RESUMO
Each essential transition metal plays a specific role in metabolic processes and has to be selectively transported. Living organisms need to discriminate between essential and non-essential metals such as cadmium (Cd(2+) ), which is highly toxic. However, transporters of the natural resistance-associated macrophage protein (NRAMP) family, which are involved in metal uptake and homeostasis, generally display poor selectivity towards divalent metal cations. In the present study we used a unique combination of yeast-based selection, electrophysiology on Xenopus oocytes and plant phenotyping to identify and characterize mutations that allow plant and mammalian NRAMP transporters to discriminate between their metal substrates. We took advantage of the increased Cd(2+) sensitivity of yeast expressing AtNRAMP4 to select mutations that decrease Cd(2+) sensitivity while maintaining the ability of AtNRAMP4 to transport Fe(2+) in a population of randomly mutagenized AtNRAMP4 cDNAs. The selection identified mutations in three residues. Among the selected mutations, several affect Zn(2+) transport, whereas only one, E401K, impairs Mn(2+) transport by AtNRAMP4. Introduction of the mutation F413I, located in a highly conserved domain, into the mammalian DMT1 transporter indicated that the importance of this residue in metal selectivity is conserved among NRAMP transporters from plant and animal kingdoms. Analyses of overexpressing plants showed that AtNRAMP4 affects the accumulation of metals in roots. Interestingly, the mutations selectively modify Cd(2+) and Zn(2+) accumulation without affecting Fe transport mediated by NRAMP4 in planta. This knowledge may be applicable for limiting Cd(2+) transport by other NRAMP transporters from animals or plants.
Assuntos
Arabidopsis/metabolismo , Cádmio/toxicidade , Proteínas de Plantas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Metais/metabolismo , Mutação , Vacúolos/metabolismoRESUMO
In most plants, NO(3)(-) constitutes the major source of nitrogen, and its assimilation into amino acids is mainly achieved in shoots. Furthermore, recent reports have revealed that reduction of NO(3)(-) translocation from roots to shoots is involved in plant acclimation to abiotic stress. NPF2.3, a member of the NAXT (nitrate excretion transporter) sub-group of the NRT1/PTR family (NPF) from Arabidopsis, is expressed in root pericycle cells, where it is targeted to the plasma membrane. Transport assays using NPF2.3-enriched Lactococcus lactis membranes showed that this protein is endowed with NO(3)(-) transport activity, displaying a strong selectivity for NO(3)(-) against Cl(-). In response to salt stress, NO(3)(-) translocation to shoots is reduced, at least partly because expression of the root stele NO(3)(-) transporter gene NPF7.3 is decreased. In contrast, NPF2.3 expression was maintained under these conditions. A loss-of-function mutation in NPF2.3 resulted in decreased root-to-shoot NO(3)(-) translocation and reduced shoot NO(3)(-) content in plants grown under salt stress. Also, the mutant displayed impaired shoot biomass production when plants were grown under mild salt stress. These mutant phenotypes were dependent on the presence of Na(+) in the external medium. Our data indicate that NPF2.3 is a constitutively expressed transporter whose contribution to NO(3)(-) translocation to the shoots is quantitatively and physiologically significant under salinity.
Assuntos
Proteínas de Transporte de Ânions/metabolismo , Proteínas de Transporte de Ânions/fisiologia , Proteínas de Arabidopsis/fisiologia , Arabidopsis/metabolismo , Proteínas de Membrana Transportadoras/fisiologia , Proteínas de Plantas/fisiologia , Tolerância ao Sal/fisiologia , Lactococcus lactis , Transportadores de NitratoRESUMO
We have investigated OsHKT2;1 natural variation in a collection of 49 cultivars with different levels of salt tolerance and geographical origins. The effect of identified polymorphism on OsHKT2;1 activity was analysed through heterologous expression of variants in Xenopus oocytes. OsHKT2;1 appeared to be a highly conserved protein with only five possible amino acid substitutions that have no substantial effect on functional properties. Our study, however, also identified a new HKT isoform, No-OsHKT2;2/1 in Nona Bokra, a highly salt-tolerant cultivar. No-OsHKT2;2/1 probably originated from a deletion in chromosome 6, producing a chimeric gene. Its 5' region corresponds to that of OsHKT2;2, whose full-length sequence is not present in Nipponbare but has been identified in Pokkali, a salt-tolerant rice cultivar. Its 3' region corresponds to that of OsHKT2;1. No-OsHKT2;2/1 is essentially expressed in roots and displays a significant level of expression at high Na⺠concentrations, in contrast to OsHKT2;1. Expressed in Xenopus oocytes or in Saccharomyces cerevisiae, No-OsHKT2;2/1 exhibited a strong permeability to Na⺠and Kâº, even at high external Na⺠concentrations, like OsHKT2;2, and in contrast to OsHKT2;1. Our results suggest that No-OsHKT2;2/1 can contribute to Nona Bokra salt tolerance by enabling root K⺠uptake under saline conditions.
Assuntos
Proteínas de Transporte de Cátions/genética , Oryza/genética , Proteínas de Plantas/genética , Tolerância ao Sal/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Proteínas de Transporte de Cátions/metabolismo , Dados de Sequência Molecular , Oryza/metabolismo , Proteínas de Plantas/metabolismo , Polimorfismo de Nucleotídeo Único , Potássio/metabolismo , Saccharomyces cerevisiae , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/metabolismo , Sódio/metabolismo , Simportadores/metabolismo , XenopusRESUMO
Soil salinity is an increasing menace that affects agriculture across the globe. Plant adaptation to high salt concentrations involves integrated functions, including control of Na+ uptake, translocation and compartmentalization. Na+ transporters belonging to the HKT family have been shown to be involved in tolerance to mild salt stress in glycophytes such as Arabidopsis, wheat and rice by contributing to Na+ exclusion from aerial tissues. Here, we have analysed the role of the HKT transporter HKT2;1, which is permeable to K+ and Na+, in barley, a relatively salt-tolerant crop that displays a salt-including behaviour. In Xenopus oocytes, HvHKT2;1 co-transports Na+ and K+ over a large range of concentrations, displaying low affinity for Na+, variable affinity for K+ depending on external Na+ concentration, and inhibition by K+ (K(i) approximately 5 mm). HvHKT2;1 is predominantly expressed in the root cortex. Transcript levels are up-regulated in both roots and shoots by low K+ growth conditions, and in shoots by high Na+ growth conditions. Over-expression of HvHKT2;1 led to enhanced Na+ uptake, higher Na+ concentrations in the xylem sap, and enhanced translocation of Na+ to leaves when plants were grown in the presence of 50 or 100 mm NaCl. Interestingly, these responses were correlated with increased barley salt tolerance. This suggests that one of the factors that limits barley salt tolerance is the capacity to translocate Na+ to the shoot rather than accumulation or compartmentalization of this cation in leaf tissues. Thus, over-expression of HvHKT2;1 leads to increased salt tolerance by reinforcing the salt-including behaviour of barley.
Assuntos
Proteínas de Transporte de Cátions/metabolismo , Hordeum/fisiologia , Proteínas de Plantas/metabolismo , Plantas Tolerantes a Sal/fisiologia , Sódio/metabolismo , Animais , Proteínas de Transporte de Cátions/genética , Clonagem Molecular , Regulação da Expressão Gênica de Plantas , Hordeum/genética , Hordeum/metabolismo , Potenciais da Membrana , Oócitos , Proteínas de Plantas/genética , Raízes de Plantas/metabolismo , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Plantas Geneticamente Modificadas/fisiologia , Potássio/metabolismo , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/metabolismo , Cloreto de Sódio/farmacologia , Xenopus , Xilema/metabolismoRESUMO
⢠Apart from their antifungal role, plant defensins have recently been shown to be involved in abiotic stress tolerance or in inhibition of root growth when added in plant culture medium. We studied the subcellular localization of these proteins, which may account for these different roles. ⢠Stable and transient expression of AhPDF1.1::GFP (green fluorescent protein) fusion proteins were analysed in yeast and plants. Functional tests established that the GFP tag did not alter the action of the defensin. Subcellular localization of AhPDF1.1 was characterized: by imaging AhPDF1.1::GFP together with organelle markers; and by immunolabelling AhPDF1.1 in Arabidopsis halleri and Arabidopsis thaliana leaves using a polyclonal serum. ⢠All our independent approaches demonstrated that AhPDF1.1 is retained in intracellular compartments on the way to the lytic vacuole, instead of being addressed to the apoplasm. ⢠These findings challenge the commonly accepted idea of secretion of defensins. The subcellular localization highlighted in this study could partly explain the dual role of plant defensins on plant cells and is of major importance to unravel the mechanisms of action of these proteins at the cellular level.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Compartimento Celular , Defensinas/metabolismo , Espaço Intracelular/metabolismo , Folhas de Planta/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Sequência de Aminoácidos , Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Proteínas de Arabidopsis/química , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Quitosana/farmacologia , Defensinas/química , Proteínas de Fluorescência Verde/metabolismo , Imunoensaio , Espaço Intracelular/efeitos dos fármacos , Dados de Sequência Molecular , Folhas de Planta/citologia , Folhas de Planta/efeitos dos fármacos , Sinais Direcionadores de Proteínas , Transporte Proteico/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/efeitos dos fármacos , Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Nicotiana/efeitos dos fármacos , Nicotiana/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Zinco/toxicidade , Rede trans-Golgi/efeitos dos fármacos , Rede trans-Golgi/metabolismoRESUMO
The ability of metal hyperaccumulating plants to tolerate and accumulate heavy metals results from adaptations of metal homeostasis. NRAMP metal transporters were found to be highly expressed in some hyperaccumulating plant species. Here, we identified TcNRAMP3 and TcNRAMP4, the closest homologues to AtNRAMP3 and AtNRAMP4 in Thlaspi caerulescens and characterized them by expression analysis, confocal imaging and heterologous expression in yeast and Arabidopsis thaliana. TcNRAMP3 and TcNRAMP4 are expressed at higher levels than their A. thaliana homologues. When expressed in yeast TcNRAMP3 and TcNRAMP4 transport the same metals as their respective A. thaliana orthologues: iron (Fe), manganese (Mn) and cadmium (Cd) but not zinc (Zn) for NRAMP3; Fe, Mn, Cd and Zn for NRAMP4. They also localize at the vacuolar membrane in A. thaliana protoplasts. Inactivation of AtNRAMP3 and AtNRAMP4 in A. thaliana results in strong Cd and Zn hypersensitivity, which is fully rescued by TcNRAMP3 or TcNRAMP4 expression. However, metal tolerance conferred by TcNRAMP expression in nramp3nramp4 mutant does not exceed that of wild-type A. thaliana. Our data indicate that the difference between TcNRAMP3 and TcNRAMP4 and their A. thaliana orthologues does not lie in a different protein function, but probably resides in a different expression level or expression pattern.
Assuntos
Metais/metabolismo , Proteínas de Plantas/metabolismo , Thlaspi/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Transporte Biológico/efeitos dos fármacos , Cádmio/toxicidade , Clonagem Molecular , DNA Complementar/genética , DNA de Plantas/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Teste de Complementação Genética , Genoma de Planta/genética , Proteínas de Fluorescência Verde/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Dados de Sequência Molecular , Mutação/genética , Proteínas de Plantas/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Thlaspi/efeitos dos fármacos , Thlaspi/genética , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Zinco/toxicidadeRESUMO
Granule size is an important parameter when using starch in industrial applications. An artificial tandem repeat of a family 20 starch-binding domain (SBD2) was engineered by two copies of the SBD derived from Bacillus circulans cyclodextrin glycosyltransferase via the Pro-Thr-rich linker peptide from Xyn10A from Cellulomonas fimi. SBD2 and a single SBD were introduced into the amylose-free potato mutant, amf, using appropriate signal sequences. The accumulation of SBD2 into transgenic starch granules was much higher than that of SBD. In a number of transformants, particularly amfSS3, the starch granules were much smaller than in control plants. The amfSS3 mean granule size was 7.8 microm, compared with 15.2 microm in the control, whereas other starch properties were unaltered. This new starch combines the advantage of the high purity of potato starch with that of the small granule size of other crop species, such as cassava, taro and wheat. This starch may find application in the manufacture of biodegradable plastic films. Both genes were also expressed in Escherichia coli and the affinity for soluble starch of the purified recombinant proteins was determined. SBD2 had an approximately 10-fold higher affinity for starch than SBD, indicating that the two appended SBDs act in synergy when binding to their target polysaccharide ligand.
RESUMO
Four potato cellulose synthase (CesA) homologs (StCesA1, 2, 3 and 4) were isolated by screening a cDNA library made from developing tubers. Based on sequence comparisons and the fact that all four potato cDNAs were isolated from this single cDNA-library, all four StCesA clones are likely to play a role in primary cell wall biosynthesis. Several constructs were generated to modulate cellulose levels in potato plants in which the granule-bound starch synthase promoter was used to target the modification to the tubers. The StCesA3 was used for up- and down-regulation of the cellulose levels by sense (SE-StCesA3) and antisense (AS-StCesA3) expression of the complete cDNA. Additionally, the class-specific regions (CSR) of all four potato cellulose synthase genes were used for specific down-regulation (antisense) of the corresponding CesA genes (csr1, 2, 3 and 4). None of the transformants showed an overt developmental phenotype. Sections of tubers were screened for altered cell wall structure by Fourier Transform Infrared microspectroscopy (FTIR) and exploratory Principal Component Analysis (PCA), and those plants discriminating from WT plants were analysed for cellulose content and monosaccharide composition. Several transgenic lines were obtained with mainly decreased levels of cellulose. These results show that the cellulose content in potato tubers can be reduced down to 40% of the WT level without affecting normal plant development, and that constructs based on the CSR alone are specific and sufficient to down-regulate cellulose biosynthesis.
Assuntos
Parede Celular/metabolismo , Celulose/biossíntese , Glucosiltransferases/genética , RNA Antissenso/biossíntese , Solanum tuberosum/metabolismo , Celulose/metabolismo , Colorimetria , DNA Complementar/genética , Expressão Gênica , Glucosiltransferases/metabolismo , Monossacarídeos/química , Família Multigênica , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , RNA Antissenso/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Solanum tuberosum/enzimologia , Solanum tuberosum/genética , Espectroscopia de Infravermelho com Transformada de Fourier , Transformação GenéticaRESUMO
Zinc (Zn) is an essential element for plants that can be accumulated to very high levels in shoots of some special plant species named hyperaccumulators. Various strategies have been used in recent years to unravel the molecular bases of such an unusual Zn transport and storage, especially in Brassicacea species. In these studies, several Zn transporters and chelators have been identified that exist both in sensitive and hyperaccumulating Brassicacea species allowing the construction of a general model for Zn homeostasis. However, some determinants involved in shoot Zn tolerance are still missing. We have previously shown that defensins confer Zn tolerance and have recently studied the sub-cellular localisation of a leaf A. halleri defensin. In this mini review, we explain why we propose that family 1 defensins could play a role in the protection of the endoplasmic reticulum functioning in leaves during a Zn overload.
Assuntos
Defensinas/metabolismo , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Plantas/metabolismo , Zinco/metabolismo , Brassicaceae/genética , Brassicaceae/metabolismo , Biologia Computacional , Defensinas/genética , Fenótipo , Folhas de Planta/genética , Proteínas de Plantas/genética , Plantas/genéticaRESUMO
We report the generation of Solanum tuberosum transformants expressing Cicer arietinum betaIII-Gal. betaIII-Gal is a beta-galactosidase able to degrade cell wall pectins during cell wall loosening that occurs prior to cell elongation. cDNA corresponding to the gene encoding this protein was identified among several chickpea beta-galactosidase cDNAs, and named CanBGal-3. CanBGal-3 cDNA was expressed in potato under the control of the granule-bound starch synthase promoter. Three betaIII-Gal transformants with varying levels of expression were chosen for further analysis. The transgenic plants displayed no significant altered phenotype compared to the wild type. However, beta-galactanase and beta-galactosidase activities were increased in the transgenic tuber cell walls and this affected the potato tuber pectins. A reduction in the galactosyl content of up to 50% compared to the wild type was observed in the most extreme transformant, indicating a reduction of 1,4-beta-galactan side-chains, as revealed by analysis with LM5 specific antibodies. Our results confirm the notion that the pectin-degrading activity of chickpea betaIII-Gal reported in vitro also occurs in vivo and in other plants, and confirm the involvement of betaIII-Gal in the cell wall autolysis process. An increase in the homogalacturonan content of transgenic tuber cell walls was also observed by Fourier transform infrared spectroscopy (FTIR) analysis.
Assuntos
Parede Celular/metabolismo , Cicer/enzimologia , Galactanos/química , Pectinas/metabolismo , Solanum tuberosum/genética , beta-Galactosidase/genética , Northern Blotting , DNA Complementar , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Pectinas/química , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , RNA Mensageiro/genética , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Rhamnogalacturonan (RG) I is a branched pectic polysaccharide in plant cell walls. Rhamnogalacturonan lyase (eRGL) from Aspergillus aculeatus is able to cleave the RG I backbone at specific sites. Transgenic potato (Solanum tuberosum L.) plants were made by the introduction of the gene encoding eRGL, under the control of the granule-bound starch synthase promoter. The eRGL protein was successfully expressed and translated into an active form, demonstrated by eRGL activity in the tuber extracts. The transgenic plants produced tubers with clear morphological alterations, including radial swelling of the periderm cells and development of intercellular spaces in the cortex. Sugar compositional analysis of the isolated cell walls showed a large reduction in galactosyl and arabinosyl residues in transgenic tubers. Immunocytochemical studies using the LM5 (galactan) and LM6 (arabinan) antibodies also showed a large reduction in galactan and arabinan side-chains of RG I. Most of the remaining LM5 epitopes were located in the expanded middle lamella at cell corners of eRGL tubers, which is in contrast to their normal location in the primary wall of wild type tubers. These data suggest that RG I has an important role in anchoring galactans and arabinans at particular regions in the wall and in normal development of the periderm.