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Doctor of Philosophy (PhD)
Year Degree Awarded
Month Degree Awarded
Plant cell walls are complex structures that contain a matrix of cellulose, lignin and hemicellulose. The regulation of the biosynthesis of these components has been well-studied in the eudicot plant Arabidopsis thaliana, and a transcriptional network has been elucidated. Several NAC and MYB family transcription factors are key regulators of secondary cell wall biosynthesis, and their functional characterization provides significant insight into the complex underlying transcriptional network. Genetic and structural evidence suggests that genes controlling this process might be different between eudicots and monocots. Here, the model grass Brachypodium distachyon has been selected to characterize the function of GNRF (GRASS NAC REPRESSOR OF FLOWERING), SWAM1 (SECONDARY WALL ASSOCIATED MYB1), and SWAM4 in the regulation of secondary cell wall biosynthesis. Phylogenetic analysis identified that GNRF and SWAM4 as the respectively AtSND2 and AtMYB61 transcription factors in B. distachyon. Co-expression analysis showed that both, GNRF and SWAM4, clustered with putative cell-wall-associated genes. Functional characterization was performed by using the overexpression plants GNRF-OE and SWAM4-OE; sodium azide mutant plants from a TILLING (Targeting Induced Local Lesion IN Genome) collection for gnrf-1, gnrf-2, gnrf-3, gnrf-4, gnrf-5, swam4-1, and swam4-2; a T-DNA insertional mutant plant, gnrf-6; and a dominant repressor plant, SWAM4-DR. GNRF-OE plants remained at juvenile stage and exhibited persistent vegetative growth, and some gnrf mutant plants were late flowering. SWAM4-DR plants were severely dwarfed. Stems of all genotypes were subjected to lignin quantification, cell wall thickness measurements, Q-RT-PCR, and RNA-seq analysis. Cell wall and transcriptomic analysis revealed that GNRF is a repressor of SWAM1, a MYB activator of cell wall thickening, and represses genes encoding cellulose, lignin, and xylan biosynthetic enzymes. GNRF was found to function as a pleiotropic repressor of cell wall biosynthesis, flowering, and transport proteins. Protein-DNA interactions were revealed in yeast by yeast-one-hybrid assays; the GNRF binding site (CT/GTA/G/CA/TNNNNT/G/CAA/CA/T/GA/TA/T) was identified by DNA affinity purification sequencing (DAP-seq) assay. SWAM4 is a putative regulator of cell wall biosynthetic genes (CESA4, CESA7, CESA8, CAD, and COMT), and other proteins associated with cell wall formation. Collectively, GNRF, SWAM4, and SWAM1 were characterized as secondary cell wall regulators in B. distachyon.
Romero-Gamboa, Sandra, "Brachypodium distachyon GNRF, SWAM1 and SWAM4 are transcriptional regulators of secondary cell wall biosynthesis" (2019). Doctoral Dissertations. 1577.