A chromosome conformation capture ordered sequence of the barley genome.

Cereal grasses of the Triticeae tribe have been the major food source in temperate regions since the dawn of agriculture. Their large genomes are characterized by a high content of repetitive elements and large pericentromeric regions that are virtually devoid of meiotic recombination. Here we present a high-quality reference genome assembly for barley (Hordeum vulgare L.). We use chromosome conformation capture mapping to derive the linear order of sequences across the pericentromeric space and to investigate the spatial organization of chromatin in the nucleus at megabase resolution. The composition of genes and repetitive elements differs between distal and proximal regions. Gene family analyses reveal lineage-specific duplications of genes involved in the transport of nutrients to developing seeds and the mobilization of carbohydrates in grains. We demonstrate the importance of the barley reference sequence for breeding by inspecting the genomic partitioning of sequence variation in modern elite germplasm, highlighting regions vulnerable to genetic erosion.

Genetics, Transcriptional Profiles, and Catalytic Properties of the UDP-Arabinose Mutase Family from Barley.

Four members of the UDP-Ara mutase (UAM) gene family from barley have been isolated and characterized, and their map positions on chromosomes 2H, 3H, and 4H have been defined. When the genes are expressed in Escherichia coli, the corresponding HvUAM1, HvUAM2, and HvUAM3 proteins exhibit UAM activity, and the kinetic properties of the enzymes have been determined, including Km, Kcat, and catalytic efficiencies. However, the expressed HvUAM4 protein shows no mutase activity against UDP-Ara or against a broad range of other nucleotide sugars and related molecules. The enzymic data indicate therefore that the HvUAM4 protein may not be a mutase. However, the HvUAM4 gene is transcribed at high levels in all the barley tissues examined, and its transcript abundance is correlated with transcript levels for other genes involved in cell wall biosynthesis. The UDP-l-Arap → UDP-l-Araf reaction, which is essential for the generation of the UDP-Araf substrate for arabinoxylan, arabinogalactan protein, and pectic polysaccharide biosynthesis, is thermodynamically unfavorable and has an equilibrium constant of 0.02. Nevertheless, the incorporation of Araf residues into nascent polysaccharides clearly occurs at biologically appropriate rates. The characterization of the HvUAM genes opens the way for the manipulation of both the amounts and fine structures of heteroxylans in cereals, grasses, and other crop plants, with a view toward enhancing their value in human health and nutrition, and in renewable biofuel production.

Development of genome-wide InDel markers and their integration with SSR, DArT and SNP markers in single barley map.

BACKGROUND: Development of molecular markers such as SSR (simple sequence repeat), DArT (diversity arrays technology) and SNP (single nucleotide polymorphism) is fundamental for linkage map construction and QTL mapping. However, DArT and SNP genotyping require special tools, and detection of SSR polymorphisms requires time-consuming polyacrylamide electrophoresis. Furthermore, many markers have been mapped in different populations such that their genetic positions are inconsistent. Recently, InDel (insertion and deletion) markers have become popular in genetic map construction and map-based cloning. RESULTS: Aligning genomic DNA sequences in two barley cultivars (Morex and Barke) identified 436,640 InDels. We designed 1140 InDel markers across the barley genome with an average genetic distance of 1 cM, each having a unique location in the barley genome. High-resolution melting (HRM) technology was used to genotype 55 InDel markers; those PCR amplicons with melting temperature differences >0.3 °C were ideal for HRM genotyping. The 1140 InDel markers together with 383 SSRs, 3909 gene-based SNPs and 1544 DArT markers were integrated into single barley genetic map according to their physical map positions. CONCLUSIONS: High-density InDel markers with specific genome locations were developed, with 6976 molecular markers (SSRs, DArTs, SNPs and InDels) integrated into single barley genetic map. HRM genotyping of the InDel markers each with single PCR band will facilitate quick map construction and gene fine-mapping.

Mapping a major QTL for malt extract of barley from a cross between TX9425 × Naso Nijo.

