Available online at www.sciencedirect.com ScienceDirect Rice Science, 2016, 23(6): 334−338 Investigation of Polymorphisms in Coding Region of OsHKT1 in Relation to Salinity in Rice PHAM Quynh-Hoa1, #, TRAN Xuan-An1, #, NGUYEN Thi-Nha-Trang1, TRAN Thi-Thuy-Anh1, HOANG Hai-Yen1, NGUYEN Thi-Hong-Van1, TANG Thi-Hanh2, DO Thi-Phuc1 (1Faculty of Biology, VNU University of Science, Hanoi, 334 Nguyen Trai, Thanh Xuan, Hanoi, Vietnam; 2Faculty of Agronomy, Vietnam National University of Agriculture, Ngo Xuan Quang Street, Trau Quy, Gia Lam, Hanoi, Vietnam; #These authors contribute equally to this study) Abstract: Rice (Oryza sativa) is sensitive to salinity, but the salt tolerance level differs among cultivars, which might result from natural variations in the genes that are responsible for salt tolerance High-affinity potassium transporter (HKTs) has been proven to be involved in salt tolerance in plants Therefore, we screened for natural nucleotide polymorphism in the coding sequence of OsHKT1, which encodes the HKT protein in eight Vietnamese rice cultivars differing in salt tolerance level In total, seven nucleotide substitutions in coding sequence of OsHKT1 were found, including two non-synonymous and five synonymous substitutions Further analysis revealed that these two non-synonymous nucleotide substitutions (G50T and T1209A) caused changes in amino acids (Gly17Val and Asp403Glu) at signal peptide and the loop of the sixth transmembrane domain, respectively To assess the potential effect of these substitutions on the protein function, the 3D structure of HKT protein variants was modelled by using PHYRE2 webserver The results showed that no difference was observed when compared those predicted 3D structure of HKT protein variants with each other In addition, the codon bias of synonymous substitutions cannot clearly show correlation with salt tolerance level It might be interesting to further investigate the functional roles of detected non-synonymous substitutions as it might correlate to salt tolerance in rice Key words: polymorphism; salt stress; OsHKT1 gene; rice Salinity can be stressful to most crop plants and result in severe agricultural loss In addition to hyperosmotic damage (Tarczynski et al, 1993), elevated Na+ concentrations can disrupt cellular processes by interfering with vital Na+-sensitive enzymes (Tester and Davenport, 2003; Munns et al, 2006) and affecting ion transport (Rains and Epstein, 1965; Schroeder et al, 1994) Na+ uptake occurs via multiple Na+-permeable channels/transporters under the saline conditions, and ion toxicity is triggered when a high level of Na+ is accumulated in the cytosol (Volkmar et al, 1999; Munns, 2002; Horie and Schroeder, 2004) Therefore, it is very important for cells to maintain a low concentration of cytosolic Na+ or to maintain a low Na+/K+ ratio in the cytosol under NaCl stress The most important way to maintain a low cytosolic Na+ concentration is to minimize the influx of Na+ into the cytosol (Horie and Schroeder, 2004; Chinnusamy et al, 2005) Na+ influx can be restricted by means of selective ion uptake It has been suggested that high-affinity potassium transporters (HKTs) mediate a substantial Na+ influx in some species (Uozumi et al, 2000; Horie et al, 2001; Golldack et al, 2002; Garciadeblás et al, 2003) While only one copy of HKT is present in Arabidopsis, 7–9 HKT genes (OsHKT1, OsHKT2, OsHKT3, OsHKT4, OsHKT5, OsHKT6, OsHKT7, OsHKT8 and OsHKT9) have been identified in rice (Oryza sativa) depending on cultivars (Uozumi et al, 2000; Garciadeblás et al, 2003) These functional genes encode proteins with distinct transport activities, which might be expressed in various tissues and/or organs (Jabnoune et al, 2009) It has been suggested that OsHKT1 is a Na+ transporter (Horie et al, 2001; Maser et al, 2002; Garciadeblás et al, 2003) and OsHKT2 is a Na+/K+ cotransporter (Horie et al, 2001; Maser et al, 2002) OsHKT8 was Received: 25 February 2016; Accepted: May 2016 Corresponding author: DO Thi-Phuc (dothiphuc13380@gmail.com; phucthido@vnu.edu.vn) Copyright © 2016, China National Rice Research Institute Hosting by Elsevier B V This is an open access article under the CC BY-NC-ND license B.V This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/) (http://creativecommons.org/licenses/by-nc-nd/4.0/) Peer review under responsibility of China National Rice Research Institute http://dx.doi.org/ http://dx.doi.org/10.1016/j.rsci.2016.05.004 PHAM Quynh-Hoa, et al Polymorphism of OsHKT1 in Rice recently shown to be a Na+ transporter that contributes to increased salt tolerance by maintaining K+ homeostasis in the shoot under salt stress (Ren et al, 2005; Rus et al, 2005) This transporter is thought to be analogous to the function of AtHKT1 in Arabidopsis, which is a Na+ transporter and plays a crucial role in controlling cytosolic Na+ detoxification (Berthomieu et al, 2003; Rus et al, 2005) Therefore, it is likely that the HKT family plays an important role in Na+/K+ homeostasis in rice, and some of its members are evidently Na+ transporters Analyses of natural genetic polymorphism can provide insight into the mechanisms of plant adaptation to environmental conditions (Brady et al, 2005; Baxter et al, 2010) Although rice is considered to be a salt-sensitive species, several varieties, such as Pokkali and Nona Bokra, display a certain level of salt tolerance Therefore, in this study, we focused on the analysis of natural polymorphism in OsHKT1 in eight Vietnamese rice cultivars, namely, Nep Non Tre, Chiem Cu, Re Nuoc, Hom Rau, Nep Oc, Ngoi, Dau An Do and Nep Deo Dang, and several single nucleotide polymorphisms were identified However, further in silico analysis revealed no difference in predicted 3D protein structures and in codon usage bias of these variants in relation to salt tolerance MATERIALS AND METHODS Rice materials Seeds of nine rice cultivars were provided by Vietnam National University of Agriculture (Hanoi, Vietnam), including Nipponbare, Nep Non Tre, Chiem Cu, Re Nuoc, Hom Rau, Nep Oc, Ngoi, Dau An Do and Nep Deo Dang The six Vietnamese rice cultivars were collected from different costal region in Vietnam (Nep Non Tre, Re Nuoc, Hom Rau, Ngoi and Dau An Do were collected in Nam Dinh Province, while Chiem Cu was collected in Quang Binh Province), and Nep Deo Dang in Tuyen Quang Province and Nep Oc in Ha Noi City These cultivars showed differences in salt tolerance The highly salt tolerant cultivars are Nep Non Tre, Hom Rau and Nep Oc, whereas the moderate salt tolerant cultivars are Chiem Cu, Re Nuoc and Ngoi, and the sensitive cultivars are Dau An Do and Nipponbare (Tran et al, 2015) The seedlings were grown in soil for 14 d, then the leaves were collected and stored at -80 °C for further analysis DNA extraction The DNA extraction was performed by using the cetyl trimethylammonium bromide (CTAB) method About 200 mg leaf powder was mixed with 500 µL CTAB buffer and incubated at 65 °C for 20 Then 500 µL CI 24:1 (chloroform: isoamylalcohol) was added and centrifuged at 14 000 r/min at °C for 15 The supernatant was transferred into a new tube, and the DNA was precipitated by cold isopropanol for 15 The DNA pellet was collected by centrifuging at 10 000 r/min at °C for and washed with 70% ethanol Finally, 335 after drying at room temperature, the pellet was dissolved in Tris-EDTA buffer and kept at -20 °C The quality and quantity