Light-induced Chloroplast Development Research Articles

Proteomic Responses of Dark-Adapted Euglena gracilis and Bleached Mutant Against Light Stimuli.

Euglena gracilis (E. gracilis) has secondary endosymbiotic chloroplasts derived from ancient green algae. Its chloroplasts are easily lost under numerous conditions to become permanently bleached mutants. Green cells adapted in the dark contain undeveloped proplastids and they will develop into mature chloroplasts after 3 days of light exposure. Thus, E. gracilis is an ideal model species for a chloroplast development study. Previous studies about chloroplast development in E. gracilis focused on morphology and physiology, whereas few studies have addressed the regulatory processes induced by light in the proteome. In this study, the whole-genome proteome of dark-adapted E. gracilis (WT) and permanently ofloxacin-bleached mutant (B2) was compared under the light exposure after 0, 12, and 72 h. The results showed that the photosynthesis-related proteins were up-regulated over time in both WT and B2. The B2 strain, with losing functional chloroplasts, seemed to possess a complete photosynthetic function system. Both WT and B2 exhibited significant light responses with similar alternation patterns, suggesting the sensitive responses to light in proteomic levels. The main metabolic activities for the utilization of carbon and energy in WT were up-regulated, while the proteins with calcium ion binding, cell cycle, and non-photosynthetic carbon fixation were down-regulated in B2. This study confirmed light-induced chloroplast development in WT from dark, and also for the first time investigates the light responses of a bleached mutant B2, providing more information about the unknown functions of residual plastids in Euglena bleached mutants.

Chloroplast thylakoid ascorbate peroxidase PtotAPX plays a key role in chloroplast development by decreasing hydrogen peroxide in Populus tomentosa.

Chloroplast development is a complex process that is critical to the growth and development of plants. However, the detailed mechanism of chloroplast development in woody plants remains unclear. In this study, we showed that chloroplasts with elaborate thylakoids could develop from proplastids in the cells of calli derived from leaf tissues of Populus tomentosa upon exposure to light. Chloroplast development was confirmed at the molecular and cellular levels. Transcriptome analysis revealed that genes related to photoreceptors and photosynthesis were significantly up-regulated during chloroplast development in a time-dependent manner. In light-induced chloroplast development, a key process was the removal of hydrogen peroxide, in which thylakoid-localized PtotAPX played a major role; light-induced chloroplast development was enhanced in PtotAPX-overexpressing transgenic P. tomentosa callus with lower levels of hydrogen peroxide, but was suppressed in PtotAPX antisense transgenic callus with higher levels of hydrogen peroxide. Moreover, the suppression of light-induced chloroplast development in PtotAPX antisense transgenic callus was relieved by the exogenous reactive oxygen species scavenging agent N,N'-dimethylthiourea (DMTU). Based on these results, we propose that PtotAPX-mediated removal of reactive oxygen species plays a key role in chloroplast development from proplastids upon exposure to light in P. tomentosa.

Highly Resolved Systems Biology to Dissect the Etioplast-to-Chloroplast Transition in Tobacco Leaves.

Upon exposure to light, plant cells quickly acquire photosynthetic competence by converting pale etioplasts into green chloroplasts. This developmental transition involves the de novo biogenesis of the thylakoid system and requires reprogramming of metabolism and gene expression. Etioplast-to-chloroplast differentiation involves massive changes in plastid ultrastructure, but how these changes are connected to specific changes in physiology, metabolism, and expression of the plastid and nuclear genomes is poorly understood. Here, we describe a new experimental system in the dicotyledonous model plant tobacco (Nicotiana tabacum) that allows us to study the leaf deetiolation process at the systems level. We have determined the accumulation kinetics of photosynthetic complexes, pigments, lipids, and soluble metabolites and recorded the dynamic changes in plastid ultrastructure and in the nuclear and plastid transcriptomes. Our data describe the greening process at high temporal resolution, resolve distinct genetic and metabolic phases during deetiolation, and reveal numerous candidate genes that may be involved in light-induced chloroplast development and thylakoid biogenesis.

