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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.
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
3.
Plant Mol Biol ; 101(1-2): 203-220, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31297725

RESUMO

KEY MESSAGE: Here, a functional characterization of a wheat MSR has been presented: this protein makes a contribution to the plant's tolerance of abiotic stress, acting through its catalytic capacity and its modulation of ROS and ABA pathways. The molecular mechanism and function of certain members of the methionine sulfoxide reductase (MSR) gene family have been defined, however, these analyses have not included the wheat equivalents. The wheat MSR gene TaMSRA4.1 is inducible by salinity and drought stress and in this study, we demonstrate that its activity is restricted to the Met-S-SO enantiomer, and its subcellular localization is in the chloroplast. Furthermore, constitutive expression of TaMSRA4.1 enhanced the salinity and drought tolerance of wheat and Arabidopsis thaliana. In these plants constitutively expressing TaMSRA4.1, the accumulation of reactive oxygen species (ROS) was found to be influenced through the modulation of genes encoding proteins involved in ROS signaling, generation and scavenging, while the level of endogenous abscisic acid (ABA), and the sensitivity of stomatal guard cells to exogenous ABA, was increased. A yeast two-hybrid screen, bimolecular fluorescence complementation and co-immunoprecipitation assays demonstrated that heme oxygenase 1 (HO1) interacted with TaMSRA4.1, and that this interaction depended on a TaHO1 C-terminal domain. In plants subjected to salinity or drought stress, TaMSRA4.1 reversed the oxidation of TaHO1, activating ROS and ABA signaling pathways, but not in the absence of HO1. The aforementioned properties advocate TaMSRA4.1 as a candidate for plant genetic enhancement.


Assuntos
Heme Oxigenase-1/metabolismo , Metionina Sulfóxido Redutases/metabolismo , Transdução de Sinais , Estresse Fisiológico , Triticum/enzimologia , Ácido Abscísico/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Arabidopsis/fisiologia , Secas , Perfilação da Expressão Gênica , Heme Oxigenase-1/genética , Metionina Sulfóxido Redutases/genética , Oxirredução , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Salinidade , Tolerância ao Sal , Plântula/enzimologia , Plântula/genética , Plântula/fisiologia , Triticum/genética , Triticum/fisiologia , Técnicas do Sistema de Duplo-Híbrido
4.
Plant Sci ; 285: 1-13, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31203874

RESUMO

Bioactive gibberellins (GAs) play multiple roles in plant development and stress responses. GA2-oxidases (GA2oxs) are a class of 2-oxoglutarate-dependent dioxygenases that regulate the deactivation of bioactive GAs. In this study, we investigated the phylogeny and domain structures of the seven GA2ox genes present in the Arabidopsis thaliana genome. Comprehensive expression analysis using translational reporter lines showed that the seven GA2ox genes are differentially expressed during Arabidopsis growth and development: GA2ox1 is specifically expressed in the hypocotyl and lateral root primordium; GA2ox2 is highly expressed in aboveground tissues; GA2ox3 is expressed in the chalazal endosperm of the early embryo sac and inflorescences; GA2ox4 is expressed in the shoot apical meristem and during lateral root initiation; GA2ox6 is expressed in the maturation zone, but not in the meristem or elongating zone of the root; GA2ox7 is constitutively expressed during almost all developmental stages; and GA2ox8 is exclusively expressed in stomatal cells. Overexpression of each of these GA2ox genes inhibited high temperature-induced hypocotyl elongation in both wild-type and elongated hypocotyl 5 plants, which have an elongated hypocotyl phenotype, suggesting that these genes negatively regulate hypocotyl elongation by reducing bioactive GA levels. This study provides a valuable resource for further elucidating the roles of GA2ox genes during different stages of development.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Genes de Plantas/fisiologia , Giberelinas/metabolismo , Oxirredutases/genética , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Giberelinas/fisiologia , Hipocótilo/crescimento & desenvolvimento , Hipocótilo/metabolismo , Oxirredutases/metabolismo , Oxirredutases/fisiologia , Filogenia , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Transcriptoma
5.
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
6.
Plant Physiol Biochem ; 141: 206-214, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31176880

