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Expression of Two Self-enhancing Antifreeze Proteins from the Beetle Dendroides canadensis in Arabidopsis thaliana

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Abstract

Antifreeze proteins depress the non-equilibrium freezing point of aqueous solutions, but only have a small effect on the equilibrium melting point. This difference between the freezing and melting points has been termed thermal hysteresis activity (THA). THA identifies the presence and relative activity of antifreeze proteins. Two antifreeze protein cDNAs, dafp-1 and dafp-4, encoding two self-enhancing (have a synergistic effect on THA) antifreeze proteins (DAFPs) from the beetle Dendroides canadensis, were introduced into the genome of Arabidopsis thaliana via Agrobacterium-mediated floral dip transformation. Southern blot analysis indicated multiple insertions of transgenes. Both DAFP-1 and/or DAFP-4 were expressed in transgenic A. thaliana as shown by RT-PCR and Western blot. Apoplastic fluid from T 3 DAFP-1 + DAFP-4-producing transgenic A. thaliana exhibited THA in the range of 1.2–1.35°C (using the capillary method to determine THA), demonstrating the presence of functioning antifreeze proteins (with signal peptides for extracellular secretion). The freezing temperature of DAFP-1 + DAFP-4-producing transgenic A. thaliana was lowered by approximately 2–3°C compared with the wild type.

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References

  • Bennett VA, Sformo T, Walters K, Toien O, Jeannet K, Hochstrasser R, Pan Q, Serianni AS, Barnes BM, Duman JG (2005) Comparative overwintering physiology of Alaska and Indiana populations of the beetle Cucujus clavipes (Fabricius): roles of antifreeze proteins, polyols, dehydration and diapause. J Exp Biol 208:4467–4477

    Article  PubMed  CAS  Google Scholar 

  • Cheng LB, Li SY, Yang GX, Jing XM, He GY, Mones NG (2010) Overexpression of soybean (Glycine max (L.) Meer.) L34 gene leads to reduced survival to cold stress in transgenic Arabidopsis. Plant Mol Biol Report 28:41–48

    Article  CAS  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  PubMed  CAS  Google Scholar 

  • Davies PL, Baardsnes J, Kuiper MJ, Walker VK (2002) Structure and function of antifreeze proteins. Philos Trans R Soc Lond B Biol Sci 357:927–935

    Article  PubMed  CAS  Google Scholar 

  • DeVries AL (1983) Antifreeze peptides and glycopeptides in cold-water fishes. Annu Rev Physiol 45:245–260

    Article  PubMed  CAS  Google Scholar 

  • DeVries AL (1986) Antifreeze glycopeptides and peptides: interactions with ice and water. Meth Enzymol 127:293–303

    Article  PubMed  CAS  Google Scholar 

  • DeVries AL (2005) Ice, antifreeze proteins and antifreeze genes in polar fishes. In: Barnes BM, Carey HV (eds) Life in the cold. University of Alaska Press, Alaska, pp 307–315

    Google Scholar 

  • Duman JG, Olsen TM (1993) Thermal hysteresis protein activity in bacteria, fungi, and phylogenetically diverse plants. Cryobiology 30:322–328

    Article  Google Scholar 

  • Duman JG (1994) Purification and characterization of a thermal hysteresis protein from a plant, the bittersweet nightshade Solanum dulcamara. Biochim Biophys Acta 1206:129–135

    Article  PubMed  CAS  Google Scholar 

  • Duman JG, Li N, Verleye D, Goetz FW, Wu DW, Andorfer CA, Benjamin T, Parmelee DC (1998) Molecular characterization and sequencing of antifreeze proteins from larvae of the beetle Dendroides canadensis. J Comp Physiol B 168:225–232

    Article  PubMed  CAS  Google Scholar 

  • Duman JG (2001) Antifreeze and ice nucleator proteins in terrestrial arthropods. Annu Rev Physiol 63:327–357

    Article  PubMed  CAS  Google Scholar 

  • Duman JG (2002) The inhibition of ice nucleators by insect antifreeze proteins is enhanced by glycerol and citrate. J Comp Physiol B 172:163–168

