Forests under climate change and air pollution: gaps in understanding and future directions for research.

Forests in Europe face significant changes in climate, which in interaction with air quality changes, may significantly affect forest productivity, stand composition and carbon sequestration in both vegetation and soils. Identified knowledge gaps and research needs include: (i) interaction between changes in air quality (trace gas concentrations), climate and other site factors on forest ecosystem response, (ii) significance of biotic processes in system response, (iii) tools for mechanistic and diagnostic understanding and upscaling, and (iv) the need for unifying modelling and empirical research for synthesis. This position paper highlights the above focuses, including the global dimension of air pollution as part of climate change and the need for knowledge transfer to enable reliable risk assessment. A new type of research site in forest ecosystems ("supersites") will be conducive to addressing these gaps by enabling integration of experimentation and modelling within the soil-plant-atmosphere interface, as well as further model development.

Metabolic-dependent changes in plant cell redox power after ozone exposure.

The tropospheric level of the phytotoxic air pollutant ozone has increased considerably during the last century, and is expected to continue to rise. Long-term exposure of higher plants to low ozone concentrations affects biochemical processes prior to any visible symptoms of injury. The current critical level of ozone used to determine the threshold for damaging plants (biomass loss) is still based on the seasonal sum of the external concentration above 40 nl.l(-1) (AOT40). Taking into account stomatal conductance and the internal capacity of leaf defences, a more relevant concept should be based upon the 'effective ozone flux', the balance between the stomatal flux and the intensity of cellular detoxification. The large decrease in the Rubisco/PEPc ratio reflects photosynthetic damage from ozone, and a large increase in activity of cytosolic PEPc, which allows increased malate production. Although the direct detoxification of ozone (and ROS produced from its decomposition) is carried out primarily by cell wall ascorbate, the existing level of this antioxidant is not sufficient to indicate the degree of cell sensitivity. In order to regenerate ascorbate, NAD(P)H is needed as the primary supplier of reducing power. It is hypothesised that increased activity of the catabolic pathways and associated shunts (glucose-6-phosphate dehydrogenase, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase, isocitrate dehydrogenase and malic enzyme) can provide sufficient NAD(P)H to maintain intracellular detoxification. Thus, measurement of the level of redox power would contribute to determination of the 'effective ozone dose', serving ultimately to improve the ozone risk index for higher plants.

Interactions between drought and O3 stress in forest trees.

Temperature increase and altered precipitation are facets of "Global Change", along with enhanced tropospheric ozone (O3) and CO2 levels. Both O3 and drought may curtail the probably limited capacity of "extra" carbon fixation in forest trees under a CO2-enriched atmosphere. In view of the exceptionally dry year of 2003 in Central Europe, this mini-review highlights O3/drought interactions in biochemical and ecophysiological responses of trees. Such interactions appear to vary, depending on the genotype and factorial scenarios. If O3 perturbs stomatal regulation, tolerance to both drought and persisting O3 exposure may be weakened, although drought preceding O3 stress may "harden" against O3 impact. Stomatal closure under drought may shield trees against O3 uptake and injury, which indeed was the case in 2003. However, the trees' "tuning" between O3 uptake and defence capacity is crucial in stress tolerance. Defence may be constrained due to limited carbon fixation, which results from the trade-off with O3 exclusion upon stomatal closure. Drought may cause a stronger reduction in stem growth than does ozone on an annual basis.

Compensation processes of Aleppo pine (Pinus halepensis Mill.) to ozone exposure and drought stress.

A long-term experiment was performed to study the effects of O3 and drought-stress (DS) on Aleppo pine seedlings (Pinus halepensis Mill.) exposed in open-top chambers. Ozone reduced gas exchange rates, ribulose-1,5-biphosphate carboxylase/oxygenase activity (Rubisco), aboveground C and needle N concentrations and C/N ratio and Ca concentrations of the twigs under 3 mm (twigs<3) and the aerial biomass. Also it increased phosphoenolpyruvate carboxylase (PEPc) and N and K concentrations of the twigs<3. Water stress decreased gas exchange rates, predawn needle water potential (PsiPd), C/N ratio, twigs<3 Ca, plant growth, aerial biomass and increased N, twigs with a diameter above 3 mm P and Mg concentrations. The combined exposure to both stresses increased N concentrations of twigs<3 and roots and aboveground biomass K content and decreased root C, maximum daily assimilation rate and instantaneous water use efficiency. The sensitivity of Aleppo pine to both stresses is determined by plant internal resource allocation and compensation mechanisms to cope with stress.

