Acetylation of polysaccharides in plant cell wall

Plant cell wall in woody tissues is a complex matrix, which consists of cellulose, matrix polysaccharides and lignin. The matrix polysaccharides are substituted with acetyl group that are hypothesised to play important roles in determining properties of these polysaccharides. The aim of this thesis was to understand the role of O-acetylation in plants and investigate possibilities for improvement of woody lignocellulose for biorefinery applications by reducing wood O-acetylation. To alter acetylation specifically in woody tissues, a promoter from Glycosyl Transferase 43 family (GT43) in Populus was isolated that had a very specific expression in secondary cell wall forming cells. This xylem specific promoter (pGT43B) was more effective in modification of wood acetylation by overexpression and suppression strategies as compared to 35S promoter (Paper I). To reduce xylan acetylation using transgenic approach, acetyl xylan esterase from Aspergillus niger, AnAXE1, was targetted specifically to the cell wall in Arabidopsis (Paper II) and in Populus (Paper III). Plants expressing AnAXE1 grew as well as wild type and had increased acetyl esterase activity. This has led to reduction in cell wall acetyl content and in xylan O-acetylation. Moreover, transgenic Arabidopsis exhibited increased resistance against a biotrophic pathogen Hyaloperonospora arabidopsidis. Both transgenic plants had improved sugar yields in saccharification with different pretreatments and without pretreatment. To reduce acetylation using cisgenic approach, Populus Reduced Wall Acetylation (RWA) gene family was characterised by suppression of the two clades RWA-AB, and RWA-CD (Paper IV). Both clades were shown to be involved in xylan acetylation in the wood. Both clades were therefore suppressed under control of xylem specific promoter pGT43B to improve wood saccharification potential. Transgenic plants had reduced wood acetyl content, normal growth, and increased sugar yield and glucose conversion % in saccharification without pretreatment. Glucose yield was also slightly increased in saccharification after acid pretreatment. These results show that reduction of cell wall acetylation by 10-30% does not alter plant growth and development, but improves yields in lignocellulose saccharification (with and without pretreatment) (Papers II, III and IV). To identify Quantitative Trait Loci (QTLs) related to cell wall acetyl content and other chemical traits in Salix, the mapping population of 463 progenies of S. viminalis and S. schwerinii was analysed by FT-IR and acetyl content assay (Paper V). 28 QTLs were identified for different cell wall chemical traits, which were co-located with several cell wall related genes and gene clusters. These QTLs and genes can be used in the future to improve wood chemical traits in Salix and Populus for biofuel production by breeding

Hybrid Aspen Expressing a Carbohydrate Esterase Family 5 Acetyl Xylan Esterase under Control of a Wood-Specific Promoter Shows Improved Saccharification

Fast-growing broad-leaf tree species can serve as feedstocks for production of bio-based chemicals and fuels through biochemical conversion of wood to monosaccharides. This conversion is hampered by the xylan acetylation pattern. To reduce xylan acetylation in the wood, the Hypocrea jecorina acetyl xylan esterase (HjAXE) from carbohydrate esterase (CE) family 5 was expressed in hybrid aspen under the control of the wood-specific PtGT43B promoter and targeted to the secretory pathway. The enzyme was predicted to deacetylate polymeric xylan in the vicinity of cellulose due to the presence of a cellulose-binding module. Cell-wall-bound protein fractions from developing wood of transgenic plants were capable of releasing acetyl from finely ground wood powder, indicative of active AXE present in cell walls of these plants, whereas no such activity was detected in wild-type plants. The transgenic lines grew in height and diameter as well as wild-type trees, whereas their internodes were slightly shorter, indicating higher leaf production. The average acetyl content in the wood of these lines was reduced by 13%, mainly due to reductions in di-acetylated xylose units, and in C-2 and C-3 mono-acetylated xylose units. Analysis of soluble cell wall polysaccharides revealed a 4% reduction in the fraction of xylose units and an 18% increase in the fraction of glucose units, whereas the contents of cellulose and lignin were not affected. Enzymatic saccharification of wood from transgenic plants resulted in 27% higher glucose yield than for wild-type plants. Brunauer-Emmett-Teller (BET) analysis and Simons' staining pointed toward larger surface area and improved cellulose accessibility for wood from transgenic plants compared to wood from wild-type plants, which could be achieved by HjAXE deacetylating xylan bound to cellulose. The results show that CE5 family can serve as a source of enzymes for in planta reduction of recalcitrance to saccharification.Peer reviewe

