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International audience; Ozone, one of the major atmospheric pollutants, alters tree growth, mainly by decreasing carbon assimilation and allocation to stems and roots. To date, the mechanisms of O-3 impact at the cellular level have been investigated mainly on young trees grown in controlled or semi-controlled conditions. In the context of climate change, it is necessary to introduce a valuable defence parameter in the models that currently predict O-3 impact on mature trees and the carbon sequestration capacity of forest ecosystems.Air pollution is an important factor that affects negatively forest ecosystems. Among oxidative air pollutants, ozone is considered as the most toxic in terms of impact on vegetation.This paper focuses on the negative impacts of ozone on trees in controlled conditions or in their natural environment. The current knowledge of the responses at cell level is presented and ways to improve their use for ozone risk assessment of forest stands are discussed.Information was collected from original papers or reviews, providing an overview of the research conducted over the last 60 years.The negative effects of ozone on carbon assimilation and tree biomass production were reviewed and discussed, with a focus on effects on cell processes implied in cell defence, including stomatal regulation, detoxification, signalling, and biosynthesis of wood compound.In the context of increasing significance of O-3 flux approach, this review intends to shed light into the black box of defence processes, which are playing a crucial part within the effective O-3 dose modelling. Today, it is recognized that tropospheric ozone inhibits tree growth and its role on the future carbon sink of the forest ecosystem is discussed along with the combination of other environmental factors like elevated temperature, water, and nitrogen supply, likely to be modified in the context of climate change.

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Ozone induces a stimulation of the phenyl- propanoid and lignin biosynthesis pathways in leaves but the response of wood, the main lignin-producing tissue, is not well documented. The purpose of this study was to compare the responses of phenylpropanoid and lignin pathways in leaves and stem wood by a simultaneous analysis of both organs. Young poplars (Populus tremula 9 alba) were subjected either to daylight ozone (200 nL L -1 during light period) or continuous ozone (200 nL L -1 during light and dark periods) in controlled chambers. The trees were tilted so as to limit the formation of tension wood to the upper side of the stem and that of opposite wood to the lower side. Continuous ozone fumigation induced more pronounced effects in leaves than daylight ozone. Tension wood and opposite wood displayed similar responses to ozone. Enzyme activities involved in phe- nylpropanoid and lignin biosynthesis increased in the leaves of ozone-treated poplars and decreased in the wood. All steps involved in phenylpropanoid and monolignol synthesis in leaves and stem wood, were also altered at the transcript level (except coniferyl aldehyde 5-hydroxylase in leaves) suggesting that the responses were tightly coordinated. The response occurred rapidly in the leaves and much later in the wood. Phenylpropanoid and lignin biosynthesis is probably first involved in a defensive role against ozone in the leaves, which would lead to consid- erable rerouting of the carbon skeletons. The later response of phenylpropanoid and lignin metabolism in wood seemed to result from readjustment to the reduced carbon supply.

International audience; Trees will have to cope with increasing levels of CO2 and ozone in the atmosphere. The purpose of this work was to assess whether the lignification process could be altered in the wood of poplars under elevated CO2 and/or ozone. Young poplars were exposed either to charcoal-filtered air (control), to elevated CO2 (800 μl l−1), to ozone (200 nl l−1) or to a combination of elevated CO2 and ozone in controlled chambers. Lignification was analysed at different levels: biosynthesis pathway activities (enzyme and transcript), lignin content, and capacity to incorporate new assimilates by using 13C labelling. Elevated CO2 and ozone had opposite effects on many parameters (growth, biomass, cambial activity, wood cell wall thickness) except on lignin content which was increased by elevated CO2 and/or ozone. However, this increased lignification was due to different response mechanisms. Under elevated CO2, carbon supply to the stem and effective lignin synthesis were enhanced, leading to increased lignin content, although there was a reduction in the level of some enzyme and transcript involved in the lignin pathway. Ozone treatment induced a reduction in carbon supply and effective lignin synthesis as well as transcripts from all steps of the lignin pathway and some corresponding enzyme activities. However, lignin content was increased under ozone probably due to variations in other major components of the cell wall. Both mechanisms seemed to coexist under combined treatment and resulted in a high increase in lignin content.

