Temperature is a key factor in the growth and development of all organisms1,2. Plants have to interpret temperature fluctuations, over hourly to monthly timescales, to align their growth and development with the seasons. Much is known about how plants respond to acute thermal stresses3,4, but the mechanisms that integrate long-term temperature exposure remain unknown. The slow, winter-long upregulation of VERNALIZATION INSENSITIVE 3 (VIN3)5,6,7, a PHD protein that functions with Polycomb repressive complex 2 to epigenetically silence FLOWERING LOCUS C (FLC) during vernalization, is central to plants interpreting winter progression5,6,8,9,10,11. Here, by a forward genetic screen, we identify two dominant mutations of the transcription factor NTL8 that constitutively activate VIN3 expression and alter the slow VIN3 cold induction profile. In the wild type, the NTL8 protein accumulates slowly in the cold, and directly upregulates VIN3 transcription. Through combining computational simulation and experimental validation, we show that a major contributor to this slow accumulation is reduced NTL8 dilution due to slow growth at low temperatures. Temperature-dependent growth is thus exploited through protein dilution to provide the long-term thermosensory information for VIN3 upregulation. Indirect mechanisms involving temperature-dependent growth, in addition to direct thermosensing, may be widely relevant in long-term biological sensing of naturally fluctuating temperatures.
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Code is available in Supplementary Data and at https://github.com/ReaAntKour/NTL8TemperatureGrowth/.
Garrity, P. A., Goodman, M. B., Samuel, A. D. & Sengupta, P. Running hot and cold: behavioral strategies, neural circuits, and the molecular machinery for thermotaxis in C. elegans and Drosophila. Genes Dev. 24, 2365–2382 (2010).
McClung, C. R. & Davis, S. J. Ambient thermometers in plants: from physiological outputs towards mechanisms of thermal sensing. Curr. Biol. 20, R1086–R1092 (2010).
Ding, Y., Shi, Y. & Yang, S. Advances and challenges in uncovering cold tolerance regulatory mechanisms in plants. New Phytol. 222, 1690–1704 (2019).
Zhang, J., Li, X.-M., Lin, H.-X. & Chong, K. Crop improvement through temperature resilience. Annu. Rev. Plant Biol. 70, 753–780 (2019).
De Lucia, F., Crevillen, P., Jones, A. M., Greb, T. & Dean, C. A PHD-Polycomb repressive complex 2 triggers the epigenetic silencing of FLC during vernalization. Proc. Natl Acad. Sci. USA 105, 16831–16836 (2008).
Sung, S. & Amasino, R. M. Vernalization in Arabidopsis thaliana is mediated by the PHD finger protein VIN3. Nature 427, 159–164 (2004).
Wood, C. C. et al. The Arabidopsis thaliana vernalization response requires a Polycomb-like protein complex that also includes VERNALIZATION INSENSITIVE 3. Proc. Natl Acad. Sci. USA 103, 14631–14636 (2006).
Michaels, S. D. & Amasino, R. M. FLOWERING LOCUS C encodes a novel MADS domain protein that acts as a repressor of flowering. Plant Cell 11, 949–956 (1999).
Sheldon, C. C. et al. The FLF MADS box gene: a repressor of flowering in Arabidopsis regulated by vernalization and methylation. Plant Cell 11, 445–458 (1999).
Hepworth, J. et al. Absence of warmth permits epigenetic memory of winter in Arabidopsis. Nat. Commun. 9, 639 (2018).
Antoniou-Kourounioti, R. L. et al. Temperature sensing is distributed throughout the regulatory network that controls FLC epigenetic silencing in vernalization. Cell Syst. 7, 643–655.e9 (2018).
Kim, S. G., Kim, S. Y. & Park, C. M. A membrane-associated NAC transcription factor regulates salt-responsive flowering via FLOWERING LOCUS T in Arabidopsis. Planta 226, 647–654 (2007).
Kim, S. G., Lee, A. K., Yoon, H. K. & Park, C. M. A membrane-bound NAC transcription factor NTL8 regulates gibberellic acid-mediated salt signaling in Arabidopsis seed germination. Plant J. 55, 77–88 (2008).
