Abstract
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 INSENSITIVE3 (VIN3)5–7, 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 progression5,6,8–11. 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 up-regulation. Indirect mechanisms involving temperature-dependent growth, in addition to direct thermosensing, may be widely relevant in long-term biological sensing of naturally fluctuating temperatures.
Multiple thermosensory pathways have been shown to regulate VIN3 expression over different timescales10,11, but what accounts for the slow dynamics of VIN3 as plants experience weeks of cold was not known. To investigate this, we identified mutants that showed early cold responses, but could not upregulate VIN3 over longer cold. A VIN3-luciferase translational reporter (Extended Data Fig.1a) was transformed into a Landsberg vin3-6 mutant. A single-copy transgenic line, with VIN3-luciferase expression similar to endogenous VIN3 (Extended Data Fig.1b,c), was selected as the mutagenesis progenitor. The VIN3-luciferase reporter substantially rescued the vernalization defect of vin3-6 (Extended Data Fig.1d,e).
Two dominant mutants were identified showing high VIN3 expression in the warm (Fig.1a,b). The mutations, named ntl8-D1 and ntl8-D2, prematurely terminated NTL8 translation and showed morphological defects (with ntl8-D1 showing more extreme dwarfism and leaf deformation) and high NTL8 expression (Extended Data Fig.2a-c), in addition to VIN3 mis-expression. The ntl8-D allele dominance suggested that the short NTL8 proteins without the C-terminal transmembrane domain are constitutively active, as found in studies linking these NAC transmembrane proteins with salt signalling, ER stress and trichome development12–17. However, protein truncation is not the only path to ectopic activity as T-DNA insertions in the NTL8 promoter also led to NTL8 overexpression and constitutive VIN3 expression prior to cold, but without morphological defects (Extended Data Fig.2d-i). NTL8 binds to NTL8 and VIN3 promoter regions in vitro 18 and in vivo (Fig.1d,Extended Data Fig.2j,k). High VIN3 and NTL8 expression prior to cold for ntl8-D is thus likely through direct NTL8 activation.
VIN3 expression was unaffected in the loss-of-function ntl8-1. NTL8 belongs to a 14-member NAC transcription factor subfamily14,19–22. NTL14 is the closest homologue to NTL8 and conditionally induced expression causes ectopic VIN3 expression (microarray data,15). Indeed, we identified an ntl14-D mutation as constitutively expressing VIN3 in our VIN3-Luciferase screen (Extended Data Fig.3a). We therefore analysed an ntl8-1, ntl14-1 double loss-of-function mutant, and found VIN3 cold-induction significantly attenuated (Extended Data Fig.2e,3b-d). Interestingly, ntl8-D can overcome the vernalization requirement for FLC silencing, while ntl8-1 showed compromised FLC silencing and cold-acceleration of flowering (Extended Data Fig.3e-h). These data suggest the extensive NAC-TM family (Extended Data Fig.3i,j) function as a redundant network in vernalization and other abiotic responses.
ntl8-D pre-activated VIN3 expression with limited further increase beyond 1 week cold exposure (Fig.1c,Extended Data Fig.4a). Cold stress response genes were not consistently affected in ntl8-D (Extended Data Fig.4b-d). Thus, NTL8’s predominant vernalization role is in long-term VIN3 temperature regulation. To assess NTL8 protein dynamics, we generated transgenics carrying translational GFP fusions to both wild-type and truncated NTL8 proteins. During cold (including fluctuating temperatures), GFP-NTL8 signal increased in leaves and root meristem regions, coincident with high VIN3 meristem expression (Fig.1e,Extended Data Fig.4e-h,5a-g). The truncated protein was nuclear-localized in stable transgenics and highly expressed in all cells, likely via NTL8 transcriptional auto-activation (Extended Data Fig.2c,j,5b). Wild-type GFP-NTL8 protein was predominantly plasma membrane and ER-localized (around the nucleus) in stable transgenics and N. benthamiana transfection assays (Extended Data Fig.5b,h,19). In stable transgenics, there was no change to this subcellular localization in response to cold (Extended Data Fig.5b). Therefore, long-term cold increased overall NTL8 protein amount, rather than changing subcellular localization. A fraction of wild-type NTL8 protein must translocate to the nucleus to activate VIN3, potentially through cleavage12,19 or alternative splicing (Extended Data Fig.6a-e).
