An RNA thermoswitch regulates daytime growth in Arabidopsis

Abstract

Temperature is a major environmental cue affecting plant growth and development. Plants often experience higher temperatures in the context of a 24 h day–night cycle, with temperatures peaking in the middle of the day. Here, we find that the transcript encoding the bHLH transcription factor PIF7 undergoes a direct increase in translation in response to warmer temperature. Diurnal expression of PIF7 transcript gates this response, allowing PIF7 protein to quickly accumulate in response to warm daytime temperature. Enhanced PIF7 protein levels directly activate the thermomorphogenesis pathway by inducing the transcription of key genes such as the auxin biosynthetic gene YUCCA8, and are necessary for thermomorphogenesis to occur under warm cycling daytime temperatures. The temperature-dependent translational enhancement of PIF7 messenger RNA is mediated by the formation of an RNA hairpin within its 5′ untranslated region, which adopts an alternative conformation at higher temperature, leading to increased protein synthesis. We identified similar hairpin sequences that control translation in additional transcripts including WRKY22 and the key heat shock regulator HSFA2, suggesting that this is a conserved mechanism enabling plants to respond and adapt rapidly to high temperatures.

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Fig. 1: Arabidopsis responds rapidly to daytime warm temperature cycles and this is mirrored by changes in translational efficiency of genes such as PIF7 within 15 min.
Fig. 2: PIF7 is necessary for thermomorphogenesis in response to warm daytime temperature cycles.
Fig. 3: PIF7 directly activates the warm temperature transcriptome in response to daytime thermal cycles.
Fig. 4: Thermosensitive hairpin structures in the HSFA2 and PIF7 5′-UTRs enhance translation in response to warm temperature.

Data availability

Raw and processed data are available from ribo-seq/RNA-seq series E-MTAB-7717, RNA-seq series GSE124003 and ChIP–seq series GSE127745.

Code availability

Code is available from this Github repository: https://github.com/shouldsee/thermoPIF7.

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Acknowledgements

We thank C. Fankhauser for discussions of unpublished results. This work was supported by: Wellcome Trust grant no. 096082 and Medical Research Council grant no. MR/R021821/1 to B.Y.W.C.; EMBO long-term postdoctoral fellowship grant no. ALTF 1418-2015 to M.B.; Wellcome Trust grant no. 106207 to A.E.F.; Gates Foundation Studentship to K.F.; and BBSRC David Phillips Fellowship grant no. BB/R011605/1 to M.D.A. P.A.W.’s laboratory was supported by a Fellowship from the Gatsby Foundation grant no. GAT3273/GLB. P.A.W’s Department is supported by the Leibniz Association.

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Contributions

B.Y.W.C. and P.A.W. conceived the research. B.Y.W.C., M.B. and P.A.W. designed experiments and wrote the manuscript. B.Y.W.C. and M.B. performed most of the experiments. B.Y.W.C. performed ribosome profiling and RNA-seq. B.Y.W.C., M.B. and P.A.W. performed RNA structure analysis, and identified and characterized RNA thermometers. M.B. performed RNA-seq, phenotypic and molecular analyses. M.D.A. performed CD and FRET analysis. K.E.J. performed ChIP–seq. M.B. and P.M. generated tagged PIF7 transgenic plants. B.Y.W.C., K.F. and F.G. performed bioinformatics analysis. A.E.F., M.D.A. and I.B. commented on and revised the manuscript.

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Correspondence to Betty Y. W. Chung or Philip A. Wigge.

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Peer review information Nature Plants thanks Jorge Casal, Meng Chen, Ive De Smet 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 PIF7, HSFA2 and WRKY22 show enhanced translation at warm temperature.

a, Scatter plot of log fold changes in TE and mRNA abundance observed in Ribo-seq with parallel RNA-seq. bd, Histograms of 5′ end positions of normalized 28-nucleotide RPF reads (blue, green and red for frames 0, 1 and 2, left axis) and RNA-Seq reads (grey, right axis) mapped to the HSP70 (b), HSFA2 (c) and WRKY22 (d) transcript.

