Figure 3.

PA binds to MPK3 and MPK6 and enhances submergence-induced MPK3 and MPK6 activity. A, Lipid binding specificity of recombinant MPK3 and MPK6 proteins on membrane filters. About 50 μM of various lipids (PA, PC, PE, PI, PG, and PS dissolved in chloroform; natural lipid mixtures purified from soy, Avanti Polar Lipids) were spotted onto nitrocellulose membrane and incubated with 10 μg of purified GST-MPK3, GST-MPK6, or GST protein. Binding was detected by immunoblotting using an anti-GST antibody. Equal volume of chloroform was spotted as negative control (Blank). B, Pull-down assay showing the physical interaction between PA and recombinant MPK3 and MPK6 proteins. Recombinant proteins were incubated with PA beads, and the precipitated GST-MPK3 and GST-MPK6 were detected with anti-GST antibody. GST-PYL4 was used as a negative control. C, Dissociation constant (K_d) for the binding of recombinant MPK3 and MPK6 proteins to liposomes of PA, PC, PI, and PS (natural lipid mixtures purified from soy, Avanti Polar Lipids). A serial dilution of various liposomes ranging from 1.5 nM to 50 μM was prepared for mixing with the labeled proteins, and their binding affinities were measured by MST analysis. K_d, dissociation constant. ND, not detected. D and E, MPK3 and MPK6 are activated by submergence. Ten-day-old WT seedlings were exposed to light submergence (LS, D) or in the dark submergence (DS, E). MPK3/MPK6 kinase activities were detected with anti-pTEpY antibody. MPK3 and MPK6 proteins were detected with anti-MPK3 and anti-MPK6 antibodies, respectively. Actin, detected with an anti-actin antibody, was used as loading control. F, Quantification of MPK phosphorylation activity shown in (D) and (E). Data were calculated according to relative intensity from three independent experiments and the average values ± sd are shown. G, MPK3 and MPK6 kinase activities in WT and pldα1 pldδ plants and following submergence for 0.5, 1, 3, and 6 h. Total proteins were extracted and immunoblotting assays were performed using anti-pTEpY, anti-MPK3, and anti-MPK6 antibodies, with Ponceau S-stained total protein as loading control. hpt, hour posttreatment. Relative intensity of each p-MPK3 or pMPK6 band normalized to the loading control is shown below. H, PA induces MPK3 and MPK6 activity in planta. Ten-day-old WT seedlings were treated without (Mock) or with 50 μM PA or PS liposomes (natural lipid mixtures purified from soy, Avanti Polar Lipids) for 0.5, 1, and 3 h, and immunoblotting assays were performed using anti-pTEpY, anti-MPK3, anti-MPK6, and anti-actin antibodies, with actin as loading control. Relative intensity of each p-MPK3 or pMPK6 band normalized to the loading control is shown below. All experiments were performed on three biological replicates with similar results. Data in (C) and (F) are means ± sd of three biological replicates. Asterisks indicate significant differences from WT at 0 h (*P < 0.05 by Student’s t test).