Hordoindolines are associated with a major endosperm-texture QTL in barley (Hordeum vulgare).

Endosperm texture has a tremendous impact on the end-use quality of wheat (Triticum aestivum L.). Cultivars of barley (Hordeum vulgare L.), a close relative of wheat, also vary measurably in grain hardness. However, in contrast to wheat, little is known about the genetic control of barley grain hardness. Puroindolines are endosperm-specific proteins found in wheat and its relatives. In wheat, puroindoline sequence variation controls the majority of wheat grain texture variation. Hordoindolines, the puroindoline homologs of barley, have been identified and mapped. Recently, substantial allelic variation was found for hordoindolines among commercial barley cultivars. Our objective was to determine the influence of hordoindoline allelic variation upon grain hardness and dry matter digestibility in the 'Steptoe' x 'Morex' mapping population. This population is segregating for hordoindoline allele type, which was measured by a HinA/HinB/Gsp composite marker. One-hundred and fifty lines of the 'Steptoe' x 'Morex' population were grown in a replicated field trial. Grain hardness was estimated by near-infrared reflectance (NIR) and measured using the single kernel characterization system (SKCS). Variation attributable to the HinA/HinB/Gsp locus averaged 5.7 SKCS hardness units (SKCS U). QTL analysis revealed the presence of several areas of the genome associated with grain hardness. The largest QTL mapped to the HinA/HinB/Gsp region on the short arm of chomosome 7 (5H). This QTL explains 22% of the SKCS hardness difference observed in this study. The results indicate that the Hardness locus is present in barley and implicates the hordoindolines in endosperm texture control.     

Kernel texture and hordoindoline patterns in barley (Hordeum vulgare)

Hordoindolines, the tryptophan-rich polypeptides affecting grain hardness in barley, appeared as three pairs of polypeptides in the acidic polyacrylamide gel electrophoresis (A-PAGE) and two-dimensional A-PAGE?×?SDS-PAGE patterns of starch-granule proteins from 18 barley cultivars. On capillary RP-HPLC/nESI-MS/MS spectrometry, one pair of polypeptides was found to correspond to hordoindoline A (HINA), one to hordoindoline B1 (HINB1) and one to hordoindoline B2 (HINB2), the two polypeptides of each pair deriving from post-translational cleavage of a native hordoindoline at different positions at the N-terminus and/or C-terminus. Amongst the barley cultivars analyzed, cvs Hart and Sundance, which were claimed to be unique in lacking the Hina gene coding for HINA, revealed similar Hina coding sequences and accumulated hordoindoline HINA on their starch granules. The amount of total hordoindolines (HINA?+?HINB1?+?HINB2) on the starch granules, as quantified by densitometric scanning of A-PAGE gels, was comparable with that of puroindolines (PINA?+?PINB) in soft-textured wheat. By contrast, the amount of B-type hordoindolines (HINB1 and HINB2 combined) was 50?% lower than that of PINB, suggesting that the absence of barley cultivars with soft kernels is likely due to the reduced amount of B-type hordoindolines accumulated on the starch granules. Approximately 22 and 27?% of the phenotypic variation for kernel hardness in 56 barley cultivars analyzed by the Single Kernel Characterization System (SKCS) were explained by differences in kernel weight and B-type hordoindoline level, respectively. By contrast, the outer husk of barley grain showed no effect on the SKCS index.     

Puroindolines: the molecular genetic basis of wheat grain hardness

The variation in grain hardness is the single most important trait that determines end-use quality of wheat. Grain texture classification is based primarily on either the resistance of kernels to crushing or the particle size distribution of ground grain or flour. Recently, the molecular genetic basis of grain hardness has become known, and it is the focus of this review. The puroindoline proteins a and b form the molecular basis of wheat grain hardness or texture. When both puroindolines are in their `functional' wild state, grain texture is soft. When either one of the puroindolines is absent or altered by mutation, then the result is hard texture. In the case of durum wheat which lacks puroindolines, the texture is very hard. Puroindolines represent the molecular-genetic basis of the Hardness locus on chromosome 5DS and the soft (Ha) and hard (ha) alleles present in hexaploid bread wheat varieties. To date, seven discrete hardness alleles have been described for wheat. All involve puroindoline a or b and have been designated Pina-D1b and Pinb-D1b through Pinb-D1g. A direct role of a related protein, grain softness protein (as currently defined), in wheat grain texture has yet to be demonstrated.     

