The 14-3-3 gene family in Nicotiana tabacum: Genome-wide identification, expression profiling, subcellular localization, and protein interaction analysis in response to cadmium stress

Plant 14-3-3 proteins play a crucial role in the fine-tuning of growth and development, stress resistance, and nutrient metabolism. Tobacco is recognized as a hyperaccumulator of cadmium (Cd), yet the understanding of the Nt14-3-3 gene family remains limited in tobacco (Nicotiana tabacum L.). This study systematically identified 28 members (designated as NtGRF1 through NtGRF28) of the Nt14-3-3 family in the cultivated variety TN90 and categorized them into ε and non-ε groups. Genes within the same evolutionary branch exhibited similar conserved motif patterns and intron-exon structures. Promoter cis-acting element analysis indicated that the expression of NtGRF genes is regulated by plant hormones, particularly abscisic acid (ABA) and jasmonic acid (JA), as well as various stress factors. RNA-seq-based analysis revealed three expression patterns for the Nt14-3-3 family genes: constitutively high-expressing, low-expressing, and tissue-specific expression groups, corroborating these findings with qPCR results. Cd stress significantly enhanced the expression levels of six out of eight randomly screened NtGRF genes (NtGRF8, NtGRF15, NtGRF16, NtGRF21, NtGRF22, and NtGRF28). Subcellular localization analysis showed that NtGRF8, NtGRF16, and NtGRF22 were expressed in the cytoplasm and nucleus, while NtGRF26 was restricted to the cytoplasm. Yeast two-hybrid assays demonstrated that NtGRF16 and NtGRF22 formed homodimers, with heterodimer formation also prevalent. These results offer important insights into the potential biological functions of the Nt14-3-3 gene family, emphasizing their role in various biological processes, particularly in the response of tobacco to cadmium stress.