Andrew J. Harvey

Cellulose Synthase-Like CslF Genes Mediate the Synthesis of Cell Wall (1,3;1,4)-ß-d-Glucans

A characteristic feature of grasses and commercially important cereals is the presence of (1,3;1,4)-β-d-glucans in their cell walls. We have used comparative genomics to link a major quantitative trait locus for (1,3;1,4)-β-d-glucan content in barley grain to a cluster of cellulose synthase–like CslF genes in rice. After insertion of rice CslF genes into Arabidopsis, we detected (1,3;1,4)-β-d-glucan in walls of transgenic plants using specific monoclonal antibodies and enzymatic analysis. Because wild-type Arabidopsis does not contain CslF genes or have (1,3;1,4)-β-d-glucans in its walls, these experiments provide direct, gain-of-function evidence for the participation of rice CslF genes in (1,3;1,4)-β-d-glucan biosynthesis.

The CesA Gene Family of Barley. Quantitative Analysis of Transcripts Reveals Two Groups of Co-Expressed Genes

Sequence data from cDNA and genomic clones, coupled with analyses of expressed sequence tag databases, indicate that the CesA (cellulose synthase) gene family from barley (Hordeum vulgare) has at least eight members, which are distributed across the genome. Quantitative polymerase chain reaction has been used to determine the relative abundance of mRNA transcripts for individual HvCesA genes in vegetative and floral tissues, at different stages of development. To ensure accurate expression profiling, geometric averaging of multiple internal control gene transcripts has been applied for the normalization of transcript abundance. Total HvCesA mRNA levels are highest in coleoptiles, roots, and stems and much lower in floral tissues, early developing grain, and in the elongation zone of leaves. In most tissues, HvCesA1, HvCesA2, and HvCesA6 predominate, and their relative abundance is very similar; these genes appear to be coordinately transcribed. A second group, comprising HvCesA4, HvCesA7, and HvCesA8, also appears to be coordinately transcribed, most obviously in maturing stem and root tissues. The HvCesA3 expression pattern does not fall into either of these two groups, and HvCesA5 transcript levels are extremely low in all tissues. Thus, the HvCesA genes fall into two general groups of three genes with respect to mRNA abundance, and the co-expression of the groups identifies their products as candidates for the rosettes that are involved in cellulose biosynthesis at the plasma membrane. Phylogenetic analysis allows the two groups of genes to be linked with orthologous Arabidopsis CesA genes that have been implicated in primary and secondary wall synthesis.

The Genetics and Transcriptional Profiles of the Cellulose Synthase-Like HvCslF Gene Family in Barley

Cellulose synthase-like CslF genes have been implicated in the biosynthesis of (1,3;1,4)-β-d-glucans, which are major cell wall constituents in grasses and cereals. Seven CslF genes from barley (Hordeum vulgare) can be divided into two classes on the basis of intron-exon arrangements. Four of the HvCslF genes have been mapped to a single locus on barley chromosome 2H, in a region corresponding to a major quantitative trait locus for grain (1,3;1,4)-β-d-glucan content. The other HvCslF genes map to chromosomes 1H, 5H, and 7H, and in two cases the genes are close to other quantitative trait loci for grain (1,3;1,4)-β-d-glucan content. Spatial and temporal patterns of transcription of the seven genes have been defined through quantitative polymerase chain reaction. In developing barley coleoptiles HvCslF6 mRNA is most abundant. Transcript levels are maximal in 4- to 5-d coleoptiles, at a time when (1,3;1,4)-β-d-glucan content of coleoptile cell walls also reaches maximal levels. In the starchy endosperm of developing grain, HvCslF6 and HvCslF9 transcripts predominate. Two peaks of transcription are apparent. One occurs just after endosperm cellularization, 4 to 8 d after pollination, while the second occurs much later in grain development, more than 20 d after pollination. Marked varietal differences in transcription of the HvCslF genes are observed during endosperm development. Given the commercial importance of cereal (1,3;1,4)-β-d-glucans in human nutrition, in stock feed, and in malting and brewing, the observation that only two genes, HvCslF6 and HvCslF9, are transcribed at high levels in developing grain is of potential relevance for the future manipulation of grain (1,3;1,4)-β-d-glucan levels.

