Planta
Planta (1982) 155:17 23
9 Springer-Verlag 1982
Evidence for a cytosolic-dependent light induction of chloroplastic glutamine synthetase during greening of etiolated rice leaves Bertrand Hirel*, Jean Vidal, and Pierre G a d a l Laboratoire de Physiologic Veg&ale M6tabolique, ERA CNRS n~ 799, Bfitiment 430, Universit~ de Paris Sud, Centre d'Orsay, F-91405 Orsay-Cedex, France
Abstract. During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase ofchloroplastic glutamine synthetase (GSz) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS2. The synthesis of GS2 was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS2 protein content in green leaves is fivefold higher than in etiolated leaves. Key words: Chloroplast - Cytosol Glutamine synt h e t a s e - Greening - Light induction (glutamine synthetase) - Oryza.
1980) and pumpkin (Kretovich etal. 1981), two isoforms (GS1 and GS2) have been identified and characterized by their specific catalytic and physiochemical properties. GS1 is responsible for a m m o n i a assimilation in the cytosol (Mann et al. 1979; Hirel and Gadal 1980b), while GS2 is involved in the chloroplastic glutamine synthesis (Mann et al. 1979; Hirel and Gadal 1980b). In a previous work we reported that in etiolated rice leaves exposed to the light the activity of GS2 increased progressively, and after 48 h light exposure the pattern of green leaves was obtained (Guiz et al. 1979). Similar results were also observed in pea (Hirel and Gadal 1981 a), Sorghum (Hirel and Gadal 1982), and barley leaves (Mann et al. 1979). This general feature observed in various kinds of plants could result either from an activation of GS2 previously synthesized and stored in an inactive form or from a light-dependent " d e n o v o " synthesis of the enzyme during greening. In order to check both hypotheses, we investigated the influence of light on the increase of glutamine synthetase during greening, in etiolated rice leaves, by using inhibitors of protein synthesis, density-labeling experiments, and specific immunochemical assays in order to quantify the GS2 content in green and etiolated leaves.
Introduction Glutamine synthetase (L. Glutamate: a m m o n i a ligase, ADP, E.C. 6.3.1.2.) is present in two major molecular forms in the leaves of several vascular plants. In rice (Hirel and Gadal 1980a), barley (Mann et al.
Materials and methods
* To whom correspondence should be sent
Greening experiments. Greening experiments were conducted with rice in a controlled environment chamber at 28~C. The light fluence rate was approximately 0.2 W/cm2 (Fluorescent lights Sylvania grolux).
Abbreviations." AbH= heavy chain of antibodies; AbL- light chain of antibodies ; AP = acid phosphatase ; CH = cycloheximide; G6PDH= glucose-6-phosphate dehydrogenase; GS= glutamine synthetase; GS~ = cytosolic gtutamine synthetase; GS2= chloroplastic glutamine synthetase; LC= lincomycin; NAD-MDH= NAD malate dehydrogenase; NADP-G3PDH= NADP glyceraldehyde-3-phosphate dehydrogenase
Plant material. Etiolated rice (Oryza sativa L. cv. Delta G 2) leaves were obtained as described previously (Hirel and Gadal 1980).
])20 labeling experiments. Shoots of 8-d old etiolated rice plants were placed in small beakers containing an aqueous solution of 80% (v/v) D20 and exposed for 48 h to continuous light. In the controls, etiolated shoots were placed under illumination condi-
0032-0935/82/0155/00 t 7/$01.40
18 tions in H20 or in the dark in D20. One gram fresh shoots were used, both for the test plants and the controls. Soluble proteins were extracted as described by Guiz et al. (1979), precipitated with solid (NH,)zSO4, and desalted through a Sephadex G25 column. 1.7 ml of the extract +2 gl (0.5 units) of commercial glucose-6phosphate dehydrogenase (Sigma grade I) were added to 0.5 ml saturated CsC1 aqueous solution (20 ~ C). The mixture was then layered in a 13-ml centrifuge tube on a CsC1 solution made up of a mixture of 1.7 ml distilled water and 1.2 ml saturated CsC1 aqueous solution (20 ~ C). The tubes in the three experiments were centrifuged at 200,000 g, using a angular rotor 50 Ti in a L5 50 Beckman ultracentrifuge for 36 h at 2~ C. Fractions of 80 gl were then collected, starting from the bottom of the tubes by means of a glass capillary tube connected to a peristaltic pump. The refractive index of every six fractions was determined using an Abbe refractometer. The values were converted to density units by use of the formula of Ifft et al. (1961).
