Hydroxymethylbutenyl diphosphate accumulation reveals MEP pathway regulation for high CO2-induced suppression of isoprene emission.

Isoprene is emitted by some plants and is the most abundant biogenic hydrocarbon entering the atmosphere. Multiple studies have elucidated protective roles of isoprene against several environmental stresses, including high temperature, excessive ozone, and herbivory attack. However, isoprene emission adversely affects atmospheric chemistry by contributing to ozone production and aerosol formation. Thus, understanding the regulation of isoprene emission in response to varying environmental conditions, for example, elevated CO2, is critical to comprehend how plants will respond to climate change. Isoprene emission decreases with increasing CO2 concentration; however, the underlying mechanism of this response is currently unknown. We demonstrated that high-CO2-mediated suppression of isoprene emission is independent of photosynthesis and light intensity, but it is reduced with increasing temperature. Furthermore, we measured methylerythritol 4-phosphate (MEP) pathway metabolites in poplar leaves harvested at ambient and high CO2 to identify why isoprene emission is reduced under high CO2. We found that hydroxymethylbutenyl diphosphate (HMBDP) was increased and dimethylallyl diphosphate (DMADP) decreased at high CO2. This implies that high CO2 impeded the conversion of HMBDP to DMADP, possibly through the inhibition of HMBDP reductase activity, resulting in reduced isoprene emission. We further demonstrated that although this phenomenon appears similar to abscisic acid (ABA)-dependent stomatal regulation, it is unrelated as ABA treatment did not alter the effect of elevated CO2 on the suppression of isoprene emission. Thus, this study provides a comprehensive understanding of the regulation of the MEP pathway and isoprene emission in the face of increasing CO2.

Engineered poplar for bioproduction of the triterpene squalene.

Building sustainable platforms to produce biofuels and specialty chemicals has become an increasingly important strategy to supplement and replace fossil fuels and petrochemical-derived products. Terpenoids are the most diverse class of natural products that have many commercial roles as specialty chemicals. Poplar is a fast growing, biomassdense bioenergy crop with many species known to produce large amounts of the hemiterpene isoprene, suggesting an inherent capacity to produce significant quantities of other terpenes. Here we aimed to engineer poplar with optimized pathways to produce squalene, a triterpene commonly used in cosmetic oils, a potential biofuel candidate, and the precursor to the further diversified classes of triterpenoids and sterols. The squalene production pathways were either re-targeted from the cytosol to plastids or co-produced with lipid droplets in the cytosol. Squalene and lipid droplet co-production appeared to be toxic, which we hypothesize to be due to disruption of adventitious root formation, suggesting a need for tissue specific production. Plastidial squalene production enabled up to 0.63 mg/g fresh weight in leaf tissue, which also resulted in reductions in isoprene emission and photosynthesis. These results were also studied through a technoeconomic analysis, providing further insight into developing poplar as a production host.

High-Resolution Modeling of Summertime Biogenic Isoprene Emissions in New York City.

As cities strive for ambitious increases in tree canopy cover and reductions in anthropogenic volatile organic compound (AVOC) emissions, accurate assessments of the impacts of biogenic VOCs (BVOCs) on air quality become more important. In this study, we aim to quantify the impact of future urban greening on ozone production. BVOC emissions in dense urban areas are often coarsely represented in regional models. We set up a high-resolution (30 m) MEGAN (The Model of Emissions of Gases and Aerosols from Nature version 3.2) to estimate summertime biogenic isoprene emissions in the New York City metro area (NYC-MEGAN). Coupling an observation-constrained box model with NYC-MEGAN isoprene emissions successfully reproduced the observed isoprene concentrations in the city core. We then estimated future isoprene emissions from likely urban greening scenarios and evaluated the potential impact on future ozone production. NYC-MEGAN predicts up to twice as much isoprene emissions in NYC as the coarse-resolution (1.33 km) Biogenic Emission Inventory System version 3.61 (BEIS) on hot summer days. We find that BVOCs drive ozone production on hot summer days, even in the city core, despite large AVOC emissions. If high isoprene emitting species (e.g., oak trees) are planted, future isoprene emissions could increase by 1.4-2.2 times in the city core, which would result in 8-19 ppbv increases in peak ozone on ozone exceedance days with current NOx concentrations. We recommend planting non- or low-isoprene emitting trees in cities with high NOx concentrations to avoid an increase in the frequency and severity of future ozone exceedance events.

