Biotechnology for Crop Protection - American Chemical Society

Chapter 3

5—Enolpyruvylshikimate 3—Phosphate Synthase

Downloaded by UNIV OF CALIFORNIA SAN DIEGO on June 5, 2015 | http://pubs.acs.org Publication Date: November 22, 1988 | doi: 10.1021/bk-1988-0379.ch003

From Biochemistry to Genetic Engineering of Glyphosate Tolerance G. Kishore, D. Shah, S. Padgette, G. della-Cioppa, C. Gasser, D. Re, C. Hironaka, M. Taylor, J. Wibbenmeyer, D. Eichholtz, M. Hayford, N. Hoffmann, X. Delannay, R. Horsch, H. Klee, S. Rogers, D. Rochester, L. Brundage, P. Sanders, and R. T. Fraley Plant Molecular Biology Group, Monsanto Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198 Glyphosate, the active ingredient of the nonselective, post-emergent, systemic herbicide Roundup®, has broad spectrum a c t i v i t y against a wide range of annual and perennial plants. Roundup® is currently used i n a v a r i e t y of agronomic and nonagronomic situations for vegetation c o n t r o l . However, lack of s e l e c t i v i t y of t h i s herbicide has prevented i t s use as an over-the-crop herbicide for e f f i c i e n t weed c o n t r o l . In this a r t i c l e , we describe two methods for engineering glyphosate tolerance into crop plants. In the f i r s t method, tolerance i s achieved by overproduction of 5-enolpyruvylshikimate 3-phosphate synthase (EPSPS), the enzyme target for glyphosate, involved i n the b i o synthesis of aromatic amino acids i n both plants and microbes. The second method for engineering tolerance is based on expression of glyphosate tolerant mutant EPSPS gene. Transgenic plants producing the mutant enzyme are tolerant to Roundup®. With both methods, the tolerance of transgenic calli to glyphosate i s s i g n i f i cantly lower when the enzyme i s l o c a l i z e d i n the cytosol instead of chloroplasts. Glyphosate i s t h e a c t i v e i n g r e d i e n t o f t h e n o n s e l e c t i v e , postemergent, systemic, foliar applied herbicide, Roundup® (1 ). Roundup® k i l l s a wide range o f b o t h annual and p e r e n n i a l p l a n t s . I n a d d i t i o n t o b e i n g n o n t o x i c t o mammals and f i s h , g l y p h o s a t e i s r a p i d l y i n a c t i v a t e d b y i n t e r a c t i o n s w i t h s o i l and i s a l s o r e a d i l y m e t a b o l i z e d b y s o i l microorganisms ( 2 ) . D e s p i t e t h e s e o u t s t a n d i n g e n v i r o n m e n t a l and weed c o n t r o l c h a r a c t e r i s t i c s , g l y p h o s a t e has o n l y l i m i t e d u t i l i t y during t h e a c t i v e growth season o f crops and v e g e t a b l e s , because i t k i l l s t h e crops as w e l l as t h e weeds. The h e r b i c i d e i s p r i m a r i l y used i n v e g e t a t i o n c o n t r o l , low o r n o - t i l l f a r m i n g and i n weed c o n t r o l d u r i n g c r o p growth u s i n g special applicators f o r herbicide delivery. Recent developments i n p l a n t 0097-6156/88A)379-0037$06.00/0 « 1988 American Chemical Society

In Biotechnology for Crop Protection; Hedin, P., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1988.


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BIOTECHNOLOGY FOR CROP PROTECTION

