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.

Strigolactone-induced degradation of SMXL7 and SMXL8 contributes to gibberellin- and auxin-mediated fiber cell elongation in cotton.

Cotton (Gossypium) fiber length, a key trait determining fiber yield and quality, is highly regulated by a class of recently identified phytohormones, strigolactones (SLs). However, the underlying molecular mechanisms of SL signaling involved in fiber cell development are largely unknown. Here, we show that the SL signaling repressors MORE AXILLARY GROWTH2-LIKE7 (GhSMXL7) and GhSMXL8 negatively regulate cotton fiber elongation. Specifically, GhSMXL7 and GhSMXL8 inhibit the polyubiquitination and degradation of the gibberellin (GA)-triggered DELLA protein (GhSLR1). Biochemical analysis revealed that GhSMXL7 and GhSMXL8 physically interact with GhSLR1, which interferes with the association of GhSLR1 with the E3 ligase GA INSENSITIVE2 (GhGID2), leading to the repression of GA signal transduction. GhSMXL7 also interacts with the transcription factor GhHOX3, preventing its binding to the promoters of essential fiber elongation regulatory genes. Moreover, both GhSMXL7 and GhSMXL8 directly bind to the promoter regions of the AUXIN RESPONSE FACTOR (ARF) genes GhARF18-10A, GhARF18-10D, and GhARF19-7D to suppress their expression. Cotton plants in which GhARF18-10A, GhARF18-10D, and GhARF19-7D transcript levels had been reduced by virus-induced gene silencing (VIGS) displayed reduced fiber length compared with control plants. Collectively, our findings reveal a mechanism illustrating how SL integrates GA and auxin signaling to coordinately regulate plant cell elongation at the single-cell level.

Subgenome-aware analyses reveal the genomic consequences of ancient allopolyploid hybridizations throughout the cotton family.

Malvaceae comprise some 4,225 species in 243 genera and nine subfamilies and include economically important species, such as cacao, cotton, durian, and jute, with cotton an important model system for studying the domestication of polyploids. Here, we use chromosome-level genome assemblies from representatives of five or six subfamilies (depending on the placement of Ochroma) to differentiate coexisting subgenomes and their evolution during the family's deep history. The results reveal that the allohexaploid Helicteroideae partially derive from an allotetraploid Sterculioideae and also form a component of the allodecaploid Bombacoideae and Malvoideae. The ancestral Malvaceae karyotype consists of 11 protochromosomes. Four subfamilies share a unique reciprocal chromosome translocation, and two other subfamilies share a chromosome fusion. DNA alignments of single-copy nuclear genes do not yield the same relationships as inferred from chromosome structural traits, probably because of genes originating from different ancestral subgenomes. These results illustrate how chromosome-structural data can unravel the evolutionary history of groups with ancient hybrid genomes.

Early archaeological evidence of wheat and cotton from medieval Ile-Ife, Nigeria.

This study reports the earliest directly dated occurrence of archaeological wheat and cotton in the humid forests of West Africa. These are the first archaeobotanical results from the medieval urban center of Ile-Ife, southwestern Nigeria, best known for its famous artworks. Both wheat and cotton likely spread through trans-Saharan trade networks that laid the foundation for later European trade systems. Forty-eight (48) grains of free-threshing wheat (Triticum aestivum/durum) represent the largest assemblage of wheat recovered in sub-Saharan West Africa, which is surprising given that wheat cannot be cultivated locally. Larger quantities of cotton (Gossypium sp.) recovered from late 12th- to early 13th-century CE contexts suggest earlier and more widespread use than wheat. Cotton may have been cultivated and manufactured into cloth locally. The quick adoption of these exotic crops illustrates the active negotiation of prestige through culinary and adornment practices, as well as a high degree of agricultural experimentation.

Brassinosteroids regulate cotton fiber elongation by modulating very-long-chain fatty acid biosynthesis.