KEY MESSAGE: One major QTL-controlling malt extract was identified on 2H, based on the data from four different environments and a large number of DH lines, determining 48% of phenotypic variation. This QTL is of a high value for marker-assisted selection. Improving malting quality traits is one of the major breeding objectives for barley breeding programmes. Among different quality traits, malt extract is one of the most important, determining the yield of beer production. The use of molecular markers linked to loci affecting the quality traits can greatly improve selection efficiency. However, the discovery of closely linked markers relies on not only the availability of the loci, but the accuracy of phenotyping. In this experiment, 188 doubled-haploid lines derived from the cross between a Japanese malting barley and a Chinese feed barley were grown in four different environments (two sites × 2 years). Different quality traits were determined and used to map QTL for these traits. Several QTLs were identified for different quality traits. One major QTL-controlling malt extract was identified on 2H and determined 48% of phenotypic variation with the closest marker of GBM1121. This QTL was consistently expressed in all four environments and is of a high value for marker-assisted selection in malting barley breeding.

Spatial gradients in cell wall composition and transcriptional profiles along elongating maize internodes.

BACKGROUND: The elongating maize internode represents a useful system for following development of cell walls in vegetative cells in the Poaceae family. Elongating internodes can be divided into four developmental zones, namely the basal intercalary meristem, above which are found the elongation, transition and maturation zones. Cells in the basal meristem and elongation zones contain mainly primary walls, while secondary cell wall deposition accelerates in the transition zone and predominates in the maturation zone. RESULTS: The major wall components cellulose, lignin and glucuronoarabinoxylan (GAX) increased without any abrupt changes across the elongation, transition and maturation zones, although GAX appeared to increase more between the elongation and transition zones. Microarray analyses show that transcript abundance of key glycosyl transferase genes known to be involved in wall synthesis or re-modelling did not match the increases in cellulose, GAX and lignin. Rather, transcript levels of many of these genes were low in the meristematic and elongation zones, quickly increased to maximal levels in the transition zone and lower sections of the maturation zone, and generally decreased in the upper maturation zone sections. Genes with transcript profiles showing this pattern included secondary cell wall CesA genes, GT43 genes, some ß-expansins, UDP-Xylose synthase and UDP-Glucose pyrophosphorylase, some xyloglucan endotransglycosylases/hydrolases, genes involved in monolignol biosynthesis, and NAM and MYB transcription factor genes. CONCLUSIONS: The data indicated that the enzymic products of genes involved in cell wall synthesis and modification remain active right along the maturation zone of elongating maize internodes, despite the fact that corresponding transcript levels peak earlier, near or in the transition zone.

A cysteine in the repetitive domain of a high-molecular-weight glutenin subunit interferes with the mixing properties of wheat dough.

The quality of wheat (Triticum aestivum L.) for making bread is largely due to the strength and extensibility of wheat dough, which in turn is due to the properties of polymeric glutenin. Polymeric glutenin consists of high- and low-molecular-weight glutenin protein subunits linked by disulphide bonds between cysteine residues. Glutenin subunits differ in their effects on dough mixing properties. The research presented here investigated the effect of a specific, recently discovered, glutenin subunit on dough mixing properties. This subunit, Bx7.1, is unusual in that it has a cysteine in its repetitive domain. With site-directed mutagenesis of the gene encoding Bx7.1, a guanine in the repetitive domain was replaced by an adenine, to provide a mutant gene encoding a subunit (MutBx7.1) in which the repetitive-domain cysteine was replaced by a tyrosine residue. Bx7.1, MutBx7.1 and other Bx-type glutenin subunits were heterologously expressed in Escherichia coli and purified. This made it possible to incorporate each individual subunit into wheat flour and evaluate the effect of the cysteine residue on dough properties. The Bx7.1 subunit affected dough mixing properties differently from the other subunits. These differences are due to the extra cysteine residue, which may interfere with glutenin polymerisation through cross-linkage within the Bx7.1 subunit, causing this subunit to act as a chain terminator.

