Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 4.134
Filtrar
1.
Acta Crystallogr D Struct Biol ; 76(Pt 4): 357-365, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254060

RESUMO

Starch is a key energy-storage molecule in plants that requires controlled synthesis and breakdown for effective plant growth. ß-Amylases (BAMs) hydrolyze starch into maltose to help to meet the metabolic needs of the plant. In the model plant Arabidopsis thaliana there are nine BAMs, which have apparently distinct functional and domain structures, although the functions of only a few of the BAMs are known and there are no 3D structures of BAMs from this organism. Recently, AtBAM2 was proposed to form a tetramer based on chromatography and activity assays of mutants; however, there was no direct observation of this tetramer. Here, small-angle X-ray scattering data were collected from AtBAM2 and its N-terminal truncations to describe the structure and assembly of the tetramer. Comparison of the scattering of the AtBAM2 tetramer with data collected from sweet potato (Ipomoea batatas) BAM5, which is also reported to form a tetramer, showed there were differences in the overall assembly. Analysis of the N-terminal truncations of AtBAM2 identified a loop sequence found only in BAM2 orthologs that appears to be critical for AtBAM2 tetramer assembly as well as for activity.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Proteínas Serina-Treonina Quinases/química , Amido/metabolismo , beta-Amilase/química , Sequência de Aminoácidos , Modelos Moleculares , Multimerização Proteica , Estrutura Quaternária de Proteína , Espalhamento de Radiação , Alinhamento de Sequência , Raios X
2.
Acta Crystallogr F Struct Biol Commun ; 76(Pt 4): 182-191, 2020 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-32254052

RESUMO

Casein kinase 2 (CK2) is a ubiquitous pleiotropic enzyme that is highly conserved across eukaryotic kingdoms. CK2 is singular amongst kinases as it is highly rigid and constitutively active. Arabidopsis thaliana is widely used as a model system in molecular plant research; the biological functions of A. thaliana CK2 are well studied in vivo and many of its substrates have been identified. Here, crystal structures of the α subunit of A. thaliana CK2 in three crystal forms and of its complex with the nonhydrolyzable ATP analog AMppNHp are presented. While the C-lobe of the enzyme is highly rigid, structural plasticity is observed for the N-lobe. Small but significant displacements within the active cleft are necessary in order to avoid steric clashes with the AMppNHp molecule. Binding of AMppNHp is influenced by a rigid-body motion of the N-lobe that was not previously recognized in maize CK2.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Caseína Quinase II/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Estrutura Terciária de Proteína
3.
Nature ; 578(7794): 317-320, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31996849

RESUMO

The ability to reverse protein aggregation is vital to cells1,2. Hsp100 disaggregases such as ClpB and Hsp104 are proposed to catalyse this reaction by translocating polypeptide loops through their central pore3,4. This model of disaggregation is appealing, as it could explain how polypeptides entangled within aggregates can be extracted and subsequently refolded with the assistance of Hsp704,5. However, the model is also controversial, as the necessary motor activity has not been identified6-8 and recent findings indicate non-processive mechanisms such as entropic pulling or Brownian ratcheting9,10. How loop formation would be accomplished is also obscure. Indeed, cryo-electron microscopy studies consistently show single polypeptide strands in the Hsp100 pore11,12. Here, by following individual ClpB-substrate complexes in real time, we unambiguously demonstrate processive translocation of looped polypeptides. We integrate optical tweezers with fluorescent-particle tracking to show that ClpB translocates both arms of the loop simultaneously and switches to single-arm translocation when encountering obstacles. ClpB is notably powerful and rapid; it exerts forces of more than 50 pN at speeds of more than 500 residues per second in bursts of up to 28 residues. Remarkably, substrates refold while exiting the pore, analogous to co-translational folding. Our findings have implications for protein-processing phenomena including ubiquitin-mediated remodelling by Cdc48 (or its mammalian orthologue p97)13 and degradation by the 26S proteasome14.


