A pea chloroplast translation elongation factor that is regulated by abiotic factors

BBRC Biochemical and Biophysical Research Communications 320 (2004) 523–530 www.elsevier.com/locate/ybbrc

A pea chloroplast translation elongation factor that is regulated by abiotic factorsq,qq B.N. Singh, R.N. Mishra, Pradeep K. Agarwal, Mamta Goswami, Suresh Nair, S.K. Sopory, and M.K. Reddy* International Centre for Genetic Engineering and Biotechnology (ICGEB), Aruna Asaf Ali Marg, New Delhi 110 067, India Received 5 May 2004 Available online

Abstract We report the cloning and characterization of both the cDNA (tufA) and genomic clones encoding for a chloroplast translation elongation factor (EF-Tu) from pea. The analysis of the deduced amino acids of the cDNA clone reveals the presence of putative transit peptide sequence and four GTP binding domains and two EF-Tu signature motifs in the mature polypeptide region. Using in vivo immunostaining followed by confocal microscopy pea EF-Tu was localized to chloroplast. The steady state transcript level of pea tufA was high in leaves and not detectable in roots. The expression of this gene is stimulated by light. The differential expression of this gene in response to various abiotic stresses showed that it is down-regulated in response to salinity and ABA and up-regulated in response to low temperature and salicylic acid treatment. These results indicate that regulation of pea tufA may have an important role in plant adaptation to environmental stresses. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Confocal microscopy; Immunostaining; Pisum sativum; Plant promoter

Plant growth and development are adversely affected by abiotic stress factors due to their sessile growth habit. However, plants respond to environmental stress through altered gene expression and illicit adaptive responses, such as the production of stress adaptive proteins, synthesis of oxidative stress protectors, and accumulation of protective solutes [14]. In recent years, using cDNA microarray technology, it has been found that the number of genes that respond to different stresses is very large [16,28]. To understand the molecular regulation of these processes, the relevant subsets of differentially expressed genes must be identified and the q

The nucleotide sequence data reported here appears in the EMBL, GenBank, and DDBJ Nucleotide Sequence Databases under the Accession Nos. Y14561 and AY083613. qq Abbreviations: ABA, abscisic acid; cDNA, complementary DNA; EF-Tu, chloroplast elongation factor; mRNA, messenger RNA; PCR, polymerase chain reaction; tufA, gene coding for chloroplast elongation factor. * Corresponding author. Fax: +91-11-26162316. E-mail address: [email protected] (M.K. Reddy). 0006-291X/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2004.05.192

regulated expression mechanisms of these genes understood. Recently Guo et al. [13] showed the involvement of translation elongation factor 2 in Arabidopsis for the low temperature induced cold acclimation. Additionally, Rausell et al. [22] showed that the overexpression of sugar beet translation initiation factor increases salt tolerance in both yeast and Arabidopsis and also improves in vitro protein synthesis under salt stress condition. These studies suggest the importance of protein synthesizing machinery and its role in abiotic stress adaptation in plants. Previously, it was shown in maize that the increased thermal tolerance is associated with 45 kDa heat shock protein [26]. Subsequently, this protein was identified as EF-Tu [3]. EF-Tu has been shown to have chaperone-like property other than its role in polypeptide elongation in bacterial system [7] of refolding the denatured proteins or preventing their aggregations during heat stress. Prokaryotic and plastid specific EF-Tu proteins are strikingly similar and thus, it is possible that the plastid specific EFTu may play a role in stress adaptation.

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The translation elongation factor is an essential component for protein synthesis and plays a role in polypeptide elongation. It interacts with aminoacyl tRNA and transports the codon specific tRNA to the aminoacyl site on the ribosome (ribosomal A-site) during translation elongation step. This function is performed by EF-Tu in prokaryotes and eukaryotic organelles, and by EF-1a in eukaryotes. EF-Tu is encoded in the chloroplast of lower photosynthetic eukaryotes such as Chlamydomonas [2] and Euglena [21] while in higher plants an evolutionary transfer of these genes has occurred from the chloroplast to the nucleus [2]. Several cDNA clones encoding chloroplast EF-Tu have been identified in a number of higher plants [2,18,20,32,33]. The expression patterns of these genes have not been studied previously in relation to different environmental stresses. During the process of identifying genes that are differentially expressed in response to abiotic stresses by subtractive cDNA cloning, we identified a full-length cDNA clone encoding a chloroplast translation elongation factor (EF-Tu) in pea. Here we report its cloning, expression, and characterization, and its transcript and protein profile by Northern and Western analysis under different stress environments. Its possible role in plant stress adaptation is discussed.

