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.

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.

Natural variations in the Cis-elements of GhRPRS1 contributing to petal colour diversity in cotton.

The cotton genus comprises both diploid and allotetraploid species, and the diversity in petal colour within this genus offers valuable targets for studying orthologous gene function differentiation and evolution. However, the genetic basis for this diversity in petal colour remains largely unknown. The red petal colour primarily comes from C, G, K, and D genome species, and it is likely that the common ancestor of cotton had red petals. Here, by employing a clone mapping strategy, we mapped the red petal trait to a specific region on chromosome A07 in upland cotton. Genomic comparisons and phylogenetic analyses revealed that the red petal phenotype introgressed from G. bickii. Transcriptome analysis indicated that GhRPRS1, which encodes a glutathione S-transferase, was the causative gene for the red petal colour. Knocking out GhRPRS1 resulted in white petals and the absence of red spots, while overexpression of both genotypes of GhRPRS1 led to red petals. Further analysis suggested that GhRPRS1 played a role in transporting pelargonidin-3-O-glucoside and cyanidin-3-O-glucoside. Promoter activity analysis indicated that variations in the promoter, but not in the gene body of GhRPRS1, have led to different petal colours within the genus. Our findings provide new insights into orthologous gene evolution as well as new strategies for modifying promoters in cotton breeding.

Uncovering genomic and transcriptional variations facilitates utilization of wild resources in cotton disease resistance improvement.

BACKGROUND: Upland cotton wild/landraces represent a valuable resource for disease resistance alleles. Genetic differentiation between genotypes, as well as variation in Verticillium wilt (VW) resistance, has been poorly characterized for upland cotton accessions on the domestication spectrum (from wild/landraces to elite lines). RESULTS: To illustrate the effects of modern breeding on VW resistance in upland cotton, 37 wild/landraces were resequenced and phenotyped for VW resistance. Genomic patterns of differentiation were identified between wild/landraces and improved upland cotton, and a significant decline in VW resistance was observed in association with improvement. Four genotypes representing different degrees of improvement were used in a full-length transcriptome analysis to study the genetic basis of VW resistance. ROS signaling was highly conserved at the transcriptional level, likely providing the basis for VW resistance in upland cotton. ASN biosynthesis and HSP90-mediated resistance moderated the response to VW in wild/landraces, and loss of induction activity of these genes resulted in VW susceptibility. The observed genomic differentiation contributed to the loss of induction of some important VW resistance genes such as HSP90.4 and PR16. CONCLUSIONS: Besides providing new insights into the evolution of upland cotton VW resistance, this study also identifies important resistance pathways and genes for both fundamental research and cotton breeding.

Functional divergence of GLP genes between G. barbadense and G. hirsutum in response to Verticillium dahliae infection.

Germin-like proteins (GLPs) play important roles in plant disease resistance but are rarely reported in cotton. We compared the expression of GLPs in Verticillium dahliae inoculate G. hirsutum (susceptible) and G. barbadense (resistant) and enriched 11 differentially expressed GLPs. 2741 GLP proteins identified from 53 species determined that GLP probably originated from algae and could be classified into 7 clades according to phylogenetic analysis, among which Clade I is likely the most ancient. Cotton GLP (two allopolyploids and two diploids) genes within a shared clade were highly conserved. Intriguingly, clade VII genes were mainly located in gene clusters that derived from the expansion of LTR transposons. Clade VII members expressed mainly in root which is the first battle against Verticillium dahlia and could be induced more intensely in G. barbadense than G. hirsutum. The GLP genes are resistant to Verticillium dahliae, which can be further investigated against Verticillium wilt.

Recent progression and future perspectives in cotton genomic breeding.

