Isolation and Semi Quantitative PCR of Na+/H+ Antiporter (SOS1 and NHX) Genes under Salinity Stress in Kochia scoparia

Isolation and Semi Quantitative PCR of Na+/H+ Antiporter (SOS1 and NHX) Genes under Salinity... Background: Kochia scoparia is a dicotyledonous annual herb and belongs to the Amaranthaceae family. Genetic diversity and resistance to drought stress of this plant has made it widely scattered in different regions which contains highly genetic diversity and great potential as fodder and can grow on salty, drought affected areas. Since the soil salinity has become widely spread, environmental concern has sparked so many debates. An important limiting factor in agricultural production worldwide is the sensitivity of most of the crop to salinity caused by high concentration of salts soil. Plants use three different strategies to prevent and adapt to high Na concentrations. + + Antiporters are important category of genes that play a pivotal role in ion homeostasis in plants. Na /H antiporters (NHX1 and SOS1) are located in tonoplasts and reduce cytosolic Na concentration by pumping in the vacuole whereas SOS1 is localized at the plasma membrane and extrudes Na in apoplasts. + + + + Results: Coding sequence of plasma membrane Na /H antiporter (SOS1) and vacuole membrane Na /H antiporter (NHX) in Kochia scoparia were isolated using conserved sequences of SOS1 and NHX. Also, expression profile under salinity stress was studied in this study. The amino acid sequences (aa) of the isolated region of K.SSOS1 and K.SNHX showed the maximum identity up to 84% and 90% to its orthologous in salicornia brachiate and suede maritime, respectively. The results of semi-quantitative RT-PCR revealed that salinization has affected positively on SOS1 transcription level. The expression of K.SSOS1 and K.SNHX in leaves and roots of Kochia scoparia were progressively increased under all salinity levels compared to control. Conclusion: The results suggest that K.SSOS1 and K.SNHX play an essential role in salt tolerance of K.scoparia and they can be useful to improve salt tolerance in other crops. + + Keywords: Kochia scoparia,Na /H antiporters, Salinity tolerance, Semi-quantitative RT-PCR Background by high salt concentration, ultimately causing yield lost. Sal- Most of studies have revealed that the greatest lost in vari- inity stress can reduce the productivity of glycophytes, ous crop production is due to abiotic stresses, such as, sal- which are the majority of agricultural products. High salt inity, water deficit, low temperature and heavy metals concentrations cause hyper osmotic stress and ion imbal- adversely affect the growth and several physiological pro- ance in plants which often as a secondary effect leads to cesses such as leaf cell growth and biomass production of oxidative damage in cellular components [1]. Plants adapt plants. An important limiting factor in agricultural produc- to environmental stresses via responses, including the tion worldwide is the sensitivity of most of the crop to sal- activation of molecular networks that regulate stress per- inity caused by high concentration of salts soil. Processes ception, signal transductionand theexpressionof both such as seed germination, seedling growth and vigor, vege- stress related genes and metabolites [2]. Plants have stress tative growth, flowering and fruit set are adversely affected specific adaptive responses as well as responses which pro- tect the plants from more than one environmental stress * Correspondence: l.fahmide@gmail.com [2]. Plants employ three different strategies to prevent and Department of Plant Breeding and Biotechnology, University of Zabol, Zabol + + adapt to high Na concentrations: 1) active Na efflux, 2) 98613-35856, Iran + + Na compartmentalization in vacuoles, and 3) Na influx Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 2 of 9 prevention [3, 4]. Antiporters are important groups of structure characterized with in silico tools. Furthermore, genesthathavea keyroleinion homeostasisinplants. profiling gene expression for two gene characterized. + + Na /H antiporters (NHX1 and SOS1) maintain the K. scoparia is an attractive plant model for study the appropriate concentration of ions in the cytosol, thereby mechanism of salt tolerance. This work to gain insights minimizing cytotoxicity. NHX1 are located in tonoplasts into the role played by this transporter in K. scoparia and reduce cytosolic Na concentration by pumping it in halophyte. the vacuole [5], whereas SOS1 is localized at the plasma membrane and extrudes Na in apoplasts [6]. Both of these Methods antiporters are driven by a motive proton force generated Genetic Samples by the H -ATPase [7]. The SOS signaling pathway consists K.scoparia was collected from Sabzevar in Khorasan of three major proteins including: SOS1, SOS2, and SOS3. Razavi Agricultural Research Center (Iran) and planted + + SOS1, which encodes a plasma membrane Na /H in Biotechnology research Center University of Zabol. antiporter, is essential in regulating Na efflux at the cellular The fresh leaves were applied to isolate RNA after salin- level. It also facilitates long distance transportation of Na ity stress (plants were irrigated by 100 mM, 200 mM, from root to shoot. Over expression of this protein leads to 300 mM and 400 mM sodium chloride solutions). salt tolerance in plants [8]. Activation of SOS1 by direct phosphorylation of the self-regulation scope is possible by Primers Design serine/threonine protein kinas or SOS2 that requires calcium Seventeen SOS1 and 22 NHX coding sequences data binding protein or SOS3 [9]. C-terminal end of the protein which are available at NCBI data base(ncbi.nlm.nih.gov) causes the Na to move. At the C-terminal end, SOS1, the have been showed in Table 1, were aligned by ClustalW 764,849 region is cyclic nucleotide-binding site and in the method provided in DNASTAR Laser gene software 998–1146 region a self-regulator domain exists. In the res- (EditSeq, Meg-Align, Version 5.00), GENEDOC (Mul- pite state the self-regulator domain interacts with upstream tiple Sequence Alignment Editor and Shading Utility sequence bearing the cyclic nucleotide-binding site [9]. In Version 2.5.000). All specific primers designed based on fact, the self-regulator domain is a target location for phos- the most conservative parts of the alignments. Specific phorylation by SOS2. After SOS1 phosphorylation, the self- forward and reverse primers were designed (Table 2). regulator domain leaves upstream location and attaches at this location of cyclic nucleotide and transferring pro- RNA Isolation and cDNA Amplification in K.scoparia tein activity begins [9]. According to the above- Total RNA of samples was isolated by Total RNA isola- mentioned information, domain connected to the cyclic tion kit (DENA Zist Asia). The cDNA(s) were synthe- nucleotide can be used as one of the most important sized using Hyper script reverse transcriptase (Gene All) locations to regulate SOS1 activity, eventually its effect and oligod (T) 18mer, P.SOS.S-REV1, P.SOS.S-REV2, P. on salinity tolerance. K. scoparia,a dicotyledonous SOS.S-REV3, P.SOS.S-REV4, P.NHX.S.REV1, P.NHX.S. erect annual herb belongs to Amaranthaceae family REV2 and P.NHX.S.REV3primers (Table 1) and ampli- with high genetic