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Macrophages fight infection and ensure tissue repair, often operating at nutrient-poor wound sites. We investigated the ability of human macrophages to metabolize glycogen. We observed that the cytokines GM-CSF and M-CSF plus IL-4 induced glycogenesis and the accumulation of glycogen by monocyte-derived macrophages. Glyconeogenesis occurs in cells cultured in the presence of the inflammatory cytokines GM-CSF and IFNγ (M1 cells), via phosphoenolpyruvate carboxykinase 2 (PCK2) and fructose-1,6-bisphosphatase 1 (FBP1). Enzyme inhibition with drugs or gene silencing techniques and 13 C-tracing demonstrate that glutamine (metabolized by the TCA cycle), lactic acid, and glycerol were substrates of glyconeogenesis only in M1 cells. Tumor-associated macrophages (TAMs) also store glycogen and can perform glyconeogenesis. Finally, macrophage glycogenolysis and the pentose phosphate pathway (PPP) support cytokine secretion and phagocytosis regardless of the availability of extracellular glucose. Thus, glycogen metabolism supports the functions of human M1 and M2 cells, with inflammatory M1 cells displaying a possible dependence on glyconeogenesis., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

Carbon isotope discrimination (∆) in leaf biomass (∆ BL ) and tree rings (∆ TR ) provides important proxies for plant responses to climate change, specifically in terms of intrinsic water-use efficiency (iWUE). However, the nonphotosynthetic 12 C/ 13 C fractionation in plant tissues has rarely been quantified and its influence on iWUE estimation remains uncertain. We derived a comprehensive, ∆ based iWUE model (iWUE com ) which includes nonphotosynthetic fractionations (d) and characterized tissue-specific d-values based on global compilations of data of ∆ BL , ∆ TR and real-time ∆ in leaf photosynthesis (∆ online ). iWUE com was further validated with independent datasets. ∆ BL was larger than ∆ online by 2.53‰, while ∆ BL and ∆ TR showed a mean offset of 2.76‰, indicating that ∆ TR is quantitatively very similar to ∆ online . Applying the tissue-specific d-values (d BL  = 2.5‰, d TR  = 0‰), iWUE estimated from ∆ BL aligned well with those estimated from ∆ TR or gas exchange. ∆ BL and ∆ TR showed a consistent iWUE trend with an average CO 2 sensitivity of 0.15 ppm ppm -1 during 1975-2015. Accounting for nonphotosynthetic fractionations improves the estimation of iWUE based on isotope records in leaf biomass and tree rings, which is ultimate for inferring changes in carbon and water cycles under historical and future climate., (© 2024 The Author(s). New Phytologist © 2024 New Phytologist Foundation.)

Although primarily studied in relation to photorespiration, serine metabolism in chloroplasts may play a key role in plant CO 2 fertilization responses by linking CO 2 assimilation with growth. Here, we show that the phosphorylated serine pathway is part of a 'photosynthetic C 1 pathway' and demonstrate its high activity in foliage of a C 3 tree where it rapidly integrates photosynthesis and C 1 metabolism contributing to new biomass via methyl transfer reactions, imparting a large natural 13 C-depleted signature. Using 13 CO 2 -labelling, we show that leaf serine, the S-methyl group of leaf methionine, pectin methyl esters, and the associated methanol released during cell wall expansion during growth, are directly produced from photosynthetically-linked C 1 metabolism, within minutes of light exposure. We speculate that the photosynthetic C 1 pathway is highly conserved across the photosynthetic tree of life, is responsible for synthesis of the greenhouse gas methane, and may have evolved with oxygenic photosynthesis by providing a mechanism of directly linking carbon and ammonia assimilation with growth. Although the rise in atmospheric CO 2 inhibits major metabolic pathways like photorespiration, our results suggest that the photosynthetic C 1 pathway may accelerate and represents a missing link between enhanced photosynthesis and plant growth rates during CO 2 fertilization under a changing climate., (© 2024. The Author(s).)

Oil palm is presently the most important oil-producing crop worldwide in terms of oil production and consumption. However, oil palm cultivation faces important challenges such as adverse climatic conditions, expensive fertilization requirements, and fungal pathogens, including Ganoderma . Intense efforts in oil palm breeding are devoted to improving both oil production yield and resistance to environmental cues. Metabolomics can be of interest because it provides many quantitative traits and metabolic signatures that can be selected for to optimize oil palm performance. Here, we briefly review how metabolomics can help oil palm breeding, and to do so, we give examples of recent metabolomics analyses and provide a roadmap to use metabolomics-assisted breeding.

Little is known about the effect of nitrogen nutrition on seedling susceptibility to seed-borne pathogens. We have previously shown that seedlings grown under high nitrate (5 mM) conditions are less susceptible than those grown under low nitrate (0.1 mM) and ammonium (5 mM) in the Arabidopsis- Alternaria brassicicola pathosystem. However, it is not known how seedling metabolism is modulated by nitrogen nutrition, nor what is its response to pathogen infection. Here, we addressed this question using the same pathosystem and nutritive conditions, examining germination kinetics, seedling development, but also shoot ion contents, metabolome, and selected gene expression. Nitrogen nutrition clearly altered the seedling metabolome. A similar metabolomic profile was observed in inoculated seedlings grown at high nitrate levels and in not inoculated-seedlings. High nitrate levels also led to specific gene expression patterns (e.g., polyamine metabolism), while other genes responded to inoculation regardless of nitrogen supply conditions. Furthermore, the metabolites best correlated with high disease symptoms were coumarate, tyrosine, hemicellulose sugars, and polyamines, and those associated with low symptoms were organic acids (tricarboxylic acid pathway, glycerate, shikimate), sugars derivatives and β-alanine. Overall, our results suggest that the beneficial effect of high nitrate nutrition on seedling susceptibility is likely due to nutritive and signaling mechanisms affecting developmental plant processes detrimental to the pathogen. In particular, it may be due to a constitutively high tryptophan metabolism, as well as down regulation of oxidative stress caused by polyamine catabolism.

