OsACA6, a P-type IIB Ca2+ATPase promotes salinity and drought stress tolerance in tobacco by ROS scavenging and enhancing the expression of stress-responsive genes

Authors


Summary

Calcium (Ca2+) regulates several signalling pathways involved in growth, development and stress tolerance. Cellular Ca2+ homeostasis is achieved by the combined action of channels, pumps and antiporters, but direct evidence for a role of Ca2+ATPase pumps in stress tolerance is lacking. Here we report the characterization of a Ca2+ATPase gene (OsACA6) from Oryza sativa, and elucidate its functions in stress tolerance. OsACA6 transcript levels are enhanced in response to salt, drought, abscisic acid and heat. In vivo localization identified plasma membranes as an integration site for the OsACA6–GFP fusion protein. Using transgenic tobacco lines, we demonstrate that over-expression of OsACA6 is triggered during salinity and drought stresses. The enhanced tolerance to these stresses was confirmed by changes in several physiological indices, including water loss rate, photosynthetic efficiency, cell membrane stability, germination, survival rate, malondialdehyde content, electrolyte leakage and increased proline accumulation. Furthermore, over-expressing lines also showed higher leaf chlorophyll and reduced accumulation of H2O2 and Na+ ions compared to the wild-type. Reduced accumulation of reactive oxygen species (ROS) was observed in transgenic lines. The increased proline accumulation and ROS scavenging enzyme activities in transgenic plants over-expressing OsACA6 efficiently modulate the ROS machinery and proline biosynthesis through an integrative mechanism. Transcriptional profiling of these plants revealed altered expression of genes encoding many transcription factors, stress- and disease-related proteins, as well as signalling components. These results suggest that Ca2+ATPases have diverse roles as regulators of many stress signalling pathways, leading to plant growth, development and stress tolerance.

Introduction

Calcium (Ca2+) is a ubiquitous intracellular second messenger that is involved in stress signalling pathways (Kaplan et al., 2006; Tuteja and Mahajan, 2007; Tuteja, 2007; Mahajan et al., 2008). Stress triggers an increase in cytosolic Ca2+, leading to aggregation of proteins and nucleic acids and precipitation of phosphates, together with disintegration of membrane lipids, which lead to cell death (Knight and Knight, 2001; Case et al., 2007). Plants have developed several mechanisms to help them to maintain cytosolic Ca2+ levels and adapt to the changing environment. These include increased export of Ca2+ from the cell or other intracellular organelles to maintain its concentration (Dodd et al., 2010). Several Ca2+ sensors, including calmodulin, calcineurin B-like proteins and calcium-dependent protein kinases, sense this increase and induce physiological responses related to stress (DeFalco et al., 2010; Conde et al., 2011).

The influx of Ca2+ into the cytosol is countered by pumping Ca2+ out from the cytosol to restore the basal cytosolic level, and this may be achieved either by P-type Ca2+ATPases or antiporters. Ca2+ATPases are high-affinity but low-capacity transporters, whereas antiporters are low-affinity but high-capacity transporters (Bose et al., 2011). Both animal and plant cells use two types of Ca2+ATPases: type IIA and type IIB. Type IIB Ca2+ATPases effectively senses Ca2+ signals through an N-terminal calmodulin-binding domain (Hwang et al., 2000). In addition to efflux mechanisms, Ca2+ATPases also play widespread physiological roles in several other processes, such as pollen development, stomatal opening/closing, reproductive and pollen tube growth, vegetative and inflorescence architecture and gibberellin signalling (Schiøtt et al., 2004; George et al., 2008; Wang et al., 2011; Lucca and León, 2012). They also modulate biotic stress by activating various components of signalling pathways (Zhu et al., 2010; Shabala et al., 2011a,b; Frey et al., 2012). There are only indirect data regrading the expression of type IIB Ca2+ATPases in response to abiotic stress (Wimmers et al., 1992; Huda et al., 2013a), and direct evidence for a role of Ca2+ATPases in abiotic stress responses is lacking. The Arabidopsis type IIB Ca2+ATPase encoded by AtACA4 is a CaM-regulated Ca2+ATPase that participates in the Ca2+-dependent signal transduction pathway associated with salt stress (Geisler et al., 2000). In Physcomitrella patens, an increased PCA1 mRNA level was observed in response to salt, dehydration and abscisic acid (ABA), while analysis of mutants revealed enhanced vulnerability to salt stress, providing evidence that type IIB Ca2+ATPases generate a salt-induced Ca2+ signature (Qudeimat et al., 2008). Type IIA Ca2+ATPases, such as those encoded by AtECA1 (Wu et al., 2002) LCA1 (Johnson et al., 2009) and AtECA3 (Mills et al., 2008), maintain correct concentrations of Ca2+, Mn2+ and Zn2+ in the cytoplasm.

In this study, we report the isolation and characterization of OsACA6, which encodes a member of the type IIB Ca2+ATPase family from rice. Its expression was enhanced in response to abiotic stresses. Using transgenic plants expressing the GUS reporter gene under the control of a native OsACA6 promoter, we studied transcriptional activation of OsACA6 during abiotic stresses. The transcriptome of OsACA6 over-expressing plants was analysed by microarray analysis. The experimental data demonstrate that over-expression of OsACA6 confers tolerance to multiple stresses in transgenic plants, which correlated with reduced accumulation of reactive oxygen species (ROS) in plants.

Results

Cloning and analysis of OsACA6 from rice

Gene-specific primers were used for amplification of full-length cDNA of the OsACA6 gene (LOC_Os04 g51610) from a rice cDNA library. The sequence analysis revealed that it is a type IIB Ca2+ATPase with very high homology to those of other monocot species (Oryza sativa and Zea mays) and dicot species (Glycine max and A. thaliana). The open reading frame (ORF) of OsACA6 cDNA (3267 bases) encodes a protein of 1088 amino acid residues, with a molecular mass of 117.66 kDa and a pI of 7.00. OsACA6 has >87% identity to OsACA1, OsACA4 and OsACA9–OsACA12, and 55% identity to members of the type IIB Ca2+ATPase family from dicot species, including AtACA1, AtACA2, AtACA4, AtACA7–AtACA11 and GmSCA1. It shows very low homology to members of the type IIA Ca2+ATPase family from rice and Arabidopsis.

Multiple sequence alignments revealed that OsACA6 contains nine conserved motifs, and the encoded protein has a calmodulin-binding site in the N-terminal region (Figure S1). It also contains ten highly conserved transmembrane domains. OsACA6 also possesses the P-domain (DKTGTLT), of which ‘D’ is phosphorylated during the catalytic cycle. It also has an A-domain (TGES) and an N-domain (KGAPE) (Figure S1). The prediction of secondary structure identified ten transmembrane α-helices and 19 cytosolic β-pleated sheets (Huda et al., 2013a). OsACA6 has 34 exons, similar to AtACA8 and AtACA10, and the presence of identical exon/intron borders among them suggested that these genes have a conserved structure in land plants (Figure S2).

