*3.4. Functional Analysis of PlNCEDs*

We first used the transgenic herbaceous peony callus to identify the impact of *PlNCED1* and *PlNCED2* on in vivo ABA content. The results showed that the expression of *PlNCED1* and *PlNCED2* in transgenic herbaceous peony callus was significantly altered compared with the control groups. The expression levels of *PlNCED1* and *PlNCED2* in the overexpressed callus were about 14.5-fold higher than that in the control callus, and the expression levels in the silenced callus were about 0.35-fold lower than that in the control callus, indicating that transgenic calluses were successfully obtained (Figure S6). As shown in Figure 4, the ABA content of the overexpressed *PlNCED* transgenic callus was significantly higher than that of the wild-type callus, while that of the silenced *PlNCED* transgenic callus was much lower, indicating a positive correlation between *PlNCEDs* and endogenous ABA content. In particular, the increase in ABA content caused by *PlNCED2* overexpression was larger than that generated by *PlNCED1* overexpression. We illustrated that *PlNCED2* specifically affects endogenous ABA content by regulating its biosynthesis.

To identify the functional involvement of *PlNCEDs* in seed dormancy, we recorded the seed germination times of *A. thaliana* WT, mutant, transgenic lines, and complementation lines. The germination rates of *A. thaliana* WT and mutant seeds reached about 90% at 48 h, but the seeds of overexpression transgenic lines did not germinate at 48h (Figure 5a,b), indicating that the overexpression of *PlNCEDs* inhibited seed germination. At 68 h, the

*PlNCED1* overexpression transgenic line seeds began to germinate, while the *PlNCED2* overexpression transgenic line seeds began to germinate at 78 h (Figure 5a,b), indicating that the inhibitory effects of *PlNCEDs* on seed germination were different, with *PlNCED1* having a slightly weaker impact on seed germination. Compared to *Atnced9-1* and/or *Atnced5-2* mutants, complementation lines induced seed dormancy with a delay and a lower rate of seed germination. In particular, the *PlNCED2* complementation line had a stronger effect (Figure 5c–f). Overall, we demonstrated that *PlNCEDs* inhibited seed dormancy release, and the inhibitory effect of *PlNCED2* was stronger.

**Figure 4.** Changes in ABA content in the overexpressed callus of *PlNCEDs*. *PlNCED1* expression in the experimental group (35S::PlNCED1-flag/pTRV2-PlNCED1), *PlNCED2* expression in the experimental group (35S::PlNCED2-flag/pTRV2-PlNCED2), and empty control group (WT). Significant differences (\*\*\*\* *p* ≤ 0.0001) are indicated by asterisks. One-Way ANOVA (*F*-test) analysis was performed using GraphPad Prism 8.0. WT was used as a control.

**Figure 5.** *Cont*.

**Figure 5.** Observation of seed germination rate of different types of *A. thaliana*. (**a,b**) Seed germination rate of overexpressed *PlNCED* transgenic lines (under Col-0 background), Col-0, and mutants (*nced9-1* and *nced5-2*); (**c,d**) seed germination rate of overexpressed *PlNCED* transgenic lines (under *nced9-1* background) and *nced9-1*; (**e,f**) seed germination rate of overexpressed *PlNCED* transgenic lines (under *nced5-2* background) and *nced5-2*. Significant differences (\*\*\*\* *p* ≤ 0.0001) are indicated by asterisks. One-Way ANOVA (*F*-test) analysis was performed using GraphPad Prism 8.0. Col-0 and *Atnced5-2*-*35S::PlNCED2-flag* in (**b**) and (**f**) were used as controls, respectively.

## **4. Discussion**

*Paeonia lactiflora* is the most familiar herbaceous peony seen in gardens and produces some of the best cut flowers in the floral industry. Though herbaceous peony is one of the most easily grown hardy perennials, its complex double seed dormancy hinders seed germination and consequently imposes adverse effects on breeding and cultivar improvements [24]. Practically, breaking herbaceous peony seed dormancy can be handily achieved through physical (e.g., cold treatments, slitting the seed coat) and biological means (e.g., hormone treatment) [25,26]. The content and level of phytohormones, particularly ABA, are the key factors for natural seed dormancy release. The final concentration of endogenous ABA depends on the dynamic balance between ABA synthesis and catabolism. Therefore, it is critical to know the genes encoding ABA metabolic enzymes and their impacts on herbaceous peony seed dormancy and germination.

