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In silico genome-wide analysis of Citrus sinensis (L.) Osbeck NHX and KEA genes and their roles in abiotic stress

  • Biochemistry & Physiology - Original Article
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Abstract

Citrus sinensis (L.) Osbeck is a valuable plant belonging to the Rutaceae family. Stress is induced in plants by abiotic factors such as drought, salinity, and temperature. The monovalent cation proton antiporter (CPA) superfamily, which includes the K+ efflux antiporter (KEA) and Na+/H+ exchanger (NHX) genes, plays a crucial role in the regulation of physiological events. This study aims to identify the KEA and NHX genes of C. sinensis and elucidate the roles of these genes in the response to abiotic stress. For this purpose, phylogenetic structure, distribution of chromosomes, gene duplications, gene and protein structures, cis-acting elements, functional gene ontologies, targeted miRNAs, and in silico PCR primer searches were performed using CsNHX and CsKEA sequences. Two KEA and fifty-five NHX were identified as a result of the analysis. Nine of the fifty-five genes (CsNHX5, CsNHX11, CsNHX12, CsNHX17, CsNHX27, CsNHX28, CsNHX47,CsNHX48, and CsNHX55) have been identified as playing a role in the stress response. On the phylogenetic tree, NHX genes were observed to be divided into three distinct clusters. The existence of multiple segmental and tandem duplications in the CsNHX genes has been demonstrated. Stress-related motifs were identified in the promoter regions of CsKEA and CsNHX by cis-acting element analysis, while stress-related miRNAs were identified by miRNA analysis. Consequently, KEA genes are responsible for transport, but they may also play a role in abiotic stress, as they contain cis-acting elements involved in the stress response and are targeted by miRNAs associated with stress. In addition, it has been determined that CsNHX5, which plays a role in the stress response, has the potential to be used in future transgenic plant production studies as it satisfies the PCR in silico criteria.

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References

  • Ambrosino L, Colantuono C, Diretto G, Fiore A, Chiusano ML (2020) Bioinformatics resources for plant abiotic stress responses: state of the art and opportunities in the fast evolving -omics era. Plants 9:591

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Akram U, Song Y, Liang C, Abid MA, Askari M, Myat AA, Abbas M, Malik W, Ali Z, Guo S, Zhang R, Meng Z (2020) Genome-wide characterization and expression analysis of NHX gene family under salinity stress in Gossypium barbadense and its comparison with Gossypium hirsutum. Genes 11:803

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Anil Kumar S, Hima Kumari P, Nagaraju M, Sudhakar Reddy P, Durga Dheeraj T, Mack A, Katam R, Kavi Kishor PB (2022) Genome-wide identification and multiple abiotic stress transcript profiling of potassium transport gene homologs in Sorghum bicolor. Front Plant Sci. https://doi.org/10.3389/fpls.2022.965530

    Article  PubMed Central  PubMed  Google Scholar 

  • Arbona V, Manzi M, Zandalinas SI, Vives-Peris V, Pérez-Clemente RM, Gómez-Cadenas A (2017) Physiological, metabolic, and molecular responses of plants to abiotic stress. In: Sarwat M, Ahmad A, Abdin M, Ibrahim M (eds) Stress Signaling in Plants: Genomics and Proteomics Perspective, Vol 2. Springer, Cham. https://doi.org/10.1007/978-3-319-42183-4_1

  • Arslan B, İncili ÇY, Ulu F, Horuz E, Bayarslan AU, Öçal M, Kalyoncuoğlu E, Baloglu MC, Altunoglu YC (2021) Comparative genomic analysis of expansin superfamily gene members in zucchini and cucumber and their expression profiles under different abiotic stresses. Physiol Mol Biol Plants 27:2739–2756

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Ayadi M, Martins V, ben Ayed R, Jbir R, Feki M, Mzid R, Géros H, Aifa S, Hanana M (2020) Genome wide identification, molecular characterization, and gene expression analyses of grapevine NHX antiporters suggest their involvement in growth, ripening, seed dormancy, and stress response. Biochem Genet 58:102–128

  • Azeem F, Zameer R, Rehman Rashid MA, Rasul I, Ul-Allah S, Siddique MH, Fiaz S, Raza A, Younas A, Rasool A, Ali MA, Anwar S, Siddiqui MH (2022a) Genome-wide analysis of potassium transport genes in Gossypium raimondii suggest a role of GrHAK/KUP/KT8, GrAKT2.1 and GrAKT1.1 in response to abiotic stress. Plant Physiol Biochem 170:110–122

