Introduction

Excessive amounts of soluble salts in soil negatively affect plant growth and development, because plants subjected to salt stress uptake considerable amount of toxic ions including sodium (Na+) and chloride (Cl−). To minimize the hazardous effects of sodium ions on cell membrane, plants use three mechanisms for prevention of Na+ accumulation in cytoplasm, i.e., restriction of Na+ ion influx, active Na+ ion efflux, and compartmentalization of Na+ ions in the vacuole [1]. Various kinds of membrane proteins such as ion pumps, transporters, and channels are directly involved in maintenance of cellular ion homeostasis under salt stress [2].

In plant cells, the transporters for K+, Na+, and/or Ca2+ are important in Na+ sensation. Plants maintain their homeostasis by keeping a high K+ concentration in the cytosol with concomitant decrease in Na+ ions, achieved by regulation of the expression and/or activities of K+/Na+ transporters as well as of the proton pumps (H+ pumps), generating the force for this purpose [3]. The maintenance of Na+–K+ homeostasis showed importance in plants grown under salt stressed conditions as Na+ stress has toxic effects on enzymes and also was the basis for disruption of K+ uptake by root cells [4].

The Ca2+ ions play crucial role in activation of salt overly sensitive (SOS) pathway to maintain ionic stability in plants. It binds to the Ca2+ sensor protein SOS3, a component of SOS pathway regulating cell’s ionic homeostasis, which in turn has a physical association with serine/threonine protein kinase, SOS2 to activate it. The activated SOS2 regulates the activities of SOS1, the plasma membrane Na+/H+ antiporter and the tonoplast Na+/H+ antiporter, regulating Na+ efflux in plasma membrane and vacuolar compartmentation, respectively [5]. Here, we focused on the expression of SOS2 gene in sunflower which ultimately mediates Na+ efflux by activating SOS1 gene expression. SOS2 functions in Na+ tolerance, regulating Na+ and K+ homeostasis, but not in metabolism. In Arabidopsis thaliana, the SOS2 gene was found to be required for intracellular Na+ and K+ ion homeostasis and mutations in SOS2 render plants more sensitive to high Na+/K+ ratio in the saline environments [6].

“SOS pathway” is the direct Na+ influx/efflux controlling system in plants. Besides that, plants have also been reported to have yeast plasma membrane protein 3 (PMP3)-like proteins, plasma membrane protein 3 functioning in regulation of cellular Na+ and K+ ion accumulation under salt stress [7]. Yeast plasma membrane protein, PMP3, was identified with high-sequence similarity to AtRCI2A and AtRCI2B (Arabidopsis rare cold-inducible genes 2A and 2B) proteins [8]. The AtRCI2A- and RCI2-related proteins, WP16 from wheat [9] and AcPMP3 from sheep grass [7] have been reported to reinstate the plasma membrane potential as well as PMP3− phenotype of yeast cells [10]. With all that, little is known about the particular role of AtRCI2A and AtRCI2B in stress response [11].

APMP3 was found localized in the root cap of a monocotyledonous halophyte Aneurolepidium chinense, regulating the Na+ and K+ accumulation in that tissues [7]. However, it is still to be determined whether its analog has been found in other halophytes or glycophytes. We found, in the present work, the expression of PMP3-1 gene in sunflower, a glycophytes, by designing the primers as described by Inada et al. [7] for A. chinense (AcPMP).

Recently, it has been established that many salt-tolerant determinants are ubiquitously found in plants. This has led to the use of classical genetic models, i.e., Arabidopsis thaliana, to further dissect salt stress responses in crop plants, for example sunflower [12]. Sunflower is considered as moderately salt sensitive [13]. Here, the genes studied in this crop, SOS2 and PMP3-1, are in fact involved in Na+ efflux mechanisms in plants subjected to salt stress. In fact, this work was the extension of the previous work on identification of relatively salt-tolerant sunflower cultivars from different available potential germplasms using photosynthetic attributes, proline contents, and accumulation of inorganic nutrients as selection criteria of salt tolerance. For improvement of salt tolerance in sunflower, the expression of SOS2 (regulating Na+ efflux) and PMP3-1 (prevention of Na+ entry) genes in this crop plant may be escorted to establish the salt-tolerant lines for the crop by controlling the Na+/K+ regulatory activity of these genes. This is the first report about the expression of root-cap PMP3 in glycophytes such as sunflower that may exploit for constructing better salt adopted lines of this important edible oil-crop.