Cell wall modifications in maize pulvini in response to gravitational stress.

Changes in cell wall polysaccharides, transcript abundance, metabolite profiles, and hormone concentrations were monitored in the upper and lower regions of maize (Zea mays) pulvini in response to gravistimulation, during which maize plants placed in a horizontal position returned to the vertical orientation. Heteroxylan levels increased in the lower regions of the pulvini, together with lignin, but xyloglucans and heteromannan contents decreased. The degree of substitution of heteroxylan with arabinofuranosyl residues decreased in the lower pulvini, which exhibited increased mechanical strength as the plants returned to the vertical position. Few or no changes in noncellulosic wall polysaccharides could be detected on the upper side of the pulvinus, and crystalline cellulose content remained essentially constant in both the upper and lower pulvinus. Microarray analyses showed that spatial and temporal changes in transcript profiles were consistent with the changes in wall composition that were observed in the lower regions of the pulvinus. In addition, the microarray analyses indicated that metabolic pathways leading to the biosynthesis of phytohormones were differentially activated in the upper and lower regions of the pulvinus in response to gravistimulation. Metabolite profiles and measured hormone concentrations were consistent with the microarray data, insofar as auxin, physiologically active gibberellic acid, and metabolites potentially involved in lignin biosynthesis increased in the elongating cells of the lower pulvinus.

The genetics, transcriptional profiles, and catalytic properties of UDP-alpha-D-xylose 4-epimerases from barley.

Cell walls in the grasses contain relatively high levels of heteroxylans and, in particular, arabinoxylans. Enzymes and corresponding genes that are involved in the provision of sugar nucleotide substrates represent potential control points for arabinoxylan biosynthesis. Following expressed sequence tag database analyses, three genes encoding barley (Hordeum vulgare) UDP-d-xylose 4-epimerases (UXE; EC 5.1.3.5), designated HvUXE1, HvUXE2, and HvUXE3, were cloned and their positions on genetic maps defined. To confirm the identity of the genes, a cDNA construct encoding HvUXE1 was expressed in Pichia pastoris. The purified, recombinant HvUXE1 catalyzed the freely reversible interconversion of UDP-alpha-d-xylopyranose and UDP-beta-l-arabinopyranose, with K(m) values of 1.8 and 1.4 mm, respectively. At equilibrium, the ratio of substrate to product was approximately 1:1. Each molecule of heterologously expressed HvUXE1 enzyme contained about one molecule of noncovalently bound NAD(+). Molecular modeling provided a structural rationale for the substrate specificity of the UDP-d-xylose 4-epimerase and, in particular, explained its tight specificity for UDP-xylose compared with other sugar nucleotide epimerases. Quantitative transcript analyses performed for each of the three genes in a range of organs showed, inter alia, that in developing barley endosperm HvUXE1 and HvUXE3 mRNA levels peaked at a time when UDP-alpha-d-xylopyranose synthase (UXS) transcripts also reached a maximum and when arabinoxylan biosynthesis was initiated. Furthermore, the data revealed that the transcription of HvUXE and HvUXS gene family members is coordinated with the incorporation of pentose sugars onto cell walls in barley leaves, roots, and developing endosperm.

Expression of vacuolar H+-pyrophosphatase (OVP3) is under control of an anoxia-inducible promoter in rice.

Vacuolar H(+)-pyrophosphatase (V-PPase) expression increases in a number of abiotic stresses and is thought to play a role in adaptation to abiotic stresses. This paper reports on the regulation of six V-PPase genes in rice (Oryza sativa L.) coleoptiles under anoxia, using flood tolerant and intolerant cultivars to test our hypothesis. Quantitative PCR analysis showed that one vacuolar H(+)-pyrophosphatase (OVP3) was induced by anoxia, particularly in flood-tolerant rice. Regulation of OVP3 expression under anoxia was investigated by analysing putative OVP promoters. The putative OVP3 promoter contained more previously identified anoxia-inducible motifs than the putative promoters of the other five OVP genes. GUS activity in transgenic rice plants containing the OVP3 promoter region linked to the GUS reporter gene was induced only by anoxia. Salt and cold treatments had little effect on OVP3 promoter-driven GUS expression when compared to the anoxic treatment.

