Parts-Prospecting for a High-Efficiency Thiamin Thiazole Biosynthesis Pathway.

Plants synthesize the thiazole precursor of thiamin (cThz-P) via THIAMIN4 (THI4), a suicide enzyme that mediates one reaction cycle and must then be degraded and resynthesized. It has been estimated that this THI4 turnover consumes 2% to 12% of the maintenance energy budget and that installing an energy-efficient alternative pathway could substantially increase crop yield potential. Available data point to two natural alternatives to the suicidal THI4 pathway: (i) nonsuicidal prokaryotic THI4s that lack the active-site Cys residue on which suicide activity depends, and (ii) an uncharacterized thiazole synthesis pathway in flowers of the tropical arum lily Caladium bicolor that enables production and emission of large amounts of the cThz-P analog 4-methyl-5-vinylthiazole (MVT). We used functional complementation of an Escherichia coli ΔthiG strain to identify a nonsuicidal bacterial THI4 (from Thermovibrio ammonificans) that can function in conditions like those in plant cells. We explored whether C. bicolor synthesizes MVT de novo via a novel route, via a suicidal or a nonsuicidal THI4, or by catabolizing thiamin. Analysis of developmental changes in MVT emission, extractable MVT, thiamin level, and THI4 expression indicated that C. bicolor flowers make MVT de novo via a massively expressed THI4 and that thiamin is not involved. Functional complementation tests indicated that C. bicolor THI4, which has the active-site Cys needed to operate suicidally, may be capable of suicidal and - in hypoxic conditions - nonsuicidal operation. T. ammonificans and C. bicolor THI4s are thus candidate parts for rational redesign or directed evolution of efficient, nonsuicidal THI4s for use in crop improvement.

Contribution of Bacillus cereus ERBP in ozone detoxification by Zamioculcas zamiifolia plants: Effect of ascorbate peroxidase, catalase and total flavonoid contents for ozone detoxification.

Eighteen plant species were screened for ozone (O3) removal in a continuous system. Zamioculcas zamiifolia had the highest O3 removal efficiency. To enhance O3 removal by Z. zamiifolia, adding a compatible endophytic bacteria, Bacillus cereus ERBP into Z. zamiifolia was studied. After operating under an O3 continuous system (150-250 ppb) at a flow rate of 0.3 L min-1 for 80 h, inoculated plants (74%) exhibited higher O3 removal efficiency than non-inoculated ones (53%). In addition, after O3 exposure (80 h), the population of B. cereus ERBP in inoculated plants was significantly increased in both shoots approximately 35 folds and leaves 13 folds compared to inoculated plants without O3 exposure. The results also showed that B. cereus ERBP had the ability to protect Z. zamiifolia against O3 stress conditions. The increase in B. cereus ERBP populations was attributed to the significant increase in ascorbate peroxidase (APX) and catalase (CAT) activity. In addition, increasing B. cereus ERBP populations led to raise total flavonoid contents which is one of antioxidant compounds. Increasing APX, CAT activities, and total flavonoid contents can enhance O3 detoxification in plant tissues. The mechanism of B. cereus ERBP for enhancing O3 phytoremediation was proposed in this study. The results suggested that B. cereus ERBP was a potential tool for alleviating O3 stress on Z. zamiifolia and enhancing O3 phytoremediation efficiency.

Combined toxic effects of microcystin-LR and phenanthrene on growth and antioxidant system of duckweed (Lemna gibba L.).

