RESUMO
A mutation abolishing cytosolic glutamine synthetase1;2 (GS1;2) activity impairs assimilation of ammonium into glutamine in both roots and basal portions of shoots, and severely decreases axillary bud outgrowth (tillering) in mutant rice seedlings. Although the gs1;2 mutant phenotype is independent of strigolactone, which inhibits tillering, it also demonstrates glutamine- or related metabolite-responsive biosynthesis of cytokinin (CK), which promotes tillering. Here, we examined the connection between GS1;2 and CK biosynthesis during tillering, focusing on basal portions of the shoots as well as apical and axillary bud meristems in the gs1;2 mutant. Despite a sufficient ammonium supply, decreases in precursor CK contents and a decrease in ammonium assimilation into glutamine were observed in basal portions of mutant shoots. Reintroducing expression of OsGS1;2 cDNA driven by its own promoter restored precursor CK contents and ammonium assimilation to wild-type levels. In basal portions of the shoots, glutamine-responsive adenosine phosphate-isopentenyltransferase4 (OsIPT4), which is also predominant in rice roots, was the predominant isogene for IPT, which synthesizes CK. Cell-specific expression of OsIPT4 in phloem companion cells in nodal vascular anastomoses connected to the axillary bud vasculature also decreased in the gs1;2 mutant. Expression of CK-responsive type-A response regulator genes as local indicators of active CKs was also abolished in the axillary bud meristem of the mutant. These results suggest that the lack of GS1;2 activity decreased levels of glutamine or a related metabolite required for CK biosynthesis, causing a deficiency in active CK in the axillary bud meristem necessary for tillering.
Assuntos
Citocininas/metabolismo , Glutamato-Amônia Ligase/metabolismo , Nitrogênio/metabolismo , Oryza/crescimento & desenvolvimento , Oryza/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/metabolismo , Compostos de Amônio/metabolismo , Citosol/enzimologia , Glutamato-Amônia Ligase/genética , Glutamina/metabolismo , Isoenzimas/genética , Isoenzimas/metabolismo , Mutação , Floema/genética , Floema/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Brotos de Planta/crescimento & desenvolvimento , Brotos de Planta/metabolismo , Plântula/genética , Plântula/crescimento & desenvolvimento , Plântula/metabolismoRESUMO
The phytohormone cytokinin regulates plant growth and development. This hormone is also synthesized by some phytopathogenic bacteria, such as Agrobacterium tumefaciens, and is as a key factor in the formation of plant tumors. The rate-limiting step of cytokinin biosynthesis is catalyzed by adenosine phosphate-isopentenyltransferase (IPT). Agrobacterium IPT has a unique substrate specificity that enables it to increase trans-zeatin production by recruiting a metabolic intermediate of the host plant's biosynthetic pathway. Here, we show the crystal structures of Tzs, an IPT from A. tumefaciens, complexed with AMP and a prenyl-donor analogue, dimethylallyl S-thiodiphosphate. The structures reveal that the carbon-nitrogen-based prenylation proceeds by the SN2-reaction mechanism. Site-directed mutagenesis was used to determine the amino acid residues, Asp-173 and His-214, which are responsible for differences in prenyl-donor substrate specificity between plant and bacterial IPTs. IPT and the p loop-containing nucleoside triphosphate hydrolases likely evolved from a common ancestral protein. Despite structural similarities, IPT has evolved a distinct role in which the p loop transfers a prenyl moiety in cytokinin biosynthesis.
