RESUMO
In Arabidopsis thaliana, biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, PfCRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Glutationa/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Animais , Antimaláricos/farmacologia , Cádmio/farmacologia , Cloroquina/farmacologia , Resistência a Medicamentos , Feminino , Genes de Plantas , Homeostase , Técnicas In Vitro , Modelos Biológicos , Mutação , Oócitos/metabolismo , Plantas Geneticamente Modificadas , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , XenopusRESUMO
Glutathione (GSH) homeostasis in plants is essential for cellular redox control and efficient responses to abiotic and biotic stress. Compartmentation of the GSH biosynthetic pathway is a unique feature of plants. The first enzyme, gamma-glutamate cysteine ligase (GSH1), responsible for synthesis of gamma-glutamylcysteine (gamma-EC), is, in Arabidopsis, exclusively located in the plastids, whereas the second enzyme, glutathione synthetase (GSH2), is located in both plastids and cytosol. In Arabidopsis, gsh2 insertion mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gsh1 null mutants. This difference in phenotype may be due to partial replacement of GSH functions by gamma-EC, which in gsh2 mutants hyperaccumulates to levels 5000-fold that in the wild type and 200-fold wild-type levels of GSH. In situ labelling of thiols with bimane and confocal imaging in combination with HPLC analysis showed high concentrations of gamma-EC in the cytosol. Feedback inhibition of Brassica juncea plastidic GSH1 by gamma-EC in vitro strongly suggests export of gamma-EC as functional explanation for hyperaccumulation. Complementation of gsh2 mutants with the cytosol-specific GSH2 gave rise to phenotypically wild-type transgenic plants. These results support the conclusion that cytosolic synthesis of GSH is sufficient for plant growth. The transgenic lines further show that, consistent with the exclusive plastidic localization of GSH1, gamma-EC is exported from the plastids to supply the cytosol with the immediate precursor for GSH biosynthesis, and that there can be efficient re-import of GSH into the plastids to allow effective control of GSH biosynthesis through feedback inhibition of GSH1.
Assuntos
Arabidopsis/genética , Citosol/metabolismo , Glutationa/biossíntese , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Dipeptídeos/metabolismo , Relação Dose-Resposta a Droga , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Mostardeira/enzimologia , Mutagênese Insercional , Fenótipo , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plastídeos/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/citologia , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Coloração e Rotulagem , Compostos de Sulfidrila/farmacologiaRESUMO
Aims: Circular RNA (circRNA) is a newly validated class of single-stranded RNA, ubiquitously expressed in mammalian tissues and possessing key functions including acting as microRNA sponges and as transcriptional regulators by binding to RNA-binding proteins. While independent studies confirm the expression of circRNA in various tissue types, genome-wide circRNA expression in the heart has yet to be described in detail. Methods and results: We performed deep RNA-sequencing on ribosomal-depleted RNA isolated from 12 human hearts, 25 mouse hearts and across a 28-day differentiation time-course of human embryonic stem cell-derived cardiomyocytes. Using purpose-designed bioinformatics tools, we uncovered a total of 15 318 and 3017 cardiac circRNA within human and mouse, respectively. Their abundance generally correlates with the abundance of their cognate linear RNA, but selected circRNAs exist at disproportionately higher abundance. Top highly expressed circRNA corresponded to key cardiac genes including Titin (TTN), RYR2, and DMD. The most abundant cardiac-expressed circRNA is a cytoplasmic localized single-exon circSLC8A1-1. The longest human transcript TTN alone generates up to 415 different exonic circRNA isoforms, the majority (83%) of which originates from the I-band domain. Finally, we confirmed the expression of selected cardiac circRNA by RT-PCR, Sanger sequencing and single molecule RNA-fluorescence in situ hybridization. Conclusions: Our data provide a detailed circRNA expression landscape in hearts. There is a high-abundance of specific cardiac-expressed circRNA. These findings open up a new avenue for future investigation into this emerging class of RNA.
Assuntos
Células-Tronco Embrionárias/metabolismo , Cardiopatias/genética , Miócitos Cardíacos/metabolismo , RNA/genética , Animais , Estudos de Casos e Controles , Diferenciação Celular , Linhagem Celular , Biologia Computacional , Bases de Dados Genéticas , Regulação da Expressão Gênica no Desenvolvimento , Estudos de Associação Genética , Marcadores Genéticos , Predisposição Genética para Doença , Cardiopatias/diagnóstico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Camundongos , Fenótipo , Reação em Cadeia da Polimerase , RNA/metabolismo , RNA Circular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Imagem Individual de Molécula , Fatores de TempoRESUMO
In higher plants the L-galactose pathway represents the major route for ascorbate biosynthesis. The first committed step of this pathway is catalyzed by the enzyme GDP-L-galactose phosphorylase and is encoded by two paralogs in Arabidopsis - VITAMIN C2 (VTC2) and VTC5. The first mutant of this enzyme, vtc2-1, isolated via an EMS mutagenesis screen, has approximately 20-30% of wildtype ascorbate levels and has been reported to have decreased growth under standard laboratory conditions. Here, we show that a T-DNA insertion into the VTC2 causes a similar reduction in ascorbate levels, but does not greatly affect plant growth. Subsequent segregation analysis revealed the growth defects of vtc2-1 mutants segregate independently of the vtc2-1 mutation. These observations suggest that it is the presence of an independent cryptic mutation that affects growth of vtc2-1 mutants, and not the 70-80% decrease in ascorbate levels that has been assumed in past studies.