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Continuous far red irradiation controls molecular properties of δ-aminolevulinate dehydratase in Raphanus sativus seedlings
Plant Science Letters 32 (1983) 295--303
295
Elsevier Scientific Publishers Ireland Ltd.
CONTINUOUS F A R RED I R R A D I A T I O N C O N T R O L S M O L E C U L A R P R O P E R T I E S O F 5-AMINOLEVULINATE D E H Y D R A T A S E IN R A P H A N U S S A T I V U S SEEDLINGS
A.P. BALANGE a and C. LAMBERTb aLaboratoire de Photobiologie, Laboratoire Associ~ au CNRS (LA 203), Facult~ des Sciences B.P. 67, 76130 Mont Saint Aignan, and bU.E.R. Scientifique de Luminy, D$partement de Biologie Mol~culaire et CeUulaire, Biochimie Fonctionnelle des Plantes, 13288 MarseiUe, C~dex 2 (France)
(Received November 1st, 1982) (Revision received June 13th, 1983) (Accepted June 18th, 1983)
SUMMARY -Aminolevulinate dehydratase (EC 4.2.1.24)I(ALAD) is a p h y t o c h r o m e dependent enzyme. Under c o n t n u o u s far red light (FR), the intraceUular location of ALAD is modified: in y o u n g irradiated seedling cotyledons (48 h from sowing) it is localised in the cytoplasm, as for seedlings kept in continuous darkness or irradiated b u t treated with erythromycin (ERT). In seedlings k e p t 120 h under continuous FR light, ALAD is detected in cytoplasm too, but also in etioplasts. Studies from DEAE-cellulose chromatography show that, when ALAD is localised in the cytoplasm it has a stable charge, b u t an unstable molecular weight. If piastids are allowed to grow normally under continuous F R light, the enzyme can be purified from stroma of etioplasts from 72 h from sowing. The molecule is unstable, both in charge and in molecular weight. ALAD from etioplasts is further transformed into a specie stable b o t h in charge and in molecular weight. The relationship b e t w e e n the molecular modifications and physiological results observed previously are discussed. K e y words: R a p h a n u s sativus - - ~ -Aminolevulinate dehydmtase -- Far red
light INTRODUCTION In higher plants numerous enzymes are under p h y t o c h r o m e control [ 1 ], and the mechanisms involved are subjected to intensive investigations. Abbreviations: ALAD, 6 -aminolevulinate dehydratase; ERT, erythromycin; FR, standard far red light (k ~ 720 nm); RuBPc, ribulose bisphosphate earboxylase. 0304-4211/83/$03.00
© 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
296
ALAD (EC 4.2.1.24) is the first enzyme detectable in higher plants of the tetrapyrrole biosynthesis pathway. We have previously shown that ALAD activity is enhanced by an irradiation with continuous FR light, involving the phytochrome system, in radish seedling cotyledons in the early hours of differentiation [2]. The enzyme is always detected in the cytoplasm during growing in the dark or the light after treatment with ERT which specifically inhibits protein synthesis on 70 S ribosomes [4]. Under continuous FR light ALAD is present in the cytoplasm until 72 h, but at that time the enzymatic activity appears also in etioplasts. The presentwork reports modifications in molecular properties of partially purified ALAD or from cytoplasm etiopiast enriched fraction during seedling differentiation in various growth conditions. These results are discussed with regard to those previously obtained [2,3]. MATERIAL AND METHODS Radish seeds (Raphanus sativus L. cv. Longue Rave Saumon~e) were germinated either in the dark or under continuous light. Enzymatic measurements, chemicals and light conditions were described elsewhere [2]. ERT 250 ~g • m1-1 was given to seedlings which were 48-h-old as described previously [2]. The different manipulations were carried out under a dim green safelight at 2°C. Etioplasts were isolated from 10 g fresh wt. cotyledons as described previously [3]. Purified etioplasts were disrupted by osmotic shock in Tris- HC1 buffer 10 mM (pH 8.7), containing MgC12 10 mM and the stromal fraction was clarified by centrifngation (Berkman J21 B, Rotor JA 20, 30 000 × g for 15 min). Stromal solution was passed through a Sephadex G-50 column and ALAD was collected at V0. Active fractions were pooled and applied on a DEAE-cellulose column (1 × 20 cm) (Trisacryl, Pharmindustrie, IBF (Industrie Biologique Franqaise), Villeneuve la Garenne, France) equilibrated with TEs- HC1 buffer 10 mM (pH 8.7) containing 10 mM MgC12. The column was rinsed with the same buffer containing 50 mM NaC1. Enzyme was