The peroxidative coupling of hemigossypol to (+)- and (-)-gossypol in cottonseed extracts

Peroxidase(s) present in embryo extracts of Gossypium hirsutum cv. Texas Marker 1 catalyzed a bimolecular coupling of [4-(3)H]-hemigossypol to [4,4'-(3)H(2)]-gossypol. The reaction was dependent on the addition of H(2)O(2) and was inhibited 71-94% by 1 and 10mM sodium azide. The phenolic coupling produced 53% (+)-gossypol and 47% (-)-gossypol in close agreement to the 49% (+)-gossypol and 51% (-)-gossypol found in the intact seed. The nearly racemic mixture of (+)-and (-)-gossypol produced in these embryo extracts can be accounted for by non-enzymatic random coupling of the free radicals of hemigossypol produced by the peroxidase. In contrast, peroxidase reaction mixtures containing crude embryo extracts of G. hirsutum var. marie-galante produced 73% (+)-gossypol and 27% (-)-gossypol. These data from the marie-galante extracts and the fact that these intact seed contain 95% (+)-gossypol suggest a regio-stereoselective bimolecular coupling of hemigossypol to gossypol. The development of the peroxidative coupling of hemigossypol to gossypol in maturing seed of G. hirsutum cv. Texas Marker 1 was correlated to the formation of gossypol and suggests that peroxidative coupling of hemigossypol contributes to gossypol biosynthesis.     

棉叶中棉酚的快速提取与测定方法研究

棉酚(G)及其相关物甲氧基半棉酚(DHG)、半棉酚酮(HGQ)、半棉酚(HG)、杀实夜蛾素(H1-4)等是棉花中重要的抗虫性萜烯类次生物质.利用高效液相色谱法(HPLC)对棉酚及其相关物进行了分离,测定了棉叶中的棉酚含量,讨论了不同的提取方法和测定条件对结果的影响,给出了一套简便、快速的分析测试方法,同时与紫外-可见分光光度法(苯胺法)的结果进行了对比,认为对于棉花的抗虫性的研究来说,HPLC是比较适宜的方法.     

8-Hydroxy-(+)-delta-cadinene is a precursor to hemigossypol in Gossypium hirsutum

[(3)H](+)-delta-Cadinene and its 8-hydroxy derivative, prepared from (1RS)-[1-(3)H]FPP by the action of one and two recombinant enzymes, respectively, were infiltrated into cotyledons of bacterial blight-resistant cotton plants as they biosynthesized sesquiterpene phytoalexins in response to infection by Xanthomonas campestris pv. malvacearum. Following both treatments, tritium appeared in the HPLC fraction that contained hemigossypol. Hemigossypol was isolated from the cotyledons that had been treated with [(3)H](+)-8-hydroxy-delta-cadinene and was trimethylsilylated and purified. In two experiments, specific radioactivity of the hemigossypol derivative indicated that 5% and 10%, respectively, of the [(3)H](+)-8-hydroxy-delta-cadinene had been converted to hemigossypol.     

Exudation of terpenoids by cotton roots

Summary The terpenoid aldehydes, desoxyhemigossypol, desoxy-6-methoxyhemigossypol, hemigossypol, 6-methoxyhemigossypol, gossypol, 6-methoxygossypol, and 6,6′-dimethoxygossypol, previously reported to be present in cotton roots (Gossypium hivsutum L.) were found on absorbing surfaces adjacent to roots. Infection of hypocotyls byRhizoctonia solani increased the quantity of terpenoids exuded by roots. Because these compounds are antimicrobial, their presence in the rhizosphere should be considered in studies of the microflora of cotton roots.     

Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-delta-cadinene synthase

Cotton plants were transformed with an antisense construct of cdn1-Cl, a member of a complex gene family of delta-(+)cadinene (CDN) synthase. This synthase catalyzes the cyclization of (E,E)-farnesyl diphosphate to form CDN, and in cotton, it occupies the committed step in the biosynthesis of cadinane sesquiterpenoids and heliocides (sesterterpenoids). Southern analyses of the digestion of leaf DNA from R(o), T(o), and T(1) plants with Hind III, Pst I and Kpn I restriction enzymes show the integration of antisense cdn1-C1 cDNA driven by the CaMV 35S promoter into the cotton genome. Northern blots demonstrate the appearance of cdn synthase mRNA preceding CDN synthase activity and the formation of gossypol in developing cottonseed. T(2) cottonseed show a reduced CDN synthase activity and up to a 70% reduction in gossypol. In T(1) leaves the accumulated amounts of gossypol, hemigossypolone and heliocides are reduced 92.4, 83.3 and 68.4%, respectively. These data demonstrate that the integration of antisense cdn1-C1 cDNA into the cotton genome leads to a reduction of CDN synthase activity and negatively impacts on the biosynthesis of cadinane sesquiterpenoids and heliocides in cotton plants.     

Dimerization of the erythropoietin receptor transmembrane domain in micelles

Erythropoietin receptor (EpoR) homodimerization is an initial regulatory step in erythrocyte formation. Receptor dimers form before ligand binding, suggesting that association between receptor proteins is dependent on the receptor itself. EpoR dimerization is an essential step in erythropoiesis, and misregulation of this dimerization has been implicated in several disease states, including multi-lineage leukemias; nevertheless, how EpoR regulates its own dimerization is unclear. In vivo experiments suggest the single-pass transmembrane helix is the strongest candidate for driving ligand-independent association. To address the self-association potential of this transmembrane segment, we studied its interaction energetics in micelles by utilizing a previously successful Staphylococcal nuclease (SN-EpoR TM) fusion protein. This fusion protein strategy allows expression of the EpoR transmembrane domain in Escherichia coli independent of the other EpoR domains. Sedimentation equilibrium analytical ultracentrifugation of the detergent-solubilized SN-EpoR TM demonstrated that the murine EpoR transmembrane domain self-associates to form dimers. Although this interaction is not as stable as the dimerization of the well-studied glycophorin A transmembrane dimer, the murine EpoR transmembrane domain dimer is more stable than the interactions of the colon carcinoma kinase 4 transmembrane domain. The same experiments with the human EpoR transmembrane domain, which differs from the mouse sequence by only three residues, revealed a less favorable interaction than that of the murine sequence and is only slightly more favorable than that expected for non-preferential binding. These results suggest that the mouse and human receptor proteins may differ in the roles they play in signaling.     

Cadmium-Induced Structure Change of Pigment Glands and the Reduction of the Gossypol Content in Cottonseed Kernels

The risk of cotton production on arable land contaminated with heavy metals has increased in recent years. Cotton shows stronger and more extensive resistance to heavy metals, such as cadmium (Cd) than that of other major crops. Here, a potted plant experiment was performed to study Cd-induced alterations in the cottonseed kernel gossypol content and pigment gland structure at maturity in two transgenic cotton cultivars (ZD-90 and SGK3) and an upland cotton standard genotype (TM-1). The results showed that Cd accumulation in cottonseed kernels increased with increasing Cd levels in the soil. The seed kernel Cd content in plants grown on Cd-treated soils was 10-20 times greater than the amount in the corresponding controls. There was a significant difference in Cd accumulation in cottonseed kernels at the 400 and 600 μM Cd levels. Cd accumulation was higher in SGK3 and ZD-90 than in TM-1. However, the gossypol content in cottonseed kernels was lower in SGK3 and ZD-90 than in TM-1. There was a negative correlation (r = 0.550) between Cd accumulation and the gossypol content in cottonseed kernels. The density of cottonseed kernel pigment glands decreased under Cd stress. This is consistent with the change in gossypol content, which decreased under Cd stress. The damage of the cultivars ZD-90 and SGK3 from Cd poisoning was relatively low under Cd stress, while TM-1 was seriously affected and exhibited Cd sensitivity. Further studies are necessary to understand the cause of the reduced gossypol content in cotton seeds under Cd stress.     

