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1.
PLoS Genet ; 16(9): e1009043, 2020 09.
Artigo em Inglês | MEDLINE | ID: mdl-32960882

RESUMO

Higher plants can continuously form new organs by the sustained activity of pluripotent stem cells. These stem cells are embedded in meristems, where they produce descendants, which undergo cell proliferation and differentiation programs in a spatiotemporally-controlled manner. Under certain conditions, pluripotency can be reestablished in descending cells and this reversion in cell fate appears to be actively suppressed by the existing stem cell pool. Mutation of the putative carboxypeptidase ALTERED MERISTEM PROGRAM1 (AMP1) in Arabidopsis causes defects in the suppression of pluripotency in cells normally programmed for differentiation, giving rise to unique hypertrophic phenotypes during embryogenesis as well as in the shoot apical meristem. A role of AMP1 in the miRNA-dependent control of translation has recently been established, however, how this activity is connected to its developmental functions is not resolved. Here we identify members of the cytochrome P450 clade CYP78A to act in parallel with AMP1 to control cell fate in Arabidopsis. Mutation of CYP78A5 and its close homolog CYP78A7 in a cyp78a5,7 double mutant caused suspensor-to-embryo conversion and ectopic stem cell pool formation in the shoot meristem, phenotypes characteristic for amp1. The tissues affected in the mutants showed pronounced expression levels of AMP1 and CYP78A5 in wild type. A comparison of mutant transcriptomic responses revealed an intriguing degree of overlap and highlighted alterations in protein lipidation processes. Moreover, we also found elevated protein levels of selected miRNA targets in cyp78a5,7. Based on comprehensive genetic interaction studies we propose a model in which both enzyme classes act on a common downstream process to sustain cell fate decisions in the early embryo and the shoot apical meristem.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Carboxipeptidases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Carboxipeptidases/genética , Diferenciação Celular/fisiologia , Linhagem da Célula , Sistema Enzimático do Citocromo P-450/genética , Citocininas/metabolismo , Meristema/citologia , Meristema/genética , Meristema/metabolismo , MicroRNAs/genética , Mutação , Fenótipo , Plantas Geneticamente Modificadas/genética , Transcriptoma
2.
Nat Commun ; 11(1): 4509, 2020 09 09.
Artigo em Inglês | MEDLINE | ID: mdl-32908151

RESUMO

Glycolysis is one of the primordial pathways of metabolism, playing a pivotal role in energy metabolism and biosynthesis. Glycolytic enzymes are known to form transient multi-enzyme assemblies. Here we examine the wider protein-protein interactions of plant glycolytic enzymes and reveal a moonlighting role for specific glycolytic enzymes in mediating the co-localization of mitochondria and chloroplasts. Knockout mutation of phosphoglycerate mutase or enolase resulted in a significantly reduced association of the two organelles. We provide evidence that phosphoglycerate mutase and enolase form a substrate-channelling metabolon which is part of a larger complex of proteins including pyruvate kinase. These results alongside a range of genetic complementation experiments are discussed in the context of our current understanding of chloroplast-mitochondrial interactions within photosynthetic eukaryotes.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Cloroplastos/enzimologia , Glicólise/fisiologia , Mitocôndrias/enzimologia , Arabidopsis/citologia , Proteínas de Arabidopsis/genética , Metabolismo Energético/fisiologia , Mutação , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/metabolismo , Fosfopiruvato Hidratase/genética , Fosfopiruvato Hidratase/metabolismo , Fotossíntese/fisiologia , Plantas Geneticamente Modificadas , Mapeamento de Interação de Proteínas , Mapas de Interação de Proteínas/fisiologia , Piruvato Quinase/genética , Piruvato Quinase/metabolismo
3.
Proc Natl Acad Sci U S A ; 117(36): 22561-22571, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32839309

RESUMO

In the shoot meristem, both WUSCHEL (WUS) and SHOOT MERISTEMLESS (STM), two transcription factors with overlapping spatiotemporal expression patterns, are essential for maintaining stem cells in an undifferentiated state. Despite their importance, it remains unclear how these two pathways are integrated to coordinate stem cell development. Here, we show that the WUS and STM pathways in Arabidopsis thaliana converge through direct interaction between the WUS and STM proteins. STM binds to the promoter of CLAVATA3 (CLV3) and enhances the binding of WUS to the same promoter through the WUS-STM interaction. Both the heterodimerization and simultaneous binding of WUS and STM at two sites on the CLV3 promoter are required to regulate CLV3 expression, which in turn maintains a constant number of stem cells. Furthermore, the expression of STM depends on WUS, and this WUS-activated STM expression enhances the WUS-mediated stem cell activity. Our data provide a framework for understanding how spatial expression patterns within the shoot meristem are translated into regulatory units of stem cell homeostasis.


