Mycorrhiza-like structures in rooted microshoots of Pinus pinea L.

Pinus pinea L. (stone pine) is one of the major plantation species in Iberian Peninsula, being Portugal the largest edible seed producer in the world. The induction and improvement of in vitro rhizogenesis of microshoots of Pinus pinea was developed in our laboratory using a co-culture system with ECM fungi. In the acclimation phase in mixed substrates, or in rhizotrons, anatomical and morphological studies were done to observe the evolution of the root system in microshoots from the co-culture system vs. control plants. Extensive dichotomous and coralloid branching of lateral roots occurred spontaneously in inoculated and control plants as well. Moreover, similar branching occurred in liquid culture of excised seedling roots without the presence of ECM fungi. The striking similarity of these organs with pine ectomycorrhizas prompted their anatomical analysis; however the presence of Hartig net was not confirmed. These results suggested that the development of ECM-like structures might have occurred spontaneously.info:eu-repo/semantics/publishedVersio

O-coumaric acid ester, a potential early signaling molecule in Pinus pinea and Pisolithus arhizus symbiosis established in vitro

During ectomycorrhizal (ECM) establishment, biochemical signals lead to the development of complex structures in both the plant and the fungus that ultimately result in the formation of an ectomycorrhiza. The cross-talk between partners begins before physical contact. Our objective was to investigate the chemical nature of the signals during the first stages of in vitro mycorrhization of Pinus pinea with Pisolithus arhizus. For this purpose a double-phase solid liquid medium was expressly developed for the co-culture in order to simplify the extraction and further molecules analysis. O-coumaric acid ester was identified using HPLC UV and LC DAD MS on the second day of co-culture and its presence was detected for up to 10 days. These results contribute to the characterization of biochemical signals during pre-colonization involving conifer species and an ECM fungus, and demonstrate the suitability of the double-phase medium developed for the growth of both organisms and for the analysis of released chemical mediators.info:eu-repo/semantics/publishedVersio

Molecular approach to characterize ectomycorrhizae fungi from Mediterranean pine stands in Portugal

Stone pine (Pinus pinea L.), like other conifers, forms ectomycorrhizas (ECM), which have benefi cial impact on plant growth in natural environments and forest ecosystems. An in vitro co-culture of stone pine microshoots with pure mycelia of isolated ECM sporocarps was used to overcome the root growth cessation not only in vitro but also to improve root development during acclimation phase. Pisolithus arhizus (Scop.) Rauschert and Lactarius deliciosus (L. ex Fr.) S.F. Gray fungi, were col lected, pure cultured and used in in vitro co-culture with stone pine microshoots. Samples of P. arhizus and L. deliciosus for the in vitro co-cultures were collected from the pine stands southwest Portugal. The in situ characterization was based on their morphotypes. To confirm the identity of the collected material, ITS amplification was applied using the pure cultures derived from the sporo carps. Additionally, a molecular profile using PCR based genomic fingerprinting comparison was executed with other genera of Basidiomycetes and Ascomycetes. Our results showed the effective ness of the techniques used to amplify DNA polymorphic sequences, which enhances the characte rization of the genetic profile of ECM fungi and also provides an option to verify the fungus identity at any stage of plant mycorrhization.info:eu-repo/semantics/publishedVersio

Embryogenic potential and expression of embryogenesis-related genes in conifers are affected by treatment with a histone deacetylase inhibitor

Somatic embryogenesis is used for vegetative propagation of conifers. Embryogenic cultures can be established from zygotic embryos; however, the embryogenic potential decreases during germination. In Arabidopsis, LEAFY COTYLEDON (LEC) genes are expressed during the embryonic stage, and must be repressed to allow germination. Treatment with the histone deacetylase inhibitor trichostatin A (TSA) causes de-repression of LEC genes. ABSCISICACID3 (ABI3) and its Zeamays ortholog VIVIPAROUS1 (VP1) act together with the LEC genes to promote embryo maturation. In this study, we have asked the question whether TSA treatment in a conifer affects the embryogenic potential and the expression of embryogenesis-related genes. We isolated two conifer LEC1-type HAP3 genes, HAP3A and HAP3B, from Picea abies and Pinus sylvestris. A comparative phylogenetic analysis of plant HAP3 genes suggests that HAP3A and HAP3B are paralogous genes originating from a duplication event in the conifer lineage. The expression of HAP3A is high, in both somatic and zygotic embryos, during early embryo development, but decreases during late embryogeny. In contrast, the expression of VP1 is initially low but increases during late embryogeny. After exposure to TSA, germinating somatic embryos of P. abies maintain the competence to differentiate embryogenic tissue, and simultaneously the germination progression is partially inhibited. Furthermore, when embryogenic cultures of P. abies are exposed to TSA during embryo maturation, the maturation process is arrested and the expression levels of PaHAP3A and PaVP1 are maintained, suggesting a possible link between chromatin structure and expression of embryogenesis-related genes in conifers

