INTRODUCTION:Cotton is an important global economic crop, and enhancing yield remains a primary breeding objective. Fiber development is controlled by a complex regulatory network, with the initiation stage being pivotal because it determines fiber number and ultimately yield. However, most studies have focused on upstream transcription factors, leaving downstream functional genes largely uncharacterized. OBJECTIVES:To explore the gene function and regulatory network of the unannotated gene GhFIR_A08 (Fiber Initiation Regulator on chromosome A08), which was identified based on previous structural-variant analyses and highly expressed during fiber initiation. METHODS:Currently high-quality Gossypium genome assemblies were compared to investigate the origin of the FIRs gene family and the mechanism of transposon-mediated copy number variation. Gene function was evaluated by generating stable homozygous transgenic lines (both knockout and overexpression) in the recipient backgrounds. Interacting proteins were screened and verified by MBP pull-down combined with mass spectrometry, yeast two-hybrid, bimolecular fluorescence complementation, and pull-down assays. The regulatory network was explored by yeast one-hybrid, dual-luciferase reporter, electrophoretic mobility shift assays, RNA-seq, and measurements of related metabolites. RESULTS:Alongside the evolutionary origin and polyploidization-driven expansion of this gene family in Gossypium, a transposon-mediated, haplotype-specific loss of GhFIR_A08 was uncovered. Overexpressing GhFIR_A08 increased the number of ovule protrusions and enhanced lint percentage (LP), whereas knockout produced the opposite phenotype. An upstream regulatory hierarchy was further uncovered in which GhWRKY16 induces another activator, GhRL6; both GhWRKY16 and GhRL6 bind the respective sites of GhFIR_A08 promoter, forming a coherent GhWRKY16-GhRL6-GhFIR_A08 module to activate GhFIR_A08. Functionally, GhFIR_A08 cooperates with Gh14-3-3, GhHSP70, GhSPL13, and GhPPR to regulate cotton fiber initiation by modulating hormone signaling, protein homeostasis, and redox processes. CONCLUSION:The uncharacterized gene, GhFIR_A08 is revealed to positively regulate fiber initiation to increase LP, and provides valuable genetic resources for molecular breeding aimed at improving cotton yield.

Cotton, a globally vital crop, faces severe yield losses due to heat-induced male sterility. To decipher the thermotolerance mechanisms, we conducted multi-omics analyses (3D chromatin architecture, transcriptome, and epigenome profiling) on heat-tolerant (84021) and heat-sensitive (H05) lines across critical anther developmental stages. We identified subgenome homoeologous gene expression bias linked to thermotolerance, driven by high temperature (HT)-induced dynamic chromatin topology reorganization. The sensitive line exhibited aberrant 3D structural hyperactivation during anther dehiscence, causing deleterious gene overexpression. Central to this regulation is GhAL5, an Alfin-like transcription factor modulated through chromatin loop dynamics and TAD-like boundary reorganization under heat stress. Functional studies confirmed the pivotal role of GhAL5: overexpression enhanced thermotolerance, while RNAi/CRISPR lines showed compromised heat resilience. Remarkably, GhAL5 conferred cross-species heat protection when expressed in rice. Mechanistically, GhAL5 potentially orchestrates male thermotolerance through bidirectional chromatin structure modulation. This study establishes 3D genome plasticity and chromatin remodeling as key drivers of plant thermal adaptation, proposing chromatin-aware breeding strategies for climate-resilient crops.