Assuntos
Arabidopsis/citologia , Arabidopsis/crescimento & desenvolvimento , Regulação da Expressão Gênica de Plantas/genética , Meristema/genética , Arabidopsis/genética , Arabidopsis/fisiologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Linhagem Celular , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Meristema/metabolismo , Ligação Proteica , Células-Tronco/citologia , Células-Tronco/metabolismo
4.
Nat Commun ; 11(1): 3914, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764676

RESUMO

Cell polarity is fundamental to the development of both eukaryotes and prokaryotes, yet the mechanisms behind its formation are not well understood. Here we found that, phytohormone auxin-induced, sterol-dependent nanoclustering of cell surface transmembrane receptor kinase 1 (TMK1) is critical for the formation of polarized domains at the plasma membrane (PM) during the morphogenesis of cotyledon pavement cells (PC) in Arabidopsis. Auxin-induced TMK1 nanoclustering stabilizes flotillin1-associated ordered nanodomains, which in turn promote the nanoclustering of ROP6 GTPase that acts downstream of TMK1 to regulate cortical microtubule organization. In turn, cortical microtubules further stabilize TMK1- and flotillin1-containing nanoclusters at the PM. Hence, we propose a new paradigm for polarity formation: A diffusive signal triggers cell polarization by promoting cell surface receptor-mediated nanoclustering of signaling components and cytoskeleton-mediated positive feedback that reinforces these nanodomains into polarized domains.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Polaridade Celular/fisiologia , Ácidos Indolacéticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/química , Membrana Celular/metabolismo , Polaridade Celular/genética , Metabolismo dos Lipídeos , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Modelos Biológicos , Proteínas Monoméricas de Ligação ao GTP/química , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Mutação , Reguladores de Crescimento de Planta/metabolismo , Plantas Geneticamente Modificadas , Agregados Proteicos , Estabilidade Proteica , Proteínas Serina-Treonina Quinases/química , Transdução de Sinais
5.
Nat Commun ; 11(1): 4285, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855390

RESUMO

Plant hormone cytokinins are perceived by a subfamily of sensor histidine kinases (HKs), which via a two-component phosphorelay cascade activate transcriptional responses in the nucleus. Subcellular localization of the receptors proposed the endoplasmic reticulum (ER) membrane as a principal cytokinin perception site, while study of cytokinin transport pointed to the plasma membrane (PM)-mediated cytokinin signalling. Here, by detailed monitoring of subcellular localizations of the fluorescently labelled natural cytokinin probe and the receptor ARABIDOPSIS HISTIDINE KINASE 4 (CRE1/AHK4) fused to GFP reporter, we show that pools of the ER-located cytokinin receptors can enter the secretory pathway and reach the PM in cells of the root apical meristem, and the cell plate of dividing meristematic cells. Brefeldin A (BFA) experiments revealed vesicular recycling of the receptor and its accumulation in BFA compartments. We provide a revised view on cytokinin signalling and the possibility of multiple sites of perception at PM and ER.


Assuntos
Proteínas de Arabidopsis/metabolismo , Membrana Celular/metabolismo , Citocininas/metabolismo , Retículo Endoplasmático/metabolismo , Corantes Fluorescentes/química , Proteínas Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Brefeldina A/farmacologia , Citocininas/química , Corantes Fluorescentes/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Meristema/citologia , Meristema/metabolismo , Plantas Geneticamente Modificadas , Proteínas Quinases/genética , Receptores de Superfície Celular/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Nat Commun ; 11(1): 4284, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855409

