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The lack of a reliable in vitro system to assess reprotoxicity is an emerging problem in the context of European law for Registration, Evaluation, Authorization and Restriction of Chemicals (REACH, 2007), as it requires a reduction in animal utilization for testing. Furthermore, in vitro reprotoxicological tests would be more relevant and greatly improved by integrating both hepatic metabolism and the blood-testis barrier. Here, we took advantage of an integrated insert in a dynamic microfluidic platform (IIDMP) to co-cultivate hepatocytes in biochip and Sertoli cells in the bicameral chamber. This microfluidic tool has been previously demonstrated to be helpful in cell function and/or quality improvement. We demonstrate that permeability of the Sertoli barrier is reduced by dynamic coculture in our system. Exometabolomics analysis reveals that interactions between hepatocytes and Sertoli cells may have been mediated by the polyamines increase and/or mid-chain fatty acid decrease in the circulating medium. These metabolic changes may be involved in permeability reduction by contributing to modifying junction protein quantity and localization. The present study gives an example of IIDMP as an in vitro partitioning/transport model for cell culture and toxicological testing. Further, based on both our previous results using an intestinal-hepatic cell coculture and the present study, IIDMP seems to be well-suited for (i) assessing the dose-response effect of chemicals within the rodent or human male reproductive tract, and (ii) improving the quality of reprotoxicological assays by including hepatic metabolism. Copyright © 2016 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Rubisco activase ( RCA) is an ancillary photosynthetic protein essential for Rubisco activity. Some data suggest that post-translational modifications (such as reduction of disulphide bridges) are involved in the regulation of RCA activity. However, despite the key role of protein phosphorylation in general metabolic regulation, RCA phosphorylation has not been well characterised. We took advantage of phosphoproteomics and gas exchange analyses with instant sampling adapted to Arabidopsis rosettes to examine the occurrence and variations of phosphopeptides associated with RCA in different photosynthetic contexts ( CO2 mole fraction, light and dark). We detected two phosphopeptides from RCA corresponding to residues Thr 78 and Ser 172, and show that the former is considerably more phosphorylated in the dark than in the light, while the latter show no light/dark pattern. The CO2 mole fraction did not influence phosphorylation of either residue. Phosphorylation thus appears to be a potential mechanism associated with RCA dark inactivation, when Rubisco-catalysed carboxylation is arrested. Since Thr 78 and Ser 172 are located in the N and Walker domains of the protein, respectively, the involvement of phosphorylation in protein-protein interaction and catalysis is likely. [ABSTRACT FROM AUTHOR]

Nitrogen isotope signatures in plants might give insights in the metabolism and allocation of nitrogen. To obtain a deeper understanding of the modifications of the nitrogen isotope signatures, we determined δ15 N in transport saps and in different fractions of leaves, axes and roots during a diel course along the plant axis. The most significant diel variations were observed in xylem and phloem saps where δ15 N was significantly higher during the day compared with during the night. However in xylem saps, this was observed only in the canopy, but not at the hypocotyl positions. In the canopy, δ15 N was correlated fairly well between phloem and xylem saps. These variations in δ15 N in transport saps can be attributed to nitrate reduction in leaves during the photoperiod as well as to 15 N-enriched glutamine acting as transport form of N. δ15 N of the water soluble fraction of roots and leaves partially affected δ15 N of phloem and xylems saps. δ15 N patterns are likely the result of a complex set of interactions and N-fluxes between plant organs. Furthermore, the natural nitrogen isotope abundance in plant tissue is not constant during the diel course - a fact that needs to be taken into account when sampling for isotopic studies. [ABSTRACT FROM AUTHOR]

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.)