diversity and great foliage potential fied with a combination of primers (Table 2). The ampli- [10], reported that K. scoparia on of its Iranian variety fications were obtained in 30 cycles at defined annealing is highly tolerant to salt and could be considered as a temperature for each pair of primers using TaqDNA foliage species in cold regions of the world. Rapid vege- polymerase (AMPLIQON). The process finished after a tative growth under high salinity and temperature and final extension for 5–15 min at 720C (Fig. 1). drought and stress makes it a very valuable candidate as a non-conventional foliage crop for arid temperate Sequencing of SOS1 and NHX in K.scoparia regions [11]. K. scoparia has been widely used in Chin- PCR products were extracted and purified from 0.8% ese and Korean traditional medicine as a treatment for agarose gel using GEL recovery DNA kit (DENA Zist skin diseases, diabetes, mellitus, rheumatoid arthritis, Asia). PCR reactions were sequenced utilizing Euro fins liver disorders, and jaundice [12, 13]. Kochia seeds con- MWG Operon company service. Sequence analysis in- tain an ovi position pheromone that can be added as an cluding deletion of error in sequences, assembly of frag- attractant for mosquito pesticides [14, 15]. It has been ments, alignment with other plant species gene reported that seeds of Kochia also contain other chemi- sequences, was done using DNA STAR Laser gene soft- cals that could be beneficial for human, such as com- ware (EditSeq, SeqManII Meg-Align, MapDraw; pounds used in ulcers, rheumatoid arthritis, treatment Version5.00), GENEDOC (Multiple Sequence Alignment and some pathogenic bacteria [15–17]. The aim of this Editor and Shading Utility Version 2.5.000 and NCBI study was to investigate the presence of SOS1 and BLAST [18]. The amino acid sequences were aligned NHX1 genes and trace it using by induced salt stress in with CLUSTALW software. SOS1 and NHX nucleotides Kochia scoparia, Futures of these genes in protein and amino acid sequences aligned and analyzed with Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 3 of 9 Table 1 Plant species and accession numbers of gene sequences used for primer design alignment Gene Plant species Coding sequence length Accession number SOS1 Salicornia brachiata 3541 bp EU879059.1 Salicornia dolichostachya 3601 bp HG799054.1 Suaeda japonica 3481 bp AB198179.1 Suaeda salsa 3541 bp KF914414.1 Halogeton glomeratus 3481 bp KT759142.1 Spinacia oleracea 3301 bp HG799055.1 Mesembryanthemum 3421 bp EF207776.1 crystallinum 3541 bp JX674067.1 Sesuvium portulacastrum 3481 bp KM986873.1 Gossypium hirsutumvoucher 3421 bp JN936862.1 Aeluropus littoralis 3421 bp NM_012801211.1 Vitis vinifera 3481 bp GU177864.1 NHX Salicornia europaea 2161 bp AY131235.1 Salicornia bigelovii 2161 bp DQ157454.1 Salicornia brachiata 1681 bp EU448383.1 Kalidium foliatum 1681 bp AY825250.1 Halostachys caspica 1621 bp GU188850.1 Salsola komarovii 2161 bp AB531436.1 Suaeda pruinosa 1621 bp KJ452342.1 Salsola suaeda 1621 bp EU073422.1 Atriplex gmelini 2341 bp AB038492.1 Suaeda japonica 1681 bp AB198178.1 Suaeda salsa 1801 bp AF370358.1 Suaeda corniculata 1621 bp DQ512716.1 Atriplex halimus 1621 bp KJ452341.1 Suaeda maritima 1621 bp AY261806.1 Chenopodium glaucum 1621 bp AY371319.1 Atriplex patens 1621 bp KC608048.1 Atriplex dimorphostegia 1621 bp AY211397.1 Populuse uphratica 1621 bp DQ414512.1 Glycine max 1501 bp JN872904.1 Table 2 Primers sequences and names were developed for isolation and gene expression of SOS1, NHX in K.scoparia Gene Primers Name Primers Sequences application SOS1 P.SOS.S.FWD1 P.SOS.S.FWD2 P.SOS.S.FWD3 5- ATG GCA GCA TCT CGA A-3 P.SOS.S.FWD4 5- ACT GGA ACA CTG TTT-3 P.SOS.S.REV1 P.SOS.S.REV2 5- CAA ATG GGA TCT GGC T-3 P.SOS.S.REV3 5- CAC TTT TGG GAG ATG GT-3 P.SOS.S.REV4 5-AGA AAA CAA ACA ATG TTC C -3 P.SOS.S.FWD1 5- CCG TTT GAT ATA AGC CA-3 5- GAG ATT ACT TGG TGA ATC-3 5- CAA TAA CAC TTT CCT TCC A-3 5- ATG GCA GCA TCT CGA A-3 NHX P.NHX.S.FWD1 5-ATGTGGTCACAGTTAAGC-3 Gene sequencing P.NHX.S.REV1 5-ATAAGCCATAAGCATCAT − 3 P.NHX.S.FWD2 5-GTGAGGTTGCTTTAATG-3 P.NHX.S.REV2 5-CCAAATACAGGCCGCAT-3 P.NHX.S.FWD3 5-ACAGATTCTGTTTGCAC-3 P.NHX.S.REV3 5-CATAAGACCAGCCCACCA-3 P.NHX.S.FWD1 5-ATGTGGTCACAGTTAAGC-3 P.NHX.S.REV1 5-ATAAGCCATAAGCATCAT − 3 SOS1 P.SOS.G.F P.SOS.G.R 5-GGAAGGTTTGGGGATGGTAT-3 Gene expression 5-GTCCAGCAAGCAAACCATT-3 NHX P.NHX.G.F P.NHX.G.R 5-TTCTGGATTGCTCAGTGCTT-3 Gene expression 5-CAGCCAGCATGTAAGAGAGG-3 18srRNA Forward 5-ATGATAACTCGACGGATCGC-3 5-CTTGGATGTGGTAGCCGTTT-3 Gene expression Reverse Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 4 of 9 Molecular Docking to Predict SOS1 Protein of K.scoparia and Regulation in the Salt Stress Molecular docking of the desired fragments isolated from Kochia using SWISSDOCK was performed as followed procedure: First, tertiary structure of se- quence fragments was predicted in the by SWISS- MODEL is a fully automated protein structure hom- ology modelling server [19–22]. Then, target ligand, cAMP from Zinc dock using online service of Swiss Dock available on the Expasy site molecular docking. The best state for interaction was reported using UCSF-Chimera method [23]. Results and Discussions In this research, isolation of the coding sequence of + + Fig. 1 Regulation of ion homeostasis by ion Na+/H+ pumps plasma membrane Na /H antiporter (SOS1) and + + + + antiporters (SOS1), vacuolar Na /H exchanger (NHX) that salt vacuolar membrane Na /H exchanger (NHX) in Kochia sensors present at the plasma and vacuolar membranes [32] scparia was performed and, the consequence of salinity stress was studied on the expression profile of this gene. SOS1 and NHX from other plants (Fig. 3). Assembled We focused on SOS1 and NHX the critical genes in the sequences for gene submitted to DDBJ data base ad- SOS pathway and vacuolar membrane for the resistant dressed: ddbj.nig.ac.jp and assigned accession number to salt stress (Fig. 1). The SOS pathway and vacuolar + + (LC218450, LC218451 for SOS1 and NHX gene membrane Na /H exchanger (NHX) are currently the respectively). most extensively studied mechanisms in controlling the salt stress response in plants. The SOS and vacuolar + + RT-PCR for Analyzing of SOS1 and NHX Gene Expression membrane Na /H exchanger (NHX) pathway is in K.scoparia under Salt Stress responsible for ion homeostasis and salt tolerance in Eight-week-old seedlings Plants were treated in Salinity plants. stress. Afterward plants were irrigated by 150 mM, 300 mM sodium chloride solutions. Then, sampling was Conserved Domains, Homology and Phylogenetic done during 12, 48, 72 h after treatment. RNAs were ex- Analyses of SOS1 and NHX in K.scoparia tracted using Total RNA isolation kit (DENA Zist Asia, After sequencing the coding SOS1 and NHX genes se- Iran) from the treated seedlings according to manufac- quences in K.scoparia, Conserved domain specified using turer’s instructions. After Dnase1 treatment of RNA sam- of NCBI revealed that putative protein SOS1 belongs to the ples, 2 μgofRNAs, usingGeneAll first strandcDNA Sodium/hydrogen exchanger family; These antiporters con- Synthesis Kit, was reverse transcribed to cDNAs, that were tain 10–12 trans membrane regions (M) at the amino- used as templates for semi quantitative RT-PCR. The terminus and a large cytoplasm region at the carboxyl cDNA