Tian, H. et al. NTL8 regulates trichome formation in Arabidopsis by directly activating R3 MYB genes TRY and TCL1. Plant Physiol. 174, 2363–2375 (2017).
Yang, Z. T. et al. The membrane-associated transcription factor NAC089 controls ER-stress-induced programmed cell death in plants. PLoS Genet. 10, e1004243 (2014).
Li, P. et al. Fructose sensitivity is suppressed in Arabidopsis by the transcription factor ANAC089 lacking the membrane-bound domain. Proc. Natl Acad. Sci. USA 108, 3436–3441 (2011).
Klein, P., Seidel, T., Stocker, B. & Dietz, K. J. The membrane-tethered transcription factor ANAC089 serves as redox-dependent suppressor of stromal ascorbate peroxidase gene expression. Front. Plant Sci. 3, 247 (2012).
O'Malley, R. C. et al. Cistrome and epicistrome features shape the regulatory DNA landscape. Cell 165, 1280–1292 (2016).
Liang, M. et al. Subcellular distribution of NTL transcription factors in Arabidopsis thaliana. Traffic 16, 1062–1074 (2015).
Kim, S. Y. et al. Exploring membrane-associated NAC transcription factors in Arabidopsis: implications for membrane biology in genome regulation. Nucleic Acids Res. 35, 203–213 (2007).
Meng, X. et al. ANAC017 coordinates organellar functions and stress responses by reprogramming retrograde signaling. Plant Physiol. 180, 634–653 (2019).
Ng, D. W. K., Abeysinghe, J. K. & Kamali, M. Regulating the regulators: the control of transcription factors in plant defense signaling. Int. J. Mol. Sci. 19, 3737 (2018).
Li, L. et al. Protein degradation rate in Arabidopsis thaliana leaf growth and development. Plant Cell 29, 207–228 (2017).
Band, L. R. et al. Growth-induced hormone dilution can explain the dynamics of plant root cell elongation. Proc. Natl Acad. Sci. USA 109, 7577–7582 (2012).
Erickson, R. O. & Michelini, F. J. The plastochron index. Am. J. Bot. 44, 297–305 (1957).
Merchante, C., Stepanova, A. N. & Alonso, J. M. Translation regulation in plants: an interesting past, an exciting present and a promising future. Plant J. 90, 628–653 (2017).
Zhang, T.-Q., Xu, Z.-G., Shang, G.-D. & Wang, J.-W. A single-cell RNA sequencing profiles the developmental landscape of Arabidopsis root. Mol. Plant 12, 648–660 (2019).
Mahonen, A. P. et al. PLETHORA gradient formation mechanism separates auxin responses. Nature 515, 125–129 (2014).
Greb, T. et al. The PHD finger protein VRN5 functions in the epigenetic silencing of Arabidopsis FLC. Curr. Biol. 17, 73–78 (2007).
Mylne, J. S. et al. LHP1, the Arabidopsis homologue of HETEROCHROMATIN PROTEIN1, is required for epigenetic silencing of FLC. Proc. Natl Acad. Sci. USA 103, 5012–5017 (2006).
Liu, F., Marquardt, S., Lister, C., Swiezewski, S. & Dean, C. Targeted 3' processing of antisense transcripts triggers Arabidopsis FLC chromatin silencing. Science 327, 94–97 (2010).
Hou, X. et al. A platform of high-density INDEL/CAPS markers for map-based cloning in Arabidopsis. Plant J. 63, 880–888 (2010).
Box, M. S., Coustham, V., Dean, C. & Mylne, J. S. Protocol: a simple phenol-based method for 96-well extraction of high quality RNA from Arabidopsis. Plant Methods 7, 7 (2011).
Kodama, Y. Time gating of chloroplast autofluorescence allows clearer fluorescence imaging in planta. PLoS ONE 11, e0152484 (2016).
