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Immunogold localization of CRT (d-f, i, k) and distribution of exchangeable Ca 2+ (g, h, j, l) in Petunia transmitting cells. a-c Methylene blue stained crosssections of the pistil style showing transmitting tissue before (a, b) and after pollination (c). d, g, i, j Distributions of CRT and loosely bound Ca 2+ in transmitting cells before pollination. e, f, h, k, l Distributions of CRT and looselybound Ca 2+ in transmitting cells after pollination. cx cortex, d dictyosome, ecm extracellular matrix, er endoplasmic reticulum, m mitochondria, pl plasmodesmata, tt transmitting tissue, ttc transmitting tissue cells, pt pollen tube, vb vascular bundle. Bars 50 μm (a-c), 500 nm (d, e, g, j, l), 200 nm (f, h, i, k) 

Immunogold localization of CRT (d-f, i, k) and distribution of exchangeable Ca 2+ (g, h, j, l) in Petunia transmitting cells. a-c Methylene blue stained crosssections of the pistil style showing transmitting tissue before (a, b) and after pollination (c). d, g, i, j Distributions of CRT and loosely bound Ca 2+ in transmitting cells before pollination. e, f, h, k, l Distributions of CRT and looselybound Ca 2+ in transmitting cells after pollination. cx cortex, d dictyosome, ecm extracellular matrix, er endoplasmic reticulum, m mitochondria, pl plasmodesmata, tt transmitting tissue, ttc transmitting tissue cells, pt pollen tube, vb vascular bundle. Bars 50 μm (a-c), 500 nm (d, e, g, j, l), 200 nm (f, h, i, k) 

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Article
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Calcium (Ca(2+)) plays essential roles in generative reproduction of angiosperms, but the sites and mechanisms of Ca(2+) storage and mobilization during pollen-pistil interactions have not been fully defined. Both external and internal Ca(2+) stores are likely important during male gametophyte communication with the sporophytic and gametophytic cel...

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... linking the stigma with the ovary. To investigate this, samples of unpollinated and polli- nated Petunia styles were processed for immunogold labeling and visualized by electron microscopy. As shown in semithin sections stained with methylene blue, Petunia has a solid style with highly specialized transmitting tissue composed of secre- tory cells (Fig. 1a, b). The extracellular matrix of this tissue is enriched with exudates and forms the appropriate physical and nutritional medium for pollen tube growth in vivo (Fig. ...
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... electron microscopy. As shown in semithin sections stained with methylene blue, Petunia has a solid style with highly specialized transmitting tissue composed of secre- tory cells (Fig. 1a, b). The extracellular matrix of this tissue is enriched with exudates and forms the appropriate physical and nutritional medium for pollen tube growth in vivo (Fig. ...
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... the cytoplasm of transmitting cells, CRT was typi- cally localized in the ER, both in unpollinated and pollinated pistils ( Fig. 1d, e, respectively). However, before pollination, numerous gold traces were also detected along the edge of these cells, on the border between the protoplast and the cell wall ( Fig. 1d, arrows). After pollination, CRT was frequently observed at the cellular peripheries ( Fig. 1f, arrows) and ac- cumulated in the plasma membrane-attached ...
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... the cytoplasm of transmitting cells, CRT was typi- cally localized in the ER, both in unpollinated and pollinated pistils ( Fig. 1d, e, respectively). However, before pollination, numerous gold traces were also detected along the edge of these cells, on the border between the protoplast and the cell wall ( Fig. 1d, arrows). After pollination, CRT was frequently observed at the cellular peripheries ( Fig. 1f, arrows) and ac- cumulated in the plasma membrane-attached patches (Fig. 1f). Consistent with CRT being a Ca 2+ -binding/buffering protein, Ca 2+ -antimonate precipitates (Ca 2+ ppts corresponding to ex- changeable Ca 2+ ) were observed in ...
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... localized in the ER, both in unpollinated and pollinated pistils ( Fig. 1d, e, respectively). However, before pollination, numerous gold traces were also detected along the edge of these cells, on the border between the protoplast and the cell wall ( Fig. 1d, arrows). After pollination, CRT was frequently observed at the cellular peripheries ( Fig. 1f, arrows) and ac- cumulated in the plasma membrane-attached patches (Fig. 1f). Consistent with CRT being a Ca 2+ -binding/buffering protein, Ca 2+ -antimonate precipitates (Ca 2+ ppts corresponding to ex- changeable Ca 2+ ) were observed in the same localizations where CRT was found; there were the ER (Fig. 1g) and several patches ...
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... 1d, e, respectively). However, before pollination, numerous gold traces were also detected along the edge of these cells, on the border between the protoplast and the cell wall ( Fig. 1d, arrows). After pollination, CRT was frequently observed at the cellular peripheries ( Fig. 1f, arrows) and ac- cumulated in the plasma membrane-attached patches (Fig. 1f). Consistent with CRT being a Ca 2+ -binding/buffering protein, Ca 2+ -antimonate precipitates (Ca 2+ ppts corresponding to ex- changeable Ca 2+ ) were observed in the same localizations where CRT was found; there were the ER (Fig. 1g) and several patches adjacent to the cell wall of the transmitting cells (Fig. 1h). It should be noted ...