Morpho-biochemical characterization of a RIL population for seed parameters and identification of candidate genes regulating seed size trait in lentil (Lens culinaris Medik.)

The seed size and shape in lentil (Lens culinaris Medik.) are important quality traits as these influences the milled grain yield, cooking time, and market class of the grains. Linkage analysis was done for seed size in a RIL (F5:6) population derived by crossing L830 (20.9 g/1000 seeds) with L4602 (42.13 g/1000 seeds) which consisted of 188 lines (15.0 to 40.5 g/1000 seeds). Parental polymorphism survey using 394 SSRs identified 31 polymorphic primers, which were used for the bulked segregant analysis (BSA). Marker PBALC449 differentiated the parents and small seed size bulk only, whereas large seeded bulk or the individual plants constituting the large-seeded bulk could not be differentiated. Single plant analysis identified only six recombinant and 13 heterozygotes, of 93 small-seeded RILs (<24.0 g/1000 seed). This clearly showed that the small seed size trait is very strongly regulated by the locus near PBLAC449; whereas, large seed size trait seems governed by more than one locus. The PCR amplified products from the PBLAC449 marker (149bp from L4602 and 131bp from L830) were cloned, sequenced and BLAST searched using the lentil reference genome and was found amplified from chromosome 03. Afterward, the nearby region on chromosome 3 was searched, and a few candidate genes like ubiquitin carboxyl-terminal hydrolase, E3 ubiquitin ligase, TIFY-like protein, and hexosyltransferase having a role in seed size determination were identified. Validation study in another RIL mapping population which is differing for seed size, showed a number of SNPs and InDels among these genes when studied using whole genome resequencing (WGRS) approach. Biochemical parameters like cellulose, lignin, and xylose content showed no significant differences between parents and the extreme RILs, at maturity. Various seed morphological traits like area, length, width, compactness, volume, perimeter, etc., when measured using VideometerLab 4.0 showed significant differences for the parents and RILs. The results have ultimately helped in better understanding the region regulating the seed size trait in genomically less explored crops like lentils

Downregulation of RWA genes in hybrid aspen affects xylan acetylation and wood saccharification

High acetylation of angiosperm wood hinders its conversion to sugars by glycoside hydrolases, subsequent ethanol fermentation and (hence) its use for biofuel production. We studied the REDUCED WALL ACETYLATION (RWA) gene family of the hardwood model Populus to evaluate its potential for improving saccharification. The family has two clades, AB and CD, containing two genes each. All four genes are expressed in developing wood but only RWA-A and -B are activated by master switches of the secondary cell wall PtNST1 and PtMYB21. Histochemical analysis of promoter:: GUS lines in hybrid aspen (Populus tremula x tremuloides) showed activation of RWA-A and -B promoters in the secondary wall formation zone, while RWA-C and -D promoter activity was diffuse. Ectopic downregulation of either clade reduced wood xylan and xyloglucan acetylation. Suppressing both clades simultaneously using the wood-specific promoter reduced wood acetylation by 25% and decreased acetylation at position 2 of Xylp in the dimethyl sulfoxide-extracted xylan. This did not affect plant growth but decreased xylose and increased glucose contents in the noncellulosic monosaccharide fraction, and increased glucose and xylose yields of wood enzymatic hydrolysis without pretreatment. Both RWA clades regulate wood xylan acetylation in aspen and are promising targets to improve wood saccharification.Peer reviewe

