Control of protein formation in chloroplasts

Plant Science, 68 (1990) 131 - 149 Elsevier Scientific Publishers Ireland Ltd.

131

Review Article C O N T R O L OF P R O T E I N F O R M A T I O N IN C H L O R O P L A S T S

ARMINIO BOSCHETTI, ERIC BREIDENBACH and REGULA BL.~TTLER Institut fftr Biochemie, Universitdt Berr~ Freiestr. 3, CH-3012 Bern (Switzerland)

{Received September 14th, 1989) {Revisionreceived January 22nd, 1990) {Accepted January 22nd, 1990)

Introduction

Chloroplasts of higher plants and green algae contain their own genetic system, comprising numerous identical copies of circular chloroplast D N A and the corresponding apparatus for the expression of this genetic information [1-5]. The complete nucleotide sequences of chloroplast DNA of Nicotiana tabacum (tobacco) [6] and Marchantia polymorpha (liverwort) [7] and of Oryza sativa (rice) [8] revealed a total number of about 130 genes encoded in the chloroplast. (Table I lists the genes mentioned in this review). Besides the genes for the chloroplast rRNAs and for 30 tRNAs, about 40 identified and 11 putative genes for chloroplast proteins as well as about 40 ORFs of at least 70 codons have been found. In these and many other species some of the genes clustered

Abbreviations: CF1, coupling factor of chloroplast ATPsynthase; CFo, intrinsic part of chloroplast ATP-synthase; cyt, cytochrome; Dl-protein, herbicide-binding, rapidly labelled, 32 kDa-protein of PS II, gene product of psbA; EFTu, translational elongation factor Tu (of chloroplasts); LHCII, light-harvesting complex II; LHCP, light-harvesting chlorophyll a/b proteins; LS, large subunit of Rubisco; ORF, open reading frame; P700/chl a-protein, P700-chlorophyU a membrane protein(s) of PS I, 65-- 70 kDa apoproteins of PS I, gene product of psaA; PS I, PS II, photosystem I and II, respectively; QB-protein, same as Dl-protein; Rubisco, ribulose-l,5-bisphosphate carboxylase/oxygenase; SS, small subunit of Rubisco.

around the rRNA genes are present in two copies in inverted repeats. Plastid genes can be divided arbitrarily into three categories. (1) Genes coding for proteins of the photosynthetic apparatus. Since photosynthesis is obviously the main activity of chloroplasts, synthesis and regulation of the proteins involved in this process have been primarily studied. (2) Genes coding for products participating in the genetic system of the chloroplast, such as rRNA, tRNA, ribosomal proteins, and enzymes and factors for translation and transcription. The regulation of the formation of such gene products could play a key role in the overall regulation of chloroplast biosynthesis. (3) Putative genes, for which no translation products, but in some cases transcripts [11,12] have been identified so far. By sequence homology to known genes of other organisms, these putative genes are thought to be involved in other biochemical processes, e.g. in some sort of oxidative electron transport [13] (in tobacco: ndhA-F [6,11]; in liverwort: ndhl-6 [7,14] in maize: ndhD, ndhE [12]), or in membrane transport (in liverwort: mbpX, mbpY [14]). The expression of this third class of chloroplast genes still remains to be elucidated, as are the genes frxA,B,C (liverwort)[14], which are homologous to genes for nitrogenase. Besides the products of chloroplast origin, a considerable number of chloroplast proteins are coded for in the nucleus, synthesized in the

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132 Table I. Chloroplastgenes and their functions. Listed are only those genes mentioned in the text. For a complete list of chloroplast genes see Refs. 8-- 10. Genes

Function

atpA,B,E atpH cab frz mbp ndh petA petB pe tD psaA psaB psaC psbA psbB psbD psbE,psbF psbH rbcL rbcS rpl rps rpoA rpoCl, rpoC2

Genes for a-,/3-and E-subunit of CF1 Gene for the proton translocating subunit of CFo Nuclear genes for LHCP Genes homologousto nitrogenase of cyanobacteria;frxA is homologousto psaC. Genes homologousto membrane transport proteins Genes homologousto mitochondrial NADH-dehydrogenase Gene for cytochromef Gene for cytocbrome be Gene for subnit IV of cytochromef/b-complex Formerly also psaA1 or pslA1; gene for the large subunit A of PSI Formerly also psaA2 or pslA2; gene for the large subunit B of PS I Putative gene in maize for a bacterial ferredoxin-like protein of PS I; homologoustofrxA Gene for the herbicide-binding, rapidly turning over D1- or QB-protein of the reaction centre of PSII Gene for the 46-kDaprotein of PS II reaction centre Gene for D2-protein of PS II reaction centre Genes for apoproteins of cyt bs9 in PS II Gene for 10-kDaphosphoprotein in PS II Gene for LS Nuclear gene for SS Gene for ribosomal protein of large ribosomal subunit Gene for ribosomal protein of small ribosomal subunit Gene homologousto a-subunit of prokaryotic RNA-polymerase Gene homologousto/~-and/3'-subunits of RNA-polymerase Gene cluster for ribosomal RNA Gene for tRNA for amino acid Y (one letter code) Chloroplast gene for EF-Tu in Chlamydomonas

