Blue-green fluorescence during hypersensitive cell death arises from phenylpropanoid deydrodimers.

Infection of Arabidopsis with avirulent Pseudomonas syringae and exposure to nitrogen dioxide (NO2) both trigger hypersensitive cell death (HCD) that is characterized by the emission of bright blue-green (BG) autofluorescence under UV illumination. The aim of our current work was to identify the BG fluorescent molecules and scrutinize their biosynthesis, localization, and functions during the HCD. Compared with wild-type (WT) plants, the phenylpropanoid-deficient mutant fah1 developed normal HCD except for the absence of BG fluorescence. Ultrahigh resolution metabolomics combined with mass difference network analysis revealed that WT but not fah1 plants rapidly accumulate dehydrodimers of sinapic acid, sinapoylmalate, 5-hydroxyferulic acid, and 5-hydroxyferuloylmalate during the HCD. FAH1-dependent BG fluorescence appeared exclusively within dying cells of the upper epidermis as detected by microscopy. Saponification released dehydrodimers from cell wall polymers of WT but not fah1 plants. Collectively, our data suggest that HCD induction leads to the formation of free BG fluorescent dehydrodimers from monomeric sinapates and 5-hydroxyferulates. The formed dehydrodimers move from upper epidermis cells into the apoplast where they esterify cell wall polymers. Possible functions of phenylpropanoid dehydrodimers are discussed.

Automatization of metabolite extraction for high-throughput metabolomics: case study on transgenic isoprene-emitting birch.

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.

Predicting functions of putative fungal sesquiterpene synthase genes based on multiomics data analysis.

Sesquiterpenes (STs) are secondary metabolites, which mediate biotic interactions between different organisms. Predicting the species-specific ST repertoires can contribute to deciphering the language of communication between organisms of the same or different species. High biochemical plasticity and catalytic promiscuity of sesquiterpene synthases (STSs), however, challenge the homology-based prediction of the STS functions. Using integrated analyses of genomic, transcriptomic, volatilomic, and metabolomic data, we predict product profiles for 116 out of 146 putative STS genes identified in the genomes of 30 fungal species from different trophic groups. Our prediction method is based on the observation that STSs encoded by genes closely related phylogenetically are likely to share the initial enzymatic reactions of the ST biosynthesis pathways and, therefore, produce STs via the same reaction route. The classification by reaction routes allows to assign STs known to be emitted by a particular species to the putative STS genes from this species. Gene expression information helps to further specify these ST-to-STS assignments. Validation of the computational predictions of the STS functions using both in silico and experimental approaches shows that integrated multiomic analyses are able to correctly link cyclic STs of non-cadalane type to genes. In the process of the experimental validation, we characterized catalytic properties of several putative STS genes from the mycorrhizal fungus Laccaria bicolor. We show that the STSs encoded by the L.bicolor mycorrhiza-induced genes emit either nerolidol or α-cuprenene and α-cuparene, and discuss the possible roles of these STs in the mycorrhiza formation.

Protein expression plasticity contributes to heat and drought tolerance of date palm.

Climate change is increasing the frequency and intensity of warming and drought periods around the globe, currently representing a threat to many plant species. Understanding the resistance and resilience of plants to climate change is, therefore, urgently needed. As date palm (Phoenix dactylifera) evolved adaptation mechanisms to a xeric environment and can tolerate large diurnal and seasonal temperature fluctuations, we studied the protein expression changes in leaves, volatile organic compound emissions, and photosynthesis in response to variable growth temperatures and soil water deprivation. Plants were grown under controlled environmental conditions of simulated Saudi Arabian summer and winter climates challenged with drought stress. We show that date palm is able to counteract the harsh conditions of the Arabian Peninsula by adjusting the abundances of proteins related to the photosynthetic machinery, abiotic stress and secondary metabolism. Under summer climate and water deprivation, these adjustments included efficient protein expression response mediated by heat shock proteins and the antioxidant system to counteract reactive oxygen species formation. Proteins related to secondary metabolism were downregulated, except for the P. dactylifera isoprene synthase (PdIspS), which was strongly upregulated in response to summer climate and drought. This study reports, for the first time, the identification and functional characterization of the gene encoding for PdIspS, allowing future analysis of isoprene functions in date palm under extreme environments. Overall, the current study shows that reprogramming of the leaf protein profiles confers the date palm heat- and drought tolerance. We conclude that the protein plasticity of date palm is an important mechanism of molecular adaptation to environmental fluctuations.

Rapid conversion of isoprene photooxidation products in terrestrial plants.

Isoprene is emitted from the biosphere into the atmosphere, and may strengthen the defense mechanisms of plants against oxidative and thermal stress. Once in the atmosphere, isoprene is rapidly oxidized, either to isoprene-hydroxy-hydroperoxides (ISOPOOH) at low levels of nitrogen oxides, or to methyl vinyl ketone (MVK) and methacrolein at high levels. Here we combine uptake rates and deposition velocities that we obtained in laboratory experiments with observations in natural forests to show that 1,2-ISOPOOH deposits rapidly into poplar leaves. There, it is converted first to cytotoxic MVK and then most probably through alkenal/ one oxidoreductase (AOR) to less toxic methyl ethyl ketone (MEK). This detoxification process is potentially significant globally because AOR enzymes are ubiquitous in terrestrial plants. Our simulations with a global chemistry-transport model suggest that around 6.5 Tg yr- of MEK are re-emitted to the atmosphere. This is the single largest MEK source presently known, and recycles 1.5% of the original isoprene flux. Eddy covariance flux measurements of isoprene and MEK over different forest ecosystems confirm that MEK emissions can reach 1-2% those of isoprene. We suggest that detoxification processes in plants are one of the most important sources of oxidized volatile organic compounds in the atmosphere.

