Molecular Genetics of Amino Acid Biosynthesis in Arabidopsis

目的是确定植物激素生长素（IAA）促进植物向性的信号转导途径。在向地性中，根向地心弯曲。极性运输假说，假定一个外排转运蛋白，泵IAA从根尖到最接近地球中心的根的一侧。在那里，IAA抑制细胞的伸长，而在另一侧的细胞伸长，结果是根向下弯曲并返回路线。 对agravitropic突变体eirl和EIR 1基因的分析表明，它是由极性运输模型预测的泵。EIR 1蛋白仅在根中表达，与某些细菌外排泵相似。已经设计了实验来提供EIR 1在极性运输中的功能的直接证据。值得注意的是，携带植物EIR 1基因的酵母细胞对某些抑制剂具有抗性，大概是因为它们能泵出化合物。增强和抑制effiux的EIRl基因突变体将在酵母中产生，该系统提供了一种独特的工具来探测植物中的这一途径。EIR 1在IAA从根尖到伸长区的不对称运输中的作用将通过确定EIR 1在根细胞质膜中是否存在不对称定位来测试。对eirl抑制子和增强子的基因筛选应该确定传递向地性信号的上游和下游基因。对EIR旁系同源物的其他家族成员的分子分析将确定这些是否是负责生长素对其他组织向性影响的IAA泵。这些基因将被克隆，并且克隆用作各种插入文库的探针以鉴定突变体。此外，将使用在这些EIR 1旁系同源物中含有显性负突变的转基因植物（在酵母测定中验证）来构建转基因植物，然后分析其向性缺陷。还将通过在eIR 1 -6 ein 4 -4双突变体中通过激活标签法鉴定叶片扩展突变体来研究细胞扩展。与野生型相比，EIN 4 -4菌株具有较少的扩增细胞，这允许鉴定具有更大或更小叶片的突变体。分析叶表皮细胞的细胞大小将区分缺陷是在细胞扩张还是在细胞分裂中。利用筛选得到的突变体构建细胞扩增的遗传途径。其他旨在描述侧根形成途径的实验包括克隆编码ALF 4的基因。alf 4突变阻断内源和外源IAA引起的侧根形成。此外，描述了一种新的方法来工程化抗高盐的转基因植物。

The goal is to identify the signal transduction pathway(s) by which the plant hormone auxin (IAA) promotes plant tropisms. In gravitropism, the root bends towards the center of the earth. The polar transport hypothesis, posits an efflux transporter that pumps IAA up from the root tip to the side of the root closest to the center of the earth. There, the IAA inhibits the elongation of the cells, whereas those on the opposite side elongate, with the result that the root bends down and navigates back on course. Analysis of the agravitropic mutant, eirl, and the EIR1 gene suggests that it is the pump predicted by the polar transport model. The EIR1 protein, expressed only in the root has similarities to some bacterial efflux pumps. Experiments have been designed to provide direct evidence for the function of EIR1 in polar transport. Remarkably, yeast cells carrying the plant EIR1 gene are resistant to some inhibitors presumably because they pump out the compound. Mutants of the EIRl gene that enhance and inhibit effiux will be generated in yeast, a system which provides a unique tool to probe this pathway in plants. The role of EIR1 in asymmetric transport of IAA from the root tip to the elongation zone will be tested by determining whether there is asymmetric localization of EIR1 in the plasma membrane of root cells. Genetic screens for suppressors and enhancers of eirl should identify the upstream and downstream genes that transduce the signal for gravitropism. These suppressors will permit construction of the gravitropism signal transduction pathway.Molecular analysis of the other family members of EIR paralogs will determine whether these are the IAA pumps responsible for the effects of auxin on tropisms in other tissues. These genes will be cloned, and the clones used as probes for various insertion libraries to identify mutants. In addition transgenic plants containing dominant negative mutations in these EIR1 paralogs (validated in the yeast assay), will be used to construct transgenic plants, which will then be analyzed for tropism defects.Cell expansion will also be studied by identifying leaf expansion mutants via the activation tagging method in an etrl-6 ein4-4 double mutant. etrl-6 ein4-4 strains have less expanded cells than wild type, which permits the identification of mutants with both larger or smaller leaves. Analysis of the cell size of leaf epidermal cells will distinguish whether the defect is in cell expansion or cell division. A genetic pathway for cell expansion will be constructed using the mutants obtained from this screen.Other experiments designed to delineate the pathway of lateral root formation involve cloning the gene encoding ALF4. alf4 mutations block lateral root formation both by endogenous and exogenous IAA. In addition, a novel approach is described to engineer transgenic plants resistant to high salt.