Brassinosteroid (BR), a growth-promoting phytohormone, regulates many plant growth processes including cell development. However, the mechanism by which BR regulates fiber growth is poorly understood. Cotton (Gossypium hirsutum) fibers are an ideal single-cell model in which to study cell elongation due to their length. Here we report that BR controls cotton fiber elongation by modulating very-long-chain fatty acid (VLCFA) biosynthesis. BR deficiency reduces the expression of 3-ketoacyl-CoA synthases (GhKCSs), the rate-limiting enzymes involved in VLCFA biosynthesis, leading to lower saturated VLCFA contents in pagoda1 (pag1) mutant fibers. In vitro ovule culture experiments show that BR acts upstream of VLCFAs. Silencing of BRI1-EMS-SUPPRESOR 1.4 (GhBES1.4), encoding a master transcription factor of the BR signaling pathway, significantly reduces fiber length, whereas GhBES1.4 overexpression produces longer fibers. GhBES1.4 regulates endogenous VLCFA contents and directly binds to BR RESPONSE ELEMENTS (BRREs) in the GhKCS10_At promoter region, which in turn regulates GhKCS10_At expression to increase endogenous VLCFA contents. GhKCS10_At overexpression promotes cotton fiber elongation, whereas GhKCS10_At silencing inhibits cotton fiber growth, supporting a positive regulatory role for GhKCS10_At in fiber elongation. Overall, these results uncover a mechanism of fiber elongation through crosstalk between BR and VLCFAs at the single-cell level.

The transcription factor ERF108 interacts with AUXIN RESPONSE FACTORs to mediate cotton fiber secondary cell wall biosynthesis.

Phytohormones play indispensable roles in plant growth and development. However, the molecular mechanisms underlying phytohormone-mediated regulation of fiber secondary cell wall (SCW) formation in cotton (Gossypium hirsutum) remain largely underexplored. Here, we provide mechanistic evidence for functional interplay between the APETALA2/ethylene response factor (AP2/ERF) transcription factor GhERF108 and auxin response factors GhARF7-1 and GhARF7-2 in dictating the ethylene-auxin signaling crosstalk that regulates fiber SCW biosynthesis. Specifically, in vitro cotton ovule culture revealed that ethylene and auxin promote fiber SCW deposition. GhERF108 RNA interference (RNAi) cotton displayed remarkably reduced cell wall thickness compared with controls. GhERF108 interacted with GhARF7-1 and GhARF7-2 to enhance the activation of the MYB transcription factor gene GhMYBL1 (MYB domain-like protein 1) in fibers. GhARF7-1 and GhARF7-2 respond to auxin signals that promote fiber SCW thickening. GhMYBL1 RNAi and GhARF7-1 and GhARF7-2 virus-induced gene silencing (VIGS) cotton displayed similar defects in fiber SCW formation as GhERF108 RNAi cotton. Moreover, the ethylene and auxin responses were reduced in GhMYBL1 RNAi plants. GhMYBL1 directly binds to the promoters of GhCesA4-1, GhCesA4-2, and GhCesA8-1 and activates their expression to promote cellulose biosynthesis, thereby boosting fiber SCW formation. Collectively, our findings demonstrate that the collaboration between GhERF108 and GhARF7-1 or GhARF7-2 establishes ethylene-auxin signaling crosstalk to activate GhMYBL1, ultimately leading to the activation of fiber SCW biosynthesis.

Yield-related quantitative trait loci identification and lint percentage hereditary dissection under salt stress in upland cotton.

