UDP-glycosyltransferase UGT96C10 functions as a novel detoxification factor for conjugating the activated dinitrotoluene sulfonate in switchgrass.

Dinitrotoluene sulfonates (DNTSes) are highly toxic hazards regulated by the Resource Conservation and Recovery Act (RCRA) in the United States. The trinitrotoluene (TNT) red water formed during the TNT purification process consists mainly of DNTSes. Certain plants, including switchgrass, reed and alfalfa, can detoxify low concentrations of DNTS in TNT red water-contaminated soils. However, the precise mechanism by which these plants detoxify DNTS remains unknown. In order to aid in the development of phytoremediation resources with high DNTS removal rates, we identified and characterized 1-hydroxymethyl-2,4-dinitrobenzene sulfonic acid (HMDNBS) and its glycosylated product HMDNBS O-glucoside as the degradation products of 2,4-DNT-3-SO3Na, the major isoform of DNTS in TNT red water-contaminated soils, in switchgrass via LC-MS/MS- and NMR-based metabolite analyses. Transcriptomic analysis revealed that 15 UDP-glycosyltransferase genes were dramatically upregulated in switchgrass plants following 2,4-DNT-3-SO3Na treatment. We expressed, purified and assayed the activity of recombinant UGT proteins in vitro and identified PvUGT96C10 as the enzyme responsible for the glycosylation of HMDNBS in switchgrass. Overexpression of PvUGT96C10 in switchgrass significantly alleviated 2,4-DNT-3-SO3Na-induced plant growth inhibition. Notably, PvUGT96C10-overexpressing transgenic switchgrass plants removed 83.1% of 2,4-DNT-3-SO3Na in liquid medium after 28 days, representing a 3.2-fold higher removal rate than that of control plants. This work clarifies the DNTS detoxification mechanism in plants for the first time, suggesting that PvUGT96C10 is crucial for DNTS degradation. Our results indicate that PvUGT96C10-overexpressing plants may hold great potential for the phytoremediation of TNT red water-contaminated soils.

Cytochrome P450-catalyzed biosynthesis of furanoditerpenoids in the bioenergy crop switchgrass (Panicum virgatum L.).

Specialized diterpenoid metabolites are important mediators of plant-environment interactions in monocot crops. To understand metabolite functions in plant environmental adaptation that ultimately can enable crop improvement strategies, a deeper knowledge of the underlying species-specific biosynthetic pathways is required. Here, we report the genomics-enabled discovery of five cytochrome P450 monooxygenases (CYP71Z25-CYP71Z29) that form previously unknown furanoditerpenoids in the monocot bioenergy crop Panicum virgatum (switchgrass). Combinatorial pathway reconstruction showed that CYP71Z25-CYP71Z29 catalyze furan ring addition directly to primary diterpene alcohol intermediates derived from distinct class II diterpene synthase products. Transcriptional co-expression patterns and the presence of select diterpenoids in switchgrass roots support the occurrence of P450-derived furanoditerpenoids in planta. Integrating molecular dynamics, structural analysis and targeted mutagenesis identified active site determinants that contribute to the distinct catalytic specificities underlying the broad substrate promiscuity of CYP71Z25-CYP71Z29 for native and non-native diterpenoids.

Highly efficient detoxification of dinitrotoluene by transgenic switchgrass overexpressing bacterial nitroreductase.

Dinitrotoluene (DNT) has been extensively used in manufacturing munitions, polyurethane foams and other important chemical products. However, it is highly toxic and mutagenic to most organisms. Here, we synthesized a codon-optimized bacterial nitroreductase gene, NfsI, for plant expression. The kinetic analysis indicates that the recombinant NfsI can detoxify both 2,4-DNT and its sulfonate (DNTS), while it has a 97.6-fold higher catalytic efficiency for 2,4-DNT than DNTS. Furthermore, we overexpressed NfsI in switchgrass (Panicum virgatum L.), which is a multiple-purpose crop used for fodder and biofuel production as well as phytoremediation. The 2,4-DNT treatment inhibited root elongation of wild-type switchgrass plants and promoted reactive oxygen species (ROS) accumulation in roots. In contrast, overexpression of NfsI in switchgrass significantly alleviated 2,4-DNT-induced root growth inhibition and ROS overproduction. Thus, the NfsI overexpressing transgenic switchgrass plant removed 94.1% 2,4-DNT after 6 days, whose efficiency was 1.7-fold higher than control plants. Moreover, the comparative transcriptome analysis suggests that 22.9% of differentially expressed genes induced by 2,4-DNT may participate in NfsI-mediated 2,4-DNT detoxification in switchgrass. Our work sheds light on the function of NfsI during DNT phytoremediation for the first time, revealing the application potential of switchgrass plants engineered with NfsI.

