Functional Analyses of the Narrow Sheath and Ragged Seedling2 Mutations During Development of Maize Lateral Organs

University Of Georgia Research Foundation Inc, Athens GA

Investigators

Abstract

0213023 Scanlon Leaves are the fundamental organ of plant shoots, and develop via the morphogenetic activity of shoot apical meristems. Previous studies suggest that the precisely regulated balance of hormone concentration and hormone transport are key components in leaf organogenesis. Three mutations disrupting early events in maize leaf pattern formation will be exploited in biochemical and molecular genetic investigations probing the role of the plant hormone auxin during early stages in leaf development. The mutations narrow sheath1, narrow sheath2 (ns1, ns2) and ragged seedling2 (rgd2) all disrupt development of maize lateral organs, leading to narrow leaf and shortened stem phenotypes. Maize leaves contain at least two lateral compartments, the central and marginal domains. NS function is essential for recruitment of the marginal compartment, but is not required for the development of the central leaf domain. The ns mutant phenotype, which mimics IAA-overexpressing mutants, is controlled by the non-homologous, duplicate factor mutations ns1 and ns2. Narrow sheath 2 has been cloned, and encodes a NITRILASE that has been shown to function in the production of auxin. Transcripts of ns2 accumulate in an alternating pattern subtending the marginal compartment of maize leaf primordia. Mutations in the ragged seedling2 gene condition more extreme narrow leaf phenotypes, and may disturb development of both the central and the marginal domains. Four specific aims are proposed. 1) The NS2 RNA and protein accumulation patterns will be analyzed in all developing maize tissues by in situ hybridization and immunolocalization. NS2-specific polyclonal antibodies will be prepared. Of particular interest is the NS2 accumulation patterns in developing embryos and in tissues unaffected by the ns mutations. 2) The expression patterns of the auxin responsive Aux/IAA genes of maize will be analyzed by in situ hybridization in ns and nonmutant samples. Non-mutant apices will be treated with xogenous auxin transport inhibitors, and ns apices will be treated with auxin. These assays will test the hypothesis that the ns mutant phenotype is the result of defective auxin-mediated signalling in the ns margin domain. 3) The ns1-R mutation is linked to a genomic polymorphism at the aldehyde oxidase (ao) locus, which encodes an enzyme involved in IAA biosynthesis, and ns mutants aberrantly express AO transcripts. Reverse genetic mutagenesis of ao loci, transposon mutagenesis of new ns1 alleles, and cloning of ao genes from ns mutant plants will determine whether ns1 encodes an AO in maize. 4) Genetic and molecular analyses will test the hypothesis that RGD2 functions during early stages of maize leaf development. Double mutant analyses of rgd2 with ns and leafbladeless1, and the expression profiles of genes involved in early leaf development (ns2, terminal ear1) will be analyzed by in situ hybridization. Additional alleles of rgd2 will be obtained by transposon tagging, for use in attempts to clone the rgd2 gene.