The role of MtCEP1, a member of the CEP (C-terminally encoded peptide) signaling peptide family, was examined in Medicago truncatula root development. MtCEP1 was expressed in root tips, vascular tissue, and young lateral organs, and was up-regulated by low nitrogen levels and, independently, by elevated CO2. Overexpressing MtCEP1 or applying MtCEP1 peptide to roots elicited developmental phenotypes: inhibition of lateral root formation, enhancement of nodulation, and the induction of periodic circumferential root swellings, which arose from cortical, epidermal, and pericycle cell divisions and featured an additional cortical cell layer. MtCEP peptide addition to other legume species induced similar phenotypes. The enhancement of nodulation by MtCEP1 is partially tolerant to high nitrate, which normally strongly suppresses nodulation. These nodules develop faster, are larger, and fix more nitrogen in the absence and presence of inhibiting nitrate levels. At 25mM nitrate, nodules formed on pre-existing swelling sites induced by MtCEP1 overexpression. RNA interference-mediated silencing of several MtCEP genes revealed a negative correlation between transcript levels of MtCEP1 and MtCEP2 with the number of lateral roots. MtCEP1 peptide-dependent phenotypes were abolished or attenuated by altering or deleting key residues in its 15 amino acid domain. RNA-Seq analysis revealed that 89 and 116 genes were significantly up- and down-regulated, respectively, by MtCEP1 overexpression, including transcription factors WRKY, bZIP, ERF, and MYB, homologues of LOB29, SUPERROOT2, and BABY BOOM. Taken together, the data suggest that the MtCEP1 peptide modulates lateral root and nodule development in M. truncatula.
Keywords: Biological nitrogen fixation; CEP; environmental regulation; lateral root formation; nodulation; root development; small signalling peptides..