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1769 Advances in Environmental Biology, 6(5): 1769-1773, 2012 ISSN 1995-0756 This is a refereed journal and all articles are professionally screened and reviewed ORIGINAL ARTICLE Assessment of Na+⁄ H+ Antiporters and H+-ATPase Pumps Transcriptional Changes in Aeluropus littoralis Dealing with Salt Stress 1 Nasiri Najmeh, 1Shokri Ehsan, 2Nematzadeh Ghorbanali 1 M.Sc in Plant Breeding, Genetic & Agricultural Biotechnology Institute of Tabarestan (GABIT), University of Agriculture Sciences and Natural Resources, Sari, Iran 2 Prof. in plant genetics, Genetic & Agricultural Biotechnology Institute of Tabarestan (GABIT), University of Agriculture Sciences and Natural Resources, Sari, Iran Nasiri Najmeh, Shokri Ehsan, Nematzadeh Ghorbanali; Assessment of Na+⁄ H+ Antiporters and H+ATPase Pumps Transcriptional Changes in Aeluropus littoralis Dealing with Salt Stress ABSTRACT NHX and SOS proteins are widely regarded as key players in the sequestration and efflux of sodium (Na+) in plant cells to avert ion toxicity in the cytosol under salinity stress. These proton-coupled sodium transporters use the proton-motive force created by the vacuolar (V-type) and plasma membrane (PM-type) H+-ATPase pumps. Here, we use Aeluropus littoralis as a halophytic monocot model to study salt-related transcriptional changes in NHX1, SOS1, and subunit c genes. The study provides evidence for induced expression of the Na+⁄H+ antiporters and PM-type H+-ATPase in response to salt stress. In addition it was found that the subunit c mRNAs of V-type pump was transcripted constitutively in A.littoralis shoots that maybe a halophytic trait. Finally the results suggest that a precise coordination at transcriptional levels between antiporters and H+ pumps was crucial during plant facing to high salinity. Key words: A.littoralis, NHX1, SOS1, H+-ATPase pumps, subunit c Introduction The C4 grass, Aeluropus littoralis, grows in the salt marshes and coastal zones. It can tolerate seawater salinity and the salinity fluctuation from water evaporation and tidal inundation, approximately from 300 mM to 600 mM. The salinity tolerance mechanisms of Aeluropus species include cellular, organizational, and whole plant adaptations, such as ion compartmentation, presence of salt glands on the leaves, ion exclusion at the root, and ion partitioning in divergent organs. Under salt stress, the maintenance of K+ and Na+ homeostasis is crucial [16]. Thus, understanding how plants cope with excessive Na+ in the environment is of great agricultural importance as soil salinity accounts for large yield losses in crops worldwide. Na+ stress disrupts K+ uptake by root cells [4]. When Na+ enters cells and accumulates to high levels, it becomes toxic to enzymes. To prevent growth cessation or cell death, excessive Na+ has to be extruded or compartmentalized in the vacuole. Unlike animal cells, plant cells do not have Na+-ATPases or Na+/K+-ATPases, and they rely on H+-ATPases and H+-pyrophosphatases to create a proton-motive force that drives the transport of all other ions and metabolites. Many of the transporters of H+, K+ and Na+ have now been identified. The regulatory mechanisms that control the expression and activity of the transporters are beginning to be elucidated. Vacuolar sequestration of Na+ not only lowers Na+ concentration in the cytoplasm but also contributes to osmotic adjustment to maintain water uptake from saline solutions. In plant, the NHX family of Na+/H+ anti-porter function in Na+ compartmentation [9]. NHX1 are localized in the tonoplast membrane, and their transcript levels are up-regulated by salinity stress [18]. In addition to NHX, The plasma membrane Na+/H+ antiporter SOS1 is essential for the salt tolerance of various model plants, including Arabidopsis thaliana [1] and its halophytic relative Thellungiella salsuginea [11], Tomato [12], and the moss Physcomitrella patens [3]. SOS1 is thought to mediate Na+ efflux at the root epidermis and longdistance transport from roots to shoots [17,12] while protecting individual cells from Na+ toxicity [3-20]. SOS1 is also indirectly required for the uptake of potassium (K+) in the presence of Na+, although the mechanistic basis is not fully understood [3,20,21]. The transcript level of SOS1 is up-regulated by NaCl stress but not by drought, cold, or abscisic acid [17]. Salt up-regulation of SOS1 expression is partially under the control of the SOS2/SOS3 regulatory pathway [17,22,7,5]. The activity of NHX and SOS Corresponding Author Shokri Ehsan, M.Sc in Plant Breeding, Genetic & Agricultural Biotechnology Institute of Tabarestan (GABIT), University of Agriculture Sciences and Natural Resources, Sari, Iran E-mail: [email protected] 1770 Adv. Environ. Biol., 6(5): 1769-1773, 2012 transport system is driven by the proton-motive force generated by the vacuolar membrane H+-ATPase, H+-pyrophosphatase and plasma membrane H+ATPase respectively. The direct stimulation of the vacuolar and plasma membrane Na+/H+ antiport system may be coordinated with the increased activity of the v-type and pm-type H+ pumps, which provide the driving force for the operation of the cation exchanger. It has been shown that transcript amount of V-type H+-ATPase different subunits shows endogenous oscillations in response to salinity [14]. More ever accumulation of mRNAs of PM H+ATPase under NaCl stress and the positive correlation with salt tolerance are well documented [10,13,6,15]. Materiels and Methods Plant Samples Preparation: Seedlings of halophyte plant A.littoralis were irrigated daily with half-strength Hoagland nutrient solution according to evaporation demand. Six-weekold seedlings were treated with 0 (control), 200 and 400 mmol/L NaCl. The final salinity level was achieved by raising 100 mmol/L NaCl every 24 h in order to avoid osmotic shock. Plant samples (shoot) were taken for analysis 7 days after salt