Search

SUMMARYThe functionality of essential metabolic processes in chloroplasts depends on a balanced integration of nuclear-and chloroplast-encoded polypeptides into the plastid’s proteome. The chloroplast chaperonin machinery is an essential player in chloroplast protein folding with a more intricate structure and subunit composition compared to the orthologous GroEL/ES chaperonin ofEscherichia coli. However, its exact role in chloroplasts remains obscure, mainly because of a very limited knowledge about the folded substrates. We employed the competition immunoprecipitation method for the identification of the chaperonin’s substrates inChlamydomonas reinhardtii. Co-immunoprecipitation of the target complex in the presence of increasing amounts of isotope-labelled competitor epitope and subsequent mass spectrometry analysis specifically allowed to distinguish true interactors from unspecifically co-precipitated proteins. Besides known substrates such as RbcL, we revealed numerous new substrates with high confidence. Identified substrate proteins differ from bulk chloroplast proteins by a higher content of beta-sheets, lower alpha-helical content and increased aggregation propensity. Immunoprecipitations performed with a subunit of the co-chaperonin lid revealed the ClpP protease as a specific partner complex, with altered interactions during heat stress, pointing to a close collaboration of these machineries to maintain protein homeostasis in the chloroplast.

As part of the BioHackathon Germany 2022, we hereby report on the success of the two projects “MIAPPE Wizard: Enabling easy creation of MIAPPE-compliant ISA metadata for Plant Phenotyping Experiments” and “DataPLANT - Facilitating Research Data Management to combat the reproducibility crisis”. Shortly before the actual hackathon, it became apparent to the participants that close coordination between the projects would be very beneficial. Both projects aimed to improve the process of collecting and aggregating metadata on plant experiments, but with different approaches.

Background Cold stress causes dynamic changes in gene expression that are partially caused by small non-coding RNAs since they regulate protein coding transcripts and act in epigenetic gene silencing pathways. Thus, a detailed analysis of transcriptional changes of small RNAs (sRNAs) belonging to all known sRNA classes such as microRNAs (miRNA) and small interfering RNA (siRNAs) in response to cold contributes to an understanding of cold-related transcriptome changes. Result We subjected A. thaliana plants to cold acclimation conditions (4 °C) and analyzed the sRNA transcriptomes after 3 h, 6 h and 2 d. We found 93 cold responsive differentially expressed miRNAs and only 14 of these were previously shown to be cold responsive. We performed miRNA target prediction for all differentially expressed miRNAs and a GO analysis revealed the overrepresentation of miRNA-targeted transcripts that code for proteins acting in transcriptional regulation. We also identified a large number of differentially expressed cis- and trans-nat-siRNAs, as well as sRNAs that are derived from long non-coding RNAs. By combining the results of sRNA and mRNA profiling with miRNA target predictions and publicly available information on transcription factors, we reconstructed a cold-specific, miRNA and transcription factor dependent gene regulatory network. We verified the validity of links in the network by testing its ability to predict target gene expression under cold acclimation. Conclusion In A. thaliana, miRNAs and sRNAs derived from cis- and trans-NAT gene pairs and sRNAs derived from lncRNAs play an important role in regulating gene expression in cold acclimation conditions. This study provides a fundamental database to deepen our knowledge and understanding of regulatory networks in cold acclimation.