Search

The development of green energy is important to mitigate global warming. Jatropha (Jatropha curcas L.) is a promising candidate for the production of alternative biofuel, which could reduce the burden on the Earth's resources. Jatropha seeds contain a large quantity of lipids that can be used to produce biofuel, and the rest of the plant has many other uses. Currently, techniques for plant genetic transformation are extensively employed to study, create, and improve the specific characteristics of the target plant. Successful transformation involves the alteration of plants and their genetic materials. The aim of this study was to generate Jatropha plants that can support biofuel production by increasing their seed size using genes found via the rice FOX-hunting system. The present study improved previous protocols, enabling the production of transgenic Jatropha in two steps: the first step involved using auxins and dark incubation to promote root formation in excised shoots and the second step involved delaying the timing of antibiotic selection in the cultivation medium. Transgenic plants were subjected to PCR analysis; the transferred gene expression was confirmed via RT-PCR and the ploidy level was investigated. The results suggest that the genes associated with larger seed size in Arabidopsis thaliana, which were found using the rice FOX-hunting system, produce larger seeds in Jatropha.

Lipocalins are very important proteins for stress resistance in plants. To better understand the function of tomato lipocalins, we observed responses to oxidative stress using over-expressed SlTIL1, SlTIL2, SlCHL, and silenced-plants. Significant differences in reactive oxygen species accumulation (oxidative damage) were observed in all tested plants under heat stress. Plants with over-expressed SlTIL1, SlTIL2, and SlCHL showed less oxidative damage compared with wild-type plants under heat stress. The expression of SlSODs was induced in over-expressed SlTIL1, SlTIL2, and SlCHL plants under normal and heat stress conditions. Furthermore, silenced PDS, SlTILs, and SlCHL plants showed slightly increasing oxidative damage under heat stress alongside with lower SlSODs under normal and stress conditions. These results suggest that SlTIL1, SlTIL2, and SlCHL were involved in antioxidant defense by eliminating ROS in tomato plants.

A homolog of the bacterial ribosome rescue factor ArfB was identified in Arabidopsis thaliana. The factor, named AtArfB for Arabidopsis thaliana ArfB, showed ribosome rescue activity in both in vivo and in vitro assays based on the bacterial translation system. As has been shown for ArfB, the ribosome rescue activity of AtArfB was dependent on the GGQ motif, the crucial motif for the function of class I release factors and ArfB. The C-terminal region of AtArfB was also important for its function. The N-terminal region of AtArfB, which is absent in bacterial ArfB, functioned as a transit peptide for chloroplast targeting in tobacco cells. These results strongly suggest that AtArfB is a ribosome rescue factor that functions in chloroplasts.

A homolog of the bacterial ribosome rescue factor ArfB was identified in Arabidopsis thaliana. The factor, named AtArfB for Arabidopsis thaliana ArfB, showed ribosome rescue activity in both in vivo and in vitro assays based on the bacterial translation system. As has been shown for ArfB, the ribosome rescue activity of AtArfB was dependent on the GGQ motif, the crucial motif for the function of class I release factors and ArfB. The C-terminal region of AtArfB was also important for its function. The N-terminal region of AtArfB, which is absent in bacterial ArfB, functioned as a transit peptide for chloroplast targeting in tobacco cells. These results strongly suggest that AtArfB is a ribosome rescue factor that functions in chloroplasts.

In this study, two temperature-induced lipocalin genes SlTIL1 and SlTIL2, and a chloroplastic lipocalin gene SlCHL were isolated from ‘Micro-Tom’ tomato. The coding sequences of SlTIL1, SlTIL2 and SlCHL were 558, 558, and 1002 bp, respectively. By TargetP analysis, no characteristic transit peptides were predicted in the proteins of SlTIL1 and SlTIL2, while a chloroplastic transit peptide was predicted in the protein of SlCHL. The subcellular localization results indicated that SlTIL1 and SlTIL2 proteins were major localized in the plasma membrane, while SlCHL was localized in chloroplast. To understand the function of lipocalins, transgenic tomato over-expressed SlTIL1, SlTIL2 and SlCHL and their virus-induced gene silencing (VIGS) plants were generated. The phenotypes were significantly affected when the SlTIL1, SlTIL2 and SlCHL were over-expressed or silenced by VIGS, which suggested that the three lipocalins played important roles in regulating the growth and development of tomato. In addition, the level of ROS (O(2)(−) and H(2)O(2)) was low in SlTIL1, SlTIL2 and SlCHL over-expressed plants, while it was high in their silenced plants. The changes in the expression of SODs were consistent with the accumulations of ROS, which indicated that lipocalins might have an important role in abiotic oxidative stress tolerance in tomato plants. Especially SlTIL1 and SlTIL2 are localized around their membranes and protect them from ROS. The results will contribute to elucidating the functions of lipocalin in plants, and provide new strategies to improve the tolerance to abiotic stress in tomato plants.

