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. 2021 Nov 29;34(2):889–909. doi: 10.1093/plcell/koab289

Figure 6.

Figure 6

MPK3 and MPK6 feedback-regulate PLDα1 and PLDδ to modulate submergence-induced PA accumulation. A, Co-IP assay showing the interaction between MPKs (MPK3 and MPK6) and PLDs (PLDα1 and PLDδ) in vivo. Constructs encoding MPK3-HA and MPK6-HA and FLAG-PLDα1 and FLAG-PLDδ were transiently co-transfected in WT Arabidopsis protoplasts and immunoprecipitated with anti-FLAG beads. B, BiFC assay of MPKs (MPK3 and MPK6) and PLDs (PLDα1 and PLDδ) in Arabidopsis. Constructs encoding split YFP, nYFP and cYFP fusions MPKs-nYFP (MPK3-nYFP and MPK6-nYFP) and PLDs-cYFP (PLDα1-cYFP and PLDδ-cYFP) were co-transfected in WT Arabidopsis protoplasts for 16 h under normal light–dark conditions. Confocal images for yellow fluorescent protein (YFP), chlorophyll autofluorescence, and bright field are shown. Bars, 10 μm. C, MPK3 and MPK6 phosphorylate PLDα1 and PLDδ in vitro. Phosphorylated PLD was detected with anti-thiophosphate ester rabbit monoclonal antibody after gel electrophoresis (top), recombinant MPK3, MPK6, and PLDα1 and PLDδ were detected with anti-GST antibody (bottom). Reactions lacking the specified components (‒) were used as controls. Recombinant proteins were separated by 10% SDS–PAGE after incubation in protein kinase buffer containing ATPγS and PNBM. D, MPK3 and MPK6 negatively regulate PLDα1 protein levels in response to submergence. Ten-day-old WT and mpk3,6-es seedlings were subjected to submergence for the indicated times. The immunoblots were probed with PLDα1-specific antibodies, with Ponceau S-stained total protein as loading control. E and F, The loss of MPK3 and MPK6 results in PA accumulation in response to submergence. Levels of total PA (E) and various PA species (F) were measured from the rosettes of 4-week-old WT and mpk3,6-es plants before submergence (air) and after 48-h submergence treatment (submergence). a, indicates significant differences when compared to that of 0 h; b indicates significant differences when compared to that of WT, respectively. Lipid profiling analyses were performed on three biological replicates with similar results. Values represent means ± sd (n = 4) of four independent biological replicates, and each replicate was collected from the rosettes of at least seven plants. Asterisks with “H” indicate significantly higher than WT for each PA species (*P < 0.05, **P < 0.01 by Student’s t test).