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

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

Coral Bleaching Independent of Photosynthetic Activity

SummaryThe global decline of reef-building corals is due in part to the loss of algal symbionts, or “bleaching,” during the increasingly frequent periods of high seawater temperatures [1, 2]. During bleaching, endosymbiotic dinoflagellate algae (Symbiodinium spp.) either are lost from the animal tissue or lose their photosynthetic pigments, resulting in host mortality if the Symbiodinium populations fail to recover [3]. The >1,000 studies of the causes of heat-induced bleaching have focused overwhelmingly on the consequences of damage to algal photosynthetic processes [4–6], and the prevailing model for bleaching invokes a light-dependent generation of toxic reactive oxygen species (ROS) by heat-damaged chloroplasts as the primary trigger [6–8]. However, the precise mechanisms of bleaching remain unknown, and there is evidence for involvement of multiple cellular processes [9, 10]. In this study, we asked the simple question of whether bleaching can be triggered by heat in the dark, in the absence of photosynthetically derived ROS. We used both the sea anemone model system Aiptasia [11, 12] and several species of reef-building corals to demonstrate that symbiont loss can occur rapidly during heat stress in complete darkness. Furthermore, we observed damage to the photosynthetic apparatus under these conditions in both Aiptasia endosymbionts and cultured Symbiodinium. These results do not directly contradict the view that light-stimulated ROS production is important in bleaching, but they do show that there must be another pathway leading to bleaching. Elucidation of this pathway should help to clarify bleaching mechanisms under the more usual conditions of heat stress in the light

Analyse structurale et fonctionnelle d'une protéine LEA (Late Embryogenesis Abundant) mitochondriale exprimée dans les graines de pois

Although few organisms are desiccation tolerant, this remarkable property is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. We show here that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into alpha-helices upon desiccation. LEAM interacts with membranes in the dry state, especially at the level of the phosphate group of phospholipids. These interactions, which are dependent upon lipid composition, provide a protection to liposomes, against desiccation and freezing. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. A reverse genetic approach was engaged to confirm this axiom and precise the physiological role of LEAM.A l'exception des graines de plantes supérieures, peu d'organismes sont capables de résister à la dessiccation. La survie à l'état sec est un phénomène multifactoriel impliquant notamment l'accumulation de protéines LEA (Late Embryogenesis Abundant). Nous avons montré que la LEAM, une protéine LEA mitochondriale exprimée dans les graines, est dépliée à l'état natif, mais peut se structurer réversiblement en hélice a sous l'effet de la dessiccation. La LEAM interagit avec les membranes à l'état sec, et plus précisément avec les groupements phosphate des phospholipides. Ces interactions, qui dépendent de la composition lipidique, permettent de protéger les liposomes des effets destructeurs de la dessiccation et de la congélation. L'ensemble de ces résultats indique que la LEAM protège la membrane interne mitochondriale pendant la dessiccation. Des approches de génétique inverse ont été initiées afin de confirmer cet axiome et de préciser le rôle physiologique de la LEA

Analyse structurale et fonctionnelle d'une protéine LEA (Late Embryogenesis Abundant) mitochondriale exprimée dans les graines de pois