Protein synthesis inhibitor experiments. Cycloheximide (CH) at 20 gg/mI and lincomycin (LC) at 100 gg/ml were used as inhibitors of protein synthesis (Ellis 1969; Reger et al. 1972; Malhotra et al. 1973). Five 50 ml pulverizations were performed on etiolated rice plants at regular intervals for 12 h before switching on the light. Separation, identification, and quantification of GS 1 and GS2 were achieved by DEAE Sephacel chromatography as described in a previous paper (Hirel and Gadal 1980a). Preparation of antibodiesagainst chloroplastieglatamine synthetase. Antibodies against chloroplastic GS were obtained as described previously (Hirel et al. 198I c).
Immunoprecipitation and protein measurement in SDS acrylamide gels. After diethylaminoethyl (DEAE) Sephacel chromatography of etiolated or 48 h green leaf extracts, fractions containing GS2 activity were pooled and solid (NH4)2SO~ was added to give a finai concentration of 80%. After 30 rain of mixing, proteins were collected by centrifugation at 17,000 g for 5 min and dissolved in a total volume of 0.3 ml with trishydroxymethylaminomethane (Tris) HC1 buffer 10 mM, pH 7.6, containing MgC12 1 mM. Immunotitration reaction of glutamine synthetases from rice leaves was carried out by incubating 0.83 nkat concentrated enzyme preparation, with various amounts (0 to 0.2 ml) of rabbit antiserum raised against the chloroplastic GS from spinach leaves. The samples were incubated at 37~ C for 1 h and then overnight at 4~ C. They were centrifuged at 40,000 g for 15 min. The amount of enzyme activity remaining in the supernatant following removal of the immune complexes was then determined. Estimation of the amounts of glutamine synthetase in both etiolated and green rice leaves exposed to light for 48 h was performed by protein measurement in sodium dodecylsulphate (SDS) acrylamide gels (Gadal et al. 1980). After determination of the equivalence point by the immunoprecipitation method, 8.3 nkat concentrated rice GS2 were immunoprecipitated with 0.4 ml antiserum. The immunoprecipitates were gently rinsed three times with I ml borate NaC1 buffer, pH 8.14, and then resuspended in 0.2 ml sodium phosphate buffer 10 raM, pH 7, containing SDS (0.4%), and solubilized by heating them for 10 rain in a boiling water bath. Amounts of proteins were determined at this stage using Scope's method (Scopes 1973). Then, 20 gl /3 mercaptoethanol, 20 ~tl pure glycerol, and 10 gl bromophenol blue (0.01% in water) were added to the solubilized proteins and heated again for 5 rain. After cooling the samples were submitted to electrophoresis in polyacrylamide gels containing SDS (0.1%), in quartz tubes, according to Weber and Osborn (1969). At the end of the run the tubes were recorded at 280 nm using a Beckman Acta CII spectrophotometer equipped with an automatic scanning device. Later the gels were stained with a Coomassie Blue solution, as described
B. Hirel et al. : Light induction of chloroplastic glutamine synthetase previously (Hire1 and Gadal 1980). To estimate the proportion of each protein in the diagrams, the peak surfaces were measured from the total amounts of proteins layered on the gels.
Enzymatic assays, chlorophyl and protein measurements. NADP glyceraldehyde-3-phosphate dehydrogenase (NADP-G3PDH) was used as the marker enzyme of greening, according to Ireland and Bradbeer (1971), and assayed using the method of Heber et al. (1963). NAD malate dehydrogenase (NAD-MDH) was used as a control during greening experiments with rice (Liu et al. 1976). Glucose-6-phosphate dehydrogenase (G6PDH) was used as an internal marker is CsC1 density gradient. It was assayed according to Muto and Uritani (1970). Acid phosphatase (AP) was used as a density marker in CsC1 gradient, according to Johnson et al. (1973). The enzyme assay was performed as indicated in the Worthington enzyme manual (1972). Glutamine synthetase (GS) was assayed as previously described (Guiz et al. 1979). Chlorophyll content was measured by the method of Bruinsma (1961), and proteins were measured by the method of Scopes (1973).