Nitrogen (N) nutrition impacts on primary carbon metabolism and can lead to changes in δ 13 C of respired CO 2 . However, uncertainty remains as to whether (1) the effect of N nutrition is observed in all species, (2) N source also impacts on respired CO 2 in roots and (3) a metabolic model can be constructed to predict δ 13 C of respired CO 2 under different N sources. Here, we carried out isotopic measurements of respired CO 2 and various metabolites using two species (spinach, French bean) grown under different NH 4 + :NO 3 - ratios. Both species showed a similar pattern, with a progressive 13 C-depletion in leaf-respired CO 2 as the ammonium proportion increased, while δ 13 C in root-respired CO 2 showed little change. Supervised multivariate analysis showed that δ 13 C of respired CO 2 was mostly determined by organic acid (malate, citrate) metabolism, in both leaves and roots. We then took advantage of nonstationary, two-pool modelling that explained 73% of variance in δ 13 C in respired CO 2 . It demonstrates the critical role of the balance between the utilisation of respiratory intermediates and the remobilisation of stored organic acids, regardless of anaplerotic bicarbonate fixation by phosphoenolpyruvate carboxylase and the organ considered., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

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.

Traditional leaf gas exchange experiments have focused on net CO 2 exchange (A net ). Here, using California poplar (Populus trichocarpa), we coupled measurements of net oxygen production (NOP), isoprene emissions and δ 18 O in O 2 to traditional CO 2 /H 2 O gas exchange with chlorophyll fluorescence, and measured light, CO 2 and temperature response curves. This allowed us to obtain a comprehensive picture of the photosynthetic redox budget including electron transport rate (ETR) and estimates of the mean assimilatory quotient (AQ = A net /NOP). We found that A net and NOP were linearly correlated across environmental gradients with similar observed AQ values during light (1.25 ± 0.05) and CO 2 responses (1.23 ± 0.07). In contrast, AQ was suppressed during leaf temperature responses in the light (0.87 ± 0.28), potentially due to the acceleration of alternative ETR sinks like lipid synthesis. A net and NOP had an optimum temperature (T opt ) of 31°C, while ETR and δ 18 O in O 2 (35°C) and isoprene emissions (39°C) had distinctly higher T opt . The results confirm a tight connection between water oxidation and ETR and support a view of light-dependent lipid synthesis primarily driven by photosynthetic ATP/NADPH not consumed by the Calvin-Benson cycle, as an important thermotolerance mechanism linked with high rates of (photo)respiration and CO 2 /O 2 recycling., (© 2024 The Author(s). Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The response of mesophyll conductance (g m ) to CO 2 plays a key role in photosynthesis and ecosystem carbon cycles under climate change. Despite numerous studies, there is still debate about how g m responds to short-term CO 2 variations. Here we used multiple methods and looked at the relationship between stomatal conductance to CO 2 (g sc ) and g m to address this aspect. We measured chlorophyll fluorescence parameters and online carbon isotope discrimination (Δ) at different CO 2 mole fractions in sunflower (Helianthus annuus L.), cowpea (Vigna unguiculata L.), and wheat (Triticum aestivum L.) leaves. The variable J and Δ based methods showed that g m decreased with an increase in CO 2 mole fraction, and so did stomatal conductance. There were linear relationships between g m and g sc across CO 2 mole fractions. g m obtained from A-C i curve fitting method was higher than that from the variable J method and was not representative of g m under the growth CO 2 concentration. g m could be estimated by empirical models analogous to the Ball-Berry model and the USO model for stomatal conductance. Our results suggest that g m and g sc respond in a coordinated manner to short-term variations in CO 2 , providing new insight into the role of g m in photosynthesis modelling., (© 2024 John Wiley & Sons Ltd.)

