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1.
Microb Cell Fact ; 18(1): 163, 2019 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-31581944

RESUMO

BACKGROUND: Sustainable production of microbial fatty acids derivatives has the potential to replace petroleum based equivalents in the chemical, cosmetic and pharmaceutical industry. Most fatty acid sources for production oleochemicals are currently plant derived. However, utilization of these crops are associated with land use change and food competition. Microbial oils could be an alternative source of fatty acids, which circumvents the issue with agricultural competition. RESULTS: In this study, we generated a chimeric microbial production system that features aspects of both prokaryotic and eukaryotic fatty acid biosynthetic pathways targeted towards the generation of long chain fatty acids. We redirected the type-II fatty acid biosynthetic pathway of Escherichia coli BL21 (DE3) strain by incorporating two homologues of the beta-ketoacyl-[acyl carrier protein] synthase I and II from the chloroplastic fatty acid biosynthetic pathway of Arabidopsis thaliana. The microbial clones harboring the heterologous pathway yielded 292 mg/g and 220 mg/g DCW for KAS I and KAS II harboring plasmids respectively. Surprisingly, beta-ketoacyl synthases KASI/II isolated from A. thaliana showed compatibility with the FAB pathway in E. coli. CONCLUSION: The efficiency of the heterologous plant enzymes supersedes the overexpression of the native enzyme in the E. coli production system, which leads to cell death in fabF overexpression and fabB deletion mutants. The utilization of our plasmid based system would allow generation of plant like fatty acids in E. coli and their subsequent chemical or enzymatic conversion to high end oleochemical products.


Assuntos
Arabidopsis/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Ácido Graxo Sintases/metabolismo , Ácidos Graxos/biossíntese , Engenharia Metabólica , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/síntese química , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/genética , 3-Oxoacil-(Proteína de Transporte de Acila) Sintase/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/síntese química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Escherichia coli/enzimologia , Proteínas de Escherichia coli/genética , Ácido Graxo Sintases/genética , Ácidos Graxos/química , Isoenzimas/síntese química , Isoenzimas/genética , Isoenzimas/metabolismo , Plasmídeos/genética , Plasmídeos/metabolismo
2.
J Agric Food Chem ; 67(32): 8905-8918, 2019 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-31380641

RESUMO

NAC TFs play crucial roles in response to abiotic stresses in plants. Here, ZmNAC071 was identified as a nuclear located transcriptional repressor. Overexpression of ZmNAC071 in Arabidopsis enhanced sensitivity of transgenic plants to ABA and osmotic stress. The expression levels of SODs, PODs, P5CSs, and AtMYB61 were inhibited by ZmNAC071, which results in reduced ROS scavenging and proline content, increased ROS level, and water loss. Besides, the expression levels of some ABA or abiotic stress-related genes, like ABIs, RD29A, DREBs, and LEAs were also significantly inhibited by ZmNAC071. Yeast one-hybrid assay demonstrated that ZmNAC071 specifically bound to the cis-acting elements containing CGT[G/A] core sequences in the promoter of stress-related genes, suggesting that ZmNAC071 may participate in the regulation of transcription of these genes through recognizing the core sequences CGT[G/A]. These results will facilitate further studies concerning the cis-elements and downstream genes targeted by ZmNAC071 in maize.


Assuntos
Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Ácido Ascórbico/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas/efeitos dos fármacos , Plantas Geneticamente Modificadas/fisiologia , Fatores de Transcrição/genética , Zea mays/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação para Baixo/efeitos dos fármacos , Pressão Osmótica , Plantas Geneticamente Modificadas/genética , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico , Fatores de Transcrição/metabolismo
3.
BMC Plant Biol ; 19(1): 368, 2019 Aug 20.
Artigo em Inglês | MEDLINE | ID: mdl-31429706

