Function of Auxin-Binding Protein 1

9514306琼斯这是一个补充奖，旨在继续研究生长素受体(生长素结合蛋白1)在生长控制中的功能。这是一种不同寻常的生长激素受体，不符合任何激素受体模式。它也有不同寻常的细胞生物学，主要分布在内质网腔中，但作用部位在质膜的胞外一侧。这项拟议的工作将利用在前一次拨款期间准备的转化细胞、植物和DNA结构。它还包含了最近出现的突变植物。这三个项目是：1.拟南芥ABP1基因的cDNA将用于转基因植物的表达研究。由于多种原因，生长素受体不可能结构性过表达；因此，生长素受体基因被置于两个可诱导启动子(tet可诱导启动子和水杨酸可诱导启动子)控制下。转基因烟草和拟南芥植株的生产现在才刚刚开始。在ABP基因被诱导表达之前和之后，将对T2植株进行鉴定，以确定该激素受体过表达对植物生长发育的影响。2.与CIBA-Geigy合作，将玉米生长素受体基因转化到玉米细胞中。发现一株高水平表达生长素受体的细胞系。这些细胞具有不同寻常的壁形态，表现为转移细胞，这是一种高度分化的植物细胞类型，参与了溶质的快速移动。该细胞系是研究这一重要细胞类型的特性和生长素受体功能的有价值的工具。这是第一次获得均质的转移细胞群体。尽管这些细胞具有截然不同的形态(许多手指突起)，但似乎具有相同的细胞壁聚合物组成。目前正在进行研究，以确定不同的细胞壁形态是如何产生的，以及ABP1是否在这种转移细胞壁形式中发挥作用。3.利用突变体转座子插入的反向遗传学方法，与先锋种子合作，发现了玉米ABP基因的突变等位基因。对部分等位基因进行了测序，发现它们分别位于4个不同的ABP基因中。将对这些突变体进行鉴定，以确定ABP基因突变是否会影响植物生长。突变体已经被种植，并将进行回交，以确定表型是否与突变等位基因相关。到目前为止，细胞生物学和生化方法已经被用来研究生长素受体的功能，而没有上述转基因和突变植物等分子遗传学工具。虽然这已经有了一段时间的成果，但如果没有分子遗传学工具，新的理解是不可能推进的。用细胞生物学技术观察到突变和转基因植物中生长素受体功能的扰动，将阐明这种不寻常的激素受体控制植物细胞生长和形态的机制。***

9514306 Jones This is a supplemental award to continue work on projects which address the function of the auxin receptor (Auxin-Binding Protein 1, ABP1) in growth control. This is an unusual growth hormone receptor that does not fit any hormone receptor paradigm. It also has unusual cell biology in that it is found predominantly in the endoplasmic reticulum lumen but has its site of action on the extracytoplasmic side of the plasma membrane. The proposed work will utilize transformed cells, plants, and DNA constructs prepared during the prior funding period of the grant. It also incorporates mutant plants that recently became available. The three projects are: 1. The cDNA for the Arabidopsis ABP1 will be used for trangenic plant expression studies. For several reasons, constitutive overexpression of this receptor is not possible; therefore, the auxin receptor cDNA was placed under the control of two inducible promoters (tet inducible and salicylic acid inducible). production of transgenic tobacco and Arabidopsis plants are just now being made. T2 plants will be characterized before and after the ABP cDNA is inducibly expressed to determine the effect of overexpression of this hormone receptor on plant growth and development. 2. In collaboration with CIBA-Geigy, maize cells were transformed with the maize auxin receptor cDNA driven by a constitutive promoter. One cell line expressing the auxin receptor at high levels was found. These cells had an unusual wall morphology that phenocopied transfer cells, a highly differentiated plant cell type that is involved in rapid movement of solutes. This cell line is a valuable tool to study the properties of this important cell type and the function of the auxin receptor. It is the first time a homogeneous population of transfer cells has been obtained. These cells, despite having a drastically different morphology (numerous finger projections), appear to have the same cell wall polymer composition. Studies are underway to determine how different wall morphology can be generated and if ABP1 has a role in this transfer cell wall form. 3. In collaboration with Pioneer seeds, mutant alleles of the maize ABP gene were found by a reverse genetics approach based on Mutator transposable element insertions. Some of the alleles were sequenced and shown to be in 4 different ABP genes. These mutants will be characterized to determine if the mutation in the ABP gene affects plant growth. Mutants have been planted and will be backcrossed to determine if the phenotype correlates with the mutant allele. To date, cell biological and biochemical approaches have been used to study the function of the auxin receptor without molecular genetic tools such as the transgenic and mutant plants described above. While this has been productive for a while, new understanding can not be advanced without molecular genetic tools. Perturbations on the function of the auxin receptor in the mutant and transgenic plants, observed using cell biological techniques, will elucidate the mechanism of this unusual hormone receptor in controlling plant cell growth and morphology. ***