Effect of in vitro morphogenesis on the production of podophyllotoxin derivatives in callus cultures of Linum album.

The anticancer compound podophyllotoxin and other related lignans can be produced in Linum album in vitro cultures, although their biosynthesis varies according to the degree of differentiation of the plant material. In general, L. album cell cultures do not form the same lignans as roots or other culture systems. Our aim was to explore how the lignan-producing capacity of organogenic cell masses is affected by the conditions that promote their formation and growth. Thus, L. album biomass obtained from plantlets was cultured in darkness or light, with or without the addition of plant growth regulators, and the levels of podophyllotoxin, methoxypodophyllotoxin and other related lignans were determined in each of these conditions. The organogenic capacity of the cell biomass grown in the different conditions was studied directly and also with light and scanning electronic microscopy, leading to the observation of.several somatic embryos and well-formed shoots. The main lignan produced was methoxypodophyllotoxin, whose production was clearly linked to the organogenic capacity of the cell biomass, which to a lesser extent was also the case for podophyllotoxin.

Detection of Epigenetic Modifications During Microspore Embryogenesis: Analysis of DNA Methylation Patterns Dynamics.

Methylation of 5-deoxy-cytidines of DNA constitutes a prominent epigenetic modification of the chromatin fiber which is locked in a transcriptionally inactive conformation. Changes in global DNA methylation are involved in many plant developmental processes during proliferation and differentiation events. The analysis of the changes of global DNA methylation distribution patterns during microspore embryogenesis induction and progression will inform on the regulatory mechanisms of the process, helping in the design of protocols to improve its efficiency in different species. To investigate the DNA methylation dynamics during microspore embryogenesis in the different cell types present in the cultures, the analysis of spatial and temporal pattern of nuclear distribution of 5-methyl-deoxy-cytidine (5mdC) constitutes a potent approach. The immunolocalization of 5mdC on sections and subsequent confocal laser microscopy analysis have been developed for in situ cellular analysis of a variety of plant samples, including embryogenic microspore and anther cultures. Quantification of 5mdC immunofluorescence intensity by image analysis software also permits to estimate differences in global DNA methylation levels among different cell types during development.

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.

Dual targeting to mitochondria and plastids of AtBT1 and ZmBT1, two members of the mitochondrial carrier family.

Zea mays and Arabidopsis thaliana Brittle 1 (ZmBT1 and AtBT1, respectively) are members of the mitochondrial carrier family. Although they are presumed to be exclusively localized in the envelope membranes of plastids, confocal fluorescence microscopy analyses of potato, Arabidopsis and maize plants stably expressing green fluorescent protein (GFP) fusions of ZmBT1 and AtBT1 revealed that the two proteins have dual localization to plastids and mitochondria. The patterns of GFP fluorescence distribution observed in plants stably expressing GFP fusions of ZmBT1 and AtBT1 N-terminal extensions were fully congruent with that of plants expressing a plastidial marker fused to GFP. Furthermore, the patterns of GFP fluorescence distribution and motility observed in plants expressing the mature proteins fused to GFP were identical to those observed in plants expressing a mitochondrial marker fused to GFP. Electron microscopic immunocytochemical analyses of maize endosperms using anti-ZmBT1 antibodies further confirmed that ZmBT1 occurs in both plastids and mitochondria. The overall data showed that (i) ZmBT1 and AtBT1 are dually targeted to mitochondria and plastids; (ii) AtBT1 and ZmBT1 N-terminal extensions comprise targeting sequences exclusively recognized by the plastidial compartment; and (iii) targeting sequences to mitochondria are localized within the mature part of the BT1 proteins.

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

BACKGROUND: In 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. RESULTS: The 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. CONCLUSION: Nanoparticles 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.

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. RESULTS: In the present study, the in situ expression and subcellular localization of Ntf4 have been analysed during the tobacco pollen developmental pathway. Cryosections, freeze-substitution and cryo-embedding in Lowicryl K4M were used as processing techniques for subsequent immunofluorescence, immunogold labelling and in situ hybridization assays. During pollen maturation, Ntf4 showed an increase in expression, as demonstrated by in situ hybridization, and specific subcellular distributions. We found that the protein was expressed from mid bicellular pollen stage until the pollen was mature. In germinating pollen, the protein increased after the initiation of germination. Translocation of the protein to the nucleus was found at specific stages; the presence of Ntf4 in the nucleus was found in the last stage of the pollen maturation and in germinating pollen. Double immunofluorescence and immunogold labelling with anti-Ntf4 (AbC4) and anti-P-MAPK (phosphorylated MAPK) antibodies revealed the co-localization of both epitopes in the nucleus at late developmental stages. CONCLUSIONS: The temporal and spatial pattern of the expression sites of Ntf4 has been characterized during pollen development, indicating that Ntf4 is a 'late gene' that is upregulated during maturation and germination, with a possible role in the gametophytic function. The translocation of the Ntf4 protein from the cytoplasm to the nucleus at late pollen developmental stages, and its co-localization with the P-MAPK epitope in several nuclear sites, indicates a relationship between the Ntf4 nuclear translocation and its active state.

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.

Multicellular structures developing during maize microspore culture express endosperm and embryo-specific genes and show different embryogenic potentialities.

