The nuclei of cellular organelles and the formation of daughter organelles by the “plastid-dividing ring”

It has been established that organelles, such as mitochondria and plastids, contain organelle-specific DNA and arise from the division of pre-existing organelles (e.g., Possingham and Lawrence, 1983). We propose that organelle DNAs, such as mitochondrial DNA and plastid DNA are not naked in organellesin situ but are organized in each case to form an “organelle nucleus” with basic proteins (Kuroiwa, 1982). The concept of organelle nuclei has changed our ideas about the division of organelles. Thus, the process of organelle division must be composed of two main events: division of the organelle nucleus and organellekinesis (division of the other components of the mitochondrion or plastid). The latter term has been adopted as an appropriate analogue of cytokinesis. We were the first to identify the plastid-dividing ring (PD-ring), which is located in the cytoplasm close to the outer envelope membrane at the constricted isthmus of dividing chloroplasts in the red algaCyanidium caldirum. The PD-ring is about 60 nm in width and 25 nm in thickness, and is a circular bundle of actin-like, fine filaments, each about 4–5 nm in diameter. Since cytochalasin B, an inhibitor of polymerization of actin filaments, inhibits the formation of the PD-ring and, thus, prevents subsequent division of chloroplasts, the PD-ring is thought to be a structure that is essential for the division of plastids (plastidkinesis). The behavior of the PD-ring during a cycle of chloroplast division can be classified into the following four stages on the basis of morphological and temporal differences. The chloroplast growth stage: the small, spherical chloroplast increases in volume and becomes a football-like structure, while the PD-ring from the previous division disappears. Formation of the PD-ring: the somewhat electron-dense body (see below) is fragmented into many, somewhat electron-dense granules, which are aligned along the equatorial region of the chloroplast and fine filaments are formed from the somewhat electron-dense granules in the equatorial region. The fine filaments of the PD-ring align themselves according to the longest axis of their overall domain, i.e., circumferentially. Contraction stage: a bundle of fine filaments begins to contract and generates a deep furrow. Conversion stage: after chloroplast division, the remnants of the PD-ring are converted into somewhat electron-dense bodies. Similar events occur during the second cycle of chloroplast division. Since similar structures are observed extensively in the plastids of algae, moss and higher plants, the PD-ring appears to be an essential structure for the division of plastids in plants.     

Distribution of chloroplast coupling factor (CF1) particles on plastid membranes during development

Samples of internal membrane systems separated from lysates of intact plastids from dark grown Avena sativa L. (vars, Cooba and Mostyn) and Hordeum vulgare L. (vars, Himalaya and Deba Abed) given different periods of illumination before isolation were assayed for trypsin-activated Ca²⁺-dependent ATPase activities and also examined in the electron microscope after treatment in the manner described by Oleszko and Moudinanakis (1974) which assists the visualization of the chloroplast coupling factor (CF₁) particles. Concentrations of membrane-attached CF₁ particles were not observed on the membrane surfaces of the prolamellar bodies (PLBs) proper but only on the attached extruded lamellar membranes. Increasing lengths of illumination followed by plastid isolation and subsequent membrane separation had the effect of progressively increasing the mean distance between these individual lamellar-attached CF₁ particles. Measurements of trypsin-activated Ca²⁺-dependent ATPase activities during similar developmental regimes indicated that functions associated with CE₁ particles are relative constant and largely independent of the period of illumination if the values were expressed on a per plastid basis indicating that assembly of CF₁ particles may take place in either etioplasts, etiochloroplasts or mature chloroplasts.Abbreviations PLB prolamellar body - EDTA ethylene-diaminetetra-acetic acid - CF₁ chloroplast coupling factor particles - ATPase adenosine triphosphatase     

The role of chloroplast membranes in the location of chloroplast DNA during the greening ofPhaseolus vulgaris etioplasts

