Cell wall components and pectin esterification levels as markers of proliferation and differentiation events during pollen development and pollen embryogenesis in Capsicum annuum L.

Plant cell walls and their polymers are regulated during plant development, but the specific roles of their molecular components are still unclear, as well as the functional meaning of wall changes in different cell types and processes. In this work the in situ analysis of the distribution of different cell wall components was performed during two developmental programmes, gametophytic pollen development, which is a differentiation process, and stress-induced pollen embryogenesis, which involves proliferation followed by differentiation processes. The changes in cell wall polymers were compared with a system of plant cell proliferation and differentiation, the root apical meristem. The analysis was also carried out during the first stages of zygotic embryogenesis. Specific antibodies recognizing the major cell wall polymers, xyloglucan (XG) and the rhamnogalacturonan II (RGII) pectin domain, and antibodies against high- and low-methyl-esterified pectins were used for both dot-blot and immunolocalization with light and electron microscopy. The results showed differences in the distribution pattern of these molecular complexes, as well as in the proportion of esterified and non-esterified pectins in the two pollen developmental pathways. Highly esterified pectins were characteristics of proliferation, whereas high levels of the non-esterified pectins, XG and RGII were abundant in walls of differentiating cells. Distribution patterns similar to those of pollen embryos were found in zygotic embryos. The wall changes reported are characteristic of proliferation and differentiation events as markers of these processes that take place during pollen development and embryogenesis

Margarita Salas, a pioneer of molecular biology in Spain and a role model for female scientists

3 p.-2 fig.Peer reviewe

Changes in cell/tissue organization and peroxidase activity as markers for early detection of graft incompatibility in peach/plum combinations

9 páginas, 3 figuras, 4 tablas -- PAGS nros. 9-17Changes in cell and tissue organization and in peroxidase activity have been analyzed to find early markers to predict graft incompatibility occurrence in peach/plum combinations (Prunus persica/Prunus spp.) at 5 months after grafting in the dormancy period. Different compatible and incompatible peach/plum grafts were grown for 5 months in a nursery. The cellular study of the graft interface revealed structural changes associated with graft incompatibility symptoms. The main structural features were cambium cell disorganization, less differentiation of vascular tissues, degeneration of phloem and xylem cells, and accumulation of phenols at the graft interface after 5 months of graft development. The peroxidase study was performed during dormancy and the vegetative growth period, and revealed a significant increase in peroxidase activity in the incompatible unions, with significant differences between compatible and incompatible grafts. Analysis of gel profiles of nonbudded rootstocks and scions revealed an anodal isoperoxidase band [relative front (Rf) = 0.48] present in scions and compatible rootstocks, and another isoperoxidase band (Rf = 0.53) only present in the incompatible rootstocks. Our results show that the analysis of cell organization to detect early structural events and the evaluation of peroxidase activity at graft unions constituted feasible and convenient methods for early diagnosis of graft incompatibility. Also, it was suggested that the presence of band Rf = 0.48 in plum rootstocks and peach cultivars could be used as a marker to predict graft compatibility for peach scions and plum rootstocksFinancial support was provided by Comisión Interministerial de Ciencia y Tecnología (project nos. AGL2005-05533 and AGL2008-00283/AGR, partially founded by BFU2008-00203 and AGL2008-04255), by the CONSID-DGA A 44, and by a fellowship granted to O.Z. from the Agencia Española de Cooperación Internacional (AECI)Peer reviewe

5-azacytidine promotes microspore embryogenesis initiation by decreasing global DNA methylation,but prevents subsequent embryo development in rapeseed and barley