RESUMO

BACKGROUND: We previously reported the involvement of nitric oxide (NO) and cyclic nucleotide-gated ion channel 6 (CNGC6) in the responses of plants to heat shock (HS) exposure. To elucidate their relationship with heat tolerance in Arabidopsis thaliana, we examined the effects of HS on several groups of seedlings: wild type, cngc6, and cngc6 complementation and overexpression lines. RESULTS: After HS exposure, the level of NO was lower in cngc6 seedlings than in wild-type seedlings but significantly elevated in the transgenic lines depending on CNGC6 expression level. The treatment of seeds with calcium ions (Ca2+) enhanced the NO level in Arabidopsis seedlings under HS conditions, whereas treatment with EGTA (a Ca2+ chelator) reduced it, implicating that CNGC6 stimulates the accumulation of NO depending on an increase in cytosolic Ca2+ ([Ca2+]cyt). This idea was proved by phenotypic observations and thermotolerance testing of transgenic plants overexpressing NIA2 and NOA1, respectively, in a cngc6 background. Western blotting indicated that CNGC6 stimulated the accumulation of HS proteins via NO. CONCLUSION: These data indicate that CNGC6 acts upstream of NO in the HS pathway, which improves our insufficient knowledge of the initiation of plant responses to high temerature.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/metabolismo , Óxido Nítrico/metabolismo , Termotolerância , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Canais de Cálcio/metabolismo , Canais de Cátion Regulados por Nucleotídeos Cíclicos/genética , Citosol/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico , Mutação , Nitrato Redutase/genética , Nitrato Redutase/metabolismo , Óxido Nítrico Sintase/genética , Óxido Nítrico Sintase/metabolismo , Plântula/genética , Plântula/metabolismo
4.
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
5.
Plant Mol Biol ; 101(1-2): 183-202, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31286324

RESUMO

KEY MESSAGE: Isoforms of 2-OGDH E1 subunit are not functionally redundant in plant growth and development of A. thaliana. The tricarboxylic acid cycle enzyme 2-oxoglutarate dehydrogenase (2-OGDH) converts 2-oxoglutarate (2-OG) to succinyl-CoA concomitant with the reduction of NAD+. 2-OGDH has an essential role in plant metabolism, being both a limiting step during mitochondrial respiration as well as a key player in carbon-nitrogen interactions. In Arabidopsis thaliana two genes encode for E1 subunit of 2-OGDH but the physiological roles of each isoform remain unknown. Thus, in the present study we isolated Arabidopsis T-DNA insertion knockout mutant lines for each of the genes encoding the E1 subunit of 2-OGDH enzyme. All mutant plants exhibited substantial reduction in both respiration and CO2 assimilation rates. Furthermore, mutant lines exhibited reduced levels of chlorophylls and nitrate, increased levels of sucrose, malate and fumarate and minor changes in total protein and starch levels in leaves. Despite the similar metabolic phenotypes for the two E1 isoforms the reduction in the expression of each gene culminated in different responses in terms of plant growth and seed production indicating distinct roles for each isoform. Collectively, our results demonstrated the importance of the E1 subunit of 2-OGDH in both autotrophic and heterotrophic tissues and suggest that the two E1 isoforms are not functionally redundant in terms of plant growth in A. thaliana.


Assuntos
Arabidopsis/enzimologia , Carbono/metabolismo , Complexo Cetoglutarato Desidrogenase/metabolismo , Nitrogênio/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Dióxido de Carbono/metabolismo , Clorofila/metabolismo , Complexo Cetoglutarato Desidrogenase/genética , Mitocôndrias/enzimologia , Mutagênese Insercional , Nitratos/metabolismo , Fenótipo , Filogenia , Folhas de Planta/enzimologia , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Isoformas de Proteínas , Subunidades Proteicas , Plântula/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/enzimologia , Sementes/genética , Sementes/crescimento & desenvolvimento
6.
Molecules ; 24(13)2019 Jul 08.
Artigo em Inglês | MEDLINE | ID: mdl-31288414

