The 2005 version of the chloroplast DNA sequence from tobacco (Nicotiana tabacum)

The complete nucleotide sequence of tobacco chloroplast DNA was first determined in 1986, and then its updated gene map was reported in 1998. During the course of sequencing the chloroplast DNA ofNicotiana sylvestris, the female progenitor of tobacco, we found some sequence errors and amended the 1998 version. The tobacco chloroplast DNA comprises 155,943 bp, 4 bp longer than the 1998 version.     

Dynamic metabonomic responses of tobacco (Nicotiana tabacum) plants to salt stress

Metabolic responses are important for plant adaptation to osmotic stresses. To understand the dosage and duration dependence of salinity effects on plant metabolisms, we analyzed the metabonome of tobacco plants and its dynamic responses to salt treatments using NMR spectroscopy in combination with multivariate data analysis. Our results showed that the tobacco metabonome was dominated by 40 metabolites including organic acids/bases, amino acids, carbohydrates and choline, pyrimidine, and purine metabolites. A dynamic trajectory was clearly observable for the tobacco metabonomic responses to the dosage of salinity. Short-term low-dose salt stress (50 mM NaCl, 1 day) caused metabolic shifts toward gluconeogenesis with depletion of pyrimidine and purine metabolites. Prolonged salinity with high-dose salt (500 mM NaCl) induced progressive accumulation of osmolytes, such as proline and myo-inositol, and changes in GABA shunt. Such treatments also promoted the shikimate-mediated secondary metabolisms with enhanced biosynthesis of aromatic amino acids. Therefore, salinity caused systems alterations in widespread metabolic networks involving transamination, TCA cycle, gluconeogenesis/glycolysis, glutamate-mediated proline biosynthesis, shikimate-mediated secondary metabolisms, and the metabolisms of choline, pyrimidine, and purine. These findings provided new insights for the tobacco metabolic adaptation to salinity and demonstrated the NMR-based metabonomics as a powerful approach for understanding the osmotic effects on plant biochemistry.     

Response to oxidative stress induced by cadmium and copper in tobacco plants (Nicotiana tabacum) engineered with the trehalose-6-phosphate synthase gene (AtTPS1)

The response of tobacco plants genetically engineered with the AtTPS1 gene to stress induced by excess Cu and Cd was evaluated in hydroponic solution (100 and 400 μM Cu and 50 and 200 μM Cd) after a 48 h exposure. Two transgenic lines, transformed with the AtTPS1 (trehalose-6-phosphate synthase) gene from Arabidopsis, with different levels of trehalose-6-phosphate synthase expression (B5H, higher and B1F, lower), and a wild type (WT) were investigated. Protein content, antioxidative enzymes (CAT, POD, SOD, and APX), glucose, fructose, lipid peroxidation, hydrogen peroxide and Cd and Cu contents were determined in leaves. The two transgenic lines were differently influenced by Cd and Cu exposure as they induced a different antioxidant enzymatic defense response. B1F and B5H plants showed a better acclimation to Cd and excess Cu compared to WT. Furthermore B1F was more tolerant than B5H to Cd and excess Cu. B1F accumulated less Cd and Cu in leaves, probably due to a more efficient exclusion mechanism. Catalase was shown to be the most important enzyme in the antioxidative system of these plants.     

Distinct physiological and metabolic reprogramming by highbush blueberry (Vaccinium corymbosum) cultivars revealed during long‐term UV‐B radiation

Despite the Montreal protocol and the eventual recovery of the ozone layer over Antarctica, there are still concerns about increased levels of ultraviolet‐B (UV‐B) radiation in the Southern Hemisphere. UV‐B induces physiological, biochemical and morphological stress responses in plants, which are species‐specific and different even for closely related cultivars. In woody plant species, understanding of long‐term mechanisms to cope with UV‐B‐induced stress is limited. Therefore, a greenhouse UV‐B daily course simulation was performed for 21 days with two blueberry cultivars (Legacy and Bluegold) under UV‐B_BE irradiance doses of 0, 0.07 and 0.19 W m^?2. Morphological changes, photosynthetic performance, antioxidants, lipid peroxidation and metabolic features were evaluated. We found that both cultivars behaved differently under UV‐B exposure, with Legacy being a UV‐B‐resistant cultivar. Interestingly, Legacy used a combined strategy: initially, in the first week of exposure its photoprotective compounds increased, coping with the intake of UV‐B radiation (avoidance strategy), and then, increasing its antioxidant capacity. These strategies proved to be UV‐B radiation dose dependent. The avoidance strategy is triggered early under high UV‐B radiation in Legacy. Moreover, the rapid metabolic reprogramming capacity of this cultivar, in contrast to Bluegold, seems to be the most relevant contribution to its UV‐B stress‐coping strategy.     

