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1.
Science ; 371(6526): 255-260, 2021 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-33446550

RESUMO

Many plant specialized metabolites function in herbivore defense, and abrogating particular steps in their biosynthetic pathways frequently causes autotoxicity. However, the molecular mechanisms underlying their defense and autotoxicity remain unclear. Here, we show that silencing two cytochrome P450s involved in diterpene biosynthesis in the wild tobacco Nicotiana attenuata causes severe autotoxicity symptoms that result from the inhibition of sphingolipid biosynthesis by noncontrolled hydroxylated diterpene derivatives. Moreover, the diterpenes' defensive function is achieved by inhibiting herbivore sphingolipid biosynthesis through postingestive backbone hydroxylation products. Thus, by regulating metabolic modifications, tobacco plants avoid autotoxicity and gain herbivore defense. The postdigestive duet that occurs between plants and their insect herbivores can reflect the plant's solutions to the "toxic waste dump" problem of using potent chemical defenses.


Assuntos
Diterpenos/metabolismo , Glucosídeos/biossíntese , Herbivoria , Manduca/fisiologia , Esfingolipídeos/biossíntese , Tabaco/metabolismo , Animais , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Hidroxilação , Manduca/enzimologia , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo , Tabaco/enzimologia
2.
Science ; 369(6504): 698-702, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32764072

RESUMO

Plant grafting is conducted for fruit and vegetable propagation, whereby a piece of living tissue is attached to another through cell-cell adhesion. However, graft compatibility limits combinations to closely related species, and the mechanism is poorly understood. We found that Nicotiana is capable of graft adhesion with a diverse range of angiosperms. Comparative transcriptomic analyses on graft combinations indicated that a subclade of ß-1,4-glucanases secreted into the extracellular region facilitates cell wall reconstruction near the graft interface. Grafting was promoted by overexpression of the ß-1,4-glucanase. Using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. These findings demonstrate that the process of cell-cell adhesion is a potential target to enhance plant grafting techniques.


Assuntos
Adesão Celular/fisiologia , Comunicação Celular/fisiologia , Celulase/metabolismo , Horticultura/métodos , Proteínas de Plantas/metabolismo , Tabaco/fisiologia , Adesão Celular/genética , Comunicação Celular/genética , Celulase/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Plantas/genética , Tabaco/enzimologia , Tabaco/genética , Transcrição Genética
3.
Gene ; 753: 144809, 2020 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-32470503

RESUMO

Small GTPases function as molecular switches to active or inactive signaling cascades via binding or hydrolyzing GTP. A type of plant specific small GTPases, the ROPs are known to be involved in plant growth, development and immunity. We determined whether ROPs are conserved in Solanaceous species and whether they are involved in plant growth, development and resistance against Phytophthora capsisi. In genome-wide screening, a total of 66 ROPs in six Solanaceous species (SolROPs) were identified, including 16 ROPs in Solanum tuberosum L. (potato), 9 in Solanum lycopersicum L. (tomato), 5 in Solanum melongena L. (eggplant), 9 in Capsicum annuum L. (pepper), 13 in Nicotiana benthamiana Domin and 14 in Nicotiana tabacum L. (tobacco). Phylogenetic analysis revealed that 11 AtROPs and 66 SolROPs fall into five distinct clades (I-V) and hence a novel and systematic gene nomenclature was proposed. In addition, a comprehensive expression analysis was performed by making use of an online database. This revealed that ROP genes are differentially expressed during plant growth and development. Moreover, gene expression of SlROP-II.1 in S. lycopersicum could be significantly induced by P. capsici. Subsequently, SlROP-II.1 and its homologues in N. benthamiana and C. annuum (NbROP-II.1 and CaROP-II.1) were selected for functional analysis using virus-induced gene silencing. Infection assays with P. capsici on silenced plants revealed that SlROP-II.1, NbROP-II.1 and CaROP-II.1 play a role in P. capsici resistance, suggesting conserved function of ROP-II clade across different Solanaceous species. In addition, NbROP-II.1 is also involved in regulating plant growth and development. This study signified the diversity of Solanaceous ROPs and their potential roles in plant growth, development and immunity.


