Journal
PLANT BIOTECHNOLOGY
Volume 39, Issue 2, Pages 179-+Publisher
JAPANESE SOC PLANT CELL & MOLECULAR BIOLOGY
DOI: 10.5511/plantbiotechnology.22.0105a
Keywords
atmospheric-pressure plasma; clathrin-mediated endocytosis; plasmaized gas; protein introduction; reactive species
Funding
- research program on development of innovative technology grants from the Project of the Bio-oriented Technology Research Advancement Institution (BRAIN)
- Cooperative Research Project of the Research Center for Biomedical Engineering
- Cabinet Office, Government of Japan
- Bio-oriented Technology Research Advancement Institution, NARO
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This study investigated the mechanism of protein uptake in plant cells using temperature-controlled atmospheric-pressure plasma. The results showed that protein uptake potential remained in leaf tissue for at least 3 hours after plasma treatment. Further analysis revealed that the introduced protein reached a similar amount to that after overnight incubation at approximately 5 hours after irradiation. Inhibitor experiments confirmed that the N-2 plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.
Previously, we developed a method that uses temperature-controlled atmospheric-pressure plasma to induce protein uptake in plant cells. In the present work, we examined the mechanism underlying such uptake of a fluorescent tagged protein in tobacco leaf cells. Intact leaf tissue was irradiated with N-2 plasma generated by a multi-gas plasma jet and then exposed to the test protein (histidine-tagged superfolder green fluorescence protein fused to adenylate cyclase); fluorescence intensity was then monitored over time as an index of protein uptake. Confocal microscopy revealed that protein uptake potential was retained in the leaf tissue for at least 3 h after plasma treatment. Further examination indicated that the introduced protein reached a similar amount to that after overnight incubation at approximately 5 h after irradiation. Inhibitor experiments revealed that protein uptake was significantly suppressed compared with negative controls by pretreatment with sodium azide (inhibitor of adenosine triphosphate hydrolysis) or sucrose or brefeldin A (inhibitors of clathrin-mediated endocytosis) but not by pretreatment with genistein (inhibitor of caveolae/raft-mediated endocytosis) or cytochalasin D (inhibitor of micropinocytosis/phagocytosis), indicating that the N-2 plasma treatment induced protein transportation across the plant plasma membrane via clathrin-mediated endocytosis.
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