Planta

Planta (1988) 176:269 276

9 Springer-Verlag 1988

Light-dependent, but phytochrome-independent, translational control of the accumulation of the P700 chlorophyll-a protein of photosystem I in barley (Hordeum vulgate L.) William Laing *, Klaus Kreuz**, and Klaus Apel*** Botanisches Institut der Christian-Albrechts-Universitfit Kiel, Olshausenstrasse 40, D-2300 Kiel, Federal Republic of Germany

Abstract. This work reports on the regulation of synthesis of the P700 chlorophylI-a apoprotein of photosystem I in barley. The m R N A for the P700 apoprotein is almost exclusively confined to the plastid membrane-bound polysomes. However, the m R N A for the 32-kDa herbicide-binding protein of photosystem II is found in both the soluble and membrane-bound polysomes. The m R N A for the P700 apoprotein is found in similar amounts in dark-grown and light-grown wild-type as well as mutant xantha-181 barley. The latter mutant is deficient in chlorophyll biosynthesis. However, while wild-type leaves accumulate the P700 chlorophyll-a protein only in the light, mutant leaves never accumulate the P700 apoprotein. A more sensitive approach was taken using isolated plastids to study P700 apoprotein synthesis. Etioplasts did not synthesize detectable P700 apoprotein even when the etioplasts were exposed to light. However, only a l-min exposure of leaves to light was necessary to induce P700 apoprotein synthesis by isolated plastids. Phytochrome involvement in controlling P700 apoprotein synthesis was tested by using red/farred light treatment of leaves. These treatments showed no far-red reversibility of red-induced P700-apoprotein synthesis in isolated plastids even after 3 h of darkness after the light treatments. From these data we conclude that the accumulation of P700 apoprotein is not under the control of phytochrome and that the light induction of P700 apoprotein is most likely mediated through the protochlorophyllide/chlorophyllide system. This control, however, may also involve cytoplasDSIR, Plant Physiology Division, Palmerston North, New Zealand 9* CIBA-Geigy, Basel, Switzerland 9** To whom correspondence should be addressed P r e s e n t addresses." *

mic signals as the synthesis of the P700 apoprotein is not turned on in illuminated etioplasts.

Key words: Hordeum (P700 apoprotein synthesis) - Photosystem I P700 apoprotein- Phytochrome and P700 apoprotein synthesis Introduction The light-induced transformation of etioplasts into chloroplasts in higher plants depends on an intimate interaction of the two genetic systems within the nucleus and the plastid (Bogorad 1975; Ellis 1981). Light does not only exert its control on processes which occur within the plastid but it simultaneously triggers reactions in the cytoplasm which contribute to chloroplast formation. Therefore, it is not surprising that not only protochlorophyllide within the plastid but also at least one additional photoreceptor, phytochrome, is involved in the light-dependent control of the complex interaction between plastids and the nucleus (Harpster and Apel 1985; Tobin and Silverthorne 1985). It has been demonstrated that light-dependent changes in the concentrations of several nuclear-encoded plastid proteins during chloroplast formation are under the control of phytochrome. Phytochrome is able both to increase the transcription of certain nuclear genes and decrease the transcription of others (Gallagher and Ellis 1982; Minami and Watanabe 1984; Silverthorne and Tobin 1984; BerryLowe and Meagher 1985; M6singer et al. 1985). While a phytochrome-dependent control at the level of transcription has been well documented for nuclear genes, much less is known about the lightdependent control of plastid-gene expression. A phytochrome-dependent change in the rate of transcription, similar to that of nuclear genes, has been proposed as a major control by which

