A dominant negative mutation of GhMYB25-like alters cotton fiber initiation, reducing lint and fuzz.

Cotton (Gossypium hirsutum) fibers, vital natural textile materials, are single-cell trichomes that differentiate from the ovule epidermis. These fibers are categorized as lint (longer fibers useful for spinning) or fuzz (shorter, less useful fibers). Currently, developing cotton varieties with high lint yield but without fuzz remains challenging due to our limited knowledge of the molecular mechanisms underlying fiber initiation. This study presents the identification and characterization of a naturally occurring dominant negative mutation GhMYB25-like_AthapT, which results in a reduced lint and fuzzless phenotype. The GhMYB25-like_AthapT protein exerts its dominant negative effect by suppressing the activity of GhMYB25-like during lint and fuzz initiation. Intriguingly, the negative effect of GhMYB25-like_AthapT could be alleviated by high expression levels of GhMYB25-like. We also uncovered the role of GhMYB25-like in regulating the expression of key genes such as GhPDF2 (PROTODERMAL FACTOR 2), CYCD3; 1 (CYCLIN D3; 1) and PLD (Phospholipase D), establishing its significance as a pivotal transcription factor in fiber initiation. We identified other genes within this regulatory network, expanding our understanding of the determinants of fiber cell fate. These findings offer valuable insights for cotton breeding and contribute to our fundamental understanding of fiber development.

MERISTEM-DEFECTIVE regulates the balance between stemness and differentiation in the root meristem through RNA splicing control.

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.

Synergistic interplay of redox homeostasis and polysaccharide synthesis promotes cotton fiber elongation.

Cell polarity is the foundation of cell development and tissue morphogenesis. The investigation of polarized growth provides opportunities to gain profound insights into morphogenesis and tissue functionality in organisms. Currently, there are still many mysteries surrounding the mechanisms that regulate polarized cell growth. Cotton fiber cells serve as an excellent model for studying polarized growth, and provide important clues for unraveling the molecular mechanisms, signaling pathways, and regulatory networks of polarized growth. In this study, we characterized two functional genes, GhMDHAR1AT/DT and GhDHAR2AT/DT with predominant expression during fiber elongation. Loss of function of both genes contributed to a significant increase in fiber length. Transcriptomic data revealed up-regulated expression of antioxidant genes in CRISPR mutant lines, along with delayed expression of secondary wall-related genes and temporally prolonged expression of primary wall-related genes. Experimental evidence demonstrated that the increase in GSH content and glutathione peroxidase (GPX) enzyme activity led to enhanced total antioxidant capacity (T-AOC), resulting in reduced H2O2 levels, which contributed to the extension of fiber elongation stage in CRISPR mutant lines. Moreover, the increased polysaccharide synthesis in CRISPR mutant lines was found to provide an abundant supply of raw materials for fiber cell wall elongation, suggesting that synergistic interplay between redox homeostasis and polysaccharide synthesis in fiber cells may facilitate cell wall remodeling and fiber elongation. This study provides valuable insights for deciphering the mechanisms of cell polarized growth and improving cotton fiber quality.

GhTCE1-GhTCEE1 dimers regulate transcriptional reprogramming during wound-induced callus formation in cotton.

Wounded plant cells can form callus to seal the wound site. Alternatively, wounding can cause adventitious organogenesis or somatic embryogenesis. These distinct developmental pathways require specific cell fate decisions. Here, we identify GhTCE1, a basic helix-loop-helix family transcription factor, and its interacting partners as a central regulatory module of early cell fate transition during in vitro dedifferentiation of cotton (Gossypium hirsutum). RNAi- or CRISPR/Cas9-mediated loss of GhTCE1 function resulted in excessive accumulation of reactive oxygen species (ROS), arrested callus cell elongation, and increased adventitious organogenesis. In contrast, GhTCE1-overexpressing tissues underwent callus cell growth, but organogenesis was repressed. Transcriptome analysis revealed that several pathways depend on proper regulation of GhTCE1 expression, including lipid transfer pathway components, ROS homeostasis, and cell expansion. GhTCE1 bound to the promoters of the target genes GhLTP2 and GhLTP3, activating their expression synergistically, and the heterodimer TCE1-TCEE1 enhances this activity. GhLTP2- and GhLTP3-deficient tissues accumulated ROS and had arrested callus cell elongation, which was restored by ROS scavengers. These results reveal a unique regulatory network involving ROS and lipid transfer proteins, which act as potential ROS scavengers. This network acts as a switch between unorganized callus growth and organized development during in vitro dedifferentiation of cotton cells.