RESUMO

Cytokinins are mobile multifunctional plant hormones with roles in development and stress resilience. Although their Histidine Kinase receptors are substantially localised to the endoplasmic reticulum, cellular sites of cytokinin perception and importance of spatially heterogeneous cytokinin distribution continue to be debated. Here we show that cytokinin perception by plasma membrane receptors is an effective additional path for cytokinin response. Readout from a Two Component Signalling cytokinin-specific reporter (TCSn::GFP) closely matches intracellular cytokinin content in roots, yet we also find cytokinins in extracellular fluid, potentially enabling action at the cell surface. Cytokinins covalently linked to beads that could not pass the plasma membrane increased expression of both TCSn::GFP and Cytokinin Response Factors. Super-resolution microscopy of GFP-labelled receptors and diminished TCSn::GFP response to immobilised cytokinins in cytokinin receptor mutants, further indicate that receptors can function at the cell surface. We argue that dual intracellular and surface locations may augment flexibility of cytokinin responses.


Assuntos
Arabidopsis/metabolismo , Citocininas/metabolismo , Proteínas Recombinantes/metabolismo , Adenina/análogos & derivados , Adenina/farmacologia , Arabidopsis/citologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Líquido Extracelular/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Histidina Quinase/genética , Histidina Quinase/metabolismo , Mutação , Plantas Geneticamente Modificadas , Proteínas Recombinantes/genética , Transdução de Sinais
7.
J Vis Exp ; (160)2020 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-32658193

RESUMO

Protein phosphorylation is crucial for the regulation of enzyme activity and gene expression under osmotic condition. Mass spectrometry (MS)-based phosphoproteomics has transformed the way of studying plant signal transduction. However, requirement of lots of starting materials and prolonged MS measurement time to achieve the depth of coverage has been the limiting factor for the high throughput study of global phosphoproteomic changes in plants. To improve the sensitivity and throughput of plant phosphoproteomics, we have developed a stop and go extraction (stage) tip based phosphoproteomics approach coupled with Tandem Mass Tag (TMT) labeling for the rapid and comprehensive analysis of plant phosphorylation perturbation in response to osmotic stress. Leveraging the simplicity and high throughput of stage tip technique, the whole procedure takes approximately one hour using two tips to finish phosphopeptide enrichment, fractionation, and sample cleaning steps, suggesting an easy-to-use and high efficiency of the approach. This approach not only provides an in-depth plant phosphoproteomics analysis (> 11,000 phosphopeptide identification) but also demonstrates the superior separation efficiency (< 5% overlap) between adjacent fractions. Further, multiplexing has been achieved using TMT labeling to quantify the phosphoproteomic changes of wild-type and snrk2 decuple mutant plants. This approach has successfully been used to reveal the phosphorylation events of Raf-like kinases in response to osmotic stress, which sheds light on the understanding of early osmotic signaling in land plants.


Assuntos
Arabidopsis/citologia , Arabidopsis/metabolismo , Pressão Osmótica , Fosfoproteínas/metabolismo , Proteômica/métodos , Transdução de Sinais , Proteínas de Arabidopsis/metabolismo , Espectrometria de Massas , Fosfopeptídeos/metabolismo , Fosforilação
8.
Plant Mol Biol ; 104(1-2): 187-201, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32681357

RESUMO

KEY MESSAGE: The mutation of FAX1 (Fatty Acid Export 1) disrupts ROS homeostasis and suppresses transcription activity of DYT1-TDF1-AMS-MS188 genetic network, leading to atypical tapetum PCD and defective pollen formation in Arabidopsis. Fatty acids (FAs) have multiple important biological functions and exert diverse cellular effects through modulating Reactive Oxygen Species (ROS) homeostasis. Arabidopsis FAX1 (Fatty Acid Export 1) mediates the export of de novo synthesized FA from chloroplast and loss of function of FAX1 impairs male fertility. However, mechanisms underlying the association of FAX1-mediated FA export with male sterility remain enigmatic. In this study, by using an integrated approach that included morphological, cytological, histological, and molecular analyses, we revealed that loss of function of FAX1 breaks cellular FA/lipid homeostasis, which disrupts ROS homeostasis and suppresses transcriptional activation of the DYT1-TDF1-AMS-MS188 genetic network of anther development, impairing tapetum development and pollen wall formation, and resulting in male sterility. This study provides new insights into the regulatory network for male reproduction in plants, highlighting an important role of FA export-mediated ROS homeostasis in the process.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Membrana/metabolismo , Pólen/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Flores/citologia , Flores/genética , Flores/crescimento & desenvolvimento , Flores/metabolismo , Redes Reguladoras de Genes , Proteínas de Membrana/genética , Mutação , Fenótipo , Pólen/genética , Reprodução , Fatores de Transcrição
9.
Proc Natl Acad Sci U S A ; 117(27): 15400-15402, 2020 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-32571955

