The PARVUS gene is expressed in cells undergoing secondary wall thickening and is essential for glucuronoxylan biosynthesis

Xylan, cellulose and lignin are the three major components of secondary walls in wood, and elucidation of the biosynthetic pathway of xylan is of importance for potential modification of secondary wall composition to produce wood with improved properties. So far, three Arabidopsis glycosyltransferases, FRAGILE FIBER8, IRREGULAR XYLEM8 and IRREGULAR XYLEM9, have been implicated in glucuronoxylan (GX) biosynthesis. In this study, we demonstrate that PARVUS, which is a member of family GT8, is required for the biosynthesis of the tetrasaccharide primer sequence, beta-D-Xyl-(1 --> 3)-alpha-l-Rha-(1 --> 2)-alpha-D-GalA-(1 --> 4)-D-Xyl, located at the reducing end of GX. The PARVUS gene is expressed during secondary wall biosynthesis in fibers and vessels, and its encoded protein is predominantly localized in the endoplasmic reticulum. Mutation of the PARVUS gene leads to a drastic reduction in secondary wall thickening and GX content. Structural analysis of GX using (1)H-nuclear magnetic resonance (NMR) spectroscopy revealed that the parvus mutation causes a loss of the tetrasaccharide primer sequence at the reducing end of GX and an absence of glucuronic acid side chains in GX. Activity assay showed that the xylan xylosyltransferase and glucuronyltransferase activities were not affected in the parvus mutant. Together, these findings implicate a possible role for PARVUS in the initiation of biosynthesis of the GX tetrasaccharide primer sequence and provide novel insights into the mechanisms of GX biosynthesis.     

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.     

The poplar glycosyltransferase GT47C is functionally conserved with Arabidopsis Fragile fiber8

Xylan is the major hemicellulose in dicot wood. Unraveling genes involved in the biosynthesis of xylan will be of importance in understanding the process of wood formation. In this report, we investigated the possible role of poplar GT47C, a glycosyltransferase belonging to family GT47, in the biosynthesis of xylan. PoGT47C from the hybrid poplar Populus alba x tremula exhibits 84% sequence similarity to Fragile fiber8 (FRA8), which is involved in the biosynthesis of glucuronoxylan in Arabidopsis. Phylogenetic analysis of glycosyltransferase family GT47 in the Populus trichocarpa genome revealed that GT47C is the only close homolog of FRA8. In situ hybridization showed that the PoGT47C gene was expressed in developing primary xylem, secondary xylem and phloem fibers of stems, and in developing secondary xylem of roots. Sequence analysis suggests that PoGT47C is a type II membrane protein, and study of the subcellular localization demonstrated that fluorescent protein-tagged PoGT47C was located in the Golgi. Immunolocalization with a xylan monoclonal antibody LM10 revealed a nearly complete loss of xylan signals in the secondary walls of fibers and vessels in the Arabidopsis fra8 mutant. Expression of PoGT47C in the fra8 mutant restored the secondary wall thickness and xylan content to the wild-type level. Together, these results suggest that PoGT47C is functionally conserved with FRA8 and it is probably involved in xylan synthesis during wood formation.     

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.     

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.     

Populus GT43 family members group into distinct sets required for primary and secondary wall xylan biosynthesis and include useful promoters for wood modification

