RESUMO
Compartmentation is a key strategy enacted by plants for the storage of specialized metabolites. The saffron spice owes its red color to crocins, a complex mixture of apocarotenoid glycosides that accumulate in intracellular vacuoles and reach up to 10% of the spice dry weight. We developed a general approach, based on coexpression analysis, heterologous expression in yeast (Saccharomyces cerevisiae), and in vitro transportomic assays using yeast microsomes and total plant metabolite extracts, for the identification of putative vacuolar metabolite transporters, and we used it to identify Crocus sativus transporters mediating vacuolar crocin accumulation in stigmas. Three transporters, belonging to both the multidrug and toxic compound extrusion and ATP binding cassette C (ABCC) families, were coexpressed with crocins and/or with the gene encoding the first dedicated enzyme in the crocin biosynthetic pathway, CsCCD2. Two of these, belonging to the ABCC family, were able to mediate transport of several crocins when expressed in yeast microsomes. CsABCC4a was selectively expressed in C. sativus stigmas, was predominantly tonoplast localized, transported crocins in vitro in a stereospecific and cooperative way, and was able to enhance crocin accumulation when expressed in Nicotiana benthamiana leaves.plantcell;31/11/2789/FX1F1fx1.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Carotenoides/metabolismo , Crocus/metabolismo , Proteínas de Plantas/metabolismo , Vacúolos/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Vias Biossintéticas , Clonagem Molecular , Crocus/genética , Flores/metabolismo , Regulação da Expressão Gênica de Plantas , Cinética , Extratos Vegetais , Folhas de Planta/citologia , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Saccharomyces cerevisiae/genética , Distribuição Tecidual/fisiologia , Nicotiana/genética , Nicotiana/metabolismoRESUMO
Tip-focused accumulation of reactive oxygen species (ROS) is tightly associated with pollen tube growth and is thus critical for fertilization. However, it is unclear how tip-growing cells establish such specific ROS localization. Polyamines have been proposed to function in tip growth as precursors of the ROS, hydrogen peroxide. The ABC transporter AtABCG28 may regulate ROS status, as it contains multiple cysteine residues, a characteristic of proteins involved in ROS homeostasis. In this study, we found that AtABCG28 was specifically expressed in the mature pollen grains and pollen tubes. AtABCG28 was localized to secretory vesicles inside the pollen tube that moved toward and fused with the plasma membrane of the pollen tube tip. Knocking out AtABCG28 resulted in defective pollen tube growth, failure to localize polyamine and ROS to the growing pollen tube tip, and complete male sterility, whereas ectopic expression of this gene in root hair could recover ROS accumulation at the tip and improved the growth under high-pH conditions, which normally prevent ROS accumulation and tip growth. Together, these data suggest that AtABCG28 is critical for localizing polyamine and ROS at the growing tip. In addition, this function of AtABCG28 is likely to protect the pollen tube from the cytotoxicity of polyamine and contribute to the delivery of polyamine to the growing tip for incorporation into the expanding cell wall.
Assuntos
Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Tubo Polínico/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/química , Tubo Polínico/crescimento & desenvolvimento , Conformação Proteica , Homologia de Sequência de AminoácidosRESUMO
Cereals are a staple food for many people around the world; however, they are also a major dietary source of toxic metal(loid)s. Many agricultural regions throughout the world are contaminated with toxic metal(loid)s, which can accumulate to high levels in the grains of cereals cultivated in these regions, posing serious health risks to consumers. Arsenic (As) and cadmium (Cd) are efficiently accumulated in cereals through metal transport pathways. Therefore, there is an urgent need to develop crops that contain greatly reduced levels of toxic metal(loid)s. Vacuolar sequestration of toxic metal(loid)s is a primary strategy for reducing toxic metal(loid)s in grains. However, until recently, detailed strategies and mechanisms for reducing toxic metal(loid)s in grain were limited by the lack of experimental data. New strategies to reduce As and Cd in grain by enhancing vacuolar sequestration in specific tissues are critical to develop crops that lower the daily intake of As and Cd, potentially improving human health. This review provides insights and strategies for developing crops with strongly reduced amounts of toxic metal(loid)s without jeopardizing agronomic traits.
