Functional analysis of an auxin-responsive gene and its effects on plant development and global transcriptional profiles
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Auxin mediates multiple aspects of plant growth and development. Auxin up-regulated genes have been studied to assess their role(s) in auxin-regulated plant growth and development. Aux/IAA genes are the most studied auxin-regulated genes. Axr3-1 mutation results in an amino acid substitution in Domain II of the encoded IAA17 protein. To gain insight into how the Domain IV mutation of axr3-1, axr3-1R4, overcomes the severe pleiotropic phenotype caused by the Domain II mutation, effects on the expression of a large number of auxin-responsive genes were evaluated, and several protein-protein interactions of mutant proteins were compared to WT. The axr3-1 mutant exhibited reduced message levels of auxin up-regulated genes, while the message levels in axr3-1R4 were similar to those of WT. The revertant protein, axr3-1R4, showed no protein-protein interaction with other Aux/IAAs, with ARFs, or with itself. The axr3- 1 protein exhibited the same protein-protein interactions as that of the WT protein. To understand the function of IAA17, IAA17 and IAA19 knockouts and axr3-1R4 were analyzed in more detail. The IAA17 gene was expressed mainly in root tissue based on both Northern analysis and IAA17 promoter-driven GUS expression in transgenic Arabidopsis plants. A double knockout mutant of IAA17/IAA19 exhibited WT-like phenotypes except that early stage roots had longer root hairs with shorter root cell size than those of the single knockouts, suggesting a synergistic effect of the two genes in early root development. Affymetrix ATH1 GeneChips were used to produce global transcriptional profiles of WT, axr3-1, axr3-1R4, and IAA17K plants. A total of 524 genes were up- or down-regulated in axr3- 1 compared to WT. Expression of relatively fewer genes was changed in axr3-1R4 and IAA17K compared to axr3-1, correlating with phenotypes of the revertant and the knockout. The global transcriptional patterns of WT and axr3-1 were used to evaluate the relationship of auxin and light in their effects on plant development. A total of 169 genes were consistently up-regulated by auxin in etiolated WT seedlings. Auxin up-regulated genes were repressed by light, and auxin down-regulated genes were induced by light.