Photosynthesis Research 19:129-152 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands Minireview

Protein synthesis by isolated chloroplasts A. G N A N A M , C.C. SUBBAIAH & R. M A N N A R M A N N A N Department of Plant Sciences, School of Biological Sciences, Madurai Kamaraj University, Madurai 625021, lndia Received 6 October 1987; accepted 10 March 1988

Key words: chloroplast biogenesis, gene expression, ribosomes, transcription, translation Abstract. Isolated chloroplasts show substantial rates of protein synthesis when illuminated. This 'in organello' protein synthesis system has been advantageously utilised to elucidate the coding capacity of chloroplast and the regulation of chloroplast genes. The system is also being used recently to transcribe and translate homologous and heterologous templates. In this mini-review,, we attempt to critically evaluate the available literature and present the current and the prospective lines of research.

Introduction

It is now common knowledge that chloroplasts, the organelles that carry out photosynthesis, are genetically autonomous, though to a limited extent. They possess their own DNA, the master molecule of genetic information, and the machinery necessary for its replication, transcription, as well as the translation of the transcribed messages into functional proteins. At the time when the presence of D N A in chloroplasts was being ascertained, a distinct type of ribosomes was also discovered to be present in the plastid (Lyttleton 1962) in the form of polysomes indicating that they were functional (Clark et al. 1964, Chen and Wildman 1966). With the demonstration of an independent protein synthetic machinery, and later the presence of genetic material itself in the chloroplasts, attention was turned to an investigation of the products of this organellar genome, i.e., which of the proteins are coded for and synthesized within the chloroplasts. Until the development of the modern tools of recombinant D N A technology, this problem had been approached in three ways: (i) identification of the proteins synthesized by isolated organelles; (ii) dissecting the in vivo protein synthetic processes with specific translational inhibitors; and (iii)analysis of cytoplasmic (extra-nuclear) gene mutations - mainly in algae like C h l a m y d o m o n a s - which are amenable to analysis by the methods of classical genetics, the only technology available at that time. Initial attempts using isolated chloroplasts to analyze the products of en-

130 dogenous templates were unrewarding, particularly because of the very low rates of incorporation of radioactive precursors; and also were fraught with problems of bacterial contamination. So the other two approaches, namely the use of compartment-specific protein synthetic inhibitors and analysis of photosynthetically deficient non-mendelian mutants, were favored for a long time. However, studies using inhibitors have not always been foolproof since these antibiotics are also known to affect many metabolic processes other than protein synthesis. Also, since photosynthetic mutants are generally pleiotropic, it was not easy to point out precisely the direct effect of the mutation in terms of the product of the defective gene, even if the locus could be assigned on the plastid genome map. Hence, unequivocal proof that a particular protein is synthesized in the chloroplast and is thus coded for by the plastid genome, should be possible by simply demonstrating its synthesis in the isolated organelle. Although the first credible report that isolated spinach chloroplasts could incorporate labeled amino acids into TCA precipitable proteins appeared as early as 1965 (Spencer 1965), the rates then obtained were too low to identify any authentic product (Kirk 1970). The ability to obtain chloroplasts that were highly active in protein synthesis required a prior, slow development of procedures to isolate intact functional plastids, and Blair and Ellis (1973) were the first to exploit such methods. These authors showed that isolated pea chloroplasts, capable of high rates of CO 2 fixation, could also synthesize discrete protein products using light as an energy source. The large subunit of RubPcase was identified as a definitive component of these products for the first time in their studies. Since then, the process of chloroplast protein synthesis in terms of its mechanism as well as the products of this system have attracted great interest from many workers. So far, at least nineteen different plant species, including four algae - Euglena, Chlamydomonas, Acetabularia and Olisthodiscus besides a woody perennial namely cashew (Anacardium occidentale L.) have been shown to yield chloroplasts capable of substantial rates of protein synthesis (Table 1) although there is an inherent variation among different plant species in the rates of protein synthesis in organello. Several reviews have appeared updating the progress in this area from time to time (Ellis 1976, 1977, 1981; Ellis and Barraclough 1978, Ellis and Hartley 1982, Ellis et al. 1978, Margulies 1986). In the present article, we discuss recent aspects of this topic, besides compiling any new information in the conventional aspects. The areas focused on here are: i) identification of products of chloroplast protein synthesis; ii) attempts to use the system to understand the regulation of chloroplast gene expression during the plastid development;

131

iii) division of labor between the soluble and thylakoid bound ribosomes in the plastid; iv) assembly and turnover of newly synthesized proteins in isolated chloroplasts; and v) attempts to use chloroplasts or their lysate as an in vitro translation or coupled transcription-translation assay system for analyzing exogenous R N A or D N A templates.