g e n e t i c e n g i n e e r i n g have p r o v i d e d new t e c h n i q u e s f o r i n t r o d u c t i o n o f h e r b i c i d e t o l e r a n c e genes i n t o crop p l a n t s ( 3 ) . I n t h i s a r t i c l e , we d i s c u s s the s t a t u s o f g e n e t i c e n g i n e e r i n g o f Roundup® t o l e r a n c e t o p l a n t s . Our e f f o r t s towards e n g i n e e r i n g Roundup® t o l e r a n c e have been f a c i l i t a t e d by an u n d e r s t a n d i n g o f the mechanism o f h e r b i c i d a l a c t i o n of glyphosate. J a w o r s k i (4) r e p o r t e d t h a t growth i n h i b i t i o n o f b o t h p l a n t and microbes by g l y p h o s a t e c o u l d be r e v e r s e d by a r o m a t i c amino a c i d s . F u r t h e r work o f Amrhein and h i s coworkers r e v e a l e d t h a t g l y p h o s a t e i n h i b i t s the s h i k i m a t e pathway enzyme, 5 - e n o l p y r u v y l s h i k i m a t e 3phosphate (EPSP) synthase ( 5 ) . T h i s enzyme c a t a l y z e s the r e a c t i o n shown i n F i g u r e 1. G l y p h o s a t e - t r e a t e d p l a n t and b a c t e r i a l c u l t u r e s accumulate s h i k i m a t e and/or s h i k i m a t e 3-phosphate ( S 3 P ) , c o n f i r m i n g t h a t i n h i b i t i o n o f EPSPS i s a t l e a s t a p a r t o f the i n v i v o mechanism o f a c t i o n o f t h i s h e r b i c i d e ( 6 , 7 ) . EPSPS has been i s o l a t e d from b o t h m i c r o o r g a n i s m s and p l a n t s , and s e v e r a l o f i t s p r o p e r t i e s have been s t u d i e d . The b a c t e r i a l and p l a n t enzymes a r e mono f u n c t i o n a l w i t h m o l e c u l a r mass o f 44-48 kD (8-15). The f u n g a l enzyme i s a p a r t o f the m u l t i f u n c t i o n a l arom complex w h i c h c a t a l y z e s f o u r o t h e r r e a c t i o n s o f the shikimate pathway ( 1 6 ) . W h i l e the b a c t e r i a l enzymes show d i f f e r e n c e s w i t h r e s p e c t t o g l y p h o s a t e s e n s i t i v i t y , the p l a n t enzymes e x h i b i t a much more narrow range o f s e n s i t i v i t y (11). T h i s accounts f o r the s u s c e p t i b i l i t y o f most p l a n t s p e c i e s t o g l y p h o s a t e . Based on s t e a d y s t a t e k i n e t i c s t u d i e s , EPSPS i n i t i a l l y forms a complex w i t h S3P w h i c h i n t e r a c t s w i t h the second s u b s t r a t e , phosp h o e n o l p y r u v a t e (PEP) t o form the t e r n a r y enzyme*S3P*PEP complex (18), Anderson, K. ; S i k o r s k i , J . ; Johnson, K. Biochemistry, in press). During t u r n o v e r , i n o r g a n i c phosphate ( P i ) i s r e l e a s e d f o l l o w e d by EPSP. G l y p h o s a t e i n h i b i t s EPSPS c o m p e t i t i v e l y w i t h r e s p e c t t o PEP and u n c o m p e t i t i v e l y w i t h r e s p e c t t o S3P (19). A l t h o u g h b i n d i n g o f PEP t o EPSPS can be d e t e c t e d i n the absence o f S3P, b i n d i n g o f g l y p h o s a t e o c c u r s o n l y i n the p r e s e n c e o f S3P. In the t e r n a r y complex o f enzyme*S3P*glyphosate, the Kd f o r S3P i s lower t h a n i n the b i n a r y enzyme*S3P complex. Thus the b i n d i n g o f S3P t o the enzyme i s enhanced i n the p r e s e n c e o f g l y p h o s a t e . W h i l e g l y p h o s a t e i n h i b i t s EPSPS c o m p e t i t i v e l y w i t h r e s p e c t t o PEP and thus may occupy the PEP b i n d i n g s i t e on the enzyme, i t i s i n t e r e s t i n g t o n o t e t h a t g l y p h o s a t e does not i n h i b i t any other PEP-dependent enzymatic r e a c t i o n (19, 20). Mechanistic studies i n d i c a t e t h a t d u r i n g the EPSPS r e a c t i o n , the C-2 carbon o f PEP i n t e r a c t s w i t h a n u c l e o p h i l e g e n e r a t i n g a t e t r a h e d r a l carbon a t C-3 o f PEP (21-24). I t has a l s o been suggested t h a t the C-2 o f PEP may be p o l a r i z e d , g e n e r a t i n g a carbonium i o n and t h a t the c a t i o n i c p r o t o n a t e d n i t r o g e n o f g l y p h o s a t e may mimic t h i s carbonium i o n . T h i s a s p e c t o f the mechanism o f i n t e r a c t i o n between g l y p h o s a t e and EPSPS remains unknown. I n a d d i t i o n t o the formation of a t e t r a h e d r a l c a r b o n a t C-3 o f PEP d u r i n g the EPSPS r e a c t i o n , i t i s the C-0 bond o f PEP t h a t i s c l e a v e d , and not the 0-P bond. T h i s i s i n c o n t r a s t t o most o t h e r PEP u t i l i z i n g enzymes. These f e a t u r e s may account f o r the s p e c i f i c i t y o f g l y p h o s a t e f o r i n h i b i t i o n o f the EPSPS e n z y m a t i c r e a c t i o n ( 2 5 ) . Based on the knowledge t h a t g l y p h o s a t e i n h i b i t s EPSPS, two mechanisms were e v a l u a t e d f o r g e n e t i c e n g i n e e r i n g o f glyphosate



3.

KISHORE ET AL.

Shlkimate-3-phosphate (S3P)

39

EPSP Synthase

Phosphoenol pyruvate (PEP)

5-Enolpyruvyl-sh!kimate«3-phosphate (EPSP)

F i g u r e 1 EPSP Synthase C a t a l y z e d R e a c t i o n .



