Distribution And Differential Expression Of

Journal of Chemical Ecology, Vol. 23, No. 11, 1997

DISTRIBUTION AND DIFFERENTIAL EXPRESSION OF (E)-4,8-DIMETHYL-l,3,7-NONATRIENE IN LEAF AND FLORAL VOLATILES OF Magnolia AND Liriodendron TAXA

HIROSHI AZUMA, 1 LEONARD B. THIEN, 2 MASAO TOYOTA,3 YOSHINORI ASAKAWA, 3 and SHOICHI KAWANO 1 * 1Department

of Botany, Graduate School of Science, Kyoto University Kyoto 606-01, Japan ^Department of Cell and Molecular Biology, Tulune University New Orleans, Louisiana 70118 ^Faculty of Pharmaceutical Sciences, Tokushima Bunri University Yamashiro-cho, Tokushima 770, Japan (Received September 13, 1996; accepted July 7, 1997)

Abstract—Analyses of volatiles emitted from artificially damaged leaves attached to branches of seven Magnolia taxa revealed the presence of (Z)-3hexenyl acetate, (Z)-3-hexenol (the green odor compounds), and several mono- and sesquiterpenes, e.g., (Z)- and (E)-/3-ocimene and caryophyllene. An herbivore-induced leaf volatile, (E)-4,8-dimethyl-l,3,7-nonatriene, known as a predator attractant in agricultural plants, was emitted 4-6 hr after leaves were damaged in M. hypoleuca. The damaged leaves of M. grandiflora, however, immediately released (E)-4,8-dimethyl-l,3,7-nonatriene. Undamaged leaves of Magnolia species examined did not emit volatile compounds. In addition, detached flowers of six Magnolia taxa and Liriodendron tulipifera also emit (E)-4,8-dimemyl-l,3,7-nonatriene as a floral volatile (up to 30% in some species); the chemical was also emitted from the intact flowers of M. heptapeta and M. salicifolia. Key Words—Floral odor, green odor, monoterpene, sesquiterpene, 4,8dimethyl-l,3,7-nonatriene, attractant, deterrent, herbivore, pollination biology, plant-insect interaction. INTRODUCTION

Secondary compounds produced in plants are involved in many plant-insect relationships, e.g., protection against herbivores, pollination, and coevolution *To whom correspondence should be addressed.

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(Ehrlich and Raven, 1964; Rhoades, 1979; Wood, 1982; Williams and Whitten, 1983; Chew, 1988; Spencer, 1988; Harborne, 1993). Leaf injury of some agricultural plants by herbivores causes the delayed release of volatile secondary compounds that attract predators and parasitoids (Loughrin et al., 1994, 1995; Turlings et al., 1990, 1995; Turlings and Tumlinson, 1992; Takabayashi et al., 1994a,b). Moreover, feeding damage by herbivores not only affects the quality and quantity of released volatiles from the damaged leaves but also induces systemic emission of these volatiles from undamaged vegetative parts of the plant damaged (Rose et al., 1996; also see Farmer and Ryan, 1990; Shulaev et al., 1997). Exploitation of such chemical signals by predators and parasitoids to locate prey formed the basis for the hypothesis that plants may actively signal predators as a defensive mechanism (Price et al., 1980; Dicke and Sabelis, 1992; Turlings et al., 1990, 1995). Volatiles emitted from flowers are also important insect attractants for pollination and may function as chemical cues for food, rest, mating, and/or larval development sites (Pellmyr and Thien, 1986). The fact that volatile compounds such as monoterpenes and fatty acid derivatives commonly occur in both (damaged) leaf and floral volatiles (Knudsen et al., 1993) suggests that floral volatiles may have originated from general leaf volatiles in the meshing of the life cycles of insects and plants (Pellmyr and Thien, 1986; Pellmyr et al., 1991). In this paper we document the emissions of volatiles from artificially damaged leaves of seven Magnolia taxa and record the distributions of (E)-4,8dimethyl-l,3,7-nonatriene in floral volatiles of Magnolia and Liriodendron taxa because it is known as one of the attractants of natural enemies emitted from feeding-damaged leaves (Turlings et al., 1990; Turlings and Tumlinson, 1992; Takabayashi et al., 1994a,b). This is the first documentation of the release of (E)-4,8-dimethyl-l,3,7-nonatriene from damaged leaves in non-agricultural plants.

