Regulation of Synthesis and Accumulation of Proteinase Inhibitors in

6 Regulation of Synthesis and Accumulation of Proteinase Inhibitors in Leaves of Wounded Tomato Plants C. E. NELSON, M. WALKER-SIMMONS, D. MAKUS, G. ZUROSKE, J. GRAHAM, and C. A. R Y A N

Downloaded by AUBURN UNIV on November 15, 2016 | http://pubs.acs.org Publication Date: January 20, 1983 | doi: 10.1021/bk-1983-0208.ch006

Washington State University, Institute of Biological Chemistry and Biochemistry and Biochemistry/Biophysics Program, Pullman, WA 99164

Two proteinase i n h i b i t o r s , Inhibitors I and II, accumulate i n leaves of tomato plants when attacked by chewing insects or mechanically wounded. The accumulation of these two antinutrient proteins is apparently a defense response and is i n i t i a t e d by the release of a putative wound hormone called the proteinase i n h i b i t o r inducing factor (PIIF). The direction of flow of PIIF out of wounded leaves i s primarily towards the apex and transport occurs maximally about 120 min following wounding. After a single severe wound, the i n v i t r o translatable tomato leaf mRNA specific for Inhibitors I and II increases to a maximum within four hours and remains constant for about five hours when i t decreases rapidly to about 50% of the maximum. The rate of i n vivo accumulation of both i n h i b i t o r proteins steadily increases, reaching a steady state after nine hours. However, a second wound at nine hours results i n a tripling of the steady state rate of i n h i b i t o r accumulation over the next several hours. The data indicates that the second wound causes no change i n the apparent translat i o n a l efficiencies of the mRNA for Inhibitors I and II but causes increased rates of i n h i b i t o r accumulation by providing more translatable i n h i b i t o r messages when the plant's translation system is operating at high efficiency. A severe mechanical wound on a s i n g l e l e a f of tomato p l a n t s i n i t i a t e s a complex s e r i e s of e x t r a c e l l u l a r and i n t r a c e l l u l a r r e a c t i o n s which r e s u l t i n the s y n t h e s i s and accumulation of two p r o t e i n a s e i n h i b i t o r s , I n h i b i t o r s I and I I , i n l e a f c e l l s (1_, 2). A second wounding, w i t h i n a few hours, r e s u l t s i n a 2-3 f o l d increase i n the r a t e s of accumulation i n i t i a t e d by the 0097-6156/83/0208-0103$06.00/O © 1983 American Chemical Society Hedin; Plant Resistance to Insects ACS Symposium Series; American Chemical Society: Washington, DC, 1983.


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f i r s t wound. A p u t a t i v e wound hormone, c a l l e d the p r o t e i n a s e i n h i b i t o r i n d u c i n g f a c t o r , P I I F , i s r e l e a s e d at the wound s i t e and t r a v e l s throughout the p l a n t to i n i t i a t e s y n t h e s i s and accumulation of the two p r o t e i n a s e i n h i b i t o r s , even i n unwounded leaves s e v e r a l cm from the wound s i t e . We view t h i s process as a p r i m i t i v e immune-like response i n which the p l a n t i s responding to pest damage by producing powerful a n t i n u t r i e n t p r o t e i n s , the p r o t e i n a s e i n h i b i t o r s , to help the p l a n t discourage p e r s i s tent or f u t u r e a t t a c k s by p e s t s . This wound response has provided a novel system f o r studying the r e g u l a t i o n of the expression of the two proteinase i n h i b i t o r s by the f a c t o r P I I F , t r i g g e r e d by severe environmental s t r e s s . In t h i s chapter we report our recent data concerning the d i r e c t i o n and time course of P I I F t r a n s p o r t through the p l a n t f o l l o w ing wounding, and our progress i n i n i t i a t i n g a program to study the molecular b i o l o g y of i n h i b i t o r accumulation. D i r e c t i o n and Rate of Flow of the Wound S i g n a l , P I I F P I I F a c t i v i t y has been i s o l a t e d by v a r i o u s techniques from tomato leaves to y i e l d a s i n g l e broad peak from Sephadex G-50 that e x h i b i t s a Mw range of about 5000 to 10,000 daltons and i s p r i m a r i l y carbohydrate i n composition ( 3 ) . P r o p e r t i e s of h i g h l y p u r i f i e d P I I F p r e p a r a t i o n s , such as l o s s of a c t i v i t y upon e i t h e r prolonged a c i d h y d r o l y s i s or periodate o x i d a t i o n , and i t s monosaccharide composition suggested that i t was s i m i l a r to the p e c t i c polysaccharides found a s s o c i a t e d w i t h the p l a n t c e l l wall. In c o l l a b o r a t i o n w i t h Dr. Peter Albersheim, of the U n i v e r s i t y of Colorado, we found that a sycamore c e l l w a l l d e r i v e d p o l y s a c c h a r i d e , c a l l e d rhamnogalacturonan I , was as a c t i v e as tomato P I I F i n inducing p r o t e i n a s e i n h i b i t o r accumulat i o n i n young tomato p l a n t s ( 4 ) . This work s u b s t a n t i a t e d that tomato P I I F was a fragment of the p l a n t c e l l w a l l . In subsequent experiments we were able to e n z y m i c a l l y degrade P I I F i n t o o l i g o s a c c h a r i d e s w i t h molecular weights of about 400 to 2000 that r e t a i n e d the c a p a c i t y to induce p r o t e i n a s e i n h i b i t o r s i n detached tomato leaves ( 3 ) . A hypothesis was presented (3) i n which P I I F i s r e l e a s e d as a mixture of p o l y - and o l i g o s a c c h a r i d e s fragmented from the c e l l w a l l by h y d r o l y t i c enzymes that e i t h e r are a c t i v a t e d during wounding or are introduced by invading p e s t s . P I I F , or a product induced by i t s presence, could then be transported r a p i d l y through the p l a n t v a s c u l a r system to t a r g e t c e l l s where i t induces the s y n t h e s i s and accumulation of proteinase i n h i b i t o r proteins. D i r e c t i o n of f l o w of P I I F . The time-course of I n h i b i t o r I accumulation i n leaves of young tomato p l a n t s at the f o u r l e a f stage, wounded at 0 time and at 72 h r , i s shown i n F i g . 1. Wounding of an upper l e a f l e t ( l e f t ) d i d not cause accumulation

