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1.
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
2.
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
3.
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
4.
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
5.
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
6.
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
7.
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 Drogas , 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
8.
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
9.
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
10.
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
11.
Mol Plant Microbe Interact ; 32(9): 1229-1242, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31012804

RESUMO

The plasma membrane (PM) is at the interface of plant-pathogen interactions and, thus, many bacterial type-III effector (T3E) proteins target membrane-associated processes to interfere with immunity. The Pseudomonas syringae T3E HopZ1a is a host cell PM-localized effector protein that has several immunity-associated host targets but also activates effector-triggered immunity in resistant backgrounds. Although HopZ1a has been shown to interfere with early defense signaling at the PM, no dedicated PM-associated HopZ1a target protein has been identified until now. Here, we show that HopZ1a interacts with the PM-associated remorin protein NbREM4 from Nicotiana benthamiana in several independent assays. NbREM4 relocalizes to membrane nanodomains after treatment with the bacterial elicitor flg22 and transient overexpression of NbREM4 in N. benthamiana induces the expression of a subset of defense-related genes. We can further show that NbREM4 interacts with the immune-related receptor-like cytoplasmic kinase avrPphB-susceptible 1 (PBS1) and is phosphorylated by PBS1 on several residues in vitro. Thus, we conclude that NbREM4 is associated with early defense signaling at the PM. The possible relevance of the HopZ1a-NbREM4 interaction for HopZ1a virulence and avirulence functions is discussed.Copyright © 2019 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.


Assuntos
Proteínas de Bactérias , Proteínas de Transporte , Interações Hospedeiro-Patógeno , Fosfoproteínas , Proteínas de Plantas , Proteínas Serina-Treonina Quinases , Pseudomonas syringae , Proteínas de Bactérias/metabolismo , Proteínas de Transporte/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/genética , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Pseudomonas syringae/metabolismo , Tabaco/enzimologia , Tabaco/microbiologia
12.
Molecules ; 24(8)2019 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-31013599

RESUMO

Flavonols are important copigments that affect flower petal coloration. Flavonol synthase (FLS) catalyzes the conversion of dihydroflavonols to flavonols. In this study, we identified a FLS gene, MaFLS, expressed in petals of the ornamental monocot Muscari aucheri (grape hyacinth) and analyzed its spatial and temporal expression patterns. qRT-PCR analysis showed that MaFLS was predominantly expressed in the early stages of flower development. We next analyzed the in planta functions of MaFLS. Heterologous expression of MaFLS in Nicotiana tabacum (tobacco) resulted in a reduction in pigmentation in the petals, substantially inhibiting the expression of endogenous tobacco genes involved in anthocyanin biosynthesis (i.e., NtDFR, NtANS, and NtAN2) and upregulating the expression of NtFLS. The total anthocyanin content in the petals of the transformed tobacco plants was dramatically reduced, whereas the total flavonol content was increased. Our study suggests that MaFLS plays a key role in flavonol biosynthesis and flower coloration in grape hyacinth. Moreover, MaFLS may represent a new potential gene for molecular breeding of flower color modification and provide a basis for analyzing the effects of copigmentation on flower coloration in grape hyacinth.


Assuntos
Flavonóis/biossíntese , Flores , Hyacinthus , Oxirredutases , Pigmentação/fisiologia , Proteínas de Plantas , Antocianinas/genética , Flavonóis/genética , Flores/enzimologia , Flores/genética , Regulação Enzimológica da Expressão Gênica/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Hyacinthus/enzimologia , Hyacinthus/genética , Oxirredutases/biossíntese , Oxirredutases/genética , Proteínas de Plantas/biossíntese , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Tabaco/enzimologia , Tabaco/genética
13.
J Virol ; 93(3)2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30429349