exposure. The shoot Na+ and K+ concentrations were determined with a Shimadzu AA-680 atomic absorption / flame spectrophotometer. Semi-quantitative RT-PCR: Oligonucleotide primers for amplification of PCR products of between 350 and 600 bp from A. littoralis were designed using Oligo 7 software according to the highly conserved amino acid sequence of diverse genes (NHX, SOS1, PMATPase,VHA-c) obtained from GenBank. Total RNA extracted using TRIZOL reagents (Invitrogen, Inc.,CA,USA) and was exposured to Rnase free DnaseI treatment according to Fermentas procedure to eliminate residual genomic DNA. Single strand cDNA was then obtained using 1µg of total RNA and oligo (dT) primers, with the RevertAid Reverse Transcriptase System (Fermentas) for RT-PCR. After RT, cDNA was amplified for 19-21 cycles (94ºC 45s, 50.3ºC -45s, and 72ºC -90 s), which was determined to be within the linear range of amplification. For equal loading of the first standard cDNA in RT-PCR reaction, the concentration must be adjusted according to an internal control gene, so the housekeeping gene β-actin was used as a control for variations in the input of RNA. Result: Quantification of NHX1 transcripts in A.litorallis shoot parts via semi-quantitative RT-PCR showed that salinization was affected NHX1 levels positively, as with increasing salinity NHX1 levels increased. In comparison with control, the mRNA level of NHX was 2.9 and 3.6 fold higher in 200 mM and 400 mM stressed plants respectively (Fig. 2). In the presence of NaCl, SOS1 transcripts were also increased in shoot parts of A.litorallis when compared with normal condition. SOS1 mRNA abundance higher in 200 mM and 400 mM stressed plants while in nonstress condition no visible band was amplified for SOS1 gene one week after salt exposure (Fig. 2). More ever NHX1 and SOS1 mRNA levels in 400 mM stressed plant was higher than 200 mM treated plants in shoot parts. The transcript level for V-H+-ATPase subunit c was found to be constitutively expressed in shoot tissues of A.litorallis plant and there wasn’t a significant difference between non-stress and treated plants (Fig. 2). Finally the transcripts of plasma membrane H+-ATPase in shoot of A.litorallis plants was up-regulated in response to salinity levels but there wasn’t a significant difference between 200 and 400 mM treated plants. Also the PMA transcripts in non-stress plants were not visibly amplified (Fig. 2). Shoot K+ to Na+ ratio drastically decreased with increase in salinity levels in A.litorallis. Sodium increased considerably while K+ slightly decreased. In comparison to control, Na+ accumulation was greatly enhanced in shoot parts of salt treated plants, but when salinity severity increased no significant difference was observed between 200 and 400 mM treated plants for accumulated sodium ions. In contrast, shoot potassium content reduced in salt treated plants in comparison to control (Fig. 1A & B). Discussion: Transcriptional changes of subunits of the vacuolar H+-ATPase in response to salinity stress have been reported from a number of plants. Saltinduced transcriptional activation of V-ATPase subunits A, B, E, and c have been shown in common ice plant [2, 6, 19]. In this work we have shown that the expression of V-ATPase subunit c was not changed at transcriptional level in A.littoralis shoots one week after salt stress. The constant V-H+ATPase transcript amounts maybe had a close relationship with the activity of V-H+-ATPase, which provided the energy for the compartmentalization of sodium in response to salinity. It is apparent that salinity affects vacuolar V-ATPase subunits gene expression differently and keeps up high mRNA levels of V-ATPase subunit c constantly in A.littoralis shoots maybe halophytic trait. In addition the mRNA level of NHX was about 3.5 fold higher in salt treated plants indicated that Na+ sequestration in vacuoles play a critical role in establishing salt tolerance. This suggests that the salt sequestration capacity of shoot parts including leaves, stem, nodes and leaf sheaths allows the plant to accumulated 1771 Adv. Environ. Biol., 6(5): 1769-1773, 2012 higher amounts of sodium chloride in the early days of dealing with salt stress. Moreover, it has been well documented that salt treatment of plants induces the activities of the plasma membrane proton pumps both in Halophytes and Glycophytes. Accumulation of mRNAs of PM H+-ATPase under NaCl stress and the positive correlation with salt tolerance are well documented [10, 13, 6, 15]. In Suaeda maritima (a natural halophyte) and Oryza sativa salt-tolerant cultivar accumulation of PM H+-ATPase gene transcript was greater than that in a non-tolerant cultivar of rice treated with NaCl [15]. In this study when Aeluropus plants were treated with NaCl, plasma membrane H+-ATPase transcripts increased. Parallel to this increase in PM H+-ATPase transcripts, the SOS1 mRNAs also increased in treated plants and such an increase in SOS1 levels was more evident at 400 mM treatment. This saltinducible transcription in both PM H+-ATPase and SOS1 levels could be attributed to relatively high Na+/H+ exchange activity in plasma membrane site that efficiently extruded Na+ from cells. Fig. 1: Effect of salinity on Sodium and Potassium concentrations (A) and the ratio of K+ to Na+ in shoot parts of A.littoralis (B). Six-week-old seedlings were treated with 0 (control), 200 and 400 mM NaCl. Fig. 2: Transcripts amount of NHX1, SOS1,V- type H+ATPase (subunit c) and PM- H+ATPase genes in the shoot part of A.littoralis under different concentration of NaCl. A. littoralis β-actin gene was used as an internal control for the same quantity of total RNAs. Experiments were repeated at least three times to obtain similar results. Conclusion: Salinity tolerance of plants is a complex trait involving adaptation at the level of cells, organs and the whole plant. 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