Seed-specific expression of the wheat puroindoline genes improves maize wet milling yields

The texture of maize ( Zea mays L.) seeds is important to seed processing properties, and soft dent maize is preferred for both wet-milling and livestock feed applications. The puroindoline genes ( Pina and Pinb ) are the functional components of the wheat ( Triticum aestivum L.) Hardness locus and together function to create soft grain texture in wheat. The PINs (PINA and PINB) are believed to act by binding to lipids on the surface of starch granules, preventing tight adhesion between starch granules and the surrounding protein matrix during seed maturation. Here, maize kernel structure and wet milling properties were successfully modified by the endosperm-specific expression of wheat Pins ( Pina and Pinb ). Pins were introduced into maize under the control of a maize γ- Zein promoter. Three Pina/Pinb expression positive transgenic lines were evaluated over two growing seasons. Textural analysis of the maize seeds indicated that the expression of PINs decreased adhesion between starch and protein matrix and reduced maize grain hardness significantly. Reduction in pressure required to fracture kernels ranged from 15.65% to 36.86% compared with control seeds. Further, the PINs transgenic maize seeds had increased levels of extractable starch as characterized by a small scale wet milling method. Starch yield was increased by 4.86% on average without negatively impacting starch purity. The development of softer maize hybrids with higher starch extractability would be of value to maize processors.     

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.     

Increased Resistance to Penicillium Seed Rot in Transgenic Wheat Over‐expressing Puroindolines

Puroindolines (PINs) are the main components of the wheat grain hardness locus (Ha) and have in vitro antimicrobial activity against bacteria and fungi. Here, we examined the effect of variation in PINA and/or PINB content upon Penicillium sp. seed fungal growth inhibition. The Penicillium sp. assays were germination assays performed after incubating seeds in Penicillium sp. contaminated soil. The first set of wheat genotypes consisted of two sets of transgenic isolines created in the varieties ‘Bobwhite’ and ‘Hi‐Line’ having over‐expression of PINA and/or PINB. The second set of genotypes consisted of near‐isogenic lines (NILs) varying for mutations in PINA or PINB created in the varieties ‘Explorer’ and ‘Hank’. After incubation in Penicillium sp.‐infected soil, transgenic wheat seeds over‐expressing PINA in both ‘Hi‐Line’ and ‘Bobwhite’ and both PINs in ‘Hi‐Line’ exhibited significantly reduced fungal infection and increased germination. No significant differences in Penicillium sp. infection or germination rates were observed in seeds of the NILs. The results indicate that puroindolines native role in seeds is to increase seed viability and that when over‐expressed as transgenes, the puroindolines are effective antifungal proteins.     

Wheat puroindolines interact to form friabilin and control wheat grain hardness

Wheat grain is sold based upon several physiochemical characteristics, one of the most important being grain texture. Grain texture in wheat directly affects many end use qualities such as milling yield, break flour yield, and starch damage. The hardness (Ha) locus located on the short arm of chromosome 5D is known to control grain hardness in wheat. This locus contains the puroindoline A (pina) and puroindoline B (pinb) genes. All wheats to date that have mutations in pina or pinb are hard textured, while wheats possessing both the soft type pina-D1a and pinb-D1a sequences are soft. Furthermore, it has been shown that complementation of the pinb-D1b mutation in hard spring wheat can restore a soft phenotype. Here, our objective was to identify and characterize the effect the puroindoline genes have on grain texture independently and together. To accomplish this we transformed a hard red spring wheat possessing a pinb-D1b mutation with soft type pina and pinb, creating transgenic isolines that have added pina, pinb, or pina and pinb. Northern blot analysis of developing control and transgenic lines indicated that grain hardness differences were correlated with the timing of the expression of the native and transgenically added puroindoline genes. The addition of PINA decreased grain hardness less than the reduction seen with added PINB. Seeds from lines having more soft type PINB than PINA were the softest. Friabilin abundance was correlated with the presence of both soft type PINA and PINB and did not correlate well with total puroindoline abundance. The data indicates that PINA and PINB interact to form friabilin and together affect wheat grain texture.Communicated by J. Dvorak     

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.     

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.     

