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No. 2] Proc. Jpn. Acad., Ser. B 94 (2018) 59 Review Exploring peptide hormones in plants: identification of four peptide hormone-receptor pairs and two post-translational modification enzymes † By Yoshikatsu MATSUBAYASHI*1, (Communicated by Shigekazu NAGATA, M.J.A.) Abstract: The identification of hormones and their receptors in multicellular organisms is one of the most exciting research areas and has lead to breakthroughs in understanding how their growth and development are regulated. In particular, peptide hormones offer advantages as cell-to- cell signals in that they can be synthesized rapidly and have the greatest diversity in their structure and function. Peptides often undergo post-translational modifications and proteolytic processing to generate small oligopeptide hormones. In plants, such small post-translationally modified peptides constitute the largest group of peptide hormones. We initially explored this type of peptide hormone using bioassay-guided fractionation and later by in silico gene screening coupled with biochemical peptide detection, which led to the identification of four types of novel peptide hormones in plants. We also identified specific receptors for these peptides and transferases required for their post- translational modification. This review summarizes how we discovered these peptide hormone– receptor pairs and post-translational modification enzymes, and how these molecules function in plant growth, development and environmental adaptation. Keywords: secreted peptide, cell-to-cell communication, post-translational modification, Arabidopsis, ligand, receptor During the past 20 years, biochemical, genetic, 1. Introduction and bioinformatic analyses have identified more than Cell-to-cell signaling mediated by hormones and a dozen secreted peptide hormones and their recep- membrane-localized receptors is one of the essential tors in plants.1)–4) These peptide hormone–receptor mechanisms by which the growth and development pairs have proven to be functionally more diverse of multicellular organisms are regulated. Upon bind- than anticipated. Some of these peptides act as ing of such hormones to the extracellular domains local signals during plant growth and development, of receptors, physicochemical interactions are con- whereas others are root-to-shoot long-distance signals verted into physiological outputs activating down- required for environmental adaptation. The number stream signaling, which modulates cellular functions of functionally characterized peptide hormones now and fates through conformational changes in the exceeds the number of classical plant hormones. receptors. Because membrane-localized receptors act Secreted peptide hormones can be divided into as master switches of complex intracellular signaling two major groups based on structural characteristics processes, the identification of hormone–receptor arising from their biogenesis pathways (Fig. 1). One pairs is one of the central issues of current biological major group of peptide hormones are small post- research. translationally modified peptides characterized by the presence of post-translational modifications *1 Division of Biological Science, Graduate School of Science, mediated by specific transferases and by their small Nagoya University, Nagoya, Japan. – † size (approximately 5 20 amino acids) after proteo- Correspondence should be addressed: Y. Matsubayashi, lytic processing. The second group comprises cys- Division of Biological Science, Graduate School of Science, Nagoya University, Chikusa, Nagoya 464-8602, Japan (e-mail: matsu@ teine-rich peptides characterized by the presence of bio.nagoya-u.ac.jp). an even number of cysteine residues (typically 6 or 8) doi: 10.2183/pjab.94.006 ©2018 The Japan Academy 60 Y. MATSUBAYASHI [Vol. 94, Peptide hormone gene Prepropeptide Signal peptide Propeptide CC C C CC Post-translational modification Disulfide bond formation Proteolytic processing (Processing) X X C Mature peptide C C C C C Small post-translationally modified peptide Cysteine-rich peptide Fig. 1. Two distinct biogenesis pathways of secreted peptide hormones categorized by their structural characteristics. Peptide hormones can be categorized into the following two groups: peptides with complex post-translational modifications followed by proteolytic processing and peptides with multiple intramolecular disulfide bonds. The first group of peptides are called small post-translationally modified peptides and the latter group are defined as cysteine-rich peptides. This scheme is adapted from our previous review articles.1),73) that participate in the formation of intramolecular terminally encoded peptide (CEP), involved in disulfide bonds. In both cases, peptide hormone genes long-distance nitrogen demand signaling,8) root are initially translated as biologically inactive pre- meristem growth factor (RGF), regulating root propeptides, followed by removal of the N-terminal meristem development,9) and Casparian strip integ- signal peptide by a signal peptidase to afford a rity factor (CIF) required for contiguous Casparian propeptides. Propeptides are further structurally strip formation.10) These critical peptide hormones modified by several enzymes to give biologically had long been overlooked, probably due to their gene functional mature peptides. redundancy. We also identified receptors involved in My research career started with the purification the perception of these peptide hormones and two of a chemical factor involved in the density effect of important transferases required for post-translational plant cell proliferation in vitro as a Ph.D. student at modification of the hormones.11),12) Nagoya University in the laboratory of Prof. Youji This review offers a personal overview of how Sakagami. This bioassay-guided approach led to the we discovered these peptide hormone–receptor pairs identification of a peptide phytosulfokine (PSK), and post-translational modification enzymes, and the first small post-translationally modified peptide how these molecules contribute to plant growth hormone found in plants.5) Since then, I have been and development. Information regarding other small fascinated with the question of to what extent post-translationally modified peptides and cysteine- peptide signaling plays a role in plant growth and rich peptides is reviewed elsewhere.1)–4) development. The major challenge in this research is, fi however, how to distinguish bona fide peptide 2. Novel approaches for the identi cation of hormones from the numerous unrelated peptides peptide hormones and receptors in plants and protein fragments present in extracellular spaces. 2.1 In silico screening for peptide hormone Additionally, because no one can predict the activ- candidates. After our identification of PSK and ities of undiscovered hormones, a conventional bio- its family of precursor polypeptides by conventional assay-guided approach is not applicable. bioassay-guided purification (described in sec- To this end, my group employed an in silico tion 3.1), we noticed several structural character- gene screening approach coupled with structural istics of the amino acid sequences within this family, determination of mature peptides6) and receptor as summarized in Fig. 2A. (a) These precursor identification using a receptor expression library.7) polypeptides were approximately 100 amino acids This molecular-oriented strategy led to the identi- in length and had N-terminal secretion signal fication of three peptide hormones, namely, C- sequences that can be detectable using public web- No. 2] Exploring peptide hormones in plants 61 based software. (b) The hormones (mature peptides) 2.2 LC-MS-based structural elucidation of were encoded near the C-terminal region of the mature peptides. Small peptide hormones are precursor. Moreover, amino acid sequences corre- generated from precursor polypeptides by post-trans- sponding to the mature peptide domain were highly lational modification and proteolytic processing. conserved within the family, but other domains Because such specific modification and trimming exhibited low sequence conservation. This observa- actions are critical for the activity of each peptide tion can be interpreted as functional mature peptide hormone, determination of the mature functional regions being under strong selective pressure and structures of the peptide hormones is indispensable tending to exhibit higher sequence conservation for detailed analysis of peptide signaling. than their neutral flanking regions. (c) The mature To detect the mature peptide generated from peptide was post-translationally modified. Because target genes of interest, it is essential to extract post-translational modifications such as sulfation apoplastic peptides lacking contamination with and glycosylation require co-substrates that contain cytoplasmic molecules that hamper LC-MS-based high-energy phosphate bonds, the biosynthesis of structural analysis, even when the target peptide post-translationally modified peptides requires con- is overexpressed. To this end, we established a siderably more energy than the biosynthesis of other submerged culture system, in which Arabidopsis peptides. Nevertheless, post-translationally modified seeds are directly sown and grown in liquid culture peptides have been evolutionarily conserved, suggest- medium.6) In these conditions, plants develop hyper- ing that these modified peptides offer greater hydric true leaves, which are characterized by a lack physiological benefit to plants than the energy cost of cuticular wax formation. Hyperhydric

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