Research High-level expression of a novel chromoplast phosphate transporter ClPHT4;2 is required for flesh color development in watermelon Jie Zhang, Shaogui Guo, Yi Ren, Haiying Zhang, Guoyi Gong, Ming Zhou, Guizhang Wang, Mei Zong, Hongju He, Fan Liu and Yong Xu National Engineering Research Center for Vegetables, Beijing Academy of Agriculture and Forestry Sciences, Key Laboratory of Biology and Genetic Improvement of Horticultural Crops (North China), Beijing Key Laboratory of Vegetable Germplasm Improvement, Beijing 100097, China Summary Author for correspondence: Chromoplast development plays a crucial role in controlling carotenoid content in water- Yong Xu melon flesh. Modern cultivated watermelons with colorful flesh are believed to originate from Tel: +86 10 51503199 pale-colored and no-sweet progenitors. But the molecular basis of flesh color formation and Email: [email protected] regulation is poorly understood. Received: 23 May 2016 More chromoplasts and released carotenoid globules were observed in the red-fleshed fruit Accepted: 11 September 2016 of the 97103 cultivar than in the pale-colored fruits of the PI296341-FR line. Transcriptome profiles of these two materials identified Cla017962, predicted as ClPHT4;2, was dramatically New Phytologist (2016) up-regulated during flesh color formation. High ClPHT4;2 expression levels were closely cor- doi: 10.1111/nph.14257 related with increased flesh carotenoid contents among 198 representative watermelon accessions. Down-regulation of ClPHT4;2 expression in transgenic watermelons reduced the Key words: chromoplast, Citrullus lanatus, fruit carotenoid accumulation. flesh color, phosphate transporter, transcrip- ClPHT4;2 as a function of chromoplast-localized phosophate transporter was tested by tional regulation. heterologous expression into a yeast phosphate-uptake-defective mutant, western blotting, subcellular localization, and immunogold electron microscopy analysis. Two transcription fac- tors, ClbZIP1 and ClbZIP2, were identified, which responded to ABA and sugar signaling to regulate ClPHT4;2 transcription only in cultivated watermelon species. Our findings suggest that elevated ClPHT4;2 gene expression is necessary for carotenoid accumulation, and may help to characterize the co-development of flesh color and sweetness during watermelon development and domestication. Introduction semiwild watermelon accessions of C. lanatus ssp. mucosospermus produce a wide range of flesh colors and sugar contents (Jeffrey, Fruit flesh color is an important nutritional and sensory quality 2001; Guo et al., 2013). There are different opinions regarding that has received considerable attention from breeders and con- the origin of the genus Citrullus and the nomenclature used to sumers. Carotenogenesis in chromoplast contributes to the fruit describe them (Chomicki & Renner, 2015; Paris, 2015). How- flesh color formation (Li & Yuan, 2013). Watermelon (Citrullus ever, it is generally accepted that, during the domestication pro- lanatus), as one of the five most consumed fresh fruits grown cess, the pigmentation of watermelon flesh increased. Differences worldwide, possesses colorful flesh and provides abundant in fruit flesh color development between cultivated and wild carotenoids to worldwide consumers (Guo et al., 2013). Different watermelons make these species ideal for comparative genetic composition and concentration of carotenoids contribute to the analyses to reveal the underlying mechanisms of flesh color for- red, orange, canary yellow, salon yellow and white flesh color in mation in fruit development. cultivated watermelons (Henderson et al., 1998). As one of the Chromoplasts use unique mechanisms to synthesize and few species that accumulate a large amount of lycopene in fruits, deposit large amounts of carotenoids (Li & Yuan, 2013; Yuan red-fleshed watermelon contains more lycopene per unit FW et al., 2015). Previous studies have been devoted to clarifying the than fresh tomatoes (Solanum lycopersicum) (Collins et al., 2006). carotenoid biosynthetic pathway in watermelon (Bang et al., Compared with the colorful flesh of modern cultivated watermel- 2007; Kang et al., 2010). Amino acid substitutions of lycopene ons (C. lanatus ssp. vulgaris), fruits of the wild accessions b-cyclase were revealed to be responsible for the accumulation of C. lanatus ssp. lanatus, have pale-colored flesh that is not sweet. lycopene or b-carotene in chromoplasts, resulting in the red or They occur naturally in southern Africa. Additionally, the canary yellow color in flesh (Bang et al., 2007). In contrast to the Ó 2016 The Authors New Phytologist (2016) 1 New Phytologist Ó 2016 New Phytologist Trust www.newphytologist.com New 2 Research Phytologist significant advances made in understanding of