Tobacco Cultivars Research Articles

Full-length transcriptome sequencing and transgenic tobacco revealed the key genes in the chlorogenic acid synthesis pathway of Sambucus chinensis L.

Chlorogenic acid is a key chemical in antioxidation and antisepsis. Sambucus chinensis L. is a herbaceous plant rich in chlorogenic acid and a potential genetic resource for breeding high-chlorogenic acid plants. However, there are few studies on the synthesis pathway of chlorogenic acid in S. chinensis. Our study found chlorogenic acid accumulation in S. chinensis to be organ-specific, higher in leaves and buds but lower in roots, stems and fruits. A total number of 546,844 CCS (Circular Consensus Sequence), including 402,767 full-length non-chimeric (FLNC) and 39 annotated sequences related to the synthesis of chlorogenic acid, was obtained by single-molecule real-time sequencing technology (SMRT). qRT-PCR showed that a number of key genes involved in chlorogenic acid synthesis were differentially expressed in various tissues of S. chinensis. Transgenic tobacco revealed that ectopic expression of the HCT homologous gene HCT-45178 increased the content of chlorogenic acid. Our results should be the first report of full-length transcriptome data of S. chinensis, which help to understand the basis of chlorogenic acid synthesis and provide a novel strategy for breeding tobacco cultivars with higher levels of chlorogenic acid. This article is protected by copyright. All rights reserved.

Comparative Transcriptome Analysis Reveals the Effect of miR156a Overexpression on Mineral Nutrient Homeostasis in Nicotiana tabacum.

Mineral nutrition plays an important role in crop growth, yield and quality. MiR156 is a regulatory hub for growth and development. To date, the understanding of miR156-mediated mineral homeostasis is limited. In this study, we overexpressed Nta-miR156a in the tobacco cultivar TN90 and analyzed the effects of miR156 on mineral element homeostasis in tobacco by comparative transcriptome analysis. The results showed that the overexpression of miR156a caused significant morphological changes in transgenic tobacco. Chlorophyll and three anti-resistance markers, proline, total phenolics, and total flavonoids, were altered due to increased miR156 expression levels. Interestingly, the distribution of Cu, Mn, Zn, and Fe in different tissues of transgenic tobacco was disordered compared with that of the wild type. Comparative transcriptome analysis showed that the overexpression of miR156 resulted in 2656 significantly differentially expressed genes. The expression levels of several metal-transport-related genes, such as NtABC, NtZIP, NtHMA, and NtCAX, were significantly increased or decreased in transgenic tobacco. These results suggest that miR156 plays an essential role in regulating mineral homeostasis. Our study provides a new perspective for the further study of mineral nutrient homeostasis in plants.

NpPP2-B10, an F-Box-Nictaba Gene, Promotes Plant Growth and Resistance to Black Shank Disease Incited by Phytophthora nicotianae in Nicotiana tabacum.

Black shank, a devastating disease affecting tobacco production worldwide, is caused by Phytophthora nicotianae. However, few genes related to Phytophthora resistance have been reported in tobacco. Here, we identified NpPP2-B10, a gene strongly induced by P. nicotianae race 0, with a conserved F-box motif and Nictaba (tobacco lectin) domain, in the highly resistant tobacco species Nicotiana plumbaginifolia. NpPP2-B10 is a typical F-box-Nictaba gene. When it was transferred into the black shank-susceptible tobacco cultivar 'Honghua Dajinyuan', it was found to promote resistance to black shank disease. NpPP2-B10 was induced by salicylic acid, and some resistance-related genes (NtPR1, NtPR2, NtCHN50, and NtPAL) and resistance-related enzymes (catalase and peroxidase) were significantly upregulated in the overexpression lines after infection with P. nicotianae. Furthermore, we showed that NpPP2-B10 actively regulated the tobacco seed germination rate, growth rate, and plant height. The erythrocyte coagulation test of purified NpPP2-B10 protein showed that NpPP2-B10 had plant lectin activity, and the lectin content in the overexpression lines was significantly higher than that in the WT, which could lead to accelerated growth and improved resistance of tobacco. SKP1 is an adaptor protein of the E3 ubiquitin ligase SKP1, Cullin, F-box (SCF) complex. We demonstrated that NpPP2-B10 could interact with the NpSKP1-1A gene in vivo and in vitro through yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC), indicating that NpPP2-B10 likely participates in the plant immune response by mediating the ubiquitin protease pathway. In conclusion, our study provides some important insights concerning NpPP2-B10-mediated regulation of tobacco growth and resistance.

