Genome-wide identification and expression analysis of the CLC gene family in pomegranate (Punica granatum) reveals its roles in salt resistance

Abstract

Backgrounds

Pomegranate (Punica granatum L.) is an important commercial fruit tree, with moderate tolerance to salinity. The balance of Cl⁻ and other anions in pomegranate tissues are affected by salinity, however, the accumulation patterns of anions are poorly understood. The chloride channel (CLC) gene family is involved in conducting Cl⁻, NO₃⁻, HCO₃⁻ and I⁻, but its characteristics have not been reported on pomegranate.

Results

In this study, we identified seven PgCLC genes, consisting of four antiporters and three channels, based on the presence of the gating glutamate (E) and the proton glutamate (E). Phylogenetic analysis revealed that seven PgCLCs were divided into two clades, with clade I containing the typical conserved regions GxGIPE (I), GKxGPxxH (II) and PxxGxLF (III), whereas clade II not. Multiple sequence alignment revealed that PgCLC-B had a P [proline, Pro] residue in region I, which was suspected to be a NO₃⁻/H⁺ exchanger, while PgCLC-C1, PgCLC-C2, PgCLC-D and PgCLC-G contained a S [serine, Ser] residue, with a high affinity to Cl⁻. We determined the content of Cl⁻, NO₃⁻, H₂PO₄⁻, and SO₄²⁻ in pomegranate tissues after 18 days of salt treatments (0, 100, 200 and 300 mM NaCl). Compared with control, the Cl⁻ content increased sharply in pomegranate tissues. Salinity inhibited the uptake of NO₃⁻ and SO₄²⁻, but accelerated H₂PO₄⁻ uptake. The results of real-time reverse transcription PCR (qRT-PCR) revealed that PgCLC genes had tissue-specific expression patterns. The high expression levels of three antiporters PgCLC-C1, PgCLC-C2 and PgCLC-D in leaves might be contributed to sequestrating Cl⁻ into the vacuoles. However, the low expression levels of PgCLCs in roots might be associated with the exclusion of Cl⁻ from root cells. Also, the up-regulated PgCLC-B in leaves indicated that more NO₃⁻ was transported into leaves to mitigate the nitrogen deficiency.

Conclusions

Our findings suggested that the PgCLC genes played important roles in balancing of Cl⁻ and NO₃⁻ in pomegranate tissues under salt stress. This study established a theoretical foundation for the further functional characterization of the CLC genes in pomegranate.