Caixia Han

Dynamic development of starch granules and the regulation of starch biosynthesis in Brachypodium distachyon: comparison with common wheat and Aegilops peregrina

BackgroundThorough understanding of seed starch biosynthesis and accumulation mechanisms is of great importance for agriculture and crop improvement strategies. We conducted the first comprehensive study of the dynamic development of starch granules and the regulation of starch biosynthesis in Brachypodium distachyon and compared the findings with those reported for common wheat (Chinese Spring, CS) and Aegilops peregrina.ResultsOnly B-granules were identified in Brachypodium Bd21, and the shape variation and development of starch granules were similar in the B-granules of CS and Bd21. Phylogenetic analysis showed that most of the Bd21 starch synthesis-related genes were more similar to those in wheat than in rice. Early expression of key genes in Bd21 starch biosynthesis mediate starch synthesis in the pericarp; intermediate-stage expression increases the number and size of starch granules. In contrast, these enzymes in CS and Ae. peregrina were mostly expressed at intermediate stages, driving production of new B-granules and increasing the granule size, respectively. Immunogold labeling showed that granule-bound starch synthase (GBSSI; related to amylose synthesis) was mainly present in starch granules: at lower levels in the B-granules of Bd21 than in CS. Furthermore, GBSSI was phosphorylated at threonine 183 and tyrosine 185 in the starch synthase catalytic domain in CS and Ae. peregrina, but neither site was phosphorylated in Bd21, suggesting GBSSI phosphorylation could improve amylose biosynthesis.ConclusionsBd21 contains only B-granules, and the expression of key genes in the three studied genera is consistent with the dynamic development of starch granules. GBSSI is present in greater amounts in the B-granules of CS than in Bd21; two phosphorylation sites (Thr183 and Tyr185) were found in Triticum and Aegilops; these sites were not phosphorylated in Bd21. GBSSI phosphorylation may reflect its importance in amylose synthesis.

Biosynthesis and Regulation of Wheat Amylose and Amylopectin from Proteomic and Phosphoproteomic Characterization of Granule-binding Proteins

Waxy starch has an important influence on the qualities of breads. Generally, grain weight and yield in waxy wheat (Triticum aestivum L.) are significantly lower than in bread wheat. In this study, we performed the first proteomic and phosphoproteomic analyses of starch granule-binding proteins by comparing the waxy wheat cultivar Shannong 119 and the bread wheat cultivar Nongda 5181. These results indicate that reduced amylose content does not affect amylopectin synthesis, but it causes significant reduction of total starch biosynthesis, grain size, weight and grain yield. Two-dimensional differential in-gel electrophoresis identified 40 differentially expressed protein (DEP) spots in waxy and non-waxy wheats, which belonged mainly to starch synthase (SS) I, SS IIa and granule-bound SS I. Most DEPs involved in amylopectin synthesis showed a similar expression pattern during grain development, suggesting relatively independent amylose and amylopectin synthesis pathways. Phosphoproteome analysis of starch granule-binding proteins, using TiO2 microcolumns and LC-MS/MS, showed that the total number of phosphoproteins and their phosphorylation levels in ND5181 were significantly higher than in SN119, but proteins controlling amylopectin synthesis had similar phosphorylation levels. Our results revealed the lack of amylose did not affect the expression and phosphorylation of the starch granule-binding proteins involved in amylopectin biosynthesis.

Molecular cloning, phylogenetic analysis, and expression profiling of endoplasmic reticulum molecular chaperone BiP genes from bread wheat ( Triticum aestivum L.)

