The higher concentration of mTagBFP strain was inoculated to compensate for its slow growth

Phosphate is well-known for its ability to covalently bond to metal oxides,27,28 giving the potential to significantly alter their surface properties. Figure 5 shows that even at relatively low concentrations of PO4 3− , the ζ-potential of nano-CuO switches from positive to negative and becomes increasingly negative with increasing PO4 3− concentrations. This change in ζ-potential is likely due to the formation of a negatively charged layer of copper phosphate on the surface of the ENMs resulting in enhanced electrostatic repulsion, leading to the effects shown in Figure 4. The nonlinear effect on sedimentation seen at higher PO4 3− concentrations may be due to covalent bridging between phosphate-coated particles. While the addition of PO4 3− slightly increases the pH of the suspension , its effect on ζ potential is greater than what is predicted for the small change in pH observed . For example, Figure S2 predicts a ζ potential of 0 at pH 6, but the addition of 2.0 mgL−1 PO4 3− results in a decrease in the ζ potential from +7.79 to −12.2 mV at the same pH. Figure S2 also shows the relationship between pH and ζ potential for nano-Cu and Cu2 and may explain why the presence of PO4 3− did not have a large impact on their behavior in the waters tested. The addition of even low concentrations of PO4 3− to nano-CuO suspensions resulted in the polarity of the particle surface charges switching from positive to negative,macetas redondas grandes but both nano-Cu and Cu2 are negatively charged throughout the whole range of pH tested.

Consequently, any sorption of PO4 3− on the surfaces of these particles is not likely to influence their behavior. 3.3. Dissolution. The total amount of aqueous phase copper and the fraction of ionic copper in Cuaq for the three ENMs varied with pH and TOC, as hypothesized, but also with ionic strength. Cuaq was generally stable over the course of the experiment but tended to increase after ∼60 days, indicating that some long-term dissolution may have been occurring. For Cu2 and nano-CuO in hydroponic media , dissolution to ionic Cu is significant even in the presence of complex-forming ions. In freshwater , the presence of NOM reduces Cuaq and Cudis, possibly through a combination of chelation and by coating particle surfaces. A further decrease in Cudis is seen in storm runoff, which at 6.49 mg C L−1 has the highest TOC content of the waters tested .The rhizosphere is the area around the plant root in soil where microorganisms are densely populated and dynamic interactions between the plants and microorganisms occur. These complex interactions and the assemblage of microorganisms are established, shaped, and maintained by the plant root. Plants exude photosynthetically fixed carbons as nutrient sources, secondary metabolites, and signaling molecules to populate root ecological niches with beneficial microorganisms. This “rhizosphere effect” has important implications for plant growth and protections against biotic and abiotic stressors, and for geochemical carbon cycling in soil environments.

With the expected rise of severe, climate-change-related weather conditions such as drought and the growing need to increase plant productivity with sustainable agricultural practices, it is vital to gain a mechanistic understanding of the rhizosphere effect and engineer the rhizosphere to improve plant growth and resilience. To understand the mechanisms of the highly dynamic processes occurring in the rhizosphere, in situ interrogation of the system with high spatiotemporal resolution is necessary. However, due to the underground nature of the root system and the sheer complexity of the microbiome in the highly heterogeneous soil environment, studying root andmicrobial interactions has been challenging. For example, in a typical rhizosphere analysis experiment, the plant is uprooted from soil in the field or a pot at a defined time point, and the microbiome and other relevant chemicals are sampled in a destructive manner. This practice of uprooting the plant is limiting because the spatial information on microbial community members is lost and the same plant generally cannot be sampled over time. Further, there are many confounding variables, such as chemical reactions with minerals in soil that complicate the analysis and make the studies less reproducible and relevant across different locations and environments. To overcome these experimental hurdles, researchers have designed various types of specialized devices such as rhizotrons and microfluidics devices to improve specific aspects of sampling, analytics, and manipulation of the rhizosphere system, albeit often deviating substantially from the natural system.

Fluorescent microscopy is a promising tool for noninvasive in situ imaging of the microbial and root interactions with high spatiotemporal resolution. Developing in situ rhizosphere imaging methods is made especially more relevant as the microbial community colonization of the root and the persistence and succession patterns are likely dynamic and dependent on the developmental stage of the plant. Better knowledge in this can help guide synthetic microbial community inoculation protocols to optimize plant productivity. A notable development of in situ imaging devices is a microfluidic root chip by Massalha et al., in which real-time imaging of the microbial colonization of Arabidopsis thaliana seedlings was captured by high-resolution fluorescent confocal microscopy. The co-inoculation by the fluorescent protein expressing Bacillus subtilis and Escherichia coli showed preferential localization, where B. subtilis established more densely at the root tip while E. coli dispersed homogeneously around the root . While microfluidic devices have shown great promises in rhizosphere research, the micro-scale dimension of the root chamber limits the samples to be young seedlings of the model plants. Rhizotrons can support the growing plants in a chamber for a much longer period of time, but are not designed for high-resolution microscopy. Recently, we described the development of fabricated ecosystems that can support the various model plants for 3–4 weeks by having the bigger root chamber, which can be modified for conventional microscopy by using a glass slide as the lower plate.