Assuntos
Proteínas Monoméricas de Ligação ao GTP/genética , Proteínas de Plantas/genética , Solanaceae/enzimologia , Solanaceae/genética , Proteínas rho de Ligação ao GTP/genética , Capsicum/enzimologia , Capsicum/genética , Genoma de Planta , Estudo de Associação Genômica Ampla/métodos , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Filogenia , Doenças das Plantas/genética , Proteínas de Plantas/metabolismo , Transdução de Sinais , Tabaco/enzimologia , Tabaco/genética , Proteínas rho de Ligação ao GTP/metabolismo
4.
Sci Rep ; 9(1): 17850, 2019 11 28.
Artigo em Inglês | MEDLINE | ID: mdl-31780730

RESUMO

The enzyme browning reaction results in grey speckles on tobacco leaves, which impairs the value and industrial usability of tobacco leaves. To demonstrate the influences of different browning degrees (BDs) of tobacco leaves on the usability of different cultivars and positions and identified structure of brown (grey) matter, we selected three flue-cured tobacco cultivars (K326, Yunyan87, and Honghuadajinyuan (Hongda)) and set four different BDs (<25%, 25% to 50%, 50% to 75%, and >75%). Indices related to: economic traits, chemical components, physical properties, and sensory quality of tobacco leaves with different cultivars were evaluated. Moreover, by utilising thin-layer chromatography and high-performance liquid chromatography, we analysed and identified the structure of the grey matter in terms of chemical composition. The experimental results show that the main component of grey speckles on tobacco leaves is 3-acetyl-6,7-dimethoxycoumarin (YC-ZJF). With the increase of BD, the amount of total sugar and reducing sugar, output value, the proportion of superior tobacco, shatter resistance index, and sensory evaluation score of the three cultivars significantly decrease, while the starch content increases significantly. The changes in protein, total nitrogen, and nicotine are insignificant with changing BD. In addition, other indices show different trends for different cultivars of flue-cured tobacco. After separation and identification of the components of grey speckled leaves, it is proved that the substance derived from grey speckles on tobacco leaves is YC-ZJF. The research is important to the study of browning mechanisms in tobacco leaves and provides corresponding targets for strategies to reduce browning thereof.


Assuntos
Pigmentos Biológicos/metabolismo , Folhas de Planta/química , Produtos do Tabaco/normas , Tabaco/metabolismo , Cromatografia/métodos , Cumarínicos/análise , Pigmentos Biológicos/análise , Folhas de Planta/metabolismo , Amido/análise , Tabaco/enzimologia
5.
Appl Microbiol Biotechnol ; 103(23-24): 9479-9491, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31701198

RESUMO

The successful production of recombinant enzymes by tobacco transplastomic plants must maintain compatibility of the heterologous enzyme with chloroplast metabolism and its long-time enzyme stability. Based on previous reports, it has been taken for granted that following biolistic-transformation, homoplasticity could be obtained from the initially heteroplastic state following successive rounds of selection in the presence of the selection agent. However, several studies indicated that this procedure does not always ensure the complete elimination of unmodified wild-type plastomes. The present study demonstrates that CelK1 transplastomic plants, which were photosyntetically as active as untransformed ones, remain heteroplastomic even after repeated selection steps and that this state does not impair the relatively high-level production of the recombinant enzyme. In fact, even in the heteroplastomic state, the recombinant protein represented about 6% of the total soluble proteins (TSP). Moreover, our data also show that, while the recombinant endoglucanase undergoes phosphorylation, this post-translation modification does not have any significant impact on the enzymatic activity. Biomass storage might be required whenever the enzyme extraction process could not be performed immediately following the harvest of tobacco mature plants. In this respect, we have observed that enzyme activity in the detached leaves stored at 4 °C is maintained up to 20 weeks without significant loss of activity. These findings may have major implications in the future of chloroplast genetic engineering-based molecular farming to produce industrial enzymes in transplastomic plants.


Assuntos
Celulase/biossíntese , Cloroplastos/genética , Agricultura Molecular , Tabaco/enzimologia , Tabaco/genética , Celulase/genética , Engenharia Genética , Microbiologia Industrial/métodos , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteoma , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética
6.
Int J Mol Sci ; 20(22)2019 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-31752180

RESUMO

Carotenoid cleavage dioxygenases (CCDs) selectively catalyze carotenoids, forming smaller apocarotenoids that are essential for the synthesis of apocarotenoid flavor, aroma volatiles, and phytohormone ABA/SLs, as well as responses to abiotic stresses. Here, 19, 11, and 10 CCD genes were identified in Nicotiana tabacum, Nicotiana tomentosiformis, and Nicotiana sylvestris, respectively. For this family, we systematically analyzed phylogeny, gene structure, conserved motifs, gene duplications, cis-elements, subcellular and chromosomal localization, miRNA-target sites, expression patterns with different treatments, and molecular evolution. CCD genes were classified into two subfamilies and nine groups. Gene structures, motifs, and tertiary structures showed similarities within the same groups. Subcellular localization analysis predicted that CCD family genes are cytoplasmic and plastid-localized, which was confirmed experimentally. Evolutionary analysis showed that purifying selection dominated the evolution of these genes. Meanwhile, seven positive sites were identified on the ancestor branch of the tobacco CCD subfamily. Cis-regulatory elements of the CCD promoters were mainly involved in light-responsiveness, hormone treatment, and physiological stress. Different CCD family genes were predominantly expressed separately in roots, flowers, seeds, and leaves and exhibited divergent expression patterns with different hormones (ABA, MeJA, IAA, SA) and abiotic (drought, cold, heat) stresses. This study provides a comprehensive overview of the NtCCD gene family and a foundation for future functional characterization of individual genes.


