The induction of pluripotent callus from somatic cells is a key step in plant regeneration, with broad implications for developmental biology, biotechnology, and crop genetic improvement. While the association between auxin and cytokinin is well established as the central regulator of regeneration programs, the roles of other phytohormones remain poorly understood. Here, we elucidate a mechanism by which the brassinosteroid (BR) signaling transcription factor BES1 negatively regulates callus formation by modulating very-long-chain fatty acid (VLCFA) biosynthesis in Arabidopsis and cotton. AtBES1 directly binds to BR response elements (BRREs) in the promoter region of AtKCS1, which encodes a rate-limiting enzyme in VLCFA biosynthesis, thereby enhancing AtKCS1 expression and elevating endogenous VLCFA levels. Functional analyses using Atkcs1 and Ghkcs1 knockout mutants revealed a marked increase in callus formation, confirming the conserved negative regulatory role of KCS1 in this process. Genetic analyses further demonstrated that KCS1 functions epistatically to BES1 within this pathway. Collectively, these findings uncover an uncharacterized regulatory module in which BR signaling and VLCFA metabolism converge to repress callus formation, offering new insights into the lipid–hormone crosstalk governing cellular pluripotency during plant regeneration.
The NAC (NAM, ATAF, and CUC) transcription factors include nuclear members and membrane members like NTL (NAC with Transmembrane motif 1-like). The objective of this study was to identify the mechanism by which NACs with different localizations synergistically regulate maize salt tolerance. Knockout, overexpression, and protein interaction studies showed that ZmNAC032 and ZmNTL1 form a module that mediates maize salt tolerance by regulating ion balance and soluble sugar accumulation. Combined ChIP, Y1H, and dual-luciferase reporter assays confirm that ZmNAC032 directly induces expression of the trehalose synthesis gene ZmTPSII.5.3 and the ion transport gene ZmHAK16a via direct interaction with the promoter cis-elements CCATCT and ACTGT, respectively. After salt stress, ZmNTL1 translocates to the nucleus and interacts with ZmNAC032, forming heterodimers that enhance ZmNAC032‑mediated transcriptional activation of ZmTPSII.5.3 and ZmHAK16a, suggesting synergistic regulation of ZmNAC032 and ZmNTL1 in maize. Double overexpression of ZmNTL1 and ZmNAC032 confers the highest salt tolerance, while double knockout confers the lowest, indicating their synergistic positive regulation in maize. This study reveals the molecular mechanism by which the transcriptional module mediated by ZmNAC032-ZmNTL1 regulates salt tolerance, which may be used to improve salt tolerance in maize via advanced breeding
Understanding how environmental fluctuations shape the plasticity of rice grain quality traits, such as amylose content (AC) and chalkiness degree (CD), is essential for breeding climate-resilient, high-quality varieties. To address this, we cultivated two plastic varieties (RD15 and HXZ9) and one stable variety (D50) across overlapping light-temperature regimes and employed time-resolved transcriptomics and full-length isoform sequencing during grain filling. Integrated analysis of genetic variation, gene expression, and alternative splicing revealed a fundamental dichotomy in regulatory strategies: plastic varieties achieve adaptability through targeted reprogramming of core metabolism and condition-sensitive alternative splicing of key regulators, while the stable variety enforces robustness via a constitutive suite of genes involved in precise RNA processing and cellular compartmentalization. Notably, we identified and experimentally validated that differential transcript usage of FLO12 is strongly associated with AC plasticity, providing a direct mechanistic link between isoform-level regulation and phenotypic variation. Our multi-omics framework, from genomic variation to dynamic transcriptional and post-transcriptional control, defines a hierarchical regulatory landscape governing the environmental responsiveness and stability of AC and CD, offering actionable targets for the precision breeding of climate-resilient, high-quality rice