Cleavage of natural rubber by rubber oxygenases in Gram-negative bacteria.

Bacterial degradation of natural rubber (NR) in an oxic environment is initiated by oxidative cleavage of double bonds in the NR-carbon backbone and is catalyzed by extracellular haem-containing rubber oxygenases. NR-cleavage products of sufficiently low molecular mass are taken up by the cells and metabolized for energy and biomass formation. Gram-negative and Gram-positive NR-degrading bacteria (usually) employ different types of rubber oxygenases such as RoxA and/or RoxB (most Gram-negative NR-degraders) or latex clearing protein Lcp (most Gram-positive NR-degraders). In order to find novel orthologues of Rox proteins, we have revisited databases and provide an update of Rox-like proteins. We describe the putative evolution of rubber oxygenases and confirm the presence of a third subgroup of Rox-related proteins (RoxCs), the biological function of which remains, however, unclear. We summarize the knowledge on the taxonomic position of Steroidobacter cummioxidans 35Y and related species. Comparison of genomic and biochemical features of strain 35Y with other species of the genus Steroidobacter suggests that strain 35Y represents a species of a novel genus for which the designation Aurantibaculum gen. nov. is proposed. A short summary on the capabilities of NR-degrading consortia, that could be superior in biotechnological applications compared to pure cultures, is also provided. KEY POINTS: • Three types of rubber oxygenases exist predominantly in Gram-negative microbes • S. cummioxidans 35Y contains RoxA and RoxB which are superior in activity • S. cummioxidans 35Y represents a species of a novel genus.

Effects of aluminum (Al) stress on the isoprenoid metabolism of two Citrus species differing in Al-tolerance.

Isoprenoid metabolism and its derivatives took part in photosynthesis, growth regulation, signal transduction, and plant defense to biotic and abiotic stresses. However, how aluminum (Al) stress affects the isoprenoid metabolism and whether isoprenoid metabolism plays a vital role in the Citrus plants in coping with Al stress remain unclear. In this study, we reported that Al-treatment-induced alternation in the volatilization rate of monoterpenes (α-pinene, ß-pinene, limonene, α-terpinene, γ-terpinene and 3-carene) and isoprene were different between Citrus sinensis (Al-tolerant) and C. grandis (Al-sensitive) leaves. The Al-induced decrease of CO2 assimilation, maximum quantum yield of primary PSII photochemistry (Fv/Fm), the lower contents of glucose and starch, and the lowered activities of enzymes involved in the mevalonic acid (MVA) pathway and 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway might account for the different volatilization rate of isoprenoids. Furthermore, the altered transcript levels of genes related to isoprenoid precursors and/or derivatives metabolism, such as geranyl diphosphate (GPP) synthase (GPPS) in GPP biosynthesis, geranylgeranyl diphosphate synthase (GGPPS), chlorophyll synthase (CHS) and GGPP reductase (GGPPR) in chlorophyll biosynthesis, limonene synthase (LS) and α-pinene synthase (APS) in limonene and α-pinene synthesis, respectively, might be responsible for the different contents of corresponding products in C. grandis and C. sinensis. Our data suggested that isoprenoid metabolism was involved in Al tolerance response in Citrus, and the alternation of some branches of isoprenoid metabolism could confer different Al-tolerance to Citrus species.

Deoxyxylulose 5-Phosphate Synthase Does Not Play a Major Role in Regulating the Methylerythritol 4-Phosphate Pathway in Poplar.

