Transgenic Tobacco Lines Research Articles

Rice lectin protein r40c1 imparts drought tolerance by modulating S-adenosylmethionine synthase 2, stress-associated protein 8 and chromatin-associated proteins.

Lectin proteins play an important role in biotic and abiotic stress responses in plants. Although the rice lectin protein Osr40c1 has been reported to be regulated by drought stress, the mechanism of its drought tolerance activity has not been studied so far. In this study, it is shown that expression of the Osr40c1 gene correlates with the drought tolerance potential of various rice cultivars. Transgenic rice plants overexpressing Osr40c1 were significantly more tolerant to drought stress than the wild-type plants. Furthermore, ectopic expression of the Osr40c1 gene in tobacco yielded a similar result. Interestingly, the protein displayed a nucleo-cytoplasmic localization and was found to interact with a number of drought-responsive proteins such as S-adenosylmethionine synthase 2 (OsSAM2), stress-associated protein 8 (OsSAP8), DNA-binding protein MNB1B (OsMNB1B), and histone 4 (OsH4). Silencing of each of these protein partners led to drought sensitivity in otherwise tolerant Osr40c1-expressing transgenic tobacco lines indicating that these partners were crucial for the Osr40c1-mediated drought tolerance in planta. Moreover, the association of Osr40c1 with these partners occurred specifically under drought stress forming a multi-protein complex. Together, our findings delineate a novel role of Osr40c1 in imparting drought tolerance by regulating OsMNB1B, OsSAM2, and OsH4 proteins, which presumably enables OsSAP8 to induce downstream gene expression.

The bacterial potassium transporter gene MbtrkH improves K+ uptake in yeast and tobacco.

K+ is an essential nutrient for plant growth and is responsible for many important physiological processes. K+ deficiency leads to crop yield losses, and overexpression of K+ transporter genes has been proven to be an effective way to resolve this problem. However, current research on the overexpression of K+ transporter genes is limited to plant sources. TrkH is a bacterial K+ transporter whose function generally depends on the regulation of TrkA. To date, whether TrkH can improve K+ uptake in eukaryotic organisms is still unknown. In this study, a novel MbtrkH gene was cloned from marine microbial metagenomic DNA. Functional complementation and K+-depletion analyses revealed that MbTrkH functions in K+ uptake in the K+-deficient yeast strain CY162. Moreover, K+-depletion assays revealed that MbtrkH overexpression improves plant K+ uptake. K+ hydroponic culture experiments showed that, compared with WT tobacco lines, MbtrkH transgenic tobacco lines had significantly greater fresh weights, dry weights and K+ contents. These results indicate that MbTrkH promotes K+ uptake independently of TrkA in eukaryotes and provide a new strategy for improving K+-use efficiency in plants.

Overexpression of a multifunctional β-glucosidase gene from thermophilic archaeon Sulfolobus solfataricus in transgenic tobacco could facilitate glucose release and its use as a reporter.

The β-glucosidase, which hydrolyzes the β(1-4) glucosidic linkage of disaccharides, oligosaccharides and glucose-substituted molecules, has been used in many biotechnological applications. The current commercial source of β-glucosidase is mainly microbial fermentation. Plants have been developed as bioreactors to produce various kinds of proteins including β-glucosidase because of the potential low cost. Sulfolobus solfataricus is a thermoacidophilic archaeon that can grow optimally at high temperature, around 80°C, and pH 2-4. We overexpressed the β-glucosidase gene from S. solfataricus in transgenic tobacco via Agrobacteria-mediated transformation. Three transgenic tobacco lines with β-glucosidase gene expression driven by the rbcS promoter were obtained, and the recombinant proteins were accumulated in chloroplasts, endoplasmic reticulum and vacuoles up to 1%, 0.6% and 0.3% of total soluble protein, respectively. By stacking the transgenes via crossing distinct transgenic events, the level of β-glucosidase in plants could further increase. The plant-expressed β-glucosidase had optimal activity at 80°C and pH 5-6. In addition, the plant-expressed β-glucosidase showed high thermostability; on heat pre-treatment at 80°C for 2h, approximately 70% residual activity remained. Furthermore, wind-dried leaf tissues of transgenic plants showed good stability in short-term storage at room temperature, with β-glucosidase activity of about 80% still remaining after 1week of storage as compared with fresh leaf. Furthermore, we demonstrated the possibility of using the archaebacterial β-glucosidase gene as a reporter in plants based on alternative β-galactosidase activity.

