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1.
BMC Plant Biol ; 19(1): 326, 2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31324141

RESUMO

BACKGROUND: Autophagy is a conserved, highly-regulated catabolic process that plays important roles in growth, development and innate immunity in plants. In this study, we compared the rate of autophagy induction in Nicotiana benthamiana plants infected with Tobacco mosaic virus or the TMV 24A + UPD mutant variant, which replicates at a faster rate and induces more severe symptoms. Using a BirA* tag and proximity-dependent biotin identification (BioID) analysis, we identified host proteins that interact with the core autophagy protein, ATG8 in TMV 24A + UPD infected plants. By combining the use of a fast replicating TMV mutant and an in vivo protein-protein screening technique, we were able to gain functional insight into the role of autophagy in a compatible virus-host interaction. RESULTS: Our study revealed an increased autophagic flux induced by TMV 24A + UPD, as compared to TMV in N. benthamiana. Analysis of the functional proteome associated with ATG8 revealed a total of 67 proteins, 16 of which are known to interact with ATG8 or its orthologs in mammalian and yeast systems. The interacting proteins were categorized into four functional groups: immune system process, response to ROS, sulphur amino acid metabolism and calcium signalling. Due to the presence of an ubiquitin-associated (UBA) domain, which is demonstrated to interact with ATG8, the Huntingtin-interacting protein K-like (HYPK) was selected for validation of the physical interaction and function. We used yeast two hybrid (Y2H), bimolecular fluorescence complementation (BiFC) and subcellular localization to validate the ATG8-HYPK interaction. Subsequent down-regulation of ATG8 by virus-induced gene silencing (VIGS) showed enhanced TMV symptoms, suggesting a protective role for autophagy during TMV 24A + UPD infection. CONCLUSION: This study presents the use of BioID as a suitable method for screening ATG8 interacting proteins in planta. We have identified many putative binding partners of ATG8 during TMV 24A + UPD infection in N. benthamiana plants. In addition, we have verified that NbHYPK is an interacting partner of ATG8. We infer that autophagy plays a protective role in TMV 24A + UPD infected plants.


Assuntos
Família da Proteína 8 Relacionada à Autofagia/metabolismo , Doenças das Plantas/virologia , Proteínas de Plantas/metabolismo , Tabaco/virologia , Autofagossomos/metabolismo , Autofagia/genética , Autofagia/fisiologia , Biotinilação , Imunidade Vegetal , Tabaco/metabolismo , Vírus do Mosaico do Tabaco
2.
J Chem Ecol ; 45(8): 708-714, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31313135

RESUMO

Plants are able to sense their environment and respond appropriately to different stimuli. Vibrational signals (VS) are one of the most widespread yet understudied ways of communication between organisms. Recent research into the perception of VS by plants showed that they are ecologically meaningful signals involved in different interactions of plants with biotic and abiotic agents. We studied changes in the concentration of alkaloids in tobacco plants induced by VS produced by Phthorimaea operculella (Lepidoptera: Gelechiidae), a generalist caterpillar that naturally feeds on the plant. We measured the concentration of nicotine, nornicotine, anabasine and anatabine in four treatments applied to 11-weeks old tobacco plant: a) Co = undamaged plants, b) Eq = Playback equipment attached to the plant without VS, c) Ca = Plants attacked by P. operculella herbivory and d) Pl = playback of VS of P. operculella feeding on tobacco. We found that nicotine, the most abundant alkaloid, increased more than 2.6 times in the Ca and Pl treatments as compared with the Co and Eq treatments, which were similar between them. Nornicotine, anabasine and anatabine were mutually correlated and showed similar concentration patterns, being higher in the Eq treatment. Results are discussed in terms of the adaptive significance of plant responses to ecologically important VS stimuli.


Assuntos
Alcaloides/análise , Lepidópteros/fisiologia , Tabaco/química , Alcaloides/metabolismo , Anabasina/análise , Animais , Cromatografia Gasosa-Espectrometria de Massas , Herbivoria , Interações Hospedeiro-Parasita , Larva/fisiologia , Modelos Lineares , Nicotina/análogos & derivados , Nicotina/análise , Análise de Componente Principal , Piridinas/análise , Tabaco/metabolismo , Tabaco/parasitologia , Vibração
3.
J Agric Food Chem ; 67(26): 7399-7409, 2019 Jul 03.
Artigo em Inglês | MEDLINE | ID: mdl-31244203

