Regulation of Glandular Size and Phytoalexin Biosynthesis by a Negative Feedback Loop in Cotton.

Plants have evolved diverse defense mechanisms encompassing physical and chemical barriers. Cotton pigment glands are known for containing various defense metabolites, but the precise regulation of gland size to modulate defense compound levels remains enigmatic. Here, it is discovered that the VQ domain-containing protein JAVL negatively regulates pigment gland size and the biosynthesis of defense compounds, while the MYC2-like transcription factor GoPGF has the opposite effect. Notably, GoPGF directly activates the expression of JAVL, whereas JAVL suppresses GoPGF transcription, establishing a negative feedback loop that maintains the expression homeostasis between GoPGF and JAVL. Furthermore, it is observed that JAVL negatively regulates jasmonate levels by inhibiting the expression of jasmonate biosynthetic genes and interacting with GoPGF to attenuate its activation effects, thereby maintaining homeostatic regulation of jasmonate levels. The increased expression ratio of GoPGF to JAVL leads to enlarged pigment glands and elevated jasmonates and defense compounds, enhancing insect and pathogen resistance in cotton. These findings unveil a new mechanism for regulating gland size and secondary metabolites biosynthesis, providing innovative strategies for strengthening plant defense.

GhHB14_D10 and GhREV_D5, two HD-ZIP III transcription factors, play a regulatory role in cotton fiber secondary cell wall biosynthesis.

KEY MESSAGE: GhHB14_D10 and GhREV_D5 regulated secondary cell wall formation and played an important role in fiber development. Cotton serves as an important source of natural fiber, and the biosynthesis of the secondary cell wall plays a pivotal role in determining cotton fiber quality. Nevertheless, the intricacies of this mechanism in cotton fiber remain insufficiently elucidated. This study investigates the functional roles of GhHB14_D10 and GhREV_D5, two HD-ZIP III transcription factors, in secondary cell wall biosynthesis in cotton fibers. Both GhHB14_D10 and GhREV_D5 were found to be localized in the nucleus with transcriptional activation activity. Ectopic overexpression of GhHB14_D10 and GhREV_D5 in Arabidopsis resulted in changed xylem differentiation, secondary cell wall deposition, and expression of genes related to the secondary cell wall. Silencing of GhHB14_D10 and GhREV_D5 in cotton led to enhanced fiber length, reduced cell wall thickness, cellulose contents and expression of secondary cell wall-related genes. Moreover, GhHB14_D10's direct interaction with GhREV_D5, and transcriptional regulation of cellulose biosynthesis genes GhCesA4-4 and GhCesA7-2 revealed their collaborative roles in secondary cell wall during cotton fiber development. Overall, these results shed light on the roles of GhHB14_D10 and GhREV_D5 in secondary cell wall biosynthesis, offering a strategy for the genetic improvement of cotton fiber quality.

Transcriptional and translational landscape fine-tune genome annotation and explores translation control in cotton.

INTRODUCTION: The unavailability of intergenic region annotation in whole genome sequencing and pan-genomics hinders efforts to enhance crop improvement. OBJECTIVES: Despite advances in research, the impact of post-transcriptional regulation on fiber development and translatome profiling at different stages of fiber growth in cotton (G. hirsutum) remains unexplored. METHODS: We utilized a combination of reference-guided de novo transcriptome assembly and ribosome profiling techniques to uncover the hidden mechanisms of translational control in eight distinct tissues of upland cotton. RESULTS: Our study identified P-site distribution at three-nucleotide periodicity and dominant ribosome footprint at 27 nucleotides. Specifically, we have detected 1,589 small open reading frames (sORFs), including 1,376 upstream ORFs (uORFs) and 213 downstream ORFs (dORFs), as well as 552 long non-coding RNAs (lncRNAs) with potential coding functions, which fine-tune the annotation of the cotton genome. Further, we have identified novel genes and lncRNAs with strong translation efficiency (TE), while sORFs were found to affect mRNA transcription levels during fiber elongation. The reliability of these findings was confirmed by the high consistency in correlation and synergetic fold change between RNA-sequencing (RNA-seq) and Ribosome-sequencing (Ribo-seq) analyses. Additionally, integrated omics analysis of the normal fiber ZM24 and short fiber pag1 cotton mutant revealed several differentially expressed genes (DEGs), and fiber-specific expressed (high/low) genes associated with sORFs (uORFs and dORFs). These findings were further supported by the overexpression and knockdown of GhKCS6, a gene associated with sORFs in cotton, and demonstrated the potential regulation of the mechanism governing fiber elongation on both the transcriptional and post-transcriptional levels. CONCLUSION: Reference-guided transcriptome assembly and the identification of novel transcripts fine-tune the annotation of the cotton genome and predicted the landscape of fiber development. Our approach provided a high-throughput method, based on multi-omics, for discovering unannotated ORFs, hidden translational control, and complex regulatory mechanisms in crop plants.

