Endogenous plant hormones of the broad bean, Vicia faba L. II. Gibberellins and plant growth inhibitors in floral organs during their development

Biochem. Physiol. Pflanzen 175,

D99~610

(1980)

Endogenous Plant Hormones of the Broad Bean, V icia fa ba L. Ir. Gibberellins and Plant Growth Inhibitors in Floral Organs during their Development1 ) W. DATHE and G. SEMBDNER Institute of Plant Hiochemistry, Halle (Saale), Research Cent re for JliIolel'ular Biology and JliIedieine, Academy of Seien('cs of the GDR K e y Te r m In d e x: gibberellins, plant growth inhibitors, abscisic acid, phytohormones floral organ development; Vicia (aba.

Summary In developing flowers the oceurrenee of GAs and plant growth inhibitors was investigated and their quantities determined by bioassaying. AHA has been identified chromatographically. The predominant endogenous GA is similar to GA 20 ael'ording to its chromatographie al behaviour and biological activity. Furthermore, so me other free GAs and GA glueoside like eomponents as weil as plant growth inhibitors not identical with AHA were detected and eharacterized partly. Distribution of single GA eomponents, AHA, and further inhibitors in different floral parts (gyn, andr, per) has been studied at 4 different stages of development. Free GAs are mainly located in the andr and per at early stages, whereas at anthesis immediately before flower opening the highest GA levels occurred in gyn. ARA maximum in fl(J\vers was reaehed at anthesis, too, and ARA was mainly loealized in gyn. Occurrence and distribution of the hormones in floral parts are discussed with respect to the physiologieal role in flower development.

Introduction

Within our investigation program on the hormonal regulation of flower and fruit development in Vicia faba we studied at first ABA and its metabolites as weIl as other plant growth inhibitors in the developing seed (GRÄBNER et al. 1980). This second part deals with the endogenous GA components and plant growth inhibitors in floral parts (gyn, andr, per) at different stages of development. The endogenous GAs and inhibitors in flowers have not yet been studied extensively. The source of GAs occurring in flowers seems to be the anthers (WEAVER and POOL 1965; JEFFCOAT et al. 1969; BARENDSE et al. 1970; JEFFCOAT and CUCKSHULL 1972; MURAKAMI 1973; 1975). Identification of GA 3 was done in developing inflorescence of Hordeum vulgare (KAUFMAN et al. 1976) as weIl as in pet als and stamens of Cassia fistula (SIRCAR et al. 1970); these floral parts containing exceptional high amounts (5flg and 2flg GA3 /g fr.wt.). In dormant pollen of Pinus attenuata GA3 , GA4 , and GA7 1)

Part I see

GIÜIlXE1(

et a1. (1980).

Abbreviations: Andr ~ Androecium; gyn ~ Gyna,ecium; per ~ Perianth; GAs ~ Gibberellins; ABA ~ Abscisie aeid; CHCl a ~ Chloroform; EtOAc ~ Ethyl acetate; HOAc ~ Acetic acid; MeOH ~ :yrethallol.

w. DATIIE a,nd G. SEMBDNER

600

were detected (KA:MIE~SKA et al. 1976). Exogenous application of GAa to flowers causes various effects, f.e. promotion of flower growth, hastening of flower development towards anthesis and divertion of assimilates to application site (HARRlS et al. 1969; JE:FFCOAT and HARRIS 1972; MURAKAMI 1975). GAs have also been shown to stimulate saccharase activity in filaments of Zea mays (SCHAEVERBEKE 1967). Furthermore, GA3 can enhance germination of pollen (STANLEY and LINSKENS 1974) and pollen tube elongation (KAMIENSKA and PHARIS 1975; SHUKLA and TEWARI 1974). However, it has to be mentioned that pollen tube growth in Oalotropis procera is effectively stimulated by the plant growth inhibitor ABA(SHUKLA and TEWARl1974). The natural occurrence of ABA was proofed in the gyn of cotton (DAVIS and ADDICOTT 1972), in flowers of olive (BADR ct al. 1971), rose (MAYAK and HALEVY 1972), and pea (EEUWENS and SCHWABE 1975). Recently, ABA was isolated as its methyl ester from pollen of Pinus dens'iflora (SHIBUYA et al. 1978). Several publications report on the occurrence of not identified inhibitors in flowers of different species (STANLEY and LIi'i'SKEl\S 1974). Material and Methods Plant material From gtass hollse eultivated V1:cia {aba pbnts flowers weIe harvested (1978: 500; 1979: 1,0(0) at !lifferent stages of devclopment (1978: 5 stages, 1979: 4) and separated in gY Il, andr, and per (on!y 1979). In order to prcvcnt wilting thc material was frozen at -20 oe immediately after preparation and snbscquently lyophilized. The phys iologiea! parameters of the f]ow ers invcstigated a,re shown in

,...