RESUMO

Flavonol synthase (FLS) belongs to the 2-oxoglutarate-dependent dioxygenase (2-ODD) superfamily. We isolated OsFLS from the rice ( Oryza sativa) cultivar "Ilmi" OsFLS includes highly conserved 2-ODD-specific motifs and FLS-specific regions. Recombinant OsFLS exhibited both FLS and flavanone 3ß-hydroxylase (F3H) activities, converting dihydroflavonols into flavonols and flavanones into dihydroflavonols, respectively, and more efficiently used dihydrokaempferol than dihydroquercetin as a substrate. OsFLS was expressed in both nonpigmented and pigmented rice seeds and was developmentally regulated during seed maturation. Transgenic tobacco ( Nicotiana tabacum) plants expressing OsFLS produced pale pink or white flowers with significantly increased levels of kaempferol-3- O-rutinoside and dramatically reduced levels of anthocyanin in their petals. Additionally, pod size and weight were reduced compared to the wild type. Several early and late biosynthetic genes of flavonoid were downregulated in the transgenic flowers. We demonstrated that OsFLS is a bifunctional 2-ODD enzyme and functions in flavonol production in planta.


Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Oryza/enzimologia , Oxirredutases/genética , Oxirredutases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Antocianinas/biossíntese , Cor , Flavonóis/biossíntese , Flores/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Cetoglutáricos/metabolismo , Oryza/genética , Tabaco/genética , Tabaco/metabolismo
4.
BMC Plant Biol ; 19(1): 253, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31196035

RESUMO

BACKGROUND: Because of their broad applications in our life, nanoparticles are expected to be present in the environment raising many concerns about their possible adverse effects on the ecosystem of plants. The aim of this study was to examine the effect of different sizes and concentrations of iron oxide nanoparticles [(Fe3O4) NPs] on morphological, physiological, biochemical, and ultrastructural parameters in tobacco (Nicotiana tabacum var.2 Turkish). RESULTS: Lengths of shoots and roots of 5 nm-treated plants were significantly decreased in all nanoparticle-treated plants compared to control plants or plants treated with any concentration of 10 or 20 nm nanoparticles. The photosynthetic rate and leaf area were drastically reduced in 5 nm (Fe3O4) NP-treated plants of all concentrations compared to control plants and plants treated with 10 or 20 nm (Fe3O4) NPs. Accumulation of sugars in leaves showed no significant differences between the control plants and plants treated with iron oxide of all sizes and concentrations. In contrast, protein accumulation in plants treated with 5 nm iron oxide dramatically increased compared to control plants. Moreover, light and transmission electron micrographs of roots and leaves revealed that roots and chloroplasts of 5 nm (Fe3O4) NPs-treated plants of all concentrations were drastically affected. CONCLUSIONS: The size and concentration of nanoparticles are key factors affecting plant growth and development. The results of this study demonstrated that the toxicity of (Fe3O4) NPs was clearly influenced by size and concentration. Further investigations are needed to elucidate more about NP toxicity in plants, especially at the molecular level.


Assuntos
Nanopartículas Metálicas , Tabaco/efeitos dos fármacos , Cloroplastos/efeitos dos fármacos , Cloroplastos/ultraestrutura , Relação Dose-Resposta a Droga , Compostos Férricos/farmacologia , Microscopia Eletrônica de Transmissão , Fotossíntese/efeitos dos fármacos , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/ultraestrutura , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/ultraestrutura , Brotos de Planta/efeitos dos fármacos , Tabaco/metabolismo , Tabaco/ultraestrutura
5.
Plant Mol Biol ; 101(1-2): 95-112, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31236845

RESUMO

KEY MESSAGE: Sorghum glycine rich proline rich protein (SbGPRP1) exhibit antimicrobial properties and play a crucial role during biotic stress condition. Several proteins in plants build up the innate immune response system in plants which get triggered during the occurrence of biotic stress. Here we report the functional characterization of a glycine-rich proline-rich protein (SbGPRP1) from Sorghum which was previously demonstrated to be involved in abiotic stresses. Expression studies carried out with SbGPRP1 showed induced expression upon application of phytohormones like salicylic acid which might be the key in fine-tuning the expression level. Upon challenging the Sorghum plants with a compatible pathogen the SbGprp1 transcript was found to be upregulated. SbGPRP1 encodes a 197 amino acid polypeptide which was bacterially-expressed and purified for in vitro assays. Gram-positive bacteria like Bacillus and phytopathogen Rhodococcus fascians showed inhibited growth in the presence of the protein. The NPN assay, electrolytic leakage and SEM analysis showed membrane damage in bacterial cells. Ectopic expression of SbGPRP1 in tobacco plants led to enhanced tolerance towards infection caused by R. fascians. Though the N-terminal part of the protein showed disorderness the C-terminal end was quite capable of forming several α-helices which was correlated with CD spectroscopic analysis. Here, we have tried to determine the structural model for the protein and predicted the association of antimicrobial activity with the C-terminal region of the protein.