Yang, H. et al. Distinct phases of Polycomb silencing to hold epigenetic memory of cold in Arabidopsis. Science 357, 1142–1145 (2017).
Alon, U. An Introduction to Systems Biology (Chapman & Hall, 2007).
Grandjean, O. et al. In vivo analysis of cell division, cell growth, and differentiation at the shoot apical meristem in Arabidopsis. Plant Cell 16, 74–87 (2004).
Reddy, G. V., Heisler, M. G., Ehrhardt, D. W. & Meyerowitz, E. M. Real-time lineage analysis reveals oriented cell divisions associated with morphogenesis at the shoot apex of Arabidopsis thaliana. Development 131, 4225–4237 (2004).
Rahni, R. & Birnbaum, K. D. Week-long imaging of cell divisions in the Arabidopsis root meristem. Plant Methods 15, 30 (2019).
Bouché, F. Arabidopsis—root cell types. Figshare https://doi.org/10.6084/m9.figshare.4688752.v1 (2017).
The Arabidopsis Information Resource (TAIR). Maps for chromosome 2. TAIR, https://www.arabidopsis.org/servlets/mapper?value=f12k2&action=search (accessed 15 May 2020).
The Arabidopsis Information Resource (TAIR). Maps for chromosome 5. TAIR https://www.arabidopsis.org/servlets/mapper?value=mwd9&action=search (accessed 15 May 2020).
We thank E. Wegel for help with microscopy; S. Chen and R. Xie for genetic screening; J. Fozard for guidance on image analysis and discussion; C. Lövkvist and all other members in the Dean and Howard groups for critical discussions; S. Wang for providing the HA::NTL8 line; H. Millar for help on protein stability assessment; and M. Trick for bioinformatic analysis of genome resequencing data (ntl8-D1). This work was funded by the European Research Council grant ‘MEXTIM’ and supported by the BBSRC Institute Strategic Programmes GRO (BB/J004588/1) and GEN (BB/P013511/1).
The authors declare no competing interests.
Peer review information Nature thanks Chris Helliwell, Ari Pekka Mähönen and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
a, Schematic for the VIN3-luciferase reporter. Luciferase was fused next to the C terminus of VIN3. b, Luminescence imaging of the transgenic line carrying the VIN3-luciferase reporter. NV indicates no cold treatment (20 °C), 4WT0 indicates 4 weeks of cold treatment at 5 °C, and 4WT1 indicates 4 weeks of cold plus 1 day of warm treatment at 20 °C. Four independent repeats with similar results were performed. c, Analysis of endogenous VIN3 and transgenic VIN3-luciferase transcripts in the reporter line under different lengths of cold treatment (5 °C). Control indicates the non-transgene vin3-6 control. 4D indicates 4 days of cold treatment. nW indicates n weeks cold treatment, in which n is 1, 2, 3, 4, 6 or 8. Two biological replicates (dots) and their means (lines) are shown. d, e, Flowering phenotype of the reporter line after 4 weeks of vernalization (5 °C). JU223, a FRI transgene, was introduced into the reporter line by crossing. Error bars are s.e.m. of 12 individuals. Analysis of variance (ANOVA) with Tukey honest significant difference (HSD) post hoc test for multiple comparisons was performed, and P values for individual comparisons of interest are shown. Scale bar, 2.5 cm. Source data
