In the dark, the STM cell densities were similar to that of WT M. loti . However, high intensity blue light prevented the growth of WT M. loti, but after 72 h, the growth of the STM strains was partially restored. The A610 values of the WT and STM strains were significantly different at 144 h . We also measured growth parameters in plants inoculated with the STM strains and exposed to blue light. For the shaded roots, no significant difference between the WT M. loti and STM strains was observed in shoot length , root length , or nodule number . For the unshaded plants, the nodule number of plants inoculated with WT M. loti was 40% of that of the shaded roots. For plants inoculated with the STM strains, the nodule count in unshaded roots was >65% of the number for the shaded roots . Taken together, these results suggest that both sets of rhizobial blue light receptors are required to inhibit nodulation.Because blue light perception by both the root and rhizobia leads to an inhibition of nodulation, we inoculated RNAi plants with the STM mutants under the same growth conditions used in Table 1 and assessed nodulation status 21 dai. As shown in Table 2, root nodules were formed in cry-RNAi plants with M. loti or in EV plants inoculated with STM strains, supporting the results shown in Table 1 and Figure 5H. The nodulation of RNAi-targeted Ljcry1A and Ljcry2B plants when inoculated by mllpp1 or mlphl was increased compared with EV plants inoculated with mllpp1 or mlphl. In RNAi-targeted Ljcry1A and Ljcry2B plants inoculated with the mllpp1 strain or the mlphl strain,dutch buckets nodulation was enhanced compared with EV plants inoculated with a WT M. loti strain .

The increase in nodule number was additive, indicating that the inhibition of nodulation by light is caused by blue light perception by both the host plant roots and rhizobia.Previous studies showed that nodulation in Trifolium subterraneum L. Woogenellup is either inhibited or not inhibited in the light, depending on the rhizobial strain . On the other hand, in Pisum sativum and P. vulgaris , root nodule numbers were decreased when roots were exposed to light. Our data support both of these seemingly disparate conclusions because, in L. japonicus, inhibition of nodulation by light is caused by blue light perception by both the host plant roots and rhizobia. Bonomi et al. reported that a short time exposure of rhizobia to white light prior to inoculation enhanced nodulation in Pisum sativum. In our study, a light treatment was given after inoculating with rhizobia, which may explain the differences in results. Using a split-root system, we found that the number of nodules formed on unshaded root systems was significantly lower compared with shaded root systems and even on a single root, which showed that nodule inhibition was not systemic . Recently, Chen et al. reported that the shoot lengths of Oryza sativa seedlings were inhibited by blue light and, in Lactuca sativa, shoot biomass was also decreased under blue light . Furthermore, shoot and hypocotyl lengths of lettuce, radish, and pepper decreased in response to increasing the quantity of both blue light and red light . Although the number of nodules did not differ between the red and blue treatments, the shoot lengths of blue light–treated plants in shaded L. japonicus roots were significantly decreased compared with red light–treated plants . In lettuce, root growth was decreased by blue light irradiation . In contrast, in rice, root growth was not different between roots either exposed to blue light or not . Our observation of root growth was that no large difference occurred whether the roots were shaded or unshaded in L. japonicus .

Taken together, these results show that overall shoot growth is inhibited by blue light irradiation, whereas the effect of blue light irradiation on root growth depends on the plant species. Under both white and blue light, a significant difference was seen in the shoot lengths of inoculated plants that had their roots shaded compared with uninoculated plants grown under the same conditions. These data lead us to conclude that the shoot growth of shaded roots was positively affected by the presence of root nodules. We also found that the inhibition of nodulation in white light was caused by its blue component and that root nodule number reduction under blue light was not related to the lack of a carbon source . Similarly, for isolated P. vulgaris roots, blue light inhibited nodulation more than red light or white light did . Our data are thus consistent with the absence of a significant effect of red light on root nodule number in Pisum sativum and, also, show that number of root nodules following blue light treatment was significantly reduced . Further implicating the key role of blue light, we found that nodulation was significantly increased in roots depleted of cry1A and cry2B . Because the expression of all the Ljcry1 and Ljcry2 genes was down-regulated in both cry1A and cry2B RNAi plants, we conclude that blue light inhibits nodulation via one or both cryptochromes 1 and 2. On the other hand, reduced expression of the three phototropin genes in L. japonicus MG20 had no effect on nodulation, indicating that blue light perception affects nodule development in the roots of this legume through cryptochromes and not phototropins. We also demonstrated that M. loti growth is inhibited specifically by blue light and that the rhizobial blue light receptors LPP1 and photolyase are involved in its suppression . Moreover, the growth of the STM strains under blue light did not lead to a full recovery of the levels of the dark-grown strains , which means that both rhizobial photoreceptors are involved. Finally, when the Ljcry1A and Ljcry2B-targeted RNAi plants were inoculated with the STM strains, an additive increase in nodule number was observed .

