Introduction

The detrimental effects of high salinity levels on plants are the consequence of osmotic and ionic stresses [1]. Plants with the ability to successfully complete the life cycle under continuous exposure to higher salinity stress (> 200 mM NaCl) are categorized as halophytes, however, rest plants are known as glycophytes (salt-sensitive) [2]. The mechanisms underlying high-level salt tolerance among many halophytes are not properly understood, but the trait is often, more or less implicitly, supposed to depend on enhanced capacities of cellular Na+ and K+ compartmentalization and homeostasis, or compatible organic osmolyte synthesis, through alterations of the expression patterns of genes encoding Na+/K+ transporters or genes involved in the synthesis or breakdown of compatible solutes [2]. The Na+ transporters, SOS1, NHX1 and HKT1 have often been considered to play key roles in salt tolerance in halophytes [3], however, comparisons between their expression patterns in halophytes and glycophytes are, with few exceptions [4,5,6,7], not available to date.

The HKT is Na+/K+ transporter and gene family is increasingly studied for its role in Na+ detoxification within the plant body [8,9,10]. Plett et al. [11] showed that over-expression of AtHKT1;1 in the mature root cortex improved the shoot Na+ exclusion, and thus increased salinity tolerance in transgenics. In a recent study, Nawaz et al. [7] compared HKT1 expression among halophytic and glycophytic species of Cochlearia, and found much higher expression levels in the halophytic species than in the glycophytic, supporting the hypothesis that enhanced HKT1 expression may be crucial for high-level salt tolerance, indeed. Eutrema salsugineum (formerly known as Thellungiella halophila), thought to be a suitable halophytic model plant because of its high DNA identity with A. thaliana [12], has at least three HKT1 genes, HKT1;1, HKT1;2 and HKT1;3 [13]. EsHKT1;2, is a K+ transporter, whereas EsHKT1;1 and EsHKT1;3 are Na+ transporters, like AtHKT1;1 [13,14,15].

There are many reports suggesting the presence of an efficient transcriptional regulation of the salt-responsive genes in halophytic species. Zhang et al. [16] analyzed the expression pattern of Suaeda liaotungensis gene BADH (SlBADH) promoter. They used 300 bp fragment (upstream from ATG) and showed almost six fold higher expression of gene under 400 mM NaCl stress as compared to control. Sun et al. [17] compared the activities of two promoters from A. thaliana and T. halophila gene (Vacuolar H+-Pyrophosphatase-VP) and concluded that 130 bp of the ThVP1 promoter contains cis-acting regulatory elements (two enhancers) which are involved in strong induction of the gene under salt stress. Li et al. [18] analyzed the role of PEAMT gene promoter from Suaeda liaotungensis (a halophyte) and predicted the presence of many salt-responsive cis-element (897 bp upstream from start codon). They reported the enhanced GUS activity (18-fold) under the control of SlPEAMT promoter at 200 mM of NaCl stress, in the transgenic tobacco. These studies and many others provided a notion that even a small fragment of promoter can contain salt stress-inducible, cis-acting motifs to regulate the expression of different genes, upon salt stress.

Surprisingly, very little work has been done to explore the role of the HKT1 promoter in the transcriptional regulation of the gene. Asins et al. [19] has observed the altered expression of SlHKT1;1 gene in roots and leaves of NILs due to the difference in their promoter sequences, which affects the transport activity of the gene, as well. The cis-regulatory allelic variations among various Arabidopsis ecotypes has also been documented for the AtHKT1;1, which lead to differential salinity tolerance of the ecotypes [20, 21]. However, there is not even a single study to compare the promoter activities of a halophyte (like Eutrema salsugineum/botschantzevii) and a glycophyte (like A. thaliana). In this study, we performed an in silico analysis of three HKT1 promoter sequences, i.e., A. thaliana (ProAtHKT1;1, 1822 bp), Eutrema salsugineum (=halophila), ecotype Shandong (ProEsHKT1;2, 1822 bp), and Eutrema botschantzevii (formerly known as Thellungiella botschantzevii), ecotype Saratov (ProEbHKT1;2, 1822 bp). We compared the activities of four promoter sequences, i.e., ProAtHKT1;1 (846 bp), ProEsHKT1;2 (1822 bp), ProEbHKT1;2 (1811 bp), and the CaMV 35S promoter, through examining AtHKT1;1 and EsHKT1;2 gene expression in the A. thaliana hkt1;1 mutant background. We also compared the potential of the constructs to reverse the Na+ hypersensitivity phenotype of the Athkt1;1 mutant.

