Nicotiana Tabacum Cv Research Articles

Unraveling tobacco BY-2 protein complexes with BN PAGE/LC–MS/MS and clustering methods

To understand physiological processes, insight into protein complexes is very important. Through a combination of blue native gel electrophoresis and LC-MS/MS, we were able to isolate protein complexes and identify their potential subunits from Nicotiana tabacum cv. Bright Yellow-2. For this purpose, a bioanalytical approach was used that works without a priori knowledge of the interacting proteins. Different clustering methods (e.g., k-means and hierarchical clustering) and a biclustering approach were evaluated according to their ability to group proteins by their migration profile and to correlate the proteins to a specific complex. The biclustering approach was identified as a very powerful tool for the exploration of protein complexes of whole cell lysates since it allows for the promiscuous nature of proteins. Furthermore, it searches for associations between proteins that co-occur frequently throughout the BN gel, which increases the confidence of the putative associations between co-migrating proteins. The statistical significance and biological relevance of the profile clusters were verified using functional gene ontology annotation. The proof of concept for identifying protein complexes by our BN PAGE/LC-MS/MS approach is provided through the analysis of known protein complexes. Both well characterized long-lived protein complexes as well as potential temporary sequential multi-enzyme complexes were characterized.

EFFECT OF 1,3;1,6-β-D-GLUCAN ON THE FORMATION OF ABNORMAL TOBACCO MOSAIC VIRUS PARTICLES IN THE LEAVES OF NICOTIANA TABACUM cv. SAMSUN

The carbohydrate-containing polymer 1,3;1,6-s-Dglucan was obtained by transformation of laminaran from the alga Laminaria cichorioides with endo-s-1,3- glucanase from marine mollusks. In electron microscope observations of phosphotungstic acid-stained preparations from Nicotiana tabacum cv. Samsun leaves inoculated with a mixture of Tobacco mosaic virus (TMV) (1 µg/ml) and glucan (1 mg/ml) or with TMV alone, we found that such preparations contained, along with virus particles of normal diameter (about 18 nm), abnormal (swollen and thinner) particles. The highest number of thin viral particles was found in dips from leaves inoculated with TMV together with glucan. It is suggested that this may be caused by a glucan-mediated increase of TMV particle proteolysis in infected leaves.

Transgene excision in pollen using a codon optimized serine resolvase CinH-RS2 site-specific recombination system

Transgene escape, a major environmental and regulatory concern in transgenic crop cultivation, could be alleviated by removing transgenes from pollen, the most frequent vector for transgene flow. A transgene excision vector containing a codon optimized serine resolvase CinH recombinase (CinH) and its recognition sites RS2 were constructed and transformed into tobacco (Nicotiana tabacum cv. Xanthi). CinH recombinase recognized 119bp of nucleic acid sequences, RS2, in pollen and excised the transgene flanked by the RS2 sites. In this system, the pollen-specific LAT52 promoter from tomato was employed to control the expression of CinH recombinase. Loss of expression of a green fluorescent protein (GFP) gene under the control of the LAT59 promoter from tomato was used as an indicator of transgene excision. Efficiency of transgene excision from pollen was determined by flow cytometry (FCM)-based pollen screening. While a transgenic event in the absence of CinH recombinase contained about 70% of GFP-synthesizing pollen, three single-copy transgene events contained less than 1% of GFP-synthesizing pollen based on 30,000 pollen grains analyzed per event. This suggests that CinH-RS2 recombination system could be effectively utilized for transgene biocontainment.

Protein changes during programmed cell death in tobacco

Programmed cell death (PCD) was induced by the Yariv reagent in Nicotiana tabacum cv. Bright Yellow-2 cell suspension. The analyses of proteins extracts by 2-D electrophoresis clearly show massive protein degradation which was mainly due to cysteine protease activity. In contrast, some proteins remained unchanged up to 72 h after PCD induction. Peptide mass fingerprints of these proteins, obtained by MALDI-TOF, identified calreticulin, heat shock protein (HSP) 60, HSP70, malate dehydrogenase and mitochondrial ATP synthase β-subunit.

