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MpMLO1 controls sperm discharge in liverwort

Abstract

In bryophytes, sexual reproduction necessitates the release of motile sperm cells from a gametophyte into the environment. Since 1856, this process, particularly in liverworts, has been known to depend on water. However, the molecular mechanism underlying this phenomenon has remained elusive. Here we identify the plasma membrane protein MpMLO1 in Marchantia polymorpha, a model liverwort, as critical for sperm discharge from antheridia. The MpMLO1-expressing tip cells among the sperm-wrapping jacket cells undergo programmed cell death upon antheridium maturation to facilitate sperm discharge after the application of water and even hypertonic solutions. The absence of MpMLO1 leads to reduced cytoplasmic Ca2+ levels in tip cells, preventing cell death and consequently sperm discharge. Our findings reveal that MpMLO1-mediated programmed cell death in antheridial tip cells, regulated by cytosolic Ca2+ dynamics, is essential for sperm release, elucidating a key mechanism in bryophyte sexual reproduction and providing insights into terrestrial plant evolution.

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Fig. 1: Expression pattern of MpMLO genes in male M. polymorpha.
Fig. 2: Knockout of MpMLO1 disrupts sperm discharge.
Fig. 3: Subcellular localization of MpMLO1–Citrine in antheridia at different developmental stages.
Fig. 4: The tip cells undergo PCD before sperm discharge.
Fig. 5: Necessity of water in sperm discharge.
Fig. 6: Functional conservation of the MpMLO1–Ca2+ module in liverwort and Arabidopsis.

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Data availability

The data for the current study are available within the paper and the Supplementary Information or from the corresponding author upon request. Source data are provided with this paper.

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Acknowledgements

We thank E. Wang (CAS Center for Excellence in Molecular Plant Science) and J. Zuo (Institute of Genetics and Developmental Biology, CAS) for the WT M. polymorpha plants, W.-C. Yang for critical comments and supervision, and the public technology service centre (Institute of Genetic and Developmental Biology, CAS) for assistance in confocal microscopy. This work was supported by the National Natural Science Foundation of China (grant no. 32170343) and the CAS Project for Young Scientists in Basic Research (grant no. YSBR-078).

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Contributions

M.-X.C. and S.-Z.L. performed the experiments and analysed the data. H.-J.L. conceived the study and supervised the project. M.-X.C. S.-Z.L. and H.-J.L. wrote the paper.

Corresponding author

Correspondence to Hong-Ju Li.

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Nature Plants thanks Moritz Nowack and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Extended data

Extended Data Fig. 1 Expression pattern of MpMLO genes.

a. RT-PCR analysis of MpMLO genes in the vegetative tissues, antheridiophore and archegoniophore. The control in archegoniophores and antheridiophores are represented by PHD and CAPE, while EF1α is used as internal reference. b. Histochemical GUS assay of the MpMLO genes in rhizoids. No GUS signal was detected in the rhizoids of plants carrying the MpMLO promoter-driven GUS construct. Bar, 1 mm.

Source data

Extended Data Fig. 2 Mutation of each MpMLO gene generated by CRISPR/Cas9.

The gene-editing regions of Mpmlo2, Mpmlo3 and Mpmlo4 are all located in the first extron. Extrons are showed with black blocks. Red asterisks, deleted bases. Red letter, inserted base.

Extended Data Fig. 3 The thalli and antheridia of the wild type and each Mpmlo mutant.

a–e. Morphological characteristics of thallus in WT and Mpmlo mutants. Scale bar, 1 mm. f. Quantification of the antheridial pore numbers in the SEM images. Data are shown as the mean ± s.e.m. n = 11 and 10 antheridiophores for each genotype. Two-tailed Student’s t-test: p = 0.5668. n.s. no statistical significance. g. Quantification of the antheridia number in h and i. Data are shown as the mean ± s.e.m. n = 14 and 16 antheridiophores for each genotype. Two-tailed Student’s t-test: p = 0.4230. h and i. Transverse sections of mature antheridiophore in WT (h) and Mpmlo1 (i). Arrows, antheridia. Scale bar, 500 μm. j and k. Number and location of antheridia in wild type and Mpmlo1. Vertical section of antheridiophores in Wild type (j) and Mpmlo1 #1 (k). The growth locations of antheridia in Mpmlo1 #1 is consistent with WT. Asterisks, antheridia. Scale bar, 100 μm.

