Ectomycorrhizal symbiosis prepares its host locally and systemically for abiotic cue signaling.

Tree growth and survival are dependent on their ability to perceive signals, integrate them, and trigger timely and fitted molecular and growth responses. While ectomycorrhizal symbiosis is a predominant tree-microbe interaction in forest ecosystems, little is known about how and to what extent it helps trees cope with environmental changes. We hypothesized that the presence of Laccaria bicolor influences abiotic cue perception by Populus trichocarpa and the ensuing signaling cascade. We submitted ectomycorrhizal or non-ectomycorrhizal P. trichocarpa cuttings to short-term cessation of watering or ozone fumigation to focus on signaling networks before the onset of any physiological damage. Poplar gene expression, metabolite levels, and hormone levels were measured in several organs (roots, leaves, mycorrhizas) and integrated into networks. We discriminated the signal responses modified or maintained by ectomycorrhization. Ectomycorrhizas buffered hormonal changes in response to short-term environmental variations systemically prepared the root system for further fungal colonization and alleviated part of the root abscisic acid (ABA) signaling. The presence of ectomycorrhizas in the roots also modified the leaf multi-omics landscape and ozone responses, most likely through rewiring of the molecular drivers of photosynthesis and the calcium signaling pathway. In conclusion, P. trichocarpa-L. bicolor symbiosis results in a systemic remodeling of the host's signaling networks in response to abiotic changes. In addition, ectomycorrhizal, hormonal, metabolic, and transcriptomic blueprints are maintained in response to abiotic cues, suggesting that ectomycorrhizas are less responsive than non-mycorrhizal roots to abiotic challenges.

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.

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.

Altered stomatal dynamics of two Euramerican poplar genotypes submitted to successive ozone exposure and water deficit.

The impact of ozone (O3) pollution events on the plant drought response needs special attention because spring O3 episodes are often followed by summer drought. By causing stomatal sluggishness, O3 could affect the stomatal dynamic during a subsequent drought event. In this context, we studied the impact of O3 exposure and water deficit (in the presence or in the absence of O3 episode) on the stomatal closure/opening mechanisms relative to irradiance or vapour pressure deficit (VPD) variation. Two genotypes of Populus nigra x deltoides were exposed to various treatments for 21 days. Saplings were exposed to 80 ppb/day O3 for 13 days, and then to moderate drought for 7 days. The curves of the stomatal response to irradiance and VPD changes were determined after 13 days of O3 exposure, and after 21 days in the case of subsequent water deficit, and then fitted using a sigmoidal model. The main responses under O3 exposure were stomatal closure and sluggishness, but the two genotypes showed contrasting responses. During stomatal closure induced by a change in irradiance, closure was slower for both genotypes. Nonetheless, the genotypes differed in stomatal opening under light. Carpaccio stomata opened more slowly than control stomata, whereas Robusta stomata tended to open faster. These effects could be of particular interest, as stomatal impairment was still present after O3 exposure and could result from imperfect recovery. Under water deficit alone, we observed slower stomatal closure in response to VPD and irradiance, but faster stomatal opening in response to irradiance, more marked in Carpaccio. Under the combined treatment, most of the parameters showed antagonistic responses. Our results highlight that it is important to take genotype-specific responses and interactive stress cross-talk into account to improve the prediction of stomatal conductance in response to various environmental modifications.

Antioxidative responses of three oak species under ozone and water stress conditions.

