Ice accommodation in plant tissues pinpointed by cryo-microscopy in reflected-polarised-light

Abstract

Background: Freezing resistant plant organs are capable to manage ice formation, ice propagation, and ice accommodation down to variable temperature limits without damage. Insights in ice management strategies are essential for the fundamental understanding of plant freezing and frost survival. However, knowledge about ice management is scarce. Ice crystal localisation inside plant tissues is challenging and is mainly based on optical appearance of ice in terms of colour and shape, investigated by microscopic methods. Notwithstanding, there are major uncertainties regarding the reliability and accuracy of ice identification and localisation. Surface light reflections, which can originate from water or resin, even at non-freezing temperatures, can have a similar appearance as ice. We applied the principle of birefringence, which is a property of ice but not of liquid water, in reflected-light microscopy to localise ice crystals in frozen plant tissues in an unambiguous manner. Results: In reflected-light microscopy, water was clearly visible, while ice was more difficult to identify. With the presented polarised cryo-microscopic system, water, including surface light reflections, became invisible, whereas ice crystals showed a bright and shiny appearance. Based on this, we were able to detect loci where ice crystals are accommodated in frozen and viable plant tissues. In Buxus sempervirens leaves, large ice needles occupied and expanded the space between the adaxial and abaxial leaf tissues. In Galanthus nivalis leaves, air-filled cavities became filled up with ice. Buds of Picea abies managed ice in a cavity at the bud basis and between bud scales. By observing the shape and attachment point of the ice crystals, it was possible to identify tissue fractions that segregate intracellular water towards the aggregating ice crystals. Conclusion: Cryo-microscopy in reflected-polarised-light allowed a robust identification of ice crystals in frozen plant tissue. It distinguishes itself, compared with other methods, by its ease of ice identification, time and cost efficiency and the possibility for high throughput. Profound knowledge about ice management strategies, within the whole range of freezing resistance capacities in the plant kingdom, might be the link to applied science for creating arrangements to avoid future frost damage to crops.

Winter Nights during Summer Time: Stress Physiological Response to Ice and the Facilitation of Freezing Cytorrhysis by Elastic Cell Wall Components in the Leaves of a Nival Species

“…How ice is accommodated in viable leaves at sublethal temperatures was assessed by the recently developed method: cryo-microscopy in reflected polarized light (CM rpl ; [ 23 ]). By CM rpl , it is possible to visualize and localize ice crystals unambiguously in plant tissues.…”

“…(2) The effect of ice formation on the “after-freezing physiological performance” was determined by measurements of the chlorophyll fluorescence and photosynthetic gas exchange. (3) In frozen leaves, ice masses were localized with cryo-microscopy in reflected polarized light (CM rpl ; [ 23 ]), and the temporal- and temperature-dependent dynamic and the extent of freezing cytorrhysis were measured by cryo-microscopy. (4) Plasma membrane staining with the amphiphilic styryl dye FM 1-43 was additionally employed to monitor changes in the plasma membrane during freezing cytorrhysis.…”

Winter survival of the unicellular green alga Micrasterias denticulata: insights from field monitoring and simulation experiments

“…For higher land plants, mechanisms of freezing survival and occasionally also the freezing strain occurring in their natural habitat are quite well-known (Buchner and Neuner 2011;Kuprian et al 2014;Neuner et al 2020;Sakai and Larcher 1987;Stegner et al 2020). While trees get fully exposed to freezing air temperatures, most other species escape cold winter temperatures by soil burial (Sakai and Larcher 1987), snow coverage (Neuner et al 1999a) or in case of hydrophytes in the water body (Larcher 1983).…”

Fusion of Mitochondria to 3-D Networks, Autophagy and Increased Organelle Contacts are Important Subcellular Hallmarks during Cold Stress in Plants

“…The higher freshwater plant Lemna has already been used as a model system in different kinds of investigations [42,43] and also for comparison with Micrasterias [22]. R. glacialis is a nival plant, inhabiting sites above 2000 m elevation in the European Alps (up to 4275 m), and has been subjected to several cold stress related physiological and ultrastructural investigations [44][45][46][47][48][49][50]. The selected set of plant model systems originating from diverse habitats and belonging to various evolutionary levels is intended to provide a comprehensive insight into the structural responses of plant cells to cold stress.…”