Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 25
Filtrar
1.
Plant Cell ; 34(4): 1375-1395, 2022 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-35078237

RESUMO

Redox processes are at the heart of universal life processes, such as metabolism, signaling, or folding of secreted proteins. Redox landscapes differ between cell compartments and are strictly controlled to tolerate changing conditions and to avoid cell dysfunction. While a sophisticated antioxidant network counteracts oxidative stress, our understanding of reductive stress responses remains fragmentary. Here, we observed root growth impairment in Arabidopsis thaliana mutants of mitochondrial alternative oxidase 1a (aox1a) in response to the model thiol reductant dithiothreitol (DTT). Mutants of mitochondrial uncoupling protein 1 (ucp1) displayed a similar phenotype indicating that impaired respiratory flexibility led to hypersensitivity. Endoplasmic reticulum (ER) stress was enhanced in the mitochondrial mutants and limiting ER oxidoreductin capacity in the aox1a background led to synergistic root growth impairment by DTT, indicating that mitochondrial respiration alleviates reductive ER stress. The observations that DTT triggered nicotinamide adenine dinucleotide (NAD) reduction in vivo and that the presence of thiols led to electron transport chain activity in isolated mitochondria offer a biochemical framework of mitochondrion-mediated alleviation of thiol-mediated reductive stress. Ablation of transcription factor Arabidopsis NAC domain-containing protein17 (ANAC017) impaired the induction of AOX1a expression by DTT and led to DTT hypersensitivity, revealing that reductive stress tolerance is achieved by adjusting mitochondrial respiratory capacity via retrograde signaling. Our data reveal an unexpected role for mitochondrial respiratory flexibility and retrograde signaling in reductive stress tolerance involving inter-organelle redox crosstalk.


Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Transdução de Sinais/fisiologia , Compostos de Sulfidrila/metabolismo , Fatores de Transcrição/metabolismo
2.
Plant Cell ; 34(10): 4007-4027, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-35818121

RESUMO

Oxidative protein folding in the endoplasmic reticulum (ER) depends on the coordinated action of protein disulfide isomerases and ER oxidoreductins (EROs). Strict dependence of ERO activity on molecular oxygen as the final electron acceptor implies that oxidative protein folding and other ER processes are severely compromised under hypoxia. Here, we isolated viable Arabidopsis thaliana ero1 ero2 double mutants that are highly sensitive to reductive stress and hypoxia. To elucidate the specific redox dynamics in the ER in vivo, we expressed the glutathione redox potential (EGSH) sensor Grx1-roGFP2iL-HDEL with a midpoint potential of -240 mV in the ER of Arabidopsis plants. We found EGSH values of -241 mV in wild-type plants, which is less oxidizing than previously estimated. In the ero1 ero2 mutants, luminal EGSH was reduced further to -253 mV. Recovery to reductive ER stress induced by dithiothreitol was delayed in ero1 ero2. The characteristic signature of EGSH dynamics in the ER lumen triggered by hypoxia was affected in ero1 ero2 reflecting a disrupted balance of reductive and oxidizing inputs, including nascent polypeptides and glutathione entry. The ER redox dynamics can now be dissected in vivo, revealing a central role of EROs as major redox integrators to promote luminal redox homeostasis.


Assuntos
Arabidopsis , Isomerases de Dissulfetos de Proteínas , Arabidopsis/genética , Arabidopsis/metabolismo , Ditiotreitol , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático/genética , Glutationa/metabolismo , Hipóxia , Oxirredução , Oxigênio/metabolismo , Isomerases de Dissulfetos de Proteínas/metabolismo , Dobramento de Proteína
3.
Plant Cell ; 34(11): 4428-4452, 2022 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-35938694