Plants are frequently exposed to adverse environmental conditions such as drought and ozone (O3). Under these conditions, plants can survive due to their ability to adjust their metabolism. The aim of the present study was to compare the detoxification mechanisms of three oak species showing different O3 sensitivity and water use strategy. Two-year-old seedlings of Quercus ilex, Q. pubescens and Q. robur were grown under the combination of three levels of O3 (1.0, 1.2 and 1.4 times the ambient O3 concentration) and three levels of water availability (on average 100, 80 and 42% of field capacity i.e. well-watered, moderate drought and severe drought, respectively) in an O3 Free Air Controlled Exposure facility. Ozone and drought induced the accumulation of reactive oxygen species (ROS) and this phenomenon was species-specific. Sometimes, ROS accumulation was not associated with membrane injury suggesting that several antioxidative defence mechanisms inhibited or alleviated the oxidative damage. Both O3 and drought increased total carotenoids that were able to prevent the peroxidation action by free radicals in Q. ilex, as confirmed by unchanged malondialdehyde by-product values. The concomitant decrease of total flavonoids may be related to the consumption of these compounds by the cell to inhibit the accumulation of hydrogen peroxide. Unchanged total phenols confirmed that Q. ilex has a superior ability to counteract oxidative conditions. Similar responses were found in Q. pubescens, although the negative impact of both factors was less efficiently faced than in the sympatric Q. ilex. In Q. robur, high O3 concentrations and severe drought induced a partial rearrangement of the phenylpropanoid pathways. These antioxidative mechanisms were not able to protect the cell structure (as confirmed by ROS accumulation) suggesting that Q. robur showed a lower degree of tolerance than the other two species.

The hydrophobic segment of Arabidopsis thaliana cluster I diacylglycerol kinases is sufficient to target the proteins to cell membranes.

Diacylglycerol kinases (DGKs) catalyze the phosphorylation of diacylglycerol into phosphatidic acid. To fulfill their role in many signalling processes, DGKs must be located at, or in, membranes. Most mammalian DGKs are cytosolic and are recruited to membranes upon stimulation, except for epsilon type DGKs that are permanently membrane-associated through a hydrophobic segment. Nothing is known about the mechanism(s) involved in the membrane localization of plant DGKs. By fusion to fluorescent proteins, we show that two DGKs from cluster I in Arabidopsis thaliana possess amino-terminal hydrophobic segments that are sufficient to address them to endoplasmic reticulum membranes.

Enhanced Formation of Methylglyoxal-Derived Advanced Glycation End Products in Arabidopsis Under Ammonium Nutrition.

Nitrate (NO3-) and ammonium (NH4+) are prevalent nitrogen (N) sources for plants. Although NH4+ should be the preferred form of N from the energetic point of view, ammonium nutrition often exhibits adverse effects on plant physiological functions and induces an important growth-limiting stress referred as ammonium syndrome. The effective incorporation of NH4+ into amino acid structures requires high activity of the mitochondrial tricarboxylic acid cycle and the glycolytic pathway. An unavoidable consequence of glycolytic metabolism is the production of methylglyoxal (MG), which is very toxic and inhibits cell growth in all types of organisms. Here, we aimed to investigate MG metabolism in Arabidopsis thaliana plants grown on NH4+ as a sole N source. We found that changes in activities of glycolytic enzymes enhanced MG production and that markedly elevated MG levels superseded the detoxification capability of the glyoxalase pathway. Consequently, the excessive accumulation of MG was directly involved in the induction of dicarbonyl stress by introducing MG-derived advanced glycation end products (MAGEs) to proteins. The severe damage to proteins was not within the repair capacity of proteolytic enzymes. Collectively, our results suggest the impact of MG (mediated by MAGEs formation in proteins) in the contribution to NH4+ toxicity symptoms in Arabidopsis.

Desaturase mutants reveal that membrane rigidification acts as a cold perception mechanism upstream of the diacylglycerol kinase pathway in Arabidopsis cells.

Membrane rigidification could be the first step of cold perception in poikilotherms. We have investigated its implication in diacylglycerol kinase (DAGK) activation by cold stress in suspension cells from Arabidopsis mutants altered in desaturase activities. By lateral diffusion assay, we showed that plasma membrane rigidification with temperature decrease was steeper in cells deficient in oleate desaturase than in wild type cells and in cells overexpressing linoleate desaturase. The threshold for the activation of the DAGK pathway in each type of cells correlated with this order of rigidification rate, suggesting that cold induced-membrane rigidification is upstream of DAGK pathway activation.

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.

Insights into the role of specific lipids in the formation and delivery of lipid microdomains to the plasma membrane of plant cells.