Assuntos
Citocininas/biossíntese , Evolução Molecular , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Agrobacterium tumefaciens/química , Agrobacterium tumefaciens/genética , Agrobacterium tumefaciens/metabolismo , Aminoácidos/química , Aminoácidos/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Citocininas/química , Cinética , Modelos Moleculares , Compostos Organotiofosforados/química , Compostos Organotiofosforados/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Zinco/química , Zinco/metabolismoRESUMO
Plants provide nourishment for animals and other heterotrophs as the sole primary producer in the food chain. Glutamine synthetase (GS), one of the essential enzymes for plant autotrophy catalyzes the incorporation of ammonia into glutamate to generate glutamine with concomitant hydrolysis of ATP, and plays a crucial role in the assimilation and re-assimilation of ammonia derived from a wide variety of metabolic processes during plant growth and development. Elucidation of the atomic structure of higher plant GS is important to understand its detailed reaction mechanism and to obtain further insight into plant productivity and agronomical utility. Here we report the first crystal structures of maize (Zea mays L.) GS. The structure reveals a unique decameric structure that differs significantly from the bacterial GS structure. Higher plants have several isoenzymes of GS differing in heat stability and catalytic properties for efficient responses to variation in the environment and nutrition. A key residue responsible for the heat stability was found to be Ile-161 in GS1a. The three structures in complex with substrate analogues, including phosphinothricin, a widely used herbicide, lead us to propose a mechanism for the transfer of phosphate from ATP to glutamate and to interpret the inhibitory action of phosphinothricin as a guide for the development of new potential herbicides.
Assuntos
Glutamato-Amônia Ligase/química , Zea mays/enzimologia , Trifosfato de Adenosina/química , Sequência de Aminoácidos , Catálise , Escherichia coli/metabolismo , Ácido Glutâmico/química , Temperatura Alta , Hidrólise , Isoleucina/química , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Recombinantes/química , Homologia de Sequência de AminoácidosRESUMO
Agrobacterium tumefaciens infects plants and induces the formation of tumors called "crown galls" by integrating the transferred-DNA (T-DNA) region of the Ti-plasmid into the plant nuclear genome. Tumors are formed because the T-DNA encodes enzymes that modify the synthesis of two plant growth hormones, auxin and cytokinin (CK). Here, we show that a CK biosynthesis enzyme, Tmr, which is encoded by the Agrobacterium T-DNA region, is targeted to and functions in plastids of infected plant cells, despite having no typical plastid-targeting sequence. Evidence is provided that Tmr is an adenosine phosphate-isopentenyltransferase (IPT) that creates a new CK biosynthesis bypass by using 1-hydroxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBDP) as a substrate. Unlike in the conventional CK biosynthesis pathway in plants, trans-zeatin-type CKs are produced directly without the requirement for P450 monooxygenase-mediated hydroxylation. Consistent with the plastid localization of Tmr, HMBDP is an intermediate in the methylerythritol phosphate pathway, a plastid-localized biosynthesis route for universal isoprenoid precursors. These results demonstrate that A. tumefaciens modifies CK biosynthesis by sending a key enzyme into plastids of the host plant to promote tumorigenesis.
Assuntos
Agrobacterium tumefaciens/enzimologia , Alquil e Aril Transferases/metabolismo , Arabidopsis/microbiologia , Proteínas de Bactérias/metabolismo , Citocininas/biossíntese , Proteínas Oncogênicas/metabolismo , Plasmídeos Indutores de Tumores em Plantas/genética , Tumores de Planta/genética , Nucleotídeos de Adenina/metabolismo , Western Blotting , Vetores Genéticos , Proteínas de Fluorescência Verde , Organofosfatos/metabolismo , Tumores de Planta/microbiologiaRESUMO
We identified four genes for potential equilibrative nucleoside transporters (ENTs) from rice (Oryza sativa; designated OsENT1 through OsENT4). Growth analysis of budding yeast (Saccharomyces cerevisiae) cells expressing OsENTs showed that OsENT2 transported adenosine and uridine with high affinity (adenosine, K(m) = 3.0 microm; uridine, K(m) = 0.7 microm). Purine or pyrimidine nucleosides and 2'-deoxynucleosides strongly inhibited adenosine transport via OsENT2, suggesting that OsENT2 possesses broad substrate specificity. OsENT2-mediated adenosine transport was resistant to the typical inhibitors of mammalian ENTs, nitrobenzylmercaptopurine ribonucleoside, dilazep, and dipyridamole. The transport activity was maximal at pH 5.0 and decreased slightly at lower as well as higher pH. In competition experiments with various cytokinins, adenosine transport by OsENT2 was inhibited by isopentenyladenine riboside (iPR). Direct measurements with radiolabeled cytokinins demonstrated that OsENT2 mediated uptake of iPR (K(m) = 32 microm) and trans-zeatin riboside (K(m) = 660 microm), suggesting that OsENT2 participates in iPR transport in planta. In mature plants, OsENT2 was predominantly expressed in roots. The OsENT2 promoter drove the expression of the beta-glucuronidase reporter gene in the scutellum during germination and in vascular tissues in germinated plants, suggesting a participation of OsENT2 in the retrieval of endosperm-derived nucleosides by the germinating embryo and in the long-distance transport of nucleosides in growing plants, respectively.
