Enzymatic formation of β-citraurin from β-cryptoxanthin and Zeaxanthin by carotenoid cleavage dioxygenase4 in the flavedo of citrus fruit

Abstract

In this study, the pathway of β-citraurin biosynthesis, carotenoid contents and the expression of genes related to carotenoid metabolism were investigated in two varieties of Satsuma mandarin (Citrus unshiu), Yamashitabeni-wase, which accumulates β-citraurin predominantly, and Miyagawa-wase, which does not accumulate β-citraurin. The results suggested that CitCCD4 (for Carotenoid Cleavage Dioxygenase4) was a key gene contributing to the biosynthesis of β-citraurin. In the flavedo of Yamashitabeni-wase, the expression of CitCCD4 increased rapidly from September, which was consistent with the accumulation of β-citraurin. In the flavedo of Miyagawa-wase, the expression of CitCCD4 remained at an extremely low level during the ripening process, which was consistent with the absence of β-citraurin. Functional analysis showed that the CitCCD4 enzyme exhibited substrate specificity. It cleaved β-cryptoxanthin and zeaxanthin at the 7,8 or 7',8' position. But other carotenoids tested in this study (lycopene, α-carotene, β-carotene, all-trans-violaxanthin, and 9-cis-violaxanthin) were not cleaved by the CitCCD4 enzyme. The cleavage of β-cryptoxanthin and zeaxanthin by CitCCD4 led to the formation of β-citraurin. Additionally, with ethylene and red light-emitting diode light treatments, the gene expression of CitCCD4 was up-regulated in the flavedo of Yamashitabeni-wase. These increases in the expression of CitCCD4 were consistent with the accumulation of β-citraurin in the two treatments. These results might provide new strategies to improve the carotenoid contents and compositions of citrus fruits.

Figure 1.

Carotenoid and apocarotenoid metabolic pathway in plants. GGPP, Geranylgeranyl diphosphate. Enzymes, listed here from top to bottom, are named according to the designation of their genes: PSY, phytoene synthase; PDS, Phytoene desaturase; ZDS, ζ-carotene desaturase; ZISO, 15-cis-ζ-carotene isomerase; CRTISO, carotenoid isomerase; LCYb, lycopene β-cyclase; LCYe, lycopene ε-cyclase; HYe, ε-ring hydroxylase; HYb, β-ring hydroxylase; ZEP, zeaxanthin epoxidase; VDE, violaxanthin deepoxidase; NCED, 9-cis-epoxycarotenoid dioxygenase.

Figure 2.

Isolation and identification of β-citraurin from the flavedo of Yamashitabeni-wase. A, HPLC analysis of β-citraurin. mAU, Milliabsorbance units. B, UV-visible light spectrum of β-citraurin. C, Fast atom bombardment mass spectrometry spectrum of β-citraurin.

Figure 3.

Carotenoid accumulation in flavedos of Yamashitabeni-wase and Miyagawa-wase during the ripening process. A, Changes in the external colors of Yamashitabeni-wase and Miyagawa-wase during the ripening process. B, Changes in the carotenoid contents in flavedos of Yamashitabeni-wase and Miyagawa-wase during the ripening process. The results shown are means ± se for triplicate samples. S, September; O, October; N, November; D, December; J, January; Phy, phytoene; ζ-Car, ζ-carotene; β-Car, β-carotene; β-Cry, β-cryptoxanthin; Zea, zeaxanthin; T-vio, all-trans-violaxanthin; C vio, 9-cis-violaxanthin; α-Car, α-carotene; Lut, lutein; β-Cit, β-citraurin; Total, total carotenoids; FW, Fresh weight.

Figure 4.

Changes in the expression of genes related to carotenoid metabolism in flavedos of Yamashitabeni-wase and Miyagawa-wase during the ripening process. The results shown are means ± se for triplicate samples. S, September; O, October; N, November; D, December; J, January. The mRNA levels were analyzed by TaqMan real-time quantitative RT-PCR. Real-time RT-PCR amplification of 18S rRNA was used to normalize the expression of the genes under identical conditions.

Figure 5.

Changes in the expression of CitCCD4 in flavedos and juice sacs of Yamashitabeni-wase (A) and Miyagawa-wase (B) during the ripening process. The results shown are means ± se for triplicate samples. S, September; O, October; N, November; D, December; J, January. The mRNA levels were analyzed by TaqMan real-time quantitative RT-PCR. Real-time RT-PCR amplification of 18S rRNA was used to normalize the expression of the genes under identical conditions.

Figure 6.

Subcellular localization of the CitCCD4-GFP fusion protein. A, Tobacco SR1 leaves were transfected with the control vector (35S-GFP). B, Tobacco SR1 leaves were transfected with the recombinant vector (35S-CitCCD4-GFP).

Figure 7.

Functional analysis of the CitCCD4 enzyme in vivo. A, HPLC analysis of the cleavage products in lycopene-accumulating E. coli BL21 (DE3) cells transformed with pRSF-2 Ek/LIC-CitCCD4. B, HPLC analysis of the cleavage products in α-carotene- and β-carotene-accumulating E. coli BL21 (DE3) cells transformed with pRSF-2 Ek/LIC-CitCCD4. C, HPLC analysis of the cleavage products in zeaxanthin-accumulating E. coli BL21 (DE3) cells transformed with pRSF-2 Ek/LIC-CitCCD4. mAU, Milliabsorbance units.

Figure 8.

Functional analysis of the CitCCD4 enzyme in vitro. A, HPLC analysis of the cleavage products from the incubation of β-cryptoxanthin with recombinant CitCCD4. B, HPLC analysis of the cleavage products from the incubation of zeaxanthin with recombinant CitCCD4. C, HPLC analysis of the cleavage products from the incubation of all-trans-violaxanthin with recombinant CitCCD4. D, HPLC analysis of the cleavage products from the incubation of 9-cis-violaxanthin with recombinant CitCCD4. mAU, Milliabsorbance units.

Figure 9.

Effects of ethylene and red LED light on β-citraurin content (A) and gene expression of CitCCD4 (B) in flavedo of Yamashitabeni-wase. The results shown are means ± se for triplicate samples. The mRNA levels were analyzed by TaqMan real-time quantitative RT-PCR. Real-time RT-PCR amplification of 18S rRNA was used to normalize the expression of the genes under identical conditions. FW, Fresh weight.

Figure 10.

The pathway from β-cryptoxanthin and zeaxanthin to β-citraurin catalyzed by CitCCD4 in citrus fruits.