of extracted DNA were estimated by visualizing the band of total DNA on ethidium bromide-stained 1% agarose gel in comparison with the relative migration and intensity of the standard kb ladder (Fermentas) Primer design and amplification of OsHKT1 by PCR Four primer pairs were used for amplifying four flanking fragments of OsHKT1 from genomic DNA, including F1-FW (5′-CATACTCGTTGGCTCGTTGC-3′) and F1-RW (5′-ATCACA GTGCTTGCCGAGTT-3′); F2-FW (5′-TGAAGCCAAGCAACCC AGAA-3′) and F2-RW (5′-CAGCACCGAACAATGTGACC-3′); F3-FW (5′-TGGCCTTATGGCTTCCTTGG-3′) and F3-RW (5′ATTGGCTTGATGCCCAGTGT-3′); F4-FW (5′-GGCATTTT CACAGCTTGCCT-3′) and F4-RW (5′-GGCATTTTCACAGC TTGCCT-3′) (Do and Nguyen, 2014) The PCR reaction mixture consisted of genomic DNA (20–50 ng), Dream Taq polymerase buffer (1×), MgCl2 (1.5 mmol/L), dNTPs mixture (0.2 mmol/L), primers (0.4 µmol/L) and Dream Taq polymerase (1 U) The PCR reaction was performed with a thermocycle of 95 °C for min, 35 cycles of 95 °C for 30 s, 56 °C for 30 s and 72 °C for min, and 72 °C for Then, µL of PCR products were separated on 1% agarose gel for 28 at constant 90 V in 1× Tris-acetate-EDTA buffer If PCR product showed only one bright single band on gel, it was purified using GeneJET PCR Purification Kit (Thermo Fisher Scientific) and sequenced on ABI PRISM 3730xl Genetic Analyzer (Applied Biosystems, USA) at First BASE DNA sequencing service (Singapore) Sequence analysis Gene sequences were analyzed using Bioedit (Hall, 1999) and Multalin webserver (Corpet, 1988) The predicted peptide sequences were analyzed using Expasy webserver (http://web.expasy.org/translate/) Modelling OsHKT1 protein in rice The 3D model of OsHKT1 protein was predicted and analyzed by using PHYRE2 program (Kelley et al, 2015) PHYRE2 is a web-based tool to predict protein structure The predicted structure of protein in PDB file created by PHYRE2 was visualized by Discovery studio 4.5 visualizer RESULTS AND DISCUSSION Amplification of OsHKT1 from nine rice cultivars The leaves of 14 d rice seedlings were collected for genomic DNA extraction The extracted DNA was used as template for the amplification of OsHKT1 in the PCR assay The specific primers were designed to amplify four amplicons that covered the complete genomic sequence of OsHKT1 As shown in Fig 1, the PCR products were specific and with the correct size Thus, the OsHKT1 gene was successfully amplified in all the 336 Rice Science, Vol 23, No 6, 2016 Fig PCR products using four pairs of primers A, PCR product of 499 bp using primer pair (F1); B, PCR product of 828 bp using primer pair (F2); C, PCR product of 603 bp using primer pair (F3); D, PCR product of 668 bp using primer pair (F4) M, kb ladder marker; Lanes to 9, Nipponbare, Nep Non Tre, Chiem Cu, Re Nuoc, Hom Rau, Nep Oc, Ngoi, Dau An Do and Nep Deo Dang, respectively investigated rice cultivars Polymorphism in nucleotide sequence of OsHKT1 gene Amplified DNA fragments were purified, and sequenced and allowed the detection of seven nucleotide substitutions when compared with the reference sequence in the database (Nipponbare allele) (Fig 2) Among the seven detected nucleotide substitutions, five were synonymous and two were non-synonymous substitutions The detected five synonymous substitutions were A360G (Nep Non Tre, Dau An Do, Chiem Cu and Nep Oc), A645G (Chiem Cu), A708G (Nep Non Tre, Dau An Do, Chiem Cu and Nep Oc), G744A (Nep Non Tre, Dau An Do, Chiem Cu and Nep Oc), and G1440A (Nep Non Tre) Interestingly, the two non-synonymous substitutions were present only in Hom Rau which was classified as salt tolerant cultivar (Tran et al, 2015) These two non-synonymous substitutions (G50T and T1209A) lead to change in amino acid of