Genome-wide identification and expression analysis of NtbHLH gene family in tobacco (Nicotiana tabacum) and the role of NtbHLH86 in drought adaptation

doi:10.1016/j.pld.2020.10.004

Abstract

Abstract: The bHLH transcription factors play pivotal roles in plant growth and development, production of secondary metabolites and responses to various environmental stresses. Although the bHLH genes have been well studied in model plant species, a comprehensive investigation of the bHLH genes is required for tobacco with newly obtained high-quality genome. In the present study, a total of 309 NtbHLH genes were identified and can be divided into 23 subfamilies. The conserved amino acids which are essential for their function were predicted for the NtbHLH proteins. Moreover, the NtbHLH genes were conserved during evolution through analyzing the gene structures and conserved motifs. A total of 265 NtbHLH genes were localized in the 24 tobacco chromosomes while the remained 44 NtbHLH genes were mapped to the scaffolds due to the complexity of tobacco genome. Moreover, transcripts of NtbHLH genes were obviously tissue-specific expressed from the gene-chip data from 23 tobacco tissues, and expressions of 20 random selected NtbHLH genes were further confirmed by quantitative real-time PCR, indicating their potential functions in the plant growth and development. Importantly, overexpressed NtbHLH86 gene confers improve drought tolerance in tobacco indicating that it might be involved in the regulation of drought stress. Therefore, our findings here provide a valuable information on the characterization of NtbHLH genes and further investigation of their functions in tobacco.

Key words: bHLH gene family, Development, Genome-wide analysis, Characterization

Ge Bai, Da-Hai Yang, Peijian Chao, Heng Yao, MingLiang Fei, Yihan Zhang, Xuejun Chen, Bingguang Xiao, Feng Li, Zhen-Yu Wang, Jun Yang, He Xie. Genome-wide identification and expression analysis of NtbHLH gene family in tobacco (Nicotiana tabacum) and the role of NtbHLH86 in drought adaptation[J]. Plant Diversity, 2021, 43(06): 510-522.

References

Abe, H., Urao, T., Ito, T., et al., 2003. Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling. Plant Cell 15, 68-78. https://doi.org/10.1105/tpc.006130.
Atchley, W.R., Terhalle, W., Dress, A., 1999. Positional dependence, cliques, and predictive motifs in the bHLH protein domain. J. Mol. Evol. 48, 501-516. https://doi.org/10.1007/pl00006494.
Bai, G., Yang, D.H., Cao, P., et al., 2019. Genome-Wide identification, gene structure and expression analysis of the MADS-box gene family indicate their function in the development of tobacco (Nicotiana tabacum L.). Int. J. Mol. Sci. 20, https://doi.org/10.3390/ijms20205043.
Bailey, T.L., Boden, M., Buske, F.A., et al., 2009. Meme suite: tools for motif discovery and searching. Nucleic Acids Res. 37, W202-W208. https://doi.org/10.1093/nar/gkp335 (Web Server issue).
Bailey, P.C., Martin, C., Toledo-Ortiz, G., et al., 2003. Update on the basic helix-loophelix transcription factor gene family in Arabidopsis thaliana. Plant Cell 15, 2497-2502. https://doi.org/10.1105/tpc.151140.
Brioudes, F., Joly, C., Szecsi, J., et al., 2009. Jasmonate controls late development stages of petal growth in Arabidopsis thaliana. Plant J. : Cell Mole Bio 60, 1070-1080. https://doi.org/10.1111/j.1365-313X.2009.04023.x.
Cheng, X., Xiong, R., Liu, H., et al., 2018. Basic helix-loop-helix gene family: genome wide identification, phylogeny, and expression in Moso bamboo. Plant Physiol. Biochem. 132, 104-119. https://doi.org/10.1016/j.plaphy.2018.08.036.
Chinnusamy, V., Ohta, M., Kanrar, S., et al., 2003. ICE1: a regulator of cold-induced transcriptome and freezing tolerance in Arabidopsis. Genes Dev. 17, 1043-1054.https://doi.org/10.1101/gad.1077503.
Dombrecht, B., Xue, G.P., Sprague, S.J., et al., 2007. MYC2 differentially modulates diverse jasmonate-dependent functions in Arabidopsis. Plant Cell 19, 2225-2245. https://doi.org/10.1105/tpc.106.048017.
