RNA editing in plant organelles: machinery, physiological function and evolution.

In plants, RNA editing is a process for converting a specific nucleotide of RNA from C to U and less frequently from U to C in mitochondria and plastids. To specify the site of editing, the cis-element adjacent to the editing site functions as a binding site for the trans-acting factor. Genetic approaches using Arabidopsis thaliana have clarified that a member of the protein family with pentatricopeptide repeat (PPR) motifs is essential for RNA editing to generate a translational initiation codon of the chloroplast ndhD gene. The PPR motif is a highly degenerate unit of 35 amino acids and appears as tandem repeats in proteins that are involved in RNA maturation steps in mitochondria and plastids. The Arabidopsis genome encodes approximately 450 members of the PPR family, some of which possibly function as trans-acting factors binding the cis-elements of the RNA editing sites to facilitate access of an unidentified RNA editing enzyme. Based on this breakthrough in the research on plant RNA editing, I would like to discuss the possible steps of co-evolution of RNA editing events and PPR proteins.

Cytochrome b(6)f mutation specifically affects thermal dissipation of absorbed light energy in Arabidopsis.

Light-induced lumenal acidification controls the efficiency of light harvesting by inducing thermal dissipation of excess absorbed light energy in photosystem II. We isolated an Arabidopsis mutant, pgr1 (proton gradient regulation), entirely lacking thermal dissipation, which was observed as little non-photochemical quenching of chlorophyll fluorescence. Map-based cloning showed that pgr1 had a point mutation in petC encoding the Rieske subunit of the cytochrome b(6)f complex. Although the electron transport rate was not affected at low light intensity, it was significantly restricted at high light intensity in pgr1, indicating that the lumenal acidification was not sufficient to induce thermal dissipation. This view was supported by (i) slow de-epoxidation of violaXanthin, which is closely related to lumenal acidification, and (ii) reduced 9-aminoacridine fluorescence quenching. Although lumenal acidification was insufficient to induce thermal dissipation, growth rate was not affected under low light growth conditions in pgr1. These results suggest that thermal dissipation is precisely regulated by lumenal pH to maintain maximum photosynthetic activity. We showed that pgr1 was sensitive to changes in light conditions, demonstrating that maximum activity of the cytochrome b(6)f complex is indispensable for short-term acclimation.

Introduction of bacterial metabolism into higher plants by polycistronic transgene expression.

Multiple-gene transformation is required to improve or change plant metabolisms effectively; but this many-step procedure is time-consuming and costing. We succeeded in the metabolic engineering of tobacco plants by introducing multiple genes as a bacteria-type operon into a plastid genome. The tobacco plastid was transformed with a polycistron consisting of three bacterial genes for the biosynthesis of a biodegradable polyester, polyhydroxybutyrate (PHB). Accumulation of PHB in the leaves of the transgenic tobacco indicated that the introduced genes were polycistronically expressed. This "phyto-fermentation" system can be used in plant production of various chemical commodities and pharmaceuticals.

The chloroplast clpP gene, encoding a proteolytic subunit of ATP-dependent protease, is indispensable for chloroplast development in tobacco.

ClpP is a proteolytic subunit of the ATP-dependent Clp protease, which is found in chloroplasts in higher plants. Proteolytic subunits are encoded both by the chloroplast gene, clpP, and a nuclear multi gene family. We insertionally disrupted clpP by chloroplast transformation in tobacco. However, complete segregation was impossible, indicating that the chloroplast-encoded clpP gene has an indispensable function for cell survival. In the heteroplasmic clpP disruptant, the leaf surface was rough by clumping, and the lateral leaf expansion was irregularly arrested, which led to an asymmetric, slender leaf shape. Chloroplasts consisted of two populations: chloroplasts that were similar to the wild type, and small chloroplasts that emitted high chl fluorescence. Ultrastructural analysis of chloroplast development suggested that clpP disruption also induced swelling of the thylakoid lumen in the meristem plastids and inhibition of etioplast development in the dark. In mature leaves, thylakoid membranes of the smaller chloroplast population consisted exclusively of large stacks of tightly appressed membranes. These results indicate that chloroplast-encoded ClpP is involved in multiple processes of chloroplast development, including a housekeeping function that is indispensable for cell survival.