The determinants of grain texture in cereals

Kernel hardness is an important agronomic trait that influences end-product properties. In wheat cultivars, this trait is determined by thePuroindoline a (Pina) andPuroindoline b (Pinb) genes, located in theHardness locus (Ha) on chromosome 5DS of the D genome. Wild type alleles code puroindoline a (PINA) and puroindoline b (PINB) proteins, which form a 15-kDa friabilin present on the surface of water-washed starch granules. Both the proteins are accumulated in the starch endosperm cells and aleurone of the mature kernels.Puroindoline-like genes coding puroindoline-like proteins in the starch endosperm occur in some of the genomes of Triticeae and Aveneae cereals. Orthologs are present in barley, rye and oats. However, some genomes of these diploid and polyploid cereals, like that ofTriticum turgidum var.durum (AABB) lack thepuroindoline genes, having a very hard kernel texture. The two wild type alleles in opposition (dominant loci) control the soft pheno-type. Mutation either inPina orPinb or in both leads to a medium-hard or hard kernel texture. The most frequent types ofPin mutations are point mutations within the coding sequence resulting in the substitution of a single amino acid or a null allele. The latter is the result of a frame shift determined by base deletion or insertion or a one-point mutation to the stop codon. The lipid-binding properties of the puroindolines affect not only the dough quality but also the plants’ resistance to pathogens. Genetic modification of cereals withPuroindoline genes and/or their promoters enable more detailed functional analyses and the production of plants with the desired characteristics.     

Starch-bound 2S proteins and kernel texture in einkorn, <Emphasis Type="Italic">Triticum</Emphasis> <Emphasis Type="Italic">monococcum</Emphasis> ssp <Emphasis Type="Italic">monococcum</Emphasis>

The starch granule proteins from 113 einkorn wheat (Triticum monococcum ssp monococcum) accessions were analyzed by acidic, polyacrylamide gel electrophoresis (A-PAGE), and two-dimensional A-PAGE x SDS-PAGE. All accessions were confirmed to contain equal amounts of two polypeptide chains corresponding to puroindoline B (Pin-B), as well as a prominent component plus a faint band corresponding to puroindoline A (Pin-A). When compared with soft-textured common wheat, “monococcum” accessions showed an increase of 3.2- and 2.7-fold in Pin-A and Pin-B levels on the starch granules, respectively. In addition, all accessions contained a novel component of the 2S super-family of seed proteins named Einkorn Trypsin Inhibitor (ETI), which was found to be encoded as a pre-protein 148 residues long. Wild-type ETI encoded by allele Eti-A ^m 1a and “valine-type” ETI encoded by allele Eti-A ^m 1b, which occurred in 107 and six einkorn accessions, respectively, were found to accumulate on starch granules as a mature protein of 121 amino acids with a hydrophobic central domain. The einkorn accessions exhibited an average SKCS index as low as −2.05 ± 11.4, which is typical of extra-soft kernels. The total surface area of starch granules in “monococcum” wheat, as determined by visual assessments in counting chambers, was estimated at 764 mm²/mg of starch, and was about 1.5 times higher than that for common wheat. The results are discussed in relation to the identification of factors that cause the extra-soft texture of einkorn kernels.     

