Eur. J. Biochem. 85, 581 -588 (1978)

The Plastid Membranes of Barley (Hordeurn vulgare) Light-Induced Appearance of mRNA Coding for the Apoprotein of the Light-Harvesting Chlorophyll a/h Protein Klaus APEL and Klaus KLOPPSTECH Riologisches Institut II der Universitiit Freiburg, and Max-Planck-Institut fur Zellbiologie, Abteilung Schweiger, Wilhelmshaven (Received November 9, 1977)

Illumination of dark-grown barley plants induces a massive insertion of the light-harvesting chlorophyll a/h protein into the developing thylakoid membrane. In addition to the onset of chlorophyll synthesis, light induces specifically the appearance of a prominent mRNA species which codes for a polypeptide of M , 29500. This component was identified as a precursor of the apoprotein of the light-harvesting chlorophyll a//? protein. The precursor has an M , larger than the authentic protein by approximately 4000. Studies of the chlorophyll-h-less mutant chlorina f2 of barley offer the first clue to the mechanism which controls the light-dependent mRNA formation. The induction of the mRNA coding for the apoprotein of the light-harvesting chlorophyll a/h protein does not seem to be linked directly to the assembly process of the light-harvesting structure and does not require chlorophyll h. It is proposed that light exerts its influence on the mRNA formation by a reaction which is different from the phototransformation of protochlorophyll( ide) to chlorophyll(ide)

Leaves of higher plants grown in darkness lack chlorophyll and cannot carry out photosynthesis. Upon illumination the plastids of these leaves undergo a series of developmental changes : membrane components are reorganized and newly synthesized, chlorophyll is produced and the capacity to photo5ynthesize develops [1-31. The final stage of the lightinduced plastid membrane differentiation results in the insertion of light-harvesting structures into the chloroplast membrane [4]. A major part of the lightharvesting chlorophyll seems to be associated with the light-harvesting chlorophyll aih protein [5]. Together with the jnsertion of chlorophylls a and h a massive incorporation of the apoprotein of the lightharvesting chlorophyll a/h protein into the membrane occurs [4,6]. While the direct influence of light on the synthesis of chlorophyll in higher plants is well documented [I], such an influence on the synthesis of the apoprotein of the light-harvesting chlorophyll ulh protein has not yet been shown. There is evidence that thc apoprotein of the lightharvesting chlorophyll alb protein is coded for by nuclear DNA [7] and that it is synthesized outside the organelle on 80-S ribosomes [8]. There are a number

of possible sites at which light may control the appearance of the apoprotein within the chioroplast membrane. Light could act at the transcriptional or translational stage of protein synthesis. Light could also regulate the transport of the protein into the organelle or it could trigger the final assembly of the complete light-harvesting chlorophyll aih complex. In the present study we provide evidence that light induces the appearance of mRNA coding for a precursor of the apoprotein of the light-harvesting chlorophyll a/h protein. This induction of a new mRNA species seems to be independent of the assembly of the light-harvesting structure and does not require chlorophyll h. MATERIALS AND METHODS Growth o f Plants

Wild -type barley (Hordeurn wlgarP) and the ch 1orophyll-h-less mutant chlorina f2 were grown in the dark on moist vermiculite at 25 ‘ C for 5 days. Plants were illuminated for different lengths of time with incandescent light (1 3000 Ix).

582

I x h t r o n of Stroma-Free PlaJtid Fragments Leaves were harvested, chilled, roughly chopped and homogenized with 0.5 M sucrose, 250 mM TrisHC1, pH 8.0, at high speed for 30 s in a Waring blendor. The homogenate was passed through two layers of cheese-cloth and two layers of Miracloth (Chicopee Mills Inc., Milltown, N.J. 08850, U.S.A.). The filtrate was centrifuged at 30000 x g for 10 min. The pellet was resuspended in 5 0 m M Tris-HCI, pH 8.0, and centrifuged for 10 min at 3000 x g. The resulting pellet was resuspended in a small volume of the same buffer, layered on a 32-ml gradient of 20- SO "/I sucrose containing SO mM Tris-HCI, pH 8.0, and was centrifuged at 113000 x g in a Spinco SW21 rotor for 60 min. The band containing plastid fragments was drawn off from the sucrose gradient with a syringe, diluted 1 :5 with water and centrifuged for 10 min at 40000 x g. The pellet was resuspended in water, lyophilized and stored at - 20 'C.

