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-<i>cis</i>-epoxycarotenoid dioxygenase (NCED) is a key rate-limiting enzyme that regulates the accumulation and content of ABA. However, the role of the <i>NCED</i> gene in perennial plants with complex seed dormancy remains largely unknown. Here, we cloned two differentially expressed paralogs of herbaceous peony <i>NCED</i> genes, named <i>PlNCED1</i> and <i>PlNCED2</i>, 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 <i>NCED</i> sequences from other plants and contained the typical conserved RPE65 domain of the NCED family. Phylogenetic analysis showed that <i>PlNCED1</i> and <i>PlNCED2</i> have a close relationship with <i>PoNCED</i> in <i>Paeonia ostii</i> and <i>VvNCED6</i> in <i>Vitis vinifera</i>, respectively. A subcellular localization assay demonstrated that the <i>PlNCED1</i> protein resided within the nucleus, while the <i>PlNCED2</i> 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 <i>PlNCEDs</i> were positively correlated with ABA content. Both <i>PlNCED</i> transgenic <i>Arabidopsis</i> lines and the functional complementation of <i>Arabidopsis NCED</i> mutants found that <i>PlNCEDs</i> promoted seed dormancy and delayed seed germination. These results reveal that <i>PlNCEDs</i> participate in the seed dormancy of herbaceous peony by regulating the accumulation of endogenous ABA.