Assuntos
Anti-Infecciosos/metabolismo , Doenças das Plantas/imunologia , Reguladores de Crescimento de Planta/farmacologia , Proteínas de Plantas/metabolismo , Ácido Salicílico/farmacologia , Sorghum/genética , Bacillus/efeitos dos fármacos , Expressão Ectópica do Gene , Glicina/metabolismo , Filogenia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Prolina/metabolismo , Rhodococcus/efeitos dos fármacos , Sorghum/imunologia , Sorghum/metabolismo , Sorghum/microbiologia , Estresse Fisiológico , Tabaco/genética , Tabaco/imunologia , Tabaco/metabolismo , Tabaco/microbiologia
6.
Plant Physiol Biochem ; 141: 388-397, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31226508

RESUMO

Members of the Jasmonate ZIM domain (JAZ) proteins act as transcriptional repressors in the jasmonate (JA) hormonal response. To characterize the potential roles of JAZ gene family in plant development and abiotic stress response, fifteen JAZs were identified based on the genome of Nicotiana tabacum. Structural analysis confirmed the presence of single Jas and TIFY motif. Tissue expression pattern analysis indicated that NtJAZ-2, -3, -5, and -10 were highly expressed in roots and NtJAZ-11 was expressed only in the cotyledons. The transcript level of NtJAZ-3, -5, -9, and -10 in the stem epidermis was higher than that in the stem without epidermis. Dynamic expression of NtJAZs exposed to abiotic stress and phytohormone indicated that the expression of most NtJAZs was activated by salicylic acid, methyl jasmonate, gibberellic acid, cold, salt, and heat stresses. With abscisic acid treatment, NtJAZ-1, -2, and -3 were not activated; NtJAZ-4, -5, and -6 were up-regulated; and the remaining NtJAZ genes were inhibited. With drought stress, the expression of NtJAZ-1, -2, -3, -4, -5, -6, -7, and -8 was up-regulated, whereas the transcript of the remaining genes was inhibited. Moreover, high concentration MeJA (more than 1 mM MeJA) had an effect on secreting trichome induction, but inhabited the plant growth. Nine NtJAZs may play important role in secreting trichome induction. These results indicate that the JAZ proteins are convergence points for various phytohormone signal networks, which are involved in abiotic stress responses.


Assuntos
Proteínas de Plantas/metabolismo , Proteínas Repressoras/metabolismo , Estresse Fisiológico , Tabaco/genética , Tabaco/metabolismo , Tricomas/metabolismo , Acetatos/metabolismo , Motivos de Aminoácidos , Arabidopsis/metabolismo , Cromossomos/metabolismo , Ciclopentanos/metabolismo , Bases de Dados Genéticas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Cadeias de Markov , Oxilipinas/metabolismo , Filogenia , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Proteínas Repressoras/genética , Transdução de Sinais , Fatores de Transcrição/genética
7.
Plant Physiol Biochem ; 141: 431-445, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31238253

RESUMO

S-adenosylmethionine synthetase (SAMS) catalyzes methionine and ATP to generate S-adenosyl-L-methionine (SAM). In plants, accumulating SAMS genes have been characterized and the majority of them are reported to participate in development and stress response. In this study, two putative SAMS genes (CsSAMS1 and CsSAMS2) were identified in cucumber (Cucumis Sativus L.). They displayed 95% similarity and had a high identity with their homologous of Arabidopsis thaliana and Nicotiana tabacum. The qRT-PCR test showed that CsSAMS1 was predominantly expressed in stem, male flower, and young fruit, whereas CsSAMS2 was preferentially accumulated in stem and female flower. And they displayed differential expression profiles under stimuli, including NaCl, ABA, SA, MeJA, drought and low temperature. To elucidate the function of cucumber SAMS, the full-length CDS of CsSAMS1 was cloned, and prokaryotic expression system and transgenic materials were constructed. Expressing CsSAMS1 in Escherichia coli BL21 (DE3) improved the growth of the engineered strain under salt stress. Overexpression of CsSAMS1 significantly increased MDA content, H2O2 content, and POD activity in transgenic lines under non-stress condition. Under salt stress, however, the MDA content of transgenic lines was lower than that of the wild type, the H2O2 content remained high, the polyamine and ACC synthesis in transgenic lines exhibited a CsSAMS1-expressed dependent way. Taken together, our results suggested that both CsSAMS1 and CsSAMS2 were involved in plant development and stress response, and a proper increase of expression level of CsSAMS1 in plants is benificial to improving salt tolerance.


