PMC:4307275 / 5432-19937 JSONTXT

{"target":"http://pubannotation.org/docs/sourcedb/PMC/sourceid/4307275","sourcedb":"PMC","sourceid":"4307275","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4307275","text":"2. Results\n\n2.1. Expression of a Green Fluorescent Protein in Rice Protoplasts\nA synthetic GFP (sGFP), a derivative with an increased brightness adjusted to plant systems [19], was used for FCA using rice protoplasts. Three kinds of sGFP-expressing vectors were constructed (see Figure 1A): sGFP, as a control construct; K-sGFP, a construct with plant-type Kz sequence near a translation start codon; and KR-sGFP, a construct with a Kz sequence and a transit peptide sequence of the rice RuBisCo small subunit (rRTp, R) for N-terminal fusion to sGFP. For the transfection with each vector DNAs, rice protoplasts were isolated from the 10-day-old rice seedlings and their fluorescence was examined under a confocal microscope. The results based on the green fluorescence showed that the sGFP signals from sGFP and K-sGFP were all dispersed into the cytoplasm and those from KR-sGFP were clearly localized into chloroplasts, supported by the exact match to red autofluorescence from chlorophylls (see Figure 1B). The mean fluorescence intensity of the images was analyzed by using a Histogram tool of the image acquisition software ZEN 2009 Light Edition, and the relative ratios of the green fluorescence intensities were introduced by the yellow scale bars on the images of Green/GFP in Figure 1B, which were normalized to it of the sGFP-transformed protoplasts. The confocal microscopic analysis showed that the localization of sGFP into chloroplasts did not have any effects on the fluorescence intensity of sGFP.\nThe effect of Kz sequence on sGFP expression level was also analyzed (see Figure 1C). Western blot analysis using a GFP antibody showed that the level of expression of K-sGFP proteins was a factor of 2.8 higher than that of sGFP proteins.\nFigure 1 Subcellular localization and expression efficiency of sGFPs in rice protoplasts. (A) Three kinds of sGFP chimera were cloned into a pB2GW7 vector to construct sGFP, K-sGFP, and KR-sGFP for use in PEG-mediated transfection into rice protoplasts. Kz: Kozak sequence; rRTp: a transit peptide of rice RuBisCo small subunit; (B) Individual and merged images of GFP, chlorophyll autofluorescence, and visible protoplasts. C: chloroplast; V: vacuole. The images were acquired at 630× magnification using a confocal microscope. The yellow signals are the merged image of the green fluorescent signals from the sGFP proteins; the red fluorescent signals from chlorophyll in the chloroplasts. The PEG-transformed protoplasts with no plasmid were used as a mock sample. The yellow scale bars on the images show the mean intensity ratio of the green fluorescence signals (for 105 pixels), analyzed by a Histogram tool, and the white scale bars mean 5 μm and (C) The expression efficiency of proteins was compared using western blot analysis. Polyclonal rabbit antibody against sGFP was used at a titer of 1:5000 and the image of PAGE gel was used to show relative quantities of the loaded proteins. The graph shows the relative band intensities that were quantified using LAS4000 software.\n\n2.2. Differences on FCA between Cytoplasm- and Chloroplast Targeting of sGFP\nTo determine the effect on a FCA according to each different subcellular localization of sGFPs, the rice protoplasts transfected with each of cytoplasm (sGFP or K-sGFP) or chloroplast (KR-sGFP)-targeting construct were split into four tubes to perform four experiments: (i) a flow cytometric analysis; (ii) quantitative real-time PCR; (iii) western blot analysis; and (iv) hemocytometer measurement. Each analysis was repeated in three independent experiments (Figure 2 and Figure 4).