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.

Genome-wide identification and mining elite allele variation of the Monoacylglycerol lipase (MAGL) gene family in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Monoacylglycerol lipase (MAGL) genes belong to the alpha/beta hydrolase superfamily, catalyze the terminal step of triglyceride (TAG) hydrolysis, converting monoacylglycerol (MAG) into free fatty acids and glycerol. RESULTS: In this study, 30 MAGL genes in upland cotton have been identified, which have been classified into eight subgroups. The duplication of GhMAGL genes in upland cotton was predominantly influenced by segmental duplication events, as revealed through synteny analysis. Furthermore, all GhMAGL genes were found to contain light-responsive elements. Through comprehensive association and haplotype analyses using resequencing data from 355 cotton accessions, GhMAGL3 and GhMAGL6 were detected as key genes related to lipid hydrolysis processes, suggesting a negative regulatory effect. CONCLUSIONS: In summary, MAGL has never been studied in upland cotton previously. This study provides the genetic mechanism foundation for the discover of new genes involved in lipid metabolism to improve cottonseed oil content, which will provide a strategic avenue for marker-assisted breeding aimed at incorporating desirable traits into cultivated cotton varieties.

The functions of laccase gene GhLAC15 in fiber colouration and development in brown-colored cotton.

The monotonicity of color type in naturally colored cottons (NCCs) has become the main limiting factor to their widespread use, simultaneously coexisting with poor fiber quality. The synchronous improvement of fiber quality and color become more urgent and crucial as the demand for sustainable development increases. The homologous gene of wild cotton Gossypium stocksii LAC15 in G. hirsutum, GhLAC15, was also dominantly expressed in the developing fibers of brown cotton XC20 from 5 DPA (day post anthesis) to 25 DPA, especially at the secondary cell wall thickening stage (20 DPA and 25 DPA). In XC20 plants with downregulated GhLAC15 (GhLAC15i), a remarkable reduction in proanthocyanidins (PAs) and lignin contents was observed. Some of the key genes in the phenylpropane and flavonoid biosynthesis pathway were down-regulated in GhLAC15i plants. Notably, the fiber length of GhLAC15i plants showed an obvious increase and the fiber color was lightened. Moreover, we found that the thickness of cotton fiber cell wall was decreased in GhLAC15i plants and the fiber surface became smoother compared to that of WT. Taken together, this study revealed that GhLAC15 played an important role in PAs and lignin biosynthesis in naturally colored cotton fibers. It might mediate fiber color and fiber quality by catalyzing PAs oxidation and lignin polymerization, ultimately regulating fiber colouration and development.

Identification and molecular evolution of the GLX genes in 21 plant species: a focus on the Gossypium hirsutum.

BACKGROUND: The glyoxalase system includes glyoxalase I (GLXI), glyoxalase II (GLXII) and glyoxalase III (GLXIII), which are responsible for methylglyoxal (MG) detoxification and involved in abiotic stress responses such as drought, salinity and heavy metal. RESULTS: In this study, a total of 620 GLX family genes were identified from 21 different plant species. The results of evolutionary analysis showed that GLX genes exist in all species from lower plants to higher plants, inferring that GLX genes might be important for plants, and GLXI and GLXII account for the majority. In addition, motif showed an expanding trend in the process of evolution. The analysis of cis-acting elements in 21 different plant species showed that the promoter region of the GLX genes were rich in phytohormones and biotic and abiotic stress-related elements, indicating that GLX genes can participate in a variety of life processes. In cotton, GLXs could be divided into two groups and most GLXIs distributed in group I, GLXIIs and GLXIIIs mainly belonged to group II, indicating that there are more similarities between GLXII and GLXIII in cotton evolution. The transcriptome data analysis and quantitative real-time PCR analysis (qRT-PCR) show that some members of GLX family would respond to high temperature treatment in G.hirsutum. The protein interaction network of GLXs in G.hirsutum implied that most members can participate in various life processes through protein interactions. CONCLUSIONS: The results elucidated the evolutionary history of GLX family genes in plants and lay the foundation for their functions analysis in cotton.

