RESUMEN
Grains are the major sink of phosphorus (P) in cereal crops, accounting for 60-85% of total plant P, but the mechanisms underlying P loading into the grains are poorly understood. We functionally characterized a transporter gene required for the distribution of P to the grains in barley (Hordeum vulgare), HvSPDT (SULTR-like phosphorus distribution transporter). HvSPDT encoded a plasma membrane-localized Pi/H+ cotransporter. It was mainly expressed in the nodes at both the vegetative and reproductive stages. Furthermore, its expression was induced by inorganic phosphate (Pi) deficiency. In the nodes, HvSPDT was expressed in both the xylem and phloem region of enlarged and diffuse vascular bundles. Knockout of HvSPDT decreased the distribution of P to new leaves, but increased the distribution to old leaves at the vegetative growth stage under low P supply. However, knockout of HvSPDT did not alter the redistribution of P from old to young organs. At the reproductive stage, knockout of HvSPDT significantly decreased P allocation to the grains, resulting in a considerable reduction in grain yield, especially under P-limited conditions. Our results indicate that node-based HvSPDT plays a crucial role in loading P into barley grains through preferentially distributing P from the xylem and further to the phloem.
Asunto(s)
Hordeum , Grano Comestible , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Fósforo/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismoRESUMEN
Manganese (Mn) is an essential element for plant growth and development, but transporters required for Mn uptake have only been identified in a few plant species. Here, we functionally characterized a member of the natural resistance-associated macrophage proteins (Nramps) family, FeNramp5 in buckwheat (Fagopyrum esculentum Moench), which is known as a species well adapted to acidic soils. FeNramp5 was mainly expressed in the roots, and its expression was upregulated by the deficiency of Mn and Fe. Furthermore, spatial and tissue-specific expression analysis showed that FeNramp5 was expressed in all tissues of the basal root regions. FeNramp5-GFP protein was localized to the plasma membrane when transiently expressed in buckwheat leaf protoplast. FeNramp5 showed the transport activity for Mn2+ and Cd2+ but not for Fe2+ when expressed in yeast. Furthermore, the transport activity for Mn2+ was higher in yeast expressing FeNramp5 than in yeast expressing AtNramp1. FeNramp5 was also able to complement the phenotype of Arabidopsis atnramp1 mutant in terms of the growth and accumulation of Mn and Cd. The absolute expression level of AtNramp1 was comparable to that of FeNramp5 in the roots, but buckwheat accumulated higher Mn than Arabidopsis when grown under the same condition. Further analysis showed that at least motif B in FeNramp5 seems important for its high transport activity for Mn. These results indicate that FeNramp5 is a transporter for the uptake of Mn and Cd and its higher transport activity for Mn is probably associated with higher Mn accumulation in buckwheat.
Asunto(s)
Proteínas de Transporte de Catión/metabolismo , Fagopyrum/metabolismo , Manganeso/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/metabolismo , Secuencias de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Transporte Biológico , Proteínas de Transporte de Catión/química , Proteínas de Transporte de Catión/genética , Regulación de la Expresión Génica de las Plantas , Metales/metabolismo , Mutación , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Protoplastos/metabolismo , Levaduras/metabolismoRESUMEN
During plant growth and development mineral elements are preferentially delivered to different organs and tissues to meet the differential demand. It has been shown that the preferential distribution of mineral nutrients in gramineous plants is mediated by node-based transporters, but the mechanisms of preferential distribution in dicots are poorly understood. Here, we report a distinct mechanism for the preferential distribution of phosphorus (P) in Arabidopsis plants, revealed by detailed functional analysis of AtSPDT/AtSULTR3;4 (SULTR-like P Distribution Transporter), a homolog of rice OsSPDT. Like OsSPDT, AtSPDT is localized at the plasma membrane and showed proton-dependent transport activity for P. Interestingly, we found that AtSPDT is mainly expressed in the rosette basal region and leaf petiole, and its expression is up-regulated by P deficiency. Tissue-specific analysis showed that AtSPDT is mainly located in the vascular cambium of different organs, as well as in the parenchyma tissues of both xylem and phloem regions. Knockout of AtSPDT inhibited the growth of new leaves under low P due to decreased P distribution to those organs. The seed yields of the wild-type and atspdt mutant plants are similar, but the seeds of mutant plants contain - less P. These results indicate that AtSPDT localized in the vascular cambium is involved in preferential distribution of P to the developing tissues, through xylem-to-phloem transfer mainly at the rosette basal region and leaf petiole.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Fósforo/metabolismo , Haz Vascular de Plantas/fisiología , Transportadores de Sulfato/genética , Simportadores/genética , Arabidopsis/genética , Transporte Biológico , Regulación de la Expresión Génica de las Plantas , Desarrollo de la Planta , Hojas de la Planta/metabolismo , Raíces de Plantas/metabolismoRESUMEN
