Import of nuclear-encoded proteins into carotenoid-deficient young etioplasts

Young etioplasts with different carotenoid contents were assayed for their ability to import in vitro synthesized nuclear-encoded proteins. The plastids were isolated from the basal 1. 5cm of dark-grown wheat seedlings developed from seeds imbibed with 4 different concentrations of Norflurazon. an inhibitor of the carotenoid biosynthesis. Plastids isolated from plants treated with the two highest concentrations. 2. 8 and 28 mg l⁻¹, of Norflurazon contained approximately 10 and 5% of the carotenoid contents, respectively, compared to the control. The total amounts of proteins in these plastids were approximately 68 and 60% compared to control plastids. Translocation assays employing the precursors of the small subunit of ribulose 1. 5-bisphosphate carboxylase/oxygenase (pSS), and the non-Photosynthetic heat-shock protein 21 (pHSP21), showed that the rate of protein import was considerably lower in plastids with low carotenoid contents. The amounts of imported, processed SS were 11 and 10% after 2. 8 and 28 mg 1⁻¹, respectively, compared to the control, whereas the amounts of HSP21 at these herbicide concentrations were 20 and 18%, respectively. The low apparent import in plastids of Norflurazon-treated leaves was not an effect of intraorganellar degradation of imported proteins, nor were there any differences in the amounts of processed, protease-protected protein when Norflurazon was added to the import reaction using control plastids. The low import capabilities are therefore discussed in relation to the possible role of the carotenoids in the translocation of cytosolically synthesized proteins into the plastidic compartment.     

Developmental Regulation of the Plastid Protein Import Apparatus

Plastid development involves the programmed accumulation of proteins. Most plastid proteins are synthesized in the cytosol and imported into the organelle by an envelope-based protein import apparatus. Previous studies have shown that developmental rates of protein accumulation correspond to mRNA levels. Here, we examined the relationship between plastid development and the activity of the protein import apparatus. Developing plastids, primarily from wheat leaves, were analyzed for their protein import capability in vitro. Import capability, initially high in proplastids, declined as much as 20-fold as plastid development approached either the mature etioplast or the mature chloroplast. The observed decline was not due to senescence, nonspecific inhibitors, or protein turnover. Furthermore, the import capability of mature etioplasts, initially very low, was transiently reactivated during light-mediated redifferentiation into chloroplasts. These results suggest that plant cells regulate the import apparatus in concert with the protein demands of the developing plastids.     

Substrate-dependent transport of the NADPH:protochlorophyllide oxidoreductase into isolated plastids.

The key regulatory enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR), is a nuclear-encoded plastid protein. Its post-translational transport into plastids is determined by its substrate. The precursor of POR (pPOR) is taken up and processed to mature size by plastids only in the presence of protochlorophyllide (Pchlide). In etioplasts, the endogenous level of Pchlide saturates the demands for pPOR translocation. During the light-induced transformation of etioplasts into chloroplasts, the Pchlide concentration declined drastically, and isolated chloroplasts rapidly lost the ability to import the precursor enzyme. The chloroplasts' import capacity for the pPOR, however, was restored when their intraplastidic level of Pchlide was raised by incubating the organelles in the dark with delta-aminolevulinic acid, a common precursor of tetrapyrroles. Additional evidence for the involvement of intraplastidic Pchlide in regulating the transport of pPOR into plastids was provided by experiments in which barley seedlings were grown under light/dark cycles. The intraplastidic Pchlide concentration in these plants underwent a diurnal fluctuation, with a minimum at the end of the day and a maximum at the end of the night period. Chloroplasts isolated at the end of the night translocated pPOR, whereas those isolated at the end of the day did not. Our results imply that the Pchlide-dependent transport of the pPOR into plastids might be part of a novel regulatory circuit by which greening plants fine tune both the enzyme and pigment levels, thereby avoiding the wasteful degradation of the imported pPOR as well as photodestruction of free Pchlide.     