Use of exotic Nicotiana tabacum germplasm for confronting an inverse genetic correlation in flue‐cured tobacco

AbstractInverse genetic correlations between cured leaf yields and alkaloid accumulation in flue‐cured tobacco (Nicotiana tabacum L.) complicate the development of higher yielding cultivars while maintaining alkaloids at commercially acceptable levels. Introgression of genomic regions from exotic source material that positively affect alkaloid levels, but with less of a corresponding negative influence on yielding ability, could enhance long‐term efforts to develop improved cultivars. Germplasm accessions “TI 464” and “TI 959” were previously identified as being potentially useful for this purpose. In this study, the relationships between yield and alkaloid accumulation were investigated in BC1F1 populations derived from crosses with these materials and found to be slightly negative or slightly positive. Derived BC1F2 families were used to calculate heritability estimates for alkaloid accumulation on a single plant basis that were low to intermediate in magnitude. The relationships between alkaloid levels of leaves from individual stalk positions and alkaloid levels averaged over all stalk positions were determined. The third stalk position was found to be the most predictive of average alkaloid levels. Selection for alkaloid levels using a single leaf from this stalk position could increase breeding efficiency. BC4F6 lines carrying low genomic contributions from either TI 464 or TI 959 were generated and evaluated as lines per se and in F1 hybrid combinations with a very high‐yielding inbred line with increased leaf number. Performance of these materials indicated that TI 464 and, to a lesser extent TI 959, may be useful for developing higher yielding tobacco cultivars with acceptable alkaloid levels.

Controlling Tobacco Diseases: An Overview of Black Shank and Fusarium Wilt

Black shank, caused by Phytophthora nicotianae, and Fusarium wilt, caused by the Fusarium oxysporium f. sp. nicotianae, are major diseases affecting tobacco crops globally. Phytophthora nicotianae is primarily found in tropical and subtropical regions and infects tobacco plants by producing zoospores that swim to root tissue and form cysts. Symptoms of black shank include root and crown rot, wilting, leaf chlorosis, stem lesions, and pith necrosis. Management strategies for black shank include cultural practices, chemical treatments, and host resistance. Chemical fungicides, such as metalaxyl or mefenoxam, oxathiapiprolin, and fluopicolide, can be used but there is a risk of fungicide resistance. Fusarium wilt is characterized by yellowing, drying, and death of leaves, leading to the death of the entire plant. Control measures for Fusarium wilt include plant resistance, cleanliness, crop rotation, nutrition, nematode management, and fumigation or biofumigation. The most successful control of Fusarium wilt has been through the use of resistant tobacco cultivars. The impact of rotation crops and resistant plants on the pathogen populations in the soil must be evaluated and soil pH and calcium levels may also impact the disease. The presence of the fungus in a field can last for several years without a vulnerable host. Int. J. Appl. Sci. Biotechnol. Vol 11(1): 1-7.