RESUMO

Understanding the innate immune mechanisms of plants is necessary for the breeding of disease-resistant lines. Previously, we identified the antiviral resistance gene JAX1 from Arabidopsis thaliana, which inhibits infection by potexviruses. JAX1 encodes a unique jacalin-type lectin protein. In this study, we analyzed the molecular mechanisms of JAX1-mediated resistance. JAX1 restricted the multiplication of a potexviral replicon lacking movement-associated proteins, suggesting inhibition of viral replication. Therefore, we developed an in vitro potato virus X (PVX) translation/replication system using vacuole- and nucleus-free lysates from tobacco protoplasts, and we revealed that JAX1 inhibits viral RNA synthesis but not the translation of the viral RNA-dependent RNA polymerase (RdRp). JAX1 did not affect the replication of a resistance-breaking mutant of PVX. Blue native polyacrylamide gel electrophoresis of fractions separated by sucrose gradient sedimentation showed that PVX RdRp constituted the high-molecular-weight complex that seems to be crucial for viral replication. JAX1 was detected in this complex of the wild-type PVX replicon but not in that of the resistance-breaking mutant. In addition, JAX1 interacted with the RdRp of the wild-type virus but not with that of a virus with a point mutation at the resistance-breaking residue. These results suggest that JAX1 targets RdRp to inhibit potexviral replication.IMPORTANCE Resistance genes play a crucial role in plant antiviral innate immunity. The roles of conventional nucleotide-binding leucine-rich repeat (NLR) proteins and the associated defense pathways have long been studied. In contrast, recently discovered resistance genes that do not encode NLR proteins (non-NLR resistance genes) have not been investigated extensively. Here we report that the non-NLR resistance factor JAX1, a unique jacalin-type lectin protein, inhibits de novo potexviral RNA synthesis by targeting the huge complex of viral replicase. This is unlike other known antiviral resistance mechanisms. Molecular elucidation of the target in lectin-type protein-mediated antiviral immunity will enhance our understanding of the non-NLR-mediated plant resistance system.


Assuntos
Farmacorresistência Viral , Doenças das Plantas/prevenção & controle , Proteínas de Plantas/metabolismo , RNA Replicase/antagonistas & inibidores , Tabaco/enzimologia , Proteínas Virais/antagonistas & inibidores , Replicação Viral , Antivirais/metabolismo , Regulação Enzimológica da Expressão Gênica , Doenças das Plantas/virologia , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/virologia , Potexvirus/fisiologia , RNA Replicase/genética , RNA Replicase/metabolismo , Tabaco/virologia , Proteínas Virais/genética , Proteínas Virais/metabolismo
14.
Plant Sci ; 278: 88-95, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30471733

RESUMO

Pyridoxal 5'-phosphate (PLP), the catalytically active form of vitamin B6, is an important cofactor for many biochemical transformations. PLP is also a very reactive molecule, and the most well-established mechanism for maintaining low levels of free PLP is its dephosphorylation by phosphatases. In our previous study, the crude enzyme extract from tobacco leaves rapidly hydrolyzed PLP at a pH optimum of 5.5. Using PLP as a substrate, a novel acid phosphatase was purified from tobacco leaves and characterized. Whether there is a PLP specific phosphatase in plants is still unknown. In this study, a cDNA clone sharing 34.72% homology with human PLP phosphatase sequences was identified from N. tabacum and characterized. The cDNA encodes a polypeptide of 319 amino acid residues, and the recombinant enzyme purified from E. coli exhibited maximum catalytic activity for PLP at pH 7.5. The properties of the purified enzyme, including pH optimum, metal requirement, optimum substrate and inhibitors were similar to those of human PLP phosphatase. Subcellular localization analysis showed that the PLP phosphatase is mainly located in chloroplast. We down-regulated the gene expression with plant RNA interference technology and found that the down-regulation has a greater impact on the transcription of genes encoding vitamin B6 metabolic enzymes. Our study further suggested that the PLP phosphatase plays an important role for maintaining PLP homeostasis within the chloroplast in plants.


