Colonization of Raphanus sativus by human pathogenic microorganisms.

Contamination of vegetables with human pathogenic microorganisms (HPMOs) is considered one of the most important problems in the food industry, as current nutritional guidelines include increased consumption of raw or minimally processed organic vegetables due to healthy lifestyle promotion. Vegetables are known to be potential vehicles for HPMOs and sources of disease outbreaks. In this study, we tested the susceptibility of radish (Raphanus sativus) to colonization by different HPMOs, including Escherichia coli PCM 2561, Salmonella enterica subsp. enterica PCM 2565, Listeria monocytogenes PCM 2191 and Bacillus cereus PCM 1948. We hypothesized that host plant roots containing bactericidal compounds are less prone to HPMO colonization than shoots and leaves. We also determined the effect of selected pathogens on radish growth to check host plant-microbe interactions. We found that one-week-old radish is susceptible to colonization by selected HPMOs, as the presence of the tested HPMOs was demonstrated in all organs of R. sativus. The differences were noticed 2 weeks after inoculation because B. cereus was most abundant in roots (log10 CFU - 2.54), S. enterica was observed exclusively in stems (log10 CFU - 3.15), and L. monocytogenes and E. coli were most abundant in leaves (log10 CFU - 4.80 and 3.23, respectively). The results suggest that E. coli and L. monocytogenes show a higher ability to colonize and move across the plant than B. cereus and S. enterica. Based on fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM) approach HPMOs were detected in extracellular matrix and in some individual cells of all analyzed organs. The presence of pathogens adversely affected the growth parameters of one-week-old R. sativus, especially leaf and stem fresh weight (decreased by 47-66 and 17-57%, respectively). In two-week-old plants, no reduction in plant biomass development was noted. This observation may result from plant adaptation to biotic stress caused by the presence of HPMOs, but confirmation of this assumption is needed. Among the investigated HPMOs, L. monocytogenes turned out to be the pathogen that most intensively colonized the aboveground part of R. sativus and at the same time negatively affected the largest number of radish growth parameters.

Distribution of exchangeable Ca2+ during the process of Larix decidua Mill. pollination and germination.

The involvement of Ca2+ ions in angiosperms sexual processes is well established, while in gymnosperms, such knowledge remains limited and is still a topic of discussion. In this study, we focused on Larix decidua, using Alizarin-red S staining and the pyroantimonate method to examine the tissue and subcellular distribution of free and loosely bound Ca2+ ions at different stages of the male gametophyte's development and its interaction with the ovule. Our findings show that in larch, both the germination of pollen grains and the growth of pollen tubes occur in an environment rich in Ca2+. These ions play a crucial role in the adhesion of the pollen grain to the stigmatic tip and its subsequent movement to the micropylar canal. There is a significant presence of free and loosely bound Ca2+ ions in both the fluid of the micropylar canal and the extracellular matrix of the nucellus. As the pollen tube extends through the nucellus, we observed a notable accumulation of Ca2+ ions just above the entry to the mature archegonium, a region likely crucial for the male gametophyte's directional growth. Meanwhile, the localized presence of free and loosely bound Ca2+ ions within the egg cell cytoplasm may inhibit the pollen tubes growth and rupture, playing an important role in fertilization.

Trichoderma viride Colonizes the Roots of Brassica napus L., Alters the Expression of Stress-Responsive Genes, and Increases the Yield of Canola under Field Conditions during Drought.

In this work, we present the results of the inoculation of canola seeds (Brassica napus L.) with Trichoderma viride strains that promote the growth of plants. Seven morphologically different strains of T. viride (TvI-VII) were shown to be capable of synthesizing auxins and exhibited cellulolytic and pectinolytic activities. To gain a deeper insight into the molecular mechanisms underlying canola-T. viride interactions, we analyzed the canola stress genes metallothioneins (BnMT1-3) and stringent response genes (BnRSH1-3 and BnCRSH). We demonstrated the presence of cis-regulatory elements responsive to fungal elicitors in the promoter regions of B. napus MT and RSH genes and observed changes in the levels of the transcripts of the above-mentioned genes in response to root colonization by the tested fungal strains. Of the seven tested strains, under laboratory conditions, T. viride VII stimulated the formation of roots and the growth of canola seedlings to the greatest extent. An experiment conducted under field conditions during drought showed that the inoculation of canola seeds with a suspension of T. viride VII spores increased yield by 16.7%. There was also a positive effect of the fungus on the height and branching of the plants, the number of siliques, and the mass of a thousand seeds. We suggest that the T. viride strain TvVII can be used in modern sustainable agriculture as a bioinoculant and seed coating to protect B. napus from drought.

