Plant Molecular Biology

, Volume 95, Issue 3, pp 243–252 | Cite as

Bypassing miRNA-mediated gene regulation under drought stress: alternative splicing affects CSD1 gene expression

  • So-Yon Park
  • Elizabeth Grabau


Key message

The binding site for miR398 in an isoform of Cu/Zn superoxide dismutase (CSD1) is eliminated by alternative splicing to bypass miR398-mediated gene down-regulation under drought stress.


MicroRNA (miRNA) binding sites (MBSs) are frequently interrupted by introns and therefore require proper splicing to generate functional MBSs in target transcripts. MBSs can also be excluded during splicing of pre-messenger RNA, leading to different regulation among isoforms. Previous studies have shown that levels of Cu/Zn superoxide dismutase (CSD) are down-regulated by miR398. In this study, sequences and transcript levels of peanut CSD1 isoforms (AhCSD1-1, AhCSD1-2.1, and AhCSD1-2.2) were analyzed under the drought stress. Results demonstrated that a miR398 binding site is eliminated in AhCSD1-2.2 as a consequence of alternative splicing, which bypasses miRNA-mediated down-regulation under drought stress. This alternative isoform was not only identified in peanut but also in soybean and Arabidopsis. In addition, transgenic Arabidopsis plants expressing AhCSD1 were more tolerant to osmotic stress. We hypothesize that the level of AhCSD1 is increased to allow diverse plant responses to overcome environmental challenges even in the presence of increased miR398 levels. These findings suggest that studies on the role of alternatively spliced MBSs affecting transcript levels are important for understanding plant stress responses.


Alternatively spliced miRNA binding sites miR398 Drought Peanut CSD1 



This work was supported in part by the USDA National Institute of Food and Agriculture, Hatch Project 221820. We thank Drs. Maria Balota, Ruth Grene, Xiaofeng Wang, Eva Collakova and Qian Zhang for valuable suggestions.

Author contributions

SP and EG designed of the work, SP performed research, SP analyzed data, SP and EG wrote the paper.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflict of interest.

Supplementary material

11103_2017_642_MOESM1_ESM.pdf (853 kb)
Supplementary material 1 (PDF 853 KB)
11103_2017_642_MOESM2_ESM.pdf (301 kb)
Supplementary material 2 (PDF 300 KB)


