Chloroplast development in higher plants is light dependent, and is accompanied by the synthesis of chlorophyll and the accumulation of many chloroplast polypeptides. There is a 100-fold greater content of the photosynthetic enzyme, ribulose-l,5-bisphosphate carboxylase-oxygenase, in light-grown seedlings of Pisum sativum than in dark-grown seedlings. Following the illumination of dark-grown seedlings, there is a parallel increase in the content of both the mRNA and the polypeptide of the small subunit of the carboxylase; this subunit is a product of the nuclear genome. The increases in the mRNA and the polypeptide of the large subunit, which is a product of the chloroplast genome, show less synchronicity. Studies with isolated leaf nuclei show that the increase in small subunit mRNA is mediated primarily at the level of transcription. Three distinct effects of light on transcription of small subunit genes have been found; a rapid (∽1 h) burst, followed by a decline, when etiolated plants are first exposed to light; a slow (∽36h) development of the competence to transcribe rapidly after the initial burst; rapid (∽20 min) switches in both directions when fully greened plants are exposed to light-dark transitions.

One of the principal characteristics of green plants which set them apart from animals is that they are photoautotrophic; they use the energy of light to convert carbon dioxide into carbohydrate through the reactions of photosynthesis. This process, which is the mainstay of life on this planet, takes place in chloroplasts (Kirk & Tilney-Bassett, 1978). These organelles are bounded by a double envelope, and contain thylakoid membranes which house the photosynthetic pigments and electron transport components, and a hydrophilic matrix termed the stroma, which contains the enzymes involved in carbon dioxide fixation. Ribulose-l,5-bisphosphate carboxylase-oxygenase (Rubisco) is of pivotal importance since it catalyses the first step in the assimilation of carbon dioxide (Lorimer, 1981) ; it is also the most abundant protein in the leaves of higher plants (Ellis, 1979).

The programming of development in plants is very similar, at a fundamental level, to that in animals. In both cases the growth and differentiation of the whole organism, and of its constituent tissues and cells, involves differential gene expression. Our major interest is to understand the molecular basis of this process, and we have concentrated much of our effort on Rubisco because of its evident importance and abundance. This protein contains two types of subunit; a large subunit which is encoded in chloroplast DNA and synthesized within the organelle, and a small subunit which is encoded in nuclear DNA and synthesized on cytoplasmic ribosomes in precursor form prior to being transported into the chloroplast (Ellis, 1981, 1983). Thus an additional attraction of this protein is that it provides an opportunity to investigate the activities of two different genetic systems occurring within the same cell.

One way in which the programming of development in plants differs from that in animals is that, in general, it is influenced to a greater extent by environmental factors, one of the most important being light (Mohr & Shropshire, 1983; Jenkins, 1984). Leaf development for example, is arrested at an early stage in dark-grown plants and is completed only following exposure to light. The formation of mature, photosynthetically-active chloroplasts is also light dependent (Bradbeer, 1981). This requirement for light is hardly surprising since it would be wasteful for the plant, in view of the major investment of seed resources required, to synthesize its photosynthetic machinery under conditions in which photosynthesis could not occur. During normal leaf development in higher plants, that is under either continuous illumination or in a day-night cycle, chloroplasts develop from rudimentary organelles termed proplastids. If, however, plants are grown in darkness the proplastids develop into etioplasts, organelles which are smaller than chloroplasts, and which lack chlorophyll and the well-differentiated thylakoid membrane system typical of chloroplasts. Following illumination of dark-grown plants, etioplasts develop into chloroplasts, and it is this transformation (termed ‘greening’) which, for experimental convenience, is most frequently used in studies of chloroplast biogenesis.

The light-induced conversion of etioplasts into chloroplasts is accompanied by dramatic changes in molecular composition. Although some components decrease in abundance following illumination, the great majority, including the various pigments, quinones and proteins of the photosynthetic apparatus, accumulate (Bradbeer, 1981). Among the proteins which increase markedly in amount is Rubisco. It should be noted however, that light is not required for the accumulation of this protein in all cases; Rubisco can be easily detected in darkgrown plants of a number of species, especially cereals. The effect of light is therefore stimulatory rather than absolute, the extent of the stimulation varying between species (Ellis, 1983).

The effect of light on the abundance of Rubisco must involve some system for the detection and transduction of the light signal which results in a change in gene expression. The aim of our research is to address two broad sets of questions. First, at what levels is the expression of genes encoding the large and small subunit polypeptides of Rubisco controlled, what molecular mechanisms are involved, and to what extent are the expressions of the genes for the two subunits co-ordinated? Second, which photoreceptor pigments mediate the effect of light, and what mechanisms link photoreception to events concerned with gene expression?

