Search

SUMMARY: Cell walls are dynamic and multi‐component materials that play important roles in many areas of plant biology. The composition and interactions of the structural elements give rise to material properties, which are modulated by the activity of wall‐related enzymes. Studies of the genes and enzymes that determine wall composition and function have made great progress, but rarely take account of potential compensatory changes in wall polymers that may accompany and accommodate changes in other components, particularly for specific polysaccharides. Here, we present a method that allows the simultaneous examination of the mass distributions and quantities of specific cell wall matrix components, allowing insight into direct and indirect consequences of cell wall manipulations. The method employs gel‐permeation chromatography fractionation of cell wall polymers followed by enzyme‐linked immunosorbent assay to identify polymer types. We demonstrate the potential of this method using glycan‐directed monoclonal antibodies to detect epitopes representing xyloglucans, heteromannans, glucuronoxylans, homogalacturonans (HGs) and methyl‐esterified HGs. The method was used to explore compositional diversity in different Arabidopsis organs and to examine the impacts of changing wall composition in a number of previously characterized cell wall mutants. As demonstrated in this article, this methodology allows a much deeper understanding of wall composition, its dynamism and plasticity to be obtained, furthering our knowledge of cell wall biology. [ABSTRACT FROM AUTHOR]

Biotechnologies that aim to produce renewable fuels, chemicals, and bioproducts from residual ligno(hemi)cellulosic biomass mostly rely on enzymatic depolymerization of plant cell walls (PCW). This process requires an arsenal of diverse enzymes, including xylanases, which synergistically act on the hemicellulose, reducing the long and complex xylan chains to oligomers and simple sugars. Thus, xylanases play a crucial role in PCW depolymerization. Until recently, the largest xylanase family, glycoside hydrolase family 11 (GH11) has been exclusively represented by endo-catalytic β-1,4- and β-1,3-xylanases. Analysis of a metatranscriptome library from a microbial lignocellulose community resulted in the identification of an unusual exo-acting GH11 β-1,4-xylanase (MetXyn11). Detailed characterization has been performed on recombinant MetXyn11 including determination of its low-resolution small-angle X-ray scattering (SAXS) molecular envelope in solution. Our results reveal that MetXyn11 is a monomeric globular enzyme that liberates xylobiose from heteroxylans as the only product. MetXyn11 has an optimal activity in a pH range from 6 to 9 and an optimal temperature of 50 °C. The enzyme maintained above 65% of its original activity in the pH range 5 to 6 after being incubated for 72 h at 50 °C. Addition of the enzyme to a commercial enzymatic cocktail (CelicCtec3) promoted a significant increase of enzymatic hydrolysis yields of hydrothermally pretreated sugarcane bagasse (16% after 24 h of hydrolysis). [ABSTRACT FROM AUTHOR]

Sudangrass, Sorghum sudanense (Piper) Stapf, is a vigorous forage crop that has also been used for biogas, paper, and electricity production. Due to the large biomass yields achieved by sudangrass and the large area of potential growth in Argentina seven sudangrass accessions from a collection of S. sudanense were analyzed to evaluate their potential as feedstocks for lignocellulosic bioethanol production, and to assess whether there is an association between the response to biotic and abiotic stresses and the composition of the biomass. The biomass composition was analyzed for major cell wall polymers, monosaccharides, and elemental composition. On average, 68% of stem lignocellulosic biomass was comprised of matrix polysaccharides and crystalline cellulose, representing a potential source of sugars for bioethanol production. Xylose was the predominant matrix polysaccharide monosaccharide comprising, on average, 45% of the total sugars, followed by arabinose, glucose, galactose, galacturonic acid, mannose, glucuronic acid, and fucose. Rhamnose was not detected in any of the biomasses analyzed. Silica was the most abundant element in sudangrass stem, followed by chloride, calcium, phosphorus and sulfur. We performed saccharification analyses after pretreatments. Alkaline pretreatment was more effective than water pretreatment. Sodium hydroxide pretreatment exposed different levels of recalcitrance among sudangrass accessions, whereas the water pretreatment did not. Phenological traits were also evaluated, showing significant variability among accessions. The comparison of major cell wall polymers and monosaccharide composition between tolerant and susceptible accessions to abiotic and biotic stresses suggests an association between the composition of the biomass and the response to stress. [ABSTRACT FROM AUTHOR]