Early expression of grain hardness in the developing wheat endosperm

Seeds from near-isogenic hard and soft wheat lines were harvested at regular intervals from 5 days post-anthesis to maturity and examined for hardness using the single kernel characterisation system (SKCS). SKCS analysis revealed that hard and soft lines could be distinguished from 15 days post-anthesis (dpa). This trend continued until maturity where the difference between the hard and soft lines was most marked. SKCS could not be applied to the small 5- and 10-dpa wheat kernels. Fresh developing endosperm material was examined using light microscopy and no visible differences between the cultivars were detected. When air-dried material was examined using scanning electron microscopy (SEM) differences between soft and hard lines were visible from as early as 5 dpa. Accumulation of puroindoline a and puroindoline b was investigated in developing seeds using both Western blotting and ELISA. Low levels of puroindoline a could be detected in the soft cultivar from 10 dpa, reaching a maximum at 32 dpa. In the hard cultivar, puroindoline a levels were negligible throughout grain development. Puroindoline b accumulates in both the soft and hard cultivars from 15 dpa, but overall contents were higher in the soft cultivar. These findings indicate that endosperm hardness is expressed very early in developing grain when few starch granules and storage proteins were deposited in the endosperm cells. Further, the near-isogenic soft and hard Heron lines could be differentiated by SEM at a stage in development when the accumulation of puroindolines could not be detected by the methods used in this study.     

Characterization of wheat puroindoline proteins

Puroindoline proteins were purified from selected UK-grown hexaploid wheats. Their identities were confirmed on the basis of capillary electrophoresis mobilities, relative molecular mass and N-terminal amino acid sequencing. Only one form of puroindoline-a protein was found in those varieties, regardless of endosperm texture. Three allelic forms of puroindoline-b protein were identified. Nucleotide sequencing of cDNA produced by RT-PCR of isolated mRNA indicated that these were the 'wild-type', found in soft wheats, puroindoline-b containing a Gly-->Ser amino acid substitution (position 46) and puroindoline-b containing a Trp-->Arg substitution (position 44). The latter two were found in hard wheats. Microheterogeneity, due to short extensions and/or truncations at the N-terminus and C-terminus, was detected for both puroindoline-a and puroindoline-b. The type of microheterogeneity observed was more consistent for puroindoline-a than for puroindoline-b, and may arise through slightly different post-translational processing pathways. A puroindoline-b allele corresponding to a Leu-->Pro substitution (position 60) was identified from the cDNA sequence of the hard variety Chablis, but no mature puroindoline-b protein was found in this or two other European varieties known to possess this puroindoline-b allele. Wheats possessing the puroindoline-b proteins with point mutations appeared to contain lower amounts of puroindoline protein. Such wheats have a hard endosperm texture, as do wheats from which puroindoline-a or puroindoline-b are absent. Our results suggest that point mutations in puroindoline-b genes may confer hard endosperm texture through accumulation of allelic forms of puroindoline-b proteins with altered functional properties and/or through lower amounts of puroindoline proteins.     

Real Time RT-PCR and flow cytometry to investigate wheat kernel hardness: role of puroindoline genes and proteins

Developing seeds from Triticum aestivum (wheat) cultivars were collected after flowering and analysed for puroindoline a and b gene expression by Real Time RT-PCR. Mature seeds were investigated for the presence and the amount of starch-associated puroindoline a and b proteins by flow cytometry. Puroindoline a gene and protein were found to have a predominant role in controlling wheat kernel hardness.     

Expression of wild-type pinB sequence in transgenic wheat complements a hard phenotype

Wheat grain hardness is a major factor in the wheat end-product quality. Grain hardness in wheat affects such parameters as milling yield, starch damage and baking properties. A single locus determines whether wheat is hard or soft textured. This locus, termed Hardness ( Ha), resides on the short arm of chromosome 5D. Sequence alterations in the tryptophan-rich proteins puroindoline a and b (PINA and PINB) are inseparably linked to hard textured grain, but their role in endosperm texture has been controversial. Here, we show that the pinB-D1b alteration, common in hard textured wheats, can be complemented by the expression of wild-type pinB-D1a in transformed plants. Transgenic wheat seeds expressing wild-type pinB were soft in phenotype, having greatly increased friabilin levels, and greatly decreased kernel hardness and damaged starch. These results indicate that the pinB-D1b alteration is most likely the causative Ha mutation in the majority of hard wheats.     

