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.
Niedotlenienie (hipoksja) u roślin jest zazwyczaj wynikiem intensywnych opadów deszczu i następujących po nich powodzi. Obecne modele klimatyczne wskazują na znaczny wzrost tych zjawisk w najbliższym latach. W zależności od gatunku i miejsca występowania, rośliny podczas ewolucji wykształciły dwie strategie adaptacyjne do stresu hipoksji: ucieczki i uśpienia. Pierwsza polega na jak najszybszym wynurzeniu przynajmniej część pędu nadziemnego nad poziom wody, natomiast druga na silnym obniżeniu tempa metabolizmu w celu przeczekania niekorzystnych warunków środowiska. Procesy te są regulowane głównie przez etylen oraz czynniki transkrypcyjne ERF (ang. ethylen response factor), które aktywują geny odpowiedzi na niedotlenienie. Większość ERFów ulega konstytutywnej transkrypcji niezależnie od stężenia tlenu. Jednak potranslacyjne modyfikacje N-końca ERFów prowadzą do ich degradacji u roślin rosnących w warunkach fizjologicznych. Podczas niedotlenienia następuje wzrost poziomu ekspresji genów związanych z metabolizmem węgla, azotu, glikolizą czy oddychaniem beztlenowym. Jednak jak wykazały badania z zastosowaniem profilowania rybosomów, w celu oszczędzania energii, rośliny poddane stresowi hipoksji silnie hamują proces inicjacji translacji. Na regulację ekspresji genów w warunkach stresowych ma wpływ także kumulacja poli(A) RNA w jądrze komórkowym i w cytoplazmatycznych granulach stresowych.
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.
Introduction: The main objective was to implement the determinations of the ex vivo resistance to cyclophosphamide and to identify the genetic profile for pediatric patients with acute leukemias. Methods: In order to determine the ex vivo drug resistance profile, MTT cytotoxicity assay was performed on mononuclear cells. Gene expression profiles were prepared on the basis of cRNA hybridization to oligonucleotide arrays of the human genome (Affymetrix). We performed also array-based comparative genomic hybridization using a SurePrint G3 Human CGH Microarray. Data was analyzed by bioinformatics tools. Verification of the relative expression level of 20 genes was carried out by qRT- PCR. Results: We observed a multitude of differentially expressed genes, e.g. ANXA1 (FC=3,04), BCL2A1 (FC=2,69), SERPINA1 (FC=2,12), DHRS7 (FC=2,13), PCDH9 (FC=- 4,58), TTC28 (FC=-2,25) and DUSP1 (FC=-2,91). The expression of genes that code for inflammation mediated by chemokine and cytokine signaling, Wnt, angiogenesis and integrin signaling and T cell activation pathways genes affect the sensitivity of leukemic blasts to cyclophosphamide. Transcriptome level changes are associated with chromosomal aberrations, especially located on chromosomes 8, 10, 14, 15, 16 and 22. Conclusion: Our work delineated genes with differentiated expression and recurrent copy number changes, and revealed novel amplified loci and frequent deletions in resistant to cyclophosphamide cells, which may guide future work aimed at identifying the relevant target genes. In particular, deletion seems to be a frequent mechanism of IFIT3 gene inactivation. ANXA1, SERPINA1, TCF7 and BCL2A1 may also be included among the candidate genes of resistance (Ontological analysis).
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