Genes regulating gland development in the cotton plant.

In seeds and other parts of cultivated, tetraploid cotton (Gossypium hirsutum L.), multicellular groups of cells lysigenously form dark glands containing toxic terpenoids such as gossypol that defend the plant against pests and pathogens. Using RNA-seq analysis of embryos from near-isogenic glanded (Gl2 Gl2 Gl3 Gl3 ) versus glandless (gl2 gl2 gl3 gl3 ) plants, we identified 33 genes that expressed exclusively or at higher levels in embryos just prior to gland formation in glanded plants. Virus-induced gene silencing against three gene pairs led to significant reductions in the number of glands in the leaves, and significantly lower levels of gossypol and related terpenoids. These genes encode transcription factors and have been designated the 'Cotton Gland Formation' (CGF) genes. No sequence differences were found between glanded and glandless cotton for CGF1 and CGF2 gene pairs. The glandless cotton has a transposon insertion within the coding sequence of the GoPGF (synonym CGF3) gene of the A subgenome and extensive mutations in the promoter of D subgenome homeolog. Overexpression of GoPGF (synonym CGF3) led to a dramatic increase in gossypol and related terpenoids in cultured cells, whereas CRISPR/Cas9 knockout of GoPGF (synonym CGF3) genes resulted in glandless phenotype. Taken collectively, the results show that the GoPGF (synonym CGF3) gene plays a critical role in the formation of glands in the cotton plant. Seed-specific silencing of CGF genes, either individually or in combination, could eliminate glands, thus gossypol, from the cottonseed to render it safe as food or feed for monogastrics.

Microbial Resistance Mechanisms to the Antibiotic and Phytotoxin Fusaric Acid.

Fusaric acid (FA) produced by Fusarium oxysporum plays an important role in disease development in plants, including cotton. This non-specific toxin also has antibiotic effects on microorganisms. Thus, one expects a potential pool of diverse detoxification mechanisms of FA in nature. Bacteria and fungi from soils infested with Fusarium and from laboratory sources were evaluated for their ability to grow in the presence of FA and to alter the structure of FA into less toxic compounds. None of the bacterial strains were able to chemically modify FA. Highly FA-resistant strains were found only in Gram-negative bacteria, mainly in the genus of Pseudomonas. The FA resistance of the Gram-negative bacteria was positively correlated with the number of predicted genes for FA efflux pumps present in the genome. Phylogenetic analysis of predicted FA resistance proteins (FUSC, an inner membrane transporter component of the efflux pump) revealed that FUSC proteins having high sequence identities with the functionally characterized FA resistance protein FusC or Fdt might be the major contributors of FA resistance. In contrast, most fungi converted FA to less toxic compounds regardless of the level of FA resistance they exhibited. Five derivatives were detected, and the detoxification of FA involved either oxidative reactions on the butyl side chain or reductive reactions on the carboxylic acid group. The production of these metabolites from widely different phyla indicates that resistance to FA by altering its structure is highly conserved. A few FA resistant saprophytic or biocontrol strains of fungi were incapable of altering FA, indicating a possible involvement of efflux transporters. Deployment of both efflux and derivatization mechanisms may be a common feature of fungal FA resistance.

FUBT, a putative MFS transporter, promotes secretion of fusaric acid in the cotton pathogen Fusarium oxysporum f. sp. vasinfectum.

Fusaric acid (FA) is a key component in virulence and symptom development in cotton during infection by Fusarium oxysporum. A putative major facilitator superfamily (MFS) transporter gene was identified downstream of the polyketide synthase gene responsible for the biosynthesis of FA in a region previously believed to be unrelated to the known FA gene cluster. Disruption of the transporter gene, designated FUBT, resulted in loss of FA secretion, decrease in FA production and a decrease in resistance to high concentrations of FA. Uptake of exogenous FA was unaffected in the disruption transformants, suggesting that FA enters the cell in Fusarium by an independent mechanism. Thus, FUBT is involved both in the extracellular transport of FA and in resistance of F. oxysporum to this non-specific toxin. A potential secondary resistance mechanism, the production of FA derivatives, was observed in FUBT deletion mutants. Molecular analysis of key biochemical processes in the production of FA could lead to future host plant resistance to Fusarium pathogens.

Conversion of fusaric acid to Fusarinol by Aspergillus tubingensis: a detoxification reaction.