BackgroundThe endoplasmic reticulum chaperone binding protein (BiP) is an important functional protein, which is involved in protein synthesis, folding assembly, and secretion. In order to study the role of BiP in the process of wheat seed development, we cloned three BiP homologous cDNA sequences in bread wheat (Triticum aestivum), completed by rapid amplification of cDNA ends (RACE), and examined the expression of wheat BiP in wheat tissues, particularly the relationship between BiP expression and the subunit types of HMW-GS using near-isogenic lines (NILs) of HMW-GS silencing, and under abiotic stress.ResultsSequence analysis demonstrated that all BiPs contained three highly conserved domains present in plants, animals, and microorganisms, indicating their evolutionary conservation among different biological species. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) revealed that TaBiP (Triticum aestivum BiP) expression was not organ-specific, but was predominantly localized to seed endosperm. Furthermore, immunolocalization confirmed that TaBiP was primarily located within the protein bodies (PBs) in wheat endosperm. Three TaBiP genes exhibited significantly down-regulated expression following high molecular weight-glutenin subunit (HMW-GS) silencing. Drought stress induced significantly up-regulated expression of TaBiPs in wheat roots, leaves, and developing grains.ConclusionsThe high conservation of BiP sequences suggests that BiP plays the same role, or has common mechanisms, in the folding and assembly of nascent polypeptides and protein synthesis across species. The expression of TaBiPs in different wheat tissue and under abiotic stress indicated that TaBiP is most abundant in tissues with high secretory activity and with high proportions of cells undergoing division, and that the expression level of BiP is associated with the subunit types of HMW-GS and synthesis. The expression of TaBiPs is developmentally regulated during seed development and early seedling growth, and under various abiotic stresses.

Rapid Characterization of Wheat Low Molecular Weight Glutenin Subunits by Ultraperformance Liquid Chromatography (UPLC)

Low molecular weight glutenin subunits (LMW-GS), as important seed storage proteins, together with HMW-GS significantly define unique dough viscoelastic properties. In this study, a rapid ultraperformance liquid chromatography (UPLC) method for the separation and characterization of LMW-GS in wheat was optimized and established. The fast, reproducible, and high-resolution UPLC separation of LMW-GS could be obtained by gradually increasing eluting gradient from 21 to 47% in 30 min at flow rate of 0.55 mL/min and 60 °C for separation temperature. By this method, analysis of one sample could be completed in <20 min, significantly less time than the traditional reversed-phase high-performance liquid chromatography (RP-HPLC) method. Under the optimized conditions, the genetic features of LMW-GS and genotype × environmental interaction were successfully analyzed, leading to a fast identification of 17 main LMW-GS alleles that were related to different quality properties in wheat. The results demonstrated that UPLC could be a powerful and alternative tool for genetic and proteomic studies of wheat grain proteins and fast identification or screening of desirable LMW-GS alleles in wheat quality improvement.

Deletion of the low-molecular-weight glutenin subunit allele Glu-A3a of wheat (Triticum aestivum L.) significantly reduces dough strength and breadmaking quality

BackgroundLow-molecular-weight glutenin subunits (LMW-GS), encoded by Glu-3 complex loci in hexaploid wheat, play important roles in the processing quality of wheat flour. To date, the molecular characteristics and effects on dough quality of individual Glu-3 alleles and their encoding proteins have been poorly studied. We used a Glu-A3 deletion line of the Chinese Spring (CS-n) wheat variety to conduct the first comprehensive study on the molecular characteristics and functional properties of the LMW-GS allele Glu-A3a.ResultsThe Glu-A3a allele at the Glu-A3 locus in CS and its deletion in CS-n were identified and characterized by proteome and molecular marker methods. The deletion of Glu-A3a had no significant influence on plant morphological and yield traits, but significantly reduced the dough strength and breadmaking quality compared to CS. The complete sequence of the Glu-A3a allele was cloned and characterized, which was found to encode a B-subunit with longer repetitive domains and an increased number of α-helices. The Glu-A3a-encoded B-subunit showed a higher expression level and accumulation rate during grain development. These characteristics of the Glu-A3a allele could contribute to achieving superior gluten quality and demonstrate its potential application to wheat quality improvement. Furthermore, an allele-specific polymerase chain reaction (AS-PCR) marker for the Glu-A3a allele was developed and validated using different bread wheat cultivars, including near-isogenic lines (NILs) and recombinant inbred lines (RILs), which could be used as an effective molecular marker for gluten quality improvement through marker-assisted selection.ConclusionsThis work demonstrated that the LMW-GS allele Glu-A3a encodes a specific LMW-i type B-subunit that significantly affects wheat dough strength and breadmaking quality. The Glu-A3a-encoded B-subunit has a long repetitive domain and more α-helix structures as well as a higher expression level and accumulation rate during grain development, which could facilitate the formation of wheat with a stronger dough structure and superior breadmaking quality.