270

light may regulate the expression of plastid-specific genes (Link 1982; Thompson et al. 1983; Rodermel and Bogorad 1985; Zhu et al. 1985). However, recent studies of several plastid-specific genes including those encoding the 32000-Mr herbicidebinding polypeptide of photosystem II, the P700reaction-center chlorophyll-a protein of photosystern I and the large subunit of ribulose-l,5-bisphosphate carboxylase, have indicated that the expression of these genes during light-dependent chloroplast formation is regulated posttranscriptionally (Fromm etal. 1985; Inamine etal. 1985; Berry et al. 1986; Klein and Mullet 1986; Kreuz et al. 1986; Deng and Gruissem 1987). For instance, in the case of the P700 chlorophyll-a protein, it has been demonstrated that even though the accumulation of the protein occurs only after the beginning of illumination, high levels of specific mRNAs for this protein are found within the plastids of darkgrown plants similar to the levels found in lightgrown plants (Klein and Mullet 1986, 1987; Kreuz et al. 1986). In the present work we have tried to define more precisely the mode of light-dependent control which governs the synthesis of the P700 chlorophyll-a protein. Our results demonstrate that in barley the m R N A for the P700 apoprotein is not translated in the dark, indicating that light exerts its control at the translational level. Furthermore, we could demonstrate that this light effect is not mediated by phytochrome but appears to be under the control of the other photoreceptor, protochlorophyllide. Material and methods Growth of plants. Barley (Hordeum vulgare L.) wild-type and xantha-1 sl mutant plants were grown for 5 d either in the dark or with exposure to continuous white light (fluorescent tubes, 8000 Ix) for various lengths of time before harvesting. In some experiments dark-grown 5-d-old seedlings were exposed to monochromatic light of 655 nm or 759 nm as described earlier (Apel 1979). Isolation ofplastids. Plastids were isolated from barley leaves according to a procedure modified from that published by Klein and Mullet (1986). All buffers were filtered through a 0.45-gm filter before use. An aliquot of 1 5 ~ 0 g of leaves was ground for 2 x 5 s in an Ultraturrax (IKa-Werk, Staufen, FRG) in the presence of 0.33 M sorbitol, 50 m M (4-(2-hydroxyethyl)-l-piperazine sulfonic acid (Hepes), 2 m M ethylenediaminetetraacetic acid (EDTA), 10 mM dithiothreitol (DTT), pH 8.0 (5 v/w). The homogenate was filtered through Miracloth and centrifuged for 2 min at 2200.g. The pellet was resuspended in 4 ml grinding buffer and the resulting suspension applied on top of a step gradient consisting of 5 ml 65% Percoll and 7 ml 42% Percoll. Percoll solutions were made up in grinding buffer and also contained 3% polyethylene glycol 6000 and 1% bovine serum albumin. The gradient was centrifuged for 10 min at 2 200-g. The intact plastids were recovered from the lower band,

W. Laing et al. : Control of P700 chlorophyll-a protein in barley mixed with 4 vol. of grinding buffer, spun down for 2 min at 2200.g and finally resuspended in a minimal volume of grinding buffer. All operations were done in the dark using a green safe light. Isolation of chloroplasts to the start of [35S] methionine incorporation took approximately 30 min.

Protein synthesis in isolated plastids. Isolated plastids were incubated in the presence of 0.33 M sorbitol, 50 mM Hepes, 2 mM EDTA, 40 gM amino acids (minus methionine), 10 mM DTT, 7.2-105 Bq [35S]methionine (4.1012 Bq.mmol-1), 10 mM ATP, 5 mM NaHCO3 and 10 m M MgCI2 in a final volume of 200 gl. Incubation was carried out at 25 ~ C in the dark or in the light (200 gmol.m -2 s 1). After 30 min an excess ofunlabelled methionine was added to a concentration of 8.5 m M and the incubation continued for another 15 min. The reaction was stopped by adding sodium dodecyl sulfate (SDS) to 1% final concentration and heating the sample for 1 min at 95 ~ C.

In-vivo labelling. Leaves of 5-d-old dark-grown barley seedlings were cut under water at the base of their stems and placed in an Eppendorf cap (10 leaves per cap) which contained 500 ~tl of [35S]methionine (1.9-107 Bq.m1-1, 4.1012 Bq.mmol 1). Leaves were incubated in the dark for 4 h or were illuminated for the last 60 or 120 min prior to the end of incubation. Leaves were frozen under liquid nitrogen, ground to a fine powder and the protein was extracted with a hot SDS-containing buffer as described previously (Kreuz et al. 1986). Polypeptides were separated electrophoretically on an SDS polyacrylamide gel as described by Laemmli (1970).

Immunoprecipitation of the P700 chlorophyll-a protein. Immunoprecipitations were done according to Batschauer et al. (1986) with antisera raised against the purified P700 chlorophyll-a protein of barley (Kreuz et al. 1986) or rye (a gift of Dr. J. Feierabend, Frankfurt, FRG).

Isolation of plastid polysomes and polysomal RNA. An aliquot of 100 g of barley leaves was ground at 0 ~ C in 500 ml grinding medium (0.33 M sorbitol, 50 mM tricine-KOH, 1 mM MgC1/, 1 mM MnCI2, 7 m M /Lmercaptoethanol, 0.1% bovine serum albumin, pH 7.8) in a Waring blendor for 4 s at low speed and 3 x 3 s at high speed. The homogenate was filtered through five layers of Miracloth and centrifuged for 5 rain at 1800-g. The pellet was resuspended gently in 6 ml grinding buffer, divided into two portions and each applied to a 30-ml 5-80% linear Percoll gradient containing 0.33 M sorbitol, 50 mM tricine-KOH, 1 m M MgC12,2 mM EDTA, pH 7.8. After centrifugation for 10 min at 1800.g in the cold (4 ~ C) the fraction of intact plastids was drawn from the gradient with a syringe and mixed with a five- to sixfold excess of solution containing 0.33 M sorbitol and 50 m M tricine-KOH, pH 7.8. The plastids were sedimented by centrifugation for 5 min at 1800.g and were subsequently lysed by resuspending them in 20 mM 2amino-2(hydroxymethyl)-l,3-propanediol (Tris) HC1, 1 5 m M Mg acetate, 30 mM KC1, pH 8.5. The suspension was centrifuged for 10 min at 27000.g (4 ~ C). The membrane sediment was separated from the clear supernatant, stored at - 8 0 ~ and subsequently used for the R N A preparation of membranebound polysomes. The supernatant was centrifuged for 2 h at 340000.g and at 4 ~ C and the resulting pellet of stroma polysomes was stored under liquid nitrogen until R N A extraction. The R N A from stroma and membrane-bound polysomes was extracted as described by Kreuz et al. (1986).

