Martine Bertrand

Carotenoid biosynthesis in diatoms

Diatoms are ubiquitous and constitute an important group of the phytoplankton community having a major contribution to the total marine primary production. These microalgae exhibit a characteristic golden-brown colour due to a high amount of the xanthophyll fucoxanthin that plays a major role in the light-harvesting complex of photosystems. In the water column, diatoms are exposed to light intensities that vary quickly from lower to higher values. Xanthophyll cycles prevent photodestruction of the cells in excessive light intensities. In diatoms, the diadinoxanthin–diatoxanthin cycle is the most important short-term photoprotective mechanism. If the biosynthetic pathways of chloroplast pigments have been extensively studied in higher plants and green algae, the research on carotenoid biosynthesis in diatoms is still in its infancy. In this study, the data on the biosynthetic pathway of diatom carotenoids are reviewed. The early steps occur through the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. Then a hypothetical pathway is suggested from dimethylallyl diphosphate (DMAPP) and isopentenyl pyrophosphate (IPP). Most of the enzymes of the pathway have not been so far isolated from diatoms, but candidate genes for each of them were identified using protein similarity searches of genomic data.

Arbuscular mycorrhizal symbiosis elicits shoot proteome changes that are modified during cadmium stress alleviation in Medicago truncatula

BackgroundArbuscular mycorrhizal (AM) fungi, which engage a mutualistic symbiosis with the roots of most plant species, have received much attention for their ability to alleviate heavy metal stress in plants, including cadmium (Cd). While the molecular bases of Cd tolerance displayed by mycorrhizal plants have been extensively analysed in roots, very little is known regarding the mechanisms by which legume aboveground organs can escape metal toxicity upon AM symbiosis. As a model system to address this question, we used Glomus irregulare-colonised Medicago truncatula plants, which were previously shown to accumulate and tolerate heavy metal in their shoots when grown in a substrate spiked with 2 mg Cd kg-1.ResultsThe measurement of three indicators for metal phytoextraction showed that shoots of mycorrhizal M. truncatula plants have a capacity for extracting Cd that is not related to an increase in root-to-shoot translocation rate, but to a high level of allocation plasticity. When analysing the photosynthetic performance in metal-treated mycorrhizal plants relative to those only Cd-supplied, it turned out that the presence of G. irregulare partially alleviated the negative effects of Cd on photosynthesis. To test the mechanisms by which shoots of Cd-treated mycorrhizal plants avoid metal toxicity, we performed a 2-DE/MALDI/TOF-based comparative proteomic analysis of the M. truncatula shoot responses upon mycorrhization and Cd exposure. Whereas the metal-responsive shoot proteins currently identified in non-mycorrhizal M. truncatula indicated that Cd impaired CO2 assimilation, the mycorrhiza-responsive shoot proteome was characterised by an increase in photosynthesis-related proteins coupled to a reduction in glugoneogenesis/glycolysis and antioxidant processes. By contrast, Cd was found to trigger the opposite response coupled the up-accumulation of molecular chaperones in shoot of mycorrhizal plants relative to those metal-free.ConclusionBesides drawing a first picture of shoot proteome modifications upon AM symbiosis and/or heavy metal stress in legume plants, the current work argues for allocation plasticity as the main driving force for Cd extraction in aboveground tissues of M. truncatula upon mycorrhization. Additionally, according to the retrieved proteomic data, we propose that shoots of mycorrhizal legume plants escape Cd toxicity through a metabolic shift implying the glycolysis-mediated mobilization of defence mechanisms at the expense of the photosynthesis-dependent symbiotic sucrose sink.

Spectroscopic Properties of Protochlorophyllide Analyzed In Situ in the Course of Etiolation and in Illuminated Leaves

Abstract The spectroscopic properties of photoactive (i.e. flash-transformable) and nonphotoactive protochlorophyll(ide)s (Pchl(ide)) were reinvestigated during the development of bean leaves in darkness. Two phases in the process of Pchl(ide) accumulation were apparent from quantitative measurements of pigment content: a lag phase (first week) during which photoactive Pchl(ide) accumulated faster than nonphotoactive Pchl(ide); and a fast phase (second week), showing parallel accumulation of both types of Pchl(ide). ‘Flashed-minus-dark’ absorbance difference spectra recorded in situ at 77 K showed that P650–655 was the predominant form of photoactive protochlorophyllide regardless of developmental stage. Quantitative analysis of energy migration processes between the Pchl(ide) forms showed the existence of energy transfer units containing a 1:8 ratio of nonphotoactive and photoactive Pchl(ide)s during development. Gaussian deconvolution of in situ 77 K fluorescence spectra indicated that the 633 nm band of nonphotoactive Pchl(ide) was made of four bands, at 625, 631, 637 and 643 nm, whose relative amplitudes only slightly changed during development. The emission band of photoactive Pchlide was also analyzed using the same method. Three components were found at 644, 652 and 657 nm. The emission band of P650–655 included the last two components, which become predominant only in fully etiolated plants. Photoactive Pchlide with an emission maximum at 653 nm was detected in the light during development of leaves of photoperiodically grown plants.