Thinopyrum elongatum (2n = 4x = 28) harbors multiple valuable resistance genes and serves as a valuable genetic resource for wheat improvement. Yr1EL was primarily identified on chromosome arm 1EL of tetraploid Th. elongatum, conferring adult-plant resistance to stripe rust. To further map and utilize Yr1EL, we introduced chromosomal rearrangements by crossing the wheat–tetraploid Th. elongatum 1E(1D) substitution line and the T1BS·1EL translocation line with the common wheat Chinese Spring ph1b mutant. In total, eight wheat–Th. elongatum chromosome 1E structural variants were identified by in situ hybridization, the GenoBaits®WheatplusEE panel, and molecular markers. These variants include one large segment translocation, one terminal fragment deletion, one chromosome 1E insertion translocation, and five chromosome 1EL terminal small fragment translocations. Based on phenotyping and genotyping of these variant lines, Yr1EL was mapped to an approximately 20.91 Mb physical interval (532.45 to 553.35 Mb) on the distal long arm of chromosome 1E corresponding to the diploid Th. elongatum reference genome. Genetic analysis confirmed that stripe rust resistance was conferred by the Yr1EL locus with incomplete dominance. Five codominant molecular markers co-segregated with the Yr1EL locus and will facilitate marker-assisted selection. Furthermore, collinearity analysis showed that this interval is structurally conserved among Triticeae species, and 29 candidate genes potentially related to disease resistance were annotated. Those candidates included receptor-like proteins, kinases, nucleotide-binding and leucine-rich repeat receptors, and other disease resistance proteins. The adult-plant resistance gene Yr1EL expands the wheat disease-resistance gene pool and provides valuable germplasm for breeding durable stripe rust-resistant cultivars.
Improving lodging resistance without compromising yield is a major challenge in rapeseed (Brassica napus L.), a globally important oilseed crop. Here, we demonstrate that calcium application simultaneously enhances lodging resistance and yield of rapeseed under pot and field conditions. In pot studies, calcium treatment enhanced photosynthetic capacity, increased the number of siliques by an average of 29.4%, and improved thousand-seed weight, resulting in higher yields. Calcium treatment also increased stem bending resistance by altering stem structure and cell-wall composition, with upper- and lower-stem strength increasing by 33.0%–35.2% and 23.9%–30.6%, respectively. We confirmed these beneficial effects at multiple field sites with different soil types and planting densities. Physiological and biochemical analyses revealed that calcium treatment strengthens stem mechanical properties by promoting calcium pectate accumulation and reducing pectin methylesterification, as supported by the upregulation of pectin methylesterase (PME) genes. Furthermore, knockout of BnaPME35 abolished the effect of calcium treatment on lodging resistance, highlighting the role of pectin methylesterification levels in lodging susceptibility. These findings suggest that targeted application of calcium-containing fertilizers offers a practical strategy for improving lodging resistance and yield in rapeseed.
The microRNA399-PHOSPHATE2 (miR399-PHO2) module is known to maintain phosphate homeostasis in plants, yet its spatiotemporal control and integration of phosphate signaling under low-phosphate (LP) stress remain unclear. In this study, we show that LP induces distinct OsmiR399/OsPHO2 expression dynamics in roots versus shoots, and root-derived OsmiR399 preferentially suppresses OsPHO2 to facilitate P uptake and translocation. An OsmiR399-resistant OsPHO2-b mutant with deletion of OsmiR399-binding sites within OsPHO2 5′-UTR, significantly reduces shoot P overaccumulation in OsPHR2/OsmiR399 overexpressors under P-replete conditions. Under LP stress, both OsPHO2-b and osmir399 mutants display reduced root plasticity, enhanced shoot growth inhibition and accelerated leaf senescence. Transcriptomic and cellular analyses revealed tissue-specific dysregulation of P-starvation response (PSR) genes, with suppressed expression in roots but elevated in shoots, as well as defective root-shoot P redistribution. Root-split experiments further showed that OsPHO2-b disrupts systemic auxin signaling and root elongation response to local LP stimuli. Genetic evidence revealed that OsPHO2 functionally intersects with OsNLA1 to synergistically regulate PSRs in a P-dependent manner. Meanwhile, overexpression of OsPHO1-2 significantly enhances root elongation and P translocation in OsPHO2-b. Collectively, our findings establish the OsmiR399-OsPHO2 module as a central hub for root-shoot communication, coordinating root plasticity with systemic signaling to facilitate LP acclimation in rice.
