Temporal change of photophobic step-up responses of <i>Euglena gracilis</i> investigated through motion analysis

The adaptation to a strong light is one of the essential characteristics of green algae, yet lacking relatively the information about the photophobic responses of Eukaryotic microalgae. We investigated the photophobic step-up responses of Euglena gracilis over a time course of several hours with alternated repetition of blue-light pulse illumination and spatially patterned blue-light illumination. Four distinctive photophobic motions in response to strong blue light were identified in a trace image analysis, namely on-site rotation, running and tumbling, continuous circular swimming, and unaffected straightforward swimming. The cells cultured in autotrophic conditions under weak light showed mainly the on-site rotation response at the beginning of blue-light illumination, but they acquired more blue-light tolerant responses of running and tumbling, circular swimming, or straightforward swimming. The efficiency of escaping from a blue-light illuminated area improved markedly with the development of these photophobic motions. Time constant of 3.0 h was deduced for the evolution of photophobic responses of E. gracilis. The nutrient-rich metabolic status of the cells resulting from photosynthesis during the experiments, i.e., the accumulation of photosynthesized nutrient products in balance between formation and consumption, was the main factor responsible for the development of photophobic responses. The reduction-oxidation status in and around E. gracilis cells did not affect their photophobic responses significantly, unlike the case of photophobic responses and phototaxis of Chlamydomonas reinhardtii cells. This study shows that the evolution of photophobic motion type of E. gracilis is dominated mainly by the nutrient metabolic status of the cells. The fact suggests that the nutrient-rich cells have a higher threshold for switching the flagellar motion from straightforward swimming to rotation under a strong light.强光适应是绿藻的重要特征之一，但目前真核微藻的避光反应（photophobic responses）相关研究信息仍相对匮乏。本研究通过交替重复蓝光脉冲照射与空间图案蓝光照射的实验方案，在数小时的时间进程中探究了纤细裸藻（Euglena gracilis）的动态避光反应。经轨迹图像分析，我们鉴定出纤细裸藻针对强蓝光的四种典型避光运动模式：原地旋转、奔跑翻滚、持续环形游动，以及不受干扰的直线游动。在弱光下自养培养的细胞，在蓝光照射初期主要表现为原地旋转反应，但随着处理进程推进，细胞逐渐获得了更强的蓝光耐受型反应，包括奔跑翻滚、环形游动或直线游动。随着这些避光运动模式的完善，细胞逃离蓝光照射区域的效率显著提升。本研究推导得出纤细裸藻避光反应演化的时间常数为3.0小时。实验过程中光合作用赋予细胞的富营养代谢状态——即光合营养产物的合成与消耗处于动态平衡的积累状态——是驱动避光反应演化的核心因素。与莱茵衣藻（Chlamydomonas reinhardtii）的避光反应及趋光性（phototaxis）不同，纤细裸藻细胞内外的氧化还原状态并未对其避光反应产生显著影响。本研究表明，纤细裸藻避光运动模式的演化主要受细胞营养代谢状态调控。该结果提示，富营养状态的细胞在强光下将鞭毛运动从直线游动切换为旋转的阈值更高。