Characterization of IRX10 and IRX10-like reveals an essential role in glucuronoxylan biosynthesis in Arabidopsis

Xylan, the major hemicellulosic polysaccharide in Arabidopsis secondary cell walls, requires a number of glycosyltransferases (GT) to catalyse formation of the various glycosidic linkages found in the polymer. In this study, we characterized IRX10 and IRX10-like ( IRX10-L ), two highly homologous genes encoding members of the glycosyltransferase family 47 (GT47). T-DNA insertions in IRX10 gave a mild irregular xylem (irx) phenotype consistent with a minor defect in secondary cell-wall synthesis, whereas plants containing mutations in IRX10-L showed no change. However, irx10 irx10-L double mutant plants showed a much more severe irx and whole-plant phenotype, suggesting considerable functional redundancy between these two genes. Detailed biochemical analysis of the irx10 irx10-L double mutant showed a large reduction of xylan in the secondary cell walls, consistent with a specific defect in xylan biosynthesis. Furthermore, the irx10 irx10-L mutant retains the unique oligosaccharide found at the reducing end of Arabidopsis xylan, but shows a severe reduction in β(1,4) xylosyltransferase activity. These characteristics are similar to those of irx9 and irx14 , mutants that are believed to be defective in xylan chain elongation, and suggests that IRX10 and IRX10-L also play a role in elongation of the xylan backbone.     

Partial functional conservation of IRX10 homologs in physcomitrella patens and Arabidopsis thaliana indicates an evolutionary step contributing to vascular formation in land plants

Background

Plant cell walls are complex multicomponent structures that have evolved to fulfil an essential function in providing strength and protection to cells. Hemicelluloses constitute a key component of the cell wall and recently a number of the genes thought to encode the enzymes required for its synthesis have been identified in Arabidopsis. The acquisition of hemicellulose synthesis capability is hypothesised to have been an important step in the evolution of higher plants.

Results

Analysis of the Physcomitrella patens genome has revealed the presence of homologs for all of the Arabidopsis glycosyltransferases including IRX9, IRX10 and IRX14 required for the synthesis of the glucuronoxylan backbone. The Physcomitrella IRX10 homolog is expressed in a variety of moss tissues which were newly formed or undergoing expansion. There is a high degree of sequence conservation between the Physcomitrella IRX10 and Arabidopsis IRX10 and IRX10-L. Despite this sequence similarity, the Physcomitrella IRX10 gene is only able to partially rescue the Arabidopsis irx10 irx10-L double mutant indicating that there has been a neo- or sub-functionalisation during the evolution of higher plants. Analysis of the monosaccharide composition of stems from the partially rescued Arabidopsis plants does not show any significant change in xylose content compared to the irx10 irx10-L double mutant. Likewise, knockout mutants of the Physcomitrella IRX10 gene do not result in any visible phenotype and there is no significant change in monosaccharide composition of the cell walls.

Conclusions

The fact that the Physcomitrella IRX10 (PpGT47A) protein can partially complement an Arabidopsis irx10 irx10-L double mutant suggests that it shares some function with the Arabidopsis proteins, but the lack of a phenotype in knockout lines shows that the function is not required for growth or development under normal conditions in Physcomitrella. In contrast, the Arabidopsis irx10 and irx10 irx10-L mutants have strong phenotypes indicating an important function in growth and development. We conclude that the evolution of vascular plants has been associated with a significant change or adaptation in the function of the IRX10 gene family.     

The Arabidopsis IRX10 and IRX10-LIKE glycosyltransferases are critical for glucuronoxylan biosynthesis during secondary cell wall formation

