Hetero‐trans‐β‐glucanase,an enzyme unique to Equisetum plants,functionalizes cellulose

Cell walls are metabolically active components of plant cells. They contain diverse enzymes, including transglycanases (endotransglycosylases), enzymes that ‘cut and paste’ certain structural polysaccharide molecules and thus potentially remodel the wall during growth and development. Known transglycanase activities modify several cell‐wall polysaccharides (xyloglucan, mannans, mixed‐linkage β‐glucan and xylans); however, no transglycanases were known to act on cellulose, the principal polysaccharide of biomass. We now report the discovery and characterization of hetero‐trans‐β‐glucanase (HTG), a transglycanase that targets cellulose, in horsetails (Equisetum spp., an early‐diverging genus of monilophytes). HTG is also remarkable in predominantly catalysing hetero‐transglycosylation: its preferred donor substrates (cellulose or mixed‐linkage β‐glucan) differ qualitatively from its acceptor substrate (xyloglucan). HTG thus generates stable cellulose–xyloglucan and mixed‐linkage β‐glucan–xyloglucan covalent bonds, and may therefore strengthen ageing Equisetum tissues by inter‐linking different structural polysaccharides of the cell wall. 3D modelling suggests that only three key amino acid substitutions (Trp → Pro, Gly → Ser and Arg → Leu) are responsible for the evolution of HTG's unique specificity from the better‐known xyloglucan‐acting homo‐transglycanases (xyloglucan endotransglucosylase/hydrolases; XTH). Among land plants, HTG appears to be confined to Equisetum, but its target polysaccharides are widespread, potentially offering opportunities for enhancing crop mechanical properties, such as wind resistance. In addition, by linking cellulose to xyloglucan fragments previously tagged with compounds such as dyes or indicators, HTG may be useful biotechnologically for manufacturing stably functionalized celluloses, thereby potentially offering a commercially valuable ‘green’ technology for industrially manipulating biomass.     

The exogenous application of AtPGLR,an endo‐polygalacturonase,triggers pollen tube burst and repair

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine‐tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark‐grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo‐PG that preferentially releases non‐methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non‐methylesterified pectin epitopes are detected. Those leaks could either be repaired by new β‐glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.     

In the grass species Brachypodium distachyon,the production of mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) occurs in the Golgi apparatus

Mixed‐linkage (1,3;1,4)‐β‐glucan (MLG) is a glucose polymer with beneficial effects on human health and high potential for the agricultural industry. MLG is present predominantly in the cell wall of grasses and is synthesized by cellulose synthase‐like F or H families of proteins, with CSLF6 being the best‐characterized MLG synthase. Although the function of this enzyme in MLG production has been established, the site of MLG synthesis in the cell is debated. It has been proposed that MLG is synthesized at the plasma membrane, as occurs for cellulose and callose; in contrast, it has also been proposed that MLG is synthesized in the Golgi apparatus, as occurs for other matrix polysaccharides of the cell wall. Testing these conflicting possibilities is fundamentally important in the general understanding of the biosynthesis of the plant cell wall. Using immuno‐localization analyses with MLG‐specific antibody in Brachypodium and in barley, we found MLG present in the Golgi, in post‐Golgi structures and in the cell wall. Accordingly, analyses of a functional fluorescent protein fusion of CSLF6 stably expressed in Brachypodium demonstrated that the enzyme is localized in the Golgi. We also established that overproduction of MLG causes developmental and growth defects in Brachypodium as also occur in barley. Our results indicated that MLG production occurs in the Golgi similarly to other cell wall matrix polysaccharides, and supports the broadly applicable model in grasses that tight mechanisms control optimal MLG accumulation in the cell wall during development and growth. This work addresses the fundamental question of where mixed linkage (1,3;1,4)‐β‐glucan (MLG) is synthesized in plant cells. By analyzing the subcellular localization of MLG and MLG synthase in an endogenous system, we demonstrated that MLG synthesis occurs at the Golgi in Brachypodium and barley. A growth inhibition due to overproduced MLG in Brachypodium supports the general applicability of the model that a tight control of the cell wall polysaccharides accumulation is needed to maintain growth homeostasis during development.     