Genetically Engineered Oleaginous Yeast Lipomyces starkeyi for Sesquiterpene α-Zingiberene Production.

Oleaginous yeast, such as Lipomyces starkeyi, are logical organisms for production of higher energy density molecules like lipids and terpenes. We demonstrate that transgenic L. starkeyi strains expressing an α-zingiberene synthase gene from lemon basil or Hall's panicgrass can produce up to 17 mg/L α-zingiberene in yeast extract peptone dextrose (YPD) medium containing 4% glucose. The transgenic strain was further examined in 8% glucose media with C/N ratios of 20 or 100, and YPD. YPD medium resulted in 59 mg/L α-zingiberene accumulation. Overexpression of selected genes from the mevalonate pathway achieved 145% improvement in α-zingiberene synthesis. Optimization of the growth medium for α-zingiberene production led to 15% higher titer than YPD medium. The final transgenic strain produced 700 mg/L α-zingiberene in fed-batch bioreactor culture. This study opens a new synthetic route to produce α-zingiberene or other terpenoids in L. starkeyi and establishes this yeast as a platform for jet fuel biosynthesis.

Peroxidase from proso millet exhibits endonuclease-like activity.

In this study, the mechanism of DNA cleavage by cationic peroxidase from proso millet (PmPOD) was investigated. PmPOD cleaved supercoiled circular DNA into both nicked circular and linear forms via a cleavage mechanism that resembles those of native endonucleases. Inhibition and ligation studies demonstrated that reactive oxygen species and the ferriprotoporphyrin IX moiety in PmPOD are not involved in PmPOD-mediated DNA cleavage. Similar to other endonucleases, Mg ions considerably enhance the DNA cleavage activity of PmPOD. Further studies suggested that PmPOD can disrupt phosphodiester bonds in DNA and mononucleotides, indicating that it is a phosphatase. The phosphatase activity of PmPOD is higher than that of horseradish peroxidase (HRP), but the peroxidase activity of PmPOD was lower than that of HRP. PmPOD-mediated hydrolytic cleavage of DNA observed in this study is different from those reported for heme proteins. This study provides valuable insights into the distinct mechanisms underlying DNA cleavage by heme proteins.

Partial replacement of nitrate by ammonium increases photosynthesis and reduces oxidative stress in tanzania guinea grass exposed to cadmium.

In order to grow and effectively uptake and accumulate cadmium (Cd), plants used for phytoextraction have to cope with toxicity, which may be influenced by the supply of nitrate (NO3-) and ammonium (NH4+). Thus, we evaluated the effect of these nitrogen forms on the photosynthetic and antioxidant enzyme activities of Panicum maximum cv. Tanzania (tanzania guinea grass) under Cd stress. Plants were grown in nutrient solution under greenhouse conditions and subjected to a 3 × 3 factorial experiment. They were supplied with three NO3-/NH4+ ratios (100/0, 70/30 and 50/50) and exposed to three Cd rates (0.0, 0.5 and 1.0 mmol L-1), being arranged in a randomized complete block design with three replications. Gas exchange parameters, oxidative stress indicators, proline concentration and antioxidant enzyme activities were studied. Exposure to Cd reduced photosynthesis by causing stomatal closure and impairing electron transport. However, the simultaneous supply of NO3- and NH4+, particularly at a 50/50 ratio, restored gas exchange and improved the function of photosystem II, increasing the photosynthetic capacity of the grass. Plants grown with 50/50 showed reduced lipid peroxidation along with increased proline synthesis. Moreover, this NO3-/NH4+ ratio increased the tolerance of tanzania guinea grass to Cd by inducing high superoxide dismutase and glutathione reductase activities in shoots and roots, respectively, maintaining cellular homeostasis and reducing oxidative stress. The negative effects of Cd on photosynthesis and on the balance between oxidants and antioxidants are attenuated by the partial replacement of NO3- by NH4+ in the nutrient solution.

