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.     

Purification and Characterization of a Novel Aminopeptidase,Plastidial Alanine-Aminopeptidase,from the Cotyledons of Etiolated Sugar Beet Seedlings

During prolonged dark growth of sugar beet (Beta vulgaris L.) seedlings, etioplasts, rapidly after the proplastid-etioplast transition, undergo a degenerative process characterized by ultrastructural modifications, protein loss, and the decrease of carotenoid and chlorophyll accumulation upon illumination. Two plastidial aminopeptidase activities were identified as early markers of this degenerative process (A. El Amrani, I. Couee, J.-P. Carde, J.-P. Gaudillere, P. Raymond [1994] Plant Physiology 106: 1555-1565). The present study focuses on one of these markers and describes the purification to homogeneity and characterization of plastidial alanine-aminopeptidase. This novel aminopeptidase was found to be a metallo-type naphthylamidase particularly active with alanyl, arginyl, and leucyl substrates. Its plastidial location was confirmed by immunofluorescence with polyclonal antibodies against the purified enzyme. Its physico-chemical and enzymic properties are discussed with respect to other higher plant aminopeptidases and to its potential functions during prolonged dark growth.     

THE ULTRASTRUCTURAL MORPHOLOGY AND ONTOGENY OF OAT COLEOPTILE PLASTIDS

Structurally similar proplastids occur in the shoot, scutellum, and root of the oat embryo at the start of germination. These proplastids follow several pathways of differentiation, depending on their location within an organ and on previous exposure to light. During the first 24 hr of germination morphologically similar amyloplasts are formed from the preexisting proplastids in most of the cells of the seedling. After about 24 hr in the light, unique chloroplasts begin to develop in a subepidermal ring of small cortical parenchyma cells in the coleoptile and give the organ a pale green color. At 48 and 72 hr the coleoptile chloroplasts and etioplasts are conspicuously different from the corresponding leaf plastids in morphology and ontogeny but contain typical photosynthetic grana and prolamellar bodies. Study of the ontogeny of plastids in the epidermal and nongreening parenchymal regions of dark grown coleoptiles shows that these plastids undergo significant losses in starch content, and some increase of membranes within the plastid, related to the age of the cell. Light has little effect on the structure of these plastids. It is suggested that the ontogeny of all the plastid types of the oat seedling begins with a common precursor—a relatively simple proplastid that is present at the time of germination. Starch grains showing two distinct types of erosion, apparently enzymatic, were observed in oat coleoptile plastids. In one type (grooved appearance) the starch grains are consistently associated with plastid membranes, while in the other type (irregular, spiny appearance) the starch grains are associated with the plastid stroma only. We suggest that there are two enzyme systems for metabolizing starch in oat plastids—one membrane-bound and the other free in the stroma.     

Involvement of nitric oxide in light-mediated greening of barley seedlings

When seedlings are grown in the dark, proplastids of the developing leaf differentiate into etioplasts. Greening of etiolated plastids is stimulated by light, which is sensed by various types of photoreceptors. Nitric oxide (NO) has been shown to be a bioactive molecule that could take part in this light-mediated process in plants. In this paper, we show that emission of NO in barley seedlings increased concomitantly with increasing activities of nitric oxide synthase (NOS) during the greening. Treatment with sodium nitroprusside (SNP), a NO donor, increased the accumulation of chlorophyll contents, enhanced the accumulation of thylakoid membrane proteins, such as light harvesting complex of photosystem II (LHCII) and PSIA/B, and then improved the effective quantum yield of photosystem II (PSII) (Phi(PSII)) in the light. Instead, treatment with either NO scavenger 2-phenyl-4,4,5,5-tetramentylimidazoline-1-oxyl-3-xide (PTIO) or NOS inhibitor N(omega)-nitro-l-arginine (l-NNA) retarded the greening of etiolated-seedlings. Moreover, sodium ferrocyanide, an analog of SNP, nitrite and nitrate, two NO-decomposition products did not have any effect on the greening process. These results indicated that NO, as an endogenous signaling molecule, participates in light-mediated greening of barley seedlings, and exogenous NO accelerates this process.     

