Drought stress severely constrains cotton yield and fiber quality, but conventional evaluation methods are inefficient and time-consuming. To address this, we developed a high-throughput, non-destructive phenotyping framework by integrating UAV-based multispectral remote sensing with machine learning, using 225 upland cotton (Gossypium hirsutum L.) accessions. The accessions were subjected to well-watered (CK) and drought stress (DS) treatments at the flowering and boll-setting stage. Canopy multispectral imagery (Green/Red/Red_edge/Near-infrared bands) was acquired via DJI Mavic 3 Multispectral UAV, and 16 vegetation indices (VIs) were derived. Concurrently, 15 agronomic and fiber quality traits were measured to calculate drought resistance coefficients (DRCs), which were used for principal component analysis (PCA) and comprehensive drought tolerance index (D) construction. Hierarchical clustering categorized the accessions into 6 drought tolerance grades (Groups I-VI). Variable importance analysis identified GNDVI, NGRVI, and NDRE as the most drought-sensitive VIs (% IncMSE > 11). Among four regression models (LR, KNN, LGBM, XGBoost), XGBoost achieved the best performance for D prediction (test set: R-2 = 0.785, RMSE = 0.032, MAE = 0.024). This study demonstrates that UAV multispectral data coupled with XGBoost enables accurate, efficient drought tolerance assessment, providing a robust tool for high-throughput germplasm screening and smart agricultural management.

Chemical defoliation is essential for mechanized harvesting of cotton (Gossypium hirsutum L.), yet the molecular mechanisms governing abscission zone (AZ) cell fate and cell-wall remodeling remain unresolved. Here, single-nucleus RNA sequencing (snRNA-seq) coupled with Monocle2 trajectory analysis delineates a pseudotemporal transition of protective-layer cells from C16_0 to C16_1, which coincides with AZ maturation. We identify the cell-wall glycoprotein GhSKS6 as a central hub in this differentiation process. CRISPR/Cas9-mediated knockout of GhSKS6 impairs AZ fracture-layer formation, delays leaf abscission, and downregulates genes associated with hemicellulose and xyloglucan remodeling. Subcellular localization and plasmolysis assays confirm the residency of GhSKS6 in the cell wall, while its overexpression in protoplasts accelerates cell wall regeneration. Moreover, the NAC transcription factor GhNAC47 directly binds to the GhSKS6 promoter to activate its expression, thereby modulating cotton leaf abscission. Our findings reveal a regulatory module involving GhNAC47 and GhSKS6 that coordinates protective-layer formation and cell-wall remodeling, offering precise molecular targets for breeding machine-harvested cotton cultivars.

L-type lectin receptor-like kinases (LecRKs) are a large family of membrane-anchored receptor kinases. L-type LecRKs play key roles in plant development and disease resistance, but their functions in abiotic stress responses are poorly understood. Soil salinization is an escalating problem that severely affects cotton (Gossypium hirsutum), a crucial cash crop for fiber, oil, and protein production. Therefore, it is urgent to improve salt tolerance. In this study, we systematically and comprehensively characterized L-type LecRKs in upland cotton. We identified 93 L-type LecRKs in the cotton genome. Phylogenetic analysis revealed that LecRKs were expanded in cotton in a pattern different from that in Arabidopsis. The expression patterns of cotton L-type LecRKs were tissue- and stress-specific as revealed by heatmap analysis. Interestingly, a pair of LecRK homeologs from subgroup VI were induced by salt treatment in both roots and leaves. Silencing GhLecRK-VI significantly impaired salt tolerance and triggered overproduction of reactive oxygen species in leaves, demonstrating its critical role in the cotton salt stress response.” Our results provide the first systematic characterization of the L-type LecRK gene family in cotton and reveal the diversity of LecRKs in Gossypium. Furthermore, our findings deepen the understanding of the roles of LecRKs in salt tolerance and will be helpful for breeding salt-tolerant cotton and other plants.