RESUMO

SUMOylation is an important protein modification that regulates the properties of substrate proteins in a variety of cellular processes. SUMOylation is catalyzed via a cascade of enzymes and is usually stimulated by SUMO E3 ligases. However, the molecular functions and regulatory mechanisms of SUMOylation in forage crops are unknown. Here, we isolated and functionally characterized DiMMS21, a homolog of the Arabidopsis thaliana SUMO ligase AtMMS21, from the forage legume Desmodium intortum. DiMMS21 is expressed ubiquitously in various D. intortum organs and its encoded protein is found in the cytoplasm and nucleus. Bioinformatics analysis indicated that DiMMS21 contains a conserved SP-RING domain that is required for its activity. Biochemical evidence supports the notion that this protein is a functional SUMO ligase. When expressed in an Arabidopsis mms21 mutant, DiMMS21 completely rescued the defects in root, leaf, and silique development. The results from cotyledon greening and marker gene expression suggested that DiMMS21 can only partially complements the role of AtMMS21 in abscisic acid (ABA) responses. In summary, we characterized the molecular features of DiMMS21 and uncovered potential roles of this SUMO ligase in development and ABA responses, increasing our understanding on the function of SUMOylation in forage crops.


Assuntos
Fabaceae/enzimologia , Regulação da Expressão Gênica de Plantas , Ligases/metabolismo , Proteínas de Plantas/metabolismo , Ácido Abscísico/metabolismo , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Núcleo Celular/metabolismo , Biologia Computacional , Citoplasma/metabolismo , DNA Complementar/metabolismo , Perfilação da Expressão Gênica , Mutação , Filogenia , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismo , Sementes/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo
7.
Mol Plant Microbe Interact ; 32(11): 1487-1495, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31241412

RESUMO

Phytoplasmas are the causative agent of numerous diseases of plant species all over the world, including important food crops. The mode by which phytoplasmas multiply and behave in their host is poorly understood and often based on genomic data. We used yeast two-hybrid screening to find new protein-protein interactions between the causal agent of apple proliferation 'Candidatus Phytoplasma mali' and its host plant. Here, we report that the 'Ca. P. mali' strain PM19 genome encodes a protein PM19_00185 that interacts with at least six different ubiquitin-conjugating enzymes (UBC; E2) of Arabidopsis thaliana. An in vitro ubiquitination assay showed that PM19_00185 is enzymatically active as E3 ligase with A. thaliana E2 UBC09 and Malus domestica E2 UBC10. We show that a nonhost bacteria (Pseudomonas syringae pv. tabaci) can grow in transgenic A. thaliana plant lines expressing PM19_00185. A connection of phytoplasma effector proteins with the proteasome proteolytic pathway has been reported before. However, this is, to our knowledge, the first time that a phytoplasma effector protein with E3 ligase activity has been reported.


Assuntos
Phytoplasma , Doenças das Plantas , Ubiquitina-Proteína Ligases , Arabidopsis/enzimologia , Arabidopsis/parasitologia , Malus/parasitologia , Phytoplasma/enzimologia , Phytoplasma/genética , Doenças das Plantas/imunologia , Doenças das Plantas/parasitologia , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/imunologia , Ubiquitina-Proteína Ligases/metabolismo
8.
Enzyme Microb Technol ; 127: 70-74, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31088620

RESUMO

D-glucuronic acid (GlcUA) is an important intermediate with numerous applications in the food, cosmetics, and pharmaceutical industries. Its biological production routes which employ myo-inositol oxygenase (MIOX) as the key enzyme are attractive. In this study, five diverse MIOX-encoding genes, from Cryptococcus neoformans, Chaetomium thermophilum, Arabidopsis thaliana, Thermothelomyces thermophila, and Mus musculus were overexpressed in Escherichia coli, respectively. A novel MIOX from Thermothelomyces thermophila (TtMIOX) exhibited high specific activity, and efficiently converted myo-inositol to GlcUA. Meanwhile, the degradation of GlcUA was inhibited by inactivation of uxaC from the Escherichia coli genome. Finally, the BWΔuxaC whole-cell biocatalyst harboring TtMIOX resulted in the production of 106 g/L GlcUA within 12 h in a 1-L bioreactor, corresponding to a conversion of 91% and productivity of 8.83 g/L/h. This study provides a feasible method for the industrial production of GlcUA.


