RESUMO
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.
Assuntos
Antirrhinum/metabolismo , Flavonoides/metabolismo , Lamiales/metabolismo , Aciltransferases/metabolismo , Oxirredutases do Álcool/metabolismo , Antocianinas/metabolismo , Antirrhinum/enzimologia , Antirrhinum/crescimento & desenvolvimento , Sistema Enzimático do Citocromo P-450/metabolismo , Flores/crescimento & desenvolvimento , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Liases Intramoleculares/metabolismo , Lamiales/enzimologia , Lamiales/crescimento & desenvolvimento , Redes e Vias Metabólicas , Mapas de Interação de Proteínas , Técnicas do Sistema de Duplo-HíbridoRESUMO
KEY MESSAGE: 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.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Genes de Plantas , Genes de RNAr , Oryza/genética , RNA Ribossômico/genética , Contagem de Células , Regulação para Baixo/genética , Dosagem de Genes , Regulação da Expressão Gênica de Plantas , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Estômatos de Plantas/citologia , Estômatos de Plantas/genética , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas , Ploidias , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Transgenes , Regulação para Cima/genéticaRESUMO
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.
Assuntos
Aciltransferases/metabolismo , Sistema Enzimático do Citocromo P-450/metabolismo , Flavonoides/metabolismo , Glycine max/enzimologia , Liases Intramoleculares/metabolismo , Proteínas Recombinantes/metabolismo , Mapeamento de Interação de Proteínas/métodosRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Metaboloma , Transcriptoma , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Cloroplastos/genética , Análise por Conglomerados , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Mutação , Análise de Componente PrincipalRESUMO
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.
Assuntos
Liderança , Pesquisadores , Ciência , Direitos da Mulher , Japão , Pesquisadores/estatística & dados numéricos , Recursos HumanosRESUMO
In this study, the pathway of ß-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 ß-citraurin predominantly, and Miyagawa-wase, which does not accumulate ß-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of ß-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of ß-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 ß-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved ß-cryptoxanthin and zeaxanthin at the 7,8 or 7',8' position. But other carotenoids tested in this study (lycopene, α-carotene, ß-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of ß-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of ß-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 ß-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.
Assuntos
Carotenoides/metabolismo , Citrus/enzimologia , Dioxigenases/metabolismo , Xantofilas/metabolismo , beta Caroteno/análogos & derivados , Cromatografia Líquida de Alta Pressão , Citrus/efeitos dos fármacos , Citrus/genética , Citrus/efeitos da radiação , Criptoxantinas , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Etilenos/farmacologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Genes de Plantas/genética , Proteínas de Fluorescência Verde/metabolismo , Luz , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Redes e Vias Metabólicas/efeitos da radiação , Dados de Sequência Molecular , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/metabolismo , Folhas de Planta/efeitos da radiação , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/metabolismo , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Frações Subcelulares/efeitos da radiação , Nicotiana/efeitos dos fármacos , Nicotiana/genética , Nicotiana/metabolismo , Nicotiana/efeitos da radiação , Xantofilas/química , Zeaxantinas , beta Caroteno/química , beta Caroteno/isolamento & purificação , beta Caroteno/metabolismoRESUMO
Type I chaperonins are large, double-ring complexes present in bacteria (GroEL), mitochondria (Hsp60), and chloroplasts (Cpn60), which are involved in mediating the folding of newly synthesized, translocated, or stress-denatured proteins. In Escherichia coli, GroEL comprises 14 identical subunits and has been exquisitely optimized to fold its broad range of substrates. However, multiple Cpn60 subunits with different expression profiles have evolved in chloroplasts. Here, we show that, in Arabidopsis thaliana, the minor subunit Cpn60ß4 forms a heterooligomeric Cpn60 complex with Cpn60α1 and Cpn60ß1-ß3 and is specifically required for the folding of NdhH, a subunit of the chloroplast NADH dehydrogenase-like complex (NDH). Other Cpn60ß subunits cannot complement the function of Cpn60ß4. Furthermore, the unique C-terminus of Cpn60ß4 is required for the full activity of the unique Cpn60 complex containing Cpn60ß4 for folding of NdhH. Our findings suggest that this unusual kind of subunit enables the Cpn60 complex to assist the folding of some particular substrates, whereas other dominant Cpn60 subunits maintain a housekeeping chaperonin function by facilitating the folding of other obligate substrates.
Assuntos
Arabidopsis/metabolismo , Chaperonina 60/metabolismo , Cloroplastos/metabolismo , Dobramento de Proteína , Sequência de Aminoácidos , Chaperonina 60/química , Chaperonina 60/genética , Cromatografia de Afinidade , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Mutação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
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.