Assuntos
Monoéster Fosfórico Hidrolases/fisiologia , Proteínas de Plantas/fisiologia , Fosfato de Piridoxal/metabolismo , Tabaco/enzimologia , Cloroplastos/metabolismo , Clonagem Molecular , Regulação para Baixo , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Interferência de RNA , Alinhamento de Sequência , Tabaco/genética , Transcrição Genética
15.
J Exp Bot ; 70(3): 1005-1016, 2019 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-30476130

RESUMO

The activity of the protein kinase STN7, involved in phosphorylation of the light-harvesting complex II (LHCII) proteins, has been reported as being co-operatively regulated by the redox state of the plastoquinone pool and the ferredoxin-thioredoxin (Trx) system. The present study aims to investigate the role of plastid Trxs in STN7 regulation and their impact on photosynthesis. For this purpose, tobacco plants overexpressing Trx f or m from the plastid genome were characterized, demonstrating that only Trx m overexpression was associated with a complete loss of LHCII phosphorylation that did not correlate with decreased STN7 levels. The absence of phosphorylation in Trx m-overexpressing plants impeded migration of LHCII from PSII to PSI, with the concomitant loss of PSI-LHCII complex formation. Consequently, the thylakoid ultrastructure was altered, showing reduced grana stacking. Moreover, the electron transport rate was negatively affected, showing an impact on energy-demanding processes such as the Rubisco maximum carboxylation capacity and ribulose 1,5-bisphosphate regeneration rate values, which caused a strong depletion in net photosynthetic rates. Finally, tobacco plants overexpressing a Trx m mutant lacking the reactive redox site showed equivalent physiological performance to the wild type, indicating that the overexpressed Trx m deactivates STN7 in a redox-dependent way.


Assuntos
Tiorredoxinas de Cloroplastos/genética , Regulação da Expressão Gênica de Plantas , Fotossíntese/genética , Proteínas de Plantas/genética , Proteínas Serina-Treonina Quinases/genética , Tabaco/genética , Tiorredoxinas de Cloroplastos/metabolismo , Cloroplastos/enzimologia , Oxirredução , Complexo de Proteína do Fotossistema II/metabolismo , Proteínas de Plantas/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Tabaco/enzimologia , Tabaco/metabolismo
16.
Planta ; 249(2): 543-561, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30293202

RESUMO

MAIN CONCLUSION: This study illustrates the differences in the gene structure of 2-oxoglutarate-dependent oxygenase involved in flavonoid biosynthesis (2ODD-IFB), and their potential roles in regulating tobacco flavonoid biosynthesis and plant growth. Flavonol synthase (FLS), anthocyanidin synthase (ANS), and flavanone 3ß-hydroxylase belong to the 2-oxoglutarate-dependent (2ODD) oxygenase family, and each performs crucial functions in the biosynthesis of flavonoids. We identified two NtFLS genes, two NtANS genes, and four NtF3H genes from Nicotiana tabacum genome, as well as their homologous genes in the N. sylvestris and N. tomentosiformis genomes. Our phylogenetic analysis indicated that these three types of genes split from each other before the divergence of gymnosperms and angiosperms. FLS evolved faster in the eudicot plants, whereas ANS evolved faster in the monocot plants. Gene structure analysis revealed two fragment insertions occurred at different times in the intron one position of tobacco FLS genes. Homologous protein modeling revealed distinct structures in the N terminus of the tobacco 2ODD oxygenases. We found that the expression patterns of genes encoding tobacco 2ODD oxygenases in flavonoids biosynthesis (2ODD-IFB) did not determine the accumulation patterns of flavonoids among various tobacco tissues, but strongly affected the concentration of flavonoids in the tissues, where they were biosynthesized. More carbon resource flowed to the flavonol biosynthesis when NtANS gene was silenced, otherwise more anthocyanidin accumulated when NtFLS gene was repressed. This study illustrates the 2ODD-IFB gene structure evolution, differences among their protein structures, and provides a foundation for regulating plant development and altering flavonoid content and/or composition through the manipulation of plant 2ODD-IFB genes.


