Réponse du peuplier soumis à la combinaison simultanée de contraintes ozone et sécheresse

 Dans des conditions naturelles, les épisodes de sécheresse et d’augmentation des concentrations d’O3 troposphérique peuvent être concomitants. Cette combinaison de contraintes nécessite une attention particulière. En effet, en réduisant l’ouverture des stomates, la sécheresse peut limiter l’entrée de l’O3 dans les feuilles. Cependant, en favorisant la fermeture des stomates, le déficit hydrique du sol limite également les apports de CO2 nécessaire à la photosynthèse et augmente le risque de stress oxydant. La simultanéité des deux stress pourrait ainsi avoir un effet négatif (additif ou synergique) pour la plante ou bien un effet antagoniste dû à une tolérance croisée (Tausz et al., 2007). L’objectif de ce chapitre est de déterminer la réponse du peuplier en condition de combinaison simultanée de contraintes, à travers l’étude des similitudes et des différences existantes au sein même des mécanismes de réponse mis en place au niveau de la régulation stomatique, de la signalisation et de la détoxication cellulaire pour faire face au déficit hydrique, à l’O3 ou à une combinaison des deux stress. Pour apporter des éléments de réponse à cette question, le chapitre sera divisé en trois parties. La première partie correspond à une étude publiée impliquant les deux génotypes de peupliers (Carpaccio et Robusta) soumis à différents traitements pendant 17 jours : – un déficit hydrique modéré et progressif de 60 % à 30 % de REW – une exposition à 120 ppb d’O3 – la combinaison des deux traitements précédents Les objectifs sont i) d’identifier si la sécheresse modifie la réponse du peuplier due à l’O3 et vice et versa, et ii) de savoir s’il existe une différence de réponse entre les 2 génotypes. Pour répondre à ces interrogations, les réponses antioxydantes du peuplier seront étudiées à travers le cycle ascorbate-glutathion (teneurs en ascorbate et glutathion, activités enzymatiques et expression des isoformes des principales enzymes permettant leur régénération). La deuxième partie apportera des éléments de réflexion supplémentaires concernant le dosage des pools d’ascorbate et glutathion, grâce au développement, pendant la thèse et à l’issue d’une collaboration internationale, du dosage de l’AsA et du GSH par HPLC. Cette partie sera donc une digression utile pour comprendre l’effet de la combinaison de contraintes sur les capacités de détoxication des arbres. En effet, l’étude de trois espèces de chênes soumis à l’O3 et/ou à la sécheresse sur un dispositif O3-FACE (Free-air concentration enrichment) apporte des éléments supplémentaires permettant de mieux comprendre les mécanismes de détoxication mis en place. Chapitre IV : Réponse du peuplier soumis à la combinaison simultanée de contraintes ozone et sécheresse 87 Pour finir, la troisième partie présentera une prospection des voies de signalisation hormonale mises en place par le génotype Robusta, soumis à différents traitements pendant 11 jours : – un déficit hydrique modérée à 45 % de REW – une exposition à 120 ppb d’O3 – la combinaison des deux traitements précédents Les objectifs sont i) d’explorer la réponse hormonale sous O3 et déficit hydrique seuls et ii) de déterminer s’il existe une différence de réponse en combinaison de stress. Pour répondre à ces questions, un profilage hormonal a été réalisé en collaboration avec le Laboratory of Hormonal Regulations in Plants, Prague, République tchèque.

Analyse intégrative de la détoxication de 2 génotypes de peuplier soumis à l’ozone et la sécheresse: Focus sur le cycle ascorbateglutathione Les résultats ont été publiés dans la revue Science of The Total Environnement : Dusart N., Gérard J., Le Thiec D., Collignon C., Jolivet Y., Vaultier M.-N., 2019. Integrated analysis of the detoxification responses of two Euramerican poplar genotypes exposed to ozone and water deficit: Focus on the ascorbate-glutathione cycle. https://doi.org/10.1016/j.scitotenv.2018.09.367 Abstract Ozone (O3) and drought increase tree oxidative stress. To protect forest health, we need to improve risk assessment, using metric model such as the phytotoxic O3 dose above a threshold of y nmol.m-2.s-1 (PODy), while taking into account detoxification mechanisms and interacting stresses. The impact of drought events on the effect of O3 pollution deserves special attention. Water deficit may decrease O3 entrance into the leaves by reducing stomatal opening; however, water deficit also induces changes in cell redox homeostasis. Besides, the behaviour of the cell antioxidative charge in case of stress combination (water deficit and O3) still remains poorly investigated. To decipher the response of detoxification mechanisms relatively to the HalliwellAsada-Foyer cycle (HAF), we exposed poplar saplings (Populus nigra x deltoides) composed of two genotypes (Carpaccio and Robusta), to various treatments for 17 days, i.e. i) mild water deficit, ii) 120 ppb O3, and iii) a combination of these two treatments. Ozone similarly impacted the growth of the two genotypes, with an important leaf loss. Water deficit decreased growth by almost one third as compared to the control plants. As for the combined treatment, water deficit protected the saplings from leaf ozone injury, but with an inhibitory effect on growth. The pool of total ascorbate was not modified by the different treatments, while the pool of total glutathione increased with POD0. We noticed a few differences between the two genotypes, particularly concerning the activity of monodehydroascorbate reductase and glutathione reductase relatively to POD0. The expression profiles of genes coding for the dehydroascorbate reductase and glutathione reductase isoforms differed, probably in link with the putative localisation of ROS production in response to water deficit and ozone, respectively. Our result would argue for a major role of MDHAR, GR and glutathione in the preservation of the redox status.

