![]() Therefore, many studies have attempted to establish efficient gene expression systems in rice, including tissue-based and individual cell-based methods. For example, the encoded proteins of some Arabidopsis genes introduced in tobacco have been shown to be mis-localized. Some systems such as tobacco and onion have been used for characterization of rice genes, but they are heterologous systems the expressed proteins in heterologous systems may exhibit aberrant traits. Rice is one of the most important cereal crops and a model species for monocotyledonous plants. Accordingly, several methods for transient gene expression have been developed, such as PEG-mediated protoplast transfection, biolistic bombardment and Agrobacterium-mediated transient transformation. Arabidopsis, maize and tobacco protoplasts, tobacco leaf epidermal cells, tobacco BY-2 cells and onion epidermal cells are commonly used for transient assays in gene expression, protein subcellular localization, protein-protein interaction and protein activity studies. ![]() Transient expression assays allow rapid and high-throughput analysis of genes in plants and thus have become widely used for characterization of gene function. These rice green tissue protoplasts will be particularly useful in studies of light/chloroplast-related processes. 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. 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. Importantly, the rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF). 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). 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. 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. Green protoplasts have been successfully used in investigations of plant signal transduction pathways related to hormones, metabolites and environmental challenges. Plant protoplasts, a proven physiological and versatile cell system, are widely used in high-throughput analysis and functional characterization of genes. ![]()
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