Brian S. Beecher

Physical mapping and a new variant of Puroindoline b-2 genes in wheat

Puroindoline a and b proteins soften the endosperm of wheat kernels. When the underlying puroindoline genes are altered by mutation or are deleted, kernels become harder. Thus, puroindoline a and b (Pina and Pinb) play an important role in wheat quality and utilization. Recently, additional Pinb genes have been reported. In the present report, we provide corroborating coding and additional 5′ and 3′ flanking sequence for three Pinb variants: Pinb-2v1, Pinb-2v2, and Pinb-2v3. Additionally, a new Pinb variant, Pinb-2v4, is reported. All four variants were physically mapped using Chinese Spring (CS) diteolosomics, nullisomic–tetrasomics, and CS-Cheyenne disomic substitution lines. Results place Pinb-2v1 on 7DL, Pinb-2v2 on 7BL, Pinb-2v3 on 7B, and Pinb-2v4 on 7AL. Pinb-2v1 and Pinb-2v4 were present in all cvs. examined: CS, Cheyenne, Recital, Wichita and Winsome. Pinb-2v2 was present in CS and Recital; Pinb-2v3 was present in Cheyenne, Wichita, and Winsome. These results are not wholly consistent with prior research and additional studies will be required to reconcile discrepancies. The discovery of Pinb-2v4 and the mapping of all four variants will contribute to a better understanding of gene duplication events in wheat and their bearing on wheat kernel texture and grain utilization.

Polyphenol oxidase (PPO) in wheat and wild relatives: molecular evidence for a multigene family

Wheat polyphenol oxidase (PPO) is the major cause of browning reactions that discolor Asian noodles and other wheat products. It has been hypothesized that genes encoding wheat PPOs may have evolved by gene duplication into a multigene family. Here we characterized PPO genomic sequences from diploid (Triticum monococcum, T. urartu, Aegilops tauschii, and Ae. speltoides), tetraploid (T. turgidum, subspecies dicoccoides and durum) and hexaploid (T. aestivum cultivars Klasic and ID377s) wheat species to gain a better understanding of the structure and organization of PPO genes. DNA fragments were amplified from a highly polymorphic and phylogenetic informative region of the gene. As a result, we obtained highly discriminative sequences. Three distinct PPOs, obtained from the A genome of T. monococcum, provided evidence for gene duplication events (paralogous loci). Furthermore, the number of sequences obtained for bread and durum wheat was higher than the expected number of orthologous loci. Sequence comparison revealed nucleotide and structural diversity, and detected five sequence intron types, all with a common insertion position. This was hypothesized to be homologous to that of intron 2 of previously reported wheat PPOs. A MITE of the Stowaway family accounted for the major difference between the five intervening sequences, and was unique to T. aestivum cv. Klasic. Nucleotide and structural diversity, together with well-resolved phylogenetic trees, provided molecular evidence to support the hypothesis of a PPO multigene family structure and organization.

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.

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.

Characterization of a Unique “Super Soft” Kernel Trait in Wheat

ABSTRACT Kernel texture in wheat (Triticum sp.) is central to end-use quality and utilization. Here we report the discovery of a novel soft kernel trait in soft white winter wheat (T. aestivum L.). Two heritable kernel phenotypes were selected among F3-derived sibs, hereafter designated “normal soft” (wild-type) and “super soft.” Normal soft lines exhibited single kernel characterization system (SKCS) hardness index (HI) values typical of soft wheat (HI ≈ 20), whereas the super soft lines were unusually soft (HI ≈ 5). Under some environments, individual super soft lines exhibited HI values as low as HI = –4. The super soft trait was manifested in reduced SKCS kernel texture and higher break flour yields, with some increase in sodium carbonate SRC (solvent retention capacity) values and sponge cake volumes. Straight-grade flour yield, flour ash, milling score, and cookie diameter were largely unaffected. With the possible exception of the sodium carbonate SRC values, we observed no indication that the supe...

Associations Between Vrs1 Alleles and Grain Quality Traits in Spring Barley Hordeum vulgare L.1

ABSTRACT Barley head row type is a major trait affecting end-use quality. Six-rowed forms emerged due to mutations in the Vrs1 gene in two-rowed barleys. Whether barley is two (Vrs1) or six rowed (vrs1) directly affects a wide range of morphological traits related to seed yield and grain quality. Vrs1 has been cloned and encodes a homeodomain transcription factor with a linked leucine zipper motif. To test the association between Vrs1 alleles and grain quality, we characterized the Vrs1 alleles among a well-described collection of 81 spring barley accessions selected for divergence in head type and dry matter digestibility (DMD). The results indicated that the majority of two-rowed barleys have the Vrs1.b3-1 allele and the majority of six-rowed barleys carry the vrs1.a1-8 allele. In comparison with two-rowed barleys, six-rowed barleys were more variable in grain hardness. This divergence in hardness values was associated with specific vrs1 alleles, with barley accessions carrying the less severe amino aci...

Enhanced Rice Growth is Conferred by Increased Leaf ADP-Glucose Pyrophosphorylase Activity

Enhanced Rice Growth is Conferred by Increased Leaf ADP-Glucose Pyrophosphorylase Activity Modification of leaf starch levels may be employed in attempts to increase cereal yield. Few studies have examined leaf starch as a plant biomass limiting factor. Here we test the hypothesis that rice plant productivity may be increased by increasing leaf starch. Starch biosynthesis is controlled by the heterotetrameric rate-limiting enzyme ADP-glucose pyrophosphorylase (AGPase). Rice variety Nipponbare was transformed with a modified form of the maize endosperm AGPase large subunit gene, Sh2r6hs, as well as with the small subunit gene, Bt2, under control of a rice RuBisCO small subunit promoter. RNA sequencing results indicated that Sh2r6hs and Bt2 transcript levels were each greater than 20 times that of the native genes. Increased total AGPase activity was correlated with higher leaf starch accumulation at the end of the day.

A review of the occurrence of Grain softness protein-1 genes in wheat (Triticum aestivum L.)

Grain softness protein-1 (Gsp-1) is a small, 495-bp intronless gene found throughout the Triticeae tribe at the distal end of group 5 chromosomes. With the Puroindolines, it constitutes a key component of the Hardness locus. Gsp-1 likely plays little role in grain hardness, but has direct interest due to its utility in phylogeny and its role in arabinogalactan peptides. Further role(s) remain to be identified. In the polyploid wheats, Triticum aestivum and T. turgidum, the gene is present in a homoeologous series. Since its discovery, there have been conflicting reports and data as to the number of Gsp-1 genes and the level of sequence polymorphism. Little is known about allelic variation within a species. In the simplest model, a single Gsp-1 gene is present in each wheat and Aegilops tauschii genome. The present review critically re-examines the published and some unpublished data (sequence available in the NCBI nucleotide and MIPS Wheat Genome Databases). A number of testable hypotheses are identified, and include the level of polymorphism that may represent (and define) different Gsp-1 alleles, the existence of a fourth Gsp-1 gene, and the apparent, at times, high level of naturally-occurring or artifactual gene chimeras. In summary, the present data provide firm evidence for at most, three Gsp-1 genes in wheat, although there are numerous data that suggest a more complex model.