**1. Introduction**

Phytohormones are important regulators of seed dormancy, among which ABA plays a major role [1–3]. Studies have shown that ABA-deficient mutants of *A. thaliana*, tomato, and maize undergo early dormancy breaking and enter the germination stage, whereas plants overexpressing the ABA biosynthetic enzyme show prolonged dormancy [4–7]. Studies also demonstrated that the change in endogenous ABA content significantly positively correlates with the degree of seed dormancy [8,9].

In plants, it is known that several phytohormones are involved in seed dormancy and germination [10–12]. Among them, endogenous ABA content is significantly positively correlated with herbaceous peony seed dormancy, while low ABA content promotes seed germination [13]. The final concentration of endogenous ABA in plant seeds depends on the dynamic balance of ABA synthesis and catabolism [1,14]. *NCED* and *CYP707A* are two key enzymes in ABA anabolic and catabolic pathways, respectively. Studies show that NCED has a common role in regulating ABA synthesis and seed dormancy in plants. For example,

**Citation:** Fei, R.; Guan, S.; Duan, S.; Ge, J.; Sun, T.; Sun, X. Elucidating Biological Functions of 9-*cis*-Epoxycarotenoid Dioxygenase Genes Involved in Seed Dormancy in *Paeonia lactiflora*. *Plants* **2023**, *12*, 710. https://doi.org/10.3390/ plants12040710

Academic Editors: Aiping Song and Yu Chen

Received: 20 December 2022 Revised: 19 January 2023 Accepted: 2 February 2023 Published: 6 February 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

*A. thaliana* contains five *NCED* family members, of which *AtNCED5* is up-regulated at the late stage of seed maturation and cooperates with *AtNCED6* and *AtNCED9* to enhance seed dormancy by controlling ABA levels [15,16]. *LeNCED1* transgenic tomato plants enhanced ABA biosynthesis and increased seed dormancy [17]. The variation trend of ABA content was consistent with that of *AhNCED1* gene transcription in peanut [18]. *PtNCED1* directly regulated orchid seed dormancy and was involved in ABA content [19]. In addition, *NCED* can also affect other physiological functions of plants by changing endogenous ABA content. Overexpressing *PvNCED1* enhanced drought tolerance by manipulating ABA levels in tobacco [4]. Silencing *AcNCED1* blocked ABA biosynthesis and delayed kiwifruit softening [20]. *PpNCED1* and *PpNCED5* can cooperatively control ABA biosynthesis and affect fruit ripening and senescence in peach fruit [21]. However, not all *NCED* family members regulate ABA synthesis. For example, a total of 23 *NCED* genes were identified in cotton. Among them, only the expression of *GhNCED5*, *GhNCED6*, and *GhNCED13* was similar to the change in ABA content, which could play a role in ABA biosynthesis [22].

Herbaceous peony (*Paeonia lactiflora* Pall.) is an herbaceous perennial flower of *Paeoniaceae*. In the long-term systematic evolutionary process, herbaceous peony seeds evolved a unique double dormancy characteristic of the epicotyl and hypocotyl [23]. In the breeding process, seed dormancy is often not released or incompletely released, which greatly reduces the germination rate and seriously affects cultivation and production [24]. Thus, understanding the mechanisms associated with herbaceous peony seed dormancy is beneficial to greatly promote the breeding of new varieties. At present, research on the seed dormancy release technology for herbaceous peony mainly focuses on mechanical scarification, low temperature, and exogenous hormone treatment [25–27]. However, the knowledge of the molecular mechanisms underlying the seed dormancy of herbaceous peony is relatively limited. Using previously published transcriptomes from herbaceous peony seeds pre- and post-double dormancy release [28], here, we were able to identify candidate genes that were associated with double dormancy in herbaceous peony. Specifically, we identified ten family members of *PlNCEDs* involved in ABA biosynthesis. Among them, *PlNCED1* (c53147\_g1) and *PlNCED2* (c69372\_g1) showed significantly differential expression. We subsequently cloned and performed expressional analysis, subcellular localization analysis, and functional characterization of *PlNCED1* and *PlNCED2* in *Paeonia lactiflora*. Our results demonstrate that the genes encoding NCED1 and NCED2 regulate ABA synthesis and consequentially affect the herbaceous peony seed dormancy process.