Day respiration (R d ) is the metabolic, nonphotorespiratory process by which illuminated leaves liberate CO 2 during photosynthesis. R d is used routinely in photosynthetic models and is thus critical for calculations. However, metabolic details associated with R d are poorly known, and this can be problematic to predict how R d changes with environmental conditions and relates to night respiration. It is often assumed that day respiratory CO 2 release just reflects 'ordinary' catabolism (glycolysis and Krebs 'cycle'). Here, we carried out a pulse-chase experiment, whereby a 13 CO 2 pulse in the light was followed by a chase period in darkness and then in the light. We took advantage of nontargeted, isotope-assisted metabolomics to determine non-'ordinary' metabolism, detect carbon remobilisation and compare light and dark 13 C utilisation. We found that several concurrent metabolic pathways ('ordinary' catabolism, oxidative pentose phosphates pathway, amino acid production, nucleotide biosynthesis and secondary metabolism) took place in the light and participated in net CO 2 efflux associated with day respiration. Flux reconstruction from metabolomics leads to an underestimation of R d , further suggesting the contribution of a variety of CO 2 -evolving processes. Also, the cornerstone of the Krebs 'cycle', citrate, is synthetised de novo from photosynthates mostly in darkness, and remobilised or synthesised from stored material in the light. Collectively, our data provides direct evidence that leaf day respiration (i) involves several CO 2 -producing reactions and (ii) is fed by different carbon sources, including stored carbon disconnected from current photosynthates., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Measurements of respiratory properties have often been made at a single time point either during daytime using dark-adapted leaves or during nighttime. The influence of the day-night cycle on respiratory metabolism has received less attention but is crucial to understand photosynthesis and photorespiration. Here, we examined how CO2- and O2-based rates of leaf dark respiration (Rdark) differed between midday (after 30-min dark adaptation) and midnight in 8 C3 and C4 grasses. We used these data to calculate the respiratory quotient (RQ; ratio of CO2 release to O2 uptake), and assessed relationships between Rdark and leaf metabolome. Rdark was higher at midday than midnight, especially in C4 species. The day-night difference in Rdark was more evident when expressed on a CO2 than O2 basis, with the RQ being higher at midday than midnight in all species, except in rice (Oryza sativa). Metabolomic analyses showed little correlation of Rdark or RQ with leaf carbohydrates (sucrose, glucose, fructose, or starch) but strong multivariate relationships with other metabolites. The results suggest that rates of Rdark and differences in RQ were determined by several concurrent CO2-producing and O2-consuming metabolic pathways, not only the tricarboxylic acid cycle (organic acids utilization) but also the pentose phosphate pathway, galactose metabolism, and secondary metabolism. As such, Rdark was time-, type- (C3/C4) and species-dependent, due to the use of different substrates., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

d-amino acids are the d stereoisomers of the common l-amino acids found in proteins. Over the past two decades, the occurrence of d-amino acids in plants has been reported and circumstantial evidence for a role in various processes, including interaction with soil microorganisms or interference with cellular signalling, has been provided. However, examples are not numerous and d-amino acids can also be detrimental, some of them inhibiting growth and development. Thus, the persistence of d-amino acid metabolism in plants is rather surprising, and the evolutionary origins of d-amino acid metabolism are currently unclear. Systemic analysis of sequences associated with d-amino acid metabolism enzymes shows that they are not simply inherited from cyanobacterial metabolism. In fact, the history of plant d-amino acid metabolism enzymes likely involves multiple steps, cellular compartments, gene transfers and losses. Regardless of evolutionary steps, enzymes of d-amino acid metabolism, such as d-amino acid transferases or racemases, have been retained by higher plants and have not simply been eliminated, so it is likely that they fulfil important metabolic roles such as serine, folate or plastid peptidoglycan metabolism. We suggest that d-amino acid metabolism may have been critical to support metabolic functions required during the evolution of land plants., (© 2024 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

The 13C isotope composition (δ13C) of leaf dry matter is a useful tool for physiological and ecological studies. However, how post-photosynthetic fractionation associated with respiration and carbon export influences δ13C remains uncertain. We investigated the effects of post-photosynthetic fractionation on δ13C of mature leaves of Cleistogenes squarrosa, a perennial C4 grass, in controlled experiments with different levels of vapour pressure deficit and nitrogen supply. With increasing leaf age class, the 12C/13C fractionation of leaf organic matter relative to the δ13C of atmosphere CO2 (ΔDM) increased while that of cellulose (Δcel) was almost constant. The divergence between ΔDM and Δcel increased with leaf age class, with a maximum value of 1.6‰, indicating the accumulation of post-photosynthetic fractionation. Applying a new mass balance model that accounts for respiration and export of photosynthates, we found an apparent 12C/13C fractionation associated with carbon export of -0.5‰ to -1.0‰. Different ΔDM among leaves, pseudostems, daughter tillers, and roots indicate that post-photosynthetic fractionation happens at the whole-plant level. Compared with ΔDM of old leaves, ΔDM of young leaves and Δcel are more reliable proxies for predicting physiological parameters due to the lower sensitivity to post-photosynthetic fractionation and the similar sensitivity in responses to environmental changes., (© The Author(s) 2023. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.)

