Molecular and functional characterization of heat-shock protein 70 in Aphis gossypii under thermal and xenobiotic stresses.

Aphis gossypii, a globally distributed and economically significant pest of several crops, is known to infest a wide range of host plants. Heat shock proteins (Hsps), acting as molecular chaperones, are essential for the insect's environmental stress responses. The present study investigated the molecular characteristics and expression patterns of AgHsp70, a heat shock protein gene, in Aphis gossypii. Our phylogenetic analysis revealed that AgHsp70 shared high similarity with homologs from other insects, suggesting a conserved function across species. The developmental expression profiles of AgHsp70 in A. gossypii showed that the highest transcript levels were observed in the fourth instar nymphs, while the lowest levels were detected in the third instar nymphs. Heat stress and exposure to four different xenobiotics (2-tridecanone, tannic acid, gossypol, and flupyradifurone (4-[(2,2-difluoroethyl)amino]-2(5H)-furanone)) significantly up-regulated AgHsp70 expression. Knockdown of AgHsp70 using RNAi obviously increased the susceptibility of cotton aphids to 2-tridecanone, gossypol and flupyradifurone. Dual-luciferase reporter assays revealed that gossypol and flupyradifurone significantly enhanced the promoter activity of AgHsp70 at a concentration of 10 mg/L. Furthermore, we identified the transcription factor heat shock factor (HSF) as a regulator of AgHsp70, as silencing AgHSF reduced AgHsp70 expression. Our results shed light on the role of AgHsp70 in xenobiotic adaptation and thermo-tolerance.

Improvement of qualitative and quantitative traits in cotton under normal and stressed environments using genomics and biotechnological tools: A review.

Due to the increasing demand for high-quality and high fiber-yielding cotton (Gossypium spp.), research into the development of stress-resilient cotton cultivars has acquired greater significance. Various biotic and abiotic stressors greatly affect cotton production and productivity, posing challenges to the future of the textile industry. Moreover, the content and quality of cottonseed oil can also potentially be influenced by future environmental conditions. Apart from conventional methods, genetic engineering has emerged as a potential tool to improve cotton fiber quality and productivity. Identification and modification of genome sequences and the expression levels of yield-related genes using genetic engineering approaches have enabled to increase both the quality and yields of cotton fiber and cottonseed oil. Herein, we evaluate the significance and molecular mechanisms associated with the regulation of cotton agronomic traits under both normal and stressful environmental conditions. In addition, the importance of gossypol, a toxic phenolic compound in cottonseed that can limit consumption by animals and humans, is reviewed and discussed.

Effect of feeding increasing levels of whole cottonseed on milk and milk components, milk fatty acid profile, and total-tract digestibility in lactating dairy cows.

Dietary fat is fed to increase energy intake and provide fatty acids (FA) to support milk fat production. Oilseeds contain unsaturated FA that increase the risk for biohydrogenation-induced milk fat depression, but FA in whole cottonseed (WCS) are expected to be slowly released in the rumen and thus have a lower risk for biohydrogenation-induced milk fat depression. Our hypothesis was that increasing dietary WCS would increase milk fat yield by providing additional dietary FA without induction of milk fat depression. Four primiparous and 8 multiparous lactating Holstein cows, 136 ± 35 and 127 ± 4 DIM, respectively, were arranged in a replicated 4 × 4 Latin square design with 21-d periods. Treatments were WCS provided at 0%, 3.4%, 6.8%, and 9.9% of dietary dry matter, and WCS was substituted for cottonseed hulls and soybean meal to maintain dietary fiber and protein. Treatment did not change milk yield. There was a treatment-by-parity interaction for milk fat percent and yield with a quadratic decreased in primiparous cows but no effect of WCS in multiparous cows. Cottonseed linearly increased milk fat trans-10 18:1 in primiparous cows but not in multiparous cows. Increasing WCS increased milk preformed (18C) FA yield and partially overcame the trans-10 18:1 inhibition of de novo FA synthesis in the primiparous cows. Apparent transfer of 18C FA from feed to milk decreased in all cows as WCS increased, but the magnitude of the change was greater in primiparous cows. Increasing WCS decreased total-tract apparent dry matter, organic matter, and neutral detergent fiber digestibility. There was no change in total FA digestibility. However, 18C FA digestibility tended to be decreased in both parities and 16C FA digestibility was quadratically increased in multiparous cows but not changed in primiparous cows. Total fecal flow of intact WCS increased as WCS level increased, but fecal flow of intact seeds as a percentage consumed was similar across treatments. Fecal flow of intact seeds was greater in multiparous cows (4.3% vs. 1.1% of consumed). Plasma concentrations of glucose, nonesterified FA, triglycerides, and insulin were not changed. However, plasma urea-N increased with increasing WCS. Plasma gossypol increased with WCS (0.08-1.15 µg/mL) but was well below expected toxic levels. In conclusion, WCS maintained milk and milk component yield when fed at up to 9.9% of the diet to multiparous cows without concerns of gossypol toxicity, but primiparous cows were more susceptible to biohydrogenation-induced milk fat depression in the current trial. This highlights the interactions of parity with diet composition when feeding rumen-available unsaturated fat to dairy cows.

