Analyzing the action of evolutionarily conserved modules on HMW-GS 1Ax1 promoter activity.

KEY MESSAGE: The specific and high-level expression of 1Ax1 is determined by different promoter regions. HMW-GS synthesis occurs in aleurone layer cells. Heterologous proteins can be stored in protein bodies. High-molecular-weight glutenin subunit (HMW-GS) is highly expressed in the endosperm of wheat and relative species, where their expression level and allelic variation affect the bread-making quality and nutrient quality of flour. However, the mechanism regulating HMW-GS expression remains elusive. In this study, we analyzed the distribution of cis-acting elements in the 2659-bp promoter region of the HMW-GS gene 1Ax1, which can be divided into five element-enriched regions. Fragments derived from progressive 5' deletions were used to drive GUS gene expression in transgenic wheat, which was confirmed in aleurone layer cells, inner starchy endosperm cells, starchy endosperm transfer cells, and aleurone transfer cells by histochemical staining. The promoter region ranging from - 297 to - 1 was responsible for tissue-specific expression, while fragments from - 1724 to - 618 and from - 618 to - 297 were responsible for high-level expression. Under the control of the 1Ax1 promoter, heterologous protein could be stored in the form of protein bodies in inner starchy endosperm cells, even without a special location signal. Our findings not only deepen our understanding of glutenin expression regulation, trafficking, and accumulation but also provide a strategy for the utilization of wheat endosperm as a bioreactor for the production of nutrients and metabolic products.

Effects of high-molecular-weight glutenin subunit combination in common wheat on the quality of crumb structure.

BACKGROUND: High-molecular-weight glutenin subunits (HMW-GSs) have important effects on bread-making quality. Allelic variations of HMW-GS in bread wheat varieties contribute in different ways to dough properties and bread volume. However, no systematic analysis has been done on the effects of allelic variation on bread-crumb structure, an important parameter when evaluating bread-making quality. In this study, seven Glu-1 deletion lines and one intact line harboring different encoding loci and derived from a cross between two spring wheat cultivars were used to investigate the contribution of a single Glu-1 locus, or combination of Glu-1 loci, to the crumb structure. RESULTS: Deletion of HMW-GS locus combinations resulted in a decline in slice size, brightness, and fineness of the bread crumb. A desirable crumb structure correlated well with preferred subunit combinations: high levels of GMPs, superior dough properties, and loaf volume. The effects of the HMW-GS combinations were ranked as Dx5 + Dy10 > Bx17 + By18 > Ax1 + Null. The Ax1 + Null allele affected the crumb structure by interacting with the Bx17 + By18 or Dx5 + Dy10. CONCLUSION: High-molecular-weight glutenin subunits had significant effects on the loaf volume and crumb structure; varying effects from different subunit combinations were observed. © 2018 Society of Chemical Industry.

Interaction between serine carboxypeptidase-like protein TtGS5 and Annexin D1 in developing seeds of Triticum timopheevi.

GS5 encoding a serine carboxypeptidase-like protein positively regulates grain size and weight through the regulation of grain width and filling and is helpful in improving cereal yields. Grain width variation determined by GS5 is associated with cell number and size, but the actual underlying mechanism is still unclear. Two orthologs of GS5, TtGS5-3A-G and TtGS5-3G-G, were cloned from the Triticum timopheevi accession no. CWI17006. To identify the proteins that interacted with TtGS5-3A-G and TtGS5-3G-G in premature grains, we performed pull-down assays followed by liquid chromatography-mass spectrometry/mass spectrometry analysis. The analyses revealed 18 proteins were present in both the TtGS5-3A-G and TtGS5-3G-G interactomes. Among five candidates selected, only Annexin D1 interacted with both TtGS5-3A-G and TtGS5-3G-G in yeast. Annexin D1, TtGS5-3A-G, and TtGS5-3G-G were located on the cytoplasmic membranes of Arabidopsis protoplasts and onion epidermal cells, and interactions between Annexin D1 and TtGS5-3A-G, as well as TtGS5-3G-G, were shown by bimolecular fluorescence complementation assays. Annexin D1 was expressed widely in different tissues, and it co-expressed with TtGS5-3A-G/TtGS5-3G-G at the grain enlargement phase. These results indicated that Annexin D1 interacted with TtGS5-3A-G and TtGS5-3G-G in premature grains. Together with the structural similarities of Annexin D1 to known fiber elongation factors, we proposed that TtGS5 might regulate the cell size by interacting with Annexin D1. The results provide significant new information for understanding the roles that GS5 plays in regulating grain size, which may be useful in improving crop yields.

Genome-wide identification of internal reference genes for normalization of gene expression values during endosperm development in wheat.

Internal reference genes that are stably expressed are essential for normalization in comparative expression analyses. However, gene expression varies significantly among species, organisms, tissues, developmental stages, stresses, and treatments. Therefore, identification of stably expressed reference genes in developmental endosperm of bread wheat is important for expression analysis of endosperm genes. As the first study to systematically screen for reference genes across different developmental stages of wheat endosperm, nine genes were selected from among 76 relatively stable genes based on high-throughput RNA sequencing data. The expression stability of these candidate genes and five traditional reference genes was assessed by real-time quantitative PCR combined with three independent algorithms: geNorm, NormFinder, and BestKeeper. The results showed that ATG8d was the most stable gene during wheat endosperm development, followed by Ta54227, while the housekeeping gene GAPDH, commonly used as an internal reference, was the least stable. ATG8d and Ta54227 together formed the optimal combination of reference genes. Comparative expression analysis of glutenin genes indicated that credible quantification could be achieved by normalization against ATG8d in developmental endosperm. The stably expressed gene characterized here can act as a proper internal reference for expression analysis of wheat endosperm genes, especially nutrient- and nutrient synthesis-related genes.