A leucine to proline mutation in puroindoline b is frequently present in hard wheats from Northern Europe

Endosperm hardness in wheat (Triticum aestivum L.) is determined by one major genetic factor, the Hardness (Ha) gene on the short arm of chromosome 5D. Grain hardness has previously been reported to result from either a failure to express puroindoline a (Pina–D1b) or a glycine to serine mutation at position 46 in puroindoline b (Pinb–D1b). In this study, which involves a large survey of 343 wheat genotypes of mostly Northern European origin, we report two new mutations in puroindoline b associated with hard endosperm. These were characterized as involving a leucine to proline change at position 60 (Pinb–D1c), and a tryptophan to arginine change at position 44 (Pinb–D1d), respectively. While the former seems to be widely distributed in germplasm around the world, the latter was only found in three winter wheats from Sweden and Netherlands. As discussed in the paper, the three known mutations in puroindoline b can be considered ”loss-of-function” mutations (i.e. soft to hard), and structural analysis may serve to predict that their dramatic effect on wheat grain texture is a result of reduced lipid–binding ability. Received: 10 June 1999 / Accepted: 21 September 1999     

The Ha locus of wheat: identification of a polymorphic region for tracing grain hardness in crosses.

The grain softness proteins or friabilins are known to be composed of three main components: puroindoline a, puroindoline b, and GSP-1. cDNAs for GSP-1 have previously been mapped to group-5 chromosomes and their location on chromosome 5D is closely linked to the grain hardness (Ha) locus of hexaploid wheat. A genomic DNA clone containing the GSP-1 gene (wGSP1-A1) from hexaploid wheat has been identified by fluorescent in situ hybridization as having originated from the distal end of the short arm of chromosome 5A. A genomic clone containing the gene (wGSP1-D1) was also isolated from Aegilops tauschii, the donor of the D genome to bread wheat. There are no introns in the GSP-1 genes, and there is high sequence identity between wGSP1-A1 and wGSP1-D1 up to 1 kb 5' and 300 bp 3' to wGSP1-D1. However, regions further upstream and downstream of wGSP1-D1 share no significant sequence identity to corresponding sequences in wGSP1-A1. These regions therefore identified potentially valuable sequences for tracing the Ha locus through assaying polymorphic DNA sequences. The sequence from 300 to 500 bp 3' to wGSP1-D1 (wGSP1-D13) was mapped to the Ha locus in a mapping population. wGSP1-D13 was also tightly linked to genes for puroindoline a and puroindoline b which have been previously mapped to be at the Ha locus. In addition wGSP1-D13 was used to detect RFLPs between near isogenic soft and hard Falcon lines and in a random selection of soft and hard wheats.     

Expression of puroindoline a enhances leaf rust resistance in transgenic tetraploid wheat

The purouindoline gene (pin) coding for puroindoline proteins (PINs) is located on chromosome 5D, controls grain hardness, and the PINs have in vitro antimicrobial activity against gram-positive (G+) bacteria, gram-negative (G-) bacteria and fungi. Wheat leaf rust caused by Puccinia triticina is one of the most important fungal diseases for common wheat with AABBDD genomes. Tetraploid wheat (AABB genome) varieties Luna and Venusia were transformed with the purouindoline a (pinA) gene by bombardment, express PINA consititutively. Transgenic plants showed enhanced response to leaf rust in greenhouse and field. Comparative study of harvesting parameters showed significant differences between transgenic and control plants. These indexes were significantly lower (P < 0.05) in control plants than that in transgenic plants, which suggests that they are significantly affected by pinA gene and that the puroindoline a protein (PINA) can effectively inhibit in vivo the growth of fungal, and the transgenic tetraploid wheat can grow well in Hubei Province, Central China, where the tetraploid wheat varieties Luna and Venusia have poor yield due to their disease-sensitivity.     

