Regulation of Plant Growth and Development by Endogenous

Wilkins, M. B. (Ed.) "Advanced Plant Physiology"; Pitman: Marshfield, MA, 1984; 514 pp. 4. Rayle, D. L.; Cleland, R. Plant Physiol. 1970, 46, 250-3. 5...
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Regulation of Plant Growth and Development by Endogenous Hormones THOMAS C. MOORE Department of Botany and Plant Pathology, Oregon State University, Corvallis, OR 97331

The roles and mechanisms of action of all the major kinds of hormones i n normal regulation of growth and development of seed plants are discussed. Auxins promote growth by inducing c e l l - w a l l loosening and nucleic acid (mRNA) and protein synthesis. Like auxins, gibberellins also have a dual c e l l - w a l l p l a s t i c i z i n g effect and gene-activation function which i s manifested as synthesis of s p e c i f i c mRNAs and proteins. The mode of action of free cytokinins i s unknown; however, the cytokinin moieties which occur i n certain molecular species of tRNA may modulate protein synthesis by influencing the binding of aminoacyl-tRNAs to the mRNA-ribosomes. In the barley aleurone system and dormant t i s s u e , ABA seems to function by opposing the action of GA and i n h i b i t i n g the synthesis of RNA, whereas evocation of rapid stomatal closure i s due to an effect on membrane permeability. It also affects protein synthesis q u a l i t a t i v e l y i n responsive tissues. Ethylene part i c i p a t e s i n growth regulation of plants throughout ontogeny. The nature of the hormonal regulation of flowering i n angiosperms has never been f u l l y e l u cidated on the basis of the known hormones. There may be unique florigens or anthesins for the regulation of the flowering process.

The subject of this paper i s natural plant growth regulators, i.e., endogenous hormones, and the mechanisms by which they regulate plant growth and development. The commonly recognized plant hormones are the auxins, g i b b e r e l l i n s , cytokinins, a b s c i s i c a c i d , ethylene and the hypothetical florigens or anthesins. As Paleg (1) stated, there i s merit i n applying different connotations to two terms, "mechanism" and "mode" of a c t i o n , which often are used synonymously. When a hormone acts upon a responsive plant system, i t of course enters into some d i r e c t and s p e c i f i c molecular interaction which r e s u l t s , eventually, i n the manifestation of a 0097-6156/85/0276-0085$06.00/0 © 1985 American Chemical Society

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measurable e f f e c t ( b i o c h e m i c a l o r p h y s i o l o g i c a l r e s p o n s e , e.g., cell elongation). But t h e r e r e a l l y a r e two a s p e c t s o f t h e hormone a c t i o n involved: (1) t h e d i r e c t and s p e c i f i c m o l e c u l a r i n t e r a c t i o n ; and (2) t h e s u c c e e d i n g s e r i e s o f e v e n t s w h i c h r e s u l t i n t h e measurable biochemical o r p h y s i o l o g i c a l response. The former i s t h e "mechanism of a c t i o n ; t h e l a t t e r i s t h e "mode" c f a c t i o n . I t i s t h e former which w i l l be d i s c u s s e d h e r e .

