Tsuneyoshi Kuroiwa

Nuclear Encoding of a Chloroplast RNA Polymerase Sigma Subunit in a Red Alga

A chloroplast RNA polymerase sigma factor is encoded by a nuclear gene, sigA, in the red alga Cyanidium caldarium RK-1. The encoded protein functions as an RNA polymerase sigma factor in vitro and it is localized to the chloroplast in vivo. SigA shows high sequence similarity to the sigma factors of cyanobacteria, which is indicative of the ancestral endosymbiotic event and subsequent transfer of the sigA gene to the nuclear genome.

Three-dimensional analysis of the senescence program in rice (Oryza sativa L.) coleoptiles

Abstract. The coleoptile of rice (Oryza sativa L. cv. Nippon-bare) emerges from an imbibed seed on day 2 after sowing. Then, it matures and senesces rapidly. For analysis of the senescence pattern within individual coleoptiles, we monitored the distribution of chlorophyll (Chl) in entire coleoptiles and in cross-sections of coleoptiles by recording the autofluorescence of Chl. Degradation of Chl was apparent at the tip of the margins of opened-out coleoptiles on day 4, when the overall levels of soluble protein and Chl per coleoptile had reached maximum values. Then, senescence proceeded from the tip to the base and from the inner mesophyll cells towards the outer epidermis, excluding tissues along vascular bundles. Further analysis of cellular senescence using samples embedded in Technovit 7100 resin revealed that the senescence of each green mesophyll cell followed an identical program, which consisted of the following steps: (i) degradation of chloroplast DNA; (ii) condensation of the nucleus, decrease in the size of chloroplasts, degradation of ribulose-1,5-bisphosphate carboxylase/oxygenase and chloroplast inner membranes; (iii) disorganization of the nucleus; (iv) complete loss of cellular components, distortion of the cell wall. Although the timing of each step and the rate at which each step was completed differed among cells of different locations within the coleoptile, this sequence was observed in all mesophyll cells in the coleoptile.

Behavior of organelles and their nucleoids in the shoot apical meristem during leaf development in Arabidopsis thaliana L.

The behavior of organelle nucleoids and cell nuclei was studied in the shoot apical meristem and developing first foliage leaves of Arabidopsis thaliana. Samples were embedded in Technovit 7100 resin, cut into thin sections and stained with 4′-6-diamidino-2-phenylindole to observe DNA. Fluorimetry was performed using a video-intensified microscope photon-counting system. The DNA content of individual mitochondria was more than 1 Mbp in the shoot apical meristem and the young leaf primordium, and decreased to approximately 170 kbp in the mature foliage leaf. In contrast, the DNA content of individual plastids was low in the shoot apical meristem and increased until day 7 after sowing. Application of 5-bromo-2′-deoxyuridine, an analogue of thymidine, was usesd to investigate DNA synthesis in situ. The activities of DNA synthesis in the mitochondria and plastids changed according to the stage of development. Mitochondrial DNA was actively synthesized in the shoot apical meristem and young leaf primordia. This strongly suggests that the amount of mitochondrial DNA per mitochondrion, which has been synthesized in the shoot apical meristem and young leaf primordium, is gradually reduced due to continual divisions of the mitochondria during low levels of mitochondrial DNA synthesis. Synthesis of DNA in the plastid became active in the leaf primordia following DNA synthesis in the mitochondria, and the small plastids were filled with large plastid nucleotids. This enlargement of the plastid nucleoids occurred before the synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase and the development of thylakoids.

