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Metabolomics studies are becoming increasingly common for understanding how plant metabolism responds to changes in environmental conditions, genetic manipulations and treatments. Despite the recent advances in metabolomics workflow, the sample preparation process still limits the high-throughput analysis in large-scale studies. Here, we present a highly flexible robotic system that integrates liquid handling, sonication, centrifugation, solvent evaporation and sample transfer processed in 96-well plates to automatize the metabolite extraction from leaf samples. We transferred an established manual extraction protocol performed to a robotic system, and with this, we show the optimization steps required to improve reproducibility and obtain comparable results in terms of extraction efficiency and accuracy. We then tested the robotic system to analyze the metabolomes of wild-type and four transgenic silver birch (Betula pendula Roth) lines under unstressed conditions. Birch trees were engineered to overexpress the poplar (Populus × canescens) isoprene synthase and to emit various amounts of isoprene. By fitting the different isoprene emission capacities of the transgenic trees with their leaf metabolomes, we observed an isoprene-dependent upregulation of some flavonoids and other secondary metabolites as well as carbohydrates, amino acid and lipid metabolites. By contrast, the disaccharide sucrose was found to be strongly negatively correlated to isoprene emission. The presented study illustrates the power of integrating robotics to increase the sample throughput, reduce human errors and labor time, and to ensure a fully controlled, monitored and standardized sample preparation procedure. Due to its modular and flexible structure, the robotic system can be easily adapted to other extraction protocols for the analysis of various tissues or plant species to achieve high-throughput metabolomics in plant research., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permission@oup.com.)

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., (© 2016 The Authors. Plant, Cell & Environment published by John Wiley & Sons Ltd.)

Plasma membrane intrinsic proteins (PIPs) are one subfamily of aquaporins that mediate the transmembrane transport of water. To reveal their function in poplar, we generated transgenic poplar plants in which the translation of PIP genes was downregulated by RNA interference investigated these plants with a comprehensive leaf plasma membrane proteome and physiome analysis. First, inhibition of PIP synthesis strongly altered the leaf plasma membrane protein composition. Strikingly, several signaling components and transporters involved in the regulation of stomatal movement were differentially regulated in transgenic poplars. Furthermore, hormonal crosstalk related to abscisic acid, auxin and brassinosteroids was altered, in addition to cell wall biosynthesis/cutinization, the organization of cellular structures and membrane trafficking. A physiological analysis confirmed the proteomic results. The leaves had wider opened stomata and higher net CO2 assimilation and transpiration rates as well as greater mesophyll conductance for CO2 (gm) and leaf hydraulic conductance (Kleaf). Based on these results, we conclude that PIP proteins not only play essential roles in whole leaf water and CO2 flux but have important roles in the regulation of stomatal movement., (Copyright © 2015. Published by Elsevier B.V.)

In the present study, biogenic volatile organic compound (BVOC) emissions and photosynthetic gas exchange of salt-sensitive (Populus x canescens (Aiton) Sm.) and salt-tolerant (Populus euphratica Oliv.) isoprene-emitting and non-isoprene-emitting poplars were examined under controlled high-salinity and high-temperature and -light episode ('sunfleck') treatments. Combined treatment with salt and sunflecks led to an increased isoprene emission capacity in both poplar species, although the photosynthetic performance of P. × canescens was reduced. Indeed, different allocations of isoprene precursors between the cytosol and the chloroplast in the two species were uncovered by means of (13)CO2 labeling. Populus × canescens leaves, moreover, increased their use of 'alternative' carbon (C) sources in comparison with recently fixed C for isoprene biosynthesis under salinity. Our studies show, however, that isoprene itself does not have a function in poplar survival under salt stress: the non-isoprene-emitting leaves showed only a slightly decreased photosynthetic performance compared with wild type under salt treatment. Lipid composition analysis revealed differences in the double bond index between the isoprene-emitting and non-isoprene-emitting poplars. Four clear metabolomics patterns were recognized, reflecting systemic changes in flavonoids, sterols and C fixation metabolites due to the lack/presence of isoprene and the absence/presence of salt stress. The studies were complemented by long-term temperature stress experiments, which revealed the thermotolerance role of isoprene as the non-isoprene-emitting leaves collapsed under high temperature, releasing a burst of BVOCs. Engineered plants with a low isoprene emission potential might therefore not be capable of resisting high-temperature episodes.

