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1.
Nature ; 598(7881): 495-499, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34497423

RESUMO

Plants deploy cell-surface and intracellular leucine rich-repeat domain (LRR) immune receptors to detect pathogens1. LRR receptor kinases and LRR receptor proteins at the plasma membrane recognize microorganism-derived molecules to elicit pattern-triggered immunity (PTI), whereas nucleotide-binding LRR proteins detect microbial effectors inside cells to confer effector-triggered immunity (ETI). Although PTI and ETI are initiated in different host cell compartments, they rely on the transcriptional activation of similar sets of genes2, suggesting pathway convergence upstream of nuclear events. Here we report that PTI triggered by the Arabidopsis LRR receptor protein RLP23 requires signalling-competent dimers of the lipase-like proteins EDS1 and PAD4, and of ADR1 family helper nucleotide-binding LRRs, which are all components of ETI. The cell-surface LRR receptor kinase SOBIR1 links RLP23 with EDS1, PAD4 and ADR1 proteins, suggesting the formation of supramolecular complexes containing PTI receptors and transducers at the inner side of the plasma membrane. We detected similar evolutionary patterns in LRR receptor protein and nucleotide-binding LRR genes across Arabidopsis accessions; overall higher levels of variation in LRR receptor proteins than in LRR receptor kinases are consistent with distinct roles of these two receptor families in plant immunity. We propose that the EDS1-PAD4-ADR1 node is a convergence point for defence signalling cascades, activated by both surface-resident and intracellular LRR receptors, in conferring pathogen immunity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/imunologia , Hidrolases de Éster Carboxílico/metabolismo , Proteínas de Ligação a DNA/metabolismo , Imunidade Vegetal , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Arabidopsis/química , Hidrolases de Éster Carboxílico/química , Proteínas de Ligação a DNA/química , Domínios Proteicos , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo
2.
Genes Dev ; 32(19-20): 1332-1343, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30254107

RESUMO

Plants have evolved complex photoreceptor-controlled mechanisms to sense and respond to seasonal changes in day length. This ability allows plants to optimally time the transition from vegetative growth to flowering. UV-B is an important part intrinsic to sunlight; however, whether and how it affects photoperiodic flowering has remained elusive. Here, we report that, in the presence of UV-B, genetic mutation of REPRESSOR OF UV-B PHOTOMORPHOGENESIS 2 (RUP2) renders the facultative long day plant Arabidopsis thaliana a day-neutral plant and that this phenotype is dependent on the UV RESISTANCE LOCUS 8 (UVR8) UV-B photoreceptor. We provide evidence that the floral repression activity of RUP2 involves direct interaction with CONSTANS, repression of this key activator of flowering, and suppression of FLOWERING LOCUS T transcription. RUP2 therefore functions as an essential repressor of UVR8-mediated induction of flowering under noninductive short day conditions and thus provides a crucial mechanism of photoperiodic flowering control.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Proteínas Cromossômicas não Histona/metabolismo , Flores/crescimento & desenvolvimento , Fotoperíodo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Ligação a DNA/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Raios Ultravioleta
3.
BMC Bioinformatics ; 23(1): 61, 2022 Feb 07.
Artigo em Inglês | MEDLINE | ID: mdl-35130839

RESUMO

BACKGROUND: As technical developments in omics and biomedical imaging increase the throughput of data generation in life sciences, the need for information systems capable of managing heterogeneous digital assets is increasing. In particular, systems supporting the findability, accessibility, interoperability, and reusability (FAIR) principles of scientific data management. RESULTS: We propose a Service Oriented Architecture approach for integrated management and analysis of multi-omics and biomedical imaging data. Our architecture introduces an image management system into a FAIR-supporting, web-based platform for omics data management. Interoperable metadata models and middleware components implement the required data management operations. The resulting architecture allows for FAIR management of omics and imaging data, facilitating metadata queries from software applications. The applicability of the proposed architecture is demonstrated using two technical proofs of concept and a use case, aimed at molecular plant biology and clinical liver cancer research, which integrate various imaging and omics modalities. CONCLUSIONS: We describe a data management architecture for integrated, FAIR-supporting management of omics and biomedical imaging data, and exemplify its applicability for basic biology research and clinical studies. We anticipate that FAIR data management systems for multi-modal data repositories will play a pivotal role in data-driven research, including studies which leverage advanced machine learning methods, as the joint analysis of omics and imaging data, in conjunction with phenotypic metadata, becomes not only desirable but necessary to derive novel insights into biological processes.