Carbon dioxide concentration and nitrogen input affect the C and N storage pools in Amanita muscaria-Picea abies mycorrhizae.

We studied the influence of elevated atmospheric CO2 concentration ([CO2]) on the vacuolar storage pool of nitrogen-containing compounds and on the glycogen pool in the hyphal sheath of Amanita muscaria (L. ex Fr.) Hooker-Picea abies L. Karst. mycorrhizae grown with two concentrations of ammonium in the substrate. Mycorrhizal seedlings were grown in petri dishes on agar containing 5.3 or 53 mg N l(-1) and exposed to 350 or 700 microl CO2 l(-1) for 5 or 7 weeks, respectively. Numbers and area of nitrogen-containing bodies in the vacuoles of the mycorrhizal fungus were determined by light microscopy linked to an image analysis system. The relative concentration of nitrogen in the vacuolar bodies was measured by electron energy loss spectroscopy (EELS). Glycogen stored in the cytosol was determined at the ultrastructural level by image analysis after staining the sections (PATAg test). Shoot dry weight, net photosynthesis and relative amounts of N in vacuolar bodies were greater at the higher N and CO2 concentrations. The numbers and areas of vacuolar N-containing bodies were significantly greater at the higher N concentration only at ambient [CO2]. In the same treatment the percentage of hyphae containing glycogen declined to nearly zero. We conclude that, in the high N/low [CO2] treatment, the mycorrhizal fungus had an insufficient carbohydrate supply, partly because of increased amino acid synthesis by the non-mycorrhizal rootlets. When [CO2] was increased, the equilibrium between storage of glycogen and N-containing compounds was reestablished.

Response of clones of Eucalyptus microtheca to NaCl in vitro.

We studied the response of nodal segments of Eucalyptus microtheca F.J. Muell. to salt stress in a tissue culture system. Three clones of Eucalyptus microtheca (37, 42 and 43) were grown in vitro under saline conditions (0-140 mM NaCl) for three months. The survival of all three clones decreased with increasing concentrations of NaCl in the medium, but the presence of up to 70 mM NaCl stimulated rooting in Clones 37 and 42. Shoot elongation of Clone 43 was less affected by salt than that of Clones 37 and 42. Leaf growth, expressed as leaf length, was stimulated 40% in Clone 42 and 33% in Clone 43, but was progressively inhibited up to 27% by 70 mM NaCl in Clone 37. Massive accumulation of Na(+) and Cl(-) occurred, especially in Clones 42 and 43 (4000 and 3000 mmol Na(+) kg(DW) (-1), respectively), as the salinity of the culture solution increased, and this was correlated with inhibition of growth. Because of a reduction in the accumulation of K(+) with increasing salinity, the K(+)/Na(+) ratio decreased from a control value of 4.5 to 0.14 in shoots grown in the presence of 140 mM NaCl. The development of techniques for selecting seedlings by monitoring the physiology of shoots in vitro instead of testing whole plants in vivo will provide a relatively simple method of selection for woody trees.

Changes in Properties of Barley Leaf Mitochondria Isolated from NaCl-Treated Plants.

Treatment of barley (Hordeum vulgare) seedlings with 400 millimolar NaCl for 3 days resulted in a reduction in plant growth and an increase in the leaf content in ions (K(+) + Na(+)) and proline. Purified mitochondria were successfully isolated from barley leaves. Good oxidative and phosphorylative properties were observed with malate as substrate. Malate-dependent electron transport was found to be only partly inhibited by cyanide, the remaining oxygen uptake being SHAM sensitive. The properties of mitochondria from NaCl-treated barley were modified. The efficiency of phosphorylation was diminished with only a slight decrease in the oxidation rates. In both isolated mitochondria and whole leaf tissue of treated plants, the lower respiration rate was due to a lower cytochrome pathway activity. In mitochondria, the activity of the alternative pathway was not modified by salt treatment, whereas this activity was increased in whole leaf tissue. The possible participation of the alternative pathway in response to salt stress will be discussed.