    Article  CAS  PubMed  Google Scholar 

  • Azeem F, Ijaz U, Ali MA, Hussain S, Zubair M, Manzoor H, Abid M, Zameer R, Kim DS, Golokhvast KS, Chung G, Sun S, Nawaz MA (2022b) Genome-wide identification and expression profiling of potassium transport-related genes in Vigna radiata under abiotic stresses. Plants 11:2

    Article  CAS  Google Scholar 

  • Bassil E, Zhang S, Gong H, Tajima H, Blumwald E (2019) Cation specificity of vacuolar NHX-type cation/H + antiporters. Plant Physiol 179:616–629

    Article  CAS  PubMed  Google Scholar 

  • de Paula Santos Martins, C, Pedrosa AM, Du D, Gonçalves LP, Yu Q, Gmitter FG, Costa MGC (2015) Genome-wide characterization and expression analysis of major intrinsic proteins during abiotic and biotic stresses in sweet orange (Citrus sinensis L. Osb.). PLoS ONE 10: e0138786. https://doi.org/10.1371/journal.pone.0138786

  • Ding Y, Tao Y, Zhu C (2013) Emerging roles of microRNAs in the mediation of drought stress response in plants. J Exp Bot 64:3077–3086

    Article  CAS  PubMed  Google Scholar 

  • Dong J, Liu C, Wang Y, Zhao Y, Ge D, Yuan Z (2021) Genome-wide identification of the NHX gene family in Punica granatum L. And their expressional patterns under salt stress. Agronomy 11:264

    Article  CAS  Google Scholar 

  • Fu X, Lu Z, Wei H, Zhang J, Yang X, Wu A, Ma L, Kang M, Lu J, Wang H, Yu S (2020) Genome-wide identification and expression analysis of the NHX (sodium/hydrogen antiporter) gene family in cotton. Front Genet. https://doi.org/10.3389/fgene.2020.00964

    Article  PubMed Central  PubMed  Google Scholar 

  • Guleria P, Mahajan M, Bhardwaj J, Yadav SK (2011) Plant Small RNAs: biogenesis, mode of action and their roles in abiotic stresses. GPB 9:183–199

    CAS  PubMed  Google Scholar 

  • He F, Shi YJ, Li JL, Lin TT, Zhao KJ, Chen LH, Mi JX, Zhang F, Zhong Y, Lu MM, Niu MX, Feng CH, Ding SS, Peng MY, Huang JL, Yang HB, Wa XQ (2022) Genome-wide analysis and expression profiling of cation/h+ exchanger (CAX) family genes reveal likely functions in cadmium stress responses in poplar. Int J Biol Macromol 204:76–88

    Article  CAS  PubMed  Google Scholar 

  • Hernandez-Garcia CM, Finer JJ (2014) Identification and validation of promoters and cis-acting regulatory elements. Plant Sci 217–218:109–119

    Article  PubMed  Google Scholar 

  • Huang L, Li Z, Sun C, Yin S, Wang B, Duan T, Liu Y, Li J, Pu G (2022) Genome-wide identification, molecular characterization, and gene expression analyses of honeysuckle NHX antiporters suggest their involvement in salt stress adaptation. PeerJ 10:e13214. https://doi.org/10.7717/peerj.13214

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Hussain Z, Khan H, Imran M, Naeem MK, Shah SH, Iqbal A, Ali SS, Rizwan M, Ali S, Muneer MA, Widemann E, Shafiq S (2022) Cation/proton antiporter genes in tomato: genomic characterization, expression profiling, and co-localization with salt stress-related QTLs. Agronomy. https://doi.org/10.3390/agronomy12020245

    Article  Google Scholar 

  • Imtiaz H, Rasool Mir A, Corpas FJ, Hayat S (2023) Impact of potassium starvation on the uptake, transportation, photosynthesis, and abiotic stress tolerance. Plant Growth Regul 99:429–448

    Article  CAS  Google Scholar 

  • Isayenkov SV, Dabravolski SA, Pan TP, Shabala S (2020) Phylogenetic diversity and physiological roles of plant monovalent cation/h+ antiporters. Front Plant Sci. https://doi.org/10.3389/fpls.2020.573564

    Article  PubMed Central  PubMed  Google Scholar 

  • Kalendar R, Lee D, Schulman AH (2011) Java web tools for PCR, in silico PCR, and oligonucleotide assembly and analysis. Genomics 98:137–144

    Article  CAS  PubMed  Google Scholar 

  • Kausar R, Wang X, Komatsu S (2022) Crop proteomics under abiotic stress: from data to insights. Plants 11:2877