Microcystins and polycyclic aromatic hydrocarbons commonly co-exist in eutrophic freshwater environments. However, their combined toxicity remains unknown. The aim of this study was to evaluate the combined toxic effects of microcystin-LR (MC-LR) and phenanthrene (Phe) on duckweed (Lemna gibba L.) during a short-term exposure (7 d). L. gibba was exposed to a range of environmentally relevant concentrations of MC-LR (5, 50, 250, 500 µg/L) and Phe (0.1, 1, 5, 10 µg/L), both individually and in MC-LR + Phe mixtures (5 + 0.1, 50 + 1, 250 + 5, 500 + 10 µg/L). Subsequently, biomarkers of toxicity such as growth, chlorophyll-a, and antioxidant enzyme activity (catalase, superoxide dismutase, and peroxidase) were analyzed in L. gibba. Growth and the antioxidant system of L. gibba were not significantly inhibited by Phe alone, whereas higher concentrations of individual MC-LR (≥50 µg/L) significantly inhibited growth and induced oxidative stress. Based on Abott's formula, their interaction effects were concentration dependent. Antagonistic effects were observed when exposed to combinations of lower concentrations of MC-LR and Phe (≤50 + 1 µg/L), while additive or synergistic effects were induced at higher concentrations of both compounds (≥250 + 5 µg/L). Moreover, higher concentrations of Phe (≥5 µg/L) increased the accumulation of MC-LR in L. gibba. Our results suggested that the toxic effects of MC-LR and phenanthrene were exacerbated only when they co-exist in water bodies at relatively high concentrations. Consequently, co-existence of MC-LR and Phe at low levels are unlikely to exacerbate ecological hazards to L. gibba in most aquatic environments, at least based on responses of this plant.

Effect of exogenous catechin on alleviating O3 stress: The role of catechin-quinone in lipid peroxidation, salicylic acid, chlorophyll content, and antioxidant enzymes of Zamioculcas zamiifolia.

Ozone (O3) can cause oxidative stress in plants and humans. Catechin is an antioxidant that enriches tea and can probably increase O3 tolerance in plants. To investigate the mechanism of catechin to alleviate O3 stress in plants, Zamiocalcus zamiifolia (an efficient plant for O3 phytoremediation) was sprayed with 5 mM catechin and was used to expose O3 (150-250) under long-term operation (10 cycles). We investigated whether exogenous catechin could enhance O3 removal and alleviate O3 stress through a balanced redox state in plants. Z. zamiifolia sprayed with catechin exhibited higher O3 removal (80.27±3.12%), than Z. zamiifolia without catechin (50.03±2.68%). O3 in the range of 150-250 ppb led to stress in plants, as shown by an increased malondialdehyde content (MDA) and salicylic acid (SA). Whereas under the presence of O3, exogenous catechin could maintain the MDA content and inhibit SA accumulation. Under Z. zamiifolia+catechin+O3 conditions, catechin reacted with O3, which led to the formation of catechin-quinone. The formation of catechin-quinone was confirmed by the depletion of reduced glutathione content (GSH). This catechin-quinone could induce GST and APX genes that are up-regulated approximately 35- and 5-fold, respectively. Hence, Z. zamiifolia+catechin+O3 conditions had higher performance for coping with oxidative stress than did Z. zamiifolia+O3 conditions. This evidence demonstrates that catechin could enhance O3 removal through a balanced redox state in plant cells. Finally, the application of tea extract for enhanced O3 removal is also shown in this study.

Effects of treated industrial wastewaters and temperatures on growth and enzymatic activities of duckweed (Lemna minor L.).

The efficacy of the removal of contaminants from wastewater depends on physico-chemical properties of pollutants and the efficiency of treatment plant. Sometimes, low amounts of toxic compounds can be still present in the treated sewage. In this work we considered the effects of contaminant residues in treated wastewaters and of temperatures on Lemna minor L. Treated effluent waters were collected, analyzed and used as duckweed growth medium. In order to better understand the effects of micropollutants and seasonal variation, the plants were grown under ambient conditions for seven days in summer and winter. Relative growth rate, pigments and phenolic compounds concentrations were determined, as well as the activities of catalase (CAT), ascorbate peroxidase (APX), guaiacol peroxidase (G-POD) and polyphenol oxidase (PPO). The pollutant concentrations varied in the two seasons, depending on the industrial and municipal activities and efficiency of treatments. Treated waters contained heavy metals, nitrogenous and phosphorus compounds, surfactants and hydrocarbons. Compared to the control, duckweed growth of treated plants decreased by 25% in summer, while in the winter due to the lower temperatures and the presence of pollutants was completely impeded. The amounts of photosynthetic pigments of treated plants were not significantly affected in the summer, while they were higher than the control in the winter when the effluent had a high nitrogen amount. High CAT activity was registered in both seasons. Treated plants had significantly lower APX activity in the summer (53%) and winter (59%) respect to the controls. The observed inhibition of the peroxidase activities in the exposed plants, confirms the controversy existing in the literature about the variability of enzymatic response in stress condition.

