NIC1 cloning and gene editing generates low‐nicotine tobacco plants

Natural and induced variations in transcriptional regulator genes result in low‐nicotine phenotypes in tobacco

“…Indeed, TI1246 and TI1573 contained both the nic1‐1 and nic2‐1 alleles, as evidenced by the amplification of genomic fragments of identical sizes with the primers flanking the deleted regions (Figure S3a,b). Moreover, and consistent with known patterns of gene expression in the nic1‐1 nic2‐1 genotype (Hibi et al., 1994; Qin et al., 2021; Shoji et al., 2010; Sui et al., 2020), relative ERF199 transcript levels in TI1246 and TI1573 were 53% and 37% those of the wild type, respectively (Figure S3c). We also observed the expression of the metabolic genes PMT and A622 , which were also downregulated 13–23% of their levels in the wild type (Figure S3c).…”

“…We obtained moderate but significant effects on the alkaloid phenotype produced by the nic1‐1 and nic1‐4 alleles (Figure 5, Figure S6), which carried similar genomic deletions distal to ERF199 (Figure 4b). The expression of ERF199 was similarly downregulated in nic1‐1 and nic1‐4 (Figure 5c, Figure S6) (Qin et al., 2021; Shoji et al., 2010; Sui et al., 2020). This shared deletion between the two alleles is likely to be responsible for the observed phenotype.…”

“…The NIC1 locus is delimited to a region corresponding to an ERF199 ‐containing gene cluster on chromosome 7 (Qin et al., 2021; Sui et al., 2020). All single nucleotide polymorphisms detected in nic1‐1 around the locus during resequencing are shown in Figure 2(a), while no insertions/deletions are there.…”

“…Clustered ERF s were found to be deleted in the nic2‐1 mutant (Kajikawa et al., 2017, Shoji et al., 2010). The NIC1 locus is located on chromosome 7, in a region containing multiple ERF genes related to those present in the NIC2 locus, including ERF199 , a homolog of ERF189 (Qin et al., 2021; Sui et al., 2020). In the nic1‐1 mutant, ERF199 expression is lower than in the wild type, and the genomic regions downstream of ERF199 are deleted (Qin et al., 2021, Sui et al., 2020).…”

“…The NIC1 locus is located on chromosome 7, in a region containing multiple ERF genes related to those present in the NIC2 locus, including ERF199 , a homolog of ERF189 (Qin et al., 2021; Sui et al., 2020). In the nic1‐1 mutant, ERF199 expression is lower than in the wild type, and the genomic regions downstream of ERF199 are deleted (Qin et al., 2021, Sui et al., 2020). The mechanistic basis of the mutation has remained elusive; it is thus unknown why nic1‐1 has a stronger effect on alkaloid phenotypes than nic2‐1 (Hibi et al., 1994; Legg & Collins, 1971).…”