RESUMEN

Under sulfur deficiency (-S), plants induce expression of the sulfate transport systems in roots to increase uptake and root-to-shoot transport of sulfate. The low-affinity sulfate transporter SULTR2;1 is predominantly expressed in xylem parenchyma and pericycle cells in Arabidopsis thaliana roots under -S. The mechanisms underlying -S-inducible expression of SULTR2;1 in roots have remained unclear, despite the possible significance of SULTR2;1 for acclimation to low-sulfur conditions. In this investigation, examination of deletions and base substitutions in the 3'-intergenic region of SULTR2;1 revealed novel sulfur-responsive elements, SURE21A (5'-CAATGTATC-3') and SURE21B (5'-CTAGTAC-3'), located downstream of the SULTR2;1 3'-untranslated region. SURE21A and SULTR21B effectively induced reporter gene expression from fusion constructs under -S in combination with minimal promoters or promoters not inducible by -S, suggesting their versatility in controlling transcription. T-DNA insertions near SURE21A and SULTR21B abolished -S-inducible expression of SULTR2;1 in roots and reduced the uptake and root-to-shoot transport of sulfate. In addition, these mutations partially suppressed SULTR2;1 expression in shoots, without changing its -S-responsive expression. These findings indicate that SULTR2;1 contributes to the increase in uptake and internal translocation of sulfate driven by gene expression induced under the control of sulfur-responsive elements in the 3'-nontranscribed intergenic region of SULTR2;1.

Asunto(s)

Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Raíces de Plantas/metabolismo , Azufre/deficiencia , Proteínas de Transporte de Anión/genética , Proteínas de Transporte de Anión/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Raíces de Plantas/genética

RESUMEN

Despite recent intensive research efforts in functional genomics, the functions of only a limited number of Arabidopsis (Arabidopsis thaliana) genes have been determined experimentally, and improving gene annotation remains a major challenge in plant science. As metabolite profiling can characterize the metabolomic phenotype of a genetic perturbation in the plant metabolism, it provides clues to the function(s) of genes of interest. We chose 50 Arabidopsis mutants, including a set of characterized and uncharacterized mutants, that resemble wild-type plants. We performed metabolite profiling of the plants using gas chromatography-mass spectrometry. To make the data set available as an efficient public functional genomics tool for hypothesis generation, we developed the Metabolite Profiling Database for Knock-Out Mutants in Arabidopsis (MeKO). It allows the evaluation of whether a mutation affects metabolism during normal plant growth and contains images of mutants, data on differences in metabolite accumulation, and interactive analysis tools. Nonprocessed data, including chromatograms, mass spectra, and experimental metadata, follow the guidelines set by the Metabolomics Standards Initiative and are freely downloadable. Proof-of-concept analysis suggests that MeKO is highly useful for the generation of hypotheses for genes of interest and for improving gene annotation. MeKO is publicly available at http://prime.psc.riken.jp/meko/.

RESUMEN

To complete the metabolic map for an entire class of compounds, it is essential to identify gene-metabolite correlations of a metabolic pathway. We used liquid chromatography-mass spectrometry (LC-MS) to identify the flavonoids produced by Arabidopsis thaliana wild-type and flavonoid biosynthetic mutant lines. The structures of 15 newly identified and eight known flavonols were deduced by LC-MS profiling of these mutants. Candidate genes presumably involved in the flavonoid pathway were delimited by transcriptome coexpression network analysis using public databases, leading to the detailed analysis of two flavonoid pathway genes, UGT78D3 (At5g17030) and RHM1 (At1g78570). The levels of flavonol 3-O-arabinosides were reduced in ugt78d3 knockdown mutants, suggesting that UGT78D3 is a flavonol arabinosyltransferase. Recombinant UGT78D3 protein could convert quercetin to quercetin 3-O-arabinoside. The strict substrate specificity of UGT78D3 for flavonol aglycones and UDP-arabinose indicate that UGT78D3 is a flavonol arabinosyltransferase. A comparison of flavonol profile in RHM knockout mutants indicated that RHM1 plays a major role in supplying UDP-rhamnose for flavonol modification. The rate of flavonol 3-O-glycosylation is more affected than those of 7-O-glycosylation by the supply of UDP-rhamnose. The precise identification of flavonoids in conjunction with transcriptomics thus led to the identification of a gene function and a more complete understanding of a plant metabolic network.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Flavonoles/metabolismo , Perfilación de la Expresión Génica/métodos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Cromatografía Liquida , Flavonoles/química , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Estructura Molecular , Mutación , Pentosiltransferasa/genética , Pentosiltransferasa/metabolismo , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato

RESUMEN

Molybdenum (Mo) is a trace element essential for living organisms, however no molybdate transporter has been identified in eukaryotes. Here, we report the identification of a molybdate transporter, MOT1, from Arabidopsis thaliana. MOT1 is expressed in both roots and shoots, and the MOT1 protein is localized, in part, to plasma membranes and to vesicles. MOT1 is required for efficient uptake and translocation of molybdate and for normal growth under conditions of limited molybdate supply. Kinetics studies in yeast revealed that the K(m) value of MOT1 for molybdate is approximately 20 nM. Furthermore, Mo uptake by MOT1 in yeast was not affected by coexistent sulfate, and MOT1 did not complement a sulfate transporter-deficient yeast mutant strain. These data confirmed that MOT1 is specific for molybdate and that the high affinity of MOT1 allows plants to obtain scarce Mo from soil.

Asunto(s)

Proteínas de Transporte de Anión/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Molibdeno/metabolismo , Suelo , Proteínas de Transporte de Anión/genética , Arabidopsis/genética , Arabidopsis/crecimiento & desarrollo , Proteínas de Arabidopsis/genética , Transporte Biológico , ADN Bacteriano/genética , Regulación de la Expresión Génica de las Plantas , Mutación , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo

RESUMEN

High-affinity sulfate transporters SULTR1;1 and SULTR1;2 are expressed at epidermis and cortex of Arabidopsis (Arabidopsis thaliana) roots during sulfur limitation. Here, we report that SULTR1;1 and SULTR1;2 are two essential components of the sulfate uptake system in roots and are regulated at posttranscriptional levels together with the previously reported transcriptional control. Double knockout of SULTR1;1 and SULTR1;2 by T-DNA insertion gene disruption resulted in complete lack of sulfate uptake capacity and severely affected plant growth under low-sulfur conditions. Expression of epitope-tagged proteins SULTR1;1mycHis and SULTR1;2mycHis, under the control of the cauliflower mosaic virus 35S promoter, rescued the uptake of sulfate and the growth of the sultr1;1 sultr1;2 double knockout mutant. The recovery of the double knockout phenotypes was attributable to the posttranscriptional accumulation of sulfate transporter proteins that derive from the epitope-tagged transgenic constructs. Both SULTR1;1mycHis and SUTLR1;2mycHis mRNAs were predominantly found in roots and slightly induced by long-term sulfur limitation. SULTR1;1mycHis and SULTR1;2mycHis proteins were found exclusively in roots, and significantly accumulated by sulfur limitation, correlating with the induction of sulfate uptake activities. In the time course of short-term sulfate starvation treatment, SULTR1;1mycHis and SULTR1;2mycHis proteins were significantly accumulated during the 8- to 72-h period, causing substantial induction of sulfate uptake activities, while their corresponding mRNAs were expressed constantly around the initial levels, except for the transient induction in the first 2 h. This study suggested the importance of root-specific and sulfur deficiency-inducible accumulation of SULTR1;1 and SULTR1;2 sulfate transporter proteins for the acquisition of sulfate from low-sulfur environment.

Asunto(s)

Proteínas de Transporte de Anión/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas , Azufre/metabolismo , Proteínas de Transporte de Anión/genética , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Transporte Biológico , Eliminación de Gen , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Datos de Secuencia Molecular , Mutación , Raíces de Plantas/genética , Raíces de Plantas/metabolismo , Plantas Modificadas Genéticamente , Azufre/farmacología , Factores de Tiempo , Transcripción Genética

RESUMEN

SULTR1;1 high-affinity sulfate transporter is highly regulated in the epidermis and cortex of Arabidopsis roots responding to sulfur deficiency (-S). We identified a novel cis-acting element involved in the -S-inducible expression of sulfur-responsive genes in Arabidopsis. The promoter region of SULTR1;1 was dissected for deletion and gain-of-function analysis using luciferase (LUC) reporter gene in transgenic Arabidopsis. The 16-bp sulfur-responsive element (SURE) from -2777 to -2762 of SULTR1;1 promoter was sufficient and necessary for the -S-responsive expression, which was reversed when supplied with cysteine and glutathione (GSH). The SURE sequence contained an auxin response factor (ARF) binding sequence (GAGACA). However, SURE was not responsive to naphthalene acetic acid, indicating its specific function in the sulfur response. The base substitution analysis indicated the significance of a 5-bp sequence (GAGAC) within the conserved ARF binding site as a core element for the -S response. Microarray analysis of early -S response in Arabidopsis roots indicated the presence of SURE core sequences in the promoter regions of -S-inducible genes on a full genome GeneChip array. It is suggested that SURE core sequences may commonly regulate the expression of a gene set required for adaptation to the -S environment.

