Volatile Floral Chemistry of Heliotropium Arborescens L. 'Marine'

HORTSCIENCE 40(5):1237–1238. 2005. cymes attached to the parent plant were placed within cylindrical glass containers (497 mL) closed at one end and having a lid with an Volatile Floral Chemistry of aperture slightly larger than the diameter of the stem. The fl ower stem attached to the parent Heliotropium arborescens L. ‘Marine’ plant was placed through the aperture which 1 2 3 was sealed with closed pore neoprene. Volatile Stanley J. Kays, Jason Hatch, and Dong Sik Yang samples were collected during the day using a Department of Horticulture, Plant Sciences Building, The University of Georgia, Tenax trapping system developed for the col- Athens, GA 30602-7273 lection of headspace volatiles (Wang and Kays, 2002). Air fl ow through the glass container Additional index words. quality, odor, aroma, fl ower, fragrance holding the cyme was generated using a small, Abstract. Selection emphasis on cyme size and fl ower color of Heliotropium arborescens L. has portable vacuum pump (model 224-44XR; led to cultivars with diminished fl oral fragrance. As a preliminary inquiry into the fragrance AirChek Sampler, SKC Inc., Eighty Four, Pa.) chemistry of the species, we identifi ed 41 volatile compounds emanating from the fl owers of with the fl ow rate monitored and regulated us- ‘Marine’ via isolation (Tenax trapping) and gas chromatography–mass spectrometry. The ing a fl ow meter with value. An air fl ow rate –1 majority of the volatile compounds emanating from the fl owers were terpenes (camphene, of about 20 mL·min was used throughout the p-cymene, δ-3-carene, α-humulene, δ-1-limonene, linalool, (E)-β-ocimene, α-pinene, and experimental period. Volatiles were collected β-thujone), benzenoids of which benzaldehyde was the most abundant, aldehydes (decanal, for a 4-h period which gave about 10 container heptanal, nonanal and octanal), and hydrocarbons (decane, heneicosane, heptadecane, volume changes. Inlet air was passed through hexadecane, nonadecane, nonane, octadecane, tetradecane, tridecane and undecane) along a charcoal fi lter (Alltech Assoc. Inc. Deerfi eld, with a cross-section of other compounds. Subsequent identifi cation and quantifi cation of Ill.) (1 cm i.d., 5-cm-long Pyrex glass tube critical ordorants will facilitate selecting new cultivars with quantitative and qualitative with a 3-cm bed of charcoal). Before use, the improvements in fragrance. charcoal fi lter was cleaned and activated at 200 –1 °C for 24 h by passing purifi ed N2 (2 mL·min ) Heliotropium is a genus of about 250 represent a major source of volatiles (Char- through the trap. All connections in the volatile species of fl owering herbs and shrubs in the ron et al., 1995), releasing an estimated 1.4 × collection system were ground glass joints or Boraginaceae family, members of which are 109 t·year–1 into the biosphere (Went, 1974). Tefl on connectors. A second collection method grown in borders and greenhouses of warm A number of volatiles are known to have was employed by placing several decapitated regions for their fragrant fl owers (Bailey, critical biological roles; for example, certain fl oral cymes in the container and the volatiles 1977). H. arborescens (synonymous with H. fl oral volatiles act as synomones, attracting were collected in a similar manner. peruvianum L. and H. corymbosum Ruiz & pollinators or as kairomones or allomones Volatiles were collected using a Tenax trap Pav.) is the common heliotrope and the most (Dunsenbery, 1992). Often the interaction (Tenax-TA, 60/80) (Alltech) prepared by pack- widely cultivated, predominately for it’s violet involves a mixture of compounds in relatively ing 50 mg of the adsorbent in a Pyrex glass tube to white fragrant fl oral cymes. Though techni- specifi c proportions and the relationship among (3.5 mm i.d. × 5 cm), giving a bed length of 3 cally a shrub, the plant is typically cultivated organisms can be relatively complex. In some cm. Silylated glass wool was used to contain as an annual and is available from a number of instances, three trophic levels may respond to the adsorbent at each end. After collection, the seed companies. In the 1950s heliotrope was a air borne chemical information. For example, trapped volatiles were eluted from the Tenax popular cut fl ower in the United States due to Nicotiana attenuata Torr. ex Wats plants