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Auxins The n a t u r a l a u x i n s a r e r e p r e s e n t e d by a s i n g l e compound, i n d o l e - 3 a c e t i c a c i d (IAA), which o c c u r s a s t h e f r e e a c i d and i n v a r i o u s "bound forms, i n c l u d i n g a t h i o g l u c o s i d e , g l y c o s y l e s t e r s , and IAA peptides. F o r a r e c e n t d i s c u s s i o n s e e t h e book c h a p t e r b y B a n d u r s k i and Nonhebel (2) i n W i l k i n s ( 3 ) . When a u x i n i s s u p p l i e d t o a r e s p o n s i v e system ( e . g . , e x c i s e d c o l e o p t i l e o r stem segment), t h e r e a r e two b a s i c r e s p o n s e s : first, c e l l w a l l and media a c i d i f i c a t i o n and c e l l w a l l l o o s e n i n g ; and second, s t i m u l a t i o n o f RNA and p r o t e i n s y n t h e s i s . The mechanism o f the c e l l w a l l and media a c i d i f i c a t i o n i s unknown. However, i t h a s been p o s t u l a t e d t h a t IAA a c t i v a t e s a plasma membrane "pump" o r ATPase t h e r e b y s t i m u l a t i n g a c t i v e p r o t o n e f f l u x from t h e plasma membrane (4_>5). The lowered pH then e i t h e r causes breakage o f hydrogen bonds between c e r t a i n c e l l - w a l l polymers ( e . g . , x y l o g l u c a n s ) and c e l l u l o s e m i c r o f i b r i l s , o r a c t i v a t e s enzymes c a p a b l e o f h y d r o l y z i n g c e r t a i n w a l l p o l y s a c c h a r i d e s t o s o f t e n the w a l l and a l l o w c e l l enlargement. In a u x i n - d e p l e t e d , e x c i s e d stem s e c t i o n s ( e . g . , soybean h y p o c o t y l ) t h i s i n i t i a l b u r s t o f e l o n g a t i o n i s t r a n s i e n t , l a s t i n g o n l y some 30 to 90 min, depending upon t h e s p e c i e s and c o n d i t i o n s . The r a t e o f enlargement f i r s t i n c r e a s e s , a f t e r a l a g p e r i o d o f 15 min o r l e s s , then b e g i n s t o d e c r e a s e , w i t h k i n e t i c s r e s e m b l i n g (perhaps i d e n t i c a l t o ) a c i d - i n d u c e d growth. The o b s e r v a t i o n t h a t IAA s t i m u l a t i o n o f growth may be p r e c e d e d by an i n c r e a s e i n RNA s y n t h e s i s (6) l e d t o t h e e a r l y i d e a t h a t IAA might a c t by d e r e p r e s s i n g c e r t a i n genes, thus c a u s i n g a l t e r e d RNA and protein synthesis. Key and a s s o c i a t e s (see 7) and o t h e r s r e p o r t e d e x t e n s i v e l y on an I A A - s t i m u l a t i o n o f i n c o r p o r a t i o n o f r a d i o l a b e l e d n u c l e o t i d e s i n t o RNA. R e c e n t l y Z u r f l u h and G u i l f o y l e (8) and T h e o l o g i s and Ray (9) r e p o r t e d t h a t c e r t a i n mRNA sequences appeared f o l l o w i n g a p p l i c a t i o n o f IAA and 2,4-D t o soybean and p e a s e e d l i n g stem segments. A few mRNA sequences i n c r e a s e d i n amount o r t r a n s l a ­ t i o n a c t i v i t y w i t h i n 15 t o 20 min o f exposure o f p e a and soybean t i s s u e s t o IAA o r 2,4-D; t h a t i s , w i t h i n about t h e same time as t h e a u x i n s t i m u l a t i o n o f c e l l enlargement ( 9 ) . I n Z u r f l u h s and G u i l f o y l e s (8) work, t h e l e v e l s o f t r a n s l a t a b l e mRNA f o r a t l e a s t t e n i n v i t r o t r a n s l a t i o n p r o d u c t s were i n c r e a s e d by 2,4-D i n s e c t i o n s of soybean h y p o c o t y l . The i n d u c t i o n by a u x i n o c c u r r e d r a p i d l y ( w i t h ­ i n 15 m i n ) , and t h e amounts o f t h e i n d u c e d i n v i t r o t r a n s l a t i o n p r o ­ d u c t s i n c r e a s e d w i t h time o f a u x i n t r e a t m e n t . T h e o l o g i s and Ray (9) s t a t e d t h a t , " A l t h o u g h f o r s e v e r a l r e a s o n s , i t seems u n l i k e l y t h a t t h o s e mRNAs a r e a c t u a l l y c a u s a t i v e i n t h e a u x i n i n d u c t i o n o f c e l l enlargement, t h e i r i n c r e a s e seems t o be r e l a t i v e l y c l o s e t o p r i m a r y a u x i n a c t i o n and might w e l l s e r v e i n m a i n t a i n i n g a s t e a d y r a t e o f c e l l enlargement o v e r t h e l o n g e r term ('second phase' o f a u x i n a c t i o n i n c e l l enlargement). O t h e r mRNAs i n c r e a s e s u b s t a n t i a l l y subsequent 11

