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ABA-activated low-nanomolar Ca2+–CPK signalling controls root cap cycle plasticity and stress adaptation

Nature Plants volume 11, pages 90–104 (2025)Cite this article

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Abstract

Abscisic acid (ABA) regulates plant stress adaptation, growth and reproduction. Despite extensive ABA–Ca2+ signalling links, imaging ABA-induced increases in Ca2+ concentration has been challenging, except in guard cells. Here we visualize ABA-triggered [Ca2+] dynamics in diverse organs and cell types of Arabidopsis thaliana using a genetically encoded Ca2+ ratiometric sensor with a low-nanomolar Ca2+-binding affinity and a large dynamic range. The subcellular-targeted Ca2+ ratiometric sensor reveals time-resolved and unique spatiotemporal Ca2+ signatures from the initial plasma-membrane nanodomain, to cytosol, to nuclear oscillation. Via receptors and sucrose-non-fermenting1-related protein kinases (SnRK2.2/2.3/2.6), ABA activates low-nanomolar Ca2+ transient and Ca2+-sensor protein kinase (CPK10/30/32) signalling in the root cap cycle from stem cells to cell detachment. Surprisingly, unlike the prevailing NaCl-stimulated micromolar Ca2+ spike, salt stress induces a low-nanomolar Ca2+ transient through ABA signalling, repressing key transcription factors that dictate cell fate and enzymes that are crucial to root cap maturation and slough. Our findings uncover ABA–Ca2+–CPK signalling that modulates root cap cycle plasticity in adaptation to adverse environments.

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Fig. 1: An ultrasensitive CRS for detecting ABA-triggered [Ca2+] dynamics.
Fig. 2: ABA triggered different Ca2+ dynamics in diverse tissues and cell types.
Fig. 3: ABA-induced dynamic spatiotemporal [Ca2+] changes in the epidermal cells of the primary root tip.
Fig. 4: ABA-induced low-nanomolar [Ca2+] increases via ABA receptors and SnRK2.2/2.3/2.6.
Fig. 5: ABA delays the root cap cycle and represses genes essential to the root cap cycle.
Fig. 6: Salt stress triggers [Ca2+] transients in the root tip and inhibits the root cap cycle.

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Data availability

All data underlying the findings of this study are available from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank A. Diener for critical reading of the paper. We thank the Horticultural Plant Biology and Metabolomics Center confocal facility at Fujian Agriculture & Forestry University and life science research core services of Northwest Agriculture & Forestry University (NWAFU) for providing confocal microscope service, as well as Y. Wang at NWAFU for confocal microscope technical support. This research is supported by NIH grants (nos R01GM060493 and R01GM129093) to J.S. and by startup funds from NWAFU, NSFC grants (nos NSFC-32370433 and NSFC-32170270) and the Interdisciplinary Frontier Innovation Team Program of NWAFU (grant no. A1080524001) to K.-h.L.

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Authors and Affiliations

  1. State Key Laboratory of Crop Stress Biology for Arid Areas and College of Life Sciences, Northwest Agriculture & Forestry University, Yangling, China

    Ziwei Lin, Ying Guo, Ruiyuan Zhang, Yiming Li & Kun-hsiang Liu

  2. Department of Molecular Biology and Centre for Computational and Integrative Biology, Massachusetts General Hospital, Boston, MA, USA

    Yue Wu, Jen Sheen & Kun-hsiang Liu

  3. Department of Genetics, Harvard Medical School, Boston, MA, USA

    Yue Wu, Jen Sheen & Kun-hsiang Liu

  4. Institute of Future Agriculture, Northwest Agriculture & Forestry University, Yangling, China

    Kun-hsiang Liu

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Contributions

Z.L., J.S. and K.-h.L. conceived and designed the project. Z.L., Y.G., R.Z., Y.L., Y.W., J.S. and K.-h.L. performed the experiments and analysed the data. Z.L., J.S. and K.-h.L. wrote the paper.

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Correspondence to Jen Sheen or Kun-hsiang Liu.

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Extended data

Extended Data Fig. 1 Detecting ABA-induced Ca2+ signals by different calcium biosensors.

