18 Cotton Pest Resistance: The Role of Pigment Gland Constituents R. D. Stipanovic, A. A. Bell, and C. R. Benedict CONTENTS 18.1 Biosynthesis of the Glandular Sesquiterpenoids 18.2 Terpenoid Aldehydes and Insect Resistance 18.3 Terpenoid Aldehydes and Disease Resistance 18.3.1 Speed of Response 18.3.2 Quality of the Phytoalexins 18.4 Conclusion References ABSTRACT Gossypium species, of which G. arboreum, G. barbadense, G. herbaceum, and G. hirsutum (Upland cotton) are cultivated for production of cotton fiber, belong to the tribe Gossypieae of the family Malvaceae. The genera in the tribe are distinguished from other genera in the family by the production of lysigenous glands that contain gossypol, a polyphenolic terpenoid (Figure 18.1), in seed. 1 The glands are usually referred to as pig- ment glands because they appear as dark dots in leaves and stems and as yellow to orange dots on seeds and roots. Cottonseed is toxic to monogastric animals, such as humans, swine, poultry, fish, and rodents; the cause of this toxicity was associated with the lysige- nous glands early in the 20th century. However, it was not until the 1940s that the major toxin was identified as gossypol and its structure determined. 2 Gossypol in seed also may be accompanied by small amounts of its 6-methyl and 6,6′-dimethyl ethers, as well as their precursors, hemigossypol and hemigossypol-6-methyl ether as shown in Figure 18.1. 3 This same mixture also is found in roots. Studies on the toxicity of gossypol to animals have been reviewed. 4 18.1 Biosynthesis of the Glandular Sesquiterpenoids Terpenoids are derived via the isoprenoid pathway from mevalonic acid. In the case of the cotton sesquiterpenoids, (+)-δ-cadinene has been shown to be the first cyclized product in the pathway 5 (Figure 18.1). Gossypol and thus δ-cadinene is derived from E,E-farnesyl diphosphate 6-8 via nerolidyl diphosphate. 9 Intermediates between δ-cadinene and desoxy- hemigossypol (dHG) have not been identified. © 1999 by CRC Press LLC dHG is converted to hemigossypol (HG) which is efficiently converted in the seed to gos- sypol presumably via a peroxidase enzyme 10 (Figure 18.1). The peroxidase dimerization of HG yields a mixture of (+)- and (–)-gossypol. In G. hirsutum, the ratio of (+) to (–) is usually about 3:2, although cultivars with higher levels of the (+)-isomer have been identified. The (–)-isomer appears to be the more biologically active 11 and this isomer may be the primary cause of toxicity in nonruminant animals. Gossypol is also the predominate terpenoid alde- hyde in root glands. In foliar plant parts, dHG is converted to hemigossypolone (HGQ) 12 (Figure 18.2). HGQ presumably undergoes a Diels-Alder reaction with either myrcene or β-ocimene to form heliocides H 2 and H 3 13,14 from the former, and heliocides H 1 and H 4 15 from the latter. The compounds HGQ, heliocides H 1 , H 2 , H 3 , and H 4 together with gossypol constitute the major terpenoid aldehyde components in foliar glands of G. hirsutum plants. 16 In G. barbadense, these compounds together with a group of O-methylated derivatives are produced 17 (Figure 18.2). In G. barbadense the phenolic C-6 position of dHG is methylated via S-adenosyl-L-methionine (SAM) by an O-methyltransferase. 18 The methylated derivative, desoxyhemigossypol-6-methyl ether (dMHG), is subsequently converted to hemigossypol- 6-methyl ether (MHG). dMHG can undergo the same set of transformations as dHG giving rise to gossypol-6-methyl ether and gossypol-6,6-dimethyl ether 3 (Figure 18.1), and to hem- igossypolone-6-methyl ether (MHGQ), and heliocides B 1 , B 2 , B 3 , and B 4 17 (Figure 18.2). FIGURE 18.1 Structures and proposed biosynthetic pathway of cotton phytoalexins and cottonseed terpenoids (FDP = E,E- farnesyl diphosphate; NDP = nerolidyl diphosphate; dHG = desoxyhemigossypol; dMHG = desoxyhemigossy- pol-6-methyl ether; HG = hemigossypol; MHG = hemigossypol-6-methyl ether; G = gossypol; MG = gossypol- 6-methyl ether; DMG = gossypol-6,6′-dimethyl ether; dHG-OMT = desoxyhemigossypol-O-methyltransferase). © 1999 by CRC Press LLC Stem tissue in both G. hirsutum and G. barbadense is normally devoid of the terpenoids. However, invasion by a pathogen induces the synthesis of dHG and HG and their methyl ether derivatives. The concentrations of the methyl ether derivatives are higher in G. barbadense than in G. hirsutum. The levels of methylation vary greatly not only among Gossypium species, but also among tissues in a given species. Methylation is introduced into the terpenoid pathway at only one point, i.e., the transfer of a methyl group to desox- yhemigossypol to form desoxyhemigossypol-6-methyl ether (dMHG) as shown in Figure 18.1. All of the methylated terpenoid aldehydes, in turn, are derived from dMHG. The methyl group is transferred from SAM by desoxyhemigossypol-O-methyl transferase (dHG-OMT). 