Genome-wide characterization of DNA methyltransferase family genes implies GhDMT6 improving tolerance of salt and drought on cotton.

BACKGROUND: DNA methylation is an important epigenetic mode of genomic DNA modification and plays a vital role in maintaining epigenetic content and regulating gene expression. Cytosine-5 DNA methyltransferase (C5-MTase) are the key enzymes in the process of DNA methylation. However, there is no systematic analysis of the C5-MTase in cotton so far, and the function of DNMT2 genes has not been studied. METHODS: In this study, the whole genome of cotton C5-MTase coding genes was identified and analyzed using a bioinformatics method based on information from the cotton genome, and the function of GhDMT6 was further validated by VIGS experiments and subcellular localization analysis. RESULTS: 33 C5-MTases were identified from three cotton genomes, and were divided into four subfamilies by systematic evolutionary analysis. After the protein domain alignment of C5-MTases in cotton, 6 highly conserved motifs were found in the C-terminus of 33 proteins involved in methylation modification, which indicated that C5-MTases had a basic catalytic methylation function. These proteins were divided into four classes based on the N-terminal difference, of which DNMT2 lacks the N-terminal regulatory domain. The expression of C5-MTases in different parts of cotton was different under different stress treatments, which indicated the functional diversity of cotton C5-MTase gene family. Among the C5-MTases, the GhDMT6 had a obvious up-regulated expression. After silencing GhDMT6 with VIGS, the phenotype of cotton seedlings under different stress treatments showed a significant difference. Compared with cotton seedlings that did not silence GhDMT6, cotton seedlings silencing GhDMT6 showed significant stress resistance. CONCLUSION: The results show that C5-MTases plays an important role in cotton stress response, which is beneficial to further explore the function of DNMT2 subfamily genes.

Systematic analysis and expression of Gossypium ATG8 family reveals the roles of GhATG8f responding to salt stress in cotton.

KEY MESSAGE: Comprehensive analysis of Gossypium ATG8 family indicates that GhATG8f could improve salt tolerance of cotton by increasing SOD, POD and CAT activity and proline accumulation. In plants, autophagy is regulated by several genes that play important roles in initiating and controlling the process. ATG8, functioning as a protein similar to ubiquitin, is involved in crucial tasks throughout the autophagosome formation process. In this research, we conducted an extensive and all-encompassing investigation of 64 ATG8 genes across four varieties of cotton. According to the subcellular localization prediction results, 49 genes were found in the cytoplasm, 6 genes in the chloroplast, 1 gene in the peroxisome, 5 genes in the nucleus, and 3 genes in the extracellular region. Phylogenetic analysis categorized a total of 5 subfamilies containing sixty-four ATG8 genes. The expression of the majority of GhATG8 genes was induced by salt, drought, cold, and heat stresses, as revealed by RNA-seq and real-time PCR. Analysis of cis-elements in the promoters of GhATG8 genes revealed the predominant presence of responsive elements for plant hormones and abiotic stress, suggesting that GhATG8 genes might have significant functions in abiotic stress response. Furthermore, we additionally performed a gene interaction network analysis for the GhATG8 proteins. The salt stress resistance of cotton was reduced due to the downregulation of GhATG8f expression, resulting in decreased activity of CAT, SOD, and POD enzymes, as well as decreased fresh weight and proline accumulation. In summary, our research is the initial exploration of ATG8 gene components in cotton, providing a basis for future investigations into the regulatory mechanisms of ATG8 genes in autophagy and their response to abiotic stress.

Small particles, big effects: How nanoparticles can enhance plant growth in favorable and harsh conditions.

By 2050, the global population is projected to reach 9 billion, underscoring the imperative for innovative solutions to increase grain yield and enhance food security. Nanotechnology has emerged as a powerful tool, providing unique solutions to this challenge. Nanoparticles (NPs) can improve plant growth and nutrition under normal conditions through their high surface-to-volume ratio and unique physical and chemical properties. Moreover, they can be used to monitor crop health status and augment plant resilience against abiotic stresses (such as salinity, drought, heavy metals, and extreme temperatures) that endanger global agriculture. Application of NPs can enhance stress tolerance mechanisms in plants, minimizing potential yield losses and underscoring the potential of NPs to raise crop yield and quality. This review highlights the need for a comprehensive exploration of the environmental implications and safety of nanomaterials and provides valuable guidelines for researchers, policymakers, and agricultural practitioners. With thoughtful stewardship, nanotechnology holds immense promise in shaping environmentally sustainable agriculture amid escalating environmental challenges.