Import of lyso-phosphatidylcholine into chloroplasts likely at the origin of eukaryotic plastidial lipids

Plastids rely on the import of extraplastidial precursor for the synthesis of their own lipids. This key phenomenon in the formation of plastidial phosphatidylcholine (PC) and of the most abundant lipids on earth, namely galactolipids, is poorly understood. Various suggestions have been made on the nature of the precursor molecule(s) transferred to plastids, but despite general agreement that PC or a close metabolite plays a central role, there is no clear-cut answer to this question because of a lack of conclusive experimental data. We therefore designed experiments to discriminate between a transfer of PC, 1-acylglycero phosphorylcholine (lyso-PC), or glycerophosphorylcholine. After pulse-chase experiments with glycerol and acetate, plastids of leek (Allium porrum L.) seedlings were purified. The labels of the glycerol moiety and the sn-1- and sn-2-bound fatty acids of plastidial lipids were determined and compared with those associated with the extraplastidial PC. After import, plastid lipids contained the glycerol moiety and the fatty acids esterified to the sn-1 position originating from the extraplastidial PC; no import of sn-2-bound fatty acid was detected. These results rule out a transfer of PC or glycerophosphorylcholine, and are totally explained by an import of lyso-PC molecules used subsequently as precursor for the synthesis of eukaryotic plastid lipids.     

Chloroplast DNA levels and the control of chloroplast division in light-grown wheat leaves

Plastids at different stages of development were isolated from light-grown wheat (Triticum aestivum, var. Maris Dove) seedling leaves, and the average chloroplast DNA (cpDNA) per plastid at each developmental stage was measured directly. In the earliest stages of development, the number of plastids per cell and the amount of cpDNA per cell increased with cell age, but cpDNA per plastid remained constant at between 800 and 1,000 genome copies per plastid. After this phase, plastids per cell continued to increase, but cpDNA per plastid decreased. Subsequently, both plastids per cell and cpDNA per plastid remained constant as cell age increased, the final DNA content being approximately 300 genome copies per plastid. These results are related to previous reports of cpDNA changes during the development of dicotyledonous plants, and to theories about the regulation of chloroplast numbers per cell.     

POR hits the road: import and assembly of a plastid protein

The biosynthesis of chlorophyll is a strictly light-dependent multistep process in higher plants. The light-dependent step is catalysed by NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1), which reduces protochlorophyllide (Pchlide) to chlorophyllide (Chlide). POR is nucleus-encoded and post-translationally imported into plastids. It has been proposed that the import of a POR protein isozyme (PORA) is totally dependent on Pchlide and uses a novel import pathway. This proposal is based on findings that PORA import only occurs in the presence of Pchlide and that the presence of overexpressed precursor of Rubisco small subunit (pSS), a protein which is known to use the general import pathway, does not outcompete PORA import. Another study demonstrated that POR precursor protein (pPOR) can be cross-linked to one of the components in the translocation machinery, Toc75, in the absence of Pchlide, and that its import can be outcompeted by the addition of the pSS. This indicates that pSS and pPOR may use the same translocation mechanism. Thus, POR does not necessarily need Pchlide for import – which is in contrast to earlier observations – and the exact POR import mechanism remains unresolved. Once in the stroma, the POR transit peptide is cleaved off and the mature POR protein is associated to the plastid inner membranes. Formation of the correct membrane–associated, thermolysin-protected assembly is strictly dependent of NADPH. As a final step, the formation of the NADPH-Pchlide-POR complex occurs. When POR accumulates in the membranes of proplastids, an attraction of monogalactosyl diacylglycerol (MGDG) can occur, leading to the formation of prolamellar bodies (PLBs) and the development of etioplasts in darkness.     

