A fundamental challenge in plant physiology is independently determining the rates of gross O₂ production by photosynthesis and O₂ consumption by respiration, photorespiration, and other processes. Previous studies on isolated chloroplasts or leaves have separately constrained net and gross O₂ production (NOP and GOP, respectively) by labeling ambient O₂ with ¹⁸O while leaf water was unlabeled. Here, we describe a method to accurately measure GOP and NOP of whole detached leaves in a cuvette as a routine gas-exchange measurement. The petiole is immersed in water enriched to a d¹⁸O of ~9,000‰, and leaf water is labeled through the transpiration stream. Photosynthesis transfers ¹⁸O from H₂O to O₂. GOP is calculated from the increase in d¹⁸O of O₂ as air passes through the cuvette. NOP is determined from the increase in O₂/N₂. Both terms are measured by isotope ratio mass spectrometry. CO₂ assimilation and other standard gas-exchange parameters also were measured. Reproducible measurements are made on a single leaf for more than 15 h. We used this method to measure the light response curve of NOP and GOP in French bean (Phaseolus vulgaris) at 21% and 2% O₂. We then used these data to examine the O₂/CO₂ ratio of net photosynthesis, the light response curve of mesophyll conductance, and the apparent inhibition of respiration in the light (Kok effect) at both oxygen levels. The results are discussed in the context of evaluating the technique as a tool to study and understand leaf physiological traits.

As global climatic changes increase plant susceptibility to large-scale disturbances such as drought and pathogens, understory responses to these disturbances will become increasingly important to long-term forest dynamics. To better understand understory responses to canopy disturbance, we measured changes in the growth and physiology of the dominant understory shrub, American witch-hazel (Hamamelis virginiana L.), in response to girdling of canopy oaks in a temperate hardwood forest of the northeastern United States. Changes in the growth and physiology of H. virginiana may be important to the regeneration of northeastern temperate forests, as this common shrub largely shapes the microenvironment for seedlings on the forest floor where it occurs. Canopy disturbance by girdling resulted in significant increases in light and soil nitrogen availability. In response to these environmental changes, basal-area growth of H. virginianaincreased by an average 334%. This growth increase corresponded to significant increases in foliar nitrogen, respiration, and leaf chlorophyll and carotenoid concentrations. These findings indicate improved environmental conditions and increased growth for this understory shrub following the loss of dominant canopy trees. This study suggests that following large-scale canopy disturbance, H. virginiana and shrubs like it may play an important role in competing for soil N and shading seedlings of regenerating canopy species.

• Leaf dark respiration (R_dark) is an important yet poorly quantified component of the global carbon cycle. Given this, we analyzed a new global database of R_dark and associated leaf traits.
• Data for 899 species were compiled from 100 sites (from the Arctic to the tropics). Several woody and nonwoody plant functional types (PFTs) were represented. Mixed-effects models were used to disentangle sources of variation in R_dark.
• Area-based R_dark at the prevailing average daily growth temperature (T) of each site increased only twofold from the Arctic to the tropics, despite a 20°C increase in growing T (8–28°C). By contrast, R_dark at a standard T (25°C, R_dark²⁵) was threefold higher in the Arctic than in the tropics, and twofold higher at arid than at mesic sites. Species and PFTs at cold sites exhibited higher R_dark²⁵ at a given photosynthetic capacity (V_cmax²⁵) or leaf nitrogen concentration ([N]) than species at warmer sites. R_dark²⁵ values at any given V_cmax²⁵ or [N] were higher in herbs than in woody plants.
• The results highlight variation in R_dark among species and across global gradients in T and aridity. In addition to their ecological significance, the results provide a framework for improving representation of R_dark in terrestrial biosphere models (TBMs) and associated land-surface components of Earth system models (ESMs).