Combined transcriptomic and metabolomic analyses elucidate key salt-responsive biomarkers to regulate salt tolerance in cotton.

BACKGROUND: Cotton is an important industrial crop and a pioneer crop for saline-alkali land restoration. However, the molecular mechanism underlying the cotton response to salt is not completely understood. METHODS: Here, we used metabolome data and transcriptome data to analyze the salt tolerance regulatory network of cotton and metabolic biomarkers. RESULTS: In this study, cotton was stressed at 400 m M NaCl for 0 h, 3 h, 24 h and 48 h. NaCl interfered with cotton gene expression, altered metabolite contents and affected plant growth. Metabolome analysis showed that NaCl stress increased the contents of amino acids, sugars and ABA, decreased the amount of vitamin and terpenoids. K-means cluster analysis of differentially expressed genes showed that the continuously up-regulated genes were mainly enriched in metabolic pathways such as flavonoid biosynthesis and amino acid biosynthesis. CONCLUSION: The four metabolites of cysteine (Cys), ABA(Abscisic acid), turanose, and isopentenyladenine-7-N-glucoside (IP7G) were consistently up-regulated under salt stress, which may indicate that they are potential candidates for cotton under salt stress biomarkers. Combined transcriptome and metabolome analysis revealed accumulation of cysteine, ABA, isopentenyladenine-7-N-glucoside and turanose were important for salt tolerance in cotton mechanism. These results will provide some metabolic insights and key metabolite biomarkers for salt stress tolerance, which may help to understanding of the metabolite response to salt stress in cotton and develop a foundation for cotton to grow better in saline soil.

GhIMP10D, an inositol monophosphates family gene, enhances ascorbic acid and antioxidant enzyme activities to confer alkaline tolerance in Gossypium hirsutum L.

BACKGROUND: Inositol monophosphates (IMP) are key enzymes in the ascorbic acid (AsA) synthesis pathways, which play vital roles in regulating plant growth and development and stresses tolerance. To date, no comprehensive analysis of the expression profile of IMP genes and their functions under abiotic stress in cotton has been reported. RESULTS: In this study, the genetic characteristics, phylogenetic evolution, cis-acting elements and expression patterns of IMP gene family in cotton were systematically analyzed. A total of 28, 27, 13 and 13 IMP genes were identified in Gossypium hirsutum (G. hirsutum), Gossypium barbadense (G. barbadense), Gossypium arboreum (G. arboreum), and Gossypium raimondii (G. raimondii), respectively. Phylogenetic analysis showed that IMP family genes could cluster into 3 clades. Structure analysis of genes showed that GhIMP genes from the same subgroup had similar genetic structure and exon number. And most GhIMP family members contained hormone-related elements (abscisic acid response element, MeJA response element, gibberellin response element) and stress-related elements (low temperature response element, defense and stress response element, wound response element). After exogenous application of abscisic acid (ABA), some GhIMP genes containing ABA response elements positively responded to alkaline stress, indicating that ABA response elements played an important role in response to alkaline stress. qRT-PCR showed that most of GhIMP genes responded positively to alkaline stress, and GhIMP10D significantly upregulated under alkaline stress, with the highest up-regulated expression level. Virus-induced gene silencing (VIGS) experiment showed that compared with 156 plants, MDA content of pYL156:GhIMP10D plants increased significantly, while POD, SOD, chlorophyII and AsA content decreased significantly. CONCLUSIONS: This study provides a thorough overview of the IMP gene family and presents a new perspective on the evolution of this gene family. In particular, some IMP genes may be involved in alkaline stress tolerance regulation, and GhIMP10D showed high expression levels in leaves, stems and roots under alkaline stress, and preliminary functional verification of GhIMP10D gene suggested that it may regulate tolerance to alkaline stress by regulating the activity of antioxidant enzymes and the content of AsA. This study contributes to the subsequent broader discussion of the structure and alkaline resistance of IMP genes in cotton.