The availability of cloned DNA hybridization probes for both the large subunit (Coen, Bedbrook, Bogorad & Rich, 1977) and the small subunit (Bedbrook, Smith & Ellis, 1980) has resulted in a number of detailed studies of the effect of light on the expression of the corresponding genes in species such as Pisum sativum (Smith & Ellis, 1981; Gallagher & Ellis, 1982; Jenkins, Hartley & Bennett, 1983; Gallagher, Jenkins, Smith & Ellis, 1984; Jenkins et al. 1984; Bennett, Jenkins & Hartley, 1984; Sasaki, Sakihama, Kamikubo & Shinozaki, 1983; Thompson et al. 1983) and Lemna gibba (Tobin, 1981; Stiekema, Wimpee, Silverthorne & Tobin, 1983). In this paper we summarize the conclusions of these studies, and present the results of our latest experiments on the photo-regulation of genes involved in the synthesis of Rubisco in Pisum sativum.

Fig. 1 summarizes some of the events believed to be involved in the synthesis of Rubisco in higher plants. In all eukaryotes, and most, but not all, prokaryotes, each molecule of Rubisco is an oligomer of sixteen subunits of two basic types, termed large and small (Miziorko & Lorimer, 1983). The large subunits (relative molecular mass (Mr) about 52 000) carry the active sites for both carboxylase and oxygenase activities. These large subunits are the major products of the chloroplast genetic system; each circle of chloroplast DNA contains one gene for the large subunit, the mRNA being translated by free chloroplast ribosomes (Ellis, 1981) to produce a higher Mr precursor (Langridge, 1981). Since each chloroplast is polyploid, and each cell contains many chloroplasts, there are several hundred to several thousand genes for the large subunit in each cell.

Fig. 1.

Co-operation of nuclear and chloroplast genomes in the synthesis of Rubisco. The wavy dotted lines indicate that the stimulatory effect of light is mediated primarily at the level of transcription.

Fig. 1.

Co-operation of nuclear and chloroplast genomes in the synthesis of Rubisco. The wavy dotted lines indicate that the stimulatory effect of light is mediated primarily at the level of transcription.

The small subunit (M about 14 000) has no known specific function. In several eukaryotes the small subunit is encoded by a small multigene family in the nucleus (Berry-Lowe, McKnight, Shah & Meagher, 1982; Dunsmuir, Smith & Bedbrook, 1983; Broglie et al. 1983). The mRNA for the small subunit is translated by free cytoplasmic ribosomes to produce a higher Mr precursor possessing an aminoterminal extension of 40–60 amino acyl residues. This precursor is transported by an ATP-dependent post-translational mechanism across the chloroplast envelope, and the extension removed by a stromal metalloprotease (Ellis & Robinson, 1984). In some lower eukaryotes the small subunit is a product of the chloroplast genetic system so that protein transport is not involved in these species (Heinhorst & Shively, 1983).

One of the most striking features of Rubisco is its sheer abundance. Up to 65 % of the soluble protein in extracts of photosynthetic cells can be accounted for by this single enzyme. For this reason Rubisco has a good claim to be the most abundant protein on earth (Ellis, 1979). The reason for the abundance of Rubisco appears to be that it is such a sluggish catalyst that the organism has to synthesize many molecules in order to achieve the required rate of carboxylation. Since the rate of carboxylation restricts plant productivity in some cases, major efforts are being made to construct more efficient Rubisco molecules by mutagenesis of their cloned nucleotide sequences (Ellis & Gatenby, 1984). This abundance of Rubisco makes it ideal for molecular biological studies, and it is not too fanciful to regard it as the haemoglobin of the plant biochemist. Antibodies and cloned DNA hybridization probes are available for both subunits, and their use in the study of the synthesis of Rubisco is the subject of this article.

Besides the large and small subunits, another polypeptide appears to be involved in the synthesis of Rubisco (Fig. 1). The assembly of Rubisco from its subunits occurs in the stroma (Smith & Ellis, 1979), but attempts to dissociate Rubisco from higher plants into its subunits, and then to reassociate them with recovery of active enzymic activity, have so far been unsuccessful. Large subunits, prepared by treatment with detergents or high pH of Rubisco purified from higher plants, are insoluble in aqueous media, as are large subunits synthesized by Escherichia coli minicells containing inserted large subunit genes (Ellis & Gatenby, 1984). The holoenzyme however, is highly soluble, occurring at concentrations up to about 300mg/ml in the stroma in vivo. Large subunits synthesized by isolated intact chloroplasts are also soluble, even though unassembled into holoenzyme (Blair & Ellis, 1973). This solubility is due to the binding of these newly synthesized large subunits to another stromal protein that we have termed the large subunit binding protein (Barraclough & Ellis, 1980; Ellis, 1981).

The large subunit binding protein is currently being studied at Warwick by S. Hemmingsen, S. D. Kung, C. Robinson and C. R. Lennox. The protein has been purified to homogeneity from Pisum sativum leaves, and sediments at a value of about 25S in the ultracentrifuge. Addition of 5 mM MgATP causes complete dissociation of the protein into a number of discrete lower relative molecular mass forms; removal of ATP reverses this dissociation. Analysis on denaturing polyacrylamide gels reveals a close doublet of subunit MT about 60000, which gives a different proteolytic digestion pattern to the large subunit. The purified binding protein has been used to raise antisera in rabbits. These antisera show no cross reactivity with large subunits, and have been used to construct a quantitative assay for the binding protein by means of rocket immunoelectrophoresis (Fig. 2).

Fig. 2.

