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Induction of caspase-3-like activity in rice following release of cytochrome-f from the chloroplast and subsequent interaction with the ubiquitin-proteasome system.

Wang H, Zhu X, Li H, Cui J, Liu C, Chen X, Zhang W - Sci Rep (2014)

Bottom Line: In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo.Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts.Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biochemistry &Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.

ABSTRACT
It has been known that the process of leaf senescence is accompanied by programmed cell death (PCD), and the previous study indicated that dark-induced senescence in detached leaves from rice led to the release of cytochrome f (Cyt f) from chloroplast into the cytoplasm. In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo. In a cell-free system, purified Cyt f activated caspase-3-like protease and endonuclease OsNuc37, and induced DNA fragmentation. Furthermore, Cyt f-induced caspase-3-like activity could be inhibited by MG132, which suggests that the activity was attributed to the 26S proteasome. Conditional expression of Cyt f in the cytoplasm could also activate caspase-3-like activity and DNA fragmentation. Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts. Yeast two-hybrid screening showed that Cyt f might interact with E3-ubiquitin ligase and RPN9b, the subunits of the ubiquitin proteasome system (UPS), and other PCD-related proteins. Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD.

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Quantification analysis of PCD mesophyll protoplasts under the expression of Cyt f by flow cytometry.Representative cytograms illustrating protoplast viability following different treatments at 0, 12, and 24 h, as estimated by annexin V-FITC and PI staining. Late apoptotic-like protoplasts (annexin V+/PI+) are positioned in the upper right quadrant; Early apoptotic-like protoplasts (annexin V+/PI−) are positioned in the lower right quadrant; Normal protoplasts (annexin V−/PI−) are present in the lower left quadrants; Necrotic protoplasts (annexin V−/PI+) are present in the upper left quadrants. To depress the background fluorescence, all the protoplasts for transient expression were prepared from etiolated seedlings.
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f6: Quantification analysis of PCD mesophyll protoplasts under the expression of Cyt f by flow cytometry.Representative cytograms illustrating protoplast viability following different treatments at 0, 12, and 24 h, as estimated by annexin V-FITC and PI staining. Late apoptotic-like protoplasts (annexin V+/PI+) are positioned in the upper right quadrant; Early apoptotic-like protoplasts (annexin V+/PI−) are positioned in the lower right quadrant; Normal protoplasts (annexin V−/PI−) are present in the lower left quadrants; Necrotic protoplasts (annexin V−/PI+) are present in the upper left quadrants. To depress the background fluorescence, all the protoplasts for transient expression were prepared from etiolated seedlings.

Mentions: Since Cyt f is located in the chloroplast under normal growth conditions, we investigated whether PCD occurs if Cyt f is expressed in the cytoplasm. In order to test this hypothesis, conditional expression of C-terminus lacking Cyt f in the cytoplasm was established in the mesophyll protoplast (see details in Methods). Protoplasts bearing pTA7002-Cyt f were treated with dexamethasone (Dex) at different times, and immunoblot analysis revealed that Cyt f could be detected in the cytoplasm as early as 6 h after 10 μM Dex induction. In the following 6 h, the expression level increased significantly (Fig. 2). Following Cyt f expression in the cytoplasm, nuclei expansion occurred at 12 h and DNA fragmentation was observed at 18 h, after Dex treatment. After 24 h, the generation of large debris indicated the disintegration of the nuclei (Fig. 3). At the same time, if the Dex was substituted by an equal volume of ethanol, the solvent of Dex, no changes in nuclei were visible (Fig. 3). It should be noted that transient expression of the reference construct showed that the transformation efficiency was as high as 71% (data not shown), which inferred that Cyt f was expressed in most of the protoplasts. Similar to the observation in a cell-free system, conditional expression of Cyt f in mesophyll protoplasts induced caspase-3-like activity up to 10-fold over than that of the vector control (Fig. 4). Viability of transformed protoplasts was assessed using fluorescein diacetate (FDA) staining. As shown in Fig. 5, within 6 h after Dex induction, both the transgenic and control protoplasts exhibited bright fluorescence and normal morphology (Fig. 5A). In the following 18 h, the fluorescence intensity was gradually reduced. However, the reduction of fluorescence intensity in transgenic protoplasts was more significant than that in the vector control, and at 24 h after Dex induction, the average RFU was reduced 70% in transgenic protoplasts, compared with a 40% reduction in the vector control (Fig. 5B). Flow cytometry analysis showed that the percentage of apoptotic-like protoplasts (Annexin V-FITC+/PI+) in the transgenic group were also increased than that in the vector control (Fig. 6), which indicates that in addition to caspase-3 activation and DNA fragmentation, expression of Cyt f in cytoplasm induced PCD in mesophyll protoplasts in rice.


Induction of caspase-3-like activity in rice following release of cytochrome-f from the chloroplast and subsequent interaction with the ubiquitin-proteasome system.

