Monika Bojko

Physiological responses of Lemna trisulca L. (duckweed) to cadmium and copper bioaccumulation

Abstract Aquatic plants are known to accumulate and bioconcentrate heavy metals. In this study, several physiological responses of aquatic vascular plant Lemna trisulca L. to elevated concentrations of cadmium (up to 10 mM) and copper (up to 50 μM) were investigated. It was found that Lemna fronds were able to accumulate both cadmium and copper, but Cu-treated material showed pronounced toxic symptoms at concentrations 1000-fold lower in comparison to Cd. Lemna trisulca could tolerate elevated levels of Cd, i.e. up to 10 mM, without significant changes in photosynthetic pigments concentration. On the contrary, Cu in concentrations 25 and 50 μM promoted significant pigment degradation. The main processes affected by Cd in Lemna fronds were total gas exchange and net photosynthesis. On the contrary, the inhibition of total gas exchange and net photosynthesis caused by Cu (2–50 μM) correlated with Chl a and carotenoid concentrations decrease as well as with the decay of fluorescence from PS II. Also, an increasing impact of respiration in total oxygen exchange was observed after treatment of Lemna with increasing Cd concentrations (up to 5 mM) and with Cu in concentration range between 2 and 50 μM. In Cd-treated fronds, a dose-dependent accumulation of two polypeptides with apparent molecular weights 18 and 10 kDa, respectively as well as the appearance of two smaller polypeptides (apparent molecular weights 8 and 7 kDa) was observed in SDS-PAGE. The nature of these polypeptides remains to be determined. On the contrary, in Cu-treated fronds neither accumulation of existing proteins nor appearance of any extra protein was observed.

Plastoquinones are effectively reduced by ferredoxin:NADP+ oxidoreductase in the presence of sodium cholate micelles. Significance for cyclic electron transport and chlororespiration.

The effect of sodium cholate and other detergents (Triton X-100, sodium dodecyl sulphate, octyl glucoside, myristyltrimethylammonium bromide) on the reduction of plastoquinones (PQ) with a different length of the side-chain by spinach ferredoxin:NADP(+) oxidoreductase (FNR) in the presence of NADPH has been studied. Both NADPH oxidation and oxygen uptake due to plastosemiquinone autoxidation were highly stimulated only in the presence of sodium cholate among the used detergents. Sodium cholate at the concentration of 20 mM was found to be the most effective on both PQ-4 and PQ-9-mediated oxygen uptake. The FNR-dependent reduction of plastoquinones incorporated into sodium cholate micelles was stimulated by spinach ferredoxin but inhibited by Mg(2+) ions. It was concluded that the structure of sodium cholate micelles facilitates contact of plastoquinone molecules with the enzyme and creates favourable conditions for the reaction similar to those found in thylakoid membranes for PQ-9 reduction. The obtained results were discussed in terms of the function of FNR as a ferredoxin:plastoquinone reductase both in cyclic electron transport and chlororespiration.

Diaphorase activity of ferredoxin: NADP oxidoreductase in the presence of dibromothymoquinone

Abstract In the presence of dibromothymoquinone (10 μM) NADPH- and ferredoxin:NADP oxidoreductase-dependent oxygen uptake was observed in buffered (40 mM Tris-HCl buffer) medium. The rate of O 2 uptake depended on the pH of the reaction mixture and was about two orders of magnitude faster at pH 8.7 than at pH 6.7. In alkaline medium the rate was about 0.2 μmol O 2 min −1 cm −3 (or 9.3 nmol O 2 min −1 mg −1 FNR) at the time of tracing, whereas at a lower pH (7.7 or 6.7) oxygen consumption was 1.5–2.0-fold faster during the first minute of monitoring than during the second minute. Ferredoxin was not an obligatory component involved in this process. However, at a lower pH (7.7, 6.7) oxygen consumption by the reaction mixture was stimulated 2–8-fold by adding ferredoxin (0.6 μM). The v 2 value was also enhanced by ca 36% in the presence of 0.2 M NaCl, whereas inorganic pyrophosphate caused reduction of v 1 and v 2 to about 62 and 3% respectively, of the control. Under anaerobic conditions the Michaelis constant ( K m ) and the pseudo-first-order rate constant ( k ) for the FNR-catalysed DBMIB reduction was about 16 μM and 0.34 s −1 , respectively. The results indicate that DBMIB can accept electrons from NADPH-ferredoxin: NADP oxidoreductase, and the reduced DBMIB (presumably in the unprotonated form) can be reoxidized by molecular oxygen.

NADPH and ferredoxin:NADP+ oxidoreductase-dependent reduction of quinones and their reoxidation

Abstract Molecular oxygen uptake was initiated by adding NADPH (1 mM) to the buffered medium containing 0.6 μ M spinach ferredoxin:NADP + oxidoreductase and 20 μ M quinone (plastoquinone-2, decyl-plastoquinone, decyl-ubiquinone, or duroquinone). At pH 7.7 the rate of oxygen uptake was 2- to 12-fold higher during an initial phase (V 1 ) than in a subsequent phase (V 2 ). Except for duroquinone, the initial rate of oxygen consumption was ca. 2.7-fold higher in alkaline than in acidic medium. Ferredoxin was not essential, although it stimulated the reaction investigated. Oxygen uptake was not detectable with plastoquinone-9 or α-tocoquinone. The possible mechanisms of the NADPH and ferredoxin:NADP oxidoreductase dependent reduction of some quinones and their reoxidation are discussed.

Ferredoxin:NADP+ oxidoreductase as a target of Cd2+ inhibitory action--biochemical studies.

