Planta

Planta 138, 167-172 (1978)

9 by Springer-Verlag 1978

Inhibition of Photosynthesis and Respiration by Batatasins M o r i t o s h i Iino, T o h r u H a s h i m o t o a n d U l r i c h Heber* Institute for Physical and Chemical Research, Wako-shi, Saitama 351, Japan

Abstract. Effects o f b a t a t a s i n s I, I I I a n d V, p h e n o l i c g r o w t h i n h i b i t o r s o c c u r i n g in d o r m a n t bulbils o f Dioscorea b a t a t a s Decne., o n p h o t o s y n t h e t i c reactions o f c h l o r o p l a s t s f r o m s p i n a c h ( S p i n a c i a oleracea L.) a n d on r e s p i r a t i o n o f m i t o c h o n d r i a f r o m p o t a t o e s ( S o l a n u m t u b e r o s u m L.) were investigated. In c h l o r o plasts, the b a t a t a s i n s effectively i n h i b i t e d CO2-depend e n t oxygen e v o l u t i o n a n d electron flow f r o m w a t e r to a c c e p t o r s such as d i c h l o r o p h e n o l i n d o p h e n o l , ferric y a n i d e a n d m e t h y l v i o l o g e n . P h o t o s y s t e m - I depend e n t electron t r a n s p o r t f r o m a s c o r b a t e to oxygen was stimulated. The p r o t o n c o n d u c t i v i t y o f t h y l a k o i d m e m b r a n e s was increased a n d p h o s p h o r y l a t i o n was u n c o u p l e d f r o m electron t r a n s p o r t . I n h i b i t i o n o f elect r o n t r a n s p o r t with w a t e r as e l e c t r o n d o n o r a p p e a r e d to p r e c e d e u n c o u p l i n g . I n m i t o c h o n d r i a l , b a t a t a s i n I did n o t m u c h inhibit s u c c i n a t e - d e p e n d e n t O2 u p t a k e in the a b s e n c e o f A D P , b u t caused s t r o n g i n h i b i t i o n in the presence o f A D P . B a t a t a s i n s I I I a n d V i n h i b i t e d o x y g e n u p t a k e irrespective o f the presence or absence of ADP. Inhibition of chloroplast and mitochondrial r e a c t i o n s b y b a t a t a s i n s was s h o w n to be reversible. Key words: B a t a t a s i n -- D i s c o r e a -- D o r m a n c y -Photophosphorylation ration.

-

Photosynthesis

-

sins ( b a t a t a s i n I, II, III, IV a n d V) have so far been i s o l a t e d ( H a s h i m o t o et al., 1972, 1974, H a s h i m o t o , unpublished). In the o a t coleoptile test, these c o m p o u n d s possess g r o w t h - i n h i b i t i n g properties. Externally a p p l i e d b a t a t a s i n i n d u c e d d o r m a n c y in nond o r m a n t y a m bulbils ( H a s h i m o t o et al., 1972). The b a t a t a s i n c o n t e n t in d o r m a n t y a m bulbils was related to the d e p t h o f d o r m a n c y suggesting t h a t e n d o g e n eous b a t a t a s i n s can be r e g u l a t i n g factors in d o r m a n c y ( H a s e g a w a a n d H a s h i m o t o , 1973). There is no inform a t i o n o n m e t a b o l i c effects o f b a t a t a s i n s . In this paper, we describe effects o f b a t a t a s i n s on activities o f isolated c h l o r o p l a s t s a n d m i t o c h o n d r i a . Such a n investigation m a y serve two different purposes. It m a y increase o u r u n d e r s t a n d i n g o f the b i o c h e m i c a l p r o p erties o f c h l o r o p l a s t s o r m i t o c h o n d r i a a n d it m a y exemplify h o w b a t a t a s i n s c o u l d affect cellular activity. F o r the latter p u r p o s e , c h l o r o p l a s t s a n d m i t o c h o n d r i a are p a r t i c u l a r l y suitable as test systems as their bioc h e m i c a l activities can be easily m e a s u r e d . There is, o f course, no need to a s s u m e f r o m o b s e r v e d effects t h a t b a t a t a s i n s m a i n l y act on the cell t h r o u g h their a c t i o n on these organelles.