Assuntos
Dioxigenases/genética , Dioxigenases/metabolismo , Análise de Sequência de DNA/métodos , Tabaco/enzimologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Carotenoides/metabolismo , Mapeamento Cromossômico , Sequência Conservada , Dioxigenases/química , Evolução Molecular , Regulação da Expressão Gênica de Plantas , Família Multigênica , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Regiões Promotoras Genéticas , Seleção Genética , Tabaco/genética
7.
J Agric Food Chem ; 67(45): 12382-12392, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31635461

RESUMO

Protoporphyrinogen oxidase (PPO, EC 1.3.3.4) is a promising target for herbicide discovery. Search for new compounds with novel chemotypes is a key objective for agrochemists. Here, we describe the discovery and systematic SAR-based structure optimization of novel N-isoxazolinylphenyltriazinones 5-9 as PPO inhibitors. The in vivo herbicidal activity and in vitro Nicotiana tabacum PPO (NtPPO) inhibitory activity were explored in detail. A number of the new synthetic compounds displayed strong PPO inhibitory activity with Ki values in the nanomolar range. Some compounds exhibited excellent and broad-spectrum weed control at the rate of 9.375-37.5 g ai/ha by postemergence application and showed improved monocotyledonous weed control compared to saflufenacil. Most promisingly, ethyl 3-(2-chloro-5-(3,5-dimethyl-2,6-dioxo-4-thioxo-1,3,5-triazinan-1-yl)-4-fluorophenyl)-5-methyl-4,5-dihydroisoxazole-5-carboxylate, 5a, with a Ki value of 4.9 nM, displayed over 2- and 6-fold higher potency than saflufenacil (Ki = 10 nM) and trifludimoxazin (Ki = 31 nM), respectively. Moreover, 5a showed excellent and broad-spectrum weed control against 32 kinds of weeds at 37.5-75 g ai/ha. Rice exhibited relative tolerance to 5a at 150 g ai/ha by postemergence application, indicating that 5a could be a potential herbicide candidate for weed control in paddy fields.


Assuntos
Inibidores Enzimáticos/farmacologia , Herbicidas/química , Herbicidas/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Protoporfirinogênio Oxidase/antagonistas & inibidores , Descoberta de Drogas , Inibidores Enzimáticos/química , Cinética , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Daninhas/química , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/enzimologia , Protoporfirinogênio Oxidase/química , Protoporfirinogênio Oxidase/metabolismo , Relação Quantitativa Estrutura-Atividade , Tabaco/química , Tabaco/efeitos dos fármacos , Tabaco/enzimologia , Controle de Plantas Daninhas
8.
Plant Mol Biol ; 101(3): 325-339, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31399934

RESUMO

KEY MESSAGE: Combining genetic engineering of MPK4 activity and quantitative proteomics, we established an in planta system that enables rapid study of MPK4 signaling networks and potential substrate proteins. Mitogen activated protein kinase 4 (MPK4) is a multifunctional kinase that regulates various signaling events in plant defense, growth, light response and cytokinesis. The question of how a single protein modulates many distinct processes has spurred extensive research into the physiological outcomes resulting from genetic perturbation of MPK4. However, the mechanism by which MPK4 functions is still poorly understood due to limited data on the MPK4 networks including substrate proteins and downstream pathways. Here we introduce an experimental system that combines genetic engineering of kinase activity and quantitative proteomics to rapidly study the signaling networks of MPK4. First, we transiently expressed a constitutively active (MPK4CA) and an inactive (MPK4IN) version of a Brassica napus MPK4 (BnMPK4) in Nicotiana benthamiana leaves. Proteomics analysis revealed that BnMPK4 activation affects multiple pathways (e.g., metabolism, redox regulation, jasmonic acid biosynthesis and stress responses). Furthermore, BnMPK4 activation also increased protein phosphorylation in the phosphoproteome, from which putative MPK4 substrates were identified. Using protein kinase assay, we validated that a transcription factor TCP8-like (TCP8) and a PP2A regulatory subunit TAP46-like (TAP46) were indeed phosphorylated by BnMPK4. Taken together, we demonstrated the utility of proteomics and phosphoproteomics in elucidating kinase signaling networks and in identification of downstream substrates.