We detected alternatively spliced NTL8 and three GFP-NTL8 protein isoforms (Fig.2a, Extended Data Fig.6a,b). Isoform 1 is full-length NTL8 protein, while shorter isoforms lacked the transmembrane domain. All isoforms increased gradually with weeks of cold, and decreased in post-cold warm, consistent with the long-term VIN3 cold response. However, isoform3 showed a faster warm response, and could be involved in other temperature pathways regulating VIN3 (Fig.2b, Extended Data Fig.6b). The gradual increase occurred without a concomitant increase in NTL8 transcript levels or alternatively spliced transcripts (Fig.2b, Extended Data Fig.6c), arguing against transcriptional up-regulation or alternative splicing being major factors in gradual NTL8 accumulation in the cold.
To understand slow VIN3 and NTL8 accumulation dynamics we considered the problem theoretically. A single-step cold-induced increase in protein production with fast degradation cannot lead to slow accumulation, as the accumulation timescale is generally dictated by the degradation timescale (Fig.2c). We therefore explored whether low NTL8 turnover might contribute to the slow VIN3 increase. Inhibition of translation using cycloheximide revealed NTL8 isoforms1,2 have half-lives of >2 days in cold and warm conditions, with plants afterwards dying from cycloheximide exposure (Fig.2d,e,Extended Data Fig.6f-i). NTL8 therefore always has low turnover, potentially similar to plant proteins with half-lives of months23.
With slow degradation, the dominant factor determining NTL8 accumulation becomes growth-dependent dilution, as modelled previously for a hormone24. We reasoned that temperature-dependent growth (Extended Data Fig.7a) could give different patterns of NTL8 accumulation in warm and cold. The total NTL8 amount increases in both conditions (Extended Data Fig.7b). However, the fast growth in the warm before or after cold (Fig.2a,b,f), could cause rapid NTL8 cellular dilution, leading to lower NTL8 concentrations. Slower growth during the cold could enable NTL8 accumulation, with higher NTL8 concentrations (Fig.2a,b).
We developed a mathematical model for the NTL8 concentration in the growing plant (Fig.3a, Extended Data Fig.7c-g and Supplementary Methods). At constant temperature, total volume is assumed proportional to time25 (Extended Data Fig.7a). Based on our data, we assigned constant production and very slow degradation of NTL8 protein at all temperatures, or no degradation in the model, and found that this model of indirect temperature sensing through the growth rate could reproduce NTL8 dynamics in the warm and cold (Fig.2,3b,c). Many aspects of NTL8 biosynthesis are likely to be temperature regulated, but these would not explain the long-term accumulation. For example, changes to production (such as a global decrease in translation26) influence the final saturated NTL8 concentration, but not the slow accumulation (Fig.2c,Extended Data Fig.7c-f). Thus, a key factor for long-term, slow dynamics of NTL8 accumulation in the cold is reduced protein dilution.
A prediction from this model is that inhibiting growth by non-cold treatments would generate similar NTL8 accumulation. Treatment of seedlings with brassinazole, paclobutrazol, hygromycin and short photoperiod in the warm validated this prediction (Fig.3d-f, Extended Data Fig.8,9a-d). In contrast, NTL8 levels were lower following gibberellin treatment in the cold which accelerates growth13 (Extended Data Fig.9e). NTL8 levels increased in treated plants in the warm, with unchanged, or even reduced relative RNA levels (Fig.3e,f). The root-tip GFP-NTL8 signal also increased in all warm treatments that inhibited growth, except for (almost lethal) hydroxyurea (Extended Data Fig.8). Furthermore, NTL8 accumulated slowly after proteasome inhibitor MG132 treatment, which also strongly inhibits growth (Extended Data Fig.9f,g). That varied treatments behaved similarly strongly supports slowed growth as the basis for NTL8 accumulation.
To further explore this concept, we developed a root computational model (Fig.4a,b, Extended Data Fig.10a). We postulated that NTL8 is transcribed in a restricted region, as found in recent single cell transcriptomic analyses27. For such a gene, growth and diffusion can broaden the region where the protein is detectable28. We applied the same logic, but assumed that diffusion was unlikely to contribute due to membrane localization of wild-type NTL8 (Extended Data Fig.4h,5b), and would not affect the overall NTL8 amount. We found that the predicted GFP-NTL8 accumulation through reduced growth in the cold matched that experimentally observed (Fig.4c,d, Extended Data Fig.10b and Methods). Furthermore, this root model captured the pattern in the warm, with little NTL8 everywhere except the meristems (Fig.4c,d).
