RESUMO
The NAM, ATAF1/2, and CUC2 (NAC) domain transcription factor VND-INTERACTING2 (VNI2) negatively regulates xylem vessel formation by interacting with another NAC domain transcription factor, VASCULAR-RELATED NAC-DOMAIN7 (VND7), a master regulator of xylem vessel formation. Here, we screened interacting proteins with VNI2 using yeast two-hybrid assay and isolated two NAC domain transcription factors, Arabidopsis thaliana ACTIVATION FACTOR 2 (ATAF2) and NAC DOMAIN CONTAINING PROTEIN 102 (ANAC102). A transient gene expression assay showed that ATAF2 upregulates the expression of genes involved in leaf senescence, and VNI2 effectively inhibits the transcriptional activation activity of ATAF2. vni2 mutants accelerate leaf senescence, whereas ataf2 mutants delay leaf senescence. In addition, the accelerated leaf senescence phenotype of the vni2 mutant is recovered by simultaneous mutation of ATAF2. Our findings strongly suggest that VNI2 interacts with and inhibits ATAF2, resulting in negatively regulating leaf senescence.
RESUMO
Bax inhibitor-1 (BI-1) is a widely conserved cell death regulator that confers resistance to environmental stress in plants. Previous studies suggest that Arabidopsis thaliana BI-1 (AtBI-1) modifies sphingolipids by interacting with cytochrome b5 (AtCb5), an electron-transfer protein. To reveal how AtBI-1 regulates sphingolipid synthesis, we screened yeast sphingolipid-deficient mutants and identified yeast ELO2 and ELO3 as novel enzymes that are essential for AtBI-1 function. ELO2 and ELO3 are condensing enzymes that synthesize very-long-chain fatty acids (VLCFAs), major fatty acids in plant sphingolipids. In Arabidopsis, we identified four ELO homologs (AtELO1-AtELO4), localized in the endoplasmic reticulum membrane. Of those AtELOs, AtELO1 and AtELO2 had a characteristic histidine motif and were bound to AtCb5-B. This result suggests that AtBI-1 interacts with AtELO1 and AtELO2 through AtCb5. AtELO2 and AtCb5-B also interact with KCR1, PAS2, and CER10, which are essential for the synthesis of VLCFAs. Therefore, AtELO2 may participate in VLCFA synthesis with AtCb5 in Arabidopsis. In addition, our co-immunoprecipitation/mass spectrometry analysis demonstrated that AtBI-1 forms a complex with AtELO2, KCR1, PAS2, CER10, and AtCb5-D. Furthermore, AtBI-1 contributes to the rapid synthesis of 2-hydroxylated VLCFAs in response to oxidative stress. These results indicate that AtBI-1 regulates VLCFA synthesis by interacting with VLCFA-synthesizing enzymes.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ácidos Graxos/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/genética , Esfingolipídeos/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Ácidos Graxos/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Esfingolipídeos/metabolismoRESUMO
Some Rumex species such as sorrel are edible as baby leaf salad greens. On the other hand, Rumex plants accumulate soluble oxalate, a toxic metabolite which causes serious diseases such as renal syndrome. We attempted to produce low-oxalate plants of R. obtusifolius, a perennial weed which has higher vitamin C and amino acid content and higher tolerance to stress than many other Rumex species. Ion beams are ionising radiation with high linear energy transfer that causes a wide spectrum of mutations. Thus, in the present study we evaluated the effects of ion beams on oxalate and other primary metabolites in leaves of R. obtusifolius using CE-MS. The results showed that oxalate content was increased by irradiation with carbon ion beams. Metabolome analysis revealed that ion beams affected carbon flow to the isocitrate pathway, which is involved in oxalate synthesis. These observations suggested that modulation of carbon flow to the isocitrate pathway is important to regulate oxalate levels in plants.
