Elongation factor 1α from A. thaliana functions as molecular chaperone and confers resistance to salt stress in yeast and plants

Plant Science 177 (2009) 156–160

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Elongation factor 1a from A. thaliana functions as molecular chaperone and confers resistance to salt stress in yeast and plants Dongjin Shin, Seok-Jun Moon, Sang Ryeol Park, Beom-Gi Kim, Myung-Ok Byun * Bio-crops Development Division, National Academy of Agricultural Science, 224 Suin-ro, Suwon 441-857, Republic of Korea

A R T I C L E I N F O

A B S T R A C T

Article history: Received 30 March 2009 Accepted 4 May 2009 Available online 15 May 2009

Salt stress is a major environmental factor influencing plant growth and development and plants have many tolerance mechanisms to overcome salt stress. To identify salt tolerance determinants in higher plants, an Arabidopsis cDNA clone that encodes translation elongation factor 1 alpha (AtEF1a) was isolated by functional complementation of the salt-sensitive phenotype of a calcineurin (CaN)-deficient yeast mutant (cnbD). AtEF1a displayed a chaperone activity in a dose-dependant manner in vitro and the chaperone activity of AtEF1a was required for NaCl tolerance in cnbD cells. When compared with wildtype Arabidopsis, AtEF1a knock-out plants were more sensitive to NaCl stress. Furthermore, transgenic plants with transgene AtEF1a were more tolerant to NaCl than the wild-type. These results suggest that AtEF1a functions as molecular chaperone, and this activity enhances NaCl tolerance in yeast and plants. ß 2009 Elsevier Ireland Ltd. All rights reserved.

Keywords: Chaperone Salt stress Elongation factor

1. Introduction Plants frequently encounter environmental stresses, external conditions that adversely affect growth, development, or productivity [1]. When plants are exposed to excessive salinity, apoplastic levels of Na+ and Cl alter the aqueous and ionic thermodynamic equilibria resulting in hyperosmotic stress, ionic imbalances, and toxicity. Therefore, survival and growth depend on the ability of the plant to re-establish cellular osmotic and ionic homeostasis in order to adapt to the environmental stress [2]. Many ions found in cells adversely affect metabolic processes when present at high concentrations, possibly by binding to and altering the properties of cofactors, substrates, membranes, and enzymes. Furthermore, many ions can enter the hydration shell of a protein and promote its denaturation. Ions such as Na+ and Cl can penetrate these shells and interfere with noncovalent interactions that maintain the structure of the protein. In contrast, compatible solutes such as proline and glycine-betaine tend to be neutrally charged at physiological pH, as they are either nonionic or zwitterionic (dipolar, with spatially separated positive and negative charges). Such osmolytes are excluded from the hydration shells of macromolecules and, therefore, do not come into direct contact with proteins. The organic character of these osmolytes probably reflects the potential toxicity of concentrated inorganic solutes [3]. Some osmolytes behave as chemical chaperones by promoting the correct refolding of unfolded proteins in vitro and in the cell [4].

* Corresponding author. Tel.: +82 31 299 1720; fax: +82 31 299 1722. E-mail address: [email protected] (M.-O. Byun). 0168-9452/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2009.05.003

Osmolytes such as proline, trehalose, and glycine-betaine may also protect native proteins from heat denaturation and favor the formation of native protein oligomers. Trehalose accumulation in yeast suppresses protein aggregation during heat shock, but also interferes with chaperone-assisted protein refolding in vivo and in vitro [5,6]. Some plant small heat shock proteins (smHSPs) are produced during seed development or water stress. Accumulation of Ha-HSP17.6 and Ha-HSP17.9 has been detected in mildly water-stressed sunflowers. Expression of the At-HSP17.6A gene is regulated by heat shock and osmotic stress, and overexpression of At-HSP17.6A in Arabidopsis thaliana confers higher osmotolerance [7]. Although combined salt and heat stresses are frequent in nature, little is known about how osmolytes control protein stability and chaperone-mediated protein refolding in the cell [8]. Here, we present evidence that AtEF1a displays a dose-dependent chaperone activity in vitro and its activity is correlated with tolerance to salt stress in yeast. We further show that transgenic plants overexpressing AtEF1a are more tolerant to NaCl than wildtype plants. Therefore, we propose that AtEF1a functions as molecular chaperone in the cell, and that this activity is required for NaCl tolerance in yeast and plants. 2. Materials and methods 2.1. Plant, yeast and E. coli strains A. thaliana (ecotype Columbia) was grown in a growth chamber at 22 8C under conditions of 70% relative humidity and a 16 h light/8 h dark cycle. The Saccharomyces cerevisiae strain YP9 (cnbD, Mata ura3

