The finding indicated that activities of GR and γ-ECS were elevated to ensure sufficient GSH turnover in response to acetaminophen induced consumption of GSH through GSH conjugation.Based on the results of this study, we propose a model to illustrate the operation of the GST detoxification pathway, which involves conjugation, accumulation and processing of these metabolites . The present study provided strong evidence that transformation of acetaminophen, one of the most used antipyretic and analgesic drugs worldwide, was likely catalyzed first by cytochrome P450, and followed by GSH conjugation mediated by GSTs. The GSH conjugates were further transformed and released back to the medium. Meanwhile, enzymes involved in GSH synthesis, regeneration and transport appeared to work in concert to maintain GSH homeostasis during acetaminophen transformation and detoxification. Detoxification by GSTs is known to play an important role in the bio-transformations of a multitude of xenobiotics in plants . When emerging contaminants such as acetaminophen are introduced in agroecosystems, GST mediated detoxification may serve the purpose to minimize their potential phytotoxicity to susceptible plants. On the other hand, however, the conjugation may effectively conserve the parent compound and its biological activity, if deconjugation occurs, e.g., in the human digestive tract. Conjugates back transformation to other biologically active compounds has been reported for benzotrizole , triclosan , and naproxen . Thus, understanding the toxicological consequence of Phase II conjugates of such emerging contaminants in agricultural plants may improve risk assessment of reuse practices of treated wastewater and biosolids. Moreover, non-food plants capable of such detoxification may be used for removing such trace contaminants,dutch bucket hydroponic in settings such as storm water basins, wetlands and vegetative buffers.

Plants rely on environmental cues for survival. Light is one such cue and plants perceive its quality, intensity, and direction. Phototropins and cryptochromes are well known blue light and UV-A photoreceptors and phytochromes are red and far-red light receptors . PHOT, which bind the blue light–absorbing chromophore FMN, harbor two FMN binding domains, LOV1 and LOV2 at the N-terminus, and a serine/threonine kinase domain at the C-terminus . LOV domains belong to the PerARNT-Sim family . PHOT control phototropism in seedlings, induce stomatal opening, and regulate chloroplast movement . CRY have two recognizable domains, a DNA photolyase-like domain at the N-terminus and C-terminal DQXVP acidic-STAES domains that are distinguished mainly by their C-terminal extensions . CRY participate in the circadian clock, anthocyanin biosynthesis, anthogenesis, and plant growth . It is well-known that light, which is perceived by photoreceptors, affects plant phenotypes by influencing phytohormones. For example, blue light perceived by PHOT induces phototropism via auxin translocation , whereas light-induced germination by PHY and CRY perception of light is induced by gibberellic acid . Leguminous plants and rhizobia establish a symbiosis in which root nodules develop on a host root. Within the nodules, rhizobia fix atmospheric nitrogen into ammonia, which eventually results in the synthesis of amino acids that are utilized by the host. In return, the plants provide photosynthetic products to the rhizobia as an energy source that drives the nitrogenfixation process . Light perceived by the above ground parts of the plant is essential for the establishment of this symbiosis. Previously, we reported that not only light quantity but, also, light quality affects nodulation and, moreover, that this photomorphogenetic event is controlled by phytochrome through jasmonic acid signaling in Lotus japonicus . Recently, Weller et al. reported that ethylene signaling influences phytochrome regulation in pea, and ethyleneinsensitive mutants are known to have increased nodule numbers .

For example, previous studies reported that nodulation in Pisum sativum was decreased by root exposure to daylight and that nodulation of isolated roots of Phaseolus vulgaris was suppressed by white light . Like higher plants, many bacteria synthesize photoreceptors such as phytochrome , and the analysis of numerous bacterial genomes has shown that photoreceptor proteins are present in many prokaryotes . For example, Giraud et al. reported that the Bradyrhizobium sp. strain ORS 278 genome has genes encoding phytochromes and Thompson and Sancar noted that Mesorhizobium loti synthesizes a photolyase that participates in blue light perception. Ogura et al. described PAS/LOV proteins in Arabidopsis that have a PAS domain at the N terminus and a LOV domain at the C terminus, and Bonomi et al. found a LOV-HK/PAS protein in Rhizobium leguminosarum bv. viceae 3841. In this study, we investigated the effects of light on the roots of Lotus japonicus Miyakojima MG20 and on the rhizobial strain to clarify the mechanisms of light perception required for nodulation in this symbiosis.To study the effect of root exposure to light on nodulation in L. japonicus Miyakojima MG20, we employed three different strategies . Ten-day-old plants growing on agar plates were inoculated with M. loti MAFF303099 and the roots of some plants were shaded. Under unshaded conditions, both the shoot and root were exposed to continuous white light whereas, when the root was shaded, only the shoot was exposed. Under these conditions, shaded roots received approximately 10 µmol m_2 s _1 of light. Although root lengths were not significantly different between unshaded and shaded plants 21 days after inoculation , the shoots from the unshaded plants were significantly shorter than those of the shaded plants . Also, unshaded roots had significantly fewer root nodules per plant than shaded roots , confirming earlier investigations that showed that nodulation is inhibited by white light.

