Carotenoid components and their biosynthesis in a bud mutant of Shiranui mandarin (Citrus reticulata Blanco) with citrine flavedo

Abstract

Carotenoids are secondary metabolite responsible for colored pigments in plants and microbes (Li et al., 2022). They are a class of C₄₀ tetraterpenoids consisting of eight isoprenoid units, and can be classified into carotenes and xanthophylls on the basis of their functional groups (Saini et al., 2015). Carotenes can be linear (phytoene, phytofluene, and ζ‍-carotene) or branched (β‍-carotene and α‍-carotene). Xanthophylls comprise β,β‍-xanthophylls (β‍-cryptoxanthin, zeaxanthin, violaxanthins, and neoxanthin) and β,ε‍-xanthophylls (α-cryptoxanthin, α-carotene, and lutein). Citrus fruits are complex sources of carotenoids, which are the principal pigments responsible for the typical orange color of most types (Chen, 2020). The difference in total carotenoid content and the diversity of carotenoid isomer proportion also accounts for other colors of citrus fruits, such as yellow, red, and pink (Chen, 2020).

Fig. 1. Appearance of flavedos and total carotenoid contents. (a) The appearance of flavedos of Shiranui and Citrine Shiranui at six stages of fruit ripening. (b) The total carotenoid contents. Data were expressed as mean±standard deviation (SD), n=3. A significance test was conducted: ^*** P<0.001 vs. Citrine Shiranui. I: breaker stage; II: coloring stage; III: full coloring stage; IV: one month after full coloring; V: two months after full coloring; VI: the full ripening stage.

Fig. 2. High-performance liquid chromatography analysis (HPLC) chromatogram of carotenoids in the flavedos of Shiranui (a) and Citrine Shiranui (b) at the full ripening stage (Stage VI).

Fig. 3. Carotenoid proportions (%) in flavedos. Data were expressed as mean±standard deviation (SD), n=3. A significance test was conducted: ^*** P<0.001, ^** P<0.01 vs. Citrine Shiranui. I: breaker stage; Ⅱ: coloring stage; Ⅲ: full coloring stage; Ⅳ: one month after full coloring; Ⅴ: two months after full coloring; Ⅵ: the full ripening stage.

Fig. 4. Expression of carotenoid biosynthesis genes (the value was normalized to the minimum among all the messenger RNA (mRNA) levels in both two cultivars for each gene). Data were expressed as mean±standard deviation (SD), n=3. A significance test was conducted: ^*** P<0.001, ^** P<0.01, and ^* P<0.05 vs. Citrine Shiranui. PSY: phytoene synthase; PDS: phytoene desaturase; ZDS: ζ-carotene desaturase; LCYb: lycopene β-cyclase; HYb: β-carotene hydroxylase; ZEP: zeaxanthin epoxidase; NSY: neoxanthin synthase; LCYe: lycopene ε-cyclase; HYe: ε-carotene hydroxylase; I: breaker stage; Ⅱ: coloring stage; Ⅲ: full coloring stage; Ⅳ: one month after full coloring; Ⅴ: two months after full coloring; Ⅵ: the full ripening stage.

Fig. 5. Schematic diagram of carotenoid biosynthetic pathways. The value of carotenoid biosynthesis genes was normalized to the minimum among all messenger RNA (mRNA) levels in all genes for either Shiranui or Citrine Shiranui. The square area indicates the proportion of carotenoid component at the full ripening stage (Stage VI). PSY: phytoene synthase; PDS: phytoene desaturase; ZDS: ζ-carotene desaturase; LCYb: lycopene β‍-cyclase; HYb: β‍-carotene hydroxylase; ZEP: zeaxanthin epoxidase; NSY: neoxanthin synthase; LCYe: lycopene ε-cyclase; HYe: ε-carotene hydroxylase.