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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.)

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.)

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.)

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.)

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.)

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.)

Potassium (K) deficiency is a rather common situation that impacts negatively on biomass, photosynthesis and N assimilation, making K fertilization often unavoidable. Effects of K deficiency have been investigated for several decades and recently progress has been made in identifying metabolomics signatures thereby offering potential to monitor the K status of crops in the field. However, effects of low K conditions could also be due to the antagonism with other nutrients like calcium (Ca) and the well-known biomarker of K deficiency, putrescine, could be a response to Ca/K imbalance rather than K deficiency per se. To sort this out, we carried out experiments in sunflower grown at either low or high K, at high or low Ca, with or without putrescine added to the nutrient solution. Using metabolomics and proteomics analysis, we show that a significant part of the low K response, such as lower photosynthesis and N assimilation, is due to calcium and can be suppressed by low Ca conditions. Putrescine addition tends to restore photosynthesis and N assimilation but unlike low Ca does not suppress but aggravates the impact of low K conditions on catabolism, including the typical fall-over in pyruvate kinase. We conclude that (a) the effects of K deficiency on key metabolic processes can be partly alleviated by the use of low Ca and not only by K fertilization and (b) in addition to its role as a metabolite, putrescine participates in acclimation to low K via the regulation of the content in enzymes involved in carbon primary metabolism., (© 2021 John Wiley & Sons Ltd.)

It is common practice to manipulate CO 2 and O 2 mole fraction during gas-exchange experiments to suppress or exacerbate photorespiration, or simply carry out CO 2 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 O 2 /CO 2 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 CO 2 assimilation or liberation and thus affect net CO 2 exchange. We conclude that manipulating CO 2 and O 2 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., (© 2020 John Wiley & Sons Ltd.)

Biomarker metabolites are of increasing interest in crops since they open avenues for precision agriculture, whereby nutritional needs and stresses can be monitored optimally. Putrescine has the potential to be a useful biomarker to reveal potassium (K + ) deficiency. In fact, although this diamine has also been observed to increase during other stresses such as drought, cold or heavy metals, respective changes are comparably low. Due to its multifaceted biochemical properties, several roles for putrescine under K + deficiency have been suggested, such as cation balance, antioxidant, reactive oxygen species mediated signalling, osmolyte or pH regulator. However, the specific association of putrescine build-up with low K + availability in plants remains poorly understood, and possible regulatory roles must be consistent with putrescine concentration found in plant tissues. We hypothesize that the massive increase of putrescine upon K + starvation plays an adaptive role. A distinction of putrescine function from that of other polyamines (spermine, spermidine) may be based either on its specificity or (which is probably more relevant under K + deficiency) on a very high attainable concentration of putrescine, which far exceeds those for spermidine and spermine. putrescine and its catabolites appear to possess a strong potential in controlling cellular K + and Ca 2+ , and mitochondria and chloroplasts bioenergetics under K + stress., (© 2020 John Wiley & Sons Ltd.)

Improving seed quality is amongst the most important challenges of contemporary agriculture. In fact, using plant varieties with better germination rates that are more tolerant to stress during seedling establishment may improve crop yield considerably. Therefore, intense efforts are currently being devoted to improve seed quality in many species, mostly using genomics tools. However, despite its considerable importance during seed imbibition and germination processes, primary carbon metabolism in seeds is less studied. Our knowledge of the physiology of seed respiration and energy generation and the impact of these processes on seed performance have made limited progress over the past three decades. In particular, (isotope-assisted) metabolomics of seeds has only been assessed occasionally, and there is limited information on possible quantitative relationships between metabolic fluxes and seed quality. Here, we review the recent literature and provide an overview of potential links between metabolic efficiency, metabolic biomarkers, and seed quality and discuss implications for future research, including a climate change context., (© 2019 John Wiley & Sons Ltd.)

