Article

Physcomitrella MADS-box genes regulate water supply and sperm movement for fertilization

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Abstract

MIKC classic (MIKCC)-type MADS-box genes encode transcription factors that function in various developmental processes, including angiosperm floral organ identity. Phylogenetic analyses of the MIKCC-type MADS-box family, including genes from non-flowering plants, suggest that the increased numbers of these genes in flowering plants is related to their functional divergence; however, their precise functions in non-flowering plants and their evolution throughout land plant diversification are unknown. Here, we show that MIKCC-type MADS-box genes in the moss Physcomitrella patens function in two ways to enable fertilization. Analyses of protein localization, deletion mutants and overexpression lines of all six genes indicate that three MIKCC-type MADS-box genes redundantly regulate cell division and growth in the stems for appropriate external water conduction, as well as the formation of sperm with motile flagella. The former function appears to be maintained in the flowering plant lineage, while the latter was lost in accordance with the loss of sperm.

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Acknowledgements

We thank N. Aono for constructing the GUS insertion and deletion mutant lines, T. Nishiyama, K. Sakakibara, Y. Sakata and the members of the Division of Evolutionary Biology for discussion, T. Maeda for advice on transcriptome analysis, K. Yamaguchi and A. Akita for next-generation sequencing, and Y. Matsumoto for preliminary observation of external water conduction. Plant cultivation, microscopy and transcriptome analyses were supported by the Model Plant Research Facility, the Spectrography and Bioimaging Facility and the Data Integration and Analysis Facility in the National Institute for Basic Biology. Electron microscopy was supported by the EM facility in the National Institute for Physiological Sciences. This work was partially funded by MEXT and JSPS KAKENHI grants to M.H., T.M. and R.K. (16H06378), to M.H. (17H06390) and to Y.S.-S. and H.O. (15H04393), and a CREST JST grant to M.K.

Author information

Affiliations

  1. Division of Evolutionary Biology, National Institute for Basic Biology, Okazaki, 444-8585, Japan

    • Shizuka Koshimizu
    • , Rumiko Kofuji
    • , Yukiko Kabeya
    • , Yuji Hiwatashi
    • , Yosuke Tamada
    • , Takashi Murata
    •  & Mitsuyasu Hasebe
  2. Department of Basic Biology, School of Life Science, SOKENDAI (The Graduate University for Advanced Studies), Okazaki, 444-8585, Japan

    • Shizuka Koshimizu
    • , Shuji Shigenobu
    • , Yuji Hiwatashi
    • , Yosuke Tamada
    • , Takashi Murata
    •  & Mitsuyasu Hasebe
  3. Graduate School of Natural Science and Technology, Kanazawa University, Kanazawa, 920-1192, Japan

    • Rumiko Kofuji
  4. School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, 226-8501, Japan

    • Yuko Sasaki-Sekimoto
    • , Mie Shimojima
    •  & Hiroyuki Ohta
  5. JST CREST, Yokohama, 226-8501, Japan

    • Yuko Sasaki-Sekimoto
    •  & Hiroyuki Ohta
  6. Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Bunkyo, 113-0033, Japan

    • Masahide Kikkawa
  7. The Earth-Life Science Institute, Tokyo Institute of Technology, Meguro, 152-8550, Japan

    • Hiroyuki Ohta
  8. Functional Genomics Facility, National Institute for Basic Biology, Okazaki, 444-8585, Japan

    • Shuji Shigenobu
  9. School of Food Industrial Sciences, Miyagi University, Sendai, 982-0215, Japan

    • Yuji Hiwatashi

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Contributions

S.K., R.K., Y.T., T.M. and M.H. conceived and designed the research in general. S.K., Y.S.-S., M.S., H.O. and M.H. designed the experiments on the cuticle. S.K., R.K., Y.S.-S., S.S., Y.K. and Y.H. performed the experiments. Every author analysed the data and wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Mitsuyasu Hasebe.

Supplementary information

  1. Supplementary Information

    Supplementary Figures 1–31, Supplementary Table 6 and Supplementary References.

  2. Life Sciences Reporting Summary

  3. Supplementary Table 1

    Differentially expressed genes in gametophores between the wild type and the sextuple deletion mutant line.

  4. Supplementary Table 2

    Comparisons between differentially expressed genes of the Physcomitrella sextuple deletion mutant line and Arabidopsis mutants of MIKCC-type MADS-box genes.

  5. Supplementary Table 3

    Differentially expressed genes in antheridia between the wild type and the sextuple deletion mutant line.

  6. Supplementary Table 4

    Differentially expressed genes in antheridia between the wild type and the triple deletion mutant line.

  7. Supplementary Table 5

    Flagellum-related genes whose transcript levels decreased in both the sextuple deletion mutant line and the triple deletion mutant line.

Videos

  1. Supplementary Video 1

    Real-time movement of Evans blue solution up the wild-type gametophore. The base of the gametophore was dipped in the solution.

  2. Supplementary Video 2

    Real-time movement of Evans blue solution up the sextuple deletion mutant gametophore. The base of the gametophore was dipped in the solution.

  3. Supplementary Video 3

    Slow-motion movie of the movement of Evans blue solution in a wild-type gametophore. The base of the gametophore was dipped in the solution.

  4. Supplementary Video 4

    Real-time movement of a wild-type sperm observed using dark-field microscopy.

  5. Supplementary Video 5

    Real-time movement of a sperm from the sextuple deletion mutant line observed using dark-field microscopy.