Zinc Oxide Nanoparticles and Zinc Sulfate Alleviate Boron Toxicity in Cotton (Gossypium hirsutum L.).

Boron toxicity significantly hinders the growth and development of cotton plants, therefore affecting the yield and quality of this important cash crop worldwide. Limited studies have explored the efficacy of ZnSO4 (zinc sulfate) and ZnO nanoparticles (NPs) in alleviating boron toxicity. Nanoparticles have emerged as a novel strategy to reduce abiotic stress directly. The precise mechanism underlying the alleviation of boron toxicity by ZnO NPs in cotton remains unclear. In this study, ZnO NPs demonstrated superior potential for alleviating boron toxicity compared to ZnSO4 in hydroponically cultivated cotton seedlings. Under boron stress, plants supplemented with ZnO NPs exhibited significant increases in total fresh weight (75.97%), root fresh weight (39.64%), and leaf fresh weight (69.91%). ZnO NPs positively affected photosynthetic parameters and SPAD values. ZnO NPs substantially reduced H2O2 (hydrogen peroxide) by 27.87% and 32.26%, MDA (malondialdehyde) by 27.01% and 34.26%, and O2- (superoxide anion) by 41.64% and 48.70% after 24 and 72 h, respectively. The application of ZnO NPs increased the antioxidant activities of SOD (superoxide dismutase) by 82.09% and 76.52%, CAT (catalase) by 16.79% and 16.33%, and POD (peroxidase) by 23.77% and 21.66% after 24 and 72 h, respectively. ZnO NP and ZnSO4 application demonstrated remarkable efficiency in improving plant biomass, mineral nutrient content, and reducing boron levels in cotton seedlings under boron toxicity. A transcriptome analysis and corresponding verification revealed a significant up-regulation of genes encoding antioxidant enzymes, photosynthesis pathway, and ABC transporter genes with the application of ZnO NPs. These findings provide valuable insights for the mechanism of boron stress tolerance in cotton and provide a theoretical basis for applying ZnO NPs and ZnSO4 to reduce boron toxicity in cotton production.

Genome-wide analysis and identification of stress-responsive genes of the CCCH zinc finger family in Capsicum annuum L.

The CCCH zinc finger gene family encodes a class of proteins that can bind to both DNA and RNA, and an increasing number of studies have demonstrated that the CCCH gene family plays a key role in growth and development and responses to environmental stress. Here, we identified 57 CCCH genes in the pepper (Capsicum annuum L.) genome and explored the evolution and function of the CCCH gene family in C. annuum. Substantial variation was observed in the structure of these CCCH genes, and the number of exons ranged from one to fourteen. Analysis of gene duplication events revealed that segmental duplication was the main driver of gene expansion in the CCCH gene family in pepper. We found that the expression of CCCH genes was significantly up-regulated during the response to biotic and abiotic stress, especially cold and heat stress, indicating that CCCH genes play key roles in stress responses. Our results provide new information on CCCH genes in pepper and will aid future studies of the evolution, inheritance, and function of CCCH zinc finger genes in pepper.

Single-cell transcriptomic analysis reveals the developmental trajectory and transcriptional regulatory networks of pigment glands in Gossypium bickii.

Comprehensive utilization of cottonseeds is limited by the presence of pigment glands and its inclusion gossypol. The ideal cotton has glandless seeds but a glanded plant, a trait found in only a few Australian wild cotton species, including Gossypium bickii. Introgression of this trait into cultivated species has proved to be difficult. Understanding the biological processes toward pigment gland morphogenesis and the associated underlying molecular mechanisms will facilitate breeding of cultivated cotton varieties with the trait of glandless seeds and glanded plant. In this study, single-cell RNA sequencing (scRNA-seq) was performed on 12 222 protoplasts isolated from cotyledons of germinating G. bickii seeds 48 h after imbibition. Clustered into 14 distinct clusters unsupervisedly, these cells could be grouped into eight cell populations with the assistance of known cell marker genes. The pigment gland cells were well separated from others and could be separated into pigment gland parenchyma cells, secretory cells, and apoptotic cells. By integrating the pigment gland cell developmental trajectory, transcription factor regulatory networks, and core transcription factor functional validation, we established a model for pigment gland formation. In this model, light and gibberellin were verified to promote the formation of pigment glands. In addition, three novel genes, GbiERF114 (ETHYLENE RESPONSE FACTOR 114), GbiZAT11 (ZINC FINGER OF ARABIDOPSIS THALIANA 11), and GbiNTL9 (NAC TRANSCRIPTION FACTOR-LIKE 9), were found to affect pigment gland formation. Collectively, these findings provide new insights into pigment gland morphogenesis and lay the cornerstone for future cotton scRNA-seq investigations.

