To elucidate the functions of the wheat mitogen-activated protein kinase genes TaMAPK1-6A, TaMAPK1-6B, and TaMAPK1-6D in seedling drought tolerance and the additive effects among these homeologous genes, this study utilized the spring wheat cultivar Fielder and its CRISPR/Cas9 mediated single-gene mutants-mapk1-aa, mapk1-bb, and mapk1-dd-generated in the Fielder genetic background. Through hybridization, self-pollination, and genotyping of segregating progeny, double-gene knockout mutants and triple-gene knockout homozygous mutants (hereafter referred to as double and triple mutants) were successfully developed. The drought tolerance at the seedling stage was systematically evaluated for these single, double, and triple mutants, along with the wild-type Fielder control. The results demonstrated that loss-of-function mutations in single TaMAPK1 genes significantly reduced drought tolerance in wheat seedlings, manifested as decreased leaf water retention, reduced accumulation of osmotic adjustment substances, and enhanced membrane lipid peroxidation. Further investigation revealed that drought tolerance was further impaired in double and triple mutants, with the deterioration of physiological and biochemical indicators intensifying as the number of mutated gene copies increased. In summary, the TaMAPK1 gene positively regulates drought tolerance at the wheat seedling stage, and its homeologous genes exhibit cumulative regulatory effects.

The R2R3-MYB transcription factor family, the largest subfamily of MYB transcription factors, plays critical roles in various plant stress responses. However, its function under low-phosphate (P) stress remains poorly characterized. In this study, a total of 127 R2R3-MYB genes were identified in the barley MorexV3 genome using bioinformatic approaches and designated as Hv2RMYB-1 to Hv2RMYB-127. These genes were predominantly distributed on the 7 barley chromosomes and were found to harbor hormone-responsive cis-acting elements in their promoter regions. Physicochemical analysis revealed that most members encode acidic and unstable proteins. Phylogenetic and collinearity analyses classified the barley R2R3-MYB family into five distinct clades, including 16 pairs of tandemly duplicated genes. Based on transcriptomic data (RNA-Seq) and quantitative real-time PCR (qRT-PCR), 10 barley R2R3-MYB genes showed differential expression under low-Phosphorus stress, among which Hv2RMYB-48 was notably upregulated. Further investigation confirmed that Hv2RMYB-48 localizes to the nucleus and can bind to the MYB-ABRE-MYBcis-element.

Plant height (PH) is an important agronomic trait affecting lodging resistance and yield in wheat. To dissect the genetic basis of PH and identify stable loci, we analyzed a natural population of 339 wheat accessions from the Huang-Huai wheat region using PH phenotypes collected across nine environments and genotypes obtained with a 21K single nucleotide polymorphism (SNP) array. Genome-wide association study (GWAS) was conducted using a mixed linear model (MLM). Linkage disequilibrium (LD) and haplotype analyses were performed for the key associated interval, and the association was further validated in a natural population of 327 accessions genotyped with a 660K SNP array. Candidate genes within the target interval were annotated and prioritized. In total, 66 stable QTLs significantly associated with PH were identified. A novel major and stable QTL, QPh. nwafu-6D, was detected on chromosome 6D (490.34-492.42 Mb) and was consistently detected in eight environments, explaining 8.23% of the phenotypic variance on average. The interval was divided into two LD blocks. In Block2, three haplotypes were identified, and the favorable haplotype Block2_Hap1 (frequency 84.1%) showed significantly reduced PH compared with other haplotypes and was significantly associated with higher thousand-kernel weight in multiple environments. In the validation population, accessions carrying the Block2_Hap1 genotype at the core SNP marker SNP_Chr6D_492211566 exhibited significantly lower PH than those with the Block2_Hap2 genotype (P< 0.001). Eight candidate genes were further prioritized in this interval, mainly involved in kinase signaling, transcriptional regulation, ubiquitin-mediated protein degradation, and hormone responses.