Assuntos
Disciplinas das Ciências Biológicas , Gerenciamento de Dados , Gestão da Informação , Metadados , Software
4.
Int J Mol Sci ; 23(11)2022 Jun 03.
Artigo em Inglês | MEDLINE | ID: mdl-35682970

RESUMO

The transition to terrestrial plants was accompanied by a progressive loss of microtubule minus-end-directed dynein motors. Instead, the minus-end-directed class-XIV kinesins expanded considerably, likely related to novel functions. One of these motors, OsDLK (Dual Localisation Kinesin from rice), decorates cortical microtubules but moves into the nucleus in response to cold stress. This analysis of loss-of-function mutants in rice indicates that OsDLK participates in cell elongation during development. Since OsDLK harbours both a nuclear localisation signal and a putative leucin zipper, we asked whether the cold-induced import of OsDLK into the nucleus might correlate with specific DNA binding. Conducting a DPI-ELISA screen with recombinant OsDLKT (lacking the motor domain), we identified the Opaque2 motif as the most promising candidate. This motif is present in the promoter of NtAvr9/Cf9, the tobacco homologue of Cold-Box Factor 4, a transcription factor involved in cold adaptation. A comparative study revealed that the cold-induced accumulation of NtAvr9/Cfp9 was specifically quelled in transgenic BY-2 cells overexpressing OsDLK-GFP. These findings are discussed as a working model, where, in response to cold stress, OsDLK partitions from cortical microtubules at the plasma membrane into the nucleus and specifically modulates the expression of genes involved in cold adaptation.


Assuntos
Cinesinas , Oryza , Núcleo Celular/metabolismo , Dineínas/metabolismo , Cinesinas/genética , Microtúbulos/metabolismo , Oryza/genética , Oryza/metabolismo
5.
New Phytol ; 229(2): 979-993, 2021 01.
Artigo em Inglês | MEDLINE | ID: mdl-33070379

RESUMO

Cytokinin and auxin are key regulators of plant growth and development. During the last decade transport mechanisms have turned out to be the key for the control of local and long-distance hormone distributions. In contrast with auxin, cytokinin transport is poorly understood. Here, we show that Arabidopsis thaliana AZG2, a member of the AZG purine transporter family, acts as cytokinin transporter involved in root system architecture determination. Even though purines are substrates for both AZG1 and AZG2, we found distinct transport mechanisms. The expression of AZG2 is restricted to a small group of cells surrounding the lateral root (LR) primordia and induced by auxins. Compared to the wild-type (WT), mutants carrying loss-of-function alleles of AZG2 have higher LR density, suggesting that AZG2 is part of a regulatory pathway in LR emergence. Moreover, azg2 is partially insensitive to exogenous cytokinin, which is consistent with the observation that the cytokinin reporter TCSnpro :GFP showed lower fluorescence signal in the roots of azg2 compared to the WT. These results indicate a defective cytokinin signalling pathway in the region of LR primordia. The integration of AZG2 subcellular localization and cytokinin transport capacity data allowed us to propose a local cytokinin : auxin signalling model for the regulation of LR emergence.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Citocininas/metabolismo , Raízes de Plantas/crescimento & desenvolvimento , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ácidos Indolacéticos
6.
Plant Physiol ; 182(1): 669-678, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31641077

