Light-Activated Pesticides - American Chemical Society

Chapter 22

Photodynamic Herbicides and Chlorophyll Biosynthesis Modulators C. A. Rebeiz, A. Montazer-Zouhoor, J. M. Mayasich, B. C. Tripathy, S. M. Wu, and C. C. Rebeiz

Downloaded by UNIV LAVAL on October 30, 2015 | http://pubs.acs.org Publication Date: May 7, 1987 | doi: 10.1021/bk-1987-0339.ch022

Laboratory of Plant Pigment Biochemistry and Photobiology, University of Illinois, Urbana, IL 61801

Higher plants have been c l a s s i f i e d into four d i f ferent greening groups depending on the monovinyl and d i v i n y l protochlorophyllide biosynthetic patterns at night and i n daylight. We have succeeded i n demon s t r a t i n g that the photodynamic h e r b i c i d a l suscepti bility of a particular plant species depends on i t s greening group and on the chemical nature of the δ-aminolevulinic acid (ALA)-dependent tetrapyrroles that accumulate as a consequence of ALA-treatment. Three groups of chemicals which modulate differen tially the monovinyl and d i v i n y l monocarboxylie chlo rophyll biosynthetic routes have now been i d e n t i f i e d namely (a) enhancers of ALA conversion to monovinyl or d i v i n y l tetrapyrroles, (b) inducers of ALA forma tion and conversion to monovinyl and d i v i n y l t e t r a pyrroles and (c) i n h i b i t o r s of d i v i n y l tetrapyrrole conversion to monovinyl tetrapyrroles. By combining ALA with member(s) of one or more of the foregoing groups o f chlorophyll biosynthesis modulators, it has become possible to design h e r b i c i d a l formulations which are very s p e c i f i c to certain crop and weed plant species under a wide range of growth conditions.

I n 1984, a n o v e l approach f o r t h e d e s i g n o f u s e f u l h e r b i c i d e s was r e p o r t e d (V). The concept and phenomenology were i l l u s t r a t e d by the d e s c r i p t i o n o f an e x p e r i m e n t a l h e r b i c i d e based on a n a t u r a l l y o c c u r r i n g amino a c i d , 6 - a m i n o l e v u l i n i c a c i d ( A L A ) . S i n c e t h e n , c o n s i d e r a b l e p r o g r e s s has been a c h i e v e d i n expanding t h e scope o f t h i s e x p e r i m e n t a l h e r b i c i d a l system, i n u n d e r s t a n d i n g i t s mode o f a c t i o n and i n i t s development i n t o a v i a b l e h e r b i c i d e . Review o f t h e E x p e r i m e n t a l Photodynamic H e r b i c i d e System P r i n c i p l e s and G u i d e l i n e s . The d i s c o v e r y o f n o v e l p e s t i c i d e s has t r a d i t i o n a l l y been t h e r e s u l t o f b l i n d s c r e e n i n g , t h a t i s t h e 0097-6156/87/0339-0295$09.50/0 © 1987 American Chemical Society

In Light-Activated Pesticides; Heitz, J., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

296

LIGHT-ACTIVATED PESTICIDES


r e s u l t o f e x p e r i m e n t a t i o n i n v o l v i n g t r i a l and e r r o r . I n a t y p i c a l year an a g r i c h e m i c a l company may b l i n d - s c r e e n 20,000 t o 50,000 c h e m i c a l s f o r h e r b i c i d a l o r i n s e c t i c i d a l a c t i v i t y . The v e r y few c h e m i c a l s t h a t e x h i b i t promise a r e then i n v e s t i g a t e d f u r t h e r and t h e i r e f f i c a c y , s e l e c t i v i t y , e n v i r o n m e n t a l impact and p h y t o t o x i c i t y a r e e v a l u a t e d . I n t h i s u n d e r t a k i n g , t h e u n d e r s t a n d i n g o f t h e mode o f a c t i o n o f a p a r t i c u l a r p e s t i c i d e i s u s u a l l y a s s i g n e d a low p r i o r i t y . Sometimes i t i s n e i t h e r i n v e s t i g a t e d n o r u n d e r s t o o d . In 1982 we s t a r t e d a r e s e a r c h e f f o r t aimed a t t h e d e s i g n o f n o v e l h e r b i c i d e s by a d o p t i n g an approach t o t a l l y d i f f e r e n t from t h e c o n v e n t i o n a l i n d u s t r i a l approach. The i d e a was t o draw on b a s i c b i o l o g i c a l knowledge i n o r d e r t o d e s i g n a h e r b i c i d e which was based on a p r e c o n c e i v e d mode o f a c t i o n . Development o f t h e Concept. The c o n c e p t u a l development o f t h e h e r b i c i d e was n a t u r a l l y i n f l u e n c e d by our past r e s e a r c h e x p e r i e n c e w i t h t h e c h e m i s t r y and b i o c h e m i s t r y o f t h e g r e e n i n g p r o c e s s . The g r e e n i n g p r o c e s s i s one o f f i v e major b i o l o g i c a l phenomena i n t h e b i o s p h e r e . The o t h e r f o u r b e i n g p h o t o s y n t h e s i s r e s p i r a t i o n , r e p r o d u c t i o n , and growth d i f f e r e n t i a t i o n and development. I t i s most o b v i o u s i n t h e s p r i n g when deciduous a n n u a l and p e r e n n i a l p l a n t s a c q u i r e t h e i r green c o l o r . T h i s v i s u a l g r e e n i n g phenomenon i s a c h e m i c a l e x p r e s s i o n o f t h e b i o s y n t h e s i s and a c c u m u l a t i o n o f c h l o r o p h y l l ( C h i ) by d e v e l o p i n g c h l o r o p l a s t s . I t i s these green organ e l l e s which a r e r e s p o n s i b l e f o r t h e c o n v e r s i o n o f s o l a r energy t o c h e m i c a l energy v i a t h e p r o c e s s o f p h o t o s y n t h e s i s . Without t h e normal o c c u r r e n c e o f t h e g r e e n i n g p r o c e s s , p h o t o s y n t h e s i s i s n o t p o s s i b l e and o r g a n i c l i f e a s we know i t , i s n o t p o s s i b l e e i t h e r . S i n c e t h e g r e e n i n g phenomenon o c c u p i e s such a c e n t r a l p o s i t i o n i n t h e economy o f t h e b i o s p h e r e , we reasoned t h a t i t s h o u l d be q u i t e p o s s i b l e t o d e s i g n a h e r b i c i d e w i t h a mode o f a c t i o n r o o t e d i n t o some f a c e t s o f t h e g r e e n i n g p r o c e s s . T h i s i n t u r n r a i s e d t h e i m p o r t a n t q u e s t i o n o f which a s p e c t s o f t h e g r e e n i n g phenomenon would b e s t l e n d i t s e l f f o r such an u n d e r t a k i n g . We f i r s t c o n s i d e r e d t h e p o s s i b i l i t y o f d e s i g n i n g a h e r b i c i d e t h a t may i n t e r f e r e w i t h t h e b i o s y n t h e s i s o f C h i . Such a h e r b i c i d e would a c t by p r e v e n t i n g t h e t r e a t e d p l a n t s from r e p l e n i s h i n g t h e C h i o f t h e f u l l y developed l e a v e s and from f o r m i n g new C h i t o accommodate t h e expan s i o n o f new l e a v e s . We opted a g a i n s t t h i s s t r a t e g y because under f i e l d c o n d i t i o n s s e e d l i n g s emerge from t h e s o i l e s s e n t i a l l y green and t h e i r r a t e o f C h i b i o s y n t h e s i s i s as slow as t h e i r r a t e o f C h i t u r n o v e r . I n o t h e r words we c o n j e c t u r e d t h a t such a h e r b i c i d e would be a very slow a c t i n g h e r b i c i d e , p a r t i c u l a r l y on weeds t h a t had a l r e a d y a t t a i n e d a c e r t a i n s i z e . Another s t r a t e g y o f f e r e d more promise. We s p e c u l a t e d t h a t i f green p l a n t s c o u l d be induced t o accumulate massive amounts o f t e t r a p y r r o l e s , i . e . o f C h i p r e c u r s o r s , by s p r a y i n g them w i t h c e r t a i n c h e m i c a l s , t h e r e i s a good chance t h a t these compounds may be developed i n t o n o n - s e l e c t i v e h e r b i c i d e s . We opted f o r t h i s ap proach f o r s e v e r a l r e a s o n s . For one, t e t r a p y r r o l e s and i n p a r t i c u l a r M g - t e t r a p y r r o l e s , a r e n o t o r i o u s t y p e I I p h o t o s e n s i t i z e r s (1-3.)• They have t h e tendency t o absorb l i g h t energy and t o p h o t o s e n s i t i z e the f o r m a t i o n o f s i n g l e t oxygen. The l a t t e r i s a v e r y p o w e r f u l o x i d a n t and c a n t r i g g e r a f r e e r a d i c a l c h a i n r e a c t i o n t h a t c a n



22.

REBEIZ ET AL.

Photodynamic Herbicides

297

d e s t r o y b i o l o g i c a l membranes, n u c l e i c a c i d s , enzymes, and many o t h e r p r o t e i n s (3.)- F u r t h e r m o r e , m e t a b o l i c M g - t e t r a p y r r o l e s are e x t r e m e l y b i o d e g r a d a b l e (4.-7) and t h e i r e n v i r o n m e n t a l impact would, t h e r e f o r e , be n e g l i g i b l e . What was not known however, was whether a green p l a n t t h a t had a c q u i r e d i t s f u l l complement o f C h i and which was b i o s y n t h e s i z i n g C h i a t r a t e s commensurate w i t h i t s slow C h i t u r n - o v e r r a t e , c o u l d be i n d u c e d , by c h e m i c a l t r e a t m e n t , t o accumulate enough t e t r a p y r r o l e s t o cause photodynamic damage. The e l u c i d a t i o n o f t h i s i s s u e i n v o l v e d the d e t e r m i n a t i o n o f very s m a l l amounts o f t e t r a p y r r o l e s i n the presence o f very l a r g e amounts of C h i . F o r t u n a t e l y , t h i s d i f f i c u l t a n a l y t i c a l problem had been t a c k l e d and s o l v e d about 10 y e a r s e a r l i e r (8-11). This i n turn made i t p o s s i b l e t o t e s t and t o demonstrate the most i m p o r t a n t premise o f the proposed h e r b i c i d e concept, namely the p o s s i b l e i n d u c t i o n o f M g - t e t r a p y r r o l e a c c u m u l a t i o n and o f photodynamic damage i n green p l a n t s by c h e m i c a l t r e a t m e n t (1_). Choice of H e r b i c i d e . I n view o f the p r e c o n c e i v e d d e s i g n , the c h o i c e of h e r b i c i d e became s t r a i g h t f o r w a r d . For y e a r s i t had been known t h a t dark-grown ( i . e . e t i o l a t e d ) p l a n t s accumulated s i g n i f i c a n t amounts o f t e t r a p y r r o l e s upon treatment w i t h ALA (12-14) ( F i g . 1). T h i s b e h a v i o u r had i t s o r i g i n i n t h r e e d i s t i n c t phenomena: (a) 6 - a m i n o l e v u l i n i c a c i d , a 5-carbon amino a c i d i s the p r e c u r s o r o f heme and C h i i n n a t u r e (1_5, 1_6^, (b) the f o r m a t i o n and a v a i l a b i l i t y o f ALA f o r heme and C h i f o r m a t i o n i s h i g h l y r e g u l a t e d by l i v i n g c e l l , (1_7, 1_8) and (c) s i n c e e t i o l a t e d p l a n t s c o n t a i n e d o n l y s m a l l amounts o f p r o t o c h l o r o p h y l l s ( P c h l s ) [ t h e immediate p r e c u r s o r s o f c h l o r o p h y l l i d e ( C h i w i t h o u t p h y t o l ) and o f C h i ] , but d i d not c o n t a i n any C h i (J_9)» the C h i b i o s y n t h e t i c pathway i n such p l a n t s was e x t r e m e l y p o t e n t ( 2 0 ) . I t was p o i s e d f o r f o r m i n g mas s i v e amounts o f C h i , s h o u l d t h e demand a r i s e upon e x p o s i n g the p l a n t s t o l i g h t (21_). Upon t r e a t m e n t o f such p l a n t s w i t h ALA, an i m p o r t a n t b i o s y n t h e t i c r e g u l a t o r y step was bypassed, namely the r e g u l a t i o n o f ALA f o r m a t i o n and a v a i l a b i l i t y t o the p l a n t ( 1 8 ) . Deluged w i t h l a r g e amounts o f ALA, the C h i b i o s y n t h e t i c machinery o f the e t i o l a t e d p l a n t s was f o r c e d t o c o n v e r t the ALA t o Mg-protop o r p h y r i n s and t o P c h l s i n d a r k n e s s and t o c o n v e r t some o f the l a t t e r t o c h l o r o p h y l l i d e s and t o C h i i n the l i g h t (20-22). As a consequence o f the above c o n s i d e r a t i o n s , and o f the known photody namic e f f e c t s o f t e t r a p y r r o l e s ( v i d e s u p r a ) , ALA appeared t o be the p e r f e c t c a n d i d a t e f o r a h e r b i c i d e . Furthermore, s i n c e ALA was a n a t u r a l amino a c i d t h a t o c c u r r e d i n a l l l i v i n g c e l l s and was an i n t e g r a l p a r t o f the food c h a i n , i t s e n v i r o n m e n t a l impact was expected t o be m i n i m a l . T h e r e f o r e , what remained t o be seen was whether mature green p l a n t s would r e a c t t o ALA t r e a t m e n t l i k e e t i o l a t e d p l a n t s and accumulate enough t e t r a p y r r o l e s t o undergo photodynamic damage. The d e m o n s t r a t i o n o f t h i s phenomenon was d e s c r i b e d i n (1_). D i s c o v e r y o f the S e l e c t i v e H e r b i c i d a l E f f e c t o f ALA. As was j u s t mentioned, the ALA - based h e r b i c i d e was meant t o be a n o n - s e l e c t i v e h e r b i c i d e . S i n c e i t a c t e d v i a the C h i b i o s y n t h e t i c pathway and s i n c e the l a t t e r was such a fundamental p r o c e s s which was be l i e v e d t o be common t o a l l green p l a n t s , we had no r e a s o n t o



