Bruce M. Greenberg

Higher plants and UV-B radiation: balancing damage, repair and acclimation

Abstract Although UV-B is a minor component of sunlight, it has a disproportionately damaging effect on higher plants. Ultraviolet-sensitive targets include DNA, proteins and membranes, and these must be protected for normal growth and development. DNA repair and secondary metabolite accumulation during exposure to UV-B have been characterized in considerable detail, but little is known about the recovery of photosynthesis, induction of free-radical scavenging and morphogenic changes. A future challenge is to elucidate how UV-B-exposed plants balance damage, repair, acclimation and adaptation responses in a photobiologically dynamic environment.

Identification of a primary in vivo degradation product of the rapidly-turning-over 32 kd protein of photosystem II

The 32 kd photosystem II protein of plant chloroplasts is rapidly turned over in the light. The initial events in the degradation of the 32 kd protein were studied. A 23.5 kd breakdown product was identified in Spirodela oligorrhiza membranes using immunological analysis. The 23.5 kd polypeptide was shown to be derived from the amino‐terminal portion of the 32 kd protein using partial proteolytic fingerprinting. An in vivo precursor–product relationship between the 32 kd protein and the 23.5 kd polypeptide was kinetically demonstrated by radiolabeling and pulse‐chase experiments. The cleavage site yielding the 23.5 kd polypeptide was localized to a functionally active region (between helices IV and V) of the 32 kd protein. We propose that an alpha‐helix‐destabilizing ‘degradation’ sequence, bordered by arginine residues 225 and 238, is involved in the formation of the 23.5 kd polypeptide.

Ability of 16 priority PAHs to be directly cytotoxic to a cell line from the rainbow trout gill

Sixteen polycyclic aromatic hydrocarbons (PAHs) were screened for their ability to be directly cytotoxic to a cell line from the rainbow trout gill, RTgill-W1. Exposure times of 2 h or less were sufficient for direct cytotoxicity to be detected, which appeared to be caused by a common mechanism, the general perturbation of membranes. This was judged by the similarity of results obtained for three fluorescent indicator dyes, alamar Blue, 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM) and neutral red. Among the 16 PAHs tested, just two- and three-ring PAHs were found to be directly cytotoxic. These were naphthalene approximately = acenaphthylene approximately = acenaphthene > fluorene approximately = phenanthrene. The results suggest that water solubility and lipophilicity are the critical properties determining the direct cytotoxicity of PAHs and that they do so by influencing PAH accumulation in membranes. Only naphthalene was effective at concentrations well below its water solubility limit. Therefore, direct cytotoxicity is likely to be most environmentally relevant only with naphthalene.

Methodology for demonstrating and measuring the photocytotoxicity of fluoranthene to fish cells in culture

Methodology was developed for quantifying the photocytotoxicity of fluoranthene to a gill cell line from rainbow trout for future use in screening polycyclic aromatic hydrocarbons for their relative photocytotoxicity to fish. Solubilization in a modified culture medium was achieved with and without foetal bovine serum (FBS) and with and without dimethyl sulfoxide (DMSO). FBS caused most of the fluoranthene to remain in solution and blocked photocytotoxicity if present during UV irradiation. DMSO had little effect on fluoranthene distribution in cell cultures but caused cells to be slightly more sensitive to the phototoxicity of fluoranthene. The indicator dyes alamar Blue() and 5-carboxyfluorescein diacetate acetoxymethyl ester were used to quantify cytotoxicity in two different ways-singly in two separate assays, and mixed together in a novel single assay, which saved time and material. With UV irradiation for 2 hr at a photon fluence rate of either 1.4 mumol UV-B/m(2)/sec (UV-A:UV-B, 1.5) or 1.1 mumol UV-B/m(2)/sec (UV-A:UV-B, 9.7), both dyes indicated increasing loss of viability with increasing doses of fluoranthene. EC(50) values ranged from 18 to 44 ng/ml (89-217 nM), with the alamar Blue assay being slightly more sensitive.

