RESUMEN
Grain cadmium (Cd) is translocated from source to sink tissues exclusively via phloem, though the phloem Cd unloading transporter has not been identified yet. Here, we isolated and functionally characterized a defensin-like gene DEFENSIN 8 (DEF8) highly expressed in rice (Oryza sativa) grains and induced by Cd exposure in seedling roots. Histochemical analysis and subcellular localization detected DEF8 expression preferentially in pericycle cells and phloem of seedling roots, as well as in phloem of grain vasculatures. Further analysis demonstrated that DEF8 is secreted into extracellular spaces possibly by vesicle trafficking. DEF8 bound to Cd in vitro, and Cd efflux from protoplasts as well as loading into xylem vessels decreased in the def8 mutant seedlings compared with the wild type. At maturity, significantly less Cd accumulation was observed in the mutant grains. These results suggest that DEF8 is a dual function protein that facilitates Cd loading into xylem and unloading from phloem, thus mediating Cd translocation from roots to shoots and further allocation to grains, representing a phloem Cd unloading regulator. Moreover, essential mineral nutrient accumulation as well as important agronomic traits were not affected in the def8 mutants, suggesting DEF8 is an ideal target for breeding low grain Cd rice.
Asunto(s)
Cadmio , Oryza , Cadmio/metabolismo , Oryza/genética , Oryza/metabolismo , Floema/metabolismo , Fitomejoramiento , Grano Comestible/metabolismo , Plantones/metabolismo , Raíces de Plantas/metabolismo , Defensinas/genética , Defensinas/análisis , Defensinas/metabolismoRESUMEN
Nitrate allocation in Arabidopsis (Arabidopsis thaliana) represents an important mechanism for mediating plant environmental adaptation. However, whether this mechanism occurs or has any physiological/agronomic importance in the ammoniphilic plant rice (Oriza sativa L.) remains unknown. Here, we address this question through functional characterization of the Nitrate transporter 1/Peptide transporter Family (NPF) transporter gene OsNPF7.9. Ectopic expression of OsNPF7.9 in Xenopus oocytes revealed that the gene encodes a low-affinity nitrate transporter. Histochemical and in-situ hybridization assays showed that OsNPF7.9 expresses preferentially in xylem parenchyma cells of vasculature tissues. Transient expression assays indicated that OsNPF7.9 localizes to the plasma membrane. Nitrate allocation from roots to shoots was essentially decreased in osnpf7.9 mutants. Biomass, grain yield, and nitrogen use efficiency (NUE) decreased in the mutant dependent on nitrate availability. Further analysis demonstrated that nitrate allocation mediated by OsNPF7.9 is essential for balancing rice growth and stress tolerance. Moreover, our research identified an indica-japonica divergent single-nucleotide polymorphism occurring in the coding region of OsNPF7.9, which correlates with enhanced nitrate allocation to shoots of indica rice, revealing that divergent nitrate allocation might represent an important component contributing to the divergent NUE between indica and japonica subspecies and was likely selected as a favorable trait during rice breeding.
Asunto(s)
Arabidopsis , Oryza , Arabidopsis/genética , Arabidopsis/metabolismo , Transportadores de Nitrato , Nitratos/metabolismo , Nitrógeno/metabolismo , Oryza/metabolismo , Fitomejoramiento , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Nitrate transporters are primarily responsible for absorption of nitrate from soil and nitrate translocation among different parts of plants. They deliver nitrate to where it is needed. However, recent studies have revealed that nitrate transporters are extensively involved in coping with adverse environmental conditions besides limited nitrate/nitrogen availability. In this review, we describe the functions of the nitrate transporters related to abiotic stresses and their regulation. The expected and unexpected roles of nitrate transporters in plant abiotic stress resistance will also be discussed.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Proteínas de Plantas/metabolismo , Estrés Fisiológico , Transportadores de Nitrato , Nitratos/metabolismo , Nitrógeno/metabolismo , Raíces de Plantas/metabolismoRESUMEN
Mechanistic insight into metal hyperaccumulation is largely restricted to Brassicaceae plants; therefore, it is of great importance to obtain corresponding knowledge from system outside the Brassicaceae. Here, we constructed and screened a cDNA library of the Cd/Zn hyperaccumulator Sedum plumbizincicola and identified a novel metallothionein-like protein encoding gene SpMTL. SpMTL showed functional similarity to other known MT proteins and also to its orthologues from non-hyperaccumulators. However, three additional cysteine residues were observed in SpMTL and appeared to be hyperaccumulator specific. Removal of these three residues significantly decreased its ability to tolerate Cd and the stoichiometry of Cd against SpMTL (molar ratio of Cd/SpMTL) to a level comparable to those of Cd/SaMTL and Cd/SeMTL in the corresponding non-hyperaccumulating relatives. SpMTL expressed in S. plumbizincicola roots at a much higher level than those of its orthologues in the non-hyperaccumulator roots. Interestingly, a positive correlation was observed between transcript levels of SpMTL in roots and Cd accumulation in leaves. Taking these results together, we propose that elevated transcript levels and heterotypic variation in protein sequences of SpMTL might contribute to the trait of Cd hyperaccumulation and hypertolerance in S. plumbizincicola.