298


I PROTOCHLOROPHYLL a. R = _ C H = C H ; R 2

2

=_CH ;R

3

3

(iDE)S = F.AI; D V 7 . F A I . E , P c h l

4

f

b. R 2 = _ C H _ C H 3 ; R = _ C H ; R = F . A I i 2 _ M V 7 _ F A I . E 2

3

3

c. R 2 = - C H = C H ; R = _ C H ; R 2

3

3

d. R = _ C H = C H ; R = H ; R 2

2

3

4

4

4

2

3

2

2

3

3

3

Pchl

l

= H ; D V , 7 _ C O O H I O _ C 0 M e Pchlide l

2

l

= A l k ; D V , 7 _ A l k . E , I O _ C O O H Pchlide

e. R = — C H 2 _ C H ; R = _ C H ; R f. R = _ C H _ C H ; R = H ; R

l

3

4

4

= H, 2 _ M V , 7 _ C O O H , I O _ C 0 M e , Pchlide 2

= A l k ; 2 _ M V , 7 _ A l k . E , I O - C O O H Pchlide

F i g u r e 1. S t r u c t u r e of m o n o v i n y l (MV) and d i v i n y l (DV) Mgp r o t o p o r p h y r i n monoester (MPE) and p r o t o c h l o r o p h y 1 1 i d e (Pchlide). (Reproduced w i t h p e r m i s s i o n from R e f e r e n c e 26. C o p y r i g h t 1983 N i j h o f f / D r . W. Junk P u b l i s h e r s . )



22.

I.

299


REBEIZ ET AL.

Mg PROTO diester, Mg PROTO monoester and M.g PROTO pools a. R = _ C H = C H ; R 3 = _ C H ; R = 2

2

3

b. R = _ C H _ C H i R = - . C H 2

2

3

3

F.AI;DV,7_FAI.E,6Me.P,Mg

4

P r o t o ( D V Mg Proto diester)

; R = F.AI; 2_MV, 7.FAI.E , 6 M e . P Mg Proto ( M V Mg Proto dieste

3

4

c. R = - C H = C H ; R = _ C H i R = H ; D V , 7 _ C O O H 6 M e . P . M g

Proto(DV

MgProto 6 M E )

d. R = _ C H = C H ; R = H ; R = A l k ; D V 7 _ A l k . E , 6 _ C O O H M g

Proto (DV

MgProto 7ester)

2

2

2

3

2

3

3

4

l

4

l

l

e. R = _ C H - C H ; R = _ C H ; R = H , 2 _ M V 7 _ C O O H , 6 M e . P , M g Proto(MV 2

2

3

3

f. R = _ C H _ C H ; R 2

2

3

3

2

2

h. R = _ C H _ C H ; R 2

2

2

3

l

MgProto 6 M E )

= H ; R = A l k . 2 _ M V . 7 A l k . E , 6 - C O O H , Mg P r o t o ( M V M g P r o t o

3

g. R = _ C H = C H ; R

4

4

3

7ester)

= H ; R = H ; D V M g Proto 4

= H; = R = H ; 2 _ M V M g Proto 4

Figure 1.—Continued. S t r u c t u r e o f m o n o v i n y l (MV) and d i v i n y l (DV) M g - p r o t o p o r p h y r i n monoester (MPE) and p r o t o c h l o r o p h y 1 1 i d e (Pchlide). (Reproduced w i t h p e r m i s s i o n from R e f e r e n c e 26. C o p y r i g h t 1983 N i j h o f f / D r . W. Junk P u b l i s h e r s . )


300


s u s p e c t even t h e p o s s i b i l i t y o f an ALA h e r b i c i d a l s e l e c t i v i t y . I t was, t h e r e f o r e , o u t o f s c i e n t i f i c r o u t i n e t h a t t h e h e r b i c i d a l e f f e c t o f ALA toward g r a s s y monocotyledonous p l a n t s (monocots) such as c o r n , wheat, o a t and b a r l e y was m o n i t o r e d . To our s u r p r i s e t h e t r e a t e d g r a s s y monocots were e s s e n t i a l l y u n a f f e c t e d by t h e s p r a y . T h i s prompted us t o expand t h e scope o f t h e A L A - s u s c e p t i b i l i t y s t u d i e s t p a v a r i e t y o f monocot and d i c o t y l e d o n o u s ( d i c o t ) p l a n t s . E s s e n t i a l l y , t h r e e t y p e s o f h e r b i c i d a l r e s p o n s e s , named Type I , I I and I I I , t o ALA + 2 , 2 - d i p y r i d y l (DPy), a C h i b i o s y n t h e s i s modula t o r , were noted (1_): ( a ) t y p e I r e s p o n s e was e x h i b i t e d by p l a n t s such as cucumber, which a f t e r t r e a t m e n t , d i e d v e r y r a p i d l y , (b) t y p e I I response was e x h i b i t e d by p l a n t s such as soybean which accumulated t e t r a p y r r o l e s i n t h e l e a f y t i s s u e s b u t n o t i n t h e stems and c o t y l e d o n s . Only t h e l e a v e s e x h i b i t e d photodynamic damage b u t the p l a n t s r e c o v e r e d and regrew v i g o r o u s l y , and ( c ) type I I I r e sponse was e x h i b i t e d by monocots such as c o r n , wheat, o a t and barley. A l t h o u g h t h e s p r a y e d t i s s u e s d i d accumulate s i g n i f i c a n t amounts o f t e t r a p y r r o l e s , photodynamic damage was m i n i m a l and t h e t r e a t e d s e e d l i n g s c o n t i n u e d t o grow and developed i n t o h e a l t h y plants. A l t h o u g h a t t h e t i m e , we d i d n o t u n d e r s t a n d t h e b i o c h e m i c a l o r i g i n o f t h i s d i f f e r e n t i a l response t o t h e ALA t r e a t m e n t , we i m m e d i a t e l y r e a l i z e d t h e importance o f t h i s phenomenon, and we undertook t h e t a s k o f e l u c i d a t i n g t h e m o l e c u l a r b a s i s o f t h i s unexpected p l a n t b e h a v i o u r . T h i s i n v o l v e d r e s e a r c h d e a l i n g w i t h the c h e m i c a l and b i o c h e m i c a l h e t e r o g e n e i t y o f t h e C h i b i o s y n t h e t i c pathway as w e l l as r e s e a r c h d e a l i n g w i t h d i f f e r e n c e s i n t h e greening patterns o f various higher plant species. The r e s u l t s o f t h i s r e s e a r c h e f f o r t a r e d e s c r i b e d below.


f

The

Multibranched

C h i a. B i o s y n t h e t i c Pathway

On t h e b a s i s o f emerging e x p e r i m e n t a l e v i d e n c e , we had proposed i n 1 9 8 0 , t h a t t h e C h i b i o s y n t h e t i c pathway was n o t a s i n g l e , l i n e a r c h a i n o f r e a c t i o n s t h a t l e d t o t h e f o r m a t i o n o f one C h i a and one C h i b c h e m i c a l s p e c i e s as had been commonly b e l i e v e d ( 2 3 - 2 5 ) . I n s t e a d we s u g g e s t e d t h a t t h e e x p e r i m e n t a l e v i d e n c e was more com p a t i b l e with the operation of a multibranched Chi b i o s y n t h e t i c pathway which l e d t o t h e f o r m a t i o n o f s e v e r a l C h i a c h e m i c a l species, having d i f f e r e n t functions i n photosynthesis ( 2 5 ) . This h y p o t h e s i s was l a t e r on r e i n f o r c e d and expanded (1J3, 26). At t h a t time we had no r e a s o n t o s u s p e c t t h a t v a r i o u s p l a n t s p e c i e s may d i f f e r i n their Chi biosynthetic a c t i v i t i e s u n t i l the d i f f e r e n t i a l ALA h e r b i c i d a l r e s p o n s e was o b s e r v e d . The l a t t e r c o u l d be r e a d i l y e x p l a i n e d on t h e b a s i s o f d i f f e r e n c e s i n t h e C h i b i o s y n t h e t i c pathways among v a r i o u s p l a n t s p e c i e s . The i n v e s t i g a t i o n o f t h i s i s s u e was t h e r e f o r e c a r r i e d o u t w i t h i n t h e c o n c e p t u a l framework o f the m u l t i b r a n c h e d C h i a b i o s y n t h e t i c pathway ( 2 6 , 2 7 ) , and l e d t o the d i s c o v e r y o f t h e 4 g r e e n i n g p a t t e r n s o f p l a n t s which a r e de s c r i b e d below. The m u l t i b r a n c h e d pathway r e p r o d u c e d i n F i g . 2 , c o n s i s t s o f s i x p a r a l l e l b i o s y n t h e t i c r o u t e s numbered 1 t o 6 . Most o f t h e C h i i n n a t u r e i s a c t u a l l y formed v i a r o u t e s 2 and 5 which are t h e major m o n o v i n y l (MV) and d i v i n y l (DV) m o n o c a r b o x y l i c r o u t e s o f t h a t pathway. M o n o v i n y l m o n o c a r b o x y l i c t e t r a p y r r o l e s possess


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one v i n y l and one f r e e c a r b o x y l i c group m o n o c a r b o x y l i c t e t r a p y r r o l e s possess two b o x y l i c group per m a c r o c y c l e ( F i g . 1 ) . t i o n s t h a t h e l p e d i n the f o r m u l a t i o n o f i n (28-39).

