Transgenic and Mutation-Based Suppression of a Berberine Bridge Enzyme-Like (BBL) Gene Family Reduces Alkaloid Content in Field-Grown Tobacco

Abstract

Motivation exists to develop tobacco cultivars with reduced nicotine content for the purpose of facilitating compliance with expected tobacco product regulations that could mandate the lowering of nicotine levels per se, or the reduction of carcinogenic alkaloid-derived tobacco specific nitrosamines (TSNAs). A berberine bridge enzyme-like (BBL) gene family was recently characterized for N. tabacum and found to catalyze one of the final steps in pyridine alkaloid synthesis for this species. Because this gene family acts downstream in the nicotine biosynthetic pathway, it may represent an attractive target for genetic strategies with the objective of reducing alkaloid content in field-grown tobacco. In this research, we produced transgenic doubled haploid lines of tobacco cultivar K326 carrying an RNAi construct designed to reduce expression of the BBL gene family. Field-grown transgenic lines carrying functional RNAi constructs exhibited average cured leaf nicotine levels of 0.684%, in comparison to 2.454% for the untransformed control. Since numerous barriers would need to be overcome to commercialize transgenic tobacco cultivars, we subsequently pursued a mutation breeding approach to identify EMS-induced mutations in the three most highly expressed isoforms of the BBL gene family. Field evaluation of individuals possessing different homozygous combinations of truncation mutations in BBLa, BBLb, and BBLc indicated that a range of alkaloid phenotypes could be produced, with the triple homozygous knockout genotype exhibiting greater than a 13-fold reduction in percent total alkaloids. The novel source of genetic variability described here may be useful in future tobacco breeding for varied alkaloid levels.

Agronomic Practices Affecting Nicotine Concentration in Flue‐Cured Tobacco: A Review

“…Although nicotine accumulation in tobacco should be considered a quantitative trait controlled by numerous genes and highly influenced by the environment, several large‐effect loci have been identified that might be used to reduce nicotine content. First, recessive alleles at the Nic1 and Nic2 (also designated as A and B ) loci have been transferred from cigar to flue‐cured tobacco to reduce nicotine levels to approximately 2 to 4 g kg −1 (Chaplin and Weeks, 1976; Lewis, 2018; Lewis et al, 2015). This variability has been associated with reduced cured leaf yields and quality, however, and no flue‐cured cultivar containing this genetic variability is widely grown commercially at the current time.…”

Non-statistical 13C Fractionation Distinguishes Co-incident and Divergent Steps in the Biosynthesis of the Alkaloids Nicotine and Tropine

“…An extremely weak "nicotine synthase" has been reported (6), but other authors could not repeat this finding (5). Although it has been suggested that this is possibly catalyzed by an enzyme of the berberine-bridge type (7), no details of the nature of this key enzyme nor of its mechanism are available.…”

Effects of overexpression of jasmonic acid biosynthesis genes on nicotine accumulation in tobacco

“…The isoflavone reductase-like enzyme A622 and a berberine bridgelike (BBL) enzyme are proposed to be involved in the condensation step of the pyridine and pyrrolidine rings to form nicotine (DeBoer, Lye, Aitken, Su, & Hamill, 2009;Kajikawa, Hirai, & Hashimoto, 2009). The synthesis and accumulation of the major and minor alkaloids is closely related and dynamically regulated (Chintapakorn & Hamill, 2003;Hung et al, 2013;Kajikawa et al, 2009;Lewis et al, 2015).…”

“…The isoflavone reductase-like enzyme A622 and a berberine bridgelike (BBL) enzyme are proposed to be involved in the condensation step of the pyridine and pyrrolidine rings to form nicotine (DeBoer, Lye, Aitken, Su, & Hamill, 2009;Kajikawa, Hirai, & Hashimoto, 2009). The synthesis and accumulation of the major and minor alkaloids is closely related and dynamically regulated (Chintapakorn & Hamill, 2003;Hung et al, 2013;Kajikawa et al, 2009;Lewis et al, 2015).Previous research indicates that many factors affect nicotine biosynthesis, including mechanical wounding, topping (decapitation of the apical meristem), plant hormones, transcription factors, and negative feedback by pathway products (Baldwin, Schmelz, & Ohnmeiss, 1994;Elliot, 1966;Wasternack & Hause, 2013) In this study, we intended to gain more insights on JA regulated nicotine synthesis in tobacco by modifying expression of the JA biosynthetic genes. As we do not know which enzyme controls the rate-limiting step of JA biosynthesis, we investigated all five major genes in the pathway.…”