Asunto(s)

Proteínas de Transporte de Anión/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Raíces de Plantas/metabolismo , Regiones Promotoras Genéticas/fisiología , Azufre/metabolismo , Proteínas de Transporte de Anión/genética , Proteínas de Arabidopsis/genética , Secuencia de Bases , Secuencia Conservada , Regulación hacia Abajo , Regulación de la Expresión Génica de las Plantas/fisiología , Plantas Modificadas Genéticamente , Unión Proteica

RESUMEN

Uptake of external sulfate from the environment and use of internal vacuolar sulfate pools are two important aspects of the acquisition of sulfur for metabolism. In this study, we demonstrated that the vacuolar SULTR4-type sulfate transporter facilitates the efflux of sulfate from the vacuoles and plays critical roles in optimizing the internal distribution of sulfate in Arabidopsis thaliana. SULTR4;1-green fluorescent protein (GFP) and SULTR4;2-GFP fusion proteins were expressed under the control of their own promoters in transgenic Arabidopsis. The fusion proteins were accumulated specifically in the tonoplast membranes and were localized predominantly in the pericycle and xylem parenchyma cells of roots and hypocotyls. In roots, SULTR4;1 was constantly accumulated regardless of the changes of sulfur conditions, whereas SULTR4;2 became abundant by sulfur limitation. In shoots, both transporters were accumulated by sulfur limitation. Vacuoles isolated from callus of the sultr4;1 sultr4;2 double knockout showed excess accumulation of sulfate, which was substantially decreased by overexpression of SULTR4;1-GFP. In seedlings, the supplied [(35)S]sulfate was retained in the root tissue of the sultr4;1 sultr4;2 double knockout mutant. Comparison of the double and single knockouts suggested that SULTR4;1 plays a major role and SULTR4;2 has a supplementary function. Overexpression of SULTR4;1-GFP significantly decreased accumulation of [(35)S]sulfate in the root tissue, complementing the phenotype of the double mutant. These results suggested that SULTR4-type transporters, particularly SULTR4;1, actively mediate the efflux of sulfate from the vacuole lumen into the cytoplasm and influence the capacity for vacuolar storage of sulfate in the root tissue. The efflux function will promote rapid turnover of sulfate from the vacuoles particularly in the vasculature under conditions of low-sulfur supply, which will optimize the symplastic (cytoplasmic) flux of sulfate channeled toward the xylem vessels.

Asunto(s)

Arabidopsis/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Sulfatos/metabolismo , Vacuolas/metabolismo , Secuencia de Bases , Cartilla de ADN , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Plantas Modificadas Genéticamente/metabolismo , Transportadores de Sulfato

RESUMEN

SULTR1;1 high-affinity sulfate transporter is highly regulated by sulfur deficiency (-S) in the epidermis and cortex of Arabidopsis roots. The regulatory mechanism of SULTR1;1 expression was studied using inhibitors for transcription, translation, protein phosphorylation and dephosphorylation. The induction of SULTR1;1 mRNA during -S was blocked by the addition of actinomycin D in the medium, suggesting that SULTR1;1 is transcriptionally regulated. Cycloheximide repressed the -S induction of SULTR1;1, but enhanced the basal mRNA level of SULTR1;1 under sulfur replete (+S) condition. In addition, the induction of SULTR1;1 by -S was significantly blocked by okadaic acid (OKA) and calyculin A (CalyA). Regulation of SULTR1;1 was further confirmed in transgenic plants expressing green fluorescent protein (GFP) under the control of SULTR1;1 promoter. Accumulation of GFP during -S was dependent to SULTR1;1 promoter, and the effects of OKA and CalyA were reproducible in the SULTR1;1 promoter-GFP plants. These results suggested that the up-regulation of SULTR1;1 by -S requires protein phosphatase as an upstream regulatory factor.

