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.

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.

Dedifferentiation of Arabidopsis thaliana cells is accompanied by a strong decrease in RNA polymerase II transcription activity and poly(A+) RNA and 25S rRNA eradication from the cytoplasm.

The mechanisms of plant cell dedifferentiation and the acquisition of totipotency are poorly understood. One of the methods to induce the dedifferentiation process in plant cells is simple and requires the removal of the cell wall. After cell wall removal in protoplasts, large-scale chromatin decondensation is observed (Tessadori et al. in J Cell Sci 120:1200-1208, 2007). Here, we show that in Arabidopsis thaliana protoplasts, despite chromatin decondensation, RNA polymerase II transcriptional activity is reduced. The subsequent investigated stages displayed a clear decrease in the quantity of 25S ribosomal RNA (rRNA) first and then poly(A+) RNA, particularly in the cytoplasm. Therefore, the reduced transcription activity and the removal of these RNA transcripts from the cytoplasm is a crucial process in obtaining totipotency in plant cells. After the cytoplasm cleaning of transcripts derived from mesophyll cells, we observed the resynthesis of these RNAs. An increase in the amount of examined molecules to a level similar to that in differentiated mesophyll cells precedes the divisions of already undifferentiated cells. In this work, we show changes in RNA polymerase II transcription dynamics and the quantity of poly(A+) RNA and 25S rRNA during dedifferentiation and re-entry into the cell cycle.

The perichromatin region of the plant cell nucleus is the area with the strongest co-localisation of snRNA and SR proteins.

The spatial organisation of the splicing system in plant cells containing either reticular (Allium cepa) or chromocentric (Lupinus luteus) nuclei was studied by immunolabelling of SR proteins, snRNA, and the PANA antigen, known markers for interchromatin granule clusters in mammalian cells. Electron microscope results allowed us to determine the distribution of these molecules within the structural domains of the nucleus. Similar to animal cells, in both plant species SR proteins were localised in interchromatin granules, but contrary to animal cells contained very small amounts of snRNA. The area with the strongest snRNA and SR protein co-localisation was the perichromatin region, which may be the location of pre-mRNA splicing in the plant cell nuclei. The only observable differences in the organisation of reticular and chromocentric nuclei were the size of the speckles and the number of snRNA pools in the condensed chromatin. We conclude that, despite remarkable changes in the nuclear architecture, the organisation of the splicing system is remarkably similar in both types of plant cell nuclei.