Arabidopsis IRX10 and IRX10-LIKE (IRX10-L) proteins are closely related members of the GT47 glycosyltransferase family. Single gene knock-outs of IRX10 or IRX10-L result in plants with either a weak or no mutant phenotype. However irx10 irx10-L double mutants are severely affected in their development, with a reduced rosette size and infrequent formation of a small infertile inflorescence. Plants homozygous for irx10 and heterozygous for irx10-L have an intermediate phenotype exhibiting a short inflorescence compared with the wild type, and an almost complete loss of fertility. Stem sections of the irx10 homozygous irx10-L heterozygous or irx10 irx10-L double mutants show decreased secondary cell-wall formation. NMR analysis shows that signals derived from the reducing end structure of glucuronoxylan were detected in the irx10 single mutant, and in the irx10 homozygous irx10-L heterozygous combination, but that the degree of polymerization of the xylan backbone was reduced compared with the wild type. Additionally, xylans from irx10 stem tissues have an almost complete loss of the GlcUA side chain, whereas the level of 4- O -Me-GlcUA was similar to that in wild type. Deletion of the predicted signal peptide from the N terminus of IRX10 or IRX10-L results in an inability to rescue the irx10 irx10-L double mutant phenotype. These findings demonstrate that IRX10 and IRX10-L perform a critical function in the synthesis of glucuronoxylan during secondary cell-wall formation, and that this activity is associated with the formation of the xylan backbone structure. This contrasts with the proposed function of the tobacco NpGUT1, which is closely related to the Arabidopsis IRX10 and IRX10-L proteins, in rhamnogalacturonan II biosynthesis.     

The Arabidopsis irregular xylem8 mutant is deficient in glucuronoxylan and homogalacturonan, which are essential for secondary cell wall integrity

The secondary cell wall in higher plants consists mainly of cellulose, lignin, and xylan and is the major component of biomass in many species. The Arabidopsis thaliana irregular xylem8 (irx8) mutant is dwarfed and has a significant reduction in secondary cell wall thickness. IRX8 belongs to a subgroup of glycosyltransferase family 8 called the GAUT1-related gene family, whose members include GAUT1, a homogalacturonan galacturonosyltransferase, and GAUT12 (IRX8). Here, we use comparative cell wall analyses to show that the irx8 mutant contains significantly reduced levels of xylan and homogalacturonan. Immunohistochemical analyses confirmed that the level of xylan was significantly reduced in the mutant. Structural fingerprinting of the cell wall polymers further revealed that irx8 is deficient in glucuronoxylan. To explore the biological function of IRX8, we crossed irx8 with irx1 (affecting cellulose synthase 8). The homozygous irx1 irx8 exhibited severely dwarfed phenotypes, suggesting that IRX8 is essential for cell wall integrity during cellulose deficiency. Taken together, the data presented show that IRX8 affects the level of glucuronoxylan and homogalacturonan in higher plants and that IRX8 provides an important link between the xylan polymer and the secondary cell wall matrix and directly affects secondary cell wall integrity.     

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.     

Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis

Previous studies using co-expression analysis have identified a large number of genes likely to be involved in secondary cell-wall formation. However, the function of very few of these genes is known. We have studied the cell-wall phenotype of irx7, irx8 and irx9, three previously described irregular xylem (irx) mutants, and irx14 and parvus-3, which we now show also to be secondary cell-wall mutants. All five mutants, which have mutations in genes encoding putative glycosyltransferases, exhibited large decreases in xylan. In addition, all five mutants were found to have the same specific defect in xylan structure, retaining MeGlcUA but lacking GlcUA side branches. Polysaccharide analysis by carbohydrate gel electrophoresis (PACE) was used to determine the xylan structure in Arabidopsis, and revealed that side branches are added to approximately one in every eight xylose residues. Interestingly, this ratio is constant in all the lines analysed despite the wide variation in xylan content and the absence of GlcUA branches. Xylanase digestion of xylan from wild-type plants released a short oligosaccharide sequence at the reducing end of the xylan chain. MALDI-TOF MS analysis indicated that this sequence of sugars was absent in xylan from irx7, irx8 and parvus-3 mutants, but was present in irx9 and irx14. This is consistent with previous NMR analysis of xylan from irx7, irx8 and irx9, and suggests that PARVUS may be involved in the synthesis of a xylan primer whereas IRX14 may be required to synthesize the xylan backbone. This hypothesis is supported by assays showing that irx9 and irx14 are both defective in incorporation of radiolabel from UDP (14)C-xylose. This study has important implications for both our understanding of xylan biosynthesis and the functional analysis of cell-wall biosynthesis genes.     

