Working towards recalcitrance mechanisms: increased xylan and homogalacturonan production by overexpression of () causes increased recalcitrance and decreased growth in .

Background: The development of fast-growing hardwood trees as a source of lignocellulosic biomass for biofuel and biomaterial production requires a thorough understanding of the plant cell wall structure and function that underlie the inherent recalcitrance properties of woody biomass. Downregulation of in was recently reported to result in improved biomass saccharification, plant growth, and biomass yield. To further understand function in biomass recalcitrance and plant growth, here we report the effects of overexpression in .

Results: Increasing transcript expression by 7-49% in -overexpression (OE) lines resulted in a nearly complete opposite biomass saccharification and plant growth phenotype to that observed previously in -knockdown (KD) lines. This included significantly reduced glucose, xylose, and total sugar release (12-13%), plant height (6-54%), stem diameter (8-40%), and overall total aerial biomass yield (48-61%) in 3-month-old, greenhouse-grown -OE lines compared to controls. Total lignin content was unaffected by the gene overexpression. Importantly, selected -OE lines retained the recalcitrance and growth phenotypes upon growth for 9 months in the greenhouse and 2.8 years in the field. -OE plants had significantly smaller leaves with lower relative water content, and significantly reduced stem wood xylem cell numbers and size. At the cell wall level, xylose and galacturonic acid contents increased markedly in total cell walls as well as in soluble and insoluble cell wall extracts, consistent with increased amounts of xylan and homogalacturonan in the -OE lines. This led to increased cell wall recalcitrance, as manifested by the 9-15% reduced amounts of recovered extractable wall materials and 8-15% greater amounts of final insoluble pellet in the -OE lines compared to controls.

Conclusions: The combined phenotype and chemotype data from -OE and -KD transgenics clearly establish as a recalcitrance- and growth-associated gene in poplar. Overall, the data support the hypothesis that GAUT12.1 synthesizes either an HG-containing primer for xylan synthesis or an HG glycan required for proper xylan deposition, anchoring, and/or architecture in the wall, and the possibility of HG and xylan glycans being connected to each other by a base-sensitive covalent linkage.