Dangqun Cui

Functional characterization of a wheat plasma membrane Na+/H+ antiporter in yeast

The functional analysis of the sodium exchanger SOS1 from wheat, TaSOS1, was undertaken using Saccharomyces cerevisiae as a heterologous expression system. The TaSOS1 protein, with significant sequence homology to SOS1 sodium exchangers from Arabidopsis and rice, is abundant in roots and leaves, and is induced by salt treatment. TaSOS1 suppressed the salt sensitivity of a yeast strain lacking the major Na+ efflux systems by decreasing the cellular Na+ content while increasing K+ content. Na+/H+ exchange activity of purified plasma membrane from yeast cells expressing TaSOS1 was higher than controls transformed with empty vector. These results demonstrate that TaSOS1 contributes to plasma membrane Na+/H+ exchange.

Physical mapping of puroindoline b-2 genes and molecular characterization of a novel variant in durum wheat (Triticum turgidum L.)

The puroindoline genes (Pina and Pinb) are the functional components of the common or bread wheat (Triticum aestivum L.) grain hardness locus that are responsible for kernel texture. In this study, four puroindoline b-2 variants were physically mapped using nulli-tetrosomic lines of bread wheat cultivar Chinese Spring and substitution lines of durum wheat (Triticum turgidum L.) cultivar Langdon. Results indicated that Pinb-2v1 was on 7D of Chinese Spring, Pinb-2v2 on 7B of Chinese Spring, Pinb-2v3 on 7B of Chinese Spring and Langdon, and Pinb-2v4 on 7A of Chinese Spring and Langdon. A new puroindoline b-2 variant, designated Pinb-2v5, was identified at the puroindoline b-2 locus of durum wheat cultivar Langdon, with a difference of only five single nucelotide polymorphisms compared with Pinb-2v4. Sequencing results indicated that, in comparison with the Pinb-2v3 sequence (AM99733 and GQ496618 with one base-pair modification of G to T at 6th position, designated Pinb-2v3a) in bread wheat cultivar Witchta, the coding region of Pinb-2v3 in 12 durum wheat cultivars had a single nucleotide change from T to C at the 311th position, resulting in a corresponding amino acid change from valine to alanine at the 104th position. This new allele was designated Pinb-2v3b. The study of puroindoline b-2 gene polymorphism in CIMMYT and Italian durum wheat germplasm and discovery of a novel puroindoline b-2 variant could provide useful information for further understanding the molecular and genetic basis of kernel hardness and illustrating gene duplication events in wheat.

Genome‐wide association study for 13 agronomic traits reveals distribution of superior alleles in bread wheat from the Yellow and Huai Valley of China

Summary Bread wheat is a leading cereal crop worldwide. Limited amount of superior allele loci restricted the progress of molecular improvement in wheat breeding. Here, we revealed new allelic variation distribution for 13 yield‐related traits in series of genome‐wide association studies (GWAS) using the wheat 90K genotyping assay, characterized in 163 bread wheat cultivars. Agronomic traits were investigated in 14 environments at three locations over 3 years. After filtering SNP data sets, GWAS using 20 689 high‐quality SNPs associated 1769 significant loci that explained, on average, ~20% of the phenotypic variation, both detected already reported loci and new promising genomic regions. Of these, repetitive and pleiotropic SNPs on chromosomes 6AS, 6AL, 6BS, 5BL and 7AS were significantly linked to thousand kernel weight, for example BS00021705_51 on 6BS and wsnp_Ex_c32624_41252144 on 6AS, with phenotypic variation explained (PVE) of ~24%, consistently identified in 12 and 13 of the 14 environments, respectively. Kernel length‐related SNPs were mainly identified on chromosomes 7BS, 6AS, 5AL and 5BL. Plant height‐related SNPs on chromosomes 4DS, 6DL, 2DS and 1BL were, respectively, identified in more than 11 environments, with averaged PVE of ~55%. Four SNPs were confirmed to be important genetic loci in two RIL populations. Based on repetivity and PVE, a total of 41 SNP loci possibly played the key role in modulating yield‐related traits of the cultivars surveyed. Distribution of superior alleles at the 41 SNP loci indicated that superior alleles were getting popular with time and modern cultivars had integrated many superior alleles, especially for peduncle length‐ and plant height‐related superior alleles. However, there were still 19 SNP loci showing less than percentages of 50% in modern cultivars, suggesting they should be paid more attention to improve yield‐related traits of cultivars in the Yellow and Huai wheat region. This study could provide useful information for dissection of yield‐related traits and valuable genetic loci for marker‐assisted selection in Chinese wheat breeding programme.

