RESUMO
In order to identify genes/proteins involved in copper tolerance, the marine alga Ulva compressa was cultivated with 10 µM copper for 3 days. The activities of antioxidant enzymes ascorbate peroxidase (AP), peroxiredoxin (PRX), thioredoxin (TRX), and glutathione-S-transferase (GST) and the level of lipoperoxides were determined in the alga cultivated with and without copper addition. Antioxidant enzyme activities and lipoperoxides level increased in response to copper excess, indicating that the alga was under oxidative stress. A cDNA library was prepared using U. compressa cultivated with 10 µM copper for 3 days. A total of 3 × 10(4) clones were isolated and 480 clones were sequenced, resulting in 235 non-redundant ESTs, of which 104 encode proteins with known functions. Among them, we identified proteins involved in (1) antioxidant metabolism such as AP, PRX, TRX, GST, and metalothionein (MET), (2) signal transduction, such as calmodulin (CAM), (3) calcium-dependent protein kinase (CDPK) and nucleoside diphosphate kinase (NDK), (4) gene expression, (5) protein synthesis and degradation, and (6) chloroplast and mitochondria electron transport chains. Half of the identified proteins are potentially localized in organelles. The relative level of 18 genes, including those coding for AP, PRX, TRX, GST, MET, CAM, CDPK, and NDK were determined by quantitative RT-PCR in the alga cultivated with 10 µM copper for 0 to 7 days. Transcript levels increased in response to copper stress and most of them reached a maximum at days 3 and 5. Thus, the selected genes are induced by copper stress and they are probably involved in copper acclimation and tolerance.
Assuntos
Ascorbato Peroxidases/genética , Cobre/toxicidade , Glutationa Transferase/genética , Peroxirredoxinas/genética , Transdução de Sinais/genética , Tiorredoxinas/genética , Ulva , Análise de Variância , Ascorbato Peroxidases/metabolismo , Sequência de Bases , Chile , Clonagem Molecular , Complexo de Proteínas da Cadeia de Transporte de Elétrons/genética , Indução Enzimática/imunologia , Etiquetas de Sequências Expressas , Biblioteca Gênica , Glutationa Transferase/metabolismo , Peróxidos Lipídicos/metabolismo , Dados de Sequência Molecular , Estresse Oxidativo/efeitos dos fármacos , Peroxirredoxinas/metabolismo , Reação em Cadeia da Polimerase em Tempo Real , Análise de Sequência de DNA , Tiorredoxinas/metabolismoRESUMO
The marine alga Ulva compressa (Chlorophyta) showed a triphasic release of intracellular calcium with maximal levels at 2, 3 and 12 h and a biphasic accumulation of intracellular hydrogen peroxide with peaks at 3 and 12 h when cultivated with copper excess. Intracellular hydrogen peroxide originated exclusively in organelles. In this work, we analyzed the intracellular origin of calcium release and the type of calcium channels activated in response to copper excess. U. compressa was treated with thapsigargin, an inhibitor of endoplasmic reticulum (ER) calcium ATPase, ryanodine, an inhibitor of ryanodine-sensitive channels, and xestospongin C, an inhibitor of inositol 1, 4, 5-triphosphate (IP(3))-sensitive channels. Thapsigargin induced the depletion of calcium stored in ER at 75 min and completely inhibited calcium release at 2, 3 and 12 h of copper exposure indicating that calcium release originated in ER. In addition, ryanodine and xestospogin C inhibited calcium release at 2 and 3 h of copper exposure whereas the peak at 12 h was only inhibited by ryanodine. Thus, copper induced the activation of ryanodine-sensitive and IP(3)-sensitive calcium channels in ER of U. compressa.