A Nitrite Transporter Associated with Nitrite Uptake by Higher Plant Chloroplasts

Chloroplasts take up cytosolic nitrite during nitrate assimilation. In this study we identified a nitrite transporter located in the chloroplasts of higher plants. The transporter, CsNitr1-L, a member of the proton-dependent oligopeptide transporter (POT) family, was detected during light-induced chloroplast development in de-etiolating cucumber seedlings. We detected a CsNitr1-L-green fluorescent protein (GFP) fusion protein in the chloroplasts of leaf cells and found that an immunoreactive 51 kDa protein was present in the isolated inner envelope membrane of chloroplasts. CsNitr1-L has an isoform, CsNitr1-S, with an identical 484 amino acid core sequence; however, in CsNitr1-S the 120 amino acid N-terminal extension is missing. Saccharomyces cerevisiae cells expressing CsNitr1-S absorbed nitrite from an acidic medium at a slower rate than mock-transformed control cells, and accumulated nitrite to only one-sixth the concentration of the control cells, suggesting that CsNitr1-S enhances the efflux of nitrite from the cell. Insertion of T-DNA in a single CsNitr1-L homolog (At1g68570) in Arabidopsis resulted in nitrite accumulation in leaves to more than five times the concentration found in the wild type. These results show that it is possible that both CsNitr1-L and CsNitr1-S encode efflux-type nitrite transporters, but with different subcellular localizations. CsNitr1-L may possibly load cytosolic nitrite into chloroplast stroma in the chloroplast envelope during nitrate assimilation. The presence of genes homologous to CsNitr1-L in the genomes of Arabidopsis and rice indicates that facilitated nitrite transport is of general physiological importance in plant nutrition.

Immunogold Localization of Photosynthetic Proteins in Euglena

Summary: Changes in pyrenoid morphology and the distribution within the chloroplast of ribulose-1, 5-bisphosphate carboxylase/oxygenase(RuBisCO)of Euglena gracilis Z were followed by immunoelectron microscopy during growth and division phase. Most of the immunoreactive protein was found in the pyrenoid during the growth phase with only a small amount of gold particles localized to the stroma. During the division phase, the pyrenoid was not detected and the gold particles were dispersed throughout the stroma. A comparison between rates of photosynthetic CO2-fixation and the amount of total carboxylase activity catalyzed by RuBisCO extracted from Euglena cells in the growth phase suggests that pyrenoid localized RuBisCO is responsible for photosynthetic CO2-fixation. The precursors to the Euglena light-harvesting chlorophyll a/b binding proteins of photosystem II(LHCP II)are large polyproteins containing multiple copies of LHCP II covalently joined by a conserved decapeptide. Light induced LHCP II synthesis is controlled at the translational level in Euglena rather than the transcriptional level as found in higher plants and other algae. Under conditions promoting LHCP II synthesis and accumulation in the thylakoids, a reaction with anti-LHCP II antibody can be observed by immunogold electron microscopy in the Golgi apparatus. The kinetics of LHCP II appearance in the Golgi apparatus as measured by immunogold electron microscopy in synchronous cells and by pulse labeled with H2[35S]O4 in cells undergoing normal light-induced chloroplast development suggests that nascent LHCP II is transported to the Golgi apparatus prior to chloroplast import and thylakoid insertion.

The phytochrome-interacting transcription factor, PIF3, acts early, selectively, and positively in light-induced chloroplast development

