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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.

Manuscript provides insights into the biology of long-lived plants, different from Arabidopsis, tomato or grass species that are widely studied. In the European larch the diplotene stage lasts approximately 5 months and it is possible to divide it into several substages and to observe each of them in details. The diplotene stage is a period of intensive microsporocyte growth associated with the synthesis and accumulation of different RNA and proteins. Larch microsporocytes display changes in chromatin morphology during this stage, alternating between 4 short stages of chromatin condensation (contraction) and 5 longer diffusion (relaxation) stages. The occurrence of a diplotene diffusion stage has been observed in many plant species. Interestingly, they have also been observed during spermiogenesis and oogenesis in animals. The aim of this study was to examine whether chromatin relaxation during the diplotene is accompanied by the synthesis and maturation of mRNA. The results reveal a correlation between the diffusion and chromatin decondensation, transcriptional activity. We also found decreasing amount of poly(A) mRNA synthesis in the consecutive diffusion stages. During the early diffusion stages, mRNA is intensively synthesized. In the nuclei large amounts of RNA polymerase II, and high levels of snRNPs were observed. In the late diffusion stages, the synthesized mRNA is not directly subjected to translation but it is stored in the nucleus, and later transported to the cytoplasm and translated. In the last diffusion stage, the level of poly(A) RNA is low, but that of splicing factors is still high. It appears that the mRNA synthesized in early stages is used during the diplotene stage and is not transmitted to dyad and tetrads. In contrast, splicing factors accumulate and are most likely transmitted to the dyad and tetrads, where they are used after the resumption of intense transcription. Similar meiotic process were observed during oogenesis in animals. This indicates the existence of an evolutionarily conserved mechanism of chromatin-based regulation of gene expression during meiotic prophase I.

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.

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