Catherine Rayon

N-Glycoprotein biosynthesis in plants: recent developments and future trends

N-glycosylation is a major modification of proteins in plant cells. This process starts in the endoplasmic reticulum by the co-translational transfer of a precursor oligosaccharide to specific asparagine residues of the nascent polypeptide chain. Processing of this oligosaccharide into high-mannose-type, paucimannosidic-type, hybrid-type or complex-type N-glycans occurs in the secretory pathway as the glycoprotein moves from the endoplasmic reticulum to its final destination. At the end of their maturation, some plant N-glycans have typical structures that differ from those found in their mammalian counterpart by the absence of sialic acid and the presence of β(1,2)-xylose and α(1,3)-fucose residues. Glycosidases and glycosyltransferases that respectively catalyse the stepwise trimming and addition of sugar residues are generally considered as working in a co-ordinated and highly ordered fashion to form mature N-glycans. On the basis of this assembly line concept, fast progress is currently made by using N-linked glycan structures as milestones of the intracellular transport of proteins along the plant secretory pathway. Further developments of this approach will need to more precisely define the topological distribution of glycosyltransferases within a plant Golgi stack. In contrast with their acknowledged role in the targeting of lysosomal hydrolases in mammalian cells, N-glycans have no specific function in the transport of glycoproteins into the plant vacuole. However, the presence of N-glycans, regardless of their structures, is necessary for an efficient secretion of plant glycoproteins. In the biotechnology field, transgenic plants are rapidly emerging as an important system for the production of recombinant glycoproteins intended for therapeutic purposes, which is a strong motivation to speed up research in plant glycobiology. In this regard, the potential and limits of plant cells as a factory for the production of mammalian glycoproteins will be illustrated.

Cell Wall Metabolism in Response to Abiotic Stress

This review focuses on the responses of the plant cell wall to several abiotic stresses including drought, flooding, heat, cold, salt, heavy metals, light, and air pollutants. The effects of stress on cell wall metabolism are discussed at the physiological (morphogenic), transcriptomic, proteomic and biochemical levels. The analysis of a large set of data shows that the plant response is highly complex. The overall effects of most abiotic stress are often dependent on the plant species, the genotype, the age of the plant, the timing of the stress application, and the intensity of this stress. This shows the difficulty of identifying a common pattern of stress response in cell wall architecture that could enable adaptation and/or resistance to abiotic stress. However, in most cases, two main mechanisms can be highlighted: (i) an increased level in xyloglucan endotransglucosylase/hydrolase (XTH) and expansin proteins, associated with an increase in the degree of rhamnogalacturonan I branching that maintains cell wall plasticity and (ii) an increased cell wall thickening by reinforcement of the secondary wall with hemicellulose and lignin deposition. Taken together, these results show the need to undertake large-scale analyses, using multidisciplinary approaches, to unravel the consequences of stress on the cell wall. This will help identify the key components that could be targeted to improve biomass production under stress conditions.

