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1.
Plant Mol Biol ; 101(1-2): 149-162, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31267255

RESUMO

KEY MESSAGE: Here we describe that the regulation of MdWRKY31 on MdHIR4 in transcription and translation levels associated with disease in apple. The phytohormone salicylic acid (SA) is a main factor in apple (Malus domestica) production due to its function in disease resistance. WRKY transcription factors play a vital role in response to stress. An RNA-seq analysis was conducted with 'Royal Gala' seedlings treated with SA to identify the WRKY regulatory mechanism of disease resistance in apple. The analysis indicated that MdWRKY31 was induced. A quantitative real-time polymerase chain reaction (qPCR) analysis demonstrated that the expression of MdWRKY31 was induced by SA and flg22. Ectopic expression of MdWRKY31 in Arabidopsis and Nicotiana benthamiana increased the resistance to flg22 and Pseudomonas syringae tomato (Pst DC3000). A yeast two-hybrid screen was conducted to further analyze the function of MdWRKY31. As a result, hypersensitive-induced reaction (HIR) protein MdHIR4 interacted with MdWRKY31. Biomolecular fluorescence complementation, yeast two-hybrid, and pull-down assays demonstrated the interaction. In our previous study, MdHIR4 conferred decreased resistance to Botryosphaeria dothidea (B. dothidea). A viral vector-based transformation assay indicated that MdWRKY31 evaluated the transcription of SA-related genes, including MdPR1, MdPR5, and MdNPR1 in an MdHIR4-dependent way. A GUS analysis demonstrated that the w-box, particularly w-box2, of the MdHIR4 promoter played a major role in the responses to SA and B. dothidea. Electrophoretic mobility shift assays, yeast one-hybrid assay, and chromatin immunoprecipitation-qPCR demonstrated that MdWRKY31 directly bound to the w-box2 motif in the MdHIR4 promoter. GUS staining activity and a protein intensity analysis further showed that MdWRKY31 repressed MdHIR4 expression. Taken together, our findings reveal that MdWRKY31 regulated plant resistance to B. dothidea through the SA signaling pathway by interacting with MdHIR4.


Assuntos
Resistência à Doença , Malus/genética , Doenças das Plantas/imunologia , Reguladores de Crescimento de Planta/farmacologia , Proteínas de Plantas/metabolismo , Ácido Salicílico/farmacologia , Arabidopsis/genética , Arabidopsis/imunologia , Arabidopsis/microbiologia , Ascomicetos/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Frutas/genética , Frutas/imunologia , Frutas/microbiologia , Regulação da Expressão Gênica de Plantas , Genes Reporter , Malus/imunologia , Malus/microbiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/genética , Regiões Promotoras Genéticas/genética , Pseudomonas syringae/fisiologia , Plântula/genética , Plântula/imunologia , Plântula/microbiologia , Transdução de Sinais , Tabaco/genética , Tabaco/imunologia , Tabaco/microbiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido
2.
BMC Plant Biol ; 19(1): 314, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307397

RESUMO

BACKGROUND: LEAFY COTYLEDON 2 (LEC2) acts throughout embryo morphogenesis and maturation phase to maintain embryogenic identity. Our previous study stated that Arabidopsis thaliana LEC2 (AtLEC2) driven by glucocorticoid receptor-dexamethasone (GR-DEX) inducible system (AtLEC2-GR) triggers embryogenic callus formation in tobacco (Nicotiana tabacum). RESULTS: In this study, the adenosine phosphate isopentenyltransferase genes AtIPT3, AtIPT7 and the tRNA isopentenyltransferase gene AtIPT9 were overexpressed in the AtLEC2-GR transgenic background. In the AtIPT7-OE AtLEC2-GR and AtIPT9-OE AtLEC2-GR seedlings, high-quality embryogenic callus was obtained under the DEX condition, and the shoot regeneration efficiency was 2 to 3.5 folds higher than AtLEC2-GR alone on hormone free medium without DEX. Transcriptome analyses showed that up-regulated BBM, L1L, ABI3, and FUS3 might function during embryogenic callus formation. However, at the shoot regeneration stage, BBM, L1L, ABI3, and FUS3 were down-regulated and Type-B ARRs were up-regulated, which might contribute to the increased shoot regeneration rate. CONCLUSIONS: A novel system for inducing shoot regeneration in tobacco has been developed using the GR-DEX system. Induced expression of AtLEC2 triggers embryogenic callus formation and overexpression of AtIPT7 or AtIPT9 improves shoot regeneration without exogenous cytokinin.


