The long noncoding RNA MdLNC499 bridges MdWRKY1 and MdERF109 function to regulate early-stage light-induced anthocyanin accumulation in apple fruit

Abstract

Anthocyanin pigments contribute to plant coloration and are valuable sources of antioxidants in the human diet as components of fruits and vegetables. Their production is known to be induced by light in (Malus domestica) apple fruit; however, the underlying molecular mechanism responsible for early-stage light-induced anthocyanin biosynthesis remains unclear. Here, we identified an ERF (ethylene response factor) protein, ERF109, involved in light-induced anthocyanin biosynthesis and found that it promotes coloration by directly binding to anthocyanin-related gene promoters. Promoter::GUS (β-glucuronidase) reporter analysis and Hi-C sequencing showed that a long non-coding RNA (lncRNA), MdLNC499, located upstream from MdERF109, induces the expression of MdERF109. A W-box cis-element in the MdLNC499 promoter was found to be regulated by a transcription factor, MdWRKY1. Transient expression in apple fruit and stable transformation of apple calli allowed us to reconstruct a MdWRKY1-MdLNC499-MdERF109 transcriptional cascade in which MdWRKY1 is activated by light to increase the transcription of MdLNC499, which in turn induces MdERF109. The MdERF109 protein induces the expression of anthocyanin-related genes and the accumulation of anthocyanins in the early stages of apple coloration. Our results provide a platform for better understanding the various regulatory mechanisms involved in light-induced apple fruit coloration.

Meta-Analysis of the Effect of Overexpression of MYB Transcription Factors on the Regulatory Mechanisms of Anthocyanin Biosynthesis

“…Anthocyanins, the main secondary metabolites of plants, are a major subclass of flavonoids that are related to tissue colors and stress responses ( Xu et al, 2015 ; Xia et al, 2021 ). Anthocyanin in plants is induced by various stresses, including light, cold, drought, wounding, and methyl viologen ( Naing et al, 2018 ; Shen et al, 2019 ; Zhou et al, 2020 ; Ma et al, 2021 ). It was illustrated that the stresses could cause an increased accumulation of anthocyanidins in MYB transformation by enhancing the expression of flavonoid structural genes.…”

“…Anthocyanin accumulation in vacuoles accounts for the red to blue color range of various tissues, including roots, stems, leaves, flowers and fruits ( Ben Zvi et al, 2008 ; Kim et al, 2010 ; Fan et al, 2020 ; Zhong et al, 2020 ; Xia et al, 2021 ). They play crucial roles in plant stress resistance by acting as scavengers of excess reactive oxygen species (ROS) ( Buer et al, 2010 ; Naing et al, 2018 ; Shen et al, 2019 ; Ma et al, 2021 ). Therefore, understanding the anthocyanin biosynthesis pathways and the functions of genes that regulate these pathways will contribute to the molecular designing and breeding of plants.…”

“…Based on the number of repeats (R) in the MYB domain ( Buer et al, 2010 ), MYBs are divided into four classes (1R, R2R3, R1R2R3, and 4R-MYB proteins). The MYB TFs regulate important processes in higher plants, such as secondary metabolism, signal transduction, and biotic and abiotic stresses ( Buer et al, 2010 ; Wang et al, 2019 ; Ma et al, 2021 ; Xia et al, 2021 ). Particularly, they are the vital regulators of anthocyanidin biosynthesis.…”

“…Particularly, they are the vital regulators of anthocyanidin biosynthesis. Many MYB genes have been overexpressed in flowers, fruits, vegetables, and crops, frequently to activate anthocyanin accumulation ( Dubos et al, 2010 ; Zhang et al, 2017 ; Fan et al, 2020 ; Xu et al, 2020 ; Ma et al, 2021 ). Most of the MYB TFs can directly or indirectly activate the promoters of structural genes of flavonoid biosynthesis pathways, thereby affecting the final anthocyanin accumulation ( Schaart et al, 2013 ).…”

“…Many MYB genes have been overexpressed in flowers, fruits, vegetables, and crops, frequently to activate anthocyanin accumulation Abbreviations: PAL, phenylalanine ammonia lyase; C4H, cinnamate 4hydroxylase; 4CL, 4-coumarate-CoA ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase; FLS, flavonol synthase; F3 H, flavonoid 3 -hydroxylase; F3 5 H, flavonoid 3 , 5 -hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; UFGT, UDPflavonoid glucosyl transferase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; TFs, transcription factors; CPA1, cation/proton antiporter 1; CBF/DREB, C-repeat/dehydration-responsive element binding proteins; NAC, NAM/ATAF/CUC; TC, transformed plants; NC, non-transformed plants; CMA, Comprehensive Meta-Analysis; CI, confidence interval; CAT, catalase; MDA, malondialdehyde; DDPH, 1,1-diphenyl-2-picryl-hydrazyl. (Dubos et al, 2010;Zhang et al, 2017;Fan et al, 2020;Xu et al, 2020;Ma et al, 2021). Most of the MYB TFs can directly or indirectly activate the promoters of structural genes of flavonoid biosynthesis pathways, thereby affecting the final anthocyanin accumulation (Schaart et al, 2013).…”