Results
Influence o f light on glutamine synthetase activity during greening of etiolated rice leaves. O n a f r e s h - w e i g h t basis, a c o n t i n u o u s i l l u m i n a t i o n o f e t i o l a t e d rice leaves f o r 48 h p r o d u c e d p r o g r e s s i v e l y a t w o - f o l d inc r e a s e o f G S a c t i v i t y in w h o l e l e a f c r u d e e x t r a c t s ( T a b l e 1). A f t e r t h e g r e e n i n g p r o c e s s , the e n z y m e activity r e m a i n e d c o n s t a n t a n d was i d e n t i c a l to t h e G S a c t i v i t y d e t e c t e d in g r e e n rice leaves. T h i s e n h a n c e m e n t in e n z y m e activity, a l t h o u g h r e l a t i v e l y slow, sugg e s t e d p r o t e i n synthesis, b u t a n o t h e r m e c h a n i s m p r o m o t e d by light, e.g., a n a c t i v a t i o n o f t h e e n z y m e , c o u l d a l s o be c o n s i d e r e d . I n o r d e r to c o n t r o l t h e t w o h y p o t h e s e s , w e h a v e s u b m i t t e d e t i o l a t e d rice s h o o t s to a density-labeling treatment with DzO over a lighte x p o s u r e p e r i o d o f 48 h.
In vivo density labeling experiments of whole leaf glutamine synthetase. T w o b a t c h e s o f e t i o l a t e d rice s h o o t s w e r e t r a n s f e r r e d , o n e to a m e d i u m c o n t a i n i n g 8 0 % D 2 0 a n d the o t h e r to H 2 0 . A f t e r 48 h l i g h t e x p o s u r e , the two crude leaf extracts were centrifuged on separ a t e CsC1 g r a d i e n t s . A c o n t r o l f o r t h e t u r n o v e r o f t o t a l G S was c o n d u c t e d as d e s c r i b e d f o r 0 2 0 l a b e l i n g during greening, but etiolated shoots were kept on a m e d i u m c o n t a i n i n g t h e h e a v y i s o t o p e in t h e d a r k f o r 48 h. Table 2 shows the changes of buoyant density of GS, glucose-6-phosphate dehydrogenase (G6PDH) (control density marker), and acid phosphatase (AP) ( g r e e n i n g m a r k e r ) in the t h r e e e x p e r i m e n t s . It c a n be seen t h a t G 6 P D H e x h i b i t e d in e v e r y c a s e a c o n s t a n t d e n s i t y o f 1.284 k g 1 - t , s h o w i n g the r e p r o d u c ibility o f e a c h e x p e r i m e n t . T h e e f f i c i e n c y o f D 2 0 labeling was checked by changes of AP buoyant density
B. Hirel et al. : Light induction of chloroplastic glutamine synthetase
19
during greening, which shifted from 1.330 kg 1-t to 1.340 kg 1-1. This data was consistant with the results obtained in pea cotyledons by Johnson et al. (1973). An increase of 0.007 kg 1-1 of total GS density in the leaf during 48 h light exposure clearly demon-
strated a neosynthesis of the enzyme. The buoyant density for GS in the 48-h grown dark control (1,309 kg 1-1) showed that the enzyme turnover was not responsible for deuterium incorporation in the dark.
