Abstract
Nicotine is the predominant alkaloid in tobacco plants, accounting for ~90% of their total alkaloid content. It is the main addictive substance in cigarettes. Reducing nicotine content in tobacco leaves will aid the development of low-nicotine tobacco products. Prior work has shown that the manipulation of genes involved in nicotine biosynthesis can achieve this purpose (Hidalgo Martinez et al., 2020). Here, we focussed on the long-sought major regulator of nicotine biosynthesis, NIC1 (A). The NIC1 gene, together with a minor locus NIC2 (B), have been identified through genetic analysis of a low-nicotine trait originating from natural mutants of cigar tobacco (Legg and Collins, 1971). Introgression of the low-nicotine trait into Burley 21 (B21) generated near-isogenic lines with different alkaloid levels: high alkaloid (HA, AABB), high intermediate (HI, AAbb), low intermediate (LI, aaBB) and low alkaloid (LA, aabb) (Legg and Collins, 1971). The genes coding for nicotine biosynthetic enzymes, such as the rate-limiting PMT and QPT, are downregulated in LA, suggesting that NIC genes are transcriptional regulators orchestrating nicotine biosynthesis (Saunders and Bush, 1979). Transcriptome-based cloning of NIC2 revealed that this locus is clustered with transcription factors from the ethylene response factor (ERF) subfamily. Of these NIC2-ERFs, ERF189 is the most effective and directly targets the GC-rich P-box element in promoters of nicotine biosynthetic genes (Shoji and Hashimoto, 2012; Shoji et al., 2010). Suppression of NIC2-ERFs reduced nicotine content in tobacco, but a significant amount of nicotine remained due to the major NIC1 locus (Kajikawa et al., 2017). To isolate the NIC1 gene, we conducted map-based cloning using 600 field-growing F2s derived from a cross between HI and LA. The segregating population was first genotyped with a custom tobacco 30K Infinium iSelect HD BeadChip. Additional markers were designed through SNP identification based on RNA-seq of the B21 NILs. NIC1 congregated with SNP4 and was flanked by SNP3 and SNP5 on chromosome 7 (Figure 1a). The delimited NIC1 region was bordered by K326 scaffolds Nitab4.5_0003553 and Nitab4.5_0007027. Reciprocal BLAST comparisons were conducted between K326 and TN90 to fill gaps. Gene annotation identified at least seven full-length single-exon ERFs (JRE5L2, ERF199, ERF91, ERF210, ERF29, ERF16 and ERF130) and two truncated ERFs (ERF110 and ERF17L3ΔN) in this region (Figure 1a). BLAST analysis showed that NIC1 and NIC2 regions were syntenic and originated, respectively, from N. sylvestris (S-genome) and N. tomentosiformis (T-genome). Notably, ERF199 (Nitab4.5_0003090g0030) is homologous to ERF189, sharing an identical binding domain (Figure 1b). Driven by their native promoters, the seven complete NIC1-ERFs were transferred to LA plants using Agrobacterium tumefaciens strain GV3101. Complementation tests showed that transfer of ERF199 (n = 28) significantly increased nicotine levels in potted T0 plants growing in greenhouse. No significant phenotype changes resulted from the remaining six NIC1-ERFs (n > 20) (Figure 1c). The ERF199-mediated nicotine increase was confirmed with T1 plants (n = 35 from 7 T0s) (Figure 1d). We, therefore, concluded that ERF199 is the NIC1 gene. To determine subcellular localization, we fused ERF199 with a red fluorescent protein (RFP) under the CaMV 35S promoter. Constitutive expression of ERF199-RFP in LA significantly increased nicotine content in both T0 (n = 15) and T1 (n = 28) plants (Figure 1e, f). Co-localization of the fusion protein with 4′, 6-diamidino-2-phenylindole (DAPI)-stained nuclei demonstrated that ERF199 is localized in the nucleus, consistent with its role as a transcriptional regulator (Figure 1g). Direct binding of ERF199 to the P box in the PMT2 promoter was verified by electrophoretic mobility shift assay (EMSA). The resulted mobility shift was eliminated by the addition of excess non-labelled probe in the competition experiment, confirming the specificity of this DNA–protein interaction (Figure 1h). Furthermore, a transient gene expression assay using tobacco protoplasts revealed that ERF199 regulates both PMT2 and QPT, the two key enzymes for nicotine biosynthesis (Figure 1i, j). We compared the genomic sequences of EFR199 alleles between HI and LA (~5kb), including 2.3kb upstream of the start codon and 2kb downstream of the stop codon, but no SNPs were detected. Expression analysis indicated that Nic1 was root-specific and significantly downregulated in LA (Figure 1k), suggesting the recessive allele was epigenetically silenced. However, targeted bisulphite sequencing of the same 5kb did not reveal significantly different DNA methylation in CpG, CHG and CHH sites. A thorough epigenome sequencing may provide insight into the underlying epimutations in