Pangenome analysis reveals transposon-driven genome evolution in cotton.

BACKGROUND: Transposable elements (TEs) have a profound influence on the trajectory of plant evolution, driving genome expansion and catalyzing phenotypic diversification. The pangenome, a comprehensive genetic pool encompassing all variations within a species, serves as an invaluable tool, unaffected by the confounding factors of intraspecific diversity. This allows for a more nuanced exploration of plant TE evolution. RESULTS: Here, we constructed a pangenome for diploid A-genome cotton using 344 accessions from representative geographical regions, including 223 from China as the main component. We found 511 Mb of non-reference sequences (NRSs) and revealed the presence of 5479 previously undiscovered protein-coding genes. Our comprehensive approach enabled us to decipher the genetic underpinnings of the distinct geographic distributions of cotton. Notably, we identified 3301 presence-absence variations (PAVs) that are closely tied to gene expression patterns within the pangenome, among which 2342 novel expression quantitative trait loci (eQTLs) were found residing in NRSs. Our investigation also unveiled contrasting patterns of transposon proliferation between diploid and tetraploid cotton, with long terminal repeat (LTR) retrotransposons exhibiting a synchronized surge in polyploids. Furthermore, the invasion of LTR retrotransposons from the A subgenome to the D subgenome triggered a substantial expansion of the latter following polyploidization. In addition, we found that TE insertions were responsible for the loss of 36.2% of species-specific genes, as well as the generation of entirely new species-specific genes. CONCLUSIONS: Our pangenome analyses provide new insights into cotton genomics and subgenome dynamics after polyploidization and demonstrate the power of pangenome approaches for elucidating transposon impacts and genome evolution.

Drought response revealed by chromatin organization variation and transcriptional regulation in cotton.

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.

High-resolution sequencing of nine elite upland cotton cultivars uncovers genic variations and breeding improvement targets.

Structural variations (SVs) are critical factors affecting genome evolution and important traits. However, identification results and functional analyses of SVs in upland cotton are rare. Here, based on the genetic relationships, breeding history and cumulative planting area of upland cotton in China, nine predominant cultivars from the past 60 years (1950s-2010s) were selected for long read sequencing to uncover genic variations and breeding improvement targets for this crop. Based on the ZM24 reference genome, 0.88-1.47 × 104 SVs per cultivar were identified, and an SV set was constructed. SVs affected the expression of a large number of genes during fiber elongation, and a transposable element insertion resulted in the glandless phenotype in upland cotton. Six widespread inversions were identified based on nine draft genomes and high-throughput chromosome conformation capture data. Multiple haplotype blocks that were always associated with aggregated SVs were demonstrated to play a pivotal role in the agronomic traits of upland cotton and drove its adaptation to the northern planting region. Exotic introgression was the source of these haplotype blocks and increased the genetic diversity of upland cotton. Our results enrich the genome resources of upland cotton, and the identified SVs will promote genetic and breeding research in cotton.

Cotton 4-coumarate-CoA ligase 3 enhanced plant resistance to Verticillium dahliae by promoting jasmonic acid signaling-mediated vascular lignification and metabolic flux.

Lignins and their antimicrobial-related polymers cooperatively enhance plant resistance to pathogens. Several isoforms of 4-coumarate-coenzyme A ligases (4CLs) have been identified as indispensable enzymes involved in lignin and flavonoid biosynthetic pathways. However, their roles in plant-pathogen interaction are still poorly understood. This study uncovers the role of Gh4CL3 in cotton resistance to the vascular pathogen Verticillium dahliae. The cotton 4CL3-CRISPR/Cas9 mutant (CR4cl) exhibited high susceptibility to V. dahliae. This susceptibility was most probably due to the reduction in the total lignin content and the biosynthesis of several phenolic metabolites, e.g., rutin, catechin, scopoletin glucoside, and chlorogenic acid, along with jasmonic acid (JA) attenuation. These changes were coupled with a significant reduction in 4CL activity toward p-coumaric acid substrate, and it is likely that recombinant Gh4CL3 could specifically catalyze p-coumaric acid to form p-coumaroyl-coenzyme A. Thus, overexpression of Gh4CL3 (OE4CL) showed increasing 4CL activity that augmented phenolic precursors, cinnamic, p-coumaric, and sinapic acids, channeling into lignin and flavonoid biosyntheses and enhanced resistance to V. dahliae. Besides, Gh4CL3 overexpression activated JA signaling that instantly stimulated lignin deposition and metabolic flux in response to pathogen, which all established an efficient plant defense response system, and inhibited V. dahliae mycelium growth. Our results propose that Gh4CL3 acts as a positive regulator for cotton resistance against V. dahliae by promoting JA signaling-mediated enhanced cell wall rigidity and metabolic flux.

