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1.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2845-2855, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472302

RESUMO

Production of biofuels such as ethanol from non-grain crops may contribute to alleviating the global energy crisis and reducing the potential threat to food security. Tobacco (Nicotiana tabacum) is a commercial crop with high biomass yield. Breeding of starch-rich tobacco plants may provide alternative raw materials for the production of fuel ethanol. We cloned the small subunit gene NtSSU of ADP-glucose pyrophosphorylase (NtAGPase), which controls starch biosynthesis in tobacco, and constructed a plant expression vector pCAMBIA1303-NtSSU. The NtSSU gene was overexpressed in tobacco upon Agrobacterium-mediated leaf disc transformation. Phenotypic analysis showed that overexpression of NtSSU gene promoted the accumulation of starch in tobacco leaves, and the content of starch in tobacco leaves increased from 17.5% to 41.7%. The growth rate and biomass yield of the transgenic tobacco with NtSSU gene were also significantly increased. The results revealed that overexpression of NtSSU gene could effectively redirect more photosynthesis carbon flux into starch biosynthesis pathway, which led to an increased biomass yield but did not generate negative effects on other agronomic traits. Therefore, NtSSU gene can be used as an excellent target gene in plant breeding to enrich starch accumulation in vegetative organs to develop new germplasm dedicated to fuel ethanol production.


Assuntos
Amido , Tabaco , Biomassa , Regulação da Expressão Gênica de Plantas , Melhoramento Vegetal , Folhas de Planta/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética , Tabaco/metabolismo
2.
Plant Physiol Biochem ; 167: 831-839, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34530327

RESUMO

Thioredoxin-like protein CDSP32 (Trx CDSP32), a thioredoxin-like (Trx-like) protein located in the chloroplast, can regulate photosynthesis and the redox state of plants under stress. In order to examine the role of Trx CDSP32 in the photosynthetic apparatus of plants exposed to cadmium (Cd), the effects of Trx CDSP32 on photosynthetic function and photoprotection in tobacco leaves under Cd exposure were studied using a proteomics approach with wild-type (WT) and Trx CDSP32 overexpression (OE) tobacco plants. Cd exposure reduced stomatal conductance, blocked PSII photosynthetic electron transport, and inhibited carbon assimilation. Increased water use efficiency (WUE), cyclic electron flow (CEF) of the proton gradient regulation 5 pathway (PGR5-CEF), and regulated energy dissipation [Y(NPQ)] are important mechanisms of Cd adaptation. However, CEF of the NAD(P)H dehydrogenase pathway (NDH-CEF) was inhibited by Cd exposure. Relative to control conditions, the expression levels of violaxanthin de-epoxidase (VDE) and photosystem II 22 kDa protein (PsbS) in OE leaves were significantly increased under Cd exposure, but those in WT leaves did not change significantly. Moreover, the expression of zeaxanthin epoxidase (ZE) under Cd exposure was significantly higher than that in WT leaves. Thus, Trx CDSP32 increased Y(NPQ) and alleviated PSII photoinhibition under Cd exposure. Trx CDSP32 not only increased PGR5-like protein 1A and 1B expression, but also alleviated the down-regulation of NAD(P)H-quinone oxidoreductase subunits induced by Cd exposure. Thus, Trx CDSP32 promotes CEF in Cd-exposed tobacco leaves. Thus, Trx CDSP32 alleviates the Cd-induced photoinhibition in tobacco leaves by regulating two photoprotective mechanisms: CEF and xanthophyll cycle-dependent energy dissipation.


Assuntos
Cádmio , Tabaco , Cádmio/metabolismo , Cádmio/toxicidade , Clorofila , Transporte de Elétrons , Elétrons , Luz , Fotossíntese , Complexo de Proteína do Fotossistema I/metabolismo , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Tiorredoxinas , Tabaco/metabolismo
3.
Viruses ; 13(8)2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34452461

