Fig 2. WRKY23 acts downstream of the Aux/IAA—ARF auxin pathway and marks developing vasculature.

(A) Schematic depiction of WRKY23 promoter; AuxRE and AuxRE-like response elements are shown as triangles (B and C) WRKY23 transcript levels depend on auxin dose and treatment time. qRT-PCR analysis of WRKY23 expression after a 4 h treatment with different concentrations of NAA (B) and after different treatment times with 10 μM NAA (C). TUB2 and SLR/IAA14 are shown as negative and positive controls, respectively. Values represent relative fold change of expression. Error bars represent standard deviation (see Materials and Methods for detailed description). (D and E) WRKY23 expression depends on the SCF^TIR1-Aux/IAA-ARF signalling pathway. qRT-PCR confirmation of the microarray experiment showing the expression of WRKY23 and genes previously connected to PIN polarity in HS::arx3-1 (D), and in arf7 arf19 double mutant plants (E). Values represent relative fold change. Error bars indicate standard deviation (see Materials and Methods for detailed description). (F, G) Expression of WRKY23::GUS in the shoot apical meristem (SAM) and in the presumptive leaf vasculature (G). Besides strong activity in the SAM, GUS staining overlaps with, and partly precedes, the appearance of differentiating vascular strands in young leaves. Two representative plants in consecutive developmental stages are shown. Patchy expression of WRKY23::GUS in the vasculature of young developing true leaves (G). Arrowheads in F and G depict areas with GUS activity presumably coinciding with future vascular strands that are not morphologically discernible yet.