carotenoid and stability (Wiese et al., 2004). Glucose can regulate ABA metabolism (Nisar et al., 2015), very little is known about the biosynthesis and activate some important transcriptional factors, biogenesis and regulation of chromoplasts. As nonphotosynthetic such as ABI3 and ABI5, to regulate plant growth (Cheng et al., plastids in ripening fruit, chromoplasts are bound by double 2002; Rolland et al., 2006). However, there is little information membranes. The inner membrane functions as the primary per- about the key regulators and the related cis-elements controlling meability barrier where several proteins help transport substrates watermelon flesh color development. between the plastids and the cytosol (Egea et al., 2010). In this study, we observed that high expression of the newly Researchers have recently focused on the transport processes characterized chromoplast Pi transporter gene ClPHT4;2 is nec- across both the inner and outer envelope membranes of plastids essary in all of the colored-flesh watermelon accessions. Two and their role in connecting the metabolism of plastids with that bZIP transcription factors, ClbZIP1 and ClbZIP2, associated of the other parts of a plant cell (Fischer, 2011; Flugge€ et al., with ABA and sugar signaling pathways regulate ClPHT4;2 tran- 2011). Proteome-level analyses of chromoplast revealed an abun- scription in the analyzed red-fleshed watermelon cultivar. dance of proteins related to ATP and hexose phosphate produc- tion and transport, suggesting that anabolic processes are Materials and Methods important for chromoplast development (Quick & Neuhaus, 1996; Wang et al., 2013). Activated anabolic processes in chro- Plant materials moplasts would lead to the excessive accumulation of inorganic phosphate (Pi) if not balanced by a Pi export activity. Many plant The red-fleshed Citrullus lanatus (Thunb.) Matsum. & Nakai ssp. Pi transporters have been identified and classified into distinct vulgaris cultivar 97103 is a typical early-maturing East Asian culti- families, including PHT1-5 and plastidic Pi translocator (pPT) var with sweet, red and crispy flesh. The pale-fleshed C. lanatus (Rausch & Bucher, 2002; Liu et al., 2016). The pPT, PHT2, ssp. lanatus watermelon line PI296341-FR (also called C. lanatus and PHT4 families are involved in plastid-localized Pi transport var. citroides) is a wild watermelon with a round shape, medium systems (Guo et al., 2008). In Arabidopsis thaliana, PHT4;2 is a size, thick and hard rind, and light-green striped fruit with non- sink-specific plastidic phosphate transporter (Irigoyen et al., sweet and pale-colored (white) flesh. Roots, leaves, stems, female 2011). However, which PHT family member is involved in chro- flowers, flowers, and fruits at four critical developmental stages moplast development and carotenoid accumulation is unclear. (immature white (10 d after pollination, DAP), white-pink (18 A transcriptome analysis of watermelon fruits at different devel- DAP), red (26 DAP) and overripe (34 DAP)) were examined in opmental stages may help researchers identify genes influencing 97103. The development of PI296341-FR fruit was divided into fruit color formation. We have published our findings regarding six stages (10, 18, 26, 34, 42 and 50 DAP) for subsequent analysis. global transcriptome profiles of fruit development in the red- Fruits from 96 recombinant inbred lines (RILs) derived from a fleshed cultivar 97103 (C. lanatus ssp. vulgaris) and in the pale- cross between 97103 and PI296341-FR and of 102 representative colored (white) line PI296341-FR (C. lanatus ssp. lanatus) (Guo watermelon accessions, including 61 C. lanatus ssp. vulgaris lines, et al., 2011, 2015). We reported that the carotenoid biosynthetic 12 C. lanatus ssp. lanatus accessions and 29 C. lanatus ssp. pathway is activated in red-fleshed fruits. Phytoene synthase (PSY) mucosospermus (also called C. lanatus var. eguis) accessions, were and phytoene desaturase (PDS) in this pathway are up-regulated analyzed at 38 DAP. The watermelon juice sugar content (soluble during red-fleshed watermelon fruit development. Additionally, solid content, °Brix) was measured using a pocket refractometer several of the differentially expressed genes in 97103 are associated PAL-1 (Atago Co. Ltd, Tokyo, Japan) from a sample of juice that with transporting ions and small molecules (Guo et al., 2015). was collected from the center of each watermelon. These differentially expressed genes are probably regulated by spe- cial transcription factors (TFs), and their upstream regulatory Carotenoid extraction and analysis regions are important for mediating transcriptional activities. Numerous studies have been conducted on phytohormone- Carotenoids from watermelon flesh (0.5 g FW) were extracted related TFs and