RESUMO

Plants depend on various cell surface receptors to integrate extracellular signals with developmental programs. One of the best-studied receptors is BRASSINOSTEROID INSENSITIVE 1 (BRI1) in Arabidopsis (Arabidopsis thaliana). Upon binding of its hormone ligands, BRI1 forms a complex with a shape-complementary coreceptor and initiates a signal transduction cascade, which leads to a variety of responses. At the macroscopic level, brassinosteroid (BR) biosynthetic and receptor mutants have similar growth defects, which initially led to the assumption that the signaling pathways were largely linear. However, recent evidence suggests that BR signaling is interconnected with several other pathways through various mechanisms. We recently described that feedback from the cell wall is integrated at the level of the receptor complex through interaction with RECEPTOR-LIKE PROTEIN 44 (RLP44). Moreover, BRI1 is required for another function of RLP44: the control of procambial cell fate. Here, we report a BRI1 mutant, bri1 cnu4 , which differentially affects canonical BR signaling and RLP44 function in the vasculature. Although BR signaling is only mildly impaired, bri1 cnu4 mutants show ectopic xylem in place of procambium. Mechanistically, this is explained by an increased association between RLP44 and the mutated BRI1 protein, which prevents the former from acting in vascular cell fate maintenance. Consistent with this, the mild BR response phenotype of bri1 cnu4 is a recessive trait, whereas the RLP44-mediated xylem phenotype is semidominant. Our results highlight the complexity of plant plasma membrane receptor function and provide a tool to dissect BR signaling-related roles of BRI1 from its noncanonical functions.


Assuntos
Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Quinases/metabolismo , Alelos , Proteínas de Arabidopsis/genética , Plantas Geneticamente Modificadas/genética , Proteínas Quinases/genética , Transdução de Sinais
7.
Proc Natl Acad Sci U S A ; 115(46): 11838-11843, 2018 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-30377268

RESUMO

Multicellularity arose independently in plants and animals, but invariably requires a robust determination and maintenance of cell fate that is adaptive to the environment. This is exemplified by the highly specialized water- and nutrient-conducting cells of the plant vasculature, the organization of which is already prepatterned close to the stem-cell niche, but can be modified according to extrinsic cues. Here, we show that the hormone receptor BRASSINOSTEROID INSENSITIVE 1 (BRI1) is required for root vascular cell-fate maintenance, as BRI1 mutants show ectopic xylem in procambial position. However, this phenotype seems unrelated to canonical brassinosteroid signaling outputs. Instead, BRI1 is required for the expression and function of its interacting partner RECEPTOR-LIKE PROTEIN 44 (RLP44), which, in turn, associates with the receptor for the peptide hormone phytosulfokine (PSK). We show that PSK signaling is required for the maintenance of procambial cell identity and quantitatively controlled by RLP44, which promotes complex formation between the PSK receptor and its coreceptor. Mimicking the loss of RLP44, PSK-related mutants show ectopic xylem in the position of the procambium, whereas rlp44 is rescued by exogenous PSK. Based on these findings, we propose that RLP44 controls cell fate by connecting BRI1 and PSK signaling, providing a mechanistic framework for the dynamic balancing of signaling mediated by the plethora of plant receptor-like kinases at the plasma membrane.


Assuntos
Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Proteínas Quinases/metabolismo , Proteínas Quinases/fisiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Diferenciação Celular/fisiologia , Hormônios Peptídicos/metabolismo , Fosforilação , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Transdução de Sinais/fisiologia
8.
J Integr Plant Biol ; 62(4): 456-469, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30912278

RESUMO

Brassinosteroids (BR) are involved in the control of several developmental processes ranging from root elongation to senescence and adaptation to environmental cues. Thus, BR perception and signaling have to be precisely regulated. One regulator is BRI1-associated kinase 1 (BAK1)-interacting receptor-like kinase 3 (BIR3). In the absence of BR, BIR3 forms complexes with BR insensitive 1 (BRI1) and BAK1. However, the biophysical and energetic requirements for complex formation in the absence of the ligand have yet to be determined. Using computational modeling, we simulated the potential complexes between the cytoplasmic domains of BAK1, BRI1 and BIR3. Our calculations and experimental data confirm the interaction of BIR3 with BAK1 and BRI1, with the BAK1 BIR3 interaction clearly favored. Furthermore, we demonstrate that BIR3 and BRI1 share the same interaction site with BAK1. This suggests a competition between BIR3 and BRI1 for binding to BAK1, which results in preferential binding of BIR3 to BAK1 in the absence of the ligand thereby preventing the active participation of BAK1 in BR signaling. Our model also suggests that BAK1 and BRI1 can interact even while BAK1 is in complex with BIR3 at an additional binding site of BAK1 that does not allow active BR signaling.