RESUMO

Ca2+ signaling is central to plant development and acclimation. While Ca2+-responsive proteins have been investigated intensely in plants, only a few Ca2+-permeable channels have been identified, and our understanding of how intracellular Ca2+ fluxes is facilitated remains limited. Arabidopsis thaliana homologs of the mammalian channel-forming mitochondrial calcium uniporter (MCU) protein showed Ca2+ transport activity in vitro. Yet, the evolutionary complexity of MCU proteins, as well as reports about alternative systems and unperturbed mitochondrial Ca2+ uptake in knockout lines of MCU genes, leave critical questions about the in vivo functions of the MCU protein family in plants unanswered. Here, we demonstrate that MCU proteins mediate mitochondrial Ca2+ transport in planta and that this mechanism is the major route for fast Ca2+ uptake. Guided by the subcellular localization, expression, and conservation of MCU proteins, we generated an mcu triple knockout line. Using Ca2+ imaging in living root tips and the stimulation of Ca2+ transients of different amplitudes, we demonstrated that mitochondrial Ca2+ uptake became limiting in the triple mutant. The drastic cell physiological phenotype of impaired subcellular Ca2+ transport coincided with deregulated jasmonic acid-related signaling and thigmomorphogenesis. Our findings establish MCUs as a major mitochondrial Ca2+ entry route in planta and link mitochondrial Ca2+ transport with phytohormone signaling.


Assuntos
Arabidopsis , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Mamíferos/metabolismo
4.
Plant Cell ; 33(12): 3700-3720, 2021 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-34498076

RESUMO

Malate and citrate underpin the characteristic flexibility of central plant metabolism by linking mitochondrial respiratory metabolism with cytosolic biosynthetic pathways. However, the identity of mitochondrial carrier proteins that influence both processes has remained elusive. Here we show by a systems approach that DICARBOXYLATE CARRIER 2 (DIC2) facilitates mitochondrial malate-citrate exchange in vivo in Arabidopsis thaliana. DIC2 knockout (dic2-1) retards growth of vegetative tissues. In vitro and in organello analyses demonstrate that DIC2 preferentially imports malate against citrate export, which is consistent with altered malate and citrate utilization in response to prolonged darkness of dic2-1 plants or a sudden shift to darkness of dic2-1 leaves. Furthermore, isotopic glucose tracing reveals a reduced flux towards citrate in dic2-1, which results in a metabolic diversion towards amino acid synthesis. These observations reveal the physiological function of DIC2 in mediating the flow of malate and citrate between the mitochondrial matrix and other cell compartments.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Ácido Cítrico/metabolismo , Transportadores de Ácidos Dicarboxílicos/genética , Malatos/metabolismo , Folhas de Planta/metabolismo , Ácidos/metabolismo , Proteínas de Arabidopsis/metabolismo , Transportadores de Ácidos Dicarboxílicos/metabolismo , Mitocôndrias/metabolismo
5.
Plant Cell ; 32(10): 3324-3345, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32796121

RESUMO

NADH and NAD+ are a ubiquitous cellular redox couple. Although the central role of NAD in plant metabolism and its regulatory role have been investigated extensively at the biochemical level, analyzing the subcellular redox dynamics of NAD in living plant tissues has been challenging. Here, we established live monitoring of NADH/NAD+ in plants using the genetically encoded fluorescent biosensor Peredox-mCherry. We established Peredox-mCherry lines of Arabidopsis (Arabidopsis thaliana) and validated the biophysical and biochemical properties of the sensor that are critical for in planta measurements, including specificity, pH stability, and reversibility. We generated an NAD redox atlas of the cytosol of living Arabidopsis seedlings that revealed pronounced differences in NAD redox status between different organs and tissues. Manipulating the metabolic status through dark-to-light transitions, respiratory inhibition, sugar supplementation, and elicitor exposure revealed a remarkable degree of plasticity of the cytosolic NAD redox status and demonstrated metabolic redox coupling between cell compartments in leaves. Finally, we used protein engineering to generate a sensor variant that expands the resolvable NAD redox range. In summary, we established a technique for in planta NAD redox monitoring to deliver important insight into the in vivo dynamics of plant cytosolic redox metabolism.