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t o 0.5, 1 and 2 h r o f a u x i n t r e a t m e n t , and beyond about 2 h r c e r t a i n o t h e r mRNA sequences become r e p r e s s e d by a u x i n s . " Obviously, i t w i l l be v e r y i m p o r t a n t u l t i m a t e l y t o e l u c i d a t e the p h y s i o l o g i c a l r o l e s o f a u x i n - r e g u l a t e d mRNAs and t h e mechanism o f t h e i r r e g u l a t i o n . The model p r e s e n t e d by Vanderhoef and Dute (10) summarizes some e a r l i e r e v i d e n c e r e c o n c i l i n g t h e e f f e c t s o f w a l l a c i d i f i c a t i o n and RNA and p r o t e i n s y n t h e s i s as a two-phase a c t i o n o f a u x i n f o l l o w i n g a d d i t i o n o f t h e hormone t o a u x i n - d e p l e t e d , r e s p o n s i v e t i s s u e s . Gibberellins The more than 50 g i b b e r e l l i n s (GAs) o c c u r as f r e e 19-carbon o r 20c a r b o n d i t e r p e n o i d mono-, d i - , o r t r i - c a r b o x y l i c a c i d s and as t h e g l u c o s i d e s and g l u c o s y l e s t e r s . See Jones and M a c M i l l a n (11) f o r a recent discussion. Much o f o u r knowledge about t h e mechanism o f a c t i o n o f GAs c o n ­ c e r n s the i n d u c t i o n by GA o f de novo s y n t h e s i s o f c e r t a i n h y d r o l y t i c enzymes i n t h e a l e u r o n e l a y e r o f g e r m i n a t i n g b a r l e y g r a i n s . In the c o u r s e o f n a t u r a l g e r m i n a t i o n o f a b a r l e y g r a i n , t h e embryo i s t h e s o u r c e o f some endogenous GA. R a d l e y (12) showed i n 1967 t h a t GAl i k e s u b s t a n c e s a r e produced by t h e s c u t e l l u m d u r i n g t h e f i r s t 2 days o f g e r m i n a t i o n and t h e r e a f t e r by t h e embryo a x i s . E i t h e r the GA i t s e l f , l i b e r a t e d by t h e embryo, o r some unknown f a c t o r c a p a b l e o f s t i m u l a t i n g GA s y n t h e s i s , d i f f u s e s a c r o s s t h e endosperm t o t h e a l e u r o n e c e l l s , i n which t h e s e v e r a l h y d r o l y t i c enzymes a r e produced de novo under t h e a c t i o n o f GA. When i s o l a t e d a l e u r o n e l a y e r s o f b a r l e y a r e i n c u b a t e d i n a s o l u ­ t i o n c o n t a i n i n g GA, they produce and s e c r e t e s e v e r a l h y d r o l y t i c enzymes. GA-dependent de novo s y n t h e s i s has been demonstrated f o r α-amylase ( 1 3 ) , p r o t e a s e ( 1 4 ) , and $ - l , 3 - g l u c a n a s e and r i b o n u c l e a s e (15). I n a d d i t i o n , a GA-dependent r e l e a s e o f r i b o n u c l e a s e and 3-1,3g l u c a n a s e has been d e m o n s t r a t e d . The i n c r e a s e i n a c t i v i t y o f a t l e a s t one h y d r o l a s e , β-amylase, i n t h e p r e s e n c e o f GA i s due t o r e ­ l e a s e o f p r e f o r m e d enzyme and n o t t o de novo s y n t h e s i s . E v i d e n c e t h a t the i n c r e a s e i n a c t i v i t y o f a t l e a s t f o u r h y d r o ­ l a s e s i n d u c e d by GA a c t u a l l y o c c u r s as a r e s u l t o f de novo s y n t h e s i s , r a t h e r than by a c t i v a t i o n o f p r e f o r m e d enzyme, has been o b t a i n e d i n v a r i o u s ways. However, t h e most u n e q u i v o c a l p r o o f comes from d e n s i t y l a b e l i n g e x p e r i m e n t s , f i r s t p e r f o r m e d by F i l n e r and V a r n e r i n 1967 (13). L a t e r J a c o b s e n and V a r n e r (14) p r o v e d by t h e same p r o c e d u r e s t h a t p r o t e a s e a l s o i s s y n t h e s i z e d de novo i n r e s p o n s e t o GA. And Bennett and C h r i s p e e l s (15) p r o v e d , u s i n g D2O, GA-induced de novo s y n t h e s i s o f r i b o n u c l e a s e and 3-l>3-glucanase i n b a r l e y a l e u r o n e cells. I n d u c t i o n o f de novo s y n t h e s i s o f α-amylase by GA i n i s o l a t e d aleurone l a y e r s i s evident a f t e r a l a g period of approximately 8 hr f o l l o w i n g a d m i n i s t r a t i o n o f t h e hormone. I n k e e p i n g w i t h hormone r e s p o n s e s g e n e r a l l y , GA must be p r e s e n t c o n t i n u o u s l y i f t h e de novo s y n t h e s i s o f h y d r o l a s e s i s t o be s u s t a i n e d . S y n t h e s i s o f new RNA i s e s s e n t i a l t o t h e G A - i n d u c t i o n o f de novo s y n t h e s i s o f h y d r o l a s e s . A c t i n o m y c i n D, an i n h i b i t o r o f RNA s y n t h e s i s , i n h i b i t s t h e s y n t h e s i s and r e l e a s e o f α-amylase i f t h e i n h i b i t o r i s p r e s e n t e d d u r i n g t h e f i r s t 7 to 8 h r a f t e r treatment. I n h i b i t o r s of p r o t e i n synthesis, such as c y c l o h e x i m i d e , a l s o i n h i b i t G A - i n d u c t i o n o f h y d r o l a s e s . And, i n t e r e s t i n g l y , a b s c i s i c a c i d , a g r o w t h - i n h i b i t i n g hormone, i n h i b i t s GA-induced α-amylase s y n t h e s i s as w e l l .