Functional Characterization of a Wheat NHX Antiporter Gene TaNHX2 That Encodes a K + /H + Exchanger

The subcellular localization of a wheat NHX antiporter, TaNHX2, was studied in Arabidopsis protoplasts, and its function was evaluated using Saccharomyces cerevisiae as a heterologous expression system. Fluorescence patterns of TaNHX2-GFP fusion protein in Arabidopsis cells indicated that TaNHX2 localized at endomembranes. TaNHX2 has significant sequence homology to NHX sodium exchangers from Arabidopsis, is abundant in roots and leaves and is induced by salt or dehydration treatments. Western blot analysis showed that TaNHX2 could be expressed in transgenic yeast cells. Expressed TaNHX2 protein suppressed the salt sensitivity of a yeast mutant strain by increasing its K+ content when exposed to salt stress. TaNHX2 also increased the tolerance of the strain to potassium stress. However, the expression of TaNHX2 did not affect the sodium concentration in transgenic cells. Western blot analysis for tonoplast proteins indicated that the TaNHX2 protein localized at the tonoplast of transgenic yeast cells. The tonoplast vesicles from transgenic yeast cells displayed enhanced K+/H+ exchange activity but very little Na+/H+ exchange compared with controls transformed with the empty vector; Na+/H+ exchange was not detected with concentrations of less than 37.5 mM Na+ in the reaction medium. Our data suggest that TaNHX2 is a endomembrane-bound protein and may primarily function as a K+/H+ antiporter, which is involved in cellular pH regulation and potassium nutrition under normal conditions. Under saline conditions, the protein mediates resistance to salt stress through the intracellular compartmentalization of potassium to regulate cellular pH and K+ homeostasis.

Allelic variation at the vernalization and photoperiod sensitivity loci in Chinese winter wheat cultivars (Triticum aestivum L.)

A total of 205 wheat cultivars from the Yellow and Huai valley of China were used to identify allelic variations of vernalization and photoperiod response genes, as well as the copy number variations (CNVs) of Ppd-B1 and Vrn-A1 genes. A novel Vrn-D1 allele with 174-bp insertion in the promoter region of the recessive allele vrn-D1 was discovered in three Chinese wheat cultivars and designated as Vrn-D1c. Quantitative real-time polymerase chain reaction showed that cultivars with the Vrn-D1c allele exhibited significantly higher expression of the Vrn-D1 gene than that in cultivars with the recessive allele vrn-D1, indicating that the 174-bp insertion of Vrn-D1c contributed to the increase in Vrn-D1 gene expression and caused early heading and flowering. The five new cis-elements (Box II-like, 3-AF1 binding site, TC-rich repeats, Box-W1 and CAT-box) in the 174-bp insertion possibly promoted the basal activity level of Vrn-D1 gene. Two new polymorphism combinations of photoperiod genes were identified and designated as Ppd-D1_Hapl-IX and Ppd-D1_Hapl-X. Association of the CNV of Ppd-B1 gene with the heading and flowering days showed that the cultivars with Ppd-B1_Hapl-VI demonstrated the earliest heading and flowering times, and those with Ppd-B1_Hapl-IV presented the latest heading and flowering times in three cropping seasons. Distribution of the vernalization and photoperiod response genes indicated that all recessive alleles at the four vernalization response loci, Ppd-B1_Hapl-I at Ppd-B1 locus, and Ppd-D1_Hapl-I at the Ppd-D1 locus were predominant in Chinese winter wheat cultivars. This study can provide useful information for wheat breeding programs to screen wheat cultivars with relatively superior adaptability and maturity.