Upland cotton is an important global cash crop for its long seed fibers and high edible oil and protein content. Progress in cotton genomics promotes the advancement of cotton genetics, evolutionary studies, functional genetics, and breeding, and has ushered cotton research and breeding into a new era. Here, we summarize high-impact genomics studies for cotton from the last 10 years. The diploid Gossypium arboreum and allotetraploid Gossypium hirsutum are the main focus of most genetic and genomic studies. We next review recent progress in cotton molecular biology and genetics, which builds on cotton genome sequencing efforts, population studies, and functional genomics, to provide insights into the mechanisms shaping abiotic and biotic stress tolerance, plant architecture, seed oil content, and fiber development. We also suggest the application of novel technologies and strategies to facilitate genome-based crop breeding. Explosive growth in the amount of novel genomic data, identified genes, gene modules, and pathways is now enabling researchers to utilize multidisciplinary genomics-enabled breeding strategies to cultivate "super cotton", synergistically improving multiple traits. These strategies must rise to meet urgent demands for a sustainable cotton industry.

Efficient genotype-independent cotton genetic transformation and genome editing.

Cotton (Gossypium spp.) is one of the most important fiber crops worldwide. In the last two decades, transgenesis and genome editing have played important roles in cotton improvement. However, genotype dependence is one of the key bottlenecks in generating transgenic and gene-edited cotton plants through either particle bombardment or Agrobacterium-mediated transformation. Here, we developed a shoot apical meristem (SAM) cell-mediated transformation system (SAMT) that allowed the transformation of recalcitrant cotton genotypes including widely grown upland cotton (Gossypium hirsutum), Sea island cotton (Gossypium barbadense), and Asiatic cotton (Gossypium arboreum). Through SAMT, we successfully introduced two foreign genes, GFP and RUBY, into SAM cells of some recalcitrant cotton genotypes. Within 2-3 months, transgenic adventitious shoots generated from the axillary meristem zone could be recovered and grown into whole cotton plants. The GFP fluorescent signal and betalain accumulation could be observed in various tissues in GFP- and RUBY-positive plants, as well as in their progenies, indicating that the transgenes were stably integrated into the genome and transmitted to the next generation. Furthermore, using SAMT, we successfully generated edited cotton plants with inheritable targeted mutagenesis in the GhPGF and GhRCD1 genes through CRISPR/Cas9-mediated genome editing. In summary, the established SAMT transformation system here in this study bypasses the embryogenesis process during tissue culture in a conventional transformation procedure and significantly accelerates the generation of transgenic and gene-edited plants for genetic improvement of recalcitrant cotton varieties.

Cotton D genome assemblies built with long-read data unveil mechanisms of centromere evolution and stress tolerance divergence.

BACKGROUND: Many of genome features which could help unravel the often complex post-speciation evolution of closely related species are obscured because of their location in chromosomal regions difficult to accurately characterize using standard genome analysis methods, including centromeres and repeat regions. RESULTS: Here, we analyze the genome evolution and diversification of two recently diverged sister cotton species based on nanopore long-read sequence assemblies and Hi-C 3D genome data. Although D genomes are conserved in gene content, they have diversified in gene order, gene structure, gene family diversification, 3D chromatin structure, long-range regulation, and stress-related traits. Inversions predominate among D genome rearrangements. Our results support roles for 5mC and 6mA in gene activation, and 3D chromatin analysis showed that diversification in proximal-vs-distal regulatory-region interactions shape the regulation of defense-related-gene expression. Using a newly developed method, we accurately positioned cotton centromeres and found that these regions have undergone obviously more rapid evolution relative to chromosome arms. We also discovered a cotton-specific LTR class that clarifies evolutionary trajectories among diverse cotton species and identified genetic networks underlying the Verticillium tolerance of Gossypium thurberi (e.g., SA signaling) and salt-stress tolerance of Gossypium davidsonii (e.g., ethylene biosynthesis). Finally, overexpression of G. thurberi genes in upland cotton demonstrated how wild cottons can be exploited for crop improvement. CONCLUSIONS: Our study substantially deepens understanding about how centromeres have developed and evolutionarily impacted the divergence among closely related cotton species and reveals genes and 3D genome structures which can guide basic investigations and applied efforts to improve crops.

Integration of eQTL Analysis and GWAS Highlights Regulation Networks in Cotton under Stress Condition.

