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. 2020 Jan 6;32(3):630–649. doi: 10.1105/tpc.19.00569

Figure 7.

Figure 7.

ONAC054 Directly or Indirectly Activates OsABF4, OsABI5, and NYC1.

(A) The ratios of expression levels (onac054-1/wild type [WT]) for genes associated with ABA metabolism and signaling at 0 and 2 DDI obtained from the microarray analysis are illustrated. The relative expression value was normalized to the WT expression level.

(B) Expression patterns of OsABF4, OsABI5, and NYC1 in wild-type (WT), onac054-1, ONAC054α-OX (N54α-OX), and ONAC054β-OX (N54β-OX) plants before and after 24 h of treatment with ABA. Detached leaf discs from 1-month-old rice seedlings were used for RT-qPCR analysis. The mean and SD values were obtained from four biological samples (three leaf discs each). HT, hour(s) of treatment.

(C) The positions of the MDM variants in the promoters of OsABF4, OsABI5, and NYC1, and promoter fragments used for the ChIP assay (green horizontal lines) and transactivation assay (orange horizontal lines).

(D) ONAC054β binding affinity to the promoter regions of OsABF4, OsABI5, and NYC1 in planta examined by ChIP assays. GFP-ONAC054β was transiently expressed in protoplasts isolated from 10-d-old wild-type rice seedlings. Fold enrichment of the promoter fragments was measured by immunoprecipitation with an anti-GFP antibody (see Methods). Enrichment of a promoter region of an unrelated gene, PP2A, was used as a negative control. The mean and SD values were obtained from four reaction mixtures.

(E) Reporter and effector constructs used in the protoplast transient assay. Each construct also contained the NOS terminator.

(F) The activation of OsABF4, OsABI5, and NYC1 promoters (−1500 to −1, relative to the start codon) by GFP-ONAC054α and GFP-ONAC054β expression in protoplasts that were incubated in darkness. The 35S promoter and an empty expression vector (35S:GFP) served as negative controls. The mean and SD values were obtained from five reaction mixtures. Asterisks indicate a significant difference compared to the wild type or negative controls (Student’s t test, *P < 0.05 and **P < 0.01). These experiments were repeated at least twice with similar results.