Extended Data Fig. 7: NO3− activates a MEKK14-MKK3-MPK1/2/7/14 phospho-relay to promote LR growth.
a MEKK13/14- and MKK3-dependent activation of MPK1/2/7/14 phosphorylation by NO3−. 7-day-old seedlings precultured on high N were subjected to 4 days N starvation and then exposed to 10 mM KNO3. Recombinant GST-tagged MPK1/2/7/14 protein was incubated with total protein extracts from wild-type (Col-0), mekk13/14 and mkk3 seedlings for 1-4 h of nitrate treatment. Abundance of GST-MPK1/2/7/14 and phosphorylated GST-MPK1/2/7/14 proteins was assessed with Coomassie Brilliant Blue staining (CBB) and anti-thiophosphate ester-specific antibody (anti-TPE). Actin was used as internal control for equal protein loading. The band intensity of phosphorylated MPK was quantified with Image J (version 1.53e) and normalized to Actin with time 0 in Col-0 samples set to 1.0. b-c Appearance of plants (b), and average LR length (c) of wild-type (Col-0), mekk13/14 and two representative T3 transgenic lines expressing GFP-tagged MKK3DD under MEKK14 promoter grown under 2 mM KCl and KNO3. Number of plants analyzed in KCl/KNO3: n = 16/16, 16/15, 16/15 and 15/15 for Col-0, mekk14, MKK3DD-#1 and MKK3DD-#2, respectively. d-e Appearance of plants (d), and average LR length (e) of wild-type (Col-0), mkk3-2 and two representative T3 transgenic lines expressing GFP-tagged MPKsDD under MKK3 promoter grown under 2 mM KCl and KNO3. Number of plants analyzed in KCl/KNO3: n = 16/16, 16/16, 16/16, 15/15, 15/16, 16/15, 16/16, 16/15, 16/14 and 16/16 for Col-0, mkk3-2, MPK1DD-#1 and MPK1DD-#2, MPK2DD-#1, MPK2DD-#2, MPK7DD-#1, MPK7DD-#2, MPK14DD-#1 and MPK14DD-#2, respectively. Scale bars, 1 cm. Different letters indicate significant differences at P < 0.01 according to one-way ANOVA and post-hoc Tukey’s test.
