School of BioSciences - Theses
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ItemThe LEUNIG regulatory complex: How does it control shoot apical meristem formation and its post-embryonic activity?( 2021)Patterning along the apical-basal (A-B) axis is a crucial step during the early stages of plant embryogenesis and leads to the establishment of two poles of which each will develop their own stem cell niches. The activity of these meristems is responsible for post-embryonic growth, with the shoot apical meristem (SAM) generating the above-ground organs and the root apical meristem (RAM) producing the subterranean structures of the plant. While several transcriptional regulators governing A-B patterning have been identified, precisely how their regulatory function is orchestrated remains elusive. This study focuses on transcriptional co-regulators LEUNIG (LUG) and closely related LEUNIG_HOMOLOG (LUH) and their role in the formation of A-B patterning during embryogenesis as well as their post-embryonic maintenance. A link between the LUG regulatory complex and SAM formation and maintenance comes from the observation that lug mutants heterozygous for the luh allele (lug luh+/-) often have enlarged SAMs resulting from misregulated cell divisions. A more severe phenotype is observed in lug luh double mutants which are embryonically lethal. In this study, a detailed characterisation of lug luh embryo phenotype reveals that these mutants display aberrant cell divisions along the A-B axis, which correlates with defects in auxin distribution, complete loss of apical identity, and altered expression of transcription factors determining basal fate. Like other co-regulators, LUG and LUH lack intrinsic DNA-binding domains and instead must interact with DNA-binding cofactors to ensure recruitment to regulatory elements of target genes. This either involves direct contact between the co-regulators and transcription factors (TFs) or the formation of higher-order complexes with adaptor proteins such as SEUSS (SEU) or related SEUSS-LIKEs (SLKs), which facilitate binding to specific TFs. Results presented in this study provide insight into the molecular framework for the LUG regulatory complex activity during embryogenesis. Both yeast and in planta assays showed that LUG/LUH and SEU/SLKs physically associate with a variety of WUSCHEL-RELATED HOMEOBOX (WOX) TFs including members of the WOX2-module. Furthermore, genetic interactions between members of the WOX2-module and the LUG regulatory complex, support their mutual action during embryogenesis. Based on the reduced activity of HOMEODOMAIN LEUCINE-ZIPPER CLASS III (HD-ZIPIII) promoters in lug luh embryos, a model is proposed in which the LUG regulatory complex functions together with WOX2-module to promote apical identity and subsequent SAM initiation through regulation of the HD-ZIPIIIs. The activity of the LUG complex in promoting basal embryo identity through positive regulation of microRNA165/166 suggests that this complex also has functions that are independent of the WOX2-module. Preliminary work reported in this study further uncovered the role of the LUG regulatory complex in post-embryonic development. While the fasciated inflorescence meristems of lug luh+/- plants displayed defects in auxin transport and altered activity of stem cell markers, embryonically rescued lug luh mutants formed flat and differentiated SAMs. In addition, rescued lug luh mutants exhibited severely disorganised RAM and defects in quiescent center (QC) specification, supporting the involvement of the LUG complex in post-embryonic RAM maintenance.
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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.