Downregulation of GAUT12 in Populus deltoides by RNA silencing results in reduced recalcitrance, increased growth and reduced xylan and pectin in a woody biofuel feedstock

Abstract

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.

Network-based integration of systems genetics data reveals pathways associated with lignocellulosic biomass accumulation and processing

“…homologs of which have previously been identified as associated with hemicellulose and lignin related properties in Populus (24). IRX8/GAUT12 is known to be involved in xylan structure (41), and modulation of its expression in poplar results in improved sugar release efficiency (42). More indirect arguments can be made for TUA2 and ATCIMS/MS1.…”

Engineering of plant cell walls for enhanced biofuel production

“…Silencing of glycosyltransferases involved in xylan biosynthesis such as Irregular Xylem 8 (IRX8) in Arabidopsis leads to a reduction in wall xylan, but also collapsed xylem vessels and thus plant dwarfism [79]. A similar IRX8 silencing approach in poplar also lead to a significant reduction in xylan and in increased glucose yields through saccharification [80]. However, in contrast to Arabidopsis the Poplar transformants displayed an increase in plant growth indicating that results obtained with Arabidopsis do not necessarily translate directly to crop plants and will have to be tested in each case.…”

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2‐year comparative analysis of field‐grown lines modified for target gene or genetic element expression

“…Recalcitrance can be defined as ‘the natural resistance of plant cell walls to microbial and enzymatic deconstruction’ (Himmel et al ., 2007) due to its complex carbohydrate‐ and lignin‐based structure. Biotechnology can improve this process through the development of better microbial or enzymatic conversion agents as well as by modifying the bio‐feedstock source itself (Biswal et al ., 2015; Bryan et al ., 2016; Davison et al ., 2015; Mottiar et al ., 2016; Wuddineh et al ., 2015). …”