The development of green energy is important to mitigate global warming. Jatropha (Jatropha curcas L.) is a promising candidate for the production of alternative biofuel, which could reduce the burden on the Earth’s resources. Jatropha seeds contain a large quantity of lipids that can be used to produce biofuel, and the rest of the plant has many other uses. Currently, techniques for plant genetic transformation are extensively employed to study, create, and improve the specific characteristics of the target plant. Successful transformation involves the alteration of plants and their genetic materials. The aim of this study was to generate Jatropha plants that can support biofuel production by increasing their seed size using genes found via the rice FOX-hunting system. The present study improved previous protocols, enabling the production of transgenic Jatropha in two steps: the first step involved using auxins and dark incubation to promote root formation in excised shoots and the second step involved delaying the timing of antibiotic selection in the cultivation medium. Transgenic plants were subjected to PCR analysis; the transferred gene expression was confirmed via RT-PCR and the ploidy level was investigated. The results suggest that the genes associated with larger seed size in Arabidopsis thaliana, which were found using the rice FOX-hunting system, produce larger seeds in Jatropha.

Agrobacterium-mediated transformation represents a key innovation for plant breeding and is routinely used in research and applied biology. However, for several species, the efficacy of transformation is limited. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs (NSAIDs), including tenoxicam (TNX), enhance the efficiency of Agrobacterium-mediated transient transformation in the model species Arabidopsis thaliana via leaf infiltration and can be successfully applied in analyses of the subcellular localisation of fluorescent fusion proteins. TNX acts as an inhibitor of plant immune responses and lacks similar transient transformation efficiency in a dde2/ein2/pad4/sid2 quadruple mutant background, thereby indicating that TNX increases the efficiency of Agrobacterium infection via a transient shutdown of the immune system mediated by jasmonic acid, ethylene, and salicylic acid signalling. In addition, we found that TNX enhances the efficiency of stable transformation in crops of agricultural and economic importance, such as Jatropha and maize, indicating that TNX can enhance the integration of exogenous DNA into the plant genome via an increased introduction of DNA into plant cells. Given that treatment with oxicam compounds is simple, cost effective, and has broad utility, we anticipate that this discovery will contribute to accelerating genome-editing technologies in plants.

Lipocalins are very important proteins for stress resistance in plants. To better understand the function of tomato lipocalins, we observed responses to oxidative stress using over-expressed SlTIL1, SlTIL2, SlCHL, and silenced-plants. Significant differences in reactive oxygen species accumulation (oxidative damage) were observed in all tested plants under heat stress. Plants with over-expressed SlTIL1, SlTIL2, and SlCHL showed less oxidative damage compared with wild-type plants under heat stress. The expression of SlSODs was induced in over-expressed SlTIL1, SlTIL2, and SlCHL plants under normal and heat stress conditions. Furthermore, silenced PDS, SlTILs, and SlCHL plants showed slightly increasing oxidative damage under heat stress alongside with lower SlSODs under normal and stress conditions. These results suggest that SlTIL1, SlTIL2, and SlCHL were involved in antioxidant defense by eliminating ROS in tomato plants.

In these study, we introduced 35Sp::sGFP-SlTIL1, 35Sp::sGFP-SlTIL2, 35Sp::sGFP-SlCHL, 35Sp::SlTIL1- sGFP, 35Sp::SlTIL2-sGFP, and 35Sp::SlCHL-sGFP constructs into onion (Allium cepa) epidermal cells, Nicotiana tabacum (SR1) and Nicotiana benthamiana leaf cells by a particle bombardment PDS-1000 System (Bio-Rad, Hercules, CA, USA). Transient expression of the sGFP-SlTIL1, sGFP-SlTIL2, sGFP-SlCHL, SlTIL1-sGFP, SlTIL2-sGFP, and SlCHL-sGFP fusion proteins were observed by a confocal laser scanning microscopy (Leica SP, Solms, Germany and LSM 700, Carl Zeiss).The result showed that the fusion proteins were accumulated in the plasma membrane and around membranes of the onion (A. cepa) epidermal cells. In case of N. tabacumleaf cells, sometimes detected the fluorescence around plastids and nuclear and in reticulate structures. In the control cells expressing sGFP (pUGW5-sGFP and pUGW6-sGFP), the fluorescence was found in the cytosol and the nucleus. The fusion proteins of SlCHL-sGFP were localized in the chloroplasts of N. tabacum and N. benthamiana leaf cells.

Flavonoid metabolons (weakly-bound multi-enzyme complexes of flavonoid enzymes) are believed to occur in diverse plant species. However, how flavonoid enzymes are organized to form a metabolon is unknown for most plant species. We analyzed the physical interaction partnerships of the flavonoid enzymes from two lamiales plants (snapdragon and torenia) that produce flavones and anthocyanins. In snapdragon, protein-protein interaction assays using yeast and plant systems revealed the following binary interactions: flavone synthase II (FNSII)/chalcone synthase (CHS); FNSII/chalcone isomerase (CHI); FNSII/dihydroflavonol 4-reductase (DFR); CHS/CHI; CHI/DFR; and flavonoid 3'-hydroxylase/CHI. These results along with the subcellular localizations and membrane associations of snapdragon flavonoid enzymes suggested that FNSII serves as a component of the flavonoid metabolon tethered to the endoplasmic reticulum (ER). The observed interaction partnerships and temporal gene expression patterns of flavonoid enzymes in red snapdragon petal cells suggested the flower stage-dependent formation of the flavonoid metabolon, which accounted for the sequential flavone and anthocyanin accumulation patterns therein. We also identified interactions between FNSII and other flavonoid enzymes in torenia, in which the co-suppression of FNSII expression was previously reported to diminish petal anthocyanin contents. The observed physical interactions among flavonoid enzymes of these plant species provided further evidence supporting the long-suspected organization of flavonoid metabolons as enzyme complexes tethered to the ER via cytochrome P450, and illustrated how flavonoid metabolons mediate flower coloration. Moreover, the observed interaction partnerships were distinct from those previously identified in other plant species (Arabidopsis thaliana and soybean), suggesting that the organization of flavonoid metabolons may differ among plant species.

