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

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

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

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

Multi-omics-driven advances in the understanding of triacylglycerol biosynthesis in oil seeds.

Vegetable oils are rich sources of polyunsaturated fatty acids and energy as well as valuable sources of human food, animal feed, and bioenergy. Triacylglycerols, which are comprised of three fatty acids attached to a glycerol backbone, are the main component of vegetable oils. Here, we review the development and application of multiple-level omics in major oilseeds and emphasize the progress in the analysis of the biological roles of key genes underlying seed oil content and quality in major oilseeds. Finally, we discuss future research directions in functional genomics research based on current omics and oil metabolic engineering strategies that aim to enhance seed oil content and quality, and specific fatty acids components according to either human health needs or industrial requirements.

GhSBI1, a CUP-SHAPED COTYLEDON 2 homologue, modulates branch internode elongation in cotton.

Branch length is an important plant architecture trait in cotton (Gossypium) breeding. Development of cultivars with short branch has been proposed as a main object to enhance cotton yield potential, because they are suitable for high planting density. Here, we report the molecular cloning and characterization of a semi-dominant quantitative trait locus, Short Branch Internode 1(GhSBI1), which encodes a NAC transcription factor homologous to CUP-SHAPED COTYLEDON 2 (CUC2) and is regulated by microRNA ghr-miR164. We demonstrate that a point mutation found in sbi1 mutants perturbs ghr-miR164-directed regulation of GhSBI1, resulting in an increased expression level of GhSBI1. The sbi1 mutant was sensitive to exogenous gibberellic acid (GA) treatments. Overexpression of GhSBI1 inhibited branch internode elongation and led to the decreased levels of bioactive GAs. In addition, gene knockout analysis showed that GhSBI1 is required for the maintenance of the boundaries of multiple tissues in cotton. Transcriptome analysis revealed that overexpression of GhSBI1 affects the expression of plant hormone signalling-, axillary meristems initiation-, and abiotic stress response-related genes. GhSBI1 interacted with GAIs, the DELLA repressors of GA signalling. GhSBI1 represses expression of GA signalling- and cell elongation-related genes by directly targeting their promoters. Our work thus provides new insights into the molecular mechanisms for branch length and paves the way for the development of elite cultivars with suitable plant architecture in cotton.

Changes in carbohydrate distribution in cotton photosynthetic organs and increase in boll weight reduce yield loss under high temperature.

Yield of cotton (Gossypium hirsutum) does not always fall with high temperature (HT) even though this induces significant reductions in fruit retention. To investigate the underlying mechanisms, a greenhouse experiment was conducted with two temperature regimes [control treatment, 28 °C; high temperature (HT), 34 °C] for 7 d. Results showed HT did not significantly influence cotton yield, but reduced boll number and increased boll weight. The 13C distribution ratio of the leaf subtending the cotton boll (LSCB) decreased while that of the cotton boll increased under HT. Transcriptomic and proteomic analyses of the LSCB revealed up-regulated genes involved in cytokinin and jasmonic acid synthesis, as well as SWEET15 (GH_D01G0218), which positively regulated photosynthesis and transport photosynthate, ultimately leading to increased boll weight. After 7 d recovery from HT, the 13C distribution ratio of the LSCB increased while that of the cotton boll decreased. However, boll weight still increased, which was related to increased amylase and sucrose phosphate synthase activities and up-regulated sucrose transport genes in the main-stem leaf and capsule wall. Thus, both accelerated sucrose synthesis and transport in the LSCB under HT and increased sucrose supply ability of the main-stem leaf and capsule wall after recovery from HT contributed to an increased boll weight, which finally maintained cotton yield.

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

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

Leaf anatomical alterations reduce cotton's mesophyll conductance under dynamic drought stress conditions.

