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.     

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.     

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.     

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。     

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.     

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.     

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。     

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.     

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.     

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.     

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.     

Immunodetection and immunolocalization of tryptophanins in oat (Avena sativa L.) seeds

Tryptophanins (TRPs) are low molecular weight, tryptophan-rich, basic proteins found in oat (Avena sativa L.) seeds. Like their counterpart puroindolines (PINs) from wheat (Triticum aestivum L.), TRPs are thought to be involved in flour softness as well as disease resistance against phytopathogenic fungi. PINs are known to be the major components of ‘friabilin’ associated with the surface of water washed starch grains and possess lipid binding properties. Two polyclonal antisera against puroindoline-a (PIN-a), and puroindoline-b (PIN-b) respectively; and a monoclonal antiserum raised against ‘friabilin’ were used as primary antibodies in immunoblotting experiments. All antisera detected immunoreactive polypeptides, with approximate relative masses of 15–16 kDa, in oat, wheat, and barley (Hordeum vulgare L.) seed extracts but not in rice (Oryza sativa L.), maize (Zea mays L.), bean (Phaseolus vulgaris L.), pea (Pisum sativum L.) and lentil (Lens culinaris Medic.) seed extracts. Immunoreactive polypeptides were detected in aqueous ethanol [52% (v/v) ethanol] seed extracts. Both anti-‘friabilin’ monoclonal and anti-PIN-b polyclonal antisera recognized 15 as well as 16 kDa tryptophanins in oat seeds from different cultivars. On the other hand, anti-PIN-a polyclonal antiserum strongly cross-reacted with 16 kDa TRP and weakly with 15 kDa TRP. Tryptophanins were found to be associated with the surface of starch grains in oat endosperm tissue using both fluorescence and confocal laser scanning microscopies with anti-‘friabilin’ monoclonal antiserum. SDS-PAGE and immunoblotting assays revealed a gradual synthesis of TRPs as early as milk stage in developing oat seeds. On the other hand, TRPs tend to undergo degradation during seed germination.     

Ectopic expression of bacterial amylopullulanase enhances bioethanol production from maize grain

Key message

Heterologous expression of amylopullulanase in maize seeds leads to partial starch degradation into fermentable sugars, which enhances direct bioethanol production from maize grain.

Abstract

Utilization of maize in bioethanol industry in the United States reached ±13.3 billion gallons in 2012, most of which was derived from maize grain. Starch hydrolysis for bioethanol industry requires the addition of thermostable alpha amylase and amyloglucosidase (AMG) enzymes to break down the α-1,4 and α-1,6 glucosidic bonds of starch that limits the cost effectiveness of the process on an industrial scale due to its high cost. Transgenic plants expressing a thermostable starch-degrading enzyme can overcome this problem by omitting the addition of exogenous enzymes during the starch hydrolysis process. In this study, we generated transgenic maize plants expressing an amylopullulanase (APU) enzyme from the bacterium Thermoanaerobacter thermohydrosulfuricus. A truncated version of the dual functional APU (TrAPU) that possesses both alpha amylase and pullulanase activities was produced in maize endosperm tissue using a seed-specific promoter of 27-kD gamma zein. A number of analyses were performed at 85 °C, a temperature typically used for starch processing. Firstly, enzymatic assay and thin layer chromatography analysis showed direct starch hydrolysis into glucose. In addition, scanning electron microscopy illustrated porous and broken granules, suggesting starch autohydrolysis. Finally, bioethanol assay demonstrated that a 40.2 ± 2.63 % (14.7 ± 0.90 g ethanol per 100 g seed) maize starch to ethanol conversion was achieved from the TrAPU seeds. Conversion efficiency was improved to reach 90.5 % (33.1 ± 0.66 g ethanol per 100 g seed) when commercial amyloglucosidase was added after direct hydrolysis of TrAPU maize seeds. Our results provide evidence that enzymes for starch hydrolysis can be produced in maize seeds to enhance bioethanol production.     

Fractionation of transgenic corn seed by dry and wet milling to recover recombinant collagen‐related proteins

