Identification and Expression Characterization Analysis of the NRT2 Gene Family in Pepper (Capsicum annuum)
WU Yuan1,2,5, SU Shi-Xian1, WANG Tao1,2, LONG Cha1, WEI Jin-Jiang1,2, JIANG Su-Yan1, XING Dan3, YUAN Yuan-Guo4, LI Wei1,2,5,*
1 College of Agriculture, Guizhou University, Guiyang 550025, China; 2 Industry Technology Institute of Pepper, Guizhou University, Guiyang 550025, China; 3 Research Institute of Pepper, Guizhou Province, Zunyi 563000, China; 4 Guizhou Institute of Horticulture, Guiyang 550006, China; 5 Engineering Research Center for Protected Vegetable Crops in Higher Learning Institutions of Guizhou Province, Guiyang 550025, China
Abstract Pepper (Capsicum annuum) is an important horticultural crop. NRT2 (nitrate transporter 2) gene families play a crucial role in nitrate absorption and transport. In order to explore the role of NRT2 gene in pepper growth and development, 8 pepper NRT2 genes were identified in this study using published pepper genome data and bioinformatics analysis methods. CaNRT2s family members were distributed on 4 chromosomes. There were some differences in protein structure and physical and chemical properties. According to phylogenetic analysis, CaNRT2s members were divided into 4 subfamilies, which were close to tomato (Lycopersicon esculentum) and potato (Solanum tuberosum) in evolutionary relationship. All members had MFS-1 conservative domain. Promoters contain a large number of hormone responsive elements, stress responsive elements and plant growth and development related elements. The expression of CaNRT2s were different in the developmental stage and tissue. CaNRT2.4 and CaNRT2.5 were highly expressed in roots, and CaNRT2.2 and CaNRT2.3 were highly expressed in multiple tissues and fruits during color transformation. The study found that the expressions of CaNRT2.1/2.2/2.3/2.8 were very low under various abiotic stresses and hormone treatment. The expressions of CaNRT2.4/2.5/2.6/2.7 were down regulated in leaves and up regulated in roots. CaNRT2.4/2.5/2.6/2.7 were up-regulated in leaves and roots after nitrogen deficiency treatment. The results were deduced that these 4 genes might be the key genes regulating nitrate transport in pepper. The research results provide theoretical basis for the cloning and functional study of NRT2 gene of NO3- absorption and transport in pepper.
WU Yuan,SU Shi-Xian,WANG Tao, et al. Identification and Expression Characterization Analysis of the NRT2 Gene Family in Pepper (Capsicum annuum)[J]. 农业生物技术学报, 2023, 31(11): 2239-2253.
[1] 李婧, 左欣欣, 赵培伶, 等. 2022. 茶树高亲和硝酸盐转运蛋白家族基因NRT2的鉴定与表达[J]. 应用与环境生物学报, 28(01): 50-56. (Li J, Zuo X X, Zhao P L, et al.2022. Identification and expression analysis of high-affinity nitrate transporter family genes NRT2 in Camellia sinensis[J]. Chinese Journal of Applied & Environmental Biology, 28(01): 50-56.) [2] 熊威, 赵涵, 周玲. 2022. 玉米ZmNRT关键基因挖掘以及氮响应基因共表达网络构建[J]. 玉米科学, 30(02): 58-68. (Xiong W, Zhao H, Zhou L.2022. ZmNRT key genes mining and nitrogen responsive genes co-expression network construction in maize[J]. Journal of Maize Sciences, 30(02): 58-68.) [3] 叶玲, 张洁, 郭永正, 等. 2020. 谷子NRT2基因家族的鉴定及生物信息学分析[J]. 山西农业科学, 48(03): 283-290. (Ye L, Zhang J, Guo Y Z, et al.2020. Identification and bioinformatics analysis of NRT2 gene family in millet[J]. Journal of Shanxi Agricultural Sciences, 48(03): 283-290.) [4] 赵敬. 2012. 辣椒内参基因的筛选及NBS-LRR类抗病基因同源序列的鉴定[D]. 硕士学位论文, 南京农业大学, 导师: 侯喜林, pp. 43. (Zhao J, 2012. Screening of reference genes and inentification of NBS-LRR gene analogs in pepper (Capsicum Annuum L.)[D]. Thesis for M.S., Nanjing Agricultural University, Supervisor: Hou X L, pp. 43.) [5] Akbudak M A, Filiz E, çetin D.2022. Genome-wide identification and characterization of high-affinity nitrate transporter 2 (NRT2) gene family in tomato (Solanum lycopersicum) and their transcriptional responses to drought and salinity stresses[J]. Journal of Plant Physiology, 272: 153684. [6] Araki R, Hasegawa H.2006. Expression of rice (Oryza sativa L.) genes involved in high-affinity nitrate transport during the period of nitrate induction[J]. Breeding Science, 56(3): 295-302. [7] Arce-Rodríguez M L, Martínez O, Ochoa-Alejo N.2021. Genome-wide identification and analysis of the MYB transcription factor gene family in chili pepper (Capsicum spp.)