RESUMO
Abscisic acid (ABA) integrates internal and external signals to coordinate plant development, growth and architecture. It plays a central role in stomatal closure, and prevents germination of freshly produced seeds and germination of non-dormant seeds under unfavorable circumstances. Here, we describe a Medicago truncatula ATP-binding cassette (ABC) transporter, MtABCG20, as an ABA exporter present in roots and germinating seeds. In seeds, MtABCG20 was found in the hypocotyl-radicle transition zone of the embryonic axis. Seeds of mtabcg20 plants were more sensitive to ABA upon germination, due to the fact that ABA translocation within mtabcg20 embryos was impaired. Additionally, the mtabcg20 produced fewer lateral roots and formed more nodules compared with wild-type plants in conditions mimicking drought stress. Heterologous expression in Arabidopsis thaliana provided evidence that MtABCG20 is a plasma membrane protein that is likely to form homodimers. Moreover, export of ABA from Nicotiana tabacum BY2 cells expressing MtABCG20 was faster than in the BY2 without MtABCG20. Our results have implications both in legume crop research and determination of the fundamental molecular processes involved in drought response and germination.
Assuntos
Ácido Abscísico/metabolismo , Medicago truncatula/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Germinação/genética , Germinação/fisiologia , Medicago truncatula/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMO
Phospholipids (PLs) are emerging as important factors that initiate signal transduction cascades at the plasma membrane. Their distribution within biological membranes is tightly regulated, e.g. by ATP-binding cassette (ABC) transporters, which preferably translocate PLs from the cytoplasmic to the exoplasmic membrane leaflet and are therefore called PL-floppases. Here, we demonstrate that a plant ABC transporter, Lr34 from wheat (Triticum aestivum), is involved in plasma membrane remodeling characterized by an intracellular accumulation of phosphatidic acid and enhanced outward translocation of phosphatidylserine. In addition, the content of phosphatidylinositol 4,5-bisphosphate in the cytoplasmic leaflet of the plasma membrane was reduced in the presence of the ABC transporter. When heterologously expressed in Saccharomyces cerevisiae, Lr34 promoted oil body formation in a mutant defective in PL-transfer in the secretory pathway. Our results suggest that PL redistribution by Lr34 potentially affects the membrane-bound proteome and contributes to the previously reported stimuli-independent activation of biotic and abiotic stress responses and neutral lipid accumulation in transgenic Lr34-expressing barley plants.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Lipídeos de Membrana/metabolismo , Proteínas de Plantas/metabolismo , Triticum/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transporte Biológico , Membrana Celular/metabolismo , Gotículas Lipídicas/metabolismo , Metabolismo dos Lipídeos , Fosfolipídeos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Nicotiana/genéticaRESUMO
Arsenic (As) is a poisonous element that causes severe skin lesions and cancer in humans. Rice (Oryza sativa L.) is a major dietary source of As in humans who consume this cereal as a staple food. We hypothesized that increasing As vacuolar sequestration would inhibit its translocation into the grain and reduce the amount of As entering the food chain. We developed transgenic rice plants expressing two different vacuolar As sequestration genes, ScYCF1 and OsABCC1, under the control of the RCc3 promoter in the root cortical and internode phloem cells, along with a bacterial γ-glutamylcysteine synthetase driven by the maize UBI promoter. The transgenic rice plants exhibited reduced root-to-shoot and internode-to-grain As translocation, resulting in a 70% reduction in As accumulation in the brown rice without jeopardizing agronomic traits. This technology could be used to reduce As intake, particularly in populations of South East Asia suffering from As toxicity and thereby improve human health.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Arsênio/metabolismo , Grão Comestível/metabolismo , Oryza/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Grão Comestível/crescimento & desenvolvimento , Genes Bacterianos , Engenharia Genética , Glutamato-Cisteína Ligase/genética , Oryza/genética , Oryza/crescimento & desenvolvimento , Plantas Geneticamente Modificadas , Regiões Promotoras GenéticasRESUMO
Soil contamination by heavy metals and metalloids such as cadmium (Cd) and arsenic (As) poses a major threat to the environment and to human health. Vacuolar sequestration is one of the main mechanisms by which plants control toxic materials including Cd and As. Understanding the mechanisms of heavy metal tolerance and accumulation can be useful for both phytoremediation and safe crop development. In this review, we summarize recent advances in deciphering the molecular mechanisms underlying vacuolar sequestration of Cd and As, and discuss potential biotechnological applications of this knowledge and efforts towards attaining these goals.
