A stromal Hsp100 protein is required for normal chloroplast development and function in Arabidopsis

Molecular chaperones are required for the translocation of many proteins across organellar membranes, presumably by providing energy in the form of ATP hydrolysis for protein movement. In the chloroplast protein import system, a heat shock protein 100 (Hsp100), known as Hsp93, is hypothesized to be the chaperone providing energy for precursor translocation, although there is little direct evidence for this hypothesis. To learn more about the possible function of Hsp93 during protein import into chloroplasts, we isolated knockout mutant lines that contain T-DNA disruptions in either atHSP93-V or atHSP93-III, which encode the two Arabidopsis (Arabidopsis thaliana) homologs of Hsp93. atHsp93-V mutant plants are much smaller and paler than wild-type plants. In addition, mutant chloroplasts contain less thylakoid membrane when compared to the wild type. Plastid protein composition, however, seems to be largely unaffected in atHsp93-V knockout plants. Chloroplasts isolated from the atHsp93-V knockout mutant line are still able to import a variety of precursor proteins, but the rate of import of some of these precursors is significantly reduced. These results indicate that atHsp93-V has an important, but not essential, role in the biogenesis of Arabidopsis chloroplasts. In contrast, knockout mutant plants for atHsp93-III, the second Arabidopsis Hsp93 homolog, had a visible phenotype identical to the wild type, suggesting that atHsp93-III may not play as important a role as atHsp93-V in chloroplast development and/or function.     

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.     

Specific lipids influence the import capacity of the chloroplast outer envelope precursor protein translocon

Recent studies demonstrated that lipids influence the assembly and efficiency of membrane-embedded macromolecular complexes. Similarly, lipids have been found to influence chloroplast precursor protein binding to the membrane surface and to be associated with the Translocon of the Outer membrane of Chloroplasts (TOC). We used a system based on chloroplast outer envelope vesicles from Pisum sativum to obtain an initial understanding of the influence of lipids on precursor protein translocation across the outer envelope. The ability of the model precursor proteins p(OE33)titin and pSSU to be recognized and translocated in this simplified system was investigated. We demonstrate that transport across the outer membrane can be observed in the absence of the inner envelope translocon. The translocation, however, was significantly slower than that observed for chloroplasts. Enrichment of outer envelope vesicles with different lipids natively found in chloroplast membranes altered the binding and transport behavior. Further, the results obtained using outer envelope vesicles were consistent with the results observed for the reconstituted isolated TOC complex. Based on both approaches we concluded that the lipids sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylinositol (PI) increased TOC-mediated binding and import for both precursor proteins. In contrast, enrichment in digalactosyldiacylglycerol (DGDG) improved TOC-mediated binding for pSSU, but decreased import for both precursor proteins. Optimal import occurred only in a narrow concentration range of DGDG.     

A mutant deficient in the plastid lipid DGD is defective in protein import into chloroplasts

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol with N-terminal extensions called transit peptides. Transit peptides function as the import signal to chloroplasts. The import process requires several protein components in the envelope and stroma and also requires the hydrolysis of ATP. Lipids have been implicated in the import process based on theories or experiments with in vitro model systems. We show here that chloroplasts isolated from an Arabidopsis mutant deficient in the plastid lipid digalactosyl diacylglycerol (DGD) were normal in importing a chloroplast outer membrane protein, but were defective in importing precursor proteins targeted to the interior of chloroplasts. The impairment includes the binding, or docking, step of the import process that is supported by 100 μM ATP.     

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.     

Photosynthesis in trees: organization of chlorophyll and photosynthetic unit size in isolated gymnosperm chloroplasts

Chloroplasts have been isolated in high yield from several gymnosperms and from two deciduous trees. The organization of chlorophyll in the chloroplasts of these woody species is basically similar to that in angiosperm crop plants and green algae. The tree chloroplasts contain two chlorophyll proteins, the P700-chlorophyll a-protein and the major light-harvesting chlorophyll a/b-protein, the size, spectral characteristics, and function of which are the same as the equivalent complexes previously isolated from other classes of green plants. All the gymnosperms have chlorophyll/P700 ratios (photosynthetic unit sizes) 1.6 to 3.8 times larger than that typically found in crop plants; the deciduous trees have units of intermediary size. The presence of fewer but larger photosynthetic units in the woody species can partially account for their lower photosynthetic rate and explains why their photosynthetic processes saturate at lower light intensities. Chloroplasts of shade needles have large units containing a greater proportion of the light-harvesting chlorophyll a/b-protein than those of sun needles.     

