Plant protein inhibitors of cell wall degrading enzymes

Plant cell walls, which consist mainly of polysaccharides (i.e. cellulose, hemicelluloses and pectins), play an important role in defending plants against pathogens. Most phytopathogenic microorganisms secrete an array of cell wall degrading enzymes (CWDEs) capable of depolymerizing the polysaccharides in the plant host wall. In response, plants have evolved a diverse battery of defence responses including protein inhibitors of these enzymes. These include inhibitors of pectin degrading enzymes such as polygalacturonases, pectinmethyl esterases and pectin lyases, and hemicellulose degrading enzymes such as endoxylanases and xyloglucan endoglucanases. The discovery of these plant inhibitors and the recent resolution of their three-dimensional structures, free or in complex with their target enzymes, provide new lines of evidence regarding their function and evolution in plant-pathogen interactions.     

Plant biotechnology for lignocellulosic biofuel production

Lignocelluloses from plant cell walls are attractive resources for sustainable biofuel production. However, conversion of lignocellulose to biofuel is more expensive than other current technologies, due to the costs of chemical pretreatment and enzyme hydrolysis for cell wall deconstruction. Recalcitrance of cell walls to deconstruction has been reduced in many plant species by modifying plant cell walls through biotechnology. These results have been achieved by reducing lignin content and altering its composition and structure. Reduction of recalcitrance has also been achieved by manipulating hemicellulose biosynthesis and by overexpression of bacterial enzymes in plants to disrupt linkages in the lignin–carbohydrate complexes. These modified plants often have improved saccharification yield and higher ethanol production. Cell wall‐degrading (CWD) enzymes from bacteria and fungi have been expressed at high levels in plants to increase the efficiency of saccharification compared with exogenous addition of cellulolytic enzymes. In planta expression of heat‐stable CWD enzymes from bacterial thermophiles has made autohydrolysis possible. Transgenic plants can be engineered to reduce recalcitrance without any yield penalty, indicating that successful cell wall modification can be achieved without impacting cell wall integrity or plant development. A more complete understanding of cell wall formation and structure should greatly improve lignocellulosic feedstocks and reduce the cost of biofuel production.     

Influence of fungal infection on plant tissues: FTIR detects compositional changes to plant cell walls

Compositional change in plant cell walls as a result of infection by non-host (putative) endophytes and a host pathogen were studied by quantifying plant cell wall degrading enzymes (CWDEs) produced by these fungi, and by detecting cell wall changes via Fourier Transform Infrared spectroscopy (FTIR) and relative lignin/carbohydrate intensity ratios. Oil palm ramets were first inoculated with homogenized fungal suspension. The treated fungal suspensions were assayed for CWDEs whereas the ramets were powderized for FTIR analysis. Results revealed that putative endophytes and host pathogen expressed all CWDEs, suggesting their probable roles in infection and colonization. Following inoculation, plant cell wall composition showed missing dips in spectra depicting changes to carbohydrate, xylan and lignin constituents. The indistinguishable FTIR spectra for putative endophyte-inoculated and pathogen-inoculated ramets suggest that both endophytes and pathogen have elicited similar responses to plant cell walls. Relative lignin/carbohydrate ratios further demonstrated that the putative endophytes did not breakdown lignin and carbohydrate, further exemplifying the non-pathogenic and asymptomatic infection by the endophytes. This study presents the influence of putative endophytes on plant tissues of oil palm, and how this compared to pathogenic infection.     

Mode of Attack on Orchardgrass Leaf Blades by Rumen Protozoa

Leaf blade sections of orchardgrass were incubated with rumen fluid and examined by scanning and transmission electron microscopy for the mode of attack on tissues by rumen protozoa. Rumen protozoa resembling Epidinium ecaudatum from caudatum degraded forage tissue in diluted, whole rumen fluid suspensions of microbes containing 1.6 mg of streptomycin per ml, which inhibited bacterial fiber-digesting activity. Cell walls of mesophyll, parenchyma bundle sheath, and epidermis became swollen and frayed to reveal a microfibrillar network and loss of electron density, indicating partial degradation. Then the protozoa ingested whole cells and fragments of cell walls with the aid of their cilia. Plant cells with partially degraded walls as well as chloroplasts without walls were present within the protozoa. These entodiniomorphs digested orchardgrass leaves by partially degrading the plant cell walls apparently by extracellular enzymes and then ingestion of the plant cells and cell wall fragments.     

