Volatile Compounds from Wheat Plants: Isolation, Identification, and

16

Downloaded by UNIV OF MICHIGAN ANN ARBOR on October 30, 2014 | http://pubs.acs.org Publication Date: August 25, 1986 | doi: 10.1021/bk-1986-0317.ch016

Volatile Compounds from Wheat Plants: Isolation, Identification, and Origin Thomas R. Hamilton-Kemp1 and R. A. Andersen2 Department of Horticulture, University of Kentucky, Lexington, ΚY 40546 Agricultural Research Service, U.S. Department of Agriculture, and Department of Agronomy, University of Kentucky, Lexington, KY 40546

1 2

Volatile compounds have been reported to stimulate germination, in vitro and in vivo, of spores of the fungal pathogens which cause wheat rust diseases. Since growth and development of the pathogens disrupt plant tissue with resultant formation of volatiles, we have isolated and identified volatile compounds from disrupted wheat leaves and stems. Volatiles were isolated by steam distillation-extraction at reduced pressure and thirty-five compounds were identified by GC-MS. Among these volatiles are compounds reported to stimulate rust spore germination. Comparisons have been made among volatiles from leaves and culms, immature and mature plants and among different stages of tissue disruption. The possible origin of compounds isolated and the effects of tissue source are discussed. One of the areas in nature where volatile compounds from plants may play a role which has not been extensively investigated is the chemical interaction between plants and microorganisms, especially fungi. Many species of fungi are known which cause serious diseases in economically important plants. These diseases frequently result in the death of a plant or the destruction of a crop if conditions for their development are favorable. Early examples of the effects of volatile compounds on the development of fungi were reported by Soulanti (1) and Fries (2) who showed that the growth of wooddecomposing fungi was stimulated by volatiles. We found that volatiles from cut or crushed leaves of cucurbitaeous plants such as melon and squash increased the degree and severity of infection of stem blight disease (fungal origin) in the cucurbits (3). The most thorough studies of promotion of fungal development by volatiles was done by French and co-workers (4-6) who showed that volatile compounds in solution or their vapors were effective promotors of rust spore germination. Rusts are diseases found on many species including crop plants such as wheat, corn, beans and others. They are caused by fungi whose life cycle includes multiple development steps with the formation of rust colored uredospores in 0097 6156/86/0317 0193S06.00/ 0 © 1986 American Chemical Society

In Biogeneration of Aromas; Parliment, T., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1986.


194

BIOGENERATION OF AROMAS

pustules beneath the epidermis of a host plant. Many synthetic v o l a t i l e s have been tested and several, including nonanal and nonanol, were e f f e c t i v e at sub-parts per m i l l i o n levels in stimulating germi nation of wheat rust uredospores (4,5)· A factor to be considered in studying the v o l a t i l e s from a host plant is that the development of the fungus on i t s host causes damage or disruption of the tissue. That is, in rust diseases the invasive vegetative growth of the fungal hyphae and the formation of spore pustules on leaves and stems of an infected plant disrupts tissue and c e l l s and results in the biogenesis of v o l a t i l e compounds. The study of injured or disrupted tissue seems contrary to the objectives of most physiological studies in which the "normal" physiological functions of the plants take place. However, under the circumstances described for the interactions of a plant and an invasive fungus J. seems pertinent to determine the effects of injury or disruption. Under these conditions the compounds formed can be expected to a r i s e from enzymatic oxidations and other degradative reactions. Experimental Winter wheat plants c u l t i v a r 'Arthur 71' were harvested from f i e l d plots at various stages of maturity (Figure 1) as described below and refrigerated at 5°C or stored frozen at -20°C. Isolation and Separation. V o l a t i l e s were isolated from 1 kg of plant tissue by steam d i s t i l l a t i o n - e x t r a c t i o n in a water recycling s t i l l (7) containing 2.8L of water and operated at reduced pressure. Hexane (4ml) was placed on the water in the s i d e arm and the s t i l l was operated at 65°C and approximately 200mm of Hg for 3 hrs. The hexane was removed after a run and the combined hexane layers from eight d i s t i l l a t i o n s were dried over Na2S0^ and concentrated to approximately 100 u l under a stream of nitrogen. The concentrate was separated i n i t i a l l y on a 1.8m χ 4mm column packed with 20% SP 2100 (nonpolar phase) on Supelcoport. The column was temperature programmed from 100°C to 180°C at a rate of ^l°C/min. Fractions corresponding to chromatographic peaks were collected in U-shaped tubes cooled in a Dry-Ice-acetone bath. Fractions were further separated on either a 30m χ 0.25mm Carbowax 20M or a 30m χ 0.32mm Supelcowax 10 fused s i l i c a c a p i l l a r y column. Mass spectral studies. Mass spectral analyses were performed using a Finnegan 330-6100 GC-MS instrument equipped with the same fused s i l i c a c a p i l l a r y columns described above. Electron impact studies were carried out at 70 eV and chemical ionization studies were performed using methane. Quantitative analysis. For a l l quantitative comparisons two analyses were performed by dividing a d i s t i l l a t e into two equal portions prior to concentration and GC a n a l y s i s . The peak areas obtained from c a p i l l a r y column chromatograms were normalized to an internal standard, pentadecane, added to each f r a c t i o n . The means of two analyses are presented and the values obtained can be interpreted as approximately equivalent to parts per b i l l i o n of v o l a t i l e compound per tissue wet weight.


