Resistant Tobacco Plants Research Articles

NRAMP6c plays a key role in plant cadmium accumulation and resistance in tobacco (Nicotiana tabacum L.)

Tobacco plants (Nicotiana tabacum L.) exhibit considerable potential for phytoremediation of soil cadmium (Cd) pollutants, owing to their substantial biomass and efficient metal accumulation capabilities. The reduction of Cd accumulation in tobacco holds promise for minimizing Cd intake in individuals exposed to cigar smoking. NRAMP transporters are pivotal in the processes of Cd accumulation and resistance in plants; however, limited research has explored the functions of NRAMPs in tobacco plants. In this investigation, we focused on NtNRAMP6c, one of the three homologs of NRAMP6 in tobacco. We observed a robust induction of NtNRAMP6c expression in response to both Cd toxicity and iron (Fe) deficiency, with the highest expression levels detected in the roots. Subsequent subcellular localization and heterologous expression analyses disclosed that NtNRAMP6c functions as a plasma membrane-localized Cd transporter. Moreover, its overexpression significantly heightened the sensitivity of yeast cells to Cd toxicity. Through CRISPR-Cas9-mediated knockout of NtNRAMP6c, we achieved a reduction in Cd accumulation and an enhancement in Cd resistance in tobacco plants. Comparative transcriptomic analysis unveiled substantial alterations in the transcriptional profiles of genes associated with metal ion transport, photosynthesis, and macromolecule catabolism upon NtNRAMP6c knockout. Furthermore, our study employed plant metabolomics and rhizosphere metagenomics to demonstrate that NtNRAMP6c knockout led to changes in phytohormone homeostasis, as well as shifts in the composition and abundance of microbial communities. These findings bear significant biological implications for the utilization of tobacco in phytoremediation strategies targeting Cd pollutants in contaminated soils, and concurrently, in mitigating Cd accumulation in tobacco production destined for cigar consumption.

Anti-Tobacco Mosaic Virus Indole Alkaloids from the Nicotiana tabacum-Derived Fungus Aspergillus versicolor

To explore the active metabolites from Aspergillus, we have discovered five undescribed tricyclic or tetracyclic indole alkaloids aspergillines K–O (1–5) and four known analogues (6–9) from the strain of Nicotiana tabacum-derived Aspergillus versicolor YNCA0363. The α-amino group derived from tryptophan in isolates 1–6 was changed to an oxygen atom, which is uncommon in the reported analogues. To the best of our knowledge, 4 and 5 represent the first examples of indole alkaloids possessing an isochromene moiety. Compounds 1 and 3 showed obvious anti-tobacco mosaic virus (TMV) activities with inhibition rates of 43.6% ± 2.2 and 58.5% ± 2.8 at the concentration of 20 μM, respectively, which were significantly higher than that of the positive control. Compound 3 also showed excellent potential of inducing systemic acquired resistance. Furthermore, compound 3 can increase the activities of two reactive oxygen species-scavenging enzymes, peroxidase and superoxide dismutase, and enhance tobacco resistance to TMV. Molecular docking results showed that compound 3 exhibits a stronger interaction with TMV-helicase, and showed higher antiviral activities than others. This work revealed that the above indole alkaloids, especially 3, could induce resistance in tobacco plants against the viral pathogen TMV.

Functional characterization and comparison of lycopene epsilon-cyclase genes in Nicotiana tabacum

BackgroundLycopene epsilon-cyclase (ε-LCY) is a key enzyme in the carotenoid biosynthetic pathway (CBP) of higher plants. In previous work, we cloned two Ntε-LCY genes from allotetraploid tobacco (Nicotiana tabacum), Ntε-LCY2 and Ntε-LCY1, and demonstrated the overall effect of Ntε-LCY genes on carotenoid biosynthesis and stress resistance. However, their genetic and functional characteristics require further research in polyploid plants.ResultsHere, we used CRISPR/Cas9 to obtain Ntε-LCY2 and Ntε-LCY1 mutants in allotetraploid N.tabacum K326. Ntε-LCY2 and Ntε-LCY1 had similar promoter cis-acting elements, including light-responsive elements. The Ntε-LCY genes were expressed in roots, stems, leaves, flowers, and young fruit, and their highest expression levels were found in leaves. Ntε-LCY2 and Ntε-LCY1 genes responded differently to normal light and high light stress. Both the Ntε-LCY2 and the Ntε-LCY1 mutants had a more rapid leaf growth rate, especially ntε-lcy2-1. The expression levels of CBP genes were increased in the ntε-lcy mutants, and their total carotenoid content was higher. Under both normal light and high light stress, the ntε-lcy mutants had higher photosynthetic capacities and heat dissipation levels than the wild type, and this was especially true of ntε-lcy2-1. The reactive oxygen species content was lower in leaves of the ntε-lcy mutants.ConclusionIn summary, the expression patterns and biological functions of the Ntε-LCY genes Ntε-LCY1 and Ntε-LCY2 differed in several respects. The mutation of Ntε-LCY2 was associated with a greater increase in the content of chlorophyll and various carotenoid components, and it enhanced the stress resistance of tobacco plants under high light.