Experimental procedures Sterilized seeds of pea (Pisum sativum) were allowed to germinate in wet germination paper for 4 days at 26 °C, and were then grown for 7 days in Hoagland solution at 22/20 °C with a 14/10-h light/dark cycle. For Northern analysis 7-day-old pea seedlings were exposed to different abiotic stresses; for salinity stress pea seedlings were treated with 100, 200, and 500 mM NaCl in Hoagland solution for 4, 6, and 24 h. For ABA treatment the pea seedlings were sprayed with 50 and 100 mM ABA solution and harvested 24 h later. For SA treatment plants were exposed to10 mM salicylic acid for 2, 4, and 12 h. For low temperature stress pea seedlings were transferred to 4 °C for 1, 6, and 12 h. In the case of light treatment the etiolated pea seedlings that were grown under dark for 7 days were exposed to light, the photosynthetic photon flux density (PPFD) at leaf level was 200 lmol m
2 s
1 , provided by metal halide lamps and the seedlings were collected after exposing them to 2, 4, 6, and 8 h of light. After the treatments, the seedlings were frozen in liquid nitrogen and processed for RNA isolation and protein extraction to analyze the expression of tufA transcript and EF-Tu protein. Isolation of EF-Tu cDNA clone by cDNA subtraction. PCR amplifiable single stranded cDNA population was synthesized from both control and salt-exposed pea (Pisum sativum) seedlings by attaching known nucleotide sequences at either end of the cDNA population [23]. Different sets of primers/adapters were incorporated on either end for control and salt-exposed cDNA population to obviate the need to remove them from the reaction mix prior to subtraction. Biotinylated forward primer was used during the PCR amplification of the cDNA population that was prepared from salt-exposed pea seedlings to incorporate biotin into the 50 ends of sense strands of amplified cDNA population. The amplified sense strands of salt-exposed cDNA population were immobilized on streptavidin-linked magnetic beads and the complementary (anti-sense) strands were then