Assuntos
Flavonoides/biossíntese , Genes de Plantas/genética , Oxigenases de Função Mista/genética , Transdução de Sinais/genética , Tabaco/genética , Sequência Conservada/genética , Éxons/genética , Íntrons/genética , Oxigenases de Função Mista/metabolismo , Filogenia , Alinhamento de Sequência , Análise de Sequência de DNA , Tabaco/enzimologia , Tabaco/metabolismo
17.
Plant Biotechnol J ; 17(2): 350-361, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-29969180

RESUMO

Plants offer fast, flexible and easily scalable alternative platforms for the production of pharmaceutical proteins, but differences between plant and mammalian N-linked glycans, including the presence of ß-1,2-xylose and core α-1,3-fucose residues in plants, can affect the activity, potency and immunogenicity of plant-derived proteins. Nicotiana benthamiana is widely used for the transient expression of recombinant proteins so it is desirable to modify the endogenous N-glycosylation machinery to allow the synthesis of complex N-glycans lacking ß-1,2-xylose and core α-1,3-fucose. Here, we used multiplex CRISPR/Cas9 genome editing to generate N. benthamiana production lines deficient in plant-specific α-1,3-fucosyltransferase and ß-1,2-xylosyltransferase activity, reflecting the mutation of six different genes. We confirmed the functional gene knockouts by Sanger sequencing and mass spectrometry-based N-glycan analysis of endogenous proteins and the recombinant monoclonal antibody 2G12. Furthermore, we compared the CD64-binding affinity of 2G12 glycovariants produced in wild-type N. benthamiana, the newly generated FX-KO line, and Chinese hamster ovary (CHO) cells, confirming that the glyco-engineered antibody performed as well as its CHO-produced counterpart.


Assuntos
Anticorpos Monoclonais/metabolismo , Proteínas de Plantas/metabolismo , Polissacarídeos/metabolismo , Tabaco/enzimologia , Animais , Anticorpos Monoclonais/imunologia , Células CHO , Sistemas CRISPR-Cas , Cricetulus , Fucose/metabolismo , Fucosiltransferases/genética , Fucosiltransferases/metabolismo , Edição de Genes , Técnicas de Inativação de Genes , Glicosilação , Anticorpos Anti-HIV , Agricultura Molecular , Pentosiltransferases/genética , Pentosiltransferases/metabolismo , Proteínas de Plantas/genética , Plantas Geneticamente Modificadas , Proteínas Recombinantes , Tabaco/genética , Xilose/metabolismo
18.
Biomolecules ; 8(4)2018 12 07.
Artigo em Inglês | MEDLINE | ID: mdl-30544626

RESUMO

Solanesol is a terpene alcohol composed of nine isoprene units that mainly accumulates in solanaceous plants, especially tobacco (Nicotiana tabacum). The present study aimed to investigate the regulation of solanesol accumulation in tobacco leaves induced by moderately high temperature (MHT). Exposure to MHT resulted in a significant increase in solanesol content, dry weight, and net photosynthetic rate in tobacco leaves. In MHT-exposed tobacco leaves, 492 and 1440 genes were significantly up- and downregulated, respectively, as revealed by RNA-sequencing. Functional enrichment analysis revealed that most of the differentially expressed genes (DEGs) were mainly related to secondary metabolite biosynthesis, metabolic pathway, carbohydrate metabolism, lipid metabolism, hydrolase activity, catalytic activity, and oxidation-reduction process. Moreover, 122 transcription factors of DEGs were divided into 22 families. Significant upregulation of N. tabacum 3-hydroxy-3-methylglutaryl-CoA reductase (NtHMGR), 1-deoxy-d-xylulose 5-phosphate reductoisomerase (NtDXR), geranylgeranyl diphosphate synthase (NtGGPS), and solanesyl diphosphate synthase (NtSPS) and significant downregulation of N. tabacum 1-deoxy-d-xylulose 5-phosphate synthase (NtDXS) and farnesyl diphosphate synthase (NtFPS) transcription upon MHT exposure were monitored by quantitative real-time PCR (qRT-PCR). This study indicated that solanesol accumulation in tobacco leaves can be manipulated through regulation of the environmental temperature and established a basis for further elucidation of the molecular mechanism of temperature regulation of solanesol accumulation.