The Cotton Wall-Associated Kinase GhWAK7A Mediates Responses to Fungal Wilt Pathogens by Complexing with the Chitin Sensory Receptors.

Plant receptor-like kinases (RLKs) are important players in response to pathogen infections. Verticillium and Fusarium wilts, caused by Verticillium dahliae (Vd) and Fusarium oxysporum f. sp vasinfectum (Fov), respectively, are among the most devastating diseases in cotton (Gossypium spp). To understand the cotton response to these soil-borne fungal pathogens, we performed a genome-wide in silico characterization and functional screen of diverse RLKs for their involvement in cotton wilt diseases. We identified Gossypium hirsutum GhWAK7A, a wall-associated kinase, that positively regulates cotton response to both Vd and Fov infections. Chitin, the major constituent of the fungal cell wall, is perceived by lysin-motif-containing RLKs (LYKs/CERK1), leading to the activation of plant defense against fungal pathogens. A conserved chitin sensing and signaling system is present in cotton, including chitin-induced GhLYK5-GhCERK1 dimerization and phosphorylation, and contributes to cotton defense against Vd and Fov Importantly, GhWAK7A directly interacts with both GhLYK5 and GhCERK1 and promotes chitin-induced GhLYK5-GhCERK1 dimerization. GhWAK7A phosphorylates GhLYK5, which itself does not have kinase activity, but requires phosphorylation for its function. Consequently, GhWAK7A plays a crucial role in chitin-induced responses. Thus, our data reveal GhWAK7A as an important component in cotton response to fungal wilt pathogens by complexing with the chitin receptors.

GhN/AINV13 positively regulates cotton stress tolerance by interacting with the 14-3-3 protein.

Neutral/alkaline invertases (N/AINVs) are sucrose hydrolases with important roles in plants. In this study, 15, 15, 15, 29, and 30 N/AINVs were identified in the Gossypium species, G. raimondii, G. herbaceum, G. arboreum, G. hirsutum, and G. barbadense, respectively. Along with two previously discovered branches, α and ß, a new clade γ was first discovered in our study. Investigation of gene collinearity showed that whole-genome duplication (WGD) and polyploidization were responsible for the expansion of the N/AINV gene family in allopolyploid Gossypium. Moreover, expression patterns revealed that GhN/AINV3/13/17/23/24/28 from the ß clade is highly expressed during the period of fiber initiation. The invertase activity of GhN/AINV13 and GhN/AINV23 were confirmed by restoring defects of invertase-deficient yeast mutant SEY2102. Treatments of abiotic stress showed that most GhN/AINVs were induced in response to polyethylene glycol (PEG) or salt stress. A virus-induced gene-silencing (VIGS) experiment and yeast two-hybrid assay demonstrated that GhN/AINV13 may interact with their positive regulators Gh14-3-3 proteins and participate in the fiber initiation or stress tolerance of cotton. Our results provided fundamental information regarding N/AINVs and highlight their potential functions in cotton stress tolerance.

A type-2C protein phosphatase (GhDRP1) participates in cotton (Gossypium hirsutum) response to drought stress.

