The nicotine demethylase CYP82E4 is essential for the formation of red dapples on flue-cured leaves of cherry-red tobacco.

Common flue-cured tobacco (Nicotiana tabacum L.) primarily accumulates nicotine, and its flue-cured leaves exhibit a lemon appearance. In contrast, a spontaneous cherry-red variant (CR60) primarily accumulates nornicotine, accompanied by distinctive red dapples on the cured leaves. In this study, suppression of conversion of nicotine to nornicotine by genome editing resulted in decreased nornicotine and N-acyl nornicotines (NacNNs), and the subsequent disappearance of red dapples in CR60. Conversely, overexpression of CYP82E4 increased nornicotine and NacNNs accumulation, inducing a red dapple phenotype in common tobacco. Notably, nicotine conversion triggered significant alterations in leaf total sugars, alkaloids, and nitrogens. Metabolome analyses using 1352 identified compounds indicated nicotine conversion dramatically affected the entire metabolic network and induced unique metabolic responses across diverse genetic backgrounds. Further WGCNA analysis revealed that nicotine conversion caused substantial contents variation of alkaloids, flavonoids and amino acids and derivatives in cured leaves. Overall, this research provides valuable insights into the mechanisms underlying red dapple formation in cherry-red tobacco, elucidating profound influence of nicotine conversion on entire metabolic network.

Comparative transcriptome analysis reveals nicotine metabolism is a critical component for enhancing stress response intensity of innate immunity system in tobacco.

The pyridine alkaloid nicotine acts as one of best-studied plant resistant traits in tobacco. Previous research has shown that NtERF199 and NtERF189, acting as master regulators within the NIC1 and NIC2 locus, quantitatively contribute to nicotine accumulation levels in N. tabacum. Genome editing-created Nic1(Nterf199) and Nic2 (Nterf189) double mutant provides an ideal platform for precisely dissecting the defensive role of nicotine and the connection between the nicotine biosynthetic pathway with other putative metabolic networks. Taking this advantage, we performed a comparative transcriptomic analysis to reevaluate the potential physiological and metabolic changes in response to nicotine synthesis defect by comparing the nic1nic2 and NIC1NIC2 plants. Our findings revealed that nicotine reduction could systematically diminishes the expression intensities of genes associated with stimulus perception, signal transduction and regulation, as well as secondary metabolic flux. Consequently, this global expression reduction might compromise tobacco adaptions to environmental fitness, herbivore resistances, and plant growth and development. The up-regulation of a novel set of stress-responsive and metabolic pathway genes might signify a newly established metabolic reprogramming to tradeoff the detrimental effect of nicotine loss. These results offer additional compelling evidence regarding nicotine's critical defensive role in nature and highlights the tight link between nicotine biosynthesis and gene expression levels of quantitative resistance-related genes for better environmental adaptation.

Establishment and application of Agrobacterium-delivered CRISPR/Cas9 system for wild tobacco (Nicotiana alata) genome editing.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) system has been widely applied in cultivated crops, but limited in their wild relatives. Nicotiana alata is a typical wild species of genus Nicotiana that is globally distributed as a horticultural plant and well-studied as a self-incompatibility model. It also has valuable genes for disease resistance and ornamental traits. However, it lacks an efficient genetic transformation and genome editing system, which hampers its gene function and breeding research. In this study, we developed an optimized hypocotyl-mediated transformation method for CRISPR-Cas9 delivery. The genetic transformation efficiency was significantly improved from approximately 1% to over 80%. We also applied the CRISPR-Cas9 system to target the phytoene desaturase (NalaPDS) gene in N. alata and obtained edited plants with PDS mutations with over 50% editing efficiency. To generate self-compatible N. alata lines, a polycistronic tRNA-gRNA (PTG) strategy was used to target exonic regions of allelic S-RNase genes and generate targeted knockouts simultaneously. We demonstrated that our system is feasible, stable, and high-efficiency for N. alata genome editing. Our study provides a powerful tool for basic research and genetic improvement of N. alata and an example for other wild tobacco species.

CRISPR/Cas9-mediated seamless gene replacement in protoplasts expands the resistance spectrum to TMV-U1 strain in regenerated Nicotiana tabacum.