The genus Gossypium is one of the most economically important crops in the world. Here, we used RNA-seq to quantify gene expression in a collection of G. arboreum seedlings and performed eGWAS on 28,382 expressed genes. We identified a total of 30,089 eQTLs in 10,485 genes, of which >90% were trans-regulate target genes. Using luciferase assays, we confirmed that different cis-eQTL haplotypes could affect promoter activity. We found ~6600 genes associated with ~1300 eQTL hotspots. Moreover, hotspot 309 regulates the expression of 325 genes with roles in stem length, fresh weight, seed germination rate, and genes related to cell wall biosynthesis and salt stress. Transcriptome-wide association study (TWAS) identified 19 candidate genes associated with the cotton growth and salt stress response. The variation in gene expression across the population played an essential role in population differentiation. Only a small number of the differentially expressed genes between South China, the Yangtze River region, and the Yellow River region sites were located in different chromosomal regions. The eQTLs found across the duplicated gene pairs showed conservative cis- or trans- regulation and that the expression levels of gene pairs were correlated. This study provides new insights into the evolution of gene expression regulation in cotton, and identifies eQTLs in stress-related genes for use in breeding improved cotton varieties.

BIN2 negatively regulates plant defence against Verticillium dahliae in Arabidopsis and cotton.

Verticillium wilt is caused by the soil-borne vascular pathogen Verticillium dahliae, and affects a wide range of economically important crops, including upland cotton (Gossypium hirsutum). Previous studies showed that expression levels of BIN2 were significantly down-regulated during infestation with V. dahliae. However, the underlying molecular mechanism of BIN2 in plant regulation against V. dahliae remains enigmatic. Here, we characterized a protein kinase GhBIN2 from Gossypium hirsutum, and identified GhBIN2 as a negative regulator of resistance to V. dahliae. The Verticillium wilt resistance of Arabidopsis and cotton were significantly enhanced when BIN2 was knocked down. Constitutive expression of BIN2 attenuated plant resistance to V. dahliae. We found that BIN2 regulated plant endogenous JA content and influenced the expression of JA-responsive marker genes. Further analysis revealed that BIN2 interacted with and phosphorylated JAZ family proteins, key repressors of the JA signalling pathway in both Arabidopsis and cotton. Spectrometric analysis and site-directed mutagenesis showed that BIN2 phosphorylated AtJAZ1 at T196, resulting in the degradation of JAZ proteins. Collectively, these results show that BIN2 interacts with JAZ proteins and plays a negative role in plant resistance to V. dahliae. Thus, BIN2 may be a potential target gene for genetic engineering against Verticillium wilt in crops.

Genetic and evolution analysis of extrafloral nectary in cotton.

Extrafloral nectaries are a defence trait that plays important roles in plant-animal interactions. Gossypium species are characterized by cellular grooves in leaf midribs that secret large amounts of nectar. Here, with a panel of 215 G. arboreum accessions, we compared extrafloral nectaries to nectariless accessions to identify a region of Chr12 that showed strong differentiation and overlapped with signals from GWAS of nectaries. Fine mapping of an F2 population identified GaNEC1, encoding a PB1 domain-containing protein, as a positive regulator of nectary formation. An InDel, encoding a five amino acid deletion, together with a nonsynonymous substitution, was predicted to cause 3D structural changes in GaNEC1 protein that could confer the nectariless phenotype. mRNA-Seq analysis showed that JA-related genes are up-regulated and cell wall-related genes are down-regulated in the nectary. Silencing of GaNEC1 led to a smaller size of foliar nectary phenotype. Metabolomics analysis identified more than 400 metabolites in nectar, including expected saccharides and amino acids. The identification of GaNEC1 helps establish the network regulating nectary formation and nectar secretion, and has implications for understanding the production of secondary metabolites in nectar. Our results will deepen our understanding of plant-mutualism co-evolution and interactions, and will enable utilization of a plant defence trait in cotton breeding efforts.

A Phi-Class Glutathione S-Transferase Gene for Verticillium Wilt Resistance in Gossypium arboreum Identified in a Genome-Wide Association Study.

