*Article* **Elucidating Biological Functions of 9-***cis***-Epoxycarotenoid Dioxygenase Genes Involved in Seed Dormancy in** *Paeonia lactiflora*

**Riwen Fei 1,2, Shixin Guan 2,3,\*, Siyang Duan 2,3, Jiayuan Ge 2,3, Tianyi Sun 2,3 and Xiaomei Sun 2,3,\***


**Abstract:** Abscisic acid (ABA) is a major phytohormone affecting seed dormancy and germination in plants. ABA is synthesized mainly through the C40 carotenoid pathway. In the ABA biosynthesis pathway, 9-*cis*-epoxycarotenoid dioxygenase (NCED) is a key rate-limiting enzyme that regulates the accumulation and content of ABA. However, the role of the *NCED* gene in perennial plants with complex seed dormancy remains largely unknown. Here, we cloned two differentially expressed paralogs of herbaceous peony *NCED* genes, named *PlNCED1* and *PlNCED2*, and further identified their involvement in seed dormancy from perennial herbaceous peony experiencing complex double seed dormancy. The deduced PlNCED amino acid sequences had high sequence homology with *NCED* sequences from other plants and contained the typical conserved RPE65 domain of the NCED family. Phylogenetic analysis showed that *PlNCED1* and *PlNCED2* have a close relationship with *PoNCED* in *Paeonia ostii* and *VvNCED6* in *Vitis vinifera*, respectively. A subcellular localization assay demonstrated that the *PlNCED1* protein resided within the nucleus, while the *PlNCED2* protein was located in the cytoplasm, indicating their different roles in the biosynthesis of ABA. Furthermore, the content of endogenous ABA in transgenic calluses showed that *PlNCEDs* were positively correlated with ABA content. Both *PlNCED* transgenic *Arabidopsis* lines and the functional complementation of *Arabidopsis NCED* mutants found that *PlNCEDs* promoted seed dormancy and delayed seed germination. These results reveal that *PlNCEDs* participate in the seed dormancy of herbaceous peony by regulating the accumulation of endogenous ABA.

**Keywords:** *Paeonia lactiflora* pall.; abscisic acid; *NCEDs*; seed dormancy; germination
