, Volume 243, Issue 1–4, pp 63–71 | Cite as

Freezing cytorrhysis and critical temperature thresholds for photosystem II in the peat moss Sphagnum capillifolium

  • Othmar Buchner
  • Gilbert Neuner
Original Article


Leaflets of Sphagnum capillifolium were exposed to temperatures from −5°C to +60°C under controlled conditions while mounted on a microscope stage. The resultant cytological response to these temperature treatments was successfully monitored using a light and fluorescence microscope. In addition to the observable cytological changes during freezing cytorrhysis and heat exposure on the leaflets, the concomitant critical temperature thresholds for inactivation of photosystem II (PS II) were studied using a micro fibre optic and a chlorophyll fluorometer mounted to the microscope stage. Chlorophyllous cells of S. capillifolium showed extended freezing cytorrhysis immediately after ice nucleation at −1.1°C in the water in which the leaflets were submersed during the measurement. The occurrence of freezing cytorrhysis, which was visually manifested by cell shrinkage, was highly dynamic and was completed within 2 s. A total reduction of the mean projected diameter of the chloroplast containing area during freezing cytorrhysis from 8.9 to 3.8 μm indicates a cell volume reduction of approximately −82%. Simultaneous measurement of chlorophyll fluorescence of PS II was possible even through the frozen water in which the leaf samples were submersed. Freezing cytorrhysis was accompanied by a sudden rise of basic chlorophyll fluorescence. The critical freezing temperature threshold of PS II was identical to the ice nucleation temperature (−1.1°C). This is significantly above the temperature threshold at which frost damage to S. capillifolium leaflets occurs (−16.1°C; LT50) which is higher than observed in most higher plants from the European Alps during summer. High temperature thresholds of PS II were 44.5°C which is significantly below the heat tolerance of chlorophyllous cells (49.9°C; LT50). It is demonstrated that light and fluorescence microscopic techniques combined with simultaneous chlorophyll fluorescence measurements may act as a useful tool to study heat, low temperature, and ice-encasement effects on the cellular structure and primary photosynthetic processes of intact leaf tissues.


Chlorophyll fluorescence Cytorrhysis Freezing tolerance Heat tolerance Light microscope temperature-controlled chamber Sphagnum capillifolium 



