Puroindolines are associated with decreased polar lipid breakdown during wheat seed development

The wheat Hardness (Ha) locus consists of Puroindoline a (Pina) and Puroindoline b (Pinb) and controls whether wheat is soft or hard textured. PINA and PINB act cooperatively to create grain softness and are associated with increased polar lipids at seed maturity. Here we examined developing seeds varying in PINA or PINB content to identify when polar lipid differences occur. One population carried Wild Type (WT) PINA while varying for the presence of PINB, while the other population carried a WT PINB and segregated for PINA. For the PINB segregating population, polar lipids were maximal on a per mg dry weight basis at 7–14 days after anthesis (DAA) while for PINA segregating population, total polar lipids were maximal at 7–21 DAA. The most abundant polar lipids at maturity were mono- and di- galactosyldiglyceride. No individual polar lipid species were specific to PINA or PINB. The effect of both PINA and PINB increased throughout seed development with greater decreases in the most abundant polar lipids in genotypes lacking either PINA or PINB. The results indicate that PINA and PINB act cooperatively to prevent polar lipid breakdown during seed maturation and that this process is central to the development of grain softness.     

A new puroindoline b mutation present in Chinese winter wheat cultivar Jingdong 11

Kernel hardness is one of the most important characteristics in determining utilization and marketing of bread wheat. Genes coding for puroindoline a and b (PINA and PINB) were located at the Ha locus and designated as Pina-D1 and Pinb-D1, respectively. The coding sequence of the Pinb gene in a Chinese winter wheat cultivar Jingdong 11 (Triticum aestivum L.) was amplified with polymerase chain reaction (PCR), and the obtained 447-bp fragment sequenced from two strands, and compared with the eight known Pinb alleles. The results showed that Jingdong 11 possessed a new Pinb allele not reported previously, and was designated as Pinb-D1q. It is characterized by a single base T to G substitution, which results in a tryptophan to leucine substitution (TGG to TTG) at position 44 and is most likely the cause of hard grain texture in Jingdong 11. The characterization of Pinb-D1 alleles would be helpful in manipulating grain hardness of bread wheat in breeding programs.     

The co-operative interaction of puroindolines in wheat grain texture may involve the hydrophobic domain

The puroindoline genes are causatively associated with wheat grain hardness, a commercially significant property. The proteins puroindoline (PIN) A and B are both required in their wild-type (WT) to impart soft grain texture, and absence of/mutations in either/both PIN(s) results in hard wheat. However, there is no biochemical clarity yet that explains this interdependence. This work critically analyses the roles of the tryptophan-rich domain (TRD), the little-known hydrophobic domain (HD), and certain other residues, in the physical associations of PINs. Site-directed mutagenesis-PCR was used to delete the TRD or HD and introduce an Arg39Gly substitution in PINA. The PINB-D1c mutant (Leu60Pro) was also investigated. The yeast two-hybrid system was used to assess the protein–protein interactions (PPI) of proteins. The TRD deletion or Arg39Gly substitution in PINA did not adversely affect its PPI, while deletion of HD resulted in a significant reduction. No effect on PPI was observed for Leu60Pro PINB. The results of this expression system strongly suggest that the HD is essential (but not sufficient) in higher-order associations of PINs. We propose a two-event model that explains the co-operative action of the PINs and why mutations outside the TRD may alter grain texture.     

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.     

Puroindoline grain hardness alleles in CIMMYT bread wheat germplasm

Grain hardness is an important quality parameter of bread wheat (Triticum aestivum L.) with importance for wheat classification and end use properties, and is controlled by the genes puroindoline a (Pina) and puroindoline b (Pinb). The presence of known hardness alleles was studied in a representative sample of 373 bread wheat lines from the breeding program at CIMMYT. The PINA-null mutation (Pina-D1b) was the most frequent hardness allele and present in 283 of the 328 lines with hard endosperm. All other hard wheat had the glycine to serine mutation in PINB (Pinb-D1b). A study of historically important CIMMYT bread wheat lines showed that Pina-D1b has been the dominating hardness allele since the inception of the wheat breeding program in Mexico. New puroindoline alleles have recently been introduced through the extensive use of synthetic hexaploid wheat, and the textural effects of various Aegilops tauschii-derived Pina and Pinb alleles were studied in 92 breeding lines derived from various crosses with synthetic wheat. Progeny lines with Pina-D1j/Pinb-D1i were on average 10 SKCS hardness units softer than those carrying the allelic combination Pina-D1c/Pinb-D1h. Further investigation is needed to validate the potential of such minor allelic differences for the improvement of soft wheat quality.     