Method for hull‐less barley transformation and manipulation of grain mixed‐linkage beta‐glucan

Hull‐less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull‐less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull‐less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over‐express genes encoding synthases for the important dietary fiber component, (1,3;1,4)‐β‐glucan (mixed‐linkage glucan), primarily present in starchy endosperm cell walls. Over‐expression of the HvCslF6 gene, driven by an endosperm‐specific promoter, produced lines where mixed‐linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub‐aleurone cells. This work provides proof‐of‐concept evidence that mixed‐linkage glucan content in hull‐less barley grain can be increased by over‐expression of the HvCslF6 gene, but also indicates that hull‐less cultivars may be more sensitive to attempts to modify cell wall composition.     

Over‐expression of specific HvCslF cellulose synthase‐like genes in transgenic barley increases the levels of cell wall (1,3;1,4)‐β‐d‐glucans and alters their fine structure

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)‐β‐d ‐glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)‐β‐d ‐glucans in the Poaceae is mediated, in part at least, by the cellulose synthase‐like CslF family of genes. Over‐expression of the barley CslF6 gene under the control of an endosperm‐specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)‐β‐d ‐glucan content in grain of transgenic barley. Analyses of (1,3;1,4)‐β‐d ‐glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)‐β‐d ‐glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)‐β‐d ‐glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)‐β‐d ‐glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)‐β‐d ‐glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)‐β‐d ‐glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)‐β‐d ‐glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.     

Measurement of Cell Wall Volume using Confocal Microscopy and its Application to Studies of Forage Degradation

The distribution of cell wall material between different plantcell types may contribute significantly to the variation indegradability of plant material with a similar overall chemicalcomposition but different anatomy. Assessment of the degradabilityof cell walls in a section suitable for digestion is a three-dimensional(3-D) problem because of the thickness of section required (50–100µm). Optical sectioning of thick sections using confocallaser scanning microscopy (CLSM) provides a method of estimatingthe volume of cell wall material present in tissue sectionsbefore and after digestion, and of visualizing the plant tissueusing 3-D image reconstruction. The use of CLSM enables degradabilitymeasurements to be made on cellsin situand can provide moreimmediate and relevant information than can be obtained by mechanicalfractionation of the tissues. The CLSM method has been usedto visualize thick sections taken from maize and barley internodesbefore and after degradation with cell wall degrading enzymes.Quantitative measurements of cell wall volume and mean cellwall thickness were made on a series of optical sections, andthe potential of the method for quantitation of cell wall degradabilityis assessed. Image analysis; plant anatomy; confocal microscopy; degradation; maize     

Characterization of Aspergillus nidulans α‐glucan synthesis: roles for two synthases and two amylases

Cell walls are essential for fungal survival and growth. Fungal walls are ～ 90% carbohydrate, mostly types not found in humans, making them promising targets for anti‐fungal drug development. Echinocandins, which inhibit the essential β‐glucan synthase, are already clinically available. In contrast, α‐glucan, another abundant fungal cell wall component has attracted relatively little research attention because it is not essential for most fungi. Aspergillus nidulans has two α‐glucan synthases (AgsA and AgsB) and two α‐amylases (AmyD and AmyG), all of which affect α‐glucan synthesis. Gene deletion showed that AgsB was the major synthase. In addition, AmyG promoted α‐glucan synthesis whereas AmyD had a repressive effect. The lack of α‐glucan had no phenotypic impact on solid medium, but reduced conidial adhesion during germination in shaken liquid. Moreover, α‐glucan level correlated with resistance to Calcofluor White. Intriguingly, overexpression of agsA could compensate for the loss of agsB at the α‐glucan level, but not for phenotypic defects. Thus, products of AgsA and AgsB have different roles in the cell wall, consistent with agsA being mainly expressed at conidiation. These results suggest that α‐glucan contributes to drug sensitivity and conidia adhesion in A. nidulans, and is differentially regulated by two synthases and two amylases.     