Comparing cadmium uptake kinetics, xylem translocation, chemical forms, and subcellular distribution of two tobacco (Nicotiana tabacum L.) cultivars

Tobacco (Nicotiana tabacum L.) is a potential phytoremediator that can reduce soil cadmium (Cd) contamination. Pot and hydroponic experiments were conducted to investigate the difference in absorption kinetics, translocation patterns, accumulation capacity, and extraction amounts between two leading tobacco cultivars in China. We studied the chemical forms and subcellular distribution of Cd in the plants to understand the diversity of the detoxification mechanism of the cultivars. The concentration-dependent kinetics of Cd accumulation in leaves, stems, roots, and xylem sap for cultivars Zhongyan 100 (ZY100) and K326, fitted well with the Michaelis-Menten equation. K326 exhibited high biomass, Cd tolerance, Cd translocation, and phytoextraction abilities. The acetic acid, sodium chloride, and water-extractable fractions accounted for > 90% of Cd in all ZY100 tissues but only in K326 roots and stems. Moreover, the acetic acid and NaCl fractions were the predominant storage forms, while the water fraction was the transport form. The ethanol fraction also contributed significantly to Cd storage in K326 leaves. As the Cd treatment increased, more NaCl and water fractions were found in K326 leaves, while only NaCl fractions increased in ZY100 leaves. For subcellular distribution, > 93% Cd proportions were primarily stored in both cultivars' soluble or cell wall fraction. The proportion of Cd in the cell wall fraction of ZY100 roots was less than that of K326, while that proportion in the soluble fraction in ZY100 leaves was higher than in K326 leaves. These findings demonstrate that Cd accumulation patterns, detoxification, and storage strategies differ between the cultivars, providing a deeper understanding of Cd tolerance and accumulation mechanism in tobacco plants. It also guides the screening of germplasm resources or gene modification to improve the Cd phytoextraction efficiency of tobacco.

Rhizosphere Microbial Community and Metabolites of Susceptible and Resistant Tobacco Cultivars to Bacterial Wilt

Soil-borne diseases are closely related to rhizosphere microecosystem. While, plant species and genotypes are important factors affected rhizosphere microecosystem. In this study, the rhizosphere soil microbial community and metabolites of susceptible and resistant tobacco cultivars were investigated. The results showed that there were significant differences in the rhizosphere microbial community and metabolites between susceptible cultivar Yunyan87 and resistant cultivar Fandi3. Furthermore, the rhizosphere soil of Fandi3 showed a higher microbial diversity than that of Yunyan87. The abundance of R. solanacearum was much higher in the rhizosphere soil of Yunyan87 than in the rhizosphere soil of Fandi3, resulting in a higher disease incidence and index. While the abundance of beneficial bacteria in the rhizosphere soil of Fandi3 were higher than that of Yunyan87. Additionally, there were significant differences in metabolites between Yunyan87 and Fandi3 cultivars, and 4-hydroxybenzaldehyde, 3-hydroxy-4-methoxybenzoic acid, vamillic aldehyde, benzoic acid, 4-hydroxybenzyl alcohol, p-hydroxybenzoic acid and phthalic acid were notably high in Yunyan87. Redundancy analysis (RDA) indicated that the rhizosphere microbial community of Fandi3 and Yunyan87 were highly correlated with various environmental factors and metabolites. Overall, susceptible and resistant tobacco cultivars had different impact on rhizosphere microbial community and metabolites. The results expand our understanding of the roles of tobacco cultivars in plant-micro-ecosystem interactions, and provide a basis for the control of tobacco bacterial wilt.

Metabolome of flue-cured tobacco is significantly affected by the presence of leaf stem

BackgroundLeaves of tobacco (Nicotiana tabacum L.) are flue-cured to use as a key industrial supply in various parts of the world. The quality of tobacco leaves is dependent on chemical components and their proportions. Generally, the stem attached to tobacco leaf is detached before curing. However, the leaf stem remains green for an extended period of time (as compared to leaf) during flue-curing. Hence, it is expected to affect the quality of tobacco's final product.ResultsTo understand the impact of the green stem of leaf on the metabolome of flue-cured tobacco, we employed a broad targeted metabolomics approach. We selected two tobacco cultivars (Yun87 and K326) and cultivated them in five geographic locations in China. For flue-curing, leaves were harvested without a stem (L) or with an attached stem (SPL). After metabolome analysis, a total of 1027 metabolites were annotated in these samples. A variable number of metabolites were differentially accumulated between both types of leaves (depending on geographic location or cultivar) representing an influence of environment or genotype. Interestingly, only 68 metabolites were differentially accumulated between L and SPL samples irrespective of the cultivar or geographic location. These differentially accumulated metabolites belonged to major groups of primary and secondary metabolites. We have discussed the importance of identified metabolites in terms of carbon, nitrogen, and polyphenolic metabolism.ConclusionThe present research is the first comprehensive description of several metabolites in tobacco leaves related to the contribution of leaf stem. The current study opens novel prospects for investigating the potential of such metabolites in improving the quality of flue-cured tobacco.

