Ice Nucleation Activity in Plants: The Distribution, Characterization, and Their Roles in Cold Hardiness Mechanisms

  • Masaya IshikawaEmail author
  • Hideyuki Yamazaki
  • Tadashi Kishimoto
  • Hiroki Murakawa
  • Timothy Stait-Gardner
  • Kazuyuki Kuchitsu
  • William S. Price
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 1081)


Control of freezing in plant tissues is a key issue in cold hardiness mechanisms. Yet freeze-regulation mechanisms remain mostly unexplored. Among them, ice nucleation activity (INA) is a primary factor involved in the initiation and regulation of freezing events in plant tissues, yet the details remain poorly understood. To address this, we developed a highly reproducible assay for determining plant tissue INA and noninvasive freeze visualization tools using MRI and infrared thermography. The results of visualization studies on plant freezing behaviors and INA survey of over 600 species tissues show that (1) freezing-sensitive plants tend to have low INA in their tissues (thus tend to transiently supercool), while wintering cold-hardy species have high INA in some specialized tissues; and (2) the high INA in cold-hardy tissues likely functions as a freezing sensor to initiate freezing at warm subzero temperatures at appropriate locations and timing, resulting in the induction of tissue-/species-specific freezing behaviors (e.g., extracellular freezing, extraorgan freezing) and the freezing order among tissues: from the primary freeze to the last tissue remaining unfrozen (likely INA level dependent). The spatiotemporal distributions of tissue INA, their characterization, and functional roles are detailed. INA assay principles, anti-nucleation activity (ANA), and freeze visualization tools are also described.


Ice nucleation activity Freezing behavior Cold hardiness Ice blocking barrier Freeze regulating substances Subzero temperature sensor Freezing order Supercooling Anti-nucleation activity MRI Infrared thermography Extracellular freezing Freezing tolerance Freezing avoidance Water 



Anti-nucleation activity


Differential thermal analysis


Ice nucleation activity


Ice nucleation temperature




Magnetic resonance imaging


Scanning electron microscopy



The authors thank Ms. Kitashima, Kitanaka, Oda, Nakatani, and Ishikawa of NIAS for their technical assistance. The authors acknowledge the facilities and the scientific and technical assistance of the National Imaging Facility, Western Sydney University Node. This was partly supported by JSPS KAKENHI Grant numbers JP17H03763, JP26660030, JP23380023, and JP16380030 to M.I., IBBP Research Fund from Japan Society for the Promotion of Science to M.I., the New Technology Development Foundation (Plant Research Fund 25–23, 26-22) and Kieikai Research Foundation (2016S069) to K.K.


