Figure 1.

In vitro, Arabidopsis PFA-DSPs display Mg²⁺-dependent PP-InsP phosphohydrolase activity with high specificity for 5-InsP₇. Recombinant His-MBP-PFA-DSPs and His-MBP-Siw14 (indicated with the plus symbol in (C) and (E)) were incubated with 0.33 mM InsP₇ at 22 °C. His-MBP served as a negative control (as indicated with the minus symbol in (C) and (E)). (A) 0.4 μM His-MBP-PFA-DSP1 was incubated for 1 h with 1-InsP₇ or 5-InsP₇, and 1 mM EDTA, MnCl₂, MgCl₂, CaCl₂, or ZnCl₂ as indicated. The reaction products were then separated by 33% PAGE and visualized by toluidine blue. (B–D) The InsP₇ phosphohydrolase activity of ∼0.4 μM His-MBP-PFA-DSPs and His-MBP-Siw14 was analyzed in the presence of 1 mM MgCl₂. After 1 h, the reaction products were then (B, D) spiked with isotopic standards mixture ([¹³C₆] 1,5-InsP₈, [¹³C₆] 5-InsP₇, [¹³C₆] 1-InsP₇, [¹³C₆] InsP₆, [¹³C₆] 2-OH InsP₅) and subjected to CE-ESI-MS analyses or (C) separated by 33% PAGE and visualized by toluidine blue/DAPI staining. (D) Data represent mean ± SEM (n = 3). Representative extracted-ion electropherograms are shown in Figure S2. Asterisks indicate values that are significantly different from the MBP control reactions (according to Student’s t test, P < 0.05 (*); P < 0.01 (**)). (E) Recombinant His-MBP-PFA-DSP5 (2 μM) was incubated with 0.33 mM InsP₇ isomers for 2 h. The reaction product was separated by 33% PAGE and visualized with toluidine blue. (A, C, E) Identity of bands was determined by migration compared to TiO₂-purified mrp5 seed extract.