Degradation of Bermuda and Orchard Grass by Species of Ruminal Bacteria

Fiber degradation in Bermuda grass and orchard grass was evaluated gravimetrically and by scanning and transmission electron microscopy after incubation with pure cultures of rumen bacteria. Lachnospira multiparus D-32 was unable to degrade plant cell wall components. Butyrivibrio fibrisolvens 49 degraded 6 and 14.9% of the fiber components in Bermuda grass and orchard grass, respectively, and Ruminococcus albus 7 degraded 11.4% orchard grass fiber but none in Bermuda grass. Both B. fibrisolvens and R. albus lacked capsules, did not adhere to fiber, and degraded only portions of the more easily available plant cell walls. R. flavefaciens FD-1 was the most active fiber digester, degrading 8.2 and 55.3% of Bermuda and orchard grass fiber, respectively. The microbe had a distinct capsule and adhered to fiber, especially that which is slowly degraded, but was able to cause erosion and disorganization of the more easily digested cell walls, apparently by extracellular enzymes. Results indicated that more digestible cell walls could be partially degraded by enzymes disassociated from cellulolytic and noncellulolytic bacteria, and data were consistent with the hypothesis that the more slowly degraded plant walls required attachment. Microbial species as well as the cell wall architecture influenced the physical association with and digestion of plant fiber.     

The Casparian Strip of Clivia miniata Reg. Roots: Isolation,Fine Structure and Chemical Nature*

The root endodermis of Clivia miniata Reg. was successfully isolated using the cell wall degrading enzymes cellulase and pectinase. The enzymes did not depolymerize those regions of the primary cell walls of anticlinal endodermal root cells where the Casparian strips were located. Since the endodermis of C. miniata roots remained in its primary developmental state over the whole root length, endodermal isolates essentially represented Casparian strips. Thus, sufficient amounts of isolated Casparian strips could be obtained to allow further detailed investigations of the isolates by microscopic, histochemical and analytical methods. Scanning electron microscopy revealed the reticular structure of the Casparian strips completely surrounding the central cylinder of the roots. Whereas in younger parts of the root only the anticlinal cell walls of the endodermis remained intact in the isolates, in older parts of the root the periclinal walls also restricted enzymatic degradation due to the deposition of lignin. Extracts of the isolates with organic solvents did not reveal any wax-like substances which might have been deposited within the cell wall forming a transport barrier, as is the case with cutin and suberin. However, several histochemical and analytical methods (elemental analysis and FTIR spectroscopy) showed that the chemical nature of the Casparian strips of C. miniata roots can definitely be a lignified cell wall. These findings are in complete agreement with studies carried out at the beginning of this century on the chemical nature of the Casparian strips of several other plant species. The implications of these results concerning apoplasmatic transport of solutes and water across Casparian strips are discussed.     

Plant glycoside hydrolases involved in cell wall polysaccharide degradation.

The cell wall plays a key role in controlling the size and shape of the plant cell during plant development and in the interactions of the plant with its environment. The cell wall structure is complex and contains various components such as polysaccharides, lignin and proteins whose composition and concentration change during plant development and growth. Many studies have revealed changes in cell walls which occur during cell division, expansion, and differentiation and in response to environmental stresses; i.e. pathogens or mechanical stress. Although many proteins and enzymes are necessary for the control of cell wall organization, little information is available concerning them. An important advance was made recently concerning cell wall organization as plant enzymes that belong to the superfamily of glycoside hydrolases and transglycosidases were identified and characterized; these enzymes are involved in the degradation of cell wall polysaccharides. Glycoside hydrolases have been characterized using molecular, genetic and biochemical approaches. Many genes encoding these enzymes have been identified and functional analysis of some of them has been performed. This review summarizes our current knowledge about plant glycoside hydrolases that participate in the degradation and reorganisation of cell wall polysaccharides in plants focussing particularly on those from Arabidopsis thaliana.     