trnY tufA

c y t o p l a s m and imported, while processed, into the chloroplast [15,16]. Most multimeric chloroplast proteins contain polypeptides of both origins in a stoichiometric relationship. A m o n g t h e m are soluble proteins such as Rubisco, extrinsic m e m b r a n e proteins like CF1, and intrinsic m e m b r a n e proteins, e.g. of the p h o t o s y n t h e t i c reaction centres. F o r an efficient synthesis of such complexes d u r i n g cell g r o w t h and life, the nuclear and chloroplast genetic s y s t e m s m u s t c o o p e r a t e to keep the formation of p a r t n e r proteins in step. Such coordinated protein synthesis, even when o c c u r r i n g in different cell c o m p a r t m e n t s , will be said to be 'coupled'. F u r t h e r m o r e , d u r i n g the cell cycle, or differentiation, or as a r e s p o n s e to changes in e n v i r o n m e n t a l or g r o w t h conditions, both nuclear and chloroplast genetic s y s t e m s should

be able to a d a p t protein synthesis and hence chloroplast function to the changing requirem e n t s of the cell. To date, t h e r e is no evidence for a r e g u l a r e x c h a n g e of nucleic acids b e t w e e n chloroplast and cytoplasm, nor for an e x p o r t of chloroplast-made proteins; the r e g u l a t o r y signals m u s t be mediated either by low molecular weight metabolites or by import of proteins. This regulation is far from being fully u n d e r s t o o d (reviewed in Ref. 17).

Observations on the Coupling of Cytoplasmic and Chloroplast Protein Synthesis Studies on the coupling of cytoplasmic and chloroplast protein synthesis gave controversial results. In long t e r m experiments, if either the cytoplasmic synthesis of SS

133 or the chloroplast synthesis of LS was inhibited by antibiotics, the other subunit was also not made anymore. The two systems were said to be coupled [18--21]. In other experiments, the existence of a small pool of either of the subunits has been supposed [22,23], since some uncoupling could be achieved in Soya by antibiotics [23] and in rice deficient in chloroplast ribosomes [24]. Furthermore, in isolated chloroplasts as well as in intact cells of Chlamydomonas treated with cycloheximide to block cytoplasmic protein synthesis, LS was still synthesized normally for 2 h [25,26]. In a plastome mutant of Oenothera hookerii, which contains an LS-mRNA of normal length, but synthesizes a truncated LS of 30 kDa, the SS is expressed. However, the pool of processed SS is low, suggesting that the amount of LS controls posttranscriptionally the synthesis or stability of SS [27].

Experimental Approaches to Study Chloroplast Gene Expression An exact definition of what is meant by 'regulation of gene expression' is a prerequisite for meaningful experiments. Gene expression in plastids is regulated: (a) when the amount of a mature, functionally active protein in the plastid is modulated; (b) in response to an alteration of the environmental conditions (light, temperature, humidity) or cell internal factors (differentiation, circadian rhythm); (c) the modulation has to be considered with respect to a reference component containing the genetic information, such as plastid DNA (for transcriptional regulation) or mRNA (for translational regulation). The last point requires the determination of the reference component during the whole course of the experiment, which, especially in earlier work, was sometimes neglected. Plastid gene expression can be regulated at different levels during the flow of genetic information {Fig. 1): (a) Transcriptional regulation occurs, when the rate of transcription varies with respect to the DNA content. Experimentally, synthesis of specific RNA and

the copy number of the plastid gene have to be measured and compared. (b) To look for regulation occurring during RNA maturation or by differential stability of specific RNA, the experimental approach should include sequence comparison of processed, mature mRNA with pre-mRNA and gene structure, or pulse-chase labeling of RNA and quantitative hybridization with specific gene probes. (c) Translational regulation occurs during translation of mature mRNA into the (primary) protein. This regulation is studied by in organello or in vitro protein synthesis using chloroplast mRNA. (d) Posttranslational regulation may operate during the formation of mature proteins from the primary proteins by chemical modifications or by affecting protein assembly to form the functional protein complex. By variations in the rate of protein degradation, the total amount of active protein may be changed. (e) Regulation during alteration of plastid types is rarely studied. Whether specific genes are expressed in non-photosynthetic plastids, is not yet clear. In practice, most studies on plastid gene expression follow one of three strategies: (i) Chloroplast morphogenesis has been studied most often during light-induced greening of etiolated seedlings or cells [15,28--34]. (ii) Less dependent on the complexity of light effects is the comparison of the composition and genetic activity between plastids of the same plant, but of different age or state of differentiation; e.g. between chloroplasts in different segments of a leaf of monocotyledons, or between leaf chloroplasts, amyloplasts of roots, and chromoplasts of fruits of the same plant. (iii) During the cell cycle of synchronised, unicellular green algae the development of chloroplasts from young to adult cells can be studied independently of unphysiological manipulations. Even very early studies with synchronised cultures of Chlamydomonas showed that the incorporation of newly synthesized proteins into thylakoid membranes is activated not only by dark-tolight transfer, but also by inborn signals at work in the early Gl-phase [35]. Until now, attempts to mimic regulatory mechanisms for

