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1.
J Plant Physiol ; 265: 153486, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34388688

RESUMO

Dwindling fossil fuel reserves and poor environmental credentials of chemical synthesis means, new renewable sources for the production and manufacture of valuable chemicals and pharmaceuticals are required. Presently, tobacco is an underutilised non-food crop with the potential to act as a biofactory. In this study, metabolite profiling across vegetative development has been carried out to provide a quantitative baseline of metabolites, their formation and interaction. Two tobacco platforms have been used, Nicotiana benthamiana and Nicotiana tabacum. Our data generated has provided the quantitative and qualitative baseline levels for exploitable pathways and metabolites, across two complementary Nicotiana species. N. benthamiana is the chassis of choice for transient expression. The metabolite data obtained for N. benthamiana highlighted that before flower emergence, the increased central carbon metabolism and high amino acid levels are available for the biosynthesis of endogenous or heterologous metabolites. In the future, engineering pathways or biocatalysts into N. benthamiana could add value to the process presently used to produce low volume, high cost pharmaceuticals. Similar outputs were obtained for N. tabacum, which has the advantage of providing a large biomass and hence, high product yield. These data provide an insight into the metabolite pools available in tobacco for future exploitation by emerging New Plant Breeding Techniques.


Assuntos
Desenvolvimento Vegetal/genética , Folhas de Planta/crescimento & desenvolvimento , Folhas de Planta/metabolismo , Metabolismo Secundário/genética , Tabaco/crescimento & desenvolvimento , Tabaco/genética , Tabaco/metabolismo , Biocombustíveis , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Variação Genética , Genótipo , Plantas Geneticamente Modificadas
2.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360584

RESUMO

Trichostatin A (TSA) is a representative histone deacetylase (HDAC) inhibitor that modulates epigenetic gene expression by regulation of chromatin remodeling in cells. To investigate whether the regulation of chromatin de-condensation by TSA can affect the increase in the efficiency of Cas9 protein-gRNA ribonucleoprotein (RNP) indel formation from plant cells, genome editing efficiency using lettuce and tobacco protoplasts was examined after several concentrations of TSA treatments (0, 0.1, 1 and 10 µM). RNP delivery from protoplasts was conducted by conventional polyethylene glycol (PEG) transfection protocols. Interestingly, the indel frequency of the SOC1 gene from TSA treatments was about 3.3 to 3.8 times higher than DMSO treatment in lettuce protoplasts. The TSA-mediated increase of indel frequency of the SOC1 gene in lettuce protoplasts occurred in a concentration-dependent manner, although there was not much difference. Similar to lettuce, TSA also increased the indel frequency by 1.5 to 1.8 times in a concentration-dependent manner during PDS genome editing using tobacco protoplasts. The MNase test clearly showed that chromatin accessibility with TSA treatments was higher than that of DMSO treatment. Additionally, TSA treatment significantly increased the level of histone H3 and H4 acetylation from lettuce protoplasts. The qRT-PCR analysis showed that expression of cell division-related genes (LsCYCD1-1, LsCYCD3-2, LsCYCD6-1, and LsCYCU4-1) was increased by TSA treatment. These findings could contribute to increasing the efficiency of CRISPR/Cas9-mediated genome editing. Furthermore, this could be applied for the development of useful genome-edited crops using the CRISPR/Cas9 system with plant protoplasts.


Assuntos
Sistemas CRISPR-Cas , Edição de Genes/métodos , Ácidos Hidroxâmicos/farmacologia , Alface/metabolismo , Proteínas de Plantas/metabolismo , Protoplastos/metabolismo , Tabaco/metabolismo , Divisão Celular , Genoma de Planta , Alface/efeitos dos fármacos , Alface/genética , Alface/crescimento & desenvolvimento , Células Vegetais , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Inibidores da Síntese de Proteínas/farmacologia , Protoplastos/efeitos dos fármacos , Tabaco/efeitos dos fármacos , Tabaco/genética , Tabaco/crescimento & desenvolvimento
3.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299210