Symbiotic nitrogen (N2 ) fixation plays a vital role in sustainable agriculture. Efficient N2 fixation requires various materials, including phosphate (Pi); however, the molecular mechanism underlying the transport of Pi into nodules and bacteroids remains largely unknown. A nodule-localized Pi transporter, GmPT7, was functionally characterized in soybean (Glycine max) and its role in N2 fixation and yield was investigated via composite and whole transgenic plants. GmPT7 protein was localized to the plasma membrane and showed transport activity for Pi in yeast. Altered expression of GmPT7 changed 33 Pi uptake from rhizosphere and translocation to bacteroids. GmPT7 was mainly localized to the outer cortex and fixation zones of the nodules. Overexpression of GmPT7 promoted nodulation, and increased plant biomass, shoot nitrogen and phosphorus content, resulting in improved soybean yield by up to 36%. Double suppression of GmPT5 and GmPT7 led to nearly complete elimination of nodulation and over 50% reduction in plant biomass, shoot nitrogen and phosphorus content, indicating that both GmPT7 and GmPT5 contribute to Pi transport for N2 fixation. Taken together, our results indicate that GmPT7 is a transporter responsible for direct Pi entry to nodules and further to fixation zones, which is required for enhancing symbiotic N2 fixation and grain yield of soybean.
Asunto(s)
Glycine max/metabolismo , Fijación del Nitrógeno , Proteínas de Transporte de Fosfato/metabolismo , Proteínas de Plantas/metabolismo , Nódulos de las Raíces de las Plantas/metabolismo , Simbiosis , Transporte Biológico , Regulación de la Expresión Génica de las Plantas , Nitrógeno/metabolismo , Fijación del Nitrógeno/genética , Especificidad de Órganos , Proteínas de Transporte de Fosfato/genética , Fósforo/metabolismo , Filogenia , Proteínas de Plantas/genética , Nodulación de la Raíz de la Planta , Saccharomyces cerevisiae/metabolismo , Glycine max/genética , Glycine max/crecimiento & desarrollo , Simbiosis/genéticaRESUMEN
Buckwheat (Fagopyrum esculentum Moench) shows high tolerance to aluminum (Al) toxicity, but the molecular mechanisms underlying its high Al tolerance are poorly understood. Here, we functionally characterized two genes (FeSTAR1 and FeSTAR2), which encode a nucleotide-binding domain and a membrane domain, respectively, of a bacterial-type ATP-binding cassette (ABC) transporter. The expression of FeSTAR1 and FeSTAR2 was induced by Al in both roots and leaves with higher expression in the roots. Spatial and tissue-specific expression analysis showed that the Al-induced expression of these two genes was found in both the root tips and basal root regions with higher expression in the root outer cell layers. The expression was neither induced by other metals including Cd and La nor by low pH and phosphorus-deficiency. FeSTAR1 and FeSTAR2 were present in a single copy in the genome, but the Al-induced transcript copy number of FeSTAR1 and FeSTAR2 was much higher than their homologous genes in rice and Arabidopsis. FeSTAR1 and FeSTAR2 form a complex when co-expressed in onion epidermal cells. Introduction of FeSTAR1 and FeSTAR2 into Arabidopsis mutants atstar1 and als3/atstar2, respectively, rescued the sensitivity of the mutants to Al. Taken together, our results indicate that FeSTAR1 and FeSTAR2 are involved in Al tolerance and that their high expression level may contribute to high Al tolerance in buckwheat.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Adaptación Fisiológica/genética , Aluminio/farmacología , Bacterias/metabolismo , Fagopyrum/genética , Fagopyrum/fisiología , Genes de Plantas , Transportadoras de Casetes de Unión a ATP/metabolismo , Arabidopsis/genética , Clonación Molecular , Fagopyrum/efectos de los fármacos , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Especificidad de Órganos/genética , Oryza/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Unión Proteica , Factores de TiempoRESUMEN
High aluminum (Al) tolerance in rice (Oryza sativa) is controlled by a Cys2His2-type zinc finger transcription factor ART1 (Al resistance transcription factor 1). There are five close homologs of ART1 in the rice genome, but the role of these homologs is unknown. We functionally characterized one of the ART1 homologs, ART2, in terms of tissue and spatial expression, subcellular localization, transcriptional activation activity, and phenotypic analysis of the knockout lines. ART2 was localized to the nucleus and showed a transcriptional activation potential in yeast. ART2 was mainly expressed in the roots, but the expression level was much lower than that of ART1. The ART2 expression was rapidly induced by Al in the roots of the wild-type rice, but not in art1 mutant. Knockout of ART2 resulted in increased sensitivity to Al toxicity, but did not alter sensitivity to different pH values. Expression profile analysis by RNA-sequencing showed that ART2 was not involved in activation of genes regulated by ART1; however, four genes seems to be regulated by ART2, which are implicated in Al tolerance. These results indicate that ART1 and ART2 regulate different pathways leading to Al tolerance, and ATR2 plays a supplementary role in Al tolerance in rice.
