ELONGATED HYPOCOTYL 5 regulates steroidal glycoalkaloid biosynthesis and fungal tolerance in tomato.

Tomato (Solanum lycopersicum L.) is rich in nutrients and has been an important target for enhancing the accumulation of various metabolites. Tomato also contains cholesterol-derived molecules, steroidal glycoalkaloids (SGAs), which contribute to pathogen defence but are toxic to humans and considered anti-nutritional compounds. Previous studies suggest the role of various transcription factors in SGA biosynthesis; however, the role of light and associated regulatory factors has not been studied in tomato. Here, we demonstrated that SGA biosynthesis is regulated by light through the ELONGATED HYPOCOTYL 5 homolog, SlHY5, by binding to light-responsive G-boxes present in the promoters of structural and regulatory genes. SlHY5 complemented Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) hy5 mutants at molecular, morphological, and biochemical levels. CRISPR/Cas9-based knockout tomato plants, SlHY5CR, showed down-regulation of SGA and phenylpropanoid pathway genes, leading to a significant reduction in SGA (α-tomatine and dehydrotomatine) and flavonol contents, whereas plants overexpressing SlHY5 (SlHY5OX) showed the opposite effect. Enhanced SGA and flavonol levels in SlHY5OX lines provided tolerance against Alternaria solani fungus, while SlHY5CR lines were susceptible to the pathogen. This study advances our understanding of the HY5-dependent light-regulated biosynthesis of SGAs and flavonoids and their role in biotic stress in tomatoes.

Vascular tissue - boon or bane? How pathogens usurp long-distance transport in plants and the defence mechanisms deployed to counteract them.

Evolutionary emergence of specialised vascular tissues has enabled plants to coordinate their growth and adjust to unfavourable external conditions. Whilst holding a pivotal role in long-distance transport, both xylem and phloem can be encroached on by various biotic factors for systemic invasion and hijacking of nutrients. Therefore, a complete understanding of the strategies deployed by plants against such pathogens to restrict their entry and establishment within plant tissues, is of key importance for the future development of disease-tolerant crops. In this review, we aim to describe how microorganisms exploit the plant vascular system as a route for gaining access and control of different host tissues and metabolic pathways. Highlighting several biological examples, we detail the wide range of host responses triggered to prevent or hinder vascular colonisation and effectively minimise damage upon biotic invasions.

Novel oceanic cyanobacterium isolated from Bangaram island with profound acid neutralizing ability is proposed as Leptolyngbya iicbica sp. nov. strain LK.

An acid-neutralizing, filamentous, non-heterocytous, marine cyanobacterium named 'LK' has been isolated from the seashore of Bangaram Island, an atoll of Lakshadweep, India, and is described here as a novel species. LK has been characterized using morphological, ecological, and genomic features. Based on 16S rRNA, whole-genome sequencing, and marker gene-based analysis, LK has been identified as a new species. LK clustered with Leptolyngbya-like strains belonging to the LPP group but diverged from Leptolyngbya sensu stricto, indicating the polyphyletic nature of the Leptolyngbya genus. Leptolyngbya sp. SIOISBB and Halomicronema sp. CCY15110 were identified as LK's two closest phylogenetic neighbors in various phylogenetic studies. The analysis of 16S rRNA, ITS secondary structures, and genome relatedness indices such as AAI, ANI, and gANI strongly support LK as a novel species of the Leptolyngbya genus. The mechanism behind acid neutralization in LK has been delineated, attributing it to a surface phenomenon most likely due to the presence of salts of calcium, magnesium, sodium, and potassium. We name LK as Leptolyngbya iicbica strain LK which is a novel species with prominent acidic pH-neutralizing properties.

Interplay of Light and ABA signaling to modulate plant development.

The exogenous light cues and the phytohormone Abscisic acid (ABA) regulate several aspects of plant growth and development. In recent years, the role of the crosstalk between the light and ABA signaling pathways in regulating different physiological processes has become increasingly evident. This includes the regulation of germination and early seedling development, control of stomatal development and conductance, growth and development of roots, buds, branches, and regulation of flowering. Light and ABA signaling cascades have various convergence points at both DNA and protein levels. The molecular crosstalk involves several light signaling factors like HY5, COP1, PIFs and BBXs that integrate with ABA signaling components like the PYL receptors and ABI5. Especially, ABI5 and PIF4 promoters serve as key "hotspots" for the integration of these two pathways. Plants acquired both light and ABA signaling pathways before they colonized land almost 500 million years ago. In this review, we discuss the recent advances in the interplay of light and ABA signaling regulating plant development and provide an overview of the evolution of these two pathways.