Cryopreservation of chloroplasts and thylakoids for studies of protein import and integration

A method is presented for preservation of isolated intact chloroplasts and isolated thylakoids for use in chloroplast protein import and thylakoid protein integration studies. Chloroplasts of pea (Pisum sativum) were preserved by storage in liquid nitrogen in the presence of a cryoprotective agent. Dimethyl sulfoxide was the most effective of several cryoprotectants examined. Approximately 65 to 70% of chloroplasts stored in liquid nitrogen in the presence of dimethyl sulfoxide remained intact upon thawing and were fully functional for the import of precursor proteins. Imported proteins were correctly localized within these chloroplasts, a process that for two of the proteins tested involved transport into the thylakoids. Lysate obtained from preserved chloroplasts was functional for protein integration assays. Preserved chloroplasts retained import and localization capability for up to 6 months of storage. Thylakoids were preserved by a modification of a method previously described (Farkas DL, Malkin S [1979] Plant Physiol 64: 942-947) for preservation of photosynthetic competence. Preserved thylakoids were nearly as active for protein integration studies as freshly prepared thylakoids. The ability to store chloroplasts and subfractions for extended periods will facilitate investigations of plastid protein biogenesis.     

Chloroplast Distribution in Arabidopsis thaliana (L.) Depends on Light Conditions during Growth

Chloroplasts of Arabidopsis thaliana move in response to blue light. Sensitivity to light and the range of fluence rates to which the chloroplasts respond were found to be comparable to those of other higher plants studied. We investigated typical chloroplast distributions in Arabidopsis grown under three different light conditions:standard-light conditions, similar to natural light intensities; weak-light intensities, close to the compensation point of photosynthesis; and strong-light intensities, close to the saturation of the light-response curve of photosynthesis. We observed a striking difference in chloroplast arrangement in darkness between plants grown under weak- and strong-light conditions. There was a slight difference after weak-light pretreatment, and the arrangements of chloroplasts after strong-light pretreatment in both plant groups were very similar. These results support the ecological significance of chloroplast movements.     

Apparent Inhibition of Chloroplast Protein Import by Cold Temperatures Is Due to Energetic Considerations Not Membrane Fluidity

The transport of proteins across virtually all types of biological membranes has been reported to be inhibited by low temperatures. Paradoxically, plants are able to acclimate to growth at temperatures below which protein import into chloroplasts is known to be blocked. In examining this incongruity, we made a number of unexpected observations. First, chloroplasts isolated from plants grown at 7/1[deg]C in light/dark and from plants grown at 25[deg]C were able to import proteins with the same efficiency over a temperature range from 5 to 21[deg]C, indicating that no functional adaptation had taken place in the protein import machinery of chloroplasts in these cold-grown plants. Second, chloroplasts from warm-grown plants were able to take up proteins at temperatures as low as 4[deg]C provided that they were illuminated. We determined that light mediates the import process at 5[deg]C by driving ATP synthesis in the stroma, the site of its utilization during protein transport. Direct measurement of the envelope phase transition temperature as well as the activity of the ATP/ADP translocator in the inner envelope membrane at 5 and 25[deg]C demonstrated that the cold block of protein import into chloroplasts observed in vitro is due primarily to energetic considerations and not to decreased membrane fluidity.     

Varying competence for protein import into chloroplasts during the cell cycle in Chlamydomonas.

By studying the import of radioactively labelled small subunit of ribulose-1,5-bisphosphate carboxylase (pSS) into chloroplasts of the green alga C. reinhardtii cw-15 protein delivery to chloroplasts was found to vary during the cell cycle. Chloroplasts were isolated from highly synchronous cultures at different time points during the cell cycle. When pSS was imported into 'young' chloroplasts isolated early in the light period about three times less pSS was processed to small subunit SS than in 'mature' chloroplasts from the middle of the light period. In 'young' chloroplasts also, less pSS was bound to the envelope surface. During the second half of the light period the import competence of isolated chloroplasts decreased again when based on chlorophyll content or cell volume, but did not change significantly when related to chloroplast number. Measurements of pSS binding to the surface of chloroplasts of different age indicated that the adaptation of protein import competence during the cell cycle is due to a variation of the number of binding sites per chloroplast surface area, rather than to modulation of the binding constant.     