Extended Data Fig. 2 PIF7–MYC protein accumulation in response to warm temperature.

ad, Western blots of PIF7–MYC protein used for the quantification shown in Fig. 1m. The PIF7::PIF7–MYC line is in the Col-0 background. Actin levels are shown as loading control. e,f, Western blot (e) and quantification (f) of PIF7–MYC protein of an independent transgenic PIF7:: PIF7–MYC line in Col-0 background at ZT0 (dawn) and ZT12; seedlings were grown in LD at constant 17 °C or with a 27 °C midday for 7 d. Protein levels were normalized to actin. Bars represent the mean, error bars indicate the SEM (n = 3). The experiment was repeated once with similar results. gi, Western blots (g) and quantification (h) of PIF7–MYC protein as well as PIF7–MYC transcript levels (i) of PIF7::PIF7–MYC (Col-0) seedlings grown at constant 17 °C for 7 d and then either shifted to 27 °C at ZT4 (= 0 h) or kept at 17 °C for the indicated amount of time. Actin levels are shown as loading control. Protein levels were normalized to actin and expressed relative to levels at 0 h, transcript levels were normalized to PP2A and expressed relative to levels at 0 h. Data points represent the mean, error bars indicate the SEM (n = 3). The experiment was repeated once with similar results. j,k, Western blot of PIF7–MYC protein at ZT8 and ZT12 of PIF7::PIF7–MYC (Col-0) seedlings grown in LD at constant 17°C (j) or with a 27°C midday (k). Seedlings were treated with 100 µM cyclo- heximide (CHX), 50 µM MG132, a combination of the two or mock-treated at ZT4 on the day of sampling. Actin levels are shown as loading control. Two biological replicates are shown. The experiment was repeated once with similar results. The open arrow indicates an unspecific signal. Asterisks indicate significant differences to 17 °C control treatment (Two-sided Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001).

Extended Data Fig. 3 Additional thermomorphogenesis phenotypes in pif mutants.

ad, Hypocotyl length of 7-d-old Col-0 and pif mutant seedlings grown in LD at constant 17 °C, 22 °C and 27 °C (a) (n = 15), in SD at constant 17 °C, 22 °C and 27 °C (b) (n = 23, except for pif4 17 °C and 22 °C with n = 21 and pif7 27 °C with n = 22), in SD at constant 17 °C or with a daytime temperature at 27 °C (c) (n = 24 except for Col-0 27 °C with n= 19 and pif7 27 °C with n = 22) and in LD at constant 17 °C or with a warm midday of 27 °C (d) (n = 20 except for Col-0 17 °C and pif7 27 °C with n = 22), respectively. Seedlings were grown at 40 μmol m−2 s−1 in LD and 80 μmol m−2 s−1 in SD. e,f, Flowering time of Col-0 and pif mutant plants grown in LD at constant 17 °C or with a warm 37 °C midday (n = 12 except for pif4 pif7 with n = 11). Flowering time was scored as leaves at bolting (e) and days to bolting (f). gi, Hypocotyl length (g, h; n = 20–25) and stomatal index (SI) (i; n = 12) of 7-d-old and 14-d-old seedlings of two independent PIF7::PIF7–MYC complementation lines in the pif7-1 background, respectively. Seedlings were grown in LD at 17 °C with a warm midday of 27 °C. Box plots display the 25th and 75th percentile with the median as centre value and whiskers representing 1.5 times the IQR. Letters indicate significance groups; samples with the same letters are not significantly different (2-way ANOVA followed by two-sided Tukey test, p < 0.05). Asterisks indicate samples that are significantly different to Col-0 wild type (One-way ANOVA followed by two-sided Dunnett’s test, * p < 0.05, ** p < 0.01, *** p < 0.001). All experiments were repeated once with similar results.

Extended Data Fig. 4 The pif7 mutant lacks induction of a subset of temperature-responsive genes at 27 °C.

a,b, Average log fold change between expression at 27 °C and 17 °C for genes differentially expressed in pif7-1 (n = 1007) (a) and genes of cluster 7 identified in Fig. 3b (n = 293) (b). Box plots display the 25th and 75th percentile with the median as centre value and whiskers representing 1.5 times the IQR. Asterisks indicate significant differences (Two-sided Student’s t-test, * p < 0.05, ** p < 0.01, *** p < 0.001).

