Construction of plant expression vectors
To explore the potential application of BFPvv variants (mBFP) in plants, at least 16 plant expression vectors were constructed in which expression of mBFP or mBFP-mS1C fusion constructs were driven by any one of the three promoters (RbcS, Adh and DR5) and the expressed proteins were targeted to five different subcellular compartments such as cytosol, apoplast, ER, chloroplast, or mitochondria, respectively (Figure 1). The mBFPvvD8 is an improved variant of BFPvv with mutagenesis in 11 amino acids (Kao et al. [2011]). More recently, mBFPvvD9 which can emit at least 30% brighter blue fluorescence than mBFPvvD8 in E. coli, was identified, and carried a point mutation at Arg199. To compare the brightness of blue fluorescence between two different variants of mBFP in plant cells, the mBFPvvD8 and mBFPvvD9 were simultaneously and transiently expressed in opposite counterpart of the same tobacco leaves by agroinfiltration. In addition, the expressed mBFP variant proteins were targeted to cytosol (pBin-R-Cy-mBFP), apoplast (pBin-R-Se-mBFP) and mitochondria (pBin-R-Mt-mBFP), respectively (Figure 1). Our results showed that the mBFPvvD9 emitted much brighter blue light in all examined tissues than the mBFPvvD8 did (Figure 2A). Therefore, the mBFPvvD9 was further used in the following experiments.
Transiently expressed mBFP or mBFP-mS1C fusion proteins in tobacco leaves
To study whether mBFP is a useful reporter in leaf tissues, the mBFP gene expression driven by RbcS promoter was transiently expressed in tobacco leaves by agroinfiltration, and the expressed mBFP proteins were targeted to five different subcellular compartments (Figure 1). The results showed that the expressed mBFP proteins no matter targeted to which compartments all can emit blue fluorescence under UV excitation as compared to a control (Figure 2B), but that stronger blue fluorescence was exhibited in the cytosol, apoplast and ER (Figure 2B). To test the effect of mBFP fusion protein on blue light emission, the mBFP was fused with the C-terminal region (176 amino acids) of ARV σC protein (Figure 1), which the C-terminal domain of σC protein was protease-stable and responsible for the cellular receptor binding (Calvo et al. [2005]). Then the ER-targeted mBFP or mBFP-mS1C fusion protein was simultaneously and transiently expressed in the opposite counterpart of same tobacco leaves by agroinfiltration (Figure 2B). The results showed that mBFP-mS1C fusion protein can emit much brighter blue fluorescence than mBFP protein can in leaf tissues (Figure 2B) or in protoplasts (Figure 2C). Since both expression constructs were driven by the same promoter, posttranscriptional regulation such as mRNA or protein stability could possibly be responsible for the difference. Therefore, RT-PCR assay was carried out to investigate the relative abundance of mRNA, and the results showed that plants transiently expressed mBFP-mS1C fusion construct could accumulate about 1.8 fold more mRNA than that of non-fusion construct (Figure 2D and E). These results suggested that increasing mBFP mRNA stability could be a future strategy for increasing its expression level. Indeed, several potential cryptic introns and mRNA instability elements were present in the mBFP sequence; for instance, AGGAAGT, GTGGACGT, and GTGAACGT, the homologues of intron splicing donor sequences (AGGTAAGT or GTGTACGT), and ATTTG, the homologue of mRNA instability element (ATTTA), were found. In previous research, modifications of GFP by optimization of codon usage and elimination of a cryptic intron can greatly enhance the GFP expression in plants (Chiu et al. [1996]; Haseloff et al. [1997]). Therefore, to further improve the expression level of mBFP in plants, the codon usage might need to be optimized for plants in the future.
mBFP is a useful reporter under hypoxic stress or auxin treatment
Further experiments were conducted to investigate whether or not the mBFP could be induced and imaged in different subcellular compartments and tissue types among plant species under various conditions. Firstly, five mBFP expression vectors driven by synthetic auxin-inducible DR5 promoter were constructed, and the expressed proteins were targeted to five different subcellular compartments (Figure 1). After agroinfiltration of tobacco leaves and flowers of moth orchid, they were then treated with or without 50 μM NAA for 24 hr. The results showed that mBFP blue fluorescence could be imaged in five subcellular compartments of tobacco leaf tissues and the petals of moth orchid, but that it showed brighter blue fluorescence in the cytosol, apoplast, and ER (Figure 3A and B). Secondly, five mBFP expression vectors driven by hypoxia-inducible Adh promoter were constructed, and the expressed proteins were targeted to five different subcellular compartments (Figure 1). Previously, it has been shown that H2O2 levels is significantly increased after O2 deprivation and is required to trigger Adh gene expression (Baxter-Burrell et al. [2002]). Therefore, after agroinfiltration, the inflorescences of moth orchid were treated with or without 1 mM H2O2 for 24 hr. The results showed that although the mBFP blue fluorescence could be observed in all five subcellular compartments, the cytosol, apoplast, ER and chloroplasts showed brighter blue fluorescence (Figure 3C). Previously, it has been reported that the roots of horizontally rather than vertically grown Arabidopsis will be under hypoxia pressure upon growing into the gel of a medium plate (Chung and Ferl [1999]). To investigate if mBFP was suitable for using as a reporter under hypoxic conditions, five transgenic Arabidopsis lines in which the mBFP expression was driven by Adh promoter, along with target proteins expected to accumulate in five different subcellular compartments, were generated. When the transgenic Arabidopsis lines were grown horizontally, the mBFP expression could be induced and the blue fluorescence could be clearly imaged in the root tips of transgenic pBin-A-Cy-mBFP (mBFP accumulated in cytosol) line, pBin-A-Se-mBFP (apoplast) line, pBin-A-Cp-mBFP (chloroplast) line and pBin-A-Mt-mBFP (mitochondria) line as compared with that in corresponding vertically grown plants (Figure 3D). These results suggested that mBFP is a useful reporter under hypoxic environments.