KEY MESSAGE: GhDRP1 acts as a negatively regulator to participate in response to drought stress possibly by modulating ABA signaling pathway and flavonoid biosynthesis pathway which affects stomata movement and thus water loss, ROS scavenging enzymes, and proline accumulation in cotton. Type-2C protein phosphatases (PP2C) may play important roles in plant stress signal transduction. Here, we show the evidence that a cotton PP2C protein GhDRP1 participates in plant response to drought stress. GhDRP1 gene encodes an active type-2C protein phosphatase (PP2C) and its expression is significantly induced in cotton by drought stress. Compared with wild type, the GhDRP1 overexpression (OE) transgenic cotton and Arabidopsis displayed reduced drought tolerance, whereas GhDRP1-silenced (RNAi) cotton showed enhanced drought tolerance. Under drought stress, malondialdehyde content was lower, whereas superoxide dismutase and peroxidase activities, proline content, stomata closure and relative water content were higher in GhDRP1 RNAi plants compared with those in wild type. In contrast, GhDRP1 OE plants showed the opposite phenotype under the same conditions. Expression levels of some stress-related and flavonoid biosynthesis-related genes were altered in GhDRP1 transgenic plants under drought stress. Additionally, GhDRP1 protein could interact with other proteins such as PYLs, SNF1-related protein kinase and GLK1-like protein. Collectively, these data suggest that GhDRP1 participates in plant response to drought stress possibly by modulating ABA signaling pathway and flavonoid biosynthesis pathway which affects stomata movement and thus water loss, ROS scavenging enzymes, and proline accumulation in cotton.

Selective fertilization with phosphite allows unhindered growth of cotton plants expressing the ptxD gene while suppressing weeds.

Weeds, which have been the bane of agriculture since the beginning of civilization, are managed manually, mechanically, and, more recently, by chemicals. However, chemical control options are rapidly shrinking due to the recent rise in the number of herbicide-resistant weeds in crop fields, with few alternatives on the horizon. Therefore, there is an urgent need for alternative weed suppression systems to sustain crop productivity while reducing our dependence on herbicides and tillage. Such a development will also allay some of the negative perceptions associated with the use of herbicide-resistance genes and heavy dependence on herbicides. Transgenic plants expressing the bacterial phosphite dehydrogenase (ptxD) gene gain an ability to convert phosphite (Phi) into orthophosphate [Pi, the metabolizable form of phosphorus (P)]. Such plants allow for a selective fertilization scheme, based on Phi as the sole source of P for the crop, while offering an effective alternative for suppressing weed growth. Here, we show that, when P is supplied in the form of Phi, ptxD-expressing cotton (Gossypium hirsutum L.) plants outcompete, in both artificial substrates and natural soils from agricultural fields, three different monocot and dicot weed species intentionally introduced in the experiments, as well as weeds naturally present in the tested soils. Importantly, the ptxD/Phi system proved highly efficacious in inhibiting the growth of glyphosate-resistant Palmer amaranth. With over 250 weed species resistant to currently available herbicides, ptxD-transgenic plants fertilized with Phi could provide an effective alternative to suppressing the growth of these weeds while providing adequate nutrition to the crop.

GhTBL34 Is Associated with Verticillium Wilt Resistance in Cotton.

Verticillium wilt (VW) is a typical fungal disease affecting the yield and quality of cotton. The Trichome Birefringence-Like protein (TBL) is an acetyltransferase involved in the acetylation process of cell wall polysaccharides. Up to now, there are no reports on whether the TBL gene is related to disease resistance in cotton. In this study, we cloned a cotton TBL34 gene located in the confidence interval of a major VW resistance quantitative trait loci and demonstrated its relationship with VW resistance in cotton. Analyzing the sequence variations in resistant and susceptible accessions detected two elite alleles GhTBL34-2 and GhTBL34-3, mainly presented in resistant cotton lines whose disease index was significantly lower than that of susceptible lines carrying the allele GhTBL34-1. Comparing the TBL34 protein sequences showed that two amino acid differences in the TBL (PMR5N) domain changed the susceptible allele GhTBL34-1 into the resistant allele GhTBL34-2 (GhTBL34-3). Expression analysis showed that the TBL34 was obviously up-regulated by infection of Verticillium dahliae and exogenous treatment of ethylene (ET), and salicylic acid (SA) and jasmonate (JA) in cotton. VIGS experiments demonstrated that silencing of TBL34 reduced VW resistance in cotton. We deduced that the TBL34 gene mediating acetylation of cell wall polysaccharides might be involved in the regulation of resistance to VW in cotton.