Comparative modeling of the three-dimensional structures of family 3 glycoside hydrolases†

There are approximately 100 known members of the family 3 group of glycoside hydrolases, most of which are classified as β‐glucosidases and originate from microorganisms. The only family 3 glycoside hydrolase for which a three‐dimensional structure is available is a β‐glucan exohydrolase from barley. The structural coordinates of the barley enzyme is used here to model representatives from distinct phylogenetic clusters within the family. The majority of family 3 hydrolases have an NH2‐terminal (α/β)8 barrel connected by a short linker to a second domain, which adopts an (α/β)6 sandwich fold. In two bacterial β‐glucosidases, the order of the domains is reversed. The catalytic nucleophile, equivalent to D285 of the barley β‐glucan exohydrolase, is absolutely conserved across the family. It is located on domain 1, in a shallow site pocket near the interface of the domains. The likely catalytic acid in the barley enzyme, E491, is on domain 2. Although similarly positioned acidic residues are present in closely related members of the family, the equivalent amino acid in more distantly related members is either too far from the active site or absent. In the latter cases, the role of catalytic acid is probably assumed by other acidic amino acids from domain 1. Proteins 2000;41:257–269.

Molecular modeling of family GH16 glycoside hydrolases: Potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae

Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)‐β‐galactanases, (1,4)‐β‐galactanases/κ‐carrageenases, “nonspecific” (1,3/1,3;1,4)‐ β‐d‐glucan endohydrolases, and (1,3;1,4)‐β‐d‐glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni‐stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)‐β‐d‐glucan endohydrolases and a κ‐carrageenase. In combination with multiple sequence alignments, the models predict the three‐dimensional (3D) dispositions of amino acid residues in the substrate‐binding and catalytic sites of XTHs and (1,3/1,3;1,4)‐β‐d‐glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos × tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)‐β‐d‐xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos × tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)‐β‐d‐glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)‐β‐d‐glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)‐β‐d‐glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.

Polysaccharide hydrolases in germinated barley and their role in the depolymerization of plant and fungal cell walls

Cell wall degradation is an important event during endosperm mobilization in the germinated barley grain. A battery of polysaccharide and oligosaccharide hydrolases is required for the complete depolymerization of the arabinoxylans and (1 --> 3,1 --> 4)-beta-glucans which comprise in excess of 90% by weight of these walls. The (1 --> 3,1 --> 4)-beta-glucan endohydrolases release oligosaccharides from their substrate and are probably of central importance for the initial solubilization of the (1 --> 3,1 --> 4)-beta-glucans, but beta-glucan exohydrolases and beta-glucosidases may be important additional enzymes for the conversion of released oligosaccharides to glucose. The latter enzymes have recently been purified from germinated barley and characterized. There is an increasing body of evidence to support the notion that the (1 --> 3,1 --> 4)-beta-glucan endohydrolases from germinated barley evolved from the pathogenesis-related (1 --> 3)-beta-glucanases which are widely distributed in plants and which hydrolyse polysaccharides that are abundant in fungal cell walls. Arabinoxylan depolymerization is also mediated by a family of enzymes, but these are less well characterized. (1 --> 4)-beta-Xylan endohydrolases have been purified and the corresponding cDNAs and genes isolated. While the presence of (1 --> 4)-beta-xylan exohydrolases and alpha-L-arabinofuranosidases has been reported many times, the enzymes have not yet been studied in detail. Here, recent advances in the enzymology and physiology of cell wall degradation in the germinated barley grain are briefly reviewed.

A Customized Gene Expression Microarray Reveals that the Brittle Stem Phenotype fs2 of Barley is Attributable to a Retroelement in the HvCesA4 Cellulose Synthase Gene

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.

Substrate specificity and catalytic mechanism of a xyloglucan xyloglucosyl transferase HvXET6 from barley (Hordeum vulgare L.)