Hedin; Plant Resistance to Insects ACS Symposium Series; American Chemical Society: Washington, DC, 1983.

Hedin; Plant Resistance to Insects ACS Symposium Series; American Chemical Society: Washington, DC, 1983. TIME (hours)

Figure 1. Time course of accumulation of Inhibitor I in terminal leaflets of young tomato plants. Leaves were wounded at the uppermost leaf (left) and the lowest leaf (right) by crushing across the midrib of the terminal leaflet with a hemostat at zero time and again at 72 h. The concentrations of Inhibitor I in leaves were determined immunologically.

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of I n h i b i t o r I i n lower l e a v e s , but a second wound (at 72 hr) on the same l e a f was weakly r e g i s t e r e d by the lower leaves and r e s u l t e d i n some accumulation of the i n h i b i t o r . Wounding of the lowest l e a f ( r i g h t ) d i d cause upper leaves to accumulate I n h i b i t o r I and a second wound at 72 hr s i g n i f i c a n t l y r e i n f o r c e d the response, p a r t i c u l a r l y i n the two uppermost leaves. In Table I the average l e v e l s of I n h i b i t o r I i n a l l f o u r t e r m i n a l l e a f l e t s of tomato p l a n t s , s i n g l y wounded at the v a r i o u s l e a f l e t p o s i t i o n s , i s shown. I t i s c l e a r that i n h i b i t o r s p r e f e r e n t i a l l y accumulated i n leaves near the apex of the p l a n t and that the t r a n s p o r t of P I I F was p r i m a r i l y a c r o p e t a l l y . The lower leaves were not disposed to accumulate much I n h i b i t o r I , even when the lowest l e a f l e t s were wounded. These experiments suggest that not only are lower o l d e r leaves much l e s s responsive to P I I F , but are apparently r e c i p i e n t s of only a s m a l l amount of the hormone that i s released above them. The experiments a l s o demonstrated that a l l of the leaves are capable of r e l e a s i n g PIIF when wounded, although the lowest wounded leaves do not appear to be r e l e a s i n g as much P I I F as upper l e a v e s . Rate of flow of P I I F . A s i n g l e s l i c e of a sharp r a z o r blade through the l e a f p e t i o l e apparently does not r e l e a s e a p p r e c i a b l e PIIF i n t o the p l a n t as evidenced by the l a c k of accumulation of i n h i b i t o r i n t i s s u e s of the e x c i s e d l e a f ( 5 ) . Thus, a s i n g l e wound can be i n f l i c t e d i n a t e r m i n a l l e a f l e t and then the e n t i r e l e a f can be severed at a measured d i s t a n c e from the wound s i t e near the base of the p e t i o l e at v a r i o u s times to determine how long i t takes f o r PIIF to t r a v e l past the p o s i t i o n of the cut and out of the l e a f , as judged by accumulation of I n h i b i t o r I i n an adjacent upper l e a f l e t 24 hr l a t e r . Our previous experiments (6) suggested t h a t about 2 hr was r e q u i r e d to maximally t r a n s p o r t P I I F out of leaves (a d i s t a n c e of about 6 cm). We repeated these experiments h e r e i n w i t h the purpose of comparing the r a t e of PIIF t r a n s p o r t w i t h that of [ ^ C ] g l u c o s e that was a p p l i e d to the wound immediately f o l l o w i n g the i n j u r y . The time f o r t r a n s p o r t of [ C ] g l u c o s e from i t s a p p l i c a t i o n at the wound s i t e to the base of the p e t i o l e ( F i g . 2, r i g h t ) was n e a r l y i d e n t i c a l to that of P I I F that was r e l e a s e d upon wounding. The amount of glucose that was t r a n s p o r t e d up to the next l e a f , however, was s m a l l compared to what passed through the p e t i o l e of the wounded l e a f . Very l i t t l e r a d i o a c t i v i t y was detected i n the adjacent lower l e a f . The maximum r a d i o a c t i v i t y was reached at about 120 min f o l l o w i n g wounding, the same time that P I I F t r a n s p o r t through t h i s r e g i o n was maximal ( F i g . 2, l e f t ) . Thus, both P I I F and [ C]glucose were transported out of the wound s i t e s at approximately the same times and were both p r e f e r e n t i a l l y transported a c r o p e t a l l y . ltf

11+

Mode of t r a n s p o r t . A j e t of hot a i r (80°C) focused on a segment of the p e t i o l e near the main stem completely destroyed


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TABLE I


INHIBITOR I ACCUMULATION IN LEAVES OF YOUNG TOMATO PLANTS 120 HOURS FOLLOWING WOUNDING OF INDIVIDUAL LEAVES AT DIFFERENT LOCATIONS ON THE PLANTS Young tomato p l a n t s having four leaves were wounded a t the l e a f p o s i t i o n shown below w i t h a hemostat a t 0 and 72 h r . A f t e r 120 hr f o l l o w i n g the i n i t i a l wounding the t e r m i n a l l e a f l e t o f each l e a f was assayed f o r I n h i b i t o r I c o n c e n t r a t i o n .