The root model predicted that regions where NTL8 accumulated during cold would maintain high levels after warm transfer (Fig.4e), due to limited growth in that region combined with long-term NTL8 stability. We verified this key prediction with a high-resolution root time series (Fig.4f, Supplementary Video 1, Extended Data Fig.10c(i,ii)). Higher growth in the warm led to newly-formed tissue having low GFP-NTL8 levels, with high GFP-NTL8 accumulation only at the root tip together with those cells that had arisen during the cold and were no longer growing. Elevated GFP-NTL8 levels in this patch of cells were maintained for 24 days of further growth in the cold (Extended Data Fig.10c(iii,iv), underlining that NTL8 protein is stable over weeks.
A combination of experiments and modelling has enabled us to show that reduced protein dilution due to less growth at low temperatures creates a long-term temperature sensing system. Temperature-dependent growth itself can thus be passively utilized as a long-term thermosensor, naturally averaging temperature fluctuations over long periods. Temperature sensing is distributed broadly through regulatory networks exploiting the temperature dependency of biochemical reactions11. The reduced dilution mechanism is therefore part of this network driving the slow accumulation of NTL8, and thus VIN3 in the long-term process of vernalization. Such an effect can emerge for any long-lived protein with constant production. It will be important to explore whether other biological processes related to long-term environmental signals utilize similar mechanisms.
Methods
Statistics
No statistical methods were used to predetermine sample size. The experiments were not randomized and investigators were not blinded to allocation during experiments and outcome assessment. Sampling in all cases is performed by collecting material from new plants (not repeated sampling), for replicates and also between timepoints, with the exception of the time lapse imaging in Supplementary Video 1.
Plant material and growth conditions
Generally, plant growth conditions were described previously29. For expression analysis and protein extraction, plants were grown on Murashige and Skoog (MS) agar plates without glucose. For microscopy, plants were grown almost vertically on MS plates containing 1% agar. Experiments under the fluctuating temperatures were done as described in10.
NTL8 overexpression lines (ntl8-OE1 (Salk_866741) and ntl8-OE2 (Salk_587226)) and the NTL8 knockout line ntll8-1 (SM_3_16309) were obtained from the Nottingham Arabidopsis Stock Centre (NASC). The NTL14 knockout line ntl14-1 (GT19225) was obtained from Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (http://genetrap.cshl.org). The 35S::HA-NTL8 transgenic line was generated by14. The VIN3-luciferase translational fusions were generated by replacing the stop codon of a VIN3 genomic fragment (from 6316bp upstream of the ATG to 3095bp downstream of the stop codon) with a linker and the luciferase coding sequence. The resultant VIN3-luciferase reporter was cloned into pSLJ-7551630 and transformed into the vin3-6 (vrn7-3), a Landsberg erecta (Ler) mutant29. Transgene copy number was assayed by idnaGENETICS (Norwich Research Park). A transgenic line containing a single-copy transgene and showing a similar expression pattern to the endogenous VIN3 was selected as the progenitor line for the forward genetic screening. Relevant primers are listed in Supplementary Table 1.
Flowering time analysis
Flowering time analysis was performed as previously described31. Briefly, plants were grown in containment conditions (16 hr light / 8 hr dark; Day temperature: 23–25 °C, and night temperature: 20–22 °C). The total rossete leaf number produced by the main apical meristem before flowering initiation was counted as a measure of flowering time.
Mutagenesis, genetic screening and map-based cloning
Mutagenesis was conducted as previously described in31. Pooled M2 (mutagenesis generation 2) seeds (from 25 M1 plants) were screened. Approximately 400 seeds from each M2 pool were sown on MS medium and stratified for 3 days in the cold (5°C). After growth in a growth cabinet for 10 days, the M2 seedlings were sprayed with 1μM luciferin (Promega, E1603) and assayed for the bioluminescence with a CCD camera (NightOwl). Two mutants were identified (Fig.1a) and crossed to Col-0 to generate a mapping population. By traditional map-based cloning, both were fine-mapped to a narrow region on chromosome 2 (Extended Data Fig.10d) and found to carry a mutation in AT2G27300 and so were named ntl8-D1 and ntl8-D2. Since ntl8-D1 was dominant, the wild type allele was mapped. SSLP markers for map-based cloning were either from https://www.arabidopsis.org or http://amp.genomics.org.cn/ 32. A ntl14-D mutation was also identified (Extended Data Fig.3a) and map-based cloning was done in a similar way as for the ntl8-D mutations (Extended Data Fig.10e). The key primers for fine mapping are listed in Supplementary Table 1.