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A c l o s e l o o k a t the e v e n t s w h i c h o c c u r d u r i n g the l a g p e r i o d i n GA i n d u c t i o n o f de novo s y n t h e s i s o f new enzymes h a s p r o v i d e d some i m p o r t a n t c l u e s a s t o whether GA a c t s a t the t r a n s c r i p t i o n a l o r t r a n s l a t i o n a l l e v e l QJ6 and p a p e r s c i t e d t h e r e i n ) . An i n c r e a s e i n p o l y r i b o s o m e f o r m a t i o n and an i n c r e a s e d s y n t h e s i s o f ribosomes and e n d o p l a s m i c r e t i c u l u m membranes were found. A l l o f t h e s e e f f e c t s b e g i n w i t h i n 2 t o 4 h r a f t e r a p p l i c a t i o n o f GA. T h e i r o b s e r v a t i o n s l e d E v i n s and V a r n e r (16) t o c o n c l u d e t h a t t h e G A - s t i m u l a t e d i n ­ c r e a s e s i n the number o f monoribosomes and the p e r c e n t a g e o f p o l y ­ ribosomes p r o b a b l y a r e p r e r e q u i s i t e f o r the hormone i n d u c t i o n o f protein synthesis. H i g g i n s e t a l . (17) p r o v i d e d a q u i t e d i r e c t l i n k between GAs t i m u l a t e d de novo enzyme s y n t h e s i s and appearance o f the complemen­ t a r y mRNA. They demonstrated c o n v i n c i n g l y t h a t the l e v e l o f t r a n s ­ l a t a b l e α-amylase mRNA i n c r e a s e d i n G A - t r e a t e d t i s s u e i n p a r a l l e l w i t h the i n c r e a s e d r a t e o f enzyme s y n t h e s i s . These r e s u l t s p r o v i d e s t i l l more e v i d e n c e t h a t GA a c t s t o i n d u c e s e l e c t i v e mRNA and de novo enzyme s y n t h e s i s i n a l e u r o n e c e l l s . On the b a s i s o f c o l l e c t i v e d a t a now a v a i l a b l e , i t may c o n f i d e n t ­ l y be c o n c l u d e d t h a t i n the b a r l e y a l e u r o n e system, GA evokes s e l e c ­ t i v e l y the s y n t h e s i s o f p a r t i c u l a r m o l e c u l a r s p e c i e s o f mRNA which i n t u r n l e a d s t o de novo s y n t h e s i s o f c e r t a i n h y d r o l y t i c enzymes. Or, as Jones and M a c M i l l a n (11) p u t i t , "There i s c o m p e l l i n g e v i d e n c e t h a t GA3 a f f e c t s the s y n t h e s i s o f h y d r o l y t i c enzymes i n c e r e a l a l e u r o n e and t h a t i t does so b y c o n t r o l l i n g the t r a n s c r i p t i o n and p o s s i b l y t r a n s l a t i o n o f new mRNAs." L e f t unanswered u n e q u i v o c a l l y a t t h i s s t a g e o f our knowledge a r e two i m p o r t a n t q u e s t i o n s . One i s whether the a c t i o n o f GA j u s t d e s c r i b e d a l o n e a c c o u n t s e x c l u s i v e l y f o r the fundamental mechanism o f a c t i o n o f GA i n the a l e u r o n e system. The o t h e r q u e s t i o n i s whether the a c t i o n o f GA i n s t i m u l a t i n g de_ novo s y n t h e s i s o f p a r t i c u l a r enzymes a s d e s c r i b e d f o r the b a r l e y a l e u r o n e system i s u n i v e r s a l . That i s , i s the mechanism o f GA a c t i o n i n t h e b a r l e y a l e u r o n e system d i r e c t l y i n d i c a t i v e o f the mechanism by which GA hormones p a r t i c i p a t e g e n e r a l l y i n the r e g u l a t i o n o f the growth and development o f h i g h e r p l a n t s ? O b v i o u s l y , the answers t o t h e s e q u e s t i o n s must a w a i t a d d i t i o n a l r e s e a r c h . Cytokinins N a t u r a l c y t o k i n i n s o c c u r as f r e e b a s e s and a s the r i b o n u c l e o s i d e s and r i b o n u c l e o t i d e s o f N ^ - s u b s t i t u t e d a d e n i n e d e r i v a t i v e s and a l s o as c o n s t i t u e n t s o f p a r t i c u l a r m o l e c u l a r s p e c i e s o f tRNA ( 1 8 ) . C y t o k i n i n s a r e u n i q u e among p l a n t hormones i n t h a t a d e n i n e compounds o f i d e n t i c a l s t r u c t u r e o c c u r i n n u c l e i c a c i d s . More s p e c i ­ f i c a l l y , p a r t i c u l a r c y t o k i n i n - a c t i v e r i b o n u c l e o s i d e s o c c u r a s com­ ponents o f c e r t a i n m o l e c u l a r s p e c i e s o f tRNA. Zachau e_t a l . (19), d u r i n g t h e d e t e r m i n a t i o n o f the base sequences o f s e r i n e tRNA i n y e a s t , f i r s t r e p o r t e d an "odd" base i m m e d i a t e l y a d j a c e n t t o the 3* end o f the a n t i c o d o n . I n c o l l a b o r a t i o n w i t h Biemann e t a l . ( 2 0 ) , t h i s "odd" base was i d e n t i f i e d a s the n a t u r a l c y t o k i n i n i s o p e n t e n y l a d e n o s i n e , w h i c h i s one o f the most h i g h l y a c t i v e c y t o k i n i n s known. In f a c t , i n a l l c a s e s , where the s p e c i f i c l o c a t i o n o f a c y t o k i n i n m o i e t y i n tRNA has been d e t e r m i n e d , i t i n v a r i a b l y i s i m m e d i a t e l y a d j a c e n t t o t h e 3' end o f the a n t i c o d o n . And, i n t e r e s t i n g l y , a l l