removed by alkali hydrolysis. The immobilized sense strands of amplified cDNA population from salt-exposed tissue were hybridized to un-amplified first strand (anti-sense) control cDNA population. Following the hybridization the common double stranded cDNA hybrids and excess sense strands of salt-exposed cDNA molecules that were immobilized on streptavidin linked paramagnetic beads were removed from the hybridization mixture by using magnetic separation. The unsubtracted differentially expressed (down-regulated during salinity stress) cDNA molecules were PCR amplified using separate sets of primers that were incorporated on either side of the control cDNA population, and the amplified cDNA was cloned directionally into pBluescript plasmid vector. One of the clones thus isolated was identified to be the full-length cDNA clone for tufA gene. Expression and purification of recombinant EF-Tu protein in Escherichia coli and developing polyclonal antibodies. The complete coding sequence for the pea EF-Tu was PCR amplified and cloned into the NdeI and XhoI sites of pET-28a expression vector (Novagen, USA). The recombinant protein was purified to homogeneity on a nickel–NTA (Qiagen, Germany) column chromatography following the manufacturer’s instructions. The purified recombinant EF-Tu was immunized to rabbit and developed polyclonal antibodies. Intact chloroplasts were isolated from pea seedlings and total proteins were fractionated on a 10% polyacrylamide gel and electroblotted onto a nitrocellulose membrane. Detection of the EF-Tu polypeptide by anti-EF-Tu polyclonal antibodies was carried out by routine Western analysis. Immunofluorescence staining and confocal microscopy. Light-grown 7-day-old pea leaves were chopped into small pieces and incubated in freshly prepared buffer-P (25 mM Mes, pH 5.5; 8 mM CaCl2 , and 600 mM mannitol) containing 13% cellulase (w/v) for 1–2 h to release the protoplasts. The isolated protoplasts were aspirated onto a polylysine coated cover-slip and fixed with 3.7% formaldehyde. After two washes with phosphate buffered saline (PBS) the cover-slip was blocked with PBS containing 5% BSA (w/v) for 1 h at room temperature and then incubated at 4 °C overnight in a 1:1000 dilution of the primary antibody in PBS containing 0.1% BSA. The unbound primary antibody was removed by washing four times with PBS and incubated again in the dark for 4 h at room temperature with Alexa-fluor 488 goat anti-rabbit immunogloblin G (Molecular Probes, USA) diluted 1:3000 in PBS containing 0.1% acetylated BSA. Protoplasts were washed again four times with PBS before mounting in antifade solution (Fluroguard, Bio-Rad, USA) and observed under microscope. Confocal laser scanning (Radiance 2100, Bio-Rad, USA) was performed using Nikon microscope (objective Plane Apo 60X/1.4 oil, Japan). The optical sections were 0.45 lM thick unless stated otherwise. The excitation wavelength for Alexa fluorescence was 488 nm (argon laser) and fluorescence was detected through emission filter HQ515/30 (high quality band pass), centered on 515 with 30 nm bandwidth. DAPI fluorescence was excited by blue diode (405 nm) and detected through emission filter HQ442/45. Image processing was carried out using LazerShop and PhotoShop (5.5) (Adobe systems, San Jose, CA, USA) was used for the final image assembly. Isolation of the pea EF-Tu genomic clone. The two gene specific primers, which were used to amplify the complete open reading frame of EF-Tu from the cDNA clone, were used to PCR amplify the EF-Tu genomic fragment using pea genomic DNA as template. PCR was carried out using 150 ng each of the gene specific primer along with 200 lM of each dNTPs and 2.5 U Taq DNA polymerase with 100 ng of pea genomic DNA as template in a 50 ll reaction. PCR conditions were 94 °C, 1 min; 55 °C, 1 min; and 72 °C, 3 min for 30 cycles. The amplification product was cloned into pGEM(T) vector (Promega, USA) and the insert was completely sequenced. Expression analysis of EF-Tu transcript and its protein. Total RNA was isolated from different tissues (leaf, root tips) of pea seedlings, seedlings exposed to light for different lengths of time, and also seedlings exposed to different abiotic stress, i.e., salinity, abscisic acid (ABA), cold temperature, and salicylic acid (SA). Approximately 20 lg of total RNA was resolved on 1% agarose formaldehyde gel and

B.N. Singh et al. / Biochemical and Biophysical Research Communications 320 (2004) 523–530 transblotted onto Hybond N membrane. The blots were probed with 32 P-labeled (nick translated) full-length EF-Tu cDNA. Hybridization and post-hybridization washes were carried out as mentioned earlier [25]. The blots were autoradiographed. For Western analysis total proteins were extracted from pea, fractionated on a 10% polyacrylamide gel, and electro-blotted onto a nitrocellulose membrane. Detection of the EF-Tu polypeptide by anti-EF-Tu polyclonal antibodies followed by alkaline-phosphatase conjugated secondary antibody, the signals were developed using ECL kit following manufacturer’s instruction and exposed to X-ray film. Cloning of the 50 -flanking region of the pea EF-Tu gene. The 50 flanking region of the EF-Tu gene was PCR amplified according to Reddy et al. [24] using a gene specific primer (50 -CGTTGAGA GGGAGTGAGGTG-30 ) synthesized in anti-sense orientation and the genome walker primer (50 -CTAATACGACTCACTATAGGG-30 ). PCR was carried out using 150 ng each of the gene specific and walker primers along with 200 lM of each dNTP and 2.5 U Taq DNA polymerase with 20 ng of adapter ligated pea genomic DNA as template in a 50 ll reaction. PCR conditions were 94 °C, 1 min; 55 °C, 1 min; and 72 °C, 1 min for 30 cycles. The amplification product was gel purified, its ends polished using T4 DNA polymerase and subsequently digested with NotI before cloning into EcoRV and NotI digested pBluescript plasmid. The insert DNA was completely sequenced. Sequencing and sequence analysis. The partial and full-length cDNA and genomic clones of EF-Tu were completely sequenced using the dideoxy chain termination method using Sequenase version 2 (USB, USA). Most of the routine sequence (protein and DNA) analyses were performed using MacVector (v 7; Oxford Molecular Group). Homology searches were done using FASTA and multiple sequence alignment was done using CLUSTAL W using MacVector suite of programmes.