RESUMO

The COVID-19 pandemic, caused by SARS-CoV-2, has rapidly spread to more than 222 countries and has put global public health at high risk. The world urgently needs cost-effective and safe SARS-CoV-2 vaccines, antiviral, and therapeutic drugs to control it. In this study, we engineered the receptor binding domain (RBD) of the SARS-CoV-2 spike (S) protein and produced it in the plant Nicotiana benthamiana in a glycosylated and deglycosylated form. Expression levels of both glycosylated (gRBD) and deglycosylated (dRBD) RBD were greater than 45 mg/kg fresh weight. The purification yields were 22 mg of pure protein/kg of plant biomass for gRBD and 20 mg for dRBD, which would be sufficient for commercialization of these vaccine candidates. The purified plant-produced RBD protein was recognized by an S protein-specific monoclonal antibody, demonstrating specific reactivity of the antibody to the plant-produced RBD proteins. The SARS-CoV-2 RBD showed specific binding to angiotensin converting enzyme 2 (ACE2), the SARS-CoV-2 receptor. In mice, the plant-produced RBD antigens elicited high titers of antibodies with a potent virus-neutralizing activity. To our knowledge, this is the first report demonstrating that mice immunized with plant-produced deglycosylated RBD form elicited high titer of RBD-specific antibodies with potent neutralizing activity against SARS-CoV-2 infection. Thus, obtained data support that plant-produced glycosylated and in vivo deglycosylated RBD antigens, developed in this study, are promising vaccine candidates for the prevention of COVID-19.


Assuntos
Anticorpos Neutralizantes/sangue , Anticorpos Antivirais/sangue , SARS-CoV-2/imunologia , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/imunologia , Enzima de Conversão de Angiotensina 2/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Chlorocebus aethiops , Glicosilação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Testes de Neutralização , Plantas Geneticamente Modificadas , Ligação Proteica , Domínios Proteicos , Engenharia de Proteínas , Estabilidade Proteica , Receptores de Coronavírus/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/metabolismo , Tabaco/genética , Tabaco/metabolismo , Células Vero
4.
Phytochemistry ; 191: 112911, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34418773

RESUMO

The pleiotropic effects of zinc deficiency on ion homeostasis have already been described in several plants. Tobacco (Nicotiana tabacum) heavy metal ATPases HMA4.1 and HMA4.2 are involved in zinc and cadmium root-to-shoot translocation. In previous research, we have shown that N. tabacum HMA4 RNAi plants and HMA4 double-nonsense mutants exhibit strongly reduced zinc and cadmium levels in leaves as well as stunted growth. In this study, the ionome and transcriptome of these lines were investigated to better characterize the effect of reduced zinc levels and to understand the impaired growth phenotype. We found that, under standard greenhouse fertilization rates, these lines accumulated up to 4- to 6-fold more phosphorus, iron, manganese, and copper than their respective controls. Under field conditions, HMA4 double-mutant plants also exhibited similar accumulation phenotypes, albeit to a lower extent. In both HMA4 RNAi plants and HMA4 mutants, transcription analysis showed a local zinc-deficiency response in leaves as well as an FIT1-mediated iron-deficiency response in roots, likely contributing to iron and manganese uptake at the root level. A phosphate-starvation response involving HHO2 was also observed in HMA4-impaired plant leaves. The high level of phosphorus observed in HMA4-impaired plants is correlated with leaf swelling and necrosis. The upregulation of aquaporin genes is in line with cellular water influx and the observed leaf swelling phenotype. These results highlight the involvement of HMA4 in zinc homeostasis and related regulatory processes that balance the micro- and macroelements in above-ground organs.


Assuntos
Cádmio , Tabaco , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Cádmio/metabolismo , Regulação da Expressão Gênica de Plantas , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Tabaco/metabolismo , Zinco/metabolismo
5.
Nat Plants ; 7(8): 1037-1049, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34373604

RESUMO

The responses of plants to their environment are often dependent on the spatiotemporal dynamics of transcriptional regulation. While live-imaging tools have been used extensively to quantitatively capture rapid transcriptional dynamics in living animal cells, the lack of implementation of these technologies in plants has limited concomitant quantitative studies in this kingdom. Here, we applied the PP7 and MS2 RNA-labelling technologies for the quantitative imaging of RNA polymerase II activity dynamics in single cells of living plants as they respond to experimental treatments. Using this technology, we counted nascent RNA transcripts in real time in Nicotiana benthamiana (tobacco) and Arabidopsis thaliana. Examination of heat shock reporters revealed that plant tissues respond to external signals by modulating the proportion of cells that switch from an undetectable basal state to a high-transcription state, instead of modulating the rate of transcription across all cells in a graded fashion. This switch-like behaviour, combined with cell-to-cell variability in transcription rate, results in mRNA production variability spanning three orders of magnitude. We determined that cellular heterogeneity stems mainly from stochasticity intrinsic to individual alleles instead of variability in cellular composition. Together, our results demonstrate that it is now possible to quantitatively study the dynamics of transcriptional programs in single cells of living plants.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Resposta ao Choque Térmico/genética , Células Vegetais/metabolismo , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Tabaco/genética , Tabaco/metabolismo , Transcrição Genética , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
6.
Int J Mol Sci ; 22(16)2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34445456