El-Gebali, S., Mistry, J., Bateman, A., et al., 2019. The Pfam protein families database in 2019. Nucleic Acids Res. 47, D427-D432. https://doi.org/10.1093/nar/gky995.
Farquharson, K.L., 2016. A domain in the bHLH transcription factor DYT1 is critical for anther development. Plant Cell 28, 997-998. https://doi.org/10.1105/tpc.16.00331.
Fedorov, A., Merican, A.F., Gilbert, W., 2002. Large-scale comparison of intron positions among animal, plant, and fungal genes. Proc. Natl. Acad. Sci. U.S.A. 99, 16128-16133. https://doi.org/10.1073/pnas.242624899.
Ferre-D’Amare, A.R., Pognonec, P., Roeder, R.G., et al., 1994. Structure and function of the b/HLH/Z domain of USF. EMBO J. 13, 180-189.
Finn, R.D., Clements, J., Arndt, W., et al., 2015. HMMER web server: 2015 update. Nucleic Acids Res. 43, W30-W38. https://doi.org/10.1093/nar/gkv397.
Flagel, L.E., Wendel, J.F., 2009. Gene duplication and evolutionary novelty in plants. New Phytol. 183, 557-564. https://doi.org/10.1111/j.1469-8137.2009. 02923.x.
Fursova, O.V., Pogorelko, G.V., Tarasov, V.A., 2009. Identification of ICE2, a gene involved in cold acclimation which determines freezing tolerance in Arabidopsis thaliana. Gene 429, 98-103. https://doi.org/10.1016/j.gene.2008.10.016.
Gonzalez, A., Zhao, M., Leavitt, J.M., et al., 2008. Regulation of the anthocyanin biosynthetic pathway by the TTG1/bHLH/Myb transcriptional complex in Arabidopsis seedlings. Plant J. : Cell Mole Bio 53, 814-827. https://doi.org/10.1111/j.1365-313X.2007.03373.x.
Groszmann, M., Bylstra, Y., Lampugnani, E.R., et al., 2010. Regulation of tissuespecific expression of SPATULA, a bHLH gene involved in carpel development, seedling germination, and lateral organ growth in Arabidopsis. J. Exp. Bot. 61, 1495-1508. https://doi.org/10.1093/jxb/erq015.
Guo, X.J., Wang, J.R., 2017. Global identification, structural analysis and expression characterization of bHLH transcription factors in wheat. BMC Plant Biol. 17, 90.https://doi.org/10.1186/s12870-017-1038-y.
Hu, B., Jin, J., Guo, A.Y., et al., 2015. Gsds 2.0: an upgraded gene feature visualization server. Bioinformatics 31, 1296-1297. https://doi.org/10.1093/bioinformatics/btu817.
Jones, S., 2004. An overview of the basic helix-loop-helix proteins. Genome Biol. 5, 226. https://doi.org/10.1186/gb-2004-5-6-226.
Kondrashov, F.A., Rogozin, I.B., Wolf, Y.I., et al., 2002. Selection in the evolution of gene duplications. Genome Biol. 3. https://doi.org/10.1186/gb-2002-3-2-research0008. RESEARCH0008.
Krzywinski, M., Schein, J., Birol, I., et al., 2009. Circos: an information aesthetic for comparative genomics. Genome Res. 19, 1639-1645. https://doi.org/10.1101/gr.092759.109.
Kumar, S., Stecher, G., Tamura, K., 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol. Biol. Evol. 33, 1870-1874. https://doi.org/10.1093/molbev/msw054.
Ledent, V., Vervoort, M., 2001. The basic helix-loop-helix protein family: comparative genomics and phylogenetic analysis. Genome Res. 11, 754-770. https://doi.org/10.1101/gr.177001.
Letunic, I., Bork, P., 2018. 20 years of the SMART protein domain annotation resource. Nucleic Acids Res. 46, D493-D496. https://doi.org/10.1093/nar/gkx922.
Li, H., Gao, W., Xue, C., Zhang, Y., Liu, Z., Zhang, Y., Meng, X., Liu, M., Zhao, J., 2019. Genome-wide analysis of the bHLH gene family in Chinese jujube (Ziziphus jujuba Mill.) and wild jujube. BMC Genom. 20, 568. https://doi.org/10.1186/s12864-019-5936-2.