134 Flow o f genetic information chloroplast-D NA

I

where regulation m a y occur

P r o c e s s e s

A. Transcriptional regulation

pre-mRNA B. Regulation during R N A - m a t u r a t i o n : -- cutting o f RNA -- splicing (trans, cis) or by differential stability o f RNA -- mRNA-binding proteins mature mRNA -- polycistronic -- m o n o c i s t r o n i c C. Translational regulation -- membrane-binding o f p o l y s o m e s initiation-, elongation-, t e r m i n a t i o n - f a c t o r s -- t R N A - e f f e c t s - -

primary protein D. Posttranslational regulation -- LS-binding protein -- protein assembly -- chemical modifications rate o f degradation -

functional protein or protein c o m p l e x E. Regulation during plastid type alterations differentiation stress -- senescence - -

- -

disintegration

Fig. 1.

Putative key mechanisms for regulation of gene expression in chloroplasts.

protein synthesis in cell-free in vitro systems have not been very successful. However, in vitro transcription systems have been used to study control of mRNA-synthesis [ 3 6 - 38].

A. Transcriptional regulation The levles of nuclear gene transcripts for chloroplast proteins, such as SS and especially LHCP, are light-regulated through the action of phytochrome [29,39--41] or by still unknown factors in a circadian manner [42--45]. The amount of mRNA for these nuclear encoded proteins show a pronounced light/dark-dependent fluctuation, with almost non-detectable

levels in the night. At least in case of the mRNA of LHCP, this fluctuation is due to circadian changes in the rate of mRNA synthesis [44,45]. In contrast to light-induction of nuclear genes, an appreciable amount of transcripts of chloroplast genes, such as rbcL, psaA, psaB, psbA, psbE, atpA, atpB or tufA are present also in dark-grown and etiolated plants [44-52]. Nevertheless, photoregulation has been described also in chloroplast genes. The psbA-gene has been claimed to be a photogene (definition according to [55]), since its transcript level increases appreciably during light-

135 induced differentiation of etioplasts to chloroplasts (maize [53--55], pea and mung bean [56], barley [57,58]). Some 'minor photogenes' with only 2-3-fold higher mRNA-levels in the light as compared to the dark have also been described [12,36,55,59,60]. To study the mechanism of chloroplast gene transcription by in vitro experiments, both particulate [61- 64] and soluble [65-73] chloro~ plast enzyme preparations have been used. The different kinds of products synthesized by the two systems led to the suggestion that either two RNA polymerases exist in chloroplasts, or eventually only one operating with different ofactors. At least one polymerase is of the prokaryotic type. Partly purified preparations of the soluble enzyme consist of 7--14 major polypeptides, some of which show immunological erossreactivity with the f3-, f3'- and a-subunits and the o70-factor of E. coli polymerase [74--77]. Moreover, mRNA and DNA sequences homologous to genes for subunits of E. coil polymerase have been found in chloroplasts [6,78--80]. However, subunits of chloroplast polymerase have been also claimed to be synthesized in the nucleo-cytoplasmic compartment [81]. The prokaryotic nature of chloroplast transcription is confirmed by homologies to the prokaryotic - 3 5 and - 1 0 boxes found in several promoters of mRNA, tRNA and rRNAgenes (reviewed by Hanley-Bowden and Chua [82]). Furthermore, chloroplast o-factor stimulates DNA-binding and transcription specificity of E. coil core polymerase [76]. Therefore, when the amount of RNA polymerase is limiting, transcription of individual genes may be governed by the promoter strength, by o-factors, by the spacing between adjacent promoters [83], or, especially in chloroplast genes cloned in circular plasmids, by the promoter topolgy depending on topoisomerases [84--87]. A prokaryotic regulation mechanism may control also the transcription of the spinach chloroplast rDNA-operon via premature termination by translation-mediated attenuation [88] as described for the E. coli trp operon [89]. The accumulation of chloroplast mRNA dur-

ing greening of etiolated cells is rather small as compared to the much higher increase in the rate of protein synthesis. Also the diurnal fluctuations of the level of plastid mRNA in tomato leaves [43] and of plastid rRNA in synchronously cultured Scenedesmus are rather small [90] as compared to variations of nuclear encoded RNA. This constancy in the accumulation of chloroplast RNA could be an indication for a constitutive transcription in chloroplasts. Indeed, since in chloroplasts several essential components of the genetic apparatus are made in the plastid, a minimal level of transcriptional and translational activity in different kinds of plastids is to be expected also in the dark. Otherwise the differentiation of proplastids and the redifferentiation of amyloplasts into chloroplasts and of epidermal or phloem cells into chloroplast containing tissue cells would not be possible [10]. In different organs of plants, grown either in the dark or in the light, the relative rates of chloroplast RNAsynthesis were the same in run-off transcription experiments [91]. The relative activities correlated well with the chloroplast DNA copy number in the different organs [92]. However, when the amounts of mRNA for various gene products (rRNA, psaA, psbA, atpB/E, rbcL) were compared in roots, cotyledons, and hypocotyls of dark grown spinach seedlings, as well as in green hydroponically cultured young leaves and roots of spinach, they were found to differ considerably. This discrepancy between transcriptional activity and mRNAlevel points to posttranscriptional mechanisms influencing the half-life of mRNA.