The fungus Fusarium oxysporum causes wilt diseases of plants and produces a potent phytotoxin fusaric acid (FA), which is also toxic to many microorganisms. An Aspergillus tubingensis strain with high tolerance to FA was isolated from soil and designated as CDRAt01. HPLC analysis of culture filtrates from A. tubingensis isolate CDRAt01 grown with the addition of FA indicated the formation of a metabolite over time that was associated with a decrease of FA. Spectral analysis and chemical synthesis confirmed the compound as 5-butyl-2-pyridinemethanol, referred to here as fusarinol. The phytotoxicity of fusarinol compared to FA was measured by comparing necrosis induced in cotton (Gossypium hirsutum L. cv. Coker 312) cotyledons. Fusarinol was significantly less phytotoxic than FA. Therefore, the A. tubingensis strain provides a novel detoxification mechanism against FA which may be utilized to control Fusarium wilt.

RNAi-mediated Ultra-low gossypol cottonseed trait: performance of transgenic lines under field conditions.

Cottonseed remains a low-value by-product of lint production mainly due to the presence of toxic gossypol that makes it unfit for monogastrics. Ultra-low gossypol cottonseed (ULGCS) lines were developed using RNAi knockdown of δ-cadinene synthase gene(s) in Gossypium hirsutum. The purpose of the current study was to assess the stability and specificity of the ULGCS trait and evaluate the agronomic performance of the transgenic lines. Trials conducted over a period of 3 years show that the ULGCS trait was stable under field conditions and the foliage/floral organs of transgenic lines contained wild-type levels of gossypol and related terpenoids. Although it was a relatively small-scale study, we did not observe any negative effects on either the yield or quality of the fibre and seed in the transgenic lines compared with the nontransgenic parental plants. Compositional analysis was performed on the seeds obtained from plants grown in the field during 2009. As expected, the major difference between the ULGCS and wild-type cottonseeds was in terms of their gossypol levels. With the exception of oil content, the composition of ULGCS was similar to that of nontransgenic cottonseeds. Interestingly, the ULGCS had significantly higher (4%-8%) oil content compared with the seeds from the nontransgenic parent. Field trial results confirmed the stability and specificity of the ULGCS trait suggesting that this RNAi-based product has the potential to be commercially viable. Thus, it may be possible to enhance and expand the nutritional utility of the annual cottonseed output to fulfil the ever-increasing needs of humanity.

Lacinilene C 7-methyl ether.

The title compound, C16H20O3 [systematic name: 1-hy-droxy-7-meth-oxy-1,6-dimethyl-4-(propan-2-yl)naphthalen-2(1H)-one], is a sesquiterpene isolated from foliar tissues of the cotton plant and is of inter-est with respect to its anti-bacterial properties. Its phenyl ring is ideally planar, and the maximum of deviation in the second ring is 0.386 (3) Å. The hy-droxy group and the methyl group are oriented in an equatorial fashion and axial, respectively, to the second ring. In the crystal, inversion dimers are formed through pairs of O-H⋯O hydrogen bonds. Weak C-H⋯O hydrogen bonds link the dimers into columns along the c axis. These columns form a crystal structure with a crystal packing factor of 0.66.

Desoxyhemigossypol 6-methyl ether.

THE TITLE SESQUITERPENE [SYSTEMATIC NAME: 6-methoxy-10-methyl-7-(propan-2-yl)-2-oxatricyclo[6.3.1.0(4,12)]dodeca-1(11),4,6,8(12),9-pentaen-5-ol], C(16)H(18)O(3), was isolated from pathogen-infected stele tissue of Gossypium barbadense. There are two mol-ecules in the asymmetric unit and the dihedral angle between their naphtho-furan systems is 86.48 (2)°. In the crystal, O-H⋯O hydrogen bonds between the hy-droxy groups and etheric O atoms link the mol-ecules into centrosymmetric tetra-mers. These tetra-mers are assembled into (010) layers via stacking inter-actions between the naphtho-furan systems [inter-planar distance 3.473 (3) Å] and short C-H⋯O contacts.

Ultra-low gossypol cottonseed: generational stability of the seed-specific, RNAi-mediated phenotype and resumption of terpenoid profile following seed germination.

