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Increase in cellulose accumulation and improvement of saccharification by overexpression of arabinofuranosidase in rice.

ABSTRACT: Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have ?-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-?-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and cellulose and carry an ester-linked feruloyl substituent. Arabinose side chains are the base point for diferuloyl cross-links and lignification. We analyzed rice plants overexpressing arabinofuranosidase (ARAF) to study the role of arabinose residues in the cell wall and their effects on saccharification. Arabinose content in the cell wall of transgenic rice plants overexpressing individual ARAF full-length cDNA (OsARAF1-FOX and OsARAF3-FOX) decreased 25% and 20% compared to the control and the amount of glucose increased by 28.2% and 34.2%, respectively. We studied modifications of cell wall polysaccharides at the cellular level by comparing histochemical cellulose staining patterns and immunolocalization patterns using antibodies raised against ?-(1,5)-linked l-Ara (LM6) and ?-(1,4)-linked d-Xyl (LM10 and LM11) residues. However, they showed no visible phenotype. Our results suggest that the balance between arabinoxylan and cellulose might maintain the cell wall network. Moreover, ARAF overexpression in rice effectively leads to an increase in cellulose accumulation and saccharification efficiency, which can be used to produce bioethanol.

SUBMITTER: Sumiyoshi M

PROVIDER: S-EPMC3817243 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Increase in cellulose accumulation and improvement of saccharification by overexpression of arabinofuranosidase in rice.

Sumiyoshi Minako M Nakamura Atsuko A Nakamura Hidemitsu H Hakata Makoto M Ichikawa Hiroaki H Hirochika Hirohiko H Ishii Tadashi T Satoh Shinobu S Iwai Hiroaki H

PloS one 20131104 11

Cellulosic biomass is available for the production of biofuel, with saccharification of the cell wall being a key process. We investigated whether alteration of arabinoxylan, a major hemicellulose in monocots, causes an increase in saccharification efficiency. Arabinoxylans have β-1,4-D-xylopyranosyl backbones and 1,3- or 1,4-α-l-arabinofuranosyl residues linked to O-2 and/or O-3 of xylopyranosyl residues as side chains. Arabinose side chains interrupt the hydrogen bond between arabinoxylan and ...[more]

PMID: 24223786

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Project description:BackgroundAs a major component of plant cell walls, cellulose provides the most abundant biomass resource convertible for biofuels. Since cellulose crystallinity and polymerization have been characterized as two major features accounting for lignocellulose recalcitrance against biomass enzymatic saccharification, genetic engineering of cellulose biosynthesis is increasingly considered as a promising solution in bioenergy crops. Although several transcription factors have been identified to regulate cellulose biosynthesis and plant cell wall formation, much remains unknown about its potential roles for genetic improvement of lignocellulose recalcitrance.ResultsIn this study, we identified a novel rice mutant (Osfc9/myb103) encoded a R2R3-MYB transcription factor, and meanwhile generated OsMYB103L-RNAi-silenced transgenic lines. We determined significantly reduced cellulose levels with other major wall polymers (hemicellulose, lignin) slightly altered in mature rice straws of the myb103 mutant and RNAi line, compared to their wild type (NPB). Notably, the rice mutant and RNAi line were of significantly reduced cellulose features (crystalline index/CrI, degree of polymerization/DP) and distinct cellulose nanofibers assembly. These alterations consequently improved lignocellulose recalcitrance for significantly enhanced biomass enzymatic saccharification by 10-28% at p < 0.01 levels (n = 3) after liquid hot water and chemical (1% H2SO4, 1% NaOH) pretreatments with mature rice straws. In addition, integrated RNA sequencing with DNA affinity purification sequencing (DAP-seq) analyses revealed that the OsMYB103L might specifically mediate cellulose biosynthesis and deposition by regulating OsCesAs and other genes associated with microfibril assembly.ConclusionsThis study has demonstrated that down-regulation of OsMYB103L could specifically improve cellulose features and cellulose nanofibers assembly to significantly enhance biomass enzymatic saccharification under green-like and mild chemical pretreatments in rice. It has not only indicated a powerful strategy for genetic modification of plant cell walls in bioenergy crops, but also provided insights into transcriptional regulation of cellulose biosynthesis in plants.

Project description:Background:Genetic modification of plant cell walls has been implemented to reduce lignocellulosic recalcitrance for biofuel production. Plant glycoside hydrolase family 9 (GH9) comprises endo-?-1,4-glucanase in plants. Few studies have examined the roles of GH9 in cell wall modification. In this study, we independently overexpressed two genes from GH9B subclasses (OsGH9B1 and OsGH9B3) and examined cell wall features and biomass saccharification in transgenic rice plants. Results:Compared with the wild type (WT, Nipponbare), the OsGH9B1 and OsGH9B3 transgenic rice plants, respectively, contained much higher OsGH9B1 and OsGH9B3 protein levels and both proteins were observed in situ with nonspecific distribution in the plant cells. The transgenic lines exhibited significantly increased cellulase activity in vitro than the WT. The OsGH9B1 and OsGH9B3 transgenic plants showed a slight alteration in three wall polymer compositions (cellulose, hemicelluloses, and lignin), in their stem mechanical strength and biomass yield, but were significantly decreased in the cellulose degree of polymerization (DP) and lignocellulose crystalline index (CrI) by 21-22%. Notably, the crude cellulose substrates of the transgenic lines were more efficiently digested by cellobiohydrolase (CBHI) than those of the WT, indicating the significantly increased amounts of reducing ends of ?-1,4-glucans in cellulose microfibrils. Finally, the engineered lines generated high sugar yields after mild alkali pretreatments and subsequent enzymatic hydrolysis, resulting in the high bioethanol yields obtained at 22.5% of dry matter. Conclusions:Overproduction of OsGH9B1/B3 enzymes should have specific activity in the postmodification of cellulose microfibrils. The increased reducing ends of ?-1,4-glucan chains for reduced cellulose DP and CrI positively affected biomass enzymatic saccharification. Our results demonstrate a potential strategy for genetic modification of cellulose microfibrils in bioenergy crops.

Dataset Information

Increase in cellulose accumulation and improvement of saccharification by overexpression of arabinofuranosidase in rice.

Publications

Increase in cellulose accumulation and improvement of saccharification by overexpression of arabinofuranosidase in rice.

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