Materials and Methods

Our earlier study on discrimination of eight sunflower (Helianthus annuus) cultivars for salt tolerance, cvs. Hysun-38 and S-278 were found to be salt tolerant and moderately salt tolerant, respectively, on the basis of percent reduction in shoot biomass [14]. For the gene expression study of SOS2 and PMP3-1 proteins regulating Na+–K+ ion homeostasis, seeds of Hysun-38 (salt tolerant) and S-278 (moderately salt tolerant) lines of sunflower were sown in washed moist sand, filled in plastic pots, and grown in a growth room at 26 °C with a 12-h photoperiod. Five-day-old seedlings were fertilized with full strength Hoagland’s nutrient solution [15]. After a 2-week growth, the control plants remained in the nutrient medium, but the experimental plants were irrigated with 150 mM NaCl added to the Hoagland’s nutrient solution. The seedlings were harvested after a sudden salinity (150 mM) shock at 0, 6, 12, and 24 h. These time interval were chosen based on some previous studies which show expression of different genes soon after the salinity shock to different plant species [16–18]. Plants collected at each time interval, at least 10 per sample, were frozen in liquid N2 for expression study.

Total RNA Extraction and RT-PCR

Total RNA was extracted from the young leaf and root tissues of the plants treated with 150 mM NaCl for 24 h using the plant RNeasy system (Qiagen, Missisauga, Ontario, Canada) following the manufacturer’s instructions. The total RNA was quantified with a Gene Quant Pro (Amersham Biosciences) spectrophotometer and fractionated in a 1 % agarose gel. For cDNA synthesis and PCR amplification of the isolated fragment, One Step Reverse Transcription PCR (RT-PCR) Kit was used according to the supplier’s instructions (Novagen) for gene expression analysis, as it can replace methods for detecting and quantifying gene expression such as Northern blots, in situ hybridization, dot blots, S1 nuclease assays, and conventional two-step RT-PCR (the two enzyme/two buffer system). The primer pairs for SOS2 gene were synthesized from Genelink, USA, for the amplification of PCR products between 50 to 120 bp as described by Halfter et al. [19]. The sequence was: 5/-AATTTGGATGATATTCGTGCAGTTTTTG-3/ and 5/-TTAACATTTAAATGGAATTGACC-3/. The primer pairs used for PMP3-1 were as described by Inada et al. [7] for A. chinense (AcPMP). The sequences were 5/-CGATATTTTCAAGTCCCTCCTTGC-3/ and 5/-CTGAACAAATCAAATGGAAACCCG-3/.

These primer pairs were used to amplify the respective gene fragments by reverse transcription-PCR (RT-PCR). The primers designed were able to amplify similar-sized PCR products (∼100 bp).

RT-PCR Conditions

RT-PCR was performed according to the instructions by One-Step RT-PCR System (Novagen) with a Perkin Elmer GeneAmp PCR system 2400. RT-PCR reaction was performed with following conditions: reverse transcription, 30 min at 60 °C; initial PCR activation, 2 min at 94 °C; denature, 1 min at 94 °C; annealing/extension, 90 s at 60 °C; repeat for 40 cycles; and final extension, 7 min for 60 °C. The “Tm” for different primer pairs was chosen with an annealing temperature about 5 °C below. For each PCR reaction, 1 μg of the total RNA was added in a mixture containing 5× reaction buffer, 2.5 mM dNTPs, 25 mM Mn(OAc)2, 10 pmol/μL of each gene-specific primer pair, 10 units/μL RNase Inhibitor. Five U rTth polymerase was added to each tube with 50 μL of a total volume of the reaction mixture. The results were analyzed on a 1 % agarose gel stained with ethidium bromide using the Bio-Imaging System (Gene Genius).

Results

RT-PCR Analysis of Sunflower Seedlings

Total RNA extracted from control and NaCl-treated leaf and root tissues of two sunflower lines was measured spectrophotometrically and fractionated on a 1 % agarose gel (Fig. 1a and b). To observe the expression of SOS2 and PMP3-1 genes in sunflower, gene-specific primers were used as described in section of materials and methods. The primers designed were able to amplify similar-sized PCR products (∼100 bp). Salt-responsive cDNAs from sunflower young tissues treated with 150 mM NaCl treatment for 24 h were amplified via reverse transcription PCR (RT-PCR).