Drought stress significantly affects cotton's net photosynthetic rate (A) by restraining stomatal (gs ) and mesophyll conductance (gm ) as well as perturbing its biochemical process, resulting in yield reductions. Despite the significant progress in dissecting effects of drought on photosynthesis, the variability observed in cotton's gm , and the mechanisms contributing to that variability under dynamic drought stress conditions are poorly understood. For that reason, a controlled-environment experiment with two cotton genotypes (Dexiamian 1, Yuzaomian 9110), three water levels (soil relative water content: control [75 ± 5]%, moderate drought [60 ± 5]%, severe drought [45 ± 5]%), and two drought durations (10 and 31 days) were conducted. The results indicated that the cotton boll biomass was significantly decreased under 10-day severe drought and 31-day moderate and severe drought. Decreases in gs were later accompanied by decreases in gm and further combined with reductions in electron transport rate, as drought stress progressed in duration and severity, ultimately resulting in significant reductions in A of subtending leaf. Stomatal and mesophyll conductance constraints were the primary factors limiting photosynthesis, while biochemical constraints decreased, as drought stress progressed. Considering gm , its decline was ascribed to increases in the diffusion resistance of CO2 through cytoplasm (rcyt ), under short- or long-term drought, as well as to increases in leaf dry mass (LMA), and decreases in the chloroplast surface area exposed to intercellular air space (Sc /S), under long-term drought. It was concluded that A could be enhanced, under dynamic drought stress conditions, by increasing gm through increasing Sc /S and reducing LMA and rcyt .

Lint percentage and boll weight QTLs in three excellent upland cotton (Gossypium hirsutum): ZR014121, CCRI60, and EZ60.

BACKGROUND: Upland cotton (Gossypium hirsutum L.) is the most economically important species in the cotton genus (Gossypium spp.). Enhancing the cotton yield is a major goal in cotton breeding programs. Lint percentage (LP) and boll weight (BW) are the two most important components of cotton lint yield. The identification of stable and effective quantitative trait loci (QTLs) will aid the molecular breeding of cotton cultivars with high yield. RESULTS: Genotyping by target sequencing (GBTS) and genome-wide association study (GWAS) with 3VmrMLM were used to identify LP and BW related QTLs from two recombinant inbred line (RIL) populations derived from high lint yield and fiber quality lines (ZR014121, CCRI60 and EZ60). The average call rate of a single locus was 94.35%, and the average call rate of an individual was 92.10% in GBTS. A total of 100 QTLs were identified; 22 of them were overlapping with the reported QTLs, and 78 were novel QTLs. Of the 100 QTLs, 51 QTLs were for LP, and they explained 0.29-9.96% of the phenotypic variation; 49 QTLs were for BW, and they explained 0.41-6.31% of the phenotypic variation. One QTL (qBW-E-A10-1, qBW-C-A10-1) was identified in both populations. Six key QTLs were identified in multiple-environments; three were for LP, and three were for BW. A total of 108 candidate genes were identified in the regions of the six key QTLs. Several candidate genes were positively related to the developments of LP and BW, such as genes involved in gene transcription, protein synthesis, calcium signaling, carbon metabolism, and biosynthesis of secondary metabolites. Seven major candidate genes were predicted to form a co-expression network. Six significantly highly expressed candidate genes of the six QTLs after anthesis were the key genes regulating LP and BW and affecting cotton yield formation. CONCLUSIONS: A total of 100 stable QTLs for LP and BW in upland cotton were identified in this study; these QTLs could be used in cotton molecular breeding programs. Putative candidate genes of the six key QTLs were identified; this result provided clues for future studies on the mechanisms of LP and BW developments.

Comparative phosphoproteomic analysis reveals that phosphorylation of sucrose synthase GhSUS2 by Ca2+-dependent protein kinases GhCPK84/93 affects cotton fiber development.

Cotton fiber elongation is a critical growth phase that affects final fiber length. Morphological analysis indicated an asynchronous fiber elongation pattern between two cotton varieties, J7-1 and J14-1. Through phosphoproteomic analysis, a total of 89 differentially-phosphorylated proteins (DPPs) were identified in elongating fibers between J7-1 and J14-1. Gene ontology (GO) analysis showed that these DPPs were mainly enriched in sucrose synthase activity, transferase activity, and UDP-glycosyltransferase activity. In J14-1, the phosphorylation level of GhSUS2, a key sucrose synthase in the sucrose metabolism pathway, was significantly higher than that in J7-1. We further revealed that GhSUS2 positively regulates fiber elongation, and GhSUS2-silenced transgenic cotton displayed the phenotype of 'short fibers' compared with the controls. During fiber development, the residue Ser11 in the GhSUS2 protein is phosphorylated by the Ca2+-dependent protein kinases GhCPK84 and GhCPK93. Phosphorylated GhSUS2 is localized in the cytoplasm, whereas unphosphorylated GhSUS2 is localized in the plasma membrane. Moreover, abscisic acid (ABA) could promote the transcription and translation of GhCPK84 and GhCPK93, thereby enhancing the phosphorylation of GhSUS2 to impede fiber elongation. Thus, our data demonstrates that GhSUS2 plays a positive role in fiber development, but its phosphorylation by GhCPK84 and GhCPK93 hinders fiber elongation of cotton.