17 p.-10 fig.Microspores are reprogrammed by stress in vitro toward embryogenesis. This process is an important tool in breeding to obtain double-haploid plants. DNA methylation is a major epigenetic modification that changes in differentiation and proliferation. We have shown changes in global DNA methylation during microspore reprogramming. 5-Azacytidine (AzaC) cannot be methylated and leads to DNA hypomethylation. AzaC is a useful demethylating agent to study DNA dynamics, with a potential application in microspore embryogenesis. This work analyzes the effects of short and long AzaC treatments on microspore embryogenesis initiation and progression in two species, the dicot Brassica napus and the monocot Hordeum vulgare. This involved the quantitative analyses of proembryo and embryo production, the quantification of DNA methylation, 5-methyl-deoxy-cytidine (5mdC) immunofluorescence and confocal microscopy, and the analysis of chromatin organization (condensation/decondensation) by light and electron microscopy. Four days of AzaC treatments (2.5 μM) increased embryo induction, response associated with a decrease of DNA methylation, modified 5mdC, and heterochromatin patterns compared to untreated embryos. By contrast, longer AzaC treatments diminished embryo production. Similar effects were found in both species, indicating that DNA demethylation promotes microspore reprogramming, totipotency acquisition, and embryogenesis initiation, while embryo differentiation requires de novo DNA methylation and is prevented by AzaC. This suggests a role for DNA methylation in the repression of microspore reprogramming and possibly totipotency acquisition. Results provide new insights into the role of epigenetic modifications in microspore embryogenesis and suggest a potential benefit of inhibitors, such as AzaC, to improve the process efficiency in biotechnology and breeding programs.Work supported by projects (references BFU2008-00203, BFU2011-23752, AGL2014-52028-R) funded by the Spanish Ministry of Economy and Competitiveness (MINECO) and the European Regional Development Fund (ERDF/FEDER). AAET is recipient of a predoctoral fellowship of the JAE-Pre Program of the Spanish National Research Council, CSIC (JAEPre2010-052), cofunded by ERDF/FEDER.Peer reviewe

Differential expression patterns of Arabinogalactan Proteins in Arabidopsis thaliana reproductive tissues

36 p.-6 fig.Arabinogalactan proteins (AGPs) are heavily glycosylated proteins existing in all members of the plant kingdom and are differentially distributed through distinctive developmental stages. Here, we showed the individual distributions of specific Arabidopsis AGPs: AGP1, AGP9, AGP12, AGP15, and AGP23, throughout reproductive tissues and indicated their possible roles in several reproductive processes. AGP genes specifically expressed in female tissues were identified using available microarray data. This selection was confirmed by promoter analysis using multiple green fluorescent protein fusions to a nuclear localization signal, β-glucuronidase fusions, and in situ hybridization as approaches to confirm the expression patterns of the AGPs. Promoter analysis allowed the detection of a specific and differential presence of these proteins along the pathway followed by the pollen tube during its journey to reach the egg and the central cell inside the embryo sac. AGP1 was expressed in the stigma, style, transmitting tract, and the chalazal and funiculus tissues of the ovules. AGP9 was present along the vasculature of the reproductive tissues and AGP12 was expressed in the stigmatic cells, chalazal and funiculus cells of the ovules, and in the septum. AGP15 was expressed in all pistil tissues, except in the transmitting tract, while AGP23 was specific to the pollen grain and pollen tube. The expression pattern of these AGPs provides new evidence for the detection of a subset of specific AGPs involved in plant reproductive processes, being of significance for this field of study. AGPs are prominent candidates for male–female communication during reproduction.This work was financed by FEDER through the COMPETE programme, and by Portuguese National funds through FCT – Fundação para a Ciência e Tecnologia (Project PTDC/AGR-GPL/115358/2009) and from an FCT PhD grant SFRH/BD/60995/2009 awarded to AMP. This project also benefited from financial support from the COST Action FA0903: ‘Harnessing Plant Reproduction for Crop Improvement’.Peer reviewe

Cambios epigenéticos en ápices crioconservados de Mentha xpiperita

La crioconservación ofrece una serie de ventajas sobre otros métodos para la conservación a largo plazo del germoplasma de especies de propagación vegetativa. En comparación con la conservación in vitro tiene la ventaja de reducir los posibles cambios genéticos que pueden ocurrir

The protein kinases AtMAP3Kε1 and BnMAP3Kε1 are functional homologues of S. pombe cdc7p and may be involved in cell division