The SPIRAL genes are required for directional control of cell elongation in Aarabidopsis thaliana.

Cells at the elongation zone expand longitudinally to form the straight central axis of plant stems, hypocotyls and roots, and transverse cortical microtubule arrays are generally recognized to be important for the anisotropic growth. Recessive mutations in either of two Arabidopsis thaliana SPIRAL loci, SPR1 or SPR2, reduce anisotropic growth of endodermal and cortical cells in roots and etiolated hypocotyls, and induce right-handed helical growth in epidermal cell files of these organs. spr2 mutants additionally show right-handed twisting in petioles and petals. The spr1spr2 double mutant's phenotype is synergistic, suggesting that SPR1 and SPR2 act on a similar process but in separate pathways in controlling cell elongation. Interestingly, addition of a low dose of either of the microtubule-interacting drugs propyzamide or taxol in the agar medium was found to reduce anisotropic expansion of endodermal and cortical cells at the root elongation zone of wild-type seedlings, resulting in left-handed helical growth. In both spiral mutants, exogenous application of these drugs reverted the direction of the epidermal helix, in a dose-dependent manner, from right-handed to left-handed; propyzamide at 1 microM and taxol at 0.2-0.3 microM effectively suppressed the cell elongation defects of spiral seedlings. The spr1 phenotype is more pronounced at low temperatures and is nearly suppressed at high temperatures. Cortical microtubules in elongating epidermal cells of spr1 roots were arranged in left-handed helical arrays, whereas the highly isotropic cortical cells of etiolated spr1 hypocotyls showed microtubule arrays with irregular orientations. We propose that a microtubule-dependent process and SPR1/SPR2 act antagonistically to control directional cell elongation by preventing elongating cells from potential twisting. Our model may have implicit bearing on the circumnutation mechanism.

Reduction of Q(A) in the dark: Another cause of fluorescence F(o) increases by high temperatures in higher plants.

Increases in the chlorophyll fluorescence F(o) (dark level fluorescence) during heat treatments were studied in various higher plants. Besides the dissociation of light-harvesting chlorophyll a/b protein complexes from the reaction center complex of PS II and inactivation of PS II, dark reduction of Q(A) via plastoquinone (PQ) seemed to be related to the F(o) increase at high temperatures. In potato leaves or green tobacco cultured cells, a part of the F(o) increase was quenched by light, reflecting light-induced oxidation of Q(A) (-) which had been reduced in the dark at high temperatures. Appearance of the F(o) increase due to Q(A) reduction depended on the plant species, and the mechanisms for this are proposed. The reductants seemed to be already present and formed by very brief illumination of the leaves at high temperatures. A ndhB-less mutant of tobacco showed that complex I type NAD(P)H dehydrogenase is not involved in the heat-induced reduction of Q(A). Quite strong inhibition of the Q(A) reduction by diphenyleneiodonium suggests that a flavoenzyme is one of the electron mediator to PQ from the reductant in the stroma. Reversibility of the heat-induced Q(A) reduction suggests that an enzyme(s) involved is activated at high temperatures and mostly returns to an inactive form at room temperature (25 degrees C).

The role of chloroplastic NAD(P)H dehydrogenase in photoprotection.

After a brief exposure to supra-saturating light, leaves of a tobacco transformant, in which chloroplastic NAD(P)H dehydrogenase (NDH) was defective, showed more severe photoinhibition than the wild-type, when judged by the parameter of chlorophyll fluorescence Fv/Fm. Repeated application of supra-saturating light eventually resulted in chlorosis in the NDH-defective mutant, while the wild-type sustained less photodamage and was able to recover from it. The mechanism of the phenomena is discussed with respect to the potential role of NDH in photosynthesis.

Identification and characterization of Arabidopsis mutants with reduced quenching of chlorophyll fluorescence.

Regulation of nonradiative dissipation of absorbed light energy in PSII is an indispensable process to avoid photoinhibition in plants. To dissect molecular mechanisms of the regulation, we identified Arabidopsis mutants with reduced quenching of Chl fluorescence using a fluorescence imaging system. By analyses of Chl fluorescence induction pattern in the light and quantum yield of both photosystems, 37 mutants were classified into three groups. The first group was characterized by an extremely high level of minimum Chl fluorescence at the open PSII center possibly due to a defect in PSII. Mutants with significant reduction in the nonphotochemical quenching formation but not in quantum yield of both photosystems were classified into the second group. Mutants in the third group showed reduction in quantum yield of both photosystems possibly due to a defect in the electron transport activity. Mutants in the second and third groups were further characterized by light intensity dependence of Chl fluorescence parameters and steady state redox level of P700.

