Journal of Plant Research

, Volume 128, Issue 4, pp 697–707 | Cite as

Isolation and functional characterization of salt-stress induced RCI2-like genes from Medicago sativa and Medicago truncatula

  • Ruicai Long
  • Fan Zhang
  • Zhenyi Li
  • Mingna Li
  • Lili Cong
  • Junmei Kang
  • Tiejun Zhang
  • Zhongxiang Zhao
  • Yan Sun
  • Qingchuan YangEmail author
Regular Paper


Salt stress is one of the most significant adverse abiotic factors, causing crop failure worldwide. So far, a number of salt stress-induced genes, and genes improving salt tolerance have been characterized in a range of plants. Here, we report the isolation and characterization of a salt stress-induced Medicago sativa (alfalfa) gene (MsRCI2A), which showed a high similarity to the yeast plasma membrane protein 3 gene (PMP3) and Arabidopsis RCI2A. The sequence comparisons revealed that five genes of MtRCI2(AE) showed a high similarity to MsRCI2A in the Medicago truncatula genome. MsRCI2A and MtRCI2(AE) encode small, highly hydrophobic proteins containing two putative transmembrane domains, predominantly localized in the plasma membrane. The transcript analysis results suggest that MsRCI2A and MtRCI2(AD) genes are highly induced by salt stress. The expression of MsRCI2A and MtRCI2(AC) in yeast mutants lacking the PMP3 gene can functionally complement the salt sensitivity phenotype resulting from PMP3 deletion. Overexpression of MsRCI2A in Arabidopsis plants showed improved salt tolerance suggesting the important role of MsRCI2A in salt stress tolerance in alfalfa.


Complementation Medicago Overexpression RCI2 Salt stress Subcellular localization 



Abscisic acid


Expressed sequence tag


Green fluorescent Protein


Rare cold inducible


Plasma membrane protein



This work was supported by the China Forage and Grass Research System (CARS-35-04) and Basic Scientific Research Fund of IAS-CAAS (2014ywf-zd-2).

Supplementary material

10265_2015_715_MOESM1_ESM.doc (130 kb)
Supplementary material 1 (DOC 130 kb)
10265_2015_715_MOESM2_ESM.doc (38 kb)
Supplementary material 2 (DOC 38 kb)