Mixed linkage (1→3), (1→4)-β-D-glucans of grasses

Cereal Chem. 81(1):115–127 The mixed-linkage (1 3),(1 4)-D-glucans are unique to the Poales, the taxonomic order that includes the cereal grasses. (1 3), (1 4)-Glucans are the principal molecules associated with cellulose microfibrils during cell growth, and they are enzymatically hydrolyzed to a large extent once growth has ceased. They appear again during the developmental of the endosperm cell wall and maternal tissues surrounding them. The roles of (1 3),(1 4)-glucans in cell wall architecture and in cell growth are beginning to be understood. From biochemical experiments with active synthases in isolated Golgi membranes, the biochemical features and topology of synthesis are found to more closely parallel those of cellulose than those of all other noncellulosic -linked polysaccharides. The genes that encode part of the (1 3),(1 4)-glucan synthases are likely to be among those of the CESA/CSL gene superfamily, but a distinct glycosyl transferase also appears to be integral in the synthetic machinery. Several genes involved in the hydrolysis of (1 3),(1 4)-glucan have been cloned and sequenced, and the pattern of expression is starting to unveil their function in mobilization of -glucan reserve material and in cell growth. The starchy endosperm, a special trait of the Poales, is the fundamental reason that cereals are of such central importance in human nutrition (Langenheim and Thimann 1982). The world harvests over 1 billion tons of cereal grains annually. Rice and wheat alone provide at least half of the calories that humans ingest. The cell walls of the grasses also figure heavily in human and animal nutrition, from the mixed-linkage (1 3),(1 4)glucans that constitute a major portion of the endosperm walls to the vast amounts of xylanand cellulose-rich walls that are consumed by grazing animals. Scientific interest in (1 3),(1 4)glucans is associated partly with problems they cause in brewing and animal feed industries and partly from benefits they offer to human diets (as reviewed by Stone and Clarke 1992). The (1 3),(1 4)-glucans are the wall constituents responsible for the enhanced ability of barley and oat brans to reduce serum cholesterol in hypercholesterolemic individuals (for example, Braaten et al 1994) and to modulate the glycemic index in diabetics (Wood et al 1990). The (1 3),(1 4)-glucans and xylans of the endosperm cell walls in flours are contributing factors to bread quality (Girhammerar et al 1986). Incomplete hydrolysis of the viscous (1 3),(1 4)-glucans during the brewing process is a major production problem and contributes to hazing of beers upon storage (for review see Woodward and Fincher 1983). Because of these practical aspects of (1 3), (1 4)-glucans, they have been the focus of many studies describing their unique structure and their content and dynamics in cereal grains. These studies of (1 3),(1 4)-glucan structure and physiology and the special cell wall of grasses have been covered by several reviews and book chapters, and the readers are directed to these for more extensive coverage of the early literature (Wilkie 1979; Bacic et al 1988; Carpita and Gibeaut 1993; Carpita 1996). Grass species use (1 3),(1 4)-glucans as structural elements of the walls of growing cells and as an endosperm storage material that is hydrolyzed during germination to provide an extra source of carbon during early seedling establishment (Meier and Reid 1982). (1 3),(1 4)-Glucan synthase represents one of the few biosynthetic machines whose activity can be preserved in vitro, and the polymer synthesized is identical in size and structure to that produced by the plants. This feature gives us a unique opportunity to study all of the interacting proteins and enzymes that comprise this system. Although plant biologists have finally identified and characterized a few genes that encode the synthesis machinery, we still don’t understand the biochemical mechanisms of synthesis very well. Identification of a (1 3),(1 4)-glucan synthase genes and determination of the three-dimensional structures of their active sites will ultimately yield important clues to understanding the chemical mechanism of glycosyl transfer, not only of this specific synthase, but also for cellulose synthase and all other plant polysaccharide synthases. In this review, we summarize the features that make cell walls of cereals different from all other flowering plants and give a special focus to the special role that (1 3),(1 4)-glucans play in wall architecture and plant development. We will highlight recent advances in structurefunctional relationships, including the characterization of the enzymes that participate in the hydrolysis of (1 3),(1 4)glucan during germination, reserve mobilization, and cell growth, and the cloning of their genes. Our central effort will be to provide new insights and speculations into the mechanisms of synthesis at the Golgi apparatus and the progress toward identification of the genes that encode the synthetic machinery. Special Cell Wall of Grasses and Cereals All plant cell walls are composites of at least three independent but coextensive, interwoven networks of polymers. The cellulose and cross-linking glycan framework embedded in a matrix of pectic substances form two of them, and these networks are eventually cross-linked into a firm, inextensible structure by structural proteins or polyphenolic substances (McCann and Roberts 1991; Carpita and Gibeaut 1993). A vast majority of flowering plants possess a type I wall in which the principal cellulose cross-linking glycan is xyloglucan and as much as 35% of the wall mass is pectin (Carpita and Gibeaut 1993). The grasses and cereals possess a type II cell wall (Carpita and Gibeaut 1993; Carpita 1996), and at least two important evolutional changes resulted in their vastly different cell wall 1 Secao de Fisiologia e Bioquimica de Plantas, Instituto de Botânica CP 4005 CEP 01061-970, Sao Paulo, SP Brazil. 2 Department of Botany and Plant Pathology, Purdue University West Lafayette, IN 47907–1155. 3 Present address: UMR CNRS-UPS 5546, Pole de Biotechnologie Vegetale, BP 17, Auzeville, F-31326 Castanet Tolosan, France. 4 Present address: Department of Plant Biology, 228 Plant Science Building, Cornell University, Ithaca, NY 14853. 5 Corresponding author. Phone: +1-765-494-4653. Fax: +1-765-494-0393. Email: carpita@purdue.edu Publication no. C-2003-1216-01R.

