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Origin and function of stomata in the moss Physcomitrella patens

Nature Plants volume 2, Article number: 16179 (2016) Cite this article

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Abstract

Stomata are microscopic valves on plant surfaces that originated over 400 million years (Myr) ago and facilitated the greening of Earth's continents by permitting efficient shoot–atmosphere gas exchange and plant hydration1. However, the core genetic machinery regulating stomatal development in non-vascular land plants is poorly understood2–4 and their function has remained a matter of debate for a century5. Here, we show that genes encoding the two basic helix–loop–helix proteins PpSMF1 (SPEECH, MUTE and FAMA-like) and PpSCREAM1 (SCRM1) in the moss Physcomitrella patens are orthologous to transcriptional regulators of stomatal development in the flowering plant Arabidopsis thaliana and essential for stomata formation in moss. Targeted P. patens knockout mutants lacking either PpSMF1 or PpSCRM1 develop gametophytes indistinguishable from wild-type plants but mutant sporophytes lack stomata. Protein–protein interaction assays reveal heterodimerization between PpSMF1 and PpSCRM1, which, together with moss–angiosperm gene complementations6, suggests deep functional conservation of the heterodimeric SMF1 and SCRM1 unit is required to activate transcription for moss stomatal development, as in A. thaliana7. Moreover, stomata-less sporophytes of ΔPpSMF1 and ΔPpSCRM1 mutants exhibited delayed dehiscence, implying stomata might have promoted dehiscence in the first complex land-plant sporophytes.

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Figure 1: The genome of the moss P. patens encodes orthologues of the bHLH transcription factors regulating stomatal development in flowering plants.
Figure 2: PpSMF1 and PpSCRM1 are required for stomatal development in the moss P. patens.
Figure 3: BiFC and yeast two-hybrid assays demonstrating PpSMF1 and PpSCRM1 protein–protein interactions.
Figure 4: Loss of PpSMF1 and the PpSCRM1 gene functions results in delayed dehiscence of spore capsules.

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Acknowledgements

We thank R. Haas and T. Fulton for excellent technical assistance. R.S.C. was supported by a NERC studentship. D.C.B is a GBMF investigator of the Howard Hughes Medical Institute. D.J.B. acknowledges funding through an ERC Advanced Grant (CDREG, 322998). R.R. acknowledges funding through the Excellence Initiative of the German Federal and States Governments (EXC294). A.C.C. and Y.K. acknowledge support from BBSRC (Grant numbers BB/F001797/1 and BB/I006710/1).

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Author notes

  1. Caspar C. Chater and Robert S. Caine: These authors contributed equally to this work.

Authors and Affiliations

  1. Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de Mexico, Cuernavaca, 62210, Mexico

    Caspar C. Chater

  2. Department of Animal and Plant Sciences, University of Sheffield, Sheffield, S10 2TN, UK

    Robert S. Caine, Andrew Fleming & David J. Beerling

  3. Plant Biotechnology, Faculty of Biology, University of Freiburg, Schaenzlestraße 1, 79104 Freiburg, Germany

    Marta Tomek, Daniel Lang, Eva L. Decker & Ralf Reski

  4. Royal College of Veterinary Surgeons, Belgravia House, 62–64 Horseferry Road, London, SW1P 2AF, UK

    Simon Wallace

  5. Centre for Plant Sciences, University of Leeds, Leeds, LS2 9JT, UK

    Yasuko Kamisugi & Andrew C. Cuming

  6. Department of Molecular Cellular and Developmental Biology, University of Michigan, Ann Arbor, 48109-1048, Michigan, USA

    Cora A. MacAlister

  7. Department of Molecular Biology and Biotechnology, University of Sheffield, Sheffield, S10 2TN, UK

    Stuart Casson & Julie E. Gray

  8. HHMI and Department of Biology, Stanford University, Stanford, 94305-5020, California, USA

    Dominique C. Bergmann

  9. Plant Molecular Cell Biology, Faculty of Biology, Ludwig-Maximilians-Universität München, LMU Biocenter, Großhaderner Straße 2, 82152 Planegg-Martinsried, Germany

    Wolfgang Frank

  10. BIOSS – Centre for Biological Signalling Studies, 79104 Freiburg, Germany

    Ralf Reski

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Contributions

C.C.C., R.S.C., W.F., J.E.G., A.F., D.J.B. and R.R. designed the study, C.C.C., R.S.C., D.L. and M.T. undertook the experiments with contributions from S.W., Y.K. and A.C.C., C.A.M., S.C., D.C.B., D.L., E.L.D and W.F. contributed materials and advice. A.C.C. constructed vectors for moss targeted knockout (SMF1 and SMF2), Y.K. carried out moss transformation (SMF2-KO) and yeast two-hybrid analysis, A.C.C. and Y.K. carried out Southern blot hybridization of the knockout mutant lines. D.J.B., R.R, C.C.C. and R.S.C. wrote the paper with contributions from D.C.B. All authors read, commented on and approved the final version of the manuscript.

Corresponding authors

Correspondence to Ralf Reski or David J. Beerling.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

Supplementary Figures 1–15, Supplementary Methods, Supplementary Discussion, Supplementary Tables 1 and 2, Supplementary References, Supplementary Data Files 1–4. (PDF 3050 kb)

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Supplementary Dataset 2. (TXT 174 kb)

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Chater, C., Caine, R., Tomek, M. et al. Origin and function of stomata in the moss Physcomitrella patens. Nature Plants 2, 16179 (2016). https://doi.org/10.1038/nplants.2016.179

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