RESUMO
Diurnal dark to light transition causes profound physiological changes in plant metabolism. These changes require distinct modes of regulation as a unique feature of photosynthetic lifestyle. The activities of several key metabolic enzymes are regulated by light-dependent post-translational modifications (PTM) and have been studied at depth at the level of individual proteins. In contrast, a global picture of the light-dependent PTMome dynamics is lacking, leaving the response of a large proportion of cellular function undefined. Here, we investigated the light-dependent metabolome and proteome changes in Arabidopsis rosettes in a time resolved manner to dissect their kinetic interplay, focusing on phosphorylation, lysine acetylation, and cysteine-based redox switches. Of over 24 000 PTM sites that were detected, more than 1700 were changed during the transition from dark to light. While the first changes, as measured 5 min after onset of illumination, occurred mainly in the chloroplasts, PTM changes at proteins in other compartments coincided with the full activation of the Calvin-Benson cycle and the synthesis of sugars at later timepoints. Our data reveal connections between metabolism and PTM-based regulation throughout the cell. The comprehensive multiome profiling analysis provides unique insight into the extent by which photosynthesis reprograms global cell function and adds a powerful resource for the dissection of diverse cellular processes in the context of photosynthetic function.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/metabolismo , Fotossíntese , Processamento de Proteína Pós-Traducional , Proteínas de Arabidopsis/metabolismo , Cloroplastos/metabolismoRESUMO
Plants need to rapidly and flexibly adjust their metabolism to changes of their immediate environment. Since this necessity results from the sessile lifestyle of land plants, key mechanisms for orchestrating central metabolic acclimation are likely to have evolved early. Here, we explore the role of lysine acetylation as a post-translational modification to directly modulate metabolic function. We generated a lysine acetylome of the moss Physcomitrium patens and identified 638 lysine acetylation sites, mostly found in mitochondrial and plastidial proteins. A comparison with available angiosperm data pinpointed lysine acetylation as a conserved regulatory strategy in land plants. Focusing on mitochondrial central metabolism, we functionally analyzed acetylation of mitochondrial malate dehydrogenase (mMDH), which acts as a hub of plant metabolic flexibility. In P. patens mMDH1, we detected a single acetylated lysine located next to one of the four acetylation sites detected in Arabidopsis thaliana mMDH1. We assessed the kinetic behavior of recombinant A. thaliana and P. patens mMDH1 with site-specifically incorporated acetyl-lysines. Acetylation of A. thaliana mMDH1 at K169, K170, and K334 decreases its oxaloacetate reduction activity, while acetylation of P. patens mMDH1 at K172 increases this activity. We found modulation of the malate oxidation activity only in A. thaliana mMDH1, where acetylation of K334 strongly activated it. Comparative homology modeling of MDH proteins revealed that evolutionarily conserved lysines serve as hotspots of acetylation. Our combined analyses indicate lysine acetylation as a common strategy to fine-tune the activity of central metabolic enzymes with likely impact on plant acclimation capacity.
Assuntos
Embriófitas/enzimologia , Malato Desidrogenase/metabolismo , Processamento de Proteína Pós-Traducional , Acetilação , Embriófitas/genética , Lisina/metabolismo , Malato Desidrogenase/genética , Mitocôndrias/enzimologia , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
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/metabolismoRESUMO
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 FluorescenteRESUMO
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/metabolismoRESUMO
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 ModificadasRESUMO
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/farmacologiaAssuntos
Mitocôndrias/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Regulação da Expressão Gênica de Plantas , Mitocôndrias/genética , Óxido Nítrico/metabolismo , Oxigênio/metabolismo , Fenômenos Fisiológicos Vegetais , Proteínas de Plantas/genética , Processamento de Proteína Pós-Traducional , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Estresse FisiológicoRESUMO
Growth and development of plants is ultimately driven by light energy captured through photosynthesis. ATP acts as universal cellular energy cofactor fuelling all life processes, including gene expression, metabolism, and transport. Despite a mechanistic understanding of ATP biochemistry, ATP dynamics in the living plant have been largely elusive. Here, we establish MgATP2- measurement in living plants using the fluorescent protein biosensor ATeam1.03-nD/nA. We generate Arabidopsis sensor lines and investigate the sensor in vitro under conditions appropriate for the plant cytosol. We establish an assay for ATP fluxes in isolated mitochondria, and demonstrate that the sensor responds rapidly and reliably to MgATP2- changes in planta. A MgATP2- map of the Arabidopsis seedling highlights different MgATP2- concentrations between tissues and within individual cell types, such as root hairs. Progression of hypoxia reveals substantial plasticity of ATP homeostasis in seedlings, demonstrating that ATP dynamics can be monitored in the living plant.