Assuntos
Escherichia coli/metabolismo , Expressão Gênica , Ácido Glucurônico/metabolismo , Inositol Oxigenase/metabolismo , Inositol/metabolismo , Proteínas Recombinantes/metabolismo , Sordariales/enzimologia , Animais , Arabidopsis/enzimologia , Arabidopsis/genética , Biotransformação , Chaetomium/enzimologia , Chaetomium/genética , Cryptococcus neoformans/enzimologia , Cryptococcus neoformans/genética , Escherichia coli/genética , Inositol Oxigenase/genética , Camundongos , Proteínas Recombinantes/genética , Sordariales/genética
9.
Acta Crystallogr D Struct Biol ; 75(Pt 5): 488-497, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31063151

RESUMO

Plant-unique membrane receptor kinases with leucine-rich repeat (LRR) extracellular domains are key regulators of development and immune responses. Here, the 1.55 Šresolution crystal structure of the immune receptor kinase SOBIR1 from Arabidopsis is presented. The ectodomain structure reveals the presence of five LRRs sandwiched between noncanonical capping domains. The disulfide-bond-stabilized N-terminal cap harbours an unusual ß-hairpin structure. The C-terminal cap features a highly positively charged linear motif which was found to be largely disordered in this structure. Size-exclusion chromatography and right-angle light-scattering experiments suggest that SOBIR1 is a monomer in solution. The protruding ß-hairpin, a set of highly conserved basic residues at the inner surface of the SOBIR LRR domain and the presence of a genetic missense allele in LRR2 together suggest that the SOBIR1 ectodomain may mediate protein-protein interaction in plant immune signalling.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Leucina/química , Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/química , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo
10.
Plant Cell Rep ; 38(9): 1081-1097, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31134349

RESUMO

KEY MESSAGE: Duplicate POT1 genes must rapidly diverge or be inactivated. Protection of telomeres 1 (POT1) encodes a conserved telomere binding protein implicated in both chromosome end protection and telomere length maintenance. Most organisms harbor a single POT1 gene, but in the few lineages where the POT1 family has expanded, the duplicate genes have diversified. Arabidopsis thaliana bears three POT1-like loci, POT1a, POT1b and POT1c. POT1a retains the ancestral function of telomerase regulation, while POT1b is implicated in chromosome end protection. Here we examine the function and evolution of the third POT1 paralog, POT1c. POT1c is a new gene, unique to A. thaliana, and was derived from a duplication event involving the POT1a locus and a neighboring gene encoding ribosomal protein S17. The duplicate S17 locus (dS17) is highly conserved across A. thaliana accessions, while POT1c is highly divergent, harboring multiple deletions within the gene body and two transposable elements within the promoter. The POT1c locus is transcribed at very low to non-detectable levels under standard growth conditions. In addition, no discernable molecular or developmental defects are associated with plants bearing a CRISPR mutation in the POT1c locus. However, forced expression of POT1c leads to decreased telomerase enzyme activity and shortened telomeres. Evolutionary reconstruction indicates that transposons invaded the POT1c promoter soon after the locus was formed, permanently silencing the gene. Altogether, these findings argue that POT1 dosage is critically important for viability and duplicate gene copies are retained only upon functional divergence.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Dosagem de Genes , Homeostase do Telômero/genética , Proteínas de Ligação a Telômeros/metabolismo , Telômero/genética , Arabidopsis/enzimologia , Arabidopsis/fisiologia , Proteínas de Arabidopsis/genética , Elementos de DNA Transponíveis/genética , Evolução Molecular , Duplicação Gênica , Mutação , Regiões Promotoras Genéticas/genética , Telomerase/genética , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/genética
11.
Planta ; 250(2): 535-548, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31111205