Materials and Methods

Plant Materials and Experimental Conditions

Eutrema salsugineum and E. botschantzevii seeds, originating from a coastal area near Shandong, China, and a solonchak soil in Saratov, Russia, were sown on garden soil (Jongkind B.V., Number 6, Aalsmeer, The Netherlands). When seedlings were three-weeks old, they were transfered to hydroponics culture [22]. Mature plants were harvested which were snap-frozen. For subsequent DNA/RNA extraction, samples were stored at − 80 °C [23].

Surface sterilization of different types of seeds [Arabidopsis thaliana (Col) wild type, Athkt1;1 mutants and transgenic lines] were done using 96% ethanol and 10% bleach, followed by three times washing in distilled water. Surface-sterilized seeds were mixed in agarose (0.1%) and spread on square petri plate containing gelrite (0.8% w/v) with Murashige and Skoog (MS—0.5%) salt, whose pH was adjusted around 5.7–5.9. For transgenic lines, hygromycin (25 µg/ml) and for the Athkt1;1 mutants, 25 µg/ml kanamycin was used as selective marker. Photoperiod for seeds germination was 10 h light and 14 h dark, at 22 °C temperature. When seedlings developed their true leaves, they were transplanted to hydroponics 1-l pots, having half-strength modified Hoagland’s nutrient solution [22] in climate room. Light intensity in climate room was set 220 µmol/m/s at the plant level, along with 20 °C (day), 15 °C (night) temperature and 75% relative humidity. Hydroponics solutions were renewed two times a week. When plants get adapted to the new environment (almost after two weeks), ten plants from each type were exposed to NaCl (50 mM) using control as a reference. Salt stress was increased gradually (at first 25 mM stress was given then increased to 50 mM). Plants were harvested after two weeks of the final concentration. Fresh weight of the roots and shoots in gram was taken.

RNA/DNA Extraction and First-Strand cDNA Synthesis

Frozen shoot tissues were used for RNA extraction via Trizol™ (Invitrogen) method, as described in Jack et al. [23]. For the synthesis of single-stranded cDNA, total RNA (2.5 µg) which was boiled for 1 min, was used. Reverse transcription was accomplished with M-MLV Reverse Transcriptase (100 Units), 100 mM DTT, 2 mM dNTPs, RT buffer (10 ×) and 10 µM of oligo dT primer (Invitrogen). For DNA isolation, we used the protocol as described in Rivera et al. [24].

Es/EbHKT1;2 Promoters Sequencing, Constructs Making and the Transformation of the Athkt1;1 Mutant

The sequence of HKT1;2 promoters from E. salsugineum and E. botschantzevii were obtained by chromosome walk, using Universal Genome Walker™ kit by Clontech. Gene specific reverse primers were used for chromosome walk on DNA (genomic). 1822 bp from E. salsugineum, 1811 bp from E. botschantzevii and 846 bp from A. thaliana [25] upstream from start codon (ATG) of HKT1, were used as promoters. Following constructs were prepared: Pro35S::EsHKT1;2, ProAtHKT1;1::EsHKT1;2, ProAtHKT1;1::AtHKT1;1, ProEsHKT1;2::AtHKT1;1 and ProEbHKT1;2::AtHKT1;1. PCR’s were performed using “Phusion® DNA Polymerase” by Finnzymes, on gDNA for amplification of the promoter region and on cDNA for amplification of HKT coding sequences. Forward primers of ProAtHKT1;1, ProEbHKT1;2, ProEsHKT1;2 and ProEsHKT1;2::AtHKT1;1 contained “CACC” 5′ overhang which is necessary for directional cloning in pENTR/D Topo, while primers for ProAtHKT1;1::AtHKT1;1, ProAtHKT1;1::EsHKT1;2 and ProEbHKT1;2::AtHKT1;1 contained attB1/2 sites (Table S1: Supplementary Information). BP reaction was performed using BP Clonase® II enzyme mix between vector containing attP-site and DNA fragment having attB-flanked to generate an entry clone. This entry clone was subsequently utilized for LR recombination reaction, using LR Clonase® II, with Gateway® destination vector for the development of an expression vector. Following expression vectors; pH7WG2, pH7WG2(-Pro35S) {constructed from pH7WG2} and pHGWFS7 [26], were used. pHGWFS7 was used for the promoters analysis, however, pH7WG2 (-Pro35S) was used for the constructs (having a promoter). Agrobacterium tumefaciens strain C58 (pMP90) containing specific construct was used to transform A. thaliana hkt1;1 mutant.