Expression of OsACA6 is induced by salt, dehydration, heat and ABA

To analyse the expression profiles of OsACA6, quantitative RT-PCR was performed. The results showed an increased level of OsACA6 transcripts induced by salt, drought, ABA and heat stress (Figure 1). OsACA6 expression was significantly activated by salt, drought and ABA stresses, but weakly by heat. The expression patterns and maximum expression differed for each stress. The expression levels peaked at 3 h for NaCl, 24 h for drought, 6 h for ABA and 12 h for heat, with corresponding maximum increases of 30-, 47-, 45- and 12-fold, respectively, compared to the control (non-stress condition). Although decreases were observed at various time points, the expression levels of OsACA6 were always higher compared to control (Figure 1). These results suggest that OsACA6 may be involved in stress adaptation.

Figure 1.

Expression pattern of OsACA6 in response to various stress treatments such as salt, drought, heat and abscisic acid.

Two-week-old rice seedlings were exposed to abiotic stresses as described previously (Huda et al., 2013a). The 2−ΔΔCT method (Livak and Schmittgen, 2001) was used to measure relative expression of target genes in stressed and non-stressed leaves. Values are means ± standard errors from three independent measurements.

Expression analysis using OsACA6 promoter–GUS fusions in transgenic plants

In transgenic tobacco plants expressing a GUS reporter gene driven by the OsACA6 promoter, GUS activity was observed in the leaves, shoot and roots. Transgenic plants in which GUS reporter gene expression was driven by the CaMV 35S promoter and wild-type tobacco were used as positive and negative controls, respectively. No expression was observed in water-treated wild-type controls, but expression was detected in plants in which GUS expression was driven by the CaMV 35S promoter (Figure S3). Strong GUS expression was observed upon treatment with salt or poly(ethylene glycol) (PEG) in OsACA6 promoter driven transgenic plants, but no GUS expression was observed in wild-type plants (Figure S3).

OsACA6 is localized to the plasma membrane

Previous studies have indicated that type IIB Ca2+ATPases are localized to the plasma membrane as well as endomembranes. To assess its localization, OsACA6 was fused with GFP at its C-terminus, and a clone with GFP alone was used as a control. These constructs were transiently expressed in onion epidermal cells, which were examined by confocal microscopy 48 h after transformation. GFP expression was detected in the nucleus and cytosol in the control cells, while expression of the OsACA6–GFP fusion protein was exclusively observed in the plasma membrane of onion epidermal cells (Figure 2 and Figure S4).

Figure 2.

OsACA6 resides in the plasma membranes of onion epidermal cells.

Cells were bombarded with constructs expressing GFP or OsACA6–GFP. GFP and OsACA6–GFP fusion proteins were transiently expressed under the control of the CaMV 35S promoter in onion epidermal cells and analysed by confocal microscopy.

Over-expression of OsACA6 and analysis of transgenic plants

To investigate the role of OsACA6 in stress tolerance, the gene was over-expressed in tobacco plants using the gene construct pBinAR-OsACA6 (Figure 3a). The integration of the transgene in more than 30 independent transgenic lines were confirmed by PCR. The wild-type plants were used as control. The T1 and T2 generation plants segregated in the expected ratios (3:1 and 9:3:3:1, respectively; Table 1). Analysis of the OsACA6 transgene was performed in the T1 and T2 generations. Thirteen independently transformed T2 transgenic lines were selected and allowed to grow to maturity. However, for subsequent analysis, only three PCR-positive lines (L7, L19 and L21) were chosen (Figure 3b). Most of the transgenic lines were morphologically indistinguishable from the wild-type plants. The stability and integration of the transgene was confirmed by Southern blot analysis (Figure 3c). No bands were detected in wild-type plants (Figure 3c, lane 2), and integration of single copies was observed in over-expressing lines (Figure 3c). The RNA expression level varied in transgenic plants, and quantitative RT-PCR analysis detected 23-, 33- and 22- fold increases in mRNA expression in over-expressing lines compared to control plants (Figure 3d). All these findings confirm stable integration of the OsACA6 gene into the transgenic plants. Stress responses were also observed in the T1 generation by a leaf disk assay (Figure 3e) as well as by phenotypic analysis (Figure 3f). Interestingly, we observed that the seeds of transgenic lines germinated 2–3 days earlier than the wild-type, even under non-stressed conditions, and consequently the growth of the transgenic seedlings was more vigorous (Figure 3f).

Table 1. Comparison of the segregation ratio and seed weight of wild-type and OsACA6 T1and T2 transgenic plants grown in the presence of water, 200 mm NaCl or 20% PEG
ParameterGenerationWild-type in waterTransgenics in 200 mm NaClTransgenics in 20% PEG
L7L19L21L7L19L21
  1. Each value represents mean of three replicates ±SE of control (wild-type) and transgenic plants (L7, L19 and L21). The number of plants used is indicated in parentheses. Values with different letters are significantly different from each other at < 0.05, level as determined by Duncan's multiple range test.

  2. kr:ks = ratio between resistant and susceptible plants grown in kanamycine plate. (n) = total number of plants mentioned for each line in brackets.

Segregation ratio, kr:ks (n)T103.25:1 (175)3.06:1 (145)2.88:1 (133)2.9:1 (183)3.19:1 (137)3.21:1 (167)
Segregation ratio, kr:ks (n)T209.01:2.53:3.12:1 (245)8.82:3.04:3.78:1 (265)9.21:2.65:2.88:1 (253)9.3:3.43:3.33:1 (183)8.98:2.53:3.11:1 (233)9.18:3.07:2.71:1 (176)
Seed weight per pod (mg)T1122.2 ± 2.8a118.98 ± 1.7a116.22 ± 1.9b114.32 ± 3.1b119.55 ± 2.1a121.11 ± 1.4a115.67 ± 3.8b
Seed weight per pod (mg)T2131.2 ± 3.1b125.43 ± 2.5c134.75 ± 2.3a128.32 ± 4.1b140.16 ± 3.7a128.55 ± 2.7b135.54 ± 1.8a
Figure 3.

Molecular analysis of OsACA6 over-expressing transgenic tobacco plants.

(a) Region of pBinAR containing the OsACA6 gene (pBinAR-OsACA6).

(b) PCR analysis using gene-specific primers.

(c) Southern blot analysis. Lane 1, positive control; lane 2, wild-type; lanes 3–5 show single-copy insertions of OsACA6 in transgenic tobacco plants.

(d) Determination of relative mRNA expression by RT-PCR.

(e) Leaf disk assay for stress tolerance in T1 transgenic tobacco. Disks floated on 200 mM NaCl or 20% PEG showed chlorophyll retention in transgenic lines, whereas wild-type plants showed bleaching of leaf disks compared with the untreated control (water).

(f) Effect of OsACA6 over-expression on salinity- and PEG-induced dehydration tolerance in T1 transgenic plants.