    Article  PubMed  CAS  Google Scholar 

  • Duman JG, Verleye D, Li N (2002) Site-specific forms of antifreeze protein in the beetle Dendroides canadensis. J Comp Physiol B 172:547–552

    Article  PubMed  CAS  Google Scholar 

  • Duman JG, Bennett V, Sformo T, Hochstrasser R, Barnes BM (2004) Antifreeze proteins in Alaskan insects and spiders. J Insect Physiol 50:259–266

    Article  PubMed  CAS  Google Scholar 

  • Duman JG, Walters KR, Sformo T, Carrasco MA, Nickell P, Lin X, Barnes BM (2010) Antifreze and ice nucleator proteins. In: Denlinger DL, Lee RE (eds) Low Temperature Biology of Insects. Cambridge University Press, Cambridge, pp 59–90

    Chapter  Google Scholar 

  • Fagard M, Vaucheret H (2000) (Trans)gene silencing in plants: How many mechanisms? Annu Rev Plant Physiol Plant Mol Biol 51:167–194

    Article  PubMed  CAS  Google Scholar 

  • Francois IE, De Bolle MF, Dwyer G, Goderis IJ, Woutors PF, Verhaert PD, Proost P, Schaaper WM, Cammue BP, Broekaert WF (2002) Transgenic expression in Arabidopsis of a polyprotein construct leading to production of two different antimicrobial proteins. Plant Physiol 128:1346–1358

    Article  PubMed  CAS  Google Scholar 

  • Goderis IJ, De Bolle MF, Francois IE, Wouters PF, Broekaert WF, Cammue BP (2002) A set of modular plant transformation vectors allowing flexible insertion of up to six expression units. Plant Mol Biol 50:17–27

    Article  PubMed  CAS  Google Scholar 

  • Griffith M, Yaish MW (2004) Antifreeze proteins in overwintering plants: a tale of two activities. Trends Plant Sci 9:399–405

    Article  PubMed  CAS  Google Scholar 

  • Griffith M, Lumb C, Wiseman SB, Wisniewski M, Johnson RW, Marangoni AG (2005) Antifreeze proteins modify the freezing process in planta. Plant Physiol 138:330–340

    Article  PubMed  CAS  Google Scholar 

  • Hightower R, Baden C, Penzes E, Lund P, Dunsmuir P (1991) Expression of antifreeze proteins in transgenic plants. Plant Mol Biol 17:1013–1021

    Article  PubMed  CAS  Google Scholar 

  • Holmberg N, Farres J, Bailey JE, Kallio PT (2001) Targeted expression of a synthetic codon optimized gene, encoding the spruce budworm antifreeze protein, leads to accumulation of antifreeze activity in the apoplasts of transgenic tobacco. Gene 275:115–124

    Article  PubMed  CAS  Google Scholar 

  • Hon WC, Griffith M, Chong P, Yang D (1994) Extraction and isolation of antifreeze proteins from winter rye (Secale cereale L.) leaves. Plant Physiol 104:971–980

    PubMed  CAS  Google Scholar 

  • Huang T, Duman JG (2002) Cloning and characterization of a thermal hysteresis (antifreeze) protein with DNA-binding activity from winter bittersweet nightshade, Solanum dulcamara. Plant Mol Biol 48:339–350

    Article  PubMed  CAS  Google Scholar 

  • Huang T, Nicodemus J, Zarka DG, Thomashow MF, Wisniewski M, Duman JG (2002) Expression of an insect (Dendroides canadensis) antifreeze protein in Arabidopsis thaliana results in a decrease in plant freezing temperature. Plant Mol Biol 50:333–344

    Article  PubMed  CAS  Google Scholar 

  • Jia Z, Davies PL (2002) Antifreeze proteins: an unusual receptor-ligand interaction. Trends Biochem Sci 27:101–106

    Article  PubMed  CAS  Google Scholar 

  • Jorov A, Zhorov BS, Yang DS (2004) Theoretical study of interaction of winter flounder antifreeze protein with ice. Protein Sci 13:1524–1537

