Isolation and characterization of a cDNA encoding mitochondrial chaperonin 10 from Arabidopsis thaliana by functional complementation of an Escherichia coli groES mutant

Chaperonin (Cpn) is one of the molecular chaperones. Cpn10 is a co-factor of Cpn60, which regulates Cpn60-mediated protein folding. It is known that Cpn10 is located in mitochondria and chloroplasts in plant cells. The Escherichia coli homologue of Cpn10 is called GroES. A cDNA for the Cpn10 homologue was isolated from Arabidopsis thaliana by functional complementation of the E. coli groES mutant. The cDNA was 647 bp long and encoded a polypeptide of 98 amino acids. The deduced amino acid sequence showed approximately 50% identity to mammalian mitochondrial Cpn10s and 30% identity to GroES. A Northern blot analysis revealed that the mRNA for the Cpn10 homologue was expressed uniformly in various organs and was markedly induced by heat-shock treatment. The Cpn10 homologue was constitutively expressed in transgenic tobaccos. Immunogold and immunoblot analyses following the subcellular fractionation of leaves from transgenic tobaccos revealed that the Cpn10 homologue was localized in mitochondria and accumulated at a high level in transgenic tobaccos.     

Nuclear-encoded chloroplast ribosomal protein L12 of Nicotiana tabacum: characterization of mature protein and isolation and sequence analysis of cDNA clones encoding its cytoplasmic precursor.

Poly(A)+ mRNA isolated from Nicotiana tabacum (cv. Petite Havana) leaves was used to prepare a cDNA library in the expression vector lambda gt11. Recombinant phage containing cDNAs coding for chloroplast ribosomal protein L12 were identified and sequenced. Mature tobacco L12 protein has 44% amino acid identity with ribosomal protein L7/L12 of Escherichia coli. The longest L12 cDNA (733 nucleotides) codes for a 13,823 molecular weight polypeptide with a transit peptide of 53 amino acids and a mature protein of 133 amino acids. The transit peptide and mature protein share 43% and 79% amino acid identity, respectively, with corresponding regions of spinach chloroplast ribosomal protein L12. The predicted amino terminus of the mature protein was confirmed by partial sequence analysis of HPLC-purified tobacco chloroplast ribosomal protein L12. A single L12 mRNA of about 0.8 kb was detected by hybridization of L12 cDNA to poly(A)+ and total leaf RNA. Hybridization patterns of restriction fragments of tobacco genomic DNA probed with the L12 cDNA suggested the existence of more than one gene for ribosomal protein L12. Characterization of a second cDNA with an identical L12 coding sequence but a different 3'-noncoding sequence provided evidence that at least two L12 genes are expressed in tobacco.     

Chloroplasts have a novel Cpn10 in addition to Cpn20 as co-chaperonins in Arabidopsis thaliana

Previously, we characterized a mitochondrial co-chaperonin (Cpn10) and a chloroplast co-chaperonin (Cpn20) from Arabidopsis thaliana (Koumoto, Y., Tsugeki, R., Shimada, T., Mori, H., Kondo, M., Hara-Nishimura, I., and Nishimura, M. (1996) Plant J. 10, 1119-1125; Koumoto, Y., Shimada, T., Kondo, M., Takao, T., Shimonishi, Y., Hara-Nishimura, I., and Nishimura, M. (1999) Plant J. 17, 467-477). Here, we report a third co-chaperonin. The cDNA was 603 base pairs long, encoding a protein of 139 amino acids. From a sequence analysis, the protein was predicted to have one Cpn10 domain with an amino-terminal extension that might work as a chloroplast transit peptide. This novel Cpn10 was confirmed to be localized in chloroplasts, and we refer to it as chloroplast Cpn10 (chl-Cpn10). The phylogenic tree that was generated with amino acid sequences of other co-chaperonins indicates that chl-Cpn10 is highly divergent from the others. In the GroEL-assisted protein folding assay, about 30% of the substrates were refolded with chl-Cpn10, indicating that chl-Cpn10 works as a co-chaperonin. A Northern blot analysis revealed that mRNA for chl-Cpn10 is accumulated in the leaves and stems, but not in the roots. In germinating cotyledons, the accumulation of chl-Cpn10 was similar to that of chloroplastic proteins and accelerated by light. It was proposed that two kinds of co-chaperonins, Cpn20 and chl-Cpn10, work independently in the chloroplast.     

