Phytochrome-mediated synthesis of ascorbic acid oxidase in mustard cotyledons

258

Biochimica et Biophysica Acta, 362 (1974) 258--265

© Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands BBA 27480 PHYTOCHROME-MEDIATED SYNTHESIS OF ASCORBIC ACID OXIDASE IN MUSTARD COTYLEDONS

T.H. ATTRIDGE University of Nottingham School of Agriculture, Sutton Bonington, Loughborough (U.K.)

(Received March 25th, 1974)

Summary Density labelling experiments demonstrate that when dark-grown mustard seedlings are irradiated with far-red light a greater increase in b u o y a n t density of ascorbic acid oxidase may be found in the light treatment than in the appropriate dark control. This probably indicates that p h y t o c h r o m e controls the rate of synthesis of the oxidase although the effects of degradation cannot be entirely ignored.

Introduction When dark-grown mustard seedlings are irradiated with far-red light, the level of activity of phenylalanine ammonia-lyase and ascorbic acid oxidase increase [1,2]. These enzyme responses have been cited to support the gene depression theory for the mode of action of p h y t o c h r o m e [3]. Attridge et al. [4] however, have presented density labelling evidence which is not in keeping with the view that the increase in the level of phenylalanine ammonia-lyase in mustard cotyledons is a result of phytochrome-mediated increase in the rate of enzyme synthesis. Coupling this with other evidence [5,6], it was suggested t h a t the most plausible explanation of the results was that the phenylalanine ammonia-lyase existed in an inactive form in dark-grown tissue and the phytochrome-mediated increase originated from this form of the enzyme via activation. This paper attempts to investigate the problem of whether all phytochrome-mediated enzyme increases involve activation rather than an increased rate of synthesis or whether this effect is peculiar to mustard cotyledons or to phenylalanine ammonia-lyase. In this paper density labelling evidence is presented which demonstrates that the increase in the level of ascorbic acid oxidase in the cotyledons of dark-grown mustard seedlings irradiated with farred light is a result of an increased rate of synthesis of this enzyme.

259 Materials and Methods

Plant materials and growth conditions Seeds of Sinapis alba (Suttons, Reading, England) were germinated in covered containers in the dark on Whatman No. 1 filter paper at 25°C.

Light source and treatment The source of far-red light consisted of 4 × 250 W single coil tungsten bulbs. The light was filtered through 10 cm of running water, one layer of No. 5A Deep Orange and one layer of No. 20 Deep Blue Primary Cinemoid. During the irradiation of plant material the t e m p e r a t u r e of the plant cabinet was k ep t at 25 + 1 ° C.

Enzyme extraction 1 g o f mustard c o t y l e d o n s were ground in 4.0 ml of 0.1 M citrate--phosphate buffer, pH 5.0, and centrifuged. The supernatant was filtered through glass-fibre paper and then passed through a Sephadex G-25 column. The protein fraction was collected and 2.5 ml was used for ultracentrifugation.

Assay of acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2) To each fraction to be assayed was added 0.5 mg of p-ni t rophenyl phosphate in 1.0 ml of 0.02 M acetate buffer, pH 5.2, at 0°C. Incubation was commenced by transferring the tubes to a water bath at 35°C for a suitable time (usually between 5 and 10 min). The reaction was stopped by the addition of 2.0 ml of 10% Na2 CO3. The absorbance of the product , p-nitrophenol, was measured at 410 nm in a Pye Unicam SP 1800 s p e c t r o p h o t o m e t e r .

Assay o f ascorbic acid oxidase (EC 1.10. 3. 3) To each fraction to be assayed was added 2.5 ml of 0.1 M citrate--phosphate buffer, pH 5.0, and the tubes were placed in a water bath at 27 ° C. Since the extracts f r o m far-red light-treated plants had high levels of activity it was possible to measure the disappearance of ascorbic acid cont i nuousl y in the Pye Unicam Sp 1800 at 265 nm. The extracts f rom dark control plants had much less activity and were treated in the following manner. Each fraction, having been diluted with 2.5 ml of buffer, was read in the SP 1800 at 265 nm. 10 pl of 10 mM ascorbic acid (which has an absorbance of A = 1.10 at 265 nm) was added and t h o r o u g h l y mixed. After a suitable period, usually 1 h, the absorbance of each fraction at 265 nm was read again and by simple calculation the a m o u n t of ascorbic acid cons um ed in this time was derived.

Density gradient centrifugation Isopycnic equilibrium centrifugation was carried o u t in a Beckman L2-65B ultracentrifuge using a T y p e 75 Ti fixed angle rotor. A step gradient was prepared in the following manner. 3 ml of distilled water were added to 2.4 g of CsC1 and 2.5 ml of the ext r a c t were layered on top. The tubes were filled to the to p with paraffin oil and centrifuged at 45 000 rev./min at 4°C for 40 h. After centrifugation a narrow gauge needle was lowered to the b o t t o m of

