Cold acclimation is affected by diurnal cycles and minute-scale random temperature fluctuations via calcium signals

Abstract

Molecular and physiological processes during cold acclimation (CA) have been investigated using plants incubated under constant low-temperature conditions. However, to comprehensively characterize CA in the field, the effects of day–night temperature cycles and minute-scale random temperature fluctuations must be clarified. Thus, we developed an experimental system that can maintain diurnal cycles and random temperature fluctuations during CA treatments. On the basis of the temperature changes in the field, three CA conditions were applied: conventional CA at 2°C (con-CA), CA with a 10°C day/2°C night cycle (C-CA), and C-CA with random temperature fluctuations only during the day (FC-CA). Because cold-induced Ca2+ signals help regulate CA, the effects of Ca2+ signals during the three CA treatments were examined using Ca2+ channel blockers (LaCl3 and ruthenium red). The freezing tolerance of Arabidopsis thaliana was similar after the C-CA and con-CA treatments, but it decreased following the FC-CA treatment. The analysis of transcription factors regulating CA processes indicated CBF/DREB1 expression levels tended to be highest for the con-CA treatment, followed by the FC-CA and C-CA treatments. Moreover, the Ca2+ signals substantially contributed to the freezing tolerance of the plants that underwent the FC-CA and C-CA treatments, while also considerably modulating gene expression in the FC-CA-treated plants. Furthermore, the Ca2+ signals enhanced CBF/DREB1 expression during the FC-CA treatment, but the Ca2+ signals derived from intracellular organelles suppressed the expression of CBF2/DREB1C and CBF3/DREB1A during the C-CA treatment. Thus, diurnal temperature cycles and random temperature fluctuations affect CA through different calcium signals, implying that plants regulate CA by precisely sensing temperature changes in the field.