Most subgroup S1 basic leucine zipper (bZIP) transcription factors (TFs) play a crucial role in the abiotic stress responses. However, their functions and molecular mechanisms remain poorly characterized in wheat (Triticum aestivum L.). In this study, we identified a novel subgroup S1 bZIP gene, designated TabZIP11-D, which was transcriptionally responsive to abscisic acid (ABA), salt, and cold stresses. TabZIP11-D encodes a nuclear-localized protein that lacks transcriptional activation activity in yeast. The Ca2+ blocker LaCl3 significantly suppressed the salt-induced expression of TabZIP11-D. TabZIP11-D interacted with the Ca2+-dependent protein kinases (TaCDPK1, TaCDPK5, TaCDPK9-1, and TaCDPK30) and the CBL-interacting protein kinase TaCIPK31. Overexpression of TabZIP11-D enhanced salt and freezing tolerance by modulating soluble sugar and proline accumulation, reducing hydrogen peroxide (H2O2) and malondialdehyde (MDA) contents, and regulating the expression levels of stress-responsive genes. Furthermore, TabZIP11-D formed a homodimer with itself and heterodimers with group C bZIP proteins. Modified yeast one-hybrid assays revealed that TabZIP14 and TabZIP36 significantly enhanced TabZIP11-D binding to the G-box cis-element in the promoter region of TaCBF1 gene. These findings demonstrate that TabZIP11-D heterodimerizes with TabZIP14/36 to regulate cold signaling by promoting the TaCBF1 transcription. TabZIP11-D functions as a positive regulator in the salt stress response by interacting with TaCDPK1/5/9-1/30 and TaCIPK31.

Overexpression of OsNRT1.1A promotes early heading and increases the tolerance in wheat under nitrogen deficiency conditions. The application of inorganic nitrogen (N) fertilizers is a major driving force for crop yield improvement. However, the overuse of fertilizers significantly raises production costs and leads to environmental problems, making it critical to enhance crop nitrogen use efficiency (NUE) for the sake of sustainable agriculture. In this study, we created a series of transgenic wheat lines carrying the rice OsNRT1.1A gene, which encodes a nitrate transporter, to investigate its possible application in improving NUE in wheat. The transgenic wheat exhibited traits such as early maturation that were highly consistent with the overexpression of OsNRT1.1A in Arabidopsis and rice. However, we also observed that overexpression of the OsNRT1.1A gene in wheat can facilitate the growth of roots under low N conditions but has no effect on other aspects of growth and development under normal N conditions. Thus, it may lead to the improvement of wheat low N tolerance,which is different from the effects reported in other plants. A field trial analysis showed that transgenic wheat exhibited increased grain yield per plant under low N conditions. Moreover, transcriptome analysis indicated that OsNRT1.1A increased the expression levels of N uptake and utilization genes in wheat, thereby promoting plant growth under low N conditions. Taken together, our results indicated that OsNRT1.1A plays an important role in improving NUE in wheat with low N availability.

Nitrogen(N)fertilizer application boosts yield and quality in crops such as wheat(Triticum aestivum L.)but raises environmental and economic concerns(Liu et al.,2019).Therefore,identifying the genes associated with N use efficiency is a major objective in wheat breeding(Liu et al.,2022b).Nitrate transporters are key factors in plant N uptake and play important roles in N responses(Schroeder et al.,2013;Fan et al.,2023).However,few studies have explored the regulation of N uptake efficiency in wheat.Here,we report the functional characterization of a gene encoding a nitrate transporter,NRT1/PTR family 6.2(TaNPF6.2,also reported as Nitrate transporter 1.4[TaNRT1.4]),and describe how it affects wheat agronomic traits by enhancing N uptake efficiency.

Heading date, grain number per spike, and grain weight are crucial traits affecting yield and adaptability in wheat. The transcription factor TaMYB72 is an important regulator of wheat grain yield and its knock-out mutants can be used as germplasm resources for wheat improvement.

The 5′-open reading frame (ORF) acts as an upstream ORF to restrict overaccumulation of the 3′-ORF encoding protein of the Reduced height-B1b gene to moderately reduce plant height in wheat.