260

the tube and alternate fractions of three and two drops were collected by means o f a peristaltic pump. The 3-drop fractions were used to assay ascorbic acid oxidase whereas the 2-drop fractions were used to measure acid phosphatase activity. Every t ent h fraction was used to measure refractive index in an Abbe 60 refractometer. These values were converted to density units using the equation of Ifft et al. [ 7 ]. Results Fig. 1 demonstrates t ha t when mustard seedlings are grown on water in the dark for 48 h and then transferred to 100% 2 H2 O and far-red light, the presence of deuterium oxide does n o t prevent the p h y t o c h r o m e - m e d i a t e d increase in the level of ascorbic acid oxidase. The e x p e r i m e n t illustrated in Fig. 2 was designed to compare the b u o y a n t density o f ascorbic acid oxidase and acid phophatase in extracts of cot yl edons o f mustard seedlings which had been grown on water in the dark for 72 h (Fig. 2a) with that of mustard seedlings which had been grown on water for 48 h and then transferred to far-red light for 24 h (Fig. 2b). From this experim e n t it may be n o t e d t ha t light t r e a t m e n t itself does n o t alter the b u o y a n t density o f either enzyme. If mustard seedlings are grown on water for 48 h and then transferred to 100% 2 H 2 0 for 6 h (Fig. 3a) or transferred to 100% 2 H 2 0 and far-red light for 6 h (Fig. 3b), only the m os t marginal increase may be seen with the acid phosphatase b u o y a n t densities over the native values. The b u o y a n t density of ascorbic acid oxidase has increased during this t r e a t m e n t bot h in the far-red and dark tr eatment . Whereas the acid phophatase b u o y a n t density values have increased similarly in bot h treatments, the b u o y a n t density of ascorbic acid oxidase increases m or e in the far-red light t r e a t m e n t than in the dark. This effect is seen to a lesser e x t e n t in Fig. 4 and 5, where plants received 12 and 24 h far-red light and 2 H 2 0 t r e a t m ent , respectively. It appears that the

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Discussion The results show that the rate of ascorbic acid oxidase synthesis is enhanced by far-red light treatment. This makes an interesting contrast to recently reported results where it was demonstrated by density labelling that the increase in the level of phenylalanine ammonia-lyase activity after far-red

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light treatment was due either to the formation of a phenylalanine ammonialyase stabilizer or to activation of phenylalanine ammonia-lyase from an existing pool [ 4 ] . The latter hypothesis was favoured due to the similarity of the results to those obtained with gherkins where substantial evidence exists to suggest that dark-grown tissue contains an inactive form of phenylalanine ammonialyase [ 5 , 6 ] . In these publications [4,5] the behaviour of acid phosphatase as an in vivo enzyme marker was an integral part of the argument, indicating that

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Fig. 5. B u o y a n t densities in CsCI o f ascorbic acid o x i d a s e and acid p h o s p h a t a s e e x t r a c t e d f r o m the c o t y l e d o n s of plants g r o w n in the d a r k on w a t e r f o r 4 8 h and t h e n (a) transferred to 100% 2 H 2 0 for 24 h, (b) t r a n s f e r r e d to 100% 2 H 2 0 a n d far-red light for 24 h. S y m b o l s as for Fig. 2.

under the experimental conditions protein synthesis was possible and increases in buoyant density detectable. In the results presented here the acid phosphatase data are not as essential. Ascorbic acid oxidase becomes labelled much more rapidly than acid phosphatase in the short treatments in these experiments. Only a marginal increase in the buoyant density of acid phosphatase is seen. This contrasts with the behaviour of ascorbic acid oxidase. With far-red

265

light and deuterium oxide treatment, the oxidase reaches its maximum b u o y a n t density by 6 h under the experimental conditions. When the seedlings are treated with deuterium oxide but remain in the dark it is not until 24 h that this b u o y a n t density is approached. One possible interpretation of the rapid increase in b u o y a n t density under far-red light is that the rate of turnover of the enzyme is increased. Thus far-red light would stimulate the breakdown of enzyme synthesized before the application of 2 H 2 0 , with the result that the enzyme population would rapidly increase in b u o y a n t density. Increased turnover, however, would be expected to depress the level of activity of ascorbic acid oxidase. It could perhaps be postulated that this observed increase in the extractable activity of the oxidase could result from activation of the newly synthesized deuterated enzyme. However, no experimental evidence exists to support such an hypothesis. The most plausible explanation of these results is that far-red light markedly enhances the rate of synthesis of ascorbic acid oxidase. On the simplest assumption that turnover is n o t affected it may be suggested that the increase in the rate of synthesis would be of the order of 4-6-fold. The density labelling evidence on the light-mediated increase of phenylalanine ammonia-lyase previously reported [4,5], could not be reconciled with the gene derepression theory of p h y t o c h r o m e action [3]. However, the results presented here for ascorbic acid oxidase do n o t conflict with this hypothesis although it may be noted that the action of p h y t o c h r o m e should be considered to be quantitative rather than qualitative since a significant level of ascorbic acid oxidase may be detected in the dark-grown tissue. Further, the data presented here indicate an increase in the rate of synthesis of ascorbic acid oxidase b u t do not demonstrate the direct involvement of regulation at the genome level. The results presented here together with results recently published [4] clearly indicate that p h y t o c h r o m e can control enzyme level through at least two different mechanisms in the same tissue. Acknowledgements My thanks are due to Miss J. Elliott for technical assistance, to Mr C.B. Johnson for helpful discussion, to Professor H. Smith for encouraging me to do this work and to the S.R.C. for supporting this project. References 1 2 3 4 5 6 7

Durst, F. and Mohr, H. (1966) Naturwissenschaften 53, 531--532 Dr umm, H.° Briinning, K. and Mohr, H. (1972) Planta 106, 259--267 Mohr, H. (1966) P h o t o c h e m . Photobiol. 5, 469--483 Attridge, T.H., Jo hns on, C.B. and Smith, H. (1974) Biochim. Biophys. Acta, 343, 440--451 Attridge, T.H. and Smith, H. (1974) Biochim. Biophys. Acta 343, 452--464 Attridge, T.H. and Smith, H. (1973) Phytochem. 12, 1 5 6 9 - - 1 5 7 4 Ifft, J.B., Volt, D.H. and Vinograd, J. (1961) J. Phys. Chem. 65, 1 1 3 8 - - 1 1 4 5