The mechanism of the repression by inorganic phosphate of phytase synthesis in the germinating wheat embryo

485

BIOCFIIMICA ET BIOPHYSICA ACTA

BBA 95677

T H E MECHANISM OF T H E R E P R E S S I O N BY INORGANIC P H O S P H A T E OF P H Y T A S E SYNTHESIS IN T H E GERMINATING W H E A T EMBRYO

RENATO BIANCHETTI AND MARIA LUISA SARTIRANA

Institute o[ Plant Sciences, University o[ Milan, Milan (Italy) Centro di Studio del C.N.R. per le ossido-riduzioni nei vegetali. (Received January 23rd, 1967) (Revised manuscript received April ioth, 1967)

SUMMARY

It has been previously shown that phytase is repressed by Pl in isolated wheat embryos. The mechanism of the repression of phytase by PI was investigated separately in the germ and in the scutellum of germinating wheat embryos. I. In the scutellum, the increase of phytase during germination is completely abolished by puromycin or by actinomycin. The supply of P1 specifically represses the synthesis of the enzyme. 2. In the germ, puromycin also blocks the increase of phytase; actinomycin only delays it, while PI has no effect on the synthesis of the enzyme. 3- In the scutellum, the maximum value of the enzyme is reached at about the 3oth h of germination. Experiments with actinomycin show that phytase synthesis is completely blocked only if actinomycin is supplied within the first 6 tl of germination, while the inhibitor, when administered after the I4th h, is completely ineffective. Similar experiments show that Pl also has no effect on phytase synthesis if supplied after 6 h of germination. These results are interpreted as indicating that Pl is effective in repressing phytase synthesis only if supplied during a period coinciding with that of phytase messenger RNA synthesis. It is concluded that P1 acts at the transcription level.

INTRODUCTION

In an earlier study (SARTIRANAAND BIANCI~ETTI1) it was shown that wheat embryos, like many other seeds, or seed organs, contain an acid phosphatase activity, mostly specific for phytin. As germination progresses, this activity increases considerably and then disappears slowly when the phytin has been used up. It was also shown that: (a) in vivo the phytase activity is regulated by the concentration of Pi in the tissues; (b) the phytase synthesis is inhibited by the Pi present in the culture medium. Biochim. Biophys. Acta, 145 (1967) 485-490

486

R. BIANCHETTI, M. L. SARTIRANA

The inhibition of phytase development by PI was also found to be specific, since at the same time growth, respiration, hexose phosphate concentrations and protein synthesis (evaluated as Ecarboxy-14Clleucine incorporation and development of a number of enzymatic activities) remained unaltered. In the present research, the study of the repression of phytase synthesis by Pt was extended from the embryo in toto to its two main components: the germ and the scutellum.

MATERIAL AND METHODS

Wheat embryos (Triticum durum var. Cappelli), separated from the seed after being soaked for 2 h in distilled water at 20 °, were grown in submerged culture, in small flasks over a rotating agitator, under sterile conditions. The incubation was carried out at 28 ° and in the dark. The embryos were fed with 0.5 % glucose and P1 was supplied as sodium and potassium salts at the required concentrations. At the end of the incubation period, the germ and the scutellum were separated with the use of a small surgical lancet. Phytase activity was assayed in the dialysed supernatant of the homogenate in o.I M acetate buffer (pH 5.5), after centrifugation at 13 ooo rev./min for 20 rain, in the presence of phytin as its sodium salt (5.io-3M), by measuring the increase of PI. All the assays were carried out at p H 5.5. Sodium phytate was prepared from the calcium and magnesium salt (commercially available) according to the method of PEERS 2. The Pt was assayed according to the method of TAUSSKY AND SHORR 3, in the supernatant of a IO % trichloroacetic acid extract. The total nucleic acid assays were carried out as follows: 200 mg of tissue were ground in a mortar with 5 ml of hot 95 % ethanol (NIEMAN AND POULSENa; SMILLIE AND KROI~TOV5) and centrifuged at 20 ooo rev./min for 20 min. The precipitate was resuspended in 1. 5 ml of o.I M K2HPOd+ 3 ml of phenol saturated with water (SANDER AND KNIGHT6). The tubes were thoroughly shaken and then centrifuged at --20 ° . The aqueous phase was removed b y aspiration and the precipitate plus the interphase material were extracted again with 2 ml of o.I M K2HPO 4. The two aqueous phases, combined together, were mixed with i ml of phenol; the aqueous phase was separated and repeatedly washed with ethyl ether, then 6 ml of 95 % ethanol were added and the samples centrifuged at 20 ooo rev./min for 20 min. The precipitate was resuspended in 5 ml of o.I M K2HPO * and the nucleic acids were determined by measurement of the absorbance at 260 m#. After the spectrophotometric determination, the nucleic acids were precipitated again with ethanol and the precipitate was plated onto small MiUipore filters, washed with 5 % trichloroacetic acid and counted for the assay of the ~2Pt incorporated into the nucleic acids.

