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RESEARCH PAPER |
Phylogenetic and functional analysis of Arabidopsis RCI2 genes
1Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraría y Alimentaria (INIA), Carretera de la Coruña, Km. 7, 28040 Madrid, Spain
2Departamento de Biología de Plantas, Centro de Investigaciones Biológicas (CIB-CSIC), Ramiro de Maeztu 9, 28040 Madrid, Spain
* To whom correspondence should be addressed. E-mail: salinas{at}cib.csic.es
Received 1 August 2007; Revised 17 October 2007 Accepted 22 October 2007
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Abstract |
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Six new Arabidopsis thaliana genes (AtRCI2C-H) have been identified that show high homology to AtRCI2A and AtRCI2B. Sequence comparisons revealed that AtRCI2-related genes are widely spread among very different organisms, including other plant species, prokaryotes, fungi, and simply organized animals, and are also organized in gene families. Most RCI2 genes show a similar exon–intron organization, which indicates that they have been structurally conserved during evolution, and encode small, highly hydrophobic proteins containing two putative transmembrane domains. Consistently, the majority of AtRCI2 proteins localize in the plasma membrane. RCI2 proteins exhibit an elevated level of sequence similarity and seem to have evolved from a common ancestor. In spite of their high similarity, conserved subcellular localization, and common origin, experimental evidence is presented suggesting that different RCI2 proteins may have distinct functional roles. Thus, as previously demonstrated for AtRCI2A and AtRCI2B, the newly identified AtRCI2 genes (AtRCI2C–H) are differentially regulated in Arabidopsis organs and in response to abiotic stresses and ABA treatment. Furthermore, only the AtRCI2 proteins that do not contain the C-terminal hydrophilic tail (i.e. AtRCI2A–C and AtRCI2H) are able to complement for the loss of the yeast AtRCI2-related gene PMP3. On the basis of these results, different aspects on the evolution and roles of RCI2 genes are discussed.
Key words: ABA, abiotic stress responses, Arabidopsis, cold acclimation, dehydration, gene family, RCI2 genes
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Introduction |
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During recent years, a large effort has been made to understand the molecular mechanisms that plants have evolved to survive adverse environmental conditions. Arabidopsis thaliana transcriptome analyses have revealed that hundred of genes are regulated by low temperature, dehydration or high salt, and that different signalling pathways are involved in this regulation (Fowler and Thomashow, 2002; Jiang and Deyholos, 2006; Oono et al., 2006). Some of these genes are also regulated by developmental cues, which is not unexpected considering that environmental stresses have pleiotropic effects on developmental processes (Salinas, 2002; Shinozaki and Yamaguchi-Shinozaki, 2007). Interestingly, several genes are not induced in response to low temperature, dehydration or high salt in all plant organs (Medina et al., 2001; Chen et al., 2002; Fukuda et al., 2004), suggesting that plant responses to abiotic stresses are differentially modulated in different tissues.
Genes that respond to abiotic stresses often belong to gene families (Yokoi et al., 2002; Tran et al., 2004; Shigaki et al., 2006). Why these genes have evolved as gene families, and whether they have redundant or complementary functions are important questions that, in most cases, still remain to be answered. Gene families arise from duplication of ancestral genes and subsequent sequence divergence, which can lead to functional specialization allowing plants to adapt better to unfavourable environments (Paterson et al., 2006). The sequence of the Arabidopsis genome revealed that 60% is segmentally duplicated, significantly more than any other sequenced eukaryotic genome (Blanc et al., 2000; Simillion et al., 2002), and 17% organized in tandemly repeated segments (Bancroft, 2001). The analysis of the deduced genes unveiled the importance of gene families, in terms of both number and size, when considering the organization and evolution of the Arabidopsis genome (Arabidopsis Genome Initiative, 2000).
In an attempt to identify new genes from Arabidopsis involved in the low-temperature response, RCI2A and RCI2B (hereafter referred as AtRCI2A and AtRCI2B, respectively), two homologous genes that are tandemly organized were isolated (Capel et al., 1997). The characterization of AtRCI2A and AtRCI2B expression uncovered that they are induced not only by low temperature but also by ABA, dehydration, and salt treatments. Analysis of Arabidopsis transgenic plants harbouring AtRCI2 promoters fused to the uidA (GUS) reporter gene revealed that the expression of AtRCI2A and AtRCI2B is differentially regulated at the tissue level during plant development and in response to abiotic stresses (Medina et al., 2001). Both genes encode small, highly hydrophobic proteins with two putative transmembrane domains (Capel et al., 1997). The absence of any signal for organelle targeting allowed it to be proposed that AtRCI2A and AtRCI2B proteins might localize to the plasma membrane (Capel et al., 1997), which is considered a primary site of injury by freezing (Lyons, 1973). A possible role for AtRCI2 proteins in maintaining membrane function and/or integrity when plants have to adapt to any environmental condition that reduces water availability was suggested. Alternatively, it was proposed that they could interact with other membrane proteins to control the hydric balance of the cells (Capel et al., 1997). A yeast plasma membrane protein, PMP3, was identified that showed high sequence similarity to AtRCI2A and AtRCI2B (Navarre and Goffeau, 2000). Deletion of the PMP3 gene results in an increase in Na+ and K+ uptake due to a hyperpolarization of the plasma membrane, which suggests a role for PMP3 in regulating plasma membrane potential. Interestingly, AtRCI2A can substitute for the loss of PMP3 (Navarre and Goffeau, 2000; Nylander et al., 2001), indicating that both proteins have a similar function. However, little is known about the precise role of AtRCI2A and AtRCI2B in stress response. Recently, the isolation of Arabidopsis transgenic plants containing disrupted or overexpressed AtRCI2A have been described (Mitsuya et al., 2005, 2006). The characterization of these plants suggested that AtRCI2A may be involved in the salt response by avoiding over-accumulation of Na+ and K+ ions (Mitsuya et al., 2005, 2006).
