Gossypol Biosynthesis Research Articles

Gene cloning: exploring cotton functional genomics and genetic improvement

Cotton is the most important natural fiber plant in the world. The genetic improvement of the quality of the cotton fiber and agricultural productivity is imperative under the situation of increasing consumption and rapid development of textile technology. Recently, the study of cotton molecular biology has progressed greatly. A lot of specifically or preferentially expressed cotton fiber genes were cloned and analyzed. On the other hand, identification of stress response genes expressed in cotton was performed by other research groups. The major stress factors were studied including the wilt pathogens Verticillium dahliae, Fusarium oxysporum f. sp. vasinfectum, bacterial blight, root-knot nematode, drought, and salt stress. What is more, a few genes related to the biosynthesis of gossypol, other sesquiterpene phytoalexins and the major seed oil fatty acids were isolated from cotton. In the present review, we focused on the major advances in cotton gene cloning and expression profiling in the recent years.

Engineering cottonseed for use in human nutrition by tissue-specific reduction of toxic gossypol.

Global cottonseed production can potentially provide the protein requirements for half a billion people per year; however, it is woefully underutilized because of the presence of toxic gossypol within seed glands. Therefore, elimination of gossypol from cottonseed has been a long-standing goal of geneticists. Attempts were made to meet this objective by developing so-called "glandless cotton" in the 1950s by conventional breeding techniques; however, the glandless varieties were commercially unviable because of the increased susceptibility of the plant to insect pests due to the systemic absence of glands that contain gossypol and other protective terpenoids. Thus, the promise of cottonseed in contributing to the food requirements of the burgeoning world population remained unfulfilled. We have successfully used RNAi to disrupt gossypol biosynthesis in cottonseed tissue by interfering with the expression of the delta-cadinene synthase gene during seed development. We demonstrate that it is possible to significantly reduce cottonseed-gossypol levels in a stable and heritable manner. Results from enzyme activity and molecular analyses on developing transgenic embryos were consistent with the observed phenotype in the mature seeds. Most relevant, the levels of gossypol and related terpenoids in the foliage and floral parts were not diminished, and thus their potential function in plant defense against insects and diseases remained untouched. These results illustrate that a targeted genetic modification, applied to an underutilized agricultural byproduct, provides a mechanism to open up a new source of nutrition for hundreds of millions of people.

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- 3H]-hemigossypol to [4,4′- 3H 2]-gossypol. The reaction was dependent on the addition of H 2O 2 and was inhibited 71–94% by 1 and 10 mM 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.

Biosynthesis of gossypol in cotton.

Abstract Cotton plants accumulate gossypol and related sesquiterpenoids in pigment glands as their defensive machinery against pathogens and herbivores. However, the accumulation of gossypol, which is toxic and antispermatogenic, also makes it hard to use cottonseeds for human and animal consumption. Detailed understanding of gossypol biosynthesis and its regulation should provide better strategies for resolving this problem. Recent progresses have been made to dissect gossypol pathway at molecular level. Genes encoding farnesyl diphosphate synthase (geranyltranstransferase), (+)-δ-cadinene synthase (CDN1 or CAD1), (+)-δ-cadinene-8-hydroxylase (CYP706B1), and S -adenosyl-L-Met: desoxyhemigossypol-6-O-methyltransferase, have been isolated, and their expression patterns analysed. A WRKY transcription factor from Gossypium arboreum , GaWRKY1, was found to activate CDN1-A gene expression through binding to W-box palindrome on the CDN1-A promoter. A laccase, GaLAC1, which might be involved in the later steps of gossypol biosynthesis, was also identified. In addition, a necrosis- and ethylene-inducing protein from Verticillium dahliae , VdNEP, was found to be involved in the interaction between cotton plants and the fungal pathogen. The expression of these genes has been studied under different developmental stages, and under treatment of fungal elicitor preparation. Antisense inhibition of CDN1 gene expression has reported to result in reduced gossypol accumulation in cottonseed to a varied degree. (+)-δ-Cadinene-8-hydroxylase, encoded by a single copy gene in the cotton genome, might be a better alternative for genetic engineering to specifically deplete gossypol biosynthesis/accumulation from cottonseeds.

VdNEP, an elicitor from Verticillium dahliae, induces cotton plant wilting.