Quantitative assay of the large subunit binding protein by rocket immuno-electrophoresis. Antisera raised against purified binding protein were used in rocket immunoelectrophoresis according to the procedure of Weeke (1973). Rockets were scanned and their areas determined by weighing. Binding protein was determined by the Biorad assay.

Fig. 2.

Quantitative assay of the large subunit binding protein by rocket immuno-electrophoresis. Antisera raised against purified binding protein were used in rocket immunoelectrophoresis according to the procedure of Weeke (1973). Rockets were scanned and their areas determined by weighing. Binding protein was determined by the Biorad assay.

When Pisum leaf polysomes are run off in a wheat-germ protein-synthesizing system, the binding protein antisera precipitate a larger precursor of Mr about 62000. This precursor is taken up and processed by intact isolated Pisum chloroplasts. These observations are consistent with the view that the binding protein is encoded in the nucleus, and synthesized in the cytoplasm in precursor form prior to transport into the chloroplast.

Our working hypothesis is that the role of the large subunit binding protein is to maintain the newly-synthesized large subunits in soluble form prior to assembly into the holoenzyme. The failure to demonstrate the assembly of Rubisco from its subunits in a soluble system has so far precluded the rigorous testing of this hypothesis. Nevertheless, in view of the potentially obligatory role of this binding protein in the production of Rubisco, we are studying the effect of light on its synthesis during the greening of etiolated Pisum seedlings.

As noted in the Introduction, light is required for the completion of leaf development in higher plants and for the concomitant formation of mature, photosynthetically active chloroplasts. Our studies have centred on the greening of Pisum sativum seedlings grown in darkness for either 6 or 8 days. When these etiolated plants are transferred to continuous white light (100//moles m−2s−1, 400–00 nm) the rate of stem extension decreases markedly, and the leaf cells within the shoot apical buds begin to enlarge and divide. After 48 h of greening under these conditions the apical buds have nearly quadrupled in fresh weight, and their DNA content has increased about three-fold (G. I. Jenkins, M. R. Hartley & J. Bennett, unpublished), indicating a corresponding increase in cell number.

Some of the biochemical changes that accompany greening are shown in Fig. 3. There is an increase in the total protein content of the apical buds and in the amount of chlorophyll. Chlorophyll is formed from the protochlorophyHide which accumulates in darkness in a light-dependent reaction catalysed by the enzyme NADPH-protochlorophyllide-oxidoreductase (Griffiths, 1978). We have previously shown, using a radioimmune assay, that very little Rubisco is present in dark-grown plants but that this protein accumulates during greening, slowly at first, but then more rapidly from 36 to 48 h after exposure to light (Jenkins et al. 1983; Bennett et al. 1984). Similar results have also been reported for Pisum sativum by Sasaki, Ishiye, Sakihama & Kamikubo (1981). The increase in the amount of Rubisco over the 48-h period is about 100-fold per apical bud; this increase clearly represents a considerable increase in the amount per leaf cell. Generally the amounts of the large and small subunits increase roughly in parallel during greening in white light in Pisum sativum (Sasaki et al. 1981; Jenkins et al. 1983; Bennett et al. 1984), and in other species (Walden & Leaver, 1981; Dean & Leech, 1982). This parallel accumulation of the two subunit types is quite remarkable considering the enormous difference in gene dosage referred to earlier; some mechanism to achieve this co-ordination of expression of the large and small subunit genes must exist.

Fig. 3.

Changes in the amounts of cellular components during greening. Pisum seedlings grown in darkness for 8 days were transferred to continuous white light and apices harvested over a 48 h period. Chlorophyll content was determined according to Arnon (1949); total protein was determined by the Biorad assay of extracts made in sodium dodecyl sulphate, and the large subunit binding protein quantified as in Fig. 2.

Fig. 3.

Changes in the amounts of cellular components during greening. Pisum seedlings grown in darkness for 8 days were transferred to continuous white light and apices harvested over a 48 h period. Chlorophyll content was determined according to Arnon (1949); total protein was determined by the Biorad assay of extracts made in sodium dodecyl sulphate, and the large subunit binding protein quantified as in Fig. 2.

We have recently started to investigate changes in the abundance of the large subunit binding protein during greening, using the rocket immunoelectrophoresis assay (Fig. 2). As shown in Fig. 3, the binding protein is readily detectable in dark-grown apical buds, and accumulates steadily following illumination. One conclusion that can be drawn from these data is that the failure of Rubisco to accumulate in dark-grown Pisum seedlings is not due to a lack of binding protein needed for assembly into stable holoenzyme; some other explanation is required. A second conclusion is that the increase in the amount of binding protein during the 48 h greening period is only three- to four-fold, similar to the increase in cell number per apical bud estimated from measurements of DNA content. Thus the amount of binding protein per cell does not appear to change greatly, if at all, as a result of illumination, although further detailed measurements are required. It may be that light has no effect on the expression of the nuclear genes encoding the binding protein. Alternatively, both its rate of synthesis and its rate of breakdown could be stimulated by light, resulting in a constant level of the protein in each cell.