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tolerance. The f i r s t mechanism was based on o v e r p r o d u c t i o n o f w i l d t y p e EPSPS; g l y p h o s a t e t o l e r a n c e may t h e r e f o r e be d e r i v e d from t h e residual EPSPS a c t i v i t y o f t h e u n i n h i b i t e d enzyme ( 2 6 ) . I n a d d i t i o n , t h e reduced c e l l u l a r c o n c e n t r a t i o n o f f r e e g l y p h o s a t e due to complex f o r m a t i o n w i t h EPSPS may a l s o c o n t r i b u t e towards t o l e r a n c e t o t h e h e r b i c i d e . The second mechanism was based on isolation o f glyphosate tolerant mutant EPSPS enzymes and e x p r e s s i o n o f t h e s e mutant genes i n p l a n t s ( 2 7 , 2 8 ) . C o n t r a r y t o the f i r s t mechanism, t h e c o n t r i b u t i o n o f g l y p h o s a t e b i n d i n g t o mutant EPSPS i s m i n i m a l and t o l e r a n c e i s d e r i v e d o n l y i f EPSPS i s the s o l e t a r g e t f o r glyphosate. As d i s c u s s e d i n t h i s a r t i c l e , c h l o r o p l a s t - t a r g e t e d mutant EPSP s y n t h a s e s c o n f e r h i g h e r l e v e l o f glyphosate t o l e r a n c e t h a n t h e w i l d t y p e enzyme t o t r a n s g e n i c tobacco p l a n t s . O v e r p r o d u c t i o n o f EPSPS has been observed i n s e v e r a l p l a n t c e l l c u l t u r e s t o l e r a n t t o g l y p h o s a t e ( 1 2 , 13, 2 9 ) . I n t h e case o f g l y p h o s a t e - t o l e r a n t C o r y d a l i s c u l t u r e s , Smart e t a l . demonstrated by 2 D-gel e l e c t r o p h o r e s i s , t h e o v e r p r o d u c t i o n o f o t h e r p r o t e i n s b e s i d e s EPSPS. Since the l e v e l s o f a c t i v i t y o f several shikimate pathway enzymes were u n a l t e r e d i n t h e t o l e r a n t c e l l l i n e compared t o t h e p a r e n t c e l l l i n e , i t was c o n c l u d e d t h a t t h e s e a m p l i f i e d p r o t e i n s may n o t be i n v o l v e d i n a r o m a t i c amino a c i d b i o s y n t h e s i s . I t i s p o s s i b l e t h a t t h e o t h e r p r o t e i n s may n o t have a r o l e i n t h e t o l e r a n c e mechanism. A l t e r a t i o n s i n p r o t e i n p r o f i l e s between g l y p h o s a t e - s e n s i t i v e and t o l e r a n t p e t u n i a c e l l l i n e s have a l s o been observed. With the glyphosate tolerant carrot c e l l line, i n a d d i t i o n t o o v e r p r o d u c t i o n o f EPSPS, t h e l e v e l s o f a r o m a t i c amino a c i d s were found t o be enhanced ( 2 9 ) . Based on t h e r e s u l t s w i t h p l a n t c e l l c u l t u r e s , i t was t h e r e f o r e n o t c l e a r i f o v e r p r o d u c t i o n o f EPSPS was s u f f i c i e n t t o o b t a i n g l y p h o s a t e t o l e r a n c e i n p l a n t s . A v a i l a b i l i t y o f the petunia c e l l l i n e overproducing EPSPS significantly aided i n the purification o f t h e enzyme t o homogeneity and e l u c i d a t i o n o f i t s amino t e r m i n a l amino a c i d sequence ( 1 2 , 2 6 ) . The sequence c o r r e s p o n d i n g t o amino a c i d s 8 t o 13 was u t i l i z e d f o r s y n t h e s i s o f t h r e e f a m i l i e s o f heptadecameric oligonucleotide probes (each 32 f o l d degenerate) and were d e s i g n a t e d EPSP1, EPSP2 and EPSP3 r e s p e c t i v e l y . By N o r t h e r n b l o t analysis, EPSP1 was found t o have t h e c o r r e c t sequence o f o l i g o n u c l e o t i d e s t o encode t h e EPSP s y n t h a s e mRNA. U s i n g EPSP1, a A g t l O cDNA l i b r a r y o f t h e g l y p h o s a t e - t o l e r a n t p e t u n i a c e l l l i n e was s c r e e n e d and a f u l l l e n g t h cDNA c l o n e o f p e t u n i a EPSPS was i s o l a t e d (26). A d d i t i o n a l studies i n d i c a t e d t h a t the increased a c t i v i t y o f EPSPS i n t h e g l y p h o s a t e t o l e r a n t p e t u n i a c e l l l i n e was due t o a m p l i f i c a t i o n o f t h e EPSPS gene r e s u l t i n g i n a n i n c r e a s e d s y n t h e s i s o f EPSP s y n t h a s e mRNA and hence p r o t e i n . A s i m i l a r i n c r e a s e i n t h e gene copy number has been demonstrated f o r g l y p h o s a t e t o l e r a n t c a r r o t s o m a t i c h y b r i d c e l l s ( 3 0 ) whereas i n C o r y d a l i s , no gene amplification has been detected (Amrhein, N. Personal Communication). N u c l e o t i d e sequence d e t e r m i n a t i o n o f t h e p e t u n i a EPSPS cDNA c l o n e r e v e a l e d t h a t i t encoded a p r e c u r s o r p r o t e i n (preEPSPS) w i t h an a d d i t i o n a l 72 amino a c i d s a t t h e amino t e r m i n a l end o f t h e EPSPS amino a c i d sequence d e t e r m i n e d f o r t h e p u r i f i e d p r o t e i n . S i n c e i t had been suggested t h a t enzymes o f t h e s h i k i m a t e pathway may be