Nitrate and ammonium proportion plays a key role in copper phytoextraction, improving the antioxidant defense in Tanzania guinea grass.

Various nitrate and ammonium proportions (NO3-/NH4+) in the growth media can increase metal phytoextraction compared to supplying solely NO3-. However, there are no studies showing these effects in plants under copper (Cu) contamination as well as their consequences in plant stress tolerance. The objective was to evaluate the effect of NO3-/NH4+ proportions in Cu phytoextraction by Panicum maximum cv. Tanzania and its consequence in the oxidative stress, photosynthesis, and antioxidant system under Cu stress. The experiment was carried out in a randomized complete block design, by using a 3 × 4 factorial with six replications. Three NO3-/NH4+ proportions (100/0, 70/30, and 50/50) were combined with four Cu rates (0.3, 250, 500, and 1000 µmol L-1) in the nutrient solution. It was found that the largest Cu accumulation in the shoots occurred at the first harvest of the plants supplied with 70/30 NO3-/NH4+ and Cu 1000 µmol L-1. Such plants also displayed high concentrations of proline in the shoots as well as high superoxide dismutase activity in the roots. Malondialdehyde concentration was high in the plant parts at the Cu rate of 1000 µmol L-1. Hence, transpiration rates, stomatal conductance, quantum efficiency of photosystem II, electron transport rate, and net photosynthesis were all low at the Cu rate of 1000 µmol L-1. Catalase, guaiacol peroxidase, ascorbate peroxidase, and glutathione reductase activities in the roots were high when plants were exposed to Cu 1000 µmol L-1. In conclusion, the combination of NO3- with NH4+ increases copper phytoextraction that causes oxidative stress, but also favors the antioxidant system of Tanzania guinea grass in attempt to tolerate such stress.

Diterpene Synthase-Catalyzed Biosynthesis of Distinct Clerodane Stereoisomers.

The diterpene synthase clerodienyl diphosphate synthase 1 (PvCPS1) from the crop plant switchgrass (Panicum virgatum) stereoselectively converts (E,E,E)-geranylgeranyl diphosphate (GGPP) into the clerodane natural product, cis-trans-clerodienyl diphosphate (CLPP, 1). Structure-guided point mutations of PvCPS1 redirected product stereoselectivity toward the formation of a rare cis-clerodane diastereomer, cis-cis-CLPP (2). Additionally, an alternative cis-clerodane diastereomer, (5S,8S,9R,10R)-13Z-CLPP (3), was produced when treating PvCPS1 and select variants thereof with the cis-prenyl substrate (Z,Z,Z)-nerylneryl diphosphate (NNPP). These results support the hypothesis that substrate configuration and minor active-site alterations impact precatalysis substrate folding in the stereoselective biosynthesis of clerodane diterpenoid scaffolds, and can be employed to provide enzymatic access to a broader range of bioactive clerodane natural products.

Simultaneous regulation of F5H in COMT-RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis.

Ferulate 5-hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O-methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT-down-regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down-regulation of F5H in COMT-RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5-OH G units. Conversely, overexpression of F5H in COMT-RNAi transgenic plants reduced G units and increased 5-OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down-regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up-regulation and down-regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.

Ultrastructure and Enzymatic Hydrolysis of Deuterated Switchgrass.

Neutron scattering of deuterated plants can provide fundamental insight into the structure of lignocellulosics in plant cell walls and its deconstruction by pretreatment and enzymes. Such plants need to be characterized for any alterations to lignocellulosic structure caused by growth in deuterated media. Here we show that glucose yields from enzymatic hydrolysis at lower enzyme loading were 35% and 30% for untreated deuterated and protiated switchgrass, respectively. Lignin content was 4% higher in deuterated switchgrass but there were no significant lignin structural differences. Transmission electron microscopy showed differences in lignin distribution and packing of fibers in the cell walls that apparently increased surface area of cellulose in deuterated switchgrass, increasing cellulose accessibility and lowering its recalcitrance. These differences in lignification were likely caused by abiotic stress due to growth in deuterated media.

Cationic peroxidase from proso millet induces human colon cancer cell necroptosis by regulating autocrine TNF-α and RIPK3 demethylation.

