Modified Tobacco Plants Research Articles

Variability of leaf pubescence characteristics in transgenic tobacco lines with partial proline dehydrogenase gene suppression

Proline, an amino acid, plays an important role in plants, and it is involved in stress resistance and development. Earlier, to study the proline role in maintaining stress resistance in plants, we obtained genetically modified transgenic lines of tobacco (Nicotiana tabacum L.) with reduced activity of proline dehydrogenase (PDH, the proline degradation gene) and increased content of proline. Transgenic tobacco plants demonstrated greater resistance to high concentrations of NaCl, drought, low temperatures, and heavy metals vs. control plants. The visual assessment showed that the leaf pubescence in transgenic plants varied noticeably. Here we apply automated analysis of the tobacco leaf folds to estimate quantitative characteristics of pubescence in genetically modified tobacco plants and the control SR1 line under non-stress conditions. Our results showed differences in the number of trichomes and their length between transgenic and control plants. The trichome number significantly increased in transgenic plants (from 1.5 to 3 times). The largest differences in the trichome numbers were observed for trichomes with lengths from 0 to 380 µm. When assessing the trichome length, the opposite was observed. In all three transgenic lines, the trichome length was significantly lower than that of the control SR1 line. The data obtained indicate the effect of proline as an important metabolome component affecting the plant phenotype. Our results demonstrate perspectives of tobacco transgenic lines as promising genetic models for studying the proline role in plant morphogenesis.

Drought monitoring in tobacco plants by in-vivo electrochemical biosensor

Real-time monitoring along with early detection of drought levels was studied in transgenic tobacco plants using an in-vivo technique with a bio-electrochemical sensor. This study reports on the detection of the plant’s water deficit status as indicated by its reporter gene expression, caused due to drought stress. Plant-based in-vivo sensing provides real-time information generated by the actual hydration/dehydration status of the plant; It complements the information provided by ex-vivo sensors sampling the plant’s surroundings. In the method described here, the expression of the β-D-glucuronidase enzyme, expressed under drought conditions in genetically modified tobacco plants, is monitored using the enzyme-substrate (4-nitrophenyl β-D-glucuronide) reaction generating an electrochemically active product. The generated product is oxidized on the working electrode of a three-electrode electrochemical cell-on-chip mounted on the leaves of Nicotiana tabacum plants. The transport mechanisms and the real-time behavior of enzyme activity was studied using the Michaelis-Menten kinetic diffusion model. The obtained electrochemical signals fit the Michaelis-Menten diffusion kinetic model with the mean absolute error below 5%. The proposed bioelectrochemical sensor detected drought stress earlier than other conventional methods. The sensor applicability under dehydration periods up to 3 weeks is demonstrated showing the highlight and problems of this method.

The Potential Use of Novel Plant-Derived Recombinant Human Collagen in Aesthetic Medicine.

Recombinant human type I collagen, identical in structure and functionality to human type I collagen, was successfully expressed and extracted from genetically modified tobacco plants. Contrarily to tissue extracted protein, rhCollagen is not immunogenic and not allergenic and has an intact triple helix structure showing superior biological functionality. A photocurable rhCollagen was developed by chemically modifying the protein to allow cross-linking under illumination at various wavelengths, maintaining the protein structural and biological functions. The use of the photocurable rhCollagen in aesthetic medicine, especially as a dermal filler and as a bioink for 3D-printed breast implant is discussed in this article.

The Aconitum carmichaelii F3′5′H Gene Overexpression Increases Flavonoid Accumulation in Transgenic Tobacco Plants

Aconitum carmichaelii Debx. is a herbal species that contains many precious bioactive substances, which are alkaloids, flavonoids, steroids, and glycosides. Flavonoids, which are major secondary compounds, play an important role in maintaining redox balance in the cells of the plant body. Many flavonoids have antibacterial, antioxidant, and anticancer properties. However, studies have mainly focused on aconitine, which is a highly toxic group A poison belonging to the alkaloid group, but with little mention of flavonoids. The flavonoids in A. carmichaelii are a group of substances with high content, concentrated in leaves and flowers, including quercetin and kaempferol. F3′5′H (Flavonoid 3′5′-hydroxylase) has been identified as the key enzyme involved in the final steps of flavonoid biosynthesis in plants in general and in A. carmichaelii specifically. This study offers the first report, and demonstrates that the overexpression of the F3′5′H gene from a herbal plant, A. carmichaelii, increases flavonoid content in genetically modified tobacco plants. The A. carmichaelii gene was transformed into tobacco leaf tissue to create transgenic tobacco plants. The AcF3′5′H gene was incorporated into the tobacco genome and was expressed in four transgenic tobacco lines (T01, T03, T05, and T014). The F3′5′H content increased from 20.33% to 32.00% compared with that in non-transformed plants (P < 0.001). Therefore, the flavonoid content of four transgenic tobacco lines increased compared to the WT, from 69.23% to 122.23% (P < 0.001). The results of the successful expression of the AcF3′5′H gene in model tobacco plants are the basis for using the AcF3′5′H gene for improving flavonoid content in other medicinal plants. Thus, the AcF3′5′H gene considered in this work could be a candidate for gene technology to enhance flavonoid accumulation in plants.

