Abstract
Basic helix–loop–helix (bHLH) gene family is a gene family of transcription factors that plays essential roles in plant growth and development, secondary metabolism and response to biotic and abiotic stresses. Therefore, a comprehensive knowledge of the bHLH gene family is paramount to understand the molecular mechanisms underlying these processes and develop advanced technologies to manipulate the processes efficiently. Ginseng, Panax ginseng C.A. Meyer, is a well-known medicinal herb; however, little is known about the bHLH genes (PgbHLH) in the species. Here, we identified 137 PgbHLH genes from Jilin ginseng cultivar, Damaya, widely cultivated in Jilin, China, of which 50 are newly identified by pan-genome analysis. These 137 PgbHLH genes were phylogenetically classified into 26 subfamilies, suggesting their sequence diversification. They are alternatively spliced into 366 transcripts in a 4-year-old plant and involved in 11 functional subcategories of the gene ontology, indicating their functional differentiation in ginseng. The expressions of the PgbHLH genes dramatically vary spatio-temporally and across 42 genotypes, but they are still somehow functionally correlated. Moreover, the PgbHLH gene family, at least some of its genes, is shown to have roles in plant response to the abiotic stress of saline. These results provide a new insight into the evolution and functional differentiation of the bHLH gene family in plants, new bHLH genes to the PgbHLH gene family, and saline stress-responsive genes for genetic improvement in ginseng and other plant species.
Similar content being viewed by others
Data availability
The data are included within the article or additional files.
References
Atchley WR, Fitch WM (1997) A natural classification of the basic helix-loop-helix class of transcription factors. Proc Natl Acad Sci USA 94:5172–5176. https://doi.org/10.1073/pnas.94.10.5172
Bailey PC, Martin COG, Quail PH, Huq E, Heim MA (2003) Update on the basic helix-loop-helix transcription factor gene family in Arabidopsis thaliana. Plant Cell 15:2497–2501. https://doi.org/10.1105/tpc.151140
Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS (2009) MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res 37:W202–W208. https://doi.org/10.1093/nar/gkp335
Chen C, Chiou W, Zhang J (2008) Comparison of the pharmacological effects of Panax ginseng and Panax quinquefolium. Acta Pharmacol Sin 29:1103–1108. https://doi.org/10.1111/j.1745-7254.2008.00868.x
Chu Y, Xiao S, Su H, Liao B, Zhang J, Xu J, Chen S (2018) Genome-wide characterization and analysis of bHLH transcription factors in Panax ginseng. Acta Pharm Sin B 8:666–677. https://doi.org/10.1016/j.apsb.2018.04.004
Conesa A, Götz S, García-Gómez JM, Terol J, Talón M, Robles M (2005) Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics 21:3674–3676. https://doi.org/10.1093/bioinformatics/bti610
Cooper B, Clarke JD, Budworth P, Kreps J, Hutchison D, Park S, Guimil S, Dunn M, Luginbühl P, Ellero C, Goff SA, Glazebrook J (2003) A network of rice genes associated with stress response and seed development. Proc Natl Acad Sci USA 100:4945–4950. https://doi.org/10.1073/pnas.0737574100
Finn RD, Coggill P, Eberhardt RY, Eddy SR, Mistry J, Mitchell AL, Potter SC, Punta M, Qureshi M, Sangrador-vegas A, Salazar GA, Tate J, Bateman A (2016) The Pfam protein families database: towards a more sustainable future. Nucleic Acids Res 44:D279–285. https://doi.org/10.1093/nar/gkv1344
