Mira Cohen

Post-Transcriptional Suppression of Cytosolic Ascorbate Peroxidase Expression during Pathogen-Induced Programmed Cell Death in Tobacco

As a means to eliminate pathogen-infected cells and prevent diseases, programmed cell death (PCD) appears to be a defense strategy employed by most multicellular organisms. Recent studies have indicated that reactive oxygen species, such as O 2 ⋅ − and H2O2, play a central role in the activation and propagation of pathogen-induced PCD in plants. However, plants contain several mechanisms that detoxify O 2 ⋅ − and H2O2 and may inhibit PCD. We found that during viral-induced PCD in tobacco, the expression of cytosolic ascorbate peroxidase (cAPX), a key H2O2 detoxifying enzyme, is post-transcriptionally suppressed. Thus, although the steady state level of transcripts encoding cAPX was induced during PCD, as expected under conditions of elevated H2O2, the level of the cAPX protein declined. In vivo protein labeling, followed by immunoprecipitation, indicated that the synthesis of the cAPX protein was inhibited. Although transcripts encoding cAPX were found to associate with polysomes during PCD, no cAPX protein was detected after in vitro polysome run-off assays. Our findings suggest that viral-induced PCD in tobacco is accompanied by the suppression of cAPX expression, possibly at the level of translation elongation. This suppression is likely to contribute to a reduction in the capability of cells to scavenge H2O2, which in turn enables the accumulation of H2O2 and the acceleration of PCD.

Signals controlling the expression of cytosolic ascorbate peroxidase during pathogen-induced programmed cell death in tobacco

In plants ascorbate peroxidase (APX) is an important H2O2-detoxifying enzyme. The expression of APX is rapidly induced in response to stresses that result in the accumulation of reactive oxygen species (ROS). We have recently reported that the steady-state level of transcripts encoding cytosolic APX (cAPX) is dramatically induced during the hypersensitive response (HR) of tobacco plants infected with tobacco mosaic virus (TMV). Because cAPX expression is closely linked to the production of ROS in plant cells, studying the regulation cAPX mRNA can reveal some of the signal transduction events associated with ROS metabolism during the HR. Analysis of cAPX mRNA induction during the HR suggested that the expression of cAPX is under the control of the HR signal transduction pathway. The activation of cAPX expression followed signaling events such as changes in protein phosphorylation and induction of ion fluxes. Expression of cAPX was suppressed under conditions of low oxygen pressure, and could only be mimicked by enhancing the intracellular generation of ROS. Interestingly, salicylic acid (SA), which is thought to be involved in ROS metabolism during the HR, did not affect the induction of cAPX mRNA during TMV-induced HR. Using cAPX expression as a marker for the production of ROS, it is suggested that SA may not be involved in the formation of ROS during the HR of tobacco to TMV, and that ROS may not be involved in the induction of the pathogenesis-related protein, PR-1, during this process.

Natural plasticity in circadian rhythms is mediated by reorganization in the molecular clockwork in honeybees

Various animals naturally switch to considerable periods of around‐the‐clock activity with no apparent ill effects. Such plasticity in overt circadian rhythms might be observed because the clock is masked by the influence of external factors, is uncoupled from behavioral outputs, or results from genuine plasticity in the clock machinery. We studied honeybees in which plasticity in circadian rhythms is socially modulated and associated with the division of labor. We confirm that “nurse” bees care for the brood around‐the‐clock even when experiencing a light:dark illumination regime. However, nurses transferred from the hive to individual cages in constant conditions have robust circadian rhythms in locomotor activity with an onset of activity at the subjective morning. These data indicate that circa‐dian rhythmicity in nurses depends on their environment, and suggest that some clockwork components were entrained even in nurses active around the clock while in the hive. Brain oscillations in transcript abundance for the putative clock genes Period, Crypto‐chrome‐m, Cycle, and Timeout were attenuated or totally suppressed in nurses as compared to behaviorally rhythmic foragers, irrespective of the illumination regime. These findings provide the first support for the hypothesis that natural plasticity in circadian rhythms is associated with reorganization of the internal clockwork.—Shemesh, Y., Cohen, M., Bloch, G. Natural plasticity in circadian rhythms is mediated by reorganization in the molecular clockwork in honeybees. FASEB J. 21, 2304–2311 (2007)