Directed disruption of the tobacco ndhB gene impairs cyclic electron flow around photosystem I.

To evaluate the physiological significance of cyclic electron flow around photosystem (PS) I, we used a reverse genetic approach to focus on 11 chloroplast genes that encode homologs of mitochondrial complex I subunits (ndhA-K). Since their discovery, the exact function of the respiratory components in plant chloroplasts has been a matter of discussion. We disrupted one of these genes (ndhB) in tobacco by chloroplast transformation. Analysis of the transient increase in chlorophyll fluorescence after actinic light illumination and the redox kinetics of P700 (reaction center chlorophylls of PS I) suggest that the cyclic electron flow around PS I is impaired in the ndhB-deficient transformants. Transformants grew normally in a greenhouse, suggesting that the cyclic electron flow around PS I mediated by ndh gene products is dispensable in tobacco under mild environmental conditions.

Inhibition of ascorbate peroxidase under oxidative stress in tobacco having bacterial catalase in chloroplasts.

To analyze the potential of the active oxygen-scavenging system of chloroplasts, we introduced Escherichia coli catalase into tobacco chloroplasts. Photosynthesis of transgenic plants was tolerant to high irradiance under drought conditions, while the wild plants suffered severe damage in photosynthesis under the same conditions. Irrespective of responses to the stress, ascorbate peroxidase was completely inactivated both in the transgenic and wild-type plants. These findings are contrary to the established idea that the ascorbate peroxidase-mediated antioxidative system protects chloroplasts from oxidative stress.

Agr, an Agravitropic locus of Arabidopsis thaliana, encodes a novel membrane-protein family member.

Mutations in the Agr locus of Arabidopsis thaliana impair the root gravitropic response. Root growth of agr mutants is moderately resistant to ethylene and to an auxin transport inhibitor. Vertically placed agr roots grow into agar medium containing IAA or naphthalene-1-acetic acid, but not into medium containing 2,4-D. Positional cloning showed that AGR encodes a root-specific member of a novel membrane-protein family with limited homology to bacterial transporters.

Mitochondrial genome structure of a cytoplasmic hybrid between tomato and wild potato.

A physical map of the mitochondrial genome was constructed for a male-sterile tomato, MSA1, which had been generated by an asymmetric cell fusion between tomato (Lycopersicon esculentum) and wild potato (Solanum acaule). The entire genomic sequence of the MSA1 mitochondria (450 kb) was represented by five maps. Even if sequence duplications were taken into consideration, at least two linkage groups (maps 1-4 and map 5) were necessary to show the overall genome. The mitochondrial genome structure of MSA1 was also analyzed by comparing the Southern hybridization patterns of MSA1 and its parents (tomato and wild potato). The mitochondrial genome of MSA1 consists of a complex mixture of the parental genomes with at least 11 molecular recombination events.

Analysis of the heterogeneous transcripts of the highly edited orf206 in tomato mitochondria.

RNA editing of tomato orf206, postulated to encode a subunit of the ABC-type heme transporter involved in the biogenesis of cytochrome c, was analyzed by sequencing products of RT-PCR. While half of the transcripts were fully edited, others were heterogeneous as a result of partial or unusual editing.

A point mutation in the gene encoding the Rubisco large subunit interferes with holoenzyme assembly.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme of photosynthetic CO2 fixation, is composed of 8 large and 8 small subunits. The Rubisco-deficient Nicotiana tabacum mutant Sp25 is able to synthesize the peptides for both subunits but does not contain any active holoenzyme. The phenotype is maternally inherited and thus caused by a mutation in the chloroplast genome, which also encodes the Rubisco large subunit. A comparison of the nucleotide sequences of the large subunit gene of the Sp25 mutant with that of the wild-type tobacco revealed a single nucleotide change in the Sp25 mutant. This resulted in an amino acid substitution at Gly-322, which was replaced by serine.