Salinity is frequently mentioned as a major constraint in worldwide agricultural production. Lint percentage (LP) is a crucial yield-component in cotton lint production. While the genetic factors affect cotton yield in saline soils are still unclear. Here, we employed a recombinant inbred line population in upland cotton (Gossypium hirsutum L.) and investigated the effects of salt stress on five yield and yield component traits, including seed cotton yield per plant, lint yield per plant, boll number per plant, boll weight, and LP. Between three datasets of salt stress (E1), normal growth (E2), and the difference values dataset of salt stress and normal conditions (D-value), 87, 82, and 55 quantitative trait loci (QTL) were detectable, respectively. In total, five QTL (qLY-Chr6-2, qBNP-Chr4-1, qBNP-Chr12-1, qBNP-Chr15-5, qLP-Chr19-2) detected in both in E1 and D-value were salt related QTL, and three stable QTL (qLP-Chr5-3, qLP-Chr13-1, qBW-Chr5-5) were detected both in E1 and E2 across 3 years. Silencing of nine genes within a stable QTL (qLP-Chr5-3) highly expressed in fiber developmental stages increased LP and decreased fiber length (FL), indicating that multiple minor-effect genes clustered on Chromosome 5 regulate LP and FL. Additionally, the difference in LP caused by Gh_A05G3226 is mainly in transcription level rather than in the sequence difference. Moreover, silencing of salt related gene (GhDAAT) within qBNP-Chr4-1 decreased salt tolerance in cotton. Our findings shed light on the regulatory mechanisms underlining cotton salt tolerance and fiber initiation.

GhMYB30-GhMUR3 affects fiber elongation and secondary wall thickening in cotton.

Xyloglucan, an important hemicellulose, plays a crucial role in maintaining cell wall structure and cell elongation. However, the effects of xyloglucan on cotton fiber development are not well understood. GhMUR3 encodes a xyloglucan galactosyltransferase that is essential for xyloglucan synthesis and is highly expressed during fiber elongation. In this study, we report that GhMUR3 participates in cotton fiber development under the regulation of GhMYB30. Overexpression GhMUR3 affects the fiber elongation and cell wall thickening. Transcriptome showed that the expression of genes involved in secondary cell wall synthesis was prematurely activated in OE-MUR3 lines. In addition, GhMYB30 was identified as a key regulator of GhMUR3 by Y1H, Dual-Luc, and electrophoretic mobility shift assay (EMSA) assays. GhMYB30 directly bound the GhMUR3 promoter and activated GhMUR3 expression. Furthermore, DAP-seq of GhMYB30 was performed to identify its target genes in the whole genome. The results showed that many target genes were associated with fiber development, including cell wall synthesis-related genes, BR-related genes, reactive oxygen species pathway genes, and VLCFA synthesis genes. It was demonstrated that GhMYB30 may regulate fiber development through multiple pathways. Additionally, GhMYB46 was confirmed to be a target gene of GhMYB30 by EMSA, and GhMYB46 was significantly increased in GhMYB30-silenced lines, indicating that GhMYB30 inhibited GhMYB46 expression. Overall, these results revealed that GhMUR3 under the regulation of GhMYB30 and plays an essential role in cotton fiber elongation and secondary wall thickening. Additionally, GhMYB30 plays an important role in the regulation of fiber development and regulates fiber secondary wall synthesis by inhibiting the expression of GhMYB46.

GhHAM regulates GoPGF-dependent gland development and contributes to broad-spectrum pest resistance in cotton.

Cotton is a globally cultivated crop, producing 87% of the natural fiber used in the global textile industry. The pigment glands, unique to cotton and its relatives, serve as a defense structure against pests and pathogens. However, the molecular mechanism underlying gland formation and the specific role of pigment glands in cotton's pest defense are still not well understood. In this study, we cloned a gland-related transcription factor GhHAM and generated the GhHAM knockout mutant using CRISPR/Cas9. Phenotypic observations, transcriptome analysis, and promoter-binding experiments revealed that GhHAM binds to the promoter of GoPGF, regulating pigment gland formation in cotton's multiple organs via the GoPGF-GhJUB1 module. The knockout of GhHAM significantly reduced gossypol production and increased cotton's susceptibility to pests in the field. Feeding assays demonstrated that more than 80% of the cotton bollworm larvae preferred ghham over the wild type. Furthermore, the ghham mutants displayed shorter cell length and decreased gibberellins (GA) production in the stem. Exogenous application of GA3 restored stem cell elongation but not gland formation, thereby indicating that GhHAM controls gland morphogenesis independently of GA. Our study sheds light on the functional differentiation of HAM proteins among plant species, highlights the significant role of pigment glands in influencing pest feeding preference, and provides a theoretical basis for breeding pest-resistant cotton varieties to address the challenges posed by frequent outbreaks of pests.

