Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants
Article first published online: 22 JUL 2003
The Plant Journal
Volume 35, Issue 4, pages 545–555, August 2003
How to Cite
Tamura, K., Shimada, T., Ono, E., Tanaka, Y., Nagatani, A., Higashi, S.-i., Watanabe, M., Nishimura, M. and Hara-Nishimura, I. (2003), Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants. The Plant Journal, 35: 545–555. doi: 10.1046/j.1365-313X.2003.01822.x
- Issue published online: 22 JUL 2003
- Article first published online: 22 JUL 2003
- Received 20 March 2003; revised 18 May 2003; accepted 28 May 2003.
- green fluorescent protein;
Green fluorescent protein (GFP) makes it possible for organelles and protein transport pathways to be visualized in living cells. However, GFP fluorescence has not yet been observed in the vacuoles of any organs of higher plants. We found that the fluorescence of a vacuole-targeted GFP was stably observed in the vacuoles of transgenic Arabidopsis plants under dark conditions, and that the fluorescence rapidly disappeared under light conditions. The vacuolar GFP was rapidly degraded within 1 h in the light, especially blue light. An inhibitor of vacuolar type H+-ATPase, concanamycin A, and an inhibitor of papain-type cysteine proteinase, E-64d, abolished both the light-dependent disappearance of GFP fluorescence and GFP degradation in the vacuoles. An in vitro assay showed that bacterially expressed GFP was degraded by extracts of Arabidopsis cultured-cell protoplasts at an acidic pH in the light. These results suggest that blue light induced a conformational change in GFP, and the resulting GFP in the vacuole was easily degraded by vacuolar papain-type cysteine proteinase(s) under the acidic pH. The light-dependent degradation accounts for the failure to observe GFP fluorescence in the vacuoles of plant organs. Our results show that stable GFP-fluoresced vacuoles are achieved by transferring the plants from the light into the dark before inspection with a fluorescent microscope. This might eliminate a large hurdle in studies of the vacuolar-targeting machinery and the organ- and stage-specific differentiation of endomembrane systems in plants.