A simple method for isolating import-competent Arabidopsis chloroplasts

We present a simple, rapid and low-cost method for isolating a high yield of Arabidopsis chloroplasts that can be used to study chloroplast protein import. Efficiency of chloroplast isolation was dependent upon the ratio between amount of plant tissue and the buffer volume, the size and speed of the homogenisation equipment, and the size of the homogenisation beaker. The import method proved useful when characterising different precursor proteins, developmental stages and import-defective mutants. Time-course experiments enabled the measurement of import rates in the linear range. Compared to protoplastation, this isolation method has significant time and cost savings (approximately 80% and approximately 95%, respectively), and yields chloroplasts with a higher capacity to import proteins.     

Protein translocon at the Arabidopsis outer chloroplast membrane.

Chloroplasts are organelles essential for the photoautotrophic growth of plants. Their biogenesis from undifferentiated proplastids is triggered by light and requires the import of hundreds of different precursor proteins from the cytoplasm. Cleavable N-terminal transit sequences target the precursors to the chloroplast where translocon complexes at the outer (Toc complex) and inner (Tic complex) envelope membranes enable their import. In pea, the Toc complex is trimeric consisting of two surface-exposed GTP-binding proteins (Toc159 and Toc34) involved in precursor recognition and Toc75 forming an aequeous protein-conducting channel. Completion of the Arabidopsis genome has revealed an unexpected complexity of predicted components of the Toc complex in this plant model organism: four genes encode homologs of Toc159, two encode homologs of Toc34, but only one encodes a likely functional homolog of Toc75. The availability of the genomic sequence data and powerful molecular genetic techniques in Arabidopsis set the stage to unravel the mechanisms of chloroplast protein import in unprecedented depth.     

In vitro Protein Synthesis by Plastids of Phaseolus vulgaris. III. Formation of Lamellar and Soluble Chloroplast Protein

Chloroplasts from leaves of plants which had been grown in the dark, and then illuminated for 12 hours were isolated, and allowed to incorporate ¹⁴C-leucine into protein, and the products of this incorporation were studied. Lamellar and soluble proteins are the principal products, and are formed in about equal amounts. Only some of the soluble proteins become heavily labeled. Those with highest specific activity have a molecular weight of the order of 140,000, while the higher molecular weight Fraction I protein has a much lower specific activity. The soluble protein as a whole does not serve as a precursor for the lamellar protein, and vice-versa, although a precursor-product relationship between a minor component of the soluble fraction and the lamellar fraction has not been ruled out. The relative protein synthesizing capabilities of chloroplasts and mitochondria are discussed with reference to the data presented.     

Carotenoid-deficient young wheat etioplasts are able to bind precursor proteins on the plastid surface but are impaired in their translocation ability

Young carotenoid-deficient etioplasts, isolated from Norflurazon (NF)-treated wheat seedlings, were used to study the role of coloured carotenoids in the binding and import reactions of different nuclear-encoded plastid proteins. Plastids from control seedlings exhibited significantly higher import efficiencies than did plastids from NF-treated plants. Etioplasts containing normal levels of carotenoids imported approximately 2000 and 800 molecules per plastid of the precursors of the small Rubisco subunit (pSS) and the Rieske FeS protein (pFeS), respectively. Plastids from NF-treated plants imported approximately 100 and 70 pSS and pFeS molecules per plastid, respectively. In addition, a maximum binding capacity of NF-treated plastids of 1200 protein molecules per plastid was observed for both pSS and pFeS when assayed at 25°C: and a maximum binding capacity of approximately 1300 molecules per plastid was noted at 4°C. For control plastids, a similar amount of binding, or approximately 1400 protein molecules per plastid, could only be observed if import was inhibited by low ATP concentrations at 4°C. When these plastids were washed and transferred to conditions promoting import at 25°C and 10 mM Mg-ATP, close to 60% of the envelope-associated precursor protein molecules were imported. These results indicate that control and NF-treated young etioplasts contain similar amounts of binding sites for precursor proteins. However, only in the case of control plastids the binding was productive and lead to import and processing in the stroma upon transfer to conditions promoting import. Plastids isolated from wheat seedlings grown in weak red light and containing different amounts of carotenoids, were assayed for their ability to bind and import a protein with unusual import characteristics, the Chlamydomonas reinhardtii PsaF precursor of PSI (pPsaF) and transit peptide deletion constructs. The PsaF protein was imported in a transit peptide-dependent manner into control etioplasts, whereas import of pPsaF into young wheat etioplasts isolated from NF-treated plants was inhibited at low levels of plastid carotenoids.     

