Among European Neottieae, Limodorum abortivum is a common Mediterranean orchid. It forms small populations with a patchy distribution in woodlands, and is characterized by much reduced leaves, suggesting a partial mycoheterotrophy. We have investigated both the photosynthetic abilities of L. abortivum adult plants and the diversity of mycorrhizal fungi in Limodorum plants growing in different environments and plant communities (coniferous and broadleaf forests) over a wide geographical and altitudinal range. Despite the presence of photosynthetic pigments, CO2 fixation was found to be insufficient to compensate for respiration in adult plants. Fungal diversity was assessed by morphological and molecular methods in L. abortivum as well as in the related rare species Limodorum trabutianum and Limodorum brulloi. Phylogenetic analyses of the fungal internal transcribed spacer (ITS) sequences, obtained from root samples of about 80 plants, revealed a tendency to associate predominantly with fungal symbionts of the genus Russula. Based on sequence similarities with known species, most root endophytes could be ascribed to the species complex encompassing Russula delica, Russula chloroides, and Russula brevipes. Few sequences clustered in separate groups nested within Russula, a genus of ectomycorrhizal fungi. The morphotypes of ectomycorrhizal root tips of surrounding trees yielded sequences similar or identical to those obtained from L. abortivum. These results demonstrate that Limodorum species with inefficient photosynthesis specifically associate with ectomycorrhizal fungi, and appear to have adopted a nutrition strategy similar to that known from achlorophyllous orchids.

We used a localized ozone (O3) fumigation (LOF) system to study acute and short-term O(3) effects on physiological leaf traits. The LOF system enabled investigation of primary and secondary metabolic responses of similarly and differently aged leaves on the same plant to three different O3 concentrations ([O3]), unconfounded by other influences on O3 sensitivity, such as genetic, meteorological and soil factors. To simulate the diurnal cycle of O3 formation, current-year and 1-year-old Quercus ilex (L.) and Quercus pubescens (L.) leaves were fumigated with O3 at different positions (and hence, different leaf ages) on the same branch over three consecutive days. The LOF system supplied a high [O3] (300+/-50 ppb) on leaves appressed to the vents, and an intermediate, super-ambient [O3] (varying between 120 and 280 ppb) on leaves less than 30 cm from the vent. Leaves more than 60 cm from the O3 vent were exposed to an [O3] comparable with the ambient concentration, with a 100 ppb peak during the hottest hours of the day. Only leaves exposed to the high [O3] were affected by the 3-day treatment, confirming that Mediterranean oak are tolerant to ambient and super-ambient [O3], but may be damaged by acute exposure to high [O3]. Stomatal and mesophyll conductance and photosynthesis were all reduced immediately after fumigation with high [O3], but recovered to control values within 72 h. Both the intercellular and chloroplast CO2 concentrations ([CO2]) remained constant throughout the experiment. Thus, although treatment with a high [O3] may have induced stomatal closure and consequent down-regulation of photosynthesis, we found no evidence that photosynthesis was limited by low [CO2] at the site of fixation. One-year-old leaves of Q. ilex were much less sensitive to O3 than current-year leaves, suggesting that the low stomatal conductance observed in aging leaves limited O3 uptake. No similar effect of leaf age was found in Q. pubescens. Dark respiration decreased during the treatment period, but a similar decrease was observed in leaves exposed to low [O3], and therefore may not be an effect of O3 treatment. Light respiration, on the other hand, was mostly constant in ozone-treated leaves and increased only in leaves in which photosynthesis was temporarily inhibited by high [O3], preventing them from acting as strong sinks that recycle respiratory CO2 in the leaves. There was no evidence of photochemical damage in Q. ilex leaves, whereas Q. pubescens leaves exposed to a high [O3] showed limited photochemical damage, but recovered rapidly. Biochemical markers were affected by the high [O3], indicating accumulation of reactive oxygen species (ROS) and increased denaturation of lipid membranes, followed by activation of isoprene biosynthesis in Q. pubescens leaves. We speculate that the high isoprene emissions helped quench ROS and normalize membrane stability in leaves recovering from O3 stress.