Interaction of Benzylaminopurine with Electron Transport in Plant Mitochondria during Malate Oxidation.

The effect of 6-benzylaminopurine (BA) was assayed on malate oxidation in mitochondria isolated from fresh and aged potato (Solanum tuberosum L.) slices. Depending on the experimental pH, two pathways for malate oxidation were selected. A pH of 7.7 favored the activity of malate dehydrogenase, which is connected with a rotenone-sensitive NADH dehydrogenase, whereas at pH 6.5 malic enzyme, linked to a rotenone-resistant NADH dehydrogenase, was more active.Experimental results indicate the existence of two sites of inhibition for BA. The first site is common with the site of inhibition of rotenone. The second site is on the classical cyanide-resistant alternative pathway, but is different from the site of salicylhydroxamic acid (SHAM) inhibition, as in succinate oxidation.Moreover, a distinct cyanide-resistant pathway, sensitive to SHAM but resistant to BA, is found to coexist with the well-known alternative pathway which is sensitive to SHAM and BA. This outlet of electrons can accommodate 10% of the total electron flow in mitochondria from fresh slices, and up to 30% in mitochondria from aged slices.

Inhibition by adenine derivatives of the cyanide-insensitive electron transport pathway of plant mitochondria.

The effect of benzylaminopurine was studied on cyanide-resistant mitochondria isolated from aged slices of potato tuber (Solanum tuberosum L. var. Bintje). Benzylaminopurine specifically acted on the cyanide-resistant alternative pathway. In the case of succinate oxidation, it mimicked the action of salicylhydroxamic acid and restored a good oxidative phosphorylation. Kinetic analyses showed that inhibitions by benzylaminopurine, salicylhydroxamic acid, and disulfiram occurred at mutually exclusive sites on the alternative pathway. Cyanide-resistant malate oxidation was only partially inhibited by benzylaminopurine and this inhibition occurred for low concentrations of this compound. On the other hand, the oxidation of exogenous NADH remained unaffected.The effects of several adenine derivatives with or without cytokinin activity and that of a purine analog with anticytokinin activity were also studied. The variation in effectiveness to inhibit cyanide-resistant electron transport was: benzylaminopurine and 7-pentylamino-3-methylpyrazolo (4,3 d) pyrimidine (anticytokinin) > alpha-alpha' dimethyl-allyl-adenine > 6-benzoylamino-9-benzylpurine > kinetin > adenine. No correlation was observed between the ability to inhibit the alternative pathway and the biological activity of these compounds. Liposolubility appeared as a major factor for potential inhibitory effect on the alternative pathway.

Control of Changes in Mitochondrial Activities during Aging of Potato Slices.

Aging of slices of potato tuber (Solanum tuberosum L.) in an aerated liquid medium induces a number of changes in mitochondrial activities. A nonphosphorylative, cyanide-insensitive electron transport pathway (alternate pathway) is brought into operation. The rate of oxidation of exogenous NADH increases markedly and the efficiency of phosphorylation with this substrate remains the same as it is in mitochondria isolated from fresh tissue slices. On the contrary, the rates of oxidation of succinate and malate do not increase while lower phosphorylative efficiencies indicate that a fraction of their electrons reaches oxygen through the alternate pathway. Chloramphenicol, a specific inhibitor of the mitochondrial protein-synthesizing system, has no effect whatsoever on these events. However, cycloheximide, which acts on the corresponding cytoplasmic system, prevents both the development of the alternate pathway and the rise in the rate of oxidation of exogenous NADH. These effects are interpreted as showing a specific control of the cytoplasmic protein-synthesizing system on the changes in mitochondrial oxidations during aging.