Biogenic isoprene (2-methyl-1,3-butadiene) improves the integrity and functionality of thylakoid membranes and scavenges reactive oxygen species (ROS) in plant tissue under stress conditions. On the basis of available physiological studies, we hypothesized that the suppression of isoprene production in the poplar plant by genetic engineering would cause changes in the chloroplast protein pattern, which in turn would compensate for changes in chloroplast functionality and overall plant performance under abiotic stress. To test this hypothesis, we used a stable isotope-coded protein-labeling technique in conjunction with polyacrylamide gel electrophoresis and liquid chromatography tandem mass spectrometry. We analyzed quantitative and qualitative changes in the chloroplast proteome of isoprene-emitting and non isoprene-emitting poplars. Here we demonstrate that suppression of isoprene synthase by RNA interference resulted in decreased levels of chloroplast proteins involved in photosynthesis and increased levels of histones, ribosomal proteins, and proteins related to metabolism. Overall, our results show that the absence of isoprene triggers a rearrangement of the chloroplast protein profile to minimize the negative stress effects resulting from the absence of isoprene. The present data strongly support the idea that isoprene improves/stabilizes thylakoid membrane structure and interferes with the production of ROS.

(H.R.); 0000-0002-9825-867X (J.-P.S.).Isoprene emissions from poplar (Populus spp.) plantations can influence atmospheric chemistry and regional climate. These emissions respond strongly to temperature, [CO 2 ], and drought, but the superimposed effect of these three climate change factors are, for the most part, unknown. Performing predicted climate change scenario simulations (periodic and chronic heat and drought spells [HDSs] applied under elevated [CO 2 ]), we analyzed volatile organic compound emissions, photosynthetic performance, leaf growth, and overall carbon (C) gain of poplar genotypes emitting (IE) and nonemitting (NE) isoprene. We aimed (1) to evaluate the proposed beneficial effect of isoprene emission on plant stress mitigation and recovery capacity and (2) to estimate the cumulative net C gain under the projected future climate. During HDSs, the chloroplastidic electron transport rate of NE plants became impaired, while IE plants maintained high values similar to unstressed controls. During recovery from HDS episodes, IE plants reached higher daily net CO 2 assimilation rates compared with NE genotypes. Irrespective of the genotype, plants undergoing chronic HDSs showed the lowest cumulative C gain. Under control conditions simulating ambient [CO 2 ], the C gain was lower in the IE plants than in the NE plants. In summary, the data on the overall C gain and plant growth suggest that the beneficial function of isoprene emission in poplar might be of minor importance to mitigate predicted short-term climate extremes under elevated [CO 2 ]. Moreover, we demonstrate that an analysis of the canopy-scale dynamics of isoprene emission and photosynthetic performance under multiple stresses is essential to understand the overall performance under proposed future conditions. Climate change will lead to an increase in global temperatures of at least 2°C in the near future (IPCC, 2014). There is at present substantial evidence that this climate change is leading to an increase in the frequency and intensity of extreme events such as heat and drought waves (Feyen and Dankers, 2009;Fischer and Schär, 2010;Perkins et al., 2012;Thornton et al., 2014), creating a sequence of recurring stress and recovery cycles for plants. Coumou and Rahmstorf (2012) showed that, in the last 15 years, five extreme heat wave events have occurred worldwide, four of which were observed also in Europe. Interactions between heat and drought under predicted elevated [CO 2 ] (IPCC, 2014) generate complex, often nonadditive physiological responses. Such effects cannot be predicted by singlefactor analyses and highlight the importance of carrying out controlled, multistress scenarios to investigate plant performance under future climate conditions (Clausen et al., 2011; Alemayehu et al., 2014).Photosynthesis, respiration, and photorespiration are the three dominating processes determining carbon (C) exchange and C metabolism in plants (Bauwe et al., 2010;Mahecha et al., 2010). In addition, the emission of biogenic volatile organic c...

Over the last decades, post‐illumination bursts (PIBs) of isoprene, acetaldehyde and green leaf volatiles (GLVs) following rapid light‐to‐dark transitions have been reported for a variety of different plant species. However, the mechanisms triggering their release still remain unclear. Here we measured PIBs of isoprene‐emitting (IE) and isoprene non‐emitting (NE) grey poplar plants grown under different climate scenarios (ambient control and three scenarios with elevated CO2 concentrations: elevated control, periodic heat and temperature stress, chronic heat and temperature stress, followed by recovery periods). PIBs of isoprene were unaffected by elevated CO2 and heat and drought stress in IE, while they were absent in NE plants. On the other hand, PIBs of acetaldehyde and also GLVs were strongly reduced in stress‐affected plants of all genotypes. After recovery from stress, distinct differences in PIB emissions in both genotypes confirmed different precursor pools for acetaldehyde and GLV emissions. Changes in PIBs of GLVs, almost absent in stressed plants and enhanced after recovery, could be mainly attributed to changes in lipoxygenase activity. Our results indicate that acetaldehyde PIBs, which recovered only partly, derive from a new mechanism in which acetaldehyde is produced from methylerythritol phosphate pathway intermediates, driven by deoxyxylulose phosphate synthase activity.