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The putative mature EF-Tu contained 409 amino acid residues with an apparent molecular weight of 45 kDa with an estimated isoelectric point being 5.39. The sequence (excluding the transit peptides) analysis showed that the pea EF-Tu has 75–80% identity with Nicotiana sylvestris, Nicotiana tabacum, Glycine max, Oryza sativa, Pelargonium, and Arabidopsis and it grouped very close to G. max EF-Tu on an un-rooted phylogenetic tree (results not shown). The organellar elongation factors are more similar to their counterparts in bacteria (67% identity) than they are to homologous counterpart in the eukaryotic cytosol, EF-1a (25% identity). The GTP binding domains GXXXXGK, DXXG, NKXD, and SAL known to be involved in GTP interaction [15] were presented as GHVDHGK, DCPG, NKVD, and SAL (residues 98–104, 160–163, 215–218, and 253–255, respectively) in pea EF-Tu in the same spatial order and also contain two chloroplast EFTu signature motifs (ALMANPAIKR and KDEES). The pea EF-Tu clone shares only 56% and 25% amino acid identity with mitochondrial EF-Tu and cytosolic EF-1a, respectively, these results indicate that pea EFTu is more likely to be the chloroplast EF-Tu (tufA) and not the mitochondrial EF-Tu (tufM) or its cytosolic counterpart EF-1a (ef-A). Expression of recombinant EF-Tu and its sub-cellular localization by immunostaining

Results and discussion Primary structure of tufA cDNA and its deduced polypeptide During our screening of pea seedlings under salt stress we isolated many cDNA clones that were differentially expressed. Sequence analysis of one of the many such clones identified a full-length cDNA clone that showed a high degree of sequence homology to tufA gene that encodes for chloroplast translation elongation factor (EF-Tu). The total size of the pea tufA cDNA clone was 1731 bp with an open reading frame of 1467 and 84 bp 50 and 180 bp 30 un-translated regions (UTR). The ORF encodes for 488 amino acids, of which first 79 amino acids are rich in serine and threonine residues and basic amino acids, with a net positive charge (estimated isoelectric point is 13) showing the features typical of a transit peptide (TP). This region has no specific homology to other transit peptides of other known chloroplast elongation factors. However, a computer analysis using neural network method, ChloroP [10], predicted that this region of the peptide is necessary for protein import into the chloroplast. The predicted transit peptide cleavage site (TVR AARG) of pea EF-Tu (Fig. 1) was also well conserved among other chloroplast translation elongation factors reported so far.

We have established a heterologous expression of pea recombinant EF-Tu in E. coli. The recombinant protein was purified to homogeneity and used to develop polyclonal antibodies. The in vivo localization of EF-Tu protein by the immunostaining of the protein blot made from sub-cellular chloroplast fraction (or from total proteins extracted from pea leaves) showed a very strong reaction with a 45 kDa protein in the chloroplast fraction indicating the localization of EF-Tu in the chloroplast (Fig. 2). Compared to the positive control (recombinant EF-Tu) 55 kDa (Fig. 2, lane 1) the native chloroplast EF-Tu is smaller in size 45 kDa (Fig. 2, lane 2) indicating that the 55 kDa precursor protein is processed into the mature 45 kDa form inside the chloroplasts suggesting that the N-terminal extension of 79 amino acid residues predicted as a transit peptide may be a bona fide transit peptide for chloroplast targeting. The in vivo localization of EF-Tu protein by immunofluorescence labeling and confocal laser scanning microscopy showed the localization of EF-Tu inside the chloroplast compartment (Fig. 3). Structure of the genomic clone of the tufA gene Using specific primers designed based on cDNA sequence a 1.7 kb fragment was PCR amplified from the pea genomic DNA, and exons and introns were

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Fig. 1. Nucleotide sequence of pea tufA gene and its 50 -flanking region. The start and the end of the cDNA sequence are shown by arrows under the nucleotide sequence. The intron/exon boundaries (gt/ag) are underlined. The nucleotide sequences in italics designate the intron region. The deduced amino acids of the coding region are represented by single letter code under the nucleotide sequence. The vertical arrowhead indicates the predicted cleavage site of the precursor polypeptide. The amino acid sequences in italics designate the putative transit peptide region. The double underlined amino acid sequences represent GTP binding sites. Sequences in outlined and bold characters represent amino acid sequences of EF-Tu signature motifs. Putative regulatory elements (Myb, ASF1, GATA, and I Box) are shown. Numbers on the left and right represent position of the amino acid and nucleotide sequences, respectively.