Characterization and fine mapping of a yellow leaf gene regulating chlorophyll biosynthesis and chloroplast development in cotton (Gossypium arboreum).

Chlorophyll biosynthesis and chloroplast development are essential for photosynthesis and plant growth. Gossypium arboreum, a valuable source of genetic variation for cotton improvement, remains poorly studied for the mechanisms regulating chlorophyll biosynthesis and chloroplast development. Here we created a G. arboreum etiolated leaf and stuntedness (els) mutant that displayed a distinct yellow color of leaves, bracts and stems throughout the whole growth, where chlorophyll accumulation in leaves was reduced and chloroplast development was delayed. The GaCHLH gene, which encodes the H subunit of magnesium chelatase (Mg-chelatase), was screened by MutMap and KASP analysis. Compared to GaCHLH, the gene Gachlh of the mutant had a single nucleotide transition (G to A) at 1549 bp, which causes the substitution of a glycine (G) by a serine (S) at the 517th amino acid, resulting in an abnormal secondary structure of the Gachlh protein. GaCHLH-silenced SXY1 and ZM24 plants exhibited a lower GaCHLH expression level, a lower chlorophyll content, and the yellow-leaf phenotype. Gachlh expression affected the expression of key genes in the tetrapyrrole pathway. GaCHLH and Gachlh were located in the chloroplasts and that alteration of the mutation site did not affect the final target position. The BiFC assay result indicated that Gachlh could not bind to GaCHLD properly, which prevented the assembly of Mg-chelatase and thus led to the failure of chlorophyll synthesis. In this study, the Gachlh gene of G. arboreum els was finely localized and identified for the first time, providing new insights into the chlorophyll biosynthesis pathway in cotton.

Characterization of chromatin accessibility and gene expression reveal the key genes involved in cotton fiber elongation.

Cotton (Gossypium hirsutum L.) is an important economic crop, and cotton fiber is one of the longest plant cells, which provides an ideal model for the study of cell elongation and secondary cell wall synthesis. Cotton fiber length is regulated by a variety of transcription factors (TF) and their target genes; however, the mechanism of fiber elongation mediated by transcriptional regulatory networks is still unclear to a large extent. Here, we used a comparative assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq) assay and RNA-seq analysis to identify fiber elongation transcription factors and genes using the short-fiber mutant ligon linless-2 (Li2 ) and wild type (WT). A total of 499 differential target genes were identified and GO analysis shows that differential genes are mainly involved in plant secondary wall synthesis and microtubule-binding processes. Analysis of the genomic regions preferentially accessible (Peak) has identified a number of overrepresented TF-binding motifs, highlighting sets of TFs that are important for cotton fiber development. Using ATAC-seq and RNA-seq data, we have constructed a functional regulatory network of each TF regulatory target gene and also the network pattern of TF regulating differential target genes. Further, to obtain the genes related to fiber length, the differential target genes were combined with FLGWAS data to identify the genes highly related to fiber length. Our work provides new insights into cotton fiber elongation.

