Two Arabidopsis proteins synthesize acetylated xylan in vitro

Xylan is the third most abundant glycopolymer on earth after cellulose and chitin. As a major component of wood, grain and forage, this natural biopolymer has far‐reaching impacts on human life. This highly acetylated cell wall polysaccharide is a vital component of the plant cell wall, which functions as a molecular scaffold, providing plants with mechanical strength and flexibility. Mutations that impair synthesis of the xylan backbone give rise to plants that fail to grow normally because of collapsed xylem cells in the vascular system. Phenotypic analysis of these mutants has implicated many proteins in xylan biosynthesis; however, the enzymes directly responsible for elongation and acetylation of the xylan backbone have not been unambiguously identified. Here we provide direct biochemical evidence that two Arabidopsis thaliana proteins, IRREGULAR XYLEM 10–L (IRX10‐L) and ESKIMO1/TRICOME BIREFRINGENCE 29 (ESK1/TBL29), catalyze these respective processes in vitro. By identifying the elusive xylan synthase and establishing ESK1/TBL29 as the archetypal plant polysaccharide O‐acetyltransferase, we have resolved two long‐standing questions in plant cell wall biochemistry. These findings shed light on integral steps in the molecular pathways used by plants to synthesize a major component of the world's biomass and expand our toolkit for producing glycopolymers with valuable properties.     

The pattern of xylan acetylation suggests xylan may interact with cellulose microfibrils as a twofold helical screw in the secondary plant cell wall of Arabidopsis thaliana

The interaction between xylan and cellulose microfibrils is important for secondary cell wall properties in vascular plants; however, the molecular arrangement of xylan in the cell wall and the nature of the molecular bonding between the polysaccharides are unknown. In dicots, the xylan backbone of β‐(1,4)‐linked xylosyl residues is decorated by occasional glucuronic acid, and approximately one‐half of the xylosyl residues are O‐acetylated at C‐2 or C‐3. We recently proposed that the even, periodic spacing of GlcA residues in the major domain of dicot xylan might allow the xylan backbone to fold as a twofold helical screw to facilitate alignment along, and stable interaction with, cellulose fibrils; however, such an interaction might be adversely impacted by random acetylation of the xylan backbone. Here, we investigated the arrangement of acetyl residues in Arabidopsis xylan using mass spectrometry and NMR. Alternate xylosyl residues along the backbone are acetylated. Using molecular dynamics simulation, we found that a twofold helical screw conformation of xylan is stable in interactions with both hydrophilic and hydrophobic cellulose faces. Tight docking of xylan on the hydrophilic faces is feasible only for xylan decorated on alternate residues and folded as a twofold helical screw. The findings suggest an explanation for the importance of acetylation for xylan–cellulose interactions, and also have implications for our understanding of cell wall molecular architecture and properties, and biological degradation by pathogens and fungi. They will also impact strategies to improve lignocellulose processing for biorefining and bioenergy.     

A G protein-coupled receptor-like module regulates cellulose synthase secretion from the endomembrane system in Arabidopsis

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Expression of fungal acetyl xylan esterase in Arabidopsis thaliana improves saccharification of stem lignocellulose

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.     

Biosynthesis of pure araban and xylan

A crude particulate enzyme preparation from mung bean shoots partially freed from sugar transferases synthesized pure araban from UDP-l-¹⁴C-arabinose. The preparation thus allowed to study some properties of the UDP-arabinose transferase which was shown to require 7 mM Mn²⁺ and pH 6–6·5 for optimal activity. Pure xylan was synthesized from UDP-d-¹⁴C-xylose if a mixture of 0·06% Triton X100 and 35 mM EDTA was added to the crude enzyme preparation. In contrast to the UDP-arabinose transferase the UDP-xylose transferase does not require bivalent metal ions.     

