Biochemistry

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⁺.

This protocol is a simple colorimetric assay for internal ammonium quantification in aqueous extracts from plant tissues. The method is based on the phenol hypochlorite assay (Berthelot reaction):

NH₄⁺ + hypochlorite + OH^- + phenol → indophenol

The oxidation of indophenol caused by phenol oxidation is a blue dye that is quantified at 635 nm in a spectrophotometer. Per ammonium molecule one molecule of indophenol is formed. The protocol described here is for Arabidopsis thaliana (A. thaliana) leaves and roots, although it is also valid for other plants species.

SIET (scanning ion-electrode technique) is a new technique to study the flow rate of the ions and molecules in real time in living biomaterials by using microelectrodes and microsensor. This technique allows non-invasive, simultaneous measurement of fluxes of specific ions at the surface of an intact plant. It has high temporal and spatial resolutions. This protocol uses the SIET system for the measurement of ions flux rate in rice plants.