RESUMO

Due to their sedentary lifestyle, plants are constantly exposed to different stress stimuli. Stress comes in variety of forms where factors like radiation, free radicals, "replication errors, polymerase slippage", and chemical mutagens result in genotoxic or cytotoxic damage. In order to face "the base oxidation or DNA replication stress", plants have developed many sophisticated mechanisms. One of them is the DNA mismatch repair (MMR) pathway. The main part of the MMR is the MutS homologue (MSH) protein family. The genome of Arabidopsis thaliana encodes at least seven homologues of the MSH family: AtMSH1, AtMSH2, AtMSH3, AtMSH4, AtMSH5, AtMSH6, and AtMSH7. Despite their importance, the functions of AtMSH homologs have not been investigated. In this work, bioinformatics tools were used to obtain a better understanding of MSH-mediated DNA repair mechanisms in Arabidopsis thaliana and to understand the additional biological roles of AtMSH family members. In silico analysis, including phylogeny tracking, prediction of 3D structure, interactome analysis, and docking site prediction, suggested interactions with proteins were important for physiological development of A. thaliana. The MSH homologs extensively interacted with both TIL1 and TIL2 (DNA polymerase epsilon catalytic subunit), proteins involved in cell fate determination during plant embryogenesis and involved in flowering time repression. Additionally, interactions with the RECQ protein family (helicase enzymes) and proteins of nucleotide excision repair pathway were detected. Taken together, the results presented here confirm the important role of AtMSH proteins in mismatch repair and suggest important new physiological roles.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Simulação de Acoplamento Molecular , Proteínas MutS/metabolismo , Proteínas de Arabidopsis/química , Sítios de Ligação , Simulação por Computador , Dano ao DNA , Reparo de Erro de Pareamento de DNA , Reparo do DNA , Replicação do DNA , Proteínas MutS/química , Conformação Proteica
7.
Plant Cell Physiol ; 60(8): 1633-1645, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31292642

RESUMO

Plants respond to a rise in ambient temperature by increasing the growth of petioles and hypocotyls. In this work, we show that Arabidopsis thaliana class I TEOSINTE BRANCHED 1, CYCLOIDEA, PCF (TCP) transcription factors TCP14 and TCP15 are required for optimal petiole and hypocotyl elongation under high ambient temperature. These TCPs influence the levels of the DELLA protein RGA and the expression of growth-related genes, which are induced in response to an increase in temperature. However, the class I TCPs are not required for the induction of the auxin biosynthesis gene YUCCA8 or for auxin-dependent gene expression responses. TCP15 directly targets the gibberellin biosynthesis gene GA20ox1 and the growth regulatory genes HBI1 and PRE6. Several of the genes regulated by TCP15 are also targets of the growth regulator PIF4 and show an enrichment of PIF4- and TCP-binding motifs in their promoters. PIF4 binding to GA20ox1 and HBI1 is enhanced in the presence of the TCPs, indicating that TCP14 and TCP15 directly participate in the induction of genes involved in gibberellin biosynthesis and cell expansion by high temperature functionally interacting with PIF4. In addition, overexpression of HBI1 rescues the growth defects of tcp14 tcp15 double mutants, suggesting that this gene is a major outcome of regulation by both class I TCPs during thermomorphogenesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Giberelinas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Complexo de Endopeptidases do Proteassoma/genética , Temperatura Ambiente , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
8.
BMC Plant Biol ; 19(1): 304, 2019 Jul 10.
Artigo em Inglês | MEDLINE | ID: mdl-31291882

RESUMO

BACKGROUND: In flowering plants, proper seed development is achieved through the constant interplay of fertilization products, embryo and endosperm, and maternal tissues. Communication between these compartments is supposed to be tightly regulated at their interfaces. Here, we characterize the deposition pattern of an apoplastic lipid barrier between the maternal inner integument and fertilization products in Arabidopsis thaliana seeds. RESULTS: We demonstrate that an apoplastic lipid barrier is first deposited by the ovule inner integument and undergoes de novo cutin deposition following central cell fertilization and relief of the FERTILIZATION INDEPENDENT SEED Polycomb group repressive mechanism. In addition, we show that the WIP zinc-finger TRANSPARENT TESTA 1 and the MADS-Box TRANSPARENT TESTA 16 transcription factors act maternally to promote its deposition by regulating cuticle biosynthetic pathways. Finally, mutant analyses indicate that this apoplastic barrier allows correct embryo sliding along the seed coat. CONCLUSIONS: Our results revealed that the deposition of a cutin apoplastic barrier between seed maternal and zygotic tissues is part of the seed coat developmental program.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Lipídeos de Membrana/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Endosperma/genética , Endosperma/crescimento & desenvolvimento , Endosperma/metabolismo , Óvulo Vegetal/genética , Óvulo Vegetal/crescimento & desenvolvimento , Óvulo Vegetal/metabolismo , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo
9.
J Photochem Photobiol B ; 197: 111535, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31319267