[J]. International Journal of Molecular Sciences, 22(5): 2229. [8] Baenas N, Belović M, Ilic N, et al.2019. Industrial use of pepper (Capsicum annum L.) derived products: Technological benefits and biological advantages[J]. Food Chemistry, 274: 872-885. [9] Bailey T L, Johnson J, Grant C E, et al.2015. The MEME suite[J]. Nucleic Acids Research, 43(W1): W39-W49. [10] Biasini M, Bienert S, Waterhouse A, et al.2014. SWISS-MODEL: Modelling protein tertiary and quaternary structure using evolutionary information[J]. Nucleic Acids Research, 42(W1): W252-W258. [11] Bloom A J.2015. The increasing importance of distinguishing among plant nitrogen sources[J]. Current Opinion in Plant Biology, 25: 10-16. [12] Cerezo M, Tillard P, Filleur S, et al.2001. Major alterations of the regulation of root NO3- uptake are associated with the mutation of Nrt2.1 and Nrt2.2 genes in Arabidopsis[J]. Plant Physiology, 127(1): 262-271. [13] Chen C, Chen H, Zhang Y, et al.2020. TBtools: An integrative toolkit developed for interactive analyses of big biological data[J]. Molecular Plant, 13(8): 1194-1202. [14] Chopin F, Orsel M, Dorbe M, et al.2007. The Arabidopsis ATNRT2.7 nitrate transporter controls nitrate content in seeds[J]. The Plant Cell, 19(5): 1590-1602. [15] Crawford N M, Glass A D.1998. Molecular and physiological aspects of nitrate uptake in plants[J]. Trends in Plant Science, 3(10): 389-395. [16] Daniel V F, Filleur S, Caboche M.1998. Nitrate transport: A key step in nitrate assimilation[J]. Current Opinion in Plant Biology, 1(3): 235-239. [17] Diao W, Snyder J C, Wang S, et al.2018. Genome-wide analyses of the NAC transcription factor gene family in pepper (Capsicum annuum L.): Chromosome location, phylogeny, structure, expression patterns, cis-elements in the promoter, and interaction network[J]. International Journal of Molecular Sciences, 19(4): 1028. [18] Duvaud S, Gabella C, Lisacek F, et al.2021. Expasy, the swiss bioinformatics resource portal, as designed by its users[J]. Nucleic Acids Research, 49(W1): W216-W227. [19] Feng H, Li B, Zhi Y, et al.2017. Overexpression of the nitrate transporter, OsNRT2.3b, improves rice phosphorus uptake and translocation[J]. Plant Cell Reports, 36(8): 1287-1296. [20] Filleur S, Dorbe M, Cerezo M, et al.2001. An Arabidopsis t‐DNA mutant affected in Nrt2 genes is impaired in nitrate uptake[J]. Febs Letters, 489(2-3): 220-224. [21] Fredes I, Moreno S, Díaz F P, et al.2019. Nitrate signaling and the control of Arabidopsis growth and development[J]. Current Opinion in Plant Biology, 47: 112-118. [22] Fu Y, Yi H, Bao J, et al.2015. LeNRT2.3 functions in nitrate acquisition and long-distance transport in tomato[J]. Febs Letters, 589(10): 1072-1079. [23] Geourjon C, Deleage G.1995. SOPMA: Significant improvements in protein secondary structure prediction by consensus prediction from multiple alignments[J]. Bioinformatics, 11(6): 681-684. [24] Gonzalez D H.2016. Plant Transcription Factors: Evolutionary, Structural and Functional Aspects[M]. Academic Press, US, pp. 3-11. [25] Jarret R L, Barboza G E, Batista F, et al.2019. Capsicum-an abbreviated compendium[J]. Journal of the American Society for Horticultural Science. American Society for Horticultural Science, 144(1): 3-22. [26] Kang W, Kim S, Lee H, et al.2016. Genome-wide analysis of Dof transcription factors reveals functional characteristics during development and response to biotic stresses in pepper[J]. Scientific Reports, 6(1): 1-12. [27] Kiba T, Feria B A, Lafouge F, et al.2012. The Arabidopsis nitrate transporter NRT2.4 plays a double role in roots and shoots of nitrogen-starved plants[J]. The Plant Cell, 24(1): 245-258. [28] Krogh A, Larsson B, Von Heijne G, et al.2001. Predicting transmembrane protein topology with a hidden Markov model: Application to complete genomes[J]. Journal of Molecular Biology, 305(3): 567-580. [29] Kumar S, Stecher G, Li M, et al.2018. MEGA X: Molecular evolutionary