Plastids In Cells Research Articles

Reorganization of tobacco root plastids during arbuscule development.

In the present paper we analyzed plastid populations labeled by the green fluorescent protein in non-mycorrhizal and mycorrhizal roots of tobacco (Nicotiana tahacum L.). We show by confocal laser scanning microscopy (i) a dramatic increase in these plastids in mycorrhizal roots and (ii) the formation of dense plastid networks covering the symbiotic interface of the arbuscular mycorrhiza, the arbuscule. These cytological observations point to an important role of root cortical cell plastids in the functioning of arbuscular mycorrhizal symbiosis.

Chloroplast protein translocon components atToc159 and atToc33 are not essential for chloroplast biogenesis in guard cells and root cells.

Protein import into chloroplasts is mediated by a protein import apparatus located in the chloroplast envelope. Previous results indicate that there may be multiple import complexes in Arabidopsis. To gain further insight into the nature of this multiplicity, we analyzed the Arabidopsis ppi1 and ppi2 mutants, which are null mutants of the atToc33 and atToc159 translocon proteins, respectively. In the ppi2 mutant, in contrast to the extremely defective plastids in mesophyll cells, chloroplasts in guard cells still contained starch granules and thylakoid membranes. The morphology of root plastids in both mutants was similar to that in wild type. After prolonged light treatments, root plastids of both mutants and the wild type differentiated into chloroplasts. Enzymatic assays indicated that the activity of a plastid enzyme was reduced only in leaves but not in roots. These results indicated that both the ppi1 and ppi2 mutants had functional root and guard cell plastids. Therefore, we propose that import complexes are cell type specific rather than substrate or plastid specific.

Record-density genome enclosed by multiple membranes

Plastids of eukaryotic plant cells originated from endosymbiotic cyanobacteria. As mitochondria, higher plant plastids are bounded by double membranes and possess autonomous genomes. Most plastid genes were transferred to the nucleus during symbiotic evolution, resulting in tight coordination of the former symbionts.The evolution of such coordination can be studied in algae harboring complex plastids that are surrounded by multiple membranes. These plastids arose when heterotrophic eukaryotes engulfed plastid-bearing eukaryotic autotrophs. In the unicellular cryptomonads, endosymbiogenesis seems stuck in an early phase. The plastid, bounded by a double membrane, is enclosed in the periplastid membrane (equivalent to the plasma membrane of the symbiont), which also contains a vestigial nucleus, the nucleomorph. The whole complex is located within the endoplasmic reticulum of the host. The recently completed sequence 1xThe highly reduced genome of an enslaved algal nucleus. Douglas, S. et al. Nature. 2001; 410: 1091–1096Crossref | PubMed | Scopus (306)See all References1 of the three chromosomes of the Guillardia theta nucleomorph now reveal the highest gene density known from a eukaryotic genome. 464 putative genes are contained within not more than 551 kbp, with negligible amounts of noncoding regions. Multiple gene copies are rare. Spliceosomal introns occur in less than 4% of the genes, and 53% have homologs of known function. The vast majority of these genes have functions that are related to gene expression and chromosome replication. Only 30 genes encode plastid proteins, and even fewer have ‘cytoplasmic’ functions within the endosymbiont. Thus, at least a thousand different nuclear encoded proteins must be imported into the complex plastid 1xThe highly reduced genome of an enslaved algal nucleus. Douglas, S. et al. Nature. 2001; 410: 1091–1096Crossref | PubMed | Scopus (306)See all

Subcellular localization of beta-glucosidase in rye, maize and wheat seedlings.

The subcellular compartmentation of beta-glucosidase was studied in rye, maize and wheat seedlings by immunocytochemical methods. For detection, we used a 10 nm gold-labeled secondary antibody, and results were observed using transmission electron microscopy. In all three species, beta-glucosidase was found in plastids, cytoplasm and cell walls. In rye, gold particles were seen on cell walls and cytoplasm in epidermal cells of the root tip and shoot, in bundle sheath cells of the shoot and in all cells, except the vascular bundle cells of the coleoptile. Gold labeling was also observed in plastids of the bundle sheath cells of rye shoot tips and in cortical cells of root tips. In wheat, gold labeling was observed on cell walls and cytoplasm of epidermal cells in the shoot base and coleoptile, and on cell walls and plastids in epidermal cells of the root tip. In maize, gold labeling was mainly found in plastids or proplastids in vascular bundle cells and bundle sheath cells of the shoot, in bundle sheath cells of the coleoptile and in epidermal cells of the root. Some gold particles were also found in cell walls and cytoplasm of stomatal guard cells of the shoot base and vascular bundle cells of the shoot tip and in the cell walls of bundle sheath cells of the shoot tip and root tip epidermal cells. Results are discussed in relation to the role of beta-glucosidase in hydroxamic acid release and overall defense mechanism of monocotyledons.