Assuntos
Proteínas de Transporte de Nucleosídeo Equilibrativas/genética , Regulação da Expressão Gênica de Plantas , Oryza/genética , Adenosina/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Primers do DNA , Proteínas de Transporte de Nucleosídeo Equilibrativas/química , Regulação da Expressão Gênica no Desenvolvimento , Cinética , Dados de Sequência Molecular , Oryza/crescimento & desenvolvimento , Filogenia , Reação em Cadeia da Polimerase , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Uridina/metabolismoRESUMO
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.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Raízes de Plantas/metabolismo , Regiões Promotoras Genéticas/fisiologia , Enxofre/metabolismo , Proteínas de Transporte de Ânions/genética , Proteínas de Arabidopsis/genética , Sequência de Bases , Sequência Conservada , Regulação para Baixo , Regulação da Expressão Gênica de Plantas/fisiologia , Plantas Geneticamente Modificadas , Ligação ProteicaRESUMO
We analyzed the spatial expression pattern of Arabidopsis thaliana adenosine phosphates-isopentenyltransferase genes (AtIPT1, AtIPT3 to AtIPT8) and the effect of inorganic nitrogen sources on their regulation. In mature plants, the AtIPTs were differentially expressed in various tissues including the roots, leaves, stems, flowers and siliques. In transgenic seedlings expressing a gene for green fluorescent protein (GFP) driven by the AtIPT promoters, AtIPT1::GFP was predominantly expressed in the vascular stele of the roots, AtIPT3::GFP was in the phloem companion cells, AtIPT5::GFP was in the lateral root primordium and pericycle, and AtIPT7::GFP was in both the vascular stele and the phloem companion cells of the roots. In a long-term treatment, the accumulation level of AtIPT5 transcript was correlated with the concentrations of NO(3)(-) and NH(4)(+) in the growth medium. However, under nitrogen-limited conditions, AtIPT3 expression was rapidly induced by NO(3)(-) in the seedlings accompanying the accumulation of cytokinins, whereas AtIPT5 expression was little affected. The NO(3)(-)-dependent accumulation of both the AtIPT3 transcript and the cytokinins was markedly reduced in a Ds transposon-insertion mutant of AtIPT3. These results suggest that nitrogen availability differentially regulates expression of AtIPT3 and AtIPT5, and that AtIPT3 is a key determinant of cytokinin biosynthesis in response to rapid changes in the availability of NO(3)(-).