Assuntos
Metaboloma/efeitos da radiação , Ácido Oxálico , Folhas de Planta/metabolismo , Radiação Ionizante , Rumex/metabolismoRESUMO
Carbon dioxide (CO2), nutrient supply, and light quality are amongst the major controlling factors to improve the biomass production and nutritional outputs in plant factory. The present study employed CE-MS to investigate the effects of high CO2, nutrient formulation, and LED on the accumulation of primary metabolites in head lettuce. Results suggested that high CO2 (1000ppm) and nutrient supply enhanced both the biomass and some amino acids. Hierarchical clustering analysis was used to evaluate effects of red LED in combination with high CO2 and Hoagland's formulation; distinctive cluster formation contained 14 amino acids (mostly branched-chain and aromatic amino acids, histidine and arginine). Thus, simultaneous treatments of monochromatic LED, high CO2 and nutrient formulation improved the amino acids accumulation, and likely reduced the inorganic nitrogen sources in planta.
Assuntos
Dióxido de Carbono/metabolismo , Lactuca/crescimento & desenvolvimento , Lactuca/metabolismo , Biomassa , Análise por Conglomerados , Eletroforese Capilar , Luz , Espectrometria de MassasRESUMO
NAD is a well-known co-enzyme that mediates hundreds of redox reactions and is the basis of various processes regulating cell responses to different environmental and developmental cues. The regulatory mechanism that determines the amount of cellular NAD and the rate of NAD metabolism remains unclear. We created Arabidopsis thaliana plants overexpressing the NAD synthase (NADS) gene that participates in the final step of NAD biosynthesis. NADS overexpression enhanced the activity of NAD biosynthesis but not the amounts of NAD+, NADH, NADP+ or NADPH. However, the amounts of some intermediates were elevated, suggesting that NAD metabolism increased. The NAD redox state was greatly facilitated by an imbalance between NAD generation and degradation in response to bolting. Metabolite profiling and transcriptional analysis revealed that the drastic modulation of NAD redox homeostasis increased tricarboxylic acid flux, causing the ectopic generation of reactive oxygen species. Vascular bundles suffered from oxidative stress, leading to a malfunction in amino acid and organic acid transportation that caused early wilting of the flower stalk and shortened plant longevity, probably due to malnutrition. We concluded that the mechanism regulating the balance between NAD synthesis and degradation is important in the systemic plant response to developmental cues during the growth-phase transition.
Assuntos
Arabidopsis/metabolismo , Arabidopsis/fisiologia , Longevidade , NAD/metabolismo , Desenvolvimento Vegetal , Amida Sintases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/metabolismo , Vias Biossintéticas , Coenzimas/metabolismo , Regulação da Expressão Gênica de Plantas , Homeostase , Peróxido de Hidrogênio/metabolismo , Metabolômica , Modelos Biológicos , Oxirredução , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , ReproduçãoRESUMO
BAX INHIBITOR-1 (BI-1) is a cell death suppressor widely conserved in plants and animals. Overexpression of BI-1 enhances tolerance to stress-induced cell death in plant cells, although the molecular mechanism behind this enhancement is unclear. We recently found that Arabidopsis (Arabidopsis thaliana) BI-1 is involved in the metabolism of sphingolipids, such as the synthesis of 2-hydroxy fatty acids, suggesting the involvement of sphingolipids in the cell death regulatory mechanism downstream of BI-1. Here, we show that BI-1 affects cell death-associated components localized in sphingolipid-enriched microdomains of the plasma membrane in rice (Oryza sativa) cells. The amount of 2-hydroxy fatty acid-containing glucosylceramide increased in the detergent-resistant membrane (DRM; a biochemical counterpart of plasma membrane microdomains) fraction obtained from BI-1-overexpressing rice cells. Comparative proteomics analysis showed quantitative changes of DRM proteins in BI-1-overexpressing cells. In particular, the protein abundance of FLOTILLIN HOMOLOG (FLOT) and HYPERSENSITIVE-INDUCED REACTION PROTEIN3 (HIR3) markedly decreased in DRM of BI-1-overexpressing cells. Loss-of-function analysis demonstrated that FLOT and HIR3 are required for cell death by oxidative stress and salicylic acid, suggesting that the decreased levels of these proteins directly contribute to the stress-tolerant phenotypes in BI-1-overexpressing rice cells. These findings provide a novel biological implication of plant membrane microdomains in stress-induced cell death, which is negatively modulated by BI-1 overexpression via decreasing the abundance of a set of key proteins involved in cell death.