Plastid Damage in Photosynthetic Cells of Mizugayatsuri (Cyperus serotinus) Leaves Treated with a Pyrazole Herbicide

The chloroplasts of mesophyll cells in Cyperus serotinus Rottb. (mizugayatsuri) leaves possessed well-developed stroma and granal thylakoids. In Kranz chloroplasts, many stroma thylakoids and a considerable amount of peripheral reticula were discernible. The interposed mestome cells had a thick cell wall and poor cytoplasm. The effect of DTP, the herbicidal entity of Pyrazolate, on the development of plastids in the photosynthetic cells of C. serotinus leaves was examined by TEM. Deformation of the plastids appeared under continuous light for 7 d in the plants treated with 1 mg L-1 DTP. However, even 50 mg L-1 DTP did not cause such damage either in the plants keptin darkness or in those exposed to light for less than 2 hr after darkness. The plastids deformed by DTP under prolonged exposure to light were very poor in the inner membrane system, but the envelopes were intact. DTP treatment in the dark allowed normal development of etioplasts, in which a central lattice was composed of numerous tubules about 40 nm in distance.Exposure to light after the dark treatment caused an extrusion of prothylakoids from a central crystalline structure of the etioplasts. The ultrastructure of nuclei and mitochondria in the DTP-treated leaves was not different from those in theuntreated plants. From the above results, we draw a conclusion that DTP causes the collapse of thylakoid membranes only inlight and not in darkness.

Nectary Structure and Ultrastructure of Unisexual Flowers of Ecballium elaterium(L.) A. Rich. (Cucurbitaceae) and their Presumptive Pollinators

The structure and ultrastructure of the nectaries of the monoecious species Ecballium elaterium were studied. Large differences in size and structure of the nectaries were observed in the two genders of flowers, those of the staminate flowers being much larger and more developed than those of the pistillate flowers. The latter do not secrete measurable amounts of nectar. In the nectariferous cells, especially of the staminate flowers, numerous plasmodesmata are present. The pre-nectar originating in the phloem is stored in the plastids of the nectariferous cells primarily as starch grains. The nectar appears to be exuded from the nectary via modified stomata. Very small insects of the order Hemiptera were found to dwell inside the flowers of the two genders, but in different numbers; their number in the staminate flowers was more than twice that in the pistillate flowers. These insects may take part in the process of pollination.

Active protein transport through plastid tubules: velocity quantified by fluorescence correlation spectroscopy.

Dynamic tubular projections emanate from plastids in certain cells of vascular plants and are especially prevalent in non-photosynthetic cells. Tubules sometimes connect two or more different plastids and can extend over long distances within a cell, observations that suggest that the tubules may function in distribution of molecules within, to and from plastids. In a new application of two-photon excitation (2PE) fluorescence correlation spectroscopy (FCS), we separated diffusion of fluorescent molecules from active transport in vivo. We quantified the velocities of diffusion versus active transport of green fluorescent protein (GFP) within plastid tubules and in the cytosol in vivo. GFP moves by 3-dimensional (3-D) diffusion both in the cytosol and plastid tubules, but diffusion in tubules is about 50 times and 100 times slower than in the cytosol and an aqueous solution, respectively. Unexpectedly larger GFP units within plastid tubules exhibited active transport with a velocity of about 0.12 microm/second. Active transport might play an important role in the long-distance distribution of large numbers of molecules within the highly viscous stroma of plastid tubules.

Occurrence of plastids in rye (Poaceae) sperm cells

Studies using classic genetics as well as restriction fragment length polymorphism analysis have demonstrated that rye, unlike most flowering plants, has biparental inheritance of both plastids and mitochondria. Yet, a previous in-depth ultrastructural study found no plastids in rye sperm cells, and DNA-specific staining revealed no cytoplasmic DNA in the male gametes of this plant. In the present study, we examined serial ultrathin sections of eight rye sperm cells (four pairs) and found unambiguous examples of plastids in all cases. The number of plastids per sperm cell varies from two to 12. The sperm of a pair may vary with regard to plastid number; however, these differences are not consistent among the sperm pairs examined.

