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.

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Abstract. Natural (e.g., earthquake, flood, wildfires) and human-made (e.g., terrorism, civil strife) disasters are inevitable, can cause extensive disruption, and produce chronic and disabling psychological injuries leading to formal diagnoses (e.g., post-traumatic stress disorder [PTSD]). Following natural disasters of earthquake (Christchurch, Aotearoa/New Zealand, 2010–11) and flood (Calgary, Canada, 2013), controlled research showed statistically and clinically significant reductions in psychological distress for survivors who consumed minerals and vitamins (micronutrients) in the following months. Following a mass shooting in Christchurch (March 15, 2019), where a gunman entered mosques during Friday prayers and killed and injured many people, micronutrients were offered to survivors as a clinical service based on translational science principles and adapted to be culturally appropriate. In this first translational science study in the area of nutrition and disasters, clinical results were reported for 24 clients who completed the Impact of Event Scale – Revised (IES-R), the Depression Anxiety Stress Scales (DASS), and the Modified-Clinical Global Impression (M-CGI-I). The findings clearly replicated prior controlled research. The IES-R Cohen’s d ESs were 1.1 (earthquake), 1.2 (flood), and 1.13 (massacre). Effect sizes (ESs) for the DASS subscales were also consistently positive across all three events. The M-CGI-I identified 58% of the survivors as “responders” (i.e., self-reported as “much” to “very much” improved), in line with those reported in the earthquake (42%) and flood (57%) randomized controlled trials, and PTSD risk reduced from 75% to 17%. Given ease of use and large ESs, this evidence supports the routine use of micronutrients by disaster survivors as part of governmental response.

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