Cell wall compositional modifications of Miscanthus ecotypes in response to cold acclimation.

Miscanthus, a potential energy crop grass, can be damaged by late frost when shoots emerge too early in the spring and during the first winter after planting. The effects of cold acclimation on cell wall composition were investigated in a frost-sensitive clone of Miscanthus x giganteus compared to frost-tolerant clone, Miscanthus sinensis August Feder, and an intermediate frost-tolerant clone, M. sinensis Goliath. Cellulose and lignin contents were higher in M. x giganteus than in the M. sinensis genotypes. In ambient temperature controls, each clone displayed different glucuronoarabinoxylan (GAX) contents and degree of arabinose substitution on the xylan backbone. During cold acclimation, an increase in (1→3),(1→4)-β-D-glucan content was observed in all genotypes. Uronic acid level increased in the frost sensitive genotype but decreased in the frost tolerant genotypes in response to cold. In all clones, major changes in cell wall composition were observed with modifications in phenylalanine ammonia-lyase (PAL) and cinnamyl alcohol dehydrogenase (CAD) activities in both non- and cold-acclimated experiments. A large increase in CAD activity under cold stress was displayed in each clone, but it was largest in the frost-tolerant clone, M. sinensis August Feder. The marked increase in PAL activity observed in the frost-tolerant clones under cold acclimation, suggests a reorientation of the products towards the phenylpropanoid pathway or aromatic synthesis. How changes in cell wall physical properties can impact frost tolerance is discussed.

Topology of the Maize Mixed Linkage (1→3),(1→4)-β-D-Glucan Synthase at the Golgi Membrane

Mixed-linkage (1→3),(1→4)-β-d-glucan is a plant cell wall polysaccharide composed of cellotriosyl and cellotetraosyl units, with decreasingly smaller amounts of cellopentosyl, cellohexosyl, and higher cellodextrin units, each connected by single (1→3)-β-linkages. (1→3),(1→4)-β-Glucan is synthesized in vitro with isolated maize (Zea mays) Golgi membranes and UDP-[14C]d-glucose. The (1→3),(1→4)-β-glucan synthase is sensitive to proteinase K digestion, indicating that part of the catalytic domain is exposed to the cytoplasmic face of the Golgi membrane. The detergent {3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid} (CHAPS) also lowers (1→3),(1→4)-β-glucan synthase activity. In each instance, the treatments selectively inhibit formation of the cellotriosyl units, whereas synthesis of the cellotetraosyl units is essentially unaffected. Synthesis of the cellotriosyl units is recovered when a CHAPS-soluble factor is permitted to associate with Golgi membranes at synthesis-enhancing CHAPS concentrations but lost if the CHAPS-soluble fraction is replaced by fresh CHAPS buffer. In contrast to other known Golgi-associated synthases, (1→3),(1→4)-β-glucan synthase behaves as a topologic equivalent of cellulose synthase, where the substrate UDP-glucose is consumed at the cytosolic side of the Golgi membrane, and the glucan product is extruded through the membrane into the lumen. We propose that a cellulose synthase-like core catalytic domain of the (1→3),(1→4)-β-glucan synthase synthesizes cellotetraosyl units and higher even-numbered oligomeric units and that a separate glycosyl transferase, sensitive to proteinase digestion and detergent extraction, associates with it to add the glucosyl residues that complete the cellotriosyl and higher odd-numbered units, and this association is necessary to drive polymer elongation.

Identification of pectin methylesterase 3 as a basic pectin methylesterase isoform involved in adventitious rooting in Arabidopsis thaliana

• Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. • A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. • We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. • Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

The Structure of the Catalytic Domain of a Plant Cellulose Synthase and Its Assembly into Dimers