Assuntos
Proteínas de Arabidopsis/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Membrana/metabolismo , Oryza/fisiologia , Proteínas de Arabidopsis/genética , Morte Celular/efeitos dos fármacos , Regulação da Expressão Gênica de Plantas , Glucosilceramidas/química , Glucosilceramidas/metabolismo , Microdomínios da Membrana/química , Proteínas de Membrana/genética , Oryza/citologia , Oryza/efeitos dos fármacos , Estresse Oxidativo/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteômica/métodos , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacologia , Esfingolipídeos/química , Esfingolipídeos/metabolismo , Estresse Fisiológico/genéticaRESUMO
Bud dormancy is an adaptive strategy that perennials use to survive unfavorable conditions. Gentians (Gentiana), popular alpine flowers and ornamentals, produce overwintering buds (OWBs) that can persist through the winter, but the mechanisms regulating dormancy are currently unclear. In this study, we conducted targeted metabolome analysis to obtain clues about the metabolic mechanisms involved in regulating OWB dormancy. Multivariate analysis of metabolite profiles revealed metabolite patterns characteristic of dormant states. The concentrations of gentiobiose [ß-D-Glcp-(1â6)-D-Glc] and gentianose [ß-D-Glcp-(1â6)-D-Glc-(1â2)-d-Fru] significantly varied depending on the stage of OWB dormancy, and the gentiobiose concentration increased prior to budbreak. Both activation of invertase and inactivation of ß-glucosidase resulted in gentiobiose accumulation in ecodormant OWBs, suggesting that gentiobiose is seldom used as an energy source but is involved in signaling pathways. Furthermore, treatment with exogenous gentiobiose induced budbreak in OWBs cultured in vitro, with increased concentrations of sulfur-containing amino acids, GSH, and ascorbate (AsA), as well as increased expression levels of the corresponding genes. Inhibition of GSH synthesis suppressed gentiobiose-induced budbreak accompanied by decreases in GSH and AsA concentrations and redox status. These results indicate that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of gentian OWBs through the AsA-GSH cycle.
Assuntos
Dissacarídeos/metabolismo , Gentiana/metabolismo , Meristema/metabolismo , Metabolômica/métodos , Proteínas de Plantas/metabolismo , Aminoácidos/metabolismo , Ácido Ascórbico/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Metabolismo dos Carboidratos/genética , Dissacarídeos/farmacologia , Dissacarídeos/fisiologia , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Gentiana/genética , Gentiana/fisiologia , Glutationa/metabolismo , Hexoquinase/genética , Hexoquinase/metabolismo , Meristema/genética , Meristema/fisiologia , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/genética , Metaboloma/genética , Dados de Sequência Molecular , Fosfoglucomutase/genética , Fosfoglucomutase/metabolismo , Proteínas de Plantas/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estações do Ano , beta-Frutofuranosidase/genética , beta-Frutofuranosidase/metabolismoRESUMO
The genomic nucleotide sequences of japonica rice (Sasanishiki and Nipponbare) contained about 2.7-kb unique region at the point of 0.4-kb upstream of the OsPsbS1 gene. In this study, we found that japonica rice with a few exceptions possessing such DNA sequences [denoted to OsMULE-japonica specific sequence (JSS)] is distinct by the presence of Mutator-like-element (MULE). Such sequence was absent in most of indica cultivars and Oryza glaberrima. In OsMULE-JSS1, we noted the presence of possible target site duplication (TSD; CTTTTCCAG) and about 80-bp terminal inverted repeat (TIR) near TSD. We also found the enhancement ofOsPsbS1 mRNA accumulation by intensified light, which was not associated with the DNA methylation status in OsMULE/JSS. In addition, O. rufipogon, possible ancestor of modern rice cultivars was found to compose PsbS gene of either japonica (minor) or indica (major) type. Transient gene expression assay showed that the japonica type promoter elevated a reporter gene activity than indica type.
