ABSTRACT
Genetic methodologies for reducing nicotine accumulation in the tobacco plant (Nicotiana tabacum L.) are of interest because of potential future regulations that could mandate lowering of this alkaloid in conventional cigarettes. Inactivation of tobacco genes such as the Berberine Bridge Like (BBL) gene family believed to encode for enzymes involved in one of the latter steps of nicotine biosynthesis could be a viable strategy for producing new tobacco cultivars with ultra-low leaf nicotine accumulation. We introduced deleterious mutations generated via ethyl methanesulfonate treatment of seed or gene editing into six known members of the BBL gene family and assembled them in different combinations to assess their relative contribution to nicotine accumulation. Significant reductions (up to 17-fold) in percent leaf nicotine were observed in genotypes homozygous for combined mutations in BBL-a, BBL-b, and BBL-c. The addition of mutations in BBL-d1, BBL-d2, and BBL-e had no additional significant effect on lowering of nicotine levels in the genetic background studied. Reduced nicotine levels were associated with reductions in cured leaf yields (up to 29%) and cured leaf quality (up to 15%), evidence of physiological complexities within the tobacco plant related to the nicotine biosynthetic pathway. Further nicotine reductions were observed for a BBL mutant line cultivated under a modified production regime in which apical inflorescences were not removed, but at the expense of further yield reductions. Plants in which BBL mutations were combined with naturally occurring recessive alleles at the Nic1 and Nic2 loci exhibited further reductions in percent nicotine, but no plant produced immeasurable levels of this alkaloid. Findings may suggest the existence of a minor, alternative pathway for nicotine biosynthesis in N. tabacum. The described genetic materials may be of value for the manufacture of cigarettes with reduced nicotine levels and for future studies to better understand the molecular biology of alkaloid accumulation in tobacco.
ABSTRACT
In previous research, we discovered a favorable quantitative trait locus (QTL) in cigar tobacco cultivar 'Beinhart 1000' designated as Phn15.1, which provides a high level of partial resistance to the black shank disease caused by Phytophthora nicotianae. A very close genetic association was also found between Phn15.1 and the ability to biosynthesize Z-abienol, a labdanoid diterpene exuded by the trichomes onto above-ground plant parts, and that imparts flavor and aroma characteristics to Oriental and some cigar tobacco types. Because accumulation of Z-abienol is considered to be undesirable for cultivars of other tobacco types, we herein describe a series of experiments to gain insight on whether this close association is due to genetic linkage or pleiotropy. First, in an in vitro bioassay, we observed Z-abienol and related diterpenes to inhibit hyphal growth of P. nicotianae at concentrations between 0.01 and 100 ppm. Secondly, we field-tested transgenic versions of Beinhart 1000 carrying RNAi constructs for downregulating NtCPS2 or NtABS, two genes involved in the biosynthesis of Z-abienol. Thirdly, we also field tested a recombinant inbred line population segregating for a truncation mutation in NtCPS2 leading to an interrupted Z-abienol pathway. We observed no correlation between field resistance to P. nicotianae and the ability to accumulate Z-abienol in either the transgenic materials or the mapping population. Results suggest that, although Z-abienol may affect P. nicotianae when applied at high concentrations in in vitro assays, the compound has little effect on black shank disease development under natural field conditions. Thus, it should be possible to disassociate Phn15.1-mediated black shank resistance identified in cigar tobacco cultivar Beinhart 1000 from the ability to accumulate Z-abienol, an undesirable secondary metabolite for burley and flue-cured tobacco cultivars.