K deficiency and waterlogging are common stresses that can occur simultaneously and impact on crop development and yield. They are both known to affect catabolism, with rather opposite effects: inhibition of glycolysis and higher glycolytic fermentative flux, respectively. But surprisingly, the effect of their combination on plant metabolism has never been examined precisely. Here, we applied a combined treatment (K availability and waterlogging) to sunflower (Helianthus annuus L.) plants under controlled greenhouse conditions and performed elemental quantitation, metabolomics, and isotope analyses at different sampling times. Whereas separate K deficiency and waterlogging caused well-known effects such as polyamines production and sugar accumulation, respectively, waterlogging altered K-induced respiration enhancement (via the C 5 -branched acid pathway) and polyamine production, and K deficiency tended to suppress waterlogging-induced accumulation of Krebs cycle intermediates in leaves. Furthermore, the natural 15 N/ 14 N isotope composition (δ 15 N) in leaf compounds shows that there was a change in nitrate circulation, with less nitrate influx to leaves under low K availablity combined with waterlogging and more isotopic dilution of lamina nitrates under high K. Our results show that K deficiency and waterlogging effects are not simply additive, reshape respiration as well as nitrogen metabolism and partitioning, and are associated with metabolomic and isotopic biomarkers of potential interest for crop monitoring., (© 2018 John Wiley & Sons Ltd.)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the most widespread carboxylating enzyme in autotrophic organisms. Its kinetic and structural properties have been intensively studied for more than half a century. Yet important aspects of the catalytic mechanism remain poorly understood, especially the oxygenase reaction. Because of its relatively modest turnover rate (a few catalytic events per second) and the competitive inhibition by oxygen, Rubisco is often viewed as an inefficient catalyst for CO 2 fixation. Considerable efforts have been devoted to improving its catalytic efficiency, so far without success. In this review, we re-examine Rubisco's catalytic performance by comparison with other chemically related enzymes. We find that Rubisco is not especially slow. Furthermore, considering both the nature and the complexity of the chemical reaction, its kinetic properties are unremarkable. Although not unique to Rubisco, oxygenation is not systematically observed in enolate and enamine forming enzymes and cannot be considered as an inevitable consequence of the mechanism. It is more likely the result of a compromise between chemical and metabolic imperatives. We argue that a better description of Rubisco mechanism is still required to better understand the link between CO 2 and O 2 reactivity and the rationale of Rubisco diversification and evolution., (© 2018 John Wiley & Sons Ltd.)

Plant carbon-use-efficiency (CUE), a key parameter in carbon cycle and plant growth models, quantifies the fraction of fixed carbon that is converted into net primary production rather than respired. CUE has not been directly measured, partly because of the difficulty of measuring respiration in light. Here, we explore if CUE is affected by atmospheric CO 2 . Sunflower stands were grown at low (200 μmol mol -1 ) or high CO 2 (1000 μmol mol -1 ) in controlled environment mesocosms. CUE of stands was measured by dynamic stand-scale 13 C labelling and partitioning of photosynthesis and respiration. At the same plant age, growth at high CO 2 (compared with low CO 2 ) led to 91% higher rates of apparent photosynthesis, 97% higher respiration in the dark, yet 143% higher respiration in light. Thus, CUE was significantly lower at high (0.65) than at low CO 2 (0.71). Compartmental analysis of isotopic tracer kinetics demonstrated a greater commitment of carbon reserves in stand-scale respiratory metabolism at high CO 2 . Two main processes contributed to the reduction of CUE at high CO 2 : a reduced inhibition of leaf respiration by light and a diminished leaf mass ratio. This work highlights the relevance of measuring respiration in light and assessment of the CUE response to environment conditions., (© 2016 John Wiley & Sons Ltd.)

Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the cornerstone of photosynthetic carbon assimilation because it catalyses the fixation of CO2 onto ribulose-1,5-bisphosphate (RuBP). The enzyme also catalyses RuBP oxygenation, thereby evolving phosphoglycolate which is recycled along the photorespiratory pathway. Oxygenation is quantitatively important, because under ordinary gaseous conditions, more than one third of RuBP molecules are oxygenated rather than carboxylated. However, contrary to carboxylation, the chemical mechanism of oxygenation is not well known, and little progress has been made since the early 80s. Here, I review recent experimental data that provide some new insights into the reaction mechanism, and carry out simple calculations of kinetic parameters. Isotope effects suggest that oxygenation is less likely initiated by a redox phenomenon (such as superoxide production) and more likely involves concerted chemical events that imply interactions with protons. A possible energy profile of the reaction is drawn which suggests that the generation of the oxygenated reaction intermediate (peroxide) is irreversible. Possible changes in oxygenation-associated rate constants between Rubisco forms are discussed., (© 2015 John Wiley & Sons Ltd.)

