Takafumi Yamashino

Genome-Wide Analyses Revealing a Signaling Network of the RcsC-YojN-RcsB Phosphorelay System in Escherichia coli

In Escherichia coli, capsular colanic acid polysaccharide synthesis is regulated through the multistep RcsC-->YojN-->RcsB phosphorelay. By monitoring a hallmarked cps::lacZ reporter gene, we first searched for physiological stimuli that propagate the Rcs signaling system. The expression of cps::lacZ was activated when cells were grown at a low temperature (20 degrees C) in the presence of glucose as a carbon source and in the presence of a relatively high concentration of external zinc (1 mM ZnCl(2)). In this Rcs signaling system, the rcsF gene product (a putative outer membrane-located lipoprotein) was also an essential signaling component. Based on the defined signaling pathway and physiological stimuli for the Rcs signaling system, we conducted genome-wide analyses with microarrays to clarify the Rcs transcriptome (i.e., Rcs regulon). Thirty-two genes were identified as putative Rcs regulon members; these genes included 15 new genes in addition to 17 of the previously described cps genes. Using a set of 37 two-component system mutants, we performed alternative genome-wide analyses. The results showed that the propagation of the zinc-responsive Rcs signaling system was largely dependent on another two-component system, PhoQ/P. Considering the fact that the PhoQ/P signaling system responds to external magnesium, we obtained evidence which supports the view that there is a signaling network that connects the Rcs system with the PhoQ/P system, which coordinately regulates extracellular polysaccharide synthesis in response to the external concentrations of divalent cations.

Transcriptional repressor PRR5 directly regulates clock-output pathways

The circadian clock is an endogenous time-keeping mechanism that enables organisms to adapt to external daily cycles. The clock coordinates biological activities with these cycles, mainly through genome-wide gene expression. However, the exact mechanism underlying regulation of circadian gene expression is poorly understood. Here we demonstrated that an Arabidopsis PSEUDO-RESPONSE REGULATOR 5 (PRR5), which acts in the clock genetic circuit, directly regulates expression timing of key transcription factors involved in clock-output pathways. A transient expression assay and ChIP-quantitative PCR assay using mutated PRR5 indicated that PRR5 associates with target DNA through binding at the CCT motif in vivo. ChIP followed by deep sequencing coupled with genome-wide expression profiling revealed the direct-target genes of PRR5. PRR5 direct-targets include genes encoding transcription factors involved in flowering-time regulation, hypocotyl elongation, and cold-stress responses. PRR5-target gene expression followed a circadian rhythm pattern with low, basal expression from noon until midnight, when PRR9, PRR7, and PRR5 were expressed. ChIP-quantitative PCR assays indicated that PRR7 and PRR9 bind to the direct-targets of PRR5. Genome-wide expression profiling using a prr9 prr7 prr5 triple mutant suggests that PRR5, PRR7, and PRR9 repress these targets. Taken together, our results illustrate a genetic network in which PRR5, PRR7, and PRR9 directly regulate expression timing of key transcription factors to coordinate physiological processes with daily cycles.

The Circadian Clock Regulates the Photoperiodic Response of Hypocotyl Elongation through a Coincidence Mechanism in Arabidopsis thaliana

The plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide range of physiological and developmental oscillations in habitats under natural light/dark cycles. Among clock-controlled developmental events, the best characterized is the photoperiodic control of flowering time in Arabidopsis thaliana. Recently, it was also reported that the clock regulates a daily and rhythmic elongation of hypocotyls. Here, we report that the promotion of hypocotyl elongation is in fact dependent on changes in photoperiods in such a way that an accelerated hypocotyl elongation occurs especially under short-day conditions. In this regard, we provide genetic evidence to show that the circadian clock regulates the photoperiodic (or seasonal) elongation of hypocotyls by modulating the expression profiles of the PIF4 and PIF5 genes encoding phytochrome-interacting bHLH (basic helix-loop-helix) factors, in such a manner that certain short-day conditions are necessary to enhance the expression of these genes during the night-time. In other words, long-day conditions are insufficient to open the clock-gate for triggering the expression of PIF4 and PIF5 during the night-time. Based on these and other results, the photoperiodic control of hypocotyl elongation is best explained by the accumulation of PIF4 and PIF5 during the night-time of short days, due to coincidence between the internal (circadian rhythm) and external (photoperiod) time cues. This mechanism is a mirror image of the photoperiod-dependent promotion of flowering in that plants should experience long-day conditions to initiate flowering promptly. Both of these clock-mediated coincidence mechanisms may coordinately confer ecological fitness to plants growing in natural habitats with varied photoperiods.