The plant GT43 protein family includes xylosyltransferases that are known to be required for xylan backbone biosynthesis, but have incompletely understood specificities. RT‐qPCR and histochemical (GUS) analyses of expression patterns of GT43 members in hybrid aspen, reported here, revealed that three clades of the family have markedly differing specificity towards secondary wall‐forming cells (wood and extraxylary fibres). Intriguingly, GT43A and B genes (corresponding to the Arabidopsis IRX9 clade) showed higher specificity for secondary‐walled cells than GT43C and D genes (IRX14 clade), although both IRX9 and IRX14 are required for xylosyltransferase activity. The remaining genes, GT43E, F and G (IRX9‐L clade), showed broad expression patterns. Transient transactivation analyses of GT43A and B reporters demonstrated that they are activated by PtxtMYB021 and PNAC085 (master secondary wall switches), mediated in PtxtMYB021 activation by an AC element. The high observed secondary cell wall specificity of GT43B expression prompted tests of the efficiency of its promoter (pGT43B), relative to the CaMV 35S (35S) promoter, for overexpressing a xylan acetyl esterase (CE5) or downregulating REDUCED WALL ACETYLATION (RWA) family genes and thus engineering wood acetylation. CE5 expression was weaker when driven by pGT43B, but it reduced wood acetyl content substantially more efficiently than the 35S promoter. RNAi silencing of the RWA family, which was ineffective using 35S, was achieved when using GT43B promoter. These results show the utility of the GT43B promoter for genetically engineering properties of wood and fibres.     

Gene expression profiles in different stem internodes reveal the genetic regulation of primary and secondary stem development in <Emphasis Type="Italic">Betula platyphylla</Emphasis>

Secondary growth of stems is an important process for the radial increase of trees. To gain an insight into the molecular mechanisms underlying stem development from primary to secondary growth and to provide information for molecular research and breeding in Betula platyphylla (birch), the gene expression profiles of material from the first, third, and fifth internodes (IN) of 3-month-old seedlings were analyzed. Compared with the first IN, 177 genes were up-regulated and 157 genes down-regulated in the third IN; in the fifth IN, 180 genes were up-regulated and 275 genes were down-regulated. The expressions of 24 genes were up-regulated and 6 genes were down-regulated in the fifth IN relative to the third IN. The differentially expressed genes were annotated as having roles in cambium, xylem, and phloem development and formation; including cell wall expansion, cellulose biosynthesis, lignin biosynthesis and deposition, xylem extension, cell wall modification, and growth hormone responses. The expressions of genes related to cell wall expansion and cellulose biosynthesis in the primary cell wall were down-regulated in the third and fifth IN relative to the first IN. Genes involved in lignin biosynthesis, xylem extension, and cellulose synthesis in the secondary cell wall were up-regulated in the third and fifth IN relative to the first IN. These results described the patterns of gene expression during stem development in birch and provided candidate genes for further functional characterization.     

巴西橡胶树HbMYB52基因的克隆及其在拟南芥中的表达

为揭示Hb MYB52在巴西橡胶树(Hevea brasiliensis)木材发育过程中的功能,从其转录组中分离克隆到1个MYB转录因子G21亚组成员基因,命名为Hb MYB52,开放阅读框为726 bp,编码242个氨基酸的蛋白,在木质部中高度表达。在拟南芥(Arabidopsis thaliana)中过表达Hb MYB52,虽未改变转基因植株株型,但植株维管束间纤维细胞壁明显增厚,同时抑制了木质纤维、导管次生壁形成。转基因拟南芥株系3和株系6中纤维素和木质素含量减少,相应各组分合成的关键酶基因的表达量也不同程度下降;株系8产生了木质素异位沉积,且木质素合成关键酶基因表达活跃。因此,推测Hb MYB52参与了植物次生壁形成调控,在拟南芥次生壁形成中可能发挥了双重功能:一方面负调控维管束次生壁形成以及各组分的生物合成,另一方面具有促进束间纤维次生壁增厚的作用。     

Cloning and characterization of cellulose synthase-like gene, PtrCSLD2 from developing xylem of aspen trees