SR45a plays a key role in enhancing cotton resistance to Verticillium dahliae by alternative splicing of immunity genes.

Alternative splicing (AS) of pre-mRNAs increases the diversity of transcriptome and proteome and plays fundamental roles in plant development and stress responses. However, the prevalent changes in AS events and the regulating mechanisms of plants in response to pathogens remain largely unknown. Here, we show that AS changes are an important mechanism conferring cotton immunity to Verticillium dahliae (Vd). GauSR45a, encoding a serine/arginine-rich RNA binding protein, was upregulated expression and underwent AS in response to Vd infection in Gossypium australe, a wild diploid cotton species highly resistant to Vd. Silencing GauSR45a substantially reduced the splicing ratio of Vd-induced immune-associated genes, including GauBAK1 (BRI1-associated kinase 1) and GauCERK1 (chitin elicitor receptor kinase 1). GauSR45a binds to the GAAGA motif that is commonly found in the pre-mRNA of genes essential for PTI, ETI, and defense. The binding between GauSR45a and the GAAGA motif in the pre-mRNA of BAK1 was enhanced by two splicing factors of GauU2AF35B and GauU1-70 K, thereby facilitating exon splicing; silencing either AtU2AF35B or AtU1-70 K decreased the resistance to Vd in transgenic GauSR45a Arabidopsis. Overexpressing the short splicing variant of BAK1GauBAK1.1 resulted in enhanced Verticillium wilt resistance rather than the long one GauBAK1.2. Vd-induced far more AS events were in G. barbadense (resistant tetraploid cotton) than those in G. hirsutum (susceptible tetraploid cotton) during Vd infection, indicating resistance divergence in immune responses at a genome-wide scale. We provided evidence showing a fundamental mechanism by which GauSR45a enhances cotton resistance to Vd through global regulation of AS of immunity genes.

Promotion of apoplastic oxidative burst by artificially selected GhCBSX3A enhances Verticillium dahliae resistance in upland cotton.

Verticillium wilt (VW) is a devasting disease affecting various plants, including upland cotton, a crucial fiber crop. Despite its impact, the genetic basis underlying cotton's susceptibility or defense against VW remains unclear. Here, we conducted a genome-wide association study on VW phenotyping in upland cotton and identified a locus on A13 that is significantly associated with VW resistance. We then identified a cystathionine ß-synthase domain gene at A13 locus, GhCBSX3A, which was induced by Verticillium dahliae. Functional analysis, including expression silencing in cotton and overexpression in Arabidopsis thaliana, confirmed that GhCBSX3A is a causal gene at the A13 locus, enhancing SAR-RBOHs-mediated apoplastic oxidative burst. We found allelic variation on the TATA-box of GhCBSX3A promoter attenuated its expression in upland cotton, thereby weakening VW resistance. Interestingly, we discovered that altered artificial selection of GhCBSX3A_R (an elite allele for VW) under different VW pressures during domestication and other improved processes allows specific human needs to be met. Our findings underscore the importance of GhCBSX3A in response to VW, and we propose a model for defense-associated genes being selected depending on the pathogen's pressure. The identified locus and gene serve as promising targets for VW resistance enhancement in cotton through genetic engineering.

Phosphatidic acid interacts with an HD-ZIP transcription factor GhHOX4 to influence its function in fiber elongation of cotton (Gossypium hirsutum).

Upland cotton, the mainly cultivated cotton species in the world, provides over 90% of natural raw materials (fibers) for the textile industry. The development of cotton fibers that are unicellular and highly elongated trichomes on seeds is a delicate and complex process. However, the regulatory mechanism of fiber development is still largely unclear in detail. In this study, we report that a homeodomain-leucine zipper (HD-ZIP) IV transcription factor, GhHOX4, plays an important role in fiber elongation. Overexpression of GhHOX4 in cotton resulted in longer fibers, while GhHOX4-silenced transgenic cotton displayed a "shorter fiber" phenotype compared with wild type. GhHOX4 directly activates two target genes, GhEXLB1D and GhXTH2D, for promoting fiber elongation. On the other hand, phosphatidic acid (PA), which is associated with cell signaling and metabolism, interacts with GhHOX4 to hinder fiber elongation. The basic amino acids KR-R-R in START domain of GhHOX4 protein are essential for its binding to PA that could alter the nuclear localization of GhHOX4 protein, thereby suppressing the transcriptional regulation of GhHOX4 to downstream genes in the transition from fiber elongation to secondary cell wall (SCW) thickening during fiber development. Thus, our data revealed that GhHOX4 positively regulates fiber elongation, while PA may function in the phase transition from fiber elongation to SCW formation by negatively modulating GhHOX4 in cotton.

