The Use of RNA Sequencing and Correlation Network Analysis to Study Potential Regulators of Crabapple Leaf Color Transformation

Abstract

Anthocyanins are plant pigments that contribute to the color of leaves, flowers and fruits, and that are beneficial to human health in the form of dietary antioxidants. The study of a transformable crabapple cultivar, 'India magic', which has red buds and green mature leaves, using mRNA profiling of four leaf developmental stages, allowed us to characterize molecular mechanisms regulating red color formation in early leaf development and the subsequent rapid down-regulation of anthocyanin biosynthesis. This analysis of differential gene expression during leaf development revealed that ethylene signaling-responsive genes are up-regulated during leaf pigmentation. Genes in the ethylene response factor (ERF), SPL, NAC, WRKY and MADS-box transcription factor (TF) families were identified in two weighted gene co-expression network analysis (WGCNA) modules as having a close relationship to anthocyanin accumulation. Analyses of network hub genes indicated that SPL TFs are located in central positions within anthocyanin-related modules. Furthermore, cis-motif and yeast one-hybrid assays suggested that several anthocyanin biosynthetic or regulatory genes are potential targets of SPL8 and SPL13B. Transient silencing of these two genes confirmed that they play a role in co-ordinating anthocyanin biosynthesis and crabapple leaf development. We present a high-resolution method for identifying regulatory modules associated with leaf pigmentation, which provides a platform for functional genomic studies of anthocyanin biosynthesis.

Systematic identification of long noncoding RNAs expressed during light‐induced anthocyanin accumulation in apple fruit

“…An analysis of the relationship between modules and anthocyanin levels revealed that the 'MEpink' module (r = 0.86, P = 1e-04) and the 'MEblue' module (r = 0.89, P = 9e-04) highly correlated with anthocyanin accumulation. Many DEGs identified in the MEpink and MEblue modules were TFs, including members of the MYB (MD17G1261100, MD09G 1278600, MD05G1120600), bHLH (MD14G1182700), SPL (MD12G1060200, MD13G1120300), WRKY (MD08G1094900, MD15G1078200, MD17G1223100), and ERF (MD15 G1221100, MD02G1096500) families (Table S10), which are all known to play important roles in anthocyanin biosynthesis (Lepiniec et al, 2006;Klee and Giovannoni, 2011;Jaakola, 2013;Pesch et al, 2015;Yang et al, 2018b).…”

MdMYB8 is associated with flavonol biosynthesis via the activation of the MdFLS promoter in the fruits of Malus crabapple

“…The statistical enrichment of the DEGs in KEGG pathways was tested by KOBAS 43 . The R package WGCNA was used to identify modules of highly correlated genes based on the FPKM data 44,45 . We filtered the genes based on gene expression and variations by using the R package DCGL.…”

“…The top 100 hub genes were calculated by eigengenebased connectivity, ranked by k (k cor,i (q) = cor(xi, E(q) ) and ME (module eigengene). KOBAS 2.0 was used for gene annotation 44 .…”

“…The Y1H assay was performed as described previously 44 . The coding DNA sequences of the 11 candidate hub genes were cloned into the EcoRI and XhoI sites of the pJG4-5 vector.…”

Transcriptome Analysis Identifies Two Ethylene Response Factors That Regulate Proanthocyanidin Biosynthesis During Malus Crabapple Fruit Development

“…RNA concentration was measured using a Qubit ® RNA Assay Kit in a Qubit ® 2.0 fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using an RNA Nano 6000 Assay Kit and a Bioanalyzer 2100 system (Agilent Technologies, CA, USA) (Yang et al, 2018).…”