Isolation and functional characterization of the mitogen-activated protein kinase kinase gene MAPKK1 from Panax notoginseng

Mitogen-activated protein kinase (MAPK) cascades are important signal transduction mechanisms that connect plant cellular and nuclear responses, which play key roles in plant development and stress responses. Here, the novel MAPK kinase (MAPKK) gene MAPKK1 was isolated from Panax notoginseng (Burk) F.H. Chen. Exogenous methyl jasmonate (MeJA), salicylic acid (SA), ethylene, and hydrogen peroxide treatments induced the transcription level of PnMAPKK1 in P. notoginseng roots. Additionally, PnMAPKK1 expression actively responded to Fusarium solani infection, a major causal agent of P. notoginseng root rot disease. The PnMAPKK1 gene was further fused with the green fluorescent protein gene in a plant expression vector and transformed into onion (Allium cepa) epidermal cells. Laser scanning confocal microscopy confirmed that the PnMAPKK1 protein localized in the cytoplasm. In addition, the plant overexpression vector pCAMBIA2300s-PnMAPKK1 was constructed and transformed into tobacco (Nicotiana tabacum L. cv. Xanthi). The PnMAPKK1 transgenic tobacco lines showed much stronger resistance levels to F. solani infection than the wild type. Moreover, the JA and SA contents in the PnMAPKK1 transgenic tobacco lines were significantly higher than those in wild type during F. solani infection. The up-regulation of several marker genes (PAL4, PR1c, WRKY1, Defensin, and LOC4) involved in JA and SA signaling was correlated with the overexpression of PnMAPKK1 in tobacco. Thus, the PnMAPKK1 gene is a positive regulator of defense responses to F. solani in P. notoginseng.

Expression of Pinellia ternata leaf agglutinin under rolC promoter confers resistance against a phytophagous sap sucking aphid, Myzus persicae

Background: Piercing/sucking insect pests in the order Hemiptera causes substantial crop losses by removing photoassimilates and transmitting viruses to their host plants. Cloning and heterologous expression of plant-derived insect resistance genes is a promising approach to control aphids and other sap-sucking insect pests. While expression from the constitutive 35S promoter provides broad protection, the phloem-specific rolC promoter provides better defense against sap sucking insects. The selection of plant-derived insect resistance genes for expression in crop species will minimize bio-safety concerns. Results: Pinellia ternata leaf agglutinin gene ( pta ), encodes an insecticidal lectin, was isolated and cloned under the 35S and rolC promoters in the pGA482 plant transformation vector for Agrobacterium -mediated tobacco transformation. Integration and expression of the transgene was validated by Southern blotting and qRT-PCR, respectively. Insect bioassays data of transgenic tobacco plants showed that expression of pta under rolC promoter caused 100% aphid mortality and reduced aphid fecundity up to 70% in transgenic tobacco line LRP-9. These results highlight the better effectivity of pta under rolC promoter to control phloem feeders, aphids. Conclusions: These findings suggested the potential of PTA against aphids and other sap sucking insect pests. Evaluation of gene in tobacco under two different promoters; 35S constitutive promoter and rolC phloem-specific promoter could be successfully use for other crop plants particularly in cotton. Development of transgenic cotton plants using plant-derived insecticidal, PTA, would be key step towards commercialization of environmentally safe insect-resistant crops. Normal 0 false false false EN-US X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi;}

Ectopic expression of a poplar gene NAC13 confers enhanced tolerance to salinity stress in transgenic Nicotiana tabacum.

