Impact of arbuscular mycorrhizal fungi (AMF) and atmospheric CO2 concentration on the biomass production and partitioning in the forage legume alfalfa

The influence of mycorrhizal symbiosis, atmospheric CO₂ concentration and the interaction between both factors on biomass production and partitioning were assessed in nodulated alfalfa (Medicago sativa L.) associated or not with arbuscular mycorrhizal fungi (AMF) and grown in greenhouse at either ambient (392 μmol?mol^?1) or elevated (700 μmol?mol^?1) CO₂ air concentrations. Measurements were performed at three stages of the vegetative period of plants. Shoot and root biomass achieved by plants at the end of their vegetative period were highly correlated to the photosynthetic rates reached at earlier stages, and there was a significant relationship between CO₂ exchange rates and total nodule biomass per plant. In non-mycorrhizal alfalfa, the production of leaves, stems and nodules biomass significantly increased when plants had been exposed to elevated CO₂ concentration in the atmosphere for 4 weeks. Regardless CO₂ concentration at which alfalfa were cultivated, mycorrhizal symbiosis improved photosynthetic rates and growth of alfalfa at early stages of the vegetative period and then photosynthesis decreased, which suggests that AMF shortened the vegetative period of the host plants. At final stages of the vegetative period, AMF enhanced both area and biomass of leaves as well as the leaves to stems ratio when alfalfa plants were cultivated at ambient CO₂. The interaction of AMF with elevated CO₂ improved root biomass and slightly increased the leaves to stems ratio at the end of the vegetative growth. Therefore, AMF may favor both the forage quality of alfalfa when grown at ambient CO₂ and its perennity for next cutting regrowth cycle when grown under elevated CO₂. Nevertheless, this hypothesis needs to be checked under natural conditions in field.     

Hyaluronan and Layilin Mediate Loss of Airway Epithelial Barrier Function Induced by Cigarette Smoke by Decreasing E-cadherin

Cigarette smoke (CigS) exposure is associated with increased bronchial epithelial permeability and impaired barrier function. Primary cultures of normal human bronchial epithelial cells exposed to CigS exhibit decreased E-cadherin expression and reduced transepithelial electrical resistance. These effects were mediated by hyaluronan (HA) because inhibition of its synthesis with 4-methylumbelliferone prevented these effects, and exposure to HA fragments of <70 kDa mimicked these effects. We show that the HA receptor layilin is expressed apically in human airway epithelium and that cells infected with lentivirus expressing layilin siRNAs were protected against increased permeability triggered by both CigS and HA. We identified RhoA/Rho-associated protein kinase (ROCK) as the signaling effectors downstream layilin. We conclude that HA fragments generated by CigS bind to layilin and signal through Rho/ROCK to inhibit the E-cadherin gene and protein expression, leading to a loss of epithelial cell-cell contact. These studies suggest that HA functions as a master switch protecting or disrupting the epithelial barrier in its high versus low molecular weight form and that its depolymerization is a first and necessary step triggering the inflammatory response to CigS.     

Implication of an Asp residue in the ribonucleolytic activity of hirsutellin A reveals new electrostatic interactions at the active site of ribotoxins

Ribotoxins are fungal extracellular ribonucleases that specifically cleave ribosomes leading to cell-death via apoptosis. α-Sarcin is the ribotoxin studied in deepest detail, and therefore constitutes the referential protein for the whole family. It has been demonstrated that ribotoxin activity depends on a very precise structural microenvironment in which electrostatic interactions among residues in the active site are of the highest importance. Hirsutellin A (HtA) has been recently described as the smallest ribotoxin known to date, encompassing all the abilities of previously characterized members of this family into a shorter sequence. Comparison of HtA and α-sarcin three-dimensional structures suggested that residues presumably forming the catalytic triad of HtA would be His 42, Glu 66, and His 113. Within this same idea, the presence of an Asp residue (Asp 40) in a position equivalent to α-sarcin Tyr 48 is highlighted as a novelty in this field. In this work, substitution mutants H42Q, E66Q and H113Q, as well as double and triple mutants in all possible combinations, are studied regarding their ribonucleolytic activity and cytotoxicity. Implication of these three residues in the ribotoxin activity of HtA is confirmed, though none of them is strictly essential for ribosomal cleavage. Studies with mutants D40N and D40N/E66Q demonstrate an important role for Asp 40 in the activity of HtA and establish a new set of electrostatic interactions different from the one described for already known ribotoxins.     