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... observed at the cellular peripheries ( Fig. 1f, arrows) and ac- cumulated in the plasma membrane-attached patches (Fig. 1f). Consistent with CRT being a Ca 2+ -binding/buffering protein, Ca 2+ -antimonate precipitates (Ca 2+ ppts corresponding to ex- changeable Ca 2+ ) were observed in the same localizations where CRT was found; there were the ER (Fig. 1g) and several patches adjacent to the cell wall of the transmitting cells (Fig. 1h). It should be noted that Ca 2+ ppts were predominant- ly observed in the ER enriched peripheral cytoplasm (Fig. 1g, arrows). Epitopes binding CRT PAb were also found at plas- modesmata connecting transmitting cells. The specific linear pattern of the ...
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... membrane-attached patches (Fig. 1f). Consistent with CRT being a Ca 2+ -binding/buffering protein, Ca 2+ -antimonate precipitates (Ca 2+ ppts corresponding to ex- changeable Ca 2+ ) were observed in the same localizations where CRT was found; there were the ER (Fig. 1g) and several patches adjacent to the cell wall of the transmitting cells (Fig. 1h). It should be noted that Ca 2+ ppts were predominant- ly observed in the ER enriched peripheral cytoplasm (Fig. 1g, arrows). Epitopes binding CRT PAb were also found at plas- modesmata connecting transmitting cells. The specific linear pattern of the labeling is likely to correspond to ER in the cytoplasmic sleeve -an essential ...
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... precipitates (Ca 2+ ppts corresponding to ex- changeable Ca 2+ ) were observed in the same localizations where CRT was found; there were the ER (Fig. 1g) and several patches adjacent to the cell wall of the transmitting cells (Fig. 1h). It should be noted that Ca 2+ ppts were predominant- ly observed in the ER enriched peripheral cytoplasm (Fig. 1g, arrows). Epitopes binding CRT PAb were also found at plas- modesmata connecting transmitting cells. The specific linear pattern of the labeling is likely to correspond to ER in the cytoplasmic sleeve -an essential component of these narrow channels (Fig. 1i, arrow). Moreover, double-labeling experi- ments using both CRT PAb and Cal ...
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... that Ca 2+ ppts were predominant- ly observed in the ER enriched peripheral cytoplasm (Fig. 1g, arrows). Epitopes binding CRT PAb were also found at plas- modesmata connecting transmitting cells. The specific linear pattern of the labeling is likely to correspond to ER in the cytoplasmic sleeve -an essential component of these narrow channels (Fig. 1i, arrow). Moreover, double-labeling experi- ments using both CRT PAb and Cal MAb clearly showed that CRT co-localized tightly with callose at the neck region of the plasmodesmata (Fig. 1i, k). Numerous Ca 2+ ppts were found in the cortical ER attached to plasmodesmata (Fig. 1j, l) as well as with their central cavity and neck regions (Fig. 1j, ...
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... cells. The specific linear pattern of the labeling is likely to correspond to ER in the cytoplasmic sleeve -an essential component of these narrow channels (Fig. 1i, arrow). Moreover, double-labeling experi- ments using both CRT PAb and Cal MAb clearly showed that CRT co-localized tightly with callose at the neck region of the plasmodesmata (Fig. 1i, k). Numerous Ca 2+ ppts were found in the cortical ER attached to plasmodesmata (Fig. 1j, l) as well as with their central cavity and neck regions (Fig. 1j, l, ...
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... the cytoplasmic sleeve -an essential component of these narrow channels (Fig. 1i, arrow). Moreover, double-labeling experi- ments using both CRT PAb and Cal MAb clearly showed that CRT co-localized tightly with callose at the neck region of the plasmodesmata (Fig. 1i, k). Numerous Ca 2+ ppts were found in the cortical ER attached to plasmodesmata (Fig. 1j, l) as well as with their central cavity and neck regions (Fig. 1j, l, ...
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... (Fig. 1i, arrow). Moreover, double-labeling experi- ments using both CRT PAb and Cal MAb clearly showed that CRT co-localized tightly with callose at the neck region of the plasmodesmata (Fig. 1i, k). Numerous Ca 2+ ppts were found in the cortical ER attached to plasmodesmata (Fig. 1j, l) as well as with their central cavity and neck regions (Fig. 1j, l, ...

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... Previous work from our laboratory provided evidence that CRT may be involved in Ca 2+ homeostasis and molecular chaperoning during the key reproductive events in Petunia pistil, such as pistil transmitting tract maturation, pollen-pistil interactions, double fertilization, and early embryogenesis [17,18,22]. We also demonstrated that CRT has a critical role in pollen tube growth in vitro [20]. ...
