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Auxin Conjugates in Arabidopsis: Identification, Formation and Genetic Analysis

Most of the IAA in plants is found not as the free acid, but conjugated to carbohydrates, amino acids or peptides, forms that are thought to be biologically inactive. These conjugates are hypothesized to play roles in IAA homeostasis via transport, protection from degradation, hydrolysis to release free hormone, and detoxification of non-physiological concentrations of hormone. However, there are few mutants whose phenotype might provide insights into these processes. We are studying IAA conjugate formation in Arabidopsis using a number of approaches. By HPLC purification and GC-MS analysis of extracts from large quantities of vegetative tissue (300 g), we have detected and positively identified three conjugates, IAA-Aspartate (IAA-Asp), IAA-Glutamate (IAA-Glu) and IAA-Valine (IAA-Val) among the 8 that we surveyed, suggesting that these are the most abundant a! mino acid conjugates of IAA in Arabidopsis vegetative tissue. In feeding studies that employed high levels of exogenous IAA (50-500 mM), we have observed the formation of IAA-Glucose, IAA-Asp, IAA-Glu and a novel IAA metabolite. We used mass spectrometry techniques to identify this compound as IAA-Glutamine (IAA-Gln) and confirmed this by comparison with IAA-Gln that we synthesized chemically. Conjugate formation in wild type is affected by light and temperature. Using a single leaf HPLC screen based on these studies, we have identified three mutants with altered capacity to form IAA conjugates. One mutant that we characterized in detail shows an 8-fold increase in the accumulation of IAA-Gln, and a slight decrease in IAA-Asp formation. The mutant's appearance and responses to light and temperature suggested a defect in photorespiration. Complementation analysis revealed that this mutant is allelic to gluS mutants that lack chloroplastic glutamate synthase ! and thus accumulate glutamine. Of the two additional mutants, one showed decreases in the formation of both IAA-Asp (7-fold) and IAA-Gln (19-fold), while the second showed a 3-fold decrease in the formation of IAA-Asp and a 3-fold increase in IAA-Gln.

auxin; Arabidopsis thaliana; IAA; indole-3-acetic acid; N-(indol-3-ylacetyl)-L-aspartic acid; N-(indol-3-ylacetyl)-L-glutamic acid; N-(indol-3-ylacetyl)-L-glutamine; N-(indol-3-ylacetyl)-L-valine; IAA-glucose

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