Barley Nodulin 26-like intrinsic protein permeates water, metalloids, saccharides, and ion pairs due to structural plasticity and diversification.

Aquaporins can facilitate the passive movement of water, small polar molecules, and some ions. Here, we examined solute selectivity for the barley Nodulin 26-like Intrinsic Protein (HvNIP2;1) embedded in liposomes and examined through stopped-flow light scattering spectrophotometry and Xenopus laevis oocyte swelling assays. We found that HvNIP2;1 permeates water, boric and germanic acids, sucrose, and lactose but not d-glucose or d-fructose. Other saccharides, such as neutral (d-mannose, d-galactose, d-xylose, d-mannoheptaose) and charged (N-acetyl d-glucosamine, d-glucosamine, d-glucuronic acid) aldoses, disaccharides (cellobiose, gentiobiose, trehalose), trisaccharide raffinose, and urea, glycerol, and acyclic polyols, were permeated to a much lower extent. We observed apparent permeation of hydrated KCl and MgSO4 ions, while CH3COONa and NaNO3 permeated at significantly lower rates. Our experiments with boric acid and sucrose revealed no apparent interaction between solutes when permeated together, and AgNO3 or H[AuCl4] blocked the permeation of all solutes. Docking of sucrose in HvNIP2;1 and spinach water-selective SoPIP2;1 aquaporins revealed the structural basis for sucrose permeation in HvNIP2;1 but not in SoPIP2;1, and defined key residues interacting with this permeant. In a biological context, sucrose transport could constitute a novel element of plant saccharide-transporting machinery. Phylogenomic analyses of 164 Viridiplantae and 2993 Archaean, bacterial, fungal, and Metazoan aquaporins rationalized solute poly-selectivity in NIP3 sub-clade entries and suggested that they diversified from other sub-clades to acquire a unique specificity of saccharide transporters. Solute specificity definition in NIP aquaporins could inspire developing plants for food production.

Molecular mechanisms of processive glycoside hydrolases underline catalytic pragmatism.

Processive and distributive catalysis defines the conversion continuum, thus underpinning the transformation of oligo- and polymeric substrates by enzymes. Distributive catalysis follows an association-transformation-dissociation pattern during the formation of enzyme-reactant complexes, whereas during processive catalysis, enzymes partner with substrates and complete multiple catalytic events before dissociation from an enzyme-substrate complex. Here, we focus on processive catalysis in glycoside hydrolases (GHs), which ensures efficient conversions of substrates with high precision, and has the advantage over distributive catalysis in efficiency. The work presented here examines a recent discovery of substrate-product-assisted processive catalysis in the GH3 family enzymes with enclosed pocket-shaped active sites. We detail how GH3 ß-d-glucan glucohydrolases exploit a transiently formed lateral pocket for product displacement and reactants sliding (or translocation motion) through the catalytic site without dissociation, including movements during nanoscale binding/unbinding and sliding. The phylogenetic tree of putative 550 Archaean, bacterial, fungal, Viridiplantae, and Metazoan GH3 entries resolved seven lineages that corresponded to major substrate specificity groups. This analysis indicates that two tryptophan residues in plant ß-d-glucan glucohydrolases that delineate the catalytic pocket, and infer broad specificity, high catalytic efficiency, and substrate-product-assisted processivity, have evolved through a complex evolutionary process, including horizontal transfer and neo-functionalisation. We conclude that the definition of thermodynamic and mechano-structural properties of processive enzymes is fundamentally important for theoretical and practical applications in bioengineering applicable in various biotechnologies.

Identification and characterisation of MdUGT78T2 as a galactosyltransferase with dual activity on flavonol and anthocyanidin substrates in red-skinned apple fruit (Malus domestica L.).