Assuntos
Regulação da Expressão Gênica de Plantas , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteômica , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Brassica napus/enzimologia , Engenharia Genética , Sistema de Sinalização das MAP Quinases , Fosforilação , Imunidade Vegetal , Folhas de Planta/enzimologia , Proteoma , Transdução de Sinais , Tabaco/enzimologia
9.
Planta ; 250(5): 1703-1715, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31414205

RESUMO

MAIN CONCLUSION: The tobacco nectar proteome mainly consists of pathogenesis-related proteins with two glycoproteins. Expression of nectarins was non-synchronous, and not nectary specific. After secretion, tobacco nectar changed from sucrose rich to hexose rich. Floral nectar proteins (nectarins) play important roles in inhibiting microbial growth in nectar, and probably also tailoring nectar chemistry before or after secretion; however, very few plant species have had their nectar proteomes thoroughly investigated. Nectarins from Nicotiana tabacum (NT) were separated using two-dimensional gel electrophoresis and then analysed using mass spectrometry. Seven nectarins were identified: acidic endochitinase, ß-xylosidase, α-galactosidase, α-amylase, G-type lectin S-receptor-like serine/threonine-protein kinase, pathogenesis-related protein 5, and early nodulin-like protein 2. An eighth nectarin, a glycoprotein with unknown function, was identified following isolation from NT nectar using a Qproteome total glycoprotein kit, separation by SDS-PAGE, and identification by mass spectrometry. Expression of all identified nectarins, plus four invertase genes, was analysed by qRT PCR; none of these genes had nectary-specific expression, and none had synchronous expression. The total content of sucrose, hexoses, proteins, phenolics, and hydrogen peroxide were determined at different time intervals in secreted nectar, both within the nectar tube (in vivo) and following extraction from it during incubation at 30 °C for up to 40 h in plastic tubes (in vitro). After secretion, the ratio of hexose to sucrose substantially increased for in vivo nectar, but no sugar composition changes were detected in vitro. This implies that sucrose hydrolysis in vivo might be done by fixed apoplastic invertase. Both protein and hydrogen peroxide levels declined in vitro but not in vivo, implying that some factors other than nectarins act to maintain their levels in the flower, after secretion.


Assuntos
Néctar de Plantas/metabolismo , Proteoma , Proteômica , Tabaco/enzimologia , Eletroforese em Gel Bidimensional , Flores/genética , Flores/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Espectrometria de Massas , Proteínas de Membrana/metabolismo , Néctar de Plantas/genética , Proteínas de Plantas/metabolismo , Tabaco/genética
10.
Int J Mol Sci ; 20(15)2019 Aug 04.
Artigo em Inglês | MEDLINE | ID: mdl-31382684

RESUMO

About 15% of higher plants have acquired the ability to convert sucrose into fructans. Fructan degradation is catalyzed by fructan exohydrolases (FEHs), which are structurally related to cell wall invertases (CWI). However, the biological function(s) of FEH enzymes in non-fructan species have remained largely enigmatic. In the present study, one maize CWI-related enzyme named Zm-6&1-FEH1, displaying FEH activity, was explored with respect to its substrate specificities, its expression during plant development, and its possible interaction with CWI inhibitor protein. Following heterologous expression in Pichia pastoris and in N. benthamiana leaves, recombinant Zm-6&1-FEH1 revealed substrate specificities of levan and inulin, and also displayed partially invertase activity. Expression of Zm-6&1-FEH1 as monitored by qPCR was strongly dependent on plant development and was further modulated by abiotic stress. To explore whether maize FEH can interact with invertase inhibitor protein, Zm-6&1-FEH1 and maize invertase inhibitor Zm-INVINH1 were co-expressed in N. benthamiana leaves. Bimolecular fluorescence complementation (BiFC) analysis and in vitro enzyme inhibition assays indicated productive complex formation. In summary, the results provide support to the hypothesis that in non-fructan species FEH enzymes may modulate the regulation of CWIs.


Assuntos
Glicosídeo Hidrolases/genética , Folhas de Planta/enzimologia , Zea mays/enzimologia , beta-Frutofuranosidase/genética , Sequência de Aminoácidos , Metabolismo dos Carboidratos/genética , Frutanos/genética , Frutanos/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Glicosídeo Hidrolases/química , Folhas de Planta/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Especificidade por Substrato , Tabaco/enzimologia , Tabaco/genética , Zea mays/genética , beta-Frutofuranosidase/antagonistas & inibidores
11.
J Plant Physiol ; 240: 153011, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31357099

