A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes

Abstract

Background: Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. In rice, protoplasts are commonly prepared from suspension cultured cells or etiolated seedlings, but only a few studies have explored the use of protoplasts from rice green tissue.

Results: Here, we report a simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device. Transfections of the generated protoplasts with plasmids of a wide range of sizes (4.5-13 kb) and co-transfections with multiple plasmids achieved impressively high efficiencies and allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis by bimolecular fluorescence complementation (BiFC) and firefly luciferase complementation (FLC). Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). Transient expression of the GFP-tagged light-related transcription factor OsGLK1 markedly upregulated transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS, which were reduced to some extent by NF treatment in the rice green tissue protoplasts.

Conclusions: We show here a simplified and highly efficient transient gene expression system using photosynthetically active rice green tissue protoplasts and its broad applications in protein immunoblot, localization and protein-protein interaction assays. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes.

Figure 1

Isolation of protoplasts from rice green tissue. A, A representative healthy 8-day-old rice seedling used for protoplast isolation. Scale bar = 1 cm. B, Red markers indicate the optimal sections of seedlings (stem and sheath) yielding protoplasts. C, Cut strips were treated with 0.6 M mannitol followed by enzymatic digestion. D, Image of protoplasts obtained using a Nikon digital camera under an Olympus microscope with a 40× objective. Scale bar = 10 μm.

Figure 2

Comparison of transfection efficiencies between rice green tissue protoplasts and etiolated protoplasts. Transient expression of different constructs in rice protoplasts. Samples were visualized under a confocal microscope. A-B, The 35S::GFP construct pUC-GFP was transiently expressed in protoplasts derived from 8-day-old rice green seedlings (A) or 8-day-old etiolated rice seedlings (B). Individual and merged images of GFP and chlorophyll autofluorescence (Chl) as well as bright field images of protoplasts are shown. Scale bars = 10 μm. C, The transfection efficiency of rice green tissue protoplasts was comparable to that of etiolated protoplasts. The ratio of GFP-positive cells to the total number of protoplasts (n ≥ 100) was scored as the transfection efficiency. Values are means, with standard errors indicated by bars, representing at least 5 replicates. D, Detection of transiently expressed bZIP63-c-myc-YFP^N (lane 1) and c-myc-YFP^N (lane 2) by Western blot. Lane 3 was a negative control. The upper panel shows an immunoblot using a monoclonal mouse anti-c-myc antibody; the lower panel shows a Coomassie Brilliant Blue (CBB)-stained PVDF membrane after immunoblotting as a loading control.

Figure 3

Transient expression efficiencies of different sized plasmids in rice green tissue protoplasts. A-B, A 4.5 kb plasmid pUC-GFP (A) and a 13 kb plasmid CD3-998 (B) were transiently expressed in rice green tissue protoplasts. Merged images of GFP or YFP and chlorophyll autofluorescence (Chl) as well as bright field images of protoplasts are shown. Scale bars = 20 μm. C, Transfection efficiency of a 13 kb plasmid compared with that of a 4.5 kb plasmid, expressed as the ratio of GFP or YFP-positive cells to the total number of protoplasts (n ≥ 100). Values are means, with standard errors indicated by bars, representing at least 5 replicates.

Figure 4

Subcellular localization analysis in rice green tissue protoplasts. Rice proteins and Arabidopsis organelle markers were transiently expressed in rice green tissue protoplasts. A, Rice OsRpl6-2-YFP labeling of mitochondria. B, Rice OsTRX m5-GFP labeling of plastids. C, OsTRX m2-GFP targeted to chloroplasts. D, BAS1-GFP targeted to chloroplasts. E, CD3-998 labeling of plastids; CD3-982 labeling of peroxisomes; CD3-958 labeling of the endoplasmic reticulum (ER); CD3-966 labeling of Golgi. F, An mCherry-based plastid marker CD3-1000. Merged images are shown with YFP, mCherry or GFP and chlorophyll autofluorescence (Chl). Bright field images of protoplasts are also shown. Scale bars = 10 μm.

Figure 5

Co-localization analysis with known organelle markers. A, Plastid-YFP and plastid-mCherry markers, or B, Golgi-YFP and plastid-mCherry markers were co-expressed in rice green tissue protoplasts. Cells showing both markers in each co-transfection are indicated by arrowheads in the merged images of YFP, mCherry and chlorophyll autofluorescence (Chl) signals. Bright field images of protoplasts are also shown. Scale bar = 20 μm.

Figure 6

Protein-protein interaction assays in rice green tissue protoplasts. A, Protein-protein interaction analysis by BiFC. Construct pairs of pUC-bZIP63-YN (bZIP63-YN) and pUC-bZIP63-YC (bZIP63-YC), bZIP63-YN and pUC-SPYCE (YC), pUC-SPYNE (YN) and bZIP63-YC, or YN and YC were transiently co-expressed in rice green tissue protoplasts. BiFC fluorescence is indicated by the YFP signal. Individual and merged images of YFP and chlorophyll autofluorescence (Chl) as well as bright field images of protoplasts are shown. The upper panel contains low power images showing high co-expression efficiency. Scale bars = 10 μm. B, Protein-protein interaction analysis by FLC. Constructs as indicated plus the RNL construct as an internal control were transiently co-expressed in rice green tissue protoplasts. Firefly luciferase activity was normalized to RNL activity. Values are means, with standard errors indicated by bars, representing 3 replicates. C, Construct pairs of pUC-BAS1-YN (BAS1-YN) and pUC-OsTRX m5-YC (OsTRX m5-YC), BAS1-YN and YC, YN and OsTRX m5-YC, or BAS1-YN and pUC-OsTRX m2-YC (OsTRX m2-YC) were transiently co-expressed in rice green tissue protoplasts. BiFC fluorescence is indicated by the YFP signal. Individual and merged images of YFP and chlorophyll autofluorescence (Chl) as well as bright field images of protoplasts are shown. Scale bars = 10 μm.

Figure 7

Photosynthesis activivity of rice green tissue protoplasts. A, Color images of the maximal PS II quantum yield (F_v/F_m) of rice green tissue and etiolated protoplasts. The false color ranged from black (0) via red, orange, yellow, green, blue and violet to purple (1) as indicated at the bottom. B, F_v/F_m in rice green tissue and etiolated protoplasts. Values are means, with standard errors indicated by bars, representing 7 replicates.

Figure 8

Expression levels of OsGLK1-upregulated genes in rice green tissue protoplasts under light and NF treatments. Protoplasts transfected with/without OsGLK1-GFP or GFP were treated with 40 μmol m^-2 s^-1 light and/or 500 nM NF for 12 h. A-D, Transcript levels of OsLhcb1, OsLhcp, GADPH and RbcS were detected by quantitative real-time PCR and normalized to that of β-actin. Values are means, with standard errors indicated by bars, representing 3 independent biological samples, each with 3 technical replicates. E, Detection of transiently expressed OsGLK1-GFP (lanes 1-2) and GFP (lanes 3-4) by Western blot. CK was a negative control. The upper panel shows an immunoblot using a monoclonal rabbit anti-GFP antibody. As a loading control, a Coomassie Brilliant Blue (CBB)-stained PVDF membrane is shown in the lower panel.