301 per m a c r o c y c l e w h i l e DV v i n y l and one f r e e c a r Some o f the key o b s e r v a t h a t pathway are d e s c r i b e d


C l a s s i f i c a t i o n o f Higher P l a n t s i n t o Four D i f f e r e n t Greening Groups As we j u s t mentioned ( v i d e s u p r a ) , t h e r e was no r e a s o n t o b e l i e v e t h a t green p l a n t s growing under n a t u r a l f i e l d c o n d i t i o n s , d i f f e r e d i n t h e i r C h i f o r m i n g pathways. Indeed, u n t i l v e r y r e c e n t l y we f i r m l y b e l i e v e d t h a t a l l green p l a n t s formed t h e i r C h i s i m u l t a n e o u s l y v i a the s i x C h i b i o s y n t h e t i c r o u t e s d e p i c t e d i n F i g . 2. This n o t i o n came under q u e s t i o n as a consequence o f two observations: (a) o f the f o r e m e n t i o n e d s p e c i e s - d e p e n d e n t d i f f e r e n t i a l ALA h e r b i c i d a l s u s c e p t i b i l i t y which c o u l d be e l e g a n t l y e x p l a i n e d by the occurrence of a d i f f e r e n t i a l Chi a b i o s y n t h e t i c heterogeneity i n v a r i o u s p l a n t s p e c i e s and (b) o f an e a r l i e r o b s e r v a t i o n t h a t e t i o l a t e d ( i . e . dark-grown) p l a n t s p e c i e s d i d indeed d i f f e r i n t h e i r P c h l i d e and C h i b i o s y n t h e t i c c a p a b i l i t i e s d u r i n g t r e a t m e n t w i t h a l t e r n a t i n g l i g h t / d a r k pulses (36). B e f o r e i n v e s t i g a t i n g the d i f f e r e n t i a l o c c u r r e n c e o f v a r i o u s C h i b i o s y n t h e t i c r o u t e s among d i f f e r e n t p l a n t s p e c i e s , i t was mandatory, however, t o develop the n e c e s s a r y a n a l y t i c a l and p r e p a r a t o r y methodology. With the development o f the a p p r o p r i a t e e x p e r i m e n t a l method o l o g y (40, 4l_) i t became p o s s i b l e t o i n v e s t i g a t e the p u t a t i v e o c c u r rence o f a d i f f e r e n t i a l C h i b i o s y n t h e t i c h e t e r o g e n e i t y i n green p l a n t s . T h i s was a c h i e v e d by s i m p l y a n a l y z i n g the MV and DV t e t r a p y r r o l e c o n t e n t o f r o u t e s 1 and 6, r o u t e s 2 + 3 and r o u t e s 4+5 ( F i g . 2) i n v a r i o u s p l a n t s p e c i e s growing under n a t u r a l p h o t o p e r i o d i c growth c o n d i t i o n s . I t was c o n s i d e r e d t h a t the amount o f a s p e c i f i c MV or DV t e t r a p y r r o l e b e l o n g i n g t o a s p e c i f i c MV or DV b i o s y n t h e t i c r o u t e and which was d e t e c t a b l e a t any p a r t i c u l a r t i m e , was r e l a t e d t o the f l o w o f t e t r a p y r r o l e i n t e r m e d i a t e s v i a t h a t b i o s y n t h e t i c r o u t e a t t h a t p a r t i c u l a r t i m e . Because o f the c y c l i c a l t e r n a t i o n o f n i g h t ( d a r k n e s s ) and l i g h t ( d a y l i g h t ) i n n a t u r e , the MV and DV t e t r a p y r r o l e c o n t e n t o f the v a r i o u s p l a n t s p e c i e s was a n a l y z e d at two s t a g e s o f the p h o t o p e r i o d : (a) a t the end o f the dark phase o f the p h o t o p e r i o d and (b) i n the m i d d l e o f the l i g h t phase o f the p h o t o p e r i o d . The a n a l y s i s a t the end o f the dark phase o f the p h o t o p e r i o d was meant t o r e f l e c t the a c t i v i t y o f the b i o s y n t h e t i c r o u t e s at n i g h t , w h i l e a n a l y s i s i n the m i d d l e o f the day was meant t o r e f l e c t the a c t i v i t y o f the b i o s y n t h e t i c r o u t e s i n d a y l i g h t . I t was c o n j e c t u r e d t h a t s h o u l d d i f f e r e n c e s be observed among v a r i o u s p l a n t s p e c i e s w i t h r e s p e c t t o any two b i o s y n t h e t i c r o u t e s , as f o r example between the MV and DV r o u t e s i n d a r k n e s s (D) i . e . at n i g h t o r i n the l i g h t ( L ) , i . e . i n d a y l i g h t , f o u r meaning f u l b i o s y n t h e t i c c o m b i n a t i o n s may be o b s e r v e d , namely (a) dark d i v i n y l / l i g h t d i v i n y l (DDV/LDV), (b) DMV/LDV, ( c ) DDV/LMV and (d) DMV/LMV. I n the c o u r s e o f our i n v e s t i g a t i o n s a l l f o u r DV-MV P c h l i d e c o m b i n a t i o n s were observed ( v i d e i n f r a ) .



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F i g u r e 2. S i x - b r a n c h e d Chla b i o s y n t h e t i c pathway: DV, d i v i n y l ; MV, m o n o v i n y l ; FA1, f a t t y a l c o h o l ; Phy, p h y t o l ; E, e s t e r ; ME, methyl e s t e r ; A l k , a l k y l group of unknown c h a i n l e n g t h ; Me, m e t h y l ; ALA, 6 - a m i n o l e v u l i n i c a c i d ; PBG, porpho b i l i n o g e n ; Urogen, u r o p o r p h y r i n o g e n ; Coprogen, c o p r o p o r p h y r i n o g e n ; Protogen, p r o t o p o r p h y r i n o g e n ; P r o t o , p r o t o p o r p h y r i n IX; LWMP, longer wavelength m e t a l l o p o r p h y r i n s ( t h e p u t a t i v e i n t e r m e d i a t e s o f r i n g E f o r m a t i o n ) ; P, e s t e r i f i c a t i o n w i t h g e r a n y l g e r a n i o l f o l l o w e d by stepwise c o n v e r s i o n o f the l a t t e r t o p h y t o l ; M, m e t h y l a t i o n . (Reproduced w i t h p e r m i s s i o n from Reference 26. C o p y r i g h t 1983 N i j h o f f / D r . W. Junk P u b l i s h e r s . )

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The DDV/LDV G r e e n i n g Group. P l a n t s p e c i e s such as cucumber (Cucumis s a t i v u s L . ) , common p u r s l a n e ( P o r t u l a c a o l e r a c e a ) and mustard ( B r a s s i c a Juncea L. and B r a s s i c a kaber) b e l o n g i n t h i s group ( 3 8 ) . D u r i n g t h e dark phase o f a 10 h dark/14 h l i g h t phot o p e r i o d , t h e s e p l a n t s accumulate m a i n l y DV p r o t o c h l o r o p h y l l i d e ( P c h l i d e ) and s m a l l e r amounts o f MV P c h l i d e (42, 4 3 ) . At daybreak, C h i f o r m a t i o n proceeds v i a t h e DV-enriched P c h l i d e p o o l ( 4 2 ) . L a t e r on d u r i n g t h e day, t h e p r o p o r t i o n o f MV P c h l i d e drops do a v e r y low l e v e l and C h i f o r m a t i o n proceeds m a i n l y v i a t h e DV-en r i c h e d Pchlide pool (42). The DMV/LDV G r e e n i n g Group. T h i s group appears t o be t h e l a r g e s t g r e e n i n g group o f h i g h e r p l a n t s and i n c l u d e monocots, such as c o r n (Zea mays L.) wheat ( T r l t i c u m s e c a l e L.) and b a r l e y (Hordeum v u l g a r e ) and d i c o t s such as t h e common bean ( P h a s e o l u s v u l g a r i s L . ) , soybean ( G l y c i n e max L.) and pigweed (Amaranthus r e t r o f l e x u s L.) ( 3 8 ) . At t h e b e g i n n i n g o f t h e dark phase o f t h e p h o t o p e r i o d , t h e s e p l a n t s s h i f t very r a p i d l y from t h e DV P c h l i d e b i o s y n t h e t i c p a t t e r n (which p r e v a i l s i n d a y l i g h t ) t o a MV P c h l i d e b i o s y n t h e t i c p a t t e r n . During t h e n i g h t they accumulate m a i n l y MV P c h l i d e and very s m a l l amounts o f DV P c h l i d e (42, 43.). At daybreak, C h i forma t i o n proceeds v i a t h e MV e n r i c h e d P c h l i d e p o o l . Under n a t u r a l day l i g h t , t h e p l a n t s s h i f t back t o a DV P c h l i d e a c c u m u l a t i o n p a t t e r n and form C h i m a i n l y v i a t h e DV-enriched P c h l i d e p o o l (42, 4 3 ) . The DDV/LMV G r e e n i n g Group. T h i s r e c e n t l y d i s c o v e r e d g r e e n i n g group was f i r s t d e s c r i b e d i n 1986 (38) and (43) and so f a r i n c l u d e s fewer p l a n t s p e c i e s than t h e o t h e r t h r e e g r e e n i n g groups. I t s members i n c l u d e g i n k g o (Ginkgo b i l o b a ) and v i o l e t s p e c i e s ( V i o l a s p e c i e s ) . D u r i n g t h e dark phase o f t h e p h o t o p e r i o d , these p l a n t s accumulate m a i n l y DV P c h l i d e and s m a l l e r amounts o f MV P c h l i d e . At daybreak, they form C h i m a i n l y v i a t h e DV-enriched P c h l i d e p o o l and l a t e r on i n d a y l i g h t form C h i v i a t h e MV-enriched P c h l i d e p o o l . The DMV/LMV Greening Group. L i k e w i s e t h i s g r e e n i n g group was a l s o r e c e n t l y d e s c r i b e d (38, ^ 3 ) . I t i n c l u d e s p l a n t s p e c i e s such as a p p l e (Pyrus malus) and Johnson g r a s s (Sorghum h a l e p e n s e ) . During the dark phase o f t h e p h o t o p e r i o d t h e s e p l a n t s accumulate predomi n a n t l y MV P c h l i d e and s m a l l e r amounts o f DV P c h l i d e . At daybreak and l a t e r on d u r i n g d a y l i g h t they form C h i m a i n l y v i a t h e MVe n r i c h e d P c h l i d e p o o l (38, 4 3 ) . Molecular

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S i n c e we s t r o n g l y s u s p e c t e d t h a t t h e d i f f e r e n t i a l ALA-dependent photodynamic s u s c e p t i b i l i t y o f green p l a n t s was c l o s e l y t i e d t o t h e biochemical o r i g i n o f the d i f f e r e n t i a l greening patterns o f higher p l a n t s , t h i s r e l a t i o n s h i p was n e x t i n v e s t i g a t e d . B i o s y n t h e t i c O r i g i n o f t h e DV and MV P c h l i d e A c c u m u l a t i o n P a t t e r n s i n t h e DDV/LDV Greening Group o f P l a n t s . The o r i g i n o f t h e DV P c h l i d e a c c u m u l a t i o n p a t t e r n i n t h i s g r e e n i n g group was r e a d i l y demonstrated w i t h t h e use o f t h e DDV/LDV cucumber c e l l - f r e e system