amounts were first normalized by 18 s rRNA PCR terminus. The transmembrane regions M3-M12 share the product intensity. PCR process was performed using the same identity with other members of the which family. The following procedure: 95 0C for 5 min followed by 35 cycles M6 and M7 regions are highly conserved. Thus, this is be- of 950C for 30 s, annealing temperature for 45 s, and 720C lieved to be the region involved in the transportation of so- for 1 min, and finally 15 min at 720C for final extension. dium and hydrogen ions. The cytoplasm region has little similarity throughout the family. Conserved domain Ana- Gel Analysis for Gene Expression of SOS1 and NHX in lysis for NHX showed that family represents five trans- K.scoparia under Salt Stress membrane helices. This suggests that the paired regions Images of the RT-PCR ethidium bromide-stained agar- form a ten-helical structure, probably forming the pore, ose gels were taken with a Vilber documentation system whereas the binds a ligand for export or regulation of the (E-BOX CX5) and Band intensity was expressed as rela- pore. The development of intracellular membrane systems tive absorbance units. The ratio between the sample and compartments has led to a considerable increase in the Total RNA and 18srRNA was determined and calculated number of ion transporters in eukaryote cells. As a result, to normalize for initial variations in sample concentra- plants contain a large number of sequences encoding pro- + + tion and as a control for reaction efficiency. Mean and teins that share homology to Na /H antiporters which are standard deviation of all experiments were calculated key transporters in maintaining the pH of actively after normalization to 18srRNA. metabolizing cells. According to highly similar sequences Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 5 of 9 Table 3 Analysis sequence alignment using MegaBlast (%identity) related to NHX and SOS1 genes isolated from K.scoparia NHX SOS1 No. Plant species %Identity Plant species Identity % 1 Suaeda maritima 90% Salicornia dolichostachya %84 2 Suaeda japonica 89% Salicornia brachiata %84 3 Atriplex gmelini 89% Suaeda japonica %83 4 Suaeda comigulata 89% Suaeda salsa %82 5 Atriplex halimus 89% Beta vulgaris %80 6 Chenopodium glaucum 89% Spinica oleraceae %81 7 Suaeda salsa 89% Sesuvium portulacastum %79 8 Suaeda pruinosa 88% Mesemebryanthemum crystallinum %77 9 Atriplex dimorphostegia 88% Vitis vinifera %78 10 Atriplex patens 88% Populus trichocarpa %76 11 Halostachy scaspica 88% Populus eupharatica %76 (Mega Blast) search for sequence homology of SOS1 and example; SOS1 gene that characterized in K.scoparia NHX genes in the NCBI data base on Table 3 was had maximum homology with Beta vulgaris, Salicorni provided. Maximum identity up to90% with Suade abrachiata, Spinaceaeoleraceae and suadea salsa, While, amaritima of NHX gene and for SOS1 gene is 84% by show the lowest similarity to Helianthus tuberosus and Salicornia brachiata. Sorghum bicolor. As well as a highest similarity NHX According to phylogenic tree; Fig. 2 coding sequences gene isolated from k.scoparia related to different species SOS1 and NHX genes isolated from K.scoparia had the from suaeda genus and the lowest similarity be seen with maximum identity with Chenpodiaceae family, for Brachypodium distachyon. ab Fig. 2 The phylogenic relationship between K.scoparia SOS1(a) and NHX(b) with SOS1 and NHX from other plant species. The phylogenic tree was constructed using ClustalW method from the DNASTAR software package. The scale bar indicates substitutions per site. The protein sequences used for construction of the phylogenetic tree are showed on the branch of tree Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 6 of 9 Effects of Salinity Stress on SOS1 and NHX Genes domain in these proteins has a For prediction binding of Expression Profiles Cyclic nucleotide to cyclic nucleotide-binding domain to Studies have identified salt tolerance determinants in SOS1 protein isolated from K.scoparia: First, the tertiary organisms ranging from cyanobacteria to fungi and structure of the desired protein domain was predicted from algae to higher plants. Research with halophytic (Automated Mode, The pipeline will automatically identify species has provided information on adaptive behav- suitable templates based on Blast and HHblits. Cyclic ior but information on the molecular level is still In- nucleotide-binding domain in these proteins has a com- sufficient. Furthermore, information related to salt mon structure with 120 amino acids. The domain consists tolerance of K.scoparia at molecular level is insuffi- of three alpha helixes and eight turn structures that form cient. In this study we tried to be focused on the a pore like structure. Three protected glycine amino acids analysis of isolation, characterization and gene ex- seem important to maintain the barrel shaped structure. pression pattern of key genes involved in salinity tol- In the anticipation of the domain in the K.scoparia,struc- erance in halophytes species such as K.scoparia. tures such as antiparallel beta sheets and alpha helix asso- Gene expression profile for SOS1 and NHX were ciated with small screws. Using molecular docking (using checked in 48 h after treatment with 0, 150, 300 by Swiss Dock, a protein-small molecule docking web ser- mMNaCl.Inthisstudy,wefound abasal levelof vice based on EADock DSS [25]) of the desired fragments SOS1 and NHX in K.scoparia withoutsaltstress, separated from Kochia plant as shown in (Fig. 4). Figure 4, which is regulated with salt treatments. Gene expres- the pore-like structure created in the first part of cyclic sion Profile for SOS1 and NHX in K.scoparia shoot nucleotide binding site, provides the most likely connec- parts showed that salinization was affected SOS1 tion for cyclic nucleotide cNMP. and NHX levels positively and positive correlation with salinity levels. In other words K.scoparia compared Discussion to control like most halophytes leaves are progressively in- Studies have identified salt tolerance determinants in or- creased under all salinity stress. Amounts of mRNA in- ganisms ranging from cyanobacteria to fungi and from creased for SOS1 gene: 1.5 and 2.5 and NHX gene: 1 and algae to higher plants. In plant cell maintain a high K / 2 times higher than the control (0 mM) in 150 and Na in the cytoplasm, under normal conditions. Under 300 mM stressed plants after 48 h of exposure respectively salt stress conditions plants have several strategies and (Fig. 3). While amounts of mRNA increased for SOS1 adaptive mechanisms for tolerant to these conditions. In gene and NHX in root plant but less than the increase in these mechanisms, to be launched sensing, signal leaves, 1 and 2 times for SOS1, 0.5 and 1-fold higher than transduction, gene expression and metabolic pathways. the control in 150 and 300 mM treated plants (Fig. 3). Evidence may be these tolerance programs slow and steady adaptation in the sensitives plants. Therefore, Prediction of SOS1 Antiporter Using Bioinformatic Tools understanding the components of these mechanisms in For prediction binding of Cyclic nucleotide to cyclic halophytes can do contribute substantially to improving nucleotide-binding domain to SOS1 protein isolated from retrofitting