Background

Determination of ureides is important for characterizing N₂ fixation and assimilation in legume plants as well as stress and nutritional responses of non-ureidic plants like Arabidopsis (Brychkova et al., 2008; Watanabe et al., 2014; Irani and Todd, 2016 and 2018). The formation of allantoate is also useful for in vivo and in vitro determination of allantoinase activity (Duran and Todd, 2012). Techniques requiring expensive equipment, such as high-performance liquid chromatography (HPLC), are commonly used for ureide quantification while ethanol extraction is mostly employed for ureide recovery from plant tissues. The present protocol is based on flexible spectrometry that uses low volume of cheap reagents. The use of phosphate buffer instead of ethanol for ureide extraction and quantification greatly improved the reproducibility of the measurements, possibly by decreasing the interfering compounds that abound with the ethanol extraction.

Allantoin and allantoate present in plant extracts are converted to glyoxylate by alkaline-acidic hydrolysis (Vogels and Van der Drift, 1970). To carry out this protocol, three tubes (A, B and C) for alkaline and/or acid hydrolysis are prepared for each sample (Figure 1). The glyoxylate is converted into glycoxylic acid phenylhydrazone and is then oxidized by ferricyanide to form red-colored 1,5-diphenylformazan. The absorbance of supernatants is measured using a spectrophotometer at 520 nm. Allantoin content is obtained by subtracting the levels of glyoxylate resultant of allantoate degradation (tube B) from glyoxylate derived of allantoin alkaline-acid hydrolysis (tube C). Likewise, allantoate content can be inferred by the subtraction of basal glyoxylate levels (tube A) from glyoxylate converted from allantoate (tube B).

The described protocol was carried out for determine ureide concentration for different tissues, nutritional and stress conditions in Arabidopsis thaliana (Lescano et al., 2016 and 2020); and in roots and shoots of nodulating and non-nodulating soybean plants (Muñoz et al., 2016).


Figure 1. Alkaline-acidic hydrolysis of ureides