Analysis of the histidine kinase gene family and the role of GhHK8 in response to drought tolerance in cotton.

As an important member of the two-component system (TCS), histidine kinases (HKs) play important roles in various plant developmental processes and signal transduction in response to a wide range of biotic and abiotic stresses. So far, the HK gene family has not been investigated in Gossypium. In this study, a total of 177 HK gene family members were identified in cotton. They were further divided into seven groups, and the protein characteristics, genetic relationship, gene structure, chromosome location, collinearity, and cis-elements identification were comprehensively analyzed. Whole genome duplication (WGD) / segmental duplication may be the reason why the number of HK genes doubled in tetraploid Gossypium species. Expression analysis revealed that most cotton HK genes were mainly expressed in the reproductive organs and the fiber at initial stage. Gene expression analysis revealed that HK family genes are involved in cotton abiotic stress, especially drought stress and salt stress. In addition, gene interaction networks showed that HKs were involved in the regulation of cotton abiotic stress, especially drought stress. VIGS experiments have shown that GhHK8 is a negative regulatory factor in response to drought stress. Our systematic analysis provided insights into the characteristics of the HK genes in cotton and laid a foundation for further exploring their potential in drought stress resistance in cotton.

The resistance of the jujube (Ziziphus jujuba) to the devastating insect pest Apolygus lucorum (Hemiptera, Insecta) involves the jasmonic acid signaling pathway.

Apolygus lucorum (Hemiptera, Insecta), cosmopolitan true bug, is a major pest of the Chinese jujube (Ziziphus jujuba). To propose control measures of A. lucorum, we investigated the molecular mechanisms of resistance in two varieties of jujube (wild jujube and winter jujube) with different sensitivities to this pest. We monitored changes of two species of jujube in the transcriptome, jasmonic acid (JA) and salicylic acid (SA) content, and the expression of genes involved in signaling pathways. The preference of A. lucorum for jujube with exogenous SA and methyl jasmonate (MeJA) were also examined. The results showed that wild jujube leaves infested by A. lucorum showed stronger resistance and non-selectivity to A. lucorum than winter jujube. By comparing data from the A. lucorum infested plants with the control, A total of 438 and 796 differentially expressed genes (DEGs) were found in winter and wild jujube leaves, respectively. GO analysis revealed that biological process termed "plant-pathogen interactions", "plant hormone transduction" and "phenylpropanoid biosynthesis". Most of DEGs enriched in JA pathways were upregulated, while most DEGs of SA pathways were downregulated. A. lucorum increased the JA content but decreased the SA content in jujube. Consistently, the JA and SA contents in winter jujube were lower than those in wild jujube leaves. The key genes ZjFAD3, ZjLOX, ZjAOS, ZjAOC3 and ZjAOC4 involved in JA synthesis of jujube leaves were significantly up-regulated after A. lucorum infestation, especially the expression and up-regulation ratio of ZjFAD3, ZjLOX and ZjAOS in wild jujube were significantly higher than those in winter jujube. MeJA-treated jujube showed an obvious repellent effect on A. lucorum. Based on these findings, we conclude that A. lucorum infestation of jujube induced the JA pathway and suppressed the SA pathway. In jujube leaves the ZjFAD3, ZjLOX and ZjAOS played important roles in increasing of JA content in jujube leaves. Thus, JA played an important role in repelling and resisting against A. lucorum in jujube.

Secondary metabolite pathway of SDG (secoisolariciresinol) was observed to trigger ROS scavenging system in response to Ca2+ stress in cotton.

Ca2+ is an essential nutrient for plants and animals which plays an important role in plant signal transduction. Although the function and regulation of mechanism of Ca2+ in alleviating various biotic and abiotic stresses in plants have been studied deeply, the molecular mechanism to adapt high Ca2+ stress is still unclear in cotton. In this study, 103 cotton accessions were germinated under 200 mM CaCl2 stress, and two extremely Ca2+-resistant (Zhong 9807, R) and Ca2+-sensitive (CRI 50, S) genotypes were selected from 103 cotton accessions. The two accessions were then germinated for 5 days in 0 mM CaCl2 and 200 mM CaCl2 respectively, after which they were sampled for transcriptome sequencing. Morphological and physiological analyses suggested that PLR2 specifically expressed in R may enhance the ability of cotton to scavenge ROS by promoting the synthesis of SDG. In conclusion, this study proposed the adaptation mechanisms to response to the high Ca2+ stress in cotton which can contribute to improve the stress resistance of cotton.