A cationic peroxidase (POD) was purified from proso millet seeds (PmPOD) using ammonium sulfate fractionation, cation exchange, and size exclusion chromatography. The purified PmPOD showed toxicity to normal cells and tumor cells, but was more sensitive in HT29 cells. Furthermore, the mechanism driving HCT116 and HT29 cell death by PmPOD was the induction of receptor interacting protein kinase 1 (RIPK1)- and RIPK3-dependent necroptosis, independent of apoptosis. More importantly, PmPOD could induce tumor necrosis factor-α (TNF-α) production through transcriptional upregulation. In addition, PmPOD could restore RIPK3 expression in HCT116 cells via the demethylation of the RIPK3 genomic sequence. Taken together, these results suggest that two distinct mechanisms are involved in PmPOD-induced necroptosis: the autocrine production of TNF-α and the restoration of RIPK3 expression.

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.

A new peroxidase from leaves of guinea grass (Panicum maximum): A potential biocatalyst to build amperometric biosensors.

A new plant peroxidase was isolated from the leaves of guinea grass (Panicum maximum) and partially purified using a biphasic polymer system (poly(ethylene glycol) - ammonium sulfate) followed by size-exclusion chromatography and ultracentrifugation until obtaining a homogeneous extract containing a high peroxidase activity. The novel peroxidase was characterized as having a specific activity of 408U/mg and a molecular weight of 30kDa. The pH for its optimum activity was 8.0 and exhibited a high thermostability at 66°C with a kinact of 8.0×10-3min-1. The best substrates for peroxidase from guinea grass are o-dianisidine and 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid). POD from guinea grass was directly immobilized on the surface of a graphene screen printed electrode and cyclic voltammograms in the presence of potassium ferrocyanide ([Fe(CN)6]3-/4-) as a redox species demonstrated an increase in the electron transfer process. The graphene- modified electrode exhibits excellent electrocatalytic activity to the reduction of H2O2, with a linear response in the 100µM to 3.5mM concentration range and a detection limit of 150µM. The new peroxidase from guinea grass allowed the modification of a graphene electrode providing a potential sensor detection system for determination of H2O2 in real samples with some biomedical or environmental importance.

Characterization of Class III Peroxidases from Switchgrass.

Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ultimately lead to the formation of lignin. In general, CIIIPRX genes encode a large number of isozymes with ranges of in vitro substrate specificities. In order to elucidate the mode of substrate specificity of these enzymes, we characterized one of the CIIIPRXs (PviPRX9) from switchgrass (Panicum virgatum), a strategic plant for second-generation biofuels. The crystal structure, kinetic experiments, molecular docking, as well as expression patterns of PviPRX9 across multiple tissues and treatments, along with its levels of coexpression with the majority of genes in the monolignol biosynthesis pathway, revealed the function of PviPRX9 in lignification. Significantly, our study suggested that PviPRX9 has the ability to oxidize a broad range of phenylpropanoids with rather similar efficiencies, which reflects its role in the fortification of cell walls during normal growth and root development and in response to insect feeding. Based on the observed interactions of phenylpropanoids in the active site and analysis of kinetics, a catalytic mechanism involving two water molecules and residues histidine-42, arginine-38, and serine-71 was proposed. In addition, proline-138 and gluntamine-140 at the 137P-X-P-X140 motif, leucine-66, proline-67, and asparagine-176 may account for the broad substrate specificity of PviPRX9. Taken together, these observations shed new light on the function and catalysis of PviPRX9 and potentially benefit efforts to improve biomass conservation properties in bioenergy and forage crops.

An essential role of caffeoyl shikimate esterase in monolignol biosynthesis in Medicago truncatula.

Biochemical and genetic analyses have previously identified caffeoyl shikimate esterase (CSE) as an enzyme in the monolignol biosynthesis pathway in Arabidopsis thaliana, although the generality of this finding has been questioned. Here we show the presence of CSE genes and associated enzyme activity in barrel medic (Medicago truncatula, dicot, Leguminosae), poplar (Populus deltoides, dicot, Salicaceae), and switchgrass (Panicum virgatum, monocot, Poaceae). Loss of function of CSE in transposon insertion lines of M. truncatula results in severe dwarfing, altered development, reduction in lignin content, and preferential accumulation of hydroxyphenyl units in lignin, indicating that the CSE enzyme is critical for normal lignification in this species. However, the model grass Brachypodium distachyon and corn (Zea mays) do not possess orthologs of the currently characterized CSE genes, and crude protein extracts from stems of these species exhibit only a weak esterase activity with caffeoyl shikimate. Our results suggest that the reaction catalyzed by CSE may not be essential for lignification in all plant species.

