The ability to measure plant hormones quantitatively is important as plant hormones regulate plant growth, development and response to biotic and abiotic cues. In this protocol, we describe the quantitative analysis of major plant hormones from crude plant extracts. Plant hormones are determined using reverse-phase liquid chromatography-tandem mass spectrometry with multiple reaction monitoring. The method provides quantification of most major plant hormones in a single run from 50 mg of fresh plant tissue. Extraction and quantitative analysis of 40 samples takes 2-3 d.

Reactive oxygen species (ROS) are produced in plants under various stress conditions and serve as important mediators in plant responses to stresses. Here, we show that the cytosolic glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenases (GAPCs) interact with the plasma membrane-associated phospholipase D (PLDd) to transduce the ROS hydrogen peroxide (H 2 O 2 ) signal in Arabidopsis thaliana. Genetic ablation of PLDd impeded stomatal response to abscisic acid (ABA) and H 2 O 2 , placing PLDd downstream of H 2 O 2 in mediating ABA-induced stomatal closure. To determine the molecular link between H 2 O 2 and PLDd, GAPC1 and GAPC2 were identified to bind to PLDd, and the interaction was demonstrated by coprecipitation using proteins expressed in Escherichia coli and yeast, surface plasmon resonance, and bimolecular fluorescence complementation. H 2 O 2 promoted the GAPC-PLDd interaction and PLDd activity. Knockout of GAPCs decreased ABA-and H 2 O 2 -induced activation of PLD and stomatal sensitivity to ABA. The loss of GAPCs or PLDd rendered plants less responsive to water deficits than the wild type. The results indicate that the H 2 O 2 -promoted interaction of GAPC and PLDd may provide a direct connection between membrane lipid-based signaling, energy metabolism and growth control in the plant response to ROS and water stress.

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