Optimization of the system There have been several modifications to the initially-developed technique (Blair and Ellis 1973, also see Ellis 1977), to suit a particular plant material as well as to maximize the efficiency of the system in terms of the rate, duration and close approximation to the in vivo translation process. Though crude chloroplast preparations were enough to carry out protein synthesis in plants like pea and spinach (Blair and Ellis 1973, Hartley et al. 1975), purification of intact chloroplasts over percoll gradients was necessary to obtain substantial rates of protein synthesis in species such as cashew Table 1. Plant species yielding chloroplasts active in in organello protein synthesis Plant species

Reference

Acetabularia cliftoni Anacardium occidentale Chlamydomonas sp. Cucumis sativus

Green (1980, 1982) Subbaiah and Gnanam (1984, unpublished) Leu et al. (1984a) Walden and Leaver (1982) Uma Bai et al. (1984) Daniell et al. (1986) Vasconeelos (1976) Ortiz et al. (1980) Klein and Mullet (1986) Archer et al. (1987) Reith and Cattolico (1985) Colijn et al. (1982) Drumm and Margulies (1970) Blair and Ellis (1973) Uma Bai et al. (1984) Geetha and Gnaman (1980a, 1980b) Mannan et al. (1987) Bottomley et al. (1976) Obokata (1984) Hachtel (1982) Krishnan et al. (1987) Grebanier et al. (1979)

Euglena gracilis Hordium vulgare Nicotiana tabacum Olisthodiscus luteus Petunia hybrida Phaseolus vulgaris Pisum sativum Ricinus communis Sorghum vulgare Spinacea oleracea Triticum aestivum Vicia faba Vigna sinensis Zea mays

132 (Subbaiah and Gnanam 1984) and Euglena (Vasconcelos 1976). Even in species like spinach and sorghum, which readily yield chloroplasts active in protein synthesis, even from crude preparations (Hartley et al. 1975, Geetha and Gnanam 1980a), a purification step over percoll solutions further improved the incorporation of labeled amino acids (Morgenthaler and Mendiola-Morgenthaler 1976). In Euglena, chloroplasts obtained by gentle lysis of sphaeroplasts and purified over percoll gradients were superior (at least by 100-fold) in protein synthetic acitivity to the organelles prepared by direct homogenization of the whole cells (Ortiz et al. 1980). Work done on pea chloroplasts in the laboratory of Jagendorf requires a detailed examination (Fish and Jagendorf 1982, Fish et al. 1983, Nivison and Jagendorf 1984). Fish and Jagendorf (1982) achieved very high rates of protein synthesis (almost 200 nmoles of [3H-leucine incorporation/mg chlorophyll) by systematically working out each of the various components and conditions starting from handling of the plants to chloroplast preparation. Use of destarched young plants, rapid chilling of leaves from pre-illuminated plants after harvest, homogenization by polytron - using high tissue to buffer ratio purification of chloroplasts over linear percoll gradients, a reaction medium containing sorbitol (350 mM), K ÷ (30 mM), MgCl 2 (1 mM), MnC12 (1 mM) and EDTA (2 mM) all routinely resulted in at least 60-100 nmoles of [3H]-leucine incorporation per mg chlorophyll (Fish and Jagendorf 1982). They could also prolong the active translation from the usual 20 min cited in previous reports to almost an hour by further modifications, namely: addition of other amino acids with sufficient free Mg 2÷ ; use of lower light intensities; and addition of Pi and ATP (Nivison and Jagendorf 1984). Recent work in our laboratory has shown that addition of such polyamines as putrescine or spermidine further improves the rate of amino acid incorporation by isolated sorghum chloroplasts (Subbaiah et al. 1987). While the above work concerns the quantitative improvement in the translational activity of isolated chloroplasts, work by Mullet et al. (1986) was aimed at simulating the in vivo protein synthetic pattern of these organelles., when isolated. Using almost similar procedures to those developed by Jagendorf and his colleagues, the above authors also obtained high rates of protein synthesis as well as a large number of labeled products resolved by SDS-PAGE and fluorography. However these authors could not confirm the assumption that all the products revealed in the fluorograph represented separate polypeptides. They tested this possibility by a pulsechase experiment and found that many of the lower molecular mass polypeptides were, in reality, incomplete products paused at discrete points of mRNA when chloroplasts were labeled. These could be converted to fulllength authentic polypeptides by a subsequent chase with cold methionine. -

-

133 By introducing this chase step, these authors found that the in vitro translation profile of plastids closely resembled the in vivo labelled pattern.