OsACA6 transgenic plants have improved biological traits for abiotic stress

We measured various physiological traits important for stress responses, such as water loss, relative water content, chlorophyll fluorescence and cell membrane stability (Figure 4). At each time point, reduced water loss rate was observed in transgenic lines (Figure 4a). To evaluate photosynthetic potential, transgenic lines were used for measurement of chlorophyll fluorescence, a useful parameter for screening stress responses. A smaller decrease in the Fv/Fm ratio was observed in the transgenic lines compared to the control during stress (Figure 4b). The cell membrane is an early target of any adverse environmental event. The cell membrane stability was measured under stress conditions, and a higher cell membrane stability was recorded for OsACA6 transgenic plants compared to control plants (Figure 4c).

Figure 4.

OsACA6 plants have improved physiological traits under stress conditions.

(a) Comparison of water loss between transgenic plants and wild-type.

(b) Change in fluorescence ratios (Fv/Fm) in OsACA6 transgenic lines and wild-type upon salt stress. A smaller decrease in the Fv/Fm ratio was observed for transgenic lines compared to the controls.

(c) Comparison of cell membrane stability of OsACA6 transgenic plants and controls following salt and drought stress.

Twenty separate plants were used. Values are means ± SE from three independent experiments.

Plants over-expressing OsACA6 show enhanced tolerance to abiotic stresses

To examine the function of OsACA6, transgenic tobacco plants over-expressing OsACA6 were generated. To compare germination efficiency, the seeds of wild-type plants and OsACA6 transgenic plants were grown on medium supplemented with 200 mm NaCl or 20% PEG. In media containing NaCl or PEG, the highest germination rates for wild-type were 30.2% and 31.4%, respectively (Figure 5a,b), while >80% seeds germinated for over-expressing lines. We also measured the primary root length, and observed that OsACA6-transformed plants had longer primary roots (Figure 5c,d) that help to absorb more water from deeper soils, and hence strengthen stress tolerance. To observe the performance of seedlings, the germinating seeds of wild-type plants and over-expressing lines were transferred to half-strength MS medium containing 200 mm NaCl or 20% PEG. Physiological changes were commonly observed, and OsACA6 transgenic seedlings showed significantly better growth performance than wild-type plants (Figure 5e). After 21 days, most of the wild-type plants became yellowish, their growth had stopped and they ultimately died, while transgenic lines showed higher root and shoot growth compared to control plants (Figure 5e). In 20% PEG, all the transgenic lines showed vigorous growth compared to control plants (Figure 5e). The measurement of fresh weight revealed that these stresses had no clear effect on transgenic lines, but impaired growth of wild-type plants (Figure 5f). The post-development assays were performed, and the survival rate of plants was scored. The transgenic seedlings were consistently more resistant to high salt stress compared with wild-type plants (Figure 6a). Two-week old lines over-expressing OsACA6 and wild-type plants were grown in pots and continuously treated with 20% PEG for 10 days. The rosette leaves of transgenic plants showed greater resistance than those of wild-type plants after 5 days re-watering (Figure 6b). In the presence of stress, >80% of transgenic plants survived, compared to only 35% survival for control plants (Figure 6c). We also performed a leaf disk assay, and found that the stress-induced loss of chlorophyll was lower in OsACA6 lines compared with wild-type plants (Figure 6d). The damage caused by stress was reflected by bleaching of leaf tissue after 72 h. The measurement of chlorophyll content provided further support for a positive relationship between OsACA6 expression and tolerance towards these stresses (Figure 6e). To assess the effect of stress on growth and development of OsACA6 over-expressing lines and wild-type plants, 40-day-old-seedlings were grown in the presence of 200 mM NaCl or 20% PEG. In the presence of stress, the wild-type plants showed growth retardation, whereas OsACA6 over-expressing lines did not develop any sign of stress (Figure 6f,g). Overall, the OsACA6 over-expressing lines grew normally and set viable seeds under continuous stress, and produced a yield comparable to wild-type plants grown in water (Table 1). However, wild-type plants did not survive until maturity under continuous stresses (Figure 6f,g). These findings indicate that OsACA6 over-expression promotes salt and drought tolerance in tobacco.

Figure 5.

Effect of OsACA6 on stress tolerance in transgenic tobacco.

(a,b) Germination of OsACA6 transgenic and wild-type plants in MS medium supplemented with 200 mM NaCl (a) or 20% PEG (b). The percentage of germinated seeds was calculated based on the number of seedlings that reached the cotyledon stage by 10 days.

(c) Comparison of primary root lengths of OsACA6 transgenic plants and wild-type plants. Five seeds of each line were planted on MS agar in triplicate, and root lengths were measured after 8 days.

(d) Comparison of root measurements.

(e) Salt and PEG responses of seedling growth. Germinating seedlings were transferred from MS medium as described in Experimental procedures.

(f) Fresh weights of 21-day-old seedlings. Ten seedlings were pooled as one sample, and three samples were measured for each line. Values are means ± SE.

Figure 6.

OsACA6 transgenic tobacco plants showed enhanced tolerance to salt or PEG stress.

(a,b) Phenotypes of OsACA6 transgenic lines (T2 generation) and wild-type following salt stress (a) and PEG stress (b).

(c) The survival rate of OsACA6 transgenic and wild-type plants was recorded after treatment. Values are means ± SE, and each experiment was performed in triplicate.

(d) Leaf disk assay for stress tolerance in T2 transgenic tobacco plants. Disks floated on 200 mM NaCl or 20% PEG showed chlorophyll retention in transgenic lines, whereas wild-type plants showed bleaching of leaf disks compared with the untreated control (water).

(e) Quantification of chlorophyll content from the leaf disk assay.

(f,g) Three-month-old OsACA6 transgenic plants grown in 200 mM NaCl (f) or 20% PEG (g) compared to wild-type.

Determination of ion contents and detection of H2O2 in response to abiotic stresses

Ion content is another potential contributor to the maintenance of membrane potential under saline conditions. To observe Na+ and K+ accumulation, wild-type and OsACA6 transgenic plants were exposed to salt stress for 72 h (Figure 7a). The ion content analysis (Na+ and K+) revealed that, in the absence of salt treatment, there were no apparent changes in transgenic plants compared with wild-type plants. After salt treatment, the Na+ content (Figure 7b) increased in both the leaves and roots of the transgenic and wild-type plants, but the Na+ increase was lower in transgenic tissues than in wild-type plants. Fluorescence indicators are valuable for non-destructive monitoring of the spatial and temporal distribution of Na+. In order to measure the relative concentration of Na+ in wild-type plants and over-expressing lines, the roots were pre-treated with 200 mm NaCl before loading with 100 μm CoroNa Green dye, and Na+ fluorescence were analyzed by confocal microscopy (Figure 7c). The fluorescence increased several fold upon binding of Na+ to CoroNa Green in the roots of wild-type plants. However, the fluorescence intensity was less in transgenic plants than in control plants (Figure 7c). Salt treatment also causes an increase in K+ content in transgenic plants compared with wild-type plants (Figure 7d). Further analysis indicated that the K+/Na+ ratios in the leaves and roots of transgenic tobacco plants were six- and twofold higher than in the wild-type plants, respectively, after salt treatment (Figure 7e). High H2O2 accumulation in plants results in programmed cell death. To determine the level of cellular H2O2 in over-expressing lines, histochemical staining with diaminobenzidine was performed. After 24 h treatment, the leaves of wild-type plants showed deeper staining than OsACA6 transgenic lines (Figure 7f).