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d e s c r i b e d i n (4J_). I t was shown t o o r i g i n a t e i n b i o s y n t h e t i c routes 2 + 3 ( F i g . 2). T h i s was a c h i e v e d by d e m o n s t r a t i n g t h e c o n v e r s i o n o f ALA t o DV P c h l i d e v i a DV p r o t o p o r p h y r i n ( P r o t o ) , DV Mg-Proto and DV Mg-Proto monoester i n d a r k n e s s (38., 39). A more i m p o r t a n t i s s u e , however, was whether t h e MV P c h l i d e a c c u m u l a t i o n p a t t e r n o r i g i n a t e d i n t h e c o n v e r s i o n o f DV P c h l i d e t o MV P c h l i d e o r whether I t o r i g i n a t e d somewhere e l s e . T h i s q u e s t i o n was s e t t l e d by d e m o n s t r a t i n g t h e b i o s y n t h e s i s o f MV P c h l i d e v i a t h e MV m o n o c a r b o x y l i c r o u t e s 4 + 5 ( F i g . 2). I t was shown t h a t a l though t h e cucumber e t i o c h l o r o p l a s t s system was n o t c a p a b l e o f c o n v e r t i n g DV P c h l i d e t o MV P c h l i d e , i t d i d c o n v e r t , v e r y e f f i c i e n t l y ALA t o MV P c h l i d e v i a MV P r o t o , MV Mg-Proto and MV Mg-Proto monoester (38, 39). F u r t h e r m o r e , i t was shown t h a t i n t h i s system, DV r o u t e s 2 + 3 and MV r o u t e s 4 + 5 ( F i g . 2) were ( a ) e i t h e r n o t i n t e r c o n n e c t e d , i . e . a t t h e l e v e l o f DV P c h l i d e , o r (b) were very weakly i n t e r c o n n e c t e d a t s i t e ( s ) between DV P r o t o and DV P c h l i d e (38, 39). The r e g u l a t i o n o f t h e DV and MV m o n o c a r b o x y l i c r o u t e s i n t h i s g r e e n i n g group o f p l a n t s i s p r e s e n t l y under i n v e s t i g a t i o n . B i o s y n t h e t i c O r i g i n o f t h e DV and MV P c h l i d e A c c u m u l a t i o n P a t t e r n s i n t h e DMV/LDV Greening Group. The o r i g i n o f t h e DV P c h l i d e accumu l a t i o n p a t t e r n i n t h i s g r e e n i n g group was i n v e s t i g a t e d w i t h t h e DMV/LDV b a r l e y c e l l - f r e e system d e s c r i b e d i n (41). The o r i g i n o f the DV P c h l i d e a c c u m u l a t i o n p a t t e r n was shown t o r e s i d e i n b i o s y n t h e t i c routes 2 + 3 ( F i g . 2) by d e m o n s t r a t i n g t h e c o n v e r s i o n o f ALA t o DV P c h l i d e v i a DV P r o t o , DV Mg-Proto and DV Mg-Proto monoester (38, 39). The o r i g i n o f t h e MV P c h l i d e a c c u m u l a t i o n p a t t e r n was however c o n s i d e r a b l y more complex than i n DDV/LDV p l a n t s . About 30% o f t h e MV P c h l i d e appeared t o be formed from ALA v i a t h e MV m o n o c a r b o x y l i c routes, i . e . routes 4 + 5 ( F i g . 2). T h i s was e v i d e n c e d by t h e d a r k - c o n v e r s i o n o f ALA t o MV P c h l i d e v i a MV P r o t o , MV Mg-Proto and MV Mg-Proto monoester i n b a r l e y e t i o c h l o r o p l a s t s p o i s e d i n t h e MV P c h l i d e a c c u m u l a t i o n mode (38, 39). A s i z a b l e f r a c t i o n o f t h e MV P c h l i d e p o o l appeared t o be a l s o formed from DV P r o t o , DV Mg P r o t o and DV Mg P r o t o monoester b u t n o t from DV P c h l i d e (38, 39). This was a p p a r e n t l y a c c o m p l i s h e d v i a one o r more DV t e t r a p y r r o l e r e d u c t a s e (s) t h a t c o n v e r t e d DV t e t r a p y r r o l e s t o MV t e t r a p y r r o l e s by r e d u c t i o n o f t h e v i n y l group a t p o s i t i o n 4 o f t h e m a c r o c y c l e t o an e t h y l group ( F i g . 1). As a consequence, i n t h i s g r e e n i n g group o f p l a n t s , t h e DV and MV m o n o c a r b o x y l i c b i o s y n t h e t i c r o u t e s were very s t r o n g l y i n t e r c o n n e c t e d (38, 39). The p r e c i s e number and b i o c h e m i c a l s i t e ( s ) o f t h e DV t e t r a p y r r o l e r e d u c t a s e s i s p r e s e n t l y under i n v e s t i g a t i o n . Very r e c e n t d a t a a l s o i n d i c a t e s t h a t i n DMV/LDV p l a n t s , under c e r t a i n g r e e n i n g c o n d i t i o n s , a s m a l l f r a c t i o n o f t h e DV P c h l i d e p o o l may be c o n v e r t i b l e t o MV P c h l i d e v i a a DV P c h l i d e r e d u c t a s e (B. C. T r i p a t h y and C. A. R e b e i z , u n p u b l i s h e d ) . I n v e s t i g a t i o n o f t h e r e g u l a t i o n o f t h e MV and DV monocarboxy l i c r o u t e s i n DMV/LDV p l a n t s i s i n p r o g r e s s . L i k e w i s e , t h e b i o s y n t h e t i c o r i g i n o f t h e DV and MV P c h l i d e a c c u m u l a t i o n p a t t e r n s i n t h e o t h e r two g r e e n i n g groups o f p l a n t s , i . e . i n t h e DDV/LMV and t h e DMV/LMV group i s a l s o under i n v e s t i g a t i o n .


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B i o c h e m i c a l O r i g i n o f t h e D i f f e r e n t i a l ALA-dependent Photodynamic S u s c e p t i b i l i t y o f Green P l a n t s The m o l e c u l a r b a s i s o f t h e d i f f e r e n t i a l photodynamic s u s c e p t i b i l i t y o f v a r i o u s p l a n t t i s s u e s and p l a n t s p e c i e s t o ALA t r e a t m e n t was i n v e s t i g a t e d w i t h i n t h e framework o f t h e f o l l o w i n g h y p o t h e s i s : (a) t h a t t h e a c c u m u l a t i o n o f t e t r a p y r r o l e s by A L A - t r e a t e d t i s s u e s was a necessary but not a s u f f i c i e n t c o n d i t i o n f o r the occurrence o f photodynamic damage and (b) t h a t i n t h e event o f t e t r a p y r r o l e a c c u m u l a t i o n t h e o c c u r r e n c e and e x t e n t o f photodynamic damage was l i k e l y t o depend (a) on t h e e x t e n t o f t e t r a p y r r o l e a c c u m u l a t i o n , (B) on t h e g r e e n i n g group o f t h e t r e a t e d p l a n t and (Y) on t h e chemi c a l n a t u r e o f t h e accumulated t e t r a p y r r o l e . I t was a l s o r e c o g n i z e d t h a t t h e e x t e n t o f a p l a n t s p e c i e s photodynamic s u s c e p t i b i l i t y t o ALA t r e a t m e n t may be due t o one o r more o f t h e f o r e m e n t i o n e d c o n d i t i o n s . The l o g i s t i c s behind t h e above h y p o t h e s i s was based upon the f o l l o w i n g o b s e r v a t i o n s . The proposed n e c e s s i t y o f t e t r a p y r r o l e a c c u m u l a t i o n f o r t h e o c c u r r e n c e o f photodynamic damage i s a consequence o f t h e b a s i c mode o f a c t i o n o f ALA toward s u s c e p t i b l e p l a n t s p e c i e s . Indeed t h e r e l a t i o n s h i p between ALA t r e a t m e n t , t o t a l t e t r a p y r r o l e a c c u m u l a t i o n and photodynamic damage has a l r e a d y been demonstrated w i t h s u s c e p t i b l e p l a n t s p e c i e s such as cucumber (1_). On t h e o t h e r hand, t h e p r o p o s a l o f t h e " n o n - s u f f i c i e n c y " c o n d i t i o n was on t h e b a s i s t h a t a l t h o u g h some t r e a t e d p l a n t s p e c i e s accumulated l a r g e amounts o f t e t r a p y r r o l e s , they d i d not undergo s i g n i f i c a n t photodynamic damage (I). I n s u s c e p t i b l e p l a n t s t h a t responded t o ALA t r e a t m e n t by a c c u m u l a t i n g t e t r a p y r r o l e s , t h e proposed dependence o f photodynamic damage on t h e e x t e n t o f t e t r a p y r r o l e a c c u m u l a t i o n i s a g a i n an o b v i o u s consequence o f t h e demonstrated dependence o f photodynamic damage on t o t a l t e t r a p y r r o l e a c c u m u l a t i o n (1_). F i n a l l y i n p l a n t s p e c i e s c a p a b l e o f ALA-dependent t e t r a p y r r o l e a c c u m u l a t i o n , t h e proposed dependence o f photodynamic damage upon the g r e e n i n g group o f t h e t r e a t e d p l a n t as w e l l as upon t h e chemi c a l n a t u r e o f t h e accumulated t e t r a p y r r o l e was based on e x p e r i m e n t a l e v i d e n c e t h a t w i l l be d e s c r i b e d below. Dependence o f Photodynamic Damage on t h e E x t e n t o f T e t r a p y r r o l e A c c u m u l a t i o n : Case Study o f t h e D i f f e r e n t i a l Photodynamic S u s c e p t i b i l i t y o f Soybean C o t y l e d o n s and P r i m a r y Leaves t o ALA Treatment. T h i s case s t u d y e x p l o r e s t h e m o l e c u l a r b a s i s o f t h e d i f f e r e n t i a l photodynamic s u s c e p t i b i l i t y o f soybean c o t y l e d o n s and soybean p r i m a r y l e a v e s t o ALA-treatment. As may be r e c a l l e d , a l t h o u g h t h e p r i m a r y l e a v e s o f soybean s e e d l i n g s a r e v e r y s u s c e p t i b l e t o ALA t r e a t m e n t , soybean stems and c o t y l e d o n s a r e not (1_). As a conse quence, a l t h o u g h t h e p r i m a r y l e a v e s o f A L A - t r e a t e d s e e d l i n g s d i e w i t h i n a few hours o f exposure t o d a y l i g h t , t h e i n t a c t stems and c o t y l e d o n s s u s t a i n t h e p r o d u c t i o n o f new l e a v e s and t h e t r e a t e d s e e d l i n g s soon r e c o v e r . The r e s i s t a n c e o f soybean stems t o ALA t r e a t m e n t i s o b v i o u s l y r e l a t e d t o t h e l a c k o f t e t r a p y r r o l e a c c u m u l a t i o n by t h e t r e a t e d stems as d e s c r i b e d i n (1_). I n o r d e r t o determine whether t h e response o f soybean c o t y l e d o n s , a DMV/LDV t i s s u e ( 4 2 ) , t o ALA



22.

REBEIZ ET AL.

Photodynamic

Herbicides

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t r e a t m e n t was a l s o r o o t e d i n a l a c k o f t e t r a p y r r o l e a c c u m u l a t i o n , the f o l l o w i n g experiment was performed. Greenhouse grown soybean s e e d l i n g s were t r e a t e d w i t h a 5 mM ALA + 15 mM DPy s o l u t i o n p r e c i s e l y as d e s c r i b e d i n (1_). A f t e r wrapping t h e p l a n t s i n aluminum f o i l and dark i n c u b a t i o n f o r 17 h (1_), t e t r a p y r r o l e a c c u m u l a t i o n by the p r i m a r y l e a v e s and by t h e c o t y l e d o n s was determined and the s e e d l i n g s were exposed t o d a y l i g h t i n t h e greenhouse t o induce photodynamic damage. I n t h i s experiment t o t a l ALA-dependent t e t r a p y r r o l e a c c u m u l a t i o n by t h e p r i m a r y l e a v e s amounted t o 201 nmoles per 100 mg o f t i s s u e p r o t e i n w h i l e t h e c o t y l e d o n s accumulated o n l y 11 nmoles o f t e t r a p y r r o l e s per 100 mg p r o t e i n . A f t e r a few hours i n d a y l i g h t , photodynamic damage t o t h e l e a v e s amounted t o 100$ w h i l e t h e c o t y l e d o n s were u n a f f e c t e d . A l t o g e t h e r , t h e s e r e s u l t s i n d i c a t e d t h a t t h e l a c k o f photo dynamic damage t o soybean c o t y l e d o n s was due t o poor exogenous ALA-dependent t e t r a p y r r o l e a c c u m u l a t i o n by t h i s t i s s u e . Dependence o f Photodynamic Damage on t h e Chemical Nature o f t h e Accumulated T e t r a p y r r o l e and on t h e Greening Group o f t h e Treated P l a n t s . I n t h e s e p r e l i m i n a r y s t u d i e s , o n l y t h r e e model p l a n t systems have been used: ( a ) cucumber s e e d l i n g s , i n t h e c o t y l e d o n s t a g e as a r e p r e s e n t a t i v e o f t h e DDV/LDV group o f p l a n t s and (b) c o r n , and t o a l e s s e r e x t e n t soybean s e e d l i n g s , as monocot and d i c o t r e p r e s e n t a t i v e s o f t h e DMV/LDV g r e e n i n g group. The t e n t a t i v e c o n c l u s i o n s drawn from t h e s e s t u d i e s a r e , t h e r e f o r e , l i m i t e d i n scope and i n t h e f u t u r e may have t o be a d j u s t e d t o accpmmodate a d d i t i o n a l o b s e r v a t i o n s d e r i v e d from A L A - s u s c e p t i b i l i t y s t u d i e s w i t h DDV/LMV, DMV/LMV as w e l l as from a d d i t i o n a l DDV/LDV and DMV/LDV p l a n t s p e c i e s . In order t o c o r r e l a t e the accumulation o f s p e c i f i c t e t r a p y r r o l e s w i t h i n d u c t i o n o f photodynamic damage, we have used a group o f 13 c h e m i c a l s which a c t I n c o n c e r t w i t h ALA. The mode o f a c t i o n o f t h e s e c h e m i c a l s , which w i l l be r e f e r r e d t o as "modula t o r s " o f C h i b i o s y n t h e s i s , w i l l be d i s c u s s e d i n some d e t a i l s l a t e r on ( v i d e i n f r a ) . They were a c o n v e n i e n t t o o l i n d e m o n s t r a t i n g r e l a t i o n s h i p s between t h e a c c u m u l a t i o n o f s p e c i f i c t e t r a p y r r o l e s and photodynamic damage. Indeed, when used i n c o n c e r t w i t h ALA, they r e s u l t e d i n t h e preponderant a c c u m u l a t i o n o f s p e c i f i c MV o r DV t e t r a p y r r o l e s as d e s c r i b e d below. I n t h e s e e x p e r i m e n t s we used low c o n c e n t r a t i o n s o f ALA (5 mM) i n c o n j u n c t i o n w i t h h i g h e r c o n c e n t r a t i o n s (10 t o 30 mM) o f each one o f t h e 13 C h i b i o s y n t h e s i s m o d u l a t o r s . The i d e a was t o induce o n l y l i m i t e d photodynamic damage i n order t o c o r r e l a t e more p r e c i s e l y between t h e e x t e n t o f t h e l a t t e r and t h e a c c u m u l a t i o n o f s p e c i f i c t e t r a p y r r o l e s . The r e s u l t s o f t h e s e e x p e r i m e n t s a r e summarized below. Case Study 1: I n d u c t i o n o f Photodynamic Damage by ALA-dependent A c c u m u l a t i o n o f MV P c h l i d e i n Cucumber, a DDV/LDV P l a n t S p e c i e s b u t not i n Corn a DDMV/LDV P l a n t S p e c i e s . I n seven o f t h e t h i r t e e n d i f f e r e n t t r e a t m e n t s which used ALA i n c o n j u n c t i o n w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f i n d i v i d u a l members o f t h e 13 C h i b i o s y n t h e s i s m o d u l a t o r s , MV P c h l i d e was t h e preponderant t e t r a p y r r o l e t h a t accumulated i n t h e dark i n t h e t r e a t e d cucumber s e e d l i n g s . I n



