Genome-Wide Profiling of WRKY Genes Involved in Benzylisoquinoline Alkaloid Biosynthesis in California Poppy (Eschscholzia californica).

Yasuyuki Yamada,Shohei Nishida,Fumihiko Sato,Nobukazu Shitan

doi:10.3389/fpls.2021.699326

Abstract

Transcription factors of the WRKY family play pivotal roles in plant defense responses, including the biosynthesis of specialized metabolites. Based on the previous findings of WRKY proteins regulating benzylisoquinoline alkaloid (BIA) biosynthesis, such as CjWRKY1—a regulator of berberine biosynthesis in Coptis japonica—and PsWRKY1—a regulator of morphine biosynthesis in Papaver somniferum—we performed genome-wide characterization of the WRKY gene family in Eschscholzia californica (California poppy), which produces various BIAs. Fifty WRKY genes were identified by homology search and classified into three groups based on phylogenetic, gene structure, and conserved motif analyses. RNA sequencing showed that several EcWRKY genes transiently responded to methyl jasmonate, a known alkaloid inducer, and the expression patterns of these EcWRKY genes were rather similar to those of BIA biosynthetic enzyme genes. Furthermore, tissue expression profiling suggested the involvement of a few subgroup IIc EcWRKYs in the regulation of BIA biosynthesis. Transactivation analysis using luciferase reporter genes harboring the promoters of biosynthetic enzyme genes indicated little activity of subgroup IIc EcWRKYs, suggesting that the transcriptional network of BIA biosynthesis constitutes multiple members. Finally, we investigated the coexpression patterns of EcWRKYs with some transporter genes and discussed the diversified functions of WRKY genes based on a previous finding that CjWRKY1 overexpression in California poppy cells enhanced BIA secretion into the medium.

Highlights

Being sessile, plants have evolved a wide array of defense mechanisms to protect themselves from diverse environmental stresses
CjWRKY1 overexpression in cultured California poppy cells enhanced benzylisoquinoline alkaloid (BIA) accumulation in the culture medium. These findings suggest that the potential WRKY transcription factors (TFs)(s) involved in the regulation of the BIA biosynthetic pathway might be functionally diversified in BIA-producing plant species and gained additional functions associated with BIA production and accumulation in E. californica
After the removal of incomplete and redundant sequences, a total of 50 putative WRKY genes were identified in the California poppy draft genome, which were designated as EcWRKY1 to EcWRKY50 (Table 1)

Summary

Introduction

Plants have evolved a wide array of defense mechanisms to protect themselves from diverse environmental stresses. The WRKY family proteins harbor at least one highly conserved WRKY domain composed of 60 amino acid residues, which includes the conserved N-terminal WRKYGQK sequence followed by a C-terminal zinc finger motif. Group I proteins generally harbor two WRKY domains and a C2H2-type zinc finger motif. Group II proteins harbor a single WRKY domain and a C2H2-type zinc finger, and these can be further classified into five subgroups (IIa–IIe). Group III proteins harbor a single WRKY domain and a C2HC-type zinc finger-like motif. The WRKY proteins modulate the expression of target genes by binding to the W-box DNA motif (C/TTGACC/T) in their promoter regions (Ulker and Somssich, 2004). The structures of several WRKY proteins indicate that the conserved WRKYGQK motif with a β-sheet structure binds to the major groove of the DNA strand of W-box sequence (Yamasaki et al, 2013). The RKYGQK residues are directly involved in DNA binding through extensive hydrophobic contacts with the methyl groups of thymine (Yamasaki et al, 2012)

Methods

Results

Conclusion