Anti-TMV activity and effects of three prieurianin-type limonoids from Munronia henryi

Three prieurianin-type limonoids (1–3), including two new compounds (1 and 2) and one known compound (3) were isolated from Munronia henryi. These compounds were tested for their activities against tobacco mosaic virus (TMV) by the conventional half-leaf method and the outcomes were analyzed by western blotting and RT-PCR assays. The three tested compounds, at 100 μg/mL, showed strong antiviral activities in the pretreated tobacco plants with inhibition rates ranging from 70.5% to 81.3%, which were significantly higher than that of the positive control, ningnanmycin (55.6%). Their potential of inducing systemic acquired resistance (SAR) was also evaluated, in which compound 1 showed excellent induction activities. Furthermore, it was found that potentiation of defense-related enzyme activity and the contents of SA was increased. Compound 1 could also inhibit the expression of TMV CP and up-regulate the expression of defense-related genes. This work revealed that these limonoids, especially compound 1 could induce resistance in tobacco plants against the viral pathogen TMV. Meanwhile, compounds 1–3 could down-regulate the expression of NtHsp70–1 and Nthsp70–261 genes, indicating that these limonoids possibly inhibit TMV infection by suppressing NtHsp70–1 and Nthsp70–261 expression. This study is the first to report antiviral compounds with two different mechanisms of action.

Induction of resistance to Myzus persicae-nicotianae in Cucumber mosaic virus infected tobacco plants using silencing of CMV-2b gene

Aphids such as tobacco aphid Myzus persicae-nicotianae, are among the most important plant viral vectors and plant viruses encode genes to interact with their vectors. Cucumber mosaic virus (CMV) encodes 2b protein as a suppressor of plant immune and it plays a vital role in CMV accumulation and susceptibility to aphid vectors. In this study, the resistance of tobacco plants (Nicotiana tabacum) to M. p. nicotianae was evaluated by silencing of 2b in CMV-infected plants. However, the pFGC-C.h silencing gene construct was transiently expressed using Agrobacterium tumefacience, LBA 4404 in tobacco leaves, and four days later, the plants were mechanically inoculated by CMV (Kurdistan isolate), and then, 15 days post-inoculation 1 nonviruliferous aphid was placed on each leaf for evaluation of resistance to M. p. nicotianae. To evaluate the tobacco plants resistance and susceptibility to M. p. nicotianae, the number of aphids existent per tobacco leaf, life table and, demographic parameters were recorded and used as a comparison indicator. The obtained results were analyzed using the age-stage, two-sex life table. The highest number of aphids was recorded on the control CMV-infected plants, while the lowest number on CMV infected leaves expressing CMV-2b silencing construct (pFGC-C.h). The obtained data revealed the lowest rate for all of intrinsic rate of natural increase (rm) (0.246/day), the rate of reproduction (r0) (17.04 females/generation), and finite rate of increase (λ) (1.279/day), on the pFGC-C.h treatment. The maximum generation time (T) (11.834 days) was observed on (V) treatment. However, the collected data revealed induction of resistance to tobacco aphids by silencing of CMV-2b in CMV infected plants.