eluted by a linear NaC1 gradient (50--350 raM, 120 ml) in Tris--HC1 buffer (pH 8.7). One-mi|H]iter fractions were collected and assayed for ALAD activity. In experiments involving further DEAE chromatography, active fractions were pooled, diluted with Tris- HC1 buffer 10 mM (pH 8.7), to obtain a final 5 mM NaCI concentration, and applied on a new DEAEcenulose column as described above. As it was impossible to obtain enough enzyme from purified etioplasts in the dark or after ERT treatment, ALAD was prepared from crude cotyledons extract. Fresh weight cotyledons (10 g) were ground in 30 ml Tris--HC1 buffer 50 mM (pH 8.7) containing 10 mM MgC12 and 14 mM 2 mercaptoethanol The brei was centrifuged 30 000 × g for 30 min and the supernatant purified through Sephadex G-50 and DEAE-ceUulose as described above. For control, enzyme from FR-irradiated seedlings was purified either
297
from crude extract or from purified etioplasts. Both procedures led to identical elution profiles after ion exchange chromatography, and thus allowed us to compare ALAD of various origins. Molecular weight estimation was performed by ACa 34 gel sieving {4 × 20 cm) (Ultrogel, Pharmindustrie, IBF, Villeneuve la Garenne, France) in Tris--HCl buffe~ 10mM (pH 8.7) containing 10 mM MgC12. A one-milliliter sample of an active fraction purified after DEAE-cellulose chromatography was placed on the column and eluted at 0.5 m l . min -1. One-milliliter fractions were collected and assayed for ALAD activity. The ACa 34 column •was calibrated with: catalase 240 000, aldoiase 128 000, bovine serum albumin 67 000, ovalbumin 43 000 daltons (SERVA). V0 was estimated with elution of dextran blue and Ve/Vo plotted vs. log molecular weight to establish a calibration curve. RESULTS AND DISCUSSION In a previous work, we had investigated the intmcellular location of ALAD and had observed that it is modified by light or antibiotic treatments. In complete darkness or under FR light and ERT treatments, or in young irradiated seedlings ALAD is located in the cytosol. At 96 and 120 h from sowing under continuous Fit light the enzymatic activity appears in etic~ plasts too [3]. When etioplasts are purified from radish seedling cotyledons grown 96 or 120 h under continuous FR light, two ALAD fractions are eluted after DEAE-cellulose chromatography. These occur at 0.11 M (peak I} and 0.15 M (peak II} NaC1 concentration, respectively (Fig~ l b and 2c}. Only the first eluted peak (peak I) is observed in etioplasts 72-h-old (Fig. la}. In fully etiolated seedlings (grown in absence of FR light), or in seedlings grown under FR light but treated with ERT, it was not possible to purify ALAD from etioplasts. If the enzyme is prepared from a crude cotyledon extract, only one ALAD charge species, eluted at 0.11 M NaC1 concentration is observed (Figs. 2b and 2c}. The same elution profile is detected after chromatography of young seedlings grown 48 h under continuous FR light (Fig. 2a). Appearance of ALAD in peak II is correlated in time with the detection of enzyme activity in etioplasts from FR-irradiated seedlings observed in these previous results. This raises the possibility that peak I (observed Fig. 2) is the cytosolic enzyme. Thus, the newly observed ALAD from peak II (Figs. l b and lc) localised in etioplasts differs in electric charge from its cytoplasmic counterpart. However, peak I is observed either in seedlings when ALAD is cytoplasmic (Fig. 2) or located in etioplasts {Fig. 1). We have tried to distinguish further molecular differences between ALAD charge forms detected through ion exchange chromatography. Some authors have also shown that ALAD in green plants may be separated in two fractions after DEAE-cellulose chromatography, eluted at NaC1 concentrations
298
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Fig. 1. Changes in ALAD elution profile o n DEAE-cellulose chromatography as a function o f seedling age. • - , ALAD activity; . . . . . . , NaC1 gradient. Extracts are prepared as d e s c r i e d in Materials and Methods from etiopiasts isolated from 10 g fresh wt. radish cotyledons grown unde~ continuous F R light. (a) 72 h from sowing; (b) 96 h from sowing; (c) 120 h from sowing.
close to those reported here [5,6]. However, t h e y consider peak I as an artifact, because of its instability on passing it through a DEAE column for a second or a third time. We have checked such a possibility for each of the radish DEAE ALAD species.
