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Behavioural responses of bacteria to light and oxygen.

Motile bacteria have long been known to swim towards or away from specific environmental stimuli such as nutrients, oxygen or light. Although there has been a detailed description of chemosensory responses in enteric species for several years, there has been little information on the mechanisms involved in responses to stimuli affecting electron transport as these usually also change the electrochemical proton gradient - at least transiently - and, thus, directly change flagellar rotation. There have, however, been major advances recently. Halobacterium salinarium uses a retinal-based sensory system to sense changes in specific wavelengths of light and to signal via a transmembrane sensory protein, which turns out to be homologous to the transmembrane chemoreceptors of Escherichia coli. A FAD-binding protein, also related to these receptors, signals changes in respiratory electron transport in E. coli. Rhodobacter sphaeroides cells do not respond to light or oxygen specifically, but sense a change in the rate of electron transfer, probably again using an electron-transport-chain-linked redox sensor, signalling through a common sensory pathway. These recent studies reveal that bacteria not only sense a range of environmental stimuli but also integrate the signals through common pathways to produce a balanced flagellar response.

Bacteria↗

The calcineurin inhibitor gossypol impairs growth, cell signalling and development in Dictyostelium discoideum.

The Dictyostelium genome harbors single copy genes for both the catalytic and regulatory subunits of the Ca2+/calmodulin-dependent protein phosphatase calcineurin. Since molecular genetic approaches to reduce the expression of these genes have failed so far, we attempted to pharmacologically target calcineurin activity in vivo by using the recently described calcineurin inhibitor, gossypol. Up-regulation of expression of the gene for the Ca2+-ATPase PAT1 in conditions of Ca2+ stress was reduced by gossypol. Dictyostelium wild-type cells treated with 12.5-100 microM gossypol showed reduced growth rates and impaired development. In addition, cell signalling was affected. A cell line that overproduces the catalytic subunit of calcineurin was more resistant to gossypol.

Animals↗

Effects of calcium on rod and cone inputs to horizontal cells of the tiger salamander retina.

Effects of extracellular calcium on signal transmission between photoreceptors and horizontal cells (HCs) are studied in superfused isolated retina of the larval tiger salamander. Horizontal cell light response is optimal when extracellular Ca2+ is maintained between 1-2 mM. Ca2+ levels beyond this range in either direction significantly reduce the HC light response amplitude. When extracellular Ca2+ is lowered from 2 mM to 0.5 mM, the rod input to HCs is reduced whereas the cone input is not affected. In comparison, the peak voltage responses of rods are not changed whereas the cone voltage responses are enhanced in 0.5 mM Ca2+. The selective suppression of rod input to HCs is probably due to the interplay of three factors: (1) the photocurrents, (2) voltage- and time-dependent membrane currents in photoreceptors, and (3) the Ca(2+)-dependent synaptic gain between photoreceptors and HCs.

Ambystoma↗

Dynamics of stimulation-induced muscle adaptation: insights from varying the duty cycle.

We sought to gain insight into the dynamics of the signalling process that initiates adaptive change in mammalian skeletal muscles in response to chronic neuromuscular stimulation. Programmable miniature stimulators were implanted into rabbits and used to impose one of the following patterns on the dorsiflexors of one ankle: 10 Hz delivered in equal on/off periods of 30 s, 30 min, or 12 h (all equivalent in terms of aggregate impulse activity to continuous 5 Hz). Two further groups received continuous stimulation at 5 Hz or 10 Hz. In every case the stimulation pattern was maintained continuously for 6 weeks. Tibialis anterior muscles stimulated intermittently with equal on/off periods of 30 s, 30 min and 12 h had contractile characteristics that were significantly slower than the contralateral, unstimulated muscles but did not differ from those of muscles stimulated continuously at 5 Hz. Muscles stimulated continuously at 10 Hz were significantly slower than either contralateral muscles or muscles stimulated with any of the other patterns. Corresponding changes were seen in myosin heavy chain isoform composition. The fatigue index, defined as the fraction of tension remaining after 5 min of a standard fatigue test, was 0.4 for muscles in the contralateral group but equal to or greater than 0.85 for muscles of all the stimulated groups. These results were interpreted with the help of a simple model of the growth and decay of a putative signalling substance based on first order kinetics. The model suggests a rate constant for the accumulation of the signalling substance that is greater than 30 h(-1), and a rate constant for its removal that is greater than 50 h(-1).

Adaptation, Physiological↗

Extension of a genetic network model by iterative experimentation and mathematical analysis.