Overexpression of Maize ZmHDZIV14 Increases Abscisic Acid Sensitivity and Mediates Drought and Salt Stress in Arabidopsis and Tobacco

Homeodomain-leucine zipper (HD-Zip) IV transcription factors are integral in the plant response to abiotic stressors. In maize, few HD-Zip IV proteins have yet been reported, and the functions are not well understood. In the current study, we isolated ZmHDZIV14 from the HD-Zip IV transcription factor family from maize and characterized its role in the stress response. We conducted a phylogenetic analysis showing that there was high homology and thus likely similar functions between ZmHDZIV14 and AtHDG11. Heterologous overexpression of ZmHDZIV14 in Arabidopsis caused increased sensitivity to abscisic acid (ABA), tolerance of salt and mannitol at germination stage, and drought tolerance of seedlings. ZmHDZIV14 transgenic Arabidopsis lines placed under drought stress additionally displayed lower levels of malondialdehyde, higher proline, and relative water content, compared to the wild-type. Further, modified tobacco plants (35S::ZmHDZIV14) responded to drought conditions with appropriate stomatal changes. After drought was relieved, expression of P5CS1, RD22, RD29B, RAB18, NCED3, and ERD1 were upregulated in transgenic Arabidopsis. It can be seen ZmHDZIV14 expression resulted in physiological and molecular changes in the transgenic lines, which improved overall drought tolerance. In summary, our results indicate that ZmHDZIV14 is a transcriptional regulator that modifies an ABA-dependent signaling pathway to regulate the drought response in plants.

Engineered crops could have it made in the shade.

Modified tobacco plants quickly speed up photosynthesis when bright light dims, boosting yields.

Diet micronutrient balance matters: How the ratio of dietary sterols/steroids affects development, growth and reproduction in two lepidopteran insects

Insects lack the ability to synthesize sterols de novo so they acquire this essential nutrient from their food. Cholesterol is the dominant sterol found in most insects, but in plant vegetative tissue it makes up only a small fraction of the total sterol profile. Instead, plants mostly contain phytosterols; plant-feeding insects generate the majority of their cholesterol by metabolizing phytosterols. However, not all phytosterols are readily converted to cholesterol, and some are even deleterious when ingested above a threshold level. In a recent study we showed that caterpillars reared on tobacco accumulating novel sterols/steroids exhibited reduced performance, even when suitable sterols were present. In the current study we examined how the dominant sterols (cholesterol and stigmasterol) and steroids (cholestanol and cholestanone) typical of the modified tobacco plants affected two insect herbivores (Heliothis virescens and Helicoverpa zea). The sterols/steroids were incorporated into synthetic diets singly, as well as in various combinations, ratios and amounts. For each insect species, a range of performance values was recorded for two generations, with the eggs from the 1st-generation adults as the source of neonates for the 2nd-generation. Performance on the novel steroids (cholestanol and cholestanone) was extremely poor compared to suitable sterols (cholesterol and stigmasterol). Additionally, performance tended to decrease as the ratio of the novel dietary steroids increased. We discuss how the balance of different dietary sterols/steroids affected our two caterpillar species, relate this back to recent studies on sterol/steroid metabolism in these two species, and consider the potential application of sterol/steroid modification in crops.

