FIGURE 3.

Activity of serine proteases at pH 7.5 during leaf senescence in two genotypes of Brassica napus L. supplied with high (HN) or low (LN) nitrate for 23 days. Soluble proteins were extracted from leaf blade of source leaf (L12) of Ténor or Samouraï plants after 0, 16, and 23 days of HN (3.75 mM NO₃^-) or LN (0.375 mM NO₃^-) treatment. Samples were subjected to a labeling of protease activity with FP-Rh (specific fluorescent probe of serine proteases) (pH 7.5; 1 h labeling). The fluorescence was detected by a scanner after separation of samples by SDS-PAGE (A). Mix corresponds to a mixture of the protein extracts obtained at 0, 16, and 23 days for both treatments (HN and LN) in the presence of FP-Rh. Mix+DFP: mix and diisopropylfluorophosphate (specific inhibitor of serine proteases). NPC: no probe control (absence of FP-Rh). After incubation, the abundance of RuBisCO large subunit (RBLC) was observed after gel staining by coomassie brilliant blue (cbb). In each lane, the total amount of loaded proteins corresponds to 20 μL of protein sample. Positions of active proteases are represented by black arrowheads. Serine protease global activity characterized by the fluorescence intensity was calculated relative to the protein amount (B). The gel is representative of three biological replicates. Vertical bars indicate ± SD of the mean of three biological replicates. Statistical differences are represented by letters (P < 0.05, ANOVA, Newman–Keuls test).