Biochemical Aspects of Glandular Trichome-Mediated Insect

Chapter 10

Biochemical Aspects of Glandular Trichome-Mediated Insect Resistance

in the Solanaceae

Downloaded by UNIV OF ARIZONA on August 2, 2012 | http://pubs.acs.org Publication Date: January 9, 1991 | doi: 10.1021/bk-1991-0449.ch010

John C. Steffens and Donald S. Walters Department of Plant Breeding and Biometry, Cornell University, Ithaca, NY 14853

Glandular trichomes of w i l d Solanaceae produce an array of b i o c h e m i c a l defenses against insects. This chapter reviews our knowledge of two of these mechanisms i n the w i l d potato, Solanum berthaultii, and the w i l d tomato, Lycopersicon pennellii. Ruptured trichomes of S. berthaultii r e l e a s e a h i g h l y expressed, epidermis-specific polyphenol oxidase that polymerizes trichome exudate and entraps insects. Glandular trichomes of L. pennellii continuously secrete a viscous mixture of glucose e s t e r s which a c t as i n s e c t f e e d i n g deterrents. Glucose e s t e r a c y l c o n s t i t u e n t s are composed of branched short and medium c h a i n length f a t t y a c i d s . B i o s y n t h e t i c i n v e s t i g a t i o n s reported here show that the branched amino a c i d b i o s y n t h e t i c pathway i s used t o form 4 and 5 carbon branched acyl groups which are subsequently elongated by acetate t o form the medium chain a c y l groups. I t appears that, i n both s p e c i e s , primary metabolic enzymes have been r e c r u i t e d and m o d i f i e d f o r trichome - s p e c i f i c i n s e c t r e s i s t a n c e mechanisms.

Development o f p l a n t s f o r use as food crops has gradually stripped these species of their natural r e s i s t a n c e t o i n s e c t s and pathogens. C o n s e q u e n t l y , most modern c u l t i v a r s rely upon inputs of pesticides to produce an a c c e p t a b l e y i e l d . I n s e c t i c i d e r e s i s t a n c e and the i n c r e a s i n g s o c i a l and economic costs o f p e s t i c i d e application have prompted efforts t o reduce the requirement of crop plants f o r these inputs. Breeding f o r h o s t p l a n t r e s i s t a n c e i s one a p p r o a c h t o r e d u c i n g 0097-6156/91Λ)449-0136$06.00Α) © 1991 American Chemical Society In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


10.

