Gossypol Biosynthesis Research Articles

Insights into the role of GhCYP and GhTPS in the gossypol biosynthesis pathway via a multiomics and functional-based approach in cotton

Two Cotton Research Institute (CRI) near-isogenic lines, CRI-12 glanded and CRI-12 glandless, were used to pinpoint potential genes and metabolic pathways linked to gossypol biosynthesis through transcriptome sequencing. We discovered more than 235 million clean reads and 1,184 differentially expressed genes (DEGs). Consecutively, we conducted a weighted gene co-expression network analysis and found strong correlation of white and yellow modules containing GhTPS (GH_D09G0090) and GhCYP (GH_D05G2016) hub genes with the gossypol content. The importance of the GhTPS and GhCYP genes was demonstrated using RT-qPCR, virus-induced gene silencing (VIGS) and target metabolite analysis. Silencing of these genes resulted in fewer glands on both leaves and stems two weeks after the infection, as compared to the wild type. In addition, a total of 152 metabolites were identified through targeted metabolite profiling. Differential metabolite screening revealed 12 and 18 significantly different metabolites in TRV:GhTPS and TRV:GhCYP plants vs. control group, respectively, showing reduction in accumulation of metabolites compared to the control. The content of hemigossypol, the final product of gossypol biosynthesis, was also reduced, as revealed by target metabolite analysis, suggesting the role of these genes in the gossypol biosynthetic pathway. Furthermore, a highly significant difference in gossypol content between the glanded and glandless lines was recorded. The findings of this study reveal a strong link between the gossypol content and GhTPS and GhCYP hub genes, suggesting their role in the gossypol biosynthetic pathway to reduce the accumulation of hemigossypol, which may offer new comprehension into the regulatory checkpoints of the gossypol biosynthesis pathway in cotton.

Cotton sprouts as potential vegetable source: Nutrient compositions and alterations in metabolomics and transcriptomics between light and dark growth conditions

Cottonseed is a by-product of cotton industry, but its broad application in human diets has not been fully elucidated. This study aims to analyse the nutritional composition of cotton sprouts and investigate metabolic and transcriptomic differences under light and darkness. The findings revealed that the glanded ZM41 yellow cotton sprouts were rich in protein (2.8 %), vitamin E (0.443 mg/100 g), and minerals, with < 0.01 % free gossypol. Moreover, protein, aspartic acid (1,026 mg/100 g), and serine (136 mg/100 g) reached their highest levels in nine-day-old sprouts, while no significant differences were observed in the other nutrients on the 7th, 9th, or 11th day. The average quantitative values of 64 cottonseed varieties found gossypol (27.9 mg/kg), protein (3.6 %), potassium (1,487.3 mg/kg), calcium (506.2 mg/kg) and magnesium (330.2 mg/kg). Integrated metabolome and transcriptome analyses revealed that 317 out of 1,880 metabolites were differentially accumulated between light and dark conditions, wherein the downregulation of structural genes encoding CHI, F3H, FLS, DFR, F3′H, ANS, LAR and ANR resulted in decreased flavonoid levels in dark-cultivated sprouts, while the upregulation of HMGR and 2-ODD-1 enhanced the biosynthesis of gossypol. This study provides insights into the nutritional and metabolic aspects of cotton sprouts, offering a reference for potential commercial applications.

GhMYC1374 regulates the cotton defense response to cotton aphids by mediating the production of flavonoids and free gossypol

Myelocytomatosis (MYC) transcription factors (TFs) in plants are well-known regulators of plant defense against herbivores. However, the role and mechanism of MYC TFs in cotton (Gossypium hirsutum L.) defense against cotton aphids (Aphis gossypii Glover) remain still elusive. Herein, on the basis of aphid-induced cotton transcriptome analysis, GhMYC1374, a cotton MYC2-like TF that was highly induced by cotton aphid attack, has been identified that confers cotton aphid resistance in cotton. GhMYC1374 was an intranuclear transcription factor with three domains: bHLH-MYC_N, RBR and bHLH_AtAIB_like. GhMYC1374 was induced under cotton aphid feeding, exogenous methyl jasmonate (MeJA) and salicylic acid (SA) treatments. GhMYC1374 transient overexpression in cotton plants enhanced cotton aphid-resistance, while GhMYC1374 silence through VIGS (virus induced gene silencing) decreased cotton aphid-resistance. GhMYC1374 transient overexpression of in cotton plants activated the phenylpropane pathway and promoted the synthesis of flavonoids, and resistance to thus enhanced the cotton resistance against aphids. In contrast, GhMYC1374 silence inhibited the biosynthesis of flavonoids. In addition, GhMYC1374 also positively activated the expression of the biosynthetic genes of free gossypol, leading to the high content of free gossypol. Taken together, our results suggest that GhMYC1374 is involved in the cotton defense response against cotton aphids by regulating the biosynthesis of flavonoids and free gossypol.