We demonstrated that the EcoFAB can generate reproducible plant physiology and exudation phenotypes by conducting the multilab experiments with the model grass B. distachyon. While the EcoFAB gives much improved imaging capability over rhizotrons and the imaging of the plant root phenotypes such as root hair length and branching pattern have become much more accessible, imaging the microbial interactions with the root remains challenging. One reason is that the current design of the EcoFAB is not suitable for imaging with high-magnification and numerical aperture objectives to interrogate the micro-scale interactions at high resolution. Another reason is that the root can easily go in and out of the focal plane with the extra chamber height, a problem that would be exacerbated by using high-magnification objectives with a thinner focal plane. To overcome these imaging constraints,maceta 25l we modified our EcoFAB to be an imaging specific device that combined a larger root chamber to support longer growth times and the ability to image root and microbial interactions. By implementing the pillar structures in the root chamber, the effective chamber height for the root is reduced so that the root remains closer to the glass surface while maintaining the flow of media throughout the chamber. The polydimethylsiloxane device body is bonded with a thin cover glass to reduce the working distance between the sample and microscope objective. Consistent hydration and supplementation of nutrients become more challenging with the more mature plants transpiring at a greater rate and the thinner chamber profile reducing the volume. We modified the commercially available growth chamber with a watering port so that the Imaging EcoFAB is housed in a sterile environment with easy sterile additions of water or media . Imaging EcoFAB is designed to improve the imaging capability by reducing the effective height of the root chamber so that the plant root grows closer to the cover glass. By adding the pillar structures with dimensions of 1.5 mm × 1.5 mm × 2 mm and pillars separated by 300 µm from each other throughout the top of the root chamber , the effective chamber height became 1 mm and the B. distachyon root was forced to grow closer to the cover glass . The pillar structures allowed effective exchange of nutrients and chemicals across the root chamber and yielded plants with similar growth patterns to the original EcoFAB.

The dimensions of the pillars and the distance between them can be modified based on the plant type and experimental specifications. However, the 3D printers have limitations with the aspect ratio of the 3D structures and resolution of the features. The printability of the pillars needs to be experimentally tested for each type of printer and resin . Once the mold is printed and treated for PDMS casting , the PDMS body is bonded with the thin cover glass to reduce the distance between the objective and the root to accommodate high magnification objectives with the short working distance . The ability to spectrally distinguish colocalizing bacteria in the rhizosphere is benefi- cial to interrogate the microbial interactions, especially while imaging at high magnification and resolution to allow segmentation and cell counting for quantitative data analysis. To demonstrate this in an Imaging EcoFAB, P. simiae strains engineered to constitutively express the fluorescent proteins mTagBFP, mTurquoise2, EGFP, mVenus, mKO, mApple, mCherry, mKate2, and mCardinal were inoculated into a week-old sterile B. distachyon. After 3 days, the spectral images at 10×, 20×, and 40× magnification were acquired in the lambda mode of ZEN software as well as the linear unmixing of the spectral images . Due to the excitation and emission spectral overlaps, all 9 fluorescent proteins could not be distinctly visualized. We also observed that the blue emitting proteins mTagBFP and mTurquoise2 did not have detectable fluorescence signals, possibly due to slower growth due to greater metabolic burdens to express these proteins. We speculate that this may be due to the greater metabolic burdens to express these proteins. The spectral images analyzed using K-means clustering algorithm and the fluorescence signals were clustered into four spectral categories : green , orange , red , and background . These three colors are represented in the spectrally unmixed images from the lambda mode . In the 40× image, which is focused around root hairs , the individual bacterial strains are resolved at various densities . Using this image, the segmentation analysis was conducted by first generating the binary image with the fluorescence intensity threshold and then applying the dilation and erosion operations with the circular structuring element to separate out the individual bacterium . This initial analysis yielded the counting of 478 cells in the image. However, to improve the accuracy of cell counting in the rhizosphere, the 3D multi-spectral image using z-stack is necessary as the different orientations of the rod-shaped P. simiae yield either rod or circular shapes on 2D profile. With the improved software package of the microscope system that supports multi-spectral 3D segmentation using machine learning algorithm , the quantitative analysis of the rhizosphere microbiome will become more accurate and mainstream. Imaging EcoFAB can be used for studying the bacterial persistence and succession patterns in the rhizosphere with the same live plant in situ over a 3- to 4-week duration. To examine this application, we conducted a succession study using two fluorescent strains of P. simiae. These two strains have identical genomes except for the fluorescent protein, allowing for the demonstration of a succession experiment in the device where a first one strain is established in the rhizosphere, followed by a second stain co-locating. First, mTagBFP expressing P. simiae was inoculated to the 2-week-old sterile B. distachyon at OD600 of 0.5. After three days, establishment of the mTagBFP strain was confirmed by fluorescent imaging . Then, the P. simiae with mCherry expression was inoculated to the same plant at OD600 of 0.1 and imaged after 2 days, showing the colocalization of mTagBFP and mCherry fluorescence around the root tip of B. distachyon .In this study, we improved the rhizosphere imaging ability of the original EcoFAB by adding pillar structures on the top of the root chamber and by using a thin cover glass as the bottom substrate. The device’s improvement was demonstrated by high-magnification and -resolution confocal imaging of the entire B. distachyon root and microbial interactions while maintaining the key advantages of the EcoFAB: real-time in situ imaging using conventional microscope system, bigger chamber to support the model plants for 3 to 4 weeks, ability to pack solid substrates, and improved environmental control such as sterility of the chamber.