Screening of Transgenic Lines

Athkt1;1 mutants (Col) seeds, obtained from NASC stock center (N6531), and A. thalaina wild type seeds were sown on the garden soil (Jongkind B.V., Number 6, Aalsmeer, The Netherlands). Plants of Athkt1;1 mutants, at silique forming stage, were transformed using ‘flower dipping’ protocol as described by Clough and Bent [27]. The matured seeds were harvested from T0 plants and were sowed on square Petri plates containing MS media, after surface sterilization. The transformed plants were easily distinguished from non-transformed ones, after 2 weeks depending on root growth. Transformed plants were transplanted to hydroponics culture [22]. After 2 weeks, leaves and root samples were taken for RNA extraction, from selected T1 progeny. After selection, null segregants were never found, and selected plants were supposed to be a mixture of heterozygotes and homozygotes. Transcript levels of the genes (Actin-2 as reference gene) were measured by Real-Time quantitative PCR. Primers for RT-qPCR were designed with a G/C ratio ranging between 50 and 60% with Tm (melting temperature) range between 58 and 60 °C. Gene-specific primers for Eutrema salsugineum (EsHKT1;2), Eutrema botschantzevii (EbHKT1;2), A. thaliana (AtHKT1;1) and Act-2 (as an internal control for expression analysis) were designed separately (Table S2: Supplementary Information). RT-qPCR data were analyzed as described by Livak and Schmittgen [28]. The gene expression was normalized to the highest expression, which was assigned a value of 1.

Tolerance Index and Water Content

At the time of harvest, fresh weight of root and shoot samples were taken. Samples were air dried at 65 °C for 72 h. Tolerance index was calculated over ten biological replicates of each transgenic line using the formula:

$${\text{Tolerance}}\;{\text{Index}}\,\left( {\text{TI}} \right) = \frac{{{\text{Average}}\;{\text{fresh}}\;{\text{weight}}\;{\text{at}}\;50\;{\text{mM}}}}{{{\text{Average}}\;{\text{fresh}}\;{\text{weight}}\;{\text{at}}\;{\text{the}}\;{\text{control}}}}$$

The percentage of water was calculated using the following formula:

$$\%\,\ {\text{age}}\;{\text{of}}\;{\text{water}} = \left( {\frac{{{\text{Fresh}}\;{\text{weight}} - {\text{Dry}}\;{\text{weight}}}}{{{\text{Fresh}}\;{\text{weight}}}}} \right) \times 100$$

Measurement of Na+ and K+

After taking fresh weight, the roots of two plants were pooled together while shoots were dried and stored separately. After measuring shoot dry weight two shoots were pooled together like roots. Na+ and K+ concentrations, in roots and shoots were determined (two plants pooled, to make five samples per genotype, per treatment level) by extracting 20 mg of dry material in 2 ml eppendorfs. Two milli liters of water was added in each eppendorf then boiled it for 1 h at 90 °C in a water bath. After cooling, samples were filtered and dilutions were made, if needed. After proper dilution, Na+ and K+ analysis on Atomic Absorption Spectrophotometer (AAS100) was done.