3.

KISHORE ET AL.

EPSP Synthase

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l o c a l i z e d i n the c h l o r o p l a s t s (11., 31-35), i t was o f i n t e r e s t t o determine i f the overproduced EPSPS i n the g l y p h o s a t e t o l e r a n t petunia cell l i n e was l o c a l i z e d i n the c y t o s o l o r plastids. A n a l y s i s o f the p l a s t i d i c and c y t o s o l i c f r a c t i o n s o f the g l y p h o s a t e t o l e r a n t p e t u n i a c e l l l i n e r e v e a l e d t h a t the overproduced EPSPS was p l a s t i d - l o c a l i z e d (36). These s t u d i e s suggested t h a t the amino t e r m i n a l e x t e n s i o n o f preEPSPS may be i n v o l v e d i n the t r a n s l o c a t i o n o f the c y t o s o l i c a l l y s y n t h e s i z e d p r o t e i n t o the p l a s t i d s , analogous to the import o f o t h e r p r o t e i n s by c h l o r o p l a s t s (37-39). I n o r d e r t o v e r i f y the r o l e o f the amino t e r m i n a l e x t e n s i o n o f preEPSPS i n i t s import by i s o l a t e d c h l o r o p l a s t s , the cDNA o f p e t u n i a EPSPS was c l o n e d i n t o a T7/SP6 t r a n s c r i p t i o n system f o r the in vitro s y n t h e s i s o f EPSPS mRNA ( 4 0 ) . T r a n s l a t i o n o f the mRNA i n the p r e s e n c e o f 3 5 S - l a b e l e d m e t h i o n i n e r e s u l t e d i n the s y n t h e s i s o f r a d i o l a b e l e d preEPSPS. The m o l e c u l a r weight o f t h i s p r o t e i n was ~8kD h i g h e r t h a n the m o l e c u l a r w e i g h t o f EPSPS p u r i f i e d from p e t u n i a c e l l l i n e s (55 vs 48 kD). I n c u b a t i o n o f the preEPSPS w i t h c h l o r o p l a s t s i s o l a t e d from l e t t u c e l e a v e s , i n the presence o f ATP and l i g h t , r e s u l t e d i n a r a p i d uptake o f the p r o t e i n from the medium t o the c h l o r o p l a s t s ( 4 0 ) . The imported p r o t e i n had a lower m o l e c u l a r mass (48 kD) i n d i c a t i n g i t s r a p i d p r o c e s s i n g t o the mature form o f EPSPS, s i m i l a r t o t h a t i s o l a t e d from the g l y p h o s a t e tolerant petunia c e l l l i n e . S i m i l a r import and p r o c e s s i n g c o u l d a l s o be demonstrated w i t h c h l o r o p l a s t s prepared from the l e a v e s o f other p l a n t s . T h i s i n d i c a t e s the c o n s e r v a t i o n o f b o t h import and p r o c e s s i n g mechanisms i n c h l o r o p l a s t s o f d i f f e r e n t p l a n t s . E x a m i n a t i o n o f the EPSPS a c t i v i t y o f the preEPSP synthase r e v e a l e d t h a t the preenzyme was c a t a l y t i c a l l y a c t i v e (40). No significant differences between the preenzyme and the mature enzyme c o u l d be detected e i t h e r with respect to a c t i v i t y or glyphosate s e n s i t i v i t y . These s t u d i e s suggest t h a t the c a t a l y t i c and c h l o r o p l a s t t r a n s i t p e p t i d e domains o f preEPSP s y n t h a s e are d i s t i n c t and i n d e p e n d e n t l y folded. More r e c e n t s t u d i e s i n d i c a t e t h a t the import o f preEPSP synthase by i s o l a t e d c h l o r o p l a s t s i s i n h i b i t e d by S3P plus glyphosate ( 4 1 ) . The e x t e n t o f maximal i n h i b i t i o n i s o n l y about 70-80%. The r e s i d u a l import r a t e c o u l d be r e f l e c t i v e o f the r a t e o f import o f the preenzyme*S3P*glyphosate complex. S i n c e complex f o r m a t i o n between these l i g a n d s and the preenzyme i s c o n f o r m a t i o n a l l y r e s t r i c t i v e , i t i s evident that c o n f o r m a t i o n a l changes i n the p r e p r o t e i n o c c u r d u r i n g i t s passage t h r o u g h the c h l o r o p l a s t membranes. Whether i n h i b i t i o n o f import o f the preenzyme i s a p h y s i o l o g i c a l m o d e - o f - a c t i o n o f g l y p h o s a t e under in vivo c o n d i t i o n s has not been determined. More r e c e n t l y , the n u c l e o t i d e sequences o f tomato and A r a b i d o p s i s EPSPS genes have been determined (42, 4 3 ) . I t i s i n t e r e s t i n g to note t h a t the mature p l a n t EPSPS shows a h i g h degree o f c o n s e r v a t i o n a t the amino a c i d l e v e l (>85%). Comparison o f the p l a n t EPSPS sequences w i t h b a c t e r i a l and f u n g a l EPSPS sequences r e v e a l s t h a t t h e r e i s a 38% i d e n t i t y between p l a n t and fungal enzymes w h i l e between the b a c t e r i a l and p l a n t enzymes, t h e r e i s a 54% i d e n t i t y . The t r a n s i t p e p t i d e s o f the p l a n t enzymes a r e n o t conserved t o a s i m i l a r e x t e n t ; between the A r a b i d o p s i s and p e t u n i a t r a n s i t p e p t i d e s , ~23% i d e n t i t y i s observed. The same p a t t e r n o f