Suppressing a ß-1,3-glucanase gene expression increases the seed and fibre yield in cotton.

Seeds are initiated from the carpel margin meristem (CMM) and high seed yield is top one of breeding objectives for many crops. ß-1,3-glucanases play various roles in plant growth and developmental processes; however, whether it participates in CMM development and seed formation remains largely unknown. Here, we identified a ß-1,3-glucanase gene (GLU19) as a determinant of CMM callose deposition and seed yield in cotton. GLU19 was differentially expressed in carpel tissues between Gossypium barbadense (Gb) and Gossypium hirsutum (Gh). Based on resequencing data, one interspecies-specific InDel in the promoter of GLU19 was further detected. The InDel was involved in the binding site of the CRABS CLAW (CRC) transcription factor, a regulator of carpel development. We found that the CRC binding affinity to the GLU19 promoter of G. barbadense was higher than that of G. hirsutum. Since G. barbadense yields fewer seeds than G. hirsutum, we speculated that stronger CRC binding to the GLU19 promoter activated higher expression of GLU19 which in turn suppressed seed production. Consistent with this hypothesis was that the overexpression of GhGLU19 caused reduced seed number, boll weight and less callose formation in CMM. Conversely, GhGLU19-knockdown (GhGLU19-KD) cotton led to the opposite phenotypes. By crossing GhGLU19-KD lines with several G. hirsutum and G. barbadense cotton accessions, all F1 and F2 plants carrying GhGLU19-KD transgenic loci exhibited higher seed yield than control plants without the locus. The increased seed effect was also found in the down-regulation of Arabidopsis orthologs lines, indicating that this engineering strategy may improve the seed yield in other crops.

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.

Suppression of plant immunity by Verticillium dahliae effector Vd6317 through AtNAC53 association.

Verticillium dahliae, a soil-borne fungal pathogen, compromises host innate immunity by secreting a plethora of effectors, thereby facilitating host colonization and causing substantial yield and quality losses. The mechanisms underlying the modulation of cotton immunity by V. dahliae effectors are predominantly unexplored. In this study, we identified that the V. dahliae effector Vd6317 inhibits plant cell death triggered by Vd424Y and enhances PVX viral infection in Nicotiana benthamiana. Attenuation of Vd6317 significantly decreased the virulence of V. dahliae, whereas ectopic expression of Vd6317 in Arabidopsis and cotton enhanced susceptibility to V. dahliae infection, underscoring Vd6317's critical role in pathogenicity. We observed that Vd6317 targeted the Arabidopsis immune regulator AtNAC53, thereby impeding its transcriptional activity on the defense-associated gene AtUGT74E2. Arabidopsis nac53 and ugt74e2 mutants exhibited heightened sensitivity to V. dahliae compared to wild-type plants. A mutation at the conserved residue 193L of Vd6317 abrogated its interaction with AtNAC53 and reduced the virulence of V. dahliae, which was partially attributable to a reduction in Vd6317 protein stability. Our findings unveil a hitherto unrecognized regulatory mechanism by which the V. dahliae effector Vd6317 directly inhibits the plant transcription factor AtNAC53 activity to suppress the expression of AtUGT74E2 and plant defense.

Cotton GhNAC4 promotes drought tolerance by regulating secondary cell wall biosynthesis and ribosomal protein homeostasis.