Respi-

Introduction B a t a t a s i n s are a class o f p h e n o l i c c o m p o u n d s which o c c u r in d o r m a n t y a m bulbils. F i v e different b a t a t a * Present address: Botanisches Institut der Universit~it Dfisseldorf,

Universit~itsstral3e 1, D-4000 Dfisseldorf, Federal Republic of Germany Abbrevations: B-I=batatasin I, 6-hydroxy-2,4,7-trimethoxyphen-

anthrene; B-III=batatasin III, 3,3'-dihydroxy-5-methoxybibenzyl; B-V=batatasin V, 2'-hydroxy-3,4,5-trimethoxybibenzyl; Chl= chlorophyll; MV=methylviologen; DCPIP=2,6-dichlorophenolindophenol ; DCMU = 3-(3',4'-dichlorophenyl)-l,l-dimethylurea; PVP = polyvinylpyrrolidone

Materials and Methods Chloroplasts were isolated from leaves of freshly harvested or of market spinach according to a modification of Jensen and Bassham's method (Heber, 1973; Jensen and Bassham, 1966). The chloroplasts were suspended in a medium containing 0.33 M sorbitol, 1 mM MgCI2, 1 mM MnClz, 2 mM EDTA, 10 mM NaC1, 0.5 mM phosphate and 50 mM HEPES (pH 7.6), and kept at 0~ C. The chlorophyll concentration was measured according to Arnon (1949). CO2 fixation was determined as 02 evolution, methyl viologen (MV) photoreduction as 02 uptake. Oxygen exchange was measured polarographicall3~with a Clark-type oxygen electrode. Photophosphorylation, which is accompanied by stoichiometric OHformation, was measured by following light-dependent pH changes in a weakly buffered reaction medium with a recording pH-meter (Hitachi-Horiba, F-7ss). Reaction cells for measurements of 02 and pH were equipped with magnetic stirrers, and jacketed in water baths. For the measurement of CO2 fixation, intact chloroplasts were used. For the other measurements, chloroplasts were

0032-0935/78/0138/0167/$01.20

168

M. Iino et al. : Inhibition of Photosynthesis and Respiration by Batatasins I

|

I

l

I

I00

70O

-- 600 80

o~

I

I

!

I

!

!

!

|

B -'lTr

B-I 9

+ NH4Cl

o

- NH4CI

500

9 A

+ NH4CI _ NH4C I

B-~" 9

+ NH4Cl

_~ 400

60

0

:~ 300

>o

IJ_l 40

-X 2~176 ~ I00 12)

20

0

I

0

I

2

5

4

5

Batatasin Concentration(lO-4 M) Fig. 1. Effect of batatasins on CO2-dependent 02 evolution b y intact chloroplasts. The reaction medium (pH 7.6, 1 ml) contained: chloroplasts (50 gg Chl); 0.33 M sorbitol, 1 mM MgC12, 1 mM MnCI2, 2 mM EDTA, 10 mM NaC1, 0.5 mM KHzPO 4 and 50 mM HEPES; 2 mM NaHCO3. Reaction temperature was 25 ~ C. Control rates (100%) averaged 100 lamol Oz/mg Chl h

