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.
Highlights
Plant protoplasts, a proven physiological and versatile cell system, are widely used in highthroughput analysis and functional characterization of genes
The Agrobacterium-mediated approach yielded higher efficiency and is inexpensive [15,16,17], but it is difficult to use for subcellular localization and other fluorescence-based analysis, as this method is often associated with a high level of nonspecific autofluorescence
7 to 10-day-old rice green seedlings cultured at 26°C on 1/2 MS medium with a 12 h light (~150 μmol m-2 s-1)/12 h dark cycle, were used for protoplast isolation (Figure 1A and Additional file 1)
Summary
A proven physiological and versatile cell system, are widely used in highthroughput analysis and functional characterization of genes. Rice is one of the most important cereal crops and a model species for monocotyledonous plants [11] Some systems such as tobacco and onion have been used for characterization of rice genes [5,6,7], but they are heterologous systems; the expressed proteins in heterologous systems may exhibit aberrant traits. In tissue-based methods, rice calli, leaves and seedlings are used for transient assays by different approaches. The Agrobacterium-mediated approach yielded higher efficiency and is inexpensive [15,16,17], but it is difficult to use for subcellular localization and other fluorescence-based analysis, as this method is often associated with a high level of nonspecific autofluorescence. The waxy structure of rice tissue is difficult to observe under a fluorescence microscope
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