Pierre Dizengremel

Effects of ozone on the carbon metabolism of forest trees

Chronic long-term exposure to near-ambient concentrations of ozone could contribute to forest decline in several regions of the world, in combination with other biotic and abiotic factors. It is generally admitted that, under ozone stress, biochemical events occur before any development of visible symptoms of damage. Photosynthesis is impaired whereas respiration is increased. The activity and quantity of Rubisco and Rubisco activase are diminished as well as the transcription of the proteins. Concomitantly, there is a general increase in the functioning of the catabolic pathways (glycolysis, pentose phosphate pathway). The mitochondrial respiration is also activated with an increased transcription of the alternative oxidase. The most impressive event is the huge increase in activity of phosphoenolpyruvate carboxylase linked to a stimulation of the enzyme biosynthesis. Therefore, the high ratio between the two carboxylases, which reaches about 25 in ozone-free air, falls to about 2 under ozone fumigation. There is also an increase in the detoxifying processes (chloroplastic superoxidase isoform). All these changes in cellular metabolism are directed towards repair and maintenance of the cell structure. In this respect, a general increase in the phenylpropanoid metabolism is also observed with the production of more phenolic compounds and a stimulation of the lignin biosynthetic pathway through the activation of several enzymes (phenylalanine ammonia lyase, cinnamyl alcohol dehydrogenase, etc.). The mechanism of ozone action however still remains to be elucidated. Ozone causes an oxidative stress producing reactive oxygen species, which are the probable source for signal chains with messenger molecules such as jasmonic acid, salicylic acid and ethylene. The problem remains of the existence of a specific series of events starting from ozone penetrating through the stomata to the repression/stimulation of gene transcription in foliar cells.

Condensed Lignins Are Synthesized in Poplar Leaves Exposed to Ozone

Poplar (Populus tremula × alba) trees (clone INRA 717-1-B4) were cultivated for 1 month in phytotronic chambers with two different levels of ozone (60 and 120 nL L–1). Foliar activities of shikimate dehydrogenase (EC 1.1.1.25), phenylalanine ammonia lyase (EC 4.3.1.5), and cinnamyl alcohol dehydrogenase (CAD, EC 1.1.1.195) were compared with control levels. In addition, we examined lignin content and structure in control and ozone-fumigated leaves. Under ozone exposure, CAD activity and CAD RNA levels were found to be rapidly and strongly increased whatever the foliar developmental stage. In contrast, shikimate dehydrogenase and phenylalanine ammonia lyase activities were increased in old and midaged leaves but not in the youngest ones. The increased activities of these enzymes involved in the late or early steps of the metabolic pathway leading to lignins were associated with a higher Klason lignin content in extract-free leaves. In addition, stress lignins synthesized in response to ozone displayed a distinct structure, relative to constitutive lignins. They were found substantially enriched in carbon-carbon interunit bonds and in p-hydroxyphenylpropane units, which is reminiscent of lignins formed at early developmental stages, in compression wood, or in response to fungal elicitor. The highest changes in lignification and in enzyme activities were obtained with the highest ozone dose (120 nL L–1). These results suggest that ozone-induced lignins might contribute to the poplar tolerance to ozone because of their barrier or antioxidant effect toward reactive oxygen species.

Ozone risk assessment for plants: Central role of metabolism-dependent changes in reducing power

The combination of stomatal-dependent ozone flux and total ascorbate level is currently presented as a correct indicator for determining the degree of sensitivity of plants to ozone. However, the large changes in carbon metabolism could play a central role in the strategy of the foliar cells in response to chronic ozone exposure, participating in the supply of reducing power and carbon skeletons for repair and detoxification, and modifying the stomatal mode of functioning. To reinforce the accuracy of the definition of the threshold for ozone risk assessment, it is proposed to also consider the redox pool (NAD(P)H), the ratio between carboxylases and the water use efficiency as indicators of the differential ozone tolerance of plants.