Summary The location of DNA containing nucleoids has been studied in greening bean (Phaseolus vulgaris L.) etioplasts using electron microscopy of thin sections and the staining of whole leaf cells with the fluorochrome DAPI. At 0 hours illumination a diffuse sphere of cpDNA surrounds most of the prolamellar body. It appears to be made up of a number of smaller nucleoids and can be asymmetric in location. The DNA appears to be attached to the outside of the prolamellar body and to prothylakoids on its periphery. With illumination the nucleoid takes on a clear ring-like shape around the prolamellar body. The maximum development of the ring-like nucleoid at 5 hours illumination is associated with the outward expansion of the prolamellar body and the outward growth of the prothylakoids. At 5 hours the electron transparent areas lie in between the prothylakoids radiating out from the prolamellar body. Between 5 hours and 15 hours observations are consistent with the growing thylakoids separating the nucleoids as the prolamellar body disappears and the chloroplast becomes more elongate. At 15 hours the fully differentiated chloroplast has discrete nucleoids distributed throughout the chloroplast with evidence of thylakoid attachment. This is the SN (scattered nucleoid) distribution ofKuroiwa et al. (1981) and is also evident in 24 hours and 48 hours chloroplasts which have more thylakoids per granum. The changes in nucleoid location occur without significant changes in DNA levels per plastid, and there is no evidence of DNA or plastid replication.The observations indicate that cpDNA partitioning in dividing SN-type chloroplasts could be achieved by thylakoid growth and effectively accomplish DNA segregation, contrasting with envelope growth segregating nucleoids in PS-type (peripheral scattered nucleoids) chloroplasts. The influence of plastid development on nucleoid location is discussed.     

PLASTID DEVELOPMENT IN IOJAP- AND CHLOROPLAST MUTATOR-AFFECTED MAIZE PLANTS

Plastids affected by either iojap or chloroplast mutator fail to green, and altered plastids are maternally transmitted to subsequent generations. The ultrastructure of iojap-affected plastids indicates that these plastids contain no ribosomes and are capable of supporting little internal membrane organization in either light or dark-grown plants. Chloroplast mutator-affected plastids of light-grown plants contain some organized internal membrane structures. In dark-grown plants, chloroplast mutator-aftected plastids contain a crystalline prolamellar body, numerous vesicles, and osmiophilic granules. The chloroplast mutator-affecled etioplasts display an abnormal distribution of lamellar membranes; these membranes, rather than radiating in a spokelike pattern from the prolamellar body, are condensed into a portion of the organelle. Light causes disruption of the prolamellar body in chloroplast mutator-affected plastids without promoting the organization of a normal thylakoid membrane system. The effects of iojap and chloroplast mutator are cell autonomous and apparently influence the individual plastid, as evidenced by the persistence of heteroplastidic cells containing normal and affected plastids.     

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.     

Origin and developmental changes of envelope proteins and translocator activities from plastids of Secale cereale L.

To determine the sites of synthesis of chloroplast-envelope proteins, we have analysed several enzyme and translocator functions ascribed to the envelope membranes, and investigated the envelope polypeptide composition of plastids isolated from 70S ribosome-deficient leaves of rye (Secale cereale L.) generated by growing the plants at a temperature of 32°C. Since the ribosomedeficient plastids are also achlorophyllous in light-grown leaves, not only were chloroplasts from mature, green leaves used for comparison, but also those from yellowing, aged leaves as well as etioplasts from dark-grown leaves raised at a temperature of 22° C. A majority of the plastidenvelope polypeptides appeared to be of cytoplasmic origin. The envelopes of ribosome-deficient plastids possessed ATPase (EC 3.6.1.3) activity; this was not, however, dependent on divalent cations, in contrast to the Mn²⁺- or Mg²⁺-dependent ATPase which is associated with chloroplast envelopes. Adenylate kinase (EC 2.7.4.3) was present in the stromal fraction of ribosome-deficient plastids and the stromal form of this enzyme is, therefore, of cytoplasmic origin. In contrast to previous findings, adenylate kinase was not, however, specifically associated with the chloroplast-envelope membranes, either in rye or in spinach. Measurements of the uptake of l-[¹⁴C]-malate into ribosome-deficient plastids indicated the presence and cytoplasmic origin of the dicarboxylate translocator. Malate uptake into rye etioplasts was, however, low. The phosphate translocator was assayed by the uptake of 3-phospho-[¹⁴C]glycerate. While rapid 3-phosphoglycerate uptake was observed for rye chloroplasts and etioplasts, it was hardly detectable for ribosome-deficient, plastids and rather low for chloroplasts from aged leaves. A polypeptide of M _r approx. 30000 ascribed to the phosphate translocator was greatly reduced in the envelope patterns of ribosome-deficient plastids and of chloroplasts from aged leaves.     

A coupling factor for photosynthetic phosphorylation from plastids of light- and dark-grown maize

1. Maize chloroplasts contain a trypsin-, dithiothreitol-, and Ca²⁺-activated ATPase. This enzyme, which can serve as a coupling factor for photosynthetic phosphorylation, differs slightly in a few properties but in general resembles a similar one in spinach plastids which was described earlier by others.