A Dietary Test of Putative Deleterious Sterols for the Aphid Myzus persicae

The aphid Myzus persicae displays high mortality on tobacco plants bearing a transgene which results in the accumulation of the ketosteroids cholestan-3-one and cholest-4-en-3-one in the phloem sap. To test whether the ketosteroids are the basis of the plant resistance to the aphids, M. persicae were reared on chemically-defined diets with different steroid contents at 0.1–10 µg ml−1. Relative to sterol-free diet and dietary supplements of the two ketosteroids and two phytosterols, dietary cholesterol significantly extended aphid lifespan and increased fecundity at one or more dietary concentrations tested. Median lifespan was 50% lower on the diet supplemented with cholest-4-en-3-one than on the cholesterol-supplemented diet. Aphid feeding rate did not vary significantly across the treatments, indicative of no anti-feedant effect of any sterol/steroid. Aphids reared on diets containing equal amounts of cholesterol and cholest-4-en-3-one showed fecundity equivalent to aphids on diets containing only cholesterol. Aphids were reared on diets that reproduced the relative steroid abundance in the phloem sap of the control and modified tobacco plants, and their performance on the two diet formulations was broadly equivalent. We conclude that, at the concentrations tested, plant ketosteroids support weaker aphid performance than cholesterol, but do not cause acute toxicity to the aphids. In plants, the ketosteroids may act synergistically with plant factors absent from artificial diets but are unlikely to be solely responsible for resistance of modified tobacco plants.

Surface electric charge of thylakoid membranes from genetically modified tobacco plants under freezing stress

In the present study we characterize for the first time electrokinetic and light scattering properties of thylakoids from freezing-tolerant tobacco plants, transformed to accumulate osmoprotectants (proline: AtP5Cs, VacP5Cs; fructan: SacB; glycine betaine: codA). Tobacco plants of wild type (WT) and transformed variants were cultivated at 2°C (cold acclimated) and −2°C (freezing stressed). “Lower salt” thylakoids (I=0.0006) of WT and SacB plants exhibited a decrease in electrophoretic mobility (EPM) after (2°C) treatment. AtP5Cs thylakoids (22°C) show a substantial increase in negative electrical charge (σ) upon illumination. We observed that “low salt”SacB thylakoids at 22°C and 2°C increased the σ on their membrane surfaces during the process of acclimation. WT (22°C) and AtP5Cs thylakoids (2°C) in “low salt” media (I=0.0156) showed a substantial increase in surface electrical charge upon illumination. Cold acclimation on WT and freezing stress on transformed plants resulted in a decrease in aggregation of thylakoids at both ionic strengths.There was a large enhancement in the relaxation capacity of reverse photosynthetic reactions in codA and SacB tobacco after freezing stress. Maximal intensity of the delayed light emission following low temperature stimuli was decreased, revealing a path for tobacco transformants to improve their cold stress tolerance.Here, we suggest the EPM value as an indicator for stability of thylakoids undergone genetic transformation.

Plant sterols and host plant suitability for a phloem-feeding insect

1. Arthropods, including insects, are unique among animals in that they cannot synthesize sterols, including cholesterol, de novo. Some phytophagous insects (e.g. caterpillars, grasshoppers) generate tissue cholesterol by metabolizing plant phytosterols. Currently, little is known about sterols in plant phloem sap, and their significance to the nutritional physiology and ecology of phloem-feeding insects. 2. The sterol profiles of leaves from two plant species, Chinese cabbage and tobacco, were dominated by the phytosterols sitosterol and stigmasterol, respectively. In contrast, the principle sterol in the phloem sap of both plants was cholesterol, which is traditionally considered an animal sterol. Cholesterol was also the most abundant sterol in the carcass and honeydew of Myzus persicae aphids feeding on these plants. 3. The effect of sterol structure on M. persicae was investigated using modified tobacco plants that contained high levels of atypical steroids, specifically ketone-steroids. Aphids reared on the modified tobacco plants had a high atypical steroid content, severely reduced reproduction and high mortality. 4. Our data indicate that the total sterol composition of plants is not necessarily representative of the sterol profile available to phloem-feeding insects, and that the sterol utilization patterns of phloem-feeding insects may differ from chewing insect herbivores utilizing the same plant. Atypical steroids are naturally at insufficient concentrations for significant deleterious effects on insect herbivores, and possible reasons why plants apparently do not use them as defensive compounds are considered.