308


e v e r y one o f t h o s e 7 t r e a t m e n t s t h e b e s t c o r r e l a t i o n was observed between MV P c h l i d e d a r k - a c c u m u l a t i o n and photodynamic damage. One such experiment i s d e s c r i b e d i n Table IA. These r e s u l t s i n d i c a t e d t h a t cucumber a DDV/LDV p l a n t s p e c i e s was p h o t o d y n a m i c a l l y s u s c e p t i b l e t o ALA-dependent dark a c c u m u l a t i o n o f MV P c h l i d e . I n o r d e r t o determine whether t h i s c o n c l u s i o n was a l s o v a l i d f o r DMV/LDV p l a n t s p e c i e s , s i m i l a r experiments were performed on c o r n . I n a l l t h e s e e x p e r i m e n t s , t h e ALA-dependent dark-accumula t i o n o f MV P c h l i d e r e s u l t e d e i t h e r i n t h e absence o f photodynamic damage, as d e s c r i b e d i n Table I B o r i n very minor damage from which the s e e d l i n g s r e c o v e r e d v e r y r a p i d l y a s r e p o r t e d i n (1_). As a consequence o f t h e s e r e s u l t s we propose t h a t w h i l e DDV/LDV p l a n t s p e c i e s such as cucumber a r e p h o t o d y n a m i c a l l y s u s c e p t i b l e t o ALA-dependent MV P c h l i d e dark a c c u m u l a t i o n , DMV/LDV p l a n t s p e c i e s such as c o r n a r e e i t h e r n o t s u s c e p t i b l e o r much l e s s p h o t o d y n a m i c a l l y s u s c e p t i b l e than t h e DDV/LDV p l a n t s p e c i e s . T h i s h y p o t h e s i s i s p r e s e n t l y b e i n g t e s t e d on a l a r g e r number o f p l a n t s p e c i e s b e l o n g i n g t o t h e two forementioned g r e e n i n g groups. Case Study 2: I n d u c t i o n o f ALA-dependent DV P c h l i d e Dark Accumula t i o n Cause Less Photodynamic Damage i n Cucumber a DDV/LDV P l a n t S p e c i e s than i n Soybean, a DMV/LDV P l a n t S p e c i e s . Four o f t h e t h i r t e e n C h i b i o s y n t h e s i s modulators r e s u l t e d i n t h e dark-accumula t i o n o f more DV P c h l i d e than MV P c h l i d e i n p a r t i c u l a r a t t h e h i g h e r c o n c e n t r a t i o n range (20 and 30 mM) o f t h e m o d u l a t o r s . I n these e x p e r i m e n t s , a l t h o u g h t h e i n c i d e n c e o f photodynamic damage d i d c o r r e l a t e w i t h ALA-induced DV P c h l i d e dark a c c u m u l a t i o n ( T a b l e I C ) , the e x t e n t o f photodynamic damage was u s u a l l y l e s s pronounced than when cucumber was f o r c e d t o accumulate MV P c h l i d e ( T a b l e I I ) . I t i s n o t known a t t h i s s t a g e whether t h e reduced photodynamic damage induced by modulators t h a t cause t h e preponderant d a r k - a c c u m u l a t i o n o f DV P c h l i d e i n cucumber i s due t o a lower photodynamic s u s c e p t i b i l i t y o f DDV/LDV p l a n t s p e c i e s per u n i t o f accumulated DV P c h l i d e or t o some o t h e r c a u s e s . I n o r d e r t o determine whether DMV/LDV p l a n t s p e c i e s e x h i b i t a h i g h e r photodynamic s u s c e p t i b i l i t y t o DV P c h l i d e d a r k - a c c u m u l a t i o n than DDV/LDV p l a n t s p e c i e s , s i m i l a r experiments a r e now b e i n g performed on soybean s e e d l i n g s i n t h e p r i m a r y l e a f s t a g e . P r e l i m i n a r y r e s u l t s have so f a r i n d i c a t e d t h a t primary l e a v e s o f soybean a r e e x t r e m e l y s u s c e p t i b l e t o ALA-based t r e a t m e n t s t h a t do r e s u l t i n DV P c h l i d e a c c u m u l a t i o n i n cucumber. At t h i s s t a g e we have no r e a s o n t o doubt t h e c o r r e l a t i o n o f t h i s photodynamic s u s c e p t i b i l i t y w i t h DV P c h l i d e a c c u m u l a t i o n by t h e soybean p r i m a r y leaves. Case Study 3: I n d u c t i o n o f Photodynamic Damage by ALA-Dependent DV M g - P r o t o p o r p h y r i n (Monoester) Accumulation i n Both Cucumber and Corn. Two o f t h e 13 C h i b i o s y n t h e s i s modulators caused t h e massive ALA-dependent a c c u m u l a t i o n o f DV M g - p r o t o p o r p h y r i n (monoester) [MP(E)] i n cucumber, a DDV/LDV p l a n t s p e c i e s , and i n c o r n , a DMV/LDV p l a n t s p e c i e s . I n b o t h s p e c i e s i t i s t h e a c c u m u l a t i o n o f DV MP(E) t h a t e x h i b i t e d t h e b e s t c o r r e l a t i o n w i t h photodynamic damage ( T a b l e I , D, E ) . Corn, however, r e c o v e r e d a f t e r a few days



Experiment

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Plant Species

0

Solvent only 5 mM ALA 10 mM 2 - p y r i d i n e a l d o x i m e 5 mM ALA + 10 mM 2 - p y r i d i n e a l d o x i m e 20 mM 2 - p y r i d i n e aldoxime 5 mM ALA + 20 mM 2 - p y r i d i n e a l d o x i m e 30 mM 2 - p y r i d i n e a l d o x i m e 5 mM ALA + 30 mM 2 - p y r i d i n e a l d o x i m e Correlation coefficient Level of s i g n i f i c a n c e

Treatment

0 43 0 83 0 83 0 68

(S)

Photodynamic Damage

DV

MV

3

DV

MP(E)

0.00 51.38 4.55 44.64 12.16 35.69 17.60 65.08 0.817 5%

0.00 0.38 0.13 0.77 -0.50b 0.44 0.16 0.32

0.00 -0.60 -0.84 -0.52 -0.34 0.08 0.43 -0.20

Continued on next page

0.00 17.81 3.08 4.96 1.94 8.12 3.34 17.62 0.569 n.s.

Exogenous ALA-dependent tetrapyrrole accumulation i n nmoles p e r 100 mg p r o t e i n s

MV

Pchlide

S e e d l i n g s were sprayed i n t h e l a t e a f t e r n o o n w i t h s o l v e n t o n l y o r w i t h s o l v e n t c o n t a i n i n g 5 mM ALA (130 g/acre) and a modulator (10 t o 30 mM) a t a r a t e o f 40 g a l l o n s p e r a c r e : The s o l v e n t c o n s i s t e d o f a c e t o n e : e t h y l a c e t a t e : tween 80: H2O (45:45:1:90 v / v / v / v ) . The sprayed p l a n t s were wrapped i n aluminum f o i l and p l a c e d i n d a r k n e s s a t 28°C f o r about 17 h. The next morning, t h e t r e a t e d p l a n t s were sampled f o r t e t r a p y r r o l e a n a l y s i s t h e n were exposed t o d a y l i g h t i n t h e greenhouse f o r t h e e v a l u a t i o n o f photodynamic damage. For more d e t a i l s c o n s u l t ( 1 ) . P c h l i d e = p r o t o c h l o r o p h y l l i d e ; MP(E) - Mg p r o t o + Mg p r o t o monoester; MV = m o n o v i n y l ; DV = d i v i n y l ; n.s. = n o t s i g n i f i c a n t . Adapted from ( 2 7 ) .

Table I . D i f f e r e n c e s i n ALA-Dependent T e t r a p y r r o l e A c c u m u l a t i o n and Photodynamic Damage Between DDV/LDV and DMV/LDV P l a n t S p e c i e s



Experiment

Corn

Plant Species

Solvent only 5 mM ALA 10 mM p i c o l i n i c a c i d 5 mM ALA + 10 mM p i c o l i n i c 20 mM p i c o l i n i c a c i d 5 mM ALA + 20 mM p i c o l i n i c 30 mM p i c o l i n i c a c i d 5 mM ALA + 30 MM p i c o l i n i c Correlation coefficient Level o f s i g n i f i c a n c e

Treatment

acid

acid

acid

0 0 0 0 0 0 0 0

(%)

DV

MV

0.00 17.36 39.86 16.03 49.93 10.24 58.49 81.29 0.000 n.s.

0.00 -2.30 -2.73 -1.83 15.79 -1.69 0;20 4.10 0.000 n.s.

0.00 -5.07 -0.06 -1.13 8.14 5.07 2.52 0.04 0.000 n.s.

0.00 -1.13 0.85 1.64 8.12 17.01 20.24 20.67 0.000 n.s.

3

DV

MP(E)

Exogenous ALA-dependent tetrapyrrole accumulation i n nmoles per 100 mg p r o t e i n s

MV

Pchlide

A c c u m u l a t i o n and Photodynamic

Photodynamic Damage

Table I . C o n t i n u e d . D i f f e r e n c e s i n ALA-Dependent T e t r a p y r r o l e Damage Between DDV/LDV and DMV/LDV P l a n t S p e c i e s


n S m

H

c/3

m

no

O

1 m

r 0 3C •7*

o


Cucumber

Cucumber

l

1

d

0.00 -0.27 -0.41 8.52

0.00 12.20 40.45 98.32 0 0 0 30

0.00 -2.60 -1.85 39.04

0.00 0.18 48.47 75.20 56.77 85.38 45.92 44.43 0.861 n

0.00 0.18 -0.58 -0.22 1.48 0.41 0.13 0.92

C o n t i n u e d on next pag

0.00 9.52 1.53 20.01

0.00 -0.04 4.44 -0.11 -0.11 1.12 0.95 6.03 0.364 n.s.

