Effects of Phenolic Acids, Coumarins, and ... - ACS Publications

Chapter 23

Effects of Phenolic Acids, Coumarins, and Flavonoids on Isolated Chloroplasts and Mitochondria Donald E. Moreland and William P. Novitzky

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on February 23, 2016 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch023

Agricultural Research Service, U.S. Department of Agriculture, and Departments of Crop Science and Botany, North Carolina State University, Raleigh, NC 27695-7620

The effects of allelopathic plant phenolics and polyphenolics (benzoic and cinnamic acids, coumarins, and flavonoids) on electron transport and phosphorylation in chloroplasts and mitochondria were investigated. A l l chemicals inhibited CO-dependent oxygen evolution in intact chloroplasts. I concentrations ranged between coumarins > cinnamates = benzoates. The compounds did not act like uncouplers. In studies with mung bean mitochondria, the compounds primarily acted as electron transport inhibitors. Malate oxidation was more sensitive than either succinate or NADH oxidation. The flavonoids were most inhibitory, with I values that ranged between 10 and 80 μΜ. For the coumarins, cinnamates, and benzoates, Ι values ranged between 1 and 20 mM. The compounds did not act as uncouplers or directly inhibit ATP synthesis. However, naringenin, some of the flavones, and the cinnamates acids inhibited the hydrolysis of ATP catalyzed by mitochondrial Mg -ATPase. The inhibition of substrate oxidation appears to result from alterations and perturbations induced in the inner membrane as evidenced by interference with carrier-mediated transport processes. 2

50

50

50

2+

The biochemical mechanisms through which allelochemicals exert deleterious or toxic effects on plants are, for the most part, unknown (1)· Some phenolic acids, cinnamic acids, coumarins, and flavonoids have been reported to inhibit photosynthesis and respiration of intact plants and microorganisms. However, the mechanisms, at the molecular level, through which the compounds interfere, remain to be ascertained. Some phenolic acids, coumarins, and flavonoids were reported to inhibit CO^-dependent 0^ This chapter not subject to U.S. copyright. Published 1987 American Chemical Society

In Allelochemicals: Role in Agriculture and Forestry; Waller, George R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY

e v o l u t i o n and p h o t o p h o s p h o r y l a t i o n o f i s o l a t e d c h l o r o p l a s t s (2). K a e m p f e r o l and q u e r c e t i n were shown t o i n h i b i t c o u p l e d e l e c t r o n t r a n s p o r t and p h o t o p h o s p h o r y l a t i o n , b u t had a l i m i t e d e f f e c t on b a s a l and u n c o u p l e d e l e c t r o n t r a n s p o r t o f i s o l a t e d t h y l a k o i d s ( 3 ) . The a u t h o r s (3) p o s t u l a t e d t h a t t h e f l a v o n e s a c t e d as energy t r a n s f e r i n h i b i t o r s , but t h a t t h e a c t i o n was d i f f e r e n t from t h e r e f e r e n c e s t a n d a r d s p h l o r i z i n and c h l o r o t r i b u t y l t i n . In s t u d i e s with i s o l a t e d p l a n t m i t o c h o n d r i a , f l a v o n e s , flavan o n e s , c i n n a m i c a c i d s , and b e n z o i c a c i d s were shown t o i n h i b i t the o x i d a t i o n o f s u c c i n a t e , m a l a t e , and NADH (2>4-9.). I n h i b i t i o n was o b s e r v e d under b o t h A D P - s t i m u l a t e d and u n c o u p l e d c o n d i t i o n s . There was no e v i d e n c e t h a t t h e v a r i o u s compounds a c t e d as u n c o u p l e r s . Kaempferol was p o s t u l a t e d t o i n h i b i t t h e p h o s p h o r y l a t i o n mechanism, but t h e a c t i o n was d i f f e r e n t from t h a t o f o l i g o m y c i n ( 9 ) . In s t u d i e s w i t h b o t h c h l o r o p l a s t s and m i t o c h o n d r i a , g l y c o s i d e s , i n g e n e r a l , were l e s s i n h i b i t o r y than t h e c o r r e s p o n d i n g a g l y c o n e s . The o b j e c t i v e s o f t h e s t u d i e s r e p o r t e d h e r e i n were t o : (a) compare t h e e f f e c t s of a s e r i e s o f p h e n o l i c a c i d s , c o u m a r i n s , and f l a v o n o i d s on whole c h a i n e l e c t r o n t r a n s p o r t and p h o s p h o r y l a t i o n i n i s o l a t e d p l a n t c h l o r o p l a s t s and m i t o c h o n d r i a ; and (b) identify s p e c i f i c s i t e s o f i n h i b i t i o n w i t h p o l a r o g r a p h i c and e n z y m a t i c techniques. E x p l o r a t o r y s t u d i e s were conducted w i t h t h e 20 compounds l i s t e d i n T a b l e I . The t h r e e g l y c o s i d e s a r e shown i n d e n t e d below t h e c o r r e s p o n d i n g a g l y c o n e s . D e t a i l e d s t u d i e s were conducted w i t h t h e s i x compounds, one r e p r e s e n t a t i v e member from each chemi c a l f a m i l y , d e s i g n a t e d w i t h an a s t e r i s k . Materials

