(A) Sequence comparison for ZDRE and the new binding site motif for bZIP23 in the promoter of accession with high ratio of Pi accumulation in -Zn/+Zn (Col-0, Ang-0, CICB-5, Est-1, RRS-10) and low Pi accumulation ratio - Zn/+Zn (Sap-0, Ts-1, Br-0 and PHW-2). (B) Differential binding of bZIP19 and bZIP23 to a specific ZDRE motif of LPCAT1 (GTGTCACA). (C–D) in planta transactivation assay. (C) 35S:bZIP23 and 35S::C-YFP were used as effectors. pLPCAT1: the native Col-0 LPACT1 promoter (with « GTGTCGAA » as new ZDRE), mpLPCAT1: a modified (point mutation) version of the Col-0 promoter to only contain the new ZDRE of Sap-0 (« GTGTCACA »); pZIP4; promoter of the zinc transporter ZIP4 gene. Each pLPCAT1 (native, Col-0), mpLPCAT1 (mutated version) or pZIP4 promoter was fused to a β-glucuronidase (GUS)-encoding reporter gene (reporter). (D) The effect of bZIP23 TF on the activity of each promoter pLPCAT1, mpLPCAT1 or pZIP4 was determined by measuring GUS activity. The effect of C-YFP protein on the activity of each promoter pLPCAT1, mpLPCAT1 or pZIP4 was used as a control to determine the basal level of GUS activity for each promoter. Comparing the effect of bZIP23 TF and C-YFP protein on each promoter enabled the determination of the relative GUS activity. Error bars represent standard error from three independent experiments. The asterisks indicate that the relative GUS activity is statistically different from the YFP control (p-value<0.01, t-test). (E) Relative bZIP23 and LPCAT1 transcripts abundance in -Zn and +Zn conditions. Col-0, Ang-0, CICB-5, Est-1, RRS-10, Sap-0, Ts-1, Br-0 and PHW-2 genotypes were grown on +Zn or -Zn agar medium. The relative mRNA level was quantified by RT-qPCR and normalized to the Ubiquitin10 reference mRNA level (UBQ10: At4g05320). Values are means of six biological replicates. Individual measurements were obtained from the analysis of shoots collected from a pool of 20 plants. Error bars indicate SD; one and two asterisks indicate a significant difference with Col-0 plants (ANOVA and Tukey test) of p<0.05 and p<0.01 respectively.