STEFFENS AND WALTERS

Glandular Trichome-Mediated Resistance

insecticide use. Wild relatives of crop plants f r e q u e n t l y possess u s e f u l insect or pathogen r e s i s t a n c e t r a i t s , and are o f t e n c r o s s a b l e w i t h c r o p p l a n t s . E f f o r t s to t r a n s f e r these resistance t r a i t s to crop p l a n t s have thus f a r proceeded by c o n v e n t i o n a l p l a n t breeding approaches. The prospect of producing transgenic crop p l a n t s possessing resistance traits d e r i v e d f r o m w i l d s p e c i e s p r o v i d e s a new avenue f o r u t i l i z i n g r e s i s t a n c e t r a i t s o f w i l d s p e c i e s and removes the barrier to their u t i l i z a t i o n imposed by i n c o m p a t i b i l i t y with crop species. Knowledge of the basic biochemical processes that contribute to resistance is critical to the success of molecular b i o l o g i c a l approaches s i n c e i t i s , at p r e s e n t , necessary to c h a r a c t e r i z e s p e c i f i c p r o d u c t s of r e s i s t a n c e genes i n o r d e r t o i d e n t i f y and t r a n s f e r t h e s e genes t o other species. A l t h o u g h knowledge of r e s i s t a n c e mechanisms may n o t be n e c e s s a r y t o e m p l o y t r a d i t i o n a l breeding m e t h o d s , i n f o r m a t i o n on t h e b i o c h e m i c a l m e c h a n i s m o f r e s i s t a n c e may p e r m i t d i r e c t s c r e e n i n g f o r t h e m e c h a n i s m i n t h e a b s e n c e o f p e s t p o p u l a t i o n s , and c a n be f a r l e s s time consuming than t r a d i t i o n a l screens f o r i n s e c t or pathogen s u s c e p t i b i l i t y . We h a v e b e e n i n v o l v e d i n m e c h a n i s t i c s t u d i e s a i m e d at understanding the b a s i s of g l a n d u l a r trichome-based insect resistance in wild Solanum (potato) and Lycopersicon (tomato) s p e c i e s . Much e f f o r t has f o c u s e d on i d e n t i f i c a t i o n o f w i l d members o f t h e Solanaceae with potentially useful resistance traits for i n t r o g r e s s i o n i n t o Solanum tuberosum and Lycopersicon esculentum. I n many c a s e s r e s i s t a n c e h a s b e e n s h o w n t o be c o n f e r r e d by g l a n d u l a r t r i c h o m e s , m o d i f i e d e p i d e r m a l cells (JJ which f u n c t i o n as p h y s i c a l and/or chemical barriers against insect attack (2-10. Tingey, this volume). The w i l d t o m a t o , Lycopersicon pennellii, and the w i l d p o t a t o , Solanum b e r t h a u l t i i , a r e two s p e c i e s w h i c h exhibit insect resistance c o n f e r r e d by glandular trichomes. S. b e r t h a u l t i i a n d L. p e n n e l l i i h a v e b e e n the focus of e f f o r t s at C o r n e l l U n i v e r s i t y t o t r a n s f e r trichome-based insect resistance t r a i t s . This chapter reviews our knowledge of the b i o c h e m i s t r y of g l a n d u l a r trichome-based insect resistance i n these species.

Type h (VI) T r i c h o m e s Insect Entrapment

and

Polyphenol

Oxidase-Mediated

T h e r e a r e p r i m a r i l y two c l a s s e s of f o l i a r glandular trichomes that c o n t r i b u t e to the insect r e s i s t a n c e of S. b e r t h a u l t i i a n d L. p e n n e l l i i . Type A t r i c h o m e s o f S. berthaultii ( r e f e r r e d t o as Type VI i n t h e genus Lycopersicon (UJ ) condition resistance to small-bodied insect pests such as aphids and l e a f h o p p e r s by an

In Naturally Occurring Pest Bioregulators; Hedin, P.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

