ICE1 of Poncirus trifoliata functions in cold tolerance by modulating polyamine levels through interacting with arginine decarboxylase

Abstract

ICE1 (Inducer of CBF Expression 1) encodes a MYC-like basic helix-loop-helix transcription factor that acts as a central regulator of cold response. In this study, we elucidated the function and underlying mechanisms of PtrICE1 from trifoliate orange [Poncirus trifoliata (L.) Raf.]. PtrICE1 was upregulated by cold, dehydration, and salt, with the greatest induction under cold conditions. PtrICE1 was localized in the nucleus and could bind to a MYC-recognizing sequence. Ectopic expression of PtrICE1 in tobacco and lemon conferred enhanced tolerance to cold stresses at either chilling or freezing temperatures. Yeast two-hybrid screening revealed that 21 proteins belonged to the PtrICE1 interactome, in which PtADC (arginine decarboxylase) was confirmed as a bona fide protein interacting with PtrICE1. Transcript levels of ADC genes in the transgenic lines were slightly elevated under normal growth condition but substantially increased under cold conditions, consistent with changes in free polyamine levels. By contrast, accumulation of the reactive oxygen species, H2O2 and O2 (-), was appreciably alleviated in the transgenic lines under cold stress. Higher activities of antioxidant enzymes, such as superoxide dismutase and catalase, were detected in the transgenic lines under cold conditions. Taken together, these results demonstrated that PtrICE1 plays a positive role in cold tolerance, which may be due to modulation of polyamine levels through interacting with the ADC gene.

Keywords: Poncirus trifoliata (L.) Raf.; ROS; bHLH; cold tolerance; polyamine; protein-interacting protein; transcription factor..

Fig. 1.

Amino acid sequence alignment of PtrICE1 and ICE1 or ICE2 of other plants, comprising Populus trichocarpa (NCBI Protein no. ABN58427), PsICE1 of Populus suaveolens (ABF48720), RcICE1 of Ricinus communis (EEF51703), MdICE1 of apple (ABS50251), GmICE1 of soybean (ACJ39211), AtICE1 and AtICE2 of Arabidopsis thaliana (AAP14668 and BAC42644, respectively), and CbICE1 of Capsella bursa-pastoris (AAS79350). Identical and similar residues are shown in black and grey background, respectively. The single bold line below the sequence indicates the basic region, while double lines represent the helix regions, which are connected by a loop, indicated by the dotted line. The dashed line shows the leucine-zipper region. The asterisk and square indicate glutamate and arginine, respectively.

Fig. 2.

Nuclear localization of PtrICE1. Onion epidermis was transformed with vectors containing GFP (A), used as a control, or PtrICE1–GFP fusion protein (B). Subcellular localization was visualized by fluorescence microscopy. Representative images show cells expressing GFP or PtrICE1–GFP fusion protein under bright field (light) or UV field (dark), together with corresponding overlaid images.

Fig. 3.

Transcriptional activation assay of PtrICE1. (A) Growth of yeast cells (strain MaV203) transformed with either control vector (upper panels) or fusion vector harbouring PtrICE1 (bottom panels) on SD/–Leu/–Trp or SD/–Leu/–Trp/–His with or without 3-AT. (B) Schematic illustration of the vectors (pGADT7-PtrICE1 and pHIS2-MYCR) used for the transactivation assay. (C) Growth of yeast cells co-transformed with vectors of the positive control (P), negative control (N), and pGADT7-PtrICE1 with pHIS2-MYCR (1) on SD/–Leu/–Trp/–His with or without 15mM 3-AT.

Fig. 4.

Expression of PtrICE1 under abiotic stresses, analysed by qPCR. Time-course expression patterns of PtrICE1 in response to dehydration (A), cold (B), and salt stress (C). Error bars show the standard deviation based on four replicates.

Fig. 5.

Overexpression of PtrICE1 confers enhanced cold tolerance in tobacco. (A) Plant phenotype of tobacco WT and transgenic (TG22 and TG12) plants before and after cold treatment for 2 d at 4 °C. (B) Electrolyte leakage of WT and transgenic plants after cold treatment. (C) Plant phenotypes of WT and transgenic lines before and after cold treatment at 4 °C for 5 d. (D) Plant phenotypes of WT and transgenic lines before and after cold treatment at 0 °C for 1 d, followed by recovery growth for 5 d in an ambient environment. (F) Survival rates of WT and transgenic plants. (G) Plant phenotype of larger WT and transgenic plants before and after cold treatment at 4 °C for 1 d. Asterisks indicate that the transgenic lines and WT are significantly different from each other (***P<0.01). (This figure is available in colour at JXB online.)