Dynamic metabolome profiling reveals significant metabolic changes during grain development of bread wheat (Triticum aestivum L.)

BACKGROUND Metabolites in wheat grains greatly influence nutritional values. Wheat provides proteins, minerals, B-group vitamins and dietary fiber to humans. These metabolites are important to human health. However, the metabolome of the grain during the development of bread wheat has not been studied so far. In this work the first dynamic metabolome of the developing grain of the elite Chinese bread wheat cultivar Zhongmai 175 was analyzed, using non-targeted gas chromatography/mass spectrometry (GC/MS) for metabolite profiling. RESULTS In total, 74 metabolites were identified over the grain developmental stages. Metabolite-metabolite correlation analysis revealed that the metabolism of amino acids, carbohydrates, organic acids, amines and lipids was interrelated. An integrated metabolic map revealed a distinct regulatory profile. The results provide information that can be used by metabolic engineers and molecular breeders to improve wheat grain quality. CONCLUSION The present metabolome approach identified dynamic changes in metabolite levels, and correlations among such levels, in developing seeds. The comprehensive metabolic map may be useful when breeding programs seek to improve grain quality. The work highlights the utility of GC/MS-based metabolomics, in conjunction with univariate and multivariate data analysis, when it is sought to understand metabolic changes in developing seeds.

Molecular characterisation and evolution of HMW glutenin subunit genes in Brachypodium distachyon L.

Brachypodium distachyon, a small wild grass within the Pooideae family, is a new model organism for exploring the functional genomics of cereal crops. It was shown to have close relationships to wheat, barley and rice. Here, we describe the molecular characterisation and evolutionary relationships of high molecular weight glutenin subunits (HMW-GS) genes from B. distachyon. Sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS-PAGE), high performance capillary electrophoresis (HPCE) and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses demonstrated that there was no HMW-GS expression in the Brachypodium grains due to the silencing of their encoding genes. Through allele-specific polymerase chain reaction (AS-PCR) amplification and cloning, a total of 13 HMW-GS encoding genes from diploid, tetraploid and hexaploid Brachypodium species were obtained, and all of them had typical structural features of y-type HMW-GS genes from common wheat and related species, particularly more similar to the 1Dy12 gene. However, the presence of an in-frame premature stop codon (TAG) at position 1521 in the coding region resulted in the conversion of all the genes to pseudogenes. Further, quantitative real-time PCR (qRT-PCR) analysis revealed that HMW-GS genes in B. distachyon displayed a similar trend, but with a low transcriptional expression profile during grain development due to the occurrence of the stop codon. Phylogenetic analysis showed that the highly conserved Glu-1-2 loci were presented in B. distachyon, which displayed close phylogenetic evolutionary relationships with Triticum and related species.