Assuntos
Alquil e Aril Transferases/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Brotos de Planta/crescimento & desenvolvimento , Técnicas de Embriogênese Somática de Plantas , Tabaco/genética , Fatores de Transcrição/genética , Dexametasona/farmacologia , Plantas Geneticamente Modificadas , Receptores de Glucocorticoides/genética , Sementes , Tabaco/embriologia , Tabaco/crescimento & desenvolvimento
3.
BMC Plant Biol ; 19(1): 310, 2019 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-31307401

RESUMO

BACKGROUND: The hypersensitive defense response (HR) in plants is a fast, localized necrotic response around the point of pathogen ingress. HR is usually triggered by a pathogen recognition event mediated by a nucleotide-binding site, leucine-rich repeat (NLR) protein. The autoactive maize NLR gene Rp1-D21 confers a spontaneous HR response in the absence of pathogen recognition. Previous work identified a set of loci associated with variation in the strength of Rp1-D21-induced HR. A polygalacturonase gene homolog, here termed ZmPGH1, was identified as a possible causal gene at one of these loci on chromosome 7. RESULTS: Expression of ZmPGH1 inhibited the HR-inducing activity of both Rp1-D21 and that of another autoactive NLR, RPM1(D505V), in a Nicotiana benthamiana transient expression assay system. Overexpression of ZmPGH1 in a transposon insertion line of maize was associated with suppression of chemically-induced programmed cell death and with suppression of HR induced by Rp1-D21 in maize plants grown in the field. CONCLUSIONS: ZmPGH1 functions as a suppressor of programmed cell death induced by at least two autoactive NLR proteins and by two chemical inducers. These findings deepen our understanding of the control of the HR in plants.


Assuntos
Apoptose/fisiologia , Proteínas de Plantas/fisiologia , Poligalacturonase/fisiologia , Zea mays/fisiologia , Apoptose/genética , Mapeamento Cromossômico , Cromossomos de Plantas , Genes de Plantas , Leucina , Proteínas de Plantas/química , Proteínas de Plantas/genética , Poligalacturonase/química , Poligalacturonase/genética , Recombinação Genética , Sequências Repetitivas de Aminoácidos , Tabaco/genética , Zea mays/enzimologia , Zea mays/genética , Zea mays/imunologia
4.
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
5.
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
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.
J Agric Food Chem ; 67(27): 7738-7747, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31199650

RESUMO

Cytosinpeptidemycin (CytPM) is a microbial pesticide that displayed broad-spectrum antiviral activity against various plant viruses. However, the molecular mechanism underlying antiviral activity of CytPM is poorly understood. In this study, the results demonstrated that CytPM could effectively delay the systemic infection of tobacco mosaic virus (TMV) in Nicotiana benthamiana and significantly inhibit the viral accumulation in tobacco BY-2 protoplasts. Results of RNA-seq indicated that 210 and 120 differential expressed genes (DEGs) were significantly up- and down-regulated after CytPM treatment in BY-2 protoplasts, respectively. In addition, KEGG analysis indicated that various DEGs were involved in endoplasmic reticulum (ER) protein processing, suggesting a possible correlation between ER homeostasis and virus resistance. RT-qPCR was performed to validate the gene expression of crucial DEGs related with defense, stress responses, signaling transduction, and phytohormone, which were consistent with results of RNA-seq. Our works provided valuable insights into the antiviral mechanism of CytPM that induced host resistance to viral infection.


