Phytochrome A and B Function Antagonistically to Regulate Cold Tolerance via Abscisic Acid-Dependent Jasmonate Signaling

Abstract

Light signaling and phytohormones both influence plant growth, development, and stress responses; however, cross talk between these two signaling pathways in response to cold remains underexplored. Here, we report that far-red light (FR) and red light (R) perceived by phytochrome A (phyA) and phyB positively and negatively regulated cold tolerance, respectively, in tomato (Solanum lycopersicum), which were associated with the regulation of levels of phytohormones such as abscisic acid (ABA) and jasmonic acid (JA) and transcript levels of ABA- and JA-related genes and the C-REPEAT BINDING FACTOR (CBF) stress signaling pathway genes. A reduction in the R/FR ratio did not alter cold tolerance, ABA and JA accumulation, and transcript levels of ABA- and JA-related genes and the CBF pathway genes in phyA mutant plants; however, those were significantly increased in wild-type and phyB plants with the reduction in the R/FR ratio. Even though low R/FR treatments did not confer cold tolerance in ABA-deficient (notabilis [not]) and JA-deficient (prosystemin-mediated responses2 [spr2]) mutants, it up-regulated ABA accumulation and signaling in the spr2 mutant, with no effect on JA levels and signaling in the not mutant. Foliar application of ABA and JA further confirmed that JA functioned downstream of ABA to activate the CBF pathway in light quality-mediated cold tolerance. It is concluded that phyA and phyB function antagonistically to regulate cold tolerance that essentially involves FR light-induced activation of phyA to induce ABA signaling and, subsequently, JA signaling, leading to an activation of the CBF pathway and a cold response in tomato plants.

Figure 1.

R and FR function antagonistically in the regulation of cold tolerance. A, Plant survival rates, relative electrolyte leakage, and CBF1 transcript levels after exposure to R, FR, and dark conditions at 4°C. B, R and FR at 40 μmol m⁻² s⁻¹ induced changes in endogenous ABA and JA accumulation. The relative electrolyte leakage was determined after 7 d of cold stress; survival rates were measured by recovery at 25°C for 6 d after the chilling treatment (n = 4); each replicate had 16 plants, and CBF1 transcript levels were analyzed 6 h after chilling treatment. Data are presented as means of four biological replicates ± sd. Different letters indicate significant differences (P < 0.05) according to Tukey’s test. FW, Fresh weight.

Figure 2.

Silencing of the tomato CBF1 and CBF1/2/3 genes abolishes L-R/FR-induced cold tolerance. A and B, Changes in the maximum photochemical efficiency of PSII (F_v/F_m; A) and relative electrolyte leakage (B) in CBF-silenced plants after chilling at 4°C, as influenced by the R/FR ratio. The false color code depicted at the bottom of the image ranges from 0 (black) to 1 (purple). F_v/F_m and relative electrolyte leakage were determined 7 d after the chilling treatment. C, Western-blot detection of oxidized proteins in leaves after 3 d of cold stress under H-R/FR or L-R/FR conditions. The western-blot experiment was performed three times with three independent biological replicates with similar results. D, Leaf Pn recorded after plants had been exposed to 4°C under H-R/FR or L-R/FR conditions for 7 d, followed by recovery at 25°C for 1 d. The R/FR ratios for the H-R/FR and L-R/FR treatments were 72:28 and 33:67, respectively. Data are presented as means of four biological replicates ± sd except for F_v/F_m, which was the mean for 15 leaves from independent plants. Different letters indicate significant differences (P < 0.05) according to Tukey’s test.

Figure 3.

Dependency on phytochromes in light quality-regulated cold tolerance. A, Images of the F_v/F_m, chilling phenotypes, and survival rates in wild-type (WT) and phytochrome mutant plants after exposure to 4°C at R, FR, or dark conditions for 7 d. The false color code depicted at the bottom of the image ranges from 0 (black) to 1 (purple). Survival rates were measured by recovery at 25°C for 6 d after the chilling treatment (4°C for 7 d; n = 4), and each replicate had 16 plants. B, Relative electrolyte leakage and transcript levels of genes involved in the CBF pathway at 7 d and 6 h, respectively, after chilling treatment. C and D, ABA (C) and JA (D) levels at 12 h and transcript levels of ABA (C) and JA (D) biosynthesis- and signaling-related genes at 6 h after cold treatment at 4°C in plants grown under different light quality conditions. Data are presented as means of four biological replicates ± sd except for F_v/F_m, which was the mean for 15 leaves from independent plants. Different letters indicate significant differences (P < 0.05) according to Tukey’s test. FW, Fresh weight.

Figure 4.

