*2.5. Functional Analysis*

To silence *PlNCED* expression in the herbaceous peony callus, the fragments of *PlNCEDs* (*PlNCED1*: 565 bp; *PlNCED2*: 387 bp) were each amplified and recombined into the linearized pTRV2 empty vector. The CDSs of *PlNCEDs* were separately inserted downstream of 35S in the pCAMBIA1300-35S-flag vector. The pTRV2-PlNCED and 35S::PlNCED vectors were transformed into EHA105-competent cells. *Agrobacterium* containing pTRV1 and *Agrobacterium* containing pTRV2-PlNCEDs were mixed in a 1:1 volume ratio for the preparation of the callus infection solution. The overexpression and silencing experiment in the herbaceous peony callus was performed according to our previous infection method [33]. After the resistance screening of the culture, part of the PlNCED transgenic callus was taken for qRT-PCR identification. The extraction, purification, and determination of the endogenous levels of ABA in the positive transgenic callus of *PlNCEDs* using an enzyme-linked immunosorbent assay (ELISA) were performed as described by He [34].

We obtained two *A. thaliana* homozygous T-DNA mutants (Figure S1): *nced5-2* and *nced9-1*. *NCED* mutants have interrupted 9-*cis*-epoxycarotenoid dioxygenase genes that result in plants that are deficient in the plant growth regulator abscisic acid. To make the transgenic line and functional complementation line of *A. thaliana*, the 35S::PlNCED vectors were transformed into *Agrobacterium* strain GV3101 and then used to infect inflorescences of *A. thaliana* WT (Col-0) and homozygous mutants (*nced5-2* and *nced9-1*) using the floraldip method [35], respectively. The transgenic line and functional complementation line were screened on 1/2 MS medium plates that contained 50 mg/L kanamycin. The seed germination rate was measured in WT, homozygous mutants, functional complementation lines, and transgenic lines (stable T3-generation genetic lines) of *A. thaliana*, which were grown at the same time under 16 h light and 8 h dark conditions at 22 ◦C.
