Abstract
The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs), a plant-specific WRKY-type TF, CjWRKY1, and a basic helix-loop-helix TF, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4′OMT and CYP719A1) were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC) reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay and by a chromatin immunoprecipitation assay. In addition, CjbHLH1 also activated transcription from truncated 4′OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. japonica are discussed.
Highlights
Higher plants produce a large variety of secondary metabolites, which are commonly classified as phenylpropanoids, aromatic polyketides, terpenoids, and alkaloids
These include genes involved in the condensation of dopamine and 4-hydroxyphenylacetaldehyde to (S)-norcoclaurine by (S)-norcoclaurine synthase (NCS; Minami et al, 2007), the conversion of (S)-norcoclaurine to (S)-reticuline by the sequential reactions of (S)-norcoclaurine 6-O-methyltransferase (6OMT; Sato et al, 1994; Morishige et al, 2000), (S)-coclaurine-N-methyltransferase (CNMT; Choi et al, 2002), (S)-N-methylcoclaurine 3 -hydroxylase (CYP80B2; Ikezawa et al, 2003), and (S)-3 -hydroxy-N-methylcoclaurine4 -O-methyltransferase (4 OMT; Morishige et al, 2000), (S)-reticuline to berberine by berberine bridge enzyme (BBE; Minami et al, 2008), (S)-scoulerine-9-O-methyltransferase (SMT; Takeshita et al, 1995), (S)-canadine synthase (CYP719A1; Ikezawa et al, 2003) and (S)-tetrahydroprotoberberine oxidase (THBO; Matsushima et al, 2012; Figure 1)
transcription factors (TFs) found to be associated with alkaloid biosynthesis belong to the WRKY, ERF, and basic helix-loop-helix (bHLH) groups, which are known to be involved in defense responses
Summary
Higher plants produce a large variety of secondary metabolites, which are commonly classified as phenylpropanoids, aromatic polyketides, terpenoids, and alkaloids. The biosynthetic pathways of several alkaloids have been well investigated at the molecular level due to their chemical uniqueness as well as their economic importance (Dewey and Xie, 2013; Hagel and Facchini, 2013; Sato, 2013; Zhu et al, 2014) Examples of these alkaloids include nicotine in Nicotiana tabacum (Solananceae), monoterpenoid indole alkaloids (MIAs), vinblastine and vincristine in Catharanthus roseus (Apocynaceae), isoquinoline alkaloids (IQAs), berberine in Coptis japonica (Ranunculaceae), sanguinarine in Eschscholzia californica (Papaveraceae) and morphine in Papaver somniferum (Papaveraceae). These include genes involved in the condensation of dopamine and 4-hydroxyphenylacetaldehyde to (S)-norcoclaurine by (S)-norcoclaurine synthase (NCS; Minami et al, 2007), the conversion of (S)-norcoclaurine to (S)-reticuline by the sequential reactions of (S)-norcoclaurine 6-O-methyltransferase (6OMT; Sato et al, 1994; Morishige et al, 2000), (S)-coclaurine-N-methyltransferase (CNMT; Choi et al, 2002), (S)-N-methylcoclaurine 3 -hydroxylase (CYP80B2; Ikezawa et al, 2003), and (S)-3 -hydroxy-N-methylcoclaurine4 -O-methyltransferase (4 OMT; Morishige et al, 2000), (S)-reticuline to berberine by berberine bridge enzyme (BBE; Minami et al, 2008), (S)-scoulerine-9-O-methyltransferase (SMT; Takeshita et al, 1995), (S)-canadine synthase (CYP719A1; Ikezawa et al, 2003) and (S)-tetrahydroprotoberberine oxidase (THBO; Matsushima et al, 2012; Figure 1)
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