42


c o n s e r v a t i o n has been observed w i t h o t h e r c h l o r o p l a s t t a r g e t e d plant proteins (44). F o l l o w i n g i t s i s o l a t i o n , t h e p e t u n i a EPSP synthase cDNA c l o n e was e n g i n e e r e d i n t o t h e T i p l a s m i d v e c t o r system s u i t a b l e f o r p l a n t t r a n s f o r m a t i o n . I n t h i s c o n s t r u c t , t h e CaMV 35S promoter cons t i t u t e d t h e 5' end o f t h e gene w h i l e t h e 3' f l a n k i n g sequence i n c l u d i n g t h e p o l y a d e n y l a t i o n s i g n a l was d e r i v e d from t h e n o p a l i n e synthase gene ( 2 6 ) . T r a n s f o r m a t i o n o f p e t u n i a l e a f d i s c s u s i n g t h i s c o n s t r u c t r e s u l t e d i n t h e p r o d u c t i o n o f c a l l i which t o l e r a t e d glyphosate treatment at 0.5mM and l.OmM. Under identical c o n d i t i o n s , c a l l i transformed w i t h a c o n t r o l v e c t o r l a c k i n g the p e t u n i a EPSPS cDNA d i d n o t s u r v i v e g l y p h o s a t e t r e a t m e n t . The e x t e n t o f o v e r p r o d u c t i o n o f EPSPS i n c a l l i r e c e i v i n g t h e EPSP synthase gene was 40-80 f o l d . These experiments demonstrated t h a t o v e r p r o d u c t i o n o f EPSPS c o n s t i t u t e d a v i a b l e mechanism f o r t h e engineering of glyphosate tolerance to p l a n t c e l l s . In o r d e r t o determine i f t h i s mechanism c o u l d a l s o c o n f e r t o l e r a n c e t o Roundup® a t t h e whole p l a n t l e v e l , p e t u n i a p l a n t s were r e g e n e r a t e d from t h e s e t r a n s f o r m e d c a l l i . The t r a n s f o r m e d p l a n t s were sprayed w i t h 0.81bs/acre o f Roundup®. W i t h i n two weeks f o l l o w i n g t h e s p r a y , t h e c o n t r o l p l a n t s were k i l l e d w h i l e t h e t r a n s g e n i c p l a n t s o v e r p r o d u c i n g EPSPS were o n l y s l i g h t l y a f f e c t e d . These p l a n t s however d i s p l a y e d s l i g h t c h l o r o s i s i n t h e growing t i p s and t h e newly emerged l e a v e s . This i s not s u r p r i s i n g since g l y p h o s a t e accumulates p r i m a r i l y i n t h e m e t a b o l i c s i n k r e g i o n s o f b o t h shoots and r o o t s ( 4 5 ) . The e x t e n t o f o v e r p r o d u c t i o n o f EPSP synthase may n o t be t h e r e f o r e s u f f i c i e n t t o c o n f e r complete t o l e r a n c e i n t h e s e t i s s u e s o f t h e sprayed p l a n t . I f i n h i b i t i o n o f EPSPS i s t h e p r i m a r y e f f e c t o f g l y p h o s a t e , i t s h o u l d be p o s s i b l e t o e n g i n e e r g l y p h o s a t e t o l e r a n c e by t h e use o f mutant enzymes. The e f f e c t i v e n e s s o f t h i s approach i s n o t o n l y dependent upon t h e l e v e l o f t o l e r a n c e o f t h e mutant enzyme b u t a l s o the V/K r a t i o o f t h e mutant enzyme compared t o t h e w i l d t y p e . G l y p h o s a t e t o l e r a n t mutants w h i c h d i s p l a y v e r y h i g h l e v e l t o l e r a n c e a l s o show s i g n i f i c a n t i n c r e a s e s i n t h e Km f o r PEP ( 2 5 , 46-48). Two o f t h e most s t u d i e d g l y p h o s a t e t o l e r a n t mutant enzymes a r e those from K l e b s i e l l a pneumoniae and Escherichia coli. A third g l y p h o s a t e t o l e r a n t mutant from S a l m o n e l l a typhimurium has a l s o been d e s c r i b e d ; t h i s enzyme does n o t appear t o be as t o l e r a n t t o g l y p h o s a t e as t h e o t h e r two mutants ( 4 9 ) . The K. pneumoniae enzyme e x h i b i t s an 1-50 o f ~50 mM f o r g l y p h o s a t e . I t s appKm f o r S3P i s 193 mM and appKm f o r PEP i s 140 mM. This i s i n c o n t r a s t t o the v a l u e s o f 45 mM and 9 mM f o r t h e Km's o f S3P and PEP r e s p e c t i v e l y f o r t h e w i l d t y p e enzyme. The mutant enzyme was more a c i d i c t h a n t h e w i l d t y p e w i t h p i v a l u e o f 4.1 compared t o 4.6. Based on s p e c i f i c a c t i v i t y d e t e r m i n a t i o n s , i t appears t h a t t h e mutant enzyme has about one t h i r d o f t h e a c t i v i t y o f w i l d t y p e . I n t e r e s t i n g l y , by SDS g e l e l e c t r o p h o r e s i s , a s l i g h t i n c r e a s e i n t h e m o l e c u l a r mass o f the mutant enzyme was a l s o d e t e c t e d . The amino a c i d change r e s p o n s i b l e f o r t h e g l y p h o s a t e t o l e r a n c e w i t h t h i s mutant i s n o t known. The mutant enzyme from £. coli e x h i b i t s s i m i l a r changes i n k i n e t i c c o n s t a n t s as t h e K. pneumoniae enzyme. I t s Km f o r S3P i s a l t e r e d -4 f o l d (80 mM v s . 19 mM) w h i l e t h a t f o r PEP i s a l t e r e d -20