METHODS AND MATERIALS

Collection of Volatiles. Seven species of Magnolia: M. praecocissima Koidz. (= M. kobus), M. tomentosa Thunb., M. salicifolia (Sieb. et Zucc.) Maxim., M. hypoleuca Sieb. et Zucc. (= M. obovata), M. sieboldii K. Koch ssp. japonica Ueda, M. heptapeta (Buchoz) Dandy (= M. denudata; Chinese taxon) and M. grandiflora L. (North American taxon) (Treseder, 1978; Ueda, 1980, 1985, 1986a,b), native to or cultivated in Japan were used for GC-MS analyses of volatiles emitted from leaves. Of the seven taxa, M. grandiflora is evergreen and the other species are deciduous trees or shrubs. Leaves were damaged with a wire brush (while attached to the plant) and

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then immediately enclosed in a polyethylene bag. Five to seven leaves on a branch were used at each sampling period except for M. hypoleuca (only one leaf, approximately 30 cm, was used). Leaf volatiles were collected from three different individuals in each taxon. Six successive samples were taken every 2 hr. Volatiles in the bag were pumped through a glass tube (7 mm ID x 5 cm) containing adsorbent (50 mg Tenax GC, mesh 60-80, GL Sciences, Tokyo, Japan) with a mini-pump (model MP-2N, Shibata Scientific Instrument Co., Ltd., Tokyo, Japan). The mini-pump was driven at approximately 1 liter/min. The glass tube containing the captured leaf volatiles was sealed with aluminum foil and stored at -20°C. Empty bags, adsorbent blanks, and bags containing undamaged leaves (controls) were analyzed for volatiles. Prior to use, the adsorbent was washed successively with two volumes of distilled methanol and ether and heated for 12 hr at 150°C. Volatiles emitted from detached flowers of the 11 Magnolia and Liriodendron tulipifera species were analyzed by GC-MS. Intact flowers of five taxa, i.e., M. sieboldii ssp.japonica, M. praecocissima, M. tomentosa, M. salicifolia, and M. heptapeta were also analyzed. Throughout sampling, no floral organs were damaged. One to a few flowers were enclosed in a polyethylene bag in the field. The volatiles were collected in the same manner as described above, except sampling periods were for 2-3 hr (for detached flowers) and 9 hr (for intact flowers), respectively. GC-MS Analysis. Leaf volatiles were eluted from the adsorbent with 350 p,\ of distilled diethyl ether. As an internal quantitative standard, 5 /xl of n-octanol solution (10 pi n-octanol/l ml diethylether) was added to the eluents. The eluents were carefully concentrated by N2 flow to approximately 100 /xl. Two microliters of the eluents were used for GC-MS analyses in all cases. GCMS analysis was performed with a Hewlett-Packard 5971A mass selective detector, connected to a Hewlett-Packard 5890 Series II gas chromatograph. A fused silica capillary column (DB-17, 0.25 mm ID x 30 m, film thickness 0.25 ^im) was used. The ion-source temperature was 185°C (the ionization energy was 70 eV). The injection temperature was 250°C and oven temperature was maintained at 50°C for the first 5 min, then programmed to 150°C at 5°C/min, finally raised to 250°C at 10°C/min, and then kept for 10 min. Helium was used as the carrier gas. The chemical structures of the volatiles were identified by comparison of the GC retention times and mass spectra with those of authentic compounds, with computer data in the library, NIST PBM HP G1033A revision code A.00.00 (Hewlett-Packard), and with our own library of more than 100 mono- and sesquiterpenes. Most authentic compounds were obtained from commercial sources, but (E)-4,8-dimethyl-l,3,7-nonatriene was synthesized from citral by the Wittig reaction (Greenwald et al., 1963).