Nuclear-encoded tobacco chloroplast ribosomal protein L24. Protein identification, sequence analysis of cDNAs encoding its cytoplasmic precursor, and mRNA and genomic DNA analysis.

Using a Nicotiana tabacum leaf cDNA library in the expression vector lambda gt11, two cDNAs encoding the full-length precursor polypeptide (M(r) 20,696) of tobacco chloroplast ribosomal protein L24 were identified and sequenced. These cDNAs encode a mature protein of 146 amino acids (M(r) 16,418) with a transit peptide of 41 amino acids (M(r) 4,278). The mature tobacco L24 protein has 78, 65, 45, and 35% sequence identity with ribosomal proteins L24 of pea, spinach, Bacillus subtilis, and Escherichia coli, respectively. The transit peptide of tobacco L24 is 54 and 57% identical with that of L24 chloroplast ribosomal proteins of pea and spinach, respectively. An expressed beta-galactosidase:L24 fusion protein, bound to nitrocellulose filters, was used as affinity matrix to purify monospecific antibody to L24 protein. Using this monospecific antibody protein L24 was identified among high performance liquid chromatography (HPLC)-purified tobacco chloroplast ribosome 50 S subunit proteins. The predicted amino terminus of the mature L24 protein was confirmed by partial sequencing of the HPLC-purified L24 protein. Northern blot analysis revealed a single mRNA band (0.85-0.90 kilobase) corresponding in size to full-length L24 cDNA. The presence of multiple genes for L24 is suggested by Southern blot hybridization and characterization of two cDNAs for L24 which only differ in their 3'-noncoding sequences.     

Nuclear-encoded chloroplast ribosomal protein L27 of Nicotiana tabacum: cDNA sequence and analysis of mRNA and genes.

A tobacco (Nicotiana tabacum cv. Petite Havana) leaf cDNA library was constructed in the expression vector lambda gt11. Immunological and nucleic acid hybridization screening yielded several cDNAs encoding an M(r) 19,641 precursor to an M(r) 14,420 mature protein which is homologous to Escherichia coli ribosomal protein L27. One cDNA (L27-1; 882 nucleotides long) contains 104 bp of 5'-noncoding sequence, 51 codons for a transit peptide, 128 codons for the predicted mature L27 polypeptide, and 241 bp of 3'-noncoding sequence, including the poly(A)29 tail. A beta-galactosidase-L27 fusion protein was bound to nitrocellulose filters, expressed, and used as an affinity matrix to purify monospecific antibody to L27 protein from an antiserum of rabbits immunized with 50S chloroplast ribosomal proteins. Using this monospecific antibody, protein L27 was identified among HPLC-purified tobacco chloroplast ribosome 50S subunit proteins. The predicted amino terminus of the mature L27 protein was confirmed by partial sequencing of the HPLC-purified L27 protein. The mature L27 protein has 66%, 61%, 56%, and 48% amino acid sequence identity with the L27-type ribosomal proteins of Bacillus subtilis, E. coli, Bacillus stearo-thermophilus, and yeast mitochondria (MRP7), respectively, in the homologous overlapping regions. The transit peptide of tobacco chloroplast ribosomal protein L27 has 41% amino acid sequence similarity with the MRP7 mitochondrial targeting sequence. Tobacco chloroplast L27 protein also has a 40 amino acid long carboxyl-terminal extension (compared to its bacterial counterparts) which is similar to the corresponding portion of yeast MRP7.(ABSTRACT TRUNCATED AT 250 WORDS)     

In planta differential targeting analysis of <Emphasis Type="Italic">Thermotoga maritima</Emphasis> Cel5A and CBM6-engineered Cel5A for autohydrolysis