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leu2 his3 trp1 ade2 lys2 cnb1::HIS3, isogenic to YPH499) was used for complementation assays [9]. The Escherichia coli strain XL1-Blue MRF was used for cloning (Stratagene, La Jolla, USA) and expression of GST-fusion proteins was performed in E. coli BL21 (pLys S) DE3 cells. 2.2. Spot assay and liquid assay The full-length and truncated forms of AtEF1a were subcloned into pYES2, an expression vector containing the GAL1 galactose inducible promoter and the URA3 selection marker. The constructs were introduced into the YP9 strain using the LiOAc method [10], and the transformed cells were plated on SC-Ura plates. The transformed cells were grown in YPD medium overnight, and aliquots (2 mL) from an exponentially growing culture at an O.D.600 of 0.1 were serially diluted (1:10, 1:100, 1:1000) and spotted onto YPGal (1% yeast extract/2% peptone/2% galactose) plates containing NaCl (1.1 M) [11]. Growth was examined after 3 days at 30 8C. For liquid assays, the transformed cells were grown in YPD medium overnight and inoculated to an O.D.600 of 0.1 into YPGal medium with or without NaCl (0.8 M) at 30 8C. The growth rate was measured after 30 h [12]. 2.3. Construction and purification of recombinant proteins Constructs to express GST fusion proteins were made using a pGEX2T vector (Amersham Biosciences, USA). Full length or deleted forms of AtEF1a were obtained by PCR using primers that corresponded to each end of the desired sequence and inserted into the vector. All of the expression constructs were validated by DNA sequencing. Expression of the GST fusion proteins was induced with isopropyl-b-D-thiogalactopyranoside (IPTG) using E. coli (BL21 strain). Proteins were purified with glutathione–Sepharose 4B beads (Amersham Biosciences) following the manufacturer’s protocols [13]. The protein concentration was determined using the Bradford method. 2.4. Thermal aggregation of malate dehydrogenase The chaperone activity of the recombinant proteins was measured by using malate dehydrogenase (MDH) as a substrate. A solution containing 3 mM MDH in 50 mM HEPES, 50 mM KCl, 10 mM (NH4)2SO4, 2 mM potassium acetate (pH 7.0), was incubated in a cuvette at 43 8C in the absence or presence of recombinant protein [14]. Turbidity due to substrate aggregation was monitored by measuring the light scattering at A360 in a DU650 spectrophotometer (Beckmann, USA) equipped with a thermostatic cuvette holder. 2.5. Isolation of a T-DNA insertion mutant A T-DNA insertion mutant was obtained from the ABRC (Columbus, OH); SALK_050704. To obtain a T-DNA insertion line containing a single integration, the segregation ratio of kanamycin resistance (KanR) was examined, and homozygous sublines were established from those segregating at a 3:1 ratio of KanR:KanS. Genomic DNA was isolated from the sublines and the integration of the T-DNA element at the annotated site was confirmed by sequencing PCR fragments using GSP1 (50 -TATCTCTTATGTGATTATTGCTTCAAATTG-30 ) and GSP2 (50 -ATCTCCAAATAGGTTCAAAATTGATAAC-30 ) or by RT-PCR analysis using GSP3 (50 -TTGCGTGAGATTTGTCTGAAAAGTA-30 ) and GSP4 (50 -TCATGTTCTTGATGAAATCACGATGAC-30 ) [15]. 2.6. Generation of transgenic plants with transgene AtEF1a For generation of transgenic plants with transgene AtEF1a, AtEF1a cDNA was cloned into the binary plant expression vector