Drought stress is a key factor limiting improvement in cotton productivity and fiber quality, which necessitates the identification the key genes that can enhance cotton’s long-term drought tolerance while maintaining yield and growth under prolonged drought stress. In this study, we discovered that ectopic expression of ScALDH21 gene from Syntrichia caninervis Mitt. in cotton confers the drought tolerance via the reconstructing redox homeostasis and phenylpropanoid pathways. Field trials further revealed that the transgenic lines exhibited enhanced drought tolerance, improved growth, and stable yield performance. The drought tolerance is attributed to the transgenic lines enhanced ability to scavenge reactive oxygen species induced by oxidative and osmotic stress.Physiologically, transgenic lines displayed elevated superoxide dismutase (SOD) activity and proline accumulation, reduced malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) levels, as well as improved photosystem II maximum photochemical efficiency (Fv/Fm) and relative chlorophyll content (SPAD value), thereby maintaining efficient photosynthesis under water deficit. Multi-omics analyses further showed that differential expressed genes and metabolites were predominantly enriched in key metabolic pathways, including the pentose phosphate pathway, tryptophan metabolism, phenylalanine/tyrosine and tryptophan biosynthesis, and flavonoid biosynthesis. Moreover, DAP-seq assays of ScALDH21 binding activity showed high consistency with transcriptomic data, confirming significant enrichment of phenylalanine metabolic pathways and increased accumulation of stress-defensive metabolites in transgenic cotton. Collectively, it is inferred that ScALDH21 possessed the fundamental enzymatic activity required for aldehyde detoxification and further reveled that it may exert at transcriptional regulatory level by activating genes associated with phenylalanine pathway. The study confirms that uncovering the unique functional genes in species adapted to extreme adverse conditions is one of the most promising ways to enhance crop drought tolerance without compromising yield or quality.

Polyploidy is prominent in plant evolution and in many of the world’s most important crops, yet how domestication reshapes the regulation of duplicated genes (homoeologs) to generate superior agronomic traits remains incompletely understood. Here, we integrate population genomics, stage-resolved transcriptomics, expression quantitative trait locus (eQTL) mapping, and coexpression network analysis across 161 semiwild and 376 cultivated accessions of allotetraploid cotton ( Gossypium hirsutum ) to dissect the regulatory consequences of domestication. We show that domestication increases both the frequency and magnitude of homoeologous expression bias (HEB), with biased pairs preferentially organized into trait-associated, functionally specialized coexpression network modules. Bias-eQTL mapping identifies HEB-associated cis -regulatory variants that are enriched in open chromatin regions. Bayesian colocalization analysis further reveals that 92 bias-eQTLs colocalize with fiber quality-related genetic loci, where favorable alleles exhibit substantial frequency increases during domestication. Collectively, this work provides a mechanistic framework linking selection-driven regulatory asymmetry to coexpression network optimization in polyploids and highlights expression bias as a promising target for precision breeding in crops.

Sexual transfer of the CRISPR/Cas genome-editing system to targeted cotton cultivars could bypass their recalcitrance to regeneration from tissue culture. We used sexual hybridization to transmit a CRISPR/LbCas12a system from a regenerable Gossypium hirsutum donor to a nonregenerable G. barbadense recipient. We knocked out the GbCLA and GbPGF genes in the recipient, generating respectively albino and glandless phenotypes. Focusing on GbPGF, we detected novel mutations in the progeny across generations, and developed a set of nearly isogenic lines. The average editing efficiency of the target gene at crRNA1 exceeded 70% in the BC3F1 generation, yielding plants with agronomic traits or fiber quality nearly identical to those of the recurrent parent but lacking glands or gossypol. We introduced the CRISPR/LbCas12a system into three other nonregenerable G. hirsutum genotypes and one diploid cotton by hybridization and edited three more genes in two recipients.

Nitrogen (N) management is crucial for sustaining crop yield, improving profitability, and ensuring environmental stewardship. Multiple soil N application is facing quite a few challenges, especially when the canopy is closing, such as low efficiencies in both N use and application, disturbance or even drop of the plant fruits during application. We suppose that N foliar spraying could be a potential candidate to partially replace soil application. Therefore, the aim of this study was to verify if the foliar N application could improve cotton yield, and its possible mechanisms in N absorption, root architecture and root-shoot relationship. A 2-season (2020-2021) field experiment with a randomized complete block design was conducted to evaluate the effects of foliar N spraying on cotton yield and its components, root development, biomass accumulation and N uptake, in comparison with the multiple soil application and the conventional farmers practice as well. The results showed that multiple soil and foliar N application at flowering period improved cotton yield over the conventional N managements, while a similar performance was observed between soil and foliar strategies. Further, foliar N spraying increased shoot biomass partitioning, establishing an allometric root-shoot relationship that ultimately resulted in a 5.3 % increase in total N uptake. Meanwhile, foliar N application reduced root allocation in the topsoil while promoting deeper root development, which confirmed as a critical factor for yield improvement by SHapley Additive exPlanations (SHAP) analysis. Overall, split foliar N application increased cotton yield by higher shoot partitioning, combined with a deeper and thinner root architecture, thereby alleviating leaf senescence. These findings highlighted the potential of foliar nitrogen strategies to support mechanised cotton production by improving nitrogen use efficiency and production sustainability, particularly in regions where soil-based fertilisation is constrained by climatic or management limitations like semi-arid regions.