the corresponding protein variant (G17V and D403E, respectively) (Fig 2) Among those detected single nucleotide polymorphisms (SNPs), only one SNP at position 50 (G50T) is reported in RiceVarMap SNPs database (http://ricevarmap.ncpgr.cn/) Prediction of potential subsequence change caused by nucleotide substitution on protein structure and protein synthesis rate The protein structure of OsHKT1 transporter was predicted by A B Fig Polymorphism in OsHKT1 sequence A, Schematic representation of polymorphism locations in the protein The position of changes in amino acid are indicated with a star; MPM, transmembrane-pore loop-transmembrane domain B, Nucleotide sequence of OsHKT1 was compared among the cultivars with the Nipponbare sequence Non-synonymous substitutions are indicated in dark grey, and the encoded amino acids are listed above Synonymous substitutions are indicated in light grey G17V means that glycine is replaced by valine; D403E means that aspartic acid is replaced by glutamic acid PHAM Quynh-Hoa, et al Polymorphism of OsHKT1 in Rice 337 B A Fig 3D ribbon model of OsHKT1 protein visualized by Discovery studio 4.5 visualizer A, 3D ribbon model of Ni-OsHKT1 protein visualized from the top that shows the pore Four residues in Ser-Gly-Gly-Gly ion selection motif are labeled and Na+ ion is indicated as a black round circle at the center of the pore B, Visualization of Ho-OsHKT1 transporter from the side that shows the zoom in D403E variance position (green) (G17V substitution belongs to the signal peptide so they are not modeled in 3D structure of OsHKT1 transporter) using PHYRE2 webserver It showed that OsHKT1 contains eight potential transmembrane domains and the two polymorphisms G17V and D403E, were placed in the signal peptide and the loop of the sixth transmembrane domain, respectively (Fig 2-A) The 3D model of OsHKT1 transporter showed the presence of three glycine residues (Gly243, Gly367 and Gly468) and one serine residue (Ser87) forming a selective filter for Na+/K+ ion (Fig 3-A) The 3D structure prediction showed that the amino acid substitutions caused no influence on the structure of OsHKT1 protein (Fig 3) That might be due to the fact that the substituted amino acids have the same charge properties and bulk to former amino acids, and also the position of the altered amino acid did not appear in the filter region Our finding is in agreement with previous results reported by Oomen et al (2012) In that study, the same polymorphism was also detected in other rice cultivars and the site-directed mutagenesis on wild-type OsHKT1 protein indicated that transport activity of variant transporter was not significantly different to that of wild-type (Oomen et al, 2012) Although synonymous substitutions did not alter the amino acid sequence, they can effect on translation efficiency via codon usage bias (Yu et al, 2015) Among five synonymous substitutions, the two at position 360 and 1440 caused clear change in codon usage frequency (Table 1) SNP at position 360 probably increased the translation rate because the usage frequency of the substituted codon was 3-fold higher than the former one However, this substitution appeared in both tolerant (Nep Non Tre and Nep Oc) and sensitive (Dau An Do and Chiem Cu) cultivars In contrast, SNP at position 1440 was only present in the tolerant cultivar Nep Non Tre, and probably reduced the translation rate With our current data, it is quite difficult to point out the relationship between nucleotide polymorphism in the coding region of OsHKT1 and the salt tolerance level of the investigated rice cultivars It might be helpful to explore the nucleotide polymorphism in the promoter region of OsHKT1, which