Setsuyuki Aoki

Differential Expression on a Daily Basis of Plastid Sigma Factor Genes from the Moss Physcomitrella patens. Regulatory Interactions among PpSig5, the Circadian Clock, and Blue Light Signaling Mediated by Cryptochromes

The nuclear-encoded plastid sigma factors are supposed to be a regulatory subunit of the multisubunit bacteria-type plastid RNA polymerase. We studied here whether or not three genes, PpSig1, PpSig2, and PpSig5 encoding plastid sigma factors, are controlled by the circadian clock and/or by blue light signaling in the moss Physcomitrella patens. Among the three PpSig genes, only PpSig5 was clearly controlled by the circadian clock. In contrast to the differential regulation on a daily timescale, a pulse of blue light induced the expression of all the three PpSig genes. This induction was significantly reduced in a knockout mutant that lacked the blue light photoreceptor cryptochromes PpCRY1a and PpCRY1b, indicating that PpCRY1a and/or PpCRY1b mediate the blue light signal that induces the expression of the PpSig genes. In a daily cycle of 12-h blue light/12-h dark, the timing of peak expression of PpSig5 and a chloroplast gene psbD, encoding the D2 subunit of photosystem II, advanced in the cryptochrome mutant relative to those in the wild type, suggesting the presence of regulatory interactions among the expression of PpSig5 and psbD, the circadian clock, and the blue light signaling mediated by the cryptochrome(s).

Characterization of two genes, Sig1 and Sig2, encoding distinct plastid σ factors1 in the moss Physcomitrella patens: phylogenetic relationships to plastid σ factors in higher plants

We isolated the cDNA for a σ factor from the moss Physcomitrella patens, which possesses unusually large N‐terminal extension and the conserved subdomains 1.2–4.2. Phylogenetic analyses indicated that this novel σ factor and PpSIG1* 2, a plastid σ factor previously identified from Physcomitrella, were classified into SigA and SigB groups, two major classes of higher plant plastid σ factors, respectively. According to the nomenclature recently proposed, we renamed PpSIG1* into PpSIG2, and named the novel σ factor PpSIG1. A transient expression assay using a green fluorescent protein showed that the N‐terminal region of PpSIG1 acts as a chloroplast‐targeting signal. Reverse transcription‐PCR experiments showed that light induces the expression of the Sig1 and Sig2 genes encoding PpSIG1 and PpSIG2, respectively. Thus, PpSIG1 and PpSIG2 are likely plastid σ factors regulating plastid gene expression in response to light signals.

Cloning and characterization of the cDNA for a plastid σ factor from the moss Physcomitrella patens

Abstract We isolated a cDNA PpSig1 encoding a plastid σ factor from the moss Physcomitrella patens . The PpSIG1 protein is composed of the conserved subdomains for recognition of −10 and −35 promoter elements, core complex binding and DNA melting. Southern blot analysis showed that the moss sig1 gene is likely a member of a small gene family. Transient expression assay using green fluorescent protein demonstrated that the N-terminal region of PpSIG1 functions as a chloroplast-targeting signal peptide. These observations suggest that multiple nuclear-encoded σ factors regulate chloroplast gene expression in P. patens .

Functional characterization of CCA1/LHY homolog genes, PpCCA1a and PpCCA1b, in the moss Physcomitrella patens

The evolution of circadian clocks in land plants is not understood, because circadian rhythms have received little attention in plants other than angiosperms. We have characterized two genes, PpCCA1a and PpCCA1b, homologs of the Arabidopsis thaliana clock genes CCA1/LHY, from the moss Physcomitrella patens. PpCCA1a and PpCCA1b, together with angiosperm CCA1/LHY homologs, belong to the clock-associated single-myb gene family of green plants (including green algae and land plants). The accumulation of PpCCA1a and PpCCA1b mRNA showed rhythms with a period of approximately 1 day, phased as are those of angiosperm homologs, under 24 h light/dark cycles or in continuous dark. However, in marked contrast to angiosperm homologs, both genes showed arrhythmic profiles in continuous light. The timing of the PpCCA1b peak is determined by the time of the last light to dark transition, suggesting that the arrhythmicity in continuous light is due to dysfunction of the core clock. We generated single and double disruptants for PpCCA1a and PpCCA1b, and found that the double disruptants showed: (i) short periodicity and damped amplitude in the PpCCA1b rhythm, (ii) similar changes in the rhythmically expressed genes PpSIG5 and PpPRRa, and (iii) de-repression of PpCCA1b transcription levels, indicating negative feedback regulation. These observations indicate that the two genes are not merely structural homologs but also functional counterparts of CCA1/LHY. Together, our results illustrate similarities as well as divergence of the clock machineries between P. patens and A. thaliana, two distantly placed species in land plant phylogeny.

A promoter-trap vector for clock-controlled genes in the cyanobacterium Synechocystis sp. PCC 6803

We constructed a promoter-trap vector pPT6803-1 to isolate circadian clock-controlled promoters in the cyanobacterium Synechocystis sp. strain PCC 6803. The vector contains a promoterless luciferase gene set (luxAB) from Vibrio harveyi that is targeted to a specific site of the Synechocystis genome as a reporter for gene expression. A library was constructed in pPT6803-1 by introducing the genomic DNA fragments upstream of luxAB to transform Synechocystis cells. Of approximately 10,000 Synechocystis transformants, at least 55 (#1-55) showed circadian rhythms of bioluminescence under continuous illumination. Clones #19, #22, and #26 exhibited obviously different waveforms of bioluminescence from each other. Deletion analysis and primer extension experiments mapped the promoters for the clpP, slr1634, and rbpP genes that are responsible for bioluminescence from #19, #22, and #26, respectively.