Molecular cloning and characterization of a novel gene MsKMS1 in Medicago sativa

Vacuole membrane proteins play a critical role in the regulation of plant physiological processes including normal growth and development, and responses to stresses. The killing me slowly 1 (KMS1) gene that encodes a soluble N-ethylmaleimide-sensitive fusion attachment receptor (SNARE) domain-containing vacuole membrane protein was first reported in Arabidopsis. Currently, the function of KMS1 in other plants under stress is poorly understood. In this study, we report cloning, expression, and characterization of a novel KMS1 gene in alfalfa (Medicago sativa L.), designated MsKMS1 (GenBank accession No. JX467688). The full-length cDNA of MsKMS1 was 1 396 bp and contained a complete open reading frame of 1 257 bp, which encoded a putative protein of 418 amino acids. The BLASTp analysis showed that MsKMS1 shared high amino acid sequence similarities with KMS1 from other plants such as Medicago truncatula (99 %), Cicer arietinum (89 %), Glycine max (77 %), Prunus mume (76 %), Ricinus communis (72 %), Populus euphratica (72 %), Theobroma cacao (72 %), and Arabidopsis thaliana (67 %). Transient transformation of onion (Allium cepa) bulb scale epidermal cells by biolistic bombardment showed that MsKMS1 was localized to the plasma membrane. Quantitative real-time PCR revealed that MsKMS1 expression was upregulated under different abiotic stresses (200 mM NaCl, 20 % (m/v) polyethylene glycol 6000] and 10 mg dm-3 abscisic acid. Transgenic tobacco plants were obtained via Agrobacterium-mediated transformation and treated with 200 mM NaCl. Reverse-transcription PCR data showed that MsKMS1 was successfully transcribed and expressed in the leaves of transgenic plants. The MsKMS1-overexpressors showed a lower malondialdehyde content and maintained a higher relative water content and proline content compared with non-transgenic controls under salt stress. These results indicate that the introduction of the MsKMS1 gene could improve salt stress resistance in tobacco plants. This study reveals the role of MsKMS1 in the regulation of plant responses to abiotic stress and provides evidence for further functional studies of the KMS1 family in alfalfa.

Antiviral activity of glucosylceramides isolated from Fusarium oxysporum against Tobacco mosaic virus infection.

Natural elicitors derived from pathogenic microorganisms represent an ecologic strategy to achieve resistance in plants against diseases. Glucosylceramides (GlcCer) are classified as neutral glycosphingolipids. GlcCer were isolated and purified from Fusarium oxysporum mycelium. F. oxysporum is a plant pathogenic fungus, abundant in soil and causing severe losses in economically important crops such as corn, tobacco, banana, cotton and passion fruit. In this study we evaluate the capacity of GlcCer in inducing resistance in N. tabacum cv Xanthi plants against Tobacco mosaic virus (TMV). Spraying tobacco plants with GlcCer before virus infection reduced the incidence of necrotic lesions caused by TMV. In addition, plants already infected with the virus showed a reduction in hypersensitive response (HR) lesions after GlcCer treatment, suggesting an antiviral effect of GlcCer. Our investigations showed that GlcCer stimulates the early accumulation of H2O2 and superoxide radicals. In addition, the expression of PR-1 (pathogenesis-related 1, with suggested antifungal action), PR-2 (β-1,3-glucanase), PR-3 (Chitinase), PR-5 (Osmotin), PAL (Phenylalanine ammonia-lyase), LOX (Lipoxygenase) and POX (Peroxidase) genes was highly induced after treatment of tobacco plants with GlcCer and induction levels remained high throughout a period of 6 to 120 hours. Our experiments demonstrate that GlcCer induces resistance in tobacco plants against infection by TMV.

A novel gene of Kalanchoe daigremontiana confers plant drought resistance

Kalanchoe (K.) daigremontiana is important for studying asexual reproduction under different environmental conditions. Here, we describe a novel KdNOVEL41 (KdN41) gene that may confer drought resistance and could thereby affect K. daigremontiana development. The detected subcellular localization of a KdN41/Yellow Fluorescent Protein (YFP) fusion protein was in the nucleus and cell membrane. Drought, salt, and heat stress treatment in tobacco plants containing the KdN41 gene promoter driving β-glucuronidase (GUS) gene transcription revealed that only drought stress triggered strong GUS staining in the vascular tissues. Overexpression (OE) of the KdN41 gene conferred improved drought resistance in tobacco plants compared to wild-type and transformed with empty vector plants by inducing higher antioxidant enzyme activities, decreasing cell membrane damage, increasing abscisic acid (ABA) content, causing reinforced drought resistance related gene expression profiles. The 3,3′-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining results also showed less relative oxygen species (ROS) content in KdN41-overexpressing tobacco leaf during drought stress. Surprisingly, by re-watering after drought stress, KdN41-overexpressing tobacco showed earlier flowering. Overall, the KdN41 gene plays roles in ROS scavenging and osmotic damage reduction to improve tobacco drought resistance, which may increase our understanding of the molecular network involved in developmental manipulation under drought stress in K. daigremontiana.