Disequilibrium evolution of the Fructose-1,6-bisphosphatase gene family leads to their functional biodiversity in Gossypium species.

BACKGROUND: Fructose-1,6-bisphosphatase (FBP) is a key enzyme in the plant sucrose synthesis pathway, in the Calvin cycle, and plays an important role in photosynthesis regulation in green plants. However, no systemic analysis of FBPs has been reported in Gossypium species. RESULTS: A total of 41 FBP genes from four Gossypium species were identified and analyzed. These FBP genes were sorted into two groups and seven subgroups. Results revealed that FBP family genes were under purifying selection pressure that rendered FBP family members as being conserved evolutionarily, and there was no tandem or fragmental DNA duplication in FBP family genes. Collinearity analysis revealed that a FBP gene was located in a translocated DNA fragment and the whole FBP gene family was under disequilibrium evolution that led to a faster evolutionary progress of the members in G. barbadense and in At subgenome than those in other Gossypium species and in the Dt subgenome, respectively, in this study. Through RNA-seq analyses and qRT-PCR verification, different FBP genes had diversified biological functions in cotton fiber development (two genes in 0 DPA and 1DPA ovules and four genes in 20-25 DPA fibers), in plant responses to Verticillium wilt onset (two genes) and to salt stress (eight genes). CONCLUSION: The FBP gene family displayed a disequilibrium evolution pattern in Gossypium species, which led to diversified functions affecting not only fiber development, but also responses to Verticillium wilt and salt stress. All of these findings provide the foundation for further study of the function of FBP genes in cotton fiber development and in environmental adaptability.

Genome-wide identification of MAPK cascade genes reveals the GhMAP3K14-GhMKK11-GhMPK31 pathway is involved in the drought response in cotton.

The mitogen-activated protein kinase (MAPK) cascade pathway, which has three components, MAP3Ks, MKKs and MPKs, is involved in diverse biological processes in plants. In the current study, MAPK cascade genes were identified in three cotton species, based on gene homology with Arabidopsis. Selection pressure analysis of MAPK cascade genes revealed that purifying selection occurred among the cotton species. Expression pattern analysis showed that some MAPK cascade genes differentially expressed under abiotic stresses and phytohormones treatments, and especially under drought stress. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) experiments showed extensive interactions between different MAPK cascade proteins. Virus-induced gene silencing (VIGS) assays showed that some MAPK cascade modules play important roles in the drought stress response, and the GhMAP3K14-GhMKK11-GhMPK31 signal pathway was demonstrated to regulate drought stress tolerance in cotton. This study provides new information on the function of MAPK cascade genes in the drought response, and will help direct molecular breeding for improved drought stress tolerance in cotton.

A cotton α1,3-/4-fucosyltransferase-encoding gene, FucT4, plays an important role in cell elongation and is significantly associated with fiber quality.

Fucosylation, one of the key posttranslational modifications, plays an important role in plants. It is involved in the development, signal transduction, reproduction, and disease resistance. α1,3-/4-Fucosyltransferase is responsible for transferring L-fucose from GDP-L-fucose to the N-glycan to exert fucosylational functions. However, the roles of the fucosyltransferase gene in cotton remain unknown. This study provided a comprehensive investigation of its possible functions. A genome-wide analysis identified four, four, eight, and eight FucT genes presented in the four sequenced cotton species, diploid Gossypium raimondii, G. arboreum, tetraploid G. hirsutum acc. TM-1, and G. barbadense cv. H7124, respectively. These FucTs were classified into two groups, with FucT4 homologs alone as a group. We isolated FucT4 in TM-1 and H7124, and named it GhFucT4 and GbFucT4, respectively. Quantitative RT-PCR and transcriptome data demonstrated that GhFucT4 had the highest expression levels in fibers among all GhFucT genes. Association studies and QTL co-localization supported the possible involvement of GhFucT4 in cotton fiber development. GhFucT4 and GbFucT4 shared high sequence identities, and FucT4 had higher expression in H7124 fiber tissues compared with TM-1. Furthermore, ectopic expression of FucT4 in transgenic Arabidopsis promoted root cell elongation, upregulated expression of genes related to cell wall loosening, and led to longer primary root. These results collectively indicate that FucT4 plays an important role in promoting cell elongation and modulating fiber development, which could be utilized to improve fiber quality traits in cotton breeding.

