Optimized LC-MS method for simultaneous polyamine profiling and ADC/ODC activity quantification and evidence that ADCs are indispensable for flower development in tomato

Abstract

Polyamines (PAs) are essential for plant development and stress responses, requiring tight homeostatic regulation. Many PA enzymes are regulated post-transcriptionally, making traditional transcript-based methods ineffective in determining their abundance, highlighting the need for alternative approaches to study PA homeostasis. Here, we refined a liquid chromatography-mass spectrometry (LC-MS) based method to simultaneously quantify activities of two key PA synthesizing enzymes - arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) - from plant tissues using stable isotope substrates. By optimizing substrate concentrations, we increased assay sensitivity >10-fold in tomato leaf tissue. We further adapted this protocol for Nicotiana benthamiana, a model plant widely used for transient recombinant protein expression. Expression of epitope-tagged ADCs in this system revealed a direct correlation between protein abundance and enzymatic activity, demonstrating that ADC activity can infer its protein abundance in native tissues. Proof-of-principle experiments with the N. benthamiana expression system, confirm substrate specificity of tomato ADC and ODC enzymes and essential catalytic residues of tomato ADCs. Beyond enzymatic activities, our LCMS-based method also permits quantification of 11 PA network metabolite concentrations from the same LCMS sample. Visualizing this data as a heatmap pathway diagram, alongside ADC/ODC activities provides a comprehensive overview of PA metabolism in plant tissues. We also studied tomato CRISPR-Cas9-induced mutants deficient in ADC or ODC, complemented by phenotypic analysis. LC-MS analysis of an adc1/adc2 double mutant - an embryo lethal genotype in Arabidopsis - had no detectable agmatine, the product of ADCs. Additionally, despite a reduction in putrescine, no impact on the downstream PAs, spermidine and spermine, was found. The adc1/adc2 double mutant showed severe developmental abnormalities, including complete flower loss, demonstrating the indispensable role of ADCs in flower development. In summary, our optimized LC-MS approach for simultaneous quantification of ADC/ODC enzyme activity and PA-pathway metabolites, the ability to transiently express and functionally analyze recombinant ADC/ODC proteins in planta, and a collection of tomato CRISPR mutants deficient in these enzymes collectively establish a versatile new experimental toolkit to dissect PA homeostasis and PA-dependent developmental processes in plants.