RESUMO

The Casparian strip (CS) is a tight junction-like structure formed by lignin impregnation on the walls of endodermal cells in plant roots. The CS membrane domain (CSDM), demarked by the CASP proteins, is important for orienting lignification enzymes. Here, we report that an endodermis-expressed multicopper oxidase, LACCASE3 (LAC3) in Arabidopsis, locates to the interface between lignin domains and the cell wall during early CS development prior to CASP1 localizing to CSDM and eventually flanks the mature CS. Pharmacological perturbation of LAC3 causes dispersed localization of CASP1 and compensatory ectopic lignification. These results support the existence of a LAC3-based CS wall domain which coordinates with CSDM to provide bidirectional positional information that guides precise CS lignification.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Lacase/metabolismo , Proteínas de Membrana/metabolismo , Raízes de Plantas/metabolismo , Arabidopsis/citologia , Parede Celular/metabolismo , Lacase/genética , Lignina/metabolismo , Proteínas de Membrana/genética , Raízes de Plantas/citologia , Plantas Geneticamente Modificadas , Domínios Proteicos
10.
J Vis Exp ; (159)2020 05 23.
Artigo em Inglês | MEDLINE | ID: mdl-32510485

RESUMO

Understanding cell and tissue level regulation of growth and morphogenesis has been at the forefront of biological research for many decades. Advances in molecular and imaging technologies allowed us to gain insights into how biochemical signals influence morphogenetic events. However, it is increasingly evident that apart from biochemical signals, mechanical cues also impact several aspects of cell and tissue growth. The Arabidopsis shoot apical meristem (SAM) is a dome-shaped structure responsible for the generation of all aboveground organs. The organization of the cortical microtubule cytoskeleton that mediates apoplastic cellulose deposition in plant cells is spatially distinct. Visualization and quantitative assessment of patterns of cortical microtubules are necessary for understanding the biophysical nature of cells at the SAM, as cellulose is the stiffest component of the plant cell wall. The stereotypical form of cortical microtubule organization is also a consequence of tissue-wide physical forces existing at the SAM. Perturbation of these physical forces and subsequent monitoring of cortical microtubule organization allows for the identification of candidate proteins involved in mediating mechano-perception and transduction. Here we describe a protocol that helps investigate such processes.


Assuntos
Arabidopsis/citologia , Citoesqueleto/metabolismo , Fenômenos Mecânicos , Microtúbulos/metabolismo , Imagem Molecular , Arabidopsis/crescimento & desenvolvimento , Fenômenos Biomecânicos , Sobrevivência Celular , Parede Celular/metabolismo , Celulose/metabolismo , Meristema/citologia , Meristema/crescimento & desenvolvimento , Morfogênese
12.
PLoS One ; 15(6): e0234154, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32497144

RESUMO

Phytohormones mediate most diverse processes in plants, ranging from organ development to immune responses. Receptor protein complexes perceive changes in intracellular phytohormone levels and trigger a signaling cascade to effectuate downstream responses. The in planta analysis of elements involved in phytohormone signaling can be achieved through transient expression in mesophyll protoplasts, which are a fast and versatile alternative to generating plant lines that stably express a transgene. While promoter-reporter constructs have been used successfully to identify internal or external factors that change phytohormone signaling, the range of available marker constructs does not meet the potential of the protoplast technique for large scale approaches. The aim of our study was to provide novel markers for phytohormone signaling in the Arabidopsis mesophyll protoplast system. We validated 18 promoter::luciferase constructs towards their phytohormone responsiveness and specificity and suggest an experimental setup for high-throughput analyses. We recommend novel markers for the analysis of auxin, abscisic acid, cytokinin, salicylic acid and jasmonic acid responses that will facilitate future screens for biological elements and environmental stimuli affecting phytohormone signaling.