disrupted osmotically in the reaction cell just before the measurement by adding intact chloroplasts to an at least 10-fold volume of water containing 8 mM MgC12. Immediately afterwards an equal volume of double strength reaction medium was added. The composition of the media used is given in the legends to the figures. Illumination was provided by a slide projector (Canon, 300 Watts), and passed through a 4 cm layer of copper sulfate solution. The light intensity was 100 to 200 mW/cm 2 at the reaction cell. Mitochondria were isolated from market potato tubers (Solanum tuberosum L.) using a slight modification of the procedure of Willenbrink (private communication). 200 g peeled potatos were homogenized in the cold in 250 ml of a medium containing 0.45 M sorbitol, 10 mM phosphate, 10 mM ascorbate, 5 mM ethylenediamine tetraacetate, 6 mM cysteine, 60 mM mercaptoethanol and 750 mg PVP (average molecular weight 25,000 daltons), pH 7.5. After removal of debris and large particles including starch a mitochondrial pellet was obtained by centrifugation at 20,000 g for 5 min. After washing, mitochondria were suspended in a medium containing 0.5 M sorbitol, 5 mM phosphate, 4 mM MgC12, 1 mM reduced glutathione and 20 mM HEPES (pH 7.3), and the suspension was stored in ice. Protein was measured according to Lowry et al. (1951). Respiration activity was measured at room temperature as 02 uptake with the oxygen electrode. Succinate was used as substrate. Batatasins used in this study were synthetic batatasins I, III and V (B-I, -III and -V). They were dissolved in ethanol and added to the reaction media. Proper controls ascertained that observed effects were not caused by ethanol.

Results and Discussion

I. Effect o f Batatasins on Chloroplast Activities 1. COz-dependent Oxygen Evolution. A s s h o w n in F i g u r e 1, all t e s t e d b a t a t a s i n s e f f e c t i v e l y i n h i b i t e d C O e -

I

I

I

2

4

6

0

2

4

6

8

I0

Batatasin Concentration ( 10 -4 M) Fig. 2. Effect of batatasins on MV photoreduction by disrupted chloroplasts in the presence or absence of NHr MV photoreduction was measured as Oa uptake. The reaction medium (pH 7.8, 1 ml) contained: chloroplasts (10 gg Chl); 0.33 M sorbitol, 4 mM MgClz, 10mM NaC1, 0.5mM KHzPO4 and 50raM HEPES; 0.1 mM MV and 0.5 mM KCN; with or without 10 mM NH4C1. Batatasin was added 1 to 1.5 min after the beginning of illumination and the rate was measured 30 s after the addition of batatasin. Reaction temperature was 25~ C

d e p e n d e n t o x y g e n e v o l u t i o n . B - I w a s the m o s t a n d B - V w a s the least e f f e c t i v e i n h i b i t o r .

2. Electron Flow with Water as Electron Donor. Elect r o n f l o w f r o m w a t e r to a n a c c e p t o r w i t h a v e r y l o w r e d o x p o t e n t i a l s u c h as m e t h y l v i o l o g e n ( M V ) inv o l v e s b o t h p h o t o s y s t e m s II a n d I. F i g u r e 2 s h o w s effects o f b a t a t a s i n s o n M V p h o t o r e d u c t i o n in the presence or absence of the uncoupler NH4C1. Catalase a c t i v i t y was i n h i b i t e d by K C N . B a t a t a s i n s i n h i b i t e d the e l e c t r o n f l o w v e r y effectively. T h i s w a s p a r t i c u l a r l y c l e a r w h e n p h o t o p h o s p h o r y l a t i o n was u n c o u p l e d f r o m e l e c t r o n f l o w by NH,~C1. I n the a b s e n c e o f N H 4 C 1 , i n h i b i t i o n o f elect r o n f l o w was c l e a r a t l o w c o n c e n t r a t i o n s o f b a t a t a sins. A s the c o n c e n t r a t i o n o f b a t a t a s i n s i n c r e a s e d , h o w e v e r , the i n h i b i t e d r a t e i n c r e a s e d u p to the r a t e seen in the p r e s e n c e o f N H ~ C 1 , s u g g e s t i n g t h a t at higher concentrations batatasins exert uncoupling of p h o s p h o r y l a t i o n . T h e d u a l e f f e c t o f b a t a t a s i n s was also o b s e r v e d w h e n f e r r i c y a n i d e o r D C P I P was u s e d as e l e c t r o n a c c e p t o r i n s t e a d o f M V ( n o t s h o w n ) . W h e n 1 , 5 - d i p h e n y l c a r b a z i d e was a d d e d as elect r o n d o n o r to b a t a t a s i n - i n h i b i t e d c h l o r o p l a s t s , t h e r e w a s in s o m e e x p e r i m e n t s a p a r t i a l r e s t o r a t i o n o f elect r o n f l o w to D C P I P ( d a t a n o t s h o w n ) . A s d i p h e n y l c a r b a z i d e f e e d s e l e c t r o n s to p h o t o s y s t e m II, b y p a s s i n g the site o f w a t e r s p l i t t i n g ( Y a m a s h i t a a n d Butler, 1969), this i n d i c a t e s t h a t o n e o f the effects o f b a t a t a sins c o n s i s t s in the i n h i b i t i o n o f w a t e r o x i d a t i o n by p h o t o s y s t e m II.

