Nicla Contran

Structural and physiological responses to ozone in manna ash (Fraxinus ornus L.) leaves of seedlings and mature trees under controlled and ambient conditions.

Leaf-level microscopical symptom structure and physiological responses were investigated in seedlings experimentally exposed to ozone (O3) in indoor chambers (150 ppb, 8 h d(-1)/7 weeks), and field trees of Manna ash (Fraxinus ornus) exposed to ambient O3 (max 93 ppb/one growing season). Ozone-induced leaf injury, including leaf reddening and stippling, was observed in both seedlings and mature trees, but the morphology of injury in the stipples differed, being hypersensitive-like (HR-like) in the chamber seedlings and accelerated cell senescence (ACS) in the field trees. In both exposure conditions, the main structural impact of O3 was on the mesophyll and especially the upper assimilating cell layers. The main physiological impact was on carbon assimilation and on stomatal sluggishness. These effects were not due to stomatal structural injury and were more severe in juvenile compared to mature trees because of environmental (water availability, light) and constitutional (gas exchange capacity) factors and differences in the cell physiology processes (HR-like vs. ACS) triggered by ozone stress. Given the plasticity of plant responses to ozone stress, dose/response relationships for tree seedlings in the indoor chambers cannot be extrapolated to mature trees unless ambient conditions are closely simulated.

Use of the antiozonant ethylenediurea (EDU) in Italy: verification of the effects of ambient ozone on crop plants and trees and investigation of EDU's mode of action.

Twenty-four experiments where EDU was used to protect plants from ozone (O(3)) in Italy are reviewed. Doses of 150 and 450 ppm EDU at 2-3 week intervals were successfully applied to alleviate O(3)-caused visible injury and growth reductions in crop and forest species respectively. EDU was mainly applied as soil drench to crops and by stem injection or infusion into trees. Visible injury was delayed and reduced but not completely. In investigations on mode of action, EDU was quickly (<2h) uptaken and translocated to the leaf apoplast where it persisted long (>8 days), as it cannot move via phloem. EDU did not enter cells, suggesting it does not directly affect cell metabolism. EDU delayed senescence, did not affect photosynthesis and foliar nitrogen content, and stimulated antioxidant responses to O(3) exposure. Preliminary results suggest developing an effective soil application method for forest trees is warranted.

Protection of ash (Fraxinus excelsior) trees from ozone injury by ethylenediurea (EDU) : Roles of biochemical changes and decreased stomatal conductance in enhancement of growth

Treatments with ethylenediurea (EDU) protect plants from ozone foliar injury, but the processes underlying this protection are poorly understood. Adult ash trees (Fraxinus excelsior), with or without foliar ozone symptoms in previous years, were treated with EDU at 450 ppm by gravitational trunk infusion in May-September 2005 (32.5 ppm h AOT40). At 30-day intervals, shoot growth, gas exchange, chlorophyll a fluorescence, and water potential were determined. In September, several biochemical parameters were measured. The protective influence of EDU was supported by enhancement in the number of leaflets. EDU did not contribute its nitrogen to leaf tissue as a fertiliser, as determined from lack of difference in foliar N between treatments. Both biochemical (increase in ascorbate-peroxidase and ascorbic acid, and decrease in apoplastic hydrogen peroxide) and biophysical (decrease in stomatal conductance) processes regulated EDU action. As total ascorbic acid increased only in the asymptomatic trees, its role in alleviating O(3) effects on leaf growth and visible injury is controversial.

Ozone sensitivity and ethylenediurea protection in ash trees assessed by JIP chlorophyll a fluorescence transient analysis