Fig. 1
figure 1

Total RNA extracted from two sunflower lines. a Total RNA isolated from salt-tolerant line (T) of control and salt-treated sunflower root tissue harvested at 0, 6, 12, and 24 h after the imposition of salt stress. M is the 1 kb DNA ladder. b Total RNA extracted from control and salt-treated root tissue of moderately salt-tolerant sunflower line (MT) at different time intervals

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SOS2 Expression in Sunflower Leaf and Root Tissues Under Salt Stress

Our results of study on SOS2 expression in sunflower root showed that SOS2 was expressed at a low level in the root tissue without salt treatment while its expression increased after the imposition of salt treatment and reached its highest level after 24 h of salt treatment. This can be clearly examined in Fig. 2a. The results suggest that the expression of SOS2 was salt-inducible in the root. However, in the leaf tissue of tolerant line, SOS2 expressed minimally without salt treatment, but its expression increased after the imposition of salt treatment and reached its highest level after 12 h of postsalinity treatment. Thereafter, the expression decreased particularly at 24 h of salt treatment (Fig. 2b). The moderately salt-tolerant line showed a relatively low level of SOS2 expression consistently at all time intervals.

Fig. 2
figure 2

SOS2 gene expression in different tissues of two sunflower lines at different time intervals. a A gradually increased SOS2 expression in sunflower root tissues at different time intervals after the initiation of NaCl treatment in moderately salt-tolerant and salt-tolerant plants. M represents 1 kb DNA marker for which last band is of 250 bp. All the amplified bands are of similar size (∼100 bp). b A gradual increase in SOS2 expression from control to treated plants in the tolerant sunflower line, while the moderately-tolerant line showed a relatively lower level of expression in the leaf tissues

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PMP3-1 Expression in Sunflower Root Tissues under Salt Stress

Sodium salt (150 mM NaCl) in the nutrient medium induced the PMP3-1 expression immediately within 24 h in the root tissues. Figure 3 clearly indicates that the PMP3-1 expression level was very low in the untreated control plants (0 h) than the treated ones in the root tissues of the both sunflower lines under salt stress. The expression level of PMP3-1 in tolerant sunflower line was highest at 6 and 12 h after the initiation of salt treatment and then went to 24 h with slightly lower level of expression (Fig. 3a). In contrast, induction of PMP3-1 was weaker in the moderately salt-tolerant sunflower line than that in the tolerant one, showing the highest level at 12 and 24 h of the salt treatment (Fig. 3b).

Fig. 3
figure 3

PMP 3-1 gene expression in roots of two sunflower lines at different time intervals. a Highest level of PMP 3-1 expression was seen in the tolerant sunflower line at 6 and 12 h of the initiation of 150 mM NaCl treatment. M represents short range DNA markers for which last band is of 100 bp. All amplified bands are of similar size (∼100 bp). b The moderately-tolerant line showed higher PMP3-1 expression at 12 and 24 h of salt treatment. M is the 1 kb DNA ladder with last band of 250 bp

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Discussion

SOS2 Upregulation by Salt Stress

Our earlier studies on identification of relatively salt-tolerant sunflower cultivars/lines by screening different available potential germplasm has shown the markedly enhanced C i /C a ratio, free proline content, and leaf and root Na+ concentrations in all (eight) sunflower cultivars studied under salt stress [14]. The increased Na+ concentration in cells is compensated by SOS pathway of ion homeostasis regulation in plants. Yang et al. [20] has confirmed the overexpression of SOS genes in transgenic Arabidopsis plants in conferring the increased salt tolerance.

To investigate the gene expression pattern of one of the SOS components, SOS2 (regulator of Na+ efflux), under salt stress and to describe if sunflower SOS2 gene is salt inducible, total RNA was extracted from leaf and root tissues of sunflower seedlings and subjected to RT-PCR analysis. SOS2 was found to be expressed in both the root and leaf tissues of sunflower seedlings and found upregulated by salt stress in the root tissues. Shahbaz et al. [14] has reported the significantly increased Na+ concentrations in the leaves and roots of eight sunflower cultivars studied after application of salt (150 mM NaCl) in the rooting medium. Considerably higher Na+ concentrations were found in leaf of tolerant sunflower line (Hysun-38), while the highest Na+ concentrations were noted in roots of moderately-tolerant sunflower line (S-278) under saline conditions. We found the upregulated SOS2 expression in roots describing its enhanced role in Na+ efflux from this tissue, which may then be passed to leaves for compartmentation in vacuoles.

