Characterization of an auxin- and abscisic acid-inducible reporter gene : Dc3-GUS in reported auxin mutants, and mutant screening based on auxin responsive Dc3-GUS expression

Auxin and abscisic acid (ABA) are both important plant hormones. Although many mutants have been isolated that are related with ABA and auxin, their signal transduction pathways in plant cells are still far from clear. Dc3 is a late embryogenesis-abundant (LEA) gene from carrot and the Dc3 promoter is ABA- and drought stress-inducible. In transgenic Arabidopsis seedlings, it has been shown that Dc3-GUS reporter gene is also auxin-inducible. To understand more about inducibility and expression pattern of Dc3-GUS , different concentrations of natural and synthetic auxins, IAA, NAA and 2,4-D, were used to induce transgenic wild type and ABA-insensitive 2 (abi2) mutant seedlings and GUS expression in roots was characterized. Abi2 mutants had clearly lower Dc3-GUS activity in the root zone of elongation and root hairs, but had the same GUS expression mode with wild type in response to auxin induction. In order to prove Dc3-GUS is a valid marker to show auxin-regulated gene expression, transgenic Arabidopsis wiId type plants were crossed with reported auxin mutants aux1-7, axr4-2 and rty. Homozygous auxin mutants were picked out and those with non-segregating GUS expression were used for Dc3-GUS expression study. It is shown in my work that Dc3 promoter was down-regulated in aux1 relative to wild type when induced by IAA and 2,4-D. axr4 also showed lower Dc3 expression level in response to 2,4-D. However, aux1 and axr4 both had similar Dc3-GUS expression to wild type when induced by NAA, and axr4 also had a wild-type response to IAA induction. These Dc3-GUS expression patterns in aux1 and axr4 are in accordance with their gene functions in auxin transport and transcription, respectively. In auxin-overproducing mutant rty, Dc3-GUS expression was much higher than wild type and exogenous auxin resulted in a super-induction of Dc3-GUS. Aux1 and axr4 mutants have a novel phenotype of down-regulated ABA-inducing Dc3-GUS expression, suggesting that ABA and auxin interact to regulate Dc3-GUS expression in Arabidopsis roots. Based on auxin-induced Dc3-GUS expression, mutant screening was performed in ethyl methane sulfonate (EMS)-treated M2 seedlings and mutants with over- or down-regulated Dc3-GUS expression (dor, ddr) were isolated. One mutant Dc3-over-regulated-2 (dor2) was shown genetically to be allelic to rty. These mutants have potential to contribute to understanding complex interactions of overlapping hormone signal pathways and their phenotypic and genetic characterization deserve advanced research