RESUMO

Conjugation of phytohormones with glucose is a means of modulating their activities, which can be rapidly reversed by the action of ß-glucosidases. Evaluation of previously characterized recombinant rice ß-glucosidases found that nearly all could hydrolyze abscisic acid glucose ester (ABA-GE). Os4BGlu12 and Os4BGlu13, which are known to act on other phytohormones, had the highest activity. We expressed Os4BGlu12, Os4BGlu13 and other members of a highly similar rice chromosome 4 gene cluster (Os4BGlu9, Os4BGlu10 and Os4BGlu11) in transgenic Arabidopsis. Extracts of transgenic lines expressing each of the five genes had higher ß-glucosidase activities on ABA-GE and gibberellin A4 glucose ester (GA4-GE). The ß-glucosidase expression lines exhibited longer root and shoot lengths than control plants in response to salt and drought stress. Fusions of each of these proteins with green fluorescent protein localized near the plasma membrane and in the apoplast in tobacco leaf epithelial cells. The action of these extracellular ß-glucosidases on multiple phytohormones suggests they may modulate the interactions between these phytohormones.


Assuntos
Ácido Abscísico/farmacologia , Ésteres/química , Glucose/metabolismo , Oryza/enzimologia , Proteínas de Plantas/metabolismo , beta-Glucosidase/metabolismo , Ácido Abscísico/química , Arabidopsis/efeitos dos fármacos , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Secas , Giberelinas/farmacologia , Hidrólise , Família Multigênica , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Tabaco/efeitos dos fármacos , Tabaco/crescimento & desenvolvimento , Tabaco/metabolismo , beta-Glucosidase/genética
4.
Methods Mol Biol ; 2328: 253-259, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34251631

RESUMO

Enhancers are one of the main classes of cis-regulatory elements (CREs) in the regulation of plant gene expression. Plant enhancers can be predicted based on genomic signatures associated with open chromatin. However, predicted enhancers need to be validated experimentally. We developed an experimental system for rapid enhancer validation. Predicted enhancer candidates are cloned into a vector containing a minimal 35S promoter and a luciferase reporter gene. The construct is then agroinfiltrated into Nicotiana benthamiana leaves followed by bioluminescence signal detection and analysis. Positive bioluminescence signals indicate the enhancer function of each candidate, and the relative signal strength from different enhancers can be quantitatively measured and compared. In summary, we have developed an efficient and rapid plant enhancer validation assay based on a bioluminescent luciferase reporter and agroinfiltration-based N. benthamiana leaf transient expression. This assay can be used for the initial screening of candidate enhancers that are active in leaf tissue. The system can potentially be used to examine the activity of candidate enhancers under different environmental conditions.


Assuntos
Elementos Facilitadores Genéticos , Genes Reporter , Medições Luminescentes/métodos , Tabaco/metabolismo , Agrobacterium/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Vetores Genéticos , Luciferases/genética , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Imagem com Lapso de Tempo , Tabaco/genética , Tabaco/crescimento & desenvolvimento , Transformação Genética
5.
Methods Mol Biol ; 2288: 293-305, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34270019

RESUMO

Haploids are plants with gametophytic chromosome number, which upon chromosome duplication results in production of doubled haploids (DHs). There are several methods to obtain haploids and DHs, of which in vitro anther culture is the most effective and widely used method in tobacco. The production of haploids and DHs through androgenesis allows for a single-step development of complete homozygous lines from heterozygous genotypes, shortening the time required to produce homozygous genotypes in comparison to the conventional breeding scheme. The DH development process comprises two main steps: induction of androgenesis and duplication of the haploid genome. The critical stages of DH protocol in tobacco are determining the bud stage for anther culture, pretreatment, anther culture media, detection and identification of haploids, and chromosome doubling. Here we present an efficient anther culture protocol to get haploids and DHs in flue-cured virginia (FCV) tobacco. This optimized protocol can be used as a potential tool for generation of haploids and DHs for genetic improvement of tobacco.


Assuntos
Flores/crescimento & desenvolvimento , Melhoramento Vegetal/métodos , Técnicas de Embriogênese Somática de Plantas/métodos , Tabaco/crescimento & desenvolvimento , Meios de Cultura , Flores/genética , Haploidia , Pólen/genética , Pólen/crescimento & desenvolvimento , Tabaco/genética
6.
Int J Biol Macromol ; 184: 955-966, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-34153360

