D, Corresponding positions of FM4-64 fluorescence, vacuoles (V), and starch grains (S) in the cytoplasm of control and TFP-treated pollen tubes. and polysaccharide synthesis). Taken together, Ca2+-CaM dysfunction Ro 61-8048 induced serial cytological responses and temporal changes in protein expression profiles, indicating the pivotal role of Ca2+-CaM in the regulation of tip growth machinery. The pollen tube delivers male gametes to Ro 61-8048 the egg in flowering plants and is thus essential for sexual reproduction. Pollen tubes represent an ideal model system with which to investigate polarized growth in plant cells. They exhibit a tip-focused cytosolic Ca2+concentration ([Ca2+]c) cellular gradient that is established mainly by extracellular Ca2+influx whereby both intracellular [Ca2+]cand extracellular Ca2+influx exhibit oscillations that are accompanied by oscillations in growth rate (Cheung and Wu, 2001,2008;Gilroy and Trewavas, 2001;Feijo et al., 2004). The Ca2+signatures must be sensed, interpreted, and transduced Ro 61-8048 to downstream components by specific molecular decoders such as calmodulin (CaM), which is a protein that possesses a helix-loop-helix (EF-hand) motif. CaM is a highly conserved regulatory protein that is ubiquitous in eukaryotic cells and has a large number of physiological functions in animals and plants. Ca2+-CaM signaling is involved in pollen tube development (Ma et al., 1999;Rato et al., 2004), and pollen tube elongation can be inhibited by extracellular application of CaM antagonists such as trifluoperazine (TFP), calmidazolium, W-7, and W-12 (Picton and Steer, 1985;Rato et al., 2004). Rabbit polyclonal to THBS1 The CaM signaling pathways and the physiological responses that they elicit depend on the expression patterns and activities of the downstream molecules regulated by Ca2+-CaM. Most previous studies have focused solely on the morphology and cytology putatively related to CaM functions. However, the data available thus far are still insufficient to provide a complete picture of the functions of CaM during pollen tube development. TFP is a potent antipsychotic phenothiazine and has been widely studied in relation to its mode of binding and specific inactivation of CaM (Picton and Steer, 1985;Vandonselaar et al., 1994;Krinke et al., 2007). Therefore, we set up several pharmacological experiments including large-scale proteomic analysis using safe concentrations of TFP in order to extend our knowledge of the involvement of CaM in pollen tube growth. Transcriptomic investigations have greatly enriched our insights on Arabidopsis (Arabidopsis thaliana) pollen development in a genome-wide range (Pina et al., 2005). However, gene expression at the transcriptional level does not always directly correlate with protein level and activity. Proteomic analyses have been conducted on several tissues involved in plant sexual reproduction, such as rice (Oryza sativa) anthers containing young microspores or under early cold stress and in male gametophytes at various developmental stages (Imin et al., 2001,2004;Kerim et al., 2003), but these reports did not discriminate between gametophytic and sporophytic tissues of the anther. Studies focusing solely on the proteome of the male gametophyte have been performed in Arabidopsis andPinus strobus(Fernando, 2005;Holmes-Davis et al., 2005). Recent proteomic investigations of pollens from Arabidopsis, rice, and gymnosperms have provided opportunities to complement the information derived from transcriptomic analysis and offered new insights into the mechanisms underlying pollen functional specification (Noir et al., 2005;Dai et al., 2006,2007;Sheoran et al., 2006). However, pollen tube development in conifers (gymnosperm) differs in several ways from that in angiosperms, such as in the extended growth period, relatively slow growth rate, and extremely delayed gametogenesis, which represent a major evolutionary divergence in male gametophyte.