In this paper, the identification and characterization of six new Arabidopsis genes (AtRCI2C-H) that show high homology to AtRCI2A and AtRCI2B are reported. In addition, it is shown that AtRCI2-related genes are widespread among very different organisms, including other plant species, prokaryotes, fungi, and animals, where they are also organized as gene families. The analysis of the AtRCI2-related genes from rice and Caenorhabditis elegans, whose genomes have been completely sequenced, and their comparison with the corresponding genes from Arabidopsis provided valuable information on the structure and organization of RCI2 genes. Remarkably, these genes and the corresponding predicted proteins are highly conserved at both sequence and structural levels, and seem to have a common evolutionary origin. Subcellular localization of AtRCI2 proteins indicates that, excluding AtRCI2D, all of them localize to the plasma membrane. Expression and functional analysis suggest that, in spite of their similarity, conserved subcellular localization, and common origin, different AtRCI2 proteins may have distinct roles in response to abiotic stresses. On the basis of these results, some aspects on the evolution and functions of RCI2 genes are discussed.
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Materials and methods |
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Screening of databases, sequence analyses, and chromosome localizations
Novel Arabidopsis RCI2 genes (AtRCI2C-H) were identified by comparing AtRCI2A and AtRCI2B proteins (Capel et al., 1997) with entries in GenBank (http://www.ncbi.nlm.nih.gov) and Arabidopsis MIPS (http://mips.gsf.de/proj/thal/db/index.html) databases, using BLASTP and TBLASTN algorithms (Altschul et al., 1997). Subsequent pair-wise DNA sequence comparisons were applied to identify and exclude double annotations. The AtRCI2 proteins and the same algorithms were used to uncover AtRCI2-related genes in rice (Oryza sativa) and C. elegans. Rice AtRCI2-related genes (OsRCI2-1–12) were searched in the GenBank at the NCBI (http://www.ncbi.nlm.nih.gov/) and TIGR (http://www.tigr.org/tdb/e2k1/osa1/) databases derived from O. sativa ssp japonica, and in GenBank at the NCBI database derived from O. sativa ssp indica. In the case of C. elegans, the complete set of AtRCI2-related genes (CeRCI2-1–13) was obtained by searching in the WormBase (http://www.wormbase.org; Stein et al., 2001). The search for AtRCI2-related genes in other organisms was carried out in the different EST collections available by April 2007 in GenBank, using the TBLASTN algorithm and all AtRCI2 proteins as queries. Redundant sequences were identified by comparing BLAST results, and only the longest ones were considered for further analyses. The accession numbers of the selected ESTs are listed in Supplementary Table 1 at JXB online.
The exon–intron boundaries of the RCI2 genes were determined with the GenScan program (http://genes.mit.edu/GENSCAN.html; Burge and Karlin, 1997), and verified, when possible, with available EST and full-length cDNA sequences. The chromosome localization of the AtRCI2 genes was established with the Map Viewer Tool from The Arabidopsis Information Resource (http://www.arabidopsis.org/servlets/mapper). The chromosome localization of the OsRCI2 and CeRCI2 genes was determined according to the rice TIGR database and WormBase, respectively. Duplications of AtRCI2 genes and the presence of AtRCI2 genes on duplicated segments were investigated by using the MIPS redundancy viewer (http://mips.gsf.de/proj/thal/db/gv/rv/) and the Simillion database (http://www.psb.rug.ac.be/bioinformatics/simillion_pnas02; Simillion et al., 2002).
Protein alignments and similarity values were generated with the Clustal X program (Thompson et al., 1997). Specific targeting sequences were predicted with the PSORT programs (http://psort.nibb.ac.jp/; Nakai and Kanehisa, 1992). Hydropathy analyses were performed using the ProtScale program (http://ca.expasy.org/cgi-bin/protscale.pl) with the Kyte and Doolittle option and a window of nine amino acids (Kyte and Doolittle, 1982). The superposition of RCI2 hydropathy plots was generated after transferring the raw data with the Microsoft Excel application (Microsoft, Redmond, WA) and aligning the sequences to match the two proline residues conserved in the first transmembrane domains. The prediction of transmembrane domains was realized with the TMHMM 2.0 program (http://www.cbs.dtu.dk; Krogh et al., 2001). The compilation of RCI2 transmembrane domains was generated, as in the case of the hydropathy plots described above. The phylogenetic tree was constructed using the Neighbor–Joining method (Saitou and Nei, 1987), and the bootstrap test carried out with 1000 iterations.
