Plant Cell. 2004 May; 16(5): 1143–1162
Asc and DHA MeasurementsAsc 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).
Plant Cell. 2004 May; 16(5): 1143–1162
Determination of Leaf Water Potential and Leaf Water ContentLeaf water potential was determined at 10 am and 2 pm using a pressure chamber (Model 3000; Soilmoisture Equipment, Santa Barbara, CA) as described by Gómez-Cadenas et al. (1996). Data are presented as the average and standard deviation of six replicate leaves. Leaf water content was measured at 10 am and 2 pm. Leaf fresh weight was measured immediately, and their water saturated weight was determined after allowing the leaf to absorb water until saturation. Leaf dry weight was measured after drying at 80°C. The difference in water weight between fresh and water-saturated leaves was then calculated in the morning and afternoon and expressed on a leaf dry weight basis. Data are presented as the average and standard deviation of six replicate leaves.
