RESUMO
Cyclic adenosine 3',5'-monophosphate (cAMP) is a recognized second messenger; however, knowledge of cAMP involvement in plant physiological processes originates primarily from pharmacological studies. To obtain direct evidence for cAMP function in plants, tobacco Bright Yellow-2 (BY-2) cells were transformed with the cAMP sponge, which is a genetically encoded tool that reduces cAMP availability. BY-2 cells expressing the cAMP sponge (cAS cells), showed low levels of free cAMP and exhibited growth inhibition that was not proportional to the cAMP sponge transcript level. Growth inhibition in cAS cells was closely related to the precocious inhibition of mitosis due to a delay in cell cycle progression. The cAMP deficiency also enhanced antioxidant systems. Remarkable changes occurred in the cAS proteomic profile compared with that of wild-type (WT) cells. Proteins involved in translation, cytoskeletal organization, and cell proliferation were down-regulated, whereas stress-related proteins were up-regulated in cAS cells. These results support the hypothesis that BY-2 cells sense cAMP deficiency as a stress condition. Finally, many proteasome subunits were differentially expressed in cAS cells compared with WT cells, indicating that cAMP signaling broadly affects protein degradation via the ubiquitin/proteasome pathway.
Assuntos
AMP Cíclico/metabolismo , Regulação da Expressão Gênica de Plantas/fisiologia , Nicotiana/citologia , Estresse Fisiológico/fisiologia , Antioxidantes/metabolismo , Linhagem Celular , AMP Cíclico/genética , Plantas Geneticamente Modificadas , Proteômica , Superóxido Dismutase/metabolismo , Fatores de Tempo , TranscriptomaRESUMO
Heat stress can have deleterious effects on plant growth by impairing several physiological processes. Plants have several defense mechanisms that enable them to cope with high temperatures. The synthesis and accumulation of heat shock proteins (HSPs), as well as the maintenance of an opportune redox balance play key roles in conferring thermotolerance to plants. In this study changes in redox parameters, the activity and/or expression of reactive oxygen species (ROS) scavenging enzymes and the expression of two HSPs were studied in tobacco Bright Yellow-2 (TBY-2) cells subjected to moderate short-term heat stress (SHS) and long-term heat stress (LHS). The results indicate that TBY-2 cells subjected to SHS suddenly and transiently enhance antioxidant systems, thus maintaining redox homeostasis and avoiding oxidative damage. The simultaneous increase in HSPs overcomes the SHS and maintains the metabolic functionality of cells. In contrast the exposure of cells to LHS significantly reduces cell growth and increases cell death. In the first phase of LHS, cells enhance antioxidant systems to prevent the formation of an oxidizing environment. Under prolonged heat stress, the antioxidant systems, and particularly the enzymatic ones, are inactivated. As a consequence, an increase in H2 O2 , lipid peroxidation and protein oxidation occurs. This establishment of oxidative stress could be responsible for the increased cell death. The rescue of cell growth and cell viability, observed when TBY-2 cells were pretreated with galactone-γ-lactone, the last precursor of ascorbate, and glutathione before exposure to LHS, highlights the crucial role of antioxidants in the acquisition of basal thermotolerance.
Assuntos
Antioxidantes/metabolismo , Regulação da Expressão Gênica de Plantas , Nicotiana/fisiologia , Estresse Fisiológico , Ascorbato Peroxidases/metabolismo , Ácido Ascórbico/metabolismo , Linhagem Celular , Sobrevivência Celular , Glutationa/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Temperatura Alta , Peróxido de Hidrogênio/metabolismo , Peroxidação de Lipídeos , Oxirredução , Estresse Oxidativo , Peroxidases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Tempo , Nicotiana/enzimologia , Nicotiana/genéticaRESUMO
Nitric oxide (NO) is a small redox molecule that acts as a signal in different physiological and stress-related processes in plants. Recent evidence suggests that the biological activity of NO is also mediated by S-nitrosylation, a well-known redox-based posttranslational protein modification. Here, we show that during programmed cell death (PCD), induced by both heat shock (HS) or hydrogen peroxide (H2O2) in tobacco (Nicotiana tabacum) Bright Yellow-2 cells, an increase in S-nitrosylating agents occurred. NO increased in both experimentally induced PCDs, although with different intensities. In H2O2-treated cells, the increase in NO was lower than in cells exposed to HS. However, a simultaneous increase in S-nitrosoglutathione (GSNO), another NO source for S-nitrosylation, occurred in H2O2-treated cells, while a decrease in this metabolite was evident after HS. Consistently, different levels of activity and expression of GSNO reductase, the enzyme responsible for GSNO removal, were found in cells subjected to the two different PCD-inducing stimuli: low in H2O2-treated cells and high in the heat-shocked ones. Irrespective of the type of S-nitrosylating agent, S-nitrosylated proteins formed upon exposure to both of the PCD-inducing stimuli. Interestingly, cytosolic ascorbate peroxidase (cAPX), a key enzyme controlling H2O2 levels in plants, was found to be S-nitrosylated at the onset of both PCDs. In vivo and in vitro experiments showed that S-nitrosylation of cAPX was responsible for the rapid decrease in its activity. The possibility that S-nitrosylation induces cAPX ubiquitination and degradation and acts as part of the signaling pathway leading to PCD is discussed.
Assuntos
Apoptose , Ascorbato Peroxidases/metabolismo , Nicotiana/citologia , Nicotiana/enzimologia , Transdução de Sinais , Aldeído Oxirredutases/metabolismo , Apoptose/efeitos dos fármacos , Ascorbato Peroxidases/isolamento & purificação , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/enzimologia , Peróxido de Hidrogênio/farmacologia , Sulfeto de Hidrogênio/farmacologia , Cinética , Modelos Biológicos , Dados de Sequência Molecular , Óxido Nítrico/metabolismo , Nitrosação/efeitos dos fármacos , Proteólise/efeitos dos fármacos , S-Nitrosoglutationa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Nicotiana/efeitos dos fármacos , Ubiquitina/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
The alteration of growth patterns, through the adjustment of cell division and expansion, is a characteristic response of plants to environmental stress. In order to study this response in more depth, the effect of heat stress on growth was investigated in tobacco BY-2 cells. The results indicate that heat stress inhibited cell division, by slowing cell cycle progression. Cells were stopped in the pre-mitotic phases, as shown by the increased expression of CycD3-1 and by the decrease in the NtCycA13, NtCyc29 and CDKB1-1 transcripts. The decrease in cell length and the reduced expression of Nt-EXPA5 indicated that cell expansion was also inhibited. Since DNA methylation plays a key role in controlling gene expression, the possibility that the altered expression of genes involved in the control of cell growth, observed during heat stress, could be due to changes in the methylation state of their promoters was investigated. The results show that the altered expression of CycD3-1 and Nt-EXPA5 was consistent with changes in the methylation state of the upstream region of these genes. These results suggest that DNA methylation, controlling the expression of genes involved in plant development, contributes to growth alteration occurring in response to environmental changes.