The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

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.     

Comparative analysis of GT14/GT14-like gene family in Arabidopsis, Oryza, Populus, Sorghum and Vitis

Glycosyltransferase family14 (GT14) belongs to the glycosyltransferase (GT) superfamily that plays important roles in the biosynthesis of cell walls, the most abundant source of cellulosic biomass for bioethanol production. It has been hypothesized that DUF266 proteins are a new class of GTs related to GT14. In this study, we identified 62 GT14 and 106 DUF266 genes (named GT14-like herein) in Arabidopsis, Oryza, Populus, Sorghum and Vitis. Our phylogenetic analysis separated GT14 and GT14-like genes into two distinct clades, which were further divided into eight and five groups, respectively. Similarities in protein domain, 3D structure and gene expression were uncovered between the two phylogenetic clades, supporting the hypothesis that GT14 and GT14-like genes belong to one family. Therefore, we proposed a new family name, GT14/GT14-like family that combines both subfamilies. Variation in gene expression and protein subcellular localization within the GT14-like subfamily were greater than those within the GT14 subfamily. One-half of the Arabidopsis and Populus GT14/GT14-like genes were found to be preferentially expressed in stem/xylem, indicating that they are likely involved in cell wall biosynthesis. This study provided new insights into the evolution and functional diversification of the GT14/GT14-like family genes.     

Disruption of ATCSLD5 results in reduced growth, reduced xylan and homogalacturonan synthase activity and altered xylan occurrence in Arabidopsis

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.     

BC10, a DUF266-containing and Golgi-located type II membrane protein, is required for cell-wall biosynthesis in rice (Oryza sativa L.)

Glycosyltransferases (GTs) are one of the largest enzyme groups required for the synthesis of complex wall polysaccharides and glycoproteins in plants. However, due to the limited number of related mutants that have observable phenotypes, the biological function(s) of most GTs in cell-wall biosynthesis and assembly have remained elusive. We report here the isolation and in-depth characterization of a brittle rice mutant, brittle culm 10 ( bc10 ). bc10 plants show pleiotropic phenotypes, including brittleness of the plant body and retarded growth. The BC10 gene was cloned through a map-based approach, and encodes a Golgi-located type II membrane protein that contains a domain designated as 'domain of unknown function 266' (DUF266) and represents a multiple gene family in rice. BC10 has low sequence similarity with the domain to a core 2 β-1,6- N- acetylglucosaminyltransferase (C2GnT), and its in vitro enzymatic activity suggests that it functions as a glycosyltransferase. Monosaccharide analysis of total and fractioned wall residues revealed that bc10 showed impaired cellulose biosynthesis. Immunolocalization and isolation of arabinogalactan proteins (AGPs) in the wild-type and bc10 showed that the level of AGPs in the mutant is significantly affected. BC10 is mainly expressed in the developing sclerenchyma and vascular bundle cells, and its deficiency causes a reduction in the levels of cellulose and AGPs, leading to inferior mechanical properties.     

Integrating genes and phenotype: a wheat–Arabidopsis–rice glycosyltransferase database for candidate gene analyses

Glycosyltransferases (GTs) constitute a very large multi-gene superfamily, containing several thousand members identified in sequenced organisms especially in plants. GTs are key enzymes involved in various biological processes such as cell wall formation, storage polysaccharides biosynthesis, and glycosylation of various metabolites. GTs have been identified in rice (Oryza sativa) and Arabidopsis thaliana, but their precise function has been demonstrated biochemically for only a few. In this work we have established a repertoire of virtually all the wheat (Triticum aestivum) GT sequences, using the large publicly available banks of expressed sequences. Based on sequence similarity with Arabidopsis and rice GTs compiled in the carbohydrate active enzyme database (CAZY), we have identified and classified these wheat sequences. The results were used to feed a searchable database available on the web () that can be used for initiating an exhaustive candidate gene survey in wheat applied to a particular biological process. This is illustrated through the identification of GT families which are expressed during cell wall formation in wheat grain maturation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was funded by a grant of the French ministry of research.     

