A role for CSLD3 during cell-wall synthesis in apical plasma membranes of tip-growing root-hair cells.
Park, S., Szumlanski, A. L., Gu, F., Guo, F. & Nielsen, E. (2011). Nature Cell Biology, 13(8), 973-980.
In plants, cell shape is defined by the cell wall, and changes in cell shape and size are dictated by modification of existing cell walls and deposition of newly synthesized cell-wall material. In root hairs, expansion occurs by a process called tip growth, which is shared by root hairs, pollen tubes and fungal hyphae. We show that cellulose-like polysaccharides are present in root-hair tips, and de novo synthesis of these polysaccharides is required for tip growth. We also find that eYFP–CSLD3 proteins, but not CESA cellulose synthases, localize to a polarized plasma-membrane domain in root hairs. Using biochemical methods and genetic complementation of a csld3 mutant with a chimaeric CSLD3 protein containing a CESA6 catalytic domain, we provide evidence that CSLD3 represents a distinct (1→4)-β-glucan synthase activity in apical plasma membranes during tip growth in root-hair cells.
The synergistic action of accessory enzymes enhances the hydrolytic potential of a “cellulase mixture” but is highly substrate specific.
Hu, J., Arantes, V., Pribowo, A. & Saddler, J. N. (2013). Biotechnology for Biofuels, 6(1), 112.
Background: Currently, the amount of protein/enzyme required to achieve effective cellulose hydrolysis is still too high. One way to reduce the amount of protein/enzyme required is to formulate a more efficient enzyme cocktail by adding so-called accessory enzymes such as xylanase, lytic polysaccharide monooxygenase (AA9, formerly known as GH61), etc., to the cellulase mixture. Previous work has shown the strong synergism that can occur between cellulase and xylanase mixtures during the hydrolysis of steam pretreated corn stover, requiring lower protein loading to achieve effective hydrolysis. However, relatively high loadings of xylanases were required. When family 10 and 11 endo-xylanases and family 5 xyloglucanase were supplemented to a commercial cellulase mixture varying degrees of improved hydrolysis over a range of pretreated, lignocellulosic substrates were observed. Results: The potential synergistic interactions between cellulase monocomponents and hemicellulases from family 10 and 11 endo-xylanases (GH10 EX and GH11 EX) and family 5 xyloglucanase (GH5 XG), during hydrolysis of various steam pretreated lignocellulosic substrates, were assessed. It was apparent that the hydrolytic activity of cellulase monocomponents was enhanced by the addition of accessory enzymes although the “boosting” effect was highly substrate specific. The GH10 EX and GH5 XG both exhibited broad substrate specificity and showed strong synergistic interaction with the cellulases when added individually. The GH10 EX was more effective on steam pretreated agriculture residues and hardwood substrates whereas GH5 XG addition was more effective on softwood substrates. The synergistic interaction between GH10 EX and GH5 XG when added together further enhanced the hydrolytic activity of the cellulase enzymes over a range of pretreated lignocellulosic substrates. GH10 EX addition could also stimulate further cellulose hydrolysis when added to the hydrolysis reactions when the rate of hydrolysis had levelled off. Conclusions: Endo-xylanases and xyloglucanases interacted synergistically with cellulases to improve the hydrolysis of a range of pretreated lignocellulosic substrates. However, the extent of improved hydrolysis was highly substrate dependent. It appears that those accessory enzymes, such as GH10 EX and GH5 XG, with broader substrate specificities promoted the greatest improvements in the hydrolytic performance of the cellulase mixture on all of the pretreated biomass substrates.
Profiling the main cell wall polysaccharides of tobacco leaves using high-throughput and fractionation techniques.
Nguema-Ona, E., Moore, J. P., Fagerström, A., Fangel, J. U., Willats, W. G. T., Hugoc, A. & Vivier, M. A. (2012). Carbohydrate Polymers, 88(3), 939-949.