Automated saccharification assay for determination of digestibility in plant materials

BackgroundCell wall resistance represents the main barrier for the production of second generation biofuels. The deconstruction of lignocellulose can provide sugars for the production of fuels or other industrial products through fermentation. Understanding the biochemical basis of the recalcitrance of cell walls to digestion will allow development of more effective and cost efficient ways to produce sugars from biomass. One approach is to identify plant genes that play a role in biomass recalcitrance, using association genetics. Such an approach requires a robust and reliable high throughput (HT) assay for biomass digestibility, which can be used to screen the large numbers of samples involved in such studies.ResultsWe developed a HT saccharification assay based on a robotic platform that can carry out in a 96-well plate format the enzymatic digestion and quantification of the released sugars. The handling of the biomass powder for weighing and formatting into 96 wells is performed by a robotic station, where the plant material is ground, delivered to the desired well in the plates and weighed with a precision of 0.1 mg. Once the plates are loaded, an automated liquid handling platform delivers an optional mild pretreatment (< 100°C) followed by enzymatic hydrolysis of the biomass. Aliquots from the hydrolysis are then analyzed for the release of reducing sugar equivalents. The same platform can be used for the comparative evaluation of different enzymes and enzyme cocktails. The sensitivity and reliability of the platform was evaluated by measuring the saccharification of stems from lignin modified tobacco plants, and the results of automated and manual analyses compared.ConclusionsThe automated assay systems are sensitive, robust and reliable. The system can reliably detect differences in the saccharification of plant tissues, and is able to process large number of samples with a minimum amount of human intervention. The automated system uncovered significant increases in the digestibility of certain lignin modified lines in a manner compatible with known effects of lignin modification on cell wall properties. We conclude that this automated assay platform is of sufficient sensitivity and reliability to undertake the screening of the large populations of plants necessary for mutant identification and genetic association studies.

Tobacco as a production platform for biofuel: overexpression of Arabidopsis DGAT and LEC2 genes increases accumulation and shifts the composition of lipids in green biomass

When grown for energy production instead for smoking, tobacco can generate a large amount of inexpensive biomass more efficiently than almost any other agricultural crop. Tobacco possesses potent oil biosynthesis machinery and can accumulate up to 40% of seed weight in oil. In this work, we explored two metabolic engineering approaches to enhance the oil content in tobacco green tissues for potential biofuel production. First, an Arabidopsis thaliana gene diacylglycerol acyltransferase (DGAT) coding for a key enzyme in triacylglycerol (TAG) biosynthesis, was expressed in tobacco under the control of a strong ribulose-biphosphate carboxylase small subunit promoter. This modification led to up to a 20-fold increase in TAG accumulation in tobacco leaves and translated into an overall of about a twofold increase in extracted fatty acids (FA) up to 5.8% of dry biomass in Nicotiana tabacum cv Wisconsin, and up to 6% in high-sugar tobacco variety NC-55. Modified tobacco plants also contained elevated amounts of phospholipids. This increase in lipids was accompanied by a shift in the FA composition favourable for their utilization as biodiesel. Second, we expressed in tobacco Arabidopsis gene LEAFY COTYLEDON 2 (LEC2), a master regulator of seed maturation and seed oil storage under the control of an inducible Alc promoter. Stimulation of LEC2 expression in mature tobacco plants by acetaldehyde led to the accumulation of up to 6.8% per dry weight of total extracted FA. The obtained data reveal the potential of metabolically modified plant biomass for the production of biofuel.

Tobacco transformants expressing antisense sequence of proline dehydrogenase gene possess tolerance to heavy metals

Analysis of resistance of genetically modified tobacco plants bearing antisense suppressor of proline dehydrogenase gene and characterized with higher content of proline to elevated concentrations of heavy metals was performed. It was demonstrated that progeny of transgenic plants have high resistance to lead, nickel and cadmium ions.

Tolerance to aluminum in genetically modified tobacco plants

Genetically modified tobacco plants tolerant to high aluminum concentrations were developed by integration of constructs containing rhamnolipid genes (rhlA and rhlB). At an aluminum concentration of 200 mM in ionite soil, the control plants perished, whereas the transgenic plants, although they were inhibited, continued to grow and produced seeds.

Improvement of the pBI121 plant expression vector by leader replacement with a sequence combining a poly(CAA) and a CT motif

To improve expression levels of recombinant proteins in plants, a new leader sequence was designed. Several elements known to enhance gene translation and/or transcription were considered, including the CaMV 35S Inr site, a CT-rich motif often shared by highly expressed plant genes and a poly(CAA) region widespread in tobamovirus and plant leaders. The effect of the synthetic leader on gusA expression was evaluated in genetically modified tobacco plants by measuring the beta-glucuronidase activity and the mRNA level. When compared to the gusA leader of pBI121, the new sequence determined a 8.6-fold and a 12.5-fold increase of enzyme concentration taking into account the whole plant population or the above-average expressors, respectively. Since most pCAMBIA vectors harbour a very short 5'-UTR, identical to a fragment of the pBI121 leader, leader replacement with the sequence herein described is strongly suggested.