and Methods

Chloroplasts. I n t a c t c h l o r o p l a s t s were i s o l a t e d from f r e s h l y h a r v e s t e d growth chamber-grown s p i n a c h ( S p i n a c i a o l e r a c e a L . ) as d e s c r i b e d by L i l l e y and Walker ( 1 0 ) . T h y l a k o i d s were p r e p a r e d by the method of Armond e_t al. (11) . C h l o r o p h y l l c o n c e n t r a t i o n s were d e t e r m i n e d by the method of MacKinney ( 1 2 ) . Photochemical r e a c t i o n s were conducted a t 25°C w i t h a photon f l u e n c e r a t e o f 750 ymol/m^ · s (PAR). CO^-dependent oxygen e v o l u t i o n o f i n t a c t c h l o r o p l a s t s was measured as d e s c r i b e d by Walker ( 1 3 ) . E f f e c t s on TABLE I : Benzoic

A l l e l o c h e m i c a l s E v a l u a t e d f o r E f f e c t s on R e a c t i o n s M e d i a t e d by I s o l a t e d C h l o r o p l a s t s and M i t o c h o n d r i a acids:

gallic salicylic syringic *vanillic

Benzaldehyde:

*vanillin

Cinnamic

caffeic trans-cinnamic £-coumaric *ferulic

acids:

Coumarins:

coumarin esculetin esculin scopoletin *umbelliferone

Flavones:

flavone kaempferol *quercetin rutin

Flavanones: *Representative

compounds s e l e c t e d

for detailed

*naringenin naringin study.


23.

MORELAND AND NOVITZKY

Effects of Allelopathic

Phenolics

249

e l e c t r o n t r a n s p o r t and p h o t o p h o s p h o r y l a t i o n were measured i n a medium (2.0 ml volume) t h a t c o n t a i n e d 0.1 M s o r b i t o l , 20 mM t r i c i n e - N a O H (pH 8 . 0 ) , 1 mM K H P 0 , 1 mM ADP, 5 mM MgCl„, 10 mM N a C l , 0.1 mM m e t h y l v i o l o g e n , and t h y l a k o i d s (40 yg Chi). E s t e r i f i c a t i o n o f P i was measured by the method o f Taussky and Shorr (14). E l e c t r o n f l o w was m o n i t o r e d p o l a r o g r a p h i c a l l y w i t h a C l a r k - t y p e p l a t i n u m e l e c t r o d e as oxygen consumed d u r i n g t h e a u t o o x i d a t i o n o f reduced m e t h y l v i o l o g e n . Uncoupled e l e c t r o n transport was determined i n the same medium w i t h the a d d i t i o n o f 5 mM N H ^ C l . E f f e c t s on d i f f e r e n t segments of the e l e c t r o n t r a n s p o r t c h a i n , c h l o r o p h y l l f l u o r e s c e n c e , and b i n d i n g s t u d i e s w i t h [ C]-atrazine were performed as d e s c r i b e d p r e v i o u s l y (15). 2