Lignins, major components of the vascular plant cell wall, provided the mechanical support that allowed the development of upright plants adapted to a terrestrial habitat. Their biosynthesis through the phenylpropanoid and monolignol pathways has been extensively studied and significant advances have recently been made in understanding the regulation of this process. Lignin deposition is also modified in response to both abiotic and biotic stresses. Here, we present an overview of lignin biosynthesis in response to various abiotic stresses: drought, salinity, heavy metals, wounding, low temperature, ozone, UV-B radiation, light, elevated CO 2 and nitrogen stress. Although the stimulation of the phenylpropanoid pathway is a common feature of stress response, the subsequent synthesis of lignin is only demonstrated in some cases. The roles of lignins in different phases of abiotic stress response are discussed as well as the regulation of their synthesis under stress.

Wood formation in trees is a dynamic process that is strongly affected by environmental factors. However, the impact of ozone on wood is poorly documented. The objective of this study was to assess the effects of ozone on wood formation by focusing on the two major wood components, cellulose and lignin, and analysing any anatomical modifications. Young hybrid poplars (Populus tremula3alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 nl l21). As upright poplars usually develop tension wood in a non-set pattern, the trees were bent in order to induce tension wood formation on the upper side of the stem and normal or opposite wood on the lower side. Biosynthesis of cellulose and lignin (enzymes and RNA levels), together with cambial growth, decreased in response to ozone exposure. The cellulose to lignin ratio was reduced, suggesting that cellulose biosynthesis was more affected than that of lignin. Tension wood was generally more altered than opposite wood, especially at the anatomical level. Tension wood may be more susceptible to reduced carbon allocation to the stems under ozone exposure. These results suggested a coordinated regulation of cellulose and lignin deposition to sustain mechanical strength under ozone. The modifications of the cellulose to lignin ratio and wood anatomy could allow the tree to maintain radial growth while minimizing carbon cost.

Cinnamyl alcohol dehydrogenase (CAD) is an enzyme involved in lignin biosynthesis. We have previously isolated pure CAD enzyme from Norway spruce (Picea abies L.) cell culture. Here we report on partial protein sequences of the 42 kDa CAD polypeptide. A cDNA encoding CAD was isolated from the spruce cell culture. The open reading frame of a full-length cDNA coded for a 357 amino acid polypeptide with a calculated M(r) of 38,777 Da. The identity of the deduced polypeptide was verified by comparison with amino acid sequences of tryptic peptides from the purified enzyme. Southern blot analysis showed the presence of only one gene for CAD. Sequence comparison with CAD from tobacco and with a N-terminal protein sequence from loblolly pine CAD showed an identity of 69.7% and 91.5%, respectively. Treatment of spruce cell cultures with elicitor, as well as of seedlings with ozone both markedly increased the CAD mRNA level.

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.

Effects of ozone and/or drought stresses on phosphoenolpyruvate carboxylase (PEPc, EC 4.1.1.31) regulation in Pinus halepensis Mill. needles were assessed over 3 months in controlled conditions. Whereas moderated water stress applied to Aleppo pine had no effect on PEPc activity compared to the control, which was probably related to the high tolerance of this species to drought, ozone stress induced a dramatic increase of PEPc activity in pine needles. This stimulation of the anaplerotic pathway could provide substrates to repair processes, well known for being enhanced upon ozone exposure. The ozone-increased PEPc activity was related, to a certain extent, to an increase in protein and mRNA levels. The possible role of the stimulation of the phosphorylation status of the enzyme in the increased PEPc activity under ozone was also investigated. Following the demonstration of the existence of the phosphorylation site at the N terminal part of Aleppo pine PEPc, it was shown that, under ozone treatment, the light/dark PEPc activity ratio and the Ki (malate) for PEPc were increased. This strengthens the hypothesis of an ozone-related post-translational process, which could be part of an adaptation of the plants to prolonged stress. When ozone and water stress were applied in combination, the enhancement in PEPc activity was only related to changes in gene expression. This difference in PEPc regulation, compared to the effect of single stress, could be the consequence of the specific action of each stress on the enzyme. This study brings new insights into the regulation of PEPc in a C3 plant, Aleppo pine under these stresses. A different regulatory mechanism of PEPc is occurring according to the stress. The physiological implications of the increase in PEPc activity in response to ozone and/or water stress are discussed.

