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, mlo5 mlo9 double-mutant and mlo5 mlo9 mlo15 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.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Data availability
The data for the current study are available within the paper and the Supplementary Information or from the corresponding authors upon request.
References
Demidchik, V., Shabala, S., Isayenkov, S., Cuin, T. A. & Pottosin, I. Calcium transport across plant membranes: mechanisms and functions. New Phytol. 220, 49–69 (2018).
Dodd, A. N., Kudla, J. & Sanders, D. The language of calcium signaling. Annu. Rev. Plant Biol. 61, 593–620 (2010).
Michard, E., Simon, A. A., Tavares, B., Wudick, M. M. & Feijó, J. A. Signaling with ions: the keystone for apical cell growth and morphogenesis in pollen tubes. Plant Physiol. 173, 91–111 (2017).
Yang, W. C., Shi, D. Q. & Chen, Y. H. Female gametophyte development in flowering plants. Annu. Rev. Plant Biol. 61, 89–108 (2010).
Li, H. J. et al. Multilayered signaling pathways for pollen tube growth and guidance. Plant Reprod. 31, 31–41 (2018).
Li, H. J. & Yang, W. C. RLKs orchestrate the signaling in plant male–female interaction. Sci. China Life Sci. 59, 867–877 (2016).
Higashiyama, T. & Yang, W. C. Gametophytic pollen tube guidance: attractant peptides, gametic controls, and receptors. Plant Physiol. 173, 112–121 (2017).
Li, H. J. et al. Arabidopsis CBP1 is a novel regulator of transcription initiation in central cell-mediated pollen tube guidance. Plant Cell 27, 2880–2893 (2015).
Chen, Y. H. et al. The central cell plays a critical role in pollen tube guidance in Arabidopsis. Plant Cell 19, 3563–3577 (2007).
Takeuchi, H. & Higashiyama, T. A species-specific cluster of defensin-like genes encodes diffusible pollen tube attractants in Arabidopsis. PLoS Biol. 10, e1001449 (2012).
Zhong, S. et al. Cysteine-rich peptides promote interspecific genetic isolation in Arabidopsis. Science 364, eaau9564 (2019).
Meng, J. G., Zhang, M. X., Yang, W. C. & Li, H. J. TICKET attracts pollen tubes and mediates reproductive isolation between relative species in Brassicaceae. Sci. China Life Sci. 62, 1413–1419 (2019).
Wang, T. et al. A receptor heteromer mediates the male perception of female attractants in plants. Nature 531, 241–244 (2016).
Takeuchi, H. & Higashiyama, T. Tip-localized receptors control pollen tube growth and LURE sensing in Arabidopsis. Nature 531, 245–248 (2016).
Cheung, A. Y. & Wu, H. M. Structural and signaling networks for the polar cell growth machinery in pollen tubes. Annu. Rev. Plant Biol. 59, 547–572 (2008).
Gao, Q. F. et al. Cyclic nucleotide-gated channel 18 is an essential Ca2+ channel in pollen tube tips for pollen tube guidance to ovules in Arabidopsis. Proc. Natl Acad. Sci. USA 113, 3096–3101 (2016).
Frietsch, S. et al. A cyclic nucleotide-gated channel is essential for polarized tip growth of pollen. Proc. Natl Acad. Sci. USA 104, 14531–14536 (2007).
Michard, E. et al. Glutamate receptor-like genes form Ca2+ channels in pollen tubes and are regulated by pistil d-serine. Science 332, 434–437 (2011).
Büschges, R. et al. The barley Mlo gene: a novel control element of plant pathogen resistance. Cell 88, 695–705 (1997).
Consonni, C. et al. Conserved requirement for a plant host cell protein in powdery mildew pathogenesis. Nat. Genet. 38, 716–720 (2006).
Chen, Z. et al. Two seven-transmembrane domain MILDEW RESISTANCE LOCUS O proteins cofunction in Arabidopsis root thigmomorphogenesis. Plant Cell 21, 1972–1991 (2009).
Kessler, S. A. et al. Conserved molecular components for pollen tube reception and fungal invasion. Science 330, 968–971 (2010).
Chen, Z. et al. Expression analysis of the AtMLO gene family encoding plant-specific seven-transmembrane domain proteins. Plant Mol. Biol. 60, 583–597 (2006).
Davis, T. C. et al. Arabidopsis thaliana MLO genes are expressed in discrete domains during reproductive development. Plant Reprod. 30, 185–195 (2017).
Qin, Y. et al. Penetration of the stigma and style elicits a novel transcriptome in pollen tubes, pointing to genes critical for growth in a pistil. PLoS Genet. 5, e1000621 (2009).
Devoto, A. et al. Topology, subcellular localization, and sequence diversity of the MLO family in plants. J. Biol. Chem. 274, 34993–35004 (1999).
Kim, M. C. et al. Calmodulin interacts with MLO protein to regulate defence against mildew in barley. Nature 416, 447–451 (2002).
Yang, W. C. et al. The SPOROCYTELESS gene of Arabidopsis is required for sporogenesis and encodes a novel protein. Genes Dev. 13, 2108–2117 (1999).
