Abstract
<jats:title>Summary</jats:title> <jats:p> <jats:list list-type="bullet"> <jats:list-item> <jats:p>The 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.</jats:p> </jats:list-item> <jats:list-item> <jats:p> We employed <jats:italic>in vivo</jats:italic> CLPC1 substrate-trapping in Arabidopsis by expressing a <jats:italic>35S:CLPC1-TRAP-STREPII</jats:italic> transgene in wild-type (WT), <jats:italic>clpf, clps1,</jats:italic> and <jats:italic>clpfclps1</jats:italic> 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. </jats:p> </jats:list-item> <jats:list-item> <jats:p> Expression of the <jats:italic>35S:CLPC1-TRAP-STREPII</jats:italic> in <jats:italic>clps1cpf</jats:italic> was embryo lethal. CLPF was trapped at a reduced level in <jats:italic>clps1</jats:italic> , supporting CLPS-CLPF interactions. Chloroplast <jats:sup>1</jats:sup> O <jats:sub>2</jats:sub> sensor EXECUTER1 (EX1) was trapped in WT and <jats:italic>clps1</jats:italic> but not significantly in <jats:italic>clpf</jats:italic> . Steady-state protein accumulation of EX1 and its homolog EX2 increased 30-fold in the CLPC1-TRAP lines and <jats:italic>clpr2-1</jats:italic> , but not in <jats:italic>clpf</jats:italic> or <jats:italic>clps1</jats:italic> . <jats:italic>In planta</jats:italic> experiments showed that the half-life of EX1 is ∼3-fold longer in <jats:italic>clpc1-1</jats:italic> than in WT, but EX1 half-life was unaffected in <jats:italic>clpf</jats:italic> . </jats:p> </jats:list-item> <jats:list-item> <jats:p> We 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 <jats:sup>1</jats:sup> O <jats:sub>2</jats:sub> signaling function. </jats:p> </jats:list-item> </jats:list> </jats:p>