Technological applications of chlorophyll a fluorescence for the assessment of environmental pollutants.

Chlorophyll a fluorescence has been extensively studied over the last few years. As demonstrated, this phenomenon is closely related to the state of photosystem II, which plays a leading role in the photosynthetic process, and therefore it has become a powerful tool to investigate this complex and any damage occurring in it as a result of physical or chemical stresses. This means that by using photosynthetic organisms as biological probes, one can consider chlorophyll a fluorescence as one of the techniques of choice to reveal the presence of some hazardous toxicants widely spread in the environment. Herbicides, pesticides, and heavy metals, whose concentration in water and food products is generally subject to extremely severe restrictions, are a concrete example of compounds detectable by chlorophyll a fluorescence. These dangerous substances react with the photosystem II, modifying the fluorescence emitted and giving responses which vary in a concentration-dependent manner. The possibility of performing easy, fast, and direct measurements of the fluorescence, even under light conditions, has opened new frontiers for the analysis in situ of pollutants. The aim of this review is to give an overview of the different techniques based on chlorophyll a fluorescence spectrometry, focusing in particular on those which represented the starting point for applications addressed to the assessment of toxic compounds in environmental samples.

Chlamydomonas reinhardtii genetic variants as probes for fluorescence sensing system in detection of pollutants.

The unicellular green alga Chlamydomonas reinhardtii is employed here for the setup of a biosensor demonstrator based on multibiomediators for the detection of herbicides. The detection is based on the activity of photosystem II, the multienzymatic chlorophyll-protein complex located in the thylakoid membrane that catalyzes the light-dependent photosynthetic primary charge separation and the electron transfer chain in cyanobacteria, algae, and higher plants. Several C. reinhardtii mutants modified on the D1 photosystem II protein are generated by site-directed mutagenesis and experimentally tested for the development of a biosensor revealing the modification of the fluorescence parameter (1 - V (J)) in the presence of herbicides. The A250R, A250L, A251C, and I163N mutants are highly sensitive to the urea and triazine herbicide classes; the newly generated F255N mutant is shown to be especially resistant to the class of urea. It follows that the response of the multibiomediators is associated to a particular herbicide subclass and can be useful to monitor several species of pollutants.

A multi-biosensor based on immobilized Photosystem II on screen-printed electrodes for the detection of herbicides in river water.

A multi-biosensor for detection of herbicides and pollutants was constructed using various photosynthetic preparations as biosensing elements. The photosynthetic thylakoid from Spinacia oleracea L., Senecio vulgaris and its mutant resistant to atrazine were immobilized with (BSA-GA) on the surface of screen-printed sensors composed of a graphite-working electrode and Ag/AgCl reference electrode deposited on a polymeric substrate. The biosensor was composed of four flow cells with independent illumination of 650 nm to activate electron transfer in Photosystem II. The principle of the detection was based on the fact that herbicides selectively block electron transport activity in a concentration-dependent manner and that the four PSII biomediators show differential recognition activity toward herbicides. Changes of the activity were registered amperometrically as rate of photoreduction of the artificial electron acceptor DQ. The setup resulted in a reusable herbicide multibiosensor with a good stability (half-life of 16.7 h for spinach thylakoids) and limit of detection of about 10(-8) M for herbicides recovered in spring in river.

A bio-dosemeter that utilises isolated enzymes to detect ionising radiation.

The development of a biosensor for the detection of ionising radiation (biodosemeter) utilising the advantageous properties of the photosystem II (PSII) complex and its response to ionising radiation is reported. The transducer signal for this biosensor can be fluorescence, which is dependent on photosynthetic activity. Exposure of biological material to ionising radiation leads to a loss of function due to the destruction of critical structures. Radiation target theory predicts an exponential decrease in biochemical activity that is dependent on the absorbed radiation energy and directly proportional to the mass of the individual molecules possessing this activity. The activity is lost whenever the protein is hit since very high energy is transferred through the chain. Several approaches were used to optimise the immobilisation of PSII complexes to improve the sensitivity of the bio-dosemeter.

