Overexpression of a novel small peptide ROTUNDIFOLIA4 decreases cell proliferation and alters leaf shape in Arabidopsis thaliana

Authors

  • Noriyuki N. Narita,

    1. National Institute for Basic Biology/Center for Integrated Bioscience, Okazaki, Aichi 444-8585, Japan,
    2. Department of Molecular Biomechanics, School of Life Science, The Graduate University for Advanced Studies, Hayama, Kanagawa 240-0193, Japan,
    Search for more papers by this author
  • Sally Moore,

    1. Institute for Cellular and Molecular Biology, Rutherford Building, The King's Buildings, Edinburgh EH9 3JH, UK,
    Search for more papers by this author
  • Gorou Horiguchi,

    1. National Institute for Basic Biology/Center for Integrated Bioscience, Okazaki, Aichi 444-8585, Japan,
    Search for more papers by this author
  • Minoru Kubo,

    1. Morphogenesis Research Group, Plant Science Center, RIKEN, Yokohama, Kanagawa 230-0045, Japan,
    Search for more papers by this author
  • Taku Demura,

    1. Morphogenesis Research Group, Plant Science Center, RIKEN, Yokohama, Kanagawa 230-0045, Japan,
    Search for more papers by this author
  • Hiroo Fukuda,

    1. Morphogenesis Research Group, Plant Science Center, RIKEN, Yokohama, Kanagawa 230-0045, Japan,
    2. Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan, and
    Search for more papers by this author
  • Justin Goodrich,

    1. Institute for Cellular and Molecular Biology, Rutherford Building, The King's Buildings, Edinburgh EH9 3JH, UK,
    Search for more papers by this author
  • Hirokazu Tsukaya

    Corresponding author
    1. National Institute for Basic Biology/Center for Integrated Bioscience, Okazaki, Aichi 444-8585, Japan,
    2. Department of Biosystems Science, School of Advanced Science, The Graduate University for Advanced Studies, Hayama, Kanagawa 240-0193, Japan
    Search for more papers by this author

For correspondence (fax +81 564 55 7512; e-mail tsukaya@nibb.ac.jp).

Present address: Institute of Cell, Animal and Population Biology, Rutherford Building, The King's Buildings, Edinburgh EH9 3JT, UK.

Summary

Leaf shape is determined by polar cell expansion and polar cell proliferation along the leaf axes. However, the genes controlling polar cell proliferation during leaf morphogenesis are largely unknown. We identified a dominant mutant of Arabidopsis thaliana, rotundifolia4-1D (rot4-1D), which possessed short leaves and floral organs. We showed that the altered leaf shape is caused by reduced cell proliferation, specifically in the longitudinal (proximal–distal) axis of the leaf, suggesting that the ROT4 gene controls polar cell proliferation in lateral organs. The ROT4 open-reading frame (ORF) encodes a novel small peptide that had not been identified in the Arabidopsis genome annotation. Overexpression of a ROT4–green fluorescence protein (GFP) fusion protein in transgenic plants recapitulated the rot4 phenotype, suggesting that ROT4 acts to restrict cell proliferation. The ROT4–GFP fusion protein localized to the plasma membrane when expressed in transgenic Arabidopsis plants. Phylogenetic analysis indicates that ROT4 defines a novel seed plant-specific family of small peptides with 22 members in Arabidopsis, ROT FOUR LIKE1–22 (RTFL1–22). All RTFL members share a conserved 29-amino acid domain, the RTF domain, and overexpression of the ROT4 RTF domain alone is sufficient to confer a rot4-1D phenotype. Loss-of-function mutations in several RTFL genes were aphenotypic, suggesting that there may be some functional redundancy between family members. Analyses by reverse transcription-polymerase chain reaction (RT-PCR) and in situ hybridization revealed that ROT4 is expressed in the shoot apex and young leaves of wild-type plants, consistent with a role for ROT4 in controlling polarity-dependent cell proliferation during wild-type leaf morphogenesis.

Ancillary