Agrobacterium tumefaciens-infection Strategies for Greater Transgenic Recovery in Nicotiana tabacum cv. TAPM26

The efficiency of vir gene induction and conditions of T-DNA delivery are important parameters for developing an efficient Agrobacterium-mediated plant transformation system. Reliable selection regimes coupled with an efficient regeneration method are important for obtaining stably transformed plants. This study reported a modified protocol for greater recovery of transgenic tobacco (Nicotiana tabacum cv. TAPM 26) plant using Agrobacterium tumefaciens-mediated transformation combined with herbicide Basta as the selection agent. Effective post co-cultivation conditions were developed by investigating the selection regime in vitro and ex vitro. The effects of carbenicillin on Agrobacterium growth and plant regeneration to determine non-phytotoxic concentration of carbenicillin was also evaluated as antibiotics used for Agrobacterium growth suppression have known to adversely affect plant regeneration. It was discovered t h a t the efficient conditions that led to greater recovery of transformants includes the exposure to 400 mg L -1 of carbenicillin post-co-cultivation for Agrobacterium elimination, 1 week of recovery period prior to Basta selection and prolonged selection pressure extended to rooting process which significantly reduces chimeric events. Basta concentration of 1.5 mg L -1 at tissue culture level was most effective in maintaining selection pressure without affecting the plants' regeneration competency. Using this improved protocol, an average of 7.00±0.33 Basta resistant shoots were obtained per leaf explant transformed and a total of 371 independently transformed tobacco plants were regenerated in this study. This highly reproducible protocol could be used to produce transgenic tobacco plants expressing useful traitsor can be utilized as model system to study the expression of particular genes.

Involvement of cytosolic ascorbate peroxidase and Cu/Zn-superoxide dismutase for improved tolerance against drought stress

In order to understand the role of cytosolic antioxidant enzymes in drought stress protection, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants overexpressing cytosolic Cu/Zn-superoxide dismutase (cytsod) (EC 1.15.1.1) or ascorbate peroxidase (cytapx) (EC 1.11.1.1) alone, or in combination, were produced and tested for tolerance against mild water stress. The results showed that the simultaneous overexpression of Cu/Znsod and apx or at least apx in the cytosol of transgenic tobacco plants alleviates, to some extent, the damage produced by water stress conditions. This was correlated with higher water use efficiency and better photosynthetic rates. In general, oxidative stress parameters, such as lipid peroxidation, electrolyte leakage, and H(2)O(2) levels, were higher in non-transformed plants than in transgenic lines, suggesting that, at the least, overexpression of cytapx protects tobacco membranes from water stress. In these conditions, the activity of other antioxidant enzymes was induced in transgenic lines at the subcellular level. Moreover, an increase in the activity of some antioxidant enzymes was also observed in the chloroplast of transgenic plants overexpressing cytsod and/or cytapx. These results suggest the positive influence of cytosolic antioxidant metabolism on the chloroplast and underline the complexity of the regulation network of plant antioxidant defences during drought stress.

Rice sodium-insensitive potassium transporter, OsHAK5, confers increased salt tolerance in tobacco BY2 cells