Asunto(s)

Proteínas de Transporte de Anión , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Transporte de Membrana , Raíces de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas Portadoras/genética , Cicloheximida/farmacología , Dactinomicina/farmacología , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Proteínas Fluorescentes Verdes , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Toxinas Marinas , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Fosforilación/efectos de los fármacos , Raíces de Plantas/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transportadores de Sulfato , Azufre/deficiencia , Azufre/metabolismo

RESUMEN

Glutamine synthetase (GS; EC 6.3.1.2) is a key enzyme of nitrogen assimilation, catalyzing the synthesis of glutamine from ammonium and glutamate. In Arabidopsis, cytosolic GS (GS1) was accumulated in roots when plants were excessively supplied with ammonium; however, the GS activity was controlled at a constant level. The discrepancy between the protein content and enzyme activity of GS1 was attributable to the kinetic properties and expression of four distinct isoenzymes encoded by GLN1;1, GLN1;2, GLN1;3 and GLN1;4, genes that function complementary to each other in Arabidopsis roots. GLN1;2 was the only isoenzyme significantly up-regulated by ammonium, which correlated with the rapid increase in total GS1 protein. GLN1;2 was localized in the vasculature and exhibited low affinities to ammonium (Km = 2450 +/- 150 microm) and glutamate (Km = 3.8 +/- 0.2 mm). The expression of the counterpart vascular tissue-localizing low affinity isoenzyme, GLN1;3, was not stimulated by ammonium; however, the enzyme activity of GLN1;3 was significantly inhibited by a high concentration of glutamate. By contrast, the high affinity isoenzyme, GLN1;1 (Km for ammonium < 10 microm; Km for glutamate = 1.1 +/- 0.4 mm) was abundantly accumulated in the surface layers of roots during nitrogen limitation and was down-regulated by ammonium excess. GLN1;4 was another high affinity-type GS1 expressed in nitrogen-starved plants but was 10-fold less abundant than GLN1;1. These results suggested that dynamic regulations of high and low affinity GS1 isoenzymes at the levels of mRNA and enzyme activities are dependent on nitrogen availabilities and may contribute to the homeostatic control of glutamine synthesis in Arabidopsis roots.

Asunto(s)

Arabidopsis/enzimología , Glutamato-Amoníaco Ligasa/metabolismo , Regulación hacia Abajo , Activación Enzimática , Isoenzimas/metabolismo , Cinética , Raíces de Plantas/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Especificidad por Sustrato

RESUMEN

Sulfate is a macronutrient required for cell growth and development. Arabidopsis has two high-affinity sulfate transporters (SULTR1;1 and SULTR1;2) that represent the sulfate uptake activities at the root surface. Sulfur limitation (-S) response relevant to the function of SULTR1;2 was elucidated in this study. We have isolated a novel T-DNA insertion allele defective in the SULTR1;2 sulfate transporter. This mutant, sel1-10, is allelic with the sel1 mutants identified previously in a screen for increased tolerance to selenate, a toxic analog of sulfate (Shibagaki et al., 2002). The abundance of SULTR1;1 mRNA was significantly increased in the sel1-10 mutant; however, this compensatory up-regulation of SULTR1;1 was not sufficient to restore the growth. The sulfate content of the mutant was 10% to 20% of the wild type, suggesting that induction of SULTR1;1 is not fully complementing the function of SULTR1;2 and that SULTR1;2 serves as the major facilitator for the acquisition of sulfate in Arabidopsis roots. Transcriptome analysis of approximately 8,000 Arabidopsis genes in the sel1-10 mutant suggested that dysfunction of the SULTR1;2 transporter can mimic general -S symptoms. Hierarchal clustering of sulfur responsive genes in the wild type and mutant indicated that sulfate uptake, reductive sulfur assimilation, and turnover of secondary sulfur metabolites are activated under -S. The profiles of -S-responsive genes further suggested induction of genes that may alleviate oxidative damage and generation of reactive oxygen species caused by shortage of glutathione.

Asunto(s)

Proteínas de Arabidopsis/genética , Arabidopsis/genética , Proteínas Portadoras/genética , Perfilación de la Expresión Génica , Proteínas de Transporte de Membrana , Azufre/metabolismo , Transcripción Genética , Arabidopsis/crecimiento & desarrollo , Arabidopsis/metabolismo , Familia de Multigenes , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transportadores de Sulfato