Measurement of delta-1-pyrroline-5-carboxylic acid in plant extracts for physiological and biochemical studies

Background/Objectives: δ1-Pyrroline-5-carboxylic acid (P5C) is a key intermediate in both the pathways leading in plants to proline synthesis, as well as in the proline catabolic route that takes place in the mitochondrion. Instead of being further oxidized, the P5C released in the latter process could be transferred to the cytosol and reduced back to proline in an apparently futile proline–P5C cycle, which seems to play a role in the plant response to pathogen attack. To date, studies on this cycle and the enzymes involved have been hampered by the lack of an efficient protocol to measure P5C in plant extracts. Methods: The experimental conditions allowing P5C to be extracted from plant tissues, stabilized, concentrated by cation-exchange chromatography, and quantified by reaction with o-aminobenzaldehyde were set up and validated. Results: The optimized protocol was shown to be capable of detecting P5C accumulation in tissues of a proline-treated Arabidopsis thaliana p5cdh knock-out mutant, as well as allowing the measurement of proline dehydrogenase activity in partially purified extracts from plant cultured cells. Conclusions: The method herein described overcomes current limitations in detecting and quantifying P5C and will facilitate the study of the related enzymes and the elucidation of its debated role in plant metabolism.