M. Iino et al. : Inhibition of Photosynthesis and Respiration by Batatasins

169

700

Table 1. Effect of batatasins on electron flow from DCPIPHz

600

to MV or 02 in the presence or absence of NH4C1. The reaction medium (pH 7.8, 1 ml) contained: chloroplasts (10 lag Chl); 0.33 M sorbitol, 4 m M MgClz, 10mM NaC1, 0 . 5 m M KH2PO4, 50ram HEPES, 0.1mM DCPIP, 10raM ascorbate, 0.5raM K C N and 5 laM D C M U ; with or without 0.1 m M MV; with or without 10 mM NH4C1. Reaction temperature was 25 ~ C

500

MV NH4C1

02 Uptake in gmol O2/mg Chl h (%) Control

4oo

300

(A)

0

B-V

2x10-4M

2x10-4M

6x10-4M

+ +

+

307 (100) 869 (283) 917 (100) 1421 (155)

617 (201) 981 (107)

1268 (413) 1632 (178)

-

+

45 (100) 106 (100)

95 (213) 102 (96)

282 (630) 265 (250)

(B)

200

B-I

193 (431) 194 (183)

100

0

2 4 6 8 Batolosin Concentration(lO-4 M)

Fig. 3. Effect of batatasins on electron flow from DCPIPH2 to MV. MV photoreduction was measured as 02 uptake. The reaction medium (pH 7.8, 1 ml) contained: chloroplasts (25 lag Chl); 0.33 M sorbitol, 4raM MgCI2, 10mM NaC1, 0 . 5 m M KHaPO4, 0.1 m M DCPIP, 10 m M ascorbate, 0.1 m M MV, 0.5 m M K C N and 5 jam D C M U . Reaction temperature was 25 ~ C. Control rates (100%) were 102, 84 and 101 ~xmol O2/mg Chl h respectively for the experiments with B-I, B-III and -V

A

B

off

Control

on

C

~f

1.5 x I0-4 M B-I

I

I min I

IO.OZjJeq.H+

3 x I0-4 M B-I

Fig. 4. Effect of B-I on light-dependent proton uptake by disrupted chloroplasts. Reaction medium (pH 7.1, 3 ml) contained: chloroplasts (200lag Chl), 0.33 M sorbitol, 4 m M MgCIz, 10mM NaC1 and 0.5 m M KH2PO4. The reaction was conducted at room temperature

3. Photosystem I-dependent Electron Flow. Electron flow through photosystem I was measured by lightdependent oxygen uptake in the presence of MV. Electrons were supplied to photosystem I from ascorbate/DCPIP. Photosystem II activity was eliminated by the addition of 5 pM DCMU. As shown in Figure 3, all tested batatasins markedly stimulated this electron flow. Uncoupling was partly responsible for stimulation as shown by the decreased stimulation in the presence of the uncoupler NH4C1 (Table 1). Also, batatasins caused a change in membrane structure permitting faster reduction of electron acceptors

such as MV or oxygen. The change in membrane structure was seen as a change in 535 nm light scattering by a chloroplast suspension on addition of batatasin (not shown). Oxygen is not an effective acceptor in photosynthesis and the reduction of oxygen by the electron transport chain is usually slow. It was considerably enhanced by batatasins even in the presence of a high and uncoupling concentration of NH4C1. Batatasins thus increase the capacity of the electron transport chain to donate electrons to acceptors such as MV (Table 1 A) or oxygen (Table 1 B). As the electron flow from the couple ascorbate/ DCPIP to MV was not inhibited by batatasins, the inhibition of electron flow from water to MV shown in the previous section must be in the region of photosystem II.