Assuntos
Antivirais , Citosina/análogos & derivados , Resistência à Doença/genética , Doenças das Plantas/prevenção & controle , Vírus do Mosaico do Tabaco/fisiologia , Tabaco/virologia , Citosina/farmacologia , Resistência à Doença/efeitos dos fármacos , Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Interações Hospedeiro-Patógeno/genética , Doenças das Plantas/virologia , Reguladores de Crescimento de Planta/genética , Protoplastos/efeitos dos fármacos , Protoplastos/virologia , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genética , Tabaco/genética , Vírus do Mosaico do Tabaco/efeitos dos fármacos , Vírus do Mosaico do Tabaco/patogenicidade
8.
BMC Plant Biol ; 19(1): 231, 2019 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-31159735

RESUMO

BACKGROUND: Four-Coumarate:CoA ligase gene (4CL) plays multiple important roles in plant growth and development by catalyzing the formation of CoA ester. 4CL belongs to the plant phenylpropane derivative, which is related to the synthesis of flavonoids and lignin and is a key enzyme in the biosynthetic pathway. RESULTS: In this study, 12 4CL genes of Fraxinus mandschurica were identified and named Fm4CL1-Fm4CL12, respectively. The analysis of the expression pattern of Fm4CL genes indicate that Fm4CL-like 1 gene may play a role in the lignin synthesis pathway. Our study indicate that overexpression of Fm4CL-like 1 increases the lignin content of transgenic tobacco by 39.5% compared to WT, and the S/G ratio of transgenic tobacco increased by 19.7% compared with WT. The xylem cell layer of transgenic line is increased by 40% compared to WT, the xylem cell wall thickness increased by 21.6% compared to the WT. Under mannitol-simulated drought stress, the root length of transgenic tobacco is 64% longer than WT, and the seed germination rate of the transgenic lines is 47% higher than that of WT. In addition, the H2O2 content in the transgenic tobacco was 22% lower than that of WT, while the POD and SOD content was higher than WT by 30 and 24% respectively, which showed Fm4CL-like 1 affect the accumulation of reactive oxygen species (ROS). The MDA content and relative conductivity was 25 and 15% lower than WT, respectively. The water loss rate is 16.7% lower than that of WT. The relative expression levels of stress-related genes NtHAK, NtAPX, NtCAT, NtABF2, and NtZFP were higher than those of WT under stress treatment. The stomatal apertures of OE (Overexpression) were 30% smaller than those of WT, and the photosynthetic rate of OE was 48% higher than that of WT. These results showed that the overexpression line exhibited stronger adaptability to osmotic stress than WT. CONCLUSIONS: Our results indicate that Fm4CL-like 1 is involved in secondary cell wall development and lignin synthesis. Fm4CL-like 1 play an important role in osmotic stress by affecting cell wall and stomatal development.


Assuntos
Secas , Proteínas de Plantas/genética , Tabaco/fisiologia , Clonagem Molecular , Fraxinus/genética , Fraxinus/metabolismo , Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Estresse Fisiológico/genética , Tabaco/genética
9.
Plant Mol Biol ; 100(3): 215-230, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31053988

RESUMO

KEY MESSAGE: Two homologs PsnSuSy1 and PsnSuSy2 from poplar played largely similar but little distinct roles in modulating sink strength, accelerating vegetative growth and modifying secondary growth of plant. Co-overexpression of them together resulted in small but perceptible additive effects. Sucrose synthase (SuSy) acts as a crucial determinant of sink strength by controlling the conversion of sucrose into UDP-glucose, which is not only the sole precursor for cellulose biosynthesis but also an extracellular signaling molecule for plants growth. Therefore, modification of SuSy activity in plants is of utmost importance. We have isolated two SuSy genes from poplar, PsnSuSy1 and PsnSuSy2, which were preferentially expressed in secondary xylem/phloem. To investigate their functions, T2 tobacco transgenic lines of PsnSuSy1 and PsnSuSy2 were generated and then crossed to generate PsnSuSy1/PsnSuSy2 dual overexpression transgenic lines. SuSy activities in all lines were significantly increased though PsnSuSy1/PsnSuSy2 lines only exhibited slightly higher SuSy activities than either PsnSuSy1 or PsnSuSy2 lines. The significantly increased fructose and glucose, engendered by augmented SuSy activities, caused the alternations of many physiological, biochemical measures and phenotypic traits that include accelerated vegetative growth, thickened secondary cell wall, and increased stem breaking force, accompanied with altered expression levels of related pathway genes. The correlation relationships between SuSy activities and many of these traits were statistically significant. However, differences of almost all traits among three types of transgenic lines were insignificant. These findings clearly demonstrated that PsnSuSy1 and PsnSuSy2 had similar but little distinct functions and insubstantial additive effects on modulating sink strength and affecting allocation of carbon elements among secondary cell wall components.