  1. Ashworth EN, Davis GA (1984) Ice nucleation within peach trees. J Amer Soc Hort Sci 109:198–201Google Scholar
  2. Ashworth EN, Kieft TL (1995) Ice nucleation activity associated with plants and fungi. In: Lee RE, Warren GJ, Gusta LV (eds) Biological ice nucleation and its applications. APS Press, St. Paul, pp 137–162Google Scholar
  3. Ball MC, Wolfe J, Canny M, Hofmann M, Nicotra AB, Hughes D (2002) Space and time dependence of temperature and freezing in evergreen leaves. Func Plant Biol 29:1259–1272CrossRefGoogle Scholar
  4. Borisjuk L, Rolletschek H, Neuberger T (2012) Surveying the plant’s world by magnetic resonance imaging. Plant J 70:129–146CrossRefGoogle Scholar
  5. Brush RA, Griffith M, Mlynarz A (1994) Characterization and quantification of intrinsic ice nucleators in winter rye (Secale cereale) leaves. Plant Physiol 104:725–735CrossRefGoogle Scholar
  6. Callaghan PT (1991) Principles of nuclear magnetic resonance microscopy. Oxford University Press, OxfordGoogle Scholar
  7. Dean RJ, Stait-Gardner T, Clarke SJ, Rogiers SY, Bobek G, Price WS (2014) Use of diffusion magnetic resonance imaging to correlate the developmental changes in grape berry tissue structure with water diffusion patterns. Plant Methods 10:35–48CrossRefGoogle Scholar
  8. Fall R, Wolber PK (1995) Biochemistry of bacterial ice nuclei. In: Lee RE, Warren GJ, Gusta LV (eds) Biological ice nucleation and its applications. APS Press, St. Paul, pp 63–83Google Scholar
  9. Fletcher GL, Hew CL, Davies PL (2001) Antifreeze proteins of teleost fishes. Annu Rev Physiol 63:359–390CrossRefGoogle Scholar
  10. Franks F (1985) Biophysics and biochemistry at low temperatures. Cambridge University Press, CambridgeGoogle Scholar
  11. Fukuda K, Kawaguchi D, Aihara T, Ogasa MY, Miki NH, Haishi T, Umebayashi T (2015) Vulnerability to cavitation differs between current-year and older xylem: non-destructive observation with a compact magnetic resonance imaging system of two deciduous diffuse-porous species. Plant Cell Environ 38:2508–2518CrossRefGoogle Scholar
  12. Gross DC, Proebsting EL Jr, Maccrindle-Zimmerman H (1988) Development, distribution, and characteristics of intrinsic, nonbacterial ice nuclei in Prunus wood. Plant Physiol 88:915–922CrossRefGoogle Scholar
  13. Gupta A, Stait-Gardner T, Ghadirian B, Price WS (2014) Fundamental concepts for the theory, dynamics of MRI. In: Awojoyogbe OB (ed) Theory, dynamics and applications of magnetic resonance imaging-I. Science Publishing Group, New York, pp 3–37Google Scholar
  14. Hacker J, Neuner G (2008) Ice propagation in dehardened alpine plant species studied by infrared differential thermal analysis (IDTA). Arc Antarc Alpine Res 40:660–670CrossRefGoogle Scholar
  15. Hirano SS, Upper CD (1995) Ecology of ice nucleation-active bacteria. In: Lee RE, Warren GJ, Gusta LV (eds) Biological ice nucleation and its applications. APS Press, St. Paul, pp 41–61Google Scholar
  16. Hirano SS, Baker LS, Upper CD (1985) Ice nucleation temperature of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury. Plant Physiol 77:259–265CrossRefGoogle Scholar
  17. Ide H, Price WS, Arata Y, Ishikawa M (1998) Freezing behaviors in leaf buds of cold-hardy conifers visualized by NMR microscopy. Tree Physiol 18:451–458CrossRefGoogle Scholar
  18. Ishikawa M (2014) Ice nucleation activity in plant tissues. Cryobiol Cryotech 60:79–88Google Scholar
  19. Ishikawa M (2016) Factors contributing to freeze regulation in cold hardy plant tissues. In: Abstracts of 61st seminar for cryobiology and cryotechnology, Tokyo Denki University, Hatoyama, 25–26 June 2016Google Scholar
  20. Ishikawa M, Sakai A (1981) Freezing avoidance mechanisms by supercooling in some Rhododendron flower buds with reference to water relations. Plant Cell Physiol 22:953–967Google Scholar
  21. Ishikawa M, Sakai A (1985) Extraorgan freezing in wintering flower buds of Cornus officinalis Sieb. et Zucc. Plant Cell Environ 8:333–338CrossRefGoogle Scholar
  22. Ishikawa M, Price WS, Ide H, Arata Y (1997) Visualization of freezing behaviors in leaf and flower buds of full-moon maple by nuclear magnetic resonance microscopy. Plant Physiol 115:1515–1524CrossRefGoogle Scholar
