Marta Lenartowska

Lead deposited in the cell wall of Funaria hygrometrica protonemata is not stable – A remobilization can occur

The hypothesis that lead (Pb) can be uptake or remobilized from the cell wall (CW) by internalization withlow-esterified pectins (up to 40%--JIM5-P), was studied in tip-growing apical cell of Funaria hygrometrica protonemata. Treatment 4h with 1mM PbCl(2) caused marked vesicular traffic intensification and the common internalization of JIM5-P from the CW. Lead bound to JIM5-P was internalized from the CW, together with this compound and entered the protoplast. It showed that Pb deposited in CW is not as safe for plant cell as previously believed. However, pulse-chase experiments (recovering 4 h and 24 h) indicated that CW and its thickenings can function as the final sequestration compartments. In Pb deposition sites, a callose layer occurred. It was localized from the protoplast site, next to Pb deposits separating sequestrated to CW and its thickenings Pb from plasma membrane almost certainly protecting the plant cell from its returning into the protoplast.

Actin filament organization and polarity in pollen tubes revealed by myosin II subfragment 1 decoration

The actin cytoskeleton plays a crucial role in pollen tube growth. In elongating pollen tubes the organization and arrangement of actin filaments (AFs) differs between the shank and apical region. However, the orientation of AFs in pollen tubes has not yet been successfully demonstrated. In the present work we have used myosin II subfragment 1 (S1) decoration to determine the polarity of AFs in pollen tubes. Electron microscopy studies revealed that in the shank of the tube bundles of AFs exhibit uniform polarity with those close to the cell cortex having their barbed ends oriented towards the tip of the pollen tube while those in the cell center have their barbed ends oriented toward the base of the tube. At the subapex, some AFs are organized in closely packed and longitudinally oriented bundles and some form curved bundles adjacent to the cell membrane. In contrast, few AFs are dispersed with random orientation in the extreme apex of the pollen tube. Our results confirm that the direction of cytoplasmic streaming within pollen tubes is determined by the polarity of AFs in the bundles.

Proper Cellular Reorganization during Drosophila spermatid Individualization Depends on Actin Structures Composed of Two Domains, Bundles and Meshwork, that Are Differentially Regulated and Have Different Functions

During spermatid individualization in Drosophila, actin structures (cones) mediate cellular remodeling that separates the syncytial spermatids into individual cells. These actin cones are composed of two structural domains, a front meshwork and a rear region of parallel bundles. We show here that the two domains form separately in time, are regulated by different sets of actin-associated proteins, can be formed independently, and have different roles. Newly forming cones were composed only of bundles, whereas the meshwork formed later, coincident with the onset of cone movement. Polarized distributions of myosin VI, Arp2/3 complex, and the actin-bundling proteins, singed (fascin) and quail (villin), occurred when movement initiated. When the Arp2/3 complex was absent, meshwork formation was compromised, but surprisingly, the cones still moved. Despite the fact that the cones moved, membrane reorganization and cytoplasmic exclusion were abnormal and individualization failed. In contrast, when profilin, a regulator of actin assembly, was absent, bundle formation was greatly reduced. The meshwork still formed, but no movement occurred. Analysis of this actin structures formation and participation in cellular reorganization provides insight into how the mechanisms used in cell motility are modified to mediate motile processes within specialized cells.

Immunocytochemical localization of esterified and unesterified pectins in unpollinated and pollinated styles of Petunia hybrida Hort.

Abstract. Localization of pectins in the style of Petunia hybrida before and after pollination was investigated by immunocytochemistry using two primary monoclonal antibodies specific to highly (JIM7) and weakly (JIM5) methylesterified pectins. In the unpollinated style, esterified pectins occurred mainly in the cell walls of cortex tissue, while unesterified pectins were present mainly in the extracellular matrix (ECM) of the transmitting tract. After pollination no remarkable differences were found in pectin distribution in the ground tissue of the style. On the other hand, in the transmitting tract a reduction in the quantity of unesterified pectins was observed. Unesterified pectins in the extracellular regions of the transmitting tissue decreased before the penetration of the pollen tubes, indicating that pollination induces a reduction in the amount of unesterified pectins in the transmitting-tract ECM. The correlation between the degradation of strongly Ca2+-binding pectins and the growing level of those ions in the extracellular regions of the transmitting tract in the pollinated pistil of P. hybrida (M. Lenartowska et al. 1997) suggests that this process may constitute a mechanism for creating an optimum calcium medium for in vivo-growing pollen tubes. Both pectin categories were localized in pollen tubes. Esterified pectin epitopes were localized mainly in the vesicles of the tip cytoplasm. Unesterified pectin epitopes were found in the external fibrillar wall of pollen tubes.

Immunocytochemical evidence of calreticulin-like protein in pollen tubes and styles of Petunia hybrida Hort.

