Fig. 5: KIN10 phosphorylates and stabilizes SPCH to promote stomatal development.
From: KIN10 promotes stomatal development through stabilization of the SPEECHLESS transcription factor
a Phosphorylation of SPCH by KIN10-MBP in vitro. Upper lines showed the gel image of ATP-γ-p32 labeled protein and bottom lines showed the gel image of Coomassie brilliant blue staining. b Quantification of stomatal index in wild-type plants, and different SPCH complemented transgenic plants. Seedlings of wild-type plants, pSPCH::SPCH-RFP/spch-4, pSPCH::SPCH-4A-RFP/spch-4 and pSPCH::SPCH-4D-RFP/spch-4 transgenic plants were grown in ½ MS liquid medium with 1% sucrose for 10 days under long-day condition. Error bars indicate standard deviation (S.D.) (n = 10). c Sucrose increased the protein stability of SPCH. Seedlins of pSPCH::SPCH-GFP and pSPCH::nucGFP were grown in sugar-free liquid medium for 2.5 days, and then treated with or without 1% sucrose for 8 h. The GFP fluorescent intensity was analyzed with more then 150 cells from 10 leaves. Numbers between bars indicated the relative fold changes of average means in the indicated materials. d SPCH proteins become unstable when sucrose was removed from liquid medium. Seedlings of pSPCH::SPCH-GFP and pSPCH::nucGFP were grown in ½ MS liquid medium containing 1% sucrose for 2.5 days then tranferred to sugar-free medium for 6 h. Asterisk between bars indicated statistically significant differences between the samples (Student’s t test, ****p < 0.0001). Error bar indicates S.D. e, f Immunoblot analysis of the effects of sucrose on the protein levels of SPCH-myc and SPCH-4A-myc using anti-myc antibody. Seedlings of p35S::SPCH-myc and p35S:SPCH-4A-myc were grown in sugar-free medium for 3 days, and then treated with 1% sucrose for different times. Actin bands were used as loading control. g Analysis the SPCH, SPCH-4A and SPCH-4D protein intensities on abaxial cotyledons of 3-day-old pSPCH::SPCH-RFP/pKIN10::KIN10-YFP, pSPCH::SPCH-4A-RFP/pKIN10::KIN10-YFP and pSPCH::SPCH-4D-RFP/pKIN10::KIN10-YFP transgenic plant by ImageJ software. The red fluorescent signals of SPCH-RFP (n = 114), SPCH-4A-RFP (n = 114) or SPCH-4D-RFP (n = 144) were quantified from more then 100 cells of 5 cotyledons. Error bars indicate S.D. Different letters above the bars indicated statistically significant differences between the samples (ANOVA analysis followed by Uncorrected Fisher’s LSD multiple comparisons test, p < 0.05).