Endosperm Texture in Wheat

One of the fundamental means of classifying wheat is through its endosperm texture. It impacts significantly on the milling process affecting among other things flour particle size and milling yield. Hardness in wheat is largely controlled by genetic factors but it can be affected by the environment and factors such as moisture, lipid, and pentosan content. The principal genetic locus controlling endosperm texture in wheat, Ha, is located on the chromosome 5D. At this locus several genes, notably the puroindolines, have been identified. Puroindolines are the major components of the 15 kDa protein band associated with starch granules that is more abundant in soft wheats than in hard. Recently the puroindolines have been shown to enhance grain hardness in rice. In this review we discuss the structure of hard and soft wheat endosperm with particular emphasis on when differences in endosperm texture can be detected in the developing seed. The role of the environment and other factors that may affect the endosperm texture is also examined together with the role of the puroindoline genes at theHa locus. Finally, we compare endosperm hardness in wheat and in barley.     

A single amino acid substitution in puroindoline b impacts its self-assembly and the formation of heteromeric assemblies with puroindoline a

Puroindolines (PINs) A and B were purified from soft (Paledor) and hard (Recital, Courtot) wheat cultivars. Their purity and heterogeneity due to post-translational processing were characterized by SDS- and acid-PAGE, reversed-phase HPLC and mass spectrometry. By using dynamic light scattering (DLS), asymmetrical flow field-flow fractionation (AF4) and size exclusion chromatography (SEC), we showed that the size distributions of PINA are similar for the three varieties and that, in solution, they self-assembled into small aggregates, mainly dimers. Conversely, PINB isolated from hard varieties (PINB-D1b and PINB-D1d) are assembled into large aggregates while PINB-D1a formed small aggregates, mainly monomers. Mixed solutions of PINA and PINB formed heteromeric aggregates. The large PINB-D1b aggregates were retained even at a high (4:1) PINA/PINB weight ratio. Reversible dissociation of large aggregates into small aggregates suggested that weak interactions control the self-assembly of PINs. The aggregative properties of PINs have now to be taken into account when studying their interactions with other components to decipher the causal relationships between these proteins and grain hardness.     

Silencing of puroindoline a alters the kernel texture in transgenic bread wheat

Grain hardness is an important end-use quality parameter of bread wheat, and one of the most important characters for quality improvement. The objective of this study was to further understand the function of puroindolines and the underlying mechanism in the formation of kernel texture. The highly efficient expression vector pUBPa harboring puroindoline a (Pina) was introduced into the bread wheat cultivar Zhongyou 9507-60 via biolistic transformation and transgenic plants were obtained. The integration of the foreign Pina gene was confirmed by PCR and genomic DNA Southern blot analysis. The levels of friabilin on the surface of water-washed starch granules varied among the transgenic lines. SDS-PAGE analysis of Triton X-114 extracted protein showed that the PINA protein was absent in three transgenic lines, indicating that the endogenous Pina gene most likely had been co-suppressed by the over-expression of the Pina transgene. SKCS kernel hardness and scanning electron microscopy analysis further confirmed the changes of kernel texture in these lines.     

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.     

Transformation of Pinb-D1x to soft wheat produces hard wheat kernel texture

Kernel hardness - a key quality trait of common wheat (Triticum aestivum L.) - is mainly conditioned by the Pina and Pinb genes. Mutation or deletion of Pina or Pinb increases kernel hardness, resulting in a hard wheat kernel texture. Here, Pinb-D1x gene was cloned from a hard wheat landrace Kashibaipi and transformed into a soft wheat cultivar Yangmai19 to assess its effect on kernel hardness and flour properties. PCR, RT-PCR and Western blot data confirmed the successful transformation and overexpression of Pinb-D1x gene in transgenic offsprings. The data of single kernel characterization system and scanning electron microscopy revealed that the introduction of Pinb-D1x in soft wheat increased the kernel hardness significantly and changed the internal structure of the kernel. Similarly, transgenic lines exhibited hard wheat like flour properties; flour whiteness and pasting temperature were significantly reduced in the transgenic lines, while the total protein content, damaged starch content, and compound parameter in the Mixograph tests (PT × TW value) showed a significant increase over the wildtype. The results showed that the transformation of the Pinb variants is a powerful strategy to alter the kernel hardness and flour properties in wheat breeding.     

