Figure 5.

JA2 Regulates NCED1 Expression through Direct Interaction with Its Promoter.

(A) to (C) Basal transcript levels of JA2, NCED1, and FLA were measured in wild-type (cv M82) and JA2-OE plants. Leaves from 4-week-old plants grown under normal conditions were harvested at the same time for RNA extraction and qRT-PCR analyses. Transcript levels of each gene were normalized to Actin2 expression. Error bars represent the sd of three technical replicates. Shown are representative data from one biological replicate; three biological replicates were conducted, yielding similar results.

(D) Schematic diagram of the NCED1 promoter showing the presence of the NAC core binding site (black triangles). Short horizontal lines indicate DNA fragments (P1, P2, and P3) used for ChIP-PCR experiments. Shown are 2-kb upstream sequences of the NCED1 gene. The translational start site (ATG) is shown at position +1.

(E) Enrichment of the indicated DNA fragments (P1 and P2) following ChIP using anti-GFP antibodies. Chromatin of transgenic plants expressing GFP-JA2 was immunoprecipitated with anti-GFP antibodies, and the presence of the indicated DNA in the immune complex was determined by PCR. The DNA fragment P3 was used as a negative control. The experiment was repeated three times with similar results.

(F) EMSA showing that the MBP-JA2 fusion protein binds to DNA probes from the NCED1 promoter in vitro. Biotin-labeled probes were incubated with MBP-JA2 protein, and the free and bound DNAs (arrows) were separated on an acrylamide gel. As indicated, unlabeled probes were used as competitors. Mu, mutated probe in which the NAC core binding site (CACG) was deleted.