The elicitor VP2 from Verticillium dahliae triggers defence response in cotton.

Verticillium dahliae is a widespread and destructive soilborne vascular pathogenic fungus that causes serious diseases in dicot plants. Here, comparative transcriptome analysis showed that the number of genes upregulated in defoliating pathotype V991 was significantly higher than in the non-defoliating pathotype 1cd3-2 during the early response of cotton. Combined with analysis of the secretome during the V991-cotton interaction, an elicitor VP2 was identified, which was highly upregulated at the early stage of V991 invasion, but was barely expressed during the 1cd3-2-cotton interaction. Full-length VP2 could induce cell death in several plant species, and which was dependent on NbBAK1 but not on NbSOBIR1 in N. benthamiana. Knock-out of VP2 attenuated the pathogenicity of V991. Furthermore, overexpression of VP2 in cotton enhanced resistance to V. dahliae without causing abnormal plant growth and development. Several genes involved in JA, SA and lignin synthesis were significantly upregulated in VP2-overexpressing cotton. The contents of JA, SA, and lignin were also significantly higher than in the wild-type control. In summary, the identified elicitor VP2, recognized by the receptor in the plant membrane, triggers the cotton immune response and enhances disease resistance.

The complex hexaploid oil-Camellia genome traces back its phylogenomic history and multi-omics analysis of Camellia oil biosynthesis.

Oil-Camellia (Camellia oleifera), belonging to the Theaceae family Camellia, is an important woody edible oil tree species. The Camellia oil in its mature seed kernels, mainly consists of more than 90% unsaturated fatty acids, tea polyphenols, flavonoids, squalene and other active substances, which is one of the best quality edible vegetable oils in the world. However, genetic research and molecular breeding on oil-Camellia are challenging due to its complex genetic background. Here, we successfully report a chromosome-scale genome assembly for a hexaploid oil-Camellia cultivar Changlin40. This assembly contains 8.80 Gb genomic sequences with scaffold N50 of 180.0 Mb and 45 pseudochromosomes comprising 15 homologous groups with three members each, which contain 135 868 genes with an average length of 3936 bp. Referring to the diploid genome, intragenomic and intergenomic comparisons of synteny indicate homologous chromosomal similarity and changes. Moreover, comparative and evolutionary analyses reveal three rounds of whole-genome duplication (WGD) events, as well as the possible diversification of hexaploid Changlin40 with diploid occurred approximately 9.06 million years ago (MYA). Furthermore, through the combination of genomics, transcriptomics and metabolomics approaches, a complex regulatory network was constructed and allows to identify potential key structural genes (SAD, FAD2 and FAD3) and transcription factors (AP2 and C2H2) that regulate the metabolism of Camellia oil, especially for unsaturated fatty acids biosynthesis. Overall, the genomic resource generated from this study has great potential to accelerate the research for the molecular biology and genetic improvement of hexaploid oil-Camellia, as well as to understand polyploid genome evolution.

Tcbf: a novel user-friendly tool for pan-3D genome analysis of topologically associating domain in eukaryotic organisms.

SUMMARY: TAD boundaries are essential for organizing the chromatin spatial structure and regulating gene expression in eukaryotes. However, for large-scale pan-3D genome research, identifying conserved and specific TAD boundaries across different species or individuals is computationally challenging. Here, we present Tcbf, a rapid and powerful Python/R tool that integrates gene synteny blocks and homologous sequences to automatically detect conserved and specific TAD boundaries among multiple species, which can efficiently analyze huge genome datasets, greatly reduce the computational burden and enable pan-3D genome research. AVAILABILITY AND IMPLEMENTATION: Tcbf is implemented by Python/R and is available at https://github.com/TcbfGroup/Tcbf under the MIT license.

MNAzyme catalyzed signal amplification-mediated lateral flow biosensor for portable and sensitive detection of mycotoxin in food samples.