Characteristics of protein synthesis by isolated chloroplasts Initial characterization of the system was carried out by Ellis and his group using pea and spinach chloroplasts. We refer the reader to earlier reviews for details (Ellis 1977, 1981), while presenting here only the highlights and recent developments in this area. The special feature of protein synthesis by isolated chloroplasts is that it is light-driven, requiring no additional energy source, unlike other wellknown in vitro translation systems. High light intensities were used in early studies; however, Nivison and Jagendorf (1984) showed low light intensities (45 #mol/m 2.s) to be more effective, even if the initial rates of incorporation were low. Gomez-Silva and Schiff (1985) studied the dose response using light varying wavelengths and showed that in Euglena the chloroplast protein synthesis was saturated at 5 W/m 2 whereas photosynthetic CO2 fixation required 15-30W/m 2 for the optimal rate. Protein synthesis was more responsive to blue light but photosynthesis responded better to white or red light. The chloroplast system is also functional in the dark if ATP is supplied, although the rates of such ATP driven protein synthesis were only 50% or less than those of the light driven synthesis (Bottomley et al. 1974, Siddell and Ellis 1975). However, working with sorghum, Geetha and Gnanam (1980a) showed that these rates could be as high as 85% of the light driven incorporation (0.5 to 1.0 nmol of phenylalanine or lysine/mg of chlorophyll/ h). Fish et al. (1983) worked out the details and established the need for M g 2+ addition (since ATP would chelate endogenous Mg 2+) for rapid ATP-driven protein synthesis in the dark. By supplying equimolar amounts of Mg 2+ and ATP these authors obtained even greater rates of [3HI leucine incorporation in the dark than those driven by light. Light-dependent incorporation itself could be enhanced by 45 % at lower intensities (45/lmol/ m2.s) and 18% at high intensities (900#mol/m2.s) by added ATP in the presence of Mg 2+ . Protein synthesis in isolated chloroplasts of a chromophytic alga Olisthodiscus luteus is an exception in that it was light-independent and also non-responsive to ATP supplied externally (Reith and Cattolico 1985). The authors assumed that a storhge product in the chloroplast (most likely mannitol) would supply the necessary ATP. Though similar light-independent protein synthesis was earlier reported in the isolated chloroplasts of

134 Acetabularia (Goffeau and Brachet 1965), some work (Green 1980) showed that chloroplast protein synthesis was strictly light dependent in this species. Reith and Cattolico, in their paper (1985), also referred to an unpublished report of diatom plastids showing light-independent protein synthesis. Except in such isolated cases, light has been shown to be an absolute requirement for chloroplast protein synthesis in the absence of any added energy source. The major light-dependent reaction of chloroplasts, namely CO2 assimilation, requires both ATP and the reducing agent NADPH. However, since protein synthesis needs only ATP, the translational activity of isolated chloroplasts is expected to depend solely on their photochemical capacity to form ATP and should be independent of their potential to generate reducing action. In fact, early work by Ramirez et al. (1968) suggested that cyclic photophosphorylation (which generates only ATP but no N A D P H and is insensitive to 3-(3,4-dichlorophenyl)-l,l-dimethylurea, DCMU) was the main source of energy for light-driven protein synthesis of spinach chloroplasts. However, the later reports showed that protein synthesis by isolated chloroplasts was fully or partially sensitive to D C M U (Blair and Ellis 1973, Colijn et al. 1982, Gomez-Silva and Schiff 1985) indicating the contribution of non-cyclic photophosphorylation to this process, although there is some evidence that D C M U acts indirectly on cyclic photophosphorylation (Arnon and Chain 1975, Hosler and Yocum 1987). We also observed a total inhibition of protein synthesis by D C M U in sorghum chloroplasts (Geetha and Gnanam 1980a) but only a partial effect in castor chloroplasts (Uma Bai et al. 1984). Interestingly, Fish and Jagendorf (1982) reported no effect of D C M U on chloroplast protein synthesis in pea (compare Blair and Ellis 1973) or even a slight stimulation (15%) at a very low concentration of the inhibitor. These contradictory effects might be due to multiple sites of action of D C M U in photosynthetic electron transport. In fact this is reflected by the dual effect of this herbicide on electron transport itself, both as an inhibitor (Izawa 1968) and as a stimulator (Ramanujam et al. 1981). We attempted to resolve this problem (i.e., which of the two - cyclic or noncyclic phosphorylation - contributes to in vitro protein synthesis) by a different approach. We followed the sequence of events during greening of etioplasts isolated from cucumber cotyledons and showed that light-dependent protein synthesis commenced concomitantly with the development of cyclic phosphorylation, well before the appearance of non-cyclic phosphorylation (Uma Bai et al. 1984). However, this did not prove that cyclic phosphorylation is the one which contributes preferentially to protein synthesis when both cyclic and non-cyclic paths are operative in the fully developed chloroplast.

