GA receptor GID1, DELLA protein SLR1, and F-box protein GID2 are major components governing the early GA signaling events in rice. When GID1 binds to GA, GID1 becomes capable of interacting with SLR1, a repressor of GA action. It is thought that SCF^GID2 complex specifically recognizes the SLR1 protein within the GID1-SLR1 complex via GID2, which leads to degradation of SLR1. To elucidate the precise molecular events on SLR1 degradation, interaction between GID1, SLR1 and GID2 was studied. Our results confirmed that interaction between GID2 and SLR1 is GA and GID1 dependent. Furthermore, we identified important regions of each protein in forming GID1-SLR1-GID2 complex. Our results suggest that when DELLA/TVHYNP domains of SLR1 interact with GID1 in the presence of GA, the GRAS domain of SLR1 becomes capable of interacting with GID1, and this interaction is indispensable for GID2 to recognize SLR1.

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SLR1 acts as a repressor of gibberellin (GA) signaling. GA perception by GA receptor GID1 causes SLR1 degradation involving the F-box protein GID2. In GA-insensitive and GA-deficient mutants, SLR1 accumulates and the severity of dwarfism is usually correlated with the level of SLR1 accumulation. An exception is the GA-insensitive F box mutant gid2, which shows milder dwarfism than mutants such as gid1 and cps even though it accumulates higher levels of SLR1. Here, we investigate on such mutant phenotype in gid2.
In gid2, loss of GID1 function or treatment with a GA biosynthesis inhibitor decreased the level of SLR1 and enhanced its dwarfism. On the contrary, overproduction of GID1 or treatment with GA₃ increased the SLR1 level in gid2 and reduced its dwarfism. These results indicate that de-repression of SLR1 repressive activity can be accomplished only by GA and GID1 but not require the F-box (GID2) function.

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Gibberellin (GA) is one of the important plant hormone involving plant growth and development. GA response is controlled by a nuclear repressor DELLA protein. Once plant cells receive GA molecules, DELLA proteins are rapidly degraded by ubiquitin-proteasome pathway. GA perception and following DELLA protein recognition by GA receptor GID1 cause ubiquitination and destruction of DELLA proteins triggering the GA response. However, there are few studies of structural analysis of GA signaling components. Here, we determined crystal structures of Arabidopsis GID1 in complex with GA and DELLA N-terminal domain at 1.8 angstrom resolution. GA molecule is binding with a pocket of GID1 core domain and sealed by the GID1 N-terminal extension. DELLA and VHYNP motifs of DELLA protein mainly interact with N-terminal extension of GID1. These results suggest that GA molecule induces conformational change of GID1 N-terminal extension and formation of scaffold for DELLA protein recognition.

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Gibberellins regulate germination, elongation and flowering. DELLA proteins are members of the plant-specific GRAS protein family and act as repressors of the GA signaling pathway. DELLA proteins are rapidly degraded in the presence of GA. The degradation mechanism is beginning to be uncovered by the discovery of GA receptor and F-box protein. To understand the downstream signaling of DELLA proteins, we have identified GAF1 that interacts with GAI. GAF1 is a novel transcriptional factor that binds to DNA with sequence specificity. Arabidopsis plants that overexpress GAF1 are early flowering with larger leaves. BiFC analysis indicated that the interaction of GAF1 and GAI disappeared after GA treatment. Our trans-activation assays in yeast and plant cells suggest that GAI protein regulates the function of GAF1. These data suggest that GA controls transcriptional activity via alteration of GAF1 complex components. Based on this model, we are searching for the target genes of GAF1.

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Gibberellins (GAs) are phytohormones that regulate many aspects of plant development. RSG is a tabacco transcriptional activator with a bZIP domain that is involved in the regulation of endogenous amounts of GAs. The functional repression of RSG reduced endogenous amounts of GAs and inhibited feedback regulation of NtGA20ox1. ChIP studies demonstrated that RSG binds to the promoter of NtGA20ox1 in vivo when endogenous amount of GAs is decreased. We found that RSG is regulated by NTCDPK1 and 14-3-3; however, the relationship to DELLA proteins that play a key role in GA signaling was unknown.
In this study, we studied the role of AtRSG, Arabidopsis ortholog of RSG, in the transcriptional regulation of GA-biosynthetic enzyme genes. Transient assay showed that AtGA20ox1 is activated by AtRSG. Furthermore, we investigated possible involvement of nuclear GA signaling repressor, DELLA proteins in the transcriptional regulation of GA-biosynthetic enzyme genes by AtRSG.

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Strigolactones were previously found in root exudates acting as communication chemicals with root parasitic weeds and symbiotic arbuscular mycorrhizal (AM) fungi. Recently, we have found that strigolactones also inhibit shoot branching in the host plants. However, it is still unclear why strigolactones act as inhibitors of shoot branching as well as being chemical signals in the rhizosphere. Strigolactone production is known to increase under phosphorus deficiency. This is thought to be a strategy of plants to activate AM fungi, which facilitate the uptake of soil nutrients by host plants. On the other hand, plants need to minimize shoot branching under nutrient deficiency for efficient utilization of the nutrient. To study the possible role of strigolactones in regulating shoot branching in response to nutrient deficiency, we investigated the relationships between strigolactone production and shoot branching under varying nutrient conditions using wild type and strigolactone-deficient mutants of rice.

