Agric.

Biol.

Chem.,

50 (9),

2193-2199,

2193

1986

Germination Inhibition in Stigma Extracts of Tobacco

and Identification of MeABA, ABA, and ABA-/?-D-Glucopyranoside Toshiake Matsuzaki and Akira Koiwai

Central Research Institute, The Japan Tobacco Inc., 6-2, Umegaoka, Midori-ku, Yokohama 227, Japan Received January 21, 1986 Extracts of various flower tissues of tobacco with 70%methanol inhibited tobacco seed germination differently. Amongthem, extracts of stigma and anther were very inhibitory. Whenthe extracts were partitioned between ethyl acetate and water, the activity of the ethyl acetate layer was stronger than that of the water layer. Stigmas and anthers had more abscisic acid (ABA)than the other floral tissues, which matched the results of the germination tests well. Guided by a bioassay using

the

inhibitory

effects

on tobacco

seed germination,

MeABA, ABA, and ABA-/?-d-

glucopyranoside were isolated and identified from stigmas. All of the MeABAisolated did not seem to be an artefact produced by esterification with the solvent, for MeABAwas detected even when stigmas were extracted with other solvents than methanol. MeABAand ABAdid not inhibit tobacco pollen germination and elongation in vitro.

In the course of studying the structures of tobacco stigma lipids, multiacylglycerols con-

tone and flavonoid types have been identified in plants.3) Above all, ABA is known to be

taining co-hydroxy fatty acids and normal fatty acids were isolated.1} Their physiological roles

the most effective seed germination inhibitor and is widely distributed in plant tissues, so we

in stigma

tissues

are not known, but they

tested for ABAamong these tissues first.

might trap pollen more easily or prevent water In the stigma extracts of Brassica oleraceae, losses from stigma surfaces.2) To examine other biological activities of these lipids, the effects of these lipids on pollen germination

and on seed germination were tested. Multiacylglycerols as well as crude lipids of the Stigma did not inhibit pollen germination, but crude lipids in the stigma inhibited tobacco seed germination. Moreover, not

only the extracts of stigma but those of other tissues

of tobacco

flowers

inhibited

seed

germination. It would be interesting to iden-

tify the structures of these active substances contained in flower tissues, especially in stigmas. So far, a quite large number of growth-inhibiting substances such as abscisic acid (ABA), benzoic acid, cinnamic acid, lacAbbreviations: ABA-GE, /?-D-glucopyranosyl chromatography with electron capture detector.

abscisate;

there are inhibitors of pollen germination,4* but their chemical natures are not known. Stigma exudates also have been shown by Martin and Brewbaker5) to have pollen germination inhibitors. However, there were no reports about studying the effects of flower tissue extracts on seed germination. To study inhibitors not only on pollen germination but also of seed germination would give some clues to their biological significance. This report deals with the effects of the extracts from

various flower tissues of tobacco on seed germination, analyses of ABAcontents in various floral tissues, and identification of MeABA, ABA, and ABA-/?-D-glucopyranoside from stigma lipids.

MeABA,methyl ester of ABA; GC-ECD, gas liquid

T. Matsuzaki and A. Koiwai

2194

MATERIALS AND METHODS Material.

Tobacco

plants

(Nicotiana

tabacum

L. cv.

BY-4) were grown in soil in 0.02m2 pots in a greenhouse at 28°C. Whenabout 20 flowers had opened on a plant, the flowers were defined as follows:

(I) very small bud,

After centrifugation the solvent was decanted and 3.7 ml of chloroform and 5.6ml of water added to the solution. Thechloroform layer was separated from aqueous meth-

anol and discarded. The pH of the methanol solution was adjusted

to 4.0 with formic acid and the solution

was

extracted with 3ml of chloroform twice. The combined chloroform solution was evaporated to dryness and the dried samples were methylated with etherial diazomethane. Samples of the methylated substances were chromatographed on TLC. The spots corresponding to MeABA were located under UVlight, scraped off, and extracted with ethyl acetate. The ethyl acetate solutions were analyz-

corolla remaining inside calyx, style length 2.0± 0.3 mm, 6~7 days before anthesis; (II) small bud, corolla emerged from calyx and of a length equal to that of calyx, style length 3.9± 1.1 mm, 4~5 days before anthesis; (III) medium bud, corolla longer than calyx but still green, style length 8.2 ± 1.2 mm, 3 days before anthesis; (IV) large bud, ed by GC-ECD. Stigmas at different pink corolla tip, style length 14.2± 1.9mm, 2 days before anthesis; (V) large bud, style length 38.5±2.lmm,

anthers remaining indehiscent, 1 day before anthesis; (VI)

developmental stages (stages I ~IV) were also analyzed. Styles at anthesis were separated into three parts (upper 1.3cm, middle 1.3cm, lower 1.3cm), and each of them was analyzed.

flower just blooming, anther dehiscent, style length 44.3 ± 1.0mm, anthesis. Various flower tissues such as petal, calyx, style, stigma, stamen, and anther at Stage VI were GC-ECD. MeABAwas measured on a Shimadzu GC7A gas chromatograph equipped with a 63Ni electron mainly used for the bioassay systems and ABAanalyses. For the isolation

and the identification

of the inhibitors,

about 43,000 stigmas at flowering were dissected, lized, and stored below -20°C until extraction.

lyophi-

capture detector in a glass column (3mmx2m) packed with 5% OV17 on chromosorb W (AW-DMCS, 60-80 mesh). The oven was at 235°C with both injection port and detector

at 260°C.

The flow pressure

of N2 was 0.8

Bioassay. Seed germination assay. Seeds of BY-4 (30 mg) kg/cm2. Levels of ABAwere found from a standard curve were scattered on two sheets of filter paper put on 25 x relating the ratio of heights to pg MeABA. 35mmglass cups. About 0.5ml of deionized water was put into the cup. To examineinhibition of tobacco seed Isolation of three active compounds from stigmas. The

germination the filter paper was previously treated with

stored tissues of stigma (40g dry wt) were homogenized

Parts

(1 liter), acetone (2 liter) and methanol (2 liter). Each eluted fraction was tested on tobacco seed germination, and inhibitory activities were found in fractions 17~ 18 eluted with hexane-diethyl ether (5 : 5, v/v) and in frac-

appropriate amounts of test compounds. Samples were and extracted three times with cold chloroform-methanol the residue was re-extracted incubated at 26°C on the photoperiod of 12hr light and (1 : 2, v/v). After filtration, 12 hr dark with moisture. After 5 ~20 days of incubation, with chloroform and the combined filtrates were washed inhibition by the test compounds was examined. The with 5% aqueous NaCl in a separatory funnel. The germination indexes of tobacco seeds were as follows; - , chloroform layer was evaporated in vacuo at 35°C and 12 g no germination; +, seeds split and small green buds of crude lipids was obtained. The crude lipids were observed; + +, cotyledons sprouted; + + +, cotyledons chromatographed on a silica gel column (lOOg, Wakogel C300) and separated into 26 fractions with successive sprouted and hypocotyls well grown. Lyophilized flower with hexane (0.6 liter), hexane-diethyl ether (9 : 1, tissues such as calyx, petal, ovary, stigma, style, anther and elutions 1 liter; 8:2, 1 liter; 7:3, 1 liter; 5:5, 1 liter), diethyl ether stamen at anthesis were extracted with 70% methanol. of the extracts

were used for bioassay

and the

residues were partitioned by diethyl ether and water. Both layers were also used in the bioassay test. Pollen germination assay. Tobacco pollen grains were incubated in test tubes at 28°C in the dark. The medium contained: sucrose, lOg; Ca(NO3)2 4H2O, 30mg; MgSO4, lOmg; KNO3,lOmg; and an appropriate amount of test material in 100ml of deionized water.6) The final pH was adjusted

to 5.7.