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tRNA s p e c i e s which a r e known t o c o n t a i n a c y t o k i n i n r e c o g n i z e codons w i t h t h e i n i t i a l base as u r a c i l ( U ) , a l t h o u g h n o t a l l tRNA s p e c i e s r e c o g n i z i n g codons b e g i n n i n g w i t h U c o n t a i n a c y t o k i n i n . The c y t o k i n i n - c o n t a i n i n g tRNA s p e c i e s a r e t R N A , t R N A ™ , t R N A ^ , t R N A P , t R N A , and t R N A . I n tRNA from m i c r o b i a l s o u r c e s , c y t o k i n i n a c t i v e r i b o n u c l e o s i d e s appear t o be p r e s e n t i n most, i f n o t a l l , o f the tRNA s p e c i e s t h a t r e c o g n i z e codons b e g i n n i n g w i t h U. But i n h i g h e r o r g a n i s m s , t h e d i s t r i b u t i o n appears t o be more r e s t r i c t e d . In t h e c a s e s o f wheat germ and e t i o l a t e d bean s e e d l i n g s , f o r exam­ p l e s , c y t o k i n i n - a c t i v e r i b o n u c l e o s i d e s are l i m i t e d to only t R N A and t R N A . S e r

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S e r

L e u

An i m p o r t a n t q u e s t i o n i s : A r e t h e c y t o k i n i n s p r e s e n t i n tRNA i n c o r p o r a t e d i n t a c t i n t o tRNA, i n a manner l i k e a l l o t h e r n u c l e o s i d e t r i p h o s p h a t e s t h a t a r e p o l y m e r i z e d i n t h e s y n t h e s i s o f tRNA, o r does the p r e s e n c e o f t h e c y t o k i n i n s r e s u l t from a l k y l a t i o n ( t r a n s f e r o f i s o p r e n o i d s i d e chain) of e x i s t i n g adenosine moieties already present i n preformed tRNA? I n f a c t , e x p e r i m e n t a l e v i d e n c e has been r e p o r t e d f o r b o t h p r o c e s s e s (21,22). But the amounts o f a c t u a l i n c o r p o r a t i o n were v e r y s m a l l , and t h e o b s e r v e d i n c o r p o r a t i o n may be m e r e l y t h e r e s u l t o f t r a n s c r i p t i o n a l e r r o r s . Meanwhile, t h e e v i d e n c e f o r c y t o ­ k i n i n m o i e t i e s i n tRNA a r i s i n g by a l k y l a t i o n o f s p e c i f i c a d e n o s i n e r e s i d u e s i n preformed tRNA i s c o n c l u s i v e ( 2 3 ) . There i s good e v i d e n c e t h a t t h e c y t o k i n i n m o i e t i e s i n tRNA a r e f u n c t i o n a l l y s i g n i f i c a n t and t h a t they do a f f e c t t h e b e h a v i o r o f t h o s e tRNA m o l e c u l e s i n t h e p r o c e s s o f p r o t e i n s y n t h e s i s . One o f t h e e a r l i e s t and most c o n c l u s i v e i n v e s t i g a t i o n s from w h i c h t h i s i m p o r t a n t f a c t emerged was c o n d u c t e d by G e f t e r and R u s s e l l (24) w i t h Escherichia c o l i . G e f t e r and R u s s e l l (24) s t u d i e d t h r e e forms o f t R N A w h i c h had the same n u c l e o t i d e sequence and d i f f e r e d o n l y i n t h e degree o f modi­ f i c a t i o n o f t h e a d e n o s i n e r e s i d u e a d j a c e n t t o t h e 3' end o f t h e a n t i ­ codon: (1) u n m o d i f i e d a d e n o s i n e ( A ) , (2) N ^ - ( A 2 - i s o p e n t e n y l ) a d e n o ­ s i n e ( i A ) , and (3) t h e m e t h y l - t h i o d e r i v a t i v e o f i A (ms2i6A). A l l t h r e e forms o f t h e tRNA were then t e s t e d f o r t y r o s i n e - a c c e p t o r a c t i ­ v i t y and f o r b i n d i n g o f t h e t y r o s y l - t R N A t o an mRNA-ribosome complex. No s i g n i f i c a n t d i f f e r e n c e s were found among t h e t h r e e forms o f tRNA as r e g a r d s amino a c i d a c c e p t o r c a p a c i t y . However, t h e tRNA^y c o n t a i n i n g an u n m o d i f i e d a d e n o s i n e r e s i d u e a d j a c e n t t o t h e a n t i c o d o n was markedly l e s s e f f e c t i v e , i n t h e i n v i t r o e x p e r i m e n t s , i n the b i n d i n g of the a m i n o a c y l - t R N A t o t h e mRNA-ribosome complex than t R N A c o n t a i n i n g i ^ A o r m s i A . Thus, i n t h i s i m p o r t a n t c a s e , a c y t o k i n i n m o i e t y e v i d e n t l y must be p r e s e n t a d j a c e n t t o t h e a n t i c o d o n o f t R N A , i f t h i s tRNA s p e c i e s i s t o f u n c t i o n e f f e c t i v e l y in protein synthesis. By whatever mechanism t h e r e g u l a t i o n i s a c h i e v e d , i t thus appears t h a t c y t o k i n i n - a c t i v e b a s e s i n c e r t a i n types o f tRNA may have a m o d u l a t i n g e f f e c t on p r o t e i n s y n t h e s i s a t the t r a n s l a t i o n a l l e v e l . T v r

