Planta (1992)188:468-477
pl~Jlflt
~
~) Springer-Verlag1992
Photocontrol of thylakoid protein synthesis in Euglena: differential post-transcriptional regulation depending on nutritional conditions Catherine Weiss*, Guy Houln~, and Rodolphe Schantz** Institut de Biologie Mol6culaire des Plantes du CNRS, 12, rue du G6n6ral Zimmer, F 67084 Strasbourg Cedex, France Received 24 February; accepted 5 June 1992
Abstract. The expression of three chloroplastic genes, psbA (the gene for the reaction center D1 protein of PSII), psbC (the gene for the PT00 apoprotein of PSI) and psaB (the gene for the intermediate antema of PSII), and a nuclear gene, cab (the gene for the chlorophyll a/bbinding protein), has been investigated during chloroplast development in Euglena gracilis Klebs. The polysomal fraction, m R N A stability and protein turnover were analysed under different conditions of cell greening. The results indicate that the main regulatory step for the nuclear and chloroplastic genes was at the translational level when greening o f cells took place on a resting medium. When cell greening took place on a medium with balanced phosphate, carbon and nitrogen sources (nutritional medium), the main regulation occurred posttranscriptionally by mobilization of the transcripts onto the polysomes. These results indicate that in E. gracilis, for a given gene, regulation operates at different levels, and that although light is the principal effector in the regulation of the genes involved in chloroplast development, the effect of metabolites should also be considered. Interestingly, it appears that these various regulation levels are similar for the chloroplastic and nucleocytoplasmic compartments. Key words: Chloroplast development - Euglena (thylakoid proteins) - Gene expression (light and nutritional regulation) - m R N A stability - Polysome - Thylakoid (protein synthesis)
Abbreviations: CPI = chlorophyll-protein complex I of PSI; LHCI (II)=light-harvesting chlorophyll a/b-binding protein of PSI (II) encoded by the nuclear cab genes; psaB =gene for the PTooapoprotein of PSI; psbA = gene for the reaction center D 1 protein of PSII ; psbC=gene for the intermediate antenna of PSII * Present address: Cold Spring Harbor Laboratory, P.O. box 100, Cold Spring Harbor, NY 11725, USA ** To whom correspondence should be addressed; FAX: (33) 8861 44 42
Introduction Meristematic cells of higher plants contain small undifferentiated organelles, the proplastids. The photodevelopment of proplastids into mature chloroplasts is one of the most important morphogenetic modifications of plants (Tobin and Silverthorne 1985). Most of the thylakoid proteins are arranged in four membrane-spanning complexes, the topology and function of which are now well established (Murphy 1986). The four complexes are built up from proteins of chloroplastic or nuclear origin, the synthesis of which must be perfectly controlled in order to produce a quantitative modulation of their distribution into the membrane depending on the principal effector, light. For a number of plastid genes it has been shown that the abundance of transcripts increases following light induction (Rodermel and Bogorad 1985; Zhu et al. 1985). Photostimulation of nuclear gene transcription has also been observed (Bennet et al. 1984; Gallagher et al. 1985). However, recent work has shown that the regulation of gene expression during chloroplast development is much more complex and, in both nuclear and chloroplastic compartments, other control levels such as m R N A stabilities or translational stimulation are superimposed on the transcriptional enhancement (Mullet 1988; Gruissem 1989). The phytoflagellate Euglena gracilis provides a convenient system to study different levels of light regulation. When grown in darkness, Euglena cells have small proplastids which lack chlorophyll and contain little protein. Exposure to light induces the transformation of the proplastids into photosynthetically competent chloroplasts, a process involving active protein synthesis (reviewed in Schwartzbach 1990). But light is not the only factor capable of affecting the regulation of photosynthetic genes in Euglena. Freyssinet et al. (1972) have shown that the proportions of carbon, phosphate and nitrogen in the medium on which the cells are grown can greatly influence the development of the chloroplast.