Alleviation of cadmium toxicity in Lemna minor by exogenous salicylic acid.

Cadmium (Cd) is a significant environmental pollutant in the aquatic environment. Salicylic acid (SA) is a ubiquitous phenolic compound. The goal of this study was to assess the morphological, physiological and biochemical changes in duckweed (L. minor) upon exposure to 10µM CdCl2, 10µM CdCl2 plus 50µM SA, or 50µM SA for 7 days. Reversing the effects of Cd, SA decreased Cd accumulation in plants, improved accumulation of minerals (Ca, Mg, Fe, B, Mo) absorption, increased endogenous SA concentration, and phenylalanine ammonialyase (PAL) activity. Chlorosis-associated symptoms, the reduction in chlorophyll content, and the overproduction of reactive oxygen species induced by Cd exposure were largely reversed by SA. SA significantly decreased the toxic effects of Cd on the activities of the superoxide dismutase, peroxidase, catalase, ascorbate peroxidase, and glutathione reductase in the fronds of L. minor. Furthermore, SA reversed the detrimental effects of Cd on total ascorbate, glutathione, the ascorbic acid/oxidized dehydroascorbate and glutathione/glutathione disulphide ratios, lipid peroxidation, malondialdehyde concentration, lipoxygenase activity, and the accumulation of proline. SA induced the up-regulation of heat shock proteins (Hsp70) and attenuated the adverse effects of Cd on cell viability. These results suggest that SA confers tolerance to Cd stress in L. minor through different mechanisms.

Mercury induced oxidative stress, DNA damage, and activation of antioxidative system and Hsp70 induction in duckweed (Lemna minor).

Mercury uptake and its effects on physiology, biochemistry and genomic stability were investigated in Lemna minor after 2 and 6d of exposure to 0-30µM Hg. The accumulation of Hg increased in a concentration- and duration-dependent manner, and was positively correlated with the leaf damage. Oxidative stress after Hg exposure was evidenced in L. minor by a significant decrease in photosynthetic pigments, an increase in malondialdehyde and lipoxygenase activities (total enzyme activity and isoenzymes activity). Fronds of L. minor exposed to Hg showed an induction of peroxidase, catalase, and ascorbate peroxidase activities (total enzyme activity and some isoenzymes activities). Exposure of L. minor to Hg reduced the activity (total enzyme activity and some isoenzymes activities) of glutathione reductase, and superoxide dismutase. Exposure to Hg produced a transient increase in the content of glutathione and ascorbic acid. The content of dehydroascorbate and oxidized glutathione in L. minor were high during the entire exposure period. Exposure of L. minor to Hg also caused the accumulation of proline and soluble sugars. The amplification of new bands and the absence of normal DNA amplicons in treated plants in the random amplified polymorphic DNA (RAPD) profile indicated that genomic template stability (GTS) was affected by Hg treatment. The accumulation of Hsp70 indicated the occurrence of a heat shock response at all Hg concentrations. These results suggest that L. minor plants were able to cope with Hg toxicity through the activation of various mechanisms involving enzymatic and non-enzymatic antioxidants, up-regulation of proline, and induction of Hsp70.

Biodegradation of direct blue 129 diazo dye by Spirodela polyrrhiza: An artificial neural networks modeling.

