J. M. Martin

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.

Analysis of barley (Hordeum vulgare) leaf senescence and protease gene expression: a family C1A cysteine protease is specifically induced under conditions characterized by high carbohydrate, but low to moderate nitrogen levels.

SUMMARY Senescence is the highly regulated last developmental phase of plant organs and tissues, and is optimized to allow nutrient remobilization to surviving plant parts, such as seeds of annual crops. High leaf carbohydrate to nitrogen (C : N) ratios have been implicated in the induction or acceleration of the senescence process. *A combination of phloem interruption in mature leaves (by steam-girdling, leading to carbohydrate accumulation from photosynthesis) and varied nitrate supply was used to analyse correlations between metabolite levels, leaf senescence parameters and induction of protease genes and proteolytic activities. *Its strong induction under conditions characterized by high C : N ratios, negative correlation of its transcript levels with chlorophylls and nitrates, its strong induction during developmental leaf senescence and its predicted localization to a lytic vacuolar compartment indicate that, among the genes tested, a family C1A cysteine protease is most likely to participate in bulk protein degradation during barley leaf senescence. *While all the genes analysed were selected based on upregulation during leaf senescence in a previous transcriptomic study, a considerably more detailed picture of protease gene regulation emerged from the data presented here, underlining the usefulness of this experimental approach for further (functional) protease characterization.

Complementation of the pina (null) allele with the wild type Pina sequence restores a soft phenotype in transgenic wheat.

The tightly linked puroindoline genes, Pina and Pinb, control grain texture in wheat, with wild type forms of both giving soft, and a sequence alteration affecting protein expression or function in either giving rise to hard wheat. Previous experiments have shown that addition of wild type Pina in the presence of mutated Pinb gave intermediate grain texture but addition of wild type Pinb gave soft grain. This raises questions as to whether Pina may be less functional than Pinb. Our goal here was to develop and characterize wheat lines expressing the wild type Pina-D1a sequence in hard wheat with the null mutation (Pina-D1b) for Pina. Three transgenic lines plus Bobwhite were evaluated in two environments. Grain texture, grain protein, and kernel weight were determined for the transgenic lines and Bobwhite. The three transgenic lines had soft phenotype, and none of the transgenic lines differed from Bobwhite for grain protein or kernel weight. The soft phenotype was accompanied by increases in Pina transcript accumulation. Total Triton X-114 extractable PINA and PINB increased from 2.5 to 5.5 times those from a soft wheat reference sample, and friabilin, PINA and PINB bound to starch, increased from 3.8 to 7.8 times those of the soft wheat reference. Bobwhite showed no starch bound PINA, but transgenic lines had levels from 5.3 to 13.7 times those of the soft wheat reference sample. Starch bound PINB in transgenic lines also increased from 0.9 to 2.5 times that for the soft wheat reference sample. The transgenic expression of wild type Pina sequence in the Pina null genotype gave soft grain with the characteristics of soft wheat including increased starch bound friabilin. The results support the hypothesis that both wild type Pin genes need to be present for friabilin formation and soft grain.

Relationship of Dough Extensibility to Dough Strength in a Spring Wheat Cross

ABSTRACT A negative relationship between dough strength and dough extensibility would pose a problem for breeding hard wheats, as both dough strength and dough extensibility are desirable. We derived 77 recombinant inbred lines (RIL) from a cross between hard red spring wheat cultivars McNeal and Thatcher. McNeal produces flour with stronger dough and lower extensibility than does Thatcher. RIL were evaluated for strength-related properties using mixograph analysis and extensibility parameters using the Kieffer attachment to the TA.XT2 texture analyzer. Additionally, the RIL were test baked. Measurements using the mixograph and the Kieffer attachment were highly heritable. Maximum dough extensibility (Extmax) was negatively correlated with resistance to extension (Rmax) (r = -0.74) and with mixograph tolerance (r = -0.45). Loaf volume was correlated with both Rmax (r = 0.42) and area under the extensigraph curve (r = 0.44) based on partial correlation analysis adjusted for protein differences. Extmax was ...

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 M2 population was produced in the soft white spring cultivar Alpowa using ethylmethane sulfonate mutagenesis. The M2 population was screened to identify new alleles of Pina and Pinb. Eighteen new Pin alleles, including eight missense alleles, were identified. F2 populations for four of the new Pin alleles were developed after crossing each back to non-mutant Alpowa. Grain hardness was then measured on F2:3 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 F2 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.

Reduced Amylose Effects on Bread and White Salted Noodle Quality

ABSTRACT Amylose content in wheat endosperm is controlled by three Wx loci, and the proportion of amylose decreases with successive accumulation of Wx null alleles at the three loci. The proportion of amylose is believed to influence end-use quality of bread and Asian noodles. The objectives of this study were to determine influence of the allelic difference at Wx-B1 locus on bread quality, bread firmness, and white salted noodle texture in a spring wheat cross segregating for the Wx-B1 locus and in a set of advanced spring wheat breeding lines differing in allelic state at the Wx- B1 locus. In addition, we examined the relationship between amylose content and flour swelling properties on bread and noodle traits. Fifty-four recombinant inbred lines of hard white spring wheat plus parents were grown in replicated trials in two years, and 31 cultivars and breeding lines of hard spring wheat were grown in two locations. Bread and white salted noodles were processed from these trials. The presence of the Wx-B...

Generation mean analysis to identify and partition the components of genetic resistance to Septoria nodorum in wheat

SummaryFoliar symptom severity of seedlings artificially inoculated with S. nodorum were used to idenify the type of gne action controlling resistance to this pathogen in the early generations of two wheat crosses. In both crosses a resistant spring wheat cultivar was crossed to a susceptible cultivar. Reciprocal crosses were included in the analysis to determine if the cytoplasm contributed in any significant degree to the level of resistance present.Results indicated that resistance was polygenic and that it could be explaned prinerpally by additive gene effects. Some differences in reciprocal crosses were evident, but a significant role for the eytoplasm in resistance is not indicated.

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 gamma-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.

Effect of Variation in Amylose Content and Puroindoline Composition on Bread Quality in a Hard Spring Wheat Population

ABSTRACT Milling and breadbaking quality of hard-textured wheat may be influenced by alternative alleles at the Wx loci controlling percent amylose in the endosperm, and the puroindoline (pin) loci controlling grain hardness. For this experiment, we developed recombinant inbred lines (RIL) from a cross between Choteau spring wheat cultivar and experimental line MTHW9904. Choteau has the PinB-D1b mutation conferring grain hardness and the Wx-B1a allele at the Wx-B1 locus conferring wild-type amylose content. MTHW9904 has the PinA-D1b allele conferring grain hardness and the Wx-B1b allele conferring lower amylose content, causing a partial waxy phenotype. RIL with the PinB-D1b mutation (n = 49) had significantly softer kernels, higher break flour yield, and higher loaf volume than lines with the PinA-D1b mutation (n = 38). Lines with partial waxy phenotype due to Wx-B1b (n = 43) had significantly lower kernel weight, lower amylose content, and higher flour swelling power than lines with wild-type starch due...

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.