Takeshi Nitta

Double-stranded RNA in rice: A novel RNA replicon in plants

The entire sequence of 13952 nucleotides of a plasmid-like, double-stranded RNA (dsRNA) from rice was assembled from more than 50 independent cDNA clones. The 5′ non-coding region of the coding (sense) strand spans over 166 nucleotides, followed by one long open reading frame (ORF) of 13716 nucleotides that encodes a large putative polyprotein of 4572 amino acid residues, and by a 70-nucleotide 3′ noncoding region. This ORF is apparently the longest reported to date in the plant kingdom. Amino acid sequence comparisons revealed that the large putative polyprotein includes an RNA helicase-like domain and an RNA-dependent RNA polymerase (replicase)-like domain. Comparisons of the amino acid sequences of these two domains and of the entire genetic organization of the rice dsRNA with those found in potyviruses and the CHV1-713 dsRNA of chestnut blight fungus suggest that the rice dsRNA is located evolutionarily between potyviruses and the CHV1-713 dsRNA. This plasmid-like dsRNA in rice seems to constitute a novel RNA replicon in plants.

Tissue-specific expression of the gene for a putative plasma membrane H(+)-ATPase in a seagrass.

A cDNA clone corresponding to the gene (ZHA1) for a putative plasma membrane H+-ATPase of a seagrass (Zostera marina L.) was isolated and sequenced. Comparison of the amino acid predicted sequence from the nucleotide sequence of ZHA1 with those encoded by known genes for plasma membrane H+-ATPases from other plants indicated that ZHA1 is most similar to the gene (PMA4) for a plasma membrane H+-ATPase in a tobacco (84.4%). Northern hybridization indicated that ZHA1 was strongly expressed in mature leaves, which are exposed to seawater and have the ability of tolerate salinity; ZHA1 was weakly expressed in immature leaves, which are protected from seawater by tightly enveloping sheaths and are sensitive to salinity. In mature leaves, in situ hybridization revealed that ZHA1 was expressed specifically in epidermal cells, the plasma membranes of which were highly invaginated and morphologically similar to those of typical transfer cells. Therefore, the differentiation cell-like structures, accompanied by the high-level expression of ZHA1, in the epidermal cells of mature leaves in particular may be important for the excretion of salt by these cells.

Enigmatic double-stranded RNA in Japonica rice

We have found a linear, 16 kb, double-stranded RNA (dsRNA) in symptomless Japonica rice (Oryza sativa L.) that is not found in Indica rice (Oryza sativa L.). The dsRNA was detected in every tissue and at every developmental stage, and its copy number was approximately constant (about 20 copies/cell). Double-stranded RNA was also detected in two strains of Oryza rufipogon (an ancestor of O. sativa). Hybridization experiments indicated that the dsRNA of O. rufipogon was homologous but not identical to that of O. sativa. The sequence of about 13.2 kb of the dsRNA was determined and two open reading frames (ORFs) were found. The larger ORF (ORF B) was more than 12 351 nucleotides long and encoded more than 4 117 amino acid residues.

Stringently and developmentally regulated levels of a cytoplasmic double-stranded RNA and its high-efficiency transmission via egg and pollen in rice

A very restricted amount of high-molecular-weight double-stranded RNA (dsRNA) has been found in healthy japonica rice plants. We discriminated dsRNA-carrying rice plants from noncarriers. The endogenous dsRNA was localized in the cytoplasm (about 100 copies per cell) and was transmissible to progeny plants by mating. In crosses between carriers and noncarriers, the RNA was transmitted efficiently to F1 plants via both egg and pollen. The rice dsRNA was maintained at an almost constant level by host plant cells from generation to generation. The high-efficiency transmission of the endogenous dsRNA to progeny plants appears to depend on the autonomously controlled replication of the dsRNA localized in cytoplasmic vesicles. However, an increase in copy number (about 10-fold) of the dsRNA was observed during the suspension culture of host cells. The number of copies of dsRNA returned to the original low value in regenerated plants, suggesting that the copy number is stringently and developmentally regulated in rice cells.

Association of particles that contain double-stranded RNAs with algal chloroplasts and mitochondria.

Linear dsRNAs (double-stranded RNAs) belonging to several distinct size classes were found to be localized in chloroplasts and mitochondria of Bryopsis spp., raising the possibility that these dsRNAs are prokaryotic in nature. The algal cytosol and nuclei did not contain dsRNAs. The amount of the dsRNAs in the organelles appeared constant, and there were about 500 copies per chloroplast. The four major dsRNAs from Bryopsis chloroplasts were about 2 kbp (kilobase pairs) in length and originated from discrete isometric particles of about 25 nm diameter. These virus-like particles were purified by CsCl density gradient centrifugation after extraction from isolated chloroplasts with chloroformbutanol and subsequent precipitation with polyethylene glycol. They had a buoyant density of about 1.40 g · cm−3 and contained four major and three minor proteins. Mitochondrial dsRNAs were about 4.5 kbp in length and formed less-stable particles of about 40 nm in diameter with a buoyant density of 1.47 g · cm−3. Some observations support the hypothesis that vertical transmission of the protein-coated, non-infectious dsRNAs occurs within cell organelles. Double-stranded RNAs of various sizes were found in most green, red, and brown algae. The characteristics of the algal dsRNAs are compared with those of dsRNAs from higher plants and the biological significance of the dsRNAs in cell organelles is discussed.