Assuntos
Regulação da Expressão Gênica de Plantas , Genes de Plantas , Oryza/genética , Sequências Reguladoras de Ácido Nucleico , Sequência de Bases , Cromossomos de Plantas , Metilação de DNA , Dosagem de Genes , Expressão Gênica , Ordem dos Genes , Genes Reporter , Genoma de Planta , Dados de Sequência Molecular , Regiões Promotoras Genéticas , Sequências Repetidas Terminais , Transcrição Gênica , Ativação TranscricionalRESUMO
Bax inhibitor-1 (BI-1) is a widely conserved cell death suppressor localized in the endoplasmic reticulum membrane. Our previous results revealed that Arabidopsis BI-1 (AtBI-1) interacts with not only Arabidopsis cytochrome b 5 (Cb5), an electron transfer protein, but also a Cb5-like domain (Cb5LD)-containing protein, Saccharomyces cerevisiae fatty acid 2-hydroxylase 1, which 2-hydroxylates sphingolipid fatty acids. We have now found that AtBI-1 binds Arabidopsis sphingolipid Δ8 long-chain base (LCB) desaturases AtSLD1 and AtSLD2, which are Cb5LD-containing proteins. The expression of both AtBI-1 and AtSLD1 was increased by cold exposure. However, different phenotypes were observed in response to cold treatment between an atbi-1 mutant and a sld1sld2 double mutant. To elucidate the reasons behind the difference, we analyzed sphingolipids and found that unsaturated LCBs in atbi-1 were not altered compared to wild type, whereas almost all LCBs in sld1sld2 were saturated, suggesting that AtBI-1 may not be necessary for the desaturation of LCBs. On the other hand, the sphingolipid content in wild type increased in response to low temperature, whereas total sphingolipid levels in atbi-1 were unaltered. In addition, the ceramide-modifying enzymes AtFAH1, sphingolipid base hydroxylase 2 (AtSBH2), acyl lipid desaturase 2 (AtADS2) and AtSLD1 were highly expressed under cold stress, and all are likely to be related to AtBI-1 function. These findings suggest that AtBI-1 contributes to synthesis of sphingolipids during cold stress by interacting with AtSLD1, AtFAH1, AtSBH2 and AtADS2.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Proteínas de Membrana/genética , Esfingolipídeos/metabolismo , Sequência de Aminoácidos , Arabidopsis/fisiologia , Proteínas de Arabidopsis/metabolismo , Ceramidas/metabolismo , Temperatura Baixa , Ácidos Graxos/metabolismo , Proteínas de Membrana/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Dados de Sequência Molecular , Oxirredutases , Fenótipo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Alinhamento de Sequência , Esfingolipídeos/análise , Estresse FisiológicoRESUMO
Arabidopsis cell growth defect factor-1 (Cdf1 in yeast, At5g23040) was originally isolated as a cell growth suppressor of yeast from genetic screening. To investigate the in vivo role of Cdf1 in plants, a T-DNA insertion line was analyzed. A homozygous T-DNA insertion mutant (cdf1/cdf1) was embryo lethal and showed arrested embryogenesis at the globular stage. The Cdf1 protein, when fused with green fluorescent protein, was localized to the plastid in stomatal guard cells and mesophyll cells. A promoter-ß-glucuronidase assay found expression of Cdf1 in the early heart stage of embryogenesis, suggesting that Cdf1 was essential for Arabidopsis embryogenesis during the transition of the embryo from the globular to heart stage.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Proteínas de Transporte/metabolismo , Plastídeos/metabolismo , Proteínas Repressoras/metabolismo , Sementes/crescimento & desenvolvimento , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Teste de Complementação Genética , Heterozigoto , Mutação , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Proteínas Repressoras/genéticaRESUMO
A volatile metabolite, 2-methylisoborneol (2-MIB), causes an unpleasant taste and odor in tap water. Some filamentous cyanobacteria produce 2-MIB via a two-step biosynthetic pathway: methylation of geranyl diphosphate (GPP) by methyl transferase (GPPMT), followed by the cyclization of methyl-GPP by monoterpene cyclase (MIBS). We isolated the genes encoding GPPMT and MIBS from Pseudanabaena galeata, a filamentous cyanobacterium known to be a major causal organism of 2-MIB production in Japanese lakes. The predicted amino acid sequence showed high similarity with that of Pseudanabaena limnetica (96% identity in GPPMT and 97% identity in MIBS). P. galeata was cultured at different temperatures to examine the effect of growth conditions on the production of 2-MIB and major metabolites. Gas chromatograph-mass spectrometry (GC-MS) measurements showed higher accumulation of 2-MIB at 30 °C than at 4 °C or 20 °C after 24 h of culture. Real-time-RT PCR analysis showed that the expression levels of the genes encoding GPPMT and MIBS decreased at 4 °C and increased at 30 °C, compared with at 20 °C. Furthermore, metabolite analysis showed dramatic changes in primary metabolite concentrations in cyanobacteria grown at different temperatures. The data indicate that changes in carbon flow in the TCA cycle affect 2-MIB biosynthesis at higher temperatures.