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.

Genome-wide identification and expression analysis of terpene synthases in Gossypium species in response to gossypol biosynthesis.

Cottonseed is an invaluable resource, providing protein, oil, and abundant minerals that significantly contribute to the well-being and nutritional needs of both humans and livestock. However, cottonseed also contains a toxic substance called gossypol, a secondary metabolite in Gossypium species that plays an important role in cotton plant development and self-protection. Herein, genome-wide analysis and characterization of the terpene synthase (TPS) gene family identified 304 TPS genes in Gossypium. Bioinformatics analysis revealed that the gene family was grouped into six subgroups TPS-a, TPS-b, TPS-c, TPS-e, TPS-f, and TPS-g. Whole-genome, segmental, and tandem duplication contributed to the evolution of TPS genes. According to the analysis of selection pressure, it was predicted that TPS genes experience predominantly negative selection, with positive selection occurring subsequently. RT-qPCR analysis in TM-1 and CRI-12 lines revealed GhTPS48 gene as the candidate gene for silencing experiments. To summarize, comprehensive genome-wide studies, RT-qPCR, and gene silencing experiments have collectively demonstrated the involvement of the TPS gene family in the biosynthesis of gossypol in cotton.

Identification of MYB gene family and functional analysis of GhMYB4 in cotton (Gossypium spp.).

Myeloblastosis (MYB) transcription factors (TFs) form a large gene family involved in a variety of biological processes in plants. Little is known about their roles in the development of cotton pigment glands. In this study, 646 MYB members were identified in Gossypium hirsutum genome and phylogenetic classification was analyzed. Evolution analysis revealed assymetric evolution of GhMYBs during polyploidization and sequence divergence of MYBs in G. hirustum was preferentially happend in D sub-genome. WGCNA (weighted gene co-expression network analysis) showed that four modules had potential relationship with gland development or gossypol biosynthesis in cotton. Eight differentially expressed GhMYB genes were identified by screening transcriptome data of three pairs of glanded and glandless cotton lines. Of these, four were selected as candidate genes for cotton pigment gland formation or gossypol biosynthesis by qRT-PCR assay. Silencing of GH_A11G1361 (GhMYB4) downregulated expression of multiple genes in gossypol biosynthesis pathway, indicating it could be involved in gossypol biosynthesis. The potential protein interaction network suggests that several MYBs may have indirect interaction with GhMYC2-like, a key regulator of pigment gland formation. Our study was the systematic analysis of MYB genes in cotton pigment gland development, providing candidate genes for further study on the roles of cotton MYB genes in pigment gland formation, gossypol biosynthesis and future crop plant improvement.

Transcriptome analysis reveals the effect of grafting on gossypol biosynthesis and gland formation in cotton

BackgroundGossypol is a unique secondary metabolite and sesquiterpene in cotton, which is mainly synthesized in the root system of cotton and exhibits many biological activities. Previous research found that grafting affected the density of pigment glands and the gossypol content in cotton.ResultsThis study performed a transcriptome analysis on cotton rootstocks and scions of four grafting methods. The gene expression of mutual grafting and self-grafting was compared to explore the potential genes involved in gossypol biosynthesis. A total of six differentially expressed enzymes were found in the main pathway of gossypol synthesis-sesquiterpene and triterpene biosynthesis (map00909): lupeol synthase (LUP1, EC:5.4.99.41), beta-amyrin synthase (LUP2, EC:5.4.99.39), squalene monooxygenase (SQLE, EC:1.14.14.17), squalene synthase (FDFT1, EC:2.5.1.21), (-)-germacrene D synthase (GERD, EC:4.2.3.75), ( +)-delta-cadinene synthase (CADS, EC:4.2.3.13). By comparing the results of the gossypol content and the density of the pigment gland, we speculated that these six enzymes might affect the biosynthesis of gossypol. It was verified by qRT-PCR analysis that grafting could influence gene expression of scion and stock. After suppressing the expression of the LUP1, FDFT1, and CAD genes by VIGS technology, the gossypol content in plants was significantly down-regulated.ConclusionsThese results indicate the potential molecular mechanism of gossypol synthesis during the grafting process and provide a theoretical foundation for further research on gossypol biosynthesis.