A reference-grade genome assembly for Gossypium bickii and insights into its genome evolution and formation of pigment glands and gossypol.

The pigment gland is a morphological characteristic of Gossypium and its related genera. Gossypium bickii (G1) is characterized by delayed pigment gland morphogenesis in the cotyledons. In this study, a reference-grade genome of G1 was generated, and comparative genomics analysis showed that G1 was closest to Gossypium australe (G2), followed by A- and D-genome species. Two large fragment translocations in chromosomes 5 and 13 were detected between the G genome and other Gossypium genomes and were unique to the G1 and G2 genomes. Compared with the G2 genome, two large fragment inversions in chromosomes 12 and 13 were detected in G1. According to the phylogeny, divergence time, and similarity analysis of nuclear and chloroplast genomes, G1 was formed by hybridization between Gossypium sturtianum (C1) and a common ancestor of G2 and Gossypium nelsonii (G3). The coordinated expression patterns of pigment gland formation (GoPGF) and gossypol biosynthesis genes in G1 were verified to be consistent with its phenotype, and nine genes that were related to the process of pigment gland formation were identified. A novel gene, GbiCYP76B6, regulated by GoPGF, was found to affect gossypol biosynthesis. These findings offer insights into the origin and evolution of G1 and its mechanism of pigment gland formation and gossypol biosynthesis.

Deficiency of a peroxisomal NADP-isocitrate dehydrogenase leads to dwarf plant and defect seed in upland cotton.

The NADP-isocitrate dehydrogenase-encoded gene GH_D13G1452 with a C-terminus tripeptide Proline-Lysine-Leucine was localized in the peroxisome. It was highly expressed in stems and ovules of 15 days post-anthesis and responded to multiple external stimuli in upland cotton. An upland cotton mutant (Ghpericdh) was identified by flanking sequence amplification and genome variation detection that exogenous sequence was inserted in the middle of the 12th intron of GH_D13G1452, resulting in the deficiency of gene expression. The Ghpericdh mutant displayed a dwarf plant phenotype when grown under field or greenhouse conditions, and GH_D13G1452 functioned as an incomplete dominance on plant height. The germination rate of mutant seed from greenhouse-grown plants was dramatically lower than that from field-grown plants, which indicated that GhperICDH plays a critical role in seed maturation and germination. Therefore, GH_D13G1452 is indispensable in the development of stems and seeds and functions in the adaptability of cotton to the environment. The Ghpericdh mutant provides insight into the function of peroxisomal ICDH and may contribute to the genetic improvement in cotton.

Development of the engineered "glanded plant and glandless seed" cotton.

After fiber, cottonseed is the second most important by-product of cotton production. However, high concentrations of toxic free gossypol deposited in the glands of the cottonseed greatly hamper its effective usage as food or feed. Here, we developed a cotton line with edible cottonseed by specifically silencing the endogenous expression of GoPGF in the seeds, which led to a glandless phenotype with an ultra-low gossypol content in the seeds and nearly normal gossypol in other parts of the plants. This engineered cotton maintains normal resistance to insect pests, but the gossypol content in the seeds dropped by 98%, and thus, it can be consumed directly as food. The trait of a low gossypol content in the cottonseeds was stable and heritable, while the protein, oil content, and fiber yield or quality were nearly unchanged compared to the transgenic receptor W0. In addition, comparative transcriptome analysis showed that down-regulated genes in the ovules of the glandless cotton were enriched in terpenoid biosynthesis, indicating the underlying relationship between gland formation and gossypol biosynthesis. These results pave the way for the comprehensive utilization of cotton as a fiber, oil, and feed crop in the future.

Parental legacy versus regulatory innovation in salt stress responsiveness of allopolyploid cotton (Gossypium) species.

