RESUMO
Low bioavailable concentrations of the micronutrient zinc (Zn) limit agricultural production on 40% of cultivated land. Here, we demonstrate that plant acclimation to Zn deficiency involves systemic regulation. Physiological Zn deficiency of Arabidopsis thaliana shoots results in increased root transcript levels of the membrane transport protein-encoding genes METAL TRANSPORT PROTEIN2 (MTP2) and HEAVY METAL ATPASE2 (HMA2), which are unresponsive to the local Zn status of roots. MTP2 and HMA2 act additively in the partitioning of Zn from roots to shoots. Chimeric GFP fusion proteins of MTP2 complement an mtp2 mutant and localize in the endoplasmic reticulum (ER) membrane of the outer cell layers from elongation to root hair zone of lateral roots. MTP2 restores Zn tolerance in a hypersensitive yeast mutant. These results are consistent with cell-to-cell movement of Zn toward the root vasculature inside the ER-luminal continuum through the desmotubules of plasmodesmata, under Zn deficiency. The previously described Zn deficiency response comprises transcriptional activation of target genes, including ZINC-REGULATED TRANSPORTER IRON-REGULATED TRANSPORTER PROTEIN genes ZIP4 and ZIP9, by the F-group bZIP transcription factors bZIP19 and bZIP23. We show that ZIP4 and ZIP9 respond to the local Zn status in both roots and shoots, in contrast to the systemic regulation identified here. Our findings are relevant for crop management and improvement toward combating human nutritional Zn deficiency that affects 30 to 50% of the world's population.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Brotos de Planta/metabolismo , Zinco/metabolismo , Adenosina Trifosfatases/genética , Arabidopsis/efeitos dos fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte de Cátions/genética , Retículo Endoplasmático/genética , Retículo Endoplasmático/metabolismo , Regulação da Expressão Gênica de Plantas , Mutação , Raízes de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/genética , Plantas Geneticamente Modificadas , Zinco/farmacologiaRESUMO
In Arabidopsis thaliana, biosynthesis of the essential thiol antioxidant, glutathione (GSH), is plastid-regulated, but many GSH functions, including heavy metal detoxification and plant defense activation, depend on cytosolic GSH. This finding suggests that plastid and cytosol thiol pools are closely integrated and we show that in Arabidopsis this integration requires a family of three plastid thiol transporters homologous to the Plasmodium falciparum chloroquine-resistance transporter, PfCRT. Arabidopsis mutants lacking these transporters are heavy metal-sensitive, GSH-deficient, and hypersensitive to Phytophthora infection, confirming a direct requirement for correct GSH homeostasis in defense responses. Compartment-specific measurements of the glutathione redox potential using redox-sensitive GFP showed that knockout of the entire transporter family resulted in a more oxidized glutathione redox potential in the cytosol, but not in the plastids, indicating the GSH-deficient phenotype is restricted to the cytosolic compartment. Expression of the transporters in Xenopus oocytes confirmed that each can mediate GSH uptake. We conclude that these transporters play a significant role in regulating GSH levels and the redox potential of the cytosol.