\n\n2.2.1. A Flow Cytometric Analysis (FCA)\nIn FCA, the fluorescence intensity was normalized by the average value of K-sGFP and showed an eight-fold higher intensity in KR-sGFP as compared to sGFP and K-sGFP (Figure 2A). The transfection ratio was also higher in KR-sGFP (65.7%) than in sGFP (16.8%) and K-sGFP (14.7%), resulting in a 4.5-fold increase in KR-sGFP than sGFP and K-sGFP (see Figure 2B). As shown in Figure 2C, the large difference in the population distribution was observed on the plot data of FCA. The threshold value (103) was determined and the fluorescence below it was regarded as the background signals using mock control. In the case of K-sGFP, a large population (83.3%) was distributed below the threshold value, while the large portion of KR-sGFP (65.7%) was distributed on the overall intervals with higher fluorescence intensity than the threshold value. These results suggest that the localization of sGFP in chloroplasts largely enhances the intensity of fluorescence on FCA (\u003e8-fold) and this causes an increase in the population with higher fluorescence than the threshold level of background signals and, consequently, increases transfection ratio, from 14.7% to 65.7%. In other words, it can be supposed that the population of K-sGFP under the threshold value might contain the transfected protoplasts with lower fluorescence intensity than the background signals. Additionally, the results of FCA between sGFP and K-sGFP are shown to be not considerably different. Thus, it can be supposed that a Kz sequence might not be a much more effective factor for FCA, even if it is helpful to increase the expression level.\nTo have a deeper look into this difference, the fluorescence intensity was partitioned into P1 (103–104), P2 (104–105), and P3 (\u003e105) subpopulations (see Figure 2C); furthermore, the distribution of transfected cells was compared in those subpopulations. Figure 3 shows that both sGFP (96%) and K-sGFP (88.4%) of the transfected population (14.4% and 64.3%, respectively) are largely distributed on a P1 subpopulation, while the KR-sGFP transfected cells are overall distributed across all subpopulations, P1–P3 and apparently, 55.9% of them belong to P2–P3 subpopulations (\u003e104). It shows that the population with the high fluorescence intensity over 104 was increased by the chloroplast targeting of sGFP. In particular, the population ratio on P3 subpopulation with high fluorescence intensity (\u003e105) was 49- and 14-fold higher in KR-sGFP (29.4%) than in sGFP (0.6%) and K-sGFP (2.1%), respectively.\nFigure 2 Comparative analysis of FCA using rice protoplasts among sGFP, K-sGFP and KR-sGFP constructs. Rice protoplasts were transfected with 5 μg plasmids of sGFP, K-sGFP, and KR-sGFP constructs. The FCAs were performed using single live cells gated by the side/forward scattering. (A) The fluorescence intensities were analyzed using the fluorescein isothiocyanate (FITC-A) channel of the flow cytometer and normalized by the average fluorescence intensity of the K-sGFP construct. The results are presented in arbitrary units; (B) The ratio of protoplasts with a fluorescence intensity exceeding the threshold value (103) to the gated protoplasts was calculated on FCA. The left axis shows the percentage of the transfected protoplasts expressing sGFP and the right axis shows their arbitrary results. The error bars show the SEM (standard error of the mean) for the data acquired from three independent transfections. Mock samples were prepared by PEG-transfection with no plasmid DNA and (C) Flow cytometry histograms (left panels) and the images of hemocytometer measurements (right panels). The fluorescence intensity on FITC was partitioned into P1 (103–104), P2 (104–105), and P3 (\u003e105) subpopulations, and M means “the missing population”. For a hemocytometer analysis, 10 μL of the transformed protoplasts was mounted on a hemocytometer, and were photographed using the DIC and FITC-A channels of a confocal microscope (200× magnification). The merged images of one square (1 mm2) on the hemocytometer were shown. The sGFP-expressing cells were identified as the green fluorescent signals, and the yellow scale bars on the images show the mean intensity ratio (for 106 pixels) of the green fluorescence, analyzed by a Histogram tool. Taken together, these results suggest that the sGFP targeting into chloroplasts using rRTp plays an important role in improving the fluorescence intensity of FCA to increase the population with higher fluorescence than the threshold value, as well as to increase its average fluorescence intensity (\u003e8-fold). However, the results of hemocytometer analysis (right panels of Figure 2C) do not correspond to the data of fluorescence intensity (left panels of Figure 2C) on FCA, in the point that the histogram analyses of those images showed that the mean intensity ratios between K-sGFP and KR-sGFP were similarly determined, as shown by scale bars (Figure 2C). Supposedly, the difference of fluorescence intensity on FCA data between K-sGFP and KR-sGFP might not be derived from the difference on the molecular levels of RNA/proteins or transfection efficiency.