Our ability to predict temperature responses of leaf respiration in light and darkness (R L and R Dk ) is essential to models of global carbon dynamics. While many models rely on constant thermal sensitivity (characterized by Q 10 ), uncertainty remains as to whether Q 10 of R L and R Dk are actually similar. We measured short-term temperature responses of R L and R Dk in immature and mature leaves of two evergreen tree species, Castanopsis carlesii and Ormosia henry in an open field. R L was estimated by the Kok method, the Yin method and a newly developed Kok-iterC c method. When estimated by the Yin and Kok-iterC c methods, R L and R Dk had similar Q 10 (c. 2.5). The Kok method overestimated both Q 10 and the light inhibition of respiration. R L /R Dk was not affected by leaf temperature. Acclimation of respiration in summer was associated with a decline in basal respiration but not in Q 10 in both species, which was related to changes in leaf nitrogen content between seasons. Q 10 of R L and R Dk in mature leaves were 40% higher than in immature leaves. Our results suggest similar Q 10 values can be used to model R L and R Dk while leaf development-associated changes in Q 10 require special consideration in future respiration models., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

Oxygen and hydrogen isotopes of cellulose in plant biology are commonly used to infer environmental conditions, often from time series measurements of tree rings. However, the covariation (or the lack thereof) between δ 18 O and δ 2 H in plant cellulose is still poorly understood. We compared plant water, and leaf and branch cellulose from dominant tree species across an aridity gradient in Northern Australia, to examine how δ 18 O and δ 2 H relate to each other and to mean annual precipitation (MAP). We identified a decline in covariation from xylem to leaf water, and onwards from leaf to branch wood cellulose. Covariation in leaf water isotopic enrichment (Δ) was partially preserved in leaf cellulose but not branch wood cellulose. Furthermore, whilst δ 2 H was well-correlated between leaf and branch, there was an offset in δ 18 O between organs that increased with decreasing MAP. Our findings strongly suggest that postphotosynthetic isotope exchange with water is more apparent for oxygen isotopes, whereas variable kinetic and nonequilibrium isotope effects add complexity to interpreting metabolic-induced δ 2 H patterns. Varying oxygen isotope exchange in wood and leaf cellulose must be accounted for when δ 18 O is used to reconstruct climatic scenarios. Conversely, comparing δ 2 H and δ 18 O patterns may reveal environmentally induced shifts in metabolism., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Phloem sap transport is essential for plant nutrition and development since it mediates redistribution of nutrients, metabolites and signaling molecules. However, its biochemical composition is not so well-known because phloem sap sampling is difficult and does not always allow extensive chemical analysis. In the past years, efforts have been devoted to metabolomics analyses of phloem sap using either liquid chromatography or gas chromatography coupled with mass spectrometry. Phloem sap metabolomics is of importance to understand how metabolites can be exchanged between plant organs and how metabolite allocation may impact plant growth and development. Here, we provide an overview of our current knowledge of phloem sap metabolome and physiological information obtained therefrom. Although metabolomics analyses of phloem sap are still not numerous, they show that metabolites present in sap are not just sugars and amino acids but that many more metabolic pathways are represented. They further suggest that metabolite exchange between source and sink organs is a general phenomenon, offering opportunities for metabolic cycles at the whole-plant scale. Such cycles reflect metabolic interdependence of plant organs and shoot-root coordination of plant growth and development.

Evaluating leaf day respiration rate (RL), which is believed to differ from that in the dark (RDk), is essential for predicting global carbon cycles under climate change. Several studies have suggested that atmospheric CO2 impacts RL. However, the magnitude of such an impact and associated mechanisms remain uncertain. To explore the CO2 effect on RL, wheat (Triticum aestivum) and sunflower (Helianthus annuus) plants were grown under ambient (410 ppm) and elevated (820 ppm) CO2 mole fraction ([CO2]). RL was estimated from combined gas exchange and chlorophyll fluorescence measurements using the Kok method, the Kok-Phi method, and a revised Kok method (Kok-Cc method). We found that elevated growth [CO2] led to an 8.4% reduction in RL and a 16.2% reduction in RDk in both species, in parallel to decreased leaf N and chlorophyll contents at elevated growth [CO2]. We also looked at short-term CO2 effects during gas exchange experiments. Increased RL or RL/RDk at elevated measurement [CO2] were found using the Kok and Kok-Phi methods, but not with the Kok-Cc method. This discrepancy was attributed to the unaccounted changes in Cc in the former methods. We found that the Kok and Kok-Phi methods underestimate RL and overestimate the inhibition of respiration under low irradiance conditions of the Kok curve, and the inhibition of RL was only 6%, representing 26% of the apparent Kok effect. We found no significant long-term CO2 effect on RL/RDk, originating from a concurrent reduction in RL and RDk at elevated growth [CO2], and likely mediated by acclimation of nitrogen metabolism., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2022. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Mitochondrial complex I (CI) plays a crucial role in oxidising NADH generated by the metabolism (including photorespiration) and thereby participates in the mitochondrial electron transfer chain feeding oxidative phosphorylation that generates ATP. However, CI mutations are not lethal in plants and cause moderate phenotypes, and therefore CI mutants are instrumental to examine consequences of mitochondrial homeostasis disturbance on plant cell metabolisms and signalling. To date, the consequences of CI disruption on the lipidome have not been examined. Yet, in principle, mitochondrial dysfunction should impact on lipid synthesis through chloroplasts (via changes in photorespiration, redox homeostasis, and N metabolism) and the endoplasmic reticulum (ER) (via perturbed mitochondrion-ER crosstalk). Here, we took advantage of lipidomics technology (by LC-MS), phospholipid quantitation by 31 P-NMR, and total lipid quantitation to assess the impact of CI disruption on leaf, pollen, and seed lipids using three well-characterised CI mutants: CMSII in N. sylvestris and both ndufs4 and ndufs8 in Arabidopsis. Our results show multiple changes in cellular lipids, including galactolipids (chloroplastic), sphingolipids, and ceramides (synthesised by ER), suggesting that mitochondrial homeostasis is essential for the regulation of whole cellular lipidome via specific signalling pathways. In particular, the observed modifications in phospholipid and sphingolipid/ceramide molecular species suggest that CI activity controls phosphatidic acid-mediated signalling.

