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.     

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.     

Dual location of MAR-binding, filament-like protein 1 in Arabidopsis, tobacco, and tomato

Matrix attachment region-binding filament-like protein 1 (MFP1) is a plant-specific long coiled-coil protein that binds double-stranded DNA. While originally identified as a component of the tobacco nuclear matrix, it was subsequently shown that the majority of MFP1 resides in mature chloroplast where it is located at the stroma side of the thylakoids and is able to bind to nucleoids. On the other hand, a 90 kDa MFP1-like protein from onion has been convincingly shown to be an intrinsic component of the onion meristematic nuclear matrix. Here, we have expanded the analysis of the subcellular location of MFP1 by using high-resolution confocal immunofluorescence microscopy and immunogold electron microscopy. Two different antisera raised against MFP1 from two species were used on isolated nuclei and chloroplasts from tomato, tobacco, and Arabidopsis. Our data show that both antibodies detect a signal in both compartments in all three species. An Arabidopsis MFP1 T-DNA insertional mutation abolishes both nuclear and chloroplast signals, indicating that the nuclear and plastidic antigens are derived from the same gene. We therefore suggest that MFP1 is a protein with a dual location, in both nuclei and chloroplasts, consistent with prior findings in onion and the dicot species investigated here.     

Ultrastructural characterization of the reversible differentiation of chloroplasts in cucumber fruit

The changes in plastid ultrastructure in the pericarp of cucumber (Cucumis sativus L) fruit were studied during fruit yellowing (which accompanied maturation) and regreening. In the course of fruit maturation, the thylakoid system was progressively reduced, and only a small number of membranes remained in the plastids of mature fruit. At the same time, the plastoglobules increased in size, often remaining in close proximity to the degrading thylakoids. In pericarp tissue which turned green again, the thylakoid network in the plastids was gradually reconstituted. Morphological similarities between the plastids in mature and regreening fruit indicated that the chloroplasts in regreened tissue were redifferentiated from the plastids of mature fruit. Reconstitution of the thylakoid system appeared to start from two morphologically distinct types of membranes: from double membranes which resembled thylakoids and from membrane-bound bodies (MBBs). The latter appeared to form thylakoids by two mechanisms: by detachment of extensions from their surfaces and by fragmentation. The plastoglobules remained in the plastids during thylakoid system reconstitution and were often observed in close proximity to developing thylakoids. In the course of chloroplast redifferentiation, several types of membraneous structures were found to be associated with the plastid envelope: (i) vesicles which appeared to separate from the envelope and to fuse subsequently with the developing thylakoids, (ii) tubules, and (iii) double-membrane sheets which appeared asde novo forming thylakoids.     

The Phenotype of a Virescent Chloroplast Mutation in Tobacco Is Associated with the Absence of a 37.5 kD Thylakoid Polypeptide

The Vir-c mutation is a virescent chloroplast mutation found in a line of plants derived from protoplast fusions between a Nicotina tabacum line and a line containing N. tabacum nuclei with Nicotiana suaveolens cytoplasm. Vir-c displays a lag period in chlorophyll accumulation and granal stack formation in young leaves. We examined total chloroplast protein in young leaves and showed the mutant contains 1.3 to 2.1 times less stromal protein, and 2.9 to 4.3 times less thylakoid protein when compared to the N. tabacum var “Turkish Samsun” control. Electrophoretic patterns of total thylakoid proteins indicated three polypeptides were specifically decreased in amount within the context of the overall reduction in thylakoid protein. Electrophoresis of thylakoid proteins synthesized by chloroplasts isolated from half-expanded leaves demonstrated that mutant chloroplasts did not synthesize a 37.5 kilodalton polypeptide which was synthesized by “Samsun” chloroplasts. A polypeptide of this molecular weight was synthesized by Vir-c chloroplasts isolated from mature leaves which had recovered the normal phenotype. Restriction digestion and electrophoresis of the mutant's chloroplast DNA produced a pattern of restriction fragments different from either N. tabacum or N. suaveolens chloroplast DNA.     

Dynamics of localization and protein composition of plastid nucleoids in light-grown pea seedlings

Summary Localization and protein composition of plastid nucleoids was analyzed in light-grown pea seedlings at various stages of leaf development. In young plastids of unopened leaf buds, nucleoids were abundant and localized in the periphery of plastids, whereas, in mature leaves, chloroplasts contained nucleoids within narrow spaces restricted by thylakoids or grana. The migration of nucleoids into the interior of plastids preceded the formation of grana, and hence, the maturation of the photosynthetic apparatus. The protein composition of nucleoids was considerably different in young plastids and mature chloroplasts. Polypeptides with a molecular mass of 70–100 kDa predominated in the nucleoids of young plastids, whereas polypeptides with molecular mass of 20–30 kDa were abundant in the nucleoids of mature chloroplasts. Immuno-blot analysis with antibodies against the nucleoids of young plastids identified various polypeptides that were significantly more abundant in the nucleoids of young plastids than in the nucleoids of mature chloroplasts. These results demonstrate that plastid nucleoids are subject to dynamic changes in both localization and composition during the normal development of chloroplasts in the light.Abbreviations DAPI 4,6-diamidino-2-phenylindol - DiOC₆ 3,3-dihexyloxacarbocyanine iodide     

Thylakoid protein kinase activity and associated control of excitation energy distribution during chloroplast biogenesis in wheat

