CYSTM3 negatively regulates salt stress tolerance in Arabidopsis

  • Yang Xu
  • Zipeng Yu
  • Shizhong Zhang
  • Changai Wu
  • Guodong Yang
  • Kang Yan
  • Chengchao ZhengEmail author
  • Jinguang HuangEmail author


Key message

CYSTM3, a small mitochondrial protein, acts as a negative regulator in salt stress response by preventing Na+ efflux and disturbing reactive oxygen species (ROS) homeostasis in Arabidopsis.


Cysteine-rich transmembrane module (CYSTM) is a not well characterized small peptide family in plants. In this study, we identified a novel mitochondrion-localized CYSTM member CYSTM3 from Arabidopsis, which was ubiquitously expressed in different tissues and dramatically induced by salt stress. Transgenic plants overexpressing CYSTM3 (OE) displayed hypersensitivity to salt stress compared with wild type (WT) plants, whereas a knockout mutant cystm3 was more tolerant to high salinity than WT. Moreover, OE lines accumulated higher contents of Na+ and ROS than WT and cystm3 upon exposure to high salinity. Further analysis revealed that CYSTM3 could deter root Na+ efflux and inhibit the activities of a range of ROS scavenging enzymes in Arabidopsis. In addition, the transcripts of nuclear salt stress-responsive genes were over-activated in cystm3 than those in WT and OE lines. Taken together, Arabidopsis CYSTM3 acts as a negative regulator in salt stress tolerance.


Arabidopsis CYSTM3 Mitochondrion Na+ efflux Reactive oxygen species Salt stress 



This work was supported by the National Natural Science Foundation of China (Grant Nos. 31570271 and 31771878), and the Natural Science Foundation of Shandong Province (Grant No. ZR2016CM22).

Author contributions

J.H. and C.Z. conceived the original screening and research plans; Z.Y., and Y.X. performed experiments; Z.Y., and Y.X. conceived the project and wrote the article; S.Z., C.W., G.Y. and K.Y. provided suggestions and proofed the article; J.H. and C.Z. supervised and complemented the writing.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

11103_2019_825_MOESM1_ESM.pdf (956 kb)
Supplementary material 1 (PDF 956 KB)


