Integration of ovular signals and exocytosis of a Ca2+ channel by MLOs in pollen tube guidance

Abstract

The spatiotemporal regulation of Ca2+ channels at the plasma membrane in response to extracellular signals is critical for development, stress response and reproduction, but is poorly understood. During flowering-plant reproduction, pollen tubes grow directionally to the ovule, which is guided by ovule-derived signals and dependent on Ca2+ dynamics. However, it is unknown how ovular signals are integrated with cytosolic Ca2+ dynamics in the pollen tube. Here, we show that MILDEW RESISTANCE LOCUS O 5 (MLO5), MLO9 and MLO15 are required for pollen tube responses to ovular signals in Arabidopsis thaliana. Phenotypically distinct from the ovule-bypass phenotype of previously identified mutants, mlo5mlo9 double-mutant and mlo5mlo9mlo15 triple-mutant pollen tubes twist and pile up after sensing the ovular cues. Molecular studies reveal that MLO5 and MLO9 selectively recruit Ca2+ channel CNGC18-containing vesicles to the plasma membrane through the R-SNARE proteins VAMP721 and VAMP722 in trans mode. This study identifies members of the conserved seven transmembrane MLO family (expressed in the pollen tube) as tethering factors for Ca2+ channels, reveals a novel mechanism of molecular integration of extracellular ovular cues and selective exocytosis, and sheds light on the general regulation of MLO proteins in cell responses to environmental stimuli.

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Fig. 1: mlo5mlo9 mutant pollen tubes curl after sensing ovular signals.
Fig. 2: Ovule-derived signals induce pollen tube twisting in mlo5mlo9 mutants.
Fig. 3: MLOs function redundantly in the recruitment of CNGC18 and in pollen tube targeting to the ovule.
Fig. 4: Shift of tip-focused Ca2+, CNGC18-GFP and MLO9-GFP during pollen tube turning.
Fig. 5: MLO5 and MLO9 interact with CNGC18.
Fig. 6: Interaction between MLO5 and VAMPs and phenotype of vamp721vamp722 mutant.

Data availability

The data for the current study are available within the paper and the Supplementary Information or from the corresponding authors upon request.

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Acknowledgements

We thank S. Huang (Tsinghua University) for the G-CaMP5 plasmids, L. Zhang (Henan Normal University) for the vamp721/722 seeds, Y.-F. Wang (Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) for the cngc18-17 and cngc18-22 seeds, J. Kudla (University of Munster) and P. Lefert-Schulze (Max Planck Institute for Plant Breeding Research) for critical comments, and R. Panstruga (RWTH Aachen University) for manuscript editing. We also thank the staff of the microscopy and proteomics platform from the Institute of Genetics and Developmental Biology for assisting in imaging and mass spectrometry. This work was supported by the NSFC grant nos. 31622010, 31870295 and 31571385 to H.-J.L., 31330053 to W.-C.Y. and the MSTC 2016YFA0500500 to W.-C.Y.

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Contributions

J.-G.M. and L.L. performed the experiments. W.-C.Y. and H.-J.L. conceived and supervised the project and wrote the manuscript. H.-J.L., J.-G.M., L.L. and W.-C.Y. designed the experiments and analysed the results. P.-F.J. provided technical assistance for the microscopy experiments. Y.-C.W. provided technical assistance for the mass spectrometry experiments.

Corresponding authors

Correspondence to Hong-Ju Li or Wei-Cai Yang.

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Extended data

Extended Data Fig. 1 MLOs expression, mutant characterization and phenotyping.

a, Real-time PCR analysis showing the expression of MLO members in root and pollen. Data are mean±s.e.m. of 3 replicates. b, Phylogenetic tree of MLO family. c, T-DNA insertion sites in mlo5 and mlo9 (left panel). Arrow, site of primers used for the RT-RCR analysis in d, Right pane, protein topology and the corresponding mutation sites of the mutants. d, RT-PCR analysis of the mutants. e, Seed set of WT, mlo5-1, mlo5-3, mlo9, and double mutants. Bar, 2 mm. f, Aniline-blue stained pollen tubes of mlo5-1 and mlo9 at 8 hours after pollination on WT stigma. Bar, 200 μm. n=3 (for d) and 6 (for e and f) biological independent experiments were performed with similar results. g, Quantification of pollen germination ratio of the WT and mlo5-1 mlo9. Data are mean±s.e.m, n=1000 pollen for each genotype. Two-tailed Students’ t-test. p=0.7465. n.s. No statistical significance. Source data