A few studies have described the physiological impacts of rare earth elements in plants

To date, many rhizosphere microbiome studies and growth chambers systems focus on the impact of plant developmental stage, genotype and soil type on microbial composition and function . On the other hand, predation as a driver in the rhizosphere microbiome remains understudied. For instance, protists are abundant in the soil and are active consumers of bacteria and fungi and play a role in nutrient cycling yet remain an overlooked part of the rhizosphere . Viruses are also pivotal in modulating host communities thereby affecting bio-geochemical cycles but their influence in the rhizosphere is poorly studied . These predatorprey interactions in the rhizosphere deserve in-depth studies which can be facilitated by these specialized growth chambers. Another area worth investigating in the rhizosphere is in anaerobic microbial ecology. At microbially relevant scales, soils primarily exist as aggregates . Aggregation creates conditions different from bulk soil, particularly in terms of oxygen diffusion and water flow resulting in anoxic spaces within aggregates and influences the microbial community . The rhizosphere is also rich in a wide range of compounds which can serve as alternative electron acceptors such as nitrate, iron, sulfate and humic substances in the absence of oxygen .

However,arandanos cultivo most anaerobic studies in the rhizosphere focus only on aqueous environments such as water-logged paddy soils despite biochemical and metatranscriptomic evidence pointing to the possibility of anaerobic respiration in the rhizosphere . To fully understand biogeochemical cycles in the rhizosphere, it is imperative to investigate rhizosphere processes in the micro-scale and to include localized redox conditions as one of the influencing parameters. Microfluidic platforms with its fast prototyping capabilities can be helpful in creating growth chambers designed to stimulate these redox changes. In the study of the rhizosphere microbiome, genetic manipulation strategies are foundational in deep characterization of microbial mechanisms and current manipulation techniques require axenic isolates. However, the uncultivability of a significant portion of soil microorganisms continues to hamper efforts in gaining mechanistic knowledge. Even for culturable isolates, the process of isolation introduces selective pressure and disturbance to the community with inevitable loss of information on spatial interactions. A recent innovation in gene editing technologies using CRISPR-cas systems demonstrated in situ editing of genetically tractable bacteria within a complex community .

Coupled with the use of transparent soil-like substrates , the application of such a technique for the editing of in situ rhizosphere microbiome while preserving spatial and temporal associations would indeed bring invaluable insights. Specialized growth chambers using 3D fabrication and microfluidic technologies are primed to facilitate such innovations. Finally, this review revealed that while similarities exist among the different growth chamber systems, many of these systems are bespoke. This makes it difficult to replicate experiments and determine reproducibility which are important cornerstones of scientific advancement. The complexity of rhizosphere interactions also warrant that computational models are essential to gain a better understanding of system level processes . However, predictive modeling requires data from standardized approaches to be comparable between experiments. Thus, future growth chamber systems and designs are encouraged to follow the open science framework to enable standardization to an extent, such as in the case of EcoFABs . Cerium oxide nanoparticles are widely used in applications such as catalyst automotive industry, glass mirrors, plate glass, and ophthalmic lenses . These NPs are among the 13 engineered nanomaterials in the list of priority for immediate testing by the Organization for Economic Cooperation and Development . However, the environmental release of CeO2 NPs from factories or applications, and their behavior and effects in the environment are not well known yet .

Previous studies have shown that CeO2 NPs are stable in soil at pH values of 7 to 9 . This suggests CeO2 NPs will remain in soil for a long time. In addition, reports from recent investigations have shown a wide variety of plant responses after exposure toCeO2 NPs. For instance, Schwabe found that CeO2 NP treatments did not reduced the growth in pumpkin and wheat. However, Ma et al. reported that, at 2000 mg/L, nano-CeO2 reduced root elongation in lettuce . Van Hoecke et al. found that CeO2 NPs, at concentrations as low as 2.6 and 5.4 mg/L, produced chronic toxicity to the unicellular alga Pseudokirchneriella subcapitata. Previous results from our research group have shown that CeO2 NPs at 2000 mg/L reduced corn and tomato germination by 30% and cucumber germination by 20% . In a more recent study, we demonstrated that CeO2 NPs are taken up and stored without change in maize roots . This previous study also revealed that the uptake of CeO2 NPs by corn plants was affected by soil organic matter content and alginate surface coating . Alginates are naturally occurring polysaccharides that have been used to stabilize NPs for several applications . This suggests that excess of alginate can be released into the environment together with NPs, with unknown consequences for edible plants. Thus, more studies are needed to better understand the impact of CeO2 NPs in plants, in environments where excess alginates could be present. On the other hand, studies have shown that carbon-based nanoparticles such as single walled carbon nanotubes triggered reactive oxygen species generation in Arabidopsis and rice . In addition, multiwall carbon nanotubes have been found to induce gene expression of heat shock protein 90 in tomato leaves and roots . However, there are no reports on the effect of CeO2 NPs on heat shock protein expresion in plants. For example, at concentration higher than 89 µmol/L, cerium affected the foliar chlorophyll content, nitrate reductase activity, shoot root length and relative yield in cowpea plants . The authors suggested the effects could be produced by the substitution of Mg2+ by Ce in chlorophyll synthesis. It has also been suggested that, due to their similar chemical characteristics, Eu, a REE, may compete with Ca for organic ligands . These studies suggest that REE elements can have serious impacts on the uptake of nutritional elements in food crops. However, to the authors’ knowledge the impact of REE NPs on the uptake of nutritional elements by plants has yet to be reported. The purposes of this work were to determine the effects of alginate on: the transport of Ce within corn plants treated with CeO2 NPs, the uptake and transport of micro and macro nutrients, the chlorophyll content, and the expression of stress related heat shock protein 70. Maize was selected for this study because it is a crop widely cultivated throughout the world for direct and indirect consumption. In addition, 40% of the corn world’s harvest is produced in the United States . In this study, corn plants were grown in soil spiked with CeO2 NPs with various alginate concentrations for one month.

