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Ca2+/calmodulin-mediated desensitization of glutamate receptors shapes plant systemic wound signalling and anti-herbivore defence

Nature Plants volume 10, pages 145–160 (2024)Cite this article

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Abstract

Plants rely on systemic signalling mechanisms to establish whole-body defence in response to insect and nematode attacks. GLUTAMATE RECEPTOR-LIKE (GLR) genes have been implicated in long-distance transmission of wound signals to initiate the accumulation of the defence hormone jasmonate (JA) at undamaged distal sites. The systemic signalling entails the activation of Ca2+-permeable GLR channels by wound-released glutamate, triggering membrane depolarization and cytosolic Ca2+ influx throughout the whole plant. The systemic electrical and calcium signals rapidly dissipate to restore the resting state, partially due to desensitization of the GLR channels. Here we report the discovery of calmodulin-mediated, Ca2+-dependent desensitization of GLR channels, revealing a negative feedback loop in the orchestration of plant systemic wound responses. A CRISPR-engineered GLR3.3 allele with impaired desensitization showed prolonged systemic electrical signalling and Ca2+ waves, leading to enhanced plant defence against herbivores. Moreover, this Ca2+/calmodulin-mediated desensitization of GLR channels is a highly conserved mechanism in plants, providing a potential target for engineering anti-herbivore defence in crops.

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Fig. 1: The GLR3.3 CTD plays a dual role in systemic electrical signalling.
Fig. 2: A unique region in GLR3.3 binds CaM in a Ca2+-dependent manner.
Fig. 3: Ca2+/CaM strongly desensitizes the GLR3.3 channel.
Fig. 4: GLR3.3-T3 alteration impairs Ca2+/CaM-mediated desensitization of the GLR3.3 channel.
Fig. 5: CRISPR-engineered GLR3.3 gain-of-function allele enhances plant systemic wound responses and anti-herbivore defence.
Fig. 6: Ca2+/CaM-mediated desensitization of GLR channels is highly conserved across plant species.

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Data availability

All data supporting the findings of this study are available within the paper and its supplementary information files. The raw mass spectrometry data were searched against the TAIR10 database (https://www.arabidopsis.org/). Source data are provided with this paper.

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Acknowledgements

This work was supported by grants from the National Institutes of Health (no. R01GM138401 to S.L.) and the Youth Program of the National Natural Science Foundation of China (no. 32000204 to C.Y.).

Author information

Author notes

  1. These authors contributed equally: Chun Yan, Qifei Gao, Mai Yang.

Authors and Affiliations

  1. Department of Plant and Microbial Biology, University of California, Berkeley, Berkeley, CA, USA

    Chun Yan, Qifei Gao, Qiaolin Shao & Sheng Luan

  2. School of Life Sciences, Tsinghua University, Beijing, China

    Mai Yang

  3. School of Engineering Medicine, Beihang University, Beijing, China

    Xiaopeng Xu & Yongbiao Zhang

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Contributions

S.L. and C.Y. conceived the work and designed the experiments. C.Y., Q.G. and M.Y. performed the experiments with assistance from Q.S., X.X. and Y.Z. S.L. and C.Y. wrote the paper with contributions from all authors.

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Correspondence to Sheng Luan.

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Extended data

Extended Data Fig. 1 CRISPR-engineered GLR3.3 mutant alleles with varying lengths of CTD.

a-d Nucleotide sequence alignment of CTDs from wild-type (CT) and CRISPR-engineered GLR3.3 mutant alleles (T1–T4). Top panel shows the row sequencing chromatogram. The 20 bp target sequences recognized by sgRNAs are underlined in black, and the protospacer adjacent motifs (PAM) are shown in black frame. The inserted or deleted nucleotides are highlighted in red. e Transient expression of GLR3.3 mutant alleles in Nicotiana benthamiana leaf epidermal cells. Agrobacterium strains harboring different GLR3.3 alleles were infiltrated into N. benthamiana leaves, and Venus fluorescence was observed three days after infiltration. Non-infiltrated leaves (denoted as empty) serve as a negative control to exclude nonspecific Venus fluorescence. Scale bar = 20 μm. Images are representative of three independent biological replicates. Related to Fig. 1.