Assuntos
Endopeptidase Clp/metabolismo , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Peptídeos/química , Peptídeos/metabolismo , Agregados Proteicos , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Endopeptidase Clp/química , Proteínas de Escherichia coli/química , Fluorescência , Proteínas de Choque Térmico/química , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Pinças Ópticas , Complexo de Endopeptidases do Proteassoma/metabolismo , Multimerização Proteica , Redobramento de Proteína , Ubiquitina/metabolismo
4.
Nat Commun ; 11(1): 440, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31974402

RESUMO

p62/SQSTM1 is an autophagy receptor and signaling adaptor with an N-terminal PB1 domain that forms the scaffold of phase-separated p62 bodies in the cell. The molecular determinants that govern PB1 domain filament formation in vitro remain to be determined and the role of p62 filaments inside the cell is currently unclear. We here determine four high-resolution cryo-EM structures of different human and Arabidopsis PB1 domain assemblies and observed a filamentous ultrastructure of p62/SQSTM1 bodies using correlative cellular EM. We show that oligomerization or polymerization, driven by a double arginine finger in the PB1 domain, is a general requirement for lysosomal targeting of p62. Furthermore, the filamentous assembly state of p62 is required for autophagosomal processing of the p62-specific cargo KEAP1. Our results show that using such mechanisms, p62 filaments can be critical for cargo uptake in autophagy and are an integral part of phase-separated p62 bodies.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Transporte/química , Proteína Sequestossoma-1/química , Proteína Sequestossoma-1/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arginina/química , Autofagia/fisiologia , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Microscopia Crioeletrônica , Cristalografia por Raios X , Células HeLa , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Lisossomos/metabolismo , Polimerização , Conformação Proteica , Domínios Proteicos , Proteína Sequestossoma-1/genética
5.
BMC Genomics ; 20(Suppl 13): 932, 2019 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-31881842

RESUMO

Proteins play essential roles in almost all life processes. The prediction of protein function is of significance for the understanding of molecular function and evolution. Network alignment provides a fast and effective framework to automatically identify functionally conserved proteins in a systematic way. However, due to the fast growing genomic data, interactions and annotation data, there is an increasing demand for more accurate and efficient tools to deal with multiple PPI networks. Here, we present a novel global alignment algorithm NetCoffee2 based on graph feature vectors to discover functionally conserved proteins and predict function for unknown proteins. To test the algorithm performance, NetCoffee2 and three other notable algorithms were applied on eight real biological datasets. Functional analyses were performed to evaluate the biological quality of these alignments. Results show that NetCoffee2 is superior to existing algorithms IsoRankN, NetCoffee and multiMAGNA++ in terms of both coverage and consistency. The binary and source code are freely available under the GNU GPL v3 license at https://github.com/screamer/NetCoffee2.


Assuntos
Algoritmos , Proteínas/metabolismo , Animais , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Entropia , Humanos , Camundongos , Mapas de Interação de Proteínas , Proteínas/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo
6.
J Agric Food Chem ; 67(45): 12496-12501, 2019 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-31623438

RESUMO

Crocins are the primary coloring ingredients of saffron. The low-glycosylated members of this compound family, such as crocin-1 (crocetin mono-glucosyl ester) and crocin-2' (crocetin di-glucosyl ester), are rarely distributed in nature and attracting interest for their therapeutic uses. In the present study, a one-pot reaction system was used for efficient preparation of crocin-1 and crocin-2' with in situ regeneration of UDP-Glc by coupling Bs-GT with At-SuSy, a sucrose synthase from Arabidopsis thaliana. Noticeably, DMSO was used as a cosolvent and resulted in improvement of the solubility of the substrate crocetin and regulation of the selectivity of glycosylation. With periodic addition of crocetin, the biosynthesis of crocin-2' was performed with a high yield of 3.25 g/L in 2% DMSO aqueous solution, whereas crocin-1 (2.12 g/L) was selectively obtained in a 10% DMSO aqueous solution. The present study provided a simple approach for the biosynthesis of crocin-1 and crocin-2'.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Bacillus subtilis/enzimologia , Carotenoides/química , Crocus/química , Dimetil Sulfóxido/química , Glucosiltransferases/química , Glicosiltransferases/química , Biocatálise
7.
J Agric Food Chem ; 67(43): 12037-12043, 2019 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-31581772

