CslF6 Gene Research Articles

Is the CslF6 gene involved in the accumulation of (1,3;1,4)-β-D-glucan in wheats, their wild relatives and their hybrids?

Mixed linkage (1,3;1,4)-β-d-glucan (MLG) is a well-recognized bioactive carbohydrate and dietary fibre with expanding applications in food industry. The MLG are small components of the cell wall of vegetative tissues of cereals synthetized by members of the Cellulose Synthase-Like genes (Csl). Within the family, the CslF6 has been the major contributor in wheat. It is of significant health and economic benefits to enhance MLG content in wheat, a staple grain with naturally low MLG levels. This study investigated the role of CslF6 gene in MLG synthesis and analysed total MLG contents, cell wall monosaccharide, glycosidic linkage composition, and profile of major comprising oligosaccharides of MLG in various wheat genotypes, their wild relatives (Aegilops caudata and Dasypyrum villosum), and hybrids between them. We observed a relationship between CslF6 gene expression and MLG accumulation across the different wheat lines. While Aegilops caudata and Dasypyrum villosum exhibited higher MLG content than other genotypes, hybrid breeding led to an increase in MLG content by 24.4% in durum wheat and 43.3% in T. aestivum. Variations in the ratios of major oligosaccharides released from MLG by lichenase treatment and in the compositions of cell wall monosaccharides and glycosidic linkages were also found. This study demonstrates that HPAEC-PAD and GC–MS-based glycomics are invaluable tools to assist breeders in selecting high MLG lines.

Exploring Aegilops caudata: A Comprehensive Study of the CslF6 Gene and β-Glucan.

In the quest for sustainable and nutritious food sources, exploration of ancient grains and wild relatives of cultivated cereals has gained attention. Aegilops caudata, a wild wheatgrass species, stands out as a promising genetic resource due to its potential for crop enhancement and intriguing nutritional properties. This manuscript investigates the CslF6 gene sequence and protein structure of Aegilops caudata, employing comparative analysis with other grass species to identify potential differences impacting β-glucan content. The study involves comprehensive isolation and characterization of the CslF6 gene in Ae. caudata, utilizing genomic sequence analysis, protein structure prediction, and comparative genomics. Comparisons with sequences from diverse monocots reveal evolutionary relationships, highlighting high identities with wheat genomes. Specific amino acid motifs in the CslF6 enzyme sequence, particularly those proximal to key catalytic motifs, exhibit variations among monocot species. These differences likely contribute to alterations in β-glucan composition, notably impacting the DP3:DP4 ratio, which is crucial for understanding and modulating the final β-glucan content. The study positions Ae. caudata uniquely within the evolutionary landscape of CslF6 among monocots, suggesting potential genetic divergence or unique functional adaptations within this species. Overall, this investigation enriches our understanding of β-glucan biosynthesis, shedding light on the role of specific amino acid residues in modulating enzymatic activity and polysaccharide composition.

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

SUMMARYThe 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)‐β‐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)‐β‐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.

Characterization and absolute quantification of the Cellulose synthase-like F6 homoeologs in oats

The Cellulose synthase-like F6 (CslF6) gene has been reported as a key factor for β-glucan synthesis in grasses. However, the action of this gene on oat β-glucan content is poorly understood. The objectives of this study were: (i) to quantify the absolute expression of CslF6 transcripts in oats under field conditions; and (ii) to characterize the CslF6 homoeologs in oat genotypes with different levels of β-glucan content. Developing kernels of five oat genotypes were collected under field conditions in two planting dates. The CslF6 absolute quantification was performed using droplet digital PCR considering global and homoeolog-specific expressions. The third exon of the CslF6 gene, which covers approximately 62.2% of the coding region, was sequenced in each homoeolog. The CslF6 expression was highly influenced by planting date, kernel developmental stage, and genotype. High CslF6 transcript number was not associated with high β-glucan content, indicating the action of a complex biosynthesis pathway controlling this trait in oats. Under stressful conditions, the CslF6 expression was more pronounced and associated with β-glucan content. The CslF6 motif has 1767 bases pair and is highly conserved between A and D homoeologs. Single nucleotide polymorphisms were not identified among the assessed genotypes, regardless of the homoeolog. The results presented in this study may contribute for a better understanding of the CslF6 influence on oat β-glucan content.

GENOME‐WIDE IDENTIFICATION AND ANALYSIS OF THE CslF GENE FAMILY IN BARLEY (Hordeum vulgare L.)