Figure 7.

Ion content and detection of H2O2 in transgenic and wild-type tobacco plants.

(a) Seedlings (21 days old) were irrigated with 200 mM NaCl solution for 5 days and photographs were taken.

(b) Na+ content in leaves and roots of transgenic and wild-type plants.

(c) Comparison of Na+ contents in root tips of OsACA6 lines and wild-type plants using CoroNa Green dye. Root tips were pre-treated with 200 mM NaCl prior to observation by confocal microscopy.

(d) K+ content in leaves and roots of transgenic and wild-type plants.

(e) K+/Na+ ratio with or without salt treatment. Values are means ± SD from three independent trials.

(f) Detection of stress-induced H2O2 production by staining with diaminobenzidine.

OsACA6 over-expressing lines modulate the ROS machinery under stress

The changes in the ROS machinery induced by salinity and drought stress were confirmed by quantifying the accumulation of malondialdehyde (MDA), electrolytic leakage and H2O2 in T2 transgenic lines. This study indicates that, under stress conditions, transgenic lines show significantly less accumulation of MDA, H2O2 and ion leakage compared to wild-type plants (Figures 8a–c and 9a–c), confirming reduced damage and higher membrane integrity during the onset of stress. To further elucidate the mechanism controlling ROS machinery under stress condition, the levels of the antioxidant enzymes chloramphenicol acetyltransferase (CAT), ascorbate peroxidases (APX) and glutathione reductase (GR) were also analysed. The activities of CAT, APX and GR increased rapidly in stress-treated transgenic lines compared with wild-type plants (Figures 8d–f and 9d–f). In this study, a higher proline accumulation and relative water content were found in transgenic plants compared to wild-type, which maintains the water balance to counteract the stress (Figures 8g,h and 9g,h).

Figure 8.

Antioxidative response of OsACA6 transgenic plants under salinity stress (200 mM NaCl).

(a) Levels of lipid peroxidation expressed in terms of MDA content.

(b) Percentage of electrolytic leakage.

(c) Changes in hydrogen peroxide content.

(d) Catalase activity in transgenic plants.

(e) Changes in ascorbate peroxidase enzyme activity.

(f) Changes in glutathione reductase enzyme activity.

(g) Changes in the level of proline accumulation.

(h) Relative water content (percentage) in OsACA6 transgenic tobacco lines.

One-way analysis of variance (anova) was used to test the significance between mean values of control and transgenic plants, and comparison among mean values was performed by the Tukey–Kramer multiple comparisons test using GraphPad InStat software version 3.0 (www.graphpad.com). The difference between wild-type and transgenic lines were statistically significant at aP < 0.05, bP < 0.01 and cP < 0.001.

Figure 9.

Antioxidative response of OsACA6 transgenic plants under drought stress (20% PEG).

(a) Levels of lipid peroxidation expressed in terms of MDA content.

(b) Percentage electrolytic leakage.

(c) Changes in hydrogen peroxide content.

(d) Catalase activity in transgenic tobacco lines.

(e) Changes in ascorbate peroxidase enzyme activity.

(f) Changes in glutathione reductase enzyme activity.

(g) Changes in the level of proline accumulation.

(h) Relative water content (percentage) in OsACA6 transgenic tobacco lines.

Statistical analysis was performed on these data as described in the legend to Figure 8.

Stress-induced genes are up-regulated in transgenic plants over-expressing OsACA6

We wished to elucidate the molecular mechanism of stress tolerance mediated by OsACA6 in tobacco. Therefore, the genes that were up-regulated in OsACA6 over-expressing plants were identified using microarrays. For this assay, 21-day-old seedlings grown in a greenhouse were exposed to 200 mm NaCl. Following treatment for 6 h, mRNA levels were compared between the wild-type plants and transgenic plants. Genes predominantly encoding stress- and disease-related proteins, signalling components and transcription factors were significantly up-regulated in the transgenic plants, as summarized in Table 2. Transcripts coding two late embryogenesis abundant proteins were significantly more abundant in transgenic plants, revealing the improved tolerance of OsACA6 over-expressing lines to osmotic stress. A number of ROS-responsive genes (heat shock protein, peroxidase superfamily protein, calmodulin-binding family protein, stroma asorbate peroxidase etc.) were up-regulated, suggesting that OsACA6 expression modulates parts of ROS pathways. Interestingly, genes encoding transcription factors, such as DREB (dehydration responsive element binding), zinc finger, NAC domain, WRKY, leucine zipper (deduced from highly conserved amino acid sequence) and MYB (myeloblastosis), were also up-regulated in the transgenic plants. Genes showing enhanced salt-induced expression in the 35S:OsACA6 plants also included germine and PR-1-like proteins, an ABC transporter, H+-transporting and cadmium/zinc-transporting ATPases, ABCG33, a chromatin remodelling complex, calcium-dependent lipid-binding proteins and an NB-ARC domain-containing disease resistance protein. All these proteins are known to be involved in plant defence mechanisms.

Table 2. Transcripts significantly up-regulated in transgenic tobacco seedlings over-expressing OsACA6 compared with the wild-type under salt-stress conditions
NumberProbe nameAccession numberGene annotationFold (log2)FunctionReference
  1. The wild-type and T2 transgenic plants were grown in the greenhouse for 14 days and stressed using 200 mm NaCl for 6 h; total RNAs were extracted from seedlings to perform gene expression profiling by microarray analysis. Transcripts that were significantly elevated in transgenic plants over-expressing OsACA6 compared to wild-type plants are shown.