Extended Data Fig. 2 Quantitative comparison of wound- and Glu-triggered SWPs in the complemented plants.

a Experimental setup for recording SWPs on rosette leaves after wounding or Glu treatment of primary roots. b, c Quantitative depolarization amplitudes of wound-triggered SWPs (b) or Glu-triggered SWPs (c) recorded from WT and GLR3.3 mutants complemented with pGLR3.3::GLR3.3-EGFP transgene (n = 11, 10, 11 and 10 plants, respectively). Boxplots show all data points from minimum to maximum (whiskers), median (line), and 25th-75th percentiles (box limits). Each colored point represents an individual plant, and data points with close values are highlight in darker colors (ns, not significant). Related to Fig. 1.

Source data

Extended Data Fig. 3 CaM7 binds to GLR3.3 CTD in a Ca2+-dose dependent manner.

a MBP–C2 was immobilized with amylose beads to pull down GST–CaM7 in the presence of Ca2+ concentration gradients or 2 mM EGTA. Three independent replicates were performed with similar results. b Control titration showing no obvious heat changes when the buffer without CBD peptide was titrated into 6His–CaM7 protein. Related to Fig. 2.

Source data

Extended Data Fig. 4 Quantitative analysis of wound- and Glu-triggered SWPs in CaM7 knockout or overexpression plants.

a Distribution of CaM7 protein in primary root of 10-day-old pCaM7::CaM7–ECFP seedling. Scale bar = 20 μm. b Transcript levels of CaM7 in WT, T-DNA insertional mutants (cam7-1 and cam7-3), and overexpression lines (Flag–CaM7-3 and Flag–CaM7-8) quantified by qRT-PCR. Columns represent means ± SD, and colored points indicate different biological replicates (n = 3). P values were determined by unpaired two-tailed Student’s t-test. c-h Representative traces, as well as quantitative depolarization amplitudes and durations of wound-triggered SWPs (c-e) or Glu-triggered SWPs (f-h) recorded from different plants (n = 14, 13, 13, 12 and 12 plants, respectively). The boxplots show all data points from minimum to maximum (whiskers), median (line), and 25th-75th percentiles (box limits). Each colored point represents an individual plant, and data points with close values are highlighted in darker colors. One-way ANOVA followed by Dunnett’s multiple comparisons test was employed to evaluate statistical significance between each group and the WT group.

Source data

Extended Data Fig. 5 CaMs potently desensitize the Glu-induced activity of GLR3.3 channel.

a Representative time-lapse images of Glu-activated [Ca2+]cyt increases in COS-7 cells expressing GLR3.3, CaM2, CaM7, or their combinations. b Representative time-lapse images of Glu-activated [Ca2+]cyt increases in COS-7 cells expressing CaM7, GLR3.3, GLR3.3-CD2 (harboring CBD-deleted CTD), or their combinations. Scale bar = 50 μm. Representative images are shown from more than eight biological replicates. Related to Fig. 3.

Extended Data Fig. 6 ITC assay confirms that the mutant CBD impairs the CaM binding capacity of GLR3.3.

The mutant CBD peptide (with 5-amino acid deletion) was chemically synthesized and used to titrate 6His–CaM7 protein in the presence of 1 mM Ca2+ (a, KD = 71.30 ± 14.16 μM) or 2 mM EGTA (b, no detectable binding). Control titration was performed by titrating the buffer into 6His–CaM7 protein (c, no detectable binding). Related to Fig. 4.

Extended Data Fig. 7 The mutant CBD impairs GLR3.3 channel desensitization.

Representative time-lapse images of Glu-activated [Ca2+]cyt increases in COS-7 cells expressing CaM7, GLR3.3, GLR3.3-T3 (harboring mutant CBD), or their combinations. Scale bar = 50 μm. Representative images are shown from more than eleven biological replicates. Related to Fig. 4.

Extended Data Fig. 8 Quantitative comparison of the Glu-triggered Ca2+ waves in plants with different GLR3.3 alleles.

