Maria-Carmen Risueño

Nanoparticle penetration and transport in living pumpkin plants: in situ subcellular identification

BackgroundIn recent years, the application of nanotechnology in several fields of bioscience and biomedicine has been studied. The use of nanoparticles for the targeted delivery of substances has been given special attention and is of particular interest in the treatment of plant diseases. In this work both the penetration and the movement of iron-carbon nanoparticles in plant cells have been analyzed in living plants of Cucurbita pepo.ResultsThe nanoparticles were applied in planta using two different application methods, injection and spraying, and magnets were used to retain the particles in movement in specific areas of the plant. The main experimental approach, using correlative light and electron microscopy provided evidence of intracellular localization of nanoparticles and their displacement from the application point. Long range movement of the particles through the plant body was also detected, particles having been found near the magnets used to immobilize and concentrate them. Furthermore, cell response to the nanoparticle presence was detected.ConclusionNanoparticles were capable of penetrating living plant tissues and migrating to different regions of the plant, although movements over short distances seemed to be favoured. These findings show that the use of carbon coated magnetic particles for directed delivery of substances into plant cells is a feasible application.

Isolation and Functional Characterisation of Two New bZIP Maize Regulators of the ABA Responsive Gene rab28

The plant hormone abscisic acid regulates gene expression in response to growth stimuli and abiotic stress. Previous studies have implicated members of the bZIP family of transcription factors as mediators of abscisic acid dependent gene expression through the ABRE cis-element. Here, we identify two new maize bZIP transcription factors, EmBP-2 and ZmBZ-1 related to EmBP-1 and OsBZ-8 families. They are differentially expressed during embryo development; EmBP-2 is constitutive, whereas ZmBZ-1 is abscisic acid-inducible and accumulates during late embryogenesis. Both factors are nuclear proteins that bind to ABREs and activate transcription of the abscisic acid-inducible gene rab28 from maize. EmBP-2 and ZmBZ-1 are phosphorylated by protein kinase CK2 and phosphorylation alters their DNA binding properties. Our data suggest that EmBP-2 and ZmBZ-1 are involved in the expression of abscisic acid inducible genes such as rab28 and their activity is modulated by ABA and by phosphorylation.

Microspore-derived embryos fromQuercus suberanthers mimic zygotic embryos and maintain haploidy in long-term anther culture

Microspore-derived embryos produced from cork oak anther cultures after long-term incubations (up to 10-12 months) were analysed in order to determine the genetic variability and ploidy level stability, as well as morphology, developmental pattern and cellular organisation. Most of the embryos from long-term anther cultures were haploid (90.7%), corresponding to their microspore origin. The presence of a low percentage of diploid embryos (7.4%) was observed. Microsatellite analysis of haploid embryos, indicated different microspores origins of the same anther. In the diploid embryos, homozygosity for different alleles was detected from anther wall tissues, excluding the possibility of clonal origin. The maintenance of a high proportion of haploid embryos, in long-term anther cultures, is similar in percentage to that reported in embryos originating after 20 days of plating (Bueno et al. 1997). This suggests that no significant alterations in the ploidy level occurred during long incubations (up to 12 months). These results suggest that ploidy changes are rare in this in vitro system, and do not significantly increase during long-term cultures. Microscopical studies of the microspore embryos in various stages revealed a healthy and well developed anatomy with no aberrant or chimeric structures. The general morphology of embryos appearing at different times after plating, looked similar to that of earlier embryos, as well as the zygotic embryos, indicating that they represent high quality material for cork oak breeding.

Arabinogalactan protein profiles and distribution patterns during microspore embryogenesis and pollen development in Brassica napus

Arabinogalactan proteins (AGPs), present in cell walls, plasma membranes and extracellular secretions, are massively glycosylated hydroxyproline-rich proteins that play a key role in several plant developmental processes. After stress treatment, microspores cultured in vitro can reprogramme and change their gametophytic developmental pathways towards embryogenesis, thereby producing embryos which can further give rise to haploid and double haploid plants, important biotechnological tools in plant breeding. Microspore embryogenesis constitutes a convenient system for studying the mechanisms underlying cell reprogramming and embryo formation. In this work, the dynamics of both AGP presence and distribution were studied during pollen development and microspore embryogenesis in Brassica napus, by employing a multidisciplinary approach using monoclonal antibodies for AGPs (LM2, LM6, JIM13, JIM14, MAC207) and analysing the expression pattern of the BnAGP Sta 39–4 gene. Results showed the developmental regulation and defined localization of the studied AGP epitopes during the two microspore developmental pathways, revealing different distribution patterns for AGPs with different antigenic reactivity. AGPs recognized by JIM13, JIM14 and MAC207 antibodies were related to pollen maturation, whereas AGPs labelled by LM2 and LM6 were associated with embryo development. Interestingly, the AGPs labelled by JIM13 and JIM14 were induced with the change of microspore fate. Increases in the expression of the Sta 39–4 gene, JIM13 and JIM14 epitopes found specifically in 2–4 cell stage embryo cell walls, suggested that AGPs are early molecular markers of microspore embryogenesis. Later, LM2 and LM6 antigens increased progressively with embryo development and localized on cell walls and cytoplasmic spots, suggesting an active production and secretion of AGPs during in vitro embryo formation. These results give new insights into the involvement of AGPs as potential regulating/signalling molecules in microspore reprogramming and embryogenesis.