ORCID IDs: 0000-0002-3422-4083 (J.M.-P.); 0000-0001-8410-5624 (J.B.); 0000-0002-9825-867X (J.-P.S.).Researchers have been examining the biological function(s) of isoprene in isoprene-emitting (IE) species for two decades. There is overwhelming evidence that leaf-internal isoprene increases the thermotolerance of plants and protects them against oxidative stress, thus mitigating a wide range of abiotic stresses. However, the mechanisms of abiotic stress mitigation by isoprene are still under debate. Here, we assessed the impact of isoprene on the emission of nitric oxide (NO) and the S-nitroso-proteome of IE and non-isoprene-emitting (NE) gray poplar (Populus 3 canescens) after acute ozone fumigation. The short-term oxidative stress induced a rapid and strong emission of NO in NE compared with IE genotypes. Whereas IE and NE plants exhibited under nonstressful conditions only slight differences in their S-nitrosylation pattern, the in vivo S-nitroso-proteome of the NE genotype was more susceptible to ozone-induced changes compared with the IE plants. The results suggest that the nitrosative pressure (NO burst) is higher in NE plants, underlining the proposed molecular dialogue between isoprene and the free radical NO. Proteins belonging to the photosynthetic light and dark reactions, the tricarboxylic acid cycle, protein metabolism, and redox regulation exhibited increased S-nitrosylation in NE samples compared with IE plants upon oxidative stress. Because the posttranslational modification of proteins via S-nitrosylation often impacts enzymatic activities, our data suggest that isoprene indirectly regulates the production of reactive oxygen species (ROS) via the control of the S-nitrosylation level of ROS-metabolizing enzymes, thus modulating the extent and velocity at which the ROS and NO signaling molecules are generated within a plant cell.It has been demonstrated that isoprene protects plants against a plethora of abiotic stresses (Singsaas et al., 1997;Behnke et al., 2007;Velikova et al., 2008;Vickers et al., 2009b). Since the discovery of the positive influence of isoprene emission on plants' photosynthetic processes in the early 1990s (Sharkey and Singsaas, 1995), many efforts have been made to explain the primary mechanism of isoprene functioning. Most attention was given to the hypothesis that isoprene improves the thermotolerance of the photosynthetic machinery by stabilizing chloroplast (thylakoid) membranes during short, high-temperature episodes (Sharkey and Singsaas, 1995;Loreto and Schnitzler, 2010). Successive studies underlined that isoprene helps maintain high rates of chloroplastic electron transport and CO 2 assimilation during heat stress and accelerates recovery from stress (Singsaas and Sharkey, 2000;Velikova et al., 2006;Behnke et al., 2010b;Way et al., 2011).One mechanistic explanation is that isoprene molecules are dissolved in thylakoid membrane and prevent membrane lipid denaturation following oxidative stress (Sharkey and Yeh, 2001). It was suggested that isoprene acts directly to st...

Throughout the temperate zones, plants face combined drought and heat spells in increasing frequency and intensity. Here, we compared periodic (intermittent, i.e., high-frequency) versus chronic (continuous, i.e., high-intensity) drought-heat stress scenarios in gray poplar (Populus3 canescens) plants for phenotypic and transcriptomic effects during stress and after recovery. Photosynthetic productivity after stress recovery exceeded the performance of poplar trees without stress experience. We analyzed the molecular basis of this stress-related memory phenotype and investigated gene expression responses across five major tree compartments including organs and wood tissues. For each of these tissue samples, transcriptomic changes induced by the two stress scenarios were highly similar during the stress phase but strikingly divergent after recovery. Characteristic molecular response patterns were found across tissues but involved different genes in each tissue. Only a small fraction of genes showed similar stress and recovery expression profiles across all tissues, including type 2C protein phosphatases, the LATE EMBRYOGENESIS ABUNDANT PROTEIN4-5 genes, and homologs of the Arabidopsis (Arabidopsis thaliana) transcription factor HOMEOBOX7. Analysis of the predicted transcription factor regulatory networks for these genes suggested that a complex interplay of common and tissue-specific components contributes to the coordination of post-recovery responses to stress in woody plants.

Protein S-nitrosylation, the covalent binding of nitric oxide (NO) to protein cysteine residues, is one of the main mechanisms of NO signaling in plant and animal cells. Using a combination of the biotin switch assay and label-free LC-MS/MS analysis, we revealed the S-nitroso-proteome of the woody model plant Populus x canescens. Under normal conditions, constitutively S-nitrosylated proteins in poplar leaves and calli comprise all aspects of primary and secondary metabolism. Acute ozone fumigation was applied to elicit ROS-mediated changes of the S-nitroso-proteome. This treatment changed the total nitrite and nitrosothiol contents of poplar leaves and affected the homeostasis of 32 S-nitrosylated proteins. Multivariate data analysis revealed that ozone exposure negatively affected the S-nitrosylation status of leaf proteins: 23 proteins were de-nitrosylated and 9 proteins had increased S-nitrosylation content compared to the control. Phenylalanine ammonia-lyase 2 (log2[ozone/control] = −3.6) and caffeic acid O-methyltransferase (−3.4), key enzymes catalyzing important steps in the phenylpropanoid and subsequent lignin biosynthetic pathways, respectively, were de-nitrosylated upon ozone stress. Measuring the in vivo and in vitro phenylalanine ammonia-lyase activity indicated that the increase of the phenylalanine ammonia-lyase activity in response to acute ozone is partly regulated by de-nitrosylation, which might favor a higher metabolic flux through the phenylpropanoid pathway within minutes after ozone exposure.