New insight into puroindoline function inferred from their subcellular localization in developing hard and soft near-isogenic endosperm and their relationship with polymer size of storage proteins

Wheat endosperm texture is correlated with one major locus, Ha, located on the short arm of chromosome 5D, which comprises several genes among which are two puroindoline genes, Pina and Pinb. In this study, we used two near-isogenic lines, the hard-textured line lacking Pina and the soft-textured line containing both Pina and Pinb wild-type genes. Hard and soft endosperms were observed at four kernel developmental stages, from 180 °Cd to 750 °Cd. Puroindolines were located within protein bodies at the onset of prolamin accumulation by transmission electron microscopy and immunolabelling. Ab initio modeling showed a closer structural relationship between puroindolines and 2S storage proteins from dicots than between puroindolines and other cysteine-rich wheat proteins, i.e. LTP and amylase inhibitors. Compared to the soft line, storage protein polymers in the hard line exhibited higher molecular mass (increase of from 6 to 93%) and polydispersity indices (increase of from 26 to 63%) over the course of the 4-year experiment. This suggests that puroindolines might impact the aggregation of storage proteins. Finally, these data pave the way for investigation of the role of protein–protein interactions in the texture of wheat endosperm.     

小麦籽粒发育时期Puroindolines蛋白与硬度的关系

为探讨Puroindolines蛋白的表达特点与籽粒硬度的关系,采用改进的SDS-PAGE凝胶分析了不同硬度小麦品种的籽粒在各个发育时期Puroindolines蛋白的表达.结果表明,不同硬度的小麦籽粒中总Puroindolines(PinA和PinB)蛋白的表达量差异不大,但与胚乳淀粉颗粒结合的Puroindolines蛋白量差异非常明显:在籽粒发育的不同时期,软质小麦籽粒淀粉粒表面结合的Puroindolines蛋白量显著高于硬质小麦;基因型同为野生型但硬度有差异的品种,籽粒较软的材料其淀粉粒表面结合的Puroindolines蛋白量也明显高于较硬的材料,说明该蛋白的结合特性是决定籽粒硬度的直接原因.结果还表明,胚乳中水溶性戊聚糖与籽粒硬度关系密切.     

Genetic and Environmental Effects on Puroindoline-a and Puroindoline-b Content and their Relationship to Technological Properties in French Bread Wheats

Grain hardness (GHa) in hexaploid wheat (Triticum aestivum L.) is a major factor of end use quality. The variation of texture has been related to the Hardness locus, closely linked with the puroindoline-a (PIN-a) and puroindoline-b (PIN-b) genes. In order to study the role of puroindoline content in texture variation, the quantity of puroindolines was determined. Puroindoline-a (PIN-a) and puroindoline-b (PIN-b) content was determined and a total of 11 bread making parameters were obtained from 40 bread wheat cultivars grown in four experimental locations. The 11 parameters were significantly influenced by the genotype whereas location did not significantly affect PIN-a or PIN-b content and loaf volume. PIN-b and grain hardness displayed the highest heritability coefficients (both 0.88). PIN-a and PIN-b content were not correlated with grain protein content (Prot) and grain hardness in hard and soft wheat types. In soft samples PIN-(a+b) content was negatively correlated, with loaf volume in two locations. Multiple regression analyses, carried separately for soft and hard types, revealed that PIN-b content explained variation of dough strength (W) and loaf volume, however their influence was mostly significant in soft types. For each location, from 22 to 91% of the phenotypic variation of strength and loaf volume was explained by combining up to three flour traits. Protein content, PIN-b and the average score of high molecular weight glutenin subunits (HMW-GS) were frequently introduced by multiple regression (without GHa) as explanatory variables of strength and loaf volume. These results strengthened the significant role of PIN-b in breadmaking (loaf volume), and indicated that biochemical factors other than puroindolines are involved in the grain hardness variation.     

Mapping kernel texture in a soft durum (Triticum turgidum subsp. durum) wheat population

Kernel texture (”hardness”) is one of the major determinants of wheat quality and is primarily controlled by the Puroindoline (Pin) genes, located at the Hardness (Ha) locus. The absence of the Ha locus is responsible for the extremely hard kernels of durum wheat (T. turgidum subsp. durum). Recently, the Pin genes from a soft common wheat variety were introgressed into durum wheat through homoeologous recombination. The objective of this study was to map kernel hardness in a soft durum wheat population derived from the cross between the varieties “Creso” and “Langdon 1–678“. In all, 428 F₆ lines were evaluated for kernel hardness through the Single Kernel Characterization System; Hardness Index (HI) values ranged from −2 to 44. The same lines were genotyped using genotyping-by-sequencing, targeted amplicon sequencing, and sequence-tagged-site markers. A total of 8537 markers were used to conduct single marker-trait association analysis and two major significant regions were identified on chromosomes 3AL and 6AS each responsible for an additive effect of ∼6 HI units. Kompetitive allele specific markers targeting these regions were selected and tested in the whole population. To date, this is the first study to investigate the genetic factors behind hardness variation in durum wheat.     

