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

Abstract

Background and Aims Conservation of the genetic diversity afforded by recalcitrant seeds is achieved by cryopreservation, in which excised embryonic axes (or, where possible, embryos) are treated and stored at temperatures lower than À180 C using liquid nitrogen. It has previously been shown that intracellular ice forms in rapidly cooled embryonic axes of Acer saccharinum (silver maple) but this is not necessarily lethal when ice crystals are 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.Methods Embryonic axes of A. saccharinum, not subjected to dehydration or cryoprotection treatments (water content was 1Á9 g H 2 O g À1 dry mass), were cooled to liquid nitrogen temperatures using two methods: plunging into nitrogen slush to achieve a cooling rate of 97 C s À1 or programmed cooling at 3Á3 C s À1. Samples were thawed rapidly (177 C s À1) and cell structure was examined microscopically immediately, and at intervals up to 72 h in vitro. Survival was assessed after 4 weeks in vitro. Axes were processed conventionally for optical microscopy and ultrastructural examination.Key Results Immediately following 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 possibility for, the extent of and the vigour of regrowth.Conclusions Autophagic degradation and ultimately autolysis of cells following cryo-exposure and formation of small (0Á2-0Á4 mm) intracellular ice crystals challenges current ideas that ice causes immediate physical damage to cells. Instead, freezing stress may induce a signal for programmed cell death (PCD). Cells that form more ice crystals during cooling have faster PCD responses.

Seed storage of Brazilian cacti species in different threat categories

“…This response was also observed for seeds of Melocactus albicephalus Buining & Brederoo, which were stored for 6 months in liquid nitrogen (Veiga-Barbosa et al 2010). Reduction of the germinability after cryopreservation can be attributed to the contamination by fungi during the germination evaluation process or to a high initial moisture of the seeds (Salomão 2002;Marchi et al 2013), which facilities the formation of intracellular ice crystals that cause ruptures in the endomembrane system and compromise cellular compartmentalization (Goldfarb et al 2010;Wesley-Smith et al 2015). The acceptable moisture limit for the cryopreservation technique is specific for each species and requires individual evaluation (Salomão 2002;Pritchard et al 2017).…”

“…; Wesley‐Smith et al . ). The acceptable moisture limit for the cryopreservation technique is specific for each species and requires individual evaluation (Salomão ; Pritchard et al .…”

“…; Wesley‐Smith et al . ). Furthermore, at room temperature there is no reduction of the metabolic activity and of the chemistry reaction speed in the seeds, resulting in an increase in deterioration of the reserve compounds and in the loss of viability (Walters et al .…”

“…Seeds stored in this condition suffer significant alteration in their biochemical and physiological processes, usually associated with the production and release of compounds that alter the structure and action of the antioxidant enzymes, causing a more pronounced reduction of the seed viability (Graham 2008). When this happens, the water equilibrium content of the seeds may increase to the levels that become inadequate for their conservation, leading to the loss of their physiological quality (Walters et al 2004;Wesley-Smith et al 2015). Furthermore, at room temperature there is no reduction of the metabolic activity and of the chemistry reaction speed in the seeds, resulting in an increase in deterioration of the reserve compounds and in the loss of viability (Walters et al 2004;Abud et al 2012;Pritchard et al 2017).…”

Different ways in which CO2 can be released during the turnover of roots in soil

“…In contrast to roots which can remain alive after excision, roots exposed to low freezing temperatures undergo a catastrophic and immediate death as a result of ice crystal growth and disruption of the cell's membranes and cytoskeleton (Wesley-Smith et al 2015). At this point, CO 2 efflux can only occur due to a residual loss of HCO 3…”