Plant

4

Mitochondria. M i t o c h o n d r i a were p r e p a r e d from 3 - d a y - o l d dark-grown mung bean ( V i g n a r a d i a t a Roxb.) h y p o c o t y l s . The i s o l a t i o n p r o c e d u r e , measurements of oxygen u t i l i z a t i o n , and e f f e c t s o f the t e s t compounds on r e s p i r a t o r y s t a t e s were c o n d u c t e d as d e s c r i b e d p r e v i o u s l y (16). In a c c o r d a n c e w i t h the t e r m i n o l o g y of Chance and W i l l i a m s (17), the A D P - s t i m u l a t e d r a t e o f r e s p i r a t i o n w i l l be r e f e r r e d t o as s t a t e 3 and A D P - l i m i t e d r e s p i r a t i o n as s t a t e 4. The m i t o c h o n d r i a had r e s p i r a t o r y c o n t r o l ( s t a t e 3 / s t a t e 4) r a t i o s t h a t averaged 4.2 ± 0 . 4 , 3.4 ± 0 . 2 , and 2.3 ± 0 . 2 , and c a l c u l a t e d ADP/0 r a t i o s t h a t averaged 2.3 ± 0 . 1 , 1.3 ± 0 . 1 , and 1.5 ± 0.1 f o r the o x i d a t i o n o ^ m a l a t e , NADH, and s u c c i n a t e , r e s p e c t i v e l y . E f f e c t s on Mg -ATPase a c t i v i t y were d e t e r m i n e d w i t h m i t o c h o n d r i a t h a t were r u p t u r e d by f r e e z i n g ( - 2 0 ° C ) and subsequent thawing (room t e m p e r a t u r e ) . The a s s a y medium and p r o c e d u r e was e s s e n t i a l l y t h a t of Blackmon and Moreland (18) e x c e p t t h a t DNP was o m i t t e d and the M g C ^ c o n c e n t r a t i o n was 5 mM. Osmotic s w e l l i n g . Changes i n the o s m o t i c s t a b i l i t y o f m i t o c h o n d r i a were m o n i t o r e d s p e c t r o p h o t o m e t r i c a l l y a t 520 nm. The 2.0 ml r e a c t i o n m i x t u r e c o n t a i n e d 10 mM Hepes-NaOH (pH 7.1) and 0.15 M KC1. M i t o c h o n d r i a (0.4 mg p r o t e i n ) were added t o g i v e an i n i t i a l absorbance o f 0 . 8 A . In s t u d i e s t h a t i n v o l v e d e f f e c t s on v a l i n o m y c i n - i n d u c e d s w e l l i n g , t h e t e s t compound was added 30 s p r i o r to the i n t r o d u c t i o n o f the i o n o p h o r e (0.1 uM). E f f e c t s on the p h o s p h a t e / h y d r o x y l a n t i p o r t e r and t h e p r o l i n e u n i p o r t e r were measured by the ammonium s w e l l i n g t e c h n i q u e (19^20). The KC1 i n the r e a c t i o n m i x t u r e i d e n t i f i e d above was r e p l a c e d by ammonium phosphate (0.1125 M) and p r o l i n e (0.2 Μ ) , r e s p e c t i v e l y . S w e l l i n g was m o n i t o r e d b e g i n n i n g 6 s a f t e r i n j e c t i o n of the m i t o c h o n d r i a to t h e s t i r r e d r e a c t i o n m i x t u r e c o n t a i n i n g t h e t e s t compound. Test chemicals. The a l l e l o c h e m i c a l s were o b t a i n e d from Sigma Chemical Co. S t o c k s o l u t i o n s of the d e s i r e d c o n c e n t r a t i o n s were p r e p a r e d as f o l l o w s : most b e n z o i c and c i n n a m i c a c i d s were p r e p a r e d as the Na s a l t s i n H^O; s a l i c y l i c a c i d , t r a n s - c i n n a m i c a c i d , f l a v o n e , k a e m p f e r o l , and v a n i l l i n were d i s s o l v e d i n a c e t o n e ; the o t h e r c h e m i c a l s were d i s s o l v e d i n DMS0. The f i n a l c o n c e n t r a t i o n o f a c e t o n e and DMS0 was h e l d c o n s t a n t at 1% (v/v) i n a l l assays i n c l u d i n g the c o n t r o l s . D a t a p r e s e n t e d were averaged from d e t e r m i n a t i o n s made w i t h t h r e e s e p a r a t e r e p l i c a t i o n s and i s o l a t i o n s .