Dose effects of restorer gene modulate pollen fertility in cotton CMS-D2 restorer lines via auxin signaling and flavonoid biosynthesis.

KEY MESSAGE: Dose effects of Rf1 gene regulated retrieval mechanism of pollen fertility for CMS-D2 cotton. Cytoplasmic male sterility conditioned by Gossypium harknessii cytoplasm (CMS-D2) is an economical pollination control system for producing hybrid cotton seeds compared to artificial and chemical emasculation methods. However, the unstable restoring ability of restorer lines is a main barrier in the large-scale application of "three-line" hybrid cotton in China. Our phenotypic investigation determined that the homozygous Rf1Rf1 allelic genotype had a stronger ability to generate fertile pollen than the heterozygous Rf1rf1 allelic genotype. To decipher the genetic mechanisms that control the differential levels of pollen fertility, an integrated metabolomic and transcriptomic analysis was performed at two environments using pollen grains of four cotton genotypes differing in Rf1 alleles or cytoplasm. Totally 5,391 differential metabolite features were detected, and 369 specific differential metabolites (DMs) were identified between homozygous and heterozygous Rf1 allelic genotypes with CMS-D2 cytoplasm. In addition, transcriptome analysis identified 2,490 differentially expressed genes (DEGs) and 96 unique hub DEGs with dynamic regulation in this comparative combination. Further integrated analyses revealed that several key DEGs and DMs involved in indole biosynthesis, flavonoid biosynthesis, and sugar metabolism had strong network linkage with fertility restoration. In vitro application of auxin analogue NAA and inhibitor Auxinole confirmed that over-activated auxin signaling might inhibit pollen development, whereas suppressing auxin signaling partially promoted pollen development in CMS-D2 cotton. Our results provide new insight into how the dosage effects of the Rf1 gene regulate pollen fertility of CMS-D2 cotton.

Sequence Characteristics and Expression Analysis of the Gene Encoding Sedoheptulose-1,7-Bisphosphatase, an Important Calvin Cycle Enzyme in Upland Cotton (Gossypium hirsutum L.).

Sedoheptulose-1,7-bisphosphatase (SBPase, EC 3.1.3.37) is a key enzyme in the plant Calvin cycle and one of the main rate-limiting enzymes in the plant photosynthesis pathway. Many studies have demonstrated that the SBPase gene plays an important role in plant photosynthetic efficiency, yield, and stress responses; however, few studies have been conducted on the function and expression of the GhSBPase gene in upland cotton. In this study, our results showed that the coding sequence (CDS) of GhSBPase gene was 1182 bp, encoding a protein with 393 amino acids. The GhSBPase protein had adenosine monophosphate (AMP) binding site and a FIG (FBPase/IMPase/glpX) domain, and had six Cys residues and a CGGT(A/Q)C motif that were involved in redox regulation in plants. Evolutionarily, the GhSBPase protein clustered into the dicotyledon subgroup and was most closely related to the tomato SlSBPase protein. Western-blot analysis further indicated that the GhSBPase gene was indeed the gene encoding the SBPase protein in upland cotton. The GhSBPase protein was localized in chloroplast, which was consistent with its function as a key enzyme in photosynthesis. The GhSBPase gene was specifically highly expressed in leaves, and its expression level was significantly lower in a yellow-green leaf mutant than in the wild type. Moreover, the GhSBPase expression was in response to drought, salt, high- and low-temperature stress, and exhibits different expression patterns. The GhSBPase promoter had the cis-acting elements in response to abiotic stress, phytohormone, and light. In addition, the GhSBPase expression was positively correlated with the chlorophyll fluorescence parameters, suggesting that changes in the expression of the GhSBPase had potential applicability in breeding for enhanced cotton photosynthetic efficiency. These results will help to understand the function of the GhSBPase gene in photosynthesis and the adaptability of plants to external stress and provide important gene information for the high-yield breeding of crops in the future.

