pp 1–13 | Cite as

Inhibition of BpEIN3 causes plaques in leaves of Betula platyphylla × B. pendula

  • Xiaoyuan Li
  • Ranhong Li
  • Chu Wang
  • Qibin Yu
  • Su Chen
  • Jing JiangEmail author
  • Guifeng Liu
Original Article


Key message

In this study, constructing BpEIN3 overexpression, inhibition expression vector, we transferred to birch to obtain BpEIN3 transgenic lines and study the function of this gene.


EIN3 is an important transcription factor in the ethylene signal transduction pathway. It initiates the expression of a series of genes that regulate ethylene, positively regulating ethylene reaction, which affects the lesion mimic of plants through interacting ethylene signal and other signals in the signal transduction pathways. In order to reveal the function of BpEIN3 in Betula platyphylla, this study used birch as a genetic transformation receptor. Constructing BpEIN3 overexpression, inhibition expression vector, we transferred to birch to obtain BpEIN3 transgenic lines and study the function of this gene, which provided a reference for further revealing the molecular mechanism of BpEIN3 regulation of senescence and programmed cell death of birch leaves. In this study, the plant height, diameter and photosynthetic parameters of BpEIN3 transgenic lines were measured. The number of leaf lesions, histochemical staining, antioxidant enzymes, endogenous hormones and the expression of disease-related genes were also recorded. Results: We analyzed the expression of BpEIN3 by qRT-PCR, which was down-regulated in BpEIN3-inhibited expression lines and up-regulated in overexpression lines. The 2-year-old BpEIN3-RNAi lines exhibited brown spots on the leaves, leading to premature senescence and defoliation. The number of leaf spots is significantly different between different transgenic lines based on solid microscope. Photosynthetic parameters of BpEIN3-RNAi lines such as photosynthetic rate (Pn), stomatal conductance (Gs) and transpiration rate (Tr) were significantly lower than wild type. The accumulation of H2O2 in the leaves of transgenic lines was more than WT. The content of endogenous salicylic acid (SA), abscisic acid (ABA), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in BpEIN3-RNAi lines was higher than that in WT, except jasmonic acid (JA) content. Meanwhile, BpEIN3-RNAi lines showed strong resistance to Alternaria alternata. We analyzed the expression of genes which were involved in ABA, JA and SA biosynthesis, metabolism and senescence. The results showed that BpPYR9, BpPYL4, BpNPR1 and BpJAZ10 were up-regulated and BpSAG12, BpNAC2, BpPR1a and BpPR1b were abundantly expressed in the 6th and 7th leaves of the transgenic lines. We concluded that the suppression of BpEIN3 could lead to lesion and promote premature senescence.


Birch BpEIN3-RNAi lines Lesion Senescence 



I thank Professor Jiang Jing and Professor Liu Guifeng for their guidance and help in the experiment. At the same time, I would like to thank Wang Chu and Li Ranhong for their help in experimental skills. I also acknowledge the help of Associate Professor Chen Su and Professor Yu Qibin in writing.


This study was supported by the following foundations: 1. National Natural Science Foundation of China (NSFC) (Grant No. 31670673); 2. The 111 Project (B16010); 3. National Natural Science Foundation of China (NSFC) (Grant No. 31800558).

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.