RESUMO

Flavonoids are representative secondary metabolites with different metabolic functions in plants. Previous study found that ectopic expression of EsMYB90 from Eutremasalsugineum could strongly increase anthocyanin content in transgenic tobacco via regulating the expression of anthocyanin biosynthesis genes. In the present research, metabolome analysis showed that there existed 130 significantly differential metabolites, of which 23 metabolites enhanced more than 1000 times in EsMYB90 transgenic tobacco leaves relative to the control, and the top 10 of the increased metabolites included caffeic acid, cyanidin O-syringic acid, myricetin and naringin. A total of 50 markedly differential flavonoids including flavones (14), flavonols (13), flavone C-glycosides (9), flavanones (7), catechin derivatives (5), anthocyanins (1) and isoflavone (1) were identified, of which 46 metabolites were at a significantly enhanced level. Integrated analysis of metabolome and transcriptome revealed that ectopic expression of EsMYB90 in transgenic tobacco leaves is highly associated with the prominent up-regulation of 16 flavonoid metabolites and the corresponding 42 flavonoid biosynthesis structure genes in phenylpropanoid/flavonoid pathways. Dual luciferase assay documented that EsMYB90 strongly activated the transcription of NtANS and NtDFR genes via improving their promoter activity in transiently expressed tobacco leaves, suggesting that EsMYB90 functions as a key regulator on anthocyanin and flavonoid biosynthesis. Taken together, the crucial regulatory role of EsMYB90 on enhancing many flavonoid metabolite levels is clearly demonstrated via modulating flavonoid biosynthesis gene expression in the leaves of transgenic tobacco, which extends our understanding of the regulating mechanism of MYB transcription factor in the phenylpropanoid/flavonoid pathways and provides a new clue and tool for further investigation and genetic engineering of flavonoid metabolism in plants.


Assuntos
Antocianinas , Brassicaceae/metabolismo , Perfilação da Expressão Gênica , Metabolômica , Proteínas de Plantas , Plantas Geneticamente Modificadas , Tabaco , Antocianinas/biossíntese , Antocianinas/genética , Brassicaceae/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética , Tabaco/metabolismo
7.
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
8.
PLoS One ; 16(8): e0253574, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34379620

RESUMO

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is responsible for the ongoing coronavirus disease (COVID-19) pandemic which is characterized by respiratory illness and severe pneumonia, and currently accounts for > 2.5 million deaths worldwide. Recently, diverse mutations in the spike protein of SARS-CoV-2 were reported in United Kingdom (Alpha) and South Africa (Beta) strains which raise concerns over the potential increase in binding affinity towards the host cell receptor and diminished host neutralization capabilities. In order to study the effect of mutation in the binding efficiency of SARS-CoV-2 receptor binding domain (RBD) with anti-SARS-CoV/CoV-2 monoclonal antibodies (mAbs), we have produced SARS-CoV-2 RBD and two variants SARS-CoV-2 RBD (Alpha RBD and Beta RBD) in Nicotiana benthamiana by transient expression. Plant-produced SARS-CoV-2 RBD-Fc, Alpha RBD-Fc and Beta RBD-Fc exhibited specific binding to human angiotensin converting enzyme 2 (ACE2) receptor determined by ELISA. Intriguingly, the binding of plant-produced SARS-CoV-2 RBD proteins to plant-produced mAbs CR3022, B38, and H4 was found to be different depending on the variant mutation. In contrary to the plant-produced SARS-CoV-2 RBD-Fc and Alpha RBD-Fc, Beta RBD-Fc variant showed weak binding affinity towards the mAbs. The result suggested that the Beta RBD variant might have acquired partial resistance to neutralizing antibodies compared to other variants. However, further studies with sera from convalescent or vaccinated individuals are required to confirm this finding.