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.

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.

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.

Comparative Transcriptome Analysis Revealed Key Genes Regulating Gossypol Synthesis in Tetraploid Cultivated Cotton.

Tetraploid cultivated cotton (Gossypium spp.) produces cottonseeds rich in protein and oil. Gossypol and related terpenoids, stored in the pigment glands of cottonseeds, are toxic to human beings and monogastric animals. However, a comprehensive understanding of the genetic basis of gossypol and gland formation is still lacking. We performed a comprehensive transcriptome analysis of four glanded versus two glandless tetraploid cultivars distributed in Gossypium hirsutum and Gossypium barbadense. A weighted gene co-expression network analysis (WGCNA) based on 431 common differentially expressed genes (DEGs) uncovered a candidate module that was strongly associated with the reduction in or disappearance of gossypol and pigment glands. Further, the co-expression network helped us to focus on 29 hub genes, which played key roles in the regulation of related genes in the candidate module. The present study contributes to our understanding of the genetic basis of gossypol and gland formation and serves as a rich potential source for breeding cotton cultivars with gossypol-rich plants and gossypol-free cottonseed, which is beneficial for improving food safety, environmental protection, and economic gains of tetraploid cultivated cotton.

The Complete Genome Sequence of a Gossypol-Degrading Bacterial Strain, Raoultella sp. YL01.

Cottonseed meal is an important source of plant protein for the meal fodder materials. But its usage in animal breeding industry is limited by a type of toxic phenol, gossypol, that has toxic effects on animal health. Microbial degradation is a promising way to lower down gossypol in cottonseed meal. However, the molecular mechanisms of bio-degradation of gossypol is still unclear. In this study we isolated a gossypol-degrading bacterial strain, YL01, and sequenced its complete genome via Oxford Nanopore sequencing method. There is a chromosome (5,737,005 bp) and a plasmid (136,446 bp) in YL01. 5489 protein coding genes in total were functionally annotated. 16S rRNA analysis showed that YL01 taxonomically belongs to the genus of Raoultella. YL01 is the first published complete genome sequence of microbes capable of gossypol degradation. Gene function annotation showed that 126 protein coding genes may involve in gossypol catabolism. Sequence similarity analysis showed that, as the only gossypol-degrading strain in the genus of Raoultella, YL01 uniquely holds 260 genes that are not possessed by other Raoultella strains. Our work gives a preliminary list for genes responsible for gossypol degradation but further investigations are needed to completely disclose this molecular processes.

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.

BACKGROUND: Gossypol 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. RESULTS: This 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. CONCLUSIONS: These results indicate the potential molecular mechanism of gossypol synthesis during the grafting process and provide a theoretical foundation for further research on gossypol biosynthesis.

Effects of cottonseed meal on performance, gossypol residue, liver function, lipid metabolism, and cecal microbiota in geese.

