The interaction between legume plants and soil bacteria rhizobia results in the formation of new organs on the plant roots, symbiotic nodules, where rhizobia fix atmospheric nitrogen. Symbiotic nodules represent a perfect model to trace how the pre-existing regulatory pathways have been recruited and modified to control the development of evolutionary “new” organs. In particular, genes involved in the early stages of lateral root development have been co-opted to regulate nodule development. Other regulatory pathways, including the players of the KNOX-cytokinin module, the homologues of the miR172-AP2 module, and the players of the systemic response to nutrient availability, have also been recruited to a unique regulatory program effectively governing symbiotic nodule development. The role of the NIN transcription factor in the recruitment of such regulatory modules to nodulation is discussed in more details.

CLE (CLAVATA3/Embryo Surrounding Region) peptides regulate different aspects of plant development. In legumes, CLE peptides are known as key components of autoregulation of nodulation (AON), which systemically controls the number of nitrogen-fixing nodules formed on the root upon symbiotic interaction with soil bacteria rhizobia. CLE peptides are produced in the root in response to rhizobia inoculation and are transported via xylem to the shoot, where they are recognized by a specific receptor. As a result, a subsequent nodule development is suppressed by a negative feedback mechanism. In addition, nitrate-induced CLE genes have been identified in model legumes, which mediate nitrate-dependent inhibition of nodulation. However, little is known about the functions of nodulation-related CLE peptides, which have not been studied in Pisum sativum. Here, we studied four homologues of CLE genes in Pisum sativum, which are closely related to nodulation-suppressing CLEs from other legumes. The expression levels of these genes were increased in developing nodules. Among them, PsCLE13, PsCLE12, and the PsNIC-like genes were upregulated in response to nitrate treatment. Moreover, we found that overexpression of the PsCLE13 and PsCLE12 genes resulted in the decreased nodule number on transgenic roots. The expression levels of pea homologues of the TOO MUCH LOVE (TML) genes were upregulated in PsCLE13- and PsCLE12-overexpressing roots in comparison with the control (GUS-overexpressing) roots, suggesting that inhibitory effect of PsCLE13 and PsCLE12 is mediated through the induction of the PsTML genes.