CYP722C from Gossypium arboreum catalyzes the conversion of carlactonoic acid to 5-deoxystrigol.

MAIN CONCLUSION: CYP722C from cotton, a homolog of the enzyme involved in orobanchol synthesis in cowpea and tomato, catalyzes the conversion of carlactonoic acid to 5-deoxystrigol. Strigolactones (SLs) are important phytohormones with roles in the regulation of plant growth and development. These compounds also function as signaling molecules in the rhizosphere by interacting with beneficial arbuscular mycorrhizal fungi and harmful root parasitic plants. Canonical SLs, such as 5-deoxystrigol (5DS), consist of a tricyclic lactone ring (ABC-ring) connected to a methylbutenolide (D-ring). Although it is known that 5DS biosynthesis begins with carlactonoic acid (CLA) derived from ß-carotene, the enzyme that catalyzes the conversion of CLA remains elusive. Recently, we identified cytochrome P450 (CYP) CYP722C as the enzyme that catalyzes direct conversion of CLA to orobanchol in cowpea and tomato (Wakabayashi et al., Sci Adv 5:eaax9067, 2019). Orobanchol has a different C-ring configuration from that of 5DS. The present study aimed to characterize the homologous gene, designated GaCYP722C, from cotton (Gossypium arboreum) to examine whether this gene is involved in 5DS biosynthesis. Expression of GaCYP722C was upregulated under phosphate starvation, which is an SL-producing condition. Recombinant GaCYP722C was expressed in a baculovirus-insect cell expression system and was found to catalyze the conversion of CLA to 5DS but not to 4-deoxyorobanchol. These results strongly suggest that GaCYP722C from cotton is a 5DS synthase and that CYP722C is the crucial CYP subfamily involved in the generation of canonical SLs, irrespective of the different C-ring configurations.

Mutation of key amino acids in the polygalacturonase-inhibiting proteins CkPGIP1 and GhPGIP1 improves resistance to Verticillium wilt in cotton.

Verticillium wilt, one of the most devastating diseases of cotton (Gossypium hirsutum), causes severe yield and quality losses. Given the effectiveness of plant polygalacturonase-inhibiting proteins (PGIPs) in reducing fungal polygalacturonase (PG) activity, it is necessary to uncover the key functional amino acids to enhance cotton resistance to Verticillium dahliae. To identify novel antifungal proteins, the selectivity of key amino acids was investigated by screening against a panel of relevant PG-binding residues. Based on the obtained results, homologous models of the mutants were established. The docking models showed that hydrogen bonds and structural changes in the convex face in the conserved portion of leucine-rich repeats (LRRs) may be essential for enhanced recognition of PG. Additionally, we successfully constructed Cynanchum komarovii PGIP1 (CkPGIP1) mutants Asp176Val, Pro249Gln, and Asp176Val/Pro249Gln and G. hirsutum PGIP1 (GhPGIP1) mutants Glu169Val, Phe242Gln, and Glu169Val/Phe242Gln with site-directed mutagenesis. The proteins of interest can effectively inhibit VdPG1 activity and V. dahliae mycelial growth in a dose-dependent manner. Importantly, mutants that overproduced PGIP in Arabidopsis and cotton showed enhanced resistance to V. dahliae, with reduced Verticillium-associated chlorosis and wilting. Furthermore, the lignin content was measured in mutant-overexpressing plants, and the results showed enhanced lignification of the xylem, which blocked the spread of V. dahliae. Thus, using site-directed mutagenesis assays, we showed that mutations in CkPGIP1 and GhPGIP1 give rise to PGIP versatility, which allows evolving recognition specificities for PG and is required to promote Verticillium resistance in cotton by restricting the growth of invasive fungal pathogens.