Table 1. Influence of light on glutamine synthetase activity during greening of etiolated rice leaves. The enzyme was extracted from 10 g fresh leaves after 8 d of dark growing and at regular intervals during greening from 0 to 72 h continuous light exposure. GS activity is expressed in nkat per g of fresh weight
Influence of light on GS isoform activity during greening: effect of inhibitors of protein synthesis on their relative proportions. In a previous paper we demon-
Etiolated Greening leaves Continuous light exposure (h) GS activity (nkat g-1 FW)
0
6
12
24
48
72
18.0
22.1
25.7
31.2
40.0
40.0
Table 2. The effect of greening on the incorporation of density label (D20) into rice leaves glutamine synthetase. Changes of buoyant density of glucose-6-phosphate dehydrogenase (G6PDH) (internal marker), acid phosphatase (AP) (greening marker) and glutamine synthetase (GS) after isopicnic centrifugation of proteins in CsC1 density gradient. The values were determined from three different experiments Assay enzyme
Dark ZH20 buoyant density (kg 1-1)
Light H20 buoyant density (kg 1-~)
Light 2H20 buoyant density (kg 1-1)
Increase Light 2H2OLight H20 (kg 1-j)
G6PDH AP GS
1.284 1.330 1.309
1.284 1.330 1.308
1.284 1.340 1.315
0.000 0.010 0.007
15
15
10
GS2.
B GS 2
l0
>pro >
strated that light mediated a light-dependent change in the relative proportions of two isoforms of GS in rice leaves (Guiz et al., 1979). In this work we determined the relative activity of cytosolic GS (GS1) and chloroplastic GS (GS2) in etiolated rice leaves and in the same material submitted to 48 h of continuous light. The results are presented in Fig. 1 and quantified in Table 3. It can be seen that the two-fold increase of total GS activity observed in Table 1 was in fact due, as shown in Fig. 1, to a five-fold increase of GSz, the chtoroplastic GS isoform, and simultaneously to a decrease (40%) of GS1, the cytosolic GS isoform. This was a confirmation of our results, showing an increase of total GS activity in the leaf during greening. The density-labeling experiment showed that upon greening part of the total GS activity was synthesized. To confirm this data we investigated, taking into account the presence of two isoforms of the enzyme, the influence of inhibitors of protein synthesis on the light-dependent balance between GS1 and
5
10,4
LC
,5 il
11,2
Planta (1982) 155:17 23
9 Springer-Verlag 1982
Evidence for a cytosolic-dependent light induction of chloroplastic glutamine synthetase during greening of etiolated rice leaves Bertrand Hirel*, Jean Vidal, and Pierre G a d a l Laboratoire de Physiologic Veg&ale M6tabolique, ERA CNRS n~ 799, Bfitiment 430, Universit~ de Paris Sud, Centre d'Orsay, F-91405 Orsay-Cedex, France
Abstract. During the greening of etiolated rice leaves, total glutamine synthetase activity increases about twofold, and after 48 h the level of activity usually observed in green leaves is obtained. A density-labeling experiment with deuterium demonstrates that the increase in enzyme activity is due to a synthesis of the enzyme. The enhanced activity obtained upon greening is the result of two different phenomena: there is a fivefold increase of chloroplastic glutamine synthetase content accompanied by a concommitant decrease (twofold) of the cytosolic glutamine synthetase. The increase ofchloroplastic glutamine synthetase (GSz) is only inhibited by cycloheximide and not by lincomycin. This result indicates a cytosolic synthesis of GS2. The synthesis of GS2 was confirmed by a quantification of the protein by an immunochemical method. It was demonstrated that GS2 protein content in green leaves is fivefold higher than in etiolated leaves. Key words: Chloroplast - Cytosol Glutamine synt h e t a s e - Greening - Light induction (glutamine synthetase) - Oryza.
1980) and pumpkin (Kretovich etal. 1981), two isoforms (GS1 and GS2) have been identified and characterized by their specific catalytic and physiochemical properties. GS1 is responsible for a m m o n i a assimilation in the cytosol (Mann et al. 1979; Hirel and Gadal 1980b), while GS2 is involved in the chloroplastic glutamine synthesis (Mann et al. 1979; Hirel and Gadal 1980b). In a previous work we reported that in etiolated rice leaves exposed to the light the activity of GS2 increased progressively, and after 48 h light exposure the pattern of green leaves was obtained (Guiz et al. 1979). Similar results were also observed in pea (Hirel and Gadal 1981 a), Sorghum (Hirel and Gadal 1982), and barley leaves (Mann et al. 1979). This general feature observed in various kinds of plants could result either from an activation of GS2 previously synthesized and stored in an inactive form or from a light-dependent " d e n o v o " synthesis of the enzyme during greening. In order to check both hypotheses, we investigated the influence of light on the increase of glutamine synthetase during greening, in etiolated rice leaves, by using inhibitors of protein synthesis, density-labeling experiments, and specific immunochemical assays in order to quantify the GS2 content in green and etiolated leaves.
