Metals and ultraviolet (UV) radiation are two environmental stressors that can cause damage to plants. These two types of stressors often impact simultaneously on plants and both are known to promote reactive oxygen species (ROS) production. However, little information is available on the potential parallel stress responses elicited by metals and UV radiation. Using the aquatic plant Lemna gibba, we found that copper and simulated solar radiation (SSR, a light source containing photosynthetically active radiation (PAR) and UV radiation) induced similar responses in the plants. Both copper and SSR caused ROS formation. The ROS levels were higher when copper was combined with SSR than when applied with PAR. Higher concentrations of copper plus PAR caused toxicity as monitored by diminished growth and chlorophyll content. This toxicity was more pronounced when copper was combined with SSR. Because the generation of ROS was also higher when copper was combined with SSR, we attributed this enhanced toxicity to elevated levels of ROS. In comparison to PAR-grown plants, SSR treated plants exhibited elevated levels of superoxide dismutase (SOD) and glutathione reductase (GR). These enzyme levels were further elevated under both PAR and SSR when copper was added at concentrations that generated ROS. Interestingly, copper treatment in the absence of SSR (i.e. copper plus PAR) induced synthesis of the same flavonoids as those observed in SSR without copper. Finally, addition of either dimethyl thiourea or GSH (two common ROS scavengers) lowered in vivo ROS production, alleviated toxicity and diminished induction of GR as well as accumulation of UV absorbing compounds. Thus, the potential of ROS being a common signal for acclimation to stress by both copper and UV can be considered.

Abstract-Polycyclic aromatic hydrocarbon (PAH) toxicity is enhanced by light, especially ultraviolet (UV) radiation. To examine a potential mechanism(s) of photoinduced toxicity of PAHs to plants, the effects of anthracene and its photoproducts on photosynthesis were investigated using the aquatic higher plant Lemna gibba L. G-3 (duckweed). Photosynthetic activity was monitored both in vivo and in vitro by measuring chlorophyll a (Chl a) fluorescence, carbon fixation, and electron transport. In simulated solar radiation (a light source with a visible light : UV-A : UV-B ratio similar to sunlight), inhibition of photosynthesis was more rapid with photomodified anthracene than with intact anthracene, and intact anthracene appeared to only inhibit photosynthesis following its photomodification. The primary site of action of photomodified anthracene was found to be electron transport at or near photosystem I (PSI). This was followed by inhibition of photosystem II (PSII), probably due to excitation pressure on PSII once the downstream electron transport through PSI was blocked. Accordingly, higher chemical concentrations and/or longer exposures were required to inhibit PSII than PSI. Net photosynthesis (carbon fixation) was also inhibited, implying that the inhibition of electron transport in PSI by photomodified anthracene can lead to diminished primary productivity. A linkage between inhibition of photosynthesis and inhibition of plant growth was established in terms of the initial site of action (PSI) and primary productivity (carbon fixation), which suggested that Chl a fluorescence can be used as a bioindicator of PAH impacts on plants.

Heavy metals and polycyclic aromatic hydrocarbons (PAHs) are often cocontaminants in industrialized environments, yet little is known about either the extent or mechanisms of their cotoxicity. To address this shortfall, the combined effects of an oxygenated PAH, 1,2-dihydroxyanthraquinone (1,2-dhATQ), and a heavy metal, Cu2+, on photosynthesis and growth of the duckweed (Lemna gibba) were evaluated. Using assays of chlorophyll a fluorescence and photosystem I activity, 1,2-dhATQ inhibited electron transport at the cytochrome b6/f complex. Conversely, Cu2+ alone (at low concentrations) had little effect on photosynthesis. When Cu2+ was combined with 1,2-dhATQ, an increase in transient and steady-state chlorophyll a fluorescence quenching occurred relative to 1,2-dhATQ alone. Treatment of isolated thylakoid membranes with 1,2-dhATQ inhibited whole-chain linear electron transport, measured as O2 consumption using methyl viologen as the electron acceptor. However, Cu2+ plus 1,2-dhATQ resulted in active O2 consumption with or without methyl viologen as an electron acceptor. From these data, we conclude that 1,2-dhATQ renders the plastoquinone pool to a highly reduced state by inhibiting at cytochrome b6/f. Then, Cu2+ is able to mediate the transfer of electrons from reduced plastoquinone to O2, forming reactive oxygen species. At the whole-organism level, when Cu2+ and 1,2-dhATQ were mixed at concentrations that resulted in the above-mentioned impacts on photosynthesis, synergistic inhibition of plant growth was observed. This suggests a catalytic mechanism of toxicity for redox active metals, a process that could be instrumental in explaining their impacts at low concentrations.