Protochlorophyllide-independent import of two NADPH:Pchlide oxidoreductase proteins (PORA and PORB) from barley into isolated plastids

The enzyme catalysing the reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide), NADPH:Pchlide oxidoreductase (POR; EC 1.6.99.1), is a nuclear-encoded protein that is post-translationally imported to the plastid. In barley and Arabidopsis thaliana , the reduction of Pchlide is controlled by two different PORs, PORA and PORB. To characterise the possible Pchlide dependency for the import reaction, radiolabelled precursor proteins of barley PORA and PORB (pPORA and pPORB, respectively) were used for in vitro assays with isolated plastids of barley and pea with different contents of Pchlide. To obtain plastids with different endogenous levels of Pchlide, several methods were used. Barley plants were grown in darkness or in greenhouse conditions for 6 days. Alternatively, greenhouse-grown pea plants were incubated for 4 days in darkness before plastid isolation, or chloroplasts isolated from greenhouse-grown plants were incubated with Δ -aminolevulinic acid (ALA), an early precursor in the Chl biosynthesis resulting in elevated Pchlide contents in the plastids. Both barley pPORA and pPORB were effectively imported into barley and pea chloroplasts isolated from the differentially treated plants, including those isolated from greenhouse-grown plants. The absence or presence of Pchlide did not significantly affect the import capacity of barley pPORA or pPORB. Assays performed on stroma-enriched fractions from chloroplasts and etioplasts of barley indicated that no post-import degradation of the proteins occurred in the stroma, irrespective of whether the incubation was performed in darkness or in light.     

Members of the Toc159 import receptor family represent distinct pathways for protein targeting to plastids

Plastids represent a diverse group of organelles that perform essential metabolic and signaling functions within all plant cells. The differentiation of specific plastid types relies on the import of selective sets of proteins from among the approximately 2500 nucleus-encoded plastid proteins. The Toc159 family of GTPases mediates the initial targeting of proteins to plastids. In Arabidopsis thaliana, the Toc159 family consists of four genes: atTOC159, atTOC132, atTOC120, and atTOC90. In vivo analysis of atToc159 function indicates that it is required specifically for the import of proteins necessary for chloroplast biogenesis. In this report, we demonstrate that atToc120 and atToc132 represent a structurally and functionally unique subclass of protein import receptors. Unlike atToc159, mutants lacking both atToc120 and atToc132 are inviable. Furthermore, atToc120 and atToc132 exhibit preprotein binding properties that are distinct from atToc159. These data indicate that the different members of the Toc159 family represent distinct pathways for protein targeting to plastids and are consistent with the hypothesis that separate pathways have evolved to ensure balanced import of essential proteins during plastid development.     

Characterisation of the assembly pathway of the pea NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR), with emphasis on the role of its substrate, Pchlide

The homologous import and membrane association of a key enzyme for chlorophyll biosynthesis, the NADPH:protochlorophyllide (Pchlide) oxidoreductase (POR, EC 1.6.99.1) into pea chloroplasts was investigated in vitro. The co-factor, NADPH, decreased binding of the precursor protein (pPOR) to the envelope membranes in the presence of ATP. The decrease of the binding reaction with NADPH was not observed with the precursor of the small subunit of Rubisco (pSS).
To investigate possible substrate-dependency for the import reaction, internal Pchlide concentrations in the plastids were raised by either an addition of δ -aminolevulinic acid to isolated plastids or etiolation of the seedlings prior to plastid isolation. Increased amounts of plastid-bound Pchlide gave no observable differences in POR import.
The capacity of POR and 11 different POR mutants, carrying charged-to-alanine scanning substitutions, to form a catalytically active POR-Pchlide-NADPH complex and to associate with the thylakoid membranes in a protease-resistant way were tested. Wild-type POR, as well as the mutants with charge substitutions in the N-terminal region of the protein, exhibited higher catalytic activity than the POR mutants carrying substitutions in the C-terminal region. Formation of a catalytically active complex did not, however, increase the association efficiency onto the thylakoids. We can, therefore, postulate that the import of pea POR into pea chloroplasts was not substrate-dependent, nor did formation of catalytically active complexes stimulate or inhibit the membrane association reaction of POR.     

