Karen E. Gerhardt

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.

Tryptophan Photolysis Leads to a UVB‐lnduced 66 kDa Photoproduct of Ribulose‐1,5 Bisphosphate Carboxylase/Oxygenase (Rubisco) In Vitro and In Vivo

Abstract— Proteins are vulnerable to environmental UVB (290‐320 nm) because aromatic amino acids, particularly Trp, absorb in this spectral region. We have shown previously that UVB impacts on ribulose‐l,5‐bisphosphate carboxylase/oxygenase (Rubisco), resulting in the formation of a 66 kDa photoproduct in vivo (Wilson et al, Plant Physiol. 109,221–229, 1995). To determine if Trp photolysis is involved in the production of this specific protein photoproduct, the effects of UVB on a homogeneous preparation of Rubisco were examined. A UVB photoproduct of 66 kDa, identical to the in vivo product, was formed in vitro. The 66 kDa product was shown by immunological methods to be a cross‐link between a large subunit (53 kDa) and a small subunit (14 kDa). Time‐resolved Trp fluorescence was used to demonstrate that a Trp fluorescence signal is lost with kinetics that mirror the rate of formation of the 66 kDa photoproduct, indicating that a Trp photolysis step is involved in the mechanism of photoproduct formation. The relative rates of both Trp photolysis and 66 kDa photoproduct formation did not change with Rubisco concentration, consistent with a monomolecular reaction that would occur between sub‐units within a Rubisco holoenzyme complex. Finally, formation of the 66 kDa photoproduct was found to be pH dependent.

Plant Growth-Promoting Bacteria Facilitate the Growth of Barley and Oats in Salt-Impacted Soil: Implications for Phytoremediation of Saline Soils

Plant growth-promoting bacteria (PGPB) strains that contain the enzyme 1-amino- cyclopropane-1-carboxylate (ACC) deaminase can lower stress ethylene levels and improve plant growth. In this study, ACC deaminase-producing bacteria were isolated from a salt-impacted (∼50 dS/m) farm field, and their ability to promote plant growth of barley and oats in saline soil was investigated in pouch assays (1% NaCl), greenhouse trials (9.4 dS/m), and field trials (6–24 dS/m). A mix of previously isolated PGPB strains UW3 (Pseudomonas sp.) and UW4 (P. sp.) was also tested for comparison. Rhizobacterial isolate CMH3 (P. corrugata) and UW3+UW4 partially alleviated plant salt stress in growth pouch assays. In greenhouse trials, CMH3 enhanced root biomass of barley and oats by 200% and 50%, respectively. UW3+UW4, CMH3 and isolate CMH2 also enhanced barley and oat shoot growth by 100%–150%. In field tests, shoot biomass of oats tripled when treated with UW3+UW4 and doubled with CHM3 compared with that of untreated plants. PGPB treatment did not affect salt uptake on a per mass basis; higher plant biomass led to greater salt uptake, resulting in decreased soil salinity. This study demonstrates a method for improving plant growth in marginal saline soils. Associated implications for salt remediation are discussed.

The Effects of Far-red Light on Plant Growth and Flavonoid Accumulation in Brassica napus in the Presence of Ultraviolet B Radiation

Flavonoid induction is regulated by complex signal transduction pathways involving cryptochrome, phytochrome and UVB photoreceptors. Previously, we identified the UVB‐inducible flavonoids in Brassica napus cv. Topas leaves and showed that UVA affected accumulation of the quercetin (Q) and kaempferol (K) glycosides (Wilson et al. [2000] Photochem. Photobiol. 73, 678–684). In this study, we examined the effects of far‐red light (FR, 700–780 nm) on UVB‐mediated flavonoid accumulation in B. napus. Plants were grown under photosynthetically active radiation (PAR, 400–700 nm, 150 μmol m−2 s−1) plus a moderate level of FR (35 μmol m−2 s−1) for 14 days, and then transferred to five different irradiation regimes (PAR ± [UVA + UVB] + moderate, intermediate or low fluence FR) for 4 days. Kinetics of flavonoid accumulation were assessed via HPLC. Accumulation of flavonoids, in general, was suppressed by increasing the amount of FR in the spectrum. Furthermore, addition of UVB (290–320 nm) to the spectrum altered the flavonoid composition by causing significant changes in the quantities of individual flavonoids. The relative levels of acylated K glycosides were diminished whereas the relative levels of nonacylated Q glycosides increased dramatically. With UVB exposure there was a five‐fold increase in the Q:K ratio. In contrast, increasing the level of FR in the presence of UVB decreased the Q:K ratio by half.

Assessment of mixture toxicity of copper, cadmium, and phenanthrenequinone to the marine bacterium Vibrio fischeri.

