Alternative oxidase 1a and 1d enable metabolic flexibility during Ala catabolism in Arabidopsis.

Ala is a central metabolite in leaf cells whose abundance is related to pyruvate (Pyr) metabolism and nocturnal respiration rates. Exposure of Arabidopsis (Arabidopsis thaliana) leaf disks to certain exogenous amino acids including Ala led to substantial increases in nighttime respiration rates as well as increases in alternative oxidase (AOX) 1d transcript and protein levels. During Ala treatment, AOX1d accumulation, but not AOX1a accumulation, was dependent upon the catabolism of Ala. Complete loss of AOX expression in aox1a aox1d leaf disks did not significantly affect oxygen consumption rates (OCR) under Ala treatment, indicating that AOX capacity per se was not essential for respiratory stimulation by Ala. Rather, Ala treatments caused induction of select antioxidant mechanisms in leaf disks, including a large increase of the ascorbate pool, which was substantially more oxidized in aox1a aox1d leaf disks. Furthermore, we observed differences in the accumulation of a sequence of TCA cycle intermediates from Pyr to 2-oxoglutarate (2-OG) in wild type (WT) upon Ala treatment that did not occur in aox1a aox1d leaf disks. The results indicate that AOX induction during enhanced Ala catabolism in leaves mediates mitochondrial redox status, allowing greater metabolic flexibility in mitochondrial organic acid metabolism.

High-throughput, dynamic, multi-dimensional: an expanding repertoire of plant respiration measurements.

A recent burst of technological innovation and adaptation has greatly improved our ability to capture respiration rate data from plant sources. At the tissue level, several independent respiration measurement options are now available, each with distinct advantages and suitability, including high-throughput sampling capacity. These advancements facilitate the inclusion of respiration rate data into large-scale biological studies such as genetic screens, ecological surveys, crop breeding trials, and multi-omics molecular studies. As a result, our understanding of the correlations of respiration with other biological and biochemical measurements is rapidly increasing. Difficult questions persist concerning the interpretation and utilization of respiration data; concepts such as allocation of respiration to growth versus maintenance, the unnecessary or inefficient use of carbon and energy by respiration, and predictions of future respiration rates in response to environmental change are all insufficiently grounded in empirical data. However, we emphasize that new experimental designs involving novel combinations of respiration rate data with other measurements will flesh-out our current theories of respiration. Furthermore, dynamic recordings of respiration rate, which have long been used at the scale of mitochondria, are increasingly being used at larger scales of size and time to reflect processes of cellular signal transduction and physiological response to the environment. We also highlight how respiratory methods are being better adapted to different plant tissues including roots and seeds, which have been somewhat neglected historically.

High-Throughput Oxygen Consumption Measurements in Leaf Tissue Using Oxygen Sensitive Fluorophores.

Respiratory rate measurements are crucial assays to understand mitochondrial biochemistry as well as metabolic regulation within tissues. Several technologies currently exist that can measure plant respiratory oxygen consumption or carbon dioxide evolution rates over short durations by either isolated mitochondria or plant tissues. Here we describe recently developed alternative methods for measuring tissue oxygen consumption rates (OCRs) using systems reliant on oxygen sensitive fluorophores. The methods described have distinct experimental advantages: they can allow high-throughput and long-duration measurements; and they are particularly suited to investigating the metabolic regulation of respiration by comparing OCRs among treatments or genotypes.

Alternative oxidase (AOX) 1a and 1d limit proline-induced oxidative stress and aid salinity recovery in Arabidopsis.