Comparative Transcriptome Profiling of CMS-D2 and CMS-D8 Systems Characterizes Fertility Restoration Genes Network in Upland Cotton.

Resolving the genetic basis of fertility restoration for cytoplasmic male sterility (CMS) can improve the efficiency of three-line hybrid breeding. However, the genetic determinants of male fertility restoration in cotton are still largely unknown. This study comprehensively compared the full-length transcripts of CMS-D2 and CMS-D8 systems to identify potential genes linked with fertility restorer genes Rf1 or Rf2. Target comparative analysis revealed a higher percentage of differential genes in each restorer line as compared to their corresponding sterile and maintainer lines. An array of genes with specific expression in the restorer line of CMS-D2 had functional annotations related to floral development and pathway enrichments in various secondary metabolites, while specifically expressed genes in the CMS-D8 restorer line showed functional annotations related to anther development and pathway enrichment in the biosynthesis of secondary metabolites. Further analysis identified potentially key genes located in the target region of fertility restorer genes Rf1 or Rf2. In particular, Ghir_D05G032450 can be the candidate gene related to restorer gene Rf1, and Ghir_D05G035690 can be the candidate gene associated with restorer gene Rf2. Further gene expression validation with qRT-PCR confirmed the accuracy of our results. Our findings provide useful insights into decoding the potential regulatory network that retrieves pollen fertility in cotton and will help to further reveal the differences in the genetic basis of fertility restoration for two CMS systems.

Comparison of Mitochondrial Genomes between a Cytoplasmic Male-Sterile Line and Its Restorer Line for Identifying Candidate CMS Genes in Gossypium hirsutum.

As the core of heterosis utilization, cytoplasmic male sterility (CMS) has been widely used in hybrid seed production. Previous studies have shown that CMS is always closely related to the altered programming of mitochondrial genes. To explore candidate CMS genes in cotton (Gossypium hirsutum), sequencing and de novo assembly were performed on the mitochondrial genome of the G. hirsutum CMS line SI3A, with G. harknessii CMS-D2 cytoplasm, and the corresponding G. hirsutum restorer line 0-613-2R. Remarkable variations in genome structure and gene transcripts were detected. The mitochondrial genome of SI3A has three circle molecules, including one main circle and two sub-circles, while 0-613-2R only has one. RNA-seq and RT-qPCR analysis proved that orf606a and orf109a, which have a chimeric structure and transmembrane domain, were highly expressed in abortive anthers of SI3A. In addition, comparative analysis of RNA-seq and full-length transcripts revealed the complex I gene nad4 to be expressed at a lower level in SI3A than in its restorer and that it featured an intron retention splicing pattern. These two novel chimeric ORFs and nad4 are potential candidates that confer CMS character in SI3A. This study provides new insight into the molecular basis of the nuclear-cytoplasmic interaction mechanism, and that putative CMS genes might be important sources for future precise design cross-breeding of cotton.

Identification and Functional Characterization of Acyl-ACP Thioesterases B (GhFatBs) Responsible for Palmitic Acid Accumulation in Cotton Seeds.