E (,.)

1:.,',. ·,'"r:.,'•.·:.-

1,· .•

I

I

M.

Stages

Anthesis Fig. 1. ])eveloprnental stages of broad bean flowersinvestigatcd.

601

Endogenous Plant Hormones of Vicia. Ir.

Table 1. Physiological characierizahon of tlower stagesinvestigated. The values are the fresh weights of flower or floral organgiven in mg. Data in braekets express the per cent water eontent Stage

JI III IV

V

Flowers harvesteu ID78

I97D

Total flower

Total flower

40,0 G2.8 88.8 114.2 121.G

Gyn

not investigated 56,2 a ..2 (84.1) iU) (84.7) GG.4 GA (84.3) 9il.4 100.2 G.7 (81.0)

Andr

Per

7.8 8.5 8.8 7.6

45.2 (863) 54.0 (84.9) 78.2 (8U) 85.9 (77.1)

(78.8) (7D.2) (82.a) (81. 7)

Fig. 1 amI T~,ble 1. At stage III we harvested a further sampie for large seale extraetion (about 40,000 flowers, 2.5 kg fr. wt., 61.6 mgjflower).

Extfaction and puritication Homogenized material was extracted with 80 % :'IeOH and thc aqucous eoncentrate partitioned at pH 2.5 with EtOAc as reported previously (DATIIE et a1. IB78). The EtOAc extracts frol1l the 500 and 1,000 flowers s,tl1lples were purified on colul1lns of DEAE-Sephadex A-25 (45 x 1.1 Cl1l) according to GRÄBNER et al. (1975). The elution with a discontinuous gradient of HOAc in80% MeOH is shown in Fig. 4. Further separation oi fractions containing free GAs (subsequent to DEAE-Sephadcx A-25) was performed by partition chromatography on Sephadex LH-20 (MACMILLAN and WELS IBn; column size: ÖO 2 Cl1l) giving 25 fractions (;1 25 ml) from organic phase and 2 fnrther ones (<150 ml) from aqlleous ph,tse. The EtOAc ex~rad of the 2.5 kg sampie was adsorbed to lfJO g ehareoal: eeHte (4: G) and nsing bateh proeednre wltshed subsequently with H 2 0, H 2 0: aeetone = 1: 1 and acetone (elteh 200 ml). 1'he fractions rercived were eombined and purified by partition chromatography on silica gel L 100 --1 GO {' m (eontaining 40°" llzO, ('olll nm sizc 40 7. 2 cm). The elution and fraction.ation is shown

Fig. 2. Chromatography on Sephadex LH-20 of free GAs from se lee ted stages of different floral parts. The fraetions of free GAs weTe obtained by separation. of the EtOAc extracts on DEAE-Sephadex A-25. The GA aetivity was determilled by dwarf rite bioassay. Referenec valucs: 1 - water, 2 _. 10- 8 1\1 GA 3 , a - 10- 7 M GA 3 •

602

W. DAT/IE anel G. SEMBDNER E (J

Dwarf rice bioassay

'"

10-7 M

0>

.5

~

Q) Q) (/)

5

10- 8

'ö

-::0>

c: 1 Q)

..J

E (J

0

e'E 0

(J

C D ............. Wheat seedling bioassay

0

5

10

tt

20

Fractions, 50 ml

~

87,5755025,7, 12,5255075 080 7570 ,551020 02020 2 5 10

CHCI3 {%)

EtOAc!%J MeOH(%) HOAc(~)

Fig. 3. Chromatography o{ the EtOAc extnut 01 {lou:ers (2,5 kg) on silica gel. The GAs were eletecteel by elwarf riee and the inhibitors by wheat seeelling bioassay of aliquots (1/ao) of each fraetion. in Fig. 3. Subsequently, the hormone components were chromatographeel on DEAE-Sephaelex A-25 anel Sephaelex LH-20. Thin layer chromatography on layers (0.3 mm) of silica gel GF 254 was useel for characterization of inhibitors anel of siliea gel G for GAs using the following solvent systems: 1. C6 H6 : EtOAc: acetone: HOAc = 40: 10: 5: 1, 2. CHCl a : EtOAc = 7: 3, 3. CHCI 3 : EtOAc: HOAc!1 =5:4:1.