Proline (Pro) catabolism and reactive oxygen species production have been linked in mammals and Caenorhabditis elegans, while increases in leaf respiration rate follow Pro exposure in plants. Here, we investigated how alternative oxidases (AOXs) of the mitochondrial electron transport chain accommodate the large, atypical flux resulting from Pro catabolism and limit oxidative stress during Pro breakdown in mature Arabidopsis (Arabidopsis thaliana) leaves. Following Pro treatment, AOX1a and AOX1d accumulate at transcript and protein levels, with AOX1d approaching the level of the typically dominant AOX1a isoform. We therefore sought to determine the function of both AOX isoforms under Pro respiring conditions. Oxygen consumption rate measurements in aox1a and aox1d leaves suggested these AOXs can functionally compensate for each other to establish enhanced AOX catalytic capacity in response to Pro. Generation of aox1a.aox1d lines showed complete loss of AOX proteins and activity upon Pro treatment, yet full respiratory induction in response to Pro remained possible via the cytochrome pathway. However, aox1a.aox1d leaves displayed symptoms of elevated oxidative stress and suffered increased oxidative damage during Pro metabolism compared to the wild-type (WT) or the single mutants. During recovery from salt stress, when relatively high rates of Pro catabolism occur naturally, photosynthetic rates in aox1a.aox1d recovered slower than in the WT or the single aox lines, showing that both AOX1a and AOX1d are beneficial for cellular metabolism during Pro drawdown following osmotic stress. This work provides physiological evidence of a beneficial role for AOX1a but also the less studied AOX1d isoform in allowing safe catabolism of alternative respiratory substrates like Pro.

Rubisco lysine acetylation occurs at very low stoichiometry in mature Arabidopsis leaves: implications for regulation of enzyme function.

Multiple studies have shown ribulose-1,5-bisphosphate carboxylase/oxygenase (E.C. 4.1.1.39; Rubisco) to be subject to Lys-acetylation at various residues; however, opposing reports exist about the biological significance of these post-translational modifications. One aspect of the Lys-acetylation that has not been addressed in plants generally, or with Rubisco specifically, is the stoichiometry at which these Lys-acetylation events occur. As a method to ascertain which Lys-acetylation sites on Arabidopsis Rubisco might be of regulatory importance to its catalytic function in the Calvin-Benson cycle, we purified Rubisco from leaves in both the day and night-time and performed independent mass spectrometry based methods to determine the stoichiometry of Rubisco Lys-acetylation events. The results indicate that Rubisco is acetylated at most Lys residues, but each acetylation event occurs at very low stoichiometry. Furthermore, in vitro treatments that increased the extent of Lys-acetylation on purified Rubisco had no effect on Rubisco maximal activity. Therefore, we are unable to confirm that Lys-acetylation at low stoichiometries can be a regulatory mechanism controlling Rubisco maximal activity. The results highlight the need for further use of stoichiometry measurements when determining the biological significance of reversible PTMs like acetylation.

Metabolite Regulatory Interactions Control Plant Respiratory Metabolism via Target of Rapamycin (TOR) Kinase Activation.

Respiration rate measurements provide an important readout of energy expenditure and mitochondrial activity in plant cells during the night. As plants inhabit a changing environment, regulatory mechanisms must ensure that respiratory metabolism rapidly and effectively adjusts to the metabolic and environmental conditions of the cell. Using a high-throughput approach, we have directly identified specific metabolites that exert transcriptional, translational, and posttranslational control over the nighttime O2 consumption rate (RN) in mature leaves of Arabidopsis (Arabidopsis thaliana). Multi-hour RN measurements following leaf disc exposure to a wide array of primary carbon metabolites (carbohydrates, amino acids, and organic acids) identified phosphoenolpyruvate (PEP), Pro, and Ala as the most potent stimulators of plant leaf RN Using metabolite combinations, we discovered metabolite-metabolite regulatory interactions controlling RN Many amino acids, as well as Glc analogs, were found to potently inhibit the RN stimulation by Pro and Ala but not PEP. The inhibitory effects of amino acids on Pro- and Ala-stimulated RN were mitigated by inhibition of the Target of Rapamycin (TOR) kinase signaling pathway. Supporting the involvement of TOR, these inhibitory amino acids were also shown to be activators of TOR kinase. This work provides direct evidence that the TOR signaling pathway in plants responds to amino acid levels by eliciting regulatory effects on respiratory energy metabolism at night, uniting a hallmark mechanism of TOR regulation across eukaryotes.

Core principles which explain variation in respiration across biological scales.

