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.     

New Frame Shift Mutation in Puroindoline B in Indian Wheat Cultivars Hyb65 and NI5439

Puroindoline genes pinA and pinB are the main components of the 15 kD friabilin protein reported to be associated with kernel softness. However, grain hardness of Hyb65 and NI5439, the two Indian wheat varieties, could not be explained based on the earlier identified alleles in puroindolines in wheat. Hyb65 and NI5439 are hard but based on the earlier identified allelic forms of puroindolines both the varieties could have been soft. In this investigation, puroindolines (a and b) from Hyb65 and NI5439 were characterised to understand their role in determining grain hardness. The sequence of puroindoline genes from both the varieties indicated that there was no mutation in pinA. However, there was frame shift mutation in pinB generated by insertion of a guanine residue 126 bp downstream from the start codon in both the varieties. This created new hardness allele of pinB designated as pinb-D1h. Frame shift also culminated into stop codon (TGA) 231 bp downstream from the start codon terminating protein synthesis at 77^th amino acid position. Five more stop codons (4TGA & 1TAG) were also created to the downstream positions of the first stop codon because of frame shift. There was additional point mutation in NI5439 (transition from A to G) resulting into change of amino acid residue from thymine to arginine at 205^th nucleotide position. Thus single nucleotide change in pinB resulted into truncated pin B and consequently the harder texture.     

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.     

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

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

A microarray analysis of wheat grain hardness

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

Molecular and biochemical characterization of puroindoline a and b alleles in Chinese landraces and historical cultivars

Kernel hardness that is conditioned by puroindoline genes has a profound effect on milling, baking and end-use quality of bread wheat. In this study, 219 landraces and 166 historical cultivars from China and 12 introduced wheats were investigated for their kernel hardness and puroindoline alleles, using molecular and biochemical markers. The results indicated that frequencies of soft, mixed and hard genotypes were 42.7, 24.3, and 33.0%, respectively, in Chinese landraces and 45.2, 13.9, and 40.9% in historical cultivars. The frequencies of PINA null, Pinb-D1b and Pinb-D1p genotypes were 43.8, 12.3, and 39.7%, respectively, in hard wheat of landraces, while 48.5, 36.8, and 14.7%, respectively, in historical hard wheats. A new Pinb-D1 allele, designated Pinb-D1t, was identified in two landraces, Guangtouxianmai and Hongmai from the Guizhou province, with the characterization of a glycine to arginine substitution at position 47 in the coding region of Pinb gene. Surprisingly, a new Pina-D1 allele, designated Pina-D1m, was detected in the landrace Hongheshang, from the Jiangsu province, with the characterization of a proline to serine substitution at position 35 in the coding region of Pina gene; it was the first novel mutation found in bread wheat, resulting in a hard endosperm with PINA expression. Among the PINA null genotypes, an allele designed as Pina-D1l, was detected in five landraces with a cytosine deletion at position 265 in Pina locus; while another novel Pina-D1 allele, designed as Pina-D1n, was identified in six landraces, with the characterization of an amino acid change from tryptophan-43 to a ‘stop’ codon in the coding region of Pina gene. The study of puroindoline polymorphism in Chinese wheat germplasm could provide useful information for the further understanding of the molecular basis of kernel hardness in bread wheat.     

Wheat puroindolines enhance fungal disease resistance in transgenic rice

Antimicrobial peptides play a role in the immune systems of animals and plants by limiting pathogen infection and growth. The puroindolines, endosperm-specific proteins involved in wheat seed hardness, are small proteins reported to have in vitro antimicrobial properties. Rice, the most widely used cereal crop worldwide, normally does not contain puroindolines. Transgenic rice plants that constitutively express the puroindoline genes pinA and/or pinB throughout the plants were produced. PIN extracts of leaves from the transgenic plants reduced in vitro growth of Magnaporthe grisea and Rhizoctonia solani, two major fungal pathogens of rice, by 35 to 50%. Transgenic rice expressing pinA and/or pinB showed significantly increased tolerance to M. grisea (rice blast), with a 29 to 54% reduction in symptoms, and R. solani (sheath blight), with an 11 to 22% reduction in symptoms. Puroindolines are effective in vivo in antifungal proteins and could be valuable new tools in the control of a wide range of fungal pathogens of crop plants.     

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

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

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.     

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

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

Puroindolines: the molecular genetic basis of wheat grain hardness

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

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease.