To more easily understand the function of a protein called lipocalin, samples from ’Micro-Tom’ tomatoes and samples of the pericarp portion of SlTIL1, SlTIL2, and SlCHL over-expressed fruit was analyzed using Technovit 7100 (Heraeus Kulzer, Wehrheim, Germany). The results showed that the structure and cells of the pericarp in its fruit in the breaker phase of the over-expressed SlTILs and SlCHL showed that most of the pericarp cells were greatly expanded in light conditions 1 and light conditions 2 when compared to controls. Exocarp and mesocarp in controls have an intercellular space, but this is not found in the intercellular space in SlTILs and SlCHL pericarp cells that are over-expressed. SlTILs and SlCHL cell structure of vascular bundles which is widely shows a wider (tenuous) cell than a control vascular bundle cell. These results indicate that cells in SlTILs and SlCHL which are over-expressed are more rapidly developing than wild-type ‘Micro-Tom’ tomatoes as controls.

Forced expression of rice 45S rRNA gene conferred ca. 2-fold increase of above-ground growth in transgenic Arabidopsis . This growth increase was probably brought by cell proliferation, not by cell enlargement. Recent increase in carbon dioxide emissions is causing global climate change. The use of plant biomass as alternative energy source is one way to reduce these emissions. Therefore, reinforcement of plant biomass production is an urgent key issue to overcome both depletion of fossil energies and emission of carbon dioxide. Here, we created transgenic Arabidopsis with a 2-fold increase in above-ground growth by forced expression of the rice 45S rRNA gene using the maize ubiquitin promoter. Although the size of guard cells and ploidy of leaf-cells were similar between transgenic and control plants, numbers of stomata and pavement cells were much increased in the transgenic leaf. This data suggested that cell number, not cell expansion, was responsible for the growth increase, which might be brought by the forced expression of exogenous and full-length 45S rRNA gene. The expression level of rice 45S rRNA transcripts was very low, possibly triggering unknown machinery to enhance cell proliferation. Although microarray analysis showed enhanced expression of ethylene-responsive transcription factors, these factors might respond to ethylene induced by abiotic/biotic stresses or genomic incompatibility, which might be involved in the expression of species-specific internal transcribed spacer (ITS) sequences within rice 45S rRNA transcripts. Further analysis of the mechanism underlying the growth increase will contribute to understanding the regulation of the cell proliferation and the mechanism of hybrid vigor.

Metabolic enzymes, including those involved in flavonoid biosynthesis, are proposed to form weakly bound, ordered protein complexes, called "metabolons". Some hypothetical models of flavonoid biosynthetic metabolons have been proposed, in which metabolic enzymes are believed to anchor to the cytoplasmic surface of the endoplasmic reticulum (ER) via ER-bound cytochrome P450 isozymes (P450s). However, no convincing evidence for the interaction of flavonoid biosynthetic enzymes with P450s has been reported previously. Here, we analyzed binary protein-protein interactions of 2-hydroxyisoflavanone synthase 1 (GmIFS1), a P450 (CYP93C), with cytoplasmic enzymes involved in isoflavone biosynthesis in soybean. We identified binary interactions between GmIFS1 and chalcone synthase 1 (GmCHS1) and between GmIFS1 and chalcone isomerases (GmCHIs) by using a split-ubiquitin membrane yeast two-hybrid system. These binary interactions were confirmed in planta by means of bimolecular fluorescence complementation (BiFC) using tobacco leaf cells. In these BiFC analyses, fluorescence signals that arose from the interaction of these cytoplasmic enzymes with GmIFS1 generated sharp, network-like intracellular patterns, which was very similar to the ER-localized fluorescence patterns of GmIFS1 labeled with a fluorescent protein. These observations provide strong evidence that, in planta, interaction of GmCHS1 and GmCHIs with GmIFS1 takes place on ER on which GmIFS1 is located, and also provide important clues to understand how enzymes and proteins form metabolons to establish efficient metabolic flux of (iso)flavonoid biosynthesis.