Effect of NADPH on intensity of mBFP blue fluorescence
NADPH is essential for mBFP blue fluorescence under UV excitation because the mechanism of mBFP blue fluorescence emission is due to the augmentation of the intrinsic bound NADPH fluorescence (Chang et al. [2004b]; Kao et al. [2011]). To investigate the effect of NADPH concentration on mBFP blue fluorescence in planta, leaf tissues of wild type or transgenic Arabidopsis pBin-R-Cy-mBFP and pBin-R-Se-mBFP lines in which the expressed mBFP protein was accumulated in cytosol and apoplast, respectively, were infiltrated with different NADPH concentrations (0, 0.1, 0.5, 1 and 2 μM) before imaging. The results showed that the higher the NADPH concentration, the stronger the mBFP blue fluorescence was emitted under UV excitation (Figure 4A). Alternatively, leaf disks of transgenic Arabidopsis (pBin-R-Se-mBFP line) were incubated with different concentrations of NADPH (0, 0.1, 0.5, 1 and 2 μM) for 15 min under suction before imaging. This result is consistent that mBFP blue fluorescence was significantly amplified with increasing NADPH concentrations (Additional file 2: Figure S1). Furthermore, when the protoplasts isolated from tobacco leaves which mBFP was transiently expressed and targeted to cytosol were treated with different NADPH concentrations (0, 0.25 and 0.5 μM), brighter mBFP blue fluorescence was imaged in the presence of higher amounts of NADPH under confocal microscope analysis (Figure 4B). Although NADPH itself displays fluorescence which may interfere with the emission of fluorescence from mBFP-bounded NADPH, fluorescence from the free form of NADPH was not detectable under 0.5 μM NADPH (Figure 4B).
Applications of the fluorescent proteins as biosensors are very useful for quantitative live imaging (Okumoto et al. [2012]). Previously, it has been shown that mBFPvvD8 could be applied in a Förster resonance energy transfer (FRET) assay (Kao et al. [2011]). Although the NADPH participates in many oxido-reductive reactions, and might be universally present in plant cells, its concentration may vary significantly in different subcellular compartments and tissue types. In this study, the brightness of mBFP fluorescence was shown to be positively correlated with the NADPH concentration (Figure 4). The results suggested that the mBFP might have the potential to be further developed as an NADPH biosensor in planta.
Subcellular localization of mBFP by confocal microscope analysis
Previous study have shown that signal peptide which helps to direct the target protein into subcellular compartments may be masked by the target protein itself, and thus causes mis-targeting or multiple targeting of the protein (Tanz et al. [2013]). In this study, mBFP accumulation in apoplast was obvious in the tested tissues (Figures 2, 3 and 4). To investigate if mBFP was correctly targeted into other compartments, the protoplasts were isolated from leaves of five transgenic Arabidopsis lines in which mBFP proteins were expected to accumulate in cytosol (pBin-R-Cy-mBFP line), ER (pBin-R-Er-mBFP and pBin-R-Er-mBFP-mS1C lines), chloroplast (pBin-R-Cp-mBFP line) and mitochondria (pBin-R-Mt-mBFP line), respectively, and were imaged under confocal microscope. The ER-resident protein used the same secretory pathway as that of protein targeted to apoplast, except for having an additional ER-retention signal in the C-terminal. Although no ER-specific marker was used in this study, the mBFP or mBFP-mS1C protein was seen to be intracellularly present, suggesting that it was ER-localized (Figures 2C and 5). The mBFP targeted to chloroplast was confirmed because of co-localization of mBFP blue fluorescence with chlorophyll auto-fluorescence (Figure 5). Therefore, our result showed that mBFP and mBFP-mS1C fusion proteins were accumulated in the correct subcellular compartments except mitochondria (Figure 5), because the green fluorescence of MitoTracker was only partially co-localized with mBFP blue fluorescence in the protoplasts isolated from transgenic Arabidopsis (pBin-R-Mt-mBFP line) (Figure 5). Therefore, the protoplasts were isolated from tobacco leaves which transiently expressed pBin-R-Mt-mBFP construct for further investigation. The result was consistent with above that mBFP is partially co-localized with Mitotracker (Additional file 3: Figure S2). It suggested that the mitochondria-targeted signal peptide might have been masked by mBFP, thus making it unable to be imported or inefficiently imported into mitochondria. Therefore, it will be better to avoid fusing mBFP in the N-terminal when apply a fusion mBFP tag protein for importing to plant mitochondria in future applications.