[show abstract][hide abstract] ABSTRACT: 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.
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 beta-expansins, UDP-Xylose synthase and UDP-Glucose pyrophosphorylase, some xyloglucan endotransglycosylases/hydrolases, genes involved in monolignol biosynthesis, and NAM and MYB transcription factor genes.
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.
[show abstract][hide abstract] ABSTRACT: An important component of barley cell walls, particularly in the endosperm, is (1,3;1,4)-β- glucan, a polymer that has proven health benefits in humans and that influences processability in the brewing industry. Genes of the cellulose synthase-like (Csl) F gene family have been shown to be involved in (1,3;1,4)-β-glucan synthesis but many aspects of the biosynthesis are still unclear. Examination of the sequence assembly of the barley genome has revealed the presence of an additional three HvCslF genes (HvCslF11, HvCslF12 and HvCslF13) which may be involved in (1,3;1,4)-β-glucan synthesis. Transcripts of HvCslF11 and HvCslF12 mRNA were found in roots and young leaves, respectively. Transient expression of these genes in Nicotiana benthamiana resulted in phenotypic changes in the infiltrated leaves, although no authentic (1,3;1,4)-β-glucan was detected. Comparisons of the CslF gene families in cereals revealed evidence of intergenic recombination, gene duplications and translocation events. This significant divergence within the gene family might be related to multiple functions of (1,3;1,4)-β-glucans in the Poaceae. Emerging genomic and global expression data for barley and other cereals is a powerful resource for characterising the evolution and dynamics of complete gene families. In the case of the CslF gene family, the results will contribute to a more thorough understanding of carbohydrate metabolism in grass cell walls.
PLoS ONE 01/2014; 9(3):e90888. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: To explain the low levels of starch, high levels of (1,3;1,4)-β-glucan, and thick cell walls in grains of Brachypodium distachyon L. relative to those in other Pooideae, aspects of grain development were compared between B. distachyon and barley (Hordeum vulgare L.). Cell proliferation, cell expansion, and endoreduplication were reduced in B. distachyon relative to barley and, consistent with these changes, transcriptional downregulation of the cell-cycle genes CDKB1 and cyclin A3 was observed. Similarly, reduced transcription of starch synthase I and starch-branching enzyme I was observed as well as reduced activity of starch synthase and ADP-glucose pyrophosphorylase, which are consistent with the lowered starch content in B. distachyon grains. No change was detected in transcription of the major gene involved in (1,3;1,4)-β-glucan synthesis, cellulose synthase-like F6. These results suggest that, while low starch content results from a reduced capacity for starch synthesis, the unusually thick cell walls in B. distachyon endosperm probably result from continuing (1,3;1,4)-β-glucan deposition in endosperm cells that fail to expand. This raises the possibility that endosperm expansion is linked to starch deposition.
Journal of Experimental Botany 09/2013; · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: BACKGROUND: Endo-(1,4)-beta-glucanase (cellulase) glycosyl hydrolase GH9 enzymes have been implicated in several aspects of cell wall metabolism in higher plants, including cellulose biosynthesis and degradation, modification of other wall polysaccharides that contain contiguous (1,4)-beta-glucosyl residues, and wall loosening during cell elongation. RESULTS: The endo-(1,4)-beta-glucanase gene families from barley (Hordeum vulgare), maize (Zea mays), sorghum (Sorghum bicolor), rice (Oryza sativa) and Brachypodium (Brachypodium distachyon) range in size from 23 to 29 members. Phylogenetic analyses show variations in clade structure between the grasses and Arabidopsis, and indicate differential gene loss and gain during evolution. Map positions and comparative studies of gene structures allow orthologous genes in the five species to be identified and synteny between the grasses is found to be high. It is also possible to differentiate between homoeologues resulting from ancient polyploidizations of the maize genome. Transcript analyses using microarray, massively parallel signature sequencing and quantitative PCR data for barley, rice and maize indicate that certain members of the endo-(1,4)-beta-glucanase gene family are transcribed across a wide range of tissues, while others are specifically transcribed in particular tissues. There are strong correlations between transcript levels of several members of the endo-(1,4)-beta-glucanase family and the data suggest that evolutionary conservation of transcription exists between orthologues across the grass family. There are also strong correlations between certain members of the endo-(1,4)-beta-glucanase family and other genes known to be involved in cell wall loosening and cell expansion, such as expansins and xyloglucan endotransglycosylases. CONCLUSIONS: The identification of these groups of genes will now allow us to test hypotheses regarding their functions and joint participation in wall synthesis, re-modelling and degradation, together with their potential role in lignocellulose conversion during biofuel production from grasses and cereal crop residues.
