Three genes from Arabidopsis thaliana with high sequence similarity to gamma carbonic anhydrase (gammaCA), a Zn containing enzyme from Methanosarcina thermophila (CAM), were identified and characterized. Evolutionary and structural analyses predict that these genes code for active forms of gammaCA. Phylogenetic analyses reveal that these Arabidopsis gene products cluster together with CAM and related sequences from alpha and gamma proteobacteria, organisms proposed as the mitochondrial endosymbiont ancestor. Indeed, in vitro and in vivo experiments indicate that these gene products are transported into the mitochondria as occurs with several mitochondrial protein genes transferred, during evolution, from the endosymbiotic bacteria to the host genome. Moreover, putative CAM orthologous genes are detected in other plants and green algae and were predicted to be imported to mitochondria. Structural modeling and sequence analysis performed in more than a hundred homologous sequences show a high conservation of functionally important active site residues. Thus, the three histidine residues involved in Zn coordination (His 81, 117 and 122), Arg 59, Asp 61, Gin 75, and Asp 76 of CAM are conserved and properly arranged in the active site cavity of the models. Two other functionally important residues (Glu 62 and Glu 84 of CAM) are lacking, but alternative amino acids that might serve to their roles are postulated. Accordingly, we propose that photosynthetic eukaryotic organisms (green algae and plants) contain gammaCAs and that these enzymes codified by nuclear genes are imported into mitochondria to accomplish their biological function.

SummaryFrataxin, a protein crucial for the biogenesis of mitochondria in different organisms, was recently identified in Arabidopsis thaliana. To investigate the role of frataxin in higher plants, we analyze two knock-out and one knock-down T-DNA insertion mutants. The knock-out mutants present an embryo-lethal phenotype, indicating an essential role for frataxin. The knock-down mutant has reduced frataxin mRNA and protein levels. This mutant also presents retarded growth, reduced fresh weight of fruits and reduced number of seeds per fruit. Surprisingly, transcription of aconitase and the Fe-S subunit of succinate dehydrogenase (SDH2-1) are increased in mutant plants; however, the activity of these proteins is reduced, indicating a role for frataxin in Fe-S cluster assembly or insertion of Fe-S clusters into proteins. Mutant plants also have increased CO 2 assimilation rates, exhibit increased formation of reactive oxygen species (ROS) and have increased levels of transcripts for proteins known to be involved in the ROS stress responses. These results indicate that frataxin is an essential protein in plants, required for full activity of mitochondrial Fe-S proteins and playing a protective role against oxidative damage.

We report the identification by two hybrid screens of two novel similar proteins, called Arabidopsis thaliana gamma carbonic anhydrase like1 and 2 (AtgammaCAL1 and AtgammaCAL2), that interact specifically with putative Arabidopsis thaliana gamma Carbonic Anhydrase (AtgammaCA) proteins in plant mitochondria. The interaction region that was located in the N-terminal 150 amino acids of mature AtgammaCA and AtgammaCA like proteins represents a new interaction domain. In vitro experiments indicate that these proteins are imported into mitochondria and are associated with mitochondrial complex I as AtgammaCAs. All plant species analyzed contain both AtgammaCA and AtgammaCAL sequences indicating that these genes were conserved throughout plant evolution. Structural modeling of AtgammaCAL sequences show a deviation of functionally important active site residues with respect to gammaCAs but could form active interfaces in the interaction with AtgammaCAs. We postulate a CA complex tightly associated to plant mitochondrial complex.