Grain shape is a key agronomic trait for grain yield and quality in crops. Studies on rice have revealed several complex signaling pathways governing cell proliferation and cell expansion during grain development. Although cell division is critical for these events, the mechanistic integration of cell division into these developmental processes to determine grain shape remains unclear. In this study, we have identified Grain Shape 11 (GS11) as a positive regulator of rice grain shape. Loss of GS11 function leads to mispositioning of the preprophase band (PPB), a cortical ring composed of microtubules and actin filaments that defines cell division orientation. This misregulation results in asymmetric divisions, reduced cell size, increased cell number, and disorganized cell arrangements, ultimately producing shorter and wider grains. GS11 encodes a plant-unique protein that associates with microtubules. GS11 physically interacts with OsTON1a and OsTON2, the components of TON1-TRM-PP2A (TTP) complex, to co-regulate PPB positioning and cell division orientation during mitosis. Moreover, GS11 genetically interacts with GW7 in regulating grain shape. Altogether, our results identify GS11 as a TTP-associated protein that functions in coordinating cell proliferation and expansion likely by modulating microtubule organization and PPB positioning, providing insights into rice grain shape regulation.
The purpose of this study was to develop a method for producing high-resolution mapping populations without the disadvantages of rare alleles and population structure, combining the advantages of biparental and association mapping: Advanced Intercross-Derived Recombinant Inbred Lines (AID-RILs). We used male sterility to create an AID-RIL population in rice. Genome-wide association studies in this population achieved high detection power and low false-positive rate for identifying quantitative-trait nucleotides, with the FarmCPU method showing the best performance. The strategy could be extended to multi-parental crosses.
Heterosis is central to breeding high-yielding maize (Zea mays L.). To dissect the genetic basis of heterosis for yield-related traits, we constructed four testcross populations using four diverse inbred lines as testers and an association mapping panel of 368 inbred lines. We systematically investigated ear diameter, ear length, kernel row number, kernels per row, and kernel weight per ear, as well as their mid-parent heterosis values, across two environments. Using whole-genome sequencing and genome-wide association analysis, we mapped genomic regions and identified candidate genes associated with these traits and their heterosis. We detected a total of 94 genomic regions containing 389 candidate genes, of which Zm00001d051889 (ETHYLENE INSENSITIVE 4 [EIN4]), involved in ethylene signaling, was a key candidate locus. We identified a 59-bp insertion–deletion (InDel) variant in its promoter region, and EIN4 showed non-additive expression in hybrids. Haplotype analysis revealed that the stacking of Hap 3 of EIN4 in hybrids is associated with greater ear diameter and kernel row number. EMS-induced loss-of-function mutation of EIN4 resulted in altered ear length in hybrids, indicating an impact on ear length heterosis. These findings demonstrate that promoter variation, haplotype changes, and EMS-induced premature termination of EIN4 all affect heterosis or yield-related traits, providing convergent genetic evidence for the contribution of EIN4 to yield-related heterosis in maize. The identification of EIN4 and its natural polymorphisms provides a useful molecular resource for maize hybrid breeding, facilitating marker-assisted selection and the development of high-yielding and stable maize varieties.
Multi-environment trials (METs) are essential for maize breeding, but their implementation is often constrained by limited resources, particularly the high cost of phenotyping. To optimize resource allocation, 264 testcrosses derived from 100 inbred lines and three testers were evaluated across six locations. Four genomic prediction models, five cross-validation schemes (CV1–CV5), and five training set proportions (S1–S5) were systematically assessed for predicting grain moisture content (GMC), grain yield (GY), and plant height (PH). Together, the five cross-validation schemes and five training set proportions generated 25 sparse testing strategies. Among the four models, the reaction norm model consistently achieved the highest prediction accuracy, and was therefore selected for subsequent analyses. Averaged across S1–S5, CV1 showed the lowest prediction accuracy, with mean accuracies of 0.361 for GMC, 0.413 for GY, and 0.480 for PH. In contrast, CV2–CV5 achieved higher and comparable accuracies. Increasing the training set proportion from 1/6 (S1) to 5/6 (S5) improved prediction accuracy by 48.3% for GMC, 30.3% for GY, and 26.7% for PH, although further gains became limited once the training set proportion exceeded approximately 50% (S3). In addition, CV3 enabled reliable estimation of parental general combining ability based on predicted hybrid performance, particularly at higher training set proportions. Overall, integrating multi-environment sparse testing with genomic prediction enables efficient hybrid evaluation under limited resources, providing a cost-effective strategy to reduce phenotyping demands and accelerate breeding progress.