Summary. With a polyclonal antibody raised against calreticulin (CRT) the locations where the protein occurs in unpollinated and pollinated styles of Petunia hybrida were localized. The epitopes binding the CRT antibody were immunolocalized preferentially in pollen tubes. In transmitting tract cells, both before and after pollination, the level of CRT was low. The protein was mainly localized in the cytosol and around dictyosomes of transmitting-tract cells. In pollen tubes, a high level of CRT was found at their tips rich in endoplasmatic reticulum, cisternae piles of reticular and/or dictyosomal origin, and vesicles. Binding sites of the CRT antibody were also found in the internal callosic cell wall of the pollen tube. These results indicate a role of CRT in cells directly participating in pollen-pistil interaction.

Calreticulin expression and localization in plant cells during pollen-pistil interactions.

In this report, the distributions of calreticulin (CRT) and its transcripts in Haemanthus pollen, pollen tubes, and somatic cells of the hollow pistil were studied. Immunoblot analysis of protein extracts from mature anthers, dry and germinated pollen, growing pollen tubes, and unpollinated/pollinated pistils revealed a strong expression of CRT. Both in vitro and in situ studies confirmed the presence of CRT mRNA and protein in pollen/pollen tubes and somatic cells of the pistil transmitting tract. The co-localization of these molecules in ER of these cells suggests that the rough ER is a site of CRT translation. In the pistil, accumulation of the protein in pollen tubes, transmitting tract epidermis (tte), and micropylar cells of the ovule (mc) was correlated with the increased level of exchangeable calcium. Therefore, CRT as a Ca2+-binding/buffering protein, may be involved in mechanism of regulation calcium homeostasis in these cells. The functional role of the protein in pollen–pistil interactions, apart from its postulated function in cellular Ca2+ homeostasis, is discussed.

Pectin dynamic and distribution of exchangeable Ca2+ in Haemanthus albiflos hollow style during pollen–pistil interactions

In this report, the localization and spatial distribution of two categories of pectin, high and low methylesterified, on the background of dynamic in loosely bound calcium (Ca2+) in Haemanthus hollow style were studied before and after pollination. In the style transmitting tract of unpollinated pistil, mainly high-methylesterified pectins were present, both in the transmitting tract epidermis and in the style canal. After pollination, an increase in the level of two investigated categories of pectin was observed, but the amount of high-methylesterified one in each period of time analyzed was permanently higher. Locally, in the regions of the style canal penetrated by pollen tubes, process of pectin de-esterification was initiated. However, pollination caused an increase of loosely bound Ca2+ level in the style transmitting tract, this process appears to be not linked with pectin de-esterification and possible Ca2+ release after the lysis of Ca2+ cross-linked de-esterified pectin. Instead, it seems to be based on Ca2+ exocytosis from the transmitting tract epidermis cells providing a source of Ca2+ for pollen tubes growing in Haemanthus hollow style.

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

AbstractMain conclusionCalreticulin is involved in stabilization of the tip-focused Ca2+gradient and the actin cytoskeleton arrangement and function that is required for several key processes drivingPetuniapollen 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.

Calreticulin expression in relation to exchangeable Ca2+ level that changes dynamically during anthesis, progamic phase, and double fertilization in Petunia

Calcium (Ca2+) plays essential roles in plant sexual reproduction, but the sites and the mechanism of Ca2+ mobile storage during pollen–pistil interactions have not been fully defined. Because the Ca2+-buffering protein calreticulin (CRT) is able to bind and sequester Ca2+, it can serve as a mobile intracellular store of easily releasable Ca2+ and control its local concentration within the cytoplasm. Our previous studies showed an enhanced expression of Petunia hybrida CRT gene (PhCRT) during pistil transmitting tract maturation, pollen germination and tube outgrowth on the stigma, gamete fusion, and early embryogenesis. Here, we demonstrate that elevated expression of CRT results in the accumulation of this protein in response to anthesis, pollination, sperm cells deposition within the receptive synergid and fertilization, when the level of exchangeable Ca2+ changes dynamically. CRT localizes mainly to the endoplasmic reticulum and Golgi compartments in the pistil transmitting tract cells, germinated pollen/tubes, and sporophytic/gametophytic cells of the ovule and corresponds with loosely bound Ca2+. Additionally, the immunogold research shows, for the first time, highly selective CRT distribution in specific nuclear sub-domains. On the basis of our results, we discuss the possible functions of CRT with respect to the critical role of Ca2+ homeostasis during key events of the multi-step process of generative reproduction in angiosperms.

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

Key messageIn 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 Ca2+gradient.AbstractPollen 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 Ca2+ in discrete regions of the tube cytoplasm. Most notably, a mechanism must function to maintain a steep gradient of Ca2+ that exists at the tip of growing pollen tube. Several pieces of evidence point to calreticulin (CRT) as a key Ca2+-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 Ca2+, 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 Ca2+ gradient and thus modulating local Ca2+ concentration within the tube cytoplasm.