During maize pollen embryogenesis, a range of multicellular structures are formed. Using different approaches, the "nature" of these structures has been determined in terms of their embryogenic potential. In situ molecular identification techniques for gene transcripts and products, and a novel cell tracking system indicated the presence of embryogenic (embryo-like structures, ELS) and non-embryogenic (callus-like structures, CLS) structures that occurred for short periods within the cultures. Some multicellular structures with a compact appearance generated embryos. RT-PCR and fluorescence in situ hybridization (FISH) with confocal microscopy techniques using specific gene markers of the endosperm (ZmESR2, ZmAE3) and embryo (LTP2 and ZmOCL1, ZmOCL3) revealed "embryo" and "endosperm" potentialities in these various multicellular structures present in the cultures. The results presented here showed distinct and specific patterns of gene expression. Altogether, the results demonstrate the presence of different molecules on both embryonic and non-embryonic structures. Their possible roles are discussed in the context of a parallel between embryo/endosperm interactions in planta and embryonic and non-embryonic structure interrelations under in vitro conditions.

Three dimensional confocal and electron microscopy imaging define the dynamics and mechanisms of diploidisation at early stages of barley microspore-derived embryogenesis.

In order to determine the timing and mechanisms of the spontaneous diploidisation throughout microspore-derived embryogenesis in barley, we have estimated the ploidy level of individual nuclei within young pro-embryos, from the first androgenetic division up to multinuclear structures still surrounded by the exine. Our methodological approach was based on the measure of the intensity of fluorescence after 4,6-Diamidino-2-phenylindole dihydrochloride staining, nuclear size and number of nucleoli in the confocal microscope. This method avoids the overlapping of the fluorescence signal in multinuclear pro-embryos, which cannot be studied using cytophotometer methods based on other types of fluorescence microscopes. The identification of haploid and diploid nuclei enabled us to determine the timing of diploidisation at early stages throughout androgenetic development. We found that diploidisation is an ongoing process that can start after the first embryogenic division and continues in multinuclear pro-embryos. Reconstruction of 3D-images of entire pro-embryos and the observation of cross and longitudinal sections across stacks of optical sections, together with correlative light and electron microscopy, provided evidences of nuclear fusion as the main mechanism of diploidisation.

Thyroid hormone regulates tubulin expression in mammalian liver. Effects of deleting thyroid hormone receptor-alpha or -beta.

Microtubules are made from polymers of alpha/beta dimers. We have observed in rat liver that, on the first day after birth, alpha-subunit is relatively high and beta-subunit low with respect to adult values. In the hypothyroid neonate, both subunits were found to be low, therefore indicating that thyroid hormone (TH) regulates these developmental changes. TH was also found to activate tubulin expression in adult liver, especially beta-subunit. To investigate the role of TH receptors (TRs) in tubulin expression, we analyzed mice lacking TRalpha or TRbeta compared with the wild type in both normal and TH-deprived adult animals. The results suggest that, in vivo, beta-tubulin protein expression in the liver is primarily under TRbeta positive control. In euthyroid mice lacking TRbeta, beta-tubulin expression was low. However, in the corresponding hypothyroid animals, it was found increased, therefore suggesting that the unliganded TRalpha might also upregulate beta-tubulin expression. Accordingly, TH administration to hypothyroid TRbeta-deprived mice reduced their high beta-tubulin expression. In parallel, the relatively high messenger level observed with these hypothyroid animals was reduced to the euthyroid level after T(3) treatment. The microtubular network of the mutant livers appeared, by immunofluorescence confocal microscopy, generally disorganized and drastically reduced in beta-tubulin in mice lacking TRbeta. In conclusion, our results indicate that beta-tubulin is critically controlled by TRbeta in the liver and that both TRs are probably needed to maintain the microtubular network organization of the liver.

The MAP kinase kinase NtMEK2 is involved in tobacco pollen germination.

The tobacco ntf4 mitogen-activated protein (MAP) kinase gene (and its encoded protein p45(Ntf4)) 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 p45(Ntf4), is activated upon pollen hydration. The MAP kinase kinase NtMEK2 activates SIPK, and here we show that it can also activate p45(Ntf4). 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 p45(Ntf4) and/or SIPK, are involved in pollen germination.

Microspore-derived embryos from Quercus suber anthers 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.

Esr proteins are secreted by the cells of the embryo surrounding region.

Three highly homologous Esr genes are expressed specifically in the embryo surrounding region at the micropylar end of the maize endosperm. The proteins belong to a family of small hydrophilic proteins that share a conserved motive with Clv3, the ligand of the receptor-like kinase Clv1. In this study, co-localization of Esr proteins with their mRNAs in the embryo surrounding region was shown with polyclonal antibodies recognizing all three Esr proteins. On a subcellular level the secretion of Esr proteins and their association with the cell wall was shown independently by cell fractionation, immunohistochemistry and transient expression of Gfp fusion proteins. Furthermore, a possible interaction of Esr proteins with a 35 kDa protein present in the lower half of maize kernels was suggested by in vitro affinity chromatography. Therefore Esr proteins share two characteristics with ligands of receptor-like kinases: they are released in the extracellular space and they have the capacity to form protein-protein interactions.