A reconstituted system reveals how activating and inhibitory interactions control DDK dependent assembly of the eukaryotic replicative helicase

During G1-phase of the cell-cycle the replicative MCM2–7 helicase becomes loaded onto DNA into pre-replicative complexes (pre-RCs), resulting in MCM2–7 double-hexamers on DNA. In S-phase, Dbf4-dependent kinase (DDK) and cyclin-dependent-kinase (CDK) direct with the help of a large number of helicase-activation factors the assembly of a Cdc45–MCM2–7–GINS (CMG) complex. However, in the absence of S-phase kinases complex assembly is inhibited, which is unexpected, as the MCM2–7 double-hexamer represents a very large interaction surface. Currently it is unclear what mechanisms restricts complex assembly and how DDK can overcome this inhibition to promote CMG-assembly. We developed an advanced reconstituted-system to study helicase activation in-solution and discovered that individual factors like Sld3 and Sld2 can bind directly to the pre-RC, while Cdc45 cannot. When Sld3 and Sld2 were incubated together with the pre-RC, we observed that competitive interactions restrict complex assembly. DDK stabilizes the Sld3/Sld2–pre-RC complex, but the complex is only short-lived, indicating an anti-cooperative mechanism. Yet, a Sld3/Cdc45–pre-RC can form in the presence of DDK and the addition of Sld2 enhances complex stability. Our results indicate that helicase activation is regulated by competitive and cooperative interactions, which restrict illegitimate complex formation and direct limiting helicase-activation factors into pre-initiation complexes.     

Improving the quality of municipal organic waste compost

The effects of different municipal organic waste (MOW) management practices (shredding, addition of carbon-rich materials and inoculation with earthworms) on organic matter stabilization and compost quality were studied. Four static piles were prepared with: (i) shredded MOW; (ii) shredded MOW+woodshavings; (iii) non-shredded MOW; and (iv) non-shredded MOW+woodshavings. After 50 days, a part of each pile was separated for vermistabilization, while the rest continued as traditional thermophilic composting piles. At different sampling dates, and in the finished products, the following parameters were measured: pH, electrical conductivity, carbon dioxide evolution, and concentrations of organic matter, total nitrogen, water-soluble carbon, nitrate nitrogen, ammonium nitrogen, and extractable phosphorus. Shredded treatments exhibited faster organic matter stabilization than non-shredded treatments, evidenced specially by earlier stabilization of carbon dioxide production and shorter thermophilic phases. Woodshavings addition greatly increased quality of final products in terms of organic matter concentration, and pH and electrical conductivity values, but decreased total nitrogen and available nutrient concentrations. Vermicomposting of previously composted material led to products richer in organic matter, total nitrogen, and available nutrient concentrations than composting only, probably due to the coupled effect of earthworm activity and a shorter thermophilic phase.     

Sucrose and fructan metabolism of different wheat cultivars at chilling temperatures

Four wheat ( Triticum aestivum L.) varieties cultivated in different climates from subtropics to North Patagonia were used to study sucrose and fructan metabolism in plants when submitted to a cold period. Higher levels of sugars were found in the more cold tolerant cultivars. Sucrose synthase (EC 2.4.1.13) and sucrose phosphate synthase (EC 2.4.1.14) activities showed a 2–3 fold increase when plants were grown at 4°C for 10 days. The more cold-tolerant wheat cultivars also showed the higher levels of enzyme activities. These metabolical changes were not due to anatomical or morphological differences produced during growth at 4°C     

Transpiration and stomatal behaviour of Quercus ilex plants during the summer in a Mediterranean carbon dioxide spring

Variations in the water relations and stomatal response of Quercus ilex were analysed under field conditions by comparing trees at two locations in a Mediterranean environment during two consecutive summers (1993 and 1994). We used the heat-pulse velocity technique to estimate transpirational water use of trees during a 5 month period from June to November 1994. At the end of sap flow measurements, the trees were harvested, and the foliage and sapwood area measured. A distinct environmental gradient exists between the two sites with higher atmospheric CO₂ concentrations in the proximity of a natural CO₂ spring. Trees at the spring site have been growing for generations in elevated atmospheric CO₂ concentrations. At both sites, maximum leaf conductance was related to predawn shoot water potential. The effects of water deficits on water relations and whole-plant transpiration during the summer drought were severe. Leaf conductance and water potential recovered after major rainfall in September to predrought values. Sap flow, leaf conductance and predawn water potential decreased in parallel with increases in hydraulic resistance, reaching a minimum in mid-summer. These relationships are in agreement with the hypothesis of the stomatal control of transpiration to prevent desiccation damage but also to avoid ‘runaway embolism’. Trees at the CO₂ spring underwent less reduction in hydraulic resistance for a given value of predawn water potential. The decrease in leaf conductance caused by elevated CO₂ was limited and tended to be less at high than at low atmospheric vapour pressure deficit. Mean (and diurnal) sap flux were consistently higher in the control site trees than in the CO₂ spring trees. The degree of reduction in water use between the two sites varied among the summer periods. The control site trees had consistently higher sap flow at corresponding values of either sapwood cross-sectional area or foliage area. Larger trees displayed smaller differences than smaller trees, between the control and the CO₂ spring trees. A strong association between foliage area and sapwood cross-sectional area was found in both the control and the CO₂ spring trees, the latter supporting a smaller foliage area at the corresponding sapwood stem cross-sectional area. The specific leaf area (SLA) of the foliage was not influenced by site. The results are discussed in terms of the effects of elevated CO₂ on plant water use at the organ and whole-tree scale.     

