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Redox-dependent structural switch and CBF activation confer freezing tolerance in plants

An Author Correction to this article was published on 14 November 2022

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Abstract

The activities of cold-responsive C-repeat-binding transcription factors (CBFs) are tightly controlled as they not only induce cold tolerance but also regulate normal plant growth under temperate conditions1,2,3,4. Thioredoxin h2 (Trx-h2)—a cytosolic redox protein identified as an interacting partner of CBF1—is normally anchored to cytoplasmic endomembranes through myristoylation at the second glycine residue5,6. However, after exposure to cold conditions, the demyristoylated Trx-h2 is translocated to the nucleus, where it reduces the oxidized (inactive) CBF oligomers and monomers. The reduced (active) monomers activate cold-regulated gene expression. Thus, in contrast to the Arabidopsis trx-h2 (AT5G39950) null mutant, Trx-h2 overexpression lines are highly cold tolerant. Our findings reveal the mechanism by which cold-mediated redox changes induce the structural switching and functional activation of CBFs, therefore conferring plant cold tolerance.

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Fig. 1: Cold-mediated demyristoylation of cytoplasmic Trx-h2 enables its translocation to the nucleus and subsequent interaction with and dissociation of CBF oligomers.
Fig. 2: Trx-h2-mediated reduction and monomerization of CBF1 in vitro and in vivo under cold stress.
Fig. 3: Trx-h2-dependent binding of CBF1 to the COR15a promoter, and expression of COR genes under cold stress.
Fig. 4: Trx-h2 enhances the freezing tolerance of Arabidopsis mainly through CBF signalling.

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The datasets generated during and/or analysed during the current study are not publicly available for privacy reasons, but are available from the corresponding author on reasonable request. Source data are provided with this paper.

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Acknowledgements

We thank S. Yang for providing the seeds of transgenic CBF1-MycOE and PCBF1:CBF1-Myc Arabidopsis lines, and J. K. Zhu for providing the seeds of cbfs triple-mutant lines. This work was supported by the grants the BioGreen21 Agri-Tech Innovation Program (grant no. PJ015824, to S.Y.L.; and PJ015674, to S.K.P), RDA, Korea and by the Basic Science Research Program through the National Research Foundation (NRF) of Korea funded by the Ministry of Education (NRF-2018R1A6A3A11048274, to E.S.L; and NRF-2019R1I1A1A01040920, to J.H.P.).

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Authors and Affiliations

Authors

Contributions

E.S.L., J.H.P., S.D.W. and S.Y.L. conceptualized this study. H.B.C., C.H.K., Y.H.C., S.K.P. and M.G.K. developed the methodology. E.S.L., S.D.W., J.H.P., H.B.C., Y.H.C, M.G.J., D.-J.Y. and W.-Y.K. performed the experiments and analysed the results. E.S.L. and S.Y.L. wrote the original draft. G.S. and S.Y.L. reviewed and edited the manuscript.

Corresponding author

Correspondence to Sang Yeol Lee.

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The authors declare no competing interests.

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Peer review information Nature Plants thanks Shuhua Yang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Generation of transgenic Arabidopsis lines expressing various forms of CBF1, Trx-h2, and their mutants in trx-h2 and cbfs backgrounds.

a, A schematic diagram of CBFs genomic structure in Col-0 Arabidopsis and the DNA construct for generating cbfs null-mutant using the CRISPR-Cas9 system. b, DNA constructs used to overexpress CBF1-Myc under the control the CaMV 35S promoter (CBF1-MycOE) and to express CBF1-Myc under the control of its native promoter (PCBF1) (PCBF1:CBF1-Myc) in Col-0 and trx-h2 backgrounds; both constructs utilized the nopaline synthase (NOS) terminator. c,d, Expression analysis of CBF1 and Trx-h2 in the overexpression lines (CBF1-MycOE/Col-0 or CBF1-MycOE/trx-h2) (c) and transgenic lines (PCBF1:CBF1-Myc/Col-0 and PCBF1:CBF1-Myc/trx-h2) (d) by RT-PCR. e, Genomic structure of the Arabidopsis T-DNA insertion knockout mutant, trx-h2 (SALK_079507). The T-DNA insertion site and forward (For)/reverse (Rev) primer-binding sites are indicated. ATG and TAA represent the start and stop codons, respectively. f, Confirmation of Trx-h2 knockout in trx-h2 plant by PCR-based genotyping. g, Schematic diagrams of DNA constructs used for the overexpression of V5-tag fusions of Trx-h2, Trx-h2(G/A), and Trx-h2(C/S) under the control of the CaMV 35S promoter and octopine synthase (OCS) terminator. Agrobacterium tumefaciens strain GV3101 carrying each construct was transformed into the trx-h2 mutant to generate Trx-h2-V5OE/trx-h2, Trx-h2(G/A)-V5OE/trx-h2, and Trx-h2(C/S)-V5OE/trx-h2 overexpression lines. h,i, Expression levels of Trx-h2 mRNA (h) and Trx-h2 protein (i) in various transgenic Arabidopsis analyzed by RT-PCR and western blotting. To quantitatively compare the expression levels of Trx-h2 mRNA and the corresponding protein among various genotypes, two different blots with short (middle panel) and long (upper panel) exposure times in the ChemiDoc™ MP System were shown. jl, A schematic diagram of DNA construct for generating Trx-h2-HAOE/cbfs lines (j). Expression levels of Trx-h2 mRNA (k) and Trx-h2 protein (l) in Trx-h2-HAOE/cbfs (lines #1 and #2) plants were analyzed by RT-PCR and western blotting, respectively. In (h–l), Tubulin and Rbc L were used as loading controls.