299
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Fig. 2. Elution profiles of ALAD from cotyledon extracts on DEAE-cellulose chromatography. (a) 48 h from sowing FR-irradiated seedlings; (b) 120 h from sowing, completely etiohted seedlings; (c) 120 h from sowing, FR-irradiated seedlings treated with ERT 250 ~g oml -~ at 48 h. • . , ALAD activity; . . . . . . , NaC1 gradient. C y t o s o l i c A L A D p u r i f i e d f r o m d a r k g r o w n seedlings, f r o m y o u n g irradiated ones (48 h f r o m sowing), f r o m seedlings t r e a t e d w i t h E R T , never dissociates
300 on f u r t h e r ion c h r o m a t o g r a p h y (result n o t shown). As r e p o r t e d b y o t h e r authors, peak I purified f r o m etioplasts grown 120 h u n d e r F R light dissociates in two peaks (Fig. 3c), b o t h o f which c o n t i n u e t o yield t w o peaks o n f u r t h e r passage on D E A E (Figs. 3d and 3e). The same results are o b t a i n e d if p e a k I observed at 72 h in etioplasts (Fig. l a ) is checked. When p e a k II d e t e c t e d after the p r i m a r y c h r o m a t o g r a p h y (Fig. 3a), is subjected to the same D E A E steps, it never dissociates (Figs. 3b a n d 3f), e x c e p t a small
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Fie 3. Elution profiles of etioplast ALAD prepared from 10 g fresh wt. radish cotyledons 120 h grown under continuous FR light during successive chromatographies on DEAE cellulose. (a) Elution profile of stromal fraction; (b) chromatography of peak II; (c) chromatography of peak I; (d), (e) chromatography of peaks pooled separately from (c); (f) chromatography from peak II from (b). * • enzymatic activity. NaCI normalities for elution are given.
301
amount of ALAD which is rapidly eluted (Fig. 3b) but we consider that it is a contamination from peak I. ALAD is a multimeric enzyme, easily undergoing subunit reassociation [7] on changing buffer [8] or ionic strength [9]. Thus after the first chromatography, the subsequent dilutions, necessary to obtain binding on another gel, generates structural modifications of etioplast enzyme from peak I. This is indeed a true artifact, but neither cytoplasmic, nor etioplast ALAD from peak II undergo such transformations. Thus, we can detect after DEAE-cellulose chromatography two kinds of ALAD molecules by reference to their charge: stable enzymes are observed, in cytosolic fraction eluted at 0.11 M NaC1 concentration and in etioplasts eluted at 0.15 M NaC1 concentration. ALAD with an unstable charge is detected in etioplasts too, but is eluted at 0.11 M NaC1 concentration. Molecular weights of the different enzyme species have been estimated by gel sieving, after purification from DEAE-cellulose chromatography as described above. Table I shows that ALAD from peak II has always a mol. wt. of 245 000. The enzyme found in the cytoplasm, or ALAD in etioplasts eluted in peak I shows different aggregation states. Little work has been done on molecular properties of ALAD from plants, and results available are mainly obtained from animals. In this material, enzyme is multimeric and the monomer (40 000 daltons) is inactive [9]. We observe after gel sieving, four molecular species ranging around 340 000, 240 000, 170 000 and 120 000 daltons, which is consistent with associations of 8, 6, 4 and 3 subunits, respectively.
TABLE I ESTIMATION OF MOLECULAR WEIGHT OF ALAD BY GEL SIEVING CHROMATOGRAPHY ON AcA 34 GEL ALAD fractions are prepared from the different peaks observed after DEAE-cellulose chromatography. Column was calibrated by reference to elution of: catalase, aldolase, bovine serum albumin, ovalbumin and dextran blue. ALAD purified from seedling cotyledons grown:
120 h continuous F R light DEAE, peak I (0.11 M NaCl) 120 h continuous F R light DEAE, peak II (0.15 M NaCl) Complete darkness DEAE, peak I (0.11 M NaCl) 120 h continuous F R light E R T treatment at 48 h from sowing DEAE, peak I (0.11 M NaCl)
Estimated mol. wt.