Plant material, growth conditions, and treatments
Seeds from A. thaliana plants, ecotype Columbia (Col), were obtained from Lehle Seeds (Round Rock, TX, USA). Plant growth conditions and treatments were as described (Medina et al., 2001). The onion (Allium cepa) bulbs used for particle bombardment experiments were purchased from local markets.
Molecular biology methods
Total RNA was extracted from different Arabidopsis organs and seeds according to Logeman et al. (1987) and Lara et al. (2003), respectively. DNA digestions and cloning were performed following standard protocols (Sambrook et al., 1989). RNA-blot hybridizations were performed as previously described by Medina et al. (2001). The specific probes for AtRCI2A and AtRCI2B were those described by Capel et al. (1997). DNA fragments partially encompassing the corresponding 3'-UTRs were used as specific probes for the rest of AtRCI2 genes. These fragments were generated by PCR with primers shown in Supplementary Table 2 at JXB online. RNA samples from each experiment were analysed in at least two independent blots, and each experiment was repeated at least twice.
Expression analysis in yeast was performed by RT-PCR using 2 µg of total RNA from WT303 and transformed YR93-31 yeast strains and M-MuLV Reverse transcriptase (USB, Cleveland, USA). RNAs were isolated according to Schmitt et al. (1990). AtRCI2 transcripts were detected using gene-specific primers indicated in Supplementary Table 3 at JXB online. The yeast ACT1 transcript was used as the endogenous control and detected using specific primers 5'-GATCTTGAGTGAACGTGGT-3’ and 5'-TTCTGGGGCTCTGAATCTT-3'. PCR products were separated on 1.5% (w/v) agarose gel containing ethidium bromide and visualized on UV light.
Isolation of AtRCI2 cDNA clones and transformation of yeast strains
Full-length cDNAs corresponding to AtRCI2A–F genes were obtained by RT-PCR using 2 µg of total RNA extracted from 4-week-old plants exposed to low temperature. The full-length AtRCI2H cDNA was synthesized by PCR using the EST no. AV824849 (Asamizu et al., 2000) as a template. The specific primers employed to obtain the cDNAs are shown in Supplementary Table 3 at JXB online. The truncated AtRCI2D-
1 cDNA, which contains a stop codon that eliminates the last 18 amino acids from the AtRCI2D protein, was generated by PCR, using the full-length AtRCI2D cDNA as a template and the oligonucleotides AtRCI2D (F) and AtRCI2D-1 (see Supplementary Table 3 at JXB online) as specific primers. All primers contained BamHI restriction sites that were used for directly cloning into the pRS416 plasmid (Navarre and Goffeau, 2000) between the promoter and terminator of PMP3. Constructs were sequenced and used to transform Saccharomyces cerevisae mutant strain YR93-31 that has the PMP3 gene deleted (Navarre and Goffeau, 2000). Transformants were selected on 0.67% (w/v) yeast nitrogen base medium, containing 2% (w/v) agar, 4% (w/v) glucose and all micronutrients except tryptophan and uracil.
Yeast sensitivity assays to NaCl
Transformed YR93-31 strains as well as 303 wild-type cells were grown in YPD medium to saturation, and their OD at 600 nm determined. Cultures were normalized to the same value (OD600=0.5), and serial 10-fold dilutions plated onto agar–YPD plates supplemented with different NaCl concentrations (0, 0.25, 0.5, and 1.0 M). Yeasts were grown for 3 d at 30 °C, and the highest NaCl concentration where they were able to grow was determined.
Subcellular localization of GFP-AtRCI2 fusion proteins
To construct translational fusions between cDNAs corresponding to GFP and AtRCI2 genes, AtRCI2 cDNAs were amplified by PCR with the oligonucleotides shown in Supplementary Table 3 at JXB online. cDNAs were subsequently cloned into the BglII site of the expression vector pAVA393 (Von Arnin et al., 1998) to generate GFP-AtRCI2 fusions under the control of the CaMV 35S promoter. The GFP-AtRCI2 fusions were transiently expressed in onion epidermal cells by particle bombardment, or in Arabidopsis protoplasts by PEG-mediated transformation, essentially as described by Yokoi et al. (2002) and Milla et al. (2006), respectively.
Fluorescence was observed by confocal microscopy (Leica TCSSP2, Leica Microsystem, Wetzlar, Germany). For imaging GFP fusions alone or in combination with YFP fusions, the excitation lines were 488 nm for GFP and 515 nm for YFP. Fluorescence was detected in a 500–538 nm band for GFP, and a 560–615 nm band for YFP. Filter sets were used to visualize and to discriminate between fluorophores.
