Were class C iron-containing superoxide dismutases of trypanosomatid parasites initially imported into a complex plastid? A hypothesis based on analyses of their N-terminal targeting signals

Trypanosomatid parasites possess 2 distinct iron-containing superoxide dismutases (Fe-SODs) designated SODA and SODC, both of which are targeted to their mitochondria. In contrast to SODAs that carry typical mitochondrial transit peptides, SODCs have highly unusual mitochondrial targeting signals. Our analyses clearly show that these pre-sequences are bipartite possessing a signal peptide-like domain followed by a transit peptide-like domain. Consequently, they resemble N-terminal extensions of proteins targeted to multi-membrane plastids, suggesting that trypanosomatids once contained a eukaryotic alga-derived plastid. Further support for this hypothesis comes from striking similarities in length, hydropathy profile, and amino acid composition of SODC pre-sequences to those of Euglena and dinoflagellate plastid proteins. To account for these data, we propose that the Trypanosomatidae initially possessed a gene encoding a mitochondrial Fe-SOD with a classical mitochondrial transit peptide. Before or after plastid acquisition, a gene duplication event gave rise to SODA and SODC. In a subsequent evolutionary step a signal peptide was linked to SODC, enabling its import into the plastid. When the trypanosomatid plastid subsequently was lost, natural selection favoured adaptation of the SODC N-terminal signal as a mitochondrial transit peptide and re-targeting to the mitochondrion.     

Effect of Ce(III) on Heat Production of Mitochondria Isolated from Hybrid Rice

The effect of cerium on mitochondria isolated from hybrid rice Shanyou 63 (Oryza sativa L) was investigated. Through in vivo culture, low dose Ce³⁺ promoted, but higher dose Ce³⁺, restrained mitochondrial heat production. However, through vitro incubation, Ce³⁺ showed only inhibitory action on mitochondrial energy turnover, the concentration required for 50% inhibition being 46.7 μM. In addition, Ce³⁺, like Ca²⁺, induced rice mitochondrial swelling and decreased membrane potential (△ψ), which was inhibited by the specific permeability transition inhibitor cyclosporine A (CsA). The induction approached a constant level while mitochondrial metabolism was fully prevented by Ce³⁺. These results demonstrated that cerium influenced rice mitochondria in vivo and in vitro via different action pathways, and the latter involved the opening of rice mitochondrial permeability.     

Molecular characterization of the mitochondrial elongation factor EF-Tu gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The mitochondrial elongation factor EF-Tu (tufM) in rice (Oryza sativa L.) was isolated and characterized. The rice tufM cDNA clone contained 1,726 nucleotides and coded for a 453 amino acid protein including a putative mitochondrial transit peptide of 64 amino acid residues. This coding region was composed of 12 exons and 11 introns. The deduced amino acid sequence showed 62% and 88% identities with rice chloroplast EF-Tu (tufA) and Arabidopsis mitochondrial EF-Tu, respectively. As previously observed for the rice tufA gene, the tufM gene is likely present as one copy in rice. The mitochondrial EF-Tu gene was differentially expressed during flower development, and the other translational EF-Tu genes (chloroplast EF-Tu and cytosolic EF-1 alpha) were also distinctly expressed in a temporal manner. Phylogenetic analysis of the rice tufM gene showed that the mitochondrial tufA homologue of Reclinomonas was more closely related to the mitochondrial tufM genes of flowering plants than fungal and other mitochondrial tuf genes. In addition, the tufM encoded an N-terminal extension showing significant similarity to that of rps14 (or sdhB), which is also a nuclear-encoded rice mitochondrial gene.     

Characterization of two rice genes for nuclear-encoded chloroplast ribosomal protein L12 and phylogenetic analysis of the acquisition of transit peptides and gene duplication

 We have identified two genes coding for chloroplast ribosomal protein L12 encoded in the nuclear genome of rice (Oryza sativa). These genes were designated rpl12-1 and rpl12-2 (rpl12, ribosomal protein L12). Northern analysis with specific probes revealed that both genes are transcribed. The expression of each gene seems to have a different regulatory machinery. It is also suggested that the expression of rpl12-1 is controlled in an organ-specific manner. The deduced amino-acid sequences of the mature peptide parts are more conserved than those of the transit peptide parts in both monocotyledonous and dicotyledonous plants. A phylogenetic tree was constructed using the nucleotide sequences of the transit peptide region of the rpl12s of reported plant. The tree includes estimates of when the transit peptides were acquired, and when the genes were duplicated, in the course of evolution. According to our hypothesis, the nuclear-translocated chloroplast ribosomal protein L12 gene obtained its transit peptide after the divergence of monocots and dicots, then gene duplications occurred independently in monocots and dicots, and subsequently rice and rye branched apart. Received: 4 October 1997 / Accepted: 5 January 1998     

