The ectopic expression of the wheat Puroindoline genes increase germ size and seed oil content in transgenic corn.
ABSTRACT: 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.
Project description:In planta analysis of protein function in a crop plant could lead to improvements in understanding protein structure/function relationships as well as selective agronomic or end product quality improvements. The requirements for successful in planta analysis are a high mutation rate, an efficient screening method, and a trait with high heritability. Two ideal targets for functional analysis are the Puroindoline a and Puroindoline b (Pina and Pinb, respectively) genes, which together compose the wheat (Triticum aestivum L.) Ha locus that controls grain texture and many wheat end-use properties. Puroindolines (PINs) together impart soft texture, and mutations in either PIN result in hard seed texture. Studies of the PINs' mode of action are limited by low allelic variation. To create new Pin alleles and identify critical function-determining regions, Pin point mutations were created in planta via EMS treatment of a soft wheat. Grain hardness of 46 unique PIN missense alleles was then measured using segregating F(2):F(3) populations. The impact of individual missense alleles upon PIN function, as measured by grain hardness, ranged from neutral (74%) to intermediate to function abolishing. The percentage of function-abolishing mutations among mutations occurring in both PINA and PINB was higher for PINB, indicating that PINB is more critical to overall Ha function. This is contrary to expectations in that PINB is not as well conserved as PINA. All function-abolishing mutations resulted from structure-disrupting mutations or from missense mutations occurring near the Tryptophan-rich region. This study demonstrates the feasibility of in planta functional analysis of wheat proteins and that the Tryptophan-rich region is the most important region of both PINA and PINB.
Project description:BACKGROUND: Grain texture is one of the most important characteristics in bread wheat (Triticum aestivum L.). Puroindoline-D1 genes play the main role in controlling grain texture and are intimately associated with the milling and processing qualities in bread wheat. RESULTS: A series of diagnostic molecular markers and dCAPS markers were used to characterize Pina-D1 and Pinb-D1 in 493 wheat cultivars from diverse geographic locations. A primer walking strategy was used to characterize PINA-null alleles at the DNA level. Results indicated that Chinese landraces encompassing 12 different Puroindoline-D1 allelic combinations showed the highest diversity, while CIMMYT wheat cultivars containing 3 different Puroindoline-D1 allelic combinations showed the lowest diversity amongst wheat cultivars from the five countries surveyed. Two novel Pina-D1 alleles, designated Pina-D1s with a 4,422-bp deletion and Pina-D1u with a 6,460-bp deletion in the Ha (Hardness) locus, were characterized at the DNA level by a primer walking strategy, and corresponding molecular markers Pina-N3 and Pina-N4 were developed for straightforward identification of the Pina-D1s and Pina-D1u alleles. Analysis of the association of Puroindoline-D1 alleles with grain texture indicated that wheat cultivars with Pina-null/Pinb-null allele, possessing an approximate 33-kb deletion in the Ha locus, have the highest SKCS hardness index amongst the different genotypes used in this study. Moreover, wheat cultivars with the PINA-null allele have significantly higher SKCS hardness index than those of Pinb-D1b and Pinb-D1p alleles. CONCLUSIONS: Molecular characterization of the Puroindoline-D1 allele was investigated in bread wheat cultivars from five geographic regions, resulting in the discovery of two new alleles - Pina-D1s and Pina-D1u. Molecular markers were developed for both alleles. Analysis of the association of the Puroindoline-D1 alleles with grain texture showed that cultivars with PINA-null allele possessed relatively high SKCS hardness index. This study can provide useful information for the improvement of wheat quality, as well as give a deeper understanding of the molecular and genetic processes controlling grain texture in bread wheat.