The heterologous expression of glycosyl hydrolases in bioenergy crops can improve the lignocellulosic conversion process for ethanol production. We attempted to obtain high-level expression of an intact Thermotoga maritima endoglucanase, Cel5A, and CBM6-engineered Cel5A in transgenic tobacco plants for the mass production and autohydrolysis of endoglucanase. Cel5A expression was targeted to different subcellular compartments, namely, the cytosol, apoplast, and chloroplast, using the native form of the pathogenesis-related protein 1a (PR1a) and Rubisco activase (RA) transit peptides. Cel5A transgenic tobacco plants with the chloroplast transit peptide showed the highest average endoglucanase activity and protein accumulation up to 4.5% total soluble protein. Cel5A-CBM6 was targeted to the chloroplast and accumulated up to 5.2% total soluble protein. In terms of the direct conversion of plant tissue into free sugar, the Cel5A-CBM6 transgenic plant was 33% more efficient than the Cel5A transgenic plant. The protein stability of Cel5A and Cel5A-CBM6 in lyophilized leaf material is an additional advantage in the bioconversion process.     

Targeted overexpression of the Escherichia coli MinC protein in higher plants results in abnormal chloroplasts

Higher plant chloroplast division involves some of the same types of proteins that are required in prokaryotic cell division. These include two of the three Min proteins, MinD and MinE, encoded by the min operon in bacteria. Noticeably absent from annotated sequences from higher plants is a MinC homologue. A higher plant functional MinC homologue that would interfere with FtsZ polymerization, has yet to be identified. We sought to determine whether expression of the bacterial MinC in higher plants could affect chloroplast division. The Escherichia coli minC (EcMinC) gene was isolated and inserted behind the Arabidopsis thaliana RbcS transit peptide sequence for chloroplast targeting. This TP-EcMinC gene driven by the CaMV 35S² constitutive promoter was then transformed into tobacco (Nicotiana tabacum L.). Abnormally large chloroplasts were observed in the transgenic plants suggesting that overexpression of the E. coli MinC perturbed higher plant chloroplast division.     

Chloroplast‐targeted bacterial RecA proteins confer tolerance to chloroplast DNA damage by methyl viologen or UV‐C radiation in tobacco (Nicotiana tabacum) plants

The nature and importance of the DNA repair system in the chloroplasts of higher plants under oxidative stress or UV radiation‐induced genotoxicity was investigated via gain‐of‐functional approaches exploiting bacterial RecAs. For this purpose, transgenic tobacco (Nicotiana tabacum) plants and cell suspensions overexpressing Escherichia coli or Pseudomonas aeruginosa RecA fused to a chloroplast‐targeting transit peptide were first produced. The transgenic tobacco plants maintained higher amounts of chloroplast DNA compared with wild‐type (WT) upon treatments with methyl viologen (MV), a herbicide that generates reactive oxygen species (ROS) in chloroplasts. Consistent with these results, the transgenic tobacco leaves showed less bleaching than WT following MV exposure. Similarly, the MV‐treated transgenic Arabidopsis plants overexpressing the chloroplast RecA homologue RECA1 showed weak bleaching, while the recA1 mutant showed opposite results upon MV treatment. In addition, when exposed to UV‐C radiation, the dark‐grown E. coli RecA‐overexpressing transgenic tobacco cell suspensions, but not their WT counterparts, resumed growth and greening after the recovery period under light conditions. Measurements of UV radiation‐induced chloroplast DNA damage using DraI assays (Harlow et al. 1994) with the chloroplast rbcL DNA probe and quantitative PCR analyses showed that the transgenic cell suspensions better repaired their UV‐C radiation‐induced chloroplast DNA lesions compared with WT. Taken all together, it was concluded that RecA‐overexpressing transgenic plants are endowed with an increased chloroplast DNA maintenance capacity and enhanced repair activities, and consequently have a higher survival tolerance to genotoxic stresses. These observations are made possible by the functional compatibility of the bacterial RecAs in chloroplasts.     