Puroindolines co-localize to the starch granule surface and increase seed bound polar lipid content

Endosperm texture in wheat is controlled by the Pina and Pinb genes that comprise the Hardness (Ha) locus. Studies have shown that soft and hard varieties differ in the amount of starch bound polar lipids but have not addressed whether PINs are directly involved and whether the presence of one particular PIN affects seed polar lipid levels and cellular localization. Here, we determined the effect of overexpressing PINA or PINB on seed bound polar lipids and PIN localization. F₃ recombinants homozygous for either a Pina or Pinb null Ha locus with or lacking a transgenically added Pina or Pinb were analyzed for grain hardness, PIN abundance, and seed bound polar lipid levels. Overexpressed PINs resulted in reduced hardness, increased starch bound PINs, and increased seed bound polar lipids. Addition of PINA to the PINA nulls or PINB to the PINB nulls resulted in higher bound polar lipid levels than the addition of the alternative PIN. Both PINs localized to the starch surface in the presence or absence of the other protein. Our results indicate that PIN overexpression results in reduced endosperm texture and increased seed bound polar lipids and that PINs independently localize to the surface of starch granules.     

Development and characterization of a Triticum aestivum-H. villosa T5VS•5DL translocation line with soft grain texture

The Hardness locus on the short arm of chromosome 5D is the main determinant of grain texture in bread wheat. The Pina and Pinb genes are tightly linked at this locus, and the soft kernel texture phenotype results when both genes are present and encode the wild-type puroindoline proteins PINA and PINB. In this study a compensating T5VS•5DL Triticum aestivum-Haynaldia villosa translocation line, NAU415, was characterized by chromosome C-banding, genomic in situ hybridization and molecular markers. Single Kernel Characterization System (SKCS) analysis and scanning electron microscopy indicated that NAU415 had soft endosperm although it lacked the wheat Pina-D1a and Pinb-D1a genes, suggesting the presence of functional Pin gene orthologs on chromosome 5VS. Using a PCR approach, Pina-related (designated Dina) and Pinb-related (Dinb) genes in H. villosa and NAU415 were identified and sequenced. The nucleotide and predicted amino acid sequences showed close similarities to the wild-type puroindolines of T. aestivum cv. Chinese Spring. The tryptophan-rich regions of both Dina and Dinb showed a sequence change from lysine-42 to arginine, a feature that may have an effect on grain texture. The potential of T5VS•5DL translocation line as a source of genes that may be used for modulation of endosperm texture and other valuable traits in wheat breeding is discussed.     

Simple ELISA Detection of a New Polymorphic Ha Locus Encoded Protein

A rapid two-site sandwich ELISA was developed for detection of a previously uncharacterised protein encoded at the Ha locus on chromosome 5DS of wheat (Triticum aestivum). The assay used the combined specificity of two antibodies to detect a protein that was soluble in aqueous alcohol, salt solutions and water. It was expressed in the endosperm of all soft wheats and Triticum tauschii accessions tested. The ELISA was highly specific, with no signal obtained with varieties that did not express the protein. The presence of the 5DS-encoded protein correlated with a significant change in both water absorption and average hardness and particle size indices in a doubled haploid population derived from a cross between cvs. Cranbrook×Halberd. Only some hard varieties expressed this protein indicating that the protein is not predictive for hardness. However, it may be a new factor, or a marker for a new factor, affecting kernel texture. A polypeptide ofM_r 66 000 was purified from an extract of Halberd flour by immunoaffinity chromatography. Its N-terminal amino acid sequence identified it as an albumin with high homology to both mammalian serum albumins and sucrose synthase from a range of cereals. The assay may be valuable in wheat breeding programmes for assessing kernel texture where the parents are of different ELISA phenotype, or for varietal identification, as the expression of the polypeptide is variable in hard wheat varieties.     