Assuntos
Arsênio/metabolismo , Biodegradação Ambiental , Cádmio/metabolismo , Proteínas de Plantas/metabolismo , Vacúolos/metabolismo , Arsênio/farmacocinética , Cádmio/farmacocinética , Proteínas de Transporte de Cátions/metabolismo , Produtos Agrícolas/efeitos dos fármacos , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Plantas/efeitos dos fármacos , Plantas/metabolismo , Poluentes do Solo/metabolismo , Poluentes do Solo/farmacocinética , Vacúolos/efeitos dos fármacosRESUMO
The exact transport characteristics of the vacuolar dicarboxylate transporter tDT from Arabidopsis are elusive. To overcome this limitation, we combined a range of experimental approaches comprising generation/analysis of tDT overexpressors, 13CO2 feeding and quantification of 13C enrichment, functional characterization of tDT in proteoliposomes, and electrophysiological studies on vacuoles. tdt knockout plants showed decreased malate and increased citrate concentrations in leaves during the diurnal light-dark rhythm and after onset of drought, when compared with wildtypes. Interestingly, under the latter two conditions, tDT overexpressors exhibited malate and citrate levels opposite to tdt knockout plants. Highly purified tDT protein transports malate and citrate in a 1:1 antiport mode. The apparent affinity for malate decreased with decreasing pH, whereas citrate affinity increased. This observation indicates that tDT exhibits a preference for dianion substrates, which is supported by electrophysiological analysis on intact vacuoles. tDT also accepts fumarate and succinate as substrates, but not α-ketoglutarate, gluconate, sulfate, or phosphate. Taking tDT as an example, we demonstrated that it is possible to reconstitute a vacuolar metabolite transporter functionally in proteoliposomes. The displayed, so far unknown counterexchange properties of tDT now explain the frequently observed reciprocal concentration changes of malate and citrate in leaves from various plant species. tDT from Arabidopsis is the first member of the well-known and widely present SLC13 group of carrier proteins, exhibiting an antiport mode of transport.
Assuntos
Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ácido Cítrico/metabolismo , Malatos/metabolismo , Transportadores de Ânions Orgânicos/isolamento & purificação , Transportadores de Ânions Orgânicos/metabolismo , Vacúolos/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Proteínas de Arabidopsis/genética , Transporte Biológico , Transportadores de Ânions Orgânicos/genéticaRESUMO
Plants reorganize their root architecture to avoid growth into unfavorable regions of the rhizosphere. In a screen based on chimeric repressor gene-silencing technology, we identified the Arabidopsis thaliana GeBP-LIKE 4 (GPL4) transcription factor as an inhibitor of root growth that is induced rapidly in root tips in response to cadmium (Cd). We tested the hypothesis that GPL4 functions in the root avoidance of Cd by analyzing root proliferation in split medium, in which only half of the medium contained toxic concentrations of Cd. The wild-type (WT) plants exhibited root avoidance by inhibiting root growth in the Cd side but increasing root biomass in the control side. By contrast, GPL4-suppression lines exhibited nearly comparable root growth in the Cd and control sides and accumulated more Cd in the shoots than did the WT. GPL4 suppression also altered the root avoidance of toxic concentrations of other essential metals, modulated the expression of many genes related to oxidative stress, and consistently decreased reactive oxygen species concentrations. We suggest that GPL4 inhibits the growth of roots exposed to toxic metals by modulating reactive oxygen species concentrations, thereby allowing roots to colonize noncontaminated regions of the rhizosphere.