Small-angle x-ray scattering gives a glimpse at the solution structure of the catalytic domains of plant cellulose synthase and their dimerization. Dimerization through the plant-specific sequences of the catalytic domain provides important clues for how Zn-finger domains couple these fundamental scaffold units into large, multimeric synthase complexes. Cellulose microfibrils are para-crystalline arrays of several dozen linear (1→4)-β-d-glucan chains synthesized at the surface of the cell membrane by large, multimeric complexes of synthase proteins. Recombinant catalytic domains of rice (Oryza sativa) CesA8 cellulose synthase form dimers reversibly as the fundamental scaffold units of architecture in the synthase complex. Specificity of binding to UDP and UDP-Glc indicates a properly folded protein, and binding kinetics indicate that each monomer independently synthesizes single glucan chains of cellulose, i.e., two chains per dimer pair. In contrast to structure modeling predictions, solution x-ray scattering studies demonstrate that the monomer is a two-domain, elongated structure, with the smaller domain coupling two monomers into a dimer. The catalytic core of the monomer is accommodated only near its center, with the plant-specific sequences occupying the small domain and an extension distal to the catalytic domain. This configuration is in stark contrast to the domain organization obtained in predicted structures of plant CesA. The arrangement of the catalytic domain within the CesA monomer and dimer provides a foundation for constructing structural models of the synthase complex and defining the relationship between the rosette structure and the cellulose microfibrils they synthesize.

Arabidopsis PECTIN METHYLESTERASE17 is co-expressed with and processed by SBT3.5, a subtilisin-like serine protease

BACKGROUND AND AIMS In Arabidopsis thaliana, the degree of methylesterification (DM) of homogalacturonans (HGs), the main pectic constituent of the cell wall, can be modified by pectin methylesterases (PMEs). In all organisms, two types of protein structure have been reported for PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an N-terminal extension (PRO part), which shows similarities to PME inhibitors (PMEI domain, Pfam04043). This PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism. This study investigated the roles of a subtilisin-type serine protease (SBT) in the processing of a PME isoform. METHODS Using a combination of functional genomics, biochemistry and proteomic approaches, the role of a specific SBT in the processing of a group 2 PME was assessed together with its consequences for plant development. KEY RESULTS A group 2 PME, AtPME17 (At2g45220), was identified, which was highly co-expressed, both spatially and temporally, with AtSBT3.5 (At1g32940), a subtilisin-type serine protease (subtilase, SBT), during root development. PME activity was modified in roots of knockout mutants for both proteins with consequent effects on root growth. This suggested a role for SBT3.5 in the processing of PME17 in planta. Using transient expression in Nicotiana benthamiana, it was indeed shown that SBT3.5 can process PME17 at a specific single processing motif, releasing a mature isoform in the apoplasm. CONCLUSIONS By revealing the potential role of SBT3.5 in the processing of PME17, this study brings new evidence of the complexity of the regulation of PMEs in plants, and highlights the need for identifying specific PME-SBT pairs.

Major changes in the cell wall during silique development in Arabidopsis thaliana

Fruit development is a highly complex process, which involves major changes in plant metabolism leading to cell growth and differentiation. Changes in cell wall composition and structure play a major role in modulating cell growth. We investigated the changes in cell wall composition and the activities of associated enzymes during the dry fruit development of the model plant Arabidopsis thaliana. Silique development is characterized by several specific phases leading to fruit dehiscence and seed dispersal. We showed that early phases of silique growth were characterized by specific changes in non-cellulosic sugar content (rhamnose, arabinose, xylose, galactose and galacturonic acid). Xyloglucan oligosaccharide mass profiling further showed a strong increase in O-acetylated xyloglucans over the course of silique development, which could suggest a decreased capacity of xyloglucans to be associated with each other or to cellulose. The degree of methylesterification, mediated by the activity of pectin methylesterases (PMEs), decreased over the course of silique growth and dehiscence. The major changes in cell wall composition revealed by our analysis suggest that it could be major determinants in modulating cell wall rheology leading to growth or growth arrest.

Tuning of Pectin Methylesterification: PECTIN METHYLESTERASE INHIBITOR 7 MODULATES THE PROCESSIVE ACTIVITY OF CO-EXPRESSED PECTIN METHYLESTERASE 3 IN A pH-DEPENDENT MANNER*

Background: PME and PMEI isoforms are co-expressed in Arabidopsis. Their biochemical interaction is yet to be characterized. Results: The processive activity of AtPME3 is regulated by AtPMEI7 in a pH-dependent manner in vitro. Conclusion: AtPMEI7 is a key component of the regulation of AtPME3 activity in planta. Significance: The tuning of AtPME3 activity by AtPMEI7 brings insights into the control of homogalacturonan methylesterification in plant cell walls. Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.