CyanoBase: a large-scale update on its 20th anniversary.

The first ever cyanobacterial genome sequence was determined two decades ago and CyanoBase (http://genome.microbedb.jp/cyanobase), the first database for cyanobacteria was simultaneously developed to allow this genomic information to be used more efficiently. Since then, CyanoBase has constantly been extended and has received several updates. Here, we describe a new large-scale update of the database, which coincides with its 20th anniversary. We have expanded the number of cyanobacterial genomic sequences from 39 to 376 species, which consists of 86 complete and 290 draft genomes. We have also optimized the user interface for large genomic data to include the use of semantic web technologies and JBrowse and have extended community-based reannotation resources through the re-annotation of Synechocystis sp. PCC 6803 by the cyanobacterial research community. These updates have markedly improved CyanoBase, providing cyanobacterial genome annotations as references for cyanobacterial research.

Abscisic Acid Participates in the Control of Cell Cycle Initiation Through Heme Homeostasis in the Unicellular Red Alga Cyanidioschyzon merolae.

ABA is a phytohormone that is synthesized in response to abiotic stresses and other environmental changes, inducing various physiological responses. While ABA has been found in unicellular photosynthetic organisms, such as cyanobacteria and eukaryotic algae, its function in these organisms is poorly understood. Here, we found that ABA accumulated in the unicellular red alga Cyanidioschyzon merolae under conditions of salt stress and that the cell cycle G1/S transition was inhibited when ABA was added to the culture medium. A gene encoding heme-scavenging tryptophan-rich sensory protein-related protein (CmTSPO; CMS231C) was positively regulated by ABA, as in Arabidopsis, and CmTSPO bound heme in vitro. The intracellular content of total heme was increased by addition of ABA, but unfettered heme decreased, presumably due to scavenging by CmTSPO. The inhibition of DNA replication by ABA was negated by addition of heme to the culture medium. Thus, we propose a regulatory role for ABA and heme in algal cell cycle initiation. Finally, we found that a C. merolae mutant that is defective in ABA production was more susceptible to salt stress, indicating the importance of ABA to stress resistance in red algae.

Overproduction of stromal ferredoxin:NADPH oxidoreductase in H2O 2-accumulating Brassica napus leaf protoplasts.

The isolation of Brassica napus leaf protoplasts induces reactive oxygen species generation and accumulation in the chloroplasts. An activated isoform of NADPH oxidase-like protein was detected in the protoplasts and the protoplast chloroplasts. The purpose of this study is to define the NADH oxidase-like activities in the H2O2-accumulating protoplast chloroplasts. Proteomic analysis of this protein revealed an isoform of ferredoxin:NADPH oxidoreductase (FNR1). While leaves highly expressed the LFNR1 transcript, protoplasts decreased the expression significantly. The protoplast chloroplasts predominantly expressed soluble FNR1 proteins. While the albino leaves of white kale (Brassica oleracea var. acephala f. tricolor cv. white pigeon) expressed FNR1 protein at the same level as B. napus leaves, the protoplasts of albino leaves displayed reduced FNR1 expression. The albino leaf protoplasts of white kale generated and accumulated H2O2 in the cytoplasm and on the plasma membrane. Intracellular pH showed that the chloroplasts were acidic, which suggest that excess H(+) was generated in chloroplast stroma. NADPH content of the protoplast chloroplasts increased by over sixfold during the isolation of protoplasts. This study reports a possibility of mediating electrons to oxygen by an overproduced soluble FNR, and suggests that the FNR has a function in utilizing any excess reducing power of NADPH.

The circadian regulation of photosynthesis.

Correct circadian regulation increases plant productivity, and photosynthesis is circadian-regulated. Here, we discuss the regulatory basis for the circadian control of photosynthesis. We discuss candidate mechanisms underpinning circadian oscillations of light harvesting and consider how the circadian clock modulates CO2 fixation by Rubisco. We show that new techniques may provide a platform to better understand the signalling pathways that couple the circadian clock with the photosynthetic apparatus. Finally, we discuss how understanding circadian regulation in model systems is underpinning research into the impact of circadian regulation in crop species.

RpaB, another response regulator operating circadian clock-dependent transcriptional regulation in Synechococcus elongatus PCC 7942.