Drought has a severe impact on the quality and yield of cotton. Deciphering the key genes related to drought tolerance is important for understanding the regulation mechanism of drought stress and breeding drought-tolerant cotton cultivars. Several studies have demonstrated that NAC transcription factors are crucial in the regulation of drought stress, however, the related functional mechanisms are still largely unexplored. Here, we identified that NAC transcription factor GhNAC4 positively regulated drought stress tolerance in cotton. The expression of GhNAC4 was significantly induced by abiotic stress and plant hormones. Silencing of GhNAC4 distinctly impaired the resistance to drought stress and overexpressing GhNAC4 in cotton significantly enhanced the stress tolerance. RNA-seq analysis revealed that overexpression of GhNAC4 enriched the expression of genes associated with the biosynthesis of secondary cell walls and ribosomal proteins. We confirmed that GhNAC4 positively activated the expressions of GhNST1, a master regulator reported previously in secondary cell wall formation, and two ribosomal protein-encoding genes GhRPL12 and GhRPL18p, by directly binding to their promoter regions. Overexpression of GhNAC4 promoted the expression of downstream genes associated with the secondary wall biosynthesis, resulting in enhancing secondary wall deposition in the roots, and silencing of GhRPL12 and GhRPL18p significantly impaired the resistance to drought stress. Taken together, our study reveals a novel pathway mediated by GhNAC4 that promotes secondary cell wall biosynthesis to strengthen secondary wall development and regulates the expression of ribosomal protein-encoding genes to maintain translation stability, which ultimately enhances drought tolerance in cotton.

Evolutionary Dynamics of Chromatin Structure and Duplicate Gene Expression in Diploid and Allopolyploid Cotton.

Polyploidy is a prominent mechanism of plant speciation and adaptation, yet the mechanistic understandings of duplicated gene regulation remain elusive. Chromatin structure dynamics are suggested to govern gene regulatory control. Here, we characterized genome-wide nucleosome organization and chromatin accessibility in allotetraploid cotton, Gossypium hirsutum (AADD, 2n = 4X = 52), relative to its two diploid parents (AA or DD genome) and their synthetic diploid hybrid (AD), using DNS-seq. The larger A-genome exhibited wider average nucleosome spacing in diploids, and this intergenomic difference diminished in the allopolyploid but not hybrid. Allopolyploidization also exhibited increased accessibility at promoters genome-wide and synchronized cis-regulatory motifs between subgenomes. A prominent cis-acting control was inferred for chromatin dynamics and demonstrated by transposable element removal from promoters. Linking accessibility to gene expression patterns, we found distinct regulatory effects for hybridization and later allopolyploid stages, including nuanced establishment of homoeolog expression bias and expression level dominance. Histone gene expression and nucleosome organization are coordinated through chromatin accessibility. Our study demonstrates the capability to track high-resolution chromatin structure dynamics and reveals their role in the evolution of cis-regulatory landscapes and duplicate gene expression in polyploids, illuminating regulatory ties to subgenomic asymmetry and dominance.

Auxin resistant 2 and short hypocotyl 2 regulate cotton fiber initiation and elongation.

Auxin, a pivotal regulator of diverse plant growth processes, remains central to development. The auxin-responsive genes auxin/indole-3-acetic acids (AUX/IAAs) are indispensable for auxin signal transduction, which is achieved through intricate interactions with auxin response factors (ARFs). Despite this, the potential of AUX/IAAs to govern the development of the most fundamental biological unit, the single cell, remains unclear. In this study, we harnessed cotton (Gossypium hirsutum) fiber, a classic model for plant single-cell investigation, to determine the complexities of AUX/IAAs. Our research identified 2 pivotal AUX/IAAs, auxin resistant 2 (GhAXR2) and short hypocotyl 2 (GhSHY2), which exhibit opposite control over fiber development. Notably, suppressing GhAXR2 reduced fiber elongation, while silencing GhSHY2 fostered enhanced fiber elongation. Investigating the mechanistic intricacies, we identified specific interactions between GhAXR2 and GhSHY2 with distinct ARFs. GhAXR2's interaction with GhARF6-1 and GhARF23-2 promoted fiber cell development through direct binding to the AuxRE cis-element in the constitutive triple response 1 promoter, resulting in transcriptional inhibition. In contrast, the interaction of GhSHY2 with GhARF7-1 and GhARF19-1 exerted a negative regulatory effect, inhibiting fiber cell growth by activating the transcription of xyloglucan endotransglucosylase/hydrolase 9 and cinnamate-4-hydroxylase. Thus, our study reveals the intricate regulatory networks surrounding GhAXR2 and GhSHY2, elucidating the complex interplay of multiple ARFs in AUX/IAA-mediated fiber cell growth. This work enhances our understanding of single-cell development and has potential implications for advancing plant growth strategies and agricultural enhancements.