RESUMO

Measurement of Pulse-Amplitude-Modulated (PAM) chlorophyll a fluorescence is widely used method for obtaining information on the functional state of photosystem II (PSII). Recently, it has been shown that some of long-established fluorescence parameters must be interpreted with caution, when the light-induced chloroplast movements occur. In our work we have analyzed the effect of chloroplast movements on these parameters. We have derived new parameters that are independent of the change in PSII absorption occurring during measurement. To verify whether there is a need for new parameters or the difference between the parameters commonly used and the newly derived ones is insignificant, we conducted an experiment with Arabidopsis thaliana wild type plants and its phot1 phot2 mutant defective in chloroplast movement. Plants were exposed to light of different qualities (450, 470, 550 or 660 nm) and quantities (100, 400 or 1200 µmol m-2 s-1) for up to 40 min. Since the blue light-induced chloroplast avoidance reaction is a photoprotective mechanism, we expected that phot1 phot2 mutant will compensate the lack of this mechanism by increasing non-photochemical quenching. However, using the light at both 450 and 470 nm, the calculation of commonly used parameter, ΦNPQ (quantum yield of regulated light-induced thermal energy dissipation in PSII) based on Hendrickson et al. [L. Hendrickson, R.T. Furbank, W.S. Chow, Photosynth. Res. 82 (2004) 73-81] showed the opposite. On the other hand, the results obtained using our newly proposed formulae to determine quantum yield of PSII thermal energy dissipation were in line with our assumption. Thus, the experimental data showed that some formulae of fluorescence parameters are dependent on the change in PSII absorption and need to be interpreted carefully. On the contrary, the formulae introduced by us can remove the effect of changes in PSII absorption that occur during measurement, without additional measurements, and give the real estimate of light-induced non-photochemical quenching.


Assuntos
Proteínas de Arabidopsis/metabolismo , Clorofila A/química , Complexo de Proteína do Fotossistema II/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/fisiologia , Luz , Modelos Teóricos , Mutagênese , Complexo de Proteína do Fotossistema II/química , Complexo de Proteína do Fotossistema II/genética , Folhas de Planta/química , Teoria Quântica , Termodinâmica
10.
Nat Commun ; 10(1): 2904, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266952

RESUMO

Plant survival necessitates constant monitoring of fluctuating light and balancing growth demands with adaptive responses, tasks mediated via interconnected sensing and signaling networks. Photoreceptor phytochrome B (phyB) and plastidial retrograde signaling metabolite methylerythritol cyclodiphosphate (MEcPP) are evolutionarily conserved sensing and signaling components eliciting responses through unknown connection(s). Here, via a suppressor screen, we identify two phyB mutant alleles that revert the dwarf and high salicylic acid phenotypes of the high MEcPP containing mutant ceh1. Biochemical analyses show high phyB protein levels in MEcPP-accumulating plants resulting from reduced expression of phyB antagonists and decreased auxin levels. We show that auxin treatment negatively regulates phyB abundance. Additional studies identify CAMTA3, a MEcPP-activated calcium-dependent transcriptional regulator, as critical for maintaining phyB abundance. These studies provide insights into biological organization fundamentals whereby a signal from a single plastidial metabolite is transduced into an ensemble of regulatory networks controlling the abundance of phyB, positioning plastids at the information apex directing adaptive responses.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fitocromo B/metabolismo , Plastídeos/metabolismo , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Eritritol/análogos & derivados , Eritritol/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Ácidos Indolacéticos/metabolismo , Luz , Fitocromo B/genética , Plastídeos/genética , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
11.
Plant Mol Biol ; 101(3): 257-268, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31302867