Anthocyanidin and flavonol glycosides have been linked to the health-promoting effects associated with apple consumption. However, very few enzymes involved in flavonoid glycosylation have been characterised to date. Here, we present the identification and phylogenetic analysis of 234 putative glycosyltransferases involved in flavonoid biosynthesis, and detail the biochemical and structural characterisation of MdUGT78T2 as a strict galactosyltransferase involved in the formation of quercetin-3-O-galactoside and cyanidin-3-O-galactoside, the major glycoconjugates of flavonoids in apple. The enzyme is also active on other flavonoids but with a lower catalytic efficiency. Our data, complemented with gene expression analysis suggest that MdUGT78T2 synthesises the glycoconjugates at both the early and late stages of fruit development. This newly discovered type of catalytic activity can potentially be exploited for in vitro modification of flavonoids to increase their stability in food products and to modify apple fruits and other commercial crops through breeding approaches to enhance their health benefits.

A chromosome-level genome assembly of Plantago ovata.

Plantago ovata is cultivated for production of its seed husk (psyllium). When wet, the husk transforms into a mucilage with properties suitable for pharmaceutical industries, utilised in supplements for controlling blood cholesterol levels, and food industries for making gluten-free products. There has been limited success in improving husk quantity and quality through breeding approaches, partly due to the lack of a reference genome. Here we constructed the first chromosome-scale reference assembly of P. ovata using a combination of 5.98 million PacBio and 636.5 million Hi-C reads. We also used corrected PacBio reads to estimate genome size and transcripts to generate gene models. The final assembly covers ~ 500 Mb with 99.3% gene set completeness. A total of 97% of the sequences are anchored to four chromosomes with an N50 of ~ 128.87 Mb. The P. ovata genome contains 61.90% repeats, where 40.04% are long terminal repeats. We identified 41,820 protein-coding genes, 411 non-coding RNAs, 108 ribosomal RNAs, and 1295 transfer RNAs. This genome will provide a resource for plant breeding programs to, for example, reduce agronomic constraints such as seed shattering, increase psyllium yield and quality, and overcome crop disease susceptibility.

The evolutionary advantage of an aromatic clamp in plant family 3 glycoside exo-hydrolases.

In the barley ß-D-glucan glucohydrolase, a glycoside hydrolase family 3 (GH3) enzyme, the Trp286/Trp434 clamp ensures ß-D-glucosides binding, which is fundamental for substrate hydrolysis during plant growth and development. We employ mutagenesis, high-resolution X-ray crystallography, and multi-scale molecular modelling methods to examine the binding and conformational behaviour of isomeric ß-D-glucosides during substrate-product assisted processive catalysis that operates in GH3 hydrolases. Enzyme kinetics reveals that the W434H mutant retains broad specificity, while W434A behaves as a strict (1,3)-ß-D-glucosidase. Investigations of reactant movements on the nanoscale reveal that processivity is sensitive to mutation-specific alterations of the tryptophan clamp. While wild-type and W434H utilise a lateral cavity for glucose displacement and sliding of (1,3)-linked hydrolytic products through the catalytic site without dissociation, consistent with their high hydrolytic rates, W434A does not adopt processive catalysis. Phylogenomic analyses of GH3 hydrolases disclose the evolutionary advantage of the tryptophan clamp that confers broad specificity, high catalytic efficiency, and processivity.

Identification and spatio-temporal expression analysis of barley genes that encode putative modular xylanolytic enzymes.

Arabinoxylans are cell wall polysaccharides whose re-modelling and degradation during plant development are mediated by several classes of xylanolytic enzymes. Here, we present the identification and new annotation of twelve putative (1,4)-ß-xylanase and six ß-xylosidase genes, and their spatio-temporal expression patterns during vegetative and reproductive growth of barley (Hordeum vulgare cv. Navigator). The encoded xylanase proteins are all predicted to contain a conserved carbohydrate-binding module (CBM) and a catalytic glycoside hydrolase (GH) 10 domain. Additional domains in some xylanases define three discrete phylogenetic clades: one clade contains proteins with an additional N-terminal signal sequence, while another clade contains proteins with multiple CBMs. Homology modelling revealed that all fifteen xylanases likely contain a third domain, a ß-sandwich folded from two non-contiguous sequence segments that bracket the catalytic GH domain, which may explain why the full length protein is required for correct folding of the active enzyme. Similarly, predicted xylosidase proteins share a highly conserved domain structure, each with an N-terminal signal peptide, a split GH 3 domain, and a C-terminal fibronectin-like domain. Several genes appear to be ubiquitously expressed during barley growth and development, while four newly annotated xylanase and xylosidase genes are expressed at extremely high levels, which may be of broader interest for industrial applications where cell wall degradation is necessary.