Expression, isolation, and characterization of a chloroplast targeting peptide

For the first time a method is described in which an N-terminal targeting peptide is isolated from Escherichia coli. After overexpression, purification, and cleavage of a fusion protein the protease-sensitive transit peptide from the chloroplast precursor protein preferredoxin could be isolated by HPLC. It was characterized by N-terminal amino acid sequencing and electrospray mass spectrometry. Its functionality was suggested by in vitro import competition experiments with isolated pea chloroplasts, in which the isolated peptide inhibited the import of radioactively labeled preferredoxin. Results from import competition experiments performed with a transit peptide deletion mutant suggested that the four extreme C-terminal amino acids lack information to interact with the chloroplast import machinery.     

Overexpression of the potential herbicide target sedoheptulose-1,7-bisphosphatase from Spinacia oleracea in transgenic tobacco

Several proteins are recalcitrant to expression in Escherichiacoli. To explore transgenic plants as an alternative expressionsystem, the gene encoding the potential herbicide target sedoheptulose-1,7-bisphosphatase (SBPase, EC 3.1.3.37) was expressed in transgenic tobacco(Nicotiana tabaccum) under the control of a duplicatedCaMV 35S RNA promoter. The active protein, a key enzyme in the Calvin cycle,accumulated to approximately 1.2% of total soluble protein. In order to purifyrecombinant SBPase, a sequence encoding six histidine residues was insertedC-terminally which allows a one step purification via Ni²⁺-NTAaffinity chromatography. N-terminal amino acid sequence analysis of the purifiedprotein confirmed processing of the transit peptide and revealed the previouslyunknown cleavage site. The transit peptide consists of 67 amino acids followedby the mature SBPase subunit of 342 amino acids including the C-terminalfusion. Purified SBPase was found to be enzymatically active after reduction with DTTand showed many biochemical properties of the native enzyme such as thedependence on Mg²⁺ and a pH optimum of 8.3. Subsequently, SBPaseproduced in transgenic tobacco was used in large-scale screening for thediscovery of novel herbicides.     

Interaction of homologues of Hsp70 and Cpn60 with ferredoxin-NADP reductase upon its import into chloroplasts

A homologue of the 70-kDa heat-shock protein (Hsp70) was purified from pumpkin chloroplasts. The molecular mass of the purified protein was approximately 75 kDa and its N-terminal amino acid sequence was very similar to those of homologues of Hsp70 from bacterial cells and from the mitochondrial matrix and stroma of pea chloroplasts. The purified homologue of Hsp70 was found in the stroma of chloroplasts. To investigate the role(s) of the homologue of Hsp70 in the chloroplast stroma, we examined the possibility that the homologue of Hsp70 might interact with newly imported proteins to assist in their maturation (for example, in their folding and assembly). Ferredoxin NADP⁺ reductase (FNR) imported into chloroplasts in vitro could be immunoprecipitated with antisera raised against the homologue of Hsp70 from pumpkin chloroplasts and against GroEL from Escherichia coli, which is a bacterial homologue of chaperonin 60 (Cpn60), in an ATP-dependent manner, an indication that newly imported FNR interacts physically with homologues of Hsp70 and Cpn60 in chloroplasts. Time-course analysis of the import of FNR showed that imported FNR interacts transiently with the homologue of Hsp70 and that the association of FNR with the homologue of Hsp70 precedes that with the homologue of Cpn60. These results suggest that homologues of Hsp70 and Cpn60 in chloroplasts might sequentially assist in the maturation of newly imported FNR in an ATP-dependent manner.     