Assuntos
Regulação da Expressão Gênica de Plantas/genética , Análise de Sequência de RNA , Terpenos/metabolismo , Tabaco/genética , Aldose-Cetose Isomerases/genética , Alquil e Aril Transferases/genética , Metabolismo dos Carboidratos/genética , Farnesiltranstransferase/genética , Geraniltranstransferase/genética , Temperatura Alta , Hidroximetilglutaril-CoA Redutases/genética , Metabolismo dos Lipídeos/genética , Redes e Vias Metabólicas/genética , Terpenos/química , Tabaco/enzimologia , Tabaco/metabolismo
19.
Viruses ; 10(11)2018 11 17.
Artigo em Inglês | MEDLINE | ID: mdl-30453630

RESUMO

Tospovirus is a tripartite negative stranded RNA virus and is considered as one of the most devastating plant viruses. Successful virus infection in plant requires many host factors. To date, very few host factors have been identified as important in Tospovirus infection in plants. We reported earlier that NSm protein encoded by Tomato spotted wilt virus (TSWV), a type species of the genus Orthotospovirus, plays critical roles in viral cell-to-cell and long-distance movement. In this study, we determined that molecular co-chaperone NbSGT1 interacted with TSWV NSm in Nicotiana benthamiana. TSWV infection significantly upregulated the expression of NbSGT1 gene and transient overexpression of NbSGT1 in N. benthamiana leaves accelerated TSWV infection. In contrast, silencing the NbSGT1 gene expression using a virus-induced gene silencing (VIGS) approach strongly inhibited TSWV NSm cell-to-cell movement, as well as TSWV local and systemic infection in N. benthamiana plants. Furthermore, NbSGT1 was found to regulate the infection of both American and Euro/Asia type tospoviruses in N. benthamiana plant. Collectively, our findings presented in this paper and the results published previously indicated that molecular co-chaperone NbSGT1 plays important roles in modulating both positive stranded and tripartite negative stranded RNA virus infection in plants.


Assuntos
Glucosiltransferases/metabolismo , Interações Hospedeiro-Patógeno , Chaperonas Moleculares/metabolismo , Tabaco/enzimologia , Tospovirus/fisiologia , Internalização do Vírus , Liberação de Vírus , Proteínas do Movimento Viral em Plantas/metabolismo , Ligação Proteica , Tabaco/virologia
20.
Plant Physiol Biochem ; 132: 515-523, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30312954

RESUMO

In this study, a cDNA of ICE1 (inducer of CBF expression 1) gene, named BcICE1, was isolated from Brassica campestris 'Longyou 6'. The deduced protein has 499 amino acids containing a typical bHLH domain and is highly identical with AtICE1 (85.9%) from Arabidopsis thaliana. BcICE1 is located in the nucleus. The activities of SOD, CAT, POD, and APX and the transcriptional levels of SOD, CAT, and POD genes were higher in BcICE1-transgenic tobacco than in wild-type (WT) tobacco under cold stress. Compared with WT tobacco, proline, soluble sugar, and chlorophyll were enhanced, whereas malondialdehyde and relative conductivity were decreased in BcICE1-transgenic tobacco. The overexpression of BcICE1 in tobacco increased the expression of CBF1, CBF2, and other stress-related genes. Moreover, under salt and PEG (25%) stress, the activities of APX and GPX and content of soluble sugar and chlorophyll in BcICE1-transgenic tobacco were higher than those in WT tobacco. Our results suggest that BcICE1 plays an important role in the response to abiotic stress.


Assuntos
Adaptação Fisiológica/genética , Brassica/genética , Genes de Plantas , Proteínas de Plantas/genética , Estresse Fisiológico/genética , Tabaco/genética , Tabaco/fisiologia , Sequência de Aminoácidos , Antioxidantes/metabolismo , Núcleo Celular/metabolismo , Clorofila/metabolismo , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Prolina/metabolismo , Frações Subcelulares/metabolismo , Tabaco/enzimologia
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