Assuntos
Proteínas de Arabidopsis/metabolismo , Brassinosteroides/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas de Arabidopsis/química , Domínio Catalítico , Proteínas de Membrana/química , Simulação de Acoplamento Molecular , Ligação Proteica , Proteínas Quinases/química , Proteínas Quinases/metabolismo , Multimerização Proteica , Proteínas Serina-Treonina Quinases/química
9.
Plant J ; 95(3): 458-473, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29763523

RESUMO

Cytokinin plays diverse roles in plant growth and development, generally acting by modulating gene transcription in target tissues. The type-B Arabidopsis response regulators (ARR) transcription factors have emerged as primary targets of cytokinin signaling and are required for essentially all cytokinin-mediated changes in gene expression. The diversity of cytokinin function is likely imparted by the activity of various transcription factors working with the type-B ARRs to alter specific sets of target genes. One potential set of co-regulators modulating the cytokinin response are the BARLEY B-RECOMBINANT/BASIC PENTACYSTEINE (BBR/BPC) family of plant-specific transcription factors. Here, we show that disruption of multiple BPCs results in reduced sensitivity to cytokinin. Further, the BPCs are necessary for the induction of a subset of genes in response to cytokinin. We identified direct in vivo targets of BPC6 using ChIP-Seq and found an enrichment of promoters of genes differentially expressed in response to cytokinin. Further, a significant number of BPC6 regulated genes are also direct targets of the type-B ARRs. Potential cis-binding elements for a number of other transcription factors linked to cytokinin action are enriched in the BPC binding fragments, including those for the cytokinin response factors (CRFs). In addition, several BPCs interact with a subset of type-A ARRs. Consistent with these results, a significant number of genes whose expression is altered in bpc mutant roots are also mis-expressed in crf1,3,5,6 and type-A arr3,4,5,6,7,8,9,15 mutant roots. These results suggest that the BPCs are part of a complex network of transcription factors that are involved in the response to cytokinin.


Assuntos
Proteínas de Arabidopsis/metabolismo , Citocininas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Genes de Plantas/genética , Glucosiltransferases/metabolismo
10.
BMC Plant Biol ; 18(1): 311, 2018 Nov 29.
Artigo em Inglês | MEDLINE | ID: mdl-30497415

RESUMO

BACKGROUND: Productivity of important crop rice is greatly affected by salinity. The plant hormone jasmonate plays a vital role in salt stress adaptation, but also evokes detrimental side effects if not timely shut down again. As novel strategy to avoid such side effects, OsJAZ8, a negative regulator of jasmonate signalling, is expressed under control of the salt-inducible promoter of the transcription factor ZOS3-11, to obtain a transient jasmonate signature in response to salt stress. To modulate the time course of jasmonate signalling, either a full-length or a dominant negative C-terminally truncated version of OsJAZ8 driven by the ZOS3-11 promoter were expressed in a stable manner either in tobacco BY-2 cells, or in japonica rice. RESULTS: The transgenic tobacco cells showed reduced mortality and efficient cycling under salt stress adaptation. This was accompanied by reduced sensitivity to Methyl jasmonate and increased responsiveness to auxin. In the case of transgenic rice, the steady-state levels of OsJAZ8 transcripts were more efficiently induced under salt stress compared to the wild type, this induction was more pronounced in the dominant-negative OsJAZ8 variant. CONCLUSIONS: The result concluded that, more efficient activation of OsJAZ8 was accompanied by improved salt tolerance of the transgenic seedlings and demonstrates the impact of temporal signatures of jasmonate signalling for stress tolerance.


Assuntos
Proteínas Correpressoras/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Plantas Tolerantes a Sal/metabolismo , Proteínas Correpressoras/genética , Proteínas Correpressoras/fisiologia , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/genética , Oxilipinas/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Plantas Geneticamente Modificadas/genética , Estresse Salino , Plantas Tolerantes a Sal/genética , Transdução de Sinais , Nicotiana/genética
11.
Plant Cell ; 27(6): 1718-29, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-26071421