Prevalence, Predictors, and Prognosis of Serious Infections in Takayasu Arteritis: A Cohort Study.

OBJECTIVE: To describe the incidence, risk factors, and outcomes associated with serious infections in patients with Takayasu arteritis (TA). METHODS: Serious infections, defined as infections resulting in hospitalization or death or unusual infections like tuberculosis, were identified from a cohort of patients with TA. Corticosteroid and disease-modifying antirheumatic drug (DMARD) use at the time of serious infection was noted. Demographic characteristics, clinical presentation, angiography, and disease activity at presentation, and the use of DMARDs during follow-up were compared between patients with TA with or without serious infections. Mortality in patients with TA who developed serious infections was compared to those who did not using hazard ratios (HR; with 95% CI). RESULTS: Of 238 patients with TA, 38 (16%) had developed serious infections (50 episodes, multiple episodes in 8; 3 episodes resulted in death). Among the 38 initial episodes, 11/38 occurred in those not on corticosteroids and 14/38 in those not on DMARDs. Pneumonia (n = 19) was the most common infection, followed by tuberculosis (n = 12). Patients with TA who developed serious infections vs those who did not had higher disease activity at presentation (active disease 97.4% vs 69.5%, mean Indian Takayasu Arteritis Activity Score 2010 12.7 (SD 7.3) vs 10.2 (SD 7.0), mean Disease Extent Index in Takayasu Arteritis 11.2 (SD 6.1) vs 8.8 (SD 6.1) and were more frequently initiated on corticosteroids or DMARDs. HRs calculated using exponential parametric regression survival-time model revealed increased mortality rate in patients with TA who developed serious infections (HR 5.52, 95% CI 1.75-17.39). CONCLUSION: Serious infections, which occurred in the absence of immunosuppressive treatment in approximately one-fifth of patients with TA, were associated with increased mortality in patients with TA.

Characterization and complete genome analysis of Klebsiella phage Kp109 with lytic activity against Klebsiella pneumoniae.

Klebsiella pneumonia is a serious pathogen involved in a range of infections. The increasing frequency of infection associated with K. pneumoniae and accelerated development of antimicrobial resistance has limited the available options of antibiotics for the treatment of infection. Bacteriophages are an attractive substitute to alleviate the problem of antibiotic resistance. In this study, isolation, microbiological and genomic characterization of bacteriophage Kp109 having the ability to infect K. pneumoniae has been shown. Phage Kp109 showed good killing efficiency and tolerance to a broad range of temperatures (4-60 °C) and pH (3-9). Transmission electron microscopy and genomic analysis indicated that phage Kp109 belongs to the genus Webervirus and family Drexlerviridae. Genomic analysis showed that the Kp109 has a 51,630 bp long double-stranded DNA genome with a GC content of 51.64%. The absence of known lysogenic, virulence, and antibiotic-resistant genes (ARGs) in its genome makes phage Kp109 safer to be used as a biocontrol agent for different purposes including phage therapy. The computational analysis of the putative endolysin gene revealed a binding energy of - 6.23 kcal/mol between LysKp109 and ligand NAM-NAG showing its potential to be used as an enzybiotic. However, future research is required for experimental validation of the in silico work to further corroborate the results obtained in the present study. Overall, phenotypic, genomic, and computational characterization performed in the present study showed that phages Kp109 and LysKp109 are promising candidates for future in vivo studies and could potentially be used for controlling K. pneumoniae infection.

Comparative genomics of an extensively drug resistant strain Klebsiella pneumoniae IITR008 with international high-risk clonal lineage ST147 isolated from river water.

