Zhao and colleagues (Nature 583, 825-829; 2020) established that prolonged cold is measured in Arabidopsis by an indirect mechanism: the membrane-tethered NAC transcription factor NTL8 is long-lived and produced at an approximately constant rate, so when low temperature slows growth, reduced dilution by cell division and expansion lets NTL8 accumulate slowly over weeks, upregulating VERNALIZATION INSENSITIVE 3 (VIN3) and COOLAIR to drive Polycomb silencing of FLOWERING LOCUS C (FLC). The mechanistic account of this sensor remains incomplete in the two respects that the source paper itself left open: the molecular route by which the membrane-tethered NTL8 pool generates a nuclear, transcriptionally active fraction (proteolytic cleavage versus translation of transmembrane-lacking splice isoforms versus other routes) is undetermined; and whether other long-lived proteins exploit growth-dependent dilution as long-term integrators of fluctuating temperature is unsurveyed and unvalidated.

Background

Vernalization is the process by which prolonged winter cold enables flowering in biennial and winter-annual plants, studied most deeply in Arabidopsis thaliana. Cold progressively represses the floral repressor gene FLOWERING LOCUS C (FLC) through the Polycomb repressive complex 2 (PRC2), a conserved chromatin-modifying complex that deposits the repressive histone mark H3K27me3; the silencing is epigenetically stable once warm temperatures return in spring. A key cold-induced input to this switch is VERNALIZATION INSENSITIVE 3 (VIN3), an accessory protein containing a PHD finger (a zinc-coordinating protein domain that binds chromatin) that is expressed only after weeks of cold and associates with PRC2 at FLC. Why VIN3 induction requires weeks, rather than hours, of cold was unexplained until Zhao et al. (2020) identified the NAC-domain transcription factor NTL8 as the slow, long-term temperature input. NAC proteins (named after NAM, ATAF1/2 and CUC2) form one of the largest plant transcription-factor families; NTL8 belongs to the NTL subgroup whose members carry a C-terminal transmembrane domain that anchors them to the plasma membrane or endoplasmic-reticulum membrane. Membrane-tethered transcription factors of this kind are typically activated by proteolytic release of the soluble N-terminal DNA-binding portion, or in some cases by splice variants that never encode the transmembrane anchor. Zhao et al. showed genetically (dominant ntl8-D alleles that truncate the transmembrane domain render NTL8 constitutively nuclear and active) that removing the membrane tether activates NTL8; in the wild type, NTL8 protein is mostly membrane-localized, has a half-life greater than two days in both warm and cold, and its transcript level does not change in the cold, yet total NTL8 protein rises slowly over weeks of cold. The explanation, supported by a mathematical model and by growth-manipulation experiments, is dilution: in the warm, fast growth (cell division and expansion) dilutes the constantly produced, long-lived protein, whereas in the cold, slowed growth reduces dilution so the concentration rises, integrating the plant's temperature-dependent growth history and hence the duration of winter. Growth inhibition at warm temperature (for example by brassinazole, paclobutrazol or hygromycin — drugs that slow growth — or by short photoperiod, i.e. short days) is sufficient to cause NTL8 accumulation, and growth accelerated by gibberellin, a plant growth-promoting hormone, in the cold reduces it, confirming that growth rate, not temperature directly, is the sensed variable. Follow-up work showed NTL8 also directly activates the FLC antisense transcript COOLAIR, making it a common regulator of the parallel antisense-mediated and PRC2-mediated silencing pathways. Three NTL8 protein isoforms were detected, the shorter two lacking the transmembrane domain and plausibly arising from alternatively spliced NTL8 transcripts (isoforms 2 and 3, identified by 3'RACE — rapid amplification of cDNA ends, a PCR technique for recovering transcript termini); however, spliced transcript levels do not increase during cold, and the paper states that a fraction of wild-type NTL8 'must translocate to the nucleus to activate VIN3, potentially through cleavage or alternative splicing', without resolving which route operates. The quantitative models rest on deliberately simple assumptions: constant production confined to the root stem-cell initials (the stem cells at the root tip that generate all other root cells), zero intrinsic degradation, once-per-day (warm) or once-per-week (cold) synchronous cell divisions, and uniform cell size; the paper argues that its conclusions should be largely unaffected by these simplifications, because the observation timescale is much longer than the cell cycle duration. The paper closes by noting that the reduced-dilution effect 'can emerge for any long-lived protein with constant production' and that exploring whether other long-term environmental responses use similar mechanisms 'will be important'.

