Distribution of an enzyme system producing cis-3-hexenal and n-hexanal from linolenic and linoleic acids in some plants

Phytochemistrg.

1978.

Vol

17, pp

869-872

Q

PergamonPress

Ltd. Pm&d

m England

0031-9422/78/0501-0869

SO2.CQQ

DISTRIBUTION OF AN ENZYME SYSTEM PRODUCING cis-3-HEXENAL AND n-HEXANAL FROM LINOLENIC AND LINOLEIC ACIDS IN SOME PLANTS AK~KAZU HATANAKA, JIRO SEKIYA and TADAHIKO KAJIWARA Department of Agricultural Chemistry, Faculty of Agriculture, Yamaguchi University, Yamaguchi 753, Japan (Received 10 October 1977) Key WordIndex-Distribution trans-2-hexenal; n-hexanal.

in 4@plants;enzymesystemproducingC,-aldehydes;

c&3-hexenal;cis-3-hexenol;

Abstract-The activity of the enzyme system &,-I) producing C,-aldehydes from C,,-unsaturated fatty acids was investigated using about 40 plants. Green leaves of dicotyledonous plants belonging to the Sphenopsida, Pteropsida Theaceae and Leguminosae showed a high enzyme (E,-I) activity but edible leafy vegetables and fuits and monocotyledonous plants showed a low activity. Seasonal changes in the enzyme (E,-I) activities were observed. The concentrations of cis-3-hexenol (leaf alcohol) and trans-Zhexenal (leaf aldehyde) and the enzyme (E,-I) activities showed a correlation ; high concentrations were observed in the summer but they were low in the winter.

describes the distribution producing C,-aldehydes acids in some plants.

INTRODUCTION cis-3-Hexenol (leaf alcohol) and trans-Zhexenal (leaf aldehyde), which are both responsible for the characteristic leaf odour, are distributed in a wide range of plants. Investigations using tea [l-9] and Farfugium japonicum [lo, 1 l] clarified the biosynthesis of cis-3-hexenol, trans-Zhexenal and 12-oxo-trans-lo-dodecenoic acid as shown in Scheme 1. Volatile C,-aldehydes and C,,-0x0 acid are produced by the oxidative cleavage of linolenic acids in the presence of oxygen and an enzyme system (E,-I) which is localized in the chloroplast lamellae. It was also reported that C,-aldehydes are formed via 13-hydroperoxides from linolenic and linoleic acids in etiolated watermelon seedling [12] and tomato fruit [13] and that C,,-compounds were formed from linoleic acid [ 12,20,22]. Gn the other hand, the distribution of linoleate-oxidizing activity in various fruits and vegetables has been reported [14-163. However, it is not clear whether the oxidation of linoleic acid leads to volatile C,-aldehyde formation or not. The present paper

of the enzyme system from C,,-unsaturated

RESULTS AND

DISCUSSION

Table 1 lists the enzyme activity producing hexenals (cis-3-hexenal and rrans-2-hexenal) from linolenic acid and n-hexanal from linoleic acid in the homogenates of plant tissues (summer, 1976). Among the tissue homogenates ofthe plants tested, those from the dicotyledonous plants 2-4,6,11-14,16, 17(L), l&21-24,26 and 27, which were green leaf tissues, showed a high enzyme (E,-I) activity. In contrast, the homogenates of Musci, edible leafy vegetables, fruits and monocotyledonous plants showed a low or no activity. Marine algae, Ishigefaliacea, Ulna perdusa and Chlorella sp., had no enzyme activity producing C,-aldehydes. The enzyme (E,-I) activity in a 4000 g pellet, corresponding to the chloroplast rich fraction of green leaf tissues, showed the same distribution as that of the whole tissue homogenates. The same results were observed in

I;T--lipidJ--jE, Linolenic acid

cis-3-Hexenol s (leaf alcohol)