Sexuality of mitochondria: Fusion, recombination, and plasmids

Mitochondrial fusion, recombination, and mobile genetic elements, which are essential for mitochondrial sexuality, are well established in various organisms. The recombination of mitochondrial DNA (mtDNA) depends upon fusion between parental mitochondria, and between their mtDNA-containing areas (mt-nuclei), to allow pairing between the parental mtDNAs. Such mitochondrial fusion followed by recombination may be called mitochondrial sex. We have identified a novel mitochondrial plasmid named mF. This plasmid is apparently responsible for promoting mitochondrial fusion and crosses over with mtDNA in successive sexual crosses with mF- strains. Only in mF+ strains carrying the mF plasmid did small spherical mitochondria fuse which subsequently underwent fusion between the mt-nuclei that contained the mtDNA derived from individual mitochondria. Several successive mitochondrial divisions followed, accompanied by mt-nuclear divisions. The resulting mitochondria contained recombinant mtDNA with the mF plasmid. Such features remind us also of the bacterial conjugative plasmids such as F plasmid. Therefore, in the final part of this chapter, we discuss the origin of sex and its relationship to the sexuality of mitochondria.

Amyloplast formation in cultured tobacco cells ; effects of plant hormones on multiplication, size, and starch content

In BY-2 cultured tobacco cells (Nicotiana tabacum L.), depletion of 2,4-dichlorophenoxyacetic acid (2,4-D) and addition of benzyladenine (BA) caused amyloplast formation, a decrease in cell multiplication, and an increase in cell size. These changes were primarily triggered by the depletion of 2,4-D, and facilitated by the addition of BA. An increase in the starch content of BY-2 cells was always accompanied by a reduction in cell multiplication. However, when hormonal conditions were unsuitable for amyloplast formation, the starch content of the cells did not increase, even if cell multiplication was forcibly terminated by the addition of aphidicolin. This result indicates that the hormonal conditions themselves, and not the decrease in cell multiplication, induce amyloplast formation in BY-2 cultured tobacco cells.

Delta-9 fatty acid desaturase gene containing a carboxyl-terminal cytochrome b5 domain from the red alga Cyanidioschyzon merolae

Abstract A delta-9 fatty acid desaturase gene, homologous to animal and fungal acyl-coenzyme A (CoA) desaturases, was isolated from the red alga Cyanidioschyzon merolae using a degenerate PCR strategy. This gene, designated as CmFAD9, has no intron. The encoded delta-9 fatty acid desaturase (CmFad9p) consists of 476 amino acids and has an estimated molecular mass of 55.4 kDa. CmFad9p is a unique delta-9 fatty acid desaturase among plants, in that it is fused with the cytochrome b5 domain at its carboxyl terminus. This is characteristic of yeast acyl-CoA desaturase. Genomic Southern hybridization suggested that the C. merolae genome contains a single gene for delta-9 fatty acid desaturase of the animal and fungal type. Southern hybridization combined with pulsed-field gel electrophoresis revealed that CmFAD9 is probably located on chromosome XI of the 17 C. merolae chromosomes. A 1.6-kb product of this gene was transcribed throughout a light/dark synchronization culture. The discovery of CmFAD9 indicates the existence of a novel type of plant delta-9 fatty acid desaturase that may function in the endoplasmic reticulum, but not in the plastid.

Comparative analysis of DNA synthesis activity in plastid-nuclei and mitochondrial-nuclei simultaneously isolated from cultured tobacco cells

Abstract Plastid-nuclei (plastid-nucleoids) and mitochondrial-nuclei (mitochondrial-nucleoids) were simultaneously isolated from cultured tobacco cells (Nicotiana tabacum L., line BY-2), and their activity synthesizing DNA in vitro was examined. The isolated plastid- and mitochondrial-nuclei incorporated about 20 and 50 pmol of dCTP, respectively, into DNA per microgram of template DNA during a 60-min incubation. The DNA synthetic activity of the two organelle-nuclei exhibited similar responses to various inhibitors; it was resistant to aphidicolin and sensitive to N-ethylmaleimide and high concentrations of ddCTP, which are all characteristics of γ-like DNA polymerase. The responses of the DNA synthetic activity in the two organelle-nuclei to pH and divalent- and monovalent-metal cation concentrations were also similar. Moreover, an in situ DNA polymerase assay following SDS-polyacrylamide gel electrophoresis revealed that DNA polymerases with an apparent molecular mass of 116 kDa were present in both the isolated plastid- and mitochondrial-nuclei, and that the two 116-kDa DNA polymerases were quite similar in terms of their sensitivity to various inhibitors, optimum assay conditions, and template preferences. These results indicate that DNA replication in plastids and mitochondria may be conducted by DNA polymerases that have quite similar characteristics.