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Fig. 2. Western blot analysis of pea EF-Tu. Intact chloroplasts were isolated from pea seedlings. Total proteins from these organelles were size fractionated on a 10% polyacrylamide gel before transferring onto a nitrocellulose membrane. Western analysis was carried out using anti-pea EF-Tu rabbit serum. Lane 1, purified recombinant EF-Tu; lane 2, chloroplast extract; note the decrease in molecular weight in lane 2 as compared with lane 1. Figure on the left and right represent molecular weights in kilodalton.

identified by comparing the nucleotide sequence of genomic clone with its cDNA sequence (Fig. 1). The pea tufA gene consisted of two exons (1–612; 868–1722) and one intron (613–867). The intron was located in the region encoding the mature polypeptide. In other nuclear tufA encoding chloroplast EF-Tu proteins the open reading frame was not interrupted by any intervening sequence [2,20,32]. However, there are two small introns in chloroplast encoded tufA gene in Euglena gracilis [21]. But in Arabidopsis thaliana and maize, several introns were present in tufM gene encoding mitochondrial translation elongation factor [8]. The present study is the first report to show the presence of intervening sequence in the coding region of chloroplast EF-Tu encoded by nuclear tufA gene in pea. An approximately 0.5 kb region of pea genomic DNA fragment was PCR amplified by directional genomic walking [24]. The amplified fragment was from a region 336 bp upstream of the translation initiation codon with 200 bp overlap in the coding region. Differential expression of tufA gene The expression of tufA in pea was found to be more in leaves than in roots (data not given). When the role of light in the induction of the pea chloroplast translation

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elongation factor was studied it was found that both the transcript as well as its protein levels were less in the dark-grown leaves when compared with the light-grown leaves. The dark-grown pea plants when transferred to light resulted in a gradual increase in transcript as well as protein levels of EF-Tu (Fig. 4) indicating the role of light in the regulation of higher plant tufA expression and accumulation of EF-Tu protein. It has been previously shown that light regulates the expression of chloroplast translation apparatus, EF-G [5], EF-TS [11], EFTu [31], IF-2 [12], and IF-3 [17] in Euglena. The presence of conserved cis acting elements like I-box, GATA-box, and/or GT1 consensus sequences in the 50 -flanking region (Fig. 1) for the light regulated gene expression of tufA gene substantiates the observation of light induced up-regulation of tufA transcript as shown in Fig. 4. As we had isolated the pea tufA gene while screening for genes differentially expressed during salinity stress, we investigated the effect of salinity stress on the expression levels of EF-Tu mRNA. To compare the results expression levels of two other genes viz. Nuclear encoded plastid specific pea ribonucleoprotein (Accession No. Y14557) and pea Calnexin (Accession No. Y17329) were used as controls in Northern analysis in addition to 18S rRNA as loding control. Northern and Western analysis showed a decrease in EF-Tu transcript as well as protein level was dependent on the concentration and duration of salinity stress treatment. The decrease was noticed on the exposure of plants to 200 mM NaCl for 6 h and to 100 mM for 24 h (Fig. 5). Unlike salinity, low temperature treatment resulted in an increase in the tufA transcript and protein with increase in time. The mechanism by which salt and low temperature bring about opposite responses is not clear. However, it was found that when the pea seedlings were exposed to different concentrations of abscisic acid (ABA) the expression levels of the EF-Tu transcript as well as its protein decreased similar to the plants that were exposed to the salinity stress (Fig. 5). However, with the treatment of salicylic acid (SA) the expression of EF-Tu transcript as well as its protein increased (Fig. 5) as in cold treated tissues. ABA regulates the promotion of seed desiccation and dormancy and mediates physiological responses of plants to environmental stresses such as drought-, salt-, hypoxic-, and cold stress, and wound or pathogen responses [19,27,30]. Salicylic acid (SA) plays an important role in defense response to biotic stress in plants; however, several studies also support the role of SA in modulating the plant response to several abiotic stresses [4,9,29]. In all these experiments plastid-specific 33RNP was more or less constant in all the stress treatments but the expression levels of the calnexin were down-regulated. The expression of tufA thus provides an interesting model to study the role of ABA and SA in salinity and cold stress mediated signal pathways. The presence of the regulatory elements such

