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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.

Seed germination is considered the beginning of the spermatophyte lifecycle, and it is a crucial stage in determining subsequent plant growth and development. Although many previous studies have found that melatonin can promote seed germination, the role of melatonin in cotton germination remains unexamined. The main objective of this study is the characterization of potential promotional effects of melatonin (at doses of 0, 10, 20, 50, 100 and 200 μM) on cotton seed germination. This experiment demonstrated that low concentrations of melatonin can promote germination, while high concentrations failed to promote germination and even inhibited germination. Together, these results indicate that a 20 μM melatonin treatment optimally promotes cotton seed germination. Compared with the control, germination potential (GP), germination rate (GR), and final fresh weight (FW) increased by 16.67%, 12.30%, and 4.81%, respectively. Although low concentrations of melatonin showed some improvement in vigor index (VI), germination index (GI), and mean germination time (MGT), these effects were not statistically significant. Antioxidant enzyme activity during seed germination was most prominent under the 20 μM melatonin treatment. Superoxide dismutase (SOD) and peroxidase (POD) activities were significantly increased by 10.37-59.73% and 17.79-47.68%, respectively, compared to the melatonin-free control. Malondialdehyde (MDA) content was reduced by 16.73-40.33%. Two important plant hormones in seed germination, abscisic acid (ABA) and gibberellins (GAs), were also studied. As melatonin concentration increased, ABA content in seeds decreased first and then increased, and GA3 content showed a diametrically opposite trend, in which the 20 μM melatonin treatment was optimal. The 20 μM melatonin treatment reduced ABA content in seeds by 42.13-51.68%, while the 20 μM melatonin treatment increased GA3 content in seeds to about 1.7-2.5 times that of seeds germinated without melatonin. This study provides new evidence suggesting that low concentrations of melatonin can promote cotton seed germination by increasing the activity of antioxidant enzymes, thereby reducing the accumulation of MDA and regulating plant hormones. This has clear applications for improving the germination rate of cotton seeds using melatonin.

Yuxian Zhu and colleagues report the draft genome of a diploid cotton Gossypium raimondii . This species is a wild South American cotton, whose progenitor is thought to have been the contributor of the D subgenome of the allotetraploid commercial species Gossypium hirsutum and Gossypium barbadense , which account for ~95% of the worldwide cotton crop. We have sequenced and assembled a draft genome of G. raimondii , whose progenitor is the putative contributor of the D subgenome to the economically important fiber-producing cotton species Gossypium hirsutum and Gossypium barbadense . Over 73% of the assembled sequences were anchored on 13 G. raimondii chromosomes. The genome contains 40,976 protein-coding genes, with 92.2% of these further confirmed by transcriptome data. Evidence of the hexaploidization event shared by the eudicots as well as of a cotton-specific whole-genome duplication approximately 13–20 million years ago was observed. We identified 2,355 syntenic blocks in the G. raimondii genome, and we found that approximately 40% of the paralogous genes were present in more than 1 block, which suggests that this genome has undergone substantial chromosome rearrangement during its evolution. Cotton, and probably Theobroma cacao , are the only sequenced plant species that possess an authentic CDN1 gene family for gossypol biosynthesis, as revealed by phylogenetic analysis.

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.

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 β-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.

Drought is a key limiting factor for cotton (Gossypium spp.) production, as more than half of the global cotton supply is grown in regions with high water shortage. However, the underlying mechanism of the response of cotton to drought stress remains elusive. By combining genome-wide transcriptome profiling and a loss-of-function screen using virus-induced gene silencing, we identified Gossypium hirsutum GhWRKY59 as an important transcription factor that regulates the drought stress response in cotton. Biochemical and genetic analyses revealed a drought stress-activated mitogen-activated protein (MAP) kinase cascade consisting of GhMAP3K15–Mitogen-activated Protein Kinase Kinase 4 (GhMKK4)–Mitogen-activated Protein Kinase 6 (GhMPK6) that directly phosphorylates GhWRKY59 at residue serine 221. Interestingly, GhWRKY59 is required for dehydration-induced expression of GhMAPK3K15, constituting a positive feedback loop of GhWRKY59-regulated MAP kinase activation in response to drought stress. Moreover, GhWRKY59 directly binds to the W-boxes of DEHYDRATION-RESPONSIVE ELEMENT-BINDING PROTEIN 2 (GhDREB2), which encodes a dehydration-inducible transcription factor regulating the plant hormone abscisic acid (ABA)-independent drought response. Our study identified a complete MAP kinase cascade that phosphorylates and activates a key WRKY transcription factor, and elucidated a regulatory module, consisting of GhMAP3K15-GhMKK4-GhMPK6-GhWRKY59-GhDREB2, that is involved in controlling the cotton drought response.

