Elżbieta Bednarska

Localisation pattern of homogalacturonan and arabinogalactan proteins in developing ovules of the gymnosperm plant Larix decidua Mill

We have identified and characterised the temporal and spatial distribution of the homogalacturonan (HG) and arabinogalactan proteins (AGP) epitopes that are recognised by the antibodies JIM5, JIM7, LM2, JIM4, JIM8 and JIM13 during ovule differentiation in Larix decidua Mill. The results obtained clearly show differences in the pattern of localisation of specific HG epitopes between generative and somatic cells of the ovule. Immunocytochemical studies revealed that the presence of low-esterified HG is characteristic only of the wall of megasporocyte and megaspores. In maturing female gametophytes, highly esterified HG was the main form present, and the central vacuole of free nuclear gametophytes was particularly rich in this category of HG. This pool will probably be used in cell wall building during cellularisation. The selective labelling obtained with AGP antibodies indicates that some AGPs can be used as markers for gametophytic and sporophytic cells differentiation. Our results demonstrated that the AGPs recognised by JIM4 may constitute molecules determining changes in ovule cell development programs. Just after the end of meiosis, the signal detected with JIM4 labelling appeared only in functional and degenerating megaspores. This suggests that the antigens bound by JIM4 are involved in the initiation of female gametogenesis in L. decidua. Moreover, the analysis of AGPs distribution showed that differentiation of the nucellus cells occurs in the very young ovule stage before megasporogenesis. Throughout the period of ovule development, the pattern of localisation of the studied AGPs was different both in tapetum cells surrounding the gametophyte and in nucellus cells. Changes in the distribution of AGPs were also observed in the nucellus of the mature ovule, and they could represent an indicator of tissue arrangement to interact with the growing pollen tube. The possible role of AGPs in fertilisation is also discussed.

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.

Distribution of poly(A) RNA and splicing machinery elements in mature Hyacinthus orientalis L. pollen grains and pollen tubes growing in vitro

Summary.The localization of poly(A) mRNA and molecules participating in pre-mRNA splicing, i.e., small nuclear ribonucleoproteins (snRNPs) and the SC35 protein, in mature Hyacinthus orientalis L. pollen grains before anthesis and pollen tubes germinating in vitro were analyzed. The observations indicated a pattern of poly(A) mRNA distribution in mature pollen grains before anthesis which differed from that in germinating pollen grains. Directly before anthesis, poly(A) mRNA was homogeneously distributed throughout the whole cytoplasm, whereas after rehydration, it accumulated at one of the pollen poles. In the pollen tube, poly(A) mRNA was present in the cytoplasm, mainly in the areas beneath the cell membrane and the apical zone. Both before anthesis and during growth of the pollen tube, splicing snRNPs and SC35 protein were localized mainly in the area of the pollen nuclei. During anthesis and just after rehydration of the pollen grains, the pattern of labeling and the levels of the investigated antigens in the areas of the vegetative and generative nuclei were similar. During growth of the pollen tube, a change was observed in the distribution and an increase in the levels of trimethylguanosine snRNA and SC35 protein in the vegetative nucleus. Such a pattern of localization of the splicing machinery suggests resumption of transcription and/or maturation of pre-mRNA in the growing pollen tube.

Transcriptional state and distribution of poly(A) RNA and RNA polymerase II in differentiating Hyacinthus orientalis L. pollen grains

Spatial distribution of poly(A) RNA, hypophosphorylated Pol IIA, and hyperphosphorylated Pol IIO form of polymerase RNA II was characterized using immunofluorescence, immunogold and fluorescence in situ hybridization (FISH) techniques in relationship to transcriptional activity in the microspore and developing pollen of H. orientalis. During the course of pollen development our results reflected much higher transcriptional activity in the vegetative cell than in the generative cell. The highest levels of transcription in pollen cells were observed in young pollen grains, successively decreasing during pollen maturation, reaching a minimum just before anthesis. Levels of poly(A) RNA were higher in the vegetative cell than in the generative cell during all observed stages of pollen development. Accompanying physiological inhibition of the RNA synthesis in mature pollen cells was a strong accumulation of poly(A) RNA in the cytoplasm, especially in the vegetative cell. Alterations in transcriptional activity of differentiating pollen cells were accompanied by changes in the level and localization pattern of both forms of Pol II. During high transcriptional activity in the pollen nuclei, both forms of RNA Pol II occurred at the periphery of chromatin masses, as well as in the areas between them. A strong decrease in Pol IIO levels was observed in generative and vegetative nuclei as transcriptional activity of pollen cells apparently became inhibited. Finally, just before anthesis, an almost complete lack of the Pol IIO was observed in both pollen nuclei. In contrast, the level of Pol IIA significantly increased during the later stages of pollen development, in spite of apparent transcriptional inhibition in both pollen cells. This rich pool of the hypophosphorylated form of Pol II was located mainly over the central areas of condensed chromatin clumps, which was especially visible in the generative nucleus. Spatial and temporal aspects of RNA synthesis, including poly(A) RNA, as well as organization of transcriptional machinery appear to be closely related in developing pollen cells.

