Seed germination is a critical step in a plants life cycle that allows successful propagation and is therefore strictly controlled by endogenous and environmental signals. another grow hormone GA promotes germination (Ogawa et al., 2003) through weakening the mechanical restraint of tissues that surround the embryo and through increasing the embryo growth potential (Ogawa et al., 2003). During the germination process, the buy PD 150606 endogenous ABA content of seeds declines, while bioactive GA levels accumulate prior buy PD 150606 to radicle emergence (Ogawa et al., 2003; Weitbrecht et al., 2011). This GA-to-ABA ratio in the seeds is the primary control of germination vigor (Kucera et al., 2005; Holdsworth et al., 2008; Rajjou et al., 2012). Emerging evidence shows that other signals are also involved in seed germination. For example, reactive oxygen species act as second messengers required for germination (Bailly et al., 2008) and interact with plant hormones to coordinate germination (Mller et al., 2009). Overall, germination control, a sophisticated process, is determined by concerted actions among various endogenous signals including some yet to be discovered. Not only is calcium (Ca2+) an indispensable nutrient for plants (White and Broadley, 2003), it also acts as a versatile second messenger through changes in free cytosolic Ca2+ level ([Ca2+]cyt) to participate in numerous developmental and adaption processes in plants, such as root hair elongation, pollen tube growth, stomatal movements, plant-microbe interactions, light signaling, and hormone responses (McAinsh and Pittman, 2009; Dodd et al., 2010; Kudla et al., 2010). Colec11 The [Ca2+]cyt elevations, relative to their low levels in the resting conditions (McAinsh and Pittman, 2009), are caused by extracellular Ca2+ influx or internal Ca2+ release from the stores, both of which may encode specific cellular Ca2+ signatures to buy PD 150606 further initiate downstream gene expressions and biological events (McAinsh and Pittman, 2009; Dodd et al., 2010; Kudla et al., 2010). The spatiotemporal changes of [Ca2+]cyt in living cells are accurately controlled and sensed by an efficient Ca2+ signaling regulatory network, among which Ca2+ channels play a large role (Dodd et al., 2010; Kudla et al., 2010). In plants, several gene families are thought to encode Ca2+-permeable channels (Ward et al., 2009; Hedrich, 2012), among them Arabidopsis (and that ABI4 plays a fundamental role in Ca2+ responses in germination. Our results demonstrate and uncover the mechanism by which AtGLR3.5 regulates Ca2+ signaling in germination, thus providing a molecular explanation for the observation that Ca2+ alleviates decreasing in germination caused by stress. Therefore, the results highlight the significance of Ca2+ in this developmental process, particularly under buy PD 150606 stress conditions. RESULTS AND DISCUSSION Extracellular Calcium Enhances Seed Germination To gain insight into the role of Ca2+ in germination, we investigated whether and how Ca2+ modulates seed germination in Arabidopsis. As shown in Determine 1A, the application of CaCl2-stimulated germination (enhanced radicle emergence and subsequent root growth) compared with the control, whereas chelating Ca2+ using EGTA severely inhibited these processes. External Ca2+ at physiological levels promoted germination in a dose-dependent manner (Fig. 1B; < 0.01 at 0.1 mm, < 0.001 at 1 and 5 mm), although high concentrations of external Ca2+ (20C80 mm) delayed germination (Supplemental Fig. S1, ACC), presumably due to a cytotoxic effect of too much Ca2+ (White and Broadley, 2003). Moreover, EGTA drastically reduced germination, to as low as 30% of the control at 10 mm (Fig. 1C; < 0.001 at 5 and 10 mm). These data show that adequate external Ca2+ is required for seeds to germinate, which points to an important role of Ca2+ in the earliest phase of the plant life cycle. Determine 1. Seed germination is usually enhanced by external calcium. A, Effect of calcium on germination. Arabidopsis wild-type seeds sown on modified MS medium containing 0 or 5 mm CaCl2 or 0 mm CaCl2 supplemented with 10 mm EGTA were.