Molecular Biology Reports

, Volume 39, Issue 5, pp 5859–5866 | Cite as

Ptcorp gene induced by cold stress was identified by proteomic analysis in leaves of Poncirus trifoliata (L.) Raf.

  • Guiyou Long
  • Jinyu Song
  • Ziniu Deng
  • Jie Liu
  • Liqun Rao


A proteomic approach was employed to investigate the cold stress-responsive proteins in trifoliate orange (Poncirus trifoliata (L.) Raf.), which is a well-known cold tolerant citrus relative and widely used as rootstock in China. Two-year-old potted seedlings were exposed to freezing temperature (−6°C) for 50 min (nonlethal) and 80 min (lethal), and the total proteins were isolated from leaves of the treated plants. Nine differentially accumulated proteins over 2-fold changes in abundance were identified by two-dimensional gel electrophoresis and mass spectrometry. Among these proteins, a resistance protein induced by the nonlethal cold treatment (protein spot #2 from P. trifoliata) was selected as target sequence for degenerated primer design. By using the designed primers, a PCR product of about 700 bp size was amplified from P. trifoliata genomic DNA, which was further cloned and sequenced. A nucleotide sequence of 676 bp was obtained and named Ptcorp. Blast retrieval showed that Ptcorp shared 88% homology with an EST of cold acclimated Bluecrop (Vaccinium corymbosum) library (Accession number: CF811080), indicating that Ptcorp had association with cold acclimation. Semiquantitative RT-PCR analysis demonstrated that Ptcorp gene was up-regulated by cold stress which was consistent with the former result of protein expression profile. As the resistance protein (NBS-LRR disease resistance protein family) gene was up-regulated by cold stress in trifoliate orange and satsuma mandarin, it may imply that NBS-LRR genes might be associated with cold resistance in citrus.


Poncirus trifoliata Mass spectrometry Cold stress Resistance protein 2-D IEF/SDS-PAGE Ptcorp 



The research was supported by the National High Technology Research and Development Program of China (863 Program, no. 2011AA100205) and the project 09WD48 funded by Hunan Agricultural University, Hunan, China.