A microarray analysis of wheat grain hardness

Grain hardness is an important quality characteristic of wheat grain, and considerable research effort has focused on characterising the genetic and biochemical basis underlying the hardness phenotype. Previous research has shown that the predominant difference between hard and soft seeds is linked to the puroindoline (PIN) proteins. In this study the near-isogenic lines of Heron and Falcon, which differ only in the grain hardness character, were compared using a cDNA microarray consisting of approximately 5,000 unique cDNA clones that were isolated from wheat and barley endosperm tissue. Our analysis showed that major differences in gene expression were evident for puroindoline-a (Pina), with a minor but not consistent change in the expression of puroindoline-b (Pinb). These observations were confirmed using a 16,000 unique cDNA microarray in a comparison of hard wheats with either the Pina null or Pinb mutation.     

Puroindoline A-gene expression is involved in association of puroindolines to starch

Puroindolines largely influence cereal grain hardness. In order to understand how they exert this influence, we carried out a molecular analysis of the pina and pinb genes of many Italian wheat cultivars. On the basis of their pin genotypes they could be divided into three groups: Pina-D1a/Pinb-D1a; Pina-D1a/Pinb-D1b; and Pina-D1b/Pinb-D1a. Five cultivars from each group were chosen to be studied to examine the quantity of puroindolines associated with starch (friabilin) and the amount not associated with starch. In addition, the level of pina expression was measured using RT-PCR. Soft cultivars (Pina-D1a/Pinb-D1a) exhibited the highest level of expression of pina; among the hard cultivars, those with the Pina-D1a/Pinb-D1b genotype showed a lower level of expression, while those with the Pina-D1b/Pinb-D1a genotype did not express pina. Total puroindoline and friabilin content was then measured by flow cytometry. Soft Pina-D1a/Pinb-D1a cultivars displayed high puroindoline content that was primarily starch associated. Hard Pina-D1b/Pinb-D1a cultivars had very low puroindoline content with no puroindoline bound to starch. Hard Pina-D1a/Pinb-D1b cultivars were highly heterogeneous with respect to both the content of puroindolines and the level of association with starch. The accurate quantification of puroindolines in starch-bound and not starch-bound forms in association with molecular analysis, indicates that pina expression and presence controls the abundance of total puroindoline and its association with starch.Communicated by H.F. Linskens     

Molecular genetics of puroindolines and related genes: allelic diversity in wheat and other grasses

The hardness or texture of cereal grains is a primary determinant of their technological and processing quality. Among members of the Triticeae, most notably wheat, much of the variation in texture is controlled by a single locus comprised of the Puroindoline a, Puroindoline b and Grain Softness Protein-1 (Gsp-1) genes. Puroindolines confer the three major texture classes of soft and hard common wheat and the very hard durum wheat. The protein products of these genes interact with lipids and are associated with the surface of isolated starch (as a protein fraction known as ‘friabilin’). During the past ten years a great diversity of alleles of both Puroindoline genes have been discovered and significant advances made in understanding the relationship between the gene presence/absence, sequence polymorphism and texture of cereal grains. Efforts have also focussed on Puroindoline and Gsp-1 genes in diploid progenitors, other Triticeae grasses and synthetic wheats in order to understand the evolution of this gene family and find potentially useful variants. The puroindoline homologues in other cereals such as rye and barley are also receiving attention. This work summarises new developments in molecular genetics of puroindolines in wheat and related Triticeae grasses, and the related genes in other cereals.     