Analysis of Pina and Pinb alleles in the micro-core collections of Chinese wheat germplasm by Ecotilling and identification of a novel Pinb allele

Kernel hardness is mainly conditioned by allelic variations of Pina-D1 and Pinb-D1 genes located on the short arm of chromosome 5D. In this work, the Ecotilling approach was optimized to investigate Pina and Pinb alleles in the micro-core collections of Chinese wheat germplasm, and three Pina and eight Pinb alleles were found. Generally, more Pinb alleles were detected in the accessions coming from the regions that grow winter or a mixture of spring and winter wheats. This was particularly evident for the Southwestern winter wheat, Xinjiang winter–spring wheat and Yellow and Huai River Valley winter wheat regions. A novel variant (designated as Pinb-D1x) was discovered in one of the accessions from the Xinjiang winter–spring wheat region. Compared to wild type (WT) allele Pinb-D1a, two nucleotide substitutions occurred in the coding region of Pinb-D1x, one (at nucleotide position 257) resulting in the replacement of a WT cysteine residue by tyrosine and the other (at nucleotide position 382) creating a premature stop codon. The implications of our data to understanding the diversity of Pina and Pinb alleles in wheat and to future molecular breeding of wheat kernel hardness are discussed.     

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.     

Identification of new puroindoline genotypes and their relationship to flour texture among wheat cultivars

Puroindoline genotypes (Pina and Pinb) and their encoded proteins related to grain hardness were studied in various common wheat cultivars from Australia, China, Japan, Korea and North America. Most of the hard wheats had the Pinb-D1b genotype with a glycine to serine mutation at position 46. In addition to the known Pina and Pinb genotypes, cultivars were found with Pina and Pinb double-null mutations (Pina-D1b/Pinb-D1h (t)) and a new Pinb frameshift mutation (designated Pinb-D1i (t)) within the region encoding a tryptophan-rich domain. This new Pinb frameshift mutation was found only in Chinese cultivars. Endosperm proteins encoded by Pina and Pinb in these cultivars were analysed by 2D-gel electrophoresis (IPG×SDS-PAGE). Cultivars with Pina and Pinb double-null mutations showed no PIN-a or PIN-b protein, and cultivars with Pinb-D1i (t) had no PIN-b protein. Surprisingly, cultivars with Pinb-D1b had severely reduced amounts of PIN-b and cultivars with Pinb-D1c showed no PIN-b proteins. Grain hardness among cultivars having mutated Pinb may be explained by the amount of PIN-b protein and not by the type of amino acid substitutions.     

Development of simple and co-dominant PCR markers to genotype puroindoline a and b alleles for grain hardness in bread wheat (Triticum aestivum L.)

Grain hardness is one of the most important quality characteristics of cultivated bread wheat (Triticum aestivum L.). A large deletion in the puroindoline a (Pina) gene or single nucleotide polymorphisms (SNPs) in the puroindoline b (Pinb) gene results in hard grain texture. So far, nine Pina alleles (Pina-D1a–Pina-D1b, Pina-D1k–Pina-D1q) and seventeen Pinb alleles (Pinb-D1a–Pinb-D1g, Pinb-D1p–Pinb-D1ab) have been identified in bread wheat. The major Pina and Pinb alleles identified in hard wheat cultivars are Pina-D1b, Pinb-D1b, Pinb-D1c and Pinb-D1d. In this study, a three-primer PCR system was employed to develop nine co-dominant STS markers for genotyping Pina-D1a and Pina-D1b, whereas temperature-switch (TS) PCR was used to develop six co-dominant SNP markers for genotyping the Pinb-D1a, Pinb-D1b, Pinb-D1c and Pinb-D1d alleles. These STS and TS-PCR markers were used to verify the grain hardness genotype of 100 wheat cultivars. The reliability and genotyping accuracy of TS-PCR markers were confirmed through sequencing of PCR products and a comparison with previously published results. Therefore, STS and TS-PCR markers offer a simple, cost-effective and reliable method for high-throughput genotyping Pina and Pinb alleles to select grain hardness in wheat quality breeding programs and for wheat market classification.     