The calcineurin B-like (CBL)-interacting protein kinase (CIPK) signaling network is the core regulatory node in the response to abiotic stress in plants; it regulates plant homeostasis by regulating various proteins mediating ion transport. However, there are few reports on CIPK-mediated ion transporters in the cotton (Gossypium hirsutum) response to drought stress. Through yeast two-hybrid assays, we identified SUPPRESSOR OF K+ TRANSPORT GROWTH DEFECT 1 (GhSKD1), which interacts with GhCIPK6D1. GhSKD1 was significantly up-regulated after drought stress, while GhSKD1 localized to the cell membrane and nucleus. Functional studies revealed that GhSKD1 positively regulates K+ efflux, thus enhancing drought tolerance in cotton. Genetic and biochemical evidence showed that the phosphorylation of GhSKD1 by GhCIPK6D1 mediates K+ influx in guard cells, thereby regulating stomatal aperture and drought tolerance in cotton. GhSKD1 represents a previously uncharacterized protein that mediates potassium ion transport during the drought stress response. This finding identifies another target of CIPK regulation in the CPL-CIPK signaling network and provides insights into the mechanisms of drought tolerance in plants.

Global climate change has intensified drought stress, presenting a significant challenge to agricultural production and breeding. The root system, as the primary organ sensing stress signals, plays a crucial role in determining plants’ drought adaptability in soil conditions. Consequently, identifying optimal root architecture under drought conditions has become essential in crop breeding. This study employed a HT-ARPP to systematically analyze a natural population of 228 representative upland cotton accessions in specialized root boxes during seedling-stage drought stress. Root phenotypes were monitored 11 times across 20 days, generating over 20,000 images through an automatic root phenotypic robot, which yielded 27 image-based digital underground root traits (i-Rtraits). The drought-resistant coefficient (DRC, ratio between drought and control of i-Rtraits) was utilized to evaluate phenotypic responses. A comprehensive index of drought adaptability (CIDA) was developed through root traits analysis, and stepwise regression analysis established a model of key i-Rtraits, enabling classification of accessions into 5 groups based on root adaptability to water deficiency. An ideal drought-adaptability root architecture was proposed through combined analysis of aboveground and underground phenotypes. The findings indicate that medium and intermediate drought resistant cotton accessions represent optimal breeding materials for maintaining stable growth under variable conditions, offering a novel strategy for future breeding programs focused on optimized root architecture.

Beyond its fundamental roles in nutrient uptake and plant anchorage, the root system critically influences crop development and stress tolerance. Rhizobox enables in situ and nondestructive phenotypic detection of roots in soil, serving as a cost-effective root imaging method. However, the opacity of the soil often results in intermittent gaps in the root images, which reduces the accuracy of the root phenotype calculations. We present a root inpainting method built upon Generative Adversarial Networks (GANs) architecture In addition, we built a hybrid root inpainting dataset (HRID) that contains 1206 cotton root images with real gaps and 7716 rice root images with generated gaps. Compared with computer simulation root images, our dataset provides real root system architecture (RSA) and root texture information. Our method avoids cropping during training by instead utilizing downsampled images to provide the overall root morphology. The model is trained using binary cross-entropy loss to distinguish between root and non-root pixels. Additionally, Dice loss is employed to mitigate the challenge of imbalanced data distribution Additionally, we remove the skip connections in U-Net and introduce an edge attention module (EAM) to capture more detailed information. Compared with other methods, our approach significantly improves the recall rate from 17.35 % to 35.75% on the test dataset of 122 cotton root images, revealing improved inpainting capabilities. The trait error reduction rates (TERRs) for the root area, root length, convex hull area, and root depth are 76.07 %, 68.63 %, 48.64 %, and 88.28 %, respectively, enabling a substantial improvement in the accuracy of root phenotyping. The codes for the EU-GAN and the 8922 labeled images are open-access, which could be reused by researchers in other AI-related work. This method establishes a robust solution for root phenotyping, thereby increasing breeding program efficiency and advancing our understanding of root system dynamics. (c) 2025 The Authors. Publishing services by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Cotton is a vital textile resource; however, its productivity and fibre quality are severely affected by soil salinity. Identifying salt-tolerant genes is critical for improving cotton resilience, yet the molecular mechanisms linking photosynthesis and chlorophyll metabolism to the salt stress response remain poorly understood. In this study, the WRKY transcription factor GhWRKY41 was identified as a key regulator of salt tolerance by screening WRKY family members responsive to salinity stress. Functional validation demonstrated that GhWRKY41 overexpression significantly enhanced salt tolerance in cotton and Arabidopsis, whereas gene knockdown increased the sensitivity of cotton to salt stress. GhWRKY41 directly binds to and activates the expression of two salt-responsive genes, GhMPK3 and GhLEA3. Global transcriptomic analyses revealed that GhWRKY41 and its Arabidopsis homologues regulate a set of genes involved in photosynthesis and salt stress responses. Notably, GhWRKY41 knockdown downregulated genes encoding photosystem reaction centre proteins, impairing photosynthetic capacity under salt stress. These findings indicate that GhWRKY41 enhances salt tolerance primarily by maintaining elevated photosynthetic activity in cotton under saline conditions. This study provides novel insights into the complex regulatory network underlying the response of cotton to salt stress and presents a valuable genetic resource for breeding salt-tolerant cotton varieties.