Assuntos
Cianobactérias/genética , Cianobactérias/metabolismo , Regulação Bacteriana da Expressão Gênica , Redes e Vias Metabólicas/fisiologia , Temperatura , Canfanos/metabolismoRESUMO
Hydrogen peroxide (H2O2) is known to be a typical endogenous signaling molecule that triggers programmed cell death in plants and metazoan. In this respect, they seem to share the mechanism of cell death caused by H2O2 and other reactive oxygen species (ROS). Bax inhibitor-1 (BI-1) is a well-conserved protein in plants and animals that serves as the inhibitor of mammalian proapoptotic proteins as well as plant ROS-induced cell death. As a target of H2O2, mitochondrion is considered to be an organelle of the primary ROS generation and perception. Thus, analysis of mitochondrial behavior in relation to functional roles of regulatory proteins (e.g., BI-1) will lead us to understand the core mechanisms of cell death regulation conserved in eukaryotes. In this chapter, we first introduce techniques of analyzing H2O2- (and ROS-) mediated changes in mitochondrial behavior. Next, we describe our understanding of the functions of plant BI-1 in regulation of ROS-induced cell death, with a technical basis for assessment of tolerance to ROS-mediated cell death in model plant systems.
Assuntos
Apoptose , Proteínas de Arabidopsis/fisiologia , Peróxido de Hidrogênio/metabolismo , Proteínas de Membrana/fisiologia , Adaptação Fisiológica , Arabidopsis/citologia , Arabidopsis/metabolismo , Técnicas de Cultura de Células , Mitocôndrias/fisiologia , Oryza/citologia , Oryza/metabolismo , Estresse Oxidativo , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Nicotiana/citologia , Nicotiana/metabolismoRESUMO
cyAbrB is a transcriptional regulator unique to and highly conserved among cyanobacterial species. A gene-disrupted mutant of cyabrB2 (sll0822) in Synechocystis sp. PCC 6803 exhibited severe growth inhibition and abnormal accumulation of glycogen granules within cells under photomixotrophic conditions. Within 6 h after the shift to photomixotrophic conditions, sodium bicarbonate-dependent oxygen evolution activity markedly declined in the ΔcyabrB2 mutant, but the decrease in methyl viologen-dependent electron transport activity was much smaller, indicating inhibition in carbon dioxide fixation. Decreases in the transcript levels of several genes related to sugar catabolism, carbon dioxide fixation, and nitrogen metabolism were also observed within 6 h. Metabolome analysis by capillary electrophoresis mass spectrometry revealed that several metabolites accumulated differently in the wild-type and mutant strains. For example, the amounts of pyruvate and 2-oxoglutarate (2OG) were significantly lower in the mutant than in the wild type, irrespective of trophic conditions. The growth rate of the ΔcyabrB2 mutant was restored to a level comparable to that under photoautotrophic conditions by addition of 2OG to the growth medium under photomixotrophic conditions. Activities of various metabolic processes, including carbon dioxide fixation, respiration, and nitrogen assimilation, seemed to be enhanced by 2OG addition. These observations suggest that cyAbrB2 is essential for the active transcription of genes related to carbon and nitrogen metabolism upon a shift to photomixotrophic conditions. Deletion of cyAbrB2 is likely to deregulate the partition of carbon between storage forms and soluble forms used for biosynthetic purposes. This disorder may cause inactivation of cellular metabolism, excess accumulation of reducing equivalents, and subsequent loss of viability under photomixotrophic conditions.
