Fig. 3.

Germination and root growth assays. (A and B) T-DNA insertion sites derived from the sequencing of genomic DNA isolated from mkkk18-1 and mkkk18-2 mutant lines. Black boxes represent an open reading frame. Homozygous knockout mutants were verified by PCR-based genotyping using the following primers: MKKK18LP plus MKKK18RP plus LBb1 for mkkk18-1 analysis (product size for the WT, ∼960 bp; product for homozygous lines, 750 bp); MEK18F plus GKatTDNA (product for homozygous lines, 850 bp) and GwMK18F plus GwMK18R (product for the WT, 1,001 bp) for mkkk18-2 genotyping. Primer sequences are indicated in Supplementary Table S1. (C) qPCR analysis confirmed a lack of MKKK18 expression in homozygous lines. Each quantification was repeated twice with similar results. The results are given as log₂ of the relative MKKK18/18S rDNA expression ratio ± SE (n = 6). (D) RD29B and RAB18 transcript levels in MKKK18 mutants. RD29B and RAB18 expression levels were determined using three biological replicates and were normalized against 18S rDNA. Each quantification was repeated twice on separate plates. The results are displayed as mean log₂ fold change ± SE (n = 9) of three independent experiments with consistent results. (E and F) Germination and root growth assays. ABA-mediated inhibition of germination (E) and primary root growth (F) in WT Col-0, MKKK18oe and MKKK18 knockout lines. Both MKKK18oe #1 and #2 lines showed similar results. Values are mean ± SE for three independent experiments (n = 30). *P < 0.01; **P < 0.001; ***P < 0.0001 with respect to the control WT Col-0 line.