The plastidic 2-C-methylerythritol 4-phosphate (MEP) pathway supplies the precursors of a large variety of essential plant isoprenoids, but its regulation is still not well understood. Using metabolic control analysis (MCA), we examined the first enzyme of this pathway, 1-deoxyxylulose 5-phosphate synthase (DXS), in multiple grey poplar (Populus × canescens) lines modified in their DXS activity. Single leaves were dynamically labeled with 13CO2 in an illuminated, climate-controlled gas exchange cuvette coupled to a proton transfer reaction mass spectrometer, and the carbon flux through the MEP pathway was calculated. Carbon was rapidly assimilated into MEP pathway intermediates and labeled both the isoprene released and the IDP+DMADP pool by up to 90%. DXS activity was increased by 25% in lines overexpressing the DXS gene and reduced by 50% in RNA interference lines, while the carbon flux in the MEP pathway was 25-35% greater in overexpressing lines and unchanged in RNA interference lines. Isoprene emission was also not altered in these different genetic backgrounds. By correlating absolute flux to DXS activity under different conditions of light and temperature, the flux control coefficient was found to be low. Among isoprenoid end products, isoprene itself was unchanged in DXS transgenic lines, but the levels of the chlorophylls and most carotenoids measured were 20-30% less in RNA interference lines than in overexpression lines. Our data thus demonstrate that DXS in the isoprene-emitting grey poplar plays only a minor part in controlling flux through the MEP pathway.

Analysis of Essential Isoprene Metabolic Pathway Proteins in Variovorax sp. Strain WS11.

Isoprene monooxygenase (IsoMO, encoded by isoABCDEF) initiates the oxidation of the climate-active gas isoprene, with the genes isoGHIJ and aldH nearly always found adjacent to isoABCDEF in extant and metagenome-derived isoprene degraders. The roles of isoGHIJ and aldH are uncertain, although each is essential to isoprene degradation. We report here the characterization of these proteins from two model isoprene degraders, Rhodococcus sp. strain AD45 and Variovorax sp. strain WS11. The genes isoHIJ and aldH from Variovorax and aldH from Rhodococcus were expressed individually in Escherichia coli as maltose binding protein fusions to overcome issues of insolubility. The activity of two glutathione S-transferases from Variovorax, IsoI and IsoJ was assessed with model substrates, and the conversion of epoxyisoprene to the intermediate 1-hydroxy-2-glutathionyl-2-methyl-3-butene (HGMB) was demonstrated. The next step of the isoprene metabolic pathway of Variovorax is catalyzed by the dehydrogenase IsoH, resulting in the conversion of HGMB to 2-glutathionyl-2-methyl-3-butenoic acid (GMBA). The aldehyde dehydrogenases (AldH) from Variovorax and Rhodococcus were examined with a variety of aldehydes, with both exhibiting maximum activity with butanal. AldH significantly increased the rate of production of NADH when added to the IsoH-catalyzed conversion of HGMB to GMBA (via GMB), suggesting a synergistic role for AldH in the isoprene metabolic pathway. An in silico analysis of IsoG revealed that this protein, which is essential for isoprene metabolism in Variovorax, is an enzyme of the formyl CoA-transferase family and is predicted to catalyze the formation of a GMBA-CoA thioester as an intermediate in the isoprene oxidation pathway. IMPORTANCE Isoprene is a climate-active gas, largely produced by trees, which is released from the biosphere in amounts equivalent to those of methane and all other volatile organic compounds combined. Bacteria found in many environments, including soils and on the surface of leaves of isoprene-producing trees, can grow on isoprene and thus may represent a significant biological sink for this globally significant volatile compound and remove isoprene before it escapes to the atmosphere, thus reducing its potency as a climate-active gas. The initial oxidation of isoprene by bacteria is mediated by isoprene monooxygenase encoded by the genes isoABCDEF. In isoprene-degrading bacteria, a second gene cluster, isoGHIJ, is also present, although the exact role in isoprene degradation by the proteins encoded by these genes is uncertain. This investigation sheds new light on the roles of these proteins in the isoprene oxidation pathway in two model isoprene-degrading bacteria of the genera Rhodococcus and Variovorax.