RESUMO

Phytosulfokine (PSK) is perceived by the leucine-rich repeat receptor kinase PSKR1 and promotes growth in Arabidopsis thaliana. PSKR1 is coexpressed with the CYCLIC NUCLEOTIDE-GATED CHANNEL gene CNGC17. PSK promotes protoplast expansion in the wild type but not in cngc17. Protoplast expansion is likewise promoted by cGMP in a CNGC17-dependent manner. Furthermore, PSKR1-deficient protoplasts do not expand in response to PSK but are still responsive to cGMP, suggesting that cGMP acts downstream of PSKR1. Mutating the guanylate cyclase center of PSKR1 impairs seedling growth, supporting a role for PSKR1 signaling via cGMP in planta. While PSKR1 does not interact directly with CNGC17, it interacts with the plasma membrane-localized H(+)-ATPases AHA1 and AHA2 and with the BRI-associated receptor kinase 1 (BAK1). CNGC17 likewise interacts with AHA1, AHA2, and BAK1, suggesting that PSKR1, BAK1, CNGC17, and AHA assemble in a functional complex. Roots of deetiolated bak1-3 and bak1-4 seedlings were unresponsive to PSK, and bak1-3 and bak1-4 protoplasts expanded less in response to PSK but were fully responsive to cGMP, indicating that BAK1 acts in the PSK signal pathway upstream of cGMP. We hypothesize that CNGC17 and AHAs form a functional cation-translocating unit that is activated by PSKR1/BAK1 and possibly other BAK1/RLK complexes.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/crescimento & desenvolvimento , Canais de Cátion Regulados por Nucleotídeos Cíclicos/fisiologia , Hormônios Peptídicos/fisiologia , Reguladores de Crescimento de Plantas/fisiologia , Proteínas de Plantas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , ATPases Translocadoras de Prótons/fisiologia , Arabidopsis/fisiologia , Membrana Celular/fisiologia , Regulação da Expressão Gênica de Plantas/fisiologia , Receptores de Superfície Celular/fisiologia , Plântula/crescimento & desenvolvimento
12.
Development ; 141(24): 4831-40, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25411212

RESUMO

In multicellular organisms, cellular differences in gene activity are a prerequisite for differentiation and establishment of cell types. In order to study transcriptome profiles, specific cell types have to be isolated from a given tissue or even the whole organism. However, whole-transcriptome analysis of early embryos in flowering plants has been hampered by their size and inaccessibility. Here, we describe the purification of nuclear RNA from early stage Arabidopsis thaliana embryos using fluorescence-activated nuclear sorting (FANS) to generate expression profiles of early stages of the whole embryo, the proembryo and the suspensor. We validated our datasets of differentially expressed candidate genes by promoter-reporter gene fusions and in situ hybridization. Our study revealed that different classes of genes with respect to biological processes and molecular functions are preferentially expressed either in the proembryo or in the suspensor. This method can be used especially for tissues with a limited cell population and inaccessible tissue types. Furthermore, we provide a valuable resource for research on Arabidopsis early embryogenesis.


Assuntos
Arabidopsis/embriologia , Núcleo Celular/química , Perfilação da Expressão Gênica/métodos , RNA Nuclear/isolamento & purificação , Sementes/metabolismo , Arabidopsis/metabolismo , Clonagem Molecular , Genótipo , Hibridização In Situ , Análise em Microsséries , Microscopia de Fluorescência , Reação em Cadeia da Polimerase em Tempo Real
13.
Proc Natl Acad Sci U S A ; 111(42): 15261-6, 2014 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-25288746

RESUMO

The brassinosteroid (BR) signaling module is a central regulator of plant morphogenesis, as indicated by the large number of BR-responsive cell wall-related genes and the severe growth defects of BR mutants. Despite a detailed knowledge of the signaling components, the logic of this auto-/paracrine signaling module in growth control remains poorly understood. Recently, extensive cross-talk with other signaling pathways has been shown, suggesting that the outputs of BR signaling, such as gene-expression changes, are subject to complex control mechanisms. We previously provided evidence for a role of BR signaling in a feedback loop controlling the integrity of the cell wall. Here, we identify the first dedicated component of this feedback loop: a receptor-like protein (RLP44), which is essential for the compensatory triggering of BR signaling upon inhibition of pectin de-methylesterification in the cell wall. RLP44 is required for normal growth and stress responses and connects with the BR signaling pathway, presumably through a direct interaction with the regulatory receptor-like kinase BAK1. These findings corroborate a role for BR in controlling the sensitivity of a feedback signaling module involved in maintaining the physico-chemical homeostasis of the cell wall during cell expansion.


