Differential alternative polyadenylation of homoeologous genes of allohexaploid wheat ABD subgenomes during drought stress response.

Because allohexaploid wheat genome contains ABD subgenomes, how the expression of homoeologous genes is coordinated remains largely unknown, particularly at the co-transcriptional level. Alternative polyadenylation (APA) is an important part of co-transcriptional regulation, which is crucial in developmental processes and stress responses. Drought stress is a major threat to the stable yield of wheat. Focusing on APA, we used poly(A) tag sequencing to track poly(A) site dynamics in wheat under drought stress. The results showed that drought stress led to extensive APA involving 37-47% of differentially expressed genes in wheat. Significant poly(A) site switching was found in stress-responsive genes. Interestingly, homoeologous genes exhibit unequal numbers of poly(A) sites, divergent APA patterns with tissue specificity and time-course dynamics, and distinct 3'-UTR length changes. Moreover, differentially expressed transcripts in leaves and roots used different poly(A) signals, the up- and downregulated isoforms had distinct preferences for non-canonical poly(A) sites. Genes that encode key polyadenylation factors showed differential expression patterns under drought stress. In summary, poly(A) signals and the changes in core poly(A) factors may widely affect the selection of poly(A) sites and gene expression levels during the response to drought stress, and divergent APA patterns among homoeologous genes add extensive plasticity to this responsive network. These results not only reveal the significant role of APA in drought stress response, but also provide a fresh perspective on how homoeologous genes contribute to adaptability through transcriptome diversity. In addition, this work provides information about the ends of transcripts for a better annotation of the wheat genome.

A gain-of-function allele of a DREB transcription factor gene ameliorates drought tolerance in wheat.

Drought is a major environmental factor limiting wheat production worldwide. However, the genetic components underlying wheat drought tolerance are largely unknown. Here, we identify a DREB transcription factor gene (TaDTG6-B) by genome-wide association study that is tightly associated with drought tolerance in wheat. Candidate gene association analysis revealed that a 26-bp deletion in the TaDTG6-B coding region induces a gain-of-function for TaDTG6-BDel574, which exhibits stronger transcriptional activation, protein interactions, and binding activity to dehydration-responsive elements (DRE)/CRT cis-elements than the TaDTG6-BIn574 encoded by the allele lacking the deletion, thus conferring greater drought tolerance in wheat seedlings harboring this variant. Knockdown of TaDTG6-BDel574 transcripts attenuated drought tolerance in transgenic wheat, whereas its overexpression resulted in enhanced drought tolerance without accompanying phenotypic abnormalities. Furthermore, the introgression of the TaDTG6-BDel574 elite allele into drought-sensitive cultivars improved their drought tolerance, thus providing a valuable genetic resource for wheat breeding. We also identified 268 putative target genes that are directly bound and transcriptionally regulated by TaDTG6-BDel574. Further analysis showed that TaDTG6-BDel574 positively regulates TaPIF1 transcription to enhance wheat drought tolerance. These results describe the genetic basis and accompanying mechanism driving phenotypic variation in wheat drought tolerance, and provide a novel genetic resource for crop breeding programs.

Genome-wide association study revealed TaHXK3-2A as a candidate gene controlling stomatal index in wheat seedlings.

Stomata are important channels for the control of gas exchange between plants and the atmosphere. To examine the genetic architecture of wheat stomatal index, we performed a genome-wide association study (GWAS) using a panel of 539 wheat accessions and 450 678 polymorphic single nucleotide polymorphisms (SNPs) that were detected using wheat-specific 660K SNP array. A total of 130 SNPs were detected to be significantly associated with stomatal index in both leaf surfaces of wheat seedlings. These significant SNPs were distributed across 16 chromosomes and involved 2625 candidate genes which participate in stress response, metabolism and cell/organ development. Subsequent bulk segregant analysis (BSA), combined with GWAS identified one major haplotype on chromosome 2A, that is responsible for stomatal index on the abaxial leaf surface. Candidate gene association analysis revealed that genetic variation in the promoter region of the hexokinase gene TaHXK3-2A was significantly associated with the stomatal index. Moreover, transgenic analysis confirmed that TaHXK3-2A overexpression in wheat decreased the size of leaf pavement cells but increased stomatal density through the glucose metabolic pathway, resulting in drought sensitivity among TaHXK3-2A transgenic lines due to an increased transpiration rate. Taken together, these results provide valuable insights into the genetic control of the stomatal index in wheat seedlings.

Variation in cis-regulation of a NAC transcription factor contributes to drought tolerance in wheat.

