Abstract
It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (NpnN’s) and adenosine 5′-phosphoramidate (NH2-pA) belonging to nucleoside 5′-phosphoramidates (NH2-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that NpnN’s induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH2-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH2-pA, NH2-pG) and pyrimidine (NH2-pU, NH2-pC) nucleoside 5′-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH2-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH2-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5′-phosphoramidates could be considered as new signaling molecules.
Highlights
Continuing our long-lasting studies on uncommon nucleotides, over a decade ago [1,2,3,4,5,6], we began to investigate the biochemistry of a rather neglected nucleotide, adenosine 5 phosphoramidate (NH2-pA), since it appeared to be a very good substrate of Fhit proteins [7]
In our earlier studies on the effect of NH2-pA on the expression of the genes coding for phenylalanine ammonia-lyase (PAL) and 4-coumarate:coenzyme A ligase (4CL) in Arabidopsis seedlings, we found that of the concentrations tested in the 0.05–25 μM range, 5 μM NH2-pA appeared to be the most effective [4]
This study demonstrated that the uncommon nucleotide NH2-pA, naturally occurring in organisms, applied to a cell suspension of Vitis vinifera, induced the expression of genes that control both the biosynthesis of stilbenes (Figures 4a and 5a) and lignins (Figure 8a,b)
Summary
Continuing our long-lasting studies on uncommon nucleotides, over a decade ago [1,2,3,4,5,6], we began to investigate the biochemistry of a rather neglected nucleotide, adenosine 5 phosphoramidate (NH2-pA), since it appeared to be a very good substrate of Fhit (for fragile histidine triad) proteins [7]. Its synthesis proceeds according to the reaction SO4-pA + NH4+ → NH2-pA + SO4 2− + 2H+ catalyzed by adenylyl sulfate:ammonia adenylyltransferase (EC 2.7.7.51) (Figure 1) This activity was found in the algae Chlorella pyrenoidosa and Euglena gracilis, the amoeba Dictyostelium discoideum, the bacterium Escherichia coli, and the higher plants Hordeum vulgare, Spinacia oleracea [8], and Lupinus luteus [5]. In the latter organism, this transferase activity proved to be an inherent property of dinucleoside triphosphatase, the Fhit protein [5]. Fhits, regardless of their origin, are able to catalyze both the synthesis and cleavage of NH2-pA [5]
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