6

6

T v r

r

T v r

T v r

2

6

T v r

There a r e good r e a s o n s p r e s e n t l y f o r b e l i e v i n g a l s o t h a t f r e e c y t o k i n i n s have i m p o r t a n t b i o l o g i c a l a c t i v i t y i n d e p e n d e n t l y o f any d i r e c t a s s o c i a t i o n w i t h tRNA: (1) t h e r e a r e r e s u l t s showing d i r e c t e f f e c t s o f exogenous c y t o k i n i n i n d e p e n d e n t l y o f any a p p a r e n t i n c o r ­ p o r a t i o n i n t o tRNA; (2) e t h a n o l i c e x t r a c t s o f c o r n k e r n e l s c o n t a i n the t r a n s - i s o m e r o f z e a t i n , w h i l e t h e tRNA h y d r o l y s a t e s o f c o r n k e r n e l s contain the c i s - i s o m e r , suggesting that z e a t i n i s not a pre­ c u r s o r i n t h e s y n t h e s i s o f tRNA i n t h a t m a t e r i a l ; (3) d i h y d r o z e a t i n

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i s a major f r e e c y t o k i n i n i n b e a n s , but i t a p p a r e n t l y does not o c c u r i n bean tRNA; (4) even t i s s u e s w h i c h c o n t a i n p o t e n t c y t o k i n i n s i n t h e i r RNA s t i l l r e q u i r e exogenous c y t o k i n i n f o r growth i n v i t r o ; and (5) the d a t a on d i r e c t i n c o r p o r a t i o n o f exogenous c y t o k i n i n s i n t o tRNA, as p r e v i o u s l y mentioned, do not show c o n c l u s i v e l y t h a t t h i s p r o c e s s i s a n y t h i n g more t h a n t r a n s c r i p t i o n a l e r r o r . A p r e v a l e n t i d e a about mechanisms o f a c t i o n o f p l a n t hormones, c y t o k i n i n f r e e b a s e s and o t h e r s , i s t h a t the hormone f i r s t b i n d s — b y weak hydrogen o r i o n i c b o n d s — t o some r e c e p t o r . The r e c e p t o r common­ l y i s e n v i s a g e d t o be an a l l o s t e r i c p r o t e i n w h i c h , as a consequence o f b i n d i n g the hormone, can, as the a l t e r e d r e c e p t o r o r as a r e c e p ­ tor-hormone complex, evoke hormonal a c t i o n (see 25). S i n c e about 1970, p r o g r e s s a l o n g t h e s e l i n e s w i t h r e s p e c t t o c y t o k i n i n s has been made by Fox and a s s o c i a t e s . A basic observation t h a t Fox and E r i o n (26) made was t h a t c y t o k i n i n s would b i n d , w i t h r a t h e r h i g h s p e c i f i c i t y , to ribosomes i s o l a t e d from h i g h e r p l a n t s . L a t e r , Fox and E r i o n (27) d e s c r i b e d a c t u a l l y t h r e e c y t o k i n i n - b i n d i n g p r o t e i n s i s o l a t e d f r o m r i b o s o m a l p r e p a r a t i o n s from wheat germ. Of p a r t i c u l a r i n t e r e s t was a f r a c t i o n o f medium m o l e c u l a r w e i g h t o f 93,000 (CBF-1) w i t h h i g h b i n d i n g a f f i n i t y f o r c y t o k i n i n s . A large number o f c y t o k i n i n s , c y t o k i n i n n u c l e o t i d e s , a n a l o g s , r e l a t e d p u r i n e s , and o t h e r p l a n t growth s u b s t a n c e s were t e s t e d f o r t h e i r a b i l i t y t o compete w i t h 6 - b e n z y l a m i n o p u r i n e ( o r N ^ - b e n z y l a d e n i n e ) f o r b i n d i n g s i t e s on CBF-1. A p p a r e n t l y , wheat germ ribosomes c o n t a i n one copy o f CBF-I p e r ribosome. C o m p e t i t i o n s t u d i e s showed a h i g h degree o f s p e c i f i c i t y for cytokinin-active moieties. More r e c e n t l y , a v e r y h i g h - a f f i n i t y b i n d i n g f a c t o r has been found a s s o c i a t e d w i t h a m i t o ­ c h o n d r i a l f r a c t i o n f r o m mung bean ( P h a s e o l u s a u r e u s ) s e e d l i n g s ( 2 8 ) . U n f o r t u n a t e l y , i t has not been p o s s i b l e y e t t o a s s i g n any b i o l o g i c a l f u n c t i o n t o the c y t o k i n i n b i n d i n g d e s c r i b e d above, and t h e s e b i n d i n g f a c t o r s cannot y e t be c o n s i d e r e d t o be t r u e c y t o k i n i n r e c e p t o r s . Abscisic