Seed size is often considered to be an important trait for seed quality, i.e., vigour and germination performance. It is believed that seed size reflects the quantity of reserve material and thus the C and N sources available for post-germinative processes. However, mechanisms linking seed size and quality are poorly documented. In particular, specific metabolic changes when seed size varies are not well-known. To gain insight into this aspect, we examined seed size and composition across different accessions of barrel medic ( Medicago truncatula Gaertn.) from the genetic core collection. We conducted multi-elemental analyses and isotope measurements, as well as exact mass GC-MS metabolomics. There was a systematic increase in N content (+0.17% N mg -1 ) and a decrease in H content (-0.14% H mg -1 ) with seed size, reflecting lower lipid and higher S-poor protein quantity. There was also a decrease in 2 H natural abundance (δ 2 H), due to the lower prevalence of 2 H-enriched lipid hydrogen atoms that underwent isotopic exchange with water during seed development. Metabolomics showed that seed size correlates with free amino acid and hexoses content, and anticorrelates with amino acid degradation products, disaccharides, malic acid and free fatty acids. All accessions followed the same trend, with insignificant differences in metabolic properties between them. Our results show that there is no general, proportional increase in metabolite pools with seed size. Seed size appears to be determined by metabolic balance (between sugar and amino acid degradation vs. utilisation for storage), which is in turn likely determined by phloem source metabolite delivery during seed development.

Isotopic analyses of plant samples are now of considerable importance for food certification and plant physiology. In fact, the natural nitrogen isotope composition (δ 15 N) is extremely useful to examine metabolic pathways of N nutrition involving isotope fractionations. However, δ 15 N analysis of amino acids is not straightforward and involves specific derivatization procedures to yield volatile derivatives that can be analysed by gas chromatography coupled to isotope ratio mass spectrometry (GC-C-IRMS). Derivatizations other than trimethylsilylation are commonly used since they are believed to be more reliable and accurate. Their major drawback is that they are not associated with metabolite databases allowing identification of derivatives and by-products. Here, we revisit the potential of trimethylsilylated derivatives via concurrent analysis of δ 15 N and exact mass GC-MS of plant seed protein samples, allowing facile identification of derivatives using a database used for metabolomics. When multiple silylated derivatives of several amino acids are accounted for, there is a good agreement between theoretical and observed N mole fractions, and δ 15 N values are satisfactory, with little fractionation during derivatization. Overall, this technique may be suitable for compound-specific δ 15 N analysis, with pros and cons.

Despite considerable advances in the past 50 y, the mechanism of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) catalysis is still not well understood. In particular, the movement and exchange of protons within the active site is not well documented: typically, kinetics of H exchange during the first steps of catalysis, i.e. abstraction of the H3 atom of ribulose 1,5-bisphosphate (RuBP) and enolization, are not clearly established. Here, we took advantage of reaction assays run in heavy water ( 2 H 2 O) to monitor the appearance of deuterated RuBP and deuterated products (3-phosphoglycerate and 2-phosphoglycolate) with exact mass LC-MS. Enolization was reversible such that de-enolization generated not only monodeuterated RuBP ( 2 H-[H-3]-ribulose 1,5-bisphosphate) but also dideuterated RuBP ( 2 H 2 -[H-3,O-3]-ribulose 1,5-bisphosphate). Carboxylation yielded about one half deuterated 3-phosphoglycerate ( 2 H-[H-2]-3-phosphoglycerate) and also a small proportion of dideuterated 3-phosphoglycerate ( 2 H 2 -[H-2,O-2]-3-phosphoglycerate). Oxygenation generated a small amount of monodeuterated, but no dideuterated, products. (Di)deuterated isotopologue abundance depended negatively on gas concentration. We conclude that in addition to the first step of proton exchange at H3 occurring before gas addition (and thus influenced by the competition between de-enolization and gas addition), there is another proton exchange step between solvent water, active site residues, and the 2,3-enediol(ate) leading to deuterated OH groups in products., Competing Interests: No potential conflict of interest was reported by the author(s)., (© 2022 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.)

C 4 photosynthesis involves a series of biochemical and anatomical traits that significantly improve plant productivity under conditions that reduce the efficiency of C 3 photosynthesis. We explore how evolution of the three classical biochemical types of C 4 photosynthesis (NADP-ME, NAD-ME and PCK types) has affected the functions and properties of mitochondria. Mitochondria in C 4 NAD-ME and PCK types play a direct role in decarboxylation of metabolites for C 4 photosynthesis. Mitochondria in C 4 PCK type also provide ATP for C 4 metabolism, although this role for ATP provision is not seen in NAD-ME type. Such involvement has increased mitochondrial abundance/size and associated enzymatic capacity, led to changes in mitochondrial location and ultrastructure, and altered the role of mitochondria in cellular carbon metabolism in the NAD-ME and PCK types. By contrast, these changes in mitochondrial properties are absent in the C 4 NADP-ME type and C 3 leaves, where mitochondria play no direct role in photosynthesis. From an eco-physiological perspective, rates of leaf respiration in darkness vary considerably among C 4 species but does not differ systematically among the three C 4 types. This review outlines further mitochondrial research in key areas central to the engineering of the C 4 pathway into C 3 plants and to the understanding of variation in rates of C 4 dark respiration., (© 2021 The Authors. New Phytologist © 2021 New Phytologist Foundation.)

Phosphoglycerate mutases (PGAMs) catalyse the reversible isomerisation of 3-phosphoglycerate and 2-phosphoglycerate, a step of glycolysis. PGAMs can be sub-divided into 2,3-bisphosphoglycerate-dependent (dPGAM) and -independent (iPGAM) enzymes. In plants, phosphoglycerate isomerisation is carried out by cytosolic iPGAM. Despite its crucial role in catabolism, little is known about post-translational modifications of plant iPGAM. In Arabidopsis thaliana, phosphoproteomics analyses have previously identified an iPGAM phosphopeptide where serine 82 is phosphorylated. Here, we show that this phosphopeptide is less abundant in dark-adapted compared to illuminated Arabidopsis leaves. In silico comparison of iPGAM protein sequences and 3D structural modelling of AtiPGAM2 based on non-plant iPGAM enzymes suggest a role for phosphorylated serine in the catalytic reaction mechanism. This is confirmed by the activity (or the lack thereof) of mutated recombinant Arabidopsis iPGAM2 forms, affected in different steps of the reaction mechanism. We thus propose that the occurrence of the S82-phosphopeptide reflects iPGAM2 steady-state catalysis. Based on this assumption, the metabolic consequences of a higher iPGAM activity in illuminated versus darkened leaves are discussed., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

The natural abundance of heavy stable isotopes ( 13 C, 15 N, 18 O, etc.) is now of considerable importance in many research fields, including human physiology. In fact, it varies between tissues and metabolites due to isotope effects in biological processes, that is, isotope discriminations between heavy and light isotopic forms during enzyme or transporter activity. The metabolic deregulation associated with many diseases leads to alterations in metabolic fluxes, resulting in changes in isotope abundance that can be identified easily with current isotope ratio technologies. In this review, we summarize the current knowledge on changes in natural isotope composition in samples (including various tissues, hair, plasma, saliva) found in patients compared to controls, caused by human diseases. We discuss the metabolic origin of such isotope fractionations and highlight the potential of using isotopes at natural abundance for medical diagnosis and/or prognostic.