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RESULTS

Three aliphatics, six monoterpenes, and seven sesquiterpenes were identified in the volatiles from artificially damaged leaves of Magnolia (Table 1). The volatiles of M. praecocissima, M. tomentosa, M. salicifolia, M. hypoleuca and M. sieboldii ssp. japonica, were mainly composed of (Z)-3-hexenol, (Z)3-hexenyl acetate, (Z)-/3-ocimene, (E)-)3-ocimene and caryophyllene. In contrast, the volatiles of the evergreen M. grandiflora were dominated by sesquiterpenes, especially /3-elemene, a-bisabolene, bicyclogermacrene, caryophyllene, -y-cadinene, and (E)-4,8-dimethyl-l,3,7-nonatriene. /3-Myrcene, limonene, linalool, /3-elemene, and a-humulene were also found in the volatiles of some Magnolia taxa as minor components. (E)-4,8-Dimethyl-l,3,7-nonatriene occurred in the volatiles of M. praecocissima, M. hypoleuca, and M. grandiflora, although the amount in M. praecocissima was very low. These compounds were not found in volatiles from undamaged leaves (Table 2), in which no characteristic ion was detected in the GC-MS ion chromatograms, except a small amount of (Z)-3-hexenyl acetate was present in volatiles from M. hypoleuca. The temporal pattern of the emissions of (Z)-3-hexenyl acetate, (E)-|3ocimene, caryophyllene, and (E)-4,8-dimethyl-l,3,7-nonatriene from artificially damaged leaves of selected taxa are shown in Figure 1. Generally, most volatiles, including (Z)-3-hexenyl acetate and (E)-/3-ocimene, were emitted immediately after damage and then the amounts decreased rapidly, although (E)-@ocimene from M. hypoleuca was emitted more abundantly at a later stage (Figure 1). In addition, caryophyllene was constantly emitted from the damaged leaves of M. tomentosa, M. hypoleuca, and M. grandiflora (Figure 1). The timing of emission of (E)-4-8-dimethy 1-1,3,7-nonatriene from artificially damaged leaves

TABLE 2. VOLATILE COMPOUND EMITTED FROM UNDAMAGED LEAVES OF SEVEN Magnolia TAXA DURING 12 HOURS" Amount of compound collected (/ig/12 hr/100 cm2 leaf area)

Compound Aliphatic (Z)-3-Hexenyl acetate

M. pra M. torn. M. sal. M. hyp. M. s.j. M. hep. (W= 1) (N = 1) (N= 1) (N = 1) (N = 1) (A/= 1)

b

b

h

0.68

_b

M. gra. (N = 1)

b

"Amount is the sum of six successive 2-hr samplings. See Table I for taxon abbreviations. kNo characteristic ion was detected in GC-MS ion chromatogram.

l>

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FIG. 1. Emissions of (Z)-3-hexenyl acetate, (E)-/3-ocimene, caryophyllene, and (E)-4,8dimethyl-l,3,7-nonatriene from artificially damaged leaves of four Magnolia taxa over time. Data represents the mean of three samples + SD. of M. hypoleuca was very different from those of (Z)-3-hexenyl acetate and other terpenes (Figure 1). In the early stages, it was not emitted from the damaged leaves at all. The emission began after 4-6 hr and peaked after 6-8 hr. Although (E)-4,8-dimethyl-l,3,7-nonatriene was emitted from the damaged leaves of M. praecocissima in a similar pattern as in M. hypoleuca, the quantity was very low. In contrast, the damaged evergreen leaves of M. grandiflora immediately released (E)-4,8-dimethyl-l,3,7-nonatriene after damage (Figure 1). GC-MS analyses of floral volatiles of Magnolia and Liriodendron indicate the flowers emit various chemical compounds such as terpenoids, benzenoids, and fatty acid derivatives (H. Azuma, M. Toyota, Y. Asakawa, L. B. Thien, R. Yamaoka, and S. Kawano, unpublished data). (E)-4,8-Dimethyl-l,3,7-nonatriene also was detected in the volatiles of detached flowers of M. virginiana, M. grandiflora, M. acuminata, M. praecocissima, M. tomentosa, M. heptapeta,