Potential detoxification of gossypol by UDP-glycosyltransferases in the two Heliothine moth species Helicoverpa armigera and Heliothis virescens

The cotton bollworm Helicoverpa armigera and the tobacco budworm Heliothis virescens are closely related generalist insect herbivores and serious pest species on a number of economically important crop plants including cotton. Even though cotton is well defended by its major defensive compound gossypol, a toxic sesquiterpene dimer, larvae of both species are capable of developing on cotton plants. In spite of severe damage larvae cause on cotton plants, little is known about gossypol detoxification mechanisms in cotton-feeding insects. Here, we detected three monoglycosylated and up to five diglycosylated gossypol isomers in the feces of H. armigera and H. virescens larvae fed on gossypol-supplemented diet. Candidate UDP-glycosyltransferase (UGT) genes of H. armigera were selected by microarray studies and in silico analyses and were functionally expressed in insect cells. In enzymatic assays, we show that UGT41B3 and UGT40D1 are capable of glycosylating gossypol mainly to the diglycosylated gossypol isomer 5 that is characteristic for H. armigera and is absent in H. virescens feces. In conclusion, our results demonstrate that gossypol is partially metabolized by UGTs via glycosylation, which might be a crucial step in gossypol detoxification in generalist herbivores utilizing cotton as host plant.     

Recovery of soluble proteins from glanded cotton tissues with amines

A simple soluble protein extraction method was developed for glanded cotton (Gossypium hirsutum L.) tissues. Gossypol, a major component of glands, is known to crosslink and precipitate proteins in cotton tissue homogenates. Established phenolic removal reagents were evaluated as gossypol binding agents and found to be less than effective in enhancing cotton leaf-soluble protein recovery. Several other amines, including a number of affinity support bound amines, were tested and found relatively ineffectual when compared with urea as cotton protein protectants. Urea and (NH4)2SO4, the next most active agent found in the study, were compared on both whole-leaf homogenates and artificial mixtures containing known quantities of poly-L-lysine and a clathrate of gossypol and acetic acid. Urea treatment resulted in both an increased number of stained bands and more concentrated staining of bands than any other treatment on polyacrylamide gels. Selected enzymes demonstrated increased activities in urea homogenates compared with other treatments.     

棉酚对棉铃虫生长及其寄生物齿唇姬蜂发育的影响(英文)

以中国北方棉区的主要害虫棉铃虫Helicoverpaarmigera（Hbner）及其内寄生蜂棉铃虫齿唇姬蜂Campoletischlorideae（Uchida）为试虫,研究了棉花重要抗虫次生性物质棉酚对棉铃虫生长和齿唇姬蜂发育的影响。0．1％的棉酚对棉铃虫幼虫有生长刺激作用,0．5％的棉酚则对棉铃虫幼虫生长有抑制作用。齿唇姬蜂成虫在寄生时对寄主大小有较严格的选择性,92％的被寄生幼虫体重小于18mg。0．1％的棉酚使寄主适合寄生期缩短10．75％;0．5％棉酚使寄主适合寄生期延长28．15％。棉酚对齿唇姬蜂发育有负效应。0．1％棉酚对寄主的生长刺激作用,不能使蜂成虫体重显著增加,但可显著延长蜂卵和幼虫期,缩短蛹期;高浓度的生长抑制作用,导致蜂成虫体重显著减轻,蛹期缩短,蜂卵和幼虫期亦显著延长。据此,对棉花抗虫性与生物防治在害虫综合防治实践中应用的协调性进行了讨论。     

Gossypol toxicity and detoxification in Helicoverpa armigera and Heliothis virescens

