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.
Highlights
Root development is mediated through complex pathways involving endogenous signalling molecules and external environmental stimuli (Van Norman et al, 2011; Matsubayashi, 2012; Murphy et al, 2012; Meng, 2012; Czyzewicz et al, 2013)
The domain 1 peptide hydroxylated at Pro4 and Pro11 (D1:HyP4,11) imparted the strongest inhibition of lateral root emergence when grown with 5 mM KNO3 and stimulated the highest increase in nodule number when grown with 0 mM KNO3
To validate that biologically active secreted peptides were present in the culture medium, the root exudates from MtCEP1ox and vector control liquid cultures were collected, concentrated and added to the growth medium supporting the growth of wild-type M. truncatula seedlings to assay for their biological activities
Summary
Root development is mediated through complex pathways involving endogenous signalling molecules and external environmental stimuli (Van Norman et al, 2011; Matsubayashi, 2012; Murphy et al, 2012; Meng, 2012; Czyzewicz et al, 2013) These endogenous signalling molecules include small secreted peptides which regulate lateral root development, nodule formation and root meristem maintenance (Matsubayashi, 2011; Delay et al, 2013a; Mohd-Radzman et al, 2013). CEPs (C-TERMINALLY ENCODED PEPTIDEs) negatively regulate lateral and primary root development and positively regulate nodulation (Ohyama et al, 2008; Delay et al, 2013b; Imin et al, 2013; Roberts et al, 2013; Tabata et al, 2014) These small, secreted peptides are cleaved from a precursor, post-translationally modified and secreted to the apoplast (Matsubayashi, 2012). Both the final size of processed peptide and its post-translational modification (PTM) greatly influence biological activity (Ohyama et al, 2008, 2009; Matsuzaki et al, 2010; Whitford et al, 2012)
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