Investigating downstream targets of CEP peptide signaling that controls root architecture and nodulation in Medicago truncatula

Abstract

Nitrogen (N) is an essential element for plant growth and productivity. Plants cannot directly fix atmospheric N. Legumes can fix atmospheric N in root nodules through their symbiotic relationship with rhizobial bacteria. In legume Medicago truncatula, CEPs (C-TERMINALLY ENCODED PEPTIDEs), through their putative shoot receptor CRA2 (COMPACT ROOT ARCHITECTURE2), positively regulate root nodulation. This process involves long distance signaling between the root and the shoot. CEP peptides and their receptors are highly conserved and specific to seed plants. In dicot Arabidopsis, perception of root-derived CEP peptides by its receptor CEPR1 (CRA2 orthologue) in the shoot mediates systemic N-demand signaling via regulating nitrate transporters (NRTs) in the root. The regulation of NRT2.1 expression involves CEP downstream effectors CEPDs (CEP DOWNSTREAMs) traveling from the shoot to the root. RNA-Seq analysis in Imin lab has unveiled two CEPD genes in Medicago as putative downstream targets of CEP signaling. However, it is not clear if and how CEPDs regulate the expression of NRT2.1 homologues, root architecture and/or nodulation in Medicago. In this study, gene expression analysis combined with reverse genetics were utilised to investigate the role of MtCEPD genes. In particular, how CEPD genes convey CEP-CRA2 signaling to regulate gene expression, root architectural changes and nodule formation in Medicago was analysed. Expression analysis of MtCEPDs on M. truncatula wild type (WT) and cra2 plants upon treatment with MtCEP peptides showed that MtCEPD1A and MtCEPD1B are highly induced by MtCEP peptide addition in CRA2 dependent manner. 35S promoter driven overexpression of MtCEPD genes increased total nodule number in WT, but not in cra2. Lateral roots were not affected by overexpression of MtCEPDs . The results suggest that MtCEPD1A and MtCEPD1B do not involve in the regulation of lateral root development but enhance nodulation as part of MtCEP-CRA2 signaling pathway. However, overexpression of MtCEPDs alone was not sufficient to rescue cra2 defects including severe reduction in nodule numbers, indicating other CRA2 downstream factors also required for successful completion of CEP-CRA2 signaling processes to positively regulate nodulation. Further studies such as gene silencing and protein interactions of MtCEPDs may allow us to understand and unravel the characteristics of MtCEPDs and MtCEP induced systemic signaling pathway controlling nodule numbers. The findings from this research may provide insights into how CEP-CRA2 signaling pathway work in legumes and may help to improve the efficiency of symbiotic N-fixation in legume crops.