ABAextraction and purification procedures in various parts offlower organs. ABAwas measured by GC-ECDby a slight modification of the method of Beardsell and Cohen.7) Briefly, after adding known amounts of ±, cis, trans ABA (Sigma) to samples of calyx, petal, ovary, stamen, anther, stigma, and style at anthesis (about 100

mg fresh wt) were homogenized with 5 ml solvent (meth-

anol-chloroform-2 n ammonium hydroxide, 12 : 5 : 3, v/v precooled below - 10°C), and stored - 15°C overnight.

tions 23~26 eluted with acetone and methanol. The combined solution of fractions 17 and 18 was condensed and rechromatographed on a silica gel column. The final purification of the inhibitory compound in this fraction was done by HPLC (Waters /zporasil 6mmx300mm,

hexane-ethyl acetate, 8 : 2, v/v 1.5 ml/min). A peak eluting at 6min was collected (CompoundI). Twoother active fractions were obtained by rechromatography of fractions 23 ~ 26. These two fractions were separately purified with HPLCand TLC, and compounds II and III were obtained. These three compounds were single

spots

on TLC

developed with the solvent system of benzene-ethyl acetate, 5 : 5, v/v (i?/0.68, compound I and RfOA, compound II) and the solvent system of chloroform-methanol-water, 75 : 25 : 3, v/v (Rf0.39,

compound III).

Germination Inhibitor in Stigma Extracts Preparation of authentic MeABA.Authentic MeABA was prepared by methylation of the corresponding free ABA(Calbiochem.) with etherial diazomethane.

Table I. Inhibitory Effects of 70% Methanol Extracts from Various Flower Tissues of Nicotiana tabacum (I) and Their Partitioning Fractions with Ethyl

Acetate and Water (II) on Tobacco Seed Germination

Sugar analysis. Sugars of ABA-conjugate were analyzed by the method of Albersheim et al.8) Spectroscopy. lowing

Brucker,

The spectra were recorded with the fol-

spectrometers;

MS, Hitachi

M-80;

500 MHz; 13C-NMR, Brucker,

^-NMR,

125 MHz.

2195

(I)

Germination _,. Concentration Tissue , ,, x t i. à" à"/j x

(mg/glass

cup)

Incubation

5

RESULTS

Petal

effects

of the

70%methanol extracts from various flower tissues of Nicotiana tabacum (I) and their fractions

with ethyl

acetate

and

water (II) on tobacco seed germination. After

5 days of incubation the 70% methanol extracts all inhibited seed germination at 1 mg/ glass cup. The inhibitory effects of the extracts diminished gradually in all tissues except stigma and anther, whoseextracts were strongly inhibitory for more than 10 days at 250 /ig/glass cup. However, seeds began to germinate after 20 days of incubation even in the extracts of stigma and anther at that concentration. This indicated that the inhibitors from stigma and anther only stopped germination at an appropriate time and were not completely toxic to seed germination. The ethyl acetate

layer had higher activity than the water layer of the extracts. Table II shows the ABAcontents in various flower tissues and upper leaves of tobacco. The amount of ABAper g fresh weight is the highest in the stigma, and next in the anther, followed by the style. There was less ABAin the calyx, ovary, stamen, and leaf

than in the stigma and anther. These results seem to explain the results obtained in Table I where the extracts of stigma and anther had higher germination inhibiting activities than

those of other tissues. The ABAcontent of stigmas increased

time (days)

10

20

AND DISCUSSION

Table I shows the inhibitory

partitioning

index*

dramatically

toward anthe-

Calyx Ovary Style Stigma Stamen Anther Control

1 0.25 1 0.25 1 0.25 1 0.25 1 0.25 1 0.25 1 0.25 0

+

++-+++ ++ +++ + ++~+++ ++ +++ + +++ + +++ + +++ ++ +++ + + +++ ++ +++ + ++ +++

+++ +++ +++ +++ +++ +++ +++ +++ - ~ + + + +++ +++ h + + +++

(II) Tissue

Concentration (mg/glass cup)