Inhibitory effects of sodium silicate on the fungal growth and secretion of cell wall‐degrading enzymes by Trichothecium roseum

Trichothecium roseum causes decay in muskmelons, apples, tomatoes and mangoes, which leads to economic losses. In this study, we investigated the effect of sodium silicate on the growth of T. roseum and the cell wall‐degrading enzymes (CWDEs) secreted by the hyphae. The results indicated that sodium silicate significantly inhibited mycelial growth and spore germination of T. roseum. The sodium silicate treatment also retarded the secretion of several CWDEs, including pectate lyase (PL), polygalacturonic acid transeliminase (PGTE), pectin methyltranseliminase (PMTE), pectin methylgalacturonase (PMG), polygalacturonase (PG), cellulase (Cx) and β‐glucosidase. These results suggest that sodium silicate exerts its effects on T. roseum through direct inhibition of its growth and secretion of CWDEs.     

Effect of High Temperature During Grain-filling on the Structure of Developing and Malted Barley Grains

High temperatures (up to 35 °C) were applied to plants ofmalting barley,Hordeum vulgareL. (‘Schooner’) fora period of 5 d during grain-filling. Heat treatment had a profoundeffect on the structure of the mature barley grain. There wasevidence of degradation of endosperm storage products in heat-treatedgrain. Starch granule development was reduced in sub-aleuronecells following heat treatment and alterations to starch granuledistribution and growth were observed in the endosperms of thesegrains. Endosperm cell wall and crushed cell layer (CCL) developmentwere sensitive to high temperatures, with the reduced thicknessof the CCL and generally patchy Calcofluor fluorescence of endospermcell walls indicative of partial hydrolysis of ß-glucans.Increased growth of the embryo took place in heat-treated grainscompared with control grains. Endosperm texture was generallymore friable in heat-treated grains than in control grains,and these grains overmodified during malting, with considerabledegradation of starch in the form of extensive pitting of A-typestarch granules. Evidence is presented for developmental andgermination events occurring simultaneously within the developinggrain.Copyright 1998 Annals of Botany Company Barley,Hordeum vulgareL., starch granules, crushed cell layer, scutellum, embryo, fluorescence microscopy, scanning electron microscopy, confocal microscopy, malting quality.     

Coordinated action of β‐galactosidases in the cell wall of embryonic axes during chickpea germination and seedling growth

The plant cell wall is a dynamic structure whose constant modification is necessary for plant cells to grow and divide. In the cell walls of chickpea (Cicer arietinum) there are at least four β‐galactosidases, whose presence and location in embryonic axes during the first 48 h of seed imbibition are discussed in this paper. We examined their roles as cell wall‐modifying enzymes in germinative and/or post‐germinative events. At the start of germination, only βV‐Gal, and to a lesser extent βIV‐Gal, appear in the axes before rupture of the testa, suggesting they are related to germination sensu stricto. Once the testa has broken, the four β‐galactosidases are involved in growth and differentiation of the axes. Immunolocation of the different proteins in axes, which in part confirms previous results in seedlings and plants, allows assignment of post‐germinative roles to βI‐Gal and βIII‐Gal as cell wall modifiers in vascular tissue elements. βIV‐Gal and βV‐Gal participate in the initial events of germination in which cell walls are involved: βV‐Gal in cell proliferation, detachment of root cap cells and initial vascular tissue differentiation; both of them in xylem maturation; and βIV‐Gal in thickening of the primary cell wall. Together with other cell wall‐modifying enzymes, such as expansins and XTH, chickpea galactosidases might function in a sequential order in turnover of the primary cell wall, allowing the elongation of embryonic axes during seed germination.     