Asunto(s)
Adaptación Fisiológica/efectos de los fármacos , Aluminio/toxicidad , Oryza/fisiología , Proteínas de Plantas/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Genes de Plantas , Estudios de Asociación Genética , Concentración de Iones de Hidrógeno , Mutación/genética , Especificidad de Órganos/genética , Oryza/efectos de los fármacos , Fenotipo , Filogenia , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente , Saccharomyces cerevisiae/metabolismo , Factores de Tiempo , Activación Transcripcional/genéticaRESUMEN
Buckwheat (Fagopyrum esculentum) shows high tolerance to aluminum (Al) toxicity, but the molecular mechanisms responsible for this high Al tolerance are still poorly understood. Here, we investigated the involvement of two MATE (multi-drug and toxic compound extrusion) genes in Al tolerance. Both FeMATE1 and FeMATE2 showed efflux transport activity for citrate, but not for oxalate when expressed in Xenopus oocytes. A transient assay with buckwheat leaf protoplasts using green fluorescent protein (GFP) fusion showed that FeMATE1 was mainly localized to the plasma membrane, whereas FeMATE2 was localized to the trans-Golgi and Golgi. The expression of FeMATE1 was induced by Al only in the roots, but that of FeMATE2 was up-regulated in both the roots and leaves. Furthermore, the expression of both genes only responded to Al toxicity, but not to other stresses including low pH, cadmium (Cd) and lanthanum (La). Heterologous expression of FeMATE1 or FeMATE2 in the Arabidopsis mutant atmate partially rescued its Al tolerance. Expression of FeMATE1 also partially recovered the Al-induced secretion of citrate in the transgenic lines, whereas expression of FeMATE2 did not complement the citrate secretion. Further physiological analysis showed that buckwheat roots also secreted citrate in addition to oxalate in response to Al in a dose-responsive manner. Taken together, our results indicate that FeMATE1 is involved in the Al-activated citrate secretion in the roots, while FeMATE2 is probably responsible for transporting citrate into the Golgi system for the internal detoxification of Al in the roots and leaves of buckwheat.
Asunto(s)
Aluminio/toxicidad , Fagopyrum/efectos de los fármacos , Fagopyrum/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Proteínas de Plantas/metabolismo , Animales , Arabidopsis/genética , Membrana Celular/metabolismo , Ácido Cítrico/metabolismo , Fagopyrum/citología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Prueba de Complementación Genética , Aparato de Golgi/metabolismo , Mutación , Oocitos/metabolismo , Proteínas de Transporte de Catión Orgánico/genética , Proteínas de Plantas/genética , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , XenopusRESUMEN
Buckwheat (Fagopyrum esculentum Moench) is able to detoxify high aluminium (Al) internally by sequestering it to the vacuoles in the leaves; however, the molecular mechanisms underlying this sequestration are unknown. We performed proteomic analysis with the leaf tonoplast-rich fraction and identified two half-size ABC transporters; FeASL1.1 and FeALS1.2. We investigated the gene expression patterns and subcellular localization. To demonstrate their physiological role, we expressed FeALS1.1 or FeALS1.2 in the Arabidopsis atals1 mutant under the control of AtALS1 promoter. FeALS1.1 expression was upregulated by Al in both the leaves and the roots, and its expression level in the roots was six times higher than its homologous gene (AtALS1) of Arabidopsis. FeALS1.2 expression, however, was not affected by Al but showed a 39 times higher expression level than AtALS1 in the leaves. When FeALS1.1 or FeALS1.2 was expressed in atals1, both of them recovered their Al tolerance through altering the subcellular localization of Al in root cells. Taken together, our results indicate that FeALS1.1 and FeALS1.2 are involved in the internal detoxification of Al in the roots and leaves, respectively, by sequestering Al into the vacuoles. Their high expression is probably required for high Al tolerance in buckwheat.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Aluminio/metabolismo , Fagopyrum/genética , Fagopyrum/metabolismo , Genes de Plantas , Proteínas de Plantas/genética , Arabidopsis/genética , Clonación Molecular , Regulación de la Expresión Génica de las Plantas , Prueba de Complementación Genética , Membranas Intracelulares/metabolismo , Mutación/genética , Especificidad de Órganos/genética , Filogenia , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteómica , ARN Mensajero/genética , ARN Mensajero/metabolismo , Fracciones Subcelulares/metabolismo , Vacuolas/metabolismoRESUMEN
Investigations into the epigenetic status of individual cells within tissues can produce both epigenetic data for different cell types and positional information of the cells. Thus, these investigations are important for understanding the intra- and inter-cellular control systems of developmental and environmental responses in plants. However, a simple method to detect epigenetic modifications of individual cells in plant tissues is not yet available because detection of the modifications requires immunohistochemistry using specific antibodies. In this study, we developed a simple immunohistochemical method that does not require sectioning to investigate epigenetic modifications. This method uses a clearing system to detect methylated histones, acetylated histones, methylated DNA and/or centromeric histone H3 variants. Analyses of four dicots and five monocots indicated that this method provides a universal technique to investigate epigenetic modifications in diverse plant species.