Measuring the carbon flux through metabolic pathways in intact illuminated leaves remains challenging because of, e.g., isotopic dilution by endogenous metabolites, the impossibility to reach isotopic steady state, and the occurrence of multiple pools. In the case of photorespiratory intermediates, our knowledge of the partitioning between photorespiratory recycling, storage, and utilization by other pathways is thus rather limited. There has been some controversy as to whether photorespiratory glycine and serine may not be recycled, thus changing the apparent stoichiometric coefficient between photorespiratory O 2 fixation and CO 2 release. We describe here an isotopic method to trace the fates of glycine, serine and glycerate, taking advantage of positional 13 C content with NMR and isotopic analyses by LC-MS. This technique is well-adapted to show that the proportion of glycerate, serine and glycine molecules escaping photorespiratory recycling is very small.

Carbon isotope discrimination (Δ) has been used widely to infer intrinsic water-use efficiency (iWUE) of C 3 plants, a key parameter linking carbon and water fluxes. Despite the essential role of mesophyll conductance (g m ) in photosynthesis and Δ, its effect on Δ-based predictions of iWUE has generally been neglected. Here, we derive a mathematical expression of iWUE as a function of Δ that includes g m (iWUE mes ) and exploits the g m -stomatal conductance (g sc ) relationship across drought-stress levels and plant functional groups (deciduous or semideciduous woody, evergreen woody and herbaceous species) in a global database. iWUE mes was further validated with an independent dataset of online-Δ and CO 2 and H 2 O gas exchange measurements with seven species. Drought stress reduced g sc and g m by nearly one-half across all plant functional groups, but had no significant effect on the g sc  : g m ratio, with a well supported value of 0.79 ± 0.07 (95% CI, n = 198). g m was negatively correlated to iWUE. Incorporating the g sc  : g m ratio greatly improved estimates of iWUE, compared with calculations that assumed infinite g m . The inclusion of the g sc  : g m ratio, fixed at 0.79 when g m was unknown, proved desirable to eliminate significant errors in estimating iWUE from Δ across various C 3 vegetation types., (© 2020 The Authors New Phytologist © 2020 New Phytologist Foundation.)

The Kok effect is a well-known phenomenon in which the quantum yield of photosynthesis changes abruptly at low light. This effect has often been interpreted as a shift in leaf respiratory metabolism and thus used widely to measure day respiration. However, there is still no formal evidence that the Kok effect has a respiratory origin. Here, both gas exchange and isotopic labeling were carried out on sunflower leaves, using glucose that was 13 C-enriched at specific C-atom positions. Position-specific decarboxylation measurements and NMR analysis of metabolites were used to trace the fate of C-atoms in metabolism. Decarboxylation rates were significant at low light (including above the Kok break point) and increased with decreasing irradiance below 100 µmol photons m -2  s -1 . The variation in several metabolite pools such as malate, fumarate or citrate, and flux calculations suggest the involvement of several decarboxylating pathways in the Kok effect, including the malic enzyme. Our results show that day respiratory CO 2 evolution plays an important role in the Kok effect. However, the increase in the apparent quantum yield of photosynthesis below the Kok break point is also probably related to malate metabolism, which participates in maintaining photosynthetic linear electron flow., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Foundation.)

Root oxygen deficiency that is induced by flooding (waterlogging) is a common situation in many agricultural areas, causing considerable loss in yield and productivity. Physiological and metabolic acclimation to hypoxia has mostly been studied on roots or whole seedlings under full submergence. The metabolic difference between shoots and roots during waterlogging, and how roots and shoots communicate in such a situation is much less known. In particular, the metabolic acclimation in shoots and how this, in turn, impacts on roots metabolism is not well documented. Here, we monitored changes in the metabolome of roots and shoots of barrel clover ( Medicago truncatula ), growth, and gas-exchange, and analyzed phloem sap exudate composition. Roots exhibited a typical response to hypoxia, such as γ-aminobutyrate and alanine accumulation, as well as a strong decline in raffinose, sucrose, hexoses, and pentoses. Leaves exhibited a strong increase in starch, sugars, sugar derivatives, and phenolics (tyrosine, tryptophan, phenylalanine, benzoate, ferulate), suggesting an inhibition of sugar export and their alternative utilization by aromatic compounds production via pentose phosphates and phospho enol pyruvate. Accordingly, there was an enrichment in sugars and a decline in organic acids in phloem sap exudates under waterlogging. Mass-balance calculations further suggest an increased imbalance between loading by shoots and unloading by roots under waterlogging. Taken as a whole, our results are consistent with the inhibition of sugar import by waterlogged roots, leading to an increase in phloem sugar pool, which, in turn, exert negative feedback on sugar metabolism and utilization in shoots., Competing Interests: The authors declare no conflict of interest.

While the general effect of CO2 enrichment on photosynthesis, stomatal conductance, N content, and yield has been documented, there is still some uncertainty as to whether there are interactive effects between CO2 enrichment and other factors, such as temperature, geographical location, water availability, and cultivar. In addition, the metabolic coordination between leaves and grains, which is crucial for crop responsiveness to elevated CO2, has never been examined closely. Here, we address these two aspects by multi-level analyses of data from several free-air CO2 enrichment experiments conducted in five different countries. There was little effect of elevated CO2 on yield (except in the USA), likely due to photosynthetic capacity acclimation, as reflected by protein profiles. In addition, there was a significant decrease in leaf amino acids (threonine) and macroelements (e.g. K) at elevated CO2, while other elements, such as Mg or S, increased. Despite the non-significant effect of CO2 enrichment on yield, grains appeared to be significantly depleted in N (as expected), but also in threonine, the S-containing amino acid methionine, and Mg. Overall, our results suggest a strong detrimental effect of CO2 enrichment on nutrient availability and remobilization from leaves to grains., (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the cornerstone of atmospheric CO 2 fixation by the biosphere. It catalyzes the addition of CO 2 onto enolized ribulose 1,5-bisphosphate (RuBP), producing 3-phosphoglycerate which is then converted to sugars. The major problem of this reaction is competitive O 2 addition, which forms a phosphorylated product (2-phosphoglycolate) that must be recycled by a series of biochemical reactions (photorespiratory metabolism). However, the way the enzyme activates O 2 is still unknown. Here, we used isotope effects (with 2 H, 25 Mg, and 18 O) to monitor O 2 activation and assess the influence of outer sphere atoms, in two Rubisco forms of contrasted O 2 /CO 2 selectivity. Neither the Rubisco form nor the use of solvent D 2 O and deuterated RuBP changed the 16 O/ 18 O isotope effect of O 2 addition, in clear contrast with the 12 C/ 13 C isotope effect of CO 2 addition. Furthermore, substitution of light magnesium ( 24 Mg) by heavy, nuclear magnetic 25 Mg had no effect on O 2 addition. Therefore, outer sphere protons have no influence on the reaction and direct radical chemistry (intersystem crossing with triplet O 2 ) does not seem to be involved in O 2 activation. Computations indicate that the reduction potential of enolized RuBP (near 0.49 V) is compatible with superoxide (O 2 •- ) production, must be insensitive to deuteration, and yields a predicted 16 O/ 18 O isotope effect and energy barrier close to observed values. Overall, O 2 undergoes single electron transfer to form short-lived superoxide, which then recombines to form a peroxide intermediate., Competing Interests: The authors declare no competing interest.