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and Liriodendron tulipifera ranging from < 1 % to more than 50% of the floral volatiles (Table 3). Two Asiatic Magnolia taxa belonging to the subgenus Yulania, i.e., M. tomentosa and M. heptapeta, emitted especially high relative amounts of the compound. Intact flowers of M. heptapeta and M. also emitted (E)-4,8-dimethyl-l,3,7-nonatriene (Table 3).

salicifolia

TABLE 3. DISTRIBUTION AND AMOUNT OF (E)-4,8-DIMETHYL-l,3,7-NoNATRiENE IN ARTIFICIALLY DAMAGED AND UNDAMAGED LEAF VOLATILES AND RELATIVE AMOUNT IN FLORAL VOLATILES OF Magnolia AND Liriodendron TAXA"

Flower' Leaf71 (ngl\2 hr/100 cm2 leaf area) Taxa

Magnolia Subgenus Magnolia M. virginiana M. virginiana1 M. grandifloras M. pyramidata M, tripetala M. hypoleuca M. sieboldii spp. japanica Subgenus Yulania M, acuminata M. praecocixsiina M. tomentosa M. salicifolia M. heptapeta" Liriodendron L. tulipifera

Damaged

2.48

1.74

%/2 hr/ 1-3 flowers

% (^g)/9 hr/flower

Undamaged

Detached ''

Intact

-

-'' 2.5 0.5

p

-''

-e

0.03

f

-e

-'

c

-''

-''

_''

_<: -r -r

e

0.1 3.1 37.9 -'' 51.8

-'• -•' 8.3 (0.48) 28.9 (39.37)

14.7

"From H. Azuma, M. Toyota, Y. Asakawa, L. B. Thien, R. Yamaoka, and S. Kawano (unpublished data). ''Both damaged and undamaged leaves remained attached to branches during sampling. c Floral organs were not damaged. ''Amounts of compounds compared with an internal standard were not examined. 'No characteristic ion indicating the compound was detected in GC-MS ion chromatogram. 'A cultivated plant in Tulane University was used. * Cultivated plants in Kyoto University were used.

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DISCUSSION

The artificially damaged leaves of Magnolia emitted the green odor compounds, (Z)-3-hexenol, (Z)-3-hexenyl acetate, and mono- and sesquiterpenes, particularly (Z)-j3-ocimene, (£)-/3-ocimene, and caryophyllene, in various combinations and quantities. Most of these volatiles are also emitted from the leaves of artificially damaged crop plants or plants attacked by herbivores (Takabayashi et al., 1994a,b; Loughrin et al., 1994, 1995; Rose et al., 1996). These volatiles also occur in floral fragrance of a wide range of angiosperms (Knudsen et al., 1993). The green odor compounds, (Z)-3-hexenol, (Z)-3-hexenyl acetate, and other C6 compounds, are synthesized from a-linolenic or linoleic acids from plastid membranes in green leaves when the leaves are damaged artificially or by herbivores (Hatanaka, 1993). Moreover, these compounds may elicit greater antennal olfactory responses than some monoterpenes in the parasitoid Microplitis croceipes (Hymenoptera) (Li et al., 1992). Corn, cotton, and other plants emit the green odors immediately after leaves are damaged, and then their release rapidly decreases. Large amounts of terpenoids such as (E)-4,8-dimethyl-l,3,7nonatriene, linalool, and (E)-3-ocimene are induced in herbivore-damaged leaves after several hours of damage and attract natural enemies of the herbivores (Turlings and Tumlinson, 1992; Takabayashi et al., 1994a,b; Turlings et al., 1995; Rose et al., 1996). Emission of these chemicals by artificially damaged leaves of Magnolia species suggests similar signaling between first and third trophic levels may occur. The composition of volatiles of damaged leaves of M. grandiflora differed considerably from the other Magnolia taxa, especially in the presence of relatively large amounts of several sesquiterpenes, the small amounts of green odors, and in the immediate emission of (E)-4,8-dimethyl-l,3,7-nonatriene. These differences suggest that the evergreen leaves of M. grandiflora store large amounts of volatiles, including a range of sesquiterpenes as (E)-4,8-dimethyl-l,3,7-nonatriene. The damaged leaves of M. heptapeta did not emit the green odors, which may be due to physiological conditions. Analyses of the GC-MS data clearly show that the undamaged leaves of seven Magnolia taxa emit no volatiles, except for M. hypoleuca, which produced a small amount of (Z)-3-hexenyl acetate, probably caused by small scratches on the leaf surface. Many investigations, however, report that undamaged leaves emit similar quantities of volatile compounds as the damaged leaves (Andersen et al., 1988; Turlings and Tumlinson, 1992; Jakobsen et al., 1994; Loughrin et al., 1994, 1995; Mattiacci et al., 1994; Takabayashi et al., 1994a,b; Rose et al., 1996). In these investigations, undamaged leaf volatiles were collected from leaves cut from the plants or from potted plants, and very young plants were utilized as plant materials. The reasons for the absence of volatiles in the

