*3.1. Cloning and Sequence Analysis of PlNCEDs*

The full-length cDNA sequences of *PlNCEDs* were isolated and deposited in Gen-Bank (GenBank accession numbers—*PlNCED1*: OL744236; *PlNCED2*: OL744237). *PlNCED1* and *PlNCED2* contained 1518 bp and 1326 bp open reading frames, encoding 505 and 441 amino acids, respectively (Figure S2). NCBI tblastx results displayed their homology to the *NCED* genes of other plant species. PoNCED from *Paeonia ostii* (74.70%) and JrNCED from *Juglans regia* (62.43%) had the highest identity with PlNCED1 and PlNCED2, respectively (Supplementary Tables S2 and S3). This similarity demonstrated that PlNCEDs are relatively conserved among diverse plant species. PlNCEDs had the typical conserved RPE65 domain of the NCED family (Figure S3), which is related to the degradation of carotenoids in plants. Phylogenetic analysis results indicated that PlNCED1 and PlNCED2 are closely related to the NCED proteins in *Paeonia ostii*, *Vitis riparia*, and *Vitis vinifera*, respectively (Figure 2). In addition, based on DNA sequences, we identified putative microRNA (miRNA) and transcription factor binding sites in the 3'UTR and promoter regions of *PlNCEDs* (Supplementary Tables S4–S7). Among the miRNAs that may target the 3'UTRs of *PlNCEDs* genes, six miR-NAs, namely, miR837-5p, miR5640, miR319c, miR6425a/b/c/d/e-5p, miR168a, and miR5304, are related to plant development (Supplementary Tables S4 and S5). Among the *cis*-acting elements that may bind to the promoter sequences of *PlNCED* genes, six cis-acting elements, namely, TCA-element, ABRE, AuxRR-core, TGA-box, TGACG-motif, and CGTCA-motif, are related to phytohormone responses (Supplementary Tables S6 and S7). These results suggest that *PlNCEDs* may be involved in plant development mediated by ABA and other phytohormones.

**Figure 2.** Neighbor-joining phylogenetic tree of *PlNCEDs*. PlNCED1 and PlNCED2 are indicated with red dots. A neighbor-joining tree was constructed based on amino acid sequences of NCED protein from different plants using MEGA 7.0 software. The bootstrap values of the branches were obtained by testing the tree 1000 times.

#### *3.2. Expression Analysis of PlNCEDs*

To explore the correlation between the expression of *PlNCED* genes and ABA accumulation in herbaceous peony seeds, we performed qRT-PCR using seeds at stages 1-6. Our results indicated that the expression levels and trends of *PlNCED1* and *PlNCED2* dynamically varied during seed dormancy release (Figure S4). The expression level of *PlNCED1* increased sharply from stage 1 to stage 2 and then decreased significantly at stage 3, where seeds experienced hypocotyl dormancy release (Figure S4). After the completion of the hypocotyl dormancy release process, the expression level of *PlNCED1* increased incrementally from stage 4 to stage 6 (Figure S4). Conversely, we observed different trajectories for the expression of the *PlNCED2* gene. Overall, *PlNCED2* maintained a high level of expression at stages 1-2, and it displayed a decreasing trend from stages 2 to 6 (Figure S4). Correspondingly, the ABA content in each of the six seed dormancy release stages largely declined from S1 to S6 (Figure S5) [36]. By comparing the dynamics of *PlNCED* expression and ABA content during the process of dormancy release, we demonstrated that only *PlNCED2* expression was positively associated with ABA accumulation.
