Spectrophotometric Assays for Measuring Photorespiratory Glutamate:Glyoxylate and Serine:Glyoxylate Aminotransferase Reactions.

Glutamate:glyoxylate aminotransferase (GGAT; EC 2.6.1.4) and serine:glyoxylate aminotransferase activities (SGAT; EC 2.6.1.45) are central photorespiratory reactions within plant peroxisomes. Both enzymatic reactions convert glyoxylate, a product of glycolate oxidase, to glycine, a substrate of the mitochondrial glycine decarboxylase complex. The GGAT reaction uses glutamate as an amino group donor and also produces α-ketoglutarate, which is recycled to glutamate in plastids by ferredoxin-dependent glutamate synthase. Using serine, a product of mitochondrial serine hydroxymethyltransferase, as an amino group donor, the SGAT reaction also produces hydroxypyruvate, a substrate of hydroxypyruvate reductase. The activities of these photorespiratory aminotransferases can be measured using indirect, coupled, spectrophotometric assays, detailed herein.

Spectrophotometric Assays for Measuring Hydroxypyruvate Reductase Activity.

Hydroxypyruvate reductase (HPR; EC 1.1.1.81) activity is integral to the photorespiratory pathway. Within photorespiration, HPR catalyzes the reduction of hydroxypyruvate, a product of the serine:glyoxylate aminotransferase reaction to glycerate, a substrate for glycerate kinase, using NADH as cofactor. Here we detail a spectrophotometric assay for measuring HPR activity in vitro by following the consumption of NADH at 340 nm.

Crystal Structure Of Photorespiratory Alanine:Glyoxylate Aminotransferase 1 (AGT1) From Arabidopsis thaliana.

Photorespiration is an energetically costly metabolic pathway for the recycling of phosphoglycolate produced by the oxygenase activity of ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) to phosphoglycerate. Arabidopsis alanine:glyoxylate aminotransferase 1 (AGT1) is a peroxisomal aminotransferase with a central role in photorespiration. This enzyme catalyzes various aminotransferase reactions, including serine:glyoxylate, alanine:glyoxylate, and asparagine:glyoxylate transaminations. To better understand structural features that govern the specificity of this enzyme, its crystal structures in the native form (2.2-Å resolution) and in the presence of l-serine (2.1-Å resolution) were solved. The structures confirm that this enzyme is dimeric, in agreement with studies of the active enzyme in solution. In the crystal, another dimer related by noncrystallographic symmetry makes close interactions to form a tetramer mediated in part by an extra carboxyl-terminal helix conserved in plant homologs of AGT1. Pyridoxal 5'-phosphate (PLP) is bound at the active site but is not held in place by covalent interactions. Residues Tyr35' and Arg36', entering the active site from the other subunits in the dimer, mediate interactions between AGT and l-serine when used as a substrate. In comparison, AGT1 from humans and AGT1 from Anabaena lack these two residues and instead position a tyrosine ring into the binding site, which accounts for their preference for l-alanine instead of l-serine. The structure also rationalizes the phenotype of the sat mutant, Pro251 to Leu, which likely affects the dimer interface near the catalytic site. This structural model of AGT1 provides valuable new information about this protein that may enable improvements to the efficiency of photorespiration.

The CELLULOSE SYNTHASE-LIKE A and CELLULOSE SYNTHASE-LIKE C families: recent advances and future perspectives.

The CELLULOSE SYNTHASE (CESA) superfamily of proteins contains several sub-families of closely related CELLULOSE SYNTHASE-LIKE (CSL) sequences. Among these, the CSLA and CSLC families are closely related to each other and are the most evolutionarily divergent from the CESA family. Significant progress has been made with the functional characterization of CSLA and CSLC genes, which have been shown to encode enzymes with 1,4-ß-glycan synthase activities involved in the biosynthesis of mannan and possibly xyloglucan backbones, respectively. This review examines recent work on the CSLA and CSLC families from evolutionary, molecular, and biochemical perspectives. We pose a series of questions, whose answers likely will provide further insight about the specific functions of members of the CSLA and CSLC families and about plant polysaccharide biosynthesis is general.