Extended Data Fig. 4 Sperm discharge assay in Mpmlo1, Mpmlo2, Mpmlo3 and Mpmlo4 mutants.

af. Sperm discharge after application of water drops. Red arrows, released sperm masses. Scale bar, 1 mm. g. Thickened jacket layer in the two Mpmlo1 mutant lines. White arrows indicate the jacket cell layer; Scale bar, 100 µm. h. Quantification of the width of different Mpmlo1 mutant lines. Data are shown as the mean ± s.e.m. n = 12, 11 and 12 antheridia for each line (from left to right). Significant differences were determined using one-way ANOVA, different letters indicate values with statistically significant (p < 0.05; Tukey honest significant difference [HSD]) and non-significant (p > 0.05; Tukey HSD) differences, respectively.

Extended Data Fig. 5 Development of jacket layer in wild type and Mpmlo mutants.

Morphological characteristics of antheridia isolated at immature and mature stages in Mpmlo mutants. Scale bar, 20 μm.

Extended Data Fig. 6 Development of antheridia in wild type, Mpmlo2, Mpmlo3 and Mpmlo4.

Vertical section of antheridia at different stages in wild type and the mutants of Mpmlo2, Mpmlo3, and Mpmlo4. Antheridia in Mpmlo2, Mpmlo3, and Mpmlo4 show no obvious difference with wild type during the developmental stages. Scale bar, 20 μm.

Extended Data Fig. 7 Expression of MpMLO1-Citrine in antheridia.

a–c. MpMLO1-Citrine expressed in whole-mount antheridia. Arrow, jacket layer. Scale bar, 50 μm. d–g. MpMLO1-Citrine expressed in flagella of sperm. Nuclei were stained by DAPI. BF, bright field. Scale bar, 5 μm.

Extended Data Fig. 8 TUNEL and FDA-PI staining of antheridia in wild type and Mpmlo1 mutant.

a. TUNEL assay for the WT and Mpmlo1 mutants. The images of TUNEL staining are representative of 141 antheridia. Three independent experiments were performed. Positive control, Dnase I treatment prior to TUNEL staining; Negative control, TUNEL staining performed without adding TdT enzyme. Details shown in Method part. Scale bar, 100 µm. b. FDA-PI staining of immature antheridia. In a and b, the white arrows indicate the antheridial tip cells. Scale bar, 100 µm. For a and b, three independent experiments were performed.

Extended Data Fig. 9 Histochemical staining of antheridia in wild type and Mpmlo1 mutant.

a. Aniline blue staining of antheridia in MpMLO1-Citrine-expressing WT and Mpmlo1 #1. Red, callose. b. Calcofluor White staining of antheridia in MpMLO1-Citrine-expressing WT and Mpmlo1 #1. Magenta, cellulose. c. JIM7 immunostaining of antheridia in MpMLO1-Citrine-expressing WT and Mpmlo1 #1. Red, esterified pectin. Green, fluorescence of MpMLO1-Citrine. For each staining, three independent experiments were performed. Arrows, tip cells. Scale bar, 50 μm.

Supplementary information

Supplementary Information

Supplementary Table 1.

Reporting Summary

Supplementary Video 1

Swimming WT sperm cells.

Supplementary Video 2

Swimming Mpmlo1 sperm cells.

Supplementary Video 3

3D imaging of MpMLO1–Citrine in the immature antheridia.

Supplementary Video 4

3D imaging of MpMLO1–Citrine in the mature antheridia.

Supplementary Video 5

Time-lapse imaging of Ca2+ in the WT antheridium.

Supplementary Video 6

Time-lapse imaging of Ca2+ in the Mpmlo1 #1 antheridium.

Supplementary Video 7

Time-lapse imaging of Ca2+ in the Mpmlo1 #2 antheridium.

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Cao, MX., Li, SZ. & Li, HJ. MpMLO1 controls sperm discharge in liverwort. Nat. Plants 10, 1027–1038 (2024). https://doi.org/10.1038/s41477-024-01703-1

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