Characterization of a novel zinc metalloprotease involved in degrading targeting peptides in mitochondria and chloroplasts

We have recently isolated and identified a novel mitochondrial metalloprotease, pre-sequence protease (PreP) from potato and shown that it degrades mitochondrial pre-sequences. PreP belongs to the pitrilysin protease family and contains an inverted zinc-binding motif. To further investigate the degradation of targeting peptides, we have overexpressed the Arabidopsis thaliana homologue of PreP, zinc metalloprotease (Zn-MP), in Escherichia coli . We have characterized the recombinant Zn-MP with respect to its catalytic site, substrate specificity and intracellular localization. Mutagenesis studies of the residues involved in metal binding identified the histidines and the proximal glutamate as essential residues for the proteolytic activity. Substrate specificity studies showed that the Zn-MP has the ability to degrade both mitochondrial pre-sequences and chloroplastic transit peptides, as well as other unstructured peptides. The Zn-MP does not recognize an amino acid sequence per se . Immunological studies and proteolytic activity measurements in isolated mitochondria and chloroplasts revealed the presence of the Zn-MP in both organelles. Furthermore, the Zn-MP was found to be dually imported to both mitochondria and chloroplasts in vitro . In summary, our data show that the Zn-MP is present and serves the same function in chloroplasts as in mitochondria – degradation of targeting peptides.     

Expression of BrD1, a plant defensin from Brassica rapa, confers resistance against brown planthopper (Nilaparvata lugens) in transgenic rices

Plant defensins are small (5-10 kDa) basic peptides thought to be an important component of the defense pathway against fungal and/or bacterial pathogens. To understand the role of plant defensins in protecting plants against the brown planthopper, a type of insect herbivore, we isolated the Brassica rapa Defensin 1 (BrD1) gene and introduced it into rice (Oryza sativa L.) to produce stable transgenic plants. The BrD1 protein is homologous to other plant defensins and contains both an N-terminal endoplasmic reticulum signal sequence and a defensin domain, which are highly conserved in all plant defensins. Based on a phylogenetic analysis of the defensin domain of various plant defensins, we established that BrD1 belongs to a distinct subgroup of plant defensins. Relative to the wild type, transgenic rices expressing BrD1 exhibit strong resistance to brown planthopper nymphs and female adults. These results suggest that BrD1 exhibits insecticidal activity, and might be useful for developing cereal crop plants resistant to sap-sucking insects, such as the brown planthopper.     

DCW11, down-regulated gene 11 in CW-type cytoplasmic male sterile rice, encoding mitochondrial protein phosphatase 2c is related to cytoplasmic male sterility

Causes of cytoplasmic male sterility (CMS) in plants have beenstudied for two decades, and mitochondrial chimeric genes havebeen predicted to induce CMS. However, it is unclear what happensafter CMS-associated proteins accumulate in mitochondria. Inour previous study of microarray analysis, we found that 140genes are aberrantly regulated in anthers of CW-type CMS ofrice (Oryza sativa L.). In the present study, we investigatedDCW11, one of the down-regulated genes in CW-CMS encoding aprotein phosphatase 2C (PP2C). DCW11 mRNA was preferentiallyexpressed in anthers, with the highest expression in maturepollen. As predicted by the N-terminal sequence, DCW11 signalpeptide–green fluorescent protein (GFP) fusion proteinwas localized in mitochondria. Knockdown of DCW11 in wild-typerice by RNA interference caused a major loss of seed-set fertility,without visible defect in pollen development. Since this knockdownphenotype resembled that of CW-CMS, we concluded that the down-regulationof DCW11 is correlated with CW-CMS. This idea was supportedby the up-regulation of alternative oxidase 1a (AOX1a), whichis known to be regulated by mitochondrial retrograde signaling,in DCW11 knockdown lines. Down-regulation of DCW11 and up-regulationof AOX1a were also observed in two other types of rice CMS.Our result indicates that DCW11 could play a role as a mitochondrialsignal transduction mediator in pollen germination.     