3.

KISHORE ET AL.

EPSP Synthase

43

f o l d (220 mM v s . 10 mM). The K i f o r g l y p h o s a t e o f t h e mutant enzyme was 4 mM compared t o 0.5 mM f o r t h e w i l d t y p e . The Vmax o f the mutant enzyme was - 6 0 % o f w i l d type EPSPS. Unlike the K. pneumoniae mutant, t h e Ε. coli mutant d i d n o t e x h i b i t any changes i n e i t h e r t h e m o l e c u l a r weight o r i s o e l e c t r i c p o i n t . Based on t h e E. coli mutant EPSPS, a number o f p l a n t g l y p h o s a t e t o l e r a n t EPSPS enzymes have been c o n s t r u c t e d by s i t e d i r e c t e d mutagenesis o f p l a n t EPSPS genes. One o f t h e k i n e t i c a l l y - c h a r a c t e r i z e d p e t u n i a mutant EPSPS showed no change i n Km f o r S3P, an 80 f o l d i n c r e a s e i n i t s Km f o r PEP and - 6 5 % o f t h e Vmax o f t h e w i l d t y p e . In the r e v e r s e r e a c t i o n , t h i s mutant d i s p l a y e d no change i n Km f o r EPSP but a 1 0 - f o l d i n c r e a s e i n i t s Km f o r P i . The mutant had a K i (glyphosate) o f 3 mM ( 2 5 ) . Based on t h e s e r e s u l t s , i t can be c o n c l u d e d t h a t i n t h i s mutant t h e b i n d i n g o f t h e phosphate m o i e t y o f PEP i s a l t e r e d . T h i s r e g i o n o f t h e enzyme must a l s o be c r i t i c a l for i n t e r a c t i o n of glyphosate w i t h EPSPS s i n c e t h e b i n d i n g o f g l y p h o s a t e i s a f f e c t e d t o a g r e a t e r e x t e n t than t h a t o f PEP. I t i s not c l e a r as t o w h i c h m o i e t y o f g l y p h o s a t e i s i n v o l v e d i n i n t e r a c t i o n w i t h t h i s region of the a c t i v e s i t e although i t i s tempting t o s p e c u l a t e t h a t i t i s t h e phosphonate m o i e t y . In addition, i t i s not understood i f t h e d i f f e r e n c e s i n b i n d i n g o f PEP versus glyphosate a r e due t o s e l e c t i v e s t e r i c problems a s s o c i a t e d w i t h b i n d i n g o f g l y p h o s a t e o r l o s s o f a few r e c o g n i t i o n s i t e s due t o c o n f o r m a t i o n a l changes. S i n c e t h e k i n e t i c c o n s t a n t s f o r t h e mutant enzymes w i t h r e s p e c t t o PEP a r e a f f e c t e d , i t i s t o be e x p e c t e d t h a t t h e s e enzymes w i l l have t o be overproduced depending on t h e i n t r a c e l l u l a r c o n c e n t r a t i o n o f PEP and t h e Km(PEP) f o r t h e mutant enzyme. The E. coli mutant EPSPS gene was engineered i n t o a p l a n t e x p r e s s i o n v e c t o r (pMON8078) and used f o r t r a n s f o r m a t i o n o f t o b a c c o l e a f d i s c s . I n t h i s c o n s t r u c t , t h e mutant EPSPS gene was d r i v e n by the CaMV 35S promoter and t h e 3 - p o l y a d e n y l a t i o n s i g n a l was d e r i v e d from t h e n o p a l i n e s y n t h a s e gene. C a l l i producing the mutant E. coli EPSPS d i d n o t show any s i g n i f i c a n t g l y p h o s a t e t o l e r a n c e compared t o untransformed c o n t r o l s (G. K i s h o r e e t a l . U n p u b l i s h e d d a t a ) . A n a l y s i s o f t h e c a l l i however r e v e a l e d t h e p r e s e n c e o f t h e mutant enzyme (Table I ) i n d