In vitro propagation and cryopreservation of Romanian endemic and rare Hypericum species

Efficient micropropagation and cryopreservation of Hypericum richeri ssp. transsilvanicum, an endemic species in Romania, and Hypericum umbellatum, a rare and endangered Daco-Balkan species, was achieved. The effects of type of explant and cytokinin on in vitro plant regeneration were investigated. Shoot organogenesis was achieved in all explants, but stem nodes regenerated best. Organogenesis from nodal segments was promoted by incubating these explants on Murashige and Skoog (MS) medium in the presence of cytokinins (6-benzyladenine, thidiazuron, kinetin or 6-??,??-dimethylallylaminopurine), each tested at four concentrations. The best morphogenic response for both Hypericum species (number of shoots per explant, shoot length, axillary branching of shoot, and frequency of shoot organogenesis) was observed when explants were incubated on MS medium containing 0.44 or 1.11???M 6-benzyladenine. Root induction was achieved only when regenerated shoots were transferred to fresh medium with or without auxin. Maximum rooting was recorded on MS medium supplemented with 2.45???M indole-3-butyric acid. Plantlets grown in vitro were successfully acclimatized in the greenhouse and showed normal development. Shoot tips and axillary buds excised from the in vitro regenerated plants were successfully cryopreserved in liquid nitrogen by the droplet-vitrification method. Following preculture in 0.25?M sucrose, dehydration and cryopreservation, the highest regeneration rates were obtained in both species by using axillary buds (68?% for H. richeri ssp. transsilvanicum and 71?% for H. umbellatum).     

Responses and structural recovery of periphytic diatom communities after short-term disturbance in some rivers (Hanoi, Vietnam)

Field transfer experiments of periphytic diatom assemblages developed on artificial substrates were set up to assess the responses of those communities to environmental disturbances. The glass slides were positioned for colonization at the relatively unpolluted site (Red, in the Red River) and at the heavily polluted site (TL, in the To Lich River) in the beginning of the experiment. After a period of 2?weeks, the colonized glass slides were concomitantly transferred from the unpolluted Red site to the heavily polluted TL site and to the moderate polluted site (NT₂, in the Nhue River) and, conversely, from the TL site to the Red site, and then to the NT₂ site. The responses and the adapting capacity of periphytic diatom communities to environmental changes were assessed through the cell density, diversity index, species richness, taxonomic composition, and diatom indices after 2 and 4?weeks of transfer periods. For all transfers except for the transfer from the Red to the TL site in which the growth inhibition of diatom cells was found, the diatom density significantly increased until the end of the experiment. Thus, the diatom communities have expressed their pollution tolerance or sensitivities by changing their composition to adapt themselves to environmental changes. Characteristic species of the Red site (Gyrosigma scalproides, Navicula recens) were replaced by Nitzschia palea, Nitzschia umbonata, Aulacoseira granulate typical species of the NT₂ site, in the biofilm transferred from the Red site to the NT₂ site. The relative abundances of typical diatom species of the Red site proliferated in the biofilm transferred from the TL site to the Red site. The replacement of periphytic diatom communities appeared after the transfer from the second week at the different sites. The slow shift of the species towards the typical species at the TL site could result from the organized structure of diatoms within biofilm before the transfer from the Red site to the TL site. The shifts in values of the Index of Specific Polluosensitivity and Diatom Assemblage Index to organic pollution throughout the experiment indicated the clear sensitivity of these indices to water quality changes.     

Brassica carinata CIL1 mediates extracellular ROS production during auxin- and ABA-regulated lateral root development

In the plant cytoplasm signals from multiple sources interact to control development. Lateral root proliferation is controlled by the antagonistic action of the hormones auxin and abscisic acid (ABA). Auxin stimulates the separation of pericycle initials and cell expansion. ABA is required for lateral root initiation and elongation and acts as an antagonist through auxin-dependent pathways. These hormones also mediate reactive oxygen species (ROS) accumulation in growing roots and promote cell expansion. We describe the isolation and characterization of a copper- and auxin-induced gene, COPPER INDUCED in LEAVES (CIL1), from Brassica carinata. Transgenic B. carinata seedlings expressing antisense CIL1 were used to determine the biological function of this gene. Lines with reduced CIL1 expression showed a decrease in lateral root development, as well as reduced sensitivity to auxin and ABA. Steady-state analysis of redox components showed a decrease in NADPH oxidase, superoxide dismutase, and catalase activity, accompanied by an increase in hydrogen peroxide concentration. GFP-tagged CIL1 accumulated at the plasma membrane and in the apoplast indicating CIL1 is likely extracellular. From these data we propose that CIL1 is an extracellular protein involved in ROS cycling mediating auxin and ABA signals.     