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Fig. 3. Immuno-cytological localization of EF-Tu in protoplasts isolated from pea leaves. Following isolation, protoplasts were fixed with 3.7% formaldehyde on poly-lysine-coated cover slip and subjected to immuno-staining with rabbit polyclonal antibodies, raised against purified EF-Tu, and Alexa-fluor 568-conjugated goat antirabbit IgG using standard procedures. The cells were counterstained with DAPI. Confocal laser scanning (Radiance 2100, Bio-Rad, USA) was performed using Nikon microscope (objective Plane Apo 60X/1.4 oil, Japan) (C). DAPI fluorescence (B) was detected through red diode (nm). Auto-fluorescence of chlorophyll (red) was excited by blue diode (405 nm) and detected through emission filter HQ442/45 (A). The superimposition of images of A, B, and C resulted in D. Note that the immunofluorescence is localized in the chloroplasts only (D). Images were processed using LazerShop and PhotoShop 5.0 (Adobe Systems). c, chloroplasts; n, nucleus.

Fig. 4. Northern and Western analysis of pea tufA transcript and EFTu protein expression in pea seedlings exposed to light for different durations. (A) Effect of light on the expression of pea tufA transcript. Pea seedlings were grown in the dark and then exposed to light for different durations. Lane ‘0,’ etiolated seedlings grown continuously in the dark; lane ‘L,’ green seedlings grown continuously in light. Lanes ‘2,’ ‘4,’ ‘6,’ and ‘8’ etiolated seedlings grown in dark for 8 days and then exposed to light for 2, 4, 6, and 8 h, respectively. In each panel tufA represents the tufA transcript signal (after probing with tufA cDNA) and 18S rRNA represents the signal obtained after stripping off the probe and rehybridizing the same blot with 18S rDNA from pea. (B) Western analysis of total proteins extracted from leaf samples, which were collected at various time intervals after exposing to light, using EF-Tu antibodies.

as MYB and ASF1 in the 50 -flanking region (Fig. 1) of pea tufA is also found in the upstream sequences of many genes that show regulated expression in response to dehydration, abscisic acid, and salicylic acid [1,6]. The modulated differential expression of tufA gene under different abiotic stresses (Fig. 5) probably indicates the concerted action of different regulatory elements found in the upstream region of tufA gene (Fig. 1).

Conclusions We have sequenced and characterized the cDNA and genomic clones encoding for a chloroplast translation elongation factor (EF-Tu) from pea. The deduced amino acid sequence analysis revealed that pea EF-Tu has 75–80% identity with other chloroplast EF-Tu

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Fig. 5. Northern and Western analysis of pea tufA transcript and EF-Tu protein expression in pea seedlings exposed to different stress treatments (A) Northern analysis of total RNA isolated from leaf samples, which were collected at various time intervals after the stress treatment and probed using 32 P-labeled DNA probes for pea tufA, 33RNP (Accession No. Y14557), Calnexin (Accession No. Y17329), and 18S rRNA. Pea plants were exposed to different levels of sodium chloride (NaCl), abscisic acid (ABA), cold temperature, 4 °C (cold), and salicylic acid (SA). Figures below represent the length of exposure of the pea plants to NaCl. Figures on the top represent the concentration of NaCl and ABA used for NaCl- and ABA-stressed plants, respectively. For Cold- and SA-stressed plants the figures on the top represent length of exposure, in hours, to the respective stresses. (B) Western analysis of total proteins extracted from leaf samples, which were collected at various time intervals after the stress treatment, using EF-Tu antibodies.

sequences reported in the database. Using polyclonal antibodies raised against the recombinant EF-Tu and employing immunofluorescence and confocal laser scanning microscopy EF-Tu was localized within the chloroplast. The transcription of tufA gene was differentially regulated under different environmental conditions. Northern and Western analysis revealed that under higher salt and ABA concentration the expression of tufA decreased whereas under light, low temperature, and in the presence of salicylic acid the tufA expression levels increased indicating that EF-Tu could play an important role in plant adaptation to environmental stress in addition to its role in peptide elongation.

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Acknowledgments The Confocal Microscope Facility at ICGEB, New Delhi, is funded through an International Senior Research Fellowship of the Wellcome Trust (UK).

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