B. Regulation during mRNA-maturation and by differential RNA-s tability Chloroplast genes are generally organized into multigene transcriptional units and are often cotranscribed. Processing of the primary transcripts results in a set of overlapping transcripts consisting of small monocistronic and larger polycistronic RNAs of different sizes, the latter being more abundant in plants exposed only for 24 h to white light than in plants grown in continuous light (pea [93]).

136 These complex maturation events are the first steps where posttranscriptional regulation would be possible. They involve mechanisms, such as processing at the 5'- and 3'-ends, endonucleolysis and cis-splicing as well as transsplicing of exons transcribed from plastid DNA onto separate primary transcripts. Examples of cotranscribed genes are rps2atpI~tpH~tpF-atpA; rpoA-rpoB-rpoC 1-rpoC2 (spinach, pea [46,94--96]); trnE-trnY-trnD; atpB-atpE; rp/12-cluster; 3'-rpsl2-rps7; rrn (tobacco [9]); psbB-psbH-petB-petD; ndhD-psaC (maize [12,97]; spinach [46,98]); psaA-psaB-rpsl4 (tobacco [99,100]; spinach [46]); psbE-psbF (barley [101,102]). Other genes such as rbcL and psbA are transcribed monocistronically. Some of the chloroplast genes contain introns, which often resemble class II introns of mitochondria. Their sequence and localization, however, can vary between different species, e.g., in the photogene psbA an intron is present in Chlamydomonas, but not in higher plants. Class III introns have also been found and a class I intron is present in tRNALe"-UAA in cyanelles, liverwort, and some higher plants [4,103]. An interesting observation [104] brings up the idea for the concerted light-induction of intron-containing chloroplast mRNA. The primary transcript of the trnK-gene, coding for tRNA Lys, rises transiently and very rapidly upon illumination of etiolated mustard seedlings. It contains an intron with an ORF similar to the gene for the mitochondrial maturase, which is thought to be responsible for correct splicing [105]. Such ORFs have been identified also in an intron of tobacco chloroplast DNA and in a ribosomal intron of Chlamydomonas [106]. It has been speculated that an early expressed putative maturase could play a key role in the production of mature chloroplast transcripts during light-induced chloroplast differentiation. However, a general mechanism of regulation can not be deduced from these findings. In plastids, processing of the polycistronic into monocistronic mRNA is not a prerequisite for their translatability. Proteins encoded by psbB, petB and petD are all translated as well from processed monocistronic as from polycis-

tronic mRNA [107]. However, in a plastid mutant of Oenothera hookeri the E- and f3subunits of CF1 are expressed as a fusion protein, although the heterologous in vitro translation of the atpB-atpE cotranscript results in normal polypeptides. Here, a defect in a translational signal or posttranslational event is responsible for the mutant phenotype [108]. Indeed, recently a translational coupling of these two genes has been demonstrated by the finding that atpE translation depends on successful translation of atpB [109]. Short inverted repeats, which could form stem and loop structures in the RNA, have been found immediately preceding transcription stop sites [9]. However, within the polycistronic transcription unit the 3'-ends of protein genes are flanked also by such inverted repeats, which obviously do not act as transcription terminators. The function of these palindromic structures could be to confer differential stability to chloroplast mRNA or to act as signals for cleavage of polycistronic into monocistronic mRNA [10,38]. Until now two genes have been found in chloroplasts, which are trans-spliced. In Marchantia polymorpha [110], in Nico tiana tabaccum [111113] and in Oryza sativa [8] the rpsl2 gene is split into three exons. While exons 2 and 3 are cotranscribed and joined by cis-splicing, exon 1 is located on a separate transcription unit. In Chlamydomonas, but not in other plants, the psaA-gene is split into three exons, widely scattered around the circular chloroplast DNA [114]. Exon 2 is transcribed in the direction opposite to the others. The analysis of mutants with impaired maturation of the psaA-transcript showed that trans splicings are affected by a number of genes, which may be regulated also; e.g. mutants with impaired joining of exon 1 to 2 can be allied to the chloroplast DNA and to 5 different nuclear complementation groups, demonstrating the complexity of the transsplicing mechanism [115,116].