Introduction

 Global warming is expected to cause wet regions to become wetter and dry regions drier (Liu & Allan, 2013). The models including an increase in temperature and vapor pressure deficit (VPD) in the warm season in addition to lower rainfall in the cold season predict an increased probability of future drought events (Park Williams et al., 2013). Drought link with an increase of forest stand mortality was already reported, along with a greater risk of forest decline (Allen et al., 2010). Water deficit is known to reduced gas exchanges and plant biomass (Chaves et al., 2003; Monclus et al., 2006). Better predicting the impact of drought on forests requires further research on the molecular and physiological mechanisms underlying plant responses and tolerance (Chaves et al., 2003; Rengel et al., 2012). Moreover, trees have to deal with atmospheric pollution like ozone pollution. Tropospheric ozone (O3) is a phytotoxic air pollutant that can damage plant cells due to its strong oxidative power (Baier et al., 2005). Plants exposed to a large amount of O3 gas show macroscopic symptoms like chlorosis or necrosis (Vollenweider et al., 2003). Moreover, the photosynthetic assimilation rate and growth both decrease (Wittig et al., 2007), and as a result the net primary production of forests decreases too (Proietti et al., 2016; Subramanian et al., 2015). A geographical analysis of tropospheric ozone showed that the ozone monthly average concentration is currently stable (Oltmans et al., 2013) even though differences are visible on a local scale. From 2000 to 2014, tropospheric ozone concentration trends decrease in North America, stabilise in Europe and increase in Asia (Gaudel et al., 2018; Mills et al., 2018). In natural environments, it is well admitted that drought events are frequently linked to heat and low cloud cover in the summer time. These conditions are optimal for the photochemical reaction responsible for ground-level ozone production to be triggered. Therefore, it is obvious that trees will have to cope with more frequent episodes of combined drought and tropospheric ozone in the future. In this context, while plant responses to these constraints applied separately have been extensively studied, a general understanding of the effects of the combination of such constraints is still lacking (Matyssek et al., 2012). Stomatal closure can be expected under water deficit, hampering ozone entrance into the leaf, and resulting in a kind of “protective effect” of water deficit. This protective or antagonistic effect was observed in birch in control chamber (Pääkkönen et al., 1998), in Picea abies (Kronfußs et al., 1998), Fagus sylvatica (Dixon et al., 1998) and more recently in poplar (Gao et al., 2017). Besides, the biomass of Norway spruce submitted to water deficit decreased as a result of simultaneous ozone treatment (Dixon et al., 1998). A synergistic effect with a disturbance of stomatal closure occurred in similar conditions Chapitre IV : Réponse du peuplier soumis à la combinaison simultanée de contraintes ozone et sécheresse 90 of combined ozone and drought (Bohler et al., 2013; Pollastrini et al., 2014). Furthermore, combined drought and ozone could affect signalling pathways and impact crosstalks between physiological and metabolic adjustments, e.g. in Quercus sp. (Cotrozzi et al., 2017a, 2017b; Pellegrini et al., 2019). Combined stresses may have a worse impact on plants or a crosstolerance effect (Tausz et al., 2007). Most abiotic constraints result in oxidative stress for the cells. The formation of reactive oxygen species (ROS) could induce an uncontrolled oxidative rate exceeding the metabolic capacity of the plant (Foyer & Noctor, 2011; Noctor et al., 2014). At the leaf scale, ozone damage occurs when the instantaneous stomatal ozone uptake of leaves overwhelms the ability of the leaf to detoxify ozone. Cell homeostasis is disrupted, and the increase in ROS leads to cell death. Among abiotic stress factors, O3 and drought admittedly produce oxidative stress (Edreva, 2005; Iriti & Faoro, 2007). Plants have two defence levels for both constraints (Castagna & Ranieri, 2009). The first is avoidance via stomatal closure. Regarding drought, this action limits evapotranspiration and in turn water loss. As for O3, this strategy prevents the gas from entering the leaf. Nevertheless, in both cases it is necessary for the plant to keep letting CO2 in because it is essential for photosynthesis. The second level of defence gathers all the detoxification and cell repair processes. The present study focuses on glutathione (GSH, γ-L-glutamyl-Lcysteinyl-glycine), a major antioxidant. GSH cannot be dissociated from ascorbate (ASC, Lthreo-hexenon-1,4-lactone), the most important antioxidant in plants. Both compounds differ from other antioxidants for three reasons. Firstly, specific enzymes like ascorbate peroxidase or glutathione peroxidase couple them to the peroxide metabolism. Secondly, oxidised forms of glutathione and ascorbate are relatively stable. Thirdly, they can be recycled by enzymes using NADPH as a reductant. Foyer and Noctor (2011) considered these three properties to define the ascorbate-glutathione cycle as “the heart of the redox hub”. Even though ascorbate is considered as the major antioxidant in plant cells, its reaction with ROS in the apoplasm and the symplasm is not sufficient to explain the differences between poplar genotypes in response to O3 (Ranieri et al., 1999; Van Hove et al., 2001). Among fast-growing tree species, poplar has a substantial economic interest. Nevertheless, its culture requires large amounts of water, and it is relatively drought-sensitive (Monclus et al., 2006; Silim et al., 2009). As a plant model, poplar species have been intensively studied under ozone exposure. Different species or genotypes showed distinct responses to ozone regarding leaf damage, biomass loss, stomatal behaviour, detoxification, and carbon allocation (Hu et al., 2015; Jolivet et al., 2016; Renaut et al., 2009). Understanding poplar responses to Chapitre IV : Réponse du peuplier soumis à la combinaison simultanée de contraintes ozone et sécheresse 91 environmental factors is an important issue for future wood biomass production. Genotype selection will have to focus on genotypes combining an optimal water use with maximum biomass production (Isebrands & Richardson, 2013). The present article investigates the responses to ozone or/and water deficit of Carpaccio and Robusta, two genotypes of Euramerican poplar (Populus deltoides x Populus nigra). These two genotypes were previously selected by our team based on their different responses to ozone as regards the stomatal response and NADPH regeneration (Dghim et al., 2013; Dumont et al., 2013). Moreover, O3 had strong effects on gene expression, especially the genes responsible for glutathione biosynthesis, and on the GSH content in the two genotypes (Dumont et al., 2014b). Our specific goals were to determine whether water deficit could modify the ozone-induced physiological response of poplar and vice versa, and whether a difference in detoxification processes could explain different responses of each genotype. We would except that ozone and water deficit interaction could impact trees in three different ways (Bansal et al., 2013; Matyssek et al., 2006; Pääkkönen et al., 1998): i) an additive effect, corresponding to the expected theoretical addition of the effects of each single stress; ii) an antagonistic effect, corresponding to a lower impact of the additive one; iii) a synergistic effect, greater than the expected additive one. These effects could differ according to the parameters taken into account. To do that, we investigated the ascorbate and glutathione contents and the redox balance, with special focus on the three enzymes implied in ascorbate and glutathione regeneration, i.e. i) monodehydroascorbate reductase (MDHAR), ii) dehydroascorbate reductase (DHAR), and iii) glutathione reductase (GR). Changes in gene expression coding for DHAR and GR isoforms were also investigated. 

