The biosynthesis of two naturally-occurring auxins in Pisum sativum L.

Auxins are a group of phytohormones, including indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-Cl-IAA), that are involved in many aspects of plant growth, including phototropism, gravitropism, shoot elongation, root initiation and flowering. The structure of the primary auxin‚ÄövÑvÆIAA‚ÄövÑvÆwas determined over 70 years ago, but the biosynthesis of this molecule, and the other naturally-occurring auxins, has remained unclear. Five pathways have been proposed for IAA biosynthesis, one tryptophan (Trp)-independent pathway and four Trp-dependent pathways. Each of the Trp-dependent pathways is named after its primary intermediate, giving rise to the indole-3-acetaldoxime, tryptamine, indole-3-acetamide and indole-3-pyruvic acid pathways. At the outset of this research, the evidence for each of these pathways was incomplete, even in the model organism Arabidopsis thaliana. This thesis reports the determination of the primary auxin biosynthesis route in the seeds of Pisum sativum (pea). The indole-3-acetaldoxime pathway was not investigated, as it has been shown previously that pea lacks the necessary enzymes for this pathway and is devoid of endogenous indole-3-acetaldoxime. First, the tryptamine pathway was examined. The mass spectral characterisation of authentic N-hydroxytryptamine (synthesised in our laboratory), a reported intermediate in the tryptamine pathway and the alleged in vitro product of the YUCCA enzymes, cast serious doubts over the role of this compound in auxin biosynthesis. It was also shown, through injection of labelled `[D_4]Trp` and \\([D_5]tryptamine\\), that tryptamine is not converted to IAA in pea seeds, indicating that this pathway does not function in these organs. This left three possible pathways to auxin. Through in vivo feeding experiments, the Trp-independent and the indole-3-acetamide pathways were eliminated as possibilities in pea seeds. The remaining pathway, the indole-3-pyruvic acid pathway, was investigated using a number of lines of enquiry. First, the functional activity of two Trp aminotransferase genes from pea (PsTAR1 and PsTAR2) was investigated using purified recombinant PsTAR1 and PsTAR2. This showed that these proteins convert Trp to indole-3-pyruvic acid and 4-chlorotryptophan to 4-chloroindole-3-pyruvic acid. A standard of the latter compound was synthesised in our laboratory, for comparison with the enzyme product. Secondly, feeding studies using \\([^{13}C_6]IAA\\) and \\([D_5]Trp\\) indicated that Trp is converted to 4-Cl-Trp, which is then converted to 4-Cl-IAA. Taken together, the evidence presented in this thesis supports parallel biosynthesis of IAA and 4-Cl-IAA by the IPyA pathway and its chlorinated version.