  1. Berrocal-Lobo M, Molina A (2004) Ethylene response factor 1 mediates arabidopsis resistance to the soilborne fungus Fusarium oxysporum. Mol Plant Microb Interact 17:763–770CrossRefGoogle Scholar
  2. Bruggeman Q, Raynaud C, Benhamed M, Delarue M (2015) To die or not to die? Lessons from lesion mimic mutants. Front Plant Sci 6:24CrossRefGoogle Scholar
  3. Chen H, Xue L, Chintamanani S et al (2009) Ethylene insensitive 3 and ethylene insensitive 3-like 1 repress salicylic acid induction deficient 2 expression to negatively regulate plant innate immunity in Arabidopsis. Plant Cell 21:2527–2540CrossRefGoogle Scholar
  4. Chini A, Fonseca S, Fernández G et al (2007) The JAZ family of repressors is the missing link in jasmonate signalling. Nature 448:666–671CrossRefGoogle Scholar
  5. Coll NS, Epple P, Dangl JL (2011) Programmed cell death in the plant immune system. Cell Death Differ 18:1247–1256CrossRefGoogle Scholar
  6. Dangl JL, Dietrich RA, Richberg MH (1996) Death don’t have no mercy: cell death programs in plant-microbe interactions. Plant Cell 8:1793–1807CrossRefGoogle Scholar
  7. Gan SS, Hörtensteiner S (2013) Frontiers in plant senescence research: from bench to bank. Plant Mol Biol 82:503–504CrossRefGoogle Scholar
  8. Harkenrider M, Sharma R, De Vleesschauwer D, Tsao L, Zhang X, Chern M, Canlas P, Zuo S, Ronald PC, Wang Z (2016) Overexpression of rice wall-associated kinase 25 (OsWAK25) alters resistance to bacterial and fungal pathogens. PLOS ONE 11(1):e0147310CrossRefGoogle Scholar
  9. Hibi T, Ohashi Y (2007) Involvement of EIN3 homologues in basic PR gene expression and flower development in tobacco plants. J Exp Bot 58:3671–3678CrossRefGoogle Scholar
  10. Huang QN, Yang Y, Shi YF et al (2010) Spotted-leaf mutants of rice (Oryza sativa). Rice Sci 17:247–256CrossRefGoogle Scholar
  11. Huang H, Wang S, Jiang J et al (2014) Overexpression of BpAP1 induces early flowering and produces dwarfism in Betula platyphylla × B. pendula. Physiol Plant 151:495–506CrossRefGoogle Scholar
  12. Hyo JK, Sung HH, You WK (2014) Gene regulatory cascade of senescence-associated NAC transcription factors activated by ethylene-insensitive2-mediated leaf senescence signalling in Arabidopsis. J Exp Bot 65:4023–4036CrossRefGoogle Scholar
  13. Lawton KA (1994) Acquired resistance signal transduction in Arabidopsis is ethylene independent. Plant Cell Online 6:581–588CrossRefGoogle Scholar
  14. Li H, Jianzhen L, Qin P et al (2003) Research progress on NPR1 gene. J Guangxi Normal Univ 21Google Scholar
  15. Li R, Chen S, Liu G et al (2017) Characterization and identification of a woody lesion mimic mutant lmd, showing defence response and resistance to Alternaria alternate in birch. Sci Rep 7:11308CrossRefGoogle Scholar
  16. Lin ZF, Li SS, Lin GZ, Sun GC, Guo JY (1984) Superoxide dismutase activity and lipid peroxidation in relation to senescence of rice leaves. Acta Bot Sin 26:605–615Google Scholar
  17. Liu JG, Wang YQ, Zhang HS et al (2013) Research progress of ERF transcription factors in plant biotic and abiotic stress responses. Acta Agric Boreali SinGoogle Scholar
  18. Liu J, Shen J, Xu Y et al (2016) Ghd2, a CONSTANS-like gene, confers drought sensitivity through regulation of senescence in rice. J Exp Bot 67:5785–5798CrossRefGoogle Scholar
  19. Mcgrann GRD, Steed A, Burt C et al (2015) Differential effects of lesion mimic mutants in barley on disease development by facultative pathogens. J Exp Bot 66:3417–3428CrossRefGoogle Scholar
  20. Miura K, Tada Y (2014) Regulation of water, salinity, and cold stress responses by salicylic acid. Front Plant Sci 5:4CrossRefGoogle Scholar