Hydrolysis Characterization of conjugated GAs after rhromatography on DEAE-Sephadex A-25 was performeel by enzymatical hyelrolysis using cellulase anel rechromatography on DEAE-Sephaelex A-25 (proceelure see DATHE et al. 1978). Free GAs afforded by hydrolysis were further separated on Sephadex LH-20.

Bioassays For detection anel quantitative determination of GA activity we used the dwarf riee (Oryza sativa L. cv. "Tan ginbozu") and dwarf pea bioassay (Pisum sativum L. ev. "Insignis") as described by SEMBDNEU et al. (1976). Inhibitor activities were determined by wheat seedling bioassay (Triticum aestivum L. cv. "Carola") according to DATHE et al. (1978).

Results

Changes of GA levels in flowers and their different parts In the EtOAc extracts of total flowers and floral parts GA activity which was detected by dwarf rice bioassay occurred after chromatography on DEAE-Sephadex A-25

603

Endogenous Plant Hormones of Vicia. II.

-s

BI

E

Cl

<: Q)

10-6 M GA.,

I

10-7

Jl

5·

"

B2

5. 1 0

g1

10-8 H20

10-7 M GÄ.:!

C2

10

8 H20

1 "0

n

D

Q)

7 10- M

G~

Q)

Cf)

5

10-8

m ~

f-1 0

15

10

5

H20

Fractions Elution with:

, °,0,25/0,25", 0,5", 0,75", 1,0" , HOAc in

80% MeOH

Fig.4. Chrornatography of GA fractions (B, C, D, cf. Fig. 3) on DEAE-Sephadex A-25. The GAs were detected by dwarf rice bioassay of aliquots (H - 1/30; C, V, - 1/10) of each fraction.

Table 8. Content of free GAs in developing flowers. The GA levels were determined as ng GA 3 -equivalents/g fr.wt. (a) or as pg GAg-equiv,tlents/fioral part or flower (b) by bioassaying (rice seedlings) of fraetions after chromatography of the EtOAc extraets on DEAE-Sephadex A-25 Stage

I II 1Il

Investigation 1978

Investigation

Total flower

Total flower

Gyn a

a

b

a

O.G 7.7

2ö 48G 208 2H1 147

not investigated (;.8 4.;) 381 8.~l 191 GA ,121 i\. 4 23.8 (l.H 88 2.1

.)

.)

'-'.'J

IV

2.G

V

1.2

41

197~J

Hioehern. Physiol. Pflanzen, Ud. 175

b

Per

Andr b

a

b

a

b

14 25 lö2 14

1ö

118 14 4"C) 14

(J.ö 2.8 loG 0.7

24H 152 12G

1.G 4.9

1.8

GO

604

w.

DATIIE

and G.

SEMBDNER

mainly in fractions of free GAs (fract. 7 -10, cf. Fig. 4). Additionally, GA conjugate like activities were found only in traces, which could not be quantified. The levels of free GAs changing in flowers and floral organs at different stages are given in Table 2. According to these data both GA concentration (a) and absolute GA amount (b) oftotal flowers showed its highest values already at an early stage (11) and a sm aller second increase just before flower opening (stage IV). In the single floral parts the following changes were observed: GA concentration (a) in the gyn and andr is alw~ys higher than in the per. However, the total amount of GAs (b) is at most stages higher in the per than in gyn and andr. The per reaches its maximal GA level already at stage H. Androeceum showed 2 GA maxima: a higher one at stage Hand a sm aller one immediately before flower opening (IV). At this stage gyn has its maximal GA content, which surmounts that of the per.