Asunto(s)
Adaptación Fisiológica , Metalotioneína/metabolismo , Metales/metabolismo , Proteínas de Plantas/metabolismo , Sedum/fisiología , Secuencia de Aminoácidos , Arabidopsis/genética , Transporte Biológico , Cadmio/metabolismo , Quelantes/metabolismo , Cisteína/metabolismo , Regulación de la Expresión Génica de las Plantas , Inactivación Metabólica , Metalotioneína/química , Metalotioneína/aislamiento & purificación , Proteínas de Plantas/química , Proteínas de Plantas/aislamiento & purificación , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Saccharomyces cerevisiae/metabolismo , Sedum/genética , Sedum/metabolismo , Especificidad de la Especie , Zinc/metabolismoRESUMEN
Stresses decouple nitrate assimilation and photosynthesis through stress-initiated nitrate allocation to roots (SINAR), which is mediated by the nitrate transporters NRT1.8 and NRT1.5 and functions to promote stress tolerance. However, how SINAR communicates with the environment remains unknown. Here, we present biochemical and genetic evidence demonstrating that in Arabidopsis thaliana, ethylene (ET) and jasmonic acid (JA) affect the crosstalk between SINAR and the environment. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that ethylene response factors (ERFs), including OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59, bind to the GCC boxes in the NRT1.8 promoter region, while ETHYLENE INSENSITIVE3 (EIN3) binds to the EIN3 binding site motifs in the NRT1.5 promoter. Genetic assays showed that cadmium and sodium stresses initiated ET/JA signaling, which converged at EIN3/EIN3-Like1 (EIL1) to modulate ERF expression and hence to upregulate NRT1.8. By contrast, ET and JA signaling mediated the downregulation of NRT1.5 via EIN3/EIL1 and other, unknown component(s). SINAR enhanced stress tolerance and decreased plant growth under nonstressed conditions through the ET/JA-NRT1.5/NRT1.8 signaling module. Interestingly, when nitrate reductase was impaired, SINAR failed to affect either stress tolerance or plant growth. These data suggest that SINAR responds to environmental conditions through the ET/JA-NRT signaling module, which further modulates stress tolerance and plant growth in a nitrate reductase-dependent manner.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Ciclopentanos/metabolismo , Etilenos/metabolismo , Nitratos/metabolismo , Oxilipinas/metabolismo , Adaptación Fisiológica , Proteínas de Transporte de Anión/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Inmunoprecipitación de Cromatina , Proteínas de Unión al ADN , Ensayo de Cambio de Movilidad Electroforética , Ambiente , Regulación de la Expresión Génica de las Plantas , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , Unión Proteica , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Estrés Fisiológico , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Elevations in extracellular calcium ([Ca(2+)]o) are known to stimulate cytosolic calcium ([Ca(2+)]cyt) oscillations to close stomata. However, the underlying mechanisms regulating this process remain largely to be determined. Here, through the functional characterization of the calcium underaccumulation mutant cau1, we report that the epigenetic regulation of CAS, a putative Ca(2+) binding protein proposed to be an external Ca(2+) sensor, is involved in this process. cau1 mutant plants display increased drought tolerance and stomatal closure. A mutation in CAU1 significantly increased the expression level of the calcium signaling gene CAS, and functional disruption of CAS abolished the enhanced drought tolerance and stomatal [Ca(2+)]o signaling in cau1. Map-based cloning revealed that CAU1 encodes the H4R3sme2 (for histone H4 Arg 3 with symmetric dimethylation)-type histone methylase protein arginine methytransferase5/Shk1 binding protein1. Chromatin immunoprecipitation assays showed that CAU1 binds to the CAS promoter and modulates the H4R3sme2-type histone methylation of the CAS chromatin. When exposed to elevated [Ca(2+)]o, the protein levels of CAU1 decreased and less CAU1 bound to the CAS promoter. In addition, the methylation level of H4R3sme2 decreased in the CAS chromatin. Together, these data suggest that in response to increases in [Ca(2+)]o, fewer CAU1 protein molecules bind to the CAS promoter, leading to decreased H4R3sme2 methylation and consequent derepression of the expression of CAS to mediate stomatal closure and drought tolerance.