Effect of Soil Temperature on Reproduction of Root-knot Nematodes in Flue-cured Tobacco with Homozygous Rk1 and/or Rk2 Resistance Genes.

Most commercial flue-cured tobacco cultivars contain the Rk1 resistance gene, which provides resistance to races 1 and 3 of Meloidogyne incognita and race 1 of M. arenaria. A number of cultivars now possess a second root-knot resistance gene, Rk2. High soil temperatures have been associated with a breakdown of root-knot resistance genes in a number of crops. Three greenhouse trials were performed from 2014 to 2015 investigate the effect of high soil temperature on the efficacy of Rk1 and/or Rk2 genes in reducing parasitism by a population of M. incognita race 3. Trials were arranged in randomized complete block design in open-top growth chambers set at 25°, 30°, and 35°C. Plants were inoculated with 3,000 eggs and data were collected 35 days post-inoculation. Galling, numbers of egg masses and eggs, and reproductive index were compared across cultivar entries. Nematode reproduction was reduced at 25°C and 30°C on entries possessing Rk1 and Rk1Rk2 compared to the susceptible entry and the entry possessing only Rk2. However, there were often no significant differences in reproduction at 35°C between entries with Rk1 and/or Rk2 compared to the susceptible control, indicating an increase of root-knot nematode parasitism on resistant entries at higher temperatures. Although seasonal differences in nematode reproduction were observed among experiments, relative differences among tobacco genotypes remained generally consistent.

Effects of cadmium on transcription, physiology, and ultrastructure of two tobacco cultivars

Cadmium (Cd) is one of the most toxic heavy metal pollutants worldwide. Tobacco is an important cash crop; however, the accumulation of Cd in its biomass is very high. Cadmium may enter the body of smokers with contaminated tobacco and the surrounding environment via smoke. Therefore, it is important to understand the mechanisms of Cd accumulation and tolerance in tobacco plants, especially in the leaves. In this study, the effects of Cd on the growth, accumulation, and biochemical indices of two tobacco varieties, K326 (Cd resistant) and NC55 (Cd sensitive), were studied through transcriptomic and physiological experiments. Transcriptome and physiological analyses showed differences in the expression of Cd transport and Cd resistance related genes between NC55 and K326 under Cd stress. The root meristem cells of NC55 were more severely damaged. The antioxidant enzyme activity, ABA and ZT content, chlorophyll content, photosynthetic rate, and nitrogen metabolism enzyme activity in K326 leaves were higher than in NC55. These data elucidate the mechanisms of low Cd accumulation and high Cd tolerance in K326 leaves and provide a theoretical basis for cultivating tobacco varieties with low Cd accumulation and high Cd resistance.

Genome-Wide Identification and Expression Analysis of the Phytocyanin Gene Family in Nicotiana tabacum

Phytocyanin (PC) is a class of plant-specific blue copper proteins involved in electron transport, plant growth, development, and stress resistance. However, PC proteins have not been systematically evaluated in tobacco plants. We determined the whole-genome sequences of the PC family in the tobacco cultivar ‘K326.’ The transcriptome data were used to analyze the expression of the NtPC family at different development stages and tissue-specific genes. Real-time fluorescence quantitative analysis was used to analyze the expression of the NtPC gene family under low temperature and methyl jasmonate stress. The tobacco NtPC family contained 110 members and was divided into four subfamilies: early nodulin-like protein (NtENODL), uclacyanin-like protein, stellacyanin1-like protein, and plantacyanin-like protein. According to phylogenetic and structural analyses, the NtPC family could be divided into eight structural types. Fifty-three NtPCs were randomly distributed on 22 of 24 tobacco chromosomes. Collinearity analysis revealed 33 pairs of genes belonging to the NtPC family. Gene ontology analysis showed that the PC genes are components of the plasma membrane and may participate in plasma membrane-related functions. The NtPC family contained numerous elements related to hormonal and abiotic stress responses and was specifically expressed in the tobacco prosperous, maturation, and budding periods. Tissue-specific expression analysis showed that some genes were tissue specific. The expression of NtENODL58 and other genes was significantly induced by low-temperature and methyl jasmonate stress. Thus, the NtPC gene family plays an important role in plant stress response.