R N A Extraction Poly(A)-containing RNA from 6 g leaf tissue (1 g leaf tissue/lO ml buffer) was prepared as described [9] except that the EDTA concentration was lowered to 10 mM and the pH of the Tris buffer adjusted to 9.0 instead of 7.5. The poly(A)-containing RNA was separated on oligo(dT)-cellulose (1 g cellulose/l .5 g leaf tissue) [9] and was stored under liquid nitrogen.

Wheat Germ Cell-Free System The translation of poly(A)-containing RNA in a cell-free wheat germ system was performed according to Roberts and Paterson [lo].

Electrophoretic Analysis of Polypeptides The lyophilized membrane material was solubilized in the presence of sodium dodecylsulfate and separated electrophoretically as described by Nelville [ll]. Coomassie blue staining of the gel was done according to the method of Fairbanks et al. [12]. Gels in which the translation products in vitro or the immunoprecipitates had been separated, were stained and treated for fluorography as described by Bonner and Laskey [ 131.

Labelling the Light-Harvesting Chlorophyll a/b Protein in vivo

of

Barley plants were grown on moist paper in testtubes for 4 days in the dark at 25 "C. Immediately after the onset of illumination [35S]methioninewas pipetted on to the paper at the bottom of the test-tube and

Light-Induced Appearance of mRNA

incubation in the light was continued for 48 h. The leaves were harvested and the thylakoid membranes isolated as described above. The membrane proteins were separated electrophoretically in the presence of dodecylsulfate. The gel was stained with Coomassie blue and the band of the 25000-M, apoprotein of the light-harvesting chlorophyll u/b protein was cut out of the gel. The stained and radioactive protein material was extracted from the gel and lyophilized as previously described [141.

Indirect Immunoprecipitation Products Synthesized in vitro

of

For the indirect immunoprecipitation an antiserum raised in rabbits against the light-harvesting chlorophyll alh protein of the green alga Acetubuluviu mediterruneu was used [14]. The chlorophyll proteins of barley and the green alga showed immunochemical identity as indicated by Ouchterlony double-diffusion tests. Goat antiserum to rabbit immunoglobulin was purchased from Behringwerke (Marburg, F.R.G.). The IgG fraction of the serum was purified by DEAEcellulose chromatography and ammonium sulfate precipitation. The indirect immunoprecipitatioii was done in the presence of low concentration of dodecylsulfate (Dobberstein, personal communication). The cell-free translation mixture was diluted 1 : 2 with 20 mM Hepes/ KOH, pH 7.1,lOO mM KCI and 2 mM Mg(CH3COO)Z and centrifuged at 164000 x g for 30 min in a Spinco SW50.1 rotor. The supernatant was adjusted to 0.1 "1,; dodecylsulfate. 1 pl of the rabbit antiserum directed against the light-harvesting chlorophyll a/h protein was added to 100 pl of the supernatant. After incubation for 30 min at 25 r C and 1 h at 4 T , 60 p1 of the goat antiserum to rabbit IgG fraction were added and the mixture was incubated for I h at 25 "C and thereafter for 16-20 h at 4 "C. The immunoprecipitate was centrifuged through a cushion of 1 M sucrose, 171 Triton X-100 and 10 mM sodium phosphate buffer, pH 7.1. The pellet was frozen and stored at - 20 "C.

Peptide Mapping of the Product in vitro and the Authentic Light-Harvesting Chlorophyll alb Protein The peptide mapping by limited proteolysis in dodecylsulfate was done essentially as described by Cleveland et al. [15]. Following the indirect immunoprecipitation the protein material was separated electrophoretically. The [ 3 5 Slmethionine-labelled 29500-M, product in vitro was eluted from the gel and was mixed with the isolated unlabelled authentic apoprotein of the light-harvesting chlorophyll a/b protein. In a separate experiment the light-harvesting chlorophyll u/b protein was labelled in vivo with [35S]methio-

583

K. Ape1 and K. Kloppstech

0

i.;

Time of illumination ( h ) 3 6 12 24 markers

130

69

T 3

0

29

13.7 12.5

00

5

10 15 20 Time of illumination ( h )

25

Fig. 2. Lighf-dependent changes It7 the umounl of p d y i A)-cotiluitiing R N A l g leaf fresh ,ceight. Dark-grown barley was illuminated for various lengths of time and the poly(A)-containing RNA wa\ isolated from the leaf tissue of these plants as described under Methods. (0) Poly(A)-containing R N A ; (A) weight o f 50 frcsh leaves