RESUMO

Despite remarkable contribution of green fluorescent protein and its variants for better understanding of various biological functions, its application for anaerobic microorganisms has been limited because molecular oxygen is essential for chromophore formation. To overcome the limitation, we engineered a plant-derived light, oxygen, or voltage (LOV) domain containing flavin mononucleotide for enhanced spectral properties. The resulting LOV variants exhibited improved fluorescence intensity (20 and 70% higher for SH3 and 70% for BR1, respectively) compared to iLOV, an LOV variant isolated in a previous study, and the quantum yields of the LOV variants (0.40 for SH3 and 0.45 for BR1) were also improved relative to that of iLOV (Q = 0.37). In addition to fluorescence intensity, the identified mutations of SH3 enabled an improved thermostability of the protein. The engineered LOV variants with enhanced spectral properties could provide a valuable tool for fluorescent molecular probes under anaerobic conditions.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/química , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Mononucleotídeo de Flavina/metabolismo , Fluorescência , Luz , Domínios Proteicos , Estabilidade Proteica
8.
PLoS Pathog ; 15(9): e1008035, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31557268

RESUMO

Phytoplasmas are insect-transmitted bacterial pathogens that colonize a wide range of plant species, including vegetable and cereal crops, and herbaceous and woody ornamentals. Phytoplasma-infected plants often show dramatic symptoms, including proliferation of shoots (witch's brooms), changes in leaf shapes and production of green sterile flowers (phyllody). Aster Yellows phytoplasma Witches' Broom (AY-WB) infects dicots and its effector, secreted AYWB protein 11 (SAP11), was shown to be responsible for the induction of shoot proliferation and leaf shape changes of plants. SAP11 acts by destabilizing TEOSINTE BRANCHED 1-CYCLOIDEA-PROLIFERATING CELL FACTOR (TCP) transcription factors, particularly the class II TCPs of the CYCLOIDEA/TEOSINTE BRANCHED 1 (CYC/TB1) and CINCINNATA (CIN)-TCP clades. SAP11 homologs are also present in phytoplasmas that cause economic yield losses in monocot crops, such as maize, wheat and coconut. Here we show that a SAP11 homolog of Maize Bushy Stunt Phytoplasma (MBSP), which has a range primarily restricted to maize, destabilizes specifically TB1/CYC TCPs. SAP11MBSP and SAP11AYWB both induce axillary branching and SAP11AYWB also alters leaf development of Arabidopsis thaliana and maize. However, only in maize, SAP11MBSP prevents female inflorescence development, phenocopying maize tb1 lines, whereas SAP11AYWB prevents male inflorescence development and induces feminization of tassels. SAP11AYWB promotes fecundity of the AY-WB leafhopper vector on A. thaliana and modulates the expression of A. thaliana leaf defence response genes that are induced by this leafhopper, in contrast to SAP11MBSP. Neither of the SAP11 effectors promote fecundity of AY-WB and MBSP leafhopper vectors on maize. These data provide evidence that class II TCPs have overlapping but also distinct roles in regulating development and defence in a dicot and a monocot plant species that is likely to shape SAP11 effector evolution depending on the phytoplasma host range.


Assuntos
Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Phytoplasma/patogenicidade , Zea mays/microbiologia , Sequência de Aminoácidos , Animais , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Interações entre Hospedeiro e Microrganismos/genética , Interações entre Hospedeiro e Microrganismos/fisiologia , Especificidade de Hospedeiro , Insetos Vetores/microbiologia , Phytoplasma/genética , Phytoplasma/fisiologia , Doenças das Plantas/microbiologia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Domínios e Motivos de Interação entre Proteínas , Estabilidade Proteica , Homologia de Sequência de Aminoácidos , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Zea mays/crescimento & desenvolvimento , Zea mays/metabolismo
9.
Mol Cells ; 42(9): 646-660, 2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31480825

RESUMO

Abscisic acid (ABA) is a phytohormone essential for seed development and seedling growth under unfavorable environmental conditions. The signaling pathway leading to ABA response has been established, but relatively little is known about the functional regulation of the constituent signaling components. Here, we present several lines of evidence that Arabidopsis Raf-like kinase Raf10 modulates the core ABA signaling downstream of signal perception step. In particular, Raf10 phosphorylates subclass III SnRK2s (SnRK2.2, SnRK2.3, and SnRK2.6), which are key positive regulators, and our study focused on SnRK2.3 indicates that Raf10 enhances its kinase activity and may facilitate its release from negative regulators. Raf10 also phosphorylates transcription factors (ABI5, ABF2, and ABI3) critical for ABAregulted gene expression. Furthermore, Raf10 was found to be essential for the in vivo functions of SnRK2s and ABI5. Collectively, our data demonstrate that Raf10 is a novel regulatory component of core ABA signaling.


