Integration of low temperature and light signaling during cold acclimation response in Arabidopsis

Abstract

Certain plants increase their freezing tolerance in response to low nonfreezing temperatures, an adaptive process named cold acclimation. Light has been shown to be required for full cold acclimation, although how light and cold signals integrate and cross-talk to enhance freezing tolerance still remains poorly understood. Here, we show that HY5 levels are regulated by low temperature transcriptionally, via a CBF- and ABA-independent pathway, and posttranslationally, via protein stabilization through nuclear depletion of COP1. Furthermore, we demonstrate that HY5 positively regulates cold-induced gene expression through the Z-box and other cis-acting elements, ensuring the complete development of cold acclimation. These findings uncover unexpected functions for HY5, COP1, and the Z-box in Arabidopsis response to low temperature, provide insights on how cold and light signals integrate to optimize plant survival under freezing temperatures, and reveal the complexity of the molecular mechanisms plants have evolved to respond and adapt to their fluctuating natural environment.

Fig. 1.

HY5 activates cold-induced gene expression through the Z-box. GUS expression and activity in WT etiolated seedlings containing different CAB1 promoter:GUS fusions (A) or the Z:NOS:GUS and NOS:GUS fusions (B), and in hy5-1 etiolated seedlings containing the Z:NOS:GUS fusion (B) grown at 20 °C or exposed 24 h to 4 °C. Data from GUS activity are expressed as means of three independent experiments with 10 plants each. Bars indicate SD. The two Z-box elements included in the −318/−222 CAB1 promoter fragment are displayed in A. (C) ChIP of DNA associated with HY5:YFP expressed under the control of its own promoter in c-hy5 etiolated seedlings growth at 20 °C or exposed 24 h to 4 °C. ChIP-qPCR was performed with an anti-GFP antibody for the −318/−222 CAB1 promoter fragment (Pro_CAB1) and an intergenic control region between genes At4g26900 and At4g26910 (At4g26900). A ChIP-qPCR assay in cold treated seedlings without anti-GFP antibody was also included as a control (-). Data are from two independent biological replicates (1, 2) and are presented as the percentage recovered from the total input DNA before immunoprecipitation (% input). (D) Expression analysis of CAB1 determined by qPCR in WT, hy5-1, and c-hy5 etiolated seedlings grown at 20 °C or exposed 24 h to 4 °C. (E) Expression analysis of At2g36580, RAP2.1, CYS-L, and LEA18 genes determined by qPCR in WT, hy5-1, and c-hy5 plants grown at 20 °C or exposed 24 h to 4 °C. In D and E, data were normalized to the expression levels of the control gene At4g26410. In C–E, bars indicate SD of triplicates.

Fig. 2.

HY5 positively regulates cold acclimation. Two-week-old WT, hy5-1, and c-hy5 plants were exposed to the indicated freezing temperatures for 6 h after being acclimated 7 d at 4 °C. Freezing tolerance was estimated as the percentage of plants surviving each specific temperature after 7 d of recovery under control conditions. Data are expressed as means of three independent experiments with 50 plants each. Bars indicate SD. (A) Freezing tolerance of cold-acclimated WT, hy5-1, and c-hy5. (B) Representative cold-acclimated plants 7 d after being exposed to −8 °C for 6 h.

Fig. 3.

HY5 promotes anthocyanin biosynthesis and restrain ROS accumulation in response to low temperature. (A) Anthocyanin levels in WT, hy5-1, and c-hy5 plants grown at 20 °C or exposed 7 d to 4 °C. (B) Representative WT, hy5-1, and c-hy5 plants exposed to 4 °C. (C) Expression analysis of CHI, CHS, and FLS genes determined by qPCR in WT, hy5-1, and c-hy5 plants grown at 20 °C or exposed 24 h to 4 °C. Data were normalized to the expression levels of the control gene At4g26410. Bars indicate SD of triplicates. (D) ROS levels quantified with DCFH₂-DA in WT, hy5-1, and c-hy5 plants exposed 7 d to 4 °C. AU, arbitrary units; FW, fresh weight. (E) ROS levels in representative leaves of WT, hy5-1, and c-hy5 plants exposed to 4 °C and stained with NBT. In A and D, data are expressed as means of three independent experiments with 25 and 10 plants each, respectively. Bars indicate SD.

Fig. 4.

The expression of HY5 is regulated at the transcriptional level by low temperature independently of ABA and CBFs. Expression analysis of HY5 determined by qPCR in Col plants exposed to 4 °C in the light or in the dark (A), and in cold-exposed WT, cbf2 mutant, CBF1-AS3 transgenic, and aba2-11 mutant plants (B). (C) Expression analysis of LUC and HY5 genes determined by qPCR in Pro_HY5:LUC plants exposed to 4 °C at the indicated times. In A–C, data were normalized to the expression levels of the control gene At4g26410. Bars indicate SD of triplicates. (D) LUC activity in etiolated Pro_HY5:LUC seedlings exposed 24 h to 4 °C. (E) Quantification of LUC activity shown in D. Data are expressed in arbitrary units as means of three independent experiments with 10 plants each. Bars indicate SD.

Fig. 5.

HY5 is stabilized in response to low temperature by nuclear depletion of COP1. (A) Levels of HY5:3HA protein (30 kDa) in 35S:HY5:3HA-complemented hy5-1 seedlings treated with MG132 and subsequently with cycloheximide (CHX), and exposed to 20 °C or 4 °C in the dark or in the light for the indicated times. The large subunit of Rubisco (55 kDa) was used as a loading control. (B) Confocal laser scanning micrographs of c-hy5 plants exposed to 20 °C or 4 °C in the dark or in the light. (C) Confocal laser scanning micrographs of cop1-4 mutants complemented with Pro_35S:YFP:COP1 exposed to 20 °C or 4 °C in the dark or in the light. B Lower and C Lower show overlays of the YFP fluorescence and the transmission images. (Scale bars: 75 μm.)

Fig. 6.

Proposed model for Z-box, HY5, and COP1 function in cold acclimation. A model in which HY5 would promote full development of cold acclimation, integrating low temperature and light signaling is suggested. The involvement of other light signaling components, including COP1 and the Z-box, is also included. Solid and dotted arrows represent established and theoretical pathways, respectively.