While the effect of CO2 enrichment on wheat (Triticum spp.) photosynthesis, nitrogen content or yield has been well-studied, the impact of elevated CO2 on metabolic pathways in organs other than leaves is poorly documented. In particular, glumes and awns, which may refix CO2 respired by developing grains and be naturally exposed to higher-than-ambient CO2 mole fraction, could show specific responses to elevated CO2 . Here, we took advantage of a free-air CO2 enrichment experiment and performed multilevel analyses, including metabolomics, ionomics, proteomics, major hormones and isotopes in Triticum durum . While in leaves, elevated CO2 tended to accelerate amino acid metabolism with many significantly affected metabolites, the effect on glumes and awns metabolites was modest. There was a lower content in compounds of the polyamine pathway (along with uracile and allantoin) under elevated CO2 , suggesting a change in secondary N metabolism. Also, cytokinin metabolism appeared to be significantly affected under elevated CO2 . Despite this, elevated CO2 did not affect the final composition of awn and glume organic matter, with the same content in carbon, nitrogen and other elements. We conclude that elevated CO2 mostly impacts on leaf metabolism but has little effect in awns and glumes, including their composition at maturity.

Nitrogen is the most limiting nutrient for plants, and it is preferentially absorbed in the form of nitrate by roots, which adapt to nitrate fluctuations by remodelling their architecture. Although core mechanisms of the response to nitrate availability are relatively well-known, signalling events controlling root growth and architecture have not all been identified, in particular in Legumes. However, the developmental effect of nitrate in Legumes is critical since external nitrate not only regulates root architecture but also N 2 -fixing nodule development. We have previously shown that in barrel medic (Medicago truncatula), the nitrate transporter MtNPF6.8 is required for nitrate sensitivity in root tip. However, uncertainty remains as to whether nitrogen metabolism itself is involved in the MtNPF6.8-mediated response. Here, we examine the metabolic effects of MtNPF6.8-dependent nitrate signalling using metabolomics and proteomics in WT and mtnpf6.8 root tips in presence or absence of nitrate. We found a reorchestration of metabolism due to the mutation, in favour of the branched chain amino acids/pantothenate metabolic pathway, and lipid catabolism via glyoxylate. That is, the mtnpf6.8 mutation was likely associated with a specific rerouting of acetyl-CoA production (glyoxylic cycle) and utilisation (pantothenate and branched chain amino acid synthesis). In agreement with our previous findings, class III peroxidases were confirmed as the main protein class responsive to nitrate, although in an MtNPF6.8-independent fashion. Our data rather suggest the involvement of other pathways within mtnpf6.8 root tips, such as Ca 2+ signalling or cell wall methylation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