Optimization of IspSib stability through directed evolution to improve isoprene production.

Enzyme stability is often a limiting factor in the microbial production of high-value-added chemicals and commercial enzymes. A previous study by our research group revealed that the unstable isoprene synthase from Ipomoea batatas (IspSib) critically limits isoprene production in engineered Escherichia coli. Directed evolution was, therefore, performed in the present study to improve the thermostability of IspSib. First, a tripartite protein folding system designated as lac'-IspSib-'lac, which could couple the stability of IspSib to antibiotic ampicillin resistance, was successfully constructed for the high-throughput screening of variants. Directed evolution of IspSib was then performed through two rounds of random mutation and site-saturation mutation, which produced three variants with higher stability: IspSibN397V A476V, IspSibN397V A476T, and IspSibN397V A476C. The subsequent in vitro thermostability test confirmed the increased protein stability. The melting temperatures of the screened variants IspSibN397V A476V, IspSibN397V A476T, and IspSibN397V A476C were 45.1 ± 0.9°C, 46.1 ± 0.7°C, and 47.2 ± 0.3°C, respectively, each of which was higher than the melting temperature of wild-type IspSib (41.5 ± 0.4°C). The production of isoprene at the shake-flask fermentation level was increased by 1.94-folds, to 1,335 mg/L, when using IspSibN397V A476T. These findings provide insights into the optimization of the thermostability of terpene synthases, which are key enzymes for isoprenoid production in engineered microorganisms. In addition, the present study would serve as a successful example of improving enzyme stability without requiring detailed structural information or catalytic reaction mechanisms.IMPORTANCEThe poor thermostability of IspSib critically limits isoprene production in engineered Escherichia coli. A tripartite protein folding system designated as lac'-IspSib-'lac, which could couple the stability of IspSib to antibiotic ampicillin resistance, was successfully constructed for the first time. In order to improve the enzyme stability of IspSib, the directed evolution of IspSib was performed through error-PCR, and high-throughput screening was realized using the lac'-IspSib-'lac system. Three positive variants with increased thermostability were obtained. The thermostability test and the melting temperature analysis confirmed the increased stability of the enzyme. The production of isoprene was increased by 1.94-folds, to 1,335 mg/L, using IspSibN397V A476T. The directed evolution process reported here is also applicable to other terpene synthases key to isoprenoid production.

Nanoscale simulation of the thylakoid membrane response to extreme temperatures.

The thylakoid membrane is in a temperature-sensitive equilibrium that shifts repeatedly during the life cycle in response to ambient temperature or solar irradiance. Plants respond to seasonal temperature variation by changing their thylakoid lipid composition, while a more rapid mechanism for short-term heat exposure is required. The emission of the small organic molecule isoprene has been postulated as one such possible rapid mechanism. The protective mechanism of isoprene is unknown, but some plants emit isoprene at high temperature. We investigate the dynamics and structure for lipids within a thylakoid membrane across temperatures and varied isoprene content using classical molecular dynamics simulations. The results are compared with experimental findings for temperature-dependent changes in the lipid composition and shape of thylakoids. The surface area, volume, and flexibility of the membrane, as well as the lipid diffusion, increase with temperature, while the membrane thickness decreases. Saturated thylakoid 34:3 glycolipids derived from eukaryotic synthesis pathways exhibit altered dynamics relative to lipids from prokaryotic synthesis paths, which could explain the upregulation of specific lipid synthesis pathways at different temperatures. Increasing isoprene concentration was not observed to have a significant thermoprotective effect on the thylakoid membranes, and that isoprene readily permeated the membrane models tested.

Origin, evolution, and future of isoprene and nitric oxide interactions within leaves.