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Strigolactones released into rhizosphere are important host-recognition signals to commence parasitism by root parasitic plants and mutualism by arbuscular mycorrhizal fungi. Effects of phosphate fertilization on production and exudation of strigolactones were examined in a split-root system of sorghum. Phosphate fertilization to one side of the split-root reduced strigolactone exudation in the other side indicating that the exudation was regulated systemically. In addition, an increase of phosphate level in shoots but not the level itself appeared to reduce production and exudation of strigolactones.

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In the cyanobacterium Synechococcus elongatus PCC 7942, the temporal information from the KaiABC-based circadian oscillator is transmitted to the regulatory machinery of gene expression. A number of mutants that are deficient in circadian gene expression have been identified and analyzed. Among them, rpaA, sasA and labA are shown to play key roles in the output pathways from the circadian oscillator. It was proposed that RpaA acts as a transcriptional factor to regulate the circadian kaiBC expression through the SasA-dependent positive pathway and the LabA-dependent negative pathway. However, the labA/sasA double mutant did show a low-amplitude circadian kaiBC expression, suggesting that there are additional circadian output pathways to regulate the kaiBC expression. We performed further screening to identify those genes that are involved in the additional output pathways.

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Luciferase genes have been used as a tool to monitor circadian rhythms of promoter activity in many organisms. We here tried to monitor circadian rhythmicities in the activities of clock gene promoters in Chlamydomonas. The promoter regions of the clock genes ROC15 and ROC75 were fused to a codon-optimized firefly luciferase coding sequence. Since ROC15 and ROC75 mRNA levels oscillate with an 8-hour phase-angle-difference, we expected that bioluminescence rhythms of the two reporters oscillate with a similar phase-angle-difference. Surprisingly, however, bioluminescence rhythms of ROC15 and ROC75 reporters oscillated with essentially the same phase angles. Do activities of the promoter regions not reflect in luciferase gene expressions? The circadian rhythms of luciferase mRNA level in the ROC15 and ROC75 reporter strains oscillated with a phase-angle-difference similar to that of endogenous ROC15 and ROC75 mRNAs, suggesting that circadian rhythmicities of luciferase reporters are regulated at posttranscriptional steps in Chlamydomonas.

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In land plants, the mechanisms underlying circadian clock have so far been analyzed mainly on angiosperm lineages, whereas there are only a few studies concerning relatively primitive species such as ferns and bryophytes. Therefore, it is unknown how similar or diverged are the mechanisms in plant kingdom, and the evolution of plant circadian systems remains to be elucidated. For these reasons, we started to study the mechanisms of the clock in the moss Physcomitrella patens, which is one of basal land plants. The genome of P. patens contains many homologs of A. thaliana clock genes such as CCA1/LHY and PRR family genes. We cloned two CCA1/LHY homolog genes PpCCA1a and PpCCA1b, and also constructed single and double knockout strains for these genes. We will report the results of recent analyses in this conference.

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One core group of clock components in Arabidopsis that controls the pace of the central oscillator is comprised of five PRR proteins whose biochemical function in the clock remains unclear. The PRRs are related to Response Regulators (RRs) of His-Asp phospho-relay signal transduction systems. We have identified and characterized genes encoding PRR homologs from Physcomitrella (Designated as PpPRR1, PpPRR2, PpPRR3 and PpPRR4).
It should be noted that the Arabidopsis PRRs lack phospho-accepting aspartate
(D) residue.Instead, each pesudo-receiver has a glutamate (E) residue at this particular position. Interestingly, Physcomitrella PRR does not show this property, instead all PpPRRs retain the aspartate (D) at the potential phosphorylation site.
Expression of PpPRRs under DD, LL and LD conditions shows circadian, arrhythmic and diurnal pattern respectively. These data have allowed us to interpret how plant PRRs evolved during evolution.

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With regard to the Arabidopsis circadian clock, an interlocking multi-loop clock model has been currently acknowledged. This tentative model consists of three interactive feedback loops, namely, core CCA1/LHY-TOC1/X loop, morning CCA1/LHY-PRR9/PRR7 loop, and evening Y-TOC1 loop, in which the undefined Y gene might be GI and/or PRR5. To evaluate this model, we already characterized a prr9 prr7 double loss-of-function mutant that had lost the morning loop, and a cca1 lhy toc1 triple mutant that lacks the core loop. To extend these lines of genetic studies, here we characterized a gi toc1 and gi prr5 double loss-of-function mutants that are expected to have no evening loop, and a prr9 prr7 toc1 triple mutant that lacks both the morning and evening loops. The results will be discussed extensively with reference to the current clock model, including the possible functions of GI and PRR.

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The circadian clock is implicated in the several cellular and physiological activities in plants. It must be synchronized to local time by environmental time cues. In Arabidopsis, although many clock-associated components have been uncovered, the clock-associated elements that are involved in entrainment are largely unknown. We have been characterizing one core group of clock components that control the pace of the central oscillator, including PRR9, PRR7, PRR5, and TOC1. In this study, the diurnal oscillation profiles of clock-controlled genes in the presence of environmental time cues were examined in a set of prr mutants. As an extreme phenotype, the prr9-10 prr7-11 prr5-11 toc1-2 quadruple mutant showed an arrhythmia phenotype even under light/dark and hot/cold cycles. Here, we will propose that the clock components PRR9, PRR7, and PRR5 together might represent elements necessary for the circadian clock to properly entrain to local time in response to environmental time cues.