The Discriminatory Transfer Routes of tRNA Genes Among Organellar and Nuclear Genomes in Flowering Plants: A Genome-Wide Investigation of <Emphasis Type="Italic">indica</Emphasis> Rice

The transfer and integration of tRNA genes from organellar genomes to the nuclear genome and between organellar genomes occur extensively in flowering plants. The routes of the genetic materials flowing from one genome to another are biased, limited largely by compatibility of DNA replication and repair systems differing among the organelles and nucleus. After thoroughly surveying the tRNA gene transfer among organellar genomes and the nuclear genome of a domesticated rice (Oryza sativa L. ssp. indica), we found that (i) 15 mitochondrial tRNA genes originate from the plastid; (ii) 43 and 80 nuclear tRNA genes are mitochondrion-like and plastid-like, respectively; and (iii) 32 nuclear tRNA genes have both mitochondrial and plastid counterparts. Besides the native (or genuine) tRNA gene sets, the nuclear genome contains organelle-like tRNA genes that make up a complete set of tRNA species capable of transferring all amino acids. More than 97% of these organelle-like nuclear tRNA genes flank organelle-like sequences over 20 bp. Nearly 40% of them colocalize with two or more other organelle-like tRNA genes. Twelve of the 15 plastid-like mitochondrial tRNA genes possess 5′- and 3′-flanking sequences over 20 bp, and they are highly similar to their plastid counterparts. Phylogenetic analyses of the migrated tRNA genes and their original copies suggest that intergenomic tRNA gene transfer is an ongoing process with noticeable discriminatory routes among genomes in flowering plants. Electronic Supplementary Material Electronic Supplementary material is available for this article at and accessible for authorised users. Reviewing Editor: Dr. David Guttman     

The two-component signal system in rice (Oryza sativa L.): a genome-wide study of cytokinin signal perception and transduction

In this report we define the genes of two-component regulatory systems in rice through a comprehensive computational analysis of rice (Oryza sativa L.) genome sequence databases. Thirty-seven genes were identified, including 5 HKs (cytokinin-response histidine protein kinase) (OsHK1–4, OsHKL1), 5 HPs (histidine phosphotransfer proteins) (OsHP1–5), 15 type-A RRs (response regulators) (OsRR1–15), 7 type B RR genes (OsRR16–22), and 5 predicted pseudo-response regulators (OsPRR1–5). Protein motif organization, gene structure, phylogenetic analysis, chromosomal location, and comparative analysis between rice, maize, and Arabidopsis are described. Full-length cDNA clones of each gene were isolated from rice. Heterologous expression of each of the OsHKs in yeast mutants conferred histidine kinase function in a cytokinin-dependent manner. Nonconserved regions of individual cDNAs were used as probes in expression profiling experiments. This work provides a foundation for future functional dissection of the rice cytokinin two-component signaling pathway.     

Characterization of two cDNA clones encoding isozymes of the F1F0-ATPase inhibitor protein of rice mitochondria

Two cDNA clones encoding F₁F₀-ATPase inhibitor proteins, which are loosely associated with the F₁ part of the mitochondrial F₁F₀-ATPase, were characterized from rice (Oryza sativa L. cv. Nipponbare). A Northern hybridization showed that the two genes (designated as IF ₁ -1 and IF ₁ -2) are transcribed in all the organs examined. However, the steady-state mRNA levels varied among organs. A comparison of the deduced amino acid sequences of the two IF ₁ genes and the amino acid sequence of the mature IF₁ protein from potato revealed that IF₁-1 and IF₁-2 have N-terminal extensions with features that are characteristic of a mitochondrial targeting signal. To determine the subcellular localization of the gene products, the IF₁-1 or IF₁-2 proteins were fused in frame to the green fluorescent protein (GFP) or the fused GFP-β-glucuronidase, and expressed transiently in onion or dayflower epidermal cells. Localized fluorescence was detected in mitochondria, confirming that the two IF₁ proteins are targeted to mitochondria. Received: 9 July 1999 / Accepted: 17 August 1999     

The Arabidopsis thaliana genome encodes at least four thioredoxins m and a new prokaryotic-like thioredoxin

Screening of cDNA libraries at low stringency and complete sequencing of EST clones with homology to thioredoxins allowed us to characterize five new prokaryotic type Arabidopsis thaliana thioredoxins. All present N-terminal extensions with characteristics of transit peptides. Four are clustered in a phylogenetic tree with the chloroplastic thioredoxin m from red and green algae and higher plants, and their transit peptides have typical characteristics of chloroplastic transit peptides. One is clearly divergent and defines a new prokaryotic thioredoxin type that we have named thioredoxin x. Its transit peptide sequence presents characteristics of both chloroplastic and mitochondrial transit peptides. The five corresponding genes are expressed at different levels, but mostly in green tissues and in in-vitro cultivated cells.     

