Rosano, G. L. | Search bioRxiv

bioRxiv2025

A canonical chloroplast unfolded protein response triggered by misfolded polypeptides

Photosynthetic efficiency and plant viability rely on chloroplast protein homeostasis. While unfolded protein responses (UPRs) in the endoplasmic reticulum and mitochondria have been extensively characterized, the chloroplast UPR (cpUPR) remains less defined, partly due to the off-target effects of traditional stress-inducing methods. In this study, we provide direct evidence for the existence of the cpUPR by expressing engineered, folding-defective variants of ferredoxin-NADP reductase (FNR) in plant chloroplasts. The expression of aggregation-prone proteins inside the organelle triggered a robust upregulation of chloroplast quality control components, including CLPB3, CLPC1/C2, and HSP90C, as revealed by immunodetection and quantitative proteomics. The proteomic response scaled with the severity of the folding defect, with the fully insoluble FNR {Delta}20 variant inducing broader changes than the partially soluble {Delta}3 variant. Network analysis showed that most differentially abundant proteins were chloroplastic and clustered into functional groups related to proteostasis and photosynthesis. Comparative analysis with lincomycin-treated plants highlighted the specificity and advantages of using misfolded proteins to study the cpUPR. Expression of the folding variants conferred tolerance to heat treatment, suggesting that the response can enhance plant fitness. Taken together, our work establishes the cpUPR as a specific stress response in chloroplasts and provides new tools for its characterization.

plant biology↗

bioRxiv2021

Arabidopsis Mediator subunit 17 connects transcription with DNA repair after UV-B exposure

Mediator 17 (MED17) is a subunit of the Mediator complex that regulates transcription initiation in eukaryotic organisms. In yeast and humans, MED17 also participates in DNA repair, physically interacting with proteins of the Nucleotide Excision DNA Repair system. We here analyzed the role of MED17 in Arabidopsis plants exposed to UV-B radiation, which role has not been previously described. Comparison of med17 mutant transcriptome to that of WT plants showed that almost one third of transcripts with altered expression in med17 plants are also changed by UV-B exposure in WT plants. To validate the role of MED17 in UV-B irradiated plants, plant responses to UV-B were analyzed, including flowering time, DNA damage accumulation and programmed cell death in the meristematic cells of the root tips. Our results show that med17 and OE MED17 plants have altered responses to UV-B; and that MED17 participates in various aspects of the DNA damage response (DDR). Increased sensitivity to DDR after UV-B in med17 plants can be due to altered regulation of UV-B responsive transcripts; but additionally MED17 physically interacts with DNA repair proteins, suggesting a direct role of this Mediator subunit during repair. Finally, we here also show that MED17 is necessary to regulate the DDR activated by ATR, and that PDCD5 overexpression reverts the deficiencies in DDR shown in med17 mutants. Together, the data presented demonstrates that MED17 is an important regulator of the DDR after UV-B radiation in Arabidopsis plants. One sentence summaryIn Arabidopsis, MED17 regulates the DNA damage response after UV-B exposure transcriptionally modulating the expression of genes and possibly also physically interacting with DNA repair proteins.

plant biology↗