Proteomic analysis of middle and late stages of bread wheat (Triticum aestivum L.) grain development.

Proteomic approaches were applied in four grain developmental stages of the Chinese bread wheat Yunong 201 and its ethyl methanesulfonate (EMS) mutant line Yunong 3114. 2-DE and tandem MALDI-TOF/TOF-MS analyzed proteome characteristics during middle and late grain development of the Chinese bread wheat Yunong 201 and its EMS mutant line Yunong 3114 with larger grain sizes. We identified 130 differentially accumulated protein spots representing 88 unique proteins, and four main expression patterns displayed a dynamic description of middle and late grain formation. Those identified protein species participated in eight biochemical processes: stress/defense, carbohydrate metabolism, protein synthesis/assembly/degradation, storage proteins, energy production and transportation, photosynthesis, transcription/translation, signal transduction. Comparative proteomic characterization demonstrated 12 protein spots that co-accumulated in the two wheat cultivars with different expression patterns, and six cultivar-specific protein spots including serpin, small heat shock protein, β-amylase, α-amylase inhibitor, dimeric α-amylase inhibitor precursor, and cold regulated protein. These cultivar-specific protein spots possibly resulted in differential yield-related traits of the two wheat cultivars. Our results provide valuable information for dissection of molecular and genetics basis of yield-related traits in bread wheat and the proteomic characterization in this study could also provide insights in the biology of middle and late grain development.

A Single-Nucleotide Polymorphism of TaGS5 Gene Revealed its Association with Kernel Weight in Chinese Bread Wheat

TaGS5 genes were cloned from bread wheat and were physically mapped on 3AS and 3DS. Sequencing results revealed that a SNP was found in the sixth exon of TaGS5-A1 gene. The SNP resulted in amino acid change from alanine to serine at the 303 bp position of TaGS5-A1. These two alleles were designated as TaGS5-A1a (alanine at the 303 bp position) and TaGS5-A1b genes (serine at the 303-bp position). Analysis of association of TaGS5-A1 alleles with agronomic traits indicated that cultivars with TaGS5-A1b possessed wider kernel width and higher thousand-kernel weight, as well as significantly lower plant height, spike length, and internode length below spike than those of cultivars with TaGS5-A1a over 3 years. These trait differences between TaGS5-A1a and TaGS5-A1b genotypes were larger in landraces than in modern cultivars. This finding suggested that TaGS5 gene played an important role in modulating yield-related traits in the landraces, which possibly resulted from numerous superior genes gathering in modern cultivars after strong artificial selection. The preferred TaGS5-A1b haplotype underwent very strong positive selection in Chinese modern wheat breeding, but not in Chinese landraces. Expression analysis of the TaGS5-A1 gene indicated that TaGS5-A1b allele possessed significantly higher expression level than TaGS5-A1b allele in differently developmental seeds. This study could provide relatively superior genotype in view of agronomic traits in wheat breeding programs. Likewise, this study could offer important information for the dissection of molecular and genetic basis of yield-related traits.

Allelic variation and distribution independence of Puroindoline b-B2 variants and their association with grain texture in wheat

In this study, we identify the allelic variation of the Pinb-B2v3 variant, which could be divided into three different alleles, Pinb-B2v3a, Pinb-B2v3b and Pinb-B2v3c. The result of χ2 tests showed that the distribution of Puroindoline b-2 variants has different frequencies in common and durum wheats. Analysis of the association of Pinb-B2v with grain hardness indicated that wheat cultivars with Pinb-B2v3b possessed relatively higher single kernel characterization system (SKCS) hardness indices in soft wheat in the 2006–2007 cropping season. Further analysis of SKCS hardness among different Puroindoline B-b2 variants by an F8 recombinant inbred line (RIL) population containing 350 RILs indicated that lines with Pinb-2v3b were on average 5.4 SKCS hardness index units harder than those carrying the Pinb-2v2 haplotype. Derived cleaved amplified polymorphic sequence markers were developed for identification of Pinb-B2v3b and Pinb-B2v3c alleles and will be useful for screening early generation materials by marker-assisted selection during wheat breeding. The results of quantitative real-time PCR indicated that the relative expression level of Pinb-B2v3b was significantly higher than those of Pinb-B2v2, Pinb-B2v3a and Pinb-B2v3c, that four Pinb-B2 alleles showed the highest relative expression level on the 14th day after anthesis during grain development, and that relative expression levels of Pinb-B2v3b and Pinb-B2v2 in leaf were significantly higher than those in root, suggesting that PINB-2 are possibly not seed-specific proteins and that the expression level of Pinb-B2v3 was possibly positively correlated with grain hardness.