Main conclusion: Methyl-jasmonate induces large increases in p-coumarate linked to arabinoxylan in Brachypodium and in abundance of GT61 and BAHD family transcripts consistent with a role in synthesis of this linkage.Jasmonic acid (JA) signalling is required for many stress responses in plants, inducing large changes in the transcriptome, including up-regulation of transcripts associated with lignification. However, less is known about the response to JA of grass cell walls and the monocot-specific features of arabinoxylan (AX) synthesis and acylation by ferulic acid (FA) and para-coumaric acid (pCA). Here, we show that methyl-jasmonate (MeJA) induces moderate increases in FA monomer, > 50% increases in FA dimers, and five-sixfold increases in pCA ester-linked to cell walls in Brachypodium callus. Direct measurement of arabinose acylated by pCA (Araf-pCA) indicated that most or all the increase in cell-wall pCA was due to pCA ester-linked to AX. Analysis of the RNA-seq transcriptome of the callus response showed that these cell-wall changes were accompanied by up-regulation of members of the GT61 and BAHD gene families implicated in AX decoration and acylation; two BAHD paralogues were among the most up-regulated cell-wall genes (seven and fivefold) after 24 h exposure to MeJA. Similar responses to JA of orthologous BAHD and GT61 transcripts are present in the RiceXPro public expression data set for rice seedlings, showing that they are not specific to Brachypodium or to callus. The large response of AX-pCA to MeJA may, therefore, indicate an important role for this linkage in response of primary cell walls of grasses to JA signalling. [ABSTRACT FROM AUTHOR]

Background: Miscanthus sinensis is a high yielding perennial grass species with great potential as a bioenergy feedstock. One of the challenges that currently impedes commercial cellulosic biofuel production is the technical difficulty to efficiently convert lignocellulosic biomass into biofuel. The development of feedstocks with better biomass quality will improve conversion efficiency and the sustainability of the value-chain. Progress in the genetic improvement of biomass quality may be substantially expedited by the development of genetic markers associated to quality traits, which can be used in a marker-assisted selection program. Results: To this end, a mapping population was developed by crossing two parents of contrasting cell wall composition. The performance of 182 F1 offspring individuals along with the parents was evaluated in a field trial with a randomized block design with three replicates. Plants were phenotyped for cell wall composition and conversion efficiency characters in the second and third growth season after establishment. A new SNP-based genetic map for M. sinensis was built using a genotyping-by-sequencing (GBS) approach, which resulted in 464 short-sequence uniparental markers that formed 16 linkage groups in the male map and 17 linkage groups in the female map. A total of 86 QTLs for a variety of biomass quality characteristics were identified, 20 of which were detected in both growth seasons. Twenty QTLs were directly associated to different conversion efficiency characters. Marker sequences were aligned to the sorghum reference genome to facilitate cross-species comparisons. Analyses revealed that for some traits previously identified QTLs in sorghum occurred in homologous regions on the same chromosome. Conclusion: In this work we report for the first time the genetic mapping of cell wall composition and bioconversion traits in the bioenergy crop miscanthus. These results are a first step towards the development of marker-assisted selection programs in miscanthus to improve biomass quality and facilitate its use as feedstock for biofuel production. [ABSTRACT FROM AUTHOR]