16. HAMILTON-KEMP AND ANDERSEN

Volatile Compounds from Wheat Plants

195

Results and Discussion I d e n t i f i c a t i o n of compounds and o r i g i n . The v o l a t i l e compounds i s o lated by steam d i s t i l l a t i o n - e x t r a c t i o n of a wheat sample are l i s t e d in Table I.


Table I.

V o l a t i l e Compounds Isolated from Vegetative Wheat Tissue by Reduced Pressure Steam D i s t i l l a t i o n Extraction

Compound

Compound

Hexanal t-2-Hexenal Heptanal t-2-Heptenal t-2-0ctenal Nonanal t-2-Nonenal t,c-2,6-Nonadienal Decanal t-2-Decenal Undecanal t-2-Undecenal Tridecanal Tetradecanal Pentadecanal c,c-8,11-Heptadecadienal c, c,c-8,11,14-Heptadecatr ienal Benzaldehyde

1-Hexanol t-2-Hexen-l-ol c-3-Hexen-l-ol 1-Heptanol 1-Octanol 1-Nonanol t-2-Nonen-l-ol c-3-Nonen-l-ol t,c-2,6-Nonadien-l-ol c,c-3,6-Nonadien-l-ol 1- Decanol c-3-Hexenyl Acetate Eugenol B-Ionone Pentadecane Hexadecane 2- 0ctadecanone

Compound i d e n t i f i c a t i o n s were based on comparison of mass spectral data and co-chromatography of plant components with standards as reported (8,9). A sample of 3,6-nonadien-l-ol was isolated from melon (10) whereas 8,11-heptadecadienal and 8,11,14-heptadecatrienal were obtained from cucumber f r u i t (11). Of primary interest in the present study is the i d e n t i f i c a t i o n of C9 aldehydes and alcohols including nonanal and nonanol which are e f f e c t i v e promotors of wheat rust spore germination. The unsaturated C9 compounds have not been evaluated for a c t i v i t y but t h e i r close structural relationships to nonanal and nonanol make them candidates for germination promotors. The unsaturated Cg aldehydes and alcohols probably a r i s e in wheat from cleavage of the 9-10 double bond of unsaturated C^e f a t t y acids, l i n o l e i c and l i n o l e n i c acids (Figure 2). G a l l i a r d and coworkers (12,13) have p a r t i a l l y p u r i f i e d enzyme systems capable of catalyzing these transformations. Lipoxygenase i n i t i a l l y converts l i n o l e i c and l i n o l e n i c acids to 9-hydroperoxides which are subsequently cleaved by hydroperoxide lyase to v o l a t i l e Cg unsaturated aldehydes and 9-oxo-nonanoic a c i d . The 3-enals are the primary v o l a t i l e cleavage products from the f a t t y acids and these are transformed by an isomerase to the more stable 2-enals (14)· The 3-enals are rather unstable but Hatanaka et a l . (15) have confirmed their presence in plant tissue with authentic samples. The Cg



HH

HH

HH

(LlNOLENIC ACID)

HH

HH

Φ ENZ.