P o s i t i o n of Wounded Leaf

1

2

Leaf # 3

4

I n h i b i t o r I Accumulation (pg/g t i s s u e )


Hedin; Plant Resistance to Insects ACS Symposium Series; American Chemical Society: Washington, DC, 1983. 14

Figure 2. Left: Inhibitor I accumulation in leaves of wounded young tomato plants. The middle leaf of young tomato plants at the three leaf stage was wounded at zero time and excised at the times shown. Inhibitor I in the upper and lower intact leaves was determined immunologically 48 h following wounding. Right: 5 /xL of U-[ C]glucose (specific activity, 28 pCi/mM) was applied directly to the wounds of the middle leaf immediately upon injury. Radioactivity was determined in 0.5-cm segments at the base of each of the three petioles at the times indicated.

TIME( min)



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the phloem but not the xylem, as evidenced by the d r y i n g of the t r e a t e d area w i t h i n an hour t o form a f i n e strand o f xylem w i t h the l e a f s t i l l i n t a c t w i t h good t u r g o r . This treatment caused the l e a f i t s e l f t o accumulate I n h i b i t o r I over the next 24 h r , but i t minimally a f f e c t e d the leaves of the r e s t of the p l a n t , i n d i c a t i n g that the d e s t r u c t i o n of the phloem d i d not i n i t i a t e P I I F t r a n s p o r t out of the l e a f but only i n t o the l e a f i t s e l f . We subjected a segment of the p e t i o l e of the lowest l e a f of s e v e r a l 3 l e a f stage tomato p l a n t s , t o a hot a i r j e t t o destroy the phloem. W i t h i n an hour the i n j u r e d p e t i o l e segments had d r i e d t o form the t h i n strands of xylem. As b e f o r e , t h i s treatment d i d not r e l e a s e PIIF i n t o the p l a n t (Table I I , t r e a t ment 3) w h i l e the l e a f i t s e l f ( l e a f #1) accumulated considerable I n h i b i t o r I . Subsequent wounding of leaves whose phloem had been destroyed (Table I I , treatment 4) d i d not r e s u l t i n I n h i b i t o r I accumulation i n adjacent l e a v e s , i n d i c a t i n g that the phloem d e s t r u c t i o n had blocked i t s t r a n s p o r t out of the wounded leaf. While the evidence presented h e r e i n c u m u l a t i v e l y supports the involvement of the phloem i n t r a n s l o c a t i n g PIIF out o f wounded tomato p l a n t s , the v e l o c i t y of t r a n s p o r t appeared to be much slower than expected f o r phloem t r a n s p o r t . When c a r e f u l l y measured from p o i n t t o p o i n t i n p e t i o l e t i s s u e the v e l o c i t y of a s s i m i l a t e out of l e a v e s , i n g e n e r a l , i s i n the order of 1-5 cm/min (_7, 8 ) . Our techniques do not a l l o w d i r e c t measurements w i t h PIIF i t s e l f , but from our i n d i r e c t measurements we c a l c u l a t e a gross movement of P I I F from the time of wounding t o time of maximum t r a n s p o r t of P I I F (as w i t h glucose) t o the base of the p e t i o l e , 6 cm from the wound s i t e , of about 0.05 cm/min. This i s over twenty times too slow f o r a process i n v o l v i n g j u s t phloem t r a n s p o r t . Thus, i f phloem t r a n s p o r t i s o c c u r r i n g , then a time of n e a r l y 2 h r must be r e q u i r e d t o d e l i v e r a maximum q u a n t i t y of PIIF i n t o the phloem a t o r near the wound s i t e . This r a t e i s much slower than normal phloem l o a d i n g , f o r example i n healthy soybeans ( 9 ) . The data seems t o i n d i c a t e that the o r i g i n s of P I I F and i t s i n t r o d u c t i o n i n t o the t r a n s p o r t system of the p l a n t i s a complex system. We can however speculate about the nature of the process from a v a i l a b l e i n f o r m a t i o n . The 2 h r p e r i o d r e q u i r e d t o reach maximum P I I F l e v e l s i n the phloem could be a consequence of enzymic degradation of the c e l l w a l l f o l l o w i n g wounding, and/or the production and a s s o c i a t i o n of some type of chemical to the c e l l w a l l fragments, e v e n t u a l l y e n t e r i n g the t r a n s p o r t system. Thus, the phloem, which i s i n v o l v e d i n t r a n s l o c a t i n g carbohydrates, would be a l o g i c a l candidate t o t r a n s p o r t s m a l l p e c t i c fragments, p r e f e r e n t i a l l y t o the upper leaves. This type of mechanism of PIIF production and r e l e a s e would r e q u i r e some type of l o a d i n g mechanism that would a l l o w the fragments t o enter the phloem as, o r a f t e r , they were produced. As a monitoring system f o r i n s e c t o r pathogen damage, t h i s process would be


