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.
Genetic transformation of creeping bentgrass mediated by Agrobacterium tumefaciens has been achieved. Embryogenic callus initiated from seeds (cv. Penn-A-4) was infected with an A. tumefaciens strain (LBA4404) harboring a super-binary vector that contained an herbicide-resistant bar gene driven either by the CaMV 35S promoter or a rice ubiquitin promoter. Plants were regenerated from 219 independent transformation events. The overall stable transformation efficiency ranged from 18% to 45%. Southern blot and genetic analysis confirmed transgene integration in the creeping bentgrass genome and normal transmission and stable expression of the transgene in the T1 generation. All independent transformation events carried one to three copies of the transgene, and a majority (60-65%) contained only a single copy of the foreign gene with no apparent rearrangements. We report here the successful use of Agrobacterium for the large-scale production of transgenic creeping bentgrass plants with a high frequency of a single-copy transgene insertion that exhibit stable inheritance patterns.
A tapetum-specific gene, RTS, has been isolated by differential screening of a cDNA library from rice panicles. RTS is a unique gene in the rice genome. RNA blot analysis and in situ hybridization indicates that this gene is predominantly expressed in the anther's tapetum during meiosis and disappears before anthesis. RTS has no introns and encodes a putative polypeptide of 94 amino acids with a hydrophobic N-terminal region. The nucleotide and deduced amino acid sequence of the gene do not show significant homology to any known sequences. However, a sequence in the promoter region, GAATTTGTTA, differs only by one or two nucleotides from one of the conserved motifs in the promoter region of two pollen-specific genes of tomato. Several other sequence motifs found in other anther-specific promoters were also identified in the promoter of the RTS gene. Transgenic and antisense RNA approaches revealed that RTS gene is required for male fertility in rice. The promoter region of RTS, when fused to the Bacillus amyloliquefaciens ribonuclease gene, barnase, or the antisense of the RTS gene, is able to drive tissue-specific expression of both genes in rice, creeping bentgrass (Agrostis stolonifera L.) and Arabidopsis, conferring male sterility to the transgenic plants. Light and near-infrared confocal microscopy of cross-sections through developing flowers of male-sterile transgenics shows that tissue-specific expression of barnase or the antisense RTS genes interrupts tapetal development, resulting in deformed non-viable pollen. These results demonstrate a critical role of the RTS gene in pollen development in rice and the versatile application of the RTS gene promoter in directing anther-specific gene expression in both monocotyledonous and dicotyledonous plants, pointing to a potential for exploiting this gene and its promoter for engineering male sterility for hybrid production of various plant species.
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.
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