Microscopic analysis showed that 75% TP-Effluent and 50% TP-Effluent cultures had the highest cell densities . The TP-Effluent and 25% TP-Effluent cultures more closely resembled the negative control TP cultures, with poor growth relative to the other cultures. Over longer growth periods, there could be a larger difference between the different acetate concentration, but for the purposes of this experiment, it was discovered that using smaller doses of effluent was practical and could increase the use efficiency of the costly effluent. This experiment in conjunction with the Drop-Out experiment also helped our collaborators at the University of Delaware understand how to optimize the chemical composition of the effluent. For the growth experiment where the effluent produced from the electrocatalytic process was procured and incorporated into the media, heterotrophic growth of algae was demonstrated successfully. Figure 4A shows that all three cultures grown with effluent media exhibit clear growth after 4 days. This growth is comparable to TAP, as shown in Figures 4B-D. It was also found that performing cell counts through hemocytometry, although more labor intensive, significantly decreased the errors between triplicates. From this final experiment, the first instance of algal growth completely decoupled from photosynthesis was achieved. For future continuation of this project,raspberry plant pot the next steps are to optimize the growing process by media treatment or to employ the use of highly controlled bioreactors.
The use of other algal species or other strains of Chlamydomonas reinhardtii can be considered as well. A more robust strain could have higher resistances to salts present in the effluent. By doing so, there is an opportunity to develop a system that exceeds the efficiency of conventional photosynthetic systems and be applied to agriculture for food and biotechnology industries such as biofuel production. This project was presented as an online presentation at the 2021 Undergraduate Research and Creative Activity Symposium at the University of California, Riverside .Pattern recognition receptors recognize conserved microbial signatures, activating immune responses . The rice PRR XA21 confers robust resistance to diverse strains of Xanthomonas oryzae pv. oryzae , the causal agent of blight disease in rice . XA21 recognizes a tyrosine-sulfated protein called RaxX derived from Xoo. Tyrosine sulfation is required for RaxX’sactivity. In rice plants expressing XA21, sulfated RaxX activates XA21-mediated immune responses, including production of reactive oxygen species , ethylene, and induction of defense gene expression . We previously reported that transgenic plants over expressing XA21 produce XA21 cleavage products . For example, a 110-kDa amino-terminal XA21 cleavage product was detected in Ubi-Myc-XA21 transgenic plants and a 70-kDa carboxyterminal cleavage product was observed in Ubi-XA21-CFP and Ubi-XA21-GFP plants . A predicted nuclear localization sequence located between the XA21 transmembrane and juxtamembrane domain was identified.
In rice protoplasts, the predicted NLS is able to direct transiently expressed GFP-tagged XA21 C-terminal domain to the nucleus. In contrast, a construct carrying alanine substitutions in the predicted NLS fails to direct the nuclear localization of XA21 C-terminal domain . To assess the biological relevance of the predicted XA21 nuclear localization sequence in planta, we generated transgenic plants expressing an XA21 variant with alanine substitutions in the predicted NLS . The Ubi-XA21nls-GFP transgenic lines displayed partial resistance to Xoo infection, as refilected in slightly longer lesion lengths and higher Xoo bacterial populations compared with Ubi-Xa21-GFP control plants. A tyrosine-sulfated 21-amino acid derivative of RaxX triggered ROS production in Ubi-XA21nlsGFP plants. The expressed protein levels of XA21nls-GFP in Ubi-XA21nls-GFP lines were lower than those observed in Ubi-XA21-GFP plants. These results suggest that lower levels of expressed protein account for the slightly longer lesions observed in Ubi-XA21nls-GFP transgenic plants and indicate that the predicted NLS is not critical for XA21-mediated immunity.Xanthomonas oryzae pv. oryzaePhilippines race 6 PXO99A was used in this study. Xoo was cultured on peptone sucrose agar plates supplemented with 20 mg L−1 cephalexin for two days, then washed off and re-suspended in sterilized water. The concentration of bacterial suspension was adjusted to an OD600 of 0.5 for inoculation. Before inoculation, 5-week-old greenhouse-grown rice plants were transferred to a walk-in growth chamber to acclimate the chamber conditions for one week.