Function of Cajal Bodies in Nuclear RNA Retention in A. thaliana Leaves Subjected to Hypoxia.

Retention of RNA in the nucleus precisely regulates the time and rate of translation and controls transcriptional bursts that can generate profound variability in mRNA levels among identical cells in tissues. In this study, we investigated the function of Cajal bodies (CBs) in RNA retention in A. thaliana leaf nuclei during hypoxia stress was investigated. It was observed that in ncb-1 mutants with a complete absence of CBs, the accumulation of poly(A+) RNA in the leaf nuclei was lower than that in wt under stress. Moreover, unlike in root cells, CBs store less RNA, and RNA retention in the nuclei is much less intense. Our results reveal that the function of CBs in the accumulation of RNA in nuclei under stress depends on the plant organ. Additionally, in ncb-1, retention of introns of mRNA RPB1 (largest subunit of RNA polymerase II) mRNA was observed. However, this isoform is highly accumulated in the nucleus. It thus follows that intron retention in transcripts is more important than CBs for the accumulation of RNA in nuclei. Accumulated mRNAs with introns in the nucleus could escape transcript degradation by NMD (nonsense-mediated mRNA decay). From non-fully spliced mRNAs in ncb-1 nuclei, whose levels increase during hypoxia, introns are removed during reoxygenation. Then, the mRNA is transferred to the cytoplasm, and the RPB1 protein is translated. Despite the accumulation of isoforms in nuclei with retention of introns in reoxygenation, ncb-1 coped much worse with long hypoxia, and manifested faster yellowing and shrinkage of leaves.

Epigenetic mechanisms of sexual reproduction in Angiosperms / Epigenetyczne mechanizmy generatywnego rozmnazania roslin okrytonasiennych.

Epigenetic mechanisms, such as DNA methylation, RNA interference, posttranslational histone modifications and rearrangements of chromatin structure play an important role during genome reprogramming in both animals and plants. The correct epigenetic pattern of eu- and heterochromatin marks allows for maintaining chromatin in an active or transcriptionally silenced state. In the life cycle of angiosperms, epigenetic mechanisms participate in genome reprogramming during: 1) differentiation of sporophyte cells into spore mother cells (SMC) that undergo meiosis, 2) development of female and male gametophytes, within which the gametes differentiate and 3) after double fertilization during the embryo and endosperm development. SMC speciation and control of meiosis, followed by reprogramming of the sperm cells and egg cell genome, are non-cell-autonomous and require RdDM pathway. These processes involve companion cells, which produce "mobile" siRNAs signal molecules. Epigenetic control of gene expression through siRNAs also participates in maintenance of gametes and embryo genome integrity and in the parental imprinting.

MicroRNA biogenesis and activity in plant cell dedifferentiation stimulated by cell wall removal.

BACKGROUND: Despite the frequent use of protoplast-to-plant system in in vitro cultures of plants, the molecular mechanisms regulating the first and most limiting stages of this process, i.e., protoplast dedifferentiation and the first divisions leading to the formation of a microcallus, have not been elucidated. RESULTS: In this study, we investigated the function of miRNAs in the dedifferentiation of A. thaliana mesophyll cells in a process stimulated by the enzymatic removal of the cell wall. Leaf cells, protoplasts and CDPs (cells derived from protoplasts) cultured for 24, 72 and 120 h (first cell division). In protoplasts, a strong decrease in the amount of AGO1 in both the nucleus and the cytoplasm, as well as dicing bodies (DBs), which are considered to be sites of miRNA biogenesis, was shown. However during CDPs division, the amounts of AGO1 and DBs strongly increased. MicroRNA transcriptome studies demonstrated that lower amount of differentially expressed miRNAs are present in protoplasts than in CDPs cultured for 120 h. Then analysis of differentially expressed miRNAs, selected pri-miRNA and mRNA targets were performed. CONCLUSION: This result indicates that miRNA function is not a major regulation of gene expression in the initial but in later steps of dedifferentiation during CDPs divisions. miRNAs participate in organogenesis, oxidative stress, nutrient deficiencies and cell cycle regulation in protoplasts and CDPs. The important role played by miRNAs in the process of dedifferentiation of mesophyll cells was confirmed by the increased mortality and reduced cell division of CDPs derived from mutants with defective miRNA biogenesis and miR319b expression.