Whole-plant Ca2+ imaging shows that Glu-triggered systemic Ca2+ signaling is enhanced in plants with GLR3.3 gain-of-function allele (3.3T3) but reduced in plants with GLR3.3 loss-of-function allele (3.3T4). a Representative images of WT, 3.3T3, and 3.3T4 plants expressing genetically encoded intracellular Ca2+ indicator MatryoshCaMP6s at the indicated time points after 100 mM L-Glu application to wound site of primary roots. Scale bar, 5 mm. b Close-up of systemic rosette leaves from whole-plant Ca2+ imaging shown in (a). Scale bar, 2 mm. Representative images are shown from more than ten independent plants. c-f Average time-course curves (c) and quantitative [Ca2+]cyt levels measured at peak (d), 180 s (e), 360 s (f) in systemic rosette leaves after Glu application to wound site of primary roots. Relative change (∆F) or maximum change (∆Fmax) of fluorescence was normalized to initial fluorescence (F0) before Glu application. Data are means ± SEM, and each colored point represents an individual plant (n = 16, 14 and 10 plants, respectively). P values were determined by unpaired two-tailed Student’s t-test, and P < 0.05 or lower was considered statistically significant. Related to Fig. 4.

Source data

Extended Data Fig. 9 Phylogenetic analysis and sequence alignment of GLR3.3 homologs.

a Phylogenetic analysis of Arabidopsis GLR3 members. The phylogenetic tree was constructed using neighbor-joining (NJ) method with 1,000 bootstrap replicates. b Sequence alignment of CTDs of Arabidopsis GLR3 members. c Sequence alignment of CTDs of GLR3.3 homologs from various plant species. Red background marks amino acids with 100% identity; blue box marks amino acids with more than 70% similarity; red text marks conserved amino acids. Underlined sequence indicates the CBD in GLR3.3. Related to Fig. 6.

Extended Data Fig. 10 Ca2+/CaM-mediated desensitization of GLR channels is highly conserved.

a Representative time-lapse images of Glu-activated [Ca2+]cyt increases in COS-7 cells expressing CaM7, GLR3 members (GLR3.1, GLR3.3, or GLR3.6), or their combinations. b Representative time-lapse images of Glu-activated [Ca2+]cyt increases in COS-7 cells expressing CaM7, GLR3.3 homologs (GLR3.3, BoGLR3.3, or OsGLR3.1), or their combinations. Scale bar = 50 μm. Representative images are shown from more than nine biological replicates. Related to Fig. 6.

Supplementary information

Supplementary Information

Supplementary Tables 1 and 2.

Reporting Summary

Supplementary Video 1

Whole-plant Ca2+ imaging of a wild-type plant upon wounding the primary root.

Supplementary Video 2

Whole-plant Ca2+ imaging of a 3.3T3 plant upon wounding the primary root.

Supplementary Video 3

Whole-plant Ca2+ imaging of a 3.3T4 plant upon wounding the primary root.

Supplementary Video 4

Whole-plant Ca2+ imaging of a wild-type plant upon Glu application to the wound site of the primary root.

Supplementary Video 5

Whole-plant Ca2+ imaging of a 3.3T3 plant upon Glu application to the wound site of the primary root.

Supplementary Video 6

Whole-plant Ca2+ imaging of a 3.3T4 plant upon Glu application to the wound site of the primary root.

Source data

Source Data Figs. 1 and 3–6 and Extended Data Figs. 2, 4 and 8

Statistical source data for Figs. 1 and 3–6 and Extended Data Figs. 2, 4 and 8.

Source Data Figs. 2, 4 and 6 and Extended Data Fig. 3

Unprocessed western blots for Figs. 2, 4 and 6 and Extended Data Fig. 3.

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Yan, C., Gao, Q., Yang, M. et al. Ca2+/calmodulin-mediated desensitization of glutamate receptors shapes plant systemic wound signalling and anti-herbivore defence. Nat. Plants 10, 145–160 (2024). https://doi.org/10.1038/s41477-023-01578-8

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  • DOI: https://doi.org/10.1038/s41477-023-01578-8

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