Corn continues to be considered an attractive transgenic host for producing recombinant therapeutic and industrial proteins because of its potential for producing recombinant proteins at large volume and low cost as coproducts of corn seed‐based biorefining. Efforts to reduce production costs have been primarily devoted to increasing accumulation level, optimizing protein extraction conditions, and simplifying the purification. In the present work, we evaluated two grain fractionation methods, dry milling and wet milling, to enrich two recombinant collagen‐related proteins; thereby, reducing the amount and type of corn‐derived impurities in subsequent protein extraction and purification steps. The two proteins were a full‐length human recombinant collagen type I alpha 1(rCIα1) chain with telopeptides and peptide foldon to effect triple helix formation and a 44‐kDa rCIα1 fragment. For each, ～60% of the rCIα1s in the seed was recovered in the dry‐milled germ‐rich fractions making up ca. 25% of the total kernel mass. For wet milling, ～60% of each was recovered in three fractions accounting for 20–25% of the total kernel mass. The rCIα1s in the dry‐milled germ‐rich fractions were enriched three to six times compared with the whole corn kernel, whereas the rCIα1s were enriched 4–10 times in selected wet‐milled fractions. The recovered starch from wet milling was almost free of rCIα1. Therefore, it was possible to generate rCIα1‐enriched fractions by both dry and wet milling along with rCIα1‐free starch using wet milling. Because of its simplicity, the dry milling procedure could be accomplished on‐farm thus minimizing the risk of inadvertent release of viable transgenic seeds. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Increasing maize seed weight by enhancing the cytoplasmic ADP-glucose pyrophosphorylase activity in transgenic maize plants

ADP-glucose pyrophosphorylase (AGPase) plays a key role in regulating starch biosynthesis in cereal seeds and is likely the most important determinant of seed strength. The Escherichia coli mutant glgC gene (glgC16), which encodes a highly active and allosterically insensitive AGPase, was introduced into maize (Zea mays L.) under the control of an endosperm-specific promoter. Developing seeds from transgenic maize plants showed up to 2–4-fold higher levels of AGPase activity in the presence of 5 mM inorganic phosphate (Pi). Transgenic plants with higher cytoplasmic AGPase activity under Pi-inhibitory conditions showed increases (13–25%) in seed weight over the untransformed control. In addition, in all transgenic maize plants, the seeds were fully filled, and the seed number of transgenic plants had no significant difference compared with that of untransformed control. These results indicate that increasing cytoplasmic AGPase activity has a marked effect on sink activity and, in turn, seed weight in transgenic maize plants.     

牡山羊草Aegilops juvenalis Puroindoline b基因的克隆

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,对六倍体牡山羊草Aegilops juvenalis（UUMMDD）的基因组DNA和胚乳eDNA进行Pinb基因扩增、克隆和序列测定,发现了两个新型Pinb等位基因Pinb-allele-1和Pinb-allele-2。该基因全长360bp,编码119个氨基酸残基。它编码的蛋白和麦类作物Puroindoline B（PinB）的成熟蛋白有非常高的同源性,具有麦类作物PinB蛋白所特有的WPTKWWK的色氨酸结构域和10个半胱氨酸所形成的5个二硫键结构。与软粒小麦ev．Capitole的Pinb-D1a相比较,其核苷酸同源性为93．1％、93．3％,氨基酸同源性为90．8％、92．4％。Pinb。allele。1和Pinb—allele一2分别含有11和9个氨基酸变异位点。RT—PCR证实了Pinb—allele一2基因在籽粒胚乳中的表达。SouthernBlot分析结果表明,牡山羊草中含有两个拷贝的Pinb基因,其中包含着与小麦差异较大的籽粒硬度控制基因。     

Soft and Hard Textured Wheat Differ in Starch Properties as Indicated by Trimodal Distribution,Morphology, Thermal and Crystalline Properties

Starch and proteins are major components in the wheat endosperm that affect its end product quality. Between the two textural classes of wheat i.e. hard and soft, starch granules are loosely bound with the lipids and proteins in soft wheat due to higher expression of interfering grain softness proteins. It might have impact on starch granules properties. In this work for the first time the physiochemical and structural properties of different sized starch granules (A-, B- and C-granules) were studied to understand the differences in starches with respect to soft and hard wheat. A-, B- and C-type granules were separated with >95% purity. Average number and proportion of A-, B-, and C-type granules was 18%, 56%, 26% and 76%, 19%, 5% respectively. All had symmetrical birefringence pattern with varied intensity. All displayed typical A-type crystallites. A-type granules also showed V-type crystallinity that is indicative of starch complexes with lipids and proteins. Granules differing in gelatinization temperature (ΔH) and transition temperature (ΔT), showed different enthalpy changes during heating. Substitution analysis indicated differences in relative substitution pattern of different starch granules. Birefringence, percentage crystallinity, transmittance, gelatinization enthalpy and substitution decreased in order of A>B>C being higher in hard wheat than soft wheat. Amylose content decreased in order of A>B>C being higher in soft wheat than hard wheat. Reconstitution experiment showed that starch properties could be manipulated by changing the composition of starch granules. Addition of A-granules to total starch significantly affected its thermal properties. Effect of A-granule addition was higher than B- and C-granules. Transmittance of the starch granules paste showed that starch granules of hard wheat formed clear paste. These results suggested that in addition to differences in protein concentration, hard and soft wheat lines have differences in starch composition also.     

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.