Disentangling loosening from softening: insights into primary cell wall structure

How cell wall elasticity, plasticity, and time‐dependent extension (creep) relate to one another, to plant cell wall structure and to cell growth remain unsettled topics. To examine these issues without the complexities of living tissues, we treated cell‐free strips of onion epidermal walls with various enzymes and other agents to assess which polysaccharides bear mechanical forces in‐plane and out‐of‐plane of the cell wall. This information is critical for integrating concepts of wall structure, wall material properties, tissue mechanics and mechanisms of cell growth. With atomic force microscopy we also monitored real‐time changes in the wall surface during treatments. Driselase, a potent cocktail of wall‐degrading enzymes, removed cellulose microfibrils in superficial lamellae sequentially, layer‐by‐layer, and softened the wall (reduced its mechanical stiffness), yet did not induce wall loosening (creep). In contrast Cel12A, a bifunctional xyloglucanase/cellulase, induced creep with only subtle changes in wall appearance. Both Driselase and Cel12A increased the tensile compliance, but differently for elastic and plastic components. Homogalacturonan solubilization by pectate lyase and calcium chelation greatly increased the indentation compliance without changing tensile compliances. Acidic buffer induced rapid cell wall creep via endogenous α‐expansins, with negligible effects on wall compliances. We conclude that these various wall properties are not tightly coupled and therefore reflect distinctive aspects of wall structure. Cross‐lamellate networks of cellulose microfibrils influenced creep and tensile stiffness whereas homogalacturonan influenced indentation mechanics. This information is crucial for constructing realistic molecular models that define how wall mechanics and growth depend on primary cell wall structure.     

Mutations of the secondary cell wall

It has not been possible to isolate a number of crucial enzymes involved in plant cell wall synthesis. Recent progress in identifying some of these steps has been overcome by the isolation of mutants defective in various aspects of cell wall synthesis and the use of these mutants to identify the corresponding genes. Secondary cell walls offer numerous advantages for genetic analysis of plant cell walls. It is possible to recover very severe mutants since the plants remain viable. In addition, although variation in secondary cell wall composition occurs between different species and between different cell types, the composition of the walls is relatively simple compared to primary cell walls. Despite these advantages, relatively few secondary cell wall mutations have been described to date. The only secondary cell wall mutations characterised to date, in which the basis of the abnormality is known, have defects in either the control of secondary cell wall deposition or secondary cell wall cellulose or lignin biosynthesis. These mutants have, however, provided essential information on secondary cell wall biosynthesis.     

A family of glycosyl hydrolase family 45 cellulases from the pine wood nematode Bursaphelenchus xylophilus

We have characterized a family of GHF45 cellulases from the pine wood nematode Bursaphelenchus xylophilus. The absence of such genes from other nematodes and their similarity to fungal genes suggests that they may have been acquired by horizontal gene transfer (HGT) from fungi. The cell wall degrading enzymes of other plant parasitic nematodes may have been acquired by HGT from bacteria. B. xylophilus is not directly related to other plant parasites and our data therefore suggest that horizontal transfer of cell wall degrading enzymes has played a key role in evolution of plant parasitism by nematodes on more than one occasion.     

GH62 arabinofuranosidases: Structure,function and applications

Motivated by industrial demands and ongoing scientific discoveries continuous efforts are made to identify and create improved biocatalysts dedicated to plant biomass conversion. α-1,2 and α-1,3 arabinofuranosyl specific α-l-arabinofuranosidases (EC 3.2.1.55) are debranching enzymes catalyzing hydrolytic release of α-l-arabinofuranosyl residues, which decorate xylan or arabinan backbones in lignocellulosic and pectin constituents of plant cell walls. The CAZy database classifies α-l-arabinofuranosidases in Glycoside Hydrolase (GH) families GH2, GH3, GH43, GH51, GH54 and GH62. Only GH62 contains exclusively α-l-arabinofuranosidases and these are of fungal and bacterial origin. Twenty-two GH62 enzymes out of 223 entries in the CAZy database have been characterized and very recently new knowledge was acquired with regard to crystal structures, substrate specificities, and phylogenetics, which overall provides novel insights into structure/function relationships of GH62. Overall GH62 α-l-arabinofuranosidases are believed to play important roles in nature by acting in synergy with several cell wall degrading enzymes and members of GH62 represent promising candidates for biotechnological improvements of biofuel production and in various biorefinery applications.     