CRISPR/Cas-based genome editing is now extensively used in plant breeding and continues to evolve. Most CRISPR/Cas current applications in plants focus on gene knock-outs; however, there is a pressing need for new methods to achieve more efficient delivery of CRISPR components and gene knock-ins to improve agronomic traits of crop cultivars. We report here a genome editing system that combines the advantages of protoplast technologies with recent CRISPR/Cas advances to achieve seamless large fragment insertions in the model Solanaceae plant Nicotiana tabacum. With this system, two resistance-related regions of the N' gene were replaced with homologous fragments from the N'alata gene to confer TMV-U1 resistance in the T0 generation of GMO-free plants. Our study establishes a reliable genome-editing tool for efficient gene modifications and provides a detailed description of the optimization process to assist other researchers adapt this system for their needs.

Partial desensitization of MYC2 transcription factor alters the interaction with jasmonate signaling components and affects specialized metabolism.

The activity of bHLH transcription factor MYC2, a key regulator in jasmonate signaling and plant specialized metabolism, is sensitive to repression by JASMONATE-ZIM-domain (JAZ) proteins and co-activation by the mediator subunit MED25. The substitution of a conserved aspartic acid (D) to asparagine (N) in the JAZ-interacting domain (JID) of Arabidopsis MYC2 affects interaction with JAZ, although the mechanism remained unclear. The effects of the conserved residue MYC2D128 on interaction with MED25 have not been investigated. Using tobacco as a model, we generated all possible substitutions of aspartic acid 128 (D128) in NtMYC2a. NtMYC2aD128N partially desensitized the repression by JAZ proteins, while strongly interacting with MED25, resulting in increased expression of nicotine pathway genes and nicotine accumulation in tobacco hairy roots overexpressing NtMYC2aD128N compared to those overexpressing NtMYC2a. The proline substitution, NtMYC2aD128P, negatively affected transactivation and abolished the interaction with JAZ proteins and MED25. Structural modeling and simulation suggest that the overall stability of the JID binding pocket is a predominant cause for the observed effects of substitutions at D128. The D128N substitution has an overall stabilizing effect on the binding pocket, which is destabilized by D128P. Our study offers an innovative tool to increase the production of plant natural products.

Ethylene inhibits ABA-induced stomatal closure via regulating NtMYB184-mediated flavonol biosynthesis in tobacco.

Stomatal movement can be regulated by ABA signaling through synthesis of reactive oxygen species (ROS) in guard cells. By contrast, ethylene triggers the biosynthesis of antioxidant flavonols to suppress ROS accumulation and prevent ABA-induced stomatal closure; however, the underlying mechanism remains largely unknown. In this study, we isolated and characterized the tobacco (Nicotiana tabacum) R2R3-MYB transcription factor NtMYB184, which belongs to the flavonol-specific SG7 subgroup. RNAi suppression and CRISPR/Cas9 mutation (myb184) of NtMYB184 in tobacco caused down-regulation of flavonol biosynthetic genes and decreased the concentration of flavonols in the leaves. Yeast one-hybrid assays, transactivation assays, EMSAs, and ChIP-qPCR demonstrated that NtMYB184 specifically binds to the promoters of flavonol biosynthetic genes via MYBPLANT motifs. NtMYB184 regulated flavonol biosynthesis in guard cells to modulate ROS homeostasis and stomatal aperture. ABA-induced ROS production was accompanied by the suppression of NtMYB184 and flavonol biosynthesis, which may accelerate ABA-induced stomatal closure. Furthermore, ethylene stimulated NtMYB184 expression and flavonol biosynthesis to suppress ROS accumulation and curb ABA-induced stomatal closure. In myb184, however, neither the flavonol and ROS concentrations nor the stomatal aperture varied between the ABA and ABA+ethylene treatments, indicating that NtMYB184 was indispensable for the antagonism between ethylene and ABA via regulating flavonol and ROS concentrations in the guard cells.

Structure Elucidation and Quantitation of 11 N'-n-Acylnornicotines in Cherry-Red Tobacco.

