Antoine Danon

Rapid Induction of Distinct Stress Responses after the Release of Singlet Oxygen in Arabidopsis

The conditional fluorescent (flu) mutant of Arabidopsis accumulates the photosensitizer protochlorophyllide in the dark. After a dark-to-light shift, the generation of singlet oxygen, a nonradical reactive oxygen species, starts within the first minute of illumination and was shown to be confined to plastids. Immediately after the shift, plants stopped growing and developed necrotic lesions. These early stress responses of the flu mutant do not seem to result merely from physicochemical damage. Peroxidation of chloroplast membrane lipids in these plants started rapidly and led to the transient and selective accumulation of a stereospecific and regiospecific isomer of hydroxyoctadecatrieonic acid, free (13S)-HOTE, that could be attributed almost exclusively to the enzymatic oxidation of linolenic acid. Within the first 15 min of reillumination, distinct sets of genes were activated that were different from those induced by superoxide/hydrogen peroxide. Collectively, these results demonstrate that singlet oxygen does not act primarily as a toxin but rather as a signal that activates several stress-response pathways. Its biological activity in Arabidopsis exhibits a high degree of specificity that seems to be derived from the chemical identity of this reactive oxygen species and/or the intracellular location at which it is generated.

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.

Plants, 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.

Plant programmed cell death: A common way to die

In the last few years programmed cell death in plants inspired many studies in development and environmental stresses. Some of these studies showed that hallmarks of animal programmed cell death were found at cellular or molecular level in plant cells in different experimental systems. Additionally the effect of over-expression in plants of animal genes implicated in programmed cell death has been tested, and some plant homologues of these genes have been found. This suggests that, despite some differences, plants and animals could share at least some common components of a core mechanism used to carry out programmed cell death in eukaryotes. In this review, we will concentrate on the last findings that suggest similarity between plant programmed cell death and its better known counterpart in animals.

UV-C radiation induces apoptotic-like changes in Arabidopsis thaliana

With a view to studying programmed cell death in plants at the molecular level, we report here for the first time that apoptotic‐like changes are induced by UV radiation in plant nuclei. In Arabidopsis thaliana seedlings a UV‐C dose of 10–50 kJ/m2 induces an oligonucleosomal DNA fragmentation which is reminiscent of the apoptotic DNA ladder described in animal cells. This DNA fragmentation was also detected in situ in protoplast nuclei as soon as 2 h after UV‐C treatment. Moreover, UV‐C induced a nuclear morphology characteristic of animal apoptotic nuclei. We propose that UV‐C induction constitutes a powerful tool to compare the cellular response to irreversible UV damage in plants to that in animals and to study programmed cell death in A. thaliana.

Redox regulation of the Calvin–Benson cycle: something old, something new

Reversible redox post-translational modifications such as oxido-reduction of disulfide bonds, S-nitrosylation, and S-glutathionylation, play a prominent role in the regulation of cell metabolism and signaling in all organisms. These modifications are mainly controlled by members of the thioredoxin and glutaredoxin families. Early studies in photosynthetic organisms have identified the Calvin–Benson cycle, the photosynthetic pathway responsible for carbon assimilation, as a redox regulated process. Indeed, 4 out of 11 enzymes of the cycle were shown to have a low activity in the dark and to be activated in the light through thioredoxin-dependent reduction of regulatory disulfide bonds. The underlying molecular mechanisms were extensively studied at the biochemical and structural level. Unexpectedly, recent biochemical and proteomic studies have suggested that all enzymes of the cycle and several associated regulatory proteins may undergo redox regulation through multiple redox post-translational modifications including glutathionylation and nitrosylation. The aim of this review is to detail the well-established mechanisms of redox regulation of Calvin–Benson cycle enzymes as well as the most recent reports indicating that this pathway is tightly controlled by multiple interconnected redox post-translational modifications. This redox control is likely allowing fine tuning of the Calvin–Benson cycle required for adaptation to varying environmental conditions, especially during responses to biotic and abiotic stresses.

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

Mitochondrial 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.