Carbapenem resistant Klebsiella pneumoniae causing severe infection resulting in morbidity and mortality have become a global health concern. K. pneumoniae with sequence type ST147 is an international high-risk clonal lineage, genomic studies have been done on K. pneumoniae ST147 isolated from clinical origin but genomic data for environmental K. pneumoniae ST147 is very scarce. Herein, K. pneumoniae IITR008, an extensively drug resistant and potentially hypervirulent bacterium, was isolated from Triveni Sangam, the confluence of three rivers where religious congregations are organized. Phenotypic, genomic and comparative genomic analysis of strain IITR008 was performed. Antibiotic susceptibility profiling revealed resistance to 9 different classes of antibiotics including ß-lactams, ß-lactam combination agents, carbapenem, aminoglycoside, macrolide, quinolones, cephams, phenicol, and folate pathway antagonists and was found to be susceptible to only tetracycline. The strain IITR008 possesses hypervirulence genes namely, iutA and iroN in addition to numerous virulence factors coding for adherence, regulation, iron uptake, secretion system and toxin. Both the IITR008 chromosome and plasmid pIITR008_75 possess a plethora of clinically relevant antibiotic-resistant genes (ARGs) including blaCTX-M-15, blaTEM-1, and blaSHV-11, corroborating the phenotypic resistance. Comparative genomic analysis with other ST147 K. pneumoniae provided insights on the phylogenetic clustering of IITR008 with a clinical strain isolated from a patient in Czech with recent travel history in India and other clinical strains isolated from India and Pakistan. According to the 'One Health' perspective, surveillance of antibiotic resistance in the environment is crucial to impede its accelerated development in diverse ecological niches.

Genetic circuits in microbial biosensors for heavy metal detection in soil and water.

With the rapid population growth, the world is witnessing an ever-increasing demand for energy and natural resources. Consequently, soil, air, and water are polluted with diverse pollutants, including heavy metals (HM). The detection of heavy metals is necessary to remediate them, which is achieved with biosensors. Initially, these HM were detected using atomic absorption spectroscopy (AAS), emission spectroscopy, mass spectrometry, gas chromatography etc., but these were costly and time consuming which further paved a way for microbe-based biosensors. The development of genetic circuits for microbe-based biosensors has become more popular in recent years for heavy metal detection. In this review, we have especially discussed the various types of genetic circuits such as toggle switches, logic gates, and amplification modules used in these biosensors as they are used to enhance sensitivity and specificity. Genetic circuits also allow for rapid and multiple analyte detection at the same time. The use of microbial biosensors for the detection of HM in the soil as well as the water is also described below. Although with a higher success rate than classical biosensors, these microbial biosensors still have some drawbacks like bioavailability and size of the analyte which are needed to be addressed.

BBX30/miP1b and BBX31/miP1a form a positive feedback loop with ABI5 to regulate ABA-mediated postgermination seedling growth arrest.

In plants, the switch to autotrophic growth involves germination followed by postgermination seedling establishment. When environmental conditions are not favorable, the stress hormone abscisic acid (ABA) signals plants to postpone seedling establishment by inducing the expression of the transcription factor ABI5. The levels of ABI5 determine the efficiency of the ABA-mediated postgermination developmental growth arrest. The molecular mechanisms regulating the stability and activity of ABI5 during the transition to light are less known. Using genetic, molecular, and biochemical approach, we found that two B-box domain containing proteins BBX31 and BBX30 alongwith ABI5 inhibit postgermination seedling establishment in a partially interdependent manner. BBX31 and BBX30 are also characterized as microProteins miP1a and miP1b, respectively, based on their small size, single domain, and ability to interact with multidomain proteins. miP1a/BBX31 and miP1b/BBX30 physically interact with ABI5 to stabilize it and promote its binding to promoters of downstream genes. ABI5 reciprocally induces the expression of BBX30 and BBX31 by directly binding to their promoter. ABI5 and the two microProteins thereby form a positive feedback loop to promote ABA-mediated developmental arrest of seedlings.

Curcumin nanoformulations for antimicrobial and wound healing purposes.

The development and spread of resistance to antimicrobial drugs is hampering the management of microbial infectious and wound healing processes. Curcumin is the most active and effective constituent of Curcuma longa L., also known as turmeric, and has a very long and strong history of medicinal value for human health and skincare. Curcumin has been proposed as strong antimicrobial potentialities and many attempts have been made to determine its ability to conjointly control bacterial growth and promote wound healing. However, low aqueous solubility, poor tissue absorption and short plasma half-life due its rapid metabolism needs to be solved for made curcumin formulations as suitable treatment for wound healing. New curcumin nanoformulations have been designed to solve the low bioavailability problem of curcumin. Thus, in the present review, the therapeutic applications of curcumin nanoformulations for antimicrobial and wound healing purposes is described.

Light signaling and UV-B-mediated plant growth regulation.

