Updated project metadata. In the present study, a differential screening following heat stress acclimation was performed in A. thaliana WT and ROF-FKBP mutated plants, using mass spectrometry, and the results were used to understand and analyze the effect of the ROF PPIases during thermotolerance acquisition in plants. Our data highlight the central role of these two PPIases in heat stress and point to their direct or indirect effect on other proteins participating in cellular functions such as protein folding and quality control, cell division, photosynthesis, and other metabolic and signaling processes. 1) Specifically, the heat stress response, protein folding and protein ER processing pathways are enhanced following a 37 oC acclimation period independently to the mutation state. 2) However, at 37 oC and in the double mutated rof1–/2 –plants, a higher accumulation of proteins belonging to the above pathways is observed compared to all other conditions (WT, mutants, control and heat stressed). 3) Furthermore, the proteasomal pathway including the common member of both the protasomal and lysosomal degradation pathway CDC48, is over-represented in the extracts of both untreated and heat stressed rof1–/2 – mutants compared to the other extracts. 4) In contrast, in the single rof1 – mutation the heat acclimation (including ROF2), protein folding and proteasomal pathways are significantly suppressed compared to the WT, at 37 oC. 5) Protein accumulation related to the heat stress and protein quality control pathways follow the rof1–/2 – > rof2 – > rof1 – pattern suggesting a differential but also synergistic role of the two proteins. 6) Protein complexes of other biochemical pathways such as the Lhc of the photosynthetic pathway and the phosphoinositide binding proteins involved in membrane-trafficking events during cell plate formation and cytokinesis (Patellin-1, 2 and 3) are negatively regulated by the rof1–/2 – mutants. Our results suggest that ROF1 and ROF2 FKBPs regulate stress response, developmental and metabolic pathways via a complex feedback mechanism that ensures the continuation of protein quality control at their absence.