNA coding for these proteins. The specificresponse of SOD-2 and SOD-4 to 90% oxygen treatments contrastswith the increase in all SOD isozymes in maize leaves treatedwith the herbicide paraquat. Key words: Air pollutants, maize, oxidative stress, oxygen, superoxide dismutase     

Two barley catalase genes respond differentially to light

Cloned catalase probes from barley ( Hordeum vulgare L.) and maize ( Zea mays L.) were used to examine catalase gene expression in greened and etiolated leaves of several barley lines. Etiolated leaves had greater levels of an mRNA detected by barley Cat1 , compared with greened leaves, in all lines. In contrast, a Cat2 -like mRNA (homologous to Cat2 of maize) was induced by light and accumulated to high levels in greened leaves, compared to the negligible levels detected in etiolated leaves. This suggests that barley contains light-inducible and light-repressible catalase genes. In the catalase-deficient barley mutant RPr 79/4, no hybridization signal was detected when RNA from greened or etiolated leaves was probed with maize Cat2 , indicating that this mutant is deficient for the light-induced Cat mRNA. In etiolated seedlings of both RPr 79/4 and its motherline, the level of the Cat1 mRNA increased coordinately with a steady increase in catalase activity. Even though the mutant RPr 79/4 was able to grow to maturity in normal air, it sustained chlorosis and significant head sterility, probably due to the lack of a light-inducible catalase. Although the mutant RPr 79/4 is not completely lacking catalase (EC 1.11.1.6), the loss of the CAT-1 isozyme is evidently harmful. This observation underscores the protective role of catalases in plants.