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

叶片是植物新梢的基本器官，通过新梢顶端分生组织的形态发生活动而发育。以往的研究表明，激素浓度和激素运输的精确调节平衡是叶片器官发生的关键组成部分。在生化和分子遗传学研究中，将利用三个破坏玉米叶片模式形成早期事件的突变，探索植物激素生长素在叶片发育早期阶段的作用。窄鞘1、窄鞘2(ns1、ns2)和粗籽粒2(Rgd2)突变均扰乱了玉米侧器官的发育，导致叶片变窄，茎变短。玉米叶片至少包含两个侧向隔室，即中央区域和边缘区域。NS功能对边缘区室的补充是必需的，但对中心叶域的发育不是必需的。Ns突变表型模仿IAA过表达突变体，由非同源的重复因子突变NS1和NS2控制。窄鞘2已经被克隆，它编码了一个NITRILASE，已经被证明在生长素的产生中起作用。NS2的转录本以交替的方式积累，位于玉米叶原基的边缘隔室。参差不齐的种子2基因突变使叶片的表型更加狭窄，并可能干扰中央和边缘区域的发育。提出了四个具体目标：1)通过原位杂交和免疫定位技术，分析NS2、RNA和蛋白质在所有发育过程中玉米组织中的积累模式。将制备NS2特异性多克隆抗体。特别令人感兴趣的是NS2在发育中的胚胎和组织中的积累模式，不受ns突变的影响。2)玉米生长素响应AUX/IAA基因的表达模式将通过原位杂交在ns和非突变样本中进行分析。非突变的顶端将用外源生长素运输抑制剂处理，而ns的顶端将用生长素处理。这些分析将检验这一假设，即ns突变表型是ns边缘区域生长素介导的信号缺陷的结果。3)NS1-R突变与乙醛氧化酶(Ao)基因座的基因组多态性有关，该基因编码参与IAA生物合成的一种酶，ns突变突变异常地表达AO转录本。Ao基因座的反向遗传突变、新的NS1等位基因的转座子突变以及来自ns突变株的ao基因的克隆将决定NS1是否编码玉米的Ao基因。4)遗传和分子分析将验证RGD2在玉米叶片发育早期发挥作用的假说。Rgd2与ns和无叶1的双突变分析，以及与叶片早期发育相关的基因(NS2，末端ear1)的表达谱将通过原位杂交进行分析。Rgd2的其他等位基因将通过转座子标签获得，用于尝试克隆rgd2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.