  1. 1.
    Kender JW (2003) Citrus. HortScience 38:1043–1047Google Scholar
  2. 2.
    Shen DX, Wang YY, Chen LG (1997) Genetics and breeding in citrus. Science Press, Beijing, p 289Google Scholar
  3. 3.
    Cao Q, Kong WF, Wen PF (2004) Plant freezing tolerance and genes express in cold acclimation. Acta Ecol Sin 24:806–811Google Scholar
  4. 4.
    Cushman JC, Bohnert HJ (2000) Genomics approaches to plant stress. Curr Opin Plant Biol 3:117–124PubMedCrossRefGoogle Scholar
  5. 5.
    Breyne P, Zabeau M (2001) Genome-wide expression analysis of plant cell cycle modulated genes. Curr Opin Plant Biol 4:136–142PubMedCrossRefGoogle Scholar
  6. 6.
    Long GY, Liu J, Rao LQ, Deng ZN, Xiong XY, Jiao L (2006) Application of mRNA differentially display technique in gene expression in plants. Sci Res Mon 21:25–27Google Scholar
  7. 7.
    Jia Y, del Rio HS, Robbins AL, Louzada ES (2004) Cloning and sequence analysis of a low temperature-induced gene from trifoliate orange with unusual pre-mRNA processing. Plant Cell Rep 23:159–166PubMedCrossRefGoogle Scholar
  8. 8.
    Cai Q, Moore GA, Guy CL (1995) An unusual group 2 LEA gene family in citrus responsive to low temperature. Plant Mol Biol 29:11–23PubMedCrossRefGoogle Scholar
  9. 9.
    Hara M, Wakasugi Y, Ikoma Y, Yano M, Ogawa K, Kuboi T (1999) cDNA sequence and expression of a cold-responsive gene in Citrus unshiu. Biosci Biotechnol Biochem 63:433–437PubMedCrossRefGoogle Scholar
  10. 10.
    Porat R, Pavoncello D, Lurie S, McCollum TG (2002) Identification of a grapefruit cDNA belonging to a unique class of citrus dehydrins and characterization of its expression patterns under temperature stress conditions. Physiol Plant 115:598–603PubMedCrossRefGoogle Scholar
  11. 11.
    Sanchez-Ballesta MT, Rodrigo MJ, Lafuente MT, Granell A, Zacarias L (2004) Dehydrin from citrus, which confers in vitro dehydration and freezing protection activity, is constitutive and highly expressed in the Flavedo of fruit but responsive to cold and water stress in leaves. J Agric Food Chem 52:1950–1957PubMedCrossRefGoogle Scholar
  12. 12.
    Sahin-Cevik M, Moore GA (2006) Identification and expression analysis of cold-regulated genes from the cold-hardy Citrus relative Poncirus trifoliata (L.) Raf. Plant Mol Biol 62:83–97PubMedCrossRefGoogle Scholar
  13. 13.
    Zivy M, de Vienne D (2000) Proteomics: a link between genomics, genetics and physiology. Plant Mol Biol 44:575–580PubMedCrossRefGoogle Scholar
  14. 14.
    van Wijk KJ (2001) Challenges and prospects of plant proteomics. Plant Physiol 126:501–508PubMedCrossRefGoogle Scholar
  15. 15.
    Salekdeh GH, Siopongco J, Ghareyazie B (2002) Proteomic analysis of rice leaves during drought stress and recovery. Proteomics 2:1131–1145PubMedCrossRefGoogle Scholar
  16. 16.
    Majoul T, Chahed K, Zamiti E, Ouelhazi L, Ghri R (2000) Analysis by two-dimensional electrophoresis of the effect of salt stress on the polypeptide patterns in roots of a salt-tolerant and a salt-sensitive cultivar of wheat. Electrophoresis 21:2562–2565PubMedCrossRefGoogle Scholar
  17. 17.
    Ouerghi Z, Remy R, Ouelhazi L, Ayadi A, Brulfert J (2000) Two-dimensional electrophoresis of soluble leaf proteins isolated from two wheat species (Triticum durum and Triticum aestivum) differing in sensitivity towards NaCl. Electrophoresis 21:2487–2491PubMedCrossRefGoogle Scholar
  18. 18.
    Hajduch M, Rakwal R, Agrawal GK, Yonekura M, Pretova A (2001) High-resolution two-dimentional electrophoresis separation of proteins from metal-stressed rice (Oryza sativa L.) leaves: drastic reductions/fragmentations of ribulose-1, 5-bisphosphate carbosylate/oxygenase and induction of stress-related proteins. Electrophoresis 22:2824–2831PubMedCrossRefGoogle Scholar
  19. 19.
    Majoul T, Bancel E, Triboï E, Hamida JB, Branlard G (2004) Proteomic analysis of the effect of heat stress on hexaploid wheat grain: characterization of heat responsive proteins from total endosperm. Proteomics 3:175–183CrossRefGoogle Scholar
  20. 20.
    Agrawal GK, Rakwal R, Yonekura M, Kubo A, Saji H (2002) Proteome analysis of differentially displayed proteins as a tool for investigating ozone stress in rice (Oryza sativa L.) seedlings. Proteomics 2:947–959PubMedCrossRefGoogle Scholar
  21. 21.
    Peck SC, Nuhse TS, Hess D, Iglesias A, Meins F, Boller T (2001) Directed proteomics identifies a plant-specific protein rapidly phosphorylated in response to bacterial and fungal elicitors. Pant Cell 13:1467–1475CrossRefGoogle Scholar
  22. 22.
    Konishi H, Ishiguro K, Komatsu S (2001) A Proteomics approach toward understanding blast fungus infection of rice grown under different levels of ritrogen fertilization. Proteomics 1:1162–1171PubMedCrossRefGoogle Scholar
  23. 23.
    Long GY, Liu J, Rao LQ (2005) Methods of plant proteome research. J Hunan Agric Univ 31:342–346Google Scholar
  24. 24.
