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SUMMARY: The biogenesis and differentiation (B&D) of amyloplasts contributes to fruit flavor and color. Here, remodeling of starch granules, thylakoids and plastoglobules was observed during development and ripening in two kiwifruit (Actinidia spp.) cultivars – yellow‐fleshed 'Hort16A' and green‐fleshed 'Hayward'. A protocol was developed to purify starch‐containing plastids with a high degree of intactness, and amyloplast B&D was studied using label‐free‐based quantitative proteomic analyses in both cultivars. Over 3000 amyloplast‐localized proteins were identified, of which >98% were quantified and defined as the kfALP (kiwifruit amyloplast proteome). The kfALP data were validated by Tandem‐Mass‐Tag (TMT) labeled proteomics in 'Hort16A'. Analysis of the proteomic data across development and ripening revealed: 1) a conserved increase in the abundance of proteins participating in starch synthesis/degradation during both amyloplast B&D; 2) up‐regulation of proteins for chlorophyll degradation and of plastoglobule‐localized proteins associated with chloroplast breakdown and plastoglobule formation during amyloplast differentiation; 3) constitutive expression of proteins involved in ATP supply and protein import during amyloplast B&D. Interestingly, two different pathways of amyloplast B&D were observed in the two cultivars. In 'Hayward', significant increases in abundance of photosynthetic‐ and tetrapyrrole metabolism‐related proteins were observed, but the opposite trend was observed in 'Hort16A'. In conclusion, analysis of the kfALP provides new insights into the potential mechanisms underlying amyloplast B&D with relevance to key fruit quality traits in contrasting kiwifruit cultivars. [ABSTRACT FROM AUTHOR]