Friedrichsen DM, Nemhauser J, Muramitsu T, Maloof JN, Alonso J, Ecker JR, Furuya M, Chory J (2002) Three redundant brassinosteroid early response genes encode putative bHLH transcription factors required for normal growth. Genetics 162:1445–1456. https://doi.org/10.1023/A:1021280325474
Gabriela TO, Enamul H, Quail PH (2003) The Arabidopsis basic/helix–loop–helix transcription factor family. Plant Cell 15:1749–1770. https://doi.org/10.1105/tpc.013839
Gao L, Gonda I, Sun H, Ma Q, Bao K, Tieman DM, Burzynski-Chang EA, Fish TL, Stromberg KA, Sacks GL, Thannhauser TW, Foolad MR, Diez MJ, Blanca J, Canizares J, Xu Y, Van der Knaap E, Huang S, Klee HJ, Giovannoni JJ, Fei Z (2019) The tomato pan-genome uncovers new genes and a rare allele regulating fruit flavor. Nat Genet 51:1044–1051. https://doi.org/10.1038/s41588-019-0410-2
Hong GJ, Xue XY, Mao YB, Wang LJ, Chen XY (2012) Arabidopsis MYC2 interacts with DELLA proteins in regulating sesquiterpene synthase gene expression. Plant Cell 24:2635–2648. https://doi.org/10.1105/tpc.112.098749
Hua S, Fan H, Ling H (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
Huntley R, Dimmer E, Barrell D, Binns D, Apweiler R (2009) The gene ontology annotation (GOA) database. Natureprecedings 10:429–438. https://doi.org/10.1038/npre.2009.3154.1
Jayakodi M, Choi BS, Lee SC, Kim NH, Park JY, Jang W, Lakshmanan M, Mohan SVG, Lee DY, Yang TJ (2018) Ginseng genome database: an open-access platform for genomics of Panax ginseng. BMC Plant Biol 18:62. https://doi.org/10.1186/s12870-018-1282-9
Jin J, Tian F, Yang DC, Meng YQ, Kong L, Luo J, Gao G (2017) PlantTFDB 4.0: toward a central hub for transcription factors and regulatory interactions in plants. Nucleic Acids Res 45:D1040–D1045. https://doi.org/10.1093/nar/gkw982
Kanaoka MM, Pillitteri LJ, Fujii H, Yoshida Y, Bogenschutz NL, Takabayashi J, Zhu JK, Torii KU (2008) SCREAM/ICE1 and SCREAM2 specify three cell-state transitional steps leading to Arabidopsis stomatal differentiation. Plant Cell 20:1775–1785. https://doi.org/10.1105/tpc.108.060848
Kim NH, Jayakodi M, Lee SC, Choi BS, Jang W, Lee J, Kim HH, Waminal NE, Lakshmanan M, Van Nguyen B, Lee YS, Park HS, Koo HJ, Park JY, Perumal S, Joh HJ, Lee H, Kim J, Kim IS, Kim K, Koduru L, Kang KB, Sung SH, Yu Y, Park DS, Choi D, Seo E, Kim S, Kim YC, Hyun DY, Park YI, Kim C, Lee TH, Kim HU, Soh MS, Lee Y, In JG, Kim HS, Kim YM, Yang DC, Wing RA, Lee DY, Paterson AH, Yang TJ (2018) Genome and evolution of the shade-requiring medicinal herb Panax ginseng. Plant Biotechnol J 16:1904–1917. https://doi.org/10.1111/pbi.12926
Kim SK, Park JH (2011) Trends in Ginseng research in 2010. J Ginseng Res 35:389–398. https://doi.org/10.5142/jgr.2011.35.4.389
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
Li H, Sun J, Xu Y, Jiang H, Xu X, Li C (2007) The bHLH-type transcription factor AtAIB positively regulates ABA response in Arabidopsis. Plant Mol Biol 65:655–665. https://doi.org/10.1007/s11103-007-9230-3
Li X, Duan X, Jiang H, Sun Y, Tang Y, Zheng Y, Guo J, Liang W, Chen L, Yin J (2006) Genome-wide analysis of basic/helix–loop–helix transcription factor family in rice and Arabidopsis. Plant Physiol 141:1167. https://doi.org/10.1104/pp.106.080580