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Metais Pesados/toxicidade , Raízes de Plantas/metabolismo , Fatores de Transcrição/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , Transporte Biológico/efeitos dos fármacos , Biomassa , Contagem de Células , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Glutationa/farmacologia , Meristema/citologia , Meristema/efeitos dos fármacos , Meristema/metabolismo , Modelos Biológicos , Estresse Oxidativo/efeitos dos fármacos , Estresse Oxidativo/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/crescimento & desenvolvimento , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Espécies Reativas de Oxigênio/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacosRESUMO
Petunia hybrida is a popular bedding plant that has a long history as a genetic model system. We report the whole-genome sequencing and assembly of inbred derivatives of its two wild parents, P. axillaris N and P. inflata S6. The assemblies include 91.3% and 90.2% coverage of their diploid genomes (1.4 Gb; 2n = 14) containing 32,928 and 36,697 protein-coding genes, respectively. The genomes reveal that the Petunia lineage has experienced at least two rounds of hexaploidization: the older gamma event, which is shared with most Eudicots, and a more recent Solanaceae event that is shared with tomato and other solanaceous species. Transcription factors involved in the shift from bee to moth pollination reside in particularly dynamic regions of the genome, which may have been key to the remarkable diversity of floral colour patterns and pollination systems. The high-quality genome sequences will enhance the value of Petunia as a model system for research on unique biological phenomena such as small RNAs, symbiosis, self-incompatibility and circadian rhythms.
Assuntos
Evolução Molecular , Genoma de Planta , Hibridização Genética , Petunia/genética , PoliploidiaRESUMO
MAIN CONCLUSION: This review presents the role of strigolactone transport in regulating plant root and shoot architecture, plant-fungal symbiosis and the crosstalk with several phytohormone pathways. The authors, based on their data and recently published results, suggest that long-distance, as well local strigolactone transport might occur in a cell-to-cell manner rather than via the xylem stream. Strigolactones (SLs) are recently characterized carotenoid-derived phytohormones. They play multiple roles in plant architecture and, once exuded from roots to soil, in plant-rhizosphere interactions. Above ground SLs regulate plant developmental processes, such as lateral bud outgrowth, internode elongation and stem secondary growth. Below ground, SLs are involved in lateral root initiation, main root elongation and the establishment of the plant-fungal symbiosis known as mycorrhiza. Much has been discovered on players and patterns of SL biosynthesis and signaling and shown to be largely conserved among different plant species, however little is known about SL distribution in plants and its transport from the root to the soil. At present, the only characterized SL transporters are the ABCG protein PLEIOTROPIC DRUG RESISTANCE 1 from Petunia axillaris (PDR1) and, in less detail, its close homologue from Nicotiana tabacum PLEIOTROPIC DRUG RESISTANCE 6 (PDR6). PDR1 is a plasma membrane-localized SL cellular exporter, expressed in root cortex and shoot axils. Its expression level is regulated by its own substrate, but also by the phytohormone auxin, soil nutrient conditions (mainly phosphate availability) and mycorrhization levels. Hence, PDR1 integrates information from nutrient availability and hormonal signaling, thus synchronizing plant growth with nutrient uptake. In this review we discuss the effects of PDR1 de-regulation on plant development and mycorrhization, the possible cross-talk between SLs and other phytohormone transporters and finally the need for SL transporters in different plant species.