Introduction Glutamine synthetase (L. Glutamate: a m m o n i a ligase, ADP, E.C. 6.3.1.2.) is present in two major molecular forms in the leaves of several vascular plants. In rice (Hirel and Gadal 1980a), barley (Mann et al.
Materials and methods
* To whom correspondence should be sent
Greening experiments. Greening experiments were conducted with rice in a controlled environment chamber at 28~C. The light fluence rate was approximately 0.2 W/cm2 (Fluorescent lights Sylvania grolux).
Abbreviations." AbH= heavy chain of antibodies; AbL- light chain of antibodies ; AP = acid phosphatase ; CH = cycloheximide; G6PDH= glucose-6-phosphate dehydrogenase; GS= glutamine synthetase; GS~ = cytosolic gtutamine synthetase; GS2= chloroplastic glutamine synthetase; LC= lincomycin; NAD-MDH= NAD malate dehydrogenase; NADP-G3PDH= NADP glyceraldehyde-3-phosphate dehydrogenase
Plant material. Etiolated rice (Oryza sativa L. cv. Delta G 2) leaves were obtained as described previously (Hirel and Gadal 1980).
])20 labeling experiments. Shoots of 8-d old etiolated rice plants were placed in small beakers containing an aqueous solution of 80% (v/v) D20 and exposed for 48 h to continuous light. In the controls, etiolated shoots were placed under illumination condi-
0032-0935/82/0155/00 t 7/$01.40
18 tions in H20 or in the dark in D20. One gram fresh shoots were used, both for the test plants and the controls. Soluble proteins were extracted as described by Guiz et al. (1979), precipitated with solid (NH,)zSO4, and desalted through a Sephadex G25 column. 1.7 ml of the extract +2 gl (0.5 units) of commercial glucose-6phosphate dehydrogenase (Sigma grade I) were added to 0.5 ml saturated CsC1 aqueous solution (20 ~ C). The mixture was then layered in a 13-ml centrifuge tube on a CsC1 solution made up of a mixture of 1.7 ml distilled water and 1.2 ml saturated CsC1 aqueous solution (20 ~ C). The tubes in the three experiments were centrifuged at 200,000 g, using a angular rotor 50 Ti in a L5 50 Beckman ultracentrifuge for 36 h at 2~ C. Fractions of 80 gl were then collected, starting from the bottom of the tubes by means of a glass capillary tube connected to a peristaltic pump. The refractive index of every six fractions was determined using an Abbe refractometer. The values were converted to density units by use of the formula of Ifft et al. (1961).
Protein synthesis inhibitor experiments. Cycloheximide (CH) at 20 gg/mI and lincomycin (LC) at 100 gg/ml were used as inhibitors of protein synthesis (Ellis 1969; Reger et al. 1972; Malhotra et al. 1973). Five 50 ml pulverizations were performed on etiolated rice plants at regular intervals for 12 h before switching on the light. Separation, identification, and quantification of GS 1 and GS2 were achieved by DEAE Sephacel chromatography as described in a previous paper (Hirel and Gadal 1980a). Preparation of antibodiesagainst chloroplastieglatamine synthetase. Antibodies against chloroplastic GS were obtained as described previously (Hirel et al. 198I c).