Asunto(s)
Epigénesis Genética , Inmunohistoquímica/métodos , Proteínas de Plantas/genética , Urea/química , Xilitol/química , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Proteína A Centromérica/genética , Proteína A Centromérica/metabolismo , Ajo/genética , Ajo/crecimiento & desarrollo , Ajo/metabolismo , Helianthus/genética , Helianthus/crecimiento & desarrollo , Helianthus/metabolismo , Histonas/genética , Histonas/metabolismo , Hordeum/genética , Hordeum/crecimiento & desarrollo , Hordeum/metabolismo , Solanum lycopersicum/genética , Solanum lycopersicum/crecimiento & desarrollo , Solanum lycopersicum/metabolismo , Oryza/genética , Oryza/crecimiento & desarrollo , Oryza/metabolismo , Hojas de la Planta/genética , Hojas de la Planta/crecimiento & desarrollo , Hojas de la Planta/metabolismo , Proteínas de Plantas/metabolismo , Nicotiana/genética , Nicotiana/crecimiento & desarrollo , Nicotiana/metabolismo , Triticum/genética , Triticum/crecimiento & desarrollo , Triticum/metabolismo , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismo , Zea mays/genética , Zea mays/crecimiento & desarrollo , Zea mays/metabolismoRESUMEN
Phosphorus is an important nutrient for crop productivity. More than 60% of the total phosphorus in cereal crops is finally allocated into the grains and is therefore removed at harvest. This removal accounts for 85% of the phosphorus fertilizers applied to the field each year. However, because humans and non-ruminants such as poultry, swine and fish cannot digest phytate, the major form of phosphorus in the grains, the excreted phosphorus causes eutrophication of waterways. A reduction in phosphorus accumulation in the grain would contribute to sustainable and environmentally friendly agriculture. Here we describe a rice transporter, SULTR-like phosphorus distribution transporter (SPDT), that controls the allocation of phosphorus to the grain. SPDT is expressed in the xylem region of both enlarged- and diffuse-vascular bundles of the nodes, and encodes a plasma-membrane-localized transporter for phosphorus. Knockout of this gene in rice (Oryza sativa) altered the distribution of phosphorus, with decreased phosphorus in the grains but increased levels in the leaves. Total phosphorus and phytate in the brown de-husked rice were 20-30% lower in the knockout lines, whereas yield, seed germination and seedling vigour were not affected. These results indicate that SPDT functions in the rice node as a switch to allocate phosphorus preferentially to the grains. This finding provides a potential strategy to reduce the removal of phosphorus from the field and lower the risk of eutrophication of waterways.