RESUMO

MAIN CONCLUSION: ACOS5, OsACOS12 and PpACOS6 are all capable of fatty acyl-CoA synthetase activity but exhibit different substrate preferences. The transcriptional regulation of ACOS for sporopollenin synthesis appears to have been conserved in Physcomitrella, rice and Arabidopsis during evolution. Sporopollenin is the major constituent of spore and pollen exines. In Arabidopsis, acyl-CoA synthetase 5 (ACOS5) is an essential enzyme for sporopollenin synthesis, and its orthologues are PpACOS6 from the moss Physcomitrella and OsACOS12 from monocot rice. However, knowledge regarding the evolutionary conservation and divergence of the ACOS gene in sporopollenin synthesis remains limited. In this study, we analysed the function and regulation of PpACOS6 and OsACOS12. A complementation test showed that OsACOS12 driven by the ACOS5 promoter could partially restore the male fertility of the acos5 mutant in Arabidopsis, while PpACOS6 did not rescue the acos5 phenotype. ACOS5, PpACOS6 and OsACOS12 all complemented the acyl-CoA synthetase-deficient yeast strain (YB525) phenotype, although they exhibited different substrate preferences. To understand the conservation of sporopollenin synthesis regulation, we constructed two constructs with ACOS5 driven by the OsACOS12 or PpACOS6 promoter. Both constructs could restore the fertility of acos5 plants. The MYB transcription factor MS188 from Arabidopsis directly regulates ACOS5. We found that MS188 could also bind the promoters of OsACOS12 and PpACOS6 and activate the genes driven by the promoters, suggesting that the transcriptional regulation of these genes was similar to that of ACOS5. These results show that the ACOS gene promoter region from Physcomitrella, rice and Arabidopsis has been functionally conserved during evolution, while the chain lengths of fatty acid-derived monomers of sporopollenin vary in different plant species.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Bryopsida/enzimologia , Coenzima A Ligases/metabolismo , Oryza/enzimologia , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/ultraestrutura , Proteínas de Arabidopsis/genética , Biopolímeros/biossíntese , Bryopsida/genética , Bryopsida/crescimento & desenvolvimento , Bryopsida/ultraestrutura , Carotenoides/biossíntese , Coenzima A Ligases/genética , Genes Reporter , Mutação , Oryza/genética , Oryza/crescimento & desenvolvimento , Oryza/ultraestrutura , Filogenia , Infertilidade das Plantas , Proteínas de Plantas/genética , Pólen/enzimologia , Pólen/genética , Pólen/crescimento & desenvolvimento , Pólen/ultraestrutura , Alinhamento de Sequência , Especificidade por Substrato , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
12.
Planta ; 250(2): 573-588, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31127375

RESUMO

MAIN CONCLUSION: The information on core components in maize polycomb repressive complex 2 (PRC2) are updated at a genome-wide scale, and the protein-protein interaction networks of PRC2 components are further provided in maize. The evolutionarily conserved polycomb group (PcG) proteins form multi-subunits polycomb repressive complexes (PRCs) that repress gene expression via chromatin condensation. In Arabidopsis, three distinct PRC2s have been identified, each determining a specific developmental program with partly functional redundancy. However, the core components and biological functions of PRC2 in cereals remain obscure. Here, we updated the information on maize PRC2 components at a genome-wide scale. Maize PRC2 subunits are highly duplicated, with five MSI1, three E(z), two ESC and two Su(z)12 homologs. ZmFIE1 is preferentially expressed in the endosperm, whereas the remaining are broadly expressed in many tissues. ZmCLF/MEZ1 and ZmFIE1 are maternally expressed imprinted genes, in contrast to the paternal-dominantly expression of ZmFIE2 in the endosperm. In maize, E(z) members likely provide a scaffold for assembling PRC2 complexes, whereas Su(z)12 and p55/MSI1-like proteins together reinforce the complex; ESC members probably determine its specificity: FIE1-PRC2 regulates endosperm cell development, whereas FIE2-PRC2 controls other cell types. The duplicated Brassicaceae-specific MEA and FIS2 also directly interact with maize PRC2 members. Together, this study establishes a roadmap for protein-protein interactions of maize PRC2 components, providing new insights into their functions in the growth and development of cereals.


Assuntos
Complexo Repressor Polycomb 2/metabolismo , Zea mays/enzimologia , Alelos , Arabidopsis/enzimologia , Arabidopsis/genética , Endosperma/enzimologia , Endosperma/genética , Endosperma/ultraestrutura , Epigenômica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Complexo Repressor Polycomb 2/genética , Domínios Proteicos , Técnicas do Sistema de Duplo-Híbrido , Zea mays/genética , Zea mays/ultraestrutura
13.
Plant Sci ; 283: 355-365, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128706