Oil palm has now become one of the most important crops, palm oil representing nearly 25% of global plant oil consumption. Many studies have thus addressed oil palm ecophysiology and photosynthesis-based models of carbon allocation have been used. However, there is a lack of experimental data on carbon fixation and redistribution within palm trees, and important C-sinks have not been fully characterized yet. Here, we carried out extensive measurement of natural (13) C-abundance (δ(13) C) in oil palm tissues, including fruits at different maturation stages. We find a (13) C-enrichment in heterotrophic organs compared to mature leaves, with roots being the most (13) C-enriched. The δ(13) C in fruits decreased during maturation, reflecting the accumulation in (13) C-depleted lipids. We further used observed δ(13) C values to compute plausible carbon fluxes using a steady-state model of (13) C-distribution including metabolic isotope effects ((12) v/(13) v). The results suggest that fruits represent a major respiratory loss (≈39% of total tree respiration) and that sink organs such as fruits are fed by sucrose from leaves. That is, glucose appears to be a quantitatively important compound in palm tissues, but computations indicate that it is involved in dynamic starch metabolism rather that C-exchange between organs., (© 2015 John Wiley & Sons Ltd.)

C sink/source balance and N assimilation have been identified as target processes conditioning crop responsiveness to elevated CO2 . However, little is known about phenology-driven modifications of C and N primary metabolism at elevated CO2 in cereals such as wheat. Here, we examined the differential effect of elevated CO2 at two development stages (onset of flowering, onset of grain filling) in durum wheat (Triticum durum, var. Sula) using physiological measurements (photosynthesis, isotopes), metabolomics, proteomics and (15) N labelling. Our results show that growth at elevated CO2 was accompanied by photosynthetic acclimation through a lower internal (mesophyll) conductance but no significant effect on Rubisco content, maximal carboxylation or electron transfer. Growth at elevated CO2 altered photosynthate export and tended to accelerate leaf N remobilization, which was visible for several proteins and amino acids, as well as lysine degradation metabolism. However, grain biomass produced at elevated CO2 was larger and less N rich, suggesting that nitrogen use efficiency rather than photosynthesis is an important target for improvement, even in good CO2 -responsive cultivars., (© 2015 John Wiley & Sons Ltd.)

[FeFe]-hydrogenase-like genes encode [Fe4 S4]-containing proteins that are ubiquitous in eukaryotic cells. In humans, iron-only hydrogenase-like protein 1 (IOP1) represses hypoxia inducible factor-1α subunit (HIF1-α) at normal atmospheric partial O2 pressure (normoxia, 21 kPa O2). In yeasts, the nar1 mutant cannot grow at 21 kPa O2, but can develop at a lower O2 pressure (2 kPa O2). We show here that plant [FeFe]-hydrogenase-like GOLLUM genes are essential for plant development and cell cycle progression. The mutant phenotypes of these plants are seen in normoxic conditions, but not under conditions of mild hypoxia (5 kPa O2). Transcriptomic and metabolomic experiments showed that the mutation enhances the expression of some hypoxia-induced genes under normal atmospheric O2 conditions and changes the cellular content of metabolites related to energy metabolism. In conclusion, [FeFe]-hydrogenase-like proteins play a central role in eukaryotes including the adaptation of plants to the ambient O2 partial pressure., (© 2013 John Wiley & Sons Ltd.)

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 δ(15)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 δ(15)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, δ(15)N was correlated fairly well between phloem and xylem saps. These variations in δ(15)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. δ(15)N of the water soluble fraction of roots and leaves partially affected δ(15)N of phloem and xylems saps. δ(15)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., (© 2013 John Wiley & Sons Ltd.)

Although ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) was discovered nearly 60 years ago, the associated chemical mechanism of the reaction is still incompletely understood. The catalytic cycle consists of four major steps: ribulose-1,5-bisphosphate binding, enolization, CO₂ or O₂ addition and hydration, and cleavage of the intermediate. The use of individual rate constants for these elemental steps yields mathematical expressions for usual kinetic constants (k(cat), K(m)), CO₂ versus O₂ specificity (S(c/o)) as well as other chemical parameters such as the ¹²C/¹³C isotope effect. That said, most of them are not simple and thus the interpretation of experimental and observed values of kcat , Km and Sc/o may be more complicated than expected. That is, Rubisco effective catalysis depends on several kinetic parameters that are influenced by both the biological origin and the cellular medium (which, in turn, can vary with environmental conditions). In this brief review, we present the basic model of Rubisco kinetics and describe how subtle biochemical changes (which may have occurred along Evolution) can easily modify Rubisco catalysis., (© 2013 John Wiley & Sons Ltd.)