RESUMO

KEY MESSAGE: The C-terminal cysteine-rich motif of NYE1/SGR1 affects chlorophyll degradation likely by mediating its self-interaction and conformational change, and somehow altering its Mg-dechelating activity in response to the changing redox potential. During green organ senescence in plants, the most prominent phenomenon is the degreening caused by net chlorophyll (Chl) loss. NON-YELLOWING1/STAY-GREEN1 (NYE1/SGR1) was recently reported to be able to dechelates magnesium (Mg) from Chl a to initiate its degradation, but little is known about the domain/motif basis of its functionality. In this study, we carried out a protein truncation assay and identified a conserved cysteine-rich motif (CRM, P-X3-C-X3-C-X-C2-F-P-X5-P) at its C terminus, which is essential for its function. Genetic analysis showed that all four cysteines in the CRM were irreplaceable, and enzymatic assays demonstrated that the mutation of each of the four cysteines affected its Mg-dechelating activity. The CRM plays a critical role in the conformational change and self-interaction of NYE1 via the formation of inter- and intra-molecular disulfide bonds. Our results may provide insight into how NYE1 responds to rapid redox changes during leaf senescence and in response to various environmental stresses.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Clorofila/química , Proteínas de Cloroplastos/metabolismo , Motivos de Aminoácidos , Quelantes/química , DNA Complementar/metabolismo , Dissulfetos , Regulação da Expressão Gênica de Plantas , Magnésio/química , Oxirredução , Fenótipo , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Conformação Proteica , Domínios Proteicos , Estresse Fisiológico
12.
Nat Commun ; 10(1): 2378, 2019 05 30.
Artigo em Inglês | MEDLINE | ID: mdl-31147541

RESUMO

Developmental plasticity of root system architecture is crucial for plant performance in nutrient-poor soils. Roots of plants grown under mild nitrogen (N) deficiency show a foraging response characterized by increased root length but mechanisms underlying this developmental plasticity are still elusive. By employing natural variation in Arabidopsis accessions, we show that the brassinosteroid (BR) signaling kinase BSK3 modulates root elongation under mild N deficiency. In particular, a proline to leucine substitution in the predicted kinase domain of BSK3 enhances BR sensitivity and signaling to increase the extent of root elongation. We further show that low N specifically upregulates transcript levels of the BR co-receptor BAK1 to activate BR signaling and stimulate root elongation. Altogether, our results uncover a role of BR signaling in root elongation under low N. The BSK3 alleles identified here provide targets for improving root growth of crops growing under limited N conditions.


Assuntos
Proteínas de Arabidopsis/genética , Nitrogênio/deficiência , Raízes de Plantas/crescimento & desenvolvimento , Proteínas Serina-Treonina Quinases/genética , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Solo/química , Regulação para Cima
13.
Plant Cell Rep ; 38(9): 1165-1180, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31161264

RESUMO

KEY MESSAGE: Arabidopsis photorespiratory gene AtAGT1 is important for the growth and development of root, the non-photosynthetic organ, and it is involved in a complex metabolic network and salt resistance. AtAGT1 in Arabidopsis encodes an aminotransferase that has a wide range of donor:acceptor combinations, including Asn:glyoxylate. Although it is one of the photorespiratory genes, its encoding protein has been suggested to function also in roots to metabolize Asn. However, experimental data are still lacking. In this study, we investigated experimentally the function of AtAGT1 in roots and our results uncovered its importance in root development during seedling establishment after seed germination. Overexpression of AtAGT1 in roots promoted both the growth of primary root and outgrowth of lateral roots. To further elucidate the molecular mechanisms underlying, amino acid content and gene expression in roots were analyzed, and results revealed that AtAGT1 is involved in a complex metabolic network and salt resistance of roots.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Reguladores de Crescimento de Planta/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Expressão Gênica , Germinação , Plantas Geneticamente Modificadas , Tolerância ao Sal , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/fisiologia , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/fisiologia , Transaminases/genética , Transaminases/metabolismo
14.
Nat Commun ; 10(1): 2834, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249292

RESUMO

Environmental information perceived by chloroplasts can be translated into retrograde signals that alter the expression of nuclear genes. Singlet oxygen (1O2) generated by photosystem II (PSII) can cause photo-oxidative damage of PSII but has also been implicated in retrograde signaling. We previously reported that a nuclear-encoded chloroplast FtsH2 metalloprotease coordinates 1O2-triggered retrograde signaling by promoting the degradation of the EXECUTER1 (EX1) protein, a putative 1O2 sensor. Here, we show that a 1O2-mediated oxidative post-translational modification of EX1 is essential for initiating 1O2-derived signaling. Specifically, the Trp643 residue in DUF3506 domain of EX1 is prone to oxidation by 1O2. Both the substitution of Trp643 with 1O2-insensitive amino acids and the deletion of the DUF3506 domain abolish the EX1-mediated 1O2 signaling. We thus provide mechanistic insight into how EX1 senses 1O2 via Trp643 located in the DUF3506 domain.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastídeos/metabolismo , Oxigênio Singlete/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Arabidopsis/química , Arabidopsis/genética , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Oxirredução , Plastídeos/química , Plastídeos/genética , Domínios Proteicos , Processamento de Proteína Pós-Traducional , Transdução de Sinais
15.
Nat Commun ; 10(1): 2828, 2019 06 27.
Artigo em Inglês | MEDLINE | ID: mdl-31249301

