Abstract/Summary

Response regulators (RRs) are crucial signalling components that allow plants to respond to fluctuations in their environment. ARR22 is a unique type-C RR previously identified in Arabidopsis that is hypothesised to be post-transcriptionally up-regulated in response to wounding at the seed:funiculus junction and hence has a predicted role in assimilate partitioning. A putative orthologue known as SAC29 has been isolated in the economically important allotetraploid crop Brassica napus (B. napus). A total of 83 putative RRs in B. napus (BnRRs) have been identified which can be classified into type-A, -B and –C RRs comparable to Arabidopsis. A subset of putative type-A and type-B BnRRs were examined further and expression was detected in early seed development stages which may reveal novel functions for these genes in B. napus. In silico and expression analyses have identified and characterised four putative ARR22 orthologues (BnRR76 – BnRR79) that exhibit 81.25% amino acid similarity. Distinct differences in nucleotide and amino acid sequence were observed in BnRR76 and BnRR78 that originate from B. rapa and B. oleracea parental genomes respectively. All genes contain two introns, one located within the 5’UTR and one in the ORF, similar to ARR22. RT-PCR analysis revealed differences in spatial and temporal expression of BnRR76 and BnRR79 during seed development. Retention of an intron located within the open reading frame in BnRR77 and BnRR79 was also observed at different stages of seed maturation.Mechanical wounding of seeds did not elicit a change in seed storage protein or cysteine protease expression even after 120 mins and hence does not support the hypothesis that putative B. napus orthologues of ARR22 are necessarily involved in assimilate partitioning. An antibody was designed to recognise an amino acid sequence present in ARR22, and BnRR76 – BnRR79, and was subsequently used in Western blot analysis. Expression of BnRR76 – BnRR79 proteins in seeds was rapidly up-regulated at 60 mins post-wounding while gene expression levels remained at a baseline level until 120 mins when protein level decreased suggesting that a rapid wound response occurs at the protein level rather than at the level of gene expression. Using a dexamethasone (DEX) inducible system, physiological effects of ARR22 overexpression were elucidated. DEX-induced overexpression resulted in severe phenotypes comparable to cytokinin receptor mutants such as reduced rosette area and stunted inflorescence. Transgenic lines in which a predicted phosphorylation site, hypothesised to be critical for protein function during stress response, had been mutated exhibited comparable phenotypic effects and hence suggests a possible different mode of mechanism of ARR22 when ectopically expressed. This project explores and characterises response regulators, with particular focus on their involvement in seed development, for the first time in the economically important oilseed crop B. napus. Future work should examine wounding effects at longer time points as well as aim to elucidate downstream components and targets of ARR22 and its putative B. napus orthologues BnRR76 – BnRR79.