Insecticidal fern protein Tma12 is possibly a lytic polysaccharide monooxygenase.

MAIN CONCLUSION: Amino acid sequence and crystal structure analyses of Tma12, an insecticidal protein isolated from the fern Tectaria macrodonta, identify it as a carbohydrate-binding protein belonging to the AA10 family of lytic polysaccharide monooxygenases, and provide the first evidence of AA10 proteins in plants. Tma12, isolated from the fern Tectaria macrodonta, is a next-generation insecticidal protein. Transgenic cotton expressing Tma12 exhibits resistance against whitefly and viral diseases. Beside its insecticidal property, the structure and function of Tma12 are unknown. This limits understanding of the insecticidal mechanism of the protein and targeted improvement in its efficacy. Here we report the amino acid sequence analysis and the crystal structure of Tma12, suggesting that it is possibly a lytic polysaccharide monooxygenase (LPMO) of the AA10 family. Amino acid sequence of Tma12 shows 45% identity with a cellulolytic LPMO of Streptomyces coelicolor. The crystal structure of Tma12, obtained at 2.2 Å resolution, possesses all the major structural characteristics of AA10 LPMOs. A H2O2-based enzymatic assay also supports this finding. It is the first report of the occurrence of LPMO-like protein in a plant. The two facts that Tma12 possesses insecticidal activity and shows structural similarity with LPMOs collectively advocate exploration of microbial LPMOs for insecticidal potential.

GhCPK33 Negatively Regulates Defense against Verticillium dahliae by Phosphorylating GhOPR3.

Verticillium wilt, caused by the soil-borne fungus Verticillium dahliae, is a destructive vascular disease in plants. Approximately 200 dicotyledonous plant species in temperate and subtropical regions are susceptible to this notorious pathogen. Previous studies showed that jasmonic acid (JA) plays a crucial role in plant-V. dahliae interactions. V. dahliae infection generally induces significant JA accumulation in local and distal tissues of the plant. Although JA biosynthesis and the associated enzymes have been studied intensively, the precise mechanism regulating JA biosynthesis upon V. dahliae infection remains unknown. Here, we identified the calcium-dependent protein kinase GhCPK33 from upland cotton (Gossypium hirsutum) as a negative regulator of resistance to V. dahliae that directly manipulates JA biosynthesis. Knockdown of GhCPK33 by Agrobacterium tumefaciens-mediated virus-induced gene silencing constitutively activated JA biosynthesis and JA-mediated defense responses and enhanced resistance to V dahliae Further analysis revealed that GhCPK33 interacts with 12-oxophytodienoate reductase3 (GhOPR3) in peroxisomes. GhCPK33 phosphorylates GhOPR3 at threonine-246, leading to decreased stability of GhOPR3, which consequently limits JA biosynthesis. We propose that GhCPK33 is a potential molecular target for improving resistance to Verticillium wilt disease in cotton.

Laccase GhLac1 Modulates Broad-Spectrum Biotic Stress Tolerance via Manipulating Phenylpropanoid Pathway and Jasmonic Acid Synthesis.

