Figure 3. Effect of sirtinol treatment on the expression of RAM and SAM markers.

(a,b) Sirtinol treatment causes ectopic expression of QC markers - WOX5:GFP-ER and QC184. Seeds of marker lines were germinated and grown on 10 μM sirtinol, GFP fluorescence (a) and GUS staining (b) was observed in seedlings at 2 dag. Scale bar: 50 μm. White dotted line in confocal image indicates a shift in the domain of WOX5 expression. Insets in (a) show GFP fluorescence of same marker in DIC images. Bold red arrow marks root-shoot junction. (c,d) Sirtinol causes ectopic expression of PLT1:PLT1-YFP and PLT2:PLT2-YFP reporters. Seeds were germinated and grown on 10 μM sirtinol and YFP fluorescence marking the localization of PLT1/2-YFP proteins was observed in seedlings at 2 dag. Scale bar: 50 μm. (e) Sirtinol causes ectopic expression of SCR:GFP, an endodermis and QC specific marker. Seeds were germinated and grown on 10 μM sirtinol containing media, GFP fluorescence was observed in seedlings at 2 dag. Scale bar: 50 μm. White dotted lines in confocal images indicate a shift in the domain of SCR expression, which normally is restricted to the QC and endodermis. Insets in (e) show GFP fluorescence of same marker in DIC images. (f) Sirtinol increases WUS and CLV3 expression in SAM. Seeds of WUS:DsRed-N7 CLV3:GFP-ER reporter line was grown on 10 μM sirtinol and fluorescence was observed in seedlings at 2 dag. Scale bar: 50 μm. (g,h) Sirtinol alters the expression level of genes involved in root and shoot meristem maintenance. Sirtinol treated (10 μM) seedlings (at 2 dag) were used for analysis expression level of root and shoot meristem regulatory genes (as named in labels) using real time qRT-PCR. Error bars indicate ± standard error (SE) of three independent experiments. One-way ANOVA was performed for statistical analysis. Asterisks indicate significant statistical differences, ***P < 0.001, **P < 0.01, *P < 0.05.