C. Weiss et al. : Effect of nutritional conditions on thylakoid protein synthesis in Euglena I n a p r e v i o u s s t u d y (Weiss et al. 1988), we d e m o n s t r a t e d t h a t d u r i n g c h l o r o p l a s t d e v e l o p m e n t the synthesis o f the p o l y p e p t i d e s i n v o l v e d in the f o r m a t i o n o f the r e a c t i o n centers P S I a n d P S I I o f c h l o r o p l a s t i c origin, a n d o f the n u c l e a r - e n c o d e d l i g h t - h a r v e s t i n g p r o t e i n s , is m a i n l y c o n t r o l l e d a t a p o s t - t r a n s c r i p t i o n a l level. A n exc e p t i o n was psbA, the gene e n c o d i n g the D1 p r o t e i n o f the P S I I r e a c t i o n center, w h e r e light i n d u c e d a l i n e a r i n c r e a s e o f t r a n s c r i p t level in b o t h t o t a l a n d p o l y s o m a l R N A . P o s t - t r a n s c r i p t i o n a l r e g u l a t i o n involves different levels o f p o s s i b l e c o n t r o l such as m R N A stability, rec r u i t m e n t o n t o the p o l y s o m e s , t r a n s l a t i o n (initiation, elongation of polypeptides, termination) and postt r a n s l a t i o n a l ( m o d i f i c a t i o n o r b r e a k d o w n o f n e w l y synthesized p e p t i d e s ) processes. I n a n a t t e m p t f u r t h e r to c h a r a c t e r i z e this p o s t - t r a n s c r i p t i o n a l r e g u l a t i o n step, we h a v e e x a m i n e d the effect o f light o n the d i s t r i b u t i o n o f t r a n s c r i p t s e n c o d i n g several c h l o r o p h y l l - b i n d i n g p r o t e i n s . W e have also invest i g a t e d m R N A stabilities a n d p r o t e i n t u r n o v e r . By using different c o n d i t i o n s for cell g r e e n i n g we h a v e o b t a i n e d evidence t h a t , d e p e n d i n g o n the n u t r i t i o n a l e n v i r o n m e n t , a given gene, c h l o r o p l a s t i c o r nuclear, m a y be r e g u l a t e d either b y m o b i l i z a t i o n o f the c o r r e s p o n d i n g m R N A o n t o polysomes or by activation of translation.
Material and methods
Greening conditions. Euglena gracilis Klebs (Z strain from the University of G6ttingen, FRG) cells were grown heterotrophically in darkness on a glutamate-malate growth medium (Ortiz et al. 1980), and then harvested and resuspended either in a resting medium (25 mM KH2PO4/NazHPO4, pH 7; 1.25 mM MgSO4) as previously described (Schantz et al. 1981), or in a modified glutamate-malate growth medium (the nutritional medium). This medium lacks glucose and the concentration of vitamin B12 is increased to 500 ng 9 1-1. The optimal ratio of carbon: nitrogen: phosphorus is maintained. The cells were allowed to adapt to the greening for 12 h in darkness and then subjected to light for various periods of time. Greening experiments using the resting medium in the presence of clindamycin were done according to Dubertret and Pineau (1984). Briefly, after a preillumination period of 16 h, clindamycin was added to a final concentration of 500 mg - ml- 1 and the culture exposed to a light flux of 5000 lx for 35 h. To remove clindamycin, cells were centrifuged, washed, resuspended in fresh resting medium and exposed to low irradiance (300 lx) in order to induce a high level of chloroplastic protein synthesis. In experiments concerning transcript stability, cells in either nutritional or resting medium were kept in the dark or greened for 24 h and then exposed to a temperature of 34 ~ C. At this temperature, the chloroplastic RNA polymerase is selectively inhibited (Brandt and Wiessner 1977 ; Brandt 1988). The cultures reached this temperature within 10 min. For in-vivo labelling experiments with psS] sulphate, darkgrown cells were harvested and resuspended either in a resting medium in which MgSO4 was replaced by MgC12, or in a nutritional medium in which all sulphur salts were replaced by chlorides. Isolation and analysis of RNA. Total and polysomal RNA was isolated as described elsewhere (Weiss et al. 1988). Polysomes were fractionated on continuous 15-60% sucrose gradients in 50 mM Tris-HC1, pH 8.5; 25 mM KC1; 10mM MgCI2. Gradients were centrifuged for 2 h at 40000 rpm in an SW 41 rotor (Beckman, Palo Alto, Cal., USA). All operations were performed at 4 ~ C. After centrifugation, the gradients were analysed by a UV analyzer
469
(UV-1 detector; Pharmacia, Saint-Quentin en Yvelines, France) with continuous monitoring at 254 nm, and fractionated. Sodium dodecyl sulfate (SDS) was added to each fraction to a final concentration of 1% (w/v), and the polysomal RNA was purified by two phenol extractions and precipitated with ethanol. The copy-DNA probes for the different chloroplastic genes and for LHCI (the light-harvesting chlorophyll a/b-binding protein of PSI encoded by the nuclear cab genes), and the procedures for Northern and dot-blot analysis, have been described previously (Houln6 and Schantz 1988; Weiss et al. 1988). Autoradiographs were scanned with a laser scanning densitometer (CS-9000; Shimadzu Corporation, Tokyo, Japan) and the relative intensity of a given band or dot determined by integration of the area under the peak. For the histograms, data are averages of two or three measurements_+ standard errors are indicated by vertical bars).
