A. Ballarin-Denti

The proton pumps of the plasmalemma and the tonoplast of higher plants

Studies on intact cells, membrane vesicles, and reconstituted proteoliposomes have demonstrated in higher plants the existence of an ATP-driven electrogenic proton pump operating at the plasmalemma. There is also evidence of a second ATP-driven H+ pump localized at the tonoplast. The characteristics of both these ATP-driven pumps closely correspond to those of the plasmalemma and tonoplast proton pumps ofNeurospora and yeasts.

Effects of orthovanadate on H+ secretion, K+ uptake, electric potential difference and membrane ATPase activities of higher plant tissues.

Abstract Orthovanadate at concentrations between 5 and 20 μM inhibited the K + -stimulated, DCCD-sensitive ATPase activity of membrane preparations (fraction sedimenting between 13 000 and 80 000 g ) from radish seedlings while it had no effect on either the oligomycin-sensitive ATPase activity of mitochondrial preparations, or the phosphorylating activity of isolated mitochondria from radish seedlings or from pea internode segments. In whole germinating radish seeds orthovanadate inhibited the increase in fresh weight, net K + uptake and H + extrusion without inducing any detectable change of respiration nor of ATP and ADP levels, and it induced a rapid, partial depolarisation of the transmembrane electric potential difference. These effects in vivo were interpreted as indicating that orthovanadate inhibits an ATP-driven electrogenic proton pump corresponding to a plasmalemma ATPase.

Micrometeorological determination of time-integrated stomatal ozone fluxes over wheat : a case study in Northern Italy

Abstract Ozone deposition fluxes have been measured over a wheat field in Northern Italy in May–June 2001 with the eddy-correlation method in order to evaluate the amount of ozone taken up by plants during the whole grain filling period, from anthesis to harvest. Ozone uptake by plants is due to its penetration through the stomata; for this reason stomatal ozone fluxes were determined, using the analogy with water vapour fluxes (Penman–Monteith approach), which are easily measured. The total ozone dose was obtained by integrating the stomatal ozone fluxes over time. The observational results showed that ozone fluxes decrease with time, following the maturation and the senescence of plants. On the average, the stomatal flux was found to be 50–60% of the total flux, but this fraction decreased during the senescence, emphasizing the importance of non-stomatal ozone deposition pathways. The approach consisting on evaluating the total ozone dose by integrating stomatal fluxes was then compared to the currently used procedure based on the evaluation of the exposure index AOT40, which uses ozone concentrations. Important differences between these two methods are highlighted. The integration of stomatal ozone fluxes appears more suitable for the evaluation of physiological uptake than the use of the exposure index.

Ozone sensitivity of Fagus sylvatica and Fraxinus excelsior young trees in relation to leaf structure and foliar ozone uptake.

During the summer of 2001, 2-year-old Fraxinus excelsior and Fagus sylvatica plants were subjected to ozone-rich environmental conditions at the Regional Forest Nursery at Curno (Northern Italy). Atmospheric ozone concentrations and stomatal conductance were measured, in order to calculate the foliar fluxes by means of a one-dimensional model. The foliar structure of both species was examined (thickness of the lamina and of the individual tissues, leaf mass per area, leaf density) and chlorophyll a fluorescence was determined as a response parameter. Stomatal conductance was always greater in Fraxinus excelsior, as was ozone uptake, although the highest absorption peaks did not match the peaks of ozone concentration in the atmosphere. The foliar structure can help explain this phenomenon: Fraxinus excelsior has a thicker mesophyll than Fagus sylvatica (indicating a greater photosynthesis potential) and a reduced foliar density. This last parameter, related to the apoplastic fraction, suggests a greater ability to disseminate the gases within the leaf as well as a greater potential detoxifying capacity. As foliar symptoms spread, the parameters relating to chlorophyll a fluorescence also change. PI (Performance Index, Strasser, A., Srivastava, A., Tsimilli-Michael, M., 2000. The fluorescence transient as a tool to characterize and screen photosynthetic samples. In: Yunus, M., Pathre, U., Mohanty, P., (Eds.) Probing Photosynthesis: Mechanisms, Regulation and Adaptation. Taylor & Francis, London, UK, pp. 445-483.) has proved to be a more suitable index than Fv/Fm (Quantum Yield Efficiency) to record the onset of stress conditions.

