Search

Background: Dissection of complex plant cell wall structures demands a sensitive and quantitative method. FTIR is used regularly as a screening method to identify specific linkages in cell walls. However, quantification and assigning spectral bands to particular cell wall components is still a major challenge, specifically in crop species. In this study, we addressed these challenges using ATR-FTIR spectroscopy as it is a high throughput, cost-effective and non-destructive approach to understand the plant cell wall composition. This method was validated by analysing different varieties of mungbean which is one of the most important legume crops grown widely in Asia. Results: Using standards and extraction of a specific component of cell wall components, we assigned 1050–1060 cm−1 and 1390–1420 cm−1 wavenumbers that can be widely used to quantify cellulose and lignin, respectively, in Arabidopsis, Populus, rice and mungbean. Also, using KBr as a diluent, we established a method that can relatively quantify the cellulose and lignin composition among different tissue types of the above species. We further used this method to quantify cellulose and lignin in field-grown mungbean genotypes. The ATR-FTIR-based study revealed the cellulose content variation ranges from 27.9% to 52.3%, and the lignin content variation ranges from 13.7% to 31.6% in mungbean genotypes. Conclusion: Multivariate analysis of FT-IR data revealed differences in total cell wall (600–2000 cm−1), cellulose (1000–1100 cm−1) and lignin (1390–1420 cm−1) among leaf and stem of four plant species. Overall, our data suggested that ATR-FTIR can be used for the relative quantification of lignin and cellulose in different plant species. This method was successfully applied for rapid screening of cell wall composition in mungbean stem, and similarly, it can be used for screening other crops or tree species. [ABSTRACT FROM AUTHOR]

Background: Lignin and xylan are important determinants of cell wall structure and lignocellulosic biomass digestibility. Genetic manipulations that individually modify either lignin or xylan structure improve polysaccharide digestibility. However, the effects of their simultaneous modifications have not been explored in a similar context. Here, both individual and combinatorial modification in xylan and lignin was studied by analysing the effect on plant cell wall properties, biotic stress responses and integrity sensing. Results: Arabidopsis plant co-harbouring mutation in FERULATE 5-HYDROXYLASE (F5H) and overexpressing Aspergillus niger acetyl xylan esterase (35S:AnAXE1) were generated and displayed normal growth attributes with intact xylem architecture. This fah1-2/35S:AnAXE1 cross was named as hyper G lignin and hypoacetylated (HrGHypAc) line. The HrGHypAc plants showed increased crystalline cellulose content with enhanced digestibility after chemical and enzymatic pre-treatment. Moreover, both parents and HrGHypAc without and after pre-treating with glucuronyl esterase and alpha glucuronidase exhibited an increase in xylose release after xylanase digestion as compared to wild type. The de-pectinated fraction in HrGHypAc displayed elevated levels of xylan and cellulose. Furthermore, the transcriptomic analysis revealed differential expression in cell wall biosynthetic, transcription factors and wall-associated kinases genes implying the role of lignin and xylan modification on cellular regulatory processes. Conclusions: Simultaneous modification in xylan and lignin enhances cellulose content with improved saccharification efficiency. These modifications loosen cell wall complexity and hence resulted in enhanced xylose and xylobiose release with or without pretreatment after xylanase digestion in both parent and HrGHypAc. This study also revealed that the disruption of xylan and lignin structure is possible without compromising either growth and development or defense responses against Pseudomonas syringae infection. [ABSTRACT FROM AUTHOR]