Cottonseed, containing 22.5% protein, remains an under-utilized and under-valued resource because of the presence of toxic gossypol. RNAi-knockdown of δ-cadinene synthase gene(s) was used to engineer plants that produced ultra-low gossypol cottonseed (ULGCS). In the original study, we observed that RNAi plants, a month or older, maintain normal complement of gossypol and related terpenoids in the roots, foliage, floral organs, and young bolls. However, the terpenoid levels and profile of the RNAi lines during the early stages of germination, under normal conditions and in response to pathogen exposure, had not been examined. Results obtained in this study show that during the early stages of seed germination/seedling growth, in both non-transgenic and RNAi lines, the tissues derived directly from bulk of the seed kernel (cotyledon and hypocotyl) synthesize little, if any new terpenoids. However, the growing root tissue and the emerging true leaves of RNAi seedlings showed normal, wild-type terpenoid levels. Biochemical and molecular analyses showed that pathogen-challenged parts of RNAi seedlings are capable of launching a terpenoid-based defence response. Nine different RNAi lines were monitored for five generations. The results show that, unlike the unstable nature of antisense-mediated low seed-gossypol phenotype, the RNAi-mediated ULGCS trait exhibited multi-generational stability.

Phylogeny and pathogenicity of Fusarium oxysporum isolates from cottonseed imported from Australia into California for dairy cattle feed.

A unique biotype of the Fusarium wilt pathogen, Fusarium oxysporum Schlecht. f.sp. vasinfectum (Atk) Sny. & Hans., found in Australia in 1993 is favored by neutral or alkaline heavy soils and does not require plant parasitic nematodes to cause disease. This makes it a threat to 4-6 million acres of USA Upland cotton ( Gossypium hirsutum L.) that is grown on heavy alkaline soil and currently is not affected by Fusarium wilt. In 2001-2002, several shiploads of live cottonseed were imported into California for dairy cattle feed. Thirteen F. oxysporum f.sp. vasinfectum isolates and four isolates of a Fusarium spp. that resembled F. oxysporum were isolated from the imported cottonseed. The isolates, designated by an AuSeed prefix, formed four vegetative compatibility groups (VCG) all of which were incompatible with tester isolates for 18 VCGs found in the USA. Isolate AuSeed14 was vegetatively compatible with the four reference isolates of Australian biotype VCG01111. Phylogenetic analyses based on EF-1α, PHO, BT, Mat1-1, and Mat1-2 gene sequences separated the 17 seed isolates into three lineages (race A, race 3, and Fusarium spp.) with AuSeed14 clustering into race 3 lineage or race A lineage depending on the genes analyzed. Indel analysis of the EF-1α gene sequences revealed a close evolutionary relationship among AuSeed14, Australian biotype reference isolates, and the four Fusarium spp. isolates. The Australian seed isolates and the four Australian biotype reference isolates caused disease with root-dip inoculation, but not with stem-puncture inoculation. Thus, they were a vascular incompetent pathotype. In contrast, USA race A lineage isolates readily colonized vascular tissue and formed a vascular competent pathotype when introduced directly into xylem vessels. The AuSeed14 isolate was as pathogenic as the Australian biotype, and it or related isolates could cause a severe Fusarium wilt problem in USA cotton fields if they become established.

Stereoselective coupling of hemigossypol to form (+)-gossypol in moco cotton is mediated by a dirigent protein.

The terpenoid gossypol, a secondary metabolite found in the cotton plant, is synthesized by a free radical dimerization of hemigossypol. Gossypol exists as an atropisomeric mixture because of restricted rotation around the central binaphthyl bond. The dimerization of hemigossypol is regiospecific in cotton. In the case of some moco cotton, the dimerization also exhibits a high level of stereoselectivity. The mechanism that controls this stereoselective dimerization is poorly understood. In this paper, we demonstrate that a dirigent protein controls this stereoselective dimerization process. A partially purified protein preparation from cotton flower petals, which by itself is unable to convert hemigossypol to gossypol, converts hemigossypol with a 30% atropisomeric excess into (+)-gossypol when combined with an exogenous laccase, which by itself produces racemic gossypol.

Effect of racemic, (+)- and (-)-gossypol on survival and development of Heliothis virescens larvae.

Gossypol is a constituent of the lysigenous foliar glands of cotton plants and is also found in glands in cottonseed. Gossypol exists as enantiomers because of restricted rotation around the binaphthyl bond. The biological activities of the enantiomers differ. For example, (+)-gossypol can be fed safely to nonruminants such as chickens, but (-)-gossypol cannot. Most commercial cottonseed contain a (+)- to (-)-gossypol ratio of approximately 3:2. Conventional breeding techniques can be used to develop cottonseed that contains >95% (+)-gossypol. Notably, gossypol protects the plant from insect herbivores. Herein, we report the effect of various forms of gossypol on Heliothis virescens (Fabricius) larvae. Three levels (0.16, 0.24, and 0.32%) of racemic, (+)-, and (-)-gossypol were added to artificial rearing diets and were fed to H. virescens larvae. All 0.24 and 0.32% gossypol diets significantly lengthened days-to-pupation and decreased pupal weight compared with the control. Percent survival was significantly less for larvae reared on diets containing 0.24% of all three forms of gossypol as compared with the control diet. (+)-Gossypol was superior or equivalent to racemic gossypol as measured by the three parameters studied. Higher concentrations of all gossypol forms were required to reduce survival and pupal weights and increase days-to-pupation for larvae of H. virescens larvae compared with the concentration needed to affect larvae of Helicoverpa zea (Boddie), which was studied previously. These results indicate that current efforts to breed cotton lines containing mostly (+)-gossypol in seed should not significantly impair the plant's natural defenses against insects.