The existence of sphingolipid- and sterol-enriched microdomains, known as lipid rafts, in the plasma membrane (PM) of eukaryotic cells is well documented. To obtain more insight into the lipid molecular species required for the formation of microdomains in plants, we have isolated detergent (Triton X-100)-resistant membranes (DRMs) from the PM of Arabidopsis (Arabidopsis thaliana) and leek (Allium porrum) seedlings as well as from Arabidopsis cell cultures. Here, we show that all DRM preparations are enriched in sterols, sterylglucosides, and glucosylceramides (GluCer) and depleted in glycerophospholipids. The GluCer of DRMs from leek seedlings contain hydroxypalmitic acid. We investigated the role of sterols in DRM formation along the secretory pathway in leek seedlings. We present evidence for the presence of DRMs in both the PM and the Golgi apparatus but not in the endoplasmic reticulum. In leek seedlings treated with fenpropimorph, a sterol biosynthesis inhibitor, the usual Delta(5)-sterols are replaced by 9beta,19-cyclopropylsterols. In these plants, sterols and hydroxypalmitic acid-containing GluCer do not reach the PM, and most DRMs are recovered from the Golgi apparatus, indicating that Delta(5)-sterols and GluCer play a crucial role in lipid microdomain formation and delivery to the PM. In addition, DRM formation in Arabidopsis cells is shown to depend on the unsaturation degree of fatty acyl chains as evidenced by the dramatic decrease in the amount of DRMs prepared from the Arabidopsis mutants, fad2 and Fad3+, affected in their fatty acid desaturases.

The cold-induced early activation of phospholipase C and D pathways determines the response of two distinct clusters of genes in Arabidopsis cell suspensions.

In plants, a temperature downshift represents a major stress that will lead to the induction or repression of many genes. Therefore, the cold signal has to be perceived and transmitted to the nucleus. In response to a cold exposure, we have shown that the phospholipase D (PLD) and the phospholipase C (PLC)/diacylglycerol kinase pathways are simultaneously activated. The role of these pathways in the cold response has been investigated by analyzing the transcriptome of cold-treated Arabidopsis (Arabidopsis thaliana) suspension cells in the presence of U73122 or ethanol, inhibitors of the PLC/diacylglycerol kinase pathway and of the phosphatidic acid produced by PLD, respectively. This approach showed that the expression of many genes was modified by the cold response in the presence of such agents. The cold responses of most of the genes were repressed, thus correlating with the inhibitory effect of U73122 or ethanol. We were thus able to identify 58 genes that were regulated by temperature downshift via PLC activity and 87 genes regulated by temperature downshift via PLD-produced phosphatidic acid. Interestingly, each inhibitor appeared to affect different cold response genes. These results support the idea that both the PLC and PLD pathways are upstream of two different signaling pathways that lead to the activation of the cold response. The connection of these pathways with the CBF pathway, currently the most understood genetic system playing a role in cold acclimation, is discussed.

Structure and expression profile of the sucrose synthase multigene family in Arabidopsis.

The release of the complete genome sequence of Arabidopsis enabled the largest sucrose synthase family described to date, comprising six distinct members, for which expression profiles were not yet available, to be identified. Aimed at understanding the precise function of each AtSUS member among the family, a comparative study of protein structure was performed, together with an expression profiling of the whole gene family using the technique of real-time quantitative reverse transcriptase-polymerase chain reaction. Transcript levels were analysed in several plant organs, including both developing and germinating seeds. A series of treatments such as oxygen deprivation, dehydration, cold treatment, or various sugar feedings were then carried out to characterize the members of the family further. The AtSUS genes exhibit distinct but partially redundant expression profiles. Under anaerobic conditions, for instance, both AtSUS1 and AtSUS4 mRNA levels increase, but in a distinct manner. AtSUS2 is specifically and highly induced in seeds at 12 d after flowering and appears as a marker of seed maturation. AtSUS3 seems to be induced in various organs under dehydration conditions including leaves deprived of water or submitted to osmotic stress as well as late-maturing seeds. AtSUS5 and AtSUS6 are expressed in nearly all plant organs and do not exhibit any transcriptional response to stresses. These results add new insights on the expression of SUS genes and are discussed in relation to distinct functions for each member of the AtSUS family.