C,C-3/6-NONADIENAL

HH H CH3-CH2-C=C-CH2-CH2-C=C-aiO I/C-2/5-NONADIENAL

i ADH ENZ HH H CHCH2-C=C-CH2-CH2-C=^-CH2-0H

CC-3,6-NONADIEN-l-OL

r

I/£-2.6-N0NADIEhhl-0L

Figure 2 . Pathways for biosynthesis of C9 aldehydes and alcohols from l i n o l e n i c a c i d .


16.

HAMILTON-KEMP A N D ANDERSEN


197


a l c o h o l s p r o b a b l y a r i s e from r e d u c t i o n o f t h e a l d e h y d e s by a l c o h o l dehydrogenase enzymes. Another major group o f compounds found in wheat a r e t h e a l d e h y d e s and a l c o h o l s . These compounds a r e formed from t h e C±q f a t t y a c i d s by pathways s i m i l a r t o t h o s e f o r t h e Cg compounds except t h a t t h e 12-13 double bond o f t h e f a t t y a c i d s is c l e a v e d (13,14,16). Comparison o f f r e s h and f r o z e n immature p l a n t s . To o b t a i n an adequate amount o f f i e l d grown wheat f o r i n i t i a l i d e n t i f i c a t i o n o f compounds, p l a n t s were h a r v e s t e d and s t o r e d f r o z e n p r i o r t o d i s t i l l a t i o n extraction. A f t e r i d e n t i f i c a t i o n s were c o n f i r m e d a comparison was made between t h e v o l a t i l e s from f r e s h and f r o z e n t i s s u e t o d e t e r m i n e i f t h e compounds i s o l a t e d from f r o z e n samples were a l s o p r e s e n t in f r e s h wheat t i s s u e . F o r t h e s e comparisons immature p l a n t s ( F i g u r e 1) 20-30cm t a l l were h a r v e s t e d and macerated in a Waring b l e n d e r p r i o r to d i s t i l l a t i o n - e x t r a c t i o n . From T a b l e I I J. can be seen t h a t t h e

T a b l e I I . Comparison o f R e l a t i v e Q u a n t i t i e s o f V o l a t i l e s from F r e s h V e r s u s F r o z e n V e g a t a t i v e T i s s u e o f Immature Wheat P l a n t s Wheat T i s s u e Compound Hexanal 1-Hexanol 2-Hexenal 2-Hexen-l-ol 3-Hexen-l-ol Nonanal 1-Nonanol 2-Nonenal 2-Nonen-l-ol 3-Nonen-l-ol 2,6-Nonadienal 2,6-Nonadien-l-ol 3,6-Nonadien-l-ol Tetradecanal Pentadecanal 8,11-Heptadecadienal 8,11,14-Heptadecatrienal B-Ionone Eugenol

Fresh

Frozen

3.2 290 2090 2050 1820 4.4 1.0 0.4 0.2 0.6 0.9 0.4 1.2 0.1 3.0 0.04 0.1 32.9 0.2

0.4 1.2 29.0 3.2 0.4 10.6 2.5 1.4 2.0 0.9 4.5 5.7 1.4 0.2 4.0 0.2 1.0 11.5 0.2

same compounds i n c l u d i n g t h e Cg compounds were p r e s e n t in t h e f r e s h and f r o z e n wheat. By f a r t h e g r e a t e s t q u a n t i t a t i v e d i f f e r e n c e s were found f o r t h e C^ compounds w h i c h were i s o l a t e d in amounts up t o s e v e r a l thousand f o l d g r e a t e r from f r e s h p l a n t s t h a n from f r o z e n p l a n t s o f t h e same a g e . Thus f r e e z i n g causes a marked l o s s o f C 5 compounds. I t a p p e a r s t h a t one o r more enzyme systems r e s p o n s i b l e f o r t h e f o r m a t i o n o f t h e s e compounds is i n a c t i v a t e d by f r e e z i n g . As p a r t o f a s t u d y o f t h e compounds t h a t a t t r a c t i n s e c t s t o i n t a c t wheat p l a n t s , B u t t e r y , e t a l . (17) r e c e n t l y i d e n t i f i e d v o l a t i l e s o b t a i n e d from u n d i s r u p t e d immature p l a n t s by headspace