Plants are constantly challenged by a multitude of pathogens and pests, which causes massive yield and quality losses annually. A promising approach to reduce such losses is to enhance the immune system of plants through genetic engineering. Previous work has shown that laccases (p-diphenol:dioxygen oxidoreductase, EC 1.10.3.2) function as lignin polymerization enzymes. Here we demonstrate that transgenic manipulation of the expression of the laccase gene GhLac1 in cotton (Gossypium hirsutum) can confer an enhanced defense response to both pathogens and pests. Overexpression of GhLac1 leads to increased lignification, associated with increased tolerance to the fungal pathogen Verticillium dahliae and to the insect pests cotton bollworm (Helicoverpa armigera) and cotton aphid (Aphis gosypii). Suppression of GhLac1 expression leads to a redirection of metabolic flux in the phenylpropanoid pathway, causing the accumulation of JA and secondary metabolites that confer resistance to V. dahliae and cotton bollworm; it also leads to increased susceptibility to cotton aphid. Plant laccases therefore provide a new molecular tool to engineer pest and pathogen resistance in crops.

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.

Comparative physiological analysis in the tolerance to salinity and drought individual and combination in two cotton genotypes with contrasting salt tolerance.

Soil salinity and drought are the two most common and frequently co-occurring abiotic stresses limiting cotton growth and productivity. However, physiological mechanisms of tolerance to such condition remain elusive. Greenhouse pot experiments were performed to study genotypic differences in response to single drought (4% soil moisture; D) and salinity (200 mM NaCl; S) stress and combined stresses (D + S) using two cotton genotypes Zhongmian 23 (salt-tolerant) and Zhongmian 41 (salt-sensitive). Our results showed that drought and salinity stresses, alone or in combination, caused significant reduction in plant growth, chlorophyll content and photosynthesis in the two cotton genotypes, with the largest impact visible under combined stress. Interestingly, Zhongmian 23 was more tolerant than Zhongmian 41 under the three stresses and displayed higher plant dry weight, photosynthesis and antioxidant enzymes activities such as superoxide dismutase (SOD), peroxidase (POD) catalase (CAT) and ascorbate peroxidase (APX) activities compared to control, while those parameters were significantly decreased in salt-stresses Zhongmian 41 compared to control. Moreover, Na+ /K+ -ATPase activity was more enhanced in Zhongmian 23 than in Zhongmian 41 under salinity stress. However, under single drought stress and D + S stress no significant differences were observed between the two genotypes. No significant differences were detected in Ca2+ /Mg2+ -ATPase activity in Zhongmian 41, while in Zhongmian 23 it was increased under salinity stress. Furthermore, Zhongmian 23 accumulated more soluble sugar, glycine-betaine and K+ , but less Na+ under the three stresses compared with Zhongmian 41. Obvious changes in leaf and root tips cell ultrastructure was observed in the two cotton genotypes. However, Zhongmian 23 was less affected than Zhongmian 41 especially under salinity stress. These results give a novel insight into the mechanisms of single and combined effects of drought and salinity stresses on cotton genotypes.

The role of gibberellin synthase gene GhGA2ox1 in upland cotton (Gossypium hirsutum L.) responses to drought and salt stress.

Gibberellins (GAs) is one kind of important endogenous hormone in plants that regulates vegetative and reproductive growth of plants. GA2ox is a class of oxidase that plays a regulatory role in the third stage of GAs synthesis. In this paper, we cloned the GhGA2ox1 gene from an upland cotton (Gossypium hirsutum L. var. CCRI49). The results showed that the CDS of GhGA2ox1 is 996 bp, which encode 331 amino acids, which has high homology with GhGA2ox2 and NtGA2ox1. The quantitative real-time PCR showed that under the conditions of salt and drought stress, the expression of GhGA2ox1 had a higher upregulation in root, stem, and leaf of transgenic plant, compared with non-transgenic plant. Cotton plant that overexpressed the GhGA2ox1 gene showed higher drought and salt tolerance than non-transgenic cotton plant, and these results were supported by data of higher free proline, chlorophyll, and relative water content in transgenic plant compared with control plant. The expression level of antiretroviral genes, including GhEREB2, GhDREB1, GhWRKY5, and GhP5CS, was upregulated to varying degrees in transgenic plant. The above results indicate that overexpressed GhGA2ox1 gene can increases the tolerance to respond to drought and salt stress in upland cotton.