EXPERIMENTAL

The germ and the scutellum show different metabolic features and evolve differently. Biochim. Biophys. Acta, 145 (i967) 485-49 °

487

REPRESSION OF PHYTASE SYNTHESIS IN WHEAT EMBRYOS

Scutellum Under our experimental conditions, the scutellum showed an increase of 7 ° % to 80 % of its initial fresh weight, the m a x i m u m weight being reached between the 3oth and the 4oth h of germination. The m a x i m u m level of the total nucleic acids was also reached at about the same time, with an increase of about 30 % of its initial value (Fig. I). The phytase activity also reached its m a x i m u m at approximately the 3oth h of germination, doubling its initial value, while in the following period it tended toward a slow decrease. Puromycin, in a concentration of 3" lO-4 M (WILLIAMSON AND SCHWEETT),completely abolished the increase in enzyme activity. A similar effect was obtained when 8 o # g actinomycin per ml ( H U R W l T Z et al.S; GOLDBERG, RABINOWITZ AND RICH9) was already present in the culture medium since the first hours of germination. It should be noted that lower concentrations of both these inhibitors (10 -4 M puromycin and IO or 3 ° #g actinomycin per ml) were found to be partially effective. A supply of 2. I0 -* M P1 induced a complete block of the increase in phytase activity in the scutellum. All these data are shown in Fig. 2. 14 13. 30-

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Fig. I. G r o w t h a n d nucleic acid c o n t e n t s in g e r m s a n d s c u t e l l a of isolated w h e a t e m b r y o s . 0 - 0 , m g f r e s h w e i g h t p e r 5 g e r m s ; 0 - © , / ~ g nucleic acid p e r Io g e r m s ; A - A , m g f r e s h w e i g h t p e r 5 scutella; & - & , / ~ g nucleic acid p e r io scutella. G e r m s a n d s c u t e l l a were s e p a r a t e d before t h e determination. Fig. 2. P h y t a s e a c t i v i t y in t h e s c u t e l l u m . Control ( 0 - 0 ) ; 2 " I o - ~ M Pi ( O - G ) ; 3 " I o - 4 M p u r o m y c i n ([[]-[3); 8o/~g a c t i n o m y c i n p e r m l ( & - & ) . A c t i n o m y c i n , p u r o m y c i n a n d Pi were p r e s e n t in t h e c u l t u r e m e d i u m f r o m t h e 2 n d h to t h e e n d of t h e e x p e r i m e n t .

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The fresh weight increase was considerable and remained almost linear for a period up to 7 ° h. The total nucleic acid level (which after only 2 h of germination was already double that in the scutellum) also increased steadily during the same period (Fig. I). The phytase development during germination in the germ followed a course almost similar to that of the enzyme in the scutellum: in the germ also, 5" lO-4 M puromycin blocked the enzyme synthesis. Actinomycin, however, inhibited the increase in enzyme activity during the first 20 h of treatment only, while during the following period the enzyme concentration increased rapidly, to reach about the Biochim. Biophys. Acta, i45 (1967) 4 8 5 - 4 9 o

488

R. BIANCHETTI, M. L. SATIRANA

s a m e values as the controls at the 5oth h. Likewise 2" IO -2 M Pi (which effectively b l o c k e d the increase in p h y t a s e a c t i v i t y in the scutellum) d i d n o t significantly alter t h e d e v e l o p m e n t of the p h y t a s e a c t i v i t y in t h e germ. All these d a t a are r e p o r t e d in Fig. 3. The l a c k of i n h i b i t i o n b y a c t i n o m y c i n a n d b y PI m i g h t be i n t e r p r e t e d in t w o different ways: (a) preexistence of the R N A messenger for t h e enzyme; (b) actinom y c i n a n d PI are n o t active if supplied e x t e r n a l l y a n d c a n n o t block the e n z y m e s y n t h e s i s because t h e y c a n n o t reach the site of action a n d be m a i n t a i n e d t h e r e at a n effective c o n c e n t r a t i o n .