Gossypol and related compounds are produced and accumulate in the aboveground parts of the cotton plant, independent of roots as the source.

Use of Ultra-low gossypol cottonseed event as a scion in a graft combination confirmed that roots are not a source of terpenoids in the aboveground parts of a cotton plant. Gossypol and related terpenoids, derived from the same basic biosynthetic pathway, are present in the numerous lysigenous glands in the aboveground parts of a cotton plant. Roots, with sparse presence of such glands, do produce significant amount of gossypol and a different set of terpenoids. These compounds serve a defensive function against various pests and pathogens. This investigation was undertaken to examine whether gossypol produced in the roots can replenish the gossypol content of the cottonseed-glands that are largely devoid of this terpenoid in a genetically engineered event. Graft unions between a scion derived from the RNAi-based, Ultra-low gossypol cottonseed (ULGCS) event, TAM66274, and a rootstock derived from wild-type parental genotype, Coker 312 (Coker), were compared with various other grafts that served as controls. The results showed that the seeds developing within the scion of test grafts (ULGCS/Coker) continued to maintain the ultra-low gossypol levels found in the TAM66274 seeds. Molecular analyses confirmed that while the key gene involved in gland development showed normal activity in the developing embryos in the scion, two genes encoding the enzymes involved in gossypol biosynthesis were suppressed. Thus, the gene expression data confirmed the results obtained from biochemical measurements and collectively demonstrated that roots are not a source of gossypol for the aboveground parts of the cotton plant. These findings, combined with the results from previous investigations, support the assertion that gossypol and related terpenoids are produced in a highly localized manner in various organs of the cotton plant and are retained therein.

Grafting‐induced seed gossypol levels by demethylation of (+)‐delta‐cadinene synthase genes in upland cotton

AbstractTwo‐year‐grafting experiments within and between Gossypium barbadense and Gossypium hirsutum accessions were conducted to study the source, transfer, and alteration of gossypol and methylation of (+)‐δ‐cadinene synthase (CDN) genes along with whole chromosomes of A01, A04, A11, D01, D05, and D11, harbouring the cotton CDNs, using the whole genome bisulfite sequencing (WGBS) method. The results revealed that although a glandless line used in this study had a low level of gossypol in the seeds, it did not contain any pigment glands, indicating that expression pathways of gland and gossypol were under genetic control of different loci in cotton. Grafts with glandless rootstocks produced significant amounts of total gossypol in the seeds, clearly demonstrating that the source of gossypol biosynthesis was not specific to the root. Heterografts consisting of scion GN, a glandless line, and scion TM‐1 grafted on Pima 3‐79 rootstocks caused increased total seed gossypol levels whereas homograft TT had decreased total seed gossypol levels. Alteration of total gossypol levels was also associated with gland density, and DNA demethylation levels of (+)‐δ‐cadinene synthase genes.

GhMYC2 activates cytochrome P450 gene CYP71BE79 to regulate gossypol biosynthesis in cotton.

GhMYC2 regulates the gossypol biosynthesis pathway in cotton through activation of the expression of gossypol synthesis gene CYP71BE79, CDNC, CYP706B1, DH1, and CYP82D113. Cotton is one of the main cash crops globally. Cottonseed contains fiber, fat, protein, and starch, and has important economic value. However, gossypol in cottonseed seriously affects the development and utilization of cottonseed. Nonetheless, gossypol has great application potential in agriculture, medicine, and industry. Therefore, it is very important to study gossypol biosynthesis and its upstream regulatory pathways. It has been reported that the content of gossypol in hairy roots of cotton is regulated through jasmonic acid signaling; however, the specific molecular mechanism has not been revealed yet. We found that the expression of basic helix-loop-helix family transcription factor GhMYC2 was significantly upregulated after exogenous administration of methyl jasmonate to cotton seedlings, and the content of gossypol changed significantly with the variation of GhMYC2 expression. Further studies revealed that GhMYC2 could specifically bind to the G-Box in the promoter region of CDNC, CYP706B1, DH1, CYP82D113, CYP71BE79 to activate its expression and regulate gossypol synthesis, and its activation of CYP71BE79 promoter was inhibited by GhJAZ2. Not only that GhMYC2 could also interact with GoPGF. In this work, the molecular mechanisms of gossypol biosynthesis regulated by GhMYC2 were analyzed. The results provide a theoretical basis for cultivating new varieties of low-gossypol or high-gossypol cotton and creating excellent germplasm resources.