BarleyVarDB: a database of barley genomic variation.

Barley (Hordeum vulgare L.) is one of the first domesticated grain crops and represents the fourth most important cereal source for human and animal consumption. BarleyVarDB is a database of barley genomic variation. It can be publicly accessible through the website at http://146.118.64.11/BarleyVar. This database mainly provides three sets of information. First, there are 57 754 224 single nuclear polymorphisms (SNPs) and 3 600 663 insertions or deletions (InDels) included in BarleyVarDB, which were identified from high-coverage whole genome sequencing of 21 barley germplasm, including 8 wild barley accessions from 3 barley evolutionary original centers and 13 barley landraces from different continents. Second, it uses the latest barley genome reference and its annotation information publicly accessible, which has been achieved by the International Barley Genome Sequencing Consortium (IBSC). Third, 522 212 whole genome-wide microsatellites/simple sequence repeats (SSRs) were also included in this database, which were identified in the reference barley pseudo-molecular genome sequence. Additionally, several useful web-based applications are provided including JBrowse, BLAST and Primer3. Users can design PCR primers to asses polymorphic variants deposited in this database and use a user-friendly interface for accessing the barley reference genome. We envisage that the BarleyVarDB will benefit the barley genetic research community by providing access to all publicly available barley genomic variation information and barley reference genome as well as providing them with an ultra-high density of SNP and InDel markers for molecular breeding and identification of functional genes with important agronomic traits in barley. Database URL: http://146.118.64.11/BarleyVar.

Gene expression patterns and catalytic properties of UDP-D-glucose 4-epimerases from barley (Hordeum vulgare L.).

UGE (UDP-Glc 4-epimerase or UDP-Gal 4-epimerase; EC 5.1.3.2) catalyses the interconversion of UDP-Gal and UDP-Glc. Both nucleotide sugars act as activated sugar donors for the biosynthesis of cell wall polysaccharides such as cellulose, xyloglucans, (1,3;1,4)-beta-D-glucan and pectins, together with other biologically significant compounds including glycoproteins and glycolipids. Three members of the HvUGE (barley UGE) gene family, designated HvUGE1, HvUGE2 and HvUGE3, have been characterized. Q-PCR (quantitative real-time PCR) showed that HvUGE1 mRNA was most abundant in leaf tips and mature roots, but its expression levels were relatively low in basal leaves and root tips. The HvUGE2 gene was transcribed at significant levels in all organs examined, while HvUGE3 mRNA levels were very low in all the organs. Heterologous expression of a near full-length cDNA confirmed that HvUGE1 encodes a functional UGE. A non-covalently bound NAD+ was released from the enzyme after denaturing with aqueous ethanol and was identified by its spectrophotometric properties and by electrospray ionization MS. The K(m) values were 40 microM for UDP-Gal and 55 muM for UDP-Glc. HvUGE also catalyses the interconversion of UDP-GalNAc and UDP-GlcNAc, although it is not known if this has any biological significance. A three-dimensional model of the HvUGE revealed that its overall structural fold is highly conserved compared with the human UGE and provides a structural rationale for its ability to bind UDP-GlcNAc.

Comparisons of Copy Number, Genomic Structure, and Conserved Motifs for α-Amylase Genes from Barley, Rice, and Wheat.

