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 (H₂O₂) operates as a signaling molecule in eukaryotes, but the specificity of its signaling capacities remains largely unrevealed. Here, we analyzed whether a moderate production of H₂O₂ from two different plant cellular compartments has divergent effects on the plant transcriptome. Arabidopsis thaliana overexpressing glycolate oxidase in the chloroplast (Fahnenstich et al., 2008; Balazadeh et al., 2012) and plants deficient in peroxisomal catalase (Queval et al., 2007; Inzé et al., 2012) were grown under non-photorespiratory conditions and then transferred to photorespiratory conditions to foster the production of H₂O₂ in both organelles. We show that H₂O₂ originating in a specific organelle induces two types of responses: one that integrates signals independently from the subcellular site of H₂O₂ production and another that is dependent on the H₂O₂ production site. H₂O₂ produced in peroxisomes induces transcripts involved in protein repair responses, while H₂O₂ produced in chloroplasts induces early signaling responses, including transcription factors and biosynthetic genes involved in production of secondary signaling messengers. There is a significant bias towards the induction of genes involved in responses to wounding and pathogen attack by chloroplastic-produced H₂O₂, including indolic glucosinolates-, camalexin-, and stigmasterol-biosynthetic genes. These transcriptional responses were accompanied by the accumulation of 4-methoxy-indol-3-ylmethyl glucosinolate and stigmasterol.

The photorespiratory pathway helps illuminated C₃-plants under conditions of limited CO₂ availability by effectively exporting reducing equivalents in form of glycolate out of the chloroplast and regenerating glycerate-3-P as substrate for RubisCO. On the other hand, this pathway is considered as probably futile because previously assimilated CO₂ is released in mitochondria. Consequently, a lot of effort has been made to reduce this CO₂ loss either by reducing fluxes via engineering RubisCO or circumventing mitochondrial CO₂ release by the introduction of new enzyme activities. Here we present an approach following the latter route, introducing a complete glycolate catabolic cycle in chloroplasts of Arabidopsis thaliana comprising glycolate oxidase (GO), malate synthase (MS), and catalase (CAT). Results from plants bearing both GO and MS activities have already been reported (Fahnenstich et al., 2008). This previous work showed that the H₂O₂ produced by GO had strongly negative effects. These effects can be prevented by introducing a plastidial catalase activity, as reported here. Transgenic lines bearing all three transgenic enzyme activities were identified and some with higher CAT activity showed higher dry weight, higher photosynthetic rates, and changes in glycine/serine ratio compared to the wild type. This indicates that the fine-tuning of transgenic enzyme activities in the chloroplasts seems crucial and strongly suggests that the approach is valid and that it is possible to improve the growth of A. thaliana by introducing a synthetic glycolate oxidative cycle into chloroplasts.

Photorespiration is one of the major carbon metabolism pathways in oxygen-producing photosynthetic organisms. This pathway recycles 2-phosphoglycolate (2-PG), a toxic metabolite, to 3-phosphoglycerate when ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) uses oxygen instead of carbon dioxide. The photorespiratory cycle is in competition with photosynthetic CO₂ fixation and it is accompanied by carbon, nitrogen and energy losses. Thus, photorespiration has become a target to improve crop yields. Moreover, during the photorespiratory cycle intermediate metabolites that are toxic to Calvin–Benson cycle and RuBisCO activities, such as 2-PG, glycolate and glyoxylate, are produced. Thus, the presence of an efficient 2-PG/glycolate/glyoxylate ‘detoxification’ pathway is required to ensure normal development of photosynthetic organisms. Here we review our current knowledge concerning the enzymes that carry out the glycolate–glyoxylate metabolic steps of photorespiration from glycolate production in the chloroplasts to the synthesis of glycine in the peroxisomes. We describe the properties of the proteins involved in glycolate–glyoxylate metabolism in Archaeplastida and the phenotypes observed when knocking down/out these specific photorespiratory players. Advances in our understanding of the regulation of glycolate–glyoxylate metabolism are highlighted.

In the C₄ pathway of photosynthesis two types of malate decarboxylases release CO₂ in bundle sheath cells, NADP- and NAD-dependent malic enzyme (NADP-ME and NAD-ME), located in the chloroplasts and the mitochondria of these cells, respectively. The C₄ decarboxylases involved in C₄ photosynthesis did not evolve de novo; they were recruited from existing housekeeping isoforms. NADP-ME housekeeping isoforms would function in the control of malate levels during hypoxia, pathogen defence responses, and microspore separation, while NAD-ME participates in the respiration of malate in the tricarboxylic acid cycle. Recently, the existence of three enzymatic NAD-ME entities in Arabidopsis, occurring by alternative association of two subunits, was described as a novel mechanism to regulate NAD-ME activity under changing metabolic environments. The C₄ NADP-ME is thought to have evolved from a C₃ chloroplastic ancestor, which in turn would have evolved from an ancient cytosolic enzyme. In this way, the C₄ NADP-ME would have emerged through gene duplication, acquisition of a new promoter, and neo-functionalization. In contrast, there would exist a unique NAD-ME in C₄ plants, which would have been adapted to perform a dual function through changes in the kinetic and regulatory properties of the C₃ ancestors. In addition to this, for the evolution of C₄ NAD-ME, insertion of promoters or enhancers into the single-copy genes of the C₃ ancestors would have changed the expression without gene duplication.

Glycolate, malate, lactate, and 2-hydroxyglutarate are important 2-hydroxy acids (2HA) in plant metabolism. Most of them can be found as D- and L-stereoisomers. These 2HA play an integral role in plant primary metabolism, where they are involved in fundamental pathways such as photorespiration, tricarboxylic acid cycle, glyoxylate cycle, methylglyoxal pathway, and lysine catabolism. Recent molecular studies in Arabidopsis thaliana have helped elucidate the participation of these 2HA in in plant metabolism and physiology. In this chapter, we summarize the current knowledge about the metabolic pathways and cellular processes in which they are involved, focusing on the proteins that participate in their metabolism and cellular/intracellular transport in Arabidopsis.