Characterization of soldat8, a Suppressor of Singlet Oxygen-Induced Cell Death in Arabidopsis Seedlings

The flu mutant of Arabidopsis thaliana overaccumulates in the dark the immediate precursor of chlorophyllide, protochlorophyllide (Pchlide), a potent photosensitizer, that upon illumination generates singlet oxygen (1O2). Once 1O2 has been released in plastids of the flu mutant, mature plants stop growing, while seedlings die. Several suppressor mutations, dubbed singlet oxygen-linked death activator (soldat), were identified that specifically abrogate 1O2-mediated stress responses in young flu seedlings without grossly affecting 1O2-mediated stress responses of mature flu plants. One of the soldat mutations, soldat8, was shown to impair a gene encoding the SIGMA6 factor of the plastid RNA polymerase. Reintroduction of a wild-type copy of the SOLDAT8 gene into the soldat8/flu mutant restored the phenotype of the flu parental line. In contrast to flu, seedlings of soldat8/flu did not bleach when grown under non-permissive dark/light conditions, despite their continuous overaccumulation of the photosensitizer Pchlide in the dark. The activity of SIGMA6 is confined primarily to the very early stage of seedling development. Inactivation of SIGMA6 in soldat8 mutants disturbed plastid homeostasis, drastically reduced the non-photochemical quenching capacity and enhanced the light sensitivity of young soldat8 seedlings. Surprisingly, after being grown under very low light, soldat8 seedlings showed an enhanced resistance against a subsequent severe light stress that was significantly higher than in wild-type seedlings. In order to reach a similar enhanced stress resistance, wild-type seedlings had to be exposed to a brief higher light treatment that triggered an acclimatory response. Such a mild pre-stress treatment did not further enhance the stress resistance of soldat8 seedlings. Suppression of 1O2-mediated cell death in young flu/soldat8 seedlings seems to be due to a transiently enhanced acclimation at the beginning of seedling development caused by the initial disturbance of plastid homeostasi

Human Cryptochrome-1 Confers Light Independent Biological Activity in Transgenic Drosophila Correlated with Flavin Radical Stability

Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism

Birth of a Photosynthetic Chassis: A MoClo Toolkit Enabling Synthetic Biology in the Microalga Chlamydomonas reinhardtii.

Microalgae are regarded as promising organisms to develop innovative concepts based on their photosynthetic capacity that offers more sustainable production than heterotrophic hosts. However, to realize their potential as green cell factories, a major challenge is to make microalgae easier to engineer. A promising approach for rapid and predictable genetic manipulation is to use standardized synthetic biology tools and workflows. To this end we have developed a Modular Cloning toolkit for the green microalga Chlamydomonas reinhardtii. It is based on Golden Gate cloning with standard syntax, and comprises 119 openly distributed genetic parts, most of which have been functionally validated in several strains. It contains promoters, UTRs, terminators, tags, reporters, antibiotic resistance genes, and introns cloned in various positions to allow maximum modularity. The toolkit enables rapid building of engineered cells for both fundamental research and algal biotechnology. This work will make Chlamydomonas the next chassis for sustainable synthetic biology

Caractérisation de la mort cellulaire programmée induite par les UV-C chez Arabidopsis thaliana

Dans l'optique d'étudier la mort cellulaire programmée (PCD) chez Arabidopsis, nous avons mis au point un système qui couple induction et quantification de la PCD. Notre système repose sur l'induction de la PCD par les UV-C. L'irradiation de protoplastes couplée à l'utilisation de la réaction TUNEL, nous a permis de quantifier la proportion de cellules en voie de PCD. La tranfection des protoplastes a ensuite permis de mettre en évidence la conservation du rôle de suppresseur de PCD des deux gènes At-DAD (Defender against apoptotic death) d'Arabidopsis ainsi que du gène viral p35 impliqué dans l'inhibition des caspases chez les animaux. Les caspases sont des proteinases spécifiquement induites au cours de la PCD chez les animaux, et qui sont responsables de la dégradation de composants importants pour la cellule. Nous avons pu détecter une activité de type caspase induite par les UV-C et spécifiquement inhibée par des inhibiteurs synthétiques de caspase. Ces mêmes inhibiteurs sont capable de protéger les protoplastes de la PCD induite par les UV-C. Nous avons purifié cette activité de type caspase par FPLC et chromatographie d'affinité et sommes en attente d'identifier par MS/MS les bandes protéiques obtenues par électrophorèse.PERPIGNAN-BU Sciences (661362101) / SudocSudocFranceF