NTL8 constructs and transformation
The NTL8prom::GFP-NTL8, NTL8prom::GFP-NTL8-D1 and NTL8prom::GFP-NTL8-D2 translational fusions contain a Ler NTL8 genomic fragment (from 2278bp upstream of the ATG to 703bp downstream of the stop codon), amplified separately from the progenitor line and the ntl8-D1 and ntl8-D2 mutants. The GFP and linker coding sequences were inserted ahead of the ATG of NTL8 by In-Fusion cloning (Takara, 638909). The resulting NTL8prom::GFP-NTL8, NTL8prom::GFP-NTL8-D1 and NTL8prom::GFP-NTL8-D2 were cloned into the binary vector pSLJ-7551630, and transformed into Agrobacterium C58. All of the 10 randomly selected NTL8prom::GFP-NTL8 transgenic lines show NTL8 accumulation under cold conditions, and the slow accumulation behaviour is independent of the transgene expression level (Extended Data Fig.4e-h). The high expression line S4, which carries a single copy NTL8prom::GFP-NTL8 transgene, was used in the paper to facilitate conducting the experiments. Relevant primers are listed in Supplementary Table 1.
Transient assays in Nicotiana benthamiana were conducted as previously described29. Imaging was done 2 days after infiltration with Agrobacterium. For short-term cold treatment, the Nicotiana benthamiana plants were kept in at 5°C for 2 days after infiltration with Agrobacterium. Stable transgenic lines were also generated in Col-0 and plants with a single copy transgene selected. Homozygous lines at the T4 generation were used for further experiments.
RNA analysis
Samples were collected at 3pm. Total RNA was prepared as previously described33. Genomic DNA was removed with TURBO DNA-free (Ambion Turbo DNase kit, AM1907) following the manufacturer’s guidelines, before reverse transcription was performed. The reverse transcription was performed with the SuperScript III First-strand Synthesis System (Invitrogen, 18080-051) according to the manufacturer’s protocol using either gene-specific primers or Oligo(dT) 12-18 (Invitrogen, 18418-012). Relevant primers are listed in Supplementary Table 1.
Cycloheximide (CHX) treatment
For the western blot assay of Fig.2d,e, 4-week vernalized 35S::HA-NTL8 seedlings were soaked in liquid MS medium supplemented with 100μM cycloheximide (Sigma-Aldrich, C1988) in warm conditions (normal growth cabinet at 20°C) and cold conditions (vernalization cabinet at 5°C). The seedlings were sampled at the timepoints of 0hr, 4hr, 8hr, 16hr, 24hr and 48hr. NTL8 proteins were detected as described in the section ‘Protein extraction and western blot assay’.
For the western blot assay of Extended Data Fig.6f,g, 10-day old NTL8prom::GFP-NTL8 seedlings were soaked in liquid MS medium supplemented with 100μM cycloheximide (Sigma-Aldrich, C1988) in warm conditions (normal growth cabinet at 20°C) and cold conditions (vernalization cabinet at 5°C). Then approximately 1.0g seedlings were sampled at the timepoints of 24hr and 48hr. NTL8 proteins were detected following the procedures in the section ‘Protein extraction and western blot assay’.
For fluorescence imaging, NTL8prom::GFP-NTL8 seeds were sown on MS medium and grown for 8 days before they were transferred to new MS medium supplemented with 100μM cycloheximide (CHX). After 48hr treatment with CHX, seedlings were imaged with the standard fluorescence microscope Leica DM6000 and confocal microscope Leica SP5.
MG132 treatment
NTL8prom::GFP-NTL8 seeds were sown on MS medium and grown for 8 days in the warm (20°C) before they were transferred to new MS medium supplemented with 100μM MG132 (Sigma-Aldrich, 474787). After 24hr or 48hr treatment with MG132 in the warm (20°C), roots of seedlings were imaged with the standard fluorescence microscope Leica DM6000. To assess root growth, images were taken with (Alphaimager). Images prior to treatment were taken immediately after the transfer and images after treatment were taken just before the fluorescence imaging.