CHLOROPLAST DEVELOPMENT DURING SPOROGENESIS IN SIX SPECIES OF MOSSES

Chloroplast development during sporogenesis in Mnium cuspidatum, M. medium, M. rostratum, Aulacomnium heterostichum, Bartramia pomiformis, and Timmia megapolitana is as follows: During the early mitotic divisions in the sporogenous area of the capsule the number of plastids is reduced from many to one cup-shaped plastid per sporogenous cell. This single plastid divides during the early spore-mother-cell stage. A second division of plastids produces four plastids within each spore-mother-cell. A massive accumulation of starch occurs within each of the four plastids. Following meiosis, the single plastid allocated to each spore produces distinct lobes that are “blebbed” off as proplastids. A photosynthetic membrane system is established within the many proplastids as each spore matures.     

Preferential digestion of chloroplast nuclei (nucleoids) during senescence of the coleoptile ofOryza sativa

Summary The coleoptile ofOryza sativa develops, grows and ages within 4 days that follow imbibition. It is, thus, a very useful system for experimental analysis of the life cycle of organelles, for example, the development, growth and aging of plastids in higher plants. We examined the behavior and levels of DNA and chlorophyll in the plastid by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified-photon counting system (VIMPCS). The whitish yellow coleoptile appeared soon after imbibition and, between the first 24 and 60 h that followed imbibition, it grew markedly in a longitudinal direction, with concomitant elongation of the cells, and an increase in the volume of plastids and in the amount of DNA in the plastids. The chlorophyll content per plastid began to increase when the coleoptile turned green, 48 h after imbibition, and reached a plateau value when the coleoptile was 3.5 mm in length, 72 h after imbibition. More than 12 h later, the chlorophyll disappeared just before the breakdown of chloroplasts was initiated. Proplastids in young coleoptiles, contained a plastid nucleus which was located in the central area of the plastids and each nucleus consisted of approximately 6 copies of plastid DNA (ptDNA). The number of copies of ptDNA per plastid increased gradually, with a concomitant increase in the volume of the plastids after imbibition, and reached approximately 130 times the value in the young proplastids, 60 h after imbibition, when the plastid developed into a chloroplast. However, each plastid nucleus did not scatter throughout the entire interior region of each chloroplast. The disappearance of each plastid nucleus occurred more than 12 h before the degeneration of the chloroplasts. The number of plastids per cell increased from 10 to 15 in young coleoptiles within 12 h after imbibition. Yet the number remained constant throughout subsequent growth and aging of the coleoptile. Thus the preferential reduction in the amount of chloroplast DNA was not due to the division of the plastid but could, perhaps, be associated directly with the aging of the cells of the coleoptile which precedes senescence of the coleoptiles.     

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.     

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.     

Stromules: a characteristic cell-specific feature of plastid morphology

Stromules (stroma-filled tubules) are highly dynamic structures extending from the surface of all plastid types examined so far, including proplastids, chloroplasts, etioplasts, leucoplasts, amyloplasts, and chromoplasts. Stromules are usually 0.35-0.85 microm in diameter and of variable length, from short beak-like projections to linear or branched structures up to 220 mum long. They are enclosed by the inner and outer plastid envelope membranes and enable the transfer of molecules as large as Rubisco (approximately 560 kDa) between interconnected plastids. Stromules occur in all cell types, but stromule morphology and the proportion of plastids with stromules vary from tissue to tissue and at different stages of plant development. In general, stromules are more abundant in tissues containing non-green plastids, and in cells containing smaller plastids. The primary function of stromules is still unresolved, although the presence of stromules markedly increases the plastid surface area, potentially increasing transport to and from the cytosol. Other functions of stromules, such as transfer of macromolecules between plastids and starch granule formation in cereal endosperm, may be restricted to particular tissues and cell types.     