Contents Summary 670 I. Introduction 671 II. Principle 1 - Plant respiration performs three distinct functions 673 III. Principle 2 - Metabolic pathway flexibility underlies plant respiratory performance 676 IV. Principle 3 - Supply and demand interact over time to set plant respiration rate 677 V. Principle 4 - Plant respiratory acclimation involves adjustments in enzyme capacities 679 VI. Principle 5 - Respiration is a complex trait that helps to define, and is impacted by, plant lifestyle strategies 680 VII. Future directions 680 Acknowledgements 682 References 682 SUMMARY: Respiration is a core biological process that has important implications for the biochemistry, physiology, and ecology of plants. The study of plant respiration is thus conducted from several different perspectives by a range of scientific disciplines with dissimilar objectives, such as metabolic engineering, crop breeding, and climate-change modelling. One aspect in common among the different objectives is a need to understand and quantify the variation in respiration across scales of biological organization. The central tenet of this review is that different perspectives on respiration can complement each other when connected. To better accommodate interdisciplinary thinking, we identify distinct mechanisms which encompass the variation in respiratory rates and functions across biological scales. The relevance of these mechanisms towards variation in plant respiration are explained in the context of five core principles: (1) respiration performs three distinct functions; (2) metabolic pathway flexibility underlies respiratory performance; (3) supply and demand interact over time to set respiration rates; (4) acclimation involves adjustments in enzyme capacities; and (5) respiration is a complex trait that helps to define, and is impacted by, plant lifestyle strategies. We argue that each perspective on respiration rests on these principles to varying degrees and that broader appreciation of how respiratory variation occurs can unite research across scales.

Variation in Leaf Respiration Rates at Night Correlates with Carbohydrate and Amino Acid Supply.

Plant respiration can theoretically be fueled by and dependent upon an array of central metabolism components; however, which ones are responsible for the quantitative variation found in respiratory rates is unknown. Here, large-scale screens revealed 2-fold variation in nighttime leaf respiration rate (RN) among mature leaves from an Arabidopsis (Arabidopsis thaliana) natural accession collection grown under common favorable conditions. RN variation was mostly maintained in the absence of genetic variation, which emphasized the low heritability of RN and its plasticity toward relatively small environmental differences within the sampling regime. To pursue metabolic explanations for leaf RN variation, parallel metabolite level profiling and assays of total protein and starch were performed. Within an accession, RN correlated strongly with stored carbon substrates, including starch and dicarboxylic acids, as well as sucrose, major amino acids, shikimate, and salicylic acid. Among different accessions, metabolite-RN correlations were maintained with protein, sucrose, and major amino acids but not stored carbon substrates. A complementary screen of the effect of exogenous metabolites and effectors on leaf RN revealed that (1) RN is stimulated by the uncoupler FCCP and high levels of substrates, demonstrating that both adenylate turnover and substrate supply can limit leaf RN, and (2) inorganic nitrogen did not stimulate RN, consistent with limited nighttime nitrogen assimilation. Simultaneous measurements of RN and protein synthesis revealed that these processes were largely uncorrelated in mature leaves. These results indicate that differences in preceding daytime metabolic activities are the major source of variation in mature leaf RN under favorable controlled conditions.

Early changes in apoplast composition associated with defence and disease in interactions between Phaseolus vulgaris and the halo blight pathogen Pseudomonas syringae Pv. phaseolicola.

The apoplast is the arena in which endophytic pathogens such as Pseudomonas syringae grow and interact with plant cells. Using metabolomic and ion analysis techniques, this study shows how the composition of Phaseolus vulgaris leaf apoplastic fluid changes during the first six hours of compatible and incompatible interactions with two strains of P. syringae pv. phaseolicola (Pph) that differ in the presence of the genomic island PPHGI-1. Leaf inoculation with the avirulent island-carrying strain Pph 1302A elicited effector-triggered immunity (ETI) and resulted in specific changes in apoplast composition, including increases in conductivity, pH, citrate, γ-aminobutyrate (GABA) and K(+) , that are linked to the onset of plant defence responses. Other apoplastic changes, including increases in Ca(2+) , Fe(2/3+) Mg(2+) , sucrose, ß-cyanoalanine and several amino acids, occurred to a relatively similar extent in interactions with both Pph 1302A and the virulent, island-less strain Pph RJ3. Metabolic footprinting experiments established that Pph preferentially metabolizes malate, glucose and glutamate, but excludes certain other abundant apoplastic metabolites, including citrate and GABA, until preferred metabolites are depleted. These results demonstrate that Pph is well-adapted to the leaf apoplast metabolic environment and that loss of PPHGI-1 enables Pph to avoid changes in apoplast composition linked to plant defences.