4. Proton Transport. In thylakoids, vectorial transport of protons is coupled to electron flow. On illumination, thylakoids take up protons from the medium creating a transmembrane proton gradient. Protons are released on darkening. The proton gradient created in the light is believed to be part of the driving force for the endergonic phosphorylation of ADP (Mitchell 1966). Figure 4 shows light-dependent proton uptake in the absence (A) and in the presence (B, C) of B-I. Oxygen was the only electron acceptor present. A low concentration of B-I (1 • 10 4 M) increased the maximal proton gradient; a high concentration (3 • 10 . 4 M) decreased it. The rates of both proton uptake on illumination and proton release on darkening were stimulated by B-I. As photosystem-I dependent electron flow to oxygen was increased by B-I (Table 1 B) and electron flow from water to oxygen was also stimulated (not shown), the increase in the rate of proton uptake by batatasin can be explained by increased electron flow. At low batatasin concentrations, an increase

170

M. Iino et al.: Inhibition of Photosynthesis and Respiration by Batatasins

i00~l' '

,

,

,

(Fig. 4, B, C). It can explain the uncoupling effects of batatasins described above (Fig. 2, Table 1).

5. Photophosphorylation. Photosystem-I dependent cyclic photophosphorylation catalyzed by phenazine methosulfate (PMS) was inhibited by the tested batatasins (Fig. 5). As electron transport through photosystem I was actually stimulated (Fig. 3, Table 1), this clearly shows that the batatasins, in addition to inhibiting electron flow in the region of photosystem II, act as uncouplers of phosphorylation in chloroplasts.

"~o~ ~ 80~~~,~,

40

a_ 20

6. Effectiveness of the Batatasins. The extent of the = 9

0

2

4

I

6

I

8

Batatasin ConcentrotionO0 -4M) Fig. 5. Effect of batatasins on PMS-mediated photophosphorylation by disrupted chloroplasts. Photophosphorylation was measured as OH- formation9 The reaction medium (3 ml) contained: chloroplasts (100 gg Chl); 0.33 M sorbitol, 4 mM MgClz, 10 mM NaC1, 0.5raM KHzPO,, 1.2raM HEPES; 0 9 ADP and 43 gM PMS. The reaction was conducted at room temperature, between pH 7.6 and 7.9. Control rates (100%) averaged 318 ~tmol OH-/rag Chl h

t + ADP o -- ADP

._= 80 k"~

E

B-~

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o

p,

60 ~ .

e7 40~

,e, -- ADP

B-I.

B-'V ~

~

I -I- ADP

E

II. Effect of Batatasins on Mitochondrial Oxygen Uptake

20

9 ,Y

0

inhibition of electron transport by batatasins increased with time. This makes it difficult to directly compare inhibition of different chloroplast activities as recorded in the figures. Also, owing to the lipid solubility of the batatasins, inhibitory effects on chloroplasts are a function of the membrane concentration. The inhibition of CO2 reduction demonstrated in Figure 1 can be caused by inhibition of electron flow and/or uncoupling of phosphorylation. Two observations suggest that inhibition of electron flow is the more important factor: In Figure 2, inhibition of electron flow is seen before uncoupling effects become apparent. Also, B-V was more effective than B-III as uncoupler of phosphorylation (Fig. 5). Photosynthesis, on the other hand, was more effectively inhibited by B-III than by B-V (Fig. 1). Electron transport was also more sensitive to B-III than to B-V (Fig. 2). All chloroplast activities were highly sensitive to

3

6

9

12

15

Batatasin Concentrotion(lO-4 M)