Lin Y, Wang K, Li X, Sun C, Yin R, Wang Y, Wang Y, Zhang MP (2018) Evolution, functional differentiation, and co-expression of the RLK gene family revealed in Jilin ginseng, Panax ginseng C.A Meyer. Mol Genet Genom 293:845–859. https://doi.org/10.1007/s00438-018-1425-6
Liu W, Tai H, Li S, Gao W, Zhao M, Xie C, Li WX (2014) bHLH122 is important for drought and osmotic stress resistance in Arabidopsis and in the repression of ABA catabolism. New Phytol 201:1192–1204. https://doi.org/10.1111/nph.12607
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT Method. Methods 25:402–408. https://doi.org/10.1006/meth.2001
Lorenzo CP, Anahit G, Irma RV, Martínez-García JF, Bilbao-Castro JR, Robertson DL (2010) Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae. Plant Physiol 153:1398–1412. https://doi.org/10.1104/pp.110.153593
Massari ME, 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
Mertens J, Pollier J, Vanden BR, López-Vidriero I, Franco-Zorrilla JM, Goossens A (2015) The bHLH transcription factors TSAR1 and TSAR2 regulate triterpene saponin biosynthesis in Medicago truncatula. Plant Physiol 170:194. https://doi.org/10.1104/pp.15.01645
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410. https://doi.org/10.1016/S1360-1385(02)02312-9
Murre C, Bain G, Dijk MAV, Engel I, Furnari BA, Massari ME, Matthews JR, Quong MW, Rivera RR, Stuiver MH (1994) Structure and function of helix–loop–helix proteins. BBA Biomembr 1218:129–135. https://doi.org/10.1016/0167-4781(94)90001-9
Ogawa S, Miyamoto K, Nemoto K, Sawasaki T, Yamane H, Nojiri H, Okada K (2017) OsMYC2, an essential factor for JA-inductive sakuranetin production in rice, interacts with MYC2-like proteins that enhance its transactivation ability. Sci Rep 7:40175. https://doi.org/10.1038/srep40175
O'Hara M, Kiefer D, Farrell K, Kemper K (1998) A review of 12 commonly used medicinal herbs. Arch Fam Med 7:523–536. https://doi.org/10.1001/archfami.7.6.523
Riechmann JL, Heard J, Martin G, Reuber L, Jiang C, Keddie J, Adam L, Pineda O, Ratcliffe OJ, Samaha RR (2000) Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes. Science 290:2105–2110. https://doi.org/10.1126/science.290.5499.2105
Sazegari S, Niazi A, Shahriari-Ahmadi F, Moshtaghi N, Ghasemi Y (2018) CrMYC1 transcription factor overexpression promotes the production of low abundance terpenoid indole alkaloids in Catharanthus roseus. Plant Omics J 11:30–36. https://doi.org/10.21475/poj.11.01.18.pne1020
Seo JS, Joo J, Kim MJ, Kim YK, Nahm BH, Song SI, Cheong JJ, Lee JS, Kim JK, Choi YD (2011) OsbHLH148, a basic helix-loop-helix protein, interacts with OsJAZ proteins in a jasmonate signaling pathway leading to drought tolerance in rice. Plant J 65:907–921. https://doi.org/10.1111/j.1365-313X.2010.04477.x
Shakirova FM (2007) Role of hormonal system in the manifestation of growth promoting and antistress action of salicylic acid. Salicylic acid: a plant hormone. Springer, Dordrecht, pp 69–89. https://doi.org/10.1007/1-4020-5184-0_4
Simionato E, Ledent V, Richards G, Thomas-Chollier M, Kerner P, Coornaert D, Degnan BM, Vervoort M (2007) Origin and diversification of the basic helix-loop-helix gene family in metazoans: insights from comparative genomics. BMC Evol Biol 7:33. https://doi.org/10.1186/1471-2148-7-33
Syed NH, Kalyna M, Marquez Y, Barta A, Brown JW (2012) Alternative splicing in plants—coming of age. Trends Plant Sci 17:616–623. https://doi.org/10.1016/j.tplants.2012.06.001