We investigated the function of ASN2, one of the three genes encoding asparagine synthetase (EC 6.3.5.4), which is the most highly expressed in vegetative leaves of Arabidopsis thaliana. Expression of ASN2 and parallel higher asparagine content in darkness suggest that leaf metabolism involves ASN2 for asparagine synthesis. In asn2-1 knockout and asn2-2 knockdown lines, ASN2 disruption caused a defective growth phenotype and ammonium accumulation. The asn2 mutant leaves displayed a depleted asparagine and an accumulation of alanine, GABA, pyruvate and fumarate, indicating an alanine formation from pyruvate through the GABA shunt to consume excess ammonium in the absence of asparagine synthesis. By contrast, asparagine did not contribute to photorespiratory nitrogen recycle as photosynthetic net CO(2) assimilation was not significantly different between lines under both 21 and 2% O(2). ASN2 was found in phloem companion cells by in situ hybridization and immunolocalization. Moreover, lack of asparagine in asn2 phloem sap and lowered (15) N flux to sinks, accompanied by the delayed yellowing (senescence) of asn2 leaves, in the absence of asparagine support a specific role of asparagine in phloem loading and nitrogen reallocation. We conclude that ASN2 is essential for nitrogen assimilation, distribution and remobilization (via the phloem) within the plant., (© 2012 Blackwell Publishing Ltd.)

Nitrogen isotope composition (δ(15) N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the δ(15) N value of whole leaf material does not properly reflect the way in which N is assimilated because isotope fractionations along metabolic reactions may cause substantial differences among leaf compounds. In other words, any change in metabolic composition or allocation pattern may cause undesirable variability in leaf δ(15) N. Here, we investigated the δ(15) N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in δ(15) N between nitrogenous compounds (up to 30‰) and the content in ((15) N enriched) nitrate had a clear influence on leaf δ(15) N. Using a simple steady-state model of day metabolism, we suggest that the δ(15) N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme-catalysed reactions in primary nitrogen metabolism. δ(15) N values were further influenced by light versus dark conditions and the probable occurrence of alternative biosynthetic pathways. We conclude that both biochemical pathways (that fractionate between isotopes) and nitrogen sources (used for amino acid production) should be considered when interpreting the δ(15) N value of leaf nitrogenous compounds., (© 2012 Blackwell Publishing Ltd.)

Although there is now a considerable literature on the inhibition of leaf respiration (CO(2) evolution) by light, little is known about the effect of other environmental conditions on day respiratory metabolism. In particular, CO(2) and O(2) mole fractions are assumed to cause changes in the tricarboxylic acid pathway (TCAP) but the amplitude and even the direction of such changes are still a matter of debate. Here, we took advantage of isotopic techniques, new simple equations and instant freeze sampling to follow respiratory metabolism in illuminated cocklebur leaves (Xanthium strumarium L.) under different CO(2) /O(2) conditions. Gas exchange coupled to online isotopic analysis showed that CO(2) evolved by leaves in the light came from 'old' carbon skeletons and there was a slight decrease in (13) C natural abundance when [CO(2) ] increased. This suggested the involvement of enzymatic steps fractionating more strongly against (13) C and thus increasingly limiting for the metabolic respiratory flux as [CO(2) ] increased. Isotopic labelling with (13) C(2) -2,4-citrate lead to (13) C-enriched Glu and 2-oxoglutarate (2OG), clearly demonstrating poor metabolism of citrate by the TCAP. There was a clear relationship between the ribulose-1,5-bisphosphate oxygenation-to-carboxylation ratio (v(o) /v(c) ) and the (13) C commitment to 2OG, demonstrating that 2OG and Glu synthesis via the TCAP is positively influenced by photorespiration., (© 2012 Blackwell Publishing Ltd.)

