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.

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.

The role of ethylene and transcriptome alterations in response to hypoxia stress in plants / Rola etylenu i zmiany na poziomie transkryptomu w odpowiedzi na stres niedotlenienia u roslin.

Hypoxia in plants is a usually the result of heavy rainfall and the subsequent flooding. All current climate models indicate a notable increase in extreme weather over the coming years. Depending on the species and geographical location, plants have developed two distinct strategies for hypoxia stress adaptation: escape and quiescence. The escape strategy involves rapid growth of part of the shoot above the water level whe­reas the second strategy requires a significant reduction in the metabolic rate of the plant in order to survive until the negative environmental conditions pass. These processes are primarily regulated by ethylen in addition to the transcription factor, ERF (ethylen response factor), which enables the transcription of hypoxia response genes. These processes are primarily regulated by ethylen in addition to the transcription factors, ERFs (ethylen response factors), which enables the transcription of hypoxia response genes. Most ERF genes are constitutively trans­cribed independently of oxygen concentration. However, post-translational modification of the N-terminus of ERFs leads to their degradation in plants growing under physiological conditions. During hypoxia there is an increase in the expression level of genes associated with carbon, nitrogen, glycolysis or anaerobic respiration. However, as shown by studies using ribosome profiling, in order to save energy, plants under hypoxic stress strongly inhibit the process of initiating translation. The regulation of gene expression under stress conditions is also influen­ced by the accumulation of poly(A) RNA in the cell nucleus and cytoplasmic stress granules.