Rings or arcs of fungus-regulated plant growth occurring on the floor of woodlands and grasslands are commonly called “fairy rings”. Fairy chemicals, 2-azahypoxanthine (AHX), imidazole-4-carboxamide (ICA), and 2-aza-8-oxohypoxanthine (AOH), are plant growth regulators involved in the phenomenon. The endogeny and biosynthetic pathways of AHX and AOH in plants have already been proven, however, those of ICA have remained unclear. We developed a high-sensitivity detection method for FCs including ICA and the endogenous ICA was detected in some plants for the first time. The quantitative analysis of the endogenous level of ICA in rice and Arabidopsis were performed using 13C-double labeled ICA. In addition, the incorporation experiment and enzyme assay using the labeled compound into rice and partially purified fraction of rice indicated that ICA is biosynthesized from 5-aminoimidazole-4-carboxamide (AICA), a metabolite on the purine metabolic pathway. The relationship between ICA and AHX was also discussed based on quantitative analysis and gene expression analysis.

To examine the effect of 2-azahypoxanthine (AHX) on rice plant (Oryza sativa L. cv. Nipponbare) growth, we carried out pot and field experiments. AHX was applied at 50 μM for two weeks at four growth stages (transplanting, tillering, panicle formation and ripening stages) in the pot experiment, and 1 mM AHX at three stages [at rising of seedling in nursery boxes (seedling treatment), transplanting and panicle formation stages] in the field experiment. Both pot and field experiments showed a tendency toward increased panicle number (PN), culm length (CL) and plant dry weights with AHX treatments. Brown rice yields were also improved by AHX treatments, especially when applying at stages of tillering and panicle formation and seedling and transplanting during pot and field experiments, respectively. In the latter, yield increased drastically up to 18.7, 15.8, 9.6 and 5.8% of control. However, panicle length and 1000-grain weight were not affected by AHX application. These results suggested that AHX increased the brown rice yield through its effects on PN and/or CL.

Jatropha, Jatropha curcas, is known as the oilseed plant that yields biofuel. An average of 30–40% of a single Jatropha seed is made of oils and fats and can be processed for use as diesel engine fuel. This chapter is concerned with using transgenic approaches to increase oil production in Jatropha. At present, we are attempting to make larger Jatropha seeds by transferring rice genes coupled with the CaMV35S promoter into Jatropha plants. We found candidate rice genes that produce increased seed size in other plants (Arabidopsis thaliana) by using rice Full-Length cDNA OvereXpressing gene 14 hunting system. We are transferring four genes (LOC_Os08g41910 encoding Sua5/YciO/YrdC/YwlC family protein, LOC_Os04g43210 encoding probable inositol transporter 2-like, LOC_Os03g49180 encoding alkaline ceramidase, and LOC_Os10g40934 encoding putative flavonol synthase/flavanone 3-hydroxylase or 2OG-Fe(II) oxygenase containing protein) to attempt to make larger Jatropha seeds. We already made some transgenic Jatropha. Here we also discuss an improved method of Agrobacterium-mediated transformation to increase seed growth efficiency. We used Gamborg’B5 medium containing 2% sucrose and 250 mg/l active charcoal to improve the condition for root induction.

We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants. Electronic supplementary material The online version of this article (doi:10.1007/s11103-014-0194-9) contains supplementary material, which is available to authorized users.

In this study, the pathway of β-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of β-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of β-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of β-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved β-cryptoxanthin and zeaxanthin at the 7,8 or 7′,8′ position. But other carotenoids tested in this study (lycopene, α-carotene, β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of β-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of β-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of β-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.

In order to examine the current status of gender equality in academic societies in Japan, we inquired about the number of women involved in leadership activities at society conferences and annual meetings, as these activities are critical in shaping scientific careers. Our findings show a clear bias against female scientists, and a need to raise consciousness and awareness in order to move closer to equality for future generations.

Research interest in proteomics is increasingly shifting toward the reverse genetic characterization of gene function at the proteome level. In plants, several distinct gene defects perturb photosynthetic capacity, resulting in the loss of chlorophyll and an albino or pale-green phenotype. Because photosynthesis is interconnected with the entire plant metabolism and its regulation, all albino plants share common characteristics that are determined by the switch from autotrophic to heterotrophic growth. Reverse genetic characterizations of such plants often cannot distinguish between specific consequences of a gene defect from generic effects in response to perturbations in photosynthetic capacity. Here, we set out to define common and specific features of protein accumulation in three different albino/pale-green plant lines. Using quantitative proteomics, we report a common molecular phenotype that connects the loss of photosynthetic capacity with other chloroplast and cellular functions, such as protein folding and stability, plastid protein import, and the expression of stress-related genes. Surprisingly, we do not find significant differences in the expression of key transcriptional regulators, suggesting that substantial regulation occurs at the posttranscriptional level. We examine the influence of different normalization schemes on the quantitative proteomics data and report all identified proteins along with their fold changes and P values in albino plants in comparison with the wild type. Our analysis provides initial guidance for the distinction between general and specific adaptations of the proteome in photosynthesis-impaired plants.