Assuntos
Parede Celular/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Glucosiltransferases/genética , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/genética , Tabaco/genética , Parede Celular/ultraestrutura , Celulose/biossíntese , Clorofila/análise , Clonagem Molecular , Perfilação da Expressão Gênica , Glucosiltransferases/metabolismo , Lignina/metabolismo , Floema/metabolismo , Fotossíntese , Polissacarídeos/metabolismo , Populus/genética , Análise de Sequência , Sacarose/metabolismo , Tabaco/citologia , Tabaco/crescimento & desenvolvimento , Xilema/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.
Nat Commun ; 10(1): 2292, 2019 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-31123263

RESUMO

The wheat Pm3 resistance gene against the powdery mildew pathogen occurs as an allelic series encoding functionally different immune receptors which induce resistance upon recognition of isolate-specific avirulence (AVR) effectors from the pathogen. Here, we describe the identification of five effector proteins from the mildew pathogens of wheat, rye, and the wild grass Dactylis glomerata, specifically recognized by the PM3B, PM3C and PM3D receptors. Together with the earlier identified AVRPM3A2/F2, the recognized AVRs of PM3B/C, (AVRPM3B2/C2), and PM3D (AVRPM3D3) belong to a large group of proteins with low sequence homology but predicted structural similarities. AvrPm3b2/c2 and AvrPm3d3 are conserved in all tested isolates of wheat and rye mildew, and non-host infection assays demonstrate that Pm3b, Pm3c, and Pm3d are also restricting the growth of rye mildew on wheat. Furthermore, divergent AVR homologues from non-adapted rye and Dactylis mildews are recognized by PM3B, PM3C, or PM3D, demonstrating their involvement in host specificity.


Assuntos
Ascomicetos/fisiologia , Proteínas Fúngicas/imunologia , Especificidade de Hospedeiro , Doenças das Plantas/imunologia , Proteínas de Plantas/imunologia , Triticum/imunologia , Ascomicetos/isolamento & purificação , Ascomicetos/patogenicidade , Dactylis/microbiologia , Resistência à Doença/imunologia , Grão Comestível/imunologia , Grão Comestível/microbiologia , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genoma Fúngico , Estudo de Associação Genômica Ampla , Proteínas NLR/imunologia , Proteínas NLR/metabolismo , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Secale/microbiologia , Tabaco/genética , Tabaco/microbiologia , Triticum/microbiologia
12.
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
13.
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
14.
Planta ; 250(2): 589-601, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31134341

RESUMO

MAIN CONCLUSION: The absence of state transitions in a Nt(Hn) cybrid is due to a cleavage of the threonine residue from the misprocessed N-terminus of the LHCII polypeptides. The cooperation between the nucleus and chloroplast genomes is essential for plant photosynthetic fitness. The rapid and specific interactions between nucleus-encoded and chloroplast-encoded proteins are under intense investigation with potential for applications in agriculture and renewable energy technology. Here, we present a novel model for photosynthesis research in which alien henbane (Hyoscyamus niger) chloroplasts function on the nuclear background of a tobacco (Nicotiana tabacum). The result of this coupling is a cytoplasmic hybrid (cybrid) with inhibited state transitions-a mechanism responsible for balancing energy absorption between photosystems. Protein analysis showed differences in the LHCII composition of the cybrid plants. SDS-PAGE analysis revealed a novel banding pattern in the cybrids with at least one additional 'LHCII' band compared to the wild-type parental species. Proteomic work suggested that the N-terminus of at least some of the cybrid Lhcb proteins was missing. These findings provide a mechanistic explanation for the lack of state transitions-the N-terminal truncation of the Lhcb proteins in the cybrid included the threonine residue that is phosphorylated/dephosphorylated in order to trigger state transitions and therefore crucial energy balancing mechanism in plants.


