Cornelia Klose

Origin of cadmium‐induced reactive oxygen species production: mitochondrial electron transfer versus plasma membrane NADPH oxidase

* Cadmium (Cd(2+)) is an environmental pollutant that causes increased reactive oxygen species (ROS) production. To determine the site of ROS production, the effect of Cd(2+) on ROS production was studied in isolated soybean (Glycine max) plasma membranes, potato (Solanum tuberosum) tuber mitochondria and roots of intact seedlings of soybean or cucumber (Cucumis sativus). * The effects of Cd(2+) on the kinetics of superoxide (O2*-), hydrogen peroxide (H(2)O(2)) and hydroxyl radical ((*OH) generation were followed using absorption, fluorescence and spin-trapping electron paramagnetic resonance spectroscopy. * In isolated plasma membranes, Cd(2+) inhibited O2*- production. This inhibition was reversed by calcium (Ca(2+)) and magnesium (Mg(2+)). In isolated mitochondria, Cd(2+) increased and H(2)O(2) production. In intact roots, Cd(2+) stimulated H(2)O(2) production whereas it inhibited O2*- and (*)OH production in a Ca(2+)-reversible manner. * Cd(2+) can be used to distinguish between ROS originating from mitochondria and from the plasma membrane. This is achieved by measuring different ROS individually. The immediate (<or= 1 h) consequence of exposure to Cd(2+) in vivo is stimulation of ROS production in the mitochondrial electron transfer chain and inhibition of NADPH oxidase activity in the plasma membrane.

Phytochromes function as thermosensors in Arabidopsis

Combining heat and light responses Plants integrate a variety of environmental signals to regulate growth patterns. Legris et al. and Jung et al. analyzed how the quality of light is interpreted through ambient temperature to regulate transcription and growth (see the Perspective by Halliday and Davis). The phytochromes responsible for reading the ratio of red to far-red light were also responsive to the small shifts in temperature that occur when dusk falls or when shade from neighboring plants cools the soil. Science, this issue p. 897, p. 886; see also p. 832 Red-light photoreceptors also act as temperature sensors in plants. Plants are responsive to temperature, and some species can distinguish differences of 1°C. In Arabidopsis, warmer temperature accelerates flowering and increases elongation growth (thermomorphogenesis). However, the mechanisms of temperature perception are largely unknown. We describe a major thermosensory role for the phytochromes (red light receptors) during the night. Phytochrome null plants display a constitutive warm-temperature response, and consistent with this, we show in this background that the warm-temperature transcriptome becomes derepressed at low temperatures. We found that phytochrome B (phyB) directly associates with the promoters of key target genes in a temperature-dependent manner. The rate of phyB inactivation is proportional to temperature in the dark, enabling phytochromes to function as thermal timers that integrate temperature information over the course of the night.

Phytochrome B integrates light and temperature signals in Arabidopsis

Combining heat and light responses Plants integrate a variety of environmental signals to regulate growth patterns. Legris et al. and Jung et al. analyzed how the quality of light is interpreted through ambient temperature to regulate transcription and growth (see the Perspective by Halliday and Davis). The phytochromes responsible for reading the ratio of red to far-red light were also responsive to the small shifts in temperature that occur when dusk falls or when shade from neighboring plants cools the soil. Science, this issue p. 897, p. 886; see also p. 832 Red-light photoreceptors also act as temperature sensors in plants. Ambient temperature regulates many aspects of plant growth and development, but its sensors are unknown. Here, we demonstrate that the phytochrome B (phyB) photoreceptor participates in temperature perception through its temperature-dependent reversion from the active Pfr state to the inactive Pr state. Increased rates of thermal reversion upon exposing Arabidopsis seedlings to warm environments reduce both the abundance of the biologically active Pfr-Pfr dimer pool of phyB and the size of the associated nuclear bodies, even in daylight. Mathematical analysis of stem growth for seedlings expressing wild-type phyB or thermally stable variants under various combinations of light and temperature revealed that phyB is physiologically responsive to both signals. We therefore propose that in addition to its photoreceptor functions, phyB is a temperature sensor in plants.

