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.

BackgroundThe 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 GAUT12.1 in Populus deltoides was recently reported to result in improved biomass saccharification, plant growth, and biomass yield. To further understand GAUT12.1 function in biomass recalcitrance and plant growth, here we report the effects of P. trichocarpa GAUT12.1 overexpression in P. deltoides.ResultsIncreasing GAUT12.1 transcript expression by 7–49% in P. deltoides PtGAUT12.1-overexpression (OE) lines resulted in a nearly complete opposite biomass saccharification and plant growth phenotype to that observed previously in PdGAUT12.1-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 PtGAUT12.1-OE lines compared to controls. Total lignin content was unaffected by the gene overexpression. Importantly, selected PtGAUT12.1-OE lines retained the recalcitrance and growth phenotypes upon growth for 9 months in the greenhouse and 2.8 years in the field. PtGAUT12.1-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 PtGAUT12.1-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 PtGAUT12.1-OE lines compared to controls.ConclusionsThe combined phenotype and chemotype data from P. deltoides PtGAUT12.1-OE and PdGAUT12.1-KD transgenics clearly establish GAUT12.1 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.Electronic supplementary materialThe online version of this article (10.1186/s13068-017-1002-y) contains supplementary material, which is available to authorized users.

BackgroundThe effective use of plant biomass for biofuel and bioproduct production requires a comprehensive glycosyl residue composition analysis to understand the different cell wall polysaccharides present in the different biomass sources. Here we compared four methods side-by-side for their ability to measure the neutral and acidic sugar composition of cell walls from herbaceous, grass, and woody model plants and bioenergy feedstocks.ResultsArabidopsis, Populus, rice, and switchgrass leaf cell walls, as well as cell walls from Populus wood, rice stems, and switchgrass tillers, were analyzed by (1) gas chromatography–mass spectrometry (GC–MS) of alditol acetates combined with a total uronic acid assay; (2) carbodiimide reduction of uronic acids followed by GC–MS of alditol acetates; (3) GC–MS of trimethylsilyl (TMS) derivatives; and (4) high-pressure, anion-exchange chromatography (HPAEC). All four methods gave comparable abundance ranking of the seven neutral sugars, and three of the methods were able to quantify unique acidic sugars. The TMS, HPAEC, and carbodiimide methods provided comparable quantitative results for the specific neutral and acidic sugar content of the biomass, with the TMS method providing slightly greater yield of specific acidic sugars and high total sugar yields. The alditol acetate method, while providing comparable information on the major neutral sugars, did not provide the requisite quantitative information on the specific acidic sugars in plant biomass. Thus, the alditol acetate method is the least informative of the four methods.ConclusionsThis work provides a side-by-side comparison of the efficacy of four different established glycosyl residue composition analysis methods in the analysis of the glycosyl residue composition of cell walls from both dicot (Arabidopsis and Populus) and grass (rice and switchgrass) species. Both primary wall-enriched leaf tissues and secondary wall-enriched wood/stem tissues were analyzed for mol% and mass yield of the non-cellulosic sugars. The TMS, HPAEC, and carbodiimide methods were shown to provide comparable quantitative data on the nine neutral and acidic sugars present in all plant cell walls.Electronic supplementary materialThe online version of this article (doi:10.1186/s13068-017-0866-1) contains supplementary material, which is available to authorized users.

BackgroundPlant cell walls contribute the majority of plant biomass that can be used to produce transportation fuels. However, the complexity and variability in composition and structure of cell walls, particularly the presence of lignin, negatively impacts their deconstruction for bioenergy. Metabolic and genetic changes associated with secondary wall development in the biofuel crop switchgrass (Panicum virgatum) have yet to be reported.ResultsOur previous studies have established a cell suspension system for switchgrass, in which cell wall lignification can be induced by application of brassinolide (BL). We have now collected cell wall composition and microarray-based transcriptome profiles for BL-induced and non-induced suspension cultures to provide an overview of the dynamic changes in transcriptional reprogramming during BL-induced cell wall modification. From this analysis, we have identified changes in candidate genes involved in cell wall precursor synthesis, cellulose, hemicellulose, and pectin formation and ester-linkage generation. We have also identified a large number of transcription factors with expression correlated with lignin biosynthesis genes, among which are candidates for control of syringyl (S) lignin accumulation.ConclusionTogether, this work provides an overview of the dynamic compositional changes during brassinosteroid-induced cell wall remodeling, and identifies candidate genes for future plant genetic engineering to overcome cell wall recalcitrance.Electronic supplementary materialThe online version of this article (10.1186/s13068-017-0954-2) contains supplementary material, which is available to authorized users.

The higher plant genomes sequenced to date include numerous genes encoding proteins classifi ed as belonging to CAZy family GT8. The large number and diversity of GT8 proteins in higher plants, which currently constitute more than 65% of the identifi ed eukaryotic GT8 genes, highlight the importance of these proteins in plants. Here we summarize a detailed phylogenetic study of GT8 proteins from three monocot and four dicot plant genomes that clearly divides higher plant GT8 proteins into two distantly related sets of clades, many of which are further divided into statistically well -supported subclades. One set, the GAUT1 ( GA lact U ronosyl T ransferase1) -related family, includes the GAUT and GA U T -L ike (GATL) proteins, comprising one proven galacturonsyltransferase and multiple additional members strongly implicated in the synthesis of pectins and xylan, two major types of polysaccharides present in plant cell walls. The second set, which includes P lant G lycogenin -like S tarch I nitiation P rotein s (PGSIPs) and G alactin ol S ynthase s (GolSs), appears not to be directly involved in plant cell wall synthesis. The PGSIPs have been suggested to play a role in priming starch biosynthesis, while the GolSs are key enzymes in the synthesis of

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The higher plant genomes sequenced to date include numerous genes encoding proteins classified as belonging to CAZy family GT8. The large number and diversity of GT8 proteins in higher plants, which currently constitute more than 65% of the identified eukaryotic GT8 genes, highlight the importance of these proteins in plants. Here we summarize a detailed phylogenetic study of GT8 proteins from three monocot and four dicot plant genomes that clearly divides higher plant GT8 proteins into two distantly related sets of clades, many of which are further divided into statistically well‐supported subclades. One set, the GAUT1 (GAlactUronosylTransferase1)‐related family, includes the GAUT andGAUT‐Like (GATL) proteins, comprising one proven galacturonsyltransferase and multiple additional members strongly implicated in the synthesis of pectins and xylan, two major types of polysaccharides present in plant cell walls. The second set, which includesPlantGlycogenin‐likeStarchInitiationProteins(PGSIPs) andGalactinolSynthases(GolSs), appears not to be directly involved in plant cell wall synthesis. The PGSIPs have been suggested to play a role in priming starch biosynthesis, while the GolSs are key enzymes in the synthesis of the raffinose family of oligosaccharides that play important roles in plant responses to environmental stress. This chapter also summarizes data from biochemical, transcriptional, and mutational studies that provide additional insights into possible functions of higher plant GT8 proteins. However, much work is needed to fully define the roles of GT8 proteins in plants, particularly with respect to their enzymatic function in plant cell wall biosynthesis.