The ferredoxin:NADP+ oxidoreductase (FNR) catalyses the ferredoxin-dependent reduction of NADP+ to NADPH in linear photosynthetic electron transport. The enzyme also transfers electrons from reduced ferredoxin (Fd) or NADPH to the cytochrome b(6)f complex in cyclic electron transport. In vitro, the enzyme catalyses the NADPH-dependent reduction of various substrates, including ferredoxin, the analogue of its redox centre - ferricyanide, and the analogue of quinones, which is dibromothymoquinone. This paper presents results on the cadmium-induced inhibition of FNR. The K(i) value calculated for research condition was 1.72 mM. FNR molecule can bind a large number of cadmium ions, as shown by the application of cadmium-selective electrode, but just one ion remains bound after dialysis. The effect of cadmium binding is significant disturbance in the electron transfer process from flavin adenine dinucleotide (FAD) to dibromothymoqinone, but less interference with the reduction of ferricyanide. However, it caused a strong inhibition of Fd reduction, indicating that Cd-induced changes in the FNR structure disrupt Fd binding. Additionally, the protonation of the thiol groups is shown to be of great importance in the inhibition process. A mechanism for cadmium-caused inhibition is proposed and discussed with respect to the in vitro and in vivo situation.

Effects of Amino and Thiol Group Reagents on the Ferredoxin:NADP+ Oxidoreductase Catalysed Reduction of Dibromothymoquinone

Effects of selective reagents of amino groups (fluorescamine, Fc) and thiol [5,5′-dithio-bis(2-nitrobenzoic) acid, DTNB] groups on the diaphorase activity of spinach ferredoxin:NADP+ oxidoreductase (FNR, E.C 1.18.1.2) in the presence of dibromothymoquinone (DBMIB) as an electron acceptor were studied. The incubation of FNR with 250 μM Fc in the time range from 0 to 120 min led to the gradual decrease of FNR activity according to biphasic kinetics. At the initial phase the activity (defined as the rate of NADPH oxidation) decreased about 4-time faster than at the subsequent second slower phase. Incubation of FNR simultaneously with Fc and DBMIB for more than 20 min caused restoration of the activity to about 80 % of the control. The inhibitory effect of Fc on the FNR-catalysed DBMIB reduction had non-competitive character. Incubation of FNR with DTNB led also to a gradual decrease of the enzyme activity, which reached about 45 % of the control after 2 h of incubation. Thus neither amino nor thiol groups in the FNR molecule are involved directly in the DBMIB reduction. However, the presence of DBMIB in the incubation medium influenced the inhibitory pattern of Fc and DTNB, and this suggests that DBMIB modified the conformational state of the FNR molecule.

Cadmium inhibitory action leads to changes in structure of ferredoxin:NADP+ oxidoreductase

This study deals with the influence of cadmium on the structure and function of ferredoxin:NADP+ oxidoreductase (FNR), one of the key photosynthetic enzymes. We describe changes in the secondary and tertiary structure of the enzyme upon the action of metal ions using circular dichroism measurements, Fourier transform infrared spectroscopy and fluorometry, both steady-state and time resolved. The decrease in FNR activity corresponds to a gentle unfolding of the protein, caused mostly by a nonspecific binding of metal ions to multiple sites all over the enzyme molecule. The final inhibition event is most probably related to a bond created between cadmium and cysteine in close proximity to the FNR active center. As a result, the flavin cofactor is released. The cadmium effect is compared to changes related to ionic strength and other ions known to interact with cysteine. The complete molecular mechanism of FNR inhibition by heavy metals is discussed.

Effect of Pyrophosphate on the Ferredoxin: NADP+ Oxidoreductase Activity in vitro.

Under physiological conditions chloroplastic ferredoxin:NADP+ oxidoreductase (EC. 1.18.12; FNR) is involved in NADP+ reduction by photoreduced ferredoxin (Fd) [1,2,3]. The isolated enzyme catalyses electron transport from NADPH to many nonphysiological electron acceptors, including cytochrome c (the reaction is mediated by ferredoxin,) ferricyanide, dichlorophenol indophenol (DCPIP), some quinones, NAD+ and many others [4,5,6]. The data published by Kai-Tai Chang et al. [7] suggest that the site for DCPIP reduction differs from the binding sites of ferredoxin and NADP+ (NADPH). We hypothesised [8] that the site of DCPIP reduction on FNR molecule is also engaged in quinones (including dibromothymoquinone, DBMIB) reduction and in vivo it may be a site of plastoquinone pool reduction by electrons derived from reduced ferredoxin. Confirmation of this supposition may contribute essentially to elucidation the mechanism of FNR involvement in the cyclic electron transport around photosystem 1. We attempted to find specific inhibitors of diaphorase activity of FNR either with DCPIP (or DBMIB) or ferredoxin/cytochrome c. The results of this study indicate that inorganic pyrophosphate at the concentration of 10 mM inhibits substantially diaphorase activity of FNR in the presence of ferredoxin/ cytochrome c, but it does not influence on FNR activity with DCPIP or DBMIB.

Phaeodactylum Tricornutum as a Potential Phytoremediator of Sea and Fresh Waters

Phaeodactylum tricornutum is commonly spotted marine diatom species. It is known of its advantages as model organism used in many scientific applications, such as sequenced genome and ease culture. Toxic elements pollution is frequent issue in developing countries since elimination of such contamination is expensive, and dangerous to human health. Bioremediation is often seen as a solution of this problem. In this work P. tricornutum is analyzed as a potential bioremediating agent in sea and freshwater. Ability to growth in different concentrations of arsenic, cadmium and mercury is tested. It is investigated if P. tricornutum is able to proliferate in freshwater samples taken from river. At last concentration of every listed element is evaluated before and after culture. It is concluded that P. tricornutum has huge potential as a bioremediating organism.