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Morphology (a–h) and callose deposition (i–l) of/in Petunia WT (a, i), si1PhCRT (c–f, j, k), scr_si1PhCRT (g, l), and ssDNA (h) elongating pollen tubes. b Outgrowing pollen tube, the stage when si1PhCRT duplex was added to the standard culture medium. c Swollen tip of shorter si1PhCRT pollen tube (arrow). d Transparent elements in the cytoplasm of shorter si1PhCRT pollen tube (arrows). e Widely and highly vacuolated tip (arrow), and strongly twisted/highly vacuolated distal shank (arrowheads) of si1PhCRT extended pollen tube. f Twisted shank (arrowheads) and rupture of the membrane/cell wall at the growing domain (arrow) of si1PhCRT fully elongated pollen tube. i–l Callose was not present at the apical tips of WT and si1PhCRT pollen tubes. k Increased callose deposition in the subapical zone of elongated si1PhCRT pollen tube (double arrows and inset). Bars 50 μm

Morphology (a–h) and callose deposition (i–l) of/in Petunia WT (a, i), si1PhCRT (c–f, j, k), scr_si1PhCRT (g, l), and ssDNA (h) elongating pollen tubes. b Outgrowing pollen tube, the stage when si1PhCRT duplex was added to the standard culture medium. c Swollen tip of shorter si1PhCRT pollen tube (arrow). d Transparent elements in the cytoplasm of shorter si1PhCRT pollen tube (arrows). e Widely and highly vacuolated tip (arrow), and strongly twisted/highly vacuolated distal shank (arrowheads) of si1PhCRT extended pollen tube. f Twisted shank (arrowheads) and rupture of the membrane/cell wall at the growing domain (arrow) of si1PhCRT fully elongated pollen tube. i–l Callose was not present at the apical tips of WT and si1PhCRT pollen tubes. k Increased callose deposition in the subapical zone of elongated si1PhCRT pollen tube (double arrows and inset). Bars 50 μm

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Main conclusion: Calreticulin is involved in stabilization of the tip-focused Ca (2+) gradient and the actin cytoskeleton arrangement and function that is required for several key processes driving Petunia pollen tube tip growth. Although the precise mechanism is unclear, stabilization of a tip-focused calcium (Ca(2+)) gradient seems to be critica...

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... We previously cloned and characterized the PhCRT gene from Petunia, belonging to the subgroup CRT1/2 [20], and showed its elevated expression during pollen development [21] and pollen-pistil interactions [20]. Moreover, using the RNA interference (RNAi) strategy, we found that post-transcriptional silencing of PhCRT1/2 expression strongly impaired pollen tube growth in vitro [22]. Therefore, we proposed an important role for CRT belonging to the subgroup CRT1/2 during the key reproductive events in angiosperms, including pollen tube growth. ...
... Our previous work demonstrating that siRNA-mediated post-transcriptional silencing of the PhCRT gene belonging to the subgroup CRT1/2 strongly impairs pollen tube elongation in vitro suggested that plant CRT plays a significant role in the tube growth [22]. Here, using the same experimental strategy, we confirm that diverse CRT isoforms are involved in the complex process of the pollen tubes' apical growth. ...
... Moreover, it was shown that the tip-growing germ tubes of fungal spores could take up siRNA directly from the culture medium [36], and the microspores of water ferns (Marsilea) were able to take up dsRNA from the medium at the time of their hydration [37]. Our previous [22] and present work demonstrated that PhCRT-specific siRNA was efficiently taken into cultured Petunia pollen tubes, causing a significant decrease in PhCRT mRNA levels without affecting the other genes. These results indicate that siRNA can overcome the barrier of the primary cell wall at the tube apex to enter the cytoplasm and induce the gene-silencing effect. ...
