RESUMEN
Glycine betaine (GB) is an important osmoprotectant and synthesized by two-step oxidation of choline. Choline monooxygenase (CMO) catalyzes the first step of the pathway and is believed to be a rate limiting step for GB synthesis. Recent studies have shown the importance of choline-precursor supply for GB synthesis. In order to investigate the role of CMO for GB accumulation in sugar beet (Beta vulgaris), transgenic plants carrying the antisense BvCMO gene were developed. The antisense BvCMO plants showed the decreased activity of GB synthesis from choline compared to wild-type (WT) plants which is well related to the suppressed level of BvCMO protein. However, GB contents were similar between transgenic and WT plants with the exception of young leaves and storage roots. Transgenic plants showed enhanced susceptibility to salt stress than WT plants. These results suggest the importance of choline-precursor-supply for GB accumulation, and young leaves and storage root are sensitive sites for GB accumulation.
Asunto(s)
Beta vulgaris/enzimología , Betaína/metabolismo , Oxigenasas/metabolismo , Plantas Modificadas GenéticamenteRESUMEN
Glutamate is a major free amino acid in cyanobacteria, but its transport properties remain largely unknown. In this study, we found that a halotolerant cyanobacterium, Aphanothece halophytica, contained a sodium dependent glutamate transporter (ApGltS). The deduced amino acid sequence of ApGltS exhibited low homology (18-19% identity) to GltS from Synechocystis sp. PCC 6803 (slr1145) and Escherichia coli. The predicted ApGltS consisted of 476 amino acid residues with a molecular weight of 50,976 Da. As analysed by hydropathy profiling, ApGltS contains 11 transmembrane segments. The ApgltS gene was isolated and expressed in E. coli ME9107, which is deficient in glutamate uptake. ME9107, expressing ApGltS, took up glutamate and its rates increased with increasing concentrations of NaCl. Kinetics studies revealed that ApGltS is a high-affinity glutamate transporter with a K(m) of about 5 µM. The presence of 0.5 M NaCl in the assay medium increased V(max) by about 3-fold. Competition experiments revealed that glutamate, glutamine, aspartate, and asparagine inhibited glutamate uptake. The level of mRNA for ApgltS was higher in A. halophytica grown at high salinity. Under high salinity conditions supplemented with glutamate, A. halophytica showed a significant increase in intracellular glycine betaine.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG/metabolismo , Proteínas Bacterianas/metabolismo , Cianobacterias/metabolismo , Ácido Glutámico/metabolismo , Cloruro de Sodio/farmacología , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG/genética , Sistemas de Transporte de Aminoácidos Acídicos/genética , Sistemas de Transporte de Aminoácidos Acídicos/metabolismo , Proteínas Bacterianas/genética , Betaína/metabolismo , Cianobacterias/efectos de los fármacos , Cianobacterias/genética , Cianobacterias/crecimiento & desarrollo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Datos de Secuencia Molecular , Peso Molecular , Filogenia , Estructura Terciaria de Proteína , ARN Mensajero/biosíntesis , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Salinidad , Tolerancia a la Sal/fisiología , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Simportadores/genética , Simportadores/metabolismoRESUMEN
Proline transporters (ProTs) originally described as highly selective transporters for proline, have been shown to also transport glycinebetaine (betaine). Here we examined and compared the transport properties of Bet/ProTs from betaine accumulating (sugar beet, Amaranthus, and Atriplex,) and non-accumulating (Arabidopsis) plants. Using a yeast mutant deficient for uptake of proline and betaine, it was shown that all these transporters exhibited higher affinity for betaine than proline. The uptake of betaine and proline was pH-dependent and inhibited by the proton uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP). We also investigated choline transport by using a choline transport-deficient yeast mutant. Results revealed that these transporters exhibited a higher affinity for choline uptake rather than betaine. Uptake of choline by sugar beet BvBet/ProT1 was independent of the proton gradient and the inhibition by CCCP was reduced compared with that for uptake of betaine, suggesting different proton binding properties between the transport of choline and betaine. Additionally, in situ hybridization experiments revealed the localization of sugar beet BvBet/ProT1 in phloem and xylem parenchyma cells.