Assuntos
Lactonas/metabolismo , Desenvolvimento Vegetal , Reguladores de Crescimento de Plantas/metabolismo , Transporte Biológico , Comunicação Celular , Sequência Conservada , Filogenia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Análise de Sequência de Proteína , SimbioseRESUMO
The distribution of the N-glycoproteome in integral membrane proteins of the vacuolar membrane (tonoplast) or the plasma membrane of Arabidopsis thaliana and, for further comparison, of the Rattus norvegicus lysosomal and plasma membranes, was analyzed. In silico analysis showed that potential N-glycosylation sites are much less frequent in tonoplast proteins. Biochemical analysis of Arabidopsis subcellular fractions with the lectin concanavalin A, which recognizes mainly unmodified N-glycans, or with antiserum against Golgi-modified N-glycans confirmed the in silico results and showed that, unlike the plant plasma membrane, the tonoplast is almost or totally devoid of N-glycoproteins with Golgi-modified glycans. Lysosomes share with vacuoles the hydrolytic functions and the position along the secretory pathway; however, our results indicate that their membranes had a divergent evolution. We propose that protection against the luminal hydrolases that are abundant in inner hydrolytic compartments, which seems to have been achieved in many lysosomal membrane proteins by extensive N-glycosylation of the luminal domains, has instead been obtained in the vast majority of tonoplast proteins by limiting the length of such domains.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Glicoproteínas/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Polissacarídeos/metabolismo , Vacúolos/metabolismo , Animais , Proteínas de Arabidopsis/química , Membrana Celular/metabolismo , Simulação por Computador , Retículo Endoplasmático/metabolismo , Glicoproteínas/química , Glicosilação , Proteínas de Membrana/metabolismo , Microssomos/metabolismo , Oligossacarídeos/metabolismo , Peptídeos/metabolismo , Proteoma/metabolismo , RatosRESUMO
Stomata control the exchange of CO2 and water vapor in land plants. Thus, whereas a constant supply of CO2 is required to maintain adequate rates of photosynthesis, the accompanying water losses must be tightly regulated to prevent dehydration and undesired metabolic changes. Accordingly, the uptake or release of ions and metabolites from guard cells is necessary to achieve normal stomatal function. The AtQUAC1, an R-type anion channel responsible for the release of malate from guard cells, is essential for efficient stomatal closure. Here, we demonstrate that mutant plants lacking AtQUAC1 accumulated higher levels of malate and fumarate. These mutant plants not only display slower stomatal closure in response to increased CO2 concentration and dark but are also characterized by improved mesophyll conductance. These responses were accompanied by increases in both photosynthesis and respiration rates, without affecting the activity of photosynthetic and respiratory enzymes and the expression of other transporter genes in guard cells, which ultimately led to improved growth. Collectively, our results highlight that the transport of organic acids plays a key role in plant cell metabolism and demonstrate that AtQUAC1 reduce diffusive limitations to photosynthesis, which, at least partially, explain the observed increments in growth under well-watered conditions.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiologia , Células do Mesofilo/fisiologia , Transportadores de Ânions Orgânicos/metabolismo , Fotossíntese/fisiologia , Estômatos de Plantas/fisiologia , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Carbono/metabolismo , Dióxido de Carbono/metabolismo , Secas , Enzimas/genética , Enzimas/metabolismo , Fumaratos/metabolismo , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Malatos/metabolismo , Mutação , Transportadores de Ânions Orgânicos/genética , Folhas de Planta/genética , Folhas de Planta/crescimento & desenvolvimento , Estômatos de Plantas/genéticaRESUMO
Approximately 0.2% of all angiosperms are classified as metal hyperaccumulators based on their extraordinarily high leaf metal contents, for example >1% zinc, >0.1% nickel or >0.01% cadmium (Cd) in dry biomass. So far, metal hyperaccumulation has been considered to be a taxon-wide, constitutively expressed trait, the extent of which depends solely on available metal concentrations in the soil. Here we show that in the facultative metallophyte Arabidopsis halleri, both insect herbivory and mechanical wounding of leaves trigger an increase specifically in leaf Cd accumulation. Moreover, the Cd concentrations accumulated in leaves can serve as an elemental defense against herbivory by larvae of the Brassicaceae specialist small white (Pieris rapae), thus allowing the plant to take advantage of this non-essential trace element and toxin. Metal homeostasis genes are overrepresented in the systemic transcriptional response of roots to the wounding of leaves in A. halleri, supporting that leaf Cd accumulation is preceded by systemic signaling events. A similar, but quantitatively less pronounced transcriptional response was observed in A. thaliana, suggesting that the systemically regulated modulation of metal homeostasis in response to leaf wounding also occurs in non-hyperaccumulator plants. This is the first report of an environmental stimulus influencing metal hyperaccumulation.
