Comparison of the MpEF1 alpha and CaMV35 promoters for application in Marchantia polymorpha overexpression studies

Abstract

Constitutive promoters are essential tools for analyses of gene functions by transgenic approaches. For overexpression and silencing studies of genes, a ubiquitous and strong expression of genes under investigation as well as selection markers is preferred. For future applications in the emerging basal plant model system Marchantia polymorpha, a liverwort, activities of the viral 35S cauliflower mosaic virus promoter and the endogenous elongation factor 1 alpha (MpEF1 alpha) promoter were analyzed. Expression of the reporter gene beta-glucuronidase (GUS), driven by the CaMV35 and MpEF1 alpha promoters, was compared throughout plant development. Significant differences were observed between the two promoter activities. The CaMV35 promoter yields a weak reporter gene expression in the meristematic zones but drives a strong expression in the thallus. The MpEF1 alpha promoter causes a strong meristematic GUS expression and is more active in female sexual tissues. Overall, the MpEF1 alpha promoter seems to be the better option for obtaining a strong and ubiquitous transgene expression. Furthermore, a whole mount in situ hybridization protocol for Marchantia was established. Analysis of MpEF1 alpha mRNA transcript in intact, whole tissues showed an expression pattern that is overall similar to the pattern of the GUS reporter gene expression driven by the MpEF1 alpha promoter, including strong expression in meristematic zones. The whole mount technique reported here can be used to determine the mRNA expression in intact gemmae and archegonia, and has the potential to be applied for screening large numbers of transgenic plants, for instance to identify knock-down mutants.