The light-harvesting chlorophyll-binding (LHC) proteins are major constituents of eukaryotic photosynthetic machinery. In plants, six different groups of proteins, LHC-like proteins, share a conserved motif with LHC. Although the evolution of LHC and LHC-like proteins is proposed to be a key for the diversification of modern photosynthetic eukaryotes, our knowledge of the evolution and functions of LHC-like proteins is still limited. In this study, we aimed to understand specifically the function of one type of LHC-like proteins, LIL3 proteins, by analyzing Arabidopsis mutants lacking them. The Arabidopsis genome contains two gene copies for LIL3, LIL3:1 and LIL3:2 . In the lil3:1/lil3:2 double mutant, the majority of chlorophyll molecules are conjugated with an unsaturated geranylgeraniol side chain. This mutant is also deficient in α-tocopherol. These results indicate that reduction of both the geranylgeraniol side chain of chlorophyll and geranylgeranyl pyrophosphate, which is also an essential intermediate of tocopherol biosynthesis, is compromised in the lil3 mutants. We found that the content of geranylgeranyl reductase responsible for these reactions was severely reduced in the lil3 double mutant, whereas the mRNA level for this enzyme was not significantly changed. We demonstrated an interaction of geranylgeranyl reductase with both LIL3 isoforms by using a split ubiquitin assay, bimolecular fluorescence complementation, and combined blue-native and SDS polyacrylamide gel electrophoresis. We propose that LIL3 is functionally involved in chlorophyll and tocopherol biosynthesis by stabilizing geranylgeranyl reductase.

Rings or arcs of fungus-regulated plant growth occur often on the floor of woodlands, in agricultural areas, and in grasslands worldwide. These rings are commonly called "fairy rings". A plant-growth regulating compound was isolated from a fairy ring forming fungus, Lepista sordida , and its chemical structure was identified as imidazole-4-carboxamide (ICA) by spectroscopic analyses including single-crystal X-ray diffraction techniques. ICA inhibited the growth of turfgrass and rice seedling. On the other hand, in a greenhouse experiment, this compound increased rice grain yield by 26% compared with control.

Several hundred nucleus-encoded factors are required for regulating gene expression in plant organelles. Among them, the most numerous are the members of the pentatricopeptide repeat (PPR) protein family. We found that PPR protein OTP82 is essential for RNA editing of the ndhB-9 and ndhG-1 sites within transcripts encoding subunits of chloroplast NAD(P)H dehydrogenase. Despite the defects in RNA editing, otp82 did not show any phenotypes in NDH activity, stability or interaction with photosystem I, suggesting that the RNA editing events mediated by OTP82 are functionally silent even though they induce amino acid alterations. In agreement with this result, both sites are partially edited even in the wild type, implying the possibility that a single gene produces heterogeneous proteins that are functionally equivalent. Although only five nucleotides separate the ndhB-8 and ndhB-9 sites, the ndhB-8 site is normally edited in otp82 mutants, suggesting that both sites are recognized by different PPR proteins. OTP82 falls into the DYW subclass containing conserved C-terminal E and DYW motifs. As in CRR22 and CRR28, the DYW motif present in OTP82 is not essential for RNA editing in vivo.

The chloroplast NAD(P)H dehydrogenase (NDH) complex functions in PSI cyclic and chlororespiratory electron transport in higher plants. Eleven plastid-encoded and three nuclear-encoded subunits have been identified so far, but the entire subunit composition, especially of the putative electron donor-binding module, is unclear. We isolated Arabidopsis thaliana crr23 (chlororespiratory reduction) mutants lacking NDH activity according to the absence of a transient increase in Chl fluorescence after actinic light illumination. Although CRR23 shows similarity to the NdhL subunit of cyanobacterial NDH-1, it has three transmembrane domains rather than the two in cyanobacterial NdhL. Unlike cyanobacterial NdhL, CRR23 is essential for stabilizing the NDH complex, which in turn is required for the accumulation of CRR23. Furthermore, CRR23 and NdhH, a subunit of chloroplast NDH, co-localized in blue-native gel. All the results indicate that CRR23 is an ortholog of cyanobacterial ndhL in Arabidopsis, despite its diversity of structure and function.

The pentatricopeptide repeat (PPR) proteins form one of the largest families in higher plants and are believed to be involved in the posttranscriptional processes of gene expression in plant organelles. It has been shown by using a genetic approach focusing on NAD(P)H dehydrogenase (NDH) activity that a PPR protein CRR4 is essential for a specific RNA editing event in chloroplasts. Here, we discovered Arabidopsis crr21 mutants that are specifically impaired in the RNA editing of the site 2 of ndhD (ndhD-2), which encodes a subunit of the NDH complex. The CRR21 gene encodes a member of the PPR protein family. The RNA editing of ndhD-2 converts the Ser-128 of NdhD to leucine. In crr21 , the activity of the NDH complex is specifically impaired, suggesting that the Ser128Leu change has important consequences for the function of the NDH complex. Both CRR21 and CRR4 belong to the E+ subgroup in the PLS subfamily that is characterized by the presence of a conserved C-terminal region (the E/E+ domain). This E/E+ domain is highly conserved and exchangeable between CRR21 and CRR4, although it is not essential for the RNA binding. Our results suggest that the E/E+ domain has a common function in RNA editing rather than of recognizing specific RNA sequences.