After harvest, the concentration of Ce and many nutrient elements were determined by ICP-OES in the root and shoots tissues. Cyclopentenone oxylipins regulate diverse biological processes. In animals, prostaglandins formed by cylcooxygenases have hormone-like effects that sustain homeostatic functions and mediate pathogen interactions, including inflammation responses.Plant cyclopentenones also have important roles in signaling and defense. While distinct functions for various cyclopentenones have been elucidated,maceteros grandes reciclados the vast majority of plant studies have focused on the 13-lipoxygenase product 12-oxo-phytodienoic acid and its derivatives, collectively termed jasmonates. A vast number of physiological and genetic analyses have demonstrated diverse roles for jasmonates including developmental processes and inducible defenses against biotic threats.In maize , genetic evidence highlights roles for jasmonic acid in defense against biotic threats, senescence, and developmental processes that control male sex determination.As an essential jasmonate precursor, 12-OPDA can trigger development and defense signaling distinct from JA itself or its bio-active amino acid conjugate -7-iso-jasmonoyl-L-isoleucine.During physiological stress in Arabidopsis, free 12-OPDA binds cyclophilin 20–3 to promote the formation of a cysteine synthase complex leading to elevated glutathione levels and cellular redox homeostasis.In general, 12-OPDA signaling is consistent with a role in cell protection and inhibition of cell death.In addition to the 13-LOX jasmonate pathway, 9-LOX genes also function in plant development and plant-biotic interactions. Specific 9-LOXs have been shown to influence cell death, root growth,senescence,and to promote either susceptibility or resistance to pathogens and pests in host-species interactions. Despite evidence linking 9-LOX genes to important biological roles, many of the specific metabolites that promote these functions have remained elusive. 10- oxo-11-phytoenoic acid and 10-oxo-11,15-phytodienoic acid have been long considered candidates for biologically active compounds given their similarity to jasmonates .First isolated from potato , 10-OPEA and 10-OPDA were identified as products of 9-allene oxide synthase enzymes and subsequent nonenzymatic cyclization, resulting in racemic mixtures of low yield stereoisomers.In contrast, our empirical findings in maize silks and a concurrent study done in maize roots showed that cis-10-OPEA synthesis in maize is consistent with full enzymatic biosynthesis, yielding predominantly – 10-OPEA as a result of an as-yet unidentified enzyme with novel 9-AOC activity.

Together, these results suggest pathway diversity in 9-cyclopentenone formation between dicot and monocot species. In a recent study, we initiated the functional characterization of 10-OPEA, 10-OPDA and derived C14 and C12 metabolites, collectively termed death acids.10-OPEA is inducible at the local site of pathogen and insect attack and has antimicrobial and herbivore growth inhibitor activity. The capacity of 10-OPEA as a plant defense signal/metabolite is similar to 12-OPDA, yet it demonstrates specificity, with only 60%overlap in the co-expression of over 5000 genes differentially expressed in response to these 2 conceptually related oxylipins. One distinction from 12-OPDA activity is the weak ability of 10-OPEA to promote accumulation of transcripts coding for protease inhibitors. Furthermore, exogenous 10-OPEA application strongly induces cysteine protease activation and programmed cell death, as evidenced by immunoblot protease labeling, lesion development, ion leakage, and DNA fragmentation.Collectively these results suggest that 9- and 13-LOXderived cyclopentenones can have distinct and separate functions, despite their structural similarities. To further elucidate and contrast the established roles of 12-OPDA in insect related defense responses, and the more specific role of 10-OPEA in fungal-infected tissues, we examined the induction of 9-LOX and 13-LOX derived cyclopentenones in additional maize tissues under varied stress conditions. To explore 9- and 13-LOX derived cyclopentenone production in maize in response to pathogen and insect attack, we first infected the inner-whorl leaf tissue and developing upper node and internode stem tissue with Cochliobolus heterostrophus and measured 10-OPEA, 10- OPDA, and 12-OPDA over a 3 day time course. At 24 h post inoculation, 10-OPEA concentrations in inner-whorl leaf tissues were significantly higher than 10-OPDA or 12-OPDA and remained higher over the duration of the time course . Similar results were observed in node/internode stem tissues, collectively indicating a bias toward 10-OPEA production in both leaf and developing node/internode tissues. Previous work has routinely demonstrated that all of these metabolites are near or at the limits of detection in healthy untreated control tissues.To mimic insect feeding, regurgitant from Spodoptera frugiperda was applied to fully expanded maize leaves. Basal levels of 12-OPDA were signifi- cantly higher than 10-OPEA and 10-OPDA with 3- and 10- fold differences, respectively . By 1 h post treatment, FAW-R had elicited a strong 13-LOX response with 12-OPDA levels exceeding 10-OPEA and 10-OPDA production by >65 fold, demonstrating a strong preference for the jasmonate pathway in response to herbivory on mature leaves. Both insect feeding and pathogen attack can occur on diverse maize organs and cause the damage-induced collapse of cells. To build upon our previous results showing abundant levels of 9-LOX cyclopentenones in the silks13 and to better understand the capacity of different organs to produce 10-OPEA and 12-OPDA, hydroponic root and silk tissues were crush damaged and metabolites were measured over a time course. Both hydroponic roots and silks displayed a rapid induction of 10-OPEA upon damage, producing approximately 3 and 40ug/ g FW within 20 min, respectively, and sustaining high levels for at least 120 min . Both 10-OPDA and 12- OPDA were at least 2-fold less than 10-OPEA in response to crush-damage and remained significantly lower throughout both time courses. Together, these results demonstrate that 10- OPEA is the predominant LOX-derived cyclopentenone in silks and roots in response to cell damage. The maize scutellum is an important developmental organ in early maize ontogeny, acting to absorb, store, and transfernutrients from the endosperm to the growing seedling while preventing microbial infections.