RESUMO

Hemoglobin-based oxygen carriers have long been pursued to meet clinical needs by using native hemoglobin (Hb) from human or animal blood, or recombinantly produced Hb, but the development has been impeded by safety and toxicity issues. Herewith we report the successful production of human fetal hemoglobin (HbF) in Nicotiana benthamiana through Agrobacterium tumefaciens-mediated transient expression. HbF is a heterotetrameric protein composed of two identical α- and two identical γ-subunits, held together by hydrophobic interactions, hydrogen bonds, and salt bridges. In our study, the α- and γ-subunits of HbF were fused in order to stabilize the α-subunits and facilitate balanced expression of α- and γ-subunits in N. benthamiana. Efficient extraction and purification methods enabled production of the recombinantly fused endotoxin-free HbF (rfHbF) in high quantity and quality. The transiently expressed rfHbF protein was identified by SDS-PAGE, Western blot and liquid chromatography-tandem mass spectrometry analyses. The purified rfHbF possessed structural and functional properties similar to native HbF, which were confirmed by biophysical, biochemical, and in vivo animal studies. The results demonstrate a high potential of plant expression systems in producing Hb products for use as blood substitutes.


Assuntos
Hemoglobina Fetal/genética , Oxigênio/metabolismo , Tabaco/genética , Hemoglobina Fetal/isolamento & purificação , Hemoglobina Fetal/metabolismo , Humanos , Ligação de Hidrogênio , Proteínas de Plantas/isolamento & purificação , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Tabaco/crescimento & desenvolvimento , Tabaco/metabolismo
7.
Methods Mol Biol ; 2317: 135-153, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028766

RESUMO

The protocol we report here is based on biolistic delivery of transforming DNA to tobacco leaves, selection of transplastomic clones by spectinomycin or kanamycin resistance and regeneration of plants with uniformly transformed plastid genomes. Because the plastid genome of Nicotiana tabacum derives from Nicotiana sylvestris, and the two genomes are highly conserved, vectors developed for N. tabacum can be used in N. sylvestris. The tissue culture responses of N. tabacum cv. Petit Havana and N. sylvestris accession TW137 are similar. Plastid transformation in a subset of N. tabacum cultivars and in Nicotiana benthamiana requires adjustment of the tissue culture protocol. We describe updated vectors targeting insertions in the unique and repeated regions of the plastid genome, vectors suitable for regulated gene expression by the engineered PPR10 RNA binding protein as well as systems for marker gene excision.


Assuntos
Genoma de Cloroplastos , Genomas de Plastídeos , Resistência a Canamicina/genética , Plastídeos/genética , Tabaco/genética , Transformação Genética , Transgenes , Marcadores Genéticos , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Espectinomicina/farmacologia , Tabaco/crescimento & desenvolvimento
8.
Methods Mol Biol ; 2317: 155-166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028767

RESUMO

Stable plastid transformation in Nicotiana tabacum has been achieved by using two different methods, the biolistic method, using a particle gun, and the polyethylene glycol (PEG)-mediated transformation. PEG-mediated plastid transformation involves the treatment of isolated protoplasts (plant cells without cell wall) with PEG in the presence of DNA. We have previously shown that in Nicotiana tabacum both methods are equally efficient. The PEG-mediated transformation efficiencies range between 20 and 50 plastid transformants per experiment (106 viable treated protoplasts). One advantage of the PEG method is that no expensive equipment such as a particle gun is required. The only crucial points are the handling and the cultivation of protoplasts. Furthermore, markers for the selection of transformed plastids are required. One of the most often used selection markers is the aadA gene which encodes for spectinomycin and streptomycin resistance. Here we describe a simplified and inexpensive protocol for the transformation of plastids in Nicotiana tabacum using an optimized protoplast culture protocol. PEG-mediated plastid transformation has the potential to be developed into a high-throughput, automated pipeline.


Assuntos
DNA de Plantas/genética , Resistência a Medicamentos , Plantas Geneticamente Modificadas/genética , Plastídeos/genética , Polietilenoglicóis/farmacologia , Tabaco/genética , Transformação Genética , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Tabaco/efeitos dos fármacos , Tabaco/crescimento & desenvolvimento
9.
Methods Mol Biol ; 2317: 195-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028770

RESUMO

The assimilation of CO2 within chloroplasts is catalyzed by the bifunctional enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase, Rubisco. Within higher plants the Rubisco large subunit gene, rbcL, is encoded in the plastid genome, while the Rubisco small subunit gene, RbcS is coded in the nucleus by a multigene family. Rubisco is considered a poor catalyst due to its slow turnover rate and its additional fixation of O2 that can result in wasteful loss of carbon through the energy requiring photorespiratory cycle. Improving the carboxylation efficiency and CO2/O2 selectivity of Rubisco within higher plants has been a long term goal which has been greatly advanced in recent times using plastid transformation techniques. Here we present experimental methodologies for efficiently engineering Rubisco in the plastids of a tobacco master line and analyzing leaf Rubisco content.