Comparative Transcriptome of Diurnally Oscillating Genes and Hormone-Responsive Genes in Arabidopsis thaliana: Insight into Circadian Clock-Controlled Daily Responses to Common Ambient Stresses in Plants

By adopting two distinct types of comprehensive transcriptome data sets, which are available to the public, we asked the critical question as to whether or not there is any significant correlation between diurnally oscillating genes during the light/dark daily cycle and hormone-responsive genes. The hormones studied here were ABA, ethylene, brassinosteroid, cytokinin, gibberellin, auxin and jasmonate. It was revealed that the expression of a significantly large number of ABA-responsive and/or methyl jasmonate (MJ)-responsive genes oscillate diurnally and robustly during the light/dark cycle. The results of this study implied that some aspects of plant stress responses, basically mediated by ABA and/or MJ, might be regulated daily though the circadian clock function at the level of transcription in order to prepare properly for action against common ambient stresses by anticipating the diurnal day/night cycle.

Phytochrome-interacting factor 4 and 5 (PIF4 and PIF5) activate the homeobox ATHB2 and auxin-inducible IAA29 genes in the coincidence mechanism underlying photoperiodic control of plant growth of Arabidopsis thaliana.

The plant circadian clock generates rhythms with a period close to 24 h, and it controls a wide variety of physiological and developmental events. Among clock-controlled developmental events, the best characterized is the photoperiodic control of flowering time, which is mediated through the CONSTANS (CO)-FLOWERING LOCUS T (FT) pathway in Arabidopsis thaliana. The clock also regulates the diurnal plant growth including the elongation of hypocotyls in a short day (SDs)-specific manner. In this mechanism, phytochromes (mainly phyB) and the PHYTOCHROME-INTERACTING FACTOR4 (PIF4) and PIF5, encoding phytochrome-interacting basic helix-loop-helix (bHLH) transcription factors, play crucial roles. The time of day-specific and photoperiodic control of hypocotyl elongation is best explained by the accumulation of the PIF4 and PIF5 proteins during night-time before dawn, especially under SDs, due to coincidence between the internal (circadian rhythm) and external (photoperiod) time cues. However, the PIF4- and/or PIF5-controlled downstream factors have not yet been identified. Here, we provide evidence that ARABIDOPSIS THALIANA HOMEOBOX PROTEIN2 (ATHB2), together with auxin-inducible IAA29, is diurnally expressed with a peak at dawn under the control of PIF4 and PIF5 specifically in SDs. This coincidentally expressed transcription factor serves as a positive regulator for the elongation of hypocotyls. The expression profiles of ATHB2 were markedly altered in certain clock and phytochrome mutants, all of which show anomalous phenotypes with regard to the photoperiodic control of hypocotyl elongation. Taken together, we propose that an external coincidence model involving the clock-controlled PIF4/PIF5-ATHB2 pathway is crucial for the diurnal and photoperiodic control of plant growth in A. thaliana.

Effect of Subinhibitory Concentrations of Macrolides on Expression of Flagellin in Pseudomonas aeruginosa and Proteus mirabilis

ABSTRACT In the present study we showed by molecular analysis that the inhibition of motility by macrolides in Proteus mirabilisand Pseudomonas aeruginosa was well correlated with the loss of the expression of flagellin. Erythromycin, clarithromycin, and azithromycin at subinhibitory concentrations (sub-MICs) suppressed the expression of flagellin dose dependently. Azithromycin had the strongest inhibitory effect on the expression of P. aeruginosa flagellin, whereas 16-membered rokitamycin had only a weak inhibitory effect. These results indicate the potential effectiveness of sub-MICs of erythromycin, clarithromycin, and azithromycin for the treatment of patients with P. mirabilis and P. aeruginosa infections.

The SskA and SrrA Response Regulators Are Implicated in Oxidative Stress Responses of Hyphae and Asexual Spores in the Phosphorelay Signaling Network of Aspergillus nidulans

Histidine-to-Aspartate (His-Asp) phosphorelay (or two-component) systems are common signal transduction mechanisms implicated in a wide variety of cellular responses to environmental stimuli in both prokaryotes and eukaryotes. For a model filamentous fungi, Aspergillus nidulans, in this study we first compiled a complete list of His-Asp phosphorelay components, including 15 genes for His-kinase (HK), four genes for response regulator (RR), and only one for histidine-containing phosphotransfer intermediate (HPt). For these RR genes, a set of deletion mutants was constructed so as to create a null allele for each. When examined these mutant strains under various conditions stressful for hyphal growth and asexual spore development, two of them (designated ΔsskA and ΔsrrA) showed a marked phenotype of hypersensitivity to oxidative stresses (particularly, to hydrogen peroxide). In this respect, expression of the vegetative-stage specific catB catalase gene was severely impaired in both mutants. Furthermore, conidia from ΔsskA were hypersensitive not only to treatment with H2O2, but also to treatment at aberrantly low (4 °C) and high (50 °C) temperatures, resulting in reduced germination efficiency. In this respect, not only the catA catalase gene specific for asexual development, but also a set of genes encoding the enzymes for synthesis of certain stress tolerant compatible solutes, such as trehalose and glycerol, were markedly downregulated in conidia from ΔsskA. These results together are indicative of the physiological importance of the His-Asp phosphorelay signaling network involving the SskA and SrrA response regulators.