Source data

Extended Data Fig. 2 Amino acid sequence characteristics of Trx-h2 and its specific interaction with CBF1 at low temperature.

a, Comparison of N-terminal amino acid sequences (~80 residues) of 11 cytoplasmic Trx-hs in Arabidopsis, and their putative acylation sites predicted by the TermiNator program. Based on the modification pattern of fatty acids, yellow, magenta, green, and cyan boxes on the left represent subgroup I, Sub-II, Sub-III, and Sub-IV Trx-hs, respectively. Critical amino acid residues required for the myristoylation, N-α-acetylation, and palmitoylation of Trx-hs are outlined by a red box (Gly2 of Sub-II), blue box (Ala2 of Sub-I), and green box (Cys5 of Sub-III & IV), respectively. The active site Cys residues (CXXC motif) are outlined by a maroon box. b, Schematic representation of Trx-h2 and its point mutation variants, Trx-h2(G/A) and Trx-h2(C/S). c, Sequence features of Trx-h2. Active site Cys residues of Trx-h2 (at amino acid positions 59 and 62) are indicated in bold blue font, and the Gly2 residue required for myristoylation (G2) is indicated in green. The conserved Trx motif (122 amino acids) is indicated in red. The bipartite nuclear localization signal (NLS) sequence identified from the NLS-mapper program18 is underlined. The asterisk at the end of the protein sequence indicates the stop codon. d, Interacted specificity of CBF1 with Trx-h2, but not with Trx-h3 (control), analyzed by BiFC at 4 °C. Scale bars = 20 μm.

Extended Data Fig. 3 Analysis of anti-Trx-h2 antibody specificity, Trx-h2 gene expression and Trx-h2 protein abundance in Col-0 plants at warm and low temperatures.

a, Specificity of anti-Trx-h2 antibody prepared in our laboratory. Total protein extracts of Col-0, trx-h2, and Trx-h2-V5OE/trx-h2 plants were separated by SDS-PAGE on a 12% polyacrylamide gel and subjected to western blotting using anti-Trx-h2 antibody. Rbc L stained with Ponceau S was used as loading controls. b, Expression level of Trx-h2 gene transcripts analyzed by qRT-PCR at 22 °C and 4 °C. Data are expressed as mean ± s.e.m (n = 3 biologically independent samples). c,d, Expression level of Trx-h2 protein analyzed by western blot in Col-0 plants at 22 °C (c) and 4 °C (d).

Source data

Extended Data Fig. 4 Protocol used for the detection of myristoylated Trx-h2 in Arabidopsis.

Azidomyristate was vacuum-infiltrated into 2-week-old Trx-h2-V5OE/trx-h2 and Trx-h2(G/A)-V5OE/trx-h2 plants incubated at 22 °C for 24 h. Total proteins extracted from these plants were incubated with phosphine-PEG3-biotin. The biotinyl-myristoylated Trx-h2-V5 was immunoprecipitated with anti-V5 antibody and separated by SDS-PAGE on a reducing gel. Then, the biotinyl-myristoylated Trx-h2-V5 was detected by western blot analysis using anti-biotin antibody. The protocol was established by modifying the method used for the detection of myristoyl proteins in cell cultures21.

Extended Data Fig. 5 Subcellular localization of Trx-h2 in Arabidopsis protoplasts at warm temperature.

a, A schematic diagram of the construct used to express Trx-h2-YFP under the control of the CaMV 35S promoter in Arabidopsis protoplasts. b, Subcellular location of Trx-h2 in Arabidopsis protoplasts at warm temperature (22 °C). YFP signal was detected by confocal microscopy. Other fluorescent makers including mCherry-HDEL, soybean α-1,2-mannosidase 1-RFP, and NLS-RFP were used to label the endoplasmic reticulum (ER), Golgi complex, and nucleus, respectively, in the second lane (RFP). The panel labeled ‘Merge’ represents overlapped images of YFP and RFP signals. Bright field images are presented in the panel labeled ‘Bright’. Scale bars = 20 μm.

