Molecular evidence that rough endoplasmic reticulum is the site of calreticulin translation in Petunia pollen tubes growing in vitro

Suwińska, Anna; Lenartowski, Robert; Smoliński, Dariusz Jan; Lenartowska, Marta

doi:10.1007/s00299-015-1777-x

Cited by 10 publications

(14 citation statements)

References 41 publications

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“…One important aspect of this optimized cellular growth is the presence of local pH gradients with an acidic apex region and an alkaline band in the clear zone followed by a subapical region of acidic pH (15,16). This complex organization of events demands an optimized protein synthesis machinery (17), as critical protein components like calreticulin have been shown to be continuously synthesized during pollen tube growth (18).…”

Section: Introductionmentioning

confidence: 99%

Transcriptome and translatome changes in germinated pollen under heat stress uncover roles of transporter genes involved in pollen tube growth

Poidevin

Forment

Ünal

et al. 2020

Preprint

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Plant reproduction is one key biological process very sensitive to heat stress and, as a consequence, enhanced global warming poses serious threats to food security worldwide. In this work we have used a high-resolution ribosome profiling technology to study how heat affects both the transcriptome and the translatome of Arabidopsis thaliana pollen germinated in vitro. Overall, a high correlation between transcriptional and translational responses to high temperature was found, but specific regulations at the translational level were also present. We show that bona fide heat shock genes are induced by high temperature indicating that in vitro germinated pollen is a suitable system to understand the molecular basis of heat responses. Concurrently heat induced significant downregulation of key membrane transporters required for pollen tube growth, thus uncovering heatsensitive targets. We also found that a large subset of the heat-repressed transporters is specifically up-regulated, in a coordinated manner, with canonical heat-shock genes in pollen tubes grown in vitro and semi in vivo, based on published transcriptomes from Arabidopsis thaliana. Ribosome footprints were also detected in gene sequences annotated as non-coding, highlighting the potential for novel translatable genes and translational dynamics.

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Section: Introductionmentioning

confidence: 99%

Transcriptome and translatome changes in germinated pollen under heat stress uncover roles of transporter genes involved in pollen tube growth

Poidevin

Forment

Ünal

et al. 2020

Preprint

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“…Although the precise mechanism is still unclear, stabilization of a tip-focused Ca 2+ gradient is critical for pollen germination and pollen tube growth (see reviews by Hepler et al 2012 ; Steinhorst and Kudla 2013 ). We previously showed that in Petunia pollen tubes growing in vitro, CRT is translated on ER membrane-bound ribosomes and accumulated in the ER at the subapical zone of the tube, where plays a role in stabilizing Ca 2+ homeostasis that is required for actomyosin-dependent cytoplasmic streaming, organelle positioning, vesicle trafficking, and cell wall biogenesis (Suwińska et al 2015 , 2017 ). Thus, we concluded that internal Ca 2+ stores involving CRT activity are crucial for proper pollen tube elongation.…”

Section: Discussionmentioning

confidence: 99%

Calreticulin localizes to plant intra/extracellular peripheries of highly specialized cells involved in pollen-pistil interactions

et al. 2017

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Calcium (Ca2+) plays essential roles in generative reproduction of angiosperms, but the sites and mechanisms of Ca2+ storage and mobilization during pollen-pistil interactions have not been fully defined. Both external and internal Ca2+ stores are likely important during male gametophyte communication with the sporophytic and gametophytic cells within the pistil. Given that calreticulin (CRT), a Ca2+-buffering protein, is able to bind Ca2+ reversibly, it can serve as a mobile store of easily releasable Ca2+ (so called an exchangeable Ca2+) 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 Ca2+ storage and mobilization during the key reproductive events. Here, we demonstrate that both CRT and exchangeable Ca2+ 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 Ca2+ storage and mobilization during sexual reproduction of angiosperms.

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“…For detailed ultrastructural analysis, elongated WT and si1 PhCRT pollen tubes (cultivated for about 4–5 h) were fixed and prepared for transmission electron microscopy as previously described (Suwińska et al 2015 ). In brief, pollen tubes were fixed with 2% glutaraldehyde (Sigma-Aldrich) in 0.1 M PBS, pH 7.2, for 2 h at room temperature followed by overnight at 4 °C, and then post-fixed with 2% osmium tetroxide (Sigma-Aldrich) in PBS for 30 min at room temperature.…”

Section: Methodsmentioning

confidence: 99%

“…Immunolocalization and FISH of CRT and its transcripts in pollen tubes were performed exactly according to the protocols described previously (Suwińska et al 2015 ). In brief, for FISH, elongated WT, si1 PhCRT , and scr_si1 PhCRT pollen tubes (cultivated for about 4–5 h) were fixed in freshly prepared 4% formaldehyde (Polysciences) in PBS, pH 7.2, enzymatically digested, and permeabilized with 0.1% saponin.…”

Section: Methodsmentioning

confidence: 99%

“…We previously suggested that the key molecule controlling Ca 2+ gradient in growing Petunia pollen tubes is the Ca 2+ -binding protein CRT, which is translated on membrane-bound ribosomes and accumulates in the abundant ER in the subapical zone of the tube (Suwińska et al 2015 ). In this region, the high [Ca 2+ ] c at the apex drops off sharply to the basal level observed throughout the shank (see reviews by Holdaway-Clarke and Hepler 2003 ; Hepler et al 2011 ; Steinhorst and Kudla 2013 ).…”

Section: Introductionmentioning

confidence: 99%

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Calreticulin is required for calcium homeostasis and proper pollen tube tip growth in Petunia

Suwińska

Wasąg

Zakrzewski

et al. 2017

Planta

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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 (Ca2+) gradient seems to be critical for pollen germination and pollen tube growth. We hypothesize that calreticulin (CRT), a Ca2+-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 Ca2+-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 Ca2+ 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, Ca2+ 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 Ca2+ homeostasis and acting as a chaperone to assure quality control of glycoproteins passing through the ER.Electronic supplementary materialThe online version of this article (doi:10.1007/s00425-017-2649-0) contains supplementary material, which is available to authorized users.

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Molecular evidence that rough endoplasmic reticulum is the site of calreticulin translation in Petunia pollen tubes growing in vitro

Cited by 10 publications

References 41 publications

Transcriptome and translatome changes in germinated pollen under heat stress uncover roles of transporter genes involved in pollen tube growth

Transcriptome and translatome changes in germinated pollen under heat stress uncover roles of transporter genes involved in pollen tube growth

Calreticulin localizes to plant intra/extracellular peripheries of highly specialized cells involved in pollen-pistil interactions

Calreticulin is required for calcium homeostasis and proper pollen tube tip growth in Petunia

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