  1. Afonso-Grunz F, Müller S (2015) Principles of miRNA–mRNA interactions: beyond sequence complementarity. Cell Mol Life Sci 72:3127–3141. doi: 10.1007/s00018-015-1922-2 CrossRefPubMedGoogle Scholar
  2. Axtell MJ (2013) Classification and comparison of small RNAs from plants. Annu Rev Plant Biol 64:137–159. doi: 10.1146/annurev-arplant-050312-120043 CrossRefPubMedGoogle Scholar
  3. Azevedo Neto AD, Nogueira RJMC, Melo Filho PA, Santos RC (2009) Physiological and biochemical responses of peanut genotypes to water deficit. J Plant Interact 5:1–10 doi: 10.1080/17429140902999243 CrossRefGoogle Scholar
  4. Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743CrossRefPubMedGoogle Scholar
  5. Dang P, Chen C (2013) Modified method for combined DNA and RNA isolation from peanut and other oil seeds. Mol Biol Rep 40:1563–1568. doi: 10.1007/s11033-012-2204-9 CrossRefPubMedGoogle Scholar
  6. Ding Y, Tao Y, Zhu C (2013) Emerging roles of microRNAs in the mediation of drought stress response in plants. J Exp Bot 64(11):3077–3086. doi: 10.1093/jxb/ert164 CrossRefPubMedGoogle Scholar
  7. Fang Y, Xie K, Xiong L (2014) Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice. J Exp Bot 65:2119–2135CrossRefPubMedPubMedCentralGoogle Scholar
  8. Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem 48:909–930. doi: 10.1016/j.plaphy.2010.08.016 CrossRefPubMedGoogle Scholar
  9. Guan Q, Lu X, Zeng H, Zhang Y, Zhu J (2013) Heat stress induction of miR398 triggers a regulatory loop that is critical for thermotolerance in Arabidopsis. Plant J 74:840–851. doi: 10.1111/tpj.12169 CrossRefPubMedGoogle Scholar
  10. Jing X et al (2015) Overexpression of copper/zinc superoxide dismutase from mangrove Kandelia candel in tobacco enhances salinity tolerance by the reduction of reactive oxygen species in chloroplast. Front Plant Sci. doi: 10.3389/fpls.2015.00023 Google Scholar
  11. Jones-Rhoades MW, Bartel DP (2004) Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 14:787–799. doi: 10.1016/j.molcel.2004.05.027 CrossRefPubMedGoogle Scholar
  12. Jones-Rhoades MW, Bartel DP, Bartel B (2006) MicroRNAs and their regulatory roles in plants. Annu Rev Plant Biol 57:19–53. doi: 10.1146/annurev.arplant.57.032905.105218 CrossRefPubMedGoogle Scholar
  13. Kitagawa N et al (2005) Computational analysis suggests that alternative first exons are involved in tissue-specific transcription in rice (Oryza sativa). Bioinformatics 21:1758–1763. doi: 10.1093/bioinformatics/bti253 CrossRefPubMedGoogle Scholar
  14. Krapovickas A, Walton CG, Williams DE, Simpson CE (2007) Taxonomy of the genus Arachis (Leguminosae). Bonplandia 16:7–205. doi: 10.2307/41941433 Google Scholar
  15. Kulcheski FR, Marcelino-Guimaraes FC, Nepomuceno AL, Abdelnoor RV, Margis R (2010) The use of microRNAs as reference genes for quantitative polymerase chain reaction in soybean. Anal Biochem 406:185–192. doi: 10.1016/j.ab.2010.07.020 CrossRefPubMedGoogle Scholar
  16. Leal-Bertioli SCM et al (2012) The effect of tetraploidization of wild Arachis on leaf morphology and other drought-related traits. Environ Exp Bot 84:17–24. doi: 10.1016/j.envexpbot.2012.04.005 CrossRefGoogle Scholar
  17. Llave C, Kasschau KD, Rector MA, Carrington JC (2002) Endogenous and silencing-associated small RNAs in plants. Plant Cell 14:1605–1619CrossRefPubMedPubMedCentralGoogle Scholar
  18. Marquez Y, Brown JWS, Simpson C, Barta A, Kalyna M (2012) Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res 22:1184–1195. doi: 10.1101/gr.134106.111 CrossRefPubMedPubMedCentralGoogle Scholar
  19. Mazzucotelli E, Mastrangelo AM, Crosatti C, Guerra D, Stanca AM, Cattivelli L (2008) Abiotic stress response in plants: when post-transcriptional and post-translational regulations control transcription. Plant Sci 174:420–431. doi: 10.1016/j.plantsci.2008.02.005 CrossRefGoogle Scholar
  20. Mehta R et al (2013) Coat protein-mediated transgenic resistance of peanut (Arachis hypogaea L.) to peanut stem necrosis disease through Agrobacterium-mediated genetic transformation Indian. J Virol 24:205–213. doi: 10.1007/s13337-013-0157-9 Google Scholar
  21. Ni Z, Hu Z, Jiang Q, Zhang H (2012) Overexpression of gma-MIR394a confers tolerance to drought in transgenic Arabidopsis thaliana. Biochem Biophys Res Commun 427:330–335CrossRefPubMedGoogle Scholar
  22. Ni Z, Hu Z, Jiang Q, Zhang H (2013) GmNFYA3, a target gene of miR169, is a positive regulator of plant tolerance to drought stress. Plant Mol Biol 82:113–129. doi: 10.1007/s11103-013-0040-5 CrossRefPubMedGoogle Scholar
  23. Park W, Li J, Song R, Messing J, Chen X (2002) CARPEL FACTORY, a dicer homolog, and HEN1, a novel protein, act in microRNA metabolism in Arabidopsis thaliana. Curr Biol 12:1484–1495CrossRefPubMedPubMedCentralGoogle Scholar
  24. Park SY et al (2007) The senescence-induced staygreen protein regulates chlorophyll degradation. Plant Cell 19:1649–1664. doi: 10.1105/tpc.106.044891 CrossRefPubMedPubMedCentralGoogle Scholar