Assuntos
Dióxido de Carbono/metabolismo , Fotossíntese , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Plastídeos/genética , Ribulose-Bifosfato Carboxilase/metabolismo , Tabaco/metabolismo , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Ribulose-Bifosfato Carboxilase/genética , Tabaco/genética , Tabaco/crescimento & desenvolvimento
10.
Methods Mol Biol ; 2317: 177-193, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34028769

RESUMO

Here we describe a protocol for the excision of plastid marker genes directly in tobacco (Nicotiana tabacum) plants by the Cre recombinase. The example of the marker gene is the barau gene flanked by loxP sites in the plastid genome. For marker excision Agrobacterium encoding the recombinase on its T-DNA is injected at an axillary bud site of a decapitated plant, forcing shoot regeneration at the injection site. The excised plastid marker, the barau gene, confers a visual aurea leaf phenotype, thus marker excision via the flanking recombinase target sites is recognized by the restoration of normal green color of the leaves. The success of in planta plastid marker excision proves that manipulation of the plastid genomes is feasible within an intact plant. Extension of the protocol to in planta plastid transformation depends on the development of new protocols for the delivery of transforming DNA and the availability of visual marker genes.


Assuntos
Agrobacterium/metabolismo , Marcadores Genéticos , Integrases/metabolismo , Brotos de Planta/genética , Plastídeos/genética , Tabaco/genética , Transformação Genética , Agrobacterium/genética , Genes de Plantas , Engenharia Genética , Genomas de Plastídeos , Integrases/genética , Brotos de Planta/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Recombinação Genética , Tabaco/crescimento & desenvolvimento
11.
BMC Plant Biol ; 21(1): 226, 2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34020584

RESUMO

BACKGROUND: Abscission is an active, organized, and highly coordinated cell separation process enabling the detachment of aerial organs through the modification of cell-to-cell adhesion and breakdown of cell walls at specific sites on the plant body known as abscission zones. In Arabidopsis thaliana, abscission of floral organs and cauline leaves is regulated by the interaction of the hormonal peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), a pair of redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE2 (HSL2), and SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) co-receptors. However, the functionality of this abscission signaling module has not yet been demonstrated in other plant species. RESULTS: The expression of the pair of NbenIDA1 homeologs and the receptor NbenHAE.1 was supressed at the base of the corolla tube by the inoculation of two virus-induced gene silencing (VIGS) constructs in Nicotiana benthamiana. These gene suppression events arrested corolla abscission but did not produce any obvious effect on plant growth. VIGS plants retained a higher number of corollas attached to the flowers than control plants, an observation related to a greater corolla breakstrength. The arrest of corolla abscission was associated with the preservation of the parenchyma tissue at the base of the corolla tube that, in contrast, was virtually collapsed in normal corollas. In contrast, the inoculation of a viral vector construct that increased the expression of NbenIDA1A at the base of the corolla tube negatively affected the growth of the inoculated plants accelerating the timing of both corolla senescence and abscission. However, the heterologous ectopic overexpression of citrus CitIDA3 and Arabidopsis AtIDA in N. benthamiana did not alter the standard plant phenotype suggesting that the proteolytic processing machinery was unable to yield active peptides. CONCLUSION: Here, we demonstrate that the pair of NbenIDA1 homeologs encoding small peptides of the IDA-like family and the receptor NbenHAE.1 control cellular breakdown at the base of the corolla tube awhere an adventitious AZ should be formed and, therefore, corolla abscission in N. benthamiana flowers. Altogether, our results provide the first evidence supporting the notion that the IDA-HAE/HSL2 signaling module is conserved in angiosperms.