UVB radiation and 17‐hydroxygeranyllinalool diterpene glycosides provide durable resistance against mirid (Tupiocoris notatus) attack in field‐grown Nicotiana attenuata plants

Depending on geographical location, plants are exposed to variable amounts of UVB radiation and herbivore attack. Because the role(s) of UVB in the priming and/or accumulation of plant defence metabolites against herbivores are not well understood, we used field‐grown Nicotiana attenuata plants to explore the effects of UVB on herbivore performance. Consistent with previous reports, UVB‐exposed plants accumulated higher levels of ultraviolet (UV)‐absorbing compounds (rutin, chlorogenic acid, crypto‐chlorogenic acid and dicaffeoylspermidine). Furthermore, UVB increased the accumulation of jasmonic acid, jasmonoyl‐L‐isoleucine and abscisic acid, all phytohormones which regulate plant defence against biotic and abiotic stress. In herbivore bioassays, N. attenuata plants experimentally protected from UVB were more infested by mirids in three consecutive field seasons. Among defence metabolites measured, 17‐hydroxygeranyllinalool diterpene glycosides (HGL‐DTGs) showed strongly altered accumulation patterns. While constitutive HGL‐DTGs levels were higher under UVB, N. attenuata plants exposed to mirid bugs (Tupiocoris notatus) had still more HGL‐DTGs under UVB, and mirids preferred to feed on HGL‐DTGs‐silenced plants when other UVB protecting factors were eliminated by UVB filters. We conclude that UVB exposure not only stimulates UV protective screens but also affects plant defence mechanisms, such as HGL‐DTGs accumulation, and modulates ecological interactions of N. attenuata with its herbivores in nature.     

Phytodetoxification of TNT by transplastomic tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>) expressing a bacterial nitroreductase

Key message

Expression of the bacterial nitroreductase gene, nfsI, in tobacco plastids conferred the ability to detoxify TNT.

Abstract

The toxic pollutant 2,4,6-trinitrotoluene (TNT) is recalcitrant to degradation in the environment. Phytoremediation is a potentially low cost remediation technique that could be applied to soil contaminated with TNT; however, progress is hindered by the phytotoxicity of this compound. Previous studies have demonstrated that plants transformed with the bacterial nitroreductase gene, nfsI have increased ability to tolerate and detoxify TNT. It has been proposed that plants engineered to express nfsI could be used to remediate TNT on military ranges, but this could require steps to mitigate transgene flow to wild populations. To address this, we have developed nfsI transplastomic tobacco (Nicotiana tabacum L.) to reduce pollen-borne transgene flow. Here we have shown that when grown on solid or liquid media, the transplastomic tobacco expressing nfsI were significantly more tolerant to TNT, produced increased biomass and removed more TNT from the media than untransformed plants. Additionally, transplastomic plants expressing nfsI regenerated with high efficiency when grown on medium containing TNT, suggesting that nfsI and TNT could together be used to provide a selectable screen for plastid transformation.

     

Artificial elevation of glutathione contents in salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) reduces susceptibility to the powdery mildew pathogen Euoidium longipes