Circadian clocks involve feedback loops that generate rhythmic expression of key genes. Molecular genetic studies in the higher plant Arabidopsis thaliana have revealed a complex clock network. The first part of the network to be identified, a transcriptional feedback loop comprising TIMING OF CAB EXPRESSION 1 (TOC1), LATE ELONGATED HYPOCOTYL (LHY) and CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), fails to account for significant experimental data. We develop an extended model that is based upon a wider range of data and accurately predicts additional experimental results. The model comprises interlocking feedback loops comparable to those identified experimentally in other circadian systems. We propose that each loop receives input signals from light, and that each loop includes a hypothetical component that had not been explicitly identified. Analysis of the model predicted the properties of these components, including an acute light induction at dawn that is rapidly repressed by LHY and CCA1. We found this unexpected regulation in RNA levels of the evening-expressed gene GIGANTEA (GI), supporting our proposed network and making GI a strong candidate for this component.

Arabidopsis↗

Tuning in to the signals controlling photoregulated gene expression in plants.

Plants have developed flexible mechanisms to respond appropriately to environmental signals. These stimuli are transduced by largely unknown signalling pathways that are likely to be modulated by endogenous developmental signals to produce an integrated response that coordinately regulates gene expression. Light is a critical environmental signal that controls many aspects of plant development via a series of photoreceptors that are able to respond to different light wavelengths. Light is also the principal energy source for photosynthesis. The photosynthetic products are carbohydrates which are translocated in the form of sucrose from the photosynthetic (source) to non-photosynthetic (sink) organs. Consequently, the control of photoregulated genes must integrate developmental inputs with signals derived from the photoreceptors, from the photosynthetic apparatus and from metabolites such as sucrose.

Carbohydrates↗

Light, phytochrome signalling and photomorphogenesis in Arabidopsis.

The phytochromes is a family of plant photoreceptors that control growth and development in response to environmental cues. Red and far-red light are the most efficient wavebands to induce conformational changes of phytochromes and consequently modify their kinetics, nuclear/cytoplasmic partitioning, ability to phosphorylate substrates, and physical interaction with proteins that bind DNA. Many players in phytochrome signalling have been identified and a complex, highly regulated network is envisaged. Here we describe the connection between different features of the phytochrome signalling network and the versatile relationship between light signals and physiological outputs shown by phytochromes.

Arabidopsis↗

Light-induced intramolecular charge movements in microbial rhodopsins in intact E. coli cells.

Microbial rhodopsins undergo cyclic photochemical reactions (photocycles) in which proton transfers and conformational changes result in charge displacements during transitions between photocycle intermediates. We report a new photoelectric method to monitor charge movements during rhodopsin photocycling with fast kinetic resolution in suspensions of intact E. coli cells. The method monitors electrical currents resulting from asymmetric photoexcitation of microbial rhodopsins by a unilateral laser flash, and kinetically resolves intramolecular charge movements. We investigated E. coli-expressed proton-transporting rhodopsins, specifically green- and blue-absorbing proteorhodopsins (GPR and BPR, respectively) from uncultivated marine plankton, and sensory rhodopsins, namely receptors from Natronomonas pharaonis and Anabaena (Nostoc) sp. PCC7120. Kinetic components of the currents correlate with photochemical transformations of the pigments, and the integrated current measures net transport by the proton-pumping rhodopsins. The photoelectric measurements distinguish between known light-driven transporters and photosensors, and reveal differences in proton transfer reactions in the two tested proton pumps. Screening of nine newly identified proteorhodopsins reveals two with GPR-type charge movements, five with BPR-type, and two with the characteristics of the sensory rhodopsins. The approach developed in the present work provides a direct, rapid and informative method for studying electrogenic events in rhodopsin photocycles and also gives a clue to functions of newly found microbial rhodopsins in nature.

Anabaena↗

Photomorphogenesis of leaves: shade-avoidance and differentiation of sun and shade leaves.

Leaf shape is an important factor in optimal plant growth, because leaves are the main photosynthetic organs. Plants exhibit plasticity in leaf shape and structure, allowing them to optimize photosynthetic efficiency. In Arabidopsis thaliana(L.) Heynh., several types of leaves develop differentially, according to light intensity and quality. When shaded, the expansion of leaf lamina is inhibited, while the petiole elongation is enhanced. This phenomenon is part of the so-called shade-avoidance syndrome. Under low light, A. thaliana develops shade leaves with only one layer of palisade tissue, whereas under high light, it develops sun leaves that have nearly two complete layers of palisade tissue. Although the molecular mechanisms of these photomorphogenic phenomena in leaves are not well understood, recent studies of A. thaliana have provided some insight. For example, some cytochrome P450s may be involved in the specific control of the petiole length during photomorphogenesis. On the other hand, switching between sun and shade leaves is regulated by long-distance signaling from mature leaves in Chenopodium album. Here we provide an overview of the mechanisms of photomorphogenesis in leaves based on recent findings.

