Ante- and post-mortem cellular injury dynamics in hybrid poplar foliage as a function of phytotoxic O3 dose.

After reaching phytotoxic levels during the last century, tropospheric ozone (O3) pollution is likely to remain a major concern in the coming decades. Despite similar injury processes, there is astounding interspecific-and sometimes intraspecific-foliar symptom variability, which may be related to spatial and temporal variation in injury dynamics. After characterizing the dynamics of physiological responses and O3 injury in the foliage of hybrid poplar in an earlier study, here we investigated the dynamics of changes in the cell structure occurring in the mesophyll as a function of O3 treatment, time, phytotoxic O3 dose (POD0), leaf developmental stage, and mesophyll layer. While the number of Hypersensitive Response-like (HR-like) lesions increased with higher O3 concentrations and POD0, especially in older leaves, most structural HR-like markers developed after cell death, independent of the experimental factors. The pace of degenerative Accelerated Cell Senescence (ACS) responses depended closely on the O3 concentration and POD0, in interaction with leaf age. Changes in total chlorophyll content, plastoglobuli and chloroplast shape pointed to thylakoid membranes in chloroplasts as being especially sensitive to O3 stress. Hence, our study demonstrates that early HR-like markers can provide reasonably specific, sensitive and reliable quantitative structural estimates of O3 stress for e.g. risk assessment studies, especially if they are associated with degenerative and thylakoid-related injury in chloroplasts from mesophyll.

The response to daylight or continuous ozone of phenylpropanoid and lignin biosynthesis pathways in poplar differs between leaves and wood.

Ozone induces a stimulation of the phenylpropanoid 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×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 phenylpropanoid 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 considerable 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.

Elevated CO2 and/or ozone modify lignification in the wood of poplars (Populus tremula x alba).

Trees will have to cope with increasing levels of CO(2) 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 CO(2) and/or ozone. Young poplars were exposed either to charcoal-filtered air (control), to elevated CO(2) (800 µl l(-1)), to ozone (200 nl l(-1)) or to a combination of elevated CO(2) 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 (13)C labelling. Elevated CO(2) 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 CO(2) and/or ozone. However, this increased lignification was due to different response mechanisms. Under elevated CO(2), 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.

Cellulose and lignin biosynthesis is altered by ozone in wood of hybrid poplar (Populus tremula × alba).

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 tremula × alba) were cultivated under different ozone concentrations (50, 100, 200, and 300 l l(-1)). 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.

Dynamics of Foliar Responses to O3 Stress as a Function of Phytotoxic O3 Dose in Hybrid Poplar.

With background concentrations having reached phytotoxic levels during the last century, tropospheric ozone (O3) has become a key climate change agent, counteracting carbon sequestration by forest ecosystems. One of the main knowledge gaps for implementing the recent O3 flux-based critical levels (CLs) concerns the assessment of effective O3 dose leading to adverse effects in plants. In this study, we investigate the dynamics of physiological, structural, and morphological responses induced by two levels of O3 exposure (80 and 100 ppb) in the foliage of hybrid poplar, as a function of phytotoxic O3 dose (POD0) and foliar developmental stage. After a latency period driven by foliar ontological development, the gas exchanges and chlorophyll content decreased with higher POD0 monotonically. Hypersensitive response-like lesions appeared early during exposure and showed sigmoidal-like dynamics, varying according to leaf age. At current POD1_SPEC CL, notwithstanding the aforementioned reactions and initial visible injury to foliage, the treated poplars had still not shown any growth or biomass reduction. Hence, this study demonstrates the development of a complex syndrome of early reactions below the flux-based CL, with response dynamics closely determined by the foliar ontological stage and environmental conditions. General agreement with patterns observed in the field appears indicative of early O3 impacts on processes relevant, e.g., biodiversity ecosystem services before those of economic significance - i.e., wood production, as targeted by flux-based CL.

Regulation of phosphoenolpyruvate carboxylase in Pinus halepensis needles submitted to ozone and water stress.

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.

Condensed lignins are synthesized in poplar leaves exposed to ozone.

Poplar (Populus tremula x 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.