Comprehensive genomic characterization of cotton cationic amino acid transporter genes reveals that GhCAT10D regulates salt tolerance.

BACKGROUND: The cationic amino acid transporters (CAT) play indispensable roles in maintaining metabolic functions, such as synthesis of proteins and nitric oxide (NO), biosynthesis of polyamine, and flow of amino acids, by mediating the bidirectional transport of cationic amino acids in plant cells. RESULTS: In this study, we performed a genome-wide and comprehensive study of 79 CAT genes in four species of cotton. Localization of genes revealed that CAT genes reside on the plasma membrane. Seventy-nine CAT genes were grouped into 7 subfamilies by phylogenetic analysis. Structure analysis of genes showed that CAT genes from the same subgroup have similar genetic structure and exon number. RNA-seq and real-time PCR indicated that the expression of most GhCAT genes were induced by salt, drought, cold and heat stresses. Cis-elements analysis of GhCAT promoters showed that the GhCAT genes promoters mainly contained plant hormones responsive elements and abiotic stress elements, which indicated that GhCAT genes may play key roles in response to abiotic stress. Moreover, we also conducted gene interaction network of the GhCAT proteins. Silencing GhCAT10D expression decreased the resistance of cotton to salt stress because of a decrease in the accumulation of NO and proline. CONCLUSION: Our results indicated that CAT genes might be related with salt tolerance in cotton and lay a foundation for further study on the regulation mechanism of CAT genes in cationic amino acids transporting and distribution responsing to abiotic stress.

Molecular characterization, expression and interaction of MAPK, MAPKK and MAPKKK genes in upland cotton.

Mitogen-activated protein kinase (MAPK) signaling cascades, consisting of three types of sequentially phosphorylated kinases (MAPKKK, MAPKK, and MAPK), play vital roles in various processes including plant development and stress response. In this study, 52 GhMAPKs, 23 GhMAPKKs, and 166 GhMAPKKKs were identified in upland cotton. Chromosomal locations, gene duplication and structure, motifs, cis-regulatory elements, and protein subcellular localization were further analyzed. With the identified MAPK cascade genes in G. arboretum and G. raimondii, a syntenic diagram of three cotton species was constructed. The interactions of seven GhMAPK cascade genes were investigated. Two complete signaling modules were defined: The GhMEKK24/GhMEKK31-GhMAPKK9-GhMAPK10 and GhMEKK3/GhMEKK24/GhMEKK31-GhMAPKK16-GhMAPK10/GhMAPK11 cascades. Moreover, interaction networks and the interaction pairs were combined with their expression patterns and demonstrated that the network mediated by the MAPK signaling cascade participates in abiotic stress signaling. Our research provides a foundation for studying the molecular mechanism of the MAPK signaling pathway under abiotic stress.

Molecular Traits and Functional Exploration of BES1 Gene Family in Plants.

The BES1 (BRI1-EMSSUPPRESSOR1) gene family is a unique class of transcription factors that play dynamic roles in the Brassinosteroids (BRs) signaling pathway. The published genome sequences of a large number of plants provide an opportunity to identify and perform a comprehensive functional study on the BES1 gene family for their potential roles in developmental processes and stress responses. A total of 135 BES1 genes in 27 plant species were recognized and characterized, which were divided into five well-conserved subfamilies. BES1 was not found in lower plants, such as Cyanophora paradoxa and Galdieria sulphuraria. The spatial expression profiles of BES1s in Arabidopsis, rice, and cotton, as well as their response to abiotic stresses, were analyzed. The overexpression of two rice BES1 genes, i.e., OsBES1-3 and OsBES1-5, promotes root growth under drought stress. The overexpression of GhBES1-4 from cotton enhanced the salt tolerance in Arabidopsis. Five protein interaction networks were constructed and numerous genes co-expressed with GhBES1-4 were characterized in transgenic Arabidopsis. BES1 may have evolved in the ancestors of the first land plants following its divergence from algae. Our results lay the foundation for understanding the complex mechanisms of BES1-mediated developmental processes and abiotic stress tolerance.

