Characterization of an Arabidopsis aminotransferase that participates in tryptophan metabolism and auxin homeostasis
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Abstract
The phytohormone indole-3-acetic acid (IAA) plays a critical role in regulating numerous aspects of plant growth and development. Biochemical evidence indicates that IAA can be synthesized both from tryptophan [Trp; Trp-dependent (D) pathways] and from an indolic precursor of Trp [Trp-independent (I) pathways]. At the genetic level, there is much support for Trp-D pathways, but there is little genetic evidence for Trp-I pathways.
Mutants with altered Trp-I IAA synthesis were identified from genetic screens using the model plant Arabidopsis thaliana. From these screens, the allelic iss1-1 and iss1-2 mutants were identified that displayed an indole-dependent IAA overproduction phenotype consisting of fused leaves and increased lateral and adventitious root growth. iss1 mutants appear wild type (WT) when grown on medium supplemented with Trp.
Using stable isotope labeling studies, iss1 and WT were both found to use primarily Trp-D synthesis when grown on unsupplemented medium. In contrast, iss1, but not WT, used primarily Trp-I synthesis, when grown on indole-supplemented medium. Surprisingly, iss1 seedlings also have a 170-fold increase in Trp when grown on indole, indicating that the increase in Trp-I IAA is not due to a loss of indole to Trp conversion but is suggestive of a role for ISS1 in Trp catabolism.
Using map-based cloning, ISS1 was identified as an uncharacterized aminotransferase that is distantly related to aromatic aminotransferase (AroATs). ISS1 is highly conserved across the plant kingdom; however none of these ISS-related genes have been characterized. To demonstrate that ISS1 is an AroAT, heterologous expression of the ISS1 cDNA was found to fully rescue the yeast aro8 aro9 double mutant that is defective in redundant AroATs needed for the production of phenylalanine (Phe) and tyrosine (Tyr) in yeast.
Based on the data presented in this thesis, the most likely role for ISS1 in planta is in Trp catabolism. However, the iss1 mutant also showed a decrease in the Phe-derived metabolite, coniferin, suggesting an additional role (either direct or indirect) for ISS1 in Phe metabolism. Although the precise role of ISS1 remains to be determined, these results presented provide genetic evidence for the existence of the Trp-I IAA pathway.