Barley is an important crop for the production of malt and beer. However, crops such as rice and wheat are rarely used for malting. α-amylase is the key enzyme that degrades starch during malting. In this study, we compared the genomic properties, gene copies, and conserved promoter motifs of α-amylase genes in barley, rice, and wheat. In all three crops, α-amylase consists of four subfamilies designated amy1, amy2, amy3, and amy4. In wheat and barley, members of amy1 and amy2 genes are localized on chromosomes 6 and 7, respectively. In rice, members of amy1 genes are found on chromosomes 1 and 2, and amy2 genes on chromosome 6. The barley genome has six amy1 members and three amy2 members. The wheat B genome contains four amy1 members and three amy2 members, while the rice genome has three amy1 members and one amy2 member. The B genome has mostly amy1 and amy2 members among the three wheat genomes. Amy1 promoters from all three crop genomes contain a GA-responsive complex consisting of a GA-responsive element (CAATAAA), pyrimidine box (CCTTTT) and TATCCAT/C box. This study has shown that amy1 and amy2 from both wheat and barley have similar genomic properties, including exon/intron structures and GA-responsive elements on promoters, but these differ in rice. Like barley, wheat should have sufficient amy activity to degrade starch completely during malting. Other factors, such as high protein with haze issues and the lack of husk causing Lauting difficulty, may limit the use of wheat for brewing.

Construction of a map-based reference genome sequence for barley, Hordeum vulgare L.

Barley (Hordeum vulgare L.) is a cereal grass mainly used as animal fodder and raw material for the malting industry. The map-based reference genome sequence of barley cv. 'Morex' was constructed by the International Barley Genome Sequencing Consortium (IBSC) using hierarchical shotgun sequencing. Here, we report the experimental and computational procedures to (i) sequence and assemble more than 80,000 bacterial artificial chromosome (BAC) clones along the minimum tiling path of a genome-wide physical map, (ii) find and validate overlaps between adjacent BACs, (iii) construct 4,265 non-redundant sequence scaffolds representing clusters of overlapping BACs, and (iv) order and orient these BAC clusters along the seven barley chromosomes using positional information provided by dense genetic maps, an optical map and chromosome conformation capture sequencing (Hi-C). Integrative access to these sequence and mapping resources is provided by the barley genome explorer (BARLEX).

Involvement of Alternative Splicing in Barley Seed Germination.

Seed germination activates many new biological processes including DNA, membrane and mitochondrial repairs and requires active protein synthesis and sufficient energy supply. Alternative splicing (AS) regulates many cellular processes including cell differentiation and environmental adaptations. However, limited information is available on the regulation of seed germination at post-transcriptional levels. We have conducted RNA-sequencing experiments to dissect AS events in barley seed germination. We identified between 552 and 669 common AS transcripts in germinating barley embryos from four barley varieties (Hordeum vulgare L. Bass, Baudin, Harrington and Stirling). Alternative 3' splicing (34%-45%), intron retention (32%-34%) and alternative 5' splicing (16%-21%) were three major AS events in germinating embryos. The AS transcripts were predominantly mapped onto ribosome, RNA transport machineries, spliceosome, plant hormone signal transduction, glycolysis, sugar and carbon metabolism pathways. Transcripts of these genes were also very abundant in the early stage of seed germination. Correlation analysis of gene expression showed that AS hormone responsive transcripts could also be co-expressed with genes responsible for protein biosynthesis and sugar metabolisms. Our RNA-sequencing data revealed that AS could play important roles in barley seed germination.

Changes in cell wall polysaccharide composition, gene transcription and alternative splicing in germinating barley embryos.