Model-based estimation of the leaf area of ozone-indicator tobacco (Nicotiana tabacum L.) plants under ambient ozone conditions

Bel-W3 is an ozone-sensitive tobacco (Nicotiana tabacum L.) cultivar widely used worldwide for ozone biomonitoring. Despite its extensive use, there is no comprehensive predictive model to non-destructively estimate the leaf area using only a common ruler, yet leaf area is a major evaluative trait in plants under ozone stress and of economic value in tobacco plants. In this method, we aimed at developing a predictive model to estimate leaf area using the product between leaf length and leaf width. To this end, we conducted a field experiment with ground-grown Bel-W3 plants treated with different solutions under ambient ozone conditions. The solutions were water, the antiozonant ethylenediurea (EDU; 500 ppm), and the antitranspirant pinolene (Vapor Gard; 1%, 5%, 10%). The chemical treatments were introduced to enhance leaves pool and capture different conditions that can occur in ozone biomonitoring projects.•A simple linear predictive model was developed and validated using data from a previous chamber experiment with small seedlings.•Overestimation of the model led to the integration of data from both experiments and development of another simple linear predictive model.•This integrated model provides improved estimation of leaf area and can be used for representative estimation of the area of Bel-W3 leaves of any sizes.

Control Efficiency of Biochar Loaded with Bacillus subtilis Tpb55 against Tobacco Black Shank

Black shank caused by Phytophthora nicotianae has become a destructive soil-borne disease to different flue-cured tobacco cultivars in Southwest China. The use of biochar amendments for microorganism synergy is a promising effective strategy for P. nicotianae development control. In this study, biochar samples were prepared from tamarisk with different pyrolization temperatures (300–500 °C). The effect of pyrolytic temperatures on the bacteria immobilization efficiency of biochar was investigated. B. subtilis Tpb55 was successfully loaded on different biochars as biocontrol composites. The survival investigation of the inoculum suggested that biochar pyrolized at 300 °C (BC300), with a large pore opening diameter; a greater pore volume exhibited a better Tpb55 immobilization. A pot experiment indicated that Tpb55-loaded BC300 had a more pronounced decrease in the disease severity index of black shank disease and an increase in the soil pH, alkali-hydrolyzable nitrogen, soil-available phosphorus, and available potassium. BC300 inoculated with Tpb55 showed the highest control effect (79.60%) against tobacco black shank in the pot experiments, with the lowest copy number of P. nicotianae DNA. In conclusion, biochar-immobilized Tpb55 may provide a new strategy for preventing and controlling tobacco black shank.

NtbHLH49, a jasmonate-regulated transcription factor, negatively regulates tobacco responses to Phytophthora nicotianae.

