Plants under oxidative stress suffer from damages that have been interpreted as unavoidable consequences of injuries inflicted upon plants by toxic levels of reactive oxygen species (ROS). However, this paradigm needs to be modified. Inactivation of a single gene, EXECUTER1, is sufficient to abrogate stress responses of Arabidopsis thaliana caused by the release of singlet oxygen: External conditions under which these stress responses are observed and the amounts of ROS that accumulate in plants exposed to these environmental conditions do not directly cause damages. Instead, seedling lethality and growth inhibition of mature plants result from genetic programs that are activated after the release of singlet oxygen has been perceived by the plant.

Shortly after the release of singlet oxygen ( 1 O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of 1 O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of 1 O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of 1 O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all 1 O2-responsive genes. Retrograde control of 1 O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.oxidative stress ͉ retrograde signaling ͉ singlet oxygen ͉ chloroplast

Phytochrome photoreceptors play a central role in the regulation of plant development. Phytochromes are red ⁄ far-red photochromic proteins with a covalently bound linear tetrapyrrole (bilin) prosthetic group. In the Pr ground state the chromophore preferentially absorbs red light, this leading to a Z fi E isomerization around the C 15 -C 16 double bond between rings C and D. Further conformational changes culminate in the formation of Pfr, the signalling state. This preferably absorbs far-red light which converts the pigment back to Pr [1]. The active photoreceptor is formed by the apoprotein taking up and covalently attaching an We have investigated mutants of phytochrome Cph1 from the cyanobacterium Synechocystis PCC6803 in order to study chromophore-protein interactions. Cph1D2, the 514-residue N-terminal sensor module produced as a recombinant His6-tagged apoprotein in Escherichia coli, autoassembles in vitro to form a holoprotein photochemically indistinguishable from the full-length product. We generated 12 site-directed mutants of Cph1D2, focusing on conserved residues which might be involved in chromophoreprotein autoassembly and photoconversion. Folding, phycocyanobilin-binding and Pr fi Pfr photoconversion were analysed using CD and UV-visible spectroscopy. MALDI-TOF-MS confirmed C259 as the chromophore attachment site. C259L is unable to attach the chromophore covalently but still autoassembles to form a red-shifted photochromic holoprotein. H260Q shows UV-visible properties similar to the wild-type at pH 7.0 but both Pr and Pfr (reversibly) bleach at pH 9.0, indicating that the imidazole side chain buffers chromophore protonation. Mutations at E189 disturbed folding but the residue is not essential for chromophore-protein autoassembly. In D207A, whereas red irradiation of the ground state leads to bleaching of the red Pr band as in the wild-type, a Pfr-like peak does not arise, implicating D207 as a proton donor for a deprotonated intermediate prior to Pfr. UV-Vis spectra of both H260Q under alkaline conditions and D207A point to a particular significance of protonation in the Pfr state, possibly implying proton migration (release and re-uptake) during Pr fi Pfr photoconversion. The findings are discussed in relation to the recently published 3D structure of a bacteriophytochrome fragment [Wagner JR, Brunzelle JS, Forest KT & Vierstra RD (2005) Nature 438, 325-331].Abbreviations BV, biliverdin IXa; Cph1D2, the N-terminal 1-514 residue sensory module of Cph1 from Synechocystis PCC6803; e, extinction coefficient; FTRR, Fourier transform resonance Raman spectroscopy; FWHM, full width half maximum; IPTG, isopropyl thio-b-D-galactoside; LED, lightemitting diode; MeOH, methanol; Pr ⁄ Pfr, red ⁄ far-red absorbing form of phytochrome; PCB, phycocyanobilin; PFB, phytochromobilin; SAR, specific absorbance ratio; SEC, size-exclusion chromatography; k max , wavelength of the absorbance maximum.