Drought is a major environmental factor limiting wheat production worldwide, and developing drought-tolerant cultivars is a central challenge for wheat breeders globally. Therefore, it is important to identify genetic components determining drought tolerance in wheat. In this study, we identified a wheat NAC gene (TaNAC071-A) that is tightly associated with drought tolerance by a genome-wide association study. Knockdown of TaNAC071-A in wheat attenuated plant drought tolerance, whereas its overexpression significantly enhanced drought tolerance through improved water-use efficiency and increased expression of stress-responsive genes. This heightened water-saving mechanism mitigated the yield loss caused by water deficit. Further candidate gene association analysis showed that a 108-bp insertion in the promoter of TaNAC071-A alters its expression level and contributes to variation in drought tolerance among wheat accessions. This insertion contains two MYB cis-regulatory elements (CREs) that can be directly bound by the MYB transcription activator, TaMYBL1, thereby leading to increased TaNAC071-A expression and plant drought tolerance. Importantly, introgression of this 108-bp insertion allele, TaNAC071-AIn-693, into drought-sensitive cultivars could improve their drought tolerance, demonstrating that it is a valuable genetic resource for wheat breeding. Taken together, our findings highlight a major breakthrough in determining the genetic basis underlying phenotypic variation in wheat drought tolerance and showcase the potential of exploiting CRE-containing indels for improving important agronomical traits.

The wheat ABA receptor gene TaPYL1-1B contributes to drought tolerance and grain yield by increasing water-use efficiency.

The role of abscisic acid (ABA) receptors, PYR1/PYL/RCAR (PYLs), is well established in ABA signalling and plant drought response, but limited research has explored the regulation of wheat PYLs in this process, especially the effects of their allelic variations on drought tolerance or grain yield. Here, we found that the overexpression of a TaABFs-regulated PYL gene, TaPYL1-1B, exhibited higher ABA sensitivity, photosynthetic capacity and water-use efficiency (WUE), all contributed to higher drought tolerance than that of wild-type plants. This heightened water-saving mechanism further increased grain yield and protected productivity during water deficit. Candidate gene association analysis revealed that a favourable allele TaPYL1-1BIn-442 , carrying an MYB recognition site insertion in the promoter, is targeted by TaMYB70 and confers enhanced expression of TaPYL1-1B in drought-tolerant genotypes. More importantly, an increase in frequency of the TaPYL1-1BIn-442 allele over decades among modern Chinese cultivars and its association with high thousand-kernel weight together demonstrated that it was artificially selected during wheat improvement efforts. Taken together, our findings illuminate the role of TaPYL1-1B plays in coordinating drought tolerance and grain yield. In particular, the allelic variant TaPYL1-1BIn-442 substantially contributes to enhanced drought tolerance while maintaining high yield, and thus represents a valuable genetic target for engineering drought-tolerant wheat germplasm.

Overexpression of the wheat NAC transcription factor TaSNAC4-3A gene confers drought tolerance in transgenic Arabidopsis.

NAC transcription factors (TFs) play critical roles in plant abiotic stress responses. However, information on the roles of NAC TFs is limited in wheat (Triticum aestivum L.). In this study, we isolated three wheat TaSNAC4 homeologous genes, TaSNAC4-3A, TaSNAC4-3B, and TaSNAC4-3D, and characterized the function of TaSNAC4-3A in plant drought tolerance. TaSNAC4 is highly expressed in seedling leaves, and expression is induced by various abiotic stresses. Transient expression and transactivation assays showed that TaSNAC4-3A is localized to the nucleus, and the C-terminal region has transcriptional activation activity. Overexpression of TaSNAC4-3A in Arabidopsis led to stimulated germination and root growth when exposed to salt and osmotic stresses, and drought stress tolerance was significantly increased in the TaSNAC4-3A transgenic lines. When compared to the control plants, the transgenic lines overexpressing TaSNAC4-3A exhibited reduced stomatal aperture size under drought stress, and therefore had lower water loss rates. In addition, the overexpression of TaSNAC4-3A led to abscisic acid (ABA) hypersensitivity at the root elongation and seed germination stages. Further transcriptomic analysis demonstrated that there was a significant up-regulation of stress responsive genes in the TaSNAC4-3A transgenic lines. Our findings have revealed the important role of TaSNAC4-3A in plant drought tolerance.

Genome-wide analysis of the AREB/ABF gene lineage in land plants and functional analysis of TaABF3 in Arabidopsis.