Characterization of the t"lower GAs So me characterization of the GAs occurring in the different floral parts at selected stages was performed using partition chromatography on Sephadex LH-20 subsequent to DEAE-Sephadex A-25. Results are shown in Fig. 2. The per contains both at stage III and IV a GA component similar to GAs/zo (fract. 7 -9) and another more polar one (fract. 12 and 13). In the andr and gyn the GAs/2o -like component occurred only at stage IV. The predominant GA in the female and male floral parts is a more polar one (fract. 17 -19) corresponding to GAI/29 • Additionally to these 2 compounds which apparently can be assumed to be the main floral GAs some further GA like activities could be found in the different floral parts at special stages. Large scale extraction of total flowers (2.5 kg, about stage IU) gave after chromatography on silica gel and subsequent dwarf rice bioassay 3 GA fractions (B, C, D; Fig. 3), which were further chromatographed on DEAE-Sephadex A-25 (Fig. 4). Fraction B yielded 2 GA peaks (BI = fract. 4; BI = fract. 7 -9). BI showed an unusual chromatographie behaviour corresponding neither to free GAs nor to neutral conjugates. Though biological activity of BI seems to be rather high the amount was too low for further characterization. Bz corresponding to free acidic GAs was subsequently cln·omatographed on Sephadex LH-20 (Fig. 5) and one GA peak similar to GA5/ 2o was obtained. Thin layer chromatography(silica gel G, system 3) gave a similar result (R F = 0.6-0.75 = GA5/ 2o). Like GAzo this flower GA is active in the dwarf riee and nearly inactive in the dwarf pea bioassay (REEVE and CROZIER 1974). Thus, B2 is assumed to be GA2o • Fraction C (cf. Fig. 3) was also purified on DEAE-Sephadex A-25 yielding a neutral (ester like; Cl) and apolar acidic (glucoside like) GA compound (C 2), both of low biological activity (Fig. 4). Cl was hydrolyzed by cellulase, rechromatographed on DEAESephadex A-25 and bioassayed giving low activity in fractions of free GAs. Because of its low amount further chromatographical characterization on Sephadex LH-20 was not possible. Hydrolysis oI C2 using cellulase and subsequent chromatography of the hydrolysate on DEAE-Sephadex A-25 showed that the GA activity shifted into fractions corresponding to free GAs. Rechromatography on Sephadex LH-20 yielded 2 GA peaks (Fig. 5), one of which (fract. 17 -19) being similar to GA I/29 and the other one being less polar.

Endogenous Plant Hormones of Vicia . II.

605

~pwart rice bioassay

~A5j20

10 E

&29

-

10- 6 M GA 3

0

...

10-7

~

Cl

c:

.!

5-

r--

10- 6

~

H.,O

Cl

c:

" 1Q) Q)

cn 0 E

0

rn'

5-

~

B Pwart pea bioassay 2

Q0005 jJg GA 3/plant .... ...., n Tween

Q)

"c:... 0

n-

....GI 1-= 0

]1 1IIIIIIIInninlU

C;pwarf rice bioassay

E

0

...

10-7 M GA 3

~

Cl

c:

.!

5-

10- 8 H20

Cl

c:

"

GI GI

CIl

10

5

10

15

Fra c t organic p h ase

20 25 o n s

Fig.5. Chrornatograph y on Sephadex LH-20 01 GA fra ctions 132 and C2 • (cf. Fig. 4). Aliquots e/5 ) of the B2 fr act ions obtained were bioassayed using dwarf ri ce and dwarf peas. - C2 was hydrolyzed by cellul ase and rc chromatographed in DEAE-Sephadex A-25 prigor to Sephade x LH-20. GA activity was deteeted by dwarf ri te bioassay.

Fraction D gavc after chromatography on DEAE-Sephadex A-25 apolar acidic GA compound (fract. 11, Fig. 4), which could bc converted by enzymatical hydrolysis to a less polar one (fract. 7 and 8), after rechromatography on Sephadex LH-20 located in fraction 20 (cL Fig. [». The n-butanol extract obtained at plI 2.5 after EtOAc partition was investigated in the same manner. GA activity was detected only in such small amounts that further characterization could not be performcd.

606

W.

DATHE

a nd G.

Fractions V1 .v2V 1 5 1 O OV

-1

E tI

Lf1

15

lJ

l.l

ABA

-

X

SEMßD:'
10- 6 M ABA

Y 1Ö

5

Elution with: I

0 .o.25~0,25~ O,5n, 0,751) 1,0n r HOAc in 80% MeOH

Fig. 6. Chromatography ofinhibitor fraetion A (cf. Fig. 3) on DEA 8 -8ephadex A-25. Inhibitor aetivity was deteeted by wheat seetlling bioassay of aliquots (1/30 ) of eaeh fmdion.