Assuntos
Flores/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tabaco/genética , Sequência de Aminoácidos , Flores/genética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Alinhamento de Sequência , Transdução de Sinais/genética , Tabaco/crescimento & desenvolvimento
12.
BMC Plant Biol ; 21(1): 236, 2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34044782

RESUMO

BACKGROUND: Lateral branches vigorously proliferate in tobacco after the topping of the inflorescence portions of stems for the maturation of the leaves to be harvested. Therefore, tobacco varieties with inhibited lateral shoot formation are highly desired by tobacco farmers. RESULTS: Genetic inhibition of lateral shoot formation was attempted in tobacco. Two groups of genes were examined by RNA interference. The first group comprised homologs of the genes mediating lateral shoot formation in other plants, whereas the second group included genes highly expressed in axillary bud primordial stages. Although "primary" lateral shoots that grew after the plants were topped off when flower buds emerged were unaffected, the growth of "secondary" lateral shoots, which were detected on the abaxial side of the primary lateral shoot base, was significantly suppressed in the knock-down lines of NtLs, NtBl1, NtREV, VE7, and VE12. Chemically induced mutations to NtLs, NtBl1, and NtREV similarly inhibited the development of secondary and "tertiary" lateral shoots, but not primary lateral shoots. The mutations to NtLs and NtBl1 were incorporated into an elite variety by backcrossing. The agronomic characteristics of the backcross lines were examined in field trials conducted in commercial tobacco production regions. The lines were generally suitable for tobacco leaf production and may be useful as new tobacco varieties. CONCLUSION: The suppressed expression of NtLs, NtBl1, NtREV, VE7, or VE12 inhibited the development of only the secondary and tertiary lateral shoots in tobacco. The mutant lines may benefit tobacco farmers by minimizing the work required to remove secondary and tertiary lateral shoots that emerge when farmers are harvesting leaves, which is a labor-intensive process.


Assuntos
Tabaco/genética , Inflorescência/enzimologia , Inflorescência/genética , Meristema/genética , Meristema/crescimento & desenvolvimento , Mutação , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Brotos de Planta/genética , Brotos de Planta/fisiologia , Interferência de RNA , Tabaco/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
13.
Gene ; 788: 145637, 2021 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-33848571

RESUMO

The pleiotropic drug resistance (PDR) proteins of the ATP-binding cassette (ABC) family play essential roles in physiological processes and have been characterized in many plant species. However, no comprehensive investigation of tobacco (Nicotiana tabacum), an important economic crop and a useful model plant for scientific research, has been presented. We identified 32 PDR genes in the tobacco genome and explored their domain organization, chromosomal distribution and evolution, promoter cis-elements, and expression profiles. A phylogenetic analysis revealed that tobacco has a significantly expanded number of PDR genes involved in plant defense. It also revealed that two tobacco PDR proteins may function as strigolactone transporters to regulate shoot branching, and several NtPDR genes may be involved in cadmium transport. Moreover, tissue expression profiles of NtPDR genes and their responses to several hormones and abiotic stresses were assessed using quantitative real-time PCR. Most of the NtPDR genes were regulated by jasmonate or salicylic acid, suggesting the important regulatory roles of NtPDRs in plant defense and secondary metabolism. They were also responsive to abiotic stresses, like drought and cold, and there was a strong correlation between the presence of promoter cis-elements and abiotic/biotic stress responses. These results provide useful clues for further in-depth studies on the functions of the tobacco PDR genes.


Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Perfilação da Expressão Gênica/métodos , Mapeamento Físico do Cromossomo/métodos , Tabaco/crescimento & desenvolvimento , Transportadores de Cassetes de Ligação de ATP/química , Cromossomos de Plantas/genética , Ciclopentanos/farmacologia , Evolução Molecular , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Família Multigênica , Oxilipinas/farmacologia , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Domínios Proteicos , Cimentos de Resina , Ácido Salicílico/farmacologia , Análise de Sequência de RNA , Estresse Fisiológico , Tabaco/efeitos dos fármacos , Tabaco/genética
14.
Environ Pollut ; 282: 117032, 2021 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-33831628