Extended Data Fig. 6 Amino acid sequence characteristics of CBFs containing five conserved Cys residues and the effect of Trx-h2 or Trx-h2(G/A) on CBFs expression and their protein structures at different temperatures.

a, Alignment of the amino acid sequences of CBF1–3 in Arabidopsis. Five conserved Cys residues (at amino acid positions 23, 30, 100, 117, and 137) are outlined in red boxes. Numbers on the right hand side indicate amino acid positions. b,c, Domain structures of CBF1 (b) and CBF1(C/S) (c); in CBF1(C/S), five conserved Cys residues were replaced by Ser residues. Red box at the N-terminus indicates the nuclear localization signal (NLS); green box indicates the AP2 domain; blue box indicates the activation domain. Numbers listed below indicate the amino acid positions. d, Expression analysis of CBF proteins and their structures in trx-h2 mutant Arabidopsis at 4 °C by western blotting on reducing (lower panel) and non-reducing (upper panel) SDS-PAGE gels. Rbc L stained with Ponceau S was used as loading controls. e, Effect of Trx-h2(G/A) on CBFs protein structures in various Arabidopsis genotypes at warm temperature (22 °C) analyzed by western blotting. d,e, ‘O’ and ‘M’ indicate CBF oligomers and monomers, respectively.

Extended Data Fig. 7 Effect of Trx-h2 on the binding of CBF1 to the COR15a promoter and on the expression of CBF mRNAs and proteins at 4 °C.

a, Nucleotide sequence of the COR15a promoter containing two CRT/DRE core motifs at nucleotide positions -439 to -444 (blue box) and -267 to -262 (green box) upstream of the transcription start site (+1 bp). b, Schematic representation of the COR15a promoter containing two CRT/DRE cis-elements indicated as blue and green boxes. c, Oligonucleotide sequence of biotin-labeled EMSA probe (-275 to -253 bp). d, Schematic representation of effector, internal control, and reporter constructs used in the luciferase (LUC) assay. e, Comparison of LUC activity measured in Nicotiana benthamiana leaves expressing P35S:CBF1 under oxidizing (X/XO) or reducing (GSH) conditions at 22 °C. f, Comparison of LUC activity in N. benthamiana leaves expressing P35S:Trx-h2 or P35S:Trx-h2(C/S) incubated at 22 °C or 4 °C. g, Analysis of CBF1–3 transcript levels in Col-0, trx-h2, and trx-h3 (control) plants incubated at 4 °C by qRT-PCR. Data are expressed as mean ± s.e.m (n = 3 biologically independent samples). Significant differences between means are indicated by asterisks (Unpaired two-tailed Student’s t test P values: *P < 0.05, **P < 0.01, ***P < 0.001). NS indicates not significant. h, Expression analysis of CBF proteins by western blot on reducing SDS-PAGE gels in Col-0, trx-h2, and cbfs Arabidopsis during the cold treatment at 4 °C. Rbc L stained with Ponceau S was used as loading controls.

Source data

Extended Data Fig. 8 Trx-h2 enhances the freezing tolerance of Arabidopsis plants grown in soil through CBF signaling.

ac, Comparison of freezing tolerance among plants of various Arabidopsis genotypes based on their recovery (a), survival rate (b), and electrolyte leakage (%) (c) after the freezing test. To prepare non-acclimated (NA) and cold-acclimated (CA) plants, 18-day-old plants grown in soil at 22 °C were placed in a freezing chamber cooled from 0 °C to the desired temperature at a rate of 2 °C decline per 30 min using a gradient cooling system. The desired temperature was held for 1 h. Then, plants were incubated at 4 °C for 12 h. The CA plants were pre-incubated at 4 °C for 5 days before the freezing test. Genetic relationship between Trx-h2 and CBFs in cold signaling was analyzed using cbfs mutant and Trx-h2-HAOE/cbfs (#1 and #2) plants. Data are expressed as mean ± s.e.m (n = 5 biologically independent samples). Significant differences between means are indicated by asterisks (Unpaired two-tailed Student’s t test P values: *P < 0.05, **P < 0.01, ***P < 0.001).

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Source Data Extended Data Fig. 1

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Lee, E.S., Park, J.H., Wi, S.D. et al. Redox-dependent structural switch and CBF activation confer freezing tolerance in plants. Nat. Plants 7, 914–922 (2021). https://doi.org/10.1038/s41477-021-00944-8

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