EDL3 is an F-box protein involved in the regulation of abscisic acid signalling in Arabidopsis thaliana

The EID1-like protein 3 (EDL3) shows high similarity to EID1 (Empfindlicher im dunkelroten Licht 1), an F-box protein that functions as a negative regulator in the signalling cascade downstream of the phytochrome A photoreceptor in Arabidopsis thaliana. Analyses revealed a strong and rapid induction of EDL3 gene expression under osmotic stress, high salinity, and upon abscisic acid (ABA) application. Therefore, it was speculated that EDL3 is involved in the regulation of responses controlled by this plant hormone, which not only regulates many aspects of plant development but also integrates responses towards temperature, drought, osmotic, and salt stresses. Physiological data obtained with over-expresser lines and a conditional knock-down mutant demonstrated that EDL3 functions as a positive regulator in ABA-dependent signalling cascades that control seed germination, root growth, greening of etiolated seedlings, and transition to flowering. Results further demonstrate that EDL3 regulates anthocyanin accumulation under drought stress. The observed effects on physiological responses fit to tissue-specific expression patterns obtained with EDL3-promoter:GUS lines. Bimolecular Fluorescence Complementation assays and yeast two-hybrid analyses showed that EDL3 carries a functional F-box domain. Thus, the protein is presumed to act as a component of a ubiquitin ligase complex that specifically directs negatively acting factors in ABA signalling to degradation via the proteasome.

The Mediator Complex Subunit PFT1 Interferes with COP1 and HY5 in the Regulation of Arabidopsis Light Signaling

Arabidopsis (Arabidopsis thaliana) mutants hypersensitive to far-red light were isolated under a light program of alternating red and far-red light pulses and were named eid (for empfindlicher im dunkelroten Licht). The dominant eid3 mutant carries a missense mutation in a conserved domain of PHYTOCHROME AND FLOWERING TIME1 (PFT1), an important component of the plant mediator coactivator complex, which links promoter-bound transcriptional regulators to RNA polymerase II complexes. Epistatic analyses were performed to obtain information about the coaction between the mutated PFT1eid3 and positively and negatively acting components of light signaling cascades. The data presented here provide clear evidence that the mutation mainly enhances light sensitivity downstream of phytochrome A (phyA) and modulates phyB function. Our results demonstrate that the Mediator component cooperates with CONSTITUTIVE PHOTORMORPHOGENIC1 in the regulation of light responses and that the hypersensitive phenotype strictly depends on the presence of the ELONGATED HYPOCOTYL5 transcription factor, an important positive regulator of light-dependent gene expression. Expression profile analyses revealed that PFT1eid3 alters the transcript accumulation of light-regulated genes even in darkness. Our data further indicate that PFT1 regulates the floral transition downstream of phyA. The PFT1 missense mutation seems to create a constitutively active transcription factor by mimicking an early step in light signaling.

SUMOylation of phytochrome-B negatively regulates light-induced signaling in Arabidopsis thaliana.

Significance The photoreceptor phytochrome-B (phyB) cycles between its active Pfr [far red light (FRL)-absorbing state λmax, 730 nm] and inactive Pr [red light (RL)-absorbing state λmax, 660 nm] forms and regulates as red/far red light-activated/inactivated molecular switch plant growth and development. Here we show that conjugation of small ubiquitin-like modifier to the photoreceptor inhibits interaction of phyB Pfr with its immediate signaling partner PHYTOCHROME INTERACTING FACTOR 5 (PIF5). The impaired interaction of these proteins negatively affects photomorphogenic responses; thus, SUMOylation similar to phosphorylation plays a role in desensitizing phyB-mediated signaling. OVERLY TOLERANT TO SALT 1 and 2 (OTS1 and OTS2) are involved in regulating phyB action as these SUMO proteases mediate deconjugation of SUMO from phyB. The red/far red light absorbing photoreceptor phytochrome-B (phyB) cycles between the biologically inactive (Pr, λmax, 660 nm) and active (Pfr; λmax, 730 nm) forms and functions as a light quality and quantity controlled switch to regulate photomorphogenesis in Arabidopsis. At the molecular level, phyB interacts in a conformation-dependent fashion with a battery of downstream regulatory proteins, including PHYTOCHROME INTERACTING FACTOR transcription factors, and by modulating their activity/abundance, it alters expression patterns of genes underlying photomorphogenesis. Here we report that the small ubiquitin-like modifier (SUMO) is conjugated (SUMOylation) to the C terminus of phyB; the accumulation of SUMOylated phyB is enhanced by red light and displays a diurnal pattern in plants grown under light/dark cycles. Our data demonstrate that (i) transgenic plants expressing the mutant phyBLys996Arg-YFP photoreceptor are hypersensitive to red light, (ii) light-induced SUMOylation of the mutant phyB is drastically decreased compared with phyB-YFP, and (iii) SUMOylation of phyB inhibits binding of PHYTOCHROME INTERACTING FACTOR 5 to phyB Pfr. In addition, we show that OVERLY TOLERANT TO SALT 1 (OTS1) de-SUMOylates phyB in vitro, it interacts with phyB in vivo, and the ots1/ots2 mutant is hyposensitive to red light. Taken together, we conclude that SUMOylation of phyB negatively regulates light signaling and it is mediated, at least partly, by the action of OTS SUMO proteases.

