Genetic and biochemical characterization of a Cat2 catalase null mutant of zea mays

Summary In all maize inbred lines examined to date, the Cat2 gene which codes for the CAT-2 catalase is expressed primarily in the scutellum upon seed imbibition. The activity of CAT-2 increases dramatically during the initial four days after germination and subsequently declines. In contrast, we have recently identified and inbred strain (A16) of maize which does not express the Cat2 gene (i.e., the CAT-2 catalase is undetectable). Electrophoretic and immunological analyses indicate that the CAT-2 protein is not present in either an active or inactive form in line A16. Genetic analysis suggests that the absence of CAT-2 expression in line A16 is due to a null allele at the Cat2 gene locus although the possibility of a mutation at a regulatory locus, closely linked to the structural gene has not been excluded. Two other enzymes involved in H₂O₂ metabolism (superoxide dismutase and peroxidase) were also compared in W64A and A16 with no significant differences being observed. Aminotriazole (AT), a known inhibitor of catalase, has been used to simulate the A16 phenomenon by inhibiting catalase activity in line W64A (which has normal expression of CAT-2). AT, in very low concentrations, effectively inhibits the expression of CAT-2 in the scutellum. This inhibition of catalase by AT does not result in changes of the developmental time-course of superoxide dismutase and peroxidase.Research supported by National Institutes of Health Grant No. GM 22733-05 to J.G.S.Paper No. 6601 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

Expression of the developmentally regulated catalase (cat) genes in maize

The catalase (H₂O₂:H₂O₂ oxidoreductase; E.C.1.11.1.6; CAT) gene-enzyme system in Zea mays L (maize) represents an ideal model for studying the molecular basis of developmental gene regulation in higher eukaryotes. This system comprises a family of structural genes that are highly regulated, both temporally and spatially, during maize development. In maize, there are four distinct forms (isozymes) of catalase that are readily discernible by convetional separation procedures. Three of the catalases have been studied in detail from a genetic and biochemical viewpoint. The catalases CAT-1, CAT-2, and CAT-3 are encoded by the distinct, unlinked genes Cat1, Cat2, and Cat3, respectively. Each of the structural genes is highly regulated both spatially and temporally in its expression. Cat1 is expressed primarily in the endosperm, aleurone, pericarp, and scutellum of developing kernels, and in the root, shoot, and scutellum of very young seedlings. Cat2 is expressed primarily in the scutellum and leaf during postgerminative sporophytic development. Cat3 is expressed, for the most part, in the shoot and pericarp of young seedlings. A number of regulatory variants have been recovered that affect the developmental program of expression of the catalases. Analysis of one variant allowed for the identification of a temporal regulatory gene (Car1) that specifically alters the developmental program of the Cat2 structural gene by acting to regulate the rate of CAT-2 protein synthesis. Cat1 has been mapped on chromosome 1S, 37 map units (m.u.) from the Cat2 structural gene. Another variant line has been isolated which lacks expression of the Cat2 gene in its tissues at all stages of development. Isolated polysomes from this line (A16) were translated in vitro, and the products were immunoprecipitated with CAT-2-specific antibodies. No CAT-2 was detectable in the A16 labeled immunoprecipitates, whereas CAT-2 was readily detected in the normal line, W64A, under similar conditions. The temporal and spatial expression of the Cat structural genes is not only influenced by genetic factors (as above), but is also responsive to exogenously applied environmental signals: light, hormones, and temperature. The mechanisms by which such signals specifically affect CAT-2 expression will be discussed.     

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.     

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).     

Chromosomal location of the catalase structural genes in Zea mays using B-A translocations

Summary B-A translocations have been used to map the catalase genes, Cat1, Cat2, and Cat3 of Zea mays. Cat1 was found to be on the short arm of chromosome 5, 9.1 map units from brittle endosperm (bt ₁). Cat2 was located on chromosome 1S, while Cat3 was located on the distal half of chromosome 1L. There was no linkage between Cat2 and Cat3. The significance of mapping the catalase structural genes is discussed.This research was supported by Grant No. GM22733 from the National Institute of General Medical Sciences, National Institutes of Health, USPHS to JGS.This is Paper No. 6437 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC     

Differential expression of the maize catalase genes during kernel development: the role of steady-state mRNA levels

In maize three isozymic forms of catalase, CAT-1, CAT-2, and CAT-3 are encoded by three distinct and unliked structural genes (Cat1, Cat2 and Cat3). Catalase activity profiles and zymogram analysis were used to examine the spatial and temporal expression of the three genes during kernel maturation. Three developmental stages of catalase expression were observed in the growing kernel. During stage 1 (6-12 days after pollination), both Cat1 and Cat3 were expressed; during stage 2 (15-18 days after pollination) only Cat1 expression was observed; and during stage 3 (21-30 days after pollination), Cat1 and Cat2 were expressed. The major constituent tissues of the kernel were examined to determine their contribution to total kernel catalase expression. Each of the tissues was found to have a unique pattern of catalase gene expression. RNA blot analysis, using catalase gene-specific nucleic acid probes, suggests that the differential expression of the three catalase genes observed in the kernel is regulated by controlling the distribution of steady-state mRNA species for the three genes.     