Acid

The mechanism o f a c t i o n o f a b s c i s i c a c i d (ABA) has been s t u d i e d t o the g r e a t e s t e x t e n t i n the b a r l e y a l e u r o n e system (29) , i n which ABA c o u n t e r a c t s the e f f e c t of GA i n the i n d u c t i o n o f h y d r o l a s e s . This a c t i o n o f ABA has l a r g e l y been t h e b a s i s f o r s p e c u l a t i n g t h a t ABA may a c t s p e c i f i c a l l y t o i n h i b i t , by some unknown mechanism, DNAdependent RNA s y n t h e s i s . Much e v i d e n c e i n d i c a t e s t h a t ABA a c t s a t the t r a n s c r i p t i o n a l l e v e l , but i t a l s o has been p r o p o s e d t h a t the i n h i b i t i o n o f i n d u c t i o n of α-amylase s y n t h e s i s i s c a u s e d , a t l e a s t i n p a r t , by an e f f e c t on t r a n s l a t i o n because ABA s t i l l i n h i b i t e d the f o r m a t i o n o f α-amylase a t 12 h r when c o r d y c e p i n (an i n h i b i t o r o f RNA s y n t h e s i s ) no l o n g e r had an e f f e c t (30). Some o t h e r r e c e n t work i n d i c a t e s an a p p a r e n t q u a l i t a t i v e e f f e c t on p r o t e i n s y n t h e s i s . Ho (31) and J a c o b s e n e t a l . (32) d e t e c t e d new peptides when a l e u r o n e l a y e r s were t r e a t e d w i t h ABA. These p e p t i d e s t u r n e d o v e r r a p i d l y , and the q u a n t i t i e s formed were r e d u c e d by i n ­ h i b i t o r s o f t r a n s c r i p t i o n and t r a n s l a t i o n . L i t t l e e l s e i s known c u r r e n t l y about the p e p t i d e s . Some p h y s i o l o g i c a l e f f e c t s o f ABA a r e c o r r e l a t e d w i t h a l t e r a t i o n o f plasma membranes. T h i s e f f e c t i s m a n i f e s t e d i n a change i n b i o ­ e l e c t r i c p o t e n t i a l a c r o s s the membranes and a l e a k i n e s s and e f f l u x of K+ w h i c h i s i n v o l v e d i n some a c t i o n s o f ABA, e.g., s t o m a t a l

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regulation. A t t h i s time the e f f e c t s on n u c l e i c a c i d and p r o t e i n s y n t h e s i s cannot be r e c o n c i l e d w i t h t h e s e e f f e c t s i n v o l v i n g changes i n membrane p e r m e a b i l i t y . Ethylene E t h y l e n e , C2H4, has many d i v e r s e e f f e c t s on p l a n t growth and d e v e l o p ­ ment. Yet the mechanism o f a c t i o n o f t h i s hormone i s not u n d e r s t o o d . A major o b s t a c l e to d i s c o v e r i n g i t s mechanism o f a c t i o n has been the l a c k o f an i s o l a t e d s u b c e l l u l a r system t h a t responds t o e t h y l e n e i n a way t h a t c l e a r l y r e f l e c t s i t s a c t i o n i n v i v o ( 3 3 ) . F r u i t t i s s u e s r e s p o n d t o e t h y l e n e by e x h i b i t i n g i n c r e a s e s i n the a c t i v i t i e s o f enzymes t h a t c a t a l y z e r i p e n i n g r e a c t i o n s , and i n some c a s e s , the i n c r e a s e s i n enzyme a c t i v i t y p r o b a b l y a r e the r e s u l t o f de novo s y n t h e s i s , r a t h e r than a c t i v a t i o n o f p r e e x i s t i n g enzymes. Other t a r g e t t i s s u e s respond s i m i l a r l y to ethylene. But i t i s n o t known whether e t h y l e n e a c t s d i r e c t l y t o evoke new enzyme p r o d u c t i o n . I n t e r p r e t a t i o n o f r e s u l t s w i t h i n h i b i t o r s o f RNA and p r o t e i n s y n t h e ­ s i s i s i n c o n c l u s i v e , because i t c o u l d be m e r e l y t h a t RNA and p r o t e i n s y n t h e s i s a r e e s s e n t i a l t o m a i n t a i n the c e l l s i n a s t a t e competent to respond to ethylene. Moreover, t h e r e a r e some r e s p o n s e s t o e t h y l ­ ene, b e s i d e s f r u i t r i p e n i n g , which o c c u r under c o n d i t i o n s w h i c h a p p a r e n t l y do not d i r e c t l y i n v o l v e RNA and p r o t e i n s y n t h e s i s ( e . g . , membrane p e r m e a b i l i t y c h a n g e s ) . I t has been p r o p o s e d t h a t the i n v i v o e t h y l e n e r e c e p t o r s i t e c o n t a i n s a m e t a l such as copper (34,35). Anthesins W i t h i n l e s s t h a n two decades a f t e r the d i s c o v e r y o f p h o t o p e r i o d i s m i n 1920, the h y p o t h e s i s d e v e l o p e d t h a t one o r more s p e c i f i c f l o w e r i n g hormones a r e r e s p o n s i b l e f o r f l o r a l i n i t i a t i o n (23,36-39). C h a i l a k h y a n (36) c o i n e d the word " f l o r i g e n " f o r the h y p o t h e t i c a l f l o w e r i n g hormone. A s h o r t time l a t e r , M e l c h e r s (40) s u g g e s t e d the term " v e r n a l i n " f o r the h y p o t h e t i c a l s t i m u l u s thought t o d e v e l o p dur­ i n g v e r n a l i z a t i o n of c o l d - r e q u i r i n g p l a n t s . Many y e a r s l a t e r , C h a i l a k h y a n p r o p o s e d the term " a n t h e s i n s " f o r h y p o t h e t i c a l f l o w e r i n g hormones (38) and gave emphasis t o the r o l e GAs p l a y i n f l o w e r i n g o f many p l a n t s . E a r l y e x p e r i m e n t s , c o n d u c t e d i n the 1930s and 1940s, w i t h c l a s s i c a l o b l i g a t e s h o r t - d a y p l a n t s such as c o c k l e b u r (Xanthium strumarium) y i e l d e d r e s u l t s w h i c h p r o v i d e d a v e r y l o g i c a l b a s i s f o r the f l o r i g e n ( a n t h e s i n ) c o n c e p t . Experiments i n v o l v i n g a l a r g e number o f p h o t o p e r i o d i c a l l y s e n s i t i v e s p e c i e s s u g g e s t e d s t r o n g l y t h a t the l e a f i s the o r g a n w h i c h p e r c e i v e s t h e p h o t o p e r i o d i c stimulus, t h a t phytochrome i s the p h o t o r e c e p t o r , and t h a t a s u b s t a n c e o r sub­ s t a n c e s i s formed i n the l e a v e s of s h o r t - d a y p l a n t s w h i c h i s t r a n s ­ l o c a t e d to v e g e t a t i v e meristems and c a u s e s t h e i r m o r p h o g e n e t i c t r a n s ­ f o r m a t i o n i n t o f l o r a l m e r i s t e m s . From numerous g r a f t i n g e x p e r i m e n t s between two s p e c i e s ( e . g . , Kalanchoë b l o s s f e l d i a n a , SDP, and Sedum s p e c t a b i l e , LDP), i t appeared t h a t t h e r e was o n l y one f l o r i g e n , o r i f two or more s u b s t a n c e s , t h e y e v i d e n t l y were p h y s i o l o g i c a l l y e q u i ­ v a l e n t among many s p e c i e s . The v a r i o u s p h o t o p e r i o d i c r e s p o n s e t y p e s would d i f f e r n o t i n t h e i r r e q u i r e m e n t f o r f l o r i g e n , but i n the en­ vironmental requirements f o r f l o r i g e n production. W i t h such e v i d e n c e c o m p a t i b l e w i t h a f l o r i g e n c o n c e p t , a l o g i c a l

