Measuring CO2 and HCO3- permeabilities of isolated chloroplasts using a MIMS-18O approach

To support photosynthetic CO2 fixation by Rubisco, the chloroplast must be fed with inorganic carbon in the form of CO2 or bicarbonate. However, the mechanisms allowing the rapid passage of this gas and this charged molecule through the bounding membranes of the chloroplast envelope are not yet completely elucidated. We describe here a method allowing us to measure the permeability of these two molecules through the chloroplast envelope using a membrane inlet mass spectrometer and 18O-labelled inorganic carbon. We established that the internal stromal carbonic anhydrase activity is not limiting for this technique, and precisely measured the chloroplast surface area and permeability values for CO2 and bicarbonate. This was performed on chloroplasts from several plant species, with values ranging from 2.3 × 10-4 m s-1 to 8 × 10-4 m s-1 permeability for CO2 and 1 × 10-8 m s-1 for bicarbonate. We were able to apply our method to chloroplasts from an Arabidopsis aquaporin mutant, and this showed that CO2 permeability was reduced 50% in the mutant compared with the wild-type reference.This work was supported by the University of Illinois as part of the Bill and Melinda Gates Foundation-funded Realizing Increased Photosynthetic Efficiency (RIPE) consortium, and the Australian Research Council’s Centre of Excellence for Translational Photosynthesis

Measuring CO2 and HCO3 - permeabilities of isolated chloroplasts using a MIMS-18O approach

To support photosynthetic CO2 fixation by Rubisco, the chloroplast must be fed with inorganic carbon in the form of CO2 or bicarbonate. However, the mechanisms allowing the rapid passage of this gas and this charged molecule through the bounding membranes of the chloroplast envelope are not yet completely elucidated. We describe here a method allowing us to measure the permeability of these two molecules through the chloroplast envelope using a membrane inlet mass spectrometer and 18O-labelled inorganic carbon. We established that the internal stromal carbonic anhydrase activity is not limiting for this technique, and precisely measured the chloroplast surface area and permeability values for CO2 and bicarbonate. This was performed on chloroplasts from several plant species, with values ranging from 2.3 × 10–4 m s–1 to 8 × 10–4 m s–1 permeability for CO2 and 1 × 10–8 m s–1 for bicarbonate. We were able to apply our method to chloroplasts from an Arabidopsis aquaporin mutant, and this showed that CO2 permeability was reduced 50% in the mutant compared with the wild-type reference.This work was supported by the University of Illinois as part of the Bill and Melinda Gates Foundation-funded Realizing Increased Photosynthetic Efficiency (RIPE) consortium, and the Australian Research Council’s Centre of Excellence for Translational Photosynthesis. The authors declare no conflict of interest

The combination of gas-phase fluorophore technology and automation to enable high-throughput analysis of plant respiration

Background Mitochondrial respiration in the dark (R dark) is a critical plant physiological process, and hence a reliable, efficient and high-throughput method of measuring variation in rates of R dark is essential for agronomic and ecological studies. However, currently methods used to measure R dark in plant tissues are typically low throughput. We assessed a high-throughput automated fluorophore system of detecting multiple O2 consumption rates. The fluorophore technique was compared with O2-electrodes, infrared gas analysers (IRGA), and membrane inlet mass spectrometry, to determine accuracy and speed of detecting respiratory fluxes. Results The high-throughput fluorophore system provided stable measurements of R dark in detached leaf and root tissues over many hours. High-throughput potential was evident in that the fluorophore system was 10 to 26-fold faster per sample measurement than other conventional methods. The versatility of the technique was evident in its enabling: (1) rapid screening of R dark in 138 genotypes of wheat; and, (2) quantification of rarely-assessed whole-plant R dark through dissection and simultaneous measurements of above- and below-ground organs. Discussion Variation in absolute R dark was observed between techniques, likely due to variation in sample conditions (i.e. liquid vs. gas-phase, open vs. closed systems), indicating that comparisons between studies using different measuring apparatus may not be feasible. However, the high-throughput protocol we present provided similar values of R dark to the most commonly used IRGA instrument currently employed by plant scientists. Together with the greater than tenfold increase in sample processing speed, we conclude that the high-throughput protocol enables reliable, stable and reproducible measurements of R dark on multiple samples simultaneously, irrespective of plant or tissue type.The support of the Australian Research Council (CE140100008) to OKA and AHM is acknowledged

Implication des réserves carbonées dans le photoproduction d'hydrogène chez l'algue verte Chlamydomonas reinhardtii