A family 16 glycoside hydrolase, xyloglucan xyloglucosyl transferase (EC 2.4.1.207), also known as xyloglucan endotransglycosylase (XET), and designated isoenzyme HvXET6, was purified approximately 400‐fold from extracts of young barley seedlings. The complete amino acid sequence of HvXET6 was deduced from the nucleotide sequence of a near full‐length cDNA, in combination with tryptic peptide mapping. An additional five to six isoforms or post‐translationally modified XET enzymes were detected in crude seedling extracts of barley. The HvXET6 isoenzyme was expressed in Pichia pastoris, characterized and compared with the previously purified native HvXET5 isoform. Barley HvXET6 has a similar apparent molecular mass of 33–35 kDa to the previously purified HvXET5 isoenzyme, but the two isoenzymes differ in their isoelectric points, pH optima, kinetic properties and substrate specificities. The HvXET6 isoenzyme catalyses transfer reactions between xyloglucans and soluble cellulosic substrates, using oligo‐xyloglucosides as acceptors, but at rates that are significantly different from those observed for HvXET5. No hydrolytic activity could be detected with either isoenzyme. Comparisons of the reaction rates using xyloglucan or hydroxyethyl cellulose as donors and a series of cellodextrins as acceptors indicated that the acceptor site of HvXET can accommodate five glucosyl residues. Molecular modelling supported this conclusion and further confirmed the ability of the enzyme’s active site to accommodate xyloglucan and cellulosic substrates. The two HvXETs followed a ping‐pong (Bi, Bi) rather than a sequential reaction mechanism.

Rice family GH1 glycoside hydrolases with β-D-glucosidase and β-D-mannosidase activities

Plant beta-D-mannosidases and a rice beta-D-glucosidase, Os3BGlu7, with weak beta-D-mannosidase activity, cluster together in phylogenetic analysis. To investigate the relationship between substrate specificity and amino acid sequence similarity in family GH1 glycoside hydrolases, Os3BGlu8 and Os7BGlu26, putative rice beta-D-glucosidases from this cluster, and a beta-D-mannosidase from barley (rHvBII), were expressed in Escherichia coli and characterized. Os3BGlu8, the amino acid sequence and molecular model of which are most similar to Os3BGlu7, hydrolysed 4-nitrophenyl-beta-D-glucopyranoside (4NPGlc) faster than 4-nitrophenyl-beta-D-mannopyranoside (4NPMan), while Os7BGlu26, which is most similar to rHvBII by these criteria, hydrolysed 4NPMan faster than 4NPGlc. All the enzymes hydrolyzed cellooligosaccharides with increased hydrolytic rates as the degree of polymerization increased from 3-6, but only rHvBII hydrolyzed cellobiose with a higher k(cat)/K(m) value than cellotriose. This was primarily due to strong binding of glucosyl residues at the+2 subsite for the rice enzymes, and unfavorable interactions at this subsite with rHvBII.

Evolutionary Dynamics of the Cellulose Synthase Gene Superfamily in Grasses

Variable selection pressure in the cellulose synthase gene superfamily reveals positions of amino acids under selection and unexpected evolutionary histories for key genes. Phylogenetic analyses of cellulose synthase (CesA) and cellulose synthase-like (Csl) families from the cellulose synthase gene superfamily were used to reconstruct their evolutionary origins and selection histories. Counterintuitively, genes encoding primary cell wall CesAs have undergone extensive expansion and diversification following an ancestral duplication from a secondary cell wall-associated CesA. Selection pressure across entire CesA and Csl clades appears to be low, but this conceals considerable variation within individual clades. Genes in the CslF clade are of particular interest because some mediate the synthesis of (1,3;1,4)-β-glucan, a polysaccharide characteristic of the evolutionarily successful grasses that is not widely distributed elsewhere in the plant kingdom. The phylogeny suggests that duplication of either CslF6 and/or CslF7 produced the ancestor of a highly conserved cluster of CslF genes that remain located in syntenic regions of all the grass genomes examined. A CslF6-specific insert encoding approximately 55 amino acid residues has subsequently been incorporated into the gene, or possibly lost from other CslFs, and the CslF7 clade has undergone a significant long-term shift in selection pressure. Homology modeling and molecular dynamics of the CslF6 protein were used to define the three-dimensional dispositions of individual amino acids that are subject to strong ongoing selection, together with the position of the conserved 55-amino acid insert that is known to influence the amounts and fine structures of (1,3;1,4)-β-glucans synthesized. These wall polysaccharides are attracting renewed interest because of their central roles as sources of dietary fiber in human health and for the generation of renewable liquid biofuels.