Oil palm is an oil-producing crop of major importance at the global scale. Oil palm mesocarp lipids are used for myriads industrial applications, and market demand has been growing for decades. In addition, oil palm seeds are oleaginous, and the oil extracted therefrom can be used for several purposes, from food to cosmetics. As such, there is a huge need in oil palm seeds to maintain the global cohort of more than 2 billion trees. However, oil palm seed germination is a rather difficult process, not only to break dormancy, but also because it is long and often reaches lower-than-expected germination rates. Surprisingly, despite the crucial importance of germination for oil palm plantation management, our knowledge is still rather limited, in particular about germinating oil palm seed metabolism. The present review incorporates different pieces of information that have been obtained in the past few years, in oil palm and in other palm species, in order to provide an overview of germination metabolism and its control. Further insights can also be gained from other oleaginous model plants, such as Arabidopsis or canola, however, palm seeds have peculiarities that must be accounted for, to gain a better understanding of germinating seed metabolism.

Nitrogen (N) assimilation is associated with 14 N/ 15 N fractionation such that plant tissues are generally 15 N-depleted compared to source nitrate. In addition to nitrate concentration, the δ 15 N value in plants is also influenced by isotopic heterogeneity amongst organs and metabolites. However, our current understanding of δ 15 N values in nitrate is limited by the relatively small number of compound-specific data. We extensively measured δ 15 N in nitrate at different time points, in sunflower and oil palm grown at fixed nitrate concentration, with nitrate circulation being varied using potassium (K) conditions and waterlogging. There were strong interorgan δ 15 N differences for contrasting situations between the two species, and a high 15 N-enrichment in root nitrate. Modelling shows that this 15 N-enrichment can be explained by nitrate circulation and compartmentalisation whereby despite a numerically small flux value, the backflow of nitrate to roots via the phloem can lead to a c. 30‰ difference between leaves and roots. Accordingly, waterlogging and low K conditions, which down-regulate sap circulation, cause a decrease in the leaf-to-root isotopic difference. Our study thus suggests that plant δ 15 N can be used as a natural tracer of N fluxes between organs and highlights the potential importance of δ 15 N of circulating phloem nitrate., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Intense efforts have been devoted to describe the biochemical pathway of plant sulphur (S) assimilation from sulphate. However, essential information on metabolic regulation of S assimilation is still lacking, such as possible interactions between S assimilation, photosynthesis and photorespiration. In particular, does S assimilation scale with photosynthesis thus ensuring sufficient S provision for amino acids synthesis? This lack of knowledge is problematic because optimization of photosynthesis is a common target of crop breeding and furthermore, photosynthesis is stimulated by the inexorable increase in atmospheric CO 2 . Here, we used high-resolution 33 S and 13 C tracing technology with NMR and LC-MS to access direct measurement of metabolic fluxes in S assimilation, when photosynthesis and photorespiration are varied via the gaseous composition of the atmosphere (CO 2 , O 2 ). We show that S assimilation is stimulated by photorespiratory metabolism and therefore, large photosynthetic fluxes appear to be detrimental to plant cell sulphur nutrition., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2019.)

Oil palm is by far the major oil-producing crop on the global scale, with c. 62 Mt oil produced each year. This species is a strong potassium (K)-demanding species cultivated in regions where soil K availability is generally low and waterlogging due to tropical heavy rains can limit further nutrient absorption. However, the metabolic effects of K and waterlogging have never been assessed precisely. Here, we examined the metabolic response of oil palm saplings in the glasshouse under controlled conditions (nutrient composition with low or high K availability, with or without waterlogging), using gas exchange, metabolomics and proteomics analyses. Our results showed that both low K and waterlogging have a detrimental effect on photosynthesis but stimulate leaf respiration, with differential accumulation of typical metabolic intermediates and enzymes of Krebs cycle and alternative catabolic pathways. In addition, we found a strong relationship between metabolic composition, the rate of leaf dark respiration, and cumulated respiratory loss. Advert environmental conditions (here, low K and waterlogging) therefore have an enormous effect on respiration in oil palm. Leaf metabolome and proteome appear to be good predictors of carbon balance, and open avenues for cultivation biomonitoring using functional genomics technologies., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Net photosynthetic assimilation in C 3 plants is mostly viewed as a simple balance between CO 2 fixation by Rubisco-catalyzed carboxylation and CO 2 production by photorespiration (and to a lower extent, by day respiration) that can be easily manipulated during gas exchange experiments using the CO 2  : O 2 ratio of the environment. However, it now becomes clear that it is not so simple, because the photosynthetic response to gaseous conditions involves 'ancillary' metabolisms, even in the short-term. That is, carbon and nitrogen utilization by pathways other than the Calvin cycle and the photorespiratory cycle, as well as rapid signaling events, can influence the observed rate of net photosynthesis. The potential impact of such ancillary metabolisms is assessed as well as how it must be taken into account to avoid misinterpretation of photosynthetic CO 2 response curves or low O 2 effects in C 3 leaves., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Pathogenic variants of OPA1, which encodes a dynamin GTPase involved in mitochondrial fusion, are responsible for a spectrum of neurological disorders sharing optic nerve atrophy and visual impairment. To gain insight on OPA1 neuronal specificity, we performed targeted metabolomics on rat cortical neurons with OPA1 expression inhibited by RNA interference. Of the 103 metabolites accurately measured, univariate analysis including the Benjamini-Hochberg correction revealed 6 significantly different metabolites in OPA1 down-regulated neurons, with aspartate being the most significant (p < 0.001). Supervised multivariate analysis by OPLS-DA yielded a model with good predictive capability (Q 2 cum  = 0.65) and a low risk of over-fitting (permQ2 = -0.16, CV-ANOVA p-value 0.036). Amongst the 46 metabolites contributing the most to the metabolic signature were aspartate, glutamate and threonine, which all decreased in OPA1 down-regulated neurons, and lysine, 4 sphingomyelins, 4 lysophosphatidylcholines and 32 phosphatidylcholines which were increased. The phospholipid signature may reflect intracellular membrane remodeling due to loss of mitochondrial fusion and/or lipid droplet accumulation. Aspartate and glutamate deficiency, also found in the plasma of OPA1 patients, is likely the consequence of respiratory chain deficiency, whereas the glutamate decrease could contribute to the synaptic dysfunction that we previously identified in this model.