the NIC1 locus. We predicted that lower nicotine than LA could be attained by eliminating ERF199. Using CRISPR technology, we generated a mutated allele caused by an ‘A’ insertion in HI (Figure 1l) and evaluated its phenotypic effect at T1. The genotypes of wild-type (WT-T1), heterozygous (Het-T1) and homozygous mutant (Mut-T1) plants were determined by DNA sequencing. Total alkaloid levels in WT-T1 plants (n = 22) were comparable with HI plants, and Het-T1s (n = 29) had intermediate levels. However, alkaloid content was barely discernible in Mut-T1 plants (n = 35); approximately 1/10 of that in LA plants (Figure 1m). Thus, manipulation of the NIC1 gene provides a new strategy for nicotine control. Furthermore, the ultra-low-nicotine levels in Mut-T1 confirmed that ERF199 is the only causal gene for nicotine biosynthesis within the NIC1 locus. Transcriptional regulation of secondary metabolite production can be controlled by a single ERF. Indeed, GAME9 locates within an ERF cluster and is the only functional regulator of steroidal glycoalkaloid biosynthesis in tomatoes (Cardenas et al., 2016). Although both ERF189 and ERF199 share the same DNA-binding domain and directly bind to P-box elements within the promoters, ERF199 is more effective. A plausible explanation may be the presence of cofactor/coactivator-recruiting activation domains. Further investigation is required to determine whether a unique activation domain or additional transcriptional cofactors play critical roles in the ERF199-regulatory network. Significant endeavours have been made to attenuate tobacco nicotine content (Hidalgo Martinez et al., 2020). Our study genetically and functionally validated ERF199 as the NIC1 gene. The NIC1 locus, originating from the S-genome, is homologous to the T-genome-donated NIC2 locus. Constitutive expression of ERF199 caused increased nicotine levels, and disruption of ERF199 function in the absence of NIC2 dramatically reduced nicotine accumulation in leaves. Thus, genetic regulation of nicotine levels in tobacco plants can be achieved by manipulating the NIC1 gene. We are thankful to Dr. Sitakanta Pattanaik and Dr. Ling Yuan for helpful discussions and critical comments. This work was supported by British American Tobacco (to SY). The authors declare that there are no conflicts of interest. MH, TG, AF, DL and SY conceived and designed the experiments. All authors performed the experiments. SY wrote the first draft of the manuscript. All authors revised the manuscript. Sequences in this article can be found in GenBank under accession numbers MZ541068 and MZ768806-MZ768811.
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Claims 17
Claim · model-written summary
NIC1 mapped with SNP4 between SNP3 and SNP5 on chromosome 7.
- Taxon
- tobacco
Nicotiana tabacumt0 - Gene
- NIC1e0
- Outcome
- congregated with SNP4 and was flanked by SNP3 and SNP5 on chromosome 7e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“NIC1 congregated with SNP4 and was flanked by SNP3 and SNP5 on chromosome 7”
Claim · model-written summary
The NIC1 region contained seven full length and two truncated ERFs.
- Taxon
- tobaccoNicotiana tabacumt0
- Outcome
- seven full-length single-exon ERFse0
- Value
- at least sevene0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“Gene annotation identified at least seven full-length single-exon ERFs (JRE5L2, ERF199, ERF91, ERF210, ERF29, ERF16 and ERF130) and two truncated ERFs (ERF110 and ERF17L3ΔN) in this region”
Claim · model-written summary
NIC1 and NIC2 regions were syntenic and derived from different ancestral genomes.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- NIC1 and NIC2e0
- Outcome
- syntenice0
- Qualifier
- N. sylvestris (S-genome) and N. tomentosiformis (T-genome)e0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“NIC1 and NIC2 regions were syntenic and originated, respectively, from N. sylvestris (S-genome) and N. tomentosiformis (T-genome)”
Claim · model-written summary
ERF199 was homologous to ERF189 and shared its binding domain.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- ERF199e0
- Outcome
- homologous to ERF189, sharing an identical binding domaine0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“ERF199 (Nitab4.5_0003090g0030) is homologous to ERF189, sharing an identical binding domain”
Claim · model-written summary
Transfer of ERF199 increased nicotine levels in greenhouse T0 plants.
- Taxon
- tobaccoNicotiana tabacumt0
- Treatment
- transfer of ERF199e0
- Gene
- ERF199e0
- Outcome
- nicotine levelse0
- Direction
- increasede0
- Setting
- greenhousee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“transfer of ERF199 (n = 28) significantly increased nicotine levels in potted T0 plants growing in greenhouse”
Claim · model-written summary
The other six NIC1 ERFs produced no significant phenotype changes.