The Cellulose synthase-like (Csl) F family has been considered as one of the most crucial genes regulating β-glucan synthesis. It is a cereal cell wall component, holding advantages for human nutrition, but disadvantages in animal nutrition, malting and brewing industries. Based on a genome-wide search method, present study identified barley CslF gene family members by considering the importance of (1,3;1,4)-β-D-glucan and newly completed barley genome. A sum of Eighteen CslF genes were recognized in the barley genome. Then, phylogenetic analyses classified them into 3 groups, that shared conserved motif compositions. A new motif, D,D,WQxxD was also found, which was responsible for the cellulose synthase. Furthermore, using RNA-seq data, the HvCslFs expression profiles were systematically examined in different tissues and tissue-specific candidates were found. Lastly, interaction network analysis identified 11 CslF genes involved in the interaction network. All together, prsent task provides valuable evidence about the genomic organization and evolutionary relationship of the CslF gene family in barley, and facilitate the functional surveys of CslF genes in barley and beyond.

Expression analysis of cellulose synthase-like genes in durum wheat

Cellulose synthase-like CslF and CslH genes have been implicated in the biosynthesis of β-glucans, a major cell wall constituents in grasses and cereals. The low β-glucan content of durum wheat and lack of information of the biosynthesis pathway make the expression analysis in different developmental stages of grain endosperm an interesting tool for the crop genetic improvement. Specific genome sequences of wheat CslF6 and CslH were isolated and the genomic sequence and structure were analysed in the cv. Svevo. In starchy endosperm at five developmental stages (6, 12, 21, 28 and 40 days after pollination) CslF6 and CslH transcripts were differentially expressed. A peak of CslF6 transcription occurred at 21 dap, while CslH was abundant at 28 dap. Significant variations were detected for both the genes in the genotypes. Significant and positive correlation were detected between β-glucan content and CslF6 gene expression at 21 dap and 40 dap, while no significant correlation was observed for CslH gene. On the overall, our correlation analysis reflected data from previous studies on other species highlighting how the abundance of transcripts encoding for CslF6 and CslH enzymes were not necessarily a good indicator of enzyme activity and/or β-glucan deposition in cell wall.

A Trihelix Family Transcription Factor Is Associated with Key Genes in Mixed-Linkage Glucan Accumulation.

Mixed-linkage glucan (MLG) is a polysaccharide that is highly abundant in grass endosperm cell walls and present at lower amounts in other tissues. Cellulose synthase-like F (CSLF) and cellulose synthase-like H genes synthesize MLG, but it is unknown if other genes participate in the production and restructuring of MLG. Using Brachypodium distachyon transcriptional profiling data, we identified a B distachyon trihelix family transcription factor (BdTHX1) that is highly coexpressed with the B distachyon CSLF6 gene (BdCSLF6), which suggests that BdTHX1 is involved in the regulation of MLG biosynthesis. To determine the genes regulated by this transcription factor, we conducted chromatin immunoprecipitation sequencing (ChIP-seq) experiments using immature B distachyon seeds and an anti-BdTHX1 polyclonal antibody. The ChIP-seq experiment identified the second intron of BdCSLF6 as one of the most enriched sequences. The binding of BdTHX1 to the BdCSLF6 intron sequence was confirmed using electrophoretic mobility shift assays (EMSA). ChIP-seq also showed that a gene encoding a grass-specific glycoside hydrolase family 16 endotransglucosylase/hydrolase (BdXTH8) is bound by BdTHX1, and the binding was confirmed by EMSA. Radiochemical transglucanase assays showed that BdXTH8 exhibits predominantly MLG:xyloglucan endotransglucosylase activity, a hetero-transglycosylation reaction, and can thus produce MLG-xyloglucan covalent bonds; it also has a lower xyloglucan:xyloglucan endotransglucosylase activity. B distachyon shoots regenerated from transformed calli overexpressing BdTHX1 showed an abnormal arrangement of vascular tissue and seedling-lethal phenotypes. These results indicate that the transcription factor BdTHX1 likely plays an important role in MLG biosynthesis and restructuring by regulating the expression of BdCSLF6 and BdXTH8.