• Depending on the atmospheric composition, isoprene emissions from plants can have a severe impact on air quality and regional climate. For the plant itself, isoprene can enhance stress tolerance and also interfere with the attraction of herbivores and parasitoids. • Here, we tested the growth performance and fitness of Populus × canescens in which isoprene emission had been knocked down by RNA interference technology (PcISPS-RNAi plants) for two growing seasons under outdoor conditions. • Neither the growth nor biomass yield of the PcISPS-RNAi poplars was impaired, and they were even temporarily enhanced compared with control poplars. Modelling of the annual carbon balances revealed a reduced carbon loss of 2.2% of the total gross primary production by the absence of isoprene emission, and a 6.9% enhanced net growth of PcISPS-RNAi poplars. However, the knock down in isoprene emission resulted in reduced susceptibility to fungal infection, whereas the attractiveness for herbivores was enhanced. • The present study promises potential for the use of non- or low-isoprene-emitting poplars for more sustainable and environmentally friendly biomass production, as reducing isoprene emission will presumably have positive effects on regional climate and air quality., (© 2011 The Authors. New Phytologist © 2011 New Phytologist Trust.)

Background: Globally plants are the primary sink of atmospheric CO(2), but are also the major contributor of a large spectrum of atmospheric reactive hydrocarbons such as terpenes (e.g. isoprene) and other biogenic volatile organic compounds (BVOC). The prediction of plant carbon (C) uptake and atmospheric oxidation capacity are crucial to define the trajectory and consequences of global environmental changes. To achieve this, the biosynthesis of BVOC and the dynamics of C allocation and translocation in both plants and ecosystems are important., Methodology: We combined tunable diode laser absorption spectrometry (TDLAS) and proton transfer reaction mass spectrometry (PTR-MS) for studying isoprene biosynthesis and following C fluxes within grey poplar (Populus x canescens) saplings. This was achieved by feeding either (13)CO(2) to leaves or (13)C-glucose to shoots via xylem uptake. The translocation of (13)CO(2) from the source to other plant parts could be traced by (13)C-labeled isoprene and respiratory (13)CO(2) emission., Principal Finding: In intact plants, assimilated (13)CO(2) was rapidly translocated via the phloem to the roots within 1 hour, with an average phloem transport velocity of 20.3±2.5 cm h(-1). (13)C label was stored in the roots and partially reallocated to the plants' apical part one day after labeling, particularly in the absence of photosynthesis. The daily C loss as BVOC ranged between 1.6% in mature leaves and 7.0% in young leaves. Non-isoprene BVOC accounted under light conditions for half of the BVOC C loss in young leaves and one-third in mature leaves. The C loss as isoprene originated mainly (76-78%) from recently fixed CO(2), to a minor extent from xylem-transported sugars (7-11%) and from photosynthetic intermediates with slower turnover rates (8-11%)., Conclusion: We quantified the plants' C loss as respiratory CO(2) and BVOC emissions, allowing in tandem with metabolic analysis to deepen our understanding of ecosystem C flux.

The X-ray crystal structure of recombinant PcISPS (isoprene synthase from gray poplar hybrid Populus×canescens) has been determined at 2.7 Å resolution, and the structure of its complex with three Mg(2+) and the unreactive substrate analogue dimethylallyl-S-thiolodiphosphate has been determined at 2.8 Å resolution. Analysis of these structures suggests that the generation of isoprene from substrate dimethylallyl diphosphate occurs via a syn-periplanar elimination mechanism in which the diphosphate-leaving group serves as a general base. This chemical mechanism is responsible for the annual atmospheric emission of 100 Tg of isoprene by terrestrial plant life. Importantly, the PcISPS structure promises to guide future protein engineering studies, potentially leading to hydrocarbon fuels and products that do not rely on traditional petrochemical sources., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In plants, isoprene plays a dual role: (a) as thermo-protective agent proposed to prevent degradation of enzymes/membrane structures involved in photosynthesis, and (b) as reactive molecule reducing abiotic oxidative stress. The present work addresses the question whether suppression of isoprene emission interferes with genome wide transcription rates and metabolite fluxes in grey poplar (Populus x canescens) throughout the growing season. Gene expression and metabolite profiles of isoprene emitting wild type plants and RNAi-mediated non-isoprene emitting poplars were compared by using poplar Affymetrix microarrays and non-targeted FT-ICR-MS (Fourier transform ion cyclotron resonance mass spectrometry). We observed a transcriptional down-regulation of genes encoding enzymes of phenylpropanoid regulatory and biosynthetic pathways, as well as distinct metabolic down-regulation of condensed tannins and anthocyanins, in non-isoprene emitting genotypes during July, when high temperature and light intensities possibly caused transient drought stress, as indicated by stomatal closure. Under these conditions leaves of non-isoprene emitting plants accumulated hydrogen peroxide (H(2)O(2)), a signaling molecule in stress response and negative regulator of anthocyanin biosynthesis. The absence of isoprene emission under high temperature and light stress resulted transiently in a new chemo(pheno)type with suppressed production of phenolic compounds. This may compromise inducible defenses and may render non-isoprene emitting poplars more susceptible to environmental stress.

