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Why green fluorescent fusion proteins have not been observed in the vacuoles of higher plants
Author(s) -
Tamura Kentaro,
Shimada Tomoo,
Ono Eiichiro,
Tanaka Yoshikazu,
Nagatani Akira,
Higashi Shoich,
Watanabe Masakatsu,
Nishimura Mikio,
HaraNishimura Ikuko
Publication year - 2003
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2003.01822.x
Subject(s) - vacuole , green fluorescent protein , fluorescence microscope , arabidopsis , arabidopsis thaliana , fluorescence , organelle , biology , microbiology and biotechnology , biophysics , biochemistry , chemistry , cytoplasm , mutant , gene , physics , quantum mechanics
Summary 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.