  21. Morris K, Mackerness SAH, Page T et al (2000) Salicylic acid has a role in regulating gene expression during leaf senescence. Plant J 23:677–685CrossRefGoogle Scholar
  22. Noushina I, Khan NA, Antonio F et al (2017) Ethylene role in plant growth, interaction with other phytohormones. Frontiers in Plant Science, Development and SenescenceGoogle Scholar
  23. Qi T, Wang J, Huang H, Liu B, Gao H, Liu Y, Song S, Xie D (2015) Regulation of jasmonate-induced leaf senescence by Antagonism between bHLH subgroup IIIe and IIId factors in Arabidopsis. Plant Cell 27(6):1634–1649CrossRefGoogle Scholar
  24. Santino A, Taurino M, Domenico SD et al (2013) Jasmonate signaling in plant development and defense response to multiple (a)biotic stresses. Plant Cell Rep 32:1085–1098CrossRefGoogle Scholar
  25. Séverine L, Vailleau F, Claudine B et al (2003) Lesion mimic mutants: keys for deciphering cell death and defense pathways in plants? Trends Plant Sci 8:0–271Google Scholar
  26. Shoumin X (1997) Changes in photosynthetic capacity and related enzyme activities during soybean (Glycine max) leaf senescence. J Northeast Normal Univ (Nat Sci Ed) 4:70–73Google Scholar
  27. Solano R, Stepanova A, Chao Q et al (1998) Nuclear events in ethylene signaling: a transcriptional cascade mediated by ETHYLENE-INSENSITIVE3 and ETHYLENE-RESPONSE-FACTOR1. Genes Dev 12:3703–3711CrossRefGoogle Scholar
  28. Thines B, Katsir L, Melotto M et al (2007) JAZ repressor proteins are targets of the SCFCOI1 complex during jasmonate signalling. Nature 448:661–665CrossRefGoogle Scholar
  29. Thomma BP, Penninckx IA, Cammue BP et al (2001) The complexity of disease signaling in Arabidopsis. Curr Opin Immunol 13:63–68CrossRefGoogle Scholar
  30. Vos MD, Zaanen WV, Koornneef A et al (2006) Herbivore-induced resistance against microbial pathogens in Arabidopsis. Plant Physiol 142:352–363CrossRefGoogle Scholar
  31. Wang Pengjie W, Dianxing SX (2018) Research progress on rice senescence-related mutants. Nucl Agron J 32:0497–0505Google Scholar
  32. Williams B, Dickman M (2008) Plant programmed cell death: can’t live with it; can’t live without it. Mol Plant Pathol 9:531–544CrossRefGoogle Scholar
  33. Yan Y, Stolz S, Chetelat A et al (2007) A downstream mediator in the growth repression limb of the jasmonate pathway. Plant Cell Online 19:2470–2483CrossRefGoogle Scholar
  34. Yang G, Chen S, Wang S et al (2015) BpGH3.5, an early auxin-response gene, regulates root elongation in Betula platyphylla × Betula pendula. Plant Cell Tissue Org Cult 120(1):239–250CrossRefGoogle Scholar
  35. Zhang CL, Guo HW (2012) Study on ethylene signal transduction pathway. Nature 34(4):219–227 (In Chinese)Google Scholar
  36. Zhang YJ, Zhao JZ, Zhang KW (2014) Research progress on mechanisms of phytohormones regulating leaf senescence. Plant Physiol JGoogle Scholar
  37. Zhang W, Wei R, Chen S et al (2014) Functional characterization of Cinnamoyl-CoA Reductase (CCR) in birch (Betula platyphylla × Betula pendula) through overexpression and suppression analysis. Physiol Plant 154Google Scholar
  38. Zhu ZQ, An FY, Feng Y (2011) Derepression of ethylene-stabilized transcription factors (EIN3/EIL1) mediates jasmonate and ethylene signaling synergy in Arabidopsis. PNAS 108:12539–12544CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.State Key Laboratory of Tree Genetics and BreedingNortheast Forestry UniversityHarbinChina
  2. 2.Department of Life Science and TechnologyMudanjiang Normal UniversityMudanjiangChina
  3. 3.University of FloridaGainesvilleUSA

Personalised recommendations