Disruption of cellulose synthesis by 2,6‐dichlorobenzonitrile affects the structure of the cytoskeleton and cell wall construction in Arabidopsis

Cellulose is the major component of plant cell walls and is an important source of industrial raw material. Although cellulose biosynthesis is one of the most important biochemical processes in plant biology, the regulatory mechanisms of cellulose synthesis are still unclear. Here, we report that 2,6‐dichlorobenzonitrile (DCB), an inhibitor of cellulose synthesis, inhibits Arabidopsis root development in a dose‐ and time‐dependent manner. When treated with DCB, the plant cell wall showed altered cellulose distribution and intensity, as shown by calcofluor white and S4B staining. Moreover, pectin deposition was reduced in the presence of DCB when immunostained with the monoclonal antibody JIM5, which was raised against pectin epitopes. This result was confirmed using Fourier transform infrared (FTIR) analysis. Confocal microscopy revealed that the organisation of the microtubule cytoskeleton was significantly disrupted in the presence of low concentrations of DCB, whereas the actin cytoskeleton only showed changes with the application of high DCB concentrations. In addition, the subcellular dynamics of Golgi bodies labelled with N‐ST‐YFP and TGN labelled with VHA‐a1‐GFP were both partially blocked by DCB. Transmission electron microscopy indicated that the cell wall structure was affected by DCB, as were the Golgi bodies. Scanning electron microscopy showed changes in the organisation of cellulose microfibrils. These results suggest that the inhibition of cellulose synthesis by DCB not only induced changes in the chemical composition of the root cell wall and cytoskeleton structure, but also changed the distribution of cellulose microfibrils, implying that cellulose plays an important role in root development in Arabidopsis.     

Engineering temporal accumulation of a low recalcitrance polysaccharide leads to increased C6 sugar content in plant cell walls

Reduced cell wall recalcitrance and increased C6 monosaccharide content are desirable traits for future biofuel crops, as long as these biomass modifications do not significantly alter normal growth and development. Mixed‐linkage glucan (MLG), a cell wall polysaccharide only present in grasses and related species among flowering plants, is comprised of glucose monomers linked by both β‐1,3 and β‐1,4 bonds. Previous data have shown that constitutive production of MLG in barley (Hordeum vulgare) severely compromises growth and development. Here, we used spatio‐temporal strategies to engineer Arabidopsis thaliana plants to accumulate significant amounts of MLG in the cell wall by expressing the rice CslF6 MLG synthase using secondary cell wall and senescence‐associated promoters. Results using secondary wall promoters were suboptimal. When the rice MLG synthase was expressed under the control of a senescence‐associated promoter, we obtained up to four times more glucose in the matrix cell wall fraction and up to a 42% increase in saccharification compared to control lines. Importantly, these plants grew and developed normally. The induction of MLG deposition at senescence correlated with an increase of gluconic acid in cell wall extracts of transgenic plants in contrast to the other approaches presented in this study. MLG produced in Arabidopsis has an altered structure compared to the grass glucan, which likely affects its solubility, while its molecular size is unaffected. The induction of cell wall polysaccharide biosynthesis in senescing tissues offers a novel engineering alternative to enhance cell wall properties of lignocellulosic biofuel crops.     