Triticum monococcum ssp. aegilopoides (syn. T. boeoticum) is a wild diploid species that harbors the AbAb genome containing many disease resistance genes useful for wheat improvement. Genetic analysis conducted on an F2 population from a cross between T. boeoticum accessions PI 427741 and PI 427560, which are contrasting in powdery mildew responses, indicated that the resistance in PI 427741 was controlled by two dominant genes, PmNCA6 and another unknown gene. Bulked segregant exome capture sequencing (BSE-Seq) analysis of a segregating F3 family without PmNCA6 demonstrated a new powdery mildew resistance gene, Pm72, on the long arm of chromosome 6A. Through linkage analysis, Pm72 was mapped to a 1.28-cM genetic interval defined by markers XTb6AL04 and XTb6AL06. According to the reference genome of T. boeoticum accession TA299, the Pm72 locus corresponded to a 940-kb physical region (Chr6A: 635,963,867–636,888,696), containing 21 nucleotide-binding leucine-rich repeat receptor (NLR)-like disease resistance genes. Phenotyping showed that Pm72 confers effective resistance to powdery mildew at both the seedling and the adult-plant stages in the hexaploid wheat background. The co-segregating marker XTb6AL05 was useful for marker-assisted selection of Pm72. The identification of the new powdery mildew resistance gene Pm72 will contribute to its positional cloning and breeding application in wheat.
Kernel moisture content (KMC) and kernel dehydration rate (KDR) strongly influence grain quality and suitability for mechanical harvesting in maize (Zea mays), yet the underlying genetic architecture is unclear. In this study, using a recombinant inbred line population phenotyped across four environments at 35, 42, 49, and 56 d after pollination (DAP), we identified 38 QTL associated with KMC and KDR, explaining 1.16%–20.88% of the phenotypic variance. The major QTL qKDR3.06 accounted for 4.88%–20.88% of the variance and was fine-mapped to a 219-kb interval in bin 3.06 of chromosome 3. Integrated parental sequence comparison, expression profiling, and regional association analysis identified ZmBAK1a (GRMZM5G815009), which encodes a brassinosteroid (BR) BAK1 family co-receptor, as the leading candidate gene in this interval. Five significant KDR-associated SNPs were located within this gene. A 202-bp InDel in the ZmBAK1a promoter distinguished the two parents, suggesting that this InDel is a causal regulatory variant. RT-qPCR revealed stage-specific differences in gene expression: the PH4CV allele (carrying the 202-bp deletion) showed lower ZmBAK1a expression at 28–40 DAP but significantly higher expression at 45 DAP than the KB020 allele. Consistent with this pattern, genotypes harboring the 202-bp deletion exhibited faster kernel dehydration in the AMP1 and AMP2 maize populations. Bioinformatic analysis identified a canonical salicylic acid (SA)-responsive cis-element within the deleted region. Given that BAK1 family genes regulate cell wall remodeling and water transport during grain maturation, we propose that ZmBAK1a influences late-stage kernel dehydration through BR-mediated signaling, with SA as a potential additional regulator. Our findings provide a functional marker and a novel gene target for breeding maize hybrids with rapidly dehydrating kernels.
Some introns can act as cis-elements to enhance gene expression without altering promoter specificity. Here, we engineered the castor bean catalase 1 first intron (CatI) by mutagenesis and identified CatI-mu7, which exhibited enhanced fusion-gene expression and improved splicing accuracy in Nicotiana benthamiana, Arabidopsis, Solanum lycopersicum, and rice. Knock-in of CatI-mu7 into HSP101 increased male fertility under heat stress in rice and Arabidopsis compared with wild-type and CatI knock-in controls, highlighting its potential applications in crop breeding.