An evolutionary view of functional diversity in family 1 glycosyltransferases

Glycosyltransferases (GTs) (EC 2.4.x.y) catalyze the transfer of sugar moieties to a wide range of acceptor molecules, such as sugars, lipids, proteins, nucleic acids, antibiotics and other small molecules, including plant secondary metabolites. These enzymes can be classified into at least 92 families, of which family 1 glycosyltransferases (GT1), often referred to as UDP glycosyltransferases (UGTs), is the largest in the plant kingdom. To understand how UGTs expanded in both number and function during evolution of land plants, we screened genome sequences from six plants (Physcomitrella patens, Selaginella moellendorffii, Populus trichocarpa, Oryza sativa, Arabidopsis thaliana and Arabidopsis lyrata) for the presence of a conserved UGT protein domain. Phylogenetic analyses of the UGT genes revealed a significant expansion of UGTs, with lineage specificity and a higher duplication rate in vascular plants after the divergence of Physcomitrella. The UGTs from the six species fell into 24 orthologous groups that contained genes derived from the common ancestor of these six species. Some orthologous groups contained multiple UGT families with known functions, suggesting that UGTs discriminate compounds as substrates in a lineage-specific manner. Orthologous groups containing only a single UGT family tend to play a crucial role in plants, suggesting that such UGT families may have not expanded because of evolutionary constraints.     

Genomic basis for cell-wall diversity in plants. A comparative approach to gene families in rice and Arabidopsis

Monocotyledons and dicotyledons are distinct, not only in their body plans and developmental patterns, but also in the structural features of their cell walls. The recent completion of the rice (Oryza sativa) genomic sequence and publication of the sequence data, together with the completed database of the Arabidopsis thaliana genome, provide the first opportunity to compare the full complement of cell-wall-related genes from the two distinct classes of flowering plants. We made this comparison by exploiting the fact that Arabidopsis and rice have type I and type II walls, respectively, and therefore represent the two extremes in terms of the structural features of plant cell walls. In this review article, we classify all cell-wall-related genes into 32 gene families, and generate their phylogenetic trees. Using these data, we can phylogenetically compare individual genes of particular interest between Arabidopsis and rice. This comparative genome approach shows that the differences in wall architecture in the two plant groups actually mirror the diversity of the individual gene families involved in the cell-wall dynamics of the respective plant species. This study also identifies putative rice orthologs of genes with well-defined functions in Arabidopsis and other plant species.     

Carbon Flux and Carbohydrate Gene Families in Pineapple

The recently sequenced pineapple genome was used to identify and analyze some of the key gene families involved in carbohydrate biosynthesis, breakdown and modification. Gene products were grouped into glycosyltransferases (GT), glycoside hydrolases (GH), carbohydrate esterases (CE), and polysaccharide lyases (PL) based upon predicted catalytic activity. Non-catalytic carbohydrate-binding modules (CBM) and enzymes involved in lignification were also identified. The pineapple genes were compared with those from two and five monocot and eudicots species, respectively. The complement of pineapple sugar and cell wall metabolism genes is similar to that found in rice and sorghum, though the numbers of GTs and GHs is often fewer. This applies to a lesser extent to the genes involved in nucleotide-sugar interconversion, with both pineapple and papaya having a minimum complement. Interestingly, pineapple does not appear to contain mixed linkage β-glucan in its walls while possessing cellulose synthase-like (Csl), J and H genes. Pineapple and papaya have less than half the number of GT1 genes involved in small molecule glycosylation compared to Arabidopsis and tomato, and fewer members in GH families than Arabidopsis. The ratio of rice and sorghum to pineapple genes in GH families was more variable than in the case of GTs and it is unclear why pineapple GH gene numbers are so low. Rice, sorghum and pineapple have far fewer CE8, PL1 and GH28 genes related to pectin metabolism than most eudicots. The general lower number of cell wall genes in pineapple possibly reflects the absence of a genome duplication event. The data also suggests that pineapple straddles the boundary between grasses (family Poaceae) and eudicots in terms of genes involved in carbohydrate metabolism, which is also reflected in its cell wall composition.     