In our previous experiments we have attempted to define the levels at which light controls the expression of genes encoding Rubisco. Smith & Ellis (1982) demonstrated, by hybridization analysis using specific cloned DNA probes, that light induces an increase in the abundance of both large and small subunit mRNAs in total RNA extracts of Pisum apical buds. Subsequently we investigated whether the increase in mRNA contents, measured by quantitative dot-blot hybridization, is correlated with the accumulation of the large and small subunit polypeptides, measured by radioimmunoassay (Jenkins et al. 1984; Bennett et al. 1984). There is a very close correlation between the rate of accumulation of the small subunit polypeptide and its mRNA, but less so for the large subunit. Accumulation of the large subunit polypeptide lags behind that of its mRNA during greening, and less polypeptide is present in dark-grown plants than might be expected from its content of mRNA for the large subunit. Thus for the large, but not for the small, subunit polypeptide, it is necessary to propose that accumulation is controlled at some level other than the availability of mRNA transcripts for translation. The data (Fig. 3) do not support the suggestion that large subunits are synthesized in dark-grown seedlings and then degraded as a result of an insufficiency of the large subunit binding protein. It is possible that complexes of the large subunit with the binding protein are turned over in the absence of small subunit polypeptides or that other post-transcriptional controls influence the accumulation of the large subunit.

Information has been recently obtained regarding the photoreceptors which mediate the effect of light on the expression of the large and small subunit genes. In plants a number of different photoreceptors are believed to function in controlling development, the most extensively studied being phytochrome (Pratt, 1982; Mohr & Shropshire, 1983). Phytochrome is a chromoprotein which exists in two photointerconvertible forms, one (Pr) absorbing maximally in the red (λmax 660 nm), and the other (Pfr) in the far-red (λmax 730 nm). Brief illumination with a low intensity of red light is sufficient to convert a large proportion of phytochrome into the Pfr form, which is regarded as the biologically active form in many responses, while illumination with far-red light converts Pfr back into Pr. This photoreversibility of phytochrome provides the basis of a simple test to establish its involvement in a given response; the inductive effect of a brief exposure to red light should be reversed by a subsequent far-red treatment, provided that insufficient time has elapsed between the light treatments for Pfr to initiate the train of events leading to the response in question. Application of this test has shown that phytochrome controls the expression of a number of plant genes (Jenkins, 1984).

In Pisum the expression of the small subunit genes is clearly under phytochrome control, although a brief red-light treatment produces only a small amount of the polypeptide (Jenkins et al. 1983) and hybridizable mRNA (Thompson et al. 1983; Jenkins et al. 1983) relative to that found under continuous white light. Phytochrome also controls the abundance of hybridizable small subunit mRNA in Lemna gibba (Stiekema et al. 1983). The situation with regard to the large subunit is more complex, since both red and far-red light treatments are equally effective in increasing the amount of both the large subunit polypeptide and its mRNA in 8-day-old dark-grown seedlings of Pisum (Jenkins et al. 1983). Similar results for large subunit mRNA have been reported for Sinapsis alba (Link, 1982). Phytochrome is probably involved, since no other known plant photoreceptor absorbs in the far red to any great extent; the most likely explanation is that even the small amount of Pfr formed in far-red light is sufficient to saturate the response for the large subunit, although not for the small. There are reports from other laboratories that red light is more effective than far-red at increasing the content of large subunit mRNA in Pisum (Thompson et al. 1983; Sasaki et al. 1983), but these experiments were performed on younger seedlings (5 days old), and this difference might be important.

We have recently started to investigate in greater detail the time course of small subunit mRNA accumulation during greening of etiolated Pisum seedlings. Fig. 4 shows the hybridization of a 32P-labelled cloned cDNA probe specific for the small subunit gene to total RNA extracts of apical buds fractionated by agarose gel electrophoresis and then transferred to nitrocellulose. The content of small subunit mRNA in RNA from dark-grown apical buds is below the level of detection by this method; when measured by dot-blot hybridization the content is about 1 % of the amount present in RNA from seedlings after 48 h greening (Jenkins et al. 1983). An increase in the abundance of small subunit mRNA is observed after 6 h of illumination with continuous white light, but this is followed by an actual decrease in the detectable amount of mRNA prior to the final, substantial increase up to 48 h (Fig. 4). This slight transient increase in small subunit mRNA content is variable, both in its extent and its timing, but it is repeatedly observed. Possible mechanisms for this effect will be mentioned in the next section. It is clear however, that the effect of light on small subunit mRNA accumulation is more complex than we at first envisaged.

Fig. 4.

Hybridization analysis of the abundance of small subunit mRNA during greening of Pisum apical buds. Pea plants were grown in darkness for 8 days (a), and then transferred to continuous white light (100 μ molesm−2s−1; 400 – 700 nm) for either 6 h (b), 9h (c), 12 h (d), 24 h (e), 36 h (f) or 48 h (g). Control plants were kept in darkness for 48 h (h). Total RNA was extracted from apical buds harvested at these times, fractionated by denaturing agarose gel electrophoresis and transferred to nitrocellulose filters (Jenkins et al. 1983). The immobilized RNA was hybridized to 32P-labelled cDNA specific for the small subunit gene (Bedbrook et al. 1980). The arrow indicates the small subunit mRNA.

