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Browsing by Author "Jordana, X"

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Editing status of mat-r transcripts in mitochondria from two plant species
(1998) Bégu, D; Mercado, A; Farré, JC; Moenne, A; Holuigue, L; Araya, A; Jordana, X
The intronic mat-r ORF encodes a protein with significant homology to retroviral reverse transcriptases. Here, we describe the nucleotide sequence of potato mat-r and study the editing status of mat-r transcripts in two systems, potato and wheat, where the mat-r ORF is part of the trans-introns but in two different configurations relative to nadl exons d and e. In potato and wheat, 13 and 15 C-to-U transitions respectively were observed. Most transcripts were partially edited, but potato transcripts were edited more efficiently than wheat transcripts. As in functional mitochondrial genes, RNA editing increased the similarity between plant mat-r proteins and their homologous non-plant counterparts. Interestingly, editing of mat-r was clustered in the reverse-transcriptase (RT) and the maturase (X) domains, two well defined regions having known functions in other systems. These results, together with the integrity and sequence conservation of mat-r, strongly suggest that the encoded protein plays a functional role in plant mitochondria.
Enhanced resistance to bacterial infection by Erwinia carotovora subsp atroseptica in transgenic potato plants expressing the attacin or the cecropin SB-37 genes
(1999) Arce, P; Moreno, M; Gutierrez, M; Gebauer, M; Dell'Orto, P; Torres, H; Acuña, I; Oliger, P; Venegas, A; Jordana, X; Kalazich, J; Holuigue, L
Blackleg and soft rot diseases, caused by the bacterium Erwinia carotovora, are among the diseases that cause important losses in culture and storage of potato. In this paper, we introduced bacterial resistance into potato, via genes encoding for proteins with antibacterial activity. For this purpose, potato clones were transformed either with the gene encoding the acidic attacin protein from Hyalophora cecropia, or with the gene encoding the cecropin analog peptide SB-37. These clones were evaluated for soft rot and blackleg resistance, after inoculation with the bacterial strain Erwinia carotovora subsp. atroseptica T7. Results reported in this paper indicate that a considerable percentage of the potato clones (15-22%) showed increased resistance to bacterial infection, revealed by reduced severity of blackleg or soft rot symptoms. Expression of the transgenes was demonstrated in some of the clones by Northern blot analysis. This is the first report indicating that. expression of the gene encoding for an attacin protein and for the cecropin SB-37 peptide in transgenic potato confers increased resistance to bacterial infection.
Expression of the chicken lysozyme gene in potato enhances resistance to infection by Erwinia carotovora subsp, Atroseptica
(2000) Serrano, C; Arce-Johnson, P; Torres, H; Gebauer, M; Gutierrez, M; Moreno, M; Jordana, X; Venegas, A; Kalazich, J; Holuigue, L
Infection of potato plants and tubers with the bacterium Erwinia carotovora subsp, atroseptica produces blackleg and soft rot diseases, which cause significant losses to crops and stored potatoes. In order to obtain resistance against this bacterium, the gene chly encoding the enzyme lysozyme from chicken was introduced into potato plants (cv. Desiree) via Agrobacterium-mediated transformation. Sixty-three and 69 transgenic potato clones were evaluated in the greenhouse for resistance to blackleg and soft rot diseases, respectively. Results reported in this paper indicate that 21%-29% of the potato clones showed increased resistance to infection by the bacterium E. c, subsp, atroseptica T7, as revealed by a reduced severity of blackleg or soft rot symptoms. Nine clones showing different levels of resistance were selected for further molecular analysis. The number of copies of the transgene integrated in the plant genome of these clones was estimated by Southern blot analysis. The level of transgene expression, detected by Northern blot analysis, correlated with the level of resistance detected in these clones.