Alveograph and Mixolab parameters associated with Puroindoline-D1 genes in Chinese winter wheats

BACKGROUND Grain texture is one of the most important characteristics of bread wheat and has a significant influence on end-use qualities. RESULTS Forty-three Chinese cultivars were tested under three environments and used to characterise kernel hardness, Puroindoline-D1 alleles and Alveograph and Mixolab parameters. The results indicated that SKCS hardness was positively correlated with Alveograph tenacity and P/L and Mixolab protein weakening (C2) and water absorption and negatively correlated with Mixolab starch gelatinisation (C3), amylasic activity (C4) and starch gelling (C5). Variance analysis showed that Puroindoline-D1 had a significant impact on SKCS hardness and most Alveograph and Mixolab parameters. Furthermore, among three Puroindoline-D1 genotypes, PINA-null/Pinb-D1a possessed the highest SKCS hardness, Alveograph tenacity and W and Mixolab stability and water absorption but the lowest Alveograph extensibility and G and Mixolab C3, C4 and C5. Pina-D1a/Pinb-D1a had the lowest SKCS hardness, Alveograph tenacity and W and Mixolab C2, water absorption and stability but the highest Alveograph extensibility and G and Mixolab C3, C4 and C5. Pina-D1a/Pinb-D1b possessed the lowest Mixolab C2 - C1, C3 - C2, C4 - C3 and C5 - C4. CONCLUSION Pina-D1a/Pinb-D1a was softer and had lower tenacity and water absorption. PINA-null/Pinb-D1a was harder and had higher tenacity and water absorption. Pina-D1a/Pinb-D1b had lower difference values among Mixolab parameters.

Identification of Winter-Responsive Proteins in Bread Wheat Using Proteomics Analysis and Virus-Induced Gene Silencing (VIGS)

Proteomic approaches were applied to identify protein spots involved in cold responses in wheat. By comparing the differentially accumulated proteins from two cultivars (UC1110 and PI 610750) and their derivatives, as well as the F10 recombinant inbred line population differing in cold-tolerance, a total of 20 common protein spots representing 16 unique proteins were successfully identified using 2-DE method. Of these, 14 spots had significantly enhanced abundance in the cold-sensitive parental cultivar UC1110 and its 20 descendant lines when compared with the cold-tolerant parental cultivar PI 610750 and its 20 descendant lines. Six protein spots with reduced abundance were also detected. The identified protein spots are involved in stress/defense, carbohydrate metabolism, protein metabolism, nitrogen metabolism, energy metabolism, and photosynthesis. The 20 differentially expressed protein spots were chosen for quantitative real-time polymerase chain reaction (qRT-PCR) to investigate expression changes at the RNA level. The results indicated that the transcriptional expression patterns of 11 genes were consistent with their protein expression models. Among the three unknown proteins, Spot 20 (PAP6-like) showed high sequence similarities with PAP6. qRT-PCR results implied that cold and salt stresses increased the expression of PAP6-like in wheat leaves. Furthermore, VIGS (virus-induced gene silencing)-treated plants generated for PAP6-like were subjected to freezing stress, these plants had more serious droop and wilt, an increased rate of relative electrolyte leakage, reduced relative water content (RWC) and decreased tocopherol levels when compared with viral control plants. However, the plants that were silenced for the other two unknown proteins had no significant differences in comparison to the BSMV0-inoculated plants under freezing conditions. These results indicate that PAP6-like possibly plays an important role in conferring cold tolerance in wheat.