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

Chloride (Cl^-) is an important micronutrient and usually accumulates to a macronutrient level in many plant species. Tobacco (<i>Nicotiana tabacum</i> 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 (<i>P</i> < 0.01) was identified using weighted gene coexpression network (WGCNA) analysis. In the red module, gene ontology (GO) analysis identified two key genes, <i>Ntab22g013620.1</i> (<i>NtGCN1</i>) and <i>Ntab08g002050.1</i> (<i>NtV-ATPase c''</i>), which might regulate leaf Cl^- accumulation. The role of <i>NtGCN1</i> 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 <i>NtGCN1</i>-overexpressing plants, whereas they were remarkably decreased in VIGS-<i>NtGCN1</i>-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.