In-vivo labelling. For the pulse-chase experiments, the cells were labelled for 3 h with carrier-free [3sS]sulphate (28 GBq. m1-1, 14 kBq 9mol 1) and the chase was initiated by addition of MgSO4 to a final concentration of 0.1 M. Cells were harvested by centrifugation at 1000 9g for 2 min, and total cell proteins were extracted by resuspending the pellet in 60 mM Tris-HCl, pH 6.8; 2% /w/v) SDS, and boiling for 2 min. The chlorophyll-protein complex I of PSI (CPI) was immunoprecipitated using polyclonal antibodies (Devic and Schantz 1984) essentially according to Rikin and Schwartzbach (1988) but using Staphylococcus membranes (Westhoff and Zetsche 1981) instead of protein A-Sepharose. The immunoprecipitated proteins were separated on a 7.5-15% SDS-polyacrylamide gel (Chua 1980) and the labelled polypeptides analyzed by fluorography.
Results W e h a v e s h o w n in e a r l i e r e x p e r i m e n t s (Weiss et al. 1988) t h a t d u r i n g c h l o r o p l a s t d e v e l o p m e n t in E. graeilis, n o c o r r e l a t i o n exists b e t w e e n a c c u m u l a t i o n o f c h l o r o p h y l l b i n d i n g p r o t e i n s a n d levels o f their c o r r e s p o n d i n g m R N A s . It a p p e a r s t h a t , w i t h the e x c e p t i o n ofpsbA, all the o t h e r genes s t u d i e d in the n u c l e u s a n d the c h l o r o p l a s t are m a i n l y r e g u l a t e d at a p o s t - t r a n s c r i p t i o n a l level. T o c h a r a c t e r i z e f u r t h e r the effect o f light a n d the influence o f n u t r i t i o n a l c o n d i t i o n s o n the r e g u l a t i o n o f the synthesis o f c h l o r o p l a s t m e m b r a n e p r o t e i n s , we h a v e r e - e x a m i n e d the different p o s s i b l e c o n t r o l steps o n t w o different cell-greening m e d i a : a resting m e d i u m c o n t a i n ing o n l y a p h o s p h a t e source a n d a n u t r i t i o n a l m e d i u m having an optimal ratio of carbon, nitrogen and phosphorus.
Greenin 9 on restin 9 medium Stability o f m R N A . Several r e p o r t s ( D e n g a n d G r u i s s e m 1987; M u l l e t a n d K l e i n 1987) h a v e s h o w n t h a t the t r a n s c r i p t i o n a l c o n t r o l o f p l a s t i d genes d u r i n g c h l o r o p l a s t d e v e l o p m e n t in h i g h e r p l a n t s m i g h t n o t be as i m p o r t a n t as p r e v i o u s l y believed, a n d t h a t the s t a b i l i t y o f t r a n scripts m a y p l a y a n a p p r e c i a b l e role. T o i n v e s t i g a t e the s t a b i l i t y o f the c h l o r o p l a s t t r a n s c r i p t s we h a v e u s e d a n e l e v a t e d t e m p e r a t u r e (34-35 ~ C) w h i c h i n h i b i t s the c h l o r o p l a s t R N A p o l y m e r a s e ( B r a n d t 1981, 1988) t h u s p r o v i d i n g a n i n d i r e c t b u t efficient w a y to f o l l o w c h l o r o p l a s t t r a n s c r i p t stability.