C. Translational regulation C.1. Evidence for translational regulation. Since some polycistronic chloroplast mRNAs,

137

e.g., the psbB-psbH-petB-petD transcript, contain information for proteins of different membrane complexes which are not coordinately synthesized in the dark, transcription and translation are obviously uncoupled and control of transcription does not seem to play the dominant role in membrane biosynthesis [46]. Other evidence for chloroplast gene expression being regulated to a great extent at the translational or posttranslational level, is based mainly on the comparison of the rates of protein synthesis with the amount of mRNA present during light-induced greening of etiolated plants or during the cell cycle of green algae [37,47,49,60,117--119]. While, as mentioned previously, the transcript levels of nuclear genes fluctuate considerably upon transfer from dark to light and may not even be detectable in the dark, the transcript levels of chloroplast genes are more stationary. The chloroplast genes seem to be transcribed constitutively also in the dark and to be stimulated only slightly by light. In etiolated pea, the level of mRNA for LS increases 3 times upon illumination. A simultaneous 3-fold increase of DNA-copy number may account for this variation. However, the amount of LS-protein, which in the dark is barley detectable, rises about 50fold during illumination [117]. Moreover, during the light-induced development of dark grown Euglena gracilis, protein synthesis in isolated chloroplasts increases about 100-fold, whereas specific mRNA increases no more than 3 times [120]. Similarly, in Spirodela oligorhizza, when light-grown fronds are returned to darkness, the mRNA-level remains constant for the D1protein and declines slightly for LS, while the rates of synthesis for both proteins drop to 15 and about 40%, respectively, of the rates in the light [118]. Also in amaranth seedlings, darkness stops LS-synthesis while the mRNA is conserved [49]. In synchronized cultures of the unicellular green alga Chlamydomonas reinhardii the rate of chloroplast protein synthesis increases dramatically to a maximal level with the beginning of the light period, then decreases in the second half of the light period to about 10%.

The amounts of mRNA, however, vary to a much smaller degree [121--123]. Presumably, according to the physiological or developmental stage of the cell some mRNA for chloroplast proteins can be conserved in a silent form and reactivated by some unknown signal. The first indications for the involvement of specific nuclear gene products in the translation of individual chloroplast mRNA and/or in protein processing come from the examination of nuclear mutants of Chlamydomonas and barley [124,125]. These mutants are deficient in PS II-activity due to the absence or reduction of one or more PS II-core proteins (genes psbA-D). In contrast, the levels of the mRNAs in the mutant plastids are equal to or greater than in wild-type. Pulse-labeling during protein synthesis show that different nuclear mutations affect the synthesis of individual proteins, e.g., of the Dl-protein (psbA) [125], the D2-protein {psbD) [126] or the P6-protein (psbC) [127]. The molecular mechanism of action of these nuclear factors is unknown. However, studies with a chloroplast suppressor mutant with altered 5'-untranslated region of psbC mRNA indicate that mRNA-binding proteins may activate translation of the P6-protein [127]. C.2. Membrane binding of polysomes. Upon the first report on thylakoid-bound polysomes 20 years ago [128], subsequent experiments indicated that in synchronized cultures of Chlamydomonas reinhardii [129,130] and in greening peas [131] light induces a rather rapid binding of ribosomes and polysomes to thylakoids, whereas in the dark the amount of membrane-bound ribosomes and polysomes is low. Furthermore, chloroplast proteins are synthesized predominantly during the lightperiod of the cell cycle in synchronized Chlamydomonas [18,122,132] or Euglena [123,133]. Whether the thylakoid-bound polysomes specifically synthesize membrane proteins, is still a matter of debate. Therefore, studies on the partition of ribosomes, polysomes and especially of mRNA between stroma and thylakoid membranes become important with regard to the regulation of protein synthesis. Determination of mRNA extracted from thylakoids and

138 stroma fractions by quantitative hybridization with cloned chloroplast genes or by heterologous in vitro translation demonstrated that thylakoid-bound polysomes contain not only mRNA for hydrophobic membrane proteins (psbA, psbD, psaA-psaB), but also a significant amount of mRNA for soluble stroma proteins (rbcL, tufA in Chlamydomonas) and for extrinsic membrane proteins (petA, petB) [25,122,134 -138]. Therefore, thylakoid-bound polysomes seem to be involved also in translation of soluble proteins. Indeed, chloroplast polysomes, although their ribosomes are of the prokaryotic type, are bound to membranes not only by the growing apolar peptide chains, as in bacteria, but also by some other, mainly polar forces acting probably between ribosomes and some as yet unidentified membrane proteins [140--142]. However, most of the investigated mRNAs are found also in the stroma fraction, with the exception of the mRNA for the P700/ chl a-protein (psaA-psaB-gene), which in barley seems to be almost exclusively confined to membrane-bound polysomes [60,139]. The topic of thylakoid-bound polysomes has been recently reviewed [143].

C.3. Proteins translated by in vitro elongation. The conventional in vivo or in organello translation experiments have not contributed much to our understanding of the role of thylakoids in translation of soluble proteins in particular, nor in regulation of translation in general. Studies using a homologous in vitro chloroplast translation system, capable of translation initiation and in which the translational activity of thylakoid-bound and free stromal polysomes could be compared would yield more information in this area. Unfortunately, in spite of a report in the literature [144], no such system exists. Therefore, systems performing only the elongation step have been used, such as homologous run-off translation (by supplementing the polysomecontaining thylakoids or stromal fraction with radiolabeled amino acids and 'energy'), or heterologous run-off translation (where in addition a postribosomal supernatant (S100) from E. coli was added).