Tobacco black shank caused by Phytophthora nicotianae is a devastating disease that causes huge losses to tobacco production across the world. Investigating the regulatory mechanism of tobacco resistance to P. nicotianae is of great importance for tobacco resistance breeding. The jasmonate (JA) signaling pathway plays a pivotal role in modulating plant pathogen resistance, but the mechanism underlying JA-mediated tobacco resistance to P. nicotianae remains largely unclear. This work explored the P. nicotianae responses of common tobacco cultivar TN90 using plants with RNAi-mediated silencing of NtCOI1 (encoding the perception protein of JA signal), and identified genes involved in this process by comparative transcriptome analyses. Interestingly, the majority of the differentially expressed bHLH transcription factor genes, whose homologs are correlated with JA-signaling, encode AtBPE-like regulators and were up-regulated in NtCOI1-RI plants, implying a negative role in regulating tobacco response to P. nicotianae. A subsequent study on NtbHLH49, a member of this group, showed that it's negatively regulated by JA treatment or P. nicotianae infection, and its protein was localized to the nucleus. Furthermore, overexpression of NtbHLH49 decreased tobacco resistance to P. nicotianae, while knockdown of its expression increased the resistance. Manipulation of NtbHLH49 expression also altered the expression of a set of pathogen resistance genes. This study identified a set of genes correlated with JA-mediated tobacco response to P. nicotianae, and revealed the function of AtBPE-like regulator NtbHLH49 in regulating tobacco resistance to this pathogen, providing insights into the JA-mediated tobacco responses to P. nicotianae.

Antivirus isoindolinone alkaloids with rare oxocyclopenta[f]isoindole frameworks isolated from the stems of flue cured tobacco

BackgroundSince Nicotiana tabacum (tobacco) has important significance to humans for their medicinal uses, to find antivirus activities inhibitors from tobacco, increase its medicinal value, and comprehensive utilization of its by-products, our group had investigated the chemical constituents of the stems of Y-202, a cultivar of tobacco which high resistance to tobacco mosaic virus (TMV).ResultsFour new isoindolinone-type alkaloids, nicoisoindoles A–D (1–4), along with four known isoindole derivatives (5–8) were isolated. Compounds 1–4 represent a new subclass of isoindolinone alkaloids with rare cyclopenta[f]isoindole-1-one frameworks. Among them, nicoisoindole C (3) possesses an unusual N-2-(5-methoxy-6-methylpyridin-2-yl) ethyl moiety, while nicoisoindole D (4) has a novel a N-(3-methyl-6-oxo-1,6-dihydropyridin-2-yl)methyl substituent. Interestingly, compounds 1, 3, and 4 showed high anti-TMV activities with inhibition rates of 43.8%, 58.8%, and 67.8% at the concentration of 20 μM, and IC50 values of 23.6, 19.5 and 15.4 μM, respectively, even more potent than that of positive control.ConclusionsThe successful isolation and structure identification of new oxocyclopenta[f]isoindole-1-ones provide materials for the screening of antivirus activities inhibitors, and contribute to the development and utilization of the waste from tobacco cultivation.Graphical

Plant-produced recombinant cytokines IL-37b and IL-38 modulate inflammatory response from stimulated human PBMCs

Affordable therapeutics are vitally needed for humans worldwide. Plant-based production of recombinant proteins can potentially enhance, back-up, or even substitute for the manufacturing capacity of the conventional, fermenter-based technologies. We plastome-engineered a tobacco cultivar to express high levels of two “plantakines” — recombinant human cytokines, interleukins IL-37b and IL-38, and confirmed their native conformation and folding. Assessment of their biological functionality was performed ex vivo by analyzing the effects exerted by the plantakines on levels of 11 cytokines secreted from human peripheral blood mononuclear cells (PBMCs) challenged with an inflammatory agent. Application of the plant-produced IL-37b and IL-38 in PBMCs stimulated with Lipopolysaccharide or Phytohaemagglutinin resulted in significant, and in particular cases—dose-dependent modulation of pro-inflammatory cytokines secretion, showing attenuation in two-thirds of significant level modulations observed. Plantakine treatments that increased inflammatory responses were associated with the higher dosage. Our results demonstrate feasibility of manufacturing functional recombinant human proteins using scalable, cost-effective and eco-friendly plant-based bioreactors.

Transcriptomics and iTRAQ-proteomics analyses provide novel insights into the defense mechanism of black shank disease in tobacco.

