Biochemical and Molecular Characterization of PvNTD2, a Nucleotidase Highly Expressed in Nodules from Phaseolus vulgaris

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 Metabolism Is Induced in Common Bean During Early Seedling Development

“…In embryonic axes of common bean, a sharp increase in ssDNAse activity occurs when the radicle emerges in parallel to the increase in nucleosidase activity, ureides, and some nucleosides. An S1/P1 nuclease with a proposed role in development and two nucleotidases are also induced after the radicle protrusion ( Lambert et al, 2014 ; Cabello-Diaz et al, 2015 ; Galvez-Valdivieso et al, 2020 ). Therefore, the coordinated expression of nucleases, nucleotidases, and nucleosidases in developing axes suggests that the nucleic acids stored during seed formation could serve as a potential reserve of nitrogen and phosphorus.…”

“…The inserts were released by digestion with Sal I and Not I, cloned in frame into pET30b+ (Novagen), and used to transform the Escherichia coli strain BL21 (DE3). Overexpression and purification of the recombinant proteins were performed as described in Galvez-Valdivieso et al (2020) except that after the addition of IPTG, the culture was incubated at 28°C for 6 h, the binding buffer was 20 mM Na 2 HPO 4 , 500 mM NaCl, and 10 mM imidazole, (pH 7.4), supplemented with DNAase, RNAse, and 1 mM phenylmethylsulfonyl fluoride (PMSF) for the bacterial lysis, and the washing and the elution buffers contained 10 and 300 mM of imidazole, respectively.…”

“…In addition, direct dephosphorylation of xanthosine monophosphate (XMP) by an XMP phosphatase also contributes to the pool of xanthosine ( Baccolini and Witte, 2019 ). Genes encoding GMP or XMP phosphatase have not been identified in Arabidopsis yet, but two phosphatases with high affinity for nucleosides monophosphate have been recently overexpressed and characterized in Phaseolus vulgaris ( Cabello-Diaz et al, 2015 ; Galvez-Valdivieso et al, 2020 ), and an XMP phosphatase activity was identified in cowpea nodules ( Atkins, 1981 ). The pyrimidine nucleotides UMP and CMP are dephosphorylated to the nucleosides uridine and cytidine, respectively, and the latter is deaminated to uridine ( Zrenner et al, 2006 ).…”

“…In addition, several studies demonstrated that nucleotide metabolism is strictly regulated at the early stages of seedling development to the point of being critical for the germination success ( Stasolla et al, 2003 ; Jung et al, 2009 ; Cornelius et al, 2011 ). In common bean, nucleotidase activity and expression of two nucleotidase genes are induced during seedling development ( Cabello-Diaz et al, 2012 , 2015 ; Galvez-Valdivieso et al, 2020 ) coincidently with the induction of nucleases ( Lambert et al, 2014 , 2016 ).…”

Methyl jasmonate elicitation of common bean seedlings induces nucleotidase activity and the expression of several nucleotidase genes in radicles

“…However, it has not been elucidated if this reaction is carried out by different enzymes or if there are specific nucleotidases for each. A phosphatase with elevated affinity for nucleoside monophosphate was purified from embryonic axes of common bean (Cabello-Díaz et al 2012), and the gene encoding the protein was identified (Cabello-Díaz et al 2015), as well as another gene whose expression is higher in the radical nodules where ureides are synthesized (Galvez-Valdivieso et al 2020).…”