Extended Data Fig. 2 Expression and subcellular localization of MLO5 and MLO9.

a, b, Confocal imaging of pollen grains from MLO5:MLO5-GFP transgenic plants. a, GFP channel; b, Merged GFP and bright channels. c, Confocal imaging of pollen tubes from MLO5:MLO5-GFP transgenic plants. df, The mutated MLO5 fails to target to the plasma membrane (d and e) and cannot rescue the shortened mlo5/9 pollen tubes (f). e, Fluorescence intensity of d. Bar in ad, 5 μm; f, 1 mm. n=6 independent biological experiments were performed for af with similar results.

Extended Data Fig. 3 mlo5/9 pollen tubes turn to LURE1.2-embedded beads normally.

WT (a) and mlo5/9 (b) pollen tubes respond to the LURE1.2-embedded beads at a similar frequency (c). n=69 and n=79 pollen tubes for WT and mlo5/9. Asterisk, LURE1-embedded beads. Data are mean±s.e.m. Two-tailed Students’ t-test, p=0.8527. n.s. No statistical significance. n=6 biologically independent experiments were repeated with similar results. Bar, 100 μm. Source data

Extended Data Fig. 4 Ca2+ influx in mlo5/9 is disrupted.

a, Pollen tube growth of mlo5/9 is sensitive to low extracellular Ca2+ concentration and insensitive to Ca2+ channel blocker LaCl3. b, Statistics of a. Data are mean±s.e.m. Two-tailed Students’ t-test, p*=0.018 for comparison between 1.5 mM Ca2+ and 10 μM LaCl3 treatments of WT. p**=0.0035 for comparison between 1.5 mM Ca2+ and 0.5 μM Ca2+ treatments of mlo5 mlo9. n=10, 13, 11, 9, 13, 15, 16, 14, 13 and 14 pistils for each treatment from left to right. p=0.0543, p=0.7471, p=0.3322 for comparison between different concentration of LaCl3 and 1.5 mM Ca2+ treatments of mlo5 mlo9. n.s. No statistical significance. Bar, 400 μm. Source data

Extended Data Fig. 5 Cl- and K+ flux is normal, but Ca2+ is defective in mlo5/9 pollen tubes.

The flux at the pollen tube tip was measured by NMT. Each plot represents the net flux values of multiple pollen tubes. n=3 biologically independent experiments for a-c were repeated with similar results. Source data

Extended Data Fig. 6 Localization of different proteins in mlo5/9 pollen tubes.

a, Confocal images showing CNGC18-mCherry in mlo5/9 pollen tubes (left panel) and fluorescence intensity (right panel). b, c, Confocal images showing that the localization of GLR1.2-GFP, PIP2;7-GFP MDIS1-GFP, PRK6-GFP, ANX1-GFP, Lti6b-GFP and RabA4D is not impaired in mlo5/9 pollen tubes. Bar, 10 μm. n=6 biologically independent experiments for ac were repeated with similar results.

Extended Data Fig. 7 The phenotyping of cngc18 site mutations.

a, b, The pollen from the mutants were pollinated onto the WT stigma and stained at 20 hours after pollination. Arrow, twisted pollen tubes. c, d, Attraction frequency of cngc18-17 and cngc18-22 pollen tubes to LURE1.2-embedded beads is no difference with the WT. Data are mean±s.e.m. Two-tailed Students’ t-test, p=0.3205, p=0.7364, respectively. n.s. No statistical significance. Bar, 50 μm. n=78, n=69 and n=71 for each genotype. Source data

Extended Data Fig. 8 Function and expression analysis of CNGC18 in mlo5/9.