(a) Fluorescence signals of GCaMP6s stimulated by ABA in the root tip of 7-day-old plants. (F-F0)/F0 represents the relative fluorescence intensity. The red-dotted box on the left indicates the area where the Ca2+ signal was detected. Dual peaks (green arrow) were visible in the ABA-induced Ca2+ transient in the root tip at a concentration of 10 μM ABA. Error bars denote ±s.e.m., n=7 plants. (b) Box plot of highest fluorescence signal ratio of CRS induced by ABA or nitrate in Fig. 1e, g. Error bars denote ±s.e.m., n=13 protoplasts. Upper and lower box boundaries represent the first and third quantiles, respectively, horizontal lines mark the median and whiskers mark the highest and lowest values. (c) Fluorescence signals of CRS stimulated by 0.1, 1, or 10 μM ABA in the root tip of 7-day-old plants. (F-F0)/F0 represents the relative fluorescence intensity. The ratio represents the relative fluorescence ratio of GCaMP6s to dTomato, with error bars denoting ±s.e.m., n=7 plants. Fluorescence signals in the root tips of 7-day-old plants were monitored after ABA stimulation using YC3.6 (d), CGf (e), and MatryoshCaMP6s (f). Error bars denote ±s.e.m., data from at least three independent experiments (total number of plants: YC3.6 mock, n=7; YC3.6 ABA, n=7; CGf mock, n=10; CGf ABA, n=25; MatryoshCaMP6s (+ABA), n=6). All results were conducted in at least three biological repeats with similar outcomes. YC3.6 and CGf did not detect ABA-induced Ca2+ signals in root tips. MatryoshCaMP6s detected ABA-induced signals from both green (calcium signal) and orange (control) channels in root tips. ABA did not affect the green/orange ratio change in the root tips of MatryoshCaMP6s. (g) CRS protein localization in the Arabidopsis cytoplasm but not in the nucleus fraction. Proteins from the CRS transgenic line were analyzed by immunoblots with anti-HA, anti-Tubulin (cytoplasm marker), and anti-Histone (nucleus marker) antibodies. All experiments were conducted in at least three biological repeats with similar results. (h) Summary of available genetically encoded Ca2+ indicators used for detecting cytosolic [Ca2+] of Arabidopsis plants. Hill coeff indicates Hill coefficience.

Source data

Extended Data Fig. 2 ABA triggers Ca2+ transients in different cell types.

Five indepedent repeats for single-cell detection of Ca2+ signals of CRS stimulated by ABA or mock treatment in guard cells (a), mesophyll protoplasts (b), mesophyll cells (c) and root tip cells (d). Ratio, relative fluorescence ratio of GCaMP6s to dTomato. Ca2+ oscillations were stimulated by ABA. We observed very few guard cells displaying different patterns of spontaneous Ca2+ oscillations featured with low amplitude (Ratio <0.5) and 1-2 peaks in 7 out of 99 guard cells in the mock treatment experiments. All Ca2+ signals were recorded at the single cell level. e, Five independent repeats for single-cell detection of Ca2+ signals of CRS-NLS stimulated by ABA or mock treatment in the nucleus of the epidermal cells in the root meristem of 7-day-old transgenic plants. Ratio, relative fluorescence ratio of GCaMP6s to dTomato. ABA, 10 μM. Left, ABA. Right, Mock.

Extended Data Fig. 3 The CRS variants do not exhibit overt growth defect phenotypes.

a, WT, CRS, CRS-NLS and CRS-PM transgenic plants were grown in soil for 21 days. Scale bars, 1 cm. b,c,d,e, Resembling WT, expression of the CRS variants did not alter plant growth. Fresh weight (b), primary root length (c) and leaf area (d) of 7-day-old Arabidopsis seedlings were measured when grown on 1/2 MS solid medium. e, CRS, CRS-NLS, CRS-PM, and WT plants were grown in soil for statistical flowering time measurements. Error bars denote ±s.e.m., data from at least three independent experiments (total number of plants: WT, n=28; CRS, n=23; CRS-NLS, n=25; CRS-PM, n=24 (b), WT, n=28; CRS, n=26; CRS-NLS, n=27; CRS-PM, n=25 (c), WT, n=16; CRS, n=16; CRS-NLS, n=16; CRS-PM, n=16 (d), WT, n=30; CRS, n=30; CRS-NLS, n=30; CRS-PM, n=30 (d)). ns (not significant) P > 0.05, (statistical significance determined by two-tailed non-paired Student’s t test). All experiments were conducted in at least three biological repeats with similar results.

Extended Data Fig. 4 Differential ABA-triggered subcellular Ca2+ dynamics within 50 s.

Ca2+ signals of of CRS-PM, CRS, CRS-NLS within 50 s in response to ABA in the epidermal cell of the root meristem zone in 7-day-old transgenic plants. The signals were subtracted from the mock control presented in Fig. 3b–d. CRS-PM detected the ABA-induced Ca2+ transient first but the nuclear Ca2+ oscillation revealed by CRS-NLS did not start until 100 s (Fig. 3d).

Extended Data Fig. 5 ABA initiates a calcium signals from extracellular sources.

a, Time-lapse images of ABA-induced Ca2+ signals near nanodomains of the plasma membrane in mesophyll cells of 7-day-old transgenic CRS plants were captured in three independent experiments. Black or white dotted circles outline the cell. White arrows indicate elevated Ca2+ signals. b, A non-selective ion channel blocker (GdCl3) or Ca2+ chelators (BAPTA and EGTA) abolished ABA-triggered Ca2+ changes in Arabidopsis root tips (Extended Data Fig. 1 and Fig. 3). Error bars denote ±s.e.m., n=8 plants. Ratio, relative fluorescence ratio of GCaMP6s to dTomato. ABA, 10 μM. Scale bar, 10 μm. All experiments were conducted in at least three biological repeats with similar results.

