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.