Tamura Kentaro, Shimada Tomoo, Ono Eiichiro, Tanaka Yoshikazu, Nagatani Akira, Higashi Sho-Ich, Watanabe Masakatsu, Nishimura Mikio, Hara-Nishimura Ikuko
Department of Botany, Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
Plant J. 2003 Aug;35(4):545-55. doi: 10.1046/j.1365-313x.2003.01822.x.
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.
绿色荧光蛋白（GFP）使细胞器和蛋白质运输途径在活细胞中可视化成为可能。然而，在高等植物的任何器官的液泡中尚未观察到GFP荧光。我们发现，在黑暗条件下，在转基因拟南芥植物的液泡中稳定观察到液泡靶向GFP的荧光，而在光照条件下荧光迅速消失。液泡GFP在光照下1小时内迅速降解，尤其是蓝光。液泡型H + -ATP酶抑制剂 concanamycin A和木瓜蛋白酶型半胱氨酸蛋白酶抑制剂E-64d消除了GFP荧光的光依赖性消失和液泡中GFP的降解。体外试验表明，细菌表达的GFP在光照下在酸性pH值下被拟南芥培养细胞原生质体提取物降解。这些结果表明，蓝光诱导GFP构象变化，并且在酸性pH下，液泡中产生的GFP容易被液泡木瓜蛋白酶型半胱氨酸蛋白酶降解。光依赖性降解解释了在植物器官的液泡中未观察到GFP荧光的原因。我们的结果表明，通过在荧光显微镜检查之前将植物从光照转移到黑暗中，可以实现稳定的GFP荧光液泡。这可能消除了植物液泡靶向机制以及内膜系统的器官和阶段特异性分化研究中的一个大障碍。
