This thesis describes a molecular-based study undertaken to analyse the expression of the RAMOSUS1 (RMS1) and RAMOSUS5 (RMS5) genes in pea (Pisum sativum). Both genes encode carotenoid cleavage dioxygenase (CCD) enzymes that are together proposed to control the synthesis of an inhibitor of bud outgrowth termed SMS (Shoot Multiplication Signal). SMS was recently identified as strigolactone. Expression analyses of RMS1 presented here have built upon earlier experiments which demonstrate it to be a highly regulated transcript. RMS1 mRNA levels are known to be rapidly decreased following removal of the shoot apex but are subsequently restored to that of intact plants by auxin (indole-3-acetic acid or IAA). This regulatory mechanism is retained in all five ramosus mutants tested to date. Together with physiological data, this indicates RMS1, and therefore SMS, are required in IAA-mediated suppression of bud outgrowth. Another significant aspect of RMS1 regulation identified in previous studies involves a graft-transmissible, long-distance feedback signal that moves from shoot to root. This feedback regulation is dependent on the RMS2 gene and enhances RMS1 expression levels. Prior to the cloning of RMS5 and its discovery as a second CCD enzyme in the RMS network, reciprocal grafting studies with the rms mutants indicated RMS5 may act in the same pathway as RMS1 to produce SMS. Multiple studies presented here demonstrate that these two CCD genes are expressed in similar tissues and are regulated by the same signals, specifically IAA and the RMS2-dependent feedback signal. Like RMS1, the RMS5 gene also retains its IAA response in the rms mutants. However, RMS5 is generally less responsive to changes in IAA and RMS2-dependent feedback, as it exhibits smaller fluctuations than RMS1 in its expression levels. Together these findings support a general view that RMS1 is more likely to control a rate-limiting step in SMS synthesis. A previous study indicated that RMS1 expression may be up-regulated by IAA through a posttranscriptional mechanism. This thesis sought to more closely examine the RMS1 and RMS5 IAA response by separately observing the effect of IAA on subsequent transcription. New transcripts, termed heterogenous nuclear RNAs (hnRNAs), were relatively quantified in parallel with existing mRNAs in the steady-state cytoplasmic pool. The experiments conducted here provide further evidence that IAA may act post-transcriptionally to stabilise RMS1 mRNA because the changes in hnRNA are not proportional to the changes in mRNA following IAA-modifying treatments. IAA may still function to induce transcription of RMS1, but this does not appear to be a significant mechanism by which IAA regulates RMS1 expression. In contrast, the IAA induction of RMS5 occurs predominantly via new transcription and RMS5 either lacks or is not as strongly subjected to the IAA-mediated mRNA stabilisation mechanism proposed for RMS1. Initial studies described in this thesis also suggest that IAA could act to regulate the expression of the Arabidopsis orthologues MORE AXILLARY BRANCHING (MAX) genes via a post-transcriptional mechanism. Analyses of MAX hnRNA and mRNA levels in Arabidopsis to date indicate it is the RMS5 orthologue MAX3 which exhibits an IAA response most like RMS1. Additional studies into the regulation of RMS1 and RMS5 presented in this thesis provide further insights into the molecular mechanisms controlling their expression levels. In vitro experiments with the translation inhibitor cycloheximide demonstrate that RMS5 expression levels are increased when protein synthesis is reduced, as previously shown for RMS1. Relative quantification of RMS1 and RMS5 hnRNA levels further demonstrate that the induction by cycloheximide is due primarily to an increase in new transcription, indicating that RMS1 and RMS5 are negatively regulated by a rapidly turned-over transcriptional repressor. Tissue specific effects on RMS1 expression were also observed which are consistent with a protein degradation function of the RMS4 F-box in the shoot. This thesis provides further evidence to suggest that SMS acts in concert with IAA to inhibit the sustained outgrowth of axillary buds. RMS1 and RMS5 expression levels are not regulated by a hypothetical fast decapitation signal which is proposed to cause the initial bud outgrowth occurring prior to decapitation-induced IAA depletion. RMS1, RMS5 and SMS are therefore unlikely to control the initial exit of buds from dormancy to an intermediate transition state. Studies here also suggest that enhanced shoot auxin transport and cytokinin biosynthesis are associated with axillary bud outgrowth because the rms mutants contain elevated shoot expression levels of a gene encoding the auxin efflux carrier PIN1 and two genes controlling cytokinin biosynthesis. Several approaches described in this study were used to characterise the RMS1 and RMS5 proteins. Anti-peptide antibodies were generated against both proteins and the results obtained show that although the antibodies are likely to recognise the full-length proteins, further work is required to effectively detect RMS1 and RMS5 in plant tissues via western blotting. Preliminary in situ immunolocalisation results indicate the RMS1 and RMS5 proteins are localised to the vasculature, consistent with gene expression analyses.
| Identifer | oai:union.ndltd.org:ADTP/254217 |
| Creators | Tanya Brcich |
| Source Sets | Australiasian Digital Theses Program |
| Detected Language | English |
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