Light plays an important role in plants' growth and development throughout their life cycle. Plants alter their morphological features in response to light cues of varying intensity and quality. Dedicated photoreceptors help plants to perceive light signals of different wavelengths. Activated photoreceptors stimulate the downstream signaling cascades that lead to extensive gene expression changes responsible for physiological and developmental responses. Proteins such as ELONGATED HYPOCOTYL5 (HY5) and CONSTITUTIVELY PHOTOMORPHOGENIC 1 (COP1) act as important factors which modulate light-regulated gene expression, especially during seedling development. These factors function as central regulatory intermediates not only in red, far-red, and blue light pathways but also in the UV-B signaling pathway. UV-B radiation makes up only a minor fraction of sunlight, yet it imparts many positive and negative effects on plant growth. Studies on UV-B perception, signaling, and response in plants has considerably surged in recent times. Plants have developed different strategies to use UV-B as a developmental cue as well as to withstand high doses of UV-B radiation. Plants' responses to UV-B are an integration of its cross-talks with both environmental factors and phytohormones. This review outlines the current developments in light signaling with a major focus on UV-B-mediated plant growth regulation.

Virus-Induced Silencing of Key Genes Leads to Differential Impact on Withanolide Biosynthesis in the Medicinal Plant, Withania somnifera.

Withanolides are a collection of naturally occurring, pharmacologically active, secondary metabolites synthesized in the medicinally important plant, Withania somnifera. These bioactive molecules are C28-steroidal lactone triterpenoids and their synthesis is proposed to take place via the mevalonate (MVA) and 2-C-methyl-d-erythritol-4-phosphate (MEP) pathways through the sterol pathway using 24-methylene cholesterol as substrate flux. Although the phytochemical profiles as well as pharmaceutical activities of Withania extracts have been well studied, limited genomic information and difficult genetic transformation have been a major bottleneck towards understanding the participation of specific genes in withanolide biosynthesis. In this study, we used the Tobacco rattle virus (TRV)-mediated virus-induced gene silencing (VIGS) approach to study the participation of key genes from MVA, MEP and triterpenoid biosynthesis for their involvement in withanolide biosynthesis. TRV-infected W. somnifera plants displayed unique phenotypic characteristics and differential accumulation of total Chl as well as carotenoid content for each silenced gene suggesting a reduction in overall isoprenoid synthesis. Comprehensive expression analysis of putative genes of withanolide biosynthesis revealed transcriptional modulations conferring the presence of complex regulatory mechanisms leading to withanolide biosynthesis. In addition, silencing of genes exhibited modulated total and specific withanolide accumulation at different levels as compared with control plants. Comparative analysis also suggests a major role for the MVA pathway as compared with the MEP pathway in providing substrate flux for withanolide biosynthesis. These results demonstrate that transcriptional regulation of selected Withania genes of the triterpenoid biosynthetic pathway critically affects withanolide biosynthesis, providing new horizons to explore this process further, in planta.

Acute inhalation toxicity study of n-heneicosane and its combination with diflubenzuron: An attracticide of Aedes aegypti.

OBJECTIVE: Combination of an oviposition pheromone and an insect growth regulator for the control of vectors is an effective approach. There is a need for toxicological evaluation before its introduction. The present study evaluates the acute inhalation toxicity of n-heneicosane and its combination with diflubenzuron in a head-only inhalation exposure chamber made of glass. MATERIALS AND METHODS: A head-only inhalation exposure chamber made of glass (volume: 3.5 l) was used for exposing four rats at a time. A glass nebulizer was used for aerosolization of n-heneicosane and its combination with diflubenzuron (1:10 w/w). Nebulization pressure was 10 and 15 psi and the air flow of exposure the chamber was adjusted to 30 lpm. Male Wistar rats were acclimatized in whole body plethysmographs that were connected to volumetric flow pressure transducers by silicon tubes. The transducers were connected to an amplifier and a digitized response was recorded through an oscillograph and personal computers. Respiratory variables were recorded online. After inhalation exposure, various other parameters like survival, body weight, organ body weight index and biochemical changes were recorded for analysis. RESULTS AND DISCUSSION: Particle size determination proved that the aerosol particles were within the respirable range. LC50 of n-heneicosane and its combination with diflubenzuron was found to be more than 5 g/m3. There were minimal changes observed during exposure to n-heneicosane and also its combination with diflubenzuron on the respiratory variables. The changes were not consistent with the dose. CONCLUSION: n-Heneicosane and its combination with diflubenzuron showed low mammalian toxicity.