Fig. 6. Effect of batatasins on succinate oxidation by mitochondria. The reaction medium (pH 79 1 ml) contained: 0.4 to 0.8 mg mitochondrial protein, 0.5 M sorbitol, 5 mM KHzPO4, 4 mM MgC12, 1 mM reduced glutathione and 20 mM HEPES; 4 mM succinate; with or without 0.5 mM ADP. The batatasin-inhibited rate of 02 uptake was measured 1 rain after batatasin addition9 Reaction temperature was 25~ C

in the rate of proton pumping can produce a larger proton gradient (Fig. 4, B) even if batatasins increase the proton conductivity of the thylakoids. The latter effect is apparent from the increased rate of proton efflux seen in the presence of batatasin after darkening

Figure 6 shows the effects of batatasins on 02 uptake by potato mitochondria with succinate as substrate. B-I did not very significantly inhibit 02 uptake in the absence of ADP, whereas in the presence of A D P it strikingly inhibited 02 uptake nearly to the level observed in the absence of ADP. This effect is reminiscent of that of kaempferol, a flavonoid compound, which is reported to inhibit phosphorylation specifically, not influencing electron flow (Koeppe and Miller 1974). ]n contrast, B-III and B-V inhibited Oa uptake irrespective of the presence or absence of ADP. No uncoupling effect became evident. Even in the absence of ADP, where mitochondrial 02 uptake was reduced and respiration was in the controlled state, addition of batatasins failed to stimulate O2 uptake 9 Only inhibition was observed.

M. Iino et al. : Inhibition of Photosynthesis and Respiration by Batatasins 4 mM succinate

~

1.5 x I0 -3 M

B-W

B

~

2 rain

o PVP I0.02 jJmoles 02 ,.5go o

off

o~t on ~///-on

I

j

off OH-

IO- 4 M B - I

t

0.5% PVP

A

Jl rain

Fig. 7A and B. Reversal of batatasin inhibition by PVP. A PMSmediated cyclic photophosphorylation by disrupted chloroplasts. Photophosphorylation was measured as OH- formation. The reaction medium was as given for Figure 5. Numbers along the trace indicate the rates of OH- formation in gmol OH /mg Chl h. B Succinate oxidation by mitochondria. The reaction medium was as given for Figure 6 (without ADP). Numbers along the trace indicate rates of O2 uptake in nanomoles O2/mg protein, min

IlL Reversibility of Batatasin Inhibition Figure 7A shows hydroxyl ion formation, which is stoichiometrically linked to ATP synthesis, in PMSmediated cyclic photophosphorylation of thylakoids. On turning on the light, there was a rapid alkalization of the medium corresponding to a rate of ATP synthesis of 2 4 0 g m o l m g Chl l h 1. 10- CM B-I was sufficient for complete inhibition of the reactionl When 0.5% polyvinylpyrrolidone (PVP), which complexes phenols (Schneider and Hallier 1970), was added to the reaction mixture, a slow restoration of photophosphorylation was observed. Though there was no complete recovery of the reaction even after the addition of 1.5% PVP, the data clearly indicate that inhibition by batatasin can be reversed, i.e. that the membrane alteration induced by batatasin is reversible. Batatasin-inhibited electron flow responded in a fashion very similar to the response of photophosphorylation to PVP (not shown). The effect of PVP on mitochondrial oxygen uptake is shown in Figure 7 B. Succinate-dependent oxygen consumption was completely inhibited by 1.5 • l 0 - 3 M B-III. After the addition of 1.5% PVP, fast recovery of oxygen uptake was observed.

Conclusion

The results presented above show clearly that batatasins are highly effective in modifying membrane properties. Their lipid solubility makes it very unlikely that they can be confined to a particular location