Li, X., Duan, X., Jiang, H., Sun, Y., Tang, Y., Yuan, Z., Guo, J., Liang, W., Chen, L., Yin, J., Ma, H., Wang, J., Zhang, D., 2006. Genome-wide analysis of basic/helix-loophelix transcription factor family in rice and Arabidopsis. Plant Physiol. 141, 1167-1184. https://doi.org/10.1104/pp.106.080580.
Li, F., Zhang, H., Wang, S., et al., 2016. Identification of topping responsive proteins in tobacco roots. Front. Plant Sci. 7, 582. https://doi.org/10.3389/fpls.2016. 00582.
Lu, R., Zhang, J., Liu, D., et al., 2018. Characterization of bHLH/HLH genes that are involved in brassinosteroid (BR) signaling in fiber development of cotton(Gossypium hirsutum). BMC Plant Biol. 18, 304. https://doi.org/10.1186/s12870-018-1523-y.
Mandaokar, A., Kumar, V.D., Amway, M., et al., 2003. Microarray and differential display identify genes involved in jasmonate-dependent anther development. Plant Mol. Biol. 52, 775-786. https://doi.org/10.1023/a:1025045217859.
Mao, T.Y., Liu, Y.Y., Zhu, H.H., et al., 2019. Genome-wide analyses of the bHLH gene family reveals structural and functional characteristics in the aquatic plant Nelumbo nucifera. PeerJ 7, e7153. https://doi.org/10.7717/peerj.7153.
Massari, M.E., Murre, C., 2000. Helix-loop-helix proteins: regulators of transcription in eucaryotic organisms. Mol. Cell Biol. 20, 429-440. https://doi.org/10.1128/mcb.20.2.429-440.2000.
McWilliam, H., Li, W., Uludag, M., et al., 2013. Analysis tool web services from the EMBL-EBI. Nucleic Acids Res. 41, W597-W600. https://doi.org/10.1093/nar/gkt376 (Web Server issue).
Moore, A.W., Barbel, S., Jan, L.Y., et al., 2000. A genomewide survey of basic helixloop-helix factors in Drosophila. Proc. Natl. Acad. Sci. U.S.A. 97, 10436-10441.https://doi.org/10.1073/pnas.170301897.
Murre, C., McCaw, P.S., Baltimore, D., 1989. A new DNA binding and dimerization motif in immunoglobulin enhancer binding, daughterless, MyoD, and myc proteins. Cell 56, 777-783. https://doi.org/10.1016/0092-8674(89)90682-x.
Nakata, M., Mitsuda, N., Herde, M., et al., 2013. A bHLH-type transcription factor, ABA-INDUCIBLE BHLH-TYPE TRANSCRIPTION FACTOR/JA-ASSOCIATED MYC2-LIKE1, acts as a repressor to negatively regulate jasmonate signaling in Arabidopsis. Plant Cell 25, 1641-1656. https://doi.org/10.1105/tpc.113.111112.
Niu, X., Guan, Y., Chen, S., Li, H., 2017. Genome-wide analysis of basic helix-loophelix (bHLH) transcription factors in Brachypodium distachyon. BMC Genom. 18, 619. https://doi.org/10.1186/s12864-017-4044-4.
Oh, E., Kim, J., Park, E., et al., 2004. PIL5, a phytochrome-interacting basic helix-loophelix protein, is a key negative regulator of seed germination in Arabidopsis thaliana. Plant Cell 16, 3045-3058. https://doi.org/10.1105/tpc.104.025163.
Oh, E., Yamaguchi, S., Hu, J., et al., 2007. PIL5, a phytochrome-interacting bHLH protein, regulates gibberellin responsiveness by binding directly to the GAI and RGA promoters in Arabidopsis seeds. Plant Cell 19, 1192-1208. https://doi.org/10.1105/tpc.107.050153.
Oh, E., Yamaguchi, S., Kamiya, Y., et al., 2006. Light activates the degradation of PIL5 protein to promote seed germination through gibberellin in Arabidopsis. Plant J. 47, 124-139. https://doi.org/10.1111/j.1365-313X.2006.02773.x.
Pires, N., Dolan, L., 2010. Origin and diversification of basic-helix-loop-helix proteins in plants. Mol. Biol. Evol. 27, 862-874. https://doi.org/10.1093/molbev/msp288.
Riechmann, J.L., Heard, J., Martin, G., et al., 2000. Arabidopsis transcription factors:genome-wide comparative analysis among eukaryotes. Science 290, 2105-2110. https://doi.org/10.1126/science.290.5499.2105.