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AtRCI2A and AtRCI2B belong to a gene family
To investigate the presence of AtRCI2A and AtRCI2B homologues in the Arabidopsis genome, BLAST searches (Altschul et al., 1997) were conducted with the predicted AtRCI2A and AtRCI2B proteins (Capel et al., 1997). Six putative genes, AtRCI2C–H, were identified that would encode proteins showing 53–94% sequence similarity to AtRCI2A and AtRCI2B, and together with AtRCI2A and AtRCI2B constitute the AtRCI2 gene family (Table 1; Fig. 1). The sequence of AtRCI2G, which was not recognized as a putative gene during the Arabidopsis automated annotation process, has been submitted to GenBank and registered with the accession number AT4G28088 (Table 1). The chromosome localization of the eight AtRCI2 genes is indicated in Table 1 and displayed in Fig. 2A. In summary, AtRCI2E, AtRCI2F, and AtRCI2G localize to chromosome 4, AtRCI2E and AtRCI2F being arranged as a tandem. In a similar way, AtRCI2A and AtRCI2B are organized as a tandem in chromosome 3, while AtRCI2D and AtRCI2H localize to chromosome 2 and AtRCI2C in chromosome 1. Chromosome 5 does not contain any AtRCI2 gene. Comparison among genomic and deduced coding sequences revealed that, as in the case of AtRCI2A and AtRCI2B (Medina et al., 2001), AtRCI2C–H genes harbour two exons separated by an intron of variable size (82–275 bp). Nevertheless, they do not seem to have the additional intron that AtRCI2A and AtRCI2B contain into their 3'-UTRs (Medina et al., 2001) (Fig. 2B).
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Table 1 shows some features of deduced AtRCI2 proteins, namely number of amino acids, molecular weights, and isoelectric points. According to their lengths, AtRCI2 proteins can be divided into two different groups. The first one includes AtRCI2A, AtRCI2B, AtRCI2H (54 amino acids each) and AtRCI2C (52 amino acids) (Table 1; Fig. 1). The second group comprises AtRCI2D, AtRCI2E, AtRCI2F, and AtRCI2G (75, 73, 74 and 77 amino acids, respectively), all of them having an extra C-terminal tail of about 20 amino acids rich in charged residues (Table 1; Fig. 1). Hydropathy analyses (Kyte and Doolittle, 1982) indicated that all AtRCI2 proteins are highly hydrophobic with two clear hydrophobic stretches and a region of lower hydrophobicity between them (Fig. 3A). Proteins belonging to the second group (AtRCI2D–G) present an additional hydrophilic tract corresponding to their C-terminal tails (Fig. 3A). The membrane topology prediction program TMHMM (Krogh et al., 2001) anticipates that AtRCI2 proteins contain two putative transmembrane domains corresponding to the hydrophobic stretches mentioned above (Fig. 3B). The sequence connecting the transmembrane domains would form a putative turn (Fig. 3B). These results are consistent with those obtained from the PSORT program (Nakai and Kanehisa, 1992), which unveiled that deduced AtRCI2 sequences do not contain any obvious consensus organelle targeting or retention signals (data not shown), suggesting, therefore, plasma membrane localization.
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AtRCI2-related genes are widespread in plants
To explore the occurrence of AtRCI2-related genes in plant species other than Arabidopsis, the EST sequences available in plant databases were analysed. A BLAST search using all the deduced AtRCI2 proteins as queries identified more than 4200 ESTs coding for putative proteins with significant sequence similarity (>35%) to them. Redundant sequences were identified, and only the longest one in each case was considered for further analyses. The accession numbers of the selected ESTs (1373) are displayed in Supplementary Table 1 at JXB online. These sequences belong to more than 150 different species, including eudicots, monocots, gymnosperms, and bryophytes. The comparison of representative examples of deduced AtRCI2-related proteins from these phyla with AtRCI2 proteins revealed a high level of sequence similarity (49–92%) (Fig. 1). The hydropathy profiles of the representative AtRCI2-related proteins displayed in Fig. 1 were very similar to those of AtRCI2 proteins (see Supplementary Fig. 1A at JXB online), which suggests they are also located in the plasma membrane. In fact, the prediction program TMHMM showed that they also contain two putative transmembrane domains corresponding to hydrophobic regions, even if the first domain is not perfectly conserved in some cases (see Supplementary Fig. 1B at JXB online).
A detailed analysis of the data obtained from the BLAST search indicated that, as in Arabidopsis, AtRCI2-related genes may be organized in gene families as well. For instance, ESTs corresponding to at least seven different AtRCI2-related genes were identified from wheat (Triticum aestivum), five from maize (Zea mays), and four from soybean (Glycine max). The genome of rice (Oryza sativa), which has been sequenced (Yu et al., 2002; Goff et al., 2002), allowed the study of the structure and organization of RCI2 genes in a plant species other than Arabidopsis. The available genome sequence for the japonica and indica subspecies was analysed separately. A total of 11 genes were identified in japonica and another 11 in indica, allowing the compilation of 12 non-redundant AtRCI2-related genes in rice (OsRCI2-1-12) (Table 2). Comparison among genomic and coding sequences revealed that the structure of OsRCI2 genes is similar as that of AtRCI2 genes (Fig. 2). OsRCI2 genes are distributed in at least six out of the 12 rice chromosomes, OsRCI2-3 and OsRCI2-4 being arranged as a tandem in the indica genome (Table 2).