0.00 3.15 9.09 23.38 8.78 32.69 7.92 14.12 0.623 20% 0.00 15.48 25.25 34.35 12.93 33.16 10.20 8.08 0.413 n.s.

0 10 73 90 93 93 100 100

Solvent only 5 mM ALA 10 mM 1 , 1 0 - p h e n a n t h r o l i n e 5 mM ALA + 10 mM 1 , 1 0 - p h e n a n t h r o l i n e 20 mM 1 , 1 0 - p h e n a n t h r o l i n e 5 mM ALA + 20 mM 1 , 1 0 - p h e n a n t h r o l i n e 30 mM 1 , 1 0 - p h e n a n t h r o l i n e 5 mM ALA + 30 mM 1 , 1 0 - p h e n a n t h r o l i n e Correlation coefficient Level of s i g n i f i c a n c e

Solvent only 5 mM ALA 10 mM 2 , 2 - d i p y r i d y l 5 mM ALA + 10 mM 2 , 2 - d i p y r i d y l

0.00 0.33 1.44 0.45 -0.34 -0.16 0.36 1.88

0.00 6.41 6.94 8.45 4.26 9.37 8.72 15.30 0.753 5%

0.00 30.62 10.50 11.07 1.49 7.32 2.52 1.73 0.29 n.s.

0 55 5 45 33 50 43 63

Solvent only 5 mM ALA 10 mM 1 , 7 - p h e n a n t h r o l i n e 5 mM ALA + 10 mM 1 , 7 - p h e n a n t h r o l i n e 20 mM 1 , 7 - p h e n a n t h r o l i n e 5 mM ALA + 20 mM 1 , 7 - p h e n a n t h r o l i n e 30 mM 1 , 7 - p h e n a n t h r o l i n e 5 mM ALA + 30 mM 1 , 7 - p h e n a n t h r o l i n e Correlation coefficient Level of s i g n i f i c a n c e



Corn

Plant Species

,

20 mM 2 , 2 - d i p y r i d y l 5 mM ALA + 2 , 2 • - d i p y r i d y l 30 mM 2 , 2 ' - d i p y r i d y l 5 mM ALA + 30 mM 2 , 2 • - d i p y r i d y l Correlation coefficient Level of s i g n i f i c a n c e

Treatment

0 31 40 80

{%)

Photodynamic Damage DV

MV

3

DV

11.01 34.70 104.28 12.87 0.377 n.s.

1.01 -0.65 -1.18 -0.39 0.068 n.s.

4. 79 5. 12 7. 26 13. 54 0. 557 n .s.

1.60 7.33 9.41 25.13 0.700 10$

Exogenous ALA-dependent tetrapyrrole accumulation i n nmoles per 100 mg p r o t e i n s

MV

MP(E)

d

c

I s t h e d i f f e r e n c e between t h e t e t r a p y r r o l e c o n t e n t o f t h e ALA o r ALA + m o d u l a t o r - t r e a t e d p l a n t s and t h a t o f the c o n t r o l p l a n t s w h i c h were sprayed w i t h s o l v e n t o n l y . ^ N e g a t i v e v a l u e s i n d i c a t e a drop i n c o n t e n t i n comparison t o t h e c o n t e n t o f t h e c o n t r o l p l a n t s . R e f e r s t o t h e p r o b a b i l i t y t h a t f o r a p o p u l a t i o n f o r which t h e c o r r e l a t i o n c o e f f i c i e n t ( r ) i s e q u a l t o z e r o , a sample o f s i z e n can be t a k e n , f o r w h i c h t h e c o r r e l a t i o n e q u a l s o r exceeds t h e c a l c u l a t e d v a l u e o f r which i s r e p o r t e d i n t h e t a b l e f o r a g i v e n sample. S i n c e c o r n p l a n t s r e c o v e r e d from photodynamic damage, t h e v a l u e s r e p o r t e d f o r c o r n were d e t e r m i n e d two days after spraying. Those r e p o r t e d f o r cucumber were d e t e r m i n e d 10 days a f t e r s p r a y i n g .

a

Experiment

Pchlide

Table I . C o n t i n u e d . D i f f e r e n c e s i n ALA-Dependent T e t r a p y r r o l e A c c u m u l a t i o n and Photodynamic Damage Between DDV/LDV and DMV/LDV P l a n t S p e c i e s


C/3

5 m

n

H

C/J

m

O

1

H

0


2-pyridine aldehyde

Chlorophyll biosynthesis modulator

Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e

Treatment

0 0 0 0 0 0 0 0

0 40 0 43 0 60 0 70

Soybean Cucumber

Photodynamic damage (*)

DV

MV

DV

0.00 -0.67 -0.90 -0:60 -1.19 -0.98 0.06 1.30

-

0.00 0.83 0.97 1.70 1.36 0.87 0.88 0.43

-

C o n t i n u e d on next page

0.00 4.62 3.13 15.34 •3:48 15.73 3.67 21.96 0.907 1$

Exogenous ALA-induced t e t r a p y r r o l e accumulation (nmoles per 100 mg p r o t e i n )

0.00 9.69 3.99 21.24 4.27 22.33 3.49 32.89 0.945 0.1$

MV

Pchlide

Cucumber

MPE

Response o f Cucumber, a DDV/LDV P l a n t S p e c i e s and o f Soybean, a DMV/LDV P l a n t S p e c i e s to Enhancers o f ALA C o n v e r s i o n t o MV P r o t o c h l o r o p h y l l i d e

Treatment c o n d i t i o n s , a b b r e v i a t i o n s and d e f i n i t i o n s a r e as i n T a b l e I . Adapted from ( 2 7 ) .

Table I I .


I

S3-

!

5

m CD m N m H > r

to to


picolinic acid


Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator

Treatment

0 3 3 6 3

0 34 0 71 0

Soybean Cucumber

Photodynamic damage «)

0.00 12.05 2.92 16.87 11.08

MV

DV

MV

MPE

DV

0.00 4.03 4.20 12.65 6.20

0.00 -0.15 -0.18 1.76 -0.33

0.00 -0.51 -0.69 -0.59 0.70

Exogenous ALA-induced tetrapyrrole accumulation (nmoles per 100 mg protein)

Pchlide

Cucumber

Table I I . Continued. Response o f Cucumber, a DDV/LDV P l a n t S p e c i e s , and of Soybean, a DMV/LDV P l a n t S p e c i e s , t o Enhancers of ALA C o n v e r s i o n t o MV P r o t o c h l o r o p h y l 1 i d e


5 m

m H n

o

m

1

H

X

r 0


,2'-dipyridyl disulfide

5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e 0 12 8 16 17 19 15 21

12 6 21

0 80 0 70 0 70 0 73

80 10 90 39.87 2.68 21 .47 0.828 5t 0.00 11.94 -2.94 18.08 -2.73 17.32 6.14 41.80 0.779 5%


17.35 2.35 9.06 0.811 5% 0.00 0.19 -0.96 1.96 0.31 3.70 1.89 4.87 0.598 20*

-

2.06 0.27 0.92 0.47 -0.16 0.28 -0.01 0.42 0.20 -1 .01 0.18 -0.43 0.75 1.07

-

0.00

0.00

-

0.55 0.69 3.99

-0.87 -0.72 -0.92

316



o f growth. These r e s u l t s i n d i c a t e d t h a t t h e a c c u m u l a t i o n o f DV MP(E) by a p l a n t was l i k e l y t o cause photodynamic damage, i r r e s p e c t i v e o f t h e g r e e n i n g group t o which t h e p l a n t b e l o n g e d . O r i g i n o f t h e D i f f e r e n t i a l Photodynamic S u s c e p t i b i l i t y o f V a r i o u s P l a n t S p e c i e s t o ALA-Dependent T e t r a p y r r o l e A c c u m u l a t i o n : A Working H y p o t h e s i s . On t h e b a s i s o f t h e above, a l b e i t l i m i t e d , o b s e r v a t i o n s we now propose t h e f o l l o w i n g w o r k i n g h y p o t h e s i s : ( a ) t h a t P c h l i d e i s t h e most u b i q u i t o u s o f t h e damage-causing p h o t o dynamic t e t r a p y r r o l e s t h a t accumulate as a consequence o f ALA-based t r e a t m e n t s , (b) t h a t DDV/LDV p l a n t s p e c i e s a r e l i k e l y t o be more p h o t o d y n a m i c a l l y s u s c e p t i b l e t o ALA-based dark t r e a t m e n t s t h a t l e a d t o MV P c h l i d e a c c u m u l a t i o n , than t o those t h a t l e a d t o DV P c h l i d e a c c u m u l a t i o n . However, i t remains t o be determined whether t h i s i s due t o d i f f e r e n c e s i n t h e photodynamic damage-causing p o t e n t i a l between e q u i m o l a r amounts o f MV and DV P c h l i d e o r whether i t i s due t o o t h e r f a c t o r s , ( c ) t h a t DMV/LDV p l a n t s p e c i e s a r e photodynami c a l l y more s u s c e p t i b l e t o DV P c h l i d e than t o MV P c h l i d e accumula t i o n , and (d) t h a t both DDV/LDV and DMV/LDV p l a n t s p e c i e s a r e h i g h l y s u s c e p t i b l e t o DV MP(E) a c c u m u l a t i o n . As was a l r e a d y p o i n t e d o u t , t h e p r e m i s e s o f t h i s h y p o t h e s i s are l i k e l y t o be expanded and/or r e f i n e d i n o r d e r t o accommodate a d d i t i o n a l o b s e r v a t i o n s d e r i v e d from a d d i t i o n a l photodynamic s u s c e p t i b i l i t y s t u d i e s o f t h e f o u r g r e e n i n g groups o f p l a n t s . Fur thermore, i t would be very d e s i r a b l e t o determine t h e r e a s o n why DDV/LDV p l a n t s p e c i e s appear t o be more s u s c e p t i b l e t o ALA-depen dent MV P c h l i d e d a r k - a c c u m u l a t i o n w h i l e DMV/LDV p l a n t s p e c i e s appear t o be more s u s c e p t i b l e t o DV t e t r a p y r r o l e d a r k - a c c u m u l a t i o n . M o d u l a t i o n o f AlA-dependent T e t r a p y r r o l e A c c u m u l a t i o n and Concommltant M o d u l a t i o n o f Photodynamic Damage by C h l o r o p h y l l Biosynthesis Modulators The o b s e r v a t i o n t h a t t h e photodynamic s u s c e p t i b i l i t y o f a p l a n t s p e c i e s depended on t h e g r e e n i n g group o f t h e p a r t i c u l a r p l a n t s p e c i e s a s w e l l as on t h e n a t u r e o f t h e accumulated t e t r a p y r r o l e s had o b v i o u s b i o t e c h n o l o g i c a l i m p l i c a t i o n s . I t suggested t h a t c h e m i c a l s t h a t may be a b l e t o induce A L A - t r e a t e d p l a n t s , b e l o n g i n g t o a c e r t a i n g r e e n i n g group, t o accumulate t h e "wrong" t y p e o f MV or DV t e t r a p y r r o l e , w h i l e i n d u c i n g o t h e r p l a n t s p e c i e s , b e l o n g i n g t o o t h e r g r e e n i n g groups, t o accumulate t h e " r i g h t " type o f MV o r DV t e t r a p y r r o l e may a c t as photodynamic h e r b i c i d e m o d u l a t o r s . I n o t h e r words, such c h e m i c a l s when used i n c o n j u n c t i o n w i t h ALA had the p o t e n t i a l t o expand t h e ALA h e r b i c i d e i n t o a h i g h l y s e l e c t i v e system o f photodynamic h e r b i c i d e s . With t h i s i n mind we undertook a l i t e r a t u r e s e a r c h f o r chemi c a l s and b i o c h e m i c a l s known t o a f f e c t i n a g e n e r a l way, C h i and P c h l f o r m a t i o n ( 4 4 - 4 6 ) . We then determined t h e s p e c i f i c e f f e c t o f t h e s e c h e m i c a l s on t h e v a r i o u s C h i a b i o s y n t h e t i c r o u t e s d e s c r i b e d i n F i g . 2. This research e f f o r t r e s u l t e d i n the i d e n t i f i c a t i o n o f a t o t a l o f 13 c h e m i c a l s which a c t e d i n c o n c e r t w i t h ALA and which were c a p a b l e o f m o d u l a t i n g t h e C h i a b i o s y n t h e t i c pathway. These chemi c a l s were, t h e r e f o r e , d e s i g n a t e d c o l l e c t i v e l y as C h i a b i o s y n t h e s i s


22.