sensitive plants. We focused on isolation, K.scoparia: First, the tertiary structure of the desired pro- characterization and gene expression pattern analysis of tein domain was predicted (Automated Mode, The pipe- main genes involved in salinity tolerance in halophytes line will automatically identify suitable templates based on from K.scoparia. In the present study, the relatively high Blast [18] and HHblits [24]. Cyclic nucleotide-binding basal expression level of V-NHX indicated the important Fig. 3 Semi quantitative RT-PCR analysis showing differential gene expression in leaf tissues and roots of 12-day-old seedlings for SOS1 and NHX gene of K.scoparia. The expression of each gene was compared relative to its expression in control gene (18 s rRNA). Samplings were carried out at 24 h after treatments with 0, 150, 300 mM salt stress Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 7 of 9 Fig. 4 Molecular docking was performed to locate the meeting point of cyclic nucleotide binding to SOS1 protein separated from K.scoparia using the online service of Swiss Dock and UCSF Chimera software. The placed mark on Fig is the pore-like structure available in the area for suitable connection between cyclic nucleotide and the desired location in SOS1 protein isolated from K.scoparia physiological function of NHX in K.scoparia, even in the treatments in A. thaliana and T. halophila.Ithas been re- absence of stress NHX levels positively have correlation ported that A. thaliana AtSOS1is expressed at low basal with salinity levels. In other words, compared to control levels but is up-regulated significantly by salt stress in both K.scoparia like most halophytes leaves progressively in- roots and shoots. Moreover, it regulates other genes creases under salinity stress. Amounts of mRNA in- in response to salt stress [8, 30]. Based on the con- creased for NHX gene: 1 and 2 times higher than the ducted docking; there is a possibility of hydrogen and control (0 mM) in 150 and 300 mM stressed plants after hydrophobic connection in the porous structure. The 48 h of exposure respectively (Fig. 4), the higher NHX1 amino acid phenylalanine, lysine, threonine, glycine expression in the leaves was a prompt response to NaCl and arginine have the most connection and position- treatment which could have helped decrease the Na ing in the porous structure according to their charge, content in the cytoplasm and maintain water concentrations polar, non-polar and structure features. Meanwhile, [26]. Previously transgenic studies have shown that the over the conserved glycine amino acids, which are involved expression of the NHX1 gene significantly enhanced plant in the formation of pore-like structure, are also effect- salt tolerance abilities, in transgenic Arabidopsis over ive in hydrophobic connections, with further investi- + + expressing AtNHX1, higher activities of the vacuolar Na /H gation on this domain, these factors affecting it can antiporter were observed and enabling it to grow in the be determined. In the other plants antiporter activity, presence of 200 mMNaCl [27]. The over expression of the long cytosolic C-Terminal tail of SOS1 in thought to + + + cotton Na /H antiporter gene GhNHX1 in tobacco be involved in the sensing of Na [8]. Furthermore, improved salt tolerance in comparison with wild-type plants SOS1 has been demonstrated to be a target of SOS + + [28]. Na /H antiporter is an important membrane protein pathway, relationships between SOS1 and SOS2/SOS3 + + responsible for pumping Na into the vacuole to reduce Na can be a way of regulating the activity of SOS1. toxicity and alleviate the adverse effects of salt stress [28]. Certain domains of SOS1 reacted with SOS2/SOS3 Theexpressionofthe K. sSOS1genein L. fusca was that characterization of these domains can be helped regulated by Na and to characterize the engagement of to the use of this protein in process will create SOS1 in kochia response to saline conditions, results showed resistance plants. To provide factors will be affected that a basal level of K.scoparia SOS1 transcripts in plants theSOS1activityand to determinesuitablemethods without salt stress, which up-regulated significantly with salt to activate these proteins in Glycophytic plant. On treatments. Amounts of mRNA increased for SOS1 gene the other NHX exchanger acts as a mediator of K and NHX in roots plant but less than the increase in leaves, transport between cytosol and vacuole, SOS2 also + + once and twice times for SOS1, 0.5 and 1 time higher than activates the vacuolar-ATPase and vacuolar Na /K the control in 150 and 300 mM treated plants. These results antiporter NHX exchangers, which compartmentalization + + + are in agreement with the Oh et al. [29], report about the in- Na /K into vacuoles. K compartmentalization in the crease of the SOS1 expression level in response to salt vacuole could result in a cytosolic K deficiency [31]. So, Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 8 of 9 determine how to communicate this antiporter can be 2. Huang GT, Ma SL, Bai LP, Zhang L, Ma H, Jia P, Liu J, Zhong M, Guo ZF. Signal transduction during cold, salt, and drought stresses in plants. Mol controlled out ways of increasing salt tolerance to be Biol Rep. 2011;39:969–87. properly. Sequencing of SOS2 and prediction of interact 3. Rajendran KA, Tester M, Roy SJ. Quantifying the three main components of with NHX can be used for controlling of SOS pathway. salinity tolerance in cereals. Plant Cell Environ. 2009;32:237–49. 4. Niu X, Bressan RA, Hasegawa PM, Pardo JM. Ion homeostasis in NaCl stress environments. Plant Physiol. 1995;109:735–42. Conclusion 5. Gaxiola RA, Rao R, Sherman A, Grifasi P, Alpier SL, Fink GR. The Arabidopsis Conclusion In my study SOS1 and NHX genes se- thaliana proton transporters, AtNHX1 and Avp1, can function in cation detoxification in yeast. Proc Natl Acad Sci U S A. 1999;96:1480–5. quenced and determined Proteins characteristics with + + 6. Shi H, Quintero FJ, Pardo JM, Zhu JK. The putative plasma membrane Na /H Insilco tools. Characterization of other genes involved antiporter SOS1 controls long-distance Na+ transport in plants. Plant these pathways and signaling pathway and investigation Cell. 2002;14:465–77. 7. Blumwald E, Aharon GS, Apse MP. Sodium transport in plant cells. invitro of proteins are a promising area of research that Biochimica Biophysica Acta. 2000;1465:140–51. may lead to improvements in the biomass production of 8. Shi H, Ishitani M, Kim C, Zhu JK. The Arabidopsis thaliana salt tolerance + + crop with external applications materials and genetic gene SOS1 encodes a putative Na /H antiporter. Proc Natl Acad Sci USA. 2000;12:6896–901. manipulation. 9. Quintero FJ, Martinez-Atienza J, Villalta I, Jiang X, Kim WY, Ali Z, Fujii H, Mendoza I, Yun DJ, Zhu JK, Pardo JM. Activation of the plasma membrane Abbreviations + + Na/H antiporter salt-overly sensitive 1 (SOS1) by phosphorylation of an cDNA: Complementary deoxyribonucleic acid; NHX: Tonoplast Na /H auto-inhibitory C-terminal domain. 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Isolation and Semi Quantitative PCR of Na+/H+ Antiporter (SOS1 and NHX) Genes under Salinity Stress in Kochia scoparia

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Copyright © 2018 by The Author(s).
