Journal: The Plant Cell
Article Title: VIH2 Regulates the Synthesis of Inositol Pyrophosphate InsP8 and Jasmonate-Dependent Defenses in Arabidopsis [OPEN]
Figure Lengend Snippet: VIH1 and VIH2 Are Functional Vip1-Type PPIP5 Kinases. (A) and (B) Structural model of the VIH2 ATP-grasp kinase domain (left) and the hPPIP5K2 ATP-grasp kinase domain (PDB ID: 3T9D, right) depicting the 5-InsP 7 binding sites and key catalytic residues. Residues coordinating substrate via polar contacts are shown as sticks, polar interactions are highlighted by dashed lines, α-helices are rendered in blue, β-sheets in orange, substrate (5-InsP 7 ) is rendered in magenta, and Mg 2+ ions are presented as green spheres. Three carbon atoms on the inositol ring are numbered. The ATP analog AMP-PNP (in [B] ) is depicted with gray carbon and orange and red phosphate groups. (C) and (D) Complementation of vip1 Δ-associated growth defects in yeast by ectopic expression of inositol pyrophosphate synthetases. The vip1 Δ yeast strain transformed with episomal pDR195( URA3 ) plasmids carrying either VIP1 , VIH1 , or VIH2 , sequences encoding their respective ATP-grasp kinase domains ( KD ) or designated kinase domain mutants, or carrying KCS1 were spotted in 8-fold serial dilutions onto uracil-free minimal medium in presence or absence of 6-azauracil, as indicated. Rescue on medium supplemented with 6-azauracil (right) reports Vip1 activity. (E) and (F) Normalized HPLC profiles of inositol phosphates of extracts from designated [ 3 H] inositol-labeled yeast transformants. Extracts were resolved by Partisphere SAX HPLC and fractions collected each minute for subsequent determination of radioactivity as indicated. Changes in elution times in independent experiments were observed and can be explained by subtle changes in column properties or column change. Experiments were repeated three times with similar results. (G) Complementation assays of kcs1 Δ-associated growth defects on high salt by ectopic expression inositol pyrophosphate synthetases. Wild-type (wt) or kcs1 Δ yeast transformants (both DDY1810 background) carrying designated plasmids were spotted in 8-fold serial dilutions onto solid minimal media (MM, uracil deficient CSM media with YNB and appropriate supplements) in presence or absence of NaCl and onto solid YPDA media incubated at 37°C.
Article Snippet: Inositol polyphosphates were extracted as described previously ( ) and resolved by strong anion exchange chromatography HPLC (using the partisphere SAX 4.6 × 125 mm column; Whatman) at a flow rate of 0.5 mL min−1 with the gradient of buffers A (1 mM EDTA) and B [1 mM EDTA and 1.3 M (NH4 )2 HPO4 , pH 3.8, with H3 PO4 ] following the standard protocol mentioned above.
Techniques: Functional Assay, Binding Assay, Expressing, Transformation Assay, Activity Assay, High Performance Liquid Chromatography, Labeling, Radioactivity, Incubation