4. Materials and Methods

4.1. Vector Constructs
The DNA fragments of both rRTp and sGFP were amplified from a pSB-RTG plasmid [14]; the sGFP is identical to commercially available eGFP (Clontech, USA). The Kz sequence (AACAATGGC) identified for plants [16,17,18] and the recombination sites (attB1 and attB2) for a gateway cloning system [30] were introduced as a non-homologous overhang in the designed PCR primers, and the chimeric fragments of sGFP, K-sGFP and KR-sGFP with attB1-B2 sites were prepared by an overlap extension-PCR [31]. The PCR primers used in this study are listed in the Table S1. All PCR reactions were performed using Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, USA).
The three chimeric gene fragments (sGFP, K-sGFP, and KR-sGFP) were cloned into the pB2GW7 vector [32] containing a 35S promoter for constitutive expression using a gateway cloning system. Gateway cloning procedures were performed using Gateway® BP Clonase® II Enzyme Mix and Gateway® LR Clonase® II Enzyme Mix following the manufacturer’s instructions (Invitrogen, Waltham, MA, USA).

4.2. Plant Materials
The dehulled seeds of rice (Oryza sativa L. Japonica cv. “Ilmi”) were sterilized by treatment once in 70% ethanol for 1 min and twice in 2% sodium hypochlorite for 20 min and then planted on Murashige and Skoog (MS) agar medium after washing five times with distilled water. The rice seedlings were grown in the dark for 10 days, and then illuminated with white light for 20 h prior to protoplast isolation.
The restricted exposure of the plants to light (20 h) was used to reduce the autofluorescence background resulting from the presence of chlorophyll. The formation of RuBisCo and the biosynthesis of chlorophyll in rice leaves have been reported to be the greatest during greening of etiolated leaf tissues [33,34]. During this process, photosynthesis-related proteins are detected within 2 h of illumination with white light [35], the stromal structures of chloroplast are evident following illumination for 16 h [36], and chlorophyll formation begins subsequently [33,36]. In several preliminary experiments, we established that the optimum illumination time was 16–20 h, which resulted in chlorophyll formation but not to a level where autofluorescence was evident in the images using a fluorescein isothiocyanate (FITC) filter (data not shown). Consequently, 20 h of illumination was used for the FCA analysis of protoplasts in this study.

4.3. Protoplast Isolation and Transfection
With minor modifications, protoplasts were isolated as described by Bart [37]. Strips (0.5 mm) were cut with new razor blades from 0.5 g leaf and stem tissue sections and then immediately immersed in 15 mL of enzyme solution (Cellulase R-10 and Macerozyme R-10; Yakult Honsha, Japan) for the digestion of the cell walls. The plate was put into a vacuum desiccator and the vacuum infiltration for 10 min was applied to increase digestion efficiency. Following the incubation with gentle shaking of 50 rpm at RT in the dark for 4 h, the enzyme reaction was stopped by the addition of 15 mL W5 solution [37]. Cell debris were filtered twice through 70 μm and 40 μm BD Falcon™ cell strainers (BD, Franklin Lakes, NJ, USA); furthermore, the protoplasts were pelleted and suspended in Mmg buffer solution [37] at 107 protoplasts·mL−1 for PEG-transfection. The number of protoplasts was analyzed using a Marienfeld hemocytometer counting system (Marienfeld-Superior, Berlin, Germany). For transfection, an equal volume of 40% PEG-3350 (Sigma, St. Louis, MO, USA) solution [37] in 0.6 M mannitol and 100 mM CaCl2 was added to 110 µL of the protoplasts (around 106 cells) and DNA solution; the mixture was incubated for 15 min. The protoplasts were washed twice with each of the two volumes of W5 and 1 mL of incubation solution and were finally resuspended in 1 mL of incubation solution and incubated at 28 °C in the dark overnight. All plasticware used for protoplasts was coated with 5% calf serum by swirling for 10 s and all buffers were filtered using 0.45 µm syringe filter (Sartorius, Gottingen, Germany).

4.4. Microscopy
For the analysis of the subcellular localization, a 5 μg aliquot of each plasmid DNA was transfected into rice protoplasts using PEG-mediated transfection and GFP signals from transfected protoplasts were observed using a Carl Zeiss LSM700 inverted confocal microscope and the image acquisition software ZEN 2009 Light Edition (Carl Zeiss, Oberkochen, Germany). A sGFP fluorescence was detected with 488 nm excitation and 505–530 nm emission wavelengths; the chlorophyll fluorescence was analyzed with 555 nm excitation and >650 nm emission. The fluorescence intensities of images were analyzed using a Histogram tool of the image acquisition software ZEN 2009 Light Edition (Carl Zeiss) following the manufacturer’s instructions. The mean intensities of images were normalized using a sGFP construct, and the ratios of the mean intensity were introduced using scale bars in Figure 1B and Figure 2C.