The effect of ethylenediurea (EDU) was tested using the chlorophyll (Chl) a fluorescence transient analysis, performed with JIP-test, to assess ambient ozone (O3) effects on photosynthesis of adult trees under natural conditions. Twelve adult European ash (Fraxinus excelsior L.) trees, known to be sensitive or tolerant to O3, determined by presence symptomatic (S) or absence asymptomatic (AS) trees of foliar symptoms in previous years, were treated either with distilled water containing 450 g m−3 EDU or with distilled water. Once a month across the growing season [the accumulated exposure over a threshold of 40 nmol(O3) mol−1 was 32.49 µmol mol−1 h−1], Chl a fluorescence transients were measured in vivo on dark-adapted leaves of 1-year-old labeled shoots, from the lower crown part. Twenty-five parameters were calculated. The maximum quantum yield of primary photochemistry (ϕPo or Fv/Fm) did not differentiate between S-and AS-trees, while increased Chl content and de-excitation rates suggested compensation of O3 injury in S-trees. Seasonal reductions in absorbing fluxes and increase in heat and fluorescence dissipation processes was due to leaf ageing and drought, the latter suggesting water deficit influenced Chl a fluorescence stronger than ambient O3 exposure. AS-trees showed elevated probability of connectivity among photosystem 2 units, a mechanism to stimulate energy dissipation and reduce photo-oxidative injury. EDU prevented the inactivation of reaction centers. This slight effect does not warrant EDU as a tool to assess O3 effects on photosynthesis, while the JIP-test is suggested for a quantitative assessment in adult trees.

Visible Foliar Injury and Physiological Responses to Ozone in Italian Provenances of Fraxinus excelsior and F. ornus

We compared leaf visible injury and physiological responses (gas exchange and chlorophyll a fluorescence) to high O3 exposure (150 nmol mol–1 h, 8 h day–1, 35–40 days) of two woody species of the same genus with different ecological features: the mesophilic green ash (Fraxinus excelsior) and the xerotolerant manna ash (F. ornus). We also studied how provenances from northern (Piedmont) and central (Tuscany) Italy, within the two species, responded to O3 exposure. Onset and extent of visible foliar injury suggested that F. excelsior was more O3 sensitive than F. ornus. The higher stomatal conductance in F. ornus than in F. excelsior suggested a larger potential O3 uptake, in disagreement to lower visible foliar injury. The higher carbon assimilation in F. ornus suggested a higher potential of O3 detoxification and/or repair. Contrasting geographical variations of ash sensitivity to O3 were recorded, as Piedmont provenances reduced gas exchange less than Tuscan provenances in F. excelsior and more in F. ornus. Visible injury was earlier and more severe in F. excelsior from Piedmont than from Tuscany, while the provenance did not affect visible injury onset and extent in F. ornus.

Ethylenediurea (EDU) Affects the Growth of Ozone-Sensitive and Tolerant Ash (Fraxinus excelsior) Trees under Ambient O3 Conditions

Adult ash trees (Fraxinus excelsior L.), known to be sensitive or tolerant to ozone, determined by presence or absence of foliar symptoms in previous years, were treated with ethylenediurea (EDU) at 450 ppm by gravitational trunk infusion over the 2005 growing season (32.5 ppm h AOT40). Tree and shoot growth were recorded in May and September. Leaf area, ectomycorrhizal infection, and leaf and fine root biomass were determined in September. EDU enhanced shoot length and diameter, and the number and area of leaves, in both O3-sensitive and tolerant trees. However, no EDU effects were recorded at the fine root and tree level. Therefore, a potential for EDU protection against O3-caused growth losses of forest trees should be evaluated during longer-term experiments.

Antioxidant system in programmed cell death of sycamore (Acer pseudoplatanus L.) cultured cells

Reactive oxygen species (ROS) have pleiotropic effects in plants. ROS can lead to cellular damage and death or play key roles in control and regulation of biological processes, such as programmed cell death (PCD). This dual role of ROS, as toxic or signalling molecules, is possible because plant antioxidant system (AS) is able to achieve a tight control over ROS cellular levels, balancing properly their production and scavenging. AS response in plant PCD has been clearly described only in the hypersensitive response in incompatible plant–pathogen interactions and in the senescence process and has not been completely unravelled. In sycamore (Acer pseudoplatanus L.) cultured cells PCD can be induced by Fusicoccin (Fc), Tunicamycin (Tu), and Brefeldin A (Ba). These chemicals induce comparable PCD time course and extent, while H2O2 production is detectable only in Fc- and, to a lesser extent, in Ba-treated cells. In this paper the AS has been investigated during PCD of sycamore cells, measuring the effects of the three inducers on the cellular levels of non-enzymatic and enzymatic antioxidants. Results show that the AS behaviour is different in the PCD induced by the three chemicals. In Fc-treated cells AS is mainly devoted to decrease the concentration of toxic intracellular H2O2 levels. On the contrary, in cells treated with Tu and Ba, the cell redox state is shifted to a more reduced state and the enzymatic AS is partially down-regulated, allowing ROS to act as signalling molecules.