Polyploidy provides an opportunity for evolutionary innovation and species diversification, especially under stressful conditions. In allopolyploids, the conditional dynamics of homoeologous gene expression can be either inherited from ancestral states pre-existing in the parental diploids or novel upon polyploidization, the latter potentially permitting a wider range of phenotypic responses to stresses. To gain insight into regulatory mechanisms underlying the diversity of salt resistance in Gossypium species, we compared global transcriptomic responses to modest salinity stress in two allotetraploid (AD-genome) cotton species, Gossypium hirsutum and G. mustelinum, relative to their model diploid progenitors (A-genome and D-genome). Multivariate and pairwise analyses of salt-responsive changes revealed a profound alteration of gene expression for about one third of the transcriptome. Transcriptional responses and associated functional implications of salt acclimation varied across species, as did species-specific coexpression modules among species and ploidy levels. Salt responsiveness in both allopolyploids was strongly biased toward the D-genome progenitor. A much lower level of transgressive downregulation was observed in the more salt-tolerant G. mustelinum than in the less tolerant G. hirsutum. By disentangling inherited effects from evolved responses, we show that expression biases that are not conditional upon salt stress approximately equally reflect parental legacy and regulatory novelty upon allopolyploidization, whereas stress-responsive biases are predominantly novel, or evolved, in allopolyploids. Overall, our work suggests that allopolyploid cottons acquired a wide range of stress response flexibility relative to their diploid ancestors, most likely mediated by complex suites of duplicated genes and regulatory factors.

Root Illumination Promotes Seedling Growth and Inhibits Gossypol Biosynthesis in Upland Cotton.

Gossypol, a terpenoid compound mainly synthesized in the cotton roots, acts as a phytoalexin in protecting the plants from biotic stress. Roots are critical for both the secondary metabolism and the growth of the plant. Light plays an important role in plant growth and material metabolism, however, the effect of root illumination (RI) on the cotton seedling growth and gossypol metabolism remains unclear. In the present study, the cotton genetic standard line TM-1 and four pairs of near-isogenic lines (NILs) were used as materials to study the impact of RI on cotton seedlings. Results showed that, compared with the cotton seedlings cultivated without RI, the photosynthetic rate, leaf area, and dry weight of roots and leaves were significantly increased, while the gossypol content in leaves and roots was significantly reduced in seedlings cultivated with RI. GO and KEGG enrichment analysis of the differentially expressed genes (DEGs) with and without RI both indicated that photosynthesis and terpenoid biosynthesis-related GO terms and pathways were significantly enriched, the expression profile confirmed that RI positively regulated the photosynthesis system and negatively affected the gossypol biosynthesis pathway in roots. This study revealed the effects of RI on seedlings' growth and gossypol biosynthesis in upland cotton, and provided important insights for the engineering of cotton with low gossypol accumulation.

Genome-wide characterization on MT family and their expression in response to environmental cues in upland cotton (Gossypium hirsutum L.).

Metallothioneins (MTs) are believed as key metal chelators and scavengers of reactive oxygen species (ROS), which are involved in tolerance and de-toxicity to multiple environmental stresses in plants. The MT gene family was characterized from upland cotton (Gossypium hirsutum L.), compared with its putative genome donors G. arboretum and raimondii. Subsequently, gene functions were predicted by promoter analysis. Moreover, gene expressions subjecting to exogenous stimuli, as well as in terms of developments, were studied. The main findings were shown as follows: 1) 19 GhMTs were identified from G. hirsutum, and the family completely included all four sub-types, namely p1, p2, p3, and pec. Sub-type p2 GhMTs were most conservative in protein motif compositions, gene structures, phylogenic relationships, and group numbers, while p3 GhMTs demonstrated much more diversiform and distant genetic relationships. 2) The GhMT family experienced apparent gene expansion, and the members from the D sub-genome were subjected to stronger environmental selection. 3) GhMTs played differential and overlapped roles in response to environmental cues. 4) GhMT6, GhMT8, and GhMT14 were involved in both vegetative and reproductive developments. These findings must provide valuable insights into understanding the plant MT gene family and novel gene resources for cotton breeding for environmental stresses, phytoremediation, and beyond.

Mercury-Induced Phytotoxicity and Responses in Upland Cotton (Gossypium hirsutum L.) Seedlings.

