Crosstalk of PIF4 and DELLA modulates CBF transcript and hormone homeostasis in cold response in tomato

Abstract

The ability to interpret daily and seasonal fluctuations, latitudinal and vegetation canopy variations in light and temperature signals is essential for plant survival. However, the precise molecular mechanisms transducing the signals from light and temperature perception to maintain plant growth and adaptation remain elusive. We show that far-red light induces PHYTOCHROME-INTERACTING TRANSCRIPTION 4 (SlPIF4) accumulation under low-temperature conditions via phytochrome A in Solanum lycopersicum (tomato). Reverse genetic approaches revealed that knocking out SlPIF4 increases cold susceptibility, while overexpressing SlPIF4 enhances cold tolerance in tomato plants. SlPIF4 not only directly binds to the promoters of the C-REPEAT BINDING FACTOR (SlCBF) genes and activates their expression but also regulates plant hormone biosynthesis and signals, including abscisic acid, jasmonate and gibberellin (GA), in response to low temperature. Moreover, SlPIF4 directly activates the SlDELLA gene (GA-INSENSITIVE 4, SlGAI4) under cold stress, and SlGAI4 positively regulates cold tolerance. Additionally, SlGAI4 represses accumulation of the SlPIF4 protein, thus forming multiple coherent feed-forward loops. Our results reveal that plants integrate light and temperature signals to better adapt to cold stress through shared hormone pathways and transcriptional regulators, which may provide a comprehensive understanding of plant growth and survival in a changing environment.

Keywords: GAI4; PIF4; Solanum lycopersicum (tomato); cold stress; hormone; light signalling.

Figure 1

SlPIF4 is regulated by both light and low temperature. (a) and (b) Expression of SlPIFs in tomato wild‐type (WT) plants (a) and REL in SlPIF‐silenced plants (b) grown at white light (120 µmol m⁻² s⁻¹) after exposure to 4 °C for 6 h and 7 days, respectively. (c) Expression of PHYA, PHYB1 and PHYB2 in tomato plants after exposure to 25 °C or 4 °C for 6 h. (d) Expression of SlPIF4 in tomato WT plants and phytochrome mutants (phyA, phyB1B2 and phyAB1B2) after exposure to 25 °C or 4 °C for 6 h under white light conditions (120 µmol m⁻² s⁻¹). (e) Expression of SlPIF4 in WT plants after exposure to 25 °C or 4 °C for 6 h, which grown under dark (D), white light (WL), red light (R) or FR light conditions. The light intensity is 120 µmol m⁻² s⁻¹. (f) Accumulation of SlPIF4 protein in tomato SlPIF4‐overexpressing (SlPIF4‐OE) plants after exposure to 25 °C or 4 °C for 12 h, which grown under D, WL, R or FR conditions. The light intensity is 120 µmol m⁻² s⁻¹. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey’s test.

Figure 2

SlPIF4 positively regulates cold tolerance in tomato and directly activates SlCBF1 expression. (a) Fv/Fm in tomato wild‐type (WT), pif4 mutant (pif4#3, pif4#10) and SlPIF4‐OE (OE#87, OE#89) plants after exposure to 4 °C under high R/FR (H‐R/FR, 2.5) light or low R/FR (L‐R/FR, 0.5) light for 7 days. The false‐colour code depicted at the bottom of the image ranges from 0 (black) to 1.0 (purple), representing the level of damage in the leaves. (b) and (c) REL (b) and SlCBF1 gene expression (c) in tomato WT, pif4 mutant (pif4#3, pif4#10) and SlPIF4‐OE (OE#87, OE#89) plants after exposure to 25 °C or 4 °C under H‐R/FR or L‐R/FR conditions for 7 days and 6 h, respectively. (d) and (e) G‐, E‐ and PBE‐box elements in the promoter of tomato SlCBF1 gene (d) and oligonucleotide used in the EMSA (e). Numbering is from predicted transcriptional start sites. The G‐box core sequence was mutated in the CBF1‐G2‐mut probe. The wt and mutated G‐box sequences are underlined. The mutated bases were indicated in red. (f) EMSA. The His‐SlPIF4 recombinant protein was incubated with biotin‐labelled wild‐type (CBF1‐G2‐wt) or mutant (CBF1‐G2‐mut) oligos. The protein purified from the empty vector was used as a negative control. (g) ChIP‐qPCR assay. WT and 35S:SlPIF4‐HA tomato plants were grown at 4 °C under L‐R/FR light for 6 h, and samples were precipitated with an anti‐HA antibody. A control reaction was processed simultaneously using mouse IgG. The ChIP results are presented as percentages of the input DNA. For light‐quality treatments, plants were maintained at R conditions (120 µmol m⁻² s⁻¹) and supplemented with different intensities of FR. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey’s test.