Phytoremediation potential of the aquatic plant Spirodela polyrrhiza was examined for direct blue 129 (DB129) azo dye. The dye removal efficiency was optimized under the variable conditions of the operational parameters including removal time, initial dye concentration, pH, temperature and amount of plant. The study reflected the significantly enhanced dye removal efficiency of S. polyrrhiza by increasing the temperature, initial dye concentration and amount of plant. Intriguingly, artificial neural network (ANN) predicted the removal time as the most dominant parameter on DB129 removal efficiency. Furthermore, the effect of dye treatment on some physiologic indices of S. polyrrhiza including growth rate, photosynthetic pigments content, lipid peroxidation and antioxidant enzymes were studied. The results revealed a reduction in photosynthetic pigments content and in multiplication of fronds after exposure to dye solution. In contrast, malondialdehyde content as well as catalase (CAT) and peroxidase (POD) activities significantly increased that was probably due to the ability of plant to overcome oxidative stress. As a result of DB129 biodegradation, a number of intermediate compounds were identified by gas chromatography-mass spectroscopy (GC-MS) analysis. Accordingly, the probable degradation pathway of DB129 in S. polyrrhiza was postulated.

Probing the ubiquinol-binding site of recombinant Sauromatum guttatum alternative oxidase expressed in E. coli membranes through site-directed mutagenesis.

In the present paper we have investigated the effect of mutagenesis of a number of highly conserved residues (R159, D163, L177 and L267) which we have recently shown to line the hydrophobic inhibitor/substrate cavity in the alternative oxidases (AOXs). Measurements of respiratory activity in rSgAOX expressed in Escherichia coli FN102 membranes indicate that all mutants result in a decrease in maximum activity of AOX and in some cases (D163 and L177) a decrease in the apparent Km (O2). Of particular importance was the finding that when the L177 and L267 residues, which appear to cause a bottleneck in the hydrophobic cavity, are mutated to alanine the sensitivity to AOX antagonists is reduced. When non-AOX anti-malarial inhibitors were also tested against these mutants widening the bottleneck through removal of isobutyl side chain allowed access of these bulkier inhibitors to the active-site and resulted in inhibition. Results are discussed in terms of how these mutations have altered the way in which the AOX's catalytic cycle is controlled and since maximum activity is decreased we predict that such mutations result in an increase in the steady state level of at least one O2-derived AOX intermediate. Such mutations should therefore prove to be useful in future stopped-flow and electron paramagnetic resonance experiments in attempts to understand the catalytic cycle of the alternative oxidase which may prove to be important in future rational drug design to treat diseases such as trypanosomiasis. Furthermore since single amino acid mutations in inhibitor/substrate pockets have been found to be the cause of multi-drug resistant strains of malaria, the decrease in sensitivity to main AOX antagonists observed in the L-mutants studied in this report suggests that an emergence of drug resistance to trypanosomiasis may also be possible. Therefore we suggest that the design of future AOX inhibitors should have structures that are less reliant on the orientation by the two-leucine residues. This article is part of a Special Issue entitled: 18th European Bioenergetic Conference.

Using proteomic analysis to investigate uniconazole-induced phytohormone variation and starch accumulation in duckweed (Landoltia punctata).

BACKGROUND: Duckweed (Landoltia punctata) has the potential to remediate wastewater and accumulate enormous amounts of starch for bioethanol production. Using systematical screening, we determined that the highest biomass and starch percentage of duckweed was obtained after uniconazole application. Uniconazole contributes to starch accumulation of duckweed, but the molecular mechanism is still unclear. RESULTS: To elucidate the mechanisms of high starch accumulation, in the study, the responses of L. punctata to uniconazole were investigated using a quantitative proteomic approach combined with physiological and biochemical analysis. A total of 3327 proteins were identified. Among these identified proteins, a large number of enzymes involved in endogenous hormone synthetic and starch metabolic pathways were affected. Notably, most of the enzymes involved in abscisic acid (ABA) biosynthesis showed up-regulated expression, which was consistent with the content variation. The increased endogenous ABA may up-regulate expression of ADP-glucose pyrophosphorylase to promote starch biosynthesis. Importantly, the expression levels of several key enzymes in the starch biosynthetic pathway were up-regulated, which supported the enzymatic assay results and may explain why there is increased starch accumulation. CONCLUSIONS: These generated data linked uniconazole with changes in expression of enzymes involved in hormone biosynthesis and starch metabolic pathways and elucidated the effect of hormones on starch accumulation. Thus, this study not only provided insights into the molecular mechanisms of uniconazole-induced hormone variation and starch accumulation but also highlighted the potential for duckweed to be feedstock for biofuel as well as for sewage treatment.

