Quantification of ABA, PA and conjugates
Samples for hormone analysis were processed according to McAdam (2015). 15 ng of [2H6]ABA and [2H3]PA (OlChemim Ltd, Czech Republic) were added to each sample as an internal standard. To measure ABA-GE levels, an additional aliquot was taken and an alkaline hydrolysis method was used (Hansen & Dörffling, 1999). Hormone samples were resuspended in 200 µl of 2% acetic acid and 10% acetonitrile in H2O (v v-1). Each sample was then centrifuged at 14800 RPM for 3 minutes and a 100 µl aliquot was taken for analysis. Hormone levels were quantified using an Agilent 6460 series triple quadrupole LC/MS (Agilent, CA, USA) fitted with an xBridge HPLC column (C18, 2.1 x 100 mm, 3.5 μm, Waters Corporation, MA, USA). Solvents used were 2% acetic acid in H2O (v v-1, Solvent A) and acetonitrile (Solvent B) at a flow of 0.3mL min-1. The running gradient went from 90% Solvent A and 10% Solvent B to 5% Solvent A and 95% Solvent B at 5 minutes and then back to initial values. An aliquot of 10 μL of sample was injected. The LC/MS was operated in negative ion electrospray mode with the needle running at 3.5 kV. To detect each metabolite and respective internal standard we used selected reaction monitoring. We used an ion source temperature of 325 ºC and nitrogen as the desolvation gas flowing at 8 l min-1. The tandem transitions were m/z 263.1 to 153, 204 and 219, for ABA; for [2H6]ABA the transitions monitored were m/z 269.1 to 159, 207 and 225. For PA, the tandem transitions were m/z 279.3 to 139.1 and 205; and m/z 282.3 to 142.1 and 208, for [2H3]PA. The cone voltage was 100V. For all transitions, the collision energy was 5V. Dwell time was set to 50 ms for each channel. Hormone levels were analyzed in the Agilent Quantitative Analysis software. Quantification was done using the m/z 263.1 to 153 and corresponding labelled channel for ABA, and m/z 279.3 to 139.1 and corresponding labelled channel for PA. Hormone levels in terms were calculated as the ratio of endogenous to labelled hormone peak areas, multiplied by the amount labelled ABA added to the sample (in all cases 15 ng), divided by the fresh weight of the sample collected. ABA-GE levels were determined as the difference between ABA levels in a quantified unhydrolyzed sample and the hydrolysed sample (Hansen & Dörffling, 1999).