Baker, S. C., Robinson-Beers, K., Villanueva, J. M., Gaiser, J. C. & Gasser, C. S. Interactions among genes regulating ovule development in Arabidopsis thaliana. Genetics 145, 1109–1124 (1997).
Boisson-Dernier, A. et al. Disruption of the pollen-expressed FERONIA homologs ANXUR1 and ANXUR2 triggers pollen tube discharge. Development 136, 3279–3288 (2009).
Gao, Q. F., Fei, C. F., Dong, J. Y., Gu, L. L. & Wang, Y. F. Arabidopsis CNGC18 is a Ca2+-permeable channel. Mol. Plant 7, 739–743 (2014).
Tunc-Ozdemir, M. et al. A cyclic nucleotide-gated channel CNGC16 in pollen is critical for stress tolerance in pollen reproductive development. Plant Physiol. 161, 1010–1020 (2013).
Filippini, F. et al. Longins: a new evolutionary conserved VAMP family sharing a novel SNARE domain. Trends Biochem. Sci. 26, 407–409 (2001).
Bassham, D. C. et al. The secretory system of Arabidopsis. Arabidopsis Book 6, e0116 (2008).
Daste, F. et al. Structure and function of longin SNAREs. J. Cell Sci. 128, 4263–4272 (2015).
Bhat, R. A. et al. Recruitment and interaction dynamics of plant penetration resistance components in a plasma membrane microdomain. Proc. Natl Acad. Sci USA 102, 3135–3140 (2005).
Zhang, L. et al. Arabidopsis R-SNARE proteins VAMP721 and VAMP722 are required for cell plate formation. PLoS ONE 6, e26129 (2011).
Devoto, A. et al. Molecular phylogeny and evolution of the plant-specific seven-transmembrane MLO family. J. Mol. Evol. 56, 77–88 (2003).
Kusch, S. et al. Comprehensive phylogenetic analysis sheds light on the diversity and origin of the MLO family of integral membrane proteins. Genome Biol. Evol. 8, 878–895 (2016).
Ngo, Q. A., Vogler, H., Lituiev, D. S., Nestorova, A. & Grossniklaus, U. A calcium dialog mediated by the FERONIA signal transduction pathway controls plant sperm delivery. Dev. Cell 29, 491–500 (2014).
Yan, L. et al. High-efficiency genome editing in Arabidopsis using YAO promoter-driven CRISPR/Cas9 system. Mol. Plant 8, 1820–1823 (2015).
Li, H. J. et al. POD1 regulates pollen tube guidance in response to micropylar female signaling and acts in early embryo patterning in Arabidopsis. Plant Cell 23, 3288–3302 (2011).
Diao, M., Qu, X. & Huang, S. Calcium imaging in Arabidopsis pollen cells using G-CaMP5. J. Integr. Plant Biol. 60, 897–906 (2018).
Fang, K. et al. Boron toxicity causes multiple effects on Malus domestica pollen tube growth. Front. Plant Sci. 7, 208 (2016).
Gao, F. et al. The NDH-1L-PSI supercomplex is important for efficient cyclic electron transport in cyanobacteria. Plant Physiol. 172, 1451–1464 (2016).
Reisinger, V. & Eichacker, L. A. Analysis of membrane protein complexes by blue native PAGE. Proteomics S2, 6–15 (2006).
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.
Author information
Authors and Affiliations
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
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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.
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. d–f, 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 a–d, 5 μm; f, 1 mm. n=6 independent biological experiments were performed for a–f 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.
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.
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.
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 a–c 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.
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 a–b 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.
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.
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.
Supplementary Video 1
3D imaging of WT pollen tubes on the septum by confocal microscopy.
Supplementary Video 2
3D imaging of mlo5 mlo9 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.
Source data
Source Data Fig. 1
Statistical Source Data.
Source Data Fig. 2
Statistical Source Data.
Source Data Fig. 3
Statistical Source Data.
Source Data Fig. 5
Unprocessed western blots.
Source Data Fig. 6
Statistical Source Data.
Source Data Fig. 6
Unprocessed western blots.
Source Data Extended Data Fig. 1
Statistical Source Data.
Source Data Extended Data Fig. 3
Statistical Source Data.
Source Data Extended Data Fig. 4
Statistical Source Data.
Source Data Extended Data Fig. 5
Statistical Source Data.
Source Data Extended Data Fig. 7
Statistical Source Data.
Source Data Extended Data Fig. 8
Statistical Source Data.
Source Data Extended Data Fig. 9
Statistical Source Data.
Rights and permissions
About this article
Cite this article
Meng, JG., Liang, L., Jia, PF. 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
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/s41477-020-0599-1
This article is cited by
-
RALF signaling pathway activates MLO calcium channels to maintain pollen tube integrity
Cell Research (2023)
-
VAMP726 and VAMP725 regulate vesicle secretion and pollen tube growth in Arabidopsis
Plant Cell Reports (2023)
-
Nitrogen nutrition contributes to plant fertility by affecting meiosis initiation
Nature Communications (2022)
-
A receptor–channel trio conducts Ca2+ signalling for pollen tube reception
Nature (2022)
-
POD1-SUN-CRT3 chaperone complex guards the ER sorting of LRR receptor kinases in Arabidopsis
Nature Communications (2022)