Pathway of anthracene modification under simulated solar radiation

Exposure of polycyclic aromatic hydrocarbons (PAHs) to sunlight results in rapid structural photomodification generally via oxidation reactions. These PAH modification products are in many cases more toxic than their parent compounds. In this study, anthracene (ANT), a rapidly photooxidized PAH, was irradiated with simulated solar radiation (SSR, 100 micromol m(-2) s(-1)) in aqueous solution to examine the photomodification pathway. The photoproducts formed were identified by HPLC. The ANT product profile after 9 h in SSR was very complex, with more than 20 compounds detected. The photoproducts formed were anthraquinones, benzoic acids, benzaldehydes and phenols showing the process to be oxidative in nature. Some of the anthraquinones were themselves subject to photooxidation, and were thus intermediates in the product pathway. The kinetics of ANT photooxidation revealed a pseudo first-order reaction with a half-life of 2 h under the SSR source used. The kinetics of product formation allowed deduction of a probable photomodification pathway. This study indicates that PAH photooxidation products are likely to exist as complex, dynamically changing mixtures in PAH contaminated aquatic environments.

PROTECTION OF THE D1 PHOTOSYSTEM II REACTION CENTER PROTEIN FROM DEGRADATION IN ULTRAVIOLET RADIATION FOLLOWING ADAPTATION OF Brassica napus L. TO GROWTH IN ULTRAVIOLET‐B

As depletion of the stratospheric ozone layer continues, the biosphere will most likely be exposed to higher levels of ultraviolet‐B (UV‐B) irradiation (290–320nm). For plants, damage from UV‐B can occur at several molecular targets with the photosynthetic apparatus being especially vulnerable. We are interested both in the mechanisms of UV‐B‐induced damage and identifying adaptation processes that can confer protection from UV‐B. Toward this end, Brassica napus (oil seed rape) plants grown under visible light plus a low level of UV‐B radiation (adapted plants) were compared to plants grown under visible light alone (control plants). Relative to the control plants, the adapted plants showed little evidence of damage at the levels of morphology or photosynthesis, indicating that B. napus has some tolerance of UV‐B and that the plants may have protection mechanisms. Consistent with this, a strong UV‐B adaptation process was observed in the plants‐accumulation of flavonoids in the epidermis. These pigments seemed to screen a molecular target in the mesophyll. Namely, the D1 photosystem II reaction center protein, which is rapidly degraded in UV‐B, was partially protected from degradation in UV‐B in the adapted plants. Moreover, the extent that the half‐life of the D1 protein increased in the adapted plants was on par with the elevation in total flavonoid concentrations. These experiments demonstrate that degradation of the D1 protein can be used as an in vivo assay of penetration of UV‐B photons to the mesophyll.

Photoinduced toxicity of polycyclic aromatic hydrocarbons to Daphnia magna: ultraviolet-mediated effects and the toxicity of polycyclic aromatic hydrocarbon photoproducts.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants known for their photoinduced toxicity. This toxicity may occur through two mechanisms: Photosensitization, and photomodification. Photosensitization generally leads to the production of singlet oxygen, a reactive oxygen species that is highly damaging to biological molecules. Photomodification of PAHs, usually via oxidation, results in the formation of new compounds and can occur under environmentally relevant levels of actinic radiation. The toxicities of 16 intact PAHs to Daphnia magna were assessed under two ultraviolet radiation conditions. The toxicity of intact PAHs generally increased in the presence of full-spectrum simulated solar radiation relative to that in the presence of visible light plus ultraviolet A only. Despite the knowledge of a bipartite mechanism of phototoxicity that includes photosensitization and photomodification, few studies have examined the effects of PAH photoproducts on animals. To expand the existing data, 14 PAH photoproducts (oxy-PAHs) also were assayed, most of which were highly toxic without further photomodification. Two photoproducts of benzo[a]pyrene, 1,6- and 3,6-benzo[a]pyrenequinone, were the most toxic compounds tested, followed closely by benz[a]anthraquinone. Each of these three compounds had a median effective concentration in the low nanomolar range. The data presented highlight the effects of ultraviolet radiation on mediating PAH toxicity and the need to analyze absorption spectra of contaminants in the prediction of photoinduced toxicity. The importance of the role of photomodification also is stressed, because several oxy-PAHs, an unregulated group of contaminants, were highly toxic to D. magna, a key bioindicator species in aquatic ecosystems.