Plants were inoculated using the scissor clipping method . For each line, 8-12 plants were inoculated and in each plant two fully-expanded leaves from 3-6 tillers were clipped using scissors with the Xoo inoculum. The lesion lengths were measured 14 days post-inoculation. In planta bacterial growth was assessed as described previously . Briefly, inoculated leaves were collected at indicated time points, cut into 5-mm pieces and incubated in 10 mL sterile water with shaking at 28 ◦C for 1 h. The suspension was diluted accordingly and spread out on PSA plates with 20 mg L−1 cephalexin. The bacterial colonies were counted after a two-day culture at 28 ◦C. Protein extraction from rice leaves and western blot assays were performed as previously described . Briefly, total protein was extracted from 100 mg of rice leaf tissue in 200 µL of pre-chilled extraction buffer and separated in an 8% SDS-polyacrylamide gel. A mouse anti-GFP antibody was used as the primary antibody for detection of GFP-tagged XA21 and XA21nls. ROS assays were performed as previously described . Fully-expanded leaves were harvested from 4-week-old hydroponically grown rice plants, cut into 2-mm2 pieces and floated on water overnight. Leaf pieces were treated with water, 1 µM non-sulfated 21-amino acid synthetic RaxX peptides or tyrosine sulfated RaxX21 peptides . For each treatment, four biological replicates were included and in each replicate two leaf pieces were used. Chemiluminescence was recorded every 30 s for 3 h in a high-sensitivity TriStar plate reader . The predicted NLS in XA21 is a basic amino acid-rich sequence localized between the transmembrane and juxtamembrane domains . An XA21 variant with alanine substitutions in the predicted NLS was generated using site-directed mutagenesis and introduced into the rice Kitaake cultivar. Five independent Ubi-XA21nls-GFP lines were obtained, and three lines that expressed detectable full-length XA21nls-GFP protein were selected for further analysis. We observed reduced gene and protein expression levels of XA21-GFP in these transgenic lines as compared with those in the Ubi-XA21-GFP control plants . We previously reported that XA21 is cleaved in transgenic plants over expressing XA21 with a GFP tag , blueberry production that the released GFP tagged C-terminal domain is localized to the nucleus and that a predicted NLS directs this domain to the nucleus in transient assays . To investigate the biological relevance of these observations, here we used a genetic approach to assess the resistance of transgenic plants expressing XA21 with mutations in the predicted NLS to Xoo. We observed that the three Ubi-XA21nls-GFP transgenic lines displayed slightly reduced resistance to Xoo, as illustrated by slightly longer lesion lengths and higher bacterial populations compared with the Ubi-XA21-GFP control plants . Sulfated, but not non-sulfated, RaxX21 is able to trigger ROS production in the Ubi-XA21nls-GFP plants, to slightly lower levels than that observed in the Ubi-XA21-GFP control . Considering the relatively lower XA21 transcript and protein levels of XA21 in Ubi-XA21nls-GFP plants as compared with the Ubi-XA21-GFP control plants , we hypothesize that the reduced level of XA21 protein accumulation is responsible for the slightly reduced resistance and reduced ROS levels observed in the Ubi-XA21nls-GFP plants.