Barley (Hordeum vulgare L.) seed germination initiates many important biological processes such as DNA, membrane and mitochondrial repairs. However, little is known on cell wall modifications in germinating embryos. We have investigated cell wall polysaccharide composition change, gene transcription and alternative splicing events in four barley varieties at 24h and 48 h germination. Cell wall components in germinating barley embryos changed rapidly, with increases in cellulose and (1,3)(1,4)-ß-D-glucan (20-100%) within 24h, but decreases in heteroxylan and arabinan (3-50%). There were also significant changes in the levels of type I arabinogalactans and heteromannans. Alternative splicing played very important roles in cell wall modifications. At least 22 cell wall transcripts were detected to undergo either alternative 3' splicing, alternative 5' splicing or intron retention type of alternative splicing. These genes coded enzymes catalyzing synthesis and degradation of cellulose, heteroxylan, (1,3)(1,4)-ß-D-glucan and other cell wall polymers. Furthermore, transcriptional regulation also played very important roles in cell wall modifications. Transcript levels of primary wall cellulase synthase, heteroxylan synthesizing and nucleotide sugar inter-conversion genes were very high in germinating embryos. At least 50 cell wall genes changed transcript levels significantly. Expression patterns of many cell wall genes coincided with changes in polysaccharide composition. Our data showed that cell wall polysaccharide metabolism was very active in germinating barley embryos, which was regulated at both transcriptional and post-transcriptional levels.

Identification and Fine Mapping of a White Husk Gene in Barley (Hordeum vulgare L.).

Barley is the only crop in the Poaceae family with adhering husks at maturity. The color of husk at barely development stage could influence the agronomic traits and malting qualities of grains. A barley mutant with a white husk was discovered from the malting barley cultivar Supi 3 and designated wh (white husk). Morphological changes and the genetics of white husk barley were investigated. Husks of the mutant were white at the heading and flowering stages but yellowed at maturity. The diastatic power and α-amino nitrogen contents also significantly increased in wh mutant. Transmission electron microscopy examination showed abnormal chloroplast development in the mutant. Genetic analysis of F2 and BC1F1 populations developed from a cross of wh and Yangnongpi 5 (green husk) showed that the white husk was controlled by a single recessive gene (wh). The wh gene was initially mapped between 49.64 and 51.77 cM on chromosome 3H, which is syntenic with rice chromosome 1 where a white husk gene wlp1 has been isolated. The barley orthologous gene of wlp1 was sequenced from both parents and a 688 bp deletion identified in the wh mutant. We further fine-mapped the wh gene between SSR markers Bmac0067 and Bmag0508a with distances of 0.36 cM and 0.27 cM in an F2 population with 1115 individuals of white husk. However, the wlp1 orthologous gene was mapped outside the interval. New candidate genes were identified based on the barley genome sequence.

Construction of High-Density Genetic Map in Barley through Restriction-Site Associated DNA Sequencing.

Genetic maps in barley are usually constructed from a limited number of molecular markers such as SSR (simple sequence repeat) and DarT (diversity arrays technology). These markers must be first developed before being used for genotyping. Here, we introduce a new strategy based on sequencing progeny of a doubled haploid population from Baudin × AC Metcalfe to construct a genetic map in barley. About 13,547 polymorphic SNP tags with >93% calling rate were selected to construct the genetic map. A total of 12,998 SNP tags were anchored to seven linkage groups which spanned a cumulative 967.6 cM genetic distance. The high-density genetic map can be used for QTL mapping and the assembly of WGS and BAC contigs. The genetic map was evaluated for its effectiveness and efficiency in QTL mapping and candidate gene identification. A major QTL for plant height was mapped at 105.5 cM on chromosome 3H. This QTL with LOD value of 13.01 explained 44.5% of phenotypic variation. This strategy will enable rapid and efficient establishment of high-density genetic maps in other species.

Draft genome sequence, and a sequence-defined genetic linkage map of the legume crop species Lupinus angustifolius L.

