The cellulose synthase-like F3 (CslF3) gene mediates cell wall polysaccharide synthesis and affects root growth and differentiation in barley.

The barley cellulose synthase-like F (CslF) genes encode putative cell wall polysaccharide synthases. They are related to the cellulose synthase (CesA) genes involved in cellulose biosynthesis, and the CslD genes that influence root hair development. Although CslD genes are implicated in callose, mannan and cellulose biosynthesis, and are found in both monocots and eudicots, CslF genes are specific to the Poaceae. Recently the barley CslF3 (HvCslF3) gene was shown to be involved in the synthesis of a novel (1,4)-ß-linked glucoxylan, but it remains unclear whether this gene contributes to plant growth and development. Here, expression profiling using qRT-PCR and mRNA in situ hybridization revealed that HvCslF3 accumulates in the root elongation zone. Silencing HvCslF3 by RNAi was accompanied by slower root growth, linked with a shorter elongation zone and a significant reduction in root system size. Polymer profiling of the RNAi lines revealed a significant reduction in (1,4)-ß-linked glucoxylan levels. Remarkably, the heterologous expression of HvCslF3 in wild-type (Col-0) and root hair-deficient Arabidopsis mutants (csld3 and csld5) complemented the csld5 mutant phenotype, in addition to altering epidermal cell fate. Our results reveal a key role for HvCslF3 during barley root development and suggest that members of the CslD and CslF gene families have similar functions during root growth regulation.

Ovule cell wall composition is a maternal determinant of grain size in barley.

In cereal species, grain size is influenced by growth of the ovule integuments (seed coat), the spikelet hull (lemma and palea) and the filial endosperm. Whether a highly conserved ovule tissue, the nucellus, has any impact on grain size has remained unclear. Immunolabelling revealed that the barley nucellus comprises two distinct cell types that differ in terms of cell wall homogalacturonan (HG) accumulation. Transcriptional profiling of the nucellus identified two pectin methylesterase (PME) genes, OVULE PECTIN MODIFIER 1 (OPM1) and OPM2, which are expressed in the unfertilized ovule but absent from the seed. Ovules from an opm1 opm2 mutant and plants expressing an ovule-specific pectin methylesterase inhibitor (PMEI), exhibit reduced HG accumulation. This results in changes to ovule cell size and shape and ovules that are longer than wild-type (WT) controls. At grain maturity, this is manifested as significantly longer grain. These findings indicate that cell wall composition during ovule development acts to limit ovule and seed growth. The investigation of ovule PME and PMEI activity reveals an unexpected role of maternal tissues in controlling grain growth before fertilization, one that has been lacking from models exploring improvements in grain size.

HvSL1 and HvMADS16 promote stamen identity to restrict multiple ovary formation in barley.

Correct floral development is the result of a sophisticated balance of molecular cues. Floral mutants provide insight into the main genetic determinants that integrate these cues, as well as providing opportunities to assess functional variation across species. In this study, we characterize the barley (Hordeum vulgare) multiovary mutants mov2.g and mov1, and propose causative gene sequences: a C2H2 zinc-finger gene HvSL1 and a B-class gene HvMADS16, respectively. In the absence of HvSL1, florets lack stamens but exhibit functional supernumerary carpels, resulting in multiple grains per floret. Deletion of HvMADS16 in mov1 causes homeotic conversion of lodicules and stamens into bract-like organs and carpels that contain non-functional ovules. Based on developmental, genetic, and molecular data, we propose a model by which stamen specification in barley is defined by HvSL1 acting upstream of HvMADS16. The present work identifies strong conservation of stamen formation pathways with other cereals, but also reveals intriguing species-specific differences. The findings lay the foundation for a better understanding of floral architecture in Triticeae, a key target for crop improvement.

HvLEAFY controls the early stages of floral organ specification and inhibits the formation of multiple ovaries in barley.

