Effect of cycloheximide on the freezing tolerance and ultrastructure of cortical parenchyma cells from mulberry twigs

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Abstract
Experiments were performed using cycloheximide (CHI) on cortical parenchyma cells from mulberry twigs (Morus bombyciz cv. Gorogi) to understand the relationship between the sequence of cytological changes suggesting replacement of the plasmamembrane and the rapid increase in freezing tolerance. Cortical cells from twigs collected on October 16 and 27, which were hardy to −10 and −15 °C, respectively, were still alive after cooling to −70 °C if they had been exposed to −3 °C for 10 to 12 days. In these hardened cells, appearance of microvesicles (MVs) in the peripheral cytoplasm and fusion of these MVs with plasmamembrane were characteristically observed. Neither cytological change nor increase of freezing tolerance was observed in cells treated by CHI before their exposure to −3 °C for 10 to 12 days. However, the suppression of such changes by CHI treatment was effective only in cells which were hardy at −10 to −15 °C and not in the cells of twigs collected on November 4, which were hardy to −20 °C. Consequently, synthesis of some functional proteins during hardening was required for the further development of freezing tolerance, and synthesis had been accomplished before the cells became hardy to −20 °C. Ultrastructural changes brought about by CHI treatment suggest that synthesis of some functional proteins related to the presumed changes of plasmamembrane occurs during hardening.

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