Asunto(s)
Agricultura/métodos , Proteínas de Transporte de Membrana/deficiencia , Proteínas de Transporte de Membrana/metabolismo , Oryza/anatomía & histología , Oryza/metabolismo , Fósforo/metabolismo , Proteínas de Plantas/metabolismo , Animales , Transporte Biológico , Grano Comestible/metabolismo , Eutrofización , Fertilizantes , Técnicas de Inactivación de Genes , Germinación , Humanos , Proteínas de Transporte de Membrana/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutación , Especificidad de Órganos , Oryza/genética , Oryza/crecimiento & desarrollo , Ácido Fítico/metabolismo , Células Vegetales/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Plantones/crecimiento & desarrollo , Xilema/metabolismoRESUMEN
Buckwheat (Fagopyrum esculentum Moench) is able to detoxify aluminum (Al) both externally and internally, but the molecular mechanisms underlying its high Al tolerance are not understood. We functionally characterized a gene (FeIREG1) belonging to IRON REGULATED/ferroportin in buckwheat, which showed high expression in our previous genome-wide transcriptome analysis. FeIREG1 was mainly expressed in the roots, and its expression was up-regulated by Al, but not by other metals and low pH. Furthermore, in contrast to AtIREG1 and AtIREG2 in Arabidopsis, the expression of FeIREG1 was not induced by Fe deficiency. Spatial expression analysis showed that the Al-induced expression of FeIREG1 was found in the root tips and higher expression was detected in the outer layers of this part. Immunostaining also showed that FeIREG1 was localized at the outer cell layers in the root tip. A FeIREG1-green fluorescent protein (GFP) fusion protein was localized to the tonoplast when transiently expressed in onion epidermal cells. Overexpression of FeIREG1 in Arabidopsis resulted in increased Al tolerance, but did not alter the tolerance to Cd, Co and Fe. The tolerance to Ni was slightly enhanced in the overexpression lines. Mineral analysis showed that the accumulation of total root Al and other essential mineral elements was hardly altered in the overexpression lines. Taken together, our results suggest that FeIREG1 localized at the tonoplast plays an important role in internal Al detoxification by sequestering Al into the root vacuoles in buckwheat.
Asunto(s)
Aluminio/metabolismo , Aluminio/toxicidad , Fagopyrum/genética , Genes de Plantas , Proteínas de Plantas/genética , Adaptación Fisiológica/efectos de los fármacos , Adaptación Fisiológica/genética , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Fagopyrum/efectos de los fármacos , Fagopyrum/metabolismo , Fagopyrum/fisiología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Concentración de Iones de Hidrógeno , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/genética , Fenotipo , Proteínas de Plantas/metabolismo , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Plantas Modificadas Genéticamente , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
The multidrug and toxic compound extrusion (MATE) transporters represent a large transporter family in plants, but the role of most genes in this family has not been examined. We functionally characterized a MATE family member, OsFRDL2, in rice (Oryza sativa). OsFRDL2 showed an efflux transport activity for citrate when it was expressed in both Xenopus oocytes and cultured tobacco cells. OsFRDL2 was mainly expressed in the roots and its expression was not induced by iron (Fe) deficiency, but it was rapidly up-regulated by aluminum (Al). Furthermore, the expression of OsFRDL2 was regulated by ART1, a C2H2-type zinc-finger transcription factor for Al tolerance. OsFRDL2 protein was localized at unidentified vesicles in the cytosol, but not co-localized with either mitochondria or peroxisomes when expressed in both onion epidermal cells and cultured tobacco cells. Knockout of OsFRDL2 decreased Al-induced secretion of citrate from the roots, but did not affect the internal citrate concentration. The Al-induced inhibition of root elongation was similar between the OsFRDL2 knockout line and its wild-type rice. Knockout of OsFRDL2 did not affect the translocation of Fe from the roots to the shoots. A double mutant between osfrdl2 and osfrdl4 or osfrdl1 did not further decrease the Al-induced citrate secretion and Fe translocation compared with the single mutant. Collectively, our results indicate that although OsFRDL2 is involved in the Al-induced secretion of citrate, its contribution to high Al tolerance is relatively small in rice.
Asunto(s)
Aluminio/toxicidad , Ácido Cítrico/metabolismo , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Oryza/fisiología , Proteínas de Plantas/metabolismo , Animales , Transporte Biológico , Expresión Génica , Técnicas de Inactivación de Genes , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismo , Cebollas/genética , Cebollas/metabolismo , Oocitos , Oryza/citología , Oryza/efectos de los fármacos , Oryza/genética , Proteínas de Plantas/genética , Raíces de Plantas/citología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Plantas Modificadas Genéticamente , Nicotiana/genética , Nicotiana/metabolismo , XenopusRESUMEN
Requirement of mineral elements in different plant tissues is not often consistent with their transpiration rate; therefore, plants have developed systems for preferential distribution of mineral elements to the developing tissues with low transpiration. Here we took silicon (Si) as an example and revealed an efficient system for preferential distribution of Si in the node of rice (Oryza sativa). Rice is able to accumulate more than 10% Si of the dry weight in the husk, which is required for protecting the grains from water loss and pathogen infection. However, it has been unknown for a long time how this hyperaccumulation is achieved. We found that three transporters (Lsi2, Lsi3, and Lsi6) located at the node are involved in the intervascular transfer, which is required for the preferential distribution of Si. Lsi2 was polarly localized to the bundle sheath cell layer around the enlarged vascular bundles, which is next to the xylem transfer cell layer where Lsi6 is localized. Lsi3 was located in the parenchyma tissues between enlarged vascular bundles and diffuse vascular bundles. Similar to Lsi6, knockout of Lsi2 and Lsi3 also resulted in decreased distribution of Si to the panicles but increased Si to the flag leaf. Furthermore, we constructed a mathematical model for Si distribution and revealed that in addition to cooperation of three transporters, an apoplastic barrier localized at the bundle sheath cells and development of the enlarged vascular bundles in node are also required for the hyperaccumulation of Si in rice husk.