Irregular xylem 7 (IRX7) is required for anchoring seed coat mucilage in Arabidopsis

Large quantities of mucilage are synthesized in seed coat epidermis cells during seed coat differentiation. This process is an ideal model system for the study of plant cell wall biosynthesis and modifications. In this study, we show that mutation in Irregular Xylem 7 (IRX7) results in a defect in mucilage adherence due to reduced xylan biosynthesis. IRX7 was expressed in the seeds from 4 days post-anthesis (DPA) to 13 DPA, with the peak of expression at 13 DPA. The seed coat epidermis cells of irx7 displayed no aberrant morphology during differentiation, and these cells synthesized and deposited the same amount of mucilage as did wild type (WT) cells. However, the distribution of the water-soluble vs. adherent mucilage layers was significantly altered in irx7 compared to the WT. Both the amount of xylose and the extent of glycosyl linkages of xylan was dramatically decreased in irx7 water-soluble and adherent mucilage compared to the WT. The polymeric structure of water-soluble mucilage was altered in irx7, with a total loss of the higher molecular weight polymer components present in the WT. Correspondingly, whole-seed immunolabeling assays and dot-immunoassays of extracted mucilage indicated dramatic changes in rhamnogalacturonan I (RG I) and xylan epitopes in irx7 mucilage. Furthermore, the crystalline cellulose content was significantly reduced in irx7 mucilage. Taken together, these results indicate that xylan synthesized by IRX7 plays an essential role in maintaining the adhesive property of seed coat mucilage, and its structural role is potentially implemented through its interaction with cellulose.     

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.     

The irregular xylem9 mutant is deficient in xylan xylosyltransferase activity

Xylan is the second most abundant polysaccharide in dicot wood, and thus elucidation of the xylan biosynthetic pathway is required to understand the mechanisms controlling wood formation. Genetic and chemical studies in Arabidopsis have implicated three genes, FRAGILE FIBER8 (FRA8), IRREGULAR XYLEM8 (IRX8) and IRREGULAR XYLEM9 (IRX9), in the biosynthesis of glucuronoxylan (GX), but the biochemical functions of the encoded proteins are not known. In this study, we determined the effect of the fra8, irx8 and irx9 mutations on the activities of xylan xylosyltransferase (XylT) and glucuronyltransferase (GlcAT). We show that microsomes isolated from the stems of wild-type Arabidopsis exhibit XylT and GlcAT activities in the presence of exogenous 1,4-linked beta-d-xylooligomers. Xylooligomers ranging in size from two to six can be used as acceptors by XylT to form xylooligosaccharides with up to 12 xylosyl residues. We provide evidence that the irx9 mutation results in a substantial reduction in XylT activity but has no discernible effect on GlcAT activity. In contrast, neither XylT nor GlcAT activity is affected by fra8 and irx8 mutations. Our results provide biochemical evidence that the irx9 mutation results in a deficiency in xylan XylT activity, thus leading to a defect in the elongation of the xylan backbone.     

Multiple cellulose synthase catalytic subunits are required for cellulose synthesis in Arabidopsis

The irregular xylem 1 (irx1) mutant of Arabidopsis has a severe deficiency in the deposition of cellulose in secondary cell walls, which results in collapsed xylem cells. This mutation has been mapped to a 140-kb region of chromosome 4. A cellulose synthase catalytic subunit was found to be located in this region, and genomic clones containing this gene complemented the irx1 mutation. IRX1 shows homology to a previously described cellulose synthase (IRX3). Analysis of the irx1 and irx3 mutant phenotypes demonstrates that both IRX1 and IRX3 are essential for the production of cellulose in the same cell. Thus, IRX1 and IRX3 define distinct classes of catalytic subunits that are both essential for cellulose synthesis in plants. This finding is supported by coprecipitation of IRX1 with IRX3, suggesting that IRX1 and IRX3 are part of the same complex.     

Arabidopsis irregular xylem8 and irregular xylem9: implications for the complexity of glucuronoxylan biosynthesis

Mutations of Arabidopsis thaliana IRREGULAR XYLEM8 (IRX8) and IRX9 were previously shown to cause a collapsed xylem phenotype and decreases in xylose and cellulose in cell walls. In this study, we characterized IRX8 and IRX9 and performed chemical and structural analyses of glucuronoxylan (GX) from irx8 and irx9 plants. IRX8 and IRX9 are expressed specifically in cells undergoing secondary wall thickening, and their encoded proteins are targeted to the Golgi, where GX is synthesized. 1H-NMR spectroscopy showed that the reducing end of Arabidopsis GX contains the glycosyl sequence 4-beta-D-Xylp-(1-->4)-beta-D-Xylp-(1-->3)-alpha-L-Rhap-(1-->2)-alpha-D-GalpA-(1-->4)-D-Xylp, which was previously identified in birch (Betula verrucosa) and spruce (Picea abies) GX. This indicates that the reducing end structure of GXs is evolutionarily conserved in woody and herbaceous plants. This sequence is more abundant in irx9 GX than in the wild type, whereas irx8 and fragile fiber8 (fra8) plants are nearly devoid of it. The number of GX chains increased and the GX chain length decreased in irx9 plants. Conversely, the number of GX chains decreased and the chain length heterodispersity increased in irx8 and fra8 plants. Our results suggest that IRX9 is required for normal GX elongation and indicate roles for IRX8 and FRA8 in the synthesis of the glycosyl sequence at the GX reducing end.     

