Paul P. G. Gauthier

In Folio Respiratory Fluxomics Revealed by 13C Isotopic Labeling and H/D Isotope Effects Highlight the Noncyclic Nature of the Tricarboxylic Acid “Cycle” in Illuminated Leaves

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.

In folio isotopic tracing demonstrates that nitrogen assimilation into glutamate is mostly independent from current CO2 assimilation in illuminated leaves of Brassica napus

*Nitrogen assimilation in leaves requires primary NH(2) 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 commonly seen as a cornerstone for nitrogen assimilation into glutamate in illuminated leaves. However, both glycolysis and day respiratory CO(2) evolution are known to be inhibited by light, thereby compromising the input of recent photosynthetic carbon for glutamate production. *In this study, we carried out isotopic labelling experiments with (13)CO(2) and (15)N-ammonium nitrate on detached leaves of rapeseed (Brassica napus), and performed (13)C- and (15)N-nuclear magnetic resonance analyses. *Our results indicated that the production of (13)C-glutamate and (13)C-glutamine under a (13)CO(2) atmosphere was very weak, whereas (13)C-glutamate and (13)C-glutamine appeared in both the subsequent dark period and the next light period under a (12)CO(2) atmosphere. Consistently, the analysis of heteronuclear ((13)C-(15)N) interactions within molecules indicated that most (15)N-glutamate and (15)N-glutamine molecules were not (13)C labelled after (13)C/(15)N double labelling. That is, recent carbon atoms (i.e. (13)C) were hardly incorporated into glutamate, but new glutamate molecules were synthesized, as evidenced by (15)N incorporation. *We conclude that the remobilization of night-stored molecules plays a significant role in providing 2-oxoglutarate for glutamate synthesis in illuminated rapeseed leaves, and therefore the natural day : night cycle seems critical for nitrogen assimilation.

A new anaplerotic respiratory pathway involving lysine biosynthesis in isocitrate dehydrogenase-deficient Arabidopsis mutants

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 (13)C-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.

Drought increases heat tolerance of leaf respiration in Eucalyptus globulus saplings grown under both ambient and elevated atmospheric [CO2] and temperature

Summary This study highlights the dynamic nature of the temperature dependence of leaf respiration in plants experiencing future climate change scenarios, particularly with respect to drought and elevated [CO2].

Tracking the origins of the Kok effect, 70 years after its discovery

Introduction The 18 New Phytologist Workshop was dedicated to possible causes of the Kok effect, the typical break in the light response curve of net photosynthesis. Available data obtained since its discovery in 1948 show that the effect is not purely caused by a down-regulation of respiration, contrary to the commonly accepted view. However, estimates of leaf respiratory rates obtained in various ecosystems with techniques including the Kok method appear to be widely consistent across different studies, suggesting that Kok-derived values can be used as a surrogate for actual day respiration values. Gross CO2 assimilation of photosynthetic organs of plants is accompanied by concurrent efflux of CO2 by photorespiration and day respiration (i.e. nonphotorespiratory CO2 evolution in the light).While the rate of photorespiration can be predicted using the internal CO2 mole fraction and equations that describe gas exchange (taking into account the stoichiometry of CO2 liberation with respect to O2 fixation by ribulose-1,5-bisphosphate carboxylase/oxygenase), estimating day respiration is much more challenging because there is no equation that can predict its rate as a function of net photosynthesis, CO2 mole fraction or other environmental parameters. That is, in equations describing gas exchange (or isotopic mass balance), day respiration (Rd) has to be determined separately or simply assumed to model net carbon (C) exchange. At the leaf level, day respiration represents a C loss of c. 5% of gross-fixed CO2 but this proportion is highly variable, depending on species and conditions (see, e.g. Atkin et al., 1997). Estimates of day respiratory CO2 loss rely on specific techniques used tomeasure Rd: amongst them, the Kokmethod is certainly the most popular, because it is easy to implement in the laboratory or in the field using classical gas-exchange systems. This method takes advantage of the ‘Kok effect’, a phenomenon first described in the 1940s in unicellular algae (Kok, 1948, 1949). This effect is further described later, and in Fig. 1. The Kok effect is believed to be primarily caused by the inhibition of respiration by light and thus provides a direct way to estimate Rd. At the present date, c. 800 published works have used, or cited, the Kokmethod, representing c. 40% of articles that involve ameasurement of Rd or deal with day respiration. However, some persisting doubt remains about the validity of this method, simply because the Kok effect is inconstant and influenced by environmental conditions (such as O2 mole fraction) in ways that may not be consistent with day respiratory metabolism. Considering the wide range of applications, and the considerable number of articles that have been published, there is an urgent need to clarify the origin of the Kok effect and to evaluate its relevance to measure Rd. This was the objective of the 18 th New Phytologist Workshop that took place in July 2016 in Angers (France).