Ozone‐induced changes in photosynthesis and photorespiration of hybrid poplar in relation to the developmental stage of the leaves

Young poplar trees (Populus tremula Michx. x Populus alba L. clone INRA 717-1B4) were subjected to 120 ppb of ozone for 35 days in phytotronic chambers. Treated trees displayed precocious leaf senescence and visible symptoms of injury (dark brown/black upper surface stippling) exclusively observed on fully expanded leaves. In these leaves, ozone reduced parameters related to photochemistry (Chl content and maximum rate of photosynthetic electron transport) and photosynthetic CO(2) fixation [net CO(2) assimilation, Rubisco (ribulose-1,5-bisphosphate carboxylase oxygenase) activity and maximum velocity of Rubisco for carboxylation]. In fully expanded leaves, the rate of photorespiration as estimated from Chl fluorescence was markedly impaired by the ozone treatment together with the activity of photorespiratory enzymes (Rubisco and glycolate oxidase). Immunoblot analysis revealed a decrease in the content of serine hydroxymethyltransferase in treated mature leaves, while the content of the H subunit of the glycine decarboxylase complex was not modified. Leaves in the early period of expansion were exempt from visible symptoms of injury and remained unaffected as regards all measured parameters. Leaves reaching full expansion under ozone exposure showed potential responses of protection (stimulation of mitochondrial respiration and transitory stomatal closure). Our data underline the major role of leaf phenology in ozone sensitivity of photosynthetic processes and reveal a marked ozone-induced inhibition of photorespiration.

The impact of atmospheric composition on plants: a case study of ozone and poplar.

Tropospheric ozone is the main atmospheric pollutant that causes damages to trees. The estimation of the threshold for ozone risk assessment depends on the evaluation of the means that this pollutant impacts the plant and, especially, the foliar organs. The available results show that, before any visible symptom appears, carbon assimilation and the underlying metabolic processes are decreased under chronic ozone exposure. By contrast, the catabolic pathways are enhanced, and contribute to the supply of sufficient reducing power necessary to feed the detoxification processes. Reactive oxygen species delivered during ozone exposure serve as toxic compounds and messengers for the signaling system. In this review, we show that the contribution of genomic tools (transcriptomics, proteomics, and metabolomics) for a better understanding of the mechanistic cellular responses to ozone largely relies on spectrometric measurements.

Carbon Metabolism Enzyme Activities and Carbon Partitioning in Pinus halepensis Mill, exposed to Mild Drought and Ozone

Summary Since several years, accelerated decline of Aleppo pine ( Pinus halepensis ) forests has been observed in mediterranean areas. In fact, the combination of various environmental factors (photochemical oxidants, drought, high light, ...) was suspected to cause this decline. In this study, three year-old Aleppo pines were exposed during 3 months to ozone fumigation (100 ppb) combined or not with mild drought to study the effects of these combined factors on some sequences linked to carbon partitioning and primary carbon metabolism within the tree. After a cumulative ozone exposure of 132ppm·h, ozone induced a significant decrease in specific activity of the whole-plant (−38%) combined with a disequilibrium of the carbon transfer between root and shoot in favour of the shoots (non significant). Moreover, while the same cumulative dose of ozone had no effect on total Rubisco activity in one year-old needles, mitochondfial NAD malic enzyme activity increased significantly (+32%). By combining ozone with mild drought, the ozone-induced responses of all the parameters were significantly amplified and Rubisco activity was significantly decreased (by 44%). These results allowed us to conclude that at 132ppm·h, ozone alone led to an increase in dark respiration. Moreover, by the combination of ozone and mild drought, a decrease carbon fixation capacity was associated to a decrease of the carbon transfered to the toots, leading to a reduced root growth. Thus, there are indications that high levels of ozone during the summer months may impair the ability of Pinus halepensis to withstand severe water stress in its natural environment.

Effect of Salt Stress on Growth and on the Detoxifying Pathway of Pedunculate Oak Seedlings (Quercus robur L.)

Summary The effect of NaCl salinity on young pedunculate oak trees was studied. Growth of the stem was affected after a 40 mM NaCl stress was applied. The leaf water potential was decreased from a 20 mM NaCl concentration in the nutrient solution. The Na + and Cl - contents were increased in plants submitted to salt stress, especially in the first flush leaves. Superoxide dismutase activity was increased under stress conditions whereas no change could be detected in the activities of ascorbate peroxidase and glutathione reductase. Moreover, the SOD isozyme pattern of oak leaves was modified. Taking these results into account, pedunculate oak could be classified as a highly salt sensitive species. However, modifications recorded in the SOD isozyme pattern suggested differences between organelles at the level of salt stress tolerance. In particular, mitochondria seemed to be less sensitive than chloroplasts, which were strongly affected.