Assuntos
Ácido Abscísico/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Transdução de Sinais , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , MAP Quinase Quinase Quinases/química , Fenótipo , Fosforilação , Fosfosserina/metabolismo , Fosfotreonina/metabolismo , Multimerização Proteica
10.
Nat Commun ; 10(1): 4216, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31527679

RESUMO

CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) is a highly conserved E3 ubiquitin ligase from plants to animals and acts as a central repressor of photomorphogenesis in plants. SUPPRESSOR OF PHYA-105 1 family members (SPA1-SPA4) directly interact with COP1 and enhance COP1 activity. Despite the presence of a kinase domain at the N-terminus, no COP1-independent role of SPA proteins has been reported. Here we show that SPA1 acts as a serine/threonine kinase and directly phosphorylates PIF1 in vitro and in vivo. SPAs are necessary for the light-induced phosphorylation, ubiquitination and subsequent degradation of PIF1. Moreover, the red/far-red light photoreceptor phyB interacts with SPA1 through its C-terminus and enhances the recruitment of PIF1 for phosphorylation. These data provide a mechanistic view on how the COP1-SPA complexes serve as an example of a cognate kinase-E3 ligase complex that selectively triggers rapid phosphorylation and removal of its substrates, and how phyB modulates this process to promote photomorphogenesis.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Arabidopsis/efeitos da radiação , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Ciclo Celular/metabolismo , Fitocromo B/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Proteínas de Ciclo Celular/genética , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Luz , Fitocromo B/química , Fitocromo B/genética , Ligação Proteica/efeitos da radiação , Domínios Proteicos
11.
J Zhejiang Univ Sci B ; 20(9): 713-727, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31379142

RESUMO

Production of reactive oxygen species (ROS) is a conserved immune response primarily mediated by NADPH oxidases (NOXs), also known in plants as respiratory burst oxidase homologs (RBOHs). Most microbe-associated molecular patterns (MAMPs) trigger a very fast and transient ROS burst in plants. However, recently, we found that lipopolysaccharides (LPS), a typical bacterial MAMP, triggered a biphasic ROS burst. In this study, we isolated mutants defective in LPS-triggered biphasic ROS burst (delt) in Arabidopsis, and cloned the DELT1 gene that was shown to encode RBOHD. In the delt1-2 allele, the antepenultimate residue, glutamic acid (E919), at the C-terminus of RBOHD was mutated to lysine (K). E919 is a highly conserved residue in NADPH oxidases, and a mutation of the corresponding residue E568 in human NOX2 has been reported to be one of the causes of chronic granulomatous disease. Consistently, we found that residue E919 was indispensable for RBOHD function in the MAMP-induced ROS burst and stomatal closure. It has been suggested that the mutation of this residue in other NADPH oxidases impairs the protein's stability and complex assembly. However, we found that the E919K mutation did not affect RBOHD protein abundance or the ability of protein association, suggesting that the residue E919 in RBOHD might have a regulatory mechanism different from that of other NOXs. Taken together, our results confirm that the antepenultimate residue E is critical for NADPH oxidases and provide a new insight into the regulatory mechanisms of RBOHD.


Assuntos
Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , NADPH Oxidases/química , Agrobacterium tumefaciens/metabolismo , Alelos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Técnicas Genéticas , Humanos , Lipopolissacarídeos/metabolismo , Luminescência , Mutação , NADPH Oxidase 2/química , NADPH Oxidases/genética , Estômatos de Plantas/metabolismo , Domínios Proteicos , Espécies Reativas de Oxigênio/metabolismo , Tabaco/metabolismo
12.
Int J Mol Sci ; 20(15)2019 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-31382643