SOS2 was found to be expressed gradually in seedlings of tolerant sunflower line with low intense band at 0 h to highest expression at 24 h post-NaCl treatment. The results presented in Fig. 2a suggest that the SOS2 expression was salt-inducible in root tissues of sunflower seedlings. This was parallel to the results obtained for the expression of AtSOS2 in A. thaliana by Liu et al. [6]. They found the SOS2 expression in both the root and shoot of A. thaliana, but the gene expression was up-regulated by salt stress in the root tissues. In A. thaliana, the expression of AtSOS2 was found to be upregulated in the root at salt (200 mM NaCl) treatment. Wang et al. [16] observed the upregulated expression of BnSOS2 by NaCl stress in the root, but the significance of this upregulation was not understood. They proposed that during salt stress, the slight upregulation of the gene expression may be important to maintain a sufficient level of SOS2 protein in root tissues.

In the leaf tissue of tolerant line, SOS2 expressed minimally particularly under control (nonstress) conditions, but its expression increased after the imposition of salt treatments and reached its highest level at 12 h postsalinity treatment. The expression then decreased at 24 h of salt treatment (Fig. 2b). These results are parallel to those obtained for the expression of AtSOS2 in A. thaliana [6]. Liu et al. [6] found in A. thaliana shoot tissue, a slight upregulation of SOS2 transcript after 3 or 6 h of salt treatment, but the AtSOS2 expression began to decrease after 12 h postsalt treatment. Similar results were also found for BnSOS2 in Brassica napus [16]. BnSOS2 expression was low in plants without salt treatment, but it reached at its highest level after 12 h of salt treatment and then decreased similar to that observed for the expression of AtSOS2 in A. thaliana [6]. We found in moderately salt-tolerant sunflower line (S278) the relatively lower level of SOS2 expression consistently at all time intervals. Therefore, the expression pattern of sunflower SOS2 was found similar to that of AtSOS2 from A. thaliana and BnSOS2 from B. napus, indicating the salt inducible nature of sunflower SOS2 expression with increased induction level in tolerant plants to better adapt their metabolism under salt stress conditions.

PMP3-1 Induction Pattern in Sunflower Roots Under Salinity Stress

PMP3 is a small hydrophobic peptide first described in yeast and is involved in preventing Na+ entry [8]. Its deletion was shown to cause higher sensitivity of yeast cells to salt stress due to Na+ over-accumulation. Plant cells also show similar regulation of ion homeostasis and have yeast PMP3 like proteins [7]. Inada et al. [7] suggested the role of AcPMP3-1 as a regulator of accumulation of both Na+ and K+ in the cells. The in situ hybridization studies carried out by Inada et al. [7], showed the root cap and root epidermis localized expression of AcPMP3-1 transcript, which strongly suggests the essential regulatory role of AcPMP3-1 in Na+/K+ transportation between plant roots and the outer environment under salt stress.

In this study, we observed the PMP3-1 induction pattern in root tissues of two contrasting sunflower lines under NaCl stress (150 mM). The results showed the PMP3-1 induction within 24 h of salt stress with a very low expression level in control (0 h) plants than that in the treated ones. In the tolerant sunflower line the PMP3-1 expression level was highest at 6 and 12 h of salt treatment; however, it was slightly reduced at 24 h of stress (Fig. 3a). The induction of PMP3-1 was weaker in the moderately-tolerant sunflower line than that in the tolerant one showing the highest level of expression at 12 and 24 h of the salt treatment (Fig. 3b). Inada et al. [7] found the highest amount of AcPMP3-1 transcript at 1 and 3 h after salt treatment but the induction was transient (gone by 24 h). They observed the highly upregulated expression levels of the two AcPMP3 genes under cold and drought stresses. Likewise, they also observed the early induction of AcPMP3 expression by H2O2 after 15 min of treatment. The difference in PMP3-1 expression in A. chinense (sheep grass) and sunflower (oil seed crop) as observed in the present study may mainly be due to the difference in the plant species. The slightly earlier expression of PMP3-1 in the tolerant line suggests the more protective effect of this protein on regulation of ion homeostasis in root tissues. In contrast, the moderately-tolerant sunflower line required some lagged time for PMP3-1 expression under saline environment, which may be involved in processes to make this line more adapted to the prevailing stress conditions.

In conclusion, the results presented here clearly show that the salt-induced tolerance would be conferred in sunflower by enhanced activities of two important ion homeostasis-regulating proteins “SOS2” and “PMP3-1”. Present results may be significant for the understanding of the mechanisms that determine intracellular ion homeostasis especially the Na+ and K+ ion homeostasis regulated by both of these proteins in plants to improve salinity tolerance.