Verticillium wilt disease is one of the most destructive biotic stresses faced by cotton plants. Here, we performed a genome-wide association study (GWAS) in 215 Chinese Gossypium arboreum accessions inoculated as seedlings with Verticillium dahliae to identify candidate loci involved in wilt resistance. We identified 309 loci that had a significant association with Verticillium wilt resistance and - log(P) values >5.0; the highest signal appeared on Ca3 in a 74 kb haplotype block. Five genes were also located within this haplotype block. One of these genes, CG05, was positioned close to the most significant SNP Ca3_23037225 (14 kb); expression of the gene was induced by V. dahliae or by treatment with salicylic acid (SA). Therefore, we suggest that CG05 may respond to invasion by V. dahliae via an SA-related signaling pathway, and we designated this gene as GaGSTF9. We showed that GaGSTF9 was a positive regulator of Verticillium wilt through the use of virus-induced gene silencing (VIGS) and overexpression in Arabidopsis. In addition, the glutathione S-transferase (GST) mutant gstf9 of Arabidopsis was found to be more susceptible to Verticillium wilt than wild-type plants. The levels of endogenous SA and hydrogen peroxide had a significant effect on Arabidopsis plants that overexpressed GaGSTF9, indicating that GST may regulate reactive oxygen species content via catalytic reduction of the tripeptide glutathione (GSH), and then affect SA content. Our data demonstrated that GaGSTF9 was a key regulator mediating cotton responses to V. dahliae and a potential candidate gene for cotton genetic improvement.

Genome-wide characterization and phylogenetic analysis of GSK gene family in three species of cotton: evidence for a role of some GSKs in fiber development and responses to stress.

BACKGROUND: The glycogen synthase kinase 3/shaggy kinase (GSK3) is a serine/threonine kinase with important roles in animals. Although GSK3 genes have been studied for more than 30 years, plant GSK genes have been studied only since the last decade. Previous research has confirmed that plant GSK genes are involved in diverse processes, including floral development, brassinosteroid signaling, and responses to abiotic stresses. RESULT: In this study, 20, 15 (including 5 different transcripts) and 10 GSK genes were identified in G. hirsutum, G. raimondii and G. arboreum, respectively. A total of 65 genes from Arabidopsis, rice, and cotton were classified into 4 clades. High similarities were found in GSK3 protein sequences, conserved motifs, and gene structures, as well as good concordance in gene pairwise comparisons (G. hirsutum vs. G. arboreum, G. hirsutum vs. G. raimondii, and G. arboreum vs. G. raimondii) were observed. Whole genome duplication (WGD) within At and Dt sub-genomes has been central to the expansion of the GSK gene family. Furthermore, GhSK genes showed diverse expression patterns in various tissues. Additionally, the expression profiles of GhSKs under different stress treatments demonstrated that many are stress-responsive genes. However, none were induced by brassinolide treatment. Finally, nine co-expression sub-networks were observed for GhSKs and the functional annotations of these genes suggested that some GhSKs might be involved in cotton fiber development. CONCLUSION: In this present work, we identified 45 GSK genes from three cotton species, which were divided into four clades. The gene features, muti-alignment, conversed motifs, and syntenic blocks indicate that they have been highly conserved during evolution. Whole genome duplication was determined to be the dominant factor for GSK gene family expansion. The analysis of co-expressed sub-networks and tissue-specific expression profiles suggested functions of GhSKs during fiber development. Moreover, their different responses to various abiotic stresses indicated great functional diversity amongst the GhSKs. Briefly, data presented herein may serve as the basis for future functional studies of GhSKs.

Genome-wide analysis of cotton GH3 subfamily II reveals functional divergence in fiber development, hormone response and plant architecture.