135 There is some evidence that photosynthetic C O 2 fixation and amino acid incorporation into proteins are competing reactions (for ATP) in isolated chloroplasts when these are illuminated; although in some species chloroplasts active in protein synthesis show poor CO2 fixation, e.g. Euglena (Ortiz et al. 1980, Reith and Cattolico 1985). Substrates of Calvin cycle such as 3-PGA, ribose-5-P, NaHCO3 and ~-ketoglutarate plus glutamate showed substantial inhibition of protein synthesis in pea chloroplasts (Fish and Jagendorf 1982). Though bicarbonate was also inhibitory to spinach chloroplasts, 3-PGA and oxaloacetate stimulated protein synthesis (Gnanam et al. 1981). We explained the promotor effect of organic acids through faster regeneration of NADP ÷ needed for the maintenance of electron transport and attendant phosphorylation. DL-glyceraldehyde, an inhibitor of the Calvin cycle, did not promote but inhibited protein synthesis (Fish and Jagendorf 1982). Though the site of action was not defined, Ca ÷ ÷ was also shown to inhibit amino acid incorporation by isolated chloroplasts (Bouthyette and Jagendorf 1981). Isolated proplastids and etioplasts were shown to be proficient in protein synthesis; but with an absolute dependence on added ATP as energy source, since they lacked machinery for light-harvesting and attendant photochemistry (Siddell and Ellis 1975, Dockerty and Merrett 1979, Obokata 1984, Cushman and Price 1986, Krishnan et al. 1987). However, rates of amino acid incorporation by immature plastids were extremely low (Drumm and Margulies 1980, Siddell and Ellis 1975, Dockerty and Merret 1979, Miller and Price 1982, Miller et al. 1983) and in some cases not even detectable, e.g. cucumber etioplasts (Walden and Leaver 1981). Cushman and Price (1986), by taking advantage of the optimization experiments carried out on pea chloroplasts by Jagendorf et al. (see above), have obtained greatly increased rates of protein synthesis from proplastids of Euglena, even though the rates were still below those observed with chloroplasts. In this organism, more differentiated plastids (etioplasts?) showed detectable (though low) rates of light-dependent protein synthesis which greatly increased if plastids were isolated from cells exposed to light for even 1 h (Miller and Price 1982, Miller et al. 1983). On the contrary, plastids capable of light-driven protein synthesis could be obtained from etiolated wheat seedlings only after 3 h of greening (Obokata 1984). Work in our laboratory showed that etioplasts from hormone pretreated cucumber cotyledons were effecient either in ATP- or light-driven protein synthesis (Uma Bai et al. 1984); and the light-dependent protein synthesis of these plastids was linear for 8 h (Daniell et al. 1986).

136 Products of chloroplast protein synthesis Though chloroplasts are made up of approximately 400 proteins - including both structural and functional ones, any contribution to these by the genome itself is very small. Theoretically a D N A molecule, the size of chloroplast genome, can code for only 20-30% of the total polypeptides found in this organelle, the rest of them being contributed by the nuclear genome. In fact, complete nucleotide sequence data of two chloroplast genomes, one from a lower plant, Merchantiapolymorpha, and another from tobacco (Ohyama et al. 1986, Shinozaki et al. 1986) could also predict that the plastid genome can make only about 120-130 products, including stable RNAs. An updated list of polypeptides identified and later confirmed to be the products of authentic chloroplast genes is given in Table 2. So far, 15 membrane polypeptides, 2 soluble products, 18 ribosomal proteins and 3 envelope polypeptides are identified from the products of in organello protein synthesis. We have found that isolated chloroplasts or etioplasts of Vigna sinensis would synthesize a set of high molecular weight proteins following heat shock. These chloroplast-produced heat shock proteins (hsps) were not synthesized in vivo if the heat shock was given to the whole leaf (Krishnan et al. 1987). However, in our recent experiments, we have observed the in vivo synthesis of these polypeptides if the leaves were exposed to a gradual temperature rise (Krishnasamy et al. 1988). We are currently looking at the origin of the message for these chloroplast-made hsps as well as their functional significance. Kloppstech et al. (1986) have also reported that an hsp in Acetabularia is coded for outside the nuclear genome. From their experiments using specific protein synthetic inhibitors, the authors presumed that the above polypeptide was synthesized within the chloroplast. This possibility can be tested by analyzing the products of protein synthesis of heat shocked chloroplasts in vitro in this species, as was done in Vigna by Krishnan et al. (1987).

Division of labor between the soluble and thylakoid-bound ribosomes in the plastid Among the products of chloroplast protein synthesis, there appeared to be a certain specificity regarding their site of synthesis within the organelle. Such a suggestion emerged early in the 1970s from a previous observation from many laboratories that a substantial fraction of chloroplast ribosomes occurred bound to chloroplast membranes, at least a part of which were in

137 Table 2. Identified products of in vitro chloroplast protein synthesis. Polypeptide

Plant species

References

Pea Spinach

Blair and Ellis (1973) Bottomtey et al. (1974) Vasconcelos (1976) Green (1980) Leu et al. (1984)

Soluble products LSU of RuBisCO

Euglena Acetabularia Chlamydomonas Protein synthetic elongation factors T and G Ef-Tu

Spinach

Euglena

Ciferri et al. (1979) Spermulli (1972) Miller et al. (1983)

Thylakoid polypeptides • , fl and e subunits of CF~ Subunit III of CF0 Cytochro .rnef

Cytochrom b557 PT00chlorophyll a apoproteins of PSI Chlorophyll a apoproteins of PS II D~ protein

Pea Spinach Pea Pea Spinach Sorghum

Acetabularia Viciafaba Pea Spinach

Euglena Maize

D2 protein

Chlamydomonas Chlamydomonas

Mendiola-Morganthaler et al. (1976) Nelson et al. (1980) Doherty and Gray (1980) Doherty and Gray (1979) Zielenski and Price (1980) Geetha and Gnanam (1980b) Mannan et al. (1987) Green (1982) Hachtel (1987) Blair and Ellis (1973) Bottomley et al. (1974) Vasconcelos (1976) Grebanier et al. (1978) Steinback et al. (1981) ~eu et al. (1984a) Herrin et al. (1981)*

Envelope membrane polypeptide Pea Spinach

Joy and Ellis (1975) Morganthaler and Mendiola-Morgenthaler (1976)