To study the functions of nuclear genes involved in chloroplast development, we systematically analyzed albino and pale green Arabidopsis thaliana mutants by use of the Activator/Dissociation (Ac/Ds) transposon tagging system. In this study, we focused on one of these albino mutants, designated apg3-1 (for a lbino or p ale g reen mutant 3). A gene encoding a ribosome release factor 1 (RF1) homologue was disrupted by the insertion of a Ds transposon into the APG3 gene; a T-DNA insertion into the same gene caused a similar phenotype (apg3-2). The APG3 gene (At3g62910) has 15 exons and encodes a protein (422-aa) with a transit peptide that functions in targeting the protein to chloroplasts. The amino acid sequence of APG3 showed 40.6% homology with an RF1 of Escherichia coli, and complementation analysis using the E. coli rf1 mutant revealed that APG3 functions as an RF1 in E. coli, although complementation was not successful in the RF2-deficient (rf2) mutants of E. coli. These results indicate that the APG3 protein is an orthologue of E. coli RF1, and is essential for chloroplast translation machinery; it was accordingly named AtcpRF1. Since the chloroplasts of apg3-1 plants contained few internal thylakoid membranes, and chloroplast proteins related to photosynthesis were not detected by immunoblot analysis, AtcpRF1 is thought to be essential for chloroplast development.

To better understand the mechanism of plastid differentiation from chloroplast to chromoplast, we examined proteome and plastid changes over four distinct developmental stages of 'Micro-Tom' fruit. Additionally, to discover more about the relationship between fruit color and plastid differentiation, we also analyzed and compared 'Micro-Tom' results with those from two other varieties, 'Black' and 'White Beauty'. We confirmed that proteins related to photosynthesis remain through the orange maturity stage of 'Micro-Tom', and also learned that thylakoids no longer exist at this stage. These results suggest that at a minimum there are changes in plastid morphology occurring before all related proteins change. We also compared 'Micro-Tom' fruits with 'Black' and 'White Beauty' using two-dimensional gel electrophoresis. We found a decrease of CHRC (plastid-lipid-associated protein) and HrBP1 (harpin binding protein-1) in the 'Black' and 'White Beauty' varieties. CHRC is involved in carotenoid accumulation and stabilization. HrBP1 in Arabidopsis has a sequence similar to proteins in the PAP/fibrillin family. These proteins have characteristics and functions similar to lipocalin, an example of which is the transport of hydrophobic molecules. We detected spots of TIL (temperature-induced lipocalin) in 2D-PAGE results, however the number of spots and their isoelectric points differed between 'Micro-Tom' and 'Black'/'White Beauty'. Lipocalin has various functions including those related to environmental stress response, apoptosis induction, membrane formation and fixation, regulation of immune response, cell growth, and metabolism adjustment. Lipocalin related proteins such as TIL and HrBP1 could be related to the accumulation of carotenoids, fruit color and the differentiation of chromoplast.

We have developed a novel gain-of-function system that we have named the FOX hunting system (Full-length cDNA Over-eXpressing gene hunting system). We used normalized full-length cDNA and introduced each cDNA into Arabidopsis by in planta transformation. About 10 000 independent full-length Arabidopsis cDNAs were expressed independently under the CaMV 35S promoter in Arabidopsis. Each transgenic Arabidopsis contained on average 2.6 cDNA clones and was monitored under various categories such as morphological changes, fertility and leaf color. We found 1487 possible morphological mutants from 15 547 transformants. When 115 pale green T(1) mutants were analyzed, 59 lines represented the mutant phenotypes in more than 50% of the T(2) progeny. Characterization of two leaf color mutants revealed the significance of this approach. We also document mutants from several categories and their corresponding full-length cDNAs.

We report here the generation of an additional collection of Dissociation (Ds) transposon-tagged, sequence-indexed lines of Arabidopsis thaliana. Our RIKEN Ds insertion collection now totals 17,668 lines. Our collection has preferential insertions in chromosomes 1 and 5, because Ds was transposed from start loci on those chromosomes (11,854 and 5,814 lines, respectively). We describe here features of the latter 5,814 lines. The former 11,854 lines have been described previously. We have created a searchable database of the insertion sites and mutated genes (http://rarge.gsc.riken.jp/), and are depositing these lines in the RIKEN BioResource Center (http://www.brc.riken.go.jp/lab/epd/Eng/). Our collection of these mutants will contribute to progress in functional genomics of plants.

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.

To study the functions of the nuclear genes involved in chloroplast development, we systematically analyzed albino and pale-green Arabidopsis thaliana mutants by using a two-component transposon system based on the Ac/Ds element of maize as a mutagen. One of the pale-green mutants, albino or pale green mutant 1 (designated as apg1), did not survive beyond the seedling stage, when germinated on soil. The chloroplasts of the apg1 plants contained decreased numbers of lamellae with reduced levels of chlorophyll. A gene encoding a 37 kDa polypeptide precursor of the chloroplast inner envelope membrane was disrupted by insertion of the Ds transposon in apg1. The 37 kDa protein had partial sequence similarity to the S-adenosylmethionine-dependent methyltransferase. The apg1 plants lacked plastoquinone (PQ), suggesting that the APG1 protein is involved in the methylation step of PQ biosynthesis, which is localized at the envelope membrane. Our results demonstrate the importance of the 37 kDa protein of the chloroplast inner envelope membrane for chloroplast development in Arabidopsis.