Involvement of chlororespiration in chilling stress in the tropical species Spathiphyllum wallisii.

Spathiphyllum wallisii plants were used to study the effect of chilling stress under high illumination on photosynthesis and chlororespiration. Leaves showed different responses that depended on root temperature. When stem, but not root, was chilled, photosystem II (PSII) was strongly photoinhibited. However, when the whole plant was chilled, the maximal quantum yield of PSII decreased only slightly below the normal values and cyclic electron transport was stimulated. Changes were also observed in the chlororespiration enzymes and PGR5. In whole plants chilled under high illumination, the amounts of NADH dehydrogenase (NDH) complex and plastid terminal oxidase (PTOX) remained similar to control and increased when only stem was chilled. In contrast, the amount of PGR5 polypeptide was higher in plants when both root and stem were chilled than in plants in which only stem was chilled. The results indicated that the contribution of chlororespiration to regulating photosynthetic electron flow is not relevant when the whole plant is chilled under high light, and that another pathway, such as cyclic electron flow involving PGR5 polypeptide, may be more important. However, when PSII activity is strongly photoinhibited in plants in which only stem is chilled, chlororespiration, together with other routes of electron input to the electron transfer chain, is probably essential.

ß-Radiation Stress Responses on Growth and Antioxidative Defense System in Plants: A Study with Strontium-90 in Lemna minor.

In the following study, dose dependent effects on growth and oxidative stress induced by ß-radiation were examined to gain better insights in the mode of action of ß-radiation induced stress in plant species. Radiostrontium (9°Sr) was used to test for ß-radiation induced responses in the freshwater macrophyte Lemna minor. The accumulation pattern of 90Sr was examined for L. minor root and fronds separately over a seven-day time period and was subsequently used in a dynamic dosimetric model to calculate ß-radiation dose rates. Exposing L. minor plants for seven days to a 9°Sr activity concentration of 25 up to 25,000 kBq·L⁻¹ resulted in a dose rate between 0.084 ± 0.004 and 97 ± 8 mGy·h⁻¹. After seven days of exposure, root fresh weight showed a dose dependent decrease starting from a dose rate of 9.4 ± 0.5 mGy·h⁻¹. Based on these data, an EDR10 value of 1.5 ± 0.4 mGy·h⁻¹ was estimated for root fresh weight and 52 ± 17 mGy·h⁻¹ for frond fresh weight. Different antioxidative enzymes and metabolites were further examined to analyze if ß-radiation induces oxidative stress in L. minor.

A molecular assessment of the genetic model of spathe color inheritance in Anthurium andraeanum (Hort.).

Past genetic studies have shown three independent loci designated O, R and M control spathe color in Anthurium andraeanum (Hort.). To evaluate the genetic model and to understand the control of anthocyanin biosynthesis at the molecular level, the expression of the anthocyanin biosynthetic genes, CHS, F3H, DFR, ANS and F3'H, was examined at the mRNA and protein levels and correlated to anthocyanin content and spathe color in eight genetically characterized anthurium cultivars representing different states of the O, R and M loci. The results showed that the expression of F3H and ANS was co-regulated by a putative transcription factor encoded by the R locus, and the expression of DFR was regulated by a putative transcription factor encoded by the O locus. White cultivars, which were in the homozygous recessive state for either O or R or both, exhibited reduced expression of the anthocyanin biosynthetic genes and hence had negligible levels of anthocyanin. Cultivars that were mm displayed reduced expression of F3'H suggesting that it may either encode a defective form of the F3'H gene or a regulator that controls its expression. Additionally, a correlation between anthocyanin abundance and the expression of F3'H in the red cultivars suggested that F3'H expression may be a key control point in the regulation of anthocyanin biosynthesis in anthurium and hence plays a major role in influencing the shade intensity in red cultivars. These findings provide evidence in support of the genetic model for color inheritance in the spathe.