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Beta vulgaris/metabolismo , Betaína/metabolismo , Proteínas Portadoras/metabolismo , Colina/metabolismo , Prolina/metabolismo , Amaranthus/genética , Amaranthus/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inhibidores , Sistemas de Transporte de Aminoácidos Neutros/genética , Arabidopsis/genética , Arabidopsis/metabolismo , Atriplex/genética , Atriplex/metabolismo , Secuencia de Bases , Beta vulgaris/genética , Transporte Biológico , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Proteínas Portadoras/antagonistas & inhibidores , Proteínas Portadoras/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática , Concentración de Iones de Hidrógeno , Datos de Secuencia Molecular , Mutación , Floema/metabolismo , Hojas de la Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Ionóforos de Protónes/farmacología , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Especificidad por Sustrato , Xilema/metabolismoRESUMEN
Oil production from oil palm is adversely affected by drought and salt. Under drought and salt stress, proline content increases in oil palm; the mechanism for this is unknown. Here, an 8319-nucleotide sequence including cDNA, genomic DNA and the promoter region of proline transporter gene from oil palm Elaeis guineensis was determined. The transporter gene exhibited high similarity to Bet/ProT genes from several plants, but the highest homology was found with rice ProT1. The exon-intron structure of genomic DNA was unique, and numerous stress-response cis-elements were found in the promoter region. Expression of cDNA EgProT1 in Escherichia coli mutant exhibited uptake activities for glycinebetaine and choline as well as proline. Under salt-stressed conditions, exogenously applied glycinebetaine was taken up into the root more rapidly than the control. These data indicate that oil palm has a unique Pro/T1 gene. Nucleotide sequence data for the cDNA and genomic DNA of proline transporter gene from Elaeis guineensis are available in the DDJB database under accession numbers AB597035 and AB597036, respectively.
Asunto(s)
Sistemas de Transporte de Aminoácidos Neutros/genética , Arecaceae/genética , Arecaceae/metabolismo , Betaína/metabolismo , Proteínas Portadoras/genética , Prolina/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/efectos de los fármacos , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Arecaceae/efectos de los fármacos , Secuencia de Bases , Betaína/análisis , Transporte Biológico , Proteínas Portadoras/efectos de los fármacos , Proteínas Portadoras/metabolismo , Colina/metabolismo , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , ADN de Plantas/química , ADN de Plantas/genética , Proteínas Transportadoras de GABA en la Membrana Plasmática , Proteínas Fluorescentes Verdes , Datos de Secuencia Molecular , Filogenia , Prolina/análisis , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN de Planta/genética , Semillas/genética , Semillas/metabolismo , Análisis de Secuencia de ADN , Cloruro de Sodio/farmacología , Estrés FisiológicoRESUMEN
It has been reported that glycinebetaine (betaine) is synthesized in response to abiotic stresses via a two-step oxidation of choline in which choline monooxygenase (CMO) and betaine aldehyde dehydrogenase (BADH) are involved. Here we show that significant amounts of betaine, > 20 micromol/gFW, accumulated in young leaves of Beta vulgaris even under normal growth conditions, whereas levels in old leaves, cotyledons, hypocotyls, and roots were low. Under the same conditions, CMO accumulates exclusively in old leaves and is difficult to be detected in young leaves. By contrast, the levels of BADH were high in all tissues. Exogenously supplied choline was converted into betaine in old leaves, but levels were significantly lower in young leaves under the same conditions. When d(11)-betaine was applied exogenously to old leaves, it was translocated preferentially into young leaves and roots. In response to salt stress, betaine levels increased in all tissues, but most significantly increased in young leaves. The levels of CMO increased in various tissues, but were low in young leaves. A betaine transporter gene was isolated. Its expression was more strongly induced in old leaves than in young leaves. Based on these data, we discussed the role of CMO and betaine transporter under stress and non-stress conditions.