We transposed Dissociation (Ds) elements from three start loci on chromosome 5 in Arabidopsis (Nossen ecotype) by using a local transposition system. We determined partial genomic sequences flanking the Ds elements and mapped the elements' insertion sites in 1,173 transposed lines by comparison with the published genomic sequence. Most of the lines contained a single copy of the Ds element. One-half of the lines contained Ds on chromosome 5; in particular, insertion "hot spots" near the three start loci were clearly observed. In the other lines, the Ds elements were transposed across chromosomes. We found other insertion hot spots at the tops of chromosomes 2 and 4, near nucleolus organizer regions 2 and 4, respectively. Another characteristic feature was that the Ds elements tended to transpose near the chromosome ends and rarely transposed near centromeres. The distribution patterns differed among the three start loci, even though they possessed the same Ds construct. More than one-half of the Ds elements were inserted irregularly into the genome; that is, they did not retain the perfect inverted repeat sequence of Ds nor leave perfect target site duplications. This precise analysis of distribution patterns will contribute to a comprehensive understanding of the transposing mechanism. From these Ds insertion sites, we have constructed a database for screening gene-knockout mutants in silico. In 583 of the 1,173 lines, the Ds elements were inserted into protein-coding genes, which suggests that these lines are gene-knockout mutants. The database and individual lines will be available freely for academic use from the RIKEN Bio-Resource Center (http://www.brc.riken.go.jp/Eng/index.html).

We determined copy numbers of p-SINE1, a short interspersed element (SINE) in rice, and found that p-SINE1 was distributed in the Oryza sativa genome with an average spacing of about 1 member per 70 kb. We identified 31 p-SINE1 members located at different loci by cloning and characterized them by sequencing. Comparison with a consensus sequence derived from their sequences revealed that all of the p-SINE1 members contained base substitution mutations at various positions. In addition to the substitutions, some members contained deletions, insertions and tandem duplications of a few bases or of a large DNA segment. These mutations seem to have occurred to inhibit transcription from each p-SINE1 member by RNA polymerase III. PCR using a pair of primers that hybridize with the sequences flanking each p-SINE1 member revealed that many of the p-SINE1 members were present at corresponding loci in strains belonging to all rice species carrying the AA genome. Several of them were, however, present at corresponding loci in strains belonging to a limited number of species or in a limited number of the strains belonging to a rice species. These p-SINE1 members are supposed to be useful in the identification and classification of various rice strains with the AA genome. Simple tandem repeats of a trinucleotide (CAT) or dinucleotide (AG) sequence existed in the flanking regions of 2 p-SINE1 members. Such repeats, called microsatellite DNA, varied in number even in the cultivated rice strains examined, suggesting that microsatellite DNA is useful for the identification and classification of cultivars.

Rings or arcs of fungus-stimulated plant growth occur worldwide; these are commonly referred to as "fairy rings". In 2010, we discovered 2-azahypoxanthine (AHX), a compound responsible for the fairy-ring phenomenon caused by fungus; AHX stimulated the growth of all the plants tested. Herein, we reveal the isolation and structure determination of a common metabolite of AHX in plants, 2-aza-8-oxohypoxanthine (AOH). AHX is chemically synthesized from 5-aminoimidazole-4-carboxamide (AICA), and AHX can be converted into AOH by xanthine oxidase. AICA is one of the members of the purine metabolic pathway in animals, plants, and microorganisms. However, further metabolism of AICA remains elusive. Based on these results and facts, we hypothesized that plants themselves produce AHX and AOH through a pathway similar to the chemical synthesis. Herein, we demonstrate the existence of endogenous AHX and AOH and a novel purine pathway to produce them in plants.