Germination incubation

index*

time (days)

5

(Ethyl acetate layer) Petal 1 Calyx

1

Style Stigma Stamen Anther (Water

10

+

+ +

+

++~+++

1 1 1

+ + -

+ + ++~+++ -

1 1

++~+++ ++~+++

1

layer)

Petal Calyx

Ovary Style Stigma Stamen

Anther

1

1 1 1

1

+++ +++

++ + +

+ ++ + + + + ~ ++

++~+++

+++

+

++~+++

0 + + + + + sis. Further, the upper part of the style (upper Control 1.3cm from the edge of the style) had more * See,thetext. ABAthan the middle or the lower part. These tissues of tobacco stigma were results suggested than an ABAgradient existed lyophilized in the pistil from ovary to stigma. chromatographed on silica gel. Twofractions, Crude lipids (12g) obtained from 40g of one of which was the combined fraction eluted

T. Matsuzaki and A. Koiwai

2196

with hexane-diethyl

NMR and 13C-NMR of authentic

ether (5 : 5, v/v) and an-

other of which was the combined eluted with acetone and methanol, inhibited tobacco seed germination.

^H-NMR;

fraction showed The in-

7.88

(d,

7=16.1Hz),

16.1 Hz), 5.95(br.

s.),

5.75(br.

(d,

2.28

(d,

/=17.2Hz),

/=1.3Hz),

hibitor was purified from the eluates of hex-

ane-diethyl ether (5 : 5, v/v) by column chromatography and HPLC, and Compound I (about 600/xg) was obtained. The chemical

1.93

(d,

MeABA 6.15

s.),

(d,

3.71 (s),2.48

/=17.2Hz),

2.01

/=1.3Hz),

1.ll

(s),

(s) ppm in CDC13, 13C-NMR; 199.56, 149.25, 51.16,

J= (d, 1.02

162.22,

136.18, 128.19, 127.10, 118.36, 49.79, 41.51, 24.33, 23.01, 21.13,

79.65, 18.84

data for CompoundI all agreed with those of ppm in CDCI3). Further, two other inhibitors, synthetic MeABAand the literature.9) GC-MS Compound II (about 400^g) and Compound showed 278 M+, 260 M+-18,

which were characteristic assignment

III (about 120jug) were obtained from the eluates of acetone and methanol. The 1H-

190 and 162

for MeABA.9)The

was further

confirmed

by 1H-

NMRof CompoundII agreed with that of authentic

NMR;

Table II. The ABAContents in Various Flower Tissues and Leaf of Nicotiana tabacum

Hz),

s.),

2.29

5.78

literature10) 6.18

(br.

(d,J=17.1Hz),

s.),

(1H-

(d,

/=16.1

2.49

2.05

(d,

(d,

/=

/=1.0),

nmol/g fresh wt

198 96 3 360 4 500 7869 2739 2889 209 16 383 640 25 16 1 0 63 4 207 7720

0.75 3.64 1.36 17.0 3 29.77 10 .36 10 .93 79 .14 1.4 5 2 .42 9 .52 4 0. 23 0.78 29.2 1

ed by GC-MSof the methylated Compound II. The Rfvalue of CompoundIII was 0.39 on silica gel G TLCdeveloped with the solvent

0.78

anomeric hydrogen at C-l of 6.19the hexose (XHNMR; 7.79 (d, /=16.4Hz), (d, 7=16.1

20 7

system

of chloroform-methanol-water

reagent.

By sugar

analyses,6)

only

resonance Hz),

at

on

20

( p p m)

(br.

2

5.80

S tig ma

A BA

1 00

s.),

assigned

Table III. Effects of MeABAand ABAat Different Concentrations Tobacco Pollen Germination and Elongation M eA B A

5.95

5.62ppm

s.),

C o n ce n tra tio n

D-glucose

was detected in CompoundIII. The iH-NMRof Compound III showed all the resonances of ABA,and a single proton (br.