In spite of increasing use in the food industry, high relative levels of palmitic acid (C16:0) in cottonseed oil imposes harmful effects on human health when overconsumed in the diet. The limited understanding of the mechanism in controlling fatty acid composition has become a significant obstacle for breeding novel cotton varieties with high-quality oil. Fatty acyl-acyl carrier protein (ACP) thioesterase B (FatBs) are a group of enzymes which prefer to hydrolyze the thioester bond from saturated acyl-ACPs, thus playing key roles in controlling the accumulation of saturated fatty acids. However, FatB members and their roles in cotton are largely unknown. In this study, a genome-wide characterization of FatB members was performed in allotetraploid upland cotton, aiming to explore the GhFatBs responsible for high accumulations of C16:0 in cotton seeds. A total of 14 GhFatB genes with uneven distribution on chromosomes were identified from an upland cotton genome and grouped into seven subfamilies through phylogenetic analysis. The six key amino acid residues (Ala, Trys, Ile, Met, Arg and Try) responsible for substrate preference were identified in the N-terminal acyl binding pocket of GhFatBs. RNA-seq and qRT-PCR analysis revealed that the expression profiles of GhFatB genes varied in multiple cotton tissues, with eight GhFatBs (GhA/D-FatB3, GhA/D-FatB4, GhA/D-FatB5, and GhA/D-FatB7) having high expression levels in developing seeds. In particular, expression patterns of GhA-FatB3 and GhD-FatB4 were positively correlated with the dynamic accumulation of C16:0 during cotton seed development. Furthermore, heterologous overexpression assay of either GhA-FatB3 or GhD-FatB4 demonstrated that these two GhFatBs had a high substrate preference to 16:0-ACP, thus contributing greatly to the enrichment of palmitic acid in the tested tissues. Taken together, these findings increase our understanding on fatty acid accumulation and regulation mechanisms in plant seeds. GhFatBs, especially GhA-FatB3 and GhD-FatB4, could be molecular targets for genetic modification to reduce palmitic acid content or to optimize fatty acid profiles in cotton and other oil crops required for the sustainable production of healthy edible oil.

GhKNL1 controls fiber elongation and secondary cell wall synthesis by repressing its downstream genes in cotton (Gossypium hirsutum).

Cotton which produces natural fiber materials for the textile industry is one of the most important crops in the world. Class II KNOX proteins are often considered as transcription factors in regulating plant secondary cell wall (SCW) formation. However, the molecular mechanism of the KNOX transcription factor-regulated SCW synthesis in plants (especially in cotton) remains unclear in details so far. In this study, we show a cotton class II KNOX protein (GhKNL1) as a transcription repressor functioning in fiber development. The GhKNL1-silenced transgenic cotton produced longer fibers with thicker SCWs, whereas GhKNL1 dominant repression transgenic lines displayed the opposite fiber phenotype, compared with controls. Further experiments revealed that GhKNL1 could directly bind to promoters of GhCesA4-2/4-4/8-2 and GhMYB46 for modulating cellulose synthesis during fiber SCW development in cotton. On the other hand, GhKNL1 could also suppress expressions of GhEXPA2D/4A-1/4D-1/13A through binding to their promoters for regulating fiber elongation of cotton. Taken together, these data revealed GhKNL1 functions in fiber elongation and SCW formation by directly repressing expressions of its target genes related to cell elongation and cellulose synthesis. Thus, our data provide an effective clue for potentially improving fiber quality by genetic manipulation of GhKNL1 in cotton breeding.

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.

Molecular cloning and characterization of GhERF105, a gene contributing to the regulation of gland formation in upland cotton (Gossypium hirsutum L.).

BACKGROUND: Gossypium hirsutum L. (cotton) is one of the most economically important crops in the world due to its significant source of fiber, feed, foodstuff, oil and biofuel products. However, the utilization of cottonseed was limited due to the presence of small and darkly pigmented glands that contain large amounts of gossypol, which is toxic to human beings and non-ruminant animals. To date, some progress has been made in the pigment gland formation, but the underlying molecular mechanism of its formation was still unclear. RESULTS: In this study, we identified an AP2/ERF transcription factor named GhERF105 (GH_A12G2166), which was involved in the regulation of gland pigmentation by the comparative transcriptome analysis of the leaf of glanded and glandless plants. It encoded an ERF protein containing a converved AP2 domain which was localized in the nucleus with transcriptional activity, and showed the high expression in glanded cotton accessions that contained much gossypol. Virus-induced gene silencing (VIGS) against GhERF105 caused the dramatic reduction in the number of glands and significantly lowered levels of gossypol in cotton leaves. GhERF105 showed the patterns of spatiotemporal and inducible expression in the glanded plants. CONCLUSIONS: These results suggest that GhERF105 contributes to the pigment gland formation and gossypol biosynthesis in partial organs of glanded plant. It also provides a potential molecular basis to generate 'glandless-seed' and 'glanded-plant' cotton cultivar.

A histone deacetylase, GhHDT4D, is positively involved in cotton response to drought stress.