Using heterologous run-off translations, the a- and/~-subunits of CF1 were found to be produced by both thylakoid-bound and free polysomes from pea chloroplasts [145]. In a similar experiment [146] LS was synthesized from thylakoid-bound polysomes. In contrast, in a heterologous run-off translation system, thylakoidbound ribosomes from spinach produced only membrane proteins (P700/chl a-protein, a- and ~-subunit of CF1, Dl-protein), whereas free stromal polysomes made only soluble proteins (LS and other stromal proteins), although both preparations contained mRNA for soluble and membrane proteins [135]. Since the product pattern of such an in vitro translation depends on the method used for the preparation of the chloroplast fractions [147] and on the composition of the reaction mixture [148,149], some of these contradictory results could probably be eliminated by optimizing the reaction, not for maximal incorporation of amino acids, but for faithful translation. When the stromal fraction of spinach was subsequently analyzed by sucrose density gradient centrifugation, most of the rbcL-transcript was found in the polysome fraction, whereas the psbA-transcript was not associated with ribosomes, but with some other, not identified protein [136]. This mRNAprotein complex could represent a silent mRNA-pool in the stroma, since this mRNAsequence was not modified as shown by correct translation in a retriculocyte lysate in vitro system. An interesting regulation mechanism has been found in homologous run-off translations using lysed etioplasts of barley. The chloroplast-synthesized chlorophyll a-binding proteins were elongated in the dark, only when chlorophyllide a was simultaneously transformed by phytylation into chlorophyll a [150]. C.4. Translational arrest. Pulse-labeling of isolated pea chloroplasts with [35S]methionine resulted in several low molecular weight products crossreacting with anti-LS immunesera. After a chase period, they were converted into full length LS, suggesting that ribosomes were pausing at several points during transla-

139

tion of mRNA [151]. Some experiments, showing a rapid response of protein synthesis to light-dark variations, point to a rather direct effect of light on translation. In amaranth seedlings, a f t e r a light-to-dark transition, the translation of the LS is immediately arrested, but the mRNA remains bound to the polysomes and conserved for several hours [49]. In barley seedlings, although similar levels of mRNA for the P700/chl a-protein (psaA/B-genes) are present in etioplasts and chloroplasts, no synthesis of these proteins could be detected in etioplasts. However, a very short exposure to light induced protein synthesis [60]. Also the two chlorophyll-binding core proteins of PSII (psbB, psbC) and the Dl-protein (psbA) are not synthesized in etioplasts of barley, in spite of the presence of their mRNAs. Fifteen minutes after illumination, protein synthesis was significant, but not accompanied by an increase of mRNA [47,48]. Homologous run-off translation of thylakoid-bound polysomes from barley etioplasts resulted only in low level synthesis of the P700/chl a-proteins. However, when etioplast membranes were disrupted with detergents, in vitro synthesis of these proteins increased to levels observed with polysomes from illuminated plants [139]. These findings suggest that protein synthesis in chloroplasts can be arrested at the level of polypeptide chain elongation, whereby the mRNA on membranebound polysomes is protected from nuclease attack.

D. Pos ttranslational regulation The amount of a peptide accumlated in the chloroplast depends not only on the rate of its synthesis, but also on its degradation, which in turn may be faster when the protein is prevented from being incorporated into a functional protein complex. Indeed, in isolated chloroplasts of pea 20-- 30% of the radioactively labeled, newly synthesized peptides have been shown to be degraded within half an hour in the light or in the presence of ATP [152]. It has been suggested that an ATPdependent protease is responsible for the hydrolysis of mature, but not integrated plas-

tid-synthesized polypeptides, while an ATPindependent protease in the stroma may remove incomplete or incorrectly translated proteins [153]. Indeed, in a mutant of Chlamydomonas reinhardii, which is unable to synthesize mRNA for the core-protein D2 of PSII due to a defect in the psbD-gene, the 32kDa protein (Dl-protein from psbA-gene) is also not made, although the mRNA for this protein is present at a normal level. Two other proteins of PSII, i.e. the chlorophyll a-binding core proteins, are found in thylakoids of the mutant, but are not accumulated [154]. Several chloroplast-made proteins are not directly incorporated into the functional enzyme. The Dl-protein, which is synthesized as a 2 kDa larger precursor peptide, is incorporated into the PSII-core complex via several steps, during which it may be sensitive to proteolytic degradation. In vivo, membrane association and cleavage of the presursor occur in the unstacked stroma lamellae. The peptide is then translocated to the stacked grana lamellae, where it becomes integrated into the reaction centre. During this traffic the mature protein becomes transiently acylated by palmitic acid [155]. However, isolated chloroplast membranes of Chlamydomonas reinhardii were also found to process and incorporate in vitro synthesized precursor protein [156]. Furthermore, the LS is not directly assembled with SS into the (LSsSSs)-holoenzyme of Rubisco. Most of the newly formed LS is found at first in a 29 S (or 7 S) complex together with 60 and 61 kDa binding proteins [22]. This complex may function not only as an intermediate for non-autonomous assembly of Rubisco, but also as a pool of protease-protected LS in the chloroplast. Antibodies against the binding protein prevents LS from being incorporated into the holoenzyme [157]. Similar binding proteins, called 'chaperonins' [158] have been described for a number of assembly processes.