\nFigure 3 The relative proportional subpopulation distribution of protoplast expressing sGFP. The fluorescence intensity was partitioned into P1, P2, and P3 subpopulations (P1: 103–104; P2: 104–105; P3: \u003e105). The total percentage of protoplasts expressing sGFP on FCA is shown by the sum of percentages of transfected cells on each subpopulation. The circular charts above each bar graph show the ratio of transfected cells on each subpopulation. The transfected protoplast proportion of each subpopulations relative to all transfected protoplasts with a fluorescence intensity exceeding a threshold value (103) was calculated on FCA. The error bars show the SEM (standard error of the mean) for the data acquired from three independent transfections. Mock samples were prepared by PEG-transfection with no plasmid DNA.\n\n2.2.2. Non-FCA-Based Analyses\nThe sGFP expression level and transfection ratio were analyzed using a non-FCA-based analysis (see Figure 4). The sGFP expressions were measured on molecular levels; mRNA transcripts by quantitative real-time PCR (see Figure 4A) and proteins by western blot analysis (see Figure 4B). As a result, both transcripts and proteins of K-sGFP FP did not considerably differ from those of the KR-sGFP. The KR-sGFP formed two protein bands of a premature-polypeptide of 32 kDa and a mature-polypeptide of 27 kDa produced after the cleavage of rRTp-transit peptide. This smaller one from KR-sGFP was supposed to be a functional form as a fluorophore emitter (see Figure 4B). The transfection ratio analysis using the hemocytometer showed no difference between K-sGFP and KR-sGFP (see Figure 4C). These results suggest that the localization of sGFP into chloroplasts does not affect the expression level of sGFP or transfection efficiency and support the presumption that the large population of the K-sGFP transfected protoplasts was analyzed as the non-transfected population with background signals because their fluorescence intensity was lower than the threshold value on FCA. The population with the lower fluorescence intensity would henceforth be called “the missing population” (M; see Figure 2C for its interval). This could also be supported by the images of hemocytometer analysis in the right panel of Figure 2C, on which fluorescence intensity and transfection ratio are not much different between K-sGFP and KR-sGFP.\nFigure 4 Comparison of K-sGFP and KR-sGFP constructs based on non-FCA analysis. (A) The mRNA transcripts of sGFP and rRTp-sGFP were compared using real-time PCR. The rice ubiquitin gene was used as an internal reference for quantitative normalization; (B) The expression efficiency of proteins compared by western blot analysis. Polyclonal rabbit antibody against sGFP was used at a titer of 1:5000 and the image of PAGE gel was used to show relative quantities of the loaded proteins. The graph shows the relative band intensities quantified using LAS4000 software and (C) The ratio of protoplast cells expressing sGFP proteins was analyzed by hemocytometer measurements. A volume of 10 μL of the transformed protoplasts was mounted on a hemocytometer and the images were obtained using the DIC and FITC-A channels of a confocal microscope (200× magnification). The sGFP-expressing cells were identified as the green fluorescent signals. The error bars show the SEM of data acquired from three independent transfections. Control samples were prepared by PEG transfection with no plasmid DNA.\n\n2.3. Optimization of the Amount of Plasmid DNAs\nTo optimize the amount of plasmid DNAs for FCA, various quantities (0, 2, 5, 10, 20, and 40 μg) of the KR-sGFP plasmid DNAs were used for rice protoplast transformation. The PEG-transfected protoplasts were split into three tubes for three experiments, respectively: (i) FCA (see Figure 5A); and (ii) western blot analysis (see Figure 5B).