Rogozin, I.B., Wolf, Y.I., Sorokin, A.V., et al., 2003. Remarkable interkingdom conservation of intron positions and massive, lineage-specific intron loss and gain in eukaryotic evolution. Curr. Biol. 13, 1512-1517. https://doi.org/10.1016/s0960-9822(03)00558-x.
Rushton, P.J., Bokowiec, M.T., Han, S., et al., 2008. Tobacco transcription factors:novel insights into transcriptional regulation in the Solanaceae. Plant Physiol. 147, 280-295. https://doi.org/10.1104/pp.107.114041.
Schweizer, F., Fernandez-Calvo, P., Zander, M., et al., 2013. Arabidopsis basic helixloop-helix transcription factors MYC2, MYC3, and MYC4 regulate glucosinolate biosynthesis, insect performance, and feeding behavior. Plant Cell 25, 3117-3132. https://doi.org/10.1105/tpc.113.115139.
Shen, W., Cui, X., Li, H., et al., 2019. Genome-wide identification and analyses of bHLH family genes in Brassica napus. Can. J. Plant Sci. 99, 589-598. https://doi.org/10.1139/cjps-2018-0230.
Shoji, T., Hashimoto, T., 2011. Tobacco MYC2 regulates jasmonate-inducible nicotine biosynthesis genes directly and by way of the NIC2-locus ERF genes. Plant Cell Physiol. 52, 1117-1130. https://doi.org/10.1093/pcp/pcr063.
Smaczniak, C., Immink, R.G., Angenent, G.C., et al., 2012. Developmental and evolutionary diversity of plant MADS-domain factors: insights from recent studies. Development 139, 3081-3098. https://doi.org/10.1242/dev. 074674.
Sun, H., Fan, H.J., Ling, H.Q., 2015. Genome-wide identification and characterization of the bHLH gene family in tomato. BMC Genom. 16, 9. https://doi.org/10.1186/s12864-014-1209-2.
Szecsi, J., Joly, C., Bordji, K., et al., 2006. BIGPETALp, a bHLH transcription factor is involved in the control of Arabidopsis petal size. EMBO J 25 (16), 3912-3920.https://doi.org/10.1038/sj.emboj.7601270.
Tepperman, J.M., Hudson, M.E., Khanna, R., et al., 2004. Expression profiling of phyB mutant demonstrates substantial contribution of other phytochromes to red-light-regulated gene expression during seedling de-etiolation. Plant J. 38, 725-739. https://doi.org/10.1111/j.1365-313X.2004.02084.x.
Toledo-Ortiz, G., Huq, E., Quail, P.H., 2003. The Arabidopsis basic/helix-loop-helix transcription factor family. Plant Cell 15, 1749-1770. https://doi.org/10.1105/tpc.013839.
Wang, Y., Liu, A., 2020. Genomic characterization and expression analysis of basic helix-loop-helix (bHLH) family genes in traditional Chinese herb Dendrobium officinale. Plants 9. https://doi.org/10.3390/plants9081044.
Yang, J., Gao, M., Huang, L., et al., 2017. Identification and expression analysis of the apple (Malus x domestica) basic helix-loop-helix transcription factor family. Sci. Rep. 7, 28. https://doi.org/10.1038/s41598-017-00040-y.
Zhang, H.B., Bokowiec, M.T., Rushton, P.J., et al., 2012. Tobacco transcription factors NtMYC2a and NtMYC2b form nuclear complexes with the NtJAZ1 repressor and regulate multiple jasmonate-inducible steps in nicotine biosynthesis. Mol. Plant 5, 73-84. https://doi.org/10.1093/mp/ssr056.
Zhang, T., Lv, W., Zhang, H., et al., 2018. Genome-wide analysis of the basic HelixLoop-Helix (bHLH) transcription factor family in maize. BMC Plant Biol. 18, 235.https://doi.org/10.1186/s12870-018-1441-z.
Zhao, K., Li, S., Yao, W., et al., 2018a. Characterization of the basic helix-loop-helix gene family and its tissue-differential expression in response to salt stress in poplar. PeerJ 6, e4502. https://doi.org/10.7717/peerj.4502.