Metabolic Flux Engineering of Cembratrien-ol Production in Both the Glandular Trichome and Leaf Mesophyll in Nicotiana tabacum.

Cembratrien-ol synthase (CBTS) catalyzes the first step in cembranoid biosynthesis, producing cembratrien-ols in plant trichomes. In our previous study, microarray transcriptomes between leaves with trichomes and leaves without trichomes showed that an NtCBTS2 gene was expressed exclusively and abundantly in trichomes. Here, two NtCBTS2 isogenes (NtCBTS2a and NtCBTS2b), derived from a diploid genome donor, Nicotiana sylvestris, were identified from N. tabacum. Both genes were expressed primarily in trichomes, with relatively decreased transcription in flowers and stems, and faint expression in roots, and no expression was detected in leaves lacking trichomes. To demonstrate the feasibility of producing natural product cembratrien-ols in tobacco mesophylls, the mesophylls of 35S:NtCBTS2b transgenic tobacco plants were used in the analysis, suggesting that constitutive expression of NtCBTS2b led to the cembratrien-ol production in mesophylls. Overexpression of NtCBTS2b using either Cauliflower mosaic virus (CaMV) 35S or trichome-specific Cyt P450 oxygenase (CYP) promoters greatly increased aphid resistance by promoting the accumulation of CBT-ols, increased the secretory cell growth in glandular trichomes and increased the levels of various physiological measures, including sugar esters, gibberellins, and cembranoid production. Meanwhile, specifically overexpressing NtCBTS2b in glandular trichomes could most efficiently promote aphid resistance in tobacco plants. Notably, our results indicate the feasibility of utilizing bio-engineering to produce large amounts of CBT-ols, and modify significantly the composition of naturally produced CBT-ols and CBT-diols, thereby promoting aphid resistance in plants.

Tobacco methyl salicylate esterase mediates nonhost resistance

Nonhost resistance is a type of broad-spectrum resistance exhibited by a given plant species to most strains of a pathogen which are generally pathogenic to other plant species. In this study, we have examined the role of tobacco SABP2 (Salicylic acid-Binding Protein 2) in nonhost resistance. SABP2, a methyl salicylate esterase is a critical component of SA-signaling pathway in tobacco plants. The transgenic tobacco SABP2-silenced lines treated with tetraFA, a known inhibitor of esterase activity of SABP2 exhibited enhanced susceptibility to nonhost pathogen, Pseudomonas syringae pv. phaseolicola compared to the control plants. The increased accumulation of SABP2 transcripts upon Psp infection supports the involvement of SABP2 in nonhost resistance. The tetra-FA treated plants also showed delayed expression of pathogenesis related-1 gene upon Psp inoculations. The expression of nonhost marker genes CDM1 and HIN1 was also monitored in tobacco plants infected with host-pathogen P.s. pv. tabaci and P.s. pv. phaseolicola. Overall, results presented in this manuscript suggest that SABP2 has a role in nonhost resistance in tobacco plants.

Expression of an engineered synthetic cry2Aa (D42/K63F/K64P) gene of Bacillus thuringiensis in marker free transgenic tobacco facilitated full-protection from cotton leaf worm (S. littoralis) at very low concentration

