Figure 6: The pub12 pub13 mutant exhibits reduced sensitivity to ABA.

(a) pub12 pub13 mutant reduces ABA-activated OST1 activity in an in-gel assay. Total proteins were isolated from 2-week-old seedlings treated with 50 μM ABA or without ABA for 30 min, and separated by SDS–PAGE gel containing 0.1 mg ml⁻¹ MBP substrate. RUBISCO was used as a loading control. Three independent experiments were done with similar results. (b) Quantitative analysis of the band intensity in a. The band intensity of OST1 in the wild type with ABA treatment was set to 1, which was compared with the band intensity from abi1-1 (Col) or pub12 pub13 with ABA treatment. Error bars are means±s.e.m. (n=3 independent experiments). (c) Seed germination greening analysis of pub12, pub13, pub12 pub13, abi1-1 (Col) and the wild-type (Columbia, Col) seeds on MS medium or MS medium supplemented with 0.1 and 0.3 μM ABA. (d) Seed germination greening ratio in b. Three independent experiments were done, each with three replicates. Error bars are means±s.d., n=3 (**P<0.01, Student’s t-test). (e) Reduced sensitivity to ABA in root growth of pub12, pub13, and pub12 pub13 mutants compared with the wild type. abi1-1 (Col, abi1-1 in Columbia accession) was included as a positive control. (f) Statistical analysis of ABA-inhibited root growth in e. Root length is relative to the control (without ABA). Three independent experiments were conducted, each with three replicates. Error bars mean±s.d., n=3 (**P<0.01, Student’s t-test). (g) RNA-seq analyses of the ABA-responsive genes in pub12 pub13, abi1-1 (Col) and the wild type (Col). Data are obtained from two independent experiments. The genes that are induced by 50 μM ABA at 1 h or 3 h were selected. The heat map was drew according to the expression levels in the wild type, pub12 pub13 and abi1-1 (Col) at ABA treatment for 1 h or 3 h (See Methods for detail). The overlapped numbers (right) between pub12 pub13 and abi1-1 (Col) are the ABA-induced genes that have lower expression levels in both pub12 pub13 and abi1-1 than in the wild type at ABA treatment for 1 or 3 h.