Zhao, Q., Xiang, X., Liu, D., et al., 2018b. Tobacco transcription factor NtbHLH123 confers tolerance to cold stress by regulating the NtCBF pathway and reactive oxygen species homeostasis. Front. Plant Sci. 9, 381. https://doi.org/10.3389/fpls.2018.00381.

[1]	Wen-Bo Wang, Tao Ao, Yan-Yu Zhang, Di Wu, Wei Xu, Bing Han, Ai-Zhong Liu. Genome-wide analysis of the B3 transcription factors reveals that RcABI3/VP1 subfamily plays important roles in seed development and oil storage in castor bean (Ricinus communis) [J]. Plant Diversity, 2022, 44(02): 201-212.
[2]	Zaiqing Wang, Jingge Kuang, Bing Han, Suiyun Chen, Aizhong Liu. Genomic characterization and expression profiles of stress-associated proteins (SAPs) in castor bean (Ricinus communis) [J]. Plant Diversity, 2021, 43(02): 152-162.
[3]	Anuj Choudhary, Antul Kumar, Nirmaljit Kaur. ROS and oxidative burst: Roots in plant development [J]. Plant Diversity, 2020, 42(01): 33-43.
[4]	JIANG Zi-Wei, LIU Bao-Dong, CHENG Xiao, LI Xiao-Dong. The Embryonic Development Study on Isoetes yunguiensis, an Endangered and Relict Pteridophyta [J]. Plant Diversity, 2015, 37(05): 531-536.
[5]	XUE Peng, WEN Bin. Effects of Drying Rates on the Desiccation Tolerance of Citrus maxima ‘Feizhouyou’ Seeds [J]. Plant Diversity, 2015, 37(03): 293-300.
[6]	GUO Yan-Dong, WANG Quan-Xi, LI Bao-Gui, CAO Jian-Guo. Studies on the Development of Gametophyte and Oogenesis of the Fern Microlepia platyphylla [J]. Plant Diversity, 2014, 36(06): 747-754.
[7]	LONG Chun-Lin. Modern Ethnobotany: An Introduction [J]. Plant Diversity, 2013, 35(4): 438-442.
[8]	SONG Juan-, CHEN Mao-Sheng-, LI Jia-Long-, NIU Long-Jian-, XU Zeng-Fu. Effects of Soilapplied Paclobutrazol on the Vegetative and Reproductive Growth of Biofuel Plant Jatropha curcas [J]. Plant Diversity, 2013, 35(2): 173-179.
[9]	ZHANG Juan-Juan-, YAN Ning-, HU Hong. The Seed Development of Three Paphiopedilum Species in Relation to Asymbiotic Germination [J]. Plant Diversity, 2013, 35(1): 33-40.
[10]	CHENG Zhi-Peng, CHANG Hai-Shuang, YANG Zhong-Nan, ZHANG Sen. ST273 Is Essential for Tapetum and Microspore Development in Arabidopsis thaliana* [J]. Plant Diversity, 2012, 34(5): 502-508.
[11]	ZHANG Qiu-Ju-, ZHANG Ai-Hua-, YANG He-, ZHANG Lian-Xue. Allelopathic Effects of Ginsenosides from Panax ginseng on the Root Development of Four Cultivar Plants in Their Early Growth Stages [J]. Plant Diversity, 2012, 34(4): 391-.
[12]	ZHANG Yan, ZHU Zhu, YANG Ji. Evolutionary Innovation Based on Co-option and Integration of Developmental Modules [J]. Plant Diversity, 2012, 34(3): 222-230.
[13]	ZHANG Li-Xia-, LAN Qin-Ying-, LI Hai-Tao-, TAN Yun-Hong-, GUAN Yan-Hong-, LI Xue-Lan. Developmental Changes in Relation to Desiccation Tolerance and Storage Characteristics of Aquilaria sinensis (Thymelaeaceae) Seeds [J]. Plant Diversity, 2011, 33(4): 458-464.
[14]	ZHOU Que, HUANG Xue-Yong, YANG Zhong-Nan, ZHANG Sen. MS1521 Gene is Required for Anther Development of Arabidopsis thaliana [J]. Plant Diversity, 2010, 32(6): 528-534.
[15]	WANG WeiQing , ,CHENG Xiao*, JIAO Yu ,CHEN GuiJu. Substratum Affects the Gametophyte Development and Reproduction of Sinopteris grevilleoides(Sinopteridaceae), an Endangered Fern Endemic to China [J]. Plant Diversity, 2010, 32(01): 25-31.