A chromosome fragment influencing wheat heading and grain size was identified using mapping of m406 mutant. The study of TaFPF1 in this fragment provides more insights into wheat yield improvement. In recent years, wheat production has faced formidable challenges driven by rapid population growth and climate change, emphasizing the importance of improving specific agronomic traits such as heading date, spike length, and grain size. To identify potential genes for improving these traits, we screened a wheat EMS mutant library and identified a mutant, designated m406, which exhibited a significantly delayed heading date compared to the wild-type. Intriguingly, the mutant also displayed significantly longer spike and larger grain size. Genetic analysis revealed that a single recessive gene was responsible for the delayed heading. Surprisingly, a large 46.58 Mb deletion at the terminal region of chromosome arm 2DS in the mutant was identified through fine mapping and fluorescence in situ hybridization. Thus, the phenotypes of the mutant m406 are controlled by a group of linked genes. This deletion encompassed 917 annotated high-confidence genes, including the previously studied wheat genes Ppd1 and TaDA1, which could affect heading date and grain size. Multiple genes in this region probably contribute to the phenotypes of m406. We further investigated the function of TaFPF1 using gene editing. TaFPF1 knockout mutants showed delayed heading and increased grain size. Moreover, we identified the direct upstream gene of TaFPF1 and investigated its relationship with other important flowering genes. Our study not only identified more genes affecting heading and grain development within this deleted region but also highlighted the potential of combining these genes for improvement of wheat traits.

Main conclusionTabZIP60 is found to interact with TaCDPK30 and act as a positive regulator of ABA synthesis-mediated salt tolerance in wheat.Wheat basic leucine zipper (bZIP) transcription factor (TabZIP60) was previously found to act as a positive regulator of salt resistance. However, its molecular mechanism in response to salt stress in wheat is still unclear. In this study, TabZIP60 was found to interact with wheat calcium-dependent protein kinase (TaCDPK30), which belonged to group III of CDPK family, and was induced by salt, polyethylene glycol, and abscisic acid (ABA) treatments. This mutation of serine 110 in TabZIP60 resulted in no interaction with TaCDPK30. Moreover, TaCDPK30 was involved in interactions with wheat protein phosphatase 2C clade A (TaPP2CA116/TaPP2CA121). TabZIP60-overexpressing wheat plants showed increased salt tolerance, as exhibited by better growth status, higher soluble sugar, and lower malonaldehyde contents of transgenic plants than wild-type wheat cv. Kenong 199 under salt stress. Moreover, transgenic lines showed high ABA content by upregulating ABA synthesis-related gene expression levels. TabZIP60 protein could bind and interact with the promoter of the wheat nine-cis epoxycarotenoid dioxygenase (TaNCED2) gene. Furthermore, TabZIP60 upregulated several stress response gene expression levels, which could also increase the plant's ability to resist salt stress. Thus, these results suggest that TabZIP60 could function as a regulator of ABA synthesis-mediated salt tolerance through interacting with TaCDPK30 in wheat.

One of the most significant transcription factors in plants, WRKYs, are crucial for plant growth and stress response. In this study, we analyzed the physicochemical properties, evolutionary relationships, conservation structure, and expression of the WRKY gene family in S. baicalensis. The WRKY family has highly conserved structural domains, which have been classified into three major categories, I, II, and III, based on the number of WRKY structural domains and zinc finger structural features. SbWRKYs of the same subgroup are functionally similar and essentially contain the same motif. Additionally, different drought stress situations resulted in varying levels of SbWRKYs expression, with the majority of these factors being up-regulated in moderate drought stress settings, and fewer of them were up-regulated under severe drought stress conditions. Under moderate drought stress, the expression of key enzymes increased, while under severe drought stress, the expression of key enzymes decreased. Mild drought stress resulted in a 26.42% increase in baicalin accumulation, while severe drought stress led to a 22.88% decrease. The protein interaction analysis of key enzyme genes and SbWRKYs revealed that the expression of key enzyme genes affected the expression of SbWRKYs. We screened nine SbWRKYs with a significant relationship with baicalin accumulation, and SbWRKY8 and SbWRKY16 showed the highest correlation with the baicalin content. These findings offer a theoretical framework for more research on the roles of SbWRKYs and show that SbWRKYs can respond to drought stress in S. baicalensis.