RESUMO

Phytochelatin synthase (PCS) is an enzyme that synthesizes phytochelatins, which are metal-binding peptides. Despite the important role of PCS in heavy metal detoxification or tolerance, the functional role of PCS with respect to other abiotic stresses remains largely unknown. In this study, we determined the function of Arabidopsis thaliana phytochelatin synthase 2 (AtPCS2) in the salt stress response. Expression of AtPCS2 was significantly increased in response to 100 and 200 mM NaCl treatment. AtPCS2-overexpressing transgenic Arabidopsis and tobacco plants displayed increased seed germination rates and seedling growth under high salt stress. In addition, transgenic Arabidopsis subjected to salt stress exhibited enhanced proline accumulation and reduced Na+/K+ ratios compared to wild type plants. Furthermore, decreased levels of hydrogen peroxide (H2O2) and lipid peroxidation were observed in transgenic Arabidopsis compared to wild type specimens. Salt stress greatly reduced transcript levels of CuSOD2, FeSOD2, CAT2, and GR2 in wild type but not transgenic Arabidopsis. Notably, levels of CAT3 in transgenic Arabidopsis were markedly increased upon salt stress, suggesting that low accumulation of H2O2 in transgenic Arabidopsis is partially achieved through induction of CAT. Collectively, these results suggest that AtPCS2 plays a positive role in seed germination and seedling growth under salt stress through a series of indirect effects that are likely involved in H2O2 scavenging, regulation of osmotic adjustment and ion homeostasis.


Assuntos
Aminoaciltransferases/genética , Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Plantas Geneticamente Modificadas/fisiologia , Tolerância ao Sal/genética , Cloreto de Sódio/farmacologia , Tabaco/fisiologia , Aminoaciltransferases/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/enzimologia , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Relação Dose-Resposta a Droga , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Tabaco/efeitos dos fármacos , Tabaco/enzimologia , Tabaco/genética
12.
Mol Biotechnol ; 61(9): 703-713, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31286381

RESUMO

Lycopene ε-cyclases (LCYEs) are key enzymes in carotenoid biosynthesis converting red lycopene to downstream lutein. The flowers of marigold (Tagetes erecta) have been superior sources to supply lutein. However, the transcriptional regulatory mechanisms of LCYe in lutein synthesis are still unclear in marigold. In this work, the expression pattern of the TeLCYe gene in marigold indicated that TeLCYe mainly expressed in floral organs. To gain a better understanding of the expression and regulatory mechanism of TeLCYe gene, the TeLCYe promoter was isolated, sequenced, and analyzed through bioinformatics tools. The results suggested that the sequence of TeLCYe promoter contained various putative cis-acting elements responsive to exogenous and endogenous factors. The full-length TeLCYe promoter and three 5'-deletion fragments were fused to the GUS reporter gene and transferred into tobacco to test the promoter activities. A strong GUS activity was observed in stems of seedlings, leaves of seedlings, middle stems, top leaves, petals, stamens, and stigmas in transgenic tobacco containing full-length TeLCYe promoter LP0-2086. Deletion of - 910 to - 429 bp 5' to ATG significantly increased the GUS activity in chloroplast-rich tissues and floral organs, while deletion occurring from 1170 to 910 bp upstream ATG decreased the TeLCYe promoter strength in stems of seedlings, leaves of seedlings, top leaves and sepals. Functional characterization of the full-length TeLCYe promoter and its' deletion fragments in stable transgenic tobacco indicated that the LP0-2086 contains some specific cis-acting elements, which might result in the high-level expression of in floral organs, and LP2-910 might contain some specific cis-acting elements which improved GUS activities in vegetable tissues.


Assuntos
Regulação da Expressão Gênica de Plantas , Liases Intramoleculares/genética , Proteínas de Plantas/genética , Regiões Promotoras Genéticas , Tagetes/genética , Tabaco/genética , Biologia Computacional/métodos , Flores/enzimologia , Flores/genética , Genes Reporter , Glucuronidase/genética , Glucuronidase/metabolismo , Liases Intramoleculares/metabolismo , Luteína/biossíntese , Licopeno/metabolismo , Folhas de Planta/enzimologia , Folhas de Planta/genética , Proteínas de Plantas/metabolismo , Caules de Planta/enzimologia , Caules de Planta/genética , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Plântula/enzimologia , Plântula/genética , Tagetes/enzimologia , Tabaco/enzimologia
13.
Proc Natl Acad Sci U S A ; 116(29): 14651-14660, 2019 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-31262827