[show abstract][hide abstract] ABSTRACT: Barley (Hordeum vulgare L.) is among the world's earliest domesticated and most important crop plants. It is diploid with a large haploid genome of 5.1 gigabases (Gb). Here we present an integrated and ordered physical, genetic and functional sequence resource that describes the barley gene-space in a structured whole-genome context. We developed a physical map of 4.98 Gb, with more than 3.90 Gb anchored to a high-resolution genetic map. Projecting a deep whole-genome shotgun assembly, complementary DNA and deep RNA sequence data onto this framework supports 79,379 transcript clusters, including 26,159 'high-confidence' genes with homology support from other plant genomes. Abundant alternative splicing, premature termination codons and novel transcriptionally active regions suggest that post-transcriptional processing forms an important regulatory layer. Survey sequences from diverse accessions reveal a landscape of extensive single-nucleotide variation. Our data provide a platform for both genome-assisted research and enabling contemporary crop improvement.
[show abstract][hide abstract] ABSTRACT: The plant cell wall is a chemically complex structure composed mostly of polysaccharides. Detailed analyses of these cell wall polysaccharides are essential for our understanding of plant development and for our use of plant biomass (largely wall material) in the food, agriculture, fabric, timber, biofuel and biocomposite industries. We present analytical techniques not only to define the fine chemical structures of individual cell wall polysaccharides but also to estimate the overall polysaccharide composition of cell wall preparations. The procedure covers the preparation of cell walls, together with gas chromatography-mass spectrometry (GC-MS)-based methods, for both the analysis of monosaccharides as their volatile alditol acetate derivatives and for methylation analysis to determine linkage positions between monosaccharide residues as their volatile partially methylated alditol acetate derivatives. Analysis time will vary depending on both the method used and the tissue type, and ranges from 2 d for a simple neutral sugar composition to 2 weeks for a carboxyl reduction/methylation linkage analysis.
[show abstract][hide abstract] ABSTRACT: Immunolabeling, combined with chemical analyses and transcript profiling, have provided a comprehensive temporal and spatial picture of the deposition and modification of cell wall polysaccharides during barley (Hordeum vulgare) grain development, from endosperm cellularization at 3 d after pollination (DAP) through differentiation to the mature grain at 38 DAP. (1→3)-β-D-Glucan appears transiently during cellularization but reappears in patches in the subaleurone cell walls around 20 DAP. (1→3, 1→4)-β-Glucan, the most abundant polysaccharide of the mature barley grain, accumulates throughout development. Arabino-(1-4)-β-D-xylan is deposited significantly earlier than we previously reported. This was attributable to the initial deposition of the polysaccharide in a highly substituted form that was not recognized by antibodies commonly used to detect arabino-(1-4)-β-D-xylans in sections of plant material. The epitopes needed for antibody recognition were exposed by pretreatment of sections with α-L-arabinofuranosidase; this procedure showed that arabino-(1-4)-β-D-xylans were deposited as early as 5 DAP and highlighted their changing structures during endosperm development. By 28 DAP labeling of hetero-(1→4)-β-D-mannan is observed in the walls of the starchy endosperm but not in the aleurone walls. Although absent in mature endosperm cell walls we now show that xyloglucan is present transiently from 3 until about 6 DAP and disappears by 8 DAP. Quantitative reverse transcription-polymerase chain reaction of transcripts for GLUCAN SYNTHASE-LIKE, Cellulose Synthase, and CELLULOSE SYNTHASE-LIKE genes were consistent with the patterns of polysaccharide deposition. Transcript profiling of some members from the Carbohydrate-Active Enzymes database glycosyl transferase families GT61, GT47, and GT43, previously implicated in arabino-(1-4)-β-d-xylan biosynthesis, confirms their presence during grain development.