Emergence of resistant insects limits the sustainability of Bacillus thuringiensis (Bt) transgenic crop plants for insect management. Beside this, the presence of unwanted marker gene(s) in the transgenic crops is also a major environmental and health concern. Thus, development of marker free transgenic crop plants expressing a new class of toxin having a different mortality mechanism is necessary for resistance management. In a previous study, we generated an engineered Cry2Aa (D42/K63F/K64P) toxin which has a different mortality mechanism as compared to first generation Bt toxin Cry1A, and this engineered toxin was found to enhance 4.1-6.6-fold toxicity against major lepidopteran insect pests of crop plants. In the present study, we have tested the potency of this engineered synthetic Cry2Aa (D42/K63F/K64P) toxin as a candidate in the development of insect resistant transgenic tobacco plants. Simultaneously, we have eliminated the selectable marker gene from the Cry2Aa (D42/K63F/K64P) expressing tobacco plants by exploiting the Cre/lox mediated recombination methodology, and successfully developed marker free T2 transgenic tobacco plants expressing the engineered Cry2Aa toxin. Realtime and western blot analysis demonstrated the expression of engineered toxin gene in transgenic plants. Insect feeding assays revealed that the marker free T2 progeny of transgenic plants expressing Cry2Aa (D42/K63F/K64P) toxin showed 82-92 and 52-61% mortality to cotton leaf worm (CLW) and cotton bollworm (CBW) respectively. Thus, this engineered Cry2Aa toxin could be useful for the generation of insect resistant transgenic Bt lines which will protect the crop damages caused by different insect pests such as CLW and CBW.

Targeting chitinase gene of Helicoverpa armigera by host-induced RNA interference confers insect resistance in tobacco and tomato.

Helicoverpa armigera Hübner (Lepidoptera: Noctuidae) is a devastating agricultural insect pest with broad spectrum of host range, causing million dollars crop loss annually. Limitations in the present conventional and transgenic approaches have made it crucial to develop sustainable and environmental friendly methods for crop improvement. In the present study, host-induced RNA interference (HI-RNAi) approach was used to develop H. armigera resistant tobacco and tomato plants. Chitinase (HaCHI) gene, critically required for insect molting and metamorphosis was selected as a potential target. Hair-pin RNAi construct was prepared from the conserved off-target free partial HaCHI gene sequence and was used to generate several HaCHI-RNAi tobacco and tomato plants. Northern hybridization confirmed the production of HaCHI gene-specific siRNAs in HaCHI-RNAi tobacco and tomato lines. Continuous feeding on leaves of RNAi lines drastically reduced the target gene transcripts and consequently, affected the overall growth and survival of H. armigera. Various developmental deformities were also manifested in H. armigera larvae after feeding on the leaves of RNAi lines. These results demonstrated the role of chitinase in insect development and potential of HI-RNAi for effective management of H. armigera.

Activation of Pathogenesis-related Genes by the Rhizobacterium, Bacillus sp. JS, Which Induces Systemic Resistance in Tobacco Plants.

Plant growth promoting rhizobacteria (PGPR) are known to confer disease resistance to plants. Bacillus sp. JS demonstrated antifungal activities against five fungal pathogens in in vitro assays. To verify whether the volatiles of Bacillus sp. JS confer disease resistance, tobacco leaves pre-treated with the volatiles were damaged by the fungal pathogen, Rhizoctonia solani and oomycete Phytophthora nicotianae. Pre-treated tobacco leaves had smaller lesion than the control plant leaves. In pathogenesis-related (PR) gene expression analysis, volatiles of Bacillus sp. JS caused the up-regulation of PR-2 encoding β-1,3-glucanase and acidic PR-3 encoding chitinase. Expression of acidic PR-4 encoding chitinase and acidic PR-9 encoding peroxidase increased gradually after exposure of the volatiles to Bacillus sp. JS. Basic PR-14 encoding lipid transfer protein was also increased. However, PR-1 genes, as markers of salicylic acid (SA) induced resistance, were not expressed. These results suggested that the volatiles of Bacillus sp. JS confer disease resistance against fungal and oomycete pathogens through PR genes expression.

Systemic Activation of Defensive Enzymes and Protection in Tobacco Plantlets against <i>Phytophthora nicotianae</i> Induced by Oligosaccharins