338 000
228 000
169 000
110 000
245 000 319 000
240 000
148 000
120 000
319 000
260 000
178 000
120 000
302 Thus, with respect to growth conditions, it is possible to isolate 3 diffe~ rent ALAD species: (1) an enzyme with constant charge, but undergoing different aggregation states appears in complete darkness, in young irradiated seedlings, or after ERT treatment. This molecule is probably cytoplasmic. Two other enzyme forms are obtained from etioplasts: (2) one form always has the same charge and the same mol. wt. (245 000). It is found in etioplasts of light grown seedlings at 96 and 120 h from sowing and is eluted at 0.15 M NaC1 concentration on DEAE-cellulose chromatography. (3) The final form appears earlier in etioplasts (72 h from sowing), is eluted at 0.11 M NaC1 concentration, but is found with variable molecular weight and charge. At that time, two hypotheses can explain the relationships between ALAD localisation and its molecular properties. It can be assumed that ALAD is firstly synthesized on cytoplasmic ribosomes as identical subunits, with constant charge but undergoing easily different aggregation states, at least in vitro, which is also reported for cytoplasmic precursors of some mitochondrial proteins [101. The cytoplasmically localised enzyme is observed in complete darkness, and in young irradiated seedlings. If seedlings are grown under continuous FR light, at least during 72 h ALAD is exported into etioplasts with structural change, giving it a complete unstable charge. This modification may'be a processing effect of unknown protease, as for a small subunit of ribulose bisphosphate carboxylase (RuBPc) [11], or another less known mechanism. At last in mature etioplasts a stabilization mechanism takes place which gives rise to ALAD molecules having stable charge and tool. wt. of 245 000. This hypothesis involves a two-step mechanism into etioplasts, which is reported neither for small subunit of RuBPc [11] nor for other chloroplastic molecules. However recent findings from Daum et al. show also a two-step transformation of cytochrome b2 in intermembrane mitochondrial space [ 12 ]. Another hypothesis may be a complete lack of correlation between ALAD in cytoplasm and enzyme in etioplast. If we consider this explanation, ALAD is synthesized both on cytoplasmic ribosomes and on etioplastic ones. In organeUes, enzyme is built as an unstable molecule from 72 h from sowing, and then stabilized from 96 to 120 h from sowing under continuous FR light by an unknown mechanism. In both hypotheses, if etioplasts are not allowed to grow normally under FR light (after ERT treatment) or if they stay in a preliminary state (young seedlings) no ALAD appears in etioplasts, either after translocaticn or after synthesis, and only the cytoplasmic enzyme is observed. It would be operating in heine pathways other than in chlorophyll biosynthesis, and is usually shaded by the presence of its etioplast counterpart. At that time results obtained previously cannot completely discard any of the proposed hypotheses. Further work based on enzyme purification and antibodies preparations will give more precise information on ALAD regulation through phytochrome system.
303
Note added in Proof D u r i n g t h e w r i t i n g o f t h e revised version, M.V. B l o o m , P. Milos and H. R o y , Proc. Natl. Acad. Scl. U.S.A., 80 ( 1 9 8 3 ) 1 0 1 3 - 1 0 1 7 , s h o w several i n t e r m e d i a t e s in t h e large R u B P c s u b u n i t a s s e m b l y , w h i c h are light d e p e n d e n t . This finding m a y s u p p o r t in p a r t o u r s e c o n d a s s u m p t i o n . REFERENCES 1 2 3 4 5 6 7 8 9 10
11 12
P. Schopfer, Annu. Rev. Plant Physiol., 28 (1973) 233. A.P. Balang~ and P. RoUin, Physiol. Veg., 17 (1) (1979) 153. A.P. Balang~ and C. Lambert, Phytochem., 19 (1980) 2541. R. Langlois, C.R. Cantor, R. Vince and S. Pestka, Biochemistry, 16 (1977) 2349. Hj.A.W. Schneider, Z. PflanzenphysioL, 62 (1970) 328. H. Tamai, Y. Shioi and T. Sasa, 20 (2) (1979) 435. D. Shemin, in: P.D. Boyer (Ed.), The Enzymes, VoL 7 Academic Press, New York, 1972, pp. 323--337. S. Van Heyningen and D. Shemin, Biochemistry, 10 (25) (197~) 4676. N. Despaux, E. Comoy, C. Bohuon and C. Boudene, Biochimie, 61 (1979) 1021. W. Neupert and G. Schatz, Trends Biochem. Sci., 1--4 (1981). ~ R.J. Ellis, Annu. Rev. Plant Physiol., 32 (1981) 111. G. Daum, S.M. Gasser and G. Schatz, J. Biol. Chem., 257 (21) (1982) 13075.