NACs are one of the major transcription factor families in plants which play an important role in plant growth and development, as well as in adverse stress responses. In this study, we cloned a salt-inducible NAC transcription factor gene (NAC13) from a poplar variety 84K, followed by transforming it into both Nicotiana tabacum and Arabidopsis thaliana. Stable expression analysis of 35S::NAC13-GFP fusion protein in Arabidopsis indicated that NAC13 protein was localized to the nucleus. We also obtained five transgenic tobacco lines. Evidence from morphological and physiological characterization and salt treatment analyses indicated that in the transgenic tobacco the salt tolerance was enhanced, suggesting that NAC13 gene may function as a positive regulator in tobacco responses to salt stress.

A Lobularia maritima LmSAP protein modulates gibberellic acid homeostasis via its A20 domain under abiotic stress conditions.

Stress-associated proteins (SAPs) are favorable targets to improve stress tolerance in plants, owing to their roles in developmental processes and stress responses. However, the role of SAPs and the molecular mechanisms by which they regulate plant stress responses remain poorly understood. Previously, it was reported that LmSAP expression was upregulated by various abiotic stressors in Lobularia maritima, and that transgenic tobacco lines with constitutively expressed LmSAPΔA20 and LmSAPΔA20-ΔAN1 showed dwarf phenotypes due to the deficiency of cell elongation under salt and osmotic stresses. In this study, we examined the function of A20 domain in the GA pathway in response to abiotic stresses. Transient expression of acGFP-LmSAPΔA20 and acGFP-LmSAPΔA20-ΔAN1 in onion epidermal cells demonstrated that these fused proteins were localized in the nucleo–cytoplasm. However, the truncated form acGFP-LmSAPΔAN1 was localized in the nucleus. Moreover, comparison of native and truncated LmSAP showed dramatic structural changes caused by the deletion of the A20 domain, leading to loss of function and localization. Interestingly, overexpression LmSAP and truncated LmSAPΔAN1 led to up-regulation of GA biosynthetic genes and increased total gibberellins (GAs) content, corresponding with accelerated development in transgenic tobacco plants. Moreover, the dwarf phenotype of the transgenic lines that express LmSAPΔA20 and LmSAPΔA20-ΔAN1 under stress conditions was fully restored by the application of exogenous GA3. These findings improve our understanding of the role of LmSAP in regulating GA homeostasis, which is important for regulating plant development under abiotic stress conditions.

Contribution of NtZIP1-like, NtZIP11 and a WAK-pectin based mechanism to the formation of Zn-related lesions in tobacco leaves

Previous studies on tobacco (Nicotiana tabacum var. Xanthi) have shown that in the presence of 200 μM Zn, lesions develop on the leaf blades from Zn accumulating mesophyll cells. This was interpreted as a tolerance mechanism against high Zn, protecting non-accumulating photosynthesizing cells. Underlying this phenomenon is the distinct capacity of mesophyll cells for Zn accumulation. To learn more about the involved mechanisms, here we searched for two key elements. First, for Zn transporters contributing to Zn accumulation in groups of mesophyll cells. Tissue-specific expression of two ZIP transporters previously identified in the leaves of tobacco plants exposed to high Zn was examined. Transgenic tobacco lines expressing GUS reporter gene under 2156 bp and 1922 bp of the promoter region of NtZIP1-like and NtZIP11, respectively, were used for analysis. Promoter activity of both genes in the leaves of high Zn-exposed plants was detected in groups of mesophyll cells, which correlates with the pattern of Zn accumulation. Second, we searched for putative components signalling the status of the cell wall. The WAKs/WAKLs and pectin fractions were the focus in the study, as it was known that upon binding pectin, WAK/WAKLs directly signals from the cell wall to the cytoplasm. Pectin microarray identified rhamnogalacturonan I (RG I) as the only pectin fraction that increased in the apoplast at high Zn (relative to control conditions), and immunocytochemistry demonstrated its presence in groups of cells. This was accompanied by increased expression of NtWAK2P-2 (putative NtWAK2), NtWAK4P-4, and NtWAK4P-2. Based on these data, a hypothesis was forwarded that RG I generated in the cell wall upon exposure to high Zn might bind to the extracellular domain of NtWAK2P-2, NtWAK4P-4, and/or NtWAK4P-2, which subsequently could transmit a signal on Zn status into the cytoplasm. It was also considered that regulation of NtZIP1-like and NtZIP11 expression by Zn availability might depend on the interaction between the RG I and/or NtWAK2P-2-, NtWAK4P-4, NtWAK4P-2.