Genetic improvement of cell wall polymer synthesis in forest trees is one of the major goals of forest biotechnology that could possibly impact their end product utilization. Identification of genes involved in cell wall polymer biogenesis is essential for achieving this goal. Among various candidate cell wall-related genes, cellulose synthase-like D (CSLD) genes are intriguing due to their hitherto unknown functions in cell wall polymer synthesis but strong structural similarity with cellulose synthases (CesAs) involved in cellulose deposition. Little is known about CSLD genes from trees. In the present article PtrCSLD2, a first CSLD gene from an economically important tree, aspen (Populus tremuloides) is reported. PtrCSLD2 cDNA was isolated from an aspen xylem cDNA library and encodes a protein that shares 90% similarity with Arabidopsis AtCSLD3 protein involved in root hair tip growth. It is possible that xylem fibers that also grow by intrusive tip growth may need expression of PtrCSLD2 for controlling the length of xylem fibers, a wood quality trait of great economical importance. PtrCSLD2 protein has a N-terminal cysteine-rich putative zinc-binding domain; eight transmembrane domains; alternating conserved and hypervariable domains; and a processive glycosyltransferases signature, D, D, D, QXXRW; all similar to aspen CesA proteins. However, PtrCSLD2 shares only 43-48% overall identity with the known aspen CesAs suggesting its distinct functional role in cell wall polymer synthesis perhaps other than cellulose biosynthesis. Based on Southern analysis, the aspen CSLD gene family consists of at least three genes and this gene copy estimate is supported by phylogenetic analysis of available CSLDs from plants. Moreover, gene expression studies using RT-PCR and in situ mRNA hybridization showed that PtrCSLD2 is expressed at a low level in all aspen tissues examined with a slightly higher expression level in secondary cell wall-enriched aspen xylem as compared to primary cell wall enriched tissues. Together, these observations suggest that PtrCSLD2 gene may be involved in the synthesis of matrix polysaccharides that are dominant in secondary cell walls of poplar xylem. Future molecular genetic analyses will clarify the functional significance of CSLD genes in the development of woody trees.     

The irregular xylem3 locus of Arabidopsis encodes a cellulose synthase required for secondary cell wall synthesis.

The irregular xylem3 (irx3) mutant of Arabidopsis has a severe deficiency in secondary cell wall cellulose deposition that leads to collapsed xylem cells. The irx3 mutation has been mapped to the top arm of chromosome V near the marker nga106. Expressed sequence tag clone 75G11, which exhibits sequence similarity to cellulose synthase, was found to be tightly linked to irx3, and genomic clones containing the gene corresponding to clone 75G11 complemented the irx3 mutation. Thus, the IRX3 gene encodes a cellulose synthase component that is specifically required for the synthesis of cellulose in the secondary cell wall. The irx3 mutant allele contains a stop codon that truncates the gene product by 168 amino acids, suggesting that this allele is null. Furthermore, in contrast to radial swelling1 (rsw1) plants, irx3 plants show no increase in the accumulation of beta-1,4-linked glucose in the noncrystalline cell wall fraction. IRX3 and RSW1 fall into a distinct subgroup (Csa) of Arabidopsis genes showing homology to bacterial cellulose synthases.     

Overexpression of <Emphasis Type="Italic">PSP1</Emphasis> enhances growth of transgenic Arabidopsis plants under ambient air conditions

Cellulose biosynthesis is mediated by cellulose synthases (CesAs), which constitute into rosette-like cellulose synthase complexe (CSC) on the plasma membrane. Two types of CSCs in Arabidopsis are believed to be involved in cellulose synthesis in the primary cell wall and secondary cell walls, respectively. In this work, we found that the two type CSCs participated cellulose biosynthesis in differentiating xylem cells undergoing secondary cell wall thickening in Populus. During the cell wall thickening process, expression of one type CSC genes increased while expression of the other type CSC genes decreased. Suppression of different type CSC genes both affected the wall-thickening and disrupted the multilaminar structure of the secondary cell walls. When CesA7A was suppressed, crystalline cellulose content was reduced, which, however, showed an increase when CesA3D was suppressed. The CesA suppression also affected cellulose digestibility of the wood cell walls. The results suggest that two type CSCs are involved in coordinating the cellulose biosynthesis in formation of the multilaminar structure in Populus wood secondary cell walls.