Deep sequencing of voodoo lily (Amorphophallus konjac): an approach to identify relevant genes involved in the synthesis of the hemicellulose glucomannan.

A Roche 454 cDNA deep sequencing experiment was performed on a developing corm of Amorphophallus konjac--also known as voodoo lily. The dominant storage polymer in the corm of this plant is the polysaccharide glucomannan, a hemicellulose known to exist in the cell walls of higher plants and a major component of plant biomass derived from softwoods. A total of 246 mega base pairs of sequence data was obtained from which 4,513 distinct contigs were assembled. Within this voodoo lily expressed sequence tag collection genes representing the carbohydrate related pathway of glucomannan biosynthesis were identified, including sucrose metabolism, nucleotide sugar conversion pathways for the formation of activated precursors as well as a putative glucomannan synthase. In vivo expression of the putative glucomannan synthase and subsequent in vitro activity assays unambiguously demonstrate that the enzyme has indeed glucomannan mannosyl- and glucosyl transferase activities. Based on the expressed sequence tag analysis hitherto unknown pathways for the synthesis of GDP-glucose, a necessary precursor for glucomannan biosynthesis, could be proposed. Moreover, the results highlight transcriptional bottlenecks for the synthesis of this hemicellulose.

Arabidopsis mannan synthase CSLA9 and glucan synthase CSLC4 have opposite orientations in the Golgi membrane.

Several proteins encoded by the cellulose synthase-like (CSL) gene family are known to be processive glycan synthases involved in the synthesis of cell-wall polysaccharides. These include CSLA proteins, which synthesize ß-(1→4)-linked mannans found in the walls of many plant species, and CSLC proteins, which are thought to synthesize the ß-(1→4)-linked glucan backbone of xyloglucan, an abundant polysaccharide in the primary walls of many plants. CSLA and CSLC proteins are predicted to have multiple membrane spans, and their products (mannan and xyloglucan) accumulate in the Golgi lumen. Knowing where these proteins are located in the cell and how they are orientated in the membrane is important for understanding many aspects of mannan and xyloglucan biosynthesis. In this study, we investigate the subcellular localization and membrane protein topology of CSLA9 and CSLC4, the members of these two families that are most highly expressed in Arabidopsis. CSLA9 and CSLC4 are found predominantly in Golgi membranes, based on co-localization with the known ER/Golgi marker ERD2-YFP. The topology of epitope-tagged proteins was examined using protease protection experiments. Experiments were designed to determine the positions of both the protein termini and the active loop of the CSL proteins investigated. The topology of CSLA9 is characterized by an odd number of transmembrane domains (probably five) and an active site that faces the Golgi lumen. In contrast, CSLC4 has an even number of transmembrane domains (probably six) and an active site that faces the cytosol. The implications of these topologies on various aspects of hemicellulose biosynthesis are discussed.

Arabidopsis - a powerful model system for plant cell wall research.

Plant cell walls are composites of various carbohydrates, proteins and other compounds. Cell walls provide plants with strength and protection, and also represent the most abundant source of renewable biomass. Despite the importance of plant cell walls, comparatively little is known about the identities of genes and functions of proteins involved in their biosynthesis. The model plant Arabidopsis and the availability of its genome sequence have been invaluable for the identification and functional characterization of genes encoding enzymes involved in plant cell-wall biosynthesis. This review covers recent progress in the identification and characterization of genes encoding proteins involved in the biosynthesis of Arabidopsis cell-wall polysaccharides and arabinogalactan proteins. These studies have improved our understanding of both the mechanisms of cell-wall biosynthesis and the functions of various cell-wall polymers, and have highlighted areas where further research is needed.

Cell wall glucomannan in Arabidopsis is synthesised by CSLA glycosyltransferases, and influences the progression of embryogenesis.

