Characterization of the pH preference of RNS2. (A) Recombinant Arabidopsis RNases RNS1 (lane 1), RNS2 (lane 2), and RNS3 (lane 3) were obtained from a yeast expression system and analyzed by an RNase activity in gel assay. Each lane contains 1 μg of protein. (B) RNase activity of yeast-expressed RNS1 (●), RNS2 (■ and ◆), and RNS3 (▲) was assayed in vitro at different pHs as indicated. The activity of each enzyme relative to the maximum activity (at optimal pH) is reported. RNS2 activity (solid lines) near the pH optimum was assayed using two different buffers, Tris (◆) or phosphate (■), to discard any buffer artifact. (C) pH preference of native RNS2. Leaf protein extracts (10 μg) from WT and rns2-2 mutant plants were analyzed by an RNase activity in gel assay, with incubations at different pHs as indicated (below the panels). The position of RNS2 is indicated. At pH 6, it is possible to detect activity of RNS1 as an RNase band below the RNS2 activity.