Deoxy-xylulose phosphate synthase (DXS) catalyzes the first step of the methylerythritol phosphate (MEP) pathway and it might regulate the metabolic flux in plastidic isoprenoid biosynthesis. We developed a sensitive assay suitable for plant extracts that is based on the decarboxylation of labeled pyruvate (1-(13)C)-PYR and detection of (13)CO(2) by isotope ratio mass spectrometry. We tested our method investigating the DXS activity in poplar leaves. Apparent DXS activity showed Michaelis constants of 111 and 158 microM for glyceraldehyde phosphate and pyruvate, respectively; pH and temperature optima were found at pH 8.6 and 45 degrees C. DXS activity was inhibited when the competitive inhibitor beta-fluoropyruvate was added to the reaction mixture. DXS activity strongly depended on leaf development with higher activity in young leaves and correlated fairly well with leaf isoprene emission potential. In mature poplar leaves, isoprene emission is the main metabolic sink of plastidic isoprenoid intermediates. Consequently, we found lower DXS activity in non-isoprene-emitting lines of poplar than in emitting plants as indicator of a lower demand of metabolic flux within the MEP pathway., (Copyright 2010 Elsevier Ltd. All rights reserved.)

In the present study, we combined transient temperature and light stress (sunfleck) and comparably analyzed photosynthetic gas exchange in Grey poplar which has been genetically modified in isoprene emission capacity. Overall, we demonstrate that for poplar leaves the ability to emit isoprene is crucial to maintain photosynthesis when exposed to sunflecks. Net CO2 assimilation and electron transport rates were strongly impaired in sunfleck-treated non-isoprene emitting poplars. Similar impairment was not detected when the leaves were exposed to high light (lightflecks) only. Within 10 h non-isoprene emitting poplars recovered from sunfleck-related impairment as indicated by chlorophyll fluorescence and microarray analysis. Unstressed leaves of non-isoprene emitting poplars had higher ascorbate contents, but also higher contents of malondialdehyde than wild-type. Microarray analyses revealed lipid and chlorophyll degradation processes in the non-isoprene emitting poplars. Thus, there is evidence for an adjustment of the antioxidative system in the non-isoprene emitting poplars even under normal growth conditions.

The aerobic formation of methane in plants has been reported previously, but has been questioned by a number of researchers. Recently, isotopic evidence demonstrated that ultraviolet irradiation and heating lead to photochemical or thermal aerobic methane formation mainly from plant pectin in the absence of microbial methane production. However, the origin of aerobic methane formation from plant material observed under low temperature and low-light/dark conditions is still unclear. Here we show that Grey poplar (Populus × canescens, syn. Populus tremula × Populus alba) plants derived from cell cultures under sterile conditions released 13C-labeled methane under low-light conditions after feeding the plants with 13CO2. Molecular biological analysis proved the absence of any microbial contamination with known methanogenic microorganisms and ruled out the possibility that methane emission from our poplar shoot cultures under aerobic low-light/dark and ambient temperature conditions could be of microbial origin. The CH4 release rates in our experiment were in the range of 0.16-0.7 ng g-1 DW h-1, adding evidence to the growing opinion that the quantitative role of aerobic methane emissions from plants in the global methane budget, at least from cold temperate or boreal regions, is only of minor importance.