Comprehensive assessment of the genes involved in withanolide biosynthesis from Withania somnifera: chemotype-specific and elicitor-responsive expression.

Withania somnifera (L.) Dunal (Family, Solanaceae), is among the most valuable medicinal plants used in Ayurveda owing to its rich reservoir of pharmaceutically active secondary metabolites known as withanolides. Withanolides are C28-steroidal lactones having a triterpenoidal metabolic origin synthesised via mevalonate (MVA) pathway and methyl-D-erythritol-4-phosphate (MEP) pathway involving metabolic intermediacy of 24-methylene (C30-terpenoid) cholesterol. Phytochemical studies suggest differences in the content and/or nature of withanolides in different tissues of different chemotypes. Though development of genomic resources has provided information about putative genes encoding enzymes for biosynthesis of intermediate steps of terpenoid backbone, not much is known about their regulation and response to elicitation. In this study, we generated detailed molecular information about genes catalysing key regulatory steps of withanolide biosynthetic pathway. The full-length sequences of genes encoding enzymes for intermediate steps of terpenoid backbone biosynthesis and their paralogs have been characterized for their functional and structural properties as well as phylogeny using bioinformatics approach. The expression analysis suggests that these genes are differentially expressed in different tissues (with maximal expression in young leaf), chemotypes and in response to salicylic acid (SA) and methyl jasmonate (MJ) treatments. Sub-cellular localization studies suggest that both paralogs of sterol ∆-7 reductase (WsDWF5-1 and WsDWF5-2) are localized in the endoplasmic reticulum (ER) thus supporting their indispensible role in withanolide biosynthesis. Comprehensive information developed, in this study, will lead to elucidation of chemotype- as well as tissue-specific withanolide biosynthesis and development of new tools for functional genomics in this important medicinal plant.

Recent advances in plant translational genomics for crop improvement.

The growing population, climate change, and limited agricultural resources put enormous pressure on agricultural systems. A plateau in crop yields is occurring and extreme weather events and urbanization threaten the livelihood of farmers. It is imperative that immediate attention is paid to addressing the increasing food demand, ensuring resilience against emerging threats, and meeting the demand for more nutritious, safer food. Under uncertain conditions, it is essential to expand genetic diversity and discover novel crop varieties or variations to develop higher and more stable yields. Genomics plays a significant role in developing abundant and nutrient-dense food crops. An alternative to traditional breeding approach, translational genomics is able to improve breeding programs in a more efficient and precise manner by translating genomic concepts into practical tools. Crop breeding based on genomics offers potential solutions to overcome the limitations of conventional breeding methods, including improved crop varieties that provide more nutritional value and are protected from biotic and abiotic stresses. Genetic markers, such as SNPs and ESTs, contribute to the discovery of QTLs controlling agronomic traits and stress tolerance. In order to meet the growing demand for food, there is a need to incorporate QTLs into breeding programs using marker-assisted selection/breeding and transgenic technologies. This chapter primarily focuses on the recent advances that are made in translational genomics for crop improvement and various omics techniques including transcriptomics, metagenomics, pangenomics, single cell omics etc. Numerous genome editing techniques including CRISPR Cas technology and their applications in crop improvement had been discussed.

Pattern recognition receptors as potential therapeutic targets for developing immunological engineered plants.

There is an urgent need to combat pathogen infestations in crop plants to ensure food security worldwide. To counter this, plants have developed innate immunity mediated by Pattern Recognition Receptors (PRRs) that recognize pathogen-associated molecular patterns (PAMPs) and damage- associated molecular patterns (DAMPs). PRRs activate Pattern-Triggered Immunity (PTI), a defence mechanism involving intricate cell-surface and intracellular receptors. The diverse ligand-binding ectodomains of PRRs, including leucine-rich repeats (LRRs) and lectin domains, facilitate the recognition of MAMPs and DAMPs. Pathogen resistance is mediated by a variety of PTI responses, including membrane depolarization, ROS production, and the induction of defence genes. An integral part of intracellular immunity is the Nucleotide-binding Oligomerization Domain, Leucine-rich Repeat proteins (NLRs) which recognize and respond to effectors in a potent manner. Enhanced understanding of PRRs, their ligands, and downstream signalling pathways has contributed to the identification of potential targets for genetically modified plants. By transferring PRRs across plant species, it is possible to create broad-spectrum resistance, potentially offering innovative solutions for plant protection and global food security. The purpose of this chapter is to provide an update on PRRs involved in disease resistance, clarify the mechanisms by which PRRs recognize ligands to form active receptor complexes and present various applications of PRRs and PTI in disease resistance management for plants.