a, Schematic of the predicted proteins with the identified mutations in ntl8-D1 and ntl8-D2 mutants. The light blue box indicates the NAC (NAM/ATAF/CUC) domain, and the red box indicates the transmembrane domain (TM). R indicates arginine, W indicates tryptophan, and the asterisk indicates the stop codon. aa, amino acid. b, Developmental phenotypes of ntl8-D1 (indicated by the red arrow) and ntl8-D2 mutants. Scale bar, 2.5 cm. Three independent repeats with similar results were performed. c, NTL8 transcript quantification in WT and ntl8-D1 and ntl8-D2 mutants in the warm (20 °C). Errors are s.e.m. of three biological replicates. d, No morphological phenotype is observed in the ntl8-OE2 mutant. Scale bar, 2.5 cm. Five independent repeats with similar results were performed. e, Schematic of the T-DNA mutant SM_3_16309(ntl8-1), Salk_866741(ntl8-OE1), Salk_587226 (ntl8-OE2) and GT19225 (ntl14-1). f, Analysis of the NTL8 transcript by qPCR in ntl8-OE1 and ntl8-OE2 in the warm (20 °C). g, Analysis of the VIN3 transcript by qPCR in ntl8-OE1 and ntl8-OE2 in the warm (20 °C). For f, g, error bars are s.e.m. of four biological replicates. h, Analysis of the NTL8 transcript in the 35S::HA-NTL8 transgenic line. i, Analysis of the VIN3 transcript in the 35S::HA-NTL8 transgenic line in the warm (20 °C). For h, i, error bars are s.e.m. of three biological replicates. j, Analysis of NTL8 binding at the NTL8 locus by ChIP. Col-0 was used as a background control. Error bars are s.e.m. of three replicates. Primer positions are shown in the NTL8 schematic below (−30: 30 bp upstream, +1,000: 1,000 bp downstream of the TSS). k, Analysis of NTL8 binding at the VIN3 locus by ChIP. Primer positions are shown in the VIN3 schematic below (−900: 900 bp upstream, 30: 30 bp downstream of the TSS, as in Fig. 1d). Two biological replicates are shown. Source data
a, Forward genetics identified ntl14-D. A schematic of NTL14 is shown below. The light blue box indicates the NAC domain, and the red box indicates TM. Q indicates glutamine, and the asterisk indicates the stop codon. Seven independent repeats with similar results were performed. b, Analysis of VIN3 transcript level by qPCR in the ntl8-1 mutant and the Col-0 control, at NV (20 °C) and 4 weeks of cold treatment (4W) at 5 °C. Error bars show s.e.m. of three biological replicates. c, As for b, but in ntl14-1 and the Ler control. Error bars show s.e.m. of four biological replicates. d, VIN3 transcript in ntl8-1ntl14-1 and WT control, at NV (20 °C) and 3 weeks of cold treatment (5 °C). WT, WT plants in the F2 population of the cross between ntl8-1 and ntl14-1. For comparison between WT and the ntl8-1ntl14-1 double mutant, we performed a two-tailed t-test: t = 4.634, d.f. =10, P = 0.0009. Error bars show s.e.m. of six biological replicates. e, f, Analysis of FLC transcripts in Ler JU223, vin3-6 JU223, ntl8-D2 JU223 and vin3-6 ntl8-D2 JU223 mutants under NV (e), 4WT0 (f) and 4WT14 (f) conditions. NV indicates the warm (20 °C) before cold treatment, 4W indicates 4 weeks of cold treatment at 5 °C, and 4WT14 indicates 4 weeks of cold plus 14 days of warm treatment at 20 °C. In f, data are normalized to the corresponding NV treatment. Error bars are s.e.m. of three biological replicates. g, Unspliced FLC levels after 4 weeks of vernalization in Col FRI and ntl8-1 FRI, normalized to the NV levels in the same genotype. Error bars are s.e.m. of three biological replicates. Unpaired t-test, two-tailed: t = 5.303, d.f. = 4, P = 0.0061. h, Flowering time, counted by the number of rosette leaves at flowering, for plants vernalized for 4 weeks. Error bars are s.e.m. of 12 individuals. Unpaired t-test, two-tailed: t = 4.241, d.f. = 22, P = 0.00031. i, Potential cross-regulation targets of NTL14 based on microarray data17. j, Potential cross-regulation targets of NTL8 based on in vitro data20. RNA was isolated from whole seedlings, and values are all normalized to UBC. Source data
Extended Data Fig. 4 Analysis of VIN3 and short-term cold stress-inducible genes in ntl8-D mutants, and characterization of independent NTL8prom::GFP-NTL8transgenic lines.