L algue verte unicellulaire Chlamydomonas reinhardtii, est capable de produire de l hydrogène en utilisant l eau comme donneur d électrons et la lumière solaire comme source d énergie. Bien que cette propriété offre des perspectives biotechnologiques intéressantes, une limitation majeure est liée à la sensibilité de l hydrogénase à l oxygène, sous produit de l activité photosynthétique. Il avait été précédemment montré qu en condition de carence en soufre, C. reinhardtii peut produire de l hydrogène pendant plusieurs jours (Melis et al. 2000). Au cours de ce processus, deux voies de production, l une directe impliquant le photosystème II (PSII) et l autre indirecte impliquant le seul PSI cohabitent, les réserves d amidon étant supposées jouer un rôle dans le fonctionnement de chacune de ces voies. L objectif de ce travail de thèse visait à élucider les mécanismes reliant le catabolisme de l amidon au processus de photo production d hydrogène. Dans un premier temps, l analyse de mutants affectés dans la biosynthèse de l amidon (sta6 et sta7) a permis de montrer que, si les réserves d amidon étaient essentielles au fonctionnement de la voie indirecte, elles ne participaient pas à la voie directe. Dans un second temps, dans le but d identifier les étapes métaboliques et les régulations impliquées dans le catabolisme de l amidon, nous avons développé une approche génétique basée sur la recherche de mutants affectés dans la mobilisation des réserves. Huit mutants (std1 à std8) affectés à des degrés divers dans leurs capacités à dégrader l amidon suite à une phase d accumulation préalable ont été isolés à partir d une banque de 15 000 mutants d insertion. Un des ces mutants, std1 est affecté dans une kinase apparentée à la famille des DYRK (dualspecificity tyrosine regulated serine threonine kinase). Bien que les cibles de cette enzyme restent à déterminer, l analyse du protéome lié aux grains d amidon indique de profondes modifications dans l expression de phosphorylases potentiellement impliquées dans la dégradation de l amidon. STD1 représente le premier élément régulateur du catabolisme de l amidon identifié à ce jour chez les végétauxThe unicellular green alga Chlamydomonas reinhardtii is able to produce hydrogen, using water as an electron donor, and sunlight as an energy source. Although this property offers interesting biotechnological perspectives, a major limitation is related to the sensitivity of hydrogenase to oxygen which is produced by photosynthesis. It had been previously shown that in conditions of sulfur deprivation, C. reinhardtii is able to produce hydrogen during several days (Melis et an. 2000). During this process, two pathways, one direct depending on photosystem II (PSII) activity and the other involving only the PSI, are involved, starch reserves being supposed to play a role in both of these pathways. The purpose of this phD thesis was to elucidate the mechanisms linking starch catabolism to the hydrogen photoproduction process. Firstly, the analysis of mutants affected in starch biosynthesis (sta6 and sta7) showed that if starch reserves are essential to the functioning of the indirect pathway, they are not involved in the direct one. Secondly, in order to identify metabolic steps and regulatory processes involved in starch breakdown, we developed a genetic approach based on the search of mutants affected in starch reserves mobilization. Eight mutant (std1 to std8) diversely affected in their ability to degrade starch after an accumulation phase have been isolated from an insertional mutant library of 15,000 clones. One of these mutants, std1, is affected in a kinase related to the DYRK family (dual specificity tyrosine regulated serine threonine kinase). Although the targets of this putative kinase remain to be identified, the analysis of the granulebound proteome displayed profound alterations in the expression profile of starch phosphorylases, potentially involved in starch breakdown. STD1 represents the first starch catabolism regulator identified to date in plantsAIX-MARSEILLE2-BU Sci.Luminy (130552106) / SudocSudocFranceF

A novel robust and high‐throughput method to measure cell death in Nicotiana benthamiana leaves by fluorescence imaging

International audienceAssessing immune responses and cell death in Nicotiana benthamiana leaf agro-infiltration assays is a powerful and widely used experimental approach in molecular plant pathology. Here, we describe a reliable high-throughput protocol to quantify strong, macroscopically visible cell death responses in N. benthamiana agro-infiltration assays. The method relies on measuring the reduction of leaf autofluorescence in the red spectrum upon cell death induction and provides quantitative data suitable for straightforward statistical analysis. Two different well-established model nucleotide-binding and leucine-rich repeat domain proteins (NLRs) were used to ensure the genericity of the approach. Its accuracy and versatility were compared to visual scoring of the cell death response and standard methods commonly used to characterize NLR activities in N. benthamiana. A discussion of the advantages and limitations of our method compared to other protocols demonstrates its robustness and versatility and provides an effective means to select the best-suited protocol for a defined experiment

Tolleter et al_lowly enriched

COPASI file of numerical modelling used for the generation of times courses for changes in singly labelled CO2 species (lowly enriched 18O bicarbonate

Tolleter et al_highly enriched

COPASI file of numerical modelling used for the the generation of time courses for changes in dually labelled CO2 (highly enriched 18O bicarbonate