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Predicted OsRCI2 proteins exhibit 41–92% of sequence similarity to AtRCI2s and can also be classified in two groups depending on their predicted sizes (Table 2; see Supplementary Fig. 2A at JXB online). Moreover, OsRCI2 proteins are also anticipated to have two transmembrane domains that would correspond to their hydrophobic stretches (see Supplementary Fig. 2B at JXB online). Other characteristics of OsRCI2s, including their molecular weights and isoelectric points, are similar to those of AtRCI2 proteins too (Table 2).
AtRCI2-related genes are present in organisms other than plants
To extend the study on AtRCI2-related genes further, the available EST databases were searched for similar sequences in other organisms than plants using all the AtRCI2 proteins as queries. Results revealed that AtRCI2-related genes are present in different species, including metazoans (nematodes and protostomes), fungi (ascomycota and basidiomycota), and prokaryotes (cyanobacteria, proteobacteria, planctomycetes, and firmicutes). Interestingly, AtRCI2-related sequences were not found in animals having more complex organization such as arthropods and vertebrates. The accession numbers of the longest non-redundant ESTs identified (340) are listed in Supplementary Table 1 at JXB online. Figure 1 displays the comparison among some representative examples of deduced AtRCI2-related proteins from nematodes, protostomes, fungi, and prokaryotes, and RCI2 proteins from Arabidopsis and other plant species. Sequence similarity ranges between 41% to 78%, indicating that RCI2 proteins are conserved during evolution. The hydropathy profiles of these representative AtRCI2-related proteins resemble those of AtRCI2 proteins (see Supplementary Fig. 1A at JXB online), and also suggest a plasma membrane localization. Furthermore, the TMHMM program predicts two putative transmembrane domains corresponding to their hydrophobic regions (see Supplementary Fig. 1B at JXB online).
The analysis of non-redundant identified ESTs (see Supplementary Table 1 at JXB online) indicated that AtRCI2-related genes from organisms other than plants can also be organized into gene families. Additional information on the structure and genome organization of these genes was obtained from the sequenced genome of Caenorhabditis elegans (C. elegans sequencing consortium, 1998). BLAST searches performed in the WormBase (Stein et al., 2001) identified 13 AtRCI2-related genes (CeRCI2-1–13) (Table 3). The predicted exon–intron organization of these genes resembles that of Arabidopsis and rice RCI2 genes, except CeRCI2-5 and CeRCI2-12 that contain two introns into their coding regions (Fig. 2). CeRCI2 genes are distributed on six chromosomes of C. elegans, CeRCI2-7-8 and CeRCI2-11-13 genes being organized in tandem on chromosomes 5 and X, respectively (Table 3).
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The deduced CeRCI2 proteins can be arranged, as their Arabidopsis and rice RCI2 counterparts, into two groups with different predicted lengths (Table 3; see Supplementary Fig. 3A at JXB online). In addition, they are also anticipated to have two putative transmembrane domains, although the first domain in CeRCI2-6 and CeRCI2-11-13 is not fully conserved (see Supplementary Fig. 3B at JXB online). CeRCI2 proteins have similar molecular weights and isoelectric points as those of RCI2 proteins from Arabidopsis and rice (Table 3).
Most AtRCI2 proteins localize to the plasma membrane
Results described above indicate that RCI2 genes encode small, highly hydrophobic proteins, containing two conserved putative transmembrane domains. Furthermore, in some cases, RCI2 proteins hold additional hydrophilic tails with charged residues. Consistent with these data, our in silico analyses suggest that most RCI2 proteins should localize in the plasma membrane. In an attempt to confirm this prediction experimentally, the subcellular localization of AtRCI2 isoforms was investigated. Translational fusions between full-length cDNAs corresponding to GFP and AtRCI2 genes were generated and placed under the control of the CaMV 35S promoter. In the case of AtRCI2G, we repeatedly failed to obtain the corresponding cDNA by RT-PCR (data not shown) and the GFP-AtRCI2G fusion could not be generated. This result is consistent with the absence of ESTs for AtRCI2G in databases and suggests that this gene must be expressed at very low levels if at all. The constructs encoding GFP–AtRCI2 fusion proteins were transiently expressed in onion epidermal cells by particle bombardment. The patterns of fluorescence observed by confocal microscopy revealed that AtRCI2A–C and AtRCI2E–H fusion proteins are localized in the plasma membrane, confirming the in silico analyses described above (Fig. 4A). In addition, in several cells transformed with AtRCI2C, AtRCI2E, and AtRCI2F fusion proteins fluorescence was also detected in punctate structures and dots of different sizes (Fig. 4A). Interestingly, the GFP–AtRCI2D fusion protein did not localize in the plasma membrane but mostly in intracellular membranes and numerous small dots (Fig. 4A), suggesting it can be associated to membranes of endoplasmic reticulum (ER) and Golgi apparatus. Similar results were obtained when the subcellular localization of GFP–AtRCI2 fusion proteins was analysed in Arabidopsis protoplasts transiently transformed with GFP–AtRCI2 constructs (see Supplementary Fig. 4 at JXB online). To evaluate the hypothesis that AtRCI2D associates to membranes of the ER and the Golgi apparatus, the GFP–AtRCI2D construct was cotransformed together with the Erd2–YFP translational fusion into onion epidermal cells. Erd2 encodes the Arabidopsis HDEL receptor, an ER and Golgi marker (Brandizzi et al., 2002). Figure 4B shows that the GFP-AtRCI2D fusion protein mainly colocalized with the Erd2 marker (Fig. 4B, arrowheads), although some fluorescence that localized independently from Erd2 could also be discerned in some cells (Fig. 4B, arrows). From all these results, it is concluded that AtRCI2A–C and AtRCI2E–H proteins are localized in the plasma membrane while AtRCI2D is mainly associated to ER and Golgi organelles.