Eleven consecutive N'-n-acylnornicotines from cherry-red tobacco were structurally elucidated and quantitively analyzed using chromatography and mass spectrometry. All of these N'-n-acylnornicotines are first reported in cherry-red tobacco, whereas N'-propionylnornicotine, N'-n-valerylnornicotine, N'-n-nonanoylnornicotine and N'-n-undecanoylnornicotine are first reported in natural products. The concentration distribution of the identified N'-n-acylnornicotines was studied and it was found that N'-n-octanoylnornicotine and N'-n-hexanoylnornicotine showed the highest concentration, accounting for 94% of the detected N'-n-acylnornicotines. The cherry-red color density of the related tobacco leaves was found to be positively correlated with the concentration of the N'-n-acylnornicotines, whereas the ultraviolet-visible spectra of the N'-n-acylnornicotines showed no absorption larger than 300 nm, indicating the discovered compounds are not responsible for the cherry-red color appearance.

Identification and Characterization of Transcription Factors Regulating Terpenoid Indole Alkaloid Biosynthesis in Catharanthus roseus.

Biosynthesis of the therapeutically valuable terpenoid indole alkaloids (TIAs), in the medicinal plant Catharanthus roseus, is one of the most elaborate and complex metabolic processes. Although genomic and transcriptomic resources have significantly accelerated gene discovery in the TIA pathway, relatively few genes of transcription factors (TFs) have been identified and characterized thus far. Systematic identification of TFs and elucidation of their functions are crucial for understanding TIA pathway regulation. The successful discovery of TFs in the TIA pathway has relied mostly on three different approaches, (1) identification of cis-regulatory motifs (CRMs) present in the pathway gene promoters as they often provide clues on potential TFs that bind to the promoters, (2) co-expression analysis, based on the assumption that TFs regulating a metabolic or developmental pathway exhibit similar spatiotemporal expression as the pathway genes, and (3) isolation of homologs of TFs known to regulate structurally similar or diverse specialized metabolites in different plant species. TFs regulating TIA pathway have been isolated using either an individual or a combination of the three approaches. Here we describe transcriptome-based coexpression analysis and cis-element determination to identify TFs in C. roseus. In addition, we describe the protocols for generation of transgenic hairy roots, Agrobacterium infiltration of flowers, and electrophoretic mobility shift assay (EMSA). The methods described here are useful for the identification and characterization of potential TFs involved in the regulation of special metabolism in other medicinal plants.

Physiological and Molecular Changes in Cherry Red Tobacco in Response to Iron Deficiency Stress.

The genotype CR60 is a spontaneous Cherry Red variant (containing granular red dapples on flue-cured leaves) of the Yunyan 87 (Y87) tobacco; it accumulates higher concentration of iron (Fe) in leaves than Y87, but the physiological differences between them remain largely unknown. We investigated the physiological and molecular mechanisms of CR60 in response to Fe deficiency under hydroponic conditions. Our results showed no significant phenotypic difference between Y87 and CR60 at optimal (40 µM) and high Fe (160 and 320 µM) concentrations. By contrast, CR60 exhibited higher tolerance to Fe deficiency (0 µM) than Y87, as shown by higher concentrations of chlorophyll in CR60 leaves after 21-day Fe-deficiency stress. Transcriptome profiling coupled with RT-PCR analyses found that the expression of IRT1 and several genes associated with chlorophyll biosynthesis and photosynthesis (e.g., PRO, GSA, FD1, PsbO, and PC) was higher in CR60 than Y87. These results indicated that CR60 maintains sufficient Fe uptake, chlorophyll biosynthesis and photosynthetic rate when subjected to Fe starvation.

NtMYB305a binds to the jasmonate-responsive GAG region of NtPMT1a promoter to regulate nicotine biosynthesis.