18 HG does not act as substrate for this enzyme. dHG-OMT has been partially purified. 18 All Gossypium species contain the structural gene for the synthesis of dHG-OMT. However, in G. hirsutum this structural gene is under the control of a dominant regulator gene, designated TM1, which apparently restricts the synthesis of the enzyme in leaf, stem, FIGURE 18.2 Structures and proposed biosynthetic pathway of the terpenoid in cotton leaves and flower buds (dHG = desoxyhemigossypol; dMHG = desoxyhemigossypol-6-methyl ether; HGQ = hemigossypolone; MHGQ = hem- igossypolone-6-methyl ether; HH 1 , HH 2 , HH 3 , HH 4 = Heliocides H 1 , H 2 , H 3 , H 4 ; HB 1 , HB 2 , HB 3 , HB 4 = Heliocides B 1 , B 2 , B 3 , B 4 ). © 1999 by CRC Press LLC and young boll tissues. 19,20 Regulator genes also have evolved in many of the other Gossyp- ium species to restrict methylation of the terpene aldehydes to some degree, especially in leaves. 17,20,21 The cultivated species G. barbadense does not contain a regulator and conse- quently has relatively high percentages of methylated terpene aldehydes in all tissues. The TM1 regulator gene in G. hirsutum usually does not function well in juvenile tissues that serve as ports of entry for most pathogens. Thus, there is little restriction of methyla- tion in root tips where fungal wilt pathogens, root rot pathogens, and nematodes usually penetrate to gain access to less protected tissues. 22 Hunter 23 reported that there is little restriction of methylation in the young hypocotyl and root that is attacked by various seed- ling pathogens. There is some degree of regulation in xylem tissue but even here as much as 20% of the terpene is methylated in some cultivars of G. hirsutum. 24 18.2 Terpenoid Aldehydes and Insect Resistance The discovery that gossypol in seed was localized strictly in glands and was responsible for cottonseed toxicity led to the search for glandless mutant cottons. It was hoped that cot- tonseed from glandless cottons could be used in human foods and in greater amounts in animal feeds. Such cottons were developed by crossing lines that contained very few glands. The completely glandless character was shown to be due to two recessive genes designated gl2 and gl3. These genes were soon incorporated into many different commer- cial breeding lines. When the glandless plants were planted in the field, they were dam- aged more severely by insects and rodents known to feed on cotton. In addition, the glandless plants were attacked by various herbivores, such as beetles, rodents, and birds, that previously were not known to attack cotton. 25-27 These studies show that the lysige- nous glands provide protection against a wide range of herbivores. In Jenkin’s 28 1995 study of 56 accessions of G. hirsutum resistant to Heliothis spp., 33 were high in terpenoid aldehydes. The experiences with glandless cottons led to the development of “high gossypol” or highly glanded cotton lines for increased resistance to insects. While highly glanded breed- ing lines were indeed more resistant to insects, the level of resistance could not always be explained by the gossypol content of leaves and flower buds. Efforts to explain this discrep- ancy led to the discovery that the major terpenoid aldehydes in glands of leaves and young bolls of Upland cotton were the terpenoid aldehyde quinone, hemigossypolone, and its derivatives, heliocides H 1 -H 4 , which are formed by a Diels-Alder reaction of the quinone with the volatile monoterpenes β-ocimene or myrcene as shown in Figure 18.2. Bell 17 showed that several Gossypium species also contain the 6-methyl ethers of hemigossy- polone and its heliocides as major terpenes in leaves and bolls. The methyl ethers of helio- cides H 1 -H 4 are referred to as heliocides B 1 -B 4 because of their original discovery in G. barbadense. Elzen et al. 29 and Bell et al. 30 subsequently showed that all of the volatile monoterpenes and sesquiterpenes, such as β-ocimene, myrcene, α- and β-pinenes, γ-ter- pinene, β-caryophyllene, humulene, and β-bisabolene, also are stored in the lysigenous glands located in green tissues. Thus, the glands in aerial parts of the plant that contain chlorophyll contain unique terpenoid aldehydes, in addition to gossypol, dissolved in essential oils. Resistance of leaves or bolls to insects correlates best with the concentrations of hemigossypolone and heliocides H 1 and H 2 . 