Temporal salt stress-induced transcriptome alterations and regulatory mechanisms revealed by PacBio long-reads RNA sequencing in Gossypium hirsutum.

BACKGROUND: Cotton (Gossypium hirsutum) is considered a fairly salt tolerant crop however, salinity can still cause significant economic losses by affecting the yield and deteriorating the fiber quality. We studied a salt-tolerant upland cotton cultivar under temporal salt stress to unfold the salt tolerance molecular mechanisms. Biochemical response to salt stress (400 mM) was measured at 0 h, 3 h, 12 h, 24 h and 48 h post stress intervals and single-molecule long-read sequencing technology from Pacific Biosciences (PacBio) combined with the unique molecular identifiers approach was used to identify differentially expressed genes (DEG). RESULTS: Antioxidant enzymes including, catalase (CAT), peroxidase (POD), superoxide dismutase (SOD) were found significantly induced under temporal salt stress, suggesting that reactive oxygen species scavenging antioxidant machinery is an essential component of salt tolerance mechanism in cotton. We identified a wealth of novel transcripts based on the PacBio long reads sequencing approach. Prolonged salt stress duration induces high number of DEGs. Significant numbers of DEGs were found under key terms related to stress pathways such as "response to oxidative stress", "response to salt stress", "response to water deprivation", "cation transport", "metal ion transport", "superoxide dismutase", and "reductase". Key DEGs related to hormone (abscisic acid, ethylene and jasmonic acid) biosynthesis, ion homeostasis (CBL-interacting serine/threonine-protein kinase genes, calcium-binding proteins, potassium transporter genes, potassium channel genes, sodium/hydrogen exchanger or antiporter genes), antioxidant activity (POD, SOD, CAT, glutathione reductase), transcription factors (myeloblastosis, WRKY, Apetala 2) and cell wall modification were found highly active in response to salt stress in cotton. Expression fold change of these DEGs showed both positive and negative responses, highlighting the complex nature of salt stress tolerance mechanisms in cotton. CONCLUSION: Collectively, this study provides a good insight into the regulatory mechanism under salt stress in cotton and lays the foundation for further improvement of salt stress tolerance.

Resequencing of cv CRI-12 family reveals haplotype block inheritance and recombination of agronomically important genes in artificial selection.

Although efforts have been taken to exploit diversity for yield and quality improvements, limited progress on using beneficial alleles in domesticated and undomesticated cotton varieties is limited. Given the complexity and limited amount of genomic information since the completion of four cotton genomes, characterizing significant variations and haplotype block inheritance under artificial selection has been challenging. Here we sequenced Gossypium hirsutum L. cv CRI-12 (the cotton variety with the largest acreage in China), its parental cultivars, and progeny cultivars, which were bred by the different institutes in China. In total, 3.3 million SNPs were identified and 118, 126 and 176 genes were remarkably correlated with Verticillium wilt, salinity and drought tolerance in CRI-12, respectively. Transcriptome-wide analyses of gene expression, and functional annotations, have provided support for the identification of genes tied to these tolerances. We totally discovered 58 116 haplotype blocks, among which 23 752 may be inherited and 1029 may be recombined under artificial selection. This survey of genetic diversity identified loci that may have been subject to artificial selection and documented the haplotype block inheritance and recombination, shedding light on the genetic mechanism of artificial selection and guiding breeding efforts for the genetic improvement of cotton.

Genome-wide characterization and expression profiling of the MAPKKK genes in Gossypium arboreum L.

Mitogen-activated protein kinase kinase kinases (MAPKKKs) are important components of MAPK cascades, which have different functions during developmental processes and stress responses. To date, there has been no systematic investigation of this gene family in the diploid cotton Gossypium arboreum L. In this study, a genome-wide survey was performed that identified 78 MAPKKK genes in G. arboreum. Phylogenetic analysis classified these genes into three subgroups: 14 belonged to ZIK, 20 to MEKK, and 44 to Raf. Chromosome location, phylogeny, and the conserved protein motifs of the MAPKKK gene family in G. arboreum were analyzed. The MAPKKK genes had a scattered genomic distribution across 13 chromosomes. The members in the same subfamily shared similar conserved motifs. The MAPKKK expression patterns were analyzed in mature leaves, stems, roots, and at different ovule developmental stages, as well as under salt and drought stresses. Transcriptome analysis showed that 76 MAPKKK genes had different transcript accumulation patterns in the tested tissues and 38 MAPKKK genes were differentially expressed in response to salt and drought stresses. These results lay the foundation for understanding the complex mechanisms behind MAPKKK-mediated developmental processes and abiotic stress-signaling transduction pathways in cotton.