Differential impact of chronic ozone exposure on expanding and fully expanded poplar leaves.

Populus tremula L. × Populus alba L. (Populus ×c anescens (Aiton) Smith) - clone INRA 717-1-B4 saplings (50 cm apex to base and carrying 19 leaves on average) - were followed for 28 days. Half of the trees were grown in charcoal-filtered air while the other half were exposed to 120 ppb ozone for 11 h a day during the light period. The expanding leaf number 4 was tagged at the beginning of the experiment and finished expansion between 7 and 14 days. These leaves were harvested weekly for biochemical and proteome analyses using quantitative bidimensional electrophoresis (DiGE). Independent of the ozone treatment, all the analyses allowed a distinction between expanding and adult leaves. The results indicate that during the expansion phase (Days 0-7) the enzymatic machinery of the leaves is set up, and remains dynamically stable in the adult leaves (Days 14-28). Although ozone had no apparent effect on expanding leaves, the metabolic stability in fully expanded leaves observed in ozone-free plants was disturbed after 2 weeks of exposure and a stress-induced response became apparent.

Elevated C02 Does not Provide Protection against Ozone Considering the Activity of Several Antioxidant Enzymes in the Leaves of Sugar Maple

Summary Seedlings of sugar maple ( Acer saccharum Marsch.) were exposed for 46 days to 700 ppm of C0 2 , 200 ppb of ozone, and 700 ppm of C0 2 + 200 ppb of ozone. A significant increase in the activity of H 2 0 2 scavenging enzymes, i.e. ascorbate peroxidase [EC 1.11.1.11] and catalase [EC 1.11.1.6], was measured due to the action of 0 3 . This increase was rather negatively affected by elevated C0 2 . A tendency of decreased activity of glutathione reductase [EC 1.6.4.2] and superoxide dismutase [EC 1.15.1.1] due to the action of 0 3 was detected. Elevated C0 2 does not provide enhanced tolerance to oxidative stress in the seedlings of sugar maple. Changes in the activity of antioxidant enzymes were more pronounced in the young leaves (developed during the experiment) than in the old leaves (developed before starting the experiment). Stimulation of chloroplastic FeSOD by elevated C0 2 was observed, indicating oxidative stress in chloroplasts evoked by elevated C0 2 level. This effect did not result in enhanced protection against the detrimental effect of ozone, most probably due to compartmentation of C0 2 and 0 3 effects within the cell.

The alternative respiratory pathway allows sink to cope with changes in carbon availability in the sink-limited plant Erythronium americanum

Mechanisms that allow plants to cope with a recurrent surplus of carbon in conditions of imbalance between source and sink activity has not received much attention. The response of sink growth and metabolism to the modulation of source activity was investigated using elevated CO(2) and elevated O(3) growth conditions in Erythronium americanum. Sink activity was monitored via slice and mitochondrial respiratory rates, sucrose hydrolysis activity, carbohydrates, and biomass accumulation throughout the growth season, while source activity was monitored via gas exchanges, rubisco and phosphoenolpyruvate carboxylase activities, carbohydrates, and respiratory rates. Elevated CO(2) increased the net photosynthetic rate by increasing substrate availability for rubisco. Elevated O(3) decreased the net photosynthetic rate mainly through a reduction in rubisco activity. Despite this modulation of the source activity, neither plant growth nor starch accumulation were affected by the treatments. Sucrose synthase activity was higher in the sink under elevated CO(2) and lower under elevated O(3), thereby modulating the pool of glycolytic intermediates. The alternative respiratory pathway was similarly modulated in the sink, as seen with both the activity and capacity of the pathway, as well as with the alternative oxidase abundance. In this sink-limited species, the alternative respiratory pathway appears to balance carbon availability with sink capacity, thereby avoiding early feedback-inhibition of photosynthesis in conditions of excess carbon availability.