RESUMO

Despite recent evidence that HDACs are involved in the environmental stress responses of plants, their roles in the abiotic stress responses of monocot plants remain largely unexplored. We investigated a HDAC gene, Bradi3g08060 (BdHD1), in Brachypodium distachyon. The Brachypodium BdHD1-overexpression plants displayed a hypersensitive phenotype to ABA and exhibited better survival under drought conditions. On the other hand, the RNA-interference plants were insensitive to ABA and showed low survival under drought stress. At the genome-wide level, overexpression of BdHD1 led to lower H3K9 acetylation at the transcriptional start sites of 230 genes than in the wild type plants under the drought treatment. We validated our ChIP-Seq data on 10 selected transcription factor genes from the 230 drought-specific genes. These genes exhibited much lower expression in the BdHD1-overexpression plants compared to the wild type plants under drought stress. We further identified an ABA-inducible transcription factor gene BdWRKY24 that was repressed in BdHD1-OE plants, but highly expressed in RNA-interference plants under drought stress. These results indicate that BdHD1 plays a positive role in ABA sensitivity and drought stress tolerance and they provide a link between the role of BdHD1 and the drought stress response at a genome-wide level in Brachypodium.


Assuntos
Ácido Abscísico/fisiologia , Brachypodium/metabolismo , Histona Desacetilases/fisiologia , Reguladores de Crescimento de Planta/fisiologia , Proteínas de Plantas/fisiologia , Ácido Abscísico/metabolismo , Arabidopsis/enzimologia , Arabidopsis/genética , Brachypodium/enzimologia , Brachypodium/genética , Brachypodium/fisiologia , Desidratação , Regulação da Expressão Gênica de Plantas , Técnicas de Silenciamento de Genes , Histona Desacetilases/metabolismo , Filogenia , Reguladores de Crescimento de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Dedos de Zinco/genética , Dedos de Zinco/fisiologia
14.
Plant Sci ; 283: 366-374, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31128707

RESUMO

The tau (U) and phi (F) classes of glutathione transferase (GST) enzymes reduce the glutathione (GSH) pool using GSH as a co-substrate, thus influence numerous redox-dependent processes including hormonal and stress responses. We performed detailed analysis of the redox potential and reactive oxygen species levels in longitudinal zones of 7-day-old roots of Arabidopsis thaliana L. Col-0 wild type and Atsgtf8 and Atgstu19 insertional mutants. Using redox-sensitive cytosolic green fluorescent protein (roGFP2) the redox status of the meristematic, transition, and elongation zones was determined under control and salt stress (3-hour of 75 or 150 mM NaCl treatment) conditions. The Atgstu19 mutant had the most oxidized redox status in all root zones throughout the experiments. Using fluorescent dyes significantly higher superoxide radical (O2-) levels was detected in both Atgst mutants than in the Col-0 control. Salt treatment resulted in the highest O2- increase in the Atgstf8 root, while the amount of H2O2 elevated most in the case of Atgstu19. Moreover, vitality decreased in Atgstu19 roots more than in wild type under salt stress. Our results indicate that AtGSTF8 and especially the AtGSTU19 proteins function in the root fine-tuning the redox homeostasis both under control and salt stress conditions.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Glutationa Transferase/fisiologia , Meristema/fisiologia , Raízes de Plantas/fisiologia , Arabidopsis/enzimologia , Arabidopsis/metabolismo , Homeostase , Peróxido de Hidrogênio/metabolismo , Meristema/metabolismo , Oxirredução , Raízes de Plantas/enzimologia , Raízes de Plantas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Estresse Salino , Superóxidos/metabolismo
15.
Science ; 364(6435): 57-62, 2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948546

RESUMO

Rho guanosine triphosphatases (GTPases) are master regulators of cell signaling, but how they are regulated depending on the cellular context is unclear. We found that the phospholipid phosphatidylserine acts as a developmentally controlled lipid rheostat that tunes Rho GTPase signaling in Arabidopsis Live superresolution single-molecule imaging revealed that the protein Rho of Plants 6 (ROP6) is stabilized by phosphatidylserine into plasma membrane nanodomains, which are required for auxin signaling. Our experiments also revealed that the plasma membrane phosphatidylserine content varies during plant root development and that the level of phosphatidylserine modulates the quantity of ROP6 nanoclusters induced by auxin and hence downstream signaling, including regulation of endocytosis and gravitropism. Our work shows that variations in phosphatidylserine levels are a physiological process that may be leveraged to regulate small GTPase signaling during development.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Fosfatidilserinas/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/química , Membrana Celular/metabolismo , Endocitose/genética , Regulação da Expressão Gênica de Plantas , Gravitropismo/genética , Ácidos Indolacéticos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/genética , Fosfatidilserinas/farmacologia , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Raízes de Plantas/crescimento & desenvolvimento , Transdução de Sinais , Imagem Individual de Molécula
16.
Plant Cell Physiol ; 60(7): 1487-1503, 2019 Jul 01.
Artigo em Inglês | MEDLINE | ID: mdl-31004494