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Plant circadian clock controls many physiological events under diurnal and seasonal changes in natural light/dark cycles. The best characterized is the photoperiodic control of flowering time in A. thaliana. Recently, it was reported that the clock regulates daily timing of rhythmic hypocotyl elongation.
Here, we found that this regulation of hypocotyl elongation is dependent on the photoperiod in a non-linear fashion. Genetic evidence will be presented for that the clock regulates the photoperiodic control of hypocotyl elongation by modulating the expression of PIF4/PIF5 (bHLH factors), which act positively hypocotyl elongation, particularly, in a manner that certain lengths of dark-period are necessary to enhance their transcription during late nighttime. Taken together, we propose the model that the photoperiodic control of hypocotyl elongation is best explained by the accumulation of PIF4/PIF5 during nighttime of short-days, due to coincidence between the internal (circadian rhythm) and external (photoperiod) time cues.

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Arabidopsis Pseudo Response Regulator (PRR) genes are components of the circadian clock mechanism. In order to understand the scope of genome-wide transcriptional regulation by PRR genes, a microarray analysis was performed for a prr9-11 prr7-10 prr5-10 triple mutant (d975) using mRNA collected during late daytime. Night genes expression were increased and day genes expression were decreased toward the dusk, but expression of these genes was constant in d975. Night genes expression levels were lower, whereas day genes were higher in d975 than wild-type. Bioinformatics approaches indicated that up-regulated genes in d975 and cold-responsive genes have significant overlap. We found that d975 are more tolerant to cold stresses than wild-type. These results suggest that PRR9, PRR7, and PRR5 are involved in the mechanism that anticipates diurnal cold stress coming, and prepares stress response, by mediating cyclic expression of stress response genes.

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One core group of circadian clock components in Arabidopsis that controls the pace of the central oscillator is comprised of five PRR (Pseudo-Response Regulator) proteins whose biochemical function in the clock remains unclear. PRR1/TOC1 and PRR5 are the only likely proteolytic substrates of the E3 ubiquitin ligase SCF^ZTL within this PRR family. We demonstrate a functional significance for the phosphorylated forms of PRR5, TOC1, and PRR3. Each PRR protein is differentially phosphorylated over the circadian cycle. The more highly phosphorylated forms of PRR5 and TOC1 interact best with the F-box protein ZTL (ZEITLUPE). TOC1 and PRR3 interact in vivo and phosphorylation of both is necessary for their optimal binding in vitro. TOC1/PRR3 phosphorylation-dependent interaction may protect TOC1 from ZTL-mediated degradation, resulting in an enhanced amplitude of TOC1 cycling.

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The establishment of single-celled leaf hairs (trichomes) from pluripotent epidermal (protodermal) cells provides a powerful system to determine the gene regulatory networks involved in plant cell fate determination. Two transcription factors, GL1 and GL3, have been associated with the initiation of trichome formation. In this study, we combined expression analyses performed in a number of different genotypes to identify a minimal set of ~500 genes associated with trichome formation. We also used ChIP-chip to identify a set of ~20 genes that are immediate targets of GL3 and GL1. As predicted for genes involved in the initiation of epidermal cell fate determination, several of the GL3/GL1 direct targets are expressed early during trichome formation. Co-expression analyses permitted to identify sets of target genes likely downstream of the GL3/GL1 regulated transcription factors, providing the first steps towards building the regulatory network associated with trichome formation.

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EDM2 is required for R-protein-mediated immunity as well as several developmental processes in Arabidopsis. It bears typical features of transcription factors and epigenetic regulators, but does not belong to any established class of proteins controlling transcription. We found EDM2 to interact in nuclei with a WNK-type protein kinase (WTK) that phosphorylates EDM2. wtk mutants do not exhibit reduced immunity, but show edm2-related developmental phenotypes. Thus, EDM2 appears to be controlled in part by WTK. Furthermore, EDM2 seems to affect the status of chromatin as well as expression of multiple genes encoding defense components or developmental regulators. We are currently examining in vivo-functions of multiple chromatin-associated proteins that we identified as EDM2 interactors to understand putative roles of EDM2 as part of regulatory complexes differentially controlling expression of gene clusters required for defense and developmental processes. We are also addressing possibly intriguing connections between its roles in immunity and development.

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ICE1 induces expression of DREB1A/CBF3 to regulate cold signaling and cold tolerance. However, it is unknown how ICE1 is activated by cold signaling. Recently, we have found that sumoylation of ICE1 at K393 regulates cold signaling. In this study, we have evaluated roles of serine/threonine residues around the K393 to find the residues for activation of ICE1. We replaced 6 serine/threonine residues around the K393 to alanine and the substitution of a certain serine enhanced transactivation activity of ICE1. Overexpression of the ICE1 (S to A) conferred more freezing tolerance than that of ICE1 (WT) in Arabidopsis. These results suggest that the serine residue has roles in the activation of ICE1.

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Modulation of histone N-tail modifications correlates with transcriptional activation and repression in eukaryotes. We reported that the histone H3 modifications clearly correlate with gene activation of the stress-inducible genes under drought stress conditions ¹⁾.
We have studied alterations of the histone modifications during the process from dehydration to rehydration. From the Arabidopsis tiling array data, we selected several gene regions with the characteristic expression pattern in the dehydration to rehydration process. Chromatin immunoprecipitation assay showed that the temporal changes in enrichment of several histone modifications occur on the drought-inducible gene regions during the process. We will discuss about the correlation between gene regulation and status of histone modifications in the dehydration to rehydration process.
¹⁾ Kim et al. (2008) Plant Cell Physiol.: 49: 1580-1588.