Assuntos
Arabidopsis/metabolismo , Técnicas Biossensoriais/métodos , Citosol/metabolismo , Proteínas Luminescentes/genética , NAD/metabolismo , Arabidopsis/genética , Carbono/metabolismo , Fluorometria/métodos , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/metabolismo , Malatos/metabolismo , Mitocôndrias/metabolismo , NAD/análise , Oxirredução , Plantas Geneticamente Modificadas , Plântula/genética , Plântula/metabolismo , Proteína Vermelha Fluorescente
6.
Proc Natl Acad Sci U S A ; 117(1): 741-751, 2020 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-31871212

RESUMO

Seeds preserve a far developed plant embryo in a quiescent state. Seed metabolism relies on stored resources and is reactivated to drive germination when the external conditions are favorable. Since the switchover from quiescence to reactivation provides a remarkable case of a cell physiological transition we investigated the earliest events in energy and redox metabolism of Arabidopsis seeds at imbibition. By developing fluorescent protein biosensing in intact seeds, we observed ATP accumulation and oxygen uptake within minutes, indicating rapid activation of mitochondrial respiration, which coincided with a sharp transition from an oxidizing to a more reducing thiol redox environment in the mitochondrial matrix. To identify individual operational protein thiol switches, we captured the fast release of metabolic quiescence in organello and devised quantitative iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. The redox state across all Cys peptides was shifted toward reduction from 27.1% down to 13.0% oxidized thiol. A large number of Cys peptides (412) were redox switched, representing central pathways of mitochondrial energy metabolism, including the respiratory chain and each enzymatic step of the tricarboxylic acid (TCA) cycle. Active site Cys peptides of glutathione reductase 2, NADPH-thioredoxin reductase a/b, and thioredoxin-o1 showed the strongest responses. Germination of seeds lacking those redox proteins was associated with markedly enhanced respiration and deregulated TCA cycle dynamics suggesting decreased resource efficiency of energy metabolism. Germination in aged seeds was strongly impaired. We identify a global operation of thiol redox switches that is required for optimal usage of energy stores by the mitochondria to drive efficient germination.


Assuntos
Arabidopsis/fisiologia , Ciclo do Ácido Cítrico/fisiologia , Germinação/fisiologia , Mitocôndrias/metabolismo , Sementes/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Oxirredução , Oxigênio/metabolismo , Plantas Geneticamente Modificadas , Proteômica/métodos , Sementes/citologia , Sementes/crescimento & desenvolvimento , Tiorredoxina h/genética , Tiorredoxina h/metabolismo , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo
7.
Plant Physiol ; 187(4): 2451-2468, 2021 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-34599589

RESUMO

Plant glutathione S-transferases (GSTs) are glutathione-dependent enzymes with versatile functions, mainly related to detoxification of electrophilic xenobiotics and peroxides. The Arabidopsis (Arabidopsis thaliana) genome codes for 53 GSTs, divided into seven subclasses; however, understanding of their precise functions is limited. A recent study showed that class II TGA transcription factors TGA2, TGA5, and TGA6 are essential for tolerance of UV-B-induced oxidative stress and that this tolerance is associated with an antioxidative function of cytosolic tau-GSTs (GSTUs). Specifically, TGA2 controls the expression of several GSTUs under UV-B light, and constitutive expression of GSTU7 in the tga256 triple mutant is sufficient to revert the UV-B-susceptible phenotype of tga256. To further study the function of GSTU7, we characterized its role in mitigation of oxidative damage caused by the herbicide methyl viologen (MV). Under non-stress conditions, gstu7 null mutants were smaller than wild-type (WT) plants and delayed in the onset of the MV-induced antioxidative response, which led to accumulation of hydrogen peroxide and diminished seedling survival. Complementation of gstu7 by constitutive expression of GSTU7 rescued these phenotypes. Furthermore, live monitoring of the glutathione redox potential in intact cells with the fluorescent probe Grx1-roGFP2 revealed that GSTU7 overexpression completely abolished the MV-induced oxidation of the cytosolic glutathione buffer compared with WT plants. GSTU7 acted as a glutathione peroxidase able to complement the lack of peroxidase-type GSTs in yeast. Together, these findings show that GSTU7 is crucial in the antioxidative response by limiting oxidative damage and thus contributes to oxidative stress resistance in the cell.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/fisiologia , Glutationa Transferase/genética , Herbicidas/efeitos adversos , Estresse Oxidativo , Paraquat/efeitos adversos , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Glutationa Transferase/metabolismo
8.
Plant Physiol ; 186(1): 125-141, 2021 05 27.
Artigo em Inglês | MEDLINE | ID: mdl-33793922