We identified an Arabidopsis thaliana gene, AtMAP3Kε1, and a Brassica napus cDNA, BnMAP3Kε1, encoding functional protein serine/threonine kinases closely related to cdc7p and Cdc15p from Schizosaccharomyces pombe and Saccharomyces cerevisiae, respectively. This is the first report of cdc7-related genes in non-fungal eukaryotes; no such genes have as yet been identified in Metazoans. The B. napus protein is able to partially complement a cdc7 loss of function mutation in S. pombe. RT–PCR and in situ hybridisation revealed that the A. thaliana and B. napus genes are expressed in both the sporophytic and the gametophytic tissues of the respective plant species and revealed further that expression is highest in dividing cells. Moreover, AtMAP3Kε1 gene expression is cell cycle-regulated, with higher expression in G2-M phases. Our results strongly suggest that the plant cdc7p-related protein kinases are involved in a signal transduction pathway similar to the SIN pathway, which positively regulates cytokinesis in S. pombe.This work was mainly supported by a EU grant (SIME project BIOTEC-RTD-CEE PL 960275). The authors also acknowledge the financial support of the MERS and CNRS to UMR 8618, and DGESIG PB98–0678

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

Póster presentado en la The Annual Plant Biology Meeting, celebrada en Minneapolis del 6 al 10 de agosto de 2011.Peer Reviewe

Magnetic nanoparticles penetration and transport in planta

Resumen del póster presentado en la 9th International Conference on the Scientific and Clinical Applications of Magnetic Carriers, celebrada en Minneapolis (Estados Unidos) del 22 al 26 de mayo de 2012.Magnetic nanoparticles are very suitable for a broad range of applications, like those involving synthesis and use of ferrofluids for bio-applications in general. In medicine the aim is to use them in diagnosis as well as in therapy. The ongoing research and results obtained up to now in these fields open a wide range of possibilities for using magnetic nanoparticles in other disciplines, for example in general plant research and agronomy. To study the use of nanoparticles in agriculture the first stage is to work out the penetration and transport into living plants and plant cells. We present here an overview of the research carried out within the scope of an interdisciplinary collaboration, on how inorganic nanoparticles interact with plant cells and tissues.

NO, ROS, and cell death associated with caspase-like activity increase in stress-induced microspore embryogenesis of barley

Under specific stress treatments (cold, starvation), in vitro microspores can be induced to deviate from their gametophytic development and switch to embryogenesis, forming haploid embryos and homozygous breeding lines in a short period of time. The inductive stress produces reactive oxygen species (ROS) and nitric oxide (NO), signalling molecules mediating cellular responses, and cell death, modifying the embryogenic microspore response and therefore, the efficiency of the process. This work analysed cell death, caspase 3-like activity, and ROS and NO production (using fluorescence probes and confocal analysis) after inductive stress in barley microspore cultures and embryogenic suspension cultures, as an in vitro system which permitted easy handling for comparison. There was an increase in caspase 3-like activity and cell death after stress treatment in microspore and suspension cultures, while ROS increased in non-induced microspores and suspension cultures. Treatments of the cultures with a caspase 3 inhibitor, DEVD-CHO, significantly reduced the cell death percentages. Stress-treated embryogenic suspension cultures exhibited high NO signals and cell death, while treatment with S-nitrosoglutathione (NO donor) in control suspension cultures resulted in even higher cell death. In contrast, in microspore cultures, NO production was detected after stress, and, in the case of 4-day microspore cultures, in embryogenic microspores accompanying the initiation of cell divisions. Subsequent treatments of stress-treated microspore cultures with ROS and NO scavengers resulted in a decreasing cell death during the early stages, but later they produced a delay in embryo development as well as a decrease in the percentage of embryogenesis in microspores. Results showed that the ROS increase was involved in the stress-induced programmed cell death occurring at early stages in both non-induced microspores and embryogenic suspension cultures; whereas NO played a dual role after stress in the two in vitro systems, one involved in programmed cell death in embryogenic suspension cultures and the other in the initiation of cell division leading to embryogenesis in reprogrammed microspores