Lupin (Lupinus angustifolius L.) is the most recently domesticated crop in major agricultural cultivation. Its seeds are high in protein and dietary fibre, but low in oil and starch. Medical and dietetic studies have shown that consuming lupin-enriched food has significant health benefits. We report the draft assembly from a whole genome shotgun sequencing dataset for this legume species with 26.9x coverage of the genome, which is predicted to contain 57,807 genes. Analysis of the annotated genes with metabolic pathways provided a partial understanding of some key features of lupin, such as the amino acid profile of storage proteins in seeds. Furthermore, we applied the NGS-based RAD-sequencing technology to obtain 8,244 sequence-defined markers for anchoring the genomic sequences. A total of 4,214 scaffolds from the genome sequence assembly were aligned into the genetic map. The combination of the draft assembly and a sequence-defined genetic map made it possible to locate and study functional genes of agronomic interest. The identification of co-segregating SNP markers, scaffold sequences and gene annotation facilitated the identification of a candidate R gene associated with resistance to the major lupin disease anthracnose. We demonstrated that the combination of medium-depth genome sequencing and a high-density genetic linkage map by application of NGS technology is a cost-effective approach to generating genome sequence data and a large number of molecular markers to study the genomics, genetics and functional genes of lupin, and to apply them to molecular plant breeding. This strategy does not require prior genome knowledge, which potentiates its application to a wide range of non-model species.

The mechanism of boron tolerance for maintenance of root growth in barley (Hordeum vulgare L.).

Cultivar differences in root elongation under B toxic conditions were observed in barley (Hordeum vulgare L.). A significant increase in the length and width of the root meristematic zone (RMZ) was observed in Sahara 3771 (B tolerant) when it was grown under excessive B concentration, compared to when grown at adequate B supply. This coincided with an increase in cell width and cell numbers in the meristematic zone (MZ), whereas a significant decrease in the length and no significant effect on the width of the MZ was observed in Clipper (B intolerant) when it was grown under excessive B supply. This was accompanied by a decrease in cell numbers, but an increase in the length and width of individual cells present along the MZ. Excessive B concentrations led to a significantly lower osmotic potential within the cell sap of the root tip in SloopVic (B tolerant) and Sahara 3771, while the opposite was observed in Clipper. Enhanced sugar levels in the root tips of SloopVic were observed between 48 and 96 h after excess B was applied. This coincided with an increase in the root elongation rate and with a 2.7-fold increase in sucrose level within mature leaf tissue. A significant decrease in reducing sugar levels was observed in the root tips of Clipper under excessive B concentrations. This coincided with significantly lower root elongation rates and lower sucrose levels in leaf tissues. Results indicate a B tolerance mechanism associated with a complex control of sucrose levels between leaf and root tip that assist in maintaining root growth under B toxicity.

Characterization and expression patterns of UDP-D-glucuronate decarboxylase genes in barley.

UDP-D-glucuronate decarboxylase (EC 4.1.1.35) catalyzes the synthesis of UDP-D-xylose from UDP-D-glucuronate in an essentially irreversible reaction that is believed to commit glycosyl residues to heteroxylan and xyloglucan biosynthesis. Four members of the barley (Hordeum vulgare) UDP-D-glucuronate decarboxylase gene family, designated HvUXS1 to HvUXS4, have been cloned and characterized. Barley HvUXS1 appears to be a cytosolic enzyme, while the others are predicted to be membrane-bound proteins with single transmembrane helices. Heterologous expression of a barley HvUXS1 cDNA in Escherichia coli yields a soluble enzyme that converts UDP-d-glucuronate to UDP-D-xylose, is associated with a single molecule of bound NAD+, and is subject to feedback inhibition by UDP-D-xylose. Quantitative PCR shows that the HvUXS1 mRNA is most abundant among the 4 HvUXS genes, accounting for more than 80% of total HvUXS transcripts in most of the tissues examined. The abundance of HvUXS1 mRNA is 10-fold higher in mature roots and stems than in leaves, developing grains, or floral tissues. Transcriptional activities of HvUXS2 and HvUXS4 genes are relatively high in mature roots, coleoptiles, and stems compared with root tips, leaves, and floral tissues, while HvUXS3 mRNA is low in all tissues. In barley leaf sections, levels of the most abundant mRNA, encoding HvUXS1, reflect the amount of soluble enzymic protein and activity. In selected tissues where HvUXS1 transcript levels are high, cell walls have higher arabinoxylan contents.