Fast identification of wheat 1BL.1RS translocation by reversed-phase ultra-performance liquid chromatography (RP-UPLC)

Abstract. The 1BL.1RS chromosomal translocation in wheat is the result of replacement of the short arm of chromosome 1B of wheat by the short arm of chromosome 1R of rye, which had been widely used as a parental line in worldwide wheat breeding, resulting in a high percentage of wheat cultivars containing this translocation. A fast and reliable approach to identify this translocation is highly desirable in modern wheat breeding. This study compared reversed-phase ultra-performance liquid chromatography (RP-UPLC), acidic polyacrylamide gel electrophoresis (A-PAGE), liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), allelic-specific PCR, and reversed-phase high-performance liquid chromatography (RP-HPLC) approaches to identify the 1BL.1RS translocation in 76 bread wheat cultivars. Two gliadin bands in the Gli-B1 region of A-PAGE separation were confirmed by LC-MS/MS to be omega secalins from the 1BL.1RS translocation, and they can be used as reliable protein markers for identifying the translocation. A few specific minor peaks eluted at 12–13 min on the RP-UPLC patterns can readily differentiate the 1BL.1RS translocation. Of the 76 wheat cultivars tested, 40 were identified as carrying the 1BL.1RS translocation by RP-UPLC, which was consistent with the results of A-PAGE, HPLC, and PCR. Compared with other established methods, RP-UPLC showed a clear advantage in fast identification of the 1BL.1RS translocation with higher reliability and lower costs, and it is therefore ideal for large-scale screening of the 1BL.1RS translocation in wheat breeding.

Characterization of HMW glutenin subunit Bx7 OE and its distribution in common wheat and related species

The overexpression of wheat Bx7 subunit (Bx7 OE ) encoded by the Glu-B1al allele is originated from a duplication event of the Bx7 gene, and has a positive effect on gluten strength. Thus, it is an important genetic resource for wheat quality improvement. In this study, the Bx7 OE subunit from a large number of bread wheat and related species was characterized by sodium dodecyl sulphate–polyacrylamide gel electrophoresis, reversed-phase high-performance liquid chromatography (RP-HPLC) and Sequence-Tagged sites (STS) markers. Only 31 bread wheat varieties were found to carry Bx7 OE . RP-HPLC quantification analysis revealed that the mean proportion of the Bx7 OE subunit to the total amount of high-molecular-weight glutenin subunits among the 31 bread wheat varieties was 41.8%, which is much higher than that of varieties with the normal Bx7 subunit (generally at 30%). Flour quality analysis of seven representative varieties with Bx7 OE and three with the normal Bx7 subunit showed that the varieties with Bx7 OE generally displayed better gluten strength than those with the normal Bx7 subunit. STS markers demonstrated that, in addition to the 31 bread wheat varieties with Bx7 OE , no PCR products were obtained from the related Triticum and Aegilops species. This suggests that the retroelement-mediated recombination event at the Glu-B1 locus could have occurred more recently, later than the formation of hexaploid wheat. The Bx7 OE subunit is mainly distributed in some bread wheat varieties from American countries with a low frequency, which is of particular importance for the quality improvement of wheat gluten.

Low molecular weight glutenin subunit gene Glu-B3h confers superior dough strength and breadmaking quality in wheat ( Triticum aestivum L.)

Low molecular weight glutenin subunit is one of the important quality elements in wheat (Triticum aestivum L.). Although considerable allelic variation has been identified, the functional properties of individual alleles at Glu-3 loci are less studied. In this work, we performed the first comprehensive study on the molecular characteristics and functional properties of the Glu-B3h gene using the wheat cultivar CB037B and its Glu-B3 deletion line CB037C. The results showed that the Glu-B3h deletion had no significant effects on plant morphological or yield traits, but resulted in a clear reduction in protein body number and size and main quality parameters, including inferior mixing property, dough strength, loaf volume, and score. Molecular characterization showed that the Glu-B3h gene consists of 1179 bp, and its encoded B-subunit has a longer repetitive domain and an increased number of α-helices, as well as higher expression, which could contribute to superior flour quality. The SNP-based allele-specific PCR markers designed for the Glu-B3h gene were developed and validated with bread wheat holding various alleles at Glu-B3 locus, which could effectively distinguish the Glu-B3h gene from others at the Glu-B3 locus, and have potential applications for wheat quality improvement through marker-assisted selection.