Cyril Abadie

In vivo stoichiometry of photorespiratory metabolism

Photorespiration is a major light-dependent metabolic pathway that consumes oxygen and produces carbon dioxide. In the metabolic step responsible for carbon dioxide production, two molecules of glycine (equivalent to two molecules of O2) are converted into one molecule of serine and one molecule of CO2. Here, we use quantitative isotopic techniques to determine the stoichiometry of this reaction in sunflower leaves, and thereby the O2/CO2 stoichiometry of photorespiration. We find that the effective O2/CO2 stoichiometric coefficient at the leaf level is very close to 2 under normal photorespiratory conditions, in line with expectations, but increases slightly at high rates of photorespiration. The net metabolic impact of this imbalance is likely to be modest.

Differential CO2 effect on primary carbon metabolism of flag leaves in durum wheat (Triticum durum Desf.).

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.

Metabolomics analysis of postphotosynthetic effects of gaseous O2 on primary metabolism in illuminated leaves

The response of underground plant tissues to O2 limitation is currently an important topic in crop plants since adverse environmental conditions (e.g. waterlogging) may cause root hypoxia and thus compromise plant growth. However, little is known on the effect of low O2 conditions in leaves, probably because O2 limitation is improbable in these tissues under natural conditions, unless under complete submersion. Nevertheless, an O2-depleted atmosphere is commonly used in gas exchange experiments to suppress photorespiration and estimate gross photosynthesis. However, the nonphotosynthetic effects of gaseous O2 depletion, particularly on respiratory metabolism, are not well documented. Here, we used metabolomics obtained under contrasting O2 and CO2 conditions to examine the specific effect of a changing O2 mole fraction from ambient (21%) to 0%, 2% or 100%. In addition to the typical decrease in photorespiratory intermediates (glycolate, glycine and serine) and a build-up in photosynthates (sucrose), low O2 (0% or 2%) was found to trigger an accumulation of alanine and change succinate metabolism. In 100% O2, the synthesis of threonine and methionine from aspartate appeared to be stimulated. These responses were observed in two species, sunflower (Helianthus annuus L.) and Arabidopsis thaliana (L.) Heynh. Our results show that O2 causes a change in the oxygenation : carboxylation ratio and also alters postphotosynthetic metabolism: (i) a hypoxic response at low O2 mole fractions and (ii) a stimulation of S metabolism at high O2 mole fractions. The latter effect is an important piece of information to better understand how photorespiration may control S assimilation.

Leaf green-white variegation is advantageous under N deprivation in Pelargonium×hortorum

Variegation (patchy surface area with different colours) is a common trait of plant leaves. In green-white variegated leaves, two tissues with contrasted primary carbon metabolisms (autotrophic in green and heterotrophic in white tissues) are juxtaposed. It is generally believed that variegation is detrimental to growth due to the lower photosynthetic surface area. However, the common occurrence of leaf variegation in nature raises the question of a possible advantage under certain circumstances. Here, we examined growth and metabolism of variegated Pelargonium×hortorum L.H.Bailey using metabolomics techniques under N deprivation. Our results showed that variegated plants tolerate N deficiency much better, i.e. do not stop leaf biomass production after 9 weeks of N deprivation, even though the growth of green plants is eventually arrested and leaf senescence is triggered. Metabolic analysis indicates that white areas are naturally enriched in arginine, which decreases a lot upon N deprivation, probably to feed green areas. This process may compensate for the lower proteolysis enhancement in green areas and thus contribute to maintaining photosynthetic activity. We conclude that under our experimental conditions, leaf variegation was advantageous under prolonged N deprivation.

Carbon allocation to major metabolites in illuminated leaves is not just proportional to photosynthesis when gaseous conditions (CO2 and O2) vary

In gas-exchange experiments, manipulating CO2 and O2 is commonly used to change the balance between carboxylation and oxygenation. Downstream metabolism (utilization of photosynthetic and photorespiratory products) may also be affected by gaseous conditions but this is not well documented. Here, we took advantage of sunflower as a model species, which accumulates chlorogenate in addition to sugars and amino acids (glutamate, alanine, glycine and serine). We performed isotopic labelling with 13 CO2 under different CO2 /O2 conditions, and determined 13 C contents to compute 13 C-allocation patterns and build-up rates. The 13 C content in major metabolites was not found to be a constant proportion of net fixed carbon but, rather, changed dramatically with CO2 and O2 . Alanine typically accumulated at low O2 (hypoxic response) while photorespiratory intermediates accumulated under ambient conditions and at high photorespiration, glycerate accumulation exceeding serine and glycine build-up. Chlorogenate synthesis was relatively more important under normal conditions and at high CO2 and its synthesis was driven by phosphoenolpyruvate de novo synthesis. These findings demonstrate that carbon allocation to metabolites other than photosynthetic end products is affected by gaseous conditions and therefore the photosynthetic yield of net nitrogen assimilation varies, being minimal at high CO2 and maximal at high O2 .

Responses to K deficiency and waterlogging interact via respiratory and nitrogen metabolism: Metabolic effects of K deficiency and waterlogging

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 C5 -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 (δ15 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.

Interactions Between Day Respiration, Photorespiration, and N and S Assimilation in Leaves

Respiration of illuminated leaves (day respiration) represents a minor carbon flux as compared to photosynthesis and photorespiration under usual gaseous conditions. However, it is crucial for leaf primary metabolism, since it sustains N assimilation and provides ATP that can be used for sucrose synthesis in the light. Although available data on interactions between photosynthesis, photorespiration, respiration, and nutrient assimilation are still rather limited, recent works taking advantage of isotopic labeling and metabolomics suggest that changes in photosynthetic and photorespiratory conditions (or the carboxylation-to-oxygenation ratio, v c /v o ) influence N assimilation, S incorporation into methionine, and possibly, displace the equilibrium between C1-metabolites. The crossroad of all of these pathways is mitochondrial metabolism. Therefore, day respiration is probably of considerable importance not only for nutrient assimilation but also for cellular metabolic coordination. This view agrees with data obtained in respiratory mutants.

Isotopic evidence for nitrogen exchange between autotrophic and heterotrophic tissues in variegated leaves

Many plant species or cultivars form variegated leaves in which blades are made of green and white sectors. On the one hand, there is little photosynthetic CO2 assimilation in white tissue simply because of the lack of functional chloroplasts and thus, leaf white tissue is heterotrophic and fed by photosynthates exported by leaf green tissue. On the other hand, it has been previously shown that the white tissue is enriched in nitrogenous compounds such as amino acids and polyamines, which can, in turn, be remobilised upon nitrogen deficiency. However, the origin of organic nitrogen in leaf white tissue, including the possible requirement for N-reduction in leaf green tissue before export to white tissue, has not been examined. Here, we took advantage of isotopic methods to investigate the source of nitrogen in the white tissue. A survey of natural isotope abundance (δ15N) and elemental composition (%N) in various variegated species shows no visible difference between white and green tissues, suggesting a common N source. However, there is a tendency for N-rich white tissue to be naturally 15N-enriched whereas in the model species Pelargonium×hortorum, white sectors are naturally 15N-depleted, indicating that changes in metabolic composition and/or N-partitioning may occur. Isotopic labelling with 15N-nitrate on illuminated leaf discs clearly shows that the white tissue assimilates little nitrogen and thus relies on nitrate reduction and metabolism in the green tissue. The N-sink represented by the white tissue is considerable, accounting for nearly 50% of total assimilated nitrate.