Pri 201 an vector

For MDH overexpression (OE), a kanamycin resistance cassette of the pRI 201-AN vector (Takara) was replaced by a hygromycin resistance cassette. Four cysteine residues of the N- and C-terminal redox switches of MDH gene (cysteine 77, 82, 418, and 430) in the pET-23c vector were replaced by serine (CtoS) by site-directed mutagenesis. Each mature MDH (WT, ΔC, or CtoS)-coding region in the pET-23c vector was fused to the transit peptide of MDH through overlap PCR and integrated into the plant transformation vector via Gibson assembly. An MDH-deficient plant line (nadp-mdh, Salk_012655C) was obtained from the Arabidopsis Biological Resource Center and transformed with the resulting vector via the Agrobacterium-mediated floral dip method. Homozygous mutant lines were selected and used for the experiments. Primers used for this study are shown in SI Appendix, Table S2.

The A. elata callus line was originally developed from the leaves of A. elata and was stably maintained in vitro on modified MS media supplemented with 3.0% sucrose, 0.01% myoinositol, 0.2 mg L⁻¹ 2,4-dichlorophenoxyacetic acid (2,4-D), 50 mM lipoic acid, and 200 μM acetosyringone. The cDNA of the PgDDS gene was cloned from P. ginseng roots and cloned into the PRI201-AN vector (TaKaRa, Shiga, Japan), generating plasmid PRI201-AN-PgDDS. PgDDS was transiently expressed in P. ginseng callus cells using Agrobacterium rhizogenes GV3101 (pSoup) (Weidi Biotechnology, Shanghai, China). A bacterial absorbance at A600 nm of 0.6 was used to infiltrate the Agrobacterium strain. The cells were cultured with Agrobacterium medium containing 200 μM acetosyringone in the dark for 2 days. Then, the cells were washed five times with water, transferred to selective MS agar medium containing 30.0 mg L⁻¹ kanamycin and 200 mg L⁻¹ timentin, and subcultured for two weeks in the dark at approximately 28 °C. Fresh callus cells (200 mg) were collected, and triterpenes were extracted using saponification solution as described above. The samples were dried and resolved using 200 μL of 80% methanol and analyzed by HPLC–MS as described above.

The BiFC assay was performed as previously described (Nagata and Abe 2021 (link)). The appropriate plasmids transformed into Agrobacterium tumefaciens strain GV3101, and the resulting strains were used for agroinfiltration of Nicotiana benthamiana leaves. Images were obtained on day 2 after infiltration. The empty plasmid (pRI201AN vector (TaKaRa)) was used as a negative control. All microscopic images were acquired using identical settings. The mean signal intensity of EYFP fluorescence in the nuclei was measured using ImageJ software.

To compare the level of ATML1, ATML1^W471L, PDF2 or PDF2^W463L protein expressed under the constitutive CaMV promoter in the Nicotiana benthamiana leaves (i.e., transient overexpression assay), the appropriate plasmids were introduced into Agrobacterium tumefaciens strain GV3101, and the resulting strains were used for agroinfiltration of Nicotiana benthamiana leaves. The empty plasmid (pRI201AN vector (TaKaRa)) was used as a negative control. Samples were collected on day 2 after infiltration, flash frozen in liquid nitrogen, and stored at − 80 °C prior to protein extraction. Protein extractions were performed as previously described (Mukherjee et al. 2022 (link)). Briefly, frozen samples were homogenized in liquid nitrogen, and hot sodium dodecyl sulfate (SDS) buffer (8 M urea, 2% SDS, 0.1 M DTT, 20% glycerol, 0.1 M Tris–HCl pH 6.8, 0.004% bromophenol blue, and 5 × Protease Inhibitor Cocktail for General Use (Nacalai tesque)) was added prior to SDS–polyacrylamide gel electrophoresis (PAGE) and Western blotting. Proteins were detected via anti-GFP primary antibody [MBL (598MS); 1:2000] and anti-rabbit IgG HRP-conjugate secondary antibody [Promega (W4018); 1:5000]. Detection of secondary antibodies were performed with the Ez West Lumi One (ATTO) using the Image Quant LAS 4000 mini (GE Healthcare).