Drought affects over 60% of wheat cultivation areas and reduces wheat yields considerably. To ensure global food security, it is imperative to decode the molecular basis of wheat drought tolerance and adaptation. In this study, we characterized a drought‑inducible heat shock transcription factor, designated TaHsfA18. Overexpression of TaHsfA18 markedly enhanced seedling survival under drought conditions, water retention, and post-drought recovery, whereas CRISPR/Cas9-mediated mutation caused heightened sensitivity to drought. We found that TaHsfA18 promoted the accumulation of osmolytes such as proline and soluble sugars and mitigated oxidative damage, as evidenced by lower levels of H2O2 and malondialdehyde. TaHsfA18 directly bound to a Heat Shock Element in the promoter of TaERF073, repressing its transcription and thereby alleviating its suppression of stress-adaptive pathways. In field trials, TaHsfA18-OE lines showed superior performance in terms of photosynthetic rate, water use efficiency, and grain yield under drought conditions. We determined that the Hap-4B-A haplotype of TaHsfA18 is associated with increased spikelet number per spike and could be useful for marker-assisted selection. Together, our data demonstrate that TaHsfA18 is an important integrator of osmotic and oxidative stress responses that shows promise as a target for engineering drought-tolerant wheat varieties.
Seed oil content is a key agronomic trait that defines the quality and economic value of soybean. Although regulatory mechanisms of seed oil accumulation have been investigated using single-omics strategies, the metabolic dynamics during seed development remain poorly understood from a multi-omics perspective. Here, we performed an integrated analysis of metabolomics and transcriptomics across multiple seed developmental stages to dissect the regulatory landscape underlying oil biosynthesis and accumulation in soybean. In total, more than 1000 differential metabolites and 7000 differentially expressed genes (DEGs) were identified in nine soybean cultivars with contrasting oil contents. Through correlation analysis linking metabolic pathways, differential metabolites, and DEGs, we characterized 10 core DEGs significantly associated with seed oil content, which are involved in amino acid metabolism, flavonoid biosynthesis, lipid biosynthesis, and phenylpropanoid biosynthesis. Further integration of oil metabolism pathway analysis and natural population haplotype analysis verified that GmADH1 and GmCrRLK1L34 function as the core hub genes in the regulatory network governing soybean seed oil accumulation. Integrating multi-omics data, our study uncovers the core regulatory circuits controlling oil accumulation in soybean seeds and provides a valuable framework for investigating key agronomic traits across diverse crop species.
Intercropping maize (Zea mays) and soybean (Glycine max) is an appealing strategy for improving land-use efficiency. However, the shade stress imposed by maize severely constrains soybean growth and yield. Most intercropping studies conducted to date have been based on artificial shading or relay intercropping, and neither the characteristics of the shade environment under simultaneous intercropping conditions nor the changes in soybean architectural traits have been systematically studied. In this study, we evaluated 907 soybean accessions grown in a simultaneous maize–soybean intercropping system over two consecutive years to identify phenotypic traits associated with yield performance. Intercropping with maize produced a dynamic and heterogeneous shade environment within the soybean canopy, characterized by reductions in the red-to-far red light (R:FR) ratio, blue light fluence, and total photosynthetically active radiation. Shading began after the soybean stage V3 and persisted throughout reproductive development, triggering the shade avoidance response and resulting in substantial yield losses in soybean. Stem diameter showed the strongest positive correlation with yield per plant under intercropping conditions, outperforming the other architectural traits tested. In addition, general shade tolerance was positively associated with intercropping yield but negatively associated with monocropping yield. We compared a shade-tolerant soybean genotype (BX11) with a shade-sensitive genotype (72021). BX11 maintained upright growth under intercropping conditions and exhibited larger stem diameter with a well-developed vascular system while maintaining high expression levels of genes involved in radial growth and vascular development. By contrast, 72021 showed excessively elongated thin stems and lodging under intercropping conditions, exhibiting extensive intercropping-induced transcriptional reprogramming consistent with the repression of secondary cell wall biosynthesis and the induction of auxin-responsive cell elongation pathways. We conclude that shade tolerance in simultaneous intercropping systems depends on the ability to sustain radial stem development and mechanical strength, providing important insights to guide the breeding of soybean varieties optimized for intercropping.