Identification of Putative Rhamnogalacturonan-II Specific Glycosyltransferases in Arabidopsis Using a Combination of Bioinformatics Approaches

Rhamnogalacturonan-II (RG-II) is a complex plant cell wall polysaccharide that is composed of an α(1,4)-linked homogalacturonan backbone substituted with four side chains. It exists in the cell wall in the form of a dimer that is cross-linked by a borate di-ester. Despite its highly complex structure, RG-II is evolutionarily conserved in the plant kingdom suggesting that this polymer has fundamental functions in the primary wall organisation. In this study, we have set up a bioinformatics strategy aimed at identifying putative glycosyltransferases (GTs) involved in RG-II biosynthesis. This strategy is based on the selection of candidate genes encoding type II membrane proteins that are tightly coexpressed in both rice and Arabidopsis with previously characterised genes encoding enzymes involved in the synthesis of RG-II and exhibiting an up-regulation upon isoxaben treatment. This study results in the final selection of 26 putative Arabidopsis GTs, including 10 sequences already classified in the CAZy database. Among these CAZy sequences, the screening protocol allowed the selection of α-galacturonosyltransferases involved in the synthesis of α4-GalA oligogalacturonides present in both homogalacturonans and RG-II, and two sialyltransferase-like sequences previously proposed to be involved in the transfer of Kdo and/or Dha on the pectic backbone of RG-II. In addition, 16 non-CAZy GT sequences were retrieved in the present study. Four of them exhibited a GT-A fold. The remaining sequences harbored a GT-B like fold and a fucosyltransferase signature. Based on homologies with glycosyltransferases of known functions, putative roles in the RG-II biosynthesis are proposed for some GT candidates.     

Functional conservation of the glycosyltransferase gene GT47A in the monocot rice

Glucuronoarabinoxylan is the major hemicellulose in grass cell walls, yet the mechanism of xylan synthesis in monocot plants is still unclear. Unraveling the genes involved in the biosynthesis of xylan in rice will be very important for the utilization of rice straw as a source of bioenergy in the future. In this report, we investigated the functional role of a rice gene homologous to Arabidopsis IRREGULAR XYLEM10 (IRX10), belonging to the glycosyl transferase (GT) gene family 47 (GT47), in the biosynthesis of xylan. The protein sequence of OsGT47A from rice exhibits a 93.49 % similarity to IRX10, which is involved in the biosynthesis of glucuronoxylan in Arabidopsis. Phylogenetic analysis of the GT47 glycosyl transferase family in the rice genome revealed that OsGT47A is a closely related homolog of IRX10 and IRX10L. Expression pattern analysis showed that the OsGT47A gene is highly expressed in the rice stem. Overexpression of OsGT47A in the irx10 irx10L double mutant rescued the plant growth phenotype and restored secondary wall thickness. Analysis of monosaccharides indicated that the rescued plants had levels of xylose identical to those of the wild type plants, and the fluorescence signals were restored in the complementation plants by xylan immunolocalization. The OsGT47A complementation under the native promoter of Arabidopsis IRX10L (ProIRX10L) partially rescued the double mutant, indicating that OsGT47A is functionally equivalent to IRX10L. Together, these results suggest that the IRX10 homolog OsGT47A exhibits functional conservation and is most likely involved in xylan synthesis in rice.     

Arabidopsis genes IRREGULAR XYLEM (IRX15) and IRX15L encode DUF579-containing proteins that are essential for normal xylan deposition in the secondary cell wall

There are 10 genes in the Arabidopsis genome that contain a domain described in the Pfam database as domain of unknown function 579 (DUF579). Although DUF579 is widely distributed in eukaryotic species, there is no direct experimental evidence to assign a function to it. Five of the 10 Arabidopsis DUF579 family members are co‐expressed with marker genes for secondary cell wall formation. Plants in which two closely related members of the DUF579 family have been disrupted by T‐DNA insertions contain less xylose in the secondary cell wall as a result of decreased xylan content, and exhibit mildly distorted xylem vessels. Consequently we have named these genes IRREGULAR XYLEM 15 (IRX15) and IRX15L. These mutant plants exhibit many features of previously described xylan synthesis mutants, such as the replacement of glucuronic acid side chains with methylglucuronic acid side chains. By contrast, immunostaining of xylan and transmission electron microscopy (TEM) reveals that the walls of these irx15 irx15l double mutants are disorganized, compared with the wild type or other previously described xylan mutants, and exhibit dramatic increases in the quantity of sugar released in cell wall digestibility assays. Furthermore, localization studies using fluorescent fusion proteins label both the Golgi and also an unknown intracellular compartment. These data are consistent with irx15 and irx15l defining a new class of genes involved in xylan biosynthesis. How these genes function during xylan biosynthesis and deposition is discussed.     