Assuntos
Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Glutationa/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Animais , Antimaláricos/farmacologia , Cádmio/farmacologia , Cloroquina/farmacologia , Resistência a Medicamentos , Feminino , Genes de Plantas , Homeostase , Técnicas In Vitro , Modelos Biológicos , Mutação , Oócitos/metabolismo , Plantas Geneticamente Modificadas , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/genética , Plasmodium falciparum/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Estresse Fisiológico , XenopusRESUMO
HMA2, HMA4, and HMA7 are three of the eight heavy metal transporting P(1B)-type ATPases in the simple plant Arabidopsis thaliana. The first two transport Zn(2+), and the third transports Cu(+). Each protein contains soluble N-terminal metal-binding domains (MBDs) that are essential for metal transport. While the MBD of HMA7 features a CxxC sequence motif characteristic of Cu(I) binding sites, those of HMA2 and HMA4 contain a CCxxE motif, unique for plant Zn(2+)-ATPases. The three MBDs HMA2n (residues 1-79), HMA4n (residues 1-96), and HMA7n (residues 56-127) and an HMA7/4n chimera were expressed in Escherichia coli. The chimera features the ICCTSE motif from HMA4n inserted in place of the native MTCAAC motif of HMA7n. Binding affinities for Zn(II) and Cu(I) of each MBD were determined by ligand competition with a number of chromophoric probes. The challenges of using these probes reliably were evaluated, and the relative affinities of the MBDs were verified by independent cross-checks. The affinities of HMA2n and HMA4n for Zn(II) are higher than that of HMA7n by a factor of 20-30, but the relative affinities for Cu(I) are inverted by a factor of 30-50. These relativities are consistent with their respective roles in metal selection and transportation. Chimera HMA7/4n binds Cu(I) with an affinity between those of HMA4n and HMA7n but binds Zn(II) more weakly than either parent protein does. The four MBDs bind Cu(I) more strongly than Zn(II) by factors of >10(6). It is apparent that the individual MBDs are not able to overcome the large thermodynamic preference for Cu(+) over Zn(2+). This information highlights the potential toxicity of Cu(+) in vivo and why copper sensor proteins are approximately 6 orders of magnitude more sensitive than zinc sensor proteins. Metal speciation must be controlled by multiple factors, including thermodynamics (affinity), kinetics (including protein-protein interactions), and compartmentalization. The structure of Zn(II)-bound HMA4n defined by NMR confirmed the predicted ferredoxin betaalphabetabetaalphabeta fold. A single Zn atom was modeled onto a metal-binding site with protein ligands comprising the two thiolates and the carboxylate of the CCxxE motif. The observed (113)Cd chemical shift in [(113)Cd]HMA4n was consistent with a Cd(II)S(2)OX (X = O or N) coordination sphere. The Zn(II) form of the Cu(I) transporter HMA7n is a monomer in solution but crystallized as a polymeric chain [(Zn(II)-HMA7n)(m)]. Each Zn(II) ion occupied a distorted tetrahedral site formed from two Cys ligands of the CxxC motif of one HMA7n molecule and the amino N and carbonyl O atoms of the N-terminal methionine of another.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Cobre/metabolismo , Zinco/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/isolamento & purificação , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/isolamento & purificação , Proteínas de Transporte/genética , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte de Cátions/genética , Proteínas de Transporte de Cátions/isolamento & purificação , Cobre/química , Cristalografia por Raios X , Regulação da Expressão Gênica de Plantas , Espectroscopia de Ressonância Magnética , Ligação Proteica/genética , Estrutura Terciária de Proteína/genética , Zinco/químicaRESUMO
Glutathione (GSH) homeostasis in plants is essential for cellular redox control and efficient responses to abiotic and biotic stress. Compartmentation of the GSH biosynthetic pathway is a unique feature of plants. The first enzyme, gamma-glutamate cysteine ligase (GSH1), responsible for synthesis of gamma-glutamylcysteine (gamma-EC), is, in Arabidopsis, exclusively located in the plastids, whereas the second enzyme, glutathione synthetase (GSH2), is located in both plastids and cytosol. In Arabidopsis, gsh2 insertion mutants have a seedling lethal phenotype in contrast to the embryo lethal phenotype of gsh1 null mutants. This difference in phenotype may be due to partial replacement of GSH functions by gamma-EC, which in gsh2 mutants hyperaccumulates to levels 5000-fold that in the wild type and 200-fold wild-type levels of GSH. In situ labelling of thiols with bimane and confocal imaging in combination with HPLC analysis showed high concentrations of gamma-EC in the cytosol. Feedback inhibition of Brassica juncea plastidic GSH1 by gamma-EC in vitro strongly suggests export of gamma-EC as functional explanation for hyperaccumulation. Complementation of gsh2 mutants with the cytosol-specific GSH2 gave rise to phenotypically wild-type transgenic plants. These results support the conclusion that cytosolic synthesis of GSH is sufficient for plant growth. The transgenic lines further show that, consistent with the exclusive plastidic localization of GSH1, gamma-EC is exported from the plastids to supply the cytosol with the immediate precursor for GSH biosynthesis, and that there can be efficient re-import of GSH into the plastids to allow effective control of GSH biosynthesis through feedback inhibition of GSH1.