genetics analysis across computing platforms[J]. Molecular Biology and Evolution, 35(6): 1547-1549. [30] Lescot M, Déhais P, Thijs G, et al.2002. PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences[J]. Nucleic Acids Research, 30(1): 325-327. [31] Lezhneva L, Kiba T, Feria Bourrellier A B, et al.2014. The Arabidopsis nitrate transporter NRT 2.5 plays a role in nitrate acquisition and remobilization in nitrogen-starved plants[J]. The Plant Journal, 80(2): 230-241. [32] Li W, Wang Y, Okamoto M, et al.2007. Dissection of the AtNRT2.1:AtNRT2.2 inducible high-affinity nitrate transporter gene cluster[J]. Plant Physiology, 143(1): 425-433. [33] Li Y, Li J, Yan Y, et al.2018. Knock-down of CsNRT2.1, a cucumber nitrate transporter, reduces nitrate uptake, root length, and lateral root number at low external nitrate concentration[J]. Frontiers in Plant Science, 9: 722. [34] Lillo C.2008. Signalling cascades integrating light-enhanced nitrate metabolism[J]. Biochemical Journal, 415(1): 11-19. [35] Naz M, Luo B, Guo X, et al.2019. Overexpression of nitrate transporter OsNRT2.1 enhances nitrate-dependent root elongation[J]. Genes, 10(4): 290. [36] Nazoa P, Vidmar J J, Tranbarger T J, et al.2003. Regulation of the nitrate transporter gene AtNRT2.1 in Arabidopsis thaliana: Responses to nitrate, amino acids and developmental stage[J]. Plant Molecular Biology, 52(3): 689-703. [37] Ohyama T.2010. Nitrogen as A Major Essential Element of Plants[M]. Research Signpost, pp. 2-17. [38] Orsel M, Krapp A, Daniel-Vedele F.2002. Analysis of the NRT2 nitrate transporter family in Arabidopsis. Structure and gene expression[J]. Plant Physiology, 129(2): 886-896. [39] Remans T, Nacry P, Pervent M, et al.2006. A central role for the nitrate transporter NRT2.1 in the integrated morphological and physiological responses of the root system to nitrogen limitation in Arabidopsis[J]. Plant Physiology, 140(3): 909-921. [40] Tahir M M, Wang H, Ahmad B, et al.2021. Identification and characterization of NRT gene family reveals their critical response to nitrate regulation during adventitious root formation and development in apple rootstock[J]. Scientia Horticulturae, 275: 109642. [41] Tegeder M, Masclaux D C.2018. Source and sink mechanisms of nitrogen transport and use[J]. New Phytologist, 217(1): 35-53. [42] Tong J, Walk T C, Han P, et al.2020. Genome-wide identification and analysis of high-affinity nitrate transporter 2 (NRT2) family genes in rapeseed (Brassica napus L.) and their responses to various stresses[J]. Bmc Plant Biology, 20(1): 1-16. [43] Tsay Y, Chiu C, Tsai C, et al.2007. Nitrate transporters and peptide transporters[J]. Febs Letters, 581(12): 2290-2300. [44] Wang J, Li Y, Zhu F, et al.2019. Genome-wide analysis of nitrate transporter (NRT/NPF) family in sugarcane Saccharum spontaneum L.[J]. Tropical Plant Biology, 12(3): 133-149. [45] Wang X, Cai X, Xu C, et al.2021. Identification and characterization of the NPF, NRT2 and NRT3 in spinach[J]. Plant Physiology and Biochemistry, 158: 297-307. [46] Wang Y, Cheng Y, Chen K, et al.2018. Nitrate transport, signaling, and use efficiency[J]. Annual Review of Plant Biology, 69: 85-122. [47] Xu G, Fan X, Miller A J.2012. Plant nitrogen assimilation and use efficiency[J]. Annual Review of Plant Biology, 63: 153-182. [48] You L, Wang Y, Zhang T, et al.2022. Genome-wide identification of nitrate transporter 2 (NRT2) gene family and functional analysis of MeNRT2.2 in cassava (Manihot esculenta Crantz)[J]. Gene, 809: 146038. [49] Zhang J, Han Z, Lu Y, et al.2021. Genome-wide identification, structural and gene expression analysis of the nitrate transporters (NRTs) family in potato (Solanum tuberosum L.)[J]. PLOS ONE, 16(10): e257383. [50] Zhao Z, Li M, Xu W, et al.2022. Genome-wide identification of NRT gene family and expression analysis of nitrate transporters in response to salt stress in Poncirus trifoliata[J]. Genes, 13(7): 1115. [51] Zheng D, Han X, An Y I, et al.2013. The nitrate transporter NRT2.1 functions in the ethylene response to nitrate deficiency in Arabidopsis[J]. Plant, Cell & Environment, 36(7): 1328-1337.