Problem Statement

The growth-dilution mechanism of long-term cold sensing established by Zhao et al. (2020) is incomplete, and the problem is to complete its mechanistic account and determine its scope, specifically: (1) Identify the molecular route, or routes, by which the membrane-tethered wild-type NTL8 protein generates the nuclear, transcriptionally active fraction that upregulates VIN3 and COOLAIR during prolonged cold — proteolytic cleavage of the full-length protein, translation of the transmembrane-domain-lacking isoforms produced from alternatively spliced NTL8 transcripts (isoforms 2 and 3), or other activation routes. (2) Determine whether other long-lived, approximately constantly produced proteins in Arabidopsis (or other organisms) exploit growth-dependent dilution as a long-term integrator of fluctuating temperature in developmental or environmental-response pathways, and if so which; the sub-question is resolved by identifying and substantiating at least one additional such protein, or by establishing that none is known.

References

  1. Zhao Y, Antoniou-Kourounioti RL, Calder G, Dean C, Howard M. Temperature-dependent growth contributes to long-term cold sensing. Nature 583, 825-829 (2020). DOI: 10.1038/s41586-020-2485-4, https://doi.org/10.1038/s41586-020-2485-4
  2. Antoniou-Kourounioti RL, Zhao Y, Dean C, Howard M. Feeling Every Bit of Winter - Distributed Temperature Sensitivity in Vernalization. Frontiers in Plant Science 12, 628726 (2021). DOI: 10.3389/fpls.2021.628726, https://doi.org/10.3389/fpls.2021.628726
  3. Zhao Y, Zhu P, Hepworth J, Bloomer R, Antoniou-Kourounioti RL, Doughty J, Heckmann A, Xu C, Yang H, Dean C. Natural temperature fluctuations promote COOLAIR regulation of FLC. Genes & Development 35, 888-898 (2021). DOI: 10.1101/gad.348362.121, https://doi.org/10.1101/gad.348362.121
  4. Nielsen M, Menon G, Zhao Y, Mateo-Bonmati E, Wolff P, Zhou S, Howard M, Dean C. COOLAIR and PRC2 function in parallel to silence FLC during vernalization. Proceedings of the National Academy of Sciences USA 121, e2311474121 (2024). DOI: 10.1073/pnas.2311474121, https://doi.org/10.1073/pnas.2311474121
  5. Maple R, Zhu P, Hepworth J, Wang JW, Dean C. Flowering time: From physiology, through genetics to mechanism. Plant Physiology 195, 190-212 (2024). DOI: 10.1093/plphys/kiae109, https://doi.org/10.1093/plphys/kiae109
  6. Coen E, Prusinkiewicz P. Developmental timing in plants. Nature Communications 15, 2674 (2024). DOI: 10.1038/s41467-024-46941-1, https://doi.org/10.1038/s41467-024-46941-1
  7. De Backer J, Van Breusegem F, De Clercq I. Proteolytic Activation of Plant Membrane-Bound Transcription Factors. Frontiers in Plant Science 13, 927746 (2022). DOI: 10.3389/fpls.2022.927746, https://doi.org/10.3389/fpls.2022.927746
  8. Dean C, Howard M. Graded versus ON/OFF control in quantitative gene expression and epigenetic memory. EMBO Journal 45, 2869-2881 (2026). DOI: 10.1038/s44318-026-00750-y, https://doi.org/10.1038/s44318-026-00750-y
  9. Montez M, Schulten A, Menon G, Dean C. Molecular integration of seasonal temperature signals in flowering time control. Nature Reviews Genetics 27, 695-707 (2026). DOI: 10.1038/s41576-026-00979-7, https://doi.org/10.1038/s41576-026-00979-7
  10. Kim SG, Lee AK, Yoon HK, Park CM. A membrane-bound NAC transcription factor NTL8 regulates gibberellic acid-mediated salt signaling in Arabidopsis seed germination. The Plant Journal 55, 77-88 (2008). DOI: 10.1111/j.1365-313X.2008.03493.x, https://doi.org/10.1111/j.1365-313X.2008.03493.x
  11. Cheng YJ, Shang GD, Xu ZG, Yu S, Wu LY, Zhai D, Tian SL, Gao J, Wang L, Wang JW. Cell division in the shoot apical meristem is a trigger for miR156 decline and vegetative phase transition in Arabidopsis. Proceedings of the National Academy of Sciences USA 118, e2115667118 (2021). DOI: 10.1073/pnas.2115667118, https://doi.org/10.1073/pnas.2115667118
  12. Maple R, Menon G, Duncan S, Schulten A, Sindalovskaya M, Yang H, Bloomer R, Howard M, Dean C. Co-transcriptional splicing changes combine with reduced productive transcription initiation for cold-induced repression of FLC. Nucleic Acids Research 54 (2026). DOI: 10.1093/nar/gkaf1462, https://doi.org/10.1093/nar/gkaf1462
  13. Jin Y, Tang X, Wei B, Zhi L, Mao Y, García-Caparrós P, Hu X. A Transcriptional Suppressor NTL8 Interacts with CONSTANS to Control Photoperiod-Mediated Flowering Time in Arabidopsis thaliana. Phyton - International Journal of Experimental Botany 95, 1-10 (2026). DOI: 10.32604/phyton.2026.067796, https://doi.org/10.32604/phyton.2026.067796