The flavonoid metabolic pathway plays a pivotal role in plant growth, development, and environmental adaptation and has undergone significant selection during cotton (Gossypium hirsutum) domestication. However, the functional role of flavonoid metabolism in fiber development remains poorly understood. In this study, we explore its functional significance in cotton fiber development by identifying expression quantitative trait loci (eQTLs) that regulate flavonoid biosynthesis and fiber-related traits. Through integrative eQTL mapping and transcriptomic analyses, we identified regulatory variants associated with key transcription factor and biosynthesis genes, including GhMYB46, GhMYB111, chalcone synthase (GhCHS), and dihydroflavonol 4-reductase (GhDFR). Functional characterization revealed that GhCHS and GhDFR play distinct roles in fiber development: in GhCHS-RNAi lines, excessive reactive oxygen species accumulation during fiber elongation impaired cell expansion, resulting in significantly shorter fibers; in contrast, GhDFR suppression disrupted cellulose biosynthesis and secondary cell wall thickening, leading to fibers with increased micronaire values. Together, these findings indicate that eQTL-mediated regulatory variation reshapes the expression of flavonoid pathway genes, thereby influencing both fiber elongation and secondary cell wall formation. Such regulatory remodeling underscores the functional integration of flavonoid metabolism into fiber development and highlights its potential as a valuable genetic resource for future fiber improvement.

Expansins are pivotal cell wall-loosening proteins that facilitate turgor-driven extension of plant cell walls. Expansin-like A (EXLA) proteins represent a subfamily, but their interaction with polysaccharides remains poorly understood during primary cell wall growth, hindered by challenges in achieving active heterologous expression for in vitro analysis. Using an insect secretion-based expression system, we successfully expressed and purified EXLA proteins. Screening 8 different polysaccharides showed that EXLAs exhibit a preference for binding to negatively charged polygalacturonic acid (PGA) and rhamnogalacturonan I (RG-I), pivotal components of pectin in the primary cell wall matrix. The crystal structure of EXLA1 was resolved at 2.5 Å resolution, revealing 3 crucial positively charged surfaces for pectin electrostatic interaction, and mutating these basic amino acids to alanine significantly reduced the binding ability. Moreover, recombinant EXLA1 promoted the extension of heat-inactivated cucumber hypocotyl walls under acidic conditions, indicating its intrinsic wall-loosening activity in vitro. EXLA1 overexpression resulted in a remodeled cell wall structure, suggesting EXLAs affect cell wall growth. These findings unveil EXLAs function during cell wall development by binding pectin through electrostatic interactions.

Escalating pressures of global climate change necessitate developing agricultural systems and crop varieties with enhanced resilience. Polyploidy, the state of possessing multiple complete sets of chromosomes arising from whole genome duplication (WGD), is a major evolutionary force in plants, often conferring novel genetic and regulatory capacities that facilitate adaptation. Allotetraploid cotton ( Gossypium spp.), which formed through the merger of distinct A and D subgenomes approximately 1 to 1.6 Mya, is an exemplary model for elucidating polyploid genome evolution and molecular mechanisms underlying stress adaptation. Whereas combining divergent genomes introduces genetic novelty and hybrid vigor, long-term adaptive success and enhanced resilience rely on complex regulatory reprogramming subsequent to the merger event. In cotton, and by extension other polyploids, this reprogramming involves dynamic structural genomic rearrangements, functional diversification of duplicated genes, and pervasive alterations in epigenetic landscapes. These processes reshape transcriptional networks, leading to homoeologous expression bias and novel regulatory interactions. These polyploidy-specific phenomena underpin differential subgenome contributions to key developmental processes and adaptive responses to major abiotic stresses including drought, salinity, and extreme temperature. A comprehensive understanding of these interconnected genetic and epigenetic control mechanisms, the resulting landscape of subgenome coordination or independence, and associated physiological consequences, are essential for designing effective strategies to breed climate-resilient crops. Here, we synthesize current insights from cotton, emphasizing their broader significance for harnessing polyploidy as a tool for future crop improvement amid global environmental change.