250

ALLELOCHEMICALS: ROLE IN AGRICULTURE AND


Results and

FORESTRY

Discussion

Chloroplast and thylakoid responses. In exploratory studies, a l l of the allelochemicals i n h i b i t e d CO^-dependent 0^ evolution of intact spinach chloroplasts. 1^ values for the s i x representative compounds are shown i n Table I I . The flavones represented by quercetin were the most i n h i b i t o r y with the flavanone naringenin being somewhat less active. The coumarins, cinnamic acids, benzoic acids, and the benzaldehyde were considerably less active. The glycosides were also i n h i b i t o r y , but at higher concentrations than the corresponding aglycones. The exploratory studies, as conducted, did not distinguish between e f f e c t s imposed on the stromal-associated CO^ f i x a t i o n (Calvin cycle) reactions or on the l i g h t reactions associated with the thylakoids. Consequently, studies were conducted on l i g h t induced electron transport and ATP synthesis associated with isolated spinach thylakoid membranes. Electron transport and photophosphorylation. Shown i n Table II are 1^ values for i n h i b i t i o n by the s i x representative compounds of coupled electron transport (column 3) and phosphory l a t i o n (column 4), and of uncoupled electron transport (last column) with spinach thylakoids. The flavonoids were the most i n h i b i t o r y with naringenin being somewhat less active than quercetin. The coumarins, cinnamic acids, and v a n i l l i n were equally i n h i b i t o r y at somewhat higher concentrations. The benzoic acids were the weakest i n h i b i t o r s . For a l l compounds, the phosphorylation reaction was most s e n s i t i v e , followed by the coupled electron transport. Uncoupled electron transport was least sensitive. I n h i b i t i o n of coupled electron transport can r e s u l t i n d i r e c t l y from an e f f e c t imposed on the phosphorylation pathway or d i r e c t l y by action on a component of the electron transport pathway. The two e f f e c t s can be d i f f e r e n t i a t e d by using an uncoupler such as NH^Cl or FCCP (carbonyl cyanide £-trifluoromethoxyphenylhydrazone) to dissipate the energized state of the membrane (21) . If the i n h i b i t i o n of electron transport coupled with ATP generation i s caused by energy transfer i n h i b i t i o n , the i n h i b i t i o n should be circumvented by the uncoupler. However, i f electron transport i n h i b i t i o n i s the cause, the addition of an uncoupler w i l l not circumvent the i n h i b i t i o n . As shown i n Table III (column 3 versus column 5), i n h i b i t i o n of the uncoupled elec tron transport rate was only p a r t i a l l y r e l i e v e d . Thus, the compounds appear to have two e f f e c t s : (a) the more sensitive i s an effect on the ATP-generating pathway; and (b) a second, but weaker, effect involved the electron-transport pathway. Energy transfer and uncoupling. Because of the suggestion that the allelochemicals might i n t e r f e r e with energy transfer, their action was compared to that of known energy transfer i n h i b i t o r s of thylakoid-synthesized ATP (22). A series of 02-consumption polarographic traces i n which H 0 served as the electron donor and methyl viologen served as the electron acceptor are shown i n Figure 1. C h l o r o t r i b u t y l t i n (Trace A), p h l o r i z i n (Trace Β ) , and DCCD (N,N -dicyclohexylcarbodiimide) (Trace C) strongly i n h i b i t e d the rate of ADP-stimulated oxygen uptake. For a l l compounds, ?

f



± ± ± ± ± ±

1.46 0.64 1.46 0.59 0.01 0.07

35 2.67 2.97 2.63 0.07 0.26

@ ± ± ± ± ±

25° .06 .12 .06 .01 .03

-I 5 0

(mM)-

19 2.20 2.13 1.87 0.04 0.21

@ ± ± ± ± ±

25 .26 .15 .31 .01 .05

Coupled^ Phosphorylation

0 14 6.30 7.03 0.20 0.84

@ @ ± ± ± ±

25 4 .52 .12 .02 .05

Uncoupled 0^ uptake

0^ evolution was measured with a Clark-type electrode. S p e c i f i c a c t i v i t y of the untreated controls averaged 40 ± 9 ymoles 0^ evolved/mg Chi · h. Data shown are arithmetic averages ± SD of determinations made with a minimum of three d i f f e r e n t i s o l a t i o n s of chloroplasts.

4.63 4.77 4.47 4.23 0.05 0.56

0^ uptake

Compounds d i d not i n h i b i t by 50%. The data presented are the percentages of i n h i b i t i o n achieved at the highest concentration (mM) that could be tested.

C

η

^H^O served as the electron donor and methyl viologen as the electron acceptor. 0^ consumption was measured with a Clark-type electrode and phosphorylation was measured c o l o r i m e t r i c a l l y . Data are presented as averaged 1 umbelliferone > v a n i l l i n = f e r u l i c acid > v a n i l l i c acid. For a l l compounds, malate oxidation was the most sensitive. In general, succinate oxidation was less sensitive and NADH oxidation was least s e n s i t i v e . The order of inhibitory potency for the chemical families was similar to that obtained against the thylakoid system. The glycosides except for r u t i n were ineffective. Uncoupling and energy transfer. Representative oxygen consumption polarographic traces that compare the e f f e c t s of standard i n h i b i t o r s with that of quercetin on the oxidation of malate are



23.