RESUMO

Plant volatile organic compounds (VOCs) mediate many interactions, and the function of common VOCs is especially likely to depend on ecological context. We used a genetic mapping population of wild tobacco, Nicotiana attenuata, originating from a cross of 2 natural accessions from Arizona and Utah, separated by the Grand Canyon, to dissect genetic variation controlling VOCs. Herbivory-induced leaf terpenoid emissions varied substantially, while green leaf volatile emissions were similar. In a field experiment, only emissions of linalool, a common VOC, correlated significantly with predation of the herbivore Manduca sexta by native predators. Using quantitative trait locus mapping and genome mining, we identified an (S)-(+)-linalool synthase (NaLIS). Genome resequencing, gene cloning, and activity assays revealed that the presence/absence of a 766-bp sequence in NaLIS underlies the variation of linalool emissions in 26 natural accessions. We manipulated linalool emissions and composition by ectopically expressing linalool synthases for both enantiomers, (S)-(+)- and (R)-(-)-linalool, reported to oppositely affect M. sexta oviposition, in the Arizona and Utah accessions. We used these lines to test ovipositing moths in increasingly complex environments. The enantiomers had opposite effects on oviposition preference, but the magnitude of the effect depended strongly both on plant genetic background, and complexity of the bioassay environment. Our study reveals that the emission of linalool, a common VOC, differs by orders-of-magnitude among geographically interspersed conspecific plants due to allelic variation in a linalool synthase, and that the response of a specialist herbivore to linalool depends on enantiomer, plant genotype, and environmental complexity.


Assuntos
Monoterpenos Acíclicos/toxicidade , Hidroliases/genética , Manduca/efeitos dos fármacos , Comportamento Predatório/efeitos dos fármacos , Tabaco/genética , Monoterpenos Acíclicos/metabolismo , Animais , Arizona , Feminino , Genótipo , Geografia , Interações Hospedeiro-Parasita/genética , Hidroliases/metabolismo , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Masculino , Manduca/fisiologia , Oviposição/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/parasitologia , Proteínas de Plantas , Locos de Características Quantitativas , Estereoisomerismo , Tabaco/enzimologia , Tabaco/parasitologia , Utah , Compostos Orgânicos Voláteis
14.
Protein Expr Purif ; 163: 105452, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31301428

RESUMO

General control nonderepressible 2 (GCN2) can phosphorylate the α subunit of eukaryotic initiation factor eIF2 (eukaryotic translation initiation factor 2) to down-regulateprotein synthesis in response to various biotic and abiotic stresses. However, the kinase activity of plant GCN2 has not been well-characterized in vitro. In this study, the kinase domain of Nicotiana tabacum GCN2 (NtGCN2) was inserted into the pET15b vector for prokaryotic expressionin Escherichia coli BL21-CodonPlus-(DE3)-RIPL after induction by 0.5 mmol L-1 IPTG for 13 h at 16 °C. The soluble protein was collected and purified by Ni2+-NTA agarose column, anion exchange, and molecular sieve, and the purified proteinwas used for kinase assays and the preparation of a polyclonal antibody. Enzyme-linked immunosorbent assay results showed that the titer of the antiserum was 1:520K. Western blot analysis showed that the prepared antibody reacted with GCN2 in tobacco. Additionally, the kinase activity of NtGCN2 was characterized by using recombinant NteIF2α protein as a substrate in vitro. The results showed that NtGCN2 phosphorylated NteIF2α in vitro, with the level of phosphorylation positively correlated with the NtGCN2 concentration and reaction time. Our study has prepared a specific antibody, and proves NtGCN2 can phosphorylate NteIF2α in vitro, which lays a foundation for further study of the function and interaction network of NtGCN2.


Assuntos
Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/isolamento & purificação , Tabaco/enzimologia , Anticorpos/imunologia , Clonagem Molecular , Ensaio de Imunoadsorção Enzimática , Escherichia coli , Fator de Iniciação 2 em Eucariotos/metabolismo , Vetores Genéticos , Fosforilação , Domínios Proteicos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/imunologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo
15.
J Agric Food Chem ; 67(33): 9254-9264, 2019 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-31356740

RESUMO

In continuation of our search for potent protoporphyrinogen IX oxidase (PPO, EC 1.3.3.4) inhibitors, we designed and synthesized a series of novel herbicidal cycloalka[d]quinazoline-2,4-dione-benzoxazinones. The bioassay results of these synthesized compounds indicated that most of the compounds exhibited very strong Nicotiana tabacum PPO (NtPPO) inhibition activity. More than half of the 37 synthesized compounds displayed over 80% control of all three tested broadleaf weeds at 37.5-150 g ai/ha by postemergent application, and a majority of them showed no phytotoxicity toward at least one kind of crop at 150 g ai/ha. Promisingly, 17i (Ki = 6.7 nM) was 6 and 4 times more potent than flumioxazin (Ki = 46 nM) and trifludimoxazin (Ki = 31 nM), respectively. Moreover, 17i displayed excellent, broad-spectrum herbicidal activity, even at levels as low as 37.5 g ai/ha, and it was determined to be safe for wheat at 150 g ai/ha in postemergent application, indicating the great potential for 17i development as a herbicide for weed control in wheat fields.


