Integrative role of plant mitochondria facing oxidative stress: The case of ozone.

Ozone is a secondary air pollutant, which causes oxidative stress in plants by producing reactive oxygen species (ROS) starting by an external attack of leaf apoplast. ROS have a dual role, acting as signaling molecules, regulating different physiological processes and response to stress, but also inducing oxidative damage. The production of ROS in plant cells is compartmented and regulated by scavengers and specific enzyme pathways. Chronic doses of ozone are known to trigger an important increase of the respiratory process while decreasing photosynthesis. Mitochondria, which normally operate with usual levels of intracellular ROS, would have to play a prominent role to cope with an enhanced ozone-derived ROS production. It is thus needed to compile the available literature on the effects of ozone on mitochondria to precise their strategy facing oxidative stress. An overview of the mitochondrial fate in three steps is proposed, i) starting with the initial responses of the mitochondria for alleviating the overproduction of ROS by the enhancement of existing antioxidant metabolism and adjustments of the electron transport chain, ii) followed by the setting up of detoxifying processes through exchanges between mitochondria and the cell, and iii) ending by an accelerated senescence initiated by mitochondrial membrane permeability and leading to programmed cell death.

Ozone exposure and flux-based response functions for photosynthetic traits in wheat, maize and poplar.

Ozone exposure- and dose-response relationships based on photosynthetic leaf traits (CO2 assimilation, chlorophyll content, Rubisco and PEPc activities) were established for wheat, maize and poplar plants grown in identical controlled conditions, providing a comparison between crop and tree species, as well as between C3 and C4 plants. Intra-specific variability was addressed by comparing two wheat cultivars with contrasting ozone tolerance. Depending on plant models and ozone levels, first-order, second-order and segmented linear regression models were used to derive ozone response functions. Overall, flux-based functions appeared superior to exposure-based functions in describing the data, but the improvement remained modest. The best fit was obtained using the POD0.5 for maize and POD3 for poplar. The POD6 appeared relevant for wheat, although intervarietal differences were found. Our results suggest that taking into account the dynamics of leaf antioxidant capacity could improve current methods for ozone risk assessment for plants.

Ozone affects ascorbate and glutathione biosynthesis as well as amino acid contents in three Euramerican poplar genotypes.

Ozone is an air pollutant that causes oxidative stress by generation of reactive oxygen species (ROS) within the leaf. The capacity to detoxify ROS and repair ROS-induced damage may contribute to ozone tolerance. Ascorbate and glutathione are known to be key players in detoxification. Ozone effects on their biosynthesis and on amino acid metabolism were investigated in three Euramerican poplar genotypes (Populus deltoides Bartr. × Populus nigra L.) differing in ozone sensitivity. Total ascorbate and glutathione contents were increased in response to ozone in all genotypes, with the most resistant genotype (Carpaccio) showing an increase of up to 70%. Reduced ascorbate (ASA) concentration at least doubled in the two most resistant genotypes (Carpaccio and Cima), whereas the most sensitive genotype (Robusta) seemed unable to regenerate ASA from oxidized ascorbate (DHA), leading to an increase of 80% of the oxidized form. Increased ascorbate (ASA + DHA) content correlated with the increase in gene expression in its biosynthetic pathway, especially the putative gene of GDP-l-galactose phosphorylase VTC2. Increased cysteine availability combined with increased expression of γ-glutamylcysteine synthetase (GSH1) and glutathione synthetase (GSH2) genes allows higher glutathione biosynthesis in response to ozone, particularly in Carpaccio. In addition, ozone caused a remobilization of amino acids with a decreased pool of total amino acids and an increase of Cys and putrescine, especially in Carpaccio. In addition, the expression of genes encoding threonine aldolase was strongly induced only in the most tolerant genotype, Carpaccio. Reduced ascorbate levels could partly explain the sensitivity to ozone for Robusta but not for Cima. Reduced ascorbate level alone is not sufficient to account for ozone tolerance in poplar, and it is necessary to consider several other factors including glutathione content.

Distinct responses to ozone of abaxial and adaxial stomata in three Euramerican poplar genotypes.

Ozone induces stomatal sluggishness, which impacts photosynthesis and transpiration. Stomatal responses to variation of environmental parameters are slowed and reduced by ozone and may be linked to difference of ozone sensitivity. Here we determine the ozone effects on stomatal conductance of each leaf surface. Potential causes of this sluggish movement, such as ultrastructural or ionic fluxes modification, were studied independently on both leaf surfaces of three Euramerican poplar genotypes differing in ozone sensitivity and in stomatal behaviour. The element contents in guard cells were linked to the gene expression of ion channels and transporters involved in stomatal movements, directly in microdissected stomata. In response to ozone, we found a decrease in the stomatal conductance of the leaf adaxial surface correlated with high calcium content in guard cells compared with a slight decrease on the abaxial surface. No ultrastructural modifications of stomata were shown except an increase in the number of mitochondria. The expression of vacuolar H(+) /Ca(2+) -antiports (CAX1 and CAX3 homologs), ß-carbonic anhydrases (ßCA1 and ßCA4) and proton H(+) -ATPase (AHA11) genes was strongly decreased under ozone treatment. The sensitive genotype characterized by constitutive slow stomatal response was also characterized by constitutive low expression of genes encoding vacuolar H(+) /Ca(2+) -antiports.