Mortality was determined using a generalized linear model with a binomial family

Interestingly, many chemicals used by humans, which are not intended for use on microbial communities, have been shown to affect microbes. For example, caffeine, a common mental stimulant, alters biofilm respiration, and an antihistamine, diphenhydramine, has been demonstrated to modify the microbial community and respiration of lake biofilms. Because of unexpected pharmaceutical effects, it is relatively difficult to predict what will occur in model organisms. This problem is exacerbated by a lack of information regarding pharmaceuticals’ effects on terrestrial insects: no available publications report the effects on any terrestrial insects’ microbial community. Arthropods, such as insects and crustaceans, rely on hormones to grow, develop, mate and even produce pigmentation. However, many pharmaceuticals, especially hormones, resemble chemicals that these organisms rely on for growth and development. These pharmaceuticals then bind to receptors and either over-express or suppress their counterparts’ natural function. This has been reported in birds, reptiles, macetas redondas and arthropods where endocrine disruption occurs, primary and secondary sexual characteristics are modified, and courtship behaviors change.

While most arthropod hormones do not closely match those of mammals, their molting hormone , is very similar to 17β-estradiol . In crustaceans, mammalian hormones have been known to cause both increased molting events and inhibition of chitobiase, the enzyme responsible for digestion of the cuticle during insect molting. In insects, 17α-ethinylestradiol, a common synthetic birth control hormone, has been shown to alter molting and lead to deformities of C. riparius. Also, BisphenolA, a common plasticizer, can bind and activate estrogen receptors in humans, and the ecdysone-binding protein in insects. In addition to these effects, pharmaceuticals have been shown to cause effects to insects over multiple generations. Megaselia scalarisis a common saprophagous pest. They are known to infect living humans , provide important ecological roles as detritivores, and because they often feed on human corpses are commonly used in forensic entomology to determine time of death. This species will generally feed on a variety of decomposing plant and animal tissues, and acts as a vector of pathogens. These insects are both fecund and hardy because females can lay over 650 eggs in 16 days and are tolerant of heavy metals. The white, roughly football-shaped eggs, hatch after approximately 24 hours into white translucent larvae. When they have matured to third instar they pupariate. Their detritivorous larval life history exposes them to a wide diversity of microorganisms that may act as pathogens, commensals, and symbionts. There is currently no record of how M. scalaris acquires their microbiota or if any symbionts are required. However, it stands to reason that they, like so many other insects, would rely on microbial symbionts.

There are many ways insects acquire symbionts: from their diet, the environment, their social network, or vertical transmittance. Currently there is little to no information regarding pharmaceutical effects at the concentrations found in reclaimed water on the growth or microbial community composition of any terrestrial detritivore. These detritvores become exposed to contaminants after the CECs enter surface waters, soil, and plants from overflow and wastewater reuse. There are studies involving antibiotics at high doses to determine necessity of microbiota in several insects, but these have not tested relevant concentrations found in reclaimed water or joint effects of other pharmaceuticals, which often coexist with antibiotics. To assess potential effects of common pharmaceuticals, we used a series of bio-assays to determine the possibility of individual and joint contamination on development, mortality and population sex ratios of M. scalaris. Any effects would have potentially important implications from medical, ecological, and forensic perspectives. Also, as there is currently no information on M. scalaris’ microbial community, information generated from this study could serve as novel information into the role possible symbionts play in M. scalaris development. Test compounds included: acetaminophen, caffeine, three antibiotics, and four estrogenic steroidal hormones. Six treatments were examined: acetaminophen, caffeine, an antibiotic mixture , a hormone mixture , a mixture of all chemicals , and a control, consisting of only distilled water. Distilled water was tested for CECs and found to not contain any. Treatment groups were chosen as representative compounds for pain relievers, mental stimulants, antibiotics commonly used on humans and livestock, hormones normally either produced or prescribed to humans, and as a mixture that would be simple, yet representative of wastewater effluent or reclaimed wastewater. Artificial diets were prepared at room temperature to negate any decomposition of the CECs.