i c a t i n g t h a t the l a c k o f t o l e r a n c e was n o t due t o inadequate e x p r e s s i o n o f t h e b a c t e r i a l mutant gene. Tobacco p l a n t s e x p r e s s i n g t h e b a c t e r i a l mutant EPSP s y n t h a s e were r e g e n e r a t e d from t r a n s f o r m e d c a l l i and s p r a y e d w i t h 0.4 l b s / a c o f Roundup®. W h i l e t h e s e p l a n t s d i s p l a y e d t o l e r a n c e t o t h e h e r b i c i d e , t h e l e v e l o f t o l e r a n c e was not s i g n i f i cant. The h e r b i c i d e caused s i g n i f i c a n t a p i c a l damage, growth o f l a t e r a l shoots and i n h i b i t i o n o f growth r a t e . These experiments e s t a b l i s h e d t h a t t h e c y t o s o l i c p r o d u c t i o n o f t h e E. coli mutant EPSPS d i d n o t c o n f e r adequate g l y p h o s a t e t o l e r a n c e t o tobacco c a l l i . Since aromatic amino acid biosynthesis occurs i n the c h l o r o p l a s t o f p l a n t s , and t h e p e t u n i a EPSPS was demonstrated t o c o n t a i n the i n f o r m a t i o n f o r i t s t r a n s l o c a t i o n t o the c h l o r o p l a s t , i t was o f i n t e r e s t t o determine i f d e l i v e r y o f t h e b a c t e r i a l mutant enzyme t o c h l o r o p l a s t o f t r a n s g e n i c p l a n t s a f f e c t e d t h e l e v e l o f t o l e r a n c e t o Roundup®. A h y b r i d gene was s y n t h e s i z e d c o n t a i n i n g !


44



Table I EPSPS s p e c i f i c activités o f c a l l i t r a n s f o r m e d w i t h EPSPS genes w i t h and w i t h o u t c h l o r o p l a s t t r a n s i t sequences

ESPS S p e c i f i c a c t i v i t y , nmol/min*mg

0 mM g l y p h o s a t e i n assay m i x t u r e

Construct

pM0N542 pM0N8078 pM0N546 pMON8083 pM0N505

ι r. + gip tp_ glP tp glp tp_ glp t p Control r

s

g

+

60 71 198 44 8

0.5 mM g l y p h o s a t e i n assay m i x t u r e

33 68 0 0 0

the t r a n s i t p e p t i d e o f p e t u n i a EPSP synthase p l u s t h e f i r s t 27 amino a c i d s o f t h e mature p e t u n i a enzyme f u s e d t o t h e remainder o f E. coli mutant EPSP s y n t h a s e . T h i s gene was e x p r e s s e d i n vitro under t h e c o n t r o l o f T7 promoter. The i n vitro synthesized polypeptide had EPSP synthase activity w h i c h was glyphosate t o l e r a n t and was r a p i d l y imported by i s o l a t e d c h l o r o p l a s t s ( 5 0 ) . The imported c h i m e r i c p r o t e i n was p r o c e s s e d by p r o t e o l y s i s t o a mature, c a t a l y t i c a l l y a c t i v e enzyme. Tobacco l e a f d i s c s t r a n s f o r m e d w i t h t h e h y b r i d p e t u n i a / E . c o l i mutant EPSP synthase gene produced c a l l i w h i c h were t o l e r a n t t o glyphosate. I n t h i s c o n s t r u c t (pM0N542) t h e h y b r i d gene was d r i v e n by t h e CaMV 35S promoter. The l e v e l o f e x p r e s s i o n o f t h e mutant EPSPS was comparable t o t h a t o b t a i n e d w i t h c a l l i t r a n s f o r m e d w i t h the pMON8078 v e c t o r (Table I ) . However u n l i k e pMON8078 c a l l i w h i c h d i d n o t s u r v i v e 0.5 mM g l y p h o s a t e , t h e pM0N542 c a l l i c o u l d s u r v i v e



3.