1A_95_P146672 EB450771 Fasciclin-like protein6.33Plant growth and developmentJohnson et al. (2011)
2A_95_P136437 EB440199 DREB-like protein5.75Regulation of expression of many stress-inducible genesReddy et al. (2011)
3A_95_P313963 FG162654 Zinc finger protein5.48Resistance mechanism for various biotic and abiotic stressesZhang et al. (2012)
4A_95_P085750 BP529365 Leucine-rich receptor-like kinase5.41Protein phosphorylationMoscatiello et al. (2006)
5A_95_P100173 BP535250 Nuclear transcription factor5.28Regulation of the defence response in ArabidopsisRen et al. (2004)
6A_95_P287248 FG195446 Methyltransferase-like protein4.72Defence responses to pathogen attackZubieta et al. (2003)
7A_95_P274218 AM798326 LEA protein4.5Plant stress toleranceOrellana et al. (2010)
8A_95_P086643 BP529592 LEA protein4.31Plant stress toleranceOrellana et al. (2010)
9A_95_P007906 EB451191 Germine-like protein4.28Defence against pathogen attackLu et al. (2010)
10A_95_P051326 BP132615 Zinc finger protein4.26Resistance mechanism for various biotic and abiotic stressesZhang et al. (2012)
11A_95_P305443 FG637875 MADS box protein4.25Regulator of developmental processesTardif et al. (2007)
12A_95_P292488 FG132900 Protein phosphatase4.16Protein phosphorylationMoscatiello et al. (2006)
13A_95_P017201 EH617604 PR-1-like protein4.11Roles in the defence responseRosales et al. (2012)
14A_95_P296488 FG160322 NAC domain protein4.1Responses to biotic and abiotic stressZhang et al. (2012)
15A_95_P189242 EB447764 MADS box protein3.92Regulator of developmental processesTardif et al. (2007)
16A_95_P064125 BP135965 RPP-13-like protein3.79Resistant to different avirulence determinantsBittner-Eddy et al. (2000)
17A_95_P304263 FG159594 Heat shock protein3.76Protection of plants against stressReddy et al. (2011)
18A_95_P052011 BP132803 WRKY DNA binding protein3.69Response to biotic and abiotic stressZhang et al. (2012)
19A_95_P269636 FG169507 Leucine zipper transcription family3.63Plant stress-responsive and hormone signal transductionReddy et al. (2011)
20A_95_P159442 EH617652 PR-1-like protein3.47Important roles in the defence responseRosales et al. (2012)
21A_95_P228269 FG157616 12-oxophytodienoate reductases3.45Role in xenobiotic detoxificationRosales et al. (2012)
22A_95_P216542 EB684025 Peroxidase superfamily protein3.34Defence and stress responsesMoscatiello et al. (2006)
23A_95_P291468 FG138785 ABC transporter protein3.33Plant development and responses to abiotic stressYazaki (2006)
24A_95_P059535 BP134765 Stroma ascorbate peroxidase3.26Defence and stress responsesMoscatiello et al. (2006)
25A_95_P284958 FG196981 MYB transcription factor3.18Plant growth, development and stress responseZhang et al. (2012)
26A_95_P305273 FG640894 Pectin methylesterase inhibitor3.18Anti-fungal activity disease resistance and stress toleranceAn et al. (2008)
27A_95_P039711 BP129626 MYB domain protein3.16Plant growth, development and stress responseZhang et al. (2012)
28A_95_P173657 EH665660 Leucine-rich receptor-like kinase3.16Protein phosphorylationMoscatiello et al. (2006)
29A_95_P122097 DW002143 CCCH-type zinc finger protein3.13Resistance mechanism for various biotic and abiotic stressesZhang et al. (2012)
30A_95_P033724 AJ632915 NAC domain protein3.13Plant responses to biotic and abiotic stressNakashima et al. (2012)
31A_95_P139712 EB443586 Receptor-like kinase3.05Protein phosphorylationMoscatiello et al. (2006)
32A_95_P157477 FS394284 DEAD box RNA helicase3.04mRNA export, development and stress responsesGong et al. (2005)
33A_95_P300558 FG170736 Nitrate transporter3.02Cadmium toleranceLi et al. (2010)
34A_95_P292633 EB427846 H+-transporting ATPase in plants3.02Stress toleranceRybchenko and Palladina (2011)
35A_95_P081950 BP528392 Lectin receptor kinase3.01Protein phosphorylationMoscatiello et al. (2006)
36A_95_P227984 FG155158 Peroxidase superfamily protein3.01Defence and stress responseMoscatiello et al. (2006)
37A_95_P294708 FG157080 Calmodulin-binding family protein2.98Cellular signalling cascades by regulation of target proteinsReddy et al. (2011)
38A_95_P232994 FG134438 Serine/threonine protein kinase2.96Protein phosphorylationMoscatiello et al. (2006)
39A_95_P139607 EB443414 Receptor-like kinases2.92Protein phosphorylationMoscatiello et al. (2006)
40A_95_P080390 BP527993 ABCG332.91Plant development, responses to abiotic stressYazaki (2006)
41A_95_P040186 BP129757 Trehalose-6-phosphate synthase2.9Responses to stressMoscatiello et al. 2006
42A_95_P040536 BP129857 Leucine-rich receptor-like kinase2.9Protein phosphorylationMoscatiello et al. (2006)
43A_95_P009246 EB451953 MADS box protein2.84Regulators of developmental processesTardif et al. (2007)
44A_95_P133547 EB435100 Leucine-rich repeat protein kinase2.84Protein phosphorylationMoscatiello et al. (2006)
45A_95_P106142 CV017771 MYB102.8Plant growth, development and stress responseZhang et al. (2012)
46A_95_P307543 FG638665 O-methyltransferase-like protein2.78Defence responses to pathogen attackZubieta et al. (2003)
47A_95_P056171 BP133890 Chromatin remodelling complex2.78Transcriptional programmes in response to environmental cuesBezhani et al. (2007)
48A_95_P090148 BP530828 MAP3Ka2.77Protein phosphorylationMoscatiello et al. (2006)
49A_95_P105342 FG132880 Catalase 22.76Plant protection against various abiotic stressesBhattacharjee (2012)
50A_95_P039756 BP129643 Calcium-dependent lipid binding2.75Acts as transcriptional regulator in response to abiotic stressReddy et al. (2011)
51A_95_P260771 FG167493 Cadmium/zinc-transporting ATPase2.7Detoxification of Zn and Cd in plantsTakahashi et al. (2012)
52A_95_P073735 BP526287 NB-ARC resistance protein2.68Plant disease resistance proteinsVan-Ooijen et al. (2008)
53A_95_P150297 EB680396 MYB124 transcription factor2.67Plant growth, development and stress responseZhang et al. (2012)
54A_95_P069850 BP137498 Global transcription factor A22.65Regulator of several types of environmental stressNishizawa et al. (2006)

Discussion

Stress alters the Ca2+ level, which triggers diverse signalling mechanisms leading to stress adaptation in plants. Plant Ca2+ATPases are assumed to be a major component in determining the shape of transient cytosolic calcium [(Ca2+)cyt] elevation in response to environmental stimuli and thus provide cellular homeostasis. Like the Arabidopsis (Huda et al., 2013a) and Physcomitrella PCA1 proteins (Qudeimat et al., 2008), the OsACA6 protein contains all the motifs of plant type IIB Ca2+ATPases, including the calmodulin-binding domain at its N-terminus. Expression of OsACA6 was induced by salt, drought, ABA and heat stresses. This is consistent with previous reports showing that Ca2+ATPase transcripts are induced by abiotic stress (Perez-Prat et al., 1992; Wimmers et al., 1992; Chung et al., 2000; Schiøtt and Palmgren, 2005; Cerana et al., 2006). Mutations in the AtACA9 gene resulted in partial male sterility, and AtACA10 mutants showed defects in growth and inflorescence architecture (Schiøtt et al., 2004; George et al., 2008). Mutant of PCA1 showed susceptibility to salt stress (Qudeimat et al., 2008). In Arabidopsis seedlings the expression of plasma membrane Ca2+ ATPases are reported to be stimulated by ABA (Cerana et al., 2006). Likewise, OsACA6 is regulated by ABA-dependent stress signalling pathways. Plant Ca2+ATPases act as positive regulators of signalling pathways as previously established in rice aleurone cells (Chen et al., 1997).