Comparative Analysis of Extremely Thermophilic Caldicellulosiruptor Species Reveals Common and Unique Cellular Strategies for Plant Biomass Utilization.

Microbiological, genomic and transcriptomic analyses were used to examine three species from the bacterial genus Caldicellulosiruptor with respect to their capacity to convert the carbohydrate content of lignocellulosic biomass at 70°C to simple sugars, acetate, lactate, CO2, and H2. Caldicellulosiruptor bescii, C. kronotskyensis, and C. saccharolyticus solubilized 38%, 36%, and 29% (by weight) of unpretreated switchgrass (Panicum virgatum) (5 g/liter), respectively, which was about half of the amount of crystalline cellulose (Avicel; 5 g/liter) that was solubilized under the same conditions. The lower yields with C. saccharolyticus, not appreciably greater than the thermal control for switchgrass, were unexpected, given that its genome encodes the same glycoside hydrolase 9 (GH9)-GH48 multidomain cellulase (CelA) found in the other two species. However, the genome of C. saccharolyticus lacks two other cellulases with GH48 domains, which could be responsible for its lower levels of solubilization. Transcriptomes for growth of each species comparing cellulose to switchgrass showed that many carbohydrate ABC transporters and multidomain extracellular glycoside hydrolases were differentially regulated, reflecting the heterogeneity of lignocellulose. However, significant differences in transcription levels for conserved genes among the three species were noted, indicating unexpectedly diverse regulatory strategies for deconstruction for these closely related bacteria. Genes encoding the Che-type chemotaxis system and flagellum biosynthesis were upregulated in C. kronotskyensis and C. bescii during growth on cellulose, implicating motility in substrate utilization. The results here show that capacity for plant biomass deconstruction varies across Caldicellulosiruptor species and depends in a complex way on GH genome inventory, substrate composition, and gene regulation.

Identification and overexpression of gibberellin 2-oxidase (GA2ox) in switchgrass (Panicum virgatum L.) for improved plant architecture and reduced biomass recalcitrance.

Gibberellin 2-oxidases (GA2oxs) are a group of 2-oxoglutarate-dependent dioxygenases that catalyse the deactivation of bioactive GA or its precursors through 2ß-hydroxylation reaction. In this study, putatively novel switchgrass C20 GA2ox genes were identified with the aim of genetically engineering switchgrass for improved architecture and reduced biomass recalcitrance for biofuel. Three C20 GA2ox genes showed differential regulation patterns among tissues including roots, seedlings and reproductive parts. Using a transgenic approach, we showed that overexpression of two C20 GA2ox genes, that is PvGA2ox5 and PvGA2ox9, resulted in characteristic GA-deficient phenotypes with dark-green leaves and modified plant architecture. The changes in plant morphology appeared to be associated with GA2ox transcript abundance. Exogenous application of GA rescued the GA-deficient phenotypes in transgenic lines. Transgenic semi-dwarf lines displayed increased tillering and reduced lignin content, and the syringyl/guaiacyl lignin monomer ratio accompanied by the reduced expression of lignin biosynthetic genes compared to nontransgenic plants. A moderate increase in the level of glucose release in these transgenic lines might be attributed to reduced biomass recalcitrance as a result of reduced lignin content and lignin composition. Our results suggest that overexpression of GA2ox genes in switchgrass is a feasible strategy to improve plant architecture and reduce biomass recalcitrance for biofuel.

Transgenic switchgrass (Panicum virgatum L.) biomass is increased by overexpression of switchgrass sucrose synthase (PvSUS1).

Sucrose synthase (SUS) converts sucrose and uridine di-phosphate (UDP) into UDP-glucose and fructose. UDP-glucose is used by the cellulose synthase to produce cellulose for cell wall biosynthesis. For lignocellulosic feedstocks such as switchgrass, the manipulation of cell walls to decrease lignin content is needed to reduce recalcitrance of conversion of biomass into biofuels. Of perhaps equal importance for bioenergy feedstocks is increasing biomass. Four SUS genes were identified in switchgrass. Each gene contained 14 or 15 introns. PvSUS1 was expressed ubiquitously in the tissues tested. PvSUS2 and PvSUS6 were highly expressed in internodes and roots, respectively. PvSUS4 was expressed in low levels in the tissues tested. Transgenic switchgrass plants overexpressing PvSUS1 had increases in plant height by up to 37%, biomass by up to 13.6%, and tiller number by up to 79% compared to control plants. The lignin content was increased in all lines, while the sugar release efficiency was decreased in PvSUS1-overexpressing transgenic switchgrass plants. For switchgrass and other bioenergy feedstocks, the overexpression of SUS1 genes might be a feasible strategy to increase both plant biomass and cellulose content, and to stack with other genes to increase biofuel production per land area cultivated.