Modelling the surface conductance of a broad-leaf canopy: effects of partial decoupling from the atmosphere

The transpiration of a mature beech ( Fagus sylvatica L.) forest was measured over a whole season by the heat pulse velocity technique and the results analysed in terms of a new analytical canopy conductance model, which takes into account the effects of partial decoupling from the atmosphere on the local humidity environment experienced by the canopy. Stand daily transpiration ranged from 0·62 to 2·97 mm d^–1, with a seasonal mean value of 1·97 mm d^–1. Maximum canopy conductance was 18·5 mm s^–1, with a mean estimated value of 5·0 mm s^–1; computed values were little affected by the assumption of neutral atmospheric conditions. The decoupling coefficient Ω varied greatly on a daily and seasonal basis, with an average value of 0·28. As a result of partial decoupling, the estimated vapour pressure deficit at the notional canopy surface exceeded the values measured above the canopy by 380 Pa on average. When correctly expressed in terms of humidity at the canopy surface, the model explained 80% of the variance in half-hourly transpiration measurements. Upon cross-validation it still explained 72% of the variance, as compared to only 40% when correction for partial decoupling was not introduced. A baseline canopy conductance of 0·7 mm s^–1, not modulated by the environment, was estimated. The implications of the model are discussed for the representation of canopy conductance and transpiration of broad-leaf forests.     

Combating stress with flavodoxin: a promising route for crop improvement

Environmental stresses and iron limitation are the primary causes of crop losses worldwide. Engineering strategies aimed at gaining stress tolerance have focused on overexpression of endogenous genes belonging to molecular networks for stress perception or responses. Based on the typical response of photosynthetic microorganisms to stress, an alternative approach has been recently applied with considerable success. Ferredoxin, a stress-sensitive target, was replaced in tobacco chloroplasts by an isofunctional protein, a cyanobacterial flavodoxin, which is absent in plants. Resulting transgenic lines showed wide-range tolerance to drought, chilling, oxidants, heat and iron starvation. The survival of plants under such adverse conditions would be an enormous agricultural advantage and makes this novel strategy a potentially powerful biotechnological tool for the generation of multiple-tolerant crops in the near future.     

Responses of Two Olive Tree (Olea Europaea L.) Cultivars to Elevated CO2 Concentration in the Field

Five-year-old plants of two olive cultivars (Frantoio and Moraiolo) grown in large pots were exposed for 7 to 8 months to ambient (AC) or elevated (EC) CO₂ concentration in a free-air CO₂ enrichment (FACE) facility. Exposure to EC enhanced net photosynthetic rate (P _N) and decreased stomatal conductance, leading to greater instantaneous transpiration efficiency. Stomata density also decreased under EC, while the ratio of intercellular (C _i) to atmospheric CO₂ concentration and chlorophyll content did not differ, except for the cv. Moraiolo after seven months of exposure to EC. Analysis of the relationship between photosynthesis and C _i indicated no significant change in carboxylation efficiency of ribulose-1,5-bisphosphate carboxylase/oxygenase after five months of exposure to EC. Based on estimates derived from the P _N-C _i relationship, there were no apparent treatment differences in daytime respiration, CO₂ compensation concentration, CO₂-saturated photosynthetic rate, or photosynthetic rate at the mean C _i, but there was a reduction in stomata limitation to P _N at EC. Thus 5-year-old olive trees did not exhibit down regulation of leaf-level photosynthesis in their response to EC, though some indication of adjustment was evident for the cv. Frantoio with respect to the cv. Moraiolo.     

Carbon sequestration by forests in the National Parks of Italy

Abstract

Recent attempts to mitigate global change have brought forestry-based carbon (C) sequestration into sharp focus due to its potential to absorb CO₂ from the atmosphere. However, the consequences of actual forest management practices on C storage capacity are still controversial to a certain extent. Under such a perspective, a distinctive relevant issue concerns the management of forest ecosystems within areas specifically designated for nature conservation. From the analysis of biomass data from forests in the National Parks of Italy, we found that the average forest C stock and sink per unit area is relatively higher within National Parks (81.21 and 2.18 tons ha^?1, respectively) than on the overall national territory (76.11 and 1.12 tons ha^?1 year^?1, respectively). The analysis confirms the influence of ecological conditions and management approach on C sequestration capacity. Although the results of the proposed assessment approach have to be considered as rough estimates, the trial proves interesting, given the relative lack of specific information, at least on a large scale, about C stocks and sinks within forest areas designated for nature conservation, and the direct comparison with those forest areas not designated to such an end. The C storage capacity can be enhanced by increasing the productivity of forests, minimizing the disturbance to stand structure and composition. Extending conservation strategies adopted in National Parks to other forest areas of the national territory would allow the restoration of C sequestration potential, where unsustainable management practices have degraded relatively large stocks of biomass.