YS
Yuuki Sakai
  • Department of Biological Sciences, Graduate School of Science, Osaka University, Japan
Research fields
  • Plant science
In vitro Phosphorylation Assay of Putative Blue-light Receptor Phototropins Using Microsomal and Plasma-membrane Fractions Prepared from Vallisneria Leaves
Authors:  Yuuki Sakai, Shin-ichiro Inoue and Shingo Takagi, date: 11/05/2015, view: 8202, Q&A: 0
An aquatic angiosperm Vallisneria (Alismatales: Hydrocharitaceae) has been used as an excellent experimental material over a century to study the light regulation of dynamic intracellular movements including chloroplast redistribution and cytoplasmic streaming (Senn, 1908; Seitz, 1987; Takagi, 1997). However, understanding of the molecular mechanisms lagged behind because of difficulty in applying modern techniques such as gene transformation to this plant. Especially, which kind of photoreceptors function in these intriguing responses has long been an unsolved topic. Recently, genes encoding plant-specific blue-light receptor phototropins were isolated in Vallisneria, for the first time from aquatic plants (Sakai et al., 2015). Phototropins were identified first as the photoreceptor for hypocotyl phototropism in Arabidopsis thaliana, and now known to regulate many responses including chloroplast photorelocation movements in various plant species (Christie, 2007). Phototropins are localized mainly on the plasma membrane and their auto-phosphorylation induced by blue light is the critical step of signal transduction pathway (Sakamoto and Briggs, 2002; Kong et al., 2006; Kong et al., 2013; Inoue et al., 2010). Here we describe a protocol for in vitro protein phosphorylation assay using crude-microsomal and plasma-membrane-enriched fractions of Vallisneria, which enabled us to verify the presence of phototropins and characterize their auto-phosphorylation responses. After these analyses, Sakai et al. (2015) proposed that Vallisneria phototropins mediate the high-intensity-blue-light-induced chloroplast avoidance response.
Observation of Chloroplast Movement in Vallisneria
Authors:  Yuuki Sakai and Shingo Takagi, date: 11/05/2015, view: 8841, Q&A: 0
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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