ACCUMULATION OF A MISFOLDED PROTEIN IN CHLOROPLASTS TRIGGERS AN ORGANELLE-SPECIFIC RESPONS

CANTOIA, ALEJO; BERROCAL, RODRIGO; BERTERO, FABRICIO; BLANCO, NICOLÁS E.; CECCARELLI, EDUARDO; ROSANO, GERMAN

Mendoza

Congreso; SAIB 2022; 2022

A quality control system of heat shock proteins, chaperones, and proteases ensures that proteins entering the chloroplast are properly folded. Yet, the system may be overloaded in stressful situations and can getoverwhelmed by unfolded proteins. In this case, a genetic program called the unfolded protein response (UPR) is unleashed and consists of increased levels of the components of the quality control network. In a laboratory setting, the UPR can be triggered by chemicals, but it is suspected that the molecular signature of the chemically induced UPR is different from other more physiological stresses. In this work, we used aggregation-prone versions of ferredoxin NADP+ reductase (Taq3-FNR and ΔC-FNR) that show a marked reduction in solubility. Genetic constructs containing the Taq3-FNR, ΔC-FNR, or wild-type FNRcoding sequences were used to transform Nicotiana benthamiana and Arabidopsis thaliana. We confirmed that Taq3-FNR protein accumulates in chloroplasts at a lower concentration than WT-FNR, suggesting increased turnover. The levels of the chloroplastic ClpB3 disaggregase were used as a marker for the UPR. Leaves overexpressing Taq3-FNR and ΔC-FNR showed a significant accumulation of ClpB3. We then analyzed alterations in the leaf proteome by proteomics using a Q-Exactive HF mass spectrometer. Data were analyzed by MaxQuant/Perseus and Proteome Discoverer. We then performed network and GO enrichment analysis using String and Cytoscape. We found that, in the presence of Taq3 and ΔC-FNR, the plant cell responds by activating the expression of molecular chaperones (such as ClpB3), proteases (FtzH), and proteins involved in retrograde signaling to the nucleus (GUN5). Our results suggest that Taq3-FNR and ΔC-FNR generate a specific response in chloroplasts, due to their presence in the stroma. Also, since most proteins with elevated levels during the UPR are nuclear-encoded, the response should involve chloroplasts-to-nucleus communication, a common feature in other UPRs.