Large-grain traits are important in high-yield breeding of wheat. In order to identify the genetic characteristics of grain traits in the large-grain wheat, we used the large-grain type Luomai 76 as the male parent and the small-grain type L529 as the female parent to construct six pedigrees of four generation populations（P 1 ,P 2 ,F 1 ,F 2 ,B 1 and B 2 ）,and applied mixed major gene plus polygenes inheritance model to identify the genetic characteristics of grain traits based on SmartGrain. The results showed that the best genetic model for grain weight was MX2-ADI-ADI; The best genetic model for grain area and grain length were PG-ADI; The best genetic model for grain perimeter length was 2MG-ADI; The best genetic model for grain width was MX2-ADI-AD. Taken together, for the selection of large-grain character, materials with high grain weight should be adopted as the parents to construct F 2 （single cross） and B 2 （backcross with large-grain parents） selection population, identified in multiple environments. The large grain characters are suggested to be rigorously selected in the high generations.

Plant height and grain size are two important agronomic traits that are closely related to crop yield. Numerous dwarf and grain-shape mutants have been studied to identify genes that can be used to increase crop yield and improve breeding programs.In this study, we characterized a dominant mutant, dwarf and round grain 1 (drg1-D), in bread wheat (Triticum aestivum L.). drg1-D plants exhibit multiple phenotypic changes, including dwarfism, round grains, and insensitivity to brassinosteroids (BR). Cell structure observation in drg1-D mutant plants showed that the reduced organ size is due to irregular cell shape.Using map-based cloning and verification in transgenic plants, we found that a Glu209Lys substitution in the DRG1 protein is responsible for the irregular cell size and arrangement in the drg1-D mutant. DRG1/TaACT7 encodes an actin family protein that is essential for polymerization stability and microfilament (MF) formation. In addition, the BR response and vesicular transport were altered by the abnormal actin cytoskeleton in drg1-D mutant plants.Our study demonstrates that DRG1/TaACT7 plays an important role in wheat cell shape determination by modulating actin organization and intracellular material transport, which could in the longer term provide tools to better understand the polymerization of actin and its assembly into filaments and arrays.

Despite recent progress in crop genomics studies, the genomic changes brought about by modern breeding selection are still poorly understood, thus hampering genomics-assisted breeding, especially in polyploid crops with compound genomes such as common wheat (Triticum aestivum). In this work, we constructed genome resources for the modern elite common wheat variety Aikang 58 (AK58). Comparative genomics between AK58 and the landrace cultivar Chinese Spring (CS) shed light on genomic changes that occurred through recent varietal improvement. We also explored subgenome diploidization and divergence in common wheat and developed a homoeologous locus-based genome-wide association study (HGWAS) approach, which was more effective than single homoeolog-based GWAS in unraveling agronomic trait-associated loci. A total of 123 major HGWAS loci were detected using a genetic population derived from AK58 and CS. Elite homoeologous haplotypes (HHs), formed by combinations of subgenomic homoeologs of the associated loci, were found in both parents and progeny, and many could substantially improve wheat yield and related traits. We built a website where users can download genome assembly sequence and annotation data for AK58, perform blast analysis, and run JBrowse. Our work enriches genome resources for wheat, provides new insights into genomic changes during modern wheat improvement, and suggests that efficient mining of elite HHs can make a substantial contribution to genomics-assisted breeding in common wheat and other polyploid crops.

The utilization of stabilized DELLA proteins Rht-B1b and Rht-D1b was crucial for increasing wheat (Triticum aestivum) productivity during the Green Revolution. However, the underlying mechanisms remain to be clarified. Here, we cloned a gain-of-function allele of the GSK3/SHAGGY-like kinase-encoding gene GSK3 by characterizing a dwarf wheat mutant. Furthermore, we determined that GSK3 interacts with and phosphorylates the Green Revolution protein Rht-B1b to promote it to reduce plant height in wheat. Specifically, phosphorylation by GSK3 may enhance the activity and stability of Rht-B1b, allowing it to inhibit the activities of its target transcription factors. Taken together, we reveal a positive regulatory mechanism for the Green Revolution protein Rht-B1b by GSK3, which might have contributed to the Green Revolution in wheat.

