Knockout of a Key Gene of the Nicotine Biosynthetic Pathway Severely Affects Tobacco Growth Under Field, but Not Greenhouse Conditions

Objective: There is great interest in developing tobacco plants containing minimal amounts of the addictive compound nicotine. Quinolate phosphoribosyltransferase (QPT) is an important enzyme both for primary (NAD production) and secondary (pyridine alkaloid biosynthesis) metabolism in tobacco. The duplication of an ancestral QPT gene in Nicotiana species has resulted in two closely related QPT gene paralogs: QPT1 which is expressed at modest levels throughout the plant, and QPT2 which is coordinately regulated with genes dedicated to alkaloid biosynthesis. This study evaluated the utility of knocking out QPT2 function as a means for producing low alkaloid tobacco plants.

Results: CRISPR/Cas9 vectors were developed to specifically mutate the tobacco QPT2 genes associated with alkaloid production. Greenhouse-grown qpt2 plants accumulated dramatically less nicotine than controls, while displaying only modest growth differences. In contrast, when qpt2 lines were transplanted to a field environment, plant growth and development was severely inhibited. Two conclusions can be inferred from this work: (1) QPT1 gene function alone appears to be inadequate for meeting the QPT demands of the plant for primary metabolism when grown in a field environment; and (2) the complete knockout of QPT2 function is not a viable strategy for producing agronomically useful, low nicotine tobaccos.