Role of Thylakoid ATP/ADP Carrier in Photoinhibition and Photoprotection of Photosystem II in Arabidopsis

The chloroplast thylakoid ATP/ADP carrier (TAAC) belongs to the mitochondrial carrier superfamily and supplies the thylakoid lumen with stromal ATP in exchange for ADP. Here, we investigate the physiological consequences of TAAC depletion in Arabidopsis (Arabidopsis thaliana). We show that the deficiency of TAAC in two T-DNA insertion lines does not modify the chloroplast ultrastructure, the relative amounts of photosynthetic proteins, the pigment composition, and the photosynthetic activity. Under growth light conditions, the mutants initially displayed similar shoot weight, but lower when reaching full development, and were less tolerant to high light conditions in comparison with the wild type. These observations prompted us to study in more detail the effects of TAAC depletion on photoinhibition and photoprotection of the photosystem II (PSII) complex. The steady-state phosphorylation levels of PSII proteins were not affected, but the degradation of the reaction center II D1 protein was blocked, and decreased amounts of CP43-less PSII monomers were detected in the mutants. Besides this, the mutant leaves displayed a transiently higher nonphotochemical quenching of chlorophyll fluorescence than the wild-type leaves, especially at low light. This may be attributed to the accumulation in the absence of TAAC of a higher electrochemical H+ gradient in the first minutes of illumination, which more efficiently activates photoprotective xanthophyll cycle-dependent and independent mechanisms. Based on these results, we propose that TAAC plays a critical role in the disassembly steps during PSII repair and in addition may balance the trans-thylakoid electrochemical H+ gradient storage.

Plastids of marine phytoplankton produce bioactive pigments and lipids.

Phytoplankton is acknowledged to be a very diverse source of bioactive molecules. These compounds play physiological roles that allow cells to deal with changes of the environmental constrains. For example, the diversity of light harvesting pigments allows efficient photosynthesis at different depths in the seawater column. Identically, lipid composition of cell membranes can vary according to environmental factors. This, together with the heterogenous evolutionary origin of taxa, makes the chemical diversity of phytoplankton compounds much larger than in terrestrial plants. This contribution is dedicated to pigments and lipids synthesized within or from plastids/photosynthetic membranes. It starts with a short review of cyanobacteria and microalgae phylogeny. Then the bioactivity of pigments and lipids (anti-oxidant, anti-inflammatory, anti-mutagenic, anti-cancer, anti-obesity, anti-allergic activities, and cardio- neuro-, hepato- and photoprotective effects), alone or in combination, is detailed. To increase the cellular production of bioactive compounds, specific culture conditions may be applied (e.g., high light intensity, nitrogen starvation). Regardless of the progress made in blue biotechnologies, the production of bioactive compounds is still limited. However, some examples of large scale production are given, and perspectives are suggested in the final section.

Separation of photosynthetic pigments and their precursors by reversed-phase high-performance liquid chromatography using a photodiode-array detector

Abstract A reversed-phase HPLC method is described for the separation of photosynthetic pigments, allowing the rapid and efficient separation of protochlorophyll(ide)s, chlorophyll(ide)s and cis- and trans-carotenoid isomers. The pigment contents of non-illuminated and illuminated bean leaves were determined and compared. Both sample types present the same carotenoid pattern. The main carotenoids are always trans-violaxanthin, trans-lutein and β-carotene. The tetrapyrrole pigment contents are very different. Non-illuminated leaves mainly contain protochlorophyllide and protochlorophyll esters whereas illuminated leaves contain mainly chlorophylls.

Responses of the marine bacterium Pseudomonas fluorescens to an excess of heavy metals : Physiological and biochemical aspects