Potassium ion (K(+)) plays vital roles in many aspects of cellular homeostasis including competing with sodium ion (Na(+)) during potassium starvation and salt stress. Therefore, one way to engineer plant cells with improved salt tolerance is to enhance K(+) uptake activity of the cells, while keeping Na(+) out during salt stress. Here, in search for Na(+)-insensitive K(+) transporter for this purpose, bacterial expression system was used to characterize two K(+) transporters, OsHAK2 and OsHAK5, isolated from rice (Oryza sativa cv. Nipponbare). The two OsHAK transporters are members of a KT/HAK/KUP transporter family, which is one of the major K(+) transporter families in bacteria, fungi and plants. When expressed in an Escherichia coli K(+) transport mutant strain LB2003, both OsHAK transporters rescued the growth defect in K(+)-limiting conditions by significantly increasing the K(+) content of the cells. Under the condition with a large amount of extracellular Na(+), we found that OsHAK5 functions as a Na(+)-insensitive K(+) transporter, while OsHAK2 is sensitive to extracellular Na(+) and exhibits higher Na(+) over K(+) transport activities. Moreover, constitutive expression of OsHAK5 in cultured-tobacco BY2 (Nicotiana tabacum cv. Bright Yellow 2) cells enhanced the accumulation of K(+) but not Na(+) in the cells during salt stress and conferred increased salt tolerance to the cells. Transient expression experiment indicated that OsHAK5 is localized to the plant plasma membrane. These results suggest that the plasma-membrane localized Na(+) insensitive K(+) transporters, similar to OsHAK5 identified here, could be used as a tool to enhance salt tolerance in plant cells.

Characterization and Expression Analysis of Four Glycine-Rich RNA-Binding Proteins Involved in Osmotic Response in Tobacco ( Nicotiana tabacum cv. Xanthi)

Plants have developed many signals and specific genes' regulations at both transcriptional and post-transcriptional levels in order to tolerate and adapt to various environmental stresses. RNA-binding proteins (RBPs) play crucial roles in the post-transcriptional regulation via mRNA splicing, polyadenylation, sequence editing, transport, mRNA stability, mRNA localization, and translation. In this paper, four cDNAs of glycine-rich RNA-binding proteins (GR-RBPs), named NtRGP-1a, -1b, -2, and -3, were isolated from Nicotiana tabacum by RT-PCR analysis, and special emphases were given to the sequences alignment, phylogenetic analysis and gene expression. Sequences alignment revealed minor difference of cDNA sequences, but no difference of deduced proteins between N. sylvestris and N. tabacum. Phylogenetic alignment revealed that four cDNAs in tobacco were clustered into two different groups. NtRGP-2 and -3 were evolutionarily closest to Arabidopsis GR-RBPs genes and related to animal GR-RBPs genes, while NtRGP-1a and -1b were closest to Gramineae GR-RBPs genes. The expression analyses of these four NtRGPs in response to different abiotic stressess showed the similar expression pattern. Moreover, the four NtRGPs, especially NtRGP-1a and NtRGP-3, were strongly induced by stresses including water, wound, cold, and high temperature, weakly induced by PEG, drought and SA, while reduced by NaCl and unaffected by ABA treatment. The fact that all of these abiotic stresses included in our experiments affected the water balance and resulted in osmotic stress on cellular level, suggests that NtRGPs in tobacco should be a family of crucial osmosis-related proteins, and may play a key role in signal transduction with ABA-independent pathway under abiotic stresses.

Enhanced Glutathione Metabolism Is Correlated with Sulfur-Induced Resistance in Tobacco mosaic virus–Infected Genetically Susceptible Nicotiana tabacum Plants

Sulfur-induced resistance, also known as sulfur-enhanced defense (SIR/SED) was investigated in Nicotiana tabacum cv. Samsun nn during compatible interaction with Tobacco mosaic virus (TMV) in correlation with glutathione metabolism. To evaluate the influence of sulfur nutritional status on virus infection, tobacco plants were treated with nutrient solutions containing either sufficient sulfate (+S) or no sulfate (-S). Sufficient sulfate supply resulted in a suppressed and delayed symptom development and diminished virus accumulation over a period of 14 days after inoculation as compared with -S conditions. Expression of the defense marker gene PR-1a was markedly upregulated in sulfate-treated plants during the first day after TMV inoculation. The occurrence of SIR/SED correlated with a higher level of activity of sulfate assimilation, cysteine, and glutathione metabolism in plants treated with sulfate. Additionally, two key genes involved in cysteine and glutathione biosynthesis (encoding adenosine 5'-phosphosulfate reductase and γ-glutamylcysteine synthetase, respectively) were upregulated within the first day after TMV inoculation under +S conditions. Sulfate withdrawal from the soil was accelerated at the beginning of the infection, whereas it declined in the long term, leading to an accumulation of sulfur in the soil of plants grown with sulfate. This observation could be correlated with a decrease in sulfur contents in TMV-infected leaves in the long term. In summary, this is the first study that demonstrates a link between the activation of cysteine and glutathione metabolism and the induction of SIR/SED during a compatible plant-virus interaction in tobacco plants, indicating a general mechanism behind SIR/SED.