REBEIZ ET AL.

Photodynamic

Herbicides

317


modulators. They were c l a s s i f i e d i n t o t h r e e major groups depending on t h e i r mode o f a c t i o n . One group c o n s i s t e d o f enhancers o f ALA c o n v e r s i o n t o t e t r a p y r r o l e s . Another group c o n s i s t e d o f i n d u c e r s o f ALA b i o s y n t h e s i s and o f t e t r a p y r r o l e a c c u m u l a t i o n w h i l e a t h i r d group c o n s i s t e d o f i n h i b i t o r s o f MV p r o t o c h l o r o p h y l l i d e accumulation. The e f f e c t o f these v a r i o u s groups o f C h i a b i o s y n t h e s i s modulators on t h e C h i a b i o s y n t h e t i c pathway and on induced photo dynamic damage i s d e s c r i b e d below. Enhancers o f ALA C o n v e r s i o n t o T e t r a p y r r o l e s . To q u a l i f y as an enhancer o f ALA c o n v e r s i o n t o a p a r t i c u l a r MV o r DV t e t r a p y r r o l e i t was c o n s i d e r e d t h a t : (a) a p a r t i c u l a r C h i b i o s y n t h e s i s modulator s h o u l d n o t r e s u l t I n a s i g n i f i c a n t a c c u m u l a t i o n o f t h e MV o r DV t e t r a p y r r o l e i n q u e s t i o n , when a p p l i e d t o a p l a n t i n t h e absence o f exogenous ALA, but (b) a t c e r t a i n c o n c e n t r a t i o n s o f t h e modulator, when t h e l a t t e r i s used j o i n t l y w i t h exogenous ALA, i t s h o u l d enhance t h e dark t e t r a p y r r o l e c o n v e r s i o n o f exogenous ALA, i n t o t h a t p a r t i c u l a r MV o r DV t e t r a p y r r o l e , over and beyond t h e ALA c o n t r o l . A s i g n i f i c a n t accumulation o f a p a r t i c u l a r t e t r a p y r r o l e was i n t u r n d e f i n e d a r b i t r a r i l y as an amount o f t h a t t e t r a p y r r o l e t h a t approached o r exceeded t h e n e t d a r k - c o n v e r s i o n r a t e o f a 5 mM exogenous ALA t r e a t m e n t i n t o t h a t t e t r a p y r r o l e . Enhancers o f ALA c o n v e r s i o n t o t e t r a p y r r o l e s were observed t o f a l l i n t o two d i s t i n c t groups namely ( a ) enhancers o f ALA c o n v e r s i o n t o MV P c h l i d e and (b) enhancers o f ALA c o n v e r s i o n t o DV P c h l i d e . These two subgroups o f enhancers w i l l now be d i s c u s s e d separately. Enhancers o f ALA C o n v e r s i o n t o MV P c h l i d e . 2 - P y r i d i n e a l d e h y d e , p i c o l i n i c a c i d , 2 , 2 - d i p y r i d y l d i s u l f i d e ( T a b l e I I ) and 2 - p y r i d i n e a l d o x i m e , t h e l a t t e r i n t h e h i g h e r c o n c e n t r a t i o n range ( T a b l e I , A) were found t o enhance p r e f e r e n t i a l l y t h e d a r k - c o n v e r s i o n o f exo genous ALA t o MV P c h l i d e i n DDV/LDV p l a n t s p e c i e s such as cucumber. I t s h o u l d be emphasized, however, t h a t a l t h o u g h t h e s e compounds enhanced p r e f e r e n t i a l l y t h e d a r k - c o n v e r s i o n o f ALA t o MV P c h l i d e , some o f them a l s o enhanced s i g n i f i c a n t l y , but t o a l e s s e r e x t e n t , the d a r k - c o n v e r s i o n o f exogenous ALA t o DV P c h l i d e . I n DDV/LDV p l a n t s p e c i e s such as cucumber, a h i g h e r c o r r e l a t i o n was observed between photodynamic damage and t h e dark-accumula t i o n o f MV P c h l i d e , than between photodynamic damage and t h e accumu l a t i o n o f DV P c h l i d e . No s i g n i f i c a n t a c c u m u l a t i o n o f e i t h e r MV o r DV M g - p r o t o p o r p h y r i n s was observed (Table I I ) . Treatment o f soybean w i t h t h e s e same enhancers o f exogenous ALA c o n v e r s i o n t o MV P c h l i d e r e s u l t e d i n m i n i m a l o r no photodynamic damage ( T a b l e I I ) . T h i s i s f u l l y c o m p a t i b l e w i t h t h e proposed d i f f e r e n t i a l s u s c e p t i b i l i t y h y p o t h e s i s . P a r t i c u l a r l y i f soybean, a DMV/LDV p l a n t s p e c i e s r e a c t e d t o treatment w i t h ALA and 2 - p y r i d i n e aldehyde, p i c o l i n i c a c i d , 2 , 2 ' - d i p y r i d y l d i s u l f i d e o r 2 - p y r i d i n e a l d o x i m e , as d i d cucumber a DDV/LDV p l a n t s p e c i e s , by a c c u m u l a t i n g MV P c h l i d e . T h i s q u e s t i o n i s p r e s e n t l y under i n v e s t i g a t i o n . ,

Enhancers o f ALA C o n v e r s i o n t o DV P c h l i d e . 4,4»-dipyridyl, 2,2'd i p y r i d y l amine and p h e n a n t h r i d i n e were observed t o f a l l i n t o t h i s



318


group o f C h i b i o s y n t h e s i s m o d u l a t o r s . At t h e h i g h e r - c o n c e n t r a t i o n range, they enhanced p r e f e r e n t i a l l y t h e dark c o n v e r s i o n o f exo genous ALA t o DV P c h l i d e i n t r e a t e d cucumber s e e d l i n g s . S i n c e cucumber i s a DDV/LDV p l a n t s p e c i e s , i t was l e s s p h o t o d y n a m i c a l l y s e n s i t i v e t o f l u c t u a t i o n s i n i t s DV P c h l i d e than t o f l u c t u a t i o n s i n i t s MV P c h l i d e c o n t e n t . T h i s i n t u r n was r e f l e c t e d by a b e t t e r c o r r e l a t i o n between photodynamic damage and MV P c h l i d e a c c u m u l a t i o n t h a n between photodynamic damage and DV P c h l i d e a c c u m u l a t i o n ( T a b l e III). No s i g n i f i c a n t a c c u m u l a t i o n o f e i t h e r MV o r DV Mg-protoporp h y r i n s was o b s e r v e d . I n c o n t r a s t t o cucumber, d a r k - t r e a t m e n t o f soybean w i t h ALA t o g e t h e r w i t h t h e f o r e m e n t i o n e d enhancers o f ALA c o n v e r s i o n t o DV P c h l i d e , r e s u l t e d i n e x t e n s i v e photodynamic damage ( T a b l e I I I ) . T h i s was t h e e x p e c t e d phenomenology i f t h e dark t r e a t m e n t o f soy bean, a DMV/LDV p l a n t s p e c i e s , w i t h ALA and 4 , 4 ' - d i p y r i d y l , 2,2'd i p y r i d y l amine o r p h e n a n t h r i d i n e had t r i g g e r e d an enhancement o r an i n d u c t i o n o f DV t e t r a p y r r o l e a c c u m u l a t i o n . T h i s matter i s p r e s e n t l y under i n v e s t i g a t i o n . I n d u c e r s o f T e t r a p y r r o l e A c c u m u l a t i o n . To q u a l i f y a s an i n d u c e r o f t e t r a p y r r o l e a c c u m u l a t i o n , i t was c o n s i d e r e d t h a t a p a r t i c u l a r C h i b i o s y n t h e s i s modulator s h o u l d , a t c e r t a i n c o n c e n t r a t i o n s , r e s u l t i n a s i g n i f i c a n t a c c u m u l a t i o n o f a p a r t i c u l a r MV o r DV t e t r a p y r r o l e , when a p p l i e d t o a p l a n t i n t h e absence o f exogenous ALA. Here a g a i n , s i g n i f i c a n t a c c u m u l a t i o n o f a p a r t i c u l a r t e t r a p y r r o l e was a r b i t r a r i l y d e f i n e d as an amount o f t h a t t e t r a p y r r o l e t h a t a p proaches o r exceeds t h e n e t d a r k - c o n v e r s i o n r a t e o f a 5 mM exogen ous ALA t r e a t m e n t i n t o t h a t t e t r a p y r r o l e . Furthermore, a t c e r t a i n c o n c e n t r a t i o n s o f t h e i n d u c e r , t h e l a t t e r , i n c o m b i n a t i o n w i t h ALA, should r e s u l t i n the accumulation o f higher l e v e l s o f the p a r t i c u l a r MV o r DV t e t r a p y r r o l e than when ALA o r t h e i n d u c e r a r e a p p l i e d to t h e p l a n t s e p a r a t e l y . 1,1O-phenanthroline ( i . e . O - p h e n a n t h r o l i n e ) ( T a b l e I , D) and 2 , 2 - d i p y r i d y l ( T a b l e I V ) were observed t o a c t p r e f e r e n t i a l l y as i n d u c e r s o f DV M g - p r o t o p o r p h y r i n + DV M g - p r o t o p o r p h y r i n monoester [DV MP(E)] a c c u m u l a t i o n . I t s h o u l d be noted t h a t w h i l e 1,1O-phenan t h r o l i n e p r e f e r e n t i a l l y induced t h e b i o s y n t h e s i s and a c c u m u l a t i o n of DV MP(E), i t a l s o i n d u c e d , t o a l e s s e r e x t e n t , t h e a c c u m u l a t i o n o f DV P c h l i d e ( T a b l e I D). 2 , 2 • - d i p y r i d y l ( T a b l e I V ) d i d n o t e x h i b i t t h i s p r o p e r t y . I n cucumber t h e h i g h e s t c o r r e l a t i o n was observed between DV MP(E) a c c u m u l a t i o n and photodynamic damage ( T a b l e s I , D and I V ) . Soybean a DMV/LDV p l a n t s p e c i e s was e q u a l l y s u s c e p t i b l e t o t r e a t m e n t w i t h 2 , 2 - d i p y r i d y l ( T a b l e I V ) and t o 1,1O-phenanthroline ( d a t a n o t shown). T h i s i n t u r n was c o m p a t i b l e w i t h t h e proposed mode o f a c t i o n h y p o t h e s i s . I n v e s t i g a t i o n s o f t h e q u a n t i t a t i v e r e l a t i o n s h i p s between t h e i n d u c t i o n o f s p e c i f i c t e t r a p y r r o l e accumula t i o n and t h e i n c i d e n c e o f photodynamic damage i n DMV/LDV p l a n t s p e c i e s such a s soybean a r e i n p r o g r e s s . f

f

I n h i b i t o r s o f MV P r o t o c h l o r o p h y l l l d e A c c u m u l a t i o n . To q u a l i f y a s an i n h i b i t o r o f MV P c h l i d e a c c u m u l a t i o n , i t was c o n s i d e r e d t h a t a p a r t i c u l a r C h i b i o s y n t h e s i s modulator (a) when used a l o n e , s h o u l d r e s u l t i n t h e i n h i b i t i o n o f MV P c h l i d e a c c u m u l a t i o n , i n comparison



4,4'-Dipyridyl



Treatment

0 10 15 50 58

0 20 0 15 0

Soybean Cucumber

Photodynamic damage

0.00 19.66 -1.82 11.09 -1.20

MV

DV

MV

MPE

DV

0.00 0.00 4.06 -1.09 -1.90 -1.09 4.44 -0.31 -0.44 -0.56 Continued on next

0.00 -0.82 -1.02 -0.69 -0.85 page


Pchlide

Cucumber

Response o f Cucumber, a DDV/LDV P l a n t S p e c i e s and o f Soybean, a DMV/LDV P l a n t S p e c i e s t o Enhancers o f ALA C o n v e r s i o n t o DV P r o t o c h l o r o p h y l l i d e

Treatment a b b r e v i a t i o n s and d e f i n i t i o n s a r e as i n T a b l e I . Adapted from ( 2 7 ) .

