Exploring Candidate Genes for Chloride Accumulation in Leaves of Tobacco ( Nicotiana tabacum L.)

Chloride (Cl^-) is an important micronutrient and usually accumulates to a macronutrient level in many plant species. Tobacco (Nicotiana tabacum L.) is a Cl^--rich plant species, with its leaf Cl^- concentration reaching 50 mg/g of DW, but its underlying physiological mechanisms remain unclear. In this study, four-week-old post-transplant tobacco seedlings were separately grown in a hydroponics nutrient solution supplied with four exogenous Cl^- doses (0, 5, 15, and 25 mM) for 4 weeks. Leaf Cl^- concentration increased gradually and significantly from 0.9 to 45.5 mg/g DW along the exogenous Cl^- gradients. A comparative transcriptomic analysis of the leaves was then performed, and one coexpression network (red module) positively correlated with leaf Cl^- concentration (P < 0.01) was identified using weighted gene coexpression network (WGCNA) analysis. In the red module, gene ontology (GO) analysis identified two key genes, Ntab22g013620.1 (NtGCN1) and Ntab08g002050.1 (NtV-ATPase c''), which might regulate leaf Cl^- accumulation. The role of NtGCN1 in tobacco leaf Cl^- accumulation was confirmed via both transient overexpression and virus-induced gene silencing (VIGS) methods. Cl^- concentrations were significantly increased in the leaves of NtGCN1-overexpressing plants, whereas they were remarkably decreased in VIGS-NtGCN1-infected tobacco plants. Overall, this study provides some novel insights into the complex molecular mechanisms of leaf Cl^- accumulation in tobacco, and the identified candidate genes could act as target genes to breed tobacco cultivars with low leaf Cl^- accumulation.