Arabidopsis↗

Dissection of stress-activated glucose transport from insulin-induced glucose transport in mammalian cells using wortmannin and ML-9.

The signaling pathways responsible for the activation of glucose transport by insulin and by metabolic stress in mammalian cells were studied in Clone 9 cells and 3T3-L1 adipocytes. Exposure of both cell types to azide or insulin markedly increased their glucose uptake capacity (Vmax.) without affecting their apparent affinity for glucose (Km). The effects of azide and insulin were not additive. Wortmannin, a selective inhibitor of phosphatidylinositol (PI) 3-kinase, did not affect stimulation of transport by azide but inhibited insulin-induced glucose transport with a Ki of < 10 nM. ML-9, a putative mitogen-activated protein kinase inhibitor, was equipotent in its inhibition of azide- and insulin-stimulated glucose transport. These findings suggest that multiple signalling cascades are involved in the stimulation of glucose transport in mammalian cells and that PI 3-kinase, an essential link in the pathway by which insulin stimulates glucose transport, is not necessary for the activation of glucose uptake by metabolic stress.

3T3 Cells↗

Retrograde signaling from the brain to the retina modulates the termination of the light response in Drosophila.

A critical factor in visual function is the speed with which photoreceptors (PRs) return to the resting state when light intensity dims. Several elements subserve this process, many of which promote the termination of the phototransduction cascade. Although the known elements are intrinsic to PRs, we have found that prompt restoration to the resting state of the Drosophila electroretinogram can require effective communication between the retina and the underlying brain. The requirement is seen more dramatically with long than with short light pulses, distinguishing the phenomenon from gross disruption of the termination machinery. The speed of recovery is affected by mutations (in the Hdc and ort genes) that prevent PRs from transmitting visual information to the brain. It is also affected by manipulation (using either drugs like neostigmine or genetic tools to inactivate neurotransmitter release) of cholinergic signals that arise in the brain. Intracellular recordings support the hypothesis that PRs are the target of this communication. We infer that signaling from the retina to the optic lobe prompts a feedback signal to retinal PRs. Although the mechanism of this retrograde signaling remains to be discerned, the phenomenon establishes a previously unappreciated mode of control of the temporal responsiveness of a primary sensory neuron.

Animals↗

Modulation of chloroplast movement in the green alga Mougeotia by the Ca2+ ionophore A23187 and by calmodulin antagonists.

The Ca2+ ionophore A23187 can induce chloroplast rotation within a single nonirradiated Mougeotia cell. The induced turning was dependent on the position of ionophore application and Ca2+ in the external medium. The role of calmodulin in mediating light-induced chloroplast rotation in the alga Mougeotia was investigated by using the paired calmodulin-antagonist drugs W5-W7 and W12-W13. In each pair, the antagonist with the greater affinity for calmodulin had the greater inhibitor effect on the phytochrome-controlled light response. These results support the hypothesis that calcium functions as a chemical messenger to couple the stimulus of phytochrome photoactivation with physiological responses in plants.

Actin Cytoskeleton↗

Expression and biochemical properties of a ferredoxin-dependent heme oxygenase required for phytochrome chromophore synthesis.

The HY1 gene of Arabidopsis encodes a plastid heme oxygenase (AtHO1) required for the synthesis of the chromophore of the phytochrome family of plant photoreceptors. To determine the enzymatic properties of plant heme oxygenases, we have expressed the HY1 gene (without the plastid transit peptide) in Escherichia coli to produce an amino terminal fusion protein between AtHO1 and glutathione S-transferase. The fusion protein was soluble and expressed at high levels. Purified recombinant AtHO1, after glutathione S-transferase cleavage, is a hemoprotein that forms a 1:1 complex with heme. In the presence of reduced ferredoxin, AtHO1 catalyzed the formation of biliverdin IXalpha from heme with the concomitant production of carbon monoxide. Heme oxygenase activity could also be reconstituted using photoreduced ferredoxin generated through light irradiation of isolated thylakoid membranes, suggesting that ferredoxin may be the electron donor in vivo. In addition, AtHO1 required an iron chelator and second reductant, such as ascorbate, for full activity. These results show that the basic mechanism of heme cleavage has been conserved between plants and other organisms even though the function, subcellular localization, and cofactor requirements of heme oxygenases differ substantially.

Arabidopsis↗

Light- and carbon-signaling pathways. Modeling circuits of interactions.