... However, localization of this protein outside the ER has also been observed in eukaryotic cells. Plant CRT was detected in many different compartments and structures, including dictyosomes, vesicles, protein bodies, nucleus, plasma membrane, and the cell wall [22]. Although, some previous work from our lab and others indicated location of CRT in several extracellular regions were somewhat controversial, an elegant work of Luczak et al. [34] provided clear evidence that CRT, similar to several other proteins, is present in the cell walls of few plant species including maize, Lupinus and Arabidopsis. ...
... Although, some previous work from our lab and others indicated location of CRT in several extracellular regions were somewhat controversial, an elegant work of Luczak et al. [34] provided clear evidence that CRT, similar to several other proteins, is present in the cell walls of few plant species including maize, Lupinus and Arabidopsis. We have previously demonstrated that both CRT and exchangeable Ca 2+ were localized to the extracellular peripheries of highly specialized plant cells, such as pollen tubes and synergids [14,16,18,22]. In pollen tubes, CRT labeling was detected in the peripheral ER-rich cytoplasm of the tube adjacent to the cell membrane and in the callosic cell wall. ...
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Background Pollen development in the anther in angiosperms depends on complicated cellular interactions associated with the expression of gametophytic and sporophytic genes which control fundamental processes during microsporo/gametogenesis, such as exo/endocytosis, intracellular transport, cell signaling, chromatin remodeling, and cell division. Most if not all of these cellular processes depend of local concentration of calcium ions (Ca ²⁺ ). Work from our laboratory and others provide evidence that calreticulin (CRT), a prominent Ca ²⁺ -binding/buffering protein in the endoplasmic reticulum (ER) of eukaryotic cells, may be involved in pollen formation and function. Here, we show for the first time the expression pattern of the PhCRT1 gene and CRT accumulation in relation to exchangeable Ca ²⁺ in Petunia hybrida developing anther, and discuss probable roles for this protein in the male gametophyte development. Results Using northern hybridization, western blot analysis, fluorescent in situ hybridization (FISH), immunocytochemistry, and potassium antimonate precipitation, we report that PhCRT1 is highly expressed in the anther and localization pattern of the CRT protein correlates with loosely bound (exchangeable) Ca ²⁺ during the successive stages of microsporo/gametogenesis. We confirmed a permanent presence of both CRT and exchangeable Ca ²⁺ in the germ line and tapetal cells, where these factors preferentially localized to the ER which is known to be the most effective intracellular Ca ²⁺ store in eukaryotic cells. In addition, our immunoblots revealed a gradual increase in CRT level from the microsporocyte stage through the meiosis and the highest CRT level at the microspore stage, when both microspores and tapetal cells show extremely high secretory activity correlated with the biogenesis of the sporoderm. Conclusion Our present data provide support for a key role of CRT in developing anther of angiosperms – regulation of Ca ²⁺ homeostasis during pollen grains formation. This Ca ²⁺ -buffering chaperone seems to be essential for pollen development and maturation since a high rate of protein synthesis and protein folding within the ER as well as intracellular Ca ²⁺ homeostasis are strictly required during the multi-step process of pollen development.
... A C-terminally truncated calreticulin was observed (CaCl2 spot 1274). Although regarded as an ER-resident protein, calreticulin was described in other subcellular compartments [43]. Animal calreticulin is linked with the recognition of tumor and apoptotic cells, and wound healing in plants it is linked to growth and stress responses [44]. ...
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... It is known that in the tip, there is a high concentration of calcium (Steinhorst and Kudla 2013); in addition, CRT has been reported in mitotic cells in the spindle apparatus and the nuclear envelope (Denecke et al. 1995). CRT could also be influencing the storage of Ca 2+ and cytosolic calcium elevations (Krause and Michalak 1997), and actively participating during the reproductive process of angiosperm plants as recently proposed by Wasąg et al. (2018). In addition, Suwińska et al. (2015) proposed that during the development of the pollen tube, the CRT keeps calcium at the tip of the PT and the suppression of this gene directly affects the cytoskeleton organization of actin (Suwińska et al. 2017). ...
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... Large numbers of CRT proteins are localized near the transmitting tract and synergid cells, cells surrounding the embryo sac, and pollen tubes. Because CRT regulates calcium storage, it may be involved in the accumulation of calcium used for pollen tube elongation (Wasag et al. 2017). CNGC18 is involved in calcium ion uptake into pollen tubes and pollen tube guidance (Gao et al. 2016). ...
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Sexual reproduction is achieved by precise interactions between male and female reproductive organs. In plant fertilization, sperm cells are carried to ovules by pollen tubes. Signals from the pistil are involved in elongation and control of the direction of the pollen tube. Genetic, reverse genetic, and cell biological analyses using model plants have identified various factors related to the regulation of pollen tube growth and guidance. In this review, I summarize the mechanisms and molecules controlling pollen tube growth to the ovule, micropylar guidance, reception of the guidance signal in the pollen tube, rupture of the pollen tube to release sperm cells, and cessation of the tube guidance signal. I also briefly introduce various techniques used to analyze pollen tube guidance in vitro.