Effects of drought and the presence of the 1BL/1RS translocation on grain vitreosity, hardness and protein content in winter wheat

Grain texture is an important component of end-use quality in wheat. The effects of water availability on the components of texture; vitreosity, determined using a Light Transflectance meter (LTm), grain hardness measured using the single-kernel characterisation system (SKCS), and protein content, were studied in field experiments of winter wheat in the UK in 2001/2002 and 2002/2003. Experiments were grown on a drought prone soil and employed a mapping population of 46 doubled haploid (DH) lines and their parents, Beaver (+1BL/1RS, soft wheat) and Soissons (1B, hard wheat). The results showed that drought increased hardness in both seasons, but the effect was never sufficient to move a line from the soft class into the hard class. Puroindoline (PIN)-a:b peak height ratio explained ca. 78% of the variation in hardness, and drought also appeared to increase the amounts of PINs in the grain. Minor quantitative trait loci (QTLs) were found for hardness on chromosomes 2A, 2D, 3A and 6D, also associated with QTLs for PINs. Vitreosity also increased in response to drought in both seasons. Variation in vitreosity explained 7–11% of the overall variation in texture within a hardness class, with hardness increasing on average by 2.2 SKCS units for each 10% increase in the proportion of vitreous grains. The relationship between vitreosity and protein content was poor, despite the fact that protein content also increased in response to drought. Minor QTLs associated with both protein content and vitreosity were found on chromosomes 1B, 4D and 5D. A minor QTL for vitreosity was also found on chromosome 2D. However, there appeared to be no direct relationship between alleles at the Ha locus, the gene which controls the difference between hard and soft wheats, and vitreosity. A positive relationship between the presence of the 1BL/1RS translocation and the proportion of vitreous grains was identified, suggesting that vitreosity was strongly linked to changes in protein quality.     

White salted noodle characteristics from transgenic isolines of wheat over expressing puroindolines

The closely linked genes puroindoline a (Pina) and puroindoline b (Pinb) control most of the variation in wheat (Triticum aestivum) grain texture. Mutations in either Pina or Pinb result in hard grain with wild type forms of both genes giving soft grain. Asian noodles are prepared from both hard and soft classes of wheat. Our objective was to examine color and texture characteristics of white salted noodles processed from flours of transgenic isolines of Hi-Line hard red spring wheat over expressing Pina-D1a, Pinb-D1a or both and a control giving a range in grain texture from very soft to hard. White salted noodles were prepared and color and texture characteristics were measured. The three softer textured transgenic isolines showed greater change in L^* with time than Hi-Line. The noodles were more adhesive (more negative value), firmer, and chewier as the grain texture became successively softer when cooked at 5 min. These texture differences were not as apparent when noodles were cooked for an optimum time. Starch pasting properties did not explain the noodle textural differences. A possible explanation for the noodle texture differences may be related to starch damage which ranged from 2.2% for HGAB to 6.7% for Hi-Line, flour particle size differences and subsequent water absorption differences among the four genotypes. Over expression of puroindolines did not enhance quality of white salted noodles when prepared under these conditions.     

Genetic and environmental factors affecting grain texture in common wheat

Thirteen wheat cultivars grown in six locations were compared for kernel weight, protein content and grain texture, as determined by the Single Kernel Characterization System (SKCS). Moreover, puroindolines a (Pin-A) and b (Pin-B) bound to starch were quantified by densitometric scanning of A-PAGE fractionations. All cultivars shared allele Pina-D1a coding for wild-type Pin-A, and differed from each other in allele composition at Pinb-D1 coding for Pin-B. Cultivars with Pinb-D1a exhibited soft grain and high amounts of Pin-A and Pin-B compared to cultivars with Pinb-D1b or Pinb-D1d. Significant genetic variation for grain hardness and Pin-A level was detected in soft cultivars. The ratio between Pin-A and Pin-B levels in soft cultivars was approximately 6:5, whereas it varied between 9:5 and 10:1 in hard cultivars. Protein content was significantly correlated with Pin-B content (r=0.34) and SKCS value (r=0.36) in soft wheats. Significant correlations (0.68 and 0.73 for soft and hard wheats, respectively) were observed between Pin-A and Pin-B levels. Grain hardness was not correlated with puroindoline levels and Pin-A/Pin-B ratio in both textural classes. By contrast, kernel weight was found to act as a major environmental factor affecting grain texture in both soft and hard wheats.     