Fluorescent antibiotic resistance marker for tracking plastid transformation in higher plants.

Plastid transformation in higher plants is accomplished through a gradual process, during which all the 300-10,000 plastid genome copies are uniformly altered. Antibiotic resistance genes incorporated in the plastid genome facilitate maintenance of transplastomes during this process. Given the high number of plastid genome copies in a cell, transformation unavoidably yields chimeric tissues, which requires the identification of transplastomic cells in order to regenerate plants. In the chimeric tissue, however, antibiotic resistance is not cell autonomous: transplastomic and wild-type sectors both have a resistant phenotype because of phenotypic masking by the transgenic cells. We report a system of marker genes for plastid transformation, termed FLARE-S, which is obtained by translationally fusing aminoglycoside 3"-adenyltransferase with the Aequorea victoria green fluorescent protein. 3"-adenyltransferase (FLARE-S) confers resistance to both spectinomycin and streptomycin. The utility of FLARE-S is shown by tracking segregation of individual transformed and wild-type plastids in tobacco and rice plants after bombardment with FLARE-S vector DNA and selection for spectinomycin and streptomycin resistance, respectively. This method facilitates the extension of plastid transformation to nongreen plastids in embryogenic cells of cereal crops.

Diatom plastids: Secondary endocytobiosis, plastid genome and protein import

Plastids of diatoms and other chromophytic algae have four surrounding membranes. In contrast to plastids of green algae, higher plants and red algae chromophytic cells are thought to have evolved by secondary endocytobiosis, i.e. by uptake of a eukaryotic photosynthetic organism by a eukaryotic host cell. This review gives a brief summary of the current views about the origin of diatom plastids and discusses possible mechanisms the cells might employ to transport nucleus‐encoded plastid proteins into these organelles.

Limonene Synthase, the Enzyme Responsible for Monoterpene Biosynthesis in Peppermint, Is Localized to Leucoplasts of Oil Gland Secretory Cells1

Circumstantial evidence based on ultrastructural correlation, specific labeling, and subcellular fractionation studies indicates that at least the early steps of monoterpene biosynthesis occur in plastids. (4S)-Limonene synthase, which is responsible for the first dedicated step of monoterpene biosynthesis in mint species, appears to be translated as a preprotein bearing a long plastidial transit peptide. Immunogold labeling using polyclonal antibodies raised to the native enzyme demonstrated the specific localization of limonene synthase to the leucoplasts of peppermint (Mentha x piperita) oil gland secretory cells during the period of essential oil production. Labeling was shown to be absent from all other plastid types examined, including the basal and stalk cell plastids of the secretory phase glandular trichomes. Furthermore, in vitro translation of the preprotein and import experiments with isolated pea chloroplasts were consistent in demonstrating import of the nascent protein to the plastid stroma and proteolytic processing to the mature enzyme at this site. These experiments confirm that the leucoplastidome of the oil gland secretory cells is the exclusive location of limonene synthase, and almost certainly the preceding steps of monoterpene biosynthesis, in peppermint leaves. However, succeeding steps of monoterpene metabolism in mint appear to occur outside the leucoplasts of oil gland cells.

Plastid sedimentation kinetics in roots of wild-type and starch-deficient mutants of Arabidopsis.

Sedimentation and movement of plastids in columella cells of the root cap were measured in seedlings of wild-type, a reduced starch mutant, and a starchless mutant of Arabidopsis. To assay for sedimentation, we used both linear measurements and the change of angle from the cell center as indices in vertical and reoriented plants with the aid of computer-assisted image analysis. Seedlings were fixed at short periods after reorientation, and plastid sedimentation correlated with starch content in the three strains of Arabidopsis. Amyloplasts of wild-type seedlings showed the greatest sedimentation, whereas plastids of the starchless mutant showed no significant sedimentation in the vertically grown and reoriented seedlings. Because previous research has shown that a full complement of starch is needed for full gravitropic sensitivity, this study correlates increased sensitivity with plastid sedimentation. However, although plastid sedimentation contributed to gravisensitivity, it was not required, because the gravitropic starchless mutant had plastids that did not sediment. This is the first study, to our knowledge, to measure plastid sedimentation in Arabidopsis roots after reorientation of seedlings. Taken together, the results of this study are consistent with the classic plastid-based and protoplast-based models of graviperception and suggest that multiple systems of perception exist in plant cells.

Green fluorescent protein as a marker to investigate targeting of organellar RNA polymerases of higher plants in vivo.

