Dataset Information

Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

ABSTRACT: Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.

SUBMITTER: Zhang J

PROVIDER: S-EPMC10762510 | biostudies-literature | 2023 Dec

REPOSITORIES: biostudies-literature

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Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

Zhang Jin J Wang Xiaqin X Wang Hsin-Tzu HT Qiao Zhenzhen Z Yao Tao T Xie Meng M Urbanowicz Breeanna R BR Zeng Wei W Jawdy Sara S SS Gunter Lee E LE Yang Xiaohan X Czarnecki Olaf O Regan Sharon S Seguin Armand A Rottmann William W Winkeler Kimberly A KA Sykes Robert R Lipzen Anna A Daum Chris C Barry Kerrie K Lu Meng-Zhu MZ Tuskan Gerald A GA Muchero Wellington W Chen Jin-Gui JG

Plant physiology 20231201 1

Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases ...[more]

PMID: 37399189

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Project description: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.

Project description: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.

Project description:Brachypodium distachyon (Brachypodium) has emerged as a useful model system for studying traits unique to graminaceous species including bioenergy crop grasses owing to its amenability to laboratory experimentation and the availability of extensive genetic and germplasm resources. Considerable natural variation has been uncovered for a variety of traits including flowering time, vernalization responsiveness, and above-ground growth characteristics. However, cell wall composition differences remain underexplored. Therefore, we assessed cell wall-related traits relevant to biomass conversion to biofuels in seven Brachypodium inbred lines that were chosen based on their high level of genotypic diversity as well as available genome sequences and recombinant inbred line (RIL) populations. Senesced stems plus leaf sheaths from these lines exhibited significant differences in acetyl bromide soluble lignin (ABSL), cell wall polysaccharide-derived sugars, hydroxycinnamates content, and syringyl:guaiacyl:p-hydroxyphenyl (S:G:H) lignin ratios. Free glucose, sucrose, and starch content also differed significantly in senesced stems, as did the amounts of sugars released from cell wall polysaccharides (digestibility) upon exposure to a panel of thermochemical pretreatments followed by hydrolytic enzymatic digestion. Correlations were identified between inbred line lignin compositions and plant growth characteristics such as biomass accumulation and heading date (HD), and between amounts of cell wall polysaccharides and biomass digestibility. Finally, stem cell wall p-coumarate and ferulate contents and free-sugars content changed significantly with increased duration of vernalization for some inbred lines. Taken together, these results show that Brachypodium displays substantial phenotypic variation with respect to cell wall composition and biomass digestibility, with some compositional differences correlating with growth characteristics. Moreover, besides influencing HD and biomass accumulation, vernalization was found to affect cell wall composition and free sugars accumulation in some Brachypodium inbred lines, suggesting genetic differences in how vernalization affects carbon flux to polysaccharides. The availability of related RIL populations will allow for the genetic and molecular dissection of this natural variation, the knowledge of which may inform ways to genetically improve bioenergy crop grasses.

Project description:High acetylation of xylan in hardwoods decreases their value as biorefinery feedstocks. To counter this problem, we have constitutively suppressed RWA genes encoding acetyl-CoA transporters using the 35S promoter, or constitutively and wood-specifically (using the WP promoter) expressed fungal acetyl xylan esterases of families CE1 (AnAXE1) and CE5 (HjAXE), to reduce acetylation in hybrid aspen. All these transformations improved the saccharification of wood from greenhouse-grown trees. Here, we describe the chemical properties and saccharification potential of the resulting lines grown in a five-year field trial, and one type of them (WP:AnAXE1) in greenhouse conditions. Chemically, the lignocellulose of the field- and greenhouse-field-grown plants slightly differed, but the reductions in acetylation and saccharification improvement of engineered trees were largely maintained in the field. The main novel phenotypic observation in the field was higher lignification in lines with the WP promoter than those with the 35S promoter. Following growth in the field, saccharification glucose yields were higher from most transformed lines than from wild-type (WT) plants with no pretreatment, but there was no improvement in saccharification with acid pretreatment. Thus, acid pretreatment removes most recalcitrance caused by acetylation. We found a complex relationship between acetylation and glucose yields in saccharification without pretreatment, suggesting that other variables, for example, the acetylation pattern, affect recalcitrance. Bigger gains in glucose yields were observed in lines with the 35S promoter than in those with the WP promoter, possibly due to their lower lignin content. However, better lignocellulose saccharification of these lines was offset by a growth penalty and their glucose yield per tree was lower. In a comparison of the best lines with each construct, WP:AnAXE1 provided the highest glucose yield per tree from saccharification, with and without pretreatment, WP:HjAXE yields were similar to those of WT plants, and yields of lines with other constructs were lower. These results show that lignocellulose properties of field-grown trees can be improved by reducing cell wall acetylation using various approaches, but some affect productivity in the field. Thus, better understanding of molecular and physiological consequences of deacetylation is needed to obtain quantitatively better results.

Dataset Information

Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

Publications

Overexpression of REDUCED WALL ACETYLATION C increases xylan acetylation and biomass recalcitrance in Populus.

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