Auxin biosynthesis in Pisum sativum : a physico-chemical perspective

There are a number of putative tryptophan-dependent pathways leading to the bioactive auxin, indole-3-acetic acid (IAA) in plants, although none of these is fully characterised in terms of the isolation of genes for enzymatic reactions, and identification and quantification of precursor compounds. Herein, the tryptamine pathway (tryptophan – tryptamine - indole-3-acetaldehyde - IAA), the indole-3-pyruvic acid pathway (tryptophan - indole-3-pyruvic acid- IAA) and the indoleacetamide pathway (tryptophan – indoleacetamide - IAA) to IAA are examined in Pisum sativum (pea) using a compound-based approach, detecting potential precursors by gas chromatography/tandem-mass spectrometry (GC/MS/MS) and ultra performance liquid chromatography/mass spectrometry (UPLC/MS), in order to complement the genetic findings of others. Synthesised deuterated forms of the intermediates involved have been used to quantify the endogenous compounds, and to investigate their metabolic fates. Methods for the isolation and quantification of the following compounds were devised and tested. Tryptophan, tryptamine, indole-3-acetaldehyde (IAAld), indoleacetamide, indole-3-ethanol (IEt) and IAA were detected as endogenous constituents, whereas indole-3-acetaldoxime and one of its products, indole-3-acetonitrile, were not detected. Metabolism experiments indicated that the tryptamine pathway to IAA in pea roots proceeds in the sequence tryptophan – tryptamine – IAAld – IAA, with IEt as a side-branch product of IAAld. The endogenous levels of IAA precursors, along with their abundance in tissue types, of a low-auxin mutant (bushy) have also been studied in order to characterise the mutation, and to assess the involvement of different IAA biosynthesis pathways. The effects of wounding, a synthetic auxin and an inhibitor of auxin action on the regulation of the biosynthesis pathways have also been studied. It appears that the tryptamine pathway is operative in pea roots; however, it cannot be ruled out that other tryptophan-dependent pathways to IAA are also operative, nor can we rule out the possibility of a tryptophan-independent pathway operating in these organs