Molecular characterization of apoplastic nucleosidases in common bean

Trabajo presentado en la XXIV Reunión de la Sociedad Española de Biología de Plantas y XVII Congreso Hispano-Luso de Biología de Plantas, celebrados en Vigo (online) del 7 al 9 de julio de 2021.Nucleotides catabolism implies the action of several enzymes, including nucleotidases (nucleotide phosphatases) and nucleosidases (nucleoside hydrolases, NSH), which dephosphorylate nucleotides and degrade nucleosides to nucleobases by removing the ribose moiety, respectively. Nucleotides, nucleosides and nucleobases produced in the catabolic pathway can be transported to other tissues. In addition, nucleotides and their derivatives have been identified in the extracellular space, although their origin is not clear; they may be released by wounded or intact cells, to come from the degradation of the extracellular ATP or be imported form the rhizosphere. In common bean, we have identified four genes encoding nucleosidases, two of which, PvNSH3 and 4, have two nucleosidase domains and are predicted to be located in the apoplastic space. Phylogenetic analysis of these sequences indicated that these genes are only present in some groups of plants, which suggests that they could have a relatively recent origin. PvNSH3 and 4 have been fussed to the eYPF which has allowed to confirm that both proteins located in the apoplastic space. PvNSH4 have been overexpressed in Nicotiana benthamiana and the recombinant protein purified by affinity chromatography. The purified protein showed higher affinity with inosine, an optimum pH of 5 and a Km with inosine of 54 uM. Analysis of a fragment of 1000 pb upstream of the start codon revealed several motifs related with the response to pathogens and wounding. Therefore, we analysed the expression of PvNSH3 and 4 in response to methyl jasmonate, a hormone that regulates the response to wounding and other biotic stresses. Results showed an increase in the levels of PvNSH4 in radicles of seedlings, whereas PvNSH3 levels were not altered. Concomitantly, nucleosidase activity was also induced in the same conditions, suggesting a role of PvNSH4 in the response to methyl jasmonate