Microbial endoxylanases: effective weapons to breach the plant cell-wall barrier or, rather, triggers of plant defense systems?

Endo-beta-1,4-xylanases (EC 3.2.1.8) are key enzymes in the degradation of xylan, the predominant hemicellulose in the cell walls of plants and the second most abundant polysaccharide on earth. A number of endoxylanases are produced by microbial phytopathogens responsible for severe crop losses. These enzymes are considered to play an important role in phytopathogenesis, as they provide essential means to the attacking organism to break through the plant cell wall. Plants have evolved numerous defense mechanisms to protect themselves against invading pathogens, amongst which are proteinaceous inhibitors of cell wall-degrading enzymes. These defense mechanisms are triggered when a pathogen-derived elicitor is recognized by the plant. In this review, the diverse aspects of endoxylanases in promoting virulence and in eliciting plant defense systems are highlighted. Furthermore, the role of the relatively recently discovered cereal endoxylanase inhibitor families TAXI (Triticum aestivum xylanase inhibitor) and XIP (xylanase inhibitor protein) in plant defense is discussed.     

The gene task1 is involved in morphological development,mycoparasitism and antibiosis of Trichoderma asperellum

Competitive activity, mycoparasitism and antibiosis of Trichoderma asperellum are considered essential mechanisms in its suppressive activity against soil-borne plant pathogens. The role of the mitogen-activated protein kinase encoding gene task1 on morphological development, mycoparasitic interaction and the production of cell wall degrading enzymes and secondary metabolites were examined in T. asperellum. The Δtask1 mutant had altered growth morphology, lost its ability to parasitise plant pathogens and showed increased expression of several cell wall degrading enzymes during confrontation with Rhizoctonia solani. T. asperellum task1 expression was negatively correlated with cell wall degrading enzyme activities during inducing experiments using pathogen cell wall compounds. In antibiosis assays, task1 deletion caused increased output of 6-pentyl-α-pyrone and inhibition of pathogen growth.     

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     

An Efficient Proteomic Approach to Analyze Agriculture Crop Biomass

While a plant cell wall is formed by a complex of various components, including polysaccharides and structural proteins, its composition and representation may vary during cell growth. Currently, plant research targets the proteins participating in wall loosening. Multiple classes of enzymes, including various hemicellulases and cellulases, are required for plant material degradation to achieve the maximum decomposition. Identifying the set of proteins involved in the breakdown of cell-wall polymers is important to understand plant material conversion into suitable products. The objective of this study was to describe a method which can be used to carry out proteomics analysis of complex plant samples and identify enzymes degrading biomass. For this purpose we used proteomic techniques including gel electrophoresis, high pressure liquid chromatography combinated with mass spectrometry followed by data evaluation using databases searching. Results show that more than 50 % of these activities correspond to enzymes with proteolytic function. This study was focused primarily on enzymes able to breakdown the lignocellulosic and hemicellulosic parts that are very important for the material conversion into required products of degradation.     

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.     

The secretome of the maize pathogen Ustilago maydis

Ustilago maydis establishes a biotrophic relationship with its host plant, i.e. plant cells stay alive despite massive fungal growth in infected tissue. The genome sequence has revealed that U. maydis is poorly equipped with plant cell wall degrading enzymes and uses novel secreted protein effectors as crucial determinants for biotrophic development. Many of these effector genes are clustered and differentially regulated during plant colonization. In this review, we analyze the secretome of U. maydis by differentiating between secreted enzymes, likely structural proteins of the fungal cell wall (excluding GPI-anchored proteins) as well as likely effectors with either apoplastic or cytoplasmic function. This classification is based on the presence of functional domains, general domain structure and cysteine pattern. In addition, we discuss possible functions of selected protein classes with a special focus on disease development.