Cortical microtubules optimize cell-wall crystallinity to drive unidirectional growth in Arabidopsis

The shape of plants depends on cellulose, a biopolymer that self-assembles into crystalline, inextensible microfibrils (CMFs) upon synthesis at the plasma membrane by multi-enzyme cellulose synthase complexes (CSCs). CSCs are displaced in directions predicted by underlying parallel arrays of cortical microtubules, but CMFs remain transverse in cells that have lost the ability to expand unidirectionally as a result of disrupted microtubules. These conflicting findings suggest that microtubules are important for some physico-chemical property of cellulose that maintains wall integrity. Using X-ray diffraction, we demonstrate that abundant microtubules enable a decrease in the degree of wall crystallinity during rapid growth at high temperatures. Reduced microtubule polymer mass in the mor1-1 mutant at high temperatures is associated with failure of crystallinity to decrease and a loss of unidirectional expansion. Promotion of microtubule bundling by over-expressing the RIC1 microtubule-associated protein reduced the degree of crystallinity. Using live-cell imaging, we detected an increase in the proportion of CSCs that track in microtubule-free domains in mor1-1, and an increase in the CSC velocity. These results suggest that microtubule domains affect glucan chain crystallization during unidirectional cell expansion. Microtubule disruption had no obvious effect on the orientation of CMFs in dark-grown hypocotyl cells. CMFs at the outer face of the hypocotyl epidermal cells had highly variable orientation, in contrast to the transverse CMFs on the radial and inner periclinal walls. This suggests that the outer epidermal mechanical properties are relatively isotropic, and that axial expansion is largely dependent on the inner tissue layers.     

Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579-containing proteins that are essential for normal xylan deposition in the secondary cell wall

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.     

Cryo-electron tomography of the onion cell wall shows bimodally oriented cellulose fibers and reticulated homogalacturonan networks

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A mutation in an Arabidopsis ribose 5-phosphate isomerase reduces cellulose synthesis and is rescued by exogenous uridine

The Arabidopsis radial swelling mutant rsw10 showed ballooning of root trichoblasts, a lower than wild-type level of cellulose and altered levels of some monosaccharides in non-cellulosic polysaccharides. Map-based cloning showed that the mutated gene (At1g71100) encodes a ribose 5-phosphate isomerase (RPI) and that the rsw10 mutation replaces a conserved glutamic acid residue with lysine. Although RPI is intimately involved with many biochemical pathways, media supplementation experiments suggest that the visible phenotype results from a defect in the production of pyrimidine-based sugar-nucleotide compounds, most likely uridine 5'-diphosphate-glucose, the presumed substrate of cellulose synthase. Two of three RPI sequences in the nuclear genome are cytoplasmic, while the third has a putative chloroplast transit sequence. The sequence encoding both cytoplasmic enzymes could complement the mutation when expressed behind the CaMV 35S promoter, while fusion of the RSW10 promoter region to the GUS reporter gene established that the gene is expressed in many aerial tissues as well as the roots. The prominence of the rsw10 phenotype in roots probably reflects RSW10 being the only cytosolic RPI in this tissue and the gene encoding the plastid RPI being relatively weakly expressed. We could not, however, detect a decrease in total RPI activity in root extracts. The rsw10 phenotype is prominent near the root tip where cells undergo division, endoreduplication and cell expansion and so are susceptible to a restriction in de novo pyrimidine production. The two cytosolic RPIs probably arose in an ancient duplication event, their present expression patterns representing subfunctionalization of the expression of the original ancestral gene.     

Secondary cell wall patterning—connecting the dots,pits and helices

All plant cells are encased in primary cell walls that determine plant morphology, but also protect the cells against the environment. Certain cells also produce a secondary wall that supports mechanically demanding processes, such as maintaining plant body stature and water transport inside plants. Both these walls are primarily composed of polysaccharides that are arranged in certain patterns to support cell functions. A key requisite for patterned cell walls is the arrangement of cortical microtubules that may direct the delivery of wall polymers and/or cell wall producing enzymes to certain plasma membrane locations. Microtubules also steer the synthesis of cellulose—the load-bearing structure in cell walls—at the plasma membrane. The organization and behaviour of the microtubule array are thus of fundamental importance to cell wall patterns. These aspects are controlled by the coordinated effort of small GTPases that probably coordinate a Turing''s reaction–diffusion mechanism to drive microtubule patterns. Here, we give an overview on how wall patterns form in the water-transporting xylem vessels of plants. We discuss systems that have been used to dissect mechanisms that underpin the xylem wall patterns, emphasizing the VND6 and VND7 inducible systems, and outline challenges that lay ahead in this field.     

