Limits...
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.

Show MeSH

Related in: MedlinePlus

Induction of Cyt f PCD hallmarks in a cell-free system.(a) DNA laddering and (b) endonuclease activity in the nuclear extracts, which were induced by different concentrations of Cyt f. M, marker; N, isolated nucleus; C, cell-free system including nucleus and cytosolic fraction; C + f1, cell-free system plus 0.2 μM Cyt f; C + f2, cell-free system plus 1 μM Cyt f. Arrowhead points to a 37 kDa dark bands, which reflected the absence of denatured salmon sperm DNA due to DNase activity. (c) Caspase-3-like activity in cytosolic fraction. Co, cell-free system without treatment. Co + f, cell-free system plus 1 μM Cyt f. Co + f + MG-132, cell-free system plush 1 μM Cyt f and 10 μM MG-132. Co + f + Ac-DEVD-CHO, cell-free system plus 1 μM Cyt f and 100 μM Ac-DEVD-CHO. RFU/mg, Relative fluorescence units per mg protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4125987&req=5

f1: Induction of Cyt f PCD hallmarks in a cell-free system.(a) DNA laddering and (b) endonuclease activity in the nuclear extracts, which were induced by different concentrations of Cyt f. M, marker; N, isolated nucleus; C, cell-free system including nucleus and cytosolic fraction; C + f1, cell-free system plus 0.2 μM Cyt f; C + f2, cell-free system plus 1 μM Cyt f. Arrowhead points to a 37 kDa dark bands, which reflected the absence of denatured salmon sperm DNA due to DNase activity. (c) Caspase-3-like activity in cytosolic fraction. Co, cell-free system without treatment. Co + f, cell-free system plus 1 μM Cyt f. Co + f + MG-132, cell-free system plush 1 μM Cyt f and 10 μM MG-132. Co + f + Ac-DEVD-CHO, cell-free system plus 1 μM Cyt f and 100 μM Ac-DEVD-CHO. RFU/mg, Relative fluorescence units per mg protein.

Mentions: Degradation of the thylakoid membrane and photosynthetic complexes has been demonstrated in dark-induced leaf senescence in rice16. In our previous study, we also found that Cyt f was released to the cytoplasm prior to PCD in senescent rice leaves (data not shown). As Cyt f is released in senescent tissue prior to death it is of interest to evaluate if it could be involved in the actual induction of the cell death program. It has been known that DNA laddering is an important biochemical hallmark in plant PCD. In order to study the effect of Cyt f on DNA fragmentation, we established a cell-free system consisting of cytosolic fraction and nuclei from rice suspension cells. Purified Cyt f was incubated in the cell-free system, and DNA and proteins were extracted from the nuclei. Compared to a cell-free system only, Cyt f induced DNA laddering in nuclei (Fig. 1A). The nuclear protein extracts were further subjected to SDS-PAGE for the detection of OsNuc37 (Oryza sativa nuclear endonucleases of 37 kDa) activity, which was responsible for DNA fragmentation in rice2. As shown in Fig. 1B, Cyt f also induced OsNuc37 activity, which was identified as 37 kDa dark bands reflecting the absence of denatured salmon sperm DNA, indicating that Cyt f could induce DNA fragmentation through activating OsNuc37. Since nuclear alteration is one of the later stage events in the PCD process, we asked whether this event is dependent on the caspase-like proteases, and detected caspase-3-like activity in the cytosolic fractions of the cell-free system. As shown in Fig. 1C, exogenous Cyt f activated a DEVDase activity, and this activity could be inhibited by the caspase-3 specific inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO). Interestingly, it was also inhibited by MG132, the specific 26S proteasome inhibitor, which indicated that Cyt f-induced cytosolic DEVDase activity was related to 26S proteasome 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)

Induction of Cyt f PCD hallmarks in a cell-free system.(a) DNA laddering and (b) endonuclease activity in the nuclear extracts, which were induced by different concentrations of Cyt f. M, marker; N, isolated nucleus; C, cell-free system including nucleus and cytosolic fraction; C + f1, cell-free system plus 0.2 μM Cyt f; C + f2, cell-free system plus 1 μM Cyt f. Arrowhead points to a 37 kDa dark bands, which reflected the absence of denatured salmon sperm DNA due to DNase activity. (c) Caspase-3-like activity in cytosolic fraction. Co, cell-free system without treatment. Co + f, cell-free system plus 1 μM Cyt f. Co + f + MG-132, cell-free system plush 1 μM Cyt f and 10 μM MG-132. Co + f + Ac-DEVD-CHO, cell-free system plus 1 μM Cyt f and 100 μM Ac-DEVD-CHO. RFU/mg, Relative fluorescence units per mg protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f1: Induction of Cyt f PCD hallmarks in a cell-free system.(a) DNA laddering and (b) endonuclease activity in the nuclear extracts, which were induced by different concentrations of Cyt f. M, marker; N, isolated nucleus; C, cell-free system including nucleus and cytosolic fraction; C + f1, cell-free system plus 0.2 μM Cyt f; C + f2, cell-free system plus 1 μM Cyt f. Arrowhead points to a 37 kDa dark bands, which reflected the absence of denatured salmon sperm DNA due to DNase activity. (c) Caspase-3-like activity in cytosolic fraction. Co, cell-free system without treatment. Co + f, cell-free system plus 1 μM Cyt f. Co + f + MG-132, cell-free system plush 1 μM Cyt f and 10 μM MG-132. Co + f + Ac-DEVD-CHO, cell-free system plus 1 μM Cyt f and 100 μM Ac-DEVD-CHO. RFU/mg, Relative fluorescence units per mg protein.
Mentions: Degradation of the thylakoid membrane and photosynthetic complexes has been demonstrated in dark-induced leaf senescence in rice16. In our previous study, we also found that Cyt f was released to the cytoplasm prior to PCD in senescent rice leaves (data not shown). As Cyt f is released in senescent tissue prior to death it is of interest to evaluate if it could be involved in the actual induction of the cell death program. It has been known that DNA laddering is an important biochemical hallmark in plant PCD. In order to study the effect of Cyt f on DNA fragmentation, we established a cell-free system consisting of cytosolic fraction and nuclei from rice suspension cells. Purified Cyt f was incubated in the cell-free system, and DNA and proteins were extracted from the nuclei. Compared to a cell-free system only, Cyt f induced DNA laddering in nuclei (Fig. 1A). The nuclear protein extracts were further subjected to SDS-PAGE for the detection of OsNuc37 (Oryza sativa nuclear endonucleases of 37 kDa) activity, which was responsible for DNA fragmentation in rice2. As shown in Fig. 1B, Cyt f also induced OsNuc37 activity, which was identified as 37 kDa dark bands reflecting the absence of denatured salmon sperm DNA, indicating that Cyt f could induce DNA fragmentation through activating OsNuc37. Since nuclear alteration is one of the later stage events in the PCD process, we asked whether this event is dependent on the caspase-like proteases, and detected caspase-3-like activity in the cytosolic fractions of the cell-free system. As shown in Fig. 1C, exogenous Cyt f activated a DEVDase activity, and this activity could be inhibited by the caspase-3 specific inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO). Interestingly, it was also inhibited by MG132, the specific 26S proteasome inhibitor, which indicated that Cyt f-induced cytosolic DEVDase activity was related to 26S proteasome 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