Through a 5-d exposure at 14 degrees C/8 degrees C (day/night), soybean (Glycine max [L.] Merr.) was acclimated to a lower temperature of 8 degrees C. In order to assess changes in protein synthesis related to chilling acclimation, proteins were labeled in vivo with [35S]methionine, separated by two-dimensional gel electrophoresis, and the derived autoradiograms were subjected to computer analysis. Two sets of chilling-acclimation-related proteins were characterized following exposure and labeling at 8 degrees C. One set corresponded to proteins whose synthesis was stimulated in acclimated plants in comparison with non-acclimated plants after transfer to 8 degrees C for 2 d. The other set also displayed an enhanced synthesis in the acclimated plants versus the non-acclimated plants but after 7 d of exposure at 8 degrees C. Most of these chilling-acclimation-related proteins were not increased during the acclimation period at 14 degrees C. Using microsequence analysis, one of these proteins was shown to have a high sequence homology with members of the heat-shock protein (HSP 70) family.

The diversity of the reactions of plants to low temperatures is illustrated by the classical distinction between chilling-sensitive and freezing-sensitive species. In an attempt to improve our understanding of the cellular and molecular basis underlying this diversity, we have investigated the fluidity of specific membranes in relation to cold resistance in Eucalyptus gunnii, a freezing-sensitive tree, and the changes in protein synthesis induced by cold acclimation in soybean (Glycine max), a chilling-sensitive species. We have been able to obtain isolated vacuoles and to purify, by free-flow electrophoresis, specific membranes (tonoplast and plasmalemma) from two cell lines of Eucalyptus gunnii which exhibit differential frost tolerance. The lateral and rotational mobilities of lipids in these different systems were studied by two biophysical techniques: fluorescence recovery after photobleaching (FRAP) and fluorescence polarisation. Our results show, for the first time, that intrinsically the tonoplast exhibits a higher fluidity than the plasma membrane. In addition, the membranes from the frost tolerant line or from acclimated lines were always more fluid than those from the sensitive line. These data strongly suggest a correlation between membrane fluidity and freezing tolerance in Eucalyptus. Through a progressive exposure to low temperatures, soybean plants were acclimated to a temperature of 8°C. As is the case with freezing-sensitive plants, acclimation improved the cold tolerance of chilling-sensitive plants. In order to assess changes in protein synthesis related to cold acclimation, proteins were labelled in vivo with 35S methionine, separated by two dimensional gel electrophoresis and the derived autoradiograms were subjected to computer analysis. The comparison of soluble proteins stimulated during acclimation and at low temperatures during the post-acclimation phase revealed that no new polypeptides were synthesised. The effects of acclimation on protein synthesis are essentially quantitative. Of the soluble proteins, the synthesis of a polypeptide homologous to the heat shock protein 70 family is stimulated. This polypeptide could protect soybean proteins from denaturation and aggregation at low, non-freezing temperatures.

Highly purified tonoplast and plasmamembrane vesicles were isolated from microsomes of Catharanthus roseus (L.) G. Don. by preparative free-flow electrophoresis. The relative amounts of tonoplast and plasma-membrane vesicles in the total microsomes varied with the pH of the grinding medium. The most electronegative fractions were identified as tonoplast using nitrate-inhibited, azide-resistant Mg(2+)-ATPase and pyrophosphatase activities as enzyme markers. The least electronegative fractions were identified as plasma membrane using glucan-synthase-II and UDPG:sterolglucosyl-transferase activities as enzyme markers. Other membrane markers, latent inosine-5'-diphosphatase (Golgi), NADPH-cytochrome-c reductase (ER) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane and did not contaminate either the tonoplast or the plasma-membrane fractions. In the course of searching for a reliable marker for tonoplast, the pyrophosphatase activity was found to be essentially associated with the tonoplast fractions purified by free-flow electrophoresis from C. roseus and other plant materials. The degree of sealing of the tonoplast and plasmamembrane vesicles was probed by their ability to pump protons (measurements of quinacrine quenching) and to generate a membrane potential (absorption spectroscopy of Oxonol VI). A critical evaluation of vesicles sidedness is presented.