To search for genes related to ABA biosynthesis, we identified two *NCED* genes (*PlNCED1* and *PlNCED2*) with differential transcription pre- and post-germination (Figures S4 and S7) based on previously published transcriptome data. Studies have shown that *NCED* genes are the key factors that control the responses of endogenous ABA content to environmental stimuli [37]. The Conserved Domain Database (CDD) search for protein sequences of *PlNCED1* and *PlNCED2* in the NCBI database indicates that *PlNCED1* and *PlNCED2* proteins belong to the RPE65 family, a characteristic conserved domain of enzymes involved in carotenoid cleavage dioxygenase [38,39]. Furthermore, the phylogenetic analysis of *NCED2* clearly revealed its intimate genetic relationship among *P. lactiflora, P. ostii*, and *V. vinifera*. This conclusion suggests that *PlNCED1* and *PlNCED2* have similar functions to *PoNCED and VvNCED*, which play an important rate-limiting role in ABA biosynthesis [40,41].

Previous reports indicate that most NCED proteins are located in chloroplasts [19,42,43]. However, our data show that PlNCED1 is located in the nucleus, but it has no Nuclear Localization Signal (NLS), a short peptide acting as a signal fragment and mediating the transport of proteins from the cytoplasm into the nucleus. Previous studies have shown that not all nucleus-expressed proteins require an NLS, and multiple additional pathways can also mediate their nuclear import [44,45]. One of these pathways is that these proteins without NLSs enter the nucleus by interacting with proteins with NLSs or with other nuclear localization proteins [46]. Therefore, our experiments imply that PlNCED1 may enter the nucleus by relying on the NLSs of other proteins. Additionally, our results suggest that the expression of *PlNCEDs* may be regulated by several miRNAs located in the 3'UTR regions of *PlNCEDs*, as well as *cis*-acting elements located upstream of transcripts (Supplementary Tables S4 and S5). Given the characteristics of these miRNAs, we further demonstrated that *PlNCED1* and *PlNCED2* might play a certain role in seed development and biotic and abiotic stresses, which is consistent with the role of ABA in plant growth. These *cis*-acting elements from the promoter sequences of *PlNCED* genes were presumed to be involved in salicylic acid, ABA, auxin, and jasmonic acid (Supplementary Tables S6 and S7). By combining *PlNCED* genes and their associated transcriptional regulatory binding site predictions, our data provide a preliminary path to explore the molecular mechanisms of ABA and other phytohormones involved in seed dormancy and germination.

The ABA content is proportionally related to the process of herbaceous peony seed dormancy and germination, while ABA accumulation in seeds gradually decreases from dormancy to germination (Figure S5) [36]. By comparing the transcription levels of *PlNCED* genes with ABA contents, we clearly show that the expression dynamics of *PlNCED2* are directly associated with ABA biosynthesis and accumulation after seeds imbibe water (Figures S4 and S5) [36], suggesting that *PlNCED2* is the crucial causal factor for ABA-mediated seed dormancy release in herbaceous peony. The deviation of *PlNCED* expression from ABA content in the imbibition stage may be due to the selfregulation of ABA metabolism genes to adapt to the dynamic balance among endogenous phytohormones after seed imbibition (Figures S4 and S5) [36]. Lee et al. (2018) also found that the level of *NCED1* in orchids (*PtNCED1*) was low in the early stage of seed development but gradually increased and then declined slightly when seeds germinated, and the resulting changes in the seed's endogenous ABA content played a key role in seed germination [19]. In contrast, the expression level of *NCED* in habanero pepper was high during seed germination, but the content of ABA gradually decreased during the same time span. This indicates that *NCED* has less of an effect on ABA synthesis and a weaker impact on seed germination in habanero pepper [47]. All of these reports indicate that *NCED* has different regulatory effects on ABA content in different plant species. Lastly, our transgenic experiment in herbaceous peony has also proved that *PlNCED2* plays a major role in ABA biosynthesis and accumulation and subsequently contributes to seed dormancy maintenance.