Differences in Active Defense Responses of Two Gossypium barbadense L. Cultivars Resistant to Fusarium oxysporum f. sp. vasinfectum Race 4.

A highly virulent race 4 genotype of Fusarium oxysporum f. sp. vasinfectum (Fov) was identified for the first time in the western hemisphere in 2002 in cotton fields in the San Joaquin Valley of California. The Gossypium barbadense L. cotton cultivars 'Seabrook Sea Island 12B2' ('SBSI') and 'Pima S-6' are resistant to Fov race 4. Active defense responses were quantitated by monitoring the accumulation of antimicrobial terpenoids (i.e., phytoalexins) in inoculated stem stele tissue in these cultivars. The increase in the concentration of the most toxic phytoalexins was statistically faster after 24 h in 'SBSI' compared to 'Pima S-6'. The sesquiterpenoid hemigossylic acid lactone, which was observed for the first time in nature, also accumulated in diseased plants. Neither hemigossylic acid lactone nor the disesquiterpenoids gossypol, gossypol-6-methyl ether, and gossypol-6,6'-dimethyl ether showed toxicity to Fov. Segregation of F2 progeny from 'SBSI' × 'Pima S-6' crosses gave a few highly susceptible plants and a few highly resistant plants, indicating separate genes for resistance in the two cultivars.

Detoxification of Fusaric Acid by the Soil Microbe Mucor rouxii.

Fusarium oxysporum f. sp. vasinfectum race 4 (VCG0114), which causes root rot and wilt of cotton (Gossypium hirsutum and G. barbadense), has been identified recently for the first time in the western hemisphere in certain fields in the San Joaquin Valley of California. This pathotype produces copious quantities of the plant toxin fusaric acid (5-butyl-2-pyridinecarboxylic acid) compared to other isolates of F. oxysporum f. sp. vasinfectum (Fov) that are indigenous to the United States. Fusaric acid is toxic to cotton plants and may help the pathogen compete with other microbes in the soil. We found that a laboratory strain of the fungus Mucor rouxii converts fusaric acid into a newly identified compound, 8-hydroxyfusaric acid. The latter compound is significantly less phytotoxic to cotton than the parent compound. On the basis of bioassays of hydroxylated analogues of fusaric acid, hydroxylation of the butyl side chain of fusaric acid may affect a general detoxification of fusaric acid. Genes that control this hydroxylation may be useful in developing biocontrol agents to manage Fov.

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.

The absolute configuration of (-)-3-hydroxy-alpha-calacorene.

3-Hydroxy-alpha-calacorene was identified in extracts from cold-shocked seedlings of cotton (Gossypium hirsutum L.) and kenaf (Hibiscus cannabinus L.), both of which are members of the Malvaceae family. (-)-3-Hydroxy-alpha-calacorene was isolated from Heterotheca inuloides Cass. (Asteraceae). HPLC on a chiral stationary phase column showed that the 3-hydroxy-alpha- calacorene from cotton and kenaf had the same relative configuration, while that from H. inuloides was of the opposite configuration. X-ray crystallographic analysis established the absolute configuration of the compound in H. inuloides as (8R)-(-)-3-hydroxy-alpha-calacorene.

Malvone A, a phytoalexin found in Malva sylvestris (family Malvaceae).

The isolation and structure of a phytoalexin, malvone A (2-methyl-3-methoxy-5,6-dihydroxy-1,4-naphthoquinone) is reported. Malvone A formation is induced in Malva sylvestris L. by the plant pathogen Verticillium dahliae. In a turbimetric assay for toxicity to V. dahliae, it had an ED50 value of 24 microg/ml. The structure of malvone A was determined by MS and NMR spectroscopy, and by X-ray crystallographic analysis. The X-ray analysis showed water molecules were located in channels that run along the a-axis.

Ratios of (+)- and (-)-gossypol in leaves, stems, and roots of selected accessions of Gossypium hirsutum var. marie galante (Watt) Hutchinson.

