New Insights into the Organization, Recombination, Expression and Functional Mechanism of Low Molecular Weight Glutenin Subunit Genes in Bread Wheat

The bread-making quality of wheat is strongly influenced by multiple low molecular weight glutenin subunit (LMW-GS) proteins expressed in the seeds. However, the organization, recombination and expression of LMW-GS genes and their functional mechanism in bread-making are not well understood. Here we report a systematic molecular analysis of LMW-GS genes located at the orthologous Glu-3 loci (Glu-A3, B3 and D3) of bread wheat using complementary approaches (genome wide characterization of gene members, expression profiling, proteomic analysis). Fourteen unique LMW-GS genes were identified for Xiaoyan 54 (with superior bread-making quality). Molecular mapping and recombination analyses revealed that the three Glu-3 loci of Xiaoyan 54 harbored dissimilar numbers of LMW-GS genes and covered different genetic distances. The number of expressed LMW-GS in the seeds was higher in Xiaoyan 54 than in Jing 411 (with relatively poor bread-making quality). This correlated with the finding of higher numbers of active LMW-GS genes at the A3 and D3 loci in Xiaoyan 54. Association analysis using recombinant inbred lines suggested that positive interactions, conferred by genetic combinations of the Glu-3 locus alleles with more numerous active LMW-GS genes, were generally important for the recombinant progenies to attain high Zeleny sedimentation value (ZSV), an important indicator of bread-making quality. A higher number of active LMW-GS genes tended to lead to a more elevated ZSV, although this tendency was influenced by genetic background. This work provides substantial new insights into the genomic organization and expression of LMW-GS genes, and molecular genetic evidence suggesting that these genes contribute quantitatively to bread-making quality in hexaploid wheat. Our analysis also indicates that selection for high numbers of active LMW-GS genes can be used for improvement of bread-making quality in wheat breeding

The expression of a bean PGIP in transgenic wheat confers increased resistance to the fungal pathogen Bipolaris sorokiniana

A possible strategy to control plant pathogens is the improvement of natural plant defense mechanisms against the tools that pathogens commonly use to penetrate and colonize the host tissue. One of these mechanisms is represented by the host plant\u2019s ability to inhibit the pathogen\u2019s capacity to degrade plant cell wall polysaccharides. Polygalacturonase-inhibiting proteins (PGIP) are plant defense cell wall glycoproteins that inhibit the activity of fungal endopolygalacturonases (endo-PGs). To assess the effectiveness of these proteins in protecting wheat from fungal pathogens, we produced a number of transgenic wheat lines expressing a bean PGIP (PvPGIP2) having a wide spectrum of specificities against fungal PGs. Three independent transgenic lines were characterized in detail, including determination of the levels of PvPGIP2 accumulation and its subcellular localization and inhibitory activity. Results show that the transgene-encoded protein is correctly secreted into the apoplast, maintains its characteristic recognition specificities, and endows the transgenic wheat with new PG recognition capabilities. As a consequence, transgenic wheat tissue showed increased resistance to digestion by the PG of Fusarium moniliforme. These new properties also were confirmed at the plant level during interactions with the fungal pathogen Bipolaris sorokiniana. All three lines showed significant reductions in symptom progression (46 to 50%) through the leaves following infection with this pathogen. Our results illustrate the feasibility of improving wheat\u2019s defenses against pathogens by expression of proteins with new capabilities to counteract those produced by the pathogens

Wheat transgenic plants expressing a bean PGIP support a role for polygalacturonase activity in the initial stage of wheat infection by Fusarium graminearum

Fusarium graminearum is one of the predominant causal agents of Fusarium Head Blight (FHB) of wheat worldwide. This fungal pathogen produces trichothecene mycotoxins, including deoxynivalenol (DON). Transgenic wheat plants expressing the FsTRI101 gene, which encodes a DON acetyltransferase, were partially protected against the spread of F. graminearum. F. graminearum also secretes an array of enzymes to degrade cell wall polymers. Since some of these enzymes are controlled by apoplastic inhibitor proteins, we are analyzing the feasibility of increasing host resistance by manipulating the activity of these cell wall components. We report the effect of the ectopic expression of a bean polygalacturonase-inhibiting protein (PvPGIP2) in transgenic wheat plants, alone and in combination with the product of the FsTri101 gene, in protecting wheat plants against F. graminearum. We monitor FHB symptom progression in inoculated heads for 23 days. We show that transgenic plants expressing PvPGIP2 show a significant reduction of FHB symptoms from 9 to 23 dpi. A parallel analysis performed on wheat transgenic lines expressing FsTri101 showed a delayed protective effect starting from 15 to 23 dpi. We also evaluated the effects of combining the Pvpgip2 and FsTRI101 transgenes on the spread of the FHB symptoms. The hybrid lines carrying both transgenes show symptom reductions equal to either parental line, but an earlier reduction of FHB symptoms (from 11 to 23 dpi) than the parental line expressing only FsTRI101. These results demonstrate that PvPGIP2 can slow Fusarium infection and support a role for polygalacturonase activity in the initial stage of colonization

