At the same time, it is noteworthy that the regiospecificity of some Lepidopteran IDIs are low, because they transform HIPP to not only HDMAPP but also the -isomer of HDMAPP and isomers with a γ-δ double bond . After the isomerization, ideally, one molecule of HDMAPP is supposed to condense with different molecules of HIPP into homo-GPP , homo-FPP , and homoGGPP . To our knowledge, all the characterized FPPSs that produce homo-FPP also produce FPP, with varied substrate preferences. This substrate promiscuity could explain why our previous work only produced C16 homosesquiterpenes as low, or non-existent HDMAPP levels may hamper homo-FPP analog production. The overwhelming production of HIPP to IPP in the optimized platform here may increase the HIPP incorporation to produce more C16, C17, and even C18 FPPs. For other prenyl diphosphates, reported point mutations in prenyltransferases that change the product profiles could be applied on the lepidopteran FPPS to produce homo-GPP . Also, the structural basis of substrate preference for HIPP/HDMAPP derived prenyl diphosphates has been analyzed, the results of which are proposed to direct the engineering of non-lepidopteran prenyltransferases to accept HIPP/HDMAPP . Finally, terpene synthases cyclized the homo prenyl pyrophosphates to terpene scaffolds. This step is the most challenging due to the lack of natural enzymes using homo prenyl pyrophosphates as the substrates. Future studies will focus on using rational design and directed evolution to alter the substrate specificity of canonical terpene synthases. Introducing extra carbon in the terpenes can significantly change their properties, exemplified by the optimized fuel properties of isoprenol biofuels.
With comparable RON boosting effects and energy densities to isopentenols, C6- and C7-isoprenols have decreased water solubilities,vertical grow making them better ingredients for fuel blends . In particular, C6-isoprenol derives from valeric acid, a key intermediate in the valerate biofuel platform . Numerous chemical reactions/processes have been developed to transform lignocellulose to valeric acid via levulinic acid , making our pathway promising to produce C6- isoprenol as a next-generation biofuel. Besides the simple terpenes we produce here, another example is -germacrene D, whose analog with two extra carbons, -14,15-dimethylgermacrene D shows a reversal in insect behavioral activity . Addressing those challenges in the homoterpene biosynthesis will enable the efficient production of various terpene analogs, leading to more diversified structures in the chemical portfolio for downstream applications.Humans are mandated to not exceed 1–2 mg/L copper in their drinking water , while some freshwater animals and plants experience acute toxic effects at concentrations as low as 10 µg/L . Because the human food chain begins with plants, it is critical to understand how plants tolerate heavy metals including copper, which is frequently concentrated in soils as a result of pesticide application, sewage sludge deposition, mining, smeltering, and industrial activities. This issue is also at the crux of applying phytoremediation approaches, which use green plants to decontaminate or contain polluted soils and sediments and to purify waste waters and landfill leachates . Metal-tolerant plants inhibit incorporation of excess metal into photosynthetic tissue by restricting transport across the root endodermis and by storage in the root cortex . In contrast, hyper accumulating plants extract metals from soils and concentrate excess amounts in harvestable parts such as leaves. Copper detoxification seems to be linked to mechanisms that bind Cu to molecular thiol groups. Cysteine-rich peptides, such as phytochelatins which transport copper to the shoot, increase in response to high cellular levels of Cu , and Cu-S binding occurs in roots and leaves of Larrea tridentata.