Two NADPH:Protochlorophyllide Oxidoreductases in Barley: Evidence for the Selective Disappearance of PORA during the Light-Induced Greening of Etiolated Seedlings

Chlorophyll synthesis in barley is controlled by two different light-dependent NADPH:protochlorophyllide oxidoreductases, termed PORA and PORB. PORA is present abundantly in etioplasts but selectively disappears soon after the beginning of illumination. This negative light effect is mediated simultaneously at three different levels. First, the concentration of porA mRNA declines drastically during illumination of dark-grown seedlings. Second, the plastids' ability to import the precursor of PORA (pPORA) is reduced during the transition from etioplasts to chloroplasts. This effect is due to a rapid decline in the plastidic level of protochlorophyllide (Pchlide), which is required for the translocation of the pPORA. Third, PORA becomes selectively destabilized in illuminated seedlings. When illuminated, PORA-Pchlide-NADPH complexes formed in the dark photoreduce their Pchlide to Chlide and become simultaneously susceptible to attack by plastid proteases. The PORA-degrading protease activity is not detectable in etioplasts but is induced during illumination. In contrast to PORA, the second Pchlide-reducing enzyme, PORB, remains operative in both illuminated and green plants. Its translocation into plastids does not depend on its substrate, Pchlide.     

Root Graviresponsiveness and Columella Cell Structure in Carotenoid-deficient Seedlings of Zea mays

Root graviresponsiveness in normal and carotenoid-deficientmutant seedlings of Zea mays was not significantly different.Columella cells in roots of mutant seedlings were characterizedby fewer, smaller, and a reduced relative volume of plastidsas compared to columella cells of normal seedlings. Plastidsin columella cells of mutant seedlings possessed reduced amountsof starch. Although approximately 10 per cent of the columellacells in mutant seedlings lacked starch, their plastids werelocated at the bottom of the cell. These results suggest that(i) carotenoids are not necessary for root gravitropism, (ii)graviresponsiveness is not necessarily proportional to the size,number, or relative volume of plastids in columella cells, and(iii) sedimentation of plastids in columella cells may not resultdirectly from their increased density due to starch content.Plastids in columella cells of normal and mutant seedlings wereassociated with bands of microtubule-like structures, suggestingthat these structures may be involved in ‘positioning’plastids in the cell. Zea mays, graviperception, graviresponsiveness, carotenoids, vp-9 mutant, columella cell, roots     

Cytoplasmic kinase and phosphatase activities can induce PsaF gene expression in the absence of functional plastids: evidence that phosphorylation/dephosphorylation events are involved in interorganellar crosstalk

PsaF is a nuclear gene for subunit III of the reaction center of photosystem I, and its expression is stimulated by cytokinins and light, when monitored at the mRNA level or at the level of GUS activity directed by chimeric promoter::uidA gene fusions in transgenic tobacco. These inductive effects can be mimicked by pertussis toxin, serotonin, phorbol acetate myristate or Ca2+, suggesting the involvement of heterotrimeric G proteins, phospholipids and Ca2+-dependent processes. Both breakdown products of the phosphatidylinositol cycle, inositol triphosphate (IP3) and diacylglycerol (or its homolog phorbol myristate acetate, PMA) appear to be involved. The IP3-dependent pathway requires kinase activity, and the signal operates via a 42-bp Ca2+-responsive element located between positions -220 and -178, while the PMA-dependent pathway requires phosphatase activity and a binding element that lies further upstream in the promoter. The effects of G proteins, phospholipids and Ca2+ on GUS gene expression are restricted to tissues with functional plastids, while modulation of phosphatase and kinase activities activates the responsive PsaF promoter regions even in photobleached material. Thus, activation of kinases and phosphatases can bypass the plastid-mediated inhibition of PsaF gene expression in tobacco seedlings. One cytoplasmic target which reflects the functional state of the plastids is protein kinase C. The enzyme can be efficiently phosphorylated in protein extracts from seedlings in which plastid function is impaired, but not in extracts from green tissue.     