Assuntos
Cucumis sativus/enzimologia , Cucumis sativus/fisiologia , Metionina Adenosiltransferase/metabolismo , Tolerância ao Sal , Antioxidantes/metabolismo , Arabidopsis/metabolismo , Secas , Perfilação da Expressão Gênica , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/química , Metionina Adenosiltransferase/genética , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Poliaminas/química , Sais , Estresse Mecânico , Estresse Fisiológico/genética , Tabaco/metabolismo
8.
Environ Sci Pollut Res Int ; 26(22): 22529-22550, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31161543

RESUMO

Widespread application of silver nanoparticles (AgNPs), due to their antibacterial and antifungal properties, increases their release into the environment and potential detrimental impact on living organisms. Plants may serve as a potential pathway for AgNPs bioaccumulation and a route into the food chain, hence investigation of AgNP phytotoxic effects are of particular importance. Since proteins are directly involved in stress response, studies of their abundance changes can help elucidate the mechanism of the AgNP-mediated phytotoxicity. In this study, we investigated proteomic changes in tobacco (Nicotiana tabacum) exposed to AgNPs and ionic silver (AgNO3). A high overlap of differently abundant proteins was found in root after exposure to both treatments, while in leaf, almost a half of the proteins exhibited different abundance level between treatments, indicating tissue-specific responses. Majority of the identified proteins were down-regulated in both tissues after exposure to either AgNPs or AgNO3; in roots, the most affected proteins were those involved in response to abiotic and biotic stimuli and oxidative stress, while in leaf, both treatments had the most prominent effect on photosynthesis-related proteins. However, since AgNPs induced higher suppression of protein abundance than AgNO3, we conclude that AgNP effects can, at least partially, be attributed to nanoparticle form.


Assuntos
Nanopartículas Metálicas/toxicidade , Prata/toxicidade , Tabaco/fisiologia , Íons , Estresse Oxidativo/efeitos dos fármacos , Folhas de Planta/metabolismo , Proteoma/metabolismo , Proteômica , Nitrato de Prata/toxicidade , Tabaco/metabolismo
9.
Ecotoxicol Environ Saf ; 182: 109369, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31238115

RESUMO

Growth and developmental changes in plants induced by pharmaceuticals reflect changes in processes at the cellular and subcellular levels. Due to their growth and cellular characteristics, plant cell suspension cultures can be a suitable model for assessing toxicity. In this study, 10-1000 µg/L of the non-steroidal anti-inflammatory drug diclofenac (DCF) decreased the viability of Nicotiana tabacum BY-2 cells after 24 h of treatment. Further, 0.1-10 mg/L DCF diminished the density of the cell suspension by 9-46% after 96 h of treatment, but at 1 and 10 µg/L, DCF increased the density by 13% and 5%, respectively, after 120 h. These changes were accompanied by increased production of total reactive oxygen species (ROS) and mitochondrial superoxide (up to 17-fold and 5-fold, respectively), and a decrease in the mitochondrial membrane potential (by ∼64%) especially at 1000 µg/L DCF. The increased ROS production was accompanied by decrease in level of reactive nitrogen species (RNS; by 36%) and total thiols (by 61%). Damage to BY-2 cells was evidenced by accumulation of neutral red in acidic compartments (up to 10-fold at 1000 µg/L DCF), and increase of autophagic vacuole formation (up to 8-fold at 1000 µg/L DCF). Furthermore, irregular or stretched nuclei were observed in nearly 27% and 50% of cells at 100 and 1000 µg/L DCF, respectively. Highest levels of chromatin condensation (11% of cells) and apoptotic DNA fragmentation (7%) were found at 10 µg/L DCF. The results revealed a significant effect of DCF on BY-2 cells after 24 h of exposure. Changes in the growth and viability parameters were indisputably related to ROS and RNS production, changes in mitochondrial function, and possible activation of processes leading to cell death.


