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

Abstract

BackgroundPlant 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.ResultsHere, 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.ConclusionsWe 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.

SND1 Transcription Factor-Directed Quantitative Functional Hierarchical Genetic Regulatory Network in Wood Formation in Populus trichocarpa

“…The total time needed for processing every 2.5 g of SDX to generate protoplasts is ;4 h (;1 h for preparing tissue strips and ;3 h for cell wall digestion). The efficiency of this system is comparable to or better than many others (Manders et al, 1992;He et al, 2007;Riazunnisa et al, 2007;Yoo et al, 2007;Sonntag et al, 2008;Tang et al, 2010;Zhang et al, 2011;Guo et al, 2012;Huddy et al, 2012;Pitzschke and Persak, 2012); however, the throughput (;4 h for every 2.5 g SDX) was still low. We then found that a debarked stem segment (Figure 1; see Supplemental Figure 1 online) with intact SDX can be used directly to release SDX protoplasts by submerging the stem into the enzyme digestion solution.…”

Expression of the Nitrate Transporter Gene OsNRT1.1A/OsNPF6.3 Confers High Yield and Early Maturation in Rice

“…12143) following the manufacturer's manual. The rice protoplasts were isolated and transformed according to the published methods described previously (Bart et al, 2006;Zhang et al, 2011). For vacuole releasing, intact rice protoplasts were treated by vacuole lysis buffer (10 mM EDTA, 10 mM EGTA, and 95 mM mannitol or sorbitol, pH 8.0 adjusted by 1 M Tris) for 3 to 5 min, and ready for microscopy observation.…”

SALT-RESPONSIVE ERF1 Regulates Reactive Oxygen Species-Dependent Signaling during the Initial Response to Salt Stress in Rice

“…Sequences of oligonucleotides used for cloning of SERF1 and MAPK5 are listed in Supplemental Table 6 online. Subsequently, plasmids were transformed into rice shoot protoplasts according to Zhang et al (2011). After transformation and incubation, the protoplasts were lysed and the renilla luciferase (RLUC) signal was measured with the Dual-Luciferase reporter assay kit using a GloMax 20/ 20 luminometer (Promega).…”

“…Rice shoot protoplasts were obtained and transformed with the 35S: SERF1-CFP construct according to Zhang et al (2011). Fluorescence imaging of the protoplasts was performed using a confocal laser scanning microscope (SP5; Leica Microsystems).…”

“…Rice shoot protoplasts were isolated and transformed according to Zhang et al (2011). Constructs for trans-activation assays were generated by amplifying 1-kb upstream promoter sequences of DREB2A, ZFP179, SERF1, and MAPK5 by PCR (see Supplemental Table 6 online).…”