Immunoprecipitation and protein measurement in SDS acrylamide gels. After diethylaminoethyl (DEAE) Sephacel chromatography of etiolated or 48 h green leaf extracts, fractions containing GS2 activity were pooled and solid (NH4)2SO~ was added to give a finai concentration of 80%. After 30 rain of mixing, proteins were collected by centrifugation at 17,000 g for 5 min and dissolved in a total volume of 0.3 ml with trishydroxymethylaminomethane (Tris) HC1 buffer 10 mM, pH 7.6, containing MgC12 1 mM. Immunotitration reaction of glutamine synthetases from rice leaves was carried out by incubating 0.83 nkat concentrated enzyme preparation, with various amounts (0 to 0.2 ml) of rabbit antiserum raised against the chloroplastic GS from spinach leaves. The samples were incubated at 37~ C for 1 h and then overnight at 4~ C. They were centrifuged at 40,000 g for 15 min. The amount of enzyme activity remaining in the supernatant following removal of the immune complexes was then determined. Estimation of the amounts of glutamine synthetase in both etiolated and green rice leaves exposed to light for 48 h was performed by protein measurement in sodium dodecylsulphate (SDS) acrylamide gels (Gadal et al. 1980). After determination of the equivalence point by the immunoprecipitation method, 8.3 nkat concentrated rice GS2 were immunoprecipitated with 0.4 ml antiserum. The immunoprecipitates were gently rinsed three times with I ml borate NaC1 buffer, pH 8.14, and then resuspended in 0.2 ml sodium phosphate buffer 10 raM, pH 7, containing SDS (0.4%), and solubilized by heating them for 10 rain in a boiling water bath. Amounts of proteins were determined at this stage using Scope's method (Scopes 1973). Then, 20 gl /3 mercaptoethanol, 20 ~tl pure glycerol, and 10 gl bromophenol blue (0.01% in water) were added to the solubilized proteins and heated again for 5 rain. After cooling the samples were submitted to electrophoresis in polyacrylamide gels containing SDS (0.1%), in quartz tubes, according to Weber and Osborn (1969). At the end of the run the tubes were recorded at 280 nm using a Beckman Acta CII spectrophotometer equipped with an automatic scanning device. Later the gels were stained with a Coomassie Blue solution, as described
B. Hirel et al. : Light induction of chloroplastic glutamine synthetase previously (Hire1 and Gadal 1980). To estimate the proportion of each protein in the diagrams, the peak surfaces were measured from the total amounts of proteins layered on the gels.
Enzymatic assays, chlorophyl and protein measurements. NADP glyceraldehyde-3-phosphate dehydrogenase (NADP-G3PDH) was used as the marker enzyme of greening, according to Ireland and Bradbeer (1971), and assayed using the method of Heber et al. (1963). NAD malate dehydrogenase (NAD-MDH) was used as a control during greening experiments with rice (Liu et al. 1976). Glucose-6-phosphate dehydrogenase (G6PDH) was used as an internal marker is CsC1 density gradient. It was assayed according to Muto and Uritani (1970). Acid phosphatase (AP) was used as a density marker in CsC1 gradient, according to Johnson et al. (1973). The enzyme assay was performed as indicated in the Worthington enzyme manual (1972). Glutamine synthetase (GS) was assayed as previously described (Guiz et al. 1979). Chlorophyll content was measured by the method of Bruinsma (1961), and proteins were measured by the method of Scopes (1973).
Results
Influence o f light on glutamine synthetase activity during greening of etiolated rice leaves. O n a f r e s h - w e i g h t basis, a c o n t i n u o u s i l l u m i n a t i o n o f e t i o l a t e d rice leaves f o r 48 h p r o d u c e d p r o g r e s s i v e l y a t w o - f o l d inc r e a s e o f G S a c t i v i t y in w h o l e l e a f c r u d e e x t r a c t s ( T a b l e 1). A f t e r t h e g r e e n i n g p r o c e s s , the e n z y m e activity r e m a i n e d c o n s t a n t a n d was i d e n t i c a l to t h e G S a c t i v i t y d e t e c t e d in g r e e n rice leaves. T h i s e n h a n c e m e n t in e n z y m e activity, a l t h o u g h r e l a t i v e l y slow, sugg e s t e d p r o t e i n synthesis, b u t a n o t h e r m e c h a n i s m p r o m o t e d by light, e.g., a n a c t i v a t i o n o f t h e e n z y m e , c o u l d a l s o be c o n s i d e r e d . I n o r d e r to c o n t r o l t h e t w o h y p o t h e s e s , w e h a v e s u b m i t t e d e t i o l a t e d rice s h o o t s to a density-labeling treatment with DzO over a lighte x p o s u r e p e r i o d o f 48 h.