RESUMO

Metabolic fluctuations in chloroplasts and mitochondria can trigger retrograde signals to modify nuclear gene expression. Mobile signals likely to be involved are reactive oxygen species (ROS), which can operate protein redox switches by oxidation of specific cysteine residues. Redox buffers, such as the highly reduced glutathione pool, serve as reservoirs of reducing power for several ROS-scavenging and ROS-induced damage repair pathways. Formation of glutathione disulfide and a shift of the glutathione redox potential (EGSH) toward less negative values is considered as hallmark of several stress conditions. Here we used the herbicide methyl viologen (MV) to generate ROS locally in chloroplasts of intact Arabidopsis (Arabidopsis thaliana) seedlings and recorded dynamic changes in EGSH and H2O2 levels with the genetically encoded biosensors Grx1-roGFP2 (for EGSH) and roGFP2-Orp1 (for H2O2) targeted to chloroplasts, the cytosol, or mitochondria. Treatment of seedlings with MV caused rapid oxidation in chloroplasts and, subsequently, in the cytosol and mitochondria. MV-induced oxidation was significantly boosted by illumination with actinic light, and largely abolished by inhibitors of photosynthetic electron transport. MV also induced autonomous oxidation in the mitochondrial matrix in an electron transport chain activity-dependent manner that was milder than the oxidation triggered in chloroplasts by the combination of MV and light. In vivo redox biosensing resolves the spatiotemporal dynamics of compartmental responses to local ROS generation and provides a basis for understanding how compartment-specific redox dynamics might operate in retrograde signaling and stress acclimation in plants.


Assuntos
Arabidopsis/metabolismo , Cloroplastos/metabolismo , Glutationa/metabolismo , Peróxido de Hidrogênio/metabolismo , Estresse Oxidativo , Arabidopsis/efeitos dos fármacos , Técnicas Biossensoriais , Cloroplastos/efeitos dos fármacos , Herbicidas/efeitos adversos , Oxirredução , Paraquat/efeitos adversos , Plântula/efeitos dos fármacos , Plântula/metabolismo
9.
Plant J ; 101(2): 420-441, 2020 01.
Artigo em Inglês | MEDLINE | ID: mdl-31520498

RESUMO

Mitochondria host vital cellular functions, including oxidative phosphorylation and co-factor biosynthesis, which are reflected in their proteome. At the cellular level plant mitochondria are organized into hundreds of discrete functional entities, which undergo dynamic fission and fusion. It is the individual organelle that operates in the living cell, yet biochemical and physiological assessments have exclusively focused on the characteristics of large populations of mitochondria. Here, we explore the protein composition of an individual average plant mitochondrion to deduce principles of functional and structural organisation. We perform proteomics on purified mitochondria from cultured heterotrophic Arabidopsis cells with intensity-based absolute quantification and scale the dataset to the single organelle based on criteria that are justified by experimental evidence and theoretical considerations. We estimate that a total of 1.4 million protein molecules make up a single Arabidopsis mitochondrion on average. Copy numbers of the individual proteins span five orders of magnitude, ranging from >40 000 for Voltage-Dependent Anion Channel 1 to sub-stoichiometric copy numbers, i.e. less than a single copy per single mitochondrion, for several pentatricopeptide repeat proteins that modify mitochondrial transcripts. For our analysis, we consider the physical and chemical constraints of the single organelle and discuss prominent features of mitochondrial architecture, protein biogenesis, oxidative phosphorylation, metabolism, antioxidant defence, genome maintenance, gene expression, and dynamics. While assessing the limitations of our considerations, we exemplify how our understanding of biochemical function and structural organization of plant mitochondria can be connected in order to obtain global and specific insights into how organelles work.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Mitocôndrias/metabolismo , Organelas/metabolismo , Proteômica , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Bases de Dados de Proteínas , Mitocôndrias/genética , Biogênese de Organelas , Organelas/genética , Proteoma/metabolismo
10.
Proc Natl Acad Sci U S A ; 115(45): E10778-E10787, 2018 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-30352850