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pCAMBIA1302 under control of the cauliflower mosaic virus 35S promoter. Wild-type A. thaliana (Col-0) plants were transformed with an Agrobacterium tumefaciens strain LBA4404 harboring a 35S:AtEF1a:GFP construct. Transformed seedlings were selected on MS plates with hygromycin. And to confirm the expression of transgene, total protein in Arabidopsis leaves were extracted in 30 mM Tris, pH 8.8, containing 1 mM EDTA and 1 plant protease inhibitor mixture (Sigma) and separated on a SDS/10% PAGE gel. Then they were blotted onto a Nylon membrane (Hybond-C Extra, Amersham Pharmacia) by using a semidry electro-blotter. AntiGFP monoclonal antibody was used for detection by using a standard protocol [10]. 2.7. Root growth assay To measure root length, seeds of Wild-type (ecotype Col-0), TDNA insertion mutant, and overexpressing AtEF1a transgenic (T2) A. thaliana plants were plated on MS medium. Plates containing seeds were held at 4 8C for 4 days and then transferred to a growth chamber (22 8C under a 16 h light/8 h dark regime) in a vertical orientation. Seedlings grown for 5 days were transferred to plates containing MS medium with or without NaCl. Seedlings were incubated in the growth chamber for an additional 7 days and then root length was measured [16]. Three replicate plates were used for each treatment to ensure reproducibility of the data. 3. Results 3.1. Isolation of a translation elongation factor 1a clone that confers increased salt tolerance to a yeast CaN null mutant To identify plant genes that can suppress the salt-sensitive phenotype of yeast cnbD, we transformed the cells with an A. thaliana cDNA library containing inserts under the ADH1 promoter. After plating 2.5  105 transformants on YPD medium supplemented with 1.1 M NaCl, a total of 34 colonies that showed remarkable NaCl tolerance were obtained [9]. Through this functional screening analysis, a truncated form of an Arabidopsis cDNA clone that encodes translation elongation factor 1a (AtEF1a, accession no. At1g07930) was isolated. In order to analyze whether AtEF1a was able to confer tolerance to salt stress in yeast cells, we constructed full length and truncated (24 amino acid deletion) AtEF1a in the episomal plasmid pYES2. Tranformants harboring the full length or truncated form of AtEF1a suppressed the NaClsensitive phenotype of cnbD cells (Fig. 1). 3.2. AtEF1a functions as a molecular chaperone and this property is important for tolerance to salt stress in yeast Some reports have suggested that prokaryotic translation elongation factors function as chaperones in vitro [17] and that some osmolytes behave as chemical chaperones by promoting the correct refolding of unfolded proteins in vitro and in the cell under stress conditions [4,6]. We hypothesized that Arabidopsis AtEF1a functions as a molecular chaperone. To examine this possibility, we purified a GST–AtEF1a fusion protein and in vitro chaperone activity was measured using MDH as a substrate [8]. The recombinant GST–AtEF1a displayed a dose-dependent chaperone activity in vitro, whereas GST alone did not (Fig. 2). Next, to test which domain is critical for the chaperone function, we constructed a series of deletion clones of AtEF1a, purified these proteins, and measured their chaperone activity. All the deletion forms of AtEF1a functioned as molecular chaperones. Interestingly, DS3, in which both the N- and C-terminal regions of AtEF1a are truncated, displayed a significantly higher chaperone activity than the full length AtEF1a or other deleted forms of

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of deletion clones of AtEF1a in the pYES2 vector. We tested for a galactose-dependent salt tolerant phenotype and found that transformants harboring full length and deleted forms of AtEF1a rescued the NaCl-sensitive phenotype of cnbD cells. Moreover, expression of DS3, which had higher in vitro chaperone activity than other forms, strongly increased the tolerance to salt stress (Fig. 3). 3.3. AtEF1a knock-out plant has a salt-sensitive phenotype whereas transgenic plants with transgene AtEF1a have a salt tolerant phenotype Fig. 1. Expression of AtEF1a rescues the salt-sensitive phenotype of cnbD mutant. The YP9 yeast strain was transformed with control vector (pYES2), wild-type AtEF1a, or truncated form of AtEF1a. Transformed cells were tested for tolerance to 1.1 M NaCl as described in Section 2, and plates were photographed after incubation at 30 8C for 3 days.