RNA editing in higher plant mitochondria modifies mRNA sequences by means of C-to-U conversions at highly specific sites. To determine the cis elements involved in recognition of an editing site in plant mitochondria, deletion and site-directed mutation constructs containing the cognate cox II mitochondrial gene were introduced into purified mitochondria by electroporation. The RNA editing status was analyzed for precursor and spliced transcripts from the test construct. We found that only a restricted number of nucleotides in the vicinity of the target C residue were necessary for recognition by the editing machinery and that the nearest neighbor 3 residues were crucial for the editing process. We provide evidence that two functionally distinguishable sequences can be defined: the 16-nucleotide 5 region, which can be replaced with the same region from another editing site, and a 6-nucleotide 3 region specific to the editing site. The latter region may play a role in positioning the actual editing residue.RNA editing refers to a process whereby the genetic message is changed at single nucleotides in a very specific manner. This process involves a variety of genetic systems and occurs by different mechanisms (reference 6 and references therein). In trypanosome kinetoplasts, RNA editing proceeds by insertion and deletion of uridine nucleotides in mRNAs (2); the insertion of C residues has been described for Physarum polycephalum mitochondria, and the insertion of some G residues occurs in paramyxovirus (25,36). Another type of RNA editing is base conversion, occurring in mammalian nuclei (31) and plant organelles. C-to-U conversions have been described for higher plant mitochondria (9, 13, 16) and to a lesser extent for chloroplasts (18,21).RNA editing is a posttranscriptional event in plant organelles. It is essential in plant mitochondrion gene expression processes such as the maturation step of organellar transcripts (26,27) or the synthesis of functional proteins, since the nucleotide conversions usually alter the coding properties of the mRNA (1). The editing systems in higher plant organelles, mitochondria, and chloroplasts share many similar features, but promiscuous chloroplast sequences are not edited in mitochondria (39); conversely, a mitochondrial sequence carrying an editing site does not sustain editing when transcribed into chloroplasts (35). These results indicate that editing recognition signals are specific to each organelle. The sequences flanking target C residues lack any apparent conserved consensus elements at the primary or secondary structure level. An essential problem is to define the signals that determine the specific recognition of every editing site.A number of in vivo studies of transgenic chloroplasts have demonstrated that mRNA sequences flanking the editing site are involved in RNA editing (4, 5). RNA editing has been determined to proceed by deamination of the C residue in wheat (3) and pea (38). However, the molecular determinants for editing-site recognition have not yet been ...

Three different nuclear genes encode the essential iron-sulfur subunit of mitochondrial complex II (succinate dehydrogenase) in Arabidopsis (Arabidopsis thaliana), raising interesting questions about their origin and function. To find clues about their role, we have undertaken a detailed analysis of their expression. Two genes (SDH2-1 and SDH2-2) that likely arose via a relatively recent duplication event are expressed in all organs from adult plants, whereas transcripts from the third gene (SDH2-3) were not detected. The tissue-and cell-specific expression of SDH2-1 and SDH2-2 was investigated by in situ hybridization. In flowers, both genes are regulated in a similar way. Enhanced expression was observed in floral meristems and sex organ primordia at early stages of development. As flowers develop, SDH2-1 and SDH2-2 transcripts accumulate in anthers, particularly in the tapetum, pollen mother cells, and microspores, in agreement with an essential role of mitochondria during anther development. Interestingly, in contrast to the situation in flowers, only SDH2-2 appears to be expressed at a significant level in root tips. Strong labeling was observed in all cell layers of the root meristematic zone, and a cell-specific pattern of expression was found with increasing distance from the root tip, as cells attain their differentiated state. Analysis of transgenic Arabidopsis plants carrying SDH2-1 and SDH2-2 promoters fused to the b-glucuronidase reporter gene indicate that both promoters have similar activities in flowers, driving enhanced expression in anthers and/or pollen, and that only the SDH2-2 promoter is active in root tips. These b-glucuronidase staining patterns parallel those obtained by in situ hybridization, suggesting transcriptional regulation of these genes. Progressive deletions of the promoters identified regions important for SDH2-1 expression in anthers and/or pollen and for SDH2-2 expression in anthers and/or pollen and root tips. Interestingly, regions driving enhanced expression in anthers are differently located in the two promoters.The mitochondrial electron transport chain of eukaryotes consists of four major multimeric enzyme complexes, one of which is succinate:ubiquinone oxidoreductase (succinate dehydrogenase; EC 1.3.5.1), commonly referred to as complex II. This important membrane-associated complex is a functional part of both the citric acid cycle and the aerobic respiratory chain, catalyzing the oxidation of succinate to fumarate and the reduction of ubiquinone to ubiquinol.Complex II has been well characterized in bacteria, fungi, and mammals and is known to be the simplest of all the complexes of the electron transport chain, with four subunits (Lemire and Oyedotun, 2002;Yankovskaya et al., 2003). It contains two peripheral membrane proteins, a flavoprotein (SDH1) and an iron-sulfur protein (SDH2), and two small integral membrane proteins (SDH3 and SDH4). The succinatebinding site is formed by the SDH1 polypeptide, which is linked covalently to a FAD molecule acting as acceptor o...