B,

(E,-I) fatty

Linoleic acid

c*s-3-Flex&o-c&-l& dodecenoic acid R, L 1R3 12-0x0-trans-lotrans-2Hexenal dodecenoic acid (leaf aldehyde)

n-I-Iexanol

Scheme 1. Biosynthetic pathway for production of C,- and C,,-compounds from linolenic and linoleic acids. E, : lipolytic acyl hydrolase. E,-I : enzyme system producing C,-aldehydes E, : enzymic and/or nonenzymic isomerization factor. E, : alcohol dehydrogenase. 869

A. HATANAKA. J. SEKIYAand T. KAJIWARA

870

Table 1. Distribution and activity of enzyme system producing C,-aldehydes in summer

Plant no. and common name

1. 2. 3. 4. 5. 6. 7. 8.

Hair moss Horsetad Osmund Bracken Ginkgo Chinese black pme .Melon Cucumber

9

Cabbage

IO. 11. 12. 13. 14. 15. 16. 17

Caullnower Radish Sakaki Tea Camellia Sasanqua Japanese persimmon strawberry

18 19. 20 21. 22. 23. 24. 25. 26. 27.

Japanese apricot Peach Japanese wistana False acacia Alfalfa White clover Soybean Kidney bean Mulberry Japanese maple

Class

Family

Genus and species

organ*

Musci Sphenopslda Pteropsida

Polytrichaceae Eqtisetaceae Osmundaceae Polypodmceae Gmkgoaceae Podocarpaceae Cucurbitaceae

Pogonatum lnj7eSum Equisetum orwnsnse Osmunda jnponica Pteridium aquilinum Ginkgo bdoba Podocarpus macrophylla Cucumrs melo

I.

Gmkgopsida Coniferopsida Dicotyledoneae

Cucumis Cruciferae

Theaceae

Ebenaceae Rosaceae Amygdalaoeae Leguminosae

Moraceae

Aceraceae

28. Spinach 29. Potato 30. Egg plant 31. Tomato 32. 33. 34. 35. 36 37.

Lettuce Banana Onion Duckweed Rice Wheat

Monocotyledoneae

satws

Brassxa oleracza var. capztata Brass~a oleracea var botrytu Rapkanus satious Cleyera japonica Thea sinensis Camellia laponrca Camellia sasanqua Dwspyros kakl Fragana grandi@ora Prunus mume Prunus persul Wisteriafloribunda Robinio pseudoacacia Medicago .xxtna 7kifolium repens Glycina max Phaseolus vulgaris Morw bombycls Acer pa/ma&m

Chenopodiaceae Solanaceae

Spinacia oleracea Solanum tuberosum Solanum melongena Lycopersrcon esculentum

Compositae MllsUXac Liliaceae LemnaC%% Gramineae

Lactuca satroa Muss paradisiae Allwm cepa Lemna polyrhraa Oryza satica Trrticum aestivum

-

L L L L L L F L L(green) L(pale green) floret L L L L L L Fbxd) I. L L I. L L L L L L L(green) L(red) L tuber L F(Pmk) L L F L L L L

*L : leaf. F : fruit. tPlant materials were harvested

in late Mav-Julv 1976. exceot for alfalfa in September, ’ fTotal hexenals : c&3- and trans-2-hexenal: _

the experiment performed in June-July 1977. The result obtained in green leaves is similar to that obtained in tea leaves; the enzyme system (E,-I) is localized in chloroplast lamellae of green leaves [l-9]. Ginkgo leaf, strawberry fruit and banana fruit showed only a slight enzyme activity, though these tissues were often used for experiments on C,-aldehyde formation [17-201. Rhee er al. [14] and Pinsky et al. [16] reported that some plants belonging to Leguminosae and Solanaceae had a high linoleate-oxidizing enzyme activity but that most of the leafy vegetables and fruits had little or no activity. Holden [ 151 also described a linoleate-oxidizing activity in some green leaves. Although the distribution of the enzyme (E,-I) activity producing C,-aldehydes was partially similar to that of linoleate-oxidizing activity (e.g.