137


138

NATURALLY OCCURRING PEST BIOREGULATORS

a d h e s i v e entrapment mechanism. The t y p e A t r i c h o m e i s s t a l k e d w i t h a t e t r a l o b u l a t e head, a p p r o x i m a t e l y 50-65 m i c r o n s i n d i a m e t e r , and f o u n d a t h i g h d e n s i t i e s on t h e l e a f s u r f a c e o f many s o l a n a c e o u s s p e c i e s . Contact with t h i s t r i c h o m e by an i n s e c t c a u s e s t h e m e m b r a n e - e n c l o s e d head o f t h e t r i c h o m e t o r u p t u r e , c o a t i n g t h e l e g s and mouthparts of the insect with the trichome contents. Immediately t h i s c o a t i n g b e g i n s t o brown and harden; as the insect ruptures additional trichomes, the dark masses on t h e l e g s and m o u t h p a r t s i n c r e a s e d r a m a t i c a l l y . These a c c r e t i o n s d i s r u p t i n s e c t f e e d i n g by restricting movement, by o c c l u d i n g t h e m o u t h p a r t s , o r by e n t r a p m e n t o f t h e i n s e c t on t h e l e a f (12). B r o w n i n g and h a r d e n i n g o f e x u d a t e on t h e i n s e c t ' s m o u t h p a r t s a n d t a r s i i s due t o t h e e n z y m a t i c a c t i v i t y o f an 0 r e q u i r i n g o x i d a s e (13.) , a n d h a s b e e n a s s o c i a t e d w i t h b o t h p e r o x i d a s e and p o l y p h e n o l o x i d a s e activities (£, 14_f 1_5.) . We r e c e n t l y showed t h a t t h e s e enzymatic a c t i v i t i e s a r e d u e t o a 59 kD p o l y p h e n o l o x i d a s e (PPO) which i s present i n the type A trichomes of S . berthaultii at approximately 1 ng/trichome head (a concentration of approximately 0.3 mM) (1£) . The s p e c i a l i z a t i o n of the glandular trichome i s r e f l e c t e d i n t h e f a c t t h a t PPO c o m p r i s e s a p p r o x i m a t e l y 60% o f t h e t o t a l s o l u b l e p r o t e i n of the organ. T r i c h o m e PPO appears t o be an example o f an e v o l u t i o n a r y m o d i f i c a t i o n o f an e x i s t i n g p l a n t enzyme f o r use as an i n s e c t d e f e n s e mechanism. PPO e n z y m e s , o f m o l e c u l a r w e i g h t 45,000, a r e l o c a l i z e d i n t h e t h y l a k o i d a n d a r e n e a r l y u b i q u i t o u s i n t i s s u e s o f p l a n t s {12., 18.) . U n l i k e o t h e r known n u c l e a r e n c o d e d c h l o r o p l a s t p r o t e i n s , t h e 45 kD t h y l a k o i d PPO, whose f u n c t i o n i s unknown, i s t r a n s l a t e d a t i t s m a t u r e s i z e o f 45 kD, a n d d o e s n o t p o s s e s s a t r a n s i t p e p t i d e s e q u e n c e (1_&) . In contrast, the 59 kD trichome PPO is translated as a 67 kD p r e c u r s o r and l o c a l i z e d i n t h e l e u c o p l a s t s o f t r i c h o m e and o u t e r e p i d e r m a l c e l l s . Immunological and primary s e q u e n c e s i m i l a r i t i e s b e t w e e n t h e 59 kD t r i c h o m e PPO and the 45 kD thylakoid PPO underscore the close e v o l u t i o n a r y r e l a t i o n s h i p b e t w e e n t h e s e two proteins. The a p p a r e n t a d v a n t a g e o f t h e v e r y h i g h c o n c e n t r a t i o n o f PPO i n the trichome i s the high initial rate of catalysis which results upon trichome rupture, f a c i l i t a t i n g the entrapment of mobile i n s e c t s . We h a v e c l o n e d t h e 5 9 kD t r i c h o m e PPO f r o m a cDNA l i b r a r y c o n s t r u c t e d f r o m e p i d e r m a l mRNA (H. Y u a n d J . S t e f f e n s , u n p u b l i s h e d data) and a r e c u r r e n t l y u s i n g t h i s c l o n e a s a p r o b e t o i s o l a t e t h e g e n o m i c DNA e n c o d i n g t h e S. b e r t h a u l t i i t r i c h o m e PPO ( S . Newman a n d J . S t e f f e n s , unpublished data). T h e s e s t u d i e s may a l l o w us to increase the insect entrapping a b i l i t i e s of c u l t i v a t e d p o t a t o , w h i c h has r e t a i n e d low Type A t r i c h o m e d e n s i t i e s and no longer possesses the biochemical machinery 2


10.

STEFFENS AND WALTERS

Glandular Trichome-Mediated Resistance

necessary for insect entrapment. Study of the regulatory sequences c o n t r o l l i n g expression of this e n z y m e may p r o v i d e a n u n d e r s t a n d i n g o f how glandular trichomes are able to synthesize the high l e v e l s of s p e c i f i c products which f r e q u e n t l y c h a r a c t e r i z e s these organs in plants. Alternatively, the regulatory s e q u e n c e s o f g l a n d u l a r t r i c h o m e g e n e s may b e exploited to express other i n s e c t i c i d a l p r o t e i n s i n the trichome and e p i d e r m i s as a f i r s t l i n e o f defense a g a i n s t i n s e c t pests.


