The dual role of GoPGF reveals that the pigment glands are synthetic sites of gossypol in aerial parts of cotton.

Gossypol and the related terpenoids are stored in the pigment gland to protect cotton plants from biotic stresses, but little is known about the synthetic sites of these metabolites. Here, we showed that GoPGF, a key gene regulating gland formation, was expressed in gland cells and roots. The chromatin immunoprecipitation sequencing (ChIP-seq) analysis demonstrated that GoPGF targets GhJUB1 to regulate gland morphogenesis. RNA-sequencing (RNA-seq) showed high accumulation of gossypol biosynthetic genes in gland cells. Moreover, integrated analysis of the ChIP-seq and RNA-seq data revealed that GoPGF binds to the promoter of several gossypol biosynthetic genes. The cotton callus overexpressing GoPGF had dramatically increased the gossypol levels, indicating that GoPGF can directly activate the biosynthesis of gossypol. In addition, the gopgf mutant analysis revealed the existence of both GoPGF-dependent and -independent regulation of gossypol production in cotton roots. Our study revealed that the pigment glands are synthetic sites of gossypol in aerial parts of cotton and that GoPGF plays a dual role in regulating gland morphogenesis and gossypol biosynthesis. The study provides new insights for exploring the complex relationship between glands and the metabolites they store in cotton and other plant species.

Profile of cotton flavonoids: Their composition and important roles in development and adaptation to adverse environments.

Cotton is a commercial crop that is cultivated in more than 50 countries. The production of cotton has severely diminished in recent years owing to adverse environments. Thus, it is a high priority of the cotton industry to produce resistant cultivars to prevent diminished cotton yields and quality. Flavonoids comprise one of the most important groups of phenolic metabolites in plants. However, the advantage and biological roles of flavonoids in cotton have yet not been studied in depth. In this study, we performed a widely targeted metabolic study and identified 190 flavonoids in cotton leaves that span seven different classes with flavones and flavonols as the dominant groups. Furthermore, flavanone-3-hydroxylase was cloned and silenced to knock down flavonoid production. The results show that the inhibition of flavonoid biosynthesis affects the growth and development of cotton and causes semi-dwarfing in cotton seedlings. We also revealed that the flavonoids contribute to cotton defense against ultraviolet radiation and Verticillium dahliae. Moreover, we discuss the promising role of flavonoids in cotton development and defense against biotic and abiotic stresses. This study provides valuable information to study the variety and biological functions of flavonoids in cotton and will help to profile the advantages of flavonoids in cotton breeding.

Tomato and cotton G protein beta subunit mutants display constitutive autoimmune responses.

Heterotrimeric G protein Gß-deficient mutants in rice and maize display constitutive immune responses, whereas Arabidopsis Gß mutants show impaired defense, suggesting the existence of functional differences between monocots and dicots. Using CRISPR/Cas9, we produced one hemizygous tomato line with a mutated SlGB1 Gß gene. Homozygous slgb1 knockout mutants exhibit all the hallmarks of autoimmune mutants, including development of necrotic lesions, constitutive expression of defense-related genes, and high endogenous levels of salicylic acid (SA) and reactive oxygen species, resulting in early seedling lethality. Virus-induced silencing of Gß in cotton reproduced the symptoms observed in tomato mutants, confirming that the autoimmune phenotype is not limited to monocot species but is also shared by dicots. Even though multiple genes involved in SA and ethylene signaling are highly induced by Gß silencing in tomato and cotton, co-silencing of SA or ethylene signaling components in cotton failed to suppress the lethal phenotype, whereas co-silencing of the oxidative burst oxidase RbohD can repress lethality. Despite the autoimmune response observed in slgb1 mutants, we show that SlGB1 is a positive regulator of the pathogen-associated molecular pattern (PAMP)-triggered immunity (PTI) response in tomato. We speculate that the phenotypic differences observed between Arabidopsis and tomato/cotton/rice/maize Gß knockouts do not necessarily reflect divergences in G protein-mediated defense mechanisms.

Optimizing the Protein Fluorescence Reporting System for Somatic Embryogenesis Regeneration Screening and Visual Labeling of Functional Genes in Cotton.

Protein fluorescence reporting systems are of crucial importance to in-depth life science research, providing systematic labeling tools for visualization of microscopic biological activities in vivo and revolutionizing basic research. Cotton somatic cell regeneration efficiency is low, causing difficulty in cotton transformation. It is conducive to screening transgenic somatic embryo using the fluorescence reporting system. However, available fluorescence labeling systems in cotton are currently limited. To optimize the fluorescence reporting system of cotton with an expanded range of available fluorescent proteins, we selected 11 fluorescent proteins covering red, green, yellow, and cyan fluorescence colors and expressed them in cotton. Besides mRuby2 and G3GFP, the other nine fluorescent proteins (mCherry, tdTomato, sfGFP, Clover, EYFP, YPet, mVenus, mCerulean, and ECFP) were stably and intensely expressed in transgenic callus and embryo, and inherited in different cotton organs derive from the screened embryo. In addition, transgenic cotton expressing tdTomato appears pink under white light, not only for callus and embryo tissues but also various organs of mature plants, providing a visual marker in the cotton genetic transformation process, accelerating the evaluation of transgenic events. Further, we constructed transgenic cotton expressing mCherry-labeled organelle markers in vivo that cover seven specific subcellular compartments: plasma membrane, endoplasmic reticulum, tonoplast, mitochondrion, plastid, Golgi apparatus, and peroxisome. We also provide a simple and highly efficient strategy to quickly determine the subcellular localization of uncharacterized proteins in cotton cells using organelle markers. Lastly, we built the first cotton stomatal fluorescence reporting system using stomata-specific expression promoters (ProKST1, ProGbSLSP, and ProGC1) to drive Clover expression. The optimized fluorescence labeling system for transgenic somatic embryo screening and functional gene labeling in this study offers the potential to accelerating somatic cell regeneration efficiency and the in vivo monitoring of diverse cellular processes in cotton.