Microscopy
Confocal imaging was performed using a 20X/0.7 NA multi-immersion lens, with water as the immersion fluid on a Leica TCS SP5 confocal microscope. For stable transgenic plants, roots were immersed in 2μg/mL propidium iodide (Sigma-Aldrich, P4864) to label the cell wall. To allow comparison between treatments, the same settings were used for all GFP-NTL8 images, with the exception of Extended Data Fig.4h which was imaged with a higher gain value and laser power.
For detecting the fluorescence of GFP-NTL8 in the leaves, confocal imaging was performed using a 63X/1.2 water immersion lens on a Leica TCS SP8X confocal microscope. The GFP was excited using 514nm laser line from a white light laser set to 80MHz pulse frequency (repeats every 12.5ns) and fluorescence emission was captured between 525-550nm using hybrid detector. Time gating was used to remove chloroplast autofluorescence in the green/yellow spectrum34, after an excitation laser pulse the fluorescence emission of GFP-NTL8 was only detected between 0.47-10.80ns. The chloroplast autofluorescence was used as a marker of the cell, as the propidium iodide does not work in the leaf. A z-stack was done to be sure that GFP-NTL8 shows no signal in the leaf under warm conditions. To allow comparison between treatments, the same settings were used for all GFP-NTL8 images.
To compare the fluorescence intensity between roots, imaging was performed using a 20X lens on a Zeiss Axio Imager fluorescence microscope, which allowed us to collect the total intensity of each root tip. Images taken with the fluorescence microscope Leica DM6000 are specified in the figure legend. To allow comparison between treatments, the same settings were used for all images. Of note, in the comparison of GFP-NTL8 and VIN3-GFP in Extended Data Fig.5e, VIN3-GFP was imaged with a longer exposure time and higher light intensity.
For imaging the GFP-NTL8 dynamics post-cold (Fig.4F and Supplementary Video 1), Leica205FA stereo-microscopy was used. Images were taken sequentially around every 10 hours to monitor root growth. To allow the root to grow normally, plants were kept in the growth cabinet in between imaging. To allow comparison between treatments, the same settings were used for all images.
The image processing was conducted with ImageJ: the intensity was linearly adjusted separately for each channel. For visual comparison of the fluorescence intensity between different treatments in the GFP-NTL8 images, the same adjustment was used in ImageJ.
Protein extraction and western blot assay
For NTL8prom::GFP-NTL8 detection, approximately 1.0g seedlings were ground to a fine powder in liquid nitrogen. Total protein was extracted by suspending the powder with 2mL IP buffer (50mM Tris-Cl pH8.0, 154mM NaCl, 10% glycerol, 5mM MgCl2, 1% Triton, 0.3% NP-40, 5mM DTT, protease inhibitor (Roche, 04693159001)). After clearing by centrifugation at 16000 g at 4°C for 20min, 100μL total protein was taken as input before enrichment with Magnetic GFP-trap beads (ChromoTek GMBH, GTMA-20). The input samples were stained with Ponceau buffer and used as a loading control for the initial level for each sample, while enriched GFP-NTL8 protein amounts were detected by western blot. GFP-tagged protein was detected with anti-GFP antibody (Roche, 11814460001). Signals were visualized by chemiluminescence (SuperSignal West Femto; Pierce, 34095) using secondary antibodies coupled to horseradish peroxidase (anti-mouse IgG, GE healthcare, NXA931V). The chemiluminescence signal was captured by exposure to the FUJI Medical X-ray film (FUJI, 4741019289). X-ray films for quantification were all scanned with printer scanner (RICOH). Images of X-ray films shown in Fig.2 were taken with a high-resolution camera (Canon). Multiple exposures with different times were performed to avoid signal saturation, and a mildly-exposed image was always selected for signal quantification with ImageJ. A constant sized rectangle was drawn in ImageJ to enclose the band and the intensity inside it was measured. For each blot, the measured values were normalised to the average intensity of all the measurements, to remove systematic variability. Ponceau stainings using Ponceau buffer (Sigma-Aldrich, P7170) for all GFP-NTL8 were from separate gels and used as same processing controls. (Sigma-Aldrich, P7170). For 35S::HA-NTL8 detection, total protein was extracted from 200mg seedlings with 200μL IP buffer (same procedure as above), 20μL aliquot of each sample was taken for western blot detection with anti-HA-peroxidase (Roche, 12013819001) or anti-HA-TAG (Cell signalling technology, 3724s). The signal visualization was as above except with secondary antibodies coupled to horseradish peroxidase (anti-rabbit IgG, GE healthcare, NA934V) for anti-HA-TAG (Cell signalling technology, 3724s). Ponceau stainings for all HA-NTL8 from the same gel were reversibly stained with Ponceau buffer (Sigma-Aldrich, P7170). All of the western blot assays were carried out with equal weight of whole seedlings, except Extended Data Fig.7b (details in the section ‘Assay of the absolute amount of NTL8’).