RNA dot blotting. Serial dilutions of R N A were denatured and applied to nitrocellulose membranes as described previously (Batschauer et al. 1986).

W. Laing et al. : Control of P700 chlorophyll-a protein in barley

271

Plasmid clones. Recombinant plasmids containing restriction fragments of plastid DNA, which code for 16S rRNA, the 32000 Mr herbicide-bindingprotein and the P700 chlorophytl-a apoprotein of photosystem-I reaction center, were used as described earlier (Kreuz et al. 1986).

Results

During illumination of dark-grown barley seedlings a rapid accumulation of the P700 chlorophylla-binding protein of photosystem I (PSI) occurs (Vierling and Alberte 1983; Kreuz et al. 1986). This change in protein concentration, however, is not accompanied by significant changes in the concentration of specific mRNA, indicating that the light-dependent accumulation of the PSI chlorophyll-binding protein in barley is regulated posttranscriptionally (Klein and Mullet 1986, 1987; Kreuz et al. 1986). Even in plastids of dark-grown barley plants the m R N A encoding the P700 apoprotein is part of a polysomal fraction as shown by the data in Fig. 1. Polysomes of plastids of dark-grown and 2-h-illuminated barley seedlings were separated into soluble and membrane-bound polysomes, and in both polysomal fractions the relative content o f m R N A s encoding the 32000-Mr herbicide-binding protein of PSII and the P700 apoproteins of PSI were determined by dot-blot hybridization using 32p-labelled gene-specific DNA-fragments as hybridization probes. As a control an identical blot was hybridized to a 16SrRNA-specific plastid-DNA fragment (Fig. 1). In all four RNA samples the same amounts of plastidspecific 16S rRNA were present, indicating that similar quantities of polysomal material had been applied to the nitrocellulose sheets. While substantial amounts of m R N A encoding the 32000-Mr herbicide-binding polypeptide were present in both polysomal fractions, the m R N A encoding the P700 apoprotein was almost exclusively confined to the fraction of membrane-bound polysomes. The relative concentration of the m R N A encoding the P700 apoprotein within the fraction of membranebound polysomes did not appear to be altered during an exposure of etiolated barley seedlings to continuous white light for up to 12 h (Fig. 1). The uptake of a given m R N A into the polysomes might be regarded as indicative for the active translation of this mRNA. Thus, it was important to see if the P700 apoprotein had been synthesized in dark-grown seedlings in the absence of concomittant chlorophyll synthesis. We tested this possibility by measuring the synthesis of the P700 apoprotein in dark-grown and illuminated barley seedlings. [35S]Methionine was fed to cut leaves

Fig. 1. Relative amounts of mRNA encoding the P700 chlorophyll-a protein, the 32000-Mr herbicide-binding protein and of the 16S rRNA in membrane-bound (M) and soluble (S) plastid polysomes of dark-grown (/9) and 6-h-illuminated (L) barley seedlings. RNA levels of polysomal fractions were determined by dot-blot hybridization using radioactively labelled gene-specific probes (Batschauer et al. 1986). For measurements of the 16S rRNA, only 5% of the amount of RNA used in the other experiments was dotted onto nitrocellulose

for brief periods of time and the radioactively labelled apoprotein was immunoprecipitated, separated electrophoretically on an SDS 10-15% polyacrylamide gradient gel and visualized by autoradiography. In addition to wild-type barley plants, the chlorophyll-deficient mutant xantha-181 was also used. In this mutant the biosynthetic pathway of chlorophyll is blocked after Mg-protoporphyrin (Fig. 2; Henningsen and Stummann 1982; v. Wettstein et al. 1971). In the mutant the relative concentration of transcripts coding for the P700 apoprotein was the same as in the wild-type seedlings and was not affected by light (Fig. 2). Synthesis of the P700 apoprotein was detectable only after wild-type plants had been exposed to continuous white light for at least 1 h (Fig. 2). In the xantha mutant kept under identical conditions no labelled apoprotein could be found, even if the length of illumination was extended further (Fig. 2). This result indicates that synthesis of the P700 apoprotein requires concomittant chlorophyll synthesis and does not occur in the dark. However, we cannot rule out that our experimental approach was not sensitive enough to detect low levels of freshly synthesized P700 apoprotein which may be rapidly degraded in the absence of chlorophyll. Thus, a different approach was chosen to increase the sensitivity of the assay for the biosynthesis of the P700 apoprorein. Plastids were isolated from etiolated barley seedlings kept in the dark or exposed for 20 min to continuous white light prior to plastid isolation. Both fractions of isolated plastids were incubated