BACKGROUND: Auxin-induced genes regulate many aspects of plant growth and development. The Gretchen Hagen 3 (GH3) gene family, one of three major early auxin-responsive families, is ubiquitous in the plant kingdom and its members function as regulators in modulating hormonal homeostasis, and stress adaptations. Specific Auxin-amido synthetase activity of GH3 subfamily II genes is reported to reversibly inactivate or fully degrade excess auxin through the formation of amino acid conjugates. Despite these crucial roles, to date, genome-wide analysis of the GH3 gene family has not been reported in cotton. RESULTS: We identified a total of 10 GH3 subfamily II genes in G. arboreum, 10 in G. raimondii, and 20 in G. hirsutum, respectively. Bioinformatic analysis showed that cotton GH3 genes are conserved with the established GH3s in plants. Expression pattern analysis based on RNA-seq data and qRT-PCR revealed that 20 GhGH3 genes were differentially expressed in a temporally and spatially specific manner, indicating their diverse functions in growth and development. We further summarized the organization of promoter regulatory elements and monitored their responsiveness to treatment with IAA (indole-3-acetic acid), SA (salicylic acid), GA (gibberellic acid) and BL (brassinolide) by qRT-PCR in roots and stems. These hormones seemed to regulate the expression of GH3 genes in both a positive and a negative manner while certain members likely have higher sensitivity to all four hormones. Further, we tested the expression of GhGH3 genes in the BR-deficient mutant pag1 and the corresponding wild-type (WT) of CCRI24. The altered expression reflected the true responsiveness to BL and further suggested possible reasons, at least in part, responsible for the dramatic dwarf and shriveled phenotypes of pag1. CONCLUSION: We comprehensively identified GH3 subfamily II genes in cotton. GhGH3s are differentially expressed in various tissues/organs/stages. Their response to IAA, SA, BL and GA and altered expression in pag1 suggest that some GhGH3 genes might be simultaneously involved in multiple hormone signaling pathways. Taken together, our results suggest that members of the GhGH3 gene family could be possible candidate genes for mechanistic study and applications in cotton fiber development in addition to the reconstruction of plant architecture.

Genome-Wide Analysis of the NF-YB Gene Family in Gossypium hirsutum L. and Characterization of the Role of GhDNF-YB22 in Embryogenesis.

Members of the NF-YB transcription factor gene family play important roles in diverse processes related to plant growth and development, such as seed development, drought tolerance, and flowering time. However, the function of NF-YB genes in cotton remains unclear. A total of 23, 24, and 50 NF-YB genes were identified in Gossypium arboreum (G. arboreum), Gossypium raimondii (G. raimondii), and G. hirsutum, respectively. A systematic phylogenetic analysis was carried out in G. arboretum, G. raimondii, G. hirsutum, Arabidopsis thaliana, cacao, rice and, sorghum, where the 150 NF-YB genes were divided into five groups (α-ε). Of these groups, α is the largest clade, and γ contains the LEC1 type NF-YB proteins. Syntenic analyses revealed that paralogues of NF-YB genes in G. hirsutum exhibited good collinearity. Owing to segmental duplication within the A sub-genome (At) and D sub-genome (Dt), there was an expanded set of NF-YB genes in G. hirsutum. Furthermore, we investigated the structures of exons, introns, and conserved motifs of NF-YB genes in upland cotton. Most of the NF-YB genes had only one exon, and the genes from the same clade exhibited a similar motif pattern. Expression data show that most NF-YB genes were expressed ubiquitously, and only a few genes were highly expressed in specific tissues, as confirmed by quantitative real-time PCR (qRT-PCR) analysis. The overexpression of GhDNF-YB22 gene, predominantly expressed in embryonic tissues, indicates that GhDNF-YB22 may affect embryogenesis in cotton. This study is the first comprehensive characterization of the GhNF-YB gene family in cotton, and showed that NF-YB genes could be divided into five clades. The duplication events that occurred over the course of evolution were the major impetus for NF-YB gene expansion in upland cotton. Collectively, this work provides insight into the evolution of NF-YB in cotton and further our knowledge of this commercially important species.

Genome-Wide Analysis Elucidates the Role of CONSTANS-like Genes in Stress Responses of Cotton.