Chromatin Immunoprecipitation (ChIP)
NTL8 protein ChIP experiments were conducted following the ChIP protocol described in35. For GFP-tagged NTL8, the immunoprecipitation used GFP-trap beads (Chromotek GTMA-20). For HA-tagged NTL8, the immunoprecipitation used anti-HA Magnetic beads (Pierce, 88836). DNA was purified and analysed as previously described35. Relevant primers are listed in Supplementary Table 1.
3’RACE for detecting alternative splicing
3’RACE was performed with the FirstChoice® RLM-RACE Kit (Thermal Fisher Scientific, AM1700) according to the procedures for 3’RACE. Briefly, total RNA extracted from wild type seedlings, grown for 8 days in standard growth conditions, was used to perform the reverse transcription with the 3’RACE adaptor provided in the kit. Two rounds of PCR were performed with nested Primer to provide more materials and better specificity. The second PCR product was then cloned into the PGEM-T vector system1 (A3600, Promega UK Ltd). Multiple single clones were sequenced. Relevant primers are listed in Supplementary Table 1. Sequence information of NTL8 isoform 2 and isoform 3 is listed in Supplementary Table 2.
Growth rate assay by measuring fresh weight
For measuring growth rate in the warm (20°C), bulk fresh weight of 50 seedlings was measured at a series of timepoints (8, 12, 16 and 20 days after moving into the growth cabinet (20°C)). For measuring growth rate in the cold, seedlings were firstly grown in the normal growth cabinet for either 7 days or 12 days before moving into the vernalization cabinet (5°C). Then bulk fresh weight of 50 seedlings was measured at a series of timepoints in the cold (5°C) (shown in Extended Data Fig.7a). Residual water on the seedling surface was carefully removed for accuracy. In addition, a fixed number of seeds (150 seeds per petri dish (140mm Triple vent petri dish, Sterili)) were evenly dispersed on the medium with enough space for growth, in order to avoid variability caused by density.
Growth inhibition by different treatments
To inhibit the growth rate in the warm (20°C), we used phytohormones and inhibitors including 200μg/L kanamycin (Sigma-Aldrich, 60615), Aminoethoxyvinylglycine (AVG, 1μM or 10μM) (Sigma-Aldrich, A6685), 1μM 2,4-Dichlorophenoxyacetic acid (2,4-D) (Sigma-Aldrich, D7299), 1mM Abscisic acid (ABA) (Sigma-Aldrich, A1049), 10μM Indole-3-acetic acid (IAA) (Sigma-Aldrich, I2886), brassinazole (Brz, 1μM or 10μM) (Sigma-Aldrich, SML1406), 20μg/L hygromycin (Sigma-Aldrich, H9773), 1-Aminocyclopropane-1-carboxylic Acid (ACC, 1μM, 10μM, or 100μM) (Sigma-Aldrich, 149101-M), Hydroxyurea (HU, 10mM or 20mM) (Sigma-Aldrich, H8627) and paclobutrazol (PAC, 2μM, 20μM, or 100μM) (Sigma-Aldrich, 43900). For imaging GFP-NTL8 in the root tips, approximately 25 seedlings, grown vertically for 6-days in the warm (20°C), were transferred to new medium supplemented with the above drugs in the warm (20°C). To assess root growth, images were taken twice with (Alphaimager), the first one immediately after the transfer and the second one 2 days later, just before the fluorescence imaging. Fluorescence imaging was performed with the Zeiss Axio Imager as described above. To detect the protein level with western blot, seeds were sown on filter paper to reduce damage during transfer for the treatment. Around 1.5g of 6-day old seedlings were transferred to new medium supplemented with the above chemicals. The seedlings were then grown for another 8 days in warm conditions before sampling. The western blot assay was performed following the procedures in the section ‘Protein extraction and western blot assay’.