under with 500 µL of purifi ed hexane containing 1 it’s fragrance as much as to the striking appear- attack by several herbivores release volatile µL·L–1 1-octadecene (Sigma, St. Louis, Mo.) ance of the fl owers. More recently, however, phytochemicals that attract natural predators of as an internal standard. The eluted solvent was breeding emphasis has focused upon larger, the insects, greatly reducing (i.e., up to 90%) concentrated to 30 µL by slowly fl ushing with more brightly colored fl owers and much of the the herbivore population (Kessler and Baldwin, purifi ed N2. fragrance has been lost (Armitage, 2001). The 2001). Floral volatiles are also key elements in GC and GC–MS of volitiles. Gas chro- common heliotrope remains a relatively obscure our enjoyment of certain species (e.g., Gardenia matographic analyses were performed using fl oricultural crop on which there has been little jasminoides Ellis). a Hewlett-Packard 5890 gas chromatograph published research. An initial attempt to assess Due to the past importance of the fragrance (Hewlett-Packard, Wilmington, Del.) equipped the volatiles produced by H. aborescens fl owers of H. arborescens and the potential for breed- with a fl ame ionization detector (FID). The GC using solid phase microextraction identifi ed ing new cultivars with improved aroma, as a column was a 30 m × 0.32 mm (i.d.) fused silica, three major compounds (benzaldehyde, benzyl preliminary inquiry we isolated and identifi ed DB-5, 0.25 µm fi lm thickness capillary column acetate and p-anisaldehyde) (Hisano et al., the major volatile compounds emanating from (J&W Scientifi c, Folsom, Calif.). Samples were 1995). The cultivar Marine is day neutral with the fl owers. injected in the splitless mode. Injector and fl ower initiation being advanced by exposure detector temperatures were 180 and 250 °C, to cool temperatures (i.e., 10 °C) before forcing Materials and Methods respectively. Oven temperature was initially (Park and Simons, 2000). It also has minimal held at 35 °C for 3 min and then programmed fragrance. Plant material. Plants of the cultivar Marine from 35 to 240 °C at 5 °C·min–1 and held 5 min. Biologically active volatile compounds such (Ball FloraPlant, West Chicago, Ill.) growing Helium was used for the carrier gas at a fl ow rate as fragrances and pheromones originate from in the Department of Horticulture ornamental of 53.1 mL·min–1. Air and hydrogen fl ow rates a myriad of sources and exert a diverse range display garden at the University of Georgia were to the detector were 370 to 380 mL·min–1 and of effects on living organisms (Kays and Paull, used for volatile collection. Individual cymes (7 28 mL·min–1, respectively. Nitrogen was used 2004). Some act as semiochemicals, functioning to 9 cm in diameter) comprised typically of 250 as the makeup gas at a fl ow rate of 30 mL·min–1. in the communication and interaction within or to 300 individual fl owers (4 to 6 mm diameter) An HP3392A integrator was connected to the among organisms (Dunsenbery, 1992). Plants supported by a many branched fl oral stem were GC for peak area measurement. used; thus some tissue other than fl oral may have Mass spectral analysis was performed us- Received for publication 6 Jan. 2005. Accepted contributed to the volatiles collected. Cymes, ing a Hewlett Packard mass spectrometer (MS for publication 20 Mar. 2005. The authors would composed of closely arranged individual fl owers like to thank Allan Armitage for providing access 5970) interfaced with a HP 5890 GC. The GC to the plant material and background information of differing maturities, were selected in which column used was a 30 m × 0.32 mm i.d. fused on the species. most fl owers had reached anthesis and relatively silica column coated with DB-5. GC and tem- 1Professor. few had senesenced. The fragrance of ‘Marine’ perature program conditions were as previously 2Undergraduate student. was perceptible but diminutive. mentioned. The GC–MS was programmed with 3Graduate student. Collection of headspace volatiles. Individual a 3 min solvent delay. Electron ionization was