RESUMO

Plant cells sheath themselves in a complex lattice of polysaccharides, proteins and enzymes forming an integral matrix known as the cell wall. Cellulose microfibrils, the primary component of cell walls, are synthesized at the plasma membrane by CELLULOSE SYNTHASE A (CESA) proteins throughout cellular growth and are responsible for turgor-driven anisotropic expansion. Associations between hormone signaling and cell wall biosynthesis have long been suggested, but recently direct links have been found revealing hormones play key regulatory roles in cellulose biosynthesis. The radially swollen 1 (rsw1) allele of Arabidopsis thaliana CESA1 harbors a single amino acid change that renders the protein unstable at high temperatures. We used the conditional nature of rsw1 to investigate how auxin contributes to isotropic growth. We found that exogenous auxin treatment reduces isotropic swelling in rsw1 roots at the restrictive temperature of 30�C. We also discovered decreases in auxin influx between rsw1 and wild-type roots via confocal imaging of AUX1-YFP, even at the permissive temperature of 19�C. Moreover, rsw1 displayed mis-expression of auxin-responsive and CESA genes. Additionally, we found altered auxin maxima in rsw1 mutant roots at the onset of swelling using DII-VENUS and DR5:vYFP auxin reporters. Overall, we conclude disrupted cell wall biosynthesis perturbs auxin transport leading to altered auxin homeostasis impacting both anisotropic and isotropic growth that affects overall root morphology.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Parede Celular/metabolismo , Glucosiltransferases/metabolismo , Ácidos Indolacéticos/metabolismo , Alelos , Arabidopsis/enzimologia , Arabidopsis/genética , Benzamidas/farmacologia , Celulose/biossíntese , Genes de Plantas/genética , Glucosiltransferases/genética , Mutação/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo
17.
Science ; 364(6435)2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948526

RESUMO

Pathogen recognition by nucleotide-binding (NB), leucine-rich repeat (LRR) receptors (NLRs) plays roles in plant immunity. The Xanthomonas campestris pv. campestris effector AvrAC uridylylates the Arabidopsis PBL2 kinase, and the latter (PBL2UMP) acts as a ligand to activate the NLR ZAR1 precomplexed with the RKS1 pseudokinase. Here we report the cryo-electron microscopy structures of ZAR1-RKS1 and ZAR1-RKS1-PBL2UMP in an inactive and intermediate state, respectively. The ZAR1LRR domain, compared with animal NLRLRR domains, is differently positioned to sequester ZAR1 in an inactive state. Recognition of PBL2UMP is exclusively through RKS1, which interacts with ZAR1LRR PBL2UMP binding stabilizes the RKS1 activation segment, which sterically blocks ZAR1 adenosine diphosphate (ADP) binding. This engenders a more flexible NB domain without conformational changes in the other ZAR1 domains. Our study provides a structural template for understanding plant NLRs.


Assuntos
Difosfato de Adenosina/química , Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Arabidopsis/microbiologia , Proteínas de Transporte/química , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas NLR/química , Fosfoproteínas/química , Proteínas Serina-Treonina Quinases/química , Difosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Ligantes , Núcleosídeo-Fosfato Quinase/metabolismo , Domínios Proteicos , Proteínas Serina-Treonina Quinases/metabolismo , Xanthomonas campestris/enzimologia
18.
Science ; 364(6435)2019 04 05.
Artigo em Inglês | MEDLINE | ID: mdl-30948527