MYB transcription factors play essential roles in regulating plant secondary metabolism and jasmonate (JA) signaling. Putrescine N-methyltransferase is a key JA-regulated step in the biosynthesis of nicotine, an alkaloidal compound highly accumulated in Nicotiana spp. Here we report the identification of NtMYB305a in tobacco (Nicotiana tabacum) as a regulatory component of nicotine biosynthesis and demonstrate that it binds to the JA-responsive GAG region, which comprises a G-box, an AT-rich motif, and a GCC-box-like element, in the NtPMT1a promoter. Yeast one-hybrid analysis, electrophoretic mobility shift assay and chromatin immunoprecipitation assays showed that NtMYB305a binds to the GAG region in vitro and in vivo. Binding specifically occurs at the ∼30-bp AT-rich motif in a G/C-base-independent manner, thus defining the AT-rich motif as previously unknown MYB-binding element. NtMYB305a localized in the nucleus of tobacco cells where it is capable of activating the expression of a 4×GAG-driven GUS reporter in an AT-rich motif-dependent manner. NtMYB305a positively regulates nicotine biosynthesis and the expression of NtPMT and other nicotine pathway genes. NtMYB305a acts synergistically with NtMYC2a to regulate nicotine biosynthesis, but no interaction between these two proteins was detected. This identification of NtMYB305a provides insights into the regulation of nicotine biosynthesis and extends the roles played by MYB transcription factors in plant secondary metabolism.

Transcriptomic analysis provides insights into the AUXIN RESPONSE FACTOR 6-mediated repression of nicotine biosynthesis in tobacco (Nicotiana tabacum L.).

KEY MESSAGE: NtARF6 overexpression represses nicotine biosynthesis in tobacco. Transcriptome analysis suggests that NtARF6 acts as a regulatory hub that connect different phytohormone signaling pathways to antagonize the jasmonic acid-induced nicotine biosynthesis. Plant specialized metabolic pathways are regulated by a plethora of molecular regulators that form complex networks. In Nicotiana tabacum, nicotine biosynthesis is regulated by transcriptional activators, such as NtMYC2 and the NIC2-locus ERFs. However, the underlying molecular mechanism of the regulatory feedback is largely unknown. Previous research has shown that NbARF1, a nicotine synthesis repressor, reduces nicotine accumulation in N. benthamiana. In this study, we demonstrated that overexpression of NtARF6, an ortholog of NbARF1, was able to reduce pyridine alkaloid accumulation in tobacco. We found that NtARF6 could not directly repress the transcriptional activities of the key nicotine pathway structural gene promoters. Transcriptomic analysis suggested that this NtARF6-induced deactivation of alkaloid biosynthesis might be achieved by the antagonistic effect between jasmonic acid (JA) and other plant hormone signaling pathways, such as ethylene (ETH), salicylic acid (SA), abscisic acid (ABA). The repression of JA biosynthesis is accompanied by the induction of ETH, ABA, and SA signaling and pathogenic infection defensive responses, resulting in counteracting JA-induced metabolic reprogramming and decreasing the expression of nicotine biosynthetic genes in vivo. This study provides transcriptomic evidence for the regulatory mechanism of the NtARF6-mediated repression of alkaloid biosynthesis and indicates that this ARF transcription factor might act as a regulatory hub to connect different hormone signaling pathways in tobacco.

R2R3-MYB Transcription Factor NtMYB330 Regulates Proanthocyanidin Biosynthesis and Seed Germination in Tobacco (Nicotiana tabacum L.).

Proanthocyanidins (PAs) are important phenolic compounds and PA biosynthesis is regulated by a ternary MBW complex consisting of a R2R3-MYB regulator, a bHLH factor and a WDR protein. In this study, a tobacco R2R3-MYB factor NtMYB330 was characterized as the PA-specific regulator in which the PA biosynthesis was promoted in the flowers of NtMYB330-overexpressing lines while decreased in the flowers of ntmyb330 mutants. NtMYB330 can interact with flavonoid-related bHLH partner NtAn1b and WDR protein NtAn11-1, and the NtMYB330-NtAn1b complex is required to achieve strong transcriptional activation of the PA-related structural genes NtDFR1, NtANS1, NtLAR1 and NtANR1. Our data reveal that NtMYB330 regulates PA biosynthesis in seeds and affects seed germination, in which NtMYB330-overexpressing lines showed higher PA accumulations in seed coats and inhibited germination, while ntmyb330 mutants had reduced seed coat PAs and improved germination. NtMYB330 affects seed germination possibly through two mechanisms: modulating seed coat PAs to affect coat-imposed dormancy. In addition, NtMYB330 regulates the expressions of abscisic acid (ABA) and gibberellin acid (GA) signaling-related genes, affecting ABA-GA crosstalk and seed germination. This study reveals that NtMYB330 specifically regulates PA biosynthesis via formation of the MBW complex in tobacco flowers and affects germination through adjustment of PA concentrations and ABA/GA signaling in tobacco seeds.