A role of Toc33 in the protochlorophyllide-dependent plastid import pathway of NADPH:protochlorophyllide oxidoreductase (POR) A

NADPH:protochlorophyllide oxidoreductase (POR) A is a key enzyme of chlorophyll biosynthesis in angiosperms. It is nucleus-encoded, synthesized as a larger precursor in the cytosol and imported into the plastids in a substrate-dependent manner. Plastid envelope membrane proteins, called protochlorophyllide-dependent translocon proteins, Ptcs, have been identified that interact with pPORA during import. Among them are a 16-kDa ortholog of the previously characterized outer envelope protein Oep16 (named Ptc16) and a 33-kDa protein (Ptc33) related to the GTP-binding proteins Toc33 and Toc34 of Arabidopsis. In the present work, we studied the interactions and roles of Ptc16 and Ptc33 during pPORA import. Radiolabeled Ptc16/Oep16 was synthesized from a corresponding cDNA and imported into isolated Arabidopsis plastids. Crosslinking experiments revealed that import of 35S-Oep16/Ptc16 is stimulated by GTP. 35S-Oep16/Ptc16 forms larger complexes with Toc33 but not Toc34. Plastids of the ppi1 mutant of Arabidopsis lacking Toc33, were unable to import pPORA in darkness but imported the small subunit precursor of ribulose-1,5-bisphosphate carboxylase/oxygenase (pSSU), precursor ferredoxin (pFd) as well as pPORB which is a close relative of pPORA. In white light, partial suppressions of pSSU, pFd and pPORB import were observed. Our results unveil a hitherto unrecognized role of Toc33 in pPORA import and suggest photooxidative membrane damage, induced by excess Pchlide accumulating in ppi1 chloroplasts because of the lack of pPORA import, to be the cause of the general drop of protein import.     

Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts.     

Influence of Cell Age on Chlorophyll Formation in Light-grown and Etiolated Wheat Seedlings

A method is described for relating the age of a cereal leaf cell to its distance from the leaf base. The rates of chlorophyll synthesis per plastid in the first leaf of light-grown and of greening etiolated seedlings of wheat (Triticum aestivum, var. Maris Dove) increase with cell age. Normally developing plastids of light-grown wheat take over 24 hours to reach the chlorophyll a/b ratio characteristic of mature wheat chloroplasts (4.5), but mature etioplasts need only 8 hours light to achieve this a/b ratio. Plastid greening potential depends only on cell age, whereas the chlorophyll a/b ratio is influenced both by cell age and by light.     

Influence of nitrogen sources on chloroplast development in wheat seedlings

The effect of different nitrogen sources (ammonium, nitrate or both ions together) on plastid development in dark-grown and illuminated seedlings of wheat ( Triticum vulgare L. cv. Yecora) has been investigated. Plastids of plants grown in ammonium showed even in the dark a larger internal membrane length, higher ribulose bisphos-phate carboxylase activity and greater content of soluble proteins than plastids of plants grown in nitrate. After the first hour of illumination rudimentary thylakoids showing some joining points were observed in the ammonium plastids. After 10 h no prolamellar bodies were seen in the ammonium plastids, and the internal plastid membrane length was greater than in the other treatments. There was no light-induced increase in protein synthesis after illumination for 1 h. After 10 h the increase observed in protein synthesis was not followed by a response in the enzyme activity in any of the treatments. After 20 h the lag in the induction of ribulose bisphosphate carboxylase ceased, the enzyme activity and soluble proteins being higher in the leaves of ammonium seedlings than in those from nitrate. From the correlation obtained between the ultrastructural electron microscope observations and the enzymatic studies, it appears that ammonium nutrition has a positive influence on the formation of the plastid membrane system and on the onset of photosynthesis and, consequently, on the development of chloroplasts.     