Plant

Phenolics

255

shown i n F i g u r e 2. U n c o u p l e r s , such as FCCP, s t i m u l a t e s t a t e 4 r e s p i r a t i o n ( T r a c e A ) , but under the same c o n d i t i o n s q u e r c e t i n had no e f f e c t on s t a t e 4 r e s p i r a t i o n ( T r a c e B). Energy t r a n s f e r i n h i b i t o r s , such as o l i g o m y c i n , i n h i b i t s t a t e 3 r e s p i r a t i o n much like electron-transport inhibitors. However, the i n h i b i t i o n imposed by energy t r a n s f e r i n h i b i t o r s , but not by e l e c t r o n t r a n s p o r t i n h i b i t o r s , i s c i r c u m v e n t e d by u n c o u p l e r s . I n h i b i t i o n of s t a t e 3 r e s p i r a t i o n by o l i g o m y c i n and r e l i e f by FCCP i s shown i n Trace C. The f a i l u r e of FCCP t o c i r c u m v e n t i n h i b i t i o n of s t a t e 3 r e s p i r a t i o n by a n t i m y c i n A , an e l e c t r o n - t r a n s p o r t i n h i b i t o r , i s shown i n T r a c e D. FCCP a l s o d i d not r e l i e v e the i n h i b i t i o n o f s t a t e 3 r e s p i r a t i o n imposed by q u e r c e t i n ( T r a c e E ) . The e v i d e n c e o b t a i n e d from the above s t u d i e s s u g g e s t s t h a t q u e r c e t i n a c t e d p r i m a r i l y as an e l e c t r o n - t r a n s p o r t i n h i b i t o r i n m i t o c h o n d r i a . No i n d i c a t i o n s were o b t a i n e d f o r a c t i o n e i t h e r as an u n c o u p l e r or as an energy t r a n s f e r i n h i b i t o r . The o t h e r a l l e l o c h e m i c a l s a c t e d s i m i l a r l y to q u e r c e t i n . In the experiments r e p o r t e d above, no s t r o n g i n d i c a t i o n s were o b t a i n e d t h a t t h e a l l e l o c h e m i c a l s i n t e r f e r e d d i r e c t l y w i t h the s y n t h e s i s of ATP by a c t i n g on the energy t r a n s f e r pathway ( F Q - F - i complex). However, some of t h e compounds d i d i n h i b i t ATP h y d r o l y s i s , as measured w i t h p r e p a r a t i o n s i n which the m i t o c h o n d r i a had been r u p t u r e d by f r e e z e - t h a w i n g ( T a b l e I I I ) . The f l a v o n e s ( e x c e p t f l a ^ o n e i t s e l f ) and c i n n a m i c a c i d s s t r o n g l y i n h i b i t e d the Mg - A T P a s e , whereas the b e n z o i c a c i d s and coumarins were weak i n h i b i t o r s , i . e . , l e s s than 15% i n h i b i t i o n a t 10 mM c o n c e n t r a t i o n s . R e s u l t s obtained with q u e r c e t i n (Table III) agree w i t h p u b l i s h e d r e p o r t s i n which t h e compound was shown n o t t o a f f e c t ATP s y n t h e s i s ^ but to i n h i b i t the h y d r o l y s i s o f ATP by t h e m i t o c h o n d r i a l Mg -ATPase (30). I n h i b i t i o n o f whole c h a i n e l e c t r o n t r a n s p o r t can r e s u l t f r o m : (a) i n t e r a c t i o n o f the i n h i b i t o r w i t h a redox component of the pathway; or (b) i n t e r a c t i o n w i t h c a r r i e r systems t h a t t r a n s p o r t s u b s t r a t e m o l e c u l e s a c r o s s the i n n e r membrane. The l a t t e r i n t e r a c t i o n c o u l d be d i r e c t or i n d i r e c t . Because e l e c t r o n t r a n s p o r t a s s o c i a t e d w i t h t h e o x i d a t i o n of m a l a t e , s u c c i n a t e , and exogenous NADH were a l l i n h i b i t e d , but to d i f f e r i n g e x t e n t s , a s p e c i f i c i n t e r a c t i o n w i t h a s i n g l e redox component of the i n n e r m i t o c h o n d r i a l membrane does not seem to be i n v o l v e d . Transport systems. P a r t i t i o n i n g o f v a r i o u s t y p e s of m o l e c u l e s such as a l l e l o c h e m i c a l s i n t o the l i p i d b i l a y e r of the m i t o c h o n d r i a l i n n e r membrane can p e r t u r b t h e membrane and a l t e r t h e c o n f o r m a t i o n , p r o p e r t i e s , and f u n c t i o n o f components of the membranes. U n f o r t u n a t e l y , i t i s n o t always p o s s i b l e to demonstrate d i r e c t l y the e x i s t e n c e of c a r r i e r s y s t e m s , but i n d i r e c t e v i d e n c e can be obtained. A l t e r a t i o n s i n d u c e d to the membrane a r e sometimes r e f l e c t e d i n the o s m o t i c b e h a v i o r of m i t o c h o n d r i a . The i n n e r membrane i s r e l a t i v e l y impermeable to many c a t i o n s , i n c l u d i n g Κ and Η , and many s o l u t e s (31) . Hence, the o r g a n e l l e s a r e o s m o t i c a l l y s t a b l e under c e r t a i n c o n d i t i o n s . I n d i c a t i o n s were o b t a i n e d t h a t the a l l e l o c h e m i c a l s i n h i b i t e d the a c t i o n of c a r r i e r - m e d i a t e d t r a n s p o r t p r o c e s s e s a s s o c i a t e d w i t h the m i t o c h o n d r i a l i n n e r mem brane (as r e f l e c t e d i n the o s m o t i c b e h a v i o r ) . Responses o b t a i n e d w i t h q u e r c e t i n a r e shown' i n F i g u r e 3 . Mitochondria are o s m o t i c a l l y +