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We show here that the EAR-motif repression domain (SRDX) can covert a transcriptional complex into a repressor by transrepression activity through protein-protein interaction. We propose here that this transrepression activity can be used for detection of protein-protein interactions in plants. We demonstrated transrepression in combination of FOS and JUN, and PI and AP3, which are known to form a heterodimer, respectively, in our transient studies. Furthermore, we showed that transgenic Arabidopsis that expressed TTG1-SRDX, which is a WD40 protein and does not has DNA binding activity, resulted in the phenotype similar to ttg1 mutants possibly due to suppression of expression of genes that are regulated by protein complex including TTG1, as similar manner in the transient study. These results indicate that the transrepressive activity by SRDX can be used to detect and confirm protein-protein interaction in plants.

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TFIIB and its related protein, BRF, are general transcription factors (GTFs) for eukaryotic RNA polymerases II and III, respectively, and have important functions in transcriptional initiation. In this study, the third type of TFIIB-related protein, pBrp, found in plant lineages was characterized in the red alga Cyanidioschyzon merolae. Chromatin immunoprecipitation analysis revealed that CmpBrp specifically occupied the rDNA promoter region in vivo. Consistently, CmpBrp and CmTBP cooperatively bound the rDNA promoter region in vitro. In vitro transcription from the rDNA promoter in crude cell lysate was severely inhibited by the CmpBrp antibody, and was also inhibited when DNA template with a mutated CmpBrp-CmTBP binding site was used. CmpBrp was shown to co-immunoprecipitate with the RNA polymerase I subunit, CmRPA190, and to co-localize in the nucleolus.Thus, together with comparative studies of Arabidopsis pBrp, we concluded that pBrp is a GTF for RNA polymerase I in plant cells.

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Al-tolerant cultivars of barley detoxify Al by secretion of citrate from the roots. Recently, a gene responsible for Al-activated citrate secretion (HvAACT1) has been identified (Furukawa et al., 2007). The expression of this gene is not induced by Al and Al-tolerant cultivars constitutively have higher expression than Al-sensitive cultivars. HvAACT1 protein is located at the epidermal cell. In the present study, to examine the mechanism regulating the expression of HvAACT1, we compared the sequence of UTR regions between Al-sensitive and tolerant cultivars. There was no correlation between Al tolerance and the sequence difference in the 3'-UTR region. In the 5'-region, we found that a specific region (about 1kb) was inserted in the genome of ORF upstream only in the Al-tolerant cultivars. 5'-RACE analysis showed that in Al-tolerant cultivars, there were multiple transcription start sites. These results suggest that this region may be involved in the higher expression of HvAACT1.

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Many genes related to iron uptake and translocation are up-regulated by iron deficiency and possess iron deficiency-responsive cis-elements, IDE1 and IDE2. We identified a novel transcription factor of rice, IDEF2, which specifically binds to IDE2 by yeast one-hybrid screening. IDEF2 recognized a part of IDE2 as the binding core sequence, which is different from any reported cis-elements bound by NAC domain transcription factors. Interestingly, IDEF2 also recognized a part of CaMV 35S promoter, which is not similar to IDE2. Repression of IDEF2 by RNAi caused aberrant iron homeostasis in rice. Among the iron-deficiency inducible genes, several genes including OsYSL2 were less induced by iron deficiency in the RNAi rice. OsYSL2 is Fe(II)-nicotianamine transporter and important for iron translocation. IDEF2 bound to OsYSL2 promoter region containing the IDEF2 binding core sequence, suggesting direct regulation of OsYSL2 expression. These results reveal novel cis-element/trans-factor interactions functionally associated with iron homeostasis.

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To predict putative cis-elements in promoters, we developed a novel in silico method with Microarray Associated Motif Analysis program (MAMA), and applied it to the microarray analysis of iron deficient and iron sufficient rice. As a result, sequences including binding core sequence (CATGC) of IDEF1, ABI3/VP1 transcription factor involved in response to iron deficiency in rice, were predicted as 2nd, 4th and 5th probable candidates of cis-elements. Sequence including binding core sequence (CA[AC]G[TC][TCA][TCA]) of IDEF2, NAC transcription factor involved in iron distribution in rice, was predicted as 12th probable cis-element candidate. Sequence similar to binding core sequence (CACGTGG) of OsIRO2, iron deficiency inducible bHLH transcription factor, was predicted as 10th probable candidate. Most of these predicted cis-elements were predicted as one of numerous candidates by existing methods. This result demonstrated that MAMA is useful for finding new functional cis-elements.

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Unicellular green alga Chlamydomonas reinhardtii synthesizes de novo a high-CO2 inducible periplasmic 43 kDa protein (H43). The gene for H43 is known to be up-regulated under high-CO₂ conditions at the transcriptional level. In this study, we intended to reveal the regulatory mechanisms for H43 induction. First, we identified the transcriptional starting site of the H43 gene. To find high-CO₂ responsible cis-element, various lengths of upstream regions of the H43 were connected to a reporter gene, aryl-sulfatase2, and the constructs were introduced into the cells. We found at least two putative high-CO₂ responsible cis-elements, -151 bp to -176 bp and -657 bp to -687 bp, that locate at upstream region of the H43 gene.

