Characterization of a NAC transcription factor involved in the regulation of pomegranate seed hardness (Punica granatum L.)

The pomegranate, Punica granatum L., which has been cultivated since antiquity, is known to be a superfruit, possessing an array of functional anti-oxidants and various other health benefits. The hardness of pomegranate seeds is an important indicator of fruit quality, which in turn affects economic value and market demand. However, the molecular mechanism underlying pomegranate seed hardness remains to be fully understood. In this study, we found a positive correlation between seed hardness and lignin content in two pomegranate varieties: “Tunisia” and “Sanbai”. Specifically, genes associated with lignin biosynthesis were differentially expressed in soft-seed and hard-seed pomegranate varieties. Among these differential genes, we cloned and characterized the NAC transcription factor PgSND1-like. Sequence alignment found a single base replacement at the 166-bp position of CDS in the PgSND1-like gene from “Tunisia” and “Sanbai”. Both PgSND1-like (Sanbai) and PgSND1-like (Tunisia) proteins are localized in the cell nucleus and have a transcription activation domain in the C-terminus. Yeast two-hybrid analysis indicated that PgSND1-like protein interacts with itself to form a homodimer. Overexpression of PgSND1-like (Sanbai) in Arabidopsis showed a higher lignin content in inflorescence stem and mature seed compared with wild-type Arabidopsis. Accordingly, the expression levels of several lignin biosynthesis-associated genes were upregulated in stem cells and mature seeds of transgenic plants. However, PgSND1-like (Tunisia) transgenic Arabidopsis showed no phenotypic differences with wild-type Arabidopsis. Taken together, we suggest that PgSND1-like may regulate at least two different functions in two pomegranate varieties, promoting lignin biosynthesis and seed hardness of pomegranate.