• The effects of elevated glutathione levels on defence responses to powdery mildew (Euoidium longipes) were investigated in a salicylic acid‐deficient tobacco (Nicotiana tabacum cv. Xanthi NahG) and wild‐type cv. Xanthi plants, where salicylic acid (SA) contents are normal.
• Aqueous solutions of reduced glutathione (GSH) and its synthetic precursor R‐2‐oxothiazolidine‐4‐carboxylic acid (OTC) were injected into leaves of tobacco plants 3 h before powdery mildew inoculation.
• SA‐deficient NahG tobacco was hyper‐susceptible to E. longipes, as judged by significantly more severe powdery mildew symptoms and enhanced pathogen accumulation. Strikingly, elevation of GSH levels in SA‐deficient NahG tobacco restored susceptibility to E. longipes to the extent seen in wild‐type plants (i.e. enhanced basal resistance). However, expression of the SA‐mediated pathogenesis‐related gene (NtPR‐1a) did not increase significantly in GSH or OTC‐pretreated and powdery mildew‐inoculated NahG tobacco, suggesting that the induction of this PR gene may not be directly involved in the defence responses induced by GSH.
• Our results demonstrate that artificial elevation of glutathione content can significantly reduce susceptibility to powdery mildew in SA‐deficient tobacco.

     

Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro‐Tom) plants to cadmium‐induced stress

The effects of varying concentrations of cadmium (Cd) on the development of Lycopersicon esculentum cv. Micro‐Tom (MT) plants were investigated after 40 days (vegetative growth) and 95 days (fruit production), corresponding to 20 days and 75 days of exposure to CdCl₂, respectively. Inhibition of growth was clearly observed in the leaves after 20 days and was greater after 75 days of growth in 1 mM CdCl₂, whereas the fruits exhibited reduced growth following the exposure to a concentration as low as 0.1 mM CdCl₂. Cd was shown to accumulate in the roots after 75 days of growth but was mainly translocated to the upper parts of the plants accumulating to high concentrations in the fruits. Lipid peroxidation was more pronounced in the roots even at 0.05 mM CdCl₂ after 75 days, whereas in leaves, there was a major increase after 20 days of exposure to 1 mM CdCl₂, but the fruit only exhibited a slight significant increase in lipid peroxidation in plants subjected to 1 mM CdCl₂ when compared with the control. Oxidative stress was also investigated by the analysis of four key antioxidant enzymes, which exhibited changes in response to the increasing concentrations of Cd tested. Catalase (EC 1.11.1.6) activity was shown to increase after 75 days of Cd treatment, but the major increases were observed at 0.1 and 0.2 mM CdCl₂, whereas guaiacol peroxidase (EC 1.11.1.7) did not vary significantly from the control in leaves and roots apart from specific changes at 0.5 and 1 mM CdCl₂. The other two enzymes tested, glutathione reductase (EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1), did not exhibit any significant changes in activity, apart from a slight decrease in SOD activity at concentrations above 0.2 mM CdCl₂. However, the most striking results were obtained when an extra treatment was used in which a set of plants was subjected to a stepwise increase in CdCl₂ from 0.05 to 1 mM, leading to tolerance of the Cd applied even at the final highest concentration of 1 mM. This apparent adaptation to the toxic effect of Cd was confirmed by biomass values being similar to the control, indicating a tolerance to Cd acquired by the MT plants.     

In vitro Antioxidant and Anti‐inflammatory Activities of Extracts from Nicotiana tabacum L. (Solanaceae) Leafy Galls Induced by Rhodococcus fascians

Plant galls are widely distributed, and their extracts are used in traditional medicine worldwide. Traditional remedies containing extracts of plant galls in China, India and some African countries have effective in the treatment of various pathologies. To open a new promising procedure for screening bioactive compounds from plant galls, standardized plant materials were generated in vitro and used for phytochemical and biological investigations. Methanol aqueous chloroform and hexane extracts of Nicotiana tabacum leafy galls induced by Rhodococcus fascians were used to evaluate phenolic and flavonoid contents, and to investigate antioxidant activity by 2,2‐diphenyl‐1‐picrylhydrazyl radical scavenging and ferric reducing antioxidant/power assays and anti‐inflammatory activity by the lipoxygenase inhibition assay. Infection by R. fascians modifies significantly the phytochemical profile of N. tabacum as well as its biological properties. The total polyphenolic content was increased (120–307%), and that of flavonoids was reduced (20–42.5%). Consequently, antioxidant and anti‐inflammatory activities of non‐infected tobacco extracts are significantly modified compared to plants treated with leafy gall extracts. This shows that infection by R. fascians favoured the production of anti‐inflammatory and antioxidant compounds in N. tabacum. The study indicates the benefit of plant galls used in traditional medicines against various pathologies.     