Mannans are hemicellulosic polysaccharides that have previously been implicated as structural constituents of cell walls and as storage reserves but which may serve other functions during plant growth and development. Several members of the Arabidopsis cellulose synthase-like A (CSLA) family have previously been shown to synthesise mannan polysaccharides in vitro when heterologously expressed. It has also been found that CSLA7 is essential for embryogenesis, suggesting a role for the CSLA7 product in development. To determine whether the CSLA proteins are responsible for glucomannan synthesis in vivo, we characterised insertion mutants in each of the nine Arabidopsis CSLA genes and several double and triple mutant combinations. csla9 mutants showed substantially reduced glucomannan, and triple csla2csla3csla9 mutants lacked detectable glucomannan in stems. Nevertheless, these mutants showed no alteration in stem development or strength. Overexpression of CSLA2, CSLA7 and CSLA9 increased the glucomannan content in stems. Increased glucomannan synthesis also caused defective embryogenesis, leading to delayed development and occasional embryo death. The embryo lethality of csla7 was complemented by overexpression of CSLA9, suggesting that the glucomannan products are similar. We conclude that CSLA2, CSLA3 and CSLA9 are responsible for the synthesis of all detectable glucomannan in Arabidopsis stems, and that CSLA7 synthesises glucomannan in embryos. These results are inconsistent with a substantial role for glucomannan in wall strength in Arabidopsis stems, but indicate that glucomannan levels affect embryogenesis. Together with earlier heterologous expression studies, the glucomannan deficiency observed in csla mutant plants demonstrates that the CSLA family encodes glucomannan synthases.

A gene from the cellulose synthase-like C family encodes a beta-1,4 glucan synthase.

Despite the central role of xyloglucan (XyG) in plant cell wall structure and function, important details of its biosynthesis are not understood. To identify the gene(s) responsible for synthesizing the beta-1,4 glucan backbone of XyG, we exploited a property of nasturtium (Tropaeolum majus) seed development. During the last stages of nasturtium seed maturation, a large amount of XyG is deposited as a reserve polysaccharide. A cDNA library was produced from mRNA isolated during the deposition of XyG, and partial sequences of 10,000 cDNA clones were determined. A single member of the C subfamily from the large family of cellulose synthase-like (CSL) genes was found to be overrepresented in the cDNA library. Heterologous expression of this gene in the yeast Pichia pastoris resulted in the production of a beta-1,4 glucan, confirming that the CSLC protein has glucan synthase activity. The Arabidopsis CSLC4 gene, which is the gene with the highest sequence similarity to the nasturtium CSL gene, is coordinately expressed with other genes involved in XyG biosynthesis. These and other observations provide a compelling case that the CSLC gene family encode proteins that synthesize the XyG backbone.

Functional genomic analysis supports conservation of function among cellulose synthase-like a gene family members and suggests diverse roles of mannans in plants.

Mannan polysaccharides are widespread among plants, where they serve as structural elements in cell walls, as carbohydrate reserves, and potentially perform other important functions. Previous work has demonstrated that members of the cellulose synthase-like A (CslA) family of glycosyltransferases from Arabidopsis (Arabidopsis thaliana), guar (Cyamopsis tetragonolobus), and Populus trichocarpa catalyze beta-1,4-mannan and glucomannan synthase reactions in vitro. Mannan polysaccharides and homologs of CslA genes appear to be present in all lineages of land plants analyzed to date. In many plants, the CslA genes are members of extended multigene families; however, it is not known whether all CslA proteins are glucomannan synthases. CslA proteins from diverse land plant species, including representatives of the mono- and dicotyledonous angiosperms, gymnosperms, and bryophytes, were produced in insect cells, and each CslA protein catalyzed mannan and glucomannan synthase reactions in vitro. Microarray mining and quantitative real-time reverse transcription-polymerase chain reaction analysis demonstrated that transcripts of Arabidopsis and loblolly pine (Pinus taeda) CslA genes display tissue-specific expression patterns in vegetative and floral tissues. Glycan microarray analysis of Arabidopsis indicated that mannans are present throughout the plant and are especially abundant in flowers, siliques, and stems. Mannans are also present in chloronemal and caulonemal filaments of Physcomitrella patens, where they are prevalent at cell junctions and in buds. Taken together, these results demonstrate that members of the CslA gene family from diverse plant species encode glucomannan synthases and support the hypothesis that mannans function in metabolic networks devoted to other cellular processes in addition to cell wall structure and carbohydrate storage.