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.

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.

Genome-wide identification and characterization of terpene synthase genes in Gossypium hirsutum

Terpenoids are widely distributed in plants and play important roles in the regulation of plant growth and development and in the interactions between plants and both the environment and other organisms. However, terpene synthase (TPS) genes have not been systematically investigated in the tetraploid Gossypium hirsutum. In this study, whole genome identification and characterization of the TPS family from G. hirsutum were carried out. Eighty-five TPS genes, including 47 previously unidentified genes, were identified in the G. hirsutum genome and classified into 5 subfamilies according to protein sequence similarities, as follows: 43 GhTPS-a, 29 GhTPS-b, 4 GhTPS-c, 7 GhTPS-e/f, and 2 GhTPS-g members. These 85 TPS genes were mapped onto 19 chromosomes of the G. hirsutum genome. Segmental duplications and tandem duplications contributed greatly to the expansion of TPS genes in G. hirsutum and were followed by intense purifying selection during evolution. Indentification of cis-acting regulatory elements suggest that the expression of TPS genes is regulated by a variety of hormones. RNA sequencing (RNA-seq) expression profile analysis revealed that the TPS genes had distinct spatiotemporal expression patterns, and several genes were highly and preferentially expressed in the leaves of cotton with gossypol glands (glanded cotton) versus a glandless strain. Virus-induced gene silencing (VIGS) of three TPS genes yielded plants characterized by fewer, smaller, and lighter gossypol glands, which indicated that these three genes were responsible for gland activity. Taken together, our results provide a solid basis for further elucidation of the biological functions of TPS genes in relation to gland activity and gossypol biosynthesis to develop cotton cultivars with low cottonseed gossypol contents.

GhCDNC and GhCYP706B1 genes mediate gossypol biosynthesis in upland cotton.

In cotton the identification and characterization of natural defense is a cost-effective, sustainable, and environment-friendly strategy to combat cotton pests. The secondary metabolites traits in cotton plant i.e., toxic gossypol glands play significant role for development and self-defense mechanism. To utilize gossypol in breeding implements, the understanding of gossypol initiation biosynthesis genes has vital importance at reproductive organ development stages. Cotton germplasm of 100 genotypes screened visually based on gossypol glandedness and a core set of ten genotypes was developed. Further three genotypes FH-330 (high glanding), F-280 (low glanding) and IRMA-197 (glandless) were used for determining the transcript abundance of twelve gossypol biosynthesis genes. Out of 100, germplasm categorized as (76) high glanding, (22) medium glanding), one genotype for each (low glanding) and (glandless) category. Real-time qPCR analysis revealed varied expression patterns among selected three genotypes. Out of twelve, three genes CYP706B1, CDNC and 2ODD-1 had strong expression levels in all tested tissues in high glanded genotype, while, slight or no expression of these genes was recorded in low glanding and glandless genotype, respectively. The shell of developing boll (10, 20, 30 DPA), and developing embryo (20, 30 DPA) showed substantially medium to maximum expression, respectively while high to medium expression was recorded in sepals and leaf tissue. Our study demonstrated that CYP706B1, CDNC and 2ODD-1 are the most promising genes involved in gossypol biosynthesis. Developing boll shell, developing embryo, leaf and sepal also have significant ability to synthesize gossypol. This will provide scientists a way to manipulate gossypol contents in economically important organs of cotton plant for targeted breeding.

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.

