Cytological and molecular characterization of carotenoid accumulation in normal and high-lycopene mutant oranges

Abstract

Ripe Cara Cara sweet orange contains 25 times as much carotenoids in flesh as Newhall sweet orange, due to high accumulation of carotenes, mainly phytoene, lycopene and phytofluene. Only yellow globular chromoplasts were observed in Newhall flesh. Distinct yellow globular and red elongated crystalline chromoplasts were found in Cara Cara but only one type of chromoplast was present in each cell. The red crystalline chromoplasts contained lycopene as a dominant carotenoid and were associated with characteristic carotenoid sequestering structures. The increased accumulation of linear carotenes in Cara Cara is not explained by differences in expression of all 18 carotenogenic genes or gene family members examined, or sequence or abundance of mRNAs from phytoene synthase (PSY) and chromoplast-specific lycopene β-cyclase (CYCB) alleles. 2-(4-Chlorophenylthio)-triethylamine hydrochloride (CPTA) enhanced lycopene accumulation and induced occurrence of red crystalline chromoplasts in cultured Newhall juice vesicles, indicating that carotenoid synthesis and accumulation can directly affect chromoplast differentiation and structure. Norflurazon (NFZ) treatment resulted in high accumulation of phytoene and phytofluene in both oranges, and the biosynthetic activity upstream of phytoene desaturase was similar in Newhall and Cara Cara. Possible mechanisms for high carotene accumulation and unique development of red crystalline chromoplasts in Cara Cara are discussed.

Figure 1

Fruit shape, size, color, carotenoids and chlorophylls in Newhall and Cara Cara oranges. (A) External and internal colors of Newhall and Cara Cara fruits during development and ripening at different stages (S1–S8). Bar, 2 cm. (B) Changes in peel CCI (citrus colour index), fruit size (transverse diameter), content of flesh total carotenoids and flesh chlorophylls during fruit development and ripening in Newhall and Cara Cara oranges. The bars represent SE from three biological replicates.

Figure 2

Profiles of carotenoids in Newhall and Cara Cara oranges. (A) Composition and amount of total carotenoids or total xanthophylls in flesh of Newhall and Cara Cara ripe fruit. (B) Changes in content of carotenoids in flesh of Newhall and Cara Cara oranges during fruit development and ripening. The bars represent SE from three biological replicates.

Figure 3

Cells of Newhall and Cara Cara orange flesh at early stage S3, middle stage S5, ripe stage S8 under DIC (differential interference contrast) microscopy. Bar, 20 μm.

Figure 4

Microscopic observation of cells from different colored juice vesicles of Cara Cara ripe fruit. (A) Photograph of dissected Cara Cara fruit with different colored juice vesicles located close to each other. (B) Cells from red to yellow juice vesicles under DIC (differential interference contrast) microscope.

Figure 5

Raman microscope spectra of yellow globular chromoplast in Newhall (1), yellow globular chromoplast in Cara Cara (2), red crystalline chromoplast in Cara Cara (3). Bar, 50 μm. Line 1, 2 represent carotenoids with 9 carbon-carbon double bond; Line 3 represent lycopene with unique 11 carbon-carbon double bond.

Figure 6

Ultrastructure of yellow globular chromoplast in Newhall, and yellow globular and red crystalline chromoplasts in Cara Cara viewed in the transmission electron microscope. Cr, crystal; CrR, crystalloid remnant; Cw, cell wall; Pg, plastoglobuli; S, starch; Th, thylakoid; UM, undulating membrane.

Figure 7

Expression of carotenogenic genes in flesh of Newhall and Cara Cara oranges during fruit development and ripening. The bars represent SE from three biological replicates_. Asterisks indicate significant differences, P < 0.05 and P < 0.01 are designated by * and **, respectively. The abbreviation for the genes is the same as listed in Fig. 1.

Figure 8

Expression of PSY1 and CYCB alleles in different colored juice vesicles at S6. (A) Different color of yellow and red juice vesicles from the same Cara Cara fruit. (B) Expression level of two alleles of PSY1 in Newhall juice vesicles and Cara Cara yellow, red juice vesicles. (C) Expression level of two alleles of CYCB in Newhall juice vesicles and Cara Cara yellow, red juice vesicles. The expression level of each of the PSY1 and CYCB allele was measured by quantitative real-time PCR using allele-specific PCR primers (Supplementary Table S1). The bars represent SE from three biological replicates. A significance test was conducted and values with the same letter indicates no significant difference at P < 0.05.

Figure 9

In vitro cultured flesh tissues of Newhall and Cara Cara following CPTA and norflurazon treatments. The 0 week tissues were separated from fruit at the S5 stage. CPTA, 2-(4-chlorophenylthio)-triethylamine hydrochloride.

Figure 10

Amount and composition of carotenoids in in vitro cultured flesh tissues of Newhall and Cara Cara following CPTA and norflurazon treatments. The 0 week tissues were separated from fruit at the S5 stage. CPTA, 2-(4-chlorophenylthio)-triethylamine hydrochloride.

Figure 11

Occurrence of red crystalline chromoplasts in cells from CPTA treated Newhall flesh tissues. The juice vesicle was from flesh tissues separated from fruit at the S5 stage and then treated with CPTA for four weeks. CPTA, 2-(4-chlorophenylthio)-triethylamine hydrochloride.