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Nitrate is an important precursor of tobacco-specific nitrosamines (TSNAs) and a remarkable difference in nitrate accumulation between lamina and midrib of flue-cured tobacco has long been observed. However, the physiological and molecular mechanisms underpinning this difference remain poorly understood. In this study, physiological and genetic factors impacting nitrate accumulation were identified in pot experiments using flue-cured tobacco K326 with contrasting nitrate content between lamina and midrib. The results showed that three times higher NO3-N content was observed in midrib than that in the lamina, along with lower pigment, NH4-N content, nitrate reductase activity (NRA), sucrose synthetase activity (SSA), and glutamine synthetase activity (GSA) in midrib. Transcriptome analysis revealed that expression of genes involved in porphyrin and chlorophyll metabolism, carotenoid biosynthesis, photosynthesis-antenna proteins, photosynthesis, carbon fixation in photosynthetic organisms, starch and sucrose metabolism, nitrogen metabolism, and biosynthesis of amino acids were significantly lower in midrib than in lamina. qRT-PCR results showed that the expression level of nitrate transporter genes LOC107782967, LOC107806749, LOC107775674, LOC107829632, LOC107799198, LOC107768465 decreased by 2.74, 1.81, 49.5, 3.5, 2.64 and 2.96-folds while LOC107789301 increased by 8.23-folds in midrib but not in lamina. Reduced chlorophyll content might result in low carbohydrate formation which is the source of energy and carbon skeleton supply, then the low capacity of nitrogen reduction, assimilation and transportation, and the poor ability of nitrate reallocation but the high capacity of accumulation might lead to nitrate accumulation in midrib. The results laid the foundation for reducing nitrate content and TSNA formation in tobacco midribs and their products. [ABSTRACT FROM AUTHOR]