Design, Synthesis, and Bioactivities of Phthalide and Coumarin Derivatives Based on the Biosynthesis and Structure Simplification of Gossypol

Because gossypol and hemigossypol show antiviral activity but are structurally complex, we designed and synthesized a series of structurally simpler phthalide and coumarin derivatives. The phthalide derivatives were synthesized by opening the naphthalene ring of hemigossypol, and the coumarin derivatives were synthesized by ring-opening reactions of the phthalide derivatives with the goal of investigating the effect of the lactone ring size on bioactivity. The bioassay results showed that the two series of target compounds possessed moderate to good activities against tobacco mosaic virus, One of the compounds showed in vivo inactivation, curative, and protection activities of 50 ± 1, 53 ± 3, and 48 ± 2% at 500 mg/L, values which are higher than those of gossypol (32 ± 1, 35 ± 1, 29 ± 1%, respectively) and comparable to those of hemigossypol (55 ± 1, 49 ± 1, and 48 ± 1%, respectively) and the commercial antiviral agent ningnanmycin (56 ± 2, 54 ± 1, 58 ± 1%) at the same dose. Thus, this compound is a promising candidate for the development of new anti-plant-virus agents. In addition, most of the synthesized compounds showed broad-spectrum activity when tested against 14 kinds of phytopathogenic fungi and showed selectivity against Sclerotinia sclerotiorum, Physalospora piricola, and Rhizoctonia cerealis. Moreover, some of the compounds exhibited activity against Plutella xylostella larvae; the two most active compounds exhibited larvicidal activities (LC50) of 4.10 and 5.47 mg/L, respectively. Further studies showed that these compounds also exhibited insecticidal activities against Mythimna separata, Helicoverpa armigera, and Pyrausta nubilalis larvae.

Identification and characterization of DIR gene family in Schisandra chinensis

Dirigent(DIR) proteins are involved in the biosynthesis of lignin, lignans, and gossypol in plants and respond to biotic and abiotic stresses. Based on the full-length transcriptome of Schisandra chinensis, bioinformatics methods were used to preliminarily identify the DIR gene family and analyze the physico-chemical properties, subcellular localization, conserved motifs, phylogeny, and expression patterns of the proteins. The results showed that a total of 34 DIR genes were screened and the encoded proteins were 156-387 aa. The physico-chemical properties of the proteins were different and the secondary structure was mainly random coil. Half of the DIR proteins were located in chloroplast, while the others in extracellular region, endoplasmic reticulum, cytoplasm, etc. Phylogenetic analysis of DIR proteins from S. chinensis and the other 8 species such as Arabidopsis thaliana, Oryza sativa, and Glycine max demonstrated that all DIR proteins were clustered into 5 subfamilies and that DIR proteins from S. chinensis were in 4 subfamilies. DIR-a subfamily has the unique structure of 8 β-sheets, as verified by multiple sequence alignment. Finally, through the analysis of the transcriptome of S. chinensis fruit at different development stages, the expression pattern of DIR was clarified. Combined with the accumulation of lignans in fruits at different stages, DIR might be related to the synthesis of lignans in S. chinensis. This study lays a theoretical basis for exploring the biological functions of DIR genes and elucidating the biosynthesis pathway of lignans in S. chinensis.

Identification and characteristics of a novel gland-forming gene in cotton.

The cotton (Gossypium hirsutum) pigment gland is a distinctive structure that functions as the main deposit organ of gossypol and its derivatives. It is also an ideal system in which to study cell differentiation and organogenesis. However, only a few genes that determine the process of gland formation have been reported, including GoPGF, CGP1, and CGFs; the molecular mechanisms underlying gland initiation are still largely unclear. Here, we report the discovery of the novel stem pigment gland-forming gene GoSPGF by map-based cloning; annotated as a GRAS transcription factor, this gene is responsible for the glandless trait specifically on the stem. In the stem glandless mutant T582, a point mutation (C to A) was found to create a premature stop codon and truncate the protein. Similarly, virus-induced gene silencing of GoSPGF resulted in glandless stems and dramatically reduced gossypol content. Comparative transcriptomic data showed that loss of GoSPGF significantly suppressed expression of many genes involved in gossypol biosynthesis and altered expression of genes involved in gibberellic acid signaling/biosynthesis. Overall, these findings provide more insight into the networks regulating glandular structure differentiation and formation in cotton, which will be helpful for understanding other plants bearing special gland structures such as tobacco (Nicotiana benthamiana), artemisia annua, mint (Mentha spp.), and rubber (Hevea brasiliensis).

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

BackgroundGossypium 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.ResultsIn 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.ConclusionsThese 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.

Cotton roots are the major source of gossypol biosynthesis and accumulation

BackgroundGossypol 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.ResultsIn 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.ConclusionsOur 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.