Simultaneous isolation of cell-nuclei, plastid-nuclei and mitochondrial-nuclei from cultured tobacco cells; comparative analysis of their transcriptional activities in vitro

Abstract A procedure was established for simultaneously isolating cell-nuclei, plastid-nuclei (plastid-nucleoids) and mitochondrial-nuclei (mitochondrial-nucleoids) from cultured tobacco cells ( Nicotiana tabacum L., line BY-2). Quantitative Southern hybridization analysis revealed cross-contamination to be minimal (less than 1% in most cases). The isolated cell-, plastid- and mitochondrial-nuclei incorporated about 17, 15 and 7-pmoles of UTP, respectively, into RNA per microgram of DNA during a 60 min incubation under appropriate conditions. The isolated cell-, plastid- and mitochondrial-nuclei exhibited different sensitivities to various transcriptional inhibitors, and synthesized RNAs that are selectively hybridizable to their own DNA. The three isolated nuclei also incorporated [ γ - 32 P]ATP radioactivity into the DEAE-paper bound fraction: Although most of this incorporation was due to protein phosphorylation, a small fraction of the incorporated radioactivities was recovered as RNA, suggesting that de novo transcription initiation may occur in our assay system. The simultaneous isolation of these three nuclei and the assay of their transcriptional activities in vitro were useful for comparative analysis of their transcription apparatus. This system may also be a useful means of analyzing the transcriptional states of cell nuclear-, plastid- and mitochondrial-genomes simultaneously.

Decrease in mitochondrial DNA and concurrent increase in plastid DNA in generative cells of Pharbitis nil during pollen development

The amount of organellar DNA in a generative cell of Pharbitis nil was observed when squashed pollen grains collected on the day of flowering were stained with the DNA-specific fluorochrome 4,6-diamidino-2-phenylindole (DAPI). Using both DAPI-fluorescence microscopy and electron microscopy, observation of the same thin section of Technovit 7100 resin-embedded material revealed that all of the organellar DNA in mature generative cells is plastid DNA, and there is no mitochondrial DNA. During pollen development, we observed organellar DNA in fluorescence microscopic images using double-staining with DAPI and 3,3-dihexyloxacarbocyanine iodide (DiOC6) and quantified the DNA using a video-intensified microscope photon counting system (VIMPCS). In the vegetative cells, the amounts of both mitochondrial and plastid DNA progressively decreased and had disappeared by 2 days before flowering. In the generative cells, mitochondrial DNA disappeared sooner than in the vegetative cells, indicating a more active mechanism for the decrease in mitochondrial DNA in the generative cells. In contrast, plastid DNA in the generative cells increased markedly. The DNA content per plastid was at a minimum value (corresponding to one copy of the plastid genome) 7 days before flowering, but it increased to a maximum value (corresponding to over 10 copies of the plastid genome) 2 days before flowering. Similar results were also obtained with immunogold electron microscopy using an anti-DNA antibody. These results suggest that the DNA content of mitochondria and plastids in P. nil is controlled independently during pollen development.

Amyloplast formation in cultured tobacco cells. III Determination of the timing of gene expression necessary for starch accumulation

Abstract When BY-2 cultured tobacco (Nicotiana tabacum L.) cells were transferred to auxin-depleted culture medium containing cytokinin (benzyladenine, 1 mg/l), the starch content per cell started increasing from 18 h of culture and amyloplasts had formed by 48 h. Pulse-treatment of the cells with actinomycin D and cycloheximide for the first 12 h (or longer) of culture significantly decreased the cellular starch content after 48 h, whereas the starch content did not decrease significantly when the cells were released from the inhibition within 6 h. This suggests that nuclear gene expression necessary for amyloplast formation begins 6–12 h after the transfer. Immunoblotting analysis of the accumulation of ADP-glucose starch glycosyl transferase (starch synthase) supported this inference.