Integrated analysis of the detoxification responses of two Euramerican poplar genotypes exposed to ozone and water deficit: Focus on the ascorbate-glutathione cycle.

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 Halliwell-Asada-Foyer cycle (HAF), we exposed poplar saplings (Populus nigra × 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.

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.

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.

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.