In vivo density labeling experiments of whole leaf glutamine synthetase. T w o b a t c h e s o f e t i o l a t e d rice s h o o t s w e r e t r a n s f e r r e d , o n e to a m e d i u m c o n t a i n i n g 8 0 % D 2 0 a n d the o t h e r to H 2 0 . A f t e r 48 h l i g h t e x p o s u r e , the two crude leaf extracts were centrifuged on separ a t e CsC1 g r a d i e n t s . A c o n t r o l f o r t h e t u r n o v e r o f t o t a l G S was c o n d u c t e d as d e s c r i b e d f o r 0 2 0 l a b e l i n g during greening, but etiolated shoots were kept on a m e d i u m c o n t a i n i n g t h e h e a v y i s o t o p e in t h e d a r k f o r 48 h. Table 2 shows the changes of buoyant density of GS, glucose-6-phosphate dehydrogenase (G6PDH) (control density marker), and acid phosphatase (AP) ( g r e e n i n g m a r k e r ) in the t h r e e e x p e r i m e n t s . It c a n be seen t h a t G 6 P D H e x h i b i t e d in e v e r y c a s e a c o n s t a n t d e n s i t y o f 1.284 k g 1 - t , s h o w i n g the r e p r o d u c ibility o f e a c h e x p e r i m e n t . T h e e f f i c i e n c y o f D 2 0 labeling was checked by changes of AP buoyant density
B. Hirel et al. : Light induction of chloroplastic glutamine synthetase
19
during greening, which shifted from 1.330 kg 1-t to 1.340 kg 1-1. This data was consistant with the results obtained in pea cotyledons by Johnson et al. (1973). An increase of 0.007 kg 1-1 of total GS density in the leaf during 48 h light exposure clearly demon-
strated a neosynthesis of the enzyme. The buoyant density for GS in the 48-h grown dark control (1,309 kg 1-1) showed that the enzyme turnover was not responsible for deuterium incorporation in the dark.
Table 1. Influence of light on glutamine synthetase activity during greening of etiolated rice leaves. The enzyme was extracted from 10 g fresh leaves after 8 d of dark growing and at regular intervals during greening from 0 to 72 h continuous light exposure. GS activity is expressed in nkat per g of fresh weight
Influence of light on GS isoform activity during greening: effect of inhibitors of protein synthesis on their relative proportions. In a previous paper we demon-
Etiolated Greening leaves Continuous light exposure (h) GS activity (nkat g-1 FW)
0
6
12
24
48
72
18.0
22.1
25.7
31.2
40.0
40.0
Table 2. The effect of greening on the incorporation of density label (D20) into rice leaves glutamine synthetase. Changes of buoyant density of glucose-6-phosphate dehydrogenase (G6PDH) (internal marker), acid phosphatase (AP) (greening marker) and glutamine synthetase (GS) after isopicnic centrifugation of proteins in CsC1 density gradient. The values were determined from three different experiments Assay enzyme
Dark ZH20 buoyant density (kg 1-1)
Light H20 buoyant density (kg 1-~)
Light 2H20 buoyant density (kg 1-1)
Increase Light 2H2OLight H20 (kg 1-j)
G6PDH AP GS
1.284 1.330 1.309
1.284 1.330 1.308
1.284 1.340 1.315
0.000 0.010 0.007
15
15
10
GS2.
B GS 2
l0
>pro >
strated that light mediated a light-dependent change in the relative proportions of two isoforms of GS in rice leaves (Guiz et al., 1979). In this work we determined the relative activity of cytosolic GS (GS1) and chloroplastic GS (GS2) in etiolated rice leaves and in the same material submitted to 48 h of continuous light. The results are presented in Fig. 1 and quantified in Table 3. It can be seen that the two-fold increase of total GS activity observed in Table 1 was in fact due, as shown in Fig. 1, to a five-fold increase of GSz, the chtoroplastic GS isoform, and simultaneously to a decrease (40%) of GS1, the cytosolic GS isoform. This was a confirmation of our results, showing an increase of total GS activity in the leaf during greening. The density-labeling experiment showed that upon greening part of the total GS activity was synthesized. To confirm this data we investigated, taking into account the presence of two isoforms of the enzyme, the influence of inhibitors of protein synthesis on the light-dependent balance between GS1 and
5
10,4
LC
,5 il
11,2