Assuntos
Reguladores de Crescimento de Planta/metabolismo , Protoplastos/citologia , Transdução de Sinais , Arabidopsis/citologia , Biomarcadores/metabolismo
13.
Nat Commun ; 11(1): 2965, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528082

RESUMO

Trajectories of cellular ontogeny are tightly controlled and often involve feedback-regulated molecular antagonism. For example, sieve element differentiation along developing protophloem cell files of Arabidopsis roots requires two antagonistic regulators of auxin efflux. Paradoxically, loss-of-function in either regulator triggers similar, seemingly stochastic differentiation failures of individual sieve element precursors. Here we show that these patterning defects are distinct and non-random. They can be explained by auxin-dependent bistability that emerges from competition for auxin between neighboring cells. This bistability depends on the presence of an auxin influx facilitator, and can be triggered by either flux enhancement or repression. Our results uncover a hitherto overlooked aspect of auxin uptake, and highlight the contributions of local auxin influx, efflux and biosynthesis to protophloem formation. Moreover, the combined experimental-modeling approach suggests that without auxin efflux homeostasis, auxin influx interferes with coordinated differentiation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Ácidos Indolacéticos/metabolismo , Raízes de Plantas/citologia , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Transformação Genética/genética
14.
PLoS Genet ; 16(4): e1008661, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32294082

RESUMO

In the Arabidopsis thaliana shoot apical meristem (SAM) the expression domains of Class III Homeodomain Leucine Zipper (HD-ZIPIII) and KANADI (KAN) genes are separated by a narrow boundary region from which new organs are initiated. Disruption of this boundary through either loss of function or ectopic expression of HD-ZIPIII and KAN causes ectopic or suppression of organ formation respectively, raising the question of how these transcription factors regulate organogenesis at a molecular level. In this study we develop a multi-channel FACS/RNA-seq approach to characterize global patterns of gene expression across the HD-ZIPIII-KAN1 SAM boundary. We then combine FACS, RNA-seq and perturbations of HD-ZIPIII and KAN expression to identify genes that are both responsive to REV and KAN1 and normally expressed in patterns that correlate with REV and KAN1. Our data reveal that a significant number of genes responsive to REV are regulated in opposite ways depending on time after induction, with genes associated with auxin response and synthesis upregulated initially, but later repressed. We also characterize the cell type specific expression patterns of auxin responsive genes and identify a set of genes involved in organogenesis repressed by both REV and KAN1.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Proteínas de Homeodomínio/metabolismo , Meristema/citologia , Meristema/metabolismo , Fatores de Transcrição/metabolismo , Análise por Conglomerados , Citocininas/metabolismo , Citometria de Fluxo , Ontologia Genética , Genes de Plantas , Ácidos Indolacéticos/metabolismo , Inflorescência , Reguladores de Crescimento de Planta/metabolismo , RNA-Seq , Transdução de Sinais , Transcriptoma
15.
Int J Mol Sci ; 21(5)2020 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-32121556

RESUMO

Nitric oxide (NO) plays an important role in stomata closure induced by environmental stimuli including pathogens. During pathogen challenge, nitric oxide (NO) acts as a second messenger in guard cell signaling networks to activate downstream responses leading to stomata closure. One means by which NO's action is achieved is through the posttranslational modification of cysteine residue(s) of target proteins. Although the roles of NO have been well studied in plant tissues and seedlings, far less is known about NO signaling and, more specifically, protein S-nitrosylation (SNO) in stomatal guard cells. In this study, using iodoTMTRAQ quantitative proteomics technology, we analyzed changes in protein SNO modification in guard cells of reference plant Arabidopsis thaliana in response to flg22, an elicitor-active peptide derived from bacterial flagellin. A total of 41 SNO-modified peptides corresponding to 35 proteins were identified. The proteins cover a wide range of functions, including energy metabolism, transport, stress response, photosynthesis, and cell-cell communication. This study creates the first inventory of previously unknown NO responsive proteins in guard cell immune responses and establishes a foundation for future research toward understanding the molecular mechanisms and regulatory roles of SNO in stomata immunity against bacterial pathogens.


