ERF5.1 modulates carotenoid accumulation by interacting with CCD4.1 in Lycium

Abstract

Carotenoids are important natural pigments and have medical and health functions for humans. Carotenoid cleavage dioxygenase 4 (CCD4) and ethylene responsive factor (ERF) participate in carotenoid metabolism, but their roles in Lycium have not been discovered. Here, we annotated LbCCDs from the Lycium reference genome and found that LbCCD4.1 expression was significantly correlated with the carotenoid metabolites during Lycium five fruit developmental stages. Over-expression of LbCCD4.1 in NQ’s leaves resulted in a series of significantly lower contents of carotenoid metabolites, including β-carotene and β-cryptoxanthin. Moreover, LbERF5.1, a transcription factor belonging to the ERF family that was located in the nucleus, was isolated. Significant reductions in the carotenoids, especially lutein, violaxanthin and their derivatives, were observed in over-expressing ERF5.1 transgenic NQ’s leaves. Over-expression or virus-induced gene silencing of LbERF5.1 in NQ’s leaves induced a consistent up- or down-expression, respectively, of LbCCD4.1. Furthermore, yeast one-hybrid and dual-luciferase reporter assays showed that ERF5.1 interacted with the promoter of CCD4.1 to increase its expression, and LbERF5.1 could bind to any one of the three predicted binding sites in the promoter of LbCCD4.1. A transcriptome analysis of LbERF5.1 and LbCCD4.1 over-expressed lines showed similar global transcript expression, and geranylgeranyl diphosphate synthase, phytoene synthase, lycopene δ-cyclase cytochrome, cytochrome P450-type monooxygenase 97A, cytochrome P450-type monooxygenase 97C, and zeaxanthin epoxidase in the carotenoid biosynthesis pathway were differentially expressed. In summary, we uncovered a novel molecular mechanism of carotenoid accumulation that involved an interaction between ERF5.1 and CCD4.1, which may be used to enhance carotenoid in Lycium.

Acknowledgements

This work was sponsored by the National Natural Science Foundation of China (No. 32060359), the Key Research & Development Program of Ningxia Hui Autonomous Region (No. 2021BEF02002,2022BBF01001), the Innovative Research Group Project of Ningxia Hui Autonomous Region (No. 2021AAC01001) and the Innovation Team for Genetic Improvement of Economic Forests (No. 2022QCXTD04).

Author contributions

J.Z. and W.A. conceived and designed the research. H.L., Y.Y., X. Zhang and X. Zhu. prepared the population material. H.L., L.D., X.L., T.H., and B.Z. performed sampling, sequencing, and transcriptome analyses. J.Z., Z.S., X.Q., and Y.C. contributed to the project discussion. J.Z., Y.X., and H.L. wrote the manuscript. J.Z., Y.X., and Y.C. revised the manuscript. All the authors read and approved the final manuscript.

Data availability statement

Data supporting the findings of this work are available within the paper and the Supplementary Tables and Figures. The transcriptome clean sequencing data have been deposited into the National Center for Biotechnology Information Sequence Read Archive database (PRJNA936937).

Conflict of interests

All the authors declare that there is no conflict of interest.

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