Wheat (Triticum aestivum L.) is a major staple food for more than one-third of the world's population. Tiller number is an important agronomic trait in wheat, but only few related genes have been cloned. Here, we isolate a wheat mutant, tiller number1 (tn1), with much fewer tillers. We clone the TN1 gene via map-based cloning: TN1 encodes an ankyrin repeat protein with a transmembrane domain (ANK-TM). We show that a single amino acid substitution in the third conserved ankyrin repeat domain causes the decreased tiller number of tn1 mutant plants. Resequencing and haplotype analysis indicate that TN1 is conserved in wheat landraces and modern cultivars. Further, we reveal that the expression level of the abscisic acid (ABA) biosynthetic gene TaNCED3 and ABA content are significantly increased in the shoot base and tiller bud of the tn1 mutants; TN1 but not tn1 could inhibit the binding of TaPYL to TaPP2C via direct interaction with TaPYL. Taken together, we clone a key wheat tiller number regulatory gene TN1, which promotes tiller bud outgrowth probably through inhibiting ABA biosynthesis and signaling.

GLYCOGEN SYNTHASE KINASE 3 physically interacts with VRN1 and regulates its accumulation to mediate flowering in wheat.

Wheat (Triticum aestivum, BBAADD) is an allohexaploid species that originated from two polyploidization events. The progenitors of the A and D subgenomes have been identified as Triticum urartu and Aegilops tauschii, respectively. Current research suggests that Aegilops speltoides is the closest but not the direct ancestor of the B subgenome. However, whether Ae. speltoides has contributed genomically to the wheat B subgenome and which chromosome regions are conserved between Ae. speltoides and the B subge-nome remain unclear. Here, we assembled a high-quality reference genome for Ae. speltoides, rese-quenced 53 accessions from seven species (Aegilops bicornis, Aegilops longissima, Aegilops searsii, Aegi-lops sharonensis, Ae. speltoides, Aegilops mutica [syn. Amblyopyrum muticum], and Triticum dicoccoides) and revealed their genomic contributions to the wheat B subgenome. Our results showed that centromeric regions were particularly conserved between Aegilops and Triticum and revealed 0.17 Gb of conserved blocks between Ae. speltoides and the B subgenome. We classified five groups of conserved and non-conserved genes between Aegilops and Triticum, revealing their biological characteristics, differenti-ation in gene expression patterns, and collinear relationships between Ae. speltoides and the wheat B sub-genome. We also identified gene families that expanded in Ae. speltoides during its evolution and 789 genes specific to Ae. speltoides. These genes can serve as genetic resources for improvement of adaptability to biotic and abiotic stress. The newly constructed reference genome and large-scale resequencing data for Sitopsis species will provide a valuable genomic resource for wheat genetic improvement and genomic studies.

Wheat (Triticum aestivum L.) is a staple food crop that provides 20% of total human calorie consumption. Gene duplication has been considered to play an important role in evolution by providing new genetic resources. However, the evolutionary fates and biological functions of the duplicated genes in wheat remain to be elucidated. In this study, the resulting data showed that the duplicated genes evolved faster with shorter gene lengths, higher codon usage bias, lower expression levels, and higher tissue specificity when compared to non-duplicated genes. Our analysis further revealed functions of duplicated genes in various biological processes with significant enrichment to environmental stresses. In addition, duplicated genes derived from dispersed, proximal, tandem, transposed, and whole-genome duplication differed in abundance, evolutionary rate, gene compactness, expression pattern, and genetic diversity. Tandem and proximal duplicates experienced stronger selective pressure and showed a more compact gene structure with diverse expression profiles than other duplication modes. Moreover, genes derived from different duplication modes showed an asymmetrical evolutionary pattern for wheat A, B, and D subgenomes. Several candidate duplication hotspots associated with wheat domestication or polyploidization were characterized as potential targets for wheat molecular breeding. Our comprehensive analysis revealed the evolutionary trajectory of duplicated genes and laid the foundation for future functional studies on wheat.