Gene expression studies in isolated mitochondria
(2005) Choury, D; Farré, JC; Jordana, X; Araya, A
The complex gene expression mechanisms that occur in plant mitochondria, such as RNA editing and splicing, are not yet well understood. RNA editing in higher plant mitochondria is a highly specific process which modifies mRNA sequences by C-to-U conversions. It has been suggested that in some cases this process is required for splicing. Here, we use an experimental model based on the introduction of DNA into isolated mitochondria by electroporation to study organellar gene expression events. Our aim was to compare processing and editing of potato small ribosomal protein 10 gene (rps10) transcripts in heterologous (wheat mitochondria) and homologous (potato mitochondria) contexts. rps10 is a suitable model because it contains a group II intron, is absent in wheat mitochondria but is actively expressed in potato mitochondria, where transcripts are spliced and undergo five C-to-U editing events. For this purpose, conditions for electroporating isolated potato mitochondria were established. rps10 was placed under the control of either potato or wheat cox2 promoters. We found that rps10 was only transcribed under the control of a cognate promoter. In wheat mitochondria, rps10 transcripts were neither spliced nor edited while they are correctly processed in potato mitochondria. Interestingly, a wheat editing site grafted into rps10 was not recognized by wheat mitochondria but was correctly edited in potato mitochondria. Taken together, these results suggest that editing might occur only when the transcripts are engaged in processing and that they would not be available to editing factors outside of a putative RNA maturation machinery complex.
cis recognition elements in plant mitochondrion RNA editing
(2001) Farré, JC; Leon, G; Jordana, X; Araya, A
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.
Identification of NPR1-dependent and independent genes early induced by salicylic acid treatment in Arabidopsis
(2005) Blanco, F; Garretón, V; Frey, N; Dominguez, C; Pérez-Acle, T; Van Der Straeten, D; Jordana, X; Holuigue, L
Salicylic acid (SA) plays a crucial role in stress resistance in plants by modifying the expression of a battery of genes. In this paper, we report the identification of a group of early SA-regulated genes of Arabidopsis (activated between 0.5-2.5 h), using the cDNA-amplified fragment length polymorphism technique (cDNA-AFLP). Using 128 different primer combinations, we identified several genes based on their differential expression during SA treatment. Among these, we identified 12 genes up-regulated by SA whose patterns of induction were confirmed by Northern analysis. The identified genes can be grouped into two functional groups: Group 1: genes involved in cell protection (i.e. glycosyltransferases, glutathion S-transferases), and Group 2: genes involved in signal transduction (protein kinases and transcription factors). We also evaluated NPR1 requirement for the induction of the 12 up-regulated genes, and found that only those belonging to Group 2 require this co-activator for their expression. In silico analysis of the promoter sequences of the up-regulated genes, allowed us to identify putative cis-elements over-represented in these genes. Interestingly, as-1-like elements, previously characterized as SA-responsive elements, were specifically over-represented in Group 1 genes. The identification of early SA-regulated genes is an important step towards understanding the complex role of this hormone in plant stress resistance.
Identification of the RPS10 polypeptide encoded by the mitochondrial genome in Solanum tuberosum
(2000) Zanlungo, S; Quiñones, V; Holuigue, L; Jordana, X
We have previously reported the identification of a gene encoding ribosomal protein S10 (rps10) in the Solanum tuberosum mitochondrial genome and shown that its transcripts undergo processing in the form of splicing and editing. Specific antibodies were raised against recombinant potato RPS10 obtained in Escherichia call from the corresponding cDNA. Protein blot analysis revealed that the RPS10 polypeptide is present in potato mitochondria and is assembled into the mitochondrial ribosome. Potato rps10 mRNA translation may initiate at a genomic-encoded AUG codon, or at a downstream in-frame AUG codon which is created by RNA editing and coincides with liverwort and pea initiation codons. A peptide corresponding to the 19 amino acid sequence between both putative initiation codons was used to raise antibodies specific for the potato RPS10 N-terminal extension. These antibodies were able to recognise a polypeptide of 16 kDa in a potato mitochondrial fraction enriched in ribosomes. This molecular mass corresponds to that of the polypeptide detected with antibodies against recombinant RPS10 and is in agreement with the molecular weight calculated from the cDNA sequence. These results shelved that potato mitochondrial rps10 gene is expressed at the protein level and that translation initiates at the first genomic-encoded AUG codon. Potato RPS10 has an N-terminal extension of unknown function when compared to RPS10 from other organisms.