Here, an enzyme-free lateral flow aptasensor was designed by target-induced strand-displacement effect and followed by the activation of multi-component nucleic acid enzyme (MNAzyme)-mediated cleavage to enable rapid and portable ochratoxin A (OTA) detection. The substrate was prepared as an oligonucleotide strand modified with magnetic beads (MB) and human chorionic gonadotropin (hCG). The interaction of OTA with the aptamer induces the release of blocking DNA, which hybridized with three separated subunits of DNA, forming a sequence-specific MNAzyme catalytic core. This core subsequently initiated an enzyme-free MNAzyme cleavage reaction in the presence of the Mg2+ cofactor, cleaving a special substrate and releasing both the incomplete MNAzyme catalytic core and hCG-DNA probe. The incomplete MNAzyme catalytic core was then recognized by substrates once again, triggering a cascade recycling cleavage and resulting in the generation of a larger number of hCG-DNA probes. After magnetic enrichment, the free hCG-DNA probes flow through the pregnancy test strip (PTS) to the T line, generating a colorimetric readout that unequivocally confirms the presence of the target OTA. This work leverages the efficient enzyme-free cleavage amplification of MNAzyme and the PTS-based portable detection device, presenting a biosensing strategy with significant potential for sensitive and portable OTA detection. This method exhibited remarkable sensitivity and selectivity for OTA detection, boasting a detection limit of 5 nM. The present study successfully demonstrated the practical application of this method on real samples, offering a viable alternative for rapid and portable detection of mycotoxins.

Comprehensive analysis of MAPK gene family in upland cotton (Gossypium hirsutum) and functional characterization of GhMPK31 in regulating defense response to insect infestation.

KEY MESSAGE: The transcriptomic, phenotypic and metabolomic analysis of transgenic plants overexpressing GhMPK31 in upland cotton revealed the regulation of H2O2 burst and the synthesis of defensive metabolites by GhMPK31. Mitogen-activated protein kinases (MAPKs) are a crucial class of protein kinases, which play an essential role in various biological processes in plants. Upland cotton (G. hirsutum) is the most widely cultivated cotton species with high economic value. To gain a better understanding of the role of the MAPK gene family, we conducted a comprehensive analysis of the MAPK gene family in cotton. In this study, a total of 55 GhMPK genes were identified from the whole genome of G. hirsutum. Through an investigation of the expression patterns under diverse stress conditions, we discovered that the majority of GhMPK family members demonstrated robust responses to abiotic stress, pathogen stress and pest stress. Furthermore, the overexpression of GhMPK31 in cotton leaves led to a hypersensitive response (HR)-like cell death phenotype and impaired the defense capability of cotton against herbivorous insects. Transcriptome and metabolomics data analysis showed that overexpression of GhMPK31 enhanced the expression of H2O2-related genes and reduced the accumulation of defensive related metabolites. The direct evidence of GhMPK31 interacting with GhRBOHB (H2O2-generating protein) were found by Y2H, BiFC, and LCI. Therefore, we propose that the increase of H2O2 content caused by overexpression of GhMPK31 resulted in HR-like cell death in cotton leaves while reducing the accumulation of defensive metabolites, ultimately leading to a decrease in the defense ability of cotton against herbivorous insects. This study provides valuable insights into the function of MAPK genes in plant resistance to herbivorous insects.

Lipid parameters, adipose tissue distribution and prognosis prediction in chronic kidney Disease patients.

BACKGROUND: Lipid management in clinic is critical to the prevention and treatment of Chronic kidney disease (CKD), while the manifestations of lipid indicators vary in types and have flexible association with CKD prognosis. PURPOSE: Explore the associations between the widely used indicators of lipid metabolism and their distribution in clinic and CKD prognosis; provide a reference for lipid management and inform treatment decisions for patients with non-dialysis CKD stage 3-5. METHODS: This is a retrospective cohort study utilizing the Self-Management Program for Patients with Chronic Kidney Disease Cohort (SMP-CKD) database of 794 individuals with CKD stages 3-5. It covers demographic data, clinical diagnosis and medical history collection, laboratory results, circulating lipid profiles and lipid distribution assessments. Primary endpoint was defined as a composite outcome(the initiation of chronic dialysis or renal transplantation, sustained decline of 40% or more in estimated glomerular filtration rate (eGFR), doubled of serum creatinine (SCr) from the baseline, eGFR less than 5 mL/min/1.73m2, or all-cause mortality). Exposure variables were circulating lipid profiles and lipid distribution measurements. Association were assessed using Relative risks (RRs) (95% confidence intervals (CIs)) computed by multivariate Poisson models combined with least absolute shrinkage and selection operator (LASSO) regression according to categories of lipid manifestations. The best model was selected via akaike information criterion (AIC), area under curve (AUC), receiver operating characteristic curve (ROC) and net reclassification index (NRI). Subgroup analysis and sensitivity analysis were performed to assess the interaction effects and robustness.. RESULTS: 255 individuals reached the composite outcome. Median follow-up duration was 2.03 [1.06, 3.19] years. Median age was 58.8 [48.7, 67.2] years with a median eGFR of 33.7 [17.6, 47.8] ml/min/1.73 m2. Five dataset were built after multiple imputation and five category-based Possion models were constructed for each dataset. Model 5 across five datasets had the best fitness with smallest AIC and largest AUC. The pooled results of Model 5 showed that total cholesterol (TC) (RR (95%CI) (per mmol/L) :1.143[1.023,1.278], P = 0.018) and percentage of body fat (PBF) (RR (95%CI) (per percentage):0.976[0.961,0.992], P = 0.003) were significant factors of composite outcome. The results indicated that comprehensive consideration of lipid metabolism and fat distribution is more critical in the prediction of CKD prognosis.. CONCLUSION: Comprehensive consideration of lipid manifestations is optimal in predicting the prognosis of individuals with non-dialysis CKD stages 3-5.