In OsACA6 promoter–GUS assays, high GUS activity was observed in tissues after induction of stress. The promoter sequence of the rice Ca2+ATPase gene harbours multiple stress-related cis-elements (Huda et al., 2013a,b), and it has been reported that over-expression of these genes mediates responses to abiotic stresses (Kasuga et al., 1999; Gilmour et al., 2000; Sakuma et al., 2006; Jung et al., 2007). Our previous findings showed that rice Ca2+ATPase promoters are responsive to abiotic stress, and are highly active in reproductive organs as well as in vascular tissue. Analysis of the promoter regions of PCA1 (Qudeimat et al., 2008) and a transcriptomic study in Arabidopsis (Kaplan et al., 2006) showed that the PCA1 gene has an over-representation of the ABRE motif, which is known to confer ABA-induced gene expression. This again suggests that Ca2+ATPases participate in ABA-mediated responses to abiotic stresses.

OsACA6 contains ten transmembrane domains, and belongs to the same phylogenetic clade as the Arabidopsis genes ACA10, ACA8 and ACA9, all of which showed exclusive localization in the plasma membrane (Huda et al., 2013a). The present study shows high expression of OsACA6 in the plasma membrane, consistent with the localization of the Arabidopsis type IIB Ca2+-ATPases ACA8, ACA9 and ACA10 (Schiøtt et al., 2004; Bonza and De Michelis, 2011). A tonoplast localization of OsACA6 cannot be excluded, but this would not change the proposed hypothesis for the role of OsACA6 in stress tolerance. Its expression in tonoplast would probably lead to lower cytosolic Ca2+ levels, the only difference being that, when localized to the tonoplast, Ca2+ would be sequestered to vacuoles instead of the extracellular space as in the case of plasma membrane localization. Additional subcellular localization sites for plant type IIB Ca2+ATPases include the ER for ACA2 (Hong et al., 1999), the small vacuole for ACA4 and PCA1 (Geisler et al., 2000; Qudeimat et al., 2008) and the central vacuole for ACA11 (Lee et al., 2007), indicating functional diversity of these pumps in relation to Ca2+ homeostasis and Ca2+ signalling.

Enhanced tolerance to environmental stresses in plant is associated with a higher cell membrane stability and photosynthetic capacity and lower rates of water loss (Mao et al., 2012). In the present study, a reduced water loss rate was observed for over-expressing lines, indicating higher water retention capacity of the transgenic plants. The maintenance of Fv/Fm ratio, membrane integrity and stability under adverse conditions are the most important constituents of stress tolerance in plants (Strasser et al., 2002). Photosystem II is affected during stress, resulting in a decreased Fv/Fm ratio (Krause and Weis, 1991). We observed a reduced decrease in the Fv/Fm ratio and higher cell membrane stability in OsACA6 transgenic plants, suggesting that OsACA6 plants had more dynamic photosynthetic capabilities and less membrane damage. Cell membrane stability has been used for evaluating tolerance to salt, frost, heat, desiccation and PEG (Farooq and Azam, 2006), and has a positive association with several physiological and biochemical parameters. In this study the functional assays in tobacco confirmed that OsACA6 over-expressing plants have abilities to tolerate a changing environment.

OsACA6 transgenic plants displayed increased seed germination and exhibited enhanced tolerance to salt and PEG stress. OsACA6 transgenic plants had longer primary roots, and therefore are capable of producing more biomass and show improved drought tolerance under water-deficit conditions. The longer root length of OsACA6 plants was associated with a higher relative water content over a long period of withholding of water. These observations suggest that the improved root architecture permits efficient use of water, which may be responsible for enhanced stress tolerance. Transgenic plants also showed increased tolerance to stress as specified by higher chlorophyll content and photosynthesis and a better survival rate, allowing the transgenic plants to grow faster, flower and set viable seeds.

Salt stress-induced appropriate ion levels are essential for cellular health, and an optimal cytosolic K+/Na+ ratio is an important indicator of plant salt tolerance (Cuin et al., 2008). In this study, OsACA6 over-expressing tobacco plants accumulated less Na+ compared to wild-type plants. However, the roots accumulated more Na+ than the leaves in transgenic plants. The sequestration of Na+ in the vacuole may inhibit Na+ toxicity in the cell and protect leaves from photosynthesis inhibition and keeping seeds free from Na+ (Blumwald et al., 2000; Apse and Blumwald, 2007; Møller et al., 2009; Cuin et al., 2011; Dang et al., 2011). Increased cytoplasmic Na+ usually causes membrane injury and affects MDA accumulation in the cell. Our findings support the hypothesis that the enhanced accumulation of Na+ in vacuoles was mainly due to over-expression of OsACA6. Therefore, over-expression of OsACA6 acts as safeguard for the cell membrane from stress-induced injuries, alleviating oxidative stress. Under stress conditions, low cytosolic K+ levels cause leaf senescence by controlling caspase-like proteases and endonucleases (Shabala, 2009). OsACA6 transgenic plants maintained a higher level of K+ in leaves and roots than the wild-type plants under salt stress. This higher K+ level in transgenic plants may not trigger caspase-like protease activity leading to programmed cell death, thus delaying senescence. Previous studies reported that salt-tolerant transgenic plants possess a greater ability to retain, or even increase, K+ contents (Espinosa-Ruiz et al., 1999; Volkov and Amtmann, 2006; Chen et al., 2007). An increase in proline accumulation is linked with adaptive mechanisms in plants (Bais and Ravishankar, 2002; Urano et al., 2009; Gill and Tuteja, 2010a; Hussain et al., 2011). The observed increase in proline content in transgenic plants reported in the present study further suggests that proline may also be a factor in enabling better K+ retention via control of K+-permeable plasma membrane channels. Thus, targeting various mechanistic modules responsible for cellular K+/Na+ homeostasis may be an effective way to improve stress tolerance in crops.

In this study, over-expressing lines showed less ROS accumulation, supporting an important role for plasma membrane Ca2+ATPases in combating abiotic stress. The improvement of stress tolerance is often related to increased activities of antioxidant enzymes (Gill and Tuteja, 2010a). The increased activities of APX and GR maintain the pool of glutathione and ascorbate in the reduced state. Our results showing increased activity of antioxidant enzymes are in agreement with previously reported studies performed in other species during heat and cold stress (Wang and Li, 2006). The adaptive mechanisms reported in plants to counter oxidative stress include efflux of excess Ca2+ from the cytosol, targeting downstream components such as different transcription factors, calmodulin binding proteins (Reddy et al., 2011) or switching off the signal triggering ROS production (Zaidi and Michaelis, 1999; Romani et al., 2004; Beffagna et al., 2005). Further evidence has shown that Ca2+ acts as an important factor during the stress response and adaptation against environmental stresses (Reddy et al., 2011). It has been assumed that an increased concentration of Ca2+ maintains the membrane integrity during stress response in plants (Palta, 1996). In addition, oxidative stress and the antioxidative machinery in plants show a correlation with Ca2+ and calmodulin levels, and the improvement of salt tolerance is closely related to enhanced activities of antioxidant enzymes in plants (Foyer et al., 1997). In conclusion, our results indicate that salt and drought tolerance are associated with an increased response of the antioxidant machinery that is correlated with the increased cytoplasmic Ca2+ concentration.

