Molecular Cloning and Functional Characterization of the Lycopene ε-Cyclase Gene via Virus-Induced Gene Silencing and Its Expression Pattern in Nicotiana tabacum

Abstract

Lycopene ε-cyclase (ε-LCY) is a key enzyme that catalyzes the synthesis of α-branch carotenoids through the cyclization of lycopene. Two cDNA molecules encoding ε-LCY (designated Ntε-LCY1 and Ntε-LCY2) were cloned from Nicotiana tabacum. Ntε-LCY1 and Ntε-LCY2 are encoded by two distinct genes with different evolutionary origins, one originating from the tobacco progenitor, Nicotiana sylvestris, and the other originating from Nicotiana tomentosiformis. The two coding regions are 97% identical at the nucleotide level and 95% identical at the amino acid level. Transcripts of Ntε-LCY were detectable in both vegetative and reproductive organs, with a relatively higher level of expression in leaves than in other tissues. Subcellular localization experiments using an Ntε-LCY1-GFP fusion protein demonstrated that mature Ntε-LCY1 protein is localized within the chloroplast in Bright Yellow 2 suspension cells. Under low-temperature and low-irradiation stress, Ntε-LCY transcript levels substantially increased relative to control plants. Tobacco rattle virus (TRV)-mediated silencing of ε-LCY in Nicotiana benthamiana resulted in an increase of β-branch carotenoids and a reduction in the levels of α-branch carotenoids. Meanwhile, transcripts of related genes in the carotenoid biosynthetic pathway observably increased, with the exception of β-OHase in the TRV-ε-lcy line. Suppression of ε-LCY expression was also found to alleviate photoinhibition of Potosystem II in virus-induced gene silencing (VIGS) plants under low-temperature and low-irradiation stress. Our results provide insight into the regulatory role of ε-LCY in plant carotenoid biosynthesis and suggest a role for ε-LCY in positively modulating low temperature stress responses.

Carotenoid Metabolism in Plants

“…Similarly, ε-LCY downregulation in canola results in the enhanced accumulation of b-carotene, zeaxanthin, violaxanthin, and lutein, although it should be noted that the ratio of b-carotene to lutein increased substantially (Yu et al, 2008). Suppression of ε-LCY in sweet potato and tobacco showed increased synthesis of a b-branch-specific pathway and enhanced tolerance to abiotic stress (Kim et al, 2013;Shi et al, 2014). Enhanced levels of b-carotene were also observed in ε-LCY tuber-specific silencing of potato (Diretto et al, 2006).…”

Overexpression of Orange (OR) and OR mutant protein inChlamydomonas reinhardtiienhances carotenoid and ABA accumulation and increases resistance to abiotic stress

“…Strategies to elevate the carotenoid content in plants have focused on altering the gene expression of key carotenogenic enzymes. For instance, down-regulation of lycopeneε -cyclase (LCY-ε) in potato (Diretto et al, 2006) and tobacco (Shi et al, 2014) increased total carotenoids by 2 to 2.5-fold. Simultaneous down-regulation of LCY-ε and β -carotene hydroxylase (CHYβ ) increased total carotenoids up to 2.7-fold in sweet potato plants and 18-fold in calli (Kim et al, 2012;Ke et al, 2019).…”

Bioinformatics Analysis of the 1-Deoxy-D-Xylulose-5-Phosphate Synthase (DXS) Gene in Cabbage (Brassica Oleracea Var. Capitata)

“…Then, lycopene (colored carotenoid) is converted from phytoene (non-color carotenoid) by desaturases and isomerases, including phytoene desaturases (PDS) [9], ζ-carotene desaturase (ZDS) [10], 15-cis-ζ-carotene isomerase (Z-ISO) [11], and carotenoid isomerase (CRTISO) [4]. Hereafter, bifurcation of the carotenoid biosynthetic pathway occurs, and the production of β-carotene and α-carotene is catalyzed by lycopene β-cyclase (LCYb) and lycopene ε-cyclase (LCYe) [12][13].…”