Asunto(s)
Proteínas de Transporte de Membrana/metabolismo , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Haz Vascular de Plantas/metabolismo , Silicio/metabolismo , Secuencia de Aminoácidos , Animales , Transporte Biológico/genética , Femenino , Regulación de la Expresión Génica de las Plantas , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de Transporte de Membrana/genética , Microscopía Confocal , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Cebollas/citología , Oocitos/metabolismo , Oryza/genética , Epidermis de la Planta/citología , Epidermis de la Planta/metabolismo , Proteínas de Plantas/genética , Haz Vascular de Plantas/citología , Haz Vascular de Plantas/genética , Plantas Modificadas Genéticamente , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Aminoácido , Xenopus , Xilema/citología , Xilema/genética , Xilema/metabolismoRESUMEN
Ascorbate is an antioxidant and coenzyme for various metabolic reactions in vivo. In plant chloroplasts, high ascorbate levels are required to overcome photoinhibition caused by strong light. However, ascorbate is synthesized in the mitochondria and the molecular mechanisms underlying ascorbate transport into chloroplasts are unknown. Here we show that AtPHT4;4, a member of the phosphate transporter 4 family of Arabidopsis thaliana, functions as an ascorbate transporter. In vitro analysis shows that proteoliposomes containing the purified AtPHT4;4 protein exhibit membrane potential- and Cl(-)-dependent ascorbate uptake. The AtPHT4;4 protein is abundantly expressed in the chloroplast envelope membrane. Knockout of AtPHT4;4 results in decreased levels of the reduced form of ascorbate in the leaves and the heat dissipation process of excessive energy during photosynthesis is compromised. Taken together, these observations indicate that the AtPHT4;4 protein is an ascorbate transporter at the chloroplast envelope membrane, which may be required for tolerance to strong light stress.
Asunto(s)
Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Ácido Ascórbico/metabolismo , Cloroplastos/metabolismo , Proteínas de Transporte de Membrana/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cartilla de ADN/genética , ADN Complementario/genética , Fluorescencia , Técnicas de Inactivación de Genes , Inmunohistoquímica , Técnicas In Vitro , Luz , Proteínas de Transporte de Membrana/metabolismo , Reacción en Cadena de la Polimerasa , Estrés Fisiológico/genéticaRESUMEN
Buckwheat (Fagopyrum esculentum Moench) is a species with high aluminum (Al) tolerance and accumulation. Although the physiological mechanisms for external and internal detoxification of Al have been well studied, the molecular mechanisms responsible are poorly understood. Here, we conducted a genome-wide transcriptome analysis of Al-responsive genes in the roots and leaves using RNA sequencing (RNA-Seq) technology. RNA-Seq generated reads ranging from 56×10(6) to 93×10(6). A total of 148,734 transcript contigs with an average length of 1,014 bp were assembled, generating 84,516 unigenes. Among them, 31,730 and 23,853 unigenes were annotated, respectively, in the NCBI plant database and TAIR database for Arabidopsis. Of the annotated genes, 4,067 genes in the roots and 2,663 genes in the leaves were up-regulated (>2-fold) by Al exposure, while 2,456 genes in the roots and 2,426 genes in the leaves were down-regulated (<2-fold) A few STOP1/ART1 (SENSITIVE TO PROTON RHIZOTOXICITY1/AL RESISTANCE TRANSCRIPTION FACTOR1)-regulated gene homologs including FeSTAR1, FeALS3 (ALUMINUM SENSITIVE3), FeALS1 (ALUMINUM SENSITIVE1), FeMATE1 and FeMATE2 (MULTIDRUG AND TOXIC COMPOUND EXTRUSION1 and 2) were also up-regulated in buckwheat, indicating some common Al tolerance mechanism across the species, although most STOP1/ART1-regulated gene homologs were not changed. Most genes involved in citric and oxalic acid biosynthesis were not significantly altered. Some transporter genes were highly expressed in the roots and leaves and responded to Al stress, implicating their role in Al tolerance and accumulation. Overall, our data provide a platform for further characterizing the functions of genes involved in Al tolerance and accumulation in buckwheat.
