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. 2022 Feb 25;119(9):e2026355119. doi: 10.1073/pnas.2026355119

Fig. 3.

Fig. 3.

The genetic regulation of seed vigor through transactivation of PER1A by bZIP23. (A) The Y1H analysis of the binding of bZIP23 and bZIP42 to the PER1 promoter. The PER1A promoter (−3 to −1,545 bp from the start codon ATG) was inserted upstream of the LacZ reporter gene. bZIP23 and bZIP42 were infused with GAD. Vectors containing GAD along were used as the control. (B) A schematic diagram indicating fragments in the PER1A promoter for ChIP-qPCR and EMSA analysis. The green and purple lines indicate the targeted fragments for ChIP-qPCR and EMSA analyses, respectively. The indicated fragments contain an ABRE element binding by the bZIP family protein (boxed sequence) and were mutated (the red colored sequence) for EMSA assay. (C) The ChIP-qPCR analysis of relative binding of bZIP23 and bZIP42 to PER1A promoter. The anti-GFP IgG affinity bead was used for DNA immunoprecipitation from embryos. The ACTIN fragment was used as an internal control. The relative enrichment was calculated as Input% of the PER1A fragment/Input% of ACTIN fragment. (D) The EMSA identification of bZIP42- and bZIP23-binding element in the PER1 promoter. The arrow indicates the shift bands, and the asterisk indicates the free probe. (E) The transient DLR assay of bZIP23 and bZIP42 binding to PER1A promoter. The constructs of pGreenII 0800-LUC containing the PER1A promoter and of pGreenII 62-SK with or without bZIP23 or bZIP42 were transiently transformed into the rice protoplasts. The LUC/REN ratio represents the relative activity of transactivation. (F) The relative expression of PER1A in seeds of WT, bZIP23 T-DNA insertion (bzip23), and overexpression transgenic (35S:bZIP23#6 and 35S:bZIP23#9) plants. (G) The relative expression of bZIP23 in seeds of WT, CRISPR knockout (per1a), and overexpressed (35S::PER1A#2 and 35S::PER1A#3) plants. (H) The germination of WT, 35S::bZIP23, per1a, 35S::bZIP23/per1a, and per1a/35S::bZIP23 seeds after being aged for different days. The seeds were aged under 42 °C and 80% RH for 15 and 18 d. ns, no significant difference. *P < 0.05, **P < 0.01, ***P < 0.001 (unpaired Student’s t test). Error bars indicate ± SE (n = 3).