Cell cycle-dependent kinase inhibitor GhKRP6, a direct target of GhBES1.4, participates in BR regulation of cell expansion in cotton.

The steroidal hormone brassinosteroid (BR) has been shown to positively regulate cell expansion in plants. However, the specific mechanism by which BR controls this process has not been fully understood. In this study, RNA-seq and DAP-seq analysis of GhBES1.4 (a core transcription factor in BR signaling) were used to identify a cotton cell cycle-dependent kinase inhibitor called GhKRP6. The study found that GhKRP6 was significantly induced by the BR hormone and that GhBES1.4 directly promoted the expression of GhKRP6 by binding to the CACGTG motif in its promoter region. GhKRP6-silenced cotton plants had smaller leaves with more cells and reduced cell size. Furthermore, endoreduplication was inhibited, which affected cell expansion and ultimately decreased fiber length and seed size in GhKRP6-silenced plants compared with the control. The KEGG enrichment results of control and VIGS-GhKRP6 plants revealed differential expression of genes related to cell wall biosynthesis, MAPK, and plant hormone transduction pathways - all of which are related to cell expansion. Additionally, some cyclin-dependent kinase (CDK) genes were upregulated in the plants with silenced GhKRP6. Our study also found that GhKRP6 could interact directly with a cell cycle-dependent kinase called GhCDKG. Taken together, these results suggest that BR signaling influences cell expansion by directly modulating the expression of cell cycle-dependent kinase inhibitor GhKRP6 via GhBES1.4.

N6 -Methyladenosine mRNA modification regulates transcripts stability associated with cotton fiber elongation.

N6 -Methyladenosine (m6 A) is the most abundant methylation modification in eukaryotic mRNA. The discovery of the dynamic and reversible regulatory mechanism of m6 A has greatly promoted the development of m6 A-led epitranscriptomics. However, the characterization of m6 A in cotton fiber is still unknown. Here, we reveal the potential link between m6 A modification and cotton fiber elongation by parallel m6 A-immunoprecipitation-sequencing (m6 A-seq) and RNA-seq analysis of fibers from the short fiber mutants Ligonliness-2 (Li2 ) and wild-type (WT). This study demonstrated a higher level of m6 A in the Li2 mutant, with the enrichment of m6 A modifications in the stop codon, 3'-untranslated region and coding sequence regions than in WT cotton. In the correlation analysis between genes containing differential m6 A modifications and differentially expressed genes, we identified several genes that could potentially regulate fiber elongation, including cytoskeleton, microtubule binding, cell wall and transcription factors (TFs). We further confirmed that the methylation of m6 A affected the mRNA stability of these fiber elongation-related genes including the TF GhMYB44, which showed the highest expression level in the RNA-seq data and m6 A methylation in the m6 A-seq data. Next, the overexpression of GhMYB44 reduces fiber elongation, whereas the silencing of GhMYB44 produces longer fibers. In summary, these results uncover that m6 A methylation regulated the expression of genes related to fiber development by affecting mRNA's stability, ultimately affecting cotton fiber elongation.

Functional characterization of TBL genes revealed the role of GhTBL7 and GhTBL58 in cotton fiber elongation.

TBL (Trichome Birefringence Like) gene family members are involved in trichome initiation and xylan acetylation in several plant species. In our research, we identified 102 TBLs from G. hirsutum. The phylogenetic tree classified TBL genes into five groups. Collinearity analysis of TBL genes indicated 136 paralogous gene pairs in G. hirsutum. Gene duplication indicated that WGD or segmental duplication contributed to the GhTBL gene family expansion. Promoter cis-elements of GhTBLs were related to growth and development, seed-specific regulation, light, and stress responses. GhTBL genes (GhTBL7, GhTBL15, GhTBL21, GhTBL25, GhTBL45, GhTBL54, GhTBL67, GhTBL72, and GhTBL77) exhibited upregulated response under exposure to cold, heat, NaCl, and PEG. GhTBL genes exhibited high expression during fiber development stages. Two GhTBL genes (GhTBL7 and GhTBL58) showed differential expression at 10 DPA fiber, as 10 DPA is a fast fiber elongation stage and fiber elongation is a very important stage of cotton fiber development. Subcellular localization of GhTBL7 and GhTBL58 revealed that these genes reside inside the cell membrane. Promoter GUS activity of GhTBL7 and GhTBL58 exhibited deep staining in roots. To further validate the role of these genes in cotton fiber elongation, we silenced these genes and observed a significant reduction in the fiber length at 10 DPA. In conclusion, the functional study of cell membrane-associated genes (GhTBL7 and GhTBL58) showed deep staining in root tissues and potential function during cotton fiber elongation at 10 DPA fiber.

