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Nitrogen (N) nutrition impacts on primary carbon metabolism and can lead to changes in δ13C of respired CO2. However, uncertainty remains as to whether (1) the effect of N nutrition is observed in all species, (2) N source also impacts on respired CO2 in roots and (3) a metabolic model can be constructed to predict δ13C of respired CO2 under different N sources. Here, we carried out isotopic measurements of respired CO2 and various metabolites using two species (spinach, French bean) grown under different NH4+:NO3− ratios. Both species showed a similar pattern, with a progressive 13C‐depletion in leaf‐respired CO2 as the ammonium proportion increased, while δ13C in root‐respired CO2 showed little change. Supervised multivariate analysis showed that δ13C of respired CO2 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 δ13C in respired CO2. 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. Summary statement: Phaseolus vulgaris and Spinacia oleracea grown under varying NH4+:NO3− ratio show 13C‐depletion in leaf‐respired CO2 as NH4+ proportion increases. Supervised multivariate analysis shows that δ13C of respired CO2 is mostly determined by malate‐citrate metabolism in leaves and roots of both species. [ABSTRACT FROM AUTHOR]

Summary statement: Day respiration is a net flux resulting from several CO2‐generating and CO2‐fixing reactions, not only related to catabolism but also to anabolism. We review pieces of evidence that decarboxylating reactions are partly fed by carbon sources disconnected from current photosynthesis and how they reflect various metabolic pathways. [ABSTRACT FROM AUTHOR]

Day respiration (Rd) is the metabolic, nonphotorespiratory process by which illuminated leaves liberate CO2 during photosynthesis. Rd is used routinely in photosynthetic models and is thus critical for calculations. However, metabolic details associated with Rd are poorly known, and this can be problematic to predict how Rd changes with environmental conditions and relates to night respiration. It is often assumed that day respiratory CO2 release just reflects 'ordinary' catabolism (glycolysis and Krebs 'cycle'). Here, we carried out a pulse‐chase experiment, whereby a 13CO2 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 13C 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 CO2 efflux associated with day respiration. Flux reconstruction from metabolomics leads to an underestimation of Rd, further suggesting the contribution of a variety of CO2‐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 CO2‐producing reactions and (ii) is fed by different carbon sources, including stored carbon disconnected from current photosynthates. Summary statement: Day respiration (Rd) is the metabolic, nonphotorespiratory process by which illuminated leaves liberate CO2 during photosynthesis. It is often assumed that day respiratory CO2 release just reflects 'ordinary' catabolism (glycolysis and Krebs 'cycle'). Using isotope pulse‐chase, we show that leaf day respiration involves several CO2‐producing reactions and is fed by different carbon sources, including stored carbon disconnected from current photosynthates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plant metabolomics has been used widely in plant physiology, in particular to analyse metabolic responses to environmental parameters. Derivatization (via trimethylsilylation and methoximation) followed by GC‐MS metabolic profiling is a major technique to quantify low molecular weight, common metabolites of primary carbon, sulphur and nitrogen metabolism. There are now excellent opportunities for new generation analyses, using high resolution, exact mass GC‐MS spectrometers that are progressively becoming relatively cheap. However, exact mass GC‐MS analyses for routine metabolic profiling are not common, since there is no dedicated available database. Also, exact mass GC‐MS is usually dedicated to structural resolution of targeted secondary metabolites. Here, we present a curated database for exact mass metabolic profiling (made of 336 analytes, 1064 characteristic exact mass fragments) focused on molecules of primary metabolism. We show advantages of exact mass analyses, in particular to resolve isotopic patterns, localise S‐containing metabolites, and avoid identification errors when analytes have common nominal mass peaks in their spectrum. We provide a practical example using leaves of different Arabidopsis ecotypes and show how exact mass GC‐MS analysis can be applied to plant samples and identify metabolic profiles. [ABSTRACT FROM AUTHOR]

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 2H natural abundance (δ2H), due to the lower prevalence of 2H-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. [ABSTRACT FROM AUTHOR]

Water deficit causes substantial yield losses that climate change is going to make even more problematic. Sustainable agricultural practices are increasingly developed to improve plant tolerance to abiotic stresses. One innovative solution amongst others is the integration of plant biostimulants in agriculture. In this work, we investigate for the first time the effects of the biostimulant –Leafamine®–a protein hydrolysate on greenhouse lettuce (Lactuca sativa L.) grown under well-watered and water-deficit conditions. We examined the physiological and metabolomic water deficit responses of lettuce treated with Leafamine® (0.585 g/pot) or not. Root application of Leafamine® increased the shoot fresh biomass of both well-watered (+40%) and deficit-irrigated (+20%) lettuce plants because the projected leaf area increased. Our results also indicate that Leafamine® application could adjust the nitrogen metabolism by enhancing the total nitrogen content, amino acid (proline) contents and the total protein level in lettuce leaves, irrespective of the water condition. Osmolytes such as soluble sugars and polyols, also increased in Leafamine®-treated lettuce. Our findings suggest that the protective effect of Leafamine is a widespread change in plant metabolism and could involve ABA, putrescine and raffinose. [ABSTRACT FROM AUTHOR]

The natural 13C abundance (δ13C) 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 δ13C values in leaves and grains. We find a linear relationship between the apparent isotope discrimination between leaves and grain (denoted as Δδcorr), 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 13C natural abundance in grains could be useful to find genotypes with better carbon allocation properties and assist current wheat breeding technologies. Summary Statement: Finding isotope‐based markers of optimal carbon partitioning to grains would be extremely useful since isotope analyses are inexpensive and can be performed routinely at high throughput. Here, we show there is a linear relationship between the apparent isotope discrimination between leaves and grain (denoted as Δδcorr), and the respiration use efficiency‐to‐harvest index ratio. 13C natural abundance in grains has thus some potential to help finding genotypes with better carbon allocation properties and assisting current wheat breeding technologies. [ABSTRACT FROM AUTHOR]