Verapamil, a Calcium Channel Blocker, Induces Systemic Antiviral Resistance in Susceptible Plants

Plant viruses can cause serious crop losses. Calcium homoeostasis is involved in the movement of animal viruses. We have examined whether intracellular calcium flux can interfere with spread of virus in plants. The calcium channel blocker verapamil, applied to Nicotiana tabacum cv. Xanthi-nc plant leaves, interfered with Tobacco mosaic virus infection in treated and untreated leaves, reducing TMV lesion number by 68 and 71%, respectively. Verapamil interfered with calcium homoeostasis of leaf cells, evident by increased calcium efflux from leaf segments. This is a first effort to use calcium channel blockers as an inducer of systemic virus resistance in plants.

An Easy Method for Agrobacterium tumefaciens-Mediated Gene Transfer to Nicotiana tabacum cv. TAPM26

Research in Agrobacterium tumefaciens mediated gene transfer has advanced and opened new avenue for plant improvement via introduction of various beneficial genes. Current study investigates transformation parameters during co-cultivation to improve T-DNA delivery into Nicotiana tabacum cv. TAPM 26 by monitoring GUS expression level. The techniques involved basic plant tissue culture and establishment of plant transformation systems. Conditions assessed were bacterial inoculum concentration, infection period, wounding and pre-culture of explants, acetosyringone (AS) concentration and co-cultivation temperature. Optimized conditions resulted in high transformation efficiency at transient level were as follows; Agrobacterium tumefaciens growth phase of A600nm 0.8, infection period of 30 min, pre-culture of wounded explants prior to infection, addition of acetosyringone (AS) in bacterial growth culture (100 µM) and in co-cultivation medium (200 µM) and co-cultivation temperature of 22°C. Although higher density bacterial culture used for the infection process gave higher transformation rate, however, it compromised the viability of the explants. On the other hand, dilution of bacterial suspension reduced necrosis in explants and improved transformation events greatly. The transformation efficiency was increased 9 fold when the infection process was carried out at low temperature of 22°C. Current study has proven among the parameters assessed, temperature was the critical factor during co-cultivation process in Agrobacterium tumefaciens mediated gene transfer.

Biochemical properties of the matrix metalloproteinase NtMMP1 from Nicotiana tabacum cv. BY-2 suspension cells

A zinc-dependent matrix metalloproteinase (NtMMP1) found in the plasma membrane of Nicotiana tabacum cv. Bright Yellow 2 (BY-2) suspension cells is thought to be responsible for the degradation of recombinant proteins secreted into the culture supernatant. We have characterized the proteolytic activity of NtMMP1 by expressing a recombinant derivative lacking the C-terminal transmembrane domain in yeast. After purifying the protein by affinity chromatography, its autocatalytic activity was analyzed using monoclonal antibodies raised against its N-terminal and C-terminal portions. Both the unprocessed and processed forms of NtMMP1 displayed caseinolytic activity and N-terminal sequencing identified an autocatalytic cleavage site within the sequence motif HFSFFP, which is similar to the corresponding sequences of the human matrix metalloproteinases stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10). Unlike all other matrix metalloproteinases investigated so far, NtMMP1 contains a disulfide bond within its propeptide thus rendering the proenzyme catalytically active. Kinetic analysis of NtMMP1 with a synthetic substrate revealed a K(m) of 10.55 +/- 0.9 microM, a k(cat) of 0.6 +/- 0.01 s(-1) and maximum activity at pH 7.5. We found that NtMMP1 degrades Desmodus rotundus salivary plasminogen activator alpha 1 (DSPAalpha1), a biopharmaceutical protein, that has proven difficult to produce in tobacco BY-2 cells. This provides a likely explanation for the frequent instability of secreted recombinant biopharmaceuticals produced in plant suspension cell cultures. Our data suggest new avenues that can be explored to improve the production of pharmaceutical proteins in plants and plant cells.

