Yoko Ishizaki

Eukaryotic-type plastid nucleoid protein pTAC3 is essential for transcription by the bacterial-type plastid RNA polymerase

Plastid transcription is mediated by two distinct types of RNA polymerases (RNAPs), bacterial-type RNAP (PEP) and phage-type RNAP (NEP). Recent genomic and proteomic studies revealed that higher plants have lost most prokaryotic transcription regulators and have acquired eukaryotic-type proteins during plant evolution. However, in vivo dynamics of chloroplast RNA polymerases and eukaryotic-type plastid nucleoid proteins have not been directly characterized experimentally. Here, we examine the association of the α-subunit of PEP and eukaryotic-type protein, plastid transcriptionally active chromosome 3 (pTAC3) with transcribed regions in vivo by using chloroplast chromatin immunoprecipitation (cpChIP) assays. PEP α-subunit preferentially associates with PEP promoters of photosynthesis and rRNA genes, but not with NEP promoter regions, suggesting selective and accurate recognition of PEP promoters by PEP. The cpChIP assays further demonstrate that the peak of PEP association occurs at the promoter-proximal region and declines gradually along the transcribed region. pTAC3 is a putative DNA-binding protein that is localized to chloroplast nucleoids and is essential for PEP-dependent transcription. Density gradient and immunoprecipitation analyses of PEP revealed that pTAC3 is associated with the PEP complex. Interestingly, pTAC3 associates with the PEP complex not only during transcription initiation, but also during elongation and termination. These results suggest that pTAC3 is an essential component of the chloroplast PEP complex. In addition, we demonstrate that light-dependent chloroplast transcription is mediated by light-induced association of the PEP–pTAC3 complex with promoters. This study illustrates unique dynamics of PEP and its associated protein pTAC3 during light-dependent transcription in chloroplasts.

Ferritin 2 domain-containing protein found in lacquer tree (Toxicodendron vernicifluum) sap has negative effects on laccase and peroxidase reactions

Lacquer tree sap, a raw material of traditional paints in East Asia, is hardened through laccase-catalyzed oxidation and the following polymerization of phenolic compound urushiol. In the sap’s water-insoluble fraction, we found two plantacyanins and a ferritin 2 domain-containing protein (TvFe2D, a homolog of Arabidopsis AT1G47980 and AT3G62730). The recombinant TvFe2D protein suppressed the accumulation of laccase-catalyzed oxidation products of a model substrate syringaldazine without decreasing oxygen consumption, the second substrate of laccase. The suppression was also observed when another substrate guaiacol or another oxidizing enzyme peroxidase was used. The functional domain of the suppression was the C-terminal half, downstream of the ferritin 2 domain. The results suggest that this protein may be involved in regulating the sap polymerization/hardening. We also discuss the possibility that homologous proteins of TvFe2D in other plants might be involved in the laccase- or peroxidase-mediated polymerization of phenolic compounds, such as lignin and flavonoids. Graphical abstract Sap exuded from the wound site of lacquer tree. The ferritin 2 domain protein in it is a novel candidate regulating urushiol polymerization/hardening.

Cacao plantations on Sulawesi Island, Indonesia: I—an agro-ecological analysis of conventional and organic farms

The cacao plantation largely contributes to the Indonesian agricultural economy, and the systems with less environment impact have become fundamental for the local farmers. The current research made a general agro-ecological evaluation of six cacao farms in Sulawesi-Indonesia cultivated under conventional systems and organic management, here referred to as environmental friendly systems (EFS). Ten agro-ecological parameters, the number of fruits per area, the rate of infection (RI), number of seeds per fruit, and seed weight per fruit were evaluated. Furthermore, plant mineral nutrition was also analyzed, including the estimation of diagnosis recommendation integrated systems. The overall data indicated that RI in the conventional systems was lower than that in the EFS. The density of dead plant materials showed negative correlations with the area of weeds and density of weeds. The density of dead plant materials, concentrations of Mg and Fe showed a positive correlation with productivity. In addition, Fe showed negative correlations with weed area and density. The nutrient balance index also showed that in Parigi and Palolo areas, the plants were well stabled in terms of mineral nutrition. These results suggest that some agro-ecological parameters can function as cacao production indicators, especially the biomass and dead leaves together with plant mineral nutrition (Mg and Fe).