Assuntos
Arabidopsis/genética , Citosol/metabolismo , Glutationa/biossíntese , Arabidopsis/enzimologia , Arabidopsis/crescimento & desenvolvimento , Dipeptídeos/metabolismo , Relação Dose-Resposta a Droga , Glutationa Sintase/genética , Glutationa Sintase/metabolismo , Mostardeira/enzimologia , Mutagênese Insercional , Fenótipo , Raízes de Plantas/citologia , Raízes de Plantas/crescimento & desenvolvimento , Raízes de Plantas/metabolismo , Plastídeos/enzimologia , Plântula/genética , Plântula/crescimento & desenvolvimento , Sementes/citologia , Sementes/crescimento & desenvolvimento , Sementes/metabolismo , Coloração e Rotulagem , Compostos de Sulfidrila/farmacologiaRESUMO
The Zn/Cd-transporting ATPases, HMA2 and HMA4, essential for root-to-shoot Zn translocation, are also able to transport Cd. Phytochelatins (PCs) are a major mechanism of Cd detoxification through the sequestration of PC-Cd complexes in vacuoles. The roles of HMA2 and HMA4 in root-to-shoot Cd translocation and Cd tolerance were investigated in the PC-deficient, cad1-3 mutant and CAD1 backgrounds. Six lines, with all possible combinations of hma2, hma4 and cad1 mutations, were constructed. The lines were tested for Cd-sensitivity on agar medium, and radioactive (109)Cd was used to measure Cd uptake and translocation from root to shoot over periods of up to 6 d. In hma4 and hma2,hma4, but not hma2, root-to-shoot Cd translocation was decreased to about 60 and 2%, respectively, of that in the wild-type. Cd sensitivity increased approximately twofold in the hma2,hma4 mutant in both CAD1 and cad1 backgrounds. PC deficiency resulted in an increase in shoot Cd concentrations. The near-complete abolition of root-to-shoot Cd translocation resulting from the loss of function of HMA2 and HMA4 demonstrates they are the major mechanism for Cd translocation in Arabidopsis thaliana.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Cádmio/farmacocinética , Fitoquelatinas/metabolismo , Adenosina Trifosfatases/genética , Arabidopsis/genética , Transporte Biológico/fisiologia , Genótipo , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismoRESUMO
The Zn/Cd-transporting ATPase, HMA2, has N- and C-terminal domains that can bind Zn ions with high affinity. Mutant derivatives were generated to determine the significance of these domains to HMA2 function in planta. Mutant derivatives, with and without a C-terminal GFP tag, were expressed from the HMA2 promoter in transgenic hma2,hma4, Zn-deficient, plants to test for functionality. A deletion mutant lacking the C-terminal 244 amino acids rescued most of the hma2,hma4 Zn-deficiency phenotypes with the exception of embryo or seed development. Root-to-shoot Cd translocation was fully rescued. The GFP-tagged derivative was partially mis-localized in the root pericycle cells in which it was expressed. Deletion derivatives lacking the C-terminal 121 and 21 amino acids rescued all phenotypes and localized normally. N-terminal domain mutants localized normally but failed to complement the hma2,hma4 phenotypes. These observations suggest that the N-terminal domain of HMA2 is essential for function in planta while the C-terminal domain, although not essential for function, may contain a signal important for the subcellular localization of the protein.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Arabidopsis/química , Arabidopsis/enzimologia , Estrutura Terciária de Proteína , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Transporte Biológico Ativo , Cádmio/farmacocinética , Mutação , Raízes de Plantas/metabolismo , Brotos de Planta/química , Plantas Geneticamente Modificadas , Radioisótopos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Zinco/análiseRESUMO
In higher plants the L-galactose pathway represents the major route for ascorbate biosynthesis. The first committed step of this pathway is catalyzed by the enzyme GDP-L-galactose phosphorylase and is encoded by two paralogs in Arabidopsis - VITAMIN C2 (VTC2) and VTC5. The first mutant of this enzyme, vtc2-1, isolated via an EMS mutagenesis screen, has approximately 20-30% of wildtype ascorbate levels and has been reported to have decreased growth under standard laboratory conditions. Here, we show that a T-DNA insertion into the VTC2 causes a similar reduction in ascorbate levels, but does not greatly affect plant growth. Subsequent segregation analysis revealed the growth defects of vtc2-1 mutants segregate independently of the vtc2-1 mutation. These observations suggest that it is the presence of an independent cryptic mutation that affects growth of vtc2-1 mutants, and not the 70-80% decrease in ascorbate levels that has been assumed in past studies.
