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.     

Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides

Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.     

Aspergillus enzymes involved in degradation of plant cell wall polysaccharides.

Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.     

Identification and characterization of Arabidopsis thaliana genes involved in xylem secondary cell walls

The xylem of higher plants offers support to aerial portions of the plant body and serves as conduit for the translocation of water and nutrients. Terminal differentiation of xylem cells typically involves deposition of thick secondary cell walls. This is a dynamic cellular process accompanied by enhanced rates of cellulose deposition and the induction of synthesis of specific secondary-wall matrix polysaccharides and lignin. The secondary cell wall is essential for the function of conductive and supportive xylem tissues. Recently, significant progress has been made in identifying the genes responsible for xylem secondary cell wall formation. However, our present knowledge is still insufficient to account for the molecular processes by which this complex system operates. To acquire further information about xylem secondary cell walls, we initially focused our research effort on a set of genes specifically implicated in secondary cell wall formation, as well as on loss-of-function mutants. Results from two microarray screens identified several key candidate genes responsible for secondary cell wall formation. Reverse genetic analyses led to the identification of a glycine-rich protein involved in maintaining the stable structure of protoxylem, which is essential for the transport of water and nutrients. A combination of expression analyses and reverse genetics allows us to systematically identify new genes required for the development of physical properties of the xylem secondary wall.     

Recent advances in the understanding of the <Emphasis Type="Italic">Aspergillus fumigatus</Emphasis> cell wall

Over the past several decades, research on the synthesis and organization of the cell wall polysaccharides of Aspergillus fumigatus has expanded our knowledge of this important fungal structure. Besides protecting the fungus from environmental stresses and maintaining structural integrity of the organism, the cell wall is also the primary site for interaction with host tissues during infection. Cell wall polysaccharides are important ligands for the recognition of fungi by the innate immune system and they can mediate potent immunomodulatory effects. The synthesis of cell wall polysaccharides is a complicated process that requires coordinated regulation of many biosynthetic and metabolic pathways. Continuous synthesis and remodeling of the polysaccharides of the cell wall is essential for the survival of the fungus during development, reproduction, colonization and invasion. As these polysaccharides are absent from the human host, these biosynthetic pathways are attractive targets for antifungal development. In this review, we present recent advances in our understanding of Aspergillus fumigatus cell wall polysaccharides, including the emerging role of cell wall polysaccharides in the host-pathogen interaction.     

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 cell wall-oriented genomic approach reveals a new and unexpected complexity of the softening in peaches

During ripening, fleshy fruits undergo textural changes that lead to loss of tissue firmness and consequent softening. It is a common idea that this process is the consequence of cell wall dismantling carried out by different and orderly expressed enzymes. For this purpose, by using a single enzyme family approach many enzymes and related genes have been characterized in different fruits. In this work, the softening of the climacteric peach fruits (Prunus persica (L.) Batsch.) has been studied by using a genomic approach, and the results obtained are novel and partly unexpected. The genes analysed encode proteins involved in the main metabolic aspects of a primary cell wall: degradation, synthesis, structure. In addition, some genes encoding cell-wall-related proteins with an unknown function have been studied. The gene expression profiles show that the softening actually begins well before the climacteric rise and continues thereafter. Genes whose expression starts before the climacteric rise are mostly down-regulated by ethylene, while genes with a ripening-specific expression are mostly up-regulated by the hormone. A few other genes are apparently insensitive to ethylene. Besides the expected parietal degradation, the softening that results from this study also comprises some repairing of the cell wall performed by enzymes involved in the synthesis of parietal polysaccharides and, especially, by proteins with structural functions. The newly synthesized polysaccharides and the structural proteins would thus help to hold together the fruit cell wall while not preventing the softening.     

The genome of the mustard leaf beetle encodes two active xylanases originally acquired from bacteria through horizontal gene transfer

The primary plant cell wall comprises the most abundant polysaccharides on the Earth and represents a rich source of energy for organisms which have evolved the ability to digest them. Enzymes able to degrade plant cell wall polysaccharides are widely distributed in micro-organisms but are generally absent in animals, although their presence in insects, especially phytophagous beetles from the superfamilies Chrysomeloidea and Curculionoidea, has recently begun to be appreciated. The observed patchy distribution of endogenous genes encoding these enzymes in animals has raised questions about their evolutionary origins. Recent evidence suggests that endogenous plant cell wall degrading enzymes-encoding genes have been acquired by animals through a mechanism known as horizontal gene transfer (HGT). HGT describes how genetic material is moved by means other than vertical inheritance from a parent to an offspring. Here, we provide evidence that the mustard leaf beetle, Phaedon cochleariae, possesses in its genome genes encoding active xylanases from the glycoside hydrolase family 11 (GH11). We also provide evidence that these genes were originally acquired by P. cochleariae from a species of gammaproteobacteria through HGT. This represents the first example of the presence of genes from the GH11 family in animals.     