First Report of Tomato spotted wilt virus in Sweet Pepper in Taiwan.

A new disorder on pepper showing symptoms of chlorosis and chlorotic spots on leaves was observed in sweet pepper (Capsicum annuum cv. Andalus) fields in Ren-Ai Township, Nantou County in July, 2009. The disorder occurred in more than 30% of the pepper plants, with a height of approximately 40 cm (1.5 feet), which was approximately one-half the size of the asymptomatic ones. Symptomatic plants bore much smaller fruits with abnormal shapes. Three symptomatic sweet pepper plants were collected and tested for potential viruses. Reverse transcription (RT)-PCR was performed for the detection using three degenerate primer pairs, gL3637/gL4435c for tospoviruses (2), Hrp5/Pot1 for potyviruses (1,3), and Tob-Uni1/Tob-Uni2 for tobamoviruses (4), and specific primers, FJJ2001-7/FJJ2001-8 (5'-TATGTCCATGGACAAATCCGAATCA and 5'-TCTCTGGATCCACGAGTTCAAACTGGGAG) for the coat protein gene of Cucumber mosaic virus (CMV). An 819-nt DNA fragment containing the partial L RNA of tospovirus was amplified from the total RNA isolated from each of these three samples by RT-PCR with primer pair gL3637/gL4435c. One amplified fragment was cloned and sequenced. A homology search in GenBank indicated that the new pepper-infecting virus in Taiwan was Tomato spotted wilt virus (TSWV) since the partial L RNA shared more than 94.5% nucleotide and 98.2% amino acid identity with five TSWV isolates (Accession Nos. AB190813, AB198742, AY070218, D10066, and NC_002052). No DNA fragment was obtained by RT-PCR using primer pairs for CMV, potyviruses, or tobamoviruses. A virus culture (TwPep1) isolated from one of the symptomatic sweet pepper plants was then established in Nicotiana tabacum cv. White Burley and N. benthamiana through triple single-lesion isolation. TWPep1 reacted positively only to the antiserum against TSWV by indirect-ELISA but not to those of Watermelon silver mottle virus, Capsicum chlorosis virus, Tobacco mosaic virus, Tomato mosaic virus, and CMV. Partial L RNA and the full-length nucleocapsid (N) gene of TWPep1 were obtained by RT-PCR with primer pairs gL3637/gL4435c and FJJ2002-74/FJJ2002-75 (5'-GCGCGCGGATCCTAATTTAACTTACARCTGCT 5'-TGCTGCCTCGAGCATACGGTCAAAGCATATAA), respectively. The 819-nt L RNA conserved region of TwPep1 (Accession No. GU222652) shared 94.4 to 97.7% nucleotide and 98.2 to 100% amino acid identity with those available in GenBank. The 777-bp N gene of TwPep1 (Accession No. GU222651) shared 96.7 to 99.1% nucleotide and 97.3 to 99.6% amino acid identity with 37 TSWV isolates available in GenBank. Sequence comparisons indicated that TwPep1 is an isolate of TSWV. TSWV was later detected by RT-PCR in all 10 symptomatic samples of sweet pepper plants collected from five fields in August 2009. To our knowledge, this is the first report of TSWV in sweet pepper in Taiwan. This is also the first demonstration of isolation and characterization of TSWV in Taiwan although TSWV was once detected in lisianthus (Eustoma rusellianum) by RT-PCR (1) but the isolation was not successful then. The occurrence of TSWV in pepper will have a direct economic impact on the important vegetable and floral industry in Taiwan because TSWV reportedly comprises a wide host range.