Isotopic analyses of plant samples are now of considerable importance for food certification and plant physiology. In fact, the natural nitrogen isotope composition (δ15N) is extremely useful to examine metabolic pathways of N nutrition involving isotope fractionations. However, δ15N 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 δ15N 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 δ15N values are satisfactory, with little fractionation during derivatization. Overall, this technique may be suitable for compound-specific δ15N analysis, with pros and cons. [ABSTRACT FROM AUTHOR]

It is common practice to manipulate CO2 and O2 mole fraction during gas‐exchange experiments to suppress or exacerbate photorespiration, or simply carry out CO2 response curves. In doing so, it is implicitly assumed that metabolic pathways other than carboxylation and oxygenation are altered minimally. In the past few years, targeted metabolic analyses have shown that this assumption is incorrect, with changes in the tricarboxylic acid cycle, anaplerosis (phosphoenolpyruvate carboxylation), and nitrogen or sulphur assimilation. However, this problem has never been tackled systematically using non‐targeted analyses to embrace all possible affected metabolic pathways. Here, we exploited combined NMR, GC–MS, and LC–MS data and conducted non‐targeted analyses on sunflower leaves sampled at different O2/CO2 ratios in a gas exchange system. The statistical analysis of nearly 4,500 metabolic features not only confirms previous findings on anaplerosis or S assimilation, but also reveals significant changes in branched chain amino acids, phenylpropanoid metabolism, or adenosine turn‐over. Noteworthy, all of these pathways involve CO2 assimilation or liberation and thus affect net CO2 exchange. We conclude that manipulating CO2 and O2 mole fraction has a broad effect on metabolism, and this must be taken into account to better understand variations in carboxylation (anaplerotic fixation) or apparent day respiration. It is generally assumed that metabolic pathways other than carboxylation and oxygenation are altered minimally by changing CO2 and O2 conditions during gas exchange. Using isotope‐assisted metabolomics analyses, we show that manipulating CO2 and O2 has a broad effect on different major metabolic pathways, and this must be taken into to better interpret variations in carboxylation (anaplerotic fixation) or apparent day respiration. [ABSTRACT FROM AUTHOR]

In the quest for biomarkers of onset and progression of Alzheimer's disease, a 1H NMR-based metabolomic study was performed on the simple single-transgenic Tg2576 mouse model. These mice develop a slow cognitive decline starting by 6 months and express amyloid plaques from 10 months of age. The metabolic profiles of extracts from five brain regions (frontal cortex, rhinal cortex, hippocampus, midbrain, and cerebellum) of Tg2576 male mice were compared to those of controls, at 1, 3, 6 and 11 months of age. The most obvious differences were due to brain regions. Age was also a discriminating parameter. Metabolic perturbations were already detected in the hippocampus and the rhinal cortex of transgenic mice as early as 1 month of age with decreased concentrations of glutamate (Glu) and N-acetylaspartate (NAA) compared to those in wild-type animals. Metabolic changes were more numerous in the hippocampus and the rhinal cortex of 3 month-old transgenic mice and involved Glu, NAA, myo-inositol, creatine, phosphocholine, and γ-aminobutyric acid (only in the hippocampus) whose concentrations decreased. A metabolic disruption characterized by an increase in the hippocampal concentrations of Glu, creatine, and taurine was detected in 6 month-old transgenic mice. At this time point, the chemical profile of the cerebellum was slightly affected. At 11 months, all the brain regions analyzed (except the frontal cortex) were metabolically altered, with mainly a marked increase in the formation of the neuroprotective metabolites creatine and taurine. Our findings demonstrate that metabolic modifications occur long before the onset of behavioral impairment. [ABSTRACT FROM AUTHOR]

This study focuses on the foraminiferal distribution on intertidal mudflats of two contrasted estuaries (Auray and Vie) along the French Atlantic coast. In both estuaries, the foraminiferal communities are dominated by Haynesina germanica and the Ammonia tepida group. Stations located near the outlets show a high diversity and abundance of species of the genus Elphidium. Stations in the inner estuary show a higher proportion of agglutinated species (Ammotium salsum, Ammobaculites agglutinans). Multivariate statistical analysis suggests that the distance to the sea and the percentage of fine sediment (<63 µm) are the two main parameters explaining the foraminiferal distribution. Chemical analyses of the sediment show that the two studied estuaries are not affected by major anthropogenic pollution, so that the faunas should mainly reflect the natural controlling parameters. Three indices of environmental quality commonly used in coastal areas show counter-intuitive differences between stations, suggesting that these indices may be less reliable for use in intertidal estuarine mudflats. The newly developed Marine Influence Index (MII) integrates three major ecological factors: the position of the sampling point on the salinity gradient, the emergence time at low tide and the relative importance of fresh water discharge. In our dataset, MII shows significant correlations with the controlling environmental parameters (distance to the sea, percentage grains < 63 µm), as well as with the foraminiferal patterns (PCA axis 1, species richness, percentage of Elphidium spp. and Quinqueloculina spp.). These results suggest that the MII explains a substantial part of the faunal variability on estuarine intertidal mudflats, and can be used to detect deviations from the natural distribution patterns in response to anthropogenic pollution. [ABSTRACT FROM AUTHOR]