Spatial and Temporal Distribution of Arabinogalactan Proteins during Larix decidua Mill. Male Gametophyte and Ovule Interaction.

The role of ArabinoGalactan Proteins (AGPs) in the sexual reproduction of gymnosperms is not as well documented as that of angiosperms. In earlier studies, we demonstrated that AGPs play important roles during ovule differentiation in Larix decidua Mill. The presented results encouraged us to carry out further studies focused on the functions of these unique glycoproteins during pollen/pollen tube and ovule interactions in Larix. We identified and analyzed the localization of AGPs epitopes by JIM4, JIM8, JIM13 and LM2 antibodies (Abs) in male gametophytes and ovule tissue during pollination, the progamic phase, and after fertilization and in vitro growing pollen tubes. Our results indicated that (1) AGPs recognized by JIM4 Abs play an essential role in the interaction of male gametophytes and ovules because their appearance in ovule cells is induced by physical contact between reproductive partners; (2) after pollination, AGPs are secreted from the pollen cytoplasm into the pollen wall and contact the extracellular matrix of stigmatic tip cells followed by micropylar canal cells; (3) AGPs synthesized in nucellus cells before pollen grain germination are secreted during pollen tube growth into the extracellular matrix, where they can directly interact with male gametophytes; (4) in vitro cultured pollen tube AGPs labeled with LM2 Abs participate in the germination of pollen grain, while AGPs recognized by JIM8 Abs are essential for pollen tube tip growth.

Dynamic distribution of ARGONAUTE1 (AGO1) and ARGONAUTE4 (AGO4) in Hyacinthus orientalis L. pollen grains and pollen tubes growing in vitro.

The transcriptional and posttranscriptional AGO-mediated control of gene expression may play important roles during male monocot gametophyte development. In this report, we demonstrated dynamic changes in the spatiotemporal distribution of AGO1 and AGO4, which are key proteins of the RNA-induced silencing complex (RISC) in Hyacinthus orientalis male gametophyte development. During maturation of the bicellular pollen grains and in vitro pollen tube growth, the pattern of AGO1 localization was correlated with previously observed transcriptional activity of the cells. During the period of high transcriptional activity, AGO1 is associated with chromatin while the clustered distribution of AGO1 in the interchromatin areas is accompanied by condensation of chromatin and the gradual transcriptional silencing of both cells in mature, dehydrated pollen. During pollen tube growth and the restarting of RNA synthesis in the vegetative nucleus, AGO1 is dispersed in the chromatin. Additionally, the gradual increase in the cytoplasmic pool of AGO1 in the elongating pollen tube indicates the activation of the posttranscriptional gene silencing (PTGS) pathway. During pollen tube growth in the generative cell and in the sperm cells, AGO1 is present mainly in the areas between highly condensed chromatin clusters. Changes in the distribution of AGO4 that indicated the possibility of spatiotemporal organization in the RNA-directed DNA methylation (RdDM) process (cytoplasmic and nuclear steps) were also observed during hyacinth male gametophyte development. Based on our findings, we propose that in the germinating pollen tube, the cytoplasmic assembly of AGO4/siRNA takes place and that the mature complexes could be transported to the nucleus to carry out their function during the next steps of pollen tube growth.

Salt Stress Reveals a New Role for ARGONAUTE1 in miRNA Biogenesis at the Transcriptional and Posttranscriptional Levels.

Plants as sessile organisms have developed prompt response mechanisms to react to rapid environmental changes. In addition to the transcriptional regulation of gene expression, microRNAs (miRNAs) are key posttranscriptional regulators of the plant stress response. We show here that the expression levels of many miRNAs were regulated under salt stress conditions. This regulation occurred at the transcriptional and posttranscriptional levels. During salinity stress, the levels of miRNA161 and miRNA173 increased, while the expression of pri-miRNA161 and pri-miRNA173 was down-regulated. Under salt stress conditions, miRNA161 and miRNA173 were stabilized in the cytoplasm, and the expressions of MIR161 and MIR173 were negatively regulated in the nucleus. ARGONAUTE1 (AGO1) participated in both processes. We demonstrated that AGO1 cotranscriptionally controlled the expression of MIR161 and MIR173 in the nucleus. Our results suggests that AGO1 interacts with chromatin at MIR161 and MIR173 loci and causes the disassembly of the transcriptional complex, releasing short and unpolyadenylated transcripts.

Epigenetic marks in the Hyacinthus orientalis L. mature pollen grain and during in vitro pollen tube growth.