Cotton is a potential and excellent candidate to balance both agricultural production and remediation of mercury-contained soil, as its main production fiber hardly involves into food chains. However, in cotton, there is known rarely about the tolerance and response to mercury (Hg) environments. In this study, the biochemical and physiological damages, in response to Hg concentrations (0, 1, 10, 50 and 100 µM), were investigated in upland cotton seedlings. The results on germination of cottonseeds indicated the germination rates were suppressed by high Hg levels, as the decrease of percentage was more than 10% at 1000 µM Hg. Shoots and roots' growth were significantly inhibited over 10 µM Hg. The inhibitor rates (IR) in fresh weight were close in values between shoots and roots, whereas those in dry weight the root growth were more obviously influenced by Hg. In comparison of organs, the growth inhibition ranked as root > leaf > stem. The declining of translocation factor (TF) opposed the Hg level as even low to 0.05 at 50 µM Hg. The assimilation in terms of photosynthesis, of cotton plants, was affected negatively by Hg, as evidenced from the performances on pigments (chlorophyll a and b) and gas exchange (Intercellular CO2 concentration (Ci), CO2 assimilation rate (Pn) and stomatal conductance (Gs)). Sick phenotypes on leaf surface included small white zone, shrinking and necrosis. Membrane lipid peroxidation and leakage were Hg dose-dependent as indicated by malondialdehyde (MDA) content and relative conductivity (RC) values in leaves and roots. More than 10 µM Hg damaged antioxidant enzyme system in both leaves and roots (p < 0.05). Concludingly, 10 µM Hg post negative consequences to upland cotton plants in growth, physiology and biochemistry, whereas high phytotoxicity and damage appeared at more than 50 µM Hg concentration.

Whole-genome resequencing of 240 Gossypium barbadense accessions reveals genetic variation and genes associated with fiber strength and lint percentage.

KEY MESSAGE: Genetic variation in a G. barbadense population was revealed using resquencing. GWAS on G.barbadense population identified several candidate genes associated with fiber strength and lint percentage. Gossypium barbadense is the second-largest cultivated cotton species planted in the world, which is characterized by high fiber quality. Here, we described the global pattern of genetic polymorphisms for 240 G. barbadense accessions based on the whole-genome resequencing. A total of 3,632,231 qualified single-nucleotide polymorphisms (SNPs) and 221,354 insertion-deletions (indels) were obtained. We conducted a genome-wide association study (GWAS) on 12 traits under four environments. Two traits with more stable associated variants, fiber strength and lint percentage, were chosen for further analysis. Three putative candidate genes, HD16 orthology (GB_D11G3437), WDL2 orthology (GB_D11G3460) and TUBA1 orthology (GB_D11G3471), on chromosome D11 were found to be associated with fiber strength, and one gene orthologous to Arabidopsis Receptor-like protein kinase HERK 1 (GB_A07G1034) was predicated to be the candidate gene for the lint percentage improvement. The identified genes may serve as promising targets for genetic engineering to accelerate the breeding process for G. barbadense and the high-density genome variation map constructed in this work may facilitate our understanding of the genetic architecture of cotton traits.

Determination of Oxidative Stress and Antioxidant Enzyme Activity for Physiological Phenotyping During Heavy Metal Exposure.

The overproduction of reactive oxygen species during abiotic stress in plants causes oxidative stress that damage the cell normal functions. For reactive oxygen species (ROS) scavenging, plants developed a defense system with antioxidant enzymes. To measure the oxidative stress and antioxidant enzymes activity spectral enzymatic analysis was used, that is material-intensive, time-consuming, and inefficient. In the present study, the four more studied and main antioxidant enzymatic assays are miniaturized in a 96-well plate system and monitored the activity of enzymes by spectrophotometry. This method has obvious advantages over the standard cuvette analysis method because the miniaturization of the 96-well microplate system decreases the amount of reaction mixture and enzymes extract, thrifts working time, and consumable costs as well.

Physiological, ultrastructural, biochemical, and molecular responses of glandless cotton to hexavalent chromium (Cr6+) exposure.

