School of BioSciences - Theses
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ItemThe role of LEUNIG_HOMOLOG in regulating mucilage release from the Arabidopsis testa( 2016)Upon hydration, Arabidopsis seeds release pectinaceous mucilage from the seed coat, which is primarily composed of rhamnogalacturonan I (RG-I). Mutations in the transcriptional regulator LEUNIG_HOMOLOG (LUH) result in mucilage extrusion defects that are associated with increased galactose (Gal) residues present on the RG-I backbone. This structural modification is correlated with reduced expression of MUCILAGE MODIFIED 2 (MUM2), a gene encoding a β-galactosidase belonging to the Glycoside Hydrolase Family 35, suggesting that BGAL6/MUM2 is positively regulated by LUH. In considering how LUH promotes BGAL6/MUM2 expression in the seed coat, two contrasting models of regulation have been proposed. According to the ‘direct’ model, LUH and associated proteins, bind to BGAL6/MUM2 regulatory sequences and promote transcription of the gene. In the ‘indirect’ model, the LUH complex regulates activity of a transcription factor (TF) that then targets BGAL6/MUM2. In one version of this model, the intermediary TF is an activator and thus LUH promotes BGAL6/MUM2 expression indirectly by activating this TF. However, as LUH shares significant sequence similarity and functional redundancy with the transcriptional co-repressor LEUNIG (LUG), it has been proposed that LUH functions as a transcriptional repressor. According to this possibility, LUH promotes BGAL6/MUM2 indirectly by limiting the activity of a TF(s) that represses BGAL6/MUM2. In order to resolve the molecular mechanism by which LUH is regulating BGAL6/MUM2, various molecular approaches were undertaken in this study. Firstly, RNA sequencing (RNA-seq) was performed on developing wildtype and luh seed, which together with extensive bioinformatic analysis, led to a number of seed coat-expressed genes that are responsive to LUH being identified. Within the group of genes that were elevated in luh mutant seeds were TFs that are known to play a role in epidermal differentiation, raising the possibility that mis-expression of one or more of these genes interferes with expression of BGAL6/MUM2. Another interesting finding was that MADS-box TFs are elevated in the seed coat of luh mutant seeds, suggesting that these transcriptional regulators may play a role in the mucilage synthesis/modification process. To help identify the TF(s) involved in BGAL6/MUM2 regulation, regulatory elements in the MUM2 gene were characterised. Initial experiments showed that robust expression of BGAL6/MUM2 in the seed coat requires the presence of both the BGAL6/MUM2 promoter as well as the large first intron. Use of phylogenetic shadowing coupled with deletion analysis of the intron identified a conserved block of sequence in the 5’ region of the intron that contains both enhancer and repressor elements. Binding sites for MADS-box TFs were subsequently found in this region and, based on deletion experiments, categorized as enhancers that ensure robust seed coat expression. Finally, this study used a combination of techniques to determine whether LUH functions as an activator or repressor in the seed coat, and whether LUH activity in the seed coat is mediated through interactions with the co-regulators SEUSS and/or SEUSS-LIKE; proteins that are required for LUG activity. None of these results proved conclusive, but were consistent with LUH being a repressor that probably forms a complex with these LUG-associated factors. Overall this study provides clear evidence that LUH is a global regulator of genes active in the developing seed and that BGAL6/MUM2 regulation occurs indirectly via an as yet unidentified repressor.