  1. Bechtold N, Pelletier G (1998) In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol Biol 82:259–266PubMedGoogle Scholar
  2. Capel J, Jarillo JA, Salinas J, MartinezZapater JM (1997) Two homologous low-temperature-inducible genes from Arabidopsis encode highly hydrophobic proteins. Plant Physiol 115:569–576PubMedCentralPubMedCrossRefGoogle Scholar
  3. Deinlein U, Stephan AB, Horie T, Luo W, Xu GH, Schroeder JI (2014) Plant salt-tolerance mechanisms. Trends Plant Sci 19:371–379PubMedCentralPubMedCrossRefGoogle Scholar
  4. Duzan HM, Zhou X, Souleimanov A, Smith DL (2004) Perception of Bradyrhizobium japonicum Nod factor by soybean [Glycine max (L.) Merr.] root hairs under abiotic stress conditions. J Exp Bot 55:2641–2646PubMedCrossRefGoogle Scholar
  5. Fernandez P, Di Rienzo J, Fernandez L, Hopp HE, Paniego N, Heinz RA (2008) Transcriptomic identification of candidate genes involved in sunflower responses to chilling and salt stresses based on cDNA microarray analysis. BMC Plant Biol 8:11PubMedCentralPubMedCrossRefGoogle Scholar
  6. Galvez AF, Gulick PJ, Dvorak J (1993) Characterization of the early stages of genetic salt-stress responses in salt-tolerant Lophopyrum elongatum, salt-sensitive wheat, and their amphiploid. Plant Physiol 103:257–265PubMedCentralPubMedGoogle Scholar
  7. Geravandi M, Farshadfar E, Kahrizi D (2011) Evaluation of some physiological traits as indicators of drought tolerance in bread wheat genotypes. RUSS J Plant Physiol 58:69–75CrossRefGoogle Scholar
  8. Goddard N, Dunn M, Zhang L, White A, Jack P, Hughes M (1993) Molecular analysis and spatial expression pattern of a low-temperature-specific barley gene, blt101. Plant Mol Biol 23:871–879PubMedCrossRefGoogle Scholar
  9. Gulick PJ, Shen W, An H (1994) ESI3, a stress-induced gene from Lophopyrum elongatum. Plant Physiol 104:799PubMedCentralPubMedCrossRefGoogle Scholar
  10. Ismail A, Takeda S, Nick P (2014) Life and death under salt stress: same players, different timing? J Exp Bot 65:2963–2979PubMedCrossRefGoogle Scholar
  11. Jiang Y, Deyholos MK (2006) Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes. BMC Plant Biol 6:25PubMedCentralPubMedCrossRefGoogle Scholar
  12. Jiang Y, Yang B, Harris NS, Deyholos MK (2007) Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots. J Exp Bot 58:3591–3607PubMedCrossRefGoogle Scholar
  13. Jin H, Sun Y, Yang Q, Chao Y, Kang J, Li Y, Margaret G (2010) Screening of genes induced by salt stress from Alfalfa. Mol Biol Rep 37:745–753PubMedCrossRefGoogle Scholar
  14. Kahrizi D, Salmanian AH, Afshari A, Moieni A, Mousavi A (2007) Simultaneous substitution of Gly96 to Ala and Ala183 to Thr in 5-enolpyruvylshikimate-3-phosphate synthase gene of E-coli (k12) and transformation of rapeseed (Brassica napus L.) in order to make tolerance to glyphosate. Plant Cell Rep 26:95–104PubMedCrossRefGoogle Scholar
  15. Kawaura K, Mochida K, Ogihara Y (2008) Genome-wide analysis for identification of salt-responsive genes in common wheat. Funct Integr Genomic 8:277–286CrossRefGoogle Scholar
  16. Lichtenthaler HK (1987) Chlorophylls and carotenoids: pigments of photosynthetic biomembranes. Method Enzymol 148:350–382Google Scholar
  17. Liu B, Feng DR, Zhang BP, Mu PQ, Zhang Y, He YM, Qi KB, Wang JF, Wang HB (2012) Musa paradisica RCI complements AtRCI and confers Na+ tolerance and K+ sensitivity in Arabidopsis. Plant Sci 184:102–111PubMedCrossRefGoogle Scholar
  18. Mahajan S, Tuteja N (2005) Cold, salinity and drought stresses: an overview. Arch Biochem Biophys 444:139–158PubMedCrossRefGoogle Scholar
  19. Mansour MM (2014) The plasma membrane transport systems and adaptation to salinity. J Plant Physiol 171:1787–1800PubMedCrossRefGoogle Scholar
  20. Mantri NL, Ford R, Coram TE, Pang EC (2007) Transcriptional profiling of chickpea genes differentially regulated in response to high-salinity, cold and drought. BMC Genom 8:303CrossRefGoogle Scholar