Background CRISPR/Cas-derived base editor enables precise editing of target sites and has been widely used for basic research and crop genetic improvement. However, the editing efficiency of base editors at different targets varies greatly. Results Here, we develop a set of highly efficient base editors in cotton plants. GhABE8e, which is fused to conventional nCas9, exhibits 99.9% editing efficiency, compared to GhABE7.10 with 64.9%, and no off-target editing is detected. We further replace nCas9 with dCpf1, which recognizes TTTV PAM sequences, to broaden the range of the target site. To explore the functional divergence of TERMINAL FLOWER 1 (TFL1), we edit the non-coding and coding regions of GhTFL1 with 26 targets to generate a comprehensive allelic population including 300 independent lines in cotton. This allows hidden pleiotropic roles for GhTFL1 to be revealed and allows us to rapidly achieve directed domestication of cotton and create ideotype germplasm with moderate height, shortened fruiting branches, compact plant, and early-flowering. Further, by exploring the molecular mechanism of the GhTFL1 L86P and GhTFL1 K53G+S78G mutations, we find that the GhTFL1 L86P mutation weakens the binding strength of the GhTFL1 to other proteins but does not lead to a complete loss of GhTFL1 function. Conclusions This strategy provides an important technical platform and genetic information for the study and creation of ideal plant architecture.

BACKGROUND:Cotton is a major world cash crop and an important source of natural fiber, oil, and protein. Drought stress is becoming a restrictive factor affecting cotton production. To facilitate the development of drought-tolerant cotton varieties, it is necessary to study the molecular mechanism of drought stress response by exploring key drought-resistant genes and related regulatory factors.RESULTS:In this study, two cotton varieties, ZY007 (drought-sensitive) and ZY168 (drought-tolerant), showing obvious phenotypic differences under drought stress, were selected. A total of 25,898 drought-induced genes were identified, exhibiting significant enrichment in pathways related to plant stress responses. Under drought induction, At subgenome expression bias was observed at the whole-genome level, which may be due to stronger inhibition of Dt subgenome expression. A gene co-expression module that was significantly associated with drought resistance was identified. About 90% of topologically associating domain (TAD) boundaries were stable, and 6613 TAD variation events were identified between the two varieties under drought. We identified 92 genes in ZY007 and 98 in ZY168 related to chromatin 3D structural variation and induced by drought stress. These genes are closely linked to the cotton response to drought stress through canonical hormone-responsive pathways, modulation of kinase and phosphatase activities, facilitation of calcium ion transport, and other related molecular mechanisms.CONCLUSIONS:These results lay a foundation for elucidating the molecular mechanism of the cotton drought response and provide important regulatory locus and gene resources for the future molecular breeding of drought-resistant cotton varieties.