Rice is the staple food of almost half of the world population, and in excess 90% of it is grown and consumed in Asia, but the disposal of rice straw poses a problem for farmers, who often burn it in the fields, causing health and environmental problems. However, with increased focus on the development of sustainable biofuel production, rice straw has been recognized as a potential feedstock for non-food derived biofuel production. Currently, the commercial realization of rice as a biofuel feedstock is constrained by the high cost of industrial saccharification processes needed to release sugar for fermentation. This study is focused on the alteration of lignin content, and cell wall chemotypes and structures, and their effects on the saccharification potential of rice lignocellulosic biomass. A recombinant inbred lines (RILs) population derived from a cross between the lowland rice variety IR1552 and the upland rice variety Azucena with 271 molecular markers for quantitative trait SNP (QTS) analyses was used. After association analysis of 271 markers for saccharification potential, 1 locus and 4 pairs of epistatic loci were found to contribute to the enzymatic digestibility phenotype, and an inverse relationship between reducing sugar and lignin content in these recombinant inbred lines was identified. As a result of QTS analyses, several cell-wall associated candidate genes are proposed that may be useful for marker-assisted breeding and may aid breeders to produce potential high saccharification rice varieties. [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 Arabidopsis genome includes seven family 34 glycosyltransferase (GT34) encoding genes. XXT1 and XXT2 have previously been shown to encode XyG α-1,6-xylosyltransferases, while knockout mutants of a third, XXT5, exhibit decreased XyG content, suggesting a similar activity. Here, we extend the study to the rest of the Arabidopsis GT34 genes in terms of biochemical activity and their roles in XyG biosynthesis., The enzyme activity of XXTs was investigated using recombinant protein expressed in E. coli. XyG analysis of single and double T-DNA insertion knockouts, together with overexpression of GT34s in selected mutant lines, provided detailed function of each gene., We reveal the activity of the third member of the GT34 gene family (XXT4) that exhibits xylosyltransferase activity. Double mutants for either xxt2 or xxt5 had a large impact on XyG content, structure and size distribution. Overexpression of the remaining member, XXT3, was able to restore XyG epitopes in xxt2, xxt5 and xxt2 xxt5 double knockouts, suggesting that it also encodes a protein with XXT activity., Our work demonstrates that five of the seven Arabidopsis GT34 genes encode XXT enzymes. [ABSTRACT FROM AUTHOR]

There are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental evidence to assign a function to it. Five of the 10 Arabidopsis DUF579 family members are co-expressed with marker genes for secondary cell wall formation. Plants in which two closely related members of the DUF579 family have been disrupted by T-DNA insertions contain less xylose in the secondary cell wall as a result of decreased xylan content, and exhibit mildly distorted xylem vessels. Consequently we have named these genes IRREGULAR XYLEM 15 ( IRX15) and IRX15L. These mutant plants exhibit many features of previously described xylan synthesis mutants, such as the replacement of glucuronic acid side chains with methylglucuronic acid side chains. By contrast, immunostaining of xylan and transmission electron microscopy (TEM) reveals that the walls of these irx15 irx15l double mutants are disorganized, compared with the wild type or other previously described xylan mutants, and exhibit dramatic increases in the quantity of sugar released in cell wall digestibility assays. Furthermore, localization studies using fluorescent fusion proteins label both the Golgi and also an unknown intracellular compartment. These data are consistent with irx15 and irx15l defining a new class of genes involved in xylan biosynthesis. How these genes function during xylan biosynthesis and deposition is discussed. [ABSTRACT FROM AUTHOR]

Background: Cell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies. Results: We developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared. Conclusions: The automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies. [ABSTRACT FROM AUTHOR]

Expansins are cell wall proteins associated with the process of plant growth. However, investigations in which expansin gene expression has been manipulated throughout the plant have often led to inconclusive results. In this article, we report on a series of experiments in which overexpression of expansin was targeted to specific phases of leaf growth using an inducible promoter system. The data indicate that there is a restricted window of sensitivity when increased expansin gene expression leads to increased endogenous expansin activity and an increase in leaf growth. This phase of maximum expansin efficacy corresponds to the mid phase of leaf growth. We propose that the effectiveness of expansin action depends on the presence of other modulating factors in the leaf and we suggest that it is the control of expression of these factors (in conjunction with expansin gene expression) that defines the extent of leaf growth. These data help to explain some of the previously observed variation in growth response following manipulation of expansin gene expression and highlight a potential linkage of the expression of modifiers of expansin activity with the process of exit from cell division. [ABSTRACT FROM AUTHOR]