Efforts to understand the cause of ¹²C versus ¹³C isotope fractionation in plants during photosynthesis and post-photosynthetic metabolism are frustrated by the lack of data on the intramolecular ¹³C-distribution in metabolites and its variation with environmental conditions. We have exploited isotopic carbon-13 nuclear magnetic resonance (¹³C NMR) spectrometry to measure the positional isotope composition (δ¹³C(i) , ‰) in ethanol samples from different origins: European wines, liquors and sugars from C₃, C₄ and crassulacean acid metabolism (CAM) plants. In C₃-ethanol samples, the methylene group was always ¹³C-enriched (∼2‰) relative to the methyl group. In wines, this pattern was correlated with both air temperature and δ(18)O of wine water, indicating that water vapour deficit may be a critical defining factor. Furthermore, in C₄-ethanol, the reverse relationship was observed (methylene-C relatively ¹³C-depleted), supporting the concept that photorespiration is the key metabolic process leading to the ¹³C distribution in C₃-ethanol. By contrast, in CAM-ethanol, the isotopic pattern was similar to but stronger than C₃-ethanol, with a relative ¹³C-enrichment in the methylene-C of up to 13‰. Plausible causes of this ¹³C-pattern are briefly discussed. As the intramolecular δ¹³C(i) -values in ethanol reflect that in source glucose, our data point out the crucial impact on the ratio of metabolic pathways sustaining glucose synthesis., (© 2011 Blackwell Publishing Ltd.)

Leaf intrinsic water-use efficiency (WUE), the ratio of photosynthetic rate to stomatal conductance (A/g(s) ), is a key plant trait linking terrestrial carbon and water cycles. A rapid, integrative proxy for A/g(s) is of benefit to crop breeding programmes aiming to improve WUE, but also for ecologists interested in plant carbon-water balance in natural systems. We hypothesize that the carbon isotope composition of leaf-respired CO(2) (δ(13) C(Rl) ), two hours after leaves are transferred to the dark, records photosynthetic carbon isotope discrimination and so provides a proxy for A/g(s) . To test this hypothesis, δ(13) C(Rl) was measured in four barley cultivars grown in the field at two levels of water availability and compared to leaf-level gas exchange (the ratio of leaf intercellular to ambient CO(2) partial pressure, C(i) /C(a) , and A/g(s) ). Leaf-respired CO(2) was more (13) C-depleted in plants grown at higher water availability, varied between days as environmental conditions changed, and was significantly different between cultivars. A strong relationship between δ(13) C(Rl) and δ(13) C of sucrose was observed. δ(13) C(Rl) was converted into apparent photosynthetic discrimination (Δ(13) C(Rl) ) revealing strong relationships between Δ(13) C(Rl) and C(i) /C(a) and A/g(s) during the vegetative stage of growth. We therefore conclude that δ(13) C(Rl) may provide a rapid, integrative proxy for A/g(s) in barley., (© 2011 Blackwell Publishing Ltd.)

In leaves, although it is accepted that CO(2) evolved by dark respiration after illumination is naturally (13) C-enriched compared to organic matter or substrate sucrose, much uncertainty remains on whether day respiration produces (13) C-depleted or (13) C-enriched CO(2). Here, we applied equations described previously for mesocosm CO(2) exchange to investigate the carbon isotope composition of CO(2) respired by autotrophic and heterotrophic tissues of Pelargonium × hortorum leaves, taking advantage of leaf variegation. Day-respired CO(2) was slightly (13) C-depleted compared to organic matter both under 21% O(2) and 2% O(2). Furthermore, most, if not all CO(2) molecules evolved in the light came from carbon atoms that had been fixed previously before the experiments, in both variegated and green leaves. We conclude that the usual definition of day respiratory fractionation, that assumes carbon fixed by current net photosynthesis is the respiratory substrate, is not valid in Pelargonium leaves under our conditions. In variegated leaves, total organic matter was slightly (13) C-depleted in white areas and so were most primary metabolites. This small isotopic difference between white and green areas probably came from the small contribution of photosynthetic CO(2) refixation and the specific nitrogen metabolism in white leaf areas., (© 2010 Blackwell Publishing Ltd.)

While there is currently intense effort to examine the (13)C signal of CO(2) evolved in the dark, less is known on the isotope composition of day-respired CO(2). This lack of knowledge stems from technical difficulties to measure the pure respiratory isotopic signal: day respiration is mixed up with photorespiration, and there is no obvious way to separate photosynthetic fractionation (pure c(i)/c(a) effect) from respiratory effect (production of CO(2) with a different delta(13)C value from that of net-fixed CO(2)) at the ecosystem level. Here, we took advantage of new simple equations, and applied them to sunflower canopies grown under low and high [CO(2)]. We show that whole mesocosm-respired CO(2) is slightly (13)C depleted in the light at the mesocosm level (by 0.2-0.8 per thousand), while it is slightly (13)C enriched in darkness (by 1.5-3.2 per thousand). The turnover of the respiratory carbon pool after labelling appears similar in the light and in the dark, and accordingly, a hierarchical clustering analysis shows a close correlation between the (13)C abundance in day- and night-evolved CO(2). We conclude that the carbon source for respiration is similar in the dark and in the light, but the metabolic pathways associated with CO(2) production may change, thereby explaining the different (12)C/(13)C respiratory fractionations in the light and in the dark.