Here, we report the systematic exploration and modeling of interactions between light and sugar signaling. The data set analyzed explores the interactions of sugar (sucrose) with distinct light qualities (white, blue, red, and far-red) used at different fluence rates (low or high) in etiolated seedlings and mature green plants. Boolean logic was used to model the effect of these carbon/light interactions on three target genes involved in nitrogen assimilation: asparagine synthetase (ASN1 and ASN2) and glutamine synthetase (GLN2). This analysis enabled us to assess the effects of carbon on light-induced genes (GLN2/ASN2) versus light-repressed genes (ASN1) in this pathway. New interactions between carbon and blue-light signaling were discovered, and further connections between red/far-red light and carbon were modeled. Overall, light was able to override carbon as a major regulator of ASN1 and GLN2 in etiolated seedlings. By contrast, carbon overrides light as the major regulator of GLN2 and ASN2 in light-grown plants. Specific examples include the following: Carbon attenuated the blue-light induction of GLN2 in etiolated seedlings and also attenuated the white-, blue-, and red-light induction of GLN2 and ASN2 in light-grown plants. By contrast, carbon potentiated far-red-light induction of GLN2 and ASN2 in light-grown plants. Depending on the fluence rate of far-red light, carbon either attenuated or potentiated light repression of ASN1 in light-grown plants. These studies indicate the interaction of carbon with blue, red, and far-red-light signaling and set the stage for further investigation into modeling this complex web of interacting pathways using systems biology approaches.

Arabidopsis↗

Defects in the cytochrome b6/f complex prevent light-induced expression of nuclear genes involved in chlorophyll biosynthesis.

Mutants with defects in the cytochrome (cyt) b6/f complex were analyzed for their effect on the expression of a subgroup of nuclear genes encoding plastid-localized enzymes participating in chlorophyll biosynthesis. Their defects ranged from complete loss of the cytb6/f complex to point mutations affecting specifically the quinone-binding QO site. In these seven mutants, light induction of the tetrapyrrole biosynthetic genes was either abolished or strongly reduced. In contrast, a normal induction of chlorophyll biosynthesis genes was observed in mutants with defects in photosystem II, photosystem I, or plastocyanin, or in wild-type cells treated with 3-(3'4'-dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-methyl-6-isopropyl benzoquinone. We conclude that the redox state of the plastoquinone pool does not control light induction of these chlorophyll biosynthetic genes. The signal that affects expression of the nuclear genes appears to solely depend on the integrity of the cytb6/f complex QO site. Since light induction of these genes in Chlamydomonas has recently been shown to involve the blue light receptor phototropin, the results suggest that cytb6/f activity regulates a plastid-derived factor required for their expression. This signaling pathway differs from that which regulates state transitions, since mutant stt7, lacking a protein kinase involved in phosphorylation of the light-harvesting complex II, was not altered in the expression of the chlorophyll biosynthetic genes.

Animals↗

Phytochrome A null mutants of Arabidopsis display a wild-type phenotype in white light.

Phytochrome is a family of photoreceptors that regulates plant photomorphogenesis; the best-characterized member of this family is phytochrome A. Here, we report the identification of novel mutations at three Arabidopsis loci (fhy1, fhy2, and fhy3) that confer an elongated hypocotyl in far-red but not in white light. fhy2 mutants are phytochrome A deficient, have reduced or undetectable levels of PHYA transcripts, and contain structural alterations within the PHYA gene. When grown in white light, fhy2 mutants are morphologically indistinguishable from wild-type plants. Thus, phytochrome A appears to be dispensable in white light-grown Arabidopsis plants. fhy2 alleles confer partially dominant phenotypes in far-red light, suggesting that the relative abundance of phytochrome A can affect the extent of the far-red-mediated hypocotyl growth inhibition response. Plants homozygous for the recessive fhy1 and fhy3 mutations have normal levels of functional phytochrome A. The FHY1 and FHY3 gene products may be responsible for the transduction of the far-red light signal from phytochrome A to downstream processes involved in hypocotyl growth regulation.

Arabidopsis↗

Evidence that the repellent receptor form of sensory rhodopsin I is an attractant signaling state.

The lifetime of the Halobacterium halobium sensory rhodopsin I (SR-I) photocycle intermediate S373 was modulated by incorporating retinal analogs into SR-I apoprotein in vitro and in vivo. Photocycles by SR-I analog pigments exhibit the same reaction scheme and similar formation rates, but different decay rates, of their S373-like species as monitored by flash spectroscopy in membrane vesicle suspensions. The attractant receptor signaling efficiencies determined by physiological measurements are proportional to the lifetimes of the S373-like intermediates, indicating that S373 is a physiological active conformation (signaling state) of the receptor. A model incorporating this finding into the SR-I photocycle is presented.

Halobacterium↗