Global water scarcity and extreme weather intensify drought stress, significantly reducing cotton yield and quality worldwide. Drought treatments are conducted using a population of chromosome segment substitution lines generated from E22 (G. hirsutum) and 3-79 (G. barbadense) as parental lines either show superior yields or fiber quality under both control and drought conditions. Fourteen datasets, covering 4 yields and 4 quality traits, are compiled and assessed for drought resistance using the drought resistance coefficient (DRC) and membership function value of drought resistance (MFVD). Genome-wide association studies, linkage analysis, and bulked segregant analysis are combined to analyze the DR-related QTL. A total of 121 significant QTL are identified by DRC and MFVD of the 8 traits. CRISPR/Cas9 and virus-induced gene silencing techniques verified DRR1 and DRT1 as pivotal genes in regulating drought resistant of cotton, with hap3-79 exhibiting greater drought resistance than hapE22 concerning DRR1 and DRT1. Moreover, 14 markers with superior yield and fiber quality are selected for drought treatment. This study offers valuable insights into yield and fiber quality variations between G. hirsutum and G. barbadense amid drought, providing crucial theoretical and technological backing for developing cotton varieties resilient to drought, with high yield and superior fiber quality.

The occurrence of whole-genome duplication or polyploidy may promote plant adaptability to harsh environments. Here, we clarify the evolutionary relationship of eight GhCIPK6 homologous genes in upland cotton (Gossypium hirsutum). Gene expression and interaction analyses indicate that GhCIPK6 homologous genes show significant functional changes after polyploidy. Among these, GhCIPK6D1 and GhCIPK6D3 are significantly up-regulated by drought stress. Functional studies reveal that high GhCIPK6D1 expression promotes cotton drought sensitivity, while GhCIPK6D3 expression promotes drought tolerance, indicating clear functional differentiation. Genetic and biochemical analyses confirm the synergistic negative and positive regulation of cotton drought resistance through GhCBL1A1-GhCIPK6D1 and GhCBL2A1-GhCIPK6D3, respectively, to regulate stomatal movement by controlling the directional flow of K+ in guard cells. These results reveal differentiated roles of GhCIPK6 homologous genes in response to drought stress in upland cotton following polyploidy. The work provides a different perspective for exploring the functionalization and subfunctionalization of duplicated genes in response to polyploidization.

AbstractN6‐methyladenosine (m6A) is the most prevalent internal modification of mRNA and plays an important role in regulating plant growth. However, there is still a lack of effective tools to precisely modify m6A sites of individual transcripts in plants. Here, programmable m6A editing tools are developed by combining CRISPR/dCas13(Rx) with the methyltransferase GhMTA (Targeted RNA Methylation Editor, TME) or the demethyltransferase GhALKBH10 (Targeted RNA Demethylation Editor, TDE). These editors enable efficient deposition or removal of m6A modifications at targeted sites of endo‐transcripts GhECA1 and GhDi19 within a broad editing window ranging from 0 to 46 nt. TDE editor significantly decreases m6A levels by 24%–76%, while the TME editor increases m6A enrichment, ranging from 1.37‐ to 2.51‐fold. Furthermore, installation and removal of m6A modifications play opposing roles in regulating GhECA1 and GhDi19 mRNA transcripts, which may be attributed to the fact that their m6A sites are located in different regions of the genes. Most importantly, targeting the GhDi19 transcript with TME editor plants results in a significant increase in root length and enhanced drought resistance. Collectively, these m6A editors can be applied to study the function of specific m6A modifications and have the potential for future applications in crop improvement.

Plants respond to environmental stresses through controlled stem cell maintenance and meristem activity. One level of gene regulation is RNA alternative splicing. However, the mechanistic link between stress, meristem function and RNA splicing is poorly understood. The MERISTEM-DEFECTIVE (MDF) Arabidopsis gene encodes an SR-related family protein, required for meristem function and leaf vascularization, and is the likely orthologue of the human SART1 and yeast Snu66 splicing factors. MDF is required for the correct splicing and expression of key transcripts associated with root meristem function. We identified RSZ33 and ACC1, both known to regulate cell patterning, as splicing targets required for MDF function in the meristem. MDF expression is modulated by osmotic and cold stress, associated with differential splicing and specific isoform accumulation and shuttling between nucleus and cytosol, and acts in part via a splicing target SR34. We propose a model in which MDF controls splicing in the root meristem to promote stemness and to repress stress response, cell differentiation and cell death pathways.