Another building block in the plant cell wall: Barley xyloglucan xyloglucosyl transferases link covalently xyloglucan and anionic oligosaccharides derived from pectin.

We report on the homo- and hetero-transglycosylation activities of the HvXET3 and HvXET4 xyloglucan xyloglucosyl transferases (XET; EC 2.4.1.207) from barley (Hordeum vulgare L.), and the visualisation of these activities in young barley roots using Alexa Fluor 488-labelled oligosaccharides. We discover that these isozymes catalyse the transglycosylation reactions with the chemically defined donor and acceptor substrates, specifically with the xyloglucan donor and the penta-galacturonide [α(1-4)GalAp]5 acceptor - the homogalacturonan (pectin) fragment. This activity is supported by 3D molecular models of HvXET3 and HvXET4 with the docked XXXG donor and [α(1-4)GalAp]5 acceptor substrates at the -4 to +5 subsites in the active sites. Comparative sequence analyses of barley isoforms and seed-localised TmXET6.3 from nasturtium (Tropaeolum majus L.) permitted the engineering of mutants of TmXET6.3 that could catalyse the hetero-transglycosylation reaction with the xyloglucan/[α(1-4)GalAp]5 substrate pair, while wild-type TmXET6.3 lacked this activity. Expression data obtained by real-time quantitative polymerase chain reaction of HvXET transcripts and a clustered heatmap of expression profiles of the gene family revealed that HvXET3 and HvXET6 co-expressed but did not share the monophyletic origin. Conversely, HvXET3 and HvXET4 shared this relationship, when we examined the evolutionary history of 419 glycoside hydrolase 16 family members, spanning monocots, eudicots and a basal Angiosperm. The discovered hetero-transglycosylation activity in HvXET3 and HvXET4 with the xyloglucan/[α(1-4)GalAp]5 substrate pair is discussed against the background of roles of xyloglucan-pectin heteropolymers and how they may participate in spatial patterns of cell wall formation and re-modelling, and affect the structural features of walls.

Co-evolution of Enzymes Involved in Plant Cell Wall Metabolism in the Grasses.

There has been a dramatic evolutionary shift in the polysaccharide composition of cell walls in the grasses, with increases in arabinoxylans and (1,3;1,4)-ß-glucans and decreases in pectic polysaccharides, mannans, and xyloglucans, compared with other angiosperms. Several enzymes are involved in the biosynthesis of arabinoxylans, but the overall process is not yet defined and whether their increased abundance in grasses results from active or reactive evolutionary forces is not clear. Phylogenetic analyses reveal that multiple independent evolution of genes encoding (1,3;1,4)-ß-glucan synthases has probably occurred within the large cellulose synthase/cellulose synthase-like (CesA/Csl) gene family of angiosperms. The (1,3;1,4)-ß-glucan synthases appear to be capable of inserting both (1,3)- and (1,4)-ß-linkages in the elongating polysaccharide chain, although the precise mechanism through which this is achieved remains unclear. Nevertheless, these enzymes probably evolved from synthases that originally synthesized only (1,4)-ß-linkages. Initially, (1,3;1,4)-ß-glucans could be turned over through preexisting cellulases, but as the need for specific hydrolysis was required, the grasses evolved specific (1,3;1,4)-ß-glucan endohydrolases. The corresponding genes evolved from genes for the more widely distributed (1,3)-ß-glucan endohydrolases. Why the subgroups of CesA/Csl genes that mediate the synthesis of (1,3;1,4)-ß-glucans have been retained by the highly successful grasses but by few other angiosperms or lower plants represents an intriguing biological question. In this review, we address this important aspect of cell wall polysaccharide evolution in the grasses, with a particular focus on the enzymes involved in noncellulosic polysaccharide biosynthesis, hydrolysis, and modification.