However, an unidentified copper species, concentrated in electron-dense granules on cell walls and some vacuole membranes, appears to be the main morphological form of copper sequestered in Oryza sativa , Cannabis sativa , Allium sativum , and Astragalus sinicus . Plants take in and exclude elements largely at the soil-root interface within the rhizosphere, i.e. the volume of soil influenced by roots, mycorrhizal fungi, and bacterial communities . Deciphering processes that control the bio-availability of metals in the field is difficult because the rhizosphere is compositionally and structurally complex. Here we report on using synchrotron-based micro-analytical and imaging tools to resolve processes by which metal tolerant plants defend themselves against excess cationic copper. We have mapped the distribution of copper in self-standing thin sections of unperturbed soils using micro-Xray fluorescence and identified structural forms of copper at points-of-interest using micro-extended X-ray absorption fine structure spectroscopy and X-ray diffraction . Because only a few small areas could be analyzed in reasonable times with micro-analyses, the uniqueness of the micro-analytical results was tested by recording the bulk EXAFS spectrum from a sample representing the entire rhizosphere and by simulating this spectrum by linear combination of copper species spectra from POIs. We investigated copper speciation in rhizospheres of Phragmites australisand Iris pseudoacorus, two widespread wetland species with high tolerances to heavy metals .P. australisis frequently used to treat wastewaters because it can store heavy metals as weakly soluble or insoluble forms. Its roots can be enriched in Cu 5-60 times relative to leaves, with large differences among ecotypes and between field-grown versus hydroponically grown plants . To take into account natural complexity, including any influence of bacteria, fungi, or climate variation, our experiment was conducted outdoors, rather than in a greenhouse on seedlings using ex-solum pots or hydroponic growth methods. The soil was from the Pierrelaye plain, a 1200 ha truck-farming area about 30 km northwest of Paris, France.
From 1899 to 1999, regular irrigation of the Pierrelaye plain with untreated sewage water from Paris caused contamination with heavy metals, mainly Zn, Pb, and Cu . Such pollution is pervasive worldwide because increasing populations and associated economic growth are diminishing available freshwater, thus leading to increased irrigation of farmlands with waste waters.In the initial soil, copper occurs in two morphological forms . One form decorates coarse organic particles that have some recognizable structures from reticular tissue , and the other occurs in the fine clayey matrix in areas that show organic particulate shapes only at high µ-XRF resolution. In the two phytoremediated soils, similar Cu-organic particulate associations, but also, hot spots of Cu grains 5-20 µm in size were observed in the thin-section maps . In the rhizosphere of P. australis, the Cu hot spots exist outside and in roots and specifically in cortical parenchyma, but not in central vascular cylinders from the stele that contain vascular bundles through which micro-nutrients are transported to reproductive and photosynthetic tissues. In contrast, the main roots and rhizome of I. pseudoacorus do not contain detectable Cu grains, but in the surrounding soil Cu grains are aligned, suggesting that they are associated with biological structures. Under an optical microscope filamentous and ramified organic structures,vertical outdoor farming similar to root hairs or hyphae from endomycorrhizal fungi, are visible in places where the Cu spots were observed by µ-XRF . Fungal forms are more likely because mycorrhizal hyphae typically are anastomosing, whereas root hairs are not. Fungi may also be implicated in the formation of Cu grains in the cortex of P. australis since roots of this plant are known to be colonized by arbuscular endomycorrhizae in contaminated environments . These hypotheses are consistent with the capacity of mycorrhizae to accumulate metals and with the storage of Cu in secondary feeder roots of the water hyacinth Eichhornia crassipes . The Cu grains have about the same size as the electron-dense Cu granules in cells of E. splendens placed in a CuSO4 solution for 30 days .iginal distribution of Cu in the rhizospheres, the Cu species could not be identified from the µ-XRF maps but instead were elucidated using EXAFS spectroscopy. All eight µ-EXAFS spectra from areas in the original soil containing the particle morphologies and chemical compositions observed with µ-XRF can be superimposed on the soil’s bulk EXAFS spectrum , indicating that the initial Cu speciation occurred uniformly.