RESUMO

Among emerging organic contaminants (EOCs), triclosan (TCS) is an antibacterial agent and frequently detected in sludge. In this study, RNA sequencing (RNA-seq) was used to obtain the first transcriptomic profile of tobacco with TCS treatment in comparison with control. The results of transcriptome profiling indicated that salicylic acid (SA) signalling pathway actively participated in the tobacco's response to TCS treatment. The accumulation of endogenous SA in transgene tobacco lines transformed with a homologous gene of SA binding protein (LcSABP) was significantly enhanced. The resistance of transgenic tobacco lines to TCS was markedly enhanced revealed by morphological and physiological indexes while the total Chl level and Pn of transgenic individuals showed about 180% and 250% higher than that of WT on average, and the accumulation of H2O2 and O2- induced by TCS in SABP overexpressing tobacco was 35.3%-37.3% and 53.0%-56.0% lower than that of WT. In order to further explore the mechanism of TCS tolerance in transgenic plants, RNA-seq was then performed to obtain the second transcriptomic profile between wild type and transgenic samples with TCS exposure. The results indicated that differentially expressed genes (DEGs) were most highly enriched in MAPK signalling pathway, amino acid synthesis pathway and plant hormone transduction pathway. Especially, genes encoding key proteins such as cytochrome P450, laccase, peroxidase, glycosyl transferase, glutathione S-transferase and ATP-binding cassette were considered to be related to the increased tolerance ability of transgenic tobacco to the treatment of TCS stress. This research will likely provide novel insights into the molecular mechanism of SA-mediated amelioration of TCS stress on tobacco.


Assuntos
Ácido Salicílico , Tabaco , Triclosan/toxicidade , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Estresse Fisiológico , Tabaco/efeitos dos fármacos , Tabaco/crescimento & desenvolvimento
15.
Plant Signal Behav ; 16(5): 1899672, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33704006

RESUMO

When synchronized with the light/dark cycle the circadian rhythm is termed a diurnal rhythm and this organizes an organism's daily life cycle in relation to the metabolic shifts during the day/night cycles. This is a complex task, particularly under stress conditions. Accurate maintenance of the diurnal rhythm becomes an issue under environmental extremes, such as drought due to the impairment of metabolism, redox balance, and structural integrity. In plants, the non-proteinogenic amino acid GABA accumulates to high levels in response to several stress factors but this is not always dependent on the activation of its biosynthesis. Here we propose a regulatory role to GABA during the diurnal rhythm in plants which is similar to its function in animals where it adjusts the circadian rhythm. Here we investigated whether GABA-biosynthesis was affected by drought stress during the diurnal cycle. For this, we took samples from leaves of N. tabacum plants subjected to PEG-mediated drought stress (-0.73 MPa) during the day and night cycle during a 24 hour period. Glutamate, GABA, and proline contents, along with GDH, GAD enzyme activities and transcript profiles were analyzed. Overall, we conclude that the oscillations in GABA biosynthesis during day and night cycle have an impact on drought stress responses which needs to be elucidated by further analysis.


Assuntos
Ritmo Circadiano/fisiologia , Secas , Tabaco/fisiologia , Ácido gama-Aminobutírico/biossíntese , Glutamato Descarboxilase/metabolismo , Glutamato Desidrogenase/metabolismo , Ácido Glutâmico/metabolismo , Prolina/metabolismo , Tabaco/crescimento & desenvolvimento
16.
BMC Plant Biol ; 21(1): 131, 2021 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-33685400

RESUMO

BACKGROUND: Weather change in high-altitude areas subjects mature tobacco (Nicotiana tabacum L.) to cold stress, which damages tobacco leaf yield and quality. A brupt diurnal temperature differences (the daily temperature dropping more than 20 °C) along with rainfall in tobacco-growing areas at an altitude above 2450 m, caused cold stress to field-grown tobacco. RESULTS: After the flue-cured tobacco suffered cold stress in the field, the surface color of tobacco leaves changed and obvious large browning areas were appeared, and the curing availability was extremely poor. Further research found the quality of fresh tobacco leaves, the content of key chemical components, and the production quality were greatly reduced by cold stress. We hypothesize that cold stress in high altitude environments destroyed the antioxidant enzyme system of mature flue-cured tobacco. Therefore, the quality of fresh tobacco leaves, the content of key chemical components, and the production quality were greatly reduced by cold stress. CONCLUSION: This study confirmed that cold stress in high-altitude tobacco areas was the main reason for the browning of tobacco leaves during the tobacco curing process. This adverse environment seriously damaged the quality of tobacco leaves, but can be mitigated by pay attention to the weather forecast and pick tobacco leaves in advance.


