Summary 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.

Hydrogen peroxide (H 2 O 2 ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H 2 O 2 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 H 2 O 2 sensing in living plants.We established H 2 O 2 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 H 2 O 2 , show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H 2 O 2 , pharmacologically-induced H 2 O 2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H 2 O 2 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 H 2 O 2 monitoring in planta and showed that intracellular H 2 O 2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H 2 O 2 dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.

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.DOI: http://dx.doi.org/10.7554/eLife.26770.001

ORCID IDs: 0000-0001-5369-7911 (S.W.); 0000-0003-4024-968X (O.V.A.); 0000-0003-0796-8308 (M.S.).Cells of complex organisms typically rely on mitochondria for energy provision. The amount of energy required to sustain cellular activity can vary strongly depending on external conditions. Vice versa, constraints on respiratory activity due to metabolic status or stress insult require mitochondrial signaling to coordinate cellular physiology with the function of the organelle. In this update, we review recent insights into plant mitochondrial energy signaling in the light of their significance to stress acclimation. First, we focus on the characteristic adjustments of the nuclear transcriptome that occur after pharmacological inhibition of the mitochondrial electron transport chain as the output of mitochondrial retrograde signaling. Second, we discuss the proteins that have recently been identified as regulators of the transcript responses and the emerging picture of their action as a signaling network. We then pose the question of how well our current models of inducing mitochondrial dysfunction relate to conditions that plants face naturally. We reason that low-oxygen stress shows striking similarities with electron transport inhibitors with respect to their impact on mitochondrial energy physiology upstream, as well as the cellular transcriptomic response. Finally, we highlight and discuss changes in mitochondrial physiology that are common to both stimuli as candidates for upstream signals. The aim of this update is to better define the physiological context in which mitochondrial signaling operates to provide new directions for future research. RESPONSES TO MITOCHONDRIAL ENERGY SIGNALING AT THE TRANSCRIPT LEVEL