Development of a nornicotine-reduced flue-cured tobacco line via EMS mutagenesis of nicotine N-demethylase genes.

Substantial progress had been made in reducing nornicotine accumulation in burley tobacco, as nornicotine is a precursor of the carcinogen N-nitrosonornicotine (NNN). Three members of the CYP82E2 family encoding nicotine N-demethylase (NND) have been reported to be responsible for the majority of nicotine demethylation that forms nornicotine in burley tobacco. We had obtained a nonsense mutant of each NND member in flue-cured tobacco from an ethyl methanesulfonate (EMS)-mutagenized population. In this study, we developed dCAPS markers for each nonsense mutation. Using marker-assisted selection, NND mutants were crossed with each other to generate a triple mutant GP449. In line with previous reports, the triple knockout caused significantly decreased levels of nornicotine and NNN in flue-cured tobacco. With the decreased nornicotine, the nicotine level was expected to accumulate. However, the nicotine level in GP449 was significantly decreased to 72.80% of wild type. Realtime RT-PCR analysis showed that the nicotine reduction was correlated with inhibited expression of nicotine biosynthetic pathway genes. The triple mutant and dCAPS markers can be utilized to develop new flue-cured tobacco varieties with lower levels of nornicotine and NNN.

Ethnobotany and diversity of medicinal plants used by the Buyi in eastern Yunnan, China.

The Buyi are a socio-linguistic group in Yunnan Province of southwest China that have a long history of using medicinal plants as part of their indigenous medical system. Given the limited written documentation of the Buyi indigenous medical system, the objective of this paper is to document the medicinal plants of the Buyi and associated traditional knowledge and transmission. Field research was conducted in four villages in Lubuge Township of Luoping County in Yunnan Province using ethnobotanical methodologies including participatory observation, semi-structured interviews, key informant interviews, and focus group discussions to elicit information on medicinal plants. In total, 120 informants (including 15 key informants who are healers) were interviewed. This study found that a total of 121 medicinal plant species belonging to 64 families are used by the Buyi including by local healers to treat different diseases. Among the medicinal plants recorded in this study, 56 species (46%) have not previously been documented in the scientific literature as having medicinal value, highlighting the pressing need for ethnobotanical documentation in indigenous communities. The most frequently used medicinal part was the leaf (24.9% of documented plants), and the most common preparation method was decoction (62.8% of medicinal). Medicinal plants were mainly used to treat rheumatism (12.4% of plants), trauma and injuries (9.6%). The documented plants are also used for other non-medicinal purposes including food, fodder, fencing, and ornamental. In addition, 35 of the medicinal plants are considered poisonous and are used by local Buyi healers for medicine. The traditional Buyi beliefs and practices associated with the documented medicinal plants likely contributes to their conservation in the environments and around Buyi communities. This study further highlights that ethnomedicinal knowledge of the Buyi is at risk of disappearing due to increased introduction and use of modern medicine in Buyi communities, livelihood changes, rapid modernization, and urbanization. Research, policy, and community programs are urgently needed to conserve the biocultural diversity associated with the Buyi medical system including ethnobotanical knowledge towards supporting both environmental and human wellbeing.

Ethylene response factor NtERF91 positively regulates alkaloid accumulations in tobacco (Nicotiana tabacum L.).

Tobacco alkaloid metabolism is regulated by various transcription factors (TFs). Here, we have characterized a non-NIC2 locus gene, Ethylene Response Factor 91 (ERF91), function in regulation of alkaloid accumulation in tobacco. NtERF91 was preferentially expressed in roots and induced by jasmonic acid. Additionally, NtERF91 was able to in vitro bind to the NtPMT2 and NtQPT2 promoters via directly targeting the GCC-box elements and transactivate NtQPT2 gene expression. Ectopic overexpression of NtERF91 not only increased the expression of most nicotine biosynthetic genes, but also altered alkaloid accumulation profile, resulting in dramatically anatabine accumulation. We conclude that NtERF91 plays an overlapped but distinct role in regulating tobacco alkaloid accumulations.