Isolation and functional validation of stress tolerant EaMYB18 gene and its comparative physio-biochemical analysis with transgenic tobacco plants overexpressing SoMYB18 and SsMYB18.

MYB transcription factors are one of the most important mediators for the survival of plants under multiple stress responses. In the present study, EaMYB18, encoding a single R3 repeat MYB DNA binding domain was isolated from stress-tolerant wild relative species of sugarcane Erianthus arundinaceus. In silico analysis of 948bp coding mRNA sequence of EaMYB18 exhibited the presence of four exons and three introns. Further, the EaMYB18 gene was transformed in tobacco and its stable inheritance was confirmed through antibiotic resistance screening, PCR amplification and Southern hybridization blotting. Results of the estimation of MDA, proline, total chlorophyll and antioxidant activities of EaMYB18 transgenic tobacco lines exhibited least oxidative damage under drought and cold stress over the untransformed ones, the over-expression of EaMYB18 has improved drought and cold stress tolerance ability in tobacco. The comparative physiological and biochemical analysis of transgenic tobacco plants overexpressing SoMYB18, SsMYB18 and EaMYB18, revealed that the EaMYB18 and SsMYB18 transgenic plants demonstrated effective tolerance to drought and cold stresses, while SoMYB18 showed improved tolerance to salt stress alone. Amongst these three genes, EaMYB18 displayed the highest potential for drought and cold stress tolerances as compared to SoMYB18 and SsMYB18 genes.

Physiological and quantitative proteomic analysis of NtPRX63-overexpressing tobacco plants revealed that NtPRX63 functions in plant salt resistance

High salinity is harmful to crop yield and productivity. Peroxidases (PRXs) play crucial roles in H2O2 scavenging. In our previous study, PRX63 significantly upregulated in tobacco plants under salt stress. Thus, in order to understand the function of PRX63 in tobacco salt response, we overexpressed this gene in tobacco (Nicotiana tabacum L.), investigated the morphological, physiological and proteomic profiles of NtPRX63-overexpressing tobacco transgenic lines and wild type. The results showed that, compared with the wild type, the transgenic tobacco plants presented enhanced salt tolerance and displayed lower ROS (reactive oxygen species), malondialdehyde (MDA) and Na+ contents; higher biomass, potassium content, soluble sugar content, and peroxidase activity; and higher expression levels of NtSOD, NtPOD and NtCAT. Protein abundance analysis revealed 123 differentially expressed proteins between the transgenic and wild-type plants. These proteins were functionally classified into 18 categories and are involved in 41 metabolic pathways. Furthermore, among the 123 proteins, eight proteins involved in the ROS-scavenging system, 12 involved in photosynthesis and energy metabolism processes, two stress response proteins, one signal transduction protein and one disulfide isomerase were significantly upregulated. Furthermore, three novel proteins that may be involved in the plant salt response were also identified. The results of our study indicate that an enhanced ROS-scavenging ability, together with the expression of proteins related to energy mobilization and the stress response, functions in the confirmed salt resistance of transgenic tobacco plants. Our data provide valuable information for research on the function of NtPRX63 in tobacco in response to abiotic stress.

Alfalfa MsCBL4 enhances calcium metabolism but not sodium transport in transgenic tobacco under salt and saline-alkali stress.