Assuntos
Genoma de Cloroplastos/genética , Genoma de Planta/genética , Complexos de Proteínas Captadores de Luz/metabolismo , Tabaco/genética , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Complexos de Proteínas Captadores de Luz/genética , Fosforilação , Fotossíntese , Complexo de Proteína do Fotossistema II/genética , Complexo de Proteína do Fotossistema II/metabolismo , Proteômica , Treonina/metabolismo , Tabaco/fisiologia
15.
J Plant Res ; 132(4): 461-471, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31115709

RESUMO

Reproductive isolation, including prezygotic and postzygotic barriers, is a mechanism that separates species. Many species in the Nicotiana section Suaveolentes exhibit reproductive isolation in crosses with Nicotiana tabacum. In this study, we investigated whether the chromosome numbers and ploidy levels of eight Nicotiana suaveolens accessions are related to the reproductive isolation after crosses with N. tabacum by flow cytometry and chromosome analyses. Additionally, the internal transcribed spacer (ITS) regions of the eight N. suaveolens accessions were sequenced and compared with the previously reported sequences of 22 Suaveolentes species to elucidate the phylogenetic relationships in the section Suaveolentes. We revealed that four N. suaveolens accessions comprised 64 chromosomes, while the other four accessions carried 32 chromosomes. Depending on the ploidy levels of N. suaveolens, several types of reproductive isolation were observed after crosses with N. tabacum, including decreases in the number of capsules and the germination rates of hybrid seeds, as well as hybrid lethality and abscission of enlarged ovaries at 12-17 days after pollination. A phylogenetic analysis involving ITS sequences divided the eight N. suaveolens accessions into three distinct clades. Based on the results, we confirmed that N. suaveolens accessions vary regarding ploidy levels and reproductive isolation mechanisms in crosses with N. tabacum. These accessions will be very useful for revealing and characterizing the reproductive isolation mechanisms in interspecific crosses and their relationships with ploidy levels.


Assuntos
Ploidias , Isolamento Reprodutivo , Tabaco/genética , Cromossomos de Plantas/genética , Cruzamentos Genéticos , DNA Intergênico/genética , Citometria de Fluxo , Flores/anatomia & histologia , Germinação/genética , Filogenia , Folhas de Planta/anatomia & histologia , Análise de Sequência de DNA , Tabaco/anatomia & histologia , Tabaco/fisiologia
16.
BMC Plant Biol ; 19(1): 218, 2019 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-31133003

RESUMO

BACKGROUND: An eukaryotic translation elongation factor-2 (eEF-2) plays an important role in protein synthesis, however, investigation on its role in abiotic stress responses is limited. A cold responsive eEF2 named as MfEF2 was isolated from yellow-flowered alfalfa [Medicago sativa subsp. falcata (L.) Arcang, thereafter M. falcata], a forage legume with great cold tolerance, and transgenic tobacco (Nicotiana tabacum L.) plants overexpressing MfEF2 were analyzed in cold tolerance and proteomic profiling was conducted under low temperature in this study. RESULTS: MfEF2 transcript was induced and peaked at 24 h and remained at the high level during cold treatment up to 96 h. Overexpression of MfEF2 in trasngenic tobacco plants resulted in enhanced cold tolerance. Compared to the wild type, transgenic plants showed higher survival rate after freezing treatment, higher levels of net photosynthetic rate (A), maximum photochemical efciency of photosystem (PS) II (Fv/Fm) and nonphotochemical quenching (NPQ) and lower levels of ion leakage and reactive oxygen species (ROS) production after chilling treatment. iTRAQ-based quantitative proteomic analysis identified 336 differentially expressed proteins (DEPs) from leaves of one transgenic line versus the wild type after chilling treatment for 48 h. GO and KEGG enrichment were conducted for analysis of the major biological process, cellular component, molecular function, and pathways of the DEPs involving in. It is interesting that many down-regulated DEPs were grouped into "photosynthesis" and "photosynthesis-antenna", such as subunits of PSI and PSII as well as light harvesting chlorophyll protein complex (LHC), while many up-regulated DEPs were grouped into "spliceosome". CONCLUSIONS: The results suggest that MfEF2 confers cold tolerance through regulating hundreds of proteins synthesis under low temperature conditions. The elevated cold tolerance in MfEF2 transgenic plants was associated with downregulation of the subunits of PSI and PSII as well as LHC, which leads to reduced capacity for capturing sunlight and ROS production for protection of plants, and upregulation of proteins involving in splicesome, which promotes alternative splicing of pre-mRNA under low temperature.