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Biomedicine; Biomedicine, general; Biological Techniques
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Abstract

Background: Kochia scoparia is a dicotyledonous annual herb and belongs to the Amaranthaceae family. Genetic diversity and resistance to drought stress of this plant has made it widely scattered in different regions which contains highly genetic diversity and great potential as fodder and can grow on salty, drought affected areas. Since the soil salinity has become widely spread, environmental concern has sparked so many debates. An important limiting factor in agricultural production worldwide is the sensitivity of most of the crop to salinity caused by high concentration of salts soil. Plants use three different strategies to prevent and adapt to high Na concentrations. + + Antiporters are important category of genes that play a pivotal role in ion homeostasis in plants. Na /H antiporters (NHX1 and SOS1) are located in tonoplasts and reduce cytosolic Na concentration by pumping in the vacuole whereas SOS1 is localized at the plasma membrane and extrudes Na in apoplasts. + + + + Results: Coding sequence of plasma membrane Na /H antiporter (SOS1) and vacuole membrane Na /H antiporter (NHX) in Kochia scoparia were isolated using conserved sequences of SOS1 and NHX. Also, expression profile under salinity stress was studied in this study. The amino acid sequences (aa) of the isolated region of K.SSOS1 and K.SNHX showed the maximum identity up to 84% and 90% to its orthologous in salicornia brachiate and suede maritime, respectively. The results of semi-quantitative RT-PCR revealed that salinization has affected positively on SOS1 transcription level. The expression of K.SSOS1 and K.SNHX in leaves and roots of Kochia scoparia were progressively increased under all salinity levels compared to control. Conclusion: The results suggest that K.SSOS1 and K.SNHX play an essential role in salt tolerance of K.scoparia and they can be useful to improve salt tolerance in other crops. + + Keywords: Kochia scoparia,Na /H antiporters, Salinity tolerance, Semi-quantitative RT-PCR Background by high salt concentration, ultimately causing yield lost. Sal- Most of studies have revealed that the greatest lost in vari- inity stress can reduce the productivity of glycophytes, ous crop production is due to abiotic stresses, such as, sal- which are the majority of agricultural products. High salt inity, water deficit, low temperature and heavy metals concentrations cause hyper osmotic stress and ion imbal- adversely affect the growth and several physiological pro- ance in plants which often as a secondary effect leads to cesses such as leaf cell growth and biomass production of oxidative damage in cellular components [1]. Plants adapt plants. An important limiting factor in agricultural produc- to environmental stresses via responses, including the tion worldwide is the sensitivity of most of the crop to sal- activation of molecular networks that regulate stress per- inity caused by high concentration of salts soil. Processes ception, signal transductionand theexpressionof both such as seed germination, seedling growth and vigor, vege- stress related genes and metabolites [2]. Plants have stress tative growth, flowering and fruit set are adversely affected specific adaptive responses as well as responses which pro- tect the plants from more than one environmental stress * Correspondence: l.fahmide@gmail.com [2]. Plants employ three different strategies to prevent and Department of Plant Breeding and Biotechnology, University of Zabol, Zabol + + adapt to high Na concentrations: 1) active Na efflux, 2) 98613-35856, Iran + + Na compartmentalization in vacuoles, and 3) Na influx Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 2 of 9 prevention [3, 4]. Antiporters are important groups of structure characterized with in silico tools. Furthermore, genesthathavea keyroleinion homeostasisinplants. profiling gene expression for two gene characterized. + + Na /H antiporters (NHX1 and SOS1) maintain the K. scoparia is an attractive plant model for study the appropriate concentration of ions in the cytosol, thereby mechanism of salt tolerance. This work to gain insights minimizing cytotoxicity. NHX1 are located in tonoplasts into the role played by this transporter in K. scoparia and reduce cytosolic Na concentration by pumping it in halophyte. the vacuole [5], whereas SOS1 is localized at the plasma membrane and extrudes Na in apoplasts [6]. Both of these Methods antiporters are driven by a motive proton force generated Genetic Samples by the H -ATPase [7]. The SOS signaling pathway consists K.scoparia was collected from Sabzevar in Khorasan of three major proteins including: SOS1, SOS2, and SOS3. Razavi Agricultural Research Center (Iran) and planted + + SOS1, which encodes a plasma membrane Na /H in Biotechnology research Center University of Zabol. antiporter, is essential in regulating Na efflux at the cellular The fresh leaves were applied to isolate RNA after salin- level. It also facilitates long distance transportation of Na ity stress (plants were irrigated by 100 mM, 200 mM, from root to shoot. Over expression of this protein leads to 300 mM and 400 mM sodium chloride solutions). salt tolerance in plants [8]. Activation of SOS1 by direct phosphorylation of the self-regulation scope is possible by Primers Design serine/threonine protein kinas or SOS2 that requires calcium Seventeen SOS1 and 22 NHX coding sequences data binding protein or SOS3 [9]. C-terminal end of the protein which are available at NCBI data base(ncbi.nlm.nih.gov) causes the Na to move. At the C-terminal end, SOS1, the have been showed in Table 1, were aligned by ClustalW 764,849 region is cyclic nucleotide-binding site and in the method provided in DNASTAR Laser gene software 998–1146 region a self-regulator domain exists. In the res- (EditSeq, Meg-Align, Version 5.00), GENEDOC (Mul- pite state the self-regulator domain interacts with upstream tiple Sequence Alignment Editor and Shading Utility sequence bearing the cyclic nucleotide-binding site [9]. In Version 2.5.000). All specific primers designed based on fact, the self-regulator domain is a target location for phos- the most conservative parts of the alignments. Specific phorylation by SOS2. After SOS1 phosphorylation, the self- forward and reverse primers were designed (Table 2). regulator domain leaves upstream location and attaches at this location of cyclic nucleotide and transferring pro- RNA Isolation and cDNA Amplification in K.scoparia tein activity begins [9]. According to the above- Total RNA of samples was isolated by Total RNA isola- mentioned information, domain connected to the cyclic tion kit (DENA Zist Asia). The cDNA(s) were synthe- nucleotide can be used as one of the most important sized using Hyper script reverse transcriptase (Gene All) locations to regulate SOS1 activity, eventually its effect and oligod (T) 18mer, P.SOS.S-REV1, P.SOS.S-REV2, P. on salinity tolerance. K. scoparia,a dicotyledonous SOS.S-REV3, P.SOS.S-REV4, P.NHX.S.REV1, P.NHX.S. erect annual herb belongs to Amaranthaceae family REV2 and P.NHX.S.REV3primers (Table 1) and ampli- with high genetic diversity and great foliage potential fied with a combination of primers (Table 2). The ampli- [10], reported that K. scoparia on of its Iranian variety fications were obtained in 30 cycles at defined annealing is highly tolerant to salt and could be considered as a temperature for each pair of primers using TaqDNA foliage species in cold regions of the world. Rapid vege- polymerase (AMPLIQON). The process finished after a tative growth under high salinity and temperature and final