Verticillium wilt is a vascular disease of cotton. The causal fungus, Verticillium dahliae, secretes elicitors in culture. We have generated approximately 1,000 5'-terminal expressed sequence tags (ESTs) from a cultured mycelium of V. dahliae. A number of ESTs were found to encode proteins harboring putative signal peptides for secretion, and their cDNAs were isolated. Heterologous expression led to the identification of a protein with elicitor activities. This protein, named V. dahliae necrosis- and ethylene-inducing protein (VdNEP), is composed of 233 amino acids and has high sequence identities with fungal necrosis- and ethylene-inducing proteins. Infiltration of the bacterially expressed His-VdNEP into Nicotiana benthamiana leaves resulted in necrotic lesion formation. In Arabidopsis thaliana, the fusion protein also triggered production of reactive oxygen species and induced the expression of PR genes. When added into suspension cultured cells of cotton (Gossypium arboreum), the fusion protein elicited the biosynthesis of gossypol and related sesquiterpene phytoalexins at low concentrations, and it induced cell death at higher concentrations. On cotton cotyledons and leaves, His-VdNEP induced dehydration and wilting, similar to symptoms caused by a crude preparation of V. dahliae elicitors. Northern blotting showed a low level of VdNEP expression in the mycelium during culture. These data suggest that VdNEP is a wilt-inducing factor and that it participates in cotton-V. dahliae interactions.

Gene expression profile changes in cotton root and hypocotyl tissues in response to infection with Fusarium oxysporum f. sp. vasinfectum.

Microarray analysis of large-scale temporal and tissue-specific plant gene expression changes occurring during a susceptible plant-pathogen interaction revealed different gene expression profile changes in cotton root and hypocotyl tissues. In hypocotyl tissues infected with Fusarium oxysporum f. sp. vasinfectum, increased expression of defense-related genes was observed, whereas few changes in the expression levels of defense-related genes were found in infected root tissues. In infected roots, more plant genes were repressed than were induced, especially at the earlier stages of infection. Although many known cotton defense responses were identified, including induction of pathogenesis-related genes and gossypol biosynthesis genes, potential new defense responses also were identified, such as the biosynthesis of lignans. Many of the stress-related gene responses were common to both tissues. The repression of drought-responsive proteins such as aquaporins in both roots and hypocotyls represents a previously unreported response of a host to pathogen attack that may be specific to vascular wilt diseases. Gene expression results implicated the phytohormones ethylene and auxin in the disease process. Biochemical analysis of hormone level changes supported this observation.

Toxicity of (+)- and (-)-gossypol to the plant pathogen, Rhizoctonia solani.

The dimeric sesquiterpene gossypol occurs naturally in cottonseed and other parts of the cotton plant. Gossypol exists as enantiomers because of the restricted rotation around the central binaphthyl bond. The (-)-enantiomer is toxic to nonruminant animals while the (+)-enantiomer exhibits little, if any, toxicity to these animals. Developing cotton plants with low levels of the (-)-gossypol could expand the use of cottonseed as a feed source. Gossypol also may play a role in protecting the plant from pathogens. The relative toxicity of (+)- and (-)-gossypol to plant pathogens has not been reported. We measured the concentration of (+)- and (-)-gossypol in roots from cotton seedlings that were treated with the biocontrol agent Trichoderma virens that induces biosynthesis of gossypol and related terpenoids in cotton roots. (-)-Gossypol was the minor enantiomer in control and treated roots, but levels were slightly higher in roots from T. virens-treated seed. We also determined the toxicity of the gossypol enantiomers and the racemate to the seedling disease pathogen Rhizoctonia solani. The (+)- and (-)-enantiomers of gossypol and the racemate are equally effective in inhibiting growth of this pathogen. The lethal doses of the gossypols required to kill the pathogen appeared to be similar, but their toxicities are significantly less than those of related cotton and kenaf sesquiterpenes. The results indicate that altering the enantiomeric ratio in cotton roots will not adversely affect the resistance of seedlings to the seedling pathogen R. solani.

Molecular cloning and functional identification of (+)-delta-cadinene-8-hydroxylase, a cytochrome P450 mono-oxygenase (CYP706B1) of cotton sesquiterpene biosynthesis.

