Auxin biosynthesis in pea : a characterisation of mutants

The optimisation of auxin concentrations, or auxin homeostasis, is modulated through several mechanisms including de novo biosynthesis, transport and inactivation, mainly conjugation and catabolism. These mechanisms are thought to work in concert to spatiotemporally regulate auxin content at a cellular and tissue level. The principal focus of this thesis will be on auxin biosynthesis and inactivation. At present, the indole-3-pyruvic acid (IPyA) pathway is the only fully characterised route for auxin biosynthesis. In the first step, the amino acid tryptophan (Trp) is converted to IPyA by members of the TRYPTOPHAN AMINOTRANSFERASE (TAA1/TAR) family. In the second step, IPyA is converted to indole-3-acetic acid (IAA) by the YUC family of flavin-containing monooxygenases (FMO). Members of the two gene families are widely distributed across plant species and the IPyA pathway is now considered to be the principal biosynthetic source of IAA, the main bioactive auxin in plants. However, the evidence in support for this route is primarily based on findings from Arabidopsis. Interestingly, previous data from Pisum sativum (garden pea) suggest that other auxin biosynthetic pathways may also be active in root tissue. These results expose the necessity of investigating the auxin biosynthesis pathway in a broader range of species. Loss-of-function mutants are invaluable tools to establish links between gene function, hormone content and morphological outputs. However, auxin-biosynthesis mutants are relatively rare in species other than Arabidopsis. Novel recessive mutants relating to the IPyA pathway, only recently available in pea, are the basis of this thesis. These comprise two alleles with disruptions in PsTAR1 (Pstar1-1 and Pstar1-2) and four alleles affecting PsYUC1 (the crispoid mutants crd-1 to crd-4). The principal focus of this thesis will be two-fold. First, to investigate the potential contributions that the IPyA pathway may have on auxin biosynthesis in pea by monitoring auxin precursors, conjugates and auxins content from several tissue types. Second, to examine the impacts that IPyA-derived auxin may have on pea morphology by characterising the phenotypes of the auxin mutants. In the first experimental chapter, the two Pstar1 mutant lines are isolated. No phenotypes were observed in the novel mutants and despite PsTAR1 being previously reported to be highly expressed in young pea seeds, the mutant analyses suggested that PsTAR1 is not required for auxin biosynthesis during early seed development. Complications experienced in the process of identifying the homozygous recessive mutants, lead to the hypothesis that a second gene, a duplicate of PsTAR1, nearly identical in sequence, is present in the pea genome, further suggesting the possibility of a fifth PsTAR member. In Chapter 3, the PsYUC1 enzyme structure is characterised using in silico techniques. Important domains and the potential binding residues of PsYUC1 are predicted. A comparative approach using the previously reported leaflet phenotypes of the crd alleles and the 3-dimentional structure of PsYUC1, a novel C-terminal motif is uncovered and proposed to be critical for the functioning of the pea enzyme. In Chapter 4, a survey of the crd phenotypes is conducted to investigate the influence of the PsYUC1 gene on pea development in general. Severe phenotypes are exhibited in the mutants demonstrating the importance of PsYUC1 for lateral organ formation and suggests a role in meristematic and/or primordial cell developmental modules. Supporting this, an auxin-reporter construct revealed that auxin activity is adversely affected in the mutants. However, IAA content while only mildly reduced in the apical bud, was not affected in several tissues despite the strong phenotypes. Interestingly, the reduction in IAA aspartate conjugate (IAAsp) content in all tissues tested, suggests that the rate of conjugation is a likely mechanism for auxin homeostasis. The last experimental chapter investigates potential explanations for the apparent decoupling of the crd phenotype and IAA content. Results indicated that IAAsp is the main conjugate in the shoot of pea with 2-oxoindole-3-acetic acid (oxIAA) not being detected. In addition, root growth assays refuted a previous proposition that IAAsp is biologically active per se in pea roots. Furthermore, the labile IAA precursor, IPyA, is demonstrated to break down non-enzymatically and to contribute substantially to the IAA pool post-extraction. Variations on well-established extraction protocols support the use of derivatisation for the stabilisation of labile compounds in order to reliably quantify auxin.