Glandless cotton can be grown to obtain cotton seeds free of toxic gossypol for use as both food and feed. However, they are not grown normally due to their lesser productivity and higher susceptibility to biotic stress. Great attention has been paid to biotic stresses rather than abiotic stresses on glandless cotton. Chromium (Cr) is a common pollutant of soil and considered a serious threat to plants due to its adverse effects on different functions. Although numerous studies are available on the toxicity of Cr6+ in various plants. However, its adverse effects and mechanism of toxicity in glandless cotton can seldom be found in the literature. This study examined the Cr6+ effect on glandless cotton in comparison to glanded cotton. Four pairs of glanded and glandless cotton near-isogenic lines (NILs) were exposed to different doses (0, 10, 50, and 100 µM/L) of Cr6+ for seven days, and biochemical, physiological, molecular, and ultrastructure changes were observed, which were significantly affected by Cr6+ at high concentrations in all NILs. The effect of Cr6+ on ionic contents shows the same trend in glanded and glandless NILs except for manganese (Mn2+) that show inhibition in glandless (ZMS-12w and Coker-312w) and enhance in the glanded NIL (ZMS-17). The gene expression of superoxide dismutase (SOD) and peroxidase (POD) revealed similar trends as enzyme activities in glandless NILs. The principal component analysis (PCA) and Agglomerative hierarchical clustering (AHC) results of all NILs from morpho-physiological traits, cluster ZMS-16, and ZMS-17 into Cr6+ sensitive group. While the glandless NILs have the potential to cope with the Cr toxicity by increasing the antioxidant enzyme activity and their gene expression. This study also revealed that Cr6+ tolerance in cotton is genotypic and has an independent mechanism in the root that not related to low gossypol.

Reduced Glutathione Protects Subcellular Compartments From Pb-Induced ROS Injury in Leaves and Roots of Upland Cotton (Gossypium hirsutum L.).

Heavy metals-based changes in the plants and their alleviation through eco-friendly agents including reduced glutathione (GSH) have been widely studied. In the present experiment, we tested the alleviatory role of reduced glutathione (GSH) in seedlings of upland cotton cultivar, TM-1 under lead (Pb) toxicity. Plants were grown in the Hoagland solution containing Pb (0 µM), Pb (500 µM), GSH (50 µM), and GSH + Pb (50 µM + 500 µM). Lead exposure exacerbated hydrogen peroxide (H2O2) and hydroxyl radical (OH•) levels, induced lipid peroxidation (MDA), and decreased the activities of catalase (CAT) and ascorbate peroxidase (APX) in the terminal and median leaves of 28-days old cotton seedlings stressed for 10 days. However, in the primary and secondary roots, CAT activity was increased but APX decreased. Similarly, peroxidase (POD) and superoxide dismutase (SOD) activities were enhanced in the median leaves but a declining trend was observed in the terminal leaves, primary roots and secondary roots. Glutathione reductase (GR) activity, ascorbic acid (AsA) contents and GSH concentrations were increased in all parts except AsA in the median leaves. Transmission electron micrographs of Pb-treated plants exhibited deformed cell wall and cell membrane, disfigured chloroplasts and irregularly shaped mitochondria in the terminal and median leaves. Further, cell membrane, mitochondria, nucleus and other cell organelles in root cells were severely affected by the Pb. Thus their identification was little bit difficult through ultramicroscopy. External GSH stabilized leaf and root ultramorphology by stabilizing cell membranes, stimulating formation of multivesicular body vesicles, and by maintaining structural integrity of other organelles. Evidently, GSH played major alleviatory role against Pb toxicity in upland cotton.

Comprehensive characterization and gene expression patterns of LBD gene family in Gossypium.

MAIN CONCLUSION: A comprehensive account of the LBD gene family of Gossypium was provided in this work. Expression analysis and functional characterization revealed that LBD genes might play different roles in G. hirsutum and G. barbadense. The Lateral Organ Boundaries Domain (LBD) proteins comprise a plant-specific transcription factor family, which plays crucial roles in physiological processes of plant growth, development, and stress tolerance. In the present work, a systematical analysis of LBD gene family from two allotetraploid cotton species, G. hirsutum and G. barbadense, together with their genomic donor species, G. arboreum and G. raimondii, was conducted. There were 131, 128, 62, and 68 LBDs identified in G. hirsutum, G. barbadense, G. arboreum and G. raimondii, respectively. The LBD proteins could be classified into two main classes, class I and class II, based on the structure of their lateral organ boundaries domain and traits of phylogenetic tree, and class I was further divided into five subgroups. The gene structure and motif composition analyses conducted in both G. hirsutum and G. barbadense revealed that LBD genes kept relatively conserved within the subfamilies. Synteny analysis suggested that segmental duplication acted as an important mechanism in expansion of the cotton LBD gene family. Cis-element analysis predicated the possible functions of LBD genes. Public RNA-seq data were investigated to analyze the expression patterns of cotton LBD genes in various tissues as well as gene expression under abiotic stress treatments. Furthermore, RT-qPCR results found that GhLBDs had various expression regulation under MeJA treatments. Expression analysis indicated the differential functions of cotton LBD genes in response to abiotic stress and hormones.