RESUMO
Arabidopsis is remarkable for having eight members of the type 1B heavy metal-transporting P-type ATPase subfamily. Sequence analyses indicate that four, two of which may be targeted to plastids, are related to known Cu(I) transporters and contain N-terminal metal-binding site (MBS) motifs similar to those identified in other organisms. The remaining four are more closely related to known divalent cation transporters of prokaryotes. Three of these form a closely related group and are believed to be Zn(II) transporters. These contain a predicted N-terminal MBS that is a variant of those found in Cu transporters in addition to extended C-terminal regions that contain likely metal-binding sequences. Our current limited knowledge of the physiological roles of these transporters is reviewed and their evolutionary relationships are explored, including an hypothesis that some, particularly the putative divalent cation transporters, are derived from horizontal gene transfer events.
RESUMO
A complete inventory of metallochaperone-like proteins containing a predicted HMA domain in Arabidopsis revealed a large family of 67 proteins. 45 proteins, the HIPPs, have a predicted isoprenylation site while 22 proteins, the HPPs, do not. Sequence comparisons divided the proteins into seven major clusters (I-VII). Cluster IV is notable for the presence of a conserved Asp residue before the CysXXCys, metal binding motif, analogous to the Zn binding motif in E. coli ZntA. HIPP20, HIPP21, HIPP22, HIPP26 and HIPP27 in Cluster IV were studied in more detail. All but HIPP21 could rescue the Cd-sensitive, ycf1 yeast mutant but failed to rescue the growth of zrt1zrt2, zrc1cot1 and atx1 mutants. In Arabidopsis, single and double mutants did not show a phenotype but the hipp20/21/22 triple mutant was more sensitive to Cd and accumulated less Cd than the wild-type suggesting the HIPPs can have a role in Cd-detoxification, possibly by binding Cd. Promoter-GUS reporter expression studies indicated variable expression of these HIPPs. For example, in roots, HIPP22 and HIPP26 are only expressed in lateral root tips while HIPP20 and HIPP25 show strong expression in the root vasculature.
Assuntos
Arabidopsis/genética , Genes de Plantas , Metalochaperonas/metabolismo , Sequência de Aminoácidos , Proteínas de Arabidopsis/classificação , Proteínas de Arabidopsis/genética , Metalochaperonas/química , Metais Pesados/metabolismo , Dados de Sequência Molecular , Filogenia , Plantas Geneticamente Modificadas , Regiões Promotoras Genéticas , Homologia de Sequência de AminoácidosRESUMO
Metal specificities of zinc and copper transport proteins HMA4 and HMA7 from the simple plant Arabidopsis thaliana match the relative, but not the absolute, affinities of their amino-terminal domains.
Assuntos
Adenosina Trifosfatases/química , Proteínas de Arabidopsis/química , Arabidopsis/metabolismo , Cobre/química , Zinco/química , Adenosina Trifosfatases/metabolismo , Proteínas de Arabidopsis/metabolismo , Cinética , Estrutura Terciária de ProteínaRESUMO
Although cell wall remodeling is an essential feature of plant growth and development, the underlying molecular mechanisms are poorly understood. This work describes the characterization of Arabidopsis (Arabidopsis thaliana) plants with altered expression of ARAF1, a bifunctional alpha-L-arabinofuranosidase/beta-D-xylosidase (At3g10740) belonging to family 51 glycosyl-hydrolases. ARAF1 was localized in several cell types in the vascular system of roots and stems, including xylem vessels and parenchyma cells surrounding the vessels, the cambium, and the phloem. araf1 T-DNA insertional mutants showed no visible phenotype, whereas transgenic plants that overexpressed ARAF1 exhibited a delay in inflorescence emergence and altered stem architecture. Although global monosaccharide analysis indicated only slight differences in cell wall composition in both mutant and overexpressing lines, immunolocalization experiments using anti-arabinan (LM6) and anti-xylan (LM10) antibodies indicated cell type-specific alterations in cell wall structure. In araf1 mutants, an increase in LM6 signal intensity was observed in the phloem, cambium, and xylem parenchyma in stems and roots, largely coinciding with ARAF1 expression sites. The ectopic overexpression of ARAF1 resulted in an increase in LM10 labeling in the secondary walls of interfascicular fibers and xylem vessels. The combined ARAF1 gene expression and immunolocalization studies suggest that arabinan-containing pectins are potential in vivo substrates of ARAF1 in Arabidopsis.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/enzimologia , Parede Celular/metabolismo , Polissacarídeos/metabolismo , Xilosidases/metabolismo , Arabidopsis/genética , Arabidopsis/crescimento & desenvolvimento , DNA Bacteriano , Expressão Gênica , Perfilação da Expressão Gênica , Glucuronidase/metabolismo , Imuno-Histoquímica , Monossacarídeos/metabolismo , Mutagênese Insercional , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Caules de Planta/crescimento & desenvolvimentoRESUMO
Essential metals are required for healthy plant growth but can be toxic when present in excess. Therefore plants have mechanisms of metal homeostasis which involve coordination of metal ion transporters for uptake, translocation and compartmentalization. However, very little metal in plants is thought to exist as free ions. A number of small, organic molecules have been implicated in metal ion homeostasis as metal ion ligands to facilitate uptake and transport of metal ions with low solubility and also as chelators implicated in sequestration for metal tolerance and storage. Ligands for a number of essential metals have been identified and proteins involved in the transport of these ligands and of metal-ligand complexes have been characterized. Here we review recent advances in understanding the role of mugineic acid, nicotianamine, organic acids (citrate and malate), histidine and phytate as ligands for iron (Fe), zinc (Zn), copper (Cu), manganese (Mn) and nickel (Ni) in plants, and the proteins identified as their transporters.
Assuntos
Proteínas de Transporte/metabolismo , Metais/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Transporte Biológico , LigantesRESUMO
Zinc (Zn) is an essential micronutrient required by all cells but is toxic in excess. We have identified three allelic Zn-sensitive mutants of Arabidopsis (Arabidopsis thaliana). The gene, designated ZINC-INDUCED FACILITATOR1 (ZIF1), encodes a member of the major facilitator superfamily of membrane proteins, which are found in all organisms and transport a wide range of small, organic molecules. Shoots of zif1 mutants showed increased accumulation of Zn but not other metal ions. In combination with mutations affecting shoot-to-root Zn translocation, zif1 hma2 hma4 triple mutants accumulated less Zn than the wild type but remained Zn sensitive, suggesting that the zif1 Zn-sensitive phenotype is due to altered Zn distribution. zif1 mutants were also more sensitive to cadmium but less sensitive to nickel. ZIF1 promoter-beta-glucuronidase fusions were expressed throughout the plant, with strongest expression in young tissues, and predominantly in the vasculature in older tissues. ZIF1 expression was highly induced by Zn and, to a lesser extent, by manganese. A ZIF1-green fluorescent protein fusion protein localized to the tonoplast in transgenic plants. MTP1 has been identified as a tonoplast Zn transporter and a zif1-1 mtp1-1 double mutant was more sensitive to Zn than either of the single mutants, suggesting ZIF1 influences a distinct mechanism of Zn homeostasis. Overexpression of ZIF1 conferred increased Zn tolerance and interveinal leaf chlorosis in some transgenic lines in which ZIF1 expression was high. We propose that ZIF1 is involved in a novel mechanism of Zn sequestration, possibly by transport of a Zn ligand or a Zn ligand complex into vacuoles.
