Huanju Qin

Transgenic expression of MYB15 confers enhanced sensitivity to abscisic acid and improved drought tolerance in Arabidopsis thaliana

Abiotic stresses cause serious crop losses. Knowledge on genes functioning in plant responses to adverse growth conditions is essential for developing stress tolerant crops. Here we report that transgenic expression of MYB15, encoding a R2R3 MYB transcription factor in Arabidopsis thaliana, conferred hypersensitivity to exogenous abscisic acid (ABA) and improved tolerance to drought and salt stresses. The promoter of MYB15 was active in not only vegetative and reproductive organs but also the guard cells of stomata. Its transcript level was substantially upregulated by ABA, drought or salt treatments. Compared with wild type (WT) control, MYB15 overexpression lines were hypersensitive to ABA in germination assays, more susceptible to ABA-elicited inhibition of root elongation, and more sensitive to ABA-induced stomatal closure. In line with the above findings, the transcript levels of ABA biosynthesis (ABA1, ABA2), signaling (ABI3), and responsive genes (AtADH1, RD22, RD29B, AtEM6) were generally higher in MYB15 overexpression seedlings than in WT controls after treatment with ABA. MYB15 overexpression lines displayed improved survival and reduced water loss rates than WT control under water deficiency conditions. These overexpression lines also displayed higher tolerance to NaCl stress. Collectively, our data suggest that overexpression of MYB15 improves drought and salt tolerance in Arabidopsis possibly by enhancing the expression levels of the genes involved in ABA biosynthesis and signaling, and those encoding the stress-protective proteins.

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.

Association analysis of genomic loci important for grain weight control in elite common wheat varieties cultivated with variable water and fertiliser supply.

Grain weight, an essential yield component, is under strong genetic control and markedly influenced by the environment. Here, by genome-wide association analysis with a panel of 94 elite common wheat varieties, 37 loci were found significantly associated with thousand-grain weight (TGW) in one or more environments differing in water and fertiliser levels. Five loci were stably associated with TGW under all 12 environments examined. Their elite alleles had positive effects on TGW. Four, two, three, and two loci were consistently associated with TGW in the irrigated and fertilised (IF), rainfed (RF), reduced nitrogen (RN), and reduced phosphorus (RP) environments. The elite alleles of the IF-specific loci enhanced TGW under well-resourced conditions, whereas those of the RF-, RN-, or RP-specific loci conferred tolerance to the TGW decrease when irrigation, nitrogen, or phosphorus were reduced. Moreover, the elite alleles of the environment-independent and -specific loci often acted additively to enhance TGW. Four additional loci were found associated with TGW in specific locations, one of which was shown to contribute to the TGW difference between two experimental sites. Further analysis of 14 associated loci revealed that nine affected both grain length and width, whereas the remaining loci influenced either grain length or width, indicating that these loci control grain weight by regulating kernel size. Finally, the elite allele of Xpsp3152 frequently co-segregated with the larger grain haplotype of TaGW2-6A, suggesting probable genetic and functional linkages between Xpsp3152 and GW2 that are important for grain weight control in cereal plants. Our study provides new knowledge on TGW control in elite common wheat lines, which may aid the improvement of wheat grain weight trait in further research.

HRS1 Acts as a Negative Regulator of Abscisic Acid Signaling to Promote Timely Germination of Arabidopsis Seeds

In this work, we conducted functional analysis of Arabidopsis HRS1 gene in order to provide new insights into the mechanisms governing seed germination. Compared with wild type (WT) control, HRS1 knockout mutant (hrs1-1) exhibited significant germination delays on either normal medium or those supplemented with abscisic acid (ABA) or sodium chloride (NaCl), with the magnitude of the delay being substantially larger on the latter media. The hypersensitivity of hrs1-1 germination to ABA and NaCl required ABI3, ABI4 and ABI5, and was aggravated in the double mutant hrs1-1abi1-2 and triple mutant hrs1-1hab1-1abi1-2, indicating that HRS1 acts as a negative regulator of ABA signaling during seed germination. Consistent with this notion, HRS1 expression was found in the embryo axis, and was regulated both temporally and spatially, during seed germination. Further analysis showed that the delay of hrs1-1 germination under normal conditions was associated with reduction in the elongation of the cells located in the lower hypocotyl (LH) and transition zone (TZ) of embryo axis. Interestingly, the germination rate of hrs1-1 was more severely reduced by the inhibitor of cell elongation, and more significantly decreased by the suppressors of plasmalemma H+-ATPase activity, than that of WT control. The plasmalemma H+-ATPase activity in the germinating seeds of hrs1-1 was substantially lower than that exhibited by WT control, and fusicoccin, an activator of this pump, corrected the transient germination delay of hrs1-1. Together, our data suggest that HRS1 may be needed for suppressing ABA signaling in germinating embryo axis, which promotes the timely germination of Arabidopsis seeds probably by facilitating the proper function of plasmalemma H+-ATPase and the efficient elongation of LH and TZ cells.