Isoprene reduces visible damage (necrosis) of leaves caused by exposure to ozone but the mechanism is not known. Here we show that in Phragmites leaves isoprene emission was stimulated after a 3-h exposure to high ozone levels. The photosynthetic apparatus of leaves in which isoprene emission was inhibited by fosmidomycin became more susceptible to damage by ozone than in isoprene-emitting leaves. Three days after ozone fumigation, the necrotic leaf area was significantly higher in isoprene-inhibited leaves than in isoprene-emitting leaves. Isoprene-inhibited leaves also accumulated high amounts of nitric oxide (NO), as detected by epifluorescence light microscopy. Our results confirm that oxidative stresses activate biosynthesis and emission of chloroplastic isoprenoid, bringing further evidence in support of an antioxidant role for these compounds. It is suggested that, in nature, the simultaneous quenching of NO and reactive oxygen species by isoprene may be a very effective mechanism to control dangerous compounds formed under abiotic stress conditions, while simultaneously attenuating the induction of the hypersensitive response leading to cellular damage and death.

Quercus ilex (L.) leaves emit monoterpenes, particularly alpha-pinene, beta-pinene and sabinene. Apart from the monoterpene pools that are stored in specialized structures and have a clear defensive or attractive role, the function of monoterpenes in Q. ilex leaves is unknown. We tested whether monoterpenes have an antioxidant role, as has previously been found for isoprene in isoprene-emitting leaves. We exposed Q. ilex leaves to either mild and repeated ozone exposure (Experiment I) or to a single acute ozone exposure (Experiment II) at temperatures ranging between 20 and 32 degrees C. Both ozone treatments rapidly stimulated monoterpene synthesis, but had no effect on photosynthesis and caused no visible damage to leaves maintained at 25, 30 or 32 degrees C. Ozone inhibited both photosynthesis and monoterpene synthesis in leaves maintained at 20 degrees C. To characterize the relationship between monoterpenes and ozone-induced damage, we fed detached leaves fosmidomycin, a selective inhibitor of isoprene synthesis. Fosmidomycin caused rapid and complete inhibition of monoterpene emissions in leaves maintained at 30 degrees C, confirming that monoterpenes are synthesized by the same biochemical pathway as isoprene. However, over the experimental period, fosmidomycin did not affect concentrations of compounds that are formed from chloroplastic isoprenoids and that might have conferred antioxidant protection, either directly (carotenoids) or indirectly (chlorophylls, xanthophylls). In leaves whose monoterpene synthesis had been inhibited by fosmidomycin, ozone rapidly and significantly inhibited photosynthesis and increased the production of hydrogen peroxide and malonyldialdehyde. We conclude that monoterpenes produced by Q. ilex leaves share the same biosynthetic pathway and function as isoprene. Furthermore, all volatile isoprenoids may have similar antioxidant properties and may be stimulated by the same stress-inducing conditions.