The cotton bollworm Helicoverpa armigera (Lepidoptera: Noctuidae) is a major pest of numerous crops. Plant responses to cotton bollworm attack are generally attributed to two primary factors: physical wounding and components of insect oral secretions. Discriminating between these responses is critical for understanding plant defense and developing pest control strategies. In this study, we found that cotton bollworm infestation specifically upregulated the expression of NtNAC29 and NtNAC94, whereas mechanical wounding did not. Functional analyses revealed that silencing either gene promoted bollworm growth, whereas overexpression suppressed larval development. These results demonstrated that NtNAC29 and NtNAC94 had pivotal roles in enhancing plant resistance to bollworm. Further analyses showed that both NAC transcription factors regulated the expression of Cysteine Protease Inhibitor 8 (NtCPI8) by binding to specific sites within the NtCPI8-1 and NtCPI8-2 promoters. Consistently, NtCPI8 exhibited a similar defensive role, significantly inhibiting cotton bollworm growth as reflected by reduced larval weight gain and shorter body length. Overall, our findings suggest that tobacco plants recognize cotton bollworm attack and activate downstream defense responses, including the induction of NtNAC29 and NtNAC94. These transcription factors in turn upregulate NtCPI8 expression, thereby strengthening plant resistance against insect herbivory. Notably, this NAC-CPI regulatory module is conserved among different crop species, providing a promising target for improving crop protection against herbivorous insects.
The objective of this study was to determine whether magnetized ionized water increases the yield, water productivity, and culm lodging-resistance potential of semi-arid winter wheat, and whether these effects depend on irrigation timing. In a two-year field experiment, we compared ordinary water with magnetized ionized water (MIW) under three supplemental irrigation timings and a rainfed control. MIW increased desirable root, leaf, and stem traits. MIW with dual-stage irrigation gave the highest grain yield, water productivity, and lodging-resistance potential.
The objective of this study was to assess the effects of saline water irrigation and fertilization regimes on soil water and salinity dynamics, grain-filling characteristics, yield formation, and crop water productivity. In a two-year field experiment in the North China Plain during the winter wheat growing season, irrigation water salinity (electrical conductivity, EC) above 7.9 dS m−1 increased soil EC and water content and reduced grain yield by as much as 52%. Compared with conventional chemical fertilization (F0), fertilization combined with organic fertilizer and microbial inoculants (F1) reduced soil EC and increased grain yield by up to 12%. Compared with F0, F1 strengthened the contribution of yield components to grain yield while reducing the influence of soil water-salinity conditions. The combined application of organic fertilizer and microbial inoculants improved wheat grain filling and crop water productivity under saline water irrigation when irrigation water EC ≤ 7.9 dS m−1.
Accurate assessment of plant disease severity is a pivotal component of agricultural management, directly assisting in pesticide application strategies and yield estimation. However, achieving automated grading in complex field environments remains a significant challenge, primarily attributed to visual confusion arising from cluttered backgrounds, leaf occlusions, and minute lesions. To overcome these limitations, we propose AgriMamba, a text-guided multimodal framework based on pathology-aware alignment, which employs a three-stage architecture. This coarse-to-fine approach uses fine-grained pathological descriptions to enhance visual lesion features with attribute-level cues during segmentation. In the first stage, a Localization-aware mamba leaf segmenter (LMLS) leverages the linear complexity of State Space Models to efficiently isolate the target leaf and filter background noise. In the second stage, a Text-guided mamba lesion segmenter (TMLS) uses pathological descriptions as explicit semantic cues to guide the precise segmentation of minute lesions. In the third stage, the framework quantitatively calculates the plant disease severity grade by utilizing the segmentation results from the previous two stages. We construct a comprehensive multimodal dataset spanning 16 crop species to evaluate performance in plant disease segmentation and grading. This dataset is currently the largest multimodal plant disease dataset, comprising 10,350 samples with RGB images, leaf masks, lesion masks, disease grades, and pathological descriptions. AgriMamba significantly outperforms state-of-the-art baselines and achieves an accuracy of 97.20% on the disease severity grading task. Our code and dataset are available at http://agrimamba.samlab.cn/.