295
Elsevier Scientific Publishers Ireland Ltd.
CONTINUOUS F A R RED I R R A D I A T I O N C O N T R O L S M O L E C U L A R P R O P E R T I E S O F 5-AMINOLEVULINATE D E H Y D R A T A S E IN R A P H A N U S S A T I V U S SEEDLINGS
A.P. BALANGE a and C. LAMBERTb aLaboratoire de Photobiologie, Laboratoire Associ~ au CNRS (LA 203), Facult~ des Sciences B.P. 67, 76130 Mont Saint Aignan, and bU.E.R. Scientifique de Luminy, D$partement de Biologie Mol~culaire et CeUulaire, Biochimie Fonctionnelle des Plantes, 13288 MarseiUe, C~dex 2 (France)
(Received November 1st, 1982) (Revision received June 13th, 1983) (Accepted June 18th, 1983)
SUMMARY -Aminolevulinate dehydratase (EC 4.2.1.24)I(ALAD) is a p h y t o c h r o m e dependent enzyme. Under c o n t n u o u s far red light (FR), the intraceUular location of ALAD is modified: in y o u n g irradiated seedling cotyledons (48 h from sowing) it is localised in the cytoplasm, as for seedlings kept in continuous darkness or irradiated b u t treated with erythromycin (ERT). In seedlings k e p t 120 h under continuous FR light, ALAD is detected in cytoplasm too, but also in etioplasts. Studies from DEAE-cellulose chromatography show that, when ALAD is localised in the cytoplasm it has a stable charge, b u t an unstable molecular weight. If piastids are allowed to grow normally under continuous F R light, the enzyme can be purified from stroma of etioplasts from 72 h from sowing. The molecule is unstable, both in charge and in molecular weight. ALAD from etioplasts is further transformed into a specie stable b o t h in charge and in molecular weight. The relationship b e t w e e n the molecular modifications and physiological results observed previously are discussed. K e y words: R a p h a n u s sativus - - ~ -Aminolevulinate dehydmtase -- Far red
light INTRODUCTION In higher plants numerous enzymes are under p h y t o c h r o m e control [ 1 ], and the mechanisms involved are subjected to intensive investigations. Abbreviations: ALAD, 6 -aminolevulinate dehydratase; ERT, erythromycin; FR, standard far red light (k ~ 720 nm); RuBPc, ribulose bisphosphate earboxylase. 0304-4211/83/$03.00
© 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
296
ALAD (EC 4.2.1.24) is the first enzyme detectable in higher plants of the tetrapyrrole biosynthesis pathway. We have previously shown that ALAD activity is enhanced by an irradiation with continuous FR light, involving the phytochrome system, in radish seedling cotyledons in the early hours of differentiation [2]. The enzyme is always detected in the cytoplasm during growing in the dark or the light after treatment with ERT which specifically inhibits protein synthesis on 70 S ribosomes [4]. Under continuous FR light ALAD is present in the cytoplasm until 72 h, but at that time the enzymatic activity appears also in etioplasts. The presentwork reports modifications in molecular properties of partially purified ALAD or from cytoplasm etiopiast enriched fraction during seedling differentiation in various growth conditions. These results are discussed with regard to those previously obtained [2,3]. MATERIAL AND METHODS Radish seeds (Raphanus sativus L. cv. Longue Rave Saumon~e) were germinated either in the dark or under continuous light. Enzymatic measurements, chemicals and light conditions were described elsewhere [2]. ERT 250 ~g • m1-1 was given to seedlings which were 48-h-old as described previously [2]. The different manipulations were carried out under a dim green safelight at 2°C. Etioplasts were isolated from 10 g fresh wt. cotyledons as described previously [3]. Purified etioplasts were disrupted by osmotic shock in Tris- HC1 buffer 10 mM (pH 8.7), containing MgC12 10 mM and the stromal fraction was clarified by centrifngation (Berkman J21 B, Rotor JA 20, 30 000 × g for 15 min). Stromal solution was passed through a Sephadex G-50 column and ALAD was collected at V0. Active fractions were pooled and applied on a DEAE-cellulose column (1 × 20 cm) (Trisacryl, Pharmindustrie, IBF (Industrie Biologique Franqaise), Villeneuve la Garenne, France) equilibrated with TEs- HC1 buffer 10 mM (pH 8.7) containing 10 mM