PRINTED IN ARGENTINA Isolation and molecular characterization of a cax gene from

A new cation exchangers (CAXs) gene was cloned and characterized from Capsella bursa-pastoris by rapid amplification of cDNA ends (RACE). The full-length cDNA sequence of cax from C. bursa-pastoris (designated as Cbcax51) was 1754 bp containing a 1398 bp open reading frame encoding a polypeptide of 466 amino-acid residues with a calculated molecular mass of 50.5 kDa and an isoelectric point of 5.69. The predicted CbCAX51 contained an IMP dehydrogenase/GMP reductase domain, two Na+/Ca2+ exchanger protein domains. Comparative and bioinformatics analyses revealed that CbCAX51 showed extensive homology with CAX from other plant species. The expression analysis by different treatments indicated that Cbcax51 could be activated by cold triggering and was related to the cold acclimation process, but its expression is regulated negatively by drought and not affected by ABA or salt.


Introduction
Plants have multiple mechanisms to maintain appropriate intracellular levels of various ions (Fox and Guerinot, 1998), while plant vacuole plays a major role in the intracellular sequestration of various compounds (Marty, 1999).There are several kinds of transporters on the vacuolar membrane: Na + /H + , Ca 2+ /H + , Mn 2+ /H + , etc., helping to establish a concentration gradient of Na + , Ca 2+ and Mn 2+ across the vacuolar membrane.Calcium (Ca 2+ ) is used by most cells to convert external signals into cytosolic information, which can drive processes that are required for full responses to a particular stimulus (Catala et al., 2003).Change in the cytosolic concentration of free Ca 2+ is the basis for Ca 2+ serving as a second messenger (Sze et al., 2000).Ca 2+ levels in the cytosol fluctuate in response to growth, development, and environmental perturbations (Sanders et al., 1999;Curran et al., 2000).The alteration of Ca 2+ partition is associated with the plants being more sensitive to environmental perturbations, such as cold.Since Ca 2+ is an extremely common second messenger, localized Ca 2+ spikes around the plant vacuole are also believed to play a pivotal role in determining signal specificity (Cheng and Hirschi, 2003).Therefore, the study of cation exchangers is important.
In plants, it has been established that Ca 2+ plays an important role as a second messenger in many important physiological processes.Precise control of the amount of Ca 2+ in the cytosol is essential for plants' tolerance to some biotic or abiotic stresses.Cation exchangers are part of the ensemble of transporters that help modulate the duration of these Ca 2+ signaling events (Ueoka-Nakanishi et al., 2000).CAX can function as Ca 2+ /H + and/or heavy metal/H + exchangers (Hirschi et al., 2000).The CAX1 from Arabidopsis was the first plant gene encoding cation exchangers to be cloned.It was identified by screening a cDNA library from Arabidopsis for clones able to complement a yeast mutant defective in vacuolar Ca 2+ transport.CAX1 may play a role in reducing cytosolic Ca 2+ concentration to resting levels after Ca 2+ increase in response to external stimuli.Transgenic tobacco plants overexpressing cax1 show increased sensitivity to chilling temperatures (Hirschi, 1999), suggesting that it can play a role in plant adaptation to this environmental condition.Presently, more than ten cax from Arabidopsis have been cloned (Maser et al., 2001).From other plant species, Vrcax1 (Vigna radiata) (Ueoka-Nakanishi et al., 1999) and Oscaxs (Oryza sativa) (Kamiya et al., 2006) were also cloned.All of the cloned plant cax, with the exception of Oscaxs, were determined to be localized to vacuolar membranes by expressing the cax fused to green fluorescent protein (GFP) gene and/or by immunochemical analysis (Pittman and Hirschi, 2001;Cheng et al., 2002;Cheng et al., 2003).Plasma and plastid membranes have also been reported to possess Ca 2+ /H + exchange activity (Kasai and Muto, 1990;Ettinger et al., 1999); however, an exchanger has not yet been identified.
However, there has been no report on the cloning of cax from Capsella bursa-pastoris, which is a wild grass belonging to the Cruciferae like Arabidopsis.It is considered to be a potential vegetable crop and can grow and set seeds normally at low temperatures.In this paper, we report the cloning of cax from C. bursapastoris by rapid amplification of cDNA ends (RACE)-PCR technology.Bioinformatics analysis and expression analysis revealed that the C. bursa-pastoris cax (Cbcax51) strongly resembles cax from other species, and is relevant to cold acclimation.

Plant materials
Seed of C. bursa-pastoris were germinated in sterilized soil.The plants were grown greenhouse at Fudan University, Shanghai, China, under continuous light at 28ºC.

Isolation of total RNA
About 1.0g materials of C. bursa-pastoris were powdered using liquid nitrogen with mortar and pestle and the total RNA was extracted using TRIZOL Reagent (GIBCO BRL) according to the manufacturer's instructions.