Short Day and Long Day treatments were applied to avoid the widespread side-effects of chemical treatments. Seeds were evenly dispersed on the medium sufficiently distant from one another. After a 3-day stratification in the cold (5°C), 6-day old seedlings were transferred to a growth cabinet (Panasonic MLR-352 series), set to either Short Day (8hr day/16hr night, light setting 3) or Long Day (16hr day/8hr night, light setting 5) conditions for two weeks in the warm (20°C). Images to assess phenotype were taken immediately before growth measurements. Growth under Short Day and Long Day conditions was measured by bulk weight of 50 fresh seedlings. The western blot assay was performed following the procedures in the section ‘Protein extraction and western blot assay’. As different light conditions have a strong effect on Rubisco abundance, as shown by Ponceau staining, MPK6 (Sigma-Aldrich, A7104) was used as the loading control.
Growth acceleration in the cold (5°C) with gibberellin (GA) treatment
To accelerate the growth rate in the cold (5°C), we used gibberellin (GA, G7645). For imaging GFP-NTL8 in the root tips, approximately 25 seedlings, grown vertically for 6 days in the warm (20°C), were transferred to new medium supplemented with the above drugs and placed in the cold (5°C) for 4 weeks. Fluorescence imaging was performed with the Zeiss Axio Imager and quantified as described above.
Assay of the absolute amount of NTL8
To test the absolute amount of NTL8 in plants between warm and cold temperatures, bulk NTL8 protein was measured by western blot from the same number of seedlings with no treatment (8-day seedlings grown in the warm (20°C)), or 8-day cold treatment (5°C, following the no treatment pregrowth), or 8-day warm treatment (20°C, following the no treatment pregrowth). The western blot assay was performed following the procedures in the section ‘Protein extraction and western blot assay’.
Automatic Fluorescence Intensity Quantification
Fluorescence microscopy images (such as in (Fig.1e)) were analyzed to quantify the fluorescence in the root tip region. A threshold (2600) was applied to the intensity of the fluorescence of GFP-NTL8 and a mask was created based on this threshold. The second largest connected area was selected (the largest being the no-signal area), and the sum intensity of its fluorescence was quantified. The selection was manually inspected, and a small number of roots were excluded (23 images from a total of 629). This simple algorithm was implemented in Python (http://www.python.org) using the czifile (https://www.lfd.uci.edu/~gohlke/) and scipy.ndimage (https://www.scipy.org) libraries. Code is available in Supplementary File Code and at https://github.com/ReaAntKour/NTL8TemperatureGrowth/
NTL8 Ordinary Differential Equation model
Effective degradation (or protein removal) has been reported as the sum of decay and dilution36. This statement is usually applied to proteins in cell populations where all cells divide, giving an exponentially increasing number of cells with time, and where production continues in all daughter cells. However, in multicellular organisms not all cells continue to divide. In particular for plants, we have an approximately linear growth in time (Extended Data Fig.7a). Furthermore, the overall rate of protein production over the whole plant can be constant. Here, we modelled the total NTL8 protein amount, the total volume of the cell population approximated by the fresh weight of the plants, and the overall protein concentration, using the equations described in the Supplementary Methods.
Computational root simulation
We then developed a model for the root to try to capture the spatial pattern observed in our imaging. We made simple assumptions for root structure and growth37–38. We assumed that all cells in the division zone divide once per day, consistent with recent measurements of the times between two divisions39. According to our growth measurements in warm and cold (Extended Data Fig.7a), we assumed that the division rate slows down to once per week in the cold. In both cases we simulated divisions occurring simultaneously for simplicity. All cells are assumed to have the same size and the elongation zone was not simulated. These assumptions significantly simplify our model and, as we are primarily interested in the division zone and our observation timescale is much longer than the cell cycle duration, our conclusions should be largely unaffected by these simplifications.
Four cell files are simulated, one each from the stele, endodermis, cortex and epidermis layers (Extended Data Fig.10a), represented by a rectangular array of cells, with a variable number of rows. As the root is an axisymmetric structure, we simulate only half a cross-section of the root but additionally show a mirror image in the figures. The lateral root cap and columella cells are ignored by the model but are also shown in (Fig.4c), together with two quiescent centre cells (the latter with constant protein amount of 1), as visual reference points. We assume that there is no degradation of the protein, in either the cold or warm.