Assuntos
Hipoxantinas/metabolismo , Oryza/metabolismo , Purinas/metabolismo , Cristalografia por Raios X , Hipoxantinas/síntese química , Hipoxantinas/química , Conformação Molecular , Purinas/química , Xantina Oxidase/metabolismoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Mutação/genética , Pigmentação/genética , Proteoma/metabolismo , Regulon/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas , Fenótipo , Fotossíntese/genética , Plastídeos/metabolismo , Subunidades Proteicas/metabolismo , Transporte Proteico/genética , Estresse Fisiológico/genética , Regulação para Cima/genéticaRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Clorofila/biossíntese , Proteínas de Cloroplastos/metabolismo , Complexos de Proteínas Captadores de Luz/metabolismo , Tocoferóis/metabolismo , Sequência de Aminoácidos , Arabidopsis/genética , Arabidopsis/efeitos da radiação , Proteínas de Arabidopsis/genética , Sequência de Bases , Proteínas de Cloroplastos/genética , Primers do DNA/genética , Estabilidade Enzimática , Genes de Plantas , Complexos de Proteínas Captadores de Luz/genética , Dados de Sequência Molecular , Mutagênese Insercional , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Oxirredutases/metabolismo , Filogenia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA de Plantas/genética , RNA de Plantas/metabolismo , Homologia de Sequência de AminoácidosRESUMO
Agrobacterium-mediated transformation is a key innovation for plant breeding, and routinely used in basic researches and applied biology. However, the transformation efficiency is often the limiting factor of this technique. In this study, we discovered that oxicam-type nonsteroidal anti-inflammatory drugs, including tenoxicam (TNX), increase the efficiency of Agrobacterium-mediated transient transformation. TNX treatment increased the transformation efficiency of Agrobacterium-mediated transformation of Arabidopsis thaliana mature leaves by agroinfiltration. The increase of efficiency by TNX treatment was not observed in dde2/ein2/pad4/sid2 quadruple mutant, indicating that TNX inhibits the immune system mediated by jasmonic acid, ethylene, and salicylic acid against to Agrobacterium. We also found that TNX-treatment is applicable for the transient expression and subcellular localization analysis of fluorescent-tagged proteins in Arabidopsis leaf cells. In addition, we found that TNX increases the efficiency of Agrobacterium-mediated transient transformation of Jatropha. Given that treatment with oxicam compounds is a simple and cost effective method, our findings will provide a new option to overcome limitations associated with Agrobacterium-mediated transformation of various plant species.
RESUMO
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.
Assuntos
Proteínas de Arabidopsis/análise , Arabidopsis/genética , Núcleo Celular/genética , Cloroplastos/genética , Elementos de DNA Transponíveis , DNA Bacteriano/análise , Bases de Dados de Ácidos Nucleicos , Alelos , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , DNA Bacteriano/genética , Mutação , Fenótipo , Plântula/genética , Plântula/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , NADH Desidrogenase/genética , Edição de RNA , RNA de Cloroplastos/genética , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/genética , Sequência de Bases , Cloroplastos/genética , Cloroplastos/metabolismo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Immunoblotting , Dados de Sequência Molecular , Mutação , NADH Desidrogenase/metabolismo , Hibridização de Ácido Nucleico , Complexo de Proteína do Fotossistema I/genética , Complexo de Proteína do Fotossistema I/metabolismo , Plantas Geneticamente Modificadas , Ligação Proteica , Proteínas de Ligação a RNA/genética , Homologia de Sequência de AminoácidosRESUMO
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.
RESUMO
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.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas de Arabidopsis/metabolismo , Ribossomos/metabolismo , Fatores de Ribosilação do ADP/química , Fatores de Ribosilação do ADP/genética , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Cloroplastos/metabolismo , Domínios Proteicos , Sinais Direcionadores de Proteínas , Transporte ProteicoRESUMO
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.
RESUMO
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.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Arabidopsis/metabolismo , Redes e Vias Metabólicas/genética , Oryza/metabolismo , Purinas/metabolismo , Aminoimidazol Carboxamida/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Oryza/genética , Oryza/crescimento & desenvolvimentoRESUMO
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.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Cloroplastos/enzimologia , NADPH Desidrogenase/metabolismo , Subunidades Proteicas/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Clorofila/metabolismo , Cianobactérias/enzimologia , Transporte de Elétrons , Eletroforese em Gel Bidimensional , Fluorescência , Dados de Sequência Molecular , Mutação/genética , NADPH Desidrogenase/química , Subunidades Proteicas/químicaRESUMO
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 (O2 - and H2O2) 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.