MgC12. The column was rinsed with the same buffer containing 50 mM NaC1. Enzyme was eluted by a linear NaC1 gradient (50--350 raM, 120 ml) in Tris--HC1 buffer (pH 8.7). One-mi|H]iter fractions were collected and assayed for ALAD activity. In experiments involving further DEAE chromatography, active fractions were pooled, diluted with Tris- HC1 buffer 10 mM (pH 8.7), to obtain a final 5 mM NaCI concentration, and applied on a new DEAEcenulose column as described above. As it was impossible to obtain enough enzyme from purified etioplasts in the dark or after ERT treatment, ALAD was prepared from crude cotyledons extract. Fresh weight cotyledons (10 g) were ground in 30 ml Tris--HC1 buffer 50 mM (pH 8.7) containing 10 mM MgC12 and 14 mM 2 mercaptoethanol The brei was centrifuged 30 000 × g for 30 min and the supernatant purified through Sephadex G-50 and DEAE-ceUulose as described above. For control, enzyme from FR-irradiated seedlings was purified either
297
from crude extract or from purified etioplasts. Both procedures led to identical elution profiles after ion exchange chromatography, and thus allowed us to compare ALAD of various origins. Molecular weight estimation was performed by ACa 34 gel sieving {4 × 20 cm) (Ultrogel, Pharmindustrie, IBF, Villeneuve la Garenne, France) in Tris--HCl buffe~ 10mM (pH 8.7) containing 10 mM MgC12. A one-milliliter sample of an active fraction purified after DEAE-cellulose chromatography was placed on the column and eluted at 0.5 m l . min -1. One-milliliter fractions were collected and assayed for ALAD activity. The ACa 34 column •was calibrated with: catalase 240 000, aldoiase 128 000, bovine serum albumin 67 000, ovalbumin 43 000 daltons (SERVA). V0 was estimated with elution of dextran blue and Ve/Vo plotted vs. log molecular weight to establish a calibration curve. RESULTS AND DISCUSSION In a previous work, we had investigated the intmcellular location of ALAD and had observed that it is modified by light or antibiotic treatments. In complete darkness or under FR light and ERT treatments, or in young irradiated seedlings ALAD is located in the cytosol. At 96 and 120 h from sowing under continuous Fit light the enzymatic activity appears in etic~ plasts too [3]. When etioplasts are purified from radish seedling cotyledons grown 96 or 120 h under continuous FR light, two ALAD fractions are eluted after DEAE-cellulose chromatography. These occur at 0.11 M (peak I} and 0.15 M (peak II} NaC1 concentration, respectively (Fig~ l b and 2c}. Only the first eluted peak (peak I) is observed in etioplasts 72-h-old (Fig. la}. In fully etiolated seedlings (grown in absence of FR light), or in seedlings grown under FR light but treated with ERT, it was not possible to purify ALAD from etioplasts. If the enzyme is prepared from a crude cotyledon extract, only one ALAD charge species, eluted at 0.11 M NaC1 concentration is observed (Figs. 2b and 2c}. The same elution profile is detected after chromatography of young seedlings grown 48 h under continuous FR light (Fig. 2a). Appearance of ALAD in peak II is correlated in time with the detection of enzyme activity in etioplasts from FR-irradiated seedlings observed in these previous results. This raises the possibility that peak I (observed Fig. 2) is the cytosolic enzyme. Thus, the newly observed ALAD from peak II (Figs. l b and lc) localised in etioplasts differs in electric charge from its cytoplasmic counterpart. However, peak I is observed either in seedlings when ALAD is cytoplasmic (Fig. 2) or located in etioplasts {Fig. 1). We have tried to distinguish further molecular differences between ALAD charge forms detected through ion exchange chromatography. Some authors have also shown that ALAD in green plants may be separated in two fractions after DEAE-cellulose chromatography, eluted at NaC1 concentrations
298
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close to those reported here [5,6]. However, t h e y consider peak I as an artifact, because of its instability on passing it through a DEAE column for a second or a third time. We have checked such a possibility for each of the radish DEAE ALAD species.