Fig. 1. Light-dq~c.ndm/chunges in the polypeptide composition of plasfid twrnhrurre.sof Mild-type barley. The plastid membranes were isolated from dark-grown barley which had been illuminated for different lengths of time. The membrane proteins were solubilized in the presence of dodecylsulfate and separated electrophoretically as described under Methods. The arrow indicates the position of the apoprotein of the light-harvesting chlorophyll u/b protein. Marker proteins: p-galactosidase (130000), bovine serum albumin (69000), carbonic anhydrase (29000), ribonuclease (1 3700) and cytochrorne c (12500)

nine and isolated as described above. 20 pg of the isolated authentic apoprotein labelled in vivo and 20 pg of a mixture of the unlabelled apoprotein and the putative precursor labelled in vifro, both solubilized in 50 pl sample buffer [15], were mixed with 1 pg Stuphylococcus U U Y ~ U S V8 protease (Miles Laboratories, 36.900-0877). Both samples were separated electrophoretically at room temperature. Proteolytic digestion proceeded directly in the stacking gel as described by Cleveland et al. [15]. Following the electrophoretic separation of the resulting peptides, the 15‘x polyacrylamide gel was stained and treated for fluorography as described [13].

RESULTS

Light-Induced Itwvtion of the Light-Harvesting Chlorophyll alb Protein into the Plustid Memhrune More than 40 polypeptide bands could be electrophoretically resolved from plastid membrane proteins, solubilized in the presence of I”/, dodecylsulfate, on polyacrylamide gels (Fig. 1). Light induced some significant changes in the protein composition of the different membrane samples (Fig. 1 ) (see also [16]).

Some of the proteins decreased or disappeared after the onset of illumination, while the appearance of other proteins in the plastid membrane fractions was triggered by light. The major membrane component, a polypeptide of M , 25000, was among the lightinduced proteins. As shown previously [I71 this polypeptide is part of the light-harvesting chlorophyll a/h protein of barley. The Ejjwt of Light on the Poly(A)-Containing R N A of Burlei, Lravc~s

One possible site at which light could control the appearance of the light-harvesting chlorophyll aih protein is the level of transcription. Most eukaryotic mRNA contains poly(A) sequences [18] which facilitate isolation of this RNA fraction by affinity chromatography. In barley the effect of light on poly(A)-containing RNA can be seen during the first 6 h of illumination, when the total amount of poly(A)-containing RNA increases from approximately 4- 6 pg/g leaf fresh weight. After the initial increase, poly(A)-containing RNA levels remained constant during 18 h of illumination (Fig. 2). During the same time the leaf fresh weight did not change significantly. The different poly(A)-containing RNA fractions isolated after various periods of illumination were translated in a heterologous cell-free wheat gerni system. Increasing amounts of RNA of the various samples were included in a series of reaction mixtures. Over a certain concentration range (0-- 15 pg RNA/ml) the amount of incorporated [35S]methionineincreased linearly in proportion to the amount of RNA before a saturation level was reached. For the analysis of the different poly(A)-containing RNA fractions amounts

584

Light-Induced Appearance of mRNA

24

Time of illumination ( h ) 0 3 6 12 24

Fig. 3. Light-dtyendcntchariges in the pattern of 35S-lahe~~edproducl r .synthesized in vitro in rhe presence of poly(A)-containing RNA fractions, which were isolured from barley illuminuted f b r d(ffrrcnr lengths of time. The poly(A)-containing R N A was translated in a cell-free system derived from wheat germ. The products in virro were separated by dodecylsulfate/polyacrylamide gel electrophoresis and visualized by autoradiography

of RNA from this concentration range were used which were calculated to have come from identical quantities of leaf tissues. The 35S-labelled products of the protein synthesis in vitro were analyzed autoradiographically after dodecylsulfatejpolyacrylamide gel electrophoresis. The major part of the translation products in vitro had molecular weights lower than 60000. A general enhancement in the amount of most RNA species following the light treatment could be observed (Fig. 3). Most of the products were found in all samples if equal amounts of poly(A)-containing RNA were used as templates (Fig. 4). There were a few products, however, whose appearance seemed to be totally dependent on the light treatment. The major polypeptide among the translation products from poly(A)-containing RNA isolated from dark-grown barley plants had an apparent molecular weight of 15000. This component disappeared during illumination (Fig. 3). On the other hand, light also induced the appearance of a new mRNA species. The RNA coding for the prominent polypeptide of 29 500 could only be detected after 2-3 h of illumination. The concentration of this RNA increased rapidly and reached a constant high level after approximately 6 h of illumination (Fig. 3). The light dependency and the similarity in the apparent molecular weights suggested to us that this light-induced mRNA might be a possible candidate for the mRNA coding for the