Edible oils with high unsaturated fatty acids, particularly oleic acid, are beneficial to human health. Cotton is one of the top five oil crops in the world, but the mechanism of high-quality oil synthesis and regulatory networks in cotton are largely unclear. Here, we identified Leafy cotyledon1-like 1 (GhL1L1), a NF-YB subfamily gene that is specifically expressed during somatic embryogenesis and seed maturation in cotton. Overexpression of GhL1L1 regulates the contents of unsaturated fatty acids in cotton, especially in the seeds, which is associated with altered expression of the cotton fatty acid biosynthesis-related genes. GhL1L1 synergistically enhanced the expression of GhFAD2-1A by binding to the G-box in its promoter, leading to an increase in the content of linoleic acid. Furthermore, this activation could be enhanced by GhNF-YC2 and GhNF-YA1 by form a transcriptional complex. Collectively, these results contribute to provide new insights into the molecular mechanism of oil biosynthesis in cotton and can facilitate genetic manipulation of cotton varieties with enhanced oil content.

Cotton is a crucial economic crop, serving as a natural fiber source for the textile industry. However, drought stress poses a significant threat to cotton fiber quality and productivity worldwide. Pyrabactin Resistance 1-Like (PYL) proteins, as abscisic acid (ABA) receptors, play a crucial role in adverse stress responses, but knowledge about the PYLs in cotton remains limited. In our study, we identified 40 GhPYL genes in Gossypium hirsutum through a genome-wide analysis of the cotton genome database. Our analysis revealed that the PYL family formed three distinct subfamilies with typical family characteristics in G. hirsutum. Additionally, through quantitative expression analysis, including transcriptome dataset and qRT-PCR, we found that all GhPYLs were expressed in all tissues of G. hirsutum, and all GhPYLs were differentially expressed under drought stress. Among them, GhPYL4A1, GhPY5D1, GhPY8D2, and a member of the type 2C protein phosphatases clade A family in Gossypium hirsutum (GhPP2CA), GhHAI2D, showed significant differences in expression levels within 12 h after stress treatment. Our protein interaction analysis and BiFC demonstrated the complex regulatory network between GhPYL family proteins and GhPP2CA proteins. We also found that there is an interaction between GhPYL8D2 and GhHAI2D, and through drought treatment of transgenic cotton, we found that GhPYL8D2 played a vital role in the response of G. hirsutum to drought through stomatal control via co-regulation with GhHAI2D. Our findings provide useful insights into the regulation of GhPYL family genes that occur in response to abiotic stresses in cotton.

SummaryN6‐methyladenosine (m6A) is the most prevalent internal modification present in mRNAs, and is considered to participate in a range of developmental and biological processes. Drought response is highly regulated at the genomic, transcriptional and post‐transcriptional levels. However, the biological function and regulatory mechanism of m6A modification in the drought stress response is still poorly understood. We generated a transcriptome‐wide m6A map using drought‐resistant and drought‐sensitive varieties of cotton under different water deficient conditions to uncover patterns of m6A methylation in cotton response to drought stress. The results reveal that m6A represents a common modification and exhibit dramatic changes in distribution during drought stress. More 5'UTR m6A was deposited in the drought‐resistant variety and was associated with a positive effect on drought resistance by regulating mRNA abundance. Interestingly, we observed that increased m6A abundance was associated with increased mRNA abundance under drought, contributing to drought resistance, and vice versa. The demethylase GhALKBH10B was found to decrease m6A levels, facilitating the mRNA decay of ABA signal‐related genes (GhZEP, GhNCED4 and GhPP2CA) and Ca2+ signal‐related genes (GhECA1, GhCNGC4, GhANN1 and GhCML13), and mutation of GhALKBH10B enhanced drought resistance at seedling stage in cotton. Virus‐induced gene silencing (VIGS) of two Ca2+‐related genes, GhECA1 and GhCNGC4, reduced drought resistance with the decreased m6A enrichment on silenced genes in cotton. Collectively, we reveal a novel mechanism of post‐transcriptional modification involved in affecting drought response in cotton, by mediating m6A methylation on targeted transcripts in the ABA and Ca2+ signalling transduction pathways.