RESUMO

H2A.Z variant has emerged as a critical player in regulating plant responses to environment; however, the mechanism by which H2A.Z mediates this regulation remains unclear. In Arabidopsis, H2A.Z has been proposed to have opposite effects on transcription depending on its localization within the gene. These opposite roles have been assigned by correlating gene expression and H2A.Z enrichment analyses but without considering the impact of possible H2A.Z post-translational modifications. Here, we show that H2A.Z can be monoubiquitinated by the PRC1 components AtBMI1A/B/C. The incorporation of this modification is required for H2A.Z-mediated transcriptional repression through a mechanism that does not require PRC2 activity. Our data suggest that the dual role of H2A.Z in regulating gene expression depends on the modification that it carries, while the levels of H2A.Z within genes depend on the transcriptional activity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Histonas/metabolismo , Complexo Repressor Polycomb 1/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Histonas/genética , Complexo Repressor Polycomb 1/genética , Ubiquitinação
16.
BMC Plant Biol ; 19(1): 244, 2019 Jun 07.
Artigo em Inglês | MEDLINE | ID: mdl-31174473

RESUMO

BACKGROUND: Even though the roles of pentatricopeptide repeat (PPR) proteins are essential in plant organelles, the function of many chloroplast-targeted PPR proteins remains unknown. Here, we characterized the function of a chloroplast-localized PPR protein (At3g59040), which is classified as the 287th PPR protein among the 450 PPR proteins in Arabidopsis ( http://ppr.plantenergy.uwa.edu.au ). RESULTS: The homozygous ppr287 mutant with the T-DNA inserted into the last exon displayed pale-green and yellowish phenotypes. The microRNA-mediated knockdown mutants were generated to further confirm the developmental defect phenotypes of ppr287 mutants. All mutants had yellowish leaves, shorter roots and height, and less seed yield, indicating that PPR287 is crucial for normal Arabidopsis growth and development. The photosynthetic activity and chlorophyll content of ppr287 mutants were markedly reduced, and the chloroplast structures of the mutants were abnormal. The levels of chloroplast rRNAs were decreased in ppr287 mutants. CONCLUSIONS: These results suggest that PPR287 plays an essential role in chloroplast biogenesis and function, which is crucial for the normal growth and development of Arabidopsis.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas de Cloroplastos/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , RNA de Cloroplastos/genética , RNA de Cloroplastos/metabolismo
17.
Nat Plants ; 5(6): 589-594, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182839

RESUMO

Brassinosteroids (BRs) are essential plant steroid hormones that regulate plant growth and development1. The most potent BR, brassinolide, is produced by addition of many oxygen atoms to campesterol by several cytochrome P450 monooxygenases (CYPs). CYP90B1 (also known as DWF4) catalyses the 22(S)-hydroxylation of campesterol and is the first and rate-limiting enzyme at the branch point of the biosynthetic pathway from sterols to BRs2. Here we show the crystal structure of Arabidopsis thaliana CYP90B1 complexed with cholesterol as a substrate. The substrate-binding conformation explains the stereoselective introduction of a hydroxy group at the 22S position, facilitating hydrogen bonding of brassinolide with the BR receptor3-5. We also determined the crystal structures of CYP90B1 complexed with uniconazole6,7 or brassinazole8, which inhibit BR biosynthesis. The two inhibitors are structurally similar; however, their binding conformations are unexpectedly different. The shape and volume of the active site pocket varies depending on which inhibitor or substrate is bound. These crystal structures of plant CYPs that function as membrane-anchored enzymes and exhibit structural plasticity can inform design of novel inhibitors targeting plant membrane-bound CYPs, including those involved in BR biosynthesis, which could then be used as plant growth regulators and agrochemicals.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Brassinosteroides/biossíntese , Sistema Enzimático do Citocromo P-450/química , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Brassinosteroides/antagonistas & inibidores , Cristalografia por Raios X , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Relação Estrutura-Atividade , Triazóis/química
18.
Nat Plants ; 5(6): 581-588, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182842