Fig. 6.

Overexpression of PtrICE1 confers enhanced cold tolerance in lemon. (A) Plant phenotypes of lemon WT and transgenic lines (#17 and #21) before and after freezing treatment (–3 °C for 4h), followed by recovery growth for 5 d in an ambient environment. (B–D) Electrolyte leakage (B), cell death (C), and chlorophyll content (D) in WT and transgenic lines after freezing treatment. Asterisks indicate a significant difference between transgenic lines and WT (**P<0.01; ***P<0.001). Ca, chlorophyll a; Cb, chlorophyll b; Ct, total chlorophyll. (E) Tolerance assay of WT and transgenic lines subjected to freezing treatment (–6 °C for 1.5h), followed by 4 d recovery in an ambient environment. The upper panels are plants before treatment, while the bottom panels are plants after recovery. (This figure is available in colour at JXB online.)

Fig. 7.

Analysis of the interaction between PtrICE1 and PtADC by Y2H and BiFC assays. (A) Growth of yeast cells of the positive control, negative control, and co-transformants of PtrICE1 and PtADC on SD/–Leu/–Trp/–Ura or SD/–Leu/–Trp/–His added with or without 3-AT. The blue colour indicates X-gal activity on SD/–Leu/–Trp medium. (B) BiFC assay using tobacco leaf epidermis. Representative images of the epidermal cells under bright field and fluorescence microscopy are shown. Positive and negative controls were bZIP63–cYFP+bZIP–nYFP and PtrICE1–nYFP+cYFP, respectively.

Fig. 8.

Analysis of ADC expression and free polyamines in tobacco. (A, B) Expression patterns of NtADC1 (A) and NtADC2 (B) in tobacco WT and transgenic lines before and after cold stress. (C, D) Free polyamine contents in WT and transgenic lines before (C) and after (D) cold stress. Put, putrescine; Spd, spermidine; Spm, spermine. Asterisks indicate a significant difference between transgenic lines and the WT at the same time point (*P<0.05; **P<0.01; ***P<0.001).

Fig. 9.

Analysis of ADC expression and free polyamines in lemon. (A) Expression patterns of ClADC in lemon WT and transgenic lines before and after 5h of cold stress. (B) Free polyamine contents in WT and transgenic lines before and after cold stress. Put, putrescine; Spd, spermidine; Spm, spermine. Asterisks indicate a significant difference between transgenic lines and the WT at the same time point (*P<0.05; **P<0.01; ***P<0.001).

Fig. 10.

Analysis of H₂O₂ and O₂ ^– in tobacco and lemon under cold stress. (A, B) Representative images indicating in situ accumulation of H₂O₂ and O₂ ^– in tobacco WT and transgenic lines (TG22 and TG12) after cold stress at chilling (A) and freezing (B) temperatures. (C) Representative images indicating in situ accumulation of H₂O₂ and O₂ ^– in lemon WT and transgenic lines (#17 and #21) after cold stress. (D, E) Levels of H₂O₂ (D) and O₂ ^– (E) in tobacco WT and transgenic lines (TG22 and TG12) after cold treatment. The insets in (D) and (E) are histochemical staining patterns using DAB and NBT, respectively. (F, G) Levels of H₂O₂ (F) and O₂ ^– (G) in lemon WT and transgenic lines (#17 and #21) after cold stress at freezing temperatures. Asterisks indicate significant difference between transgenic lines and the WT (*P<0.05; **P<0.01; ***P<0.001).

Fig. 11.

Analysis of SOD and CAT activities in tobacco and lemon. (A, B) Activity of SOD (A) and CAT (B) in tobacco WT and transgenic lines (TG22 and TG12) before and after cold treatment. (C, D) Activity of SOD (C) and CAT (D) in lemon WT and transgenic lines (#17 and #21) before and after cold stress. Asterisks indicate a significant difference between transgenic lines and the WT (*P<0.05; **P<0.01).