Abstract
Nucleoside hydrolases (NSH; nucleosidases) catalyze the cleavage of nucleosides into ribose and free nucleobases. These enzymes have been postulated as key elements controlling the ratio between nucleotide salvage and degradation. Moreover, they play a pivotal role in ureidic legumes by providing the substrate for the synthesis of ureides. Furthermore, nucleotide metabolism has a crucial role during germination and early seedling development, since the developing seedlings require high amount of nucleotide simultaneously to the mobilization of nutrient in cotyledons. In this study, we have cloned two nucleosidases genes from Phaseolus vulgaris, PvNSH1 and PvNSH2, expressed them as recombinant proteins, and characterized their catalytic activities. Both enzymes showed a broad range of substrate affinity; however, PvNSH1 exhibited the highest activity with uridine, followed by xanthosine, whereas PvNSH2 hydrolyses preferentially xanthosine and shows low activity with uridine. The study of the regulation of nucleosidases during germination and early postgerminative development indicated that nucleosidases are induced in cotyledons and embryonic axes just after the radicle emergence, coincident with the induction of nucleases activity and the synthesis of ureides in the embryonic axes, with no remarkable differences in the level of expression of both nucleosidase genes. In addition, nucleosides and nucleobase levels were determined as well in cotyledons and embryonic axes. Our results suggest that PvNSH1 and PvNSH2 play an important role in the mobilization of nutrients during this crucial stage of plant development.
Highlights
Nucleotides are molecules of crucial relevance in all living organisms
Purine and pyrimidine nucleotides can be synthesized de novo or through the salvage pathways, or been taken up from the soil, where they are found in significant amounts (Phillips et al, 1997; Jenkins et al, 2017)
Purine nucleotide catabolism is relevant in ureidic legumes; xanthine is the precursor of the ureides allantoin and allantoate, molecules that play an important role in the transport and storage of nitrogen in these legumes (Todd et al, 2006)
Summary
Nucleotides are molecules of crucial relevance in all living organisms. Besides being elementary components of RNA and DNA, they participate in bioenergetic processes, are part of cofactors for enzymes, and are components of secondary messengers and metabolites (Zrenner et al, 2006). The knowledge of purine and pyrimidine metabolism during germination and postgerminative development is limited and based mainly on studies with radiolabeled precursors (Ashihara et al, 2018) These works showed that at the initial phase of germination, the salvage of nucleosides and nucleobases supports the synthesis of nucleotides and nucleic acids, whereas in the later stages, the demand of nucleic acid is mainly fulfilled by the de novo pathways, and the catabolic pathways increase to provide carbon and nitrogen to the growing seedling (Ashihara et al, 2020c). Two cytosolic NSHs have been identified and cloned, both ORFs have been overexpressed, and the recombinant enzymes have been characterized Their regulation during germination and early postgerminative development in cotyledons and embryonic axes has been analyzed, as well as the level of nucleosides, nucleobases, ureides, and nuclease activity during these developmental stages. This work constitutes the first comprehensive analysis of nucleoside metabolism during germination and early seedling development in plants
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