[show abstract][hide abstract] ABSTRACT: Arabinoxylan arabinofuranohydrolases (AXAHs) are family GH51 enzymes that have been implicated in the removal of arabinofuranosyl residues from the (1,4)-β-xylan backbone of heteroxylans. Five genes encoding barley AXAHs range in size from 4.6 kb to 7.1 kb and each contains 16 introns. The barley HvAXAH genes map to chromosomes 2H, 4H, and 5H. A small cluster of three HvAXAH genes is located on chromosome 4H and there is evidence for gene duplication and the presence of pseudogenes in barley. The cDNAs corresponding to barley and wheat AXAH genes were cloned, and transcript levels of the genes were profiled across a range of tissues at different developmental stages. Two HvAXAH cDNAs that were successfully expressed in Nicotiana benthamiana leaves exhibited similar activities against 4-nitrophenyl α-L-arabinofuranoside, but HvAXAH2 activity was significantly higher against wheat flour arabinoxylan, compared with HvAXAH1. HvAXAH2 also displayed activity against (1,5)-α-L-arabinopentaose and debranched arabinan. Western blotting with an anti-HvAXAH antibody was used to define further the locations of the AXAH enzymes in developing barley grain, where high levels were detected in the outer layers of the grain but little or no protein was detected in the endosperm. The chromosomal locations of the genes do not correspond to any previously identified genomic regions shown to influence heteroxylan structure. The data are therefore consistent with a role for AXAH in depolymerizing arabinoxylans in maternal tissues during grain development, but do not provide compelling evidence for a role in remodelling arabinoxylans during endosperm or coleoptile development in barley as previously proposed.
Journal of Experimental Botany 02/2012; 63(8):3031-45. · 5.24 Impact Factor
[show abstract][hide abstract] ABSTRACT: Plant cell walls consist predominantly of polysaccharides and lignin. There has been a surge of research activity in plant cell wall biology in recent years, in two key areas. Firstly, in the area of human health it is now recognized that cell wall polysaccharides are key components of dietary fiber, which carries significant health benefits. Secondly, plant cell walls are major constituents of lignocellulosic residues that are being developed as renewable sources of liquid transport biofuels. In both areas, the cell walls of the Poaceae, which include the cereals and grasses, are particularly important. The non-cellulosic wall polysaccharides of the Poaceae differ in comparison with those of other vascular plants, insofar as they contain relatively high levels of heteroxylans as "core" polysaccharide constituents and relatively smaller amounts of heteromannans, pectic polysaccharides, and xyloglucans. Certain grasses and cereals walls also contain (1,3;1,4)-β-glucans, which are not widely distributed outside the Poaceae. Although some genes involved in cellulose, heteroxylan, and (1,3;1,4)-β-glucan synthesis have been identified, mechanisms that control expression of the genes are not well defined. Here we review current knowledge of cell wall biology in plants and highlight emerging technologies that are providing new and exciting insights into the most challenging questions related to the synthesis, re-modeling and degradation of wall polysaccharides.