Fig. 4.

Hybridization analysis of the abundance of small subunit mRNA during greening of Pisum apical buds. Pea plants were grown in darkness for 8 days (a), and then transferred to continuous white light (100 μ molesm−2s−1; 400 – 700 nm) for either 6 h (b), 9h (c), 12 h (d), 24 h (e), 36 h (f) or 48 h (g). Control plants were kept in darkness for 48 h (h). Total RNA was extracted from apical buds harvested at these times, fractionated by denaturing agarose gel electrophoresis and transferred to nitrocellulose filters (Jenkins et al. 1983). The immobilized RNA was hybridized to 32P-labelled cDNA specific for the small subunit gene (Bedbrook et al. 1980). The arrow indicates the small subunit mRNA.

The results discussed in the previous section demonstrate unequivocally that light induces an increase in the steady state concentrations of both small and large subunit mRNAs in Pisum apical buds, and that this increase is of major importance in accounting for the accumulation of the polypeptides during greening.

The question thus arises as to how this increase in mRNA content is produced. One possible explanation is that light increases the rates of transcription of the large and small subunit genes; another would be that the rate of mRNA synthesis is unchanged in the light, but that light causes a decrease in the rate of mRNA degradation. In order to distinguish between these possibilities for the small subunit genes we have undertaken measurements of small subunit gene transcription by nuclei isolated from Pisum apical buds (Gallagher & Ellis, 1982). Corresponding studies of large subunit gene transcription in isolated chloroplasts are now being initiated.

Nuclei are isolated from apical buds by Percoll density gradient centrifugation following homogenization. The nuclei incorporate labelled UMP, supplied as UTP, into RNA, which is heterodisperse in size up to about 25S. Fig. 5 shows that the incorporation proceeds linearly over the first 20 min of incubation, and then slowly decreases in rate. No incorporation is observed in the absence of nucleosidetriphosphates. The addition of α -aminitin at 10μg/ml, a concentration which inhibits completely transcription by RNA polymerase II, inhibits RNA synthesis by about 36%. Studies with other species have shown that transcription by isolated nuclei represents elongation of already engaged polymerases rather than the combined result of elongation and initiation (Tsai et al. 1978; Dermann et al. 1981). Thus measurement of small subunit gene transcription in isolated nuclei can be used to distinguish between changes in either transcription or mRNA turnover as mechanisms which regulate the steady state amount of small subunit mRNA.

Fig. 5.

Time course of RNA synthesis by isolated Pisum nuclei. Nuclei were incubated in a buffer containing 50mM-tris-HCl, pH7·8, 75mM-NH4Cl, 10 μM-MgCl2,20 % glycerol, 500μM-ATP, GTP & CTP, 10μM [5,6-3H]-UTP at 2μCi/μl, and 106 nuclei (10 μg DNA) per 25 μl. At various times aliquots were removed and TCA-insoluble counts determined in duplicate. Symbols: •, complete incubation mixture; ○, unlabelled nucleotides omitted.

Fig. 5.

Time course of RNA synthesis by isolated Pisum nuclei. Nuclei were incubated in a buffer containing 50mM-tris-HCl, pH7·8, 75mM-NH4Cl, 10 μM-MgCl2,20 % glycerol, 500μM-ATP, GTP & CTP, 10μM [5,6-3H]-UTP at 2μCi/μl, and 106 nuclei (10 μg DNA) per 25 μl. At various times aliquots were removed and TCA-insoluble counts determined in duplicate. Symbols: •, complete incubation mixture; ○, unlabelled nucleotides omitted.

Gallagher & Ellis (1982) used cloned cDNA hybridization probes to detect small subunit gene transcripts in the labelled RNA synthesized by isolated nuclei. Small subunit transcripts could barely be detected in the RNA products of nuclei obtained from dark-grown apical buds; DNA-excess hybridization showed that the transcripts were 18 times more abundant in RNA synthesized by nuclei isolated from light-grown buds. Although these experiments suggest that small subunit transcripts are synthesized at a greater rate in nuclei isolated from illuminated plants, it is possible that there is no dark-light difference in the rate of transcription, but that newly synthesized transcripts are rapidly degraded in nuclei from dark-grown plants. To test this possibility Gallagher & Ellis (1982) undertook a pulse-chase experiment with the isolated nuclei. Nuclei from dark-grown buds were incubated with 32P-UTP for 10 min, and aliquots incubated for a further period in the presence of excess unlabelled UTP and actinomycin D. The low amount of small subunit transcript synthesized during the initial 10 min pulse persisted during the chase period for at least 120 min, indicating the stability of the transcripts. Thus there is strong evidence that the increase in the small subunit mRNA content during greening in Pisum is the result of transcriptional control.

Our aim is to understand how a light signal detected by phytochrome, and possibly other photoreceptors, is able to effect a change in transcriptional activity. To do this it is necessary to develop an in vitro transcriptional system in which the action of light can be reproduced over a short period. In an attempt to find rapid effects of light we have monitored the rate of transcription of small subunit genes in isolated nuclei following the transfer of dark-grown plants to continuous white light. The results (Fig. 6) correspond well with those for the accumulation of the small subunit mRNA during greening (Fig. 4) ; that is, the rate of transcription is initially very low, and increases slowly to a maximum after about 36 h illumination. Moreover, a transient increase in the rate of small subunit gene transcription is observed within 1 h of exposure to light, which is consistent with the transient increase in small subunit mRNA content during the early stages of greening (Fig. 4).