The chromosome-scale reference genome of mirid bugs (Adelphocoris suturalis) genome provides insights into omnivory, insecticide resistance, and survival adaptation.

BACKGROUND: Adelphocoris suturalis (Hemiptera: Miridae) is a notorious agricultural pest, which causes serious economic losses to a diverse range of agricultural crops around the world. The poor understanding of its genomic characteristics has seriously hindered the establishment of sustainable and environment-friendly agricultural pest management through biotechnology and biological insecticides. RESULTS: Here, we report a chromosome-level assembled genome of A. suturalis by integrating Illumina short reads, PacBio, 10x Chromium, and Hi-C mapping technologies. The resulting 1.29 Gb assembly contains twelve chromosomal pseudomolecules with an N50 of 1.4 and 120.6 Mb for the contigs and scaffolds, respectively, and carries 20,010 protein-coding genes. The considerable size of the A. suturalis genome is predominantly attributed to a high amount of retrotransposons, especially long interspersed nuclear elements (LINEs). Transcriptomic and phylogenetic analyses suggest that A. suturalis-specific candidate effectors, and expansion and expression of gene families associated with omnivory, insecticide resistance and reproductive characteristics, such as digestion, detoxification, chemosensory receptors and long-distance migration likely contribute to its strong environmental adaptability and ability to damage crops. Additionally, 19 highly credible effector candidates were identified and transiently overexpressed in Nicotiana benthamiana for functional assays and potential targeting for insect resistance genetic engineering. CONCLUSIONS: The high-quality genome of A. suturalis provides an important genomic landscape for further investigations into the mechanisms of omnivory, insecticide resistance and survival adaptation, and for the development of integrated management strategies.

Histone ubiquitination controls organ size in cotton (Gossypium hirsutum).

Ubiquitination plays a vital role in modifying protein activity and destiny. Ub-conjugating enzyme E2 is one of the enzymes that participates in this precise process. There are at least 169 E2 proteins in the allotetraploid cotton (Gossypium hirsutum), but their function remains unknown. Here we identify an E2 gene GhUBC2L and show its positive role in cell proliferation and expansion. Complete knock-down of GhUBC2L in cotton resulted in retarded growth and reduced organ size. Conversely, overexpression of GhUBC2L promoted cotton growth, generating enlarged organs in size. Monoubiquitination of H2A and H2B was strongly impaired in GhUBC2L-suppressed cotton but slightly enhanced in GhUBC2L-overexpressed plant. GhUbox8, a U-box type E3 ligase protein, was found to interact with GhUBC2L both in vivo and in vitro, indicating their synergistical function in protein ubiquitination. Furthermore, GhUbox8 was shown to interact with a series of histone proteins, including histone H2A and H2B, indicating its potential monoubiquitination on H2A and H2B. Expression of genes relating to cell cycle and organ development were altered when the expression of GhUBC2L was changed. Our results show that GhUBC2L modulates histone monoubiquitination synergistically with GhUbox8 to regulate the expression of genes involved in organ development and cell cycle, thus controlling organ size in cotton. This research provides new insights into the role of protein ubiquitination in organ size control. Histone monoubiquitination plays an important role in plant development. Here, we identified an E2 enzyme GhUBC2L that modulates histone monoubiquitination synergistically with an E3 ligase GhUbox8 to mediate organ size control in cotton.

High-temperature stress in crops: male sterility, yield loss and potential remedy approaches.

