Seed is the basic guarantee for the continuation of plant species (Bewley, et al. 2013). Seed dormancy is an ecophysiological characteristic of plants that adapt to the environment during long-term growth and development. From a biological perspective, seed dormancy can ensure the survival of species in harsh environments, reduce the competition between individuals in the same species, and prevent seed germination in unsuitable seasons (Bewley 1997). It plays a positive role in plant individuals, survival, evolution, and the protection of plant germplasm resources (Willis, et al. 2014). But in terms of the production of cultivated species, seeds often need to germinate quickly, neatly and grow quickly to obtain high economic yield (Lafta and Mou 2013; Mutlu, et al. 2020). At the same time, an early sprouting phenomenon caused by the lack of seed dormancy has a negative impact on the production of cereal crops (Finkelstein, et al. 2008). Therefore, based on the characteristics of seed dormancy and the actual needs of agricultural and forestry production, scholars have discussed this issue from different scientific perspectives. Seed dormancy release is accompanied by a series of physiological and biochemical reactions, including the repair of metabolic changes, decomposition and utilization of storage substances and energy metabolism (Li and Min 2020; Liu, et al. 2020; Vigliocco, et al. 2020). Seed dormancy is coordinately regulated by external stimulation (such as light, temperature and humidity, etc.) and endogenous factors (such as phytohormones, sugars and nitrogen compounds, etc.) (Chen, et al. 2020a; Chen, et al. 2021; Klupczynska and Pawlowski 2021; Malavert, et al. 2020; Sano and Marion-Poll 2021; Wala, et al. 2021). Phytohormones are important growth regulators in this process. Ethylene (ETH) can negatively regulate seed dormancy by inhibiting ABA synthesis and signal transduction, or affect seed germination and early seedling growth by interacting with sugar signals (Naing, et al. 2021; Xia, et al. 2018). Brassinosteroid (BR) can reduce the sensitivity of seeds to ABA, thereby stimulating seed germination (Ha, et al. 2018; Kim, et al. 2019). Cytokinin (CTK) promotes seed germination by indirectly antagonizing with ABA (Shen, et al. 2020). Auxin also cannot independently regulate seed dormancy and germination, but positively regulate ABA signal by enhancing the sensitivity of seeds to ABA, thereby affecting seed dormancy and germination (Liu, et al. 2013; Munguia-Rodriguez, et al. 2020).
ABA content was positively correlated with seed dormancy (Tognacca and Botto 2021). The final concentration of endogenous ABA in plants depends on the dynamic balance of ABA synthesis and catabolism (Eggels, et al. 2018; Yan, et al. 2022). ABA biosynthesis pathway mainly includes C15 direct pathway and C40 indirect pathway. In higher plants, ABA is mainly synthesized through an indirect pathway. In the indirect pathway, zeaxanthin is used as the starting point to form violaxanthin catalyzed by zeaxanthin epoxidase (ZEP), and the violaxanthin is converted into 9'-cis-Neoxanthin and 9'-cis-Violaxanthin. Then these two by-products are oxidized to xanthoxin under the action of 9-cis-epoxycarotenoid dioxygenase. Subsequently, xanthoaldehydes leave the plastid and enter the cytoplasm, which is catalyzed by short-chain dehydrogenase/reductase (SDR) to form ABA aldehyde, and finally oxidized by aldehyde oxidase (AAO) to form ABA (Taylor, et al. 2005). The biological decomposition pathways of ABA mainly include: oxidative binding inactivation and oxidative inactivation. In higher plants, ABA is mainly decomposed through oxidative inactivation. The oxidative inactivation pathway of ABA is divided into 7'-hydroxylation, 8'-hydroxylation and 9'-hydroxylation according to the methyl site, which generate 7'-hydroxy-ABA, 8'-hydroxy-ABA and 9'-hydroxy-ABA, respectively, and causing oxidative inactivation of ABA. Among the three oxidative inactivation modes, 8’-hydroxylation reaction is proved to be the most important metabolic pathway of ABA in higher plants. ABA is catalyzed by 8'-hydroxylase to form 8'-hydroxy-ABA, and its spontaneous isomerization generates phaseic acid (PA). PA eventually generates diammine phaseic acid under the action of PA reductase (PAR) (Okamoto, et al. 2006; Saito, et al. 2004; Weng, et al. 2016). Studies have shown that ABA-deficient mutants of A. thaliana, tomato and maize show early dormancy breaking and turning into germination stage, whereas ABA-overexpressing plants show delayed dormancy (Jia, et al. 2021; Martin-Rodriguez, et al. 2016; Qin and Zeevaart 2002; Wu, et al. 2014). It can be verified that the change of endogenous hormone ABA content is significantly positively correlated with the degree of seed dormancy (Wang, et al. 2015).