  1. Anjum NA et al (2016) Catalase and ascorbate peroxidase-representative H2O2-detoxifying heme enzymes in plants. Environ Sci Pollut Res Int 23:19002–19029. CrossRefGoogle Scholar
  2. Apse MP, Aharon GS, Snedden WA, Blumwald E (1999) Salt tolerance conferred by overexpression of a vacuolar Na+/H + antiport in Arabidopsis. Science 285:1256–1258CrossRefGoogle Scholar
  3. Arnon DI (1949) Copper ENZYMES in isolated chloroplasts. polyphenoloxidase in Beta Vulgaris. Plant Physiol 24:1–15CrossRefGoogle Scholar
  4. Barrero JM, Rodriguez PL, Quesada V, Piqueras P, Ponce MR, Micol JL (2006) Both abscisic acid (ABA)-dependent and ABA-independent pathways govern the induction of NCED3, AAO3 and ABA1 in response to salt stress. Plant Cell Environ 29:2000–2008. CrossRefGoogle Scholar
  5. Beers RF Jr, Sizer IW (1952) A spectrophotometric method for measuring the breakdown of hydrogen peroxide by catalase. J Biol Chem 195:133–140Google Scholar
  6. Boudsocq M, Lauriere C (2005) Osmotic signaling in plants: multiple pathways mediated by emerging kinase families. Plant Physiol 138:1185–1194. CrossRefGoogle Scholar
  7. Butow RA, Avadhani NG (2004) Mitochondrial signaling: the retrograde response. Mol Cell 14:1–15CrossRefGoogle Scholar
  8. Choudhury FK, Rivero RM, Blumwald E, Mittler R (2016) Reactive oxygen species, abiotic stress and stress combination. Plant J. Google Scholar
  9. Choudhury FK, Rivero RM, Blumwald E, Mittler R (2017) Reactive oxygen species, abiotic stress and stress combination. Plant J 90:856–867. CrossRefGoogle Scholar
  10. Darley CP, van Wuytswinkel OC, van der Woude K, Mager WH, de Boer AH (2000) Arabidopsis thaliana and Saccharomyces cerevisiae NHX1 genes encode amiloride sensitive electroneutral Na+/H + exchangers. Biochem J 351:241–249CrossRefGoogle Scholar
  11. Davletova S, Schlauch K, Coutu J, Mittler R (2005) The zinc-finger protein Zat12 plays a central role in reactive oxygen and abiotic stress signaling in Arabidopsis. Plant Physiol 139:847–856. CrossRefGoogle Scholar
  12. De Clercq I et al (2013) The membrane-bound NAC transcription factor ANAC013 functions in mitochondrial retrograde regulation of the oxidative stress response in Arabidopsis. Plant Cell 25:3472–3490. CrossRefGoogle Scholar
  13. Gaxiola RA, Li J, Undurraga S, Dang LM, Allen GJ, Alper SL, Fink GR (2001) Drought- and salt-tolerant plants result from overexpression of the AVP1 H+-pump. Proc Natl Acad Sci USA 98:11444–11449. CrossRefGoogle Scholar
  14. Goyal K, Walton LJ, Tunnacliffe A (2005) LEA proteins prevent protein aggregation due to water stress. Biochem J 388:151–157. CrossRefGoogle Scholar
  15. Huseynova IM, Aliyeva DR, Aliyev JA (2014) Subcellular localization and responses of superoxide dismutase isoforms in local wheat varieties subjected to continuous soil drought. Plant Physiol Biochem PPB 81:54–60. CrossRefGoogle Scholar
  16. Inze A, Vanderauwera S, Hoeberichts FA, Vandorpe M, Van Gaever T, Van Breusegem F (2012) A subcellular localization compendium of hydrogen peroxide-induced proteins. Plant Cell Environ 35:308–320. CrossRefGoogle Scholar
  17. Ji H, Pardo JM, Batelli G, Van Oosten MJ, Bressan RA, Li X (2013) The salt overly sensitive (SOS) pathway: established and emerging roles. Mol Plant 6:275–286 CrossRefGoogle Scholar
  18. Jia F, Wang C, Huang J, Yang G, Wu C, Zheng C (2015) SCF E3 ligase PP2-B11 plays a positive role in response to salt stress in Arabidopsis. J Exp Bot 66:4683–4697. CrossRefGoogle Scholar
  19. Kuzniak E, Sklodowska M (2004) The effect of Botrytis cinerea infection on the antioxidant profile of mitochondria from tomato leaves. J Exp Bot 55:605–612. CrossRefGoogle Scholar
  20. Li D et al (2011) Transcriptional profiling of Medicago truncatula under salt stress identified a novel CBF transcription factor MtCBF4 that plays an important role in abiotic stress responses. BMC Plant Biol 11:109. CrossRefGoogle Scholar
  21. Marklund S, Marklund G (1974) Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47:469–474CrossRefGoogle Scholar
  22. Mir R, Leon J (2014) Pathogen and circadian controlled 1 (PCC1) protein is anchored to the plasma membrane and interacts with subunit 5 of COP9 signalosome in Arabidopsis. PLoS ONE 9:e87216. CrossRefGoogle Scholar
  23. Mir R, Hernandez ML, Abou-Mansour E, Martinez-Rivas JM, Mauch F, Metraux JP, Leon J (2013) Pathogen and circadian controlled 1 (PCC1) regulates polar lipid content, ABA-related responses, and pathogen defence in Arabidopsis thaliana. J Exp Bot 64:3385–3395. CrossRefGoogle Scholar
  24. Mittler R, Vanderauwera S, Gollery M, Van Breusegem F (2004) Reactive oxygen gene network of plants. Trends Plant Sci 9:490–498. CrossRefGoogle Scholar
  25. Moller IM (2001) Plant mitochondria and oxidative stress: electron transport, NADPH turnover, and metabolism of reactive oxygen species. Annu Rev Plant Physiol Plant Mol Biol 52:561–591. CrossRefGoogle Scholar