Measurement of Gross Photosynthesis, Respiration in the Light and Mesophyll Conductance Using H218O Labeling

H218O labeling and IRMS can be used to measure gross photosynthesis, mesophyll conductance, and respiratory uptake in the light in leaflets exposed to 21% and 2% O2. 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 δ18O 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 δ18O 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.

Dew-induced transpiration suppression impacts the water and isotope balances of Colocasia leaves

Foliar uptake of water from the surface of leaves is common when rainfall is scarce and non-meteoric water such as dew or fog is more abundant. However, many species in more mesic environments have hydrophobic leaves that do not allow the plant to uptake water. Unlike foliar uptake, all species can benefit from dew- or fog-induced transpiration suppression, but despite its ubiquity, transpiration suppression has so far never been quantified. Here, we investigate the effect of dew-induced transpiration suppression on the water balance and the isotope composition of leaves via a series of experiments. Characteristically, hydrophobic leaves of a tropical plant, Colocasia esculenta, are misted with isotopically enriched water to reproduce dew deposition. This species does not uptake water from the surface of its leaves. We measure leaf water isotopes and water potential and find that misted leaves exhibit a higher water potential and a more depleted water isotope composition than dry leaves, suggesting a ∼ 30% decrease in transpiration rate compared to control leaves. We propose three possible mechanisms governing the interaction of water droplets with leaf energy balance: increase in albedo from the presence of dew droplets, decrease in leaf temperature from the evaporation of dew, and local decrease in vapor pressure deficit. Comparing previous studies on foliar uptake to our results, we conclude that transpiration suppression has an effect of similar amplitude, yet opposite sign to foliar uptake on leaf water isotopes.

Phosphorus deficiency alters scaling relationships between leaf gas exchange and associated traits in a wide range of contrasting Eucalyptus species

Phosphorus (P) limitation is known to have substantial impacts on leaf metabolism. However, uncertainty remains around whether P deficiency alters scaling functions linking leaf metabolism to associated traits. We investigated the effect of P deficiency on leaf gas exchange and related leaf traits in 17 contrasting Eucalyptus species that exhibit inherent differences in leaf traits. Saplings were grown under controlled-environment conditions in a glasshouse, where they were subjected to minus and plus P treatments for 15 weeks. P deficiency decreased P concentrations and increased leaf mass per area (LMA) of newly-developed leaves. Rates of photosynthesis (A) and respiration (R) were also reduced in P-deficient plants compared with P-fertilised plants. By contrast, P deficiency had little effect on the temperature sensitivity of R. Irrespective of P treatment, on a log-log basis A and R scaled positively with increasing leaf nitrogen concentration [N] and negatively with increasing LMA. Although P deficiency had limited impact on A-R-LMA relationships, rates of CO2 exchange per unit N were consistently lower in P-deficient plants. Our results highlight the importance of P supply for leaf carbon metabolism and show how P deficiencies (i.e. when excluding confounding genotypic and environmental effects) can have a direct effect on commonly used leaf trait scaling relationships.

The impact of dew-induced transpiration suppression on the water and isotope balances of Colocasia esculenta leaves

Foliar uptake of water from the surface of leaves is common when rainfall is scarce and non-meteoric water such as dew or fog is more abundant. However, many species in more mesic environments have hydrophobic leaves that do not allow the plant to uptake water. Unlike foliar uptake, all species can benefit from dew- or fog-induced transpiration suppression, but despite its ubiquity, transpiration suppression has so far never been quantified. Here, we investigate the effect of dew-induced transpiration suppression on the water balance and the isotope composition of leaves via a series of experiments. Characteristically hydrophobic leaves of a tropical plant, Colocasia esculenta, are misted with isotopically enriched water to reproduce dew deposition. This species does not uptake water from the surface of its leaves. We measure leaf water isotopes and water potential and find that misted leaves exhibit a higher water potential (p < 0.05) and a more depleted water isotope composition than dry leaves (p < 0.001), suggesting a ~30% decrease in transpiration rate (p < 0.001) compared to control leaves. We propose three possible mechanisms governing the interaction of water droplets with leaf energy balance: increase in albedo from the presence of dew droplets, decrease in leaf temperature from the evaporation of dew, and local decrease in vapor pressure deficit. Comparing previous studies on foliar uptake to our results, we conclude that transpiration suppression has an effect of similar amplitude, yet opposite sign to foliar uptake on leaf water isotopes.