The ERECTA family of receptor-like kinases (RLKs) plays a crucial regulatory role in plant stomatal development and responses to abiotic stresses. This study therefore focused on investigating the barley HvERECTA gene, aiming to clarify its regulatory roles in these biological processes and provide potential targets for barley drought resistance breeding. We first cloned the HvERECTA gene from barley and conducted comprehensive bioinformatics analyses to characterize its sequence and protein properties. Subsequently, we constructed a heterologous overexpression vector carrying the HvERECTA gene and transformed it into Arabidopsis thaliana. For the transgenic Arabidopsis lines, we systematically determined stomatal phenotypic traits, expression levels of the key related genes, and physiological indicators under drought stress conditions to evaluate the gene function. The bioinformatics analysis results showed that the full-length coding sequence (CDS) of HvERECTA is 2 934 bp, encoding a protein of 977 amino acids. The HvERECTA protein is localized to the plasma membrane and contains a typical serine/threonine protein kinase domain, which is a hallmark of the receptor-like kinase family. Sequence alignment and phylogenetic analysis revealed that HvERECTA is highly conserved among gramineous plants, with the highest homology to those from wheat (Triticum aestivum) and ryegrass (Lolium perenne). Additionally, analysis of cis-acting elements in the HvERECTA promoter region indicated that it is rich in regulatory elements associated with abiotic stress responses and plant hormone signaling, such as abscisic acid (ABA)-responsive elements (ABRE) and drought-responsive elements (MBS). Phenotypic observations on the transgenic Arabidopsis lines showed that overexpression of HvERECTA significantly reduced stomatal density compared with the wild-type control. Molecular analysis demonstrated that the expression levels of genes encoding key stomatal development transcription factors, namely SPCH (SPEECHLESS), MUTE, and FAMA, were all downregulated by more than 50% in the transgenic lines. In contrast, the expression level of YODA, a core gene in the mitogen-activated protein kinase (MAPK) cascade pathway that is involved in stomatal development regulation, was upregulated by 50%-80% in the transgenic plants. Under drought stress, the transgenic Arabidopsis lines showed enhanced drought resistance-related physiological performance. Specifically, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) in the transgenic lines were increased by 25%-40% compared with the control. Meanwhile, the content of malondialdehyde (MDA), which is an indicator of lipid peroxidation and cellular damage under stress, was decreased by approximately 30% in the transgenic lines. Additionally, the water loss rate of the transgenic lines was reduced by 20%, and their relative water content was higher than that of the control. Furthermore, the expression levels of stress-responsive genes, including AtKIN2, AtABF3, and AtRD22, showed no abnormal changes under normal growth conditions but were significantly upregulated under drought stress in the transgenic lines. In conclusion, the barley HvERECTA gene enhances plant drought resistance by regulating the MAPK signaling pathway to reduce stomatal density and by improving the activity of the plant antioxidant defense system. These two regulatory pathways collectively enhance the water retention capacity of plants under drought stress, thereby improving their stress tolerance. This study clarifies the important regulatory role of HvERECTA in stomatal development and abiotic stress response, and provides an important functional gene resource for drought-resistant molecular breeding of barley.

To investigate genome-wide mRNA secondary structure reprogramming and its correlation with translation efficiency under heat stress in wheat, this study performed a multi-omics analysis on wheat seedlings (cv. Chinese Spring) under different conditions, utilizing SHAPE-MaP and polysome profiling. The results showed that the SHAPE reactivity of start and stop codon positions in wheat transcripts was significantly higher than that of flanking regions, exhibiting conserved structural features. Compared with the control, heat stress resulted in a decrease in the average SHAPE reactivity across transcripts, indicating that mRNA tends to form double-stranded structures. This change exhibited reversibility during recovery. At the early stage of heat stress, structural changes in the start codon region were the most significant. Polysome profiling showed that heat stress caused a decrease in polysome peaks, and translation was inhibited at the genome-wide level. Correlation analysis revealed that there was no significant correlation between SHAPE reactivity changes and translation efficiency changes of various functional regions at the genome-wide level. In summary, the heat-induced increase in mRNA folding may be a proactive defense strategy, which presents a non-linear complex relationship with translational regulation.

