Background

Pip, a non-protein amino acid derivative of lysine, accumulates in local and distal tissue of pathogen infected plants and in body fluids of patients with peroxisomal disorders (Schutgens et al., 1986; Yoon and An, 2010; Návarová et al., 2012; Wang et al., 2018). In plants, Pip is synthesized from lysine via ALD1 (AGD2 like defense response protein) encoded aminotransferase (Návarová et al., 2012). ALD1 localizes at the chloroplast and converts lysine to ϵ-amino-α-keto caproic acid, which then cyclizes to form 1-piperideine-2 carboxylic acid (P2C) (Ding et al., 2016; Hartmann et al., 2017). The P2C intermediate is subsequently converted to Pip by ornathine cyclodeaminase (encoded by SARD4) (Ding et al., 2016; Hartmann et al., 2017). Pip accumulation upon pathogen infection is associated with the induction of systemic acquired resistance (SAR), a form of broad-spectrum defense that protects uninfected parts of the plant against secondary infections. Pip confers SAR by increasing levels of the free radicals, nitric oxide (NO) and reactive oxygen species (ROS), which act upstream of glycerol-3-phosphate (G3P). Thus, a linear pathway comprising Pip→NO↔ROS→AzA→G3P functions in parallel with salicylic acid-derived signaling, and that both pathways are essential for the induction of SAR (Chanda et al., 2011; Yu et al., 2013; Gao et al., 2014; Wang et al., 2014; Bernsdorff et al., 2016; Lim et al., 2016; Wenig et al., 2019). Pip and G3P have also been suggested to operate in a positive feedback loop that functions upstream of volatile pinenes (Wenig et al., 2019). Notably, de novo synthesis of Pip in distal tissues is dependent on both SA and G3P (Wang et al., 2018). These results suggest that metabolites in a signaling cascade can stimulate biosynthesis of each other depending on their relative levels and their site of action. Notably, ALD1-derived factors (such as Pip) also contribute to SAR-associated transcriptional reprograming in the systemic tissue since pathogen-responsive transcriptional changes in the distal tissue are almost completely absent in ald1 mutant plants (Gruner et al., 2013). Exogenous application of pipecolic acid also enhances disease resistance against bacterial and viral pathogens in Nicotiana plants (Vogel-Adghough et al., 2013; Wang et al., 2019). Recent work has shown that Pip levels are regulated by calmodulin-binding transcription factors (Kim et al., 2020; Sun et al., 2020), calcium-dependent protein kinase 5 (Guerra et al., 2020), and Jumonji domain containing K3K4 demethylase (Li et al., 2019). Thus, determination of pipecolic acid in the plant tissues is an integral part of research on plant defense.

In the present method, we used propyl chloroformate based one-step derivatization procedure for quantification of Pip from Arabidopsis leaves. Norvaline was used as an internal standard and the derivatized products were analysed by gas chromatography (GC)-mass spectrometry (MS) using selective ion monitoring (SIM) mode. Our method is based on two earlier procedures that either used methyl chloroformate to derivatize amino acids (Villas-Bôas et al., 2003) or propyl chloroformate based derivatization of amino acids which were extracted using a commercial kit (EZ:faast free amino acid analysis kit, Phenomenex) (Kugler et al., 2006). One other GC-MS based method for Pip quantification from human plasma involved trimethylsilyl- and trifluoroacyl-derivatizations (Yoon and An, 2010). This method is laborious and moreover uses [2H⁹]-Pip as an internal standard, which is ~1,000 times more expensive compared to norvaline. The reaction scheme for internal standard norvaline (Figure 1A) and the analyte pipecolic acid (Figure 1B) are shown in Figure 1. The method reported here is simple, cost effective, and can be used to process 100-200 samples in a day.


Figure 1. Derivatization reactions for internal standard L-norvaline (A) and analyte L-pipecolic acid (B) with propyl chloroformate.Propyl chloroformate reacts with both carboxyl and amino groups in basic conditions releasing HCl for respective condensation reactions. The HCl is neutralized by NaOH in the reaction mixture to produce H₂O and NaCl. The mixed anhydride formed from the carboxyl group and propyl chloroformate is lost as CO₂.