Assuntos
Benzoxazinas/química , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Herbicidas/química , Herbicidas/farmacologia , Proteínas de Plantas/antagonistas & inibidores , Protoporfirinogênio Oxidase/antagonistas & inibidores , Quinazolinas/química , Benzoxazinas/farmacologia , Desenho de Fármacos , Cinética , Proteínas de Plantas/química , Plantas Daninhas/efeitos dos fármacos , Plantas Daninhas/enzimologia , Protoporfirinogênio Oxidase/química , Relação Quantitativa Estrutura-Atividade , Quinazolinas/farmacologia , Tabaco/efeitos dos fármacos , Tabaco/enzimologia , Controle de Plantas Daninhas
16.
Plant J ; 100(2): 237-250, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31215085

RESUMO

Methylated inositol, d-pinitol (3-O-methyl-d-chiro-inositol), is a common constituent in legumes. It is synthesized from myo-inositol in two reactions: the first reaction, catalyzed by myo-inositol-O-methyltransferase (IMT), consists of a transfer of a methyl group from S-adenosylmethionine to myo-inositol with the formation of d-ononitol, while the second reaction, catalyzed by d-ononitol epimerase (OEP), involves epimerization of d-ononitol to d-pinitol. To identify the genes involved in d-pinitol biosynthesis in a model legume Medicago truncatula, we conducted a BLAST search on its genome using soybean IMT cDNA as a query and found putative IMT (MtIMT) gene. Subsequent co-expression analysis performed on publicly available microarray data revealed two potential OEP genes: MtOEPA, encoding an aldo-keto reductase and MtOEPB, encoding a short-chain dehydrogenase. cDNAs of all three genes were cloned and expressed as recombinant proteins in E. coli. In vitro assays confirmed that putative MtIMT enzyme catalyzes methylation of myo-inositol to d-ononitol and showed that MtOEPA enzyme has NAD+ -dependent d-ononitol dehydrogenase activity, while MtOEPB enzyme has NADP+ -dependent d-pinitol dehydrogenase activity. Both enzymes are required for epimerization of d-ononitol to d-pinitol, which occurs in the presence of NAD+ and NADPH. Introduction of MtIMT, MtOEPA, and MtOEPB genes into tobacco plants resulted in production of d-ononitol and d-pinitol in transformants. As this two-step pathway of d-ononitol epimerization is coupled with a transfer of reducing equivalents from NADPH to NAD+ , we speculate that one of the functions of this pathway might be regeneration of NADP+ during drought stress.


Assuntos
Medicago truncatula/metabolismo , Secas , Regulação da Expressão Gênica de Plantas/fisiologia , Glicosídeos/metabolismo , Medicago truncatula/enzimologia , Metiltransferases/metabolismo , NADP/metabolismo , Proteínas de Plantas/metabolismo , Tabaco/enzimologia , Tabaco/metabolismo
17.
Plant J ; 99(6): 1031-1046, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31215089

RESUMO

Mesophyll conductance (gm ), the diffusion of CO2 from substomatal cavities to the carboxylation sites in the chloroplasts, is a highly complex trait driving photosynthesis (net CO2 assimilation, AN ). However, little is known concerning the mechanisms by which it is dynamically regulated. The apoplast is considered as a 'key information bridge' between the environment and cells. Interestingly, most of the environmental constraints affecting gm also cause apoplastic responses, cell wall (CW) alterations and metabolic rearrangements. Since CW thickness is a key determinant of gm , we hypothesize that other changes in this cellular compartiment should also influence gm . We study the relationship between the antioxidant apoplastic system and CW metabolism and the gm responses in tobacco plants (Nicotiana sylvestris L.) under two abiotic stresses (drought and salinity), combining in vivo gas-exchange measurements with analyses of antioxidant activities, CW composition and primary metabolism. Stress treatments imposed substantial reductions in AN (58-54%) and gm (59%), accompanied by a strong antioxidant enzymatic response at the apoplastic and symplastic levels. Interestingly, apoplastic but not symplastic peroxidases were positively related to gm . Leaf anatomy remained mostly stable; however, the stress treatments significantly affected the CW composition, specifically pectins, which showed significant relationships with AN and gm . The treatments additionally promoted a differential primary metabolic response, and specific CW-related metabolites including galactose, glucosamine and hydroxycinnamate showed exclusive relationships with gm independent of the stress. These results suggest that gm responses can be attributed to specific changes in the apoplastic antioxidant system and CW metabolism, opening up more possibilities for improving photosynthesis using breeding/biotechnological strategies.