Each cell has an associated NTL8 amount. The step size of the simulation is a day and, every day, a constant amount of NTL8 is made (⅓ per day) in each initial cell only (Fig.4a), excluding the epidermis initial. The total production rate in the simulated half root is therefore 1 per day, and the average production per cell file is ¼ per day matching the production rate in the ODE model.
Following production, every day in the warm or every 7th day in the cold, synchronous cell division occurs in the division zone (Fig.4a, b, Extended Data Fig.10a). We assumed that the division zone is made up of 32 cells in each cell file, including the initial cells. This is an approximation since the cell numbers vary, but it is the equivalent of assuming that a cell that comes from the division of an initial cell divides a further 5 times (Fig.4b). At each division, for the bottom 32 rows of cells, each row is turned into 2 adjacent rows (therefore the growth rate in the warm is Vd = 32 per day, and the amount present in the parent cell is split equally between the daughter cells. It is straightforward to calculate the relative amount of protein in a simulated cell a given distance from the root tip under constant conditions (Fig.4b). For all rows of cells above 32, there is no change to the cell or to the simulated amount of protein contained within.
Each day, after production and (in case it occurs) division, we calculate the amount of NTL8 in each cell and the overall concentration in the root. The latter is defined as the total amount of protein, divided by the number of cells in the simulated half root (including the epidermis).
Initially, the root has 64 rows of cells. Also, at t = 0, the amount of NTL8 in the initial cells and those immediately above (excluding the epidermis cell file which has no NTL8) is chosen to be equal to 2/21. The two rows of cells above those, each have half that amount initially (1/21), and the four above those each have a quarter (1/42) and so forth. Therefore, at t = 0, the relative amounts in the 64 cells of a cell file are the same as in (Fig.4b), the total amount in each cell file (excluding the epidermis which has no NTL8) is equal to ⅔ (two times its production rate) and the overall concentration in the root is (1/128).
For the comparison with the ODE model (Extended Data Fig.10b), the concentration from the computational simulation model was multiplied by Vd /Vg = 32/0.0274 and then directly compared to the output of the ODE model that was run with the same pregrowth and vernalization parameters as the other. Therefore, the root average concentration in the computational simulation matches the ODE model results, apart from changes due to the discontinuity of cell divisions in the simulation (Extended Data Fig.10b).
Code is available in Supplementary File Code and at https://github.com/ReaAntKour/NTL8TemperatureGrowth/
Extended Data
Supplementary Material
Acknowledgements
We thank Eva Wegel for help with microscopy; Shuqin Chen and Rongrong Xie for genetic screening; Dr John Fozard (JIC) for guidance on image analysis and discussion; Cecilia Lövkvist and all other members in the Dean and Howard groups for critical discussions; Shucai Wang (Northeast Normal University) for providing the HA::NTL8 line; Prof Harvey Millar (The University of Western Australia) for help on protein stability assessment; and Martin Trick (John Innes Centre) 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).
Footnotes
Author contributions: Conceptualization: YZ, RLAK, CD, MH. Experimental methodology: RLAK performed RNA extraction and qPCR for Fig. 1c, 3f and Extended Data Fig. 3g, YZ performed all other experiments, GC developed fluorescence imaging protocol and assisted with image acquisition. Theoretical methodology: RLAK performed all model development and analysis. Data analysis: YZ performed image processing for Western quantification, RLAK performed image analysis of fluorescence intensity. Funding acquisition: CD, MH. Project administration: CD. Supervision: CD, MH. Writing: YZ, RLAK, CD, MH.
Competing interests: The authors declare no competing interests.
Additional information:
Supplementary Information is available for this paper.
Reprints and permissions information is available at www.nature.com/reprints.
Data and code availability
Quantitative source data for figures and full scanned images of western blots are provided with the paper. Raw images that support the findings of this study are available at doi: 10.6084/m9.figshare.12283970. Code is available as a supplementary file and at https://github.com/ReaAntKour/NTL8TemperatureGrowth/
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
Quantitative source data for figures and full scanned images of western blots are provided with the paper. Raw images that support the findings of this study are available at doi: 10.6084/m9.figshare.12283970. Code is available as a supplementary file and at https://github.com/ReaAntKour/NTL8TemperatureGrowth/