Gossypol is an allelochemical that occurs naturally throughout the cotton plant as an enantiomeric mixture. Gossypol and related terpenoids protect the plant from some insect herbivores. Cottonseed has a high protein content, but it is underutilized because (-)-gossypol, which is toxic to nonruminants, occurs in the seed along with (+)-gossypol. Commercial Upland cottons usually have an approximate 3:2 (+)- to (-)-gossypol ratio in the seed, but plants can be bred with <8% (-)-gossypol using accessions of Gossypium hirsutum var. marie galante as parents. We report the (+)- and (-)-gossypol ratios and the concentration of related terpenoids in the stems, leaves, and roots of four accessions of marie galante that show high, moderate, and near normal levels of (+)-gossypol in the seed; we compare these values to the commercial cultivar Stoneville 474, which has 62% (+)-gossypol in the seed. In the marie galante accessions 2452 and 2425 that have the highest levels of (+)-gossypol in the seed, the percent (+)-gossypol and the concentration of gossypol and the related terpenoids were significantly higher (P = 0.05) in the stems and leaves as compared to Stoneville 474. Our analysis indicates that progeny from accessions 2452 and 2425 that retain these traits should not be overly susceptible to herbivorous insects.

Occurrence of (+)- and (-)-gossypol in wild species of cotton and in Gossypium hirsutum Var. marie-galante (Watt) Hutchinson.

Gossypol occurs as a mixture of enantiomers in cottonseed. These enantiomers exhibit different biological activities. The (-)-enantiomer is toxic to animals, but it has potential medicinal uses. Therefore, cottonseed with >95% (-)-gossypol could have biopharmaceutical applications. The (+)-enantiomer shows little, if any, toxicity to nonruminant animals. Thus, cottonseed with >95% (+)-gossypol could be more readily utilized as a feed for nonruminants. The (+)- to (-)-gossypol ratio in commercial Upland (Gossypium hirsutum) cottonseed is usually about 3:2, whereas that in commercial Pima cottonseed (Gossypium barbadense) is approximately 2:3. Herein are reported the (+)- to (-)-gossypol ratios in the seed from 28 wild species of cotton (194 accessions), 94 accessions of G. hirsutum var. marie-galante, and 3 domesticated species (11 accessions). It was found that some or all of the accessions of Gossypium darwinii, Gossypium sturtianum, Gossypium areysianum, Gossypium longicalyx, Gossypium harknessii, and Gossypium costulatum produce an excess of (-)-gossypol but none >65%. At least one accession of Gossypium anomalum, Gossypium mustelinum, Gossypium gossypioides, and Gossypium capitis-viridis contained >94% (+)-gossypol. One of the 94 accessions of G. hirsutum var. marie-galante (i.e., no. 2469) contained 97% (+)-gossypol.

RNAi construct of a cytochrome P450 gene CYP82D109 blocks an early step in the biosynthesis of hemigossypolone and gossypol in transgenic cotton plants.

Naturally occurring terpenoid aldehydes from cotton, such as hemigossypol, gossypol, hemigossypolone, and the heliocides, are important components of disease and herbivory resistance in cotton. These terpenoids are predominantly found in the glands. Differential screening identified a cytochrome P450 cDNA clone (CYP82D109) from a Gossypium hirsutum cultivar that hybridized to mRNA from glanded cotton but not glandless cotton. Both the D genome cotton Gossypium raimondii and A genome cotton Gossypium arboreum possessed three additional paralogs of the gene. G. hirsutum was transformed with a RNAi construct specific to this gene family and eight transgenic plants were generated stemming from at least five independent transformation events. HPLC analysis showed that RNAi plants, when compared to wild-type Coker 312 (WT) plants, had a 90% reduction in hemigossypolone and heliocides levels, and a 70% reduction in gossypol levels in the terminal leaves, respectively. Analysis of volatile terpenes by GC-MS established presence of an additional terpene (MW: 218) from the RNAi leaf extracts. The (1)H and (13)C NMR spectroscopic analyses showed this compound was δ-cadinen-2-one. Double bond rearrangement of this compound gives 7-hydroxycalamenene, a lacinilene C pathway intermediate. δ-Cadinen-2-one could be derived from δ-cadinene via a yet to be identified intermediate, δ-cadinen-2-ol. The RNAi construct of CYP82D109 blocks the synthesis of desoxyhemigossypol and increases the induction of lacinilene C pathway, showing that these pathways are interconnected. Lacinilene C precursors are not constitutively expressed in cotton leaves, and blocking the gossypol pathway by the RNAi construct resulted in a greater induction of the lacinilene C pathway compounds when challenged by pathogens.

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.