Assuntos
Adaptação Fisiológica/genética , Regulação da Expressão Gênica de Plantas , Medicago/fisiologia , Fator 2 de Elongação de Peptídeos/genética , Proteínas de Plantas/genética , Tabaco/fisiologia , Temperatura Baixa , Medicago/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/fisiologia , Estresse Fisiológico/genética , Tabaco/genética
17.
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
18.
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
19.
BMC Plant Biol ; 19(1): 145, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30991965

RESUMO

BACKGROUND: This study aimed to identify the transcription factors of nitrate reductase genes (NIA1 and NIA2) promoters and hypothetical cis-element of NRE2. Based on the constructed cDNA library of Nicotiana tabacum K326, a yeast one-hybrid system was established using the Matchmaker® Gold Yeast One-Hybrid Library Screening System from Clontech. The transcription factors of NIA1 andNIA2 promoters and NRE2 cis-elements were screened. RESULTS: After sequencing and bioinformatics analysis, 15 cDNA sequences were identified: 9 for NIA1 (including XP_016503563.1 and NP_001312236.1), 3 for NIA2 (including XP_016510250.1), and 3 for NRE2 (including XM_016576899.1). XP_016503563.1 was annotated in PREDICTED: CRM-domain containing factor CFM3, and NP_001312236.1chloroplastic/mitochondrial-like in Nicotiana tabacum. NP_001312236.1 was annotated in Sulfite oxidase-like of Nicotiana tabacum. XP_016510250.1 was annotated as PREDICTED: uncharacterized protein LOC107827596 in Nicotiana tabacum. XM_016576899.1 was annotated in PREDICTED: Nicotiana tabacum RING-H2 finger protein ATL16-like (LOC107759033). CONCLUSION: A yeast one-hybrid library was successfully constructed. The identified transcription factors may provide a theoretical basis for the study of plant nitrate reductase.


Assuntos
Genes de Plantas , Nitrato Redutase/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Elementos de Resposta/genética , Tabaco/genética , Fatores de Transcrição/metabolismo , Técnicas do Sistema de Duplo-Híbrido , Clonagem Molecular , Biblioteca Gênica , Glucuronidase/metabolismo , Nitrato Redutase/metabolismo , Proteínas de Plantas/metabolismo
20.
Science ; 364(6436)2019 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-30975858

RESUMO

Plants and animals recognize conserved flagellin fragments as a signature of bacterial invasion. These immunogenic elicitor peptides are embedded in the flagellin polymer and require hydrolytic release before they can activate cell surface receptors. Although much of flagellin signaling is understood, little is known about the release of immunogenic fragments. We discovered that plant-secreted ß-galactosidase 1 (BGAL1) of Nicotiana benthamiana promotes hydrolytic elicitor release and acts in immunity against pathogenic Pseudomonas syringae strains only when they carry a terminal modified viosamine (mVio) in the flagellin O-glycan. In counter defense, P. syringae pathovars evade host immunity by using BGAL1-resistant O-glycans or by producing a BGAL1 inhibitor. Polymorphic glycans on flagella are common to plant and animal pathogenic bacteria and represent an important determinant of host immunity to bacterial pathogens.


Assuntos
Flagelina/imunologia , Flagelina/metabolismo , Interações Hospedeiro-Patógeno/imunologia , Polímeros/metabolismo , Pseudomonas syringae/imunologia , Tabaco/enzimologia , Tabaco/microbiologia , beta-Galactosidase/metabolismo , Glicosilação , Hidrólise , Polissacarídeos/química , Polissacarídeos/metabolismo , Pseudomonas syringae/patogenicidade , Tabaco/genética , Tabaco/imunologia , beta-Galactosidase/genética
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