Inhibition of protease activity by antisense RNA improves recombinant protein production in Nicotiana tabacum cv. Bright Yellow 2 (BY‐2) suspension cells

Recombinant proteins produced in plant suspension cultures are often degraded by endogenous plant proteases when secreted into the medium, resulting in low yields. To generate protease‐deficient tobacco BY‐2 cell lines and to retrieve the sequence information, we cloned four different protease cDNAs from tobacco BY‐2 cells (NtAP, NtCP, NtMMP1, and NtSP), which represent the major catalytic classes. The simultaneous expression of antisense RNAs against these endogenous proteases led to the establishment of cell lines with reduced levels of endogenous protease expression and activity at late stages of the cultivation cycle. One of the cell lines showing reduced proteolytic activity in the culture medium was selected for the expression of the recombinant full‐length IgG1(κ) antibody 2F5, recognizing the gp41 surface protein of HIV‐1. This cell line showed significantly reduced degradation of the 2F5 heavy chain, resulting in four‐fold higher accumulation of the intact antibody heavy chain when compared to transformed wild type cells expressing the same antibody. N‐terminal sequencing data revealed that the antibody has two cleavage sites within the CDR‐H3 and one site at the end of the H4‐framework region. These cleavage sites are found to be vulnerable to serine proteases. The data provide a basis for further improvement of plant cells for the production of recombinant proteins in plant cell suspension cultures.     

Genetic polymorphism in Caulerpa taxifolia (Ulvophyceae) chloroplast DNA revealed by a PCR‐based assay of the invasive Mediterranean strain

Abstract An invasive, cold‐tolerant strain of the tropical green alga Caulerpa taxifolia was introduced recently in the Mediterranean Sea and along the Californian coast. We screened 50 aquarium and open‐sea C. taxifolia specimens for the presence/absence of an intron located in the rbcL gene of chloroplast DNA. We also reanalysed a total of 229 sequences of the Internal Transcribed Spacer (ITS) of ribosomal DNA, combining previously published sequences from different studies with 68 new sequences to complement rbcL data. The introduced Mediterranean strain was found to be characterized by the absence of the rbcL intron and by the occurrence of a particular monomorphic ITS type. A PCR assay based on rbcL gene was developed to detect new introductions of the invasive strain of C. taxifolia. This rapid and inexpensive test could be useful to assist environment managers in the preservation of coastal marine ecosystems.     

He–Ne laser (632.8 nm) pre‐irradiation gives protection against DNA damage induced by a near‐infrared trapping beam

We report the results of a study carried out to investigate the effect of He–Ne laser (632.8 nm) pre‐irradiation on DNA damage induced by continuous wave 1064 nm trapping beam exposure in MCF‐7 cells. A significant decrease in % tail DNA (p < 0.05) was observed in MCF‐7 cells pre‐exposed to He–Ne laser beam. The dependence of the induced protection against 1064 nm trapping beam irradiation induced DNA damage on the time interval between the two irradiations as well as the He–Ne laser pre‐irradiation parameters is presented. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Male‐sterile maize plants produced by targeted mutagenesis of the cytochrome P450‐like gene (MS26) using a re‐designed I–CreI homing endonuclease

The I–CreI homing endonuclease from Chlamydomonas reinhardti has been used as a molecular tool for creating DNA double‐strand breaks and enhancing DNA recombination reactions in maize cells. The DNA‐binding properties of this protein were re‐designed to recognize a 22 bp target sequence in the 5th exon of MS26, a maize fertility gene. Three versions of a single‐chain endonuclease, called Ems26, Ems26+ and Ems26++, cleaved their intended DNA site within the context of a reporter assay in a mammalian cell line. When the Ems26++ version was delivered to maize Black Mexican Sweet cells by Agrobacterium‐mediated transformation, the cleavage resulted in mutations at a co‐delivered extra‐chromosomal ms26‐site in up to 8.9% of the recovered clones. Delivery of the same version of Ems26 to immature embryos resulted in mutations at the predicted genomic ms26‐site in 5.8% of transgenic T₀ plants. This targeted mutagenesis procedure yielded small deletions and insertions at the Ems26 target site consistent with products of double‐strand break repair generated by non‐homologous end joining. One of 21 mutagenized T₀ plants carried two mutated alleles of the MS26 gene. As expected, the bi‐allelic mutant T₀ plant and the T₁ progeny homozygous for the ms26 mutant alleles were male‐sterile. This paper described the second maize chromosomal locus (liguless‐1 being the first one) mutagenized by a re‐designed I–CreI–based endonuclease, demonstrating the general utility of these molecules for targeted mutagenesis in plants.     

