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, 57 (2), 291-9

A Cyan Fluorescent Reporter Expressed From the Chloroplast Genome of Marchantia Polymorpha

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A Cyan Fluorescent Reporter Expressed From the Chloroplast Genome of Marchantia Polymorpha

Christian R Boehm et al. Plant Cell Physiol.

Abstract

Recently, the liverwort Marchantia polymorpha has received increasing attention as a basal plant model for multicellular studies. Its ease of handling, well-characterized plastome and proven protocols for biolistic plastid transformation qualify M. polymorpha as an attractive platform to study the evolution of chloroplasts during the transition from water to land. In addition, chloroplasts of M. polymorpha provide a convenient test-bed for the characterization of genetic elements involved in plastid gene expression due to the absence of mechanisms for RNA editing. While reporter genes have proven valuable to the qualitative and quantitative study of gene expression in chloroplasts, expression of green fluorescent protein (GFP) in chloroplasts of M. polymorpha has proven problematic. We report the design of a codon-optimized gfp varian, mturq2cp, which allowed successful expression of a cyan fluorescent protein under control of the tobacco psbA promoter from the chloroplast genome of M. polymorpha. We demonstrate the utility of mturq2cp in (i) early screening for transplastomic events following biolistic transformation of M. polymorpha spores; (ii) visualization of stromules as elements of plastid structure in Marchantia; and (iii) quantitative microscopy for the analysis of promoter activity.

Keywords: Chloroplast; Codon bias; GFP; Marchantia polymorpha; Reporter gene; Stromules.

Figures

Fig. 1
Fig. 1
Comparison of the mturquoise2 and mturq2cp coding regions. The amino acid sequence is shown below the aligned nucleotide sequences. Changed nucleotides are shaded.
Fig. 2
Fig. 2
Generation and verification of homoplasmic chloroplast transformants. (A) Maps of the pCS CL0*b transformation vector (top), target region in the wild type (WT) cpDNA (middle) and the same region after integration of the cassette embracing the aadA and mturq2cp genes (bottom). Filled triangles indicate the rRNA operon promoter (black) and the psbA promoter (white) of the tobacco chloroplast genome, respectively. Filled rectangles indicate the psbA terminator of the tobacco chloroplast genome (black) and a hybrid 3′-untranslated region composed of the prokaryotic double terminator BBa_B0015 and the rps16 terminator of the tobacco chloroplast genome (white), respectively. Black arrows indicate the position and orientation of the PCR primers pHP f and pHP r used for the detection of WT or transplastomic (CL0*b) cpDNA. Gray arrows indicate the position and orientation of the PCR primers pB f and pB r used for confirmation of the integrity of the reporter gene in transplastomic (CL0*b) lines. Primer sequences are provided in the Materials and Methods. (B) PCR analysis of genomic DNA isolated from WT and transplastomic (CL0*b) plants. Homoplasmy (top) and integrity of the reporter gene (bottom) were confirmed for transplastomic (CL0*b) lines after 4 months of repetitive subculture under selective conditions.
Fig. 3
Fig. 3
Analysis of chloroplast fluorescence in vivo. (A) Widefield micrographs of wild type (WT) and transplastomic (CL0*b) thalli imaged under GFPlongpass and CFP filter settings. Scale bar = 1 mm. (B) Confocal micrographs of WT and transplastomic (CL0*b) thalli imaged under Autofluorescence and CFP channel emission settings. Scale bar = 10 µm. (C) Stromules visualized by chloroplast-localized mTurquoise2 in transplastomic (CL0*b) M. polymorpha (white arrows). Shown is a subsection of image CL0*b Merge from (B). Scale bar = 10 µm. (D) Normalized chloroplast fluorescence465–495 nm of transplastomic (CL0*b) lines relative to the WT. Three different thallus sections from each line were subjected to confocal imaging to capture micrographs under Autofluorescence and CFP channel emission settings upon excitation using the argon laser at 458 nm. Chloroplast-localized CFP channel intensity was normalized for tissue depth via the corresponding Autofluorescence channel intensity. Error bars represent the SD of normalized chloroplast fluorescence over the cyan spectral window 465–495 nm between three different thallus sections.
Fig. 4
Fig. 4
Detection of chloroplast-expressed mTurquoise2 in transgenic lines of Marchantia polymorpha. (A) M. polymorpha protein extracts separated by SDS–PAGE. Protein extracts from wild type (WT) and transplastomic (CL0*b) lines of M. polymorpha were separated by 4–12% SDS–PAGE, and visualized by in-gel fluorescence (left) and subsequent Coomassie stain (right). The fluorescence image was generated using a custom imaging device described in the Materials and Methods for visualization of CFP bands (emission 486/10 nm, green) and marker (emission 540/10 nm, red). (B) Untagged and His6-tagged mTurquoise2 separated by SDS–PAGE. Untagged mTurquoise2 was expressed from plasmids pCS CL0*b (psbA promoter) in BL21 E. coli and pCRB SREI (T7 promoter) in T7 Express E. coli. His6-tagged mTurquoise2 was expressed from plasmid pCRB SREI6his (T7 promoter) and purified by affinity chromatography. The rightmost lanes contain 50 ng of unboiled and boiled purified protein, respectively. Visualization by in-gel fluorescence and Coomassie stain was conducted as described above. (C) Standard curve for quantification of mTurquoise2 based on in-gel fluorescence. Serial dilutions of 1 µg purified of mTurquoise2 were separated by 4–12% SDS–PAGE and the band intensity quantified by in-gel fluorescence as described in the Materials and Methods. Levels of mTurquoise2 extracted from transplastomic (CL0*b) M. polymorpha (red cross) were estimated by linear regression analysis on densities of fluorescent target bands as shown in (A). Error bars represent the SD of average band density between three different experiments.

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