Forward genetic analysis of cellulose biosynthesis inhibitor resistance and wall hydrolysis sensitivity.
The functional analysis of components involved in cellulose biosynthesis is central in understanding cell wall assembly and structure in plants. We conducted screens using the herbicides, isoxaben and flupoxam which inhibit cellulose biosynthesis in higher plants. Mutations resulting in a high degree of resistance to isoxaben (ixr) or flupoxam (fxr) were attributed to single amino acid substitutions in primary wall CESAs. Twelve novel resistance alleles were isolated and no cross-resistance was observed. Point mutations were mostly clustered around the C-terminal regions of CESA1 and CESA3, and CESA3 and CESA6 for fxr and ixr respectively. Resistance to isoxaben was also conferred by modification to the putative catalytic regions of CESA3. This resulted in cellulose deficient phenotypes characterized by reduced crystallinity and dwarfism. These results provide genetic evidence supporting CESA1-CESA3, and CESA3-CESA6 association with flupoxam and isoxaben respectively targeting and disrupting these interactions. The ixr and fxr mutants also exhibited enhanced saccharification under enzymatic degradation schemes which is consistent with the observed reduction in cellulose crystallinity. A second forward genetic screen was performed using mild acid hydrolysis to isolate mutants with enhanced saccharification. This screen identified sixty-three responsive to acid hydrolysis (rah) lines. Unconventional strategies to increase sugar yields from plant biomass where highlighted. These included starch hyper-accumulators such as starch excess 4 (sex4) loss-of-function mutants and the perturbation of polar auxin transport. Disruption of the serine/threonine kinase positive regulator of auxin efflux, PINOID (PID) was found to significantly enhance sugar release in Arabidopsis and similar effects were observed in the maize orthologue, BARREN INFLORESENCE 2 (BIF2). Furthermore, the application of N-1-naphthylphthalamic acid (NPA) in Arabidopsis, maize, Miscanthus and switchgrass phenocopied the enhanced wall saccharification effects of PID. This study attempted to elucidate some of the interactions of seemingly unrelated pathways in the context of wall biosynthesis and saccharification enhancement.