The recent identification of phage-type RNA polymerases encoded in the nuclear genome of higher plants has provided circumstantial evidence for functioning of these polymerases in the transcription of the mitochondrial and plastid genomes, as demonstrated by sequence analysis and in vitro import experiments. To determine the subcellular localization of the phage-type organellar RNA polymerases in plants, the putative transit peptides of the RNA polymerases RpoT;1 and RpoT;3 from Arabidopsis thaliana and RpoT from Chenopodium album were fused to the coding sequence of a green fluorescent protein (GFP). The constructs were used to stably transform A. thaliana. Transgenic plants were examined for green fluorescence with epifluorescence and confocal laser scanning microscopy. Plants expressing the GFP fusions under control of the CaMV35S promoter exhibited a distinct subcellular localization of the GFP fluorescence for each of the fusion constructs. In plants expressing GFP fusions with the putative transit peptides of ARAth;RpoT;1 and CHEal;RpoT, fluorescence was found exclusively in mitochondria, both in root and leaf cells. In contrast, GFP fluorescence in plants expressing the ARAth;RpoT;3-GFP construct accumulated in chloroplasts of leaf cells and nongreen plastids (leucoplasts) of root cells. By demonstrating targeting in plants, the data add substantial evidence for the phage-type RNA polymerases from C. album and A. thaliana to function in the transcriptional machinery of mitochondria and plastids.

Anatomical and ultrastructural changes of the floral nectary ofPisum sativum L. during flower development

The floral nectary ofPisum sativum L. is situated on the receptacle at the base of the gynoecium. The gland receives phloem alone which departed the vascular bundles supplying the staminal column. Throughout the nectary, only the companion cells of the phloem exhibited wall ingrowths typical of transfer cells. Modified stomata on the nectary surface served as exits for nectar, but stomatal pores developed well before the commencement of secretion. Furthermore, stomatal pores on the nectary usually closed by occlusion, not by guard-cell movements. Pore occlusion was detected most frequently in post-secretory and secretory glands, and less commonly in pre-secretory nectaries. A quantitative stereological study revealed few changes in nectary fine structure between buds, flowers secreting nectar, and post-secretory flowers. Dissolution of abundant starch grains in plastids of subepidermal secretory cells when secretion commenced suggests that starch is a precursor of nectar carbohydrate production. Throughout nectary development, mitochondria were consistently the most plentiful organelle in both epidermal and subepidermal cells, and in addition to the relative paucity of dictyosomes, endoplasmic reticulum, and their associated vesicles, the evidence suggests that floral nectar secretion inP. sativum is an energy-requiring (eccrine) process, rather that granulocrine.

Temporal and Spatial Coordination of Cells with Their Plastid Component

Careful coordination of cell multiplication with plastid multiplication and partition at cytokinesis is required to maintain the universal presence of plastids in the major photosynthetic lines of evolution. However, no cell cycle control points are known that might underlie this coordination. We review common properties, and their variants, of plastids and plastid DNA in germline, multiplying, and mature cells of phyla capable of photosynthesis. These suggest a basic level of control dictated perhaps by the same mechanisms that coordinate cell size with the nuclear ploidy level. No protein synthesis within the plastid appears to be necessary for this system to operate successfully at the level that maintains the presence of plastids in cells. A second, and superimposed, level of controls dictates expansion of the plastid in both size and number in response to signals associated with differentiation and with the environment. We also compare the germane properties of plastids with those of mitochondria. With the advent of genomics and new cell and molecular techniques, the players in these control mechanisms should now be identifiable.

Plastid Gene Expression during Amyloplast Formation in Cultured Tobacco Cells

Summary We analyzed changes in plastid gene expression during amyloplast formation in cultured tobacco (Nicotiana tabacum L.) cells. When cultured tobacco cells (line BY 2) in the stationary phase were transferred to auxin-depleted culture medium that contained cytokinin, leucoplast-like plastids in the BY-2 cells were converted to amyloplasts within two days. The cell number, number of plastids per cell, and the relative amount of plastid DNA per cell remained nearly constant throughout this process. During amyloplast formation, transcripts for most plastid genes increased only transiently, and stopped increasing during the late phase of amyloplast development, while transcript levels for psbA (a gene for D 1 protein of photosystem 11) and psaAlB (genes for P700 apoprotein Al/A2 of photosystem 1) continued to increase. Assay of transcription in vitro using isolated plastid-nuclei (nucleoids) showed that the transcriptional activities of most plastid genes decreased dramatically during this process, whereas the transcription of psbA and psaAl B continued at a considerable rate, showing a correlation between transcription and transcript accumulation. However, the fact that increases in transcript abundance were observed for all the plastid genes investigated here in spite of the marked decrease in their transcriptional activity suggests that the degradation of transcripts may also be inactive in the amyloplasts of BY-2 cells.