Extended Data Fig. 5 PIF7 affects auxin biosynthesis.

a,b, IGV browser view of PIF7–MYC binding at the YUC8 (a) and YUC9 (b) promoters. c,d, Relative expression of YUC8 (c) and YUC9 (d) observed in the RNA-seq experiment displayed in Fig. 3. Data are expressed relative to Col-0 27 °C at ZT8. e,f, Hypocotyl length of 7-d-old Col-0 or pif7-1 seedlings treated with 100 or 500 nM picloram (PIC) or mock-treated (n = 24). Box plots display the 25th and 75th percentile with the median as centre value and whiskers representing 1.5 times the IQR. Letters indicate significance groups; samples with the same letters are not significantly different (2-way ANOVA followed by two-sided Tukey test, p < 0.05). The experiment was repeated once with similar results.

Extended Data Fig. 6 PIF7 and PIF4 are likely to interact.

a, Venn diagram showing the overlap between PIF7–MYC and PIF4-HA ChIP–seq peaks. p-value was obtained by Fisher’s exact test for the independence of the two gene sets in comparison with the genomic background (n = 33554). b, IGV browser view of PIF7–MYC and PIF4-HA binding in the ATHB2 promoter. c, Yeast-2-hybrid assay testing interaction of PIF4 and PIF7 proteins expressed as fusions to a GAL4 binding domain (BD) or activation domain (AD). Empty vectors expressing BD and AD served as negative controls. The experiment was repeated once with similar results.

Extended Data Fig. 7 Hairpin structures in the 5′ UTR confer responsiveness to warm temperature.

a, mfe plot of the WRKY22 5′ UTR using a 40 nt sliding window. b, Predicted hairpin structure in the WRKY22 5′ UTR; mutated sequences used in in vitro studies are indicated in boxes. ce, In vitro translation of 5′ UTR hairpin::FLUC RNA fusions at different temperatures, using FLUC activity as read-out. Translation assays with WRKY22 (c) and 5′ -capped PIF7 (d) wild-type (WT), 3′ and 5′ disrupted (d3, d5), reconstituted (r) and stabilized (st) hairpin constructs as well as with PIF7 WT and mutated hairpin loop (mLoop) constructs (e) were performed. Data points represent the mean of two technical replicates. The experiments were repeated twice with similar results.

Extended Data Fig. 8 Mutations in the 5′ UTR hairpin affect PIF7–MYC protein accumulation.

Western blots of PIF7–MYC protein of independent PIF7::PIF7–MYC transgenic lines harbouring wild-type (WT), 3′ and 5′ disrupted (d3, d5), reconstituted (r) and stabilized (st) hairpin sequences. Seedlings were grown in LD at constant 17°C or with a 27°C midday. Actin levels are shown as loading control. Blots were used for quantifications shown in Fig. 4j–l and Extended Data Fig. 9b. The experiment was repeated once with similar results.

Extended Data Fig. 9 PIF7–MYC protein accumulation and hypocotyl elongation of transgenic PIF7::PIF7–MYC lines harbouring mutant hairpin sequences.

a, Quantification of PIF7–MYC protein at ZT12 in independent PIF7::PIF7–MYC transgenic lines har- bouring wild-type (WT), 3′ and 5′ disrupted (d3, d5), reconstituted (r) and stabilized (st) hairpin sequences. Seedlings were grown in LD at constant 17°C (left) or with a 27°C midday (right). Protein levels were normalized to actin and levels were expressed relative to the levels of the PIF7::PIF7–MYC (Col-0) line used in previous experiments to allow for comparisons across blots. Data points represent the mean, error bars indicate the SEM (n = 3). b, Hypocotyl length of the transgenic lines analysed in (a) (n = 25). Seedlings were grown in LD at constant 17°C (left) or with a 27°C midday (right) for 7 d. Box plots display the 25th and 75th percentile with the median as centre value and whiskers representing 1.5 times the IQR. The experiment was repeated once with similar results.

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Chung, B.Y.W., Balcerowicz, M., Di Antonio, M. et al. An RNA thermoswitch regulates daytime growth in Arabidopsis. Nat. Plants 6, 522–532 (2020). https://doi.org/10.1038/s41477-020-0633-3

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