Phototactic tails: Evolution and molecular basis of a novel sensory trait in sea snakes.

Dermal phototaxis has been reported in a few aquatic vertebrate lineages spanning fish, amphibians and reptiles. These taxa respond to light on the skin of their elongate hind-bodies and tails by withdrawing under cover to avoid detection by predators. Here, we investigated tail phototaxis in sea snakes (Hydrophiinae), the only reptiles reported to exhibit this sensory behaviour. We conducted behavioural tests in 17 wild-caught sea snakes of eight species by illuminating the dorsal surface of the tail and midbody skin using cold white, violet, blue, green and red light. Our results confirmed phototactic tail withdrawal in the previously studied Aipysurus laevis, revealed this trait for the first time in A. duboisii and A. tenuis, and suggested that tail photoreceptors have peak spectral sensitivities between blue and green light (457-514 nm). Based on these results, and an absence of photoresponses in five Aipysurus and Hydrophis species, we tentatively infer that tail phototaxis evolved in the ancestor of a clade of six Aipysurus species (comprising 10% of all sea snakes). Quantifying tail damage, we found that the probability of sustaining tail injuries was not influenced by tail phototactic ability in snakes. Gene profiling showed that transcriptomes of both tail skin and body skin lacked visual opsins but contained melanopsin (opn4x) in addition to key genes of the retinal regeneration and phototransduction cascades. This work suggests that a nonvisual photoreceptor (e.g., Gq rhabdomeric) signalling pathway underlies tail phototaxis, and provides candidate gene targets for future studies of this unusual sensory innovation in reptiles.

A Novel (1,4)-ß-Linked Glucoxylan Is Synthesized by Members of the Cellulose Synthase-Like F Gene Family in Land Plants.

As a significant component of monocot cell walls, (1,3;1,4)-ß-glucan has conclusively been shown to be synthesized by the cellulose synthase-like F6 protein. In this study, we investigated the synthetic activity of other members of the barley (Hordeum vulgare) CslF gene family using heterologous expression. As expected, the majority of the genes encode proteins that are capable of synthesizing detectable levels of (1,3;1,4)-ß-glucan. However, overexpression of HvCslF3 and HvCslF10 genes resulted in the synthesis of a novel linear glucoxylan that consists of (1,4)-ß-linked glucose and xylose residues. To demonstrate that this product was not an aberration of the heterologous system, the characteristic (1,4)-ß-linkage between glucose and xylose was confirmed to be present in wild type barley tissues known to contain HvCslF3 and HvCslF10 transcripts. This polysaccharide linkage has also been reported in species of Ulva, a marine green alga, and has significant implications for defining the specificity of the cell wall content of many crop species. This finding supports previous observations that members of a single CSL family may not possess the same carbohydrate synthetic activity, with the CSLF family now associated with the formation of not only (1,3)- and (1,4)-ß-glucosidic linkages, but also (1,4)-ß-glucosidic and (1,4)-ß-xylosidic linkages.

Composition and biosynthetic machinery of the Blumeria graminis f. sp. hordei conidia cell wall.

Infection of barley with the powdery mildew causal agent, Blumeria graminis f. sp. hordei (Bgh), can lead to devastating damage to barley crops. The recent emergence of fungicide resistance imposes a need to develop new antifungal strategies. The enzymes involved in cell wall biosynthesis are ideal targets for the development of fungicides. However, in order to narrow down any target proteins involved in cell wall formation, a greater understanding of the cell wall structure and composition is required. Here, we present a detailed carbohydrate analysis of the Bgh conidial cell wall, a full annotation of Carbohydrate Active enZymes (CAZy) in the Bgh genome, and a comprehensive expression profile of the genes involved in cell wall metabolism. Glycosidic linkage analysis has revealed that the cell wall polysaccharide fraction of Bgh conidia predominantly consists of glucosyl residues (63.1%) and has a greater proportion of galactopyranosyl residues compared to other species (8.5%). Trace amounts of xylosyl residues were also detected, which is unusual in ascomycetes. Transcripts of the genes involved in cell wall metabolism show high expression of chitin deacetylases, which assist fungi in evading the host defence system by deacetylating chitin to chitosan. The data presented suggest that the cell wall components of the conidia and the corresponding obligate biotrophic CAZy gene profile play a key role in the infection process.

