Transcriptional Analysis of Carotenoids Accumulation and Metabolism in a Pink-Fleshed Lemon Mutant

Abstract

Pink lemon is a spontaneous bud mutation of lemon (Citrus limon, L. Burm. f) characterized by the production of pink-fleshed fruits due to an unusual accumulation of lycopene. To elucidate the genetic determinism of the altered pigmentation, comparative carotenoid profiling and transcriptional analysis of both the genes involved in carotenoid precursors and metabolism, and the proteins related to carotenoid-sequestering structures were performed in pink-fleshed lemon and its wild-type. The carotenoid profile of pink lemon pulp is characterized by an increased accumulation of linear carotenoids, such as lycopene, phytoene and phytofluene, from the early stages of development, reaching their maximum in mature green fruits. The distinctive phenotype of pink lemon is associated with an up-regulation and down-regulation of the genes upstream and downstream the lycopene cyclase, respectively. In particular, 9-cis epoxycarotenoid dioxygenase genes were overexpressed in pink lemon compared with the wild-type, suggesting an altered regulation of abscisic acid biosynthesis. Similarly, during early development of the fruits, genes of the carotenoid-associated proteins heat shock protein 21, fibrillin 1 and 2 and orange gene were overexpressed in the pulp of the pink-fleshed lemon compared to the wild-type, indicating its increased capacity for sequestration, stabilization or accumulation of carotenes. Altogether, the results highlighted significant differences at the transcriptomic level between the pink-fleshed lemon and its wild-type, in terms of carotenoid metabolism and the capacity of stabilization in storage structures between the two accessions. Such changes may be either responsible for the altered carotenoid accumulation or in contrast, a metabolic consequence.

Keywords: Citrus limon; carotenoids; citrus; fruit quality; gene expression; lycopene; pigments.

Figure 1

Schematic representation of carotenoid biosynthesis in citrus fruits, indicating main enzymes and genes of the pathway. 1-deoxy-D-xylulose-5-phosphate synthase (DXS), hydroxymethylbutenyl diphosphate synthase (HDS) and reductase (HDR), geranylgeranyl diphosphate synthase (GGPSS), phytoene synthase (PSY), phytoene desaturase (PDS), ζ-carotene isomerase (ZISO), ζ-carotene desaturase (ZDS), ε-lycopene cyclase (ε-LCY), β-lycopene cyclase (β-LCY1/2), ε-carotene hydroxylase (ε-CHX), β-carotene hydroxylase (β-CHX), zeaxanthin epoxidase (ZEP) and 9-cis-epoxy-carotenoid dioxygenase (NCED). Green and red arrows represent carotenoid biosynthesis flux in green and mature tissues, respectively.

Figure 2

External and internal appearance of pink lemon (PL) (A) and wild-type (WT) (B) lemon fruit during development and maturation. Immature green (IG), mature green (MG), breaker (BR) and fully mature (FM) stages. Changes in color (a/b Hunter) of the pulp of PL and WT (C) during fruit maturation. Data are the mean ± SD of at least 10 fruits.

Figure 3

Changes in the expression of genes involved in the MEP pathway: GGPS11, geranylgeranyl diphosphate synthase 11 (A); DXS, 1-deoxy-D-xylulose-5-phosphate synthase (B); HDS, hydroxymethylbutenyl diphosphate synthase (C); HDR, hydroxymethylbutenyl diphosphate reductase (D), in the pulp of the PL and WT lemon fruit at four developmental stages: IG (immature green), MG (mature green), BR (breaker), FM (fully mature). Asterisks indicate significant differences between genotypes for each developmental stage (p < 0.01) by one-way ANOVA (p < 0.01).

Figure 4

Changes in the expression of genes involved in carotenoids biosynthesis: PSY, phytoene synthase (A); PSY3a, phytoene synthase 3a (B); PDS, phytoene desaturase (C); ZDS1, Ƶ-carotene desaturase 1 (D); ZISO, Ƶ-carotene isomerase (E); ε-LCY, ε-cyclase (F); β-LCY1, β-lycopene cyclase 1 (G); β-LCY2b, β-lycopene cyclase 2b (H); β-CHX, β-carotene hydroxylase (I), in the pulp of PL and WT lemon fruit at four developmental stages: IG (immature green), MG (mature green), BR (breaker), FM (fully mature). Asterisks indicate significant differences between genotypes for each developmental stage (p < 0.01) by one-way ANOVA (p < 0.01).

Figure 5

Changes in the expression of genes involved in abscisic acid biosynthesis: NCED1, 9-cis-epoxy-carotenoid dioxygenase 1 (A); NCED2, 9-cis-epoxy-carotenoid dioxygenase 2 (B), in the pulp of the PL and WT lemon fruit at four developmental stages: IG (immature green), MG (mature green), BR (breaker), FM (fully mature). Asterisks indicate significant differences between genotypes for each developmental stage (p < 0.01) by one-way ANOVA (p < 0.01).

Figure 6

Changes in the expression of genes of carotenoid-associated proteins: HSP20-3, heat shock protein 20-3 (A); HSP20-4, heat shock protein 20-4 (B); HSP21, heat shock protein 21 (C); FIB1, fibrillin 1 (D); FIB2, fibrillin 2 (E); Or, orange protein gene (F), in the pulp of the PL and WT lemon fruit at four developmental stages: IG (immature green), MG (mature green), BR (breaker), FM (fully mature). Asterisks indicate significant differences between genotypes for each developmental stage (p < 0.01) by one-way ANOVA (p < 0.01).