Molecular Genetics of Amino Acid Biosynthesis in Arabidopsis

目的是确定植物激素生长素（IAA）促进植物趋向性的信号转导途径。在地心引力说中，根向地心弯曲。极地运输假说假设有一个外排转运体将IAA从根尖泵到离地球中心最近的根部。在那里，IAA抑制了细胞的伸长，而另一边的细胞则伸长，结果是根向下弯曲，并沿着路线返回。对该突变体eirl和EIR1基因的分析表明，它是由极性输运模型预测的泵。仅在根中表达的EIR1蛋白与一些细菌外排泵相似。已经设计了实验，为EIR1在极性输运中的作用提供直接证据。值得注意的是，携带植物EIR1基因的酵母细胞对某些抑制剂具有抗性，这可能是因为它们会排出这种化合物。在酵母菌中会产生增强和抑制分泌的EIRl基因突变体，这一系统为探索植物中的这一途径提供了独特的工具。通过确定EIR1在根细胞质膜中是否存在不对称定位，来检验EIR1在IAA从根尖向伸长区不对称运输中的作用。对eirl的抑制因子和增强因子的遗传筛选应该能识别出转导向地倾向信号的上游和下游基因。这些抑制因子将允许构造向地性信号转导通路。对EIR相似物的其他家族成员的分子分析将确定这些是否是IAA泵负责生长素对其他组织趋向性的影响。这些基因将被克隆，克隆的基因将被用作各种插入文库的探针，以识别突变体。此外，在这些EIR1类似物中含有显性负突变的转基因植物（在酵母试验中得到验证）将用于构建转基因植物，然后分析其趋向性缺陷。在etrl-6 ein4-4双突变体中，通过激活标记法鉴定叶片扩增突变体，研究细胞扩增。Etrl-6 ein4-4菌株比野生型细胞扩增较少，这允许鉴定叶片较大或较小的突变体。分析叶表皮细胞的细胞大小可以区分是细胞扩增缺陷还是细胞分裂缺陷。利用从该筛选中获得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ALF4的基因。内源和外源IAA均可阻断alf4突变的侧根形成。此外，本文还提出了一种新的抗高盐转基因植物的设计方法。

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.