Phytotoxicity of amoxicillin to the duckweed Spirodela polyrhiza: Growth, oxidative stress, biochemical traits and antibiotic degradation.

The increasing availability of antibiotics in wastewater has created a serious threat to non-target organisms in the environment. The aim of this study was to evaluate the potential toxicity of amoxicillin on duckweed Spirodela polyrhiza during a short-term exposure (7 d). The duckweed was exposed to a range of environmentally relevant (0.0001-0.01 mg L-1) and high (0.1 and 1 mg L-1) concentrations of amoxicillin. Subsequently, biomarkers of toxicity such as growth, pigments (Chl a, Chl b and carotenoids), antioxidative enzyme activity (catalase, CAT; superoxide dismutase, SOD; and ascorbate peroxidases, APX), and biochemical content (protein, lipid and starch) were analysed in their fronds. The high dose (1 mg L-1) of amoxicillin caused a significant (p < 0.05) decrease in photopigments, protein, starch and lipid content and an increase in carotenoids/total Chl and Chl a/Chl b ratios in fronds of Spirodela polyrhiza. The results showed a shift in biomarkers: a decrease in frond growth and relative growth rate (RGR) (16.2-53.8%) and an increase in the activities (mmol mg protein-1) of CAT (0.021-0.041), APX (0.84-2.49) and SOD (0.12-0.23) in fronds. The significantly (p < 0.05) greater reduction in amoxicillin content in duckweed setups (84.6-100%) than in the control (62.1-73%) suggested that phytodegradation is an important mechanism in removing antibiotics from water, apart from hydrolysis and photodegradation, which occur in control setups. Overall, the results suggested a toxic effect of amoxicillin on Spirodela polyrhiza, even at low concentrations, and nonetheless, the duckweed contributed directly to the degradation of antibiotics in the water and throughout the phytoremediation process.

Uranium and cadmium provoke different oxidative stress responses in Lemna minor L.

Common duckweed (Lemna minor L.) is ideally suited to test the impact of metals on freshwater vascular plants. Literature on cadmium (Cd) and uranium (U) oxidative responses in L. minor are sparse or, for U, non-existent. It was hypothesised that both metals impose concentration-dependent oxidative stress and growth retardation on L. minor. Using a standardised 7-day growth inhibition test, the adverse impact of these metals on L. minor growth was confirmed, with EC50 values for Cd and U of 24.1 ± 2.8 and 29.5 ± 1.9 µm, respectively, and EC10 values of 1.5 ± 0.2 and 6.5 ± 0.9 µm, respectively. The metal-induced oxidative stress response was compared through assessing the activity of different antioxidative enzymes [catalase, glutathione reductase, superoxide dismutase (SOD), ascorbate peroxidase (APOD), guaiacol peroxidase (GPOD) and syringaldizyne peroxidase (SPOD)]. Significant changes in almost all antioxidative enzymes indicated their importance in counteracting the U- and Cd-imposed oxidative burden. However, some striking differences were also observed. For activity of APODs and SODs, a biphasic but opposite response at low Cd compared to U concentrations was found. In addition, Cd (0.5-20 µm) strongly enhanced plant GPOD activity, whereas U inhibited it. Finally, in contrast to Cd, U up to 10 µm increased the level of chlorophyll a and b and carotenoids. In conclusion, although U and Cd induce similar growth arrest in L. minor, the U-induced oxidative stress responses, studied here for the first time, differ greatly from those of Cd.

Calcium-mediated responses and glutamine synthetase expression in greater duckweed (Spirodela polyrhiza L.) under diethyl phthalate-induced stress.

This study was carried out to assess the influence of diethyl phthalate (DEP) alone or associated with calcium chloride (CaCl2) on greater duckweed plants, emphasizing the implications of calcium in amelioration of DEP-induced stress on plant growth. Greater duckweed were treated with DEP in variable concentrations, as 0, 0.25, 0.5, 1.0 and 2.0mM for 7 days, or treated with the same concentration either 2mM DEP or 2mM DEP plus 10mM CaCl2·2H2O in different duration 0-7 days. Treatment with 2mM DEP resulted in increasing proline content, protease activity, and ammonia accumulation in duckweed tissues. NADH-glutamate dehydrogenase (NADH-GDH; EC 1.4.1.2) and Δ(1)-pyrroline-5-carboxylate reductase (P5CR; EC 1.5.1.2), two key enzymes in the glutamate pathway of proline synthesis, showed increase in activity with DEP treatment and positively correlated with proline accumulation. No further increase in proline accumulation was observed with addition of calcium chloride to the DEP-treated cultures. However, supplementation of Ca(2+) can mitigate the adverse effect of DEP, at least in part to decrease the DEP-induced superoxide accumulation and increase in GDH activity for ammonia assimilation in duckweed fronds. In addition, effects of calcium on mitigation of DEP injury were also observed in glutamine synthetase (GS; EC 6.3.1.2) expression. Both GS1 and GS2 polypeptide accumulation and the level of total GS activity were nearly equivalent to the control. Exogenous proline protects GS2 from DEP-modulated redox damage in the chloroplast lysates but there is no remarkable protection effects on D1 (the 32kDa protein in photosystem II reaction center) degradation. In conclusion, the glutamate pathway of proline synthesis might be involved in mitigation of DEP-induced injury, and calcium plays an important role in increasing GDH, P5CR, and GS expression.

Crystal structure and statistical coupling analysis of highly glycosylated peroxidase from royal palm tree (Roystonea regia).

Royal palm tree peroxidase (RPTP) is a very stable enzyme in regards to acidity, temperature, H(2)O(2), and organic solvents. Thus, RPTP is a promising candidate for developing H(2)O(2)-sensitive biosensors for diverse applications in industry and analytical chemistry. RPTP belongs to the family of class III secretory plant peroxidases, which include horseradish peroxidase isozyme C, soybean and peanut peroxidases. Here we report the X-ray structure of native RPTP isolated from royal palm tree (Roystonea regia) refined to a resolution of 1.85A. RPTP has the same overall folding pattern of the plant peroxidase superfamily, and it contains one heme group and two calcium-binding sites in similar locations. The three-dimensional structure of RPTP was solved for a hydroperoxide complex state, and it revealed a bound 2-(N-morpholino) ethanesulfonic acid molecule (MES) positioned at a putative substrate-binding secondary site. Nine N-glycosylation sites are clearly defined in the RPTP electron-density maps, revealing for the first time conformations of the glycan chains of this highly glycosylated enzyme. Furthermore, statistical coupling analysis (SCA) of the plant peroxidase superfamily was performed. This sequence-based method identified a set of evolutionarily conserved sites that mapped to regions surrounding the heme prosthetic group. The SCA matrix also predicted a set of energetically coupled residues that are involved in the maintenance of the structural folding of plant peroxidases. The combination of crystallographic data and SCA analysis provides information about the key structural elements that could contribute to explaining the unique stability of RPTP.