Phosphenolpyruvate carboxylase (PEPC)-catalysed fixation of bicarbonate to C 4 acids is commonly believed to represent a rather small flux in illuminated leaves. In addition, its potential variation with O 2 and CO 2 is not documented and thus is usually neglected in gas-exchange studies. Here, we used quantitative NMR analysis of sunflower leaves labelled with 13 CO 2 (99% 13 C) under controlled conditions and measured the amount of 13 C found in the four C-atom positions in malate, the major product of PEPC activity. We found that amongst malate 13 C-isotopomers present after labelling, most molecules were labelled at both C-1 and C-4, showing the incorporation of 13 C at C-4 by PEPC fixation and subsequent redistribution to C-1 by fumarase (malate-fumarate equilibrium). In addition, absolute quantification of 13 C content showed that PEPC fixation increased at low CO 2 or high O 2 , and represented up to 1.8 μmol m -2  s -1 , that is, 40% of net assimilation measured by gas exchange under high O 2 /CO 2 conditions. Our results show that PEPC fixation represents a quantitatively important CO 2 -fixing activity that varies with O 2 and/or CO 2 mole fraction and this challenges the common interpretation of net assimilation in C 3 plants, where PEPC activity is often disregarded or considered to be constant at a very low rate., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Mutants affected in complex I are useful to understand the role played by mitochondrial electron transport and redox metabolism in cellular homeostasis and signaling. However, their respiratory phenotype is incompletely described and a specific examination of day respiration (R d ) is lacking. Here, we used isotopic methods and metabolomics to investigate the impact of complex I dysfunction on R d in two respiratory mutants of forest tobacco (Nicotiana sylvestris): cytoplasmic male sterile II (CMSII) and nuclear male sterile 1 (NMS1), previously characterized for complex I disruption. R d was higher in mutants and the inhibition of leaf respiration by light was lower. Higher R d values were caused by increased (phosphoenol)pyruvate (PEP) metabolism at the expense of anaplerotic (PEP carboxylase (PEPc) -catalyzed) activity. De novo synthesis of Krebs cycle intermediates in the light was larger in mutants than in the wild-type, although numerically small in all genotypes. Carbon metabolism in mutants involved alternative pathways, such as alanine synthesis, and an increase in amino acid production with the notable exception of aspartate. Our results show that the alteration of NADH re-oxidation activity by complex I does not cause a general inhibition of catabolism, but rather a re-orchestration of fluxes in day respiratory metabolism, leading to an increased CO 2 efflux., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Quantification of leaf respiration is important for understanding plant physiology and ecosystem biogeochemical processes. Leaf respiration continues in the light (R L ) but supposedly at a lower rate than in the dark (R D k ). However, there is no method for direct measurement of R L and the available methods require nonphysiological measurement conditions. A method based on isotopic disequilibrium quantified R L (R L 13C ) and mesophyll conductance of young and old fully expanded leaves of six species. R L 13C was compared to R L determined by the Laisk method (R L  Laisk ) on the very same leaves with a minimum time lag. R L  13C and R L  Laisk were generally lower than R D k , and were not significantly affected by leaf ageing. R L  Laisk and R L  13C were positively correlated (r 2  = 0.35), and both were positively correlated with R D k (r 2  ≥ 0.6). R L  Laisk was systematically lower than R L  13C by 0.4 μmol m -2  s -1 . Using A/C c instead of A/C i curves, a higher photocompensation point Γ* (by 5 μmol mol -1 ) was found but no influence on R L  Laisk estimates was observed. The results imply that the Laisk method underestimates actual R L significantly, probably related to the measurement condition of low CO 2 and irradiance. The isotopic disequilibrium method is useful for assessing responses of R L to irradiance and CO 2 , improving our mechanistic understanding of R L ., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

In gas-exchange experiments, manipulating CO 2 and O 2 is commonly used to change the balance between carboxylation and oxygenation. Downstream metabolism (utilization of photosynthetic and photorespiratory products) may also be affected by gaseous conditions but this is not well documented. Here, we took advantage of sunflower as a model species, which accumulates chlorogenate in addition to sugars and amino acids (glutamate, alanine, glycine and serine). We performed isotopic labelling with 13 CO 2 under different CO 2 /O 2 conditions, and determined 13 C contents to compute 13 C-allocation patterns and build-up rates. The 13 C content in major metabolites was not found to be a constant proportion of net fixed carbon but, rather, changed dramatically with CO 2 and O 2 . Alanine typically accumulated at low O 2 (hypoxic response) while photorespiratory intermediates accumulated under ambient conditions and at high photorespiration, glycerate accumulation exceeding serine and glycine build-up. Chlorogenate synthesis was relatively more important under normal conditions and at high CO 2 and its synthesis was driven by phosphoenolpyruvate de novo synthesis. These findings demonstrate that carbon allocation to metabolites other than photosynthetic end products is affected by gaseous conditions and therefore the photosynthetic yield of net nitrogen assimilation varies, being minimal at high CO 2 and maximal at high O 2 ., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

