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Title: Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum

Author
item WESLEY-SMITH, JAMES - Council For Scientific And Industrial Research (CSIR)
item Walters, Christina
item PAMMENTER, N.W. - University Of Kwazulu-Natal
item BERJAK, P. - University Of Kwazulu-Natal

Submitted to: Annals of Botany
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/13/2015
Publication Date: 6/1/2015
Citation: Wesley-Smith, J., Walters, C.T., Pammenter, N., Berjak, P. 2015. Why is intracellular ice lethal? A microscopical study showing evidence of programmed cell death in cryo-exposed embryonic axes of recalcitrant seeds of Acer saccharinum. Annals of Botany. 115(6):991-1000. DOI:10.1093/aob/mcv009.

Interpretive Summary: This manuscript presents a unique perspective on the nature of lethality from intracellular ice formation – a vital question that remains unanswered after a century of investigation. Traditionally, intracellular ice has been postulated to cause massive cellular trauma (often at the site of cell membranes) which leads to necrosis. Immediately after thawing, we show excellent integrity of cells that experienced ice crystals too small to disrupt structure. However, despite great cell integrity, the stress of ice crystal formation induced ‘self-destruct’ metabolism commonly called programmed cell death (PCD) and affected cells consumed themselves through autophagy. Our interpretation is that the formation of ice crystals compresses cytoplasm and induces PCD metabolism. This contributes to a greater body of literature on the effect of mechanical stress on PCD. Therapies that reduce PCD appear to be the obvious next step in enhancing survival of cryopreserved plant tissues, especially those from tropical areas.

Technical Abstract: Intracellular ice formed in rapidly cooled embryonic axes of Acer saccharinum and was not necessarily lethal when ice crystals were small. This study seeks to understand the nature and extent of damage from intracellular ice, and the course of recovery and regrowth in surviving tissues. Embryonic axes of A. saccharinum, receiving no dehydration or cryoprotection treatments (water content was 1.9 g H2O g-1 dry mass), were cooled to liquid nitrogen temperatures using two methods, plunging into nitrogen slush to achieve a cooling rate of 97oC sec-1or programmed cooling at 3.3oC sec-1. Samples were thawed rapidly (177oC sec-1) and cell structure was examined microscopically immediately and up to 72 h in vitro. Survival was assessed after four weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination. Immediately post-thaw after cryogenic exposure, cells from axes did not show signs of damage at an ultrastructural level. Signs that cells had been damaged were apparent after several hours of in vitro culture and appeared as autophagic decomposition. In surviving tissues, dead cells were sloughed off and pockets of living cells were the origin of regrowth. In roots, regrowth occurred from the ground meristem and procambium, not the distal meristem which became lethally damaged. Regrowth of shoots occurred from isolated pockets of surviving cells of peripheral and pith meristems. The size of these pockets may determine the extent and vigour of regrowth. Autophagic degradation of cells following cryoexposure and formation of tiny intracellular ice crystals challenges current ideas that ice causes irreparable trauma to cells. Rather freezing stress may induce a signal for programmed cell death (PCD). Cells that formed more ice crystals during cooling had faster PCD response.