KISHOREETAL.

EPSP Synthase

45

glyphosate concentrations up t o 10 mM. Plants expressing the h y b r i d gene were g e n e r a t e d from t r a n s f o r m e d c a l l i and s p r a y e d w i t h 0.4 l b s / a c o f Roundup®. These p l a n t s d i d not show the a p i c a l damage o b s e r v e d w i t h p l a n t s e x p r e s s i n g the b a c t e r i a l gene b y i t s e l f and were n o t s i g n i f i c a n t l y a f f e c t e d b y t h e h e r b i c i d e . Subcellular fractionation studies revealed that the hybrid p r o t e i n was localized i n the chloroplasts o f transgenic plants while the bacterial mutant protein was cytosol-localized (50). I n t e r e s t i n g l y , the l e v e l o f e x p r e s s i o n o f mutant EPSP s y n t h a s e was similar i n both cytosol-targetted and c h l o r o p l a s t - t a r g e t t e d transgenic plants. Thus, t h e c h l o r o p l a s t l o c a l i z e d mutant EPSPS p r o v i d e s s i g n i f i c a n t l y improved g l y p h o s a t e t o l e r a n c e o v e r t h a t o f the c y t o s o l - l o c a l i z e d mutanty. iz i s c l e a r trom tne above d i s c u s s i o n ensure t h a t g l y p h o s a t e t o l e r a n c e may be c o n f e r r e d t o p l a n t s b o t h b y o v e r p r o d u c t i o n o f w i l d type EPSPS as w e l l as mutant EPSP s y n t h a s e s . I t has been suggested t h a t g l y p h o s a t e may have m u l t i p l e s i t e s o f a c t i o n i n plant cells (51-56). I f t h i s i s t r u e , mutant EPSPS enzymes would not c o n f e r g l y p h o s a t e t o l e r a n c e t o p l a n t s , w h i c h i s e v i d e n t l y not the case. I t appears, t h e r e f o r e , t h a t reports concerning t h e e f f e c t o f g l y p h o s a t e on o t h e r a s p e c t s o f p l a n t m e t a b o l i s m a r e due t o secondary e f f e c t s o f the h e r b i c i d e a r i s i n g as a consequence o f the i n h i b i t i o n o f a r o m a t i c amino a c i d b i o s y n t h e s i s . More r e c e n t l y , the p e t u n i a EPSPS gene has been demonstrated t o c o n f e r Roundup® t o l e r a n c e i n t r a n s g e n i c tomato p l a n t s . The t r a n s g e n i c tomato p l a n t s were t e s t e d under f i e l d c o n d i t i o n s f o r t h e i r p r o d u c t i v i t y and v i g o r . No s i g n i f i c a n t d i f f e r e n c e s were o b s e r v e d i n crop p r o d u c t i v i t y between t r a n s g e n i c and c o n t r o l tomato p l a n t s . T h i s demonstrates t h a t i n t r o d u c t i o n and e x p r e s s i o n o f genes i n t o p l a n t s does not a f f e c t crop y i e l d . Tfte commercial implications of engineering herbicide r e s i s t a n c e t o crop p l a n t s a r e s i g n i f i c a n t . W h i l e e x t e n d i n g crop t o l e r a n c e t o the h e r b i c i d e i s one a s p e c t o f t h i s development, o t h e r significant factors t o note a r e : i . reduced dependency on h e r b i c i d e s , s i n c e t h e f a r m e r c a n use t h e h e r b i c i d e o n l y when needed, i i . use o f e n v i r o n m e n t a l l y s a f e h e r b i c i d e s , and i i i . development o f new, s a f e herbicides ( 5 7 ) . The t o o l s o f biochemistry and m o l e c u l a r b i o l o g y have p r o g r e s s e d s i g n i f i c a n t l y w i t h i n the l a s t decade t o p e r m i t a more r i g o r o u s s t r u c t u r e - f u n c t i o n study of the herbicide-target protein interaction. This i s e x p e c t e d t o have a s i g n i f i c a n t impact on development o f new herbicides. Acknowledgments We thank Ms. Jamie Wehrheim f o r t y p i n g t h i s m a n u s c r i p t . We a r e g r a t e f u l t o D r . E. J a w o r s k i f o r h i s s u p p o r t and encouragement t h r o u g h o u t the course o f t h e s e i n v e s t i g a t i o n s .

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