Asunto(s)
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Señalización del Calcio/efectos de los fármacos , Proteínas de Unión al Calcio/genética , Calcio/farmacología , Epigénesis Genética/efectos de los fármacos , Estomas de Plantas/fisiología , Proteína-Arginina N-Metiltransferasas/metabolismo , Ácido Abscísico/farmacología , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arginina/metabolismo , Señalización del Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Clonación Molecular , Sequías , Espacio Extracelular/efectos de los fármacos , Espacio Extracelular/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas/genética , Genes Supresores , Histonas/metabolismo , Homeostasis/efectos de los fármacos , Homeostasis/genética , Metilación/efectos de los fármacos , Modelos Biológicos , Mutación/genética , Estomas de Plantas/citología , Estomas de Plantas/efectos de los fármacosRESUMEN
The plant vacuole is an important organelle for storing excess iron (Fe), though its contribution to increasing the Fe content in staple foods remains largely unexplored. In this study we report the isolation and functional characterization of two rice genes OsVIT1 and OsVIT2, orthologs of the Arabidopsis VIT1. Transient expression of OsVIT1:EGFP and OsVIT2:EGFP protein fusions revealed that OsVIT1 and OsVIT2 are localized to the vacuolar membrane. Ectopic expression of OsVIT1 and OsVIT2 partially rescued the Fe(2+) - and Zn(2+) -sensitive phenotypes in yeast mutant Δccc1 and Δzrc1, and further increased vacuolar Fe(2+) , Zn(2+) and Mn(2+) accumulation. These data together suggest that OsVIT1 and OsVIT2 function to transport Fe(2+) , Zn(2+) and Mn(2+) across the tonoplast into vacuoles in yeast. In rice, OsVIT1 and OsVIT2 are highly expressed in flag leaf blade and sheath, respectively, and in contrast to OsVIT1, OsVIT2 is highly responsive to Fe treatments. Interestingly, functional disruption of OsVIT1 and OsVIT2 leads to increased Fe/Zn accumulation in rice seeds and a corresponding decrease in the source organ flag leaves, indicating an enhanced Fe/Zn translocation between source and sink organs, which might represent a novel strategy to biofortify Fe/Zn in staple foods.
Asunto(s)
Regulación de la Expresión Génica de las Plantas/genética , Hierro/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Oryza/genética , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Transporte Biológico , Expresión Génica , Membranas Intracelulares/metabolismo , Manganeso/metabolismo , Proteínas de Transporte de Membrana/genética , Mutagénesis Insercional , Especificidad de Órganos , Oryza/citología , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Fenotipo , Floema/citología , Floema/genética , Floema/crecimiento & desarrollo , Floema/metabolismo , Hojas de la Planta/citología , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusión , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Plantones/citología , Plantones/genética , Plantones/crecimiento & desarrollo , Plantones/metabolismo , Semillas/citología , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Alineación de Secuencia , Vacuolas/metabolismo , Zinc/metabolismoRESUMEN
Nitrate reallocation to plant roots occurs frequently under adverse conditions and was recently characterized to be actively regulated by Nitrate Transporter1.8 (NRT1.8) in Arabidopsis (Arabidopsis thaliana) and implicated as a common response to stresses. However, the underlying mechanisms remain largely to be determined. In this study, characterization of NRT1.5, a xylem nitrate-loading transporter, showed that the mRNA level of NRT1.5 is down-regulated by salt, drought, and cadmium treatments. Functional disruption of NRT1.5 enhanced tolerance to salt, drought, and cadmium stresses. Further analyses showed that nitrate, as well as Na(+) and Cd(2+) levels, were significantly increased in nrt1.5 roots. Important genes including Na(+)/H(+) exchanger1, Salt overly sensitive1, Pyrroline-5-carboxylate synthase1, Responsive to desiccation29A, Phytochelatin synthase1, and NRT1.8 in stress response pathways are steadily up-regulated in nrt1.5 mutant plants. Interestingly, altered accumulation of metabolites, including proline and malondialdehyde, was also observed in nrt1.5 plants. These data suggest that NRT1.5 is involved in nitrate allocation to roots and the consequent tolerance to several stresses, in a mechanism probably shared with NRT1.8.