Acetaminophen , caffeine , estrone , 19-norethindrone , 17β- estradiol , 17α- ethynylestradiol , lincomycin , and oxytetracycline concentrations were chosen based on the maximum concentrations measured by Kolpin et al. 5 . Ciprofloxacin concentration was chosen from the maximum lake water concentration reported by Mutiyar and Mittal6 . The chemicals used were purchased as follows: acetaminophen with a purity of ≥ 90%; ; caffeine at laboratory grade purity ; lincomycin, oxytetracycline, and ciprofloxacin with purities of ≥98% ; estrone, 19-norethindrone, 17β- estradiol, and 17α- ethynylestradiol at ≥98% purity . Blue formula 4-24® instant Drosophila medium, hereafter known as ‘blue diet’, was purchased from Carolina Biological Supply Company . Hydrochloric acid was obtained from Fisher Scientific. Sodium hydroxide was acquired from Sigma-Aldrich as anhydrous pellets. Stock solutions were prepared by adding powdered chemicals to deionized water. Approximately 5 mL 80% ethanol was added to 250 mL steroidal hormone solutions to facilitate dissolution. Hydrochloric acid was added to antibiotic chemical solutions to facilitate dissolution and pH was adjusted using NaOH to pH 4.00. Compounds were added to distilled water to the desired concentrations for each treatment and then an equal amount of blue diet flakes was added as described by the manufacturer. In all experiments, preparations and concentrations of treatment groups were prepared as stated previously.Eggs were transferred, by microspatula, to the blue diet of each 9 cm Petri dish. There were 3 replicates per life stage for each of the 6 treatments . Lids contained a size 7 cork-borer hole affixed with 2 layers of fine organza mesh to allow for moisture and gas exchange. Following egg placement, Petri dish lids and bottoms were aligned and secured with parafilm. Petri dishes were monitored daily for development. Six individuals were collected at third instar, pupa, and adult life-stages,maceta de 10 litros triple washed with 200 proof ethanol, and stored in clean 200 proof ethanol at -60 °C until DNA extraction. During the collection of each treatment group and life-stage, blanks in triplicate of DDI H2O were used to monitor contamination. Before extraction, triplicate blanks were pooled. All genomic information was processed using macQIIME ver. 1.9.1-20150604 . We used USEARCH v6.1 to identify and remove chimeric sequences, and SUMACLUST to cluster OTUs and remove any with less than two reads per sample. We used 97% sequence identity to bin OTUs and choose representative OTUs. We then performed standard alpha and beta diversity analyses in QIIME. To assign taxonomy to OTUs, Greengenes taxonomy and the RDP Naïve Bayesian Classifier were utilized, and we also performed BLASTN searches against NCBI’s online Nucleotide Collection and 16S ribosomal RNA sequences databases . Taxonomy was then used to identify any mitochondria or chloroplast OTUs, which were removed from the dataset as in McFrederick & Rehan . We aligned the quality-filtered dataset using the pynast aligner and the Greengenes database . We then reconstructed the phylogeny of the bacterial OTUs using FASTTREE version 2.1.3. Next we performed weighted and unweighted UniFrac analyses using the generated phylogeny and OTU tables. Using the generated distance matrices, we performed Adonis and created PCA 74 graphs in R version 3.3.1 .

Foralpha diversity, we plotted rarefaction curves in GraphPad Prism version 6.00 software , and used gplots to create a heatmap of the most abundant bacterial families; a 0.025 proportional abundance in at least one sample was used as the cutoff.All statistical analyses were performed using R . Normality was determined using Shapiro-Wilk normality tests. Differences in days to pupariation were determined using the ‘survival’ and the ‘OIsurv’ packages. In all cases, when data were not considered normal, either a Poisson distribution or a negative binomial generalized linear model was used and the best fitting model was determined from Akaike’s ‘An Information Criterion’. Adonis within the R package “vegan” was used for all PERMANOVA analyses. As there is no post-hoc test for Adonis, we used adjusted p values obtained from metagenomeHIT_zig in R through QIIME to determine differentially abundant OTUs in treatments between life stages.Megaselia scalaris, a common detritivore, has been known to develop on substances as diverse as human wounds and corpses, modeling clay, and emulsion paint. Their ability to grow and mature on these diets, with minimal effect on their survival, and their tolerance to heavy metals 145 makes any effect of pharmaceuticals at very low doses found in reclaimed water even more surprising. In our study, the females had no preference for untreated diets versus any treated diets. This poses a problem for the insect population, as there was higher larval mortality when developing on a caffeinecontaminated food source. Because females require an additional 24 hours 142 afteremergence in order to be receptive to males, populations exposed to hormones or antibiotics would be adversely affected. If females require an extra six days to emerge and become receptive, there is a reasonable possibility the males would leave the area or perish before mating. In addition, the suitability of decaying food sources tends to be temporary . Collectively, these factors could likely negatively influence population growth. Also, these changes in population growth rate could hinder forensic scientists from determining an accurate time of death if there were long lasting or even moderate concentrations of these pharmaceuticals in the body at death. Sex ratios of emergent adults were also affected in the caffeine and mixture treatments. The sex ratios found in control treatments in our study are similar to those reported in Benner & Ostermeyer of a male: female sex ratio at 25° C of 1.18:1. However, sex ratios from the acetaminophen, caffeine, and mixture treatments differed significantly from the controls. A major difference in sex ratio would change the reproductive capacity of a population. It is unclear why acetaminophen and caffeine would alter sex ratios, however acetaminophen as been recorded to hinder the production of arachadonic acid in mosquitoes, another Dipteran, and it could be playing a similar role here . Ibuprofen, another analgesic and antipyretic has been shown to alter the sex ratio in another . Many insects rely on their microbial communities and endosymbionts to grow and develop . However, Adonis, the statistical method used to analyze these data, does not have a post hoc test available that would allow direct pairwise comparisons between treatments. Nonetheless, there are changes in the bacterial community based on adjusted p-values evaluating differential abundance. We found significant shifts in the microbial community in the various life stages examined within the control treatments. A similar result has been reported for mosquitoes and other insects. Not surprisingly, insects that undergo complete metamorphosis and also rely on a different food source as adults would require a different bacterial community; however there is one family, Pseudomonadaceae, which appears in all treatments and life-stages. Species in this family are gram-negative Proteobacteria that cannot survive in acidic environments . They are fairly common in insects , and can be responsible for 90+% of the bacterial community. They are resistant to antibiotics , which potentially explains why they are so prevalent in many of our treatments. Pseudomonadaceae is responsible for ~ 50% of the bacteria in all life-stages, followed by Alcaligenaceae, Enterobacteriaceae, and Xanthomonadaceae. Pseudomonadaceae and Enterobacteriaceae families contain known symbionts in insects and could be filling the same role in M. scalaris. When Pseudomonadaceae is removed from the heatmap , it becomes clear how the next three highly proportional families change with life-stage. Alcaligenaceae tends to become more proportionally abundant in pupae and adults than in larvae. Species in the family Alcaligenaceae are oxidase- and catalase-positive and utilize a variety of organic and amino acids as carbon sources . Enterobacteriaceae has higher proportions in larvae than in adults. Species of Enterobacteriaceae are likely to be either symbionts or a pathogen to their hosts . Enterobacteriaceae includes Buchnera, an important endosymbiont of aphids , and other species that inhabit various insects to provide facultative benefits.