Assuntos
Alquil e Aril Transferases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Citocininas/biossíntese , Nitratos/metabolismo , Transferases/metabolismo , Alquil e Aril Transferases/genética , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Regulação para Baixo/genética , Regulação da Expressão Gênica de Plantas/genética , Mutação/genética , Nitrogênio/deficiência , Raízes de Plantas/enzimologia , Raízes de Plantas/genética , Regiões Promotoras Genéticas/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Plântula/enzimologia , Plântula/genética , Transferases/genéticaRESUMO
Cytokinins (CKs), a group of phytohormones, are adenine derivatives that carry either an isoprene-derived or an aromatic side chain at the N(6) terminus. trans-Zeatin (tZ), an isoprenoid CK, is assumed to play a central physiological role because of its general occurrence and high activity in bioassays. Although hydroxylation of isopentenyladenine-type CKs is a key step of tZ biosynthesis, the catalyzing enzyme has not been characterized yet. Here we demonstrate that CYP735A1 and CYP735A2 are cytochrome P450 monooxygenases (P450s) that catalyze the biosynthesis of tZ. We identified the genes from Arabidopsis using an adenosine phosphate-isopentenyltransferase (AtIPT4)/P450 co-expression system in yeast. Co-expression of AtIPT4 and CYP735A enabled yeast to excrete tZ and the nucleosides to the culture medium. In vitro, both CYP735As preferentially utilized isopentenyladenine nucleotides rather than the nucleoside and free base forms and produced tZ nucleotides but not the cis-isomer. The expression of CYP735A1 and CYP735A2 was differentially regulated in terms of organ specificity and response to CK. Root-specific induction of CYP735A2 expression by CK suggests that the trans-hydroxylation is involved in the regulation of CK metabolism and signaling in roots.
Assuntos
Sistema Enzimático do Citocromo P-450/genética , Citocininas/metabolismo , Oxigenases de Função Mista/metabolismo , Zeatina/metabolismo , Proteínas de Arabidopsis , Catálise , Regulação da Expressão Gênica de Plantas , Hidroxilação , Oxigenases de Função Mista/genética , Reguladores de Crescimento de Plantas/metabolismo , Raízes de Plantas/metabolismo , Estruturas Vegetais/metabolismoRESUMO
Plants produce the common isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP) through the methylerythritol phosphate (MEP) pathway in plastids and the mevalonate (MVA) pathway in the cytosol. To assess which pathways contribute DMAPP for cytokinin biosynthesis, metabolites from each isoprenoid pathway were selectively labeled with (13)C in Arabidopsis seedlings. Efficient (13)C labeling was achieved by blocking the endogenous pathway genetically or chemically during the feed of a (13)C labeled precursor specific to the MEP or MVA pathways. Liquid chromatography-mass spectrometry analysis demonstrated that the prenyl group of trans-zeatin (tZ) and isopentenyladenine is mainly produced through the MEP pathway. In comparison, a large fraction of the prenyl group of cis-zeatin (cZ) derivatives was provided by the MVA pathway. When expressed as fusion proteins with green fluorescent protein in Arabidopsis cells, four adenosine phosphate-isopentenyltransferases (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) were found in plastids, in agreement with the idea that the MEP pathway primarily provides DMAPP to tZ and isopentenyladenine. On the other hand, AtIPT2, a tRNA isopentenyltransferase, was detected in the cytosol. Because the prenylated adenine moiety of tRNA is usually of the cZ type, the formation of cZ in Arabidopsis seedlings might involve the transfer of DMAPP from the MVA pathway to tRNA. Distinct origins of large proportions of DMAPP for tZ and cZ biosynthesis suggest that plants are able to separately modulate the level of these cytokinin species.
Assuntos
Arabidopsis/metabolismo , Terpenos/metabolismo , Zeatina/química , Zeatina/metabolismo , Nucleotídeos de Adenina/metabolismo , Isótopos de Carbono , Citocininas/metabolismo , Eritritol/metabolismo , Proteínas de Fluorescência Verde , Proteínas Luminescentes/genética , Ácido Mevalônico/farmacocinética , Pentosefosfatos/farmacocinética , Plastídeos/metabolismo , Plântula/metabolismo , Transferases/metabolismoRESUMO
We describe a new enzymatic reaction method for the preparation of the radioisotope-labeled cytokinins isopentenyladenine (iP), trans-zeatin (tZ), and their ribosides. The method is based on the three enzyme activities of an adenylate isopentenyltransferase (IPT; EC 2.5.1.27) from Arabidopsis thaliana, an alkaline phosphatase (EC 3.1.3.1) from calf intestine, and a purine-nucleoside phosphorylase (EC 2.4.2.1) from Escherichia coli. The A. thaliana IPT, AtIPT7, utilized both dimethylallyldiphosphate and 4-hydroxy-3-methyl-2-( E)-butenyl diphosphate as isoprenoid donors. The dual specificity of the substrates enabled us to produce iP-type and tZ-type cytokinins separately in the same system simply by switching the substrates. Our method affords a much higher yield of the labeled products than the chemical reaction methods previously used. These labeled compounds will be useful tools for cytokinin research, such as receptor-ligand assays and cell metabolism studies.