Summary Fire is often associated with episodes of air pollution, possibly involving the release of biogenic isoprenoids (isoprene and monoterpenes). The direct and indirect impacts of fire on isoprenoid emission by plants of the Mediterranean vegetation were studied. Leaves of Arbutus unedo, Phillyrea latifolia, Cistus incanus, Cistus mospeliensis, Pistacia lentiscus, Quercus ilex, Quercus suber, Quercus pubescens, Myrtus communis and Pinus halepensis were exposed to direct fire or to the fire‐consequent wave of elevated temperature. Half the tested plant species did not emit isoprenoids and the treatments did not induce isoprenoid emission. In contrast, isoprene was emitted by intact leaves of Q. pubescens and M. communis, while monoterpenes were emitted by intact leaves of Q. ilex, Q. suber and P. halepensis. The two treatments rapidly reduced isoprene emission by isoprene‐emitting species and monoterpene emission by Quercus spp. This inhibition was associated with photosynthetic inhibition, and recovery was seen in Quercus spp. within days of treatment. Recovery was also associated with the recovery of photosynthesis, suggesting that emitted isoprenoids continue to be formed predominantly from photosynthetic intermediates after a fire episode. In Q. pubescens leaves, however, recovery from the elevated‐temperature treatment caused a sustained increase of isoprene emission which was not mirrored by a similar increase in photosynthesis. Whether this represents the induction of alternative metabolic pathways or an increase of the flux of photosynthetic carbon in the isoprene pathway is not known. Isoprene‐emitting species in areas surrounding fire may emit a substantially larger hydrocarbon flux for several days after fire. The elevated‐temperature treatment induced the emission of α‐pinene from Myrtus leaves, and the fire treatment stimulated the emission of several monoterpenes from Pinus needles. The emission began to decrease within minutes in Myrtus, while it increased within the first 100 min in Pinus, where it was detectable the day after the event although the flux was smaller than in prestressed needles. Exposure to fire and to the associated elevated temperature may induce bursts of monoterpenes from plants that regularly do not emit these compounds and temporarily increase the load of monoterpenes in the atmosphere by pine species. These emissions may contribute to photochemical reactions involved in smog and ozone formation.

Water scarcity is one of the most detrimental effects on crop yield. We studied the effect of water stress on carbon acquisition and allocation to monoterpenes of commercial importance in spearmint and rosemary plants. Increasing levels of water stress reduced photosynthesis and plant biomass. Under increasing water-stress levels photosynthesis was limited by low CO2 availability due to reduced stomatal and mesophyll conductance, while photochemical and biochemical processes were not affected. At the balsamic period, significantly higher amounts of monoterpenes per unit mass were formed in water-stressed leaves with respect to irrigated controls. However, because of plant biomass reduction, water stress reduced the total yield of monoterpenes. Appropriate irrigation strategies may, therefore, potentially improve yield of monoterpenes in field grown spearmint and rosemary crops.

Isoprene emitted from plants is made in chloroplasts from dimethylallyl pyrophosphate (DMAPP). Leaves of Populus nigra and Phragmites australis exposed to (13)CO(2) for 15 min emitted isoprene that was about 90% (13)C, but DMAPP isolated from those leaves was only 28% and 36% (13)C, respectively. The labeled DMAPP is likely to represent chloroplastic DMAPP contributing to isoprene emission. A substantial (13)C labeling was also found in both emission and DMAPP pool of low-emitting, young leaves of Phragmites. This confirms that low emission of young leaves is not caused by absence of chloroplastic DMAPP but rather by enzyme characteristics. A very low (13)C labeling was found in the DMAPP pool and in the residual isoprene emission of leaves previously fed with fosmidomycin to inhibit isoprene formation. This shows that fosmidomycin is a very effective inhibitor of the chloroplastic biosynthetic pathway of isoprene synthesis, that the residual isoprene is formed from extra-chloroplastic sources, and that chloroplastic and extrachloroplastic pathways are not cross-linked, at least following inhibition of the chloroplastic pathway. Refixation of unlabeled respiratory CO(2) in the light may explain incomplete labeling of isoprene emission, as we found a good association between these two parameters.

ABSTRACTIsoprene basal emission (the emission of isoprene from leaves exposed to a light intensity of 1000 µmol m−2 s−1 and maintained at a temperature of 30 °C) was measured in Phragmites australis plants growing under elevated CO2 in the Bossoleto CO2 spring at Rapolano Terme, Italy, and under ambient CO2 at a nearby control site. Gas exchange and biochemical measurements were concurrently taken. Isoprene emission was lower in the plants growing at elevated CO2 than in those growing at ambient CO2. Isoprene emission and isoprene synthase activity (IsoS) were very low in plants growing at the bottom of the spring under very rich CO2 and increased at increasing distance from the spring (and decreasing CO2 concentration). Distance from the spring did not significantly affect photosynthesis making it therefore unlikely that there is carbon limitation to isoprene formation. The isoprene emission rate was very quickly reduced after rapid switches from elevated to ambient CO2 in the gas‐exchange cuvette, whereas it increased when switching from ambient to elevated CO2. The rapidity of the response may be consistent with post‐translational modifications of enzymes in the biosynthetic pathway of isoprene formation. Reduction of IsoS activity is interpreted as a long‐term response. Basal emission of isoprene was not constant over the day but showed a diurnal course opposite to photosynthesis, with a peak during the hottest hours of the day, independent of stomatal conductance and probably dependent on external air temperature or temporary reduction of CO2 concentration. The present experiments show that basal emission rate of isoprene is likely to be reduced under future elevated CO2 levels and allow improvement in the modelling of future isoprene emission rates.