Discrete subcellular localization of membrane-bound ATPase activity in marine angiosperms and marine algae

The subcellular distribution of membrane-bound ATPases was compared among terrestrial plants, seagrasses and marine algae by cytochemical techniques. High ATPase activity was detected in the copiously invaginated plasma membrane that was characteristic of transfer cells but not in the tonoplast of epidermal cells in mature leaves of seagrasses. Magnesium- or Ca2+-dependent ATPase activity was induced together with the characteristics of transfer cells during the development of leaf tissues able to resist seawater. Northern hybridization revealed the effective induction of the synthesis of mRNA for plasma-membrane H+-ATPase during the development of leaves. Such high ATPase activity was not detected in the smooth plasma membranes of marine macro-algae but was found in the membranes of some cytoplasmic vesicles or microvacuoles, providing evidence of the excretion of salts by exocytosis. It appears, therefore, that two essentially different methods for excreting excess salts have developed separately in these two classes of marine plants. The evolution of mechanisms of salt tolerance in the plant kingdom is discussed in terms of the differential subcellular distribution of ATPase activity.

Selective occurrence of endogenous double-stranded RNAs in insects

Abstract Noninfectious endogenous double-stranded RNAs (dsRNAs) of 1–15 kb in length are widely distributed in both the plant and fungal kingdoms. Here we report the first evidence of the selective occurrence of such plasmid-like dsRNAs in the animal kingdom, a variety of symptomless insects. A considerable portion of larval, pupal and adult individuals of butterflies were found to harbor characteristic sets of dsRNA components at defined levels. Some cultured cell lines from the cabbage armyworm, Mamestra brassicae , also harbored indigenous dsRNAs, which had no lytic effects on the host cells and did not interfere with their normal growth. The moth dsRNAs were incapable of naturally infecting noncarrier cells during culture, a result that suggests efficient transmission of the dsRNAs to progeny cells through cell division. The total copy number of the dsRNAs in the cultured moth cells was estimated to be about 3000. The moth dsRNAs were found broadly around the mitochondrial fraction after subcellular fractionation, and they did not appear to form distinct virus-like particles.

Culture of protoplasts isolated from somatic embryos of carrot

Abstract Protoplasts were enzymatically released from somatic embryos of carrot and were cultured in a liquid medium and an agar plate. Addition of 10−7 M 2,4-dichlorophenoxyacetic acid (2,4-D) to the modified Lin and Staba medium kept most of the isolated protoplasts more healthy. About 10% of the isolated protoplasts divided once within a week, but they did not proliferate continuously. In a medium without 2,4-D, on the other hand, about 0.1% of the embryo-protoplasts began to divide continuously after 2–3 weeks. They again formed cell clusters and somatic embryos, but slowly. Cell contact or aggregation was found to be effective for isolated embryo- protoplasts to maintain their original appearance and to proliferate continuously. Without contact with other cells, most of the cultured protoplasts formed highly vacuolated, very large cells without proliferation.

Iron stress response in chickpea (Cicer arietinum L.): morphological changes and levels of H+-ATPase mRNA in root tips.

As a response to iron (Fe) deficiency, subapical swelling with formation of transfer cells was observed in root tips of a tolerant chickpea cultivar. Levels of H+-ATPase mRNA in the root tips within apical 10 mm were not influenced by the Fe supply to the medium. The findings indicate that the activity of H+-ATPase under Fe deficiency might be regulated at the post-transcriptional levels.

Three Size Classes of Higher Plant DNA-like RNA Grouped by MAK Column Chromatography I. Fractionation and Characterization

Summary Rapidly labeled DNA-like RNA from broad bean shoots was separated with salt concentration gradients into three distinct size classes utilizing MAK 1 ) column chromatography. It was found that two minor classes were eluted just before light and heavy components of rRNAs, respectively, and a major one after them. Proportions of these three newly-formed RNA classes observed in the chromatographic profiles depend significantly upon the tissues used. The two minor classes of DNA-like RNA sedimented rather monodispersedly at about 13 S and 20 S but another of higher molecular weight was largely heterogeneous and sedimented more slowly than the heavy component of rRNA. These DNA-like RNA components were distinguishable from each other by SDS-polyacrylamide gel electrophoresis, and showed high hybridization with single-stranded homologous DNA. Hybridization of the 13 S RNA was effectively inhibited by the high-molecular-weight DNA-like RNA. In contrast, hybridization of the high-molecular-weight RNA class was only incompletely inhibited by the 13 S RNA. The poly(A) content in various classes of DNA-like RNA was found to be rather low. These observations suggest that the three classes of DNA-like RNA exist in the cell as entities of different molecular weights and that the MAK fractionation profiles result from grouping of heterogeneous DNA-like RNAs. The 13 S and 20 S RNA classes are most likely fractions of mRNA , most of which are deficient in sufficiently long poly(A)-segments. The grouping of DNA-like RNA into three size classes by MAK column chromatography is a general phenomenon in various kinds of organisms.