An integrative approach to the identification of Arabidopsis and rice genes involved in xylan and secondary wall development

Xylans constitute the major non-cellulosic component of plant biomass. Xylan biosynthesis is particularly pronounced in cells with secondary walls, implying that the synthesis network consists of a set of highly expressed genes in such cells. To improve the understanding of xylan biosynthesis, we performed a comparative analysis of co-expression networks between Arabidopsis and rice as reference species with different wall types. Many co-expressed genes were represented by orthologs in both species, which implies common biological features, while some gene families were only found in one of the species, and therefore likely to be related to differences in their cell walls. To predict the subcellular location of the identified proteins, we developed a new method, PFANTOM (plant protein family information-based predictor for endomembrane), which was shown to perform better for proteins in the endomembrane system than other available prediction methods. Based on the combined approach of co-expression and predicted cellular localization, we propose a model for Arabidopsis and rice xylan synthesis in the Golgi apparatus and signaling from plasma membrane to nucleus for secondary cell wall differentiation. As an experimental validation of the model, we show that an Arabidopsis mutant in the PGSIP1 gene encoding one of the Golgi localized candidate proteins has a highly decreased content of glucuronic acid in secondary cell walls and substantially reduced xylan glucuronosyltransferase activity.     

A collection of glycosyltransferases from rice (Oryza sativa) exposed to atrazine

The rice (Oryza sativa) GTs belong to a super family possibly with hundreds of members. However, which GTs are involved in plant response to toxic chemicals is unknown. Here, we demonstrated 59 novel GT genes screened from our recent genome-wide sequencing datasets of rice crops exposed to atrazine (a herbicide persistent in ecosystems). Analysis of GT genes showed that most of the GTs contain functional domains typically found in proteins transferring glycosyl moieties to their target compounds. A phylogenetic analysis revealed that many GT genes from different families have diverse cis-elements necessary for response to biotic and environmental stresses. Experimental validation for the GTs was undertaken through a microarray, and 36 GT genes were significantly detected with an expression pattern similar to that from deep-sequencing datasets. Furthermore, 12 GT genes were randomly selected and confirmed by quantitative real-time RT-PCR. Finally, the special activity of total GTs was determined in rice roots and shoots, with an increased activity under the atrazine exposure. This response was closely associated with atrazine absorption in the rice tissues. These results indicate that exposure to atrazine can trigger specific GT genes and enzyme activities in rice.     

Diverse roles of PtrDUF579 proteins in Populus and PtrDUF579-1 function in vascular cambium proliferation during secondary growth

DUF579 (domain of unknown function 579) family proteins contain a DUF579 domain structure but vary greatly in their overall sequence similarity. Several DUF579 proteins have been found to play a role in cell wall biosynthesis in Arabidopsis, while DUF579 family genes have not yet been systematically investigated in Populus. In this study, the Populus DUF579 family proteins were found to be localized in different cell types and subcellular locations. The diverse expression patterns of the proteins indicate that they may perform different functions in Populus. Among the DUF579 family members, PtrDUF579-1 is found to be specifically expressed in vascular cambium zone cells where it is localized in the Golgi apparatus. Suppression of PtrDUF579-1 expression reduced plant height and stem diameter size. Cambium cell division and xylem tissue growth was inhibited while secondary cell wall formation was unchanged in PtrDUF579-1 suppressed plants. Cell walls analysis showed that the composition of the pectin fraction of the cambium cell wall was altered while other polysaccharides were not affected in PtrDUF579-1 suppressed plants. This observation suggest cambium expressed PtrDUF579-1 may affect cell wall biosynthesis and be involved in cambium cell proliferation in Populus. Overall, DUF579 family proteins play a diverse set of roles in Populus.     

Unraveling the functions of glycosyltransferase family 47 in plants

The function of glycosyltransferases (GTs) from family GT47 was first identified in animal exostosins as β-glucuronyltransferase involved in the synthesis of heparan sulfate. Two recent papers report the functions of two plant members in this family as a pectin β-glucuronyltransferase and a xyloglucan β-galactosyltransferase. These findings greatly extend our understanding of the biological functions of family GT47 and also represent an important leap toward the molecular dissection of cell wall biosynthesis.