The B-type cyclin gene, CycB2, serves as a negative regulator of glandular trichome initiation. Through targeted knockout of NtCycB2 in Nicotiana tabacum cv. K326 using the CRISPR/Cas9 system, we created a variety, HK326, which exhibits significantly increased density and larger glandular heads of long glandular trichomes. Under Cd-stress, HK326 exhibited enhanced Cd tolerance, as demonstrated by a robust root system, strengthened cell membrane stability, and higher photosynthetic parameters. HK326 exhibited enhanced Cd-stress tolerance due to a strong excretion capacity of long glandular trichomes by forming calcium oxalate crystals. Cd mainly accumulated in tobacco shoots rather than remained in roots. Specifically, Cd levels of the HK326 shoot surface were nearly two-fold of those of K326, resulting in less Cd internally in the roots and shoots. Gene expression patterns revealed 11 Cd transporter genes that were upregulated after Cd-stress in shoots, roots, and trichomes. Among them, the NtHMA2 gene encoding heavy metal ATPases and involved in the transport of divalent heavy metal cations was expressed consistently and significantly higher in HK326 than K326, both before and after Cd-stress. NtHMA2 expression was strong in trichomes, moderate in shoots, while weak in roots. The results indicate that NtHMA2 may be involved in Cd excretion from glandular trichomes. Our findings suggest HK326 may be an appropriate candidate plant for Cd-stress tolerance.

Nicotine (Nic) exposed to the environment which comes from tobacco products is the main addictive agent and specific classes of hazardous compound that merit concern. In this study, we have established a fast and reliable method to achieve specific detection of Nic in natural nicotiana tabacum within 30 s through a miniaturized platform based on screen printed gold electrode (SPE). A simple electrochemical pretreatment mean was employed on gold surface that led to the exposure of Au (111) facet and a convenient sample pretreatment method was adopted to realize the extraction of Nic in tobacco. The present electrochemical sensor exhibits an ample range of sensing from 10 μg/g to 200 μg/g, which is able to compliance with tobacco industry testing standards of actual samples. Over 60 sampling points from different origins in China or other countries were performed with direct analysis using this method and satisfactory results have been obtained. The proposed approach was demonstrated to be a very promising platform for significantly improving analytical efficiency in laboratories as well as for monitoring the source reduction control of Nic in the environment.

Tobacco is the most widespread non-food crop in the world. In Brazil, tobacco cultivation is one of the main commodities of the southern region. However, its production is associated with environmental impacts and risks to human health, which have yet to be quantified. This paper uses midpoint and endpoint life cycle assessment (LCA) to analyze the potential environmental damage and human health risk associated with agricultural production of conventional Virginia (CV), organic Virginia (OV), and Burley (BU) tobacco varieties. Organic tobacco production substitutes synthetic fertilizers and pesticides with organic compounds in the cropping stage. The results show that for one ton of dried tobacco, BU, CV, and OV emit 1,610, 1,426, and 1,091 kg CO₂ eq, respectively. For organic production, greater impacts are linked to the land use (LU) impact category. The endpoint results showed that OV production resulted in a higher potential for human and environmental damage than BU and CV. The drying of green OV and CV tobacco requires the burning of firewood, which emits high levels of particulate matter and is associated with human health (HH) damage. Overall, the HH damage category accounts for 68%, 82%, and 78% of the total score points associated with the production of BU, CV, and OV.

Air pollution has been identified as a major cause of environmental and human health damage. O₃ is an oxidative pollutant that causes leaf symptoms in sensitive plants. This study aims to adjust a multilinear model for the monitoring of O₃ in subtropical climatic conditions by associating O₃ concentrations with measurements of morphological leaf traits in tobacco plants and different environmental variables. The plants were distributed into five areas (residential, urban or industrial) in the southern region of Brazil and exposed during 14 periods, of 14 days each, during the years of 2014 and 2015. The environmental variables and leaf traits during the exposure periods were described by mean, median, standard deviation and minimum and maximum values. Spearman correlation and multiple linear regression analyses were applied on data from exposure periods. Leaf injury index, leaf area, leaf dry mass, temperature, relative humidity, global solar radiation and accumulated rainfall were used in the regression analyses to select the best models for predicting O₃ concentrations. Leaf injury characteristically caused by O₃ was verified in all areas and periods of plant exposure. Higher values of leaf injury (24.5% and 27.7%) were registered in the 13th and 12th exposure periods during spring and in areas influenced by urban and industrial clutches. The VPD, temperature, global solar radiation and O₃ were correlated to leaf injury. Environmental variables [leaf area, leaf dry mass, global solar radiation and accumulated rainfall] and primarily the VPD were fundamental to improve the adjustments done in the bioindicator model (R² ≥ 0.73). Our research shows that biomonitoring employing the tobacco “Bel-W3” can be improved by measuring morphological leaf traits and meteorological parameters. Additionally, O₃ fumigation experiment should be performed with biomonitoring as conducted in this study, which are useful in understanding the role of other environmental factors.