Photolytic generation of nitric oxide (NO), isoprene, and reactive oxygen species (ROS) pre-dated life on Earth (~4 billion years ago). However, isoprene-ROS-NO interactions became relevant to climate chemistry ~50 million years ago, after aquatic and terrestrial ecosystems became dominated by isoprene-emitting diatoms and angiosperms. Today, NO and NO2 (together referred to as NOx) are dangerous biogenic gaseous atmospheric pollutants. In plants, NO, with its multiple sources and sinks, acts as a secondary messenger that regulates development at low doses and induces cell death at high doses. Likewise, biogenic isoprene is a putative antioxidant and hormone 'enabler' that hastens plant (and leaf) growth and reproduction, and improves plant tolerance to transient abiotic stresses. Using examples from controlled-chamber simulation and field studies of isoprene oxidation, we discuss the likely nature and extent of isoprene oxidation within leaves. We argue that isoprene-NO interactions vary greatly among plant species, driven by differences in isoprene emission rate and nitrate assimilation capacity (i.e. NO sink strength), ROS availability, and the within-leaf ratio between free-NO and isoprene. In a warmer and CO2-fertilized future climate, antagonism between isoprene and NO within leaves will probably occur in a NO-rich (relative to present) environment, yielding a greater proportion of isoprene oxidation products, and inducing major changes in NO-mediated growth and stress responses.

Differential impact of crown rust (Puccinia coronata) infection on photosynthesis and volatile emissions in the primary host Avena sativa and the alternate host Rhamnus frangula.

Rust infection results in decreases in photosynthesis and stress volatile emissions, but how these changes vary among host species has not been studied. We demonstrated that the impact of the obligate biotrophic fungus, Puccinia coronata f. sp. avenae, on foliage physiological processes is stronger in the primary host, Avena sativa (cultivated oat), than in the alternate host, Rhamnus frangula (alder buckthorn). Photosynthesis decreased with increasing percentage of damaged leaf area (DA) in both species, but reductions were greater in A. sativa. In A. sativa, photosynthetic reductions resulted from reductions in stomatal conductance and photosynthetic capacity; in R. frangula, reductions were due to reduced capacity. Infection reduced photosynthetic biomass and key nutrients in A. sativa, but not in R. frangula. In A. sativa, stress-elicited emissions (methyl jasmonate, green leaf volatiles, long-chain saturated aldehydes, mono- and sesquiterpenes, benzenoids, and carotenoid breakdown products) increased with increasing DA from 0% to 40%, but decreased with further increases in DA. In R. frangula, volatile emissions were slightly elicited but, surprisingly, constitutive isoprene emissions were enhanced. Different hosts had characteristic volatile fingerprints, indicating differential activation of biochemical pathways. Fungal-elicited reductions in photosynthesis scale uniformly with stress severity. In the sensitive host, biphasic scaling of volatiles indicates that heavy spread of chlorosis/necrosis leads to an overall cessation of physiological functioning.

Mapping competitive pathways to terpenoid biosynthesis in Synechocystis sp. PCC 6803 using an antisense RNA synthetic tool.

BACKGROUND: Synechocystis sp. PCC 6803 utilizes pyruvate and glyceraldehyde 3-phosphate via the methylerythritol 4-phosphate (MEP) pathway for the biosynthesis of terpenoids. Considering the deep connection of the MEP pathway to the central carbon metabolism, and the low carbon partitioning towards terpenoid biosynthesis, significant changes in the metabolic network are required to increase cyanobacterial production of terpenoids. RESULTS: We used the Hfq-MicC antisense RNA regulatory tool, under control of the nickel-inducible PnrsB promoter, to target 12 different genes involved in terpenoid biosynthesis, central carbon metabolism, amino acid biosynthesis and ATP production, and evaluated the changes in the performance of an isoprene-producing cyanobacterial strain. Six candidate targets showed a positive effect on isoprene production: three genes involved in terpenoid biosynthesis (crtE, chlP and thiG), two involved in amino acid biosynthesis (ilvG and ccmA) and one involved in sugar catabolism (gpi). The same strategy was applied to interfere with different parts of the terpenoid biosynthetic pathway in a bisabolene-producing strain. Increased bisabolene production was observed not only when interfering with chlorophyll a biosynthesis, but also with carotenogenesis. CONCLUSIONS: We demonstrated that the Hfq-MicC synthetic tool can be used to evaluate the effects of gene knockdown on heterologous terpenoid production, despite the need for further optimization of the technique. Possible targets for future engineering of Synechocystis aiming at improved terpenoid microbial production were identified.