Sites of toxicity of polycyclic aromatic hydrocarbons (PAHs) were examined to determine if inhibition of photosynthetic activity could be correlated to whole-organism toxicity. The inhibition of photosynthesis was observed by detecting the induction kinetics of endogenous chlorophyll a (Chl a) fluorescence. Anthracene (ANT) photooxidation products were applied to the aquatic higher plant Lemna gibba L. G-3 at concentrations ranging from 0.01 to 10 ppm. The impact on Chl a fluorescence was found to correlate with whole-organism toxicity for the 13 PAH compounds tested in this in vivo study. The mechanism of toxic action starts with inhibition of photosystem I (PSI) or the cytochrome-b6/f complex, followed by photooxidative damage to photosystem II (PSII). To study the effects of oxygenated ANTs on photosynthesis in vivo, the IC(50)s for F(V)/F(M) (PSII activity) and F(Q)/F(M) (activity downstream from PSII) were determined. The IC(50)s for a decrease of F(Q)/F(M) for all 13 chemicals were on average twofold lower than those for F(V)/F(M). F(V)/F(M) was found to be a measure of acute toxicity, whereas F(Q)/F(M) was found to be a measure of chronic toxicity. Thus, Chl a fluorescence by use of the whole organism was able to detect the impacts of photomodified ANT products and indicate a site of action for the chemicals.

lncreased levels of solar ultraviolet (290-320 nm) (UV-B) radiation could have profound effects on plant proteins because the aromatic amino acids in proteins absorb strongly i n this spectral region. We have investigated the effects of UV-B radiation on plant proteins and have observed a nove1 66-kD protein. This product was formed i n vivo when Brassica napus 1. plants grown for 21 d in 65 pmol m-* s-' photosynthetically active radiation were subsequently exposed to 65 pmol m-' s-' photosynthetically active radiation plus UV-B radiation (1.5 pmol m-' s-'). l h e protein appeared after 4 h of UV-B irradiation and accumulated during the next 16 h in UV-B. The 66-kD protein cross-reacted with an antiserum against the ribulose-1,s-bisphosphate carboxylase/oxygenase (Rubisco) holoenzyme. Analysis of soluble leaf proteins revealed that the 66-kD product had a number of isoforms corresponding closely to those of the large subunit of Rubisco (LSU). Partia1 proteolytic digests of the LSU and the 66-kD protein resulted in an equivalent pattern of protein fragments, leading to the conclusion that the 66-kD protein was a photomodified form of the LSU. A similar high molecular m a s variant of Rubisco was observed in soluble protein extracts from leaves of tomato (Lycopersicon esculentum), tobacco (Nicotiana tabacum), and pea (Pisum sativum L.) plants treated in vivo with UV-B, suggesting that it might be a common product, at least among C, plants. It is interesting that the 66-kD product appears to be generated after incorporation of the LSU into holoenzyme complexes. This conclusion was drawn from two lines of evidence. First, the LSU variant co-purified with holoenzyme complexes isolated by nondenaturing polyacrylamide gel electrophoresis. Second, a UV-B-specific 66-kD protein did not accumulate in a tobacco mutant that synthesizes the Rubisco subunits but does not assemble them into normal holoenzyme complexes.