This protocol can be used to inhibit the biosynthesis of polyamines, specifically putrescine, in tomato plants grown with NH4+ as a solely N source. In general, polyamines are positively charged small metabolites implicated in physiological processes, including organogenesis, embryogenesis, floral initiation and development, leaf senescence, pollen tube growth, fruit development and ripening and participate in the response to abiotic and biotic stresses (Tiburcio et al., 2014). Polyamines are synthesized from amino acids by decarboxylation of ornithine or arginine by ornithine decarboxylase (ODC) or arginine decarboxylase (ADC), respectively (Walters, 2003). Tomato plants grown with NH4+ as the sole N source presented an increase of putrescine content in leaves (Fernández-Crespo et al., 2015). To assess the importance of putrescine accumulation, DL-α-(Difluoromethyl)arginine (DFMA) and DL-α-(Difluoromethyl)ornithine (DFMO), inhibitors of putrescine synthesis, were used as irreversible inhibitors of ADC and ODC enzymes, respectively (Fallon and Phillips, 1988), with the purpose of reducing cellular putrescine accumulation induced by NH4+ nutrition.
The inhibitor solution containing 2 mM DFMA and 5 mM DFMO was applied directly to each pot during the week prior to sample collection. Putrescine content was reduced by 35.3% in tomato plants grown with NH4+.
[Background] The application of the inhibitors DFMA and DFMO was normally performed in MS medium and in vitro assays (Perez-Amador et al., 2002; Stes et al., 2011). However, we needed to test effectiveness of these inhibitors in vivo with the purpose to maintain natural growth conditions. Moschou et al. (2008) demonstrated the inhibition effect of DFMA and DFMO when applied in hydroponic cultures at 0.1 mM and 1 mM respectively. In this work, we used similar approaches with some modifications: the hydroponic culture was changed by vermiculite growing medium and the concentration applied for the inhibitors was modified.
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