Mechanisms and Functions of Long Non-Coding RNAs at Multiple Regulatory Levels.

Long non-coding (lnc) RNAs are non-coding RNAs longer than 200 nt. lncRNAs primarily interact with mRNA, DNA, protein, and miRNA and consequently regulate gene expression at the epigenetic, transcriptional, post-transcriptional, translational, and post-translational levels in a variety of ways. They play important roles in biological processes such as chromatin remodeling, transcriptional activation, transcriptional interference, RNA processing, and mRNA translation. lncRNAs have important functions in plant growth and development; biotic and abiotic stress responses; and in regulation of cell differentiation, the cell cycle, and the occurrence of many diseases in humans and animals. In this review, we summarize the functions and mechanisms of lncRNAs in plants, humans, and animals at different regulatory levels.

Identification, Characterization, and Expression Patterns of TCP Genes and microRNA319 in Cotton.

The TEOSINTE BRANCHED 1, CYCLOIDEA, and PROLIFERATING CELL FACTORS (TCP) gene family is a group of plant-specific transcription factors that have versatile functions in developmental processes and stress responses. In this study, a total of 73 TCP genes in upland cotton were identified and characterizated. Phylogenetic analysis classified them into three subgroups: 50 belonged to PCF, 16 to CIN, and 7 to CYC/TB1. GhTCP genes are randomly distributed in 22 of the 26 chromosomes in cotton. Expression patterns of GhTCPs were analyzed in 10 tissues, including different developmental stages of ovule and fiber, as well as under heat, salt, and drought stresses. Transcriptome analysis showed that 44 GhTCP genes exhibited varied transcript accumulation patterns in the tested tissues and 41 GhTCP genes were differentially expressed in response to heat, salt, and drought stresses. Furthermore, three GhTCP genes of the CIN clade were found to contain miR319-binding sites. An anti-correlation expression of GhTCP21 and GhTCP54 was analyzed with miR319 under salt and drought stress. Our results lay the foundation for understanding the complex mechanisms of GhTCP-mediated developmental processes and abiotic stress-signaling transduction pathways in cotton.

Genome-wide Identification and analysis of the stress-resistance function of the TPS (Trehalose-6-Phosphate Synthase) gene family in cotton.

BACKGROUND: Trehalose (a-D-glucopyranosyl a-D-glucopyranoside) is a nonreducing disaccharide and is widely distributed in bacteria, fungi, algae, plants and invertebrates. In the study, the identification of trehalose-6-phosphate synthase (TPS) genes stress-related in cotton, and the genetic structure analysis and molecular evolution analysis of TPSs were conducted with bioinformatics methods, which could lay a foundation for further research of TPS functions in cotton. RESULTS: The genome information of Gossypium raimondii (group D), G. arboreum L. (group A), and G. hirsutum L. (group AD) was used in the study. Fifty-three TPSs were identified comprising 15 genes in group D, 14 in group A, and 24 in group AD. Bioinformatics methods were used to analyze the genetic structure and molecular evolution of TPSs. Real-time PCR analysis was performed to investigate the expression patterns of gene family members. All TPS family members in cotton can be divided into two subfamilies: Class I and Class II. The similarity of the TPS sequence is high within the same species and close within their family relatives. The genetic structures of two TPS subfamily members are different, with more introns and a more complicated gene structure in Class I. There is a TPS domain(Glyco transf_20) at the N-terminal in all TPS family members and a TPP domain(Trehalose_PPase) at the C-terminal in all except GrTPS6, GhTPS4, and GhTPS9. All Class II members contain a UDP-forming domain. The responses to environmental stresses showed that stresses could induce the expression of TPSs but the expression patterns vary with different stresses. CONCLUSIONS: The distribution of TPSs varies with different species but is relatively uniform on chromosomes. Genetic structure varies with different gene members, and expression levels vary with different stresses and exhibit tissue specificity. The upregulated genes in upland cotton TM-1 is significantly more than that in G. raimondii and G. arboreum L. Shixiya 1.