Japan, although well qualified as a country that promotes scientific advancement, lags behind other developed nations in gender equality. The percentage of female professionals trained in science, technology, engineering, and mathematics (STEM) is at 13.8%, the lowest among developed countries ([ 1 ][1]). Why is Japan so slow in maximizing the potential of female scientists in STEM? Survey results ([ 2 ][2], [ 3 ][3]) indicate that there are too few women in positions of authority who can help younger women with career enhancement. Furthermore, male scientists show an unconscious bias when evaluating their female colleagues. Finally, female scientists often avoid competition and underestimate their ability, leading to passivity when seeking leadership roles. In 2009, the Ministry of Education, Culture, Sports, Science and Technology in Japan initiated the program “Supporting Positive Activities for Female Researchers.” The 5-year program's goal is to accelerate the numbers of female scientists and their promotion rate at 12 research universities. A midterm evaluation of the program's impact showed a substantial increase in female faculty, particularly in positions of greater responsibility in several universities ([ 4 ][4], [ 5 ][5]). The program gives university leaders a clear path to unlocking the potential of female scientists and helps pave the way with affirmative action, reserving positions for women. These government measures will only have a long-term effect on the ratio and roles of women in STEM fields if the academic climate and leadership changes in Japan. This challenge will require proper enforcement of regulations by deans and department leaders. 1. [↵][6] “Women and Men in Japan (2012)” (Gender Equality Bureau Cabinet Office, Government of Japan, 2012); [http://www.gender.go.jp/english\_contents/pr\_act/pub/pamphlet/women-and-men.html][7]. 2. [↵][8] EPMEWSE Survey Report: Diverse Visions of Scientists and Engineers in the 21st Century: For the Promotion of Gender Equality: . 3. [↵][9] EPMEWESE, Large-Scale Survey of Actual Conditions of Gender Equality in Scientific and Technological Professions (2008); [http://annex.jsap.or.jp/renrakukai/doc\_pdf/h19enquete\_report_en.pdf][10]. 4. [↵][11] Office for Women Researchers, Tohoku University, Tohoku Leading Women's Jump Up Project (2013); [www.morihime.tohoku.ac.jp/english/en\_jump\_up.html][12]. 5. [↵][13] Kyushu University Women Researchers, Promotion Project (2012); . [1]: #ref-1 [2]: #ref-2 [3]: #ref-3 [4]: #ref-4 [5]: #ref-5 [6]: #xref-ref-1-1 "View reference 1 in text" [7]: http://www.gender.go.jp/english_contents/pr_act/pub/pamphlet/women-and-men.html [8]: #xref-ref-2-1 "View reference 2 in text" [9]: #xref-ref-3-1 "View reference 3 in text" [10]: http://annex.jsap.or.jp/renrakukai/doc_pdf/h19enquete_report_en.pdf [11]: #xref-ref-4-1 "View reference 4 in text" [12]: http://www.morihime.tohoku.ac.jp/english/en_jump_up.html [13]: #xref-ref-5-1 "View reference 5 in text"

A majority of the proteins of the chloroplast are encoded by the nuclear genome, and are post-translationally targeted to the chloroplast. From databases of tagged insertion lines at international seed stock centers and our own stock, we selected 3246 Ds/Spm (dissociator/suppressor-mutator) transposon- or T-DNA-tagged Arabidopsis lines for genes encoding 1369 chloroplast proteins (about 66% of the 2090 predicted chloroplast proteins) in which insertions disrupt the protein-coding regions. We systematically observed 3-week-old seedlings grown on agar plates, identified mutants with abnormal phenotypes and collected homozygous lines with wild-type phenotypes. We also identified insertion lines for which no homozygous plants were obtained. To date, we have identified 111 lines with reproducible seedling phenotypes, 122 lines for which we could not obtain homozygotes and 1290 homozygous lines without a visible phenotype. The Chloroplast Function Database presents the molecular and phenotypic information obtained from this resource. The database provides tools for searching for mutant lines using Arabidopsis Genome Initiative (AGI) locus numbers, tagged line numbers and phenotypes, and provides rapid access to detailed information on the tagged line resources. Moreover, our collection of insertion homozygotes provides a powerful tool to accelerate the functional analysis of nuclear-encoded chloroplast proteins in Arabidopsis. The Chloroplast Function Database is freely available at http://rarge.psc.riken.jp/chloroplast/. The homozygous lines generated in this project are also available from the various Arabidopsis stock centers. We have donated the insertion homozygotes to their originating seed stock centers.

Several hundred nucleus-encoded factors are required for regulating gene expression in plant organelles. Among them, the most numerous are the members of the pentatricopeptide repeat (PPR) protein family. We found that PPR protein OTP82 is essential for RNA editing of the ndhB-9 and ndhG-1 sites within transcripts encoding subunits of chloroplast NAD(P)H dehydrogenase. Despite the defects in RNA editing, otp82 did not show any phenotypes in NDH activity, stability or interaction with photosystem I, suggesting that the RNA editing events mediated by OTP82 are functionally silent even though they induce amino acid alterations. In agreement with this result, both sites are partially edited even in the wild type, implying the possibility that a single gene produces heterogeneous proteins that are functionally equivalent. Although only five nucleotides separate the ndhB-8 and ndhB-9 sites, the ndhB-8 site is normally edited in otp82 mutants, suggesting that both sites are recognized by different PPR proteins. OTP82 falls into the DYW subclass containing conserved C-terminal E and DYW motifs. As in CRR22 and CRR28, the DYW motif present in OTP82 is not essential for RNA editing in vivo.

Scientific Research on Priority Areas16085206 Ministry of Education, Culture, Sports, Science, and Technology of Japan 17GS0316 Ministry of Agriculture, Forestry, and Fisheries of Japan GPN0008 Australian Research Council CE0561495; International audience; The plant-specific DYW subclass of pentatricopeptide repeat proteins has been postulated to be involved in RNA editing of organelle transcripts. We discovered that the DYW proteins CHLORORESPIRATORY REDUCTION22 (CRR22) and CRR28 are required for editing of multiple plastid transcripts but that their DYW motifs are dispensable for editing activity in vivo. Replacement of the DYW motifs of CRR22 and CRR28 by that of CRR2, which has been 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 functionally replace that of CRR2. We propose that different DYW family members have acquired distinct functions in the divergent processes of RNA maturation, including RNA cleavage and RNA editing.