Transcriptional activity and distribution of splicing machinery elements during Hyacinthus orientalis pollen tube growth

Summary.The localization of newly formed transcripts and molecules participating in pre-mRNA splicing, i.e., small nuclear ribonucleoproteins (snRNPs) and SC35 protein, in growing pollen tubes of Hyacinthus orientalis L. were analyzed in vitro and in vivo. The results indicated that the restart of RNA synthesis occurred first in the vegetative and then in the generative nucleus of both in vitro and in vivo growing pollen tubes. Changes in RNA synthesis were accompanied by the redistribution of splicing machinery elements in both vegetative and generative nuclei of the growing pollen tube. At stages of pollen tube growth when the vegetative and generative nuclei were transcriptionally active, clear differences in the distribution pattern of the splicing system components were observed in both pollen nuclei. While both small nuclear RNA with a trimethylguanosine cap on the 5′ end and SC35 protein were diffusely distributed in the nucleoplasm in the vegetative nucleus, the studied antigens were only present in the areas between condensed chromatin in the generative nucleus. When the transcriptional activity of both pollen nuclei could no longer be observed at later stages of pollen tube growth, snRNPs and SC35 protein were still present in the vegetative nuclei but not in the generative nuclei. We, therefore, investigated potential differences in the spatial organization of splicing system elements during pollen tube growth. They clearly reflect differences in gene expression patterns in the vegetative and the generative cells, which may be determined by the different biological roles of angiosperm male gametophyte cells.

Intracellular organization of the pre-mRNA splicing machinery during Hyacinthus orientalis L. pollen development

Spatial organization of splicing machinery elements in metabolically and functionally different pollen cells during Hyacinthus orientalis pollen grain development was examined by localization of trimethylguanosine (TMG) snRNA and Sm proteins, representing splicing small nuclear ribonucleoproteins, as well as SR splicing factors was investigated. In young pollen grains the level of all labeled antigens was the highest displaying essentially uniform distribution in the vegetative and generative nucleus. In the polarized microspore, as well as in the vegetative cell of the young pollen grain, both TMG snRNA and Sm proteins were also found highly concentrated in Cajal bodies. After detachment of the generative cell from the sporoderm, the redistribution of splicing machinery elements into speckled-shape clusters was observed in both nuclei in the pollen. In the mature pollen grain, labeled antigens were still present, both in the vegetative and the generative nucleus. The results reflected that in differentiating H. orientalis pollen cells, the intracellular organization pre-mRNA splicing machinery undergoes significant and characteristic changes during the course of pollen grain development. Changes in the distribution of spliceosomal components relate to the transcriptional activity of both pollen cells during their maturation.

Localization of calcium on the stigma surface of Ruscus aculeatus L. : Studies using chlorotetracycline and X-ray microanalysis.

By use of chlorotetracycline and X-ray microanalysis it is demonstrated that the receptive surface of the stigma of Ruscus aculeatus is rich in calcium. The high level of calcium is found in the epidermal cells and in the exudate covering the stigma. These results indicate that in vivo, as in vitro, calcium takes part in the regulation of pollen grain germination.

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.

Changes in poly(A) RNA and TMG snRNA distribution in the embryo sac of Hyacinthus orientalis L. before and after fertilization

The content and distribution of the poly(A) RNA and splicing machinery element––TMG snRNA in cells of the Hyacinthus orientalis L. mature embryo sac, during the progamic phase and after fertilization, were investigated. Using fluorescence in situ hybridization and immunofluorescence methods we showed that in the mature unfertilized embryo sac strong signal indicating poly(A) RNA and TMG snRNA appeared only in synergids and antipodal cells. In synergids accumulation of poly(A) RNA was shown in cytoplasm around the filiform apparatus. In the egg cell and central cell of the embryo sac accumulation of poly(A) RNA was very low and only low pool of TMG snRNA was observed in the nuclei of these cells. During the progamic phase, dramatic changes in the accumulation and distribution of poly(A) RNA and TMG snRNA were observed in the synergids. In these cells a considerable decrease in accumulation of TMG snRNA in the nucleus and poly(A) RNA in the region of the filiform apparatus occurred. In the egg cell and central cell the content of polyadenylated transcripts and TMG snRNA was still low. After fertilization a drastic increase in polyadenylated transcripts and TMG snRNA content was observed in the cells which undergo a fusion with the sperm cells (the zygote and the fertilized central cells). In contrast, a progressive decrease in poly(A) RNA and splicing snRNAs accumulation was observed in degenerating antipodal cells and synergids.

Nuclear activity of sperm cells during Hyacinthus orientalis L. in vitro pollen tube growth

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.