RESUMO

Matching ATP:NADPH provision and consumption in the chloroplast is a prerequisite for efficient photosynthesis. In terms of ATP:NADPH ratio, the amount of ATP generated from the linear electron flow does not meet the demand of the Calvin-Benson-Bassham (CBB) cycle. Several different mechanisms to increase ATP availability have evolved, including cyclic electron flow in higher plants and the direct import of mitochondrial-derived ATP in diatoms. By imaging a fluorescent ATP sensor protein expressed in living Arabidopsis thaliana seedlings, we found that MgATP2- concentrations were lower in the stroma of mature chloroplasts than in the cytosol, and exogenous ATP was able to enter chloroplasts isolated from 4- and 5-day-old seedlings, but not chloroplasts isolated from 10- or 20-day-old photosynthetic tissues. This observation is in line with the previous finding that the expression of chloroplast nucleotide transporters (NTTs) in Arabidopsis mesophyll is limited to very young seedlings. Employing a combination of photosynthetic and respiratory inhibitors with compartment-specific imaging of ATP, we corroborate the dependency of stromal ATP production on mitochondrial dissipation of photosynthetic reductant. Our data suggest that, during illumination, the provision and consumption of ATP:NADPH in chloroplasts can be balanced by exporting excess reductants rather than importing ATP from the cytosol.


Assuntos
Trifosfato de Adenosina/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Mitocôndrias/metabolismo , Fotossíntese/genética , Folhas de Planta/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Transporte Biológico , Técnicas Biossensoriais/métodos , Cloroplastos/genética , Citosol/metabolismo , Transferência Ressonante de Energia de Fluorescência , Regulação da Expressão Gênica no Desenvolvimento , Genes Reporter , Luz , NADP/metabolismo , Proteínas de Transporte de Nucleotídeos/genética , Proteínas de Transporte de Nucleotídeos/metabolismo , Oxirredução , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismo , Transdução de Sinais
13.
New Phytol ; 221(3): 1649-1664, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30347449

RESUMO

Hydrogen peroxide (H2 O2 ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H2 O2 notoriously hard to detect dynamically, specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour in vivo have hampered any meaningful H2 O2 sensing in living plants. We established H2 O2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2-Orp1 using confocal microscopy and multiwell fluorimetry. We confirmed sensor oxidation by H2 O2 , show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H2 O2 , pharmacologically-induced H2 O2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H2 O2 dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants. We provided a well defined toolkit for H2 O2 monitoring in planta and showed that intracellular H2 O2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H2 O2 dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.


Assuntos
Arabidopsis/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Peróxido de Hidrogênio/metabolismo , Espaço Intracelular/metabolismo , Explosão Respiratória , Compostos de Sulfidrila/metabolismo , Arabidopsis/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/metabolismo , Glutationa/metabolismo , Concentração de Íons de Hidrogênio , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Oxirredução , Explosão Respiratória/efeitos dos fármacos , Plântula/efeitos dos fármacos , Plântula/metabolismo , Transdução de Sinais/efeitos dos fármacos , Vitamina K 3/farmacologia
14.
New Phytol ; 224(4): 1668-1684, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31386759