171

in the cell such as the vacuole. Rather, this property makes them well suited for membrane interactions. The presented results show that their incorporation into membranes alters membrane activities. If fully reversible, membrane modification by a normal cell constituent must be considered ~t0~ be a physiological effect. Batatasins were found to be effective in inhibiting electron flow of both chloroplasts and mitochondria. Reversibility of inhibition could be demonstrated. Though it would be highly speculative to suggest that batatasins regulate dormancy of yam bulbils by suppressing respiration and related processes, it is safe to conclude more neutrally that membrane modification by batatasins must affect the activity of a cell. The observation, that the batatasins modify thylakoid membranes so as to stimulate electron flow to oxygen is interesting in regard to a physiological role of electron flow from water to oxygen in photosynthesis. Oxygen reduction by the electron transport chain of chloroplasts (Mehler, 1951) has been shown to occur not only in isolated chloroplasts (Egneus et al., 1975) but also in leaves (Heber, 1969). It is linked to ATP synthesis (Forti and Jagendorf, 1961). Usually, oxygen reduction is a slow process. However, its rate might be subject to modification by phenolic compounds. The increase in electron flow to oxygen under the influence of batatasins serves to illustrate this possibility. A role of phenolic compounds in the regulation of oxygen reduction by the electron transport chain chloroplasts has already been suggested by Elstner and Heupel (1974). We are grateful to Prof. K. Shibata for valuabIe advice and criticism and to Dr. Y. Inoue, Dr. Y. Kobayashi and Miss A. Suzuki for their kind help. U.H. acknowledges support from the Japan Society for the Promotion of Science and from the Deutsche Forschungsgemeinschaft.

References Arnon, D. : Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol. 24, 1 15 (1949) Egneus, H., Heber, U., Matthiesen, U., Kirk, M.: Reduction of oxygen by the electron transport chain of intact chloroplasts during assimilation of carbon dioxide. Biochim. Biophys. Acta 408, 252 268 (1975) Elstner, E.F., Heupel, A.: On the mechanism of photosynthetic oxygen reduction by isolated chloroplast lamellae. Z. Naturforschg. 29c, 564-571 (1974) Forti, G., Jagendorf, A.T. : Photosynthetic phosphorylation in the absence of redox dyes : oxygen and ascorbate effects. Biochim. Biophys. Acta 54, 322-330 (1961) Hasegawa, K., Hashimoto, T.: Quantitative changes of batatasins and abscisic acid in relation to the development of dormancy in Yam bulbils. Plant & Cell Physiol. 14, 369-377 (1973) Hashimoto, T., Hasegawa, K., Kawarada, A.: New dormancyinducing substances of Yam bulbils. Planta 108, 369 374 (1972)

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M. Iino et al.: Inhibition of Photosynthesis and Respiration by Batatasins

Hashimoto, T., Hasegawa, K., Yamagnchi, H. : Structure and synthesis of batatasins, dormancy-inducing substances of Yam bulbils. Phytochemistry 13, 2849-2852 (1974) Heber, U.: Conformational changes of chloroplasts induced by illumination of leaves in vivo. Biochim. Biophys. Acta 180, 302-319 (1969) Heber, U. : Stoichiometry of reduction and phosphorylation during illumination of intact chloroplasts. Biochim. Biophys. Acta 305, 140 152 (1973) Jensen, R.G., Bassham, J.A.: Photosynthesis by isolated chloroplasts. Proc. Natl. Acad. Sci. USA 56, 1095 1101 (1966) Koeppe, D.E., Miller, R.J.: Kaempferol inhibition of Corn mitochondrial phosphorylation. Plant Physiol. 54, 374-378 (1974) Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. : Protein measurements with the Folin phenol reagent. J. Biol. Chem. 193, 265-275 (1951)

Mehler, A.H. : Reactions of illuminated chloroplasts. L Mechanism of the reduction of oxygen and other Hill reagents. Arch. Biochem. Biophys. 33, 65 77 (1951) Mitchell, P. : Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Biol. Rev. 41, 445-502 (1966) Schneider, V., Hallier, U. : Polyvinylpyrrolidon als Schutzstoff bei der Untersuchung gerbstoffgehemmter Enzymreaktionen. Planta 94, 134 139 (1970) Yamashita, T., Butler, W.L.: Inhibition of the Hill reaction by Tris and restoration by electron donation to photosystem II. Plant Physiol. 44, 435438 (1969)

Received 29 September; accepted 26 October 1977