Fig. 6.

Changes in the rate of transcription of small subunit genes during greening of Pisum seedlings. Pea plants were grown from seed in darkness for 6 days and then transferred to continuous white light as in Fig. 4. Apices were harvested and nuclei isolated at various times over a 48 h period. The rate of transcription of small subunit genes was determined as described by Gallagher & Ellis (1982).

Fig. 6.

Changes in the rate of transcription of small subunit genes during greening of Pisum seedlings. Pea plants were grown from seed in darkness for 6 days and then transferred to continuous white light as in Fig. 4. Apices were harvested and nuclei isolated at various times over a 48 h period. The rate of transcription of small subunit genes was determined as described by Gallagher & Ellis (1982).

A number of models for the control of transcription of specific genes have been proposed. These include the methylation of non-expressed genes (Razin & Riggs, 1980) and alterations in chromatin configuration (Weintraub & Goudine, 1976). It is also possible that various members of the small subunit multigene family are controlled differently by light. Our present speculation is that initiation occurs in dark-grown plants, but only slowly, and that illumination causes engaged polymerases to produce a small amount of small subunit transcript; a relatively long period of illumination is then required before re-initiation is completed, and transcription can proceed at its maximal rate. It is also evident from Fig. 4 that the small subunit transcripts produced initially are unstable and subject to degradation.

Although this initial burst of transcription occurs rapidly following illumination, it is too small and variable to provide the basis of an in vitro analysis. We have found however, that large rapid changes in small subunit gene transcription are observed if plants which are capable of a high rate of small subunit transcription are subjected to brief light-dark transitions. As shown in Fig. 7, when competent plants are transferred to darkness the rate of transcription decreases to a low level within 20 min. The rate of transcription then rapidly increases over a similar time period if these dark-treated plants are returned to continuous white light. It should be noted that the initial stages of nuclear isolation from dark-treated plants are carried out in darkness.

Fig. 7.

Rapid light-induced changes in the rate of transcription of Pisum small subunit genes. Pea plants were grown in darkness for 6 days, transferred to continuous white light (see Fig. 4) for 36 h, and returned to darkness for either 20 min, 1 h or 5 h. Plants left in darkness for 5 h were then transferred to white light for either 20min, 1 h or 4h. Apical buds were harvested from the plants at these times and nuclei were isolated. The harvesting and initial stages of isolation of the nuclei were performed in darkness. The rate of transcription of small subunit genes was determined as described by Gallagher & Ellis (1982).

Fig. 7.

Rapid light-induced changes in the rate of transcription of Pisum small subunit genes. Pea plants were grown in darkness for 6 days, transferred to continuous white light (see Fig. 4) for 36 h, and returned to darkness for either 20 min, 1 h or 5 h. Plants left in darkness for 5 h were then transferred to white light for either 20min, 1 h or 4h. Apical buds were harvested from the plants at these times and nuclei were isolated. The harvesting and initial stages of isolation of the nuclei were performed in darkness. The rate of transcription of small subunit genes was determined as described by Gallagher & Ellis (1982).

We are at present attempting to identify the photoreceptors which mediate these different effects of light; that is, the rapid initial increase in transcription, the slow development of the competence to transcribe rapidly and lastly, the rapid switch induced by a dark-light transition. We hope that these studies will provide information that will lead to the development of a biochemical analysis of the molecular basis of the photoregulation of transcription of the small subunit genes.