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Fig. 2. Elution profiles of ALAD from cotyledon extracts on DEAE-cellulose chromatography. (a) 48 h from sowing FR-irradiated seedlings; (b) 120 h from sowing, completely etiohted seedlings; (c) 120 h from sowing, FR-irradiated seedlings treated with ERT 250 ~g oml -~ at 48 h. • . , ALAD activity; . . . . . . , NaC1 gradient. C y t o s o l i c A L A D p u r i f i e d f r o m d a r k g r o w n seedlings, f r o m y o u n g irradiated ones (48 h f r o m sowing), f r o m seedlings t r e a t e d w i t h E R T , never dissociates
300 on f u r t h e r ion c h r o m a t o g r a p h y (result n o t shown). As r e p o r t e d b y o t h e r authors, peak I purified f r o m etioplasts grown 120 h u n d e r F R light dissociates in two peaks (Fig. 3c), b o t h o f which c o n t i n u e t o yield t w o peaks o n f u r t h e r passage on D E A E (Figs. 3d and 3e). The same results are o b t a i n e d if p e a k I observed at 72 h in etioplasts (Fig. l a ) is checked. When p e a k II d e t e c t e d after the p r i m a r y c h r o m a t o g r a p h y (Fig. 3a), is subjected to the same D E A E steps, it never dissociates (Figs. 3b a n d 3f), e x c e p t a small
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Fie 3. Elution profiles of etioplast ALAD prepared from 10 g fresh wt. radish cotyledons 120 h grown under continuous FR light during successive chromatographies on DEAE cellulose. (a) Elution profile of stromal fraction; (b) chromatography of peak II; (c) chromatography of peak I; (d), (e) chromatography of peaks pooled separately from (c); (f) chromatography from peak II from (b). * • enzymatic activity. NaCI normalities for elution are given.
301
amount of ALAD which is rapidly eluted (Fig. 3b) but we consider that it is a contamination from peak I. ALAD is a multimeric enzyme, easily undergoing subunit reassociation [7] on changing buffer [8] or ionic strength [9]. Thus after the first chromatography, the subsequent dilutions, necessary to obtain binding on another gel, generates structural modifications of etioplast enzyme from peak I. This is indeed a true artifact, but neither cytoplasmic, nor etioplast ALAD from peak II undergo such transformations. Thus, we can detect after DEAE-cellulose chromatography two kinds of ALAD molecules by reference to their charge: stable enzymes are observed, in cytosolic fraction eluted at 0.11 M NaC1 concentration and in etioplasts eluted at 0.15 M NaC1 concentration. ALAD with an unstable charge is detected in etioplasts too, but is eluted at 0.11 M NaC1 concentration. Molecular weights of the different enzyme species have been estimated by gel sieving, after purification from DEAE-cellulose chromatography as described above. Table I shows that ALAD from peak II has always a mol. wt. of 245 000. The enzyme found in the cytoplasm, or ALAD in etioplasts eluted in peak I shows different aggregation states. Little work has been done on molecular properties of ALAD from plants, and results available are mainly obtained from animals. In this material, enzyme is multimeric and the monomer (40 000 daltons) is inactive [9]. We observe after gel sieving, four molecular species ranging around 340 000, 240 000, 170 000 and 120 000 daltons, which is consistent with associations of 8, 6, 4 and 3 subunits, respectively.
TABLE I ESTIMATION OF MOLECULAR WEIGHT OF ALAD BY GEL SIEVING CHROMATOGRAPHY ON AcA 34 GEL ALAD fractions are prepared from the different peaks observed after DEAE-cellulose chromatography. Column was calibrated by reference to elution of: catalase, aldolase, bovine serum albumin, ovalbumin and dextran blue. ALAD purified from seedling cotyledons grown:
120 h continuous F R light DEAE, peak I (0.11 M NaCl) 120 h continuous F R light DEAE, peak II (0.15 M NaCl) Complete darkness DEAE, peak I (0.11 M NaCl) 120 h continuous F R light E R T treatment at 48 h from sowing DEAE, peak I (0.11 M NaCl)
Estimated mol. wt.