Assuntos
Arabidopsis/metabolismo , Arabidopsis/parasitologia , Cádmio/metabolismo , Folhas de Planta/metabolismo , Folhas de Planta/parasitologia , Animais , Borboletas/patogenicidade , Regulação da Expressão Gênica de Plantas , Herbivoria , Larva/patogenicidadeRESUMO
Grape (Vitis vinifera) accumulates various polyphenolic compounds, which protect against environmental stresses, including ultraviolet-C (UV-C) light and pathogens. In this study, we looked at the transcriptome and metabolome in grape berry skin after UV-C irradiation, which demonstrated the effectiveness of omics approaches to clarify important traits of grape. We performed transcriptome analysis using a genome-wide microarray, which revealed 238 genes up-regulated more than 5-fold by UV-C light. Enrichment analysis of Gene Ontology terms showed that genes encoding stilbene synthase, a key enzyme for resveratrol synthesis, were enriched in the up-regulated genes. We performed metabolome analysis using liquid chromatography-quadrupole time-of-flight mass spectrometry, and 2,012 metabolite peaks, including unidentified peaks, were detected. Principal component analysis using the peaks showed that only one metabolite peak, identified as resveratrol, was highly induced by UV-C light. We updated the metabolic pathway map of grape in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and in the KaPPA-View 4 KEGG system, then projected the transcriptome and metabolome data on a metabolic pathway map. The map showed specific induction of the resveratrol synthetic pathway by UV-C light. Our results showed that multiomics is a powerful tool to elucidate the accumulation mechanisms of secondary metabolites, and updated systems, such as KEGG and KaPPA-View 4 KEGG for grape, can support such studies.
Assuntos
Vias Biossintéticas , Frutas/genética , Perfilação da Expressão Gênica , Metabolômica , Estilbenos/metabolismo , Raios Ultravioleta , Vitis/genética , Vias Biossintéticas/efeitos da radiação , Calibragem , Escuridão , Fluorescência , Frutas/metabolismo , Frutas/efeitos da radiação , Ontologia Genética , Genes de Plantas , Metaboloma/genética , Metaboloma/efeitos da radiação , Anotação de Sequência Molecular , Análise de Componente Principal , Metabolismo Secundário/genética , Metabolismo Secundário/efeitos da radiação , Vitis/metabolismo , Vitis/efeitos da radiaçãoRESUMO
Arsenic (As) is a chronic poison that causes severe skin lesions and cancer. Rice (Oryza sativa L.) is a major dietary source of As; therefore, reducing As accumulation in the rice grain and thereby diminishing the amount of As that enters the food chain is of critical importance. Here, we report that a member of the Oryza sativa C-type ATP-binding cassette (ABC) transporter (OsABCC) family, OsABCC1, is involved in the detoxification and reduction of As in rice grains. We found that OsABCC1 was expressed in many organs, including the roots, leaves, nodes, peduncle, and rachis. Expression was not affected when plants were exposed to low levels of As but was up-regulated in response to high levels of As. In both the basal nodes and upper nodes, which are connected to the panicle, OsABCC1 was localized to the phloem region of vascular bundles. Furthermore, OsABCC1 was localized to the tonoplast and conferred phytochelatin-dependent As resistance in yeast. Knockout of OsABCC1 in rice resulted in decreased tolerance to As, but did not affect cadmium toxicity. At the reproductive growth stage, the As content was higher in the nodes and in other tissues of wild-type rice than in those of OsABCC1 knockout mutants, but was significantly lower in the grain. Taken together, our results indicate that OsABCC1 limits As transport to the grains by sequestering As in the vacuoles of the phloem companion cells of the nodes in rice.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , Arsênio/metabolismo , Regulação da Expressão Gênica de Plantas , Oryza/metabolismo , Sementes/metabolismo , Regulação para Cima , Transportadores de Cassetes de Ligação de ATP/genética , Transporte Biológico Ativo/genética , Cádmio/metabolismo , Oryza/citologia , Oryza/genética , Floema/citologia , Floema/metabolismo , Sementes/citologia , Sementes/genéticaRESUMO
The aluminum-activated malate transporters (ALMTs) form a membrane protein family exhibiting different physiological roles in plants, varying from conferring tolerance to environmental Al(3+) to the regulation of stomatal movement. The regulation of the anion channels of the ALMT family is largely unknown. Identifying intracellular modulators of the activity of anion channels is fundamental to understanding their physiological functions. In this study we investigated the role of cytosolic nucleotides in regulating the activity of the vacuolar anion channel AtALMT9. We found that cytosolic nucleotides modulate the transport activity of AtALMT9. This modulation was based on a direct block of the pore of the channel at negative membrane potentials (open channel block) by the nucleotide and not by a phosphorylation mechanism. The block by nucleotides of AtALMT9-mediated currents was voltage dependent. The blocking efficiency of intracellular nucleotides increased with the number of phosphate groups and ATP was the most effective cellular blocker. Interestingly, the ATP block induced a marked modification of the current-voltage characteristic of AtALMT9. In addition, increased concentrations of vacuolar anions were able to shift the ATP block threshold to a more negative membrane potential. The block of AtALMT9-mediated anion currents by ATP at negative membrane potentials acts as a gate of the channel and vacuolar anion tune this gating mechanism. Our results suggest that anion transport across the vacuolar membrane in plant cells is controlled by cytosolic nucleotides and the energetic status of the cell.