A total of 240 28-d-old male goslings were used to investigate the effects of cottonseed meal (CSM) on performance, gossypol residue, liver function, lipid metabolism, and cecal microbiota. All birds were randomly allotted into five groups (eight goslings/replicate, six replicates/group) and subjected to a 35-d experiment. Five isonitrogenous and isoenergetic diets were formulated to produce diets in which 0% (control), 25% (CSM25), 50% (CSM50), 75% (CSM75), and 100% (CSM100) of protein from soybean meal was replaced by protein from CSM. The free gossypol contents in the five diets were 0, 44, 92, 135, and 183 mg/kg, respectively. Dietary CSM did not affect the growth performance from 29 to 63 d and carcass traits at 63 d (P > 0.05). Liver gossypol residues were influenced (P < 0.05) by dietary CSM and increased linearly (P < 0.05) and quadratically (P < 0.05) as dietary CSM increased. The malondialdehyde content of the liver was lower in the CSM100 group than in the other groups (P < 0.05). Serum triglyceride and low-density lipoprotein cholesterol were influenced (P < 0.05) by dietary CSM and increased linearly (P < 0.05) with increasing dietary CSM. Dietary CSM altered (P < 0.05) the composition of some fatty acids in the liver and breast muscle. The concentration of linolenic acid and Σn-3 polyunsaturated fatty acid (PUFA) in the liver and breast muscle decreased linearly, but the Σn-6/Σn-3 PUFA ratio increased linearly with increasing dietary CSM (P < 0.05). Dietary CSM affected (P < 0.05) the hepatic gene expression of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC), and apolipoprotein B (ApoB). As the dietary CSM concentration increased, the hepatic gene expression of FAS increased linearly (P < 0.05) and quadratically (P < 0.05), but the hepatic gene expression of ACC and ApoB increased linearly (P < 0.05). The CSM diet decreased the relative abundance of the Bacteroidota and Bacteroides (P < 0.05), and the CSM50 diet increased the relative abundance of the Firmicutes and Colidextribacter (P < 0.05) compared to the control group. Overall, these results show that dietary CSM has no adverse effects on the performance of goslings from 29 to 63 d. However, CSM affected organismal lipid metabolism, reduced products' edible value, and adaptively altered cecum microbiota.

The shortage of feed resources and the rising price have become one of the significant challenges for animal husbandry worldwide. Considering the strong tolerance and adaptability to roughage of geese, less expensive crop byproducts are used in goose feed by animal nutritionists. Cottonseed meal (CSM) is a potential substitute for soybean meal, and the main concern for its use in poultry feed is free gossypol. This study aimed to investigate the effects of CSM on the performance, gossypol residue, liver function, lipid metabolism, and cecal microbiota in geese. Results showed that dietary CSM has no adverse effects on the performance and liver function of goslings. However, gossypol residue in goose liver increased with increasing dietary CSM. Besides, CSM affected organismal lipid metabolism, altered the tissue fatty acid composition, and adaptively changed cecum microbial microbiota. In summary, CSM is a good dietary protein source for geese, but further attention may be needed to its use for the edible value of goose products.

(-)- Gossypol Inhibition of Musashi-Mediated Forgetting Improves Memory and Age-Dependent Memory Decline in Caenorhabditis elegans.

Musashi RNA-binding proteins (MSIs) retain a pivotal role in stem cell maintenance, tumorigenesis, and nervous system development. Recently, we showed in C. elegans that Musashi (MSI-1) actively promotes forgetting upon associative learning via a 3'UTR-dependent translational expression of the Arp2/3 actin branching complex. Here, we investigated the evolutionary conserved role of MSI proteins and the effect of their pharmacological inhibition on memory. Expression of human Musashi 1 (MSI1) and Musashi 2 (MSI2) under the endogenous Musashi promoter fully rescued the phenotype of msi-1(lf) worms. Furthermore, pharmacological inhibition of human MSI1 and MSI2 activity using (-)- gossypol resulted in improved memory retention, without causing locomotor, chemotactic, or learning deficits. No drug effect was observed in msi-1(lf) treated worms. Using Western blotting and confocal microscopy, we found no changes in MSI-1 protein abundance following (-)- gossypol treatment, suggesting that Musashi gene expression remains unaltered and that the compound exerts its inhibitory effect post-translationally. Additionally, (-)- gossypol suppressed the previously seen rescue of the msi-1(lf) phenotype in worms expressing human MSI1 specifically in the AVA neuron, indicating that (-)- gossypol can regulate the Musashi pathway in a memory-related neuronal circuit in worms. Finally, treating aged worms with (-)- gossypol reversed physiological age-dependent memory decline. Taken together, our findings indicate that pharmacological inhibition of Musashi might represent a promising approach for memory modulation.