Photosystem II-based biosensors for the detection of pollutants.

Photosystem II (PSII) is the supramolecular pigment-protein complex in the chloroplast, which catalyses the light-induced transfer of electrons from water to plastoquinone (PQ) in a process that evolves oxygen. The PSII complex is also known to bind some groups of (photosynthetic) herbicides, heavy metals and other chemical substances that affect its activity. The objective of this study is to provide an overview of the systems available for the bioassay of pollutants using biosensors that are based on the photochemical activity of PSII. Some applications of the PSII-based biosensors including herbicide, heavy metal monitoring and the detection of radiation in space experiments are reported.

A device to study the effect of space radiation on photosynthetic organisms.

This research concerns the study of the effects of ionising space radiation on the oxygen-evolving activity of algae and cyanobacteria, focusing our attention on Photosystem II (PS-II), the oxygen-evolving complex. These microorganisms as higher plants, can use light energy to split water molecules and evolve oxygen in a process that produce storable energy-rich products from atmospheric carbon dioxide. Algae and cyanobacteria which can grow in the presence of nutrients and carbonate are expected to be utilised to maintain an oxygen-atmosphere and to constitute biomass in space shuttles. Irradiation was performed in gamma 60Co-sources of different activities; fluorescence techniques in vivo and SDS-PAGE analysis in vitro were used to determine PS-II efficiency during radiation stress. We determined the radiation target on PS-II by immunoblot. We built a miniaturised growth box that preserves constant pressure and temperature to measure automatically photosynthetic activity by a fluorescence sensor, directly in space during a mission in an ASI balloon.

A sensitive photosystem II-based biosensor for detection of a class of herbicides.

We have developed a biosensor for the detection of residual triazine-, urea- and phenolic-type herbicides, using isolated photosystem II (PSII) particles from the thermophilic cyanobacterium, Synechococcus elongatus, as biosensing elements. The herbicide detection was based on the fact that, in the presence of artificial electron acceptors, the light-induced electron transfer through isolated PSII particles is accompanied by the release of oxygen, which is inhibited by the herbicide in a concentration-dependent manner. The PSII particles were immobilized between dialysis membrane and the Teflon membrane of the Clark oxygen electrode mounted in a flow cell that was illuminated. Inclusion of the antibiotic chloramphenicol in the reaction mixtures prolonged, by 50%, the lifetime of the biosensor. The use of highly active PSII particles in combination with the flow system resulted in a reusable herbicide biosensor with good stability (50% of initial activity was still remaining after 35-h use at 25 degrees C) and high sensitivity (detection limit for diuron was 5 x 10(-10) M).

Decreased Photosystem II Core Phosphorylation in a Yellow-Green Mutant of Wheat Showing Monophasic Fluorescence Induction Curve.

In the present work we study the regulation of the distribution of the phosphorylated photosystem II (PSII) core populations present in grana regions of the thylakoids from several plant species. The heterogeneous nature of PSII core phosphorylation has previously been reported (M.T. Giardi, F. Rigoni, R. Barbato [1992] Plant Physiol 100: 1948-1954; M.T. Giardi [1993] Planta 190: 107-113). The pattern of four phosphorylated PSII core populations in the grana regions appears to be ubiquitous in higher plants. In the dark, at least two phosphorylated PSII core populations are always detected. A mutant of wheat (Triticum durum) that shows monophasic room-temperature photoreduction of the primary quinone electron acceptor of PSII as measured by chlorophyll fluorescence increase in the presence and absence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea and by fluorescence upon flash illumination in intact leaves also lacks the usual distribution of phosphorylated PSII core populations. In this mutant, the whole PSII core population pattern is changed, probably due to altered threonine kinase activity, which leads to the absence of light-induced phosphorylation of CP43 and D2 proteins. The results, correlated to previous experiments in vivo, support the idea that the functional heterogeneity observed by fluorescence is correlated in part to the PSII protein phosphorylation in the grana.