extension for 5–15 min at 720C (Fig. 1). drought and stress makes it a very valuable candidate as a non-conventional foliage crop for arid temperate Sequencing of SOS1 and NHX in K.scoparia regions [11]. K. scoparia has been widely used in Chin- PCR products were extracted and purified from 0.8% ese and Korean traditional medicine as a treatment for agarose gel using GEL recovery DNA kit (DENA Zist skin diseases, diabetes, mellitus, rheumatoid arthritis, Asia). PCR reactions were sequenced utilizing Euro fins liver disorders, and jaundice [12, 13]. Kochia seeds con- MWG Operon company service. Sequence analysis in- tain an ovi position pheromone that can be added as an cluding deletion of error in sequences, assembly of frag- attractant for mosquito pesticides [14, 15]. It has been ments, alignment with other plant species gene reported that seeds of Kochia also contain other chemi- sequences, was done using DNA STAR Laser gene soft- cals that could be beneficial for human, such as com- ware (EditSeq, SeqManII Meg-Align, MapDraw; pounds used in ulcers, rheumatoid arthritis, treatment Version5.00), GENEDOC (Multiple Sequence Alignment and some pathogenic bacteria [15–17]. The aim of this Editor and Shading Utility Version 2.5.000 and NCBI study was to investigate the presence of SOS1 and BLAST [18]. The amino acid sequences were aligned NHX1 genes and trace it using by induced salt stress in with CLUSTALW software. SOS1 and NHX nucleotides Kochia scoparia, Futures of these genes in protein and amino acid sequences aligned and analyzed with Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 3 of 9 Table 1 Plant species and accession numbers of gene sequences used for primer design alignment Gene Plant species Coding sequence length Accession number SOS1 Salicornia brachiata 3541 bp EU879059.1 Salicornia dolichostachya 3601 bp HG799054.1 Suaeda japonica 3481 bp AB198179.1 Suaeda salsa 3541 bp KF914414.1 Halogeton glomeratus 3481 bp KT759142.1 Spinacia oleracea 3301 bp HG799055.1 Mesembryanthemum 3421 bp EF207776.1 crystallinum 3541 bp JX674067.1 Sesuvium portulacastrum 3481 bp KM986873.1 Gossypium hirsutumvoucher 3421 bp JN936862.1 Aeluropus littoralis 3421 bp NM_012801211.1 Vitis vinifera 3481 bp GU177864.1 NHX Salicornia europaea 2161 bp AY131235.1 Salicornia bigelovii 2161 bp DQ157454.1 Salicornia brachiata 1681 bp EU448383.1 Kalidium foliatum 1681 bp AY825250.1 Halostachys caspica 1621 bp GU188850.1 Salsola komarovii 2161 bp AB531436.1 Suaeda pruinosa 1621 bp KJ452342.1 Salsola suaeda 1621 bp EU073422.1 Atriplex gmelini 2341 bp AB038492.1 Suaeda japonica 1681 bp AB198178.1 Suaeda salsa 1801 bp AF370358.1 Suaeda corniculata 1621 bp DQ512716.1 Atriplex halimus 1621 bp KJ452341.1 Suaeda maritima 1621 bp AY261806.1 Chenopodium glaucum 1621 bp AY371319.1 Atriplex patens 1621 bp KC608048.1 Atriplex dimorphostegia 1621 bp AY211397.1 Populuse uphratica 1621 bp DQ414512.1 Glycine max 1501 bp JN872904.1 Table 2 Primers sequences and names were developed for isolation and gene expression of SOS1, NHX in K.scoparia Gene Primers Name Primers Sequences application SOS1 P.SOS.S.FWD1 P.SOS.S.FWD2 P.SOS.S.FWD3 5- ATG GCA GCA TCT CGA A-3 P.SOS.S.FWD4 5- ACT GGA ACA CTG TTT-3 P.SOS.S.REV1 P.SOS.S.REV2 5- CAA ATG GGA TCT GGC T-3 P.SOS.S.REV3 5- CAC TTT TGG GAG ATG GT-3 P.SOS.S.REV4 5-AGA AAA CAA ACA ATG TTC C -3 P.SOS.S.FWD1 5- CCG TTT GAT ATA AGC CA-3 5- GAG ATT ACT TGG TGA ATC-3 5- CAA TAA CAC TTT CCT TCC A-3 5- ATG GCA GCA TCT CGA A-3 NHX P.NHX.S.FWD1 5-ATGTGGTCACAGTTAAGC-3 Gene sequencing P.NHX.S.REV1 5-ATAAGCCATAAGCATCAT − 3 P.NHX.S.FWD2 5-GTGAGGTTGCTTTAATG-3 P.NHX.S.REV2 5-CCAAATACAGGCCGCAT-3 P.NHX.S.FWD3 5-ACAGATTCTGTTTGCAC-3 P.NHX.S.REV3 5-CATAAGACCAGCCCACCA-3 P.NHX.S.FWD1 5-ATGTGGTCACAGTTAAGC-3 P.NHX.S.REV1 5-ATAAGCCATAAGCATCAT − 3 SOS1 P.SOS.G.F P.SOS.G.R 5-GGAAGGTTTGGGGATGGTAT-3 Gene expression 5-GTCCAGCAAGCAAACCATT-3 NHX P.NHX.G.F P.NHX.G.R 5-TTCTGGATTGCTCAGTGCTT-3 Gene expression 5-CAGCCAGCATGTAAGAGAGG-3 18srRNA Forward 5-ATGATAACTCGACGGATCGC-3 5-CTTGGATGTGGTAGCCGTTT-3 Gene expression Reverse Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 4 of 9 Molecular Docking to Predict SOS1 Protein of K.scoparia and Regulation in the Salt Stress Molecular docking of the desired fragments isolated from Kochia using SWISSDOCK was performed as followed procedure: First, tertiary structure of se- quence fragments was predicted in the by SWISS- MODEL is a fully automated protein structure hom- ology modelling server [19–22]. Then, target ligand, cAMP from Zinc dock using online service of Swiss Dock available on the Expasy site molecular docking. The best state for interaction was reported using UCSF-Chimera method [23]. Results and Discussions In this research, isolation of the coding sequence of + + Fig. 1 Regulation of ion homeostasis by ion Na+/H+ pumps plasma membrane Na /H antiporter (SOS1) and + + + + antiporters (SOS1), vacuolar Na /H exchanger (NHX) that salt vacuolar membrane Na /H exchanger (NHX) in Kochia sensors present at the plasma and vacuolar membranes [32] scparia was performed and, the consequence of salinity stress was studied on the expression profile of this gene. SOS1 and NHX from other plants (Fig. 3). Assembled We focused on SOS1 and NHX the critical genes in the sequences for gene submitted to DDBJ data base ad- SOS pathway and vacuolar membrane for the resistant dressed: ddbj.nig.ac.jp and assigned accession number to salt stress (Fig. 1). The SOS pathway and vacuolar + + (LC218450, LC218451 for SOS1 and NHX gene membrane Na /H exchanger (NHX) are currently the respectively). most extensively studied mechanisms in controlling the salt stress response in plants. The SOS and vacuolar + + RT-PCR for Analyzing of SOS1 and NHX Gene Expression membrane Na /H exchanger (NHX) pathway is in K.scoparia under Salt Stress responsible for ion homeostasis and salt tolerance in Eight-week-old seedlings Plants were treated in Salinity plants. stress. Afterward plants were irrigated by 150 mM, 300 mM sodium chloride solutions. Then, sampling was Conserved Domains, Homology and Phylogenetic done during 12, 48, 72 h after treatment. RNAs were ex- Analyses of SOS1 and NHX in K.scoparia tracted using Total RNA isolation kit (DENA Zist Asia, After sequencing the coding SOS1 and NHX genes se- Iran) from the treated seedlings according to manufac- quences in K.scoparia, Conserved domain specified using turer’s instructions. After Dnase1 treatment of RNA sam- of NCBI revealed that putative protein SOS1 belongs to the ples, 2 μgofRNAs, usingGeneAll first strandcDNA Sodium/hydrogen exchanger family; These antiporters con- Synthesis Kit, was reverse transcribed to cDNAs, that were tain 10–12 trans membrane regions (M) at the amino- used as templates for semi quantitative RT-PCR. The terminus and a large cytoplasm region at the carboxyl cDNA amounts were first normalized by 18 s rRNA PCR terminus. The transmembrane regions M3-M12 share the product intensity. PCR process was performed using the same identity with other members of the which family. The following procedure: 95 0C for 5 min followed by 35 cycles M6 and M7 regions are highly conserved. Thus, this is be- of 950C for 30 s, annealing temperature for 45 s, and 720C lieved to be the region involved in the transportation of so- for 1 min, and finally 15 min at 720C for final extension. dium and hydrogen ions. The cytoplasm region has little similarity throughout the family. Conserved domain Ana- Gel Analysis for Gene Expression of SOS1 and NHX in lysis for NHX showed that family represents five trans- K.scoparia under Salt Stress membrane helices. This suggests that the paired regions Images of the RT-PCR ethidium bromide-stained agar- form a ten-helical structure, probably forming the