    Article  PubMed  CAS  Google Scholar 

  • Kawahara H, Fujii A, Inoue M, Kitao S, Fukuoka J, Obata H (2009) Antifreeze activity of cold acclimated Japanese radish and purification of antifreeze peptide. Cryo Lett 30:119–31

    CAS  Google Scholar 

  • Kenward KD, Altschuler M, Hildebrand D, Davies PL (1993) Accumulation of type I fish antifreeze protein in transgenic tobacco is cold-specific. Plant Mol Biol 23:377–385

    Article  PubMed  CAS  Google Scholar 

  • Kenward KD, Brandle J, McPherson J, Davies PL (1999) Type II fish antifreeze protein accumulation in transgenic tobacco does not confer frost resistance. Transgenic Res 8:105–117

    Article  PubMed  CAS  Google Scholar 

  • Lin X, O’Tousa JE, Duman JG (2010) Expression of two self-enhancing antifreeze proteins from Dendroides canadensis in Drosophila melanogaster. J Insect Physiol 56:341–349

    Article  PubMed  CAS  Google Scholar 

  • Lütcke HA, Chow KC, Mickel FS, Moss KA, Kern HF, Scheele GA (1987) Selection of AUG initiation codons differs in plants and animals. EMBO J 6:43–48

    PubMed  Google Scholar 

  • Meyer K, Keil M, Naldrett MJ (1999) A leucine-rich repeat protein of carrot that exhibits antifreeze activity. FEBS Lett 447:171–178

    Article  PubMed  CAS  Google Scholar 

  • Olsen TM, Duman JG (1997a) Maintenance of the supercooled state in overwintering Pyrochroid beetle larvae Dendroides canadensis: role of hemolymph ice nucleators and antifreeze proteins. J Comp Physiol B 167:105–113

    Article  Google Scholar 

  • Olsen TM, Duman JG (1997b) Maintenance of the supercooled state in the gut of the Pyrochroid beetle larvae Dendroides canadensis: role of gut ice nucleators and antifreeze proteins. J Comp Physiol B 116:114–122

    Article  Google Scholar 

  • Olsen T, Sass S, Li N, Duman JG (1998) Factors contributing to seasonal increases in inoculative freezing resistance in overwintering fire-colored beetle larvae Dendroides canadensis (Pyrochroidae). J Exp Biol 201:1585–1594

    PubMed  CAS  Google Scholar 

  • Pihakaski-Maunsbach K, Moffatt B, Testillano P, Risueno M, Yeh S, Griffith M, Maunsbach AB (2001) Genes encoding chitinase-antifreeze proteins are regulated by cold and expressed by all cell types in winter rye shoots. Physiol Plant 112:359–371

    Article  PubMed  CAS  Google Scholar 

  • Pudney PD, Buckley SL, Sidebottom CM, Twigg SN, Sevilla MP, Holt CB, Roper D, Telford JH, McArthur AJ, Lillford PJ (2003) The physico-chemical characterization of a boiling stable antifreeze protein from a perennial grass (Lolium perenne). Arch Biochem Biophys 410:238–245

    Article  PubMed  CAS  Google Scholar 

  • Rangan L, Vogel C, Srivastava A (2008) Analysis of context sequence surrounding translation initiation site from complete genome of model plants. Mol Biotechnol 39:207–213

    Article  PubMed  CAS  Google Scholar 

  • Raymond JA, DeVries AL (1977) Adsorption inhibition as a mechanism of freezing resistance in polar fishes. Proc Natl Acad Sci USA 74:2589–2593

    Article  PubMed  CAS  Google Scholar 

  • Simpson DJ, Smallwood M, Twigg S, Doucet CJ, Ross J, Bowles DJ (2005) Purification and characterisation of an antifreeze protein from Forsythia suspensa (L.). Cryobiology 51:230–234

    Article  PubMed  CAS  Google Scholar 

  • Smallwood M, Worrall D, Byass L, Elias L, Ashford D, Doucet CJ, Holt C, Telford J, Lillford P, Bowles DJ (1999) Isolation and characterization of a novel antifreeze protein from carrot (Daucus carota). Biochem J 340:385–391