Asunto(s)
Adaptación Fisiológica , Proteínas de Transporte de Anión/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/fisiología , Nitratos/metabolismo , Estrés Fisiológico , Adaptación Fisiológica/genética , Proteínas de Transporte de Anión/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Cadmio/metabolismo , Regulación hacia Abajo/genética , Regulación de la Expresión Génica de las Plantas , Genes de Plantas/genética , Metaboloma/genética , Mutación/genética , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Sodio/metabolismo , Estrés Fisiológico/genéticaRESUMEN
Much of our dietary uptake of heavy metals is through the consumption of plants. A long-sought strategy to reduce chronic exposure to heavy metals is to develop plant varieties with reduced accumulation in edible tissues. Here, we describe that the fission yeast (Schizosaccharomyces pombe) phytochelatin (PC)-cadmium (Cd) transporter SpHMT1 produced in Arabidopsis (Arabidopsis thaliana) was localized to tonoplast, and enhanced tolerance to and accumulation of Cd2+, copper, arsenic, and zinc. The action of SpHMT1 requires PC substrates, and failed to confer Cd2+ tolerance and accumulation when glutathione and PC synthesis was blocked by L-buthionine sulfoximine, or only PC synthesis is blocked in the cad1-3 mutant, which is deficient in PC synthase. SpHMT1 expression enhanced vacuolar Cd2+ accumulation in wild-type Columbia-0, but not in cad1-3, where only approximately 35% of the Cd2+ in protoplasts was localized in vacuoles, in contrast to the near 100% found in wild-type vacuoles and approximately 25% in those of cad2-1 that synthesizes very low amounts of glutathione and PCs. Interestingly, constitutive SpHMT1 expression delayed root-to-shoot metal transport, and root-targeted expression confirmed that roots can serve as a sink to reduce metal contents in shoots and seeds. These findings suggest that SpHMT1 function requires PCs in Arabidopsis, and it is feasible to promote food safety by engineering plants using SpHMT1 to decrease metal accumulation in edible tissues.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/metabolismo , Arabidopsis/metabolismo , Cadmio/metabolismo , Fitoquelatinas/farmacología , Schizosaccharomyces/metabolismo , Semillas/metabolismo , Vacuolas/metabolismo , Adaptación Fisiológica/efectos de los fármacos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico/efectos de los fármacos , Butionina Sulfoximina/farmacología , Cadmio/toxicidad , Citosol/efectos de los fármacos , Citosol/metabolismo , Glutatión/metabolismo , Datos de Secuencia Molecular , Mutación/genética , Especificidad de Órganos/efectos de los fármacos , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Brotes de la Planta/efectos de los fármacos , Brotes de la Planta/metabolismo , Plantas Modificadas Genéticamente , Schizosaccharomyces/efectos de los fármacos , Semillas/efectos de los fármacos , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismo , Vacuolas/efectos de los fármacosRESUMEN
Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-beta-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd(2+)) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd(2+)-sensitive phenotype. Further analyses showed that Cd(2+) stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd(2+) tolerance.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Proteínas de Transporte de Anión/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimología , Cadmio/toxicidad , Nitratos/metabolismo , Exudados de Plantas/metabolismo , Xilema/enzimología , Adaptación Fisiológica/genética , Proteínas de Transporte de Anión/genética , Arabidopsis/citología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Concentración de Iones de Hidrógeno/efectos de los fármacos , Mutación/genética , Transportadores de Nitrato , Exudados de Plantas/genética , Transporte de Proteínas/efectos de los fármacos , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , Regulación hacia Arriba/efectos de los fármacos , Xilema/citología , Xilema/efectos de los fármacos , Xilema/genéticaRESUMEN