Chlamydomonas

Schmidt et al. (1983) Dorne et al. (1983) Hachtel (t985)

Ribosomal proteins Spinach

Viciafaba * Thylakoid ribosomes.

the form ofpolysomes (Chen and Wildman 1970, Chua et al. 1973). Furthermore, the dependence of their release from the membranes on puromycin, an elongation inhibitor, indicated the involvement of these bound polysomes in the synthesis of membrane proteins which in turn helped

138 the ribosomes to bind the membranes (Margulies and Michaels 1974). Margulies et al. (1975) tested this possibility and demonstrated in Chlamydomonas that these polysomes were active in protein synthesis in vitro when supplemented with cell extract and the newly synthesized proteins were vectorially discharged into the membrane. The authors also proposed that these membrane bound polysomes could be important in the synthesis of thylakoid polypeptides. Jagendorf and co-workers (Alscher et al. 1978, Alscher-Herman et al. 1979, Yamamoto et al. 1981, Fish and Jagendorf 1982, Hurewitz and Jagendorf 1987) made detailed studies on the binding of ribosomes to thylakoid membrane and concluded that i) the binding was both ionic and by insertion of the nascent polypeptide chains; ii) high salt and puromycin were necessary to release them; iii) anoxia and transfer of plants or organelles to darkness would also release the bound polysomes; iv) binding of ribosomes onto the membrane in light, required a product of 70S ribosomal protein synthesis as well as non-cyclic electron flow; v) the major effect of light on ribosome binding could be due to higher stromal pH; and vi) ribosomes would get redistributed between stroma and thylakoids. They also showed that the isolated membrane-bound polysomes would incorporate amino acids into proteins if supplemented with the S-30 fraction from E. coli and majority of the products were associated with the membranes (Alscher et al. 1978). Using this soluble factor-supplemented system, Michaels and his colleagues tested the products of these bound polysomes in Chlamydomonas and showed that these ribosomes synthesized ~ and fl subunits of coupling factor, and D1 and D2 polypeptides of the PS II reaction center (Herrin and Michaels 1985a, 1985b, Herrin et al. 1981). Other workers also demonstrated that the thylakoid bound polysomes synthesized only membrane polypeptides such as the reaction center polypeptides of PS I (Minami and Watanabe 1984, Margulies et al. 1987) and PS II including cyt b559 (Minami et al. 1986), coupling factor subunits ~ and fl and the 32 kDa herbicide binding protein (Minami and Watanabe 1984) while soluble proteins like the large subunit of RuBisCO were manufactured only by stromal ribosomes (Minami and Watanabe 1984, Leu et al. 1984b). However there was no compartmentalization of messengers for soluble and membrane polypeptides and both the populations of polysomes had similar m R N A members (Minami and Watanabe 1984, Leu et al. 1984b). Hence it was suggested that the thylakoid membranes might play a role in the expression of chloroplast mRNAs leading to a division of labor between the stromal and membrane bound polysomes (Leu et al. 1984b). However,

139 Bhaya and Jagendorf (1984, 1985a, 1985b), working with their optimized translation systems of chloroplast sub-fractions, did not find any such specificity in the synthesis of large subunit (LSU) of RuBisCO and ct and fl subunits of coupling factor CFI, both being synthesized by stromal and thylakoid bound polysomes in pea. But they did observe such a specificity in the synthesis of the highly hydrophobic protein - subunit III of CF0 which was primarily the product of thylakoid ribosomes (Bhaya and Jagendorf 1984b). Hattori and Margulies (1986) also found that LSU of RuBisCO was an important product of thylakoid bound ribosomes in spinach and suggested that such a thylakoid association of LSU synthesis might be significant in view of its hydrophobic nature. The reverse of this argument may hold true with respect to the synthesis of CF1 subunits on stromal ribosomes, since this multisubunit complex (CF1) is hydrophilic and only lossely attached to the membrane. These reports caution us against generalizing the proposal of division of labor between the two populations of chloroplast polysomes, and further studies in this direction are warranted.

Gene expression during chloroplast biogenesis and by inducing agents Many workers have utilized greening etiolated tissues or dark grown algal cells to study gene expression during chloroplast biogenesis, since the process of de-etiolation, though much telescoped in time, is almost similar to the normal chloroplast development (Silverthrone and Ellis 1980). Ellis and his group carried out pioneering studies in this area, utilizing the in organello protein synthesis system they had developed to monitor differential expression of plastid genome during light-induced development of etiolated pea shoots (Siddell and Ellis 1975) and during the normal development of spinach leaves (Silverthrone and Ellis 1980). They observed that, in chloroplasts isolated from different stages of greening, synthesis of LSU declined faster than that of peak D. These observations made in organello reflected the changes in vivo in the level of these two proteins and their transcripts during greening (Silverthrone and Ellis 1980). Walden and Leaver (1981) found that only quantitative changes occur in the rate of incorporation by developing plastids of cucumber cotyledons, without any evidence of differential translation of specific mRNAs. However, both qualitative as well as quantitative changes were observed in the activity of protein synthesis by plastids isolated at different periods of illumination of dark-grown Euglena cells (Miller and Price 1982, Miller et al. 1983, Cushman and Price 1986). Notable among them were i) a gradual increase in the synthesis of LSU and 32 kDa polypeptides;

