The C-TERMINALLY ENCODED PEPTIDE(CEP) peptides play crucial roles in plant growth and response to environmental factors. These peptides were characterized as positive regulators of symbiotic nodule development in legume plants. However, little is known about the CEP peptide family in pea. Here, we discovered in pea genome 21 CEP genes (PsCEPs), among which three genes contained additional conserved motifs corresponding to the PIP (PAMP-induced secreted peptides) consensus sequences. We characterized the expression patterns of pea PsCEP genes based on transcriptomic data, and for six PsCEP genes with high expression levels in the root and symbiotic nodules the detailed expression analysis at different stages of symbiosis and in response to nitrate treatment was performed. We suggest that at least three PsCEP genes, PsCEP1, PsCEP7 and PsCEP2, could play a role in symbiotic nodule development, whereas the PsCEP1 and PsCEP13 genes, downregulated by nitrate addition, could be involved in regulation of nitrate-dependent processes in pea. Further functional studies are required to elucidate the functions of these PsCEP genes.

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.

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Legumes are important suppliers of vegetable protein. In symbiotic interactions with soil bacteria rhizobia, legume plants form nitrogen-fixing nodules on their roots, where molecular nitrogen is fixed and incorporated into organic compounds. A host plant controls the number of symbiotic nodules to meet its nitrogen demands. The presence of high amount of nitrate in the soil suppresses the formation of nodules. CLE (CLAVATA3/EMBRYO SURROUNDING REGION) peptides produced in the root in response to rhizobial inoculation and/or nitrate were shown to control the number of symbiotic nodules. Previously, we have identified the MtCLE35 gene upregulated by the rhizobia and the nitrate treatment in Medicago truncatula, which systemically inhibited nodulation when overexpressed. Using genetic engineering approaches we increased its transcriptional activity in transgenic roots, which almost completely prevented the formation of symbiotic nodules. Moreover, we obtained stable transgenic lines overproducing the MtCLE35 peptide, and found that their had lower shoot and root biomass in comparison to the wild-type plants. The study of metabolome of MtCLE35-overproducing plants revealed the increased level of amino acids in the roots, suggesting the stimulating effect of MtCLE35 on amino acid synthetic pathways. In addition, we obtained several knock-out lines, where the MtCLE35 gene was edited using the CRISPR/Cas9-mediated system. The detailed analysis of these plants will allow us to understand the mechanisms of MtCLE35 action in the regulation of plant nitrogen status and symbiotic interaction with rhizobia. This work was supported by the grant from Saint Petersburg State University ID 93020341 and by the Russian Foundation for Basic Research project 20-016-00129.