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Singlet oxygen- and EXECUTER1-mediated signaling is initiated in grana margins and depends on the protease FtsH2
Saturday, 2016/07/02 | 03:27:26

Liangsheng Wang, Chanhong Kim, Xia Xu, Urszula Piskurewicz, Vivek Dogra, Somesh Singh, Hanno Mahler, and Klaus Apel

Significance

Singlet oxygen (1O2)- and EXECUTER1 (EX1)-dependent signaling triggers programmed cell death in seedlings and inhibits growth of mature plants of the fluorescent (flu) mutant of Arabidopsis. The EX1 protein has been located in chloroplasts to the grana margins close to where chlorophyll is synthesized and the disassembly of damaged photosystem II (PSII) and reassembly of active PSII take place. With the onset of 1O2-mediated signaling there is a rapid decline of EX1 that depends on the ATP-dependent zinc metalloprotease FtsH. Generation of 1O2 without the decline of EX1 is not sufficient to activate 1O2 signaling. As FtsH cleaves also the D1 reaction center protein of damaged PSII, EX1-dependent signaling seems not only spatially but also functionally linked to the repair of PSII.

Abstract

Formation of singlet oxygen (1O2) has been implicated with damaging photosystem II (PSII) that needs to undergo continuous repair to maintain photosynthetic electron transport. In addition to its damaging effect, 1O2 has also been shown to act as a signal that triggers stress acclimation and an enhanced stress resistance. A signaling role of 1O2 was first documented in the fluorescent (flu) mutant of Arabidopsis. It strictly depends on the chloroplast protein EXECUTER1 (EX1) and happens under nonphotoinhibitory light conditions. Under severe light stress, signaling is initiated independently of EX1 by 1O2 that is thought to be generated at the acceptor side of active PSII within the core of grana stacks. The results of the present study suggest a second source of 1O2 formation in grana margins close to the site of chlorophyll synthesis where EX1 is localized and the disassembly of damaged and reassembly of active PSII take place. The initiation of 1O2 signaling in grana margins depends on EX1 and the ATP-dependent zinc metalloprotease FtsH. As FtsH cleaves also the D1 protein during the disassembly of damaged PSII, EX1- and 1O2-mediated signaling seems to be not only spatially but also functionally associated with the repair of PSII.

 

See http://www.pnas.org/content/113/26/E3792.full

PNAS June 28 2016; vol.113; no. 26: E3792–E3800

 

Fig. 1. The biological activity and localization of EX1-FLAG. (A) Complementation of flu/ex1 with a genomic DNA construct that encodes EX1 fused to the FLAG-tag (gEX1-Flag) under control of the native EX1 promoter (pEX1). WT, flu, flu/ex1, and the complemented flu/ex1 line (pEX1::gEX1-Flag/flu/ex1) were grown for 13 d under continuous light and either kept for another 32 h under continuous light (14 dL) or transferred to the dark for 8 h and re-exposed to light for 24 h (13 dL/8 hD/24 hL). Singlet oxygen-mediated growth inhibition and photoinhibition of PSII as revealed by transient Chl fluorescence changes in flu are suppressed in flu/ex1 and restored in the complemented flu/ex1 line. R.F.U.: Relative Fluorescence Unit. (B) Singlet oxygen-mediated changes of the maximum efficiency of PSII (Fv/Fm). Seedlings were grown for 5 d under continuous light, shifted to the dark for 8 h, and re-exposed to light for various lengths of time. PSII activity is inhibited in flu and the complemented flu/ex1 but not in flu/ex1 and WT, indicating that 1O2-mediated and EX1-dependent signaling occurs under nonphotoinhibitory light and 1O2 generated in seedlings with a flu background does not directly damage PSII. The results represent the mean and SD of Fv/Fm measurements of at least 30 individual seedlings. (C) Localization of EX1-FLAG in chloroplast membranes. Chloroplasts (Chloro) isolated from flu/ex1 complemented with pEX1::gEX1-Flag (+) and noncomplemented flu/ex1 control lines (−) were lysed and separated by centrifugation into membrane (TM) and stroma (Stroma) fractions. Proteins were solubilized, separated by SDS/PAGE, and analyzed on Western blots using antisera against FLAG, LHCP, and the large subunit of ribulose-1,5-bisphospate carboxylase (RbcL). (D) Localization of EX1-FLAG in grana stacks that retain their margin parts. Chloroplast membranes were treated with the detergent digitonin and separated by differential centrifugation into three subfractions that were pelleted at 10,000 × g, 40,000 × g and 144,000 × g. Proteins of these subfractions were analyzed by immunoblotting as shown in C, and the PSII RC proteins D1 and D2, the PSI-specific PsaF, ATPase, FtsH2, and the margin-specific CURT1 were used as markers to locate EX1-FLAG. Colocalization of EX1-FLAG and CURT1 indicates that EX1 is highly enriched in grana stacks that retain their margin parts. Cytochrome f (Cytf) was used as a loading control. (E) Localization of EX1-FLAG in grana margins. Detergent-treated chloroplast membranes were separated into grana core (GC), grana margins (GM), and stroma lamellae (SL). The same marker proteins as shown in D were used to locate EX1-FLAG. The various subfractions were also characterized by their Chl a/b ratios shown below D and E.

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