Plant Cell Wall Is a Stumbling Stone for Molecular Biologists

Cell wall is a key structure of the plant organism engaged in numerous functions, and plants spend enormous resources on cell wall formation. Cell wall components are the most widespread organic substances on the Earth. However important is assembling plant cell wall polysaccharides, this process has been insufficiently studied by the methods of molecular genetics; in particular, too little is known of the genes that code for the relevant enzymes (glycosyltransferases, GT). The review addresses the current situation by expounding on GT classification, describing the characteristics of enzymes that synthesize cell wall polysaccharides, and summing up the existing knowledge of already identified and putative cellulose and callose synthases and GT localized in the Golgi apparatus. The methodology for searching and characterizing new genes that participate in cell wall formation is under discussion.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 3, 2005, pp. 443–462.Original Russian Text Copyright © 2005 by Gorshkova, Nikolovski, Finaev.     

Approaches to understanding the functional architecture of the plant cell wall.

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.     

Pectin: cell biology and prospects for functional analysis

Pectin is a major component of primary cell walls of all land plants and encompasses a range of galacturonic acid-rich polysaccharides. Three major pectic polysaccharides (homogalacturonan, rhamnogalacturonan-I and rhamnogalacturonan-II) are thought to occur in all primary cell walls. This review surveys what is known about the structure and function of these pectin domains. The high degree of structural complexity and heterogeneity of the pectic matrix is produced both during biosynthesis in the endomembrane system and as a result of the action of an array of wall-based pectin-modifying enzymes. Recent developments in analytical techniques and in the generation of anti-pectin probes have begun to place the structural complexity of pectin in cell biological and developmental contexts. The in muro de-methyl-esterification of homogalacturonan by pectin methyl esterases is emerging as a key process for the local modulation of matrix properties. Rhamnogalacturonan-I comprises a highly diverse population of spatially and developmentally regulated polymers, whereas rhamnogalacturonan-II appears to be a highly conserved and stable pectic domain. Current knowledge of biosynthetic enzymes, plant and microbial pectinases and the interactions of pectin with other cell wall components and the impact of molecular genetic approaches are reviewed in terms of the functional analysis of pectic polysaccharides in plant growth and development.     

A novel bioinformatics approach identifies candidate genes for the synthesis and feruloylation of arabinoxylan

Arabinoxylans (AXs) are major components of graminaceous plant cell walls, including those in the grain and straw of economically important cereals. Despite some recent advances in identifying the genes encoding biosynthetic enzymes for a number of other plant cell wall polysaccharides, the genes encoding enzymes of the final stages of AX synthesis have not been identified. We have therefore adopted a novel bioinformatics approach based on estimation of differential expression of orthologous genes between taxonomic divisions of species. Over 3 million public domain cereal and dicot expressed sequence tags were mapped onto the complete sets of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana) genes, respectively. It was assumed that genes in cereals involved in AX biosynthesis would be expressed at high levels and that their orthologs in dicotyledonous plants would be expressed at much lower levels. Considering all rice genes encoding putative glycosyl transferases (GTs) predicted to be integral membrane proteins, genes in the GT43, GT47, and GT61 families emerged as much the strongest candidates. When the search was widened to all other rice or Arabidopsis genes predicted to encode integral membrane proteins, cereal genes in Pfam family PF02458 emerged as candidates for the feruloylation of AX. Our analysis, known activities, and recent findings elsewhere are most consistent with genes in the GT43 families encoding beta-1,4-xylan synthases, genes in the GT47 family encoding xylan alpha-1,2- or alpha-1,3-arabinosyl transferases, and genes in the GT61 family encoding feruloyl-AX beta-1,2-xylosyl transferases.     

