Figure 1.

Confirmation of Target Gene Silencing in Transgenic K. fedtschenkoi RNAi Lines rPPCK1-1 and rPPCK1-3.

Downregulation of the target endogenous KfPPCK1 gene was confirmed at the level of KfPPCK1 transcript abundance (A), and target protein PPC phosphorylation and the apparent K_i of PPC for l-malate in rapidly desalted leaf extracts, measured as l-malate (mM) required for a 50% reduction in extractable PPC activity (B). Gene transcript abundance was measured using RT-qPCR for the wild type, rPPCK1-1, and rPPCK1-3 for target genes: KfPPCK1 (A), KfPPCK2 (C), and KfPPCK3 (D). Mature leaves (leaf pair 6) were sampled from three individual, clonal, biological replicate plants collected every 4 h across the 12-h-light/12-h-dark cycle. A thioesterase/thiol ester dehydrase-isomerase superfamily gene (KfTEDI) was amplified from the same cDNAs as a reference gene. Gene transcript abundance data represent the mean of three technical replicates of each of three biological replicates and were normalized to reference gene KfTEDI; error bars represent the se of the mean calculated for each biological replicate. In all cases, plants were entrained under 12-h-light/12-h-dark cycles for 7 d prior to sampling. In (B), protein abundance (right panel) and the phosphorylation state of PPC (left panel) were determined by immunoblot analyses, and the apparent K_i of PPC for l-malate was measured using rapidly desalted leaf extracts (K_i values in mM below the corresponding left panel immunoblot time points). Total leaf protein (leaf pair 6) was isolated from leaves sampled every 4 h across the 12-h-light/12-h-dark cycle, separated using SDS-PAGE, and used for immunoblot analyses with antibodies raised to a phospho-PPC peptide (left panel). Sample loading was normalized according to total protein and confirmed using the immunoblot for total PPC protein, which is stable over the 12-h-light/12-h-dark cycle (right panel). The white bar below each panel represents the 12-h-light period, and the black bar below each panel represents the 12-h-dark period. For (A), (C), and (D), black data are for the wild type, blue for rPPCK1-1, and red for rPPCK1-3.