31 Also, in artificial diets hemigossypolone- 6-methyl ether and the methylated heliocides are less than one half as toxic as their unme- thylated counterparts. 32 © 1999 by CRC Press LLC 18.3 Terpenoid Aldehydes and Disease Resistance In the U.S. two species of cotton are grown commercially, G. hirsutum or Upland cottons and G. barbadense or pima cotton. The overwhelming majority of acreage is devoted to Upland cotton production. Traditionally, the G. barbadense cultivar Seabrook Sea Island (SBSI) has been considered to be the premiere cultivar in resistance to Verticillium dahliae. However, even this cultivar can be rendered susceptible by growing the plants at slightly cooler temperatures. It is dur- ing the growing season when night time temperatures are cooler that this pathogen is most devastating. Thus, no cotton is immune to this pathogen and strategies are required to pro- vide breeders with the tools to increase resistance to the wilt pathogens that are applicable to the diverse G. hirsutum germplasm which is grown across the cotton belt. Early experiments by Bell 33 showed that xylem tissues and boll endocarp tissue, which normally are devoid of terpenoids, rapidly synthesize gossypol and related terpenoid alde- hydes in response to infection by fungal pathogens. This reaction occurred both in glanded and glandless cottons, showing that the gland alleles affect the storage of gossypol but not its biosynthesis. These studies also showed that terpenoid aldehydes are synthesized as part of the active defense against microbial infections and thus should be considered as phytoalexins. Bell 34 then showed that when xylem vessels were infected with V. dahliae more than 50% of the terpenoid aldehydes were exuded into the xylem vessels. Many of the details of the role of terpenoid aldehydes as phytoalexins (active defense agents) in response to infection by wilt fungi have been reviewed. 35-38 The terpenoids are synthesized by the perivascular cells 39-41 appressed to the xylem vessels and are exuded first into the vessels and then into the surrounding intercellular spaces. The most abundant compound formed in G. hirsutum is hemigossypol. Its biosynthetic precursor, desoxyhem- igossypol (Figure 18.1) occurs at about one third the concentration of hemigossypol 48 to 72 h after inoculation. In G. barbadense, the 6-methyl ethers of these compounds (Figure 18.1) are usually the predominant compounds and the desoxyhemigossypol-6- methyl ether concentration is 2 to 3 times greater than that of hemigossypol-6-methyl ether. Accumulation of terpenoid aldehydes in xylem vessels occurs more rapidly in resistant than susceptible cultivars in response to the wilt pathogen Fusarium oxysporum f. sp. vasin- fectum (F.o.v.) as well as to V. dahliae. 42,43 The onset of rapid phytoalexin accumulation in resistant cultivars coincides almost perfectly with the time that fungal spread is curtailed in the xylem vessels. 44 The above observations along with the demonstration that hemi- gossypol is deposited on fungal hyphae in vessels in situ 39 strongly indicate a determina- tive role for terpenoid aldehydes and their naphthofuran precursors in resistance to wilt fungi. Two recent experiments further support this conclusion. Bell et al. 45 introduced recessive and dominant genes from G. barbadense and G. sturtianum, respectively, into G. hirsutum to increase the percentage of the 6-methyl ethers from less than 5% to more than 50% in the leaves of one set of cotton near-isolines while leaving the normal levels in the sister set of near-isolines. The increase in methylation decreased the toxicity of the total ter- penoids because the less toxic ethers replaced their more toxic unmethylated counterparts. In all four pairs, the lines with enhanced methylation had decreased resistance to Verticil- lium