Isolation of Plastids from Sunflower Cotyledons during Germination

Plastids from cotyledons of sunflower (Helianthus annus L.) seedlings, germinated in the dark or in the light, were isolated by isopycnic sucrose density gradient centrifugation. At all stages of development the whole plastids contained triose phosphate isomerase, NADPH-glyoxylate reductase, and l-dihydroxyphenylalanine oxidase, which were used as marker enzymes. At the beginning of germination the isopycnic density of whole plastids (proplastids) was about 1.22 g cm⁻³. During development of proplastids into etioplasts in the dark, their isopycnic density increased to 1.26 g cm⁻³. During exposure of germinating seedlings to white light for 2 days, the isopycnic density of whole plastids decreased from 1.26 to 1.22 g cm⁻³. These changes in isopycnic density of plastids on sucrose density gradients are consistent with changes in the plastid ultrastructure caused by the protein-rich prolamellar body or by the lipid-rich thylakoids. Broken plastids (thylakoids), determined by the main peak of chlorophyll, increased in isopycnic density from less than 1.14 to about 1.17 g cm⁻³ during illumination. During germination no major changes occurred in the isopycnic density of mitochondria. Microbodies had an isopycnic density of 1.24 g cm⁻³ in very early stages of germination, and their density increased to 1.265 g cm⁻³, when glyoxysomal enzymes reached maximum development.     

Protoporphyrinogen oxidation in chloroplasts and plant mitochondria,a step in heme and chlorophyll synthesis

The oxidation of protoporphyrinogen to protoporphyrin was demonstrated in greening plastids and mitochondria from greening barley shoots. The plastids, purified by sucrose gradient centrifugation, were essentially free of a mitochondrial marker enzyme. The plastid activity was destroyed by mild heating and was proportional to plastid concentration suggesting, an enzymatic reaction. Uroporphyrinogen I was not oxidized at an appreciable rate. Activity was also demonstrated in etioplasts and mitochondria from dark-grown barley, and in chloroplasts from commercial spinach leaves. The chelating agent 1,10-phenanthroline partially decreased activity in plant organelles, but cyanide did not. The plastid activity, like the activity in liver mitochondria, was readily demonstrable at pH 8.4 in the presence of glutathione as reducing agent. However, the plastid activity was markedly enhanced by assay at pH 7.0 and the absence of reducing agents. These properties distinguish the activity in plants from that previously described in mammalian mitochondria and photosynthetic bacteria.     

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.     

Ultrastructure of Protein Bodies and Plastids in Cotyledon of Nelumbo nucifera Gaertn.at Seed Germination

The present article deals mainly with the formation and dissolution of protein bodies and development of plastids in cotyledon cells of Nelumbo nucifera during seed germination. Electron microscopic studies reveal that protein bodies are formed after imbibition of the cotyledons before germination. They are produced through accumulation of protein material in small vacuoles delivered from the exudates of endoplasmic reticulum or by fragmentation of endoplasmic reticulum itself. In the period of germination, most of the material in the protein bodies dissolute and they coalesce with each other forming large vacuoles. The protein residue of the vacuoles condenses into small blocks with high electron density adhering to the tonoplast or freely floating in the vacuole. Thus, it suggests that the protein bodies of the germinating N. nucifera cotyledons are originated from vacuoles formed by endoplasmic reticulum. Part of the plastids found in cotyledonous cells of mature N. nucifera seeds exists as proplastids. They develop continuously after imbibition of the cotyledons. During the period of seed germination, many concentric lamellae are developed along the plastid membrane on which they later coalesce with the neighboring concentric lameUae forming loosely organized prolamellar bodies which condense into paracrystalline lattices. No ribosomes are present in the inter spaces of paracrystatline lattice. One to several prolamellar bodies can be developed in one plastid.     

Fatty acid synthesis and the oxidative pentose phosphate pathway in developing embryos of oilseed rape (Brassica napus L.)

