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.
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.
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.