Survey of the total fatty acid and triacylglycerol composition and content of 30 duckweed species and cloning of a Δ6-desaturase responsible for the production of γ-linolenic and stearidonic acids in Lemna gibba.

BACKGROUND: Duckweeds, i.e., members of the Lemnoideae family, are amongst the smallest aquatic flowering plants. Their high growth rate, aquatic habit and suitability for bio-remediation make them strong candidates for biomass production. Duckweeds have been studied for their potential as feedstocks for bioethanol production; however, less is known about their ability to accumulate reduced carbon as fatty acids (FA) and oil. RESULTS: Total FA profiles of thirty duckweed species were analysed to assess the natural diversity within the Lemnoideae. Total FA content varied between 4.6% and 14.2% of dry weight whereas triacylglycerol (TAG) levels varied between 0.02% and 0.15% of dry weight. Three FA, 16:0 (palmitic), 18:2Δ9,12 (Linoleic acid, or LN) and 18:3Δ9,12,15 (α-linolenic acid, or ALA) comprise more than 80% of total duckweed FA. Seven Lemna and two Wolffiela species also accumulate polyunsaturated FA containing Δ6-double bonds, i.e., GLA and SDA. Relative to total FA, TAG is enriched in saturated FA and deficient in polyunsaturated FA, and only five Lemna species accumulate Δ6-FA in their TAG. A putative Δ6-desaturase designated LgDes, with homology to a family of front-end Δ6-FA and Δ8-spingolipid desaturases, was identified in the assembled DNA sequence of Lemna gibba. Expression of a synthetic LgDes gene in Nicotiana benthamiana resulted in the accumulation of GLA and SDA, confirming it specifies a Δ6-desaturase. CONCLUSIONS: Total accumulation of FA varies three-fold across the 30 species of Lemnoideae surveyed. Nine species contain GLA and SDA which are synthesized by a Δ6 front-end desaturase, but FA composition is otherwise similar. TAG accumulates up to 0.15% of total dry weight, comparable to levels found in the leaves of terrestrial plants. Polyunsaturated FA is underrepresented in TAG, and the Δ6-FA GLA and SDA are found in the TAG of only five of the nine Lemna species that produce them. When present, GLA is enriched and SDA diminished relative to their abundance in the total FA pool.

Analysis of ADP-glucose pyrophosphorylase expression during turion formation induced by abscisic acid in Spirodela polyrhiza (greater duckweed).

BACKGROUND: Aquatic plants differ in their development from terrestrial plants in their morphology and physiology, but little is known about the molecular basis of the major phases of their life cycle. Interestingly, in place of seeds of terrestrial plants their dormant phase is represented by turions, which circumvents sexual reproduction. However, like seeds turions provide energy storage for starting the next growing season. RESULTS: To begin a characterization of the transition from the growth to the dormant phase we used abscisic acid (ABA), a plant hormone, to induce controlled turion formation in Spirodela polyrhiza and investigated their differentiation from fronds, representing their growth phase, into turions with respect to morphological, ultra-structural characteristics, and starch content. Turions were rich in anthocyanin pigmentation and had a density that submerged them to the bottom of liquid medium. Transmission electron microscopy (TEM) of turions showed in comparison to fronds shrunken vacuoles, smaller intercellular space, and abundant starch granules surrounded by thylakoid membranes. Turions accumulated more than 60% starch in dry mass after two weeks of ABA treatment. To further understand the mechanism of the developmental switch from fronds to turions, we cloned and sequenced the genes of three large-subunit ADP-glucose pyrophosphorylases (APLs). All three putative protein and exon sequences were conserved, but the corresponding genomic sequences were extremely variable mainly due to the invasion of miniature inverted-repeat transposable elements (MITEs) into introns. A molecular three-dimensional model of the SpAPLs was consistent with their regulatory mechanism in the interaction with the substrate (ATP) and allosteric activator (3-PGA) to permit conformational changes of its structure. Gene expression analysis revealed that each gene was associated with distinct temporal expression during turion formation. APL2 and APL3 were highly expressed in earlier stages of turion development, while APL1 expression was reduced throughout turion development. CONCLUSIONS: These results suggest that the differential expression of APLs could be used to enhance energy flow from photosynthesis to storage of carbon in aquatic plants, making duckweeds a useful alternative biofuel feedstock.