Assuntos
Anti-Inflamatórios não Esteroides/toxicidade , Diclofenaco/toxicidade , Testes de Toxicidade/métodos , Mitocôndrias/metabolismo , Espécies Reativas de Nitrogênio , Espécies Reativas de Oxigênio/metabolismo , Superóxidos , Suspensões , Tabaco/metabolismo
10.
Plant Sci ; 284: 16-24, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31084868

RESUMO

In this paper, we evaluated the genotoxicity of cadmium (Cd) in plants by performing a methylation-sensitive amplification polymorphism (MSAP) on the model plant Nicotiana benthamiana. Among 255 loci examined, 14 genes were found to show altered cytosine methylation patterns in response to Cd stress. Four of those genes (NbMORC3, NbHGSNAT, NbMUT, and NbBG) were selected for further analysis due to their predicted roles in plant development. Cd-induced changes of cytosine methylation status in MSAP fragments of selected genes were confirmed using bisulfite sequencing polymerase chain reaction (BSP). In addition, the expression levels of these genes were found to correlate with cadmium dosage, and a knock-down of these four genes via virus-induced genes silencing (VIGS) led to abnormal development and elevated sensitivity to cadmium stress. Silencing of these four genes resulted in altered cadmium accumulation in different parts of the experimental plants. Our data indicate that cadmium exposure causes dramatic changes in the cytosine methylation status of the plant genome, thus affecting the expression of many genes that are vital for plant growth and are involved in cadmium stress response.


Assuntos
Cádmio/toxicidade , Citosina/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Tabaco/efeitos dos fármacos , DNA de Plantas/efeitos dos fármacos , Genes de Plantas/efeitos dos fármacos , Metilação/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Estresse Fisiológico/efeitos dos fármacos , Tabaco/genética , Tabaco/metabolismo
11.
Environ Pollut ; 251: 13-21, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31071628

RESUMO

Cigarette smoking (CS) and betel quid (BQ) chewing are two known risk factors that have synergistic potential for the enhancing the development of oral squamous cell carcinoma (OSCC) in Taiwan. Most mutagens and carcinogens are metabolically activated by cytochrome P450 (CYP450) to exert their mutagenicity or carcinogenicity. Previous studies have shown that metabolic activation of the tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), by CYP2A6 activity determines NNK-induced carcinogenesis. In addition, safrole affects cytochrome P450 activity in rodents. However, the effect of BQ safrole on the metabolism of tobacco-specific NNK and its carcinogenicity remains elusive. This study demonstrates that safrole (1 mg/kg/d) induced CYP2A6 activity, reduced urinary 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) levels, and increased NNK-induced DNA damage, including N7-methylguanine, 8-OH-deoxyguanosine and DNA strand breaks in a Syrian golden hamster model. Furthermore, altered NNK metabolism and increased NNK-induced DNA damage were also observed in healthy subjects with CS and BQ chewing histories compared to healthy subjects with CS histories. In conclusion, BQ containing safrole induced tobacco-specific NNK metabolic activation, resulting in higher NNK-induced genotoxicity. This study provides valuable insight into the synergistic mechanisms of CS- and BQ-induced OSCC.


Assuntos
Nitrosaminas/urina , Safrol/toxicidade , Uso de Tabaco/urina , Tabaco/metabolismo , Ativação Metabólica/efeitos dos fármacos , Animais , Carcinoma de Células Escamosas/induzido quimicamente , Carcinoma de Células Escamosas/metabolismo , Cricetinae , Citocromo P-450 CYP2A6/metabolismo , Feminino , Humanos , Fígado/efeitos dos fármacos , Fígado/enzimologia , Neoplasias Bucais/induzido quimicamente , Neoplasias Bucais/metabolismo , Taiwan , Tabaco/toxicidade
12.
Environ Sci Pollut Res Int ; 26(18): 18053-18070, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31093913

RESUMO

The successful phytoextraction of potentially toxic elements (PTEs) from polluted soils can be achieved by growing non-food and industrial crops. Tobacco (Nicotiana tabacum L.) is one of the main industrial crops and is widely grown in many countries. Tobacco can uptake high concentrations of PTEs especially in aboveground biomass without suffering from toxicity. This review highlighted the potential of tobacco for the phytoextraction of heavy metals and tolerance mechanisms under metal stress. Different management practices have been discussed which can enhance the potential of this plant for metal extraction. Finally, suitable options for the management/disposal of biomass enriched in excess metal have been elaborated to prevent secondary pollution.