Focus Issue on Plant Cell Walls: The Arabidopsis Family GT43 Glycosyltransferases Form Two Functionally Nonredundant Groups Essential for the Elongation of Glucuronoxylan Backbone

There exist four members of family GT43 glycosyltransferases in the Arabidopsis (Arabidopsis thaliana) genome, and mutations of two of them, IRX9 and IRX14, have previously been shown to cause a defect in glucuronoxylan (GX) biosynthesis. However, it is currently unknown whether IRX9 and IRX14 perform the same biochemical function and whether the other two GT43 members are also involved in GX biosynthesis. In this report, we performed comprehensive genetic analysis of the functional roles of the four Arabidopsis GT43 members in GX biosynthesis. The I9H (IRX9 homolog) and I14H (IRX14 homolog) genes were shown to be specifically expressed in cells undergoing secondary wall thickening, and their encoded proteins were targeted to the Golgi, where GX is synthesized. Overexpression of I9H but not IRX14 or I14H rescued the GX defects conferred by the irx9 mutation, whereas overexpression of I14H but not IRX9 or I9H complemented the GX defects caused by the irx14 mutation. Double mutant analyses revealed that I9H functioned redundantly with IRX9 and that I14H was redundant with IRX14 in their functions. In addition, double mutations of IRX9 and IRX14 were shown to cause a loss of secondary wall thickening in fibers and a much more severe reduction in GX amount than their single mutants. Together, these results provide genetic evidence demonstrating that all four Arabidopsis GT43 members are involved in GX biosynthesis and suggest that they form two functionally nonredundant groups essential for the normal elongation of GX backbone.Secondary walls constitute the bulk of cellulosic biomass produced by vascular plants. Cellulosic biomass in the form of fibers and wood is an important raw material for a myriad of industrial uses, such as timber, pulping, papermaking, and textiles. Due to the dwindling of nonrenewable fossil fuels and the detrimental effects of burning fossil fuels on the global environment, there has been an urgent call to develop alternative renewable energy sources, and the lignocellulosic biomass from plants is considered to be an attractive renewable source for biofuel production (Somerville, 2006). However, lignocellulosic biomass is recalcitrant to the enzymatic conversion of cellulose into sugars, because cellulose is embedded in a complex mixture of polysaccharides and lignin polymers that block the accessibility of degrading enzymes. It has been shown that reduction of lignin and xylan by chemical or enzymatic treatment or by the transgenic approach reduces the recalcitrance of the lignocellulosic biomass to saccharification (Chen and Dixon, 2007; Himmel et al., 2007; Lee et al., 2009a). Therefore, a complete understanding of how individual components of lignocellulosic biomass are biosynthesized will potentially allow us to design novel strategies for genetic modification of cell wall composition and, hence, reduction in biomass recalcitrance to biofuel production.Xylan is the main hemicellulose that cross-links with cellulose in the secondary walls of dicot plants (Carpita and McCann, 2000). It is made of a linear backbone of β-(1,4)-linked xylosyl residues, about 10% of which are attached with side chains of single residues of glucuronic acid (GlcA) and/or 4-O-methylglucuronic acid (MeGlcA) via α-(1,2)-linkages. The backbone xylosyl residues may also be substituted with the arabinosyl group and acetylated. Based on the nature of the side chains, xylan is generally grouped as (methyl)glucuronoxylan (GX), which is the main hemicellulose in dicots, and arabinoxylan and glucuronoarabinoxylan, which are the most abundant hemicelluloses in grass cell walls (Ebringerová and Heinze, 2000). In addition to the xylosyl backbone, the reducing end of xylan from birch (Betula verrucosa), spruce (Picea abies), Arabidopsis (Arabidopsis thaliana), and poplar (Populus alba × Populus tremula) contains a unique tetrasaccharide sequence β-d-Xylp-(1→3)-α-l-Rhap-(1→2)-α-d-GalpA-(1→4)-d-Xylp (Shimizu et al., 1976; Johansson and Samuelson, 1977; Andersson et al., 1983; Peña et al., 2007; Lee et al., 2009a).The biosynthesis of xylan requires multiple glycosyltransferases and other modifying enzymes. Early biochemical studies revealed the activities of xylosyltransferases, glucuronosyltransferases, arabinosyltransferases, methyltransferases, and acetyltransferases that are likely involved in the biosynthesis of xylan (Baydoun et al., 1983, 1989; Kuroyama and Tsumuraya, 2001; Gregory et al., 2002; Porchia et al., 2002; Urahara et al., 2004; Zeng et al., 2008). However, none of the genes corresponding to these xylan biosynthetic enzymes have been identified. Recent molecular and genetic studies in Arabidopsis and poplar have led to the identification of a number of glycosyltransferases that are essential for GX biosynthesis. Among them, several members of the families GT47 and GT8 from Arabidopsis (FRA8, F8H, IRX8, and PARVUS) and poplar (GT47C, GT8D, and GT8E/8F) are implicated in the biosynthesis of the GX reducing end sequence (Aspeborg et al., 2005; Brown et al., 2005, 2007; Zhong et al., 2005; Zhou et al., 2006, 2007; Lee et al., 2007b, 2009b, 2009c; Peña et al., 2007; Persson et al., 2007). These glycosyltransferase genes are specifically expressed in vessels and fibers, and their encoded proteins are targeted to Golgi, where GX is synthesized, except for PARVUS and GT8E/8F, which are predominantly located in the endoplasmic reticulum (Lee et al., 2007b, 2009c). Mutations of the Arabidopsis FRA8, IRX8, and PARVUS genes all led to a near loss of the reducing end tetrasaccharide sequence and a reduction in GX amount (Brown et al., 2007; Lee et al., 2007b; Peña et al., 2007), indicating their essential roles in the biosynthesis of the GX reducing end sequence, although their exact enzymatic activities are still unknown.The genetic studies have also identified roles of two members of family GT43 glycosyltransferases, IRX9 and IRX14, from Arabidopsis and GT43B from poplar in the biosynthesis of the GX xylosyl backbone (Brown et al., 2007; Peña et al., 2007; Zhou et al., 2007). The expression of IRX9 has been shown to be associated with cells undergoing secondary wall biosynthesis, and its encoded protein is targeted to the Golgi. Mutation of the IRX9 gene causes a drastic reduction in xylan xylosyltransferase activity (Brown et al., 2007; Lee et al., 2007a) and concomitantly a substantial decrease in the GX chain length and GX amount (Peña et al., 2007). Mutation of IRX14 was shown to result in a reduction in the GX level and the xylosyltransferase activity (Brown et al., 2007). In addition, two functionally redundant glycosyltransferases, IRX10 and IRX10-like, which belong to family GT47, were also demonstrated to be required for the normal GX level and xylan xylosyltransferase activity, suggesting their involvement in the biosynthesis of the GX xylosyl backbone (Brown et al., 2009; Wu et al., 2009).In this report, we performed comprehensive molecular and genetic studies of the roles of all members of the Arabidopsis family GT43 glycosyltransferases in GX biosynthesis. We show that, like IRX9, the other three GT43 members, I9H (IRX9 homolog), IRX14, and I14H (IRX14 homolog), are expressed in secondary wall-containing cells and that their encoded proteins are targeted to the Golgi. We have found that the GX defects in the irx9 mutant can be rescued by overexpression of I9H but not IRX14 and I14H. Similarly, overexpression of I14H but not IRX9 and I9H is able to complement the GX defects caused by the irx14 mutation. Furthermore, genetic analysis of an array of double mutants revealed redundant and nonredundant roles of GT43 members in GX biosynthesis. Our findings demonstrate that the Arabidopsis family GT43 glycosyltransferases form two functionally nonredundant groups essential for the normal elongation of GX backbone.     