<Emphasis Type="Italic">Arabidopsis</Emphasis> <Emphasis Type="Italic">thaliana</Emphasis> Rubisco small subunit transit peptide increases the accumulation of <Emphasis Type="Italic">Thermotoga maritima</Emphasis> endoglucanase Cel5A in chloroplasts of transgenic tobacco plants

Over the past decade various approaches have been used to increase the expression level of recombinant proteins in plants. One successful approach has been to target proteins to specific subcellular sites/compartments of plant cells, such as the chloroplast. In the study reported here, hyperthermostable endoglucanase Cel5A was targeted into the chloroplasts of tobacco plants via the N-terminal transit peptide of nuclear-encoded plastid proteins. The expression levels of Cel5A transgenic lines were then determined using three distinct transit peptides, namely, the light-harvesting chlorophyll a/b-binding protein (CAB), Rubisco small subunit (RS), and Rubisco activase (RA). RS:Cel5A transgenic lines produced highly stable active enzymes, and the protein accumulation of these transgenic lines was up to 5.2% of the total soluble protein in the crude leaf extract, remaining stable throughout the life cycle of the tobacco plant. Transmission election microscopy analysis showed that efficient targeting of Cel5A protein was under the control of the transit peptide.     

Cloning, sequencing, and functional expression in Escherichia coli of chaperonin (groESL) genes from Vibrio cholerae.

Using a series of oligonucleotides synthesized on the basis of conserved nucleotide motifs in heat-shock genes, the groESL heat-shock operon from a Vibrio cholerae TSI-4 strain has been cloned and sequenced, revealing that the presence of two open reading frames (ORFs) of 291 nucleotides and 1,632 nucleotides separated by 54 nucleotides. The first ORF encoded a polypeptide of 97 amino acids, GroES homologue, and the second ORF encoded a polypeptide of 544 amino acids, GroEL homologue. A comparison of the deduced amino acid sequences revealed that the primary structures of the V. cholerae GroES and GroEL proteins showed significant homology with those of the GroES and GroEL proteins of other bacteria. Complementation experiments were performed using Escherichia coli groE mutants which have the temperature-sensitive growth phenotype. The results showed that the groES and groEL from V. cholerae were expressed in E. coli, and groE mutants harboring V. cholerae groESL genes regained growth ability at high temperature. The evolutionary analysis indicates a closer relationship between V. cholerae chaperonins and those of the Haemophilus and Yersinia species.     

Expression of recombinant proteins lacking methionine as N-terminal amino acid in plastids: human serum albumin as a case study

Removal of the N-terminal methionine of a protein could be critical for its function and stability. Post-translational modifications of recombinant proteins expressed in heterologous systems may change amino-terminal regions. We studied the expression of mature proteins lacking methionine as the N-terminal amino acid in tobacco chloroplasts, using human serum albumin (HSA) as an example. Two approaches were explored. First, we fused the Rubisco small subunit transit peptide to HSA. This chimeric protein was correctly processed in the stroma of the chloroplast and rendered the mature HSA. The second approach took advantage of the endogenous N-terminal methionine cleavage by methionine aminopeptidase. Study of this protein processing reveals a systematic cleavage rule depending on the size of the second amino acid. Analysis of several foreign proteins expressed in tobacco chloroplasts showed a cleavage pattern in accordance to that of endogenous proteins. This knowledge should be taken into account when recombinant proteins with N-terminus relevant for its function are expressed in plastids.     

Homologous cpn60 genes in Rhizobium leguminosarum are not functionally equivalent

Many bacteria possess 2 or more genes for the chaperonin GroEL and the cochaperonin GroES. In particular, rhizobial species often have multiple groEL and groES genes, with a high degree of amino-acid similarity, in their genomes. The Rhizobium leguminosarum strain A34 has 3 complete groE operons, which we have named cpn.1, cpn.2 and cpn.3. Previously we have shown the cpn. 1 operon to be essential for growth, but the two other cpn operons to be dispensable. Here, we have investigated the extent to which loss of the essential GroEL homologue Cpn60.1 can be compensated for by expression of the other two GroEL homologues (Cnp60.2 and Cpn60.3). Cpn60.2 could not be overexpressed to high levels in R. leguminosarum, and was unable to replace Cpn60.1. A strain that overexpressed Cpn60.3 grew in the absence of Cpn60.1, but the complemented strain displayed a temperature-sensitive phenotype. Cpn60.1 and Cpn60.3, when coexpressed in Escherichia coli, preferentially selfassembled rather than forming mixed heteroligomers. We conclude that, despite their high amino acid similarity, the GroEL homologues of R. leguminosarum are not functionally equivalent in vivo.     