During the sexual reproduction of flowering plants, epigenetic control of gene expression and genome integrity by DNA methylation and histone modifications plays an important role in male gametogenesis. In this study, we compared the chromatin modification patterns of the generative, sperm cells and vegetative nuclei during Hyacinthus orientalis male gametophyte development. Changes in the spatial and temporal distribution of 5-methylcytosine, acetylated histone H4 and histone deacetylase indicated potential differences in the specific epigenetic state of all analysed cells, in both the mature cellular pollen grains and the in vitro growing pollen tubes. Interestingly, we observed unique localization of chromatin modifications in the area of the generative and the vegetative nuclei located near each other in the male germ unit, indicating the precise mechanisms of gene expression regulation in this region. We discuss the differences in the patterns of the epigenetic marks along with our previous reports of nuclear metabolism and changes in chromatin organization and activity in hyacinth male gametophyte cells. We also propose that this epigenetic status of the analysed nuclei is related to the different acquired fates and biological functions of these cells.

Regulation of poly(A) RNA retention in the nucleus as a survival strategy of plants during hypoxia.

Last finding indicates that post-transcriptional processes are significant in low-oxygen conditions, but their nature is poorly understood. Here, we localized poly(A) RNA and mRNA coding proteins involved and not involved with resistance to hypoxia in Lupinus luteus and Arabidopsis thaliana during submergence and after recovery of aerobic conditions. We showed a strong nuclear accumulation of poly(A) RNA and 6 of 7 studied mRNAs with a concurrent strong reduction in RNA polymerase II transcription during hypoxia. In this study, the nucleus did not accumulate mRNA of the ADH1 (alcohol dehydrogenase 1) gene, which is a core hypoxia gene. The RNA accumulation in the nucleus is among the mechanisms of post-transcriptional gene regulation that prevents translation. However re-aeration was accompanied by a strong increase in the amount of the mRNAs in the cytoplasm and a simultaneous decrease in nuclear mRNAs. This finding indicates that the nucleus is a storage site for those of mRNAs which are not involved in the response to hypoxia for use by the plants after the hypoxic stress. In this study, the highest intensity of RNA accumulation occurred in Cajal bodies (CBs); the intensity of accumulation was inversely correlated with transcription. Under hypoxia, ncb-1 mutants of Arabidopsis thaliana with a complete absence of CBs died sooner than wild type (WT), accompanied by a strong reduction in the level of poly(A) RNA in the nucleus. These results suggest that the CBs not only participate in the storage of the nuclear RNA, but they also could take part in its stabilization under low-oxygen conditions.

Late progamic phase and fertilization affect calreticulin expression in the Hyacinthus orientalis female gametophyte.

KEY MESSAGE: Calreticulin expression is upregulated during sexual reproduction of Hyacinthus orientalis, and the protein is localized both in the cytoplasm and a highly specialized cell wall within the female gametophyte. Several evidences indicate calreticulin (CRT) as an important calcium (Ca(2+))-binding protein that is involved in the generative reproduction of higher plants, including both pre-fertilization and post-fertilization events. Because CRT is able to bind and sequester exchangeable Ca(2+), it can serve as a mobile intracellular store of easily releasable Ca(2+) and control its local cytosolic concentrations in the embryo sac. This phenomenon seems to be essential during the late progamic phase, gamete fusion, and early embryogenesis. In this report, we demonstrate the differential expression of CRT within Hyacinthus female gametophyte cells before and during anthesis, during the late progamic phase when the pollen tube enters the embryo sac, and at the moment of fertilization and zygote/early endosperm activation. CRT mRNA and the protein localize mainly to the endoplasmic reticulum (ER) and Golgi compartments of the cells, which are involved in sexual reproduction events, such as those in sister synergids, the egg cell, the central cell, zygote and the developing endosperm. Additionally, immunogold research demonstrates selective CRT distribution in the filiform apparatus (FA), a highly specific component of the synergid cell wall. In the light of our previous data showing the total transcriptional activity of the Hyacinthus female gametophyte and the results presented here, we discuss the possible functions of CRT with respect to the critical role of Ca(2+) homeostasis during key events of sexual plant reproduction. Moreover, we propose that the elevated expression of CRT within the female gametophyte is a universal phenomenon in the cells involved in double fertilization in higher plants.

Strain-specific bioaccumulation and intracellular distribution of Cd²âº in bacteria isolated from the rhizosphere, ectomycorrhizae, and fruitbodies of ectomycorrhizal fungi.