The ultimate way to dissect the biological function of *PlNCED* genes involved in seed dormancy is to generate transgenic lines of herbaceous peony. However, it is difficult to establish a stable and efficient genetic transformation system for herbaceous peony [48]. Although the genetic transformation system of herbaceous peony has been used for the functional analysis of some genes in recent years [49–51], it takes at least 3 years to obtain transgenic seeds from transgenic seedlings due to the growth characteristics of herbaceous peony. In order to efficiently verify the effect of *PlNCED* genes on the seed germination rate, we used *A. thaliana*, a typical short-growth-cycle model plant, for subsequent gene transformation experiments in this study [52]. *Arabidopsis thaliana* mutants play an important role in revealing the growth and development of different plants. *AtNCED5* and *AtNCED9* mutants in *A. thaliana* have been found to affect ABA production in the embryo and endosperm, leading to seed dormancy [15,16]. Therefore, we built *PlNCED* transgenic lines in wild-type *A. thaliana* as well as *PlNCED* complementation lines in *A. thaliana NCED* mutants to explore the role of *PlNCED* in seed dormancy and germination. Phenotyping

*PlNCED* transgenic lines indeed showed that weak seed dormancy was induced to a certain extent by the overexpression of *PlNCEDs* in *A. thaliana* (Figure 5a,b). More significantly, the seed germination rate of the *PlNCED2* complementation line was significantly lower than that of the *nced5-2* mutant (Figure 5f), indicating that overexpressed *PlNCED2* rescued the partial function of *Arabidopsis NCED* genes. Collectively, the biological function of *PlNCEDs* is consistent to that of *A. thaliana NCEDs* by promoting seed dormancy and delaying germination.

### **5. Conclusions**

In summary, we identified and cloned two genes from the *NCED* gene family, which are related to ABA synthesis in herbaceous peony. By comparing their protein sequences and phylogenetics with those of homologs in other plant species, we were able to detect their functional conserved domain. The dynamics of *PlNCED2* transcription epistatically regulated endogenous ABA biosynthesis and accumulation. Using transgenic and complementation rescue lines in *Arabidopsis*, we were able to demonstrate the phenotypic traits of *PlNCED* genes, which induce seed dormancy and hinder seed germination. Our data and analysis provide the first step to understanding the underlying molecular and genetic mechanisms of complex double seed dormancy in herbaceous peony.

**Supplementary Materials:** The following supporting information can be downloaded at https: //www.mdpi.com/article/10.3390/plants12040710/s1. Figure S1: Identification of *nced* homozygous mutants in *A. thaliana*.; Figure S2: Nucleotide and amino acid sequences of the *PlNCED* gene coding regions; Figure S3: Conserved domain prediction for the proteins encoded by *PlNCEDs*; Figure S4: *PlNCED* expression at different stages of seed dormancy release in herbaceous peony; Figure S5: Changes in ABA content at different seed dormancy release stages in herbaceous peony; Figure S6: Identification of *PlNCED* transgenic callus; Figure S7: Other *PlNCED* family members' expression at different stages of seed dormancy release in herbaceous peony; Table S1: Primers used in this study; Table S2: The identification of PlNCED1 from different plant species (%); Table S3: The identification of PlNCED1 from different plant species (%); Table S4: Prediction of miRNA targets in 3'UTR of *PlNCED1*; Table S5: Prediction of miRNA targets in 3'UTR of *PlNCED2*; Table S6: Prediction of *cis*-acting elements in promoter sequence of *PlNCED1*; Table S7: Prediction of *cis*-acting elements in promoter sequence of *PlNCED2*.

**Author Contributions:** Conceptualization, X.S. and S.G.; methodology, X.S.; software, S.D.; validation, R.F., J.G., and T.S.; formal analysis, R.F.; investigation, S.G.; resources, X.S.; data curation, S.G.; writing—original draft preparation, R.F.; writing—review and editing, S.D.; visualization, R.F.; supervision, X.S.; project administration, S.G.; funding acquisition, X.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the National Natural Science Foundation of China, grant number 32071814, and the China Agriculture Research System of MOF and MARA, grant number CARS-23.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The datasets generated and analyzed during the current study are available in the NCBI repository—*PlNCED1*: OL744236, https://www.ncbi.nlm.nih.gov/nuccore/ OL744236, *PlNCED2*: OL744237, https://www.ncbi.nlm.nih.gov/nuccore/OL744237.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.