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TABLE I I


EFFECT OF THE HOT AIR-DESTRUCTION OF PETIOLE PHLOEM ON THE MOVEMENT OF P I I F OUT OF MECHANICALLY WOUNDED LEAVES A l l treatments of young tomato p l a n t s (having three leaves) were at the lowest l e a f (#1) shown below. Hot a i r (80°C) was a p p l i e d to the base of the p e t i o l e through a window i n a t e f l o n s h i e l d to destroy a segment of phloem t i s s u e . A s i n g l e wound, perpend i c u l a r to the m i d r i b was i n f l i c t e d a t the center of the t e r m i n a l l e a f l e t of l e a f #1 three days a f t e r hot a i r treatment and I n h i b i t o r I l e v e l s were assayed i n the leaves 24 h r l a t e r .

3. Treatment

If

^•W^t

Leaf # 2

I n h i b i t o r I Accumulation (yg/g t i s s u e ) 1.

No treatment

0

0

10

2.

No phloem d e s t r u c t i o n l e a f / / l mechanically wounded

133

126

86

3.

Leaf #1 phloem destroyed, l e a f #1 t i s s u e unwounded

173

35

32

4.

Leaf #1 phloem destroyed, l e a f #1 t i s s u e wounded

165

14

27


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w e l l s u i t e d , d i r e c t i n g messages from wounded o r damaged t i s s u e s to the younger healthy t i s s u e s t o i n i t i a t e i n h i b i t o r accumulation.


In V i t r o Synthesis of P r e - P r o t e i n a s e I n h i b i t o r s w i t h mRNA from Wounded Tomato P l a n t s The two p r o t e i n a s e i n h i b i t o r s that accumulate i n leaves of wounded tomato leaves have been i s o l a t e d and c h a r a c t e r i z e d . I n h i b i t o r I has a molecular weight of 41,000 and i s composed of subunits w i t h molecular weights of about 8100 (10). It is, t h e r e f o r e , a pentamer i n i t s n a t i v e s t a t e . Each subunit possesses an a c t i v e s i t e s p e c i f i c f o r chymotrypsin, and the apparent f o r the i n h i b i t i o n of chymotrypsin i s about 10"" M (10). I n h i b i t o r I I has a molecular weight o f about 23,000, i s composed of two s u b u n i t s , and s t r o n g l y i n h i b i t s both t r y p s i n and chymotrypsin w i t h K. values of about 1 0 ~ and 10" M r e s p e c t i v e l y (10) . Messenger RNA has been prepared from leaves o f wounded and unwounded tomato p l a n t s and only leaves of wounded p l a n t s c o n t a i n t r a n s l a t a b l e mRNAs s p e c i f i c f o r I n h i b i t o r s I and I I (11) . Both p r o t e i n s have been shown t o be t r a n s l a t e d i n v i t r o i n a r e t i c u l o c y t e l y s a t e system as p r e i n h i b i t o r s , 2000-3000 daltons l a r g e r than those synthesized and accumulated i n v i v o (11). The p r e i n h i b i t o r s may be important i n the compartmentaliz a t i o n of the i n h i b i t o r s as they are stored i n the c e n t r a l v a c u o l e , o r p l a n t lysosome, of the plant c e l l s (12). We have now studied the time course of the increase i n t r a n s l a t a b l e mRNA i n leaves of wounded p l a n t s u t i l i z i n g poly(A) mRNA i s o l a t e d a t v a r i o u s times f o l l o w i n g wounding. When young tomato p l a n t s are wounded, by chewing i n s e c t s or by a severe crushing of any type, the l e v e l s of t o t a l poly(A) t r a n s l a t a b l e mRNA f o r both p r o t e i n a s e I n h i b i t o r I and I n h i b i t o r I I r i s e r a p i d l y during the f i r s t 4 h r a f t e r wounding (13). This r i s e was measured by q u a n t i f y i n g the immunoprecipitates that can be recovered s p e c i f i c a l l y from e l e c t r o p h o r e t i c gels a f t e r t r a n s l a t i o n i n a c e l l f r e e r a b b i t r e t i c u l o c y t e l y s a t e system (11, 13). An example of such g e l s i s shown i n F i g . 3. I n t h i s example the newly t r a n s l a t e d I n h i b i t o r s I and I I were i s o l a t e d as immunoprecipitates w i t h I n h i b i t o r I IgG p l u s I n h i b i t o r I I IgG and electrophoresed i n 15% acrylamide gels i n the presence of SDS and mercaptoethanol. The a n a l y s i s of r a d i o a c t i v i t y ( [ S ] m e t h i o n i n e ) i n c o r p o r a t e d i n t o each i n h i b i t o r i n the gels was taken as a measure of the c o n c e n t r a t i o n of i n h i b i t o r mRNAs. T r a n s l a t a b l e mRNAs f o r I n h i b i t o r s I and I I are present a t near maximum l e v e l s w i t h i n 4 h r f o l l o w i n g wounding ( F i g . 4A). The l e v e l s remain h i g h u n t i l 9 hr. A f t e r 9 h r the l e v e l s decrease to l e s s than h a l f t h e i r o r i g i n a l l e v e l ( F i g . 4A). In the same l e a v e s , during the same time, the r a t e s of i n v i v o accumulation of I n h i b i t o r s I and I I ( F i g . 4B) s t e a d i l y increase during the 9