The potential role of the plastidial oxidative pentose phosphate pathway (OPPP) in providing the NADPH for fatty acid synthesis in plastids from developing embryos of Brassica napus (L.) has been investigated. Measurements of distributions of enzyme activities in fractions obtained from homogenates of isolated embryos have revealed that the glucose 6-phosphate and 6-phosphogluconate dehydrogenases are present in both cytosol and plastid, as is ribose 5-phosphate isomerase. However, transketolase and transaldolase are most probably confined to the plastid, while ribulose 5-phosphate epimerase is essentially cytosolic, although a very small proportion of plastid-localized activity cannot be ruled out. The activity of the OPPP in intact plastids was measured by the release of (14)CO(2) from [1-(14)C]glucose 6-phosphate. Activity was detectable in the absence of electron sinks created by the addition of metabolites to the incubation media and was stimulated 1.3-, 3.2-, and 7.9-fold by the respective additions of glutamine plus 2-oxoglutarate, cofactors and substrates for fatty acid synthesis, or methyl viologen. An increase in OPPP activity in response to additions that are absolutely required for fatty acid synthesis in these isolated plastids provides direct evidence that these two processes are connected, most probably by NADP/NADPH metabolism. The OPPP activity with methyl viologen was more than twice that during fatty acid synthesis, suggesting that the latter is not limited by OPPP capacity. Light energy may also contribute to reductant provision and, consistent with the possibility of maintenance of a balance of NADPH from light and the OPPP, glucose 6-phosphate dehydrogenase activity in the isolated plastids was decreased by light or by DTT.     

High-frequency transformation of undeveloped plastids in tobacco suspension cells

Although leaf chloroplast transformation technology was developed more than a decade ago, no reports exist of stable transformation of undeveloped plastids or other specialized plastid types, such as proplastids, etioplasts, or amyloplasts. In this work we report development of a dark-grown tobacco suspension cell model system to investigate the transformation potential of undeveloped plastids. Electron microscope analysis confirmed that the suspension cells carry plastids that are significantly smaller (approximately 50-fold less in volume) and have a very different subcellular localization and developmental state than leaf cell chloroplasts. Using antibiotic selection in the light, we demonstrated that both plastid and nuclear transformation of these cell suspensions is efficient and reproducible, with plastid transformation frequency at least equal to that of leaf chloroplast transformation. Homoplasmic plastid transformants are readily obtained in cell colonies, or in regenerated plants, providing a more consistent and versatile model than the leaf transformation system. Because of the uniformity of the cell suspension model, we could further show that growth rate, selection scheme, particle size, and DNA amount influence the frequency of transformation. Our results indicate that the rate-limiting steps for nuclear and plastid transformation are different, and each must be optimized separately. The suspension cell system will be useful as a model for understanding transformation in those plant species that utilize dark-grown embryogenic cultures and for characterizing the steps that lead to homoplasmic plastid transformation.     

Identification of the Ndh (NAD(P)H-plastoquinone-oxidoreductase) complex in etioplast membranes of barley: changes during photomorphogenesis of chloroplasts

In the last few years the presence in thylakoid membranes of chloroplasts of a NAD(P)H-plastoquinone oxidoreductase complex (Ndh complex) homologous to mitochondrial complex I has been well established. Herein, we report the identification of the Ndh complex in barley etioplast membranes. Two plastid DNA-encoded polypeptides of the Ndh complex (NDH-A and NDH-F) were relatively more abundant in etioplast membranes than in thylakoids from greening chloroplasts. Conversion of etioplast into chloroplast, after light exposure of barley seedlings grown in the dark, was accompanied by a decrease in the NADH dehydrogenase activity associated to plastid membranes. Using native-PAGE and immunolabelling techniques we have determined that a NADH specific dehydrogenase activity associated with plastid membranes, which was more active in etioplasts than in greening chloroplasts, contained the NDH-A and NDH-F polypeptides. These results complemented by those obtained through blue-native-PAGE indicated that NDH-A and NDH-F polypeptides are part of a 580 kDa NADH dependent dehydrogenase complex present in etioplast membranes. This finding proves that accumulation of the Ndh complex is independent of light. The decrease in the relative levels and specific activity of this complex during the transition from etioplast to chloroplasts was accompanied by a parallel decrease in the specific activity of peroxidase associated to plastid membranes. Based on the mentioned observations it is proposed that an electron transport chain from NADH to H2O2 could be active in barley etioplasts.     