Ambient Temperature Signal Feeds into the Circadian Clock Transcriptional Circuitry Through the EC Night-Time Repressor in Arabidopsis thaliana

An interlocking multiloop model has been generally accepted to describe the transcriptional circuitry of core clock genes, through which robust circadian rhythms are generated in Arabidopsis thaliana. The circadian clock must have the ability to integrate ambient temperature signals into the clock transcriptional circuitry to regulate clock function properly. Clarification of the underlying mechanism is a longstanding subject in the field. Here, we provide evidence that temperature signals feed into the clock transcriptional circuitry through the evening complex (EC) night-time repressor consisting of EARLY FLOWERING 3 (ELF3, ELF4) and LUX ARRHYTHMO (LUX; also known as PCL1). Chromatin immunoprecipitation assays showed that PSEUDO-RESPONSE REGULATOR7 (PRR7), GIGANTEA (GI) and LUX are direct targets of the night-time repressor. Consequently, transcription of PRR9/PRR7, GI and LUX is commonly regulated through the night-time repressor in response to both moderate changes in temperature (Δ6°C) and differences in the steady-state growth-compatible temperature (16-28°C). A warmer temperature inhibits EC function more, whereas a cooler temperature stimulates it more. Consequently, the expression of these target genes is up-regulated in response to a warm temperature specifically during the dark period, whereas they are reversibly down-regulated in response to a cool temperature. Transcription of another EC target, the PIF4 (PHYTOCHROME-INTERACTING FACTOR 4) gene, is modulated through the same thermoregulatory mechanism. The last finding revealed the sophisticated physiological mechanism underlying the clock-controlled output pathway, which leads to the PIF4-mediated temperature-adaptive regulation of hypocotyl elongation.

Circadian-Associated Rice Pseudo Response Regulators (OsPRRs): Insight into the Control of Flowering Time

A small family of plant proteins, designated PSEUDO RESPONSE REGULATORS (PRRs), is crucial for a better understanding of the molecular link between circadian rhythm and photoperiodic control of flowering time in the dicotyledonous model plant Arabidopsis thaliana. Recently, we showed that the monocotyledonous model plant Oryza sativa also has homologous members of the OsPRR family (Oryza sativa PRR). In the previous experiments with rice, we mainly characterized a japonica variety (Nipponbare). By employing an indica variety (Kasalath), in this study we further characterized OsPRRs with reference to the photoperiod sensitivity Hd (Heading date) QTL (quantitative trait loci) implicated in the control of flowering time in rice. The circadian-controlled and sequential expression profiles of the five OsPRR genes were observed not only for Nipponbare but also for Kasalath. Then each of these OsPRR genes was mapped on the rice chromosomes. Among these OsPRR genes, OsPRR37 was mapped very closely to Hd2-QTL, which was identified as the major locus that enhances the photoperiod sensitivity of flowering in Nipponbare. Furthermore, we found that Kasalath has a severe mutational lesion in the OsPRR37 coding sequence.

The Common Function of a Novel Subfamily of B-Box Zinc Finger Proteins with Reference to Circadian-Associated Events in Arabidopsis thaliana

Over 1,600 genes encoding putative transcription factors have been identified in the Arabidopsis genome sequence, however, their physiological functions are not yet fully understood. In this study, a small subfamily of double B-box zinc finger (DBB, DOUBLE B-BOX) genes, encoding eight putative transcription factors, were characterized with reference to the circadian rhythm and the early photomorphogenic regulation of hypocotyl elongation in response to light signals. Among these, it was found that the transcriptions of five DBB genes were under the control of circadian rhythm. To gain insight into the physiological roles of these putative transcription factors, forward and reverse genetic studies were carried out. The results suggested that they are commonly implicated in light signal transduction during early photomorphogenesis, however, their functions are not totally redundant, as judged by the fact that their circadian-expression profiles (or phases) were distinctive from each other, and by the fact that some DBBs (named DBB1a, DBB1b, STO, and STH) were apparently implicated in light signal transduction in a negative manner, whereas another (named DBB3) was implicated in a positive manner with regard to light-induced inhibition of elongation of hypocotyls. We also found that homologous B-box zinc finger genes are widely conserved in higher plants (e.g., Oryza sativa). Taking this altogether, it is probable that in addition to previously characterized bZIP-type transcription factors (e.g., HY5 and HYH) and bHLH-type transcription factors (e.g., PIF4 and PIF5/PIL6), a set of B-box zinc finger transcription factors should also be taken into consideration for a better understanding of the complex molecular mechanisms underlying the early photomorphogenic development of Arabidopsis thaliana.