Brassinosteroids & hormone interactions in pea

Brassinosteroids (BRs) are now widely recognised as essential regulators of plant development. Our knowledge of BR biosynthesis and signalling has increased dramatically in the last decade, largely due to the characterisation of BR mutants. The characterisation of a novel BR mutant and the use of BR mutants to study the interactions between BRs and the 'classical' hormones were the primary aims of this thesis. The Ike mutant had previously been classified in our laboratory as a putative BR mutant. Here it is shown that the Ike mutation blocks BR biosynthesis after 6-deoxocastasterone (6-DeoxoCS). This was determined by BR quantification, and by metabolism and application studies in Ike plants. The block in BR biosynthesis after 6-DeoxoCS was confirmed by sequence analysis of Ike, which revealed a mutation in the start codon of the PsBR6ox6 (CYP85A6) gene, one of two genes controlling the oxidation of 6-DeoxoCS in pea. Previously characterised BR mutants (1k, Ike and 1kb) were used to examine interactions between BRs and other hormone groups. Studies involving BRs and gibberellins (GAs) showed that the level of GA₂₀ (the immediate precursor of the bioactive GA₁, GA₁) was consistently higher in all shoot tissues of BR mutants, compared with the wild-type (WT). Furthermore, the application of brassinolide (BL) to Ikb plants reduced GA₂₀ levels, and the conversion of GA₁₉ to GA₂₀ was reduced in epi-BL-treated Ikb plants. These results indicate that BRs negatively regulate GA₂₀ levels in pea. However, this did not consistently result in altered levels of GA₁. In addition, the effect of altered GA₁, levels on BR levels was examined. Changing GAi levels, by application studies or by the use of GA mutants, had no effect on BR content. Together, these results indicate that the effect of BR levels on GA levels (and vice versa) in pea is probably not of major biological significance. Auxin transport and production were examined in more detail before investigating the possibility of an interaction between IAA and BRs. Results showed that leaves are required to maintain normal IAA content in the plant, as defoliation significantly reduced IAA levels in the apical bud and internodes. Evidence is also presented that mature leaves are capable of IAA biosynthesis in their own right. Together, these results support the previously published idea that mature leaves are likely to be a major contributor of IAA present in the basipetal transport stream. Furthermore, results from studies involving IAA and BRs suggest that BRs are required to maintain normal IAA distribution and transport in pea plants. This is based on the fact that BR-deficiency alters IAA distribution throughout the stem, with a build-up of IAA in the apical bud and a reduction of IAA in mature internodes of BR mutants. The likely cause of this altered IAA distribution appears to be altered IAA transport, as the movement of [³H]IAA in Ikb plants differed from that in the WT. The results presented in this thesis significantly broaden our knowledge of the interactions between the three main hormones that promote stem elongation in pea. In particular they provide further insight into the regulation of plant development by BRs. The characterisation of the Ike mutant, which is the first mutant to be isolated that affects the final steps of BR biosynthesis in pea, will be a valuable tool in future BR research