This work was enabled by the Austrian Science Found (FWF-project 17992-B06 to G. Neuner) as a preliminary feasibility study in context with the general examination and visualisation of freezing phenomena in plant tissues at the tissue and the cellular level. We are thankful to Dr. Georg Gärtner for providing taxonomic expertise.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Andrews CJ (1996) How do plants survive ice? Ann Bot (Lond) 78:529–536. doi: 10.1006/anbo.1996.0157 CrossRefGoogle Scholar
  2. Ashworth EN, Pearce RS (2002) Extracellular freezing in leaves of freezing-sensitive species. Planta 214:798–805. doi: 10.1007/s00425-001-0683-3 CrossRefPubMedGoogle Scholar
  3. Balagurova N, Drozdov S, Grabovik S (1996) Cold and heat resistance of five species of Sphagnum. Ann Bot Fenn 33:33–37Google Scholar
  4. Beck EH, Fettig S, Knake C, Hartig K, Bhattarai T (2007) Specific and unspecific responses of plants to cold and drought stress. J Biosci 32:501–510. doi: 10.1007/s12038-007-0049-5 CrossRefPubMedGoogle Scholar
  5. Bilger HW, Schreiber U, Lange OL (1984) Determination of leaf heat resistance: comparative investigation of chlorophyll fluorescence changes and tissue necrosis methods. Oecologia 63:256–262. doi: 10.1007/BF00379886 CrossRefGoogle Scholar
  6. Braun V, Buchner O, Neuner G (2002) Thermotolerance of photosystem 2 of three alpine species under field conditions. Photosynthetica 40:6587–6595. doi: 10.1023/A:1024312304995 CrossRefGoogle Scholar
  7. Buchner O, Lütz C, Holzinger A (2007) Design and construction of a new temperature-controlled chamber for light and confocal microscopy under monitored conditions: biological application for plant samples. J Microsc 225:183–191. doi: 10.1111/j.1365-2818.2007.01730.x CrossRefPubMedGoogle Scholar
  8. Buiteveld H, Hakvoort JMH, Donze M (1994) The optical properties of pure water. In: Jaffe JS (ed) SPIE Proceedings on Ocean Optics XII, 2258:174–183Google Scholar
  9. Carpentier R (1999) The effect of high temperature stress on the photosynthetic apparatus. In: Pessarakli M (ed) Handbook of plant and crop stress, 2nd edn. Marcel Dekker Inc., New York, p 1254Google Scholar
  10. Chaplin M (2008) Online document In: Water structure and science. London South Bank University, Available via permanent URL: of subordinate document. Accessed 10 Dec 2008
  11. Crowe JH, Carpenter JF, Crowe LM, Anchordoguy TJ (1990) Are freezing and dehydration similar stress vectors? A comparison of modes of interaction of stabilizing solutes with biomolecules. Cryobiology 27:219–231. doi: 10.1016/0011-2240(90)90023-W CrossRefGoogle Scholar
  12. Hacker J, Neuner G (2007) Ice propagation in plants visualized at the tissue level by infrared differential thermal analysis (IDTA). Tree Physiol 27:1661–1670PubMedGoogle Scholar
  13. Hacker J, Spindelböck J, Neuner G (2008) Mesophyll freezing and effects of freeze dehydration visualized by simultaneous measurement of IDTA and differential imaging chlorophyll fluorescence. Plant Cell Environ 31:1725–1733. doi: 10.1111/j.1365-3040.2008.01881.x CrossRefPubMedGoogle Scholar
  14. Hajek T, Beckett RT (2008) Effect of water content components on desiccation and recovery in Sphagnum mosses. Ann Bot (Lond) 101:165–173. doi: 10.1093/aob/mcm287 CrossRefGoogle Scholar
  15. Hansen J, Beck E (1988) Evidence for ideal and non-ideal equilibrium freezing of leaf water in frost hardy ivy (Hedera helix) and winter barley (Hordeum vulgare). Bot Acta 101:76–82Google Scholar
  16. Körner C, Larcher W (1988) Plant life in cold climates. In: Long S, Woodward FI (eds) Plants and temperature, vol 42. The Company of Biologists Limited, Cambridge, pp 25–57Google Scholar
  17. Kouril R, Lazar D, Ilık P, Skotnica J, Krchnak P, Naus J (2004) High-temperature induced chlorophyll fluorescence rise in plants at 40–50°C: experimental and theoretical approach. Photosynth Res 81:49–66. doi: 10.1023/B:PRES.0000028391.70533.eb CrossRefPubMedGoogle Scholar
  18. Kreeb KH (1990) Hitzeresistenz. In: Kreeb KH (ed) Methoden zur Pflanzenökologie und Bioindikation. Gustav Fischer, Jena, pp 72–75Google Scholar
  19. Larcher W (1990) Kälteresistenz. In: Kreeb KH (ed) Methoden zur Pflanzenökologie und Bioindikation, 2nd edn. Gustav Fischer, Jena, pp 76–92Google Scholar
  20. Larcher W (2003) Physiological plant ecology. Ecophysiology and stress physiology of functional groups. Springer, BerlinGoogle Scholar
  21. Levitt J (1978) An overview of freezing injury and survival and its relationship to other stresses. In: Li PH, Sakai A (eds) Plant cold hardiness and freezing stress. Academic, New York, pp 3–15Google Scholar
