Functional characterization of barley betaglucanless mutants demonstrates a unique role for CslF6 in (1,3;1,4)-β-D-glucan biosynthesis

(1,3;1,4)-β-D-glucans (mixed-linkage glucans) are found in tissues of members of the Poaceae (grasses), and are particularly high in barley (Hordeum vulgare) grains. The present study describes the isolation of three independent (1,3;1,4)-β-D-glucanless (betaglucanless; bgl) mutants of barley which completely lack (1,3;1,4)-β-D-glucan in all the tissues tested. The bgl phenotype cosegregates with the cellulose synthase like HvCslF6 gene on chromosome arm 7HL. Each of the bgl mutants has a single nucleotide substitution in the coding region of the HvCslF6 gene resulting in a change of a highly conserved amino acid residue of the HvCslF6 protein. Microsomal membranes isolated from developing endosperm of the bgl mutants lack detectable (1,3;1,4)-β-D-glucan synthase activity indicating that the HvCslF6 protein is inactive. This was confirmed by transient expression of the HvCslF6 cDNAs in Nicotiana benthamiana leaves. The wild-type HvCslF6 gene directed the synthesis of high levels of (1,3;1,4)-β-D-glucans, whereas the mutant HvCslF6 proteins completely lack the ability to synthesize (1,3;1,4)-β-D-glucans. The fine structure of the (1,3;1,4)-β-D-glucan produced in the tobacco leaf was also very different from that found in cereals having an extremely low DP3/DP4 ratio. These results demonstrate that, among the seven CslF and one CslH genes present in the barley genome, HvCslF6 has a unique role and is the key determinant controlling the biosynthesis of (1,3;1,4)-β-D-glucans. Natural allelic variation in the HvCslF6 gene was found predominantly within introns among 29 barley accessions studied. Genetic manipulation of the HvCslF6 gene could enable control of (1,3;1,4)-β-D-glucans in accordance with the purposes of use.     

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.     

Method for hull‐less barley transformation and manipulation of grain mixed‐linkage beta‐glucan

Hull‐less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull‐less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull‐less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over‐express genes encoding synthases for the important dietary fiber component, (1,3;1,4)‐β‐glucan (mixed‐linkage glucan), primarily present in starchy endosperm cell walls. Over‐expression of the HvCslF6 gene, driven by an endosperm‐specific promoter, produced lines where mixed‐linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub‐aleurone cells. This work provides proof‐of‐concept evidence that mixed‐linkage glucan content in hull‐less barley grain can be increased by over‐expression of the HvCslF6 gene, but also indicates that hull‐less cultivars may be more sensitive to attempts to modify cell wall composition.     

Chromosomal loci associated with endosperm hardness in a malting barley cross

A breeding objective for the malting barley industry is to produce lines with softer, plumper grain containing moderate protein content (9–12%) as they are more likely to imbibe water readily and contain more starch per grain, which in turn produces higher levels of malt extract. In a malting barley mapping population, ‘Arapiles’ × ‘Franklin’, the most significant and robust quantitative trait locus (QTL) for endosperm hardness was observed on the short arm of chromosome 1H, across three environments over two growing seasons. This accounted for 22.6% (Horsham 2000), 26.8% (Esperance 2001), and 12.0% (Tarranyurk 2001) of the genetic variance and significantly increased endosperm hardness by 2.06–3.03 SKCS hardness units. Interestingly, Arapiles and Franklin do not vary in Ha locus alleles. Therefore, this region, near the centromere on chromosome 1H, may be of great importance when aiming to manipulate endosperm hardness and malting quality. Interestingly, this region, close to the centromere on chromosome 1H, in our study, aligns with the region of the genome that includes the HvCslF9 and the HvGlb1 genes. Potentially, one or both of these genes could be considered to be candidate genes that influence endosperm hardness in the barley grain. Additional QTLs for endosperm hardness were detected on chromosomes 2H, 3H, 6H and 7H, confirming that the hardness trait in barley is complex and multigenic, similar to many malting quality traits of interest.     