Assuntos
Proteínas de Bactérias/genética , Mutação , Synechocystis/genética , Synechocystis/metabolismo , Proteínas de Bactérias/metabolismo , Carbono/metabolismo , Deleção de Genes , Regulação Bacteriana da Expressão Gênica , Ácidos Cetoglutáricos/metabolismo , Ácidos Cetoglutáricos/farmacologia , Metaboloma , Nitrogênio/metabolismo , Fotossíntese/genética , Ácido Pirúvico/metabolismo , Bicarbonato de Sódio/metabolismo , Synechocystis/efeitos dos fármacos , Synechocystis/crescimento & desenvolvimentoRESUMO
Terrestrial plant pollen is classified into two categories based on its metabolic status: pollen with low-metabolism are termed "orthodox" and pollen with high-metabolism are termed "recalcitrant." Nicotinamide adenine dinucleotide (NAD) is crucial for a number of metabolisms in all extant organisms. It has recently been shown that NAD homeostasis plays an important role in a broad range of developmental processes and responses to environment. Recently, a reverse genetic approach shed light on the significance of NAD biosynthesis on pollen fate. In orthodox Arabidopsis pollen, NAD(+) that was accumulated in excess at dispersal dramatically decreased on rehydration. The lack of a key gene that is involved in NAD biosynthesis compromised the excess accumulation. Moreover, absence of the excess accumulation phenocopied the so-called recalcitrant pollen, as demonstrated by the germination inside anthers and the loss of desiccation tolerance. Upon rehydration, NAD(+)-consuming inhibitors impaired tube germination. Taken together, our results suggest that accumulation of NAD(+) functions as a physiochemical molecular switch for suspended metabolism and that the decrease of NAD(+) plays a very important role during transitions in metabolic states. Shifting of the redox state to an oxidizing environment may efficiently control the comprehensive metabolic network underlying the onset of pollen germination.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , NAD/metabolismo , Pólen/metabolismo , Homeostase , Nicotinamida-Nucleotídeo Adenililtransferase/metabolismo , OxirreduçãoRESUMO
Although the nicotinamide nucleotides NAD(H) and NADP(H) are essential for various metabolic reactions that play major roles in maintenance of cellular homeostasis, the significance of NAD biosynthesis is not well understood. Here, we investigated the dynamics of pollen nicotinamide nucleotides in response to imbibition, a representative germination cue. Metabolic analysis with capillary electrophoresis electrospray ionization mass spectrometry revealed that excess amount of NAD+ is accumulated in freshly harvested dry pollen, whereas it dramatically decreased immediately after contact with water. Importantly, excess of NAD+ impaired pollen tube growth. Moreover, NAD+ accumulation was retained after pollen was imbibed in the presence of NAD+-consuming reaction inhibitors and pollen germination was greatly retarded. Pollen deficient in the nicotinate/nicotinamide mononucleotide adenyltransferase (NMNAT) gene, encoding a key enzyme in NAD biosynthesis, and a lack of NAD+ accumulation in the gametophyte, showed precocious pollen tube germination inside the anther locule and vigorous tube growth under high-humidity conditions. Hence, the accumulation of excess NAD+ is not essential for pollen germination, but instead participates in regulating the timing of germination onset. These results indicate that NAD+ accumulation acts to negatively regulate germination and a decrease in NAD+ plays an important role in metabolic state transition.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/metabolismo , Germinação , NAD/metabolismo , Pólen/crescimento & desenvolvimento , Pólen/metabolismo , Vias Biossintéticas , Umidade , NAD/biossíntese , Tubo Polínico/crescimento & desenvolvimento , Tubo Polínico/metabolismo , Sobrevivência de TecidosRESUMO
2-Hydroxy fatty acids mainly contained in sphingolipids are synthesized by a sphingolipid fatty acid 2-hydroxylase (FAH). Recently, two FAH homologs in Arabidopsis thaliana (AtFAH1 and AtFAH2), without any cytochrome b5(Cb5)-like domains, which are essential for the function of Saccharomyces cerevisiae and mammalian FAH, were identified and both AtFAHs were shown to be activated by the interaction with Cb5. In this study, we compared FAHs of various plants, animals and fungi. Interestingly, only plants had two FAH homologs and none of plant FAHs had any Cb5-like domains. In addition, we showed from the interaction and expression analyses that AtFAHs interacted with multiple Cb5s probably in various tissues. Thus, plant FAHs may have evolved unlike animal and fungus FAHs.