Bioaccumulation of Cd(2+) in soil bacteria might represent an important route of metal transfer to associated mycorrhizal fungi and plants and may have potential as a tool to accelerate Cd(2+) extraction in the bioremediation of contaminated soils. The present study examined the bioaccumulation of Cd(2+) in 15 bacterial strains representing three phyla (Firmicutes, Proteobacteria, and Bacteroidetes) that were isolated from the rhizosphere, ectomycorrhizae, and fruitbody of ectomycorrhizal fungi. The strains Pseudomonas sp. IV-111-14, Variovorax sp. ML3-12, and Luteibacter sp. II-116-7 displayed the highest biomass productivity at the highest tested Cd(2+) concentration (2 mM). Microscopic analysis of the cellular Cd distribution revealed intracellular accumulation by strains Massilia sp. III-116-18, Pseudomonas sp. IV-111-14, and Bacillus sp. ML1-2. The quantities of Cd measured in the interior of the cells ranged from 0.87 to 1.31 weight % Cd. Strains originating from the rhizosphere exhibited higher Cd(2+) accumulation efficiencies than strains from ectomycorrhizal roots or fruitbodies. The high Cd tolerances of Pseudomonas sp. IV-111-16 and Bacillus sp. ML1-2 were attributed to the binding of Cd(2+) as cadmium phosphate. Furthermore, silicate binding of Cd(2+) by Bacillus sp. ML1-2 was observed. The tolerance of Massilia sp. III-116-18 to Cd stress was attributed to a simultaneous increase in K(+) uptake in the presence of Cd(2+) ions. We conclude that highly Cd-tolerant and Cd-accumulating bacterial strains from the genera Massilia sp., Pseudomonas sp., and Bacillus sp. might offer a suitable tool to improve the bioremediation efficiency of contaminated soils.

Spatial and temporal localization of homogalacturonans in Hyacinthus orientalis L. ovule cells before and after fertilization.

KEY MESSAGE: The composition of homogalacturonans (HGs) in the ovule and the female gametophyte cell walls was shown to be rearranged dynamically during sexual reproduction of H. orientalis. In angiosperms, homogalacturonans (HGs) play an important role in the interaction between the male gametophyte and the pistil transmitting tract, but little is known about the participation of these molecules at the final stage of the progamic phase and fertilization. The aim of our study was to perform immunocytochemical localization of highly (JIM7 MAb) and weakly (JIM5 MAb) methyl esterified and Ca(2+)-associated HG (2F4 MAb) in the ovule and female gametophyte cells of Hyacinthus orientalis before and after fertilization. It was found that pollination induced the rearrangement of HG in (1) the micropylar canal of the ovule, (2) the filiform apparatus of the synergids, and (3) the region of fusion between sperm cells and their target cells. Fertilization led to further changes in pectin composition of these three regions of the ovule. A new cell wall was synthesized around the zygote with a characteristic pattern of localization of all examined HG fractions, which we called "sporoderm-like". The developing endosperm prepared for cellularization by synthesizing highly methyl-esterified HG, which was stored in the cytoplasm. Pollination- and fertilization-induced changes in the composition of the HG in the micropyle of the ovule and the apoplast of female gametophyte cells are discussed in the context of: (1) micropylar pollen tube guidance, (2) preparation of the egg cell and the central cells for fusion with sperm cells, and (3) the polyspermy block.

Interactive and Single Effects of Ectomycorrhiza Formation and Bacillus cereus on Metallothionein MT1 Expression and Phytoextraction of Cd and Zn by Willows.

Single and joint ectomycorrhizal (+ Hebeloma mesophaeum) and bacterial (+ Bacillus cereus) inoculations of willows (Salix viminalis) were investigated for their potential and mode of action in the promotion of cadmium (Cd) and zinc (Zn) phytoextraction. Dual fungal and bacterial inoculations promoted the biomass production of willows in contaminated soil. Single inoculations either had no effect on the plant growth or inhibited it. All inoculated willows showed increased concentrations of nutritional elements (N, P, K and Zn) and decreased concentrations of Cd in the shoots. The lowest biomass production and concentration of Cd in the willows (+ B. cereus) were combined with the strongest expression of metallothioneins. It seems that biotic stress from bacterial invasion increased the synthesis of these stress proteins, which responded in decreased Cd concentrations. Contents of Cd and Zn in the stems of willows were combination-specific, but were always increased in dual inoculated plants. In conclusion, single inoculations with former mycorrhiza-associated B. cereus strains decreased the phytoextraction efficiency of willows by causing biotic stress. However, their joint inoculation with an ectomycorrhizal fungus is a very promising method for promoting the phytoextraction of Cd and Zn through combined physiological effects on the plant.

