Quantification of Mitochondrial Pyrroline-5-Carboxylate in Arabidopsis Under Pseudomonas Syringae Infection

Pyrroline-5 carboxylic acid (P5C) is an intermediary metabolite formed during proline synthesis and catabolism. Proline catabolism in two steps occurs in mitochondria where proline dehydrogenase (ProDH) catabolizes proline into P5C, and finally, pyrroline-5 carboxylate dehydrogenase catabolizes P5C into glutamate. Estimating mitochondrial P5C is essential to know how the metabolism of P5C in mitochondria via proline catabolism genes is responsible for causing disease and defense reaction in the case of Pseudomonas syringae-Arabidopsis interaction. There is no protocol available in the literature to estimate mitochondrial P5C due to which its role is masked and not properly deciphered. Here, we describe the protocol for estimating mitochondrial P5C from Arabidopsis leaves infected with bacterial pathogen P. syringae. Mitochondria was isolated from Arabidopsis leaf samples by percoll gradient centrifugation method. Mitochondrial amino acids and their derivatives were extracted in 80% methanol and without derivatization directly used for LC-MS analysis with standard labeled amino acids acting as internal control. P5C was detected as the daughter ion of the parent ion proline and quantified from the internally labeled proline standard.