RESUMO
Recent findings have suggested that reactive oxygen species (ROS) are important signaling molecules for regulating plant responses to abiotic and biotic stress and that there exist source- and kind-specific pathways for ROS signaling. In plant cells, a major source of ROS is chloroplasts, in which thylakoid membrane-bound ascorbate peroxidase (tAPX) plays a role in the regulation of H(2)O(2) levels. Here, to clarify the signaling function of H(2)O(2) derived from the chloroplast, we created a conditional system for producing H(2)O(2) in the organelle by chemical-dependent tAPX silencing using estrogen-inducible RNAi. When the expression of tAPX was silenced in leaves, levels of oxidized protein in chloroplasts increased in the absence of stress. Microarray analysis revealed that tAPX silencing affects the expression of a large set of genes, some of which are involved in the response to chilling and pathogens. In response to tAPX silencing, the transcript levels of C-repeat/DRE binding factor (CBF1), a central regulator for cold acclimation, was suppressed, resulting in a high sensitivity of tAPX-silenced plants to cold. Furthermore, tAPX silencing enhanced the levels of salicylic acid (SA) and the response to SA. Interestingly, we found that tAPX silencing-responsive genes were up- or down-regulated by high light (HL) and that tAPX silencing had a negative effect on expression of ROS-responsive genes under HL, suggesting synergistic and antagonistic roles of chloroplastic H(2)O(2) in HL response. These findings provide a new insight into the role of H(2)O(2)-triggered retrograde signaling from chloroplasts in the response to stress in planta.
Assuntos
Arabidopsis/fisiologia , Núcleo Celular/fisiologia , Cloroplastos/fisiologia , Peróxido de Hidrogênio/metabolismo , Transdução de Sinais , Estresse Fisiológico , Aclimatação , Antioxidantes/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Ascorbato Peroxidases/genética , Ascorbato Peroxidases/metabolismo , Núcleo Celular/metabolismo , Cloroplastos/metabolismo , Temperatura Baixa , Resistência à Doença/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas/efeitos da radiação , Técnicas de Silenciamento de Genes , Genes de Plantas , Luz , Análise de Sequência com Séries de Oligonucleotídeos , Oxirredução , Folhas de Planta/enzimologia , Folhas de Planta/metabolismo , Plantas Geneticamente Modificadas , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo Real , Ácido Salicílico/metabolismo , Ácido Salicílico/farmacologia , Proteínas das Membranas dos Tilacoides/genética , Proteínas das Membranas dos Tilacoides/metabolismo , Transativadores/genética , Transativadores/metabolismo , Transcrição GênicaRESUMO
Though two types of chloroplastic ascorbate peroxidase (APX) located in the thylakoid membrane (tAPX) and stroma (sAPX) have been thought to be key regulators of intracellular levels of H(2)O(2), their physiological significance in the response to photooxidative stress is still under discussion. Here we characterized single mutants lacking either tAPX (KO-tAPX) or sAPX (KO-sAPX). Under exposure to high light or treatment with methylviologen under light, H(2)O(2) and oxidized proteins accumulated to higher levels in both mutant plants than in the wild-type plants. On the other hand, the absence of sAPX and tAPX drastically suppressed the expression of H(2)O(2)-responsive genes under photooxidative stress. Interestingly, the most marked effect of photooxidative stress on the accumulation of H(2)O(2) and oxidized protein and gene expression was observed in the KO-tAPX plants rather than the KO-sAPX plants. The present findings suggest that both chloroplastic APXs, but particularly tAPX, are important for photoprotection and gene regulation under photooxidative stress in Arabidopsis leaves.