  26. Moller IM, Kristensen BK (2004) Protein oxidation in plant mitochondria as a stress indicator. Photochem Photobiol Sci 3:730–735. CrossRefGoogle Scholar
  27. Moller IM, Jensen PE, Hansson A (2007) Oxidative modifications to cellular components in plants. Ann Rev Plant Biol 58:459–481. CrossRefGoogle Scholar
  28. Moller IS et al (2009) Shoot Na + exclusion and increased salinity tolerance engineered by cell type-specific alteration of Na + transport. in Arabidopsis. Plant Cell 21:2163–2178. CrossRefGoogle Scholar
  29. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681. CrossRefGoogle Scholar
  30. Ng S et al (2014) Anterograde and retrograde regulation of nuclear genes encoding mitochondrial proteins during growth, development, and stress. Mol Plant 7:1075–1093. CrossRefGoogle Scholar
  31. Noctor G, Reichheld JP, Foyer CH (2018) ROS-related redox regulation and signaling in plants semin. Cell Dev Biol 80:3–12. CrossRefGoogle Scholar
  32. Osakabe Y, Osakabe K, Shinozaki K, Tran LS (2014) Response of plants to water stress. Front Plant Sci 5:86. CrossRefGoogle Scholar
  33. Rubio F, Gassmann W, Schroeder JI (1995) Sodium-driven potassium uptake by the plant potassium transporter HKT1 and mutations conferring salt tolerance. Science 270:1660–1663CrossRefGoogle Scholar
  34. Sauerbrunn N, Schlaich NL (2004) PCC1: a merging point for pathogen defence and circadian signalling in Arabidopsis. Planta 218:552–561. CrossRefGoogle Scholar
  35. Seligman AM, Karnovsky MJ, Wasserkrug HL, Hanker JS (1968) Nondroplet ultrastructural demonstration of cytochrome oxidase activity with a polymerizing osmiophilic reagent, diaminobenzidine (DAB). J Cell Biol 38:1–14CrossRefGoogle Scholar
  36. Shen Y, Li J, Gu R, Yue L, Wang H, Zhan X, Xing B (2018) Carotenoid and superoxide dismutase are the most effective antioxidants participating in ROS scavenging in phenanthrene accumulated wheat leaf. Chemosphere 197:513–525. CrossRefGoogle Scholar
  37. Sun W, Van Montagu M, Verbruggen N (2002) Small heat shock proteins and stress tolerance in plants. Biochim et Biophys Acta 1577:1–9CrossRefGoogle Scholar
  38. Sun J et al (2009) NaCl-induced alternations of cellular and tissue ion fluxes in roots of salt-resistant and salt-sensitive poplar species. Plant Physiol 149:1141–1153. CrossRefGoogle Scholar
  39. Suzuki N, Koussevitzky S, Mittler R, Miller G (2012) ROS and redox signalling in the response of plants to abiotic stress. Plant Cell Environ 35:259–270. CrossRefGoogle Scholar
  40. Tuteja N (2007) Mechanisms of high salinity tolerance in plants. Methods Enzymol 428:419–438. CrossRefGoogle Scholar
  41. Venancio TM, Aravind L (2010) CYSTM, a novel cysteine-rich transmembrane module with a role in stress tolerance across eukaryotes. Bioinformatics 26:149–152. CrossRefGoogle Scholar
  42. Xu Y et al (2018a) CYSTM, a novel non-secreted cysteine-rich peptide family, involved in environmental stresses in Arabidopsis thaliana. Plant Cell Physiol 59:423–438. CrossRefGoogle Scholar
  43. Xu Y et al (2018b) NtLTP4, a lipid transfer protein that enhances salt and drought stresses tolerance in Nicotiana tabacum. Sci Rep 8:8873. CrossRefGoogle Scholar
  44. Yao X, Li J, Liu J, Liu K (2015) An Arabidopsis mitochondria-localized RRL protein mediates abscisic acid signal transduction through mitochondrial retrograde regulation involving ABI4. J Exp Bot 66:6431–6445. CrossRefGoogle Scholar
  45. Yoshiba Y, Kiyosue T, Nakashima K, Yamaguchi-Shinozaki K, Shinozaki K (1997) Regulation of levels of proline as an osmolyte in plants under water stress. Plant Cell Physiol 38:1095–1102CrossRefGoogle Scholar
  46. Zhang JL, Shi H (2013) Physiological and molecular mechanisms of plant salt tolerance. Photosynth Res 115:1–22. CrossRefGoogle Scholar
  47. Zhao Y et al (2013) The actin-related Protein2/3 complex regulates mitochondrial-associated calcium signaling during salt stress in Arabidopsis. Plant Cell 25:4544–4559. CrossRefGoogle Scholar
  48. Zhou H et al (2014) Inhibition of the Arabidopsis salt overly sensitive pathway by 14-3-3 proteins. Plant Cell 26:1166–1182. CrossRefGoogle Scholar
  49. Zhu JK (2001) Plant salt tolerance. Trends Plant Sci 6:66–71CrossRefGoogle Scholar
  50. Zhu JK (2002) Salt and drought stress signal transduction. in plants. Annu Rev Plant Biol 53:247–273. CrossRefGoogle Scholar

Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  • Yang Xu
    • 1
  • Zipeng Yu
    • 1
  • Shizhong Zhang
    • 1
  • Changai Wu
    • 1
  • Guodong Yang
    • 1
  • Kang Yan
    • 1
  • Chengchao Zheng
    • 1
    Email author
  • Jinguang Huang
    • 1
    Email author
  1. 1.State Key Laboratory of Crop Biology, College of Life SciencesShandong Agricultural UniversityTaiʼanPeople’s Republic of China

Personalised recommendations