Fig. 2.

The second W‐box in the OsNF‐YC4 promoter region is bound by OsWRKY121, and its deletion increased OsNF‐YC4 transcription. (a) Structure of the full‐length OsNF‐YC4 promoter and deletion derivatives used for luciferase assays. RAV1A (CAACA/TGTTG) and WRKY (RGTCA/TGACY) binding sites are indicated as purple and green boxes, respectively. Deleted regions are in red with a single or string of ‘x's. (b) Relative luciferase activity (normalized against total protein content) increased upon deletion of the second and third WRKY binding sites. Promoter versions D1, D2, and D3 were fused to luciferase reporter genes and transiently expressed in tobacco leaves after infiltration. Bar graphs show mean values ± error bars indicating the SE of mean; n  ≥ 3 independent experiments. Student's t‐test was used to compare the LUC (luciferase) activity between deletions and controls. (c) The structures of OsNF‐YC4 promoter fragments (P1–P5, 198‐bp, and 193‐bp) with different combinations of W‐box motifs. These fragments were PCR‐amplified from WT plants and used for biotin labeling and EMSA (Electrophoretic Mobility Shift Assay) in (d–f). (d) EMSA demonstrated that OsWRKY121 could bind to the second W‐box (in P2) of the OsNF‐YC4 promoter, but not to the first (in P1) or the third (in P3) W‐box motifs. P4 served as a negative control without any W‐box, while P5 contained all three W‐box motifs. (e) Competition with unlabeled fragments confirmed the specific binding of OsWRKY121 to the second W‐box motif (P2) in the OsNF‐YC4 promoter. (f) OsWRKY121 bound to the 198‐bp native fragment containing all three W‐box motifs, but not the 193‐bp synthesized fragment lacking the second 5‐bp W‐box (shown in a red box in c), confirming the importance of the second W‐box for OsWRKY121 binding. Twenty femtomole of biotin‐labeled DNA fragments were used in (d–f). EBNA (Epstein–Barr nuclear antigen) from the EMSA kit served as a positive control in (d–f).