Overexpression of Puroindoline a gene in transgenic durum wheat (Triticum turgidum ssp. durum) leads to a medium–hard kernel texture

Durum wheat is the second-most widely grown wheat species, and is primarily used in the production of pasta and couscous. The grain utilization of durum wheat is partly related to its very hard kernel texture because of the lack of the D genome and consequentially the Puroindoline genes. Our previous study reported the transformation of durum wheat with the Puroindoline a (Pina) gene. Here, we characterized the transgenic durum wheat lines expressing the Pina gene, and studied the effects of PINA on grain texture and other kernel characteristics. SDS-PAGE and Western blotting results demonstrated that starch-bound PINA levels of Pina-overexpressing lines were lower than that of Pina-positive control, common wheat cv. Chinese Spring, suggesting a weak association of PINA protein with starch granules in the absence of Pinb. Grain hardness analysis and flour milling tests indicated that the overexpression of PINA resulted in decreased grain hardness and increased flour yield in transgenic durum wheat lines. The agronomic performance of the transgenic and control lines was also examined and it was found that no significant differences in measured traits were observed between Pina-overexpressing and control lines in the 2-year field trials. Since grain hardness strongly affects milling and end-use qualities, the development of medium–hard-textured durum wheat lines is not only of significance for our knowledge of grain hardness and Puroindolines, but also has practical implications for plant breeders and food technologists for the expansion of utilization of durum wheat.     

Characterization of puroindolines in the control of endosperm texture in common wheat lines with substitutions of homeologous group-5 chromosomes

The genetic control of grain hardness and its association with the specific friabilin content on starch granules of common wheat cultivars and lines with intervarietal substitutions of homeologous group-5 chromosomes were studied. A significant correlation was revealed between the technological parameters of grain hardness (mean size of flour particles) and the specific content of puroindolines on the starch surface estimated in terms of starch doses. The results obtained allowed the method of starch doses to be used to identify soft and hard wheat cultivars and lines based on an analysis of a single grain. The biochemical analysis confirmed the previously obtained estimates of flour-grinding properties of wheat cultivars and substitution lines and allowed specific genotypes to be characterized according to the composition of puroindolines. The influence of chromosomes 5D and 5A of donor wheat cultivars on the activity of the Ha loci of recipient cultivars was revealed and found to be associated with the composition of PIN products and with the expression of the Pina-D1 and Pinb-D1 genes.     

Genome organisation and retrotransposon driven molecular evolution of the endosperm Hardness (Ha) locus in Triticum aestivum cv Glenlea

Wheat endosperm texture is controlled primarily by a locus (Ha), which comprises Gsp-1, Pina and Pinb genes encoding the so-called grain softness protein, puroindoline-a and puroindoline-b, respectively. Pina and Pinb were detected only on the D-genome of hexaploid wheat and its diploid progenitors while Gsp-1 was on all three homoeologous loci. Hexaploid cultivar Glenlea has a hard phenotype due to a null Pina genotype (D-genome) but the sequence organization is not reported. This study aimed at understanding the evolution of homoeologous Ha loci. Sequencing of three BAC clones from cv Glenlea was performed and sequence analyses delimited the Ha loci which spanned 3,925, 5,330 and 31,607 bp in the A-, B- and D-genomes, respectively. A solo LTR of Angela retroelement, downstream to Gsp-A1 and a fragment of Sabrina retroelement, downstream of Gsp-B1, were discovered. We propose that the insertion of these elements into the intergenic regions have driven the deletions of genomic segments harbouring Pina and Pinb genes in the A- and B-genomes of hexaploid wheat. Similarly, fragments of Romani and Vagabond retroelements were identified between truncated Pina and Pinb genes, indicating their role in the deletion of Pina in Glenlea, leading to its hard texture. Structural differences of the Ha locus region of the A-genome between two hexaploid wheat varieties namely Glenlea and Renan (CR626929), suggested the presence of more than one tetraploid ancestor in the origin of hexaploid wheat.     

Creation and functional analysis of new Puroindoline alleles in Triticum aestivum

The Hardness (Ha) locus controls grain texture and affects many end-use properties of wheat (Triticum aestivum L.). The Ha locus is functionally comprised of the Puroindoline a and b genes, Pina and Pinb, respectively. The lack of Pin allelic diversity is a major factor limiting Ha functional analyses and wheat quality improvement. In order to create new Ha alleles, a 630 member M(2) population was produced in the soft white spring cultivar Alpowa using ethylmethane sulfonate mutagenesis. The M(2) population was screened to identify new alleles of Pina and Pinb. Eighteen new Pin alleles, including eight missense alleles, were identified. F(2) populations for four of the new Pin alleles were developed after crossing each back to non-mutant Alpowa. Grain hardness was then measured on F(2:3) seeds and the impact of each allele on grain hardness was quantified. The tested mutations were responsible for between 28 and 94% of the grain hardness variation and seed weight and vigor of all mutation lines was restored among the F(2) populations. Selection of new Pin alleles following direct phenotyping or direct sequencing is a successful approach to identify new Ha alleles useful in improving wheat product quality and understanding Ha locus function.     