Assuntos
Arabidopsis/citologia , Flagelina/farmacologia , Estômatos de Plantas/citologia , Estômatos de Plantas/metabolismo , Proteoma/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Ontologia Genética , Células do Mesofilo/citologia , Células do Mesofilo/efeitos dos fármacos , Células do Mesofilo/metabolismo , Óxido Nítrico/metabolismo , Nitrosação , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/fisiologia , Espécies Reativas de Oxigênio/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(13): 7482-7493, 2020 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-32170020

RESUMO

Plants balance their competing requirements for growth and stress tolerance via a sophisticated regulatory circuitry that controls responses to the external environments. We have identified a plant-specific gene, COST1 (constitutively stressed 1), that is required for normal plant growth but negatively regulates drought resistance by influencing the autophagy pathway. An Arabidopsis thaliana cost1 mutant has decreased growth and increased drought tolerance, together with constitutive autophagy and increased expression of drought-response genes, while overexpression of COST1 confers drought hypersensitivity and reduced autophagy. The COST1 protein is degraded upon plant dehydration, and this degradation is reduced upon treatment with inhibitors of the 26S proteasome or autophagy pathways. The drought resistance of a cost1 mutant is dependent on an active autophagy pathway, but independent of other known drought signaling pathways, indicating that COST1 acts through regulation of autophagy. In addition, COST1 colocalizes to autophagosomes with the autophagosome marker ATG8e and the autophagy adaptor NBR1, and affects the level of ATG8e protein through physical interaction with ATG8e, indicating a pivotal role in direct regulation of autophagy. We propose a model in which COST1 represses autophagy under optimal conditions, thus allowing plant growth. Under drought, COST1 is degraded, enabling activation of autophagy and suppression of growth to enhance drought tolerance. Our research places COST1 as an important regulator controlling the balance between growth and stress responses via the direct regulation of autophagy.


Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Estresse Fisiológico/fisiologia , Arabidopsis/citologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Autofagossomos/metabolismo , Autofagia/fisiologia , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas de Transporte/metabolismo , Secas , Genes de Plantas , Transdução de Sinais , Estresse Fisiológico/genética
17.
Nature ; 579(7800): 561-566, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32214247

RESUMO

Species that propagate by sexual reproduction actively guard against the fertilization of an egg by multiple sperm (polyspermy). Flowering plants rely on pollen tubes to transport their immotile sperm to fertilize the female gametophytes inside ovules. In Arabidopsis, pollen tubes are guided by cysteine-rich chemoattractants to target the female gametophyte1,2. The FERONIA receptor kinase has a dual role in ensuring sperm delivery and blocking polyspermy3. It has previously been reported that FERONIA generates a female gametophyte environment that is required for sperm release4. Here we show that FERONIA controls several functionally linked conditions to prevent the penetration of female gametophytes by multiple pollen tubes in Arabidopsis. We demonstrate that FERONIA is crucial for maintaining de-esterified pectin at the filiform apparatus, a region of the cell wall at the entrance to the female gametophyte. Pollen tube arrival at the ovule triggers the accumulation of nitric oxide at the filiform apparatus in a process that is dependent on FERONIA and mediated by de-esterified pectin. Nitric oxide nitrosates both precursor and mature forms of the chemoattractant LURE11, respectively blocking its secretion and interaction with its receptor, to suppress pollen tube attraction. Our results elucidate a mechanism controlled by FERONIA in which the arrival of the first pollen tube alters ovular conditions to disengage pollen tube attraction and prevent the approach and penetration of the female gametophyte by late-arriving pollen tubes, thus averting polyspermy.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Fertilização , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Óxido Nítrico/metabolismo , Óvulo Vegetal/metabolismo , Pectinas/metabolismo , Fosfotransferases/metabolismo , Tubo Polínico/metabolismo , Parede Celular/química , Parede Celular/metabolismo , Óvulo Vegetal/citologia , Pectinas/química , Tubo Polínico/citologia
18.
Int J Mol Sci ; 21(7)2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32218176

RESUMO

MiPEPs are short natural peptides encoded by microRNAs in plants. Exogenous application of miPEPs increases the expression of their corresponding miRNA and, consequently, induces consistent phenotypical changes. Therefore, miPEPs carry huge potential in agronomy as gene regulators that do not require genome manipulation. However, to this end, it is necessary to know their mode of action, including where they act and how they enter the plants. Here, after analyzing the effect of Arabidopsis thaliana miPEP165a on root and aerial part development, we followed the internalization of fluorescent-labelled miPEP165a into roots and compared its uptake into endocytosis-altered mutants to that observed in wild-type plants treated or not with endocytosis inhibitors. The results show that entry of miPEP165a involves both a passive diffusion at the root apex and endocytosis-associated internalization in the differentiation and mature zones. Moreover, miPEP165a is unable to enter the central cylinder and does not migrate from the roots to the aerial part of the plant, suggesting that miPEPs have no systemic effect.


