Chloroplasts accumulate to weak light and escape from strong light. These light-induced responses have been known from the 19th century (Böhm, 1856). Up to now, many scientists have developed different methods to investigate these dynamic phenomena in a variety of plant species including the model plant Arabidopsis thaliana, a terrestrial dicot (Wada, 2013). Especially, a serial recording to trace the position of individual chloroplast for the analysis of its mode of movement is critical to understand the underlying mechanism. An aquatic monocot Vallisneria (Alismatales: Hydrocharitaceae, Figure 1A) has contributed over a century to such investigation (Senn, 1908; Zurzycki, 1955; Seitz, 1967), because Vallisneria leaves have rectangular parallelepiped-shaped epidermal cells aligned orderly in a monolayer (Figure 1B), providing an excellent experimental system for microscopic studies. Here we describe a protocol for the up-to-date time-lapse imaging procedures to analyze Vallisneria chloroplast movement. Using this and prototype procedures, the relevant photoreceptor systems (Izutani et al., 1990; Dong et al., 1995; Sakai et al., 2015), association with actin cytoskeleton (Dong et al., 1996; Dong et al., 1998; Sakai and Takagi 2005; Sakurai et al., 2005), and regulatory roles of Ca2+ (Sakai et al., 2015) have been strenuously investigated.
Figure 1. Vallisneria plant. A. Whole plant body; B. A bright-field image of adaxial epidermal cells containing a large number of chloroplasts; C. Culture facilities.
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