MsCBL4 expression in tobacco enhanced its salt and saline-alkali stress tolerance by regulating calcium accumulation in roots, indicating the important role of calcium metabolism in plant saline-alkali stress tolerance The calcineurin B-like (CBL) family of proteins play important roles in plant abiotic stress tolerance and signal transduction. CBL4 is known to participate in the Salt Overly Sensitive pathway; however, little is currently known regarding the mechanisms underlying the response of CBL4 to saline-alkali stress. In this study, we cloned and characterized the alfalfa MsCBL4 gene. We found that MsCBL4 showed the highest expression in root tissues and was induced by salt and saline-alkali stress, with the latter causing higher induction. Overexpression of MsCBL4 in tobacco enhanced salt and saline-alkali stress tolerance and reduced the Na+/K+ ratio in roots of transgenic lines. Salt (30 and 300mM NaCl) and saline-alkali (30mM NaHCO3) stress assays performed for MsCBL4 transgenic tobacco lines revealed a substantial influx of sodium ions in roots under saline-alkali stress and indicated that the expression of MsCBL4 had little influence on sodium ion content reduction. In contrast, in roots subjected to saline-alkali stress, calcium accumulation occurred and was significantly enhanced by the overexpression of MsCBL4. Physiological and biochemical analyses indicated that MsCBL4 plays an important role in saline-alkali stress tolerance via its influence on the regulation of calcium transport and accumulation. These results provide novel insights into the saline-alkali stress tolerance mechanisms of plants.

Quantitative Proteomic Analysis of Alligator Weed Leaves Reveals That Cationic Peroxidase 1 Plays Vital Roles in the Potassium Deficiency Stress Response

Alligator weed is reported to have a strong ability to adapt to potassium deficiency (LK) stress. Leaves are the primary organs responsible for photosynthesis of plants. However, quantitative proteomic changes in alligator weed leaves in response to LK stress are largely unknown. In this study, we investigated the physiological and proteomic changes in leaves of alligator weed under LK stress. We found that chloroplast and mesophyll cell contents in palisade tissue increased, and that the total chlorophyll content, superoxide dismutase (SOD) activity and net photosynthetic rate (PN) increased after 15 day of LK treatment, but the soluble protein content decreased. Quantitative proteomic analysis suggested that a total of 119 proteins were differentially abundant proteins (DAPs). KEGG analysis suggested that most represented DAPs were associated with secondary metabolism, the stress response, photosynthesis, protein synthesis, and degradation pathway. The proteomic results were verified using parallel reaction monitoring mass spectrometry (PRM–MS) analysis and quantitative real-time PCR (qRT-PCR)assays. Additional research suggested that overexpression of cationic peroxidase 1 of alligator weed (ApCPX1) in tobacco increased LK tolerance. The seed germination rate, peroxidase (POD) activity, and K+ content increased, and the hydrogen peroxide (H2O2) content decreased in the three transgenic tobacco lines after LK stress. The number of root hairs of the transgenic line was significantly higher than that of WT, and net K efflux rates were severely decreased in the transgenic line under LK stress. These results confirmed that ApCPX1 played positive roles in low-K+ signal sensing. These results provide valuable information on the adaptive mechanisms in leaves of alligator weed under LK stress and will help identify vital functional genes to apply to the molecular breeding of LK-tolerant plants in the future.

Effect of Changes in Genome Ploidy on the Mosaic Character of nptII Gene Expression in Epialleles of the Transgenic Tobacco Line Nu21

The influence of changes in the genome ploidy and the gene dose on the mosaic pattern of expression of the nptII gene in epialleles of the Nu21 transgenic tobacco line was evaluated. The general preservation of the expression profile of the marker gene nptII is shown (high in the Nu5 epiallele and low in the Nu6 epiallele) in heteroploid crosses of diploid transgenic plants (2n = 48) with polyploid nontransgenic plants (3n = 72, 4n = 96), as well as in self-pollination of experimentally obtained tetraploid transgenic plants of the Nu21 line (4n = 96) and in their hybrids from crossing with nontransgenic diploid plants. It has been established that the stability of expression of the nptII marker gene is influenced by the initial epigenetic status of epialleles and the direction of crossing in hybridization of tetraploid nontransgenic tobacco plants with diploid transgenic plants of the Nu21 line.