470
C. Weiss et al. : Effect o f nutritional c o n d i t i o n s o n thylakoid protein synthesis in
a
~~L1 0 O U) C
~
8
~
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pl~Jlflt
~
~) Springer-Verlag1992
Photocontrol of thylakoid protein synthesis in Euglena: differential post-transcriptional regulation depending on nutritional conditions Catherine Weiss*, Guy Houln~, and Rodolphe Schantz** Institut de Biologie Mol6culaire des Plantes du CNRS, 12, rue du G6n6ral Zimmer, F 67084 Strasbourg Cedex, France Received 24 February; accepted 5 June 1992
Abstract. The expression of three chloroplastic genes, psbA (the gene for the reaction center D1 protein of PSII), psbC (the gene for the PT00 apoprotein of PSI) and psaB (the gene for the intermediate antema of PSII), and a nuclear gene, cab (the gene for the chlorophyll a/bbinding protein), has been investigated during chloroplast development in Euglena gracilis Klebs. The polysomal fraction, m R N A stability and protein turnover were analysed under different conditions of cell greening. The results indicate that the main regulatory step for the nuclear and chloroplastic genes was at the translational level when greening o f cells took place on a resting medium. When cell greening took place on a medium with balanced phosphate, carbon and nitrogen sources (nutritional medium), the main regulation occurred posttranscriptionally by mobilization of the transcripts onto the polysomes. These results indicate that in E. gracilis, for a given gene, regulation operates at different levels, and that although light is the principal effector in the regulation of the genes involved in chloroplast development, the effect of metabolites should also be considered. Interestingly, it appears that these various regulation levels are similar for the chloroplastic and nucleocytoplasmic compartments. Key words: Chloroplast development - Euglena (thylakoid proteins) - Gene expression (light and nutritional regulation) - m R N A stability - Polysome - Thylakoid (protein synthesis)
Abbreviations: CPI = chlorophyll-protein complex I of PSI; LHCI (II)=light-harvesting chlorophyll a/b-binding protein of PSI (II) encoded by the nuclear cab genes; psaB =gene for the PTooapoprotein of PSI; psbA = gene for the reaction center D 1 protein of PSII ; psbC=gene for the intermediate antenna of PSII * Present address: Cold Spring Harbor Laboratory, P.O. box 100, Cold Spring Harbor, NY 11725, USA ** To whom correspondence should be addressed; FAX: (33) 8861 44 42
Introduction Meristematic cells of higher plants contain small undifferentiated organelles, the proplastids. The photodevelopment of proplastids into mature chloroplasts is one of the most important morphogenetic modifications of plants (Tobin and Silverthorne 1985). Most of the thylakoid proteins are arranged in four membrane-spanning complexes, the topology and function of which are now well established (Murphy 1986). The four complexes are built up from proteins of chloroplastic or nuclear origin, the synthesis of which must be perfectly controlled in order to produce a quantitative modulation of their distribution into the membrane depending on the principal effector, light. For a number of plastid genes it has been shown that the abundance of transcripts increases following light induction (Rodermel and Bogorad 1985; Zhu et al. 1985). Photostimulation of nuclear gene transcription has also been observed (Bennet et al. 1984; Gallagher et al. 1985). However, recent work has shown that the regulation of gene expression during chloroplast development is much more complex and, in both nuclear and chloroplastic compartments, other control levels such as m R N A stabilities or translational stimulation are superimposed on the transcriptional enhancement (Mullet 1988; Gruissem 1989). The phytoflagellate Euglena gracilis provides a convenient system to study different levels of light regulation. When grown in darkness, Euglena cells have small proplastids which lack chlorophyll and contain little protein. Exposure to light induces the transformation of the proplastids into photosynthetically competent chloroplasts, a process involving active protein synthesis (reviewed in Schwartzbach 1990). But light is not the only factor capable of affecting the regulation of photosynthetic genes in Euglena. Freyssinet et al. (1972) have shown that the proportions of carbon, phosphate and nitrogen in the medium on which the cells are grown can greatly influence the development of the chloroplast.