Circadian Rhythm and cDNA Cloning of the Clock Gene period in the Honeybee Apis cerana japonica

Abstract Isolated individual foragers of Apis cerana japonica could be entrained under a light-dark cycle, and the predominant activity was concentrated to the later part of the photophase. Foragers showed circadian rhythm under conditions of constant light and constant dark with free-running periods of more and less than 24 hr, respectively. These observations indicated that A. cerana possesses a circadian clock controlling locomotor activity. To investigate the molecular mechanism underlying the circadian system we cloned cDNA for a homolog of the clock gene period (per) from the honeybee by a PCR-strategy. The cloned percDNAs consisted of two types, α and β, encoding a putative protein of 1124 amino acids and 1116 amino acids, respectively. The sequences of types α and β were identical except that the former possessed an additional 24 bp stretch corresponding to 8 amino acids in the conserved C2 block. These two types were assumed to be differentially spliced variants and found also in per cDNA of A. mellifera. In support of this idea, Southern blotting experiments showed that per of A. cerana is a single copy gene. RT-PCR analysis and subcloning of the products revealed that the both types α and β are expressed in the brain of the forager. A quantitative RT-PCR assay by which the level of per mRNA in one single brain can be detected was established. Per mRNA level showed daily oscillation under a light-dark cycle with a change of the ratio of type α to β.

Pseudo-Response Regulator (PRR) Homologues of the Moss Physcomitrella patens: Insights into the Evolution of the PRR Family in Land Plants

The pseudo-response regulators (PRRs) are the circadian clock component proteins in the model dicot Arabidopsis thaliana. They contain a receiver-like domain (RLD) similar to the receiver domains of the RRs in the His–Asp phosphorelay system, but the RLDs lack the phosphoacceptor aspartic acid residue invariably conserved in the receiver domains. To study the evolution of PRR genes in plants, here we characterize their homologue genes, PpPRR1, PpPRR2, PpPRR3 and PpPRR4, from the moss Physcomitrella patens. In the phylogenetic analysis, PpPRRs cluster together, sister to an angiosperm PRR gene subfamily, illustrating their close relationships with the angiosperm PRRs. However, distinct from the angiosperm sequences, the RLDs of PpPRR2/3/4 exhibit a potential phosphoacceptor aspartic acid–aspartic acid–lysine (DDK) motif. Consistently, the PpPRR2 RLD had phosphotransfer ability in vitro, suggesting that PpPRR2 functions as an RR. The PpPRR1 RLD, on the other hand, shows a partially diverged DDK motif, and it did not show phosphotransfer ability. All PpPRRs were expressed in a circadian and light-dependent manner, with differential regulation between PpPRR2/4 and PpPRR1/3. Altogether, our results illustrate that PRRs originated from an RR(s) and that there are intraspecific divergences among PpPRRs. Finally, we offer scenarios for the evolution of the PRR family in land plants.

The plastid sigma factor SIG5 is involved in the diurnal regulation of the chloroplast gene psbD in the moss Physcomitrella patens

We analyzed the function of a plastid sigma factor, SIG5, by targeted gene disruption in the moss Physcomitrella patens. High‐intensity light induced the chloroplast gene psbD in the wild‐type strain (WT), whereas this induction was nullified in the PpSig5‐disrupted strains (ΔSig5). Moreover, diurnally regulated changes of psbD transcription showed lowered amplitude in ΔSig5 than in WT. We concluded that the moss SIG5 mediates multiple layers of signals to intricately regulate psbD transcription.

Circadian Clocks of Synechocystis sp. Strain PCC 6803, Thermosynechococcus elongatus, Prochlorococcus spp., Trichodesmium spp. and Other Species

Abstract The cyanobacterium Synechococcus elongatus PCC 7942 has been established as the model system for studying the molecular mechanisms of the cir-cadian clock in cyanobacteria. This chapter mainly focuses on other cyanobacteria, such as SynechocystisL sp. strain PCC 6803, Thermosynechococcus elongatus and the genera Trichodesmium and Prochlorococcus. Here, we describe the research background, current status, possible problems and perspectives for studying circa-dian rhythms for each species/group and we summarize the related works of other cyanobacteria and plastids.

Heterologous Expression and Functional Characterization of a Physcomitrella Pseudo Response Regulator Homolog, PpPRR2, in Arabidopsis

Physcomitrella patens has four homologs of the pseudo-response regulator involved in the circadian clock mechanism in seed plants. To gain insight into their function, Arabidopsis transgenic lines misexpressing PpPRR2 were constructed. Phenotypic analysis of the transformants with reference to clock-related gene expression and photoperiodic responses revealed that heterologous expression of the moss PpPRR2 gene modifies the intrinsic mechanism underlying the circadian clock in Arabidopsis, suggesting that PpPRR2 serves as a clock component in P. patens.