We describe here a method to study and manipulate photorespiration in intact illuminated leaves. When the CO 2 /O 2 mole fraction ratio changes, instant sampling is critical, to quench leaf metabolism and thus trace rapid metabolic modification due to gaseous conditions. To do so, we combine 13 CO 2 labeling and gas exchange, using a large custom leaf chamber to facilitate fast sampling by direct liquid nitrogen spraying. Moreover, the use of a high chamber surface area (about 130 cm 2 ) allows one to sample a large amount of leaf material to carry out 13 C-nuclear magnetic resonance (NMR) analysis and complementary analyses, such as isotopic analyses by high-resolution mass spectrometry (by both GC and LC-MS). 13 C-NMR gives access to absolute 13 C amounts at the specific carbon atom position in the labeled molecules and thereby provides an estimate of 13 C-flux of photorespiratory intermediates. Since NMR analysis is not very sensitive and can miss minor metabolites, GC or LC-MS analyses are useful to monitor metabolites at low concentrations. Furthermore, 13 C-NMR and high-resolution LC-MS allow to estimate isotopologue distribution in response to 13 CO 2 labeling while modifying photorespiration activity., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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, velocity and allocation have been proposed to play a role in physiological limitations of crop yield, along with photosynthetic activity or water use efficiency. Although there is clear evidence that carbon allocation to grains effectively drives yield in cereals like wheat (as reflected by the harvest index), the influence of phloem transport rate and velocity is less clear. Here, we took advantage of previously published data on yield, respiration, carbon isotope composition, nitrogen content and water consumption in winter wheat cultivars grown across several sites with or without irrigation, to express grain production in terms of phloem sucrose transport and compare with xylem water transport. Our results suggest that phloem sucrose transport rate follows the same relationship with phloem N transport regardless of irrigation conditions and cultivars, and seems to depend mostly on grain weight (i.e., mg per grain). Depending on the assumption made for phloem sap sucrose concentration, either phloem sap velocity or its proportionality coefficient to xylem velocity change little with environmental conditions. Taken as a whole, phloem transport from leaves to grains seems to be homeostatic within a narrow range of values and following relationships with other plant physiological parameters across cultivars and conditions. This suggests that phloem transport per se is not a limitation for yield in wheat but rather, is controlled to sustain grain filling., (© 2023 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Quantitative nuclear magnetic resonance (NMR) for isotopic measurements, known as irm-NMR (isotope ratio measured by NMR), is well suited for the quantitation of 13 C-isotopomers in position-specific isotope analysis and thus for measuring the carbon isotope composition (δ 13 C, mUr) in C-atom positions. Irm-NMR has already been used with glucose after derivatization to study sugar metabolism in plants. However, up to now, irm-NMR has exploited a "single-pulse" sequence and requires a relatively large amount of material and long experimental time, precluding many applications with biological tissues or extracts. To reduce the required amount of sample, we investigated the use of 2D-NMR analysis. We adapted and optimized the NMR sequence so as to be able to analyze a small amount (10 mg) of a glucose derivative (diacetonide glucofuranose, DAGF) with a precision better than 1 mUr at each C-atom position. We also set up a method to correct raw data and express 13 C abundance on the usual δ 13 C scale (δ-scale). In fact, due to the distortion associated with polarization transfer and spin manipulation during 2D-NMR analyses, raw 13 C abundance is found to be on an unusual scale. This was compensated for by a correction factor obtained via comparative analysis of a reference material (commercial DAGF) using both previous (single-pulse) and new (2D) sequences. Glucose from different biological origins (CO 2 assimilation metabolisms of plants, namely, C 3 , C 4 , and CAM) was analyzed with the two sequences and compared. Validation criteria such as selectivity, limit of quantification, precision, trueness, and robustness are discussed, including in the framework of green analytical chemistry.

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.

Experimental approaches to isolate drivers of variation in the carbon-bound hydrogen isotope composition (δ 2 H) of plant cellulose are rare and current models are limited in their application. This is in part due to a lack in understanding of how 2 H-fractionations in carbohydrates differ between species. We analysed, for the first time, the δ 2 H of leaf sucrose along with the δ 2 H and δ 18 O of leaf cellulose and leaf and xylem water across seven herbaceous species and a starchless mutant of tobacco. The δ 2 H of sucrose explained 66% of the δ 2 H variation in cellulose (R 2  = 0.66), which was associated with species differences in the 2 H enrichment of sucrose above leaf water ( ε sucrose : -126% to -192‰) rather than by variation in leaf water δ 2 H itself. ε sucrose was positively related to dark respiration (R 2  = 0.27), and isotopic exchange of hydrogen in sugars was positively related to the turnover time of carbohydrates (R 2  = 0.38), but only when ε sucrose was fixed to the literature accepted value of - 171 ‰. No relation was found between isotopic exchange of hydrogen and oxygen, suggesting large differences in the processes shaping post-photosynthetic fractionation between elements. Our results strongly advocate that for robust applications of the leaf cellulose hydrogen isotope model, parameterization utilizing δ 2 H of sugars is needed., (© 2022 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Increases in terrestrial water-use efficiency (WUE) have been reported in many studies, pointing to potential changes in physiological forcing of global carbon and hydrological cycles. However, gains in WUE are of uncertain magnitude over longer (i.e. >10 years) periods of time largely owing to difficulties in accounting for structural and physiological acclimation. 13 C signatures (i.e. δ 13 C) of plant organic matter have long been used to estimate WUE at temporal scales ranging from days to centuries. Mesophyll conductance is a key uncertainty in estimated WUE owing to its influence on diffusion of CO 2 to sites of carboxylation. Here we apply new knowledge of mesophyll conductance to 464 δ 13 C chronologies in tree-rings of 143 species spanning global biomes. Adjusted for mesophyll conductance, gains in WUE during the 20th century (0.15 ppm year -1 ) were considerably smaller than those estimated from conventional modelling (0.26 ppm year -1 ). Across the globe, mean sensitivity of WUE to atmospheric CO 2 was 0.15 ppm ppm -1 . Ratios of internal-to-atmospheric CO 2 (on a mole fraction basis; c i /c a ) in leaves were mostly constant over time but differed among biomes and plant taxa-highlighting the significance of both plant structure and physiology. Together with synchronized responses in stomatal and mesophyll conductance, our results suggest that ratios of chloroplastic-to-atmospheric CO 2 (c c /c a ) are constrained over time. We conclude that forest WUE may have not increased as much as previously suggested and that projections of future climate forcing via CO 2 fertilization may need to be adjusted accordingly., (© 2022 John Wiley & Sons Ltd.)

