Molecular Genetics of Root Hair Development in Arabidopsis

在配子体自交不亲和（GSI）的茄科植物中，s等位基因花粉识别和排斥是由单个遗传位点s控制的。如果花粉的单个s等位基因与雌蕊中的任何一个s等位基因匹配，则花粉被拒绝。传递了孢子体和配子体中s位点的等位基因构成信息。s位点的产物是这些信息的载体。在雌蕊中表达的s位点产物包括一系列等位核糖核酸酶（S-RNases）。研究表明，每个s等位基因编码一个不同的S-RNase。然而，尚不清楚这些差异如何导致s等位基因特异性识别。在雄性配子体（即花粉s）中尚未发现s等位基因特异性产物。本研究旨在了解s等位基因特异性识别和排斥的分子基础。第一步将是开发一个系统，其中s等位基因特异性识别和排斥可以被操纵。从烟叶中克隆的S-RNases将被用于向转基因植物中引入新的等位基因特异性。利用该表达系统，将有可能在体外操作克隆的S-RNase序列，并评估其对体内传粉行为的影响。作为转基因受体的植物材料的选择非常重要。N. alata的变换效率太低，无法产生必要数量的变换子。因此，转基因将被引入植物中，如烟叶（Nicotiana plumbaginifolia），并与白杨杂交，或转化为具有s等位基因特异性花粉排斥能力的F1杂种。在任何一种情况下，SI反应的固有特异性提供了良好的控制。麦克卢尔博士将寻求在转基因植物中引入或修改一种新的特异性。鉴定花粉- s产物是本提案的主要目标。提出了一种遗传与分子生物学相结合的方法。产生了两个s等位基因分离的F2大群体。一个给定s等位基因的纯合子将被合并。利用这些汇集的材料，减法杂交和差异mRNA显示将用于鉴定推定的花粉- s cDNA。GSI具有帮助理解植物细胞间通讯的潜力。为了实现其潜力，GSI研究必须进入分析阶段。本课题旨在通过开发S-RNases的等位基因特异性功能的直接分析系统，以及鉴定花粉中负责等位基因特异性识别的产物来实现这一目标。*** 409Schiefelbeintio $ W ' gg!植物细胞的形态发生主要是通过精确控制细胞分裂和细胞增大来实现的。这项工作的长期目标是分析单个遗传位点对植物细胞发育复杂过程的贡献。正在研究的模式系统是拟南芥根毛的形成。拟南芥根毛发育的完整过程可细分为以下几个阶段：(1)根表皮细胞命运定形；(2)表皮细胞基部最初形成凸起；(3)根尖生长使毛膨胀；(4)根毛成熟和根尖停止生长。在本研究中，我们将研究影响根毛细胞发育的两个阶段（细胞扩增和细胞命运规范）的基因。在拟议研究的一部分中，将克隆影响根毛细胞扩增的基因，并根据它们与农杆菌T-DNA的关联来确定其特征。已经从拟南芥的T-DNA群体中鉴定出了几种根毛突变体。其中之一在先前表征的位点RHD3中具有突变，并且已被用于分离RHD3基因的推定基因组和cDNA克隆。在该研究的另一个主要部分，影响根表皮细胞命运规范的基因将被表征和鉴定。拟南芥TTG基因，影响毛状体的形成和花青素的产生，已被证明影响根表皮细胞的命运。正常情况下，只有特定位置的表皮细胞才会形成毛发。然而，在ttg突变植物中，所有的根表皮细胞似乎都形成了毛，而在异位表达ttg同源物（玉米R基因）的拟南芥植物中，根表皮细胞上没有毛。这些研究将有助于确定影响根毛细胞形成过程中两个基本过程的基因，并将为控制植物细胞发育的分子机制提供见解。同源的，同源的!!!!!g g (Times New Roman Symbol & Arial & z P R U W e $ W - ' ' h c d d k + Dana Brigham Dana Brigham

9316152 McClure In solanaceous plants with gametophytic self-incompatibility (GSI), S-allele pollen recognition and rejection are controlled by a single genetic locus, the S-locus. Pollen is rejected if its single S-allele matches either S-allele in the pistil. Information is communicated about the allelic constitution of the S-locus in the sporophyte and in the gametophyte. The products of the S-locus are the carriers of this information. The S-locus products expressed in the pistil comprise a series of allelic ribonucleases, the S-RNases. It has been shown that each S-allele encodes a different S-RNase. However, it is not known how these differences lead to S-allele specific recognition. No S-allele specific product has yet been identified in the male gametophyte (i.e., pollen-S). This proposal seeks to develop an understanding of the molecular basis for S-allele specific recognition and rejection. The first step will be to develop a system in which S-allele specific recognition and rejection can be manipulated. Cloned S-RNases from Nicotiana alata will be used to introduce new allelic specificity into transgenic plants. Using the expression system, it will be possible to manipulate cloned S-RNase sequences in vitro and assess the effects on pollination behavior in vivo. The choice of plant materials for use as transgene recipient is very important. The transformation efficiency of N. alata is too low to generate the necessary number of transformants. Therefore, transgenes will be introduced into a plant such as Nicotiana plumbaginifolia and crossed into N. alata or into a transformable F1 hybrid ca pable of S-allele specific pollen rejection. In