pore, ose gels were taken with a Vilber documentation system whereas the binds a ligand for export or regulation of the (E-BOX CX5) and Band intensity was expressed as rela- pore. The development of intracellular membrane systems tive absorbance units. The ratio between the sample and compartments has led to a considerable increase in the Total RNA and 18srRNA was determined and calculated number of ion transporters in eukaryote cells. As a result, to normalize for initial variations in sample concentra- plants contain a large number of sequences encoding pro- + + tion and as a control for reaction efficiency. Mean and teins that share homology to Na /H antiporters which are standard deviation of all experiments were calculated key transporters in maintaining the pH of actively after normalization to 18srRNA. metabolizing cells. According to highly similar sequences Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 5 of 9 Table 3 Analysis sequence alignment using MegaBlast (%identity) related to NHX and SOS1 genes isolated from K.scoparia NHX SOS1 No. Plant species %Identity Plant species Identity % 1 Suaeda maritima 90% Salicornia dolichostachya %84 2 Suaeda japonica 89% Salicornia brachiata %84 3 Atriplex gmelini 89% Suaeda japonica %83 4 Suaeda comigulata 89% Suaeda salsa %82 5 Atriplex halimus 89% Beta vulgaris %80 6 Chenopodium glaucum 89% Spinica oleraceae %81 7 Suaeda salsa 89% Sesuvium portulacastum %79 8 Suaeda pruinosa 88% Mesemebryanthemum crystallinum %77 9 Atriplex dimorphostegia 88% Vitis vinifera %78 10 Atriplex patens 88% Populus trichocarpa %76 11 Halostachy scaspica 88% Populus eupharatica %76 (Mega Blast) search for sequence homology of SOS1 and example; SOS1 gene that characterized in K.scoparia NHX genes in the NCBI data base on Table 3 was had maximum homology with Beta vulgaris, Salicorni provided. Maximum identity up to90% with Suade abrachiata, Spinaceaeoleraceae and suadea salsa, While, amaritima of NHX gene and for SOS1 gene is 84% by show the lowest similarity to Helianthus tuberosus and Salicornia brachiata. Sorghum bicolor. As well as a highest similarity NHX According to phylogenic tree; Fig. 2 coding sequences gene isolated from k.scoparia related to different species SOS1 and NHX genes isolated from K.scoparia had the from suaeda genus and the lowest similarity be seen with maximum identity with Chenpodiaceae family, for Brachypodium distachyon. ab Fig. 2 The phylogenic relationship between K.scoparia SOS1(a) and NHX(b) with SOS1 and NHX from other plant species. The phylogenic tree was constructed using ClustalW method from the DNASTAR software package. The scale bar indicates substitutions per site. The protein sequences used for construction of the phylogenetic tree are showed on the branch of tree Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 6 of 9 Effects of Salinity Stress on SOS1 and NHX Genes domain in these proteins has a For prediction binding of Expression Profiles Cyclic nucleotide to cyclic nucleotide-binding domain to Studies have identified salt tolerance determinants in SOS1 protein isolated from K.scoparia: First, the tertiary organisms ranging from cyanobacteria to fungi and structure of the desired protein domain was predicted from algae to higher plants. Research with halophytic (Automated Mode, The pipeline will automatically identify species has provided information on adaptive behav- suitable templates based on Blast and HHblits. Cyclic ior but information on the molecular level is still In- nucleotide-binding domain in these proteins has a com- sufficient. Furthermore, information related to salt mon structure with 120 amino acids. The domain consists tolerance of K.scoparia at molecular level is insuffi- of three alpha helixes and eight turn structures that form cient. In this study we tried to be focused on the a pore like structure. Three protected glycine amino acids analysis of isolation, characterization and gene ex- seem important to maintain the barrel shaped structure. pression pattern of key genes involved in salinity tol- In the anticipation of the domain in the K.scoparia,struc- erance in halophytes species such as K.scoparia. tures such as antiparallel beta sheets and alpha helix asso- Gene expression profile for SOS1 and NHX were ciated with small screws. Using molecular docking (using checked in 48 h after treatment with 0, 150, 300 by Swiss Dock, a protein-small molecule docking web ser- mMNaCl.Inthisstudy,wefound abasal levelof vice based on EADock DSS [25]) of the desired fragments SOS1 and NHX in K.scoparia withoutsaltstress, separated from Kochia plant as shown in (Fig. 4). Figure 4, which is regulated with salt treatments. Gene expres- the pore-like structure created in the first part of cyclic sion Profile for SOS1 and NHX in K.scoparia shoot nucleotide binding site, provides the most likely connec- parts showed that salinization was affected SOS1 tion for cyclic nucleotide cNMP. and NHX levels positively and positive correlation with salinity levels. In other words K.scoparia compared Discussion to control like most halophytes leaves are progressively in- Studies have identified salt tolerance determinants in or- creased under all salinity stress. Amounts of mRNA in- ganisms ranging from cyanobacteria to fungi and from creased for SOS1 gene: 1.5 and 2.5 and NHX gene: 1 and algae to higher plants. In plant cell maintain a high K / 2 times higher than the control (0 mM) in 150 and Na in the cytoplasm, under normal conditions. Under 300 mM stressed plants after 48 h of exposure respectively salt stress conditions plants have several strategies and (Fig. 3). While amounts of mRNA increased for SOS1 adaptive mechanisms for tolerant to these conditions. In gene and NHX in root plant but less than the increase in these mechanisms, to be launched sensing, signal leaves, 1 and 2 times for SOS1, 0.5 and 1-fold higher than transduction, gene expression and metabolic pathways. the control in 150 and 300 mM treated plants (Fig. 3). Evidence may be these tolerance programs slow and steady adaptation in the sensitives plants. Therefore, Prediction of SOS1 Antiporter Using Bioinformatic Tools understanding the components of these mechanisms in For prediction binding of Cyclic nucleotide to cyclic halophytes can do contribute substantially to improving nucleotide-binding domain to SOS1 protein isolated from retrofitting sensitive plants. We focused on isolation, K.scoparia: First, the tertiary structure of the desired pro- characterization and gene expression pattern analysis of tein domain was predicted (Automated Mode, The pipe- main genes involved in salinity tolerance in halophytes line will automatically identify suitable templates based on from K.scoparia. In the present study, the relatively high Blast [18] and HHblits [24]. Cyclic nucleotide-binding basal expression level of V-NHX indicated the important Fig. 3 Semi quantitative RT-PCR analysis showing differential gene expression in leaf tissues and roots of 12-day-old seedlings for SOS1 and NHX gene of K.scoparia. The expression of each gene was compared relative to its expression in control gene (18 s rRNA). Samplings were carried out at 24 h after treatments with 0, 150, 300 mM salt stress Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 7 of 9 Fig. 4 Molecular docking was performed to locate the meeting point of cyclic nucleotide binding to SOS1 protein separated from K.scoparia using the online service of Swiss Dock and UCSF Chimera software. The placed mark on Fig is the pore-like structure available in the area for suitable connection between cyclic nucleotide and the desired location in SOS1 protein isolated from K.scoparia physiological function of NHX in K.scoparia, even in the treatments