Black shank disease caused by Phytophthora nicotianae is one of the most important diseases in tobacco worldwide and can result in a devastating loss in tobacco cultivation. Many efforts have been carried out to identify the chromosome segment from Nicotiana plumbaginifolia containing a resistance locus carrying a gene named Php; however, the Php gene has not been cloned, and knowledge of the potential mechanism of the Php gene in the resistant lines is limited. To further characterize the resistance mechanism of the Php gene, we first used the resistant line "RBST" and the susceptible cultivar "Honghuadajinyuan" (HD) to obtain the near-isogenic line RBS89 containing the Php gene from RBST. RBS89 showed high resistance to black shank disease. Transcriptomic and iTRAQ analyses were applied to explore the potential defense mechanisms in RBS89 plants in comparison with HD plants with or without inoculation. Many differentially expressed genes (DEGs) and proteins were identified, and some pathogenesis-related (PR) proteins were extensively abundant in the RBS89 plants when compared with the HD plants in response to black shank disease. Importantly, overexpression of the PR gene NtPR-1B in HD plants improved the resistance of tobacco plants to black shank disease, indicating that NtPR-1B and Php genes might have similar roles in protecting tobacco from black shank disease. However, the relationship between NtPR-1B and Php genes requires further analysis. Therefore, our study provides valuable information for breeding tobacco cultivars with black shank disease resistance and sheds light on the defense mechanism of black shank disease in tobacco for enhancing Phytophthora resistance in other Solanaceae crops.

Cloning, characterization and functional analysis of NtMYB306a gene reveals its role in wax alkane biosynthesis of tobacco trichomes and stress tolerance.

Trichomes are specialized hair-like organs found on epidermal cells of many terrestrial plants, which protect plant from excessive transpiration and numerous abiotic and biotic stresses. However, the genetic basis and underlying mechanisms are largely unknown in Nicotiana tabacum (common tobacco), an established model system for genetic engineering and plant breeding. In present study, we identified, cloned and characterized an unknown function transcription factor NtMYB306a from tobacco cultivar K326 trichomes. Results obtained from sequence phylogenetic tree analysis showed that NtMYB306a-encoded protein belonged to S1 subgroup of the plants’ R2R3-MYB transcription factors (TFs). Observation of the green fluorescent signals from NtMYB306a-GFP fusion protein construct exhibited that NtMYB306a was localized in nucleus. In yeast transactivation assays, the transformed yeast containing pGBKT7-NtMYB306a construct was able to grow on SD/-Trp-Ade+X-α-gal selection media, signifying that NtMYB306a exhibits transcriptional activation activity. Results from qRT-PCR, in-situ hybridization and GUS staining of transgenic tobacco plants revealed that NtMYB306a is primarily expressed in tobacco trichomes, especially tall glandular trichomes (TGTs) and short glandular trichomes (SGTs). RNA sequencing (RNA-seq) and qRT-PCR analysis of the NtMYB306a-overexpressing transgenic tobacco line revealed that NtMYB306a activated the expression of a set of key target genes which were associated with wax alkane biosynthesis. Gas Chromatography–Mass Spectrometry (GC-MS) exhibited that the total alkane contents and the contents of n-C28, n-C29, n-C31, and ai-C31 alkanes in leaf exudates of NtMYB306a-OE lines (OE-3, OE-13, and OE-20) were significantly greater when compared to WT. Besides, the promoter region of NtMYB306a contained numerous stress-responsive cis-acting elements, and their differential expression towards salicylic acid and cold stress treatments reflected their roles in signal transduction and cold-stress tolerance. Together, these results suggest that NtMYB306a is necessarily a positive regulator of alkane metabolism in tobacco trichomes that does not affect the number and morphology of tobacco trichomes, and that it can be used as a candidate gene for improving stress resistance and the quality of tobacco.

Nitrogen metabolic rate and differential ammonia volatilization regulate resistance against opportunistic fungus Alternaria alternata in tobacco.