Abiotic stress seriously affects the growth, yield, and fiber quality of cotton. It is of great importance to cultivate drought-resistant and salt-tolerant cotton. NAC (NAM, ATAF1/2, and CUC2) is a plant-specific transcription factor, which is widely involved in the response to abiotic stress. Here, we discovered the GhNAC3 gene isolated from the expression profile of drought stress in cotton and verified its functions in cotton. First, GhNAC3 was strongly induced expression by drought and salt stresses. Gene structure analysis revealed that GhNAC3 had a conserved NAC domain and was homologous to several stress-related NAC transcription factors gene of Arabidopsis. Subcellular localization and transcriptional activation assays revealed that GhNAC3 was a nuclear protein with a C-terminal transcriptional activation domain. Overexpression of GhNAC3 enhanced Arabidopsis tolerance to drought stress with reduced sensitivity to ABA, characterized by increased germination and cotyledon rates under drought stress, and promoted root elongation. VIGS silencing of GhNAC3 reduced cotton tolerance to drought stress as indicated by the low water content of the leaves under drought treatment, significantly faster water loss and lower ABA content in detached leaves, along with the accumulation of more hydrogen peroxide (H2O2) and malondialdehyde (MDA). In conclusion, GhNAC3 plays an important role in the abiotic stress of cotton, which might have great application potential in molecular breeding of cotton varieties with drought resistance.

Root and leaf are essential organs of plants in sensing and responding to drought stress. However, comparative knowledge of non-coding RNAs (ncRNAs) of root and leaf tissues in the regulation of drought response in cotton is limited. Here, we used deep sequencing data of leaf and root tissues of drought-resistant and drought-sensitive cotton varieties for identifying miRNAs, lncRNAs and circRNAs. A total of 1531 differentially expressed (DE) ncRNAs was identified, including 77 DE miRNAs, 1393 DE lncRNAs and 61 DE circRNAs. The tissue-specific and variety-specific competing endogenous RNA (ceRNA) networks of DE lncRNA-miRNA-mRNA response to drought were constructed. Furthermore, the novel drought-responsive lncRNA 1 (DRL1), specifically and differentially expressed in root, was verified to positively affect phenotypes of cotton seedlings under drought stress, competitively binding to miR477b with GhNAC1 and GhSCL3. In addition, we also constructed another ceRNA network consisting of 18 DE circRNAs, 26 DE miRNAs and 368 DE mRNAs. Fourteen circRNA were characterized, and a novel molecular regulatory system of circ125- miR7484b/miR7450b was proposed under drought stress. Our findings revealed the specificity of ncRNA expression in tissue- and variety-specific patterns involved in the response to drought stress, and uncovered novel regulatory pathways and potentially effective molecules in genetic improvement for crop drought resistance.

Background Somatic embryogenesis is a major process for plant regeneration. However, cell communication and the gene regulatory network responsible for cell reprogramming during somatic embryogenesis are still largely unclear. Recent advances in single-cell technologies enable us to explore the mechanism of plant regeneration at single-cell resolution. Results We generate a high-resolution single-cell transcriptomic landscape of hypocotyl tissue from the highly regenerable cotton genotype Jin668 and the recalcitrant TM-1. We identify nine putative cell clusters and 23 cluster-specific marker genes for both cultivars. We find that the primary vascular cell is the major cell type that undergoes cell fate transition in response to external stimulation. Further developmental trajectory and gene regulatory network analysis of these cell clusters reveals that a total of 41 hormone response-related genes, including LAX2 , LAX1 , and LOX3 , exhibit different expression patterns in the primary xylem and cambium region of Jin668 and TM-1. We also identify novel genes, including CSEF , PIS1 , AFB2 , ATHB2 , PLC2 , and PLT3 , that are involved in regeneration. We demonstrate that LAX2 , LAX1 and LOX3 play important roles in callus proliferation and plant regeneration by CRISPR/Cas9 editing and overexpression assay. Conclusions This study provides novel insights on the role of the regulatory network in cell fate transition and reprogramming during plant regeneration driven by somatic embryogenesis.