Single-cell transcriptome atlas reveals somatic cell embryogenic differentiation features during regeneration.

Understanding somatic cell totipotency remains a challenge facing scientific inquiry today. Plants display remarkable cell totipotency expression, illustrated by single-cell differentiation during somatic embryogenesis (SE) for plant regeneration. Determining cell identity and exploring gene regulation in such complex heterogeneous somatic cell differentiation have been major challenges. Here, we performed high-throughput single-cell sequencing assays to define the precise cellular landscape and revealed the modulation mode of marker genes during embryogenic differentiation in cotton (Gossypium hirsutum L.) as the crop for biotechnology application. We demonstrated that nonembryogenic calli (NEC) and primary embryogenic calli (PEC) tissues were composed of heterogeneous cells that could be partitioned into four broad populations with six distinct cell clusters. Enriched cell clusters and cell states were identified in NEC and PEC samples, respectively. Moreover, a broad repertoire of new cluster-specific genes and associated expression modules were identified. The energy metabolism, signal transduction, environmental adaptation, membrane transport pathways, and a series of transcription factors were preferentially enriched in cell embryogenic totipotency expression. Notably, the SE-ASSOCIATED LIPID TRANSFER PROTEIN (SELTP) gene dose-dependently marked cell types with distinct embryogenic states and exhibited a parabolic curve pattern along the somatic cell embryogenic differentiation trajectory, suggesting that SELTP could serve as a favorable quantitative cellular marker for detecting embryogenic expression at the single-cell level. In addition, RNA velocity and Scissor analysis confirmed the pseudo-temporal model and validated the accuracy of the scRNA-seq data, respectively. This work provides valuable marker-genes resources and defines precise cellular taxonomy and trajectory atlases for somatic cell embryogenic differentiation in plant regeneration.

N6-methyladenosine RNA modification regulates photoperiod sensitivity in cotton.

The methylation of N6-methyladenosine (m6A) involves writers, erasers, and readers, acting synergistically in posttranscriptional regulation. These processes influence various biological processes, including plant floral transition. However, the specific role of m6A modifications in photoperiod sensitivity in cotton (Gossypium hirsutum) remains obscure. To elucidate this, in this study, we conducted transcriptome-wide m6A sequencing during critical flowering transition stages in the photoperiod-sensitive wild G. hirsutum var. yucatanense (yucatanense) and the photoperiod-insensitive cultivated cotton G. hirsutum acc. TM-1 (TM-1). Our results revealed significant variations in m6A methylation of 2 cotton varieties, with yucatanense exhibiting elevated m6A modification levels compared with TM-1 under long-day conditions. Notably, distinct m6A peaks between TM-1 and yucatanense correlated significantly with photoperiod sensitivity. Moreover, our study highlighted the role of the demethylase G. hirsutum ALKB homolog 5 (GhALKBH5) in modulating m6A modification levels. Silencing GhALKBH5 led to a decreased mRNA level of key photoperiodic flowering genes (GhADO3, GhAGL24, and GhFT1), resulting in delayed bud emergence and flowering. Reverse transcription quantitative PCR analyses confirmed that silencing GhADO3 and GhAGL24 significantly downregulated the expression of the floral integrator GhFT1. Collectively, our findings unveiled a transcriptional regulatory mechanism in which GhALKBH5-mediated m6A demethylation of crucial photoperiodic flowering transcripts modulated photoperiod sensitivity in cotton.