Assuntos
Anticorpos Monoclonais/metabolismo , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/metabolismo , Tabaco/metabolismo , Enzima de Conversão de Angiotensina 2/metabolismo , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Reações Antígeno-Anticorpo , COVID-19/patologia , COVID-19/virologia , Humanos , Ligação Proteica , Domínios Proteicos/imunologia , Proteínas Recombinantes/genética , SARS-CoV-2/isolamento & purificação , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/genética , Glicoproteína da Espícula de Coronavírus/imunologia
9.
Int J Mol Sci ; 22(16)2021 Aug 08.
Artigo em Inglês | MEDLINE | ID: mdl-34445241

RESUMO

Global warming leads to a progressive rise in environmental temperature. Plants, as sessile organisms, are threatened by these changes; the male gametophyte is extremely sensitive to high temperature and its ability to preserve its physiological status under heat stress is known as acquired thermotolerance. This latter can be achieved by exposing plant to a sub-lethal temperature (priming) or to a progressive increase in temperature. The present research aims to investigate the effects of heat priming on the functioning of tobacco pollen grains. In addition to evaluating basic physiological parameters (e.g., pollen viability, germination and pollen tube length), several aspects related to a correct pollen functioning were considered. Calcium (Ca2+) level, reactive oxygen species (ROS) and related antioxidant systems were investigated, also to the organization of actin filaments and cytoskeletal protein such as tubulin (including tyrosinated and acetylated isoforms) and actin. We also focused on sucrose synthase (Sus), a key metabolic enzyme and on the content of main soluble sugars, including UDP-glucose. Results here obtained showed that a pre-exposure to sub-lethal temperatures can positively enhance pollen performance by altering its metabolism. This can have a considerable impact, especially from the point of view of breeding strategies aimed at improving crop species.


Assuntos
Temperatura Alta , Proteínas de Plantas/metabolismo , Tubo Polínico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Termotolerância , Tabaco/metabolismo , Proteínas de Plantas/genética , Tubo Polínico/genética , Tabaco/genética
10.
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
11.
Plant Physiol Biochem ; 166: 657-667, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34214776

RESUMO

To reveal the mechanism of photosynthesis inhibition by infection and the response of the MAPK signaling pathway to pathogen infection, tobacco leaves were inoculated with Pseudomonas syringae pv. tabaci (Pst), and the effects of Pst infection on photosynthesis of tobacco leaves were studied by physiological and proteomic techniques, with a focus on MAPK signaling pathway related proteins. Pst infection was observed to lead to the degradation of chlorophyll (especially Chl b) in tobacco leaves and the down-regulation of light harvesting antenna proteins expression, thus limiting the light harvesting ability. The photosystem II and I (PSII and PSI) activities were also decreased, and Pst infection inhibited the utilization of light and CO2. Proteomic analyses showed that the number of differentially expressed proteins (DEPs) under Pst infection at 3 d were significantly higher than at 1 d, especially the number of down-regulated proteins. The KEGG enrichment of DEPs was mainly enriched in the energy metabolism processes such as photosynthesis antenna proteins and photosynthesis. The down-regulation of chlorophyll a-b binding protein, photosynthetic electron transport related proteins (e.g., PSII and PSI core proteins, the Cytb6/f complex, PC, Fd, FNR), ATP synthase subunits, and key enzymes in the Calvin cycle were the key changes associated with Pst infection that may inhibit tobacco photosynthesis. The effect of Pst infection on the PSII electron acceptor side was significantly greater than that on the PSII donor side. The main factor that decreased the photosynthetic ability of tobacco leaves with Pst infection at 1 d may be the inhibition of photochemical reactions leading to an insufficient supply of ATP, rather than decreased expression of enzymes involved in the Calvin cycle. At 1 d into Pst infection, the PSII regulated energy dissipation yield Y(NPQ) may play a role in preventing photosynthetic inhibition in tobacco leaves, but the long-term Pst infection significantly inhibited Y(NPQ) and the expression of PsbS proteins. Proteins involved in the MAPK signaling pathway were up-regulated, suggesting the MAPK signaling pathway was activated to respond to Pst infection. However, at the late stage of Pst infection (at 3 d), MAPK signaling pathway proteins were degraded, and the defense function of the MAPK signaling pathway in tobacco leaves was damaged.


Assuntos
Pseudomonas syringae , Tabaco , Clorofila , Clorofila A , Transporte de Elétrons , Fotossíntese , Complexo de Proteína do Fotossistema II/metabolismo , Folhas de Planta/metabolismo , Proteômica , Transdução de Sinais , Tabaco/metabolismo
12.
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
13.
Plant Sci ; 310: 110971, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34315589