Cryptochrome-1-dependent execution of programmed cell death induced by singlet oxygen in Arabidopsis thaliana

Programmed cell death (PCD) plays an important role during the life cycle of higher organisms. Although several regulatory mechanisms governing PCD are thought to be conserved in animals and plants, light-dependent cell death represents a form of PCD that is unique to plants. The light requirement of PCD has often been associated with the production of reactive oxygen species during photosynthesis. In support of this hypothesis, hydrogen peroxide and superoxide have been shown to be involved in triggering a PCD response. In the present work, we have used the conditional flu mutant of Arabidopsis to analyze the impact of another reactive oxygen species, singlet oxygen, on cell death. Unexpectedly, the light-dependent release of singlet oxygen alone is not sufficient to induce PCD of flu seedlings but has to act together with a second concurrent blue light reaction. This blue-light-specific trigger of PCD could not be attributed to a photosynthetic reaction or redox change within the chloroplast but to the activation of the blue light/UVA-specific photoreceptor cryptochrome. The singlet oxygen-mediated and cryptochrome-dependent cell death response differs in several ways from PCD triggered by hydrogen peroxide/superoxide.

The Importance of Cardiolipin Synthase for Mitochondrial Ultrastructure, Respiratory Function, Plant Development, and Stress Responses in Arabidopsis

CARDIOLIPIN SYNTHASE (CLS) catalyzes the synthesis of cardiolipin, the signature phospholipid of the mitochondrial inner membrane. Through characterization of a cls mutant in Arabidopsis, this study shows that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions. Cardiolipin (CL) is the signature phospholipid of the mitochondrial inner membrane. In animals and yeast (Saccharomyces cerevisiae), CL depletion affects the stability of respiratory supercomplexes and is thus crucial to the energy metabolism of obligate aerobes. In eukaryotes, the last step of CL synthesis is catalyzed by CARDIOLIPIN SYNTHASE (CLS), encoded by a single-copy gene. Here, we characterize a cls mutant in Arabidopsis thaliana, which is devoid of CL. In contrast to yeast cls, where development is little affected, Arabidopsis cls seedlings are slow developing under short-day conditions in vitro and die if they are transferred to long-day (LD) conditions. However, when transferred to soil under LD conditions under low light, cls plants can reach the flowering stage, but they are not fertile. The cls mitochondria display abnormal ultrastructure and reduced content of respiratory complex I/complex III supercomplexes. The marked accumulation of tricarboxylic acid cycle derivatives and amino acids demonstrates mitochondrial dysfunction. Mitochondrial and chloroplastic antioxidant transcripts are overexpressed in cls leaves, and cls protoplasts are more sensitive to programmed cell death effectors, UV light, and heat shock. Our results show that CLS is crucial for correct mitochondrial function and development in Arabidopsis under both optimal and stress conditions.

The Arabidopsis sweetie mutant is affected in carbohydrate metabolism and defective in the control of growth, development and senescence

SUMMARY Sugars modulate many vital metabolic and developmental processes in plants, from seed germination to flowering, senescence and protection against diverse abiotic and biotic stresses. However, the exact mechanisms involved in morphogenesis, developmental signalling and stress tolerance remain largely unknown. Here we report the characterization of a novel Arabidopsis thaliana mutant, sweetie, with drastically altered morphogenesis, and a strongly modified carbohydrate metabolism leading to elevated levels of trehalose, trehalose-6-phosphate and starch. We additionally show that the disruption of SWEETIE causes significant growth and developmental alterations, such as severe dwarfism, lancet-shaped leaves, early senescence and flower sterility. Genes implicated in sugar metabolism, senescence, ethylene biosynthesis and abiotic stress were found to be upregulated in sweetie. Our physiological, biochemical, genetic and molecular data indicate that the mutation in sweetie was nuclear, single and recessive. The effects of metabolizable sugars and osmolytes on sweetie morphogenesis were distinct; in light, sweetie was hypersensitive to sucrose and glucose during vegetative growth and a partial phenotypic reversion took place in the presence of high sorbitol concentrations. However, SWEETIE encodes a protein that is unrelated to any known enzyme involved in sugar metabolism. We suggest that SWEETIE plays an important regulatory function that influences multiple metabolic, hormonal and stress-related pathways, leading to altered gene expression and pronounced changes in the accumulation of sugar, starch and ethylene.

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 ((1)O2). Once (1)O2 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 (1)O2-mediated stress responses in young flu seedlings without grossly affecting (1)O2-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 (1)O2-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 homeostasis.