Ozone fluxes and foliar injury development in the ozone-sensitive poplar clone Oxford (Populus maximowiczii × Populus berolinensis): a dose–response analysis

Between 2004 and 2005 a combined open plot and open-top chamber (OTC) experiment was carried out at Curno (Northern Italy) with cuttings of the poplar clone Oxford (Populus maximowiczii Henry x Populus berolinensis Dippel) grown in open plots (OPs, ambient air), charcoal-filtered OTCs (CF, ozone concentration reduced to 50% of ambient) or non-filtered OTCs (NF, ozone concentration reduced to 95% of ambient). Plants in half of the chambers were kept well-watered (WET), and plants in the remaining chambers were not watered (DRY). The onset and development of visible foliar injury and the stomatal conductance to water vapor (g(w)) were assessed during each growing season. A stomatal conductance model was parameterized by the Jarvis approach, allowing the calculation of ozone stomatal fluxes of plants in each treatment. The pattern of visible symptoms was analyzed in relation to ozone exposure (AOT40, accumulated ozone over a threshold of 40 ppb) and accumulated ozone stomatal fluxes (AF(ST)). Symptoms became visible at an AOT40 between 9584 and 13,110 ppb h and an AF(ST) between 27.85 and 30.40 mmol O(3) m(-2). The development of symptoms was more widespread and faster in plants in WET plots than in DRY plots. A slightly higher dose of ozone was required to cause visible symptoms in plants in DRY plots than in WET plots. By the end of each growing season, plants in the CF OTCs had absorbed a high dose of ozone (31.60 mmol O(3) m(-2) in 2004 and 32.83 mmol O(3) m(-2) in 2005, for WET plots), without developing any visible symptoms. A reliable dose-response relationship was defined by a sigmoidal curve model. The shape of this curve expresses the change in leaf sensitivity and physiologic state over a prolonged ozone exposure. After the appearance of the first symptoms, foliar injury increased more rapidly than the increases in ozone exposure and ozone absorbed dose; however, when the injury incidence reached 75%, the plant response declined.

Ozone Risk Assessment and Mapping in the Alps Based on Data from Passive Samplers

Passive samplers (diffusion tubes with organic reagent, produced by Passam of Switzerland) were used in a sampling campaign for the detection of weekly mean ozone concentrations in 15 sites over a domain of 80 x 40 km on the southern side of the European Alps from May to August 1998. The area is characterized by vast natural terrain of complex topography, with conifer and broadleaf forests. It is difficult to access and monitor air quality there with continuous analysers. By applying geostatistical techniques (ordinary kriging), and correcting the interpolated ozone concentrations according to the altitude of each single grid cell (2 x 2 km), maps of weekly ozone concentrations were produced. The weekly ozone data were used to assess daily and hourly data by means of an iterative procedure based on a functional dependence of ozone concentrations both on altitude and on the time of day. This allowed the estimation of values with an exposure index such as AOT40 (accumulated exposure over the threshold of 40 ppb) in all 800 cells of the domain. This also allowed the mapping of risk assessment related to the effects of ozone on the regional forest vegetation. Results obtained show values that exceed the exposure standards adopted in the Kuopio protocol (1996). Excess exposure values also match values calculated over a wider territorial domain by using hourly data on ozone concentration derived from continuous automatic analysers.