Simple,rapid and reliable methods to obtain high quality RNA and genomic DNA from <Emphasis Type="Italic">Quercus ilex</Emphasis> L. leaves suitable for molecular biology studies

Isolation of high-quality RNA and genomic DNA (gDNA) from many samples is a necessary step before accomplishing molecular biology studies. The particular composition of Quercus ilex leaves, specially hard and rich in cell wall material, polyphenolics and secondary metabolites, usually results in preparations contaminated with non-nucleic acid compounds. Although many methods have been developed, each case of study demands a protocol adapted to the specific plant sample and the pursued research objectives. We have evaluated several protocols to establish the methodology that best suited to our current genetic and molecular studies on Q. ilex. Our priority was to select the simplest methods reducing the plant starting material and the time employed, without compromising yield, quality and integrity of the isolated nucleic acids. Our results point to two protocols based on silica-membrane purification, as the most convenient for Q. ilex leaf tissue, and both procedures are greatly improved by adding insoluble polyvinyl polypyrrolidone during the isolation process. The protocols optimized here can be completed at the microfuge scale and allow a researcher to process 48 samples in 1 h, producing high quality preparations suitable for the routinely molecular biology applications with higher efficiency than other more labour and time-consuming protocols.     

Improved organogenic capacity of shoot cultures from mature pedunculate oak trees through somatic embryogenesis as rejuvenation technique

Theoretically, complete rejuvenation of mature trees should occur through somatic embryogenesis, however, this has not been extensively studied. The main objective of the present study was to increase the efficiency of in vitro clonal propagation for mature Quercus robur (100–300 years old), by induction of somatic embryogenesis as rejuvenation step prior to establishment of shoot culture through micropropagation of somatic embryo-derived plantlets. Shoot culture lines of “mature” origin were established from epicormic shoots of two centenarian oak genotypes (Sainza and CR-0) and maintained by axillary shoot proliferation. Embryogenic lines were also initiated from epicormic leaf explants of the same genotypes and maintained by secondary somatic embryogenesis. Although the frequency of somatic embryo conversion into plantlets was low in pedunculate oak, shoot culture lines could be established and maintained by axillary branching from several germinated somatic embryos. For each genotype and shoot culture line of the two origins (mature tree and somatic plantlets), shoot multiplication rate and elongation as well as rooting ability parameters were compared. Compared with “mature-origin” shoot cultures and after more than one year propagation in vitro, shoot lines established from somatic plantlets produced a significantly higher proportion of elongated, rootable shoots (from 26.0–31.6 to 36.8–40.5%) with increased rooting ability (from 3.3–45.6% to 23.2–89.8%). In the case of 300-year-old Sainza genotype such a high organogenic capacity was similar to shoot cultures initiated from basal sprouts. Basal sprouts are considered as “mature” material that retains juvenile characteristics compared with epicormic shoots forced from crown branches. Somatic embryogenesis only slightly improved plant regeneration of shoot cultures from basal sprouts, thus validating their use as “juvenile control”. The present results provide evidence that some rejuvenation occurred during the process of somatic embryogenesis and resulted in improved shoot growth and rooting of somatic embryo-derived culture compared with “mature” shoot culture. The results reported in this study might be useful in embryogenic systems with low plant conversion rates. The proposed experimental model might also be useful in finding molecular markers of plant ontogeny.     

Allometric and Group Differences in the Xenarthran Femur

The aim of this study is to analyze shape variation in the xenarthran femur to gain insights into their behavior and locomotion. Specimens of both Cingulata (armadillos and glyptodonts) and Pilosa (anteaters and sloths) were studied and within each group body mass varies by several orders of magnitude. The main focus of the analysis was allometric variation in femoral shape in the three groups studied, armadillos, glyptodonts, and pilosans. Three dimensional coordinates were recorded for 40 homologous landmarks on each of 51 xenarthran femurs. The data were analyzed by geometric morphometric methods, and form space analysis was used to identify the allometric variation in each of the three groups. Across all groups, larger specimens tended to have larger articular surfaces, more robust femora generally, and the shape of the femoral condyles was more suited to extended postures. In addition, in larger specimens the medial condyle was much larger than the lateral condyle and the third trochanter was located more distally. The larger armadillo femora had a greater trochanter located considerably proximal to the femoral head and this is thought to improve femoral extension, but in glyptodonts and pilosans the larger specimens had a greater trochanter that was far lateral to the femoral head and this is interpreted as enhancing femoral rotation.