The CONSTANS (CO)-like gene family has been well studied for its role in the regulation of plant flowering time. However, their role remains poorly understood in cotton. To better understand the possible roles of CO-like in cotton, we performed a comprehensive genome-wide analysis of CO-like genes in cotton. Phylogenetic tree analysis showed that CO-like genes naturally clustered into three groups. Segmental duplication and whole genome duplication (WGD), which occurred before polyploidy, were important contributors to its expansion within the At ("t" indicates tetraploid) and Dt subgenomes, particularly in Group III. Long-terminal repeat retroelements were identified as the main transposable elements accompanying 18 genes. The genotype of GhCOL12_Dt displayed low diversity; it was a candidate involved in domestication. Selection pressure analyses indicated that relaxed purifying selection might have provided the main impetus during the evolution of CO-like genes in upland cotton. In addition, the high expression in the torus and calycle indicated that CO-like genes might affect flowering. The genes from Group II, and those from Group III involved in segmental duplication or WGD, might play important roles in response to drought and salt stress. Overall, this comprehensive genome-wide study of the CO-like gene family would facilitate further detailed studies in cotton.

Salicylic acid-related cotton (Gossypium arboreum) ribosomal protein GaRPL18 contributes to resistance to Verticillium dahliae.

BACKGROUND: Verticillium dahliae is a phytopathogenic fungal pathogen that causes vascular wilt diseases responsible for considerable decreases in cotton yields. The complex mechanism underlying cotton resistance to Verticillium wilt remains uncharacterized. Identifying an endogenous resistance gene may be useful for controlling this disease. RESULTS: We cloned the ribosomal protein L18 (GaRPL18) gene, which mediates resistance to Verticillium wilt, from a wilt-resistant cotton species (Gossypium arboreum). We then characterized the function of this gene in cotton and Arabidopsis thaliana plants. GaRPL18 encodes a 60S ribosomal protein subunit important for intracellular protein biosynthesis. However, previous studies revealed that some ribosomal proteins are also inhibitory toward oncogenesis and congenital diseases in humans and play a role in plant disease defense. Here, we observed that V. dahliae infections induce GaRPL18 expression. Furthermore, we determined that the GaRPL18 expression pattern is consistent with the disease resistance level of different cotton varieties. GaRPL18 expression is upregulated by salicylic acid (SA) treatments, suggesting the involvement of GaRPL18 in the SA signal transduction pathway. Virus-induced gene silencing technology was used to determine whether the GaRPL18 expression level influences cotton disease resistance. Wilt-resistant cotton species in which GaRPL18 was silenced became more susceptible to V. dahliae than the control plants because of a significant decrease in the abundance of immune-related molecules. We also transformed A. thaliana ecotype Columbia (Col-0) plants with GaRPL18 according to the floral dip method. The plants overexpressing GaRPL18 were more resistant to V. dahliae infections than the wild-type Col-0 plants. The enhanced resistance of transgenic A. thaliana plants to V. dahliae is likely mediated by the SA pathway. CONCLUSION: Our findings provide new insights into the role of GaRPL18, indicating that it plays a crucial role in resistance to cotton "cancer", also known as Verticillium wilt, mainly regulated by an SA-related signaling pathway mechanism.

GaMYB85, an R2R3 MYB gene, in transgenic Arabidopsis plays an important role in drought tolerance.

BACKGROUND: MYB transcription factors (TFs) are one of the largest families of TFs in higher plants and are involved in diverse biological, functional, and structural processes. Previously, very few functional validation studies on R2R3 MYB have been conducted in cotton in response to abiotic stresses. In the current study, GaMYB85, a cotton R2R3 MYB TF, was ectopically expressed in Arabidopsis thaliana (Col-0) and was functionally characterized by overexpression in transgenic plants. RESULTS: The in-silico analysis of GaMYB85 shows the presence of a SANT domain with a conserved R2R3 MYB imperfect repeat. The GaMYB85 protein has a 257-amino acid sequence, a molecular weight of 24.91 kD, and an isoelectric point of 5.58. Arabidopsis plants overexpressing GaMYB85 exhibited a higher seed germination rate in response to mannitol and salt stress, and higher drought avoidance efficiency than wild-type plants upon water deprivation. These plants had notably higher levels of free proline and chlorophyll with subsequent lower water loss rates and higher relative water content. Germination of GaMYB85 transgenics was more sensitive to abscisic acid (ABA) and extremely liable to ABA-induced inhibition of primary root elongation. Moreover, when subjected to treatment with different concentrations of ABA, transgenic plants with ectopically expressed GaMYB85 showed reduced stomatal density, with greater stomatal size and lower stomatal opening rates than those in wild-type plants. Ectopic expression of GaMYB85 led to enhanced transcript levels of stress-related marker genes such as RD22, ADH1, RD29A, P5CS, and ABI5. CONCLUSIONS: Our results indicate previously unknown roles of GaMYB85, showing that it confers good drought, salt, and freezing tolerance, most probably via an ABA-induced pathway. These findings can potentially be exploited to develop improved abiotic stress tolerance in cotton plants.