Changes in high molecular weight glutenin subunit composition can be genetically engineered without affecting wheat agronomic performance

The genomes of modern cultivars have been painstakingly selected for the presence of favorable alleles at multiple loci, which interact to produce superior phenotypes. Genetic transformation provides a tool to introduce new genes without altering the original gene combinations. However, the random genetic and epigenetic changes sometimes generated by the transformation process have been associated with losses in agronomic performance. The agronomic performance of 50 transgenic wheat (Triticum aestivum L.) lines containing additional copies of native or modified high molecular weight glutenin subunit (HMW-GS) genes and the selectable marker bar, their untransformed parent 'Bobwhite', four lines containing only bar, and 10 null segregant lines were assessed in small plot trials over 2 yr and three locations. Most of the transgenic lines did not show significant changes in performance relative to Bobwhite, although the transgenic lines as a group tended toward lower performance. Null-segregant and bar-only lines performed similarly to Bobwhite. No relationship could be established between performance and particular transgenes or their expression levels. Despite the overall lower performance of the transgenic lines, many with agronomic performance equivalent to Bobwhite were identified. These findings suggest that extant techniques for genetic engineering of wheat are capable of producing agronomically competitive lines for use as cultivars or parents in breeding programs. © Crop Science Society of America

Changes in high molecular weight glutenin subunit composition can be genetically engineered without affecting wheat agronomic performance

The genomes of modern cultivars have been painstakingly selected for the presence of favorable alleles at multiple loci, which interact to produce superior phenotypes. Genetic transformation provides a tool to introduce new genes without altering the original gene combinations. However, the random genetic and epigenetic changes sometimes generated by the transformation process have been associated with losses in agronomic performance. The agronomic performance of 50 transgenic wheat (Triticum aestivum L.) lines containing additional copies of native or modified high molecular weight glutenin subunit (HMW-GS) genes and the selectable marker bar, their untransformed parent 'Bobwhite', four lines containing only bar, and 10 null segregant lines were assessed in small plot trials over 2 yr and three locations. Most of the transgenic lines did not show significant changes in performance relative to Bobwhite, although the transgenic lines as a group tended toward lower performance. Null-segregant and bar-only lines performed similarly to Bobwhite. No relationship could be established between performance and particular transgenes or their expression levels. Despite the overall lower performance of the transgenic lines, many with agronomic performance equivalent to Bobwhite were identified. These findings suggest that extant techniques for genetic engineering of wheat are capable of producing agronomically competitive lines for use as cultivars or parents in breeding programs. © Crop Science Society of America

The structure and evolution of the human beta-globin gene family.

We present the results of a detailed comparison of the primary structure of human beta-like globin genes and their flanking sequences. Among the sequences located 5' to these genes are two highly conserved regions which include the sequences ATA and CCAAT located 31 +/- 1 and 77 +/- 10 bp, respectively, 5' to the mRNA capping site. Similar sequences are found in the corresponding locations in most other eucaryotic structural genes. Calculation of the divergence times of individual beta-like globin gene pairs provides the first description of the evolutionary relationships within a gene family based entirely on direct nucleotide sequence comparisons. In addition, the evolutionary relationship of the embryonic epsilon-globin gene to the other human beta-like globin genes is defined for the first time. Finally, we describe a model for the involvement of short direct repeat sequences in the generation of deletions in the noncoding and coding regions of beta-like globin genes during evolution

Transformation of wheat with high molecular weight subunit genes results in improved functional properties

The high molecular weight (HMW) subunits of wheat glutenin are major determinants of the elastic properties of gluten that allow the use of wheat doughs to make bread, pasta, and a range of other foods. There are both quantitative and qualitative effects of HMW subunits on the quality of the grain, the former being related to differences in the number of expressed HMW subunit genes. We have transformed bread wheat in order to increase the proportions of the HMW subunits and improve the functional properties of the flour. A range of transgene expression levels was obtained with some of the novel subunits present at considerably higher levels than the endogenous subunits. Analysis of T2 seeds expressing transgenes for one or two additional HMW subunits showed stepwise increases in dough elasticity, demonstrating the improvement of the functional properties of wheat by genetic engineering.&nbsp