In cotton, gossypol and related sesquiterpene aldehydes are present in the glands of aerial tissues and in epidermal cells of roots. A cytochrome P450 was found to be expressed in aerial tissues of glanded cotton cultivars, but not or at an extremely low level in the aerial tissues of a glandless cultivar. Its cDNA was then isolated from Gossypium arboreum L. After expression in Saccharomyces cerevisiae, the P450 was found to catalyse the hydroxylation of (+)-delta-cadinene, forming 8-hydroxy-(+)-delta-cadinene. This P450 mono-oxygenase has been classified as CYP706B1, and is the first member of the CYP706 family for which a function has been determined. Sesquiterpene aldehydes and CYP706B1 transcripts were detected in roots of both the glanded and glandless cultivars and in aerial tissues of the glanded cultivar. In suspension cultured cells of G. arboreum, elicitors prepared from the phytopathogenic fungus Verticillium dahliae caused a dramatic induction of CYP706B1 expression. The expression pattern of CYP706B1 and the position at which it hydroxylates (+)-delta-cadinene suggest that it catalyses an early step in gossypol biosynthesis. Southern blotting revealed a single copy of CYP706B1 in the genome of G. arboreum. CYP706B1 holds good potential for manipulation of gossypol levels in cottonseed via genetic engineering.

A revised scheme for the biosynthesis of gossypol

Chemical degradation of gossypol samples biosynthesized in cotton seeding roots form three differently labelled mevalonate precursors has revealed that, in contrast to previous claims, formation of this compound occurs as with other sesquiterpenes of the cadalane type via ten-membered cations correlated by a 1,3-hydride shift.

The biosynthesis of 2,7-dihydroxycadalene in infected cotton cotyledons: the folding pattern of the farnesol precursor and possible implications for gossypol biosynthesis

Incorporation of [1,2-13C2]acetate into the cotton phytoalexin 2,7-dihydroxycadalene by bacteria-inoculated cotton seedling has established a folding pattern for its carbon skeleton which differs from that deduced for the other cotton phytoalexin gossypol by earlier 14C studies.

Account of Dr. Lyman’s research on gossypol and its significance and impact on the cottonseed industry

AbstractCarl M. Lyman and his associates made highly significant contributions to the development of basic and applied knowledge of gossypol and gossypol‐like pigments and their relationship to the utilization of cottonseed protein. Included among their studies were investigations of physiological activity, effect upon protein quality for monogastric animal nutrition, elimination and inactivation, biosynthesis of gossypol in the cotton root, isolation and characterization of a previously unidentified gossypol‐like compound, and ration formulation procedures for efficient feeding of both glanded and glandless cottonseed protein.

Biosynthesis of Gossypol: INCORPORATION OF MEVALONATE-2-14C AND ISOPRENYL PYROPHOSPHATES

Abstract Based on the previous observations that gossypol is biosynthetically formed from acetate, and from the location of the incorporated 14C atoms, gossypol biosynthesis via the isoprenoid pathway has been postulated. To support this hypothesis, the incorporation of mevalonate-2-14C (a key intermediate in the isoprenoid pathway) into gossypol as well as the distribution of the radioactivity in the gossypol molecule was studied. The results showed that 6 molecules of mevalonate-2-14C are stereospecifically incorporated into 1 molecule of gossypol. The monoterpene alcohols, geraniol-2-14C and nerol-2-14C, were chemically synthesized and the incorporation of their respective pyrophosphate ester into gossypol was tested. The incorporation of geranyl-2-14C pyrophosphate into gossypol was equal to the incorporation of mevalonate-2-14C, whereas neryl-2-14C pyrophosphate was utilized twice as efficiently as mevalonate-2-14C. The location of the 14C atoms of these substrates in the gossypol molecule was consistent with the isoprenoid pathway. Similarly, the cis-cis isomer of the sesquiterpene alcohol, farnesyl-2-14C-PP, was 4 times as efficient as substrate compared to the trans-cis isomer. In constrast, the incorporation of trans-trans- and cis-trans-farnesyl-2-14C-PP into gossypol was negligible. From the labeling pattern of gossypol synthesized from mevalonate-2-14C, neryl-2-14C-PP, and trans-cis-farnesyl-2-14C-PP, it is proposed that gossypol is produced by a specific cyclization of cis-cis-farnesyl pyrophosphate.

Biosynthesis of C14-labeled Gossypol

Biosynthesis of C14-labeled Gossypol

Biosynthesis of Gossypol by Excised Cotton Roots

GOSSYPOL, a toxic phenol, occurs primarily in the resin glands of cotton seed but also has been isolated from the tap-root bark of the cotton plant1. A study of the distribution of gossypol in the cotton plant revealed that the roots, especially the feeder roots, contained high levels of gossypol. This was true for strains of cotton in which the plants and seed were free of resin glands as well as for strains which contained many glands in plants and seed. The high levels of gossypol in the roots and the minute amounts in the seed of the glandless plants suggest that the gossypol was synthesized in the root tissue.