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The blue fluorescent protein from Vibrio vulnificus CKM-1 is a useful reporter for plant research
Jin-Min Tu1, Ming-Chung Chang2, Lynn Lh Huang3
1Institute of Tropical Plant Sciences, National Cheng Kung University, 1 University Rd, Tainan, 701, Taiwan.
Background:
The mBFP is an improved variant of NADPH-dependent blue fluorescent protein that was originally identified from the non-bioluminescent pathogenic bacteria Vibrio vulnificus CKM-1. To explore the application of mBFP in plants, the mBFP gene expression was driven by one of the three promoters, namely, leaf-specific (RbcS), hypoxia-inducible (Adh) or auxin-inducible (DR5) promoters, in different plant tissues such as leaves, roots and flowers under diverse treatments. In addition, the expressed mBFP protein was targeted to five subcellular compartments such as cytosol, endoplasmic reticulum, apoplast, chloroplast and mitochondria, respectively, in plant cells.
Results:
When the mBFP was transiently expressed in the tobacco leaves and floral tissues of moth orchid, the cytosol and apoplast exhibited brighter blue fluorescence than other compartments. The recombinant mBFP-mS1C fusion protein exhibited enhanced fluorescence intensity that was correlated with more abundant RNA transcripts (1.8 fold) as compared with a control. In the root tips of horizontally grown transgenic Arabidopsis, mBFP could be induced as a reporter under hypoxia condition. Furthermore, the mBFP was localized to the expected subcellular compartments, except that dual targeting was found when the mBFP was fused with the mitochondria-targeting signal peptide. Additionally, the brightness of mBFP blue fluorescence was correlated with NADPH concentration.
Conclusion:
The NADPH-dependent blue fluorescent protein could serve as a useful reporter in plants under aerobic or hypoxic condition. However, to avoid masking the mitochondrial targeting signal, fusing mBFP as a fusion tag in the C-terminal will be better when the mBFP is applied in mitochondria trafficking study. Furthermore, mBFP might have the potential to be further adopted as a NADPH biosensor in plant cells. Future codon optimization of mBFP for plants could significantly enhance its brightness and expand its potential applications.
Insights
The improved NADPH-dependent blue fluorescent protein (mBFP) functions as a reporter in plants under varying oxygen levels. Its brightness correlates with NADPH concentration, suggesting potential as a biosensor.
Area of Science:
- Plant molecular biology
- Biochemistry
- Fluorescent protein technology
Background:
- The study investigates an improved variant of NADPH-dependent blue fluorescent protein (mBFP), derived from Vibrio vulnificus.
- mBFP gene expression was explored in plants using leaf-specific, hypoxia-inducible, and auxin-inducible promoters.
- The protein was targeted to various subcellular compartments, including cytosol, endoplasmic reticulum, apoplast, chloroplast, and mitochondria.
Purpose of the Study:
- To assess the utility of mBFP as a reporter in plant tissues under different conditions.
- To investigate the subcellular localization and fluorescence characteristics of mBFP in plant cells.
- To explore the potential of mBFP as a biosensor for NADPH levels in plants.
Main Methods:
- Transient expression of mBFP in tobacco leaves and moth orchid floral tissues.
- Utilizing specific promoters (RbcS, Adh, DR5) to drive mBFP expression in transgenic Arabidopsis.
- Targeting mBFP to different subcellular compartments using signal peptides.
- Analyzing fluorescence intensity and correlating it with RNA transcripts and NADPH concentration.
Main Results:
- Brighter blue fluorescence was observed in the cytosol and apoplast of transiently expressed mBFP.
- Recombinant mBFP-mS1C fusion protein showed enhanced fluorescence intensity, correlating with increased RNA transcripts.
- mBFP acted as an inducible reporter in Arabidopsis root tips under hypoxia and localized to expected compartments, with dual targeting observed for mitochondria-fused protein.
- mBFP fluorescence brightness was directly correlated with cellular NADPH concentration.
Conclusions:
- NADPH-dependent blue fluorescent protein (mBFP) is a viable reporter in plants under both aerobic and hypoxic conditions.
- For mitochondrial studies, C-terminal fusion of mBFP is recommended to avoid masking targeting signals.
- mBFP shows potential as a NADPH biosensor in plant cells, with future codon optimization expected to enhance its performance and applications.