a, Analysis of VIN3 transcript level under different lengths of cold treatment by qPCR. 4WTn indicates 4 weeks of cold at 5 °C plus n days of warm at 20 °C, in which n is 1, 3 or 7. NV: 20 °C. Data are normalized to UBC. Error bars are s.e.m. of four biological replicates. b–d, RD29A (b), COR47 (c) and COR15 (d) transcript quantification in WT and ntl8-D mutants. NV indicates no cold treatment (20 °C), and 2D indicates 2-day cold treatment at 5 °C. Error bars are s.e.m. of three biological replicates. e, All ten randomly selected NTL8prom::GFP-NTL8 transgenic lines show NTL8 accumulation in the cold. 7W refers to 7 weeks of cold at 5 °C. Scale bars, 100 μm. The schematic of GFP-NTL8 is shown above. A GFP linker was fused at the N terminus of NTL8. Three roots were assayed with similar results for each line in both NV and 7W. f, Two representative lines showing the slow accumulation behaviour of NTL8 in the cold. 1W, 2W and 4W refer to 1, 2 and 4 weeks of cold, respectively, at 5 °C. Scale bars, 100 μm. Two independent repeats with similar results were performed. g, The transcript level of the NTL8prom::GFP-NTL8 transgene in the two representative lineS4 and lineS8. Values are normalized to UBC. Error bars are s.e.m. of three biological replicates. RNA was isolated from whole seedlings. h, Detection of low GFP-NTL8 levels in NV plants after long exposure. Propidium iodide staining was used to mark the root structure. Scale bar, 50 μm. Five roots were assayed with similar results. Source data
a, Schematics of the GFP-NTL8, GFP-NTL8-D1 and GFP-NTL8-D2 proteins. A GFP linker was fused at the N terminus of NTL8, NTL8-D1 and NTL8-D2. b, Localization of GFP-NTL8 and GFP-NTL8-D2 in the root in stable transgenic plants (top) and only the propidium iodide staining channel for the same roots (bottom), indicating that they are the same optical section. Eight-day-old roots were imaged with Leica SP5 confocal microscopy. NV: 20 °C, and 4W indicates 4 weeks of treatment in the vernalization room at 5 °C. Propidium iodide staining was used to mark the root structure. Scale bars, 50 μm. Four independent repeats with similar results were performed. c, d, NTL8 accumulation after exposure to fluctuating temperatures. In c, imaging of the GFP-NTL8 fluorescence signal in the root tip with a Leica DM6000 microscope, under exposure to different lengths of fluctuating temperatures (Fluct; with an average temperature of 14 °C) and constant temperature of 14 °C (Con) is shown. Scale bar, 50 μm. Independent roots (n = 6 for NV, 12 for Con-2W, 13 for Fluct-2W, 4 for Con-4W and 5 for Fluct-4W) were assayed with similar results. In d, analysis of the GFP-NTL8 protein using western blot after the same conditions as in c is shown. One replicate is represented. Ponceau staining of the input on a separate gel was used for the loading control. Quantification of band intensity is shown on each gel. For gel source data, see Supplementary Fig. 1. e, Domains of NTL8 (i and ii) and VIN3 (iii and iv) in the root tip after 7 weeks of cold. i and iii show the merged image of bright-field and fluorescence channels, and ii and iv show the fluorescence channel only. Scale bars, 100 μm. Independent roots (n = 5 for GFP-NTL8 and 13 for VIN3-GFP) were assayed with similar results. f, Detection of the GFP-NTL8 fluorescence signal in the shoot in warm conditions (20 °C) or after 6 weeks of cold (5 °C). Green indicates the signal of GFP-NTL8, red indicates the signal from chlorophyll autofluorescence, and grey indicates the bright-field. Scale bars, 50 μm. g, i and ii are the side view of the 3D projection of f(i) and f(ii), respectively. Three independent repeats with similar results were performed. h, Subcellular localization of GFP-NTL8, GFP-NTL8-D1 and GFP-NTL8-D2 with a transient assay in N. benthamiana. GFP-NTL8 (i), GFP-NTL8-D1 (iii) and GFP-NTL8-D2 (iv) were kept in warm (20 °C) conditions for 2 days before imaging; GFP-NTL8 (ii) was kept in the cold (5 °C) for 2 days before imaging. The arrows point to nuclei. Scale bar, 25 μm. Two independent repeats with similar results were performed.