Abiotic stress causes increases in [Ca2+]cyt, which are maintained by the combined activity of Ca2+ channels, Ca2+ exchangers, and Ca2+ATPases present on the plasma membrane, plus receptors and channels present in the intracellular storage compartments. We speculate that over-expression of OsACA6 enhanced the efflux of excess Ca2+ from cytoplasm. As intracellular [Ca2+]cyt often increases during stress conditions, increased efficiency in returning the [Ca2+]cyt to normal resting levels during such stressful conditions may lead to improved stress adaptation. Furthermore, the Ca2+/CaM binding ability of OsACA6 and suggest feedback attenuation of [Ca2+]cyt during abiotic stress by activating CaM-mediated signal transduction pathways and other related signalling pathways. Plants possess several CaM-related proteins, but only plasma membrane Ca2+ATPases are present in animals (Yang and Poovaiah, 2003). These observations suggest that plants utilize a more complex regulatory mechanism for Ca2+ efflux via Ca2+/CaM-regulated Ca2+ATPases. The various CaM isoforms may interact differently with target proteins, and such regulation may be one mechanism enabling cells to fine-tune Ca2+ signalling (Reddy, 2001; Reddy et al., 2011). CaM regulates both Ca2+ influx through Ca2+ channels and Ca2+ efflux by calcium transporters. Previous studies have shown that, upon Ca2+ binding, CaM tightly interacts with two K+ channel domains of Ca2+-activated potassium channels. Therefore, we believe that over-expression of OsACA6 may block the Ca2+ influx channels present on the plasma membrane. The over-expression of OsACA6 may also remove other divalent cations and prevent mineral toxicity to the cell. Several studies in non-plant systems have shown that increased Ca2+ pump activity alters the signal transduction system (Camacho and Lechleiter, 1993; Lechleiter et al., 1998; Roderick et al., 2000). These studies suggest that OsACA6 may have a role in shaping the calcium spike and thereby regulate signalling mechanisms in plants. These combined features may contribute to the improved stress tolerance mediated by OsACA6 over-expression as observed in our study.

Downstream targets for Ca2+ATPases have not been identified in plants. The microarray analysis shows up-regulation of many stress-related and growth/development-related proteins, signalling components, oxidative burst proteins and transcription factors, as well as proteins involved in protein phosphorylation (Table 2). Transcription factors were also up-regulated in transgenic plants. Various studies have confirmed that over-expression of these transcription factors in transgenic plants confers abiotic stress tolerance of crops (Kasuga et al., 1999; Cattivelli et al., 2008; Hussain et al., 2011; Morran et al., 2011; Zhang et al., 2012). Interestingly, transgenic plants showed fourfold increase in expression of LEA proteins, which have been reported to be involved in stress tolerance (Orellana et al., 2010). Members of the MADS box family bind to CaM (Popescu et al., 2007) and are associated with vegetative organs (Huang et al., 1995) as well as flower development (Tardif et al., 2007). Previously, a differential expression profile was observed for these family members in response to salt, desiccation and cold stress (Arora et al., 2007), and a 2.8–4.3-fold up-regulation of MADS box proteins was detected in the present study (Table 2). Peroxidase superfamily proteins were also up-regulated, suggesting a defensive role of OsACA6 by modulating the ROS machinery. Recent findings indicated involvement of Ca2+ATPases in receptor kinases-mediated signalling pathways (Frey et al., 2012) and a prominent role in plant biotic stress (Nemchinov et al., 2008; Zhu et al., 2010). We observed up-regulation of various kinases and disease-related proteins in transgenic plants (Table 2). Calmodulin also binds to various calcineurin B-like proteins, which act as calcium sensors for specific signal responses in plants. A hypothetical model for the role of OsACA6 is proposed in Figure 10, combining all the observations of the present study. Cellular Ca2+ and ROS production increase in response to abiotic stress. The over-expression of Ca2+ ATPases leads to an increase in Ca2+ efflux and antioxidant defence, thus contributing to cellular homeostasis and improved tolerance against oxidative stress.

Figure 10.

Hypothetical model for the role of OsACA6 in plant stress adaptation and signal transduction mechanisms.

Stress causes an increase in cytosolic Ca2+ and ROS. Over-expression of OsACA6 switches off the ROS production by activating the antioxidant defence system, and Ca2+ binds to the calmodulin (CaM) binding site of OsACA6. The Ca2+–CaM complex then activates various transcription factors, protein kinases and defence- and growth-related proteins, as well as membrane transporters, and regulates their expression, resulting in stress responses. Overall, the stress response involves the co-ordination action of many gene products, which may cross-talk with each other.

Overall, in the present study, the expression of OsACA6 was assessed, and over-expression resulted in enhanced tolerance against salinity and drought stress. Our morphological, physiological and biochemical evidence showed that transgenic lines are more tolerant to drought and salinity compared to wild-type. These results provide direct evidence for a function of OsACA6 in imparting salinity/drought stress tolerance without yield loss by strengthening photosynthesis and antioxidant machinery in transgenic plants as well as by regulating stress-responsive genes. Bioengineering of members of this gene family may allow creation of crops with improve stress tolerance.

Experimental Procedures

Plasmid construction, bioinformatics analysis and generation of transgenic plants

Full-length cDNA for OsACA6 (Genbank accession number KF240826, locus ID LOC_Os04 g51610) was amplified from a rice cDNA library by PCR using primers 5′-GTCGACATGGAGTCCGCGTCGTCTTCACTAG-3′ and 5′-GTCGACGTAGCTTCATACATCGC TGTGATCAGC-3′ (SalI site underlined), cloned into the TA cloning vector (pGEMT, Promega, Madison, WI, USA) and verified by sequencing. Multiple sequence alignment was performed to identify conserved motifs, domains and transmembrane regions among members of the type II Ca2+ATPases. The MSU Rice Genome Annotation Project database (http://rice.plantbiology.msu.edu/) was used for calculation of the isoelectric point and molecular weight. The cDNA was also cloned into the pBinAR vector (Hoefgen and Willmitzer, 1990) under the control of the CaMV 35S promoter for generation of transgenic plants. Tobacco (Nicotiana tabacum) leaf disks were transformed using a standard procedure (Horsch et al., 1985), and positive transgenic lines were screened for using kanamycin plates.

Analysis of gene expression by quantitative real-time PCR

To assess the expression of OsACA6, 2-week-old rice seedlings were treated with various stresses. The RNA from stressed and unstressed samples was isolated, and cDNA was prepared. RT-PCR analysis was performed as previously described (Huda et al., 2013a) using primers 5′-GCTTGGCATAGTTGGGATAAAGG-3′ (forward) and 5′- CCACACTCCAAGGCAATGG-3′ (reverse). The α-tubulin gene was used to normalize expression of OsACA6, and was transcribed using primers 5′- GGTGGAGGTGATGATGCTTT-3′ (forward) and 5′-ACCACGGGCAAAGTTGTTAG-3′ (reverse). Biological and technical replicates of each sample were used for analysis.