338 000
228 000
169 000
110 000
245 000 319 000
240 000
148 000
120 000
319 000
260 000
178 000
120 000
302 Thus, with respect to growth conditions, it is possible to isolate 3 diffe~ rent ALAD species: (1) an enzyme with constant charge, but undergoing different aggregation states appears in complete darkness, in young irradiated seedlings, or after ERT treatment. This molecule is probably cytoplasmic. Two other enzyme forms are obtained from etioplasts: (2) one form always has the same charge and the same mol. wt. (245 000). It is found in etioplasts of light grown seedlings at 96 and 120 h from sowing and is eluted at 0.15 M NaC1 concentration on DEAE-cellulose chromatography. (3) The final form appears earlier in etioplasts (72 h from sowing), is eluted at 0.11 M NaC1 concentration, but is found with variable molecular weight and charge. At that time, two hypotheses can explain the relationships between ALAD localisation and its molecular properties. It can be assumed that ALAD is firstly synthesized on cytoplasmic ribosomes as identical subunits, with constant charge but undergoing easily different aggregation states, at least in vitro, which is also reported for cytoplasmic precursors of some mitochondrial proteins [101. The cytoplasmically localised enzyme is observed in complete darkness, and in young irradiated seedlings. If seedlings are grown under continuous FR light, at least during 72 h ALAD is exported into etioplasts with structural change, giving it a complete unstable charge. This modification may'be a processing effect of unknown protease, as for a small subunit of ribulose bisphosphate carboxylase (RuBPc) [11], or another less known mechanism. At last in mature etioplasts a stabilization mechanism takes place which gives rise to ALAD molecules having stable charge and tool. wt. of 245 000. This hypothesis involves a two-step mechanism into etioplasts, which is reported neither for small subunit of RuBPc [11] nor for other chloroplastic molecules. However recent findings from Daum et al. show also a two-step transformation of cytochrome b2 in intermembrane mitochondrial space [ 12 ]. Another hypothesis may be a complete lack of correlation between ALAD in cytoplasm and enzyme in etioplast. If we consider this explanation, ALAD is synthesized both on cytoplasmic ribosomes and on etioplastic ones. In organeUes, enzyme is built as an unstable molecule from 72 h from sowing, and then stabilized from 96 to 120 h from sowing under continuous FR light by an unknown mechanism. In both hypotheses, if etioplasts are not allowed to grow normally under FR light (after ERT treatment) or if they stay in a preliminary state (young seedlings) no ALAD appears in etioplasts, either after translocaticn or after synthesis, and only the cytoplasmic enzyme is observed. It would be operating in heine pathways other than in chlorophyll biosynthesis, and is usually shaded by the presence of its etioplast counterpart. At that time results obtained previously cannot completely discard any of the proposed hypotheses. Further work based on enzyme purification and antibodies preparations will give more precise information on ALAD regulation through phytochrome system.
303
Note added in Proof D u r i n g t h e w r i t i n g o f t h e revised version, M.V. B l o o m , P. Milos and H. R o y , Proc. Natl. Acad. Scl. U.S.A., 80 ( 1 9 8 3 ) 1 0 1 3 - 1 0 1 7 , s h o w several i n t e r m e d i a t e s in t h e large R u B P c s u b u n i t a s s e m b l y , w h i c h are light d e p e n d e n t . This finding m a y s u p p o r t in p a r t o u r s e c o n d a s s u m p t i o n . REFERENCES 1 2 3 4 5 6 7 8 9 10
11 12
P. Schopfer, Annu. Rev. Plant Physiol., 28 (1973) 233. A.P. Balang~ and P. RoUin, Physiol. Veg., 17 (1) (1979) 153. A.P. Balang~ and C. Lambert, Phytochem., 19 (1980) 2541. R. Langlois, C.R. Cantor, R. Vince and S. Pestka, Biochemistry, 16 (1977) 2349. Hj.A.W. Schneider, Z. PflanzenphysioL, 62 (1970) 328. H. Tamai, Y. Shioi and T. Sasa, 20 (2) (1979) 435. D. Shemin, in: P.D. Boyer (Ed.), The Enzymes, VoL 7 Academic Press, New York, 1972, pp. 323--337. S. Van Heyningen and D. Shemin, Biochemistry, 10 (25) (197~) 4676. N. Despaux, E. Comoy, C. Bohuon and C. Boudene, Biochimie, 61 (1979) 1021. W. Neupert and G. Schatz, Trends Biochem. Sci., 1--4 (1981). ~ R.J. Ellis, Annu. Rev. Plant Physiol., 32 (1981) 111. G. Daum, S.M. Gasser and G. Schatz, J. Biol. Chem., 257 (21) (1982) 13075.