Transition to the reproductive phase, inflorescence formation and flower development are crucial elements that ensure maximum reproductive success in a plant's life cycle. To understand the regulatory mechanisms underlying correct flower development in barley (Hordeum vulgare), we characterized the multiovary 5 (mov5.o) mutant. This mutant develops abnormal flowers that exhibit mosaic floral organs typified by multiple carpels at the total or partial expense of stamens. Genetic mapping positioned mov5 on the long arm of chromosome 2H, incorporating a region that encodes HvLFY, the barley orthologue of LEAFY from Arabidopsis. Sequencing revealed that, in mov5.o plants, HvLFY contains a single amino acid substitution in a highly conserved proline residue. CRISPR-mediated knockout of HvLFY replicated the mov5.o phenotype, suggesting that HvLFYmov5 represents a loss of function allele. In heterologous assays, the HvLFYmov5 polymorphism influenced protein-protein interactions and affinity for a putative binding site in the promoter of HvMADS58, a C-class MADS-box gene. Moreover, molecular analysis indicated that HvLFY interacts with HvUFO and regulates the expression of floral homeotic genes including HvMADS2, HvMADS4 and HvMADS16. Other distinct changes in expression differ from those reported in the rice LFY mutants apo2/rfl, suggesting that LFY function in the grasses is modulated in a species-specific manner. This pathway provides a key entry point for the study of LFY function and multiple ovary formation in barley, as well as cereal species in general.

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.

Auxin Treatment Enhances Anthocyanin Production in the Non-Climacteric Sweet Cherry (Prunus avium L.).

Abscisic acid (ABA) is a key signaling molecule promoting ripening of non-climacteric fruits such as sweet cherry (Prunus avium L.). To shed light on the role of other hormones on fruit development, ripening and anthocyanin production, the synthetic auxin 1-naphthaleneacetic acid (NAA) was applied to sweet cherry trees during the straw-color stage of fruit development. NAA-treated fruits exhibited higher concentrations of 1-aminocyclopropane-1-carboxylic acid (ACC) and ABA-glucose ester (ABA-GE), which are a precursor of ethylene and a primary storage form of ABA, respectively. Consistent with these observations, transcript levels of genes encoding ACC synthase and ACC oxidase, both involved in ethylene biosynthesis, were increased after 6 days of NAA treatment, and both ABA concentration and expression of the regulator gene of ABA biosynthesis (NCED1 encoding 9-cis-epoxycarotenoid dioxygenase) were highest during early fruit ripening. In addition, transcript levels of key anthocyanin regulatory, biosynthetic and transport genes were significantly upregulated upon fruit exposure to NAA. This was accompanied by an increased anthocyanin concentration and fruit weight whilst fruit firmness and cracking index decreased. Altogether our data suggest that NAA treatment alters ethylene production, which in turn induces ripening in sweet cherry and enhanced anthocyanin production, possibly through ABA metabolism. The results from our study highlight the potential to use a single NAA treatment for manipulation of cherry ripening.

Overexpression of HvCslF6 in barley grain alters carbohydrate partitioning plus transfer tissue and endosperm development.

In cereal grain, sucrose is converted into storage carbohydrates: mainly starch, fructan, and mixed-linkage (1,3;1,4)-ß-glucan (MLG). Previously, endosperm-specific overexpression of the HvCslF6 gene in hull-less barley was shown to result in high MLG and low starch content in mature grains. Morphological changes included inwardly elongated aleurone cells, irregular cell shapes of peripheral endosperm, and smaller starch granules of starchy endosperm. Here we explored the physiological basis for these defects by investigating how changes in carbohydrate composition of developing grain impact mature grain morphology. Augmented MLG coincided with increased levels of soluble carbohydrates in the cavity and endosperm at the storage phase. Transcript levels of genes relating to cell wall, starch, sucrose, and fructan metabolism were perturbed in all tissues. The cell walls of endosperm transfer cells (ETCs) in transgenic grain were thinner and showed reduced mannan labelling relative to the wild type. At the early storage phase, ruptures of the non-uniformly developed ETCs and disorganization of adjacent endosperm cells were observed. Soluble sugars accumulated in the developing grain cavity, suggesting a disturbance of carbohydrate flow from the cavity towards the endosperm, resulting in a shrunken mature grain phenotype. Our findings demonstrate the importance of regulating carbohydrate partitioning in maintenance of grain cellularization and filling processes.