Results

In Silico Analysis of EsHKT1;2, EbHKT1;2 Promoters in Comparison with the AtHKT1;1 Promoter

The sequences of the EsHKT1;2, EbHKT1;2 and AtHKT1;1 promoters (each was 1822 bp in length) were used for in silico analysis via web-based tools (PLACE/Signal Scan database-http://www.dna.affrc.go.jp/) [29]. The promoters from EsHKT1;2 and EbHKT1;2 have 95% identity among themselves, while both promoters showed 38% identity with the AtHKT1;1 promoter, on nucleotide basis (sequence alignment S1: Supplementary Information).

We identified several putative cis-acting regulatory biotic/abiotic stress-responsive elements which include the core motif for DRE/CRT (dehydration-responsive element/C-repeat) and putative GT-1 cis-elements (2 motifs in EsHKT1;2 and EbHKT1;2 and 3 in the AtHKT1;1 promoter sequences; Table S3: Supplementary Information). A common cis-acting element, CAAT-box, which is mostly present in promoters and enhancer regions, was identified at several places in three promoter sequences. The ACGT motif was found 3, 4 and 5 times in AtHKT1;1, EbHKT1;2 and EsHKT1;2 promoter sequences, respectively. Potential MYB-, MYC- and MYBCORE recognition sites have been identified at several places in the analyzed sequences (Table S3: Supplementary Information).

Selection and Molecular Analysis of T 0/T 1 Transgenic Plants

The amplified E. salsugineum cDNA sequence appeared to be EsHKT1;2. It shared 83% identity, on a nucleotide basis, with A. thaliana HKT1;1 (sequence alignment S2: Supplementary Information). On a protein basis, EsHKT1;2 and AtHKT1;1 were 79% identical (sequence alignment S3: Supplementary Information). The EbHKT1;2 (1811 bp) and EsHKT1;2 (1822 bp) promoters were cloned for this experiment. We successively performed two experiments with two sets of independent transgenic lines.

PCR and Real-Time PCR analyses were performed on all the T0 plants and 3–4 randomly selected plants from the T1 progeny (the first generation of transgenic plants) to determine the expression levels (Fig. 1) of the transgenes, under control condition. Only those transgenic lines were selected which showed the similar transcript expression level of AtHKT1;1 and/or EsHKT1;2, for the subsequent experiments (at least two independent lines per genotype). EsHKT1;2 was most strongly expressed from ProAtHKT1;1, in comparison with any other promoter (Fig. 1). Also AtHKT1;1 was well expressed, approximately at the level of wild type A. thaliana, when expressed under its native promoter. The ProEbHKT1;2::AtHKT1;1 and ProEsHKT1;2::AtHKT1;1 constructs were not detectably expressed, under un-stressed condition.

Fig. 1
figure 1

Expression of HKT1 in transgenic lines (T1) harbouring Pro35S::EsHKT1;2, ProAtHKT1;1::EsHKT1;2, ProAtHKT1;1::AtHKT1;1, ProEsHKT1;2::AtHKT1;1, ProEbHKT1;2::AtHKT1;1 construct, using Real-Time quantitative PCR. A. thaliana wild type (At-Wt) and Athkt1;1 mutant were used as positive and negative controls, respectively. Transgenic lines with comparable levels of HKT1 expression were used in subsequent experiments. Each value is an average of 3–4 independent biological replicate ± SE. Significant differences (p < 0.05) between means are indicated by different superscripted letters

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Tolerance Index

There were evident differences in salt tolerance among various transgenic lines. A. thaliana wild type and transgenic under the control of ProAtHKT1;1 and Pro35S looked green and remained healthy. There were no apparent foliar chlorosis/necrosis, however, they showed senescence of the cotyledons and the oldest leaves (~ 10 to 20%). These transgenic lines did not exhibit any mortality upon exposure to 50 mM NaCl. Transgenic lines under the control of ProEsHKT1;2 or ProEbHKT1;2 proved to be sensitive with much smaller leaves. These lines exhibited chlorosis/necrosis with enhanced senescence of the older leaves (~ 40%), as well.