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AtRCI2 genes are differentially regulated in Arabidopsis organs and in response to abiotic stresses
The presence of multiple isoforms of RCI2 proteins in different organisms, most of them showing a relevant level of similarity and a predicted common subcellular localization, raises the question as to whether or not they have redundant functions. Previously, it was demonstrated that AtRCI2A and AtRCI2B are differentially regulated during Arabidopsis development and in response to abiotic stresses such as low temperature, dehydration, and high salt (Capel et al., 1997; Medina et al., 2001), which suggested that all AtRCI2 isoforms do not play exactly the same role. To investigate this possibility further, the expression of AtRCI2C–F in seeds and different organs of Arabidopsis plants grown under control conditions was analysed first by RNA-blot hybridizations with specific probes. In contrast to AtRCI2A and AtRCI2B, whose transcripts mainly accumulate in stems and roots, Fig. 5A shows that AtRCI2C transcripts were found at low levels in all the organs analysed except seeds. AtRCI2D expression was mainly detected in roots and flowers, and moderately in leaves and stems. As in the case of AtRCI2A, AtRCI2B, and AtRCI2C, the expression of AtRCI2D was not detected in seeds (Fig. 5A). AtRCI2E was expressed at low levels in all organs, including seeds, while AtRCI2F expression was principally disclosed in stems, roots, and seeds (Fig. 5A). It was not possible to detect AtRCI2G transcripts by RNA-blot hybridizations (data not shown), which is consistent with the absence of corresponding ESTs in databases. Regarding AtRCI2H, its expression is restricted to seeds (Fig. 5A).
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The expression of AtRCI2 genes in response to different treatments (Fig. 5B) was also examined. As previously reported, AtRCI2A and AtRCI2B were induced by low temperature, dehydration, high salt, and exogenous ABA (Capel et al., 1997; Medina et al., 2001; Nylander et al., 2001). On the other hand, while AtRCI2C expression was unaffected by these treatments, AtRCI2D–F messengers accumulated to different extents in response to them (Fig. 5B). Transcripts corresponding to AtRCI2G and AtRCI2H could not be detected (data not shown). Taken together, all these data demonstrate that AtRCI2 genes are differentially regulated in Arabidopsis organs and in response to abiotic stresses, indicating that the activity of the different AtRCI2 isoforms is confined to particular tissues under control and stressed conditions, and, therefore, that most likely they do not have redundant functions.
A subset of AtRCI2 genes complement the PMP3 deletion in yeast
The functional analysis of RCI2 proteins was more directly approached by investigating the capacity of AtRCI2 genes to complement the deletion of the yeast AtRCI2-related gene PMP3. cDNAs corresponding to AtRCI2A–F and AtRCI2H were cloned into a yeast expression vector containing the promoter and terminator of PMP3, and used to transform PMP3-defficient yeast cells which exhibit high sensitivity to Na+ (Navarre and Goffeau, 2000). Adequate expression of all transformed cDNAs in yeast cells was assessed by RT-PCR (data not shown). The sensitivity of transformed cells to increasing Na+ concentrations was subsequently estimated. Figure 6 shows that, as reported by Navarre and Goffeau (2000), the cDNA corresponding to AtRCI2A was able to complement the deletion of PMP3. In spite of the high similarity existing among AtRCI2 genes, only AtRCI2B, AtRCI2C, and AtRCI2H were also able to complement the PMP3 deletion though to a lesser extent than AtRCI2A (Fig. 6).