Improved phytoaccumulation of cadmium by genetically modified tobacco plants ( Nicotiana tabacum L.). Physiological and biochemical response of the transformants to cadmium toxicity

The response of tobacco plants ( Nicotiana tabacum L.)—non-transformed and transformed with a metallothionein gene MThis from Silene vulgaris L.—to increase cadmium supply in the nutrient solution was compared. The transgenic plants accumulated significantly more Cd both in the roots and the leaves. Visual toxicity symptoms and disturbance in water balance were correlated with Cd tissue content. Treatment with 300 μM CdCl 2 resulted in inhibition of photosynthesis and mobilization of the ascorbate-glutathione cycle. Treatment with 500 μM CdCl 2 led to irreversible damage of photosynthesis and oxidative stress. An appearance of a new peroxidase isoform and changes in the leaf polypeptide pattern were observed at the highest Cd concentration. The level of non-protein thiols gradually increased following the Cd treatment both in transgenic and non-transformed plants.

Accumulation of Cadmium by Transgenic Tobacco

Especially tailored transgenic plants were prepared for the purpose of phytoremediation. In the case of heavy metals, high accumulation in plants is the main requirement, particularly in the harvestable parts. Improvement in a plant's ability to tolerate heavy metals via the introduction of metallothionein (MT) genes has already been demonstrated by different authors. In this study, an additional metal binding domain to the implemented protein was introduced in order to further enhance the metal binding capacity. For this reason, gene coding a peptide containing six histidine moieties was combined with a CUP1 yeast metallothionein gene originating from Saccharomyces cerevisiae, which was introduced into tobacco plants using an Agrobacterium tumefaciens vector. Transformants selected on the basis of the kanamycine resistance were checked for the presence of transgenes by RNA isolation of aseptic plants and RT-PCR. The effectiveness of the obtained lines of genetically modified tobacco plants to accumulate heavy metals was tested on sand media with the addition of KNOP'S nutrient solution modified by the addition of cadmium (0.2 mg Cd/l as Cd (NO 3 ) 2 x 4 H 2 O), and compared with non-transformed control plants (the average of the controls was 16.9 mg Cd/kg of dry matter (DM) above ground and 14.3 mg Cd/kg DM in roots). The best performing line HisCUP-X accumulated 90% more Cd in above-ground parts (31.3 mg Cd/kg DM above ground, 10.6 mg Cd/kg DM in roots) during the six-week test period without visible differences in the growth characteristics. Increased translocation into the harvestable parts of the plant is considered as especially important.

Reassessment of qualitative changes in lignification of transgenic tobacco plants and their impact on cell wall assembly

In tobacco plants the effect of antisense down-regulation of various genes encoding enzymes of the monolignol biosynthetic pathway resulted in quantitative and qualitative changes in lignin distribution and in diverse alterations of the secondary wall assembly of modified tobacco plants. Total lignin content, composition in syringyl and guaiacyl units, and absolute proportions of condensed and non-condensed substructures occurring in the cell walls, were differentially modified according to the repressed gene. Immunocytochemical characterisation and visualisation of the distribution of condensed and non-condensed lignin substructure epitopes in transmission electron microscopy (TEM) revealed that some transformations entailed profound and specific alterations in the secondary wall biogenesis. Correlation between micro-morphological cell wall alterations and semi-quantitative immuno-analysis of the topochemical distribution of lignin sub-units suggests that the mode of polymerisation of monolignols into non-condensed units, favoured by the microfibril matrix of the secondary wall, plays an important part in the lignified cell wall assembly.

The Growth Response of the Stems of Genetically Modified Tobacco Plants (Nicotiana tabacum‘Samsun’) to Flexural Stimulation

Genetically modified tobacco plants (Nicotiana tabacum‘Samsun’) with antisense cinnamyl alcohol dehydrogenase DNA, produce secondary xylem of a reduced tensile stiffness. These plants were grown alongside control plants. The stems of the plants were flexed or protected from flexing over a period of several weeks. The tensile moduli and second moments of areas of the different tissues inside the stems were measured and used to calculate the bending stiffness of the plants. In tobacco, the cylinder of xylem was found to be the most important tissue in determining the bending stiffness of the plants. The thickness of the xylem tissue cylinder increased when plants were subjected to flexural stimulation. This increased the bending stiffness of the stems. The response to mechanical stimulation was found to be correlated with tissue strain and the genetically modified plants were able to exactly compensate for the reduced modulus of their xylem tissue by increasing the thickness of the xylem tissue cylinder more than in control plants.