  23. Ishikawa M, Ide H, Price WS, Arata Y, Nakamura T, Kishimoto T (2009) Freezing behaviours in plant tissues: visualization using NMR micro-imaging and biochemical regulatory factors involved. In: Gusta LV, Tanino KK, Wisniewski ME (eds) Plant cold hardiness: from the laboratory to the field. CABI, Cambridge, pp 19–28CrossRefGoogle Scholar
  24. Ishikawa M, Ishikawa M, Toyomasu T, Aoki T, Price WS (2015) Ice nucleation activity in various tissues of Rhododendron flower buds: their relevance to extraorgan freezing. Front Plant Sci 6:149CrossRefGoogle Scholar
  25. Ishikawa M, Ide H, Yamazaki H, Murakawa H, Kuchitsu K, Price WS, Arata Y (2016) Freezing behaviors in wintering Cornus florida flower bud tissues revisited using MRI. Plant Cell Environ 39:2663–2675CrossRefGoogle Scholar
  26. Ishikawa M, Ide H, Tsujii T, Kuchitsu K, Price WS, Arata Y (2018) Preferential freezing avoidance localized in anthers and embryo sacs in wintering Daphne kamtschatica var. jezoensis flower buds visualized by MRI. Plant Cell Environ (accepted)Google Scholar
  27. Kasuga J, Hashidoko Y, Nishioka A, Yoshiba M, Arakawa K, Fujikawa S (2008) Deep supercooling xylem parenchyma cells of katsura tree (Cercidiphyllum japonicum) contain flavonol glycosides exhibiting high anti-ice nucleation activity. Plant Cell Environ 31:1335–1348CrossRefGoogle Scholar
  28. Kishimoto T, Sekozawa Y, Yamazaki H, Murakawa H, Kuchitsu K, Ishikawa M (2014a) Seasonal changes in ice nucleation activity in blueberry stems and effects of cold treatments in vitro. Environ Exp Bot 106:13–23CrossRefGoogle Scholar
  29. Kishimoto T, Yamazaki H, Saruwatari A, Murakawa H, Sekozawa Y, Kuchitsu K, Price WS, Ishikawa M (2014b) High ice nucleation activity located in blueberry stem bark is linked to primary freeze initiation and adaptive freezing behavior of the bark. AoB Plants 6:plu044CrossRefGoogle Scholar
  30. Kitaura K (1967) Freezing and injury of mulberry trees by late spring frost. Bull Seric Exp Station 22:202–323Google Scholar
  31. Köckenberger W (2001) Functional imaging of plants by magnetic resonance experiments. Trends in Plant Sci 6:286–292CrossRefGoogle Scholar
  32. Kuprian E, Briceno V, Wagner J, Neuner G (2014) Ice barriers promote supercooling and prevent frost injury in reproductive buds, flowers and fruits of alpine dwarf shrubs throughout the summer. Environ Exp Bot 106:4–12CrossRefGoogle Scholar
  33. Kuroda H, Sagisaka S, Chiba K (1990) Frost induces cold acclimation and peroxide scavenging systems coupled with the pentose phosphate cycle in apple twigs under natural conditions. J Jpn Soc Hort Sci 59:409–416CrossRefGoogle Scholar
  34. Larcher W, Meindl U, Ralser E, Ishikawa M (1991) Persistent supercooling and silica deposition in cell walls of palm leaves. J Plant Physiol 139:146–154CrossRefGoogle Scholar
  35. Lindow SE (1983) The role of bacterial ice nucleation in frost injury to plants. Annu Rev Phytopathol 21:363–384CrossRefGoogle Scholar
  36. Nagata A, Kose K, Terada Y (2016) Development of an outdoor MRI system for measuring flow in a living tree. J Magn Reson 265:129–138CrossRefGoogle Scholar
  37. Neuner G (2014) Frost resistance in alpine woody plants. Front Plant Sci 5:1–13CrossRefGoogle Scholar
  38. Neuner G, Xu B, Hacker J (2010) Velocity and pattern of ice propagation and deep supercooling in woody stems of Castanea sativa, Morus nigra and Quercus robur measured by IDTA. Tree Physiol 30:1037–1045CrossRefGoogle Scholar
  39. Pearce RS (2001) Plant freezing and damage. Ann Bot 87:417–424CrossRefGoogle Scholar
  40. Price WS, Ide H, Arata Y, Ishikawa M (1997a) Visualization of freezing behaviours in flower bud tissues of cold hardy Rhododendron japonicum by nuclear magnetic resonance micro-imaging. Aust J Plant Physiol 24:599–605CrossRefGoogle Scholar
  41. Price WS, Ide H, Ishikawa M, Arata Y (1997b) Intensity changes in 1H-NMR micro-images of plant materials exposed to subfreezing temperatures. Bioimages 5:91–99Google Scholar
  42. Pruppacher HR (1967) Interpretation of experimentally determined growth rates of ice crystals in supercooled water. J Chem Phys 47:1807–1813CrossRefGoogle Scholar