Analysis of cell wall synthesis and metabolism during early germination of Blumeria graminis f. sp. hordei conidial cells induced in vitro.

As an obligate biotroph, Blumeria graminis f. sp. hordei (Bgh) cannot be grown in an axenic culture, and instead must be cultivated on its host species, Hordeum vulgare (barley). In this study an in vitro system utilizing n-hexacosanal, a constituent of the barley cuticle and known inducer of Bgh germination, was used to cultivate Bgh and differentiate conidia up to the appressorial germ tube stage for analysis. Transcriptomic and proteomic profiling of the appressorial germ tube stage revealed that there was a significant shift towards energy and protein production during the pre-penetrative phase of development, with an up-regulation of enzymes associated with cellular respiration and protein synthesis, modification and transport. Glycosidic linkage analysis of the cell wall polysaccharides demonstrated that during appressorial development an increase in 1,3- and 1,4-linked glucosyl residues and xylosyl residues was detected along with a significant decrease in galactosyl residues. The use of this in vitro cultivation method demonstrates that it is possible to analyse the pre-penetrative processes of Bgh development in the absence of a plant host.

Revised Phylogeny of the Cellulose Synthase Gene Superfamily: Insights into Cell Wall Evolution.

Cell walls are crucial for the integrity and function of all land plants and are of central importance in human health, livestock production, and as a source of renewable bioenergy. Many enzymes that mediate the biosynthesis of cell wall polysaccharides are encoded by members of the large cellulose synthase (CesA) gene superfamily. Here, we analyzed 29 sequenced genomes and 17 transcriptomes to revise the phylogeny of the CesA gene superfamily in angiosperms. Our results identify ancestral gene clusters that predate the monocot-eudicot divergence and reveal several novel evolutionary observations, including the expansion of the Poaceae-specific cellulose synthase-like CslF family to the graminids and restiids and the characterization of a previously unreported eudicot lineage, CslM, that forms a reciprocally monophyletic eudicot-monocot grouping with the CslJ clade. The CslM lineage is widely distributed in eudicots, and the CslJ clade, which was thought previously to be restricted to the Poales, is widely distributed in monocots. Our analyses show that some members of the CslJ lineage, but not the newly identified CslM genes, are capable of directing (1,3;1,4)-ß-glucan biosynthesis, which, contrary to current dogma, is not restricted to Poaceae.

A Genome Wide Association Study of arabinoxylan content in 2-row spring barley grain.

In barley endosperm arabinoxylan (AX) is the second most abundant cell wall polysaccharide and in wheat it is the most abundant polysaccharide in the starchy endosperm walls of the grain. AX is one of the main contributors to grain dietary fibre content providing several health benefits including cholesterol and glucose lowering effects, and antioxidant activities. Due to its complex structural features, AX might also affect the downstream applications of barley grain in malting and brewing. Using a high pressure liquid chromatography (HPLC) method we quantified AX amounts in mature grain in 128 spring 2-row barley accessions. Amounts ranged from ~ 5.2 µg/g to ~ 9 µg/g. We used this data for a Genome Wide Association Study (GWAS) that revealed three significant quantitative trait loci (QTL) associated with grain AX levels which passed a false discovery threshold (FDR) and are located on two of the seven barley chromosomes. Regions underlying the QTLs were scanned for genes likely to be involved in AX biosynthesis or turnover, and strong candidates, including glycosyltransferases from the GT43 and GT61 families and glycoside hydrolases from the GH10 family, were identified. Phylogenetic trees of selected gene families were built based on protein translations and were used to examine the relationship of the barley candidate genes to those in other species. Our data reaffirms the roles of existing genes thought to contribute to AX content, and identifies novel QTL (and candidate genes associated with them) potentially influencing the AX content of barley grain. One potential outcome of this work is the deployment of highly associated single nucleotide polymorphisms markers in breeding programs to guide the modification of AX abundance in barley grain.