Evidence of a significant role of glutathione reductase in the sulfur assimilation pathway.

With the objective of studying the role of glutathione reductase (GR) in the accumulation of cysteine and methionine, we generated transgenic tobacco and Arabidopsis lines overexpressing the cytosolic AtGR1 and the plastidic AtGR2 genes. The transgenic plants had higher contents of cysteine and glutathione. To understand why cysteine levels increased in these plants, we also used gr1 and gr2 mutants. The results showed that the transgenic plants have higher levels of sulfite, cysteine, glutathione and methionine, which are downstream to adenosine 5' phosphosulfate reductase (APR) activity. However, the mutants had lower levels of these metabolites, while the sulfate content increased. A feeding experiment using 34 SO42- also showed that the levels of APR downstream metabolites increased in the transgenic lines and decreased in gr1 compared with their controls. These findings, and the results obtained from the expression levels of several genes related to the sulfur pathway, suggest that GR plays an essential role in the sulfur assimilation pathway by supporting the activity of APR, the key enzyme in this pathway. GR recycles the oxidized form of glutathione (GSSG) back to reduce glutathione (GSH), which serves as an electron donor for APR activity. The phenotypes of the transgenic plants and the mutants are not significantly altered under non-stress and oxidative stress conditions. However, when germinating on sulfur-deficient medium, the transgenic plants grew better, while the mutants were more sensitive than the control plants. The results give substantial evidence of the yet unreported function of GR in the sulfur assimilation pathway.

Агробактериальная трансформация табака генетическим модулем биосинтеза антималярийного агента артемизинина

Artemisinin-based medicines are the most effective treatment for malaria. To date, the wormwood plants (Artemisia annua L.) are the main source of artemisinin. Due to the limited nature of this source, considerable efforts are directed towards the development of methods for artemisinin production via heterologous expression systems. We used in this study agrobacterial transformation to transfer the genetic module of the artemisinin biosynthesis pathway into plants and then analyzed its transcription in a heterologous host. Tobacco plants were transformed with the artemisinin biosynthesis genes encoding amorpha-4,11-diene synthase, artemisin-aldehyde All(13) reductase, amorpha-4,11-diene monooxygenase, cytochrome P450 reductase from A. annua and yeast 3-hydroxy-3-methylglutaryl-coenzyme A reductase cloned in the pArtemC vector; farnesyl diphosphate synthase and aldehyde dehydrogenase were used to transform the plants as parts of vector p2356. As a result of transformation with the pArtemC and p2356 vectors, in twos transgenic lines with all target genes were obtained. Five genes of artemisinin biosynthesis and two genes of biosynthesis of its precursors were successfully transferred into the genome of transgenic tobacco lines as a result of the co-transformation with abovementioned vectors. Thus, the entire artemisinin biosynthesis pathway was first reconstructed in heterologous plants: the transcription of the artemisinin biosynthesis genes in the tobacco plants was shown via RT-PCR. The obtained results will be used in further research on expression systems for the production of artemisinin and other non-protein substances in heterologous host plants. artemisinin, malaria, metabolic engineering, tobacco, transgenic plants This work was supported by a Grant from the Russian Science Foundation no. 19-14-00190.

Expression of Arabidopsis Hexokinase in Tobacco Guard Cells Increases Water-Use Efficiency and Confers Tolerance to Drought and Salt Stress.