Assuntos
Proteínas de Arabidopsis/metabolismo , Oxigenases de Função Mista/metabolismo , Animais , Proteínas de Arabidopsis/genética , Citocromos b5/genética , Citocromos b5/metabolismo , Oxigenases de Função Mista/genética , Ligação Proteica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
The isogamous green alga Chlamydomonas reinhardtii has emerged as a premier model for studying the genetic regulation of fertilization and sexual development. A key regulator is known to be a homeoprotein gene, GAMETE-SPECIFIC PLUS1 (GSP1), which triggers the zygotic program. In this study, we isolated a mutant, biparental31 (bp31), which lacks GSP1. bp31 mt+ gametes fuse normally to form zygotes, but the sexual development of the resulting diploid cell is arrested and pellicle/zygospore/tetrad formation is abolished. The uniparental inheritance of chloroplast (cp) and mitochondrial (mt) DNA (cytoplasmic inheritance) was also impaired. bp31 has a deletion of â¼60 kb on chromosome 2, including GSP1. The mutant phenotype was not rescued by transformation with GSP1 alone but could be rescued by the cotransformation with GSP1 and another gene, INOSITOL MONOPHOSPHATASE-LIKE1, which is involved in various cellular processes, including the phosphatidylinositol signaling pathway. This study confirms the importance of Gsp1 in mediating the zygotic program, including the uniparental inheritance of cp/mtDNA. Moreover, the results also suggest a role for inositol metabolism in the sexual developmental program.
Assuntos
Chlamydomonas reinhardtii/genética , DNA de Cloroplastos/genética , DNA Mitocondrial/genética , Proteínas de Homeodomínio/metabolismo , Proteínas de Plantas/genética , Zigoto/crescimento & desenvolvimento , Chlamydomonas reinhardtii/crescimento & desenvolvimento , Herança Extracromossômica , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Proteínas de Homeodomínio/genética , Dados de Sequência Molecular , Mutação , Proteínas de Plantas/metabolismo , Deleção de SequênciaRESUMO
2-Hydroxy fatty acids (2-HFAs) are predominantly present in sphingolipids and have important physicochemical and physiological functions in eukaryotic cells. Recent studies from our group demonstrated that sphingolipid fatty acid 2-hydroxylase (FAH) is required for the function of Arabidopsis (Arabidopsis thaliana) Bax inhibitor-1 (AtBI-1), which is an endoplasmic reticulum membrane-localized cell death suppressor. However, little is known about the function of two Arabidopsis FAH homologs (AtFAH1 and AtFAH2), and it remains unclear whether 2-HFAs participate in cell death regulation. In this study, we found that both AtFAH1 and AtFAH2 had FAH activity, and the interaction with Arabidopsis cytochrome b5 was needed for the sufficient activity. 2-HFA analysis of AtFAH1 knockdown lines and atfah2 mutant showed that AtFAH1 mainly 2-hydroxylated very-long-chain fatty acid (VLCFA), whereas AtFAH2 selectively 2-hydroxylated palmitic acid in Arabidopsis. In addition, 2-HFAs were related to resistance to oxidative stress, and AtFAH1 or 2-hydroxy VLCFA showed particularly strong responses to oxidative stress. Furthermore, AtFAH1 interacted with AtBI-1 via cytochrome b5 more preferentially than AtFAH2. Our results suggest that AtFAH1 and AtFAH2 are functionally different FAHs, and that AtFAH1 or 2-hydroxy VLCFA is a key factor in AtBI-1-mediated cell death suppression.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Ácidos Graxos/metabolismo , Oxigenases de Função Mista/metabolismo , Esfingolipídeos/metabolismo , Estresse Fisiológico , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Sistema Enzimático do Citocromo P-450/genética , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Ésteres/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Peróxido de Hidrogênio/farmacologia , Hidroxilação/efeitos dos fármacos , Oxigenases de Função Mista/genética , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Células Vegetais/efeitos dos fármacos , Células Vegetais/metabolismo , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Estresse Fisiológico/genéticaRESUMO