F i g u r e 2. R e p r e s e n t a t i v e p o l a r o g r a p h i c t r a c e s t h a t d e p i c t e f f e c t s o f q u e r c e t i n ( Q u e r . ) on oxygen u t i l i z a t i o n by mung bean m i t o c h o n d r i a w i t h m a l a t e as s u b s t r a t e . Trace A, s t i m u l a t i o n o f s t a t e 4 r e s p i r a t i o n by FCCP; t r a c e B , l a c k o f s t a t e 4 s t i m u l a t i o n by q u e r c e t i n ; t r a c e C , c i r c u m v e n t i o n o f oligomycin ( o l i g . ) - i n h i b i t e d s t a t e 3 r e s p i r a t i o n by FCCP; t r a c e D, l a c k o f c i r c u m v e n t i o n o f a n t i m y c i n A - i n h i b i t e d s t a t e 3 r e s p i r a t i o n by FCCP; and t r a c e E , l a c k o f c i r c u m v e n t i o n o f q u e r c e t i n - i n h i b i t e d s t a t e 3 r e s p i r a t i o n by FCCP. Rates o f oxygen u t i l i z a t i o n , e x p r e s s e d as nmoles O^/mg p r o t e i n · m i n , are i n d i c a t e d p a r e n t h e t i c a l l y .


23.


MEM.

A

OUT

K



+

a-

257

>K+

VAL

-? ——>a~ — •

2

Phenolics

V A L (0.1 uM)

IN

H 0

Plant

aosA H O 2

ι

1 mm

Β

1

0

Mtto /

ΡΓ

> PI"

OH
H 0 2

κ 1min-i

\ ^ *g 0

Figure 3. Diagrammatic models and representative traces of absorbance changes that show the k i n e t i c s and i n h i b i t i o n by quercetin (Quer.) of (A) valinomycin-induced swelling of mung mitochondria suspended i n isosmotic KC1; (B) swelling of mung bean mitochondria suspended i n isosmotic ammonium phosphate; and (C) swelling of mung bean mitochondria suspended i n isosmotic proline.



258


stable when suspended i n isotonic KC1. However, Κ permeability can be induced a r t i f i c a l l y by ionophores such as valinomycin (32). As depleted i n Scheme A, valinomycin forms a l i p i d - s o l u b l e complex with Κ anjjl transports i t across the membrane. An increase i n i n t e r n a l Κ w i l l be accompanied by the movement (diffusion) of the CI counter ion, i n some as yet unknown manner, to maintain e l e c t r o n e u t r a l i t y . The increase i n the matrix concentration of KC1 w i l l result i n the osmotic i n f l u x of water. Swelling i s measured as a decrease i n absorbance. The extent of swelling under control conditions i s shown i n the 0 trace. Quercetin i n h i b i t e d , i n a concentration-dependent manner, the extent of osmotic swelling. By means of the ammonium swelling technique, the existence of a P i /OH exchange c a r r i e j can be demonstrated (19). A s shown i n Figure 3B, external NH^ dissociates into NH^ and Η . The neutral ammonia (NH^) passes across the inner membrane down a concentration gradient. Jnside the matrix, NH^ associate^ to equilibrium with protons (H ) to form the ammonium 1οη_(ΝΗ^ ) and OH . The OH exchanges for phosphate on the Pi""/OH antiporter. The increased osmotic pressure induces spontaneous swelling that i s reflected as a decrease i n absorbance. This i s shown i n the 0 (control) trace. As before, quercetin, i n a concentration-dependent manner, inhibited the magnitude of the spontaneous swelling. In isosmotic solutions, movement of neutral amino acids such as proline across the inner membrane into the matrix results i n swelling of mitochondria (20). As shown i n Figure 3C, the movement of proline i s thought to occur v i a a uniport. The increased concentration of proline i n the matrix produces an osmotic-induced swelling. Kinetics of the swelling response i s shown i n the 0 (control) trace. Again, quercetin inhibited t h i s response i n a concentration-dependent manner. The other representative allelochemicals inhibited the three transport processes much as quercetin did. The concentration ranges at which the allelochemicals produced interference were similar to those that inhibited whole-chain electron transport. The i n h i b i t i o n of multisubstrate oxidation that involved complexes I and I I , and the ubiquinone pool, observed with the allelochemicals, can best be explained by alterations and perturba tions induced to the inner membrane. No clear-cut evidence was obtained f o r interactions with s p e c i f i c complexes of the membranes. Inhibition of transport processes may result from: (a) a l t e r a tions and perturbations induced to the inner membrane by the allelochemicals, or (b) prevention of the development of an electrochemical potential difference across the membrane (energization of the membrane). An energized state i s required for the transport of ions, or the cotransport of ions with other organic molecules, including substrates, across the membrane. The allelochemicals could i n t e r f e r e with transport through both mechanisms. By i n h i b i t i n g electron transport, the allelochemicals would prevent energization of the membrane. However, the membrane transport processes examined above do not require an energized membrane. The recorded interferences r e f l e c t alterations induced in the behavior of the c a r r i e r systems either through a direct interaction with the proteins themselves, or to the f l u i d i t y or i n t e g r i t y of the membrane within which the porters operate. +