Overexpressing HRS1 Confers Hypersensitivity to Low Phosphate‐Elicited Inhibition of Primary Root Growth in Arabidopsis thaliana

Phosphate (Pi) deficiency causes dramatic root system architecture (RSA) changes in higher plants. Here we report that overexpression of HRS1 leads to enhanced sensitivity to low Pi-elicited inhibition of primary root growth in Arabidopsis thaliana seedlings. Bioinformatic investigations uncovered that HRS1 and its six homologs encode putative G2-like transcription factors in Arabidopsis. Analysis of promoter::GUS reporter lines revealed that HRS1 transcripts were present mainly in the root hair region and root hair cells under Pi-sufficient conditions. Pi deprivation increased HRS1 expression level and expanded its expression domain. Although HRS1 knockout mutant did not differ from wild type (WT) control irrespective of Pi status, its overexpression lines were significantly more susceptible to low Pi-elicited primary root shortening. In both WT and HRS1 overexpression seedlings, low Pi-induced primary root shortening was accompanied by enhanced root hair cell differentiation, but this enhancement occurred to a greater extent in the latter genotype. Collectively, our data suggest that HRS1 may be involved in the modulation of primary root and root hair growth in Pi-deprived Arabidopsis seedlings, and provide useful clues for further research into the function of HRS1 and its homologs and the mechanisms behind RSA changes under Pi-deficient conditions.

Molecular analysis of phosphomannomutase (PMM) genes reveals a unique PMM duplication event in diverse Triticeae species and the main PMM isozymes in bread wheat tissues

BackgroundPhosphomannomutase (PMM) is an essential enzyme in eukaryotes. However, little is known about PMM gene and function in crop plants. Here, we report molecular evolutionary and biochemical analysis of PMM genes in bread wheat and related Triticeae species.ResultsTwo sets of homoeologous PMM genes (TaPMM-1 and 2) were found in bread wheat, and two corresponding PMM genes were identified in the diploid progenitors of bread wheat and many other diploid Triticeae species. The duplication event yielding PMM-1 and 2 occurred before the radiation of diploid Triticeae genomes. The PMM gene family in wheat and relatives may evolve largely under purifying selection. Among the six TaPMM genes, the transcript levels of PMM-1 members were comparatively high and their recombinant proteins were all enzymatically active. However, PMM-2 homoeologs exhibited lower transcript levels, two of which were also inactive. TaPMM-A1, B1 and D1 were probably the main active isozymes in bread wheat tissues. The three isozymes differed from their counterparts in barley and Brachypodium distachyon in being more tolerant to elevated test temperatures.ConclusionOur work identified the genes encoding PMM isozymes in bread wheat and relatives, uncovered a unique PMM duplication event in diverse Triticeae species, and revealed the main active PMM isozymes in bread wheat tissues. The knowledge obtained here improves the understanding of PMM evolution in eukaryotic organisms, and may facilitate further investigations of PMM function in the temperature adaptability of bread wheat.

Coexpression network analysis of the genes regulated by two types of resistance responses to powdery mildew in wheat.

Powdery mildew disease caused by Blumeria graminis f. sp. tritici (Bgt) inflicts severe economic losses in wheat crops. A systematic understanding of the molecular mechanisms involved in wheat resistance to Bgt is essential for effectively controlling the disease. Here, using the diploid wheat Triticum urartu as a host, the genes regulated by immune (IM) and hypersensitive reaction (HR) resistance responses to Bgt were investigated through transcriptome sequencing. Four gene coexpression networks (GCNs) were developed using transcriptomic data generated for 20 T. urartu accessions showing IM, HR or susceptible responses. The powdery mildew resistance regulated (PMRR) genes whose expression was significantly correlated with Bgt resistance were identified, and they tended to be hubs and enriched in six major modules. A wide occurrence of negative regulation of PMRR genes was observed. Three new candidate immune receptor genes (TRIUR3_13045, TRIUR3_01037 and TRIUR3_06195) positively associated with Bgt resistance were discovered. Finally, the involvement of TRIUR3_01037 in Bgt resistance was tentatively verified through cosegregation analysis in a F2 population and functional expression assay in Bgt susceptible leaf cells. This research provides insights into the global network properties of PMRR genes. Potential molecular differences between IM and HR resistance responses to Bgt are discussed.