Extended Data Fig. 6 Analysis of NTL8 isoforms in WT plants, and assay of NTL8 protein turnover rate under CHX treatment.
a, Schematic of NTL8 RNA isoforms identified by 3′RACE and sequence of isoforms 2 and 3. The red arrows indicate the differences of isoform 2 and isoform 3 from isoform 1. b, Quantification of protein concentration from western blots of Fig. 2a and additional replicates of experiment, showing each band separately. c, Quantification of the transcript levels of all three isoforms by qPCR. NV: 20 °C. 4W indicates 4 weeks of treatment in the vernalization room at 5 °C. Error bars are s.e.m. of eight (for isoform 1) or four (for isoform 2 and isoform 3) biological replicates. d, Comparison of the proteins produced by WT NTL8prom::GFP-NTL8 and mutant NTL8prom::GFP-NTL8-D2. The first band produced in the GFP-NTL8 line is absent in the GFP-NTL8-D2 line. On the basis of the sequence of GFP-NTL8-D2, the full-length form with the TM domain cannot be made. This suggests that the absent band corresponds to full length GFP-NTL8. The second band produced in the GFP-NTL8 line matches the size on the gel of the GFP-NTL8-D2 form. Ponceau staining of the input on a separate gel was used for the loading control. For gel source data, see Supplementary Fig. 1. Two independent repeats with similar results were performed. e, Diagram of the predicted proteins produced by alternative splicing. The NTL8 isoform 2 (286 aa) has a similar amino acid number as the NTL8-D2 (293 aa) form. Given that the second band for GFP-NTL8 in d matches the size on the gel of the NTL8-D2 form, the second band could be, at least in part, produced by NTL8 isoform 2. Furthermore, the alternative splicing site for isoform 3 is present in the ntl8-D2 allele, so NTL8 isoform 3 could still be produced by the ntl8-D2 allele, and the resulting protein probably produces the band in the GFP-NTL8-D2 sample that matches the third band in the WT. On the basis of the protein marker, all of the bands gave systematically larger molecular weights than those predicted by the amino acid sequence, possibly due to large post-translational modifications, potential measurement inaccuracy or a combination of the two. f, g, GFP-NTL8 protein concentration determined by western blot assay. Eight-day-old seedlings were treated with 100 μM CHX in the warm (20 °C; f) or in the cold (5 °C; g), as indicated for 24 h and 48 h. Ponceau staining of the input on separate gels was used for the loading control. Quantification of band intensity is shown on each gel. Two independent repeats with similar results were performed; for gel source data, see Supplementary Fig. 1. h, i, Eight-day old seedlings treated with 100 μM CHX in the warm (20 °C) or the cold (5 °C), as indicated for 48 h, were imaged with the fluorescence microscope Leica DM6000 (h) and with the Leica SP5 confocal microscope (i). Root structures and dying cells are shown in i with propidium iodide staining. Scale bars, 100 μm. Two independent repeats with similar results were performed. Source data
Extended Data Fig. 7 Changes in production and growth rate are still consistent with the mathematical model.