Figure 3

SlPIF4 promotes the gene expression and endogenous levels of ABA and JA biosynthesis in response to cold stress. (a) and (c) Expression of NCED6 (a) and AOS2 (c) in tomato WT, pif4 mutant and SlPIF4‐OE plants after exposure to 25 °C or 4 °C under low R/FR (L‐R/FR, 0.5) light conditions for 6 h. (b) and (d) Endogenous levels of ABA (b) and JA (d) biosynthesis in tomato WT, pif4 mutant and SlPIF4‐OE plants after exposure to 25 °C or 4 °C under L‐R/FR light for 12 h. For light‐quality treatments, plants were maintained at R conditions (120 µmol m⁻² s⁻¹) and supplemented with different intensities of FR. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey’s test.

Figure 4

SlPIF4 negatively regulates expression of GA biosynthesis genes and endogenous levels of GA under cold stress. (a) and (b) Expression of GA3ox2 (a) and GA20ox1 (b) in tomato WT, pif4 mutant and SlPIF4‐OE plants after exposure to 25 °C or 4 °C under low R/FR (L‐R/FR, 0.5) light conditions for 6 h. (c) to (h) Levels of active GAs (GA₁, GA₃ and GA₄; d, f and h), their precursors (GA₉, GA₁₉ and GA₂₀; c, e and g) in tomato WT, pif4 mutant and SlPIF4‐OE plants after exposure to 25 °C or 4 °C under L‐R/FR light for 12 h. For light‐quality treatments, plants were maintained at R conditions (120 µmol m⁻² s⁻¹) and supplemented with different intensities of FR. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey’s test.

Figure 5

SlPIF4 directly binds to the promoter of SlGAI4 and activates its expression under cold stress in tomato. (a) The REL in tomato wild‐type (pTRV) and SlGAI‐silenced (pTRV‐GAI1, pTRV‐GAI2, pTRV‐GAI3, pTRV‐GAI4, pTRV‐GAI5, pTRV‐GAI6, pTRV‐GAI7, pTRV‐GAI8, pTRV‐GAI9, pTRV‐GAI10) plants after exposure to 4 °C for 7 days. (b) Expression of SlGAI4 in tomato WT, pif4 mutant (pif4#3, pif4#10) and SlPIF4‐OE (OE#87, OE#89) plants after exposure to 25 °C or 4 °C under high R/FR (H‐R/FR, 2.5) light or low R/FR (L‐R/FR, 0.5) light conditions for 6 h. (c) EMSA. G‐, E‐ and PBE‐box elements in the promoter of tomato SlGAI4 gene and oligonucleotide used in the EMSA. Numbering is from predicted transcriptional start sites. The G‐box core sequence was mutated in the GAI4‐G1/2‐mut probe. The wt and mutated G‐box sequences are underlined. The mutated bases were indicated in red. The His‐SlPIF4 recombinant protein was incubated with biotin‐labelled wild‐type (GAI4‐G1/2‐wt) or mutant (GAI4‐G1/2‐mut) oligos. The protein purified from the empty vector was used as a negative control. (d) Dual‐LUC assay showing the effects of SlPIF4 on SlGAI4 promoter activation under cold stress. The SlGAI4 promoter was fused to the luciferase (LUC) reporter (pGAI4::LUC), and promoter activity was determined by transient expression of it with empty vector (EV) or 35S:SlPIF4 (PIF4) in tobacco. The tobacco plants were exposed to 25 °C or 4 °C for 24 h after infiltration at 25 °C for 24 h. Relative LUC activity was normalized to the Renilla (REN) luciferase. (e) ChIP‐qPCR assay. WT and 35S:SlPIF4‐HA tomato plants were grown at 4 °C under L‐R/FR light for 6 h, and samples were precipitated with an anti‐HA antibody. A control reaction was processed simultaneously using mouse IgG. The ChIP results are presented as percentages of the input DNA. For light‐quality treatments, plants were maintained at R conditions (120 µmol m⁻² s⁻¹) and supplemented with different intensities of FR. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey’s test.

Figure 6

SlGAI4 is a positive regulator in L‐R/FR‐induced cold tolerance in tomato. (a) The REL in tomato wild‐type (pTRV) and SlGAI4‐silenced plants (pTRV‐GAI4) after exposure to 25 °C or 4 °C under high R/FR (H‐R/FR, 2.5) light or low R/FR (L‐R/FR, 0.5) light conditions for 7 days. (b) Fv/Fm in tomato pTRV and pTRV‐GAI4 plants after exposure to 4 °C under H‐R/FR or L‐R/FR conditions for 7 days. The false‐colour code depicted at the bottom of the image ranges from 0 (black) to 1.0 (purple), representing the level of damage in the leaves. (c) Expression of SlCBF1 in tomato pTRV and pTRV‐GAI4 plants after exposure to 25 °C or 4 °C under H‐R/FR or L‐R/FR conditions for 6 h. (d) The REL in tomato wild‐type (WT) and SlGAI4‐overexpressing plants (OE#54, OE#56) after exposure to 25 °C or 4 °C under H‐R/FR or L‐R/FR conditions for 7 days. (e) Fv/Fm in tomato WT and SlGAI4‐overexpressing plants (OE#54, OE#56) plants after exposure to 4 °C under H‐R/FR or L‐R/FR conditions for 7 days. (f) Expression of SlCBF1 in tomato WT and SlGAI4‐overexpressing plants (OE#54, OE#56) plants after exposure to 25 °C or 4 °C under H‐R/FR or L‐R/FR conditions for 6 h. For light‐quality treatments, plants were maintained at R conditions (120 µmol m⁻² s⁻¹) and supplemented with different intensities of FR. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey’s test.