Abiotic stresses such as drought and saline water impose major limitations on plant growth. Modulation of stomatal behavior may help plants cope with such stresses by reducing both water loss and salt uptake. Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in guard cells’ sugar-sensing, mediating stomatal closure and coordinating photosynthesis with transpiration. We generated transgenic tobacco lines expressing the Arabidopsis hexokinase1 (AtHXK1) under the guard cell-specific promoter KST1 and examined those plants using growth room and greenhouse experiments. The expression of AtHXK1 in tobacco guard cells reduced stomatal conductance and transpiration by about 25% with no negative effects on photosynthesis or growth, leading to increased water-use efficiency. In addition, these plants exhibited tolerance to drought and salt stress due to their lower transpiration rate, indicating that improved stomatal function has the potential to improve plant performance under stress conditions.

Expression of V-PPase proton pump, singly or in combination with a NHX1 transporter, in transgenic tobacco improves copper tolerance and accumulation.

One of the most important strategies evolved by plants to tolerate heavy metals (HMs) is their sequestration into the vacuole. Recent studies have demonstrated that Cu sequestration into vacuole is dependent on the electrochemical gradient generated by vacuolar proton pumps: the V-H+-PPase and the V-H+-ATPase. In a previous study, we demonstrated that co-expression of V-H+-PPase and a sodium/proton antiporter genes, isolated from wheat, in transgenic tobacco plants significantly increases both H+ pumping activity of the endogenous V-H+-ATPase and V-H+-PPase compared to wild-type (WT) plants, all grown in the absence of stress. In the present study, we evaluated the effect of expression, in tobacco, of vacuolar proton pump, TaVP1, singly or in combination with sodium/proton antiporter, TaNHXS1, on copper (Cu) tolerance and accumulation. Results showed that, when subjected to Cu stress, TaVP1 single transgenic tobacco lines exhibited a more robust root system, greater biomass production, less chlorophyll loss, lower MDA and H2O2 production, and higher catalase activity and accumulated more Cu than did WT. Interestingly, double transgenic tobacco lines exhibited the best Cu tolerance and accumulation than either of the single TaVP1 transgenic lines or WT plants, when subjected to excess Cu. In fact, double transgenic lines accumulated 2.5-fold and 1.9-fold more Cu than did WT and single TaVP1 lines, respectively. Thus, these results clearly demonstrate the usefulness of expression of vacuolar proton pump alone or in combination with sodium/proton antiporter as novel strategy for Cu phytoremediation.

A β‐1,3‐glucanase gene from Panax notoginseng confers resistance in tobacco to Fusarium solani

β‐1,3‐glucanases are members of the pathogenesis‐related protein (PR)-2 family, and they play important roles in defense response in plants. A β‐1,3‐glucanase gene was isolated from Panax notoginseng and named as PnGlu1 in this study. This gene encodes a protein of 379 amino acids containing a potential signal peptide, and subcellular localization analysis confirmed that PnGlu1 protein was localized in the cell wall. The PnGlu1 expression level in P. notoginseng roots was evidently induced by Methyl jasmonate, salicylic acid, ethylene, and hydrogen peroxide treatment. Moreover, the PnGlu1 gene showed high transcription level after inoculation with Fusarium solani, which is the important agent of P. notoginseng root rot disease. In addition, the PnGlu1 recombinant protein expressed in Escherichia coli showed a high-level inhibitory activity against Fusarium solani and Fusarium oxysporum growth. Furthermore, the plant overexpression vector pCAMBIA2300s-PnGlu1 was constructed and overexpressed in tobacco (Nicotiana tabacum L. cv Xanthi). The transgenic tobacco plants showed strong resistance to F. solani infection. Additionally, the gene expression levels of some jasmonic acid biosynthesis genes and PRs were significantly up-regulated in the PnGlu1 transgenic tobacco lines. This study indicated that the PnGlu1 is a vital defense gene during response to F. solani.

Effects on Plant Growth and Reproduction of a Peach R2R3-MYB Transcription Factor Overexpressed in Tobacco.

