Functional characterization and comparison of lycopene epsilon-cyclase genes in Nicotiana tabacum

Abstract

Background Lycopene epsilon-cyclase (ε-LCY) is a key enzyme in the carotenoid biosynthetic pathway (CBP) of higher plants. In previous work, we cloned two Ntε-LCY genes from allotetraploid tobacco (Nicotiana tabacum), Ntε-LCY2 and Ntε-LCY1, and demonstrated the overall effect of Ntε-LCY genes on carotenoid biosynthesis and stress resistance. However, their genetic and functional characteristics require further research in polyploid plants. Results Here, we used CRISPR/Cas9 to obtain Ntε-LCY2 and Ntε-LCY1 mutants in allotetraploid N.tabacum K326. Ntε-LCY2 and Ntε-LCY1 had similar promoter cis-acting elements, including light-responsive elements. The Ntε-LCY genes were expressed in roots, stems, leaves, flowers, and young fruit, and their highest expression levels were found in leaves. Ntε-LCY2 and Ntε-LCY1 genes responded differently to normal light and high light stress. Both the Ntε-LCY2 and the Ntε-LCY1 mutants had a more rapid leaf growth rate, especially ntε-lcy2-1. The expression levels of CBP genes were increased in the ntε-lcy mutants, and their total carotenoid content was higher. Under both normal light and high light stress, the ntε-lcy mutants had higher photosynthetic capacities and heat dissipation levels than the wild type, and this was especially true of ntε-lcy2-1. The reactive oxygen species content was lower in leaves of the ntε-lcy mutants. Conclusion In summary, the expression patterns and biological functions of the Ntε-LCY genes Ntε-LCY1 and Ntε-LCY2 differed in several respects. The mutation of Ntε-LCY2 was associated with a greater increase in the content of chlorophyll and various carotenoid components, and it enhanced the stress resistance of tobacco plants under high light.

A Small Subunit of Geranylgeranyl Diphosphate Synthase Functions as an Active Regulator of Carotenoid Synthesis in Nicotiana tabacum

“…The silencing of PSY, PDS, β-LCY, and VDE caused the photobleaching of different plant organs and a significant decline in photosynthetic efficiency and stress resistance [ 35 , 38 , 39 , 40 ]. Unlike these genes in the carotenoid pathway, CRISPR/Cas9-mediated NtSSU II silencing hampered the normal growth of transgenic tobacco, with a decline in leaf area, plant height, and biomass ( Figure 6 ).…”

“…The CRISPR/Cas9 vectors pSHE401 were kindly provided by Prof. Qijun Chen from China Agricultural University, which were successfully modified to enable the precise mutation of genes in tobacco [ 36 , 40 ]. The design of target sites and the construction of CRISPR/Cas9 vectors for NtSSU II were carried out according to the Mutation Sites Based Specific Primers Polymerase Chain Reaction (MSBSP-PCR) method [ 36 ].…”

“…The optimal sgRNA sequence was selected close to the 5-terminal of the NtSSU II CDS ( Table S2 ). The double-stranded DNA was generated by a pair of primers as sgRNA-SSU II-F and sgRNA-SSU II-R ( Table S2 ) using our previous method [ 36 , 40 ]. The recombinant vectors pHSE401-SSU II were constructed and transformed individually into tobacco callus.…”

NtDREB-1BL1 Enhances Carotenoid Biosynthesis by Regulating Phytoene Synthase in Nicotiana tabacum

“…Carotenoids are essential nutrients and important health-beneficial compounds for humans. As a subgroup of isoprenoid compounds with diverse structures, carotenoids are widely distributed in plants, algae, photosynthetic bacteria, and fungi [ 1 , 2 , 3 , 4 , 5 ]. Carotenoids are well-known antioxidants in higher plants, which not only quench reactive oxygen species (ROS) but also serve to sacrifice themselves under conditions of oxidative stress [ 2 , 5 ].…”

Genome Editing of Golden SNP-Carrying Lycopene Epsilon-Cyclase (LcyE) Gene Using the CRSPR-Cas9/HDR and Geminiviral Replicon System in Rice

“…Therefore, GTs in plants, especially alleles without selectable markers, still need to be improved with many studies. Carotenoids are synthesized in plastids and accumulated as red, orange, and yellow pigments, among which β-carotene, lycopene, and lutein are industrially important components as powerful antioxidants [ 19 ]. The cyclization of lycopene ends catalyzed by lycopene β -cyclase and lycopene ε -cyclase forms β -carotene or α -carotene.…”