Gossypol is a polyphenolic secondary metabolite produced by cotton plants, which is toxic to many organisms. Gossypol's aldehyde groups are especially reactive, forming Schiff bases with amino acids of proteins and cross-linking them, inhibiting enzyme activities and contributing to toxicity. Very little is known about gossypol's mode of action and its detoxification in cotton-feeding insects that can tolerate certain concentrations of this compound. Here, we tested the toxicity of gossypol and a gossypol derivative lacking free aldehyde groups (SB-gossypol) toward Helicoverpa armigera and Heliothis virescens, two important pests on cotton plants. Larval feeding studies with these two species on artificial diet supplemented with gossypol or SB-gossypol revealed no detectable toxicity of gossypol, when the aldehyde groups were absent. A cytochrome P450 enzyme, CYP6AE14, is upregulated in H. armigera feeding on gossypol, and has been claimed to directly detoxify gossypol. However, using in vitro assays with heterologously expressed CYP6AE14, no metabolites of gossypol were detected, and further studies suggest that gossypol is not a direct substrate of CYP6AE14. Furthermore, larvae feeding on many other plant toxins also upregulate CYP6AE14. Our data demonstrate that the aldehyde groups are critical for the toxicity of gossypol when ingested by H. armigera and H. virescens larvae, and suggest that CYP6AE14 is not directly involved in gossypol metabolism, but may play a role in the general stress response of H. armigera larvae toward plant toxins.     

Effects of cotton cultivars differing in gossypol content on the quality of Aphis gossypii as prey for two species of Coccinellidae

Plants can have detrimental effects on biological control agents by affecting their prey or host quality. Thus, it is important to understand the tri-trophic interactions between plants, herbivores and natural enemies when implementing biological control programmes. Studies have shown that both morphological and chemical traits of host plants can affect the third trophic level. Cotton plants are known to produce alkaloids such as gossypol, a sesquiterpene aldehyde that can confer resistance against herbivorous arthropods. Nevertheless, little is known about the effect of gossypol on biological control agents. In this study, we investigated how three cotton cultivars (BRS Rubi, BRS Safira and BRS Verde) differing in gossypol content affect development and growth of predatory coccinellids, Eriopis connexa and Harmonia axyridis, feeding on the cotton aphid Aphis gossypii reared on those cultivars. The results show that the cultivar BRS Rubi (highest gossypol content) had a sub-lethal effect on the development and growth of both Coccinellidae species compared with the other cultivars. Overall, the cultivar BRS Rubi reduced slightly fecundity, net reproductive rate and intrinsic rate of natural increase for both Coccinellidae species. However, because aphid populations stay short periods of time in the field, and adult coccinellids may supplement its diet with alternative prey and plant material this sub-lethal indirect effect of gossypol may not have a detrimental effect on field biological control of cotton aphid by either E. connexa or H. axyridis, thus suggesting a compatibility between plant resistance and biological control agents.     

Chemical Composition of Defatted Cottonseed and Soy Meal Products

Chemical composition is critical information for product quality and exploration of new use. Hence defatted cottonseed meals from both glanded (with gossypol) and glandless (without gossypol) cotton seeds were separated into water soluble and insoluble fractions, or water soluble, alkali soluble as well as total protein isolates. The contents of gossypol, total protein and amino acids, fiber and carbohydrates, and selected macro and trace elements in these products were determined and compared with each other and with those of soy meal products. Data reported in this work improved our understanding on the chemical composition of different cottonseed meal products that is helpful for more economical utilization of these products. These data would also provide a basic reference for product standards and quality control when the production of the cottonseed meal products comes to pilot and industrial scales.     

Analysis of self-association of West Nile virus capsid protein and the crucial role played by Trp 69 in homodimerization

The understanding of capsid (C) protein interactions with itself would provide important data on how the core is organized in flaviviruses during assembly. In this study, West Nile (WN) virus C protein was shown to form homodimers using yeast two-hybrid analysis in conjunction with mammalian two-hybrid and in vivo co-immunoprecipitation assays. To delineate the region on the C protein which mediates C-C dimerization, truncation studies were carried out. The results obtained clearly showed that the internal hydrophobic segment flanked by helix I and helix III of WN virus C protein is essential for the self-association of C protein. The crucial role played by Trp 69 in stabilizing the self-association of C protein was also demonstrated by mutating Trp to Gly/Arg/Phe. Substitution of the Trp residue with Gly/Arg abolished the dimerization, whereas substitution with Phe decreased the self-association significantly. The results of this study pinpoint a critical residue in the C protein that potentially plays a role in stabilizing the homotypic interaction.