Sea Island cotton ( Gossypium barbadense ) produces premium-quality fibres, yet the genetic basis underlying its fibre development remains elusive. Here, we identify two key non-synonymous single nucleotide polymorphisms (SNPs, G/C and G/A) in the gene Gbar_D13G024080 , which encodes the TRANSMEMBRANE PROTEIN 209 (TMEM209). These SNPs resulted in amino acid changes (V/L and R/K), and are significantly correlated with the fibre length in Sea Island cotton. CRISPR-Cas9-mediated knockout of GbTMEM209 significantly enhanced fibre length and fibre strength in both G. hirsutum and G. barbadense . Conversely, overexpression of GbTMEM209 in G. hirsutum led to reduced fibre length. Further mechanistic investigation revealed that GbTMEM209 competitively interacts with GbHOX3 to impair its transcriptional activation on cell wall-loosening genes GbEXPA1 and GbRDL1 . Moreover, during the elongation stage of the fibres, GbTMEM209 and GbHOX3 exhibit an antagonistic relationship, which jointly regulate the development of cotton fibres. Virus-induced gene silencing (VIGS) of GbHOX3 , GbEXPA1 , or GbRDL1 consistently resulted in shortened fibres in Sea Island cotton, validating their critical roles in fibre development. Our findings establish GbTMEM209 as a novel negative regulator of fibre elongation and uncover a protein competition-mediated transcriptional control mechanism in cotton fibre morphogenesis. These findings provide valuable genetic targets and conceptual insights for molecular breeding programs aimed at improving cotton fibre quality.

Enhancing cotton yield remains a paramount breeding objective. Given limited arable land, increasing planting density is an effective strategy to boost cotton yield. However, the genetic basis of plant architecture suitable for high-density planting, particularly the molecular mechanism that controls fruit branch angle (FBA), remains largely unknown. Here, we identified qFBA-A11, a major quantitative trait locus associated with FBA, using a Gossypium hirsutum × Gossypium mustelinum introgression line population. Map-based cloning revealed that GhFBA1_At, whose encoded protein has no known functional domains, is the major gene positively regulating FBA. Further investigations demonstrated that GATA5, a light-responsive transcription factor, positively regulates GhFBA1_At expression. GhFBA1_At protein inhibits KNAT7 accumulation through a direct protein-protein interaction and downregulates the expression of genes related to cell wall biosynthesis. This process promotes cell expansion while reducing cell wall thickness, ultimately weakening the mechanical strength of the cell wall and leading to a loose plant architecture. A structural variation (SV) at the GhFBA1_At locus in G. mustelinum causes complete gene loss, resulting in a compact plant architecture. Phylogenetic analysis showed that this SV is unique to G. mustelinum. CRISPR-Cas9 editing of GhFBA1_At produced a compact plant architecture and enhanced cotton yield under high-density planting. Our findings establish GhFBA1_At as a crucial regulator of FBA, reveal its molecular mechanism, and provide valuable germplasm resources for ideal plant architecture breeding in cotton.

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.

Herbivore effectors play central roles in plant-insect interactions; yet, their molecular targets and modes of action remain poorly defined. Here, we performed data-independent acquisition proteomic profiling of oral secretions from cotton bollworm (Helicoverpa armigera) larvae fed on an artificial diet and four cotton cultivars. A total of 212 proteins were identified, including 39 differentially expressed proteins and 13 candidate effectors. Based on secretion characteristics and evolutionary features, six venom protein-related candidates were selected for functional validation. Transgenic cotton plants overexpressing these genes were generated, and feeding assays demonstrated that three independent 35S:PESD3 lines and three 35S:HYPB1 lines significantly enhanced bollworm performance relative to wild-type cotton. Further analyses showed that HYPB1 and PESD3 can be secreted into cotton tissues through mechanical wounds. Among these candidates, HYPB1 showed typical structural and evolutionary characteristics of venom-related proteins. Multiple complementary protein-protein interaction assays demonstrated that HYPB1 physically interacts with the cotton dirigent protein GhDIR15. Silencing of GhDIR15 via virus-induced gene silencing reduced cotton resistance to H. armigera and was accompanied by decreased lignin accumulation and reduced phenolic metabolite levels, indicating suppression of the cell wall-associated defense pathway. Together, these results identify HYPB1 as a previously uncharacterized effector that promotes bollworm feeding by targeting GhDIR15 and suppressing lignin biosynthesis, thereby further compromising cell wall-mediated defense. Although PESD3 also promoted bollworm performance in transgenic cotton, its underlying mechanism requires further investigation. This work provides mechanistic insight into how H. armigera manipulates host secondary metabolism to attenuate plant defense.