    Article  PubMed Central  PubMed  Google Scholar 

  • Khare T, Joshi S, Kaur K, Srivastav A, Shriram V, Srivastava AK, Suprasanna P, Kumar V (2021) Genome-wide in silico identification and characterization of sodium-proton (Na+/H+) antiporters in Indica rice. Plant Gene 26:100280. https://doi.org/10.1016/j.plgene.2021.100280

    Article  CAS  Google Scholar 

  • Khraiwesh B, Zhu JK, Zhu J (2012) Role of miRNAs and siRNAs in biotic and abiotic stress responses of plants. BBA-Gene Regul Mech 1819:137–148

    CAS  Google Scholar 

  • Kong M, Luo M, Li J, Feng Z, Zhang Y, Song W, Zhang R, Wang R, Wang Y, Zhao J, Tao Y, Zhao Y (2021) Genome-wide identification, characterization, and expression analysis of the monovalent cation-proton antiporter superfamily in maize, and functional analysis of its role in salt tolerance. Genomics 113:1940–1951

    Article  CAS  PubMed  Google Scholar 

  • Li Z, Wang C, Long D, Jiang Y, He L, Wang Z, Ma X, Bai F, Liu J, Wu L, Song F (2022) Genome-wide identification, bioinformatics characterization and functional analysis of pectin methylesterase inhibitors related to low temperature-induced juice sac granulation in navel orange (Citrus sinensis Osbeck). Sci Hortic. https://doi.org/10.1016/j.scienta.2022.110983

    Article  Google Scholar 

  • Liew SS, Ho WY, Yeap SK, Bin Sharifudin SA (2018) Phytochemical composition and in vitro antioxidant activities of Citrus sinensis peel extracts. PeerJ 6:e5331. https://doi.org/10.7717/peerj.5331

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Lin SB, Ouyang WZ, Hou XJ, Xie LL, Hu CG, Zhang JZ (2015) Genome-wide identification, isolation and expression analysis of auxin response factor (ARF) gene family in sweet orange (Citrus sinensis). Front Plant Sci (MAR). https://doi.org/10.3389/fpls.2015.00119

    Article  Google Scholar 

  • Liu C, Wang X, Xu Y, Deng X, Xu Q (2014) Genome-wide analysis of the R2R3-MYB transcription factor gene family in sweet orange (Citrus sinensis). Mol Biol Rep 41:6769–6785

    Article  CAS  PubMed  Google Scholar 

  • Lu XY, Huang XL (2008) Plant miRNAs and abiotic stress responses. BBRC 368:458–462

    CAS  PubMed  Google Scholar 

  • Ma W, Ren Z, Zhou Y, Zhao J, Zhang F, Feng J, Liu W, Ma X (2020) Genome-wide identification of the Gossypium hirsutum NHX genes reveals that the endosomal-type GhNHX4A Is critical for the salt tolerance of cotton. Int J Mol Sci 21:7712

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mangukia N, Rao P, Patel K, Pandya H, Rawal RM (2022) Unveiling the nature’s fruit basket to computationally identify Citrus sinensis csi-mir169–3p as a probable plant miRNA against reference and Omicron SARS-CoV-2 genome. Comput Biol Med 146:105502. https://doi.org/10.1016/j.compbiomed.2022.105502

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Mannucci C, Calapai F, Cardia L, Inferrera G, D’Arena G, di Pietro M, Navarra M, Gangemi S, Ventura Spagnolo E, Calapai G (2018) Clinical pharmacology of Citrus aurantium and Citrus sinensis for the treatment of anxiety. Evid-Based Complement Altern Med. https://doi.org/10.1155/2018/3624094

    Article  Google Scholar 

  • Manoli A, Trevisan S, Quaggiotti S, Varotto S (2018) Identification and characterization of the BZR transcription factor family and its expression in response to abiotic stresses in Zea mays L. Plant Growth Regul 84:423–436

    Article  CAS  Google Scholar 

  • Miao L, Chen C, Yao L, Tran J, Zhang H (2019) Genome-wide identification, characterization, interaction network and expression profile of GAPDH gene family in sweet orange (Citrus sinensis). PeerJ 7:e7934. https://doi.org/10.7717/peerj.7934

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Nakashima K, Takasaki H, Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) NAC transcription factors in plant abiotic stress responses. BBA-Gene Regulat Mech 1819:97–103

    CAS  Google Scholar 

  • Paul A, Chatterjee A, Subrahmanya S, Shen G, Mishra N (2021) NHX gene family in Camellia sinensis: in-silico genome-wide identification, expression profiles, and regulatory network analysis. Front Plant Sci. https://doi.org/10.3389/fpls.2021.777884