Morphological and physiological responses of Brassica napus to ultraviolet-B radiation : Photomodification of ribulose-1,5-bisphosphate carboxylase/oxygenase and potential acclimation processes

Summary As the stratospheric ozone layer is depleted, the biosphere will be exposed to higher levels of ultraviolet-B (UV-B) radiation (290–320 nm). Using laboratory light sources that simulate the spectral quality of sunlight, we are examining some of the mechanisms involved in plant responses to UV-B. It was found that exposure of ribulose-l,5-bisphosphate carboxylase/oxygenase (Rubisco) from Brassica napus to UV-B in vivo or in vitro resulted in production of a high molecular weight (HMW) variant of the large subunit. Coincident with formation of the HMW product in vitro was a loss in tryptophan fluorescence. To protect against damage, plants can acclimate to UV-B. To this end, we have studied cotyledon curling in B. napus ; a photomorphogenic response specific to UV-B. To characterize the photoreceptor for curling, inhibitors of photochemical signaling were employed. A quencher of flavin excitation, and inhibitors of Ca ++ and cyclic nucleotide signaling diminished curling. Biosynthesis of flavonoids and other UV-absorbing pigments also occurred in B. napus exposed to the levels of UV-B that caused curling. To determine which flavonoids and other UV-absorbing compounds were UV-B specific, HPLC analysis was carried out. Approximately 20 distinct UV-absorbing pigments were produced in response to UV-B radiation. Thus, using B. napus we were able to follow UV-B induced damage and acclimation.

INFLUENCE OF ULTRAVIOLET RADIATION ON GROWTH AND PHOTOSYNTHESIS OF TWO COLD OCEAN DIATOMS1

The influence of chronic exposure to UV‐B and UV‐A radiation on growth and photosynthesis of two polar marine diatoms (Pseudonitzschia seriata and Nitzschia sp.) was investigated in cultures exposed to moderate photon fluences for 3–7 days. Population growth rates were diminished 50% by UV‐B. Fluorescence induction kinetics of photo‐system II (PSII) revealed that UV‐B caused lower Fv/Fm ratios and half‐rise times, indicating damage to the reaction center of PSII and to related elements of the photosynthetic electron transport chain. Carbon assimilation rates per cell and per chlorophyll a were nonetheless highest for UV‐B—exposed populations, which also had the highest chlorophyll a content per cell. The UV‐B—exposed cells were, however, more vulnerable to visible light‐induced photoinhibition. Exposure to UV‐A in the absence of UV‐B had little effect on growth, fluorescence induction of PSII, or chlorophyll a contents but did have some inhibitory effects on carbon assimilation per chlorophyll a and per cell. The increased photosynthetic capacity of UV‐B‐exposed cells suggested some ability to compensate for damage to the photosynthetic apparatus.

Applications of the aquatic higher plant Lemna gibba for ecotoxicological assessment

Although higher plants represent a significant portion of the total biomass in some aquatic environments, their use in ecosystem evaluation has lagged behind that of other organisms. This is partly due to a lack of convenient aquatic higher plant systems that can be employed for ecotoxicological assessment. However, the aquatic C-3 monocot Lemna gibba has many attributes that makes it useful for ecosystem health assessment. In this report, using examples from the literature and our research, some of the applications Lemna has for environmental research are discussed. Toxicant impacts on Lemna can be readily assessed in terms of growth; the plants multiply quickly and changes in biomass (which doubles approximately every 2 days) can be accurately measured by counting leaves. The plants are small, allowing for simultaneous multiple replication. The small size also makes the lighting conditions easy to control; sunlight can be accurately simulated and specific spectral regions can be enhanced or deleted. Lemna is amenable to in vitro chlorophyll and photosynthesis assays, which make excellent companion endpoints for growth. The plants assimilate chemicals directly from the growth medium, facilitating controlled toxicant application. Furthermore, Lemna has a high bioconcentration capacity, indicating a potential for use in bioremediation technologies.