Photosynthesis of C3, C3-C4, and C4 grasses at glacial CO2.

Most physiology comparisons of C3 and C4 plants are made under current or elevated concentrations of atmospheric CO2 which do not reflect the low CO2 environment under which C4 photosynthesis has evolved. Accordingly, photosynthetic nitrogen (PNUE) and water (PWUE) use efficiency, and the activity of the photosynthetic carboxylases [Rubisco and phosphoenolpyruvate carboxylase (PEPC)] and decarboxylases [NADP-malic enzyme (NADP-ME) and phosphoenolpyruvate carboxykinase (PEP-CK)] were compared in eight C4 grasses with NAD-ME, PCK, and NADP-ME subtypes, one C3 grass, and one C3-C4 grass grown under ambient (400 µl l(-1)) and glacial (180 µl l(-1)) CO2. Glacial CO2 caused a smaller reduction of photosynthesis and a greater increase of stomatal conductance in C4 relative to C3 and C3-C4 species. Panicum bisulcatum (C3) acclimated to glacial [CO2] by doubling Rubisco activity, while Rubisco was unchanged in Panicum milioides (C3-C4), possibly due to its high leaf N and Rubisco contents. Glacial CO2 up-regulated Rubisco and PEPC activities in concert for several C4 grasses, while NADP-ME and PEP-CK activities were unchanged, reflecting the high control exerted by the carboxylases relative to the decarboxylases on the efficiency of C4 metabolism. Despite having larger stomatal conductance at glacial CO2, C4 species maintained greater PWUE and PNUE relative to C3-C4 and C3 species due to higher photosynthetic rates. Relative to other C4 subtypes, NAD-ME and PEP-CK grasses had the highest PWUE and PNUE, respectively; relative to C3, the C3-C4 grass had higher PWUE and similar PNUE at glacial CO2. Biomass accumulation was reduced by glacial CO2 in the C3 grass relative to the C3-C4 grass, while biomass was less reduced in NAD-ME grasses compared with NADP-ME and PCK grasses. Under glacial CO2, high resource use efficiency offers a key evolutionary advantage for the transition from C3 to C4 photosynthesis in water- and nutrient-limited environments.

Cholinesterase activity on Echinogammarus meridionalis (Pinkster) and Atyaephyra desmarestii (Millet): characterisation and in vivo effects of copper and zinc.

Metals are released into freshwater ecosystems from natural and anthropogenic sources, compromising their structural and functional equilibrium. As early warning tools, cholinesterases (ChEs) are usually used to assess the effects of organophosphate and carbamate pesticides, but are also known to be inhibited by metals. The objectives of this work were to characterise the activity of ChE present in the amphipod Echinogammarus meridionalis and the shrimp Atyaephyra desmarestii and to evaluate the in vivo effects of the metals copper and zinc in their ChE activity. To achieve this, firstly the activity of ChE forms were characterised using different in vitro assays with substrates and selective inhibitors. Then, the in vivo effects of 48 h exposures to increasing concentrations of copper and zinc on ChE activity were determined. The ChE form present in both species was acetylcholinesterase (AChE) since both revealed preference for the acetylthiocholine iodide substrate, total inhibition with eserine, the inhibitor of ChEs, and with 1,5-bis(4-allyldimethylammoniumphenyl)-pentan-3-one dibromide, the specific inhibitor of AChE, and presented insensitivity to iso-OMPA, a specific inhibitor of butyrylcholinesterase. The activity of ChEs was inhibited by zinc exposures in the amphipod species, but was not affected by copper. Exposure to copper and zinc did not affect ChEs activity in the shrimp at the concentrations tested. This work is a relevant contribution as foundation for the use of AChE in freshwater crustaceans in further studies including biomonitoring campaigns in different contamination scenarios.