Evidence of physical interactions of puroindoline proteins using the yeast two-hybrid system

The puroindoline proteins PINA and PINB play key roles in determining wheat grain texture and also have potential antimicrobial roles. Many recent studies show that their roles in grain texture involve some interaction or interdependence, and their antimicrobial activity may also involve formation of protein complexes. The issue of whether any homo- and/or heteromeric associations occur amongst the PIN proteins is thus critical for understanding their biological functions and exploiting them for grain texture modifications or antimicrobial applications, but is as yet unresolved. This work has utilised the well-established yeast two-hybrid system to directly address this issue. The results confirm occurrence of in vivo interactions between the two PIN proteins for the first time, and show that PINB interacts with itself and also interacts, although somewhat weakly, with PINA, while PINA is a weaker interactor. The results explain the many reported observations suggesting a co-operative interaction between the two proteins and provide a rapid and efficient tool for testing the effects of various alleles/mutations on the interactions and lipid binding properties of these proteins, which are of functional significance to grain texture and antimicrobial defence functions.     

Recurrent deletions of puroindoline genes at the grain hardness locus in four independent lineages of polyploid wheat

Polyploidy is known to induce numerous genetic and epigenetic changes but little is known about their physiological bases. In wheat, grain texture is mainly determined by the Hardness (Ha) locus consisting of genes Puroindoline a (Pina) and b (Pinb). These genes are conserved in diploid progenitors but were deleted from the A and B genomes of tetraploid Triticum turgidum (AB). We now report the recurrent deletions of Pina-Pinb in other lineages of polyploid wheat. We analyzed the Ha haplotype structure in 90 diploid and 300 polyploid accessions of Triticum and Aegilops spp. Pin genes were conserved in all diploid species and deletion haplotypes were detected in all polyploid Triticum and most of the polyploid Aegilops spp. Two Pina-Pinb deletion haplotypes were found in hexaploid wheat (Triticum aestivum; ABD). Pina and Pinb were eliminated from the G genome, but maintained in the A genome of tetraploid Triticum timopheevii (AG). Subsequently, Pina and Pinb were deleted from the A genome but retained in the A(m) genome of hexaploid Triticum zhukovskyi (A(m)AG). Comparison of deletion breakpoints demonstrated that the Pina-Pinb deletion occurred independently and recurrently in the four polyploid wheat species. The implications of Pina-Pinb deletions for polyploid-driven evolution of gene and genome and its possible physiological significance are discussed.     

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     

粗山羊草(Aegilops tauschii)中Pinb基因的克隆和表达分析

puroindoline a(Pina)和puroindoline b(Pinb)是控制小麦籽粒硬度的主效基因。根据已报道的小麦Pinb基因的保守序列,设计合成了一对特异性引物,对粗山羊草Aegilops tauschii(DD)的基因组DNA进行Pinb基因扩增、克隆和序列分析,发现了一个新型Pinb等位基因。该基因长447 bp,编码148个氨基酸残基,具有麦类作物PinB蛋白所特有的WPTKWWK色氨酸结构域和10个半胱氨酸所形成的5个二硫键结构。与软粒小麦cv.Capitole的Pinb-D1a相比较,该基因含有14个氨基酸变异位点,其中包括一个紧邻色氨酸结构域的变异位点(Val66Phe),其核苷酸和氨基酸同源性分别为93.3%和90.5%。RT-PCR和Western Blot证实了Pinb基因在籽粒胚乳中的表达。Southern Blot分析结果表明,粗山羊草中Pinb基因为单拷贝。研究结果表明,粗山羊草中包含着与小麦差异较大的籽粒硬度控制基因,对此基因的进一步研究将加深对小麦籽粒硬度形成分子机制的了解。     