in A. thaliana and T. halophila.Ithas been re- absence of stress NHX levels positively have correlation ported that A. thaliana AtSOS1is expressed at low basal with salinity levels. In other words, compared to control levels but is up-regulated significantly by salt stress in both K.scoparia like most halophytes leaves progressively in- roots and shoots. Moreover, it regulates other genes creases under salinity stress. Amounts of mRNA in- in response to salt stress [8, 30]. Based on the con- creased for NHX gene: 1 and 2 times higher than the ducted docking; there is a possibility of hydrogen and control (0 mM) in 150 and 300 mM stressed plants after hydrophobic connection in the porous structure. The 48 h of exposure respectively (Fig. 4), the higher NHX1 amino acid phenylalanine, lysine, threonine, glycine expression in the leaves was a prompt response to NaCl and arginine have the most connection and position- treatment which could have helped decrease the Na ing in the porous structure according to their charge, content in the cytoplasm and maintain water concentrations polar, non-polar and structure features. Meanwhile, [26]. Previously transgenic studies have shown that the over the conserved glycine amino acids, which are involved expression of the NHX1 gene significantly enhanced plant in the formation of pore-like structure, are also effect- salt tolerance abilities, in transgenic Arabidopsis over ive in hydrophobic connections, with further investi- + + expressing AtNHX1, higher activities of the vacuolar Na /H gation on this domain, these factors affecting it can antiporter were observed and enabling it to grow in the be determined. In the other plants antiporter activity, presence of 200 mMNaCl [27]. The over expression of the long cytosolic C-Terminal tail of SOS1 in thought to + + + cotton Na /H antiporter gene GhNHX1 in tobacco be involved in the sensing of Na [8]. Furthermore, improved salt tolerance in comparison with wild-type plants SOS1 has been demonstrated to be a target of SOS + + [28]. Na /H antiporter is an important membrane protein pathway, relationships between SOS1 and SOS2/SOS3 + + responsible for pumping Na into the vacuole to reduce Na can be a way of regulating the activity of SOS1. toxicity and alleviate the adverse effects of salt stress [28]. Certain domains of SOS1 reacted with SOS2/SOS3 Theexpressionofthe K. sSOS1genein L. fusca was that characterization of these domains can be helped regulated by Na and to characterize the engagement of to the use of this protein in process will create SOS1 in kochia response to saline conditions, results showed resistance plants. To provide factors will be affected that a basal level of K.scoparia SOS1 transcripts in plants theSOS1activityand to determinesuitablemethods without salt stress, which up-regulated significantly with salt to activate these proteins in Glycophytic plant. On treatments. Amounts of mRNA increased for SOS1 gene the other NHX exchanger acts as a mediator of K and NHX in roots plant but less than the increase in leaves, transport between cytosol and vacuole, SOS2 also + + once and twice times for SOS1, 0.5 and 1 time higher than activates the vacuolar-ATPase and vacuolar Na /K the control in 150 and 300 mM treated plants. These results antiporter NHX exchangers, which compartmentalization + + + are in agreement with the Oh et al. [29], report about the in- Na /K into vacuoles. K compartmentalization in the crease of the SOS1 expression level in response to salt vacuole could result in a cytosolic K deficiency [31]. So, Fahmideh and Fooladvand Biological Procedures Online (2018) 20:11 Page 8 of 9 determine how to communicate this antiporter can be 2. Huang GT, Ma SL, Bai LP, Zhang L, Ma H, Jia P, Liu J, Zhong M, Guo ZF. Signal transduction during cold, salt, and drought stresses in plants. Mol controlled out ways of increasing salt tolerance to be Biol Rep. 2011;39:969–87. properly. Sequencing of SOS2 and prediction of interact 3. Rajendran KA, Tester M, Roy SJ. Quantifying the three main components of with NHX can be used for controlling of SOS pathway. salinity tolerance in cereals. Plant Cell Environ. 2009;32:237–49. 4. Niu X, Bressan RA, Hasegawa PM, Pardo JM. Ion homeostasis in NaCl stress environments. Plant Physiol. 1995;109:735–42. Conclusion 5. Gaxiola RA, Rao R, Sherman A, Grifasi P, Alpier SL, Fink GR. The Arabidopsis Conclusion In my study SOS1 and NHX genes se- thaliana proton transporters, AtNHX1 and Avp1, can function in cation detoxification in yeast. Proc Natl Acad Sci U S A. 1999;96:1480–5. quenced and determined Proteins characteristics with + + 6. Shi H, Quintero FJ, Pardo JM, Zhu JK. The putative plasma membrane Na /H Insilco tools. Characterization of other genes involved antiporter SOS1 controls long-distance Na+ transport in plants. Plant these pathways and signaling pathway and investigation Cell. 2002;14:465–77. 7. Blumwald E, Aharon GS, Apse MP. Sodium transport in plant cells. invitro of proteins are a promising area of research that Biochimica Biophysica Acta. 2000;1465:140–51. may lead to improvements in the biomass production of 8. Shi H, Ishitani M, Kim C, Zhu JK. The Arabidopsis thaliana salt tolerance + + crop with external applications materials and genetic gene SOS1 encodes a putative Na /H antiporter. Proc Natl Acad Sci USA. 2000;12:6896–901. manipulation. 9. Quintero FJ, Martinez-Atienza J, Villalta I, Jiang X, Kim WY, Ali Z, Fujii H, Mendoza I, Yun DJ, Zhu JK, Pardo JM. Activation of the plasma membrane Abbreviations + + Na/H antiporter salt-overly sensitive 1 (SOS1) by phosphorylation of an cDNA: Complementary deoxyribonucleic acid; NHX: Tonoplast Na /H auto-inhibitory C-terminal domain. Proc Natl Acad Sci USA. 2011;108:2611–6. exchanger; PCR: Polymerase chain reaction; RT-PCR: Reverse transcription 10. Mullinex W. Kochia (Kochia spp.) biology outline and bibliography.1998. polymerase chain reaction; SOS: Salt overly sensitive; SOS1: Plasma + + 11. Kafi M, Asadi H, Ganjeali A. Possible utilization of high-salinity waters and membrane Na /H exchanger application of low amounts of water for production of the halophyte K. scoparia as alternative fodder in saline agroecosystems. Agric Water Manag. Acknowledgements 2010;97:139–47. We are grateful to all members of department of plant breeding and 12. Choi J, Lee KT, Jung HJ, Park HS, Park HJ. Anti-rheumatoid arthritis effect of biotechnology, University of Zabol for their helpful discussion and technical the Kochia. Arch Pharma Cal Res. 2002;25:336–42. assistance. 13. Kim NY, Lee MK, Park MJ, Kim SJ, Park HJ. Momordin Ic and Oleanolic acid from Kochiae Fructus reduce carbon tetrachloride induced hepatoxicity in Funding rats. J Med Food. 2005;8:177–83. Publication of this article was funded by University of Zabol. 14. Whitney HM, Sayanova JA, Pickett JA. Isolation and expression pattern of two putative acyl-ACP desaturase cDNAs from K.scoparia. J Exp Bot. 2004;55:787–9. Availability of data and materials 15. Friesen LF, Beckie HJ, Warwick SI, Van RC. The biology of Canadian weeds. The datasets measured and analyzed during the study are available from the 138. K.scoparia (L.) Schrad. Can J Plant Sci. 2009;89:141–67. corresponding authors upon reasonable request. 16. Beckie HJ, Blackshaw RE, Low R, Hall LM, Sauder CA, Martin S, Brandt RN, Shirriff SW. Glyphosate-and acetolactate synthase inhibitor–resistant kochia Authors’ contributions (Kochia scoparia) in western Canada. Weed Sci. 2013;61:310–8. LF and ZF performed the experiments, analysis and interpretation of data. LF 17. 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Biological Procedures OnlineSpringer Journals

Published: Jun 1, 2018

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