No abstract

Nucleotides are molecules of great importance in plant physiology. In addition to being elementary units of the genetic material, nucleotides are involved in bio-energetic processes, play a role as cofactors, and are also components of secondary metabolites and the hormone cytokinin. The common bean (Phaseolus vulgaris) is a legume that transports the nitrogen fixed in nodules as ureides, compounds synthetized from purine nucleotides. The first step in this pathway is the removal of the 5’-phosphate group by a phosphatase. In this study, a gene that codes for a putative nucleotidase (PvNTD2) has been identified in P. vulgaris. The predicted peptide contains the conserved domains for haloacid dehalogenase-like hydrolase superfamily. The protein has been overexpressed in Escherichia coli, and the purified protein showed molybdate-resistant phosphatase activity with nucleoside monophosphates as substrates, confirming that the identified gene codes for a nucleotidase. The optimum pH for the activity was 7–7.5. The recombinant enzyme did not show special affinity for any particular nucleotide, although the behaviour with AMP was different from that with the other nucleotides. The activity was inhibited by adenosine, and a regulatory role for this nucleoside was proposed. The expression pattern of PvNTD2 shows that it is ubiquitously expressed in all the tissues analysed, with higher expression in nodules of adult plants. The expression was maintained during leaf ontogeny, and it was induced during seedling development. Unlike PvNTD1, another NTD previously described in common bean, the high expression of PvNTD2 was maintained during nodule development, and its possible role in this organ is discussed.

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.

Germination and seedling development are crucial phases in a plant’s life cycle with economical and agronomical implications. The RNA quality in seeds is linked to seed viability, being an important agronomic trait since this leads to a loss in germination efficiency. In addition, RNA can be an important phosphorous reservoir in seeds, affecting the efficiency of the mobilisation of nutrients towards the seedlings. However, knowledge about the physiological function of ribonucleases during germination and seedling development is scarce. We analysed the ribonuclease activities of cotyledons during these processes and the expression of S-like ribonucleases T2. Ribonuclease activity was detected in cotyledons at 1 day after imbibition and the specific activity increased during germination and seedling development, reaching a maximal value at 10 days after imbibition. At this stage, the levels of proteins and RNA in cotyledons were very low. Using in-gel assays, three ribonucleases were detected with apparent molecular masses of 16, 17 and 19 kDa along cotyledon ontogeny. The S-like ribonucleases T2 family consists of four genes in common bean (PvRNS1 to PvRNS4). The expression of PvRNS1, PvRNS2 and PvRNS4 increased in the phase of nutrient mobilisation in cotyledons. The expression of PvRNS1 increased 1000 fold in cotyledons, from 1 to 6 days after imbibition. The suppression of the induction of ribonuclease activity and gene expression in decapitated seedlings suggests that the regulatory signal comes from the developing axes. These results clearly state that S-like ribonucleases T2 are involved in RNA turnover in cotyledons during seedling development.