RESUMO

Plasma membrane (PM) lipid composition and domain organization are modulated by polarized exocytosis. Conversely, targeting of secretory vesicles at specific domains in the PM is carried out by exocyst complexes, which contain EXO70 subunits that play a significant role in the final recognition of the target membrane. As we have shown previously, a mature Arabidopsis trichome contains a basal domain with a thin cell wall and an apical domain with a thick secondary cell wall, which is developed in an EXO70H4-dependent manner. These domains are separated by a cell wall structure named the Ortmannian ring. Using phospholipid markers, we demonstrate that there are two distinct PM domains corresponding to these cell wall domains. The apical domain is enriched in phosphatidic acid (PA) and phosphatidylserine, with an undetectable amount of phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the basal domain is PIP2-rich. While the apical domain recruits EXO70H4, the basal domain recruits EXO70A1, which corresponds to the lipid-binding capacities of these two paralogs. Loss of EXO70H4 results in a loss of the Ortmannian ring border and decreased apical PA accumulation, which causes the PA and PIP2 domains to merge together. Using transmission electron microscopy, we describe these accumulations as a unique anatomical feature of the apical cell wall-radially distributed rod-shaped membranous pockets, where both EXO70H4 and lipid markers are immobilized.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Lipídeos de Membrana/genética , Proteínas de Transporte Vesicular/genética , Arabidopsis/química , Proteínas de Arabidopsis/química , Membrana Celular/química , Membrana Celular/genética , Exocitose/genética , Lipídeos de Membrana/metabolismo , Fosfatidilinositol 4,5-Difosfato/química , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilserinas/química , Fosfatidilserinas/genética , Tricomas/química , Tricomas/genética , Proteínas de Transporte Vesicular/química
13.
Mol Cell ; 76(1): 177-190.e5, 2019 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-31421981

RESUMO

The phytohormone auxin plays crucial roles in nearly every aspect of plant growth and development. The auxin response factor (ARF) transcription factor family regulates auxin-responsive gene expression and exhibits nuclear localization in regions of high auxin responsiveness. Here we show that the ARF7 and ARF19 proteins accumulate in micron-sized assemblies within the cytoplasm of tissues with attenuated auxin responsiveness. We found that the intrinsically disordered middle region and the folded PB1 interaction domain of ARFs drive protein assembly formation. Mutation of a single lysine within the PB1 domain abrogates cytoplasmic assemblies, promotes ARF nuclear localization, and results in an altered transcriptome and morphological defects. Our data suggest a model in which ARF nucleo-cytoplasmic partitioning regulates auxin responsiveness, providing a mechanism for cellular competence for auxin signaling.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Ácidos Indolacéticos/farmacologia , Proteínas Intrinsicamente Desordenadas/metabolismo , Reguladores de Crescimento de Planta/farmacologia , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Regulação da Expressão Gênica de Plantas , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Ligação Proteica , Dobramento de Proteína , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Fatores de Transcrição/química , Fatores de Transcrição/genética
14.
Cell Host Microbe ; 26(2): 193-201, 2019 08 14.
Artigo em Inglês | MEDLINE | ID: mdl-31415752

RESUMO

Plant innate immunity is triggered via direct or indirect recognition of pathogen effectors by the NLR family immune receptors. Mechanistic understanding of plant NLR function has relied on structural information from individual NLR domains and inferences from studies on animal NLRs. Recent reports of the cryo-EM structures of the Arabidopsis plant immune receptor ZAR1 in monomeric inactive and transition states, as well as the active oligomeric state or the "resistosome," have afforded a quantum leap in our understanding of how plant NLRs function. In this Review, we outline the recent structural findings and examine their implications for the activation of plant immune receptors more broadly. We also discuss how NLR signaling in plants, as illustrated by the ZAR1 structure, is analogous to innate immune receptor signaling mechanisms across kingdoms, drawing particular attention to the concept of signaling by cooperative assembly formation.