NPR1-independent activation of immediate early salicylic acid-responsive genes in Arabidopsis
(2004) Uquillas, C; Letelier, I; Blanco, F; Jordana, X; Holuigue, L
Salicylic acid (SA) is a key signal for the activation of defense genes in response to stress. The activation of late defense genes by SA, such as PR-1, involves the participation of the NPR1 protein. This protein acts as coactivator of the TGA factors that recognize as-1-like elements in the PR-1 promoter. Considering that functional as-1-like elements are also found in the promoter of SA- and auxin-responsive immediate early genes, we tested the hypothesis that NPR1 is also required for activation of these genes. The expression of the immediate early genes glutathione Stransferase (GST6) and glucosyltransferase (EIGT) was studied in npr1 mutant and wild-type Arabidopsis plants. In the npr1 mutant background, SA and 2,4-dichlorophenoxyacetic acid were unable to promote transcription of PR-1 but effectively stimulated the expression of GST6 and EIGT. Furthermore, increased binding of proteins to the GST6 as-1-like promoter element was detected in nuclear extracts from npr1 and wild-type plants after treatment with SA. In summary, these results indicate that activation of immediate early genes by SA proceeds through an NPRI-independent pathway. Therefore, we propose that activation by SA of immediate early and late genes occur by different mechanisms.
Nuclear SDH2-1 and SDH2-2 genes, encoding the iron-sulfur subunit of mitochondrial complex II in arabidopsis, have distinct cell-specific expression patterns and promoter activities
(2004) Elorza, A; León, G; Gómez, I; Mouras, A; Holuigue, L; Araya, A; Jordana, X
Three different nuclear genes encode the essential iron-sulfur subunit of mitochondrial complex 11 (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 beta-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 beta-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.
Phosphorylation of nuclear proteins directs binding to salicylic acid-responsive elements
(1997) Stange, C; Ramirez, I; Gomez, I; Jordana, X; Holuigue, L
The cis-located DNA sequence as-1 (Activation Sequence-1) from CaMV 35S promoter has been previously identified as an element that can confer inducibility by salicylic acid (SA) with immediate early kinetics. This sequence specifically binds to ASF-1 (Activation Sequence Factor-1), previously characterized in tobacco nuclear extracts. To assess whether modulation of ASF-1 binding activity can explain the activation of the as-1 sequence observed in vivo, we performed electrophoretic mobility shift assays using nuclear extracts from SA-treated and water-treated tobacco plants. Our results indicate that treatment of plants with SA increases ASF-1 binding to as-1 and to ocs, an as-1-like element from the Agrobacterium octopine synthase gene. In contrast, SA treatment has no effect on the binding of GT-1 factor to its target light-inducible box II element. Furthermore, treatment of nuclear extracts from SA-treated plants with alkaline phosphatase decreases ASF-1 binding to the as-1 element. This can be reversed by pretreatment with 10 mM NaF. Accordingly, pretreatment of nuclear extracts from control water-treated plants with ATP produces an increase in ASF-1 binding activity similar to that observed with SA. This effect of ATP is reversed by treatment with alkaline phosphatase and prevented by quercetin, a casein kinase II inhibitor. These results support the hypothesis that a nuclear protein kinase is involved in the immediate early events of transcriptional activation triggered by SA.
The as-1 promoter element is an oxidative stress-responsive element and salicylic acid activates it via oxidative species
(2002) Garretón, V; Carpinelli, J; Jordana, X; Holuigue, L
The activation sequence-1 (as-1)-like element found in the promoter of some glutathione S-transferase (GST) genes, has been previously described as a salicylic acid (SA)- and auxin-responsive element. In this paper, we tested the hypothesis that the activating effect of SA on the as-1 element is mediated by oxidative species. Supporting this hypothesis, our results show that the antioxidants dimethylthiourea (DMTU) and 3-t-butyl-4-hydroxy-anizole (BHA) inhibit the SA-induced transcription of genes controlled by as-1 elements in tobacco (Nicotiana tabacum) plants [i.e. GNT35 gene coding for a GST and (as-1)(4)/beta-glucuronidase (GUS) reporter transgene]. DMTU and BHA also inhibit SA-activated as-1-binding activity in nuclear extracts. Further support for the hypothesis that the as-1 element is activated by oxidative species comes from our result showing that light potentiates the SA-induced activation of the as-1 element. Furthermore, methyl viologen, a known oxidative stress inducer in plants, also activates the as-1 element. Increasing H2O2 levels by incubation with H2O2 or with the catalase inhibitor 3-amino-1,2,5-triazole does not activate the (as-1)(4)/GUS gene. On the contrary, 3-amino-1,2,5-triazole inhibits the activating effect of SA on the (as-1)(4)/GUS gene. These results suggest that oxidative species other than H2O2 mediate the activation of the as-1 element by SA. Our results also suggest that even though the as-1 binding activity is stimulated by oxidative species, this is not sufficient for the transactivation of genes controlled by this element. The complex interplay between SA and reactive oxygen species in the transcriptional activation of defense genes is discussed.