Systematic analysis of how phytochrome B dimerization determines its specificity.

Phytochromes are red/far-red-light detecting photoreceptors that regulate plant growth and development. They photo-interconvert between an inactive Pr (red-light absorbing) and a physiologically active Pfr (far-red-light absorbing) form, acting as light-controlled molecular switches. Although the two major plant phytochromes A (phyA) and B (phyB) share similar absorption properties, they exhibit dramatic differences in their action spectra. Since both phytochromes antagonistically regulate seedling development under vegetative shade, it is essential for plants to clearly distinguish between phyA and phyB action. This discrimination is not comprehensible solely by the molecular properties of the phytochromes, but is evidently due to the dynamics of the phytochrome system. Using an integrated experimental and mathematical modelling approach we show that phytochrome dimerization is an essential element for phyB function. Our findings reveal that light-independent Pfr to Pr relaxation (dark reversion) and association/dissociation to nuclear bodies (NBs) severely depend on the conformational state of the phyB dimer. We conclude that only Pfr–Pfr homodimers of phyB can be responsible for triggering physiological responses, leading to a suppression of phyB function in the far-red range of the light spectrum.

Molecular mechanisms for mediating light-dependent nucleo/cytoplasmic partitioning of phytochrome photoreceptors

The photoreceptors phytochromes monitor the red/far-red part of the spectrum, exist in the biologically active Pfr (far-red absorbing) or inactive Pr (red absorbing) forms, and function as red/far-red light-regulated molecular switches to modulate plant development and growth. Phytochromes are synthesized in the cytoplasm, and light induces translocation of the Pfr conformer into the nucleus. Nuclear import of phytochromes is a highly regulated process and is fine-tuned by the quality and quantity of light. It appears that phytochrome A (phyA) and phytochrome B (phyB) do not possess active endogenous nuclear import signals (NLSs), thus light-induced translocation of these photoreceptors into the nucleus requires direct protein–protein interactions with their NLS-containing signaling partners. Sub-cellular partitioning of the various phytochrome species is mediated by different molecular machineries. Translocation of phyA into the nucleus is promoted by FAR-RED ELONGATED HYPOCOTYL 1 (FHY1) and FHY1-LIKE (FHL), but the identity of nuclear transport facilitators mediating the import of phyB-E into the nucleus remains elusive. Phytochromes localized in the nucleus are associated with specific protein complexes, termed photobodies. The size and distribution of these structures are regulated by the intensity and duration of irradiation, and circumstantial evidence indicates that they are involved in fine-tuning phytochrome signaling.

Characterization of photomorphogenic responses and signaling cascades controlled by phytochrome‐A expressed in different tissues

The photoreceptor phytochrome A acts as a light-dependent molecular switch and regulates responses initiated by very low fluences of light (VLFR) and high fluences (HIR) of far-red light. PhyA is expressed ubiquitously, but how phyA signaling is orchestrated to regulate photomorphogenesis is poorly understood. To address this issue, we generated transgenic Arabidopsis thaliana phyA-201 mutant lines expressing the biologically active phyA-YFP photoreceptor in different tissues, and analyzed the expression of several reporter genes, including ProHY5:HY5-GFP and Pro35S:CFP-PIF1, and various FR-HIR-dependent physiological responses. We show that phyA action in one tissue is critical and sufficient to regulate flowering time and root growth; control of cotyledon and hypocotyl growth requires simultaneous phyA activity in different tissues; and changes detected in the expression of reporters are not restricted to phyA-containing cells. We conclude that FR-HIR-controlled morphogenesis in Arabidopsis is mediated partly by tissue-specific and partly by intercellular signaling initiated by phyA. Intercellular signaling is critical for many FR-HIR induced responses, yet it appears that phyA modulates the abundance and activity of key regulatory transcription factors in a tissue-autonomous fashion.

New insights of red light-induced development

The red/far-red light absorbing photoreceptors phytochromes regulate development and growth and thus play an essential role in optimizing adaptation of the sessile plants to the ever-changing environment. Our understanding of how absorption of a red/far-red photon by phytochromes initiates/modifies diverse physiological responses has been steadily improving. Research performed in the last 5 years has been especially productive and led to significant conceptual changes about the mode of action of these photoreceptors. In this review, we focus on the phytochrome B photoreceptor, the major phytochrome species active in light-grown plants. We discuss how its light-independent inactivation (termed dark/thermal reversion), post-translational modification, including ubiquitination, phosphorylation and sumoylation, as well as heterodimerization with other phytochrome species modify red light-controlled physiological responses. Finally, we discuss how photobiological properties of phytochrome B enable this photoreceptor to function also as a thermosensor.