Genome-wide identification of ZmMYC2 binding sites and target genes in maize

Abstract

Background: Jasmonate (JA) is the important phytohormone to regulate plant growth and adaption to stress signals. MYC2, an bHLH transcription factor, is the master regulator of JA signaling. Although MYC2 in maize has been identified, its function remains to be clarified.

Results: To understand the function and regulatory mechanism of MYC2 in maize, the joint analysis of DAP-seq and RNA-seq is conducted to identify the binding sites and target genes of ZmMYC2. A total of 3183 genes are detected both in DAP-seq and RNA-seq data, potentially as the directly regulating genes of ZmMYC2. These genes are involved in various biological processes including plant growth and stress response. Besides the classic cis-elements like the G-box and E-box that are bound by MYC2, some new motifs are also revealed to be recognized by ZmMYC2, such as nGCATGCAnn, AAAAAAAA, CACGTGCGTGCG. The binding sites of many ZmMYC2 regulating genes are identified by IGV-sRNA.

Conclusions: All together, abundant target genes of ZmMYC2 are characterized with their binding sites, providing the basis to construct the regulatory network of ZmMYC2 and better understanding for JA signaling in maize.

Keywords: cis-element; Jasmonate; MYC2; Maize; Transcription factor.

Fig. 1

RNA-seq analysis of ZmMYC2 transient overexpression in maize protoplasts. (A) volcano plot of DEGs in ZmMYC2 transient overexpression maize protoplasts and the control. Log2Foldchange indicates the fold change of DEGs with upregulation or downregulation. (B) GO enrichment analysis of DEGs. BP, biological process. CC, cellular component. MF, molecular function. (C) KEGG enrichment analysis of DEGs in different pathways with upregulation (Up term) and downregulation (Down term). The specific enriched pathways only with upregulated DEGs were also shown (Specific term)

Fig. 2

Joint analysis of RNA-seq and DAP-seq for ZmMYC2. (A) Venn plot of shared and specific genes by joint analysis of RNA-seq and DAP-seq for ZmMYC2. (B) Volcano plots for target regulating genes (TRGs) and indirect regulating genes (IRGs) of ZmMYC2. (C) Predominant GO terms of TRGs and IRGs through GO enrichment analysis

Fig. 3

Cluster analysis for gene expression analysis of ZmMYC2 target regulating genes. Gene expression of ZmMYC2 target regulating genes (TRGs) were compared among the RNA-seq data in this study and the transcriptomic data on Maize eFP including root and shoot apical meristem (SAM) growth, treatments of PEG, salt, heat and cold, infection of Colletotrichum graminicola and Cercospora zeina. (A) Cluster analysis of ZmMYC2 TRGs with expression comparison among RNA-seq data in this study and the data with abiotic stress treatments in Maize eFP, biotic stress treatments (B), root and SAM growth (C). C1-4, clustered genes. C1 and C2 exhibited upregulation by ZmMYC2 but upregulation (C1) or downregulation (C2) in the corresponding treatments. C3 and C4 were repressed by ZmMYC2 but showed increasing (C3) or decreasing (C4) expression in the corresponding treatments

Fig. 4

Plant hormone related genes regulated by ZmMYC2. Graphs of eight plant hormone synthesis and signling with key genes shown. (A-H), Jasmonate (JA), abscisic acid (ABA), cytokinin, gibberellin (GA), ethylene, auxin, brassinolide (BR) and salicylic acid (SA). Plant hormone names are labeled in orange. The key genes regulated by ZmMYC2 as TRGs are highlighted in blue. Circles close to genes indicate fold enrichment in DAP-seq and fold change in RNA-seq data for specific genes, respectively. Value changes are shown by color intensity. The genes with functional characterization were labeled in purple

Fig. 5

Transcription factors regulated by ZmMYC2. (A) Bar plot of transcription factors regulated by ZmMYC2 with families and gene numbers. (B-H) Lollipop plot of seven transcription factor families regulated by ZmMYC2. Fold enrichment (green circle) by DAP-seq and log2fold change (red circle) by RNA-seq were shown for each genes. The genes highlighted in purple were functional characterized in previous studies

Fig. 6

The main motifs bound by ZmMYC2. The main motifs were detected by DAP-seq to be bound by ZmMYC2 with length of 8 (A-C), 10 (D-E) and 12 (F). % of Target is percentage of target sequences with motif. The core sequences of E-box and G-box were labeled in red (A, C). Normal distribution of motifs is shown to reveal motif centrality. The x-axis indicates the relative position of motif in the peak. The y-axis indicates the count of motif on the different position of peak. Gaussian fitting curve is highlight in dark red

Fig. 7

Motif distribution bound by ZmMYC2 on selected genes Gene location on maize chromosomes are shown with gene names. The initial codon and transcription direction are indicted with the red arrow. The peaks indicate the binding of ZmMYC2 on genes by DAP-seq analysis with fold enrichment

Fig. 8

The proposed regulating model of ZmMYC2 ZmMYC2 targets to many plant hormone signaling genes and transcription factor encoding genes, which confers the broad function of ZmMYC2 in plant growth and stress responses through regulating these genes in various pathways