The phytochrome (phy) family of sensory photoreceptors transduce informational light signals to selected nuclear genes, inducing plant growth and developmental responses appropriate to the environment. Existing data suggest that one signaling pathway by which this occurs involves direct, intranuclear interaction of the photoactivated phy molecule with PIF3, a basic helix-loop-helix transcription factor. Here, we provide evidence from recently identified pif3 mutant alleles that PIF3 is necessary for early chloroplast greening and rapid phy-induced expression of nuclear genes encoding chloroplast components upon first exposure of seedlings to light. Therefore, these data indicate that PIF3 functions to transduce phy signals to genes involved in a critical facet of the early seedling deetiolation process, the generation of a functional photosynthetic apparatus. When transgenically expressed GUS:PIF3 fusion protein constructs were used, we found that PIF3 protein levels are rapidly and reversibly modulated by the photoreceptor over diurnal cycles in Arabidopsis seedlings. The PIF3 protein declines rapidly to a basal steady-state level upon initial light exposure, but reaccumulates to preirradiation levels in darkness during the subsequent night period. These data suggest that PIF3 may function in early phy signaling at the dark-to-light transition, not only during initial seedling deetiolation, but daily at dawn under diurnal light-dark cycles.

A novel role of water-soluble chlorophyll proteins in the transitory storage of chorophyllide.

All chlorophyll (Chl)-binding proteins involved in photosynthesis of higher plants are hydrophobic membrane proteins integrated into the thylakoids. However, a different category of Chl-binding proteins, the so-called water-soluble Chl proteins (WSCPs), was found in members of the Brassicaceae, Polygonaceae, Chenopodiaceae, and Amaranthaceae families. WSCPs from different plant species bind Chl a and Chl b in different ratios. Some members of the WSCP family are induced after drought and heat stress as well as leaf detachment. It has been proposed that this group of proteins might have a physiological function in the Chl degradation pathway. We demonstrate here that a protein that shared sequence homology to WSCPs accumulated in etiolated barley (Hordeum vulgare) seedlings exposed to light for 2 h. The novel 22-kD protein was attached to the outer envelope of barley etiochloroplasts, and import of the 27-kD precursor was light dependent and induced after feeding the isolated plastids the tetrapyrrole precursor 5-aminolevulinic acid. HPLC analyses and spectroscopic pigment measurements of acetone-extracted pigments showed that the 22-kD protein is complexed with chlorophyllide. We propose a novel role of WSCPs as pigment carriers operating during light-induced chloroplast development.

Regulation of Plastid Gene Expression by High Temperature during Light Induced Chloroplast Development

Effect of high temperature on the plastid gene expression during the light induced chloroplast development in etiolated seedlings was determined by Northern hybridisation using cloned DNA fragments of wheat chloroplast genome. Based on their response to high temperature, plastid genes were grouped into three categories: (1) plastid genes whose expression was not affected by high temperature (genes for rRNA, ribosomal proteins, tRNAs, and some genes coding for putative NADH dehydrogenase); (2) plastid genes whose expression increased at high temperature (genes coding for α-subunit of RNA polymerase and some unidentified transcripts, and (3) plastid genes whose expression decreased at high temperature (genes coding for proteins involved in photosynthetic process). Loss of a number of primary transcripts originating from operons consisting of genes that code for proteins involved in the photosynthetic process was observed. The expression of all the light inducible plastid genes was inhibited suggesting that the light inducibility property was lost at high temperature.

Effects of δ-aminolevulinic acid on pigment formation and chlorophyllase activity in French bean leaf

Chloroplast development and chlorophyll biosynthesis are co-regulated. Treatment by levulinic acid resulted in a linear relation in both chlorophyll and carotenoid contents, during greening of etiolated French bean leaf discs. Chlorophyll biosynthesis appeared to control that of caroteins. In the presence of levulinic acid; at different levels, photosystem II (PS II) activity decreased when expressed on a chlorophyll basis. Chlorophyllase activity was increased progressively by increasing levulinic acid concentration. Thus, levulinic acid could be used to arrest the light-induced chloroplast development at a desired phase of greening and acts as determinator of chloroplast development in green tissues.