OCCURRENCE AND CHARACTERIZATION OF ARABINOGALACTAN‐LIKE PROTEINS AND HEMICELLULOSES IN MICRASTERIAS (STREPTOPHYTA)1

The cell wall of the green alga Micrasterias denticulata Bréb. ex Ralfs (Desmidiaceae, Zygnematophyceae, Streptophyta) was investigated to obtain information on the composition of component polysaccharides and proteoglycans to allow comparison with higher plants and to understand cell wall functions during development. Various epitopes currently assigned to arabinogalactan‐proteins (AGPs) of higher plants could be detected in Micrasterias by immuno TEM and immunofluorescence methods, but the walls did not bind the β‐d ‐glycosyl‐Yariv (β‐GlcY) reagent. Secretory vesicles and the primary wall were labeled by antibodies against AGPs (JIM8, JIM13, JIM14). Dot and Western blot experiments indicated a proteoglycan nature of the epitopes recognized, which consisted of galactose and xylose as major sugars by high performance anion exchange chromatography with pulsed amperometric detection (HPAEC‐PAD). Epitopes of alkali‐soluble polysaccharides assigned to noncellulosic polysaccharides in higher plants could be detected and located in the wall during its formation. The polyclonal anti‐xyloglucan (anti‐XG) antibody labeled primary and secondary wall of Micrasterias, whereas the monoclonal antibody CCRC‐M1, directed against the fucose/galactose side chain of xyloglucan (XyG), did not recognize any structures. Labeling by anti‐XG antibody at the trans‐sites of the dictyosomes and at wall material containing vesicles indicated that secretion of the epitopes occurred similar to higher plants. The presence of (1→3, 1→4)‐β‐glucan (mixed linked glucan) in the secondary cell wall but not in the primary cell wall of Micrasterias could be demonstrated by an antibody recognizing this glucan type, whereas (1→3)‐β‐glucan (callose) could not be detected. The analytical results revealed that alkali‐soluble polysaccharides in the secondary wall of Micrasterias consist mostly of (1→3, 1→4)‐β‐d ‐glucan.     

KRE genes are required for β‐1,6‐glucan synthesis,maintenance of capsule architecture and cell wall protein anchoring in Cryptococcus neoformans

The polysaccharide β‐1,6‐glucan is a major component of the cell wall of Cryptococcus neoformans, but its function has not been investigated in this fungal pathogen. We have identified and characterized seven genes, belonging to the KRE family, which are putatively involved in β‐1,6‐glucan synthesis. The H99 deletion mutants kre5Δ and kre6Δskn1Δ contained less cell wall β‐1,6‐glucan, grew slowly with an aberrant morphology, were highly sensitive to environmental and chemical stress and were avirulent in a mouse inhalation model of infection. These two mutants displayed alterations in cell wall chitosan and the exopolysaccharide capsule, a primary cryptococcal virulence determinant. The cell wall content of the GPI‐anchored phospholipase B1 (Plb1) enzyme, which is required for cryptococcal cell wall integrity and virulence, was reduced in kre5Δ and kre6Δskn1Δ. Our results indicate that KRE5, KRE6 and SKN1 are involved in β‐1,6‐glucan synthesis, maintenance of cell wall integrity and retention of mannoproteins and known cryptococcal virulence factors in the cell wall of C. neoformans. This study sets the stage for future investigations into the function of this abundant cell wall polymer.     

Distribution of unesterified and esterified pectins in cell walls of pollen tubes of flowering plants

Immunocytochemical localization of polygalacturonic acid (pectin) and methyl-esterified pectin in the walls of pollen tubes of 20 species of flowering plants grown in vitro was investigated by using monoclonal antibodies (MAbs) JIM5 and JIM7 and by means of confocal laser scanning microscopy (CLSM). In general, periodic annular deposits of pectins were found coating the tube wall in species possessing solid styles, and a more uniform pectin sheath in tube walls in species having hollow styles or no styles. We hypothesize that the periodic ring-like structure of the pectin sheath reinforces pollen tubes for passing through the transmitting tract in the style. Esterified pectin which prevents Ca²⁺-induced gelification of pectate is located predominantly at the apex. This implies that pectin esterification is related to tip wall loosening that is required for cell wall expansion during tip growth of pollen tubes. The occurrence of unesterified pectins in other areas of pollen tube walls suggests that de-esterification of pectin following tip expansion leads to a more rigid form of pectin that contributes to the construction of the pollen tube wall.