Genetic engineering of a Lemna isoleucine auxotroph.

Lemna, a member of the Lemnaceae or duckweed family, is a small aquatic plant that can be quickly transformed to produce recombinant proteins in a contained and controlled bioprocessing environment. The containment capability of Lemna has been further improved with the creation of an auxotroph platform that requires isoleucine supplementation for survival of transformed plant lines. Using an RNAi based approach, threonine deaminase (TD) expression was targeted and thus resulted in dramatically reduced expression of this key enzyme in the isoleucine biosynthesis pathway. Auxotrophic plants expressing RNAi for TD were generated in the presence of isoleucine and selected based on their inability to propagate without isoleucine supplementation. TD transcripts isolated from the superior auxotroph lines were shown to be less than 10% of wild type level and thus confirmed the auxotroph phenotype to be derived from the specific knock down of TD expression. When grown under optimal conditions with appropriate isoleucine supplementation, biomass accumulation of the auxotroph lines was equivalent to that of wild type plants. To demonstrate the application of this system for production of recombinant proteins, an avian influenza H5N1 hemagglutinin (HA) protein was expressed in the isoleucine auxotroph platform. The successful expression of H5N1 HA vaccine antigen, in the isoleucine auxotroph background demonstrates the applicability of using an auxotroph to express biotherapeutics and vaccines in a highly contained expression system.

Isolation and gene expression analysis of a papain-type cysteine protease in thermogenic skunk cabbage (Symplocarpus renifolius).

Skunk cabbage (Symplocarpus renifolius) spadices contain abundant transcripts for cysteine protease (CP). From thermogenic spadices, we isolated SrCPA, a highly expressed CP gene that encoded a papain-type CP. SrCPA is structurally similar to other plant CPs, including the senescence-associated CPs found in aroids. The expression of SrCPA increased during floral development, and was observed in all floral tissues except for the stamens.

Response of antioxidant defences to Zn stress in three duckweed species.

In the plants, Lemna gibba, Lemna minor and Spirodela polyrrhiza L., the effect of different concentrations of zinc (0.01, 0.05, 0.1, 0.5 and 1.5mgL(-1) Zn) applied for four day was assessed by measuring changes in the chlorophyll, protein, malondialdehyde (MDA), catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX) and guiacolperoxidase (GPX) activity of the plants. According to results, Zn contents in plants increased with increasing Zn supply levels. The level of photosynthetic pigments and soluble proteins reduced only upon exposure to high Zn concentrations. At the same time, the level of malondialdehyde (MDA) increased with increasing Zn concentration. These results suggested an alleviation of stress that was possibly the result of antioxidants such as catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX) as well as guaiacol peroxidise (GPOX), which increased linearly with increasing Zn levels. Cellular antioxidant levels showed an increase suggesting a defensive mechanism to preserve against oxidative stress given rise to by Zn. Besides, the proline amount in L. gibba, L. minor and S. polyrrhiza increased with increasing zinc levels. These conclusions proposed that L. gibba, L. minor and S. polyrrhiza are supplied with an efficient antioxidant mechanism against Zn-induced oxidative stress which saves the plant's photosynthetic machinery from damage. It is concluded that higher zinc levels cause oxidative stress in L. gibba, L. minor and S. polyrrhiza cells and may reason membrane damage through production of ROS and interferes with chlorophyll metabolism.