Genome-wide analysis of WOX genes in upland cotton and their expression pattern under different stresses.

BACKGROUND: WUSCHEL-related homeobox (WOX) family members play significant roles in plant growth and development, such as in embryo patterning, stem-cell maintenance, and lateral organ formation. The recently published cotton genome sequences allow us to perform comprehensive genome-wide analysis and characterization of WOX genes in cotton. RESULTS: In this study, we identified 21, 20, and 38 WOX genes in Gossypium arboreum (2n = 26, A2), G. raimondii (2n = 26, D5), and G. hirsutum (2n = 4x = 52, (AD)t), respectively. Sequence logos showed that homeobox domains were significantly conserved among the WOX genes in cotton, Arabidopsis, and rice. A total of 168 genes from three typical monocots and six dicots were naturally divided into three clades, which were further classified into nine sub-clades. A good collinearity was observed in the synteny analysis of the orthologs from At and Dt (t represents tetraploid) sub-genomes. Whole genome duplication (WGD) and segmental duplication within At and Dt sub-genomes played significant roles in the expansion of WOX genes, and segmental duplication mainly generated the WUS clade. Copia and Gypsy were the two major types of transposable elements distributed upstream or downstream of WOX genes. Furthermore, through comparison, we found that the exon/intron pattern was highly conserved between Arabidopsis and cotton, and the homeobox domain loci were also conserved between them. In addition, the expression pattern in different tissues indicated that the duplicated genes in cotton might have acquired new functions as a result of sub-functionalization or neo-functionalization. The expression pattern of WOX genes under different stress treatments showed that the different genes were induced by different stresses. CONCLUSION: In present work, WOX genes, classified into three clades, were identified in the upland cotton genome. Whole genome and segmental duplication were determined to be the two major impetuses for the expansion of gene numbers during the evolution. Moreover, the expression patterns suggested that the duplicated genes might have experienced a functional divergence. Together, these results shed light on the evolution of the WOX gene family, and would be helpful in future research.

Genome-wide analysis of the HD-ZIP IV transcription factor family in Gossypium arboreum and GaHDG11 involved in osmotic tolerance in transgenic Arabidopsis.

HD-ZIP IV proteins belong to the homeodomain-leucine zipper (HD-ZIP) transcription factor family and are involved in trichome development and drought stress in plants. Although some functions of the HD-ZIP IV group are well understood in Arabidopsis, little is known about their function in cotton. In this study, HD-ZIP genes were identified from three Gossypium species (G. arboreum, G. raimondii and G. hirsutum) and clustered into four families (HD-ZIP I, II, III and IV) to separate HD-ZIP IV from the other three families. Systematic analyses of phylogeny, gene structure, conserved domains, and expression profiles in different plant tissues and the expression patterns under osmotic stress in leaves were further conducted in G. arboreum. More importantly, ectopic overexpression of GaHDG11, a representative of the HD-ZIP IV family, confers enhanced osmotic tolerance in transgenic Arabidopsis plants, possibly due to elongated primary root length, lower water loss rates, high osmoprotectant proline levels, significant levels of antioxidants CAT, and/or SOD enzyme activity with reduced levels of MDA. Taken together, these observations may lay the foundation for future functional analysis of cotton HD-ZIP IV genes to unravel their biological roles in cotton.