Fig. 5. MAPKK-kinases are required for plant ABA response.

a Genome structures and T-DNA insertion sites of M3K genes are shown. b Genomic regions of CRISPR/Cas9-mediated M3Kδ1 and M3Kδ7 gene deletions are shown. These deletions were introduced in the m3kδ6-2 T-DNA knockout mutant as a background. c RT-PCR assays show transcripts of kinase domains of M3Ks in the m3kδ1crispr m3kδ6-2 m3kδ7crispr triple mutant. d m3kδ1crispr m3kδ6-2 m3kδ7crispr triple mutant seedlings were grown on 1/2 MS plates supplemented with 2 µM ABA or ethanol (control) for 16 days. e Seedlings showing green cotyledons as in d were counted. n = 3 (EtOH) and n = 4 (ABA) experiments, means ± s.d., 45 seeds per genotype were used in each experiment. f RT-PCR shows M3Kδ1, δ6, and δ7 expression in the indicated m3k T-DNA insertion mutants. δ6(KD) refers to primers that amplify the M3Kδ6 kinase domain in the m3kδ6-1 T-DNA line. g m3kδ1 m3kδ6-1 m3kδ7 T-DNA triple mutant plants were grown on 1/2MS plates supplemented with 0.8 µM ABA for 9 days. h Seedlings showing green cotyledons as in g were counted. n = 3, means ± s.d., 60–88 seeds were used per genotype in each assay. i Three amiRNA lines targeting M3Kδ1, δ6, and δ7 were grown on 1/2MS plates supplemented with EtOH (control) or 2 µM ABA for 9 days. As a control line, the amiRNA-HsMYO line²¹ was used. j Seedlings showing green cotyledons as in i were counted. n = 3 (EtOH) and 4 (ABA) experiments, means ± s.d., 81 seeds per genotype were analyzed in each experiment. (e, h, and i) Letters at the top of columns are grouped based on two-way ANOVA and Tukey’s test, P < 0.05.