Assuntos
Metais Pesados/análise , Poluentes do Solo/análise , Solo/química , Tabaco/crescimento & desenvolvimento , Biodegradação Ambiental , Biomassa , Metais Pesados/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Poluentes do Solo/metabolismo , Tabaco/efeitos dos fármacos , Tabaco/metabolismo
13.
Plant Sci ; 283: 147-156, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128684

RESUMO

Harpin proteins secreted by plant-pathogenic gram-negative bacteria induce diverse plant defenses against different pathogens. Harpin-induced 1 (HIN1) gene highly induced in tobacco after application of Harpin protein is involved in a common plant defense pathway. However, the role of HIN1 against Tobacco mosaic virus (TMV) remains unknown. In this study, we functionally characterized the Nicotiana benthamiana HIN1 (NbHIN1) gene and generated the transgenic tobacco overexpressing the NbHIN1 gene. In a subcellular localization experiment, we found that NbHIN1 localized in the plasma membrane and cytosol. Overexpression of NbHIN1 did not lead to observed phenotype compared to wild type tobacco plant. However, the NbHIN1 overexpressing tobacco plant exhibited significantly enhanced resistance to TMV infection. Moreover, RNA-sequencing revealed the transcriptomic profiling of NbHIN1 overexpression and highlighted the primary effects on the genes in the processes related to biosynthesis of amino acids, plant-pathogen interaction and RNA transport. We also found that overexpression of NbHIN1 highly induced the expression of NbRAB11, suggesting that jasmonic acid signaling pathway might be involved in TMV resistance. Taken together, for the first time we demonstrated that overexpressing a pathogenesis-related gene NbHIN1 in N. benthamiana significantly enhances the TMV resistance, providing a potential mechanism that will enable us to engineer tobacco with improved TMV resistance in the future.


Assuntos
Ciclopentanos/metabolismo , Resistência à Doença/genética , Oxilipinas/metabolismo , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Transdução de Sinais , Vírus do Mosaico do Tabaco , Tabaco/genética , Western Blotting , Clonagem Molecular , Genes de Plantas/genética , Genes de Plantas/fisiologia , Microscopia Confocal , Filogenia , Folhas de Planta/metabolismo , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas , Reação em Cadeia da Polimerase em Tempo Real , Transdução de Sinais/genética , Tabaco/metabolismo , Tabaco/virologia , Técnicas do Sistema de Duplo-Híbrido
14.
Plant Sci ; 283: 195-201, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128689

RESUMO

Methionine and threonine are two essential amino acids whose low levels limit the nutritional quality of seeds. The current objective was to define factors that regulate and might increase their levels in seeds. Feeding experiments carried out on receptacles of developing tobacco (Nicotiana tabacum) capsules showed that 1 mM of S-methylmethionine increased the level of methionine to contents similar to 2.5 mM of homoserine, an intermediate metabolite of the aspartate family of amino acids. The latter also increased the level of threonine. Based on these findings, we generated tobacco seeds that expressed a combination of bacterial feedback-insensitive aspartate kinase (bAK), which was previously reported to have a high level of threonine/methionine, and feedback-insensitive cystathionine γ-synthase (CGS), the regulatory enzyme of the methionine biosynthesis pathway. Plants expressing this latter gene previously showed having higher levels of methionine. The results of total amino acids analysis showed that the level of threonine was highest in the bAK line, which has moderate levels of methionine and lysine, while the highest level of methionine was found in seeds expressing both heterologous genes. The results suggest that the level of threonine in tobacco seeds is limited by the substrate, while that of methionine is limited also by the activity of CGS.


Assuntos
Carbono-Oxigênio Liases/metabolismo , Metionina/metabolismo , Proteínas de Plantas/metabolismo , Sementes/metabolismo , Treonina/metabolismo , Tabaco/metabolismo , Aminoácidos/metabolismo , Homosserina/metabolismo , Redes e Vias Metabólicas , Plantas Geneticamente Modificadas , Sementes/enzimologia , Tabaco/enzimologia
15.
Plant Sci ; 283: 329-342, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128704