A l'exception des graines de plantes supérieures, peu d'organismes sont capables de résister à la dessiccation. La survie à l'état sec est un phénomène multifactoriel impliquant notamment l'accumulation de protéines LEA (Late Embryogenesis Abundant). Nous avons montré que la LEAM, une protéine LEA mitochondriale exprimée dans les graines, est dépliée à l'état natif, mais peut se structurer réversiblement en hélice a sous l'effet de la dessiccation. La LEAM interagit avec les membranes à l'état sec, et plus précisément avec les groupements phosphate des phospholipides. Ces interactions, qui dépendent de la composition lipidique, permettent de protéger les liposomes des effets destructeurs de la dessiccation et de la congélation. L'ensemble de ces résultats indique que la LEAM protège la membrane interne mitochondriale pendant la dessiccation. Des approches de génétique inverse ont été initiées afin de confirmer cet axiome et de préciser le rôle physiologique de la LEAMAlthough few organisms are desiccation tolerant, this remarkable property is widespread among plant seeds. Survival without water relies on an array of mechanisms, including the accumulation of stress proteins such as the late embryogenesis abundant (LEA) proteins. We show here that LEAM, a mitochondrial LEA protein expressed in seeds, is a natively unfolded protein, which reversibly folds into alpha-helices upon desiccation. LEAM interacts with membranes in the dry state, especially at the level of the phosphate group of phospholipids. These interactions, which are dependent upon lipid composition, provide a protection to liposomes, against desiccation and freezing. The overall results provide strong evidence that LEAM protects the inner mitochondrial membrane during desiccation. A reverse genetic approach was engaged to confirm this axiom and precise the physiological role of LEAM.ANGERS-BU Lettres et Sciences (490072106) / SudocSudocFranceF

Auxiliary electron transport pathways in chloroplasts of microalgae

Microalgae are photosynthetic organisms which cover an extraordinary phylogenic diversity and have colonized extremely diverse habitats. Adaptation to contrasted environments in terms of light and nutrient's availabilities has been possible through a high flexibility of the photosynthetic machinery. Indeed, optimal functioning of photosynthesis in changing environments requires a fine tuning between the conversion of light energy by photosystems and its use by metabolic reaction, a particularly important parameter being the balance between phosphorylating (ATP) and reducing (NADPH) power supplies. In addition to the main route of electrons operating during oxygenic photosynthesis, called linear electron flow or Z scheme, auxiliary routes of electron transfer in interaction with the main pathway have been described. These reactions which include non-photochemical reduction of intersystem electron carriers, cyclic electron flow around PSI, oxidation by molecular O2 of the PQ pool or of the PSI electron acceptors, participate in the flexibility of photosynthesis by avoiding over-reduction of electron carriers and modulating the NADPH/ATP ratio depending on the metabolic demand. Forward or reverse genetic approaches performed in model organisms such as Arabidopsis thaliana for higher plants, Chlamydomonas reinhardtii for green algae and Synechocystis for cyanobacteria allowed identifying molecular components involved in these auxiliary electron transport pathways, including Ndh-1, Ndh-2, PGR5, PGRL1, PTOX and flavodiiron proteins. In this article,we discuss the diversity of auxiliary routes of electron transport inmicroalgae, with particular focus in the presence of these components in the microalgal genomes recently sequenced. We discuss how these auxiliary mechanisms of electron transport may have contributed to the adaptation of microalgal photosynthesis to diverse and changing environments

A mitochondrial late embryogenesis abundant protein stabilizes model membranes in the dry state

Late embryogenesis abundant (LEA) proteins are a highly diverse group of polypeptides expected to play important roles in desiccation tolerance of plant seeds. They are also found in other plant tissues and in some anhydrobotic invertebrates, fungi, protists and prokaryotes. The LEA protein LEAM accumulates in the matrix space of pea (Pisum sativum) mitochondria during late seed maturation. LEAM is an intrinsically disordered protein folding into amphipathic α-helix upon desiccation. This suggests that it could interact with the inner mitochondrial membrane, providing structural protection in dry seeds. Here, we have used Fourier-transform infrared and fluorescence spectroscopy to gain insight into the molecular details of interactions of LEAM with phospholipid bilayers in the dry state and their effects on liposome stability. LEAM interacted specifically with negatively charged phosphate groups in dry phospholipids, increasing fatty acyl chain mobility. This led to an enhanced stability of liposomes during drying and rehydration, but also upon freezing. Protection depended on phospholipid composition and was strongly enhanced in membranes containing the mitochondrial phospholipid cardiolipin. Collectively, the results provide strong evidence for a function of LEAM as a mitochondrial membrane protectant during desiccation and highlight the role of lipid composition in the interactions between LEA proteins and membranes

Auxiliary electron transport pathways in chloroplasts of microalgae

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Is the heat on for plant mitochondria?

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