Altered Expression of Genes Implicated in Xylan Biosynthesis Affects Penetration Resistance against Powdery Mildew.

Heteroxylan has recently been identified as an important component of papillae, which are formed during powdery mildew infection of barley leaves. Deposition of heteroxylan near the sites of attempted fungal penetration in the epidermal cell wall is believed to enhance the physical resistance to the fungal penetration peg and hence to improve pre-invasion resistance. Several glycosyltransferase (GT) families are implicated in the assembly of heteroxylan in the plant cell wall, and are likely to work together in a multi-enzyme complex. Members of key GT families reported to be involved in heteroxylan biosynthesis are up-regulated in the epidermal layer of barley leaves during powdery mildew infection. Modulation of their expression leads to altered susceptibility levels, suggesting that these genes are important for penetration resistance. The highest level of resistance was achieved when a GT43 gene was co-expressed with a GT47 candidate gene, both of which have been predicted to be involved in xylan backbone biosynthesis. Altering the expression level of several candidate heteroxylan synthesis genes can significantly alter disease susceptibility. This is predicted to occur through changes in the amount and structure of heteroxylan in barley papillae.

Alanine aminotransferase controls seed dormancy in barley.

Dormancy allows wild barley grains to survive dry summers in the Near East. After domestication, barley was selected for shorter dormancy periods. Here we isolate the major seed dormancy gene qsd1 from wild barley, which encodes an alanine aminotransferase (AlaAT). The seed dormancy gene is expressed specifically in the embryo. The AlaAT isoenzymes encoded by the long and short dormancy alleles differ in a single amino acid residue. The reduced dormancy allele Qsd1 evolved from barleys that were first domesticated in the southern Levant and had the long dormancy qsd1 allele that can be traced back to wild barleys. The reduced dormancy mutation likely contributed to the enhanced performance of barley in industrial applications such as beer and whisky production, which involve controlled germination. In contrast, the long dormancy allele might be used to control pre-harvest sprouting in higher rainfall areas to enhance global adaptation of barley.

Genetic Diversity and Genome Wide Association Study of ß-Glucan Content in Tetraploid Wheat Grains.

Non-starch polysaccharides (NSPs) have many health benefits, including immunomodulatory activity, lowering serum cholesterol, a faecal bulking effect, enhanced absorption of certain minerals, prebiotic effects and the amelioration of type II diabetes. The principal components of the NSP in cereal grains are (1,3;1,4)-ß-glucans and arabinoxylans. Although (1,3;1,4)-ß-glucan (hereafter called ß-glucan) is not the most representative component of wheat cell walls, it is one of the most important types of soluble fibre in terms of its proven beneficial effects on human health. In the present work we explored the genetic variability of ß-glucan content in grains from a tetraploid wheat collection that had been genotyped with a 90k-iSelect array, and combined this data to carry out an association analysis. The ß-glucan content, expressed as a percentage w/w of grain dry weight, ranged from 0.18% to 0.89% across the collection. Our analysis identified seven genomic regions associated with ß-glucan, located on chromosomes 1A, 2A (two), 2B, 5B and 7A (two), confirming the quantitative nature of this trait. Analysis of marker trait associations (MTAs) in syntenic regions of several grass species revealed putative candidate genes that might influence ß-glucan levels in the endosperm, possibly via their participation in carbon partitioning. These include the glycosyl hydrolases endo-ß-(1,4)-glucanase (cellulase), ß-amylase, (1,4)-ß-xylan endohydrolase, xylanase inhibitor protein I, isoamylase and the glycosyl transferase starch synthase II.