Assuntos
Brassinosteroides/química , Pectinas/química , Proteínas de Plantas/fisiologia , Proteínas de Arabidopsis/fisiologia , Parede Celular/metabolismo , Clonagem Molecular , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Homeostase , Ligantes , Microscopia Confocal , Mutação , Fenótipo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Serina-Treonina Quinases/fisiologia , Transdução de Sinais
14.
Plant Physiol ; 168(3): 776-87, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-25971551

RESUMO

Fluorescence-based protein-protein interaction techniques are vital tools for understanding in vivo cellular functions on a mechanistic level. However, only under the condition of highly efficient (co)transformation and accumulation can techniques such as Förster resonance energy transfer (FRET) realize their potential for providing highly accurate and quantitative interaction data. FRET as a fluorescence-based method unifies several advantages, such as measuring in an in vivo environment, real-time context, and the ability to include transient interactions as well as detecting the mere proximity of proteins. Here, we introduce a novel vector set that incorporates the benefit of the recombination-based 2in1 cloning system with the latest state-of-the-art fluorescent proteins for optimal coaccumulation and FRET output studies. We demonstrate its utility across a range of methods. Merging the 2in1 cloning system with new-generation FRET fluorophore pairs allows for enhanced detection, speeds up the preparation of clones, and enables colocalization studies and the identification of meaningful protein-protein interactions in vivo.


Assuntos
Arabidopsis/genética , Vetores Genéticos/metabolismo , Nicotiana/genética , Proteínas de Plantas/metabolismo , Mapeamento de Interação de Proteínas , Clonagem Molecular , DNA Bacteriano/genética , Transferência Ressonante de Energia de Fluorescência , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Microscopia de Fluorescência , Fotodegradação , Plantas Geneticamente Modificadas , Plasmídeos/genética , Transformação Genética
15.
Plant Physiol ; 168(3): 1013-24, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26025051

RESUMO

Polycomb-repressive complexes (PRCs) play key roles in development by repressing a large number of genes involved in various functions. Much, however, remains to be discovered about PRC-silencing mechanisms as well as their targeting to specific genomic regions. Besides other mechanisms, GAGA-binding factors in animals can guide PRC members in a sequence-specific manner to Polycomb-responsive DNA elements. Here, we show that the Arabidopsis (Arabidopsis thaliana) GAGA-motif binding factor protein basic pentacysteine6 (BPC6) interacts with like heterochromatin protein1 (LHP1), a PRC1 component, and associates with vernalization2 (VRN2), a PRC2 component, in vivo. By using a modified DNA-protein interaction enzyme-linked immunosorbant assay, we could show that BPC6 was required and sufficient to recruit LHP1 to GAGA motif-containing DNA probes in vitro. We also found that LHP1 interacts with VRN2 and, therefore, can function as a possible scaffold between BPC6 and VRN2. The lhp1-4 bpc4 bpc6 triple mutant displayed a pleiotropic phenotype, extreme dwarfism and early flowering, which disclosed synergistic functions of LHP1 and group II plant BPC members. Transcriptome analyses supported this synergy and suggested a possible function in the concerted repression of homeotic genes, probably through histone H3 lysine-27 trimethylation. Hence, our findings suggest striking similarities between animal and plant GAGA-binding factors in the recruitment of PRC1 and PRC2 components to Polycomb-responsive DNA element-like GAGA motifs, which must have evolved through convergent evolution.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , Motivos de Nucleotídeos/genética , Proteínas do Grupo Polycomb/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Transporte/metabolismo , Proteínas Cromossômicas não Histona/química , Regulação da Expressão Gênica de Plantas , Loci Gênicos , Pleiotropia Genética , Histonas/metabolismo , Lisina/metabolismo , Metilação , Modelos Biológicos , Mutação/genética , Proteínas Nucleares/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína
16.
Plant Cell ; 24(6): 2610-23, 2012 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-22739826