Nitrate in plants is preferentially stored in vacuoles; however, how vacuolar nitrate is reallocated and to which biological process(es) it might contribute remain largely elusive. In this study, we functionally characterized three nitrate transporters NPF5.10, NPF5.14, and NPF8.5 that are tonoplast-localized. Ectopic expression in Xenopus laevis oocytes revealed that they mediate low-affinity nitrate transport. Histochemical analysis showed that these transporters were expressed preferentially in pericycle and xylem parenchyma cells. NPF5.10, NPF5.14, and NPF8.5 overexpression significantly decreased vacuolar nitrate contents and nitrate accumulation in Arabidopsis shoots. Further analysis showed that the sextuple mutant (npf5.10 npf5.14 npf8.5 npf5.11 npf5.12 npf5.16) had a higher 15NO3-uptake rate than the wild-type Col-0, but no significant difference was observed for nitrate accumulation between them. The septuple mutant (npf5.11 npf5.12 npf5.16 npf5.10 npf5.14 npf8.5 clca) generated by using CRISPR/Cas9 showed essentially decreased nitrate reallocation compared to wild type when exposed to nitrate starvation, though no further decrease was observed when compared to clca. Notably, NPF5.10, NPF5.14, and NPF8.5 as well as NPF5.11, NPF5.12, and NPF5.16 were consistently induced by mannitol, and more nitrate was detected in the sextuple mutant than in the wild type after mannitol treatment. These observations suggest that vacuolar nitrate efflux is regulated by several functional redundant nitrate transporters, and the reallocation might contribute to osmotic stress response other than mineral nutrition.
RESUMEN
Iron (Fe) transport and reallocation are essential to Fe homeostasis in plants, but it is unclear how Fe homeostasis is regulated, especially under stress. Here we report that NPF5.9 and its close homolog NPF5.8 redundantly regulate Fe transport and reallocation in Arabidopsis. NPF5.9 is highly upregulated in response to Fe deficiency. NPF5.9 expresses preferentially in vasculature tissues and localizes to the trans-Golgi network, and NPF5.8 showed a similar expression pattern. Long-distance Fe transport and allocation into aerial parts was significantly increased in NPF5.9-overexpressing lines. In the double mutant npf5.8 npf5.9, Fe loading in aerial parts and plant growth were decreased, which were partially rescued by Fe supplementation. Further analysis showed that expression of PYE, the negative regulator for Fe homeostasis, and its downstream target NAS4 were significantly altered in the double mutant. NPF5.9 and NPF5.8 were shown to also mediate nitrate uptake and transport, although nitrate and Fe application did not reciprocally affect each other. Our findings uncovered the novel function of NPF5.9 and NPF5.8 in long-distance Fe transport and homeostasis, and further indicated that they possibly mediate nitrate transport and Fe homeostasis independently in Arabidopsis.
Asunto(s)
Arabidopsis/genética , Arabidopsis/metabolismo , Homeostasis/genética , Transporte Iónico/genética , Hierro/metabolismo , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Variación Genética , Genotipo , MutaciónRESUMEN
Apoplastic iron (Fe) in roots represents an essential Fe storage pool. Reallocation of apoplastic Fe is of great importance to plants experiencing Fe deprivation, but how this reallocation process is regulated remains elusive, likely because of the highly complex cell wall structure and the limited knowledge about cell wall biosynthesis and modulation. Here, we present genetic and biochemical evidence to demonstrate that the Cdi-mediated galactosylation of rhamnogalacturonan-II (RG-II) is required for apoplastic Fe reallocation. Cdi is expressed in roots and up-regulated in response to Fe deficiency. It encodes a putative glycosyltransferase localized to the Golgi apparatus. Biochemical and mass spectrometry assays showed that Cdi catalyzes the transfer of GDP-L-galactose to the terminus of side chain A on RG-II. Disruption of Cdi essentially decreased RG-II dimerization and hence disrupted cell wall formation, as well as the reallocation of apoplastic Fe from roots to shoots. Further transcriptomic, Fourier transform infrared spectroscopy, and Fe desorption kinetic analyses coincidently suggested that Cdi mediates apoplastic Fe reallocation through extensive modulation of cell wall components and consequently the Fe adsorption capacity of the cell wall. Our study provides direct evidence demonstrating a link between cell wall biosynthesis and apoplastic Fe reallocation, thus indicating that the structure of the cell wall is important for efficient usage of the cell wall Fe pool.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Pared Celular/metabolismo , Hierro/metabolismo , Nucleotidiltransferasas/metabolismo , Pectinas/biosíntesis , Proteínas de Arabidopsis/genética , Galactosa/metabolismo , Regulación de la Expresión Génica de las Plantas , Nucleotidiltransferasas/genética , Pectinas/metabolismo , Raíces de Plantas/metabolismoRESUMEN