Previously, a dysfunction of the SMALL ACIDIC PROTEIN1 (SMAP1) gene was identified as the cause of the anti-auxin resistant1 (aar1) mutant of Arabidopsis (Arabidopsis thaliana). SMAP1 is involved in the response pathway of synthetic auxin, 2,4-dichlorophenoxyacetic acid, and functions upstream of the auxin/indole-3-acetic acid protein degradation step in auxin signaling. However, the exact mechanism by which SMAP1 functions in auxin signaling remains unknown. Here, we demonstrate that SMAP1 is required for normal plant growth and development and the root response to indole-3-acetic acid or methyl jasmonate in the auxin resistant1 (axr1) mutation background. Deletion analysis and green fluorescent protein/glutathione S-transferase pull-down assays showed that SMAP1 physically interacts with the CONSTITUTIVE PHOTOMORPHOGENIC9 SIGNALOSOME (CSN) via the SMAP1 F/D region. The extremely dwarf phenotype of the aar1-1 csn5a-1 double mutant confirms the functional role of SMAP1 in plant growth and development under limiting CSN functionality. Our findings suggest that SMAP1 is involved in the auxin response and possibly in other cullin-RING ubiquitin ligase-regulated signaling processes via its interaction with components associated with RELATED TO UBIQUITIN modification.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/crescimento & desenvolvimento , Ubiquitinas/metabolismo , Ácido 2,4-Diclorofenoxiacético/farmacologia , Acetatos/farmacologia , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Caulimovirus/genética , Caulimovirus/metabolismo , Ciclopentanos/farmacologia , Regulação da Expressão Gênica de Plantas , Teste de Complementação Genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde/metabolismo , Ácidos Indolacéticos/farmacologia , Mutação , Oxilipinas/farmacologia , Fenótipo , Epiderme Vegetal/genética , Epiderme Vegetal/metabolismo , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Plantas Geneticamente Modificadas/metabolismo , Regiões Promotoras Genéticas , Mapeamento de Interação de Proteínas , Interferência de RNA , Sementes/genética , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Transdução de Sinais , Ubiquitinas/genéticaRESUMO
When ammonium is the sole nitrogen (N) source, plant growth is suppressed compared with the situation where nitrate is the N source. This is commonly referred to as ammonium toxicity. It is widely known that a combination of nitrate and ammonium as N source alleviates this ammonium toxicity (nitrate-dependent alleviation of ammonium toxicity), but the underlying mechanisms are still not completely understood. In plants, ammonium toxicity is often accompanied by a depletion of organic acids and inorganic cations, and by an accumulation of ammonium. All these factors have been considered as possible causes for ammonium toxicity. Thus, we hypothesized that nitrate could alleviate ammonium toxicity by lessening these symptoms. We analyzed growth, inorganic N and cation content and various primary metabolites in shoots of Arabidopsis thaliana seedlings grown on media containing various concentrations of nitrate and/or ammonium. Nitrate-dependent alleviation of ammonium toxicity was not accompanied by less depletion of organic acids and inorganic cations, and showed no reduction in ammonium accumulation. On the other hand, shoot growth was significantly correlated with the nitrate concentration in the shoots. This suggests that nitrate-dependent alleviation of ammonium toxicity is related to physiological processes that are closely linked to nitrate signaling, uptake and reduction. Based on transcript analyses of various genes related to nitrate signaling, uptake and reduction, possible underlying mechanisms for the nitrate-dependent alleviation are discussed.