Off-campus UMass Amherst users: To download campus access dissertations, please use the following link to log into our proxy server with your UMass Amherst user name and password.

Non-UMass Amherst users: Please talk to your librarian about requesting this dissertation through interlibrary loan.

Dissertations that have an embargo placed on them will not be available to anyone until the embargo expires.

Author ORCID Identifier

https://orcid.org/0000-0001-9572-8464

Document Type

Campus-Only Access for One (1) Year

Degree Name

Doctor of Philosophy (PhD)

Degree Program

Plant Biology

Year Degree Awarded

2019

Month Degree Awarded

May

First Advisor

Samuel Hazen

Subject Categories

Plant Biology

Abstract

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.

Share

COinS