RESUMO

The UV-A/blue light photoreceptor crytochrome2 (cry2) plays a fundamental role in the transition from the vegetative to the reproductive phase in the facultative long-day plant Arabidopsis thaliana. The cry2 protein level strongly decreases when etiolated seedlings are exposed to blue light; cry2 is first phosphorylated, polyubiquitinated, and then degraded by the 26S proteasome. COP1 is involved in cry2 degradation, but several cop1 mutants show only reduced but not abolished cry2 degradation. SUPPRESSOR OF PHYA-105 (SPA) proteins are known to work in concert with COP1, and recently direct physical interaction between cry2 and SPA1 was demonstrated. Thus, we hypothesized that SPA proteins could also play a role in cry2 degradation. To this end, we analyzed cry2 protein levels in spa mutants. In all spa mutants analyzed, cry2 degradation under continuous blue light was alleviated in a fluence rate-dependent manner. Consistent with a role of SPA proteins in phytochrome A (phyA) signaling, a phyA mutant had enhanced cry2 levels, particularly under low fluence rate blue light. Fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy studies showed a robust physical interaction of cry2 with SPA1 in nuclei of living cells. Our results suggest that cry2 stability is controlled by SPA and phyA, thus providing more information on the molecular mechanisms of interaction between cryptochrome and phytochrome photoreceptors.


Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ciclo Celular/metabolismo , Criptocromos/metabolismo , Fitocromo A/metabolismo , Proteínas Serina-Treonina Quinases/genética , Arabidopsis/genética , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência/métodos , Mutação , Fitocromo A/genética , Proteínas Serina-Treonina Quinases/metabolismo
17.
Nucleic Acids Res ; 41(21): 9764-78, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23975197

RESUMO

WRKY transcription factors constitute a large protein family in plants that is involved in the regulation of developmental processes and responses to biotic or abiotic stimuli. The question arises how stimulus-specific responses are mediated given that the highly conserved WRKY DNA-binding domain (DBD) exclusively recognizes the 'TTGACY' W-box consensus. We speculated that the W-box consensus might be more degenerate and yet undetected differences in the W-box consensus of WRKYs of different evolutionary descent exist. The phylogenetic analysis of WRKY DBDs suggests that they evolved from an ancestral group IIc-like WRKY early in the eukaryote lineage. A direct descent of group IIc WRKYs supports a monophyletic origin of all other group II and III WRKYs from group I by loss of an N-terminal DBD. Group I WRKYs are of paraphyletic descent and evolved multiple times independently. By homology modeling, molecular dynamics simulations and in vitro DNA-protein interaction-enzyme-linked immunosorbent assay with AtWRKY50 (IIc), AtWRKY33 (I) and AtWRKY11 (IId) DBDs, we revealed differences in DNA-binding specificities. Our data imply that other components are essentially required besides the W-box-specific binding to DNA to facilitate a stimulus-specific WRKY function.


Assuntos
Proteínas de Arabidopsis/química , Proteínas de Ligação a DNA/química , Fatores de Transcrição/química , Sequência de Aminoácidos , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/metabolismo , Proteínas de Ligação a DNA/classificação , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Simulação de Dinâmica Molecular , Dados de Sequência Molecular , Filogenia , Estrutura Terciária de Proteína , Homologia Estrutural de Proteína , Fatores de Transcrição/classificação , Fatores de Transcrição/metabolismo
18.
Plant Mol Biol ; 85(3): 301-16, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24729002

RESUMO

Rice is one of the most important staple foods worldwide, but it often contains inorganic arsenic, which is toxic and gives rise to severe health problems. Rice plants take up arsenate As(V) via the phosphate transport pathways, though it is not known how As(V), as compared to phosphate, modifies the expression of phosphate transporters (PTs). Therefore, the impact of As(V) or phosphate (Pi) on the gene expression of PTs and several Pi signaling regulators was investigated. Rice plants were grown on medium containing different As(V) or Pi concentrations. Growth was evaluated and the expression of tested genes was quantified at different time points, using quantitative RT-PCR (qPCR). The As and P content in plants was determined using inductively coupled plasma mass spectrometry (ICP-MS). As(V) elicited diverse and opposite responses of different PTs in roots and shoots, while Pi triggered a more shallow and uniform transcriptional response in several tested genes. Only a restricted set of genes, including PT2, PT3, PT5 and PT13 and two SPX-MFS family members, was particularly responsive to As(V). Despite some common reactions, the responses of the analyzed genes were predominantly ion-specific. The possible reasons and consequences are discussed.


