Abstract
Because proline accumulates rapidly in response to several stress conditions such as drought and excess salt, increased intracellular levels of free proline are considered a hallmark of adaptive reactions in plants, particularly in response to water stress. Proline quantitation is easily achievable by reaction with ninhydrin, since under acidic conditions peculiar red or yellow reaction products form with this unique cyclic amino acid. However, little attention has been paid to date to cross-reaction of ninhydrin with other amino acids at high levels, or with structurally related compounds that may also be present at significant concentrations in plant tissues, possibly leading to proline overestimation. In vitro at high pH values, δ1-pyrroline-5-carboxylate reductase, the enzyme catalyzing the second and last step in proline synthesis from glutamate, was early found to catalyze the reverse oxidation of proline with the concomitant reduction of NAD(P)+ to NAD(P)H. Here we characterized this reverse reaction using recombinant enzymes from Arabidopsis thaliana and Oryza sativa, and demonstrated its utility for the specific quantification of L-proline. By optimizing the reaction conditions, fast, easy, and reproducible measurement of L-proline concentration was achieved, with similar sensitivity but higher specificity than the commonly used ninhydrin methods.
Highlights
Water stress tolerance is in the focus of many research projects and a major goal for plant breeding to secure crop productivity in the face of ongoing climate change (Ahanger et al, 2017)
We have previously described P5CRs from Arabidopsis thaliana, Oryza sativa, and Medicago truncatula as very stable enzymes that can be stored at 4◦C over prolonged periods with negligible loss of activity (Funck et al, 2012; Giberti et al, 2014; Forlani et al, 2015a; Ruszkowski et al, 2015)
D-proline and DL-pure δ1pyrroline-5-carboxylic acid (P5C) produced about 60% and 50% of the absorbance at 520 nm compared to equivalent concentrations of L-proline, while hydroxyproline reacted with similar efficiency as pipecolate (Figure 1B)
Summary
Water stress tolerance is in the focus of many research projects and a major goal for plant breeding to secure crop productivity in the face of ongoing climate change (Ahanger et al, 2017). A modification of this method was described in which lysine and hydroxylysine do not react (Bates et al, 1973), establishing a quick assay for proline in plant extracts that has become the most widely adopted reference method (cited by more than 12,000 articles) and is used with minor modifications until today. This assay takes advantage of the formation of a red, hydrophobic reaction product from proline and ninhydrin at very low pH and high temperature (100◦C), while all other proteinogenic amino acids produce little or no color. An intermediate in the synthesis and degradation of
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