2. Maize etioplasts (immature plastids in dark-grown plants) also contain this ATPase, and it is shown that NaCl-EDTA extracts of etioplasts can restore photosynthetic phosphorylation activity to depleted green membranes of chloroplasts.

3. Electron microscopy of maize etioplast and chloroplast membranes demonstrates the presence of protruding knobs, approx. 90 Å in diameter. Removal and reassociation of knobs with membranes can be correlated with the ability to carry on photosynthetic phosphorylation.

4. Most or possibly all of the coupling factor (measured as ATPase) activity of a chloroplast may be present in the etioplast from which it develops. The photosynthetic membrane of the chloroplast can be formed in stages.

5. The significance of these observations is discussed with regard to membrane formation in general and plastid membrane development in particular.     

Visualization of plastid nucleoids in situ using the PEND-GFP fusion protein

Plastid DNA is a circular molecule of 120-150 kbp, which is organized into a protein-DNA complex called a nucleoid. Although various plastids other than chloroplasts exist, such as etioplasts, amyloplasts and chromoplasts, it is not easy to observe plastid nucleoids within the cells of many non-green tissues. The PEND (plastid envelope DNA-binding) protein is a DNA-binding protein in the inner envelope membrane of developing chloroplasts, and a DNA-binding domain called cbZIP is present at its N-terminus. We made various PEND-green fluorescent protein (GFP) fusion proteins using the cbZIP domains from various plants, and found that they were localized in the chloroplast nucleoids in transient expression in leaf protoplasts. In stable transformants of Arabidopsis thaliana, PEND-GFP fusion proteins were also localized in the nucleoids of various plastids. We have succeeded in visualizing plastid nucleoids in various intact tissues using this stable transformant. This technique is useful in root, flower and pollen, in which it had been difficult to observe plastid nucleoids. The relative arrangement of nucleoids within a chloroplast was kept unchanged when the chloroplast moved within a cell. During the division of plastid, nucleoids formed a network structure, which made possible equal partition of nucleoids.     

Comparison of the Molecular Weights of Proteins Synthesized by Isolated Chloroplasts with Those Which Appear during Greening in Zea mays

The proteins of prolamellar bodies of etioplasts and of thylakoid membranes of greening and mature chloroplasts from Zea mays were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Three classes of proteins were distinguished: those present in etioplasts and disappearing during greening, those absent in etioplasts and appearing during greening, and those present in both etioplasts and chloroplasts. The largest number of proteins belonged to this last class.The molecular weights of chloroplast thylakoid proteins were compared to the molecular weights of the membrane-associated proteins synthesized by isolated, mature chloroplasts. Thirteen of the 15 to 20 membrane-bound proteins made by isolated chloroplasts corresponded in size to proteins present in chloroplasts. Most of the 13 are present in both etioplasts and chloroplasts although a few were the same size as proteins which increase during greening. Production of most of the membrane proteins made in the plastids is not stringently regulated by light in vivo. The polypeptide subunits of the light-harvesting pigment-protein complex, the most abundant proteins of the chloroplast thylakoids, were absent from etioplasts. They were not synthesized by isolated chloroplasts.     

In Vitro Protein Synthesis by Plastids of Phaseolus vulgaris: IV. Amino Acid Incorporation by Etioplasts and Effect of Illumination of Leaves on Incorporation by Plastids 1

Protein synthesis in vitro by etioplasts and chloroplasts from Phaseolus vulgaris was examined to study the factors regulating the development of etioplasts into chloroplasts. The properties of incorporation of (14)C-leucine into protein by etioplasts from plants grown 6.5 days in darkness are similar to those of chloroplasts from plants of the same age that were illuminated for 12 hours. However, the rate of incorporation per plastid by chloroplasts is 4 times higher than the rate of amino acid incorporation by etioplasts. When 6-day-old plants are placed in light, this 4-fold increase occurs within 6 hours and is maintained up to 36 hours. The difference in rate of amino acid incorporation into protein between etioplasts and chloroplasts represents a real difference in the ability of etioplasts and chloroplasts to synthesize protein. A difference in pool size of leucine between etioplasts and chloroplasts does not account for the difference in amino acid incorporation between etioplasts and chloroplasts. Also the difference in photosynthetic capabilities of etioplasts and chloroplasts does not account for the difference in the ability to incorporate amino acid into protein. Furthermore, there are no factors in homogenates of etiolated leaves which inactivate amino acid incorporation into protein by chloroplasts. The difference in rates of amino acid incorporation between etioplasts and chloroplasts is correlated with the state of development of the plastids. The plastids have increased ability to incorporate amino acid into protein when the plastids are undergoing growth and differentiation.     