Assuntos
Arseniatos/farmacologia , Oryza/metabolismo , Proteínas de Transporte de Fosfato/metabolismo , Fosfatos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Oryza/efeitos dos fármacos , Oryza/genética , Proteínas de Transporte de Fosfato/genética , Brotos de Planta/genética , Brotos de Planta/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Plântula/crescimento & desenvolvimento , Sementes , Transdução de Sinais
19.
Plant Cell ; 23(1): 381-95, 2011 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-21278122

RESUMO

Control of energy homeostasis is crucial for plant survival, particularly under biotic or abiotic stress conditions. Energy deprivation induces dramatic reprogramming of transcription, facilitating metabolic adjustment. An in-depth knowledge of the corresponding regulatory networks would provide opportunities for the development of biotechnological strategies. Low energy stress activates the Arabidopsis thaliana group S1 basic leucine zipper transcription factors bZIP1 and bZIP53 by transcriptional and posttranscriptional mechanisms. Gain-of-function approaches define these bZIPs as crucial transcriptional regulators in Pro, Asn, and branched-chain amino acid metabolism. Whereas chromatin immunoprecipitation analyses confirm the direct binding of bZIP1 and bZIP53 to promoters of key metabolic genes, such as ASPARAGINE SYNTHETASE1 and PROLINE DEHYDROGENASE, the G-box, C-box, or ACT motifs (ACTCAT) have been defined as regulatory cis-elements in the starvation response. bZIP1 and bZIP53 were shown to specifically heterodimerize with group C bZIPs. Although single loss-of-function mutants did not affect starvation-induced transcription, quadruple mutants of group S1 and C bZIPs displayed a significant impairment. We therefore propose that bZIP1 and bZIP53 transduce low energy signals by heterodimerization with members of the partially redundant C/S1 bZIP factor network to reprogram primary metabolism in the starvation response.


Assuntos
Aminoácidos/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/metabolismo , Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Fatores de Transcrição de Zíper de Leucina Básica/genética , Escuridão , Regulação da Expressão Gênica de Plantas , Mutação , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Multimerização Proteica , Protoplastos/metabolismo , Transdução de Sinais , Estresse Fisiológico , Transcrição Gênica
20.
Microbiol Res ; 289: 127890, 2024 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-39243685

RESUMO

This study investigates the molecular mechanisms underlying salt stress responses in plants, focusing on the regulatory roles of OsNAM2, a gene influenced by the plant growth-promoting rhizobacterium Bacillus amyloliquefaciens (SN13). The study examines how SN13-modulated OsNAM2 enhances salt tolerance in Arabidopsis through physiological, biochemical, and molecular analyses. Overexpression of OsNAM2, especially with SN13 inoculation, improves germination, seedling growth, root length, and biomass under high NaCl concentrations compared to wild-type plants, indicating a synergistic effect. OsNAM2 overexpression enhances relative water content, reduces electrolyte leakage and malondialdehyde accumulation, and increases proline content, suggesting better membrane integrity and stress endurance. Furthermore, SN13 and OsNAM2 overexpression modulates essential metabolic genes involved in glycolysis, the pentose phosphate pathway, and the tricarboxylic acid cycle, facilitating metabolic adjustments crucial for salt stress adaptation. The interaction of OsNAM2 with SUS, facilitated by SN13, suggests enhanced sucrose metabolism efficiency, providing substrates for protective responses. Additionally, OsNAM2 plays a regulatory role in the ABA signaling pathway through significant protein-protein interactions like with AFP2. This study highlights the intricate interplay between SN13-responsive OsNAM2 and key signaling pathways, suggesting strategies for enhancing crop salt tolerance through targeted genetic and microbial interventions.


Assuntos
Arabidopsis , Bacillus amyloliquefaciens , Regulação da Expressão Gênica de Plantas , Tolerância ao Sal , Arabidopsis/genética , Arabidopsis/metabolismo , Arabidopsis/fisiologia , Tolerância ao Sal/genética , Bacillus amyloliquefaciens/genética , Bacillus amyloliquefaciens/metabolismo , Bacillus amyloliquefaciens/fisiologia , Raízes de Plantas/microbiologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Raízes de Plantas/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plântula/crescimento & desenvolvimento , Plântula/genética , Transdução de Sinais , Cloreto de Sódio/metabolismo , Germinação , Estresse Salino , Ácido Abscísico/metabolismo , Prolina/metabolismo
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