Subcellular localization of OsACA6

The coding sequence of OsACA6 was fused with GFP and cloned into the XbaI restriction site of the pMBPII-GFP expression vector (Sharma et al., 2012) under the control of the CaMV 35S promoter for expression of 35S::OsACA6–GFP fusion protein. Onion epidermal cells were bombarded with the above constructs using a particle gun-mediated system, (Genepulser Excell System, Bio Rad, Hercules, CA, USA) and localization of OsACA6 was detected using confocal microscopy.

Promoter isolation, tobacco transformation, abiotic stress treatment and GUS staining

A 1478 bp flanking region was amplified from genomic DNA by RT-PCR using primers 5′-CAAGCTTCGTGCTTGCATGTCACTTTTT ATG-3′ and 5′-CGGGATCCCTGGTCGCTAGTGAAGACGA-3′ (HindIII site underlined), and cloned into the pCAMBIA-1391Z vector (M1707, Marker Gene Technologies, Inc., Eugene, OR, USA). The standard procedure developed by Horsch et al., 1985) was used to create transgenic tobacco plants. Plants expressing a construct comprising the CaMV 35S promoter fused to GUS were used as a positive control and wild-type plants were used as a negative control. Positive transgenic lines were confirmed by PCR using promoter-specific primers. Various tissues of stress-treated transgenic plants were stained with X-Gluc (5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid) as described previously (Jefferson et al., 1987).

Molecular identification of transgenic plants

Genomic DNA was isolated from leaf tissue using the CTAB (hexadecyltrimethylammonium bromide) method (Edwards et al.,1991). The transgenic lines were identified by PCR using gene-specific primers. For Southern analysis, 20 μg genomic DNA from PCR-positive tobacco lines was digested with XhoI and resolved on an agarose gel. The DNA was transferred to nylon membrane (Hybond N, Amersham Pharmacia, http://www.gelifesciences.com/), and hybridized with radiolabelled OsACA6 cDNA as described previously (Pham et al., 2000). The level of expression of OsACA6 in OsACA6-positive plants was analysed by RT-PCR. Homozygous transgenic lines (T2) showing a higher level of expression were selected for further analysis.

Morphological characterization of transgenic plants

The seeds of transgenic plants and wild-type plants were grown on germination medium supplemented with 200 mm NaCl or 20% PEG to assess the stress tolerance. The germination was observed and recorded at 2-day intervals up to 10 days. The root morphologies of seedlings were studied by growth on MS medium. Seeds of wild-type and transgenic plants were grown on vertical plates in MS medium for 8 days, and primary root lengths were measured. In an additional experiment, germinating seeds of wild-type and transgenic plants were transferred to medium supplemented with 200 mm NaCl or 20% PEG and grown vertically for 21 days. The physiological changes were observed, and fresh weights were recorded. All experiments were repeated three times. Water loss rates and cell membrane stability were measured as described previously (Mao et al., 2012). Chlorophyll fluorescence (Fv/Fm) was recorded in fully expanded leaves using an infra-red gas analyser (Li-6400, Li-COR, Lincoln, NE, USA). Leaf disks assays and determination of the total chlorophyll content of transgenic and wild-type plants were performed as described by Dang et al. (2011).

Measurement of Na+ and K+ contents

Na+ ion content was measured from roots and mature leaves of wild-type and transgenic plants treated with 0 and 200 mm NaCl using argon plasma emission spectrometry. K+ was estimated using a flame photometer. All experiments were performed in triplicate.

Detection of H2O2 and Na+ in response to stress

The accumulation of H2O2 was detected by histochemical staining using the diaminobenzidine method (Wang et al., 2011). Following stress treatment (12 h), roots of wild-type and transgenic plants were separated and incubated with fluorescent dyes in liquid medium of the same composition used in the stress. Intracellular Na+ was detected in roots stained using 100 μm CoroNa Green AM dye (Invitrogen, Ltd., Carlsbad, CA, USA), and images were obtained by confocal laser scanning microscopy.

Measurements of biochemical and physiological parameters: MDA, electrolyte leakage, proline, H2O2 and relative water content and antioxidant enzyme assays

Two-week-old seedling were subjected to salinity (200 mm NaCl) or dehydration stress (20% PEG) for 1, 6, 12 and 24 h. Stressed and control samples were collected, and immediately frozen in liquid nitrogen and stored at −80°C. Electrolytic leakage, proline and H2O2 content were measured from fresh samples after stress recovery. Quantification of MDA, electrolyte leakage, proline, H2O2 and activity of antioxidant enzymes was performed as described previously (Garg et al., 2012). All experimental data were recorded as the means of three independent experiments.

Abiotic stress tolerance assays

For salt and drought tolerance assays, 50 plants of each line and wild-type were grown in pots containing vermiculite, constantly watered for 14 days, and then irrigated with 200 mm NaCl or 20% PEG solution every 2 days for 10 days. The seedlings were recovered after re-watering for 5 days. During these treatments, morphological changes were detected and photographed when variations were apparent, and the survival rate was measured. In another experiment, 35-day-old plants were also subjected to salt or PEG stress for 25 days, and photographs were taken. The treated plants were again shifted to normal growth conditions. Consistent results were observed in three independent experiments. The results of a representative set of experiments are shown.

Microarray analysis

Two-week-old seedlings of transgenic tobacco and wild-type were treated with 200 mm NaCl for 6 h. Total RNAs were isolated and sent to Genotypic Technology Pvt Ltd (Bangalore, India) for analysis. Using 10 μg of total RNA, double-stranded cDNA was synthesized with a T7 promoter -containing oligo (dT) primer followed by in vitro transcription using a Agilent's Quick-Amp labelling Kit (Agilent Technologies India Pvt. Ltd., New Delhi, India). Resulting cRNA was hybridized using Agilent's In situ Hybridization kit. Microarray analysis was performed using an Agilent microarray chip (http://www.agilent.co.in/) for Nicotiana tabaccum (4 × 44k format; product name G2514F), and the chip was scanned using an Agilent gene array scanner. Scanned images were processed and analysed using Bioconductor R software (http://www.bioconductor.org/), and values were normalized using percentile shift normalization. Genes exhibiting more than two-fold enhanced or reduced transcription level are considered to show significant alterations in expression. Additionally, the χ2 test was used to increase the statistical accuracy of the results. The genes for which < 0.05 are included in the results presented.

Data analysis

Data were evaluated statistically and the standard error was calculated. SPSS (10.0; IBM, Armonk, NY, USA) was used for analysis of variance to determine the least significant difference. The means were separated using Duncan's multiple range test. Letters indicate significant differences at < 0.05, < 0.01 and < 0.001.

Acknowledgements

K.M.K.H. and M.S.A.B. are the recipients of Arturo Falaschi International Centre for Genetic Engineering and Biotechnology pre-doctoral fellowships. The work on plant stress tolerance and signal transduction in N.T.'s laboratory is partially supported by Department of Science and Technology and Department of Biotechnology, Government of India, and partially supported by the International Centre for Genetic Engineering and Biotechnology (New Delhi, India). We would like to thank Stanley J. Roux and Greg Clark (Institute for Cell and Molecular Biology, University of Texas at Austin, TX) for their help in correcting the manuscript.

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