Isoprene is emitted by a variety of plant species, but its role remains elusive. Several experiments indicated that isoprene may have a protective role under stressful conditions. We investigated if the inhibition of endogenous isoprene synthesized by Phragmites australis leaves provokes oxidative stress, i.e. induces the accumulation of reactive oxygen species and products of membrane degradation, and reduces the physiological performances of leaves exposed to elevated temperatures. By increasing temperature from 30 to 48°C, photosynthesis decreased both in isoprene-emitting leaves and in leaves in which isoprene was inhibited by feeding 20μM fosmidomycin. However, isoprene inhibition was incomplete and the residual emission showed the same temperature-dependence of the emission from isoprene-emitting leaves, peaking at 44°C. At this temperature a significantly higher electron transport rate was observed in isoprene-emitting leaves than in isoprene-inhibited leaves suggesting that isoprene may have facilitated electron flow through the photosynthetic/photorespiratory cycles. More H2O2 and malonyldialdehyde contents and higher catalase and peroxidase activities were observed in isoprene-inhibited than in isoprene-emitting leaves. These changes were less evident after exposure to the temperature ramp up to 48°C than in leaves maintained at 30°C. This suggests that isoprene, independent of the temperature stress, effectively reduces the accumulation of reactive oxygen species and protects membrane from denaturation.

The detection of 12CO2 emission from leaves in air containing 13CO2 allows simple and fast determination of the CO2 emitted by different sources, which are separated on the basis of their labelling velocity. This technique was exploited to investigate the controversial effect of CO2 concentration on mitochondrial respiration. The 12CO2 emission was measured in illuminated and darkened leaves of one C4 plant and three C3 plants maintained at low (30-50 ppm), atmospheric (350-400 ppm) and elevated (700-800 ppm) CO2 concentration. In C3 leaves, the 12CO2 emission in the light (Rd) was low at ambient CO2 and was further quenched in elevated CO2, when it was often only 20-30% of the 12CO2 emission in the dark, interpreted as the mitochondrial respiration in the dark (Rn). Rn was also reduced in elevated CO2. At low CO2, Rd was often 70-80% of Rn, and a burst of 12CO2 was observed on darkening leaves of Mentha sativa and Phragmites australis after exposure for 4 min to 13CO2 in the light. The burst was partially removed at low oxygen and was never observed in C4 leaves, suggesting that it may be caused by incomplete labelling of the photorespiratory pool at low CO2. This pool may be low in sclerophyllous leaves, as in Quercus ilex where no burst was observed. Rd was inversely associated with photosynthesis, suggesting that the Rd/Rn ratio reflects the refixation of respiratory CO2 by photosynthesizing leaves rather than the inhibition of mitochondrial respiration in the light, and that CO2 produced by mitochondrial respiration in the light is mostly emitted at low CO2, and mostly refixed at elevated CO2. In the leaves of the C4 species Zea mays, the 12CO2 emission in the light also remained low at low CO2, suggesting efficient CO2 refixation associated with sustained photosynthesis in non-photorespiratory conditions. However, Rn was inhibited in CO2-free air, and the velocity of 12CO2 emission after darkening was inversely associated with the CO2 concentration. The emission may be modulated by the presence of post-illumination CO2 uptake deriving from temporary imbalance between C3 and C4 metabolism. These experiments suggest that this uptake lasts longer at low CO2 and that the imbalance is persistent once it has been generated by exposure to low CO2.