Carbon isotope fractionation in metabolic processes following carboxylation of ribulose-1,5-bisphosphate (RuBP) is not as well described as the discrimination during photosynthetic CO(2) fixation. However, post-carboxylation fractionation can influence the diel variation of delta(13)C of leaf-exported organic matter and can cause inter-organ differences in delta(13)C. To obtain a more mechanistic understanding of post-carboxylation modification of the isotopic signal as governed by physiological and environmental controls, we combined the modelling approach of Tcherkez et al., which describes the isotopic fractionation in primary metabolism with the experimental determination of delta(13)C in leaf and phloem sap and root carbon pools during a full diel course. There was a strong diel variation of leaf water-soluble organic matter and phloem sap sugars with relatively (13)C depleted carbon produced and exported during the day and enriched carbon during the night. The isotopic modelling approach reproduces the experimentally determined day-night differences in delta(13)C of leaf-exported carbon in Ricinus communis. These findings support the idea that patterns of transitory starch accumulation and remobilization govern the diel rhythm of delta(13)C in organic matter exported by leaves. Integrated over the whole 24 h day, leaf-exported carbon was enriched in (13)C as compared with the primary assimilates. This may contribute to the well-known--yet poorly explained--relative (13)C depletion of autotrophic organs compared with other plant parts. We thus emphasize the need to consider post-carboxylation fractionations for studies that use delta(13)C for assessing environmental effects like water availability on ratio of mole fractions of CO(2) inside and outside the leaf (e.g. tree ring studies), or for partitioning of CO(2) fluxes at the ecosystem level.

Stable isotopes and their associated mechanistic frameworks have provided the means to model biological transformations from inorganic sources to organic sinks, and their impact on long-term terrestrial carbon reservoirs. However, stable isotopes have also the potential to diagnose the mechanisms by which biological systems operate, when using 'multidimensional' analyses of the isotopic outputs. For this purpose, we suggest that isotopic signals may be treated as mathematical vectors to reveal their interrelationships. These visualizations may be achieved, thanks to multidimensional representations (the 'isotopology' method), or by appreciating the colinearity of isotopic vectors to develop a clustering analysis (the 'isotopomic' method) similar to that used in molecular biology. Both methods converge to the same mathematical form, i.e. both lead to a covariation approach. Using simple practical examples, we argue that such procedures allow the deconvolution of plant biological systems by revealing the hierarchy of contributory physiological processes.

We describe an open leaf gas exchange system coupled to a tunable diode laser (TDL) spectroscopy system enabling measurement of the leaf respiratory CO(2) flux and its associated carbon isotope composition (delta(13)C(Rl)) every 3 min. The precision of delta(13)C(Rl) measurement is comparable to that of traditional mass spectrometry techniques. delta(13)C(Rl) from castor bean (Ricinus communis L.) leaves tended to be positively related to the ratio of CO(2) produced to O(2) consumed [respiratory quotient (RQ)] after 24-48 h of prolonged darkness, in support of existing models. Further, the apparent fractionation between respiratory substrates and respired CO(2) within 1-8 h after the start of the dark period was similar to previous observations. In subsequent experiments, R. communis plants were grown under variable water availability to provide a range in delta(13)C of recently fixed carbohydrate. In leaves exposed to high light levels prior to the start of the dark period, CO(2) respired by leaves was up to 11 per thousand more enriched than phloem sap sugars within the first 10-15 min after plants had been moved from the light into the dark. The (13)C enrichment in respired CO(2) then decreased rapidly to within 3-7 per thousand of phloem sap after 30-60 min in the dark. This strong enrichment was not observed if light levels were low prior to the start of the dark period. Measurements of RQ confirmed that carbohydrates were the likely respiratory substrate for plants (RQ > 0.8) within the first 60 min after illumination. The strong (13)C enrichment that followed a high light-to-dark transition coincided with high respiration rates, suggesting that so-called light-enhanced dark respiration (LEDR) is fed by (13)C-enriched metabolites.