plays role in the regulation of gene expression CONCLUSIONS We have successfully amplified and sequenced the OsHKT1 from eight Vietnamese rice cultivars and Nipponbare (control) Our data revealed seven single nucleotide substitutions compared to the reference sequence of the rice database From these, two substitutions led to amino acid substitutions which occurred in salt tolerance cultivar Hom Rau Thus, it is deserved to further research on the functional role of the detected non-synonymous substitutions Based on the topological model of OsHKT1 protein, the two detected aminoacid variants were placed in signal peptide and the loop of the sixth transmembrane, respectively Our data suggests that they probably not have a significant impact on the structure of OsHKT1 transporter Furthermore, among the different Table Changes in codon usage bias by nucleotide substitutions in nine rice cultivars Position of Former Usage frequency of Substituted Usage frequency of Amino Cultivar nucleotide variance codon former codon (‰) substituted codon (‰) acid codon 360 CUA 7.7 CUG 21.0 Leu Nep Non Tre, Dau An Do, Nep Oc, Chiem Cu 645 UCA 12.4 UCG 12.3 Leu Chiem Cu 708 ACA 11.6 ACG 11.4 Thr Nep Non Tre, Dau An Do, Nep Oc, Chiem Cu 744 ACG 11.4 ACA 11.6 Thr Nep Non Tre, Dau An Do, Nep Oc, Chiem Cu 1440 AAG 32.3 CAA 13.5 Lys Nep Non Tre Values in bold meant that the substituted nucleotide caused an increase in codon usage frequence 338 Rice Science, Vol 23, No 6, 2016 investigated rice cultivars, two out of five detected synonymous substitutions showed an effect on the protein synthesis rate in terms of codon frequent usage However, a correlation with salt tolerance level was not found ACKNOWLEDGEMENTS This work was financially supported by the Vietnam National University, Hanoi, Vietnam (Grant No QG.14.22) Many thanks to Dr SANG Nguyen Van for his helpful suggestions and comments, and to Dr ANTONIO Carla for her great help in English improvement REFERENCES Baxter I, Brazelton J N, Yu D, Huang Y S, Lahner B, Yakubova E, Li Y, Bergelson J, Borevitz J O, Nordborg M, Vitek O, Salt D E 2010 A coastal cline in sodium accumulation in Arabidopsis thaliana is driven by natural variation of the sodium transporter AtHKT1;1 PLoS Genet, 6: e1001193 Berthomieu P, Conejero G, Nublat A, Brackenbury W J, Lambert C, Savio C, Uozumi N, Oiki S, Yamada K, Cellier F, Gosti F, Simonneau T, Essah P A, Tester M, Very A A, Sentenac H, Casse F 2003 Functional analysis of AtHKT1 in Arabidopsis shows that Na+ recirculation by the phloem is crucial for salt tolerance EMBO J, 22(9): 2004–2014 Brady K U, Kruckeberg A R, Bradshaw Jr H D 2005 Evolutionary ecology of plant adaptation to serpentine soils Annu Rev Ecol Evol Syst, 36: 243–266 Chinnusamy V, Jagendorf A, Zhu J K 2005 Understanding and improving salt tolerance in plants Crop Sci, 45: 437–448 Corpet F 1988 Multiple sequence alignment with hierarchical clustering Nucl Acids Res, 16(22): 10881–10890 Do T P, Nguyen T T T 2014 Polymorphism analysis of OsHKT1 gene in rice (Oryza sativa) VNU J Sci: Natl Sci Technol, 30: 253–259 (in Vietnamese with English abstract) Garciadeblás B, Senn M E, Banuelos M A, Rodriguez-Navarro A 2003 Sodium transport and HKT transporters: The rice model Plant J, 34: 788–801 Golldack D, Su H, Quigley F, Kamasani U R, Munoz-Garay C, Balderas E, Popova O V, Bennett J, Bohnert H J, Pantoja O 2002 Characterization of a HKT-type transporter in rice as a general alkali cation transporter Plant J, 31(4): 529–542 Hall T A 1999 BioEdit: A user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT Nucl Acids Symp Ser, 41: 95–98 Horie T, Yoshida K, Nakayama H, Yamada K, Oiki S, Shinmyo A 2001 Two types of HKT transporters