Asc was measured as described (Foyer et al., 1983). Apoplastic levels were obtained essentially as described (Pignocchi et al., 2003) in which leaves from 8-week-old tobacco were collected, the midvein removed, and 2-cm² pieces vacuum infiltrated at −70 kPa with chilled 10 mM citrate, pH 3.0, for 10 min. Leaves were blotted dry, rolled, and centrifuged in a prechilled syringe at 2000 rpm for 10 min at 4°C. An equal volume of 2.5 M HClO₄ (for the ascorbate assay) or 10% metaphoric acid (for the GSH assay) was added to the apoplastic wash fluid. Guard cells were isolated as described (Kruse et al., 1989). For whole leaf assays, leaves were ground in 2.5 M HClO₄ and centrifuged at 13,000 rpm for 10 min. Two volumes of 1.25 M Na₂CO₃ were added to the supernatant, and after centrifugation, 100 μL was added to 895 μL 100 mM K₂HPO₄/KH₂PO₄, pH 5.6. Asc was determined by the change in absorbence at 265 nm after the addition of 0.25 units of ascorbate oxidase. The total amount of reduced and oxidized ascorbic acid (i.e., Asc and DHA) was determined by reducing DHA to Asc (in a reaction containing 100 mM K₂HPO₄/KH₂PO₄, pH 6.5, 2 mM GSH, and 0.1 μg recombinant wheat DHAR protein incubated at 25°C for 20 min) before measuring Asc. The amount of DHA was determined as the difference between these two assays. The level of GSH and GSSG was determined as described (Shimaoka et al., 2000).