Time-resolved inducible knock-down of Clp protease subunits in tobacco identifies putative protease substrates

ABSTRACT: The Clp protease complex in plants is the one with the highest complexity through all life kingdoms. It is composed of 14 nuclear- and plastid-encoded subunits which are allocated in different rings across the complex. The proteolytic ring (P-ring; ClpP3-ClpP6) and non-proteolytic ring (R-ring; ClpR1-ClpR4 and ClpP1) form the core complex playing an essential role in protein degradation and complex stabilization, respectively. The chaperones, ClpC1, ClpC2 and ClpD are involved in substrate folding and unfolding while accessory proteins, ClpT1 and ClpT2, are important to stabilize the core complex assembly. Adaptor proteins of the complex, ClpS and ClpF, recognize protease targets. Finding substrates of proteases is one of the remaining open questions in the protease field and specifically in the Clp protease. Several attempts were made analyzing the change of protein abundance in null clp mutants by proteomic analysis. However, null clp mutants are associated with massive accumulation of pleiotropic effects, as for example impaired growth and delayed in plant development, making impossible the detection of potential Clp substrates. To address this question, we design an ethanol (EtOH) inducible strategy to silence the expression of several subunits of the Clp protease in tobacco, and follow the change in protein abundance through the EtOH time-course. In this way, we distinguish between primary and secondary effects and determine time-resolved changes in proteins stability. Time-resolved proteomic analysechloroplast processes affected by the repression of Clp activity (e.g., photosynthesis and protein homeostasis).

INSTRUMENT(S): Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Leaf

SUBMITTER: Silvia Martinez Jaime  

LAB HEAD: Ralph Bock

PROVIDER: PXD008305 | Pride | 2022-02-22

REPOSITORIES: Pride