Crosstalk between the chloroplast protein import and SUMO systems revealed through genetic and molecular investigation in Arabidopsis

The chloroplast proteome contains thousands of different proteins that are encoded by the nuclear genome. These proteins are imported into the chloroplast via the action of the TOC translocase and associated downstream systems. Our recent work has revealed that the stability of the TOC complex is dynamically regulated by the ubiquitin-dependent chloroplast-associated protein degradation (CHLORAD) pathway. Here, we demonstrate that the TOC complex is also regulated by the SUMO system. Arabidopsis mutants representing almost the entire SUMO conjugation pathway can partially suppress the phenotype of ppi1, a pale-yellow mutant lacking the Toc33 protein. This suppression is linked to increased abundance of TOC proteins and improvements in chloroplast development. Moreover, data from molecular and biochemical experiments support a model in which the SUMO system directly regulates TOC protein stability. Thus, we have identified a regulatory link between the SUMO system and the chloroplast protein import machinery.

Salt induced programmed cell death in rice: evidence from chloroplast proteome signature

Soil salinity, depending on its intensity, drives a challenged plant either to death, or survival with compromised productivity. On exposure to moderate salinity, plants can often survive by sacrificing some of their cells ‘in target’ following a route called programmed cell death (PCD). In animals, PCD has been well characterised, and involvement of mitochondria in the execution of PCD events has been unequivocally proven. In plants, mechanistic details of the process are still in grey area. Previously, we have shown that in green tissues of rice, for salt induced PCD to occur, the presence of active chloroplasts and light are equally important. In the present work, we have characterised the chloroplast proteome in rice seedlings at 12 and 24 h after salt exposure and before the time point where the signature of PCD was observed. We identified almost 100 proteins from chloroplasts, which were divided in to 11 categories based on the biological functions in which they were involved. Our results concerning the differential expression of chloroplastic proteins revealed involvement of some novel candidates. Moreover, we observed maximum phosphorylation pattern of chloroplastic proteins at an early time point (12 h) of salt exposure.

Methodology: an optimized, high-yield tomato leaf chloroplast isolation and stroma extraction protocol for proteomics analyses and identification of chloroplast co-localizing proteins

Background Chloroplasts are critical organelles that perceive and convey metabolic and stress signals to different cellular components, while remaining the seat of photosynthesis and a metabolic factory. The proteomes of intact leaves, chloroplasts, and suborganellar fractions of plastids have been evaluated in the model plant Arabidopsis, however fewer studies have characterized the proteomes of plastids in crops. Tomato (Solanum lycopersicum) is an important world-wide crop and a model system for the study of wounding, herbivory and fruit ripening. While significant advances have been made in understanding proteome and metabolome changes in fruit ripening, far less is known about the tomato chloroplast proteome or its subcompartments. Results With the long-term goal of understanding chloroplast proteome dynamics in response to stress, we describe a high-yielding method to isolate intact tomato chloroplasts and stromal proteins for proteomic studies. The parameters that limit tomato chloroplast yields were identified and revised to increase yields. Compared to published data, our optimized method increased chloroplast yields by 6.7- and 4.3-fold relative to published spinach and Arabidopsis leaf protocols, respectively; furthermore, tomato stromal protein yields were up to 79-fold higher than Arabidopsis stromal proteins yields. We provide immunoblot evidence for the purity of the stromal proteome isolated using our enhanced methods. In addition, we leverage our nanoliquid chromatography tandem mass spectrometry (nanoLC–MS/MS) data to assess the quality of our stromal proteome. Using strict criteria, proteins detected by 1 peptide spectral match, by one peptide, or were sporadically detected were designated as low-level contaminating proteins. A set of 254 proteins that reproducibly co-isolated with the tomato chloroplast stroma were identified. The subcellular localization, frequency of detection, normalized spectral abundance, and functions of the co-isolating proteins are discussed. Conclusions Our optimized method for chloroplast isolation increased the yields of tomato chloroplasts eightfold enabling the proteomics analysis of the chloroplast stromal proteome. The set of 254 proteins that co-isolate with the chloroplast stroma provides opportunities for developing a better understanding of the extensive and dynamic interactions of chloroplasts with other organelles. These co-isolating proteins also have the potential for expanding our knowledge of proteins that are co-localized in multiple subcellular organelles.

Crosstalk between chloroplast protein import and the SUMO system revealed through genetic and molecular investigation

The chloroplast proteome contains thousands of different proteins that are encoded by the nuclear genome. These proteins are imported into the chloroplast via the action of the TOC translocase and associated downstream systems. Our recent work has revealed that the stability of the TOC complex is dynamically regulated via the ubiquitin-dependent chloroplast-associated protein degradation (CHLORAD) pathway. Here, we demonstrate that the stability of the TOC complex is also regulated by the SUMO system. Arabidopsis mutants representing almost the entire SUMO conjugation pathway can partially suppress the phenotype of ppi1, a pale yellow mutant lacking the Toc33 protein. This suppression is linked to increased stability of TOC proteins and enhanced chloroplast development. In addition, we demonstrate using molecular and biochemical experiments that the SUMO system directly targets TOC proteins. Thus, we have identified a regulatory link between the SUMO system and chloroplast protein import.