140 ii) decrease in a 44 kDa polypeptide, possibly elongation factor Ef-Tu; and iii) gradual disappearance of a set of high molecular weight polypeptides synthesized by the proplastids (Miller and Price 1982, Miller et al. 1983). Later, using optimized conditions for in organello protein synthesis, Cushman and Price (1986) observed that undifferentiated proplastids manufactured a large number of polypeptides that were absent in the products of more mature proplastids or chloroplasts. These authors were not certain whether these additional products made by proplastids represented authentic individual polypeptides or differences in the post-translational modifications. Significant changes both in the soluble and membrane associated products of in organello synthesis were also reported during plastid biogenesis of light-grown wheat seedlings, although the exact identity of these products was not given except for LSU of RuBisCO (Obokata 1984). Klein and Mullet (1986, 1987) followed the sequence of events during chloroplast development in barley by assaying the products of chloroplast protein synthesis as well as by estimating the in vivo transcript levels for individual polypeptides. They showed that barley etioplasts, like their counterparts in pea and spinach, could synthesize LSU and ~ and fl subunits of ATPase but not apoproteins of P S I and PS II or the 32 kDa herbicide binding protein, despite the presence of significant transcript levels for these polypeptides even before greening. These polypeptides appeared rapidly once the seedlings were illuminated, thus indicating that regulation occurred at the translation level. However, during later stages of leaf development, synthesis of Chl a-apoproteins and LSU by chloroplasts declined rapidly, but the 32 kDa polypeptide continued to be synthesized - as was reported in pea and spinach by Ellis and his associates. A general observation which also summarizes the changes occurring in the in organello protein synthesis during greening is a decrease in the ratio of incorporation into the soluble fraction as compared to that in the membrane fraction (Obokata 1984, Klein and Mullet 1986, 1987). Such a rapid increase in the synthesis of membrane polypeptides, with a concomitant decrease in soluble products during illumination of etiolated tissue (Klein and Mullet 1987) is consistent with the proposed in vivo phenomenon of light-induced redistribution of plastid ribosomes from stroma onto the membranes, and so also with the proposal that polysomes bound to the membrane primarily synthesize thylakoid polypeptides (Alscher-Herman et al. 1979). Garcia et al. (1983) studied the role of chloroplast protein synthesis in the induction of leaf senescence in barley, since it had been shown earlier that chloramphenicol retarded leaf senescence (Sabater and Rodriguez 1978, Yu and Kao 1981). The authors monitored the changes in the pattern of polypeptides synthesized by isolated chloroplasts during senescence of

141 detached leaf segments. Plastid protein synthetic activity greatly increased 10-20 h after incubation of leaf segments in the dark, though there was a marked loss of rRNA. The rise in the rate of incorporation during dark incubation was more pronounced if kinetin was added to the medium. At the same time, incubation in light decreased the protein synthetic activity of isolated chloroplasts and kinetin could restore it only to a small extent. There were also qualitative differences in the newly synthesized polypeptides. Dark incubation, which promoted senescence, induced the synthesis of a 66 kDa polypeptide. A polypeptide of almost similar molecular weight (62 kDa) was found to increase during the senescence of soybean cotyledons (Bricker and Newman 1980). By contrast, treatments that retarded senescence, such as kinetin application, enhanced the synthesis of an entirely different set of proteins, one of them being the LSU of RuBisCO (Garcia et al. 1983). However it is not known whether this differential expression of plastid genes represents a cause or an effect of leaf senescence. The in organello protein synthesis was also used to study gene expression in chloroplasts induced by other environmental agents like temperature, or genotoxic agents such as ethidium bromide or herbicides (Krishnan et al. 1987, Subbaiah et al. 1987). Archer et al. (1987) have used the system to diagnose the molecular basis of a chloroplast coded virescent mutant in tobacco. From their experiments on in organello protein synthesis, they concluded that the inability of plastids from the mutant to make a 37.5 kDa polypeptide was probably the causal factor of this mutation and thus this polypeptide might be important for the assembly of pigment-protein complexes in the developing world.

Processing, assembly and turnover of in organello synthesized products The majority of polypeptides manufactured in the chloroplast are synthesized in their authentic size except for the 32kDa herbicide-binding protein. The primary amino acid sequence derived from the nucleotide sequence of the gene coding for this protein gave a sum of 353 amino acids with an expected molecular mass of 38 950 (Zurawski et al. 1982). Cohen et al. (1984) attempted to explain this discrepancy between the predicted and the authentic sizes of the polypeptide, when they located a second initiation codon, downstream to the first one and, in fact, it was at this second methionine that the translation was initiated in their in vitro experiments yielding a polypeptide of 34 000 molecular mass. Cohen et al. (1984) invoked a product-precursor relationship to explain the difference in the sizes of in vitro (heterologous), and in organello synthesized products. In fact such a