How can organellar protein N-terminal sequences be dual targeting signals? In silico analysis and mutagenesis approach

Organellar nuclear-encoded proteins can be mitochondrial, chloroplastic or localized in both mitochondria and chloroplasts. Most of the determinants for organellar targeting are localized in the N-terminal part of the proteins, which were therefore analyzed in Arabidopsis thaliana. The mitochondrial, chloroplastic and dual N-terminal sequences have an overall similar composition. However, Arg is rare in the first 20 residues of chloroplastic and dual sequences, and Ala is more frequent at position 2 of these two types of sequence as compared to mitochondrial sequences. According to these observations, mutations were performed in three dual targeted proteins and analyzed by in vitro import into isolated mitochondria and chloroplasts. First, experiments performed with wild-type proteins suggest that the binding of precursor proteins to mitochondria is highly efficient, whereas the import and processing steps are more efficient in chloroplasts. Moreover, different processing sites are recognized by the mitochondrial and chloroplastic processing peptidases. Second, the mutagenesis approach shows the positive role of Arg residues for enhancing mitochondrial import or processing, as expected by the in silico analysis. By contrast, mutations at position 2 have dramatic and unpredicted effects, either enhancing or completely abolishing import. This suggests that the nature of the second amino acid residue of the N-terminal sequence is essential for the import of dual targeted sequences.     

Characterization of Arabidopsis glutamine phosphoribosyl pyrophosphate amidotransferase-deficient mutants

Using a transgene-based screening, we previously isolated several Arabidopsis mutants defective in protein import into chloroplasts. Positional cloning of one of the loci, CIA1, revealed that CIA1 encodes Gln phosphoribosyl pyrophosphate amidotransferase 2 (ATase2), one of the three ATase isozymes responsible for the first committed step of de novo purine biosynthesis. The cia1 mutant had normal green cotyledons but small and albino/pale-green mosaic leaves. Adding AMP, but not cytokinin or NADH, to plant liquid cultures partially complemented the mutant phenotypes. Both ATase1 and ATase2 were localized to chloroplasts. Overexpression of ATase1 fully complemented the ATase2-deficient phenotypes. A T-DNA insertion knockout mutant of the ATase1 gene was also obtained. The mutant was indistinguishable from the wild type. A double mutant of cia1/ATase1-knockout had the same phenotype as cia1, suggesting at least partial gene redundancy between ATase1 and ATase2. Characterizations of the cia1 mutant revealed that mutant leaves had slightly smaller cell size but only half the cell number of wild-type leaves. This phenotype confirms the role of de novo purine biosynthesis in cell division. Chloroplasts isolated from the cia1 mutant imported proteins at an efficiency less than 50% that of wild-type chloroplasts. Adding ATP and GTP to isolated mutant chloroplasts could not restore the import efficiency. We conclude that de novo purine biosynthesis is not only important for cell division, but also for chloroplast biogenesis.     

Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts

Hsp70 family proteins function as motors driving protein translocation into mitochondria and the endoplasmic reticulum. Whether Hsp70 is involved in protein import into chloroplasts has not been resolved. We show here Arabidopsis thaliana knockout mutants of either of the two stromal cpHsc70s, cpHsc70-1 and cpHsc70-2, are defective in protein import into chloroplasts during early developmental stages. Protein import was found to be affected at the step of precursor translocation across the envelope membranes. From solubilized envelope membranes, stromal cpHsc70 was specifically coimmunoprecipitated with importing precursors and stoichiometric amounts of Tic110 and Hsp93. Moreover, in contrast with receptors at the outer envelope membrane, cpHsp70 is important for the import of both photosynthetic and nonphotosynthetic proteins. These data indicate that cpHsc70 is part of the chloroplast translocon for general import and is important for driving translocation into the stroma. We further analyzed the relationship of cpHsc70 with the other suggested motor system, Hsp93/Tic40. Chloroplasts from the cphsc70-1 hsp93-V double mutant had a more severe import defect than did the single mutants, suggesting that the two proteins function in parallel. The cphsc70-1 tic40 double knockout was lethal, further indicating that cpHsc70-1 and Tic40 have an overlapping essential function. In conclusion, our data indicate that chloroplasts have two chaperone systems facilitating protein translocation into the stroma: the cpHsc70 system and the Hsp93/Tic40 system.     