Asunto(s)
Aluminio/farmacología , Fagopyrum/genética , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Arabidopsis/fisiología , Secuencia de Bases , Fagopyrum/efectos de los fármacos , Fagopyrum/fisiología , Expresión Génica , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Anotación de Secuencia Molecular , Datos de Secuencia Molecular , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Hojas de la Planta/fisiología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/genética , Raíces de Plantas/fisiología , Análisis de Secuencia de ARNRESUMEN
KEY MESSAGE: Mid-bicellular pollen vegetative cells in tobacco escape from G1 arrest and proceed to the G1/S transition towards androgenesis within 1 day under glutamine starvation conditions in vitro. In the Nicotiana tabacum pollen culture system, immature pollen grains at the mid-bicellular stage can mature in the presence of glutamine; however, if glutamine is absent, they deviate from their native cell fate in a few days. The glutamine-starved pollen grains cannot undergo maturation, even when supplied with glutamine later. Instead, they undergo cell division towards androgenesis slowly within 10 days in a medium containing appropriate nutrients. During the culture period, they ought to escape from G1 arrest to proceed into S phase as the primary step towards androgenesis. However, this event has not been experimentally confirmed. Here, we demonstrated that the pollen vegetative cells proceeded to the G1/S transition within approximately 15-36 h after the start of culture. These results were obtained by analyzing transgenic pollen possessing a fusion gene encoding nuclear-localizing GFP under the control of an E2F motif-containing promoter isolated from a gene encoding one of DNA replication licensing factors. Observations using a 5-ethynyl-2'-deoxyuridine DNA labeling and detection technique uncovered that the G1/S transition was soon followed by S phase. These hallmarks of vegetative cells undergoing dedifferentiation give us new insights into upstream events causing the G1/S transition and also provide a novel strategy to increase the frequency of the androgenic response in tobacco and other species, including recalcitrants.
Asunto(s)
Proteínas de Ciclo Celular/genética , Regulación de la Expresión Génica de las Plantas , Nicotiana/crecimiento & desarrollo , Polen/crecimiento & desarrollo , Secuencias de Aminoácidos , Secuencia de Bases , Proteínas de Ciclo Celular/metabolismo , Desdiferenciación Celular , Células Cultivadas , Cartilla de ADN/genética , Fase G1 , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Glutamina/metabolismo , Datos de Secuencia Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Polen/citología , Polen/embriología , Regiones Promotoras Genéticas/genética , Fase S , Nicotiana/citología , Nicotiana/embriologíaRESUMEN
Manganese (Mn) is an essential micronutrient for plants, but is toxic when present in excess. The rice plant (Oryza sativa L.) accumulates high concentrations of Mn in the aerial parts; however, the molecular basis for Mn tolerance is poorly understood. In the present study, genes encoding Mn tolerance were screened for by expressing cDNAs of genes from rice shoots in Saccharomyces cerevisiae. A gene encoding a cation diffusion facilitator (CDF) family member, OsMTP8.1, was isolated, and its expression was found to enhance Mn accumulation and tolerance in S. cerevisiae. In plants, OsMTP8.1 and its transcript were mainly detected in shoots. High or low supply of Mn moderately induced an increase or decrease in the accumulation of OsMTP8.1, respectively. OsMTP8.1 was detected in all cells of leaf blades through immunohistochemistry. OsMTP8.1 fused to green fluorescent protein was localized to the tonoplast. Disruption of OsMTP8.1 resulted in decreased chlorophyll levels, growth inhibition in the presence of high concentrations of Mn, and decreased accumulation of Mn in shoots and roots. However, there was no difference in the accumulation of other metals, including Zn, Cu, Fe, Mg, Ca, and K. These results suggest that OsMTP8.1 is an Mn-specific transporter that sequesters Mn into vacuoles in rice and is required for Mn tolerance in shoots.