Quizalofop-p-ethyl-induced phytotoxicity and genotoxicity in Lemna minor and Lemna gibba.

In this study, the effects of the herbicide, quizalofop-p-ethyl, on pigment contents (total chlorophyll, chlorophyll a/b, carotenoid), antioxidant enzyme [superoxide dismutase (SOD) and guaiacol peroxidase (POD)] activities, lipid peroxidation product (malondialdehyde: MDA) and DNA profiles were investigated in Lemna gibba and Lemna minor. Laboratory-acclimatized plants were treated with quizalofop-p-ethyl at 31.375, 62.75, 125 and 250 mg L(-1) for 24 and 96 h. It was determined that quizalofop-p-ethyl affected both the physiological status and the DNA profiles of L. gibba and L. minor. The photosynthetic pigments of L. gibba were more sensitive to the herbicide than were those of L. minor at both treatment times. SOD and POD activities were elevated in both plants at 24 h. However at 96 h, SOD activity decreased in L. minor and had irregular changes in L. gibba.. Significant increases in the amounts of MDA were observed in L. gibba, whereas the levels of this compound decreased in L. minor at 24 and 96 h. Polymorphism in DNA profiles was determined using the Random Amplified Polymorphic DNA (RAPD) technique. Four primers were used for scoring (appearance and disappearance of DNA polymorphic bands), and equally weighted maximum parsimony analyses were performed. Fewer differences were observed at 24 h, and more new bands were observed at 96 h in L. gibba. The RAPD profiles of L. minor produced by all of the primers were slightly less affected by the herbicide treatment than were those of L. gibba.

Phytotoxicity and antioxidative enzymes of green microalga (Desmodesmus subspicatus) and duckweed (Lemna minor) exposed to herbicides MCPA, chloridazon and their mixtures.

In this study, we evaluate the toxicity of MCPA (auxin-like growth inhibitor), chloridazon (CHD) (PSII-inhibitor) and their mixtures to floating plants and planktonic algae. Toxicity of MCPA (4-chloro-2-methylphenoxyacetic acid) and CHD (5-amino-4-chloro-2-phenyl-3(2H)-pyridazinone) was first assessed in two growth inhibition tests with Lemna minor (ISO/DIS 20079) and Desmodesmus subspicatus (ISO 8692). Next, herbicide mixtures at concentrations corresponding to the EC values were used to assess their interactive effects, and the biomarkers were: for duckweed fresh weight, frond area, chlorophyll content and number of fronds, and for algae cell count and cell volume. The 3d EC10 and EC50 values using cell counts of D. subspicatus were 142.7 and 529.1 mg/L for MCPA and 1.7 and 5.1 mg/L for CHD. The 7d EC10 and EC50 values using frond number of L. minor amounted to 0.8 and 5.4 mg/L for MCPA and 0.7 and 10.4 mg/L for CHD. Higher sensitivity of reproductive (number of cells/fronds) than growth processes (cell volume/frond area) to herbicides applied individually and in mixtures was especially pronounced in the responses of Desmodesmus. Herbicide interactions were assessed by the two-way ANOVA and Abbott's formula. Generally, an antagonistic interaction with Lemna was revealed by MCPA and chloridazon, whereas additive effect of both herbicides was observed for Desmodesmus. A significant stimulation of SOD and APX activity by binary mixtures was noted in algal cells mainly after 24 and 48 hours of exposure. The extremely high stimulation of the activity of both enzymes was induced by the combination EC10CHD + EC50MCPA (48 h). Presumably due to oxidative stress, the treatment with CHD at concentration EC50 after 72 h was lethal for algae grown in aerated cultures, in contrast to standardized test conditions. Taking into account the consequences of risk assessment for herbicide mixtures we can state that a relatively low toxicity, as well as the lack of significant synergy between MCPA and CHD to non-target plants appears to be the most important result.