title: Border sequences of Medicago truncatula CLE36 are specifically cleaved by endoproteases common to the extracellular fluids of Medicago and soybean.
abstract: CLE (CLAVATA3/ESR-related) peptides are developmental regulators that are secreted into the apoplast. Little is known about the role of the sequences that flank CLE peptides in terms of their biological activity or how they are targeted by proteases that are known to liberate the final active CLE peptides from their precursor sequences. The biological activity of Medicago truncatula CLE36, which possesses broadly conserved border sequences flanking the putative final active CLE36 peptide product, was assessed. Using in vitro root growth assays and an in vitro root and callus formation assay it is shown that CLE36 peptides of different lengths possess differential biological activities. Using mass spectrometry, Glycine max and Medicago extracellular fluids were each shown to possess an endoproteolytic activity that recognizes and cleaves at border sequences in a synthetic 31 amino acid CLE36 'propeptide bait' to liberate biologically active peptide products. Inhibitor studies suggest that a subtilisin, in combination with a carboxypeptidase, liberated and trimmed CLE36, respectively, to form biologically relevant 11-15 amino acid cleavage products. The 15 amino acid cleavage product is more biologically potent on Arabidopsis than shorter or longer CLE peptides. In situ hybridization shows that the soybean orthologue of CLE36 (GmCLE34) is expressed in the provascular tissue. The results suggest that secreted subtilisins can specifically recognize the border sequences of CLE36 propeptides and liberate biologically active cleavage products. These secreted proteases may affect the stability and biological activity of CLE peptides in the apoplast or be involved in CLE36 processing.
authors.affiliation: Australian Research Council Centre of Excellence for Integrative Legume Research, Plant Science Division, Research School of Biology, Australian National University, Canberra ACT Australia, 0200. Michael.djordjevic@anu.edu.au
authors.forename: Michael A
authors.initials: MA
authors.lastname: Djordjevic
authors.forename: Marie
authors.initials: M
authors.lastname: Oakes
authors.forename: Chui E
authors.initials: CE
authors.lastname: Wong
authors.forename: Mohan
authors.initials: M
authors.lastname: Singh
authors.forename: Prem
authors.initials: P
authors.lastname: Bhalla
authors.forename: Lucia
authors.initials: L
authors.lastname: Kusumawati
authors.forename: Nijat
authors.initials: N
authors.lastname: Imin
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D017124:Conserved Sequence / Q000235:genetics; D045604:Extracellular Fluid / Q000187:drug effects / Q000201:enzymology*; D018506:Gene Expression Regulation, Plant / Q000187:drug effects; D017343:Genes, Plant / Q000235:genetics; D046913:Medicago truncatula / Q000187:drug effects / Q000201:enzymology*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D018517:Plant Roots / Q000187:drug effects / Q000254:growth & development; D017386:Sequence Homology, Amino Acid; D013025:Glycine max / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics; D013045:Species Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity / Q000187:drug effects
chemical_list: D010455:Peptides; D010940:Plant Proteins; D010447:Peptide Hydrolases