Haplotype Variation of Glu-D1 Locus and the Origin of Glu-D1d Allele Conferring Superior End-Use Qualities in Common Wheat

In higher plants, seed storage proteins (SSPs) are frequently expressed from complex gene families, and allelic variation of SSP genes often affects the quality traits of crops. In common wheat, the Glu-D1 locus, encoding 1Dx and 1Dy SSPs, has multiple alleles. The Glu-D1d allele frequently confers superior end-use qualities to commercial wheat varieties. Here, we studied the haplotype structure of Glu-D1 genomic region and the origin of Glu-D1d. Using seven diagnostic DNA markers, 12 Glu-D1 haplotypes were detected among common wheat, European spelt wheat (T. spelta, a primitive hexaploid relative of common wheat), and Aegilops tauschii (the D genome donor of hexaploid wheat). By comparatively analyzing Glu-D1 haplotypes and their associated 1Dx and 1Dy genes, we deduce that the haplotype carrying Glu-D1d was likely differentiated in the ancestral hexaploid wheat around 10,000 years ago, and was subsequently transmitted to domesticated common wheat and T. spelta. A group of relatively ancient Glu-D1 haplotypes was discovered in Ae. tauschii, which may serve for the evolution of other haplotypes. Moreover, a number of new Glu-D1d variants were found in T. spelta. The main steps in Glu-D1d differentiation are proposed. The implications of our work for enhancing the utility of Glu-D1d in wheat quality improvement and studying the SSP alleles in other crop species are discussed.

Genome-wide analysis of complex wheat gliadins, the dominant carriers of celiac disease epitopes

Gliadins, specified by six compound chromosomal loci (Gli-A1/B1/D1 and Gli-A2/B2/D2) in hexaploid bread wheat, are the dominant carriers of celiac disease (CD) epitopes. Because of their complexity, genome-wide characterization of gliadins is a strong challenge. Here, we approached this challenge by combining transcriptomic, proteomic and bioinformatic investigations. Through third-generation RNA sequencing, full-length transcripts were identified for 52 gliadin genes in the bread wheat cultivar Xiaoyan 81. Of them, 42 were active and predicted to encode 25 α-, 11 γ-, one δ- and five ω-gliadins. Comparative proteomic analysis between Xiaoyan 81 and six newly-developed mutants each lacking one Gli locus indicated the accumulation of 38 gliadins in the mature grains. A novel group of α-gliadins (the CSTT group) was recognized to contain very few or no CD epitopes. The δ-gliadins identified here or previously did not carry CD epitopes. Finally, the mutant lacking Gli-D2 showed significant reductions in the most celiac-toxic α-gliadins and derivative CD epitopes. The insights and resources generated here should aid further studies on gliadin functions in CD and the breeding of healthier wheat.

High-throughput mining of E-genome-specific SNPs for characterizing Thinopyrum elongatum introgressions in common wheat

Diploid Thinopyrum elongatum (EE, 2n = 2x = 14) and related polyploid species constitute an important gene pool for improving Triticeae grain and forage crops. However, the genomic and molecular marker resources are generally poor for these species. To aid the genetic, molecular, breeding and ecological studies involving Thinopyrum species, we developed a strategy for mining and validating E‐genome‐specific SNPs using Th. elongatum and common wheat (Triticum aestivum, AABBDD, 2n = 6x = 42) as experimental materials. By comparing the transcriptomes between Chinese Spring (CS, a common wheat variety) and the CS‐Th. elongatum octoploid, 35,193 candidate SNPs between E genome genes and their common wheat orthologs were computed. Through comparative genomic analysis, these SNPs were putatively assigned to the seven individual E genome chromosomes. Among 420 randomly selected SNPs, 373 could be validated. Thus, approximately 89% of the mined SNPs may be authentic with respect to their polymorphism and chromosomal location. Using 14 such SNPs as molecular markers, complex E genome introgressions were reliably identified in 78 common wheat‐Th. elongatum hybrids, and the structural feature of a novel recombinant chromosome formed by 6E and 7E was revealed. Finally, based on testing 33 SNPs assigned to chromosome 3E in multiple genotypes of Th. elongatum, Pseudoroegneria stipifolia (carrying the St genome related to E) and common wheat, we suggest that some of the SNP markers may also be applicable for genetic studies within and among the Thinopyrum species (populations) carrying E and/or St genomes in the future.