Arabinans are found in the pectic network of many cell walls, where, along with galactan, they are present as side chains of Rhamnogalacturonan l. Whilst arabinans have been reported to be abundant polymers in the cell walls of seeds from a range of plant species, their proposed role as a storage reserve has not been thoroughly investigated. In the cell walls of Arabidopsis seeds, arabinose accounts for approximately 40% of the monosaccharide composition of non-cellulosic polysaccharides of embryos. Arabinose levels decline to ∼15% during seedling establishment, indicating that cell wall arabinans may be mobilized during germination. Immunolocalization of arabinan in embryos, seeds, and seedlings reveals that arabinans accumulate in developing and mature embryos, but disappear during germination and seedling establishment. Experiments using 14C-arabinose show that it is readily incorporated and metabolized in growing seedlings, indicating an active catabolic pathway for this sugar. We found that depleting arabinans in seeds using a fungal arabinanase causes delayed seedling growth, lending support to the hypothesis that these polymers may help fuel early seedling growth. [ABSTRACT FROM PUBLISHER]

Expansins are primary agents inducing cell wall extension, and are therefore obvious targets in biotechnological applications aimed at the modification of cell size in plants. In trees, increased fibre length is a goal of both breeding and genetic engineering programmes. We used an α-expansin PttEXPA1 that is highly abundant in the wood-forming tissues of hybrid aspen ( Populus tremula L. ×  P. tremuloides Michx.) to evaluate its role in fibre elongation and wood cell development. PttEXPA1 belongs to Subfamily A of α-expansins that have conserved motifs at the N- and C-termini of the mature protein. When PttEXPA1 was over-expressed in aspen, an extract of the cell wall-bound proteins of the transgenic plants exhibited an increased expansin activity on cellulose–xyloglucan composites in vitro, indicating that PttEXPA1 is an active expansin. The transgenic lines exhibited increased stem internode elongation and leaf expansion, and larger cell sizes in the leaf epidermis, indicating that PttEXPA1 protein is capable of increasing the growth of these organs by enhancing cell wall expansion in planta. Wood cell development was also modified in the transgenic lines, but the effects were different for vessel elements and fibres, the two main cell types of aspen wood. PttEXPA1 stimulated fibre, but not vessel element, diameter growth, and marginally increased vessel element length, but did not affect fibre length. The observed differences in responsiveness to expansin of these cell types are discussed in the light of differences in their growth strategies and cell wall composition. [ABSTRACT FROM AUTHOR]

Craterostigma plantagineum is one of the few higher plants capable of surviving desiccation throughout its vegetative tissues. Water loss results in cell shrinkage and a commensurate folding of the cell wall indicating an unusual degree of wall flexibility. We show that wall extensibility undergoes a marked increase during dehydration and rehydration. Similar increases were observed in the activity of expansins in cell walls during these processes suggesting a role for these proteins in increasing wall flexibility. Three α-expansin cDNAs were cloned from dehydrating leaves and transcript levels for one correlated closely with the observed changes in expansin activity during the dehydration and rehydration of leaves. [Copyright &y& Elsevier]

Discusses data that arabinan chains play a key role in determining guard cell wall flexibility in plants. Preparation of stomata; Molecular basis of the physical properties of plant cell walls; Finding that hydrolysis of cell wall homogalacturonan increases stomatal aperture.

Expansins are cell wall proteins thought to play an important role in growth and other events involving cell wall modifications. Whereas the expression patterns of many isoforms have been characterised, the nature of their activity is still poorly understood. Large amounts of active expansins are necessary to undertake biochemical studies and identify their substrates. We report here the successful expression of a recombinant expansin [CsExp1, isolated from cucumber (Cucumis sativus L.) hypocotyls] in transgenic tomato (Lycopersicon esculentum Mill.) plants, under the control of a constitutive promoter. In some transformants, CsExp1 transcript and protein accumulated to high levels, and expansin activity extractable from the cell walls was increased up to about 20-fold the activity measured in wild-type plants. These results confirm the identity of the CsExp1 coding sequence, and will enable large quantities of active expansin to be obtained for further studies. [ABSTRACT FROM AUTHOR]

Investigates the action of expansion using cellulose composites. Increase of the total extension in xyloglucan composites; Effects of expansion on uniaxial extensions with glucomanna; Target of alpha-expansion in cell walls.

Informs about the role that expansin, an extracellular protein, plays in the increasing of plant cell wall extensibility. Expansin inducing tissue expansion in vivo; Localized control of tissue expansion possibly sufficient to induce leaf formation; Suggestion of a role for biophysical forces in the regulation of plant development.