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AAugustBook.indbugustBook.indb 11237237 66/14/05/14/05 112:10:472:10:47 PPMM used and the MS conditions were ion source p-cymene in Tilia cordata Mill. (Buchbauer Literature Cited 200 °C; electron energy 70 eV; multiplier et al., 1995), linalool in Lonicera japonica Armitage, A.M. 2001. Armitage’s manual of annuals, voltage 220 V; GC–MS interface zone 300 °C. Thunb. (Miyake et al., 1998), α-humulene in biennials, and half-hardy perennials. Timber Press, Ions from 20 to 550 atomic mass units were Alpinia chinensis Rosc. (Dung et al., 1994), Portland, Ore. monitored. The identities of compounds were and β-thujone in Lippia turbinata Griseb. and Bailey, L.H. 1977. Manual of cultivated plants. Macmil- lian, New York. based on the Wiley Spectral Library (6th ed.) L. polystachya Griseb. (Zygadlo et al., 1995). Buchbauer, G., B. Remberg, and L. Jirovetz. 1995. Compara- and the National Institute of Standards 75 K Eight benzenoid compounds were identifi ed, tive headspace analysis of living and fresh cut lime tree library and, when possible, confi rmed using one of which was previously reported from fl owers (Tiliae fl ores). Flavour Frag. J. 10:221–224. authentic standards. heliotrope fl owers (Hisano et al., 1995). Brunke, E.J., F.J. Hammerschmidt, and G. Schmaus. 1992. Scent of roses—Recent results. Flavour Frag. Quantitatively, benzaldehyde was the most J. 7:195–198. Results and Discussion abundant component. Each of the aldehydes Charron, C.S., D.J. Cantliffe, and R.R. Heath. 1995. Volatile (i.e., heptanal, octanal, nonanal and decanal) emissions from plants. Hort. Rev. 17:43–72. In total, 41 compounds were identifi ed and a number of the hydrocarbons (i.e., nonane, Dabiri, M. and F. Sefi dkon. 2001. Analysis of the essential oil from aerial parts of Perovskia triplicifolia Benth. emanating from the fl owers of H. arborescens decane, tridecane, hexadecane, heptadecane, at different stages of plant growth. Flavour Frag. J. cv. Maine (Table 1). These included a cross- octadecane and heneicosane) are known to 16:435–438. section of terpenes, which have been shown emanate from honeysuckle fl owers (Lonicera Dung, N.X., T.D. Chinh, D.D. Rang, and P.A. Leclercq. to contribute to the aroma of fl owers and other japonica Thunb.) (Ikeda et al., 1994). 1994. Chemical composition of the fl ower oil of Al- pinia chinensis Rosc. from Vietnam. J. Essential Oil plant parts of a number of species (Giamakis The data presented in Table 1 represent Res. 6:637–638. et al., 2001; Kilic et al., 2004; Nickavar et al., compounds identifi ed using both intact and Dung, N.X., N.T.B. Tuyet, P. Van Khien, A. Barthel, and 2004). Alpha-pinene is a major constituent decapitated cymes. Decapitation allowed P.A. Piet. 1998. Chemical composition of the fl ower in the volatiles from Encyclia vespa (Vell.) substantially increasing the amount of plant oil of Curcuma pierreana Gagnep. from Vietnam. J. Essential Oil Res. 10:527–528. Dressler & G. E. Pollard and E. fragrans (Sw.) material from which volatiles could be trapped, Dunsenbery, D.B. 1992. Sensory ecology: How organisms Lemee fl owers (Zoghbi et al., 2002). The other thus facilitating the identifi cation of components acquire and respond to information. W.H. Freeman and terpenes identifi ed have been reported as ema- that were found in relatively small amounts. Co., New York. nating from a variety of fl owers, e.g., camphene While we have not quantifi ed differences, de- Giamakis, A., O. Kretsi, I. Chinou, and C.G. Spyropoulos. 2001. Eucalyptus camaldulensis: Volatiles from im- in Curcuma pierreana Gagnep. (Dung et al., capitation altered the volatile profi les obtained. mature fl owers and high production of 1,8-cineole 1998), δ-3-carene in Perovskia atriplicifolia For example, p-cymene was not found in the and β-pinene by in vitro cultures. Phytochemistry Benth. (Dabiri et al., 2001), δ-1-limonene, and headspace of fl owers that were severed from the 58:351–355. parent plant. A similar repression was reported Hisano, H., K. Ishimaru, H. Tada, and Y. Ikeda. 1995. Table 1. Volatile compounds emanating from the for p-cymene in lime tree fl owers (Tilia cordata Flavors of heliotrope fl owers, analyzed by solid phase fl oral cymes of Heliotrope arborescens ‘Marine’ microextraction method. Nippon Shokuhin Kagaku Mill.) (Buchbauer et al., 1995). Decapitation, Gakkaishi 2:6–8. identifi ed by GC–MS. likewise, can result in the production of wound Ikeda, N., M. Ishihara, T. Tsuneya, M. Kawakita, and M. Compound volatiles also altering the profi le. Yoshihara. 1994. Volatile components of honeysuckle 2-Hexanone (Lonicera japonica Thunb.) fl owers. Flavour Frag. J. Although not investigated, the time of col- 9:325–331. 