Cat-2 gene expression

Summary Poly(A)⁴ RNA was isolated from maize scutella of different stages of post-germinative development and translated in vitro in a rabbit reticulocyte translation system. Immunoprecipitation of the translation products with CAT-2-specific antibody was used to quantitate the relative levels of translatable CAT-2 mRNA at each stage. The results show a close correlation between the developmental profile of Cat2 gene expression and the profile of CAT-2 mRNA levels. Evidence that the levels of CAT-2 mRNA are regulated by a temporal regulatory gene (Car1) is presented and the possible mechanism(s) of this regulation discussed.This work was supported by Research Grants No. GM22733 and No. GM33817 from the U.S. National Institutes of Health, Public Health Service to J.G.S. This is paper No. 9933 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695, USA     

Molecular evolution of maize catalases and their relationship to other eukaryotic and prokaryotic catalases

We have compared the nucleotide and protein sequences of the three maize catalase genes with other plant catalases to reconstruct the evolutionary relationship among these catalases. These sequences were also compared with other eukaryotic and prokaryotic catalases. Phylogenies based on distances and parsimony analysis show that all plant catalases derive from a common ancestral catalase gene and can be divided into three distinct groups. The first, and major, group includes maizeCatl, barleyCat1, riceCatB and most of the dicot catalases. The second group is an apparent dicot-specific catalase group encompassing the tobaccoCat2 and tomatoCat. The third is a monocot-specific catalase class including the maize Cat3, barley Cat2, and riceCatA. The maize Cat2 gene is loosely related to the first group. The distinctive features of monocot-specific catalases are their extreme high codon bias at the third position and low degree of sequence similarity to other plant catalases. Similarities in the intron positions for several plant catalase genes support the conclusion of derivation from a common ancestral gene. The similar intron position between bean catalases and human catalase implies that the animal and plant catalases might have derived from a common progenitor gene sequence. Correspondence to: J.G. Scandalios     

Non-Coordinate Genetic Alterations in the Expression of Catalase and other Glyoxysomal Enzymes during Maize Development

In the scutellum of maize during post-germinative development,the primary form of catalase expressed is the product of theCat2 structural gene, CAT-2. The developmental time-course ofCAT-2 protein follows a rapid increase with a peak at approximately4–5 d alter germination and a subsequent decline. An inbredstrain of maize, A337, has been found to exhibit a similar generalizedprofile with the significant exception that the level of CAT-2protein present in the scutellum is far above that in the ‘typical’maize lines exemplified by W64A. Our data suggest that the higherlevels of CAT-2 exhibited in A337 are due to increased synthesisand accumulation of more CAT-2 protein, and not merely to enzymeactivation. A comparison of A337 and W64A showed that the twolines are similar with respect to number of glyoxysomes andwith the exception of catalase, other microbody associated enzymesexhibit similar activity levels and developmental profiles.Thus, the results presented suggest that the catalasc developmentalprogramme characteristic of line A337 is not due to a concurrentincrease and subsequent decline in the number of glyoxysomesformed in the scutellum during this developmental period butis instead due to a greater level of CAT-2 protein. The datafurther support our earlier findings that the genes coding forglyoxysomal enzymes in maize are non-coordinately regulated. Key words: Gene regulation, glyoxysomes, catalase, glyoxysomal enzymes     

Separate molecular mechanisms regulate CAT2 gene expression before and after glyoxysome maturation in two genetic lines of maize

The temporal expression pattern of the CAT-2 catalase isozyme in scutella of Zea mays seedlings normally coincides with that of other major glyoxysomal enzymes. In standard genetic lines (e.g., W64A), the CAT-2 enzyme is synthesized de novo after imbibition, reaches a peak at approximately 4 days later, and then declines steadily. In a high CAT-2 genetic line, R6-67, the enzyme accumulates in a linear fashion for at least 8 days after imbibition and reaches a level 3-fold higher than in W64A. During the first 9 days of early seedling growth in W64A, the correlation between Cat2 mRNA levels and CAT-2 protein suggests that pretranslational control governs Cat2 gene expression. In R6-67, the steady rise in CAT-2 protein appears to result from a pretranslational control mechanism in which Cat2 mRNA apparently never declines to levels which would limit the rate of accumulation of CAT-2 protein. In addition, the amount of Cat2 mRNA bound to polysomes is 3-fold higher in R6-67 at day 9, relative to W64A at day 9, reflecting a much greater capacity to synthesize CAT-2 later in development. Despite substantial differences in Cat2 mRNA levels between genetic lines, early CAT-2 protein accumulation is similar until day 5, when other glyoxysomal enzymes also attain maximal activity levels. The early increase in CAT-2, between day 2 and day 5 post-imbibition, occurs despite a sharp decline in polysomal Cat2 mRNA. This is related to a transient decline in total extractable polysomes which paradoxically coincides with the peak in glyoxysomal enzyme activities.(ABSTRACT TRUNCATED AT 250 WORDS)