Nicotine is the most abundant alkaloid in cultivated tobacco (Nicotiana tabacum), typically constituting more than 90 % of total alkaloids. Recently, the U.S. Food and Drug Administration issued an advance notice of proposed rulemaking to obtain information for consideration in developing a tobacco product standard to set the maximum nicotine level in cigarette filler. Cigarettes that can meet a very low nicotine filler level regulation are dependent upon technical achievability. Historically, nic1nic2 mutant lines (approximately 95 % nicotine reduction) have been the unique resource for developing low alkaloid traits. However, these mutants produce plants having very poor leaf quality making them commercially unfavourable. In comparison, our data showed nicotine levels in Putrescine N-methyltransferase (PMT) RNAi experimental lines were reduced more than 95 % and had a better grade index after curing compared to nic1/nic2 mutant controls. To understand grade index/leaf quality and its correlation with nicotine levels, transcriptomics and metabolomics studies were performed using seven Burley and four flue-cured lines at different plant growth time intervals. We will discuss our findings on differential gene expression of these 11 lines and changes in key metabolite markers.