Non-isotopic mapping of ribosomal RNA synthesis and processing in the nucleolus.

Abstract. The precise location of ribosomal RNA (rRNA) synthesis within the nucleolus is the subject of recent controversy; some investigators have detected nascent RNA in the dense fibrillar components (DFCs) while others have localized transcription to the fibrillar centers (FCs). We endeavored to resolve this controversy by applying a new technique for non-isotopic labeling of RNA and examined the synthesis and movement of non-isotopically labeled rRNA within the nucleolus. We found that rRNA is synthesized only in a restricted area of DFCs, also involving the boundary region with FCs. We traced a movement of RNA from transcription sites through DFCs to granular components. Our results indicate functional compartmentalization of DFCs with respect to the synthesis and processing of precursor rRNA. In situ mapping of the 5′ leader sequence of the 5′ external transcribed spacer together with transcription labeling indicated that transcription and the first steps in processing of precursor rRNA are spatially separated. Surprisingly, the results also pointed to a partially extended conformation of newly synthesized precursor rRNA transcripts.

The MAP kinase kinase NtMEK2 is involved in tobacco pollen germination

The tobacco ntf4 mitogen‐activated protein (MAP) kinase gene (and its encoded protein p45Ntf4) is expressed at later stages of pollen maturation. We have found that the highly related MAP kinase SIPK is also expressed in pollen and, like p45Ntf4, is activated upon pollen hydration. The MAP kinase kinase NtMEK2 activates SIPK, and here we show that it can also activate p45Ntf4. In an attempt to inhibit the function of both MAP kinases simultaneously we constructed a loss‐of‐function mutant version of NtMEK2, which, in transient transformation assays, led to an inhibition of germination in the transformed pollen grains. These data indicate that NtMEK2, and by inference its substrates p45Ntf4 and/or SIPK, are involved in pollen germination.

Distribution of B-36 nucleolar protein in relation to transcriptional activity in plant cells

The Mr 34000 nucleolar protein B-36 was immunolocalized using a mouse monoclonal antibody in different plant cells by light and electron microscopy. Specific immunofluorescence was observed in nucleoli of cryofixed and cryosectioned material. Immunoelectron microscopy was performed on ultrathin cryosections and Lowicryl sections. Immunogold labelling revealed that B-36 is mainly localized in the nucleolar dense fibrillar component (DFC). The fibrillar centres and the granular component were practically devoid of gold particles. Nucleoli with different rates of transcriptional activity and, as a consequence, with different morphologies showed the same immunolocalization. This was true for both somatic and germinal tissues of the different plant species studied: Allium cepa root meristematic cells and Scilla peruviana and Capsicum annuum pollen grains. The presence of the B-36 antigen in the DFC of nucleoli with very different rates of transcriptional activity indicates that it is a constitutive protein. Moreover, the localization suggests that it could play a role in the transcription and/or early processing of rRNA. The presence of B-36 in the different plant tissues and species studied, confirms once more its high level of conservation throughout evolution. Another Mr 34000 protein called fibrillarin was also found in the DFC. Taking these and other published data together, we think that the antibodies used in the different studies are probably binding different epitopes of identical or very similar Mr 34000 nucleolar proteins.

In situ molecular identification of the Ntf4 MAPK expression sites in maturing and germinating pollen

Background information. MAPKs (mitogen‐activated protein kinases) are involved in the transduction of different signals in eukaryotes. They regulate different processes, such as differentiation, proliferation and stress response. MAPKs act through the phosphorylation cascade, being the last element that phosphorylates the final effector of the cell response. They are activated when their threonine and tyrosine residues are phosphorylated. Ntf4, a MAPK with a molecular mass of 45 kDa, has been reported to be expressed in pollen and seeds. Biochemical studies have indicated that the expression and the activation of Ntf4 is regulated during pollen maturation, although an increase of the activation is observed when the pollen is hydrated, just at the beginning of the germination. However, nothing is known about its subcellular localization.

Variations of Nucleolar Ultrastructure in Relation to Transcriptional Activity During G1 S, and G2 Periods of Microspore Interphase

The nucleolus is a highly dinamic organelle. Variations in the synthesis and/or processing of rRNA produces redistribution of the structural nucleolar components, in both physiological and experimental conditions (Goessens 1984, Hadjiolov 1985, Olmedilla et a1. 1987, Risueno and Medina 1986).

OSMIUM AMMINE : REVIEW OF CURRENT APPLICATIONS TO VISUALIZE DNA IN ELECTRON MICROSCOPY

This review has been collectively written. The contribution of the authors is mentioned for each part. References have been grouped at the end of the review. The objective of this review is to outline the principle of the method for electron microscopy, to emphasize the major applications and recent developments of this technique for DNA detection and finally to compare this technique with some other methods of DNA detection.