Forward genetic analysis of cellulose biosynthesis inhibitor resistance and wall hydrolysis sensitivity.
Date
2014-09-01
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Abstract
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.
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Keywords
Cellulose, Wall hydrolysis, Plant genetics