Molecular Dynamics and Social Regulation of Context-Dependent Plasticity in the Circadian Clockwork of the Honey Bee

The social environment influences the circadian clock of diverse animals, but little is known about the functional significance, the specifics of the social signals, or the dynamics of socially mediated changes in the clock. Honey bees switch between activities with and without circadian rhythms according to their social task. Forager bees have strong circadian rhythms, whereas “nurse” bees typically care for the brood around-the-clock with no circadian rhythms in behavior or clock gene expression. Here we show that nurse-age bees that were restricted to a broodless comb inside or outside the hive showed robust behavioral and molecular circadian rhythms. By contrast, young nurses tended brood with no circadian rhythms in behavior or clock gene expression, even under a light-dark illumination regime or when placed with brood—but no queen—in a small cage outside the hive. This behavior is context-dependent because nurses showed circadian rhythms in locomotor activity shortly after removal from the hive, and in clock gene expression after ∼16 h. These findings suggest that direct interaction with the brood modulates the circadian system of honey bees. The dynamics of rhythm development best fit models positing that at least some pacemakers continue to oscillate and be entrained by the environment in nurses that are active around the clock. These cells set the phase to the clock network when the nurse is removed from the hive. These findings suggest that despite its robustness, the circadian system exhibits profound plasticity, enabling adjustment to rapid changes in the social environment.

Microarray Analysis of Natural Socially Regulated Plasticity in Circadian Rhythms of Honey Bees

Honey bee workers care for (“nurse”) the brood around the clock without circadian rhythmicity, but then they forage outside with strong circadian rhythms and a consolidated nightly rest. This chronobiological plasticity is associated with variation in the expression of the canonical “clock genes” that regulate the circadian clock: nurse bees show no brain rhythms of expression, while foragers do. These results suggest that the circadian system is organized differently in nurses and foragers. Nurses switch to activity with circadian rhythms shortly after being removed from the hive, suggesting that at least some clock cells in their brain continue to measure time while in the hive. We performed a microarray genome-wide survey to determine general patterns of brain gene expression in nurses and foragers sampled around the clock. We found 160 and 541 transcripts that exhibited significant sinusoidal oscillations in nurses and foragers, respectively, with peaks of expression distributed throughout the day in both task groups. Consistent with earlier studies, transcripts of genes involved in circadian rhythms, including Clockwork Orange that has not been studied before in bees, oscillated in foragers but not in nurses. The oscillating transcripts also were enriched for genes involved in the visual system, “development” and “response to stimuli” (foragers), “muscle contraction” and “microfilament motor gene expression” (nurses), and “generation of precursor metabolites” and “energy” (both). Transcripts of genes encoding P450 enzymes oscillated in both nurses and foragers but with a different phase. This study identified new putative clock-controlled genes in the honey bee and suggests that some brain functions show circadian rhythmicity even in nurse bees that are active around the clock.

The expression and phylogenetics of the Inhibitor Cysteine Knot peptide OCLP1 in the honey bee Apis mellifera

