Dariusz Jan Smoliński

Additional nucleoli and NOR activity during meiotic prophase I in larch (Larix decidua Mill.).

Summary.Transcriptional activity was investigated in successive stages of prophase I (male meiosis) of larch meiocytes. Br-UTP incorporated into RNA was detected by light and electron microscopy. Two peaks of RNA synthesis were identified in the nucleolus. The first occurred during the zygotene–pachytene stage and the second (not previously described in plant meiocytes) in the diplotene. These processes correlated with a considerable increase in nucleolus volume during these periods. At the end of the zygotene, several perinucleolar structures lying close to each other and containing rRNA, argyrophilic proteins, U3 small nucleolar RNA, and fibrillarin were observed. The occurrence of newly formed RNA was also observed in these structures. This suggests that the observed perinucleolar structures correspond to the additional nucleoli known from animals.

NTR1 is required for transcription elongation checkpoints at alternative exons in Arabidopsis

The interconnection between transcription and splicing is a subject of intense study. We report that Arabidopsis homologue of spliceosome disassembly factor NTR1 is required for correct expression and splicing of DOG1, a regulator of seed dormancy. Global splicing analysis in atntr1 mutants revealed a bias for downstream 5′ and 3′ splice site selection and an enhanced rate of exon skipping. A local reduction in PolII occupancy at misspliced exons and introns in atntr1 mutants suggests that directionality in splice site selection is a manifestation of fast PolII elongation kinetics. In agreement with this model, we found AtNTR1 to bind target genes and co‐localise with PolII. A minigene analysis further confirmed that strong alternative splice sites constitute an AtNTR1‐dependent transcriptional roadblock. Plants deficient in PolII endonucleolytic cleavage showed opposite effects for splice site choice and PolII occupancy compared to atntr1 mutants, and inhibition of PolII elongation or endonucleolytic cleavage in atntr1 mutant resulted in partial reversal of splicing defects. We propose that AtNTR1 is part of a transcription elongation checkpoint at alternative exons in Arabidopsis.

Lignin and lignans in plant defence: Insight from expression profiling of cinnamyl alcohol dehydrogenase genes during development and following fungal infection in Populus

Cinnamyl alcohol dehydrogenase (CAD) catalyses the final step in the biosynthesis of monolignol, the main component of lignin. Lignins, deposited in the secondary cell wall, play a role in plant defence against pathogens. We re-analysed the phylogeny of CAD/CAD-like genes using sequences from recently sequenced genomes, and analysed the temporal and spatial expression profiles of CAD/CAD-like genes in Populus trichocarpa healthy and infected plants. Three fungal pathogens (Rhizoctonia solani, Fusarium oxysporum, and Cytospora sp.), varying in lifestyle and pathogenicity, were used for plant infection. Phylogenetic analyses showed that CAD/CAD-like genes were distributed in classes represented by all members from angiosperm lineages including basal angiosperms and Selaginella. The analysed genes showed different expression profiles during development and demonstrated that three genes were involved in primary xylem maturation while five may function in secondary xylem formation. Expression analysis following inoculation with fungal pathogens, showed that five genes were induced in either stem or leaves. These results add further evidence that CAD/CAD-like genes have evolved specialised functions in plant development and defence against various pest and pathogens. Two genes (PoptrCAD11 and PoptrCAD15), which were induced under various stresses, could be treated as universal markers of plant defence using lignification or lignan biosynthesis.

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.

Periodic expression of Sm proteins parallels formation of nuclear Cajal bodies and cytoplasmic snRNP-rich bodies

Small nuclear ribonucleoproteins (snRNPs) play a fundamental role in pre-mRNA processing in the nucleus. The biogenesis of snRNPs involves a sequence of events that occurs in both the nucleus and cytoplasm. Despite the wealth of biochemical information about the cytoplasmic assembly of snRNPs, little is known about the spatial organization of snRNPs in the cytoplasm. In the cytoplasm of larch microsporocytes, a cyclic appearance of bodies containing small nuclear RNA (snRNA) and Sm proteins was observed during anther meiosis. We observed a correlation between the occurrence of cytoplasmic snRNP bodies, the levels of Sm proteins, and the dynamic formation of Cajal bodies. Larch microsporocytes were used for these studies. This model is characterized by natural fluctuations in the level of RNA metabolism, in which periods of high transcriptional activity are separated from periods of low transcriptional activity. In designing experiments, the authors considered the differences between the nuclear and cytoplasmic phases of snRNP maturation and generated a hypothesis about the direct participation of Sm proteins in a molecular switch triggering the formation of Cajal bodies.

Poly(A) RNAs Including Coding Proteins RNAs Occur in Plant Cajal Bodies

The localisation of poly(A) RNA in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus, Arabidopsis thaliana) nuclei was studied through in situ hybridisation. In both types of nuclei, the amount of poly(A) RNA was much greater in the nucleus than in the cytoplasm. In the nuclei, poly(A) RNA was present in structures resembling nuclear bodies. The molecular composition as well as the characteristic ultrastructure of the bodies containing poly(A) RNA demonstrated that they were Cajal bodies. We showed that some poly(A) RNAs in Cajal bodies code for proteins. However, examination of the localisation of active RNA polymerase II and in situ run-on transcription assays both demonstrated that CBs are not sites of transcription and that BrU-containing RNA accumulates in these structures long after synthesis. In addition, it was demonstrated that accumulation of poly(A) RNA occurs in the nuclei and CBs of hypoxia-treated cells. Our findings indicated that CBs may be involved in the later stages of poly(A) RNA metabolism, playing a role storage or retention.