Regulation of cyclic electron flow in C3 plants: differential effects of limiting photosynthesis at ribulose‐1,5‐bisphosphate carboxylase/oxygenase and glyceraldehyde‐3‐phosphate dehydrogenase

Cyclic electron flow around photosystem I (CEF1) is thought to augment chloroplast ATP production to meet metabolic needs. Very little is known about the induction and regulation of CEF1. We investigated the effects on CEF1 of antisense suppression of the Calvin-Benson enzymes glyceraldehyde-3-phosphate dehydrogenase (gapR), and ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit (SSU), in tobacco (Nicotiana tabacum cv. Wisconsin 38). The gapR, but not ssuR, mutants showed substantial increases in CEF1, demonstrating that specific intermediates, rather than slowing of assimilation, induce CEF1. Both types of mutant showed increases in steady-state transthylakoid proton motive force (pmf) and subsequent activation of the photoprotective q(E) response. With gapR, the increased pmf was caused both by up-regulation of CEF1 and down-regulation of the ATP synthase. In ssuR, the increased pmf was attributed entirely to a decrease in ATP synthase activity, as previously seen in wild-type plants when CO₂ levels were decreased. Comparison of major stromal metabolites in gapR, ssuR and hcef1, a mutant with decreased fructose 1,6-bisphosphatase activity, showed that neither the ATP/ADP ratio, nor major Calvin-Benson cycle intermediates can directly account for the activation of CEF1, suggesting that chloroplast redox status or reactive oxygen species regulate CEF1.

First Report of Cucumber mosaic virus in Paeonia lactifera in France.

Chinese peony (Paeonia lactiflora Pall.), a hardy ornamental plant of the family Paeoniaceae cultivated in gardens and for cut flower production, is frequently infected by Tobacco rattle virus (TRV) in the field. The virus usually induces severe mosaic and chlorotic ringspot symptoms in the leaves, decreasing the commercial value of cut flowers. TRV is routinely detected by mechanical inoculation onto Nicotiana tabacum cv Xanthi, where it induces typical necrotic local ringspots in 3 to 7 days, followed by a reverse transcription (RT)-PCR test (2). In 2004, Xanthi test plants inoculated with sap extracts from 4 of 36 P. lactiflora cv. Odile plants grown in a field plot in the region of Hyères (southeast France) showed systemic mosaic symptoms in addition to the TRV-typical response. In each case, Cucumber mosaic virus (CMV) was detected by the reactions of a range of inoculated plants (1), the observation of 30 nm isometric particles in crude leaf extracts with the electron microscope, and by positive reactions in double antibody sandwich (DAS)-ELISAs with specific polyclonal antibodies. In double-immunodiffusion analysis, these isolates were shown to belong to the group II of CMV isolates (3). ELISA of the peony plants confirmed the presence of CMV and revealed two additional infected plants in the spring of 2006. Following isolation from local lesions on Vigna unguiculata and multiplication in Xanthi tobacco plants, one of the isolates was used to inoculate manually or with Myzus persicae aphids 10 CMV-free plants of P. lactiflora cv. Odile obtained from meristem culture. Three months postinoculation, only three of the aphid-inoculated plants were CMV positive by DAS-ELISA. No change was observed at 1 year postinoculation and no symptoms have been observed, even in CMV-infected plants. CMV appears to be latent in P. lactiflora, therefore detection of CMV before vegetative propagation of the plants is advised because of the risks of synergism for symptoms with other viruses such as TRV. To our knowledge this is the first report of CMV in peony.

