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).     

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     

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     

An RFLP marker in tomato linked to the Fusarium oxysporum resistance gene I2

Summary The locus, I2, which in tomato confers resistance against Fusarium oxysporum f. sp. lycopersici race 2, was introgressed into Lycopersicon esculentum from the wild species L. pimpinellifolium (P.I. 126915). We searched for restriction fragment length polymorphisms (RFLPs) between nearly isogenic lines (NILs) in clones that map to the region introgressed from the wild species. Since I2 maps to chromosome 11, we used DNA clones from this chromosome as hybridization probes to Southern blots containing bound DNA of the NILs digested with 23 restriction enzymes. Of the 14 chromosome 11 clones, 9 exhibited polymorphism. These clones were further hybridized to verification filters that contained DNA from resistant and susceptible L. esculentum varieties digested with the enzymes that gave the polymorphism. One clone, TG105, was found to be associated with I2; 19 susceptible lines showed a different RFLP with this probe than 16 resistant lines, including the original L. pimpinellifolium accession used as a source for the resistance gene. These results together with our mapping analysis indicate that TG105 is closely linked to the resistance gene.     

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.