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.     

Analysis of Pina and Pinb alleles in the micro-core collections of Chinese wheat germplasm by Ecotilling and identification of a novel Pinb allele

Kernel hardness is mainly conditioned by allelic variations of Pina-D1 and Pinb-D1 genes located on the short arm of chromosome 5D. In this work, the Ecotilling approach was optimized to investigate Pina and Pinb alleles in the micro-core collections of Chinese wheat germplasm, and three Pina and eight Pinb alleles were found. Generally, more Pinb alleles were detected in the accessions coming from the regions that grow winter or a mixture of spring and winter wheats. This was particularly evident for the Southwestern winter wheat, Xinjiang winter–spring wheat and Yellow and Huai River Valley winter wheat regions. A novel variant (designated as Pinb-D1x) was discovered in one of the accessions from the Xinjiang winter–spring wheat region. Compared to wild type (WT) allele Pinb-D1a, two nucleotide substitutions occurred in the coding region of Pinb-D1x, one (at nucleotide position 257) resulting in the replacement of a WT cysteine residue by tyrosine and the other (at nucleotide position 382) creating a premature stop codon. The implications of our data to understanding the diversity of Pina and Pinb alleles in wheat and to future molecular breeding of wheat kernel hardness are discussed.     

Wheat (Triticum aestivum L.) puroindoline functionality in bread making and its impact on bread quality

Wheat puroindolines (PINs) spontaneously adsorb at air/water interfaces and show excellent foaming properties. They can positively impact bread quality, in which the formation of stable foam is important for product quality. The impact of endogenous PINs on bread quality was studied by preparing gluten–starch blends from isolated gluten and starch fractions with different PIN levels, which allowed largely retaining the interaction between PINs and flour components. Our results indicate that blends with high PIN levels yielded more homogeneous crumb structures with fine gas cells than bread made with blends containing medium or low PIN levels. However, the mechanism by which PINs exert this crumb improving effect is not clear. Varying PIN levels impacted neither dough extensibility nor did it result in different PIN levels in dough liquor. Lipid removal yielded bread with a less homogeneous crumb gas cell distribution, indicating that lipids also are required to obtain good crumb structure.     

Variation in polar lipid composition among near-isogenic wheat lines possessing different puroindoline haplotypes

The exact mechanism underlying wheat (Triticum aestivum L.) kernel hardness is unknown. Similar to puroindoline proteins, polar lipids are present on the surface of starch granules. The objective of this research was to determine the specific polar lipid species present on the surface of wheat starch from near-isogenic wheat lines that have different puroindoline haplotypes and endosperm hardness. Four near-isogenic wheat lines were used in this study, all derived from the soft cultivar Alpowa. Direct infusion tandem mass spectrometry was used to identify the lipid species in whole-meal, flour and starch samples. Endosperm hardness had no significant effect on the polar lipid contents in wheat whole-meal, a slight influence on the polar lipid contents of the flour fractions and a significant influence on the polar lipid composition of the polar lipids located on the surface of wheat starch. The greatest quantities of polar lipids on the starch-surface occurred when both puroindoline proteins were present in their wild-type form. Starch-surface polar lipid content dramatically decreased when one of the puroindoline proteins was null or if pin-B was in the mutated form. The least amount of polar lipids was present when pin-B was in its mutated form and pin-A was in its wild-type form.     

Identification of new Pina null mutations among Asian common wheat cultivars

Wheat grain hardness is one of the most important phenotypes related to milling, baking and noodle making. Either a mutation of the Puroindoline-a (Pina) gene or Puroindoline-b (Pinb) gene results in hard grain texture. A deletion mutation of Pina (Pina-D1b) is widely distributed among common wheat cultivars. Although North/South American and Australian cultivars and their descendants have a 15-kbp deletion in common, two new types of deletion mutation were found among Asian wheat cultivars. A 4.4-kbp deletion was found in one Korean and two Chinese wheat cultivars beginning at position +371 within the Pina coding region. The other, a 10.4-kbp deletion, was found in three Chinese and nine Japanese wheat cultivars, including five Japanese landraces, beginning at position −5112. It caused the deletion of the full-length Pina gene. These findings suggest that Asian wheat cultivars are genetically distinct from those in other regions. The 4.4-kbp and 10.4-kbp deletion mutants were designated as Pina-D1r and Pina-D1s, respectively.