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Plant-specific Dof transcription factors are involved in various processes unique to plants. Here we show the presence of 19 putative Dof genes in Physcomitrella patens (PpDof1-19) and one Dof gene in the green alga Chlamydomonas reinhardtii (CrDof), suggesting that the origin of the Dof transcription factors pre-dates the divergence of the green algae and the ancestors of terrestrial plants. Phylogenic analysis classified Dof genes into three groups, one of which includes PpDof1-6, CrDof and angiosperm Dof genes. Further characterization of PpDof1-6 genes revealed intimate relationships between PpDof1 and 2, PpDof3 and 4, and PpDof5 and 6. We therefore generated single knock out lines for each gene and double knock out lines in which both closely related Dof genes were disrupted. Among them, the PpDof1 PpDof2 double knock out line was found to show abnormal growth. We are currently performing more detailed analyses of this line.

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Higher order of chromatin structures influences a variety of molecular events occurring in nuclei such as DNA replication, DNA recombination and transcription of genes. Our previous study on the Gln1;1 gene, which encodes cytosolic glutamine synthetase of radish, demonstrated that the gene promoter requires, for its proper expression in transgenic Arabidopsis, an A/T-rich structure encompassing the gene. We further carried out the study on the gene structure, and found that the A/T-rich structure of the gene introduced into Arabidopsis genome causes aberrant plant development in transformants at considerably high proportions. Examples of the aberrant plant development included abolishment of shoot apical meristems, aberrant pattern of branching, abnormal development of flowers, leaves, stems etc. Our observations thus raised a possibility that the A/T-rich structure of Gln1;1 introduced into Arabidopsis genome interferes proper chromatin function, and affects finely-tuned genetic program of plant body development.

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PPR proteins have been implicated in the control of plant organelle gene expression. Many PPR proteins contain a highly conserved C-terminal domain, so-called DYW domain. Recently, Salone et al. have proposed a hypothesis that the DYW domains are responsible for RNA editing in plant organelles and catalyze RNA editing (FEBS Lett. 581, 4132-4138, 2007). To investigate the function of the DYW domain-containing PPR proteins, we constructed the Physcomitrella patens gene knockout mutants and characterized RNA editing in the mutants. Knockout of the PpPPR_71 gene resulted in significant reduced growth of protonemata and completely impaired RNA editing in the mitochondrial ccmF transcript. This strongly suggests that PpPPR_71 is responsible for RNA editing for the ccmF editing site. To further investigate the possibility that the DYW domain is an RNA editing enzyme, we tested the RNA editing activity of PpPPR_71. The results will be presented.

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In RNA editing, specific cytidine nucleotides are frequently altered to uridine in RNA in plant organelles. The plant-specific PPR proteins with the DYW motif (DYW subclass) have been postulated to be involved in RNA editing of organelle transcripts. We discovered that the DYW proteins CRR22 and CRR28 are required for RNA editing of several specific sites in plastids, but their DYW motifs are dispensable for editing activity in vivo. Replacement of the DYW motifs of CRR22 and CRR28 by that of CRR2, shown to be capable of endonucleolytic cleavage, blocks the editing activity of both proteins. In return, the DYW motifs of neither CRR22 nor CRR28 can be functionally replaced by that of CRR2. Based on these results, we discuss the evolution of the DYW subclass and RNA editing in plant organelles.

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The plastid genome in higher plants is transcribed by NEP (T7-phage type RNA polymerase) and PEP (bacterial type RNA polymerase). Plastidic accD and rpo genes are exclusively transcribed by NEP, whereas photosynthetic genes are mainly transcribed by PEP. Gene expression is primary determined by transcription level, but in plastids post-transcriptional regulations like RNA editing, trimming and splicing of immature RNAs are also crucial for correct and enough expression of functional RNAs or active proteins. We focused in an Arabidopsis PPR gene named PPRTp, because the major NEP (RPOTp) and PPRTp show highest correlation coefficient with each other in ATTED-II database. An Arabidopsis pprTp mutant showed albino and severe morphological deficiency. We found that an RNA editing in accD mRNA did not occur in this mutant. We will discuss the critical role of PPRTp mediated by the RNA editing of accD mRNA.

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NEP is one of two heterologous transcription systems in plastids and belongs to T7 phage-type RNA polymerase. Arabidopsis has two NEP genes encoding RPOTmp (RPOT;2) and RPOTp (RPOT;3) on the nuclear genome. It has been suggested that RPOTmp probably localizes in both chloroplast and mitochondria. It is physiologically involved in root development and possibly activates a rrn16 promoter. However, the molecular detail of its functional significance is still unclear. We focus in direct and indirect function of RPOTmp in the expression of translational machineries including tRNAs and ribosomal proteins. We characterized therpoTmp mutant by (1) northern and QPCR analyses of nuclear- and plastid-encoded genes for plasidic translation system, (2) polysome analysis, (3) stress response to high light and several reagent affecting chloroplast function, and (4) quantification of plastid DNA content. From these results, we will discuss the involvement of RPOTmp in the expression of plastidic translation system.