  43. Quamme HA (1995) Deep supercooling in buds of woody plants. In: Lee RE, Warren GJ, Gusta LV (eds) Biological ice nucleation and its applications. APS Press, St. Paul, pp 183–200Google Scholar
  44. Sakai A, Larcher W (1987) Frost survival of plants: responses and adaptation to freezing stress, ecological studies 62. Springer-Verlag, BerlinGoogle Scholar
  45. Scotter AJ, Marshall CB, Graham LA, Gilbert JA, Garnham CP, Davies PL (2006) The basis for hyperactivity of antifreeze proteins. Cryobiology 53:229–239CrossRefGoogle Scholar
  46. Sekozawa Y, Sugaya S, Gemma H, Iwahori S, Ishikawa M (2002) Seasonal changes in the ice nucleation activity of various tissues in Japanese pear (Pyrus pyrifolia Nakai) in relation to their freezing behavior and frost injury. Acta Hort 587:543–547CrossRefGoogle Scholar
  47. Thomashow MF (1999) Plant cold acclimation, freezing tolerance genes and regulatory mechanisms. Ann Rev Plant Physiol Plant Mol Biol 50:571–599CrossRefGoogle Scholar
  48. Tsushima K (2015) Ice and snow physics (online textbook). Accessed 15 Sept 2017
  49. Ueda Y, Anma K, Ishikawa M (2002) Variation of Rosa and its genealogical implication in cultivated roses. 9. Ice nucleating activity of Rosa species. Proc Jpn Soc Hort Sci 71(suppl 1):293Google Scholar
  50. Upper CD, Vali G (1995) The discovery of bacterial ice nucleation and its role in the injury of plants by frosts. In: Lee RE, Warren GJ, Gusta LV (eds) Biological ice nucleation and its applications. APS Press, St. Paul, pp 29–39Google Scholar
  51. Vali G (1971) Quantitative evaluation of experimental results on the heterogeneous freezing nucleation of supercooled liquids. J Atmos Sci 28:402–409CrossRefGoogle Scholar
  52. Vali G (1995) Principles of ice nucleation. In: Lee RE, Warren GJ, Gusta LV (eds) Biological ice nucleation and its applications. APS Press, St. Paul, pp 1–28Google Scholar
  53. Vali G, Stansbury EJ (1966) Time-dependent characteristics of the heterogeneous nucleation of ice. Can J Phys 44:477–502CrossRefGoogle Scholar
  54. Venturas MD, Sperry JS, Hacke UG (2017) Plant xylem hydraulics: what we understand, current research, and future challenges. J Integ Plant Biol 59:356–389CrossRefGoogle Scholar
  55. Warren GJ (1995) Identification and analysis of ina genes and proteins. In: Lee RE, Warren GJ, Gusta LV (eds) Biological ice nucleation and its applications. APS Press, St. Paul, pp 85–99Google Scholar
  56. Wisniewski M, Lindow SE, Ashworth EN (1997) Observations of ice nucleation and propagation in plants using infrared video thermography. Plant Physiol 113:327–334CrossRefGoogle Scholar
  57. Wisniewski ME, Gusta LV, Fuller MP, Karlson D (2009) Ice nucleation, propagation and deep supercooling: the lost tribes of freezing studies. In: Gusta LV, Tanino KK, Wisniewski ME (eds) Plant cold hardiness: from the laboratory to the field. CABI, Cambridge, pp 1–11Google Scholar
  58. Yamazaki H, Ishikawa M (2010) Analysis of freezing behavior in blueberry stems visualized using differential infra-red thermography. Cryobiol Cryotech 56:91–95Google Scholar
  59. Yamazaki H, Yoshida S, Ishikawa M (2011) Freezing behavior in blueberry stems analyzed using differential infra-red thermography and differential thermal analysis. Cryobiol Cryotech 57:77–81Google Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  • Masaya Ishikawa
    • 1
    • 2
    Email author
  • Hideyuki Yamazaki
    • 3
  • Tadashi Kishimoto
    • 4
  • Hiroki Murakawa
    • 5
  • Timothy Stait-Gardner
    • 6
  • Kazuyuki Kuchitsu
    • 5
  • William S. Price
    • 6
  1. 1.Imaging Frontier CenterTokyo University of ScienceNodaJapan
  2. 2.Department of Forest Science, Graduate School of Agricultural and Life SciencesThe University of TokyoBunkyo-ku, TokyoJapan
  3. 3.International Patent Organism DepositoryNational Institute of Technology and EvaluationKisarazuJapan
  4. 4.Division of Plant SciencesNational Institute of Agrobiological SciencesTsukubaJapan
  5. 5.Department of Applied Biological ScienceTokyo University of ScienceNodaJapan
  6. 6.Nanoscale Organisation and Dynamics GroupWestern Sydney UniversityPenrithAustralia

Personalised recommendations