A comprehensive overview of cotton genomics, biotechnology and molecular biological studies.

Cotton is an irreplaceable economic crop currently domesticated in the human world for its extremely elongated fiber cells specialized in seed epidermis, which makes it of high research and application value. To date, numerous research on cotton has navigated various aspects, from multi-genome assembly, genome editing, mechanism of fiber development, metabolite biosynthesis, and analysis to genetic breeding. Genomic and 3D genomic studies reveal the origin of cotton species and the spatiotemporal asymmetric chromatin structure in fibers. Mature multiple genome editing systems, such as CRISPR/Cas9, Cas12 (Cpf1) and cytidine base editing (CBE), have been widely used in the study of candidate genes affecting fiber development. Based on this, the cotton fiber cell development network has been preliminarily drawn. Among them, the MYB-bHLH-WDR (MBW) transcription factor complex and IAA and BR signaling pathway regulate the initiation; various plant hormones, including ethylene, mediated regulatory network and membrane protein overlap fine-regulate elongation. Multistage transcription factors targeting CesA 4, 7, and 8 specifically dominate the whole process of secondary cell wall thickening. And fluorescently labeled cytoskeletal proteins can observe real-time dynamic changes in fiber development. Furthermore, research on the synthesis of cotton secondary metabolite gossypol, resistance to diseases and insect pests, plant architecture regulation, and seed oil utilization are all conducive to finding more high-quality breeding-related genes and subsequently facilitating the cultivation of better cotton varieties. This review summarizes the paramount research achievements in cotton molecular biology over the last few decades from the above aspects, thereby enabling us to conduct a status review on the current studies of cotton and provide strong theoretical support for the future direction.

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.

A brassinosteroid transcriptional regulatory network participates in regulating fiber elongation in cotton.

Brassinosteroids (BRs) participate in the regulation of plant growth and development through BRI1-EMS-SUPPRESSOR1 (BES1)/BRASSINAZOLE-RESISTANT1 (BZR1) family transcription factors. Cotton (Gossypium hirsutum) fibers are highly elongated single cells, and BRs play a vital role in the regulation of fiber elongation. However, the mode of action on how BR is involved in the regulation of cotton fiber elongation remains unexplored. Here, we generated GhBES1.4 over expression lines and found that overexpression of GhBES1.4 promoted fiber elongation, whereas silencing of GhBES1.4 reduced fiber length. DNA affinity purification and sequencing (DAP-seq) identified 1,531 target genes of GhBES1.4, and five recognition motifs of GhBES1.4 were identified by enrichment analysis. Combined analysis of DAP-seq and RNA-seq data of GhBES1.4-OE/RNAi provided mechanistic insights into GhBES1.4-mediated regulation of cotton fiber development. Further, with the integrated approach of GWAS, RNA-seq, and DAP-seq, we identified seven genes related to fiber elongation that were directly regulated by GhBES1.4. Of them, we showed Cytochrome P450 84A1 (GhCYP84A1) and 3-hydroxy-3-methylglutaryl-coenzyme A reductase 1 (GhHMG1) promote cotton fiber elongation. Overall, the present study established the role of GhBES1.4-mediated gene regulation and laid the foundation for further understanding the mechanism of BR participation in regulating fiber development.