Rheological Behaviour of Wheat Endosperm—Proposal for Classification Basedon the Rheological Characteristics of EndospermTest Samples

A simple, rapid method for the preparation of parallelepiped-shaped samples from a grain is used in the proposal of a study of the rheological behaviour of wheat endosperm. Compression rupture, creep and relaxation tests are used. A series of compression tests on mealy and vitreous endosperm of different wheat varieties (soft, hard and durum) shows that the rheological properties are influenced by both the genetic origin and grain vitreousness. The main mechanical characteristics—Young's modulus, elastic and rupture stresses, rupture energy and rupture strain—were determined at moisture contents of 12 to 17%. The influence of the moisture content on rheological behaviour is demonstrated. The vitreous endosperm of some wheat varieties displays considerable ductility before rupture. The nature of this plasticity was analysed by creep and relaxation tests on hard and soft wheats. Comparison of the different endosperm rheology values clarifies the notions of vitreousness and hardness. Wheat classification based on endosperm mechanical characteristics is proposed. It seems that Young's modulus characterises hardness whereas rupture energy is related to the vitreousness of the different varieties studied. Entering the results in a Young's modulus–rupture energy system leads to a classification of wheats according to two essential factors: hardness of varietal origin and vitreousness of cultural origin.     

江苏淮北地区小麦品种资源籽粒硬度基因等位变异的KASP检测

为了解江苏淮北地区小麦品种资源的籽粒硬度概况及硬度基因型分布规律,以74份近年来江苏淮北地区所育品种(系)和38份来自黄淮其他麦区的常用亲本为材料,采用单籽粒谷物硬度测试仪、KASP标记检测技术和基因扩增及测序技术对其SKCS硬度值及硬度基因型进行鉴定。硬度检测结果表明,供试小麦品种(系)硬度变化范围较大,但硬质麦的比例最大,为70.5%。与常用亲本相比,江苏淮北地区育成品种中软质麦比例较高,为34.3%,但在高代品系中软质麦比例下降到20.5%。基因型检测结果表明,在Puroindoline-D1位点,供试品种(系)中共检测到4种基因型,即野生型(Pina-D1a/Pinb-D1a)、Pina-D1b、Pinb-D1b和Pinb-D1p,其频率依次为25.0%、2.7%、67.9%和4.5%。其中,野生型和Pinb-D1p主要分布在江苏淮北地区。不同硬度基因型的硬度值也存在差异,其中以Pina-D1b基因型的硬度值最高,野生型(Pina-D1a/Pinb-D1a)硬度值最低,Pinb-D1b和Pinb-D1p两硬质类型的籽粒硬度没有显著性差异。在Pinb-2位点,供试品种(系)中共检测到25份材料为Pinb-B2b基因型,包含21份硬质麦、2份混合麦和2份软质麦,其平均硬度值为63.8。     

The effects of puroindoline b on the ultrastructure of endosperm cells and physicochemical properties of transgenic rice plant

Endosperm texture is an important factor governing the end-product quality of cereals. The texture of wheat (Triticum aestivum L.) endosperm is controlled by puroindoline a and b genes which are both absent in rice (Oryza sativa L.). It has been reported that the endosperm texture of rice can be modified by puroindoline genes. The mechanism, however, by which puroindolines affect the ultrastructure of rice endosperm cells remains to be investigated. In this study, we observed the ultrastructure of endosperm cells and the morphology of isolated starch granules of the transgenic rice expressing the puroindoline b gene. SEM and TEM observations indicated that compound starch granules were embedded within the matrix material in non-transgenic rice, Nipponbare, whereas they were surrounded by spaces in the transgenic rice. The morphology and size of each starch granule were not different between non-transgenic and the transgenic rice. However, the transgenic rice flour showed smaller particle size, higher starch damage, and lower viscosity during gelatinization than that of non-transgenic rice. These results confirm that puroindoline b reduces the grain hardness in rice. Moreover, the results also suggest that puroindoline b functions at the surface of compound starch granules, and not on polygonal starch granules in rice endosperm.     

Starch isolation method impacts soft wheat (Triticum aestivum L. cv. Claire) starch puroindoline and lipid levels as well as its functional properties

Wheat (Triticum aestivum L.) kernel hardness is a major quality characteristic, which has been ascribed to the presence of puroindolines a and b. These proteins occur in higher levels at the surface of water-washed starch granules from soft wheat cultivars than at that of starch from hard wheat cultivars. In the present study, prime starch was isolated from flour from soft wheat (cultivar Claire) using a dough ball or batter based separation method. Starch isolated with the dough ball method contained lower levels of puroindolines, as well as of other starch granule associated proteins and lipids than that isolated with the batter method. Similar patterns of puroindoline and lipid levels after starch isolation can presumably be related to (polar) lipid binding by puroindolines. Both isolated starch fractions showed comparable differential scanning calorimetry thermograms, whereas higher levels of starch surface associated components restricted starch swelling. Necessary controls demonstrated that the observed differences did not arise from artefacts associated with hydration, fractionation or freeze-drying in the experimental protocols. Apparently, proteins and lipids at the starch granule surface impact water absorption and, as such, starch swelling, but they do not affect starch granule internal phenomena such as melting of the crystalline amylopectin chains.     