Wheat wounding-responsive HD-Zip IV transcription factor GL7 is predominantly expressed in grain and activates genes encoding defensins.

KEY MESSAGE: Several classes of transcription factors are involved in the activation of defensins. A new type of the transcription factor responsible for the regulation of wheat grain specific defensins was characterised in this work. HD-Zip class IV transcription factors constitute a family of multidomain proteins. A full-length cDNA of HD-Zip IV, designated TaGL7 was isolated from the developing grain of bread wheat, using a specific DNA sequence as bait in the Y1H screen. 3D models of TaGL7 HD complexed with DNA cis-elements rationalised differences that underlined accommodations of binding and non-binding DNA, while the START-like domain model predicted binding of lipidic molecules inside a concave hydrophobic cavity. The 3'-untranslated region of TaGL7 was used as a probe to isolate the genomic clone of TdGL7 from a BAC library prepared from durum wheat. The spatial and temporal activity of the TdGL7 promoter was tested in transgenic wheat, barley and rice. TdGL7 was expressed mostly in ovary at fertilisation and its promoter was active in a liquid endosperm during cellularisation and later in the endosperm transfer cells, aleurone, and starchy endosperm. The pattern of TdGL7 expression resembled that of genes that encode grain-specific lipid transfer proteins, particularly defensins. In addition, GL7 expression was upregulated by mechanical wounding, similarly to defensin genes. Co-bombardment of cultured wheat cells with TdGL7 driven by constitutive promoter and seven grain or root specific defensin promoters fused to GUS gene, revealed activation of four promoters. The data confirmed the previously proposed role of HD-Zip IV transcription factors in the regulation of genes that encode lipid transfer proteins involved in lipid transport and defence. The TdGL7 promoter could be used to engineer cereal grains with enhanced resistance to insects and fungal infections.

Correction to: Wheat wounding-responsive HD-Zip IV transcription factor GL7 is predominantly expressed in grain and activates genes encoding defensins.

Due to an unfortunate turn of events, the panels O to S are missing in Fig. 8 of the original publication. The correct Fig. 8 and its caption is published here and should be treated as definitive.

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.

Asexual Female Gametogenesis Involves Contact with a Sexually-Fated Megaspore in Apomictic Hieracium.

Apomixis results in asexual seed formation where progeny are identical to the maternal plant. In ovules of apomictic species of the Hieracium subgenus Pilosella, meiosis of the megaspore mother cell generates four megaspores. Aposporous initial (AI) cells form during meiosis in most ovules. The sexual pathway terminates during functional megaspore (FM) differentiation, when an enlarged AI undergoes mitosis to form an aposporous female gametophyte. Then, the mitotically programmed FM dies along with the three other megaspores by unknown mechanisms. Transcriptomes of laser-dissected AIs, ovule cells, and ovaries from apomicts and AI-deficient mutants were analyzed to understand the pathways involved. The steps leading to AI mitosis and sexual pathway termination were determined using antibodies against arabinogalactan protein epitopes found to mark both sexual and aposporous female gametophyte lineages at inception. At most, four AIs differentiated near developing megaspores. The first expanding AI cell to contact the FM formed a functional AI that underwent mitosis soon after megaspore degeneration. Transcriptome analyses indicated that the enlarged, laser-captured AIs were arrested in the S/G2 phase of the cell cycle and were metabolically active. Further comparisons with AI-deficient mutants showed that AIs were enriched in transcripts encoding homologs of genes involved in, and potentially antagonistic to, known FM specification pathways. We propose that AI and FM cell contact provides cues required for AI mitosis and megaspore degeneration. Specific candidates to further interrogate AI-FM interactions were identified here and include Hieracium arabinogalactan protein family genes.