ABA is a key factor affecting seed dormancy and germination. Previous studies have been confirmed in many plants (Eggels, et al. 2018; Pan, et al. 2018; Shen, et al. 2018). After exogenous application of ABA inhibitors, the germination rate of A. thaliana seeds was significantly increased, and the ABA-deficient mutant seeds had no dormancy characteristics (Léon-Kloosterziel, et al. 1996; Lopez-Molina, et al. 2001). NCED and CYP707A encode key enzymes in ABA synthesis and decomposition pathways, respectively. Different members of family genes have different regulatory roles in plant seed dormancy. How they affect seed dormancy and their regulatory mechanism have been thoroughly studied in model plant - A. thaliana. AtNCED family members have different expression sites in seeds. AtNCED6 is only expressed in endosperm, while AtNCED9 is expressed in both embryo and endosperm (Lefebvre, et al. 2006). They can not only regulate ABA content in A. thaliana embryo, but also promote and maintain embryo dormancy. AtNCED5 is up-regulated at the late stage of seed maturation in A. thaliana, and cooperates with AtNCED6 and AtNCED9 to enhance seed dormancy (Frey, et al. 2012; Lefebvre, et al. 2006). AtCYP707A1 mainly express in seed coat and endosperm at the middle stage of seed maturation, and the seed dormancy of cyp707a1 mutant is significantly enhanced (Okamoto, et al. 2006; Wu, et al. 2022). AtCYP707A2 is mainly expressed in the seed coat, embryo and endosperm of A. thaliana at the late stage of seed imbibition and maturation (Ikeya, et al. 2020). The ABA content of A. thaliana cyp707a2 mutant seeds is five times that of wild-type A. thaliana seeds, and the seeds show deep dormancy (Chen, et al. 2020b; Saito, et al. 2004).
The effects of NCED and CYP707A on seed dormancy were also verified in other plants. During the imbibition process of Zoysia japonica seeds, CYP707A plays a leading role in the decrease of ABA content (Dong, et al. 2021). Overexpression of Oryza sativa OsNCED3 in wild-type A. thaliana can increase ABA content and promote seed dormancy (Liao, et al. 2021). The expression of PvNCED in Phaseolus vulgaris can increase the seed dormancy (Enomoto, et al. 2017). Overexpression of LeNCED1 in Lycopersicon esculentum delays seed germination (Thompson, et al. 2000). AhNCED2 plays a positive role in maintaining seed dormancy in Arachis hypogaea (Bo, et al. 2010).
Herbaceous peony (Paeonia lactiflora Pall.) is the herbaceous perennial flower of Paeoniaceae. In the long-term systematic evolution process, the seeds of herbaceous peony form a unique double dormancy characteristic of upper and lower hypocotyls. In the breeding process, the dormancy is often not released or incompletely released, which greatly reduces the germination rate and seriously affects the actual cultivation and production, especially the breeding of new varieties and the process of hybrid breeding (Li 1999). At present, the research on the dormancy release technology of herbaceous peony seeds mainly focuses on mechanical breaking, low temperature and hormone treatment, endogenous inhibitor determination and so on (Ren 2016; Sun, et al. 2012; Zhang 2015). However, there are few studies on the molecular mechanism of genes related to seed dormancy release. The previous study of our laboratory found that the endogenous ABA content in herbaceous peony seeds was significantly negatively correlated with seed germination (Li, et al. 2020). Herbaceous peony seeds endogenous ABA content is mainly regulated by ABA synthesis key gene PlNCEDs and metabolism key gene PlCYP707As (Li, et al. 2020). In this study, from the perspective of ABA synthesis, ten family members of PlNCED were found from transcriptome data (Supplement Figure 1 and Figure 2). Finally, c53147_g1 (PlNCED1) and c69372_g1 (PlNCED2) with significant differential expression were selected as research objects. In order to further identify the role of PlNCED1 and PlNCED2 in seed dormancy release, we first cloned the full-length cDNA of PlNCED1 and PlNCED2 from herbaceous peony seeds, and determined the specific action sites of its encoded protein in plant cells. Finally, the role of PlNCED1 and PlNCED2 in ABA metabolism and seed germination was clarified through homologous and heterologous genetic transformation. The results of this study can enrich the related theory of herbaceous peony seed dormancy and provide a scientific basis for finding effective seed dormancy breaking methods of herbaceous peony in the future.