Immunocytochemical studies on the behavior photosynthetic enzymes during the cell cycle of synchronized cells of Euglena gracilis Z

Euglena cells were grown synchronously under photoautotrophic culture conditions on a 14 h light-10 h dark alternations. Changes in morphology of the pyrenoid and those in distribution of RuBisCO in chloroplasts were followed by immunoelectron microscopy during the growth and division phases of Euglena cells. The immunoreactive protein were densely localized in the pyrenoid, and thinly distributed in the stroma during the growth phase. During the division phase, the pyrenoid could not be detected and the gold particles were dispersed throughout the stroma. From a comparison of photosynthetic CO2 -fixation with the total carboxylase activity of RuBisCO extracted from Euglena cells in the growth phase, it is suggested that the carboxylase in the pyrenoid functions in CO2 -fixation in photosynthesis. Cells of Euglena contain a LHC II. The precursors to LHC II are large polyproteins containing multiple copies of LHC II, and photocontrol of their formation is largely translational. Under conditions favoring LHC II accumulation in the thylakoids, a reaction with anti-LHC II antibody can be observed in the Golgi by immunogold electron microscopy. The timing of the immunoreaction in the Golgi in synchronous cells and in cells undergoing normal light-induced chloroplast development suggests that the nascent LHC II passes through the Golgi on the way to the thylakoids.

Immunoelectron microscopic studies on the behavior of LHCP II in Euglena

Cells of Euglena contain a light-harvesting chlorophyll a/b complex associated with photosystem II. In Euglena, the formation of the26.5kDa principal light-harvesting chlorophyll a/b binding protein of photosystem II(LHCP II)has a number of unusual features. The precursors to LHCP II are large polyproteins containing multiple copies of LHCP II, and photocontrol of their formation is largely translational. Under conditions favoring LHCP II accumulation in the thylakoids, a reaction with anti-LHCP II antibody can be observed in the Golgi by immunogold electron microscopy. The timing of the immunoreaction in the Golgi in synchronous cells and in cells undergoing normal light-induced chloroplast development suggests that the nascent LHCP II passes through the Golgi on the way to the thylakoids. The compartmentalized osmiophilic structure(COS)also shows an immunoreation. These observations, and others discussed in this paper, suggest that light permits translation of polyprotein LHCP II precursors on cytoplasmic ribosomes of the rough endoplasmic reticulum(ER)and that these pass through the ER to the Golgi where, presumably, further modifications take place. Since an LHCP II immunoreaction is found in Golgi vesicles, these may transport the nascent LHCP II to the plastid and facilitate its uptake.

Differential Transcription of Pea Chloroplast Genes during Light-Induced Leaf Development (Continuous Far-Red Light Activates Chloroplast Transcription).

Plastid gene expression was analyzed in pea (Pisum sativum L.) plants grown in darkness, continuous far-red light, and white light. Responses induced by continuous far-red light were most likely mediated by PHYA. Plastid transcription activity was low in dark-grown plants. In contrast, plastids of plants grown in white or far-red light showed a 10-fold increase in transcription activity between 4 and 6 d postimbibition (dpi) and a decrease between 6 and 9 dpi. Plastid RNAs accumulated in illuminated plants from 5 to 7 dpi. In far-red-light-illuminated plants, plastid mRNA levels remained elevated until 14 dpi. In white-light-grown plants, most plastid RNAs decreased in abundance after 7 dpi to very low levels by 14 dpi. This indicates that white light induces a general decrease in plastid RNA stability compared to far-red-light-illuminated seedlings. PsbA mRNA accumulated in older, dark-grown, far-red, and white-light-illuminated seedlings, consistent with this RNA having high stability. Transcription of genes encoding the plastid's transcription and translation apparatus increased relative to rbcL and other genes encoding proteins of the photosynthetic apparatus from 4 to 5 dpi and then declined 10-fold from 5 to 9 dpi. These data document dynamic modulation of plastid gene transcription and mRNA stability during light-induced chloroplast development in pea.

Cytosolic and plastid forms of 5-enolpyruvylshikimate-3-phosphate synthase in Euglena gracilis are differentially expressed during light-induced chloroplast development.

The enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19), the target of the herbicide glyphosate [N-(phosphonomethyl)glycine], exists in two molecular forms in Euglena gracilis. One form has previously been characterized as a monofunctional 59 kDa protein. The other form constitutes a single domain of the multifunctional 165 kDa arom protein. The two enzyme forms are inversely regulated at the protein and mRNA levels during light-induced chloroplast development, as demonstrated by the determination of their enzyme activities after non-denaturing polyacrylamide gel electrophoresis and Northern hybridization analysis with a Saccharomyces cerevisiae ARO1 gene probe. The arom protein and its mRNA predominate in dark-grown cells, and the levels of both decline upon illumination. In contrast, the monofunctional EPSP synthase and its mRNA are induced by light, the increase in mRNA abundance preceding accumulation of the protein. The two enzymes are localized in different subcellular compartments, as demonstrated by comparing total protein patterns with those of isolated organelles. Glyphosate-adapted wild-type cells and glyphosate-tolerant cells of a plastid-free mutant of E. gracilis, W10BSmL, were used for organelle isolation and protein extraction, as these cell lines overproduce EPSP synthase and the arom protein, respectively. Evidence was obtained for the cytosolic localization of the arom protein and the plastid compartmentalization of the monofunctional EPSP synthase. These conclusions are further supported by the observation that EPSP synthase precursor, produced by in vitro translation of the hybrid-selected mRNA, was efficiently taken up and processed to mature size by isolated chloroplasts from photoautotrophic wild-type E. gracilis cells, while the in vitro-synthesized arom protein was not sequestered by isolated Euglena plastids.

Observations on Components of Photosystem II During the Greening of Select Mutants of Scenedesmus

The formation of photosystem II of the photosynthetic apparatus was studied in five different developmental mutants of the green alga, Scenedesmus obliquus . These strains are characterized by their ability to synthesize chlorophyll only in the light and by specific differences in their photosystem II (PS-II) capacity and composition when fully developed. Cells from mutants C-2A, LF1-YI-RVT1 and WT-LHC 1 -Y3 form a functional PS-II upon transfer from darkness into light whereas those of strains C-2A-10 and LF1-Y1 do not develop PS-II activity under any conditions. Analysis of a variety of chloroplast reactions with the latter two strains showed that they lack the PS-II reaction center only minimally. 35 S labeling during light-induced chloroplast development revealed that only C-2A-10 and LF1-Y1 failed to develop the normal D1 protein (32 kDa polypeptide In LF1-Y1, only the unprocessed form (34 kDa) of this protein was synthesized and, as was noted for the original mutant LF-1, is inactive as a PS-II reaction center component. During development of C-2A-10, which appears to be pleiotropic in its PS-II deficiency, this protein is not synthesized and the PS-II core antenna polypeptides (the 48 and 44 kDA polypeptides) and a 14.5 kDa polypeptide also appear to be synthesized in lesser amounts or not to be synthesized at all. No other changes in PS-II chloroplast membrane components were noted in these studies, which would implicate other polypeptides in PS-II function of Scenedesmus .

Photocontrol and processing of LHCP II apoprotein in Euglena: Possible role of Golgi and other cytoplasmic sites

Like other green photosynthetic eukaryotes, cells of Euglena gracilis var. bacillaris and strain Z contain a light-harvesting chlorophyll a/b complex associated with photosystem II. In Euglena, the formation of the 26.5 kDa principal light-harvesting chlorophyll a/b binding protein of photosystem II (LHCP II) has a number of unusual features. The precursors to LHCP II are large polyproteins containing multiple copies of LHCP II, and photocontrol of their formation is largely translational. Under conditions favoring LHCP II accumulation in the thylakoids, a reaction with anti-LHCP II antibody can be observed in the Golgi by immunogold electron microscopy. The timing of the immunoreaction in the Golgi in synchronous cells and in cells undergoing normal light-induced chloroplast development suggests that the nascent LHCP II passes through the Golgi on the way to the thylakoids. The compartmentalized osmiophilic structure (COS) also shows an immunoreaction. These observations, and others discussed in this paper, suggest that light permits translation of polyprotein LHCP II precursors on cytoplasmic ribosomes of the rough endoplasmic reticulum (ER) and that these pass through the ER to the Golgi where, presumably, further modifications take place. Since an LHCP II immunoreaction is found in Golgi vesicles, these may transport the nascent LHCP II to the plastid and facilitate its uptake.

