Fig. 4

Function identification of LjZCD on CNglcs content. a Schematic diagram of reporter and effector in Arabidopsis protoplast transactivation assays. Reporter is the fusion of the CYP79D3, CYP736A2, and UGT85K3 promoter with firefly luciferase gene (LUC). The LjZCD fused with the CaMV 35S promoter respectively to from effectors. NOS, the transcriptional terminator of the nopaline synthase gene from Agrobacterium tumefaciens. b Luciferase assays of LjZCD regulate the expression of CYP79D3, CYP736A2, and UGT85K3. The empty vector was used for control. Values represent the mean ± SE of triplicate experiments. Significant differences between values are indicated with different letters (*, P < 0.05; **, P < 0.005; ***, P < 0.0001 one-way ANOVA). c Subcellular localization of LjZCD protein in Arabidopsis. LjZCD-GFP constructs were co-transformed with AtH2B-Mcherry into Arabidopsis mesophyll protoplasts and examined by confocal laser scanning microscopy. Confocal micrographs are shown brightfield, chloroplast auto-fluorescence, green fluorescent protein (GFP), and the merged images from left to right. AtH2B-Mcherry was used as nuclear localization maker. Bar, 5 μm. d The relative expression of CNglcs genes in wild type (WT) and 3 lines of OE-35S::LjZCD. e The contents of two cyanogenic glycosides in WT and 3 lines of overexpression material OE-35S::LjZCD. The white box and gray box indicate Lotaustralin and Linamarin, respectively