title: The sequence flanking the N-terminus of the CLV3 peptide is critical for its cleavage and activity in stem cell regulation in Arabidopsis.
abstract: BACKGROUND
  Although it is known that CLAVATA3 (CLV3) acts as 12- and/or 13-amino acid (AA) secreted peptides to regulate the number of stem cells in shoot apical meristems (SAMs), how functional CLV3 peptides are generated and if any particular sequences are required for the processing remain largely unknown.
  
  
  RESULTS
  We developed a mass spectrometry (MS)-based in vitro assay to monitor the cleavage of heterologously produced CLV3 fusion protein. Through co-cultivation of the fusion protein with Arabidopsis seedlings, we identified two cleavage sites: the previously reported one before Arg70 and a new one before Met39. Using synthetic peptides together with MALDI-Tof-MS analyses, we demonstrated that the non-conserved 5-AA motifs flanking N-termini of the CLV3 and its orthologous CLE1 peptides were critical for their cleavages and optimal activities in vitro. We also found that substitutions of Leu69 by Ala in fusion protein and in synthetic peptide of CLV3 compromised their cleavages, leading to significantly reduced activities in regulating the sizes of shoot and root meristems.
  
  
  CONCLUSIONS
  These results suggest that 5-AA residues flanking the N-terminus of CLV3 peptide are required for proper cleavages and optimal function in stem cell regulation.
authors.forename: Ting-Ting
authors.initials: TT
authors.lastname: Xu
authors.forename: Xiu-Fen
authors.initials: XF
authors.lastname: Song
authors.forename: Shi-Chao
authors.initials: SC
authors.lastname: Ren
authors.affiliation: Key Laboratory of Plant Molecular Physiology, Institute of Botany, Chinese Academy of Sciences, Nanxincun 20, Fragrant Hill, Beijing 100093, China. cmliu@ibcas.ac.cn.
authors.forename: Chun-Ming
authors.initials: CM
authors.lastname: Liu
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology* / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000378:metabolism*; D001681:Biological Assay; D007930:Leucine / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D018517:Plant Roots / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D036226:Seedlings / Q000254:growth & development; D016415:Sequence Alignment; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013234:Stem Cells / Q000166:cytology* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: C118167:AT2G27250 protein, Arabidopsis; D029681:Arabidopsis Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D007930:Leucine