To elucidate the molecular mechanism of miRNA in wheat's response to dry-hot wind stress, a sensitive germplasm Xinchun 14 and a tolerant variety Xinchun 46 were selected as materials. During the grain-filling stage, artificial simulation of dry-hot wind stress (daytime temperature 35 ℃/nighttime temperature 25 ℃, relative humidity 20%, wind speed 5 m·s^-1, continuous treatment for 5 days) was applied. High-throughput sequencing technology was used to screen differentially expressed miRNAs, followed by validation via qRT-PCR. The results showed that 203 miRNAs were involved in the expression under dry-hot wind stress, with 136 co-expressed miRNAs. After differential screening, six core differentially expressed miRNAs were ultimately identified, namely, novel_miR_537, novel_miR_443, novel_miR_469, novel_miR_393, novel_miR_390, and novel_miR_339. These miRNAs were significantly enriched in four pathways: the biosynthesis of mannose O-glycans, the degradation of polysaccharides, peroxisomes, and the metabolism of selenium compounds. Target gene predictions revealed that these miRNAs targeted and regulated key membrane protein-encoding genes, including β-1, 2-xyltransferase, α-1, 3-arabinosyltransferase, GDSL esterase/lipase, α-L-fucosidase, and peroxidase, and are involved in the maintenance of cell wall stability, reactive oxygen species clearance, and osmotic regulation processes. This indicates there is a miRNA regulatory network in the response of dry-hot wind in wheat, and it jointly participates in the resistance process of wheat to dry-hot wind stress in the form of a 'miRNA-targeted gene' molecular module.

High mobility group (HMG) proteins are key regulators of chromatin dynamics in eukaryotes. To investigate the dynamic responses of the wheat HMG family to drought and phosphate starvation, a genome-wide identification of the TaHMG genes was performed based on the common wheat reference genome (IWGSC Ref Seq V1.0). Bioinformatic analyses were conducted to determine the physicochemical properties, phylogeny, collinearity, and promoter cis-elements of the family members. Public transcriptome data and quantitative real-time PCR (qRT-PCR) were integrated to examine tissue-specific expression patterns and temporal expression profiles under drought stress (15% PEG6000; 0, 1, 3, 6, 12, and 24 h) and phosphate starvation (0 μmol·L^-1 Pi; 0, 1, 3, 6, 12, and 24 h). A total of 32 TaHMG genes were identified, which were phylogenetically divided into two subfamilies: HMGA (9 members) and HMGB (23 members). Collinearity analysis indicated that the family evolved predominantly under purifying selection. Promoter analysis revealed that TaHMG genes are enriched in hormone and stress-responsive elements, including abscisic acid (ABRE) and methyl jasmonate (MeJA)-responsive motifs. qRT-PCR showed that Ta-6D-HMGB1, Ta-3D-HMGB1 and Ta-7D-HMGB2 were highly expressed in leaves, whereas Ta-2D-HMGB1, Ta-5A-HMGB1, and Ta-7D-HMGB1 were highly expressed in stems. Under drought stress, five TaHMG genes were rapidly induced at early stage (3 or 6 h), while Ta-7D-HMGB2 was initially suppressed (0-3 h) and subsequently up-regulated (6 h). Under phosphate starvation, expression peaks of different members occurred at 12 or 24 h post treatment, revealing temporal specificity and functional divergence among genes. Collectively, members of the wheat HMG family adapt to abiotic stresses through differential expression, and stress-responsive genes such as Ta-7D-HMG2 and Ta-3D-HMGB1 represent promising candidates for molecular breeding of stress tolerance.

Xinjiang, especially in the southern Xinjiang region, precipitation is scarce and the climate is arid. Land in this area suffers from varying degrees of salinization, which affects wheat growth and leads to reduced yields. Screening salt-tolerant germplasm resources is an effective approach to improving grain production capacity in southern Xinjiang. In this study, 42 spring wheat varieties approved and released in the Yellow River Irrigation District of Ningxia and its surrounding areas were used as experimental materials. Under 150 mmol·L^-1 NaCl stress, germination and phenotypic indices were measured at the germination and seedling stages, and relative values of the 13 indices were calculated to comprehensively evaluate the salt tolerance of different varieties. The results showed that the relative values of most indices at the germination and seedling stages were below 100%. The coefficients of variation for germination energy, germination index, and vigor index at the germination stage exceeded 20%. At the seedling stage, the coefficients of variation for seedling height, above-ground fresh weight, above-ground dry weight, root dry weight, and root length were greater than 20%. Significant positive correlations were observed among the indices. The tested materials were classified into three categories via hierarchical cluster analysis: 15 salt-tolerant genotypes, 20 intermediate genotypes, and 7 sensitive genotypes. Ningchun 3, Ningchun 8, Ningchun 9, Ningchun 12, and Ningchun 35 exhibited superior salt tolerance and strong stress adaptability, which preliminarily verifies the feasibility of cross-regional introduction of Ningchun spring wheat germplasm resources, and providing material basis and methodological reference for breeding new salt-tolerant wheat varieties in southern Xinjiang.