272

W. Laing et al. : Control of P700 chlorophyll-a protein in barley

A Alo-----Proto~Ng Prolo~ Mg Proto MNIE~ PChlid~Chhd~ChIo--Ch{ b

t

t

xan -flO xan-f26 xon-q37

xon-[ 35 xon-181

t olb-f 17 Norflurazo~

t chlorir)a-f 2

xQn-~l.5 olb- e 16

Fig. 2A-C. Light-dependent changes in the amounts of mRNA and of the freshly synthesized P700 chlorophyll-a protein in wild-type and the xantha-181 mutant of barley. B RNA levels in the dark-grown (D) and illuminated (L) plants were determined by dot-blot hybridization as described previously (Batschauer et al. 1986; Kreuz et al. 1986). C The synthesis of the apoprotein was determined by feeding [3SS]methionine for 4 h to dark-grown leaves. During incubation, leaves were kept in the dark (0) or exposed for the last 60 (60) or 120 (120) rain to continuous white light. The labelled apoprotein was immunoprecipitated from total leaf extracts, separated electrophoretically and visualized by autoradiography. A The upper part of the figure illustrates the step of chlorophyll biosynthesis which is blocked in the xantha-181 mutant

in the presence of ATP and a mixture of unlabelled amino acids and [35S]methionine at 25~ for 30 min. Samples were kept either in the dark or exposed to continuous white light during incubation. Protein synthesis was stopped by boiling the samples in the presence of 1% SDS for 2 min. The solubilized polypeptides were separated electrophoretically on an SDS 10-15% polyacrylamide gradient gel. The 35S-labelled polypeptides were detected by autoradiography. An antiserum raised against the purified P700 apoprotein of PSI was used to immunoprecipitate the corresponding antigenic polypeptide from a mixture of total solubilized plastid proteins. The pattern of polypeptides synthesized in the four different plastid samples looked similar. Most of the labelled polypeptides were detected in all the plastid fractions from etiolated barley seedlings kept in the dark or treated with white light for 20 min pior to plastid isolation (Fig. 3). The absence or presence of light during incubation had no impact on the labelling of these polypeptides. Only a few of the major labelled polypeptides were clearly affected by light. A polypeptide of molecular weight 32000 was heavily labelled in the two plastid fractions isolated from preilluminated seedlings and in the plastid fraction of dark-grown seedlings, which had been illuminated during incubation. In isolated etioplasts incubated in the dark, this polypeptide was only weakly labelled (Fig. 3). Another major polypeptide whose synthesis was affected by light could

be immunoprecipitated by the antiserum against the P700 apoprotein. In contrast to the 32000-Mr polypeptide, this protein was not labelled in isolated plastids from dark-grown seedlings, even if the sample was exposed to white light during incubation (Fig. 3). It should be noted that under these conditions protochlorophyllide had been photoreduced to chlorophyllide which had been transformed subsequently to chlorophyll a. In plastids isolated from seedlings which had been preilluminated for 20 min, the P700 apoprotein was synthesized both in the light as well as in the dark, indicating that the translation of the m R N A in these preconditioned plastids no longer depends on the presence of a light signal. The additional labelled band detectable after electrophoretic separation of the solubilized immunoprecipitates was consistently present also in the control, and thus appeared to be unrelated to the P700 apoprotein. It migrated on the gel in the molecular-weight range of the heavy chain of immunoglobulin G and most likely was the consequence of traces of free [35S]methionine attached to this abundant protein and carried over from the immunoprecipitation step. In subsequent work the effect of shortening the white light pretreatment of dark-grown seedlings on the synthesis of P700 apoprotein in plastids isolated from these plants was analyzed. When the length of the light pretreatment of dark-grown seedlings was reduced to 1 min, labelled P700 apoprotein could still be detected following the incubation of plastids

W. Laing et al. : Control of P700 chlorophyll-a protein in barley

273

Fig. 3A, B. Synthesis of the P700 chlorophyll-a-binding protein by plastids isolated from dark-grown and illuminated barley seedlings. A Plastids isolated from etiolated seedlings kept in the dark or illuminated for 20 rain prior to plastid isolation were incubated in the dark (D) or in the light (L) in the presence of unlabelled amino acids and [35S]methionine for 30 rain at 25 ~ C. Radioactively labelled plastid membrane proteins were solubilized, separated electrophoretically on an SDS-containing polyacrylamide gel and detected by autoradiography. Arrow 1 marks the position of the P700 chlorophyll-a protein, arrow 2 marks the position of the 32000-M~ polypeptide. B Immunoprecipitation of the radioactively labelled P700 apoprotein with a monospecific antiserum from the four plastid fractions described under A. Control: Immunoprecipitation with a preimmune serum from the plastid fraction isolated from preilluminated seedlings and incubated in the light

