Fritz L, Stringher C G, Colepicolo P
Biology Department, Northern Arizona University, Flagstaff 86011-5640, USA.
Braz J Med Biol Res. 1996 Jan;29(1):111-7.
Gonyaulax polyedra is a unicellular marine photosynthetic dinoflagellate known to display numerous circadian rhythms, including bioluminescence, motility, cell division and several chloroplast-related rhythms. Due to this, Gonyaulax has become a widely used model organism for studying the cellular biological clock. In this work we describe another rhythm for Gonyaulax cells also associated with the cell's chloroplasts, a rhythm in localization of the enzyme nitrate reductase (NR). A polyclonal antibody was raised against NR purified from G. polyedra cells and used as a probe in immunogold labelling experiments on cell thin sections, comparing day- and night-phase cells. The enzyme localizes to chloroplasts in day-phase cells, while the enzyme is active, and is largely absent in night-phase cells. Counts of gold particle distribution in day- versus night-phase cells show an approximate three-fold increase in enzyme labelling in day-phase plastids. These results closely approximate the four-fold differences shown for NR activity between day and night Gonyaulax cells by biochemical studies. We conclude from the diurnal difference in labelling that NR is localized in Gonyaulax chloroplasts during the day phase and is absent (broken down) in night-phase cells. Thus NR in Gonyaulax is compartmentalized in the chloroplasts and is therefore subject to similar circadian control mechanisms exhibited for other plastid rhythms.
多面 Gonyaulax 是一种单细胞海洋光合甲藻，已知其表现出多种昼夜节律，包括生物发光、运动性、细胞分裂以及几种与叶绿体相关的节律。正因如此，Gonyaulax 已成为研究细胞生物钟的广泛使用的模式生物。在这项工作中，我们描述了 Gonyaulax 细胞的另一种与叶绿体相关的节律，即硝酸还原酶（NR）的定位节律。我们制备了一种针对从多面 Gonyaulax 细胞中纯化的 NR 的多克隆抗体，并将其用作细胞薄片免疫金标记实验中的探针，比较了白天和夜间细胞。该酶在白天细胞的叶绿体中定位，此时酶具有活性，而在夜间细胞中基本不存在。白天和夜间细胞中金颗粒分布的计数显示，白天质体中酶标记增加了约三倍。这些结果与生化研究显示的 Gonyaulax 细胞白天和夜间 NR 活性的四倍差异非常接近。我们从标记的昼夜差异得出结论，NR 在白天定位于 Gonyaulax 叶绿体中，而在夜间细胞中不存在（分解）。因此，Gonyaulax 中的 NR 在叶绿体中被分隔，因此受到与其他质体节律类似的昼夜控制机制的影响。