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next step would be to attempt to isolate and chemically characterize the flowering stimulus or stimuli. In fact, there have been many such efforts. Generally the attempts have ended in failure; occa­ sionally there have been modest successes (41,42). Thus, there is much circumstantial evidence that flower initiation is controlled, at least in part, by anthesins. Meanwhile, certain of the known hormones definitely are involved in the regulation of flowering, as indicated by numerous investigations conducted with GAs since circa 1957 and with ABA since circa 1967. Lang (43) first reported in 1957 that exogenous GA could cause flowering in numerous species of long-day and vernalization-requiring plants under noninductive environmental conditions. However, GA is not the long sought after florigen. One reason is that GAs do not cause flowering of short-day plants under noninductive conditions, or even in a l l long-day plants. Moreover, there is now good evidence that flowering and flower-bearing stem elongation (bolting) are sepa­ rate processes in plants such as Silene armeria (44), with GA direct­ ly promoting stem elongation only. While GA is not florigen, i t con­ ceivably might be vernalin. During the early investigations of the effects of exogenous ABA on plants, i t was reported that this growth-inhibiting hormone could cause flowering of certain short-day plants (e.g., Chenopodium rubrum, Pharbitis n i l , and some others) under long-day conditions (23). But ABA is not a florigen because there is no effect of exo­ genous ABA on flowering of some short-day plants or on long-day plants. It may be one of the postulated flowering inhibitors, which have long been thought to be produced in the leaves of long-day plants subjected to noninductive short-day conditions. Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Paleg, L. G. Ann. Rev. Plant Physiol. 1965, 16, 291-322. Bandurski, R. S.; Nonhebel, H. M. In "Advanced Plant Physiol­ ogy"; Wilkins, Μ. Β . , Ed.; Pitman: Marshfield, MA, 1984; pp. 1-20. Wilkins, M. B. (Ed.) "Advanced Plant Physiology"; Pitman: Marshfield, MA, 1984; 514 pp. Rayle, D. L.; Cleland, R. Plant Physiol. 1970, 46, 250-3. Hager, Α.; Menzel, H . ; Krauss, A. Planta 1971, 100, 47-75. Silberger, J.; Skoog, F. Science 1953, 118, 443-4. Key, J . L. Ann. Rev. Plant Physiol. 1969, 20, 449-74. Zurfluh, L. L.; Guilfoyle, T. J . Plant Physiol. 1982, 69, 332-7. Theologis, Α.; Ray, P. M. In "Plant Growth Substances 1982"; Wareing, P. F . , Ed.; Academic: London, 1982; pp. 43-57. Vanderhoef, L. N.; Dute, R. R. Plant Physiol. 1981, 67, 146-9. Jones, R. L.; MacMillan, J . In "Advanced Plant Physiology"; Wilkins, Μ. Β . , Ed.; Pitman: Marshfield, MA, 1984; pp. 21-52. Radley, M. Planta 1967, 75, 164-71. Filner, P.; Varner, J . E. Proc. Natl. Acad. Sci. U.S.A. 1967, 58, 1520-6. Jacobsen, J . V . ; Varner, J . E. Plant Physiol. 1967, 42, 15961600. Bennett, P. Α.; Chrispeels, M. J . Plant Physiol. 1972, 49, 445-7.

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