Dichlorodiphenyl-trichloroethane (DDT) and permethrin (PMT) are amongst most prevalent pesticides in the environment. Although their toxicity has been extensively studied, molecular mechanisms and metabolic effects remain unclear, including in liver where their detoxification occurs. Here, we used metabolomics, coupled to RT-qPCR analysis, to examine effects of DDT and PMT on hepatocytes cultivated in biochips. At 150 μM, DDT caused cell death, cytochrome P450 induction and modulation of estrogen metabolism. Metabolomics analysis showed an increase in some lipids and sugars after 6 h, and a decrease in fatty acids (tetradecanoate, octanoate and linoleate) after 24 h exposure. We also found a change in expression associated with genes involved in hepatic estrogen, lipid, and sugar metabolism. PMT at 150 μM perturbed lipid/sugar homeostasis and estrogen signaling pathway, between 2 and 6 h. After 24 h, lipids and sugars were found to decrease, suggesting continuous energy demand to detoxify PMT. Finally, at 15 μM, DDT and PMT appeared to have a small effect on metabolism and were detoxified after 24 h. Our results show a time-dependent perturbation of sugar/lipid homeostasis by DDT and PMT at 150 μM. Furthermore, DDT at high dose led to cell death, inflammatory response and oxidative stress., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Purpose: Systemic increases in reactive oxygen species, and their association with inflammation, have been proposed as an underlying mechanism linking obesity and age-related macular degeneration (AMD). Studies have found increased levels of oxidative stress biomarkers and inflammatory cytokines in obese individuals; however, the correlation between obesity and retinal inflammation has yet to be assessed. We used the leptin-deficient (ob/ob) mouse to further our understanding of the contribution of obesity to retinal oxidative stress and inflammation., Methods: Retinas from ob/ob mice were compared to age-matched wild-type controls for retinal function (electroretinography) and gene expression analysis of retinal stress ( Gfap ), oxidative stress ( Gpx3 and Hmox1 ), and complement activation ( C3 , C2 , Cfb , and Cfh ). Oxidative stress was further quantified using a reactive oxygen species and reactive nitrogen species (ROS and RNS) assay. Retinal microglia and macrophage migration to the outer retina and complement activation were determined using immunohistochemistry for IBA1 and C3, respectively. Retinas and sera were used for metabolomic analysis using QTRAP mass spectrometry., Results: Retinal function was reduced in ob/ob mice, which correlated to changes in markers of retinal stress, oxidative stress, and inflammation. An increase in C3-expressing microglia and macrophages was detected in the outer retinas of the ob/ob mice, while gene expression studies showed increases in the complement activators ( C2 and Cfb ) and a decrease in a complement regulator ( Cfh ). The expression of several metabolites were altered in the ob/ob mice compared to the controls, with changes in polyunsaturated fatty acids (PUFAs) and branched-chain amino acids (BCAAs) detected., Conclusions: The results of this study indicate that oxidative stress, inflammation, complement activation, and lipid metabolites in the retinal environment are linked with obesity in ob/ob animals. Understanding the interplay between these components in the retina in obesity will help inform risk factor analysis for acquired retinal degenerations, including AMD.

Contents 986 I. 987 II. 987 III. 988 IV. 991 V. 992 VI. 995 VII. 997 VIII. 998 References 998 SUMMARY: It has been 75 yr since leaf respiratory metabolism in the light (day respiration) was identified as a low-flux metabolic pathway that accompanies photosynthesis. In principle, it provides carbon backbones for nitrogen assimilation and evolves CO 2 and thus impacts on plant carbon and nitrogen balances. However, for a long time, uncertainties have remained as to whether techniques used to measure day respiratory efflux were valid and whether day respiration responded to environmental gaseous conditions. In the past few years, significant advances have been made using carbon isotopes, 'omics' analyses and surveys of respiration rates in mesocosms or ecosystems. There is substantial evidence that day respiration should be viewed as a highly dynamic metabolic pathway that interacts with photosynthesis and photorespiration and responds to atmospheric CO 2 mole fraction. The view of leaf day respiration as a constant and/or negligible parameter of net carbon exchange is now outdated and it should now be regarded as a central actor of plant carbon-use efficiency., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Glutamate (Glu) is the cornerstone of nitrogen assimilation and photorespiration in illuminated leaves. Despite this crucial role, our knowledge of the flux to Glu de novo synthesis is rather limited. Here, we used isotopic labelling with 13 CO 2 and 13 C-NMR analyses to examine the labelling pattern and the appearance of multi-labelled species of Glu molecules to trace the origin of C-atoms found in Glu. We also compared this with 13 C-labelling patterns in Ala and Asp, which reflect citrate (and thus Glu) precursors, that is, pyruvate and oxaloacetate. Glu appeared to be less 13 C-labelled than Asp and Ala, showing that the Glu pool was mostly formed by 'old' carbon atoms. There were modest differences in intramolecular 13 C- 13 C couplings between Glu C-2 and Asp C-3, showing that oxaloacetate metabolism to Glu biosynthesis did not involve C-atom redistribution by the Krebs cycle. The apparent carbon allocation increased with carbon net photosynthesis. However, when expressed relative to CO 2 fixation, it was clearly higher at low CO 2 while it did not change in 2% O 2 , as compared to standard conditions. We conclude that Glu production from current photosynthetic carbon represents a small flux that is controlled by the gaseous environment, typically upregulated at low CO 2 ., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Plant mutants for genes encoding subunits of mitochondrial complex I (CI; NADH:ubiquinone oxidoreductase), the first enzyme of the respiratory chain, display various phenotypes depending on growth conditions. Here, we examined the impact of photoperiod, a major environmental factor controlling plant development, on two Arabidopsis (Arabidopsis thaliana) CI mutants: a new insertion mutant interrupted in both ndufs8.1 and ndufs8.2 genes encoding the NDUFS8 subunit and the previously characterized ndufs4 CI mutant. In the long day (LD) condition, both ndufs8.1 and ndufs8.2 single mutants were indistinguishable from Columbia-0 at phenotypic and biochemical levels, whereas the ndufs8.1 ndufs8.2 double mutant was devoid of detectable holo-CI assembly/activity, showed higher alternative oxidase content/activity, and displayed a growth retardation phenotype similar to that of the ndufs4 mutant. Although growth was more affected in ndufs4 than in ndufs8.1 ndufs8.2 under the short day (SD) condition, both mutants displayed a similar impairment of growth acceleration after transfer to LD compared with the wild type. Untargeted and targeted metabolomics showed that overall metabolism was less responsive to the SD-to-LD transition in mutants than in the wild type. The typical LD acclimation of carbon and nitrogen assimilation as well as redox-related parameters was not observed in ndufs8.1 ndufs8 Similarly, NAD(H) content, which was higher in the SD condition in both mutants than in Columbia-0, did not adjust under LD We propose that altered redox homeostasis and NAD(H) content/redox state control the phenotype of CI mutants and photoperiod acclimation in Arabidopsis., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

NAD is a pyridine nucleotide that is involved in cell metabolism and signaling of plant growth and stress. Recently, we reported on the multifaceted nature of NAD-inducible immunity in Arabidopsis. We identified NAD as an integral regulator of multiple defense layers such as production of ROS, deposition of callose, stimulation of cell death and modulation of defense metabolism including the defense hormones SA, JA and ABA, and other defense-associated metabolites. Altogether, NAD-induced immune effects confer resistance to diverse pathogenic microbes. Our addendum to this work further demonstrates an impact of NAD on the cytosolic calcium pool, a well-known component of early plant defense response.