RESUMO

Chloroplasts are integral to sensing biotic and abiotic stress in plants, but their role in transducing Ca2+-mediated stress signals remains poorly understood1,2. Here we identify cMCU, a member of the mitochondrial calcium uniporter (MCU) family, as an ion channel mediating Ca2+ flux into chloroplasts in vivo. Using a toolkit of aequorin reporters targeted to chloroplast stroma and the cytosol in cMCU wild-type and knockout lines, we provide evidence that stress-stimulus-specific Ca2+ dynamics in the chloroplast stroma correlate with expression of the channel. Fast downstream signalling events triggered by osmotic stress, involving activation of the mitogen-activated protein kinases (MAPK) MAPK3 and MAPK6, and the transcription factors MYB60 and ethylene-response factor 6 (ERF6), are influenced by cMCU activity. Relative to wild-type plants, cMCU knockouts display increased resistance to long-term water deficit and improved recovery on rewatering. Modulation of stromal Ca2+ in specific processing of stress signals identifies cMCU as a component of plant environmental sensing.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Cloroplastos/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Canais de Cálcio/genética , Cloroplastos/genética , Escherichia coli , Técnicas de Inativação de Genes , Sistema de Sinalização das MAP Quinases , Pressão Osmótica
19.
Nat Plants ; 5(6): 604-615, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182845

RESUMO

During phloem unloading, multiple cell-to-cell transport events move organic substances to the root meristem. Although the primary unloading event from the sieve elements to the phloem pole pericycle has been characterized to some extent, little is known about post-sieve element unloading. Here, we report a novel gene, PHLOEM UNLOADING MODULATOR (PLM), in the absence of which plasmodesmata-mediated symplastic transport through the phloem pole pericycle-endodermis interface is specifically enhanced. Increased unloading is attributable to a defect in the formation of the endoplasmic reticulum-plasma membrane tethers during plasmodesmal morphogenesis, resulting in the majority of pores lacking a visible cytoplasmic sleeve. PLM encodes a putative enzyme required for the biosynthesis of sphingolipids with very-long-chain fatty acid. Taken together, our results indicate that post-sieve element unloading involves sphingolipid metabolism, which affects plasmodesmal ultrastructure. They also raise the question of how and why plasmodesmata with no cytoplasmic sleeve facilitate molecular trafficking.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Floema/metabolismo , Plasmodesmos/ultraestrutura , Esfingolipídeos/biossíntese , Arabidopsis/genética , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Genes de Plantas , Glucanos/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Mutação , Raízes de Plantas/metabolismo , Plasmodesmos/metabolismo , Transferases (Outros Grupos de Fosfato Substituídos)/genética , Transferases (Outros Grupos de Fosfato Substituídos)/metabolismo
20.
Nat Plants ; 5(6): 616-625, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31182849

RESUMO

The lipid-derived hormone jasmonate (JA) regulates plant immunity and adaptive growth by triggering a genome-wide transcriptional programme. In Arabidopsis thaliana, JA-triggered transcriptional programming is largely orchestrated by the master transcription factor MYC2. The function of MYC2 is dependent on its physical interaction with the MED25 subunit of the Mediator transcriptional co-activator complex. Here we report the identification of JA enhancers (JAEs) through profiling the occupancy pattern of MYC2 and MED25. JA regulates the dynamic chromatin looping between JAEs and their promoters in a MED25-dependent manner, while MYC2 auto-regulates itself through JAEs. Interestingly, the JAE of the MYC2 locus (named ME2) positively regulates MYC2 expression during short-term JA responses but negatively regulates it during constant JA responses. We demonstrate that new gene editing tools open up new avenues to elucidate the in vivo function of enhancers. Our work provides a paradigm for functional study of plant enhancers in the regulation of specific physiological processes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ciclopentanos/metabolismo , Proteínas Nucleares/metabolismo , Oxilipinas/metabolismo , Transdução de Sinais , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Arseniato Redutases/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Cromatina/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica de Plantas , Histonas/metabolismo , Regiões Promotoras Genéticas , Ligação Proteica
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