Real-time detection of isoprene marker gas based on micro-integrated chromatography system with GOQDs-modifiedµGC column and metal oxide gas detector.

Isoprene is a typical physiological marker that can be used to screen for chronic liver disease. This work developed a portable micro-integrated chromatography analysis system based on micro-electromechanical system technology, nanomaterials technology and embedded microcontroller technology. The system integrated components such as graphene oxide quantum dots modified semi-packed microcolumn, In2O3nanoflower (NF) gas-sensitive detector and 3D printed miniature solenoid valve group. The effectiveness of the separation effect of the micro-integrated system was verified by gas mixture test; the laws of the influence of carrier gas pressure and column temperature on the chromatographic separation performance, respectively, were investigated, and the working conditions (column temperature 90 °C and carrier gas pressure 7.5 kPa) for system testing were determined. The percentages of relative standard deviation of the peak areas and retention times obtained for the separated gases were in the range of 0.95%-6.06%, indicating the good reproducibility of the system. Meanwhile, the microintegrated system could detect isoprene down to 50 ppb at small injection volume (1 ml). The system response increased with increasing isoprene concentration and was linearly correlated with isoprene concentration (R2= 0.986), indicating that the system was expected to be used for trace detection of isoprene, a marker gas for liver disease, in the future.

Important Roles and Formation of Atmospheric Organosulfates in Marine Organic Aerosols: Influence of Phytoplankton Emissions and Anthropogenic Pollutants.

Organosulfates (OSs) could be potentially important compounds in marine organic aerosols, while their formation in marine atmospheres is far from clear due to a lack of cruise observations. In this work, shipboard atmospheric observations were conducted over the Yellow Sea and Bohai Sea to investigate the abundance and formation of biogenic isoprene/monoterpene-OSs in marine aerosols. The quantified OSs and NOSs accounted for 0.04-6.9% of marine organic aerosols and were 0.07-2.2% of the non-sea-salt (nss) sulfate in terms of sulfur content. Isoprene-related (nitrooxy-)OSs occupied 27-87% of the total quantified OSs, following the abundance order of summer > autumn > spring or winter. This order was driven by the marine phytoplankton biomass and sea surface temperature (SST), which controlled the seawater and atmospheric isoprene concentration levels. Under the severe impacts of anthropogenic pollutants from the East Asia continent in winter, monoterpene nitrooxy-OSs, generated with NOx involved in, increased to 34.4 ± 35.5 ng/m3 and contributed 68% of the quantified (nitrooxy-)OSs. Our results highlight the notable roles of biogenic OSs in marine organic aerosols over regions with high biological activity and high SST. The formation of biogenic OSs and their roles in altering marine aerosol properties calls for elaboration through cruise observations in different marine environments.

Nonequilibrium Behavior in Isoprene Secondary Organic Aerosol.

