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 Mp_MLO1_-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 Mp_MLO1_ 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. Access through your institution Buy or

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DEVELOPMENT OF HAPLOID REPRODUCTIVE ORGANS IN _MARCHANTIA POLYMORPHA_ Article 11 April 2024 FLUORESCENCE SHADOW IMAGING OF _HYPSIBIUS EXEMPLARIS_ REVEALS MORPHOLOGICAL DIFFERENCES BETWEEN

SUCROSE- AND CACL2-INDUCED OSMOBIOTES Article Open access 23 May 2024 DATA AVAILABILITY The data for the current study are available within the paper and the Supplementary Information or

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biological-image analysis. _Nat. Methods_ 9, 676–682 (2012). Article  CAS  PubMed  Google Scholar  Download references 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). AUTHOR INFORMATION Author notes * These authors contributed equally: Meng-Xing

Cao, Shi-Zhen Li. AUTHORS AND AFFILIATIONS * Key Laboratory of Seed Innovation, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Meng-Xing Cao, 

Shi-Zhen Li & Hong-Ju Li * Center for Molecular Agrobiology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Meng-Xing Cao, Shi-Zhen Li &

 Hong-Ju Li * University of Chinese Academy of Sciences, Beijing, China Hong-Ju Li Authors * Meng-Xing Cao View author publications You can also search for this author inPubMed Google

Scholar * Shi-Zhen Li View author publications You can also search for this author inPubMed Google Scholar * Hong-Ju Li View author publications You can also search for this author inPubMed 

Google Scholar 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. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. PEER REVIEW PEER REVIEW INFORMATION _Nature

Plants_ thanks Moritz Nowack and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. 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 EXPRESSION PATTERN OF MP_MLO_ GENES. A. RT-PCR analysis of

Mp_MLO_ 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 Mp_MLO_ promoter-driven GUS construct.

Bar, 1 mm. Source data EXTENDED DATA FIG. 2 MUTATION OF EACH MP_MLO_ GENE GENERATED BY CRISPR/CAS9. The gene-editing regions of Mp_mlo2_, Mp_mlo3_ and Mp_mlo4_ 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 MP_MLO_

MUTANT. A–E. Morphological characteristics of thallus in WT and Mp_mlo_ 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 Mp_mlo1_ (I). Arrows, antheridia. Scale bar, 500 μm. J and K. Number and location of antheridia in wild type and Mp_mlo1_. Vertical section of antheridiophores

in Wild type (J) and Mp_mlo1 #1_ (K). The growth locations of antheridia in Mp_mlo1 #1_ is consistent with WT. Asterisks, antheridia. Scale bar, 100 μm. EXTENDED DATA FIG. 4 SPERM DISCHARGE

ASSAY IN MP_MLO1_, MP_MLO2_, MP_MLO3_ AND MP_MLO4_ MUTANTS. A–F. Sperm discharge after application of water drops. Red arrows, released sperm masses. Scale bar, 1 mm. G. Thickened jacket

layer in the two Mp_mlo1_ mutant lines. White arrows indicate the jacket cell layer; Scale bar, 100 µm. H. Quantification of the width of different Mp_mlo1_ 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 MP_MLO_ MUTANTS. Morphological characteristics of antheridia isolated at immature and mature stages in Mp_mlo_ mutants. Scale bar, 20 μm. EXTENDED DATA FIG. 6

DEVELOPMENT OF ANTHERIDIA IN WILD TYPE, MP_MLO2_, MP_MLO3_ AND MP_MLO4._ Vertical section of antheridia at different stages in wild type and the mutants of Mp_mlo2_, Mp_mlo3_, and Mp_mlo4_.

Antheridia in Mp_mlo2_, Mp_mlo3_, and Mp_mlo4_ 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 MP_MLO1_ MUTANT. A. TUNEL assay for the WT and Mp_mlo1_ 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 MP_MLO1_

MUTANT. A. Aniline blue staining of antheridia in MpMLO1-Citrine-expressing WT and Mp_mlo1 #1_. Red, callose. B. Calcofluor White staining of antheridia in MpMLO1-Citrine-expressing WT and

Mp_mlo1 #1_. Magenta, cellulose. C. JIM7 immunostaining of antheridia in MpMLO1-Citrine-expressing WT and Mp_mlo1 #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 Mp_mlo1_ 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 Mp_mlo1_ #1 antheridium. SUPPLEMENTARY VIDEO 7 Time-lapse imaging of Ca2+ in the Mp_mlo1_ #2 antheridium. SOURCE DATA SOURCE DATA EXTENDED DATA FIG. 1 DNA gels 1–4. RIGHTS AND

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permissions ABOUT THIS ARTICLE CITE THIS ARTICLE 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 Download citation * Received: 27 June 2023 * Accepted: 18 April 2024 * Published: 03 June 2024 * Issue Date: June 2024 * DOI:

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