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.     

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.     

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     

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).     

Cat3, a third gene locus coding for a tissue-specific catalase in maize: Genetics, intracellular location, and some biochemical properties

Summary A new and unique catalase isozyme, CAT-3, has been found in Zea mays. It is encoded in the Cat3 nuclear structural gene which is distinct from the two previously described catalase structural genes, Cat1 and Cat2. The Cat3 gene is both tissue- and time-dependent in its expression, being expressed primarily in young leaves and in the pericarp of nearly mature kernels. Cell fractionation experiments, utilizing epicotyl (coleoptile+primary leaf) and mesocotyl cells, suggest that CAT-3 is associated with the mitochrondria where it may play a role in the alternate oxidase pathway. CAT-3 was purified and characterized with respect to some of its biochemical properties. While CAT-3 differs in some of its properties from CAT-1 and CAT-2, it is similar to these and to other catalases in most respects.Research supported by National Institutes of Health Grant No. GM 22733 to JGS.Paper No. 5255 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC.     

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     

Regulation of Glyoxysomal Enzyme Expression in Maize

The activity levels of three glyoxysomal enzymes (catalase, isocitric lyase, and malate synthase) were measured in the scutellum following germination of the inbred lines W64A, R6-67, and A16. In W64A, as in most maize lines examined, germination was accompanied by a rapid and synchronous increase in the activities of all three enzymes, and reached a peak at about day 4 and declined thereafter. In R6-67, catalase activity continues to increase past day 4 and reaches its highest activity level on later days. In A16, catalase activity is very low due to the lack of expression of the Cat2 gene. Despite these significant differences in catalase expression, the levels of the other two glyoxysomal enzymes did not differ in these inbred lines. Artificial inhibition of catalase in W64A by exogenous application of 10^–4 M aminotriazole did not inhibit germination, nor did it alter the levels of the other two glyoxysomal enzymes. Similarly, application of 10^–4 M itaconate to W64A seeds inhibited the appearance of isocitric lyase, but did not inhibit germination or alter the levels of malate synthase or catalase. Comparative cell fractionation and immunological studies were conducted with W64A and A16 and their microbodies were observed under the electron microscope. Cell fractionation studies were also conducted with W64A seeds germinated in the presence of aminotriazole or itaconate. Thus, our results suggest that the expression of these three glyoxysomal enzymes is not regulated coordinately in the maize scutellum.     

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.     

Evidence for processing of maize catalase 2 and purification of its messenger RNA aided by translation of antibody-bound polysomes

Two-dimensional gel analysis of the in vitro and in vivo labeled catalase 2 (CAT-2) isozyme protein of Zea mays L. and western gel analysis of native CAT-2 and in vitro labeled CAT-2 indicated that the protein is processed from a precursor to a lower molecular weight form in the scutellum. The CAT-2 from each source appeared on two-dimensional gels as one major species and two to three subspecies of the same molecular weight. We have also purified the mRNA encoding CAT-2 from scutella of line R6-67 using the procedure of polysome immunoadsorption. As a midcourse check on the progress of purification, we translated a small portion of the purified Cat2 mRNA-containing polysomes while they were still complexed with CAT-2 antibodies and bound to protein A-Sepharose. This revealed the presence of highly purified Cat2 polysomes. The final mRNA could not be translated in the wheat germ system but was highly active in the reticulocyte lysate system. The translation product had a molecular weight of 56 000, compared to that of 54 000 for purified CAT-2 protein. We have also enriched for Cat2 mRNA by size selection on methylmercury-agarose gels. The Cat2 resided with and slightly above the 18S ribosomal contaminant band of the total poly(A+) mRNA. It is therefore about 1805 bases long, which is 224 bases longer than the calculated coding length of 1581 bases.     

The inheritance of rye seed peroxidases

Summary Genetic analyses were conducted on peroxidase of the embryo and endosperm of seeds of one open pollinated and six inbred lines of cultivated rye (Secale cereale L.), and one line of Secale vavilovii Grossh. The analyses of the individual parts of the S. cereale seed yield a total of 14 peroxidase isozymes. Isozymes m, a, b, c, d, e, f and g (in order from faster to slower migration) were found in the embryo plus scutellum, while isozymes 1, 2, 3, 4, 5 and 6 (also from faster to slower migration) were peculiar of the endosperm. S. vavilovii has isozymes m, c₁, d, e, f and g in its embryo plus scutellum, and isozyme 2 in the endosperm. Segregation data indicated that at least 13 different loci would be controlling the peroxidase of S. cereale. Isozymes a and b are controlled by alleles of the same locus, all the other loci have one active and dominant allele coding for one isozyme, and other null and recessive allele. The estimation of linkage relationships shows that five endosperm loci are linked, and tentative maps are shown. A possible dosage effect and the existence of controlling gene(s) for endosperm isozyme 4 is reported. All these data and the high frequency of null alleles found are discussed in relation to recent reports.