    Article  PubMed Central  PubMed  Google Scholar 

  • Pu L, Lin R, Zou T, Wang Z, Zhang M, Jian S (2022) Genome-wide identification, primary functional characterization of the NHX gene family in Canavalia rosea, and their possible roles for adaptation to tropical coral reefs. Genes 13:33

    Article  CAS  Google Scholar 

  • Rehman HM, Nawaz MA, Shah ZH, Daur I, Khatoon S, Yang SH, Chung GM (2017) In-depth genomic and transcriptomic analysis of five K+ transporter gene families in soybean confirm their differential expression for nodulation. Front Plant Sci. https://doi.org/10.3389/fpls.2017.00804

    Article  PubMed Central  PubMed  Google Scholar 

  • Shafique Khan F, Zeng RF, Gan ZM, Zhang JZ, Hu CG (2021) Genome-wide identification and expression profiling of the WOX gene family in Citrus sinensis and functional analysis of a CsWUS member. Int J Mol Sci 22:4919

    Article  PubMed Central  PubMed  Google Scholar 

  • Sharma H, Taneja M, Upadhyay SK (2020) Identification, characterization and expression profiling of cation-proton antiporter superfamily in Triticum aestivum L. and functional analysis of TaNHX4-B. Genomics 112:356–370

    Article  CAS  PubMed  Google Scholar 

  • Shukla LI, Chinnusamy V, Sunkar R (2008) The role of microRNAs and other endogenous small RNAs in plant stress responses. BBA-Gene Regulat Mech 1779:743–748

    CAS  Google Scholar 

  • Siddique MH, Babar NI, Zameer R, Muzammil S, Nahid N, Ijaz U, Masroor A, Nadeem M, Rashid MAR, Hashem A, Azeem F, Abd-allah EF (2021) Genome-wide identification, genomic organization, and characterization of potassium transport-related genes in Cajanus cajan and their role in abiotic stress. Plants 10:2238

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Sun L, Nasrullah KF, Nie Z, Xu J, Huang X, Sun J, Wang P (2022) Genome-wide identification and transcript analysis during fruit ripening of ACS gene family in sweet orange (Citrus sinensis). Sci Hortic. https://doi.org/10.1016/j.scienta.2021.110786

    Article  Google Scholar 

  • Sunkar R, Chinnusamy V, Zhu J, Zhu JK (2007) Small RNAs as big players in plant abiotic stress responses and nutrient deprivation. Trends Plant Sci 12:301–309

    Article  CAS  PubMed  Google Scholar 

  • Sunkar R, Li YF, Jagadeeswaran G (2012) Functions of microRNAs in plant stress responses. Trends Plant Sci 17:196–203

    Article  CAS  PubMed  Google Scholar 

  • Tiwari R, Rajam MV (2022) RNA- and miRNA-interference to enhance abiotic stress tolerance in plants. J Plant Biochem Biotechnol 31:689–704

    Article  CAS  Google Scholar 

  • Topu M, Sesiz U, Hatipoğlu R, Toklu F, Özkan H (2019) Moleküler Markörler ve Bitki Islahında Kullanımları. In Çiftçi Y and Uncuoğlu A (eds) Bitki Biyoteknolojisinde Güncel Yaklaşımlar Palme Yayınevi, Ankara, pp 17–37

  • Tripathi A, Goswami K, Sanan-Mishra N (2015) Role of bioinformatics in establishing microRNAs as modulators of abiotic stress responses: The new revolution. Front Physiol 6:286. https://doi.org/10.3389/fphys.2015.00286

    Article  PubMed Central  PubMed  Google Scholar 

  • Tungmunnithum D, Thongboonyou A, Pholboon A, Yangsabai A (2018) Flavonoids and other phenolic compounds from medicinal plants for pharmaceutical and medical aspects: an overview. Medicines 5:93

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wang W, Liu JH (2015) Genome-wide identification and expression analysis of the polyamine oxidase gene family in sweet orange (Citrus sinensis). Gene 555:421–429

    Article  CAS  PubMed  Google Scholar 

  • Wang Y, Ying J, Zhang Y, Xu L, Zhang W, Ni M, Zhu Y, Liu L (2020) Genome-wide identification and functional characterization of the cation proton antiporter (CPA) family related to salt stress response in radish (Raphanus sativus l.). Int J Mol Sci 21:1–20

    Article  Google Scholar 

  • Wei Q, Ma Q, Ma Z, Zhou G, Feng F, Le S, Lei C, Gu Q (2019) Genome-wide identification and characterization of sweet orange (Citrus sinensis) aquaporin genes and their expression in two citrus cultivars differing in drought tolerance. Tree Genet Genomes 15:17. https://doi.org/10.1007/s11295-019-1321-1