Nicotiana species are used to study agriculturally and industrially relevant processes, but limited screening methods are available for this species. A tobacco leaf cell wall preparation was fractionated using both chemical and enzymatic methods; the fractions obtained were subsequently analysed using rapid high-throughput wall profiling tools. The results confirmed previous data showing that mature tobacco leaf cell walls are predominantly composed of pectic homogalacturonans with lesser amounts of hemicellulosic arabinoxyloglucan and glucuronoxylan polymers. This confirmation provided proof that the profiling methods could generate good-quality results and paves the way for high-throughput screening of tobacco mutants where a range of biological processes, where the cell wall profile is important, are studied. A novel enzymatic oligosaccharide fingerprinting method was optimized to rapidly analyse the structure of XXGG-rich arabinoxyloglucans characteristic of Solanaceae species such as tobacco. Digestion profiles using two available xyloglucanase-specific endoglucanases: Trichoderma reseei EGII and Paenibacillus sp. xyloglucanase were compared showing that the latter enzyme has a higher specificity toward tobacco arabinoxyloglucans, and is better-suited for screening. This methodology would be suitable for species, such as tomato (Solanum lycopersicum) or potato (Solanum tuberosum), with similar XXGG-type motifs in their xyloglucan structure.
Identification of quantitative trait loci affecting hemicellulose characteristics based on cell wall composition in a wild and cultivated rice species.
Zhang, S. J., Song, X. Q., Yu, B. S., Zhang, B. C., Sun, C. Q., Knox, J. P. & Zhou, Y. H. (2012). Molecular Plant, 5(1), 162-175.
Cell wall hemicellulosic polysaccharides are structurally complex and diverse. Knowledge about the synthesis of cell wall hemicelluloses and their biological roles is limited. Quantitative trait loci (QTL) mapping is a helpful tool for the dissection of complex phenotypes for gene identification. In this study, we exploited the natural variation in cell wall monosaccharide levels between a common wild rice, Yuanj, and an elite indica cultivar, Teqing, and performed QTL mapping with their introgression lines (ILs). Chemical analyses conducted on the culms of Yuanj and Teqing showed that the major alterations are found in glucose and xylose levels, which are correlated with specific hemicellulosic polymers. Glycosidic linkage examination revealed that, in Yuanj, an increase in glucose content results from a higher level of mixed linkage β-glucan (MLG), whereas a reduction in xylose content reflects a low level of xylan backbone and a varied arabinoxylan (AX) structure. Seventeen QTLs for monosaccharides have been identified through composition analysis of the culm residues of 95 core ILs. Four major QTLs affecting xylose and glucose levels are responsible for 19 and 21% of the phenotypic variance, respectively. This study provides a unique resource for the genetic dissection of rice cell wall formation and remodeling in the vegetative organs.
XTH31, encoding an in vitro XEH/XET-active enzyme, regulates aluminum sensitivity by modulating in vivo XET action, cell wall xyloglucan content, and aluminum binding capacity in Arabidopsis.
Zhu, W. X. F., Shi, Y. Z., Lei, G. J., Fry, S. C., Zhang, B. C., Zhou, Y. H., Braam, J., Jiang, T., Xu, X. Y., Mao, C. Z., Pan, Y. J., Yang, J. L., Wu, P. & Zheng, S. J. (2012). The Plant Cell, 24(11), 4731-4747.
Xyloglucan endohydrolase (XEH) and xyloglucan endotransglucosylase (XET) activities, encoded by xyloglucan endotransglucosylase-hydrolase (XTH) genes, are involved in cell wall extension by cutting or cutting and rejoining xyloglucan chains, respectively. However, the physiological significance of this biochemical activity remains incompletely understood. Here, we find that an XTH31 T-DNA insertion mutant, xth31, is more Al resistant than the wild type. XTH31 is bound to the plasma membrane and the encoding gene is expressed in the root elongation zone and in nascent leaves, suggesting a role in cell expansion. XTH31 transcript accumulation is strongly downregulated by Al treatment. XTH31 expression in yeast yields a protein with an in vitro XEH:XET activity ratio of >5000:1. xth31 accumulates significantly less Al in the root apex and cell wall, shows remarkably lower in vivo XET action and extractable XET activity, has a lower xyloglucan content, and exhibits slower elongation. An exogenous supply of xyloglucan significantly ameliorates Al toxicity by reducing Al accumulation in the roots, owing to the formation of an Al-xyloglucan complex in the medium, as verified by an obvious change in chemical shift of 27Al-NMR. Taken together, the data indicate that XTH31 affects Al sensitivity by modulating cell wall xyloglucan content and Al binding capacity.
Recognition of xyloglucan by the crystalline cellulose‐binding site of a family 3a carbohydrate‐binding module.