Photosystem II Core Phosphorylation Heterogeneity, Differential Herbicide Binding, and Regulation of Electron Transfer in Photosystem II Preparations from Spinach.

The effect of photosystem II core phosphorylation on the secondary quinone acceptor of photosystem II (Q(B)) domain environment was analyzed by comparative herbicide-binding studies with photosystem II preparations from spinach (Spinacia oleracea L.). It was found that phosphorylation reduces the binding affinity for most photosynthetic herbicides. The binding of synthetic quinones and of the electron acceptor 2,6-dichlorophenolindophenol is also reduced by photosystem II phosphorylation. Four photosystem II core populations isolated from membranes showed different extents of phosphorylation as well as different degrees of affinity for photosynthetic herbicides. These findings support the idea that heterogeneity of photosystem II observed in vivo could be, in part, due to phosphorylation.

Breakdown of the photosystem II reaction center D1 protein under photoinhibitory conditions: identification and localization of the C-terminal degradation products.

Illumination of a suspension of thylakoids with light at high intensity causes inhibition of the photosystem II electron transport activity and loss from the membrane of the D1 protein of the photosystem II reaction center. Impairment of the electron transport activity and depletion of D1 protein from the thylakoid membrane of pea were investigated with reference to the presence or absence of oxygen in the suspension. The breakdown products of the D1 protein were identified by immunoblotting with anti-D1 polyclonal antibodies which were proven to recognize mainly the C-terminal region of the protein. The results obtained show that (i) the light-induced inactivation of the photosystem II electron transport activity under anaerobic conditions is faster than in the presence of oxygen; (ii) depletion of D1 protein is observed on a longer time scale with respect to loss of electron transport activity and is faster when photoinhibition is performed in the presence of oxygen; (iii) C-terminal fragments of D1 are only observed when photoinhibition is carried out anaerobically and are mainly localized in the stroma-exposed regions; and (iv) the fragments observed after anaerobic photoinhibition are quickly degraded on further illumination of the thylakoid suspension in the presence of oxygen.

Relationships between heterogeneity of the PSII core complex from grana particles and phosphorylation.

Isoelectrofocusing of photosystem II enriched membranes from spinach reveals the presence of at least four different populations of PSII core complex. The four bands are neither equally populated nor equally active in electron transport from diphenylcarbazide to 2,6-dichlorophenolindophenol. Under conditions of a low and high phosphorylation level a change in the relative populations of the PSII core isoforms is observed and the amount of radiolabelled phosphate incorporated into the four types of complexes is correlated to the value of their isoelectric point suggesting that the origin of the heterogeneity evidenced in vitro is at least partially due to different levels of light-induced phosphorylation. A 9 KD phosphoprotein, previously described in PSII, is found in our core complex preparation at a concentration which decreases as the total phosphorylation level on D1/D2 polypeptides increases.

Photosynthetic properties of leaves of a yellow green mutant of wheat compared to its wild type.

We investigated several photosynthetic parameters of a virescent mutant of durum wheat and of its wild-type. Electron transport rate to ferricyanide was the same in the two genotypes when expressed on leaf area basis while O2 evolution of the leaf tissue in saturating light and CO2 was slightly higher in the yellow genotype. RuBPCase was also slightly higher. Quantum yield per absorbed light was similar in the two genotypes. P700 and Cyt f were less concentrated in the mutant while PS II was only marginally lower. The light response curve of CO2 assimilation indicated higher level of photosynthesis of the mutant in high light, which corresponded to a lower non-photochemical quenching compared to the wild-type. It is concluded that the reaction centres, cyt f and chlorophyll are not limiting factors of electron transport in wheat seedlings and that electron transport capacity is in excess with respect to that needed for driving photosynthesis. Since the differences in photosynthesis reflect differences in RuBPCase activity, it is suggested that this enzyme limits photosynthesis in wheat seedlings also at high light intensities.