The Wheat Puroindoline Genes Confer Fungal Resistance in Transgenic Corn

Puroindoline a and b (Pina and Pinb), together make up the functional components of the wheat grain hardness locus (Ha) and have antimicrobial properties. The antifungal activity of puroindoline proteins, PINA and PINB, has been demonstrated in vitro and in vivo. In this study, Pina and Pinb were introduced into corn under the control of a corn Ubiquitin promoter. Two Pina/Pinb expression–positive transgenic events were evaluated for resistance to Cochliobolus heterostrophus, the corn southern leaf blight (SLB) pathogen. Transgenic corn expressing Pins showed significantly increased tolerance to C. heterostrophus, averaging 42.1% reduction in symptoms. Pins are effective in vivo as antifungal proteins and could be valuable tools in corn SLB control.     

Kernel softness in wheat is determined by starch granule bound Puroindoline proteins

Wheat has a vital position in agriculture because it is a staple food for masses and variation in grain hardness governs its applications. Soft wheats have softer endosperm texture that mills easily, so needs less energy to mill, produces smaller particles, and small amount of starch is damaged after milling as compared to hard wheat. Soft texture results from higher level of friabilin whereas hard texture results from low level of friabilin on starch granule surface. Friabilin, a marker of kernel texture is primarily composed of Puroindolines (PINs) and its genes (Pins) are located on the Hardness (Ha) locus. The Pins are the molecular-genetic basis of kernel softness in wheat. When both Pins are in their ‘wild state’ (Pina-D1a and Pinb-D1a), wheat kernel is soft. Absence or mutation in one of the Pins results in hard grain texture with different effects on end use and milling qualities. Pina-D1b genotypes gave harder grain texture, higher protein content, water absorption of flour, damaged starch granules and greater flour yield than hard wheat. Recently, other Pins like genes, Pin b variant genes located on the long arm of chromosome 7A were reported in bread wheat with more than 70% similarity to Pinb (Pinb-D1a) at the DNA level. Other genes located on chromosomes 1A, 2A, 5A, 7A, 5B, 2D and 6D also affect kernel texture. However the main determinants are the variants in the allelic diversity of Puroindoline family genes. Contemporary studies show that Pins are multifunctional family of genes having a range of functions from grain hardness to natural defense against insects and pathogens such as viruses, bacteria and fungi.     

高大山羊草Aegilops longissima puroindoline b基因的克隆与表达分析

籽粒硬度是小麦加工品质的重要影响因素。puroindoline a（Pina）和puroindoline b（Pinb）是控制小麦籽粒硬度的主效基因。根据已报导的小麦Pinb基因的保守序列,设计合成了一对特异性引物,对高大山羊草Aegilops longissima（SS）的基因组DNA和胚乳RNA进行Pinb基因扩增、克隆、序列测定和表达分析,发现了一个新型Pinb等位基因。基因长360bp,编码119个氨基酸残基,对应于麦类作物Puroindoline B（PinB）成熟蛋白的结构区域,具有麦类作物Pinb基因特有的WPTKWWK的色氨酸结构域基因序列和10个半胱氨酸所形成的5个二硫键结构。与软粒小麦cv．Capitole的Pinb—D1a相比较,其核苷酸和氨基酸同源性分别为93．3％和92．4％。RT—PCR证实了Pinb基因在籽粒胚乳中的表达。研究结果表明,高大山羊草中包含着与小麦差异较大的籽粒硬度控制基因,为栽培小麦品质改良提供了丰富的遗传资源。     

The ectopic expression of the wheat Puroindoline genes increase germ size and seed oil content in transgenic corn

Plant oil content and composition improvement is a major goal of plant breeding and biotechnology. The Puroindoline a and b (PINA and PINB) proteins together control whether wheat seeds are soft or hard textured and share a similar structure to that of plant non-specific lipid-transfer proteins. Here we transformed corn (Zea mays L.) with the wheat (Triticum aestivum L.) puroindoline genes (Pina and Pinb) to assess their effects upon seed oil content and quality. Pina and Pinb coding sequences were introduced into corn under the control of a corn Ubiquitin promoter. Three Pina/Pinb expression positive transgenic events were evaluated over two growing seasons. The results showed that Pin expression increased germ size significantly without negatively impacting seed size. Germ yield increased 33.8% while total seed oil content was increased by 25.23%. Seed oil content increases were primarily the result of increased germ size. This work indicates that higher oil content corn hybrids having increased food or feed value could be produced via puroindoline expression.