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: PpENA1 is a membrane-spanning transporter from the moss Physcomitrella patens, and is the first type IID P-type ATPase to be reported in the plant kingdom. In Physcomitrella, PpENA1 is essential for normal growth under moderate salt stress, while in yeast, type IID ATPases provide a vital efflux mechanism for cells under high salt conditions by selectively transporting Na+ or K+ across the plasma membrane. To investigate the structural basis for cation-binding within the type IID ATPase subfamily, we used homology modeling to identify a highly conserved cation-binding pocket between membrane helix (MH) 4 and MH 6 of the membrane-spanning pore of PpENA1. Mutation of specific charged and polar residues on MHs 4-6 resulted in a decrease or loss of protein activity as measured by complementation assays in yeast. The E298S mutation on MH 4 of PpENA1 had the most significant effect on activity despite the presence of a serine at this position in fungal type IID ATPases. Activity was partially restored in an inactivated PpENA1 mutant by the insertion of two additional serine residues on MH 4 and one on MH 6 based on the presence of these residues in fungal type IID ATPases. Our results suggest that the residues responsible for cation-binding in PpENA1 are distinct from those in fungal type IID ATPases, and that a fungal-type cation binding site can be successfully engineered into the moss protein.
Biochimica et Biophysica Acta 11/2010; 1808(6):1483-92. · 4.66 Impact Factor
[show abstract][hide abstract] ABSTRACT: Higher plants resist the forces of gravity and powerful lateral forces through the cumulative strength of the walls that surround individual cells. These walls consist mainly of cellulose, noncellulosic polysaccharides and lignin, in proportions that depend upon the specific functions of the cell and its stage of development. Spatially and temporally controlled heterogeneity in the physicochemical properties of wall polysaccharides is observed at the tissue and individual cell levels, and emerging in situ technologies are providing evidence that this heterogeneity also occurs across a single cell wall. We consider the origins of cell wall heterogeneity and identify contributing factors that are inherent in the molecular mechanisms of polysaccharide biosynthesis and are crucial for the changing biological functions of the wall during growth and development. We propose several key questions to be addressed in cell wall biology, together with an alternative two-phase model for the assembly of noncellulosic polysaccharides in plants.
Nature Chemical Biology 10/2010; 6(10):724-32. · 12.95 Impact Factor
[show abstract][hide abstract] ABSTRACT: The barley (Hordeum vulgare) brittle stem mutants, fs2, designated X054 and M245, have reduced levels of crystalline cellulose compared with their parental lines Ohichi and Shiroseto. A custom-designed microarray, based on long oligonucleotide technology and including genes involved in cell wall metabolism, revealed that transcript levels of very few genes were altered in the elongation zone of stem internodes, but these included a marked decrease in mRNA for the HvCesA4 cellulose synthase gene of both mutants. In contrast, the abundance of several hundred transcripts changed in the upper, maturation zones of stem internodes, which presumably reflected pleiotropic responses to a weakened cell wall that resulted from the primary genetic lesion. Sequencing of the HvCesA4 genes revealed the presence of a 964-bp solo long terminal repeat of a Copia-like retroelement in the first intron of the HvCesA4 genes of both mutant lines. The retroelement appears to interfere with transcription of the HvCesA4 gene or with processing of the mRNA, and this is likely to account for the lower crystalline cellulose content and lower stem strength of the mutants. The HvCesA4 gene maps to a position on chromosome 1H of barley that coincides with the previously reported position of fs2.
[show abstract][hide abstract] ABSTRACT: 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 18.104.22.168), 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.
[show abstract][hide abstract] ABSTRACT: The cell walls of the starchy endosperm of barley are relatively minor components of the grain in comparison to starch and
protein, but exert considerable influence over many malting quality characteristics. Wall components such as (1,3;1,4)-β-D-gmcans, which are commonly but less precisely known as mixed linkage β-glucans, can have direct and often detrimental effects on various processes in the brewery and on the quality and shelf life
of the final beer. The same wall components can have indirect effects on other quality characteristics, such as malt extract
and fermentability. The undesirable nature of many of these direct and indirect effects has led breeders, maltsters and brewers
to develop precise assays to monitor levels of (1,3;1,4)-β-D-glucans in barley grain and malt, and in many cases to select against high levels of this polysaccharide. While commercial
interest in endosperm cell wall components has been focused on (1,3;1,4)-β-D-glucans, the walls contain other components that have similar chemical and physicochemical properties and which merit further
attention in the context of malting quality of barley. Here, we will examine the properties and roles of all components of
barley endosperm walls in malting quality and relate these to the biological functions of cell walls in grain development
and germination. There is a good deal of information available on the physiology and enzymology of wall degradation in germinated
barley grain, but only recently have we been able to identify enzymes involved in the biosynthesis of key wall constituents.