Nutritional correlations between plants and pathogens can crucially affect disease severity. As an essential macronutrient, the availability of nitrogen (N) and the types of N content play a fundamental part not only in energy metabolism and protein synthesis but also in pathogenesis. However, a direct connection has not yet been established between differences in the level of resistance and N metabolism. Pertinently, former studies hold ammonia (NH3) accountable for the development of diseases in tobacco (Nicotiana tabacum L.) and in some post-harvest fruits. With a purpose of pinpointing the function of NH3 volatilization on Alternaria alternata (Fries) Keissl pathogenesis and its correlation with both N metabolism and resistance differences to Alternaria alternata infection in tobacco, leaf tissue of two tobacco cultivars with susceptibility (Changbohuang; CBH), or resistance (Jingyehuang; JYH) were analyzed apropos of ammonia compensation point, apoplastic NH4+ concentration, pH value as well as activities of key enzymes and N status. At the leaf age of 40 to 60 d, the susceptible cultivar had a significantly higher foliar apoplastic ammonium (NH4+) concentration, pH value and NH3 volatilization potential compared to the resistant one accompanied by a significant reduction in glutamine synthetase (GS), which in particular was a primary factor causing the NH3 volatilization. The NH4+ concentration in CBH was 1.44 times higher than that in JYH, and CBH had NH3 compensation points that were 7.09, 6.15 and 4.35-fold higher than those of JYH at 40, 50 and 60 d, respectively. Moreover, the glutamate dehydrogenase (GDH) activity had an upward tendency related to an increased NH4+ accumulation in both leaf tissues and apoplast but not with the NH3 compensation point. Collectively, our results strongly suggest that the accumulation of NH3 volatilization, rather than NH4+ and total N, was the primary factor inducing the Alternaria alternata infection in tobacco. Meanwhile, the susceptible cultivar was characterized by a higher N re-transfer ability of NH3 volatilization, in contrast to the disease–resistant cultivar, and had a stronger capability of N assimilation and reutilization. This study provides a deeper understanding of the pathogenicity mechanism induced by Alternaria alternata, which is useful for breeding Alternaria alternata-resistant varieties of tobacco, at the same time, our research is also conducive to control tobacco brown spot caused by Alternaria alternata in the field.

Effects of foliar silicon (Si) applications on tobacco plant under salt stress

Tuzluluk bitkilerde verim ve kaliteyi olumsuz yönde etkileyen en önemli abiyotik stres faktörleri arasında yer almasına karşılık, silisyum (Si) bitkilerde stres faktörlerini azaltan bir element olarak bilinmektedir. Bu çalışmada tütün bitkisinde tuz stresinin neden olduğu hasara karşı yapraktan Si uygulamalarının koruyucu etkisinin araştırılması amaçlanmıştır. Çalışmada dört farklı (0, 1.5, 3, 4.5 ve 6 dS m-1) tuz dozu altında yetiştirilen Canik 190/5 tütün çeşidine yapraktan 1 ve 2 defa Si uygulamaları yapılmıştır. Tüm bitki kuru madde verimi, Na, K, Ca, Mg, P konsantrasyonu ile K/Na oranı incelenmiştir. Artan dozlarda tuz uygulamaları tütün bitkisinin yeşil aksam kuru madde veriminde azalmaya neden olurken, yapraktan Si uygulamaları kuru madde verimlerindeki azalmayı yavaşlatmıştır. Kontrol uygulamalarının ortalama bitki kuru madde verimi 6.42 g bitki-1 iken, yapraktan 1 defa Si uygulamasında bu değer 6.65 g bitki-1’e çıkmış, yapraktan 2 defa Si uygulamasında ise 7.08 g bitki-1’e çıkmıştır. Silisyum uygulamaları tütün bitkisinin Na konsantrasyonlarında azalmaya neden olurken, K konsantrasyonları ile K/Na oranlarında artışa neden olarak bitkinin tuza karşı dayanıklılığını arttırmıştır. Çalışma sonucunda; tütün bitkisinin tuz stresine karşı dayanıklılığının arttırılmasında yapraktan Si uygulamalarının pozitif etkisinin olabileceği, uygulama sayısı bakımından ise yapraktan 2 defa uygulamanın daha etkili olduğu ortaya çıkmıştır.