either case, the inherent specificity of the SI reaction provides excellent controls. Dr. McClure will look for the introduction of, or modification of, a new specificity in the transgenic plants. Identification of the pollen-S product is a major goal of this proposal. A combined genetic and molecular biological approach is proposed. A large F2 population segregating for two S-alleles has been generated. Plants homozygous for a given S-allele will be pooled. Using these pooled materials, subtractive hybridization and differential mRNA display will be used to identify a putative pollen-S cDNA. GSI has the potential to help understand plant cell-cell communication. To realize its potential, GSI research must move into an analytical phase. This proposal aims to accomplish this by developing a system for direct analysis of the allelic specificity function of S-RNases, and identifying the product responsible for allele specific recognition in pollen. *** 409Schiefelbeintio $ W ` e g g ! F The morphogenesis of plant cells is primarily achieved by the precise control of cell division and cell enlargement. The long-term objective of the proposed work is to analyze the contribution of single genetic loci to the complex process of plant cell development. The model system being studies is the formation of root hairs in Arabidopsis thaliana. The complete process of Arabidopsis root hair development can be subdivided into several phases: (1) root epidermal cell fate specification, (2) the initial formation of a bulge at the base of the epidermal cell, (3) the expansion of the hair by tip growth, and (4) the maturation of the hair and cessation of tip growth. In this proposal, genes will be studied that appear to influence two of these stages of root hair cell development (cell expansion and cell fate specification). In one part of the proposed research, genes affecting root hair cell expansion will be cloned and characterized by virtue of their association with the T-DNA of Agrobacterium. Several root hair mutants have already been identified from T-DNA populations of Arabidopsis. One of these possesses a mutation in a previously-characterized locus, RHD3, and it has been used to isolate putative genomic and cDNA clones of the RHD3 gene. In the other major part of the proposed research, genes that affect cell fate specification in the root epidermis will be characterized and identified. The Arabidopsis TTG gene, which affects trichome formation and anthocyanin production, has been shown to influence the fate of root epidermal cells. Normally, only epidermal cells in particular positions will form hairs. However, it ttg mutant plants, all root epidermal cells appear to form hairs, and in Arabidopsis plants that ectopically express a putative TTG homolog (the maize R gene), no hairs form on the root epidermal cells. These studies will servt to define genes that affect two of the basic processes that occur d uring the formation of root hair cells, and they will provide insights into the molecular mechanisms that control cell development in plants.homologueserve W W R W ! ! ! ! ! ! g g ( Times New Roman Symbol & Arial & z P R U W e $ W - ` " h c d d k + Dana Brigham Dana Brigham