Acetylation and deacetylation of histones are important for regulating a series of biological processes in plants. Histone deacetylases (HDACs) control the histone deacetylation that plays an important role in plant response to abiotic stress. In our study, we show the evidence that GhHDT4D (a member of the HD2 subfamily of HDACs) is involved in cotton (Gossypium hirsutum) response to drought stress. Overexpression of GhHDT4D in Arabidopsis increased plant tolerance to drought, whereas silencing GhHDT4D in cotton resulted in plant sensitivity to drought. Simultaneously, the H3K9 acetylation level was altered in the GhHDT4D silenced cotton, compared with the controls. Further study revealed that GhHDT4D suppressed the transcription of GhWRKY33, which plays a negative role in cotton defense to drought, by reducing its H3K9 acetylation level. The expressions of the stress-related genes, such as GhDREB2A, GhDREB2C, GhSOS2, GhRD20-1, GhRD20-2 and GhRD29A, were significantly decreased in the GhHDT4D silenced cotton, but increased in the GhWRKY33 silenced cotton. Given these data together, our findings suggested that GhHDT4D may enhance drought tolerance by suppressing the expression of GhWRKY33, thereby activating the downstream drought response genes in cotton.

Genome-wide identification of the mitogen-activated protein kinase (MAPK) family in cotton (Gossypium hirsutum) reveals GhMPK6 involved in fiber elongation.

Mitogen-activated protein kinases (MAPKs) are important in regulating plant development as well as stress response. In this study, we genome-widely identified 56 MAPK genes in upland cotton. These MAPK genes unequally distribute on 22 chromosomes of cotton genome, but no MAPK gene is located on At_Chr6, Dt_Chr6, At_Chr13 and Dt_Chr13. The exons and introns in GhMAPK gene family vary widely at the position, number and length. Furthermore, GhMAPK family can be divided into 4 groups (A, B, C and D), and the TEY type of T-loop exists in three groups (A, B and C), but the TDY type of T-loop is only in group D. Further study revealed that some GhMAPK genes (including GhMPK6) are preferentially expressed in elongating fibers. GhMPK6 maintains a high phosphorylation level in elongating fibers, and its phosphorylation was enhanced in fibers by phytohormones brassinosteroid (BR), ethylene and indole-3-acetic acid (IAA). Additionally, GhMPK6 could interact with GhMKK2-2 and GhMKK4, suggesting that GhMKK2-2/4-GhMPK6 module may be involved in phosphorylation of its downstream proteins for regulating fiber elongation of cotton.

Genome-wide identification and functional characterization of cotton (Gossypium hirsutum) MAPKKK gene family in response to drought stress.

BACKGROUND: Mitogen-activated protein kinase kinase kinases (MAPKKKs) are significant components in the MAPK signal pathway and play essential roles in regulating plants against drought stress. To explore MAPKKK gene family functioning in cotton response and resistance to drought stress, we conducted a systematic analysis of GhMAPKKKs. RESULTS: In this study, 157 nonredundant GhMAPKKKs (including 87 RAFs, 46 MEKKs and 24 ZIKs) were identified in cotton (Gossypium hirsutum). These GhMAPKKK genes are unevenly distributed on 26 chromosomes, and segmental duplication is the major way for the enlargement of MAPKKK family. Furthermore, members within the same subfamily share a similar gene structure and motif composition. A lot of cis-elements relevant to plant growth and response to stresses are distributed in promoter regions of GhMAPKKKs. Additionally, these GhMAPKKKs show differential expression patterns in cotton tissues. The transcription levels of most genes were markedly altered in cotton under heat, cold and PEG treatments, while the expressions of some GhMAPKKKs were induced in cotton under drought stress. Among these drought-induced genes, we selected GhRAF4 and GhMEKK12 for further functional characterization by virus-induced gene silencing (VIGS) method. The experimental results indicated that the gene-silenced cotton displayed decreased tolerance to drought stress. Malondialdehyde (MDA) content was higher, but proline accumulation, relative leaf water content and activities of superoxide dismutase (SOD) and peroxidase (POD) were lower in the gene-silenced cotton, compared with those in the controls, under drought stress. CONCLUSION: Collectively, a systematic survey of gene structure, chromosomal location, motif composition and evolutionary relationship of MAPKKKs were performed in upland cotton (Gossypium hirsutum). The following expression and functional study showed that some of them take important parts in cotton drought tolerance. Thus, the data presented here may provide a foundation for further investigating the roles of GhMAPKKKs in cotton response and resistance to drought stress.