Characterization of a new 4BS.7HL wheat-barley translocation line using GISH, FISH, and SSR markers and its effect on the β-glucan content of wheat

A spontaneous interspecific Robertsonian translocation was revealed by genomic in situ hybridization (GISH) in the progenies of a monosomic 7H addition line originating from a new wheat 'Asakaze komugi' × barley 'Manas' hybrid. Fluorescence in situ hybridization (FISH) with repetitive DNA sequences (Afa family, pSc119.2, and pTa71) allowed identification of all wheat chromosomes, including wheat chromosome arm 4BS involved in the translocation. FISH using barley telomere- and centromere-specific repetitive DNA probes (HvT01 and (AGGGAG)(n)) confirmed that one of the arms of barley chromosome 7H was involved in the translocation. Simple sequence repeat (SSR) markers specific to the long (L) and short (S) arms of barley chromosome 7H identified the translocated chromosome segment as 7HL. Further analysis of the translocation chromosome clarified the physical position of genetically mapped SSRs within 7H, with a special focus on its centromeric region. The presence of the HvCslF6 gene, responsible for (1,3;1,4)-β-D-glucan production, was revealed in the centromeric region of 7HL. An increased (1,3;1,4)-β-D-glucan level was also detected in the translocation line, demonstrating that the HvCslF6 gene is of potential relevance for the manipulation of wheat (1,3;1,4)-β-D-glucan levels.     

Development of a complete set of wheat–barley group-7 Robertsonian translocation chromosomes conferring an increased content of β-glucan

Key message

A complete set of six compensating Robertsonian translocation chromosomes involving barley chromosome 7H and three chromosomes of hexaploid wheat was produced. Grain β-glucan content increased in lines containing 7HL.

Abstract

Many valuable genes for agronomic performance, disease resistance and increased yield have been transferred from relative species to wheat (Triticum aestivum L.) through whole-arm Robertsonian translocations (RobT). Although of a great value, the sets of available translocations from barley (Hordeum vulgare L.) are limited. Here, we present the production of a complete set of six compensating RobT chromosomes involving barley chromosome 7H and three group-7 chromosomes of wheat. The barley group-7 long-arm RobTs had a higher grain β-glucan content compared to the wheat control. The β-glucan levels varied depending on the temperature and were higher under hot conditions. Implicated in this increase, the barley cellulose synthase-like F6 gene (CslF6) responsible for β-glucan synthesis was physically mapped near the centromere in the long arm of barley chromosome 7H. Likewise, wheat CslF6 homoeologs were mapped near the centromere in the long arms of all group-7 wheat chromosomes. With the set of novel wheat–barley translocations, we demonstrate a valuable increase of β-glucan, along with a resource of genetic stocks that are likely to carry many other important genes from barley into wheat.

     

Gibberellin like Substances in Parts of Developing Barley Grain

In husks, the activity of gibberellin-like substances extracted with aqueous methanol (M-“free” GAs) showed a maximum on the 9th day after pollination. In developing embryos, M-“free” GAs showed no biological activity, whereas biological active component(s) were obtained when the embryos were extracted with Tris buffer. The “free” GAs found in the buffer homogenates (B-“free” GAs) of developing embryos showed a maximum of activity on the 33rd day after pollination. Bound GAs recovered from the precipitated protein of the buffer homogenate (“Protein-bound” GAs) were found in embryo and endosperm. Developing endosperm generally contains the major amount of the extractable gibberellin-like substances. In this tissue, the amount of all examined fractions (M-“free” GAs, B-“free” GAs and “protein-bound” GAs) increased after pollination to reach a maximum on the 21st day, before decreasing to a minimum at grain maturity. Moreover, the curves for dry weight increase and gibberellin like substances follow a remarkably similar course, with the latter reaching its maximim slightly earlier than the former one. This result indicates that gibberellines may participate in the regulation of the accumulation process in the endosperm of barley grain.     

Auxin-induced elongation growth and expressions of cell wall-bound exo- and endo-beta-glucanases in barley coleoptiles

When auxin stimulates rapid cell elongation growth of cereal coleoptiles, it causes a degradation of 1,3:1,4-beta-glucan in hemicellulosic polysaccharides. We examined gene expressions of endo-1,3:1,4-beta-glucanase (EI) and exo-beta-glucanase (ExoII), of which optimum pH are about 5, and molecular distribution of hemicellulosic polysaccharides in barley (Hordeum vulgare L.) coleoptile segments treated with or without IAA. IAA (10(-5) M) stimulated the gene expression of EI, while it did not affect that of ExoII. IAA induced gene expression of EI after 4 h and increased wall-bound glucanase activity after 8 h. The molecular weight distribution of hemicellulosic polysaccharides from coleoptile cell walls was shifted to lower molecular weight region by 2 h of IAA treatment. Fusicoccin (10(-6) M) mimicked IAA-induced elongation growth and the decrease in molecular weight of hemicellulosic 1,3:1,4-beta-glucan of coleoptiles in the first 4 h, but it did not promote elongation growth thereafter. These facts suggest that acidification of barley cell walls by IAA action enhances pre-existing cell wall-bound glucanase activity in the early first phase of IAA-induced growth and the late second phase involves the gene expression of EI by IAA.