BackgroundThe inherent recalcitrance of woody bioenergy feedstocks is a major challenge for their use as a source of second-generation biofuel. Secondary cell walls that constitute the majority of hardwood biomass are rich in cellulose, xylan, and lignin. The interactions among these polymers prevent facile accessibility and deconstruction by enzymes and chemicals. Plant biomass that can with minimal pretreatment be degraded into sugars is required to produce renewable biofuels in a cost-effective manner.ResultsGAUT12/IRX8 is a putative glycosyltransferase proposed to be involved in secondary cell wall glucuronoxylan and/or pectin biosynthesis based on concomitant reductions of both xylan and the pectin homogalacturonan (HG) in Arabidopsis irx8 mutants. Two GAUT12 homologs exist in Populus trichocarpa, PtGAUT12.1 and PtGAUT12.2. Knockdown expression of both genes simultaneously has been shown to reduce xylan content in Populus wood. We tested the proposition that RNA interference (RNAi) downregulation of GAUT12.1 alone would lead to increased sugar release in Populus wood, that is, reduced recalcitrance, based on the hypothesis that GAUT12 synthesizes a wall structure required for deposition of xylan and that cell walls with less xylan and/or modified cell wall architecture would have reduced recalcitrance. Using an RNAi approach, we generated 11 Populus deltoides transgenic lines with 50 to 67% reduced PdGAUT12.1 transcript expression compared to wild type (WT) and vector controls. Ten of the eleven RNAi lines yielded 4 to 8% greater glucose release upon enzymatic saccharification than the controls. The PdGAUT12.1 knockdown (PdGAUT12.1-KD) lines also displayed 12 to 52% and 12 to 44% increased plant height and radial stem diameter, respectively, compared to the controls. Knockdown of PdGAUT12.1 resulted in a 25 to 47% reduction in galacturonic acid and 17 to 30% reduction in xylose without affecting total lignin content, revealing that in Populus wood as in Arabidopsis, GAUT12 affects both pectin and xylan formation. Analyses of the sugars present in sequential cell wall extracts revealed a reduction of glucuronoxylan and pectic HG and rhamnogalacturonan in extracts from PdGAUT12.1-KD lines.ConclusionsThe results show that downregulation of GAUT12.1 leads to a reduction in a population of xylan and pectin during wood formation and to reduced recalcitrance, more easily extractable cell walls, and increased growth in Populus.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-015-0218-y) contains supplementary material, which is available to authorized users.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

Summary Genome‐wide association studies (GWAS) have great promise for identifying the loci that contribute to adaptive variation, but the complex genetic architecture of many quantitative traits presents a substantial challenge. We measured 14 morphological and physiological traits and identified single nucleotide polymorphism (SNP)‐phenotype associations in a Populus trichocarpa population distributed from California, USA to British Columbia, Canada. We used whole‐genome resequencing data of 882 trees with more than 6.78 million SNPs, coupled with multitrait association to detect polymorphisms with potentially pleiotropic effects. Candidate genes were validated with functional data. Broad‐sense heritability (H2) ranged from 0.30 to 0.56 for morphological traits and 0.08 to 0.36 for physiological traits. In total, 4 and 20 gene models were detected using the single‐trait and multitrait association methods, respectively. Several of these associations were corroborated by additional lines of evidence, including co‐expression networks, metabolite analyses, and direct confirmation of gene function through RNAi. Multitrait association identified many more significant associations than single‐trait association, potentially revealing pleiotropic effects of individual genes. This approach can be particularly useful for challenging physiological traits such as water‐use efficiency or complex traits such as leaf morphology, for which we were able to identify credible candidate genes by combining multitrait association with gene co‐expression and co‐methylation data.