Cross-family transcription factor interaction between MYC2 and GBFs modulates terpenoid indole alkaloid biosynthesis.

Biosynthesis of medicinally valuable terpenoid indole alkaloids (TIAs) in Catharanthus roseus is regulated by transcriptional activators such as the basic helix-loop-helix factor CrMYC2. However, the transactivation effects are often buffered by repressors, such as the bZIP factors CrGBF1 and CrGBF2, possibly to fine-tune the accumulation of cytotoxic TIAs. Questions remain as to whether and how these factors interact to modulate TIA production. We demonstrated that overexpression of CrMYC2 induces CrGBF expression and results in reduced alkaloid accumulation in C. roseus hairy roots. We found that CrGBF1 and CrGBF2 form homo- and heterodimers to repress the transcriptional activities of key TIA pathway gene promoters. We showed that CrGBFs dimerize with CrMYC2, and CrGBF1 binds to the same cis-elements (T/G-box) as CrMYC2 in the target gene promoters. Our findings suggest that CrGBFs antagonize CrMYC2 transactivation possibly by competitive binding to the T/G-box in the target promoters and/or protein-protein interaction that forms a non-DNA binding complex that prevents CrMYC2 from binding to its target promoters. Homo- and heterodimer formation allows fine-tuning of the amplitude of TIA gene expression. Our findings reveal a previously undescribed regulatory mechanism that governs the TIA pathway genes to balance metabolic flux for TIA production in C. roseus.

A differentially regulated AP2/ERF transcription factor gene cluster acts downstream of a MAP kinase cascade to modulate terpenoid indole alkaloid biosynthesis in Catharanthus roseus.

Catharanthus roseus produces bioactive terpenoid indole alkaloids (TIAs), including the chemotherapeutics, vincristine and vinblastine. Transcriptional regulation of TIA biosynthesis is not fully understood. The jasmonic acid (JA)-responsive AP2/ERF transcription factor (TF), ORCA3, and its regulator, CrMYC2, play key roles in TIA biosynthesis. ORCA3 forms a physical cluster with two uncharacterized AP2/ERFs, ORCA4 and 5. Here, we report that (1) the ORCA gene cluster is differentially regulated; (2) ORCA4, while overlapping functionally with ORCA3, modulates an additional set of TIA genes. Unlike ORCA3, ORCA4 overexpression resulted in dramatic increase of TIA accumulation in C. roseus hairy roots. In addition, CrMYC2 is capable of activating ORCA3 and co-regulating TIA pathway genes concomitantly with ORCA3. The ORCA gene cluster and CrMYC2 act downstream of a MAP kinase cascade that includes a previously uncharacterized MAP kinase kinase, CrMAPKK1. Overexpression of CrMAPKK1 in C. roseus hairy roots upregulated TIA pathways genes and increased TIA accumulation. This work provides detailed characterization of a TF gene cluster and advances our understanding of the transcriptional and post-translational regulatory mechanisms that govern TIA biosynthesis in C. roseus.

Molecular authentication of the ethnomedicinal plant Sabia parviflora and its adulterants by DNA barcoding technique.

Sabia parviflora Wall. ex Roxb. is a traditional herb widely used by Chinese people, especially by the Buyi ethnic group which resides in Guizhou and Yunnan provinces. According to the Chinese Ethnic Pharmacopeia, the species is commonly used for soothing the liver and for the treatment of icteric hepatitis, hemostasis, and inflammation. However, due to the similar morphological characters of Sabia species and higher market demands, there are many substitutes and adulterants of S. parviflora. In this study, the differential identification of 6 Sabia species and 7 adulterants were investigated through DNA sequence analysis of three candidate DNA barcodes (trnH-psbA, rbcL-α, matK). Based on sequence alignments, we concluded that not only the trnH-psbA spacer sequence can distinguish S. parviflora from other Sabia species, but the matK + rbcL-α sequences also can differentiate it from the substitutes and adulterants. The classification tree of all samples based on rbcL-α sequences indicated that the rbcL region can identify samples into a family/genus level. Our results suggest that the three candidate barcodes can be used for the identification of S. parviflora and to distinguish it from common substitutes or adulterants.