Under exposure to 50 mM NaCl both of the constructs with the A. thaliana promoter, ProAtHKT1;1::AtHKT1;1 and ProAtHKT1;1::EsHKT1;2, complemented the Athkt1;1 mutant, approximately to wild type level (Fig. 2). As expected, the constructs with extremely low expression, did not complement the Athkt1;1 mutant to any significant extent.

Fig. 2
figure 2

Tolerance index on fresh weight basis measured in T1 progeny harbouring Pro35S::EsHKT1;2, ProAtHKT1;1::EsHKT1;2, ProAtHKT1;1::AtHKT1;1, ProEsHKT1;2::AtHKT1;1, ProEbHKT1;2::AtHKT1;1 constructs. Plants were grown hydroponically at 50 mM NaCl stress for 14 days. A. thaliana wild type (At-Wt) and Athkt1;1 mutant were used as positive and negative controls, respectively. Ten replicates were used for each genotype and two independent T1 lines were used in the experiment. Each bar is a mean of 20 plants ± SE. Significant differences (p < 0.05) between means are indicated by different superscripted letters

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The Pro35S::EsHKT1;2 constructs complemented the mutant in one of the experiments, but not in the other, in spite of the fact that in both experiments the HKT expression level was at least one order of magnitude higher under the CaMV 35S promoter than under any of the others. Overall, the results of the two experiments were consistent (Fig. 2).

The Water Content of Fresh Leaves

Consistent with their tolerance index, lines harbouring the ProAtHKT1;1::EsHKT1;2 and ProAtHKT1;1::AtHKT1;1 constructs maintained wild type-like water percentages in their leaves (> 90%) under salinity stress, whereas the plants with the Eutrema promoters desiccated to a degree comparable with that of the Athkt1;1 mutant (Fig. 3). The construct with the CaMV 35S-promoter completely complemented the mutant in the first experiment, but only incompletely in the second one. Overall, the results of the two experiments were consistent.

Fig. 3
figure 3

Water content (percentage of shoot fresh weight) in T1 progeny harbouring Pro35S::EsHKT1;2, ProAtHKT1;1::EsHKT1;2, ProAtHKT1;1::AtHKT1;1, ProEsHKT1;2::AtHKT1;1, ProEbHKT1;2::AtHKT1;1 constructs. Plants were grown hydroponically at control and 50 mM NaCl stress for 14 days. A. thaliana wild type (At-Wt) and Athkt1;1 mutant were used as positive and negative controls, respectively. Ten replicates were used for each genotype and two independent T1 lines were used in the experiment. Each bar is a mean of 20 plants ± SE. Significant differences (p < 0.05) between means are indicated by different superscripted letters

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Na+ and K+ Analysis in Transgenic T 1 Plants

We compared Na+ and K+ accumulation in shoots and roots of wild type and transgenic lines, in two sets of experiment. Average values of Na+ contents from both experiment were used to depict the results (Fig. 4). In both experiments the genotype with the Pro35S::EsHKT1;2 constructs exhibited a lower Na+ concentration in their shoots than any of the other lines. The foliar Na+ concentration in the ProAtHKT1;1::AtHKT1;1 and ProAtHKT1;1::EsHKT1;2 lines was significantly higher than in the Pro35S::EsHKT1;2 line, but significantly lower than in the lines under either Es and/or Eb promoters. Although in second experiment, the Na+ concentration was detected comparatively higher in all genotypes, however, the trend of Na+ contents remained the same as in both experiments. Overall, results from both experiments exhibited the similar trend (Fig. 4).