a, Growth rate at different temperatures estimated from the weight of 50 seedlings at different times following growth in warm (20 °C) and cold (5 °C) conditions. Seedlings were transferred to the cold (blue data points) after 7 days in the warm (data shown as empty circles) or 12 days (filled diamonds). Error bars are s.e.m. of six (8 days, 12 days warm) and three (all other time points) biological replicates. Linear regression was used, with the fitted lines shown together with the slopes corresponding to the growth rates in the different conditions. The difference in growth between warm and cold conditions was approximately sevenfold. The older seedlings grew faster in the cold. Therefore, we used the average slope between the growth rates of 7-day-old and 12-day-old plants in the cold as the cold growth rate. b, Assay of the absolute amount of NTL8 by western blot with the same number of seedlings grown for 8 days in warm (20 °C) conditions and then moved to the cold (5 °C) or kept in the warm (20 °C), showing that the amount of NTL8 per plant increases in the warm and in the cold. Error bars are s.e.m. of 13 biological replicates. For gel source data, see Supplementary Fig. 1. We performed a one-way ANOVA test: P = 8.54 × 10−10, F = 39.42, R2 = 0.6865, with the Tukey HSD post-hoc test for multiple comparisons, which showed that all three pairs (NV-Cold: P = 1.44 × 10−9, NV-Warm: P = 0.0038, Cold-Warm: P = 1.55 × 10−5) are significantly different. c, Model from Fig. 3c (no degradation) reproduced for comparison (α = 1/4, tdiv (cold) = 7 days). d, Same model with a fourfold longer division time (α = 1/4, tdiv (cold) = 28 days), showing accumulation that saturates more slowly. e, Model with decreased production (α = 1/8, tdiv (cold) = 7 days). The timescale of the accumulation does not change, but the saturated levels are decreased, thus increasing the requirement for reduced dilution to explain the experimentally observed accumulation. f, Model with decreased production and a fourfold longer division time (α = 1/8, tdiv (cold) = 28 days), showing that further reduced dilution can recover some of the effect due to decreased production. g, Table of parameters of the model from Fig. 3a–c. Source data
a, Inhibition of growth by applying Kan (200 μg/l), AVG (1 μM or 10 μM), ABA (1 mM), 24D (1 μM), IAA (10 μM), Brz (1 μM or 10 μM), ACC (1 μM, 10 μM or 100 μM), HU (10 mM or 20 mM) and PAC (2 μM, 20 μM or 100 μM). Control indicates no treatment. Seedlings grown for around 6 days were transferred to new medium supplemented with the indicated chemicals for 2 days in the warm (20 °C). Two independent experiments showed similar results. Scale bar, 0.5 cm. b, Imaging of the fluorescence signal of GFP-NTL8 in the root tip of plants from a after treatments for 2 days in the warm (20 °C). Scale bar, 100 μm. c, Quantification of the fluorescence intensity averaged over multiple roots. Two independent experiments were combined; roots were imaged per treatment (treatment order as in c from left to right): 16 roots total (6 excluded), 25 (1 excluded), 13 (0 excluded), 55 (3 excluded), 13 (0 excluded), 18 (0 excluded), 19 (0 excluded), 20 (7 excluded), 30 (3 excluded), 52 (0 excluded), 19 (0 excluded), 40 (0 excluded), 25 (0 excluded), 43 (0 excluded), 30 (1 excluded), 42 (0 excluded) and 51 (1 excluded). Error bars are s.e.m. We observed higher NTL8 in all treatments that inhibited growth without killing the plants. Seedlings treated with 10 mM or 20 mM HU for 2 days were almost dead. The 1 μM AVG treatment did not increase NTL8 levels, which is expected as growth was not slowed in that case. ACC treatments showed subtle effects, possibly due to indirect effects. Source data
a, Seedling phenotypes. Six-day-old seedlings were treated under short-day (8-h light/16-h dark) or long-day (16-h light/8-h dark) conditions for 2 weeks in the warm (20 °C). Scale bar, 0.5 cm. b, Assay of growth in a by measuring fresh weight. The bulk of 50 individual seedlings were weighed. Error bars indicate s.e.m. of ten (for short day) or eight (for long day) replicates. c, Analysis of NTL8 protein concentration from treatments in b by western blot assay with equal weight of starting material. As shown by Ponceau staining, which differs between short-day and long-day treatments. Therefore, MPK6 was used as the loading control. Ponceau staining and MPK6 antibody of the input were performed on the same gel, separate to the NTL8 gel. For gel source data, see