in Leguminosae. edible leafy vegetables and fruits) the plants with a high linoleate-oxidizing activity (e.g. Solanaceae) did not always show a high enzyme (E,-I) activity. Thus the enzyme (E,-I) activity producing C,aldehydes appears to be distributed in a different manner to the linoleate-oxidizing activity. However, it is not clear yet whether green leaf tissues with a moderate to high enzyme activity producing C,-aldehydes involve lipoxygenase in the enzyme system (E,-I) or not. The fact that the enzyme system (E,-1)is localized in the 4ooo g pellet suggests that most of these enzyme systems are a similar type of enzyme system to that in tea chloroplasts. Seasonal changes in the activities of enzyme system (E,-I) in tea leaves were described previously : they were high in summer and low in winter [6, 71. As shown in Table 2, the enzyme activity of radish and Theaceae

Distribution of the enzyme system producing C,-aldehydes

871

Activity of enzyme system producing C!,-aldehydest Fresh tissue homogenate wg fr. wt) cis-3hexenal 0 1004 214 85 0 577 2921 26 0 532 555 49 1012 854 382 389 105 621 11 692 623 386 68 1037 1029 566 299 1025 820 769 555 119 0 224 26 171 0 0 38 54 26 288

trans-Z hexenal 0 57 107 321 24 165 316 14 0 123 33 I 93 14 279 5 7 79 21 61 85 14 9 263 127 104 70 410 58 9 2 86 0 38 0 203 0 175 0 0 0 57

total hexenalsS 0 1061 321 406 24 742 3231 40 0 655 588 56 1105 868 661 392 112 700 32 753 708 400 17 1300 1156 670 369 1435 878 778 557 265 0 262 26 374 0 175 38 54 26 345

4000 g precipitate fraction @B/g PPt.) he&al 19 1507 966 702 6 109 430 42 88 52 36 12 357 292 318 150 61 141 55 256 342 52 154 439 509 110 1375 61 124 368 287 73 27 30 6 81 6 84 30 36 1 15

Isomerizatlon rate’

cis-3hexenal

trails-2hexcnal

total hexenalsf

he&nal

90 1776 3816 290 132 1710

16 481 54 458 63 284 27 8 71 341 19 101 189 134 0 102 1712 115 95 521 -

106 2257 3870 748 195 1994

230 1877 2298 1580 48 388

189 32 221 941 67 317 2733 3134 498 894 1712 223 1607 3521 -

62 23 117 184 48 781 1403 1785 604 209 3427 101 1137 1115

162 24 150 600 48 216 2544 3000 498 792 0 108 1512 3000 288 5376

27 499 -

315 5875

56 1124

36 552 360 576 2820 3372 96 96 240 348 60 360 12 510 300 0

324 402 324 133 101 303 0 16

360 954 684 709 2921 3675 96 112 -

250 995 209 144 1510 JO14 94 41

38 0 8 177 8 55 8 55

278 348 68 537 20 565 308 55

71 174 17 115 38 200 17 21

Degree Fresh of tissue activity homogenate L H H H L H H L L M M L H H H M L H L H H M L H H M H h4 M H H L L M L L L L L L L L

0 5 33 79 100 22 10 35 0 19 6 13 8 2 42 2 6 11 66 8 12 4 1 20 11 16 19 29 7 2 0 33 0 15 0 54 0 100 0 0 0 17

4OcQ9 pellet

2 21 1 61 32 14 -. 14 25 32 37 28 32 7 4 0 11 100 52 6 15 9 8

90 42 47 19 3 8 0 14 14 0 12 33 40 10 3 100

§H, M, L show high, moderate and low enzyme activity, respectively. trans-2-hexenal l[Isomerization rate (%) = total hexenals x 100.