Assuntos
Antioxidantes/metabolismo , Parede Celular/metabolismo , Células do Mesofilo/metabolismo , Fotossíntese/fisiologia , Tabaco/metabolismo , Dióxido de Carbono/metabolismo , Catalase/metabolismo , Parede Celular/química , Clorofila/metabolismo , Secas , Peróxido de Hidrogênio/metabolismo , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/metabolismo , Peroxidase/metabolismo , Folhas de Planta/anatomia & histologia , Folhas de Planta/metabolismo , Estômatos de Plantas/metabolismo , Estômatos de Plantas/fisiologia , Salinidade , Superóxido Dismutase/metabolismo , Tabaco/enzimologia
18.
Mol Plant Microbe Interact ; 32(11): 1496-1507, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31251114

RESUMO

The molecular mechanisms acting between host recognition of pathogen effectors by nucleotide-binding leucine-rich repeat receptor (NLR) proteins and mitogen-activated protein kinase (MAPK) signaling cascades are unknown. MAPKKKα (M3Kα) activates MAPK signaling leading to programmed cell death (PCD) associated with NLR-triggered immunity. We identified a tomato M3Kα-interacting protein, SlMai1, that has 80% amino acid identity with Arabidopsis brassinosteroid kinase 1 (AtBsk1). SlMai1 has a protein kinase domain and a C-terminal tetratricopeptide repeat domain that interacts with the kinase domain of M3Kα. Virus-induced gene silencing of Mai1 homologs in Nicotiana benthamiana increased susceptibility to Pseudomonas syringae and compromised PCD induced by four NLR proteins. PCD was restored by expression of a synthetic SlMai1 gene that resists silencing. Expression of AtBsk1 did not restore PCD in Mai1-silenced plants, suggesting SlMai1 is functionally divergent from AtBsk1. PCD caused by overexpression of M3Kα or MKK2 was unaffected by Mai1 silencing, suggesting Mai1 acts upstream of these proteins. Coexpression of Mai1 with M3Kα in leaves enhanced MAPK phosphorylation and accelerated PCD. These findings suggest Mai1 is a molecular link acting between host recognition of pathogens and MAPK signaling.


Assuntos
Interações Hospedeiro-Patógeno , Proteínas Quinases Ativadas por Mitógeno , Doenças das Plantas , Transdução de Sinais , Interações Hospedeiro-Patógeno/fisiologia , Lycopersicon esculentum/enzimologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Doenças das Plantas/imunologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Pseudomonas syringae/enzimologia , Tabaco/enzimologia
19.
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
20.
Metab Eng ; 55: 33-43, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31091467

RESUMO

Plants and cyanobacteria are promising heterologous hosts for metabolic engineering, and particularly suited for expression of cytochrome P450 (P450s), enzymes that catalyse key steps in biosynthetic pathways leading to valuable natural products such as alkaloids, terpenoids and phenylpropanoids. P450s are often difficult to express and require a membrane-bound NADPH-dependent reductase, complicating their use in metabolic engineering and bio-production. We previously demonstrated targeting of heterologous P450s to thylakoid membranes both in N. benthamiana chloroplasts and cyanobacteria, and functional substitution of their native reductases with the photosynthetic apparatus via the endogenous soluble electron carrier ferredoxin. However, because ferredoxin acts as a sorting hub for photosynthetic reducing power, there is fierce competition for reducing equivalents, which limits photosynthesis-driven P450 output. This study compares the ability of four electron carriers to increase photosynthesis-driven P450 activity. These carriers, three plant ferredoxins and a flavodoxin-like engineered protein derived from cytochrome P450 reductase, show only modest differences in their electron transfer to our model P450, CYP79A1 in vitro. However, only the flavodoxin-like carrier supplies appreciable reducing power in the presence of competition for reduced ferredoxin, because it possesses a redox potential that renders delivery of reducing equivalents to endogenous processes inefficient. We further investigate the efficacy of these electron carrier proteins in vivo by expressing them transiently in N. benthamiana fused to CYP79A1. All but one of the fusion enzymes show improved sequestration of photosynthetic reducing power. Fusion with the flavodoxin-like carrier offers the greatest improvement in this comparison - nearly 25-fold on a per protein basis. Thus, this study demonstrates that synthetic electron transfer pathways with optimal redox potentials can alleviate the problem of endogenous competition for reduced ferredoxin and sets out a new metabolic engineering strategy useful for producing valuable natural products.


Assuntos
Cloroplastos , Sistema Enzimático do Citocromo P-450 , Engenharia Metabólica , Fotossíntese/genética , Proteínas de Plantas , Tabaco , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cloroplastos/enzimologia , Cloroplastos/genética , Cianobactérias/genética , Cianobactérias/metabolismo , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Transporte de Elétrons/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Tabaco/enzimologia , Tabaco/genética
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