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

MAIN CONCLUSION: 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.

Biodegradation of Free Gossypol by Helicoverpa armigera Carboxylesterase Expressed in Pichia pastoris.

Gossypol is a polyphenolic toxic secondary metabolite derived from cotton. Free gossypol in cotton meal is remarkably harmful to animals. Furthermore, microbial degradation of gossypol produces metabolites that reduce feed quality. We adopted an enzymatic method to degrade free gossypol safely and effectively. We cloned the gene cce001a encoding carboxylesterase (CarE) into pPICZαA and transformed it into Pichia pastoris GS115. The target protein was successfully obtained, and CarE CCE001a could effectively degrade free gossypol with a degradation rate of 89%. When esterase was added, the exposed toxic groups of gossypol reacted with different amino acids and amines to form bound gossypol, generating substances with (M + H) m/z ratios of 560.15, 600.25, and 713.46. The molecular formula was C27H28O13, C34H36N2O6, and C47H59N3O3. The observed instability of the hydroxyl groups caused the substitution and shedding of the group, forming a substance with m/z of 488.26 and molecular formula C31H36O5. These properties render the CarE CCE001a a valid candidate for the detoxification of cotton meal. Furthermore, the findings help elucidate the degradation process of gossypol in vitro.

Effects of cottonseed meal on growth performance, liver redox status, and serum biochemical parameters in goslings at 1 to 28 days of age.

BACKGROUND: Cottonseed meal (CSM), a relatively rich source of protein and amino acids, is used as an inexpensive alternative to soybean meal (SBM) in poultry diets. However, the toxicity of free gossypol in CSM has been a primary concern. The present study was conducted to investigate the effects of CSM on growth performance, serum biochemical parameters, and liver redox status in goslings at 1 to 28 days of age. Three hundred 1-day-old male goslings were randomly divided into 5 groups (10 goslings/pen, 6 replicate pens/group) and subjected to a 28-day experiment. Five isonitrogenous and isoenergetic diets were formulated such that 0% (control), 25% (CSM25), 50% (CSM50), 75% (CSM75), and 100% (CSM100) of protein from SBM was replaced by protein from CSM. The free gossypol contents in the five diets were 0, 56, 109, 166, and 222 mg/kg, respectively. RESULTS: The results showed that dietary CSM was associated with linear decreases in body weight, average daily feed intake and average daily gain and linear increases in the feed-to-gain ratio from 1 to 28 days of age (P < 0.001). As the dietary CSM concentration increased, a numerical increase was found in the mortality of goslings. According to a single-slope broken-line model, the breakpoints for the average daily gain of dietary free gossypol concentration on days 1 to 14, 15 to 28, and 1 to 28 occurred at 23.63, 14.78, and 18.53 mg/kg, respectively. As the dietary CSM concentration increased, serum albumin (P < 0.001) concentrations decreased linearly and serum uric acid (P = 0.011) increased linearly. The hydroxyl radical scavenging ability (P = 0.002) and catalase (P < 0.001) and glutathione peroxidase (P = 0.001) activities of the liver decreased linearly with increasing dietary CSM. However, dietary CSM did not affect the concentrations of reactive oxygen metabolites, malondialdehyde, or protein carbonyl in the liver. CONCLUSIONS: The increasing dietary CSM increased the concentration of free gossypol and altered the composition of some amino acids in the diet. A high concentration of CSM reduced the growth performance of goslings aged 1 to 28 days by decreasing feed intake, liver metabolism, and antioxidant capacity. From the primary concern of free gossypol in CSM, the tolerance of goslings to free gossypol from CSM is low, and the toxicity of free gossypol has a cumulative effect over time.