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Chloroplasts have their own genome and genetic systems derived from endosymbiosis of ancestral cyanobacteria. However, during long evolution, chloroplasts lost their autonomy and most regulatory systems became under nuclear control. We here used the primitive, unicellular red alga Cyanidioschyzon merolae, which shows ancestral characteristics on many aspects including chloroplast genome and transcription systems. Therefore, transcription in C.merolae can be regulated more autonomously than that in higher plants.
To understand light-dependent transcription regulation in C.merolae chloroplasts, we performed run-on transcription and ChIP analyses. We found that the chloroplast two-component system, which is composed of the unique histidine kinase(HIK) and one of chloroplast-encoded response regulators(Ycf27), is involved in this regulation. In addition, we performed these assays using photosynthetic electron transport inhibitors, and showed that redox level of plastoquinone did not affect this regulation. Including structural characteristics of HIK, the system for light-responsive transcription in C.merolae is discussed.

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Replication of plastid DNA (ptDNA) is essential for self-duplication of plastid. To investigate cis-acting element and mode for replication, intermediates of ptDNA replication were isolated from the liverwort, Marchantia polymorpha L., suspension-culture cell. No replication bubble intermediate was detected by two-dimensional agarose gel electrophoresis, which indicates the absence of fixed active replication origin in the ptDNA. Replication forks proceeding in the direction opposite to that of rRNA transcription were accumulated at the boundary regions between inverted repeats (IR) and short single copy region. Furthermore, recombination intermediates were also detected in the same region. These intermediates disappeared after displacement of the region, demonstrating that the regions contain cis-acting elements which serve as replication fork barrier (RFB) or sites for homologous recombination. The roll of RFB may be regulation of DNA replication mediated by homologous recombination, or prevention of collision between the transcription apparatus of the rRNA operon and the replication machinery.

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Rubisco and stroma-localized fluorescent proteins of chloroplasts are mobilized to the vacuole via RCBs by an ATG-dependent autophagic process. In this study, we analyzed factors that affect the RCB-creation. RCBs are accumulated in the vacuole when excised leaves are incubated in darkness and nutrient-free medium with concanamycin A. The addition of MS medium containing sucrose remarkably suppressed the RCB-creation. Sucrose was the main constituent which suppresses the RCB-creation in MS medium. The RCB-creation was also suppressed in the light, but the suppression was retarded by DCMU. The content of soluble sugars and starch increased in the light, but decreased in darkness. These results suggested that the sugar contents in leaves were the main factors that affect the RCB-creation. We also found that the light specifically suppressed the RCB-creation, but not the other autophagosome-creation. These results indicated the presence of mechanism which specifically controls the RCB-creation in autophagy.

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During leaf senescence, chloroplast proteins are degraded as a major source of nitrogen for new growth, while chloroplasts gradually shrink and decrease in number. Autophagy is a bulk degradation of the cytosol and organelles. We previously demonstrated that vesicles called Rubisco-containing bodies (RCB) are produced by chloroplasts and transported into the vacuole by autophagy. In this study, we investigated the chloroplasts degradation pathway by autophagy in detail.
Using wild-type and autophagy defected mutant Arabidopsis, individually darkened leaves were used to observe senescence and chloroplast degradation. During the 5-d treatment, the number and size of chloroplasts decreased in wild-type, while the number remained constant and the size decrease was suppressed in the mutant. In the vacuole of wild-type cells, whole chloroplasts accumulated as well as RCB. These results demonstrate the two distinct chloroplast degradation pathways by autophagy, one for whole chloroplasts and one for RCB.

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To better understand the mechanism of phytochrome-mediated signaling, we used a basal land plant, the liverwort Marchantia polymorpha L. M. polymorpha has a single phytochrome gene, MpPHY, of which product, Mpphy, shows red/far-red photoreversibility in vitro. It has been also shown that a single amino-acid substitution in Mpphy, MpphyY241H, eliminated photoreversibility. In WT, red light inhibited cell elongation and promoted cell division after spore germination, rhizoid formation and regeneration of thalli, while far-red light canceled the effects. In transgenic plants expressing MpphyY241H, formation of rhizoid and constitutive thallus regeneration were observed regardless of light condition. Unlike WT, RNAi line of MpPHY showed transition to reproductive phase in a far-red independent manner. These results indicate that Mpphy regulates photomorphogenesis throughout the lifecycle in M. polymorpha, and that M. polymorpha can be a model plant for analyzing signaling mechanism mediated by phytochrome.

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PHYTOCHROME INTERACTING FACTOR 3 (PIF3) interacts with the active phytochrome B (phyB). To identify mutations in the phyB N-terminus that disrupt this interaction, we developed a yeast reverse-hybrid screen. Fifteen mutations identified in this screen, or in previous genetic screens for Arabidopsis mutants showing reduced sensitivity to red light, were shown to disrupt light-induced binding of phyB to PIF3 in in vitro binding assays. These phyB mutations fall into two classes: Class I (11 mutations) containing those defective in light perception, and Class II (4 mutations) containing those normal in light perception, but defective in binding to PIF3. A bioinformatics comparison of phyB to BphP, for which a crystal structure has been solved, predicts that three of the four Class II mutated residues are solvent exposed in a cleft between PAS and GAF domains, suggesting that these residues could be directly required for the physical interaction of phyB with PIF3.