A previous large-scale proteomics study in Arabidopsis also found OPT3 at the plasma membrane

We present evidence suggesting that this Cd over-accumulation may be the result of an enhanced transport of Cd through the plant, making opt3-2 a suitable background for studying long-distance transport of non-essential heavy metals. We further show that OPT3 is targeted to the plasma membrane and is preferentially expressed in the phloem. The Fe/Zn/Mn uptake transporter IRT1 and other ironstarvation-induced genes are constitutively up-regulated in opt3-2. Interestingly, shoot-specific expression of OPT3 restores metal homeostasis and IRT1 up-regulation in roots showing that OPT3 is the first identified molecular component of the network transferring information on the iron status from leaves to roots. Moreover, Fe mobilization between leaves is impaired in opt3-2, suggesting that OPT3 mediates the movement of Fe out of the leaves, and this transport is required for proper communication between leaves and roots and maintenance of the trace-metal homeostasis in Arabidopsis. Understanding phloem-mediated signaling, transport, and seed-loading mechanisms of both essential and non-essential heavy metals will help to develop strategies for excluding toxic metals from seeds and enhance the nutritional value of grains and plant-based products.Members of the Arabidopsis oligopeptide transporter family have been shown to mediate the transport of a broad spectrum of peptides .

Glutathione and phytochelatins are peptides that mediate tolerance and long-distance transport of heavy metals ; therefore, we screened mutants in the Arabidopsis OPT family for differential accumulation of Cd in seeds. A mutant of the Arabidopsis OPT3 gene, opt3-2,plants in pots ideas showed the strongest over-accumulation of Cd in seeds . To test whether this Cd over-accumulation had an effect on seedling growth, assays were performed on plates in the presence and absence of Cd. Figure 1B shows that opt3-2 is hypersensitive to Cd when grown on medium containing 50 μM CdCl2. To determine whether the increased Cd concentration in opt3-2 seeds was due to a systemic over accumulation of Cd throughout the plant, opt3-2 seedlings were grown hydroponically for 6 weeks, exposed to 20 μM CdCl2 for 72h and the metal concentration of roots and leaves was measured by ICP–OES . The roots of opt3-2 over-accumulated Cd compared to wild-type; however, unexpectedly, Cd concentrations in leaves were almost five-fold less than those of wild-type plants . Conversely, seeds of opt3-2 plants show a large increase in Cd levels compared to wild-type seeds .Previous GUS staining experiments have shown that OPT3 is expressed throughout the vasculature; however, localization at a higher resolution has not been evaluated . To identify where in the vasculature OPT3 is preferentially expressed, β-glucuronidase was expressed under the control of the native OPT3 promoter. Under standard growth conditions, GUS staining was negligible; however, under Fe-limiting conditions , staining was clearly observed in the phloem, but not in the pith or endodermis . Consistent with our findings, cell-type-specific micro-array data sets show the highest intensity values of OPT3 in the phloem, comparable to the phloem sucrose transporter SUC2 . Thus, two independent approaches show preferential expression of OPT3 in the phloem.