  22. Liénard D, Durambur G, Kiefer-Meyer MC, Nogué F, Menu-Bouaouiche L, Charlot F, Gomord V, Lassalles JP (2008) Water transport by aquaporins in the extant plant Physcomitrella patens. Plant Physiol 146:1207–1218. doi: 10.1104/pp.107.111351 CrossRefPubMedGoogle Scholar
  23. Lovelock CE, Jackson AE, Melick DR, Seppelt RD (1995) Reversible photoinhibition in antarctic moss during freezing and thawing. Plant Physiol 109:955–961PubMedGoogle Scholar
  24. Maseyk KS, Green TGA, Klinac D (1999) Photosynthetic responses of New Zealand Sphagnum species. N Z J Bot 37:155–165Google Scholar
  25. Mazur P (1963) Kinetics of water loss from cells at subzero temperatures and the likelihood of intracellular freezing. J Gen Physiol 47:347–469. doi: 10.1085/jgp.47.2.347 CrossRefPubMedGoogle Scholar
  26. Neuner G, Pramsohler M (2006) Freezing and high temperature thresholds of photosystem II compared to ice nucleation, frost and heat damage in evergreen subalpine plants. Physiol Plant 126:196–204. doi: 10.1111/j.1399-3054.2006.00605.x CrossRefGoogle Scholar
  27. Oliver MJ, Velten J, Mishler BD (2005) Desiccation tolerance in bryophytes: a reflection of the primitive strategy for plant survival in dehydrating habitats? Integr Comp Biol 45:788–799. doi: 10.1093/icb/45.5.788 CrossRefGoogle Scholar
  28. Pearce RS (2001) Plant freezing and damage. Ann Bot (Lond) 87:417–424. doi: 10.1006/anbo.2000.1352 CrossRefGoogle Scholar
  29. Proctor MCF (2000) The bryophyte paradox: tolerance of desiccation, evasion of drought. Plant Ecol 151:41–49. doi: 10.1023/A:1026517920852 CrossRefGoogle Scholar
  30. Proctor MCF, Pence VC (2002) Vegetative tissues: bryophytes, vascular resurrection plants, and vegetative propogules. In: Black M, Pritchard HW (eds) Desiccation and survival in plants: drying without dying. CABI Publishing, Wallingford, Oxon, pp 207–237CrossRefGoogle Scholar
  31. Quamme H (1995) Deep supercooling in buds of woody plants. In: Lee RE Jr, Warren GJ, Gusta LV (eds) Biological ice nucleation and its application. American Phytopathological Society, St. Paul, Minnesota, pp 183–199Google Scholar
  32. Sakai A, Larcher W (1987) Frost survival of plants. Responses and adaptation to freezing stress. In: Billings WD, Golloy F, Large DL, Olsen JS, Ramment H (eds) Ecological studies, vol 62. Springer, Berlin, GermanyGoogle Scholar
  33. Schipperges B, Rydin H (1998) Response of photosynthesis of Sphagnum species from contrasting microhabitats to tissue water content and repeated desiccation. New Phytol 140:677–684. doi: 10.1046/j.1469-8137.1998.00311.x CrossRefGoogle Scholar
  34. Schreiber U, Berry JA (1977) Heat-induced changes of chlorophyll fluorescence in intact leaves correlated with damage of the photosynthetic apparatus. Planta 136:233–238. doi: 10.1007/BF00385990 CrossRefGoogle Scholar
  35. Taschler D, Neuner G (2004) Summer frost resistance and freezing patterns measured in situ in leaves of major alpine plant growth forms in relation to their upper distribution boundary. Plant Cell Environ 27:737–746. doi: 10.1111/j.1365-3040.2004.01176.x CrossRefGoogle Scholar
  36. Warren SG (1984) Optical constants of ice from the ultraviolet to the microwave. Appl Opt 23:1206–1225. doi: 10.1364/AO.23.001206 CrossRefPubMedGoogle Scholar
  37. Warren SG, Brandt RE (2008) Optical constants of ice from the ultraviolet to the microwave: a revised compilation. J Geophys Res . doi: 10.1029/2007JD009744 Google Scholar
  38. Weis E, Berry JA (1988) Plants and high temperature stress. In: Long SP, Woodward FI (eds) Plants and temperature. The Company of Biologists Limited, Cambridge, pp 329–346Google Scholar
  39. Weng JH, Lai MF (2005) Estimating heat tolerance among plant species by two chlorophyll fluorescence parameters. Photosynthetica 43:439–444. doi: 10.1007/s11099-005-0070-6 CrossRefGoogle Scholar
  40. Williams TG, Flanagan LB (1998) Measuring and modelling environmental influences on photosynthetic gas exchange in Sphagnum and Pleurozium. Plant Cell Environ 21:555–564. doi: 10.1046/j.1365-3040.1998.00292.x CrossRefGoogle Scholar
  41. Zhu JJ, Beck E (1991) Water relations of pachysandra leaves during freezing and thawing. Plant Physiol 97:1146–1153. doi: 10.1104/pp.97.3.1146 CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  1. 1.Department of Physiology and Cell Physiology of Alpine PlantsUniversity of Innsbruck, Institute of BotanyInnsbruckAustria

Personalised recommendations