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

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.