Figure 7

SlGAI4 promotes expression of genes in ABA and JA pathway and their accumulation in response to cold stress. (a) and (c) Expression of NCED6 (a) and LOXD (c) in tomato wild‐type (WT‐pTRV), SlGAI4‐silenced plants (WT‐pTRV‐GAI4) and SlGAI4‐overexpressing plants (OE‐GAI4‐pTRV) after exposure to 25 °C or 4 °C under low R/FR (L‐R/FR, 0.5) light conditions for 6 h. (b) and (d) Endogenous levels of ABA (b) and JA (d) biosynthesis in tomato wild‐type (WT‐pTRV), SlGAI4‐silenced plants (WT‐pTRV‐GAI4) and SlGAI4‐overexpressing plants (OE‐GAI4‐pTRV) after exposure to 25 °C or 4 °C under L‐R/FR light for 12 h. For light‐quality treatments, plants were maintained at R conditions (120 µmol m⁻² s⁻¹) and supplemented with different intensities of FR. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey’s test.

Figure 8

The effects of GA₃ and PAC on cold tolerance in tomato WT, pif4 Mutant, SlPIF4‐OE and not mutant plants. (a) Fv/Fm in tomato wild‐type (WT), pif4 mutant (pif4) and SlPIF4‐overexpressing plants (SlPIF4‐OE) after exposure to 4 °C under low R/FR (L‐R/FR, 0.5) light conditions for 7 days, which pretreated with water (H₂O), GA₃ (50 μm) or PAC (GA biosynthesis inhibitor, 25 μm) for 12 h prior to exposure to cold conditions at 4 °C. The false‐colour code depicted at the bottom of the image ranges from 0 (black) to 1.0 (purple), representing the level of damage in the leaves. (b) and (c) REL (b) and SlCBF1 gene expression (c) in tomato WT, pif4 and SlPIF4‐OE plants after exposure to 25 °C or 4 °C under L‐R/FR light for 7 days and 6 h, respectively, which pretreated with H₂O, GA₃ or PAC for 12 h prior to exposure to cold conditions at 4 °C. (d) Fv/Fm in tomato wild‐type (WT) and ABA‐deficient mutant (not) plants after exposure to 4 °C under L‐R/FR light for 7 days, which pretreated with water (H₂O), GA₃ or PAC for 12 h prior to exposure to cold conditions at 4 °C. (e) and (f) REL (e) and SlCBF1 gene expression (f) in tomato WT and not plants after exposure to 25 °C or 4 °C under L‐R/FR light for 7 days and 6 h, respectively, which pretreated with H₂O, GA₃ or PAC for 12 h prior to exposure to cold conditions at 4 °C. For light‐quality treatments, plants were maintained at R conditions (120 µmol m⁻² s⁻¹) and supplemented with different intensities of FR. Data are presented as the means of three biological replicates (±SD). Different letters indicate significant differences (P < 0.05) according to Tukey’s test.

Figure 9

DELLAs negatively regulate SlPIF4 protein abundance at low‐temperature condition. (a) Levels of SlPIF4‐HA proteins in 35S:PIF43‐HA tomato plants grown at 25 °C or 4 °C for 24 h. 35S:PIF43‐HA seedlings were pretreated with water (H₂O), GA₃ (50 μm) or PAC (GA biosynthesis inhibitor, 25 μm) for 12 h before exposure to cold stress. (b) Levels of SlPIF4 proteins in tomato wild‐type (pTRV) and SlGAI4‐silenced plants (pTRV‐GAI4) after exposure to 4 °C under high R/FR (H‐R/FR, 2.5) light or low R/FR (L‐R/FR, 0.5) light conditions for 24 h. (c) Levels of SlPIF4 proteins in tomato wild‐type (WT) and SlGAI4‐overexpressing plants (OE#54, OE#56) after exposure to 4 °C under H‐R/FR or L‐R/FR conditions for 24 h. For light‐quality treatments, plants were maintained at R conditions (120 µmol m⁻² s⁻¹) and supplemented with different intensities of FR. Rubisco was used as a loading control.

Figure 10

A proposed model for SlPIF4 positively regulating tomato cold tolerance by integrating light and temperature signals. Briefly, L‐R/FR and low temperature induce SlPIF4 protein accumulation via a phyA‐dependent pathway under cold stress. SlPIF4 not only directly activates CBF expression but also associates with the promoter of the SlGAI4 gene and activates its transcription, promoting ABA and JA biosynthesis and CBF expression. Thus, SlPIF4 is a positive regulator in L‐R/FR‐induced cold tolerance in tomato. SlGAI4, a DELLA protein, acts downstream of SlPIF4 and positively regulates L‐R/FR‐induced cold tolerance. Interestingly, when large amounts of SlGAI4 protein accumulate during cold stress, it represses SlPIF4 accumulation in a negative feedback manner.