C o n tro l*

(75 :

22: 3, v/v), which was identical with that of ABA-conjugate.n) The compound was found to have a sugar moiety by anthrone sulfuric

Each value is an average of 3 replicates.

Pol len g e rm in a tio n

(br.

7=16.1Hz),

ng/g fresh wt

L eaf (U pp er)

T e st

(d,

1.93 (d,.7=1.3Hz), 1.12 (s), 1.03 (s) ppm in CDCI3). This assignment was further confirm-

T issu es

com p ound s

5.96

17.1Hz),

ABA contents

P etal C alyx O vary Style (U p per) (M idd le) (L ow er) Stigm a S tage I Stage II Stage III Stage IV Stam en A nther

ABA and the

7.83

c ru d e lip id 20

1 00

10 0

4-

+

+

5.62

to (d,

the

J=

2, 4 -D in i tr op he n ol

20

10 0

I +

+

+

+

+

in d e x *

Without test compounds.

Tobacco pollen germination and elongation was observed microscopically after 4 hr of incubation of tobacco pollen grains at 28°C in the dark. +, pollen grains were well germinated and elongated; - , no germination.

2197

Germination Inhibitor in Stigma Extracts 8.7Hz), Hz),2.31 s.),

1.ll

CDC13).

4.1-3.2 (multiplet), (d,/=17.1 Hz),2.04(br. (br.

s.),

1.ll

The presence

(s),

2.47 (d, /= 16.8 s.), 1.91 (br.

1.01

(s)

ppm

in

of a hexose is also

confirmed by the electron impact mass spectra of the acetylated CompoundIII, fragments at m/z 331, 271, 169 and 109. A key fragment at m/z 595 (M+ + 1) was obtained bythe FD-MS of the acetylated CompoundIII. These results indicated that Compound III was ABA-jS-dglucopyrano side.

So far ABAhas been isolated and identified in many plants,10~13) and it is suggested that

with methanol under neutral or alkaline conditions.

artifact

MeABA obtained

conjugates,

be an

resulting from methanolysis of ABA because

stigma

tissues

were ex-

tracted with chloroform-methanol mixed solvents. To find whether MeABAwas present whentissues were extracted with other solvents

80%acetone, 80%ethanol, and chloroform-

isopropanol (1 : 1, v/v) were chosen. Figure la and b shows the GC-ECDchromatogram of

MeABAfraction

(a) and ABAfraction

(b)

whenfresh stigma tissues were extracted with

ABAis involved in physiological phenomena such as bud dormancy, abscission, senescence, and stomatal opening.14'15* Goldschmidt first

(a)

(b)

L

found that much ABAis contained in styles of citrus,16) but its physiological role in the

stigma is not known. ABAderivatives

here might

MeABA

con-

tained in large amounts in stigma tissues of tobacco might have some physiological significance. However, neither crude lipids of

stigma nor MeABAand ABAinfluenced pol-

len germination and elongation even at 100 ppm (Table III). This result alone cannot

exclude the possibility that ABAderivatives have some physiological role in pollination. ABA-GEwas first identified by Koshimizu 0 2 4 6 8min 0 2 4 6 8min time time å etal. from the seeds of lupin17) and the existence of this compound have been demonstrat- Fig. l(a) and (b). The GC-ECD Chromatogram of ed indirectly by detecting ABAin the alkali MeABAFraction (a) and ABAFraction (b) when Stigma hydrolysates of many plant tissues.18* Recent- Tissues were Extracted with Cold 80% Acetone. ly, Boyer and Zeevaart19) identified ABA-GE The injection amountof(a) was two times higher than that from leaves of Xanthium and spinach, demon- of(b) at the range of 102x 16. strating that the glucosyl ester of abscisic acid mg ___ _cpntcQl__ _ was the only abscisic conjugate in these leaves. The ABAconjugate from the stigma lipids of 3200\ \ tobacco also found to have a glucosyl ester moiety. The total amounts of ABAderivatives » å \\ obtained