23.



Plant

Phenolics

259


Conclusions In the studies with i s o l a t e d chloroplasts and thylakoids, the primary effect of the phenolic allelochemicals was on the ATPgenerating pathway, i . e . , energy transfer i n h i b i t i o n . The compounds acted on the electron-transport pathway at higher concentrations, but the exact s i t e ( s ) remain to be i d e n t i f i e d . These may be located on the oxidizing side of PS II or around the PQ pool. The s i t e s are not associated with PS I. The compounds did not act as uncouplers. In mitochondria, the allelochemicals acted primarily as electron transport i n h i b i t o r s . Malate oxidation was more s e n s i t i v e than either succinate or NADH oxidation. No evidence for i n t e r a c t i o n with a s p e c i f i c membrane complex was obtained. Instead, i n h i b i t i o n of substrate oxidation seems to result from a l t e r a t i o n s and perturbations produced i n the inner membrane as r e f l e c t e d i n interference with the behavior of transport processes. The compounds did not act as uncouplers or d i r e c t l y i n h i b i t ATP synthesis. However, naringenin, some of the flavones, and the cinnamic acids djtjl i n h i b i t the hydrolysis of ATP catalyzed by mitochondrial Mg -ATPase. The concentrations of phenolic and polyphenolic allelochemicals that inhibited the various reactions of i s o l a t e d chloroplasts and mitochondria have been reported to occur, either s i n g u l a r l y or i n combination, i n organic l i t t e r (1). The studies conducted herein were short-term, on the order of several minutes, i n which immediate responses were measured. Preincubation of the allelochemicals with the test systems could be expected to lower the concentrations required to produce i n h i b i t i o n . E f f e c t s reported herein on interference with chloroplast and mitochondrial electron transport and phosphorylation by polyphenolic allelochemicals are i n agreement with r e s u l t s obtained by other investigators 02-9). I n h i b i t i o n of photosynthesis and r e s p i r a t i o n of intact plants and microorganisms ÇL) can be explained by the interferences measured with the isolated organelles. However, d e t a i l s of the biochemical mechanisms involved remain to be i d e n t i f i e d . Conceivably, the phenolic allelochemicals may perturb other c e l l u l a r membranes (plasmalemma, tonoplast, nuclear, and endoplasmic reticulum) as they did the mitochondrial membrane. A l t e r a t i o n s to the permeability of membranes by polyphenolic and other allelochemicals have been reported ÇL). Transport or cotransport of many ions and organic molecules across semipermeable membranes requires energy or an energized state of the membrane. The energy i s provided d i r e c t l y or i n d i r e c t l y by ATP. Phenolic allelochemicals would l i m i t the a v a i l a b i l i t y of mitochondrial and chloroplast-generated ATP by acting on e i t h e r , or both, the electron transport or energy g e n e r a t i n g pathways. Quercetin has been shown to i n h i b i t Na /K -ATPases as well as mitochondrial Mg -ATPase (30). Naringenin, other flavones, and the cinnamic acids could behave l i k e quercetin. Hence, at least some of the phenolic allelochemicals could prevent the u t i l i z a t i o n of ATP energy required for transport of materials across c e l l u l a r membranes by i n h i b i t i n g the hydrolysis of ATP. Conceivably, a l t e r a tions induced to the permeability of organelle membranes coupled


260


with a reduction in the availability of chloroplast and mitochondrially generated ATP is involved in the biochemical mechanisms of action associated with phenolic allelochemicals.


