Okazawa Atsushi, Tango Lei, Itoh Yoshiko, Fukusaki Ei'ichiro, Kobayashi Akio
Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan.
Z Naturforsch C J Biosci. 2006 Jan-Feb;61(1-2):111-7. doi: 10.1515/znc-2006-1-220.
Chlorophyllase (Chlase) catalyzes the initial step of chlorophyll (Chl)-degradation, but the physiological significance of this reaction is still ambiguous. Common understanding of its role is that Chlase is involved in de-greening processes such as fruit ripening, leaf senescence, and flowering. But there is a possibility that Chlase is also involved in turnover and homeostasis of Chls. Among the de-greening processes, autumnal coloration is one of the most striking natural phenomena, but the involvement of Chlase during autumnal coloration is not clear. Previously, it was shown that Chlase activity and expression level of the Chlase gene were not increased during autumnal coloration in Ginkgo biloba, indicating that Chlase does not work specially in the de-greening processes in G. biloba. In this study, we characterized the recombinant Chlase and analyzed its subcellular localization to understand the role of the cloned Chlase of G. biloba (GbCLH). GbCLH exhibited its highest activity at pH 7.5, 40 degrees C. Kinetic analysis revealed that GbCLH hydrolyzes pheophytin (Pheo) a and Chl a more rapidly than Pheo b and Chl b. Transient expression analysis of 40 N-terminus amino acids of GbCLH fused with GFP (green fluorescent protein) and subcellular fractionation showed that GbCLH localizes within chloroplasts. Together with our previous results, property of GbCLH and its location within the chloroplasts suggest that GbCLH plays a role in the turnover and homeostasis of Chls in green leaves of G. biloba.
叶绿素酶（Chlase）催化叶绿素（Chl）降解的初始步骤，但其该反应的生理意义仍不明确。对其作用的普遍理解是，叶绿素酶参与果实成熟、叶片衰老和开花等脱绿过程。但叶绿素酶也有可能参与叶绿素的周转和稳态维持。在脱绿过程中，秋季叶色变化是最显著的自然现象之一，但叶绿素酶在秋季叶色变化过程中的作用尚不清楚。此前研究表明，银杏秋季叶色变化过程中，叶绿素酶活性和叶绿素酶基因的表达水平并未升高，这表明叶绿素酶在银杏的脱绿过程中并无特殊作用。在本研究中，我们对重组叶绿素酶进行了特性分析，并分析了其亚细胞定位，以了解克隆的银杏叶绿素酶（GbCLH）的作用。GbCLH在pH 7.5、40℃时表现出最高活性。动力学分析表明，GbCLH水解脱镁叶绿素（Pheo）a和叶绿素a的速度比脱镁叶绿素b和叶绿素b更快。对与绿色荧光蛋白（GFP）融合的GbCLH的40个N端氨基酸进行瞬时表达分析和亚细胞分级分离表明，GbCLH定位于叶绿体中。结合我们之前的研究结果，GbCLH的特性及其在叶绿体中的定位表明，GbCLH在银杏绿叶中叶绿素的周转和稳态维持中发挥作用。