Transition metals and polycyclic aromatic hydrocarbons (PAHs) are cocontaminants at many sites. Contaminants in mixtures are known to interact with biological systems in ways that can greatly alter the toxicity of individual compounds. The toxicities (individually and as mixtures) of copper (Cu), a redox‐active metal; cadmium (Cd), a nonredox active metal; and phenanthrenequinone (PHQ), a redox‐active oxygenated PAH, were examined using the bioluminescent bacterium Vibrio fischeri. We found that the cotoxicity of Cu/PHQ was dependent on the ratio of concentrations of each chemical in the mixture. Different interaction types (synergism, antagonism, and additivity) were observed with different combinations of these toxicants. The interaction types changed from antagonism at a low Cu to PHQ ratio (1:4), to additive at an intermediate Cu to PHQ ratio (2:3), to synergistic at higher Cu to PHQ ratios (3:2 and 4:1). In contrast to Cu/PHQ mixtures, the cotoxicity of Cd/PHQ did not change at different mixture ratios and was found for the most part to be additive. For the individual chemicals and their mixtures, reactive oxygen species (ROS) production was observed in V. fischeri, suggesting that individual and mixture toxicity of Cu, Cd, and PHQ to V. fischeri involves ROS‐related mechanisms. This study shows that mixture ratios can alter individual chemical toxicity, and should be taken into account in risk assessment.

Ultraviolet Wavelength Dependence of Photomorphological and Photosynthetic Responses in Brassica napus and Arabidopsis thaliana

Abstract Among the photomorphological responses in plants induced by ultraviolet-B radiation (UVB; 290 nm–320 nm) are leaf asymmetry, leaf thickening and cotyledon curling. We constructed an action spectrum of cotyledon curling in light-grown Brassica napus to characterize the UVB photoreceptor that initiates this response. Cotyledon curling was also characterized in Arabidopsis thaliana. Peak efficiency for this response occurred between 285 and 290 nm. Additionally, UVB-induced changes in epidermal cells from A. thaliana cotyledons were assessed because they are the likely site of UVB photoreception that leads to curling. Investigation of cellular structure, chlorophyll a fluorescence and chlorophyll concentration indicated that cotyledon curling is not concomitant with gross cellular damage or inhibition of photosynthesis, which only occurred in response to wavelengths <280 nm. Many UVB effects are apparently an indirect consequence of UVB radiation, dependent on UVB-mediated increases in reactive oxygen species (ROS) that either act as a signal in the UVB transduction pathway or cause oxidative damage. The cotyledon curling response was impeded by ascorbate and cystine, ROS scavengers and was promoted by H2O2, a ROS. We suggest that following absorption by a UVB chromophore, ROS are generated via photosensitization, ultimately leading to cotyledon curling.

Examination of the mechanism of phenanthrenequinone toxicity to Vibrio fischeri: evidence for a reactive oxygen species-mediated toxicity mechanism.

Phenanthrenequinone (PHQ) is a photoproduct of phenanthrene, one of the most prevalent polycyclic aromatic hydrocarbons in the environment. Phenanthrenequinone is a compound of substantial interest, because its toxicity can be much greater than its parent chemical to aquatic organisms. The toxicity mechanisms of PHQ to the luminescent marine bacterium Vibrio fischeri were examined in the present study. Phenanthrenequinone can redox cycle in bacterial cells and transfer electrons to O2, enhancing the production of superoxide (O*2-), hydrogen peroxide (H2O2), and other reactive oxygen species (ROS). Exposure of cells to PHQ increased activity of superoxide dismutase (SOD), which detoxifies the ROS superoxide. Concentrations of PHQ that induced the production of H2O2 and other ROS, as well as the elevated levels of Fe-SOD, were correlated with its toxicity as measured by luminescence. Furthermore, toxicity of PHQ to V. fischeri was lowered under the anaerobic conditions, suggesting that the absence of oxygen, which would limit the production of ROS, alleviated toxicity of PHQ. Thus, a ROS-mediated toxicity mechanism of PHQ is highly implicated by in the present study.

Phytoremediation of Salt-Impacted Soils and Use of Plant Growth-Promoting Rhizobacteria (PGPR) to Enhance Phytoremediation

Soil salinization negatively impacts plant growth and soil structure, which leads to environmental stress and agricultural/economic losses. Improved plant growth during salt-induced ionic and osmotic plant stress is the key to successful phytoremediation of salt-impacted sites. Using plant growth-promoting rhizobacteria (PGPR) in PGPR-Enhanced Phytoremediation Systems (PEPS), positive effects of PGPR on plant biomass and health have been observed in greenhouse and field experiments. Revegetation is arguably the most important aspect of salt phytoremediation and substantial biomass increases occur in PGPR-treated plants in both sodic and saline soils. PGPR protect against inhibition of photosynthesis and plant membrane damage, which suggests that they confer tolerance to plants under salt stress. Using PEPS, decreases in soil salinity are observed due to uptake of sodium and chloride from the soil into foliar plant tissue. Although rates of uptake do not change due to PGPR inoculation, higher plant biomass due to PGPR enhancement of plant performance leads to greater salt uptake on a per area basis relative to that of untreated plants. Significant improvements in plant growth and commensurate sodium chloride uptake, and the results of mass balance studies used to assess the direct impact of ion uptake on actual observed changes in soil salinity, provide evidence that phytoremediation of salt-impacted soil is feasible within acceptable time frames using PEPS.