BACKGROUND: Proline (Pro) and γ-aminobutyric acid (GABA) play important roles in plant development and stress tolerance. However, the molecular components responsible for the transport of these molecules in rice remain largely unknown. RESULTS: Here we identified OsProT1 and OsProT3 as functional transporters for Pro and GABA. Transient expression of eGFP-OsProTs in plant protoplasts revealed that both OsProT1 and OsProT3 are localized to the plasma membrane. Ectopic expression in a yeast mutant demonstrated that both OsProT1 and OsProT3 specifically mediate transport of Pro and GABA with affinity for Pro in the low affinity range. qRT-PCR analyses suggested that OsProT1 was preferentially expressed in leaf sheathes during vegetative growth, while OsProT3 exhibited relatively high expression levels in several tissues, including nodes, panicles and roots. Interestingly, both OsProT1 and OsProT3 were induced by cadmium stress in rice shoots. CONCLUSIONS: Our results suggested that plasma membrane-localized OsProT1 and OsProT3 efficiently transport Pro and GABA when ectopically expressed in yeast and appear to be involved in various physiological processes, including adaption to cadmium stress in rice plants.
RESUMEN
A reversed-phase high-performance liquid chromatographic technique was developed to separate cadmium-phytochelatin complexes (Cd-PC2, Cd-PC3, and Cd-PC4) of interest in the plant Arapidopsis thaliana. High-performance liquid chromatography (HPLC) was coupled to an inductively coupled plasma mass spectrometric (ICP-MS) system with some modification to the interface. This was done in order to sustain the plasma with optimum sensitivity for cadmium detection in the presence of the high methanol loads used in the gradient elution of the reversed-phase separation. The detection limits were found to be 91.8 ngl(-1), 77.2 ngl(-1) and 49.2 ngl(-1) for Cd-PC2, Cd-PC3, and Cd-PC4 respectively. The regression coefficients (r2) for Cd-PC2 to Cd-PC4 detection ranged from 0.998 to 0.999. The method was then used to investigate the occurrence and effect of cadmium-phytochelatin complexes in wild-type Arabidopsis and a phytochelatin-deficient mutant cad1-3 that had been genetically modified to ectopically express the wheat TaPCS1 phytochelatin synthase enzyme. The primary complex found in both wild-type and transgenic plants was Cd-PC2. In both lines, higher levels of Cd-PC2 were found in shoots than in roots, showing that phytochelatin synthases contribute to the accumulation of cadmium in shoots, in the Cd-PC2 form. Genetic modification did, however, impact the overall accumulation of Cd. Transgenic plants contained almost two times more cadmium in the form of Cd-PC2 in their roots than did the corresponding wild-type plants. Similarly, the shoot samples of the modified species also contained more (by 1.6 times) cadmium in the form of Cd-PC2 than the wild type. The enhanced role of PC2 in the transgenic Arabidopsis correlates with data showing long-distance transport of Cd in transgenic plants. Targeted transgenic expression of non-native phytochelatin synthases may contribute to improving the efficiency of plants for phytoremediation.
Asunto(s)
Arabidopsis/química , Cadmio/química , Cromatografía Líquida de Alta Presión/métodos , Espectrometría de Masas/métodos , Fitoquelatinas/química , Arabidopsis/genética , Arabidopsis/metabolismo , Cadmio/análisis , Genotipo , Mutación , Fitoquelatinas/metabolismo , Plantas Modificadas Genéticamente , Reproducibilidad de los ResultadosRESUMEN
Pollution by heavy metals limits the area of land available for cultivation of food crops. A potential solution to this problem might lie in the molecular breeding of food crops for phytoremediation that accumulate toxic metals in straw while producing safe and nutritious grains. Here, we identify a rice quantitative trait locus we name cadmium (Cd) accumulation in leaf 1 (CAL1), which encodes a defensin-like protein. CAL1 is expressed preferentially in root exodermis and xylem parenchyma cells. We provide evidence that CAL1 acts by chelating Cd in the cytosol and facilitating Cd secretion to extracellular spaces, hence lowering cytosolic Cd concentration while driving long-distance Cd transport via xylem vessels. CAL1 does not appear to affect Cd accumulation in rice grains or the accumulation of other essential metals, thus providing an efficient molecular tool to breed dual-function rice varieties that produce safe grains while remediating paddy soils.