The MAPKKK gene family in Gossypium raimondii: genome-wide identification, classification and expression analysis.

Mitogen-activated protein kinase (MAPK) cascades are conserved signal transduction pathways in all eukaryotic organisms. MAPKKKs (MAPK kinase kinases) operate at the top levels of these cascades. Recently, this family of genes has been systematically investigated in Arabidopsis, rice and maize, but has not yet been characterized in cotton. In this study, we identified 78 putative MAPKKK genes in the genome of the diploid cotton, Gossypium raimondii. They were classified into three subfamilies, of which 12 were ZIK, 22 were MEKK and 44 were Raf. The ZIK and MEKK genes displayed a scattered genomic distribution across 11 of the 13 chromosomes, whereas Raf genes were distributed across the entire genome. Their conserved patterns observed for introns and additional domains were consistent with the evolutionary relationships inferred from the phylogenetic analysis within subfamily. Transcriptome sequencing data were used to investigate their transcript profiles in mature leaves, 0 day and 3 days post-anthesis (DPA) ovules. Sixty MAPKKK genes were expressed, of which 41 were strongly expressed in mature leaves. Twelve MAPKKK genes were more highly expressed in 3-DPA ovules than in 0-DPA ovules. Our results provide a foundation for future evolutionary and functional characterizations of MAPKKK genes in cotton and probably other Gossypium plants.

Difference in miRNA expression profiles between two cotton cultivars with distinct salt sensitivity.

MicroRNAs (miRNAs) are a class of endogenous, non-coding small RNAs that play important roles in many developmental processes and stress responses in plants and animals. Cotton (Gossypium hirsutum L.) is considered a relatively salt-tolerant non-halophytic plant species. To study the role of miRNAs in salt adaptation, a salt-tolerant cotton cultivar SN-011 and a salt-sensitive cultivar LM-6 were used to detect differentially expressed miRNAs. Using miRNA microarray analysis and a computational approach, 17 cotton miRNAs belonging to eight families were identified. Although they are conserved, 12 of them showed a genotype-specific expression model in both the cultivars. Under salt stress treatment, miR156a/d/e, miR169, miR535a/b and miR827b were dramatically down-regulated in SN-011, while miR167a, miR397a/b and miR399a were up-regulated. Only miR159 was found to be down-regulated in LM-6 under salt stress. To gain insight into their functional significance, 26 target genes were predicted and their functional similarity was further analyzed. Quantitative real-time PCR showed that the expression of seven target genes showed a significant inverse correlation with corresponding miRNAs. These differentially expressed miRNAs can help in further study into the role of transcriptome homeostasis in the adaptation responses of cotton to salt.

Molecular traits and functional analysis of Rapid Alkalinization Factors (RALFs) in four Gossypium species.

Rapid Alkalinization Factors (RALFs) are plant-secreted, cysteine-rich polypeptides which are known to play essential roles in plant developmental processes and in several defense mechanisms. So far, RALF polypeptides have not been investigated in the Gossypium genus. In this study, 42, 38, 104 and 120 RALFs were identified from diploid G. arboreum and G. raimondi and tetraploid G. hirsutum and G. barbadense, respectively. These were further divided into four groups. Protein characteristics, sequence alignment, gene structure, conserved motifs, chromosomal location and cis-element identification were comprehensively analyzed. Whole genome duplication (WGD) /segmental duplication may be the reason why the number of RALF genes doubled in tetraploid Gossypium species. Expression patterns analysis showed that GhRALFs had different transcript accumulation patterns in the tested tissues and were differentially expressed in response to various abiotic stresses. Furthermore, GhRALF41-3 over-expressing (OE) plants showed reduction in root length and developed later with short stems and small rosettes than that of the wild type. The GhRALF14-8 and GhRALF27-8 OE plants, especially the latter, showed increase in seed abortion. Both transgenic Arabidopsis and VIGS cotton demonstrate that three GhRALFs are negative regulators in response to salt stress. Our systematic analyses provided insights into the characterization of RALF genes in Gossypium, which forms genetic basis for further exploration in their potential applications in cotton production.