Compressive strength of Super Soft wheat endosperm

Wheat endosperm texture (hardness) largely determines end-product suitability. Since its development 25 years ago, the single kernel classification system (SKCS, a mechanical instrument that measures, among other properties, the force imparted on a kernel during crushing) has been used in breeding programs to differentiate soft wheats from hard wheats. Nominally, these have a soft to hard SKCS hardness index (HI) range of 25–75 (dimensionless units). However, in recent years, breeders have developed extremely soft (‘Super Soft’) lines having SKCS HI < 0. Until now, these very low SKCS HIs have not been corroborated with traditional methodologies that characterize mechanical strength. Herein, we report on the relationships between SKCS HI and three compressive strength properties (maximum stress, Young's modulus, and work) in Super Soft wheat. With respective correlation coefficients of 0.76, 0.66, and 0.75, we have found that the relationships between SKCS HI and compressive strength agree with prior research involving ordinary soft and hard wheats.     

青海小麦籽粒硬度等位变异研究

为了解小麦品种籽粒硬度的遗传多样性,利用单粒谷物硬度测定、PCR扩增和核苷酸测序技术,分析了66份青海小麦品种籽粒硬度主效基因的等位变异。结果表明,青海小麦以硬质类型为主,比例达到47.0%,混合麦比例为19.7%,软质麦比例为33.3%。硬度基因有5种组合类型:野生型、Pina-D1a/PinbD1b、Pina-D1a/Pinb-D1c、Pina-D1a/Pinb-D1x和Pina-D1b/Pinb-D1a。野生型小麦类型比例最高,占59.09%,SKCS硬度指数平均为44.12,变化范围为12.75~84.89。突变类型的品种籽粒均为硬质。因此,在青海硬质小麦可以通过突变类型的分子标记进行选育,软质小麦选育需在利用硬度基因分子标记筛选的基础上进一步考察籽粒硬度性状的表现型。     

Wheat grain softness protein (Gsp1) is a puroindoline-like protein that displays a specific post-translational maturation and does not interact with lipids

Compared to puroindolines a and b, grain softness proteins (Gsp1) remained up to now the less characterized members of this wheat specific seed protein family. Due to their low expression levels, their purification remains relatively arduous. We report in the present work on the purification of wheat Gsp, Gsp1b and its post-translational maturation. We showed that Gsp1b underwent different proteolytic cleavages both in the N and C-terminal extremities of the preproprotein. Especially, after the putative signal peptide, a 29 mer peptide is highlighted that is highly truncated in puroindolines (8–9 mer). We also showed that Gsp1b and puroindolines display similar secondary structure but Gsp1b does not interact in vitro with lipids. In addition, by using the iterative threading assembly refinement (I-TASSER) methods, we showed that Gsp1b three-dimensional structure prediction is however identical to both those of puroindolines and of the dicotyledon 2S storage proteins. In contrast with puroindolines, all these data make us question about the role of Gsp1 proteins in endosperm texture and the previously suggested function in plant defense lent to these proteins.     

Analysis of the milling reduction of bread wheat farina: Physical and biochemical characterisation

The reduction of coarse farina during milling from five representative hard and soft French bread wheat cultivars was followed step by step. Particle size distribution of the milled products and energy required to dissociate wheat farina revealed large differences between cultivars. A grinding index (required energy to produce 1 kg of flour), K′, corresponding to the energy necessary to obtain a given amount of flour, was found to be related with both grain hardness and vitreousness. Analysis of particle size distribution suggested a major influence of hardness in the production of fine particles <50 μm during the reduction process. On the other hand, vitreousness appeared to impact on the extent of coarse particle size reduction. Differences in farina reduction behaviour of the wheats analysed were related to their endosperm mechanical properties as determined by compression tests and structural characteristics as measured by Hg intrusion porosity. Vitreousness appeared to affect the endosperm extensibility and degree of porosity. The profile of the different lipid and protein classes in the reduction products of two cultivars showing opposite behaviour as well as different PINA/PINB ratio were determined. The results suggest that the most resistant parts of the grain are enriched in glycolipids, whereas phospholipids appear associated with the most friable parts, at least in the soft wheat grains tested.