Theocharidis A, Van Dongen S, Enright AJ, Freeman TC (2009) Network visualization and analysis of gene expression data using BioLayout Express3D. Nat Protoc 4:1535–1550. https://doi.org/10.1038/nprot.2009.177
Tran T, Kim YR, Yang JL, Oh DR, Dao TT, Oh WK (2014) Dammarane triterpenes from the leaves of Panax ginseng enhance cellular immunity. Bioorg Med Chem 22:499–504. https://doi.org/10.1016/j.bmc.2013.11.002
Van Moerkercke A, Steensma P, Schweizer F, Pollier J, Gariboldi I, Payne R, Vanden Bossche R, Miettinen K, Espoz J, Purnama PC, Kellner F, Seppänen-Laakso T, O'Connor SE, Rischer H, Memelink J, Goossens A (2015) The bHLH transcription factor BIS1 controls the iridoid branch of the monoterpenoid indole alkaloid pathway in Catharanthus roseus. Proc Natl Acad Sci USA 112:8130–8135. https://doi.org/10.1073/pnas.1504951112
Wang J, Cheng G, Wang C, He Z, Lan X, Zhang S, Lan H (2017) The bHLH transcription factor CgbHLH001 is a potential interaction partner of CDPK in halophyte Chenopodium glaucum. Sci Rep 7:8441. https://doi.org/10.1038/s41598-017-06706-x
Wang K, Jiang S, Sun C, Lin Y, Yin R, Wang Y, Zhang MP (2015) The spatial and temporal transcriptomic landscapes of Ginseng, Panax ginseng C. A. Meyer. Sci Rep 5:18283. https://doi.org/10.1038/srep18283
Wang R, Zhao P, Kong N, Lu R, Pei Y, Huang C, Ma H, Chen Q (2018a) Genome-wide identification and characterization of the potato bHLH transcription factor family. Genes 9:54. https://doi.org/10.3390/genes9010054
Wang Y, Li X, Lin Y, Wang Y, Wang K, Sun C, Lu T, Zhang MP (2018b) structural variation, functional differentiation, and activity correlation of the cytochrome P450 gene superfamily revealed in ginseng. Plant Genome. https://doi.org/10.3835/plantgenome2017.11.0106
Xi S, Yu W, Na S (2018) Transcriptional regulation of bHLH during plant response to stress. Biochem Biophys Res Commun 503:397–401. https://doi.org/10.1016/j.bbrc.2018.07.123
Xing B, Yang D, Yu H, Zhang B, Yan K, Zhang X, Han R, Liang Z (2018) Overexpression of SmbHLH10 enhances tanshinones biosynthesis in Salvia miltiorrhiza hairy roots. Plant Sci 276:229–238. https://doi.org/10.1016/j.plantsci.2018.07.016
Xu J, Chu Y, Liao B, Xiao S, Yin Q, Bai R, Su H, Dong L, Li X, Qian J, Zhang J, Zhang Y, Zhang X, Wu M, Zhang J, Li G, Zhang L, Chang Z, Zhang Y, Jia Z, Liu Z, Afreh D, Nahurira R, Zhang L, Cheng R, Zhu Y, Zhu G, Rao W, Zhou C, Qiao L, Huang Z, Cheng YC, Chen S (2017) Panax ginseng genome examination for ginsenoside biosynthesis. Gigascience 6:1–15. https://doi.org/10.1093/gigascience/gix093
Yamamura C, Mizutani E, Okada K, Nakagawa H, Fukushima S, Tanaka A, Maeda S, Kamakura T, Yamane H, Takatsuji H, Mori M (2016) Diterpenoid phytoalexin factor, a bHLH transcription factor, plays a central role in the biosynthesis of diterpenoid phytoalexins in rice. Plant J 84:1100–1113. https://doi.org/10.1111/tpj.13065
Yan Q, Hou H, Singer SD, Yan X, Guo R, Wang X (2014) The grape VvMBF1 gene improves drought stress tolerance in transgenic Arabidopsis thaliana. Plant Cell Tiss Org 118:571–582. https://doi.org/10.1007/s11240-014-0508-2
Yin J, Li X, Zhan Y, Li Y, Qu Z, Sun L, Wang S, Yang J, Xiao J (2017a) Cloning and expression of BpMYC4 and BpbHLH9 genes and the role of BpbHLH9 in triterpenoid synthesis in birch. BMC Plant Biol 17:214. https://doi.org/10.1186/s12870-017-1150-z