    Article  PubMed  CAS  Google Scholar 

  • Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354

    Article  PubMed  CAS  Google Scholar 

  • Urrutia ME, Duman JG, Knight CA (1992) Plant thermal hysteresis proteins. Biochim Biophys Acta 1121:199–206

    Article  PubMed  CAS  Google Scholar 

  • Verma SS, Chinnusamy V, Bansal KC (2008) A simplified floral dip method for transformation of Brassica napus and B. carinata. J Plant Biochem Biotechnol 17:197–200

    CAS  Google Scholar 

  • Wallis JG, Wang H, Guerra DJ (1997) Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures. Plant Mol Biol 35:323–330

    Article  PubMed  CAS  Google Scholar 

  • Wang W, Wei L (2003) Purification of boiling-soluble antifreeze protein from the legume Ammopiptanthus mongolicus. Prep Biochem Biotechnol 33:67–80

    Article  PubMed  CAS  Google Scholar 

  • Wang L, Duman JG (2005) Antifreeze proteins of the beetle Dendroides canadensis enhance one another’s activities. Biochemistry 44:10305–10312

    Article  PubMed  CAS  Google Scholar 

  • Wang Y, Qiu L, Dai C, Wang J, Luo J, Zhang F, Ma J (2008) Expression of insect (Microdera puntipennis dzungarica) antifreeze protein MpAFP149 confers the cold tolerance to transgenic tobacco. Plant Cell Rep 27:1349–1358

    Article  PubMed  CAS  Google Scholar 

  • Wharton DA, Barrett J, Goodall G, Marshall CJ, Ramlov H (2005) Ice-active proteins from the Antarctic nematode Panagrolaimus davidi. Cryobiology 51:198–207

    Article  PubMed  CAS  Google Scholar 

  • Wisniewski M, Lindow SE, Ashworth EN (1997) Observations of ice nucleation and propagation in plants using infrared video thermography. Plant Physiol 113:327–334

    PubMed  CAS  Google Scholar 

  • Wisniewski M, Webb R, Balsamo R, Close TJ, Yu XM, Griffith M (1999) Purification, immunolocalization, cryoprotective and antifreeze activity of PCA60: a dehydrin from peach (Prunus persica). Physiol Plant 105:600–608

    Article  CAS  Google Scholar 

  • Worrall D, Elias L, Ashford D, Smallwood M, Sidebottom C, Lillford P, Telford J, Holt C, Bowles D (1998) A carrot leucine-rich-repeat protein that inhibits ice recrystallization. Science 282:115–117

    Article  PubMed  CAS  Google Scholar 

  • Wu DW, Duman JG, Xu L (1991) Enhancement of insect antifreeze protein activity by antibodies. Biochim Biophys Acta 1076:416–420

    Article  PubMed  CAS  Google Scholar 

  • Xu W, Shi W (2008) A “nonsterile” method for selecting and growing Arabidopsis thaliana transformants (T2 transgenic lines) resistant to kanamycin. Plant Mol Biol Report 26:350–357

    Article  CAS  Google Scholar 

  • Yasmeen A, Mirza B, Inayatullah S, Safdar N, Jamil M, Ali S, Choudhry MF (2009) In planta transformation of tomato. Plant Mol Biol Report 27:20–28

    Article  CAS  Google Scholar 

  • Zhu B, Xiong AS, Peng RH, Xu J, Jin XF, Meng XR, Yao QH (2010) Over-expression of ThpI from Choristoneura fumiferana enhances tolerance to cold in Arabidopsis. Mol Biol Rep 37:961–966

    Article  PubMed  CAS  Google Scholar 

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Acknowledgments

This work was partially supported by National Science Foundation grant IOS-0618342 to JGD.

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Correspondence to John. G. Duman.

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Lin, X., Wisniewski, M.E. & Duman, J.G. Expression of Two Self-enhancing Antifreeze Proteins from the Beetle Dendroides canadensis in Arabidopsis thaliana . Plant Mol Biol Rep 29, 802–813 (2011). https://doi.org/10.1007/s11105-011-0287-4

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