wilt. Furthermore, Eldon and Hillocks 46 were able to break resistance to Verticillium wilt by using an enzyme inhibitor that disrupts an early step (i.e., HMGR-CoA reductase) in the terpenoid biosynthetic pathway. The relationship between phytoalexins and disease resistance appears to be controlled by the speed of response and quality of the phytoalexins. Thus, plants that respond to an infection © 1999 by CRC Press LLC by the rapid synthesis of phytoalexins are expected to be more resistant. Furthermore, plants that produce the highest concentration of the most toxic phytoalexin likewise are expected to be more resistant. Progress in understanding and developing these comple- mentary processes are discussed below. 18.3.1 Speed of Response Bell 34 showed that the time at which phytoalexins began to accumulate was about 24 h sooner in vessels of a resistant G. barbadense compared to a more susceptible G. hirsutum. He also showed that the fungus grew from the vessels into surrounding tissue of G. hirsutum only in the young terminal stem tissue which had very limited ability to make terpenoid aldehydes. He proposed that the speed of terpenoid aldehyde synthesis relative to the speed of secondary colonization by the fungus was a critical determinant of resis- tance. 35 In a kinetic analysis of cotton stele tissue from resistant G. barbadense (SBSI) infected with V. dahliae, Alchanati et al. 9 showed that δ-cadinene synthase mRNA, δ-cadinene syn- thase activity, and formation of sesquiterpenoid phytoalexins were induced 12 h after inoc- ulation with the fungus. mRNA was already at a peak level at 12 h, while δ-cadinene synthase activity was at 54%, with peak level occurring at 48 h. Phytoalexins were not detected until 24 h. Cui et al. 43,47 compared the mRNA levels of β -1,3-glucanase, chitinase, phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), caffeic acid O-methyltransferase (C- OMT), 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR), and δ-cadinene synthase (δ- CS) in each of the four cultivars at 12, 24, 36, 48, 72, and 96 h after inoculation with a fungal suspension or sterile water. Low levels of β-1,3-glucanase mRNA were observed in both fungal and water-treated plants. Chitinase mRNA was detected only in inoculated plants, but in relatively low levels. Thus, these enzymes may be important, but do not appear to be critical for an incompatible response in cotton. PAL mRNA was constitutively expressed in all plants from all treatments. PAL removes an amino group from phenylalanine to form cinnamic acid. Cinnamic acid is utilized by the plant to produce either flavonoids or lignin. In cotton, flavonoids are converted to condensed tannins as part of the active defense response. 48 CHS is a key enzyme needed to synthesize condensed tannins while caffeic acid o-methyltransferase (C-OMT) converts cinnamic acids to lignin precursors. A high PAL mRNA level constitutively expressed in all four cultivars suggests there is always enough enzyme activity for flavonoid and/or lignin synthesis. CHS mRNA levels were higher in the resistant plants but did not reach their highest levels until 60 h post inoculation (PI). mRNA levels of C-OMT, a key enzyme leading to lignin precursors, were higher in early samples from resistant cultivars as compared to susceptible cultivars with maximal activity measured at 12 h PI. This may in part account for xylem vessel plugging which physically restricts the fungus and prevents its spread. 41,49-53 HMGR is a key enzyme in the formation of mevalonate, the sole precursor for terpenoid biosynthesis. 54,55 δ-CS is the enzyme that catalyzes the formation of δ-cadinene, the first compound unique to the cotton terpenoid phytoalexin synthesis. 5 Maximal levels of both HMGR mRNA and δ-CS mRNA were higher in the resistant cultivars with maximal activity measured at 12 h PI. These results show that currently available resistant cotton cultivars are very quick to recognize the presence of the pathogen. At 12 h, PI, C-OMT mRNA involved in the biosyn- thesis of lignins and HMGR and δ-CS mRNA’s involved in phytoalexin biosynthesis are already at maximal levels. At 12 h, the conidia used to inoculate the plant have just begun to germinate. Thus, in new resistant G. hirsutum cotton cultivars, early recognition is not a problem. In order to further augment resistance, increasing the potency of the phytoalexins offers an attractive option. 