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

MAIN CONCLUSION: 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.

Comparative Transcriptome Analysis Reveals Genes Associated with the Gossypol Synthesis and Gland Morphogenesis in Gossypium hirsutum.

Gossypium hirsutum is an important source of natural textile fibers. Gossypol, which is a sesquiterpenoid compound mainly existing in the cotton pigment glands, can facilitate resistance to the stress from diseases and pests. The level of gossypol in the cotton is positively correlated to the quantity of pigment glands. However, the underlying regulatory mechanisms of gossypol synthesis and gland morphogenesis are still poorly understood, especially from a transcriptional perspective. The transcripts of young leaves and ovules at 30 DPA of the glanded plants and glandless plants were studied by RNA-Seq and 865 million clean reads were obtained. A total of 34,426 differentially expressed genes (DEGs) were identified through comparative transcriptome analysis. Genes related to gossypol synthesis or gland morphogenesis displayed significant differential expression between the two cultivars. Functional annotation revealed that the candidate genes related to catalytic activity, the biosynthesis of secondary metabolites, and biomolecular decomposition processes. Our work herein unveiled several potential candidate genes related to gossypol synthesis or gland morphogenesis and may provide useful clues for a breeding program of cotton cultivars with low cottonseed gossypol contents.

An Overview of Cotton Gland Development and Its Transcriptional Regulation.

Cotton refers to species in the genus Gossypium that bear spinnable seed coat fibers. A total of 50 species in the genus Gossypium have been described to date. Of these, only four species, viz. Gossypium, hirsutum, G. barbadense, G. arboretum, and G. herbaceum are cultivated; the rest are wild. The black dot-like structures on the surfaces of cotton organs or tissues, such as the leaves, stem, calyx, bracts, and boll surface, are called gossypol glands or pigment glands, which store terpenoid aldehydes, including gossypol. The cotton (Gossypium hirsutum) pigment gland is a distinctive structure that stores gossypol and its derivatives. It provides an ideal system for studying cell differentiation and organogenesis. However, only a few genes involved in the process of gland formation have been identified to date, and the molecular mechanisms underlying gland initiation remain unclear. The terpenoid aldehydes in the lysigenous glands of Gossypium species are important secondary phytoalexins (with gossypol being the most important) and one of the main defenses of plants against pests and diseases. Here, we review recent research on the development of gossypol glands in Gossypium species, the regulation of the terpenoid aldehyde biosynthesis pathway, discoveries from genetic engineering studies, and future research directions.

Enzymological, histological, and serum biomarker responses of snubnose pompano on complete replacement of fishmeal using cottonseed meal supplemented with lysine and methionine in the diet.

In a feeding experiment, cottonseed meal (CSM) was used to replace fishmeal (FM) in the diet of snubnose pompano, Trachinotus blochii, supplemented with lysine and methionine to assess the growth, nutritive profile, hematological, histological, and stress biomarker response. Experimental fishes were randomly stocked in five treatments each with triplicates. Five isonitrogenous and isolipidic diets with graded level of CSM (0, 8.7, 17.4, 26.0, and 34.7%) as replacement for FM protein (0, 25, 50, 75, and 100%) were formulated and fed to respective treatments. Comparison between various parameters among the treatments was made using orthogonal polynomial contrasts to indicate the statistical significance. Higher alkaline phosphatase, acid phosphatase, lactate dehydrogenase, malate dehydrogenase, aspartate, and alanine aminotransferase activities were observed in 0CSM group and followed by 100CSM group as higher inclusion level of CSM with higher free gossypol content did not affect the metabolic enzyme activities. The maximum muscular free gossypol accretion of 1.28 mg kg-1 (on wet basis) was recorded in 100CSM group which was very well below the critical limit set by FDA. As a conclusion, fishmeal can be completely replaced using cottonseed meal in the diet of pompano without adverse effect on growth, metabolism, and general health.

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.