Oligosaccharins are potent biomolecules which activate defense responses and resistance in tobacco plants. However, it is not known the systemic behavior of defensive enzymes activated by these elicitors. In this work, the dynamic behavior of key defensive enzymes was evaluated in tobacco plant leaves previously treated through the roots with chitosan polymer (CH), chitosan (COS) and pectic (OGAS) oligosaccharides and Spermine (Sp). All macromolecules tested activated protein levels and defense enzymatic activity in tobacco leaves but with different response dynamics among them and depending on the biochemical variable evaluated. Defense response above control levels were detected since 12 hours after treatments and it consisted in a biphasic behavior with two peaks for PAL (EC 4.3.1.5) and β 1 - 3 glucanase (EC 3.2.1.6) enzymatic activities. The highest enzymatic levels for these enzymes were achieved at 48 hours in plantlets elicited with COS and at 72 hours for those plants treated with chitosan polymer, while the highest POD (EC 1.11.1.6) activity was detected with CH between 48 and 72 hours. These results demonstrated systemic defense activation by oligosaccharins in tobacco whose dynamic of defense response is affected by the kind of oligosaccharins tested. When applying OGAS by foliar spray on tobacco, systemic resistance against Phytoththora nicotianae was induced and plantlets were protected with the low concentration tested by 46% under the bioassays conditions performed. Moreover, enzymatic determinations on roots and leaves previous to plant-pathogen interaction showed increments above 30% of control levels for PAL and POD activities. It means that oligosaccharins activate local and systemic defense responses in plants in the absent of pathogen infection.

Protein extract of tobacco expressing StoVe1 gene inhibits Verticillium dahliae proliferation

Verticillium dahliae is a principal pathogen causing verticillium wilt in Solanaceae crops. StoVe1 is a gene resisting to verticillium wilt isolated from Solanum torvum. In order to generate resistant tobacco plants, StoVe1 was inserted in the orientation behind CaMV 35S promoter of vector pGS and this construct was introduced into tobacco by Agrobacterium-mediated transformation. A total of 12 kanamycin-resistant plants were generated and 7 independent transgenic lines were identified by PCR analysis. Quantitative RT-PCR analysis showed that the levels of StoVe1 transcript in transgenic lines were up to 2–6 fold higher than in the control. Anti-fungal assay indicated that the protein extract of transgenic tobacco lines showed strong inhibition activity to V. dahliae, 2 fold or higher compared to control plants. This result reveals that StoVe1, as a V. dahliae resistance gene, has an application potential in plant breeding for verticillium wilt resistance.

Eudesmanolides from Wedelia trilobata (L.) Hitchc. as Potential Inducers of Plant Systemic Acquired Resistance

Ten eudesmanolides (1-10) including five new ones (1-4 and 6) were isolated from the whole plant of Wedelia trilobata (L.) Hitchc., a notoriously invasive weed in South China. As main constituents of W. trilobata, eight of these eudesmanolides were tested for their antitobacco mosaic virus (TMV) activities by the conventional half-leaf and leaf-disk method along with Western blot analysis. All of the tested compounds, at 10 μg/mL, showed strong antiviral activities in the pretreated tobacco plants with inhibition rates ranging from 46.7% to 76.5%, significantly higher than that of the positive control, ningnanmycin (13.5%). Their potential of inducing systemic acquired resistance (SAR) was also evaluated, and compounds 1 and 8 showed excellent induction activities. Furthermore, it was found that different concentrations of compound 1 promoted phenylalanine ammonia-lyase (PAL) activity in tobacco plants. To our knowledge, this is the first report that eudesmanolides could induce resistance in tobacco plants against the viral pathogen TMV.

Role of phospholipase D in metabolic reactions of transgenic tobacco cax1 cells under the influence of salt stress

This work was aimed at investigating the primary reactions of plant cell metabolism in response to salt stress. It was found that the phospholipase D regulatory enzyme is activated in wild-type and transgenic cax1 tobacco plants during the early stages of the influence of salt stress. We have shown that a disturbance in the intracellular homeostasis of calcium ions and oppression of phospholipase D activity decrease the resistance of tobacco plants under the influence of salinity and also indicate the involvement of such systems in signaling during stress adaptation of plants.

Morphological and biochemical responses of selected germplasm of tobacco to soil inoculation with Pythium aphanidermatum