Asunto(s)
Cadmio/metabolismo , Defensinas/metabolismo , Oryza/metabolismo , Productos Agrícolas , Citosol/metabolismo , Espacio Extracelular/metabolismo , Raíces de Plantas/metabolismo , Sitios de Carácter Cuantitativo , Contaminantes del Suelo/metabolismo , Xilema/metabolismoRESUMEN
A great proportion of nitrate taken up by plants is stored in vacuoles. Vacuolar nitrate accumulation and release is of great importance to nitrate reallocation and efficient utilization. However, how plants mediate nitrate efflux from vacuoles to cytoplasm is largely unknown. The current study identified NPF5.11, NPF5.12 and NPF5.16 as vacuolar nitrate efflux transporters in Arabidopsis. Histochemical analysis showed that NPF5.11, NPF5.12 and NPF5.16 were expressed preferentially in root pericycle cells and xylem parenchyma cells, and further analysis showed that these proteins were tonoplast-localized. Functional characterization using cRNA-injected Xenopus laevis oocytes showed that NPF5.11, NPF5.12 and NPF5.16 were low-affinity, pH-dependent nitrate uptake transporters. In npf5.11 npf5.12 npf5.16 triple mutant lines, more root-fed 15NO3- was translocated to shoots compared to the wild type control. In the NPF5.12 overexpression lines, proportionally less nitrate was maintained in roots. These data together suggested that NPF5.11, NPF5.12 and NPF5.16 might function to uptake nitrate from vacuoles into cytosol, thus serving as important players to modulate nitrate allocation between roots and shoots.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Vacuolas/metabolismo , Animales , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Femenino , Concentración de Iones de Hidrógeno , Nitratos/metabolismo , Oocitos/fisiología , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Brotes de la Planta/genética , Brotes de la Planta/metabolismo , Plantas Modificadas Genéticamente , Xenopus laevisRESUMEN
Nitrogen deficiency induces leaf senescence. However, whether or how nitrate might affect this process remains to be investigated. Here, we report an interesting finding that nitrate-instead of nitrogen-starvation induced early leaf senescence in nrt1.5 mutant, and present genetic and physiological data demonstrating that nitrate starvation-induced leaf senescence is suppressed by NRT1.5. NRT1.5 suppresses the senescence process dependent on its function from roots, but not the nitrate transport function. Further analyses using nrt1.5 single and nia1 nia2 nrt1.5-4 triple mutant showed a negative correlation between nitrate concentration and senescence rate in leaves. Moreover, when exposed to nitrate starvation, foliar potassium level decreased in nrt1.5, but adding potassium could essentially restore the early leaf senescence phenotype of nrt1.5 plants. Nitrate starvation also downregulated the expression of HAK5, RAP2.11, and ANN1 in nrt1.5 roots, and appeared to alter potassium level in xylem sap from nrt1.5. These data suggest that NRT1.5 likely perceives nitrate starvation-derived signals to prevent leaf senescence by facilitating foliar potassium accumulation.
Asunto(s)
Proteínas de Transporte de Anión/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Nitratos/metabolismo , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Potasio/metabolismo , Proteínas de Transporte de Anión/genética , Arabidopsis/citología , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Transporte Biológico , Mutación , Factores de Tiempo , Xilema/metabolismoRESUMEN
The vacuole is a pivotal organelle functioning in storage of metabolites, mineral nutrients, and toxicants in higher plants. Accumulating evidence indicates that in addition to its storage role, the vacuole contributes essentially to long-distance transport of metals, through the modulation of Vacuolar sequestration capacity (VSC) which is shown to be primarily controlled by cytosolic metal chelators and tonoplast-localized transporters, or the interaction between them. Plants adapt to their environments by dynamic regulation of VSC for specific metals and hence targeting metals to specific tissues. Study of VSC provides not only a new angle to understand the long-distance root-to-shoot transport of minerals in plants, but also an efficient way to biofortify essential mineral nutrients or to phytoremediate non-essential metal pollution. The current review will focus on the most recent proceedings on the interaction mechanisms between VSC regulation and long-distance metal transport.