here

were very small,

but

the

O)

amounts per g dry wt in stigma was in much higher level than that in other plant tissues.20* However, the physiological significance of MeABA, ABA, and ABA-GE in stigma tissues was still unknown. Milborrow and Mallaby21*

demonstrated

that MeABAwas an artefact produced from ABA-conjugates

when tissues were extracted

\\

i100"

fc

VV

$ I

°O.OO2QO20.2

2 20

Concentration

Fig. 2. Growth Inhibition

(ppm)

by MeABAand ABAof

Tobacco Seed Germination. Samples were weighed after 7 days of incubation. MeABA;

à" - à"-ABA.

O-O,

2198

T. Matsuzaki and A. Koiwai

cold 80% acetone. GC-MSanalyses of these fractions and the comparisonof the retention time of authentic MeABAand Me-trans ABA in GC-ECDindicated that the peak eluted at 6min corresponded to MeABAand the peak eluted at 7min was Me-trans ABA. These peaks were also identified whentissues were extracted with the other two solvent systems. While the ABAfraction (Fig. lb) had trace amounts of Ms-trans ABA,the MeABAfraction (Fig.

la) had a significant

amount of it.

Further, MeABAwas also identified from the extracts of lyophilized stigma tissues extracted with 80% acetone. These suggested that all of the MeABAobtained above were not produced by esterification

with the solvents.

The

reason why different conclusions were obtained from Milborrow and Mallaby21) might be caused partly by using different species and

different tissues. They used fruits of avocado (Persea gratissima).21) However, the possibility that MeABAwas produced enzymatically at extraction could not be excluded even when tissues were extracted with other solvents than

methanol.22) To demonstrate these points fur-

sugar moiety was not involved in the activ-

ity.n)

Apart from the inhibitory effects of ABA derivatives on seed germination, we succeeded in the tissue culture of stigma and also discovered that only young stigmas could form stigma- and style-like structures.26) Old stigmashad lost the ability. This raised the possibility that some inhibitory substances that stop cell division during stigma development

might be involved. ABAderivatives might be these substances, and increasing levels of ABA in stigmas during development might regulate pistil morphogenesis. There were no reports on

inhibition

by ABAderivatives

of stigma-

or style-like

The rela-

tionship between the concentrations of ABA derivatives and the stigma-like now under investigation.

outgrowth

is

Acknowledgments. Wewish to thank Dr. M. Noma and Mr. K. Koseki of this laboratory for their kind advice, and Dr. S. Eda and Mr. H. Tazaki for the sugar analyses and FD-MSof ABAderivatives. Wealso wish to express our thanks to Mr. Y. Yamashita for the GC-ECD analyses.

ther investigations using techniques such as radioimmuno assay would be necessary. Figure 2 shows the effects of ABA and

MeABAon tobacco seed germination at different concentrations. While ABAinhibited seed germination at about 2 ppm, MeABAdid

of the induction

structures.

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1) T. Matsuzaki, A. Koiwai and N. Kawashima, Agric. BioL Chem., 47, 77 (1983). 2) R. N. Konar and H. F. Linskens,

Planta,

71, 372

(1966). A. C.

Leopold and P. E. Kriedemann, "Plant at lower concentrations than ABA.The mech- 3) anism of these different activities of MeABA Growth and Development " 2nd Ed., McGrawHill, 1975. and ABAwere not examined here. Koshimizu 4) T. Hodgkin and G. D. Lyon, Ann. Bot., 52, 781

et al.ll) found similar phenomena in the bioassay of rice seedlings. They postulated that

5)

MeABApenetrated more easily into the seedlings than ABA. This might also explain the results of Fig. 2.

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MeABA and ABA affected

the

stomatal opening of Xanthium24) and sugar beet25) somewhat differently, he pointed out the different esterase activities

of the species.21)

The germination inhibition by ABA-GEwas about half of that of ABA, indicating that the

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