Targeted Switchgrass BAC Library Screening and Sequence Analysis Identifies Predicted Biomass and Stress Response-Related Genes

To identify switchgrass homologs of rice genes, known/predicted to control biomass and stress response-related traits, we screened 96,000 clones from two switchgrass bacterial artificial chromosome (BAC) libraries. Full-length sequencing of 311 BAC clones revealed sequence for ∼3.2 % (51.7 Mb) of the switchgrass genome, coding for 3948 genes. A comparison with Arabidopsis and five grass genomes revealed that switchgrass genes share the highest number of homologs with rice (95.5 %) followed by foxtail millet (91.7 %) and Sorghum (91.5 %). One hundred eighteen of the annotated genes are unique to switchgrass. Gene annotation and ontology analysis revealed 695 genes belonging to gene families targeted in the screening. These include 350 kinase, 203 glycosyltransferase (GT), 109 glycoside hydrolase (GH), and 33 ethylene responsive transcription factor (ERF) family genes. Rice homologs of 65 genes, identified here, have demonstrated roles in bioenergy-relevant traits. These include 14 GT2 family genes involved in the synthesis of cellulose and hemicelluloses. Comparative expression analysis in six switchgrass organs revealed a conserved expression pattern for three cellulose synthase (CesA1, CesA2, and CesA9) and five cellulose-synthase-like genes (CslA2, CslA11, CslC1, CslD4, and CslE6). CslF genes that encode mixed linkage glucans are expressed in wider range of tissues in switchgrass compared with rice.

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)-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.

A mutation of the cellulose-synthase-like (CslF6) gene in barley (Hordeum vulgare L.) partially affects the β-glucan content in grains

The chemical induced barley mutant m351 was first selected for its low level of mixed-linkage (1–3,1–4) beta-D-glucan (MLG) in an experimental effort to search for barley lines with varied grain MLG contents. The MLG decrease in m351 was associated with increased levels of fructans and crude fiber, but maintained the same plant characteristics under field conditions. The mutation was mapped to a genetic locus flanked by two SSR markers, Bmag369 and Bmag564, on chromosome 7H. Molecular cloning of the CslF6 gene from the m351 line revealed the presence of a point mutation, causing a substitution of an alanine for threonine at position 849 in the amino acid sequence of the corresponding protein. The resultant protein retains some functionality and affects other components in the m351 grain. Those metabolic changes associated with MLG reduction in m351 is the first case reported of a partially functional CslF6 gene in cereal grains. The results contribute to better understanding of the functional effects of the CslF6 gene and the mutant has potential implications in grain end-use quality improvement.

Genetic markers for CslF6 gene associated with (1,3;1,4)-β-glucan concentration in barley grain

The amount of (1,3;1,4)-β-D glucan accumulated in barley (Hordeum vulgare L.) cell walls is an important consideration for grain end-use. One of the major genes responsible for (1,3;1,4)-β-glucan biosynthesis is HvCslF6, which was analyzed in this study to determine the allelic variation between low (1,3;1,4)-β-glucan (∼3.3%) cultivar CDC Bold and high β-glucan (∼5.2%) line TR251. The CDC Bold HvCslF6 allele showed 16 single nucleotide polymorphisms (SNPs) and two indels when genomic region downstream of the ATG start codon was compared to TR251 allele. Both indels added 16 nucleotides to HvCslF6 first intron of CDC Bold and a single SNP in the third exon altered alanine 590 codon in the CDC Bold sequence to a threonine codon in TR251 allele. Genetic markers were developed for five polymorphic sites and confirmed useful to select low and high β-glucan lines in a previously characterized CDC Bold/TR251 mapping population and a novel F5 recombinant inbred line (RIL) population derived from a Merit/H93174006 (4.8 and 5.3% (1,3;1,4)-β-glucan) cross. An analysis of parental lines of six populations segregating for β-glucan concentration validated association between the TR251 HvCslF6 haplotype and high (1,3;1,4)-β-glucan concentration in populations showing a (1,3;1,4)-β-glucan quantitative trait locus (QTL) on chromosome 7H.

Loss of Cellulose synthase-like F6 function affects mixed-linkage glucan deposition, cell wall mechanical properties, and defense responses in vegetative tissues of rice.