The four subunits of mitochondrial respiratory complex II are encoded by multiple nuclear genes and targeted to mitochondria in Arabidopsis thaliana
(2002) Figueroa, P; Léon, G; Elorza, A; Holuigue, L; Araya, A; Jordana, X
Mitochondrial respiratory complex II contains four subunits: a flavoprotein (SDH1), an iron-sulphur subunit (SDH2) and two membrane anchor subunits (SDH3 and SDH4). We have found that in Arabidopsis thaliana SDH1 and SDH3 are encoded by two, and SDH4 by one nuclear genes, respectively. All these encoded polypeptides are found to be imported into isolated plant mitochondria. While both SDH1 proteins are highly conserved when compared to their counterparts in other organisms, SDH3 and SDH4 share little similarity with non-plant homologues. Expression of SDH1-1, SDH3 and SDH4 genes was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the second SDH1 gene (SDH1-2) is expressed at a low level.
The rpl5-rps14-cob gene arrangement in Solanum tuberosum: rps14 is a transcribed and unedited pseudogene
(1996) Quinones, V; Zanlungo, S; Moenne, A; Gomez, I; Holuigue, L; Litvak, S; Jordana, X
The L5 ribosomal protein gene (rpl5) and a S14 ribosomal protein pseudogene were identified by sequence analysis in the potato mitochondrial genome. The two genes are separated by one nucleotide and are found upstream of the apocytochrome b gene (cob), an arrangement conserved also in Arabidopsis and Brassica. The rpl5 gene has an intact open reading frame while the rps14 locus is disrupted by a five nucleotide duplication that introduces a frameshift in the reading frame. Editing of rpl5 and pseudorps14 cotranscripts has been studied by cDNA sequence analysis. Eight C residues are edited into U in the rpl5 coding region, resulting in eight amino acid changes that increase the homology between potato and other RPL5 polypeptides. Interestingly, the rps14 pseudogene sequence is not edited at any nucleotide position.
Three different genes encode the iron-sulfur subunit of succinate dehydrogenase in Arabidopsis thaliana
(2001) Figueroa, P; León, G; Elorza, A; Holuigue, L; Jordana, X
The iron-sulfur protein is an essential component of mitochondrial complex II (succinate dehydrogenase, SDH), which is a functional enzyme of both the citric acid cycle and the respiratory electron transport chain. This protein is encoded by a single-copy nuclear gene in mammals and fungi and by a mitochondrial gene in Rhodophyta and the protist Reclinomonas americana. In Arabidopsis thaliana, the homologous protein is now found to be encoded by three nuclear genes. Two genes (sdh2-1 and sdh2-2) likely arose from a relatively recent duplication event since they have similar structures, encode nearly identical proteins and show similar expression patterns. Both genes are interrupted by a single intron located at a conserved position. Expression was detected in all tissues analysed, with the highest steady-state mRNA levels found in flowers and inflorescences. In contrast, the third gene (sdh2-3) is interrupted by 4 introns, is expressed at a low level, and encodes a SDH2-3 protein which is only 67% similar to SDH2-1 and SDH2-2 and has a different N-terminal presequence. Interestingly, the proteins encoded by these three genes are probably functional because they are highly conserved compared with their homologues in other organisms. These proteins contain the cysteine motifs involved in binding the three iron-sulfur clusters essential for electron transport. Furthermore, the three polypeptides are found to be imported into isolated plant mitochondria.