Hernandez-Gomez, M. C., Rydahl, M. G., Rogowski, A., Morland, C., Cartmell, A., Crouch, L., Labourel, A., Fontes, C. M. G. A., Willats, W. G. T., Gilbert, H. J. & Knox, J. P. (2015). FEBS Letters, 589(18), 2297-2303.
Type A non-catalytic carbohydrate-binding modules (CBMs), exemplified by CtCBM3acipA, are widely believed to specifically target crystalline cellulose through entropic forces. Here we have tested the hypothesis that type A CBMs can also bind to xyloglucan (XG), a soluble β-1,4-glucan containing α-1,6-xylose side chains. CtCBM3acipA bound to xyloglucan in cell walls and arrayed on solid surfaces. Xyloglucan and cellulose were shown to bind to the same planar surface on CBM3acipA. A range of type A CBMs from different families were shown to bind to xyloglucan in solution with ligand binding driven by enthalpic changes. The nature of CBM-polysaccharide interactions is discussed.
Methods for Xyloglucan Structure Analysis in Brachypodium distachyon.
Liu, L. (2017). Brachypodium Genomics, Humana Press, New York, NY, 65-71.
Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) has become an important tool for the analysis of biomolecules, such as DNA, peptides, and oligosaccharides. This technique has been developed as a rapid, sensitive, and accurate means for analyzing cell wall polysaccharide structures. Here, we describe a method using mass spectrometry to provide xyloglucan composition and structure information of Brachypodium plants which will be useful for functional characterization of xyloglucan biosynthesis pathway in Brachypodium distachyon.
Structural Insights into the Thermophilic Adaption Mechanism of Endo-1,4-β-Xylanase from Caldicellulosiruptor owensensis.
Liu, X., Liu, T., Zhang, Y., Xin, F., Mi, S., Wen, B., Gu, T., Xinyuan Shi, X., Wang, F. & Sun, L. (2017). Journal of agricultural and food chemistry, 66(1), pp 187-193.
Xylanases (EC 18.104.22.168) are a kind of enzymes degrading xylan to xylooligosaccharides (XOS) and have been widely used in a variety of industrial applications. Among them, xylanases from thermophilic microorganisms have distinct advantages in industries that require high temperature conditions. The CoXynA gene, encoding a glycoside hydrolase (GH) family 10 xylanase, was identified from thermophilic Caldicellulosiruptor owensensis and was overexpressed in Escherichia coli. Recombinant CoXynA showed optimal activity at 90°C with a half-life of about 1 h at 80°C and exhibited highest activity at pH 7.0. The activity of CoXynA activity was affected by a variety of cations. CoXynA showed distinct substrate specificities for beechwood xylan and birchwood xylan. The crystal structure of CoXynA was solved and a molecular dynamics simulation of CoXynA was performed. The relatively high thermostability of CoXynA was proposed to be due to the increased overall protein rigidity resulting from the reduced length and fluctuation of Loop 7.
Xyloglucan Fucosylation Modulates Arabidopsis Cell Wall Hemicellulose Aluminium binding Capacity.
Wan, J. X., Zhu, X. F., Wang, Y. Q., Liu, L. Y., Zhang, B. C., Li, G. X., Zhou, Y. H. & Zheng, S. J. (2018). Scientific Reports, 8(1), 428.
Although xyloglucan (XyG) is reported to bind Aluminium (Al), the influence of XyG fucosylation on the cell wall Al binding capacity and plant Al stress responses is unclear. We show that Arabidopsis T-DNA insertion mutants with reduced AXY3 (XYLOSIDASE1) function and consequent reduced levels of fucosylated XyG are more sensitive to Al than wild-type Col-0 (WT). In contrast, T-DNA insertion mutants with reduced AXY8 (FUC95A) function and consequent increased levels of fucosylated XyG are more Al resistant. AXY3 transcript levels are strongly down regulated in response to 30 min Al treatment, whilst AXY8 transcript levels also repressed until 6 h following treatment onset. Mutants lacking AXY3 or AXY8 function exhibit opposing effects on Al contents of root cell wall and cell wall hemicellulose components. However, there was no difference in the amount of Al retained in the pectin components between mutants and WT. Finally, whilst the total sugar content of the hemicellulose fraction did not change, the altered hemicellulose Al content of the mutants is shown to be a likely consequence of their different XyG fucosylation levels. We conclude that variation in XyG fucosylation levels influences the Al sensitivity of Arabidopsis by affecting the Al-binding capacity of hemicellulose.