E. Regulation during plastid alterations As discussed until now, regulation of plastid gene expression has been studied mainly during the development of photosynthetically

140 competent chloroplasts in response to a darkto-light transition or a day-night regime (reviewed in [159]). However, other plastid types, characterized by their different morphology, pigmentation and metabolic activities, can be formed by differentiation. The small proplastids, present in embryonic and meristematic cells of higher plants, may differentiate into chloroplasts of photosynthetic tissues (in some cases, after a dark period, via etioplasts). Proplastids and chloroplasts can further differentiate into amyloplasts in roots and tubers, or into chromoplasts in flowers and some fruits. This differentiation, when not too much advanced, is reversible. Like other morphogenetic events, it is governed by a plant-inherent program and accompanied by alterations in the expression of the plastid and nuclear genomes [160-165]. Due to difficulties in their isolation, the chemical composition and the biochemical and genetic functions of the non-photosynthetic plastids have been poorly studied. E.1. Immature plastids. The few studies on gene expression in immature plastids [166170] show that the patterns of the proteins synthesized in the plastids change according to the developmental stage [171]. In a (pro)plastid preparation from dark-grown Euglena, protein synthesis is greatly stimulated by ATP and Mg 2÷. Some of the newly formed proteins are different from those synthesized by isolated chloroplasts. They have not been identified, but do not seem to be degradation or premature termination products, since pulse-chase experiments indicate similar turnover rates of the newly formed proteins in both plastid types [172]. E.2. Amyloplasts. In amyloplasts of spinach roots the relative transcription rates of 10 representative genes are comparable to those in chloroplasts. However, the accumulation of their mRNAs is different in roots and other tissues, indicating that transcription is constitutive, but transcript accumulation is governed by a posttranscriptional control mechanism. Especially mRNAs for photosynthetic proteins are significantly lacking in amyloplast poly-

somes, as compared to mRNAs for ribosomal proteins [92]. In cell cultures of the white, heterotrophic cell line of sycamore {Acer pseudoplataneus) the amyloplast DNA is heavily methylated, in contrast to the plastid DNA from an autotrophic revertant cell line. Genes, for which methylation has been detected (e.g. rbcL, atpA/B/E, psaA, rps4) are not transcribed in vitro by E. coli polymerase, whereas the nonmethylated genes are transcribed [173]. Whether DNA-methylation is a general mechanism for repressing plastid genes remains to be established. E.3. Mesophyll and bundle sheath cells. Morphologically and biochemically different chloroplasts are found in mesophyll and bundle sheath cells of C4-plants. The former are able to perform photosynthetic electron transport and C02-fixation into pyruvate, the latter contain the Calvin-cycle enzymes. The cell-specific differentiation of these chloroplast types in maize is accompanied with the accumulation of poly(A)-containing, nuclear encoded mRNAs for phosphoenolpyruvate carboxylase and pyruvate dikinase in mesophyll cells, and for SS, malic enzyme and proteins of the oxygen evolving complex in bundle sheath cells [161]. In addition, the mRNA for the chloroplast encoded LS (rbcL-gene) is missing in mesophyll and present in bundle sheath cells, while the mRNA for the Dl-protein {psbA) is distributed vice versa between both cell types [162,163]. In etiolated maize seedlings the rbcL-mRNA is present also in mesophyll cells, but the level drops rapidly during light-induced maturation of the plastids. It is not yet clear whether this is due to a reduced transcription rate or enhanced mRNA turnover. E.4. Chromoplasts. During differentiation of chloroplasts in ripening tomatoes and fruits of bell pepper (Capsicum annuum) chlorophyll and thylakoids together with their proteins disappear gradually, while fibrous and dilated membrane structures and new carotenoids, mainly xanthophyUs, appear [174]. In contrast, the plastid DNA is conserved in chromoplasts [174 --177]. During chromoplast formation the transcriptional activity in the plastid drops continu-