Diversity of chloroplast genome among local clones of cocoa (Theobroma cacao, L.) from Central Sulawesi

Chloroplast genomes typically range in size from 120 to 170 kilo base pairs (kb), which relatively conserved among plant species. Recent evaluation on several species, certain unique regions showed high variability which can be utilized in the phylogenetic analysis. Many fragments of coding regions, introns, and intergenic spacers, such as atpB-rbcL, ndhF, rbcL, rpl16, trnH-psbA, trnL-F, trnS-G, etc., have been used for phylogenetic reconstructions at various taxonomic levels. Based on that status, we would like to analysis the diversity of chloroplast genome within species of local cacao (Theobroma cacao L.) from Central Sulawesi. Our recent data showed, there were more than 20 clones from local farming in Central Sulawesi, and it can be detected based on phenotypic and nuclear-genome-based characterization (RAPD- Random Amplified Polymorphic DNA and SSR- Simple Sequences Repeat) markers. In developing DNA marker for this local cacao, here we also included analysis based on the variation of chloroplast g...

Comparative Analysis of Chloroplast psbD Promoters in Terrestrial Plants

The transcription of photosynthesis genes encoded by the plastid genome is mainly mediated by a prokaryotic-type RNA polymerase called plastid-encoded plastid RNA polymerase (PEP). Standard PEP-dependent promoters resemble bacterial sigma-70-type promoters containing the so-called -10 and -35 elements. On the other hand, an unusual light- and stress-responsive promoter (psbD LRP) that is regulated by a 19-bp AAG-box immediately upstream of the -35 element has been mapped upstream of the psbD-psbC operon in some angiosperms. However, the occurrence of the AAG-box containing psbD LRP in plant evolution remains elusive. We have mapped the psbD promoters in eleven embryophytes at different evolutionary stages from liverworts to angiosperms. The psbD promoters were mostly mapped around 500–900 bp upstream of the psbD translational start sites, indicating that the psbD mRNAs have unusually long 5′-UTR extensions in common. The -10 elements of the psbD promoter are well-conserved in all embryophytes, but not the -35 elements. We found that the AAG-box sequences are highly conserved in angiosperms and gymnosperms except for gnetaceae plants. Furthermore, partial AAG-box-like sequences have been identified in the psbD promoters of some basal embryophytes such as moss, hornwort, and lycophyte, whereas liverwort has the standard PEP promoter without the AAG-box. These results suggest that the AAG-box sequences of the psbD LRP may have evolved from a primitive type of AAG-box of basal embryophytes. On the other hand, monilophytes (ferns) use another type of psbD promoter composed of a distinct cis-element upstream of the potential -35 element. Furthermore, we found that psbD expression is not regulated by light in gymnosperms or basal angiosperms, although they have the well-conserved AAG-box sequences. Thus, it is unlikely that acquisition of the AAG-box containing psbD promoter is directly associated with light-induced transcription of the psbD-psbC operon. Light- and stress-induced transcription may have evolved independently and multiple times during terrestrial plant evolution.

Identification of a Chloroplast-Localized SAP Domain Containing Protein in Arabidopsis thaliana

Plastids are plant-specific organelles that originated from cyanobacteria, and possess their own genome and generic system. In higher plants, plastids may differentiate into several forms, such as chloroplasts and amyloplasts. Unlike cyanobacteria, plastid genes in higher plants are transcribed by two distinct RNA polymerases: plastid encoded eubacterial-type RNA Polymerase (PEP) and nuclear encoded phage-type RNA Polymerase (NEP). It has been established that plastid transcription plays an important role in plastid differentiation. However, molecular mechanism underlying plastid differentiation is still unclear. To identify genes involved in nuclear control of plastid differentiation, we screened for albino mutants in RIKEN phenome database and found 12 candidates. Among them, we focused on At3g04260, since it is predicted to be localized in chloroplasts and has been identified in several plastid transcription complex proteomes. At3g04260 contains a SAP domain that is involved in DNA binding in eukaryotic matrix attachment region binding proteins. Transient and Gel shift assays suggested that the N-terminal sequences of At3g04260 act as a plastid transit peptide and that its SAP domain had DNA binding activity. To understand the role of At3g04260 in plastid differentiation, we isolated T-DNA insertion mutant of At3g04260. As expected, the mutant showed an albino phenotype. The tilling microarray analysis showed that expression of all PEP-dependent genes including photosynthesis and rRNA genes were significantly reduced, however NEP-dependent transcripts were not decreased in the mutant plants. These results suggested that the plastid localized DNA binding protein, At3g04260 is involved in the regulation of plastid gene expression during chloroplast development.