Assuntos
Arabidopsis/efeitos dos fármacos , Endocitose , Arabidopsis/citologia , Arabidopsis/metabolismo , Transporte Biológico , Divisão Celular/efeitos dos fármacos , Difusão , Endocitose/efeitos dos fármacos , Fenótipo , Raízes de Plantas/citologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas
19.
Cell ; 180(3): 427-439.e12, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32004461

RESUMO

Cell polarity is fundamental for tissue morphogenesis in multicellular organisms. Plants and animals evolved multicellularity independently, and it is unknown whether their polarity systems are derived from a single-celled ancestor. Planar polarity in animals is conferred by Wnt signaling, an ancient signaling pathway transduced by Dishevelled, which assembles signalosomes by dynamic head-to-tail DIX domain polymerization. In contrast, polarity-determining pathways in plants are elusive. We recently discovered Arabidopsis SOSEKI proteins, which exhibit polar localization throughout development. Here, we identify SOSEKI as ancient polar proteins across land plants. Concentration-dependent polymerization via a bona fide DIX domain allows these to recruit ANGUSTIFOLIA to polar sites, similar to the polymerization-dependent recruitment of signaling effectors by Dishevelled. Cross-kingdom domain swaps reveal functional equivalence of animal and plant DIX domains. We trace DIX domains to unicellular eukaryotes and thus show that DIX-dependent polymerization is an ancient mechanism conserved between kingdoms and central to polarity proteins.


Assuntos
Arabidopsis/química , Arabidopsis/citologia , Polaridade Celular/fisiologia , Células Vegetais/fisiologia , Polimerização , Domínios Proteicos , Animais , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteína Axina/química , Proteína Axina/metabolismo , Bryopsida/química , Bryopsida/citologia , Bryopsida/genética , Bryopsida/crescimento & desenvolvimento , Células COS , Chlorocebus aethiops , Proteínas Desgrenhadas/metabolismo , Células HEK293 , Humanos , Marchantia/química , Marchantia/citologia , Marchantia/genética , Marchantia/crescimento & desenvolvimento , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Plantas Geneticamente Modificadas , Proteínas Repressoras/metabolismo , Via de Sinalização Wnt
20.
Biochem Biophys Res Commun ; 524(4): 977-982, 2020 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-32059845

RESUMO

We previously found that VAMP721/722 SNARE proteins guide secretory vesicles to pathogen-attacking sites during immune responses in Arabidopsis, which suggests that these vesicles should deliver immune molecules. However, the lethality of vamp721 vamp722 double null mutant makes it difficult to understand the nature of cargo transported via VAMP721/722 vesicles. Since VAMP721/722-depleted (VAMP721+/-VAMP722-/- and VAMP721-/-VAMP722+/-) plants show compromised resistance to extracellular pathogens, we assume that an immune protein secreted through the VAMP721/722-engaged exocytosis would be remained more in VAMP721/722-depleted plants than WT. By comparing intracellular proteins between WT and VAMP721/722-depleted plants, we found caffeoyl-CoA O-methyltransferase 1 (CCOAOMT1) involved in the lignin biosynthesis was more abundantly detected in both VAMP721/722-depleted lines than WT. Plants are well-known to deposit secondary cell walls as physical barriers at pathogen-attempting sites. Therefore, extracellular detection of CCOAOMT1 and impaired resistance to Pseudomonas syringae DC3000 in ccoaomt1 plants suggest that plants secrete cell wall-modifying enzymes at least including CCOAOMT1 to reinforce the secondary cell walls for immunity.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Metiltransferases/metabolismo , Proteínas R-SNARE/metabolismo , Arabidopsis/citologia , Parede Celular/metabolismo , Lignina/metabolismo , Vesículas Secretórias/metabolismo
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