Molecular genetics of nucleotide sugar interconversion pathways in plants

Nucleotide sugar interconversion pathways represent a series of enzymatic reactions by which plants synthesize activated monosaccharides for the incorporation into cell wall material. Although biochemical aspects of these metabolic pathways are reasonably well understood, the identification and characterization of genes encoding nucleotide sugar interconversion enzymes is still in its infancy. Arabidopsis mutants defective in the activation and interconversion of specific monosaccharides have recently become available, and several genes in these pathways have been cloned and characterized. The sequence determination of the entire Arabidopsis genome offers a unique opportunity to identify candidate genes encoding nucleotide sugar interconversion enzymes via sequence comparisons to bacterial homologues. An evaluation of the Arabidopsis databases suggests that the majority of these enzymes are encoded by small gene families, and that most of these coding regions are transcribed. Although most of the putative proteins are predicted to be soluble, others contain N-terminal extensions encompassing a transmembrane domain. This suggests that some nucleotide sugar interconversion enzymes are targeted to an endomembrane system, such as the Golgi apparatus, where they may co-localize with glycosyltransferases in cell wall synthesis. The functions of the predicted coding regions can most likely be established via reverse genetic approaches and the expression of proteins in heterologous systems. The genetic characterization of nucleotide sugar interconversion enzymes has the potential to understand the regulation of these complex metabolic pathways and to permit the modification of cell wall material by changing the availability of monosaccharide precursors.     

The functions of cell wall polysaccharides in composition and architecture revealed through mutations

The plant cell wall is a highly organized composite of many different polysaccharides, proteins and aromatic substances. These complex matrices define the shape of each individual cell, and ultimately, they are the determinants of plant morphology. The fine structures of the major angiosperm cell wall polysaccharides have been characterized, but it is not well understood how these polysaccharides are assembled into a metabolically active architecture. Cell wall biogenesis and remodeling may be partitioned into six major stages of development (precursor synthesis, polymerization, secretion, assembly, rearrangement and disassembly), and to date, a handful of mutations have been identified that affect the composition and structure in each of these stages. To greatly augment this collection, we have initiated a program to use Fourier transform infrared spectroscopy as a high through-put screen to identify a broad range of cell-wall mutants of Arabidopsis and maize. We anticipate that such mutants will be useful to probe the impact of the individual components and their metabolism on basic processes of plant growth and development. The structures of dicot and grass walls, the identification of representative cell wall mutants, and the use of a novel spectroscopic screen to identify many more cell wall mutants, are briefly reviewed.     

Pectins: structure, biosynthesis, and oligogalacturonide-related signaling.

Pectin is a family of complex polysaccharides present in all plant primary cell walls. The complicated structure of the pectic polysaccharides, and the retention by plants of the large number of genes required to synthesize pectin, suggests that pectins have multiple functions in plant growth and development. In this review we summarize the current level of understanding of pectin primary and tertiary structure, and describe new methods that may be useful to study localized pectin structure in the plant cell wall. We also discuss progress in our understanding of how pectin is biosynthesized and review the biological activities and possible modes of action of pectic oligosaccharides referred to as oligogalacturonides. We present our view of critical questions regarding pectin structure, biosynthesis, and function that need to be addressed in the coming decade. As the plant community works towards understanding the functions of the tens of thousands of genes expressed by plants, a large number of those genes are likely to be involved in the synthesis, turnover, biological activity, and restructuring of pectin. A combination of genetic, molecular, biochemical and chemical approaches will be necessary to fully understand the function and biosynthesis of pectin.     

Bioinformatics-Based Identification of Candidate Genes from QTLs Associated with Cell Wall Traits in Populus

Quantitative trait locus (QTL) studies are an integral part of plant research and are used to characterize the genetic basis of phenotypic variation observed in structured populations and inform marker-assisted breeding efforts. These QTL intervals can span large physical regions on a chromosome comprising hundreds of genes, thereby hampering candidate gene identification. Genome history, evolution, and expression evidence can be used to narrow the genes in the interval to a smaller list that is manageable for detailed downstream functional genomics characterization. Our primary motivation for the present study was to address the need for a research methodology that identifies candidate genes within a broad QTL interval. Here we present a bioinformatics-based approach for subdividing candidate genes within QTL intervals into alternate groups of high probability candidates. Application of this approach in the context of studying cell wall traits, specifically lignin content and S/G ratios of stem and root in Populus plants, resulted in manageable sets of genes of both known and putative cell wall biosynthetic function. These results provide a roadmap for future experimental work leading to identification of new genes controlling cell wall recalcitrance and, ultimately, in the utility of plant biomass as an energy feedstock.