C. Weiss et al. : Effect of nutritional conditions on thylakoid protein synthesis in Euglena I n a p r e v i o u s s t u d y (Weiss et al. 1988), we d e m o n s t r a t e d t h a t d u r i n g c h l o r o p l a s t d e v e l o p m e n t the synthesis o f the p o l y p e p t i d e s i n v o l v e d in the f o r m a t i o n o f the r e a c t i o n centers P S I a n d P S I I o f c h l o r o p l a s t i c origin, a n d o f the n u c l e a r - e n c o d e d l i g h t - h a r v e s t i n g p r o t e i n s , is m a i n l y c o n t r o l l e d a t a p o s t - t r a n s c r i p t i o n a l level. A n exc e p t i o n was psbA, the gene e n c o d i n g the D1 p r o t e i n o f the P S I I r e a c t i o n center, w h e r e light i n d u c e d a l i n e a r i n c r e a s e o f t r a n s c r i p t level in b o t h t o t a l a n d p o l y s o m a l R N A . P o s t - t r a n s c r i p t i o n a l r e g u l a t i o n involves different levels o f p o s s i b l e c o n t r o l such as m R N A stability, rec r u i t m e n t o n t o the p o l y s o m e s , t r a n s l a t i o n (initiation, elongation of polypeptides, termination) and postt r a n s l a t i o n a l ( m o d i f i c a t i o n o r b r e a k d o w n o f n e w l y synthesized p e p t i d e s ) processes. I n a n a t t e m p t f u r t h e r to c h a r a c t e r i z e this p o s t - t r a n s c r i p t i o n a l r e g u l a t i o n step, we h a v e e x a m i n e d the effect o f light o n the d i s t r i b u t i o n o f t r a n s c r i p t s e n c o d i n g several c h l o r o p h y l l - b i n d i n g p r o t e i n s . W e have also invest i g a t e d m R N A stabilities a n d p r o t e i n t u r n o v e r . By using different c o n d i t i o n s for cell g r e e n i n g we h a v e o b t a i n e d evidence t h a t , d e p e n d i n g o n the n u t r i t i o n a l e n v i r o n m e n t , a given gene, c h l o r o p l a s t i c o r nuclear, m a y be r e g u l a t e d either b y m o b i l i z a t i o n o f the c o r r e s p o n d i n g m R N A o n t o polysomes or by activation of translation.
Material and methods
Greening conditions. Euglena gracilis Klebs (Z strain from the University of G6ttingen, FRG) cells were grown heterotrophically in darkness on a glutamate-malate growth medium (Ortiz et al. 1980), and then harvested and resuspended either in a resting medium (25 mM KH2PO4/NazHPO4, pH 7; 1.25 mM MgSO4) as previously described (Schantz et al. 1981), or in a modified glutamate-malate growth medium (the nutritional medium). This medium lacks glucose and the concentration of vitamin B12 is increased to 500 ng 9 1-1. The optimal ratio of carbon: nitrogen: phosphorus is maintained. The cells were allowed to adapt to the greening for 12 h in darkness and then subjected to light for various periods of time. Greening experiments using the resting medium in the presence of clindamycin were done according to Dubertret and Pineau (1984). Briefly, after a preillumination period of 16 h, clindamycin was added to a final concentration of 500 mg - ml- 1 and the culture exposed to a light flux of 5000 lx for 35 h. To remove clindamycin, cells were centrifuged, washed, resuspended in fresh resting medium and exposed to low irradiance (300 lx) in order to induce a high level of chloroplastic protein synthesis. In experiments concerning transcript stability, cells in either nutritional or resting medium were kept in the dark or greened for 24 h and then exposed to a temperature of 34 ~ C. At this temperature, the chloroplastic RNA polymerase is selectively inhibited (Brandt and Wiessner 1977 ; Brandt 1988). The cultures reached this temperature within 10 min. For in-vivo labelling experiments with psS] sulphate, darkgrown cells were harvested and resuspended either in a resting medium in which MgSO4 was replaced by MgC12, or in a nutritional medium in which all sulphur salts were replaced by chlorides. Isolation and analysis of RNA. Total and polysomal RNA was isolated as described elsewhere (Weiss et al. 1988). Polysomes were fractionated on continuous 15-60% sucrose gradients in 50 mM Tris-HC1, pH 8.5; 25 mM KC1; 10mM MgCI2. Gradients were centrifuged for 2 h at 40000 rpm in an SW 41 rotor (Beckman, Palo Alto, Cal., USA). All operations were performed at 4 ~ C. After centrifugation, the gradients were analysed by a UV analyzer
469
(UV-1 detector; Pharmacia, Saint-Quentin en Yvelines, France) with continuous monitoring at 254 nm, and fractionated. Sodium dodecyl sulfate (SDS) was added to each fraction to a final concentration of 1% (w/v), and the polysomal RNA was purified by two phenol extractions and precipitated with ethanol. The copy-DNA probes for the different chloroplastic genes and for LHCI (the light-harvesting chlorophyll a/b-binding protein of PSI encoded by the nuclear cab genes), and the procedures for Northern and dot-blot analysis, have been described previously (Houln6 and Schantz 1988; Weiss et al. 1988). Autoradiographs were scanned with a laser scanning densitometer (CS-9000; Shimadzu Corporation, Tokyo, Japan) and the relative intensity of a given band or dot determined by integration of the area under the peak. For the histograms, data are averages of two or three measurements_+ standard errors are indicated by vertical bars).