Mixed-linkage glucan (MLG) is a cell wall polysaccharide containing a backbone of unbranched (1,3)- and (1,4)-linked β-glucosyl residues. Based on its occurrence in plants and chemical characteristics, MLG has primarily been associated with the regulation of cell wall expansion due to its high and transient accumulation in young, expanding tissues. The Cellulose synthase-like F (CslF) subfamily of glycosyltransferases has previously been implicated in mediating the biosynthesis of this polymer. We confirmed that the rice (Oryza sativa) CslF6 gene mediates the biosynthesis of MLG by overexpressing it in Nicotiana benthamiana. Rice cslf6 knockout mutants show a slight decrease in height and stem diameter but otherwise grew normally during vegetative development. However, cslf6 mutants display a drastic decrease in MLG content (97% reduction in coleoptiles and virtually undetectable in other tissues). Immunodetection with an anti-MLG monoclonal antibody revealed that the coleoptiles and leaves retain trace amounts of MLG only in specific cell types such as sclerenchyma fibers. These results correlate with the absence of endogenous MLG synthase activity in mutant seedlings and 4-week-old sheaths. Mutant cell walls are weaker in mature stems but not seedlings, and more brittle in both stems and seedlings, compared to wild type. Mutants also display lesion mimic phenotypes in leaves, which correlates with enhanced defense-related gene expression and enhanced disease resistance. Taken together, our results underline a weaker role of MLG in cell expansion than previously thought, and highlight a structural role for MLG in nonexpanding, mature stem tissues in rice.

Over‐expression of specific HvCslF cellulose synthase‐like genes in transgenic barley increases the levels of cell wall (1,3;1,4)‐β‐d‐glucans and alters their fine structure

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.

Gene silencing of barley P23k involved in secondary wall formation causes abnormal tiller formation and intercalary elongation

P23k is a monocot-unique protein that is highly expressed in barley. Our previous loss-of-function studies in barley leaves indicated that P23k, localized to tissues where cell wall polysaccharides accumulate, might contribute to secondary wall formation in the leaf. However, the P23k loss-of-function analysis was limited to the leaf, which is a vegetative organ. Considering the involvement of P23k in secondary wall formation, a dramatically altered phenotype is expected in the stem of P23k gene-silenced barley, where marked secondary wall deposition occurs during the reproductive growth stage. To test this hypothesis, barley striped mosaic virus-based virus-induced gene silencing of P23k was performed. Abnormal tiller formation and arrested intercalary elongation were observed in P23k-silenced barley. From these results, we speculated that cell wall architecture was altered by P23k gene silencing. Consistent with this idea, we observed a marked decrease in the amount of cell wall polysaccharides stained with calcofluor and down-regulation of the cellulose synthase-like CslF6 gene involved in (1,3;1,4)-β-D-glucan synthesis. Taken together, these results suggest that P23k is possibly involved in determining secondary wall architecture and contributes to tiller formation and intercalary elongation in barley.

β-1,3 : 1,4-Glucan Synthase Activity in Rice Seedlings under Water

The metabolism of beta-1,3 : 1,4-glucan regulates the mechanical properties of cell walls, and thereby changes the elongation growth of Poaceae plants. A previous study has shown that elongation growth of rice coleoptiles under water is enhanced by increased activity of beta-1,3 : 1,4-glucan hydrolases; however, the involvement of beta-1,3 : 1,4-glucan synthase activity in elongation growth under water has not yet been clarified. The beta-1,3 : 1,4-glucan synthase activity in a microsomal fraction prepared from rice seedlings grown under water was compared with that from control seedlings grown in air. The change under water in the relative expression level of CslF6, a major isoform of the beta-1,3 : 1,4-glucan synthase genes, was examined by quantitative reverse-transcriptase PCR. The level of beta-1,3 : 1,4-glucan synthase activity in submerged seedlings decreased to less than 40 % of that of the control seedlings and was accompanied by a significant reduction in the amount of beta-1,3 : 1,4-glucan in the cell walls. Under water, the expression of CslF6 was reduced to less than 20 % of the unsubmerged control. Bubble aeration partially restored both beta-1,3 : 1,4-glucan synthase activity and the expression of CslF6 under water, correlating with suppression of the submergence-induced elongation growth of coleoptiles. Submergence down-regulates the expression of the CslF6 gene, leading to a decreased level of beta-1,3 : 1,4-glucan synthase activity. Together with the increased activity of beta-1,3 : 1,4-glucan hydrolases, the decreased activity of beta-1,3 : 1,4-glucan synthase contributes to the decrease in the amount of beta-1,3 : 1,4-glucan in the cell walls under water. The suppression of beta-1,3 : 1,4-glucan synthesis under water may be mainly due to oxygen depletion.

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)-beta-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)-beta-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)-beta-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)-beta-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)-beta-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)-beta-d-glucan levels.