Extended Data Fig. 6 Ca2+ elevation induced by ABA was not homogeneously distributed at the plasma membrane.

Kymography analysis of CRS-PM in response to ABA in the epidermal cells of the root meristem zone of 7-day-old plants was performed in two more independent experiments besides the result presented in Fig. 3f. The red box indicates the detection region.

Extended Data Fig. 7 CRS, CRS-NLS and CRS-PM show similar Ca2+ dynamic range in root tip cells.

Range of ABA-induced changes in cytosolic [Ca2+]. The highest ratio represents the maximum [Ca2+] with 1mM CaCl2 in digitonin-treated cells for 20 minutes [Ca2+] in the root tip. The lowest ratio represents the minimum [Ca2+] in the BAPTA-AM and EGTA-treated cells for 15 minutes in the root tip. Error bars denote ±s.e.m., data from at least three independent experiments (total number of plants: CRS (EGTA and BAPTA-AM), n=9; CRS (Digitonin), n=9; CRS-NLS (EGTA and BAPTA-AM), n=9; CRS (Digitonin), n=10; CRS-PM (EGTA and BAPTA-AM), n=7; CRS-PM (Digitonin), n=7). Ratio, relative fluorescence ratio of GCaMP6s to dTomato. All experiments were conducted in at least three biological repeats with similar results.

Extended Data Fig. 8 A FRET-based sensor CPKaleon shows that CPK32aleon is activated in response to ABA in root tip protoplasts.

a, Schematic diagram of CPKaleons, displaying the variable ___domain, kinase ___domain, pseudosubstrate segment (PS), and calmodulin-like ___domain (CLD) containing four EF-hand motifs. eGFP and cpVenus173 sandwich the CPK PS-CLD. b, Fluorescence accumulation in root tip protoplasts expressing CPKaleons with or without digitonin treatment. Fluorescence accumulation was quantified. Error bars denote ±s.e.m., data from at least three independent experiments (total number of protoplasts: CPK2aleon mock, n=24; CPK2aleon Digitonin, n=36; CPK32aleon mock, n=25; CPK2aleon Digitonin, n=25). ns (not significant) P > 0.05, **** P < 0.0001 (statistical significance determined by two-tailed non-paired Student’s t test). c, CPK32, but not CPK2, is activated by ABA in root tip protoplasts. Error bars denote ±s.e.m., data from at least three independent experiments (total number of protoplasts: CPK2aleon mock, n=55; CPK2aleon ABA, n=29; CPK32aleon mock, n=23; CPK2aleon ABA, n=30). ns (not significant) P > 0.05, *** P < 0.001 (statistical significance determined by two-tailed non-paired Student’s t test). All experiments were conducted in at least three biological repeats with similar results.

Extended Data Fig. 9 Relative expression of genes associated with the root cap differentiation program in response to ABA.

The analysis was conducted on 5-day-old wild-type and pyrpyl112458 (a) or icpk (c) root tips in response to ABA, and 5-day-old wild-type (b) with or without 100 μM EGTA-AM pretreatment for 30 minutes in response to ABA. The RT-qPCR analyses of gene expression levels were normalized to the expression of UBQ10 in each sample. The average gene expression ratio from triplicate samples was calculated and is presented in Fig. 5d, Fig. 5e and Fig. 5f. d, The analysis was conducted on 5-day-old wild-type root tips in response to 200 mM NaCl treatment. The RT-qPCR analyses of gene expression levels were normalized to the expression of UBQ10 in each sample. The average gene expression ratio from triplicate samples was calculated and is presented in Fig. 6d.

Extended Data Fig. 10 Salt stress promotes ABA accumulation in root tips and inhibits root cap growth.

a, ABA accumulation in root cap cells and epidermal cells measured using the FRET sensor nlsABACUS2-400n with or without 200 mM NaCl treatment for 30 min. Scale bars, 10 μm. ABA accumulation was quantified. Error bars denote ±s.e.m., n=10 plants. **** P < 0.0001 (statistical significance determined by two-tailed non-paired Student’s t test). b, Treatment with 200 mM NaCl for 24 h severely inhibited root cap cycle in 5-day-old WT plants. Scale bars, 10 μm. All results are reproducible from at least three independent experiments.

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Lin, Z., Guo, Y., Zhang, R. et al. ABA-activated low-nanomolar Ca2+–CPK signalling controls root cap cycle plasticity and stress adaptation. Nat. Plants 11, 90–104 (2025). https://doi.org/10.1038/s41477-024-01865-y

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