Unravel the Mystery ofNIC1-locus on Nicotine Biosynthesis Regulation in Tobacco

Abstract

<jats:title>ABSTRACT</jats:title><jats:sec><jats:title>Background</jats:title><jats:p>Nicotine biosynthesis is mainly regulated by jasmonate (JA) signaling cascade in <jats:italic>Nicotiana tabacum</jats:italic>. As an allotetraploid species, the regulation of nicotine biosynthesis has been genetically verified via two unlinked <jats:italic>NIC</jats:italic> loci (named as <jats:italic>NIC1</jats:italic> and <jats:italic>NIC2</jats:italic>) which are possibly originated from its two ancestral diploids. Previously, a <jats:italic>N. tomentosiformis</jats:italic> originated ethylene response factor (<jats:italic>ERF</jats:italic>) gene cluster was identified as the <jats:italic>NIC2</jats:italic>-locus which has been demonstrated positively regulates nicotine accumulation in <jats:italic>N. tabacum</jats:italic>.</jats:p></jats:sec><jats:sec><jats:title>Results</jats:title><jats:p>Here, we describe the genetic mapping of <jats:italic>NIC1</jats:italic>-locus, the major nicotine regulatory locus, by using a <jats:italic>NIC1</jats:italic>-locus segregating population through bulked segregant analysis. We identified two linkage marker TM23004 and TM22038 were delimited the <jats:italic>NIC1</jats:italic>-locus within a ~34.3-Mb genomic region at pseudochromosome 07 of tobacco genome. Genomic scan within this region revealed a <jats:italic>NIC2</jats:italic>-<jats:italic>like</jats:italic> locus <jats:italic>ERF</jats:italic> gene cluster exist in. To verify this <jats:italic>ERF</jats:italic> gene cluster is the genetically called “<jats:italic>NIC1</jats:italic>-locus”, different functional experiments based on most of the <jats:italic>ERFs</jats:italic> in regulating nicotine biosynthesis and their influences on alkaloid accumulations have been carried out. Collinearity analysis showed that <jats:italic>NIC1</jats:italic>-locus <jats:italic>ERF</jats:italic> genes are originated from <jats:italic>N. sylvestris</jats:italic> and exclusively expressed in root tissues. In addition, transcriptomic results indicate that <jats:italic>NIC1</jats:italic>-locus <jats:italic>ERF</jats:italic> genes are coexpressed with the <jats:italic>NIC2</jats:italic>-locus <jats:italic>ERF</jats:italic> genes and other nicotine biosynthetic genes and regulators after JA induction. Furthermore, the suppressed expression of four <jats:italic>ERFs</jats:italic> of the <jats:italic>NIC1</jats:italic>-locus genes corresponding with decreased <jats:italic>NtPMT</jats:italic> and <jats:italic>NtQPT</jats:italic> expression in <jats:italic>NtMYC2</jats:italic>-RNAi lines indicates the selected <jats:italic>NIC1</jats:italic>-locus <jats:italic>ERFs</jats:italic> function in downstream of <jats:italic>NtMYC2</jats:italic> in the JA signaling cascades. In the meanwhile, the alkaloid levels are also determined by the amplitude of the four <jats:italic>ERF</jats:italic> gene expressions in both wild type and LA mutant. Additionally, <jats:italic>in vitro</jats:italic> binding assays, transient activation assays, and ectopic expression in transgenic plants demonstrate that these <jats:italic>ERF</jats:italic> genes are able to bind the GCC-box elements residing in the step-limiting gene promoters (such as <jats:italic>NtPMT2</jats:italic>, <jats:italic>NtQPT2</jats:italic>) and functional redundant but quantitatively transactivate nicotine biosynthetic gene expression. For <jats:italic>nic1</jats:italic>-locus mutation, two different sizes of deletions (<jats:italic>nic1-S</jats:italic> and <jats:italic>nic1-B</jats:italic>) were identified which occurred at the surrounding regions of the <jats:italic>NIC1</jats:italic>-locus gene cluster, which might disrupt, to some extent, chromosomal microenvironment and change gene expression around the deletion regions (including <jats:italic>NIC1</jats:italic>-locus <jats:italic>ERFs</jats:italic>), resulting in the decreased expression levels of <jats:italic>NIC1</jats:italic>-locus <jats:italic>ERFs</jats:italic> (such as <jats:italic>NtERF199</jats:italic>) and reduced alkaloid accumulation in the <jats:italic>nic1</jats:italic>-locus mutant.</jats:p></jats:sec><jats:sec><jats:title>Conclusions</jats:title><jats:p>Our findings not only provide insight in to the mechanism of the <jats:italic>NIC1</jats:italic>-locus <jats:italic>ERFs</jats:italic> in the regulatory network of nicotine biosynthesis, but also unraveled the theoretical basis of the <jats:italic>nic1</jats:italic>-locus mutation in low nicotine mutant. These functional verified <jats:italic>NIC1</jats:italic>-locus <jats:italic>ERF</jats:italic> genes can be further used as potential target(s) for ethyl methanesulfonate-based mutagenesis to manipulate nicotine level in tobacco variety in tobacco breeding program.</jats:p></jats:sec>

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).…”