8

7

35


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Figure 3. SDS-urea electrophoresis of proteinase Inhibitors I and IL Key: Lane 1, proteinase Inhibitor II; Lane 2, proteinase Inhibitor I (both lanes stained with Coomassie blue); and Lane 3, [ S]preproteinase Inhibitors I and II synthesized in an in vitro rabbit reticulocyte system immunoprecipitated for Inhibitors I and II. 35


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r


T


TIME (hours) Figure 4. Time course analysis of the accumulation of Inhibitors I and II protein, translatable mRNAs and apparent translational efficiencies in leaves of singly and doubly wounded tomato plants. Key: —•—, Inhibitor I, single wound; — O — , Inhibitor II, single wound; — • —, Inhibitor I, double wound; and — O — , Inhibitor II, double wound. A: In vitro translation of 4-fig quantities of tomato leaf mRNA and subsequent isolation of specific preinhibitors through the preformed antibody technique (\\). B: In vivo accumulation of Inhibitors I and II proteins in wounded tomato leaves. C: Apparent translational efficiencies.



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f i r s t 9 h r a f t e r wounding. By 14 h r the r a t e s of accumulation have reached a steady s t a t e r a t e that remains constant f o r s e v e r a l hours. This suggests that some s h i f t i n the c e l l i s o c c u r r i n g a t about 9 h r that i s r e f l e c t e d i n both the i n v i t r o t r a n s l a t i o n o f mRNA and i n the i n v i v o accumulation of i n h i b i t o r s . The cause o f the s h i f t i n l e v e l s of t r a n s l a t a b l e mRNA i s unknown and could r e s i d e i n e i t h e r some s t r u c t u r a l f e a t u r e of the mRNAs themselves that change t h e i r t r a n s l a t i o n a l r a t e s , o r the d i f f e r e n c e s could r e s u l t i n changes i n t h e i r r a t e s of s y n t h e s i s o r degradation. Nevertheless, the s h i f t r e s u l t s i n species of mRNA that are r e s p o n s i b l e f o r the more e f f i c i e n t steady s t a t e r a t e of s y n t h e s i s of the two i n h i b i t o r s a f t e r 9 h r . A second wounding of the leaves 9 h r a f t e r the i n i t i a l wounding, t r i p l e d the r a t e o f accumulation of both I n h i b i t o r s I and I I ( F i g . 4B) over those of once wounded p l a n t s . This second wound a l s o r e s u l t e d i n the maintenance of the mRNA l e v e l s present a t 9 h r so that the decrease i n mRNA noted i n s i n g l y wounded p l a n t s d i d not occur. The t r a n s l a t a b l e mRNA l e v e l s remained high through the 18th h r ( F i g . 4A). Thus, high mRNA l e v e l s a t 18 h r i n doubly wounded p l a n t s i s r e f l e c t e d i n over a doubling of I n h i b i t o r s I and I I s y n t h e s i s and accumulation. As shown i n F i g . 4C, a second wounding a f t e r 9 h r d i d not f u r t h e r increase the t r a n s l a t i o n a l e f f i c i e n c y of I n h i b i t o r s I and I I mRNA although i t s i g n i f i c a n t l y increases the r a t e s of i n h i b i t o r s y n t h e s i s and accumulation. The second wound apparently provides more mRNA when the p l a n t ' s t r a n s l a t i o n a l system i s already operating a t high e f f i c i e n c y . The apparent increase i n t r a n s l a t i o n a l e f f i c i e n c y of mRNA a f t e r 9 h r i s not the r e s u l t of an i n c r e a s e i n t o t a l poly(A) mRNA. Two separate e x t r a c t i o n s of t o t a l mRNA from leaves of wounded tomato p l a n t s showed the opposite; t h a t s u b s t a n t i a l l y l e s s poly(A) RNA was present a t 14 hr (40 ± 10.6 ug/g of l e a f ) and 18 h r (55 ± 3.5 ug/g of l e a f ) a f t e r a s i n g l e wound than a t 9 h r (96 ± 4.2 ug/g o f l e a f t i s s u e ) . A s i m i l a r time course f o r t r a n s l a t a b l e mRNA has been reported (14) i n b a r l e y aleurone l a y e r s , i n response t o g i b b e r e l l i c a c i d . T o t a l p o l y ( A ) RNA was found t o increase d r a m a t i c a l l y i n the f i r s t 12 h r a f t e r hormone a p p l i c a t i o n , f o l l o w e d by a r a p i d d e c l i n e t o 25% o f the maximum a t 18 h r . On the other hand, the accumulation of ovalbumin mRNA i n response t o progesterone i n c h i c k o v i d u c t s (15) i s an example that does not appear to behave i n t h i s manner. The p o s s i b i l i t y that the d i f f e r e n c e i n apparent t r a n s l a t i o n e f f i c i e n c i e s described here might be due t o the presence o f a d i f f e r e n t , perhaps l a r g e r species of mRNA being present a t 9 o r 18 h r , was i n v e s t i g a t e d by a n a l y s i s of the s i z e s o f mRNAs f o r I n h i b i t o r s I and I I on l i n e a r sucrose g r a d i e n t s . U l t r a c e n t r i f u g a t i o n of 200 ug a l i q u o t s of mRNA from leaves of 9- and 18-hr s i n g l e wounded p l a n t s and 18-hr double wounded p l a n t s demons t r a t e d that a l l three mRNAS migrated i d e n t i c a l l y ( F i g . 5 ) . To l o c a t e the p o s i t i o n of p o l y ( A ) RNAs s p e c i f i c f o r I n h i b i t o r s I +