Arnond
.
I.
(
1949
).
Copper enzymes in isolated chloroplasts: polyphenoloxidase in Beta vulgaris
.
Plant Physiol
.
24
,
1
15
.
Barraclough
,
R.
&
Ellis
,
R. J.
(
1980
).
Protein synthesis in chloroplasts IX. Assembly of newly-synthesized large subunits into ribulose bisphosphate carboxylase in isolated intact pea chloroplasts
.
Biochim. Biophys. Acta
608
,
19
31
.
Bedbrook
,
J. R.
,
Smith
,
S. M.
&
Ellis
,
R. J.
(
1980
).
Molecular cloning and sequencing of DNA encoding the small subunit of chloroplast ribulose-1,5-bisphosphate carboxylase
.
Nature
287
,
692
697
.
Bennett
,
J.
,
Jenkins
,
G. I.
&
Hartley
,
M. R.
(
1984
).
Differential regulation of the accumulation of the light-harvesting chlorophyll a/b-binding complex and ribulose bisphosphate carboxylase/oxygenase in greening pea leaves
.
J. cell. Biochem. (in press)
.
Berry-Lowe
,
S.
,
Mcknight
,
T. D.
,
Shah
,
D. M.
&
Meagher
,
R. B.
(
1982
).
The nucleotide sequence, expression, and evolution of one member of a multigene family encoding the small subunit of ribulose-l,5-bisphosphate carboxylase in soybean
.
J. molec. Appl. Genet
.
1
,
483
498
.
Blair
,
G. E.
&
Ellis
,
R. J.
(
1973
).
Protein synthesis in chloroplasts I. Light-driven synthesis of the large subunit of Fraction I protein by isolated pea chloroplasts
.
Biochim. Biophys. Acta
319
,
223
234
.
Bradbeer
,
J. W.
(
1981
).
Development of photosynthetic function during chloroplast biogenesis
.
In The Biochemistry of Plants (edsN. K. Boardman & M. D. Hatch)
, vol.
8
, pp.
424
472
.
New York
:
Academic Press
.
Broglie
,
R.
,
Coruzzi
,
G.
,
Lamppa
,
G.
,
Keith
,
B.
&
Chua
,
N.-H.
(
1983
).
Structural analysis of nuclear genes coding for the precursor to the small subunit of wheat ribulose-1,5-biphosphate carboxylase
.
Biotechnology
1
,
53
61
.
Coen
,
D. M.
,
Bedbrook
,
J. R.
,
Bogorad
,
L.
&
Rich
,
A.
(
1977
).
Maize chloroplast DNA encoding the large subunit of ribulose bisphosphate carboxylase
.
Proc. natn. Acad. Sci., U.S.A
.
74
,
5487
5491
.
Dean
,
C.
&
Leech
,
R. M.
(
1982
).
The co-ordinated synthesis of the large and small subunits of ribulose bisphosphate carboxylase during early cellular development within a seven-day old wheat leaf
.
FEBS Letts
.
140
,
113
116
.
Derman
,
E.
,
Krauter
,
K.
,
Walling
,
L.
,
Weinberger
,
C.
,
Ray
,
M.
&
Darnell
,
J. E.
(
1981
).
Transcriptional control in the production of liver-specific mRNAs
.
Cell
23
,
731
739
.
Dunsmuir
,
P.
,
Smith
,
S. M.
&
Bedbrook
,
J. R.
(
1983
).
A number of different nuclear genes for the small subunit of RuBPCase are transcribed in petunia
.
Nucl. Acids Res
.
11
,
4177
4183
.
Ellis
,
R. J.
(
1979
).
The most abundant protein in the world
.
Trends in Biochem. Sci
.
4
,
241
244
.
Ellis
,
R. J.
(
1981
).
Chloroplast proteins: synthesis, transport and assembly
.
Ann. Rev. Plant Physiol
.
32
,
111
137
.
Ellis
,
R. J.
(
1983
).
Chloroplast protein synthesis: principles and problems
.
In Subcellular Biochemistry
vol.
9
, (ed.
D. B.
Roodyn
), pp.
237
261
.
New York
:
Plenum Publishing Co
.
Ellis
,
R. J.
&
Gatenby
,
A. A.
(
1984
).
Ribulose bisphosphate carboxylase: properties and synthesis. In The Genetic Manipulation of Plants and its Application to Agriculture
.
Ann. Proc. Phytochem. Soc. Eur
. (eds
P.
Lea
&
G. R.
Stewart
),
Oxford University Press (in press
).
Ellis
,
R. J.
&
Robinson
,
C.
(
1984
).
Post-translational transport and processing of cytoplasmically-synthesized precursors of organellar proteins
.
In The Enzymology of the Post-Translational Modification of Proteins
(eds
R. B.
Freedman
&
H. C.
Hawkins
), Vol.
II
New York
:
Academic Press (in press
).
Gallagher
,
T. F.
&
Ellis
,
R. J.
(
1982
).
Light-stimulated transcription of genes for two chloroplast polypeptides in isolated pea leaf nuclei
.
The EM BO J
1
,
1493
1498
.
Gallagher
,
T. F.
,
Jenkins
,
G. I.
,
Smith
,
S. M.
&
Ellis
,
R. J.
(
1984
).
Photoregulation of the nuclear and chloroplast genes for ribulose bisphosphate carboxylase
.
In Chloroplast Biogenesis
(ed.
R. J.
Ellis
),
Seminar Series No. 21 of the Society for Experimental Biology
.
Cambridge University Press (in press
).
Griffiths
,
W. T.
(
1978
).
Reconstitution of chlorophyllide formation by isolated chloroplast membranes
.
Biochem. J
.
174
,
681
692