Cotton roots are the major source of gossypol biosynthesis and accumulation.

BACKGROUND: Gossypol is a specific secondary metabolite in Gossypium species. It not only plays a critical role in development and self-protection of cotton plants, but also can be used as important anti-cancer and male contraceptive compound. However, due to the toxicity of gossypol for human beings and monogastric animals, the consumption of cottonseeds was limited. To date, little is known about the gossypol metabolism in cotton plants. RESULTS: In this study, we found that cotyledon was the primary source of gossypol at the seed germination stage. But thereafter, it was mainly originated from developing roots. Grafting between glanded and glandless cotton as well as sunflower rootstocks and cotton scion revealed that gossypol was mainly synthesized in the root systems of cotton plants. And both glanded and glandless cotton roots had the ability of gossypol biosynthesis. But the pigment glands, the main storage of gossypol, had indirect effects on gossypol biosynthesis. In vitro culture of root and rootless seedling confirmed the strong gossypol biosynthesis ability in root system and the relatively weak gossypol biosynthesis ability in other organs of the seedling. Expression profiling of the key genes involved in the gossypol biosynthetic pathway also supported the root as the major organ of gossypol biosynthesis. CONCLUSIONS: Our study provide evidence that the cotton root system is the major source of gossypol in both glanded and glandless cottons, while other organs have a relatively weak ability to synthesize gossypol. Gossypol biosynthesis is not directed related to the expression of pigment glands, but the presence of pigment glands is essential for gossypol accumulation. These findings can not only clarify the complex regulation network of gossypol metabolism, but it could also accelerate the crop breeding process with enhanced commercial values.

Salt-tolerance diversity in diploid and polyploid cotton (Gossypium) species.

The development of salt-tolerant genotypes is pivotal for the effective utilization of salinized land and to increase global crop productivity. Several cotton species comprise the most important source of textile fibers globally, and these are increasingly grown on marginal or increasingly saline agroecosystems. The allopolyploid cotton species also provide a model system for polyploid research, of relevance here because polyploidy was suggested to be associated with increased adaptation to stress. To evaluate the genetic variation of salt tolerance among cotton species, 17 diverse accessions of allopolyploid (AD-genome) and diploid (A- and D-genome) Gossypium were evaluated for a total of 29 morphological and physiological traits associated with salt tolerance. For most morphological and physiological traits, cotton accessions showed highly variable responses to 2 weeks of exposure to moderate (50 mm NaCl) and high (100 mm NaCl) hydroponic salinity treatments. Our results showed that the most salt-tolerant species were the allopolyploid Gossypium mustelinum from north-east Brazil, the D-genome diploid Gossypium klotzschianum from the Galapagos Islands, followed by the A-genome diploids of Africa and Asia. Generally, A-genome accessions outperformed D-genome cottons under salinity conditions. Allopolyploid accessions from either diploid genomic group did not show significant differences in salt tolerance, but they were more similar to one of the two progenitor lineages. Our findings demonstrate that allopolyploidy in itself need not be associated with increased salinity stress tolerance and provide information for using the secondary Gossypium gene pool to breed for improved salt tolerance.

Melatonin enhances cotton immunity to Verticillium wilt via manipulating lignin and gossypol biosynthesis.