A

B

C

D

E

F

Fig. 4. Ideni(jicution of the precursor of the apoprotein qf the lightharvesting chlorophyll a/b protein of barley by dodec~l.Pulfate/polyucrylamide gel electrophoresis and autoradiography. (A, F) The isolated apoprotein of the light-harvesting chlorophyll ulh protein derived from barley labelled in vivo with [35S]methionine. (D, E) Analysis of the products synthesized in v i m in a wheat germ system in the presence of poly(A)-convaining R N A isolated from illuminated (E) and dark-grown (D) barley plants. (C) Indirect immunoprecipitation of (D) with an IgG fraction directed against the light-harvesting chlorophyll a/b protein. No radioactive precipitate was observed. (B) Indirect immunoprecipitation of (E) with an IgG fraction directed against the light-harvesting chlorophyll ajb protein

apoprotein of the light-harvesting chlorophyll a/h protein. Iden tiJication o j the Light-Induced 29 000-M, Product in vitro as n Precursor o f the Light-Harvesting Chlorophyll ajb Protein

Poly(A)-containing RNAs from dark-grown and illuminated ( 3 2 h) barley plants were isolated and equal amounts of RNA were used as templates for the protein synthesizing wheat germ system in vitro. Electrophoretic analysis of the translation products confirmed the above-mentioned results; viz., in darkgrown leaves the 29 500 M , band was absent and after 12 h light, a massive appearance of this polypeptide had been induced (Fig. 4 D, E). An IgG fraction directed against the light-harvesting chlorophyll nib protein was added to the two cellfree translation mixtures. The polypeptide of M , 29 500 was immunoprecipitated specifically by the IgG fraction (Fig.4B). In addition to the prominent 29500 M , product a minor component of M , approximately 32000 was also coprecipitated. This product has not

5x5

K. Ape1 and K. Kloppstech

yet been identified. The immunoprecipitation occurred only from the translation mixture directed by poly(A)containing R N A from illuminated plants but not from the corresponding sample of dark-grown plants (Fig. 4C). In both cases control sera gave no precipitation. For further identification of the 29 500- M , product in vitro peptide mapping of this isolated product and the isolated authentic apoprotein was made following limited proteolysis in dodecylsulfate and analysis by polyacrylamide gel electrophoresis (Fig. 5). The 29 500M , product in vitro was digested into 3 labelled peptide fragments, all of which seemed to appear in the corresponding pattern of the authentic protein. In addition to these 3 fragments the peptide pattern of the authentic protein material contained a fourth 35S-labelled fragment. By the similarity of immunological properties and of the peptide map to those of the apoprotein of the light-harvesting chlorophyll a/b protein as well as by its light dependency and its larger molecular weight (Fig. 4A,F), the 29 500-M, polypeptide was identified as a precursor to the apoprotein of the lightharvesting chlorophyll a/b protein. The Effect of Light on the Poly(A)-Containing RNA of tlze Chlorophyll-b-Less Mutant of Barley The chlorophyll-h-less mutant chlorina f2 of barley lacks both, chlorophyll b and the light-harvesting chlorophyll a/b protein [37,19,20]. In wild-type barley light induced the appearance of the prominent apoprotein of the chlorophyll protein (Fig.6). In the h- mutant a similar dramatic light-dependent insertion of this prominent polypeptide could not be observed, even after extensive illumination of the plants (Fig. 6). However, in the protein pattern of the mutant a Faint protein band appeared at approximately the same position as the prominent polypeptide of M , 25 000 (Fig. 6). As discussed previously by Machold et al. [17], it is not known whether this material is due to a small amount of the apoprotein of the light-harvesting chlorophyll a / h protein or to a different minor component that comigrates with the chlorophyll-binding protein. The absence of the light-harvesting chlorophyll a/h protein from the thylakoid membrane of the barley mutant has been used to emphasize the important role of chlorophyll b for the formation of the light-harvesting structure. It was interesting to know whether in the mutant the lack of chlorophyll b had any effect on the inducibility of the mRNA by light. Wild-type and 6-mutant plants were grown in the dark and also illuminated for 24 h. The poly(A)-containing RNA was isolated and amounts of RNA similar to those shown in Fig. 3 were translated in the

