Cold stress limits plant growth, development and yields, and the C-repeat binding factors (CBFs) function in the cold resistance in plants. However, how pomegranate CBF transcription factors respond to cold signal remains unclear. Considering the significantly up-regulated expression of PgCBF3 and PgCBF7 in cold-tolerant Punica granatum ‘Yudazi’ in comparison with cold-sensitive ‘Tunisia’ under 4 °C, the present study focused on the two CBF genes. PgCBF3 was localized in the nucleus, while PgCBF7 was localized in the cell membrane, cytoplasm, and nucleus, both owning transcriptional activation activity in yeast. Yeast one-hybrid and dual-luciferase reporter assay further confirmed that PgICE1 could specifically bind to and significantly enhance the activation activity of the promoters of PgCBF3 and PgCBF7. Compared with the wild-type plants, the PgCBF3 and PgCBF7 transgenic Arabidopsis thaliana lines had the higher survival rate after cold treatment; exhibited increased the contents of soluble sugar and proline, while lower electrolyte leakage, malondialdehyde content, and reactive oxygen species production, accompanying with elevated enzyme activity of catalase, peroxidase, and superoxide dismutase; and upregulated the expression of AtCOR15A, AtCOR47, AtRD29A, and AtKIN1. Collectively, PgCBFs were positively regulated by the upstream PgICE1 and mediated the downstream COR genes expression, thereby enhancing freezing tolerance.

Laccase (LAC) is the key enzyme responsible for lignin biosynthesis. Here, 57 PgLACs from pomegranate were identified and distributed on eight chromosomes and one unplaced scaffold. They were divided into six groups containing three typical Cu-oxidase domains. Totally, 51 cis-acting elements in the promoter region of PgLACs are involved in response to ABA, GA, light, stress, etc., indicating diverse functions of PgLACs. The expression profiles of 13 PgLACs during the seed development stage showed that most PgLACs expressed at a higher level earlier than at the later seed development stage in two pomegranate cultivars except PgLAC4. Also, PgLAC1/6/7/16 expressed at a significantly higher level in soft-seed ‘Tunisia’; on the contrary, PgLAC37 and PgLAC50 with a significantly higher expression in hard-seed ‘Taishanhong’. Combined with their distinguishing cis-acting elements, it was concluded that PgLAC1/6/7 may respond to GA via TATC-box and GARE-motif, and PgLAC16 repressed the promotor activity of embryo mid-maturation genes via RY-element so as to contribute to softer seed formation, whereas PgLAC37/50 may participate in seed formation and accelerate seed maturity via ABRE and G-box elements. Collectively, the dramatic gene expressions of PgLAC1/6/7/16/37/50 will provide valuable information to explore the formation of soft- and hard-seed in pomegranate.