The circadian clock of cyanobacteria is composed of KaiA, KaiB, and KaiC proteins, and the SasA-RpaA two-component system has been implicated in the regulation of one of the output pathways of the clock. In this study, we show that another response regulator that is essential for viability, the RpaA paralog, RpaB, plays a central role in the transcriptional oscillation of clock-regulated genes. In vivo and in vitro analyses revealed that RpaB and not RpaA could specifically bind to the kaiBC promoter, possibly repressing transcription during subjective night. This suggested that binding may be terminated by RpaA to activate gene transcription during subjective day. Moreover, we found that rpoD6 and sigF2, which encode group-2 and group-3 σ factors for RNA polymerase, respectively, were also targets of the RpaAB system, suggesting that a specific group of σ factors can propagate genome-wide transcriptional oscillation. Our findings thus reveal a novel mechanism for a circadian output pathway that is mediated by two paralogous response regulators.

Characterization of four nuclear-encoded plastid RNA polymerase sigma factor genes in the liverwort Marchantia polymorpha: blue-light- and multiple stress-responsive SIG5 was acquired early in the emergence of terrestrial plants.

The plastids of plant cells each contain their own genome, and a bacterial-type RNA polymerase called plastid-encoded plastid RNA polymerase (PEP) is involved in transcription of this genome. While the catalytic core subunits are encoded by the plastid genome, the specificity subunit of PEP, sigma, is generally encoded by the nuclear genome and imported into plastids from the cytoplasm after translation. In this study, we identified and analyzed four sigma factor genes from the nuclear genome of a liverwort, Marchantia polymorpha. Phylogenetic analysis suggested that three of the four genes were orthologous to vascular plant genes and thus they were named MpSIG1, MpSIG2 and MpSIG5. The remaining gene was named MpSIGX. The gene products were predicted to localize to the plastid, and this prediction was experimentally demonstrated by expressing yellow fluorescent protein fusion genes in vivo. As with SIG5 genes of other plant species, expression of MpSIG5 was induced by blue-light irradiation and also under various stress conditions, indicating that the regulatory mechanism responsible is conserved among divergent plant species. However, while the major role of SIG5 in vascular plants is to repair the damaged PSII reaction center through psbD gene transcription, the relevant blue-light-responsive promoter (psbD-BLRP) was not found in M. polymorpha and psbD transcript accumulation did not occur in conjunction with MpSIG5 induction. Thus, the physiological role of SIG5 is probably divergent among plant phyla.

Mitochondrial localization of ferrochelatase in a red alga Cyanidioschyzon merolae.

Ferrochelatase (FECH) is an essential enzyme for the final step of heme biosynthesis. In green plants, its activity has been reported in both plastids and mitochondria. However, the precise subcellular localization of FECH remains uncertain. In this study, we analyzed the localization of FECH in the unicellular red alga, Cyanidioschyzon merolae. Immunoblot and enzyme activity analyses of subcellular fractions localized little FECH in the plastid. In addition, immunofluorescence microscopy identified that both intrinsic and hemagglutinin (HA)-tagged FECH are localized in the mitochondrion. We therefore conclude that FECH is localized in the mitochondrion in C. merolae.

Plastid-to-nucleus retrograde signals are essential for the expression of nuclear starch biosynthesis genes during amyloplast differentiation in tobacco BY-2 cultured cells.

Amyloplasts, a subtype of plastid, are found in nonphotosynthetic tissues responsible for starch synthesis and storage. When tobacco (Nicotiana tabacum) Bright Yellow-2 cells are cultured in the presence of cytokinin instead of auxin, their plastids differentiate from proplastids to amyloplasts. In this program, it is well known that the expression of nucleus-encoded starch biosynthesis genes, such as ADP-Glucose Pyrophosphorylase (AgpS) and Granule-Bound Starch Synthase (GBSS), is specifically induced. In this study, we investigated the roles of plastid gene expression in amyloplast differentiation. Microarray analysis of plastid genes revealed that no specific transcripts were induced in amyloplasts. Nevertheless, amyloplast development accompanied with starch biosynthesis was drastically inhibited in the presence of plastid transcription/translation inhibitors. Surprisingly, the expression of nuclear AgpS and GBSS was significantly repressed by the addition of these inhibitors, suggesting that a plastid-derived signal(s) that reflects normal plastid gene expression was essential for nuclear gene expression. A series of experiments was performed to examine the effects of intermediates and inhibitors of tetrapyrrole biosynthesis, since some of the intermediates have been characterized as candidates for plastid-to-nucleus retrograde signals. Addition of levulinic acid, an inhibitor of tetrapyrrole biosynthesis, resulted in the up-regulation of nuclear AgpS and GBSS gene expression as well as starch accumulation, while the addition of heme showed opposite effects. Thus, these results indicate that plastid transcription and/or translation are required for normal amyloplast differentiation, regulating the expression of specific nuclear genes by unknown signaling mechanisms that can be partly mediated by tetrapyrrole intermediates.