RESUMO

Hypoxia regularly occurs during plant development and can be induced by the environment through, for example, flooding. To understand how plant tissue physiology responds to progressing oxygen restriction, we aimed to monitor subcellular physiology in real time and in vivo. We establish a fluorescent protein sensor-based system for multiparametric monitoring of dynamic changes in subcellular physiology of living Arabidopsis thaliana leaves and exemplify its applicability for hypoxia stress. By monitoring cytosolic dynamics of magnesium adenosine 5'-triphosphate, free calcium ion concentration, pH, NAD redox status, and glutathione redox status in parallel, linked to transcriptional and metabolic responses, we generate an integrated picture of the physiological response to progressing hypoxia. We show that the physiological changes are surprisingly robust, even when plant carbon status is modified, as achieved by sucrose feeding or extended night. Inhibition of the mitochondrial respiratory chain causes dynamics of cytosolic physiology that are remarkably similar to those under oxygen depletion, highlighting mitochondrial electron transport as a key determinant of the cellular consequences of hypoxia beyond the organelle. A broadly applicable system for parallel in vivo sensing of plant stress physiology is established to map out the physiological context under which both mitochondrial retrograde signalling and low oxygen signalling occur, indicating shared upstream stimuli.


Assuntos
Arabidopsis/metabolismo , Citosol/metabolismo , Mitocôndrias/metabolismo , Células Vegetais/metabolismo , Trifosfato de Adenosina/metabolismo , Arabidopsis/citologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Cálcio/metabolismo , Carbono/metabolismo , Transporte de Elétrons , Glutationa/metabolismo , Concentração de Íons de Hidrogênio , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , NAD/metabolismo , Oxigênio/metabolismo , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas
15.
Plant Physiol ; 173(2): 1355-1370, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28031475

RESUMO

Over the recent years, several proteins that make up the mitochondrial calcium uniporter complex (MCUC) mediating Ca2+uptake into the mitochondrial matrix have been identified in mammals, including the channel-forming protein MCU. Although six MCU gene homologs are conserved in the model plant Arabidopsis (Arabidopsis thaliana) in which mitochondria can accumulate Ca2+, a functional characterization of plant MCU homologs has been lacking. Using electrophysiology, we show that one isoform, AtMCU1, gives rise to a Ca2+-permeable channel activity that can be observed even in the absence of accessory proteins implicated in the formation of the active mammalian channel. Furthermore, we provide direct evidence that AtMCU1 activity is sensitive to the mitochondrial calcium uniporter inhibitors Ruthenium Red and Gd3+, as well as to the Arabidopsis protein MICU, a regulatory MCUC component. AtMCU1 is prevalently expressed in roots, localizes to mitochondria, and its absence causes mild changes in Ca2+ dynamics as assessed by in vivo measurements in Arabidopsis root tips. Plants either lacking or overexpressing AtMCU1 display root mitochondria with altered ultrastructure and show shorter primary roots under restrictive growth conditions. In summary, our work adds evolutionary depth to the investigation of mitochondrial Ca2+ transport, indicates that AtMCU1, together with MICU as a regulator, represents a functional configuration of the plant mitochondrial Ca2+ uptake complex with differences to the mammalian MCUC, and identifies a new player of the intracellular Ca2+ regulation network in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Canais de Cálcio/metabolismo , Arabidopsis/citologia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cálcio/metabolismo , Canais de Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Regulação da Expressão Gênica de Plantas , Mitocôndrias/genética , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Mutação , Filogenia , Proteínas de Plantas/genética , Raízes de Plantas/genética , Raízes de Plantas/metabolismo
16.
Plant Cell ; 27(11): 3190-212, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26530087