A Pseudomonas fluorescens strain was isolated from oxic marine sediments obtained from the strand zone of the St Anne Bay (a moderately metal-contaminated site to the west of Cherbourg harbour). The strain, which exhibited a high tolerance to metal contamination when cultivated (minimal inhibitory concentration=950 microM [62 mg L(-1)] for Zn, 660 microM [42 mg L(-1)] for Cu, and 505 microM [57 mg L(-1)] for Cd), was further characterized by its physiological and biochemical responses to metal additions to the culture medium. Bacterial growth was significantly disturbed by 380 microM Zn (25 mg L(-1)), 315 microM Cu (20 mg L(-1)) and 90 microM Cd (10 mg L(-1)). The Zn-containing alkaline phosphatase was studied as an intoxication biomarker. Its activity was stimulated (+9%) by an excess of Zn, but inhibited by Cd (-55%) and Cu (-10%), these two elements could displace the native Zn or/and disturb the enzyme 3D-structure. Bacterial O(2) consumption was recorded as a global physiological response to metal stress. This parameter dropped with increasing Cd and Cu contamination (-49% and -45%, respectively, at 20 mg L(-1)). By contrast, Zn increased O2 consumption (approximately +40% for the different tested concentrations). The proteomes of bacteria grown in the presence or absence of 20 mg metal L(-1) were characterized by 2D-gel electrophoresis. The number of spots exhibiting a difference in intensity between the contaminated sample and the control was 65, 68, and 103, for Zn, Cu and Cd, respectively. Among them, 45, 61 and 82 spots respectively appeared de novo or increased in intensity, indicative of metal-stimulated synthesis, particularly for Cu and Cd. In summary, whereas Cd and Cu treatments both stressed cells and slowed down primary metabolism to differing extents, Zn has a stimulating action on several physiological and biochemical parameters.

Photoactive protochlorophyllide regeneration in cotyledons and leaves from higher plants.

Abstract Chlorophyll accumulation during greening implies the continuous transformation of photoactive protochlorophyllide (Pchlide) to chlorophyllide. Since this reaction is a light-dependent step, the study of regeneration of photoactive Pchlide under a continuous illumination is difficult. Therefore this process is best studied on etiolated plants during a period of darkness following the initial photoreduction of photoactive Pchlide. In this study, the regeneration process has been studied using spinach cotyledons, as well as barley and bean leaves, illuminated by a single saturating flash. The regeneration was characterized using 77 K fluorescence emission and excitation spectra and high-performance liquid chromatography. The fluorescence data indicated that the same spectral forms of photoactive Pchlide are regenerated by different pathways: (1) photoactive Pchlide regeneration starts immediately after the photoreduction through the formation of a nonphotoactive Pchlide form, emitting fluorescence at approximately 651 nm. This form is similar to the large aggregate of photoactive Pchlide present before the illumination, but it contains oxidized form of nicotinamide adenine dinucleotide phosphate, instead of the reduced form (NADPH), in the ternary complexes; and (2) after the dislocation of the large aggregates of chlorophyllide–light-dependent NADPH:Pchlide a photooxidoreductase–NADPH ternary complexes, the regeneration occurs at the expense of the several nonphotoactive Pchlide spectral forms present before the illumination.

Changes in the photosynthetic pigments in bean leaves during the first photoperiod of greening and the subsequent dark-phase. Comparison between old (10-d-old) leaves and young (2-d-old) leaves

Chlorophyll and carotenoid variations of 2-d-old and 10-d-old bean leaves (Phaseolus vulgaris var Red Kidney) were analyzed by HPLC during the first photoperiod of greening (16 h light + 8 h dark). The HPLC method used is suitable for the separation of cis- and trans-carotenoid isomers, Pchlide a and Chlide a as well as their esters. The main results are (1) before illumination the composition of the carotenoid pool is similar at the two developmental stages; (2) non-illuminated 2-d-old leaves are devoid of Pchlide a ester; (3) chlorophyll and carotenoid accumulation in 2-d-old leaves presented a lag phase twice longer than observed in 10-d-old ones; (4) Chlide a seems directly esterified to Chl a in 2-d-old leaves whereas esterification requires four steps in 10-d-old leaves and, (5) the kinetics of Chl and carotenoid accumulation are different at the two investigated developmental stages.

Strategies developed by the marine bacterium Pseudomonas fluorescens BA3SM1 to resist metals: A proteome analysis

A global proteomic evaluation of the response of the marine bacterium Pseudomonas fluorescens BA3SM1 to Cd, Zn and Cu was performed by two dimensional gel electrophoresis followed by mass spectrometry. When stressed with Cd, the most toxic metal for P. fluorescens BA3SM1, cell growth is rapidly affected and the number of proteins up-regulated (sixteen for 0.4 mM Cd) remains low in comparison with results obtained for Zn and Cu (twenty eight for 1.5mM Zn and forty four for 1.5 mM Cu). The changes in protein expression indicate that the cell adapts to metals by inducing essentially seven defense mechanisms: cell aggregation/biofilm formation (Zn=Cu>Cd); modification of envelope properties to increase the extracellular metal biosorption and/or control the uptake of metal (Cu>Zn); metal export (Cd=Zn and probably Cu); responses to oxidative stress (Cu>Zn>Cd); intracellular metal sequestration (Zn=Cu and probably Cd); hydrolysis of abnormally folded proteins (Cd=Cu), and the over-synthesis of proteins inhibited by metal (Cd>Cu>Zn). To the best of our knowledge, this is the first report showing that a marine P. fluorescens is able to acquire a metal-resistant phenotype, making the strain BA3SM1 a promising agent for bioremediation processes.