Assuntos
Adenina/análogos & derivados , Adenina/metabolismo , Citocininas/metabolismo , Zeatina/metabolismo , Fosfatase Alcalina/metabolismo , Alquil e Aril Transferases/metabolismo , Animais , Arabidopsis/enzimologia , Bovinos , Citocininas/química , Escherichia coli/enzimologia , Regulação da Expressão Gênica , Isopenteniladenosina , Estrutura Molecular , Purina-Núcleosídeo Fosforilase/metabolismo , Trítio , Zeatina/análogos & derivadosRESUMO
The maize response regulator genes ZmRR1 and ZmRR2 respond to cytokinin, and the translated products seem to be involved in nitrogen signal transduction mediated by cytokinin through the His-Asp phosphorelay. To elucidate the physiological function of the proteins, we examined the temporal and spatial distribution in maize leaves by immunochemical analysis and use of transgenic plants. ZmRR1 and ZmRR2 polypeptides could be distinctively detected by western blotting. The polypeptides accumulated in leaves within 5 h of the supply of nitrate to nitrogen-depleted maize, and the accumulation was transient. The extent of induction was larger in the leaf tip, which is rich in photosynthetically matured cells, than elsewhere. In leaves, the polypeptides accumulated mostly in mesophyll cells. Histochemical analyses of transgenic maize harboring a ZmRR1 promoter-beta-glucuronidase fusion gene also showed most of the expression to be in these cells. These results suggest that ZmRR1 and ZmRR2 are induced in mesophyll cells and function in nitrogen signal transduction mediated by cytokinin.
Assuntos
Citocininas/farmacologia , Proteínas de Plantas/metabolismo , Zea mays/citologia , Zea mays/metabolismo , Regulação da Expressão Gênica de Plantas , Genes de Plantas/genética , Nitratos/farmacologia , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/análise , Proteínas de Plantas/genética , Proteínas de Plantas/imunologia , Plantas Geneticamente Modificadas , Transdução de Sinais/efeitos dos fármacos , Zea mays/efeitos dos fármacos , Zea mays/genéticaRESUMO
In higher plants, inorganic nitrogen has crucial effects on growth and development, providing cellular components and modulating gene expression. To date, not only nitrogen assimilatory genes but also a substantial number of genes with other functions have been shown to be selectively regulated by the availability of nitrogen. In terms of the communicating substance(s) between root and shoot, accumulating evidence suggests that nitrate itself is the primary signal molecule triggering the activation of transcription of nitrate assimilation and related genes. On the other hand, some of the genes involved in photosynthesis, cell cycling and translation machinery are also regulated, at least in part, by nitrate and other nitrogen sources and, in some cases, the effect can be mimicked by cytokinin treatment. Spatial and temporal studies on the accumulation levels and the translocation of cytokinin in response to nitrate replenishment in maize showed subsequent accumulation of various cytokinin species in the roots, xylem sap and leaves. In Arabidopsis thaliana, trans-zeatin riboside-5'-monophosphate and/or trans-zeatin riboside also accumulated in the roots in response to nitrate resupply. These studies suggest that cytokinin metabolism and translocation could be commonly modulated by nitrogen availability in higher plants. Thus, in addition to nitrate, cytokinin could be another root-to-shoot signal communicating nitrogen availability.