However, uninoculated plants did not differ in shoot length whether they were shaded or not , suggesting that the difference in shoot length of the inoculated plants grown under unshaded conditions is related to the presence of rhizobia. In a split-root system in which the two root systems were inoculated with M. loti but one side of the root system was either totally shaded or both shaded and unshaded, we found that, although root lengths were not significantly different between the two different shaded and shaded/unshaded root systems , the overall number of nodules per root system was significantly reduced in the roots grown under completely unshaded conditions compared with those in shaded conditions . We used the data from Figure 1E and, as shown in Supplementary Figure S3A, prepared a graph combining the total number of nodules per split-root systems . We next analyzed the expression of nin, a nodulation gene marker, and found that its expression was significantly reduced on the unshaded root whereas nin was highly expressed in the shaded root in S/U plants, which were better nodulated. The reduction in nin expression was, thus, directly correlated with the reduced nodule number. Finally,dutch buckets system we investigated the effects of light on nodulation in a single root. The root of an inoculated plant growing in agar in a test tube was divided into three zones . Each zone was either exposed to light from the side or kept in the dark by masking the test tube with black tape. First, we found that nodules developed only in fully shaded roots and not in fully illuminated roots , supporting the results shown in Figure 1C and E. The number of root nodules formed in the shaded zone was significantly higher than that in the unshaded zone of the USU roots. In SUU and UUS plants, nodulation was slightly but not significantly higher in the shaded zones than in the unshaded zones. In the roots with the midzone shaded , nodulation was always higher than in the other SU treatments. Because the midzone of roots represents the most susceptible region for rhizobial infection and nodulation, this may explain the higher nodule number in this zone compared with other regions under shaded conditions. These data are in agreement with previous results showing that wild-type Medicago truncatula and L. japonicus develop root nodules in the susceptible zone .To determine which wavelength of light is critical for the inhibition of nodulation, we irradiated roots with blue or red light supplied from above for 21 days and analyzed nodulation thereafter. Although the light intensity in the soil at a depth of 10 cm would most likely be around 0.1 µmol m_2 s _1 under our treatment conditions, the unshaded and shaded roots were exposed to approximately 60 µmol m_2 s _1 and approximately 10 µmol m_2 s _1 of light, respectively. Under red light, no significant differences in shoot and root length or in the number of root nodules between unshaded and shaded plants were observed . Under blue light, however, both shoot length and nodule number were significantly reduced in the unshaded plants and a slight decrease in root length was also observed . As for white light, we measured the shoot length of inoculated and uninoculated Lotus plants grown under blue light conditions to investigate whether the difference in shoot length under blue light with or without root shading was influenced by nodulation. As Supplementary Figure S4 illustrates, although no significant difference in shoot length between shaded and unshaded plants was seen for the uninoculated plants, the shoot lengths of shaded plants with inoculated roots were significantly increased, just as they were in white light. Thus, there is a strong correlation between shoot length and inoculation with rhizobia in both blue and white light.

To investigate further whether the reduction in nodulation in the illuminated roots was caused by lower photosynthetic activity under blue light, we measured the activity under both red and blue light and found that blue light did not lead to a reduced photosynthetic rate . This result strongly suggests that, in L. japonicus roots, nodulation is inhibited in response to blue light perception and not by reduced photosynthetic activity.To analyze which step of the nodulation pathway is inhibited by blue light, both infection thread presence and nodule size class were measured. As shown in Figure 2C, not only did the number of nodules differ between the shaded and unshaded blue light–grown plants, but the number of infection threads per plant, as measured by DsRed fluorescence, was also reduced . The nodule size classes also differed between the two treatments. A lower proportion, i.e., 61 vs. 48% of the total number , were large nodules and a greater number were small nodules in the unshaded plants compared with the shaded ones . Also, the fresh weight per nodule of the unshaded roots was significantly reduced compared with that of the shaded roots, reflecting nodule size . The same trend was observed in acetylene reduction activity . Thus, in response to blue light, fewer infection threads were formed on the illuminated roots, causing a major decrease in nodule number and size. The ARA assay showed that the nodules that developed in unshaded roots were functional and that light did not directly affect nitrogen fixation but had an indirect effect by decreasing nodule number and weight.To investigate whether light affects rhizobial growth, we exposed liquid cultures to blue or red light. Growth, measured by absorbance at 610 nm of M. loti MAFF303099 under red light of either low or high intensity, was similar to that of the dark . On the other hand, low-intensity blue light significantly reduced M. loti growth and high-intensity blue light decreased it even more, as measured by absorbance, compared with the dark control . These results indicated that rhizobial growth is inhibited by blue light and that the lack of rhizobial proliferation may be an explanation for the reduction in nodule number. To investigate whether blue light has a direct effect on rhizobial growth, we transferred rhizobia that had been growing for 72 h in blue light to dark conditions and monitored their growth over the following 96 h. After a lag period, rhizobial growth resumed in the dark at a rate similar to that of a culture maintained in the dark for the entire experiment . However, when the rhizobia were exposed to blue light for the entire time period, growth was negligible, whereas when cells grown in blue light were transferred to the dark at 72 h, their A610 values increased by 120 h after transer and the cells were growing at the same rate as the dark controls. These results strongly support the conclusion that rhizobial growth is inhibited specifically by blue light. We then investigated the growth of two signature-tagged mutagenesis strains in M. loti with disrupted genes homologous to LOV-HK/PAS protein and photolyase .