(1,3;1,4)-ß-Glucan Biosynthesis by the CSLF6 Enzyme: Position and Flexibility of Catalytic Residues Influence Product Fine Structure.

Cellulose synthase-like F6 (CslF6) genes encode polysaccharide synthases responsible for (1,3;1,4)-ß-glucan biosynthesis in cereal grains. However, it is not clear how both (1,3)- and (1,4)-linkages are incorporated into a single polysaccharide chain and how the frequency and arrangement of the two linkage types that define the fine structure of the polysaccharide are controlled. Through transient expression in Nicotiana benthamiana leaves, two CSLF6 orthologs from different cereal species were shown to mediate the synthesis of (1,3;1,4)-ß-glucans with very different fine structures. Chimeric cDNA constructs with interchanged sections of the barley and sorghum CslF6 genes were developed to identify regions of the synthase enzyme responsible for these differences. A single amino acid residue upstream of the TED motif in the catalytic region was shown to dramatically change the fine structure of the polysaccharide produced. The structural basis of this effect can be rationalized by reference to a homology model of the enzyme and appears to be related to the position and flexibility of the TED motif in the active site of the enzyme. The region and amino acid residue identified provide opportunities to manipulate the solubility of (1,3;1,4)-ß-glucan in grains and vegetative tissues of the grasses and, in particular, to enhance the solubility of dietary fibers that are beneficial to human health.

Genetics and physiology of cell wall polysaccharides in the model C4 grass, Setaria viridis spp.

BACKGROUND: Setaria viridis has emerged as a model species for the larger C4 grasses. Here the cellulose synthase (CesA) superfamily has been defined, with an emphasis on the amounts and distribution of (1,3;1,4)-ß-glucan, a cell wall polysaccharide that is characteristic of the grasses and is of considerable value for human health. METHODS: Orthologous relationship of the CesA and Poales-specific cellulose synthase-like (Csl) genes among Setaria italica (Si), Sorghum bicolor (Sb), Oryza sativa (Os), Brachypodium distachyon (Bradi) and Hordeum vulgare (Hv) were compared using bioinformatics analysis. Transcription profiling of Csl gene families, which are involved in (1,3;1,4)-ß-glucan synthesis, was performed using real-time quantitative PCR (Q-PCR). The amount of (1,3;1,4)-ß-glucan was measured using a modified Megazyme assay. The fine structures of the (1,3;1,4)-ß-glucan, as denoted by the ratio of cellotriosyl to cellotetraosyl residues (DP3:DP4 ratio) was assessed by chromatography (HPLC and HPAEC-PAD). The distribution and deposition of the MLG was examined using the specific antibody BG-1 and captured using fluorescence and transmission electron microscopy (TEM). RESULTS: The cellulose synthase gene superfamily contains 13 CesA and 35 Csl genes in Setaria. Transcript profiling of CslF, CslH and CslJ gene families across a vegetative tissue series indicated that SvCslF6 transcripts were the most abundant relative to all other Csl transcripts. The amounts of (1,3;1,4)-ß-glucan in Setaria vegetative tissues ranged from 0.2% to 2.9% w/w with much smaller amounts in developing grain (0.003% to 0.013% w/w). In general, the amount of (1,3;1,4)-ß-glucan was greater in younger than in older tissues. The DP3:DP4 ratios varied between tissue types and across developmental stages, and ranged from 2.4 to 3.0:1. The DP3:DP4 ratios in developing grain ranged from 2.5 to 2.8:1. Micrographs revealing the distribution of (1,3;1,4)-ß-glucan in walls of different cell types and the data were consistent with the quantitative (1,3;1,4)-ß-glucan assays. CONCLUSION: The characteristics of the cellulose synthase gene superfamily and the accumulation and distribution of (1,3;1,4)-ß-glucans in Setaria are similar to those in other C4 grasses, including sorghum. This suggests that Setaria is a suitable model plant for cell wall polysaccharide biology in C4 grasses.

Evolutionary Dynamics of the Cellulose Synthase Gene Superfamily in Grasses.

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)-ß-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)-ß-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.