Summary: Black poplar (Populus nigra L.) plants grown at 25 and 35 degrees C were subjected to drought stress to assess the combined impact of two consequences of global climate change--rising temperature and drought--on isoprene biosynthesis and emission. At both temperatures, photosynthesis was inhibited by moderate drought, but isoprene emission only decreased when drought was prolonged. The mRNA transcript level, protein concentration and activity of isoprene synthase (ISPS) changed in concert with isoprene emission during drought stress. However, ISPS activity decreased before isoprene emission during drought development, indicating a tighter control of the emission at a transcriptional or post-transcriptional level under moderate drought stress, and at both temperatures. A residual isoprene emission was measured when photosynthesis was totally inhibited after 35 days of drought. This photosynthesis-independent emission of isoprene was probably dependent on a cytosolic carbon supply as all the properties of ISPS were drastically inhibited. Isoprene emission was associated with dark respiration during the entire drought stress period, and at both temperatures, indicating that the two processes are sustained by, but do not compete for, the same carbon source. Isoprene emission was directly related to phosphoenolpyruvate carboxylase activity in plants grown at 25 degrees C and inversely related in plants grown at 35 degrees C, suggesting a strong temperature control on the regulation of the pyruvate flowing from the cytosol to the plastidic isoprenoid biosynthetic pathway under drought stress and recovery. In re-watered plants, the temperature control on isoprene emission was suppressed, despite complete recovery of photosynthesis and ISPS activity similar to levels in plants subjected to mild drought stress. Our results reveal the overriding effects of drought on isoprene emission, possibly affecting protein level or substrate supply. These effects may largely offset the predicted impact of rising temperatures on the emission of isoprene in terrestrial ecosystems.

Nitrogen nutrition and salt stress experiments were performed in a greenhouse with hydroponic-cultured, salt-sensitive Grey poplar (Populus x canescens) plants to study the combined influence of different N sources (either 1 mm NO(3) (-) or NH(4)(+)) and salt (up to 75 mm NaCl) on leaf gas exchange, isoprene biosynthesis and VOC emissions. Net assimilation and transpiration proved to be highly sensitive to salt stress and were reduced by approximately 90% at leaf sodium concentrations higher than 1,800 microg Na g dry weight (dw)(-1). In contrast, emissions of isoprene and oxygenated VOC (i.e. acetaldehyde, formaldehyde and acetone) were unaffected. There was no significant effect of combinations of salt stress and N source, and neither NO(3)(-) or NH(4)(+) influenced the salt stress response in the Grey poplar leaves. Also, transcript levels of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (PcDXR) and isoprene synthase (PcISPS) did not respond to the different N sources and only responded slightly to salt application, although isoprene synthase (PcISPS) activity was negatively affected at least in one of two experiments, despite high isoprene emission rates. A significant salt effect was the strong reduction of leaf dimethylallyl diphosphate (DMADP) content, probably due to restricted availability of photosynthates for DMADP biosynthesis. Further consequences of reduced photosynthetic gas exchange and maintaining VOC emissions are a very high C loss, up to 50%, from VOC emissions related to net CO(2) uptake and a strong increase in leaf internal isoprene concentrations, with maximum mean values up to 6.6 microl x l(-1). Why poplar leaves maintain VOC biosynthesis and emission under salt stress conditions, despite impaired photosynthetic CO(2) fixation, is discussed.

Isoprene (2-methyl-1,3-butadiene) emission varies diurnally in different species. In poplar (Populus spp.), it has recently been shown that the gene encoding the synthesizing enzyme for isoprene, isoprene synthase (ISPS), displays diurnal variation in expression. Working on shoot cultures of Grey poplar (Populus x canescens) placed under a different light regime in phytochambers, we showed that these variations in PcISPS gene expression, measured by quantitative real-time polymerase chain reaction, are not only due to day-night changes, but also are linked to an internal circadian clock. Measurement of additional selected isoprenoid genes revealed that phytoene synthase (carotenoid pathway) displays similar fluctuations, whereas 1-deoxy-d-xylulose 5-phosphate reductoisomerase, possibly the first committed enzyme of the 1-deoxy-d-xylulose 5-phosphate pathway, only shows light regulation. On the protein level, it appeared that PcISPS activity and protein content became reduced under constant darkness, whereas under constant light, activity and protein content of this enzyme were kept high. In contrast, isoprene emission rates under continuous irradiation displayed circadian changes as is the case for gene expression of PcISPS. Furthermore, binding assays with Arabidopsis (Arabidopsis thaliana) late elongated hypocotyl, a transcription factor of Arabidopsis involved in circadian regulation, clearly revealed the presence of circadian-determining regulatory elements in the promoter region of PcISPS.