Yin R, Zhao M, Wang K, Lin Y, Wang Y, Sun C, Wang Y, Zhang MP (2017b) Functional differentiation and spatial-temporal co-expression networks of the NBS-encoding gene family in Jilin ginseng Panax ginseng C.A. Meyer. PLoS ONE 12:e0181596. https://doi.org/10.1371/journal.pone.0181596
Zhai Y, Zhang L, Xia C, Fu S, Zhao G, Jia J, Kong X (2016) The wheat transcription factor, TabHLH39, improves tolerance to multiple abiotic stressors in transgenic plants. Biochem Biophys Res Commun 473:1321–1327. https://doi.org/10.1016/j.bbrc.2016.04.071
Zhao M, Lin Y, Wang Y, Li X, Han Y, Wang K, Sun C, Wang Y, Zhang M (2019) Transcriptome analysis identifies strong candidate genes for ginsenoside biosynthesis and reveals its underlying molecular mechanism in Panax ginseng C.A. Meyer. Sci Rep 9:615. https://doi.org/10.1038/s41598-018-36349-5
Zhao M, Morohashi KG, Grotewold E, Lloyd A (2008) The TTG1-bHLH-MYB complex controls trichome cell fate and patterning through direct targeting of regulatory loci. Development 135:1991–1999. https://doi.org/10.1242/dev.016873
Zhang J, Liu B, Li M, Feng D, Jin H, Wang P, Liu J, Xiong F, Wang J, Wang H (2015) The bHLH transcription factor bHLH104 interacts with IAA-LEUCINE RESISTANT3 and modulates iron homeostasis in Arabidopsis. Plant Cell 27:787–805. https://doi.org/10.1105/tpc.114.132704
Zhang M, Wu YH, Lee MK, Liu Y, Rong Y, Santos TS, Wu C, Xie F, Nelson RL, Zhang HB (2010) Numbers of genes in the NBS and RLK families vary by more than four-fold within a plant species and are regulated by multiple factors. Nucleic Acids Res 38:6513–6525. https://doi.org/10.1093/nar/gkq524
Zhang M, Liu Y-H, Chang C-S, Zhi H, Wang S, Xu W, Smith CW, Zhang H-B (2019) Quantification of gene expression while taking into account RNA alternative splicing. Genomics 111(6):1517–1528. https://doi.org/10.1016/j.ygeno.2018.10.009
Zhou J, Li F, Wang J, Ma Y, Chong K, Xu Y (2009) Basic helix-loop-helix transcription factor from wild rice (OrbHLH2) improves tolerance to salt- and osmotic stress in Arabidopsis. J Plant Physiol 166:1296–1306. https://doi.org/10.1016/j.jplph.2009.02.007
Funding
This study was funded by China 863 Project (2013AA102604-3), the Bureau of Science and Technology of Jilin Province (20170101010JC, 20180414077GH, 20180101027JC, 20190201264JC, 20190103104JH), the Development and Reform Commission of Jilin Province (2016C064, 2018C047-3).
Author information
Authors and Affiliations
Contributions
MPZ and YW designed the experiments of the study. LZ and MZZ wrote the manuscript and MPZ revised the manuscript. LZ, MZZ, MC, LL, YJ, SZL, KYW, YFW, CYS, JC, PC, JL, and YJS performed the experiments and contributed to data analysis. All authors reviewed and approved the final manuscript.
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Ethical standards
This article does not contain any studies with human participants or animals performed by any of the authors.
Additional information
Communicated by Stefan Hohmann.
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Zhu, L., Zhao, M., Chen, M. et al. The bHLH gene family and its response to saline stress in Jilin ginseng, Panax ginseng C.A. Meyer. Mol Genet Genomics 295, 877–890 (2020). https://doi.org/10.1007/s00438-020-01658-w
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00438-020-01658-w