Wang H, Zhu X, Li H, Cui J, Liu C, Chen X, Zhang W - Sci Rep (2014)

Quantification analysis of PCD mesophyll protoplasts under the expression of Cyt f by flow cytometry.Representative cytograms illustrating protoplast viability following different treatments at 0, 12, and 24 h, as estimated by annexin V-FITC and PI staining. Late apoptotic-like protoplasts (annexin V+/PI+) are positioned in the upper right quadrant; Early apoptotic-like protoplasts (annexin V+/PI−) are positioned in the lower right quadrant; Normal protoplasts (annexin V−/PI−) are present in the lower left quadrants; Necrotic protoplasts (annexin V−/PI+) are present in the upper left quadrants. To depress the background fluorescence, all the protoplasts for transient expression were prepared from etiolated seedlings.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4125987&req=5

f6: Quantification analysis of PCD mesophyll protoplasts under the expression of Cyt f by flow cytometry.Representative cytograms illustrating protoplast viability following different treatments at 0, 12, and 24 h, as estimated by annexin V-FITC and PI staining. Late apoptotic-like protoplasts (annexin V+/PI+) are positioned in the upper right quadrant; Early apoptotic-like protoplasts (annexin V+/PI−) are positioned in the lower right quadrant; Normal protoplasts (annexin V−/PI−) are present in the lower left quadrants; Necrotic protoplasts (annexin V−/PI+) are present in the upper left quadrants. To depress the background fluorescence, all the protoplasts for transient expression were prepared from etiolated seedlings.
Mentions: Since Cyt f is located in the chloroplast under normal growth conditions, we investigated whether PCD occurs if Cyt f is expressed in the cytoplasm. In order to test this hypothesis, conditional expression of C-terminus lacking Cyt f in the cytoplasm was established in the mesophyll protoplast (see details in Methods). Protoplasts bearing pTA7002-Cyt f were treated with dexamethasone (Dex) at different times, and immunoblot analysis revealed that Cyt f could be detected in the cytoplasm as early as 6 h after 10 μM Dex induction. In the following 6 h, the expression level increased significantly (Fig. 2). Following Cyt f expression in the cytoplasm, nuclei expansion occurred at 12 h and DNA fragmentation was observed at 18 h, after Dex treatment. After 24 h, the generation of large debris indicated the disintegration of the nuclei (Fig. 3). At the same time, if the Dex was substituted by an equal volume of ethanol, the solvent of Dex, no changes in nuclei were visible (Fig. 3). It should be noted that transient expression of the reference construct showed that the transformation efficiency was as high as 71% (data not shown), which inferred that Cyt f was expressed in most of the protoplasts. Similar to the observation in a cell-free system, conditional expression of Cyt f in mesophyll protoplasts induced caspase-3-like activity up to 10-fold over than that of the vector control (Fig. 4). Viability of transformed protoplasts was assessed using fluorescein diacetate (FDA) staining. As shown in Fig. 5, within 6 h after Dex induction, both the transgenic and control protoplasts exhibited bright fluorescence and normal morphology (Fig. 5A). In the following 18 h, the fluorescence intensity was gradually reduced. However, the reduction of fluorescence intensity in transgenic protoplasts was more significant than that in the vector control, and at 24 h after Dex induction, the average RFU was reduced 70% in transgenic protoplasts, compared with a 40% reduction in the vector control (Fig. 5B). Flow cytometry analysis showed that the percentage of apoptotic-like protoplasts (Annexin V-FITC+/PI+) in the transgenic group were also increased than that in the vector control (Fig. 6), which indicates that in addition to caspase-3 activation and DNA fragmentation, expression of Cyt f in cytoplasm induced PCD in mesophyll protoplasts in rice.

Bottom Line: In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo.Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts.Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD.

View Article: PubMed Central - PubMed

Affiliation: Dept. of Biochemistry &Molecular Biology, College of Life Science, Nanjing Agricultural University, Nanjing, Jiangsu 210095, China.

ABSTRACT
It has been known that the process of leaf senescence is accompanied by programmed cell death (PCD), and the previous study indicated that dark-induced senescence in detached leaves from rice led to the release of cytochrome f (Cyt f) from chloroplast into the cytoplasm. In this study, the effects of Cyt f on PCD were studied both in vitro and in vivo. In a cell-free system, purified Cyt f activated caspase-3-like protease and endonuclease OsNuc37, and induced DNA fragmentation. Furthermore, Cyt f-induced caspase-3-like activity could be inhibited by MG132, which suggests that the activity was attributed to the 26S proteasome. Conditional expression of Cyt f in the cytoplasm could also activate caspase-3-like activity and DNA fragmentation. Fluorescein diacetate staining and annexin V-FITC/PI double staining demonstrated that Cyt f expression in cytoplasm significantly increased the percentage of PCD protoplasts. Yeast two-hybrid screening showed that Cyt f might interact with E3-ubiquitin ligase and RPN9b, the subunits of the ubiquitin proteasome system (UPS), and other PCD-related proteins. Taken together, these results suggest that the released Cyt f from the chloroplast into the cytoplasm might activate or rescue caspase-3-like activity by interacting with the UPS, ultimately leading to the induction of PCD.

Show MeSH
Related in: MedlinePlus