Detection and characterization of a plastid envelope DNA-binding protein which may anchor plastid nucleoids.

Chloroplast DNA (cpDNA) binds to the envelope membrane of actively dividing chloroplasts (plastids) in young pea leaves. South-western blotting was used to identify and characterize the protein involved in the binding of cpDNA to the envelope membrane. A 130 kDa protein in the inner chloroplast (plastid) envelope membrane binds specific sequences within the cpDNA. These included a 0.41 kbp sequence located upstream of the psaAB gene, a 0.57 kbp sequence located downstream of the petA gene and a 1.2 kbp sequence located within the rpoC2 gene. The protein was detected in the envelope membrane of young pea leaves in which the cpDNA had been located by fluorescence microscopy at the chloroplast periphery, whereas it was undetectable in mature leaves. We therefore propose that the 130 kDa protein is involved in the binding of cpDNA to the envelope membrane, and named it plastid envelope DNA-binding protein.     

Variation in Plastid Number: Effect on Chloroplast and Nuclear Deoxyribonucleic Acid Complement in the Unicellular Alga Olisthodiscus luteus

Changes in the physiological state of the multiplastidic alga Olisthodiscus luteus result in a shift in chloroplast complement from 33 to 21 plastids. The effect of this induced change in organelle complement on nuclear and chloroplast DNA levels has been analyzed. Data suggest that the absolute amount of chloroplast and nuclear DNA found within a cell remains constant but that the amount of chloroplast DNA per plastid is inversely proportional to the number of chloroplasts to which that DNA must be distributed.     

Changes in distribution of nucleoids in developing and dividing chloroplasts and etioplasts ofArena sativa

Summary Nucleoid distribution in chloroplasts and etioplasts at the different developmental stages was examined with the first leaves ofAvena sativa by using a DNA-specific fluorescent probe, 46-diamidino-2-phenylindole (DAPI). In light-grown first leaves, three types of plastid nucleoid distribution were recognized. 1. Peripheral distribution in undeveloped chloroplasts which contain only a few thylakoids in the middle region of the leaf sheath. 2. Ring-like arrangement along the rim of developing and dividing young chloroplasts, of which grana were composed of four to eight layers of thylakoids, at the base of the leaf blade. The plane of the nucleoids' ring is in parallel with the face of the thylakoids. 3. Scattered distribution of 10 to 20 discrete spherular nucleoids in the stroma of fully developed chloroplasts, of which grana were composed of up to 20 thylakoids, in the regions of the middle and the tip of the leaf blade. In dark-grown first leaves two types were recognized. 1. Peripheral distribution in developing and dividing young etioplasts in the leaf sheath and the base of the leaf blade. 2. Scattered distribution of 10 or more discrete spherular nucleoids in fully developed etioplasts, containing extended prothylakoids, in the regions of the middle and the tip of the leaf blade. Ring-like arrangement of nucleoids was not observed in any etioplasts. The results indicates that spatial arrangement of plastid nucleoids dynamically changes in close relationship with the development of the inner membrane systems of plastids.     

Some unusual features of the ultrastructure of the chloroplasts of the green algal orderCaulerpales and their development

Summary The ultrastructure of developing and mature chloroplasts of members of the green algal orderCaulerpales is described. The mature chloroplasts develop from small starch containing plastids. These small starch containing plastids may also develop into the large amyloplasts characteristic of this order. The thylakoid organizing body (TOB), a system of concentric lamellae found at one end of the plastid, appears to be involved in initial thylakoid membrane synthesis. During early plastid development the first formed thylakoids, the plastid DNA and lipid are closely associated with this body. Many developing plastids also have a number of microfilaments near the chloroplast envelope. These microfilaments extend from the TOB towards the opposite end of the plastid.The size and structure of the mature caulerpalean chloroplast varies greatly between species, as does the size and structure of the TOB. The simplest type of TOB occurs inAvrainvillea erecta and the most complex inCaulerpa cactoides. The membranes of the TOB are connected by crossbridges and they are also connected with the inner chloroplast envelope membrane. The structure of the TOB, its relation to the chloroplast envelope, its association with the thylakoids and its possible functions are described.     

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.     