In plants, anthocyanin production is controlled by MYB and bHLH transcription factors. In peach, among the members of these families, MYB10.1 and bHLH3 have been shown to be the most important genes for production of these pigments during fruit ripening. Anthocyanins are valuable molecules, and the overexpression of regulatory genes in annual fast-growing plants has been explored for their biotechnological production. The overexpression of peach MYB10.1 in tobacco plants induced anthocyanin pigmentation, which was particularly strong in the reproductive parts. Pigment production was the result of an up-regulation of the expression level of key genes of the flavonoid biosynthetic pathway, such as NtCHS, NtCHI, NtF3H, NtDFR, NtANS, and NtUFGT, as well as of the proanthocyanidin biosynthetic pathway such as NtLAR. Nevertheless, phenotypic alterations in transgenic tobacco lines were not only limited to anthocyanin production. Lines showing a strong phenotype (type I) exhibited irregular leaf shape and size and reduced plant height. Moreover, flowers had reduced length of anther’s filament, nondehiscent anthers, reduced pistil length, aborted nectary glands, and impaired capsule development, but the reproductive parts including androecium, gynoecium, and petals were more pigmented that in wild type. Surprisingly, overexpression of peach MYB10.1 led to suppression of NtMYB305, which is required for floral development and, of one of its target genes, NECTARIN1 (NtNCE1), involved in the nectary gland formation. MYB10.1 overexpression up-regulated JA biosynthetic (NtAOS) and signaling (NtJAZd) genes, as well as 1-aminocyclopropane-1-carboxylate oxidase (NtACO) in flowers. The alteration of these hormonal pathways might be among the causes of the observed floral abnormalities with defects in both male and female gametophyte development. In particular, approximately only 30% of pollen grains of type I lines were viable, while during megaspore formation, there was a block during FG1 (St3-II). This block seemed to be associated to an excessive accumulation of callose. It can be concluded that the overexpression of peach MYB10.1 in tobacco not only regulates flavonoid biosynthesis (anthocyanin and proanthocyanidin) in the reproductive parts but also plays a role in other processes such as vegetative and reproductive development.

Ectopic Expression of Multiple Chrysanthemum (Chrysanthemum × morifolium) R2R3-MYB Transcription Factor Genes Regulates Anthocyanin Accumulation in Tobacco.

The generation of chrysanthemum (Chrysanthemum × morifolium) flower color is mainly attributed to the accumulation of anthocyanins. In the anthocyanin biosynthetic pathway in chrysanthemum, although all of the structural genes have been cloned, the regulatory function of R2R3-MYB transcription factor (TF) genes, which play a crucial role in determining anthocyanin accumulation in many ornamental crops, still remains unclear. In our previous study, four light-induced R2R3-MYB TF genes in chrysanthemum were identified using transcriptomic sequencing. In the present study, we further investigated the regulatory functions of these genes via phylogenetic and alignment analyses of amino acid sequences, which were subsequently verified by phenotypic, pigmental, and structural gene expression analyses in transgenic tobacco lines. As revealed by phylogenetic and alignment analyses, CmMYB4 and CmMYB5 were phenylpropanoid and flavonoid repressor R2R3-MYB genes, respectively, while CmMYB6 was an activator of anthocyanin biosynthesis, and CmMYB7 was involved in regulating flavonol biosynthesis. Compared with wild-type plants, the relative anthocyanin contents in the 35S:CmMYB4 and 35S:CmMYB5 tobacco lines significantly decreased (p < 0.05), while for 35S:CmMYB6 and 35S:CmMYB7, the opposite result was obtained. Both in the 35S:CmMYB4 and 35S:CmMYB5 lines, the relative expression of several anthocyanin biosynthetic genes in tobacco was significantly downregulated (p < 0.05); on the contrary, several genes were upregulated in the 35S:CmMYB6 and 35S:CmMYB7 lines. These results indicate that CmMYB4 and CmMYB5 negatively regulate anthocyanin biosynthesis in chrysanthemum, while CmMYB6 and CmMYB7 play a positive role, which will aid in understanding the complex mechanism regulating floral pigmentation in chrysanthemum and the functional divergence of the R2R3-MYB gene family in higher plants.