Inhibitors in floral parts In order to get information on t he nature of endogenous inhibitors alarge scale preparat ion of total flowers (2.5 kg) was performed. After chromatography of the EtOAc extract on silica gel and subsequent wheat seedling bioassay one main inhibitor fraction was found (A, Fig.3). DEAE-Sephadex-chromatography yielded an ABA like and 2 further inhibitors, X and Y (Fig. 6). ABA fraction (fract. 4) was separated using thin layer chromatography (silica gel GF 254 , system 1) and giving 2 inhibitor zoues (zone 1: R F = 0.:35 - 0.50 = ABA; zone 2: R.F = 0.50 - 0.63 = Z); inhibitor activity was about t he same in both zones. The ABA Iike compound was methylated by diazomethan and rechromatographed on t hin layers (siljca gel GF254 , system 2) obtaining a fluorescence quenching spot correspond ing to ABA-Me (R F = 0.65). Thus, identity with ABA can be assumed. Neither cOlnpound Z no1' the components X and Y could be identified bpcause of the very low quantit ies . All floral parts contained at any stage illvestigated an ABA fraction. In gyn and andr only ABA was detected in this ABA fmction by thin layer chromatography (silica gel GF254 , system L R F = 0. 35 - 0.5) subsequent to DEAE-SephadexA-25. However, in t he per ABA fraction showed by th inlayer chromatography in addition to ABA a less polar inhibitor corresponding to compoulld Z isolated by al rge scale preparation of total f1owrrs. The g ~'n contained t]1(' inhibitor Yat stage IV in addition to ABA. In the per t he cO ll\pound Y oeeurred at all stages investigated and X at stages !I I- V in addition to AHA anel eompo!1eut Z. In the andr only ABA was fO lll1 Cl. The amounts of ABA as weil a8 of inhibitors X and Y in floral parts dnrillg flower developmeut are shown in Tablp :~. T]1(' inhibitor activiti!'s w('re det<'nnined b.v wlwat seedling bioassay of fractions obta ined b~' ehrnmatography Oll DEAE-Sephadex A-2;,) of EtOAc extraets. For ealcnlatioll 01' AR\ cont(,l1t8 in pn tlw AR\ aetivities dotermined afte r DEAE-Sephadex

607

Endogenous Plant Hormones of V'icia, II.

Table 3. Contenls o{ inhibitors i n developing (towers. The inhibitor levels were determined as ng ABA-equivalentsjg fr.wt. (a) or per floral part (b) by bioas5aying (wheat seedlings) of fractions after ehromatography 01 the EtOAc extracts on DEAESephauex A-25 Stage

II III

IV

V

Antir

Gyn

ABA

ABA

Per 1nh.

a

b

a

68 100 241 166

0.5 0.8 2.1 1.2

228 0.7 134 0.5 725 4.6 119 0. 8

b

~I.y""

Total flower

ABA

1uh. "x" 1nh. "y" ABA

b

a,

b

a

b

a

0 0 215 0

0 0

82 50 126 68

3.7 2.7 9.8 6.8

o0 63 3.4 24 1.9 79 6.8

1.4

0

a

b

a

b

23 24 16 43

1.1

87 60 177 78

4.9 4.0 16.5 7.8

1.3

1.3 3.7

A-25 were corrected by thc activity of component Z after thin layer chromatography. The levels of ABA representing the predominant floral inhibitor reach in all floral organs their highest values at anthesis (stage IV) immediately before flower opening. At this stage the ABA concentration (a) in gyn surmounts that of the per and the andr. However, the absolute ABA amount(b) of floral part is highest in per at each stage investigated. The inhibitor X occurring only in the per reaches its highest level at stage V. The inhibitor Y showed its maximal amounts in the per at the same stage and in gyn somewhat earlier (IV). Discussion

