Characterization of a CLE-like peptide hormone produced in Nicotiana tabacum cell cultures

Phytohormones are chemical messengers that coordinate all the developmental and physiological processes in plants. Nine general classes of hormones are recognized as major players in cell to cell signaling. These are the “old five”classes: auxins, cytokinins, gibberellins, ethylene, and abscisic acid, to which brassinosteroids, salicylates, strigolactones and jasmonates have been added by the end of the 20th and the beginning of this century. Additionally, little signal peptides were recognized to have a critical role in plant development and interaction with the environment. Such peptides are phytohormones involved in meristems homeostasis, biotic and abiotic defense response, root growth, organ abscission and fruit repining. Despite the potential of these small peptides in controlling the production of economically interesting sources of food, many of these peptides are still structurally and functionally uncharacterized. One interesting group of peptides are the CLE Peptides. CLE peptides are processed small-signal peptides that harbor post-translational modifications that are crucial for their biological functions. The Prunus persica CLE27 peptide represents a possible candidate as a mediator in the interplay between auxin and ethylene. Initially annotated as a gene of unknown function, it drew attention because of its expression, noted by means of microarray experiments on ripening peach. Indeed, it is induced by ripening in the late phases of fruit development. In this work, a heterologous host production system with Nicotiana tabacum cell culture was characterized. The GUS reporter gene was used to better identify the cell culture lines that expressed the construct of interest more efficiently. The intensity of the response was found helpful to measure the expression of the recombinant peptide. The potential post-translational modifications of the CLE27 peptide were investigated using mass spectrometry analyses. The peptide was found to undergo proline hydroxylation and most likely tri-arabinosylation of the second Hydroxyproline of the peptide sequence. Thus, the N. tabacum host system successfully produced the post-translationally modified peptide of interest. Furthermore, the potential of using the exhausted cell medium as a bio-stimulant containing the PpCLE27 peptide was explored by means of bioassays on root development. However, further studies are necessary to clarify the function of the PpCLE27 peptide