  25. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP (2002) MicroRNAs in plants. Genes Dev 16:1616–1626. doi: 10.1101/gad.1004402 CrossRefPubMedPubMedCentralGoogle Scholar
  26. Remans T, Opdenakker K, Guisez Y, Carleer R, Schat H, Vangronsveld J, Cuypers A (2012) Exposure of Arabidopsis thaliana to excess Zn reveals a Zn-specific oxidative stress signature. Environ Exp Bot 84:61–71. doi: 10.1016/j.envexpbot.2012.05.005 CrossRefGoogle Scholar
  27. Ryan BM, Robles AI, Harris CC (2010) Genetic variation in microRNA networks: the implications for cancer research. Nat Rev Cancer 10:389–402. doi: 10.1038/nrc2867 CrossRefPubMedPubMedCentralGoogle Scholar
  28. Song JB, Gao S, Sun D, Li H, Shu XX, Yang ZM (2013) miR394 and LCR are involved in Arabidopsis salt and drought stress responses in an abscisic acid-dependent manner. BMC Plant Biol 13:210. doi: 10.1186/1471-2229-13-210 CrossRefPubMedPubMedCentralGoogle Scholar
  29. Sunkar R (2010) MicroRNAs with macro-effects on plant stress responses. Semin Cell Dev Biol 21:805–811. doi: 10.1016/j.semcdb.2010.04.001 CrossRefPubMedGoogle Scholar
  30. Sunkar R, Zhu J-K (2004) Novel and stress-regulated microRNAs and other small RNAs from Arabidopsis. Plant Cell 16:2001–2019. doi: 10.1105/tpc.104.022830 CrossRefPubMedPubMedCentralGoogle Scholar
  31. Sunkar R, Kapoor A, Zhu JK (2006) Posttranscriptional induction of two Cu/Zn superoxide dismutase genes in Arabidopsis is mediated by downregulation of miR398 and important for oxidative stress tolerance. Plant Cell 18:2051–2065. doi: 10.1105/tpc.106.041673 CrossRefPubMedPubMedCentralGoogle Scholar
  32. Sunkar R, Li Y-F, Jagadeeswaran G (2012) Functions of microRNAs in plant stress responses. Trends Plant Sci 17:196–203. doi: 10.1016/j.tplants.2012.01.010 CrossRefPubMedGoogle Scholar
  33. Trindade I, Capitao C, Dalmay T, Fevereiro MP, Santos DM (2010) miR398 and miR408 are up-regulated in response to water deficit in Medicago truncatula. Planta 231:705–716. doi: 10.1007/s00425-009-1078-0 CrossRefPubMedGoogle Scholar
  34. Varkonyi-Gasic E, Wu R, Wood M, Walton EF, Hellens RP (2007) Protocol: a highly sensitive RT-PCR method for detection and quantification of microRNAs plant. Methods 3:12. doi: 10.1186/1746-4811-3-12 Google Scholar
  35. Wang T, Chen L, Zhao M, Tian Q, Zhang WH (2011) Identification of drought-responsive microRNAs in Medicago truncatula by genome-wide high-throughput sequencing. BMC Genom 12:367. doi: 10.1186/1471-2164-12-367 CrossRefGoogle Scholar
  36. Wu C-T, Chiou C-Y, Chiu H-C, Yang U-C (2013) Fine-tuning of microRNA-mediated repression of mRNA by splicing-regulated and highly repressive microRNA recognition element. BMC Genom 14:1–12. doi: 10.1186/1471-2164-14-438 CrossRefGoogle Scholar
  37. Wu J, Zhang J, Li X, Xu J, Wang L (2016) Identification and characterization of a PutCu/Zn-SOD gene from Puccinellia tenuiflora (Turcz.) Scribn. et Merr. Plant Growth Regul 79:55–64. doi: 10.1007/s10725-015-0110-6 CrossRefGoogle Scholar
  38. Xia K et al (2012) OsTIR1 and OsAFB2 downregulation via OsmiR393 overexpression leads to more tillers, early flowering and less tolerance to salt and drought in rice. PLoS ONE 7:e30039CrossRefPubMedPubMedCentralGoogle Scholar
  39. Yamasaki H, Abdel-Ghany SE, Cohu CM, Kobayashi Y, Shikanai T, Pilon M (2007) Regulation of copper homeostasis by micro-RNA in Arabidopsis. J Biol Chem 282:16369–16378. doi: 10.1074/jbc.M700138200 CrossRefPubMedGoogle Scholar
  40. Yang X, Zhang H, Li L (2012) Alternative mRNA processing increases the complexity of microRNA-based gene regulation in Arabidopsis. Plant J 70:421–431. doi: 10.1111/j.1365-313X.2011.04882.x CrossRefPubMedGoogle Scholar
  41. Yi F, Xie S, Liu Y, Qi X, Yu J (2013) Genome-wide characterization of microRNA in foxtail millet (Setaria italica). BMC Plant Biol 13:212CrossRefPubMedPubMedCentralGoogle Scholar
  42. Zhang X et al (2015) Molecular analysis of the chloroplast Cu/Zn-SOD gene (AhCSD2) in peanut. Crop J 3:246–257. doi: 10.1016/j.cj.2015.03.006 CrossRefGoogle Scholar
  43. Zhao CZ et al (2010) Deep sequencing identifies novel and conserved microRNAs in peanuts (Arachis hypogaea L.). BMC Plant Biol 10:3. doi: 10.1186/1471-2229-10-3 CrossRefPubMedPubMedCentralGoogle Scholar
  44. Zhou M, Luo H (2014) Role of microRNA319 in creeping bentgrass salinity and drought stress response. Plant Signal Behav 9(4):e28700. doi: 10.4161/psb.28700 CrossRefPubMedCentralGoogle Scholar
  45. Zhou M, Li D, Li Z, Hu Q, Yang C, Zhu L, Luo H (2013) Constitutive expression of a miR319 gene alters plant development and enhances salt and drought tolerance in transgenic creeping bentgrass. Plant Physiol 161:1375–1391. doi: 10.1104/pp.112.208702 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  1. 1.Department of Plant Pathology, Physiology, and Weed ScienceVirginia TechBlacksburgUSA

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