Secretion

o f Sugar E s t e r s by

Glandular

Trichomes

A s i m i l a r degree of b i o c h e m i c a l s p e c i a l i z a t i o n e x i s t s i n g l a n d u l a r trichomes which s e c r e t e sugar e s t e r s . Glucose and s u c r o s e e s t e r s a r e p r o d u c e d by s e v e r a l g e n e r a w i t h i n the Solanaceae (20-25) . In c o n t r a s t to the PPOc o n t a i n i n g trichome, these sugar e s t e r s are c o n t i n u o u s l y secreted from the apex of a t a l l (750μπι) , t a p e r e d t r i c h o m e d e s i g n a t e d as t h e Type Β o r Type IV t r i c h o m e i n Solanum a n d Lycopersicon, r e s p e c t i v e l y (H) . Described i n i t i a l l y a s e p i c u t i c u l a r l i p i d s b y F o b e s e t a l . (2ϋ) , the glucose and sucrose esters of Solanum and Lycopers icon s p e c i e s have b e e n shown t o physically e n t r a p s m a l l p e s t s s u c h as s p i d e r m i t e s and a p h i d s (272_£; T i n g e y , t h i s volume) , and t o a c t as a p h i d f e e d i n g deterrents. As l i t t l e as 33 μg sucrose ester/cm s i g n i f i c a n t l y d e t e r s f e e d i n g by g r e e n p e a c h and p o t a t o aphids i n a r t i f i c i a l f e e d i n g c h a m b e r b i o a s s a y s (£.,HQ.) . L e v e l s o f s u c r o s e e s t e r s p r e s e n t on leaflets of S. b e r t h a u l t i i r a n g e a b o v e 100 mg/ cm ( u n p u b l i s h e d ) , and i n L. p e n n e l l i i a r e f r e q u e n t l y a b o v e 3 0 0 m g / c m (Z3.) . In a d d i t i o n t o d i r e c t l y reducing aphid f e e d i n g , sugar esters indirectly contribute to reduction in virus t r a n s m i s s i o n b y a p h i d s (3_1) . O t h e r r e s u l t s s u g g e s t t h a t sugar e s t e r s may also inhibit bacterial and fungal g r o w t h (1,21) . The s u g a r e s t e r s p r o d u c e d b y s o l a n a c e o u s trichomes a r e p r i m a r i l y composed o f s h o r t t o medium c h a i n l e n g t h f a t t y a c i d s (C4-C12) e s t e r i f i e d t o g l u c o s e o r s u c r o s e . Both branched and s t r a i g h t c h a i n a c y l components are present i n these esters. Each species produces a c h a r a c t e r i s t i c combination of f a t t y acids e s t e r i f i e d at 2 to 4 sugar hydroxyls ( T a b l e I) . Among t h e known species producing sucrose esters, a c y l a t i o n of every p o s s i b l e h y d r o x y l has been d e s c r i b e d e x c e p t t h e l , 4 , and 6 p o s i t i o n s (20-25). The d i v e r s i t y o f f a t t y a c i d c o m p o s i t i o n as w e l l as t h e acylation positions are p u z z l i n g phenomena. I n s t r u c t u r e / a c t i v i t y s t u d i e s we are unable to detect differences i n aphid feeding d e t e r r e n c e when c o m p a r i n g s u c r o s e and g l u c o s e esters. Esters bearing d i f f e r e n t chain lengths of f a t t y acid a l s o do n o t e x h i b i t significant activity differences (H) · The o r i g i n o f s u g a r e s t e r s t r u c t u r a l diversity 2

2

2

1

1


f

139


Group

+

Major a c y l groups,

10-methylundecanoate

dodecanoate

9-methyldecanoate_

undecanoate

*

sucrose

2,3,3',4

glucose

2,3,4

Solanum Solanum neocardensii aethiopsicum

Minor a c y l groups (

Biochemical Aspects of Glandular Trichome-Mediated Insect

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