Table I I I .


I

5

m H > r

N

2

DO

m

70

to

lO



5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator

Treatment

0 15 0 36

76 85 93

Soybean

25

-

0 25

-

75 0 25

Cucumber

Photodynamic damage

•

• -

15.37

DV

MV

MPE

DV

9.91

-

22.68 9.21 34.54 0.647 10% 0.00 3.87

-1.71

-

-

0.00 -0.69

0.31 0.73 -0.08

-0.81

0.00 -0.87

-

-0.09 -0.81 -0.39


20.69 -1.34 22.56 0.760 5% 0.00 16.23

MV

Pchlide

Cucumber

Table I I I . C o n t i n u e d . Response of Cucumber, a DDV/LDV P l a n t S p e c i e s , and of Soybean, a DMV/LDV P l a n t S p e c i e s , t o Enhancers of ALA C o n v e r s i o n t o DV P r o t o c h l o r o p h y l 1 i d e


a m

n

C/3

rn H

m a -v

1

ft

x

r 0

O


phenanthridine

2,2'-Dipyridyl amine

20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of significance 0 0 84 87 92 94 100 100

6 64 9 82

-

0 75 10 80 15 70 10 80

-

0 30 0 15

3.70 20.19 0.94 15.48 0.962 0.1$ 0.00 44.20 12.25 36.11 8.98 49.60 5.46 57.05 0.955 0.1$


0.53 20.67 0.09 10.80 0.81 5$ 0.00 16.82 5.80 17.70 6.67 29.79 3.33 72.84 0.706 10$

-

-

-

0.00 0.86 0.27 0.78 0.01 0.31 0.56 1.87

0.43 -1.97 0.60 -0.80

-

-

0.00 0.35 0.12 0.24 -0.01 -0.04 0.38 1.76

-0.36 -1.01 -0.62 -0.92


2,2'-dipyridyl


Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level o f s i g n i f i c a n c e

Treatment

0 0 6 34 46 53 80 90

0 45 0 25 0 90 25 100

Soybean Cucumber

Photodynamic damage

DV

MV

Cucumber

MPE

DV

0.00 46.36 7.69 19.60 -4.76 37.20 -3.96 29.73 0.7415 10*

0.00 1.65 -0.86 0.73 1.39 5.34 6.07 14.30 0.814 5%

0.00 -0.58 -0.09 4.50 15.29 70.39 15.32 160.04 0.846 5%


0.00 100.16 31.45 75.40 2.94 28.94 8.73 45.16 0.329 n.s.

MV

Pchlide

Treatments, a b b r e v i a t i o n s and d e f i n i t i o n s a r e a s i n T a b l e I . Adapted from ( 2 7 ) .

Table I V . Response o f Cucumber, a DDV/LDV P l a n t S p e c i e s and o f Soybean, a DMV/LDV P l a n t S p e c i e s t o I n d u c e r s o f DV M g - p r o t o p o r p h y r i n a c c u m u l a t i o n


UJ

rn

5

n

m a m C/J H

1

ft

r 0

to


22.

REBEIZ ET AL.

Photodynamic

Herbicides

323

t o t h e u n t r e a t e d c o n t r o l s , and/or (b) when used i n c o n j u n c t i o n w i t h ALA, i t s h o u l d r e s u l t i n t h e i n h i b i t i o n o f MV P c h l i d e a c c u m u l a t i o n i n comparison t o t h e A L A - t r e a t e d c o n t r o l . 4 , 7 - p h e n a n t h r o l i n e , 2 , 3 - d i p h y r i d y l and 2 , 4 - d i p y r i d y l (Table V) as w e l l as 1 , 7 - p h e n a n t h r o l i n e (Table I C) f a l l i n t o t h i s group o f C h i b i o s y n t h e s i s m o d u l a t o r s . I n most cases so f a r I n v e s t i g a t e d , when t h e i n h i b i t o r was used j o i n t l y w i t h ALA, e s p e c i a l l y a t t h e h i g h e r c o n c e n t r a t i o n l e v e l s o f i n h i b i t o r , t h e i n h i b i t i o n o f MV P c h l i d e d a r k - a c c u m u l a t i o n was accompanied by an enhancement o f DV P c h l i d e a c c u m u l a t i o n , i n comparison t o t h e A L A - t r e a t e d c o n t r o l (Table V ) . M g - p r o t o p o r p h y r i n a c c u m u l a t i o n was n o t o b s e r v e d . I n cucumber, a DDV/LDV p l a n t s p e c i e s , i n h i b i t o r - i n d u c e d photo dynamic damage over and beyond t h e A L A - t r e a t e d c o n t r o l s was e i t h e r minimal (4,7-phenanthroline, 2 , 3 - d i p y r i d y l i n Table V and 1,7p h e n a n t h r o l i n e i n T a b l e I C) or was absent ( 2 , 4 - d i p y r i d y l i n T a b l e V ) . However, i n soybean a DMV/LDV p l a n t s p e c i e s , t h e s e same ALA + i n h i b i t o r t r e a t m e n t s r e s u l t e d i n e x t e n s i v e photodynamic damage over and beyond t h e A L A - t r e a t e d c o n t r o l s (Table V ) . These r e s u l t s were i n t u r n f u l l y c o m p a t i b l e w i t h t h e proposed mode o f a c t i o n hypothesis. Epilogue The r e s e a r c h e f f o r t d e s c r i b e d i n t h i s work has a l r e a d y l e d t o t h e development o f photodynamic h e r b i c i d e f o r m u l a t i o n c a p a b l e o f con t r o l l i n g broad l e a f weeds i n Kentucky b l u e g r a s s , under f i e l d c o n d i t i o n s (47) and i n c o n t r o l l i n g s e v e r a l monocot and d i c o t weed s p e c i e s i n c o r n and soybean under greenhouse c o n d i t i o n s . I n sum mary such an e f f o r t has i n v o l v e d (a) t h e c l a s s i f i c a t i o n o f the p l a n t s p e c i e s t o be d e s t r o y e d and t h o s e t o be saved i n t o t h e i r r e s p e c t i v e g r e e n i n g groups, (b) s e l e c t i o n o f one o r more C h i b i o s y n t h e s i s m o d u l a t o r s t o a c t j o i n t l y w i t h ALA and t o Induce t h e u n d e s i r a b l e weeds t o accumulate u n d e s i r a b l e t e t r a p y r r o l e s t h a t do not b e l o n g t o a f u n c t i o n a l b i o s y n t h e t i c r o u t e , ( c ) development o f a f i e l d s o l v e n t system c a p a b l e o f d e l i v e r i n g t h e ALA and t h e C h i b i o s y n t h e s i s m o d u l a t o r ( s ) t o t h e c h l o r o p l a s t , where ALA i s c o n v e r t e d t o t e t r a p y r r o l e s and f i n a l l y (d) t e s t i n g t h e developed s o l v e n t system under t h e f i e l d c o n d i t i o n s f o r which i t had been designed (47). Because o f t h e p o s s i b i l i t y o f combining i n d i v i d u a l members o f the f o u r c l a s s e s o f C h i b i o s y n t h e s i s m o d u l a t o r s and ALA, f i v e , f o u r , t h r e e o r two a t a t i m e , i t i s p o s s i b l e t o d e s i g n a v e r y l a r g e number o f u s e f u l h e r b i c i d e s . For example w i t h t h e 13 C h i b i o s y n t h e s i s m o d u l a t o r s d e s c r i b e d i n t h i s work, i t i s a l r e a d y p o s s i b l e t o d e s i g n 3458 d i f f e r e n t h e r b i c i d a l m i x t u r e s . On t h e o t h e r hand t h e d i s c o v e r y o f one o r two a d d i t i o n a l C h i b i o s y n t h e s i s m o d u l a t o r s has the p o t e n t i a l o f r e s u l t i n g i n 1470 and 2410 a d d i t i o n a l h e r b i c i d e s respectively. Acknowledgments T h i s work was s u p p o r t e d by N a t i o n a l S c i e n c e F o u n d a t i o n Grant DMB 85-07217, by funds from t h e I l l i n o i s A g r i c u l t u r a l Experiment S t a t i o n and by t h e John P. T r e b e l l a s P h o t o b i o t e c h nology Research Endowment t o C.A.R.



4,7-phenan throline



Treatment

0 3 93 94 100

0 60 3 45 21

Soybean Cucumber

Photodynamic damage «}

0.00 41.65 -0.24 19.18 -5.33

MV

DV

MV

MPE

DV

0.00 8.04 2.66 16.00 3.09

0.00 0.92 0.20 1.05 0.99

0.00 0.91 0.34 0.19 -0.95


Pchlide

Cucumber

Response o f Cucumber a DDV/LDV P l a n t S p e c i e s and o f Soybean, a DMV/LDV P l a n t S p e c i e s t o I n h i b i t o r s o f MV P r o t o c h l o r o p h y l l i d e A c c u m u l a t i o n

Treatments, a b b r e v i a t i o n s and d e f i n i t i o n s a r e as i n T a b l e I . Adapted from ( 2 7 ) .

Table V.


r

3 m

n

m

T3

m D

I

H

X

5

to


2,3-dipyridyl

5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e Solvent only 5 mM ALA 10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level of s i g n i f i c a n c e 0 8 28 35 40 45 65 70

94 100 97

-

0 49 0 25 0 28 0 40

-

56 48 88

12.08 0.74 -4.58 0.299 n.s. 0.00 65.42 6.08 26.16 -1.01 41.56 8.46 19.50 0.869 5%


-

-

0.00 -0.25 -0.22 0.70 1.47 -0.79 -0.14 0.63

1.38 0.86 1.21

-

1.48 -0.31 0.62 3.98 1.47 2.01

6.27

0.00

—

-0.45 0.19 -0.56

C o n t i n u e d on next page

18.85 14.60 25.70 0.889 1* 0.00 30.80 5.55 31.26 6.82 58.53 14.59 38.75 0.763 10*

to

§

5

N

m H > r

m DO m

70

to to


2,4-dipyridyl


10 mM modulator 5 mM ALA + 10 mM modulator 20 mM modulator 5 mM ALA + 20 mM modulator 30 mM modulator 5 mM ALA + 30 mM modulator Correlation coefficient Level o f significance

5 mM ALA

Solvent only

Treatment

47 60

40

20

13 25

3 8

-

0 8 0 5

-

0 35 0

Soybean Cucumber

Photodynamic damage «}

8.15

5%

3.44 0.927

-5.71

9.28

-

-5.05

n.s.

0.227

-

-0.12 11.63 4.15 26.54

-0.63

0.00

0.00 -3.32

MV

MPE

DV

0.00

-0.46 0.57 0.82 0.30

-

-0.16 0.57 0.34

-

-

-1.12 -1.03 1.32

-

0.93 0.32

0.00


DV

Cucumber

19.65

MV

Pchlide

Table V. C o n t i n u e d . Response o f Cucumber, a DDV/LDV P l a n t S p e c i e s , and of Soybean, a DMV/LDV P l a n t S p e c i e s , t o I n h i b i t o r s o f MV P r o t o c h l o r o p h y 1 1 i d e A c c u m u l a t i o n


m

5

m a m H n

1

H

ft

r 0

to

22.

REBEIZ ET AL.


327

Legend o f Symbols. P c h l i d e : p r o t o c h l o r o p h y l l i d e ; P r o t o : protopor p h y r i n I X ; Mg-proto; Mg p r o t o p o r p h y r i n I X ; MPE: M g - p r o t o p o r p h y r i n monoester; MP ( E ) : a m i x t u r e o f Mg-Proto and MPE; C h i : c h l o r o p h y l l ; monocot: monocotyledonous p l a n t ; d i c o t : dicotyledonous p l a n t ; MV: m o n o v i n y l ; DV: d i v i n y l ; C h l i d e : c h l o r o p h y l l i d e ; P r o t o gen: p r o t o p o r p h y r i n o g e n ; A l k . E: a l k y l e s t e r ; P c h l : protochloro p h y l l i d e e s t e r ; P c h l ( i d e ) : p c h l i d e + P c h l ; DPy: 2 , 2 - d i p y r i d y l . 1

Literature Cited


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