RESUMO

The flowering-time gene FD encodes a bZIP transcription factor that interacts with FLOWERING LOCUS T (FT) to induce flowering in Arabidopsis. Previous research has identified two FT homologs of Platanus acerifolia, PaFT and PaFTL, which each have different expression patterns and are involved in diverse developmental processes. However, it is not known whether such FT/FD complexes participate in the flowering processes in P. acerifolia. Therefore, we isolated two closely related FD homologs, PaFDL1 and PaFDL2, and investigated their functions through the analysis of expression profiles, transgenic phenotypes, their interactions with different FT proteins, and potential cis-regulatory elements in their promoters. The PaFDL genes were found to display their maximal expression levels during the stage of floral transition, and subsequent expression patterns were also seen to be related to inflorescence developmental stage. In addition, both PaFDL1 and PaFDL2 were found to be subject to post-transcriptional alternative splicing, each gene producing two transcript forms. Transgenic tobacco overexpressing each of the four resulting transcript types displayed accelerated floral initiation and produced abnormal flowers. The results suggested that the complete PaFDL proteins may interact with different PaFT/PaFTL proteins in order to fulfill both conservative and diverse functions in floral initiation and floral development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Flores/metabolismo , Flores/fisiologia , Proteínas de Plantas/metabolismo , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Flores/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tabaco/genética , Tabaco/metabolismo
14.
Methods Mol Biol ; 2328: 203-214, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34251628

RESUMO

Plants use different regulatory modules in response to changes in their surroundings. With the transcriptomic approaches governing all research areas, an integrative, fast, and sensitive approach toward validating genes of interest becomes a critical step prior to functional studies in planta. This chapter describes a detailed method for a quantitative analysis of transcriptional readouts of defense response genes using tobacco leaves as a transient system. The method uses Luciferase reporter assays to monitor activities of defense pathway promoters. Under normal conditions, the JASMONATE ZIM-DOMAIN (JAZ) proteins repress defense genes by preventing their expression. Here, we will provide a detailed protocol on the use of a dual-luciferase system to analyze activities of various defense response promoters simultaneously. We will use two well-characterized modules from the Jasmonic acid (JA) defense pathway; the JAZ3 repressor protein and the promoters of three of JA responsive genes, MYC2, 3 and 4. This assay revealed not only differences in promoter strength but also provided quantitative insights on the JAZ3 repression of MYCs in a quantitative manner.


Assuntos
Ciclopentanos/metabolismo , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/metabolismo , Proteínas Repressoras/metabolismo , Tabaco/metabolismo , Agrobacterium tumefaciens/metabolismo , Primers do DNA , Genes myc/genética , Luciferases/genética , Plantas Geneticamente Modificadas/genética , Regiões Promotoras Genéticas , Domínios Proteicos/genética , Proteínas Repressoras/genética , Tabaco/genética
15.
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
16.
Molecules ; 26(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299447

RESUMO

Tobacco (Nicotiana tabacum Linn.) is a famous traditional herb used in folk medicine. The essential oils of tobacco have been demonstrated in modern studies to possess antioxidant, anti-inflammatory, and neuroprotective properties, while its anxiolytic effect has not been reported. The purpose of this study was to evaluate the anxiolytic effect of Yunnan tobacco essential oil (YTO) and Zimbabwe tobacco essential oil (ZTO) on mice. The constituents of YTO and ZTO were analyzed by GC/MS. The anxiolytic effect of YTO and ZTO (0.1%, 1%, and 10%, v/v) on male ICR mice was evaluated in the light-dark box test (LDB) and the elevated plus maze test (EPM) test via inhalation and transdermal administration. After the behavioral tests, salivary corticosterone levels in mice were measured. The behavioral analysis showed that the administration of both YTO and ZTO elevated the time that the mice spent in the light chamber in the LDB test compared to the untreated control. In the EPM test, YTO and ZTO increased the time spent in open arms and the number of entries into the open arms. In addition, both YTO and ZTO significantly decreased salivary corticosterone levels in mice (p ≤ 0.001). In summary, our results demonstrated that inhalation and transdermal administration of both YTO and ZTO showed anxiolytic effect on male ICR mice.