The cornerstone of carbon (C) and nitrogen (N) metabolic interactions – respiration – is presently not well understood in plant cells: the source of the key intermediate 2-oxoglutarate (2OG), to which reduced N is combined to yield glutamate and glutamine, remains somewhat unclear.
We took advantage of combined mutations of NAD- and NADP-dependent isocitrate dehydrogenase activity and investigated the associated metabolic effects in Arabidopsis leaves (the major site of N assimilation in this genus), using metabolomics and 13C-labelling techniques.
We show that a substantial reduction in leaf isocitrate dehydrogenase activity did not lead to changes in the respiration efflux rate but respiratory metabolism was reorchestrated: 2OG production was supplemented by a metabolic bypass involving both lysine synthesis and degradation.
Although the recycling of lysine has long been considered important in sustaining respiration, we show here that lysine neosynthesis itself participates in an alternative respiratory pathway. Lys metabolism thus contributes to explaining the metabolic flexibility of plant leaves and the effect (or the lack thereof) of respiratory mutations.

Nitrogen isotope composition (d15N) in plant organic matter is currently used as a natural tracer of nitrogen acquisition efficiency. However, the d15N 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 com- position or allocation pattern may cause undesirable vari- ability in leaf d15N. Here, we investigated the d15N in different leaf fractions and individual metabolites from rapeseed (Brassica napus) leaves. We show that there were substantial differences in d15N between nitrogenous com- pounds (up to 30‰) and the content in (15N enriched) nitrate had a clear influence on leaf d15N. Using a simple steady-state model of day metabolism, we suggest that the d15N value in major amino acids was mostly explained by isotope fractionation associated with isotope effects on enzyme-catalysed reactions in primary nitrogen metabo- lism. d15N 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 consid- ered when interpreting the d15N value of leaf nitrogenous compounds.

• Nitrogen assimilation in leaves requires primary NH2 acceptors that, in turn, originate from primary carbon metabolism. Respiratory metabolism is believed to provide such acceptors (such as 2-oxoglutarate), so that day respiration is com- monly seen as a cornerstone for nitrogen assimilation into glutamate in illuminated leaves. However, both glycolysis and day respiratory CO2 evolution are known to be inhibited by light, thereby compromising the input of recent photosynthetic car- bon for glutamate production.
• In this study, we carried out isotopic labelling experiments with 13CO2 and 15N- ammonium nitrate on detached leaves of rapeseed (Brassica napus), and per- formed 13C- and 15N-nuclear magnetic resonance analyses.
• Our results indicated that the production of 13C-glutamate and 13C-glutamine under a 13CO2 atmosphere was very weak, whereas 13C-glutamate and 13C-gluta- mine appeared in both the subsequent dark period and the next light period under a 12CO2 atmosphere. Consistently, the analysis of heteronuclear (13C–15N) interac- tions within molecules indicated that most 15N-glutamate and 15N-glutamine mol- ecules were not 13C labelled after 13C ⁄ 15N double labelling. That is, recent carbon atoms (i.e. 13C) were hardly incorporated into glutamate, but new glutamate mol- ecules were synthesized, as evidenced by 15N incorporation.
• We conclude that the remobilization of night-stored molecules plays a signifi- cant role in providing 2-oxoglutarate for glutamate synthesis in illuminated rape- seed leaves, and therefore the natural day : night cycle seems critical for nitrogen assimilation.

While the possible importance of the tricarboxylic acid (TCA) cycle reactions for leaf photosynthesis operation has been recognized, many uncertainties remain on whether TCA cycle biochemistry is similar in the light compared with the dark. It is widely accepted that leaf day respiration and the metabolic commitment to TCA decarboxylation are down-regulated in illuminated leaves. However, the metabolic basis (i.e. the limiting steps involved in such a down-regulation) is not well known. Here, we investigated the in vivo metabolic fluxes of individual reactions of the TCA cycle by developing two isotopic methods, 13C tracing and fluxomics and the use of H/D isotope effects, with Xanthium strumarium leaves. We provide evidence that the TCA “cycle” does not work in the forward direction like a proper cycle but, rather, operates in both the reverse and forward directions to produce fumarate and glutamate, respectively. Such a functional division of the cycle plausibly reflects the compromise between two contrasted forces: (1) the feedback inhibition by NADH and ATP on TCA enzymes in the light, and (2) the need to provide pH-buffering organic acids and carbon skeletons for nitrate absorption and assimilation.