Cellulose biosynthesis: current views and evolving concepts

* AIMS: To outline the current state of knowledge and discuss the evolution of various viewpoints put forth to explain the mechanism of cellulose biosynthesis. * SCOPE: Understanding the mechanism of cellulose biosynthesis is one of the major challenges in plant biology. The simplicity in the chemical structure of cellulose belies the complexities that are associated with the synthesis and assembly of this polysaccharide. Assembly of cellulose microfibrils in most organisms is visualized as a multi-step process involving a number of proteins with the key protein being the cellulose synthase catalytic sub-unit. Although genes encoding this protein have been identified in almost all cellulose synthesizing organisms, it has been a challenge in general, and more specifically in vascular plants, to demonstrate cellulose synthase activity in vitro. The assembly of glucan chains into cellulose microfibrils of specific dimensions, viewed as a spontaneous process, necessitates the assembly of synthesizing sites unique to most groups of organisms. The steps of polymerization (requiring the specific arrangement and activity of the cellulose synthase catalytic sub-units) and crystallization (directed self-assembly of glucan chains) are certainly interlinked in the formation of cellulose microfibrils. Mutants affected in cellulose biosynthesis have been identified in vascular plants. Studies on these mutants and herbicide-treated plants suggest an interesting link between the steps of polymerization and crystallization during cellulose biosynthesis. * CONCLUSIONS: With the identification of a large number of genes encoding cellulose synthases and cellulose synthase-like proteins in vascular plants and the supposed role of a number of other proteins in cellulose biosynthesis, a complete understanding of this process will necessitate a wider variety of research tools and approaches than was thought to be required a few years back.     

Disruption of the chitin synthase gene CHS1 from Fusarium asiaticum results in an altered structure of cell walls and reduced virulence

Natural resistance of wheat against Fusarium head blight (FHB) is inadequate and new strategies for controlling the disease are required. Chitin synthases that catalyze chitin biosynthesis would be an ideal target for antifungal agents. In this study, a class I chitin synthase gene (CHS1) from Fusarium asiaticum, the predominant species of FHB pathogens on wheat in China, was functionally disrupted via Agrobacterium tumefaciens-mediated transformation. Specific disruption of the CHS1 gene resulted in a 58% reduction of chitin synthase activity, accompanied by decreases of 35% in chitin content, 22% in conidiation, and 16% in macroconidium length. The Δchs1 mutant strain had a growth rate comparable to that of the wild-type on PDA medium but had a 35% increase in the number of nuclear cellulae and exhibited a remarkably increased sensitivity to osmosis stresses. Electron microscopy revealed substantial changes occurring in cell wall structures of the macroconidium, ascospore, and mycelium, with the most profound changes in the mycelium. Furthermore, the Δchs1 mutant displayed significantly reduced pathogenicity on wheat spikes and seedlings. Re-introduction of a functional CHS1 gene into the Δchs1 mutant strain restored the wild-type phenotype. These results reveal an important in vivo role played by a CHS1 gene in a FHB pathogen whose mycelial chitin could serve as a target for controlling the disease.     

Disruption of the cellulose synthase gene, AtCesA8/IRX1, enhances drought and osmotic stress tolerance in Arabidopsis

Two allelic Arabidopsis mutants, leaf wilting 2-1 and leaf wilting 2-2 (lew2-1 and lew2-2 ), were isolated in a screen for plants with altered drought stress responses. The mutants were more tolerant to drought stress as well as to NaCl, mannitol and other osmotic stresses. lew2 mutant plants accumulated more abscisic acid (ABA), proline and soluble sugars than the wild type. The expression of a stress-inducible marker gene RD29A, a proline synthesis-related gene P5CS (pyrroline-5-carboxylate synthase) and an ABA synthesis-related gene SDR1 (alcohol dehydrogenase/reductase) was higher in lew2 than in the wild type. Map-based cloning revealed that the lew2 mutants are new alleles of the AtCesA8/IRX1 gene which encodes a subunit of a cellulose synthesis complex. Our results suggest that cellulose synthesis is important for drought and osmotic stress responses including drought induction of gene expression.     

植物细胞壁同聚半乳糖醛酸的代谢与功能

果胶是细胞壁多糖的重要组成成分,对植物正常的生长发育十分重要。作为初生细胞壁中果胶的一种主要组成成分,同聚半乳糖醛酸（homogalacturonan,HG）是由α-D-半乳糖醛酸单体经α-（1,4）-糖苷键连接起来的一种长链大分子物质。HG的合成和降解参与了细胞壁中的多糖代谢,影响了细胞壁的结构和功能。同时,HG精确的去甲酯化以及HG所参与的细胞壁关联激酶（WAKs）和促分裂原活化蛋白激酶（MAPKs）相关的信号转导途径,在植物生长发育中也发挥着重要作用。该文主要从HG的合成、降解和循环利用以及HG的作用等方面对植物细胞壁中HG的研究进展进行了阐述。     

