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

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          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.

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          Most cited references47

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          Observations of Ice Nucleation and Propagation in Plants Using Infrared Video Thermography.

          We evaluated the use of infrared (IR) video thermography to observe directly ice nucleation and propagation in plants. An imaging radiometer with an HgCdTe long-wave (8-12 [mu]m) detector was utilized to image the thermal response of plants during freezing. IR images were analyzed in real time and recorded on videotape. Information on the videotape was subsequently accessed and analyzed utilizing IR image analysis software. Freezing of water droplets as small as 0.5 [mu]L was clearly detectable with the radiometer. Additionally, a comparison of temperature tracking data collected by the radiometer with data collected with thermocouples showed close correspondence. Monitoring of an array of plant species under different freezing conditions revealed that ice nucleation and propagation are readily observable by thermal imaging. In many instances, the ice nucleation-active bacterium Pseudomonas syringae placed on test plants could be seen to initiate freezing of the whole plant. Apparent ice nucleation by intrinsic nucleators, despite the presence of ice nucleation-active bacteria, was also evident in some species. Floral bud tissues of peach (Prunus persica) could be seen to supercool below the temperature of stem tissues, and ice nucleation at the site of insertion of the thermocouple was frequently observed. Rates of propagation of ice in different tissues were also easily measured by thermal imaging. This study demonstrates that IR thermography is an excellent method for studying ice nucleation and propagation in plants.
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            The effect of water, sugars, and proteins on the pattern of ice nucleation and propagation in acclimated and nonacclimated canola leaves.

            Infrared video thermography was used to observe ice nucleation temperatures, patterns of ice formation, and freezing rates in nonacclimated and cold acclimated leaves of a spring (cv Quest) and a winter (cv Express) canola (Brassica napus). Distinctly different freezing patterns were observed, and the effect of water content, sugars, and soluble proteins on the freezing process was characterized. When freezing was initiated at a warm subzero temperature, ice growth rapidly spread throughout nonacclimated leaves. In contrast, acclimated leaves initiated freezing in a horseshoe pattern beginning at the uppermost edge followed by a slow progression of ice formation across the leaf. However, when acclimated leaves, either previously killed by a slow freeze (2 degrees C h(-1)) or by direct submersion in liquid nitrogen, were refrozen their freezing pattern was similar to nonacclimated leaves. A novel technique was developed using filter paper strips to determine the effects of both sugars and proteins on the rate of freezing of cell extracts. Cell sap from nonacclimated leaves froze 3-fold faster than extracts from acclimated leaves. The rate of freezing in leaves was strongly dependent upon the osmotic potential of the leaves. Simple sugars had a much greater effect on freezing rate than proteins. Nonacclimated leaves containing high water content did not supercool as much as acclimated leaves. Additionally, wetted leaves did not supercool as much as nonwetted leaves. As expected, cell solutes depressed the nucleation temperature of leaves. The use of infrared thermography has revealed that the freezing process in plants is a complex process, reminding us that many aspects of freezing tolerance occur at a whole plant level involving aspects of plant structure and metabolites rather than just the expression of specific genes alone.
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              Mechanism of freeze-thaw injury and recovery: A cool retrospective and warming up to new ideas.

              Understanding cellular mechanism(s) of freeze-thaw injury (FTI) is key to the efforts for improving plant freeze-tolerance by cultural methods or molecular/genetic approaches. However, not much work has been done in the last 25+ years to advance our understanding of the nature and cellular loci of FTI. Currently, two FTI lesions are predominantly implicated: 1) structural and functional perturbations in plasma membrane; 2) ROS-induced oxidative damage. While both have stood the test of time, many questions remain unresolved and other potentially significant lesions need to be investigated. Additionally, molecular mechanism of post-thaw recovery (PTR), a critical component of frost-survival, has not been well investigated. Mechanistic understanding of repair after reversible injury could expand the options for strategies to improve frost-hardiness. In this review, without claiming to be exhaustive, I have attempted to synthesize major discoveries from last several decades on the mechanisms of FTI and the relatively little research conducted thus far on PTR mechanisms. It is followed by proposing of hypotheses for mechanism(s) for irreversible FTI or PTR involving cytosolic calcium and ROS signaling. Perspective is presented on some unresolved questions and research on new ideas to fill the knowledge gaps and advance the field.
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                Author and article information

                Contributors
                matthias.stegner@uibk.ac.at
                johanna.wagner@uibk.ac.at
                gilbert.neuner@uibk.ac.at
                Journal
                Plant Methods
                Plant Methods
                Plant Methods
                BioMed Central (London )
                1746-4811
                20 May 2020
                20 May 2020
                2020
                : 16
                : 73
                Affiliations
                GRID grid.5771.4, ISNI 0000 0001 2151 8122, Department of Botany, Unit Functional Plant Biology, , University of Innsbruck, ; Sternwartestrasse 15, 6020 Innsbruck, Austria
                Author information
                http://orcid.org/0000-0001-9330-5564
                Article
                617
                10.1186/s13007-020-00617-1
                7240938
                80084884-5cbd-4d98-a1b6-f2ef91ceeac0
                © The Author(s) 2020

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                : 31 March 2020
                : 15 May 2020
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/501100002428, Austrian Science Fund;
                Award ID: P30139-B32
                Award Recipient :
                Categories
                Methodology
                Custom metadata
                © The Author(s) 2020

                Plant science & Botany
                birefringence,ice crystal,ice localisation,ice segregation,ice management,freezing resistance

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