Assuntos
Trifosfato de Adenosina/metabolismo , Alumínio/metabolismo , Proteínas de Arabidopsis/metabolismo , Canais de Cloreto/metabolismo , Transporte de Íons , Trifosfato de Adenosina/química , Alumínio/química , Ânions/química , Ânions/metabolismo , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Canais de Cloreto/química , Citosol/química , Citosol/metabolismo , Potenciais da Membrana , Vacúolos/química , Vacúolos/metabolismoRESUMO
The (seco)iridoids and their derivatives, the monoterpenoid indole alkaloids (MIAs), form two large families of plant-derived bioactive compounds with a wide spectrum of high-value pharmacological and insect-repellent activities. Vinblastine and vincristine, MIAs used as anticancer drugs, are produced by Catharanthus roseus in extremely low levels, leading to high market prices and poor availability. Their biotechnological production is hampered by the fragmentary knowledge of their biosynthesis. Here we report the discovery of the last four missing steps of the (seco)iridoid biosynthesis pathway. Expression of the eight genes encoding this pathway, together with two genes boosting precursor formation and two downstream alkaloid biosynthesis genes, in an alternative plant host, allows the heterologous production of the complex MIA strictosidine. This confirms the functionality of all enzymes of the pathway and highlights their utility for synthetic biology programmes towards a sustainable biotechnological production of valuable (seco)iridoids and alkaloids with pharmaceutical and agricultural applications.
Assuntos
Catharanthus/metabolismo , Iridoides/metabolismo , Catharanthus/genética , Genes de Plantas , Dados de Sequência Molecular , Nicotiana/genéticaRESUMO
Aluminum-activated malate transporters (ALMTs) form an important family of anion channels involved in fundamental physiological processes in plants. Because of their importance, the role of ALMTs in plant physiology is studied extensively. In contrast, the structural basis of their functional properties is largely unknown. This lack of information limits the understanding of the functional and physiological differences between ALMTs and their impact on anion transport in plants. This study aimed at investigating the structural organization of the transmembrane domain of the Arabidopsis (Arabidopsis thaliana) vacuolar channel AtALMT9. For that purpose, we performed a large-scale mutagenesis analysis and found two residues that form a salt bridge between the first and second putative transmembrane α-helices (TMα1 and TMα2). Furthermore, using a combination of pharmacological and mutagenesis approaches, we identified citrate as an "open channel blocker" of AtALMT9 and used this tool to examine the inhibition sensitivity of different point mutants of highly conserved amino acid residues. By this means, we found a stretch within the cytosolic moiety of the TMα5 that is a probable pore-forming domain. Moreover, using a citrate-insensitive AtALMT9 mutant and biochemical approaches, we could demonstrate that AtALMT9 forms a multimeric complex that is supposedly composed of four subunits. In summary, our data provide, to our knowledge, the first evidence about the structural organization of an ion channel of the ALMT family. We suggest that AtALMT9 is a tetramer and that the TMα5 domains of the subunits contribute to form the pore of this anion channel.