Abstract

BackgroundMany methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In this context, the preservation of a defined frozen state during the entire fixation procedure is an essential prerequisite. However, current techniques are not able to fix frozen plant tissues for transmission electron microscopy (TEM) without interrupting the cold chain. Chemical fixation by glutaraldehyde and osmium tetroxide is not possible at sub-zero temperatures. Cryo-fixation methods, such as high pressure freeze fixation (HPF) representing the state-of-the-art technique for best structural preservation, are not equipped for freezing frozen samples. In order to overcome this obstacle, a novel technical approach for maintaining the cold chain of already frozen plant samples prior and during HPF is presented.ResultsDifferent algae (Micrasterias denticulata, Klebsormidium crenulatum) and higher plant tissues (Lemna sp., Ranunculus glacialis, Pinus mugo) were successfully frozen and prepared for HPF at freezing temperatures (− 2 °C, − 5 °C, − 6 °C) within a newly developed automatic freezing unit (AFU), that we manufactured from a standard laboratory freezer. Preceding tests on photosynthetic electron transport and ability to plasmolyse show that the temperatures applied did not impair electron transport in PSII nor cell vitality. The transfer of the frozen specimen from the AFU into the HPF-device and subsequently cryo-fixation were performed without intermediate thawing. After cryo-substitution and further processing, the resulting TEM-micrographs showed excellent ultrastructure preservation of the different organisms when compared to specimens fixed at ambient temperature.ConclusionsThe method presented allows preserving the ultrastructure of plant cells in the frozen state during cryo-fixation. The resulting high quality TEM-images represent an important step towards a better understanding of the consequences of extracellular ice formation on cellular ultrastructure. It has the potential to provide new insights into changes of organelle structure, identification of intracellular injuries during ice formation and may help to understand freezing and thawing processes in plant tissues. It may be combined with analytical TEM such as electron energy loss spectroscopy (EELS), X-ray analyses (EDX) and various other electron microscopic techniques.

Highlights

  • Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions

  • Sample temperature Temperature during the controlled freezing exposure prior to high pressure freeze fixation (HPF) During the experimental freezing exposure within an automatic freezing unit (AFU) that was newly developed by modifying a standard laboratory freezer, the leaf temperatures and the temperatures of the samples followed the preset-temperature course with high accuracy

  • During the cooling phase down to the target freezing temperatures (M. denticulata, K. crenulatum and Lemna sp.: − 2 °C, R. glacialis: − 5 °C, P. mugo: − 6 °C) temperature deviations occurred with maximum ± 0.1 °C (M. denticulata, K. crenulatum), ± 0.4 °C (Lemna sp.) and ± 0.5 °C (R. glacialis)

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Summary

Introduction

Many methodological approaches have focused so far on physiological and molecular responses of plant tissues to freezing but only little knowledge is available on the consequences of extracellular ice-formation on cellular ultrastructure that underlies physiological reactions. In this context, the preservation of a defined frozen state during the entire fixation procedure is an essential prerequisite. A key point for survival of freezing temperatures is the tolerance of ice formation within the plant tissue that generally represents a dramatic incident. Ice which is formed outside the cell wall (extracellularly) can principally be survived down to certain freezing temperature thresholds [1]. The causes of freezing injury to cells, the nature and cellular loci, are still unknown [8]

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