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bioRxiv · 10.64898/2026.08.05.743032

The chloroplast CLP chaperone-protease system controls the steady-state abundance of the singlet oxygen sensors EXCUTER 1 and 2

Ravenburg, C. M.·Routray, P.·Bouchnak, I.·Yuan, B.·Julkowska, M. M.·van Wijk, K. J.
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Abstract

O_LIThe chloroplast CLP chaperone-protease is essential for chloroplast biogenesis. CLP substrate selection is aided by the N-recognin CLPS1 and CLPF adaptors. They interact with each other and the CLPC1 chaperone, but their specific functions are poorly understood. C_LIO_LIWe employed in vivo CLPC1 substrate-trapping in Arabidopsis by expressing a 35S:CLPC1-TRAP-STREPII transgene in wild-type (WT), clpf, clps1, and clpfclps1 to test the consequences of the loss of these adaptors on CLPC1-trapped proteins. Immunoblotting and protein half-life experiments were carried out for identified CLP substrates. C_LIO_LIExpression of the 35S:CLPC1-TRAP-STREPII in clps1cpf was embryo lethal. CLPF was trapped at a reduced level in clps1, supporting CLPS-CLPF interactions. Chloroplast 1O2 sensor EXECUTER1 (EX1) was trapped in WT and clps1 but not significantly in clpf. Steady-state protein accumulation of EX1 and its homolog EX2 increased 30-fold in the CLPC1-TRAP lines and clpr2-1, but not in clpf or clps1. In planta experiments showed that the half-life of EX1 is [~]3-fold longer in clpc1-1 than in WT, but EX1 half-life was unaffected in clpf. C_LIO_LIWe conclude that the CLP system plays a key role in EX1,2 homeostasis by keeping their intra-chloroplast concentrations low through continuous degradation, upstream of their 1O2 signaling function. C_LI

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Ravenburg, C. M., Routray, P., Bouchnak, I., Yuan, B., Julkowska, M. M., van Wijk, K. J.. 2026-08-06. The chloroplast CLP chaperone-protease system controls the steady-state abundance of the singlet oxygen sensors EXCUTER 1 and 2. https://doi.org/10.64898/2026.08.05.743032

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