ABSTRACT
Transcription factors control many processes in plants and have high potentials to manipulate specialized metabolic pathways. Transcriptional regulation of the biosynthesis of monoterpenoid indole alkaloids (MIAs), nicotine alkaloids, and benzylisoquinoline alkaloids (BIAs) has been characterized using Catharanthus roseus, Nicotiana and Coptis plants. However, metabolic engineering in which specific transcription factors are used in alkaloid biosynthesis is limited. In this study, we characterized the effects of ectopic expression of CjWRKY1, which is a transcriptional activator with many targets in BIA biosynthesis in Coptis japonica (Ranunculaceae) and Eschscholzia californica (California poppy, Papaveraceae). Heterologous expression of CjWRKY1 in cultured California poppy cells induced increases in transcripts of several genes encoding BIA biosynthetic enzymes. Metabolite analyses indicated that the overexpression of the CjWRKY1 gene also induced increases in the accumulation of BIAs such as sanguinarine, chelerythrine, chelirubine, protopine, allocryptopine, and 10-hydroxychelerythrine in the culture medium. Previous characterization of EcbHLH1 and current results indicated that both transcription factors, WRKY1 and bHLH1, are substantially involved in the regulation of BIA biosynthesis. We discuss the function of CjWRKY1 in E. californica cells and its potential for metabolic engineering in BIA biosynthesis.
Subject(s)
Alkaloids/biosynthesis , Benzylisoquinolines/metabolism , Eschscholzia/metabolism , Plant Proteins/genetics , Eschscholzia/genetics , Genes, Plant , Transcriptional ActivationABSTRACT
Isoquinoline alkaloids (IQAs), terpenoid indole alkaloid and nicotine are some of the most studied alkaloids. Recently, several groups have reported that the biosynthesis of these alkaloids is regulated by basic helix-loop-helix (bHLH) transcription factors. Whereas the biosyntheses of nicotine and terpenoid indole alkaloid in Nicotiana plants and Catharanthus roseus are directly or indirectly regulated by Arabidopsis thaliana MYC2 homologs, a non-MYC2-type bHLH transcription factor, CjbHLH1, comprehensively regulates berberine biosynthesis in Coptis japonica. Interestingly, CjbHLH1 homologous genes were found in many IQA-producing plant species, which suggests that non-MYC2-type CjbHLH homologs are specifically associated with IQA biosynthesis. To test whether CjbHLH1 homologs are involved in the biosynthesis of IQA in a plant other than C. japonica, we isolated two genes homologous to CjbHLH1, i.e. EcbHLH1-1 and EcbHLH1-2, from Eschscholzia californica (California poppy). Stable transformants in which the expression levels of EcbHLH1 genes were constitutively suppressed by RNA interference (RNAi) showed a reduced expression of some IQA biosynthetic enzyme genes. A metabolite analysis confirmed that the suppression of EcbHLH1, particularly EcbHLH1-2, caused a decrease in sanguinarine accumulation in transgenic cultured cells. These results indicate that non-MYC2-type EcbHLH1s regulate IQA biosynthesis in California poppy like CjbHLH1 in C. japonica.
Subject(s)
Basic Helix-Loop-Helix Transcription Factors/metabolism , Benzophenanthridines/biosynthesis , Coptis/metabolism , Eschscholzia/metabolism , Plant Proteins/metabolism , Acetates/pharmacology , Benzophenanthridines/chemistry , Berberine/chemistry , Biosynthetic Pathways/drug effects , Biosynthetic Pathways/genetics , Cyclopentanes/pharmacology , Down-Regulation/drug effects , Eschscholzia/drug effects , Eschscholzia/genetics , Gene Expression Regulation, Plant/drug effects , Gene Silencing/drug effects , Isoquinolines/chemistry , Organ Specificity/drug effects , Oxylipins/pharmacology , Plants, Genetically Modified , RNA, Messenger/genetics , RNA, Messenger/metabolism , Seedlings/drug effects , Seedlings/genetics , Sequence Homology, Amino AcidABSTRACT
Chloroplasts are responsible for biosynthesis of salicylic acid (SA) an important signal molecule in plant immunity. EDS5 is a homolog of the MATE (multidrug and toxic compound extrusion) family of transporters, and is essential for SA biosynthesis. It has been speculated that EDS5 would be involved in the export of SA from chloroplasts. However, the subcellular localization of EDS5 remains largely uncharacterized. We demonstrate here that EDS5 is specifically localized to the chloroplast envelope membrane in Arabidopsis. In addition, we found that EDS5 is preferentially expressed in epidermal cells. These findings suggest that EDS5 is responsible for transport of SA from chloroplasts to the cytoplasm in epidermal cells.
