Structural basis of lactate dehydrogenase A-gossypol complex.

Lactate dehydrogenase A (LDHA) is a key enzyme in Warburg's effect, a characteristic of cancer cells. LDHA is a target of anticancer agents that inhibit the metabolism of cancer cells. Gossypol is a known cancer therapeutic agent that inhibits LDHA by competitive inhibition. However, the mechanisms of inhibition of LDHA by gossypol is unknown. Here, we elucidate the binding of gossypol and LDHA using biochemical and biophysical methods. The crystal structure of the complex between LDHA and gossypol is presented. The binding of gossypol affects LDHA activity by a conformational change in the active-site loop. Our research contributes to the structural insight into LDHA with gossypol and approaches gossypol as a novel therapeutic candidate targeting the metabolic pathways for cancer cells.

Mirids secrete a TOPLESS targeting protein to enhance JA-mediated defense and gossypol accumulation for antagonizing cotton bollworms on cotton plants.

Most coexisting insect species exhibit stunted growth compared to individual species on plants. This phenomenon reflects an interspecific antagonism drawing extensive attention, while the underlying mechanisms remain largely uncharacterized. Mirids (Apolygus lucorum) and cotton bollworms (Helicoverpa armigera) are two common cotton pests. We identified a secretory protein, ASP1, from the oral secretion of mirids, found in the nucleus of mirid-infested cotton leaves. ASP1 specifically targets the transcriptional co-repressor TOPLESS (TPL) and inhibits NINJA-mediated recruitment of TPL, promoting plant defense response and gossypol accumulation in cotton glands. ASP1-enhanced defense inhibits the growth of cotton bollworms on cotton plants, while having limited impact on mirids. The mesophyll-feeding characteristic allows mirids to avoid most cotton glands, invalidating cotton defense. Our investigation reveals the molecular mechanism by which mirids employ cotton defense to selectively inhibit the feeding of cotton bollworms.

Integrative transcriptomic and metabolomic analysis to elucidate the effect of gossypol on Enterobacter sp. GD5.

Gossypol, a yellow polyphenolic compound found in the Gossypium genus, is toxic to animals that ingest cotton-derived feed materials. However, ruminants display a notable tolerance to gossypol, attributed to the pivotal role of ruminal microorganisms in its degradation. The mechanisms of how rumen microorganisms degrade and tolerate gossypol remain unclear. Therefore, in this study, Enterobacter sp. GD5 was isolated from rumen fluid, and the effects of gossypol on its metabolism and gene expression were investigated using liquid chromatography-mass spectrometry (LC-MS) and RNA analyses. The LC-MS results revealed that gossypol significantly altered the metabolic profiles of 15 metabolites (eight upregulated and seven downregulated). The Kyoto Encyclopedia of Genes and Genomes analysis results showed that significantly different metabolites were associated with glutathione metabolism in both positive and negative ion modes, where gossypol significantly affected the biosynthesis of amino acids in the negative ion mode. Transcriptomic analysis indicated that gossypol significantly affected 132 genes (104 upregulated and 28 downregulated), with significant changes observed in the expression of catalase peroxidase, glutaredoxin-1, glutathione reductase, thioredoxin 2, thioredoxin reductase, and alkyl hydroperoxide reductase subunit F, which are related to antioxidative stress. Furthermore, Gene Ontology analysis revealed significant changes in homeostatic processes following gossypol supplementation. Overall, these results indicate that gossypol induces oxidative stress, resulting in the increased expression of antioxidative stress-related genes in Enterobacter sp. GD5, which may partially explain its tolerance to gossypol.

Structural identification and comprehension of human ALDH1L1-Gossypol complex.

The folate metabolism enzyme ALDH1L1 catalyzed 10-formyltetrahydrofolate to tetrahydrofolate and CO2. Non-small cell lung cancer cells (NSCLC) strongly express ALDH1L1. Gossypol binds to an allosteric site and disrupts the folate metabolism by preventing NADP+ binding. The Cryo-EM structures of tetrameric C-terminal aldehyde dehydrogenase human ALDH1L1 complex with gossypol were examined. Gossypol-bound ALDH1L1 interfered with NADP+ by shifting the allosteric site of the structural conformation, producing a closed-form NADP+ binding site. In addition, the inhibition activity of ALDH1L1 was targeted with gossypol in NSCLC. The gossypol treatment had anti-cancer effects on NSCLC by blocking NADPH and ATP production. These findings emphasize the structure characterizing ALDH1L1 with gossypol.

GhHAM regulates GoPGF-dependent gland development and contributes to broad-spectrum pest resistance in cotton.

Cotton is a globally cultivated crop, producing 87% of the natural fiber used in the global textile industry. The pigment glands, unique to cotton and its relatives, serve as a defense structure against pests and pathogens. However, the molecular mechanism underlying gland formation and the specific role of pigment glands in cotton's pest defense are still not well understood. In this study, we cloned a gland-related transcription factor GhHAM and generated the GhHAM knockout mutant using CRISPR/Cas9. Phenotypic observations, transcriptome analysis, and promoter-binding experiments revealed that GhHAM binds to the promoter of GoPGF, regulating pigment gland formation in cotton's multiple organs via the GoPGF-GhJUB1 module. The knockout of GhHAM significantly reduced gossypol production and increased cotton's susceptibility to pests in the field. Feeding assays demonstrated that more than 80% of the cotton bollworm larvae preferred ghham over the wild type. Furthermore, the ghham mutants displayed shorter cell length and decreased gibberellins (GA) production in the stem. Exogenous application of GA3 restored stem cell elongation but not gland formation, thereby indicating that GhHAM controls gland morphogenesis independently of GA. Our study sheds light on the functional differentiation of HAM proteins among plant species, highlights the significant role of pigment glands in influencing pest feeding preference, and provides a theoretical basis for breeding pest-resistant cotton varieties to address the challenges posed by frequent outbreaks of pests.

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.

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.

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.

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.