title: Novel MtCEP1 peptides produced in vivo differentially regulate root development in Medicago truncatula.
abstract: Small, post-translationally modified and secreted peptides regulate diverse plant developmental processes. Due to low natural abundance, it is difficult to isolate and identify these peptides. Using an improved peptide isolation protocol and Orbitrap mass spectrometry, nine 15-amino-acid CEP peptides were identified that corresponded to the two domains encoded by Medicago truncatula CEP1 (MtCEP1). Novel arabinosylated and hydroxylated peptides were identified in root cultures overexpressing MtCEP1. The five most abundant CEP peptides were hydroxylated and these species were detected also in low amounts in vector control samples. Synthetic peptides with different hydroxylation patterns differentially affected root development. Notably, the domain 1 peptide hydroxylated at Pro4 and Pro11 (D1:HyP4,11) imparted the strongest inhibition of lateral root emergence when grown with 5mM KNO3 and stimulated the highest increase in nodule number when grown with 0mM KNO3. Inhibition of lateral root emergence by D1:HyP4,11 was not alleviated by removing peptide exposure. In contrast, the domain 2 peptide hydroxylated at Pro11 (D2:HyP11) increased stage III-IV lateral root primordium numbers by 6-fold (P < 0.001) which failed to emerge. Auxin addition at levels which stimulated lateral root formation in wild-type plants had little or no ameliorating effect on CEP peptide-mediated inhibition of lateral root formation or emergence. Both peptides increased and altered the root staining pattern of the auxin-responsive reporter GH3:GUS suggesting CEPs alter auxin sensitivity or distribution. The results showed that CEP primary sequence and post-translational modifications influence peptide activities and the improved isolation procedure effectively and reproducibly identifies and characterises CEPs.
authors.affiliation: Division of Plant Sciences, Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra ACT 0200, Australia.
authors.forename: Nadiatul A
authors.initials: NA
authors.lastname: Mohd-Radzman
authors.affiliation: Thermo Fisher Scientific Pty Ltd, 5 Caribbean Drive, Scoresby, VIC 3179, Australia.
authors.forename: Steve
authors.initials: S
authors.lastname: Binos
authors.affiliation: Mass Spectrometry Facility, Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra ACT 0200, Australia.
authors.forename: Thy T
authors.initials: TT
authors.lastname: Truong
authors.affiliation: Division of Plant Sciences, Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra ACT 0200, Australia.
authors.forename: Nijat
authors.initials: N
authors.lastname: Imin
authors.affiliation: Thermo Fisher Scientific Pty Ltd, 5 Caribbean Drive, Scoresby, VIC 3179, Australia.
authors.forename: Michael
authors.initials: M
authors.lastname: Mariani
authors.affiliation: Division of Plant Sciences, Research School of Biology, College of Medicine, Biology and Environment, The Australian National University, Canberra ACT 0200, Australia michael.djordjevic@anu.edu.au.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Djordjevic
mesh_terms: D000595:Amino Acid Sequence; D018506:Gene Expression Regulation, Plant; D007210:Indoleacetic Acids / Q000494:pharmacology*; D046913:Medicago truncatula / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism; D009280:Naphthaleneacetic Acids / Q000494:pharmacology*; D010937:Plant Growth Regulators / Q000494:pharmacology*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018517:Plant Roots / Q000254:growth & development / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D053719:Tandem Mass Spectrometry
chemical_list: D007210:Indoleacetic Acids; D009280:Naphthaleneacetic Acids; D010937:Plant Growth Regulators; D010940:Plant Proteins; C034182:1-naphthaleneacetic acid
keywords: Legume root development; mass spectrometry; nodulation; peptide isolation; peptide signaling; post-translational modification; secreted peptides.