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.
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Claims 7
Claim · model-written summary
The method isolated protoplasts from young rice green tissue without extra chemicals or vacuum.
- Taxon
- rice
Oryza sativae0 - Outcome
- isolating protoplastse0
- Comparator
- unnecessary chemicals and a vacuum devicee0
Not reportedValue
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“simplified method for isolating protoplasts from normally cultivated young rice green tissue without the need for unnecessary chemicals and a vacuum device”
Claim · model-written summary
Transfection and cotransfection of rice protoplasts achieved high efficiencies.
- Taxon
- riceOryza sativat0
- Outcome
- high efficienciese0
- Value
- 4.5-13 kbe0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“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”
t0“rice green tissue protoplast”
Claim · model-written summary
The protoplasts supported immunoblotting, localization, and protein interaction assays.
- Taxon
- riceOryza sativat0
- Outcome
- protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysise0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“allowed evaluations by 1) protein immunoblotting analysis, 2) subcellular localization assays, and 3) protein-protein interaction analysis”
t0“rice green tissue protoplast”
Claim · model-written summary
Rice green tissue protoplasts were photosynthetically active.
- Taxon
- riceOryza sativae0
- Outcome
- photosynthetically activee0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“rice green tissue protoplasts were photosynthetically active”
Claim · model-written summary
Rice green tissue protoplasts were sensitive to norflurazon.
- Taxon
- riceOryza sativae0
- Treatment
- norflurazone0
- Outcome
- sensitive to the retrograde plastid signaling inducer norflurazone0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“rice green tissue protoplasts were photosynthetically active and sensitive to the retrograde plastid signaling inducer norflurazon (NF)”
Claim · model-written summary
OsGLK1 expression upregulated four photosynthetic genes.
- Taxon
- riceOryza sativat0
- Treatment
- Transient expression of the GFP-tagged light-related transcription factor OsGLK1e0
- Gene
- OsGLK1e0
- Outcome
- transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcSe0
- Direction
- upregulatede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“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”
t0“rice green tissue protoplast”
Claim · model-written summary
Norflurazon reduced those transcript levels to some extent.
- Taxon
- riceOryza sativae0
- Treatment
- NF treatmente0
- Outcome
- transcript levelse1
- Direction
- reducede0
- Qualifier
- to some extente0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“which were reduced to some extent by NF treatment in the rice green tissue protoplasts”
e1“transcript levels of the endogeneous photosynthetic genes OsLhcb1, OsLhcp, GADPH and RbcS”
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See all 32 papers →Claims are grouped by organism, treatment and what was measured or targeted. A paper counts as positive when any of its claims in the group is positive.