A physiological and proteomic study of poplar leaves during ozone exposure combined with mild drought.

The occurrence of high-ozone concentrations during drought episodes is common considering that they are partially caused by the same meteorological phenomena. It was suggested that mild drought could protect plants against ozone-induced damage by causing the closure of stomata and preventing the entry of ozone into the leaves. The present experiment attempts to create an overview of the changes in cellular processes in response to ozone, mild drought and a combined treatment based on the use of 2D-DiGE to compare the involved proteins, and a number of supporting analyses. Morphological symptoms were worst in the combined treatment, indicating a severe stress, but fewer proteins were differentially abundant in the combined treatment than for ozone alone. Stomatal conductance was slightly lowered in the combined treatment. Shifts in carbon metabolism indicated that the metabolism changed to accommodate for protective measures and changes in the abundance of proteins involved in redox protection indicated the presence of an oxidative stress. This study allowed identifying a set of proteins that changed similarly during ozone and drought stress, indicative of crosstalk in the molecular response of plants exposed to these stresses. The abundance of other key proteins changed only when the plants are exposed to specific conditions. Together this indicates the coexistence of generalized and specialized responses to different conditions.

Analysis of cytosolic isocitrate dehydrogenase and glutathione reductase 1 in photoperiod-influenced responses to ozone using Arabidopsis knockout mutants.

Oxidative stress caused by ozone (O3 ) affects plant development, but the roles of specific redox-homeostatic enzymes in O3 responses are still unclear. While growth day length may affect oxidative stress outcomes, the potential influence of day length context on equal-time exposures to O3 is not known. In Arabidopsis Col-0, day length affected the outcome of O3 exposure. In short-days (SD), few lesions were elicited by treatments that caused extensive lesions in long days (LD). Lesion formation was not associated with significant perturbation of glutathione, ascorbate, NADP(H) or NAD(H). To investigate roles of two genes potentially underpinning this redox stability, O3 responses of mutants for cytosolic NADP-isocitrate dehydrogenase (icdh) and glutathione reductase 1 (gr1) were analysed. Loss of ICDH function did not affect O3 -induced lesions, but slightly increased glutathione oxidation, induction of other cytosolic NADPH-producing enzymes and pathogenesis-related gene 1 (PR1). In gr1, O3 -triggered lesions, salicylic acid accumulation, and induction of PR1 were all decreased relative to Col-0 despite enhanced accumulation of glutathione. Thus, even at identical irradiance and equal-time exposures, day length strongly influences phenotypes triggered by oxidants of atmospheric origin, while in addition to its antioxidant function, the GR-glutathione system seems to play novel signalling roles during O3 exposure.

Effects of ozone on stomatal responses to environmental parameters (blue light, red light, CO2 and vapour pressure deficit) in three Populus deltoides × Populus nigra genotypes.

The effect of ozone (O(3)) on stomatal regulation was studied in three Euramerican poplar genotypes (Populus deltoides × Populus nigra: Carpaccio, Cima and Robusta). The impact of O(3) on stomatal conductance responses to variations in blue light, red light, CO(2) concentration and vapour pressure deficit (VPD) was studied. Upon O(3) exposure, a sluggish response of stomatal movements was observed, characterized by slower reactions to increases in blue light intensity, CO(2) concentration and VPD, and lower amplitude of the response to variations in light intensity. That sluggish response should be taken into account in stomatal conductance models for phytotoxic ozone dose (POD(Y)) calculations. The speed of the response to variations in environmental parameters appears as a determining factor of genotype-related sensitivity.

Capacity for NADPH regeneration in the leaves of two poplar genotypes differing in ozone sensitivity.