    Yan SP, Tang ZC, Su WA, Sun WN (2005) Proteomic analysis of salt stress-responsive proteins in rice root. Proteomics 5:235–244PubMedCrossRefGoogle Scholar
  25. 25.
    Shu LB, Ding W, Wu JH, Feng FJ, Luo LJ, Mei HW (2010) Proteomic analysis of rice leaves shows the different regulations to osmotic stress and stress signals. J Integr Plant Biol 52:981–995PubMedCrossRefGoogle Scholar
  26. 26.
    Yelenosky G (1985) Cold hardiness in citrus. Hortic Rev 7:201–238Google Scholar
  27. 27.
    Liu J, Long GY, Rao LQ, Fan S, Yang H, Peng GP, Jiang FJ, Li LL (2006) Study on the lethal temperature of Poncirus trifoliata Raf. and Citrus reticalata Blanco cv. Miyamoto seedlings with different cold-resistance during growth period. Life Sci Res 10:82–86Google Scholar
  28. 28.
    Yan JX, Wait R, Berkelman T, Harry RA, Westbrook JA, Wheeler CH, Dunn MJ (2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21:3666–3672PubMedCrossRefGoogle Scholar
  29. 29.
    He XH, Li YR, Guo YZ, Tang ZP, Li RB (2005) Genetic analysis of 23 mango cultivars collection in Guanxi province revealed by ISSR. Mol Plant Breed 3:829–834Google Scholar
  30. 30.
    Kreps JA, Wu Y, Chang HS, Zhu T, Wang X, Harper JF (2002) Transcriptome changes for Arabidopsis in response to salt, osmotic, and cold stress. Plant Physiol 130:2129–2141PubMedCrossRefGoogle Scholar
  31. 31.
    Beck EH, Heim R, Hansen J (2004) Plant resistance to cold stress: mechanisms and environmental signals triggering frost hardening and dehardening. J Biosci 29:449–459PubMedCrossRefGoogle Scholar
  32. 32.
    Liu ZQ, Lin DB (1993) The relationship between specific proteins regulated by ABA/GAs and cold resistance of citrus plants. Acta Hortic Sin 20:335–340Google Scholar
  33. 33.
    Lin DB, Liu ZQ (1994) Effect of cold acclimation and ABA on membrane stability and synthesis of membrane protein in citrus. J Nanjing Agric Univ 17:1–5Google Scholar
  34. 34.
    Collu G, Unver N, Peltenburg-Looman AM, van der Heijden R, Verpoorte R, Memelink J (2001) Geraniol 10-hydroxylase, a cytochrome P450 enzyme involved in terpenoid indole alkaloid biosynthesis. FEBS Lett 508:215–220PubMedCrossRefGoogle Scholar
  35. 35.
    Canto-Canche BB, Meijer AH, Collu G, Verpoorte R, Loyola-Vargas VM (2005) Characterization of a polyclonal antiserum against the monoterpene monooxygenase, geraniol 10-hydroxylase from Catharanthus roseus. J Plant Physiol 162:393–402PubMedCrossRefGoogle Scholar
  36. 36.
    Dangl JL, Jones JD (2001) Plant pathogens and integrated defence responses to infection. Nature 411:826–833PubMedCrossRefGoogle Scholar
  37. 37.
    McHale L, Tan X, Koehl P, Michelmore RW (2006) Plant NBS-LRR proteins: adaptable guards. Genome Biol 7:212PubMedCrossRefGoogle Scholar
  38. 38.
    Meyers BC, Kozik A, Griego A, Kuang H, Michelmore RW (2003) Genome-wide analysis of NBS-LRR-encoding genes in Arabidopsis. Plant Cell 15:809–834PubMedCrossRefGoogle Scholar
  39. 39.
    Zhou T, Wang Y, Chen JQ, Araki H, Jing Z, Jiang K, Shen J, Tian D (2004) Genome-wide identification of NBS genes in japonica rice reveals significant expansion of divergent non-TIR NBS-LRR genes. Mol Gen Genomics 271:402–415CrossRefGoogle Scholar
  40. 40.
    Song J, Bradeen JM, Naess SK, Raasch JA, Wielgus SM, Haberlach GT, Liu J, Kuang H, Austin-Phillips S, Buell CR (2003) Gene RB cloned from Solanum bulbocastanum confers broad spectrum resistance to potato late blight. Proc Natl Acad Sci 100:9128–9133PubMedCrossRefGoogle Scholar
  41. 41.
    van der Vossen E, Sikkema A, Hekkert BL, Gros J, Stevens P, Muskens M, Wouters D, Pereira A, Stiekema W, Allefs S (2003) An ancient R gene from the wild potato species Solanum bulbocastanum confers broad-spectrum resistance to Phytophthora infestans in cultivated potato and tomato. Plant J 36:867–882PubMedCrossRefGoogle Scholar
  42. 42.
    Sasaki T, Matsumoto T, Yamamoto K, Sakata K, Baba T, Katayose Y, Wu J, Niimura Y, Cheng Z, Nagamura Y (2002) The genome sequence and structure of rice chromosome 1. Nature 420:312–316PubMedCrossRefGoogle Scholar
  43. 43.
    Jung EH, Jung HW, Lee SC, Han SW, Heu S, Hwang BK (2004) Identification of a novel pathogen-induced gene encoding a leucine-rich repeat protein expressed in phloem cells of Capsicum annuum. Biochim Biophys Acta 1676:211–222PubMedGoogle Scholar
  44. 44.
    Chini A, Grant JJ, Seki M, Shinozaki K, Loake GJ (2004) Drought tolerance established by enhanced expression of the CC-NBS-LRR gene, ADR1, requires salicylic acid, EDS1 and ABI1. Plant J 38:810–822PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Guiyou Long
    • 1
    • 2
  • Jinyu Song
    • 2
  • Ziniu Deng
    • 2
  • Jie Liu
    • 1
  • Liqun Rao
    • 1
  1. 1.College of Bioscience and BiotechnologyHunan Agricultural UniversityChangshaChina
  2. 2.National Center for Citrus ImprovementChangshaChina

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