CottonMD: a multi-omics database for cotton biological study.

Cotton is an important economic crop, and many loci for important traits have been identified, but it remains challenging and time-consuming to identify candidate or causal genes/variants and clarify their roles in phenotype formation and regulation. Here, we first collected and integrated the multi-omics datasets including 25 genomes, transcriptomes in 76 tissue samples, epigenome data of five species and metabolome data of 768 metabolites from four tissues, and genetic variation, trait and transcriptome datasets from 4180 cotton accessions. Then, a cotton multi-omics database (CottonMD, http://yanglab.hzau.edu.cn/CottonMD/) was constructed. In CottonMD, multiple statistical methods were applied to identify the associations between variations and phenotypes, and many easy-to-use analysis tools were provided to help researchers quickly acquire the related omics information and perform multi-omics data analysis. Two case studies demonstrated the power of CottonMD for identifying and analyzing the candidate genes, as well as the great potential of integrating multi-omics data for cotton genetic breeding and functional genomics research.

GhBES1 mediates brassinosteroid regulation of leaf size by activating expression of GhEXO2 in cotton (Gossypium hirsutum).

KEY MESSAGE: We proposed a working model of BR to promote leaf size through cell expansion. In the BR signaling pathway, GhBES1 affects cotton leaf size by binding to and activating the expression of the E-box element in the GhEXO2 promoter region. Brassinosteroid (BR) is an essential phytohormone that controls plant growth. However, the mechanisms of BR regulation of leaf size remain to be determined. Here, we found that the BR deficient cotton mutant pagoda1 (pag1) had a smaller leaf size than wild-type CRI24. The expression of EXORDIUM (GhEXO2) gene, was significantly downregulated in pag1. Silencing of BRI1-EMS-SUPPRESSOR 1 (GhBES1), inhibited leaf cell expansion and reduced leaf size. Overexpression of GhBES1.4 promoted leaf cell expansion and enlarged leaf size. Expression analysis showed GhEXO2 expression positively correlated with GhBES1 expression. In plants, altered expression of GhEXO2 promoted leaf cell expansion affecting leaf size. Furthermore, GhBES1.4 specifically binds to the E-box elements in the GhEXO2 promoter, inducing its expression. RNA-seq data revealed many down-regulated genes related to cell expansion in GhEXO2 silenced plants. In summary, we discovered a novel mechanism of BR regulation of leaf size through GhBES1 directly activating the expression of GhEXO2.

Evaluation of Intracellular Gene Transfers from Plastome to Nuclear Genome across Progressively Improved Assemblies for Arabidopsis thaliana and Oryza sativa.

DNA originating from organellar genomes are regularly discovered in nuclear sequences during genome assembly. Nevertheless, such insertions are sometimes omitted during the process of nuclear genome assembly because the inserted DNA is assigned to organellar genomes, leading to a systematic underestimation of their frequency. With the rapid development of high-throughput sequencing technology, more inserted fragments from organelle genomes can now be detected. Therefore, it is necessary to be aware of the insertion events from organellar genomes during nuclear genome assembly to properly attribute the impact and rate of such insertions in the evolution of nuclear genomes. Here, we investigated the impact of intracellular gene transfer (IGT) from the plastome to the nuclear genome using genome assemblies that were refined through time with technological improvements from two model species, Arabidopsis thaliana and Oryza sativa. We found that IGT from the plastome to the nuclear genome is a dynamic and ongoing process in both A. thaliana and O. sativa, and mostly occurred recently, as the majority of transferred sequences showed over 95% sequence similarity with plastome sequences of origin. Differences in the plastome-to-nuclear genome IGT between A. thaliana and O. sativa varied among the different assembly versions and were associated with the quality of the nuclear genome assembly. IGTs from the plastome to nuclear genome occurred more frequently in intergenic regions, which were often associated with transposable elements (TEs). This study provides new insights into intracellular genome evolution and nuclear genome assembly by characterizing and comparing IGT from the plastome into the nuclear genome for two model plant species.