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Pollen tube growth depends on several complex processes, including exo/endocytosis, cell wall biogenesis, intracellular transport, and cell signaling. Our previous results provided evidence that calreticulin (CRT)—a prominent calcium (Ca2+)-buffering molecular chaperone in the endoplasmic reticulum (ER) lumen—is involved in pollen tube formation and function. We previously cloned and characterized the CRT gene belonging to the CRT1/2 subgroup from Petunia hybrida (PhCRT1/2), and found that post-transcriptional silencing of PhCRT1/2 expression strongly impaired pollen tube growth in vitro. Here, we report cloning of a new PhCRT3a homolog; we identified the full-length cDNA sequence and described its molecular characteristics and phylogenetic relationships to other plant CRT3 genes. Using an RNA interference (RNAi) strategy, we found that knockdown of PhCRT3a gene expression caused numerous defects in the morphology and ultrastructure of cultivated pollen tubes, including disorganization of the actin cytoskeleton and loss of cytoplasmic zonation. Elongation of siPhCRT3a pollen tubes was disrupted, and some of them ruptured. Our present data provide the first evidence that PhCRT3a expression is required for normal pollen tube growth. Thus, we discuss relationships between diverse CRT isoforms in several interdependent processes driving the apical growth of the pollen tube, including actomyosin-dependent cytoplasmic streaming, organelle positioning, vesicle trafficking, and cell wall biogenesis.
... The plant CRT family consists of three members: CRT1, CRT2 and CRT3; while CRT1/2 isoforms represent one subgroup and appear to work as primary proteins within a general ER chaperone framework, plantspecific CRT3 is a divergent member that seems to be co-expressed with pathogen/signal transductionrelated genes [8][9][10]. CRT and/or its mRNAs were found in dry and germinating pollen/tubes of Ginkgo [11], Liriodendron [12], Nicotiana [13], Haemanthus [14], Arabidopsis [15] and Petunia [16][17][18][19][20]. But to date, only three reports proved CRT expression or localization in developing anther of angiosperms. ...
... We have also demonstrated that Petunia pollen tubes accumulate of both PhCRT1/2 mRNA and CRT protein in the ER-rich cytoplasmic regions [19]. Moreover, we found that post-transcriptional silencing of PhCRT1/2 expression impairs pollen tube elongation [20]. Thus, we proposed an important role for CRT belonging to the subgroup CRT1/2 during the key reproductive events in angiosperms. ...
... 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]. The importance of CRT in these processes suggests that this protein play some important roles during complicated communications of developing male gametophyte with highly specialized sporophytic cells in the anther, such as the tapetal cells. ...
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... Moreover, the potential contribution of the ER Ca 2+ buffering protein calreticulin (Joshi et al., 2019;Mariani et al., 2003) in modulation of [Ca 2+ ] ER increases remains to be investigated further. Indeed, recent reports support the role of plant calreticulin in the overall cellular Ca 2+ homeostasis (Su et al., 2019;Suwi nska et al., 2017). ...
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... We predicted the function of these genes; it was found that three of these genes are related to calcium ions, namely MD08G1097600, MD08G1097700, and MD05G1147400. MD08G1097600 and MD08G1097700 are calreticulin, as shown in Figure 5. Calreticulin is found in Petunia and plays an important role in maintaining the gradient of calcium ions at the tip of the pollen tube [31]. Previous studies in Papaver found that self-incompatibility can break the gradient of calcium ions at the tip of the pollen tube [32], and E-helix and F-helix hand protein can bind calcium ions to participate in calcium signal transduction. ...
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... 10,11 CALR, by regulating Ca 2+ release from inositol three phosphate, regulates activation of NF-AT transcription factor. 7,12 It was demonstrated that mutation in CALR with disrupting the NF-AT and GATA-4 signaling pathway contributes to cardiac dysfunction. 4,7,11 It is possible to hypothesize that CVD development in younger patients is likely due to gene defects. ...
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... Moreover, identification of the unknown targets of CPKs (particularly for nuclear targets such as TFs) and depiction of the elaborate internetwork of the Ca 2+ /CPKs pathway with other signal pathways will lead to important insights into the mechanisms of PT growth. The progress of experimental techniques such as various omics techniques, Y2H screens, CRISPR/Cas gene editing, and RNAi by directly adding the siRNAs into the PT culture medium (Suwinska et al., 2017), various molecular probes (Mravec et al., 2017), microfluidics and microrobotics (Burri et al., 2020), and computational methods (Damineli et al., 2017) will provide new opportunities and boost our understanding of the Ca 2+ /CPKs signal pathway in PT growth. ...
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... When mechanical force acts on the cell surface and the mechanical signal is transmitted to the cytoskeleton, ER, and nucleus, the stimulation of mechanical force can cause the ER stress response and upregulate the expression of CRT 34 . Calreticulin is an important ER Ca2+-buffering protein participating in adjusting ER Ca2+ reserve and discharge 35,36 , acts as a molecular chaperone, which can help proteins fold correctly [37][38][39] . So far, the majority of studies have concentrated upon the expression and function of CRT in pulmonary and renal brosis. ...