Plants endure challenging environments in which they are constantly threatened by diverse pathogens. The soil-borne fungus Verticillium dahliae is a devastating pathogen affecting many plant species including cotton, in which it significantly reduces crop yield and fiber quality. Melatonin involvement in plant immunity to pathogens has been reported, but the mechanisms of melatonin-induced plant resistance are unclear. In this study, the role of melatonin in enhancing cotton resistance to V. dahliae was investigated. At the transcriptome level, exogenous melatonin increased the expression of genes in phenylpropanoid, mevalonate (MVA), and gossypol pathways after V. dahliae inoculation. As a result, lignin and gossypol, the products of these metabolic pathways, significantly increased. Silencing the serotonin N-acetyltransferase 1 (GhSNAT1) and caffeic acid O-methyltransferase (GhCOMT) melatonin biosynthesis genes compromised cotton resistance, with reduced lignin and gossypol levels after V. dahliae inoculation. Exogenous melatonin pre-treatment prior to V. dahliae inoculation restored the level of cotton resistance reduced by the above gene silencing effects. Melatonin levels were higher in resistant cotton cultivars than in susceptible cultivars after V. dahliae inoculation. The findings indicate that melatonin affects lignin and gossypol synthesis genes in phenylpropanoid, MVA, and gossypol pathways, thereby enhancing cotton resistance to V. dahliae.

Genome-wide analysis of genetic variations between dominant and recessive NILs of glanded and glandless cottons.

Cotton is an important economic crop in worldwide. It produces fiber for the textile industry and provides cottonseeds with high-quality protein and oil. However, the presence of gossypol limits the utilization of cottonseed. Two pairs of cotton near isogenic lines (NILs) with different pigment glands, i.e., Coker 312 vs Coker 312 W and CCRI12 vs CCRI12W, exhibit different gossypol contents. The glandless traits of Coker 312 W and CCRI12W are controlled by recessive and dominant genes, respectively. However, knowledge regarding the genomic variations in the NILs is limited. Therefore, the NILs genomes were resequenced and the sequencing depths were greater than 34×. Compared with the TM-1 genome, numerous SNPs, Indels, SVs, and CNVs were discovered. KEGG pathway analysis revealed that genes with SNPs and Indels from the recessive NILs and genes with Indels from the dominant NILs shared only one enriched pathway, i.e., the sesquiterpenoid and triterpenoid biosynthesis pathway, which is relevant to gossypol biosynthesis. Expression analysis revealed that key genes with variations that participate in the gossypol biosynthesis and pigment gland formation pathways had different expression patterns among the dominant, recessive glandless and glanded plants. The expression levels in the glanded organs were higher than those in their NILs. Altogether, our results provide deeper insight into cotton NILs with different pigment glands.

Identification and profiling of upland cotton microRNAs at fiber initiation stage under exogenous IAA application.

BACKGROUND: Cotton is the most essential textile crop worldwide, and phytohormones are critical for cotton fiber development. One example is the role of auxin in fiber initiation, but we know little molecular basis. MicroRNAs (miRNAs) have a significant function in cotton development; nevertheless their role in fiber initiation remains unclear. Here, exogenous IAA was applied to cotton plant before anthesis. Utilizing small RNA sequencing, the mechanism underlying miRNA-mediated regulation of fiber initiation under exogenous IAA treatment was investigated. RESULTS: With exogenous IAA application, the endogenous IAA and GA contents of IAA treated (IT) ovules were higher than control (CK) ovules at the fiber initiation stage, while endogenous ABA content was lower in IT than CK. Using scanning electron microscopy, we found the fiber number and size were significantly promoted in IT at 0 DPA. Fiber quality analysis showed that fiber length, uniformity, strength, elongation, and micronaire of IT were higher than CK, though not statistically significant, while lint percent was significantly higher in IT. We generated six small RNA libraries using - 3, 0, and 3 DPA ovules of IT and CK, and identified 58 known miRNAs and 83 novel miRNAs together with the target genes. The differential expressed miRNAs number between IT and CK at - 3, 0, 3 DPA was 34, 16 and 24, respectively. Gene ontology and KEGG pathway enrichment analyses for the target genes of the miRNAs expressed in a differential manner showed that they were significantly enriched in 30 terms and 8 pathways. QRT-PCR for those identified miRNAs and the target genes related to phytohormones and fiber development was performed, and results suggested a potential role of these miRNAs in fiber initiation. CONCLUSIONS: The exogenous IAA application affected the relative phytohormone contents in ovule and promoted fiber initiation in cotton. Identification and profiling of miRNAs and their targets at the fiber initiation stage provided insights for miRNAs' regulation function of fiber initiation. These findings not only shed light on the regulatory network of fiber growth but also offer clues for cotton fiber amelioration strategies in cotton.