Abstract
With respect to the biosynthesis of plant alkaloids, that of benzylisoquinoline alkaloids (BIAs) has been the most investigated at the molecular level. Previous investigations have shown that the biosynthesis of BIAs is comprehensively regulated by WRKY and bHLH transcription factors, while promoter analyses of biosynthesis enzyme-encoding genes have also implicated the involvement of members of the APETALA2/ethylene responsive factor (AP2/ERF) superfamily. To investigate the physiological roles of AP2/ERF transcription factors in BIA biosynthesis, 134 AP2/ERF genes were annotated using the draft genome sequence data of Eschscholzia californica (California poppy) together with transcriptomic data. Phylogenetic analysis revealed that these genes could be classified into 20 AP2, 5 RAV, 47 DREB, 60 ERF and 2 Soloist family members. Gene structure, conserved motif and orthologous analyses were also carried out. Gene expression profiling via RNA sequencing in response to methyl jasmonate (MeJA) indicated that approximately 20 EcAP2/ERF genes, including 10 group IX genes, were upregulated by MeJA, with an increase in the expression of the transcription factor-encoding gene EcbHLH1 and the biosynthesis enzyme-encoding genes Ec6OMT and EcCYP719A5. Further quantitative RT-PCR confirmed the MeJA responsiveness of the EcAP2/ERF genes, i.e., the increased expression of 9 group IX, 2 group X and 2 group III ERF subfamily genes. Transactivation activity of group IX EcAP2/ERFs was also confirmed by a luciferase reporter assay in conjunction with the promoters of the Ec6OMT and EcCYP719A5 genes. The physiological roles of AP2/ERF genes in BIA biosynthesis and their evolution in the regulation of alkaloid biosynthesis are discussed.
Highlights
Plants produce structurally divergent low-molecular-weight chemical compounds commonly classified as phenylpropanoids, aromatic polyketides, terpenoids and alkaloids to protect themselves against biotic and abiotic stresses such as pathogen or herbivore attack and UV irradiation
This analysis revealed that a GCC-box-like sequence that may be a target of APETALA2/Ethylene responsive factor (AP2/ERF) family proteins in the promoter of benzylisoquinoline alkaloids (BIAs) biosynthesis enzyme-encoding genes is possibly involved in the regulation of BIA biosynthesis
Since expression analysis indicated that many group IX EcAP2/ERF genes have methyl jasmonate (MeJA) responsiveness, we further investigated the role of these EcAP2/ERF proteins in the transactivation of BIA biosynthesis enzyme-encoding genes via a transient luciferase (LUC) reporter assay (Fig. 8)
Summary
Plants produce structurally divergent low-molecular-weight chemical compounds commonly classified as phenylpropanoids, aromatic polyketides, terpenoids and alkaloids to protect themselves against biotic and abiotic stresses such as pathogen or herbivore attack and UV irradiation. Genes have been largely identified and characterized; transcription factors involved in the regulation of BIA biosynthesis have been isolated, e.g., WRKY superfamily transcription factors such as CjWRKY1 from C. japonica and PsWRKY from P. somniferum and basic helix-loop-helix (bHLH) transcription factors such as CjbHLH1 from C. japonica and EcbHLH1-1/1-2 from E. californica[5,6,7,8] Both CjWRKY1 and CjbHLH1 function as comprehensive transcription factors; that is, they upregulate the expression of most BIA biosynthesis enzyme-encoding genes in C. japonica cells through direct interaction with their promoter region[6,9]. To elucidate the detailed regulatory mechanism of gene expression and investigate other transcription factors in BIA biosynthesis, the characterization of cis-acting elements in the promoter region of BIA biosynthesis enzyme-encoding genes was performed in C. japonica cells[9]. Ectopic expression of Arabidopsis thaliana and Glycine max AP2/ERF transcription factors in E. californica and P. somniferum cells increased the transcript levels of several BIA biosynthesis enzyme-encoding genes and influenced BIA production[14]
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