A bacteriophage T4 gene which functions to inhibit Escherichia coli Lon protease has been identified. This pin (proteolysis inhibition) gene was selected for its ability to support plaque formation by a lambda Ots vector at 40 degrees C. Southern blot experiments indicated that this T4 gene is included within the 4.9-kilobase XbaI fragment which contains gene 49. Subcloning experiments showed that T4 gene 49.1 (designated pinA) is responsible for the ability of the Ots vector to form plaques at 40 degrees C. Deficiencies in Lon protease activity are the only changes known in E. coli that permit lambda Ots phage to form plaques efficiently at 40 degrees C. lon+ lysogens of the lambda Ots vector containing pinA permitted a lambda Ots phage to form plaques efficiently at 40 degrees C. Furthermore, these lysogens, upon comparison with similar lysogens lacking any T4 DNA, showed reduced levels of degradation of puromycyl polypeptides and of canavanyl proteins. The lon+ lysogens that contained pinA exhibited other phenotypic characteristics common to lon strains, such as filamentation and production of mucoid colonies. Levels of degradation of canavanyl proteins were essentially the same, however, in null lon lysogens which either contained or lacked pinA. We infer from these data that the T4 pinA gene functions to block Lon protease activity; pinA does not, however, appear to block the activity of proteases other than Lon that are involved in the degradation of abnormal proteins.     

A conserved mechanism for nitrile metabolism in bacteria and plants

Pseudomonas fluorescens SBW25 is a plant growth-promoting bacterium that efficiently colonises the leaf surfaces and rhizosphere of a range of plants. Previous studies have identified a putative plant-induced nitrilase gene ( pinA ) in P. fluorescens SBW25 that is expressed in the rhizosphere of sugar beet plants. Nitrilase enzymes have been characterised in plants, bacteria and fungi and are thought to be important in detoxification of nitriles, utilisation of nitrogen and synthesis of plant hormones. We reveal that pinA is a NIT4-type nitrilase that catalyses the hydrolysis of β-cyano- l -alanine, a nitrile common in the plant environment and an intermediate in the cyanide detoxification pathway in plants. In plants cyanide is converted to β-cyano- l -alanine, which is subsequently detoxified to aspartic acid and ammonia by NIT4. In P. fluorescens SBW25 pinA is induced in the presence of β-cyano- l -alanine, and the β-cyano- l -alanine precursors cyanide and cysteine. pinA allows P. fluorescens SBW25 to use β-cyano- l -alanine as a nitrogen source and to tolerate toxic concentrations of this nitrile. In addition, pinA is shown to complement a NIT4 mutation in Arabidopsis thaliana , enabling plants to grow in concentrations of β-cyano- l -alanine that would otherwise prove lethal. Interestingly, over-expression of pinA in wild-type A. thaliana not only resulted in increased growth in high concentrations of β-cyano- l -alanine, but also resulted in increased root elongation in the absence of exogenous β-cyano- l -alanine, demonstrating that β-cyano- l -alanine nitrilase activity can have a significant effect on root physiology and root development.     

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。     

Relevance of physiological efficiency in wheat grain quality and the prospects of improvement

Released and pre-released bread wheat varieties evaluated in national wheat programme of India (503 genotypes) during 2005–14 under different environments were examined for the role of physiological parameters in grain quality. Genotypes with slow plant height growth but faster rate of grain filling enhanced protein content. Plants where growth in height and grain development was slow, grains were hard, provided proportionate vegetative growth phase is longer. Steady grain growth rate benefited gluten strength and gluten quality. Irrespective of total crop duration, longer reproductive phase was an effective indicator of higher flour recovery and test weight. Magnitude and significance of morphological attributes in grain quality was almost similar to that of physiological processes, therefore prospects of utilizing these field traits were examined to enhance grain properties. Early heading and longer grain filling was effective to increase test weight whereas delayed heading and shorter plant height enhanced protein content. Bold grains hampered grain hardness and delayed heading added more bran in the flour. Genotypes with poor grain bearing and quick grain ripening had lower sedimentation value. Instead of protein, it was wet gluten which expressed negative association with yield. To improvise gluten quality, extended reproductive phase but with less grain weight was helpful. Contribution of longer post-anthesis period was observed crucial in flour recovery. These useful simple field expressions can be deployed to uplift quality of wheat grains.     

Nap Hal, an Indian Landrace of Wheat, Contains Unique Genes for Better Biscuit Making Quality

Nap Hal, an Indian landrace of wheat, exhibited unique characteristics suitable for biscuit making quality. Double null trait at Glu-D1 locus in Nap Hal was associated with reduced gluten strength as reflected in low sedimentation volume (16.0 ml, 6 g test), lower Farinograph peak time (2.0 min) and lower tolerance (higher break down 140 BU) to mixing. PCR analysis of puroindoline genes showed the presence of both pinA and glycine type pinB associated with soft grain texture of wheat. This is the first report that Nap Hal contains unique combination of soft grain characteristics determined by puroindolines as well as low gluten strength determined by null alleles at Glu-D1 locus. The combination of both soft grain characteristics and weak gluten is useful in developing varieties suitable for biscuit making where the use of chemical improvers can be reduced.     

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.     

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     

Characterization of wheat puroindoline proteins

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