Assuntos
Óleos Voláteis/farmacologia , Extratos Vegetais/farmacologia , Tabaco/metabolismo , Animais , Ansiedade/tratamento farmacológico , Comportamento Animal/efeitos dos fármacos , China , Feminino , Masculino , Camundongos , Camundongos Endogâmicos ICR , Atividade Motora/efeitos dos fármacos , Óleos Voláteis/administração & dosagem , Extratos Vegetais/administração & dosagem
17.
J Biotechnol ; 336: 10-24, 2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34116128

RESUMO

An extreme temperature regime beyond desired level imposes significant stress in crop plants. The low and high temperature stresses are one of the primary constraints for plant development and yield. Finger millet, being a climate resilient crop, is a potential source of novel stress tolerant genes. In this study, functional characterization of finger millet DREB2A gene in different abiotic stress conditions was done. This novel EcDREB2A transcription factor isolated from finger millet is a truncated version of DREB2A gene compared to previously reported DREB genes from other plant species. The overexpression of EcDREB2A in transgenic tobacco exhibits improved tolerance against heat stress 42 °C for up to 7 days, by altering physiology and biochemical means. However, same transgenic lines were unable to provide tolerance to 200 mM NaCl and 200 mM Mannitol stress. Under heat stress conditions, increased seed germination with improved lateral roots, fresh and dry weight relative to wild type (WT) was observed. The EcDREB2A transgenics exposed to heat stress showed improved rate of stomatal conductance, chlorophyll and carotenoids contents, and other photosynthesis parameters compared to WT plants. EcDREB2A overexpression also resulted in increased antioxidant enzyme activity (SOD, CAT, GR, POD and, APX) with decreased electrolyte leakage (EL), H2O2, and malondialdehyde (MDA) content than WT plants under heat stress. Quantitative real time expression analysis demonstrated that all eight downstream genes were significantly upregulated in transgenic plants only after heat stress. Our data provide a clear demonstration of the positive impact of overexpression of EcDREB2A providing heat stress tolerance to plants.


Assuntos
Eleusine , Tabaco , Secas , Eleusine/genética , Eleusine/metabolismo , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/genética , Tabaco/genética , Tabaco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
18.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069632

RESUMO

In tobacco, the efficiency of Zn translocation to shoots depends on Zn/Cd status. Previous studies pointed to the specific contribution of root parts in the regulation of this process, as well as the role of NtZIP4A/B (from the ZIP family; Zrt Irt-like Proteins). Here, to verify this hypothesis, NtZIP4A/B RNAi lines were generated. Then, in plants exposed to combinations of Zn and Cd concentrations in the medium, the consequences of NtZIP4A/B suppression for the translocation of both metals were determined. Furthermore, the apical, middle, and basal root parts were examined for accumulation of both metals, for Zn localization (using Zinpyr-1), and for modifications of the expression pattern of ZIP genes. Our results confirmed the role of NtZIP4A/B in the control of Zn/Cd-status-dependent transfer of both metals to shoots. Furthermore, they indicated that the middle and basal root parts contributed to the regulation of this process by acting as a reservoir for excess Zn and Cd. Expression studies identified several candidate ZIP genes that interact with NtZIP4A/B in the root in regulating Zn and Cd translocation to the shoot, primarily NtZIP1-like in the basal root part and NtZIP2 in the middle one.


Assuntos
Proteínas de Transporte de Cátions/metabolismo , Tabaco/metabolismo , Zinco/metabolismo , Adenosina Trifosfatases/metabolismo , Transporte Biológico/genética , Cádmio/metabolismo , Proteínas de Transporte de Cátions/genética , Regulação da Expressão Gênica de Plantas/genética , Homeostase , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética
19.
Int J Mol Sci ; 22(11)2021 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-34073055

RESUMO

Plant proline-rich proteins (PRPs) are cell wall proteins that occur in the plant kingdom and are involved in plant development and stress response. In this study, 9 PRP genes were identified from the apple genome and a comprehensive analysis of the PRP family was conducted, including gene structures, phylogenetic analysis, chromosome mapping, and so on. The expression of MdPRPs varied among tissues and in response to different types of stresses. MdPRP4 and MdPRP7 were induced by five detected stress treatments, including heat, drought, abscisic acid, cold, and salt; the expression patterns of the others varied under different types of stress. Subcellular localization showed that MdPRPs mainly functioned in the cytoplasm, except for MdPRP1 and MdPRP5, which also functioned in the nucleus. When MdPRP6 was overexpressed in tobacco, the transgenic plants showed higher tolerance to high temperature (48 °C) compared with wild-type (WT) plants. The transgenic plants showed milder wilting, a lower accumulation of electrolyte leakage, MDA and ROS, and a higher level of chlorophyll and SOD and POD activity, indicating that MdPRP6 may be an important gene in apples for heat stress tolerance. Overall, this study suggested that MdPRPs are critically important for the ability of apple responses to stresses.


Assuntos
Malus/genética , Proteínas de Plantas , Domínios Proteicos Ricos em Prolina , Estresse Fisiológico , Temperatura Alta , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética , Tabaco/metabolismo
20.
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
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