Translational regulation of plastid gene expression in Euglena gracilis

Translation of plastid messenger RNAs depends on aminoacyl-tRNAs formed by charging plastid-encoded tRNAs with cognate amino acids. The enzymes involved, chloroplast aminoacyl-tRNA synthetases, are encoded in the nucleus. Both the tRNAs and the aminoacyl-tRNA synthetases are stimulated in synthesis if dark-grown cells are exposed to light. However, their accumulation during light-induced chloroplast development in Euglena gracilis starts with an appreciable lag-phase. During this period the availability of charged tRNAs probably limits protein synthesis. Due to the contemporary need of glutamyl-tRNA Glu GAA in chlorophyll synthesis this particular tRNA is very likely depleted. Based on an analysis of glutamate codon frequency in known plastid genes, the effect of a glutamyl-tRNA Glu GAA limitation on the translation of plastid messages is discussed.

Translation and stability of proteins encoded by the plastid psbA and psbB genes are regulated by a nuclear gene during light-induced chloroplast development in barley

We have characterized a nuclear mutant of barley, viridis-115, lacking photosystem II (PSII) activity and compared it to wild-type seedlings during light-induced chloroplast development. Chloroplasts isolated from wild-type and viridis-115 seedlings illuminated for 1 h synthesized similar polypeptides and had similar protein composition. After 16 h of illumination, however, mutant plastids exhibited reduced ability to radiolabel D1, CP47, and several low Mr membrane polypeptides, and by 72 h, synthesis of these proteins was undetectable. Immunoblot analysis showed that plastids of dark-grown wild-type barley lacked several PSII proteins (D1, D2, CP47, and CP43) and that 16 h of illumination resulted in the accumulation of these polypeptides. In contrast, these polypeptides did not accumulate in illuminated viridis-115 seedlings, although mutant plastids accumulated two PSII proteins that participate in oxygen evolution, oxygen-evolving enhancers 1 and 3. Northern analysis showed that the levels of psbA and psbB mRNA in mutant plastids were equal to or greater than levels in wild-type plastids throughout the developmental period examined here. These results indicate that the nuclear mutation present in viridis-115 affects the translation and stability of the chloroplast-encoded D1 and CP47 polypeptides and that its influence is expressed after the onset of light-induced chloroplast development.

Control of gene expression during higher plant chloroplast biogenesis. Protein synthesis and transcript levels of psbA, psaA-psaB, and rbcL in dark-grown and illuminated barley seedlings.

Etioplasts of 4.5-day-old dark-grown barley synthesize and accumulate most of the membrane and nearly all the soluble polypeptides of mature chloroplasts of light-grown seedlings. Etioplasts do not synthesize a limited set of chloroplast-encoded polypeptides which are major constituents of chloroplast thylakoid membranes: two chlorophyll apoproteins of photosystem I (68 and 65 kDa), two chlorophyll apoproteins of photosystem II (47 and 43 kDa), and a 32-kDa polypeptide which has now been identified as the psbA gene product. Throughout development in the dark, etioplasts were unable to synthesize the chlorophyll apoproteins of photosystem I and II or the psbA gene product despite the presence of significant transcript levels for psbA and psaA-psaB (encode for photosystem I chlorophyll apoproteins). Light was not required for the synthesis of ribulose bisphosphate carboxylase large subunit with the highest rate of large subunit synthesis occurring in young dark-grown seedlings. Illumination of 4.5-day-old dark-grown barley rapidly induced the synthesis of the chlorophyll apoproteins and the psbA gene product at a time when transcript levels for psbA and psaA-psaB did not increase appreciably. Therefore, during the early stages of light-induced development the synthesis of the chlorophyll apoproteins of photosystem I and psbA gene product is regulated at the translational level. With continued chloroplast development in the light, the synthesis of the chlorophyll apoproteins of photosystem I and II decline rapidly as does the synthesis of the large subunit of ribulose bisphosphate carboxylase. The decline in polypeptide synthesis correlated with a decline in rbcL and psaA-psaB transcript levels and a light-dependent decline in plastid rRNA content. In contrast, synthesis of the psbA gene product was maintained throughout light-induced chloroplast development which correlated with the maintenance of psbA transcript levels. However, light is not strictly required for psbA transcript accumulation since psbA transcript levels increased slightly with continued plastid development in dark-grown seedlings.