Fig. 4
figure 4

Na+ concentrations in shoots of T1 progeny from Pro35S::EsHKT1;2, ProAtHKT1;1::EsHKT1;2, ProAtHKT1;1::AtHKT1;1, ProEsHKT1;2::AtHKT1;1, ProEbHKT1;2::AtHKT1;1. Plants were grown hydroponically at control and 50 mM NaCl stress for 14 days. A. thaliana wild type (At-Wt) and Athkt1;1 mutant were used as positive and negative controls, respectively. Five replicates (two shoots were pooled together) were used for each genotype and two independent T1 lines were used in the experiment. Each bar is a mean of 10 plants ± SE. Significant differences (p < 0.05) between means are indicated by different superscripted letters

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The Athkt1;1 mutant and transgenic lines under either Es and/or Eb promoters showed similar shoot K+ concentration, however, wild type showed highest foliar K+ concentration than all the other lines, under NaCl exposure (Fig. 5). Transgenic line expressing construct ProAtHKT1;1::EsHKT1;2 also maintained significantly higher K+, upon salt stress, in its shoot as compared to the lines expressing ProAtHKT1;1::AtHKT1;1.

Fig. 5
figure 5

K+ concentration in the shoots of T1 progeny from Pro35S::EsHKT1;2, ProAtHKT1;1::EsHKT1;2, ProAtHKT1;1::AtHKT1;1, ProEsHKT1;2::AtHKT1;1, ProEbHKT1;2::AtHKT1;1. Plants were grown hydroponically at control and 50 mM NaCl stress for 14 days. A. thaliana wild type (At-Wt) and Athkt1;1 mutant were used as positive and negative controls, respectively. Five replicates (two shoots were pooled together) were used for each genotype and two independent T1 lines were used in the experiment. Each bar is a mean of 20 plants ± SE. Significant differences (p < 0.05) between means are indicated by different superscripted letters

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Na+/K+ ratios of shoots and roots of all genotypes were also calculated (Supplementary Figure S1). Transgenic lines under the control of Es and/or Eb promoter showed Na+/K+ ratio in shoot as high as found for Athkt1;1 mutants and was significantly higher as compared to all other genotypes (Supplementary Figure S1a). Root Na+/K+ ratio for lines harbouring constructs ProAtHKT1;1::EsHKT1;2, ProEsHKT1;2::AtHKT1;1, ProEbHKT1;2::AtHKT1;1 and Athkt1;1 mutant, were found significantly low as compared to other genotypes (Supplementary Figure S1b).

Discussion

In this study, we compared the expression pattern and activities of four promoters, i.e., ProAtHKT1;1, ProEsHKT1;2, ProEbHKT1;2 and CaMV 35S promoter, using AtHKT1;1 and EsHKT1;2 cDNA. As clearly shown by the tolerance index, only the ProAtHKT1;1::AtHKT1;1 and ProAtHKT1;1::EsHKT1;2 constructs more or less completely complemented the Athkt1;1 mutant regarding its salt hypersensitivity phenotype. The ProEsHKT1;2::AtHKT1;1 and ProEbHKT1;2::AtHKT1;1 constructs did not yield any detectable complementation at all, while the Pro35S::EsHKT1;2 construct only incompletely complemented the mutant. The same conclusion can be drawn on the basis of the foliar water contents in the salt treatment. The complete lack of complementation observed with the ProEsHKT1;2::AtHKT1;1 and ProEbHKT1;2::AtHKT1;1 constructs is doubtlessly owing to low AtHKT1;1 expression in the transgenic lines. The latter could be due to the absence of essential transcriptional activator/s or, more likely, the lacking an essential response element located upstream of the nucleotides that we used as promoter [30], which are required in A. thaliana. The incomplete complementation provided by the Pro35S::EsHKT1;2 construct is most probably owing to a non-tissue-specific expression of EsHKT1;2 [8].