Materials and methods*

Plant material and exposure conditions 

Cuttings of two Euramerican poplar genotypes (Populus deltoides x Populus nigra: ‘Carpaccio’ and ‘Robusta’) were planted in five-liter pots filled with loam (N/P/K 14/16/18, 1.2kg m-3 , Gramoflor SPI Universel), fertilised by adding 5 g of slow-release nutritive granules (Nutricot T-100, N/P/K/MgO 13/13/13/2, Fertil, Boulogne-Billancourt, France). The experiment was first conducted for 3 weeks on Robusta and was later duplicated on Carpaccio. The plants were grown in growth chambers for 5 weeks at 75/85 % relative humidity (day/night) with a 14-hour light period (300 µmol.m-2.s-1 PPFD at mid-leaf height from 08:00 to 22:00) and a temperature * N.D.A : Cette partie étant incluse dans l’article en anglais, elle est redondante avec le Chapitre II :Matériels et méthodes, excepté pour le Tableau 5 qui apporte des éléments nouveaux sur les gènes étudiés. Chapitre IV : Réponse du peuplier soumis à la combinaison simultanée de contraintes ozone et sécheresse 92 of 23/20 °C. Sixty-four plants were randomly distributed in eight phytotron chambers with growth conditions identical to those in the growth chamber; half of them were set for O3 treatment (120 nmol.mol-1 for 13 hours). At the end of a 7-day long acclimation period, the 17- day-long O3 treatment started, while control saplings were exposed to charcoal-filtered air. O3 was produced from pure O2 with two ozone generators (OZ500; Fischer, Bonn, Germany and CMG3-3; Innovatec II, Rheinbach, Germany) and injected directly into the chambers one hour after the beginning of the photoperiod until its end. A set of analysers (O341M; Environment S.A., Paris, France) was used to monitor O3 concentrations. Half of the saplings were submitted to water deficit. Soil moisture was determined with a TDR probe (Trime-Fm, IMKO GmbH, Ettlingen, Germany) and related to pot weight. Poplars were watered every day at 15.00, after weight measurements. For the well-watered treatment, poplars were irrigated at 85 % of relative extractable water (REW). For the water deficit treatment, irrigation was set to 55% of REW the first week, and then at 35% of REW until the end of the experiment. The number of leaves, the diameter at the collar, and height were recorded on each individual twice a week until the end of the experiment. 

