Fig 3. The amplitude and duration of MAPK activation regulate photosynthetic inhibition.

(A) Controlling the amplitude of MPK3/MPK6 activation in DD plants by spraying different concentrations of DEX. Soil-grown DD plants were spray treated with different concentrations of DEX. At 9 h after DEX spray, NtMEK2^DD expression and MPK3/MPK6 activation were detected by immunoblot analysis using anti-FLAG and anti-pTEpY, respectively. Fv/Fm was measured at 18 h after DEX treatment. Values are means ± SD, n = 6, **P ≤ 0.001. The numerical values used to construct panel A can be found in S1 Data. (B) Transient activation of MPK3/MPK6 after flg22 treatment does not induce a decrease in PSII core D1 protein. Twelve-d-old DD and Col-0 plants grown in liquid medium were treated with 5 μM DEX and 200 nM flg22 for indicated times, respectively. Thylakoid membranes were isolated and solubilized with 1% dodecyl maltoside. Samples equivalent to 2 μg of chlorophyll were loaded to a 12% SDS-PAGE with 6 M urea. Anti-D1 was used to detect D1 abundance. MAPK activation was detected by immunoblot analysis using anti-pTEpY antibody. Ponceau S staining was used to show equal loading. (C) Twelve-d-old DD and Col-0 plants grown in ½ MS agar plates were treated with 5 μM DEX or EtOH (solvent control) and then kept in dark or under light for 12 h. D1 protein abundance in thylakoid membrane preparations was detected by immunoblot analysis using anti-D1 antibody. Ponceau S staining was used to show equal loading. (D) Prolonged, but not transient, MAPK activation induces photosynthetic inhibition. Twelve-d-old DD MPK6SR plants grown in liquid medium were first treated with 5 μM DEX. Seedlings were washed three times with ½ MS medium to remove DEX before the addition of 10 μM NA-PP1. Black bars indicate the durations of DEX or NA-PP1 treatment. Thylakoid membranes were isolated and solubilized with 1% dodecyl maltoside. Samples equivalent to 8 μg of chlorophyll were loaded to a BN-PAGE. See also S3 Fig. BN-PAGE, blue native polyacrylamide gel electrophoresis; CBB, Coomassie brilliant blue; Col-0, Columbia-0; CP43, photosystem II chlorophyll protein at 43 kDa; DD, GVG-NtMEK2^DD; DEX, dexamethasone; DNH, NADH dehydrogenase-like; EtOH, ethanol; FLAG, an octapeptide; flg22, a 22 amino acids flagellin fragment; LHCII, light-harvesting complex II; mc, mega-complex; MPK, mitogen-activated protein kinase; MS, Murashige and Skoog medium; NA-PP1, 4-amino-1-tert-butyl-3-(1’-naphthyl)pyrazolo[3,4-d]pyrimidine; pMPK, phosphorylated MPK; PSI, photosystem I; PSII, photosystem II; pTEpY, dually phosphorylated Thr/Glu/Tyr peptide; sc, super-complex; SDS-PAGE, sodium dodecyl sulfate-PAGE.