Molecular Genetics of Amino Acid Biosynthesis in Arabidopsis

点击翻译按钮获取中文摘要

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.