Freezing treatment under light conditions leads to a dramatic enhancement of freezing tolerance in cold-acclimated Arabidopsis.

Overwintering plants survive subzero temperatures by cold acclimation (CA), wherein they acquire freezing tolerance through short-term exposure to low temperatures above 0°C. The freezing tolerance of CA plants increases when they are subsequently exposed to mild subzero temperatures, a phenomenon known as second-phase cold hardening (2PH). Here, we explored the molecular mechanism and physiological conditions of 2PH. The results show that, compared with supercooling, a freezing treatment during 2PH after CA enhanced the freezing tolerance of Arabidopsis. This required CA as a pretreatment, and was designated as second-phase freezing acclimation (2PFA). Light increased the effect of 2PFA to enhance freezing tolerance after CA. C-repeat binding factor and cold-regulated genes were downregulated by light during the 2PFA treatment, a different transcription profile from that during CA. The freezing tolerance of 2PFA plants was decreased by the presence of the photosynthetic electron transfer inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea during the 2PFA treatment. Compared with wild-type plants, phototropin1,2 and phyb mutants showed lower freezing tolerance after 2PFA treatment. These results show that exposure to freezing after CA increases freezing tolerance as a secondary process, and that freezing under light conditions further increases freezing tolerance via pathways involving photoreceptors and photosynthetic electron transfer.