- Taxon
- tobaccoNicotiana tabacumt0
- Treatment
- remaining six NIC1-ERFse0
- Gene
- NIC1-ERFse0
- Outcome
- phenotype changese0
- Direction
- No significante0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“No significant phenotype changes resulted from the remaining six NIC1-ERFs (n > 20)”
Claim · model-written summary
The ERF199 nicotine increase was confirmed in T1 plants.
- Taxon
- tobaccoNicotiana tabacumt0
- Organ / stage
- T1 plantse0
- Gene
- ERF199e0
- Outcome
- nicotine increasee0
- Direction
- increasee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“The ERF199-mediated nicotine increase was confirmed with T1 plants (n = 35 from 7 T0s)”
Claim · model-written summary
ERF199 RFP expression increased nicotine in T0 and T1 plants.
- Taxon
- tobaccoNicotiana tabacumt0
- Material
- LAe0
- Treatment
- Constitutive expression of ERF199-RFPe0
- Gene
- ERF199-RFPe0
- Outcome
- nicotine contente0
- Direction
- increasede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“Constitutive expression of ERF199-RFP in LA significantly increased nicotine content in both T0 (n = 15) and T1 (n = 28) plants”
Claim · model-written summary
ERF199 localized to the nucleus.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- ERF199e0
- Outcome
- localized in the nucleuse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“Co-localization of the fusion protein with 4′, 6-diamidino-2-phenylindole (DAPI)-stained nuclei demonstrated that ERF199 is localized in the nucleus”
Claim · model-written summary
ERF199 bound specifically to the P box in the PMT2 promoter.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- ERF199e0
- Outcome
- Direct binding of ERF199 to the P box in the PMT2 promotere0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“Direct binding of ERF199 to the P box in the PMT2 promoter was verified by electrophoretic mobility shift assay (EMSA). The resulted mobility shift was eliminated by the addition of excess non-labelled probe in the competition experiment, confirming the specificity of this DNA–protein interaction”
Claim · model-written summary
ERF199 regulated both PMT2 and QPT in protoplasts.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- ERF199e0
- Outcome
- regulates both PMT2 and QPTe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“a transient gene expression assay using tobacco protoplasts revealed that ERF199 regulates both PMT2 and QPT”
Claim · model-written summary
No SNPs distinguished HI and LA ERF199 alleles in the compared interval.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- EFR199e0
- Outcome
- no SNPs were detectede0
- Comparator
- HI and LAe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“We compared the genomic sequences of EFR199 alleles between HI and LA (~5kb), including 2.3kb upstream of the start codon and 2kb downstream of the stop codon, but no SNPs were detected”
Claim · model-written summary
Nic1 expression was root specific and reduced in LA.
- Taxon
- tobaccoNicotiana tabacumt0
- Material
- LAe0
- Organ / stage
- roote0
- Gene
- Nic1e0
- Outcome
- Nic1e0
- Direction
- downregulatede0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“Expression analysis indicated that Nic1 was root-specific and significantly downregulated in LA”
Claim · model-written summary
Targeted sequencing found no significant methylation differences.
- Taxon
- tobaccoNicotiana tabacumt0
- Outcome
- DNA methylation in CpG, CHG and CHH sitese0
- Direction
- did not reveal significantly differente0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“targeted bisulphite sequencing of the same 5kb did not reveal significantly different DNA methylation in CpG, CHG and CHH sites”
Claim · model-written summary
Heterozygous edited plants had intermediate alkaloid levels.
- Taxon
- tobaccoNicotiana tabacumt0
- Material
- Het-T1se0
- Outcome
- intermediate levelse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“Het-T1s (n = 29) had intermediate levels”
Claim · model-written summary
Homozygous edited plants had barely discernible alkaloids, about a tenth of LA.
- Taxon
- tobaccoNicotiana tabacumt0
- Material
- Mut-T1e0
- Outcome
- alkaloid contente0
- Direction
- barely discerniblee0
- Value
- approximately 1/10e0
- Comparator
- LA plantse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“alkaloid content was barely discernible in Mut-T1 plants (n = 35); approximately 1/10 of that in LA plants”
Claim · model-written summary
The authors identify ERF199 as NIC1 and the only causal gene at this locus.
- Taxon
- tobaccoNicotiana tabacumt0
- Gene
- ERF199e0
- Outcome
- only causal gene for nicotine biosynthesis within the NIC1 locuse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“tobacco plants”
e0“the ultra-low-nicotine levels in Mut-T1 confirmed that ERF199 is the only causal gene for nicotine biosynthesis within the NIC1 locus”
The same questions in other papers
overexpression · molecular: metabolites · Nicotiana tabacum (tobacco)Positive and negative results
8 papers in the same direction as this paper · 3 in the opposite direction
- negative
- negative
- no effect
8 papers more in this group
See all 12 papers →Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.