ABA (and apparently. some furt her inhibitors) and GAs seem to be important hormonal constituents in developing flowers. The highest GA levels in andr and per at stage II corrcspond with the beginlling of intensive elongation growth and fresh weight increase indicating a functional role of GAs in these processes. Similarly, MURAKAlIH (1973) detected in flowers of Pharbitis nil a strong GA incrcase when the petals appear horn the calyx. In Mimbilis jalapa obviously GAs derivillg from the alldr are responsible for floral tube growth (MORAKAMI 1975) and in Glechoma hedemcea corolla size also seems to be controlled by the andr (PU CK 1957). Exogenous application of GA417 increased significantly the growth of thc per in Begonia franconi s (BERGHOEF and BRUINSMA 1979) and Cleome flowers (DE JONG and BRUINS1y thc absence of gyn and andr. Possibly the cndogellous GA level in per is at tlwse stages high cllough to induce flower opening. The importance of GAs in this proccss was shown in a dwarf mutant of the cabbage variety "Dithmarscher". Flower opening in the in floreseences dcveloped after vernalization occurs in the dwarf plants only after exogenous GAaapplication(SEl\iBDNER, unpublishcd data).

608

W. DATHE aud G.

SEMßDNEJ~

In the andr of Vicia the GA concentration was at stage II 3 timcs higher than in the per. However, the less polar GAs representing early steps of GA interconversion have been found in thc per and not in the andr (cf. Fig. 2). Thus, thc site of GA biosynthesis in flowers can not be deduced from the results received. The second GA peak at stage IV (immediately before flower opening) is mainly caused by the high GA level in gyn. This GA increase in gyn might be due to the release of GAs from conjugated forms at least partly, beeause the maill GA in gyn at stage IV corresponds to the GA detected in flowers at stage III in conjugated form (cf. Figs. 2 and 5). This conjugate eould be splitted completely by cellulase indicating an equatoriallinkage between the GA and thc probable sugar residuc (SCHLIEMANN and SCHNEIDER 1979; SCHNEIDER and SCHLIEMA~.l\ 1979). Occurrenee and physiological significance of GA conjugates during flower development nood some further detailed investigation. Corresponding to the second GA maximum at stage IV ABA reaches its maximal levels at the same time. This can be related to the increase of sink aetivity. In wheat ABA injection into immature caryopses was shown to incrcase significantly the assimilate flow to application si te (DEWDNEY and MCWHA 1979). With respect to sink-sourec activity, like in other processes, ABA has to be considered as an illtegrated component of the endogenolls hormone situation. Pistil developmcnt in f10wers of Cleome is stimulatcd by cytokinins, but inhibited by GA3 and GA4/ 7 causing female abortion. ABA also reduces pistil growth, however, without complete blocking of its development, but ABA (lven removes the abortive effect of GAs (DE JONG and BRUINSMA 1974a, b). The low levels of GAs in gyn at early developmental stages of Vicia f10wers corresponds to the data from Cleome flowers mentioned. DOTl1lant pollen of Pinus and Lilium species contains large amounts of less polar GAs which disappear on germination and polar GAs increase markedly (BARENDSE etal.1970; KAlIiIENSKA and PHARIS 1975). In Vicia flowers pollen hormones have not been studied separately but Were included in the andr. The GAs 10ca.Iized in the pollen probably are involved in pollen germination (und fertilization?) but not in flower development. As a main cndogenous GA in Vicia flowcrs G~o has been identificd chromatographicaJly. Immature secda are known to containlarge amounts of G~o (SPONSEL et al. 1979). A se co nd component of Vicia f10wcrs (fract. 17-19, cf. Fig. 2) might be GAl or GA29 , tho latter being identified in immature seeds, too (SPONSEL et al. 1979). Acknowledgement We a.re gntteful tu Prof. H. 1'. I'IIAHlS, Department 01 Biology, University oI Calgary, Calga.r y , Canada for dwarf rire seen. The authoIs wish to thank Mrs. A. FRITZSCHE aud Mrs. M. KROHN for technital a.ssistante aud Mrs. S. VOßKEFELD for bioassaying.

References BADH, S. A., MAHTlN, G. C., aud HARTMANN, H. T.: A Modified Method fOT Extraction and Identification of Abscisie Arid aud Giberellin-like Substances from Olive (Olea europaea). PhysioJ. Plantarum 24, 191-198 (1971).

Endogenous Plant Hormones of V ieia. 11.

609

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Received Mareh 13, 1980. Authors ' address: Dr. WIU'RIED DATHE a nd Prof. Dr. GÜNTHER SnIBDNER, Institute of Plant Bioehemistry, Research Centre for Molecular Biology and Medieine, Academy of Sciences of thc GDR, DDR - 4020 Halle (Saale), Weinberg ß.