+


6. NELSON ET AL.




i

r

f r a c t i o n number Figure 5. Size analysis of Inhibitors I and II specific mRNA from levels of 9- and 18-h singly wounded tomato plants and 18-h doubly wounded plants. PolyfA )* RNA was applied to 15-30% linear sucrose gradients and was spun at 25,000 rpm. Twenty-five fractions were collected, the absorbency was measured, and the mRNA was precipitated by cold ethanol. In vitro translations were performed with each fraction in a rabbit reticulocyte system, and isolation of the preinhibitors with preformed antibody precipitates located the position of the two inhibitors. The gradients were calibrated by centrifugation of tomato leaf poly(A)~ RNA on an identical gradient. The locations of translatable mRNAs for Inhibitors I and II were identical with RNA obtained from 9- and 18-h singly wounded or 18-h doubly wounded plants.


115


116


and I I the e l u t e d gradient m a t e r i a l was p r e c i p i t a t e d w i t h ethanol and i n v i t r o t r a n s l a t i o n performed. F r a c t i o n s c o n t a i n i n g p r e i n h i b i t o r s I and I I were i d e n t i f i e d by s p e c i f i c immunoprecipitat i o n o f the t r a n s l a t i o n products (data not shown). A n a l y s i s of the gradient (16) w i t h known standards of tomato l e a f RNA that do not bind t o o l i g o ( d T ) - c e l l u l o s e columns ( p o l y ( A ) ~ m a t e r i a l ) showed that I n h i b i t o r s I and I I mRNAs always migrated w i t h sedimentation c o e f f i c i e n t s of 13S and 15S, r e s p e c t i v e l y . In order t o f u r t h e r p u r i f y the s p e c i f i c messengers f o r the i n h i b i t o r s , a d d i t i o n a l 15-30% g r a d i e n t s were run on mRNA samples that had been p a r t i a l l y p u r i f i e d by o l i g o ( d T ) - c e l l u l o s e . Fract i o n s corresponding t o the known p o s i t i o n of I n h i b i t o r s I and I I mRNA were combined and r e c e n t r i f u g e d i n 10-25% l i n e a r sucrose g r a d i e n t s ( F i g . 6 ) . By comparing the amount of r a d i o l a b e l s p e c i f i c a l l y immunoprecipitated t o the t o t a l number of counts incorporated i n the t o t a l t r a n s l a t i o n r e a c t i o n we c a l c u l a t e d the r e l a t i v e p u r i t y of the two messages (Table I I I ) . These p h y s i c a l techniques have thus provided a 1 5 - f o l d p u r i f i c a t i o n of the s p e c i f i c mRNA f o r I n h i b i t o r I and a 5 - f o l d p u r i f i c a t i o n of I n h i b i t o r I I mRNA. The mRNA f o r both I n h i b i t o r s I and I I appeared t o be t y p i c a l of e u k a r y o t i c messengers that code f o r s m a l l p r o t e i n s of 8-12,000 daltons having a p o l y ( A ) t a i l s i n c e both messengers bind s p e c i f i c a l l y t o o l i g o ( d T ) - c e l l u l o s e . There i s no evidence of t r a n s l a t a b l e messengers f o r the two i n h i b i t o r s i n the RNA f r a c t i o n that d i d not bind t o the o l i g o ( d T ) a f f i n i t y r e s i n (data not shown). The l e n g t h of poly(A) segments present i n poly(A) RNA from pooled sucrose d e n s i t y gradient f r a c t i o n s r i c h i n I n h i b i t o r s I and I I mRNA was determined by s u b j e c t i n g the RNA t o p a n c r e a t i c and Tj RNase d i g e s t i o n , and l a b e l i n g the 5' t e r m i n a l ends w i t h [ y - P ] A T P and p o l y n u c l e o t i d e k i n a s e . R e s u l t s of the length determination i n polyacrylamide g e l s i n d i c a t e that poly(A) fragments are about 100 n u c l e o t i d e s long. The d i s t r i b u t i o n of lengths i n the experiment represent a v a r i e t y of mRNAs and the t a i l lengths of the two I n h i b i t o r s are not known but are assumed to be w i t h i n t h i s d i s t r i b u t i o n . In a f u r t h e r experiment we assayed f o r the presence of a cap s t r u c t u r e on the mRNAs f o r both I n h i b i t o r s I and I I by competitive i n h i b i t i o n by 7-methyl-guanosine 5'-monophosphate ( m G p ) of the i n v i t r o t r a n s l a t i o n of these messengers. Concentrations of 40 uM m G p i n h i b i t e d by 50% the i n v i t r o t r a n s l a t i o n of t o t a l tomato l e a f p o l y ( A ) mRNA ( F i g . 7A). This l e v e l i s 4 0 - f o l d lower than that r e q u i r e d to s i m i l a r l y i n h i b i t r a b b i t g l o b i n mRNA t r a n s l a t e d i n a r a b b i t r e t i c u l o c y t e l y s a t e (17) and 4 - f o l d lower than that r e q u i r e d t o i n h i b i t the same mRNA i n a wheat germ system (18). I t was of i n t e r e s t that the t r a n s l a t i o n of I n h i b i t o r I i s i n h i b i t e d t o 50% by 20 uM m G 'p w h i l e 50% i n h i b i t i o n of I n h i b i t o r I I r e q u i r e s l e s s than 10 uM ( F i g . 7B). The b a s i s of t h i s d i f f e r e n c e i s not understood but +

32

7

5f

7

5?