142 precursor was identified among the products of protein synthesis byisolated maize chloroplasts as early as in 1978 (Grebanier et al. 1978) and unfortunately these chloroplasts were unable to process this polypeptide. This possibility was later tested by pulse chase experiments and it was shown that isolated chloroplasts would synthesize and post-translationally process the 34.6 kDa precursor to the authentic 32 kDa polypeptide (see e.g., Minami and Watanabe 1985). Marder et al. (1984), who carried out detailed studies on this protein, showed that the processing occurred at the C-terminus of the precursor. There were reports that other polypeptides such as LSU of RuBisCO (Langridge 1981), Cyt b559(Zielinski and Price 1980) and the fl-subunit of CF~ (Watanabe and Price 1982) might also be synthesized as higher molecular weight precursors, but this could not be confirmed by later workers. Many of the chloroplast proteins are multi-subunit complexes most of which contain polypeptides synthesized both in the cytoplasm as well as the chloroplast. Many studies have shown that polypeptides newly synthesized in isolated plastids would assemble into authentic complexes. Products such as the subunits of CF0 and CF~, LSU of RuBisCO and reaction center polypeptides could be extracted as part of their respective complexes by standard methods from the organeUes previously labelled in vitro (Mendiola-Morgenthaler et al. 1976, Ellis 1977, Barraclough and Ellis 1980, Nelson et al. 1980, Zieliniski and Price 1980, Green 1980, Hacthel 1982, 1985, 1987, Bloom et al. 1983, Mannan et al. 1987). Nelson et al. (1980), who demonstrated the in organello assembly of new synthesized CF~ subunits, suggested that isolated chloroplasts would contain pools of cytoplasmically synthesized subunits of this complex. Alternatively it could be that the newly synthesized polypeptides were exchanged with the pre-existing subunits in the complex, the only possibility that would explain the assembly of ~t and fl subunits of CF~ synthesized by ribosomes bound to washed thylakoids into authentic CF~ complex in spinach and Chlamydomonas (Minami and Watanabe 1984, Herrin and Michaels 1985a). It was, however, exceptional that, in isolated maize chloroplasts, the newly formed CF~ subunits were not correctly assembled (Grebanier et al. 1978). As mentioned earlier, these chloroplasts also failed to process the precursor of the 32 kDa polypeptide, for reasons unknown. Interestingly, the ribosomal proteins, synthesized by isolated spinach chloroplasts, assembled themselves into incomplete ribosomal particles which were stable (Dorne et al. 1984). The authors suggested that the formation of these particles could be an intermediate step in the assembly of complete ribosomes, a phenomenon similar to the stepwise assembly of ribosomes in E. coll. The assembly of LSU into holoenzyme was worked out in detail by Ellis

143 and his associates and has been shown to require binding another stromal protein before integrating with cytoplasmically formed small subunit SSU (for details see Ellis and Van der Vies 1988). Bloom et al. (1983) showed that assembly of LSU in organello was a light-dependent process mediated by ATP. The requirement for light was not verified for the assembly of other proteins. Proteins that are newly formed by isolated chloroplasts are not entirely stable. A part of the fully mature polypeptides may be degraded without assembly due to a lack of cytoplasmically synthesized complementary polypeptides. There may also be incomplete or defective polypeptides that have to be removed by proteolysis. Such an instability of in organello synthesized proteins was first reported to occur in pea chloroplasts during ATP-driven amino acid incorporation (Fish et al. 1983). This was followed by detailed studies (Liu and Jagendorf 1984, Malek et al. 1984) which confirmed that about 20-35% of the labeled protein was degraded during a subsequent incubation period of 20-30 rain after a pulse feeding of label for 10-30 min. This proteolysis was strictly ATP dependent and was either totally absent or very little in evidence in the dark. Addition of Mg 2÷ -ATP stimulated the degradation 2-3 fold in darkness or light. The protein loss was the greatest (34-40%) when ATP was added together with intense light. In Euglena proplastids or chloroplasts, the proteolytic loss was as much as 50-60% (Cushman and Price 1986). Hydrolysis was not specific for a particular protein, and thylakoid as well as soluble proteins were degraded at comparable rates (Liu and Jagendorf 1984, Malek et al. 1984, Cushman and Price 1986). Liu and Jagendorf (1985) have examined the role of this ATP-dependent proteolysis. They suggested that this system, mainly confined to the thylakoids, might hydrolyze mature but non-integrated polypeptides. This was indicated by the increased rates of ATP-dependent in organello degradation of in vivo labeled chloroplast polypeptides in the presence of cycloheximide. They could also localize another proteolytic system in the stroma, which was ATP-independent but required Mg 2÷ and attacked prematurely terminated polypeptides induced by puromycin, canavanine or kanamycin. The turnover of the 32 kDa atrazine binding protein has attracted considerable attention. Ellis and his group were the first to highlight the special features of this polypeptide (Siddell and Ellis 1975, Ellis 1981) which is, so far, the best studied chloroplast protein (Kyle 1985). This was the major product of chloroplast protein synthesis in terms of incorporation in vitro but this protein would never accumulate like the LSU of RuBisCO (Ellis 1977). Thus it has been found to have the fastest turnover of all the chloroplast proteins. This is also reflected by in vivo labeling studies in