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Because the AtRCI2 genes that encode AtRCI2 long proteins (AtRCI2D–F) were not able to complement the PMP3 deletion, the hypothesis that the extra C-terminal hydrophilic tail characteristic of these proteins could play an important role in their complementation capacities was evaluated. With this aim, the C-terminal tail of AtRCI2D, comprising leucine 58 to alanine 75 (Fig. 1), was deleted to generate the AtRCI2D-
1 truncated protein. The corresponding AtRCI2D-1 cDNA was cloned into the yeast expression vector described above, which was used to transform PMP3-deficient yeast cells. Interestingly, AtRCI2D-1 complemented the deletion of PMP3 (Fig. 6), indicating that the extra C-terminal tails of the long AtRCI2 proteins may be critical for their complementation capacities and, therefore, may determine their function(s). All these results confirm that all AtRCI2 isoforms are not functionally redundant. |
Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Six new Arabidopsis genes homologous to AtRCI2A and AtRCI2B have been identified and characterized. The eight AtRCI2 genes (AtRCI2A–H) are distributed among four out of the five Arabidopsis chromosomes, and constitute a novel gene family. Large-scale duplications have been claimed to contribute greatly to the current shape of the Arabidopsis genome (Blanc et al., 2000; Bowers et al., 2003), with deletions and frequent tandem duplications of individual genes changing the microstructure of the duplicated segments (Bancroft, 2001). Our data suggest that AtRCI2 genes have been implicated in some of these chromosome rearrangements. Thus, AtRCI2D is localized in a region of chromosome 2 that is duplicated in chromosome 4 (Blanc et al., 2000). The duplicated region contains AtRCI2E and AtRCI2F organized as a tandem. The organization of AtRCI2A and AtRCI2B in chromosome 3 indicates that, in all likelihood, they have also been originated by tandem duplication as AtRCI2E and AtRCI2F. On the other hand, AtRCI2H is localized in a region of chromosome 2 that is duplicated in chromosome 3 (Blanc et al., 2000). This duplicated region, however, does not contain any AtRCI2 gene, probably because of a subsequent partial deletion.
RCI2-related genes have been described in several plant species, including barley, rice, sheep grass, wheat, and wheat grass (Goddard et al., 1993; Gulick et al., 1994; Hughes and Dunn, 1996; Imai et al., 2005; Inada et al., 2005; Morsy et al., 2005), as well as in the moss Physcomitrella patens (Kroemer et al., 2004). Here, searching through the available databases, it is shown that AtRCI2-related genes are widespread among different organisms, including most plant species, simple metazoans, fungi, and prokaryotes. It is noteworthy that no AtRCI2-related genes were noticed in the genomes of organisms like protostomes (arthropods, molluscs) and deuterostomes (echinoderms, chordates), suggesting that the functions of the corresponding proteins are not necessary in these organisms, or have been undertaken by other proteins. At a structural level, RCI2 genes are remarkably well conserved during evolution, most of them being constituted by two exons separated by a single intron. A detailed analysis of the ESTs identified revealed the presence of several AtRCI2-related isoforms within the same species, indicating that, as AtRCI2s, AtRCI2-related genes are also organized into gene families. In rice, and C. elegans, for instance, 12 and 13 different AtRCI2-related genes were found, respectively. Some of these genes are arranged in tandem, as in Arabidopsis, which allows speculation that tandem duplications of individual genes have contributed to the evolution of RCI2 gene families.
Our data clearly show that both sequence and topology have been conserved in AtRCI2 and AtRCI2-related proteins, suggesting that they have a common evolutionary origin. To test this hypothesis, a combined phylogenetic analysis was performed with the eight AtRCI2 proteins and the representative AtRCI2-related proteins shown in Fig. 1. An unrooted tree inferred from a Neighbor–Joining analysis (Saitou and Nei, 1987) is displayed in Fig. 7. The tree indicates that, in fact, RCI2 proteins have evolved from a common ancestor to three evolutionary groups. Group I is constituted by all plant proteins, while group II is composed by the proteins from prokaryotes, and group III by a cluster of proteins belonging to different organisms including nematodes, protostomes, and fungi. Interestingly, the group of plant proteins can be subdivided in four (A–D) evolutionary branches, AtRCI2s being distributed in branches A, B, and C. Branch A is constituted by AtRCI2C and AtRCI2H isoforms, together with the representative AtRCI2-related protein from O. sativa. AtRCI2 proteins containing the extra C-terminal tail (AtRCI2D-G) compose branch B, confirming the common evolutionary pathway predicted for their corresponding genes on the basis of their structure and chromosome distribution. In a similar way, AtRCI2A and AtRCI2B, whose corresponding genes are organized in tandem, are localized in the same evolutionary branch (C), together with representative proteins from other different species. RCI2-related proteins from bryophytes constitute branch D, indicating a relatively early diversification of RCI2 families during plant evolution.
