Figure 7.

In vivo redox state of CHLM in VIGS plants and CHLM enzymatic activity assay. A, Total protein extracts from leaves of VIGS-GFP(Col-0), VIGS-TRX m2m4/m1(Col-0), VIGS-GFP(ntrc), and VIGS-TRX m2m4/m1(ntrc) plants under nonreducing conditions. Plants grew for 3 weeks after infiltration. Extracts were treated with (+) or without (−) AMS. In vivo CHLM was detected by immunoblot analysis using the anti-CHLM antibody. For loaded proteins, x = 50 μg of total proteins. Samples were separated on a nonreducing SDS-polyacrylamide gel. red, The reduced form; ox, the oxidized form. Relative oxidation (%) represents quantification of the oxidized form of CHLM, in which the immunoreacting CHLM protein bands were quantified by using the Tanon Gis software and the content of the oxidized variant was determined from the ratio between the oxidized form and the total amount of both the reduced and oxidized forms of each sample. We repeated it three times and got the similar results. n.d., Nondetectable. B, Plastid CHLM activity was measured with isolated total proteins from 5-week-old VIGS-GFP(Col-0), VIGS-TRX m2m4/m1(Col-0), VIGS-GFP(ntrc), and VIGS-TRX m2m4/m1(ntrc) plants. The extracted total proteins were treated with (+) or without (−) DTT. The data represent means ± sd of three biological replicates. Statistical significance compared with VIGS-TRX m2m4/m1(ntrc) plants treated with DTT and VIGS-TRX m2m4/m1(ntrc) plants without DTT is indicated by the asterisk (*, P ≤ 0.05 > 0.01, Student’s t test).