Assuntos
Proteínas de Arabidopsis , Proteínas de Transporte , Imunidade Vegetal/imunologia , Receptores Imunológicos , Transdução de Sinais , Difosfato de Adenosina , Trifosfato de Adenosina/metabolismo , Arabidopsis/imunologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/imunologia , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/imunologia , Proteínas de Transporte/metabolismo , Imunidade Inata , Proteínas NLR/química , Proteínas NLR/metabolismo , Imunidade Vegetal/fisiologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Domínios Proteicos , Receptores Imunológicos/química , Receptores Imunológicos/imunologia , Receptores Imunológicos/metabolismo
15.
Nucleic Acids Res ; 47(17): 9037-9052, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31372633

RESUMO

RNA-guided surveillance systems constrain the activity of transposable elements (TEs) in host genomes. In plants, RNA polymerase IV (Pol IV) transcribes TEs into primary transcripts from which RDR2 synthesizes double-stranded RNA precursors for small interfering RNAs (siRNAs) that guide TE methylation and silencing. How the core subunits of Pol IV, homologs of RNA polymerase II subunits, diverged to support siRNA biogenesis in a TE-rich, repressive chromatin context is not well understood. Here we studied the N-terminus of Pol IV's largest subunit, NRPD1. Arabidopsis lines harboring missense mutations in this N-terminus produce wild-type (WT) levels of NRPD1, which co-purifies with other Pol IV subunits and RDR2. Our in vitro transcription and genomic analyses reveal that the NRPD1 N-terminus is critical for robust Pol IV-dependent transcription, siRNA production and DNA methylation. However, residual RNA-directed DNA methylation observed in one mutant genotype indicates that Pol IV can operate uncoupled from the high siRNA levels typically observed in WT plants. This mutation disrupts a motif uniquely conserved in Pol IV, crippling the enzyme's ability to inhibit retrotransposon mobilization. We propose that the NRPD1 N-terminus motif evolved to regulate Pol IV function in genome surveillance.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , RNA Polimerases Dirigidas por DNA/genética , Regulação da Expressão Gênica de Plantas , Motivos de Aminoácidos , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Metilação de DNA/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/metabolismo , Inativação Gênica , Genoma de Planta , Plantas Geneticamente Modificadas , Domínios Proteicos/genética , RNA Replicase/metabolismo , RNA Interferente Pequeno/biossíntese , RNA Interferente Pequeno/genética , Retroelementos/genética
16.
Genes (Basel) ; 10(9)2019 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-31466344

RESUMO

The Cys2His2 (C2H2)-type zinc-finger protein (ZFP) family, which includes 176 members in Arabidopsis thaliana, is one of the largest families of putative transcription factors in plants. Of the Arabidopsis ZFP members, only 33 members are conserved in other eukaryotes, with 143 considered to be plant specific. C2H2-type ZFPs have been extensively studied and have been shown to play important roles in plant development and environmental stress responses by transcriptional regulation. The ethylene-responsive element binding-factor-associated amphiphilic repression (EAR) domain (GCC box) has been found to have a critical role in the tolerance response to abiotic stress. Many of the plant ZFPs containing the EAR domain, such as AZF1/2/3, ZAT7, ZAT10, and ZAT12, have been shown to function as transcriptional repressors. In this review, we mainly focus on the C1-2i subclass of C2H2 ZFPs and summarize the latest research into their roles in various stress responses. The role of C2H2-type ZFPs in response to the abiotic and biotic stress signaling network is not well explained, and amongst them, C1-2i is one of the better-characterized classifications in response to environmental stresses. These studies of the C1-2i subclass ought to furnish the basis for future studies to discover the pathways and receptors concerned in stress defense. Research has implied possible protein-protein interactions between members of C1-2i under various stresses, for which we have proposed a hypothetical model.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/genética , Fatores de Transcrição/fisiologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Estresse Fisiológico , Fatores de Transcrição/química , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Dedos de Zinco
17.
Phys Chem Chem Phys ; 21(34): 18727-18740, 2019 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-31424463

RESUMO

The plant stress protein COR15A stabilizes chloroplast membranes during freezing. COR15A is an intrinsically disordered protein (IDP) in aqueous solution, but acquires an α-helical structure during dehydration or the increase of solution osmolarity. We have used small- and wide-angle X-ray scattering (SAXS/WAXS) combined with static and dynamic light scattering (SLS/DLS) to investigate the structural and hydrodynamic properties of COR15A in response to increasing solution osmolarity. Coarse-grained ensemble modelling allowed a structure-based interpretation of the SAXS data. Our results demonstrate that COR15A behaves as a biomacromolecule with polymer-like properties which strongly depend on solution osmolarity. Biomacromolecular self-assembly occurring at high solvent osmolarity is initiated by the occurrence of two specific structural subpopulations of the COR15A monomer. The osmolarity dependent structural selection mechanism is an elegant way for conformational regulation and assembly of COR15A. It highlights the importance of the polymer-like properties of IDPs for their associated biological function.