Fig. 4. Light-dependent, but phytochrome-independent synthesis of the P700 chlorophyll-a protein in isolated plastids of barely seedlings. Plastids were isolated from seedlings kept in the dark (D) or treated for I min with red light (R) or for 1 rain with red light followed by 5 min of far-red light (R/FR) prior to plastid isolation. Equal aliquots of each of the isolated plastid fractions were incubated either in the dark (D) or in the light (L) for 30 min as described in Fig. 3. The radioactively labelled P700 chlorophyll-a protein was immunoprecipitated from each plastid sample, solubilized, separated electrophoretically on an SDS polyacrylamide gel and detected by autoradiography

isolated from these preilluminated plants (data not shown). Even though the sensitivity of the assay for measuring the synthesis of P700 apoprotein had been increased substantially, no P700-apoprorein synthesis could be found in plastids isolated from dark-grown seedlings which had not been preilluminated. Since the synthesis of the P700 apoprotein in isolated plastids clearly depended on the preilluruination of etiolated seedlings, we tried to characterize the photoreceptor which was involved in mediating the light signal. Phytochrome had been implicated as an important photoreceptor for the light-dependent control of plastid gone expression (Link 1982; Thompson et al. 1983; Rodermel and Bogorad 1985; Zhu et al. 1985). The possible involvement of phytochrome in the light-dependent induction of P700-apoprotein synthesis in barley was tested in the following experiment. Darkgrown seedlings were exposed either to a J-min

red-light pulse alone or a l-min red-light pulse followed by 5 min of far-red light. At the end of irradiation, plastids were isolated from the two plant samples. In addition to the plastids from preilluminated seedlings, plastids from dark-grown seedlings were also used as a control. All three plastid fractions were incubated in the presence of [3SS]methionine either in the dark or in the light as described above. At the end of incubation the amount of radioactively labelled P700 apoprotein was determined by immunoprecipitation. While in the dark sample no radioactively labelled P700 apoprotein could be detected, in the plastids isolated from seedlings pretreated with red or red/farred light, equal amounts of the labelled P700 apoprotein could be immunoprecipitated. Again, in the absence or presence of light during plastid incubation the extent of P700 apoprotein synthesis did not show any detectable difference (Fig. 4). Even thought the far red light did not reverse

274

w. Laing et al. : Control of P700 chlorophyll-aprotein in barley

the red-light effect on the synthesis of the P700 apoprotein in isolated plastids the possible importance of phytochrome for this process was not completely ruled out by our experiment. After receiving a reverting far-red pulse it may take the seedlings extra time to come to the far-red level of gene expression. Thus, a delay was introduced between ending the far-red treatment and isolating the plastids to allow the full consequence of the far-red treatment to occur (M6singer et al. 1987). During a second experiment, seedlings treated with either a red-light pulse alone or a combination of red light and far-red light were transferred back to darkness before plastids were isolated and the synthesis of the P700 apoprotein was measured. When the length of the dark period between irradiation of seedlings and the preparation of plastids was increased up to 3 h there was still no difference between the extent of P700 apoprotein labelling in plastids isolated from seedlings preirradiated with red or red/far-red light (data not shown). These results indicate that phytochrome is not involved in the photocontrol of P700-apoprotein synthesis in barley.

(Vierling and Alberte 1983 ; Klein and Mullet 1986; Kreuz et al. 1986). In our present work we had initially used in-vivo labelling techniques to monitor the synthesis of the apoprotein. In wild-type plants labelling of the protein with [3SS]methionine could be detected only after the seedlings had been exposed to continuous light. In the chlorophylldeficient barley mutant xantha-1 sl, which contains the same relative concentration of m R N A as the wild-type plants, the incorporation of [3SS]methionine into the apoprotein was not observed either in the dark or in the light. This result could indicate that the synthesis of the P700 chlorophyll-a protein requires concomittant chlorophyll synthesis and does not occur in the dark. However, we could not rule out the possibility that the experimental approach was not sensitive enough to detect low levels of freshly synthesized P700 apoprotein which might be rapidly degraded in the absence of light. Thus, as a second more sensitive approach we used isolated intact plastids to monitor the translation of the P700-apoprotein-specific mRNA. Numerous studies have demonstrated that in isolated plastids mRNAs are faithfully translated into proteins and reflect the pattern of proteins synthesized in plastids of intact plants (e.g. Ellis 1981). In isolated etioplasts of barley a large number of proteins are synthesized; however, the apoprotein of the P700 chlorophyll-a-binding protein could not be detected among these freshly synthesized polypeptides even though the sensitivity of the assay was much higher than in the preceeding in-vivo labelling experiment. The high sensitivity of our detection assay is demonstrated by the fact that a brief exposure of dark-grown barley seedlings to 1 min of light is sufficient to induce the appearance of a strongly labelled P700 apoprotein. Since we used a monospeciflc antiserum for immunoprecipitation the labelled P700 apoprotein could be identified unequivocally. Even after the autoradiogram had been overexposed there was no detectable trace of the radioactively labelled apoprotein within the immunoprecipitate from etioplasts of dark-grown plants. This result indicates quite strongly that the large amount of m R N A for the P700 apoprotein which is present in dark-grown seedlings is translated only after these plants have been exposed to light. Thus, the light-dependent control of the accumulation of the P700 chlorophyll-a protein appears to be exerted at the level of m R N A translation. At the moment little is known about how light controls the translation of a specific m R N A which has already been taken up into the fraction of membrane-bound polysomes prior to the beginning of illumination.