Arabidopsis thaliana IRX10 and two related proteins from psyllium and Physcomitrella patens are xylan xylosyltransferases

The enzymatic mechanism that governs the synthesis of the xylan backbone polymer, a linear chain of xylose residues connected by β‐1,4 glycosidic linkages, has remained elusive. Xylan is a major constituent of many kinds of plant cell walls, and genetic studies have identified multiple genes that affect xylan formation. In this study, we investigate several homologs of one of these previously identified xylan‐related genes, IRX10 from Arabidopsis thaliana, by heterologous expression and in vitro xylan xylosyltransferase assay. We find that an IRX10 homolog from the moss Physcomitrella patens displays robust activity, and we show that the xylosidic linkage formed is a β‐1,4 linkage, establishing this protein as a xylan β‐1,4‐xylosyltransferase. We also find lower but reproducible xylan xylosyltransferase activity with A. thaliana IRX10 and with a homolog from the dicot plant Plantago ovata, showing that xylan xylosyltransferase activity is conserved over large evolutionary distance for these proteins.     

Two cotton fiber‐associated glycosyltransferases,GhGT43A1 and GhGT43C1, function in hemicellulose glucuronoxylan biosynthesis during plant development

Xylan is the major hemicellulosic constituent in dicot secondary cell walls. Cell wall composition of cotton fiber changes dynamically throughout development. Not only the amounts but also the molecular sizes of the hemicellulosic polysaccharides show substantial changes during cotton fiber development. However, none of the genes encoding glycosyltransferases (GTs) responsible for synthesizing xylan have been isolated and characterized in cotton fiber. In this study, we applied a bioinformatics approach and identified two putative GTs from cotton, designated GhGT43A1 and GhGT43C1, which belong to the CAZy GT43 family and are closely related to Arabidopsis IRX9 and IRX14, respectively. We show that GhGT43A1 is highly and preferentially expressed in 15 and 20 days post‐anthesis (dpa) cotton fiber, whereas GhGT43C1 is ubiquitously expressed in most organs, with especially high expression in 15 dpa fiber and hypocotyl. Complementation analysis demonstrates that GhG43A1 and GhGT43C1 are orthologs of Arabidopsis IRX9 and IRX14, respectively. Furthermore, we show that overexpression of GhGT43A1 or GhGT43C1 in Arabidopsis results in increased xylan content. We also show that overexpression of GhGT43A1 or GhGT43C1 leads to more cellulose deposition. These findings suggest that GhGT43A1 and GhGT43C1 likely participate in xylan synthesis during fiber development.     

Arabidopsis family GT43 members are xylan xylosyltransferases required for the elongation of the xylan backbone

Xylan is the second most abundant polysaccharide in plant biomass targeted for biofuel production. Therefore, it is imperative to understand the biochemical mechanism underlying xylan biosynthesis. Although previous genetic studies have identified several genes implicated in xylan biosynthesis, biochemical proof of any of their encoded proteins as a xylan xylosyltransferase (XylT) responsible for xylan backbone biosynthesis is still lacking. In this study, we investigated the enzymatic activities of two Arabidopsis thaliana GT43 members, IRX9 (Irregular Xylem9) and IRX14, which have been genetically shown to be non-redundantly involved in the elongation of the xylan backbone. IRX9 and IRX14, alone or simultaneously, were heterologously expressed in tobacco BY2 cells, and microsomes isolated from the transgenic BY2 cells were tested for XylT activity using xylotetraose (Xyl(4)) as an acceptor and UDP-[(14)C]xylose as a donor. It was found that although microsomes with expression of IRX9 or IRX14 alone exhibited little incorporation of radiolabeled xylose, a high level of incorporation of radiolabeled xylose onto Xyl(4) was conferred by microsomes with co-expression of IRX9 and IRX14. Further analysis using fluorescent anthranilic acid-labeled xylotetraose (Xyl(4)-AA) as an acceptor revealed that up to five β-(1,4)-linked xylosyl residues were able to be transferred onto Xyl(4)-AA by microsomes with co-expression of IRX9 and IRX14. Furthermore, it was shown that xylooligomers ranging from Xyl(3)-AA to Xyl(6)-AA could all be used as acceptors for the xylosyl transfer by microsomes with co-expression of IRX9 and IRX14. Together, these findings provide the first biochemical evidence that IRX9 and IRX14 are xylosyltransferases that operate cooperatively in the elongation of the xylan backbone.