2-Hexanol lection may also quantitatively and qualitatively Kays, S.J. and R.E. Paull. 2004. Postharvest biology. Exon Ethyl benzene alter the volatile profi le (Matile and Altenburger, Press, Athens, Ga. 1,2-Dimethyl benzene 1988). For example, certain species of Nicotiana Kessler, A. and I.T. Baldwin. 2001. Defensive function of p-Xylene exhibit nocturnal rhythms in fragrance chemistry herbivore-induced plant volatile emissions in nature. ethenyl benzene Science 291:2141–2144. Nonane (Raguso et al., 2003). Kilic, A., H. Hafi zoglu, H. Kollmannsberger, and S. Nitz. Heptanal In the current study, volatiles present in the 2004. Volatile constituents and key odorants in leaves, Methoxybenzene headspace around the fl owers were sampled buds, fl owers, and fruits of Laurus nobilis L. J. Agr. Food Chem. 52:1601–1606. α-Pinene rather than using solvent extraction of the fl ow- Matich, A.J., H. Young, J.M. Allen, M.Y. Wang, S. Fielder, Camphene ers. The latter can signifi cantly increase the num- M.A. McNeilage, and E.A. MacRae. 2003. Actinidia Benzaldehyde ber of compounds identifi ed and the quantity of arguta: Volatile compounds in fruit and fl owers. Phy- 6-Methy-5-heptene-2-one higher molecular weight molecules (Matich et tochemistry 63:285–301. Decane Matile, P. and R. Altenburger. 1988. Rhythms of fragrance al., 2003), however, many components isolated emission in fl owers. Planta 174:242–247. Octanal in this manner have relatively low volatilities 1,4-Dichlorobenzene Miyake, T., R. Yamaoka, and T. Yahara. 1988. Floral scents of hawkmoth-pollinated fl owers in Japan. J. Plant Res. δ-3-Carene and are often insignifi cant contributors to the fragrance. In contrast, the Tenex trapping tech- 111:119–205. δ-1-Limonene Nickavar, B., G. Amin, and M. Yosefi . 2004. Volatile 2-Ethyl-1-hexanol nique used in the study has a higher collection constituents of the fl ower and fruit oils of Pittospo- (E)-β-Ocimene effi ciency for the more volatile, lower molecular rum tobira (Thunb.) Ait. grown in Iran. J. Biosci. Undecane weight compounds. 59(3/4):174–176. Linalool* The volatiles produced from plants, whether Park, B.H. and P. Simon. 2000. Environmental regulation of p-Cymene fl owering time in heliotrope (Heliotropium arborescens from the fl owers or other organs (Wang and L. cv. Marine). Scientia Hort. 85:231–241. β-Thujone Kays, 2003), can vary signifi cantly among Nonanal Raguso, R.A., R.A. Levin, S.E. Foose, M.W. Holmberg, and cultivars, contributing to the diverse range in L.A. McDade. 2003. Fragrance chemistry, nocturnal Decanal fragrances and aromas found in the plant king- rhythms and pollination “syndromes” in Nicotiana. Benzothiazole Phytochemistry 63:265–284. 4-Methoxy benzaldehyde dom. For example, the volatile constituents of Wang, Y. and S.J. Kays. 2002. Sweetpotato volatile chemistry Octacosane rose fl owers varied markedly among cultivars in relation to sweetpotato weevil (Cylas formicarius) Eicosane (Brunke et al., 1992). The cultivars Othello, behavior. J. Amer. Soc. Hort. Sci. 127:656–662. Tricosane Duchesse de Montebello and Lichtkönigin Wang, Y. and S.J. Kays. 2003. Analytically directed fl avor Tridecane Lucia differed markedly from Queen Elizabeth selection in breeding food crops. J. Amer. Soc. Hort. -Humulene Sci. 128:711–720. α indicating that substantial genetic variation Went, F.W. 1974. Refl ections and speculations. Ann. Rev. Tetradecane exists which could be readily exploited via Plant Physiol. 25:1–26. Hexadecane plant breeding to create new cultivars with Zoghbi, M.G.B., E.H.A. Eloisa, M.F.F. da Silva, and J.G.S. Heptadecane Maia. 2002. Flower scent analysis of Encyclia vespa Octadecane distinctive fragrances. Chemically character- (Vell.) Dressler & G. E. Pollard and E. fragrans (Sw.) 6,10,14-Trimethyl-2-pentadecanone izing the volatiles emanating from a fl oral crop Lemee. Acta Amazonica 32:65–70. 2,6,10,14-Tetramethyl pentadecane and identifying and quantifying the critical Zygadlo, J.A., A.L. Lamarque, C.A. Guzman, and N.R. Nonadecane compounds allows developing an analytical Grosso. 1995. Composition of the fl ower oils of some Heneicosane Lippia and Aloysia species from Argentina. J. Essential means of making progeny selection decisions Oil Res. 7:593–595. *Tentatively identifi ed. in fl ower breeding programs.

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