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Our in silico analysis suggested that AtRCI2 and AtRCI2-related proteins contain two conserved transmembrane domains and localize to the plasma membrane. Consistent with these data, subcellular localization of AtRCI2 proteins, as revealed by transient expression of GFP-AtRCI2 fusions in onion epidermal cells and Arabidopsis protoplasts, indicates that only AtRCI2D does not localize to the plasma membrane, being associated to the ER and Golgi compartment. In agreement with a plasma membrane localization for the majority of AtRCI2-related proteins, the yeast AtRCI2-related protein PMP3 was purified from this cellular component (Navarre and Goffeau, 2000). Moreover, a plasma membrane localization has also been recently reported for the wheat RCI2-related protein WPI6 (Imai et al., 2005), and the RCI2-related protein LTI6a was detected in the membrane protein fraction of rice cold-stressed seedlings (Morsy et al., 2005), further supporting that most RCI2 proteins are localized in the plasma membrane.
Little is known about the function of RCI2 proteins. The high sequence similarity of AtRCI2 and AtRCI2-related proteins, their predicted common evolutionary origin, and their conserved subcellular localization, suggest that RCI2 proteins may retain a conserved role. In this regard, the yeast AtRCI2-related protein PMP3 has been involved in controlling the plasma membrane potential, which, in turn, would contribute to the regulation of Na+ uptake in yeast cells (Navarre and Goffeau, 2000), and AtRCI2A has been reported to play a role in salt tolerance by avoiding over-accumulation of Na+ and K+ ions in Arabidopsis (Mitsuya et al., 2005, 2006). Moreover, complementation analysis revealed that AtRCI2A (Navarre and Goffeau, 2000; Nylander et al., 2001) and RCI2-related proteins WP16 from wheat (Imai et al., 2005) and AcPMP3 from sheep grass (Inada et al., 2005) restore the plasma membrane potential and the salt-sensitive phenotype of yeast cells lacking PMP3. These data indicate that, besides PMP3 and AtRCI2A, other RCI2 proteins can be involved in regulating plasma membrane potential and, therefore, abiotic stress responses. In this study, however, it is shown that, similarly to what has previously been demonstrated for AtRCI2A and AtRCI2B (Capel et al., 1997; Medina et al., 2001) and described for several AtRCI2-related genes from rice and sheep grass (Inada et al., 2005; Morsy et al., 2005), AtRCI2C, AtRCI2D, AtRCI2E, and AtRCI2F genes are differentially regulated in Arabidopsis organs and in response to abiotic stresses and ABA treatment. These results suggest that, in spite of their high similarity, shared evolutionary origin, and conserved subcellular localization, all RCI2 proteins do not have exactly the same function.
Additional support to the hypothesis that different RCI2 proteins may have different roles, comes from results showing that, in addition to AtRCI2A, only AtRCI2B, AtRCI2C, and AtRCI2H genes are able to complement the PMP3 deletion in yeast and, therefore, may function in membrane potential homeostasis. The fact that AtRCI2D, AtRCI2E, and AtRCI2F are unable to substitute the the absence of PMP3 is particularly striking. The main difference between these proteins and AtRCI2A–C and AtRCI2H is that they contain an extra C-terminal hydrophilic tail of around 20 amino acids. Interestingly, although this tail does not contain any sequence resembling reported functional or targeting motives, when it is removed from AtRCI2D the truncated protein acquires the capacity to substitute for the lack of PMP3, suggesting it can have a role in AtRCI2D function. Considering that extra C-terminal tails are not restricted to some RCI2 proteins from Arabidopsis but are present in other AtRCI2-related proteins (see above), it is tempting to propose that they may modulate the function of RCI2 proteins by modifying either their stability or activity. In summary, the results reported in this work indicate that different members from the RCI2 protein family have unique functions. It has been demonstrated that different AtRCI2 proteins play particular roles in the Arabidopsis response to abiotic stresses. Genetic analysis through the identification and characterization of mutants affected in AtRCI2 and AtRCI2-related genes will certainly contribute to understand further the functional redundancies and specificities of RCI2 proteins.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Supplementary data in three tables and four figures can be found at JXB online.
Supplementary Table 1. Genebank accession numbers of the identified ESTs encoding AtRCI2-related proteins.
Supplementary Table 2. Primers used for AtRCI2 probes.
Supplementary Table 3. Primers used to generate AtRCI2 cDNAs and GFP-AtRCI2 fusions, and to perform RT-PCR expression analyses in yeast.
Supplementary Fig. 1. Hydropathy profiles and transmembrane predictions for AtRCI2-related proteins.
Supplementary Fig. 2. Hydropathy profiles and transmembrane predictions for OsRCI2 proteins.
Supplementary Fig. 3. Hydropathy profiles and transmembrane predictions for CeRCI2 proteins.
Supplementary Fig. 4. Subcellular localization of GFP–AtRCI2 fusion proteins in Arabidopsis protoplasts.
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Acknowledgements |
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We thank Dr A Goffeau for the gift of yeast plasmid and strains, Dr JM Malpica and D Abia for bioinformatic assistance, M Seisdedos and S Hernandez for their invaluable help with the confocal microscope, A Redondo for technical assistance, and Dr G Salcedo and Dr JJ Sanchez-Serrano for their suggestions on the manuscript. This work was supported by grants BIO2004-00628 from CICYT and CPE03-006-C6-1 from INIA.
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