Gain-of-function activation of MPK3/MPK6 promotes ectopic overexpression of <i>GPT1</i> and loss-of-function of <i>MKK4/MKK5</i> leads to a reduced <i>GPT1</i> expression and lipid accumulation.

(A) Bright-field, DAPI-staining, and GPT1-eYFP fluorescence images of pollen from PGPT1:GPT1-eYFP DD plants treated with either ethanol control (+EtOH) or DEX. The intensity of eYFP fluorescence was quantified, and normalized to that in the +EtOH control, which was set as 100%. The difference in the intensity of eYFP fluorescence between DEX treated and EtOH control was very significant (PPGPT1:GPT1-eYFP transgenic lines in DD background were analyzed and all gave similar results. Results from one of the five lines are shown. MN, microspore nucleus. Bar = 10 μm. (B) Bright-field, DAPI-staining, and GPT1-eYFP fluorescence images of pollen from PGPT1:GPT1-eYFP and PGPT1:GPT1-eYFP mkk4 mkk5 plants. The intensity of eYFP fluorescence was quantified, and normalized to that in the Col-0 background, which was set as 100%. The difference in eYFP fluorescence intensity between mkk4 mkk5 double mutant and Col-0 control was very significant (PPGPT1:GPT1-eYFP transgenic lines in both wild-type and mkk4 mkk5 backgrounds were analyzed with similar results. One of them is shown. VN, vegetative nucleus. GN, generative nucleus. Bar = 10 μm. (C) Nile blue A lipid staining of pollen from Col-0 and mkk4 mkk5 plants. (D) Nile blue A lipid staining of pollen from chemical genetically rescued MPK6SR plants treated with either DMSO solvent or NA-PP1. (E) Quantitation of Nile blue A-stainable pollen from Col-0 or mkk4 mkk5 plants. (F) Quantitation of Nile blue A-positive pollen from DMSO- and NA-PP1 treated MPK6SR plants. At least 100 pollen grains were counted in each repeat. Error bars indicate SD (n = 3). **P ≤ 0.01. Bar = 50 μm. (G) Working model depicts the function of plastid-localized GPT1 downstream of MKK4/MKK5—MPK3/MPK6—WRKY2/WRKY34 signaling pathway in controlling lipid body accumulation during pollen maturation.