A

B

C

D

Fig. 5. Peptide mapping ofthe ZYSOO-M,product in vitro ( A J untl thr uirthentic apoprotein of the light-harvesting chlorophyll a,'b protcin ( B ) h,y limited proteolysis in sodium dodecylsulfate cind una1y.vi.v hy get elecirophoresis. The proteins were isolated and digested in the presence of Staphylococcus aureus protease as described under Methods. (C, D) The undigested 29500-M, product in vitro ( C ) and the authentic apoprotein (D). The proteins were labelled with [J5S]methionine and visualized in the gel by autoradiography

cell-free system derived from wheat germ. Electrophoretic analysis of the products in vitro revealed no significant differences between wild type and the mutant (Fig.7). In both plants light induced the appearance of mRNA coding for the apoprotein of the light-harvesting chlorophyll a/b protein. Even though chlorophyll b, which seems to be necessary for the assembly of this chlorophyll protein, was absent from the mutant, the induction of the mRNA functioned normally. Thus, the light-dependent appearance of this RNA species was linked neither to the insertion of the complete chlorophyll protein nor to the presence of chlorophyll h.

DISCUSSION Three basic facts concerning the insertion of the light-harvesting chlorophyll a / h protein and its regulation emerge from our study. Light Induces the Appearance of a New m R N A Species The total amount of poly(A)-containing R N A increased during the first 6 h of illumination and reached a constant level which did not change during the following 18 h. In the work of others with animal cells it was shown that the length of the poly(A)

586

Light-Induced Appearance of mRNA

-0

wild type 24 0

Time of illumination ( h ) 24 0 24

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wild type

6-mutant 24 0

wild type 24 0

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29

13.7 12.5 Fig. 6 . L ~ ~ h t - t k i ~ ~ ~ c~l~uriges o i r l i ~ n in ~ the /Joljpc~ptrd(~ i~ornp(~.sition (?/ pIfi.(ticlt?icmhr.atir.to f w i l d - / y p horlr?' ond the b- miituiit. The appearance of the light-harvesting chlorophyll u/h protein is induced only in the wild type as indicated by the arrow. Electrophoretic analysis of membrane proteins and staining of the gel with Coomassie blue was performed as described under Methods. Marker proteins as in Fig. 1

sequence decreases with time [21] presumably to give rise to poly(A)-free RNA [22]. Thus, the constant level of poly(A)-containing RNA after 6 h of illumination does not necessarily imply the end of the lightinduced increase of mRNA but could also reflect a steady state between the formation of poly(A)-containing RNA and its conversion to poly(A)-free mRNA. Among the RNA species of the poly(A)-containing RNA fraction observed in our experiments, the prominent mRNA coding for the polypeptide of M , 29500 required light (Fig.4B,C). Under our conditions only those mRNAs could be detected which contained the poly(A) sequence. Previous studies have revealed two mechanisms for the formation of poly(A)-containing RNAs. Firstly, stored, unadenylated RNAs could be adenylated in the cytoplasm [231. the poly(A) tract be added to the genetic message immediately after the completion of its transcription in the nucleus [18]. While the first of the two mechanisms has been reported only for highly specialized cells [23] in most other cases the poly(A) tract was found to be added to the mRNA

Fig. 7. Lighi-dependcwt induclion of m R N A c d i n g .for /lie apoprorein of the c/ilorciphj~lla/b protein in both irild-tyy,harleq' and /hi.13- mutun/. The arrows indicate the position or the precursor of the light-harvesting chlorophyll a:b protein. Wild-type and h - mutant plants were grown in the dark and also illuminated for 24 h. The poly(A)-containing R N A was isolated from these plants and translated in the wheat germ system. The products in vitro were analyzed electrophoretically and autoradiographically as described undcr Methods. The numbers indicate the lengths of time during which the various plants had been exposed to light

immediately after the synthesis of this RNA within the nucleus. Thus it is likely, although not conclusive, that the induction of a new poly(A)-containing mRNA in dark-grown barley leaves was due to a direct influence of light on its synthesis rather than an activation of a dormant template. An effect of light on the expression of genetic messages has already been described in several other plant systems [24-291 and there is evidence suggesting that this may include a specific induction of the translation of some mRNA species [24- 26,291. However, with the exception of the mRNA coding for the phenylalanine ammonia-lyase in parsley (Petroselinum hortense) cell cultures [25,26], no other mRNAs have been identified which were affected by light. Our results differ from the recent report of Giles et al. [29]. who found in bean leaves an effect of light only on the pattern of polysomal poly(A)-containing RNA but not on the composition of total poly(A)containing RNA. The Light-Induced mRNA Codes f o r a Higher Molecular weight Precursor of the Apoprotein *J'the Light-Harvesting Chlorophyll alb Protein