with different properties of Na+ and K+ transport in Oryza sativa Plant J, 27(2): 129–138 Horie T, Schroeder J I 2004 Sodium transporters in plants: Diverse genes and physiological functions Plant Physiol, 136: 2457–2462 Jabnoune M, Espeout S, Mieulet D, Fizames C, Verdeil J L, Conéjéro G, Rodríguez-Navarro A, Sentenac H, Guiderdoni E, Abdelly C, Very A A 2009 Diversity in expression patterns and functional properties in the rice HKT transporter family Plant Physiol, 150(4): 1955–1971 Kelley L A, Mezulis S, Yates C M, Wass M N, Sternberg M J E 2015 The Phyre2 web portal for protein modeling, prediction and analysis Nat Protoc, 10: 845–858 Maser P, Hosoo Y, Goshima S, Horie T, Eckelman B, Yamada K, Yoshida K, Bakker E R, Shinmyo A, Oiki S, Schroeder J I, Uozumi N 2002 Glycine residues in potassium channel-like selectivity filters determine potassium selectivity in four-loopper-subunit HKT transporters from plants Proc Natl Acad Sci USA, 99(9): 6428–6433 Munns R 2002 Comparative physiology of salt and water stress Plant Cell Environ, 25(2): 239–250 Munns R, James R A, Laeuchli A 2006 Approaches to increasing the salt tolerance of wheat and other cereals J Exp Bot, 57(5): 1025–1043 Oomen R J F J, Benito B, Sentenac H, Rodrıguez-Navarro A, Talon M, Very A A, Domingo C 2012 HKT2;2/1, a K+permeable transporter identified in a salt-tolerant rice cultivar through surveys of natural genetic polymorphism Plant J, 71(5): 750–762 Rains D W, Epstein E 1965 Transport of sodium in plant tissue Science, 148: 1611 Ren Z H, Gao J P, Li L G, Cai X L, Huang W, Chao D Y, Zhu M Z, Wang Z Y, Luan S, Lin H X 2005 A rice quantitative trait locus for salt tolerance encodes a sodium transporter Nat Genet, 37: 1141–1146 Rus A M, Bressan R A, Hasegawa P M 2005 Unraveling salt tolerance in crops Nat Genet, 37: 1029–1030 Schroeder J I, Ward J M, Gassmann W 1994 Perspectives on the physiology and structure of inward rectifying K+ channels in higher plants: Biophysical implications for K+ uptake Ann Rev Biophys Biomol Struct, 23: 441–471 Tarczynski M C, Jensen R G, Bohnert H J 1993 Stress protection of transgenic tobacco by production of the osmolyte mannitol Science, 259: 508–510 Tester M, Davenport R 2003 Na+ tolerance and Na+ transport in higher plants Ann Bot, 91(5): 503–527 Tran X A, Nguyen T N T, Do T P 2015 Evaluation of salt tolerance of some Vietnamese rice varieties VNU J Sci: Natl Sci Technol, 31: 1–7 (in Vietnamese with English abstract) Uozumi N, Kim E J, Rubio F, Yamaguchi T, Muto S, Tsuboi A, Bakker E P, Nakamura T, Schroeder J I 2000 The Arabidopsis HKT1 gene homolog mediates inward Na+ currents in Xenopus laevis oocytes and Na+ uptake in Saccharomyces cerevisiae Plant Physiol, 122(4): 1249–1260 Volkmar K M, Hu Y, Steppuhn H 1999 Physiological responses of plants to salinity: A review Can J Plant Sci, 78: 19–27 Yu C H, Dang Y K, Zhou Z P, Wu C, Zhao F Z, Sachs M S, Liu Y 2015 Codon usage influences the local rate of translation elongation to regulate co-translational protein folding Mol Cell, 59(5): 744–754 (Managing Editor: WANG Caihong) ... Quynh-Hoa, et al Polymorphism of OsHKT1 in Rice recently shown to be a Na+ transporter that contributes to increased salt tolerance by maintaining K+ homeostasis in the shoot under salt stress... (http://web.expasy.org/translate/) Modelling OsHKT1 protein in rice The 3D model of OsHKT1 protein was predicted and analyzed by using PHYRE2 program (Kelley et al, 2015) PHYRE2 is a web-based tool to predict protein structure... to point out the relationship between nucleotide polymorphism in the coding region of OsHKT1 and the salt tolerance level of the investigated rice cultivars It might be helpful to explore the nucleotide