These results thus demonstrate that the inhibition of nodulation by light is caused by blue light perception by both host plant roots and rhizobia. With regard to nodulation, the number of infection threads per plant in L. japonicus is severely reduced in plants grown under blue light . Nodule size classes also differed between the shaded and unshaded plants. In the latter, a large population of smaller-sized nodules and only a few large ones developed on the illuminated roots compared with the shaded controls , suggesting that the inhibition of L. japonicus MG20 root nodulation under blue light results from one or both reduced or abnormal infection. Nodule fresh weights are also lower in the illuminated plants. At least two hypotheses can be invoked to explain these results. The first is based on the fact that light perception reduces rhizobial growth, resulting in insufficient size of the rhizobial population needed for inducing the earliest stages of nodule formation. This scenario is consistent with the studies on Rhizobium leguminosarum bv. viciae 3841 in which white light was reported to interfere with the synthesis of bacterial surface determinants that are needed for competent infection and nodulation of pea . Inhibition of attachment to root surfaces might also result in fewer infection events with a concomitant reduction in nodule size and weight,grow bucket which we observed. An alternative or additional mechanism is based on the observations of Grobbelaar et al. that light perception affects nodule development independently of the early stages of attachment, root hair deformation, and infection thread formation, which would mean that darkness is not required for nodule initiation but for nodule organogenesis. Our data are also consistent with this mechanism because we observed a larger size class of nodules in the 0.5- to 1-mm range and a reduction in the group of 1-mm-sized nodules in the shaded vs. unshaded roots. In addition, the fresh weight per nodule under shaded conditions was greater than when the roots were illuminated. However, the fact that fewer mature nodules were observed on unshaded roots could solely be due to the abortion or lack of the initial nodulation attempts under blue light. We are currently examining which of these two mechanisms is more likely to explain the downstream effects of blue light in the M. loti–L. japonicus symbiosis. Higher plants developed avoidance mechanisms to survive under conditions of biotic or abiotic stress. It is well-recognized that plants utilize light as the trigger for these responses. For example, in the case of anthogenesis and light-induced germination, the plant determines the timing by light perception through phytochromes and cryptochromes to increase the possibility of survival of descendants . In root negative phototropism, plant roots bend opposite to the light direction via phototropins . In the shade avoidance syndrome, plants grow taller or bend toward the light to avoid shade through the action of phytochromes . In shade avoidance in the case of root nodulation and mycorrhization, host plants suppress these interactions under suboptimal light conditions through the phytochrome system to avoid expending energy . Therefore, we can consider inhibition of nodulation by light as one of several avoidance responses plants use to conserve energy in response to environmental stress. Sun et al. reported that the light from the surrounding environment of herbaceous plants enters the interior of the stem. Then, via an internal light-conducting system, light of wavelengths between 710 and 940 nm, which includes far-red light, is conducted axially and efficiently from leaves and stems toward underground roots, whereas lower wavelengths of light , such as blue, are not.

These authors also stated that this internal light environment might be of crucial importance for the phytochrome-regulated metabolic activities of plant stems and roots. Because the phytochrome system is involved in the response to FR light conducted to the root, another system, such as blue light perception, may be utilized for avoidance mechanisms in response to stress. Clearly, more studies are needed.As nanotechnology rapidly evolves,engineered nano materials are entering air, waters, soils,and sediments where they could adversely affect organisms to ecosystems.Actual environmental impacts of ENMs have not been documented, and there are uncertainties about the potential for, and how to evaluate, impacts.ENM ecotoxicology elucidates hazards and their mechanisms.The scope overlaps with conventional ecotoxicology, although ENMs are particulate and diverse,with varying cores, native or acquired surface chemistries,conditional agglomeration or dissolution,and size- plus composition-dependent electronic properties,affecting their reactivity and biological interactions.Focusing ENM ecotoxicology invokes exposure scenarios relevant to ENM production,use,disposal,and product release .Scenarios consider environmental fate and transport,bio-availability,and ENM uptake into ecological receptors.In conventional chemical toxicology, observed and perceived exposures often diverge. In ENM ecotoxicology, water is emphasized, while soil and sediment impacts have received less attention.Where do exposures occur? What ENM forms and quantities are involved? Which ecological receptors are affected?Which local exposure conditions prevail? As with conventional chemical risk assessment, such questions unite hazard and exposure assessments.Standardized test regimens do not derive from scenarios, since ENM test conditions are predefined for standardized end points.Ideally, ENM hazards are studied at realistic exposures for ecologically relevant receptors. An example would be studying real soils under controllable yet realistic conditions, that is, in greenhouses or lysimeters.However, requiring absolute realism in all ENM ecotoxicology would pose scientific challenges associated with measuring ENMs analytically in environmental media; measuring toxicity across a representative range of environmental conditions; characterizing environmental ENM forms and their transformations so that toxicity is measured for representative materials; ENMs altering physical or chemical exposure media conditions; and few efficient approaches for estimating hazards and exposures necessary to evaluate risks before ENM products develop. Another challenge is internal to the scientific community: multiple dissimilar working definitions of environmental relevance intruding on scholarship, including peer review.Environmental relevance remains undefined, leading to categorization of research around a few selected concepts.Previously, over 600 published studies were examined to compare modeled or measured environmental concentrations of ENMs versus concentrations administered in ENM ecotoxicity assessments.The study found nominal concentration disparities, but also infrequent testing at low ENM concentrations. The study noted uncertainties in ENM exposure modeling, and that other toxicity testing conditions beyond ENM concentration including aqueous chemistry, biological receptor, system complexity, and ENM form relate to real-world conditions. However, the study did not establish what constitutes environmental relevance in the ecotoxicology of ENMs.