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Fig. 3- Phytase activity in the germ. For legends see Fig. 2. Fig. 4- C&pxcity of 32pt incorporation into the nucleic acid of the germ. 3aPl incorporated into nucleic acid: O - Q , control; A - A , 8o #g actinomycin per ml. ~2P1in the alcohol-soluble fraction: Q-C), control; ~-/~, 8o/~g actinomycin per ml. 32P1in the alcohol-soluble fraction: © ©, control; & - & , 8o/~g actinomycin per ml. The inhibitor was present in the medium from the 2nd h. Groups of 4° embryos were drawn from the culture at the hours of germination indicated and incubated in 2 ml of 3" lO-3 M 32P1 (io/~C//,mole) for 45 min at 28 °. Germs were separated from scutella at the end of the incubation. As r e g a r d s a c t i n o m y c i n , a m e a n s to settle the question was offered b y a s t u d y of the action of a c t i n o m y c i n on R N A synthesis, e v a l u a t e d as 32P1 i n c o r p o r a t e d i n t o the R N A . Fig. 4 shows t h a t a c t i n o m y c i n required a considerable i n c u b a t i o n time, n o t y e t a c c u r a t e l y e v a l u a t e d , b u t exceeding 3 h, in order to p r o d u c e a c o m p l e t e inhibition a n d it can be seen t h a t an a l m o s t t o t a l i n h i b i t i o n of t h e R N A synthesis is o b t a i n e d w i t h a t r e a t m e n t l a s t i n g 3 to I8 h. Afterwards, the germ g r a d u a l l y recovered from the inhibition, a n d t h e i n c o r p o r a t i o n of 32P1 into R N A was resumed. This, t o g e t h e r with the fact t h a t a c t i n o m y c i n is i n h i b i t i n g the germ g r o w t h d u r i n g t h e first 3o h only, while s u b s e q u e n t l y t h e g r o w t h r a t e of the a c t i n o m y c i n - t r e a t e d g e r m s is equal to t h a t of the controls, leads to the a s s u m p t i o n t h a t t h e germ is develo p i n g a certain resistence to a c t i n o m y c i n . C o n t r o l e x p e r i m e n t s showed t h a t t h e a c t i n o m y c i n from t h e culture m e d i u m , even after a l m o s t 3o h of i n c u b a t i o n , r e t a i n s to the same e x t e n t its c a p a c i t y to act on the same a n d on o t h e r material. On t h e o t h e r hand, a c t i n o m y c i n c o n s i d e r a b l y modified the time-course of the e n z y m e changes. Fig. 3 shows t h a t a c t i n o m y c i n i n d u c e d a m a r k e d lag in the p h y t a s e formaBiochim. Biophys, Acre, 145 (1967) 485-49o

REPRESSION OF PHYTASE SYNTHESIS IN WHEAT EMBRYOS

489

tion as compared with the controls and it was after the 3oth h only, when the 3~P1 incorporation into the RNA was resumed, that the phytase activity rapidly increased. These observations might suggest that, in the germ as well as in the scuteJlum, the enzyme synthesis did not depend upon a preexisting RNA messenger. Even the lack of action of Pt on the germ phytase, as opposed to the case of the scutellum, may be easy to explain when it is considered that the supply of Pl affects quite differently the internal pools in the germ and in the scutellum (Fig. 5)- The scutellum has, indeed, already shown a massive increase in its Pt concentration at the very onset of germination, while in the germ the Pl concentration has remained equal to or become slightly higher than that of the controls.