Effect of growth temperature on chloroplast structure and activity in barley

Seedlings of barley (Hordeum vulgare L. cv. Abyssinian) were grown at constant temperature and light intensity and the properties and structure of chloroplasts in the primary leaf were examined. Seventeen growth temperatures ranging from 2 to 37 C were employed. Three major effects of the growth temperature were seen. (a) At very low and high growth temperatures chloroplast biogenesis was inhibited. This occurred in plants grown at temperatures above 32 C while growth at 2 C resulted in a mixed population of pale yellow, pale green, and green plants. (b) Chloroplasts were produced at all other temperatures tested but growth temperatures within a few degrees of those inhibitory to chloroplast development resulted in chloroplasts with abnormal properties and structure. Chloroplasts in the green plants grown at 2 and 5 C showed a number of structural peculiarities, including a characteristic crimping of granal thylakoids. Photoreductive activity, measured using ferricyanide as the Hill oxidant in the presence of gramicidin D, was high, but this activity in chloroplasts isolated from plants grown at 2 C showed thermal inactivation at temperatures 5 degrees lower than was the case with plants grown at higher temperatures. High growth temperatures (30 to 32 C) yielded chloroplasts with reduced photoreductive activity and a tendency toward the formation of large grana and disorientation of the lamellar systems with respect to one another. Chloroplasts of the most affected plants (grown at 32 C) frequently contained a very large elongated granum, with narrow intrathylakoid spaces. (c) Photoreductive activity was not constant at intermediate growth temperatures but steadily declined with decreasing growth temperatures between 27 and 11 C. Some alterations in chloroplast structure were also observed.

The changes in chloroplast activity and structure indicate that acclimation to temperature takes place over the entire temperature range in which chloroplast development is permitted.

     

AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis

In plant cells, chloroplasts have essential roles in many biochemical reactions and physiological responses. Chloroplasts require numerous protein components, but only a fraction of these proteins are encoded by the chloroplast genome. Instead, most are encoded by the nuclear genome and imported into chloroplasts from the cytoplasm post-translationally. Membrane proteins located in the chloroplast outer envelope membrane (OEM) have a critical function in the import of proteins into the chloroplast. However, the biogenesis of chloroplast OEM proteins remains poorly understood. Here, we report that an Arabidopsis ankyrin repeat protein, AKR2A, plays an essential role in the biogenesis of the chloroplast OEM proteins. AKR2A binds to chloroplast OEM protein targeting signals, as well as to chloroplasts. It also displays chaperone activity towards chloroplast OEM proteins, and facilitates the targeting of OEP7 to chloroplasts in vitro. AKR2A RNAi in plants with an akr2b knockout background showed greatly reduced levels of chloroplast proteins, including OEM proteins, and chloroplast biogenesis was also defective. Thus, AKR2A functions as a cytosolic mediator for sorting and targeting of nascent chloroplast OEM proteins to the chloroplast.     

Processing of a wheat light-harvesting chlorophyll a/b protein precursor by a soluble enzyme from higher plant chloroplasts

A processing activity has been identified in higher plant chloroplasts that cleaves the precursor of the light-harvesting chlorophyll a/b- binding protein (LHCP). A wheat LHCP gene previously characterized (Lamppa, G.K., G. Morelli, and N.-H. Chua, 1985. Mol. Cell Biol. 5:1370- 1378) was used to synthesize RNA and subsequently the labeled precursor polypeptide in vitro. Incubation of the LHCP precursors with a soluble extract from lysed chloroplasts, after removal of the thylakoids and membrane vesicles, resulted in the release of a single 25-kD peptide. In contrast, when the LHCP precursors were used in an import reaction with intact pea or wheat chloroplasts, two forms (25 and 26 kD) of mature LHCP were found. The peptide released by the processing activity in the organelle-free assay comigrated with the lower molecular mass form of mature LHCP produced during import. Properties of the processing activity suggest that it is an endopeptidase. Chloroplasts from both pea and wheat, two divergent higher plants, contain the processing enzyme, suggesting its physiological importance in LHCP assembly into the thylakoids. We discuss the implications of LHCP precursor processing by a soluble enzyme that may be in the stromal compartment.