RESUMO

Cold is an abiotic stress seriously threatening crop productivity by decreasing biomass production. The pollen tube is a target of cold stress, but also a useful model to address questions on cell wall biosynthesis. We here provide (immuno)cytological data relative to the impact of cold on the pollen tube cell wall. We clearly show that the growth pattern is severely affected by the stress, since the typical pulsed-growth mechanism accompanied by the periodic deposition of pectin rings is absent/severely reduced. Additionally, pectins and cellulose accumulate in bulges provoked by the stress, while callose, which colocalizes with pectins in the periodic rings formed during pulsed growth, accumulates randomly in the stressed samples. The altered distribution of the cell wall components is accompanied by differences in the localization of glucan synthases: cellulose synthase shows a more diffuse localization, while callose synthase shows a more frequent cytoplasmic accumulation, thereby denoting a failure in plasma membrane insertion. The cell wall observations are complemented by the analysis of intracellular Ca2+, pH and reactive oxygen species (ROS): while in the case of pH no major differences are observed, a less focused Ca2+ and ROS gradients are present in the stressed samples. The standard oscillatory growth of pollen tubes is recovered by transient changes of turgor pressure induced by hypoosmotic media. Overall our data contribute to the understanding of the impact that cold stress has on the normal development of the pollen tube and unveil the cell wall-related aberrant features accompanying the observed alterations.


Assuntos
Parede Celular/metabolismo , Tubo Polínico/crescimento & desenvolvimento , Tabaco/genética , Cálcio/metabolismo , Parede Celular/fisiologia , Celulose/metabolismo , Resposta ao Choque Frio , Técnica Indireta de Fluorescência para Anticorpo , Germinação , Glucanos/metabolismo , Concentração de Íons de Hidrogênio , Pressão Osmótica , Pectinas/metabolismo , Pólen/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Tabaco/metabolismo
16.
Planta ; 250(2): 463-473, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31065786

RESUMO

MAIN CONCLUSION: RDR6 gene knockout Nicotiana benthamiana plant was successfully produced using CRISPR/Cas9 technology. The production of recombinant proteins in plants has many advantages, such as safety and reduced costs. However, there are several problems with this technology, especially low levels of protein production. The dysfunction of the RNA silencing mechanism in plant cells would be effective to improve recombinant protein production because the RNA silencing mechanism efficiently degrades transgene-derived mRNAs. Therefore, to overcome this problem, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology was used to develop RNA silencing-related gene knockout transgenic Nicotiana benthamiana. We successfully produced RNA-dependent RNA polymerase 6 (RDR6), one of the most important components of the RNA silencing mechanism-knockout N. benthamiana (ΔRDR6 plants). The ΔRDR6 plants had abnormal flowers and were sterile, as with the Arabidopsis RDR6 mutants. However, a transient gene expression assay showed that the ΔRDR6 plants accumulated larger amounts of green fluorescent protein (GFP) and GFP mRNA than the wild-type (WT) plants. Small RNA sequencing analysis revealed that levels of small interfering RNA against the GFP gene were greatly reduced in the ΔRDR6 plants, as compared to that of the WT plants. These findings demonstrate that the ΔRDR6 plants can express larger amounts of recombinant proteins than WT plants and, therefore, would be useful for recombinant protein production and understanding the contributions of RDR6 to genetic and physiological events in plants.


Assuntos
Sistemas CRISPR-Cas , Plantas Geneticamente Modificadas , RNA Replicase/metabolismo , Tabaco/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Edição de Genes , Expressão Gênica , Proteínas de Fluorescência Verde , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Interferência de RNA , RNA Replicase/genética , Proteínas Recombinantes , Tabaco/metabolismo
17.
Int J Mol Sci ; 20(10)2019 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091777

RESUMO

Salinity is one of the most severe forms of abiotic stress and affects crop yields worldwide. Plants respond to salinity stress via a sophisticated mechanism at the physiological, transcriptional and metabolic levels. However, the molecular regulatory networks involved in salt and alkali tolerance have not yet been elucidated. We developed an RNA-seq technique to perform mRNA and small RNA (sRNA) sequencing of plants under salt (NaCl) and alkali (NaHCO3) stress in tobacco. Overall, 8064 differentially expressed genes (DEGs) and 33 differentially expressed microRNAs (DE miRNAs) were identified in response to salt and alkali stress. A total of 1578 overlapping DEGs, which exhibit the same expression patterns and are involved in ion channel, aquaporin (AQP) and antioxidant activities, were identified. Furthermore, genes involved in several biological processes, such as "photosynthesis" and "starch and sucrose metabolism," were specifically enriched under NaHCO3 treatment. We also identified 15 and 22 miRNAs that were differentially expressed in response to NaCl and NaHCO3, respectively. Analysis of inverse correlations between miRNAs and target mRNAs revealed 26 mRNA-miRNA interactions under NaCl treatment and 139 mRNA-miRNA interactions under NaHCO3 treatment. This study provides new insights into the molecular mechanisms underlying the response of tobacco to salinity stress.