Techniques of Ozone Monitoring in a Mountain Forest Region: Passive and Continuous Sampling, Vertical and Canopy Profiles

Ozone is the most harmful air pollutant for plant ecosystems in the Mediterranean and Alpine areas due to its biological and economic damage to crops and forests. In order to evaluate the relation between ozone exposure and vegetation injury under on-field conditions, suitable ozone monitoring techniques were investi-gated. In the framework of a 5-year research project aimed at ozone risk assessment on forests, both continuous analysers and passive samplers were employed during the summer seasons (1994—1998) in different sites of a wide mountain region (80 x 40 km2) on the southern slope of the European Alps. Continuous analysers allowed the recording of ozone hourly concentration means necessary both to calculate specific exposure indexes (such as AOT, SUM, W126) and to record daily time-courses. Passive samplers, even though supplied only weekly mean concentration values, made it possible to estimate the altitude concentration gradient useful to correct the altitude dependence of ozone concentrations to be inserted into exposure indexes. In-canopy ozone profiles were also determined by placing passive samplers at different heights inside the forest canopy. Vertical ozone soundings by means of tethered balloons (kytoons) allowed the measurement of the vertical concentration gradient above the forest canopy. They also revealed ozone reservoirs aloft and were useful to explain the ozone advection dynamic in mountain slopes where ground measurement proved to be inadequate. An intercomparison between passive (PASSAM, CH) and continuous measurements highlighted the necessity to accurately standardize all the exposure operations, particularly the pre- and postexposure conservation at cold temperature to avoid dye (DPE) activity. Advantages and disadvantages from each mentioned technique are discussed.

Environmental population and forest stress: A multidisciplinary approach study on alpine forest ecosystems

Abstract A two-years research program has been developed by the Lombardy Foundation for the Environment (FLA) which aims to investigate the effects of air and soil pollution on forest ecosystems. The project has assembled a multidisciplanry research group to study forest decline using the approaches of geobotany, genetics, plant physiology and biochemistry, plant pathology, soil chemistry and microbiology. Air and soil pollution have been monitored by measuring wet and dry deposition, gaseous pollutants and soil heavy metals and xenobiotics. Environmental pollution has also been determined by using lichens and other plant and animal bioindicators.

Ozone and air particulate measurements in mountain forest sites

Abstract Ozone and total suspended particulate (TSP) concentrations have been measured for two consecutive summer seasons (1994–1995) during a forest-survey campaign carried out in two lateral valleys of Valtellina (Italian Alps) characterized by a very different degree of novel decline symptoms. Sampling methods and experimental data relative to ozone, measured by passive samplers and continuous monitors, and particulate, determined by multielemental analysis using EDXRF techniques, are described. While PST levels were low and almost equal in the two sites, ozone concentration was much higher in the more damaged valley, where its values were above the toxicity threshold for many plant species.

Cation effluxes associated with the uptake of TPP+, TPA+, and TPMP+ by Neurospora : evidence for a predominantly electroneutral influx process

Previously observed anomalies in the transport of lipid-soluble cations (LSIs) - presumed voltage-probe ions-by intact fungal cells [1] prompted a systematic investigation of ion exchanges induced by high (millimolar) concentrations of the particular species tetraphenylphosphonium ion (TPP+), tetraphenylarsonium ion (TPA+), and triphenylmethylphosphonium ion (TPMP+). With low extracellular free Ca2+ (no calcium added to the medium), influx of the LSIs was biphasic, indicating rapid entry into the cytoplasm followed by sequestration into a subcompartment. The latter process, especially, was strongly inhibited by extracellular Ca2+ (1 mM). Contrary to the expectation for electrophoretically driven entry of LSIs into fungal cells, no major efflux of protons (acidification of the medium) could be measured; in fact, significant alkalinization of the medium was observed. The major cellular inorganic cations, K+ or Na+ (under different conditions), were released during LSI uptake, but with kinetic behavior which clearly ruled out direct coupling to the uptake of TPP+, TPA+, or TPMP+. The major mechanism for entry of these lipid-soluble cations into Neurospora appears to be electroneutral diffusion in combination with one or more hydrophilic anions. Subsequent penetration of the fungal vacuoles would result in binding of LSIs to storage polyanions (viz., polyphosphate) and concomitant displacement of the normal vacuolar cations, such as basic amino acids and polyamines, thus leading to alkalinization of the extracellular medium. The observed effluxes of cytoplasmic K+ and Na+ should result independently from energetic changes (i.e., uncoupling of the mitochondrial) and are most easily described by simple, but asynchronous, changes in the average rate constants for entry and exit of the alkali-metal cations.