Expression of wheat puroindoline genes in transgenic rice enhances grain softness

The puroindoline genes (pinA and pinB) are believed to play critical roles in wheat (Triticum aestivum L.) grain texture. Mutations in either gene are associated with hard wheat. No direct evidence exists for the ability of puroindolines to modify cereal grain texture. Interestingly, puroindolines appear to be absent in cereal species outside of the tribe Triticeae, in which the dominant form of grain texture is hard. To assess the ability of the puroindolines to modify cereal grain texture, the puroindolines were introduced into rice (Oryzae sativa L.) under the control of the maize ubiquitin promoter. Textural analysis of transgenic rice seeds indicated that expression of PINA and/or PINB reduced rice grain hardness. After milling, flour prepared from these softer seeds had reduced starch damage and an increased percentage of fine flour particles. Our data support the hypothesis that puroindolines play important roles in controlling wheat grain texture and may be useful in modifying grain texture of other cereals.     

Prevalence of Puroindoline D1 and Puroindoline b-2 variants in U.S. Pacific Northwest wheat breeding germplasm pools, and their association with kernel texture

Kernel texture is a major factor influencing the classification and end use properties of wheat (Triticum aestivum L.), and is mainly controlled by the Puroindoline a (Pina) and Puroindoline b (Pinb) genes. Recently, a new puroindoline gene, Puroindoline b-2 (Pin b-2), was identified. In this study, 388 wheat cultivars and advanced breeding lines from the U.S. Pacific Northwest were investigated for frequencies of Puroindoline D1 alleles and Pinb-2 variants 2 and 3. Results indicated that Pinb–D1b (74.0%) was the predominant genotype among hard wheats (N = 196), the only other hard allele encountered was Pina-D1b (26.0%). Across all varieties, Pinb-2v3 was the predominant genotype (84.5%) compared with Pinb-2v2 (15.5%). However, among 240 winter wheat varieties (124 soft white, 15 club, 68 hard red and 33 hard white varieties), all carried Pinb-2v3. Among spring wheats, Pinb-2v2 and Pinb-2v3 frequencies were more variable (soft white 25.0:75.0, hard red 58.2:41.8 and hard white 40.0:60.0, respectively). Kernel texture variation was analyzed using 247 of the 388 wheat varieties grown in multi-location factorial trials in up to 7 crop years. The range of variety means among the four groups, soft winter, soft spring, hard winter and hard spring, was on the order of 15–25 single kernel characterization system (SKCS) Hardness Index. The least significant difference for each of these trials ranged from 2.8 to 5.6 SKCS Hardness Index. Observations lead to the conclusion that Pinb-2 variants do not exert a prominent effect on kernel texture, however, Pinb–2 variants do identify features of wheat germ plasm structure in the U.S. Pacific Northwest.     

Chromosome 5H of <Emphasis Type="Italic">Hordeum</Emphasis> species involved in reduction in grain hardness in wheat genetic background

Grain hardness is an important factor affecting end-use quality in wheat. Mutations of the puroindoline genes, which are located on chromosome 5DS, control a majority of grain texture variations. Hordoindoline genes, which are the puroindoline gene homologs in barley, are located on chromosome 5HS and are also responsible for grain texture variation. In this study, we used three types of wheat–barley species (Hordeum vulgare, H. vulgare ssp. spontaneum, and H. chilense) chromosome addition lines and studied the effect of chromosome 5H of these species on wheat grain characteristics. The 5H chromosome addition lines showed significantly lower grain hardness and higher grain weight than the corresponding wheat parents. The effect of enhancing grain softness was largest in the wheat–H. chilense line regardless of having an increase in grain weight similar to those in the wheat–H. vulgare and wheat–H. spontaneum lines. Our results indicated that chromosome 5H of the Hordeum species plays a role in enhancing grain softness and increasing grain weight in the wheat genetic background, and the extent of effect on grain hardness depends on the type of Hordeum species. Protein analysis of hordoindolines indicated that profiles of 2D-electrophoresis of hordoindolines were different among Hordeum species and hordoindolines in the addition lines appeared to be most abundant in wheat–H. chilense line. The differences in enhancing grain softness among the Hordeum species might be attributed to the quantity of hordoindolines expressed in the 5H chromosome addition lines. These results suggested that the barley hordoindolines located on chromosome 5HS play a role in reducing grain hardness in the wheat genetic background.