RESUMEN
Tomato (Solanum lycopersicum) is a model species for studying fruit development, wounding, herbivory, and pathogen attack. Despite tomato's world-wide economic importance and the role of chloroplasts as metabolic hubs and integrators of environmental cues, little is known about the stromal proteome of tomato. Using a high-yielding protocol for chloroplast and stromal protein isolation, MudPIT nano-LC-MS/MS analyses, a robust in-house protein database (the Atlas) for predicting the plastid localization of tomato proteins, and rigorous selection criteria for inclusion/exclusion in the stromal proteome, we identified 1,278 proteins of the tomato stromal proteome. We provide one of the most robust stromal proteomes available to date with empirical evidence for 545 and 92 proteins not previously described for tomato plastids and the Arabidopsis stroma, respectively. The relative abundance of tomato stromal proteins was determined using the exponentially modified protein abundance index (emPAI). Comparison of the abundance of tomato and Arabidopsis stromal proteomes provided evidence for the species-specific nature of stromal protein homeostasis. The manual curation of the tomato stromal proteome classified proteins into ten functional categories resulting in an accessible compendium of tomato chloroplast proteins. After curation, only 91 proteins remained as unknown, uncharacterized or as enzymes with unknown functions. The curation of the tomato stromal proteins also indicated that tomato has a number of paralogous proteins, not present in Arabidopsis, which accumulated to different levels in chloroplasts. As some of these proteins function in key metabolic pathways or in perceiving or transmitting signals critical for plant adaptation to biotic and abiotic stress, these data suggest that tomato may modulate the bidirectional communication between chloroplasts and nuclei in a novel manner. The stromal proteome provides a fertile ground for future mechanistic studies in the field of tomato chloroplast-nuclear signaling and are foundational for our goal of elucidating the dynamics of the stromal proteome controlled by the solanaceous-specific, stromal, and wound-inducible leucine aminopeptidase A of tomato.
RESUMEN
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.