Transfer of RPS14 and RPL5 from the mitochondrion to the nucleus in grasses
(2004) Sandoval, P; León, G; Gómez, I; Carmona, R; Figueroa, P; Holuigue, L; Araya, A; Jordana, X
Gene transfer from the mitochondrion to the nucleus, a process of outstanding importance to the evolution of the eukaryotic cell, is an ongoing phenomenon in higher plants. After transfer, the mitochondrial gene has to be adapted to the nuclear context by acquiring a new promoter and targeting information to direct the protein back to the organelle. To better understand the strategies developed by higher plants to transfer organellar genes during evolution, we investigated the fate of the mitochondrial PPL5-RPS14 locus in grasses. While maize mitochondrial genome does not contain RPS14 and RPL5 genes, wheat mitochondrial DNA contains an intact RPL5 gene and a nonfunctional RPS14 pseudogene. RPL5 and psiRPS14 are co-transcribed and their transcripts are edited. In wheat, the functional RPS14 gene is located in the nucleus, within the intron of the respiratory complex II iron-sulfur subunit gene (SDH2). Its organization and expression mechanisms are similar to those previously described in maize and rice, allowing us to conclude that RPS14 transfer and nuclear activation occurred before divergence of these grasses. Unexpectedly, we found evidence for a more recent RPL5 transfer to the nucleus in wheat. This nuclear wheat RPL5 acquired its targeting information by duplication of an existing targeting presequence for another mitochondrial protein, ribosomal protein L4. Thus, mitochondrial and nuclear functional RPL5 genes appear to be maintained in wheat, supporting the hypothesis that in an intermediate stage of the transfer process, both nuclear and mitochondrial functional genes coexist. Finally, we show that RPL5 has been independently transferred to the nucleus in the maize lineage and has acquired regulatory elements for its expression and a mitochondrial targeting peptide from an unknown source. (C) 2003 Elsevier B.V. All rights reserved.
Transfer of rps14 from the mitochondrion to the nucleus in maize implied integration within a gene encoding the iran-sulphur subunit of succinate dehydrogenase and expression by alternative splicing
(1999) Figueroa, P; Gómez, I; Holuigue, L; Araya, A; Jordana, X
The maize mitochondrial genome does not contain a gene coding for ribosomal protein S14. In this paper we show that the functional rps14 gene was translocated to the nucleus and acquired the signals conferring expression and product targeting to the mitochondrion in a way not previously described. Transferred rps14 was found integrated between both exons of a gene encoding the iron-sulphur subunit of the respiratory complex II (sdh2). Sdh2 exon 1 and rps14 were separated by a typical plant nuclear intron that was spliced to give a mature poly(A)+ mRNA of 1.4 kb. This processed mRNA encoded a chimeric SDH2 (truncated)-RPS14 polypeptide, and we show that this chimeric polypeptide is targeted into isolated plant mitochondria, where it is proteolytically processed in a complex way. An alternative splicing event utilizing the same 5' splice site and a different downstream 3' splice site generated a second mature poly(A)+ mRNA of 1.3 kb that contained both sdh2 exons. This sdh2 transcript encoded an SDH2 polypeptide highly conserved compared with its homologues in other organisms, and it contained the three cysteine-rich clusters that made up the three nonheme iron-sulphur centres responsible for electron transport. To our knowledge, these results constitute the first evidence of alternative splicing playing a role in the expression and targeting of two mitochondrial proteins with different functions from the same gene.
Transfer of the mitochondrial rps10 gene to the nucleus in rice
(2000) Kubo, N; Jordana, X; Ozawa, K; Zanlungo, S; Harada, K; Sasaki, T; Kadowaki, K
Mitochondrial ribosomal protein S10 (rps10) is encoded by the mitochondrial genome in potato and pea. Here we show that the rps10 gene is absent from the mitochondrial genome of rice and has been transferred to the nucleus. Cloning and transcriptional analysis show that there are two rps10 genes in the rice nuclear genome and that their transcripts differ in abundance. Western analysis detected the RPS10 protein in the soluble fraction of rice mitochondrial although neither RPS10 has any obvious N-terminal presequence for targeting to mitochondria. This result suggests that targeting information is present in the internal region of rice RPS10. Genomic sequence analysis indicated that each rps10 gene has an intron in the 5' untranslated region (5' UTR) and that these intron sequences are homologous to each other. This result strongly suggests that a duplication event occurred after transfer of the rps10 gene to the nucleus. The duplicated rps10 genes have since been translocated to different chromosomes, because the two rps10 genes were mapped on chromosomes 6 and 12 by RFLP analysis. Interestingly, the 5' UTR and the intron of the rice rps10 genes are homologous to sequences found in several rice genes with various functions, such as osk4, EF-1 beta 2 and RAG1, suggesting a common origin and a functional role for the 5' UTR. Acquisition of the 5' flanking region-might have accelerated the activation of the mitochondrial rps10 gene which was transferred to the nuclear genome.

Browsing by Author "Jordana, X"

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