5' RACE of Cbcax51
According to the protocol of the SMART TM RACE cDNA Amplification Kit (Clontech), about 100ng of total RNA was reversely transcribed with primer 5'CDS primer coupled with (dC) tailing and SMART II A oligo, which annealed to the tail of the RNA and served as an extended template for PowerScript RT.The first round of PCR was performed with 5'CBCAX1 and universal primer A Mix (UPM) (provided in the kit).Subsequently, nested PCR was performed under normal PCR reaction conditions using 5'CBCAX2 and the nested universal primer A (NUP) (provided in the kit).

3' RACE of Cbcax51
According to the protocol of Rapid Amplification of cDNA Ends Kit (GIBCO BRL), an aliquot of isolated 100ng total RNA was reversely transcribed using the cDNA synthesis primer AP (provided in the kit).The gene-specific primer 3'caxGSP1 and universal amplification primer (UAP) was used to amplify the 3' end cDNA.Then, the gene-specific primer 3'caxGSP2 and abridged universal amplification primer (AUAP) was used to the nested PCR.

Generation of the full-length cDNA of Cbcax51
By comparing and aligning the sequences of the 5'-RACE and 3'-RACE products, the full-length cDNA sequence of Cbcax51 was deduced and subsequently amplified with the primers FCBCAX and RCBCAX, using One Step PCR Kit (TaKaRa).All RT-PCR products were purified and cloned into pMD 18-T vector (TaKaRa), and sequenced.All the primers used in RACE were listed in Table 1.

Comparative and bioinformatics analysis
Comparative and bioinformatics analyses of Cbcax51 were carried using computer programs available online.The nucleotide sequence, deduced amino acid sequence and open reading frame (ORF) encoded by Cbcax51 were analyzed and sequence comparison was conducted through database search using BLAST program (http://www.ncbi.nlm.nih.gov).Transmembrane analysis was carried out by TMHMM-Prediction of transmembrane helices in proteins (http:// www.cbs.dtu.dk/services/TMHMM-2.0/).

Expression analysis
To study the expression of the Cbcax51 under different abiotic stress conditions such as low temperature, ABA, salt stress and drought, total RNA was extracted from seedlings of C. bursa-pastoris under various treatments, and then subjected to semi-quantitative reverse transcription (RT)-PCR for Cbcax51 expression.For low temperature treatments, the 4-week-old seedlings grown under normal conditions were transferred to a growth chamber set to 4ºC for 4h, 8h, and 24h, respectively.For ABA treatment, after the 4-week-old seedlings were sprayed with 100 μM ABA, the seedlings grew for 4h, 8h, and 24h respectively.Salt stress was accomplished by watering plants with 250mM NaCl, after which the seedlings grew for 4h, 8h and 24h respectively.Drought was induced by removing 4-week-old seedling from soil and set them under room temperature for 0.5h, 1h and 4h respectively.After the treatments, leaves were frozen immediately in liquid nitrogen and stored at -70ºC until use.We use three plants per treatment.RT-PCRs using a One Step PCR Kit (TaKaRa) were performed at 50ºC for 30min followed by 20 cycles of amplification (94ºC for 45sec, 60ºC for 45sec, and 72ºC for 1min), using gene-specific primers Fcbcax1 and Rcbcax1 (Table 1).Two primers, 18SF and 18SR (Table 1), were also used to amplify 18S rRNA gene in the semi-quantitative RT-PCR as controls.

Cloning of the full-length cDNA of Cbcax51
By comparing and aligning the sequences of the 5'-RACE and 3'-RACE products, the full-length cDNA sequence of Cbcax51 was deduced and subsequently amplified using One Step PCR Kit with the primers FCBCAX and RCBCAX (Table 1).The full-length cDNA of Cbcax51 was 1754 bp and contained a 1398 bp complete ORF encoding a polypeptide of 466 of amino acids, a 16 bp of 5'-and 337 bp of 3'untranslated sequences (Database Accession No.: AY660870).The translation stop codon and one putative polyadenylation signal site were recognized in the 3'-untranslated region, which were followed by a short poly (A) tail (Fig. 1).

Characterization of CbCAX51
CAXs have in general 10-14 transmembrane (TM)spanning domains along 400 amino acid residues.CAXs contain a central hydrophilic motif rich in acidic amino acid residues that bisect the polypeptide into two approximately equal segments (Waditee et al., 2004).The Cbcax51 cDNA contains an open reading frame encoding a deduced 466 amino acids polypeptide with a molecular weight of 50.5 kDa and pI of 5.69 (Fig. 1).Ac-cording to the result obtained from the PROSITE database (http://www.expasy.org/prosite), the CbCAX51 possess five putative glycosylation sites (Asn-Xxx-Thr/Ser), eight putative phosphor sites and nine myristyl sites.According to the result obtained from InterProScan (http:// www.ebi.ac.uk /interpro/scan.html),the CbCAX51 contains an IMP dehydrogenase/GMP reductase domain and two Na + /Ca 2+ exchanger protein domains (Fig. 2A).The transmembrane analysis of CbCAX51, which was carried out online by SPLIT 4.0 SERVER, showed that like AtCAX1 from A. thaliana, there were eleven predicted transmembrane helix positions (Fig. 2B).The CbCAX51 contain a central hydrophilic motif, rich in acidic amino acid residues (Fig. 1).