The effect of inoculation with Pythium aphanidermatum was studied on total phenol (TP), salicylic acid (SA), chlorophylls and carotenoid contents of leaves and plant growth characteristics of five tobacco cultivars, namely RK-10 P3, RK-12 P3, RK-13 P4, RK-18 P8 and RK-26 P3, to assess cultivar response at biochemical and morphological levels. Root rot measured at 0–5 scale was 2.66 on cv. RK-10 P3, followed by 2. 33 on cv. RK-18 P8, 1.33 on cv. RK-26 P3 and 1.0 on cv. RK-13 P4. The cv. RK-12 P3 did not develop measurable root rot. The rhizosphere population of root rot fungus increased over time, being highest on the cv. RK-10 P3 (P ≤ 0.001), followed by cvs. RK-18 P8, RK-26 P3, RK-13 P4 and RK-12 P3. Inoculation with the fungus resulted in 5% (cv. RK-10 P3), 10.3% (cv. RK-18 P8, P ≤ 0.05), 10.9% (cv. RK-26 P3, P ≤ 0.05), 16.4% (cv. RK-13 P4, P ≤ 0.01) and 41.5% (cv. RK-12 P3, P ≤ 0.001) increase in the TP content of leaves. SA concentration in tobacco leaves increased marginally (0.8%–3%) in cvs. RK-10 P3, RK-18 P8 and RK-26 P3, but considerably (16%–17%, P ≤ 0.01) in cv. RK-13 and RK-12 P3 in comparison to uninoculated plants. Total chlorophyll content of leaves in response to inoculation with P. aphanidermatum decreased by 27% and 23% in tobacco cvs. RK-10 P3 and RK-18 P8 (P ≤ 0.001) and 17.6 (P ≤ 0.01) and 10.6% (P ≤ 0.05) in cv. RK-26 P3 and RK-13 P4, respectively. Reduction in chlorophylls a and b was 20% and 15% in cv. RK-10 P3 and 20% and 11% in cv. RK-18 P8. Total carotenoid contents of tobacco leaves decreased significantly in cvs. RK-10 P3 and RK-18 P8 (P ≤ 0.05). Significant and greater decrease in plant growth variables was recorded in the cultivars in which increase in TP and SA was lower and decrease in chlorophyll and carotenoids was greater. This study has revealed that greater synthesis of TP and SA may provide resistance in tobacco plants against P. aphanidermatum. The cv. RK-12 P3, in which greatest increase in the SA (17%) and TP (41.5%) was recorded, did not exhibit a significant decrease in plant growth variables and leaf pigments (P ≤ 0.05).

Cadmium resistance in tobacco plants expressing the MuSI gene

MuSI, a gene that corresponds to a domain that contains the rubber elongation factor (REF), is highly homologous to many stress-related proteins in plants. Since MuSI is up-regulated in the roots of plants treated with cadmium or copper, the involvement of MuSI in cadmium tolerance was investigated in this study. Escherichia coli cells overexpressing MuSI were more resistant to Cd than wild-type cells transfected with vector alone. MuSI transgenic plants were also more resistant to Cd. MuSI transgenic tobacco plants absorbed less Cd than wild-type plants. Cd translocation from roots to shoots was reduced in the transgenic plants, thereby avoiding Cd toxicity. The number of short trichomes in the leaves of wild-type tobacco plants was increased by Cd treatment, while this was unchanged in MuSI transgenic tobacco. These results suggest that MuSI transgenic tobacco plants have enhanced tolerance to Cd via reduced Cd uptake and/or increased Cd immobilization in the roots, resulting in less Cd translocation to the shoots.

Chitosan physico–chemical properties modulate defense responses and resistance in tobacco plants against the oomycete Phytophthora nicotianae

Enzymatic defense responses and protection against Phytophthora nicotianae were studied in tobacco plants treated with chitosan of different molecular weights (MW) and degrees of acetylation (DA). The concentration and mode of chitosan application affected enzymatic induction in tobacco leaves. β-1,3-Glucanase (EC 3.2.1.6) activity required 10 times the polymer concentration relative to the oligochitosan mixture to induce the highest activity above control when treated by foliar spray, indicating the influence of molecular weight in this response. PAL (EC 4.3.1.5) and POD (EC 1.11.1.6) activities increased above control as a result of the influence of polymer degree of acetylation when treatments were applied by foliar spray. A higher DA favored PAL activity, whereas a lower DA induced higher POD activity. Using an in vitro bioassay, it was found that the three chitosan compounds caused a reduction of the infection index of P. nicotianae in tobacco plants that was dependent on elicitor concentrations. There was a significant relationship between the reduction of the infection index and an increase in PAL activity when chitosan was applied by foliar spray and by substrate drench to the plant rhizosphere. These results demonstrate the influence of chitosan physico–chemical properties in plant-induced resistance and the relevance of particular responses in plant protection against pathogens.