AcePosF21 directly binds to the AceGGP3 promoter and positively regulates AsA synthesis. A) Y1H assay showing the interaction between AcePosF21 and AceGGP3 promoter (LacZ::AceGGP3pro). The yeast strain was grown on SD/-Trp/-Ura/BU salt/X-gal media for 3 d. LacZ::P53pro + pB42AD::P53: positive control; LacZ::AceGGP3pro + pB42AD: negative control. B) Luciferase activity analysis shows that AcePosF21 activates the transcription of AceGGP3 promoter based on the LUC/REN ratio (right) in N. benthamiana leaves. A representative photograph was shown (left picture), and LUC/REN ratio of empty vector control (EV) was taken as 1 for normalization (n = 4). C) The AcePosF21 binding on the promoter of AceGGP3 (above) and electrophoretic mobility shift assay (EMSA) analysis of the AcePosF21 (below). The biotin-labeled probe 5′-TACAGTGGTCCTCCACAACGTCCACATGAACCACATGG-3′ was replaced with a biotin-labeled mutant probe 5′-TACAGTGGTCCTCCACACCCCCCACATGAACCACATGG-3′. The unlabeled wild-type probes were used as a competitor. 50 × and 100 × represent the rates as the competitor. The purified AcePosF21-6×His protein was incubated with the biotin-labeled probes, competitor, and mutant probes. The arrows indicated a bound DNA protein complex. +: presence; −: absence. D) Chromatin immunoprecipitation (ChIP) assay using 35S::AcePosF21-FLAG transgenic kiwifruit calli. The Chromatin was immunoprecipitated with FLAG antibody, and quantitative real-time polymerase chain reaction (qPCR) was performed with the primers listed in Supplemental Table S3. Wild-type (WT) kiwifruit was used as control, and its value was set to 1. E) AsA content of kiwifruit fruits of AcePosF21 transient expression at 7 d after vector infiltration (n = 4). OX-EV/TRV-EV: empty vector; OX: transient overexpression; TRV: transient antisense-expression. F–G) RT-qPCR analysis of AceGGP3F) and AcePosF21G) in E). H) Relative AsA content (AsA content of WT was taken as 5 for normalization) of three AcePosF21-overexpression transgenic lines in kiwifruit calli. OE: stable overexpression of kiwifruit calli. I–J) RT-qPCR analysis of AceGGP3I) and AcePosF21J) expression in H). K) A schematic map of the targeting sites in the exon regions (rectangle) of AcePosF21; the PAM motifs (NGG) are shown in the last three bases. L) Schematic diagram of the CRISPR/Cas9 vector and AcePosF21 targeting sites (diamonds) within the tRNA-sgRNA fusions. M) Sequences and chromatograms of AcePosF21 in WT and two independent homozygous mutant calli (posf21#9 and posf21#27). The target sequence is underlined, the PAM sequence is contained in the rectangle after the target sequence, and the dashes indicate deletions. N) Relative AsA content of AcePosF21-editing lines. O) RT-qPCR analysis of AceGGP3 in N). Error bars denote the standard deviation (±SD), n = 3 to 4. Significant differences were analysis by t-test using GraphPad Prism 8 (*P < 0.05; **P < 0.01; ***P < 0.001).