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. 2018 May 7;115(21):5606–5611. doi: 10.1073/pnas.1722068115

Fig. 4.

Fig. 4.

ACS genes are regulated indirectly by PIF5 at night. (A) R.E.L. of PIF5 at ZT0 in F1 hybrids between different combinations of ecotypes. Asterisks indicate down-regulation in the hybrids at the statistically significant level (P < 0.05) relative to MPV. (B) R.E.L. of ACS2 and ACS9 in Col-0 (WT), PIF5-OX, and pif1345 mutant lines in diurnal conditions. Asterisks Above and Below the graphs, respectively, indicate up-regulation in PIF5-OX and down-regulation in pif1345 at the statistically significant level (P < 0.05) relative to WT. (C) Transient luciferase expression assays indicate that PIF5 directly activated expression of ACS2 and ACS9. The infiltrated constructs are ACS promoter fused with luciferase (ACSp:LUC, 1), ACSp:LUC cotransfected with 35S:PIF5 (2), ACS mutated promoter:luciferase (ACSmp:LUC, 3), and ACSmp:LUC with 35S:PIF5 (4). Luciferase activity images of N. benthamiana leaves are shown for ACS2 and ACS9. (D) Relative luminescence intensity (y axis) for each comparison in C. (E) ChIP-qPCR showed fold enrichment (y axis) of PIF5 in the promoters of ACS2, ACS7, and ACS9. Coding region of PP2A was used as a nonbinding control for normalization.