Plastid stromules are induced by stress treatments acting through abscisic acid

Stromules are highly dynamic stroma-filled tubules that extend from the surface of all plastid types in all multi-cellular plants examined to date. The stromule frequency (percentage of plastids with stromules) has generally been regarded as characteristic of the cell and tissue type. However, the present study shows that various stress treatments, including drought and salt stress, are able to induce stromule formation in the epidermal cells of tobacco hypocotyls and the root hairs of wheat seedlings. Application of abscisic acid (ABA) to tobacco and wheat seedlings induced stromule formation very effectively, and application of abamine, a specific inhibitor of ABA synthesis, prevented stromule induction by mannitol. Stromule induction by ABA was dependent on cytosolic protein synthesis, but not plastid protein synthesis. Stromules were more abundant in dark-grown seedlings than in light-grown seedlings, and the stromule frequency was increased by transfer of light-grown seedlings to the dark and decreased by illumination of dark-grown seedlings. Stromule formation was sensitive to red and far-red light, but not to blue light. Stromules were induced by treatment with ACC (1-aminocyclopropane-1-carboxylic acid), the first committed ethylene precursor, and by treatment with methyl jasmonate, but disappeared upon treatment of seedlings with salicylate. These observations indicate that abiotic, and most probably biotic, stresses are able to induce the formation of stromules in tobacco and wheat seedlings.     

Early and late plastid development in response to chill stress and heat stress in wheat seedlings

Five-day-old etiolated wheat (Triticum aestivum L.) seedlings were transferred to 7°C (chill stress), 25°C (control), and 42°C (heat stress) and were kept in the dark or light for different time periods. Plastids were isolated from the control and stressed seedlings, and their low-temperature (77 K) fluorescence emission spectra were monitored. Most of the Protochlorophyllide (Pchlide) present in heat-stressed etiolated seedlings were in nonphototransformable form. The phototransformable Pchlide (F657) rapidly decreased when 5-day-old etiolated seedlings were transferred to 42°C in the dark for 24 h. A flash illumination of 0.2 s given to etiolated heat-stressed seedlings resulted in substantial arrest of Shibata shift, while in chill-stress conditions, it was only partially affected. In high temperature, due to disaggregation of polymeric Pchlide–Pchlide oxidoreductase (POR)–nicotinamide adenine dinucleotide phosphate (NADPH) molecules, the conversion of nonphototransformable Pchlide to its phototransformable form is substantially delayed resulting in impaired Shibata shift and belated development of the core antenna CP47 Photosystem II (PSII). Chill stress, however, did not disaggregate the polymeric Pchlide–POR–NADPH molecule-suppressed Pchlide and Chl synthesis and impaired of the assembly of PSII core antenna CP47 that emits F695 and PSI that emits F735. The decreased gene/protein expression and reduced posttranslational import of plastidic proteins, importantly POR in temperature-stressed plants, may be responsible for the delay in conversion of nonphototransformable to phototransformable form of Pchlide and plastid biogenesis.     

The physical and functional borders of transit peptide-like sequences in secondary endosymbionts

Background  

Plastids rely on protein supply by their host cells. In plastids surrounded by two membranes (primary plastids) targeting of these proteins is facilitated by an N-terminal targeting signal, the transit peptide. In secondary plastids (surrounded by three or four membranes), transit peptide-like regions are an essential part of a bipartite topogenic signal sequence (BTS), and generally found adjacent to a N-terminally located signal peptide of the plastid pre-proteins. As in primary plastids, for which no wealth of functional information about transit peptide features exists, the transit peptide-like regions used for import into secondary ones show some common features only, which are also poorly characterized.