[show abstract][hide abstract] ABSTRACT: Biosynthesis of the (1,3)-beta-D: -glucan (curdlan) in Agrobacterium sp., is believed to proceed by the repetitive addition of glucosyl residues from UDP-glucose by a membrane-embedded curdlan synthase (CrdS) [UDP-glucose: (1,3)-beta-D: -glucan 3-beta-D: -glucosyltransferase; EC 22.214.171.124]. The catalytic module of CrdS (cm-CrdS) was expressed in good yield from a cDNA encoding cm-CrdS cloned into the pET-32a(+) vector, containing a coding region for thioredoxin, and from the Champion pET SUMO system that possesses a coding region of a small ubiquitin-related modifier (SUMO) partner protein. The two DNA fusions, designated pET-32a_cm-CrdS and SUMO_cm-CrdS were expressed as chimeric proteins. High yields of inclusion bodies were produced in E. coli and these could be refolded to form soluble proteins, using a range of buffers and non-detergent sulfobetaines. A purification protocol was developed, which afforded a one-step on-column refolding and simultaneous purification of the recombinant 6xHis-tagged SUMO_cm-CrdS protein. The latter protein was digested by a specific protease to yield intact cm-CrdS in high yields. The refolded SUMO_cm-CrdS protein did not exhibit curdlan synthase activity, but showed a circular dischroism spectrum, which had an alpha/beta-type-like conformation. Amino acid sequences of tryptic fragments of the SUMO_cm-CrdS fusion and free cm-CrdS proteins, determined by MALDI/TOF confirmed that the full-length proteins were synthesized by E. coli, and that no alterations in amino acid sequences occurred. A three-dimensional model of cm-CrdS predicted the juxtaposition of highly conserved aspartates D156, D208, D210 and D304, and the QRTRW motif, which are likely to play roles in donor and acceptor substrate binding and catalysis.
[show abstract][hide abstract] ABSTRACT: Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)-β-d-glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)-β-d-glucans in the Poaceae is mediated, in part at least, by the cellulose synthase-like CslF family of genes. Over-expression of the barley CslF6 gene under the control of an endosperm-specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)-β-d-glucan content in grain of transgenic barley. Analyses of (1,3;1,4)-β-d-glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)-β-d-glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)-β-d-glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)-β-d-glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)-β-d-glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)-β-d-glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)-β-d-glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.
[show abstract][hide abstract] ABSTRACT: Three barley xyloglucan endotransglycosylases (HvXETs), known as xyloglucan xyloglucosyl transferases (EC 126.96.36.199), were subjected to kinetic and computational docking studies. The k(cat) x K(m)(-1) values with the reduced [3H]-labelled XXXG, XXLG/XLXG and XLLG acceptor substrates were 0.02 x 10(-2), 0.1 x 10(-2) and 3.2 x 10(-2) s(-1) microM(-1), while the K(m) constants were 10.6, 8.6 and 5.3 mM, obtained for HvXET3, HvXET4 and HvXET6, respectively. Docking of XLLG in acceptor-binding regions revealed that at least two conformational states were likely to participate in all isoforms. The assessments of kinetic and computational data indicated that the disposition of aromatic residues at the entrance to the active sites and the flexibility of proximal COOH-terminal loops could orient acceptors more or less favourably during binding, thus leading to tighter or weaker K(m) constants. The data suggested that binding of acceptors in HvXETs is guided by contributions from the conserved residues in the active sites and by the of neighbouring loops.
Archives of Biochemistry and Biophysics 04/2010; 496(1):61-8. · 3.37 Impact Factor