To investigate the physiological function of AtEF1a in plants, a knock-out line containing a single T-DNA insertion in the AtEF1a locus was identified (SALK_050704) by searching the Arabidopsis database and homozygous plants were screened by PCR analysis and RT-PCR using specific primers (data not shown). To assess the effect of the AtEF1a knock-out on stress tolerance, knock-out mutant plants were exposed to NaCl stress. The AtEF1a knock-out plants have a slight growth defect under normal conditions, but were severely sensitive to NaCl stress (Fig. 4). An AtEF1a–GFP fusion protein was overexpressed under the constitutive CaMV 35S promoter in Arabidopsis plants for a gain-offunction analysis of AtEF1a. Two independent transgenic lines with different levels of AtEF1a–GFP expression were analyzed by Western blot analysis (Fig. 5). The growth and size of the 35S::AtEF1a:GFP plants were very similar to 35S::GFP WT plants. To assess the effect of AtEF1a overexpression on stress tolerance,

Fig. 2. AtEF1a functions as molecular chaperone. 3 mM MDH in 50 mM HEPES (pH 7.0) was incubated in a spectrophotometer cell at 43 8C with 10 mM GST (&), 3 mM (&), 6 mM (*), or 10 mM AtEF1a (*). Substrate aggregation was monitored by measuring the light scattering at A360.

AtEF1a in vitro (Fig. 3). These data suggest that the eukaryotic translation elongation factor 1a functions as a molecular chaperone, and that the central domain of AtEF1a is essential for chaperone activity in vitro. To analyze whether the chaperone activity of AtEF1a is correlated with NaCl tolerance in yeast, we constructed a series

Fig. 3. Deletion analysis of AtEF1a. (A) Illustration of deletion protein of AtEF1a. Solid bars present the deletion clones originating from AtEF1a. The number indicates the position of amino acids. (B) 3 mM MDH was incubated in a spectrophotometer cell at 43 8C with 10 mM GST, 3 mM AtEF1a, DS1, DS2, or DS3. (C) Growth rate was compared in minimal medium with or without 0.8 M NaCl. Liquid growth assays were performed after 30 h of growth and is shown as a percentage of relative growth.

Fig. 4. The AtEF1a knock-out mutant is sensitive to salt stress. (A) Schematic presentation of the gene structure of the AtEF1a knock-out obtained from the ABRC. The closed triangle indicates the T-DNA insertion position. (B) Root growth of Col-0 and AtEF1a knock-out plants (atef1a). 5-Day-old seedlings were transferred to MS medium supplemented with or without NaCl. Plates were photographed after an additional 7 days in culture. (C) 5 days seedlings grown, Col-0 (&), atef1a (*), on MS agar plates placed vertically were transferred to MS medium supplemented with or without different concentrations of NaCl. Root growth was measured after an additional 7 days of growth.

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Fig. 5. Transgenic plants overexpressing AtEF1a are salt tolerant. (A) Western blot analysis of AtEF1a. Total protein (100 mg) from wild-type and AtEF1a transgenic plants were used for Western blotting with an anti-GFP antibody. (B) Root growth of wild-type and transgenic plants. 5-Day-old seedlings were transferred to MS medium supplemented with or without 100 mM NaCl. Plates were photographed after an additional 7 days in culture. (C) 5 days seedlings grown, wild-type (&), 3-1 lines (*), 8-5 lines (*), on MS agar plates placed vertically were transferred to MS medium supplemented with or without different concentrations of NaCl. Root growth was measured after an additional 7 days of growth.

35S::AtEF1a:GFP plants were exposed to NaCl stress. Transgenic plants with transgene AtEF1a were more tolerant to NaCl than the wild-type (Fig. 5). These results indicate that one function of AtEF1a in plants may be to protect salt-sensitive proteins from salt stress conditions by acting as a molecular chaperone. 4. Discussion In this paper, we have presented evidence that AtEF1a protects cellular proteins under salt stress conditions in plants and yeast. Our data demonstrate that (i) AtEF1a specifically rescues the NaClsensitive phenotype of a CaN-deficient yeast mutant, (ii) AtEF1a functions as molecular chaperone in vitro, and (iii) transgenic plants overexpressing AtEF1a showed enhanced NaCl tolerance, whereas AtEF1a knock-out plants were severely affected by NaCl stress. EF1a and EF-Tu have been intensely studied for many years in relation to their role in translation. In this series of reactions, EF1a promotes codon-directed binding of aminoacyl-tRNA (aa-tRNA) in the ribosome [18]. Expression of AtEF1a suppressed the NaClsensitive phenotype of yeast cnbD cells. Therefore, we first hypothesized that overexpression of AtEF1a confers salt tolerance by promoting protein synthesis in yeast. To test this possibility, we used two point mutants of AtEF1a generated by site-directed mutagenesis; N153T and D156E. Previous structural and mutational studies of EF1a have supported the importance of the N135KXD138 motif in translation. Structurally, this motif is important for binding and recognizing the guanine ring in GTP, and mutations in this EF1a motif reduce both the fidelity of

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translation and the requirement for nucleotide exchange [19,20]. However, there is no relationship between this motif and salt tolerance (data not shown). This result suggests that AtEF1a suppresses the salt-sensitive phenotype of cnbD cells by a mechanism distinct from its role in protein synthesis. In animal and plant cells, under normal conditions, ER stress proteins may serve as protein chaperones for proteins exported from the ER [21,22]. During stress, the induction of ER stress proteins may block translation and limit the accumulation of abnormal proteins in the cell [23]. EF1a is a positive regulator of ER stress-induced apoptosis and expression of eEF1A-2 protects caspase-3-mediated apoptosis in myotubes [24,25]. Recent reports indicate that EF1a, the eukaryotic counterpart of EF-Tu, as well as EF-Tu itself, may participate in the degradation of N-terminally blocked proteins by the 26S proteasome [26,27]. EF-Tu has a chaperone-like activity which promotes refolding of denatured rhodanese [17]. These results are particularly intriguing in that they suggest that EF-Tu or EF1a may interact with a partially unfolded protein and that the peptide elongation factor may serve as a chaperone which binds to ubiquitin conjugated proteins. In this study, we also observed that AtEF1a has a dose-dependent chaperone activity in vitro. High temperature, salinity, and drought stress can cause denaturation and dysfunction of many proteins [28]. Some osmolytes behave as ‘‘chemical chaperones’’ by promoting the correct refolding of unfolded proteins both in vitro and in the cell [4,6]. HSP and late embryogenesis abundant (LEA) proteins help to protect against stress by controlling the proper folding and conformation of both structural and functional proteins [29]. A significant osmoprotective effect was obtained in E. coli transformed with the cytosolic chaperonin CCP1a from Bruguiera sexangula [30]. In plants, Rausell et al. demonstrated that overexpression of the sugar beet eIF1A specifically increased the tolerance of yeast to sodium and lithium salts, and transgenic Arabidopsis plants expressing BveIF1A exhibited increased tolerance to NaCl [31]. The los1–1 mutant, which encodes an EF2, affected the functionality of this elongation factor specifically at low temperatures, but not at high temperatures [32]. The chaperone activity of EF1a was correlated with its phenotype in NaCl stress in yeast in this study. Therefore, we have expected transgenic plants with transgene AtEF1a have strongly tolerance phenotype under salt stress condition. But transgenic plants with transgene AtEF1a possessed a little weak or more a salt tolerant phenotype. We thought there is a possibility that AtEF1a gene is highly expressed independent of stress conditions. However, AtEF1a knock-out plants possessed a salt-sensitive phenotype. Therefore, we propose that protein synthesis factors readily respond to stress conditions and that AtEF1a functions as ‘‘molecular chaperone’’ by protecting native proteins under salt stress conditions both in vitro and in vivo. Acknowledgments This work was supported by the National Academy of Agricultural Science, the Rural Development Administration (RDA), and a grant (CG3134) from the Crop Functional Genomics section of the 21st Century Frontier Research Program, Ministry of Science, Republic of Korea. This study was supported by 2009 Postdoctoral Course Program of the National Academy of Agricultural Science, RDA, Republic of Korea. References [1] K. Yamaguchi-Shinozaki, K. Shinozaki, Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses, Annu. Rev. Plant Biol. 57 (2006) 781–803.

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