Cell capacity for cytosolic NADPH regeneration by NADP-dehydrogenases was investigated in the leaves of two hybrid poplar (Populus deltoides × Populus nigra) genotypes in response to ozone (O3 ) treatment (120 ppb for 17 days). Two genotypes with differential O3 sensitivity were selected, based on visual symptoms and fallen leaves: Robusta (sensitive) and Carpaccio (tolerant). The estimated O3 flux (POD0 ), that entered the leaves, was similar for the two genotypes throughout the treatment. In response to that foliar O3 flux, CO2 assimilation was inhibited to the same extent for the two genotypes, which could be explained by a decrease in Rubisco (EC 4.1.1.39) activity. Conversely, an increase in PEPC (EC 4.1.1.31) activity was observed, together with the activation of certain cytosolic NADP-dehydrogenases above their constitutive level, i.e. NADP-G6PDH (EC 1.1.1.49), NADP-ME (malic enzyme) (EC 1.1.1.40) and NADP-ICDH (NADP-isocitrate dehydrogenase) (EC1.1.1.42). However, the activity of non-phosphorylating NADP-GAPDH (EC 1.2.1.9) remained unchanged. From the 11th fumigation day, NADP-G6PDH and NADP-ME profiles made it possible to differentiate between the two genotypes, with a higher activity in Carpaccio than in Robusta. At the same time, Carpaccio was able to maintain high levels of NADPH in the cells, while NADPH levels decreased in Robusta O3 -treated leaves. All these results support the hypothesis that the capacity for cells to regenerate the reducing power, especially the cytosolic NADPH pool, contributes to improve tolerance to high ozone exposure.

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.

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.

A difference gel electrophoresis study on thylakoids isolated from poplar leaves reveals a negative impact of ozone exposure on membrane proteins.

Populus tremula L. x P. alba L. (Populus x canescens (Aiton) Smith), clone INRA 717-1-B4, saplings were subjected to 120 ppb ozone exposure for 28 days. Chloroplasts were isolated, and the membrane proteins, solubilized using the detergent 1,2-diheptanoyl-sn-glycero-3-phosphocholine (DHPC), were analyzed in a difference gel electrophoresis (DiGE) experiment comparing control versus ozone-exposed plants. Extrinsic photosystem (PS) proteins and adenosine triphosphatase (ATPase) subunits were detected to vary in abundance. The general trend was a decrease in abundance, except for ferredoxin-NADP(+) oxidoreductase (FNR), which increased after the first 7 days of exposure. The up-regulation of FNR would increase NAPDH production for reducing power and detoxification inside and outside of the chloroplast. Later on, FNR and a number of PS and ATPase subunits decrease in abundance. This could be the result of oxidative processes on chloroplast proteins but could also be a way to down-regulate photochemical reactions in response to an inhibition in Calvin cycle activity.

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.

Source-sink imbalance increases with growth temperature in the spring geophyte Erythronium americanum.

Spring geophytes produce larger storage organs and present delayed leaf senescence under lower growth temperature. Bulb and leaf carbon metabolism were investigated in Erythronium americanum to identify some of the mechanisms that permit this improved growth at low temperature. Plants were grown under three day/night temperature regimes: 18/14 °C, 12/8 °C, and 8/6 °C. Starch accumulated more slowly in the bulb at lower temperatures probably due to the combination of lower net photosynthetic rate and activation of a 'futile cycle' of sucrose synthesis and degradation. Furthermore, bulb cell maturation was delayed at lower temperatures, potentially due to the delayed activation of sucrose synthase leading to a greater sink capacity. Faster starch accumulation and the smaller sink capacity that developed at higher temperatures led to early starch saturation of the bulb. Thereafter, soluble sugars started to accumulate in both leaf and bulb, most probably inducing decreases in fructose-1,6-bisphosphatase activity, triose-phosphate utilization in the leaf, and the induction of leaf senescence. Longer leaf life span and larger bulbs at lower temperature appear to be due to an improved equilibrium between carbon fixation capacity and sink strength, thereby allowing the plant to sustain growth for a longer period of time before feedback inhibition induces leaf senescence.

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.

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.

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.

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.

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.

Foliar CO2 fixation in bean (Phaseolus vulgaris L.) submitted to elevated ozone: distinct changes in Rubisco and PEPc activities in relation to pigment content.

Using open-top chambers, the impact of ozone (O(3)) on foliar carboxylases of bean (Phaseolus vulgaris L.) was investigated. From sowing, beans were exposed to non-filtered air (NF) and NF supplied with 40 (+40) and 80 (+80) nL L(-1) O(3). Twenty days after emergence, primary and first trifoliate leaves were sampled. Biochemical characteristics of leaves from +40 were quite similar to those from NF. Conversely, +80 induced distinct biochemical effects in primary and first trifoliate leaves. Regarding primary leaves, +80 reduced ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activity by 33% whereas it stimulated phosphoenolpyruvate carboxylase (PEPc) activity by 376%. The reduction in Rubisco activity was accompanied by a decrease in both Rubisco subunit amounts and a consistent oxidative modification of the Rubisco small subunit (SSU). These changes came with a drastic loss in pigmentation. Regarding first trifoliate leaves, +80 stimulated Rubisco activity by 33% while it disturbed neither PEPc activity nor pigmentation. Surprisingly, the enhanced Rubisco activity was associated with a slight decrease in Rubisco protein quantity, which was not coupled with the formation of carbonyl groups in Rubisco-SSU.