Biosynthesis of plant cell wall polysaccharides - a complex process.

Cellulose, a major component of plant cell walls, is made by dynamic complexes that move within the plasma membrane while depositing cellulose directly into the wall. On the other hand, matrix polysaccharides are made in the Golgi and delivered to the wall via secretory vesicles. Several Golgi proteins that are involved in glucomannan and xyloglucan biosynthesis have been identified, including some glycan synthases that show sequence similarity to the cellulose synthase proteins and several glycosytransferases that add sidechains to the polysaccharide backbones. Recent progress in identifying the proteins needed for polysaccharide biosynthesis should lead to an improved understanding of the molecular details of these complex processes, and eventually to an ability to manipulate them in an effort to generate plants that have improved properties for human uses.

Expression of cellulose synthase-like (Csl) genes in insect cells reveals that CslA family members encode mannan synthases.

Glucuronoarabinoxylan, xyloglucan, and galactomannan are noncellulosic polysaccharides found in plant cell walls. All consist of beta-linked glycan backbones substituted with sugar side chains. Although considerable progress has been made in characterizing the structure of these polysaccharides, little is known about the biosynthetic enzymes that produce them. Cellulose synthase-like (Csl) genes are hypothesized to encode Golgi-localized beta-glycan synthases that polymerize the backbones of noncellulosic polysaccharides. To investigate this hypothesis, we used heterologous expression in Drosophila Schneider 2 (S2) cells to systematically analyze the functions of the gene products of a group of Csl genes from Arabidopsis and rice (Oryza sativa L.), including members from five Csl gene families (CslA, CslC, CslD, CslE, and CslH). Our analyses indicate that several members of the CslA gene family encode beta-mannan synthases. Recombinant CslA proteins produce beta-linked mannan polymers when supplied GDP-mannose. The same proteins can produce beta-linked glucomannan heteropolymers when supplied both GDP-mannose and GDP-glucose. One CslA protein also produced beta-linked glucan polymers when supplied GDP-glucose alone. Heterologous expression studies of additional candidate glycan synthases in insect cells or other systems may help identify other noncellulosic polysaccharide biosynthetic enzymes.

Alanine aminotransferase homologs catalyze the glutamate:glyoxylate aminotransferase reaction in peroxisomes of Arabidopsis.

Plant peroxisomal glyoxylate aminotransferases play central roles within the photorespiratory pathway. Genes encoding glyoxylate aminotransferases have been isolated from several animals and microbes, but only recently have plant homologs been identified. Three Arabidopsis homologs of alanine (Ala):glyoxylate aminotransferase 2 (AGT2) contain a putative type 1 peroxisomal targeting signal (PTS1), but the metabolic significance of these AGT2 homologs is unknown. GGT1 and GGT2 are Ala aminotransferase (AlaAT) homologs from Arabidopsis that represent another type of glyoxylate aminotransferase. These proteins are class I aminotransferases, each containing a putative PTS1. GGT1 and GGT2 are members of a small family of AlaATs in Arabidopsis. When expressed as recombinant proteins in Escherichia coli, GGT1 and GGT2 displayed biochemical characteristics very similar to one another, and to the Arabidopsis protein purified from leaves. Four aminotransferase activities were specifically associated with GGT1 and GGT2, using the substrate pairs glutamate (Glu):glyoxylate, Ala:glyoxylate, Glu:pyruvate, and Ala:2-oxoglutarate. GGT1 and GGT2 may have partially redundant functions; transcripts of both genes were detected in many of the same tissues. Although Glu:glyoxylate aminotransferase (GGT) activity has been observed in several locations in different plants and algae, including the cytoplasm and mitochondria, our subcellular fractionation data indicate that GGT activity was exclusively peroxisomal in Arabidopsis. Thus, glyoxylate aminotransferase reactions in plant peroxisomes appear to be catalyzed by at least two distinct types of aminotransferases: an AGT1 homolog with serine:glyoxylate aminotransferase activity (A.H. Liepman, L.J. Olsen [2001] Plant J 25: 487-498), and a pair of closely related, potentially redundant AlaAT homologs with GGT activity.