Transcriptomic analysis reveals the key role of histone deacetylation via mediating different phytohormone signalings in fiber initiation of cotton.

BACKGROUND: Histone deacetylation is one of the most important epigenetic modifications and plays diverse roles in plant development. However, the detailed functions and mechanisms of histone deacetylation in fiber development of cotton are still unclear. HDAC inhibitors (HDACi) have been commonly used to study the molecular mechanism underlying histone deacetylation or to facilitate disease therapy in humans through hindering the histone deacetylase catalytic activity. Trichostatin A (TSA)-the most widely used HDACi has been extensively employed to determine the role of histone deacetylation on different developmental stages of plants. RESULTS: Through in vitro culture of ovules, we observed that exogenous application of TSA was able to inhibit the fiber initiation development. Subsequently, we performed a transcriptomic analysis to reveal the underlying mechanisms. The data showed that TSA treatment resulted in 4209 differentially expressed genes, which were mostly enriched in plant hormone signal transduction, phenylpropanoid biosynthesis, photosynthesis, and carbon metabolism pathways. The phytohormone signal transduction pathways harbor the most differentially expressed genes. Deeper studies showed that some genes promoting auxin, Gibberellic Acid (GA) signaling were down-regulated, while some genes facilitating Abscisic Acid (ABA) and inhibiting Jasmonic Acid (JA) signaling were up-regulated after the TSA treatments. Further analysis of plant hormone contents proved that TSA significantly promoted the accumulation of ABA, JA and GA3. CONCLUSIONS: Collectively, histone deacetylation can regulate some key genes involved in different phytohormone pathways, and consequently promoting the auxin, GA, and JA signaling, whereas repressing the ABA synthesis and signaling to improve the fiber cell initiation. Moreover, the genes associated with energy metabolism, phenylpropanoid, and glutathione metabolism were also regulated by histone deacetylation. The above results provided novel clues to illuminate the underlying mechanisms of epigenetic modifications as well as related different phytohormones in fiber cell differentiation, which is also very valuable for the molecular breeding of higher quality cotton.

iFeatureOmega: an integrative platform for engineering, visualization and analysis of features from molecular sequences, structural and ligand data sets.

The rapid accumulation of molecular data motivates development of innovative approaches to computationally characterize sequences, structures and functions of biological and chemical molecules in an efficient, accessible and accurate manner. Notwithstanding several computational tools that characterize protein or nucleic acids data, there are no one-stop computational toolkits that comprehensively characterize a wide range of biomolecules. We address this vital need by developing a holistic platform that generates features from sequence and structural data for a diverse collection of molecule types. Our freely available and easy-to-use iFeatureOmega platform generates, analyzes and visualizes 189 representations for biological sequences, structures and ligands. To the best of our knowledge, iFeatureOmega provides the largest scope when directly compared to the current solutions, in terms of the number of feature extraction and analysis approaches and coverage of different molecules. We release three versions of iFeatureOmega including a webserver, command line interface and graphical interface to satisfy needs of experienced bioinformaticians and less computer-savvy biologists and biochemists. With the assistance of iFeatureOmega, users can encode their molecular data into representations that facilitate construction of predictive models and analytical studies. We highlight benefits of iFeatureOmega based on three research applications, demonstrating how it can be used to accelerate and streamline research in bioinformatics, computational biology, and cheminformatics areas. The iFeatureOmega webserver is freely available at http://ifeatureomega.erc.monash.edu and the standalone versions can be downloaded from https://github.com/Superzchen/iFeatureOmega-GUI/ and https://github.com/Superzchen/iFeatureOmega-CLI/.

GRAND: An Integrated Genome, Transcriptome Resources, and Gene Network Database for Gossypium.