Recent studies have shown that instantaneous gas-particle equilibrium partitioning assumptions fail to predict SOA formation, even at high relative humidity (∼85%), and photochemical aging seems to be one driving factor. In this study, we probe the minimum aging time scale required to observe nonequilibrium partitioning of semivolatile organic compounds (SVOCs) between the gas and aerosol phase at ∼50% RH. Seed isoprene SOA is generated by photo-oxidation in the presence of effloresced ammonium sulfate seeds at <1 ppbv NOx, aged photochemically or in the dark for 0.3-6 h, and subsequently exposed to fresh isoprene SVOCs. Our results show that the equilibrium partitioning assumption is accurate for fresh isoprene SOA but breaks down after isoprene SOA has been aged for as short as 20 min even in the dark. Modeling results show that a semisolid SOA phase state is necessary to reproduce the observed particle size distribution evolution. The observed nonequilibrium partitioning behavior and inferred semisolid phase state are corroborated by offline mass spectrometric analysis on the bulk aerosol particles showing the formation of organosulfates and oligomers. The unexpected short time scale for the phase transition within isoprene SOA has important implications for the growth of atmospheric ultrafine particles to climate-relevant sizes.

Molecular characteristics of isoprene synthase and its control effects on isoprene emissions from tropical trees.

The isoprene emission rate from plants is simulated by a function of light intensity and leaf temperature, and the G-93 formula is the most extensively applied algorithm for this purpose. Isoprene is biosynthesized by the enzyme isoprene synthase (IspS), and instantly emitted from the leaf. Enzyme kinetics of IspS and substrate availability are important factors involved in the short-term leaf-level control of isoprene emissions. It is thus assumed that the parameters of G-93 may correlate with the kinetics of IspSs, however, at present there is no data available on the relationship between these two parameters. In this investigation, six IspS genes from tropical trees were cloned, their properties characterized, and the relationship between the enzyme kinetics of IspSs and the parameters of G-93 examined. There was a negative correlation between the enzyme kinetics of IspS Km and parameter CT1 of G93, which is used to define the temperature dependency of isoprene emissions. However, performance constant of IspS (kcat/Km) only showed slight positive correlation with CT1.suggesting that the enzyme kinetics of IspS has limited significance in controlling the temperature response of isoprene emissions. The molecular structure of IspS was further elucidated using a molecular dynamics simulation with a focus on the active site in the 6 α-helices bundle. The simulation of the enzyme-substrate complex of IspS from B. variegata predicted a new metal binding domain in helix F (E383) and catalytic motif FXRDRLXE in the A-C loop that could involve the deprotonation of dimethylallyl diphosphate (DMADP) to form a carbocation. Notably, after the binding of a metal ion and DMADP, the active-site closure mechanism was found to involve conformational alterations in the helix H-α1 and transition from a loose to tight enclosure of the 6 α-helices bundles to tune the active pocket size. The characteristics identified for the IspSs from tropical trees could help to explain regional isoprene emissions in tropical areas.

Rapid hydrolysis of tertiary isoprene nitrate efficiently removes NOx from the atmosphere.

The formation of a suite of isoprene-derived hydroxy nitrate (IHN) isomers during the OH-initiated oxidation of isoprene affects both the concentration and distribution of nitrogen oxide free radicals (NOx). Experiments performed in an atmospheric simulation chamber suggest that the lifetime of the most abundant isomer, 1,2-IHN, is shortened significantly by a water-mediated process (leading to nitric acid formation), while the lifetime of a similar isomer, 4,3-IHN, is not. Consistent with these chamber studies, NMR kinetic experiments constrain the 1,2-IHN hydrolysis lifetime to less than 10 s in deuterium oxide (D2O) at 298 K, whereas the 4,3-IHN isomer has been observed to hydrolyze much less efficiently. These laboratory findings are used to interpret observations of the IHN isomer distribution in ambient air. The IHN isomer ratio (1,2-IHN to 4,3-IHN) in a high NOx environment decreases rapidly in the afternoon, which is not explained using known gas-phase chemistry. When simulated with an observationally constrained model, we find that an additional loss process for the 1,2-IHN isomer with a time constant of about 6 h best explains our atmospheric measurements. Using estimates for 1,2-IHN Henry's law constant and atmospheric liquid water volume, we show that condensed-phase hydrolysis of 1,2-IHN can account for this loss process. Simulations from a global chemistry transport model show that the hydrolysis of 1,2-IHN accounts for a substantial fraction of NOx lost (and HNO3 produced), resulting in large impacts on oxidant formation, especially over forested regions.