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In higher plants, light and gravity are important parameters in seedling development. PKS1 was identified as a phytochrome interacting factor. In Arabidopsis, PKS1 is a member of a small gene family (PKS1-4) and pks mutants show abnormal phototropism. Moreover, We had shown that PKS1 interacts with PHOT1 and NPH3.
In this study, we show that hypocotyl growth orientation is more complex phenomenon than previously anticipated. Red light inhibits re-orientation of the hypocotyl growth axis when applied from the time of germination. However when applied to hypocotyls undergoing negative gravitropism, red light leads to a deviation from strait growth. Both red light effects require phytochrome signaling and PKS gene family. Moreover, the results of phototropism in pksphot1 and pksphyA combinations suggested that PKS proteins mainly act in the phot1 pathway. We thus propose that PKS proteins may coordinate hypocotyl growth orientation between phytochrome and phot1 dependent responses under low blue light.

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Earlier studies of phototropic fluence-response curves with maize coleoptiles identified first and second pulse-induced phototropisms and time-dependent phototropism (Iino, 1990). In the present study, we analyzed the phototropic fluence-response curve of rice coleoptiles and investigated causal relationships between the phototropin signaling pathway and the components resolved in fluence-response curves. Owing to weak phototropic responsiveness, it has been difficult to analyze pulse-induced phototropism in rice coleoptiles (Neumann and Iino, 1997). We were able to obtain a full phototropic fluence-response curve using lazy1 mutants (Yoshihara and Iino, 2007), which show reduced gravitropism. In this mutant, first and second pulse-induced phototropisms and time-dependent phototropism were found and an additional pulse-induced response was identified in low fluences. By using double mutants generated by crossing lazy1 and cpt1 (Haga and Iino, 2005), it was uncovered that all the multiphasic fluence-response components are dependent on phototropin signaling.

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Phototropin (phot) is a blue light photoreceptor in plants that mediates phototropism, chloroplast relocation, stomata opening. Arabidopsis has two isoforms, phot1 and phot2. A phot molecule has two photoreceptive domains named LOV1 and LOV2 binding one FMN and a Ser/Thr kinase domain. Phot is thought to be a ligh-tregulated protein kinase.
From our studies, two important amino acids involved in photoreaction of FMN and conformational changes in the LOV domains have been found. Molecular-dynamics calculation and FTIR data propose the involvements of Arg interacting with phosphate grop of FMN and Gln nearby FMN, respectively, in the intramolecuar signal transduction. We have introduced the Arg-Lys or the Gln-Lue mutation into the LOV1, LOV2 or both LOV1 and LOV2 of Arabidopsis phot2 and examined the effect on phototropic and chloroplast avoidance responses. We found that these amino acids in the LOV2 domain play crucial roles in these physiological responses.

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Chloroplast relocations are induced in response to light conditions. Chloroplasts move toward weak light (accumulation response) and avoid from strong light (avoidance response). The responses have been reported to be essential for plant survival. To date, we have screened Arabidopsis mutants and have identified several genes involved in chloroplast relocations. In this study, we isolated 2 novel mutants, web1 and web2, defective of avoidance response. Velocity of chloroplast avoidance movement in web1 and web2 were slower than that in wild-type. Both corresponding genes encode coiled-coil proteins, and their expression patterns were closely similar. Interaction between WEB1 and WEB2 were found by the yeast two-hybrid analysis. GFP-fused WEB1 localized to cell periphery and slightly in cytosol. GFP-fused WEB2 localized only in cytosol. Bimolecular fluorescence complementation analysis revealed their interaction in cytosol. These results suggest that cytosolic WEB1-WEB2 complex modulates velocity of chloroplast avoidance movement in Arabidopsis.

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Abscisic acid (ABA) inhibits blue light-dependent stomatal opening and promotes stomatal closure under drought stress in daytime. Here we show that phosphatidic acid (PA), a second messenger of ABA signaling, inhibits the signal transduction that leads to the activation of the plasma membrane H⁺-ATPase in response to blue light. PA as well as ABA inhibited both the blue light-dependent stomatal opening and the H⁺ pumping. These ABA-mediated inhibitions were partially restored by 1-butanol, an inhibitor of PA production. PA also inhibited the blue light-dependent phosphorylation of the H⁺-ATPase without affecting that of phototropins, blue light receptors for stomatal opening. Furthermore, PA bound to and inhibited the protein phosphatase 1 (PP1), a positive regulator of the blue light signaling. Taken together, these results suggest that PA is involved in the ABA-mediated inhibition of blue light-dependent stomatal opening through inhibiting signaling component(s) between phototropins and the H⁺-ATPase, possibly PP1.

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Phototropins act as blue light receptors for phototropism, chloroplast relocation, stomatal opening and leaf expansion. In the blue light-induced stomatal opening, blue light signal perceived by phototropins induces activation of the plasma membrane H⁺-ATPase through the phosphorylation of the C-terminus with subsequent binding of the 14-3-3 protein and stomatal opening. However, signaling pathway between phots and H⁺-ATPase is largely unknown. In this study, we performed the screening to isolate the suppressor mutants for closed stomata phenotype in phot1 phot2 double mutant in Arabidopsis. One of the isolated mutants, named scs1 (suppressor mutant for closed stomata phenotype in phot1 phot2), showed open stomata phenotype, but this mutant was sensitive to abscisic acid. Interestingly, the H⁺-ATPase in guard cells of scs1 showed constitutively high activity state, therefore, this mutant is likely to show the opened stomata phenotype. Now, we are trying to identify SCS1 locus by map-based cloning.