Light Intensity Affects Chlorophyll Synthesis During Greening Process by Metabolite Signal from Mitochondrial Alternative Oxidase in Arabidopsis

Although mitochondrial alternative oxidase(AOX)has been proposed to play essential roles in high light stress tolerance,the effects of AOX on chlorophyll synthesis are unclear.Previous studies indicated that during greening,chlorophyll accumulation was largely delayed in plants whose mitochondrial cyanide-resistant respiration was inhibited by knocking out nuclear encoded AOX gene.Here we show that this delay of chlorophyll accumulation was more significant under high light condition.Inhibition of cyanide-resistant respiration was also accompanied by the increase of plastid NADPH/NADP~+ratio,especially under high light treatment which subsequently blocked the import of multiple plastidial proteins,such as some components of the photosynthetic electron transport chain,the Calvin-Benson cycle enzymes and malate/oxaloacetate shuttle components.Over expression of AOXla rescued the aoxla mutant phenotype,including the chlorophyll accumulation during greening and plastidial protein import.It thus suggests that light intensity affects chlorophyll synthesis during greening process by a metabolic signal,the AOX-derived plastidial NADPH/NADP~+ratio change.And our results thus revealed a molecular mechanism of chloroplast-mitochondria interactions.     

Plastid differentiation and chlorophyll biosynthesis in different leaf layers of white cabbage (Brassica oleracea cv. capitata)

The contents of protochlorophyllide, protochlorophyll and chlorophyll together with the native arrangements of the pigments and the plastid ultrastructure were studied in different leaf layers of white cabbage (Brassica oleracea cv. capitata) using absorption, 77 K fluorescence spectroscopy and transmission electron microscopy. The developmental stage of the leaves was determined using the differentiation of the stoma complexes as seen by scanning electron microscopy and light microscopy. The pigment content showed a gradual decrease from the outer leaf layer towards the central leaves. The innermost leaves were in a primordial stage in many aspects; they were large but had typical proplastids with few simple inner membranes, and contained protochlorophyllide and its esters in a 2 : 1 ratio and no chlorophyll. Short‐wavelength, not flash‐photoactive protochlorophyllide and/or protochlorophyll forms emitting at 629 and 636 nm were dominant in the innermost leaves. These leaves also had small amounts of the 644 and 654 nm emitting, flash‐photoactive protochlorophyllide forms. Rarely prolamellar bodies were observed in this layer. The outermost leaves had the usual characteristics of fully developed green leaves. The intermediary layers contained chlorophyll a and chlorophyll b besides the protochlorophyll(ide) pigments and had various intermediary developmental stages. Spectroscopically two types of intermediary leaves could be distinguished: one with only a 680 nm emitting chlorophyll a form and a second with bands at 685, 695 and 730 nm, corresponding to chlorophyll–protein complexes of green leaves. In these leaves, a large variety of chloroplasts were found. The data of this work show that etioplasts, etio‐chloroplasts or chloro‐etioplasts as well as etiolated leaves do exist in the nature and not only under laboratory conditions. The specificity of cabbage leaves compared with those of dark‐grown seedlings is the retained primordial or intermediary developmental stage of leaves in the inner layers for very long (even for a few month) period. This opens new developmental routes leading to formation of specially developed plastids in the various cabbage leaf layers. The study of these plastids provided new information for a better understanding of the plastid differentiation and the greening process .