a, Transcription of CNGC18 in mlo5/9 is not affected. 1, 2 and 3 are pollen tubes from three different plants of the annotated genotype. b, Overexpression of CNGC18-GFP can partially rescue the impaired pollen tube growth of mlo5/9. Bar, 1 mm. n=3 biologically independent experiments for ab were repeated with similar results. c, Pollen tube length of different transgenic lines of mlo5/9 expressing CNGC18-GFP. Data are mean±s.e.m. n=17, 12, 14, 16, 11 and 9 pistils for each genotype. Two-tailed Students’t-test, p***=0.000299153, p***=0.000558313, p***=0.000566278, p***= 0.000576008 for 1# to 4#, respectively. d, e, NMT analysis showing the Ca2+ flux of two transgenic lines (1# and 2#) compared with the same mlo5/9 pollen tube. n=9 pollen tubes were measured for each genotype. n=3 independent experiments for d, e were repeated with similar results. Source data

Extended Data Fig. 9 Triple mutants generated by CRISPR technology.

a, Mutations of MLO15 in two different alleles (mlo15-1 and mlo15-2). b, Semi-in vitro pollen tube length of mlo5/9 and mlo5/9/15-1 mutants. Data are mean±s.e.m. Two-tailed Students’ t-test, p***=4.39×10-5, p***=0.000569674 for 1# and 2#. n=23, 19, 18, 14, 15 pistils for each genotype. c, Representative images of in vivo pollen tube length of mlo5/9 and mlo5/9/15-1 mutants at 3, 5 and 8 hours after pollination (HAP). Bar, 200 μm. d, Quantification of c. Data are mean±s.e.m. Two-tailed Students’t-test, p*=0.0378, p**=0.005820983, p**=0.001425173 for comparison between mlo5/9 and mlo5/9 /15-1 at 3HAP, 5HAP, 8HAP, respectively. n.s. No statistical significance. p=0.27263357 for WT and mlo5/9 at 8HAP. n=19, 17, 18, 16, 10, 18, 20, 19 and 19 pistils for each sample. Source data

Extended Data Fig. 10 The working model of MLO-mediated vesicle fusion of CNGC18 to the plasma membrane in response to ovular signals.

a, Model of the MLOs-mediated CNGC18-vesicle fusion through the syntaxin (SYP) and VAMP to the plasma membrane site where the ovular signals are sensed. b, Model of pollen tube response to the ovule. In the wild-type, perception of ovular signals and MLO5/9/15-mediated CNGC18 recruitment to the direction of ovular signals, which is necessary for Ca2+ shift, are coupled to facilitate directional growth. In mlo5/9/15 pollen tubes, background Ca2+ gradient is maintained in the pollen tube tip, but lack of MLO5/9/15 causes failed shift of CNGC18 and Ca2+ gradient to the direction of ovule signals. But some machinery for ovular signal perception and pollen tube growth are still working. In this condition, the growth and the reorientation of pollen tubes are decoupled and the resulting two potential growth-points cause the pollen tubes grow forward and backward, and even turn in circles. c, The diagram of the pollen tube twisting phenotype of mlo5/9 and mlo5/9/15 mutants.

Supplementary information

Supplementary Information

Supplementary Figs. 1 and 2, and primers used in this study.

Reporting Summary

Supplementary Video 1

3D imaging of WT pollen tubes on the septum by confocal microscopy.

Supplementary Video 2

3D imaging of mlo5mlo9 pollen tubes on the septum by confocal microscopy.

Supplementary Video 3

Dynamics of MLO5-GFP in growing pollen tubes.

Supplementary Video 4

Dynamics of MLO9-GFP in growing pollen tubes.

Supplementary Video 5

Dynamics of cytosolic Ca2+ in the WT pollen tube growing in LURE1.2 solution.

Supplementary Video 6

Dynamics of cytosolic Ca2+ in the mlo5 mlo9 pollen tube growing in LURE1.2 solution.

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Meng, J., Liang, L., Jia, P. et al. Integration of ovular signals and exocytosis of a Ca2+ channel by MLOs in pollen tube guidance. Nat. Plants 6, 143–153 (2020). https://doi.org/10.1038/s41477-020-0599-1

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