\nFCA showed the lowest sGFP fluorescence at 2 μg DNA and the highest at 10 μg DNA, however, the difference in fluorescence range was not very large among quantities ranging from 5–40 μg (Figure 5A). Similarly, the protein accumulations were not much different among 5–40 μg (Figure 5B). Comprehensively, an optimal amount of transfected DNAs resulted in 10 μg DNAs for FCA. Our results demonstrate that the use of only 5–10 μg DNA was enough for FCA using rice protoplasts and that large amounts of plasmid DNAs (\u003e40 μg) are not necessary for FCA of our protoplast system. In this study, the smallest quantitiy (5 μg DNA) of the optimized DNA quantity (5–10 μg DNA) was used for various analyses.\nFigure 5 Optimization of the amount of DNA for FCA-based analysis using a KR-sGFP reporter system. The fluorescent signals exceeding the gating threshold were detected in single live protoplasts. (A) The sGFP fluorescence was detected using the FITC-A channel of the flow cytometer and its values were normalized by the fluorescent intensity of the protoplasts transfected using 0 μg DNA. The results are presented in arbitrary units. The transfection ratio was calculated by the proportion of transfected protoplasts relative to total protoplasts gated by side scattering and forward scattering procedure on FCA. The transfected protoplasts are determined by their fluorescence intensity exceeding the threshold value (103) on FCA; The results are normalized by transfection ratio of the protoplasts transfected using 2 μg DNA and presented in arbitrary units and (B) The expression efficiency of proteins was compared using western blot analysis. Polyclonal rabbit antibody against sGFP was used at a titer of 1:5000 and the image of PAGE gel was used to show relative quantities of the loaded proteins. The graph shows the relative band intensities quantified using LAS4000 software.","divisions":[{"label":"title","span":{"begin":0,"end":10}},{"label":"sec","span":{"begin":12,"end":3042}},{"label":"title","span":{"begin":12,"end":78}},{"label":"p","span":{"begin":79,"end":1515}},{"label":"p","span":{"begin":1516,"end":1754}},{"label":"figure","span":{"begin":1755,"end":3042}},{"label":"label","span":{"begin":1755,"end":1763}},{"label":"caption","span":{"begin":1765,"end":3042}},{"label":"p","span":{"begin":1765,"end":3042}},{"label":"sec","span":{"begin":3044,"end":12232}},{"label":"title","span":{"begin":3044,"end":3120}},{"label":"p","span":{"begin":3121,"end":3605}},{"label":"sec","span":{"begin":3607,"end":9585}},{"label":"title","span":{"begin":3607,"end":3646}},{"label":"p","span":{"begin":3647,"end":5254}},{"label":"p","span":{"begin":5255,"end":6157}},{"label":"figure","span":{"begin":6158,"end":7903}},{"label":"label","span":{"begin":6158,"end":6166}},{"label":"caption","span":{"begin":6168,"end":7903}},{"label":"p","span":{"begin":6168,"end":7903}},{"label":"p","span":{"begin":7904,"end":8764}},{"label":"figure","span":{"begin":8765,"end":9585}},{"label":"label","span":{"begin":8765,"end":8773}},{"label":"caption","span":{"begin":8775,"end":9585}},{"label":"p","span":{"begin":8775,"end":9585}},{"label":"sec","span":{"begin":9587,"end":12232}},{"label":"title","span":{"begin":9587,"end":9616}},{"label":"p","span":{"begin":9617,"end":11138}},{"label":"figure","span":{"begin":11139,"end":12232}},{"label":"label","span":{"begin":11139,"end":11147}},{"label":"caption","span":{"begin":11149,"end":12232}},{"label":"p","span":{"begin":11149,"end":12232}},{"label":"title","span":{"begin":12234,"end":12281}},{"label":"p","span":{"begin":12282,"end":12621}},{"label":"p","span":{"begin":12622,"end":13317}},{"label":"label","span":{"begin":13318,"end":13326}}],"tracks":[{"project":"2_test","denotations":[{"id":"25561231-8805250-50799904","span":{"begin":172,"end":174},"obj":"8805250"},{"id":"T28969","span":{"begin":172,"end":174},"obj":"8805250"}],"attributes":[{"subj":"25561231-8805250-50799904","pred":"source","obj":"2_test"},{"subj":"T28969","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#93d7ec","default":true}]}]}}