School of BioSciences - Theses
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ItemRegulation of mucilage production in the Arabidopsis seed coat through MADS-box TF family members( 2018)During Arabidopsis seed development, the epidermal cells of the seed coat produce large quantities of pectinaceous mucilage polysaccharides. Following imbition, mucilage is released from these cells and forms a thick protective layer around the germinating seed that can be visualised with Ruthenium Red staining. The seed coat epidermis is therefore an attractive system to investigate the production of cell wall polysaccharides, particularly pectin. Previously, mutations in the transcriptional regulator LEUNIG_HOMOLOG (LUH) have been shown to be associated with a mucilage extrusion defect that is caused by reduced expression of the β-galactosidase MUCILAGE MODIFIED2 (MUM2). To better understand the role of LUH in regulating MUM2, RNA-Seq analysis was performed on whole seeds and identified genes that were differentially expressed in luh mutants. This transcriptomic analysis not only revealed elevated expression of transcription factors with a known role in epidermal cell differentiation, but also several MADS-box transcription factors that perform roles in floral development and silique shattering such SHATTERPROOF1 (SHP1) and SHP2. This thesis presents evidence that SHP1, SHP2 and SEPALLATA3 (SEP3) are a new class of regulators involved in mucilage polysaccharides modification, as loss of their activity result in mucilage extrusion defects. These mucilage defects are enhanced in shp1 shp2 double mutants and correlate with reduced MUM2 expression. While carbohydrate analysis data failed to show an expected increase in galactose residues attached to pectin in shp1 shp2 and sep3 mutants, the decrease of MUM2 in these mutants is shown to contribute to their mucilage defects as partial rescue of mucilage defects in shp1 shp2 and sep3 was achieved with rescue of MUM2 expression. This is further supported by the use of SRDX repressor motif fusions to SHP2 that not only produced mucilage extrusion defects when introduced into Col-0, but also were correlated with the level of reduced MUM2 levels. Finally, the positive regulation of MUM2 by MADS-box TFs was established through a combination of CArG-box motif mutations in the regulatory sequences of MUM2, demonstrating the requirement of MADS-box TFs for proper MUM2 seed coat expression. This was further supported by luciferase-based transactivation assays, which produced increased MUM2 promoter activity on the addition of SHP1, SHP2 and SEP3. In addition, this positive regulation of MUM2 by SHP1, SHP2 and SEP3 was shown to occur in a complex with LUH, mediated by the co-regulator SEUSS (SEU), in protein-protein interaction assays such as yeast 2-hybrid (Y2H), Y3H, and bimolecular fluorescence complementation (BiFC). Overall, this study provides novel, clear evidence that MADS-box TFs can directly bind to MUM2 and mediate its activation with LUH. Future work includes expanding on and integrating MADS-box TFs into a working model that describes the regulatory pathways controlling mucilage polysaccharides production in the Arabidopsis seed coat.