  21. Medina J, Ballesteros ML, Salinas J (2007) Phylogenetic and functional analysis of Arabidopsis RCI2 genes. J Exp Bot 58:4333–4346PubMedCrossRefGoogle Scholar
  22. Merchan F, de Lorenzo L, Rizzo SG, Niebel A, Manyani H, Frugier F, Sousa C, Crespi M (2007) Identification of regulatory pathways involved in the reacquisition of root growth after salt stress in Medicago truncatula. Plant J 51:1–17PubMedCrossRefGoogle Scholar
  23. Mitsuya S, Taniguchi M, Miyake H, Takabe T (2005) Disruption of RCI2A leads to over-accumulation of Na+ and increased salt sensitivity in Arabidopsis thaliana plants. Planta 222:1001–1009PubMedCrossRefGoogle Scholar
  24. Mitsuya S, Taniguchi M, Miyake H, Takabe T (2006) Overexpression of RC12A decreases Na+ uptake and mitigates salinity-induced damages in Arabidopsis thaliana plants. Physiol Plant 128:95–102CrossRefGoogle Scholar
  25. Morsy MR, Almutairi AM, Gibbons J, Yun SJ, de Los Reyes BG (2005) The OsLti6 genes encoding low-molecular-weight membrane proteins are differentially expressed in rice cultivars with contrasting sensitivity to low temperature. Gene 344:171–180PubMedCrossRefGoogle Scholar
  26. Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681PubMedCrossRefGoogle Scholar
  27. Navarre C, Goffeau A (2000) Membrane hyperpolarization and salt sensitivity induced by deletion of PMP3, a highly conserved small protein of yeast plasma membrane. EMBO J 19:2515–2524PubMedCentralPubMedCrossRefGoogle Scholar
  28. Nylander M, Heino P, Helenius E, Palva ET, Ronne H, Welin BV (2001) The low-temperature- and salt-induced RCI2A gene of Arabidopsis complements the sodium sensitivity caused by a deletion of the homologous yeast gene SNA1. Plant Mol Biol 45:341–352PubMedCrossRefGoogle Scholar
  29. Shabala S (2013) Learning from halophytes: physiological basis and strategies to improve abiotic stress tolerance in crops. Ann Bot 112:1209–1221PubMedCentralPubMedCrossRefGoogle Scholar
  30. Shavrukov Y (2013) Salt stress or salt shock: which genes are we studying? J Exp Bot 64:119–127PubMedCrossRefGoogle Scholar
  31. Shinozaki K, Yamaguchi-Shinozaki K (2000) Molecular responses to dehydration and low temperature: differences and cross-talk between two stress signaling pathways. Curr Opin Plant Biol 3:217–223PubMedCrossRefGoogle Scholar
  32. Thomashow MF (1999) Plant cold acclimation: freezing tolerance genes and regulatory mechanisms. Annu Rev Plant Physiol Plant Mol Biol 50:571–599PubMedCrossRefGoogle Scholar
  33. Wang LY, Shiozaki K (2006) The fission yeast stress MAPK cascade regulates the pmp3(+) gene that encodes a highly conserved plasma membrane protein. FEBS Lett 580:2409–2413PubMedCrossRefGoogle Scholar
  34. Xiong LM, Schumaker KS, Zhu JK (2002) Cell signaling during cold, drought, and salt stress. Plant Cell 14:S165–S183PubMedCentralPubMedCrossRefGoogle Scholar
  35. Yao D, Zhang X, Zhao X, Liu C, Wang C, Zhang Z, Zhang C, Wei Q, Wang Q, Yan H, Li F, Su Z (2011) Transcriptome analysis reveals salt-stress-regulated biological processes and key pathways in roots of cotton (Gossypium hirsutum L.). Genomics 98:47–55PubMedCrossRefGoogle Scholar
  36. Zhu JK (2002) Salt and drought stress signal transduction in plants. Annu Rev Plant Biol 53:247–273PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© The Botanical Society of Japan and Springer Japan 2015

Authors and Affiliations

  • Ruicai Long
    • 1
  • Fan Zhang
    • 1
  • Zhenyi Li
    • 1
  • Mingna Li
    • 2
  • Lili Cong
    • 1
  • Junmei Kang
    • 1
  • Tiejun Zhang
    • 1
  • Zhongxiang Zhao
    • 3
  • Yan Sun
    • 2
  • Qingchuan Yang
    • 1
    Email author
  1. 1.Institute of Animal SciencesChinese Academy of Agricultural SciencesBeijingChina
  2. 2.College of Animal Science and TechnologyChina Agriculture UniversityBeijingChina
  3. 3.Cangzhou Technical College and Cangzhou Academy of Agriculture and Forestry SciencesCangzhouChina

Personalised recommendations