If the initial soil contained various assemblages of Cu species that were distributed unevenly, then we might expect that the proportions of species also would have varied among analyzed areas and been detectable by µ-EXAFS spectroscopy ; however, this was not observed. These spectra match those for Cu2+ binding to carboxyl ligands in natural organic matter, as commonly observed . Elemental Cu. In contrast, only the reference spectrum of elemental copper matches the µ-EXAFS spectra of the 12 hot-spot Cu grains, which are statistically invariant . Photo reduction of Cu2+ in the X-ray beam cannot explain the formation of Cu0 because no elemental Cu was detected in the initial soil by powder and µ-EXAFS, and Cu0 was detected in the two phytore mediated soils at 10 K by bulk EXAFS, and all individual spectral scans from the same sample could be superimposed. At 10 K, radiation damage is delayed , and if Cu had been reduced in the beam, the proportion of Cu0 would have increased from scan to scan which was not observed. Spectra for rhizosphere Cu grains and reference metallic copper have the same phase and overall line shape, but they have significant differences in fine structure and amplitude, which provide details about the nature of the Cu grains. In the soil Cu grains, shoulders at 5.8 and 7.3 Å-1 are weak and the spectral amplitude is reduced by about 35% and attenuated, relative to metallic copper. The decreased amplitude of the EXAFS signal for the Cu grains relative to well-crystallized metallic copper cannot arise from over absorption because the spectra of the grains were recorded in transmission mode and because the amplitude reduction from over absorption would be uniform in R-space, as demonstrated for ZnS and MnO2 particles , which does not occur in this case. Derived radial structure–functions share the four-peak character of Cu metal . However, long-distance pair correlations are progressively diminished in the soil spectra, indicating multiple interatomic distances , reduced coordination numbers from small particles , and/or abundant micro-structural deffects, such as grain and twin boundary dislocations or atomic-scale vacancies.The rhizospheres were oxidizing as indicated by the presence of iron oxyhydroxide , absence of sulfide minerals, and the fact that P. australis and I. pseudoacorus are typical wetlands plants with aerenchyma that facilitate oxygen flow from leaves to roots . Thermodynamic calculations using compositions of soil solutions collected below the rhizosphere indicate that Cu+ and Cu2+ species should have been dominant . These points along with the occurrences of nanocrystalline Cu0 in plant cortical cells and as stringer morphologies outside the roots together suggest that copper was reduced biotically. Ecosystem ecology of the rhizosphere indicates synergistic or multiple reactions by three types of organisms: plants, endomycorrhizal fungi, and bacteria. Normally, organisms maintain copper homeostasis through cation binding to bio-active molecules such as proteins and peptides. When bound, the Cu2+/Cu1+ redox couple has elevated half-cell potentials that facilitate reactions in the electron-transport chain. Even though average healthy cell environments are sufficiently reducing , there are enough binding sites to maintain copper in its two oxidized states. Copper is also important in controlling cell-damaging free radicals produced at the end of the electron-transport chain, for example in the superoxide dismutase enzyme Cu-Zn-SOD, which accelerates the disproportionation of superoxide to O2 and hydrogen peroxide. However, unbound copper ions can catalyze the decomposition of hydrogen peroxide to water and more free radical species. To combat toxic copper and free radicals, many organisms overproduce enzymes such as catalase, chelates such as glutathione, and antioxidants . Mineralization could also be a defense against toxic copper, but reports of Cu+ and Cu2+ bio-minerals are rare; only copper sulfide in yeast and copper oxalate in lichens and fungi are known. Atacamite 3Cl) in worms does not appear to result from a biochemical defense. Biomineralization of copper metal may have occurred by a mechanism analogous to processes for metallic nanoparticle synthesis that exploit ligand properties of organic molecules. In these processes, organic molecules are used as templates to control the shape and size of metallic nanoparticles formed by adding strong reductants to bound cations. For copper nanoparticles and nanowires, a milder reductants ascorbic acidshas been used. Ascorbic acid, a well-known antioxidant, reduces Cu cations to Cu0 only when the cations are bound to organic substrates such as DNA in the presence of oxygen in the dark or via autocatalysis on Cu metal seeds in the absence of stabilizing organic ligands .