198



analysis (purge and trap technique). 3-Hexen-l-ol and 3-hexenyl acetate were found to be major components of the young whole plants (15-20 cm t a l l ) . 3-Hexen-l-ol and other compounds have "green" grass-like odors and appear to be q u a l i t a t i v e l y and quantitatively important components which contribute to the c h a r a c t e r i s t i c odor of the immature plants studied in the present work a l s o . Analysis of the Cg compounds indicated that freezing caused an increase of 2-3 f o l d of most of these v o l a t i l e s with the exception of the 2-enols which increased more than 10-fold. Since the 2-enols were s i m i l a r l y affected there may be an enzyme responsible for the reduction of the 2-enals to alcohols which is d i s t i n c t from the system responsible for reduction of 3-enals. Comparison of leaves and culms of mature plants. Comparisons were made between v o l a t i l e s steam d i s t i l l e d from leaves and culms (hollow stems) of mature plants which were 40-50 cm t a l l (Figure 1). Rusts manifest d i f f e r e n t a f f i n i t i e s for these plant parts. Plants were harvested, stored refrigerated overnight and leaf blades were cut from the culms and both parts were cut into 3-6 cm segments. The results from these comparisons are presented in Table I I I . With

Table I I I . Comparison of Relative Quantities of V o l a t i l e s from ____ Leaves and Culms of Mature Wheat Plants Compound

Leaves

Culms

Hexanal 1-Hexanol 2-Hexenal 2-Hexen-l-ol 3-Hexen-l-ol Nonanal 1-Nonanol 2-Nonenal 3-Nonen-l-ol 2,6-Nonadienal 2,6-Nonadien-l-ol 3,6-Nonadien-l-ol Tetradecanal Pentadecanal 8,11-Heptadecadienal B-Ionone 3-Hexenyl Acetate Hexadecane

0.5 0.9 1.1 0.6 3.3 39.2 17.7 0.2 0.5 0.4 0.2 1.0 0.2 2.1 0.1 4.4 1.3 0.5

0.3 0.5 0.5 0.1 1.0 7.9 9.8 0.8 11.3 1.8 1.1 19.7 0.5 6.6 0.2 1.4 0.3 0.3

regard to the C^ compounds, there was a decrease in the quantity of the major components, nonanal and nonanol, in the culms. There was a marked increase of 10-20 f o l d in the 3-enols in the culms. This suggests that the 3-enols are biosynthetically related since they both increased whereas 2,6-nonadien-l-ol did not increase as markedly. (The 2-nonen-l-ol was obscured by an adjacent peak.) The amounts of C^ compounds were greatly reduced in mature


16.

HAMILTON-KEMP AND ANDERSEN


199

plants when compared to the young plants used in the previous comparisons. The precipitous drop in these compounds may be due to a decrease in f a t t y acid substates, enzymic a c t i v i t y or both in mature plants. There was also a trend toward reduction in amounts of Cfc compounds in culms compared to leaves.


Comparison of whole and cut plants. An experiment was also done to compare v o l a t i l e s from cut plants with those from whole or intact plants. Immature plants 30-50 cm t a l l were harvested and r e f r i g e r ated overnight and then cut into 3-6 cm segments. From Table IV J.

Table IV.