RESUMO

Plant organelle function must constantly adjust to environmental conditions, which requires dynamic coordination. Ca(2+) signaling may play a central role in this process. Free Ca(2+) dynamics are tightly regulated and differ markedly between the cytosol, plastid stroma, and mitochondrial matrix. The mechanistic basis of compartment-specific Ca(2+) dynamics is poorly understood. Here, we studied the function of At-MICU, an EF-hand protein of Arabidopsis thaliana with homology to constituents of the mitochondrial Ca(2+) uniporter machinery in mammals. MICU binds Ca(2+) and localizes to the mitochondria in Arabidopsis. In vivo imaging of roots expressing a genetically encoded Ca(2+) sensor in the mitochondrial matrix revealed that lack of MICU increased resting concentrations of free Ca(2+) in the matrix. Furthermore, Ca(2+) elevations triggered by auxin and extracellular ATP occurred more rapidly and reached higher maximal concentrations in the mitochondria of micu mutants, whereas cytosolic Ca(2+) signatures remained unchanged. These findings support the idea that a conserved uniporter system, with composition and regulation distinct from the mammalian machinery, mediates mitochondrial Ca(2+) uptake in plants under in vivo conditions. They further suggest that MICU acts as a throttle that controls Ca(2+) uptake by moderating influx, thereby shaping Ca(2+) signatures in the matrix and preserving mitochondrial homeostasis. Our results open the door to genetic dissection of mitochondrial Ca(2+) signaling in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Ligação ao Cálcio/metabolismo , Motivos EF Hand , Mitocôndrias/metabolismo , Arabidopsis/genética , Cálcio , Sinalização do Cálcio , Respiração Celular , Citosol/metabolismo , DNA Bacteriano/genética , Mitocôndrias/ultraestrutura , Mutagênese Insercional/genética , Filogenia , Raízes de Plantas/metabolismo , Raízes de Plantas/ultraestrutura , Ligação Proteica , Transporte Proteico , Plântula/metabolismo , Homologia de Sequência de Aminoácidos , Frações Subcelulares/metabolismo
17.
Plant Physiol ; 172(2): 901-912, 2016 10.
Artigo em Inglês | MEDLINE | ID: mdl-27506242

RESUMO

Glycolysis generates methylglyoxal (MGO) as an unavoidable, cytotoxic by-product in plant cells. MGO scavenging is performed by the glyoxalase system, which produces d-lactate as an end product. d-Lactate dehydrogenase (d-LDH) is encoded by a single gene in Arabidopsis (Arabidopsis thaliana; At5g06580). It catalyzes in vitro the oxidation of d-lactate to pyruvate using flavin adenine dinucleotide as a cofactor; knowledge of its function in the context of the plant cell remains sketchy. Blue native-polyacrylamide gel electrophoresis of mitochondrial extracts combined with in gel activity assays using different substrates and tandem mass spectrometry allowed us to definitely show that d-LDH acts specifically on d-lactate, is active as a dimer, and does not associate with respiratory supercomplexes of the inner mitochondrial membrane. The combined use of cytochrome c (CYTc) loss-of-function mutants and respiratory complex III inhibitors showed that CYTc acts as the in vivo electron acceptor of d-LDH. CYTc loss-of-function mutants, as well as the d-LDH mutants, were more sensitive to d-lactate and MGO, indicating that they function in the same pathway. In addition, overexpression of d-LDH and CYTc increased tolerance to d-lactate and MGO Together with fine-localization of d-LDH, the functional interaction with CYTc in vivo strongly suggests that d-lactate oxidation takes place in the mitochondrial intermembrane space, delivering electrons to the respiratory chain through CYTc These results provide a comprehensive picture of the organization and function of d-LDH in the plant cell and exemplify how the plant mitochondrial respiratory chain can act as a multifunctional electron sink for reductant from cytosolic pathways.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Citocromos c/metabolismo , L-Lactato Desidrogenase/metabolismo , Aldeído Pirúvico/metabolismo , Arabidopsis/citologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Biocatálise , Western Blotting , Células Cultivadas , Citocromos c/genética , Transporte de Elétrons , L-Lactato Desidrogenase/genética , Ácido Láctico/metabolismo , Espectrometria de Massas , Microscopia Confocal , Membranas Mitocondriais/metabolismo , Mutação , Oxirredução , Consumo de Oxigênio , Plantas Geneticamente Modificadas , Ácido Pirúvico/metabolismo
19.
Phys Rev E ; 106(1-1): 014803, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35974632