In order to explore the evaluation methods and indicators of nitrogen efficiency at the maturity stage of spring wheat in Xinjiang and to screen high nitrogen-efficient germplasm resources, providing reference for breeding wheat varieties with high nitrogen-use efficiency, twenty spring wheat varieties widely cultivated in Xinjiang were used as research materials, and four nitrogen fertilizer levels were set up: 0 kg·hm^-2 (N0), 100 kg·hm^-2 (N1), 200 kg·hm^-2 (N2), and 300 kg·hm^-2 (N3). Eleven traits were investigated under different nitrogen levels, such as stem dry weight, leaf dry weight, weight per spike, grain number per spike, grain yield, plant height, and SPAD value of wheat at maturity. Principal component analysis (PCA) and membership function were used to comprehensively evaluate wheat varieties and classify nitrogen efficiency types. The results showed that, compared with N2 and N3 treatments, N0 and N1 treatments significantly resulted in reduced plant height during the wheat maturity stage, thinner stems and leaves, and lower grain yield; The coefficients of variation for most traits under N0 and N1 treatments were higher than those under N2 and N3. Correlation analysis showed that stem weight was significantly positively correlated with spike weight and grain weight under the four nitrogen levels, and spike number per unit area was negatively correlated with stem weight, spike weight, grain weight per spike, and 1 000-grain weight. Four principal components were extracted under each nitrogen level across two years, with cumulative contribution rates ranging from 75.63% to 82.20%. Based on the comprehensive nitrogen-use efficiency index (G value), the tested wheat varieties were classified into four categories: nitrogen-efficient type, nitrogen-inefficient type, low N fertilizer application nitrogen-efficient type, and high N fertilizer application nitrogen-efficient type. Combining G value and grain yield, seven high-yield and nitrogen-efficient varieties were identified, including Hechun 137, Liangchun 1242, Liangchun 1354, Xinchun 38, Xinchun 47, Xinchun 48, and Xinchun 6.

Nulli-tetrasomic materials are the important chromosomal genetic wheat materials, which have been used for target gene localization. However, fewer studies reported on the agronomic and quality traits of nulli-tetrasomic materials. In order to analyze the effects of chromosome deletions and duplications on major agronomic and quality traits of wheat, the agronomic, grain and flour processing quality traits of 38 nulli-tetrasomic materials were investigated. The results showed that coefficients of variation of agronomic traits showed grain number per spike>tiller number>spike length>spikelet number>plant height. Compared with the control（Chinese spring）, the number of spikelets of N7D-T7A increased significantly; the number of grains per spike of N2A-T2B and N6B-T6A decreased significantly; the plant height of N4B-T4A and N7D-T7A increased significantly; the plant height of N6D-T6B decreased significantly; the spike length of N4A-T4B and N7A-T7B decreased significantly; yet there was no significant difference in the number of tillers between all nulli-tetrasomic materials and the control. The coefficients of variation of grain traits of the nulli-tetrasomic materials ranked as 1 000-grain weight>grain area>length-width ratio>grain width>grain length>perimeter; compared with the control, the grain area of N3B-T3D, N3D-T3A, N3D-T3B, N6A-T6B, and N6B-T6A increased significantly; the grain width of N3B-T3A, N3B-T3D, N3D-T3B, N6A-T6B, and N6B-T6A increased significantly, and the length-width ratio of N1B-T1A, N1B-T1D, N3A-T3D, N4A-T4B, N4A-T4D, N5B-T5A, N5B-T5D, N6A-T6D, N7A-T7B, and N7A-T7D decreased significantly. The 1 000-grain weight of N2A-T2B was significantly reduced, and there was no significant difference in circumference and grain length between all nulli-tetrasomic materials and the control. The variation coefficients of flour quality traits of the nulli-tetrasomic materials ranked as Zeleny>wet gluten content>protein content>moisture content>starch content; compared with the control, the nulli-tetrasomic materials had differences in flour quality traits, which are not significant. The coefficients of variation of nulli-tetrasomic materials ranked as stability time>development time>farinograph quality number>weakening value>water absorption; compared with the control, only the first homologous group nulli-tetrasomic materials showed significant differences in dough development time and stability time. Among them, the stability time of N1A-T1B dough reached significant differences, the dough development time and the stability time of N1A-T1D reached extremely significant differences, while there was no significant difference in other indices between the nulli-tetrasomic materials and the control. The results of this study lay a foundation for further analysis of the genetic effects of nulli-tetrasomic materials.