141

ously, but differentially for various genes [164]. In ripening tomatoes the mRNA-level for photosynthesis-specific proteins (Dl-protein, LS, SS, P700/chl a-protein (psaA) and LHCP) diminish to low levels, while several nuclear encoded transcripts (aldolase, tubulin, extensin) increase [165]. Other chromoplast-specific enzymatic activities, especially for carotenoid and lipid metabolism, seem to be nuclear encoded also. In Capsicum no evidence for new chromoplast-specific transcripts was found by hybridization with labelled cDNA probes [179] and also ribosomes and rRNA are no longer detectable in fully differentiated chromoplasts [178]. E.5. Senescence. Some genetic regulation may be involved also in senescence, which often is accelerated experimentally by bringing detached leaves into the dark. Senescence consists of a complex temporal sequence of events, and is characterised by a decline in chlorophyll and protein content, photosynthetic capability, especially linear electron transport, and by reduction of carbohydrate content, leading finally to disintegration of the chloroplast and to cell death [180]. During the early steps of leaf senescence, specific alterations have been observed in the population of cytoplasmic mRNA and proteins [181,182] as well as in chloroplast protein synthesis [183-185]. However, since senescence leads to irreversible damage of the cell, it is difficult to distinguish between mechanisms of regulation and degradation. E.6. Stress. Regulation of gene expression can be induced in plants also by subjecting the organisms to stress situations, such as heat shock, cold acclimation, high light intensity, osmotic stress, desiccation, iron deprivation etc. Under stress situation the protein pattern often changes dramatically. Heat-shock induces the rapid production of a set of nuclearencoded, evolutionarily conserved heat-shock proteins, some of which are found in the chloroplast (reviewed by Vierling et al. [186]). They are synthesized as precursors and processed by removal of a 5-6.5-kDa peptide during import into chloroplasts [187]. In contrast, upon heatshock, most of the mRNA formed during normal growth are no longer expressed at the

same rate, but remain present in the cell for some time [188]. The primary cause of this translational control is not clear. However, in a heat-shocked reticulocyte system the (cytoplasmic) translational activity could be restored by addition of eukaryotic initiation factor 2 [189]. Cold acclimation is also accompanied among other changes, by the synthesis of new proteins [190] which are induced at the transcriptional level [191 -- 194]. When Fe is added to chlorotic pea seedlings, nuclear as well as chloroplast transcripts (cab, rbcS, rbcL) increase several fold after a lag phase of about 20 h, while chloroplast rRNA starts to be synthesized after 40 h. The mRNA for Dl-protein (psbA), however, shows little change during greening [195]. These few reports on regulation of gene expression in stress situations show that mainly nuclear encoded genes have been studied. The effect of stress on chloroplast gene expression remains to be determined. Conclusions

Since 1909, when Baur [196] and Correns [197] discovered extrachromosomal inheritance, geneticists have been aware of the existence of at least two genetic systems in plant cells and have addressed themselves to the question of how they are regulated reciprocally (for review see Refs. 160, 198-200}. However, it was only in the last 15 years that gene expression could be dissected at the molecular level into different steps. Treatments of cells and plants have been elaborated to manipulate experimentally gene expression in chloroplasts, mainly by dark-to-light transitions by which photosynthetically competent chloroplasts are induced. Furthermore, gene expression in chloroplasts and in plastids from non-photosynthetic tissues have been compared. From such studies, the concept emerged that, in contrast to the transcriptionally regulated nuclear genes, chloroplast genes are controlled predominantly at the posttranscriptional and/or translational level. The mechanism of this regulation

142 r e m a i n s o b s c u r e . S i n c e t r a n s c r i p t i o n of p l a s t i d DNA seems to be mainly constitutive, quest i o n s a b o u t t u r n o v e r a n d c o n s e r v a t i o n of chloroplast mRNA have to be resolved. With r e s p e c t t o t h e t r a n s l a t i o n a l r e g u l a t i o n of chlor o p l a s t p r o t e i n s y n t h e s i s , t h e r o l e of p o l y s o m e b i n d i n g t o t h y l a k o i d s a n d of m o d u l a t i o n of t h e a f f i n i t y of r i b o s o m e s t o c h l o r o p l a s t m R N A s t i l l remain to be defined. Further unresolved probl e m s c o n c e r n t h e r o l e of p l a s t i d g e n e s in t h e f o r m a t i o n a n d r e d i f f e r e n t i a t i o n of n o n - p h o t o synthetic plastids, such as amylo- and chromo° p l a s t s , t h e r e g u l a t i o n of g e n e e x p r e s s i o n through developmental and circadian factors, a n d t h e r e p r e s s i o n of p l a s t i d g e n e s d u r i n g s t r e s s s i t u a t i o n s . E v e r y e f f o r t u n d e r t a k e n in a n s w e r i n g o n e of t h e s e q u e s t i o n s will a l s o b e a s t e p t o w a r d s t h e u n d e r s t a n d i n g of t h e r e c i p r o cal r e l a t i o n s h i p a n d c o o p e r a t i o n b e t w e e n t h e t w o g e n e t i c s y s t e m s p r e s e n t in t h e nucleoc y t o p l a s m a n d in t h e p l a s t i d s of p l a n t cells. Acknowledgements

We thank Dr. Leticia Mendiola-Morgent h a l e r for r e a d i n g a n d c o m m e n t i n g on t h e manuscript. The work from the authors laboratory has been supported by the Swiss National Foundation for Scientific Research.

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