Project description:BACKGROUND:Secaloindoline a (Sina) and secaloindoline b (Sinb) genes of hexaploid triticale (x Triticosecale Wittmack) are orthologs of puroindoline a (Pina) and puroindoline b (Pinb) in hexaploid wheat (Triticum aestivum L.). It has already been proven that RNA interference (RNAi)-based silencing of Pina and Pinb genes significantly decreased the puroindoline a and puroindoline b proteins in wheat and essentially increased grain hardness (J Exp Bot 62:4025-4036, 2011). The function of Sina and Sinb in triticale was tested by means of RNAi silencing and compared to wheat. RESULTS:Novel Sina and Sinb alleles in wild-type plants of cv. Wanad were identified and their expression profiles characterized. Alignment with wheat Pina-D1a and Pinb-D1a alleles showed 95% and 93.3% homology with Sina and Sinb coding sequences. Twenty transgenic lines transformed with two hpRNA silencing cassettes directed to silence Sina or Sinb were obtained by the Agrobacterium-mediated method. A significant decrease of expression of both Sin genes in segregating progeny of tested T1 lines was observed independent of the silencing cassette used. The silencing was transmitted to the T4 kernel generation. The relative transcript level was reduced by up to 99% in T3 progeny with the mean for the sublines being around 90%. Silencing of the Sin genes resulted in a substantial decrease of secaloindoline a and secaloindoline b content. The identity of SIN peptides was confirmed by mass spectrometry. The hardness index, measured by the SKCS (Single Kernel Characterization System) method, ranged from 22 to 56 in silent lines and from 37 to 49 in the control, and the mean values were insignificantly lower in the silent ones, proving increased softness. Additionally, the mean total seed protein content of silenced lines was about 6% lower compared with control lines. Correlation coefficients between hardness and transcript level were weakly positive. CONCLUSIONS:We documented that RNAi-based silencing of Sin genes resulted in significant decrease of their transcripts and the level of both secaloindoline proteins, however did not affect grain hardness. The unexpected, functional differences of Sin genes from triticale compared with their orthologs, Pin of wheat, are discussed.
Project description:Gene silencing by RNA interference is a particularly important tool in the study of gene function in polyploid cereal species for which the collections of natural or induced mutants are very limited. Previously we have been testing small interfering RNA-based approach of gene silencing in wheat and triticale. In this research, artificial microRNAs (amiRs) were studied in the same species and the same target genes to compare effectiveness of both gene silencing pathways. amiR cassettes were designed to silence Puroindoline a (Pina) and Puroindoline b (Pinb) hardness genes in wheat and their orthologues Secaloindoline a (Sina) and Secaloindoline b (Sinb) genes in triticale. Each of the two cassettes contained 21 nt microRNA (miR) precursor derived from conserved regions of Pina/Sina or Pinb/Sinb genes, respectively. Transgenic plants were obtained with high efficiency in two cultivars of wheat and one cultivar of triticale after using the Pinb-derived amiR vector for silencing of Pinb or Sinb, respectively. Lack of transgenic plants in wheat or very low transformation efficiency in triticale was observed using the Pina-derived amiR cassette, despite large numbers of embryos attempted. Silencing of Pinb in wheat and Sinb in triticale was highly efficient in the T1 generation. The transcript level of Pinb in wheat was reduced up to 92% and Sinb in triticale was reduced up to 98%. Moreover, intended silencing of Pinb/Sinb with Pinb-derived amiR cassette was highly correlated with simultaneous silencing of Pina/Sina in the same transgenic plants. High downregulation of Pinb/Pina genes in T1 plants of wheat and Sinb/Sina genes in T1 plants of triticale was associated with strong expression of Pinb-derived amiR. Silencing of the target genes correlated with increased grain hardness in both species. Total protein content in the grains of transgenic wheat was significantly lower. Although, the Pinb-derived amiR cassette was stably inherited in the T2 generation of wheat and triticale the silencing effect including strongly decreased expression of silenced genes as well as strong expression of Pinb-derived amiR was not transmitted. Advantages and disadvantages of posttranscriptional silencing of target genes by means of amiR and siRNA-based approaches in polyploid cereals are discussed.
