A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome of<i>Marchantia polymorpha</i>

Boehm, Christian R.; Ueda, Minoru; Nishimura, Yoshiki; Shikanai, Toshiharu; Haseloff, Jim

doi:10.1093/pcp/pcv160

Cited by 22 publications

(25 citation statements)

References 52 publications

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“…In the blue/cyan range, mTurquoise2 was chosen for its very low p K a (p K a mTurquoise2 = 3.1), which was the lowest of all proteins tested in this study (Goedhart et al., ). Despite being a relatively new FP, it has been successfully expressed in several plant species such as A. thaliana , N. benthamiana, and Marchantia polymorpha (Boehm, Ueda, Nishimura, Shikanai, & Haseloff, ; Ghareeb, Laukamm, & Lipka, ; Rolland, Badger, & Price, ).…”

Section: Resultsmentioning

confidence: 99%

I see the light! Fluorescent proteins suitable for cell wall/apoplast targeting in Nicotiana benthamiana leaves

Stoddard

Rolland

2019

Plant Direct

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Correct subcellular targeting is crucial for protein function. Protein location can be visualized in vivo by fusion to a fluorescent protein ( FP ). Nevertheless, despite intense engineering efforts, most FP s are dim or completely quenched at low pH (<6). This is particularly problematic for the study of proteins targeted to acidic compartments such as vacuoles ( pH ~ 3–6) or plant cell walls ( pH ~ 3.5–8.3). Plant cell walls play important roles (e.g. structural/protective role, control of growth/morphogenesis), are diverse in structure and function, and are highly dynamic (e.g. during cell growth, in response to biotic/abiotic stresses). To study and engineer plant cell walls, it is therefore critical to identify robust tools which can be used to locate proteins expressed in the apoplast. Here we used a transient expression assay in Nicotiana benthamiana leaves to test a range of FP s in vivo , and determined which ones retained strong fluorescence in the acidic environment of the apoplast. We selected 10 fluorescent proteins with a range of in vitro properties; two historical FP s and eight FP s with in vitro properties suggesting lower pH sensitivity or improved brightness, some of which had never been tested in plants prior to our study. We targeted each FP to the cytosol or the apoplast and compared the fluorescence in both compartments, before testing the in vivo pH sensitivity of FP s across a pH 8–4 gradient. Our results suggest that mT urquoise2, mN eonGreen, and mC herry are suited to tracking proteins in the apoplast under dynamic pH conditions. These fluorescent proteins may also be useful in other acidic compartments such as vacuoles.

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Section: Resultsmentioning

confidence: 99%

I see the light! Fluorescent proteins suitable for cell wall/apoplast targeting in Nicotiana benthamiana leaves

Stoddard

Rolland

2019

Plant Direct

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“…This approach was recently extended to the expression of a fluorescent reporter gene from the Marchantia plastome (Boehm et al 2016), which allows characterization of plastid promoters in this model system (see Fig. 3).…”

Section: Synthetic Botanymentioning

confidence: 99%

Synthetic Botany

Boehm

Pollak

Purswani³

et al. 2017

Cold Spring Harb Perspect Biol

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Plants are attractive platforms for synthetic biology and metabolic engineering. Plants' modular and plastic body plans, capacity for photosynthesis, extensive secondary metabolism, and agronomic systems for large-scale production make them ideal targets for genetic reprogramming. However, efforts in this area have been constrained by slow growth, long life cycles, the requirement for specialized facilities, a paucity of efficient tools for genetic manipulation, and the complexity of multicellularity. There is a need for better experimental and theoretical frameworks to understand the way genetic networks, cellular populations, and tissue-wide physical processes interact at different scales. We highlight new approaches to the DNA-based manipulation of plants and the use of advanced quantitative imaging techniques in simple plant models such as Marchantia polymorpha. These offer the prospects of improved understanding of plant dynamics and new approaches to rational engineering of plant traits.

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“…In vitro assays of inserted genes with several synonymous codons show that translation efficiency does not always correlate with codon usage in plastid mRNAs (Nakamura and Sugiura, 2007), but they have been used in several codon optimization studies (Lutz et al, 2001;Ye et al, 2001;Franklin et al, 2002;Lenzi et al, 2008;Jabeen at al., 2010;Madesis et al, 2010;Gisby et al, 2011;Wang et al, 2015b;Boehm et al, 2016;Nakamura et al, 2016). While some studies achieved significant increases in expression (75-to 80-fold) after codon optimization (Franklin et al, 2002;Gisby et al, 2011), other studies observed negligible enhancement (Ye et al, 2001;Lenzi et al, 2008;Daniell et al, 2009;Wang et al, 2015b;Nakamura et al, 2016).…”

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confidence: 99%

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

et al. 2016

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Codon optimization based on psbA genes from 133 plant species eliminated 105 (human clotting factor VIII heavy chain [FVIII HC]) and 59 (polio VIRAL CAPSID PROTEIN1 [VP1]) rare codons; replacement with only the most highly preferred codons decreased transgene expression (77-to 111-fold) when compared with the codon usage hierarchy of the psbA genes. Targeted proteomic quantification by parallel reaction monitoring analysis showed 4.9-to 7.1-fold or 22.5-to 28.1-fold increase in FVIII or VP1 codon-optimized genes when normalized with stable isotope-labeled standard peptides (or housekeeping protein peptides), but quantitation using western blots showed 6.3-to 8-fold or 91-to 125-fold increase of transgene expression from the same batch of materials, due to limitations in quantitative protein transfer, denaturation, solubility, or stability. Parallel reaction monitoring, to our knowledge validated here for the first time for in planta quantitation of biopharmaceuticals, is especially useful for insoluble or multimeric proteins required for oral drug delivery. Northern blots confirmed that the increase of codonoptimized protein synthesis is at the translational level rather than any impact on transcript abundance. Ribosome footprints did not increase proportionately with VP1 translation or even decreased after FVIII codon optimization but is useful in diagnosing additional rate-limiting steps. A major ribosome pause at CTC leucine codons in the native gene of FVIII HC was eliminated upon codon optimization. Ribosome stalls observed at clusters of serine codons in the codon-optimized VP1 gene provide an opportunity for further optimization. In addition to increasing our understanding of chloroplast translation, these new tools should help to advance this concept toward human clinical studies.

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A Cyan Fluorescent Reporter Expressed from the Chloroplast Genome ofMarchantia polymorpha

Cited by 22 publications

References 52 publications

I see the light! Fluorescent proteins suitable for cell wall/apoplast targeting in Nicotiana benthamiana leaves

I see the light! Fluorescent proteins suitable for cell wall/apoplast targeting in Nicotiana benthamiana leaves

Synthetic Botany

Codon Optimization to Enhance Expression Yields Insights into Chloroplast Translation

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