The development of highly potent and selective small molecule correctors of Z α1-antitrypsin misfolding

α1-antitrypsin deficiency is characterised by the misfolding and intracellular polymerisation of mutant α1-antitrypsin protein within the endoplasmic reticulum (ER) of hepatocytes. Small molecules that bind and stabilise Z α1-antitrypsin were identified via a DNA-encoded library screen. A subsequent structure based optimisation led to a series of highly potent, selective and cellular active α1-antitrypsin correctors

Methylation levels of a novel genetic element, EgNB3 as a candidate biomarker associated with the embryogenic competency of oil palm

The association between DNA methylation status and embryogenic competency in oil palm tissue culture was examined through Representational Difference Analysis (RDA) approach, using methylation-sensitive restriction endonucleases. "Difference Products" (DPs) of RDA derived from palms of similar genetic backgrounds but exhibiting different embryogenesis rates during the regeneration process were isolated. The DPs were sequenced using a pyrosequencing platform. To our knowledge, this is the first study profiling partial HpaII methylation sites in oil palm young leaf tissues which are potentially associated with embryogenic amenability through a genomic subtractive approach. Quantitative real-time PCR analysis demonstrated that the methylation status of a novel fragment, EgNB3, was higher in highly embryogenic leaf explants compared to low embryogenesis rate materials. These differences are likely to be contributed by the 5′-mCCGG-3′ and/or 5′-mCmCGG-3′ methylation patterns. Our data suggest that the differentially methylated site in EgNB3 has potential as a molecular biomarker for the screening of oil palm leaf explants for their embryogenic potentials

A Third Measure-Metastable State in the Dynamics of Spontaneous Shape Change in Healthy Human's White Cells

Human polymorphonuclear leucocytes, PMN, are highly motile cells with average 12-15 µm diameters and prominent, loboid nuclei. They are produced in the bone marrow, are essential for host defense, and are the most populous of white blood cell types. PMN also participate in acute and chronic inflammatory processes, in the regulation of the immune response, in angiogenesis, and interact with tumors. To accommodate these varied functions, their behavior is adaptive, but still definable in terms of a set of behavioral states. PMN morphodynamics have generally involved a non-equilibrium stationary, spheroid Idling state that transitions to an activated, ellipsoid translocating state in response to chemical signals. These two behavioral shape-states, spheroid and ellipsoid, are generally recognized as making up the vocabulary of a healthy PMN. A third, “random” state has occasionally been reported as associated with disease states. I have observed this third, Treadmilling state, in PMN from healthy subjects, the cells demonstrating metastable dynamical behaviors known to anticipate phase transitions in mathematical, physical, and biological systems. For this study, human PMN were microscopically imaged and analyzed as single living cells. I used a microscope with a novel high aperture, cardioid annular condenser with better than 100 nanometer resolution of simultaneous, mixed dark field and intrinsic fluorescent images to record shape changes in 189 living PMNs. Relative radial roundness, R(t), served as a computable order parameter. Comparison of R(t) series of 10 cells in the Idling and 10 in the Treadmilling state reveals the robustness of the “random” appearing Treadmilling state, and the emergence of behaviors observed in the neighborhood of global state transitions, including increased correlation length and variance (divergence), sudden jumps, mixed phases, bimodality, power spectral scaling and temporal slowing. Wavelet transformation of an R(t) series of an Idling to Treadmilling state change, demonstrated behaviors concomitant with the observed transition

Development of a small molecule that corrects misfolding and increases secretion of Z α1 -antitrypsin.

Severe α1 -antitrypsin deficiency results from the Z allele (Glu342Lys) that causes the accumulation of homopolymers of mutant α1 -antitrypsin within the endoplasmic reticulum of hepatocytes in association with liver disease. We have used a DNA-encoded chemical library to undertake a high-throughput screen to identify small molecules that bind to, and stabilise Z α1 -antitrypsin. The lead compound blocks Z α1 -antitrypsin polymerisation in vitro, reduces intracellular polymerisation and increases the secretion of Z α1 -antitrypsin threefold in an iPSC model of disease. Crystallographic and biophysical analyses demonstrate that GSK716 and related molecules bind to a cryptic binding pocket, negate the local effects of the Z mutation and stabilise the bound state against progression along the polymerisation pathway. Oral dosing of transgenic mice at 100 mg/kg three times a day for 20 days increased the secretion of Z α1 -antitrypsin into the plasma by sevenfold. There was no observable clearance of hepatic inclusions with respect to controls over the same time period. This study provides proof of principle that "mutation ameliorating" small molecules can block the aberrant polymerisation that underlies Z α1 -antitrypsin deficiency