Tracking gene and protein expression during microspore embryogenesis by Confocal Laser Scanning Microscopy

9 pages, 2 figures.Confocal Laser Scanning Microscopy (CLSM) technology and bioimaging are powerful tools for three-dimensional (3D) and colocalization molecular analysis of the microspore embryogenesis. Strategies with fluorescent-labelled probes for in situ hybridization and immunofluorescence have provided unique images of the spatial and temporal pattern of the expression of genes and proteins, and of the sub-cellular rearrangements that accompany the microspore embryogenesis. Various signalling and stress proteins were differentially expressed in reprogrammed microspores and young embryos, and specific endosperm and embryo genes were expressed at different stages, supporting the existence of an endosperm-like domain, in cereals. Specific features such as changes in cell wall components and pectin esterification, presence of callose in special walls, and different behaviour of Cajal nuclear bodies were found in embryogenic microspores and young embryos, constituting early embryogenic markers. The 3D analysis of the nuclear dynamics at early stages of microspore embryogenesis has proved that the nuclear fusion was the mechanism of the spontaneous diploidization.This work was supported by projects of the Spanish Ministry of Education and Science, MEC BFU2005-01094, AGL2005-05104, BFU2008-00203 and AGL200804255.Peer reviewe

Cork oak microspore embryogenesis

In vivo, the microspore inside the anther divides and follows the gametophytic program to form the mature pollen grain. In vitro, upon the application of a stress treatment the microspore can be deviated towards a proliferation process leading to embryogenesis, the so-called microspore embryogenesis that can be induced in anther and isolated microspore cultures (Maluszynski et al. 2003).This work has been supported by projects (BFU2011-23752, AGL2014-52028-R, AGL2017-82447-R) funded by the Spanish Ministry of Economy and Competitiveness, MINECO, and the European Regional Development Fund (ERDF/FEDER) of the European Commission.Peer reviewe

H3K9 methylation patterns during somatic embryogenic competence expression in tamarillo (Solanum betaceum Cav.)

9 p.-6 fig.The capacity to regenerate is intrinsic to plants and is the basis of natural asexual propagation and artificial cloning. Despite there are different ways of plant regeneration, they all require a change in cell fate and pluripotency reacquisition, in particular somatic embryogenesis. The mechanisms underlying somatic cell reprogramming for embryogenic competence acquisition, expression and maintenance remain not fully understood. These complex processes have been often associated with epigenetic markers, mainly DNA methylation, while little is known about the possible role of histone modifications. In the present study, the dynamics of global levels and distribution patterns of histone H3 methylation at lysine 9 (H3K9), a major repressive histone modification, were analyzed in somatic embryogenesis-induced cell lines with different embryogenic capacities and during somatic embryo initiation, in the woody species Solanum betaceum. Quantification of global H3K9 methylation showed similar levels in the three types of proliferating calli (embryogenic, long-term and non-embryogenic), kept in high sucrose and auxin-containing medium. Microscopic analyzes revealed heterogeneous cell organization and different cell types, particularly evident in embryogenic callus. The H3K9 dimethylation (H3K9me2) immunofluorescence signal was lower in nuclei of cells showing embryogenic-like and proliferating features, while labeling was higher in vacuolated, non-embryogenic cells with higher proliferation rates. By auxin removal, somatic embryo development was promoted in the embryogenic cell line. During the initiation of this process, increasing levels of global H3K9 methylation were found, together with increasing H3K9me2 immunofluorescence signals, especially in cells of the developing embryo. These results suggest that H3K9 methylation is involved in somatic embryo development, a developmental pathway in which this epigenetic mark could play a role in the gene transcription variation that is associated with embryogenic competence expression in S. betaceum. Altogether, these data provide new insights into the role of this epigenetic mark in somatic embryogenesis in trees, where scarce information is available.This work was supported by Foundation for Science and Technology (Portugal), through PhD fellowship SFRH/BD/136925/2018 and COVID/BD/152914/2023 awarded to Daniela Cordeiro; Center for Functional Ecology—Science for People and the Planet (UIDB/04004/2020), financed by FCT/MCTES through national funds (PIDDAC); Cost Action 19125, through a Short-Term Scientific Mission awarded to Daniela Cordeiro; project PID2020-113018RB-I00, funded by Spanish MCIN/AEI/10.13039/501100011033 and projects TED2021-129633B-I00 and CPP2021-008750, funded by MCIN/AEI/10.13039/501100011033 and NextGenerationEU/ PRTR.Peer reviewe