Assuntos
Proteínas de Arabidopsis/química , Proteínas Intrinsicamente Desordenadas/química , Concentração Osmolar , Conformação Proteica , Espalhamento a Baixo Ângulo , Solventes/química , Raios X
18.
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
19.
EMBO J ; 38(18): e102140, 2019 09 16.
Artigo em Inglês | MEDLINE | ID: mdl-31304983

RESUMO

Plants sense different parts of the sun's light spectrum using distinct photoreceptors, which signal through the E3 ubiquitin ligase COP1. Here, we analyze why many COP1-interacting transcription factors and photoreceptors harbor sequence-divergent Val-Pro (VP) motifs that bind COP1 with different binding affinities. Crystal structures of the VP motifs of the UV-B photoreceptor UVR8 and the transcription factor HY5 in complex with COP1, quantitative binding assays, and reverse genetic experiments together suggest that UVR8 and HY5 compete for COP1. Photoactivation of UVR8 leads to high-affinity cooperative binding of its VP motif and its photosensing core to COP1, preventing COP1 binding to its substrate HY5. UVR8-VP motif chimeras suggest that UV-B signaling specificity resides in the UVR8 photoreceptor core. Different COP1-VP peptide motif complexes highlight sequence fingerprints required for COP1 targeting. The blue-light photoreceptors CRY1 and CRY2 also compete with transcription factors for COP1 binding using similar VP motifs. Thus, our work reveals that different photoreceptors and their signaling components compete for COP1 via a conserved mechanism to control different light signaling cascades.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Fotorreceptores de Plantas/química , Fotorreceptores de Plantas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Animais , Proteínas de Arabidopsis/química , Sítios de Ligação , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/metabolismo , Criptocromos/química , Criptocromos/metabolismo , Cristalografia por Raios X , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Células Sf9 , Transdução de Sinais , Ubiquitina-Proteína Ligases/química
20.
Nature ; 572(7768): 270-274, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31291642

RESUMO

Receptor kinases of the Catharanthus roseus RLK1-like (CrRLK1L) family have emerged as important regulators of plant reproduction, growth and responses to the environment1. Endogenous RAPID ALKALINIZATION FACTOR (RALF) peptides2 have previously been proposed as ligands for several members of the CrRLK1L family1. However, the mechanistic basis of this perception is unknown. Here we report that RALF23 induces a complex between the CrRLK1L FERONIA (FER) and LORELEI (LRE)-LIKE GLYCOSYLPHOSPHATIDYLINOSITOL (GPI)-ANCHORED PROTEIN 1 (LLG1) to regulate immune signalling. Structural and biochemical data indicate that LLG1 (which is genetically important for RALF23 responses) and the related LLG2 directly bind RALF23 to nucleate the assembly of RALF23-LLG1-FER and RALF23-LLG2-FER heterocomplexes, respectively. A conserved N-terminal region of RALF23 is sufficient for the biochemical recognition of RALF23 by LLG1, LLG2 or LLG3, and binding assays suggest that other RALF peptides that share this conserved N-terminal region may be perceived by LLG proteins in a similar manner. Structural data also show that RALF23 recognition is governed by the conformationally flexible C-terminal sides of LLG1, LLG2 and LLG3. Our work reveals a mechanism of peptide perception in plants by GPI-anchored proteins that act together with a phylogenetically unrelated receptor kinase. This provides a molecular framework for understanding how diverse RALF peptides may regulate multiple processes, through perception by distinct heterocomplexes of CrRLK1L receptor kinases and GPI-anchored proteins of the LRE and LLG family.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Arabidopsis/metabolismo , Proteínas Ligadas por GPI/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/química , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fragmentos de Peptídeos/metabolismo , Fosfotransferases/metabolismo , Proteínas de Arabidopsis/genética , Peptídeos e Proteínas de Sinalização Intercelular/genética , Modelos Moleculares , Mutagênese , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fosfotransferases/genética , Maleabilidade , Ligação Proteica/genética , Conformação Proteica , Multimerização Proteica
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA