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1.
Plant J ; 76(5): 766-80, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24188189

RESUMO

Triticum aestivum aluminum-activated malate transporter (TaALMT1) is the founding member of a unique gene family of anion transporters (ALMTs) that mediate the efflux of organic acids. A small sub-group of root-localized ALMTs, including TaALMT1, is physiologically associated with in planta aluminum (Al) resistance. TaALMT1 exhibits significant enhancement of transport activity in response to extracellular Al. In this study, we integrated structure-function analyses of structurally altered TaALMT1 proteins expressed in Xenopus oocytes with phylogenic analyses of the ALMT family. Our aim is to re-examine the role of protein domains in terms of their potential involvement in the Al-dependent enhancement (i.e. Al-responsiveness) of TaALMT1 transport activity, as well as the roles of all its 43 negatively charged amino acid residues. Our results indicate that the N-domain, which is predicted to form the conductive pathway, mediates ion transport even in the absence of the C-domain. However, segments in both domains are involved in Al(3+) sensing. We identified two regions, one at the N-terminus and a hydrophobic region at the C-terminus, that jointly contribute to the Al-response phenotype. Interestingly, the characteristic motif at the N-terminus appears to be specific for Al-responsive ALMTs. Our study highlights the need to include a comprehensive phylogenetic analysis when drawing inferences from structure-function analyses, as a significant proportion of the functional changes observed for TaALMT1 are most likely the result of alterations in the overall structural integrity of ALMT family proteins rather than modifications of specific sites involved in Al(3+) sensing.


Assuntos
Alumínio/metabolismo , Malatos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Plantas/metabolismo , Triticum/genética , Sequência de Aminoácidos , Animais , Oócitos , Transportadores de Ânions Orgânicos/genética , Filogenia , Proteínas de Plantas/genética , Domínios e Motivos de Interação entre Proteínas , Deleção de Sequência , Relação Estrutura-Atividade , Triticum/metabolismo , Xenopus laevis
2.
Plant Cell Environ ; 35(7): 1185-200, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22211473

RESUMO

Root efflux of organic acid anions underlies a major mechanism of plant aluminium (Al) tolerance on acid soils. This efflux is mediated by transporters of the Al-activated malate transporter (ALMT) or the multi-drug and toxin extrusion (MATE) families. ZmALMT2 was previously suggested to be involved in Al tolerance based on joint association-linkage mapping for maize Al tolerance. In the current study, we functionally characterized ZmALMT2 by heterologously expressing it in Xenopus laevis oocytes and transgenic Arabidopsis. In oocytes, ZmALMT2 mediated an Al-independent electrogenic transport product of organic and inorganic anion efflux. Ectopic overexpression of ZmALMT2 in an Al-hypersensitive Arabidopsis KO/KD line lacking the Al tolerance genes, AtALMT1 and AtMATE, resulted in Al-independent constitutive root malate efflux which partially restored the Al tolerance phenotype. The lack of correlation between ZmALMT2 expression and Al tolerance (e.g., expression not localized to the root tip, not up-regulated by Al, and higher in sensitive versus tolerance maize lines) also led us to question ZmALMT2's role in Al tolerance. The functional properties of the ZmALMT2 transporter presented here, along with the gene expression data, suggest that ZmALMT2 is not involved in maize Al tolerance but, rather, may play a role in mineral nutrient acquisition and transport.


Assuntos
Malatos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Plantas/metabolismo , Raízes de Plantas/metabolismo , Zea mays/genética , Alumínio/metabolismo , Animais , Arabidopsis/genética , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Transporte de Íons , Oócitos , Transportadores de Ânions Orgânicos/genética , Proteínas de Plantas/genética , Raízes de Plantas/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo , Xenopus laevis , Zea mays/metabolismo
3.
Plant J ; 60(3): 411-23, 2009 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-19563436

RESUMO

In this study we examined the role of protein phosphorylation/dephosphorylation in the transport properties of the wheat (Triticum aestivum) root malate efflux transporter underlying Al resistance, TaALMT1. Pre-incubation of Xenopus laevis oocytes expressing TaALMT1 with protein kinase inhibitors (K252a and staurosporine) strongly inhibited both basal and Al(3+)-enhanced TaALMT1-mediated inward currents (malate efflux). Pre-incubation with phosphatase inhibitors (okadaic acid and cyclosporine A) resulted in a modest inhibition of the TaALMT1-mediated currents. Exposure to the protein kinase C (PKC) activator, phorbol 12-myristate 13-acetate (PMA), enhanced TaALMT1-mediated inward currents. Since these observations suggest that TaALMT1 transport activity is regulated by PKC-mediated phosphorylation, we proceeded to modify candidate amino acids in the TaALMT1 protein in an effort to identify structural motifs underlying the process regulating phosphorylation. The transport properties of eight single point mutations (S56A, S183A, S324A, S337A, S351-352A, S384A, T323A and Y184F) generated in amino acid residues predicted to be phosphorylation sites and examined electrophysiologically. The basic transport properties of mutants S56A, S183A, S324A, S337A, S351-352A, T323A and Y184F were not altered relative to the wild-type TaALMT1. Likewise the sensitivity of these mutants to staurosporine resembled that observed for the wild-type transporter. However, the mutation S384A was noticeable, as in oocytes expressing this mutant protein TaALMT1-mediated basal and Al-enhanced currents were significantly inhibited, and the currents were insensitive to staurosporine or PMA. These findings indicate that S384 is an essential residue regulating TaALMT1 activity via direct protein phosphorylation, which precedes Al(3+) enhancement of transport activity.


Assuntos
Alumínio/toxicidade , Malatos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Fosfosserina/metabolismo , Proteínas de Plantas/metabolismo , Triticum/efeitos dos fármacos , Triticum/metabolismo , Alumínio/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , Mutação , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Transportadores de Ânions Orgânicos/genética , Proteínas de Plantas/genética , Inibidores de Proteínas Quinases/farmacologia , Acetato de Tetradecanoilforbol/análogos & derivados , Acetato de Tetradecanoilforbol/farmacologia , Triticum/genética , Xenopus laevis
4.
J Plant Res ; 123(1): 105-18, 2010 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-19902321

RESUMO

Although barley (Hordeum vulgare L.) is a salt-tolerant crop, the underlying physiological and molecular mechanisms of salt tolerance remain to be elucidated. Therefore, we investigated the response of salt-tolerant (K305) and salt-sensitive (I743) cultivars to salt stress at both physiological and molecular levels. Salt treatment increased xylem sap osmolarity, which was attributed primarily to a rise in Na(+) and Cl(-) concentration; enhanced accumulation of the ions in shoots; and reduced plant growth more severely in I743 than K305. The concentration of K(+) in roots and shoots decreased during 8 h of salt treatment in both cultivars but with no marked difference between cultivars. Hence, the severe growth reduction in I743 is attributed to the elevated levels of (mainly) Na(+) in shoots. Analysis of gene expression using quantitative RT-PCR showed that transcripts of K(+)-transporters (HvHAK1 and HvAKT1), vacuolar H(+)-ATPase and inorganic pyrophosphatase (HvHVA/68 and HvHVP1) were more abundant in shoots of K305 than in shoots of I743. Expression of HvHAK1 and Na(+)/H(+) antiporters (HvNHX1, HvNHX3 and HvNHX4) was higher in roots of K305 than in I743 with prolonged exposure to salt. Taken together, these results suggest that the better performance of K305 compared to I743 during salt stress may be related to its greater ability to sequester Na(+) into sub-cellular compartments and/or maintain K(+) homeostasis.


Assuntos
Hordeum/fisiologia , Tolerância ao Sal/fisiologia , Clonagem Molecular , Regulação da Expressão Gênica de Plantas/genética , Regulação da Expressão Gênica de Plantas/fisiologia , Hordeum/genética , Concentração Osmolar , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Brotos de Planta/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tolerância ao Sal/genética , Plantas Tolerantes a Sal/genética , Plantas Tolerantes a Sal/fisiologia , Trocadores de Sódio-Hidrogênio/genética , Trocadores de Sódio-Hidrogênio/fisiologia , Especificidade da Espécie , Simportadores/genética , Simportadores/fisiologia , Xilema/fisiologia
5.
Physiol Plant ; 120(4): 575-584, 2004 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-15032819

RESUMO

Exudation of organic anions is believed to be a common tolerance mechanism for both aluminium toxicity and phosphorus deficiency. Nevertheless, which of these stresses that actually elicit the exudation of organic anions from rape (Brassica napus L) remains unknown, and the combined effects of Al toxicity and P deficiency on rape have not been reported before. Therefore, in the current study, Brassica napus var. Natane nourin plants grown with or without 0.25 mM P were exposed to 0 or 50 micro M AlCl(3) and several parameters related to the exudation of organic anions from the roots were investigated. Eight days of P deficiency resulted in a significant growth reduction, but P deficiency alone did not induce exudation of organic anions. In contrast, Al strongly induced organic acid exudation, while simultaneously inhibiting root growth. Increased in-vitro activity of citrate synthase (CS, EC 4.1.3.7), malate dehydrogenase (MDH, EC 1.1.1.37) and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31), together with reduced root respiration, indicated that the Al-induced accumulation and subsequent exudation of citrate and malate were associated with both increased biosynthesis and reduced metabolism of citric and malic acid. Phosphorus-sufficient plants showed more pronounced aluminium-induced accumulation and exudation of organic anions than P-deficient plants. A divided root chamber experiment showed the necessity of direct contact between Al and roots to elicit exudation of organic anions. Prolonged exposure (10 days) to Al resulted in a decrease in the net exudation of citrate and malate, and the rate of decrease was much more rapid in P-deficient plants than in P-sufficient plants. It is concluded that P nutrition affects the level of Al-induced synthesis and exudation of organic anions. However, the mechanism needs further investigation.

6.
C R Biol ; 334(2): 127-39, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-21333943

RESUMO

In one of the most important crops, barley (Hordeum vulgare L.), gene expression and physiological roles of most major intrinsic proteins (MIPs) remained to be elucidated. Here we studied expression of five tonoplast intrinsic protein isoforms (HvTIP1;2, HvTIP2;1, HvTIP2;2, HvTIP2;3 and HvTIP4;1), a NOD26-like intrinsic protein (HvNIP2;1) and a plasma membrane intrinsic protein (HvPIP2;1) by using the quantitative real-time RT-PCR. Five-day-old seedlings were exposed to abiotic stresses (salt, heavy metals and nutrient deficiency), abscisic acid (ABA) and gibberellic acid (GA) for 24 h. Treatment with 100 mM NaCl, 0.1 mM ABA and 1 mM GA differentially regulated gene expression in roots and shoots. Nitrogen and prolonged P-deficiency downregulated expression of most MIP genes in roots. Intriguingly, gene expression was restored to the values in the control three days after nutrient supply was resumed. Heavy metals (0.2 mM each of Cd, Cu, Zn and Cr) downregulated the transcript levels by 60-80% in roots, whereas 0.2 mM Hg upregulated expressions of most genes in roots. This was accompanied by a 45% decrease in the rate of transpiration. In order to study the physiological role of the MIPs, cDNA of three genes (HvTIP2;1, HvTIP2;3 and HvNIP2;1) have been cloned and heterologous expression was performed in Xenopus laevis oocytes. Osmotic water permeability was determined by a swelling assay. However, no water uptake activity was observed for the three proteins. Hence, the possible physiological role of the proteins is discussed.


Assuntos
Regulação da Expressão Gênica de Plantas , Hordeum/genética , Proteínas de Plantas/biossíntese , Estresse Fisiológico/genética , Ácido Abscísico/farmacologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , DNA Complementar/genética , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes de Plantas , Giberelinas/farmacologia , Hordeum/efeitos dos fármacos , Hordeum/metabolismo , Proteínas de Membrana/biossíntese , Proteínas de Membrana/genética , Proteínas de Membrana/fisiologia , Metais Pesados/farmacologia , Oócitos , Osmose/efeitos dos fármacos , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/fisiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/metabolismo , Brotos de Planta/efeitos dos fármacos , Brotos de Planta/metabolismo , Transpiração Vegetal/efeitos dos fármacos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Cloreto de Sódio/farmacologia , Transcrição Gênica/efeitos dos fármacos , Xenopus laevis
7.
Plant Signal Behav ; 2(4): 255-7, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19704671

RESUMO

We have previously reported that Al-induces citrate and malate efflux from P-sufficient and P-deficient plants of rape (Brassica napus L.) and that P-deficiency alone could not induce this response. Further investigation showed that the transcript of two genes designated BnALMT1 and BnALMT2 is accumulated in roots by Al-treatment. Transgenic tobacco cells (Nicotiana tabacum) and Xenopus laevis oocytes expressing the BnALMT1 and BnALMT2 proteins released more malate than control cells in the presence of Al, indicating that the BnALMT genes encode an Al-activated malate transporter. The transgenic tobacco cells exposed to toxic level of Al grew better than control cells indicating that the genes can enhance Al-resistance of plant cells. In this study we showed the subcellular localization of BnALMT1 fused to the green fluoresce protein (GFP). The BnALMT1:: GFP construct was transiently expressed in protoplasts prepared from Arabidopsis leaves using the polyethylene glycol (PEG) method. The result showed that the BnALMT1 protein is localized in the plasma membrane. This provides further evidence that the BnALMT proteins facilitate the transport of malate across the plasma membrane (PM).

8.
Plant Physiol ; 142(3): 1294-303, 2006 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-17028155

RESUMO

The release of organic anions from roots can protect plants from aluminum (Al) toxicity and help them overcome phosphorus (P) deficiency. Our previous findings showed that Al treatment induced malate and citrate efflux from rape (Brassica napus) roots, and that P deficiency did not induce the efflux. Since this response is similar to the malate efflux from wheat (Triticum aestivum) that is controlled by the TaALMT1 gene, we investigated whether homologs of TaALMT1 are present in rape and whether they are involved in the release of organic anions. We isolated two TaALMT1 homologs from rape designated BnALMT1 and BnALMT2 (B. napus Al-activated malate transporter). The expression of these genes was induced in roots, but not shoots, by Al treatment but P deficiency had no effect. Several other cations (lanthanum, ytterbium, and erbium) also increased BnALMT1 and BnALMT2 expression in the roots. The function of the BnALMT1 and BnALMT2 proteins was investigated by heterologous expression in cultured tobacco (Nicotiana tabacum) cells and in Xenopus laevis oocytes. Both transfection systems showed an enhanced capacity for malate efflux but not citrate efflux, when exposed to Al. Smaller malate fluxes were also activated by ytterbium and erbium treatment. Transgenic tobacco cells grew significantly better than control cells following an 18 h treatment with Al, indicating that the expression of BnALMT1 and BnALMT2 increased the resistance of these plant cells to Al stress. This report demonstrates that homologs of the TaALMT1 gene from wheat perform similar functions in other species.


Assuntos
Alumínio/farmacologia , Brassica napus/citologia , Brassica napus/metabolismo , Malatos/metabolismo , Transportadores de Ânions Orgânicos/genética , Transportadores de Ânions Orgânicos/metabolismo , Proteínas de Plantas/metabolismo , Sequência de Aminoácidos , Transporte Biológico Ativo/efeitos dos fármacos , Brassica napus/efeitos dos fármacos , Brassica napus/genética , Células Cultivadas , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Proteínas de Plantas/genética , Nicotiana/citologia
9.
Plant Physiol ; 138(1): 287-96, 2005 May.
Artigo em Inglês | MEDLINE | ID: mdl-15834009

RESUMO

The aluminum (Al)-induced secretion of citrate has been regarded as an important mechanism for Al resistance in soybean (Glycine max). However, the mechanism of how Al induces citrate secretion remains unclear. In this study, we investigated the regulatory role of plasma membrane H+-ATPase on the Al-induced secretion of citrate from soybean roots. Experiments performed with plants grown in full nutrient solution showed that Al-induced activity of plasma membrane H+-ATPase paralleled secretion of citrate. Vanadate and fusicoccin, an inhibitor and an activator, respectively, of plasma membrane H+-ATPase, exerted inhibitory and stimulatory effects on the Al-induced secretion of citrate. Higher activity of plasma membrane H+-ATPase coincided with more citrate secretion in Al-resistant than Al-sensitive soybean cultivars. These results suggested that the effects of Al stress on citrate secretion were mediated via modulation of the activity of plasma membrane H+-ATPase. The relationship between the Al-induced secretion of citrate and the activity of plasma membrane H+-ATPase was further demonstrated by analysis of plasma membrane H+-ATPase transgenic Arabidopsis (Arabidopsis thaliana). When plants were grown on Murashige and Skoog medium containing 30 microM Al (9.1 microM Al3+ activity), transgenic plants exuded more citrate compared with wild-type Arabidopsis. Results from real-time reverse transcription-PCR and immunodetection analysis indicated that the increase of plasma membrane H+-ATPase activity by Al is caused by transcriptional and translational regulation. Furthermore, plasma membrane H+-ATPase activity and expression were higher in an Al-resistant cultivar than in an Al-sensitive cultivar. Al activated the threonine-oriented phosphorylation of plasma membrane H+-ATPase in a dose- and time-dependent manner. Taken together, our results demonstrated that up-regulation of plasma membrane H+-ATPase activity was associated with the secretion of citrate from soybean roots.


Assuntos
Alumínio/farmacologia , Citratos/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Glycine max/fisiologia , Raízes de Plantas/fisiologia , ATPases Translocadoras de Prótons/genética , Arabidopsis/enzimologia , Arabidopsis/genética , Sequência de Bases , Membrana Celular/metabolismo , Primers do DNA , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Dados de Sequência Molecular , Fosforilação , Fosfotreonina/metabolismo , Raízes de Plantas/efeitos dos fármacos , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Reação em Cadeia da Polimerase , Biossíntese de Proteínas/efeitos dos fármacos , ATPases Translocadoras de Prótons/metabolismo , Glycine max/efeitos dos fármacos , Glycine max/genética , Glycine max/crescimento & desenvolvimento , Transcrição Gênica/efeitos dos fármacos
10.
J Exp Bot ; 55(397): 663-71, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14754917

RESUMO

The Al-induced release of organic acid has been suggested as an important mechanism for Al resistance in plants. In this study, the effect of K-252a and abscisic acid (ABA) on the efflux of citrate was investigated in soybean (Glycine max L.) roots. Al initiated citrate efflux from the root apices 30 min after the addition of Al. The Al-triggered efflux of citrate was sensitive to metabolic inhibitors and anion channel inhibitors. Pretreatment or treatment with K-252a, an inhibitor of protein kinase, severely inhibited the Al-induced efflux of citrate accompanying an increase in Al accumulation and intensified Al-induced root growth inhibition. Al-treatment increased the endogenous level of abscisic acid (ABA) in soybean roots in a dose- and time-dependent manner, while K-252a failed to inhibit the Al-induced increase in endogenous ABA. Exogenous application of ABA increased the activity of citrate synthase (EC 4.1.3.7) by 26.2%, and decreased Al accumulation by 32.3%, respectively. ABA-induced increases in citrate efflux and root elongation were suppressed by K-252a, while ABA could not reverse the K-252a effects. Taken together, these results suggest that ABA is probably involved in the early response, after which K-252a-sensitive protein kinases play a key step in regulating the activity of an anion channel, through which citrate is released from the apical cells of soybean roots.


Assuntos
Ácido Abscísico/farmacologia , Carbazóis/farmacologia , Citratos/metabolismo , Inibidores Enzimáticos/farmacologia , Glycine max/metabolismo , Raízes de Plantas/metabolismo , Alumínio/metabolismo , Alumínio/farmacologia , Alcaloides Indólicos , Cinética , Raízes de Plantas/efeitos dos fármacos , Inibidores de Proteínas Quinases , Glycine max/efeitos dos fármacos , Verapamil/farmacologia
11.
Funct Plant Biol ; 31(11): 1075-1083, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-32688975

RESUMO

The response of greater purple lupin (Lupinus pilosus L.) to a combination of phosphorus (P) deficiency and aluminium (Al) toxicity is unknown, and the mechanisms involved in the exudation of organic anions from greater purple lupin have not been reported. Therefore, plants grown with (+P) or without (-P) 250 µm P were exposed to 0 or 50 µm AlCl3 and the amount of organic anions exuded and the activities of plasma membrane H+-ATPase (E.C 3.6.3.6) and H+-pumps were investigated. Twenty days of P deficiency resulted in significantly reduced shoot growth and increased proteoid root formation. Exposure to 50 µm AlCl3 did not induce citrate exudation but did induce some malate exudation in -P plants. In contrast, P deficiency did induce exudation of citrate. Enhanced citrate exudation was attributed to the large increase in the activity of plasma membrane H+-ATPase and associated H+ transport. This was shown by the inhibitory effect of vanadate on plasma membrane H+-ATPase activity in vitro and on citrate exudation in vivo. However, vanadate did not suppress the exudation of malate. During 9 h of Al exposure, exudation of citrate showed a continuing increase for both -P and +P plants, while malate exudation increased only during the first 3 h, after which it rapidly declined. The total amount of organic anion exudation was significantly higher for -P plants. In the presence of 50 µm anion channel blockers [anthracene-9-carboxylic acid (A-9-C), niflumic acid (NIF) and phenylglyoxal (PG)], the exudation of citrate and malate was reduced by 25-40%. It was concluded that P deficiency induces citrate exudation by enhancing the activity of plasma membrane H+-ATPase and H+ export. In L. pilosus, exudation of organic anions occurs primarily in response to P deficiency but not Al toxicity. This contrasts with previous results obtained in Brassica napus L.

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