.
Heinhorst
,
S.
&
Shively
,
J. M.
(
1983
).
Encoding of both subunits of ribulose-1,5-bisphosphate carboxylase by the organelle genome of Cyanophora paradoxa
.
Nature
304
,
373
374
.
Jenkins
,
G. I.
(
1984
).
The photoregulation of gene expression in plants
.
In Oxford Surveys of Plant Molecular and Cell Biology
(ed.
B. J.
Miflin
).
Oxford University Press (in press
).
Jenkins
,
G. L
,
Gallagher
,
T. F.
,
Hartley
,
M. R.
,
Bennett
,
J.
&
Ellis
,
R. J.
(
1984
).
Photo regulation of gene expression during chloroplast biogenesis
.
In Advances in Photosynthesis Research
(ed.
C.
Sybesma
), Vol
IV
. pp.
863
872
.
The Hague
:
Junk
.
Jenkins
,
G. L
,
Hartley
,
M. R.
&
Bennett
,
J.
(
1983
).
Photoregulation of chloroplast development: transcriptional, translational and post-translational controls?
Phil. Trans. R. Soc. London B
303
,
419
431
.
Kirk
,
J. T. O.
&
Tilney-Bassett
,
R. A. E.
(
1978
).
The Plastids; their Chemistry, Growth, Structure and Inheritance. Second
ed.
Amsterdam
:
Elsevier/North-Holland
.
Langridge
,
P.
(
1981
).
Synthesis of the large subunit of spinach ribulose bisphosphate carboxylase may involve a precursor polypeptide
.
FEBS Letts
.
123
,
85
89
.
Link
,
G.
(
1982
).
Phytochrome control of plastid mRNA in mustard
.
Planta
154
,
81
86
.
Lorimer
,
G.
(
1981
).
The carboxylation and oxygenation of ribulose-l,5-bisphosphate: the primary events in photosynthesis and photorespiration
.
Ann. Rev. Plant Physiol
.
32
,
349
383
.
Miziorko
,
H. M.
&
Lorimer
,
G.
(
1983
).
Ribulose-l,5-bisphosphate carboxylase-oxygenase
.
Ann. Rev. Plant Physiol
.
52
,
507
535
.
Mohr
,
H.
&
Shropshire
,
W.
Jr.
(
1983
).
An introduction to photomorphogenesis for the general reader
.
In Encyc. Plant Physiol. New Series Vol
.
16A
(eds.
W.
Shropshire
&
H.
Mohr
), pp.
24
38
.
Berlin
:
Springer
.
Pratt
,
L. H.
(
1982
).
Phytochrome: the protein moiety
.
Ann. Rev. Plant Physiol
.
33
,
557
582
.
Razin
,
A.
&
Riggs
,
A. D.
(
1980
).
DNA methylation and gene expression
.
Science
210
,
604
610
.
Sasaki
,
Y.
,
Ishiye
,
M.
,
Sakihama
,
T.
&
Kamikubo
,
T.
(
1981
).
Light-induced increase in mRNA activity coding for the small subunit of ribulose-l,5-bisphosphate carboxylase
.
J. biol. Chem
.
256
,
2315
2320
.
Sasaki
,
Y.
,
Sakihama
,
T.
,
Kamikubo
,
T.
&
Shinozaki
,
K.
(
1983
).
Phytochrome-mediated regulation of two mRNAs, encoded by nuclei and chloroplasts, of ribulose-1,5-bisphosphate carboxylase-oxygenase
.
Eur. J. Biochem
.
133
,
617
620
.
Smith
,
S. M.
&
Ellis
,
R. J.
(
1979
).
Processing of small subunit precursor of ribulose bisphosphate carboxylase and its assembly into whole enzyme are stromal events
.
Nature
278
,
662
664
.
Smith
,
S. M.
&
Ellis
,
R. J.
(
1981
).
Light-stimulated accumulation of transcripts of nuclear and chloroplast genes for ribulose bisphosphate carboxylase
.
J. molec. Appl. Genet
.
1
,
127
-
137
.’
Stiekema
,
W. J.
,
Wimpee
,
C. F.
,
Silverthorne
,
J.
&
Tobin
,
E. M.
(
1983
).
Phytochrome control of the expression of two nuclear genes encoding chloroplast proteins in Lemna gibba
.
Plant Physiol
.
72
,
717
724
.
Thompson
,
W. F.
,
Everett
,
M.
,
Polans
,
N. O.
,
Jorgensen
,
R. A.
&
Palmer
,
J. D.
(
1983
).
Phytochrome control of RNA levels in developing pea and mung bean leaves
.
Planta
158
,
487
500
.
Tobin
,
E. M.
(
1981
).
Phytochrome-mediated regulation of messenger RNAs for the small subunit of ribulose-l,5-bisphosphate carboxylase and the light-harvesting chlorophyll a/b binding protein in Lemna gibba
.
Plant molec. Biology
1
,
35
51
.
Tsai
,
S. Y.
,
Roop
,
D. R.
,
Tsai
,
M. J.
,
Stein
,
J. P.
,
Means
,
A. R.
&
O’Malley
,
B. W.
(
1978
).
Effect of estrogen on gene expression in the chick oviduct. Regulation of the ovomucoid gene
.
Biochemistry (Wash)
17
,
5773
5780
.
Walden
,
R.
&
Leaver
,
C. J.
(
1981
).
Synthesis of chloroplast proteins during germination and early development of cucumber
.
Plant Physiol
.
67
,
1090
1096
.
Weeke
,
B.
(
1973
).
In A Manual of Quantitative Electrophoresis: Methods and Applications
(eds
N. H.
Alexsen
,
J.
Kroll
&
B.
Weeke
),
Scand. J. Immunol
, vol.
2
,
supplement no. 1
. Publishers.
Weintraub
,
H.
&
Goudine
,
M.
(
1976
).
Chromosomal subunits in active genes have an altered conformation
.
Science
193
,
848
856
.