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When Unity Is Strength: The Strategies Used by Chlamydomonas to Survive Environmental Stresses

International audienceThe unicellular green alga Chlamydomonas reinhardtii is a valuable model system to study a wide spectrum of scientific fields, including responses to environmental conditions. Most studies are performed under optimal growth conditions or under mild stress. However, when environmental conditions become harsher, the behavior of this unicellular alga is less well known. In this review we will show that despite being a unicellular organism, Chlamydomonas can survive very severe environmental conditions. To do so, and depending on the intensity of the stress, the strategies used by Chlamydomonas can range from acclimation to the formation of multicellular structures, or involve programmed cell death

l-Galactono-1,4-lactone Dehydrogenase Is Required for the Accumulation of Plant Respiratory Complex I

International audienceMitochondrial NADH-ubiquinone oxidoreductase (complex I) is the largest enzyme of the oxidative phosphorylation system, with subunits located at the matrix and membrane domains. In plants, holocomplex I is composed of more than 40 subunits, 9 of which are encoded by the mitochondrial genome (NAD subunits). In Nicotiana sylvestris, a minor 800-kDa subcomplex containing subunits of both domains and displaying NADH dehydrogenase activity is detectable. The NMS1 mutant lacking the membrane arm NAD4 subunit and the CMSII mutant lacking the peripheral NAD7 subunit are both devoid of the holoenzyme. In contrast to CMSII, the 800-kDa subcomplex is present in NMS1 mitochondria, indicating that it could represent an assembly intermediate lacking the distal part of the membrane arm. L-galactono-1,4-lactone dehydrogenase (GLDH), the last enzyme in the plant ascorbate biosynthesis pathway, is associated with the 800-kDa subcomplex but not with the holocomplex. To investigate possible relationships between GLDH and complex I assembly, we characterized an Arabidopsis thaliana gldh insertion mutant. Homozygous gldh mutant plants were not viable in the absence of ascorbate supplementation. Analysis of crude membrane extracts by blue native and two-dimensional SDS-PAGE showed that complex I accumulation was strongly prevented in leaves and roots of Atgldh plants, whereas other respiratory complexes were found in normal amounts. Our results demonstrate the role of plant GLDH in both ascorbate biosynthesis and complex I accumulation

Ultraviolet-C Overexposure Induces Programmed Cell Death in Arabidopsis, Which Is Mediated by Caspase-like Activities and Which Can Be Suppressed by Caspase Inhibitors, p35 and Defender against Apoptotic Death

International audiencePlants, animals, and several branches of unicellular eukaryotes use programmed cell death (PCD) for defense or developmental mechanisms. This argues for a common ancestral apoptotic system in eukaryotes. However, at the molecular level, very few regulatory proteins or protein domains have been identified as conserved across all eukaryotic PCD forms. A very important goal is to determine which molecular components may be used in the execution of PCD in plants, which have been conserved during evolution, and which are plant-specific. Using Arabidopsis thaliana, we have shown that UV radiation can induce apoptosis-like changes at the cellular level and that a UV experimental system is relevant to the study of PCD in plants. We report here that UV induction of PCD required light and that a protease cleaving the caspase substrate Asp-Glu-Val-Asp (DEVDase activity) was induced within 30 min and peaked at 1 h. This DEVDase appears to be related to animal caspases at the biochemical level, being insensitive to broad-range cysteine protease inhibitors. In addition, caspase-1 and caspase-3 inhibitors and the pan-caspase inhibitor p35 were able to suppress DNA fragmentation and cell death. These results suggest that a YVADase activity and an inducible DEVDase activity possibly mediate DNA fragmentation during plant PCD induced by UV overexposure. We also report that At-DAD1 and At-DAD2, the two A. thaliana homologs of Defender against Apoptotic Death-1, could suppress the onset of DNA fragmentation in A. thaliana, supporting an involvement of the endoplasmic reticulum in this form of the plant PCD pathway