Effects of R/FR ratio and the irradiance of FR on cold tolerance in wide-type (WT) and phyA and phyB1B2 mutant plants. A, Relative electrolyte leakage and transcript levels of CBF1 gene expression after 4°C treatment for 7 d and 6 h, respectively. B and C, ABA (B) and JA (C) levels at 12 h and transcript levels of ABA (B) and JA (C) biosynthesis-related genes (NCED6 [B] and LOXD [C]) at 6 h after cold treatment at 4°C in plants grown under different R/FR ratios. D, Relative electrolyte leakage of plants after growth under white light (W) or R conditions, supplied with different intensities of FR (0, 50, 100, and 200 μmol m⁻² s⁻¹), at 4°C for 7 d. The intensity of white light and R both are 100 μmol m⁻² s⁻¹. E, ABA and JA levels at 12 h after cold treatment at 4°C in plants grown under white light conditions with different intensities of FR (0, 50, 100, and 200 μmol m⁻² s⁻¹). F, Transcript levels of CBF1, NCED6, and LOXD at 6 h after cold treatment at 4°C in plants grown under white light conditions with different intensities of FR (0, 50, 100, and 200 μmol m⁻² s⁻¹). For A, B, and C, the light intensity was 120 μmol m⁻² s⁻¹. Data are presented as means of four biological replicates ± sd. Different letters indicate significant differences (P < 0.05) according to Tukey’s test. FW, Fresh weight.

Figure 5.

Cold tolerance is regulated by the R/FR ratio during the cold treatment but not by pretreatment. A, Images of the F_v/F_m and the accumulation of hydrogen peroxide (3,3′-diaminobenzidine [DAB] staining) and superoxide (nitroblue tetrazolium [NBT] staining) in tomato leaves. Plants were grown under white light (W), H-R/FR (H), or L-R/FR (L) at 25°C for 4 d as pretreatments before cold stress and then exposed to H-R/FR or L-R/FR at 25°C or 4°C for 7 d. The false color code depicted at the bottom of the image ranges from 0 (black) to 1 (purple). B, Relative electrolyte leakage and transcript levels of genes involved in the CBF pathway in tomato leaves at different R/FR ratio conditions after 25°C or 4°C treatment for 7 d and 6 h, respectively. C and D, ABA (C) and JA (D) production at 12 h and transcript levels of ABA (C) and JA (D) biosynthesis- and signaling-related genes at 6 h after 25°C or 4°C treatment under different R/FR regimes, respectively. The R/FR ratios for the H-R/FR, L-R/FR, and white light treatments were 72:28, 33:67, and 67:33, respectively. The arrows indicate transfer between growth light conditions. Data are presented as means of four biological replicates ± sd except for F_v/F_m, which was the mean for 15 leaves from independent plants. Different letters indicate significant differences (P < 0.05) according to Tukey’s test. FW, Fresh weight.

Figure 6.

Cold tolerance as influenced by ABA and JA biosynthesis under L-R/FR and H-R/FR conditions. A, Images of the F_v/F_m in the ABA-deficient mutant not and the JA-deficient mutant spr2 after exposure to 4°C under different R/FR ratios for 7 d. The false color code depicted at the bottom of the image ranges from 0 (black) to 1 (purple). B and C, Relative electrolyte leakage at 7 d after treatment. JA (B) and ABA (C) levels at 12 h after treatment and transcript levels of CBF-related genes and JA (B) and ABA (C) biosynthesis- and signaling-related genes at 6 h after chilling at 4°C at different R/FR ratios in wild-type (WT), spr2, and not plants. The R/FRs ratios for the H-R/FR and L-R/FR treatments were 72:28 and 33:67, respectively. Data are presented as means of four biological replicates ± sd except for F_v/F_m, which was the mean for 15 leaves from independent plants. Different letters indicate significant differences (P < 0.05) according to Tukey’s test. FW, Fresh weight.

Figure 7.

ABA acts upstream of JA in the cold response. A and B, Images of the F_v/F_m in the wild type (WT) and the ABA-deficient mutant not (A) or the JA-deficient mutant spr2 (B) as influenced by foliar application of MeJA or ABA. The false color code depicted at the bottom of the image ranges from 0 (black) to 1 (purple). C and D, Relative electrolyte leakage and transcript levels of CBF1 in wild-type and not (C) or spr2 (D) mutant plants as influenced by foliar application of MeJA or ABA. E and F, ABA (E) and JA (F) levels, ABA biosynthesis gene (NCED6; E), and JA biosynthesis gene (LOXD; F) relative expression following foliar application of MeJA or ABA in wild-type plants. Fifty micromolar MeJA or ABA was applied 12 h prior to exposure to cold conditions at 4°C. Samples for the determination of relative electrolyte leakage, gene transcript levels, and phytohormone contents were collected 3 d, 6 h, and 12 h after chilling at 4°C. Data are presented as means of four biological replicates ± sd except for F_v/F_m, which was the mean for 15 leaves from independent plants. Different letters indicate significant differences (P < 0.05) according to Tukey’s test. FW, Fresh weight.