RESUMO

Nucleotide-binding, leucine-rich repeat receptors (NLRs) perceive pathogen effectors to trigger plant immunity. Biochemical mechanisms underlying plant NLR activation have until now remained poorly understood. We reconstituted an active complex containing the Arabidopsis coiled-coil NLR ZAR1, the pseudokinase RKS1, uridylated protein kinase PBL2, and 2'-deoxyadenosine 5'-triphosphate (dATP), demonstrating the oligomerization of the complex during immune activation. The cryo-electron microscopy structure reveals a wheel-like pentameric ZAR1 resistosome. Besides the nucleotide-binding domain, the coiled-coil domain of ZAR1 also contributes to resistosome pentamerization by forming an α-helical barrel that interacts with the leucine-rich repeat and winged-helix domains. Structural remodeling and fold switching during activation release the very N-terminal amphipathic α helix of ZAR1 to form a funnel-shaped structure that is required for the plasma membrane association, cell death triggering, and disease resistance, offering clues to the biochemical function of a plant resistosome.


Assuntos
Difosfato de Adenosina/química , Proteínas de Arabidopsis/química , Arabidopsis/imunologia , Arabidopsis/microbiologia , Proteínas de Transporte/química , Resistência à Doença , Interações Hospedeiro-Patógeno/imunologia , Peptídeos e Proteínas de Sinalização Intracelular/química , Proteínas NLR/química , Fosfoproteínas/química , Proteínas Serina-Treonina Quinases/química , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/metabolismo , Microscopia Crioeletrônica , Ligantes , Núcleosídeo-Fosfato Quinase/metabolismo , Domínios Proteicos , Estrutura Secundária de Proteína , Proteínas Serina-Treonina Quinases/metabolismo , Xanthomonas campestris/enzimologia
19.
Science ; 363(6433)2019 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-30898901

RESUMO

Physical damage to cells leads to the release of immunomodulatory peptides to elicit a wound defense response in the surrounding tissue. In Arabidopsis thaliana, the plant elicitor peptide 1 (Pep1) is processed from its protein precursor, PRECURSOR OF PEP1 (PROPEP1). We demonstrate that upon damage, both at the tissue and single-cell levels, the cysteine protease METACASPASE4 (MC4) is instantly and spatiotemporally activated by binding high levels of Ca2+ and is necessary and sufficient for Pep1 maturation. Cytosol-localized PROPEP1 and MC4 react only after loss of plasma membrane integrity and prolonged extracellular Ca2+ entry. Our results reveal that a robust mechanism consisting of conserved molecular components links the intracellular and Ca2+-dependent activation of a specific cysteine protease with the maturation of damage-induced wound defense signals.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Arabidopsis/imunologia , Cálcio/metabolismo , Cisteína Endopeptidases/metabolismo , Imunomodulação , Imunidade Vegetal , Precursores de Proteínas/metabolismo , Sequência de Aminoácidos , Citosol/enzimologia , Oligopeptídeos/metabolismo
20.
Proc Natl Acad Sci U S A ; 116(12): 5499-5504, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30842286

RESUMO

Fungi have the potential to produce a large repertoire of bioactive molecules, many of which can affect the growth and development of plants. Genomic survey of sequenced biofertilizer fungi showed many secondary metabolite gene clusters are anchored by iterative polyketide synthases (IPKSs), which are multidomain enzymes noted for generating diverse small molecules. Focusing on the biofertilizer Trichoderma harzianum t-22, we identified and characterized a cryptic IPKS-containing cluster that synthesizes tricholignan A, a redox-active ortho-hydroquinone. Tricholignan A is shown to reduce Fe(III) and may play a role in promoting plant growth under iron-deficient conditions. The construction of tricholignan by a pair of collaborating IPKSs was investigated using heterologous reconstitution and biochemical studies. A regioselective methylation step is shown to be a key step in formation of the ortho-hydroquinone. The responsible methyltransferase (MT) is fused with an N-terminal pseudo-acyl carrier protein (ψACP), in which the apo state of the ACP is essential for methylation of the growing polyketide chain. The ψACP is proposed to bind to the IPKS and enable the trans MT to access the growing polyketide. Our studies show that a genome-driven approach to discovering bioactive natural products from biofertilizer fungi can lead to unique compounds and biosynthetic knowledge.


Assuntos
Arabidopsis/metabolismo , Ferro/metabolismo , Policetídeos/metabolismo , Trichoderma/genética , Arabidopsis/enzimologia , Redes e Vias Metabólicas/genética , Metilação , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Família Multigênica/genética , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Trichoderma/enzimologia , Trichoderma/metabolismo
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