The identification of the precursor was based on similar immunochemical properties as well as similar peptide maps of the product in vitro and the apoprotein

587

K. Apel and K. Kloppstech

of the chlorophyll protein. Furthermore, the appearance of both polypeptides depended on the presence of light. Our finding that the precursor is larger than the authentic protein is consistent with similar results on larger precursor polypeptides in both animals and plants [30 - 331. The extended amino acid sequence of the precursor has been proposed to function in the attachment of polysomes to membranes, thus providing the topological conditions for the transfer of secretory proteins across the membrane (signal hypothesis, [31]). Since the apoprotein of the light-harvesting chlorophyll a/h protein is synthesized outside the organelle on 80-S ribosomes [8] and since it has to be transported across the outer and inner envelope membranes of the chloroplasts before its insertion into the thylakoid membrane, it would be tempting to suggest a linkage between the function of the extended sequence in the precursor and the protein transfer according to the signal hypothesis. However. this hypothesis has not yet been verified in plants and alternative mechanisms have been proposed [30]. The appearance of one mRNA species coding for the apoprotein of the light-harvesting chlorophyll ail? protein suggests that the protein moiety of this chlorophyll protein consists of only one polypeptide. However, by the methods used for its detection, no proof for the RNA homogeneity can be offered. This is important with respect to recent findings that the light-harvesting chlorophyll a/h protein complex of some green algae is composed of two different polypeptide subunits [6,14,34,35]. A similar structure has been discussed also for the light-harvesting chlorophyll a / h protein of higher plants [ l , 31. However, the reports on the existence of such a high-molecular-weight light-harvesting chlorophyll u / h protein complex in higher plants are still conflicting [36,37].

h- mutant of barley [17,20] as well as on intermittentlight-treated pea plants [4] suggests that the presence of chlorophyll b is an absolute requirement for the appearance of the light-harvesting chlorophyll a/h protein within the thylakoid membrane. Even though the light-harvesting chlorophyll a/h protein is absent from the plastid membrane of the h- mutant. light induces mRNA which codes for this protein as in the wild type. Thus, the induction of this mRNA seems to be independent of the assembly of the complete light-harvesting structure. Furthermore, the lack of chlorophyll h has no detectable impact on the lightregulated appearance of this RNA within the mutant. These results raise the possibility that a light-absorbing inductor other than chlorophyll may be responsible for the induction of thc mRNA coding for the lightharvesting chlorophyll aih protein. Investigations to characterize this postulated light mediator are in progress. The authors are grateful to Drs B. Ilobberstein. K . Miller, T. Redlinger and P. Schopfer for cooperatlon. valuable suggestions and careful reading of the manuscript and to c'. Springweilel- for ance. Some of the experiments were done i n the . C;. Schwcigcr, to wholn the authors are particularly grateful for his interest and his generous support. This investigation was supported by Doui.ychi>~c~rse/iun~.sgc~trIcin.vc.hrtfl (S.F.H. 46).

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The Induction of m R N A Coding ,fiw the Apopvotein q f the Light-Harvesting Chlorophyll alb Protein

is Independent of the Membrane Assembly and Does Not Require Chlorophyll b It is known that light is necessary for the transformation of protochlorophyll(ide) and the subsequent chlorophyll synthesis [I]. This could be the only site at which light exerts a direct influence on the formation of the light-harvesting chlorophyll u/h protein. The light-dependent induction of a new mRNA coding for the protein moiety of this chlorophyll protein could be a consequence of the presence of chlorophyll. Alternatively, light could act at several sites. In this case the light-dependent induction of mRNA would not necessarily depend on the presence of chlorophyll. A clue to the mechanism which controls the induction of light-dependent mRNA comes from the study of the 6 - mutant of barley. Work on the

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K. Apel, Biologisches Institut I1 der Albert-Ludwigs-Universitiit Freiburg, Schiinzlestrdsse 1, D-7800 Freiburg i. Br., Federal Republic of Germany K. Kloppstech, lnstitut fur Botanik, Technische Universitat Hannover, HerrenhCuser Strasse 2, D-3000 Hannover, Federal Republic of Germany