Pyridine nucleotides, such as NAD, are crucial redox carriers and have emerged as important signaling molecules in stress responses. Previously, we have demonstrated in Arabidopsis (Arabidopsis thaliana) that the inducible NAD-overproducing nadC lines are more resistant to an avirulent strain of Pseudomonas syringae pv tomato (Pst-AvrRpm1), which was associated with salicylic acid-dependent defense. Here, we have further characterized the NAD-dependent immune response in Arabidopsis. Quinolinate-induced stimulation of intracellular NAD in transgenic nadC plants enhanced resistance against a diverse range of (a)virulent pathogens, including Pst-AvrRpt2, Dickeya dadantii, and Botrytis cinerea Characterization of the redox status demonstrated that elevated NAD levels induce reactive oxygen species (ROS) production and the expression of redox marker genes of the cytosol and mitochondrion. Using pharmacological and reverse genetics approaches, we show that NAD-induced ROS production functions independently of NADPH oxidase activity and light metabolism but depends on mitochondrial respiration, which was increased at higher NAD. We further demonstrate that NAD primes pathogen-induced callose deposition and cell death. Mass spectrometry analysis reveals that NAD simultaneously induces different defense hormones and that the NAD-induced metabolic profiles are similar to those of defense-expressing plants after treatment with pathogen-associated molecular patterns. We thus conclude that NAD triggers metabolic profiles rather similar to that of pathogen-associated molecular patterns and discuss how signaling cross talk between defense hormones, ROS, and NAD explains the observed resistance to pathogens., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Breast cancer is the most common cancer in women worldwide. Despite the information provided by anatomopathological assessment and molecular markers (such as receptor expression ER, PR, HER2), breast cancer therapies and prognostics depend on the metabolic properties of tumor cells. However, metabolomics have not provided a robust and congruent biomarker yet, likely because individual metabolite contents are insufficient to encapsulate all of the alterations in metabolic fluxes. Here, we took advantage of natural 13 C and 15 N isotope abundance to show there are isotopic differences between healthy and cancer biopsy tissues or between healthy and malignant cultured cell lines. Isotope mass balance further suggests that these differences are mostly related to lipid metabolism, anaplerosis and urea cycle, three pathways known to be impacted in malignant cells. Our results demonstrate that the isotope signature is a good descriptor of metabolism since it integrates modifications in C partitioning and N excretion altogether. Our present study is thus a starting point to possible clinical applications such as patient screening and biopsy characterization in every cancer that is associated with metabolic changes.

Considerable efforts are currently devoted to understanding the regulation of primary carbon metabolism in plant leaves, which is known to change dramatically with environmental conditions, e.g. during light/dark transitions. Protein phosphorylation is believed to be a key factor in such a metabolic control. In fact, some studies have suggested modifications in the phosphorylation status of key enzymes in the dark compared with the light, or when photosynthesis varies. However, a general view of the phosphoproteome and reciprocal alterations in both the phosphoproteome and metabolome under a wide spectrum of CO 2 and O 2 conditions so as to vary both gross photosynthesis and photorespiration is currently lacking. Here, we used an instant sampling system and strictly controlled gaseous conditions to examine short-term metabolome and phosphoproteome changes in Arabidopsis rosettes. We show that light/dark, CO 2 and O 2 mole fraction have differential effects on enzyme phosphorylation. Phosphorylation events that appear to be the most important to regulate metabolite contents when photosynthesis varies are those associated with sugar and pyruvate metabolism: sucrose and starch synthesis are major phosphorylation-controlled steps but pyruvate utilization (by phosphoenolpyruvate carboxylase and pyruvate dehydrogenase) and pyruvate reformation (by pyruvate orthophosphate dikinase) are also subjected to phosphorylation control. Our results thus show that the phosphoproteome response to light/dark transition and gaseous conditions (CO 2 , O 2 ) contributes to the rapid adjustment of major pathways of primary C metabolism., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Metabolic and physiological analyses of Arabidopsis thaliana glycolate oxidase (GOX) mutant leaves were performed to understand the development of the photorespiratory phenotype after transfer from high CO2 to air. We show that two Arabidopsis genes, GOX1 and GOX2, share a redundant photorespiratory role. Air-grown single gox1 and gox2 mutants grew normally and no significant differences in leaf metabolic levels and photosynthetic activities were found when compared with wild-type plants. To study the impact of a highly reduced GOX activity on plant metabolism, both GOX1 and GOX2 expression was knocked-down using an artificial miRNA strategy. Air-grown amiRgox1/2 plants with a residual 5% GOX activity exhibited a severe growth phenotype. When high-CO2-grown adult plants were transferred to air, the photosynthetic activity of amiRgox1/2 was rapidly reduced to 50% of control levels, and a high non-photochemical chlorophyll fluorescence quenching was maintained. (13)C-labeling revealed that daily assimilated carbon accumulated in glycolate, leading to reduced carbon allocation to sugars, organic acids, and amino acids. Such changes were not always mirrored in leaf total metabolite levels, since many soluble amino acids increased after transfer, while total soluble protein, RuBisCO (ribulose-1,5-bisphosphate carboxylase/oxygenase), and chlorophyll amounts decreased in amiRgox1/2 plants. The senescence marker, SAG12, was induced only in amiRgox1/2 rosettes after transfer to air. The expression of maize photorespiratory GOX in amiRgox1/2 abolished all observed phenotypes. The results indicate that the inhibition of the photorespiratory cycle negatively impacts photosynthesis, alters carbon allocation, and leads to early senescence in old rosette leaves., (© The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)