BACKGROUND: Previous studies have shown that ABFs (abscisic acid-responsive transcription factors) are important ABA-signaling components that participate in abiotic stress response. However, little is known about the function of ABFs in Triticum aestivum. In addition, although various ABFs have been identified in other species, the phylogenetic relationship between ABF transcription factors has not been systemically investigated in land plants. RESULTS: In this study, we systemically collected ABFs from land plants and analyzed the phylogenetic relationship of these ABF genes. The ABF genes are present in all the land plants we investigated, including moss, lycophyte, monocots, and eudicots. Furthermore, these ABF genes are phylogenetically divided into seven subgroups, differentiations that are supported by variation in the gene structure, protein properties, and motif patterns. We further demonstrated that the expression of ABF genes varies among different tissues and developmental stages, and are induced by one or more environmental stresses. Furthermore, we found that three wheat ABFs (TaABF1, TaABF2, and TaABF3) were significantly induced by drought stress. Compared with wild-type (WT) plants, transgenic Arabidopsis plants overexpressing TaABF3 displayed enhanced drought tolerance. CONCLUSIONS: These results provide important ground work for understanding the phylogenetic relationships between plant ABF genes. Our results also indicate that TaABFs may participate in regulating plant response to abiotic stresses.

Characterization of wheat homeodomain-leucine zipper family genes and functional analysis of TaHDZ5-6A in drought tolerance in transgenic Arabidopsis.

BACKGROUND: Many studies in Arabidopsis and rice have demonstrated that HD-Zip transcription factors play important roles in plant development and responses to abiotic stresses. Although common wheat (Triticum aestivum L.) is one of the most widely cultivated and consumed food crops in the world, the function of the HD-Zip proteins in wheat is still largely unknown. RESULTS: To explore the potential biological functions of HD-Zip genes in wheat, we performed a bioinformatics and gene expression analysis of the HD-Zip family. We identified 113 HD-Zip members from wheat and classified them into four subfamilies (I-IV) based on phylogenic analysis against proteins from Arabidopsis, rice, and maize. Most HD-Zip genes are represented by two to three homeoalleles in wheat, which are named as TaHDZX_ZA, TaHDZX_ZB, or TaHDZX_ZD, where X denotes the gene number and Z the wheat chromosome on which it is located. TaHDZs in the same subfamily have similar protein motifs and intron/exon structures. The expression profiles of TaHDZ genes were analysed in different tissues, at different stages of vegetative growth, during seed development, and under drought stress. We found that most TaHDZ genes, especially those in subfamilies I and II, were induced by drought stress, suggesting the potential importance of subfamily I and II TaHDZ members in the responses to abiotic stress. Compared with wild-type (WT) plants, transgenic Arabidopsis plants overexpressing TaHDZ5-6A displayed enhanced drought tolerance, lower water loss rates, higher survival rates, and higher proline content under drought conditions. Additionally, the transcriptome analysis identified a number of differentially expressed genes between 35S::TaHDZ5-6A transgenic and wild-type plants, many of which are involved in stress response. CONCLUSIONS: Our results will facilitate further functional analysis of wheat HD-Zip genes, and also indicate that TaHDZ5-6A may participate in regulating the plant response to drought stress. Our experiments show that TaHDZ5-6A holds great potential for genetic improvement of abiotic stress tolerance in crops.

Regulatory changes in TaSNAC8-6A are associated with drought tolerance in wheat seedlings.

Wheat is a staple crop produced in arid and semi-arid areas worldwide, and its production is frequently compromised by water scarcity. Thus, increased drought tolerance is a priority objective for wheat breeding programmes, and among their targets, the NAC transcription factors have been demonstrated to contribute to plant drought response. However, natural sequence variations in these genes have been largely unexplored for their potential roles in drought tolerance. Here, we conducted a candidate gene association analysis of the stress-responsive NAC gene subfamily in a wheat panel consisting of 700 varieties collected worldwide. We identified a drought responsive gene, TaSNAC8-6A, that is tightly associated with drought tolerance in wheat seedlings. Further analysis found that a favourable allele TaSNAC8-6AIn-313 , carrying an insertion in the ABRE promoter motif, is targeted by TaABFs and confers enhanced drought-inducible expression of TaSNAC8-6A in drought-tolerant genotypes. Transgenic wheat and Arabidopsis TaSNAC8-6A overexpression lines exhibited enhanced drought tolerance through induction of auxin- and drought-response pathways, confirmed by transcriptomic analysis, that stimulated lateral root development, subsequently improving water-use efficiency. Taken together, our findings reveal that natural variation in TaSNAC8-6A and specifically the TaSNAC8-6AIn-313 allele strongly contribute to wheat drought tolerance and thus represent a valuable genetic resource for improvement of drought-tolerant germplasm for wheat production.