A link between sterol biosynthesis, the cell wall, and cellulose in Arabidopsis

A crucial role for sterols in plant growth and development is underscored by the identification of three Arabidopsis sterol biosynthesis mutants that exhibit embryonic defects: fackel (fk), hydra1 (hyd1), and sterol methyltransferase 1/cephalopod (smt1/cph). We have taken a dual approach of sterol profiling and ultrastructural analysis to investigate the primary defects underlying the mutant phenotypes. Comprehensive gas chromatography GC-MS analysis of hyd1 in comparison to fk reveals an abnormal accumulation of unique sterol intermediates in each case. Sterol profiling of the fk hyd1 double mutant provides genetic evidence that FK C-14 reductase acts upstream of HYD1 C-8,7 isomerase. Despite distinct differences in sterol profiles, fk and hyd1 as well as smt1/cph share ultrastructural features such as incomplete cell walls and aberrant cell wall thickenings in embryonic and post-embryonic tissues. The common defects are coupled with ectopic callose and lignin deposits. We show that all three mutants exhibit a deficiency in cellulose, but are not reduced in pectin and sugars of the cell wall and cytosol. The sterol biosynthesis inhibitors 15-azasterol and fenpropimorph also cause cell wall gaps in dividing root cells and a reduction in bulk cellulose, corroborating that the cell wall abnormalities are due to altered sterol composition. Our results demonstrate that sterols are crucial for cellulose synthesis in the building of the plant cell wall.     

Constitutive expression of a fungal glucuronoyl esterase in Arabidopsis reveals altered cell wall composition and structure

A family 15 carbohydrate esterase (CE15) from the white‐rot basidiomycete, Phanerochaete carnosa (PcGCE), was transformed into Arabidopsis thaliana Col‐0 and was expressed from the constitutive cauliflower mosaic virus 35S promoter. Like other CE15 enzymes, PcGCE hydrolyzed methyl‐4‐O‐methyl‐d ‐glucopyranuronate and could target ester linkages that contribute to lignin–carbohydrate complexes that form in plant cell walls. Three independently transformed Arabidopsis lines were evaluated in terms of nine morphometric parameters, total sugar and lignin composition, cell wall anatomy, enzymatic saccharification and xylan extractability. The transgenic lines consistently displayed a leaf‐yellowing phenotype, as well as reduced glucose and xylose content by as much as 30% and 35%, respectively. Histological analysis revealed 50% reduction in cell wall thickness in the interfascicular fibres of transgenic plants, and FT‐IR microspectroscopy of interfascicular fibre walls indicated reduction in lignin cross‐linking in plants overexpressing PcGCE. Notably, these characteristics could be correlated with improved xylose recovery in transgenic plants, up to 15%. The current analysis represents the first example whereby a fungal glucuronoyl esterase is expressed in Arabidopsis and shows that the promotion of glucuronoyl esterase activity in plants can alter the extent of intermolecular cross‐linking within plant cell walls.     

Classification and identification of Arabidopsis cell wall mutants using Fourier-Transform InfraRed (FT-IR) microspectroscopy

We have developed a novel procedure for the rapid classification and identification of Arabidopsis mutants with altered cell wall architecture based on Fourier-Transform Infrared (FT-IR) microspectroscopy. FT-IR transmission spectra were sampled from native 4-day-old dark-grown hypocotyls of 46 mutants and the wild type treated with various drugs. The Mahalanobis distance between mutants, calculated from the spectral information after compression with the Discriminant Variables Selection procedure, was used for alpha hierarchical cluster analysis. Despite the completely unsupervised nature of the classification procedure, we show that all mutants with cellulose defects appeared in the same cluster. In addition, mutant alleles of similar strength for several unrelated loci were also clustered, which demonstrates the sensitivity of the method to detect a wide array of cell wall defects. Comparing the cellulose-deficient cluster with the cluster that contained wild-type controls led to the identification of wave numbers that were diagnostic for altered cellulose content in the context of an intact cell wall. The results show that FT-IR spectra can be used to identify different classes of mutants and to characterize cell wall changes at a microscopic level in unknown mutants. This procedure significantly accelerates the identification and classification of cell wall mutants, which makes cell wall polysaccharides more accessible to functional genomics approaches.