Our observation that AtHKT1;1 and EsHKT1;2 are both able to complement the Athkt1;1 mutant is not self-evident, since AtHKT1;1 is a Na+-selective transporter [31], whereas EsHKT1;2 is a K+-selective transporter, even in the presence of NaCl [14]. AtHKT1;1 is supposed to provide salinity tolerance through resorbing Na+ from the xylem, thus preventing its accumulation in the shoot [32, 33], while EsHKT1;2 is supposed to maintain a sufficient K+ concentration in shoot, under salinity stress (Fig. 5) [14, 15]. Foliar Na+ concentrations were significantly lower for the wild type A. thaliana and lines having gene under AtHKT1;1 promoter, surprisingly regardless of the origin of the coding region, thus both genes doubtlessly complemented the Athkt1;1 mutant under our experimental conditions. Which suggested that maintenance of sufficient K+ concentration in shoot might be crucial for the salinity response of the transgenic line expressing ProAtHKT1;1::EsHKT1;2 [34, 35]. However, the lines with constructs under Es and/or Eb promoters showed the foliar Na+ contents as high as in the Athkt1;1 mutant plants and failed to revert the Na+ hypersensitivity of the mutants. Lines having construct under AtHKT1;1 promoter showed comparatively high K+ concentrations as compared to the lines with the construct under Es and/or Eb promoters, however, the difference was not too high between all lines.

Low-cytosolic Na+/K+ ratio is a major determinant for the salt stress tolerance of any plant which ultimately play important role in growth and developmental success of the plant. Lower Na+/K+ ratio in shoots of the lines harbouring construct Pro35S::EsHKT1;2, ProAtHKT1;1::EsHKT1;2 and ProAtHKT1;1::AtHKT1;1 as well as At-Wt. Positively correlate with salinity tolerance of these genotypes. Low Na+/K+ ratio in shoot depicting plants ability to restrict more Na+ contents in roots, and has been proved as a determinantal factor in Zea mays L. salt tolerance [36]. Low Na+/K+ ratio in roots of transgenic lines with ProAtHKT1;1::EsHKT1;2 indicate the higher contents of K+. Surprisingly, Pro35S::EsHKT1;2 showed high Na+/K+ ratio, which may be attributed to ectopic expression of EsHKT1;2 under CaMV 35S promoter [8]. Overall, Na+/K+ ratio in roots of other genotypes did not correlate positively with their salinity tolerance.

We identified many putative cis-acting regulatory abiotic stress-responsive elements. The identified core motif related to dehydration-responsive element/C-repeat (DRE/CRT), has been reported for its role in drought and high salt-stress conditions [37]. Another important cis-acting regulatory element, GT-1 was also found which has been predicted to be involved in gene induction (SCaM-4) under salt stress, in Glycine max [38]. Identified CAAT-box and ACGT cis-acting DNA sequences have been reported to be required for induction of various genes such as dehydration-responsive gene (AtNCED3) [39, 40]. Prabu and Parsad [41] demonstrated the putative role of Saccharum officinarum MYB gene and showed that the promoter activity was increased by 2- to 4-fold under salt, cold and dehydration stress. The indentified putative cis-acting elements (ACGT and MYC) were present in higher number in the promoter sequence of EbHKT1;2, whereas the GT-1 cis-element, CAAT-box and MYBCORE were present at higher numbers in A. thaliana. Baek et al. [30] have reported two tandem repeats (R1 and R2) in AtHKT1;1 promoter region (~ 3.9 kb upstream of the start codon) and suggest that these repeats are involved in AtHKT1;1 regulation under salt stress. Unfortunately, we could not include longer fragment of AtHKT1;1 promoter however, expression of AtHKT1;1 transcripts in transgenic lines harboring AtHKT1;1 under the control of its native promoter (0.846 kb from ATG), was comparable to wild type expression level (Fig. 1). This indicates that 0.846 kb of AtHKT1;1 promoter is functionally active and is complementing the Athkt1;1 mutant up to wild type level (Fig. 2). In any case, the role of above mentioned identified cis-acting elements in the Eutrema HKT1;2 promoters, if any, should be established by a series of promoter-deletion experiments.

Conclusions

Expression under the E. salsugineum and E. botschantzevii promoters did not yield any significant expression, either because of the lack of an essential upstream response element/s, or lacking an essential trans-acting regulator/s, in A. thaliana, and thus failed to complement the Athkt1;1 mutant. When the gene is expressed under AtHKT1;1 promoter, both AtHKT1;1 and EsHKT1;2 fully complemented the mutant, in that they restored a wild type-like salt tolerance level. Several abiotic stress-responsive element have been identified in EsHKT1;2 and EbHKT1;2 promoter regions.