Chloroplast ribosomal protein L15, like L1, L13 and L21, is significantly larger than its E. coli homologue

The purification and identification by peptide sequence and immunological data of the spinach chloroplast homologue of E. coli L15 is presented. A significant increase in its mass over the E. coli counterpart is shown and is accounted for, in part, by a sequenced 18-residue N-terminal extension. A still larger C-terminal extension or internal insertion(s) is inferred. The migration position of the L15 in a 2D gel pattern of spinach chloroplast 50S subunit proteins is shown. Lack of sequence identity with the known chloroplast genomic data confirms the nuclear coding of this protein, and the N-terminal sequence given here provides the transit peptide cleavage site of the cytoplasmic precursor.     

An Arabidopsis gene encoding a chloroplast-targeted beta-amylase

beta-Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that beta-amylases are not involved in the metabolism of chloroplast-localized transitory leaf starch. However, we have identified a novel beta-amylase gene, designated ct-Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct-Bmy encodes a precursor protein which contains a typical N-terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic beta-amylases of higher plants. Expression of the ct-Bmy cDNA in E. coli confirmed that the encoded protein possesses beta-amylase activity. CT-BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT-BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT-BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct-Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.     

Targeting of protein to chloroplasts in transgenic tobacco by fusion to mutated transit peptide

Summary Transport of foreign proteins into chloroplasts was studied in a transgenic plant expressing two different fusion proteins, the transit peptide (TP) of ribulose-bisphosphate carboxylase small subunit (SS) fused to neomycin phosphotransferase (TP-NPT II) and, the same transit peptide plus the amino-terminal 23 amino acids of mature SS linked to NPT II. The second fusion protein (TP-SS-NPT II) was found in isolated chloroplasts but accumulated to a lesser degree than the first (TP-NPT II). This finding does not support the hypothesis that the highly conserved amino acid sequence surrounding the cleavage site between the transit peptide (TP) and mature SS is required for efficient transport. This cleavage region shows a markedly higher conservation than either the mature protein or the TP sequences in SS genes from different plant species. Evidence is presented indicating that the transport of the TP-SS-NPT II precursor is diminished as a result of competition between the rate of its uptake and the rate of its degradation by cytosolic proteases. In an attempt to identify further regions in the TP involved in transport and processing, we designed derivatives of both the TP-SS-NPT II and TP-NPT II precursors. A derivative of TP-SS-NPT II lacking the amino acids at the processing site was expressed in plants and was shown to be transported and processed. A derivative of TP-NPT II comprising the first 41 amino acids (out of 57) of the transit peptide linked to NPT II was also expressed in plants. This protein was not imported into the organelles; however a significant amount of partially processed fusion protein was found to be attached to the outer membrane of the chloroplast.     

Tissue-specific,light-regulated and plastid-regulated expression of the single-copy nuclear gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana

The single-copy PetC gene encoding the chloroplast Rieske FeS protein of Arabidopsis thaliana consists of five exons interrupted by four introns and encodes a protein of 229 amino acid residues with extensive sequence similarity to the chloroplast Rieske proteins of other higher plants. The N-terminal 50 amino acid residues constitute a presequence for targeting to the chloroplast and the remaining 179 amino acid residues make up the mature protein. Three of the introns are in identical positions in the PetC gene of Chlamydomonas reinhardtii, suggesting that they are of ancient origin. RNA-blot hybridisation showed that the gene was expressed in shoots, but not roots, and was light regulated and repressed by sucrose. The expression of chimeric genes consisting of PetC promoter fragments fused to the beta-glucuronidase (GUS) reporter gene was examined in A. thaliana and tobacco. In A. thaliana, GUS activity was detected in leaves, stems, flowers and siliques, but not in roots, and showed a strong correlation with the presence of chloroplasts. In transgenic tobacco, low levels of GUS activity were also detected in light-exposed roots. GUS activity in transgenic tobacco seedlings was light regulated and was decreased by norflurazon in the light suggesting regulation of PetC expression by plastid signals.     

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.