Comparison of Relative Quantities of V o l a t i l e s from Whole and Cut Segments of Immature Wheat Plants

Compound Nonanal 1-Nonanol 2-Nonenal 3-Nonen-l-ol 2,6-Nonadienal 2,6-Nonadien-l-ol 3,6-Nonadien-l-ol B-Ionone Pentadecanal

Whole

Cut

2.1 2.2 0.1 0.1 0.1

5.0 6.0 0.2 1.4 0.2 0.1 2.6 2.2 3.0

0.1 0.7 1.6

can be seen that the y i e l d s of most of the C9 compounds increased 2-3 f o l d . In contrast, 3-nonen-l-ol and 3,6-nonadien-l-ol increased approximately 15-25 f o l d on cutting indicating that they are biosynt h e t i c a l l y related. The 2-enols were isolated in such small amounts that changes could not be quantitated. It should be noÇed that the procedures necessary for harvesting and d i s t i l l a t i o n of whole plants may have resulted in bruising which contributed to the v o l a t i l e s i s o l a t e d . However, there were clear differences in y i e l d of 3-enols between cut and whole plants. Conclusions The results obtained provide information on the types of compounds which are obtained from wheat plants by a single i s o l a t i o n method, steam d i s t i l l a t i o n - e x t r a c t i o n , and how the r e l a t i v e amounts of these compounds vary with certain treatments. Further studies are needed to expand knowledge of the types and amounts of compounds present and their relevance to the interaction of a fungal pathogen with a host plant during disease development. French and Gallimore (5) have suggested that c e r t a i n v o l a t i l e s such an nonanol, might be applied to plants to cause premature germination of spores and reduce the spread of disease. S i m i l a r l y , J. may be possible to obtain high l e v e l s of certain endogenous v o l a t i l e promotors of spore germination, in the future, through genetic manipulation after the v o l a t i l e s present in host plants and their metabolic origins have been thouroghly investigated.


200


Acknowledgments We are grateful to Lois Kemp, Pierce Fleming, and John Loughrin for assistance and to George Lovelace for mass spectral analysis. We thank Pam Wingate for typing the manuscript. Literature Cited 1.


2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17.

Soulanti, O. Publ. Tech. Forschungsanst. Finland. 1951, 21, 1-95. Fries, N. Sven. Bot. Tidskr. 1961, 55, 1-16. Pharis, V. L.; Kemp, T. R.; Knavel, D. E. Scientia Hortic. 1982, 17, 311-317. French, R. C.; Gallimore, M. D. J. Agric. Food Chem. 1971, 19, 912-915. French, R. C.; Gallimore, M. D. J. Agric. Food Chem. 1972, 20, 421-423. French, R. C.; Gale, A. W.; Graham, C. L.; Rines, H. W. J. Agric. Food Chem. 1975, 23, 4-8. Kemp, T. R.; Stoltz, L P.; Smith, W. T.; Chaplin, C. E., Proc. Amer. Soc. Hort. Sci. 1968, 93, 334-339. Hamilton-Kemp, T. R.; Andersen, R. A. Phytochemistry 1984, 23, 1176-1177. Hamilton-Kemp, T. R.; Andersen, R. A. Phytochemistry, in press. Kemp, T. R.; Knavel, D. E.; Stoltz, L. P.; Lundin, R. E. Phytochemistry 1974, 13, 1167-1170. Kemp, T. R. J. Amer. Oil Chem. Soc. 1975, 52, 300-302. Galliard, T.; Phillips, D. R. Biochem. Biophys. Acta. 1976, 431, 278-287. Galliard, T.; Phillips, D. R.; Reynolds, J. Biochem. Biophys. Acta. 1976, 441, 181-192. Phillips, D. R.; Mathew, J. Α.; Reynolds, J.; Fenwick, G. R. Phytochemistry. 1979, 18, 401-404. Hatanaka, Α.; Kajiwara, T.; Harada, T. Phytochemistry. 1975, 14, 2589-2592. Hatanaka, Α.; Kajiwara, T.; Sekiya, J. Phytochemistry 1976, 16, 1125-1126. Buttery, R. G.; Xu, C-j; Ling, L. C. J. Agric. Food Chem. 1985, 33, 115-117.

RECEIVED January 3, 1986


Volatile Compounds from Wheat Plants: Isolation, Identification, and

Recommend Documents