Mitsumasa Hanaoka

Structural features of a wheat plastome as revealed by complete sequencing of chloroplast DNA

Abstract. Structural features of the wheat plastome were clarified by comparison of the complete sequence of wheat chloroplast DNA with those of rice and maize chloroplast genomes. The wheat plastome consists of a 134,545-bp circular molecule with 20,703-bp inverted repeats and the same gene content as the rice and maize plastomes. However, some structural divergence was found even in the coding regions of genes. These alterations are due to illegitimate recombination between two short direct repeats and/or replication slippage. Overall comparison of chloroplast DNAs among the three cereals indicated the presence of some hot-spot regions for length mutations. Whereas the region with clustered tRNA genes and that downstream of rbcL showed divergence in a species-specific manner, the deletion patterns of ORFs in the inverted-repeat regions and the borders between the inverted repeats and the small single-copy region support the notion that wheat and rice are related more closely to each other than to maize.

Glutamyl-tRNA mediates a switch in RNA polymerase use during chloroplast biogenesis.

Chloroplast genes of higher plants are transcribed by two types of RNA polymerase that are encoded by nuclear (NEP (nuclear‐encoded plastid RNA polymerase)) or plastid (PEP (plastid‐encoded plastid RNA polymerase)) genomes. NEP is largely responsible for the transcription of housekeeping genes during early chloroplast development. Subsequent light‐dependent chloroplast maturation is accompanied by repression of NEP activity and activation of PEP. Here, we show that the plastid‐encoded transfer RNA for glutamate, the expression of which is dependent on PEP, directly binds to and inhibits the transcriptional activity of NEP in vitro. The plastid tRNAGlu thus seems to mediate the switch in RNA polymerase usage from NEP to PEP during chloroplast development.

Circadian Control of Chloroplast Transcription by a Nuclear-Encoded Timing Signal

Synchronizing Photosynthetic Capacity Coordination of photosynthetic activity with sunlight benefits plant productivity. Noordally et al. (p. 1316) analyzed how the Arabidopsis circadian clock keeps the chloroplasts working in tune with the Sun. SIGMA FACTOR5 (SIG5) is encoded in the cell nucleus and reflects circadian cycles with changes in its own transcript abundance. SIG5 acts, however, in the chloroplast, where it supports photosystem II production. In plants, day/night information is communicated from a nuclear-encoded circadian oscillator to the chloroplast. 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.

DNA Microarray Analysis of Plastid Gene Expression in an Arabidopsis Mutant Deficient in a Plastid Transcription Factor Sigma, SIG2

The plastid genome of higher plants contains more than one hundred genes for photosynthesis, gene expression, and other processes. Plastid transcription is done by two types of RNA polymerase, PEP and NEP. PEP is a eubacteria-type RNA polymerase that is essential for chloroplast development. In Arabidopsis thaliana, six sigma factors (SIG1-6) are encoded by the nuclear genome, and postulated to determine the transcription specificity of PEP. In this study, we constructed a DNA microarray for all of the plastid protein-coding genes, and analyzed the effects of the sig2 lesion on the global plastid gene expression. Of the 79 plastid protein genes, it was found that only the psaJ transcript was decreased in the mutant, whereas transcripts of 47 genes were rather increased. Since many of the up-regulated genes are under the control of NEP, it was suggested that the NEP activity was increased in the sig2-1 mutant.

Chinese spring wheat (Triticum aestivum L.) chloroplast genome: Complete sequence and contig clones

Libraries of plasmid clones covering the entire chloroplast (cp) genome of the common wheat,Triticum aestivum cv. Chinese Spring were constructed and assembled into contig-clones. From these, we obtained the complete nucleotide sequence of wheat chloroplast DNA—a 134,540 bp circular DNA (DDBJ accession no. AB042240) containing four species of ribosomal RNA, 30 genes for 20 species of transfer RNA, and 71 protein coding genes. Additionally, we detected five unidentified open reading frames conserved among grasses. Plasmid clones are available on request.

Induction of a Group 2 σ-Factor, RPOD3, by High Light and the Underlying Mechanism in Synechococcus elongatus PCC 7942

Among the σ70 family bacterial σ factors, group 2 σ factors have similar promoter recognition specificity to group 1 (principal) σ factors and express and function under specific environmental and physiological conditions. In general, the cyanobacterial genome encodes more than four group 2 σ factors, and the unicellular Synechococcus elongatus PCC 7942 (Synechococcus) has five group 2 σ factors (RpoD2–6). In this study, we analyzed expression of group 2 σ factors of Synechococcus at both mRNA and protein levels, and we showed that the rpoD3 expression was activated only by high light (1,500 μmol photons m–2 s–1) among the various stress conditions examined. After high light shift, rpoD3 mRNA accumulated transiently within the first 5 min and diminished subsequently, whereas RpoD3 protein increased gradually during the first several hours. We also found that the rpoD3 deletion mutant rapidly lost viability under the same conditions. Analysis of the rpoD3 promoter structure revealed the presence of an HLR1 (high light-responsive element 1) sequence, which was suggested to be responsible for the high light-induced transcription under the control of the NblS (histidine kinase)-RpaB (response regulator) two-component system (Kappell, A. D., and van Waasbergen, L. G. (2007) Arch. Microbiol. 187, 337–342), at +6 to +23 with respect to the transcriptional start site. Here we demonstrated that recombinant RpaB protein specifically bound to HLR1 of the rpoD3 and hliA genes in vitro, and overexpression of a truncated RpaB variant harboring only the phosphoreceiver domain derepressed the transcription in vivo. Thus, we have concluded that phosphorylated RpaB are repressing the rpoD3 and hliA transcription under normal growth conditions, and the RpaB dephosphorylation induced by high light stress results in transcriptional derepression.

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.

RpaB, Another Response Regulator Operating Circadian Clock-dependent Transcriptional Regulation in Synechococcus elongatus PCC 7942

Background: The circadian output pathway in cyanobacteria is mediated by a two-component system consisting of SasA/RpaA. Results: An additional response regulator, RpaB, directly binds to clock-regulated promoters during the night. Conclusion: RpaB is also a key regulator of the circadian output pathway; RpaA and RpaB function cooperatively. Significance: Clarification of output pathway details is crucial for understanding the circadian clock. 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.

Microarray profiling of plastid gene expression in a unicellular red alga, Cyanidioschyzon merolae

Plastid genomes of red algae contain more genes than those of green plant lineages, and it is of special interest that four transcription factors derived from ancestral cyanobacteria are encoded therein. However, little is known about transcriptional regulation of the red algal plastid genome. In this study, we constructed a red algal plastid DNA microarray of Cyanidioschyzon merolae covering almost all protein coding genes, and found that plastid genes are differentially activated by illumination. Run-on transcription assays using isolated plastids confirmed that activation takes place at the transcriptional level. In bacteria and plants, sigma factors determine the genes that are to be transcribed, and four plastid sigma factors (Cm_SIG1–4) encoded in the nuclear genome of C. merolae may be responsible for differential gene expression of the plastid genome. We found that transcripts for all Cm_SIG genes accumulated transiently after a shift from dark to light, whereas only the Cm_SIG2 transcript was increased after a shift from low to high light, suggesting that Cm_SIG2 is a sigma factor that responds to high light. Phylogenetic analysis of plastid sigma factors suggested that sigma factors of red and green algal plastids and the group 1 sigma factors of cyanobacteria form a monophyletic group.

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.