With the increasing amount of cotton omics data, breeding scientists are confronted with the question of how to use massive cotton data to mine effective breeding information. Here, we construct a Gossypium Resource And Network Database (GRAND), which integrates 18 cotton genome sequences, genome annotations, two cotton genome variations information, and also four transcriptomes for Gossypium species. GRAND allows to explore and mine this data with the help of a toolbox that comprises a flexible search system, BLAST and BLAT suite, orthologous gene ID, networks of co-expressed genes, primer design, Gbrowse and Jbrowse, and drawing instruments. GRAND provides important information regarding Gossypium resources and hopefully can accelerate the progress of cultivating cotton varieties.

Glucose regulates cotton fiber elongation by interacting with brassinosteroid.

In plants, glucose (Glc) plays important roles, as a nutrient and signal molecule, in the regulation of growth and development. However, the function of Glc in fiber development of upland cotton (Gossypium hirsutum) is unclear. Here, using gas chromatography-mass spectrometry (GC-MS), we found that the Glc content in fibers was higher than that in ovules during the fiber elongation stage. In vitro ovule culture revealed that lower Glc concentrations promoted cotton fiber elongation, while higher concentrations had inhibitory effects. The hexokinase inhibitor N-acetylglucosamine (NAG) inhibited cotton fiber elongation in the cultured ovules, indicating that Glc-mediated fiber elongation depends on the Glc signal transduced by hexokinase. RNA sequencing (RNA-seq) analysis and hormone content detection showed that 150mM Glc significantly activated brassinosteroid (BR) biosynthesis, and the expression of signaling-related genes was also increased, which promoted fiber elongation. In vitro ovule culture clarified that BR induced cotton fiber elongation in a dose-dependent manner. In hormone recovery experiments, only BR compensated for the inhibitory effects of NAG on fiber elongation in a Glc-containing medium. However, the ovules cultured with the BR biosynthetic inhibitor brassinazole and from the BR-deficient cotton mutant pag1 had greatly reduced fiber elongation at all the Glc concentrations tested. This demonstrates that Glc does not compensate for the inhibition of fiber elongation caused by BR biosynthetic defects, suggesting that the BR signaling pathway works downstream of Glc during cotton fiber elongation. Altogether, our study showed that Glc plays an important role in cotton fibre elongation, and crosstalk occurs between Glc and BR signaling during modulation of fiber elongation.

Identification, evolutionary analysis and functional diversification of RAV gene family in cotton (G. hirsutum L.).

MAIN CONCLUSION: Genome wide analysis, expression pattern analysis, and functional characterization of RAV genes highlight their roles in roots, stem development and hormonal response. RAV (Related to ABI3 and VP1) gene family members have been involved in tissues/organs growth and hormone signaling in various plant species. Here, we identified 247 RAVs from 12 different species with 33 RAV genes from G. hirsutum. Phylogenetic analysis classified RAV genes into four distinct groups. Analysis of gene structure showed that most GhRAVs lack introns. Motif distribution pattern and protein sequence logos indicated that GhRAV genes were highly conserved during the process of evolution. Promotor cis-acting elements revealed that promotor regions of GhRAV genes encode numerous elements related to plant growth, abiotic stresses and phytohormones. Chromosomal location information showed uneven distribution of 33 GhRAV genes on different chromosomes. Collinearity analysis identified 628 and 52 orthologous/ paralogous gene pairs in G. hirsutum and G. barbadense, respectively. Ka/Ks values indicated that GhRAV and GbRAV genes underwent strong purifying selection pressure. Selecton model and codon model selection revealed that GhRAV amino acids were under purifying selection and adaptive evolution exists among GhRAV proteins. Three dimensional structure of GhRAVs indicated the presence of numerous alpha helix and beta-barrels. Expression level revealed that some GhRAV genes exhibited high expression in roots (GhRAV3, GhRAV4, GhRAV11, GhRAV18, GhRAV20 and GhRAV30) and stem (GhRAV3 and GhRAV18), indicating their potential role in roots and stem development. GhRAV genes can be regulated by phytohormonal stresses (BL, JA and IAA). Our study provides a reference for future studies related to the functional analysis of GhRAVs in cotton.