Genome-wide analysis of the ERF Family in Stephania japonica provides insights into the regulatory role in Cepharanthine biosynthesis

Abstract

Introduction: Cepharanthine (CEP), a bisbenzylisoquinoline alkaloid (bisBIA) extracted from Stephania japonica, has received significant attention for its anti-coronavirus properties. While ethylene response factors (ERFs) have been reported to regulate the biosynthesis of various alkaloids, their role in regulating CEP biosynthesis remains unexplored.

Methods: Genome-wide analysis of the ERF genes was performed with bioinformatics technology, and the expression patterns of different tissues, were analyzed by transcriptome sequencing analysis and real-time quantitative PCR verification. The nuclear-localized ERF gene cluster was shown to directly bind to the promoters of several CEP-associated genes, as demonstrated by yeast one-hybrid assays and subcellular localization assays.

Results: In this work, 59 SjERF genes were identified in the S. japonica genome and further categorized into ten subfamilies. Notably, a SjERF gene cluster containing three SjERF genes was found on chromosome 2. Yeast one-hybrid assays confirmed that the SjERF gene cluster can directly bind to the promoters of several CEP-associated genes, suggesting their crucial role in CEP metabolism. The SjERFs cluster-YFP fusion proteins were observed exclusively in the nuclei of Nicotiana benthamiana leaves. Tissue expression profiling revealed that 13 SjERFs exhibit high expression levels in the root, and the qRT-PCR results of six SjERFs were consistent with the RNA-Seq data. Furthermore, a co-expression network analysis demonstrated that 24 SjERFs were highly positively correlated with the contents of various alkaloids and expression levels of CEP biosynthetic genes.

Conclusion: This study provides the first systematic identification and analysis of ERF transcription factors in the S.japonica genome, laying the foundation for the future functional research of SjERFs transcription factors.

Keywords: Cepharanthine biosynthesis; ERF; Stephania japonica; expression patterns; genome-wide analysis.

Figure 1

Phylogenetic tree of 59 SjERFs and 121 AtERFs. The ERF protein sequences of S. japonica and A. thaliana were used to construct the phylogenetic tree using the Neighbor-Joining (NJ) method, with 1,000 bootstrap replicates.

Figure 2

Schematic diagram of phylogenetic analysis, exon/intron distribution, and motifs analysis of SjERF TFs. (A) Phylogenetic tree of 59 SjERF proteins. (B) Motif distribution of 59 SjERF proteins. (C) The exon-intron structure of 59 SjERF genes. Yellow rectangle: UTR; black line: intron; blue rectangle: CDS.

Figure 3

Pivotal cis−elements in the promoter of SjERF TFs.

Figure 4

The chromosome distribution and synteny analysis of SjERFs. (A) Chromosomal locations and their synteny of SjERFs. The connecting lines indicate duplicated gene pairs in 59 SjERFs. (B) The phylogenetic tree of SjERFs and functional ERF cluster. (C) The topologically associating domains (TADs) region of three SjERFs.

Figure 5

Members of the SjERFs cluster specifically bind to the GCC boxes in the promoters of CEP-associated genes in vitro. (A–C) Phylogenetic tree of CEP biosynthetic genes using MEGA11 with 1000 bootstrap replicates by Neighbor-joining (NJ) method. (D) Schematic diagrams of the SjNCS4, SjNCS5, Sj6OMT2, and SjCNMT4 promoters. The positions of potential GCC boxes are shown as blue Rectangles. (E) Yeast one-hybrid (Y1H) assay indicates that the SjERFs cluster binds to the GCC box in the promoters of CEP-associated genes, including SjNCS4, SjNCS5, Sj6OMT2 and SjCNMT4. Yeast cells transformed with different combinations of constructs were grown on SD/−Ura/−Trp/+X-gal medium. Photographs were taken after 3 d of incubation at 30°C. Y1H assays were repeated three times.

Figure 6

SjERFs protein fused to a yellow fluorescent protein (YFP) transiently expressed in N. benthamiana. Scale bar: 20 μm.

Figure 7

Expression patterns of 59 SjERFs in different tissues of S. japonica. (A) Hierarchical clustering of the expression level of SjERFs with RNA-Seq. (B) The expression profiles of six SjERFs in different tissues with the qRT-PCR method.

Figure 8

Analysis of correlation between SjERFs, CEP-biosynthetic genes, and BIAs metabolites. (A) Co-expression network of SjERF genes, CEP biosynthetic genes, and BIAs metabolites with |r| > 0.9 and p-value < 0.05. Orange squares: CEP biosynthetic genes, red circles: SjERF genes. Blue octagons: BIAs metabolites. (B) The cluster heatmap shows the expression correlations between five CEP biosynthetic genes, and two BIA precursors, alongside 23 BIA-type structures, and forty-six SjERFs.