Assuntos
MicroRNAs/genética , RNA Mensageiro/genética , Tolerância ao Sal , Tabaco/genética , Transcriptoma , Carbonatos/farmacologia , Regulação da Expressão Gênica de Plantas , MicroRNAs/metabolismo , RNA Mensageiro/metabolismo , Tabaco/efeitos dos fármacos , Tabaco/metabolismo
18.
Plant Physiol Biochem ; 139: 591-599, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31030027

RESUMO

In this research, in-silico and in-vitro approaches were adopted with the aim to investigate the relationship between the tobacco leaf structures (trichomes) and the production of secondary metabolites with antimicrobial activity. Machine learning techniques were used to know the correlation between phenotypic traits and the production of secondary metabolites in Nicotiana tabacum plants. Then, an in-vitro experimental study was carried out to corroborate the proposed model. The relationship between the morphology and distribution of the different types of trichomes in the upper and lower leaves with the contrasting profiles of the chemical composition (diterpenes and sugar esters) of the leaf exudates between different lines of tobacco were found. We determined the influence of each trichome type with secondary metabolites production and the necessary concentration to achieve antimicrobial and antioxidant activity.


Assuntos
Tabaco/metabolismo , Regulação da Expressão Gênica de Plantas , Aprendizado de Máquina , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Tabaco/genética
19.
IET Nanobiotechnol ; 13(1): 23-29, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30964033

RESUMO

The stress conditions imposed by the impact of metal and non-metal oxide nanoparticles over plant systems enhances the synthesis of reactive oxygen species (ROS), resulting in oxidative damage at cellular level. The objective of this study was to synthesise the gold nanoparticles (GNps) from the leaves protein of Nicotiana tabacum L. cv. xanthi, its characterisation, and response on plant physiology and ROS scavenging activity on plants after exposure to different stresses. The authors have treated N. tabacum L. cv. xanthi plants with 100, 200, 300, 400, and 500 ppm biochemically synthesised GNps and examined physiological as well as biochemical changes. Results showed that biochemically synthesised GNps exposure significantly increased the seed germination (P < 0.001), root (P < 0.001), shoot growth (P < 0.001), and antioxidant ability (P < 0.05) of plants depending on bioengineered GNPs concentrations. Low concentrations (200-300 ppm) of GNps boosted growth by ∼50% and significantly increase in photosynthetic parameters such as total chlorophyll content (P < 0.05), membrane ion leakage (P < 0.05) as well as malondialdehyde (P < 0.05) content with respect to untreated plants under stress conditions. The high concentration (400-500 ppm) of GNps affected these parameters in a negative manner. The total antioxidant activity was also elevated in the exposed plants in a dose-dependent manner.


Assuntos
Depuradores de Radicais Livres/farmacologia , Ouro/química , Nanopartículas Metálicas/química , Estresse Fisiológico/efeitos dos fármacos , Tabaco/metabolismo , Química Verde , Folhas de Planta/química , Folhas de Planta/metabolismo , Plantas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Tabaco/química
20.
Methods Mol Biol ; 1933: 297-303, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30945194

RESUMO

RNA-protein interactions play important roles in various eukaryotic biological processes. Molecular imaging of subcellular localization of RNA-protein complexes in plants is critical for understanding these interactions. However, methods to image RNA-protein interactions in living plants have not yet been developed until now. Recently, we have developed a trimolecular fluorescence complementation (TriFC) system for in vivo visualization of RNA-protein interaction by transient expression in tobacco leaves. In this method, we combined conventional bimolecular fluorescence complementation (BiFC) system with the MS2 system (phage MS2 coat protein [MCP] and its binding RNA sequence [MS2 sequence]) to tag lncRNA. Target RNA is tagged with 6xMS2, and MCP and RNA-binding protein are fused with YFP fragments. DNA constructs encoding such fusion RNA and proteins are infiltrated into tobacco leaves with Agrobacterium suspensions. RNA-protein interaction in vivo is observed by confocal microscopy.


Assuntos
Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência/métodos , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , RNA de Plantas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Tabaco/metabolismo , Fluorescência , Regulação da Expressão Gênica de Plantas , Vetores Genéticos , Proteínas Luminescentes/genética , Microscopia Confocal , Folhas de Planta/genética , Proteínas de Plantas/genética , RNA de Plantas/genética , Proteínas de Ligação a RNA/genética , Tabaco/genética
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