Project description:Grain hardness is an important quality trait of cereal crops. In wheat, it is mainly determined by the Hardness locus that harbors genes encoding puroindoline A (PINA) and puroindoline B (PINB). Any deletion or mutation of these genes leading to the absence of PINA or to single amino acid changes in PINB leads to hard endosperms. Although it is generally acknowledged that hardness is controlled by adhesion strength between the protein matrix and starch granules, the physicochemical mechanisms connecting puroindolines and the starch-protein interactions are unknown as of this time. To explore these mechanisms, we focused on PINA. The overexpression in a hard wheat cultivar (cv. Courtot with the Pina-D1a and Pinb-D1d alleles) decreased grain hardness in a dose-related effect, suggesting an interactive process. When PINA was added to gliadins in solution, large aggregates of up to 13 ?m in diameter were formed. Turbidimetry measurements showed that the PINA-gliadin interaction displayed a high cooperativity that increased with a decrease in pH from neutral to acid (pH 4) media, mimicking the pH change during endosperm development. No turbidity was observed in the presence of isolated ?- and ?-gliadins, but non-cooperative interactions of PINA with these proteins could be confirmed by surface plasmon resonance. A significant higher interaction of PINA with ?-gliadins than with ?-gliadins was observed. Similar binding behavior was observed with a recombinant repeated polypeptide that mimics the repeat domain of gliadins, i.e., (Pro-Gln-Gln-Pro-Tyr)8. Taken together, these results suggest that the interaction of PINA with a monomeric gliadin creates a nucleation point leading to the aggregation of other gliadins, a phenomenon that could prevent further interaction of the storage prolamins with starch granules. Consequently, the role of puroindoline-prolamin interactions on grain hardness should be addressed on the basis of previous observations that highlight the similar subcellular routing of storage prolamins and puroindolines.
Project description:Kernel hardness, which has great influence on the end-use properties of common wheat, is mainly controlled by Puroindoline genes, Pina and Pinb. Using EcoTILLING platform, we herein investigated the allelic variations of Pina and Pinb genes and their association with the Single Kernel Characterization System (SKCS) hardness index in a diverse panel of wheat germplasm.The kernel hardness varied from 1.4 to 102.7, displaying a wide range of hardness index. In total, six Pina and nine Pinb alleles resulting in 15 genotypes were detected in 1787 accessions. The most common alleles are the wild type Pina-D1a (90.4%) and Pina-D1b (7.4%) for Pina, and Pinb-D1b (43.6%), Pinb-D1a (41.1%) and Pinb-D1p (12.8%) for Pinb. All the genotypes have hard type kernel hardness of SKCS index (>60.0), except the wild types of Pina and Pinb combination (Pina-D1a/Pinb-D1a). The most frequent genotypes in Chinese and foreign cultivars was Pina-D1a/Pinb-D1b (46.3 and 39.0%, respectively) and in Chinese landraces was Pina-D1a/Pinb-D1a (54.2%). The frequencies of hard type accessions are increasing from 35.5% in the region IV, to 40.6 and 61.4% in the regions III and II, and then to 77.0% in the region I, while those of soft type are accordingly decreasing along with the increase of latitude. Varieties released after 2000 in Beijing, Hebei, Shandong and Henan have higher average kernel hardness index than that released before 2000.The kernel hardness in a diverse panel of Chinese wheat germplasm revealed an increasing of kernel hardness generally along with the latitude across China. The wild type Pina-D1a and Pinb-D1a, and one Pinb mutant (Pinb-D1b) are the most common alleles of six Pina and nine Pinb alleles, and a new double null genotype (Pina-D1x/Pinb-D1ah) possessed relatively high SKCS hardness index. More hard type varieties were released in recent years with different prevalence of Pin-D1 combinations in different regions. This work would benefit the understanding of the selection and molecular processes of kernel hardness across China and different breeding stages, and provide useful information for the improvement of wheat quality in China.
Project description:Grain hardness is an important quality trait that influences product development in wheat. This trait is governed by variation in puroindoline proteins (PINA and PINB). Our study evaluated 551 Indian wheat germplasm lines for diversity in Pina and Pinb genes. Eighty-two lines were shortlisted for full length sequencing and grain hardness studies. Sequencing studies identified six unknown alleles: two for the Pina gene and four for the Pinb gene. Five of them were novel with non-synonymous changes in the corresponding amino acid sequences. Identified mutations in the deduced mature proteins and their pre- and pro-peptides influenced the hardness characteristics of the grain. We classified these 82 varieties into different hardness categories with reference to international and Indian systems of classification. The majority of Indian wheat varieties were categorized as hard. This study revealed that unexplored Indian wheat germplasm can be a good source of genetic variability for both Pina and Pinb genes, helping in marker-assisted breeding and in obtaining wheat with different textural properties.