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Mechanism o/the action o/P+ in the scutellum In the scutellum, the development of phytase appears clearly and is easily reproducible. It is totally blocked either by puromycin or by actinomycin supplied at the beginning of germination and it is almost entirely blocked by Pt. As regards the mechanism of repression by Pt, there are three possible hypotheses: (a) control of the transcription of DNA into the RNA messenger; (b) control of the translation of the RNA messenger to give the enzymatic protein; (c) acceleration of the breakdown of enzymatic protein. The choice to be made among the above-mentioned hypotheses is dependent largely upon the possibility of measuring with sufficient accuracy the time-courses of the RNA messenger, the enzyme synthesis and, particularly, the relationship between the two time-courses. Since previous experiments with actinomycin showed that development of the enzyme was controlled by the synthesis of RNA messenger at the onset of germination, the supply of actinomycin at different stages of the enzyme development should make it possible to determine, at least approximately, the time lapse during which the specific RNA messenger synthesis takes place and whether it precedes or coincides with the interval in which the enzyme increase is Biochim. Biophys. Acta, 145 (1967) 485-49o

49 °

R. BIANCHETTI, M. L. SARTIRANA

occurring. For this purpose we supplied actinomycin to different samples at 2, 4, 6, IO, 14, 16 and 20 h after the onset of germination and in all these samples the subsequent development of the enzyme was determined (Fig. 6). A total block of the enzyme formation is only obtained when actinomycin is supplied during the first 6 h after the onset of germination, while no effect is observed if actinomycin is supplied after the I4th h. As previously shown (Fig. 2), the maximum enzyme activity is only reached at approximately the 3oth h of germination, i.e. several hours after the synthesis of RNA messenger has stopped. It is clear that the translation of the phytase RNA messenger takes place several hours after the synthesis has stopped. The repression by Pt was studied in a similar experiment, in which the ion was supplied at different times of germination. Here again (Fig. 6), a complete repression of the formation of phytase was observed only when Pt was supplied during the first 6 h of germination, while Pt supplied at later stages remained entirely ineffective. The capacity of PI to block the synthesis of phytase is therefore evident only during the period in which the formation of the enzyme is sensitive to actinomycin, while no effect of P l o n the enzyme formation is observed during the subsequent period. These findings, therefore, rule out the possibility that P1 may accelerate the breakdown of enzyme or affect the translation of the RNA messenger into protein. On the other hand, our observations are in favour of the hypothesis that PI, like actinomycin but in a specific way, may inhibit the formation of the RNA messenger for phytase. This behaviour is that expected from a specific repressor of enzyme formation. As suggested in a previous report 1, it seems possible to give an interpretation of the development and subsequent disappearance of phytase in the course of germination, based on the variations of PI taking place in the tissues of the embryo. In the germ, as well as in the scutellum, it was observed that at the onset of germination a rapid and marked decrease of free Pt takes place. Subsequently, the Pt concentration gradually increases, due to phytase activity, until it reaches values higher than its initial ones (Fig. 5). As shown by the experiments with actinomycin, the first of these two metabolic situations corresponds to the period in which the synthesis of the phytase RNA messenger takes place and the second to that in which such a synthesis does not occur any longer. It should be stressed that parallel experiments showed that similar variations in the concentration of Pt in the tissues are occurring even in embryos still connected to their seed, a fact which was previously observed in germinating oat seeds (ALBAUM AND UMBREIT l ° ) . T h e s e findings therefore support the hypothesis that the concentration of P I constitutes an important factor in the control of phytase synthesis, under physiological conditions, during seed germination. REFERENCES M. L. SARTIRANA AND R. BIANCHETTI, Plant Physiol., (1967) in t h e press. F. G. PEERS, Biochem. J., 53 (1953) lO2. H. H. TAUSSKY AND E. BHORR, J. Biol. Chem., 202 (1953) 675R. H. NIEMAN AND L. L. POULSEN, Plant Physiol., 38 (1963) 31. R. 1V~.BMILLIE AND G. KROKTOV, Can. J. Bot., 38 (196o) 31. H. L. BANGER AND C. A. KNIGHT, Biochem. Biophys. Res. Commun., 13 (1963) 455. A. R. WILLIAMSON AND R. BCHWEET,Nature, 202 (1964) 435. J. HtlRWlTZ, J. J. FORTH, M. MALANY" AND M. ALEXANDER, Proc. Natl. Acad. Sci. U.S., 48 (1962) 1222. 9 I. H. GOLDBERG, i'~. RABINOWlTZ AND E. RICH, Proc. Natl. Acad. Sci. U.S., 48 (1962) 2o94. IO H. G. ALBAUM AND W. W. UMBREIT, Am. J. Botany, 3 ° (1943) 553I 2 3 4 5 6 7 8

Biochim. Biophys. Acta, 145 (i967) 485-490