Translating auxin responses into ovules, seeds and yield: Insight from Arabidopsis and the cereals.

Grain production in cereal crops depends on the stable formation of male and female gametes in the flower. In most angiosperms, the female gamete is produced from a germline located deep within the ovary, protected by several layers of maternal tissue, including the ovary wall, ovule integuments and nucellus. In the field, germline formation and floret fertility are major determinants of yield potential, contributing to traits such as seed number, weight and size. As such, stimuli affecting the timing and duration of reproductive phases, as well as the viability, size and number of cells within reproductive organs can significantly impact yield. One key stimulant is the phytohormone auxin, which influences growth and morphogenesis of female tissues during gynoecium development, gametophyte formation, and endosperm cellularization. In this review we consider the role of the auxin signaling pathway during ovule and seed development, first in the context of Arabidopsis and then in the cereals. We summarize the gene families involved and highlight distinct expression patterns that suggest a range of roles in reproductive cell specification and fate. This is discussed in terms of seed production and how targeted modification of different tissues might facilitate improvements.

Transcriptomics technologies.

Transcriptomics technologies are the techniques used to study an organism's transcriptome, the sum of all of its RNA transcripts. The information content of an organism is recorded in the DNA of its genome and expressed through transcription. Here, mRNA serves as a transient intermediary molecule in the information network, whilst noncoding RNAs perform additional diverse functions. A transcriptome captures a snapshot in time of the total transcripts present in a cell. The first attempts to study the whole transcriptome began in the early 1990s, and technological advances since the late 1990s have made transcriptomics a widespread discipline. Transcriptomics has been defined by repeated technological innovations that transform the field. There are two key contemporary techniques in the field: microarrays, which quantify a set of predetermined sequences, and RNA sequencing (RNA-Seq), which uses high-throughput sequencing to capture all sequences. Measuring the expression of an organism's genes in different tissues, conditions, or time points gives information on how genes are regulated and reveals details of an organism's biology. It can also help to infer the functions of previously unannotated genes. Transcriptomic analysis has enabled the study of how gene expression changes in different organisms and has been instrumental in the understanding of human disease. An analysis of gene expression in its entirety allows detection of broad coordinated trends which cannot be discerned by more targeted assays.

The Dynamics of Transcript Abundance during Cellularization of Developing Barley Endosperm.

Within the cereal grain, the endosperm and its nutrient reserves are critical for successful germination and in the context of grain utilization. The identification of molecular determinants of early endosperm development, particularly regulators of cell division and cell wall deposition, would help predict end-use properties such as yield, quality, and nutritional value. Custom microarray data have been generated using RNA isolated from developing barley grain endosperm 3 d to 8 d after pollination (DAP). Comparisons of transcript abundance over time revealed 47 gene expression modules that can be clustered into 10 broad groups. Superimposing these modules upon cytological data allowed patterns of transcript abundance to be linked with key stages of early grain development. Here, attention was focused on how the datasets could be mined to explore and define the processes of cell wall biosynthesis, remodeling, and degradation. Using a combination of spatial molecular network and gene ontology enrichment analyses, it is shown that genes involved in cell wall metabolism are found in multiple modules, but cluster into two main groups that exhibit peak expression at 3 DAP to 4 DAP and 5 DAP to 8 DAP. The presence of transcription factor genes in these modules allowed candidate genes for the control of wall metabolism during early barley grain development to be identified. The data are publicly available through a dedicated web interface (https://ics.hutton.ac.uk/barseed/), where they can be used to interrogate co- and differential expression for any other genes, groups of genes, or transcription factors expressed during early endosperm development.

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.

Down-regulation of the glucan synthase-like 6 gene (HvGsl6) in barley leads to decreased callose accumulation and increased cell wall penetration by Blumeria graminis f. sp. hordei.