Arabidopsis MRPs/ABCCs have been shown to remove various organic and inorganic substrates from the cytosol to other subcellular compartments. Here we first demonstrate that heterologous expression of AtMRP7 in tobacco (Nicotiana tabacum var. Xanthi) modifies cadmium accumulation, distribution and tolerance. Arabidopsis MRP7 was localized both in the tonoplast and in the plasma membrane when expressed in tobacco. Its overexpression increased tobacco Cd-tolerance and resulted in enhanced cadmium concentration in leaf vacuoles, indicating more efficient detoxification by means of vacuolar storage. Heterologous AtMRP7 expression also led to more efficient retention of Cd in roots, suggesting a contribution to the control of cadmium root-to-shoot translocation. The results underscore the use of AtMRP7 in plant genetic engineering to modify the heavy-metal accumulation pattern for a broad range of applications.

Genotoxicity of urban air has been analysed almost exclusively in airborne particulates. We monitored the genotoxic effects of airborne pollutants in the urban air of Perugia (Central Italy). Two plant bioindicators with different genetic endpoints were used: micronuclei in meiotic pollen mother cells using Tradescantia-micronucleus bioassay (Trad-MCN) and DNA damage in nuclei of Nicotiana tabacum leaves using comet assay (Nicotiana-comet). Buds of Tradescantia clone # 4430 and young N. tabacum cv. Xanthi plants were exposed for 24 h at three sites with different pollution levels. One control site (indoor control) was also used. The two bioassays showed different sensitivities toward urban pollutants: Trad-MCN assay was the most sensitive, but DNA damage in N. tabacum showed a better correlation with the pollutant concentrations. In situ biomonitoring of airborne genotoxins using higher plants combined with chemical analysis is thus recommended for characterizing genotoxicity of urban air. Plant bioassays used to explore in situ the correlation between air pollution and genotoxicity.

Ethylenediurea (EDU) is a common research tool for investigating ozone impacts on vegetation, although the role of different application routes (foliar spray vs soil drench) on EDU persistence in the leaves is unknown. We quantified EDU concentrations in leaves of the O3-sensitive Bel-W3 cultivar of tobacco treated with EDU as either foliar spray or soil drench. Foliar EDU concentrations were measured by Q-TOF LC/MS. When EDU was applied as foliar spray, 1 h was enough for reaching a measurable concentration within the leaf. EDU concentration increased over the 21-day period when the leaf was not washed after the application (treatment #1), while it decreased when the leaf was washed after the application (treatment #2). These results suggest that: a) dry deposition of EDU onto the leaf surface was gradually absorbed into the unwashed leaf, although the mechanisms of such uptake were unclear; b) concentration of EDU was decreased quickly (−35%) during the first 24 h from application and more slowly during the following three days (−20%) in the washed leaves. Degradation did not involve enzymatic reactions and was not affected by the presence of ROS. When EDU was applied as soil drench, foliar concentrations increased over time, likely due to adsorption onto soil organic matter and gradual re-solubilization by irrigation water. An analysis of EDU concentration in protoplast and intercellular washing fluid showed that EDU did not enter the cells, but was retained in the apoplast only. Possible implications of EDU in the apoplast and recommendations for EDU application are discussed.

This study intended to determine whether the redox state in plants of Nicotiana tabacum ‘Bel-W3’ fluctuates in response to the environmental factors in a sub-tropical area contaminated by ozone (São Paulo, SE – Brazil) and which environmental factors are related to this fluctuation, discussing their biomonitoring efficiency. We comparatively evaluated the indicators of redox state (ascorbic acid, glutathione, superoxide dismutase, ascorbate peroxidase, and glutathione reductase) and leaf injury in 17 field experiments performed in 2008. The redox state was explained by the combined effects of chronic levels of O₃ and meteorological variables 4–6 days prior to the plant sampling. Moderate leaf injury was observed in most cases. The redox state of tobacco decreases few days after their placement in the sub-tropical environment, causing them to become susceptible to oxidative stress imposed by chronic doses of O₃. Its bioindicator efficiency would not be diminished in such levels of atmospheric contamination.

Among emerging organic contaminants (EOCs), triclosan (TCS) is an antibacterial agent and frequently detected in sludge. In this study, RNA sequencing (RNA-seq) was used to obtain the first transcriptomic profile of tobacco with TCS treatment in comparison with control. The results of transcriptome profiling indicated that salicylic acid (SA) signalling pathway actively participated in the tobacco’s response to TCS treatment. The accumulation of endogenous SA in transgene tobacco lines transformed with a homologous gene of SA binding protein (LcSABP) was significantly enhanced. The resistance of transgenic tobacco lines to TCS was markedly enhanced revealed by morphological and physiological indexes while the total Chl level and Pₙ of transgenic individuals showed about 180% and 250% higher than that of WT on average, and the accumulation of H₂O₂ and O₂⁻ induced by TCS in SABP overexpressing tobacco was 35.3%–37.3% and 53.0%–56.0% lower than that of WT. In order to further explore the mechanism of TCS tolerance in transgenic plants, RNA-seq was then performed to obtain the second transcriptomic profile between wild type and transgenic samples with TCS exposure. The results indicated that differentially expressed genes (DEGs) were most highly enriched in MAPK signalling pathway, amino acid synthesis pathway and plant hormone transduction pathway. Especially, genes encoding key proteins such as cytochrome P450, laccase, peroxidase, glycosyl transferase, glutathione S-transferase and ATP-binding cassette were considered to be related to the increased tolerance ability of transgenic tobacco to the treatment of TCS stress. This research will likely provide novel insights into the molecular mechanism of SA-mediated amelioration of TCS stress on tobacco.