Preparation and characterization of envelope membranes from nongreen plastids

We have developed a reliable procedure for the purification of envelope membranes from cauliflower (Brassica oleracea L.) bud plastids and sycamore (Acer pseudoplatanus L.) cell amyloplasts. After disruption of purified intact plastids, separation of envelope membranes was achieved by centrifugation on a linear sucrose gradient. A membrane fraction, having a density of 1.122 grams per cubic centimeter and containing carotenoids, was identified as the plastid envelope by the presence of monogalactosyldiacylglycerol synthase. Using antibodies raised against spinach chloroplast envelope polypeptides E24 and E30, we have demonstrated that both the outer and the inner envelope membranes were present in this envelope fraction. The major polypeptide in the envelope fractions from sycamore and cauliflower plastids was identified immunologically as the phosphate translocator. In the envelope membranes from cauliflower and sycamore plastids, the major glycerolipids were monogalactosyldiacylglycerol, digalactosyldiacylglycerol, and phosphatidylcholine. Purified envelope membranes from cauliflower bud plastids and sycamore amyloplasts also contained a galactolipid:galactolipid galactosyltransferase, enzymes for phosphatidic acid and diacylglycerol biosynthesis, acyl-coenzyme A thioesterase, and acyl-coenzyme A synthetase. These results demonstrate that envelope membranes from nongreen plastids present a high level of homology with chloroplasts envelope membranes.     

The stromal protein large subunit of ribulose-1,5-bisphosphate carboxylase is translated by membrane-bound ribosomes.

Translation of the large subunit of ribulose-1,5-bisphosphate carboxylase (LSU) was investigated by labeling of isolated barley plastids with [35S]-methionine. In both chloroplasts and etioplasts, labeling of LSU was severely impaired if plastid membranes were removed from the reaction mixtures. Removal of membrane-bound polysomes with high salt or puromycin greatly decreased translation of LSU. Pulse-labeled chloroplast membranes were shown to release LSU if chased with unlabeled methionine in the presence of stroma. Immunoprecipitation detected higher amounts of labeled LSU translation intermediates associated with the membrane fraction than in the soluble fraction. We therefore conclude that, in plastids, membrane-bound polysomes are required not only for translation of membrane-intrinsic proteins but also for translation of a soluble protein.     

Characterization of the preprotein translocon at the outer envelope membrane of chloroplasts by blue native PAGE

The preprotein translocon at the outer envelope membrane of chloroplasts (Toc) mediates the recognition and import of nuclear-encoded preproteins into chloroplasts. Two receptor components, Toc159 and Toc34, and the channel Toc75 form the Toc complex. In this study, we have analyzed the molecular architecture and organization of the Toc complex by blue native PAGE (BN-PAGE), which is a high-resolution method for separating membrane protein complexes under non-denaturing conditions. Pea chloroplasts isolated in the presence of a protease inhibitor cocktail were directly solubilized in detergent solution and analyzed by BN-PAGE and size exclusion chromatography. Subsequent immunoblot analyses indicated that the complex composed of Toc75, Toc159 and Toc34 has a molecular mass of 800-1,000 kDa. Limited proteolysis revealed a core of the Toc complex, which was resistant to proteases and detergent treatments. The stoichiometry of the three Toc proteins was calculated as approximately 1 : 3 : 3 between Toc159 : Toc75 : Toc34. We have also analyzed the Toc complex of etioplasts and root plastids. These plastids were found to have essentially the same sized Toc complex as that of the chloroplast.     

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.     

POR - import and membrane association of a key element in chloroplast development

The development of proplastids or etioplasts to chloroplast is visualized by the accumulation of chlorophyll in leaves of higher plants. The biosynthesis of chlorophyll includes a light-dependent reduction of protochlorophyllide (Pchlide) to chlorophyllide (Chlide). This light-dependent step is catalysed by the nucleus-encoded NADPH:Pchlide oxidoreductase (POR, EC 1.6.99.1). POR is active within plastids and therefore has to be translocated over the plastid envelope membranes. The import of chloroplast proteins seems to follow a general import pathway using translocons at the outer and inner envelope membrane. POR cross-linking to Toc75, one of the major translocon components at the outer envelope membrane, indicates its use of the general import pathway. However, since variations exist within the so-called general import pathway one has to consider previous data suggesting a novel totally Pchlide-dependent import pathway of one POR isoform, PORA. The suggested Pchlide dependency of POR import is discussed since recent observations contradict this idea. In the stroma the POR transit peptide is cleaved off and the mature POR protein is targeted to the plastid inner membranes. The correct and stable association of POR to the membrane requires the cofactor NADPH. Functional activity of POR calls for formation of an NADPH–Pchlide–POR complex, a formation that probably takes place after the membrane association and is dependent on a phosphorylation reaction.