Riboflavin activates defense responses in tobacco and induces resistance against Phytophthora parasitica and Ralstonia solanacearum

The effects of riboflavin on defense responses and secondary metabolism in tobacco ( Nicotiana tabacum cv. NC89) cell suspensions and the effects of protecting tobacco seedlings against Phytophthora parasitica var. nicotianae and Ralstonia solanacearum were investigated. Defense responses elicited by riboflavin in tobacco cells included an oxidative burst, alkalinization of the extracellular medium, expression of 4 defense-related genes with different kinetics and intensities, and accumulation of 2 total phenolic compounds, scopoletin and lignin. When applied to tobacco plants challenged by P. parasitica and R. solanacearum, riboflavin treatment resulted in 47.9% and 48.0% protection, respectively. These results suggest that riboflavin can both induce a series of defense responses and secondary metabolism in cell suspensions and protect tobacco against P. parasitica and R. solanacearum.

Inhibition effect on tobacco mosaic virus and regulation effect on calreticulin of oligochitosan in tobacco by induced Ca 2+ influx

Oligochitosan induce plant resistance against plant virus via different signal transduction pathways. Here, the Ca 2+ signaling pathway was investigated by which oligochitosan elicited defense responses to tobacco mosaic virus (TMV) in tobacco ( Nicotiana tabacum cv. Huangmiaoyu nn) plants. The results showed that the multiplication and movement of TMV in tobacco plants were inhibited by oligochitosan. Oligochitosan also increased cytosolic free calcium ions and regulated the expression of calreticulin. Furthermore, the inhibitory effect on TMV and the regulation effect on calreticulin were depressed when blocking the Ca 2+ signaling pathway by EGTA. These results indicated that oligochitosan induced tobacco resistance to TMV through Ca 2+ signaling pathway.

Production of xylanase in transgenic tobacco for industrial use in bioenergy and biofuel applications

Xylanases are used in various agricultural and industrial applications. A synthetic, modified, codon-optimized xylanase gene (XynZ) from Clostridium thermocellum was expressed in transgenic tobacco plants. The coding sequence of XynZ was placed between the modified Mirabilis mosaic virus full-length transcript promoter with duplicated enhancer domains and the terminator sequence from the rbcSE9 gene. Three constructs were developed to evaluate XynZ expression levels by targeting gene products into the cytosol, intercellular space, or endoplasmic reticulum in transgenic plants. These chimeric genes, expressed in transgenic tobacco (Nicotiana tabacum cv. Samsun NN) were stably inherited in successive plant generations (R0, R1, and R2 progeny; primary, second, and third generation) as shown by molecular characterization (RT-PCR and qRT-PCR) and enzymatic assays. A Western blot analysis of plant extracts showed presence of a polypeptide of the expected size that cross-reacted with xylanase-specific antibodies. Transgenic plants were morphologically similar to wild-type plants and showed no deleterious effect due to transgene expression. The expressed xylanase was heat-stable, having optimum activity between 55°C and 75°C over a pH range of 5 to 5.6.

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.

Identification and charactherization of Lysobacter capsici strain PG4: a new plant health-promoting rhizobacterium.

A Gram-negative bacterial isolate, named PG4, was recovered from the rhizosphere of a tobacco plant (Nicotiana tabacum cv. Burley) and was identified as Lysobacter capsici based on the sequencing and phylogenetic analysis of 16S rRNA and characteristics in the Biolog Identification System. Strain PG4 showed chitinase and protease activity and was able to inhibit the growth of a broad range of phytopathogenic fungi. Growth of Gram-negative bacteria was not affected by PG4, but it was active against some Gram-positive bacteria such as Bacillus subtilis, Lysteria monocytogenes and Rhodococcus fascians. Fe3+ availability positively influenced PG4 antagonism towards the latter group of bacteria, even though siderophore production was not detected. Seed bacterization reduced the number of the resulting tomato seedlings infected by Fusarium oxysporum f. sp. lycopersici, F. oxysporum f. sp. radicis-lycopersici and Rhizoctonia solani. In greenhouse trials strain PG4 drastically reduced the incidence of tomato crown and root rot and led to a marked increase in plant fresh weight.