Characterization of the early leaf senescence mutant els3 and identification of its causal gene ELS3, which encodes an LRR-RLK protein in wheat. Leaf senescence is an important agronomic trait that affects both crop yield and quality. However, few senescence-related genes in wheat have been cloned and functionally analyzed. Here, we report the characterization of the early leaf senescence mutant els3 and fine mapping of its causal gene ELS3 in wheat. Compared with wild-type Yanzhan4110 (YZ4110), the els3 mutant had a decreased chlorophyll content and a degraded chloroplast structure after the flowering stage. Further biochemical assays in flag leaves showed that the superoxide anion and hydrogen peroxide contents increased, while the activities of antioxidant enzymes, including catalase, superoxide dismutase and glutathione reductase, decreased gradually after the flowering stage in the els3 mutant. To clone the causal gene underlying the phenotype of leaf senescence, a genetic map was constructed using 10,133 individuals of F2:3 populations, and ELS3 was located in a 2.52 Mb region on chromosome 2DL containing 16 putative genes. Subsequent sequence analysis and gene annotation identified only one SNP (C to T) in the first exon of TraesCS2D02G332700, resulting in an amino acid substitution (Pro329Ser), and TraesCS2D02G332700 was preliminarily considered as the candidate gene of ELS3. ELS3 encodes a leucine-rich repeat receptor-like kinase (LRR-RLK) protein that is localized on the cell membrane. We also found that the transient expression of mutant TraesCS2D02G332700 can induce leaf senescence in N. benthamiana. Taken together, TraesCS2D02G332700 is likely to be the candidate gene of ELS3 and may have a function in regulating leaf senescence.

Previously we have found that TabZIP60 from the ABF/AREB (ABRE-binding factor/ABA-responsive element-binding protein) subfamily of bZIP transcription factor (TF) was involved in salt stress response. However, the regulatory mechanism of TabZIP60 is unknown. In the present study, we identified two calcium-dependent protein kinase (CDPK) genes, TaCDPK5/TaCDPK9-1, which were clustered into group I and were induced by salt, abscisic acid (ABA), and polyethylene glycol (PEG) treatments. RT-qPCR results showed that the expression level of salt-induced TabZIP60 was drastically inhibited by Ca2+ channel blocker LaCl3. TaCDPK5/TaCDPK9-1 were involved in interaction with TabZIP60 protein in vivo and in vitro. And TaCDPK5/TaCDPK9-1 could autophosphorylate and phosphorylate TabZIP60 protein in a Ca2+-dependent way. Mutational analysis indicated that Serine-110 of TabZIP60 was essential for TaCDPK5/TaCDPK9-1-TabZIP60 interaction and was the phosphorylation site of TaCDPK5/TaCDPK9-1 kinases. Yeast two-hybrid assay results showed the interactions between TaCDPK5/TaCDPK9-1 and wheat protein phosphatase 2 C clade A TaPP2CA116/ TaPP2CA121 separately. These findings demonstrate that the phosphorylation status of TabZIP60 controlled by TaPP2CA116/ TaPP2CA121 and TaCDPK5/TaCDPK9-1 might play a crucial role in wheat during salt stress.

Wheat is one of the world's major staple food crops, and breeding for improvement of grain yield is a priority under the scenarios of climate change and population growth. WRKY transcription factors are multifaceted regulators in plant growth, development, and responses to environmental stimuli. In this study, we identify the WRKY gene TaGSNE (Grain Size and Number Enhancer) in common wheat, and find that it has relatively high expression in leaves and roots, and is induced by multiple abiotic stresses. Eleven single-nucleotide polymorphisms were identified in TaGSNE, forming two haplotypes in multiple germplasm collections, named as TaGSNE-Hap-1 and TaGSNE-Hap-2. In a range of different environments, TaGSNE-Hap-2 was significantly associated with increases in thousand-grain weight (TGW; 3.0%) and spikelet number per spike (4.1%), as well as with deeper roots (10.1%) and increased root dry weight (8.3%) at the mid-grain-filling stage, and these were confirmed in backcross introgression populations. Furthermore, transgenic rice lines overexpressing TaGSNE had larger panicles, more grains, increased grain size, and increased grain yield relative to the wild-type control. Analysis of geographic and temporal distributions revealed that TaGSNE-Hap-2 is positively selected in China and Pakistan, and TaGSNE-Hap-1 in Europe. Our findings demonstrate that TaGSNE overcomes the trade-off between TGW/grain size and grain number, leading us to conclude that these elite haplotypes and their functional markers could be utilized in marker-assisted selection for breeding high-yielding varieties.