Ozone pollution, water deficit stress and time drive poplar phyllospheric bacterial community structure.

Ground-level ozone (O3) pollution often rise in the summer and coincide with drought stress, which alters the relationships between trees and associated microbial communities in a manner that can have pronounced effects on associated biological activity and ecosystem integrity. Discerning the responses of phyllosphere microbial communities to O3 and water deficit could highlight the ability of plant-microbe interactions to either exacerbate or mitigate the effects of these stressors. Accordingly, this study was designed as the first report to specifically interrogate the impacts of elevated O3 and water deficit stress on phyllospheric bacterial community composition and diversity in hybrid poplar saplings. Significant reductions in phyllospheric bacterial alpha diversity indices were observed, with clear evidence of significant time × water deficit stress interactions. The combination of elevated O3 and water deficit stress shifted in the bacterial community composition over sampling time, resulted in significant increases in the relative abundance of the dominant Gammaproteobacteria phyla together with reductions in Betaproteobacteria. An increased prevalence of Gammaproteobacteria may represent a potential diagnostic dysbiosis-related biosignature associated with poplar disease risk. Significant positive correlations were observed between both Betaproteobacteria abundance and diversity indices and key foliar photosynthetic traits and isoprene emissions, whereas these parameters were negatively correlated with Gammaproteobacteria abundance. These findings suggest that the photosynthesis-related properties in plant leaves are closely related to the makeup of the phyllosphere bacterial community. These data provide novel insight into how plant-associated microbes can help maintain plant health and the stability of the local ecosystem in O3-polluted and dried environments.

A Novel Isoprene Synthase from the Monocot Tree Copernicia prunifera (Arecaceae) Confers Enhanced Drought Tolerance in Transgenic Arabidopsis.

The capacity to emit isoprene, among other stresses, protects plants from drought, but the molecular mechanisms underlying this trait are only partly understood. The Arecaceae (palms) constitute a very interesting model system to test the involvement of isoprene in enhancing drought tolerance, as their high isoprene emissions may have contributed to make them hyperdominant in neotropical dry forests, characterized by recurrent and extended periods of drought stress. In this study we isolated and functionally characterized a novel isoprene synthase, the gene responsible for isoprene biosynthesis, from Copernicia prunifera, a palm from seasonally dry tropical forests. When overexpressed in the non-emitter Arabidopsis thaliana, CprISPS conferred significant levels of isoprene emission, together with enhanced tolerance to water limitation throughout plant growth and development, from germination to maturity. CprISPS overexpressors displayed higher germination, cotyledon/leaf greening, water usage efficiency, and survival than WT Arabidopsis under various types of water limitation. This increased drought tolerance was accompanied by a marked transcriptional up-regulation of both ABA-dependent and ABA-independent key drought response genes. Taken together, these results demonstrate the capacity of CprISPS to enhance drought tolerance in Arabidopsis and suggest that isoprene emission could have evolved in Arecaceae as an adaptive mechanism against drought.

Overview of fungal terpene synthases and their regulation.

Terpenes and terpenoids are a group of isoprene-derived molecules that constitute the largest group of natural products and secondary metabolites produced by living things, with more than 25,000 compounds reported. These compounds are synthesized by enzymes called terpene synthases, which include several families of cyclases and enzymes. These are responsible for adding functional groups to cyclized structures. Fungal terpenoids are of great interest for their pharmacological properties; therefore, understanding the mechanisms that regulate their synthesis (regulation of the mevalonate pathway, regulation of gene expression, and availability of cofactors) is essential to direct their production. For this reason, this review addresses the detailed study of the biosynthesis of fungal terpenoids and their regulation by various physiological and environmental factors.