Supplementary Fig. 1. d, Quantification of the relative NTL8 protein concentration in c with ImageJ (normalized to short-day levels). Error bars show s.e.m. of four replicates. e, Effect of GA treatment on NTL8 accumulation in the cold. Eight-day-old seedlings grown in the warm (20 °C) were transferred to a new medium supplemented with or without GA (10 μM) and treated for 4 weeks in the cold (5 °C) before imaging. Quantification of fluorescence intensity; roots were imaged per treatment (treatment order as in e from left to right): 28 roots total (0 excluded), 28 (0 excluded), 31 (0 excluded) and 31 (1 excluded). Error bars are s.e.m. For comparison between treatments with and without GA, one-tailed t-test was performed: for lineS4: t = −3.21, d.f. = 54, P = 0.0011; for lineS8: t = −7.75, d.f. = 59, P = 7.2 × 10−11. f, Inhibition of growth by applying MG132 (100 μM). −MG132 indicates no treatment. Seedlings grown for around 6 days in the warm (20 °C) were transferred to new medium supplemented with the indicated chemicals for 2 days in the warm (20 °C). Scale bar, 0.5 cm. g, Imaging of the fluorescence signal of GFP-NTL8 in the root tip of plants from f after treatments for 24 h and 48 h. Scale bar, 50 μm. Six independent repeats with similar results were performed. Source data
Extended Data Fig. 10 Root tissue structure and computational model of the root, NTL8 protein stability, and map-based cloning of ntl8-D and ntl14-D mutations.
a, Diagram of root structure showing the division zone, elongation zone and differentiation zone, as well as the different tissue types in the meristematic region. Modified from ref. 40. b, Analytical solution of the ODE model (solid line) and the computational simulation (dashed line) of the growth dilution model give the same predicted NTL8 concentration pattern. A small difference is seen in the cold because, in the simulation, division is occurring in a single step every week, as opposed to the smooth, averaged growth of the ODE model. c, NTL8 protein is stable over timescales of weeks. 4-week cold (5 °C) root imaged after a further 2 days in the warm (20 °C; 4WT2; i and ii), or 24 days in the cold (5 °C) following the 2-day warm treatment (iii and iv). i and iii show the root tip, and ii and iv show the region of the root where NTL8 accumulated during the 4-week cold period. NTL8 is maintained at those high levels after transfer to warm (ii), due to limited further growth in that region, and persists there after transfer back to the cold for at least 24 days (iv). Root structures and dying cells are shown with propidium iodide staining. Scale bars, 100 μm. Two roots were imaged. d, Diagram of map-based cloning for ntl8-D1 and ntl8-D2. Recombination numbers are indicated separately for ntl8-D1 and ntl8-D2 mutants. The diagram was drawn according to ref. 41. e, Diagram of map-based cloning for ntl14-D. Recombination numbers are indicated as shown. The diagram was drawn according to ref. 42.
This file contains Supplementary Methods (a description of the Ordinary Differential Equation models used in this study) and Supplementary Figure 1 (the uncropped scans). The Supplementary Methods include a Model of Fig. 2c, demonstrating that the timescale of accumulation is determined by the degradation rate and a full description of model of Fig. 3a (NTL8 Ordinary Differential Equation model) with or without degradation. In Supplementary Figure 1, Ponceau stainings for all GFP-NTL8 gels were from separate gels and used as same processing controls. Ponceau stainings for MPK6 and HA-NTL8 were from the same gel and reversibly stained with Ponceau buffer.
A list of oligos used in this study.
Sequence information of NTL8 isoform 2 and isoform 3.
A zipped file containing all custom code used in this study. The folder includes the code for automatic image analysis (image_process_fluorescence.py) with associated files and test data, the code for the computational model of Fig.4 (rootSim.m), the LICENSE document and a README.md file with information on how to use the code. The same is uploaded to a GitHub repository at: https://github.com/ReaAntKour/NTL8TemperatureGrowth/.
Tracking the dynamics of GFP-NTL8 in the root in the warm following 6 weeks vernalization at 5 °C.
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Zhao, Y., Antoniou-Kourounioti, R.L., Calder, G. et al. Temperature-dependent growth contributes to long-term cold sensing. Nature 583, 825–829 (2020). https://doi.org/10.1038/s41586-020-2485-4