Market class, cooking time, quality, and milled grain yield are largely influenced by the seed size and shape of the lentil (Lens culinaris Medik.); thus, they are considered to be important quality traits. To unfold the pathways regulating seed size in lentils, a transcriptomic approach was performed using largeseeded (L4602) and small-seeded (L830) genotypes. The study has generated nearly 375 million high-quality reads, of which 98.70% were properly aligned to the reference genome. Among biological replicates, very high similarity in fragments per kilobase of exon per million mapped fragments values (R > 0.9) showed the consistency of RNA-seq results. Various differentially expressed genes associated mainly with the hormone signaling and cell division pathways, transcription factors, kinases, etc. were identified as having a role in cell expansion and seed growth. A total of 106,996 unigenes were used for differential expression (DE) analysis. String analysis identified various modules having certain key proteins like Ser/Thr protein kinase, seed storage protein, DNA-binding protein, microtubule-associated protein, etc. In addition, some growth and cell division–related micro-RNAs like miR3457 (cell wall formation), miR1440 (cell proliferation and cell cycles), and miR1533 (biosynthesis of plant hormones) were identified as having a role in seed size determination. Using RNA-seq data, 5254 EST-SSR primers were generated as a source for future studies aiming for the identification of linked markers. In silico validation using Genevestigator® was done for the Ser/Thr protein kinase, ethylene response factor, and Myb transcription factor genes. It is of interest that the xyloglucan endotransglucosylase gene was found differentially regulated, suggesting their role during seed development; however, at maturity, no significant differences were recorded for various cell wall parameters including cellulose, lignin, and xylose content. This is the first report on lentils that has unfolded the key seed size regulating pathways and unveiled a theoretical way for the development of lentil genotypes having customized seed sizes. [ABSTRACT FROM AUTHOR]

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 (Hj AXE) from carbohydrate esterase (CE) family 5 was expressed in hybrid aspen under the control of the wood-specific Pt GT43B 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 Hj AXE 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. [ABSTRACT FROM AUTHOR]

Cell wall hemicelluloses and pectins are O-acetylated at specific positions, but the significance of these substitutions is poorly understood. Using a transgenic approach, we investigated how reducing the extent of O-acetylation in xylan affects cell wall chemistry, plant performance and the recalcitrance of lignocellulose to saccharification. The Aspergillus niger acetyl xylan esterase AnAXE1 was expressed in Arabidopsis under the control of either the constitutively expressed 35S CAMV promoter or a woody-tissue-specific GT43B aspen promoter, and the protein was targeted to the apoplast by its native signal peptide, resulting in elevated acetyl esterase activity in soluble and wall-bound protein extracts and reduced xylan acetylation. No significant alterations in cell wall composition were observed in the transgenic lines, but their xylans were more easily digested by a β-1,4-endoxylanase, and more readily extracted by hot water, acids or alkali. Enzymatic saccharification of lignocellulose after hot water and alkali pretreatments produced up to 20% more reducing sugars in several lines. Fermentation by Trametes versicolor of tissue hydrolysates from the line with a 30% reduction in acetyl content yielded ∼70% more ethanol compared with wild type. Plants expressing 35S:AnAXE1 and pGT43B:AnAXE1 developed normally and showed increased resistance to the biotrophic pathogen Hyaloperonospora arabidopsidis, probably due to constitutive activation of defence pathways. However, unintended changes in xyloglucan and pectin acetylation were only observed in 35S:AnAXE1- expressing plants. This study demonstrates that postsynthetic xylan deacetylation in woody tissues is a promising strategy for optimizing lignocellulosic biomass for biofuel production. [ABSTRACT FROM AUTHOR]

The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT-q PCR and histochemical ( GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall-forming cells (wood and extraxylary fibres). Intriguingly, GT43 A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary-walled cells than GT43 C and D genes ( IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43 E, F and G ( IRX9- L clade), showed broad expression patterns. Transient transactivation analyses of GT43 A and B reporters demonstrated that they are activated by Ptxt MYB021 and PNAC085 (master secondary wall switches), mediated in Ptxt MYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43 B expression prompted tests of the efficiency of its promoter ( p GT43 B), relative to the Ca MV 35 S ( 35 S) promoter, for overexpressing a xylan acetyl esterase ( CE5) or downregulating REDUCED WALL ACETYLATION ( RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by p GT43 B, but it reduced wood acetyl content substantially more efficiently than the 35 S promoter. RNAi silencing of the RWA family, which was ineffective using 35 S, was achieved when using GT43 B promoter. These results show the utility of the GT43 B promoter for genetically engineering properties of wood and fibres. [ABSTRACT FROM AUTHOR]

The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwal, 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 rwal, 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. [ABSTRACT FROM AUTHOR]

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 toward 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. [ABSTRACT FROM AUTHOR]

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 Oacetyl 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. [ABSTRACT FROM AUTHOR]

The Reduced Wall Acetylation (RWA) proteins are involved in cell wall acetylation in plants. Previously, we described a single mutant, rwal, 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 rwal, 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. [ABSTRACT FROM AUTHOR]