A Comprehensive Analysis of Chromoplast Differentiation Reveals Complex Protein Changes Associated with Plastoglobule Biogenesis and Remodeling of Protein Systems in Sweet Orange Flesh

Globular and crystalloid chromoplasts were observed to be region specifically formed in sweet orange (Citrus sinensis) flesh and converted from amyloplasts during fruit maturation, which was associated with the composition of specific carotenoids and the expression of carotenogenic genes. Subsequent isobaric tag for relative and absolute quantitation (iTRAQ)-based quantitative proteomic analyses of purified plastids from the flesh during chromoplast differentiation and senescence identified 1,386 putative plastid-localized proteins, 1,016 of which were quantified by spectral counting. The iTRAQ values reflecting the expression abundance of three identified proteins were validated by immunoblotting. Based on iTRAQ data, chromoplastogenesis appeared to be associated with three major protein expression patterns: (1) marked decrease in abundance of the proteins participating in the translation machinery through ribosome assembly; (2) increase in abundance of the proteins involved in terpenoid biosynthesis (including carotenoids), stress responses (redox, ascorbate, and glutathione), and development; and (3) maintenance of the proteins for signaling and DNA and RNA. Interestingly, a strong increase in abundance of several plastoglobule-localized proteins coincided with the formation of plastoglobules in the chromoplast. The proteomic data also showed that stable functioning of protein import, suppression of ribosome assembly, and accumulation of chromoplast proteases are correlated with the amyloplast-to-chromoplast transition; thus, these processes may play a collective role in chromoplast biogenesis and differentiation. By contrast, the chromoplast senescence process was inferred to be associated with significant increases in stress response and energy supply. In conclusion, this comprehensive proteomic study identified many potentially new plastid-localized proteins and provides insights into the potential developmental and molecular mechanisms underlying chromoplast biogenesis, differentiation, and senescence in sweet orange flesh.Chromoplasts are special organelles with superior ability to synthesize and store massive amounts of carotenoids, bringing vivid red, orange, and yellow colors to many flowers, fruits, and vegetables (Li and Yuan, 2013). Chromoplasts exhibit various morphologies, such as crystalline, globular, tubular, and membranous structures (Egea et al., 2010). The relationship between the architecture and carotenoid composition has been well stated in diverse pepper (Capsicum annuum) and tomato (Solanum lycopersicum) fruits (Kilcrease et al., 2013; Nogueira et al., 2013). Crystalline bodies have been observed in carrot (Daucus carota; Frey-Wyssling and Schwegler, 1965) and tomato (Harris and Spurr, 1969), which predominantly consist of β-carotene and lycopene, respectively. Globular and/or tubular-globular chromoplasts, in which numerous lipid droplets (also named plastoglobules), which act as passive storage compartments for triglycerides, sterol ester, and some pigments, are accumulated, were described for yellow fruits from kiwi (Actinidia deliciosa), papaya (Carica papaya), and mango (Mangifera indica), which contain lutein, β-cryptoxanthin, and β-carotene as the major pigments, respectively (Vasquez-Caicedo et al., 2006; Montefiori et al., 2009; Schweiggert et al., 2011). Carotenoid composition has been reported to be regulated by the expression of carotenogenic genes in the flesh of various citrus fruits differing in their internal colors (Fanciullino et al., 2006, 2008). Chromoplasts are frequently derived from fully developed chloroplasts, as seen during fruit ripening from green to red or yellow fruits in tomato and pepper (Egea et al., 2010). In some cases, chromoplasts also arise from nonphotosynthetic plastids, such as colorless proplastids, leucoplasts, or amyloplasts (Knoth et al., 1986; Schweiggert et al., 2011). To date, most studies on chromoplast differentiation have been focused on the synthesis of carotenoids by combining biochemical and molecular analyses (Cazzonelli and Pogson, 2010; Egea et al., 2010; Bian et al., 2011; Li and Yuan, 2013), and little is known about the molecular mechanisms underlying chromoplast biogenesis (Li and Yuan, 2013).Recently, proteomics has become an efficient tool to study the protein composition of subcellular organelles such as chromoplasts and their dynamic changes during the development of a particular plant organ/tissue. The majority of chromoplast-related studies are concerned with the functions of these organelles in various crops, such as pepper, tomato, watermelon (Citrulis lanatus), carrot, cauliflower (Brassica oleracea), and papaya (Siddique et al., 2006; Wang et al., 2013). However, only a few of such studies addressed the mechanisms underlying plastid differentiation, such as the transition from proplastid to chloroplast in maize (Zea mays; Majeran et al., 2010), from etioplast to chloroplast in pea (Pisum sativum; Kanervo et al., 2008) and rice (Oryza sativa; Kleffmann et al., 2007), and from chloroplast to chromoplast in tomato (Barsan et al., 2012). In tomato, chromoplastogenesis appears to be associated with major metabolic shifts, including a strong decrease in abundance of the proteins involved in light reaction and an increase in terpenoid biosynthesis and stress-response proteins (Barsan et al., 2012). These changes in proteins are in agreement with the structural changes occurring in tomato during fruit ripening, which is characterized by the loss of chlorophyll and the synthesis of colored compounds. Chromoplast differentiation from nonphotosynthetic plastids occurs frequently in a number of plant tissues, such as watermelon flesh and carrot root (Kim et al., 2010; Wang et al., 2013). However, to the best of our knowledge, no large-scale proteomic study for understanding this developmental process has been reported.Citrus is one of the most economically important fruit crops in the world. Different from the model fruit tomato, which represents climacteric fruits, citrus shows nonclimacteric characteristics during fruit maturation. Additionally, citrus fruits exhibit a unique anatomical fruit structure consisting of two major sections, the pericarp and the edible flesh. Considerable progress has been made in the understanding of chromoplast differentiation in the pericarp of citrus fruits (Eilati et al., 1969; Iglesias et al., 2007), which is a process similar to that of tomato and pepper (Egea et al., 2010). However, little is known about the molecular basis of chromoplast differentiation in the edible flesh, even though there is increasing evidence suggesting an essential role of carotenoid synthesis in inducing chromoplast differentiation (Egea et al., 2010; Bian et al., 2011; Li and Yuan, 2013). Recently, we successfully isolated and purified intact chromoplasts containing a large number of plastoglobules from the flesh of sweet orange (Citrus sinensis) fruits at the maturation stage (Zeng et al., 2011). The same method has also been used successfully to isolate plastids from sweet orange flesh in earlier maturation stages (Zeng et al., 2014), thus making comparative and quantitative proteomic analyses of plastid differentiation possible. In this study, we investigated how ultrastructural changes of plastids/chromoplasts during sweet orange fruit maturation might be associated with changes in the composition of carotenoids and the expression of carotenogenic genes in red and yellow flesh of the fruits. Furthermore, we employed the isobaric tag for relative and absolute quantitation (iTRAQ)-based technology to investigate how protein compositional changes might be correlated with metabolic and structural changes in the plastids of sweet orange flesh during their transformation from amyloplasts to chromoplasts.