Gas exchange measurements and POD0 calculations

 Net CO2 assimilation (A) and stomatal conductance to water vapour (gw) were performed using a portable photosynthesis system (Li-6200, Li Cor, Lincoln, NE, USA) inside the phytotronic chamber (under phytotronic microclimate conditions). Measurements were carried out 3 times a week, one hour after light was switched on. Gas exchanges were measured on the first fully expanded leaves (the 10th leaf from the apex) at the beginning of the treatment. The air flow in the leaf chamber was set to 450 µmol.s-1. Measurements were made for 30 seconds after stabilisation. gw measurements allowed to estimate the Phytotoxic Ozone Dose above a threshold flux of 0 nmol.m-2.s-1 (POD0), according to Bagard et al. (2015). 

 Leaf sampling 

Fully expanded leaves were sampled on days 2, 11, and 17 after the beginning of fumigation. Two leaves located next to each other (the 9th and 10th leaves at the beginning of the treatment) were sampled per sapling and per chamber. Midribs and petioles were cut off from the foliar limb using a razor blade, and the leaf material was rapidly frozen in liquid nitrogen and immediately stored at -80°C. Leaves were ground to a fine powder with a mortar and pestle in liquid N2. The leaf powder was stored at -80°C until further analyses. 

Enzymatic activity assays

 Extraction, filtration, and protein determination

 Fifty mg of leaf powder extract were ground with 1.3 mL of cold extraction buffer containing 10% (w/w) of polyvinylpolypyrrolidone (PVPP), 100 mM Bicine–KOH (pH 7.5), 6% (w/w) of PVP 25, 5 mM MgCl2, 5 mM EGTA, 10% (v/v) of glycerol, and 0.28% (v/v) of a protease inhibitor mixture. Crude extracts were then centrifuged at 20,000 x g for 20 min at 4°C. One mL of each supernatant was filtered through a column (PD MiniTrap G25) equilibrated with 100 mM bicine–KOH buffer (pH 7.8) and containing 10% (v/v) of glycerol, 5 mM MgCl2, and 2 mM DTT. All the extraction steps were performed at 4°C. The filtered extracts were stored at −80°C until analysis. Total soluble proteins were determined using Bradford’s Coomassie Blue G250 method (1976), with bovine serum albumin as a standard. 

Dehydroascorbate Reductase (DHAR, EC 1.8.5.1) assay

 DHAR activity was measured as described by Hossain and Asada (1984), with slight modifications. The reaction was monitored in microplates in a final volume of 200 µL containing 100 mM HEPES-KOH buffer pH 7, 2.5 mM reduced glutathione (GSH), 1 mM EDTA, 0.2 mM DHA, and 5 µL of desalted extract. The reaction was monitored at 265 nm, at 25°C, and initiated by adding DHA. The non-enzymatic reaction of GSH was taken into account and the activity was corrected by multiplying the values by 0.98 (correction due to absorbance of GSSG at 265 nm) (Asada, 1984). Four technical replicates per biological sample were performed. DHAR activity was expressed as nkat of ascorbate produced.mg-1 protein. IV.2.2.4.c. Monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) assay MDHAR activity was measured as described by Hossain and Asada (1984), with slight modifications. The reaction was monitored in microplates in a final volume of 200 µL containing 50 mM HEPES-KOH buffer pH 7.6, 2.5 mM L-ascorbate (ASA), 0.2 mM NADPH, and 50 µL of filtrated extract. After incubation 10 min at 25°C, the reaction was initiated by adding 0.028 unit of ascorbate oxidase (EC 1.10.3.3), and the absorbance was monitored at 340 nm for 5 min. A correction was made to take into account NADPH oxidation in the absence of ASA oxidase. Three technical replicates per biological sample were performed. MDHAR activity was expressed as nkat of oxidised NADPH.mg-1 protein.