    Article  Google Scholar 

  • Wei Y, Mu H, Xu G, Wang Y, Li Y, Li S, Wang L (2021) Genome-wide analysis and functional characterization of the UDP-glycosyltransferase family in grapes. Horticulturae. https://doi.org/10.3390/horticulturae7080204

    Article  Google Scholar 

  • Wu GQ, Wang JL, Li SJ (2019) Genome-wide identification of Na+/H+ antiporter (NHX) genes in sugar beet (Beta vulgaris L.) and their regulated expression under salt stress. Genes 10:401. https://doi.org/10.3390/genes10050401

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Wu L, Wu M, Liu H, Gao Y, Chen F, Xiang Y (2020) Monovalent cation/proton antiporters (CPAs) in moso bamboo (Phyllostachys edulis): genome-wide identication, molecular evolutionary and functional description. 23 March 2020, PREPRINT (Version 1) available at Research Square. https://doi.org/10.21203/rs.3.rs-18213/v1

  • Xiaolin Z, Baoqiang W, Xian W, Xiaohong W (2022) Identification of the CIPK-CBL family gene and functional characterization of CqCIPK14 gene under drought stress in quinoa. BMC Genomics 23:1–18

    Article  Google Scholar 

  • Xiong B, Gong Y, Li Q, Li L, Mao H, Liao L, Wang X, Deng H, Zhang M, Wang Z (2022) Genome-wide analysis of the GLK gene family and the expression under different growth stages and dark stress in sweet orange (Citrus sinensis). Horticulturae 8:1076

    Article  Google Scholar 

  • Xu J, Xu H, Liu Y, Wang X, Xu Q, Deng X (2015) Genome-wide identification of sweet orange (Citrus sinensis) histone modification gene families and their expression analysis during the fruit development and fruit-blue mold infection process. Front Plant Sci. https://doi.org/10.3389/fpls.2015.00607

    Article  PubMed Central  PubMed  Google Scholar 

  • Xu X, Zhou Y, Wang B, Ding L, Wang Y, Luo L, Zhang Y, Kong W (2019) Genome-wide identification and characterization of laccase gene family in Citrus sinensis. Gene 689:114–123

    Article  CAS  PubMed  Google Scholar 

  • Yamaguchi-Shinozaki K, Shinozaki K (2005) Organization of cis-acting regulatory elements in osmotic- and cold-stress-responsive promoters. Trends Plant Sci 10:88–94

    Article  CAS  PubMed  Google Scholar 

  • Yang Y, Lee JH, Poindexter MR, Shao Y, Liu W, Lenaghan SC, Ahkami AH, Blumwald E, Stewart CN (2021) Rational design and testing of abiotic stress-inducible synthetic promoters from poplar cis-regulatory elements. Plant Biotechnol J 19:1354–1369

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  • Yarra R (2019) The wheat NHX gene family: Potential role in improving salinity stress tolerance of plants. Plant Gene 18:100178

    Article  CAS  Google Scholar 

  • Zhang F, Yang J, Zhang N, Wu J, Si H (2022a) Roles of microRNAs in abiotic stress response and characteristics regulation of plant. Front Plant Sci 13:01–21. https://doi.org/10.3389/fpls.2022.919243

    Article  Google Scholar 

  • Zhang H, Mi L, Xu L, Yu C, Li C, Chen C (2019) Genome-wide identification, characterization, interaction network and expression profile of GRAS gene family in sweet orange (Citrus sinensis). Sci Rep. https://doi.org/10.1038/s41598-018-38185-z

    Article  PubMed Central  PubMed  Google Scholar 

  • Zhang Y, Zhang Y, Luo L, Lu C, Kong W, Cheng L, Xu X, Liu J (2022b) Genome wide identification of respiratory burst oxidase homolog (Rboh) genes in Citrus sinensis and functional analysis of CsRbohD in cold tolerance. Int J Mol Sci. https://doi.org/10.3390/ijms23020648

    Article  PubMed Central  PubMed  Google Scholar 

  • Zhou H, Qi K, Liu X, Yin H, Wang P, Chen J, Wu J, Zhang S (2016) Genome-wide identification and comparative analysis of the cation proton antiporters family in pear and four other Rosaceae species. Mol Genet Genomics 291:1727–1742

    Article  CAS  PubMed  Google Scholar 

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    Ummahan Öz

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Öz, U. In silico genome-wide analysis of Citrus sinensis (L.) Osbeck NHX and KEA genes and their roles in abiotic stress. Braz. J. Bot (2024). https://doi.org/10.1007/s40415-024-00981-5

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