A comparison of the biosynthesis of indole-3-acetic acid and phenylacetic acid

The biosynthesis of the auxin, indole-3-acetic acid (IAA), has been well studied in recent years. It is now accepted that the indole-3-pyruvate (IPyA) pathway is the main IAA biosynthetic route and is responsible for the majority of IAA produced in plants. This pathway involves the transamination of the amino acid, tryptophan (Trp), to IPyA by the TAR (TRYPTOPHAN AMINOTRANSFERASE RELATED) enzymes, followed by the oxidative decarboxylation of IPyA to IAA by the YUCCA proteins. Recently it has been suggested that the TARs and the YUCCAs are also responsible for the biosynthesis of a less well-studied auxin, phenylacetic acid (PAA). Specifically, it is proposed that the TARs convert a second amino acid, phenylalanine (Phe), to phenylpyruvate, and that the YUCCAs convert phenylpyruvate to PAA. The work in this thesis addresses several aspects of the biosynthesis of PAA. Using isotopically labelled compounds, several techniques were developed and adapted to demonstrate isotopic labelling of putative PAA precursors. It is reported herein that Phe is metabolised to both phenylpyruvate and PAA in homogenised plant extracts as well as in intact plant systems, consistent with previous hypotheses. Additional experiments also demonstrate that the putative intermediate, phenylpyruvate, can be converted to PAA, as well as back to Phe. These experiments couple the biosynthesis of amino acids (Phe) and auxins (PAA), which appear to share a common mechanism. The aromatic aminotransferases (ArATs) have been characterised in several genera and catalyse the reversible conversion of Phe to phenylpyruvate. Here, an ArAT sequence is isolated from the pea gene atlas as a candidate enzyme for the biosynthesis of PAA. However, investigations into IAA biosynthetic mutants demonstrate that the TARs and the YUCs do not contribute significantly to PAA biosynthesis in pea, maize or Arabidopsis. These mutants display significantly altered IAA (or IPyA) levels, but the endogenous levels of PAA (or phenylpyruvate) are not different from their respective WTs. It is suggested that the conversion of Phe to PAA, via phenylpyruvate, may be functional in plants but is not catalysed by the enzymes of the IPyA pathway. Analyses of the distribution of IAA and PAA in major land plant divisions suggest that both auxins are present in all land plants. Applying the above-mentioned metabolism experiments also reveals that the IPyA and phenylpyruvate pathways appear to be functional in all land plants. These findings are supported by genetic evidence from several gene databases, which demonstrate that the TARs, YUCs and ArATs are present in a range of these species. Additionally, investigations on the major IAA metabolic genes, the DAOs, the GH3s and the UGTs, reveals unique distributions across the land plants and suggests that the regulation of IAA content in derived species (e.g. angiosperms) is stronger than in members of basal lineages. Finally, this thesis describes further characterisation of the bushy mutant of pea, which has reduced IAA and IPyA levels as well as a small reduction in endogenous phenylpyruvate level. In this thesis, the bushy locus is also mapped to the top of pea linkage group 1 where it is closely linked with (but not identical to) the genes encoding the aldehyde oxidases. The culmination of this thesis demonstrates that the IAA biosynthetic machinery does not function extensively in the biosynthesis of PAA and that the two auxins have unique biosynthetic pathways that are conserved throughout the land plants.