Effects of heat and drought stress on post-illumination bursts of volatile organic compounds in isoprene-emitting and non-emitting poplar.

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.

RNAi-mediated downregulation of poplar plasma membrane intrinsic proteins (PIPs) changes plasma membrane proteome composition and affects leaf physiology.

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.

The diversification of terpene emissions in Mediterranean oaks: lessons from a study of Quercus suber, Quercus canariensis and its hybrid Quercus afares.

Interspecific gene flow is common in oaks. In the Mediterranean, this process produced geographical differentiations and new species, which may have contributed to the diversification of the production of volatile terpenes in the oak species of this region. The endemic North African deciduous oak Quercus afares (Pomel) is considered to be a stabilized hybrid between the evergreen Quercus suber (L.) and the deciduous Quercus canariensis (Willd.), presumably being monoterpene and isoprene emitters, respectively. In a common garden experiment, we examined the terpene emission capacities, terpene synthase (TPS) activities and nuclear genetic markers in 52 trees of these three oak species. All but one of the Q. suber and Q. canariensis trees were found to be genetically pure, whereas most Q. afares trees possessed a mixed genotype with a predominance of Q. suber alleles. Analysis of the foliar terpene emissions and TPS activities revealed that all the Q. canariensis trees strongly produced isoprene while all the Q. suber trees were strong monoterpene producers. Quercus afares trees produced monoterpenes as well but at more variable and significantly lower rates, and with a monoterpene pattern different than that observed in Q. suber. Among 17 individuals tested, one Q. afares tree emitted only an insignificant amount of terpenes. No mixed isoprene/monoterpene emitter was detected. Our results suggest that the capacity and pattern of volatile terpene production in Algerian Q. afares populations have strongly diverged from those of its parental species and became quantitatively and qualitatively reduced, including the complete suppression of isoprene production.

Isoprene emission-free poplars--a chance to reduce the impact from poplar plantations on the atmosphere.

• 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.

Biogenic volatile organic compound and respiratory CO2 emissions after 13C-labeling: online tracing of C translocation dynamics in poplar plants.

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.

Structure of isoprene synthase illuminates the chemical mechanism of teragram atmospheric carbon emission.

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.

RNAi-mediated suppression of isoprene emission in poplar transiently impacts phenolic metabolism under high temperature and high light intensities: a transcriptomic and metabolomic analysis.

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.

Analysis of 1-deoxy-D-xylulose 5-phosphate synthase activity in Grey poplar leaves using isotope ratio mass spectrometry.

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.

Isoprene emission protects photosynthesis in sunfleck exposed Grey poplar.

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.

Determination of de novo and pool emissions of terpenes from four common boreal/alpine trees by 13CO2 labelling and PTR-MS analysis.

Boreal forests emit a large amount of monoterpenes into the atmosphere. Traditionally these emissions are assumed to originate as evaporation from large storage pools. Thus, their diurnal cycle would depend mostly on temperature. However, there is indication that a significant part of the monoterpene emission would originate directly from de novo synthesis. By applying 13CO2 fumigation and analyzing the isotope fractions with proton transfer reaction mass spectrometry (PTR-MS) and classical GC-MS, we determined the fractions of monoterpene emissions originating from de novo biosynthesis in Pinus sylvestris (58%), Picea abies (33.5%), Larix decidua (9.8%) and Betula pendula (100%). Application of the observed split between de novo and pool emissions from P. sylvestris in a hybrid emission algorithm resulted in a better description of ecosystem scale monoterpene emissions from a boreal Scots pine forest stand.

Nonmicrobial aerobic methane emission from poplar shoot cultures under low-light 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.

Modelling the drought impact on monoterpene fluxes from an evergreen Mediterranean forest canopy.

In many ecosystems drought cycles are common during the growing season but their impact on volatile monoterpene emissions is unclear. Therefore, we aimed to develop and evaluate a process-based modelling approach to explore the explanatory power of likely mechanisms. The biochemically based isoprene and monoterpene emission model SIM-BIM2 has been modified and linked to a canopy model and a soil water balance model. Simulations are carried out for Quercus ilex forest sites and results are compared to measured soil water, photosynthesis, terpene-synthase activity, and monoterpene emission rates. Finally, the coupled model system is used to estimate the annual drought impact on photosynthesis and emission. The combined and adjusted vegetation model was able to simulate photosynthesis and monoterpene emission under dry and irrigated conditions with an R(2) of 0.74 and 0.52, respectively. We estimated an annual reduction of monoterpene emission of 67% for the extended and severe drought period in 2006 in the investigated Mediterranean ecosystem. It is concluded that process-based ecosystem models can provide a useful tool to investigate the involved mechanisms and to quantify the importance of specific environmental constraints.

Isoprene emission is not temperature-dependent during and after severe drought-stress: a physiological and biochemical analysis.

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.

Circadian rhythms of isoprene biosynthesis in grey poplar leaves.

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.

Diurnal and seasonal variation of isoprene biosynthesis-related genes in grey poplar leaves.

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.