Assuntos
Adaptação Fisiológica , Proteínas de Arabidopsis/fisiologia , Arabidopsis/fisiologia , Zinco/metabolismo , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Mutação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
* The usefulness of the zinc (Zn)-fluorophore, Zinpyr-1, to examine the localization of Zn in the roots of Arabidopsis has been investigated. * In wild-type roots Zinpyr-1 fluorescence was predominantly in the xylem. The fluorescence signal was abolished by the application of the Zn-chelator, N,N,N',N-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), and was increased by increasing exogenous Zn in the medium, indicating that fluorescence reflected relative Zn concentrations. * In the hma2, hma4 double mutant, which is deficient in root to shoot Zn translocation, Zinpyr-1 fluorescence was low in the xylem and high in the adjacent pericycle cells in which HMA2 and HMA4 are specifically expressed in a wild type. Zinpyr-1 fluorescence was also increased in the endodermis. * These results show that Zinpyr-1 can be used to examine the effects of mutations in Zn transporters on the localization of Zn in Arabidopsis roots and should be a useful addition to the tools available for studying Zn homeostasis in plants.
Assuntos
Arabidopsis/metabolismo , Fluoresceínas/metabolismo , Corantes Fluorescentes/metabolismo , Raízes de Plantas/metabolismo , Zinco/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Quelantes/farmacologia , Etilenodiaminas/farmacologia , Estudos de Viabilidade , Regulação da Expressão Gênica de Plantas , Proteínas de Fluorescência Verde/genética , Homeostase , Microscopia Confocal/métodos , Plantas Geneticamente Modificadas , Xilema/metabolismoRESUMO
Glutathione (GSH) has been implicated in maintaining the cell cycle within plant meristems and protecting proteins during seed dehydration. To assess the role of GSH during development of Arabidopsis (Arabidopsis thaliana [L.] Heynh.) embryos, we characterized T-DNA insertion mutants of GSH1, encoding the first enzyme of GSH biosynthesis, gamma-glutamyl-cysteine synthetase. These gsh1 mutants confer a recessive embryo-lethal phenotype, in contrast to the previously described GSH1 mutant, root meristemless 1(rml1), which is able to germinate, but is deficient in postembryonic root development. Homozygous mutant embryos show normal morphogenesis until the seed maturation stage. The only visible phenotype in comparison to wild type was progressive bleaching of the mutant embryos from the torpedo stage onward. Confocal imaging of GSH in isolated mutant and wild-type embryos after fluorescent labeling with monochlorobimane detected residual amounts of GSH in rml1 embryos. In contrast, gsh1 T-DNA insertion mutant embryos could not be labeled with monochlorobimane from the torpedo stage onward, indicating the absence of GSH. By using high-performance liquid chromatography, however, GSH was detected in extracts of mutant ovules and imaging of intact ovules revealed a high concentration of GSH in the funiculus, within the phloem unloading zone, and in the outer integument. The observation of high GSH in the funiculus is consistent with a high GSH1-promoterbeta-glucuronidase reporter activity in this tissue. Development of mutant embryos could be partially rescued by exogenous GSH in vitro. These data show that at least a small amount of GSH synthesized autonomously within the developing embryo is essential for embryo development and proper seed maturation.
Assuntos
Arabidopsis/embriologia , Glutationa/biossíntese , Sementes/crescimento & desenvolvimento , Alelos , Sequência de Bases , Cromatografia Líquida de Alta Pressão , Primers do DNA , DNA Bacteriano/genética , Genes Letais , Homozigoto , Reação em Cadeia da Polimerase , Sementes/metabolismoRESUMO
Glycosyl hydrolases are important mediators of plant cell wall modification during plant development. These enzymes catalyse the hydrolytic release of specific sugars, such as L-arabinose, from the polysaccharide-rich cell wall matrix. The cloning and expression analysis of two genes, AtASD1 and AtASD2, encoding putative alpha-L-arabinofuranosidases in Arabidopsis thaliana are reported here. AtASD1 and AtASD2 identities were assigned on the basis of homology to plant and microbial family 51 glycoside hydrolases. Using RT-PCR, RNA gel blot analysis and reporter gene expression analysis, AtASD1 and AtASD2 were shown to have different developmental expression profiles. High levels of AtASD1 promoter activity are present in multiple tissues during vegetative and reproductive growth. AtASD1 expression is particularly intense in zones of cell proliferation, the vascular system, developing and regressing floral tissues, and floral abscission zones. By comparison, AtASD2 expression is limited to the vasculature of older root tissue and to some floral organs and floral abscission zones.
Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/genética , Regulação da Expressão Gênica de Plantas , Regulação da Expressão Gênica , Genes de Plantas , Glicosídeo Hidrolases/genética , Sequência de Aminoácidos , Arabidopsis/classificação , Arabidopsis/enzimologia , Proteínas de Arabidopsis/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Primers do DNA , Flores/enzimologia , Flores/genética , Regulação da Expressão Gênica no Desenvolvimento , Glucuronidase/genética , Glucuronidase/metabolismo , Hordeum/enzimologia , Dados de Sequência Molecular , Filogenia , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
In a process called phytoremediation, plants can be used to extract, detoxify, and/or sequester toxic pollutants from soil, water, and air. Phytoremediation may become an essential tool in cleaning the environment and reducing human and animal exposure to potential carcinogens and other toxins. Arabidopsis has provided useful information about the genetic, physiological, and biochemical mechanisms behind phytoremediation, and it is an excellent model genetic organism to test foreign gene expression. This review focuses on Arabidopsis studies concerning: 1) the remediation of elemental pollutants; 2) the remediation of organic pollutants; and 3) the phytoremediation genome. Elemental pollutants include heavy metals and metalloids (e.g., mercury, lead, cadmium, arsenic) that are immutable. The general goal of phytoremediation is to extract, detoxify, and hyperaccumulate elemental pollutants in above-ground plant tissues for later harvest. A few dozen Arabidopsis genes and proteins that play direct roles in the remediation of elemental pollutants are discussed. Organic pollutants include toxic chemicals such as benzene, benzo(a)pyrene, polychlorinated biphenyls, trichloroethylene, trinitrotoluene, and dichlorodiphenyltrichloroethane. Phytoremediation of organic pollutants is focused on their complete mineralization to harmless products, however, less is known about the potential of plants to act on complex organic chemicals. A preliminary survey of the Arabidopsis genome suggests that as many as 700 genes encode proteins that have the capacity to act directly on environmental pollutants or could be modified to do so. The potential of the phytoremediation proteome to be used to reduce human exposure to toxic pollutants appears to be enormous and untapped.
RESUMO
Arabidopsis thaliana has eight genes encoding members of the type 1(B) heavy metal-transporting subfamily of the P-type ATPases. Three of these transporters, HMA2, HMA3, and HMA4, are closely related to each other and are most similar in sequence to the divalent heavy metal cation transporters of prokaryotes. To determine the function of these transporters in metal homeostasis, we have identified and characterized mutants affected in each. Whereas the individual mutants exhibited no apparent phenotype, hma2 hma4 double mutants had a nutritional deficiency phenotype that could be compensated for by increasing the level of Zn, but not Cu or Co, in the growth medium. Levels of Zn, but not other essential elements, in the shoot tissues of a hma2 hma4 double mutant and, to a lesser extent, of a hma4 single mutant were decreased compared with the wild type. Together, these observations indicate a primary role for HMA2 and HMA4 in essential Zn homeostasis. HMA2promoter- and HMA4promoter-reporter gene constructs provide evidence that HMA2 and HMA4 expression is predominantly in the vascular tissues of roots, stems, and leaves. In addition, expression of the genes in developing anthers was confirmed by RT-PCR and was consistent with a male-sterile phenotype in the double mutant. HMA2 appears to be localized to the plasma membrane, as indicated by protein gel blot analysis of membrane fractions using isoform-specific antibodies and by the visualization of an HMA2-green fluorescent protein fusion by confocal microscopy. These observations are consistent with a role for HMA2 and HMA4 in Zn translocation. hma2 and hma4 mutations both conferred increased sensitivity to Cd in a phytochelatin-deficient mutant background, suggesting that they may also influence Cd detoxification.