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Adaptación Fisiológica/efectos de los fármacos , Manganeso/toxicidad , Oryza/fisiología , Proteínas de Plantas/metabolismo , Secuencia de Aminoácidos , Cationes , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Difusión , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Datos de Secuencia Molecular , Mutación/genética , Oryza/efectos de los fármacos , Oryza/genética , Fenotipo , Proteínas de Plantas/química , Proteínas de Plantas/genética , Transporte de Proteínas/efectos de los fármacos , Saccharomyces cerevisiae/efectos de los fármacos , Análisis de Secuencia de Proteína , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/metabolismoRESUMEN
Cu is an essential element for plant growth, but the molecular mechanisms of its distribution and redistribution within the plants are unknown. Here, we report that Yellow stripe-like16 (YSL16) is involved in Cu distribution and redistribution in rice (Oryza sativa). Rice YSL16 was expressed in the roots, leaves, and unelongated nodes at the vegetative growth stage and highly expressed in the upper nodes at the reproductive stage. YSL16 was expressed at the phloem of nodes and vascular tissues of leaves. Knockout of this gene resulted in a higher Cu concentration in the older leaves but a lower concentration in the younger leaves at the vegetative stage. At the reproductive stage, a higher Cu concentration was found in the flag leaf and husk, but less Cu was present in the brown rice, resulting in a significant reduction in fertility in the knockout line. Isotope labeling experiments with (65)Cu showed that the mutant lost the ability to transport Cu-nicotianamine from older to younger leaves and from the flag leaf to the panicle. Rice YSL16 transported the Cu-nicotianamine complex in yeast. Taken together, our results indicate that Os-YSL16 is a Cu-nicotianamine transporter that is required for delivering Cu to the developing young tissues and seeds through phloem transport.
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Cobre/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Oryza/metabolismo , Floema/metabolismo , Ácido Azetidinocarboxílico/análogos & derivados , Ácido Azetidinocarboxílico/metabolismo , Secuencia de Bases , Transporte Biológico/genética , Cationes/análisis , Cationes/metabolismo , Cobre/análisis , Fertilidad , Regulación de la Expresión Génica de las Plantas , Técnicas de Inactivación de Genes , Proteínas de Transporte de Membrana/genética , Modelos Biológicos , Datos de Secuencia Molecular , Mutación , Cebollas/genética , Cebollas/metabolismo , Especificidad de Órganos , Oryza/genética , Fenotipo , Floema/genética , Hojas de la Planta/genética , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Reproducción , Semillas/genética , Semillas/metabolismo , Análisis de Secuencia de ADNRESUMEN
Aluminum (Al) toxicity is a major factor limiting crop production in acidic soil, but the molecular mechanisms of Al tolerance are poorly understood. Here, we report that two genes, STAR1 (for sensitive to Al rhizotoxicity1) and STAR2, are responsible for Al tolerance in rice. STAR1 encodes a nucleotide binding domain, while STAR2 encodes a transmembrane domain, of a bacterial-type ATP binding cassette (ABC) transporter. Disruption of either gene resulted in hypersensitivity to aluminum toxicity. Both STAR1 and STAR2 are expressed mainly in the roots and are specifically induced by Al exposure. Expression in onion epidermal cells, rice protoplasts, and yeast showed that STAR1 interacts with STAR2 to form a complex that localizes to the vesicle membranes of all root cells, except for those in the epidermal layer of the mature zone. When expressed together in Xenopus laevis oocytes, STAR1/2 shows efflux transport activity specific for UDP-glucose. Furthermore, addition of exogenous UDP-glucose rescued root growth in the star1 mutant exposed to Al. These results indicate that STAR1 and STAR2 form a complex that functions as an ABC transporter, which is required for detoxification of Al in rice. The ABC transporter transports UDP-glucose, which may be used to modify the cell wall.
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Transportadoras de Casetes de Unión a ATP/fisiología , Aluminio/farmacología , Oryza/metabolismo , Proteínas de Plantas/fisiología , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Animales , Sitios de Unión , Transporte Biológico/fisiología , Proteínas Fluorescentes Verdes/análisis , Cebollas/genética , Oryza/efectos de los fármacos , Oryza/genética , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/análisis , Xenopus laevis/genética , Levaduras/genéticaRESUMEN
Lsi1 (OsNIP2;1) is the first silicon (silicic acid) transporter identified in plant, which belongs to the nodulin 26-like intrinsic membrane protein (NIP) subfamily. In this study, we characterized the function of this transporter by using the Xenopus laevis oocyte expression system. The transport activity of Lsi1 for silicic acid was significantly inhibited by HgCl2 but not by low temperature. Lsi1 also showed an efflux transport activity for silicic acid. The substrate specificity study showed that Lsi1 was able to transport urea and boric acid; however, the transport activity for silicic acid was not affected by the presence of equimolar urea and was decreased only slightly by boric acid. Furthermore, among the NIPs subgroup, OsNIP2;2 showed transport activity for silicic acid, whereas OsNIP1;1 and OsNIP3;1 did not. We propose that Lsi1 and its close homologues form a unique subgroup of NIP with a distinct ar/R selectivity filter, which is located in the narrowest region on the extra-membrane mouth and govern the substrate specificity of the pore.