Knockout of a key gene of the nicotine biosynthetic pathway severely affects tobacco growth under field, but not greenhouse conditions

Abstract

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.

Genetic regulation and manipulation of nicotine biosynthesis in tobacco: strategies to eliminate addictive alkaloids

“…However, efforts to reduce alkaloid contents by manipulating these early steps in the nicotine biosynthesis pathway have not always yielded favorable outcomes. There are two main reasons for these failures: (i) interference with polyamine and NAD supplies ( Sato et al , 2001 ; Dalton et al , 2016 ; Choubey and Rajam, 2017 ; Khan et al , 2017 ; Hidalgo Martinez et al , 2020 ; Smith et al , 2022 ) and (ii) the compensatory production of pyridine-derived alkaloids, anabasine and anatabine, when the supply of pyrrolidine rings is blocked ( Chintapakorn and Hamill, 2003 ; Steppuhn et al , 2004 ; Shoji and Hashimoto, 2008 ; Wang et al , 2009 ; DeBoer et al , 2011a ; Dalton et al , 2016 ). In contrast to the early steps, biochemical details of later steps in nicotine biosynthesis, including ring condensation, remain elusive.…”

“…These alterations in growth could be attributed to a decreased supply of NAD. Additionally, a QPT2 -knockout mutant generated using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) genome editing showed drastically reduced nicotine production ( Smith et al , 2022 ). It is important to note that this tobacco mutant displayed only modest growth changes in a greenhouse environment, but, when transplanted to fields, its growth and development were severely inhibited.…”