The differences in XA21 protein levels in Ubi-XA21-GFP vs. Ubi-XA21nls-GFP may be due to position effects of the transgene, as we and others have observed in previous studies. Together, these results indicate that Ubi-XA21nls-GFP is able to respond to RaxX21-sY and confers resistance to Xoo, which suggest that the predicted NLS is not required for XA21-mediated immunity. This result conflicts with our previous report that disruption of XA21 nuclear localization in Ubi-XA21-GFP-NES caused enhanced susceptibility . One possible explanation for this discrepancy is that the addition of the NES to XA21 disrupts activity of XA21. The biological role of the XA21 cleavage product remains unknown. The XA21 intracellular domain interacts with several proteins predicted to be nuclear localized, including the XB10/WRKY62 transcription factor . The nuclear localization of the XA21-GFP cleavage products in transient assays suggests a role in transcriptional regulation in the nucleus . However, here we demonstrate that nuclear localization of the XA21 intracellular domain is not critical for XA21-mediated immunity. Land-based ecosystems in the northern hemisphere appear to remove, at least temporarily, a substantial portion of anthropogenic CO# from the atmosphere . The mechanisms behind this C sink are not well understood, even though knowledge of these processes is vital to predict and interpret the responses of ecosystems to global change . Changes in plant productivity due to CO# enrichment , nitrogen deposition , land use change , andclimatic effects have been investigated as potential components . However, the response of microbial communities to these perturbations, and their potential influence on C cycling, have received scarce attention. Mycorrhizal fungi in particular might play an important role in the sequestration of C in soil under elevated CO# and N deposition. This group, which symbiotically colonizes plant roots, forms associations with 80% of plant species and is found in nearly every habitat in the world . Plants allocate an estimated 10–20% of net photosynthate to mycorrhizal fungi, although this number can range from 5 to 85% among systems . A substantial amount of C allotted to mycorrhizal tissues could be long-lived in the soil. Chitin, whichis not readily decomposed , can constitute up to 60% of fungal cell walls . Arbuscular mycorrhizal fungi are also the sole producers of glomalin, a potentially recalcitrant glycoprotein . AM hyphae in the absorptive hyphal network have lifespans of only 5–7 d , and with each cycle residual hyphal C should remain in the soil. Furthermore, some micro-arthropods prefer to graze on non-mycorrhizal fungi rather than on a variety of AM fungi , and therefore might not necessarily speed up tissue turnover significantly. As a result, glomalin alone can account for 30–60% of C in undisturbed soils , assuming that the protein is 30% C by weight M. C. Rillig, pers. comm. Likewise, portions of ectomycorrhizal biomass were responsible for approx. 15% of soil organic matter in two hardwood forests . Carbon derived from mycorrhizal tissue can account for a significantly sized pool within ecosystems and globally. Because mycorrhizal fungi acquire most or all their C directly from living plants, the nutrient status of foliage strongly affects mycorrhizal growth. As elevated CO# generally increases plant growth and root-to-shoot ratio , greater allocation of C to mycorrhizal structures might follow . Effects of elevated CO# on mycorrhizal growth have been reviewed by O’Neill , Diaz , Hodge , and Staddon & Fitter , with an emphasis on changes in percentage root length colonized and total root length colonized per plant. These reviews indicate that the percentage of roots with mycorrhizal structures might not necessarily change under elevated CO# . However, as root biomass tends to rise, total mycorrhizal biomass per plant might do so as well. This response varies among systems and does not necessarily occur universally. By contrast, increases in N availability through deposition or fertilization tend to reduce root colonization and fruit body production by ECM fungi . Effects of CO# and N availability on the biomass or production of extraradical hyphae have been less intensively studied or summarized. In this review, we address the current state of knowledge regarding the potential for mycorrhizal tissue to form a sink or source of C in response to elevated CO# or N deposition. First, we present an overview of processes and pools involved in the cycling of mycorrhizal C and the relevance of various measures of mycorrhizal dynamics .Second, we discuss known effects of CO# concentration on hyphal biomass, turnover, tissue quality and community composition. Next, we focus on the influence of N availability on these same factors, and finally we address potential interactions between elevated CO# and N availability.Processes involved in the cycling of mycorrhizal C include production, survivorship and decomposition rates of tissue. As mycorrhizal tissue grows, C is transferred from the atmosphere via plants to the pool of live hyphae. Micro-arthropods might graze a fraction of live hyphae, but grazing on AM hyphae should be low, as in feeding trials mites and collembola appear to prefer nonmycorrhizal fungi .