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.

The initial number of fibers and fiber length affect fiber yield and quality in cotton (Gossypium hirsutum). The mechanism of fiber initiation centers on the transcription factor (TF) GhMYB25-like, and the involvement of other regulatory factors remains unclear. Here, we established a GhMYB25-like-based interaction network that regulates fiber initiation, and focused on the interacting protein AGAMOUS-like 4 (GhAGL4), which is a MIKC-MADS TF, and other MIKC-MADS family members. Genome-wide identification of the cotton MIKC-MADS family members identified 2 highly co-expressed genes, GhAGL1 and GhAGL4, which are predominantly expressed in ovules at 0 d post-anthesis. CRISPR/Cas9-mediated loss of function of GhAGL1 or GhAGL4 led to a decrease in fiber initials and impeded fiber elongation, with the Ghagl1 Ghagl4 double mutant exhibiting a stronger phenotype. GhAGL1 and GhAGL4 interact in vitro and in vivo. DNA affinity purification and sequencing combined with RNA sequencing identified CCCH zinc finger 1 (GhCZF1) as a downstream gene of GhAGL1 and GhAGL4. Interestingly, GhMYB25-like enhances the ability of GhAGL1-GhAGL4 heterodimers to activate downstream genes. During the early fiber elongation stage, GhCZF1 activates the expression of the xyloglucan endotransglycosylase/hydrolase (XTH) genes GhXTH16 and GhXTH23 to regulate fiber elongation. Our findings reveal a molecular mechanism involved in fiber initiation and early elongation, providing a foundation for enhancing cotton fiber yield and quality.

Resistance to the necrotrophic pathogen Rhizoctonia solani is a major bottleneck in cotton breeding, hindered by the lack of resistance germplasm, accurate phenotyping methods, and defined resistance genes. Here, we established a precise phenotyping system and performed a genome-wide association study (GWAS) on a natural population of Upland cotton (Gossypium hirsutum). A major resistance locus on chromosome D03, which explains 19.65% of the phenotypic variation, was identified. GhGLR4.8, encoding a glutamate receptor-like protein, was pinpointed as the causal gene. Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-mediated knockout significantly compromised resistance, whereas overexpression of the elite haplotype GhGLR4.8Hap3 conferred robust protection. Functional characterization indicated that GhGLR4.8 promotes lignin deposition and strengthens cell wall structure while maintaining reactive oxygen species (ROS) balance. Loss-of-function mutants displayed excessive ROS accumulation accompanied by pronounced tissue maceration, suggesting that GhGLR4.8 restricts fungal invasion through coordinated regulation of ROS homeostasis. This study reports the first major resistance gene against R. solani in cotton, providing key insights into the balance between ROS signaling and structural defense, as well as a valuable genetic resource for molecular breeding to stabilize cotton yield.

The generation of complex traits involves the coordinated interplay of multiple gene networks. Elucidating the function of transcriptional cis-regulatory elements (CREs) in regulating gene expression is crucial for understanding complex regulatory pathways and improving our ability to modify macro-phenotypes. While traditional bulk sequencing approaches rely on tissue or cell population aggregates, single-cell transcriptomics provides a more precise perspective by capturing cell-type-specific information. The integration of single-cell technology with genome-wide genetic screening, particularly through the single-cell CRISPR (scCRISPR) system, enables the identification of critical regulatory elements and provides novel insights into gene-expression control mechanisms. Here, we summarise recent advances in diverse strategies for functional genome analysis using the scCRISPR system, with an emphasis on its potential to revolutionise single-cell genetic screening of CREs. We also explore the challenges and opportunities for applying these approaches in plant research.