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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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Screening for the putative interacting proteins of Cyt f in Arabidopsis cDNA library through the Y2H.C4-32 represents different clones in the cDNA library, and the mRNA information in these clones is shown in Table 1. PC, positive control; NC, negative control.
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f7: Screening for the putative interacting proteins of Cyt f in Arabidopsis cDNA library through the Y2H.C4-32 represents different clones in the cDNA library, and the mRNA information in these clones is shown in Table 1. PC, positive control; NC, negative control.

Mentions: In order to reveal the mechanisms of the activation of caspase-3-like activity by Cyt f, a yeast two-hybrid system (Y2H) was used to screen the possible Cyt f-interacting proteins in the cytoplasm. The C-terminus lacking Cyt f was fused to the GAL4 DNA-binding domain (BD) in pGBKT7, and used as a bait to screen the cDNA library of Arabidopsis. We got five mRNAs as putative Cyt f-interacting proteins, and all of them are PCD-related (Fig. 7 and Table 1). Among these putative interacting proteins, RPN9b and RING-H2 finger protein ATL72 (E3-ubiquitin ligase) are the subunits of the Ubiquitin-Proteasome-System (UPS), and CDPK32 might be involved in the phosphorylation of UPS subunits (Table 1). Combined with the results that Cyt f activated caspase-3-like activity could be inhibited by MG132 (Fig. 1C), our results suggested that the 26S proteasome might be responsible for the caspase-3-like activity in rice, and that Cyt f induced this activity in cytoplasm by interacting with the regulatory and/or catalytical subunits of UPS.


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)

Screening for the putative interacting proteins of Cyt f in Arabidopsis cDNA library through the Y2H.C4-32 represents different clones in the cDNA library, and the mRNA information in these clones is shown in Table 1. PC, positive control; NC, negative control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f7: Screening for the putative interacting proteins of Cyt f in Arabidopsis cDNA library through the Y2H.C4-32 represents different clones in the cDNA library, and the mRNA information in these clones is shown in Table 1. PC, positive control; NC, negative control.
Mentions: In order to reveal the mechanisms of the activation of caspase-3-like activity by Cyt f, a yeast two-hybrid system (Y2H) was used to screen the possible Cyt f-interacting proteins in the cytoplasm. The C-terminus lacking Cyt f was fused to the GAL4 DNA-binding domain (BD) in pGBKT7, and used as a bait to screen the cDNA library of Arabidopsis. We got five mRNAs as putative Cyt f-interacting proteins, and all of them are PCD-related (Fig. 7 and Table 1). Among these putative interacting proteins, RPN9b and RING-H2 finger protein ATL72 (E3-ubiquitin ligase) are the subunits of the Ubiquitin-Proteasome-System (UPS), and CDPK32 might be involved in the phosphorylation of UPS subunits (Table 1). Combined with the results that Cyt f activated caspase-3-like activity could be inhibited by MG132 (Fig. 1C), our results suggested that the 26S proteasome might be responsible for the caspase-3-like activity in rice, and that Cyt f induced this activity in cytoplasm by interacting with the regulatory and/or catalytical subunits of UPS.

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