+

7


5


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10 15 20 FRACTION NUMBER

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25

Figure 6. Partial purification of Inhibitors I and II mRNA. Fractions containing Inhibitors I and II mRNA determined by in vitro translation analyses were recovered from an initial 15-30% linear sucrose gradient, precipitated by cold ethanol, and applied to a 10-25% linear sucrose gradient. The sample was centrifuged for 36 h at 25,000 rpm. Fractions of the gradient were collected and subjected to in vitro translation analyses. The upper graph represents total [ S]methionine incorporation assayed with 1 fxL of the translation mixture as described (11). The bottom figure quantitates the radiolabel incorporated specifically into Inhibitor I (solid bars) and Inhibitor II (open bars). 35




118

TABLE I I I PARTIAL PURIFICATION OF INHIBITORS I AND I I mRNAs

% Counts Purification

Total Translatable I n h i b i t o r s Total Translatable Protein

Inhibitor I

Inhibitor I I

Oligo(dT) Chromatography

0.27%

0.22%

F i r s t Sucrose Gradient

0.42%

0.35%

Second Sucrose Gradient

4.1%

1.2%



6.

NELSON ET AL.



7-METHYL GUANOSINE 5'-MONOPHOSPHATE (mM) 7

5

Figure 7. Inhibition of in vitro translation of tomato leaf mRNA by m G 'p. Quantities of 4 fig mRNA from wounded tomato leaves were translated and analyzed. A: Total incorporation of [ S]methionine into trichloroacetic acid insoluble protein. Assays were done with 1-fiL fractions of the translation reaction as described 35

B: Incorporation of [ S]methionine into pre-Inhibitors I (O) and II (Q) as isolated by preformed antibody precipitates. 35


119

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could be explained i f there i s a d i f f e r e n c e between the a f f i n i t i e s of the two proteinase I n h i b i t o r mRNAs f o r the mRNA b i n d i n g s i t e on the 40S i n i t i a t i o n complex o r between the I n v i v o cap s t r u c t u r e s . I n h i b i t i o n of t r a n s l a t i o n by d e r i v a t i v e s of m G 'p has been used as evidence that a s p e c i f i c mRNA contains a 5 t e r m i n a l cap s t r u c t u r e (19), but t h i s i n t e r p r e t a t i o n must be t r e a t e d w i t h c a u t i o n (20). However, the f a c t that the t r a n s l a t i o n of I n h i b i t o r s I and I I mRNA i s s e v e r a l f o l d more s t r o n g l y i n h i b i t e d by m G p than g l o b i n mRNA lends support t o the presence of capped s t r u c t u r e s a t the 5 terminus of the i n h i b i t o r mRNAs. The unequivocal i d e n t i f i c a t i o n of the cap s t r u c t u r e s w i l l r e q u i r e p u r i f i c a t i o n of the mRNAs f o r each i n h i b i t o r . Thus, s t u d i e s of the p r o p e r t i e s of the mRNAs i s o l a t e d a t d i f f e r e n t times a f t e r wounding do not r e v e a l any d i f f e r e n c e s that might r e f l e c t the l a r g e changes i n t r a n s l a t i o n a l e f f i c i e n c i e s observed i n tomato leaves 9-14 h r a f t e r wounding. During t h i s time there may be changes i n 5 -end capping, p o l y a d e n y l a t i o n and i n t e r n a l methylation. I t i s a l s o p o s s i b l e that some c e l l u l a r component i n v o l v e d w i t h s y n t h e s i s and t r a n s p o r t of i n h i b i t o r s i n t o the c e n t r a l vacuole may be i n v o l v e d . The vacuole o r l y s o some i s considered t o o r i g i n a t e w i t h the G o l g i apparatus. C e l l u l a r events during the f i r s t hours f o l l o w i n g wounding may be i n v o l v e d w i t h the production of a component(s) t o f a c i l i t a t e ribosome-mRNA b i n d i n g o r some other process i n the G o l g i apparatus t o s p e c i f i c a l l y accommodate t r a n s p o r t of I n h i b i t o r s I and I I i n t o the vacuole. A l t e r n a t i v e l y , poor e f f i c i e n c i e s of i n h i b i t o r mRNAs may be due t o t h e i r i n c o r p o r a t i o n i n t o r i b o n u c l e o p r o t e i n p a r t i c l e s (RNPs) such as found i n sea u r c h i n embryos (21). Newly made mRNA i n the embryos i s found i n RNPs and they apparently have "weak" template a c t i v i t i e s w h i l e i n these p a r t i c l e s . The presence of newly synthesized tomato mRNA i n s i m i l a r p a r t i c l e s might e x p l a i n the apparently low t r a n s l a t i o n a l e f f i c i e n c i e s noted h e r e i n . The use of chaotropic b u f f e r s i n the p r e p a r a t i o n of tomato l e a f mRNA (11) would not d i f f e r e n t i a t e between f r e e o r polysome-bound mRNAs and those complexed i n RNPs. I f an RNP o r s i m i l a r p a r t i c l e i s i n v o l v e d , then i t s r o l e must be a temporal one s i n c e a second wound does not repeat the phenomenon ( F i g . 4 ) . Cloning of the mRNAs enriched i n I n h i b i t o r s I and I I messages i s underway t o provide cDNA probes t o more f u l l y explore the r e g u l a t i o n of the tomato genes coding f o r I n h i b i t o r s I and I I i n response t o wounding. With such clones not only can d i r e c t h y b r i d i z a t i o n techniques be employed t o probe the l e v e l s of PIIF-induced mRNA, but more i m p o r t a n t l y , the genes f o r the i n h i b i t o r s can h o p e f u l l y be i s o l a t e d , the s t r u c t u r a l features of the genes be s t u d i e d and the molecular b a s i s f o r the r e g u l a t i o n of gene expression i n response t o s t r e s s can be explored. 7