Changes in extranucleolar transcription during actinomycin D-induced apoptosis

Actinomycin D (AMD) inhibits DNAdependent RNA polymerases and its selectivity depends on the concentration used; at very high concentrations it may also induce apoptosis. This study investigates the effects of different concentrations (0.01 to 1 µg/ml) of AMD on RNA transcription and maturation and on the organization of nuclear ribonucleoproteins (RNPs), and their relationship with apoptosis induction. Human HeLa cells were used as a model system. At the lowest concentration used, AMD induced the segregation of the nucleolar components and impaired r-RNA synthesis, as revealed by the decreased immunopositivity for bromouridine incorporation and for DNA/RNA hybrid molecules. The synthesis of pre-mRNAs, on the contrary, was active, while the immunolabeling of snRNP proteins and of the SC-35 splicing factor strongly decreased on perichromatin fibrils (where they are involved in co-transcriptional splicing). This suggests that the post-transcriptional maturation of extranucleolar RNAs was also affected. Moreover, still in the absence of typical late morphological or biochemical signs of apoptosis (i.e. chromatin condensation), these cells displayed the early apoptotic features, i.e. the externalization of phosphatidylserine residues on the plasma membrane and propidium iodide exclusion in vivo. At the highest concentrations of AMD used, apoptosis massively occurred, with the typical morphological events (progressive chromatin condensation, clustering of snRNPs and SC-35 splicing factor, cell blebbing). However, transcription of hnRNAs was maintained in the residual areas of diffuse chromatin up to advanced apoptotic stages. The inhibition of rRNA synthesis and the defective pre-mRNA maturation seem to be part of the apoptotic process induced by AMD

Epigenetic changes accompany developmental programmed cell death in tapetum cells.

10 páginas, 8 figuras --PAGS nros. 16-29(2014)The tapetum, the nursing tissue inside anthers, undergoes cellular degradation by programmed cell death (PCD) during late stages of microspore–early pollen development. Despite the key function of tapetum, little is known about the molecular mechanisms regulating this cell death process in which profound nuclear and chromatin changes occur. Epigenetic features (DNA methylation and histone modifications) have been revealed as hallmarks that establish the functional status of chromatin domains, but no evidence on the epigenetic regulation of PCD has been reported. DNA methylation is accomplished by DNA methyltransferases, among which DNA methyl transferase 1 (MET1) constitutes one of the CG maintenance methyltransferase in plants, also showing de novo methyltransferase activity. In this work, the changes in epigenetic marks during the PCD of tapetal cells have been investigated by a multidisciplinary approach to reveal the dynamics of DNA methylation and the pattern of expression of MET1 in relation to the main cellular changes of this PCD process which have also been characterized in two species, Brassica napus and Nicotiana tabacum. The results showed that tapetum PCD progresses with the increase in global DNA methylation and MET1 expression, epigenetic changes that accompanied the reorganization of the nuclear architecture and a high chromatin condensation, activity of caspase 3-like proteases and Cyt c release. The reported data indicate a relationship between the PCD process and the DNA methylation dynamics and MET1 expression in tapetal cells, suggesting a possible new role for the epigenetic marks in the nuclear events occurring during this cell death process and providing new insights into the epigenetic control of plant PCDThis work was supported by the Spanish Ministry of Economy and Competitivity, MINECO [BFU2011-23752]; the Spanish National Research Council, CSIC [201020E038]; the Spanish Ministry of Science and Innovation, MICINN [grant to N.C. for Stays of Foreign Postdoctoral Researchers and grant to J.C.E. for Sabaticcal Stays in Spanish Research Centre]; the Spanish Ministry of Science and Innovation, MICINN [a post-doctoral Juan-de-la-Cierva grant (JCI-2007-123-1177) to M.R.S.

Genes normally expressed in the endosperm are expressed at early stages of microspore embryogenesis in maize

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