Genome-wide identification of CBL family genes in Nicotiana tabacum and the functional analysis of NtCBL4A-1 under salt stress

The CALCINEURIN B-LIKE PROTEIN family genes (CBLs) encode a group of plant-specific calcium sensor proteins that play critical roles in plant development and stress response. Although genome-wide analysis of CBL family genes has been conducted in several diploid plant species, less is known about their functions in allopolyploid plant species. This study conducted a detailed analysis of tobacco CBL family genes. Of the 24 NtCBL genes identified, 14 were derived from the maternal genome donor N. sylvestris, and 10 were from the paternal genome donor N. tomentosiformis. Phylogenetic analysis revealed that NtCBLs could be clustered into three different groups with distinct N-terminal characteristics. All NtCBLs contain EF-hand motifs, but amino acid substitutions and configuration changes may be responsible for variations in their Ca2+-binding abilities. Alternative splicing is common for NtCBL gene transcripts, which may contribute to variations in translation efficiency or protein interaction preferences. Several NtCBL genes exhibited shoot- or root-predominant expression patterns, and some were responsive to salt and/or drought stress. NtCBL4A-1, an ortholog of Arabidopsis thaliana AtCBL4 (SOS3), was predominantly expressed in roots and exhibited a lower gene expression level under salt stress. Further functional analysis showed that overexpression of NtCBL4A-1 increased the sensitivity of transgenic tobacco to Na+-induced salt stress, which is inconsistent with the role of AtCBL4 in contributing to salt tolerance. This study provides data for identifying CBL gene functions and selecting candidate stress-tolerant genes in N. tabacum. Additionally, the results may aid gene function studies in other allopolyploid plant species.