The activity of thylakoid protein kinase and the regulation of excitation energy distribution between photosystems I and II was examined during chloroplast biogenesis in light-grown Triticum aestivum (wheat) leaves. The specific activity of the thylakoid protein kinase decreased some six-fold during development from the young plastids at the base of the 7-d-old leaf to the mature chloroplasts at the leaf tip. Appreciable activity was also detected in plastids isolated from etiolated leaves. In mature chloroplasts the majority of phosphate was incorporated into the M_r=26,000 apo-proteins of the light-harvesting chlorophyll a/b-protein complex (LHCP). However, at early stages of chloroplast development and in the etioplast, the phosphate was predominantly incorporated into a polypeptide of M_r=9,000 dalton. Immature thylakoids, isolated from the base of the leaf, had relatively low concentrations of LHCP and could perform a State 1-State 2 transition, as demonstrated by ATP-induced quenching of photosystem II fluorescence. Analyses of photosystem I and photosystem II fluorescence-induction curves from intact leaf tissue demonstrated that this transition occurs in vivo at early stages of leaf development and, therefore, may play an important role in regulating energy transduction during chloroplast biogenesis.     

Heterogeneity of chloroplasts in Acetabularia mediterranea. Heterogeneous distribution and morphology of chloroplast DNA

A high percentage of chloroplasts in the siphonaceous green alga Acetabularia mediterranea lacks DNA: staining with the sensitive DNA-specific fluorochrome 4′-6-diamidino-2-phenylindole (DAPI) revealed that DNA was present only in 47–51% of the total plastid population. The distribution of DNA-containing chloroplasts appeared heterogeneous, exhibiting an apicobasal gradient. DNA could be detected in 64, 46, 35, and 36% of the plastids from the apical, subapical, middle, and basal part of the cell, respectively. DNA morphology also exhibited heterogeneity. Three types of nucleoid were distinguished: (1) round compact nucleoids; (2) long thin nucleoids characteristic of chloroplasts of the cell apex and the whorls; and (3) elaborate nucleoids appearing to consist of several subunits, which were more typical of the middle and basal part of the cell. On the basis of the nucleoid morphology and the decrease in DNA-containing plastids from the apex towards the basis of the cell, we propose a model for the development of plastids lacking DNA in relation to chloroplast replication.     

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.     

Use of the fluorochrome 4''6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA

Use of the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) makes it possible to examine in situ the structure of chloroplast DNA (chDNA) with the fluorescence microscope. This simplifies the study of genetic and developmental changes in chloroplast DNA. Three examples are presented. (a) Wild-type Euglena gracilis B contains several chloroplast DNA nucleoids per chloroplast. A yellow mutant lacking functional chloroplasts is similar, but such nucleoids are absent in an aplastidic mutant strain known from biochemical studies to have lost its chDNA. (b) In vegetative cells of the giant-celled marine algae Acetabularia and Batophora, only about a quarter of the chloroplasts have even one discernible chloroplast DNA particle, and such particles vary in size, showing a 30-fold variation in the amount of DNA-bound DAPI fluorescence detected per chloroplast. By contrast, 98% of chloroplasts in developing Acetabularia cysts contain chDNA, with as many as nine nucleoids per chloroplast. (c) DAPI-stained chloroplasts of chromophyte algae display the peripheral ring of DNA expected from electron microscope studies. However, these rings are not uniform in thickness, but are necklace-like, with the appearance of beads on a string. Since the multiple nucleoids in plastids of chlorophyte algae also appear to be interconnected throughout the chloroplast, a common structural plan may underlie chDNA morphology in both groups of algae.     

Biogenesis of water splitting by photosystem II during de‐etiolation of barley (Hordeum vulgare L.)

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de‐etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll‐binding protein complexes and development of water splitting via O₂ production in plastids (etiochloroplasts) isolated during de‐etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light‐harvesting antenna [PSII‐light‐harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de‐etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de‐etiolation, etiochloroplasts revealed the same water‐splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de‐etiolation precedes assembly of the PSII‐LHCII supercomplexes. Taken together, data show a rapid establishment of water‐splitting activity during etioplast‐to‐chloroplast transition and emphasize that assembly of the functional water‐splitting site of PSII is not the rate‐limiting step in the formation of photoactive thylakoid membranes.     

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.     

Maternal inheritance,cytology and macromolecular composition of defective chloroplasts in a variegated mutant of Nicotiana tabacum

Summary By phase microscopy of living cells the cause of a maternally-inherited variegated, spontaneous mutation of Nicotiana tabacum L. cv. Turkish Samsun was shown to be the presence of defective chloroplasts. These were intermingled with normal chloroplasts in some of the cells of the mesophyll tissue. In young, expanding leaves, the defective chloroplasts contain traces of chlorophylls a and b in the same ratio as found in normal chloroplasts, but only one-thirtieth of the quantity. As the defective chloroplasts mature, the green pigments disappear. The defective chloroplasts thus appear to be greatly deficient in thylakoid membranes. From their dynamic changes in shape, the defective chloroplasts appear to consist almost entirely of mobile phase, the structure which surrounds the thylakoid system of membranes of normal chloroplasts of higher plants. Consistent with this idea, two constitutents located in the mobile phase of normal chloroplasts—70S ribosomes and Fraction I protein—were detected in defective chloroplasts. The Fraction I protein was unchanged in specific ribulose diphosphate carboxylase activity from enzyme isolated from normal chloroplasts. Speculations are presented that the mutation in chloroplast DNA responsible for the formation of defective chloroplasts cannot be attributed to cistrons coding for the protein of Photosystem II, chloroplast ribosomal RNA or proteins, Fraction I protein, or the DNA-dependent RNA polymerase of chloroplasts.