Acknowledgments This was a cooperative investigation of the United States Department of Agriculture, Agricultural Research Service and the North Carolina Agricultural Research Service, Raleigh, N.C. Paper No. 10183 of the Journal series of the North Carolina Agricultural Research Service, Raleigh, N.C. 27695-7601. The study was sup ported in part by Public Health Service Grant ES 00044. Apprecia tion is extended to F. S. Farmer and M. A. Norman for technical assistance with several phases of the study. Literature Cited 1. Rice, E.L. "Allelopathy"; Academic Press: Orlando, FL, 1984, 422 pp. 2. Tissut, M.; Chevallier, D.; Douce, R. Phytochemistry 1980, 19, 495-500. 3. Arntzen, C.J.; Falkenthal, S.V.; Bobick, S. Plant Physiol. 1974, 53, 304-6. 4. Stenlid, G. Physiol. Plant. 1968, 21, 882-94. 5. Stenlid, G. Phytochemistry 1970, 9, 2251-6. 6. Stenlid, G. Phytochemistry 1976, 15, 911-2 7. Ravanel, P.; Tissut, M.; Douce, R. Phytochemistry 1981, 20, 2101-3. 8. Ravanel, P.; Tissut, M.; Douce, R. Plant Physiol. 1982, 69, 375-8 9. Koeppe, D.E.; Miller, R.J. Plant Physiol. 1974, 54, 374-8. 10. Lilley, P. McC.; Walker, D.A. Biochim. Biophys. Acta 1974, 368, 269-78. 11. Armond, P.Α.; Arntzen, C.J.; Briantais, J.-M.; Vernotte, C. Arch. Biochem. Biophys. 1976, 175, 54-63. 12. MacKinney, G. J. Biol. Chem. 1941, 140, 315-22. 13. Walker, D.A. In "Methods in Enzymology", Vol. 69; San Pietro, Α., Ed.; Academic Press: New York, 1980; pp. 94-104. 14. Taussky, H.H.; Shorr, E. J. Biol. Chem. 1953, 202, 675-85. 15. Moreland, D.E.; Novitzky, W.P. Chem.-Biol. Interact. 1984, 48, 153-68. 16. Moreland, D.E.; Huber, S.C. Pestic. Biochem. Physiol. 1979, 11, 247-57. 17. Chance, B.; Williams, G.R. J. Biol. Chem. 1955, 217, 409-27. 18. Blackmon, W.J.; Moreland, D.E. Plant Physiol. 1971, 47, 532-6. 19. Phillips, M.L.; Williams, G.R. Plant Physiol. 1973, 51, 667-70. 20. Cavalieri, A.J.; Huang, A.H.C. Plant Physiol. 1980, 66, 588-91. 21. Izawa, S.; Good, N.E. In "Methods in Enzymology", Vol. 24; San Pietro, A. Ed.; Academic Press: New York, 1972; pp. 355-77. 22. McCarty, R.E. In "Methods in Enzymology", Vol. 69; San Pietro, A. Ed.; Academic Press: New York, 1980; pp. 719-28. 23. Izawa, S. In "Methods in Enzymology", Vol. 69; San Pietro, A. Ed.; Academic Press: New York, 1980; pp. 413-34.


23.


24. 25. 26.


27. 28. 29. 30. 31.

32.


Plant Phenolics

261

Trebst, A. In "Methods in Enzymology", Vol. 24; San Pietro, A. Ed.; Academic Press: New York, 1972; pp. 146-65. Trebst, A. In "Methods in Enzymology", Vol. 69; San Pietro, A. Ed.; Academic Press: New York, 1980; pp. 675-715. Arntzen, C.J.; Pfister, Κ., Steinback, K.E. In "Herbicide Resistance in Plants"; LeBaron, H.M.; Gressel, J., Eds.; John Wiley & Sons: New York, 1982; pp. 185-214. Brewer, P.E.; Arntzen, C.J.; Slife, F.W. Weed Sci., 27, 300-8. Paterson, D.R.; Arntzen, C.J. In "Methods in Chloroplast Molecular Biology"; Edelman, M.; Hallick, R.B.; Chua, N.-H., Eds.; Elsevier Biomedical Press: Amsterdam, 1982; pp. 109-18. Tischer, W.; Strotman, H. Biochim. Biophys. Acta 1977, 460, 113-25. Linnett, P.E.; Beechey, R.B. In "Methods in Enzymology", Vol. 55; Fleischer, S; Packer, L., Eds.; Academic Press: New York, 1979; pp. 472-518. Chappel, J.B.; Crofts, A.R. In "Regulation of Metabolic Processes in Mitochondria"; Tager, J.M.; Pappa, S.; Quagliariello, E.; Slater, E.C., Eds.; Elsevier: Amsterdam, 1966; pp. 293-316. Pressman, B.C. Annu. Rev. Biochem. 1976, 45, 501-29.

RECEIVED December 26, 1985


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