Assuntos
Resposta ao Choque Frio/fisiologia , Fatores de Transcrição de Choque Térmico/fisiologia , Folhas de Planta/química , Folhas de Planta/crescimento & desenvolvimento , Tabaco/química , Tabaco/crescimento & desenvolvimento , China , Produtos Agrícolas/química , Produtos Agrícolas/crescimento & desenvolvimento
17.
J Vis Exp ; (167)2021 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-33522504

RESUMO

High demand for antibodies as therapeutic interventions for various infectious, metabolic, autoimmune, neoplastic, and other diseases creates a growing need in developing efficient methods for recombinant antibody production. As of 2019, there were more than 70 FDA-approved monoclonal antibodies, and there is exponential growth potential. Despite their promise, limiting factors for widespread use are manufacturing costs and complexity. Potentially, plants offer low-cost, safe, and easily scalable protein manufacturing strategies. Plants like Nicotiana benthamiana not only can correctly fold and assemble complex mammalian proteins but also can add critical post-translational modifications similar to those offered by mammalian cell cultures. In this work, by using native GFP and an acid-stable variant of green fluorescent protein (GFP) fused to human monoclonal antibodies, we were able to visualize the entire transient antibody expression and purification process from N. benthamiana plants. Depending on the experiment's purpose, native GFP fusion can ensure easier visualization during the expression phase in the plants, while acid-stable GFP fusion allows for visualization during downstream processing. This scalable and straightforward procedure can be performed by a single researcher to produce milligram quantities of highly pure antibody or antibody fusion proteins in a matter of days using only a few small plants. Such a technique can be extended to the visualization of any type of antibody purification process and potentially many other proteins, both in plant and other expression systems. Moreover, these techniques can benefit virtual instructions and be executed in a teaching laboratory by undergraduate students possessing minimal prior experience with molecular biology techniques, providing a foundation for project-based exploration with real-world applications.


Assuntos
Imunoglobulina G/biossíntese , Proteínas Recombinantes de Fusão/biossíntese , Tabaco/genética , Agrobacterium tumefaciens/metabolismo , Animais , Anticorpos Monoclonais/metabolismo , Sequência de Bases , Cromatografia , Eletroforese em Gel de Poliacrilamida , Proteínas de Fluorescência Verde/metabolismo , Humanos , Canamicina/farmacologia , Folhas de Planta/microbiologia , Plantas Geneticamente Modificadas , Tabaco/crescimento & desenvolvimento , Tabaco/microbiologia , Raios Ultravioleta
18.
Plant Mol Biol ; 106(1-2): 85-108, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33629224

RESUMO

KEY MESSAGE: Overexpression of StCaM2 in tobacco promotes plant growth and confers increased salinity and drought tolerance by enhancing the photosynthetic efficiency, ROS scavenging, and recovery from membrane injury. Calmodulins (CaMs) are important Ca2+ sensors that interact with effector proteins and drive a network of signal transduction pathways involved in regulating the growth and developmental pattern of plants under stress. Herein, using in silico analysis, we identified 17 CaM isoforms (StCaM) in potato. Expression profiling revealed different temporal and spatial expression patterns of these genes, which were modulated under abiotic stress. Among the identified StCaM genes, StCaM2 was found to have the largest number of abiotic stress responsive promoter elements. In addition, StCaM2 was upregulated in response to some of the selected abiotic stress in potato tissues. Overexpression of StCaM2 in transgenic tobacco plants enhanced their tolerance to salinity and drought stress. Accumulation of reactive oxygen species was remarkably decreased in transgenic lines compared to that in wild type plants. Chlorophyll a fluorescence analysis suggested better performance of photosystem II in transgenic plants under stress compared to that in wild type plants. The increase in salinity stress tolerance in StCaM2-overexpressing plants was also associated with a favorable K+/Na+ ratio. The enhanced tolerance to abiotic stresses correlated with the increase in the activities of anti-oxidative enzymes in transgenic tobacco plants. Overall, our results suggest that StCaM2 can be a novel candidate for conferring salt and drought tolerance in plants.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Secas , Proteínas de Plantas/metabolismo , Salinidade , Solanum tuberosum/metabolismo , Estresse Fisiológico , Tabaco/genética , Tabaco/fisiologia , Antioxidantes/metabolismo , Proteínas de Ligação ao Cálcio/genética , Calmodulina/genética , Calmodulina/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genoma de Planta , Germinação/efeitos dos fármacos , Germinação/genética , Íons , Membranas , Fotossíntese/efeitos dos fármacos , Reguladores de Crescimento de Plantas/farmacologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Prolina/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequências Reguladoras de Ácido Nucleico/genética , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/crescimento & desenvolvimento , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Tabaco/enzimologia , Tabaco/crescimento & desenvolvimento , Fatores de Transcrição/metabolismo , Água/metabolismo
19.
Int J Mol Sci ; 22(2)2021 Jan 18.
Artigo em Inglês | MEDLINE | ID: mdl-33477652