5

1

7

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1

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Summary


The wound-induced s y n t h e s i s and accumulation of p r o t e i n a s e I n h i b i t o r s I and I I i n tomato leaves has provided a model system to study the r e g u l a t i o n of p r o t e i n a s e i n h i b i t o r genes i n p l a n t s . The s i m p l i c i t y of the phenomenon has made i t p o s s i b l e t o i s o l a t e the wound-factor, o r hormone, and t o study i t s r e l e a s e , d i r e c t i o n and r a t e of t r a n s p o r t i n tomato p l a n t s . Messenger RNA has been i s o l a t e d from leaves of wounded p l a n t s and c o n t a i n s t r a n s l a t a b l e mRNAs f o r the two p r o t e i n a s e i n h i b i t o r s . Studies w i t h these mRNAs have provided a b a s i s f o r the i n i t i a t i o n of a program t o clone i n h i b i t o r cDNAs f o r s t u d i e s of the molecular b a s i s o f the wound-induced process of i n h i b i t o r s y n t h e s i s . Acknowledgements We would l i k e t o thank A l a n Rogers f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e and Richard Hamlin f o r growing the p l a n t s . Literature Cited 1. 2.

3. 4. 5. 6. 7. 8. 9 10. 11. 12. 13. 14. 15.

Green, T.; Ryan, C.A. Science 1972, 175, 776-777. Ryan, C.A. "Current Topics in Cellular R e g u l a t i o n " ; E. Stadtman; B.L. Horecker, eds., Academic P r e s s : New York, 1980; pp 1-23. Bishop, P.D.; Makus, D.J.; Pearce, G.; Ryan, C.A. Proc. N a t l . Acad. Sci. (USA) 1981, 78, 3536-3540. Ryan, C.A.; Bishop, P.; Pearce, G.; Darvill, A.; M c N e i l , M.; Albersheim, P. P l a n t P h y s i o l . 1982, 68, 616-618. Ryan, C.A. P l a n t P h y s i o l . 1974, 54, 328-332. Green, T.R.; Ryan, C.A. P l a n t P h y s i o l . 1972, 51, 19-21. Canny, M.J. "Phloem T r a n l o c a t i o n " ; Cambridge Univ. P r e s s : Cambridge, 1973; pp 301-312. M i n c h i n , P.E.H.; Troughton, J.H. Ann. Rev. P l a n t P h y s i o l . 1980, 31, 191-215. F i s h e r , D.B. P l a n t P h y s i o l . 1970, 45, 114-118. P l u n k e t t , G.; Senear, D.F.; Zuroske, G.; Ryan, C.A. Arch. Biochem. Biophys. 1982, 213, 463-472. Nelson, C.E.; Ryan, C.A. Proc. N a t l . Acad. Sci. (USA) 1980, 77, 1975-1979. Walker-Simmons, M.; Ryan, C.A. P l a n t P h y s i o l . 1977, 60, 61-63. Nelson, C.E.; Ryan, C.A. Biochem. Biophys. Res. Commun. 1980, 94, 355-359. Muthukrishnan, S.; Chanda, G.R.; Maxwell, E.S. Proc. N a t l . Acad. Sci. (USA) 1979, 76, 6181-6185. Mulvihill, E.R.; P a l m i t e r , R.D. J. Biol. Chem. 1980, 255, 2085-2091.



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17. 18. 19. 20. 21.

McConkey, E.H. "Methods in Enzymology," V o l . 12, P t . A; L. Grossman; K. Moldave, eds., Academic P r e s s : New York, 1967; pp 620-634. Fresno, M.; Vazquez, D. Eur. J. Biochem. 1980, 103, 125-132. Hickey, E.D.; Weber, L.A.; B a g l i o n i , C. Proc. N a t l . Acad. Sci. (USA) 1976, 73, 19-23. F i l i p o w i c z , W. FEBS L e t t . 1978, 96, 1-11. Takagi, S.; M o r i , T. J . Biochem. 1979, 86, 231-238. Rudensey, L.M.; I n f a n t e , A.A. Biochem. 1979, 18, 3056-3063. 1982


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Regulation of Synthesis and Accumulation of Proteinase Inhibitors in

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