Microcalorimetric Investigation on Metabolic Activity and Effects of La (III) in Mitochondria Isolated from Indica Rice 9311

Thermogenic metabolic curves were determined by the ampoule method at 303 K using a TAM air isothermal microcalorimeter in mitochondria isolated from rice 9311 (Oryza sativa L). From the thermogenic curves the activity recovery rate constant k and the maximum heat power P _m were obtained. Both were positively correlated to the protein content of rice mitochondria. The corresponding correlation coefficients were 0.9959 and 0.9950, respectively, indicating that the in vitro metabolic activity of mitochondria can be reliably expressed by these parameters. Addition of La (III) ions in concentrations ranging from 0 to 130 μg/mL resulted in significantly higher k and P _m values. Concentrations from 140 to 180 μg/mL had the opposite effect. These results are consistent with previous reports on the effects of rare earth elements on plant growth. We propose that the lanthanum-induced change of mitochondrial metabolic activity is a possible mechanism by which La (III) ions influence indica rice 9311 growth.     

The use of rice seeds to produce human pharmaceuticals for oral therapy

Rice (Oryza sativa L.) is the major staple food consumed by half of the world's population. Rice seeds have gained recent attention as bioreactors for the production of human pharmaceuticals such as therapeutic proteins or peptides. Rice seed production platforms have many advantages over animal cell or microbe systems in terms of cost-effectiveness, scalability, safety, product stability and productivity. Rice seed-based human pharmaceuticals are expected to become innovative therapies as edible drugs. Therapeutic proteins can be sequestered within natural cellular compartments in rice seeds and protected from harsh gastrointestinal environments. This review presents the state-of-the-art on the construction of gene cassettes for accumulation of pharmaceutical proteins or peptides in rice seeds, the generation of transgenic rice plants, and challenges involved in the use of rice seeds to produce human pharmaceuticals.     

Characterization of protein synthesis by isolated rice mitochondria

Summary Bacteria-free mitochondria were isolated from aseptically grown, etiolated and green seedlings of both cytoplasmic male-sterile (WA-type) and male-fertile rice (Oryza sativa L.). Protein synthesis in these isolated mitochondria was characterized by gel electrophoresis/fluorography and by the incorporation of [³⁵S]-methionine into protein. In the presence of cycloheximide, a set of some 25 discrete polypeptides and an electrophoretically unresolved population were synthesized. This pattern of protein synthesis in organello was essentially the same in mitochondria isolated from both male-fertile and malesterile cytoplasms. Our data does not preclude the possibility, however, that the WA-type CMS possesses a tissue-specific and/or a low abundance mitochondrial protein(s), whose synthesis eluded detection under our experimental conditions. The synthesis of the mitochondria-encoded polypeptides by isolated rice mitochondria was inhibited by chloramphenicol and incompletely inhibited by erythromycin. A minor chloramphenicol-insensitive, cycloheximide-sensitive translation activity was found consistently to copurify with the mitochondria. This activity generated a reproducible electrophoretic profile of a poorly resolved, weakly labelled population of polypeptides and of a few conspicuous polypeptides, including a 42 kDa species.     

Microcalorimetric Investigation on Effects of LaIII on Metabolic Activity of Mitochondria Isolated from Hybrid Rice

Rare earth elements (REEs) have beneficial influence on plant growth and are widely used in agriculture practice, but little is known about the behavior of the REEs in mitochondria of plant cell. Thermogenic metabolic curves were determined by the ampoule method at 303 K using a TAM air isothermal microcalorimeter in mitochondria isolated from hybrid rice Liangyoupeijiu (Oryza sativa L.), and the effect of La^III on its mitochondrial metabolism was investigated. From the obtained heat flux curves, the crucial parameters including as activity recovery rate constant (k) and maximum heat power (P_max) were determined. Application of La³⁺ in concentrations ranging from 0 to 120 μg/ml significantly increased k and P_max values with the high point reaching 346 and 222% of the control, respectively. Concentrations from 140–150 μg/ml had the opposite effect. These results were consistent with previous reports on the effects of REEs on plant growth. It was concluded that the La‐induced change of mitochondrial metabolic activity is a possible mechanism by which La^III ions influences hybrid rice growth.