The inhibitory effects of UV-B radiation (280–315 nm) on Gunnera magellanica growth correlate with increased DNA damage but not with oxidative damage to lipids

Damage to DNA and disruption of membrane integrity by lipid peroxidation processes are two of the proposed causes of UV‐B‐induced growth inhibition in plants. However, the relative significance of these different types of molecular damage has not been established in experiments carried out under realistic physiological conditions. Plants of Gunnera magellanica (a native herb from southern Patagonia) were exposed to a gradient of biologically effective UV‐B doses (from 0 to 6.5 kJ m^?2 d^?1 of UV‐B_be) in a greenhouse study. Leaf expansion was measured and sensitive techniques were used to detect damage to DNA (in the form of cyclobutane pyrimidine dimers; CPDs) and lipid peroxidation (via electronic‐paramagnetic resonance; EPR). Leaf expansion decreased and the CPD density increased with increasing UV‐B doses, but the degree of lipid peroxidation remained unaffected. The highest UV‐B dose induced a transient oxidative stress situation (as evaluated using the ratio of ascorbyl radical to ascorbate, A^·/AH^–), which was rapidly controlled by an increase in the ascorbate pool. The present results suggest that under a range of UV‐B_be doses that overlaps the range of doses that G. magellanica plants experience in their natural environment, growth inhibition is better explained by DNA damage than by increased lipid peroxidation.     

Over-expression of tobacco <Emphasis Type="Italic">UBC1</Emphasis> encoding a ubiquitin-conjugating enzyme increases cadmium tolerance by activating the 20S/26S proteasome and by decreasing Cd accumulation and oxidative stress in tobacco (<Emphasis Type="Italic">Nicotiana tabacum</Emphasis>)

Ubiquitin (Ub)-conjugating enzyme (UBC, E2) receives Ub from Ub-activating enzyme (E1) and transfers it to target proteins, thereby playing a key role in Ub/26S proteasome-dependent proteolysis. UBC has been reported to be involved in tolerating abiotic stress in plants, including drought, salt, osmotic and water stresses. To isolate the genes involved in Cd tolerance, we transformed WT (wild-type) yeast Y800 with a tobacco cDNA expression library and isolated a tobacco cDNA, NtUBC1 (Ub-conjugating enzyme), that enhances cadmium tolerance. When NtUBC1 was over-expressed in tobacco, cadmium tolerance was enhanced, but the Cd level was decreased. Interestingly, 20S proteasome activity was increased and ubiquitinated protein levels were diminished in response to cadmium in NtUBC1 tobacco. By contrast, proteasome activity was decreased and ubiquitinated protein levels were slightly enhanced by Cd treatment in control tobacco, which is sensitive to Cd. Moreover, the oxidative stress level was induced to a lesser extent by Cd in NtUBC1 tobacco compared with control plants, which is ascribed to the higher activity of antioxidant enzymes in NtUBC1 tobacco. In addition, NtUBC1 tobacco displayed a reduced accumulation of Cd compared with the control, likely due to the higher expression of CAX3 (Ca²⁺/H⁺ exchanger) and the lower expression of IRT1 (iron-responsive transporter 1) and HMA-A and -B (heavy metal ATPase). In contrast, atubc1 and atubc1atubc2 Arabidopsis exhibited lower Cd tolerance and proteasome activity than WT. In conclusion, NtUBC1 expression promotes cadmium tolerance likely by removing cadmium-damaged proteins via Ub/26S proteasome-dependent proteolysis or the Ub-independent 20S proteasome and by diminishing oxidative stress through the activation of antioxidant enzymes and decreasing Cd accumulation due to higher CAX3 and lower IRT1 and HMA-A/B expression in response to 50 µM Cd challenge for 3 weeks.