SIG1, a sigma factor for the chloroplast RNA polymerase, differently associates with multiple DNA regions in the chloroplast chromosomes in vivo.

Chloroplasts have their own DNA and gene expression systems. Transcription in chloroplasts is regulated by two types of RNA polymerase, nuclear-encoded plastid RNA polymerase (NEP) and plastid-encoded plastid RNA polymerase (PEP), and multiple sigma factors for PEP. To study transcriptional regulation in chloroplasts, a molecular genetic approach has extensively been used. However, this method may include indirect effects, and it cannot be applied to the analysis of factors essential to survival. These limitations make understanding specific regulation by transcription factors difficult. Chromatin immunoprecipitation (ChIP) is a powerful and useful tool for obtaining information on transcription-factor binding sites; it can directly detect dynamic changes in their interaction patterns in vivo. To further understand transcriptional regulation in chloroplasts, we here established a ChIP-based method in Arabidopsis thaliana and analyzed the binding pattern of a chloroplast sigma factor, SIG1. We found that SIG1 specifically binds to newly identified target promoters as well as to a set of promoters of genes whose mRNA expression is dependent on OsSIG1 in rice and that this binding changed in response to high-light stress. These results suggested that the ChIP-based approach is very useful in understanding transcriptional regulation of chloroplast genes and can overcome several problems posed by conventional methods.

RpaB, an essential response regulator for high-light stress, is extensively involved in transcriptional regulation under light-intensity upshift conditions in Synechococcus elongatus PCC 7942.

In cyanobacteria, transcription of a set of genes is specifically induced by high-light-stress conditions. In previous studies, RpaB, a response regulator of the two-component system, was shown to be involved in this regulation in vitro and in vivo. In this study, we examined whether RpaB-dependent transcriptional regulation was extensively observed, not only under high-light-stress conditions but also under various light intensities. Transcription of high-light-dependent genes hliA, nblA and rpoD3 was transiently and drastically induced during a dark-to-light shift in a manner similar to high-light-stress responses. Moreover, expression of these genes was activated under various light-intensity upshift conditions. Phos-tag SDS-PAGE experiments showed that the phosphorylation level of RpaB was decreased along with transcriptional induction of target genes in all of the light environments examined herein. These results suggest that RpaB may be widely involved in transcriptional regulation under dark-to-light and light-intensity upshift conditions and that high-light-responsive genes may be required in various light conditions other than high-light condition. Furthermore, it is hypothesised that RpaB is regulated by redox-dependent signals rather than by high-light-stress-dependent signals.

Dynamics of RpaB-promoter interaction during high light stress, revealed by chromatin immunoprecipitation (ChIP) analysis in Synechococcus elongatus PCC 7942.

In cyanobacteria, a series of genes are induced by, and cause tolerance to, high light stress conditions. Some of these genes share a short, repeated sequence motif known as a high light regulatory 1 (HLR1) element in their promoter regions. Previously, RpaB, a two-component response regulator, was shown to interact with the HLR1 element of several high light-responsive promoters in vitro. However, how RpaB regulates target promoters in vivo remained elusive. In this study, we analyzed the role of RpaB in transcriptional regulation of high light-responsive genes by chromatin immunoprecipitation (ChIP) analysis, which has been recently developed and utilized to study in vivo interactions between DNA-binding proteins and the relevant target DNA. One of the advantages of this method is the ability to detect dynamic interaction patterns in response to various growth and/or environmental conditions instantaneously at the time of the analysis. Here we examined the binding patterns of RpaB under various light conditions using ChIP assays. We found that strong interactions of RpaB with target promoters were weakened in a high light-dependent manner, and that the lower binding level of RpaB continued as long as the high light conditions were maintained. Thus, in regulation of high light-inducible genes, we suggest that RpaB functions as a repressor under normal light conditions, and that high light conditions result in release of the repression.

Drying the leaves of Perilla frutescens increases their content of anticancer nutraceuticals.