Ribosomal RNA of Hyacinthus orientalis L. female gametophyte cells before and after fertilization.

The nucleolar activity of Hyacinthus orientalis L. embryo sac cells was investigated. The distributions of nascent pre-rRNA (ITS1), 26S rRNA and of the 5S rRNA and U3 snoRNA were determined using fluorescence in situ hybridization (FISH). Our results indicated the different rRNA metabolism of the H. orientalis female gametophyte cells before and after fertilization. In the target cells for the male gamete, i.e., the egg cell and the central cell whose activity is silenced in the mature embryo sac (Piecinski et al. in Sex Plant Reprod 21:247-257, 2008; Niedojadlo et al. in Planta doi: 10.1007/s00425-012-1599-9 , 2011), rRNA metabolism is directed at the accumulation of rRNPs in the cytoplasm and immature transcripts in the nucleolus. In both cells, fertilization initiates the maturation of the maternal pre-rRNA and the expression of zygotic rDNA. The resumption of rRNA transcription observed in the hyacinth zygote indicates that in plants, there is a different mechanism for the regulation of RNA Pol I activity than in animals. In synergids and antipodal cells, which have somatic functions, the nucleolar activity is correlated with the metabolic activity of these cells and changes in successive stages of embryo sac development.

Transcriptional activity of Hyacinthus orientalis L. female gametophyte cells before and after fertilization.

We characterized three phases of Hyacinthus orientalis L. embryo sac development, in which the transcriptional activity of the cells differed using immunolocalization of incorporated 5'-bromouracil, the total RNA polymerase II pool and the hypo- (initiation) and hyperphosphorylated (elongation) forms of RNA Pol II. The first stage, which lasts from the multinuclear stage to cellularization, is a period of high transcriptional activity, probably related to the maturation of female gametophyte cells. The second stage, encompassing the period of embryo sac maturity and the progamic phase, involves the transcriptional silencing of cells that will soon undergo fusion with male gametes. During this period in the hyacinth egg cell, there are almost no newly formed transcripts, and only a small pool of RNA Pol II is present in the nucleus. The transcriptional activity of the central cell is only slightly higher than that observed in the egg cell. The post-fertilization stage is related to the transcriptional activation of the zygote and the primary endosperm cell. The rapid increase in the pool of newly formed transcripts in these cells is accompanied by an increase in the pool of RNA Pol II, and the pattern of enzyme distribution in the zygote nucleus is similar to that observed in the somatic cells of the ovule. Our data, together with the earlier results of Piecinski et al. (2008), indicate post-fertilization synthesis and the maturation of numerous mRNA transcripts, suggesting that fertilization in H. orientalis induces the activation of the zygote and endosperm genomes.

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.

Apaf-1 expression in human cutaneous melanoma progression and in pigmented nevi.

Malignant melanoma is notoriously refractive to therapy and resistant to apoptosis. This may reflect the downregulation of Apaf-1, an important mediator of mitochondrial-dependent apoptosis, observed in vitro in melanoma cell lines and by immunohistochemistry for Apaf-1 protein in histological samples of primary and metastatic melanomas. Although it has been suggested that loss of Apaf-1 expression may be an indicator of malignant transformation in melanoma, previous studies on Apaf-1 expression in benign pigmented nevi were performed without reference to their histologic type. Here we have evaluated the expression of Apaf-1 mRNA by fluorescence in situ hybridization and of Apaf-1 protein by immunohistochemistry in a large panel of human melanomas and in eight types of pigmented nevi, considered potential precursors for cutaneous melanoma. We observe a strong negative correlation between melanoma progression assessed according to Clark classification and the expression of Apaf-1. A significant decrease in Apaf-1 expression was observed between Clark II and Clark III lesions, the stages usually associated with a transition from horizontal to vertical growth phase of melanoma. There was also statistically significant difference in Apaf-1 mRNA expression between melanomas of Breslow thickness <1 mm and >4 mm. No Apaf-1 expression could be detected in lymph node melanoma metastases. These results suggest that Apaf-1 expression may become a prognostic marker for progress of human cutaneous melanoma and further support the notion that loss of Apaf-1 may be an important contributory factor in the development of the disease.