Analysis of nuclear sequences homologous to the B4 plasmid-like DNA of rice mitochondria; evidence for sequence transfer from mitochondria to nuclei.

Nuclear sequences homologous to the plasmid-like DNA, B4, were analyzed in the Japonica rice variety, Fujiminori. Homologous sequences existed at several positions in the nuclear genome, but each contained only a portion of the B4 sequence. It was impossible to reconstruct the entire sequence of B4 even by collating all the homologous sequences. Overlaps between some of the B4 sequences present in the nuclear genome resulted in parts of the sequence being represented more than once. These features indicate that nuclear sequences homologous to B4 are not the origin of B4 and that they have been transferred from mitochondria and integrated into the nuclear genome. Five other foreign sequences originating in the chloroplast or mitochondrial genome were found within 1 kb of the B4-homologous sequences. Structural analysis is consistent with the hypothesis that the DNA sequences were transferred via RNA.

[Two cases of anorectal malignant melanoma].

Although on anorectal malignant melanoma is relatively rare, we report two cases, that of a 88-year-old woman, and a 78-year-old man. Both patients were admitted to the hospital due to rectal bleeding and were diagnosed as having rectal carcinoma. Following an abdominoperineal rectal amputation, a pathological examination of the resected specimens revealed a malignant melanoma. Thus, it is necessary to carry out a careful examination of patients with tumors of the anorectal region when there is the least suspicion of a malignancy.

Nucleotide sequence and molecular characterization of plasmid-like DNAs from mitochondria of cytoplasmic male-sterile rice.

Two plasmid-like DNAs, B2 and B3, were isolated from mitochondria of the cytoplasmic male-sterile rice, A-58 CMS. Molecular clones having their complete sequences were constructed and used as probes of mitochondrial and nuclear genomes by Southern hybridization. No evidence was found that integrated copies of either one exist in the main mitochondrial genome, but sequences homologous to both were present in the nuclear genome. The complete nucleotide sequences of B2 and B3 were established and compared to those of rice B1 and B4 and to the 1.9- and the 1.4-kbp plasmid-like DNAs of maize. Many of the sequences were common to both plant species.

Plant regeneration from cytoplasmic hybrids of rice (Oryza sativa L.).

We obtained cybrid plants by electrofusing γ-irradiated protoplasts of a cytoplasmic male-sterile line "A-58 CMS" (Oryza sativa L.) and iodoacetamide (IOA)-treated protoplasts of the fertile (normal) rice cultivar "Fujiminori". The cybridity of the plants was confirmed by mitochondrial (mt) DNA restriction endonuclease, and plasmid-like DNA analyses, and by isozyme, cytological and morphological investigations. The chromosome number of the cybrid plants is 24.

Properties of the circular plasmid-like DNA, B4, from mitochondria of cytoplasmic male-sterile rice.

The plasmid-like DNA, B4, consisting of 969 base pairs (bp), was isolated from mitochondria of the cytoplasmic male-sterile rice, A-58 CMS. Molecular clones containing the complete B4 sequence were constructed and used in Southern hybridization to probe mitochondrial and nuclear genomes. No evidence was found for the existence of integrated copies of B4 in the main mitochondrial genomes of either the male-sterile or fertile rice. Sequences homologous to B4, however, were found in the rice nuclear genome. The complete B4 nucleotide sequence was determined, and a sequence homologous to B4 was found in the 1.9 kbp plasmid-like DNA of maize.

Asymmetric hybridization between cytoplasmic male-sterile (CMS) and fertile rice (Oryza safiva L.) protoplasts.

(60)Co-irradiated protoplasts of the cytoplasmic male-sterile line A-58 CMS (Oryza saliva L.) were electrofused with iodoacetamide (IOA)-treated protoplasts of the fertile (normal) rice cultivar 'Fujiminori'. Seven of the colonies that formed were identified as cytoplasmic hybrids (cybrids): they all had the peroxidase isozymes of the fertile 'Fujiminori' parent, but contained four plasmid-like DNAs (Bl, B2, B3 and B4) from the sterile A-58 CMS parent in their mitochondrial genomes. In addition, digestion of cybrid mtDNA gave a set of restriction fragments that differed from those of the parents.