Identification of NHXs in Gossypium species and the positive role of GhNHX1 in salt tolerance.

BACKGROUND: Plant Na+/H+ antiporters (NHXs) are membrane-localized proteins that maintain cellular Na+/K+ and pH homeostasis. Considerable evidence highlighted the critical roles of NHX family in plant development and salt response; however, NHXs in cotton are rarely studied. RESULTS: The comprehensive and systematic comparative study of NHXs in three Gossypium species was performed. We identified 12, 12, and 23 putative NHX proteins from G. arboreum, G. raimondii, and G. hirsutum, respectively. Phylogenetic study revealed that repeated polyploidization of Gossypium spp. contributed to the expansion of NHX family. Gene structure analysis showed that cotton NHXs contain many introns, which will lead to alternative splicing and help plants to adapt to high salt concentrations in soil. The expression changes of NHXs indicate the possible differences in the roles of distinct NHXs in salt response. GhNHX1 was proved to be located in the vacuolar system and intensively induced by salt stress in cotton. Silencing of GhNHX1 resulted in enhanced sensitivity of cotton seedlings to high salt concentrations, which suggests that GhNHX1 positively regulates cotton tolerance to salt stress. CONCLUSION: We characterized the gene structure, phylogenetic relationship, chromosomal location, and expression pattern of NHX genes from G. arboreum, G. raimondii, and G. hirsutum. Our findings indicated that the cotton NHX genes are regulated meticulously and differently at the transcription level with possible alternative splicing. The tolerance of plants to salt stress may rely on the expression level of a particular NHX, rather than the number of NHXs in the genome. This study could provide significant insights into the function of plant NHXs, as well as propose promising candidate genes for breeding salt-resistant cotton cultivars.

GbMPK3 overexpression increases cotton sensitivity to Verticillium dahliae by regulating salicylic acid signaling.

The soil-born vascular disease Verticillium wilt, which is caused by fungal pathogen Verticillium dahliae, is a devastating disease of cotton worldwide. In the last decade, a large number of genes have been found to participate in cotton-V. dahliae interactions, but the detailed mechanisms of cotton resistance to V. dahliae remain unclear. Here, we functionally characterized MPK3, a MAPK gene from cotton. MPK3 was induced in the roots of both resistant and susceptible cotton cultivars by V. dahliae inoculation. Transgenic cotton and tobacco with constitutively higher GbMPK3 expression conferred higher V. dahliae susceptibility, while MPK3 knockdown in cotton has limited effect on cotton resistance to V. dahliae. Expression profiling revealed that SA-mediated defense pathway genes (WRKY70, PR1, and PR5) accumulated after V. dahliae inoculation in roots of both wild-type and transgenic cotton, and the expression levels of these genes were higher in GbMPK3-overexpressing plants than in wild-type plants, indicating that GbMPK3 upregulation may reduce plant resistance to V. dahliae through regulating salicylic acid signaling transduction.

Flavonoid accumulation in spontaneous cotton mutant results in red coloration and enhanced disease resistance.

Cotton, the leading natural fiber, is cultivated worldwide, but its production is seriously threatened by pathogens. Accordingly, the selection of resistant cultivars has become a key priority of cotton breeding programs. In this study, a spontaneous mutant with red coloration (S156) and a control cultivar (S78) were used as experimental materials for a comparative analysis. Metabolomic analysis revealed the enrichment of flavonoids in S156 leaves compared with S78 leaves, and transcriptomic analysis revealed the upregulated expression of flavonoid biosynthesis genes in S156 leaves relative to S78 leaves. In addition, the red mutant showed a significantly increase in resistance to Verticillium dahliae, a fungal pathogen that poses a major threat to cotton production. The pathogen invasion process was suppressed in the red cotton cultivar. This study reveals the mechanism underlying the red coloration of S156 cotton and indicates the great potential of red cotton in pathogen- and insect-resistant breeding of cotton.

Calcium-dependent protein kinases in cotton: insights into early plant responses to salt stress.

BACKGROUND: Soil salinization is one of the major environmental constraints to plant growth and agricultural production worldwide. Signaling components involving calcium (Ca2+) and the downstream calcium-dependent protein kinases (CPKs) play key roles in the perception and transduction of stress signals. However, the study of CPKs in cotton and their functions in response to salt stress remain unexplored. RESULTS: A total of 98 predicted CPKs were identified from upland cotton (Gossypium hirsutum L. 'TM-1'), and phylogenetic analyses classified them into four groups. Gene family distribution studies have revealed the substantial impacts of the genome duplication events to the total number of GhCPKs. Transcriptome analyses showed a wide distribution of CPKs' expression among different organs. A total of 19 CPKs were selected for their rapid responses to salt stress at the transcriptional level, most of which were also incduced by the thylene-releasing chemical ethephon, suggesting a partal overlap of the salinity and ethylene responses. Silencing of 4 of the 19 CPKs (GhCPK8, GhCPK38, GhCPK54, and GhCPK55) severely compromised the basal cotton resistance to salt stress. CONCLUSIONS: Our genome-wide expression analysis of CPK genes from up-land cotton suggests that CPKs are involved in multiple developmental responses as well as the response to different abiotic stresses. A cluster of the cotton CPKs was shown to participate in the early signaling events in cotton responses to salt stress. Our results provide significant insights on functional analysis of CPKs in cotton, especially in the context of cotton adaptions to salt stress.