Discussion

Our present work on the light-dependent accumulation of the P700 chlorophyll-a protein of photosystem I in barley produced two major results. First, the translation of m R N A encoding this protein was detectable only after the beginning of illumination, even though similar high levels of this m R N A are present in dark-grown seedlings and in illuminated plants. Second, the light-dependent translation of this m R N A is not under the control of phytochrome. In previous work we found high levels of m R N A encoding the P700 chlorophyll-a protein in the polysomal fraction of etioplasts before the beginning of illumination (Kreuz et al. 1986). In our present study we extended these observations by showing that in barley this m R N A is almost exclusively confined to the fraction of membranebound polysomes. Similar results have been reported for other proteins in several species of higher plants (Herrin et al. 1981 ; Minami and Watanabe 1984; Bhaya and Jagendorf 1985; Margulies et al. 1987). The uptake of this m R N A into the m e m b r a n e - b o u n d polysome fraction in etioplasts might indicate that the active translation of this m R N A occurs in the absence of light. However, various attempts to detect the freshly synthesized apoprotein of the P700 chlorophyll-a protein in dark-grown seedlings have been unsuccessful

W. Laing et al. : Control of P700 chlorophylt-a protein in barley

275

The light-dependent control of m R N A translation operates only if intact seedlings have been illuminated. When etioplasts from dark-grown seedlings were illuminated after their isolation the translation of the P700 chlorophyll-a protein did not proceed. This failure of m R N A translation does not seem to be the consequence of an artifactual damage of plastids which might have occurred during their preparation, and which could have affected the plastid's capability to respond to a light stimulus. The synthesis of other plastid proteins, for instance a prominent 32000-Mr polypeptide, was induced after etioplasts isolated from dark-grown seedlings had been illuminated during the incubation. In previous studies of the light-dependent control of chloroplast formation in higher plants, phytochrome has been implicated as a photoreceptor which might have a direct effect on plastid development (Link 1982; Thompson et al. 1983; Rodermel and Bogorad 1985; Zhu et al. 1985). So far, phytochrome has been shown to control plastids indirectly by regulating the light-dependent transcription of several nuclear genes encoding plastid-specific proteins (Gallagher and Ellis 1982; Silverthorne and Tobin 1984; Berry-Lowe and Meagher 1985; M6singer etal. 1985). A direct phytochrome-dependent transcriptional control of plastid gene expression, as it has been proposed by some authors (Link 1982; Thompson et al. 1983; Rodermel and Bogorad 1985; Zhu et al. 1985), has not been confirmed (Fromm etal. 1985; Berry et al. 1986; Klein and Mullet 1986, 1987; Kreuz et al. 1986; Deng and Gruissem 1987). Indeed, more recent work has demonstrated that the expression of most plastid-specific genes seems not to be regulated at the transcriptional level but rather at some posttranscriptional steps (Deng and Gruissem 1987; Deng et al. 1987; Mullet and Klein 1987). In the present work we demonstrated that the light-dependent accumulation of the P700 chlorophyll-a protein is not under the control of phytochrome. This light effect is mediated most likely by the phototransformation of protochlorophyllide to chlorophyllide: in addition to phytochrome, protochlorophyllide is the only known photoreceptor of etiolated plants which absorbs light in the red light range (e.g. Shropshire and Mohr 1983). As shown in the present work, red light is able to elicit the translation of the m R N A for the P700 apoprotein in etiolated barley seedlings. The xantha_l s 1 mutant, in which the biosynthesis of chlorophyllide is blocked prior to the formation of protochlorophyllide (v. Wettstein et al. 1971; Henn-

ingsen and Stummann 1982), does not accumulate the P700 apoprotein, even though the m R N A for this protein is present in similar high levels as in the wild-type plants. Furthermore, earlier work had shown a close correlation between the extent of protochlorophyllide reduction and the amount of freshly synthesized P700 chlorophyll-a protein (Vierling and Alberte 1983; Klein and Mullet 1986). All these results indicate strongly that the photoreduction of protochlorophyllide is involved in the light-dependent control of the accumulation of the P700 chlorophyll-a protein. However, the differences in the effects of light on the translation of the m R N A in isolated etioplasts and intact etiolated seedlings indicate that the light control might be more complex. Upon illumination of isolated etioplasts, protochlorophyllide is photoreduced to chlorophyll(ide). Nevertheless, synthesis of the P700 apoprotein was not detected under these conditions and occurred ony if light was given to the intact plant prior to plastid isolation. These results could indicate that the light-dependent control of P700-apoprotein accumulation does not depend only on plastid-localized factors but may require additional light-dependent signals which are derived from the surrounding cytoplasm, and thus are available only in the intact plant but not in isolated organelles. Even though evidence for the importance of a plastid-derived factor for the control of nuclear gene expression has been presented recently by several authors (Bradbeer et al. 1979; Mayfield and Taylor 1984; Batschauer et al. 1986; Oelmfiller and Mohr 1986) a cytoplasmic factor controlling inversely the light-dependent expression of plastid genes has not yet been found. Other artifactual reasons for the failure of light to induce the translation of the P700 apoprotein m R N A in isolated etioplasts are not ruled out. We are grateful to Dr. D. von Wettstein (Carlsberg Laboratory, Copenhagen, Denmark) for his generous gift of barley mutant seeds and to Dr. J. Feierabend (Universit/it Frankfurt, F R G ) for providing us with an antiserum against P700 chlorophyll-a protein of rye. One of us (W.L.) is indebted to the Alexander v. Humboldt-Foundation for the award of a fellowship. Last but not least we are grateful to Waltraud Harmsen for typing the manuscript and to Ruth Schulz for preparing the photographs. This work has been supported by the Deutsche Forschungsgemeinschaft.

References Apel, K. (1979) Phytochrome-induced appearance of m R N A activity for the apoprotein of the light-harvesting chlorophyl1 a/b protein of barley (Hordeum vulgare). Eur. J. Biochem. 97, 183 188 Batschauer, A., M6singer, E., Kreuz, K., D6rr, I., Apel, K. (1986) The implication of a plastid-derived factor in the

276

W. Laing et al. : Control of P700 chlorophyll-a protein in barley

transcriptional control of nuclear genes encoding the lightharvesting chlorophyll a/b protein. Eur. J. Biochem. 154, 625-634 Berry, J.O., Nikolau, B.J., Carr, J.P., Klessig, D.F. (1986) Translational regulation of light-induced ribulose 1,5-bisphosphate carboxylase gene expression in Amaranth. Mol. Cell. Biol. 6, 2347-2353 Berry-Lowe, S.L., Meagher, R.B. (1985) Transcriptional regulation of a gene encoding the small subunit of ribulose-l,5bisphosphate carboxylase in soybean tissue is linked to the phytochrome response. Mol. Cell. Biol. 5, 191~1917 Bhaya, D., Jagendorf, A.T. (1985) Synthesis of the ~ and fl subunits of coupling factor I by polysomes from pea chloroplasts. Arch. Biochem. Biophys. 237, 217-223 Bogorad, L. (1975) Evolution of organelles and eukaryotic genomes. Science 188, 891-898 Bradbeer, J.W., Atkinson, Y.E., B6rner, T., Hagemann, R. (1979) Cytoplasmic synthesis of plastid polypeptides may be controlled by plastid-synthesized RNA. Nature 279, 816817 Deng, X.-W., Gruissem, W. (1987) Control of plastid gene expression during development: The limited role of transcriptional regulation. Cell 49, 379-387 Deng, X.-W., Stern, D.B., Tonkyn, J.C., Gruissem, W. (1987) Plastic run-on transcription. Application to determine the transcriptional regulation of spinach plastid genes. J. Biol. Chem. 262, 9641-9648 Ellis, R.J. (1981) Chloroplast proteins: synthesis, transport and assembly. Annu. Rev. Plant Physiol. 32, 111 137 Fromm, H., Devic, M., Fluhr, R., Edelman, M. (1985) Control of psb A gene expression: in mature Spirodela chloroplasts light regulation of 32-Kd protein synthesis is independent of transcript level. EMBO J. 4, 291 295 Gallagher, T.F., Ellis, R.J. (1982) Light-stimulated transcription of genes for two chloroplast polypeptides in isolated pea leaf nuclei. EMBO J. 1, 1493-1498 Harpster, M., Apel, K. (1985) The light-dependent regulation of gene expression during plastid development in higher plants. Physiol. Plant. 64, 142152 Henningsen, K.W., Stummann, B.M. (1982) Use of mutants in the study of chloroplast biogenesis. In: Encyclopedia of plant physiology, N.S., vol. 14B: Nucleic acids and proteins in plants II, pp. 597 644, Parthier, B., Boulter, D., eds. Springer, Berlin Heidelberg New York Herrin, D., Michaels, A., Hickey, E. (1981) Synthesis of chloroplast membrane polypeptide on thylakoid-bound ribosomes during the cell cycle of Chlamydomonas reinhardtii 137 +. Biochim. Biophys. Acta 644, 136-145 Inamine, G., Nash, B., Weissbach, H., Brot, N. (1985) Light regulation of the synthesis of the large subunit of ribulose1,5-bisphosphate carboxylase in peas: evidence for translational control. Proc. Nat. Acad. Sci. USA 82, 5690 5694 Klein, R.R., Mullet, J.E. (1986) Regulation of chloroplast-encoded chlorophyll-binding protein translation during higher plant chloroplast biogenesis. J. Biol. Chem. 261, 1113811145 Klein, R.R., Mullet, J.E. (1987) Control of gene expression during higher plant chloroplast biogenesis. Protein synthesis and transcript levels of psbA, psaA-psaB, and rbcL in darkgrown and illuminated barley seedlings. J. Biol. Chem. 262, 4341-4348 Kreuz, K., Dehesh, K., Apel, K. (1986) The light-dependent accumulation of the P700 chlorophyll a protein of the pho-

tosystem I reaction center in barley. Evidence for translational control. Eur. J. Biochem. 159, 459-467 Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680-685 Link, G. (1982) Phytochrome control of plastid mRNA in mustard (Sinapis alba L.). Planta 154, 81-86 Margulies, M.M., Tiffany, H.L., Hattori, T. (1987) Photosystern I reaction center polypeptides of spinach are synthesized on thylakoid-bound ribosomes. Arch. Biochem. Biophys. 254, 454-461 Mayfield, S.P., Taylor, W.C. (1984) Carotenoid-deficient maize seedlings fail to accumulate light-harvesting chlorophyll a/b binding protein (LHCP) mRNA. Eur. J. Biochem. 144, 7984 Minami, E.-I., Watanabe, A. (1984) Thylakoid membranes: The translational site of chloroplast DNA-regulated thylakoid polypeptides. Arch. Biochem. Biophys. 235, 56~570 M6singer, E., Batschauer, A., Sch/ifer, E., Apel, K. (1985) Phytochrome-control of in vitro transcription of specific genes in isolated nuclei from barley (Hordeum vulgate). Eur. J. Biochem. 147, 137-142 M6singer, E., Batschauer, A., Vierstra, R., Apel, K., Sch~ifer, E. (1987) Comparison of the effects of exogenous native phytochrome and in-vivo irradiation on in-vitro transcription in isolated nuclei from barley (Hordeum vulgare). Planta 170, 505-514 Mullet, J.E., Klein, R.R. (1987) Transcription and RNA stability are important determinants of higher plant chloroplast RNA levels. EMBO J. 6, 1571-1579 Oelmfiller, R., Mohr, H. (1986) Photooxidative destruction of chloroplasts and its consequences for expression of nuclear genes. Planta 167, 106-113 Rodermel, S.R., Bogorad, L. (1985) Maize plastid photogenes: Mapping and photoregulation of transcript levels during light-induced development. J. Cell Biol. 100, 463476 Shropshire, W., Jr, Mohr, H., eds. (1983) Encyclopedia of plant physiol., N.S., vol. 16A: Photomorphogenesis. Springer, Berlin Heidelberg New York Tokyo Silverthorne, J., Tobin, E. (1984) Demonstration of transcriptional regulation of specific genes by phytochrome action. Proc. Natl. Acad. Sci. USA 81, 1112-1116 Thompson, W.F., Everett, M., Polans, N.O., Jorgensen, R.A., Palmer, J.D. (1983) Phytochrome control of RNA levels of developing pea and mungbean leaves. Planta 158, 48750O Tobin, E., Silverthorne, J. (1985) Light regulation of gene expression in higher plants. Annu. Rev. Plant Physiol. 36, 569 593 Vierling, E., Alberte, R.S. (1983) Regulation of synthesis of the photosystem I reaction center. J. Cell Biol. 97, 18061814 von Wettstein, D., Henningsen, K.W., Boynton, J.E., Kannangara, G.C., Nielsen, O.F. (1971)The genetic control of chloroplast development in barley. In: Autonomy and biogenesis of mitochondria and chloroplasts, pp. 205-223, Boardman, N.K., Linnane, A.W., Smillie, R.M., eds. North-Holland Publishing Company, Amsterdam Zhu, Y.S., Kung, S.D., Bogorad, L. (1985) Phytochrome control of levels of mRNA complementary to plastid and nuclear genes of maize. Plant Physiol. 79, 371-376 Received 18 March; accepted 26 May 1988

Light-dependent, but phytochrome-independent, translational control of the accumulation of the P700 chlorophyll-a protein of photosystem I in barley (Hordeum vulgare L.).

This work reports on the regulation of synthesis of the P700 chlorophyll-a apoprotein of photosystem I in barley. The mRNA for the P700 apoprotein is ...
1MB Sizes 0 Downloads 0 Views