decreased in winter. Spinach exhibited little activity both in summer and winter. However, that of Chinese black pine almost doubled in winter. Thus enzyme (E,-I) activity in most plants changes during the seasons. As shown in Scheme 1, cis-3-hexenal is at first produced from linolenic acid, then rapidly isomerized to trans-2hexenal. The isomerization rate varied widely both in the tissue homogenate and 4000 g pellet (Table 1). Table 3 shows the concentrations of n-hexanal, trans-2-hexenal, and cis-3-hexenol in tissue homogenates in summer (cis-3-hexenal was not detected because of complete isomerization to trans-Zhexenal during steam-distillation). A high concentration of trans-2-hexenal was observed in leaf homogenates (plants 6, 11-13, 16, 18, 21 and 27) which also showed a high enzyme (E,-I) activity. A high concentration of cis-3-hexenol was found in plants 12, 13, 21 and 27.

But in plants 6, 11 and 16 little or no cis-3-hexenol was detected, although the plants contained a large amount of trans-2-hexenal. This was probably due to lack of alcohol dehydrogenase (E4) as found with F. japonicum [lo], In winter, the concentration of trans-2-hexenal was decreased and cis-3-hexenol was barely detectable. This seemed to be due to the decrease in the activity of enzyme system (E,-I) and/or lipolytic acyl hydrolase (E,) which catalyses the hydrolysis of lipids. Takei et al. [Zl] also reported the wide distribution of cis-3-hexenol and trans2-hexanal with high concentrations in summer and low amounts in winter. rr-Hexanal showed similar seasonal changes to that of trans-2-hexenal, though its concentration was low. The fact that the enzyme (E,-I) activity and the concentrations of trans-Zhexenal and cis-3-hexenol are very low in edible leafy vegetables and fruits suggests

A. HATANAKA. J. SEKIYAand T. KAJIWARA

872

a reason for the selection of these vegetables and fruits during a long period of cultivation. Table 2. Acttvity

of the enzyme system producing in the winter Enzyme

activity

producing

Fresh tissue homogenate @g/g fr. wt) Plant no. 6 11 12 13 14 23 28

Organ

L L L L L L L *Plant materials fTota1 hexenals

Total hexenalst

n-hexanal

1524 999 157 200 283 434 80

153 104 91 15 95 660 36

were harvested

C,-aldehydes

C,-aldehydes’

4000 g pellet (pg/g PPt.) Total hexenalst 4877 458 856 376 424 139 108

804 366 1109 456 811 704 192

in November-December

1976.

: c&3- and trans-2-hexenal.

Concentratton

2 6 9 11 12 13 14 16 18 21 27

Organ

L L L(green) L L L L L L L L(green) L(red) 28 L 31 F L __ __~ L1 = Lmcc.

n-hexanal 0.97 0.07 tr 0.14 tr brl0 tr 1.20 :T38 1.30 tr tr 094

1.65 121.0 3.30 20.70 8.36 6 47 7 70 16.0 41.20 26 10 208.20 39 07 0 tr 29.26

and in the

(us/g fr. wt)

trans-2-hexenal

REFERENCES

n-hexanal

Table 3. Concentrations of n-hexanal, tram=Zhexenal cis-3-hexenol in the essential oil of tissue homogenates summer

Plant no.

EXPERIMENTAL

Fresh plant materials were harvested near the Umversity immediately before use except for plants 29 and 30 whtch were obtained from a local market Enzyme activity (E,-I) producing C,-aldehydes in a crude tissue homogenate was assayed wtth linolenic or linoleic acid substrate by method I prevtously described [4.7]. Enzyme activity (E,-I) in a 4000 g pellet prepared by the method of r4. 71. was assaved bv method II oreviouslv described [4, 71: Preparatton of.essential 011 and quantttativk analysis of C,-compounds were the same as previously described [6].

cis-3-hexenol 0 tr tr 0 5.94 3 87 1.78 tr 1.85 2.82 6.34 2.86 0 tr tr

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