Project description:Kernel hardness is a key trait of wheat seeds, largely controlled by two tightly linked genes Puroindoline a and b (Pina and Pinb). Genes homologous to Pinb, namely Pinb2, have been studied. Whether these genes contribute to kernel hardness and other important seed traits remains inconclusive. Using the high-quality bread wheat reference genome, we show that PINB2 are encoded by three homoeologous loci Pinb2 not syntenic to the Hardness locus, with Pinb2-7A locus containing three tandem copies. PINB2 proteins have several features conserved for the Pin/Pinb2 phylogenetic cluster but lack a structural basis of significant impact on kernel hardness. Pinb2 are seed-specifically expressed with varied expression levels between the homoeologous copies and among wheat varieties. Using the high-quality genome information, we developed new Pinb2 allele specific markers and demonstrated their usefulness by 1) identifying new Pinb2 alleles in Triticeae species; and 2) performing an association analysis of Pinb2 with kernel hardness. The association result suggests that Pinb2 genes may have no substantial contribution to kernel hardness. Our results provide new insights into Pinb2 evolution and expression and the new allele-specific markers are useful to further explore Pinb2's contribution to seed traits in wheat.
Project description:Puroindoline (Pina and Pinb) genes control grain texture or hardness in wheat. Wild-type/soft alleles lead to softer grain while a mutation in one or both of these genes results in a hard grain. Variation in hardness in genotypes with identical Pin alleles (wild-type or mutant) is known but the molecular basis of this is not known. We now report the identification of wheat genotypes with hard grain texture and wild-type/soft Pin alleles indicating that hardness in wheat may be controlled by factors other than mutations in the coding region of the Pin genes. RNA-Seq analysis was used to determine the variation in the transcriptome of developing grains of thirty three diverse wheat genotypes including hard (mutant Pin) and soft (wild type) and those that were hard without having Pin mutations. This defined the role of pin gene expression and identified other candidate genes associated with hardness. Pina was not expressed in hard wheat with a mutation in the Pina gene. The ratio of Pina to Pinb expression was generally lower in the hard non mutant genotypes. Hardness may be associated with differences in Pin expression and other factors and is not simply associated with mutations in the PIN protein coding sequences.
Project description:Wheat end-use quality mainly derives from two interrelated characteristics: the compositions of gluten proteins and grain hardness. The composition of gluten proteins determines dough rheological properties and thus confers the unique viscoelastic property on dough. One group of gluten proteins, high molecular weight glutenin subunits (HMW-GS), plays an important role in dough functional properties. On the other hand, grain hardness, which influences the milling process of flour, is controlled by Puroindoline a (Pina) and Puroindoline b (Pinb) genes. However, little is known about the combined effects of HMW-GS and PINs on dough functional properties. In this study, we crossed a Pina-expressing transgenic line with a 1Ax1-expressing line of durum wheat and screened out lines coexpressing 1Ax1 and Pina or lines expressing either 1Ax1 or Pina. Dough mixing analysis of these lines demonstrated that expression of 1Ax1 improved both dough strength and over-mixing tolerance, while expression of PINA detrimentally affected the dough resistance to extension. In lines coexpressing 1Ax1 and Pina, faster hydration of flour during mixing was observed possibly due to the lower water absorption and damaged starch caused by PINA expression. In addition, expression of 1Ax1 appeared to compensate the detrimental effect of PINA on dough resistance to extension. Consequently, coexpression of 1Ax1 and PINA in durum wheat had combined effects on dough mixing behaviors with a better dough strength and resistance to extension than those from lines expressing either 1Ax1 or Pina. The results in our study suggest that simultaneous modulation of dough strength and grain hardness in durum wheat could significantly improve its breadmaking quality and may not even impair its pastamaking potential. Therefore, coexpression of 1Ax1 and PINA in durum wheat has useful implications for breeding durum wheat with dual functionality (for pasta and bread) and may improve the economic values of durum wheat.