HY5 and COP1 function antagonistically in the regulation of nicotine biosynthesis in Nicotiana tabacum.

Nicotine constitutes approximately 90% of the total alkaloid content in leaves within the Nicotiana species, rendering it the most prevalent alkaloid. While the majority of genes responsible for nicotine biosynthesis express in root tissue, the influence of light on this process through shoot-to-root mobile ELONGATED HYPOCOTYL 5 (HY5) has been recognized. CONSTITUTIVE PHOTOMORPHOGENIC1 (COP1), a key regulator of light-associated responses, known for its role in modulating HY5 accumulation, remains largely unexplored in its relationship to light-dependent nicotine accumulation. Here, we identified NtCOP1, a COP1 homolog in Nicotiana tabacum, and demonstrated its ability to complement the cop1-4 mutant in Arabidopsis thaliana at molecular, morphological, and biochemical levels. Through the development of NtCOP1 overexpression (NtCOP1OX) plants, we observed a significant reduction in nicotine and flavonol content, inversely correlated with the down-regulation of nicotine and phenylpropanoid pathway. Conversely, CRISPR/Cas9-based knockout mutant plants (NtCOP1CR) exhibited an increase in nicotine levels. Further investigations, including yeast-two hybrid assays, grafting experiments, and Western blot analyses, revealed that NtCOP1 modulates nicotine biosynthesis by targeting NtHY5, thereby impeding its transport from shoot-to-root. We conclude that the interplay between HY5 and COP1 functions antagonistically in the light-dependent regulation of nicotine biosynthesis in tobacco.

Advances in regulatory mechanism(s) and biotechnological approaches to modulate nicotine content in tobacco.

More than 8 million deaths are caused by tobacco-related diseases every year. A staggering 1.2 million of those fatalities occur due to second-hand smoke exposure among non-smokers, but more than 7 million are due to direct tobacco use among smokers. Nicotine acts as the key ingredient triggering the addiction. The United States Food and Drug Administration (FDA) has classified more than 90 chemical components of tobacco and related smoke as hazardous or potentially hazardous leading to cancer, cardiovascular, respiratory, and reproductive disorders. Hence, reducing nicotine content has been the foremost objective to reduce health and death risks. Therefore, various biotechnological approaches for developing tobacco varieties with low nicotine concentrations are urgently required for the welfare of humankind. In recent years, numerous advancements have been made in nicotine-based tobacco research, suggesting regulatory components involved in nicotine biosynthesis and developing nicotine-less tobacco varieties through biotechnological approaches. This review highlights the various regulatory components and major approaches used to modulate nicotine content in tobacco cultivars.

Mutation in shoot-to-root mobile transcription factor, ELONGATED HYPOCOTYL 5, leads to low nicotine levels in tobacco.

Tobacco remains one of the most commercially important crops due to the parasympathomimetic alkaloid nicotine used in cigarettes. Most genes involved in nicotine biosynthesis are expressed in root tissues; however, their light-dependent regulation has not been studied. Here, we identified the ELONGATED HYPOCOTYL 5 homolog, NtHY5, from Nicotiana tabacum and demonstrated that NtHY5 could complement the Arabidopsis thaliana hy5 mutant at molecular, morphological and biochemical levels. We report the development of CRISPR/Cas9-based knockout mutant plants of tobacco, NtHY5CR, and show down-regulation of the nicotine and phenylpropanoid pathway genes leading to a significant reduction in nicotine and flavonol content, whereas NtHY5 overexpression (NtHY5OX) plants show the opposite effect. Grafting experiments using wild-type, NtHY5CR, and NtHY5OX indicated that NtHY5 moves from shoot-to-root to regulate nicotine biosynthesis in the root tissue. Shoot HY5, directly or through enhancing expression of the root HY5, promotes nicotine biosynthesis by binding to light-responsive G-boxes present in the NtPMT, NtQPT and NtODC promoters. We conclude that the mobility of HY5 from shoot-to-root regulates light-dependent nicotine biosynthesis. The CRISPR/Cas9-based mutants developed, in this study; with low nicotine accumulation in leaves could help people to overcome their nicotine addiction and the risk of death.