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undamaged leaves of Magnolia may simply be that leaves attached to the branches were used. Induction of volatiles in agricultural plants is achieved by interactions of herbivore caterpillar regurgitant (Mattiacci et al., 1994) and/or application of (3-glucosidase to mechanically damaged leaves that results in an emission of a blend of volatiles similar to those emitted by feeding herbivores (Mattiacci et al., 1995). Thus, in some taxa, enzymes such as /3-glucosidase (an elicitor) probably activate biosynthesis of terpenoids (and other compounds), including two terpene alcohols, nerolidol and geranyl-linalool, that eventually lead to 4,8dimethyl-l,3,7-nonatriene and4,8,12-trimethyl-l,3,7,ll-tridecatetraene (Boland et al., 1992). Jasmonic acid and its derivative, methyl jasmonate, are elicitors or signal transducers in some plants (Farmer and Ryan, 1990; Boland et al., 1995). Although the activities of j3-glucosidase and jasmonic acid derivatives have not been analyzed in Magnolia, methyl jasmonate and jasmone were present as components of floral volatiles of some plants including M. grandiflora (Thien, et al., 1975; Kaiser, 1991; Yasukawa et al., 1992; Knudsen et al., 1993). Emission of (E)-4,8-dimethyl-l,3,7-nonatriene by some taxa of Magnolia and Liriodendron comprised up to 30% of the floral volatiles (Table 3). Although more than 700 chemical compounds have been isolated in floral scents of angiosperms from 441 taxa (134 families) (Knudsen et al., 1993), the presence of (E)-4,8-dimethyl-l,3,7-nonatriene in floral scents is relatively rare (ca. 20 families) (Kaiser, 1991; Boland et al., 1992; Knudsen et al., 1993; Knudsen and Mori, 1996). The emission of (E)-4,8-dimethyl-l,3,7-nonatriene may be inductively generated in the detached flowers of some Magnolia, but the intact flowers of M. heptapeta constantly emit a high amount of the compound. The family Magnoliaceae represents an ancient lineage of plants (Thorne, 1996), and the genus Magnolia is primarily composed of temperate and subtropical taxa that typically bear numerous, large, highly fragrant flowers over long periods of time and attract a wide range of insect visitors and pollinators (Thien, 1974; Yasukawa, et al., 1992). Despite the attraction of a large number of insects to the flowers, floral and leaf herbivory appears to be low on most Magnolia taxa, even though species of Papilio (Lepidoptera: Papilionidae) feed on members of the Magnoliaceae (Scriber, 1988). Although the function of (E)-4,8-dimethyl-l,3,7-nonatriene in floral volatiles is still uncertain, the fact that this compound is emitted not only by the damaged leaves but also by the flowers of some Magnolia taxa and the common occurrence of several terpenoids such as /3-ocimene, jS-myrcene, linalool, and caryophyllene in floral volatiles of many flowering plants (Knudsen et al., 1993) suggest that the interaction between flower and pollinator and the chemical communication between the first and third trophic levels may be interrelated.

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Acknowledgments—We thank Dr. Ryohei Yamaoka for valuable comments and discussions throughout the present study. This study was financially supported by the Japanese Government (Monbusho Scientific Research Grant-in-aid for Priority Areas No. 04264102 to Shoichi Kawano).

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