Small cysteine-rich peptides have diverse functions in insects including antimicrobial defense, phenoloxidase activity regulation, and toxic inhibition of ion channels of prey or predator. We combined bioinformatics and measurements of transcript abundance to start characterizing AmOCLP1, a recently discovered Inhibitor Cysteine Knot peptide in the honey bee Apis mellifera. We found that the genomes of ants, bees, and the wasp Nasonia vitripennis encode orthologous sequences indicating that OCLP1 is a conserved peptide and not unique to the honey bee. Search of available EST libraries and quantitative real time PCR analyses indicate that the transcript of AmOCLP1 is ubiquitous with expression in life stages ranging from embryos to adults and in all tested tissues. In worker honey bees AmOCLP1 expression was not associated with age or task and did not show clear enrichment in any of the tested tissues. There was however a consistent trend toward higher transcript levels in the abdomen of foragers relative to levels in the head or thorax, and compared to levels in the abdomen of younger worker bees. By contrast, in drones AmOCLP1 transcript levels appeared higher in the head relative to the abdomen. Finer analyses of the head and abdomen indicated that the AmOCLP1 transcript is not enriched in the stinger and the associated venom sac or in cephalic exocrine glands. The evolutionary conservation in the Hymenoptera, the ubiquitous expression, and the lack of enrichment in the venom gland, stinger, exocrine glands, and the brain are not consistent with the hypotheses that OCLP1 is a secreted honeybee toxin or an endotoxin acting in the central nervous system. Rather we hypothesize that OCLP1 is a conserved antimicrobial or phenoloxidase inhibitor peptide.

Detection of a protein specific to high-salt Dunaliella cells

Summary Dunaliella parva , a green unicellular alga, exists in mutually-interconvertible forms capable of growing in NaCl solutions from 0.2 M to at least 4 M. Interconversion between 0.5 M and 3.5 M NaCl takes 48 h in the upward and 1½ h in the downward direction. Cultures are grown from single cells and must therefore be genetically identical. However, SDS-PAGE analysis of the insoluble cell fraction (largely membranes) has revealed that in cells grown in 3.5 M NaCl there is an extra polypeptide (Mr 140 -160 kDa) that is not seen in cells grown in 0.5 M NaCl. The extra polypeptide has been found in all cultures growing in or above 2 M NaCl.

After-Effects of Vectorial Photo-Excitation in Solar-Tracking Leaves of Lavatera cretica L. — A Kinetic Analysis

Summary Leaves of Lavatera cretica L. were exposed to different constant levels of TO (at Al = +15° +30° or + 60°C), or BO excitation (at AI = −15°, −30°, or −60°), along their midvein. After 90, or 150min, they were transferred to light regimes that were not vectorially effective (white, red or darkness) and the time-course of the subsequent changes in LE was recorded. These changes are the expression of solute (and water) fluxes in the sector of pulvinar motor tissue subtending the midvein, whose associated photoreceptors had been vectorially excited. The diaphototropic response was non-inductive and required continuous VE. When such VE was terminated by darkness, the immediate reorientation was inertial, i.e. its direction was dictated by that of the preceding VE. This short-lived phase was replaced by the primary post-inertial phase, during which LE fell, irrespective of whether the preceding VE had been TO, or BO. However, the subsequent reorientation differed qualitatively when preceded by TO, or BO. Kinetic analysis showed that the post-inertial phases in laminar reorientation were quantitatively modified by the level (AI) and duration of the preceding VE. Thus, the laminar reorientation in darkness was not nyctinastic, but induced by the preceding VE. Non vectorial white or red light, had marked and different effects on the kinetics of both post-inertial phases, suggesting the involvement of different photoreceptors.

Factors involved in early polarization of the anterior-posterior axis in the milkweed bug Oncopeltus fasciatus

The axes of insect embryos are defined early in the blastoderm stage. Genes involved in this polarization are well known in Drosophila, but less so in other insects, such as the milkweed bug Oncopeltus fasciatus. Using quantitative PCR, we looked at differential expression of several candidate genes for early anterior‐posterior patterning and found that none of them are expressed asymmetrically in the early blastoderm. We then used an RNA‐Seq approach to identify novel candidate genes that might be involved in early polarization in Oncopeltus. We focused on transcription factors (TFs) as these are likely to be central players in developmental processes. Using both homology and domain based identification approaches, we were unable to find any TF encoding transcripts that are expressed asymmetrically along the anterior‐posterior axis at early stages. Using a GO‐term analysis of all asymmetrically expressed mRNAs, we found an enrichment of genes relating to mitochondrial function in the posterior at the earliest studied time‐point. We also found a gradual enrichment of transcription related activities, giving us a putative time frame for the maternal to zygotic transition. Our dataset provides us with a list of new candidate genes in early development, which can be followed up experimentally.