Background Superoxide dismutases (SODs) are a key antioxidant enzyme family, which have been implicated in protecting plants against the toxic effects of reactive oxygen species. Despite current studies have shown that the gene family are involved in plant growth and developmental processes and biotic and abiotic stress responses, little is known about its functional role in upland cotton. Results In the present study, we comprehensively analyzed the characteristics of the SOD gene family in upland cotton ( Gossypium hirsutum ). Based on their conserved motifs, 18 GhSOD genes were identified and phylogenetically classified into five subgroups which corroborated their classifications based on gene-structure patterns and subcellular localizations. The GhSOD sequences were distributed at different densities across 12 of the 26 chromosomes. The conserved domains, gene family evolution cis -acting elements of promoter regions and miRNA-mediated posttranscriptional regulation were predicted and analyzed. In addition, the expression pattern of 18 GhSOD genes were tested in different tissues/organs and developmental stages, and different abiotic stresses and abscisic acid, which indicated that the SOD gene family possessed temporal and spatial specificity expression specificity and may play important roles in reactive oxygen species scavenging caused by various stresses in upland cotton. Conclusions This study describes the first genome-wide analysis of the upland cotton SOD gene family, and the results will help establish a foundation for the further cloning and functional verification of the GhSOD gene family during stress responses, leading to crop improvement.

High-quality cotton fibre equates to a more comfortable textile. Fibre length is an important index of fibre quality. Hydrogen peroxide (H₂O₂) acts as a signalling molecule in the regulation of fibre elongation. Results from in vitro ovule culture suggest that the alteration of fibre cell H₂O₂ levels affects fibre development. Ascorbate peroxidase (APX) is an important reactive oxygen species (ROS) scavenging enzyme, and we found that GhAPX1AT/DT encoded one member of the previously unrealized group of cytosolic APXs (cAPXs) that were preferentially expressed during the fibre elongation stage. Transgenic cottons with up- and down-regulation of GhAPX1AT/DT were generated to control fibre endogenous levels of H₂O₂. Suppression of all cAPX (IAO) resulted in a 3.5-fold increase in H₂O₂ level in fibres and oxidative stress, which significantly suppressed fibre elongation. The fibre length of transgenic lines with over-expression or specific down-regulation of GhAPX1AT/DT did not show any obvious change. However, the fibres in the over-expression lines exhibited higher tolerance to oxidative stress. Differentially expressed genes (DEGs) in fibres at 10 days post-anthesis (DPA) of IAO lines identified by RNA-seq were related to redox homeostasis, signalling pathways, stress responses and cell wall synthesis, and the DEGs that were up-regulated in IAO lines were also upregulated in the 10 DPA and 20 DPA fibres of wild cotton compared with domesticated cotton. These results suggest that optimal H₂O₂ levels and redox state regulated by cytosolic APX are key mechanisms regulating fibre elongation, and dysregulation of the increase in H₂O₂ induces oxidative stress and results in shorter fibres by initiating secondary cell wall-related gene expression.

Background Carbon dots (CDs), an emerging class of nanomaterials, have shown great potential to modulate plant growth and development. However, the underlying molecular mechanisms and gene regulatory networks through which CDs influence physiological processes remain largely unexplored, limiting their application in agriculture. Results In this study, citric acid was used to synthesis CDs by microwave-assisted method. Here, we treat cotton leaves with different concentrations of CDs to study their effects on cotton growth and development. The findings demonstrated that, at appropriate doses, CDs could enhance cotton biomass accumulation, with concurrent increases in fresh and dry weights. At 100 mg·L − 1 , cotton exhibited optimal morphological performance: the aboveground biomass showed increases of 67.69% and 42.89% in fresh and dry weight, respectively. In addition, the belowground biomass displayed 34.48% and 46.65% increments, respectively. CDs-mediated increases in chlorophyll content and photosynthetic enzyme activities, together with improvements in photosystem performance, suggest that CDs likely enhance photosynthetic efficiency in cotton and thereby promote growth. Moreover, CDs strengthened the antioxidant defense system primarily by enhancing the activities of key enzymes (SOD and POD), despite a concurrent decrease in CAT activity. To elucidate the molecular basis underlying the observed physiological responses, RNA-sequencing (RNA-seq) was performed to provide deeper insights into the gene expression profiles associated with CD-mediated growth. In summary, transcriptomic analysis revealed that CDs significantly enriched the ‘photosynthesis-antenna proteins’ pathway, where Lhcb1 (light-harvesting complex II chlorophyll a/b binding protein 1), Lhcb2 (light-harvesting complex II chlorophyll a/b binding protein 2) and Lhcb3 (light-harvesting complex II chlorophyll a/b binding protein 3) were significantly up-regulated, and the ‘flavone and flavonol biosynthesis’ pathway, involving the up-regulation of F3′5′H (flavonoid 3’,5’-hydroxylase). These molecular findings are highly consistent with the physiological data, demonstrating that CDs promote cotton growth by enhancing light-harvesting efficiency and antioxidant defense systems. Conclusions By integrating transcriptomic profiling with physiological measurements, this study elucidates the molecular pathways by which citric acid–based CDs enhance photosynthesis and antioxidant capacity in cotton. Our findings provide a mechanistic basis for the use of CDs to optimize crop production.

Key messageGhDRP1 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.