Function of Auxin-Binding Protein 1

这是一项补充奖励，用于继续研究生长素受体（生长素结合蛋白1，ABP1）在生长控制中的功能。这是一种不寻常的生长激素受体，不适合任何激素受体范例。它也具有不同寻常的细胞生物学特性，因为它主要存在于内质网腔内，但其作用部位在质膜的胞质外侧。拟议的工作将利用在先前资助期间准备的转化细胞、植物和DNA结构。它还结合了最近可用的突变植物。这三个项目是：1；拟南芥ABP1的cDNA将用于转基因植物的表达研究。由于几个原因，这种受体的组成性过表达是不可能的；因此，将生长素受体cDNA置于两种诱导启动子（tet诱导和水杨酸诱导）的控制下。转基因烟草和拟南芥植物的生产才刚刚开始。我们将在诱导表达ABP cDNA前后对T2植株进行表征，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，用玉米生长素受体cDNA转化玉米细胞，由组成型启动子驱动。发现了一个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细胞壁形态，表型复制了转移细胞，这是一种高度分化的植物细胞类型，参与溶质的快速运动。该细胞系为研究这一重要细胞类型的特性和生长素受体的功能提供了有价值的工具。这是首次获得均匀的转移细胞群体。这些细胞，尽管具有完全不同的形态（许多手指突出），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态中起作用。3. 与Pioneer种子公司合作，通过基于Mutator转座元件插入的反向遗传学方法发现了玉米ABP基因的突变等位基因。部分等位基因在4个不同的ABP基因中被测序。这些突变体将被表征以确定ABP基因的突变是否影响植物生长。已经种植了突变体，并将进行回交以确定表型是否与突变等位基因相关。迄今为止，细胞生物学和生物化学方法已被用于研究生长素受体的功能，而没有使用分子遗传学工具，如上述转基因和突变植物。虽然这在一段时间内是有成效的，但没有分子遗传工具，新的认识就无法推进。利用细胞生物学技术观察突变体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. ***