56 © 1999 by CRC Press LLC 18.3.2 Quality of the Phytoalexins Since the quickness in recognizing the pathogen and mobilizing defense biosynthetic path- ways in new Verticillium wilt resistant G. hirsutum cultivars appear to be reaching its max- imum potential, it is appropriate to target the quality or toxicity of the phytoalexins. Two approaches can be envisioned — the introduction of foreign genes from a V. dahliae immune plant or an alteration in the biosynthetic pathway to increase the toxicity of the existing phytoalexins in cotton. We view the latter approach as more easily attainable at this time. The toxicity of the phytoalexins has been determined against a number of fungi. The ED 50 of desoxyhemigossypol, the most potent antibiotic, ranges from 5 to 30 ppm against differ- ent fungi. The naphthofurans are usually about twice as toxic as their aldehyde deriva- tives. 42,45,57-59 However, it takes twice as much of the 6-methyl ethers to be as toxic as their unmethylated counterparts. Similarly, the methylated terpenoids in leaves, hemigossy- polone-6-methyl ether and the O-methylated heliocides, are less than one half as toxic as their unmethylated counterparts. 31 Thus, methylation has an undesirable effect on toxicity to both pathogenic fungi and insects. This observation suggests that preventing or lower- ing the rate of methylation would enhance the natural defenses of the plant to many pests. The evolution of regulatory genes to “shut down” methylation in leaves in some Gossypium species such as G. hirsutum, which is discussed in the section on biosynthesis, support this conclusion. 17,21 Our immediate goal is to identify the dHG-O-methyltransferase (dHG-OMT) gene and use antisense technology to lower expression of the dHG-OMT gene. Partial purification of the dHG-OMT enzyme has been accomplished. 18 Suppressed expression of the dHG-OMT gene is expected to enhance resistance of cotton to multiple pests. 18.4 Conclusion Because of the toxicity of gossypol, its inclusion in the glands of cottonseed is viewed as an undesirable attribute by the cottonseed industry. An active research effort is currently underway to overcome this problem using molecular biology to block synthesis of gossypol or at least of its (–)-isomer which is thought to be the toxic component. However, the glan- dular terpenoid aldehydes in the foliar plant parts constitute an important component in the plant’s defense to insects. Similarly, current evidence suggests that the phytoalexins pro- duced in the xylem tissue are essential for protecting the plant from pathogenic fungi. This knowledge has led us to believe that a significant increase in resistance to pathogens can be accomplished by blocking methylation of desoxyhemigossypol using antisense constructs. References 1. Fryxell, P.A., The Natural History of the Cotton Tribe, Texas A&M University Press, College Station, 1979, 245. 2. Adams, R. and Geissman, T.A., Gossypol, a pigment of cottonseed, Chem. Rev., 60, 555, 1960. 3. Stipanovic, R.D., Bell, A.A., Mace, M.E., and Howell, C.R., Antimicrobial terpenoids of Gos- sypium: 6-methoxygossypol and 6,6′-dimethoxygossypol, Phytochemistry, 14, 1077, 1975. © 1999 by CRC Press LLC 4. Berardi, L.C. and Goldblatt, G.A., Gossypol, in Toxic Constituents of Plant Foodstuffs, 2nd ed., Liener, I. E., Ed., Academic Press, New York, 1980, 184. 5. Davis, G.D. and Essenberg, M., (+)-delta-Cadinene is a product of sesquiterpene cyclase activity in cotton, Phytochemistry, 39, 553, 1995. 6. 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Mace, M.E., Stipanovic, R.D., and Bell, A.A., Toxicity of cotton phytoalexins to zoopathogenic fungi, Natural Toxins, 1, 294, 1993. © 1999 by CRC Press LLC . of β -1 ,3-glucanase, chitinase, phenylalanine ammonia-lyase (PAL), chalcone synthase (CHS), caffeic acid O-methyltransferase (C- OMT), 3-hydroxy-3-methyl glutaryl CoA reductase (HMGR), and δ-cadinene. gossypol-6-methyl ether and gossypol-6,6-dimethyl ether 3 (Figure 18. 1), and to hem- igossypolone-6-methyl ether (MHGQ), and heliocides B 1 , B 2 , B 3 , and B 4 17 (Figure 18. 2). FIGURE 18. 1 Structures and. group of O-methylated derivatives are produced 17 (Figure 18. 2). In G. barbadense the phenolic C-6 position of dHG is methylated via S-adenosyl-L-methionine (SAM) by an O-methyltransferase. 18 The