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

叶是植物芽的基本器官，并且通过芽顶端分生组织的形态发生活性发育。 以往的研究表明，精确调节激素浓度和激素运输的平衡是叶器官发生的关键组成部分。 三个突变破坏玉米叶型形成的早期事件将被利用在生物化学和分子遗传学研究探索植物激素生长素在叶发育的早期阶段的作用。 窄鞘1、窄鞘2（ns 1、ns 2）和参差不齐的幼苗2（rgd 2）突变都破坏玉米侧生器官的发育，导致窄叶和短茎表型。 玉米叶至少含有两个侧区室，即中央区和边缘区。 NS的功能是必不可少的边缘室的招聘，但不是必需的中央叶域的发展。ns突变体表型，它模仿IAA过表达突变体，是由非同源，重复因子突变ns 1和ns 2控制。 窄鞘2已被克隆，并编码一种硝化酶，该酶已被证明在生长素的产生中起作用。 ns 2转录本以交替模式积累，对着玉米叶原基的边缘室。 在ragged seedling 2基因条件下的突变更极端的窄叶表型，并可能干扰中央和边缘域的发展。 提出了四个具体的目标。1）通过原位杂交和免疫定位分析所有发育中的玉米组织中的NS 2 RNA和蛋白质积累模式。 将制备NS 2特异性多克隆抗体。 特别感兴趣的是NS 2在发育中的胚和未受ns突变影响的组织中的积累模式。2）将通过原位杂交在ns和非突变样品中分析玉米生长素响应Aux/IAA基因的表达模式。 非突变体的顶端将用外源生长素转运抑制剂处理，ns顶端将用生长素处理。 这些分析将测试的假设，即ns突变体表型是有缺陷的生长素介导的信号传导的结果在ns边缘域。3）ns 1-R突变与基因组多态性在醛氧化酶（ao）基因座，编码一种酶参与IAA的生物合成，和ns突变体异常表达AO成绩单。 ao基因座的反向遗传诱变、新ns 1等位基因的转座子诱变以及从ns突变体植物中克隆ao基因将确定ns 1是否编码玉米中的AO。4）遗传和分子分析将检验RGD 2在玉米叶片发育的早期阶段起作用的假设。 rgd 2与ns和leafbladeless 1的双突变体分析，以及参与早期叶发育的基因（ns 2，末端ear 1）的表达谱将通过原位杂交进行分析。 rgd 2的其他等位基因将通过转座子标记获得，用于尝试克隆rgd 2基因。

0213023ScanlonLeaves 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 mutantphenotype, 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 intissues 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.