A regular intake of plant-derived bioactive agents has gained popularity because of the health benefits. Fresh leafy greens, however, normally have a low concentration of such bioactive agents. In this study, we found that drying markedly affected the accumulation of secondary metabolites and that dried leaves of Perilla frutescens L. (perilla) contained more anticancer flavonoids than fresh leaves. Drying is a major method of food preparation, particularly for plant-based foods, but the quality of the bioactive agents contained in the fresh and dried leaves of perilla has received only scant attention. Quantitative analysis of the concentrations of perillaldehyde, rosmarinic acid, apigenin, luteolin, 4-hydroxyphenyllactic acid, and 4-coumaric acid, some of which are known as nutraceuticals, revealed that the effect of drying significantly increased apigenin (28-fold) and luteolin (86-fold), but decreased rosmarinic acid in all leaf stages. We examined the positive effect on flavonoid levels on perilla leaves and confirmed that, by comparison with fresh perilla leaves, the dried leaves contained greater concentrations of anticancer flavonoids regardless of variety, form, or manner of cultivation. This indicates that drying can significantly increase the level of flavonoids in perilla leaves without a loss of flavor. Therefore, drying is a simple and effective method to improve the concentrations of bioactive agents, which increases the intake of beneficial substances derived from herbs and edible plants. This finding serves as a method for the supply of raw plant materials rich in bioactive agents that are suitable for labeling as edible nutraceuticals.

The cyanobacterial principal sigma factor region 1.1 is involved in DNA-binding in the free form and in transcription activity as holoenzyme.

Cyanobacterial principal sigma factor, sigma(A), includes a specifically conserved cluster of basic amino acids in the amino-terminal extension called region 1.1. We found that the sigma(A) in a thermophilic cyanobacterium Thermosynechococcus elongatus BP-1 binds DNA in the absence of the core RNA polymerase and that sigma(A) lacking region 1.1 is not able to bind DNA. This indicates that, in the cyanobacterium, region 1.1 participates in DNA-binding, rather than inhibiting the interaction between free sigma and DNA, as found in other principal sigma factors of eubacteria. The results of in vitro transcription assays with the reconstituted RNA polymerase showed that region 1.1 reduces transcription activity from the cpc promoter.

Molecular genetic analysis of chloroplast gene promoters dependent on SIG2, a nucleus-encoded sigma factor for the plastid-encoded RNA polymerase, in Arabidopsis thaliana.

Most photosynthesis-related genes in mature chloroplasts are transcribed by a eubacterial-type RNA polymerase (PEP) whose core subunits are encoded by the plastid genome. It has been shown previously that six putative nuclear genes (SIG1 to SIG6) encode promoter-specificity factors for PEP in Arabidopsis thaliana, and we isolated a T-DNA insertion line of SIG2 (sig2-1 mutant) that manifests aberrant chloroplast development. With the use of S1 nuclease protection and primer extension analyses, we have now characterized the SIG2-dependent chloroplast promoters in A.thaliana. The amounts of transcripts derived from one of the multiple psbD promoters (psbD -256) and from the promoters of two tRNA genes (trnE-UUC and trnV-UAC) were markedly and specifically decreased in the sig2-1 mutant. The abundance of these transcripts was restored to wild-type levels by introduction into the mutant of a SIG2 transgene. The recombinant SIG2 protein mixed with Escherichia coli core RNA polymerase could bind to a DNA fragment that contains the SIG2-dependent psbD -256, trnE-UUC or trnV-UAC promoter. Sequences similar to those of the -35 and -10 promoter elements of E.coli were identified in the regions of the SIG2-dependent chloroplast genes upstream of the transcription initiation sites.

The nuclear-encoded sigma factor SIG4 directly activates transcription of chloroplast psbA and ycf17 genes in the unicellular red alga Cyanidioschyzon merolae.

The plant organelle chloroplast originated from the endosymbiosis of a cyanobacterial-like photosynthetic bacterium, and still retains its own genome derived from this ancestor. We have been focusing on a unicellular red alga, Cyanidioschyzon merolae, as a model photosynthetic eukaryote. In this study, we analyzed the transcriptional specificity of SIG4, which is one of four nuclear-encoded chloroplast RNA polymerase sigma factors in this alga. Accumulation of the SIG4 protein was observed in response to nitrogen depletion or high light conditions. By comparing the chloroplast transcriptomes under nitrogen depletion and SIG4-overexpressing conditions, we identified several candidate genes as SIG4 targets. Together with the results of chromatin immunoprecipitation analysis, the promoters of the psbA (encoding the D1 protein of the photosystem II reaction center) and ycf17 (encoding a protein of the early light-inducible protein family) genes were shown to be direct activation targets. The phycobilisome (PBS) CpcB protein was decreased by SIG4 overexpression, which suggests the negative involvement of SIG4 in PBS accumulation.

Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3708, Which Performs Type II Complementary Chromatic Acclimation.

To explore the variation of the light-regulated genes during complementary chromatic acclimation (CCA), we determined the complete genome sequence of the cyanobacterium Geminocystis sp. strain NIES-3708. Within the light-regulated operon for CCA, we found genes for phycoerythrin but not phycocyanin, suggesting that this cyanobacterium modulates phycoerythrin composition only (type II CCA).

Complete Genome Sequence of Cyanobacterium Geminocystis sp. Strain NIES-3709, Which Harbors a Phycoerythrin-Rich Phycobilisome.

The cyanobacterium Geminocystis sp. strain NIES-3709 accumulates a larger amount of phycoerythrin than the related NIES-3708 strain does. Here, we determined the complete genome sequence of the NIES-3709 strain. Our genome data suggest that the different copy number of rod linker genes for phycoerythrin leads to the different phycoerythrin contents between the two strains.

Sense transgene-induced post-transcriptional gene silencing in tobacco compromises the splicing of endogenous counterpart genes.

Sense transgene-induced post-transcriptional gene silencing (S-PTGS) is thought to be a type of RNA silencing in which ARGONAUTE1 directs the small interfering RNA (siRNA)-mediated cleavage of a target mRNA in the cytoplasm. Here, we report that the altered splicing of endogenous counterpart genes is a main cause for the reduction of their mature mRNA levels. After the S-PTGS of a tobacco endoplasmic reticulum ω-3 fatty acid desaturase (NtFAD3) gene, 3'-truncated, polyadenylated endo-NtFAD3 transcripts and 5'-truncated, intron-containing endo-NtFAD3 transcripts were detected in the total RNA fraction. Although transcription proceeded until the last exon of the endogenous NtFAD3 gene, intron-containing NtFAD3 transcripts accumulated in the nucleus of the S-PTGS plants. Several intron-containing NtFAD3 transcripts harboring most of the exon sequences were generated when an endogenous silencing suppressor gene, rgs-CaM, was overexpressed in the S-PTGS plants. These intron-containing NtFAD3 splice variants were generated in the presence of NtFAD3 siRNAs that are homologous to the nucleotide sequences of these splice variants. The results of this study indicate that the inhibition of endo-NtFAD3 gene expression is primarily directed via the alteration of splicing and not by cytoplasmic slicer activity. Our results suggest that the transgene and intron-containing endogenous counterpart genes are differentially suppressed in S-PTGS plants.

Nuclear-encoded chloroplast RNA polymerase sigma factor SIG2 activates chloroplast-encoded phycobilisome genes in a red alga, Cyanidioschyzon merolae.

The phycobilisome (PBS) is a photosynthetic light-harvesting complex in red algae, whose structural genes are separately encoded by both the nuclear and chloroplast genomes. While the expression of PBS genes in both genomes is responsive to environmental changes to modulate light-harvesting efficiency, little is known about how gene expression of the two genomes is coordinated. In this study, we focused on the four nuclear-encoded chloroplast sigma factors to understand aspects of this coordination, and found that SIG2 directs the expression of chloroplast PBS genes in the red alga Cyanidioschyzon merolae.

Circadian control of chloroplast transcription by a nuclear-encoded timing signal.

Circadian timekeeping in plants increases photosynthesis and productivity. There are circadian oscillations in the abundance of many chloroplast-encoded transcripts, but it is not known how the circadian clock regulates chloroplast transcription or the photosynthetic apparatus. We show that, in Arabidopsis, nuclear-encoded SIGMA FACTOR5 (SIG5) controls circadian rhythms of transcription of several chloroplast genes, revealing one pathway by which the nuclear-encoded circadian oscillator controls rhythms of chloroplast gene expression. We also show that SIG5 mediates the circadian gating of light input to a chloroplast-encoded gene. We have identified an evolutionarily conserved mechanism that communicates circadian timing information between organelles with distinct genetic systems and have established a new level of integration between eukaryotic circadian clocks and organelles of endosymbiotic origin.