RESUMO

V2 proteins encoded by some whitefly-transmitted geminiviruses were reported to be functionally important proteins. However, the functions of the V2 protein of tobacco curly shoot virus (TbCSV), a monopartite begomovirus that causes leaf curl disease on tomato and tobacco in China, remains to be characterized. In our report, an Agrobacterium infiltration-mediated transient expression assay indicated that TbCSV V2 can suppress local and systemic RNA silencing and the deletion analyses demonstrated that the amino acid region 1-92 of V2, including the five predicted α-helices, are required for local RNA silencing suppression. Site-directed substitutions showed that the conserved basic and ring-structured amino acids in TbCSV V2 are critical for its suppressor activity. Potato virus X-mediated heteroexpression of TbCSV V2 in Nicotiana benthamiana induced hypersensitive response-like (HR-like) cell death and systemic necrosis in a manner independent of V2's suppressor activity. Furthermore, TbCSV infectious clone mutant with untranslated V2 protein (TbCSV∆V2) could not induce visual symptoms, and coinfection with betasatellite (TbCSB) could obviously elevate the viral accumulation and symptom development. Interestingly, symptom recovery occurred at 15 days postinoculation (dpi) and onward in TbCSV∆V2/TbCSB-inoculated plants. The presented work contributes to understanding the RNA silencing suppression activity of TbCSV V2 and extends our knowledge of the multifunctional role of begomovirus-encoded V2 proteins during viral infections.


Assuntos
Begomovirus/genética , Potexvirus/genética , Tabaco/virologia , Proteínas Virais/genética , Begomovirus/patogenicidade , China , Lycopersicon esculentum/crescimento & desenvolvimento , Lycopersicon esculentum/virologia , Doenças das Plantas/genética , Doenças das Plantas/virologia , Potexvirus/patogenicidade , Interferência de RNA , Tabaco/crescimento & desenvolvimento , Virulência/genética
20.
Plant Cell Rep ; 40(3): 491-506, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33388892

RESUMO

KEY MESSAGE: StMAPK11 overexpression promotes potato growth, physiological activities and photosynthesis under drought conditions. Mitogen-activated protein kinases (MAPKs) are import regulators of MAPK pathway in plants under drought condition. However, the critical role in potato (Solanum tuberosum L.) drought resistance is not fully understood. In this study, we aimed to explore the role of StMAPK11 under drought stress. The result of RT-qPCR for assay of StMAPKs expression demonstrated that 15 StMAPKs were differentially expressed in leaves, flowers, petioles, stamens, pistils, stems, stolons, roots, tubers and tuber peels of potato. StMAPKs was dynamically modulated by abiotic stresses and plant hormone treatments, and StMAPK11 was apparently up-regulated under drought conditions. Therefore, the vectors pCPB-StMAPK11 and pCPBI121-miRmapk11 for over-expression and down-regulation of StMAPK11 were constructed, respectively, and introduced into potato cultivar Atlantic. The result showed that StMAPK11 promoted potato growth under drought conditions, as well as the physiological activities evidenced by changes in SOD, CAT and POD activity and H2O2, proline and MDA content. StMAPK11 up-regulation intensified drought resistance of potato plant by elevating antioxidant activities and photosynthesis. Moreover, we consolidated the protective role of StMAPK11 in tobacco and Arabidopsis against drought stress. The result could provide new insights into the function of StMAPK11 in drought response and its possible mechanisms.


Assuntos
Secas , Proteína Quinase 11 Ativada por Mitógeno/metabolismo , Fotossíntese/fisiologia , Proteínas de Plantas/metabolismo , Solanum tuberosum/fisiologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Enzimas/metabolismo , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Proteína Quinase 11 Ativada por Mitógeno/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Prolina/metabolismo , Estresse Fisiológico , Tabaco/genética , Tabaco/crescimento & desenvolvimento
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