RESUMO

Minimal surface problems arise naturally in many soft matter systems whose free energies are dominated by surface or interface energies. Of particular interest are the shapes, stability, and mechanical stresses of minimal surfaces spanning specific geometric boundaries. The "catenoid" is the best-known example where an analytical solution is known which describes the form and stability of a minimal surface held between two parallel, concentric circular frames. Here we extend this problem to nonaxisymmetric, parallel frame shapes of different orientations by developing a perturbation approach around the known catenoid solution. We show that the predictions of the perturbation theory are in good agreement with experiments on soap films and finite element simulations (Surface Evolver). Combining theory, experiment, and simulation, we analyze in depth how the shapes, stability, and mechanical properties of the minimal surfaces depend on the type and orientation of elliptic and three-leaf clover shaped frames. In the limit of perfectly aligned nonaxisymmetric frames, our predictions show excellent agreement with a recent theory established by Alimov et al. [Phys. Fluids 33, 052104 (2021)1070-663110.1063/5.0047461]. Moreover, we put in evidence the intriguing capacity of minimal surfaces between nonaxisymmetric frames to transmit a mechanical torque despite being completely liquid. These forces could be interesting to exploit for mechanical self-assembly of soft matter systems or as highly sensitive force captors.

20.
Clin Cancer Res ; 27(3): 775-784, 2021 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-33060122

RESUMO

PURPOSE: Recent data have shown that single-fraction irradiation delivered to the whole brain in less than tenths of a second using FLASH radiotherapy (FLASH-RT), does not elicit neurocognitive deficits in mice. This observation has important clinical implications for the management of invasive and treatment-resistant brain tumors that involves relatively large irradiation volumes with high cytotoxic doses. EXPERIMENTAL DESIGN: Therefore, we aimed at simultaneously investigating the antitumor efficacy and neuroprotective benefits of FLASH-RT 1-month after exposure, using a well-characterized murine orthotopic glioblastoma model. As fractionated regimens of radiotherapy are the standard of care for glioblastoma treatment, we incorporated dose fractionation to simultaneously validate the neuroprotective effects and optimized tumor treatments with FLASH-RT. RESULTS: The capability of FLASH-RT to minimize the induction of radiation-induced brain toxicities has been attributed to the reduction of reactive oxygen species, casting some concern that this might translate to a possible loss of antitumor efficacy. Our study shows that FLASH and CONV-RT are isoefficient in delaying glioblastoma growth for all tested regimens. Furthermore, only FLASH-RT was found to significantly spare radiation-induced cognitive deficits in learning and memory in tumor-bearing animals after the delivery of large neurotoxic single dose or hypofractionated regimens. CONCLUSIONS: The present results show that FLASH-RT delivered with hypofractionated regimens is able to spare the normal brain from radiation-induced toxicities without compromising tumor cure. This exciting capability provides an initial framework for future clinical applications of FLASH-RT.See related commentary by Huang and Mendonca, p. 662.


Assuntos
Neoplasias Encefálicas/radioterapia , Disfunção Cognitiva/prevenção & controle , Elétrons/uso terapêutico , Glioblastoma/radioterapia , Lesões Experimentais por Radiação/prevenção & controle , Animais , Encéfalo/fisiopatologia , Encéfalo/efeitos da radiação , Disfunção Cognitiva/diagnóstico , Disfunção Cognitiva/etiologia , Disfunção Cognitiva/fisiopatologia , Feminino , Humanos , Camundongos , Órgãos em Risco/fisiopatologia , Órgãos em Risco/efeitos da radiação , Hipofracionamento da Dose de Radiação , Lesões Experimentais por Radiação/diagnóstico , Lesões Experimentais por Radiação/etiologia , Lesões Experimentais por Radiação/fisiopatologia , Dosagem Radioterapêutica , Espécies Reativas de Oxigênio
SELEÇÃO DE REFERÊNCIAS
Detalhe da pesquisa