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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., (© 2022. The Author(s).)

Key Message: In germinating pollen grains and growing pollen tubes, CRT is translated on ER membrane-bound ribosomes in the regions where its activity is required for stabilization of tip-focused Ca (2+) gradient. Pollen tube growth requires coordination of signaling, exocytosis, and actin cytoskeletal organization. Many of these processes are thought to be controlled by finely tuned regulation of cytoplasmic Ca(2+) in discrete regions of the tube cytoplasm. Most notably, a mechanism must function to maintain a steep gradient of Ca(2+) that exists at the tip of growing pollen tube. Several pieces of evidence point to calreticulin (CRT) as a key Ca(2+)-binding/-buffering protein involved in pollen germination and pollen tube growth. We previously hypothesized that in germinating pollen and growing tubes, CRT is translated on the ribosomes associated with endoplasmic reticulum (ER) in the regions where its activity might be required. In this report, we have addressed this idea by identifying the sites where CRT mRNA, CRT protein, 18S rRNA, and rough ER are localized in Petunia pollen tubes. We observed all four components in the germinal aperture of pollen grains and in subapical regions of elongating tubes. These results seem to support our idea that CRT is translated on ER membrane-bound ribosomes during pollen germination and pollen tube growth. In elongated pollen tubes, we found CRT mainly localized in the subapical zone, where ER and Golgi stacks are abundant. In eukaryotic cells, these organelles serve as mobile intracellular stores of easily releasable Ca(2+), which can be buffered by proteins such as CRT. Therefore, we postulate that subapical-localized CRT is involved in pollen tube growth by maintaining the stable tip-focused Ca(2+) gradient and thus modulating local Ca(2+) concentration within the tube cytoplasm.

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. [ABSTRACT FROM AUTHOR]

Calcium (Ca) plays essential roles in generative reproduction of angiosperms, but the sites and mechanisms of Ca storage and mobilization during pollen-pistil interactions have not been fully defined. Both external and internal Ca stores are likely important during male gametophyte communication with the sporophytic and gametophytic cells within the pistil. Given that calreticulin (CRT), a Ca-buffering protein, is able to bind Ca reversibly, it can serve as a mobile store of easily releasable Ca (so called an exchangeable Ca) in eukaryotic cells. CRT has typical endoplasmic reticulum (ER) targeting and retention signals and resides primarily in the ER. However, localization of this protein outside the ER has also been revealed in both animal and plant cells, including Golgi/dictyosomes, nucleus, plasma membrane/cell surface, plasmodesmata, and even extracellular matrix. These findings indicate that CRT may function in a variety of different cell compartments and specialized structures. We have recently shown that CRT is highly expressed and accumulated in the ER of plant cells involved in pollen-pistil interactions in Petunia, and we proposed an essential role for CRT in intracellular Ca storage and mobilization during the key reproductive events. Here, we demonstrate that both CRT and exchangeable Ca are localized in the intra/extracellular peripheries of highly specialized plant cells, such as the pistil transmitting tract cells, pollen tubes, nucellus cells surrounding the embryo sac, and synergids. Based on our present results, we propose that extracellularly located CRT is also involved in Ca storage and mobilization during sexual reproduction of angiosperms. [ABSTRACT FROM AUTHOR]

Main conclusion : Calreticulin is involved in stabilization of the tip-focused Ca 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) gradient seems to be critical for pollen germination and pollen tube growth. We hypothesize that calreticulin (CRT), a Ca-binding/buffering chaperone typically residing in the lumen of the endoplasmic reticulum (ER) of eukaryotic cells, is an excellent candidate to fulfill this role. We previously showed that in Petunia pollen tubes growing in vitro, CRT is translated on ER membrane-bound ribosomes that are abundant in the subapical zone of the tube, where CRT's Ca-buffering and chaperone activities might be particularly required. Here, we sought to determine the function of CRT using small interfering RNA (siRNA) to, for the first time in pollen tubes growing in vitro, knockdown expression of a gene. We demonstrate that siRNA-mediated post-transcriptional silencing of Petunia hybrida CRT gene ( PhCRT) expression strongly impairs pollen tube growth, cytoplasmic zonation, actin cytoskeleton organization, and the tip-focused Ca gradient. Moreover, reduction of CRT alters the localization and disturbs the structure of the ER in abnormally elongating pollen tubes. Finally, cytoplasmic streaming is inhibited, and most of the pollen tubes rupture. Our data clearly show an interplay between CRT, Ca gradient, actin-dependent cytoplasmic streaming, organelle positioning, and vesicle trafficking during pollen tube elongation. Thus, we suggest that CRT functions in Petunia pollen tube growth by stabilizing Ca homeostasis and acting as a chaperone to assure quality control of glycoproteins passing through the ER. [ABSTRACT FROM AUTHOR]

In this study, the transcriptional state and distribution of RNA polymerase II, pre-mRNA splicing machinery elements, and rRNA transcripts were investigated in the sperm cells of Hyacinthus orientalis L. during in vitro pollen tube growth. During the second pollen mitosis, no nascent transcripts were observed in the area of the dividing generative cell, whereas the splicing factors were present and their pools were divided between newly formed sperm cells. Just after their origin, the sperm cells were shown to synthesize new RNA, although at a markedly lower level than the vegetative nucleus. The occurrence of RNA synthesis was accompanied by the presence of RNA polymerase II and a rich pool of splicing machinery elements. Differences in the spatial pattern of pre-mRNA splicing factors localization reflect different levels of RNA synthesis in the vegetative nucleus and sperm nuclei. In the vegetative nucleus, they were localized homogenously, whereas in the sperm nuclei a mainly speckled pattern of small nuclear RNA with a trimethylguanosine cap (TMG snRNA) and SC35 protein distribution was observed. As pollen tube growth proceeded, inhibition of RNA synthesis in the sperm nuclei was observed, which was accompanied by a gradual elimination of the splicing factors. In addition, analysis of rRNA localization indicated that the sperm nuclei are likely to synthesize some pool of rRNA at the later steps of pollen tube. It is proposed that the described changes in the nuclear activity of H. orientalis sperm cells reflect their maturation process during pollen tube growth, and that mature sperm cells do not carry into the zygote the nascent transcripts or the splicing machinery elements. [ABSTRACT FROM PUBLISHER]