The recent characterization of the polysaccharide composition of papillae deposited at the barley cell wall during infection by the powdery mildew pathogen, Blumeria graminis f. sp. hordei (Bgh), has provided new targets for the generation of enhanced disease resistance. The role of callose in papilla-based penetration resistance of crop species is largely unknown because the genes involved in the observed callose accumulation have not been identified unequivocally. We have employed both comparative and functional genomics approaches to identify the functional orthologue of AtGsl5 in the barley genome. HvGsl6 (the barley glucan synthase-like 6 gene), which has the highest sequence identity to AtGsl5, is the only Bgh-induced gene among the HvGsls examined in this study. Through double-stranded RNA interference (dsRNAi)-mediated silencing of HvGsl6, we have shown that the down-regulation of HvGsl6 is associated with a lower accumulation of papillary and wound callose and a higher susceptibility to penetration of the papillae by Bgh, compared with control lines. The results indicate that the HvGsl6 gene is a functional orthologue of AtGsl5 and is involved in papillary callose accumulation in barley. The increased susceptibility of HvGsl6 dsRNAi transgenic lines to infection indicates that callose positively contributes to the barley fungal penetration resistance mechanism.

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.

Differences in glycosyltransferase family 61 accompany variation in seed coat mucilage composition in Plantago spp.

Xylans are the most abundant non-cellulosic polysaccharide found in plant cell walls. A diverse range of xylan structures influence tissue function during growth and development. Despite the abundance of xylans in nature, details of the genes and biochemical pathways controlling their biosynthesis are lacking. In this study we have utilized natural variation within the Plantago genus to examine variation in heteroxylan composition and structure in seed coat mucilage. Compositional assays were combined with analysis of the glycosyltransferase family 61 (GT61) family during seed coat development, with the aim of identifying GT61 sequences participating in xylan backbone substitution. The results reveal natural variation in heteroxylan content and structure, particularly in P. ovata and P. cunninghamii, species which show a similar amount of heteroxylan but different backbone substitution profiles. Analysis of the GT61 family identified specific sequences co-expressed with IRREGULAR XYLEM 10 genes, which encode putative xylan synthases, revealing a close temporal association between xylan synthesis and substitution. Moreover, in P. ovata, several abundant GT61 sequences appear to lack orthologues in P. cunninghamii. Our results indicate that natural variation in Plantago species can be exploited to reveal novel details of seed coat development and polysaccharide biosynthetic pathways.

Powerful regulatory systems and post-transcriptional gene silencing resist increases in cellulose content in cell walls of barley.

BACKGROUND: The ability to increase cellulose content and improve the stem strength of cereals could have beneficial applications in stem lodging and producing crops with higher cellulose content for biofuel feedstocks. Here, such potential is explored in the commercially important crop barley through the manipulation of cellulose synthase genes (CesA). RESULTS: Barley plants transformed with primary cell wall (PCW) and secondary cell wall (SCW) barley cellulose synthase (HvCesA) cDNAs driven by the CaMV 35S promoter, were analysed for growth and morphology, transcript levels, cellulose content, stem strength, tissue morphology and crystalline cellulose distribution. Transcript levels of the PCW HvCesA transgenes were much lower than expected and silencing of both the endogenous CesA genes and introduced transgenes was often observed. These plants showed no aberrant phenotypes. Although attempts to over-express the SCW HvCesA genes also resulted in silencing of the transgenes and endogenous SCW HvCesA genes, aberrant phenotypes were sometimes observed. These included brittle nodes and, with the 35S:HvCesA4 construct, a more severe dwarfing phenotype, where xylem cells were irregular in shape and partially collapsed. Reductions in cellulose content were also observed in the dwarf plants and transmission electron microscopy showed a significant decrease in cell wall thickness. However, there were no increases in overall crystalline cellulose content or stem strength in the CesA over-expression transgenic plants, despite the use of a powerful constitutive promoter. CONCLUSIONS: The results indicate that the cellulose biosynthetic pathway is tightly regulated, that individual CesA proteins may play different roles in the synthase complex, and that the sensitivity to CesA gene manipulation observed here suggests that in planta engineering of cellulose levels is likely to require more sophisticated strategies.

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.