Observation and isolation of the amyloplasts ribosomes in cotyledon cells of lotus

Ribosome-like particles have been found in the proplastids in young cotyledon cells of lotus (Nelumbo nucifera Gaertn L.) Following the development of young embryo, some lamellar structures and tubular complex occurred in the plastids in young cotyledon cells, and some ribosomlike particles appeared in the loose region of these membrane system and stroma. About 15–20 d after fertilization, with the further development of plastid, a large number of starch and DNA were synthesized in the plastids, and the plastids contained abundant and clear morphologically ribosomes, some of which presented spiral structure. About 16–18 d after fertilization, amyloplasts were isolated and purified from cotyledon of lotus, and ribosomes bands were obtained by use of sucrose density gradient centrifugation of ribosomes isolated from amyloplasts. RNA and protein contents of ribosomes have also been determined.

Plastid ontogeny during petal development in Arabidopsis.

Imaging of chlorophyll autofluorescence by confocal microscopy in intact whole petals of Arabidopsis thaliana has been used to analyze chloroplast development and redifferentiation during petal development. Young petals dissected from unopened buds contained green chloroplasts throughout their structure, but as the upper part of the petal lamina developed and expanded, plastids lost their chlorophyll and redifferentiated into leukoplasts, resulting in a white petal blade. Normal green chloroplasts remained in the stalk of the mature petal. In epidermal cells the chloroplasts were normal and green, in stark contrast with leaf epidermal cell plastids. In addition, the majority of these chloroplasts had dumbbell shapes, typical of dividing chloroplasts, and we suggest that the rapid expansion of petal epidermal cells may be a trigger for the initiation of chloroplast division. In petals of the Arabidopsis plastid division mutant arc6, the conversion of chloroplasts into leukoplasts was unaffected in spite of the greatly enlarged size and reduced number of arc6 chloroplasts in cells in the petal base, resulting in few enlarged leukoplasts in cells from the white lamina of arc6 petals.

The Division Apparatus of Plastids and Mitochondria

Mitochondria and plastids in eukaryotic cells contain distinct genomes and multiply in the cytoplasm by binary division of preexisting organelles. Mitochondrial and plastid nuclei are easily visualized as compartments in the matrix of organelles by high-resolution fluorescence microscopy and by immunoelectron microscopy using anti-DNA antibodies. Plastid and mitochondrial division can be clearly separated into two main events: division of the organelle nuclei, and then division of the rest of the organelles, the process of organellokinesis (mitochondriokinesis and plastidokinesis). The mechanical apparatus that regulates organellokinesis has remained undetermined. In 1986, the plastid-dividing apparatus (PD ring) for plastidokinesis was first identified by us in the primitive red alga Cyanidium caldarium RK-1. The PD ring is located in the cytoplasm outside the organelle envelope at the constricted isthmus of dividing organelles and has subsequently been found in all eukaryotic plants examined. We were also the first to identify the mitochondrion-dividing apparatus (MD ring) for mitochondriokinesis in the unicellular red alga Cyanidioschyzon merolae in 1993. Eukaryotic cell division is therefore controlled by at least three dividing apparata (rings), a contractile ring, an MD ring, and a PD ring, while bacterial division is controlled by a single bacterial contractile FtsZ ring. The aims of this review are to present the fine structure, process of formation, and contraction of the organelle-dividing apparatus, focusing on evolutionary conservation and diversion from the bacterial contractile ring.

In situPCR on Plant Material with Sub-cellular Resolution

In situPCR and reverse transcribedin situPCR have been tested on leaves of sugar cane fixed in FAA, 4% PFA or 2% PFA+2.5% GA.In situPCR amplification of the gene coding for ribulose-1,5-bisphosphate carboxylase/oxygenase (rbcL) was successfully performed following all three fixation protocols. As expected the PCR product was restricted to the plastids of all cells. Reverse transcribedin situPCR was performed onrbcL mRNA and in this case the PCR product was restricted to the plastids of the bundle sheath cells. This is the first report ofin situPCR on plant material and only the second report ofin situPCR with sub-cellular resolution.In situPCR onrbcL may prove to be a valuable positive control for futurein situPCR studies on plant material.