SummaryUpon a dark/light shift the conditional flu mutant of Arabidopsis starts to generate singlet oxygen ( 1 O 2 ) that is restricted to the plastid compartment. Distinct sets of genes are activated that are different from those induced by hydrogen peroxide/superoxide. One of the genes that is rapidly upregulated is EDS1 (enhanced disease susceptibility). The EDS1 protein has been shown to be required for the resistance to biotrophic pathogens and the accumulation of salicylic acid (SA) that enhances the defenses of a plant by inducing the synthesis of pathogen-related (PR) proteins. Because of the similarity of its N-terminal portion to the catalytic site of lipases, EDS1 has also been implicated with the release of polyunsaturated fatty acids and the subsequent formation of various oxylipins. The release of singlet oxygen in the flu mutant triggers a drastic increase in the concentration of free SA and activates the expression of PR1 and PR5 genes. These changes depend on the activity of EDS1 and are suppressed in flu/eds1 double mutants. Soon after the beginning of singlet oxygen production, the synthesis of oxylipins such as jasmonic acid (JA) and 12-oxophytodienoic acid (OPDA) also start and plants stop growing and induce a cell-death response. The inactivation of EDS1 does not affect oxylipin synthesis, growth inhibition and the initiation of cell death, but it does allow plants to recover much faster from singlet oxygen-mediated growth inhibition and it also suppresses the spread of necrotic lesions in leaves. Hence, singlet oxygen activates a complex stress-response program with EDS1 playing a key role in initiating and modulating several steps of it. This program includes not only responses to oxidative stress, but also responses known to be activated during plant-pathogen interactions and wounding.

We have successfully co-expressed two genes from the bilin biosynthetic pathway of Synechocystis together with cyanobacterial phytochrome 1 (Cph1) from the same organism to produce holophytochrome in Escherichia coli. Heme oxygenase was used to convert host heme to biliverdin IXK K which was then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase, presumably with the aid of host ferredoxin. In this host environment Cph1 apophytochrome was able to autoassemble with the phycocyanobilin in vivo to form fully photoreversible holophytochrome. The system can be used as a tool for further genetic studies of phytochrome function and signal transduction as well as providing an excellent source of holophytochrome for physicochemical studies. ß 2001 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Although sessile, plants are able to grow toward or away from an environmental stimulus. Important examples are stem or leaf orientation of higher plants in response to the direction of the incident light. The responsible photoreceptors belong to the phototropin photoreceptor family. Although the mode of phototropin action is quite well understood, much less is known of how the light signal is transformed into a bending response. Several lines of evidence indicate that a lateral auxin gradient is responsible for asymmetric cell elongation along the light gradient within the stem. However, some of the molecular key players leading to this asymmetric auxin distribution are, as yet, unidentified. Previously, it was shown that phototropin gets autophosphorylated upon illumination and binds to a scaffold protein termed NPH3 (for nonphototropic hypocotyl 3). Using a yeast three-hybrid approach with phototropin and NPH3 as a bait complex, we isolated a protein, termed EHB1 (for enhanced bending 1), with a so far unknown function, which binds to this binary complex. This novel interacting factor negatively affects hypocotyl bending under blue light conditions in Arabidopsis (Arabidopsis thaliana) and thus seems to be an important component regulating phototropism. Interestingly, it could be shown that the gravitropic response was also affected. Thus, it cannot be ruled out that this protein might also have a more general role in auxin-mediated bending toward an environmental stimulus.

Singlet oxygen is reported to have the most potent damaging effect upon the photosynthetic machinery. Usually this reactive oxygen molecule acts in concert with other ROS types under stressful conditions. To understand the specific role of singlet oxygen we took advantage of the conditional flu mutant of Arabidopsis thaliana. In flu, the negative feedback loop is abolished, which blocks chlorophyll biosynthesis in the dark. Therefore high amounts of free protochlorophyllide accumulate during darkness. If flu gets subsequently illuminated, free protochlorophyllide acts as a photosensitiser leading almost exclusively to high amounts of (1)O2. Analysing the thylakoid protein pattern by using 2D PAGE and subsequent MALDI-TOF analysis, we could show, in addition to previous described effects on photosystem II, that singlet oxygen has a massive impact on the thylakoid ATP synthase, especially on its gamma subunit. Additionally, it could be shown that the activity of the ATP synthase is reduced upon singlet oxygen exposure and that the rate of non-photochemical quenching is affected in flu mutants exposed to (1)O2.

Key Points Question Can electroencephalography (EEG), combined with clinical, biological, and magnetic resonance imaging (MRI) analyses, help to better characterize patients with neurologic coronavirus disease 2019 (COVID-19) and diagnose specific COVID-19–related encephalopathy? Findings Neurologic manifestations, biological findings, EEG findings, and brain MRI images were analyzed in a cohort study of 78 adult patients with COVID-19. Nine patients had no identified cause of brain injury, as revealed by biological and MRI findings; their injury was defined as COVID-19–related encephalopathy. Meaning This study suggests that, although neurologic manifestations, EEG findings, and MRI findings may appear heterogeneous and nonspecific, multimodal monitoring may better identify patients with COVID-19–related encephalopathy and guide treatment strategy.