Isoprene synthase (ISPS) catalyzes the elimination of pyrophosphate from dimethylallyl diphosphate (DMADP) forming isoprene, a volatile hydrocarbon emitted from many plant species to the atmosphere. In the present work, immunological techniques were applied to study and localize ISPS in poplar leaves (Populus x canescens). Immunogold labeling using polyclonal antibodies generated against His-tagged recombinant ISPS protein detected ca. 44% of ISPS in the stroma of the chloroplasts and ca. 56% of gold particles attached to the stromal-facing side of the thylakoid membranes. ISPS isolated from leaves exhibited the same biochemical properties as the recombinant ISPS without the plastid-targeting peptide heterologous expressed in E. coli, whereas an additional C- or N-terminal His-tag changed the biochemical features of the recombinant enzyme with regard to temperature, pH, and substrate dependence. In comparison to the closely related class of monoterpene synthases from angiosperms and ISPS of oaks, the most striking feature of the poplar ISPS is a cooperative substrate dependence which is characteristic to enzymes with positive substrate activation. The detection of four immunoreactive bands in poplar leaf extracts with isoelectric points from 5.0 to 5.5 and a native molecular weight of ca. 51 kDa give reason for future studies on post-translational modifications of ISPS.

Transcript levels of mRNA from 1-deoxy-D-xylulose 5-phosphate reductoisomerase (PcDXR), isoprene synthase (PcISPS), and phytoene synthase (PcPSY) showed strong seasonal variations in leaves of Grey poplar (Populus x canescens [Aiton] Sm.). These changes were dependent on the developmental stage and were strongly correlated to temperature and light. The expression rates of the genes PcDXR and PcISPS were found to be significantly correlated to each other, whereas the expression of the PcPSY gene showed a different seasonal pattern. Protein concentration and enzyme activity of PcISPS showed distinct seasonal patterns peaking in late summer, whereas highest transcription levels of PcISPS were observed in early summer. Moreover, correlation between PcISPS protein concentration and enzyme activity changed, in particular in autumn, when PcISPS protein levels remained high while enzyme activity declined, indicating posttranslational modifications of the enzyme. The positive correlation between dimethylallyl diphosphate levels and PcISPS protein content was found to be consistent with the demonstrated synchronized regulation of PcDXR and PcISPS, suggesting that metabolic flux through the 1-deoxy-D-xylulose 5-phosphate pathway and isoprene emission capacity are closely intercoordinated. Transcript levels of PcISPS showed strong diurnal variation with maximal values before midday in contrast to PcDXR, whose gene expression exhibited no clear intraday changes. During the course of a day, in vitro PcISPS activities did not change, whereas leaf dimethylallyl diphosphate levels and isoprene emission showed strong diurnal variations depending on actual temperature and light profiles on the respective day. These results illustrate that the regulation of isoprene biosynthesis in Grey poplar leaves seems to happen on transcriptional, posttranslational, and metabolic levels and is highly variable with respect to seasonal and diurnal changes in relation to temperature and light.

Isoprene synthase (ISPS) catalyzes the elimination of pyrophosphate from dimethylallyl diphosphate (DMADP) forming isoprene, a volatile hydrocarbon emitted from many plant species to the atmosphere. In the present work, immunological techniques were applied to study and localize ISPS in poplar leaves ( Populus × canescens). Immunogold labeling using polyclonal antibodies generated against His-tagged recombinant ISPS protein detected ca. 44% of ISPS in the stroma of the chloroplasts and ca. 56% of gold particles attached to the stromal-facing side of the thylakoid membranes. ISPS isolated from leaves exhibited the same biochemical properties as the recombinant ISPS without the plastid-targeting peptide heterologous expressed in E. coli, whereas an additional C- or N-terminal His-tag changed the biochemical features of the recombinant enzyme with regard to temperature, pH, and substrate dependence. In comparison to the closely related class of monoterpene synthases from angiosperms and ISPS of oaks, the most striking feature of the poplar ISPS is a cooperative substrate dependence which is characteristic to enzymes with positive substrate activation. The detection of four immunoreactive bands in poplar leaf extracts with isoelectric points from 5.0 to 5.5 and a native molecular weight of ca. 51 kDa give reason for future studies on post-translational modifications of ISPS. [ABSTRACT FROM AUTHOR]