The genes encoding chloroplast ribosomal proteins S7 and S12 are located in the inverted repeat of Spirodela oligorhiza chloroplast DNA.

We have used a variety of methods to localize the genes for ribosomal proteins S7 and S12 on Spirodela chloroplast DNA. Heterologous hybridization with a rps12 gene specific probe from Euglena has revealed the presence of rps12 homologous sequences within the inverted repeat of Spirodela chloroplast DNA on the fragment BamHI-V. In the partial nucleotide sequence of this fragment, two regions of amino acid sequence homology to Euglena S12 can be identified, separated from each other by a 542 bp intron with conserved boundary sequences. As was found for Nicotiana S12, the Spirodela S12 coding regions are for 85 amino acids homologous (79%) to E. coli S12 (starting from residue 38 to the C-terminus). Likewise, we are unable to identify the 37 5' terminal codons of rps12 in Spirodela. The functionality of the Spirodela rps12 sequence is discussed. The rps7 gene is located adjacent to rps12. Chloroplast ribosomal protein C-S11 (homologous to S7) has been detected by immunoprecipitation with both a polyspecific anti 30S serum and an anti C-S11 serum, among the in vitro translation products of mRNAs selected by Spirodela chloroplast DNA fragments BamHI-V and BamHI-P. Since in a DNA dependent E. coli cell free system, only BamHI-V appears to be capable of synthesis of C-S11, it is concluded that rps7 is located entirely within BamHI-V and is transcribed into a mRNA which extends into BamHI-P. As determined by Northern hybridization experiments, rps7 is cotranscribed with rps12; a stable transcript of approx. 1100 b is detected in total cellular Spirodela RNA with either rps12 and rps7 gene specific probes. The rps12 probe additionally detects an approx. 600 b transcript, which presumably corresponds to the excised rps12 intron RNA. Finally we have examined the expression of both rps7 and rps12 during light induced chloroplast development by Northern blotting and by immunoblotting. It is shown, that the steady-state levels of neither chloroplast ribosomal protein transcripts, nor those of the chloroplast ribosomal proteins itself, change significantly during the greening process.

Translational regulation of protein synthesis during light-induced chloroplast development in Euglena

Control of gene expression in Euglena was examined during light-induced chloroplast development. Greening was achieved under standard conditions which allowed the synthesis of all plastid proteins in both cytoplasmic and chloroplastic compartments, or under experimentally modified conditions inducing the preferential synthesis of the photosystem II (PSII) light-harvesting antenna or reaction centers. The relative composition of total mRNAs in cellular, cytoplasmic or chloroplastic fractions, as analyzed by their in-vitro translation products in cell-free systems did not significantly change during the in-vivo protein-synthesis processes which are specific to each greening system. By contrast, cytoplasmic polysomal mRNAs extracted during the selective recovery phase of PSII light-harvesting antennae provided a major in-vitro synthesis product of 28 kDa which could correspond to a precursor of the main 26-kDa apoprotein of the light-harvesting chlorophyll a/b protein complex. Similarly, the in-vivo selective synthesis of the 41-kDa and 51-kDa polypeptides of PSII reaction centers was concomitant with an enrichment of plastid polysomes in mRNA species coding for polypeptides of the same molecular weight. These observations confirm that protein synthesis during chloroplast development in Euglena is weakly regulated at the transcription level and they demonstrate that translational regulation occurs in both the cytoplasmic and the chloroplastic compartments.