4.5. Analysis of sGFP Expression in Protoplasts
For the expression analysis, protoplasts (approximately 1 × 106) were centrifuged, the supernatant was removed, and the pellet was immediately frozen in LN2 and stored at −80 °C until RNA and protein extraction.
Total RNA was extracted from rice protoplasts using the RNeasy® Plant mini kit (Qiagen, Hilden, Germany) and the 1st strand cDNA was synthesized using a mRNA selective PCR kit (AMV) ver. 1.1 (Takara, Shiga, Japan) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using a Thunderbird™ SYBR® qPCR mix (Toyobo, Osaka, Japan); the SYBR-fluorescence signals were detected and quantified using a CFX96™ Real-Time PCR system (Bio-Rad, Foster City, CA, USA). The quantified SYBR-fluorescence of sGFP transcript was analyzed using CFX Manager™ ver. 2.1 (Bio-Rad). A rice ubiquitin gene (AK061988) was used as a reference gene to normalize the expression data for each sGFP transcript.
The protoplast pellet was suspended in 160 μL of phosphate buffered saline (PBS) (Caisson Laboratories, North Logan, UT, USA) and 40 μL of 5× SDS-PAGE loading buffer (Biosesang, Seongnam, Korea) and heated in boiling water for 10 min. The boiled protoplasts were centrifuged at 4 °C; 5 μL of the supernatant was loaded to each of two 12% acrylamide gels for SDS-PAGE. Following the migration, the proteins in one gel were stained with EZ-Silver Staining Kit for Protein (Biosesang) to visualize the loaded amount and quality, while the proteins in the other gel were transferred to PVDF membrane (Whatman, Kent, UK) using Trans-Blot® SD Semi-Dry Electrophoretic Transfer Cell (Bio-Rad). The sGFP chimeric proteins were blotted using rabbit polyclonal antibody of GFP (Abcam, Cambridge, UK) at 1:5000 dilution and secondary anti-rabbit alkaline phosphatase conjugate (Promega, Madison, WI, USA) at 1:5000 dilution. The blots were incubated with Novex® AP Chemiluminescent Substrate (CDP-Star®) (Invitrogen). The developed bands were imaged using a LAS-4000 luminescence detector; the intensity of the bands was quantified using the LAS 4000 software (Fujifilm, Tokyo, Japan).

4.6. Flow Cytometric Analysis
The expression of sGFP was monitored at 24 h following transfection using a FACS Aria II flow cytometry system (BD). Protoplasts were pelleted and resuspended in an appropriate volume of PBS buffer and immediately subjected to flow cytometry. Flow cytometry analysis was performed as described by Hagenbeek and Rock [9]. The cell density and sample injection speed was adjusted to the particular experiment for the best possible yield or fastest achievable speed. Live protoplasts were gated using the forward and side scattering fields for data acquisition; irregular shaped protoplasts were regarded as dead or aggregated and were gated out during the data collection. As shown in Figure 6, non-single cells, such as cell-multiplets or cell debris, were excluded through a large initial gating of a side scattering vs. a forward scattering (FSC)-A, and two more gating steps of a SSC-height vs. -width and a FSC-height vs. -width were used more strictly to exclude non-single cells from the next analysis. The green fluorescence of sGFP was excited by a 488 nm laser and detected using the FITC-A channel. For each analysis, the sGFP fluorescence signals of 105 events in the gated population were collected and detected using the FITC-A channel and analyzed using FACS Diva software 6.0 (BD). Control protoplasts were prepared through PEG transfection with no plasmid as a negative control. The fluorescence intensities of the control samples (100–103), derived from chlorophyll in the chloroplasts, provided the autofluorescence background value for FCA of other protoplasts expressing sGFP proteins.
Figure 6  The scatter plots of representative data from FCA of rice protoplasts. The protoplast populations were initially gated through a forward scatter (FSC) area versus a side scatter (SSC) area to exclude non-single cells such as cell-multiplets or cell debris. Afterwards, further gates were performed using a SSC-height vs. SSC-width dot plot and a FSC-height vs. FSC-width to more strictly select single protoplast cells.