Assuntos
Adenosina/análogos & derivados , Citocininas/metabolismo , Isopenteniladenosina/análogos & derivados , Nitratos/farmacologia , Nitrogênio/metabolismo , Raízes de Plantas/efeitos dos fármacos , Brotos de Planta/efeitos dos fármacos , Adenosina/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Arabidopsis/metabolismo , Transporte Biológico , Regulação da Expressão Gênica de Plantas , Isopenteniladenosina/metabolismo , Nitratos/metabolismo , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Transdução de Sinais , Zea mays/efeitos dos fármacos , Zea mays/genética , Zea mays/metabolismoRESUMO
Glyoxysomes, a group of specialized peroxisomes, are organelles that degrade fatty acids by the combination of fatty acid beta-oxidation and glyoxylate cycle. However, the mechanism underlying the transport of the fatty acids across the peroxisomal membrane is still obscure in higher plant cells. We identified and analyzed the PED3 gene and its gene product, Ped3p. The phenotype of the Arabidopsis ped3 mutant indicated that the mutation in the PED3 gene inhibits the activity of fatty acid beta-oxidation. Ped3p is a 149-kDa protein that exists in peroxisomal membranes. The amino acid sequence of Ped3p had a typical characteristic for "full-size" ATP-binding cassette (ABC) transporter consisting of two transmembrane regions and two ATP-binding regions. This protein was divided into two parts, that had 32% identical amino acid sequences. Each part showed a significant sequence similarity with peroxisomal "half" ABC transporters so far identified in mammals and yeast. Ped3p may contribute to the transport of fatty acids and their derivatives across the peroxisomal membrane.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Proteínas de Arabidopsis/genética , Arabidopsis/metabolismo , Ácidos Graxos/metabolismo , Peroxissomos/metabolismo , Transportadores de Cassetes de Ligação de ATP/isolamento & purificação , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adenosina Trifosfatases , Alelos , Sequência de Aminoácidos , Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Arabidopsis/metabolismo , Mapeamento Cromossômico , Glioxilatos/metabolismo , Glioxissomos/metabolismo , Glioxissomos/ultraestrutura , Microscopia Eletrônica , Dados de Sequência Molecular , Fenótipo , Sementes/genética , Sementes/metabolismo , Homologia de Sequência de Aminoácidos , Triglicerídeos/metabolismoRESUMO
Sulfate transporters present at the root surface facilitate uptake of sulfate from the environment. Here we report that uptake of sulfate at the outermost cell layers of Arabidopsis root is associated with the functions of highly and low-inducible sulfate transporters, Sultr1;1 and Sultr1;2, respectively. We have previously reported that Sultr1;1 is a high-affinity sulfate transporter expressed in root hairs, epidermal and cortical cells of Arabidopsis roots, and its expression is strongly upregulated in plants deprived of external sulfate. A novel sulfate transporter gene, Sultr1;2, identified on the BAC clone F28K19 of Arabidopsis, encoded a polypeptide of 653 amino acids that is 72.6% identical to Sultr1;1 and was able to restore sulfate uptake capacity of a yeast mutant lacking sulfate transporter genes (K(m) for sulfate = 6.9 +/- 1.0 microm). Transgenic Arabidopsis plants expressing the fusion gene construct of the Sultr1;2 promoter and green fluorescent protein (GFP) showed specific localization of GFP in the root hairs, epidermal and cortical cells of roots, and in the guard cells of leaves, suggesting that Sultr1;2 may co-localize with Sultr1;1 in the same cell layers at the root surface. Sultr1;1 mRNA was abundantly expressed under low-sulfur conditions (50-100 microm sulfate), whereas Sultr1;2 mRNA accumulated constitutively at high levels under a wide range of sulfur conditions (50-1500 microm sulfate), indicating that Sultr1;2 is less responsive to changes in sulfur conditions. Addition of selenate to the medium increased the level of Sultr1;1 mRNA in parallel with a decrease in the internal sulfate pool in roots. The level of Sultr1;2 mRNA was not influenced under these conditions. Antisense plants of Sultr1;1 showed reduced accumulation of sulfate in roots, particularly in plants treated with selenate, suggesting that the inducible transporter Sultr1;1 contributes to the uptake of sulfate under stressed conditions.