Comparative and bioinformatics analysis
Sequence alignment using Vector NTI Suite 8.0 and BLAST research showed that the predicted amino acid sequence of CbCAX51 had high identity with CAXs from other plant species, such as AtCAX1 (NP_181352), AtCAX2 (NP_566452), AtCAX3 (AAV85729), AtCAX4 (NP_568091) from A. thaliana and OsCAX1c (BAD83661) from O. sativa, with the identity of 87.8%, 41.9%, 76.8%, 53.7% and 47.5% respectively (Fig. 3).It demonstrated that CbCAX51 has high identity in structure and sequence to the previously characterized AtCAX1 from A. thaliana in its primary sequence.AtCAX1 functions as a cation exchanger and has been analyzed using yeast heterologous expression technology and T-DNA insertion mutant experimental systems (Cheng et al., 2003).Therefore, CbCAX51 might be the counterpart of AtCAX1 that modulated plant cation exchangers.

Expression analysis
Semi-quantitative RT-PCR was carried out to investigate the expression of Cbcax51 under different abiotic stress.The Cbcax51 expression was induced transiently in leaves, reaching a maximum level after 24h of exposing plants to low temperature (Fig. 4A).This result indicated that Cbcax51 could be activated by cold triggering and was related to the cold acclimation process.It has been reported that the expression of Atcax1 from A. thaliana was induced in response to stimuli such as low temperature in order to regulate the calcium content in cytosol.The changed cytosol calcium content activated the cbf gene expression-regulated pathway and transcriptional cascade leading to the expression of coldresponsive genes under cold stress (Wang et al., 2005), by activating the promoter of cbf gene to induce its expression.The CBF subsequently binds the DRE/CRT element in the promoter of the cold-regulated genes and activated transcription of them, thereby increasing plants' freezing tolerance.Therefore, it is possible that Cbcax51, which strongly resembled Atcax1, acts as a similar transcriptional regulator in cbf gene expression-  regulated pathway in response to cold stress.Because many cold-inducible genes also are responsive to exogenous ABA and osmotic stresses (Thomashow, 1999), the effect of ABA, high salt and drought on Cbcax51 mRNA accumulation was also analyzed.The expression of Cbcax51 is not affected by ABA or high salt treatment; the gene transcripts were almost at the same level during the different time periods whether under ABA stress or high salt (not shown).Figure 4B shows that transcript level was lower than the control after the 4h dehydration treatment.Therefore, we infer that it is regulated negatively by drought.Overall, Cbcax51 expression was inhibited by drought but was not affected by ABA and high-salt treatments, which is consilient with the report about the Atcax1 in A. thaliana (Catala et al., 2003).Furthermore, although the significance of this expression pattern is not clear at present, it indicates that during C. bursa-pastoris development and in response to different adverse environmental situations, the regulation of Cbcax51 is accomplished through several ABA-independent signal transduction pathways.A: RT-PCR analysis with total RNA obtained from leaves of the seedling exposed at 4°C for 4h, 8h and 24h respectively; B: RT-PCR analysis with total RNA obtained from leaves of seedling that were removed from the soil and kept under room temperature for 0.5, 1 and 4h respectively.Equal RNA loading was used as the control, by amplifying 18S rRNA for each treatment.CK indicates the control.

FIGURE 1 .
FIGURE 1.The full-length cDNA sequence and the deduced amino acid sequence of Cbcax51.The start codon (ATG) and the stop codon (TAA) are shown in bold italically.The N-terminal regulatory region (NRR) is shown in wave underline, Ca 2+ domain is shown in double underline, and a central hydrophilic motif is underlined.

FIGURE 3 .
FIGURE 3. Multi-alignment of amino acid sequences of CbCAX51 and other CAX proteins.The identical amino acids are shown in black background and the conserved amino acids are shown in gray background.The aligned CAXs include sequences from C. bursa-pastoris (AY660870), A. thaliana (NP_181352, NP_566452, AAV85729 and NP_568091), and O. sativa (BAD83661).

FIGURE 4 .
FIGURE 4. Expression pattern of Cbcax51 by semi-quantitative RT-PCR analysis in response to different treatments.A: RT-PCR analysis with total RNA obtained from leaves of the seedling exposed at 4°C for 4h, 8h and 24h respectively; B: RT-PCR analysis with total RNA obtained from leaves of seedling that were removed from the soil and kept under room temperature for 0.5, 1 and 4h respectively.Equal RNA loading was used as the control, by amplifying 18S rRNA for each treatment.CK indicates the control.