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We used two methods and assayed to collect … We used two methods and assayed to collect the HS of Arabidopsis seedlings after a 7 day-incubation. To quench enzyme activity in Arabidopsis, we treated the seedlings in the following manner: (i) a set of HS vials wrapped in aluminum foil were incubated at 80 °C for 30 min in a windy oven (WFO-600 ND; Eyela, Tokyo, Japan) and (ii) 0.25-M EDTA-NaOH water solution (pH 7.5) was added into another set of vials to attain a final EDTA concentration of 50 mM. Solid CaCl2 was then immediately added to a final concentration of 5 M. Next, 10-μL of n-alkane standard solutions C8-20 (0.8 mg/l) was added to each vial as an internal standard. The vials were closed with magnetic screw-caps and then sonicated (US-108; NSD, Suwa, Japan) at a frequency of 38-Hz for 5 min. Vials without any treatment were prepared and used as controls.<br />
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Nine types of SPME fibers were purchased from Supelco (Supelco, PA, USA). A SPME fiber coated with a 100-μm-thick layer of polydimethylsiloxane (PDMS) metal alloy (100-μm PDMS) was finally chosen for VOC profiling (see Results and Discussion section). SPME fibers were coated with (i) 30-μm- or (ii) 7-μm-thick layers of PDMS-fused silica (FS) fiber/stainless steel (SS), (iii) 75-μm-thick layer of carboxen/polydimethylsiloxane (CAR/PDMS) FS/SS, (iv) 85-μm-thick layer of CAR/PDMS StableFlex (SF) fiber/SS, (v) 65–μm-thick layer of PDMS/divinylbenzene (DVB) SF/SS, (vi) 50/30-μm-thick layer of DVB/CAR on PDMS (DVB/CAR/PDMS) SF/SS, (vii) 85-μm-thick layer of polyacrylate (PA 85) or (viii) carbowax-polyethylene glycol (C-PEG) for HS collection. Before the analysis, all fibers were conditioned at the appropriate conditioning temperature and time as shown in Table 3. Each fiber was exposed to the vial headspace for 20 min at 60 °C with continuous agitation. 20 min at 60 °C with continuous agitation.
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