Cytoplasmic male sterility in plants is associated with mitochondrial dysfunction. We have proposed that a nuclear-encoded chimeric peptide formed by mitochondrial sequences when imported into the mitochondria may impair organelle function and induce male sterility in plants. A model developed to test this hypothesis is reported here. Assuning that the editing process in higher plant mitochondria reflects a requirement for producing active proteins, we have used edited and unedited coding sequences of wheat ATP synthase subunit 9 (atp9) fused to the coding sequence of a yeast coxlV transit peptide. Transgenic plants containing unedited atp9 exhibited either fertile, semifertile, or male-sterile phenotypes; controls containing edited atp9 or only the selectable marker gave fertile plants. Pollen fertility ranged from 31% to 75% in fertile plants, 10% to 20% in semifertile plants, and <2% in malesterile plants. Genetic and molecular data showed that the chimeric plasmid containing the transgene is inherited as a Mendelian trait. The transgenic protein is imported into the mitochondria. The production and frequency of semifertile or male-sterile transgenic plants conform to the proposed hypothesis.Male sterility in plants leads to pollen abortion. This phenomenon is often observed in alloplasmic plants combining cytoplasmic and nuclear genetic material from two different species (1). Maternally inherited cytoplasmic male sterility (CMS) has been recently reviewed (2). Molecular studies on CMS have revealed the existence of modifications in mitochondrial DNA. In a number of CMS plants, the sterile phenotype is associated with the production of proteins arising from chimeric genes (3, 4); their incorporation into the mitochondrial membrane or into multiprotein enzyme complexes may lead to the impairment of mitochondrial function.The hypothesis developed in this report is that the impairment of mitochondrial function may be obtained by introducing an altered subunit in the ATP synthase complex. The protein ATP9 represents a good candidate because it is involved in the proton channel of the ATP synthase. ATP9 is mitochondrially encoded in plants but nuclear-encoded in Neurospora (5) and mammals (6). A mitochondrially encoded protein can be introduced into nuclei and imported into mitochondria when fused to a targeting sequence (7,8). An approach similar to the one described here has been used to introduce herbicide resistance into tobacco chloroplasts (9).We have shown a discrepancy between the amino acid sequence of the ATP9 protein and the predicted sequence deduced from the gene.

The bulk of the secretion of the subcommissural organ is formed by glycoproteins that appear to be derived from two precursor forms of 540 and 320 kDa. Upon release into the ventricle, these glycoproteins aggregate to form Reissner's fiber. We report the isolation of three cDNA clones from a cDNA library prepared from bovine subcommissural organ RNA, by using an anti-Reissner's fiber serum for immunoscreening. Inserts of 0.7, 1.2, and 2.5 kb were amplified by the polymerase chain reaction, subcloned into pUC18 vector, and sequenced. Although restriction mapping of the three inserts initially suggested that all of them were derived from the same mRNA, sequence analysis showed that a short non-homologous region was present in the 0.7-kb insert when compared with the 1. 2-kb and 2.5-kb inserts, suggesting that they corresponded to two different, although highly homologous, mRNAs. Northern analyses showed a single mRNA species of approximately 9.5 kb present in the subcommissural organ and missing in the choroid plexus, brain cortex, and liver. In situ hybridization confirmed that the expression of the RNA was restricted to cells of the bovine subcommissural organ. Polyclonal antibodies raised against a synthetic peptide, whose amino-acid sequence was deduced from the 2.5-kb cDNA, reacted specifically with the bovine and rat subcommissural organ-Reissner's fiber complex. In immunoblots of bovine subcommissural organ, this antibody revealed the precursor 540-kDa form and its putative processed form of 450 kDa. It is concluded that the cloned cDNA encodes for the major constitutive glycoprotein of Reissner's fiber, here designated as RF-Gly I. The sequenced region of RF-Gly I displays a high degree of homology with some regions of the von Willebrand factor and certain mucins; it also displays two motifs homologous with repeats present in proteins of the spondin family and other proteins. A core sequence of the RF-Gly I repeats suggests that this molecule displays protein-binding properties.

Mitochondrial gene expression was studied using an electrotransformation protocol to introduce foreign DNA into purified wheat mitochondria. Optimal conditions for DNA uptake and transient gene expression were determined. We show here that a DNA plasmid containing either a cognate or a non-cognate gene under the control of a plant mitochondrial promoter is incorporated into the organelle and faithfully recognized by the transcription machinery. Transcripts generated by a plasmid bearing the intron-containing cox II gene were correctly spliced. Moreover, the transcripts were edited at the expected target C residues. The expression and maturation process of the transgene is dependent on the integrity of functional elements such as the promotor or the presence of structural domains necessary for splicing. The mitochondrial transformation described in this report is an important tool to study the multiple steps involved in plant mitochondrial gene expression at conditions closer to those found in vivo.