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Previous analysis of the mutants of Synechococcus elongatus PCC 7942 defective in glycogen biosynthesis indicated that the storage polysaccharide has the significant role for the viability under high-salt and oxidative stresses. In the present study, mutants of phosphorylase (glgP), debranching enzyme (glgX) and α-1,4-glucanotransferase (malQ) have been constructed and characterized for the catabolic activity of glycogen accumulated in the cell.
The cellular content of glycogen in wild type (WT) cells remained constant throughout the diurnal cycles (12 h/12 h) of the growth and very little decrease was observed in the dark period. In contrast, the glycogen breakdown was highly stimulated when WT cells were exposed to 0.2 M NaCl in the continuous darkness. Under the same conditions, the activity of glycogen degradation in the glgP, glgX and malQ mutants was markedly reduced as compared to WT. It is therefore concluded that these enzymes are responsible for the glycogen catabolism in cyanobacteria.

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The cyanobacterial genomes contain two homologues of the groEL gene, groEL1 and groEL2. The groEL1 gene forms an operon with groES whereas groEL2 is not accompanied by groES. Plant chloroplasts also possess two types of Cpn60 which are GroEL homologues, known to form a hetero-oligomeric assembly. The present study is being conducted to unveil whether the cyanobacterial GroELs can also show a hetero-oligomeric assembly. Native polyacrylamide gel electrophoresis showed that in Synechococcus elongatus PCC 7942, unlike in E. coli, GroEL1 and GroEL2 do not form a tetradecamer.. In vitro the cyanobacterial GroEL1 formed smaller oligomers such as a heptamer, while GroEL2 formed only a trimer. To investigate hetero-oligomeric interaction, a pull-down assay was conducted using Thermosynechococcus elongatus His-GroEL1 and S.vulcanus GroEL1 or GroEL2. The His-GroEL1 formed a hetero-oligomer with the GroEL2. The distinct in vitro properties of the cyanobacterial oligomers implicate physiological significance which may be required under environmental stress.

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More than 80 non-coding RNAs (ncRNAs) have been identified in Escherichia coli while a few ncRNAs are known in cyanobacteria. In our laboratory, the entire genome of a unicellular cyanobacterium Synechococcus elongatus PCC 6301 (hereafter PCC 6301) was sequenced and 2,525 potential protein-coding genes and 55 RNA genes were assigned. To identify novel ncRNA genes in the PCC 6301 genome, we compared the intergenic regions of the PCC 6301 genome with three freshwater cyanobacteria, Synechocystis sp. PCC 6803, Nostoc sp. PCC 7120 and Thermosynechococcus elongatus BP-1 genomes. This analysis revealed three putative ncRNA genes. One was located between syc2263_d (guaB) and syc2264_d (trxA). We have already reported as Yfr1 RNA (Nakamure et al. PCP 2007). The other two were located in the downstream of syc0182_d (rluA) and in the downstream of syc0894_d (rplL).

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The CmpR protein of Synechococcus elongatus is a homologue of CbbR, the transcription regulator of the cbb operon of photosynthetic and chemosynthetic bacteria. It is required for low-CO₂-induced activation of the cmpABCD operon encoding the ABC bicarbonate transporter. CmpR seems to involve 2-phosphoglycolate (2-PG) as a co-inducer, because its binding to the cmpA regulatory region is strongly enhanced by 2-PG. Under high-CO₂ conditions, by contrast, CmpR acted as a repressor of the low-CO₂-inducible operon ndhF3D3chpY. Gel shift assays revealed two CmpR-binding regions in the ndhF3 upstream region, one of which was 2-PG-independent and the other was 2-PG-dependent. The former was located near the promoter, suggesting its role in repression of transcription, whereas the latter was located upstream from the promoter and was likely to be involved in de-repression of transcription under low-CO₂. Thus, CmpR has different modes of action for activation and repression of gene expression.

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The psaAB genes, which encode photosystem I reaction center subunit, are the critical target of regulation in environmental responses of at least cyanobacteria. We previously reported that the Rrf2-type transcriptional regulator Slr0846 regulates psaAB expression in Synechocystis sp. PCC 6803. On the other hand, the OmpR-type response regulator RpaB, which is involved in various high-light responses, also binds to the psaAB promoter. Meanwhile, target genes of RpaA, which has strong sequence homology to the RpaB, are unknown. In this study, we examined the binding of His-tagged RpaA to the psaAB promoter by EMSA and DNaseI footprinting assay. It was found that RpaA binds to one of the three RpaB binding motifs in the psaAB promoter, although the binding affinity of RpaA was weaker than that of RpaB. To further study the role of RpaA for the psaAB transcription in vivo, we constructed RpaA deletion and over-expression strains.

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We recently demonstrated a self-sustainable robust circadian oscillation of KaiC phosphorylation by reconstituting KaiA, KaiB and KaiC proteins with ATP in vitro. The temperature-compensated ATPase activity of KaiC oscillates in a circadian manner in vitro. The activities of KaiC mutant variants without KaiA and KaiB were directly proportional to their cycle frequencies, indicating that the ATPase activity defines the circadian oscillation period. The mechanism of circadian clock is intrinsic to KaiC itself. The crystal structure of hexameric KaiC revealed that ATP molecules are wedged between individual KaiC subunits and that two phosphorylation sites are near the ATP binding regions. We will discuss the temperature compensation of ATPase activity and the conformational change of ATP-binding domain of KaiC.

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