Global food security is one of the utmost essential challenges in the 21st century in providing enough food for the growing population while coping with the already stressed environment. High temperature (HT) is one of the main factors affecting plant growth, development and reproduction and causes male sterility in plants. In male reproductive tissues, metabolic changes induced by HT involve carbohydrates, lipids, hormones, epigenetics and reactive oxygen species, leading to male sterility and ultimately reducing yield. Understanding the mechanism and genes involved in these pathways during the HT stress response will provide a new path to improve crops by using molecular breeding and biotechnological approaches. Moreover, this review provides insight into male sterility and integrates this with suggested strategies to enhance crop tolerance under HT stress conditions at the reproductive stage.

N6-methyladenosine RNA modification regulates cotton drought response in a Ca2+ and ABA-dependent manner.

N6 -methyladenosine (m6 A) 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 m6 A modification in the drought stress response is still poorly understood. We generated a transcriptome-wide m6 A map using drought-resistant and drought-sensitive varieties of cotton under different water deficient conditions to uncover patterns of m6 A methylation in cotton response to drought stress. The results reveal that m6 A represents a common modification and exhibit dramatic changes in distribution during drought stress. More 5'UTR m6 A 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 m6 A abundance was associated with increased mRNA abundance under drought, contributing to drought resistance, and vice versa. The demethylase GhALKBH10B was found to decrease m6 A 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 m6 A 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 m6 A methylation on targeted transcripts in the ABA and Ca2+ signalling transduction pathways.

GhWRKY41 forms a positive feedback regulation loop and increases cotton defence response against Verticillium dahliae by regulating phenylpropanoid metabolism.

Despite the established significance of WRKY proteins and phenylpropanoid metabolism in plant immunity, how WRKY proteins modulate aspects of the phenylpropanoid pathway remains undetermined. To understand better the role of WRKY proteins in plant defence, we identified a cotton (Gossypium hirsutum) protein, GhWRKY41, that is, universally and rapidly induced in three disease-resistant cotton cultivars following inoculation with the plant pathogenic fungus, Verticillium dahliae. We show that overexpression of GhWRKY41 in transgenic cotton and Arabidopsis enhances resistance to V. dahliae, while knock-down increases cotton more susceptibility to the fungus. GhWRKY41 physically interacts with itself and directly activates its own transcription. A genome-wide chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq), in combination with RNA sequencing (RNA-seq) analyses, revealed that 43.1% of GhWRKY41-binding genes were up-regulated in cotton upon inoculation with V. dahliae, including several phenylpropanoid metabolism master switches, receptor kinases, and disease resistance-related proteins. We also show that GhWRKY41 homodimer directly activates the expression of GhC4H and Gh4CL, thereby modulating the accumulation of lignin and flavonoids. This finding expands our understanding of WRKY-WRKY protein interactions and provides important insights into the regulation of the phenylpropanoid pathway in plant immune responses by a WRKY protein.

High temperature induces male sterility via MYB66-MYB4-Casein kinase I signaling in cotton.

High temperature (HT) causes male sterility and decreases crop yields. Our previous works have demonstrated that sugar and auxin signaling pathways, Gossypium hirsutum Casein kinase I (GhCKI), and DNA methylation are all involved in HT-induced male sterility in cotton. However, the signaling mechanisms leading to distinct GhCKI expression patterns induced by HT between HT-tolerant and HT-sensitive cotton anthers remain largely unknown. Here, we identified a GhCKI promoter (ProGhCKI) region that functions in response to HT in anthers and found the transcription factor GhMYB4 binds to this region to act as an upstream positive regulator of GhCKI. In the tapetum of early-stage cotton anthers, upregulated expression of GhMYB4 under HT and overexpressed GhMYB4 under normal temperature both led to severe male sterility phenotypes, coupled with enhanced expression of GhCKI. We also found that GhMYB4 interacts with GhMYB66 to form a heterodimer to enhance its binding to ProGhCKI. However, GhMYB66 showed an expression pattern similar to GhMYB4 under HT but did not directly bind to ProGhCKI. Furthermore, HT reduced siRNA-mediated CHH DNA methylations in the GhMYB4 promoter, which enhanced the expression of GhMYB4 in tetrad stage anthers and promoted the formation of the GhMYB4/GhMYB66 heterodimer, which in turn elevated the transcription of GhCKI in the tapetum, leading to male sterility. Overall, we shed light on the GhMYB66-GhMYB4-GhCKI regulatory pathway in response to HT in cotton anthers.