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.

The natural 13 C abundance (δ 13 C) in plant leaves has been used for decades with great success in agronomy to monitor water-use efficiency and select modern cultivars adapted to dry conditions. However, in wheat, it is also important to find genotypes with high carbon allocation to spikes and grains, and thus with a high harvest index (HI) and/or low carbon losses via respiration. Finding isotope-based markers of carbon partitioning to grains would be extremely useful since isotope analyses are inexpensive and can be performed routinely at high throughput. Here, we took the advantage of a set of field trials made of more than 600 plots with several wheat cultivars and measured agronomic parameters as well as δ 13 C values in leaves and grains. We find a linear relationship between the apparent isotope discrimination between leaves and grain (denoted as Δδ corr ), and the respiration use efficiency-to-HI ratio. It means that overall, efficient carbon allocation to grains is associated with a small isotopic difference between leaves and grains. This effect is explained by postphotosynthetic isotope fractionations, and we show that this can be modelled by equations describing the carbon isotope composition in grains along the wheat growth cycle. Our results show that 13 C natural abundance in grains could be useful to find genotypes with better carbon allocation properties and assist current wheat breeding technologies., (© 2022 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

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.)

The prevalence of phylogenetic constraints in Rubisco evolution has been emphasised recently by (Bouvier et al., 2021), who argued that phylogenetic inheritance limits Rubisco adaptation much more than the biochemical trade-off between specificity, CO 2 affinity and turn-over. In this Opinion, we have critically examined how a phylogenetic signal can be computed with Rubisco kinetic properties and phylogenetic trees, and we arrive at a different conclusion. In particular, Rubisco's adaptation is partly driven by C 4 vs. C 3 photosynthetic conditions in Angiosperms, apparent phylogenetic signals being mostly due to either homoplasy, computation artefacts or the use of nearly identical sister species. While phylogenetic inheritance of an ancestral enzyme form probably has some role in Rubisco's adaptation landscape, it is a minor player, at least compared to microenvironmental conditions such as CO 2 and O 2 concentrations., (Copyright © 2021 Elsevier GmbH. All rights reserved.)

Potassium is a macroelement essential to many aspects of plant life, such as photosynthesis, phloem transport or cellular electrochemistry. Many enzymes in animals or microbes are known to be stimulated or activated by potassium (K + ions). Several plant enzymes are also strictly K + -dependent, and this can be critical when plants are under K deficiency and thus intracellular K + concentration is low. Although metabolic effects of low K conditions have been documented, there is presently no review focusing on roles of K + for enzyme catalysis or activation in plants. In this mini-review, we compile the current knowledge on K + -requirement of plant enzymes and take advantage of structural data to present biochemical roles of K + . This information is instrumental to explain direct effects of low K + content on metabolism and this is illustrated with recent metabolomics data., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

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.)