In-vivo labelling. For the pulse-chase experiments, the cells were labelled for 3 h with carrier-free [3sS]sulphate (28 GBq. m1-1, 14 kBq 9mol 1) and the chase was initiated by addition of MgSO4 to a final concentration of 0.1 M. Cells were harvested by centrifugation at 1000 9g for 2 min, and total cell proteins were extracted by resuspending the pellet in 60 mM Tris-HCl, pH 6.8; 2% /w/v) SDS, and boiling for 2 min. The chlorophyll-protein complex I of PSI (CPI) was immunoprecipitated using polyclonal antibodies (Devic and Schantz 1984) essentially according to Rikin and Schwartzbach (1988) but using Staphylococcus membranes (Westhoff and Zetsche 1981) instead of protein A-Sepharose. The immunoprecipitated proteins were separated on a 7.5-15% SDS-polyacrylamide gel (Chua 1980) and the labelled polypeptides analyzed by fluorography.
Results W e h a v e s h o w n in e a r l i e r e x p e r i m e n t s (Weiss et al. 1988) t h a t d u r i n g c h l o r o p l a s t d e v e l o p m e n t in E. graeilis, n o c o r r e l a t i o n exists b e t w e e n a c c u m u l a t i o n o f c h l o r o p h y l l b i n d i n g p r o t e i n s a n d levels o f their c o r r e s p o n d i n g m R N A s . It a p p e a r s t h a t , w i t h the e x c e p t i o n ofpsbA, all the o t h e r genes s t u d i e d in the n u c l e u s a n d the c h l o r o p l a s t are m a i n l y r e g u l a t e d at a p o s t - t r a n s c r i p t i o n a l level. T o c h a r a c t e r i z e f u r t h e r the effect o f light a n d the influence o f n u t r i t i o n a l c o n d i t i o n s o n the r e g u l a t i o n o f the synthesis o f c h l o r o p l a s t m e m b r a n e p r o t e i n s , we h a v e r e - e x a m i n e d the different p o s s i b l e c o n t r o l steps o n t w o different cell-greening m e d i a : a resting m e d i u m c o n t a i n ing o n l y a p h o s p h a t e source a n d a n u t r i t i o n a l m e d i u m having an optimal ratio of carbon, nitrogen and phosphorus.
Greenin 9 on restin 9 medium Stability o f m R N A . Several r e p o r t s ( D e n g a n d G r u i s s e m 1987; M u l l e t a n d K l e i n 1987) h a v e s h o w n t h a t the t r a n s c r i p t i o n a l c o n t r o l o f p l a s t i d genes d u r i n g c h l o r o p l a s t d e v e l o p m e n t in h i g h e r p l a n t s m i g h t n o t be as i m p o r t a n t as p r e v i o u s l y believed, a n d t h a t the s t a b i l i t y o f t r a n scripts m a y p l a y a n a p p r e c i a b l e role. T o i n v e s t i g a t e the s t a b i l i t y o f the c h l o r o p l a s t t r a n s c r i p t s we h a v e u s e d a n e l e v a t e d t e m p e r a t u r e (34-35 ~ C) w h i c h i n h i b i t s the c h l o r o p l a s t R N A p o l y m e r a s e ( B r a n d t 1981, 1988) t h u s p r o v i d i n g a n i n d i r e c t b u t efficient w a y to f o l l o w c h l o r o p l a s t t r a n s c r i p t stability.
470
C. Weiss et al. : Effect o f nutritional c o n d i t i o n s o n thylakoid protein synthesis in
a
~~L1 0 O U) C
~
8
~
6
I---
0 c ..O