A role for mesenchyme dynamics in mouse lung branching morphogenesis

Mammalian airways are highly ramified tree-like structures that develop by the repetitive branching of the lung epithelium into the surrounding mesenchyme through reciprocal interactions. Based on a morphometric analysis of the epithelial tree, it has been recently proposed that the complete branching scheme is specified early in each lineage by a programme using elementary patterning routines at specific sites and times in the developing lung. However, the coupled dynamics of both the epithelium and mesenchyme have been overlooked in this process. Using a qualitative and quantitative in vivo morphometric analysis of the E11.25 to E13.5 mouse whole right cranial lobe structure, we show that beyond the first generations, the branching stereotypy relaxes and both spatial and temporal variations are common. The branching pattern and branching rate are sensitive to the dynamic changes of the mesoderm shape that is in turn mainly dependent upon the volume and shape of the surrounding intrathoracic organs. Spatial and temporal variations of the tree architecture are related to local and subtle modifications of the mesoderm growth. Remarkably, buds never meet after suffering branching variations and continue to homogenously fill the opening spaces in the mesenchyme. Moreover despite inter-specimen variations, the growth of the epithelial tree and the mesenchyme remains highly correlated over time at the whole lobe level, implying a long-range regulation of the lung lobe morphogenesis. Together, these findings indicate that the lung epithelial tree is likely to adapt in real time to fill the available space in the mesenchyme, rather than being rigidly specified and predefined by a global programme. Our results strongly support the idea that a comprehensive understanding of lung branching mechanisms cannot be inferred from the branching pattern or behavior alone. Rather it needs to be elaborated upon with the reconsideration of mesenchyme-epithelium coupled growth and lung tissues mechanics.     

SCANNING ELECTRON MICROSCOPY OBSERVATIONS OF DEFORMITIES IN SMALL PENNATE DIATOMS EXPOSED TO HIGH CADMIUM CONCENTRATIONS1

Different types of malformations are likely to affect the morphology of diatoms when exposed to particularly unstable environmental conditions, the most easily identifiable being distortion of the whole frustule. In the present study, we investigated, by means of SEM, valve abnormalities induced by high cadmium contamination (100 μg · L^?1) in small pennate diatoms. Changes in the shape of Amphora pediculus (Kütz.) Grunow and anomalous sculpturing of the cell wall of many species, such as Encyonema minutum (Hilse) D. G. Mann, Mayamaea agrestris (Hust.) Lange‐Bert., Gomphonema parvulum (Kütz.) Kütz., or Eolimna minima (Grunow) Lange‐Bert., were observed, which were not, or almost not, noticeable in the LM. With consideration to current knowledge of diatom morphogenesis, metal uptake by the cell would induce, directly or indirectly, damage to many cytoplasmic components (e.g., microtubules, cytoskeleton, Golgi‐derived vesicles) involved in the precisely organized silica deposition. This study confirms that many species, whatever their size, are likely to exhibit morphological abnormalities under cadmium stress, and that this indicator may be valuable for the biomonitoring of metal contamination, even if SEM observations are not necessary for routine studies.     

Consequences of altered eicosanoid patterns for nociceptive processing in mPGES-1-deficient mice

Cyclooxygenase-2 (COX-2)-dependent prostaglandin (PG) E(2) synthesis in the spinal cord plays a major role in the development of inflammatory hyperalgesia and allodynia. Microsomal PGE(2) synthase-1 (mPGES-1) isomerizes COX-2-derived PGH(2) to PGE(2). Here, we evaluated the effect of mPGES-1-deficiency on the nociceptive behavior in various models of nociception that depend on PGE(2) synthesis. Surprisingly, in the COX-2-dependent zymosan-evoked hyperalgesia model, the nociceptive behavior was not reduced in mPGES-1-deficient mice despite a marked decrease of the spinal PGE(2) synthesis. Similarly, the nociceptive behavior was unaltered in mPGES-1-deficient mice in the formalin test. Importantly, spinal cords and primary spinal cord cells derived from mPGES-1-deficient mice showed a redirection of the PGE(2) synthesis to PGD(2), PGF(2alpha) and 6-keto-PGF(1alpha) (stable metabolite of PGI(2)). Since the latter prostaglandins serve also as mediators of nociception they may compensate the loss of PGE(2) synthesis in mPGES-1-deficient mice.     

Sphingosine 1-phosphate modulates spinal nociceptive processing

Sphingosine 1-Phosphate (S1P) modulates various cellular functions such as apoptosis, cell differentiation, and migration. Although S1P is an abundant signaling molecule in the central nervous system, very little is known about its influence on neuronal functions. We found that S1P concentrations were selectively decreased in the cerebrospinal fluid of adult rats in an acute and an inflammatory pain model. Pharmacological inhibition of sphingosine kinases (SPHK) decreased basal pain thresholds and SphK2 knock-out mice, but not SphK1 knock-out mice, had a significant decrease in withdrawal latency. Intrathecal application of S1P or sphinganine 1-phosphate (dihydro-S1P) reduced the pain-related (nociceptive) behavior in the formalin assay. S1P and dihydro-S1P inhibited cyclic AMP (cAMP) synthesis, a key second messenger of spinal nociceptive processing, in spinal cord neurons. By combining fluorescence resonance energy transfer (FRET)-based cAMP measurements with Multi Epitope Ligand Cartography (MELC), we showed that S1P decreased cAMP synthesis in excitatory dorsal horn neurons. Accordingly, intrathecal application of dihydro-S1P abolished the cAMP-dependent phosphorylation of NMDA receptors in the outer laminae of the spinal cord. Taken together, the data show that S1P modulates spinal nociceptive processing through inhibition of neuronal cAMP synthesis.