Suppressors of EIR1: elements of a root-specific auxin signal transduction cascade

During the past years Arabidopsis thaliana has been widely used as experimental organism in advancing our understanding of the biology of plants. The most exciting progress in the regulation of plant development has been made by the identification of informative mutations in order to assign functions to genes that play a specific role in plant growth and development. Plant hormones are quite different in structure and in action from animal hormones, but despite decades of effort, the mechanisms of their actions remain unknown. The plant hormone auxin is central to the regulation of cell division, cell elongation and differentiation. In an effort to identify genes involved in auxin signal transduction, a collection of auxin-related mutants have been isolated from Arabidopsis. Phenotype analysis of one of these mutants, called EIRl, demonstrated that the EIR1 gene is required for auxin transport. The defect in auxin transport is manifested by reduced auxin levels in the root. Hence, the EIRl plants develop longer roots which are defective in tropic responses. This project aims to use the EIR1 mutant to isolate further components of the auxin signal transduction pathway. For this purpose the EIRl plants will be used for a second round of mutagenesis. It is expected that suppressors of EIRl result in the reversion of the root phenotype. Suppressors exhibiting an auxin response phenotype in the absence of auxin in EIRl background therefore are prime candidates for being involved in auxin mediated regulation of root growth. During a second round of screening the putative EIRl suppressors will be tested for their auxin response by crosses to several auxin up-regulated reporter lines. In order to facilitate the subsequent cloning and characterisation of EIRl suppressors, gene tagging will be used. Ac transposon mutagenesis will be performed for the isolation of loss of function and T-DNA activation tagging for gain of function mutants, respectively. Characterisation and integration of new genes into the already existing framework of auxin signal transduction will be a major contribution toward the understanding of auxin involvement in plant development and adaptation processes.

Exciting progress has been made by the identification of informative mutations in the model plant Arabidopsis thaliana in order to assign functions to genes that play a specific role in plant growth and development. The plant hormone auxin is central to the regulation of cell division, cell elongation and differentiation. In an effort to identify genes involved in auxin signal transduction, a collection of auxin-related mutants were isolated. Phenotypic analyses of one of these mutants, called eir1, demonstrated that the EIR1 gene is required for auxin transport. To identify further components of the auxin signal transduction pathway a genetic trick was used and eir1 plants were mutagenized a second time. Suppressors of eir1 phenotypes, i.e. mutants exhibiting an auxin response phenotype in the absence of auxin are prime candidates for being involved in auxin mediated signal transduction. About 14.000 mutated plants were generated and screened. After further characterisation of 200 candidates four were analysed on a molecular level. The first gene was involved in the regulation of the quantity of free auxin. The second gene indicates that auxin and a second plant hormone, cytokinin, influences the chromatin structure in cells which ceases proliferation and start to differentiate. Further support that auxin signal transduction is coupled with other regulatory pathways came from the isolation of the third gene. This gene demonstrates that auxin together with sucrose is influencing flowering time. Mutants which accumulate sucrose and starch resulted in plants with an extreme late flowering phenotype. The last gene that was isolated within the project turned out to be a plant specific new protein which is involved in the regulation of cell expansion. The four isolated genes illustrate exemplary at which molecular levels auxin is regulating plant morphogenesis. The project proofed that the chosen methodology was successful and provides the fundament for the isolation and characterisation of further genes which are regulating morphogenesis in plants - a prerequisite for the adaptation processes in a changing environment.