Acetylation of woody lignocellulose: significance and regulation

Non-cellulosic cell wall polysaccharides constitute approximately one quarter of usable biomass for human exploitation. In contrast to cellulose, these components are usually substituted by O-acetyl groups, which affect their properties and interactions with other polymers, thus affecting their solubility and extractability. However, details of these interactions are still largely obscure. Moreover, polysaccharide hydrolysis to constituent monosaccharides, is hampered by the presence of O-acetyl groups, necessitating either enzymatic (esterase) or chemical de-acetylation, increasing the costs and chemical consumption. Reduction of polysaccharide acetyl content in planta is a way to modify lignocellulose towards improved saccharification. In this review we: 1) summarize literature on lignocellulose acetylation in different tree species, 2) present data and current hypotheses concerning the role of O-acetylation in determining woody lignocellulose properties, 3) describe plant proteins involved in lignocellulose O-acetylation, 4) give examples of microbial enzymes capable to de-acetylate lignocellulose, and 5) discuss prospects for exploiting these enzymes in planta to modify xylan acetylation

Reduced Wall Acetylation Proteins Play Vital and Distinct Roles in Cell Wall O-Acetylation in Arabidopsis

The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwa2, which has about 20% lower level of O-acetylation in leaf cell walls and no obvious growth or developmental phenotype. In this study, we generated double, triple, and quadruple loss-of-function mutants of all four members of the RWA family in Arabidopsis (Arabidopsis thaliana). In contrast to rwa2, the triple and quadruple rwa mutants display severe growth phenotypes revealing the importance of wall acetylation for plant growth and development. The quadruple rwa mutant can be completely complemented with the RWA2 protein expressed under 35S promoter, indicating the functional redundancy of the RWA proteins. Nevertheless, the degree of acetylation of xylan, (gluco)mannan, and xyloglucan as well as overall cell wall acetylation is affected differently in different combinations of triple mutants, suggesting their diversity in substrate preference. The overall degree of wall acetylation in the rwa quadruple mutant was reduced by 63% compared with the wild type, and histochemical analysis of the rwa quadruple mutant stem indicates defects in cell differentiation of cell types with secondary cell walls

In muro deacetylation of xylan affects lignin properties and improves saccharification of aspen wood

Background: Lignocellulose from fast growing hardwood species is a preferred source of polysaccharides for advanced biofuels and “green” chemicals. However, the extensive acetylation of hardwood xylan hinders lignocellulose saccharification by obstructing enzymatic xylan hydrolysis and causing inhibitory acetic acid concentrations during microbial sugar fermentation. To optimize lignocellulose for cost-effective saccharification and biofuel production, an acetyl xylan esterase AnAXE1 from Aspergillus niger was introduced into aspen and targeted to cell walls. Results: AnAXE1-expressing plants exhibited reduced xylan acetylation and grew normally. Without pretreatment, their lignocellulose yielded over 25% more glucose per unit mass of wood (dry weight) than wild-type plants. Glucose yields were less improved (+7%) after acid pretreatment, which hydrolyses xylan. The results indicate that AnAXE1 expression also reduced the molecular weight of xylan, and xylan–lignin complexes and/or lignin co-extracted with xylan, increased cellulose crystallinity, altered the lignin composition, reducing its syringyl to guaiacyl ratio, and increased lignin solubility in dioxane and hot water. Lignin-associated carbohydrates became enriched in xylose residues, indicating a higher content of xylo-oligosaccharides. Conclusions: This work revealed several changes in plant cell walls caused by deacetylation of xylan. We propose that deacetylated xylan is partially hydrolyzed in the cell walls, liberating xylo-oligosaccharides and their associated lignin oligomers from the cell wall network. Deacetylating xylan thus not only increases its susceptibility to hydrolytic enzymes during saccharification but also changes the cell wall architecture, increasing the extractability of lignin and xylan and facilitating saccharification.Bio4Energ