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... Other Ca 2+ sensor proteins include calmodulin, calmodulin-like proteins, and calcineurin B-proteins that act as "sensor relays" transmitting the Ca 2+ signal via altered protein-protein interactions. Several of these sensor relays have been described in connection with tube growth, actin organisation, and regulation of K + transmembrane transport [117][118][119][120][121]. Ca 2+ -mediated stabilisation and destabilisation of actin filaments could also play a role in establishing actin structure, as high Ca 2+ concentrations cause disassembly of filamentous actin, acting via actin-binding proteins [122]. ...
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Plants display a complex life cycle, alternating between haploid and diploid generations. During fertilisation, the haploid sperm cells are delivered to the female gametophyte by pollen tubes, specialised structures elongating by tip growth, which is based on an equilibrium between cell wall-reinforcing processes and turgor-driven expansion. One important factor of this equilibrium is the rate of pectin secretion mediated and regulated by factors including the exocyst complex and small G proteins. Critically important are also non-proteinaceous molecules comprising protons, calcium ions, reactive oxygen species (ROS), and signalling lipids. Among the latter, phosphatidylinositol 4,5-bisphosphate and the kinases involved in its formation have been assigned important functions. The negatively charged headgroup of this lipid serves as an interaction point at the apical plasma membrane for partners such as the exocyst complex, thereby polarising the cell and its secretion processes. Another important signalling lipid is phosphatidic acid (PA), that can either be formed by the combination of phospholipases C and diacylglycerol kinases or by phospholipases D. It further fine-tunes pollen tube growth, for example by regulating ROS formation. How the individual signalling cues are intertwined or how external guidance cues are integrated to facilitate directional growth remain open questions.
... 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). On the other hand, in the female gametophyte, the presence of CTR in nuclei has been proposed as a protein that regulates the supply of calcium to calcium-dependent scaffolding proteins , and after double fertilization, the highest CTR accumulation in the ES is present in the zygote and the early development of the endosperm (Niedojadło et al. 2015). ...
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Calcium is a secondary messenger that regulates and coordinates the cellular responses to environmental cues. Despite calcium being a key player during fertilization in plants, little is known about its role during the development of the endosperm. For this reason, the distribution, abundance, and dynamics of cytosolic calcium during the first stages of endosperm development of Agave tequilana and Agave salmiana were analyzed. Cytosolic calcium and actin filaments detected in the embryo sacs of Agave tequilana and A. salmiana revealed that they play an important role during the division and nuclear migration of the endosperm. After fertilization, a relatively high concentration of cytosolic calcium was located in the primary nucleus of the endosperm, as well as around migrating nuclei during the development of the endosperm. Cytosolic calcium participates actively during the first mitosis of the endosperm mother cell and interacts with the actin filaments that generate the motor forces during the migration of the nuclei through the large cytoplasm of the central cell.
... Another class of calcium-sensitive protein, calreticulin (CRT), has been implicated in pollen tube growth in petunia. Knockdown of PhCRT expression in growing tubes alters the cytoskeletal actin dynamics as well as associated cytoplasmic streaming and the Ca 2+ gradient, supporting a role for this protein in stabilizing ion fluxes coupled to the regulation of actin [54]. The function of CRT was previously investigated in tobacco pollen and hypothesized to play an important role in Ca 2+ homeostasis [55]. ...
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The coordinated growth of pollen tubes through floral tissues to deliver the sperm cells to the egg and facilitate fertilization is a highly regulated process critical to the Angiosperm life cycle. Studies suggest that the concerted action of a variety of signaling pathways underlies the rapid polarized tip growth exhibited by pollen tubes. Ca2+ and small GTPase-mediated pathways have emerged as major players in the regulation of pollen tube growth. Evidence suggests that these two signaling pathways not only integrate with one another but also with a variety of other important signaling events. As we continue to elucidate the mechanisms involved in pollen tube growth, there is a growing importance in taking a holistic approach to studying these pathways in order to truly understand how tip growth in pollen tubes is orchestrated and maintained. This review considers our current state of knowledge of Ca2+-mediated and GTPase signaling pathways in pollen tubes, how they may intersect with one another, and other signaling pathways involved. There will be a particular focus on recent reports that have extended our understanding in these areas.