144 which very short pulse labeling (5-30min) in the light led to the rather exclusive visualization of this protein on the autotradiogram (Hoffman-Falk et al. 1983) whereas long labeling (> 12h) generated an autoradiograph pattern similar to the Coomassie Blue pattern. The turnover is dependent on light and is proportional to the light intensity. Electron transport inhibitors such as DCMU would inhibit the degradation. The actual mechanism of hydrolysis is still being worked out, however. There is also evidence that cytoplasmic protein synthesis regulates the translational activity of chloroplasts. Reardon and Price (1983) demonstrated that in organello protein synthesis in plastids isolated from Euglena was substantially reduced (40-90%) if the cells were pretreated for 2-4h with cycloheximide (CHI), an inhibitor of cytoplasmic protein synthesis. The synthesis of soluble products was more sensitive to the treatment than that of membrane polypeptides, though there were no qualitative changes in the polypeptide patterns of the soluble fraction. However, qualitative changes were seen in thylakoid associated products - especially those that required a processing step - such as the precursor of QB or herbicide binding protein. In chloroplasts isolated from CHI treated cells, the precursor for this protein accumulated and so also did a few novel polypeptides larger than 65 kDa. These changes could be partly reversed if the chloroplasts from treated cells were incubated with cytoplasm from control cells. These data suggested that products of cytoplasmic protein synthesis were involved in the processing of chloroplast translation products. The severe decrease in the synthesis of soluble products in plastids from CHI treated cells could be due to the depletion of their cytoplasmically synthesized partners for assembly (Spreitzer et al. 1985). Alternatively, it could be due to rapid degradation of newly synthesized proteins, known to be triggered during such conditions (Liu and Jagendorf 1985).

Isolated chloroplasts as a potential in vitro transcription and/or translation system

Until the early 1970s, protein synthesis by isolated chloroplasts was used only to identify plastome (plastid genome) coded products. Later, attempts were made to exploit the system for translating exogenous messengers of both homologous and heterologous origin. More recent work has shown that the usefulness of the system can be further extended to the transcription of exogenous DNA templates as well as the coupling of this process to translation, thus qualifying it as a complete assay system for gene expression.

145 We have used pre-illuminated chloroplasts (in order to exhaust the endogenous templates) to translate exogenously provided mRNAs (Indira and Gnanam 1976, Geetha and Gnanam 1980a). We have been able to show that pre-incubation completely eliminates the background incorporation due to native transcripts and renders the organelles an efficient in vitro translation assay system for transcripts of different origin - both prokaryotic and eukaryotic. We effectively substituted this system for other well-known in vitro protein synthesis systems such as wheat germ or rabbit reticulocyte lysate in our studies to establish the origin of messages for different chloroplast proteins such as P S I apoprotein, castorbean chloroplast glycosidase and pyruvate dikinase (Geetha and Gnanam 1980a, 1980b, Geetha et al. 1981, Mariappan 1984, Valliammai 1985, Uma Bai 1985, Ananda Krishnan and Gnanam 1987). Cammerino et al. (1982), working basically on the same idea, used lysed chloroplasts after treatment with micrococcal nuclease to remove pre-existing mRNAs, since the lysates could not stand the preincubation step we used in our experiments. These lysates faithfully translated both exogenously supplied homologous and heterologous mRNAs. The lysed chloroplast system is more precise than the whole chloroplast preparations in terms of availabiiity of exogenously added RNA and is also more open to manipulation to keep the requirements of cofactors etc., optimal. In spite of such a clear demonstration of the efficacy of isolated chloroplasts or their lysates for translating exogenous RNA templates and also the obvious advantages of the technique over other in vitro systems (as outlined in earlier publications, see Geetha and Gnanam 1980a) such as the ease and inexpensive nature of the preparation of material, others have continued using other in vitro translation systems such as wheat germ or E. coli S-30 fraction, even to test RNA from chloroplasts. Bard et al. (1985) demonstrated that lysates of chloroplasts can be used as a DNA-dependent coupled transcription-translation system as efficient as a similar system established in E. coli (Bottomley and Whitfield 1979). The new system, according to the authors' claim, responds well to total ct-DNA or its cloned fragments, plasmid vectors and also E. coli chromosomal D N A as well. However confirmatory reports on both the in vitro systems (Cammerino et al. 1982, Bard et al. 1985) are lacking, both from other laboratories and from their own. An immediate application of such a system, if it were well standardized, could be the identification of many open reading frames revealed by the sequence data of chloroplast genomes in Marchantia and tobacco (Ohyama et al. 1985, Shinozaki et al. 1985). Another important task is to study the expression of chloroplast genes, not just the developmental regulation in leaves but also in all the other parts of the plant. It is now known that

146 plastids in other tissues also contain identical genomes but develop differentially to perform divergent functions depending on the tissue. The in vitro systems can be used to study the processes and factors involved in such tissue-specific regulation of gene expression.

Acknowledgements The authors thank Ms. Mary Andrews for her dedicated typing of the manuscript. Part of the work reviewed here is supported by a grant no. 21(7)/84-STP-II from the Department of Science and Technology.

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