To gain insight into the subcellular localization of OPT3, an N-terminal YFP–OPT3 translational fusion was infiltrated into Nicotiana benthamiana leaves. Fluorescence was detected along the cell periphery, indicative of plasma membrane localization . A weaker perinuclear fluorescence and transvacuolar strands were also observed in some cells , indicating that a fraction of the YFP–OPT3 localizes to the endoplasmic reticulum . The ER fluorescence pattern, however, was not present in all cells. Furthermore, Hechtian strands were clearly present connecting the cell wall to the plasma membrane of plasmolyzed leaf cells . These results suggest that OPT3 is a plasma membrane transporter preferentially expressed in the phloem.The Arabidopsis mutant opt3-2 shows a constitutive Fe-deficiency response in roots including the up-regulation of the Fe/Zn/Mn transporter IRT1 . Despite this Fe-deficiency response, Fe sensing in shoots remains intact . The molecular mechanisms mediating shoot-to-root signaling of iron status in plants remain largely unknown. The impaired iron sensing in roots but not shoots of opt3-2, in conjunction with phloem localization, suggests a possible role of OPT3 in shoot-to-root transport of a signal reporting metal status. To test this hypothesis, the OPT3 coding sequence was expressed in opt3-2 under the control of the shoot-specific chlorophyll a/b binding protein promoter . Shoot specificity of the CAB2 promoter was determined by GUS staining . RT–PCR analyses confirmed that OPT3 is preferentially expressed in the shoots of three independent transgenic lines . The residual OPT3 transcript in opt3-2 roots expressing CAB2pro:OPT3 plants is consistent with the knockdown nature of the opt3-2 allele. Thus, the low level of OPT3 transcript in roots is not sufficient to properly regulate metal homeostasis in roots .

Two of the major phenotypes described in opt3-2 are the constitutive iron-deficiency response in roots, as illustrated by high IRT1 expression , and the over-accumulation of Cd in seeds . Thus, we tested whether shoot-specific expression of OPT3 was able to complement both phenotypes. As shown by RT– PCR, IRT1 transcript levels were greatly reduced in the roots of CAB2pro:OPT3-expressing plants compared to the opt3-2 mutant . These results show that shoot-specific expression of OPT3 is sufficient for proper regulation of metal homeostasis, including communication between leaves and roots. Furthermore, the Cd accumulation in CAB2pro:OPT3 seeds was reduced to wild-type levels . Seedling hypersensitivity to Cd was also rescued in the three independent CAB2pro:OPT3– expressing lines . Collectively, these results demonstrate that shoot-specific OPT3 expression is sufficient to complement opt3-2 root phenotypes, suggesting that OPT3 may mediate the long-distance transport of a signaling molecule from leaves to relay information about metal status, thus contributing to whole-plant metal homeostasis.To test whether OPT3 functions in the mobilization of Fe or other molecules, we first assessed the capacity of wild-type and opt3-2 to remobilize Fe from one leaf to other leaves using the radio tracer 59Fe. In these experiments, 59Fe was loaded into a mature leaf as Fe2+ at a slightly acidic pH to resemble the apoplastic pH. The addition of ascorbic acid was used to reduce Fe3+ to Fe2+ and maintain it in the reduced form. Figure 7A shows that Fe can be re-mobilized from one leaf to adjacent leaves in wild-type. In contrast,container size for blueberries opt3-2 shows negligible movement of 59Fe between leaves. Figure 7B shows the 59Fe activity in the four leaves adjacent to the leaf where the 59Fe was originally applied. Compared to wild-type, opt3-2 shows a severe reduction in the quantity of 59Fe mobilized from one leaf to the adjacent leaves , suggesting that OPT3 is required for the reallocation of Fe between plant tissues. In fact, opt3-2 plants over-accumulate Fe in mature leaves compared to wild-type, as visualized by Perls’ staining . Interestingly, over-accumulation of Fe in opt3-2 occurs only in mature leaves but not in young leaves . Moreover, accumulation of Fe in opt3-2 is more evident at the base of the trichomes and near the vasculature in the minor veins but not in the main vasculature, suggesting that, in opt3-2, the reallocation of Fe between leaves is impaired, particularly at advanced stages of leaf development . To test whether OPT3 functions as a Fe2+ transporter similarly to IRT1, we expressed OPT3 in a yeast strain deficient in Fe2+ uptake . As previously shown, IRT1 expression in yeast allows the fet3fet4 strain to grow on minimal media without the addition of extra Fe . OPT3 was unable to rescue the fet3fet4 strain , suggesting that, in yeast, OPT3 does not mediate the uptake of Fe2+ like IRT1. Subcellular localization studies, however, show that OPT3–YFP protein fusions do not localize to the plasma membrane in yeast in contrast to in planta .

This mislocalization of OPT3 in yeast precluded further characterization of OPT3 using yeast as a heterologous system. Note that, if fet3fet4 yeast cells were not sufficiently pre-starved of iron, growth of the fet3fet4 mutant was observed, and therefore long-term starvation of yeast was required for these complementation tests. We attempted complementation with different yeast promoters, starting with the strong GAL promoter . Using the phosphoglycerate kinase yeast promoter, OPT3 also did not complement the pre-iron-starved fet3fet4 yeast mutant, consistently with previous studies showing that OPT3 does not complement this yeast mutant . Nevertheless, Fe re-mobilization studies suggest that OPT3 is essential for the remobilization of Fe within plant tissues; whether this transport occurs as Fe2+ or as a Fe-ligand complex remains to be determined. OPT3 is a member of the oligopeptide transporter family and some members of this family have been found to have broad substrate specificity for peptides of different length and amino acid composition . To test whether OPT3 mediates the long-distance transport of GSH in planta, we pursued radio tracer experiments to assess the movement of 35S-GSH from one leaf to adjacent leaves . No differences were found between wild-type and opt3-2, suggesting that OPT3 does not participate in the mobilization of GSH between plant tissues. Interestingly, opt3- 2 rosette leaves from plants exposed to 20 μM CdCl2 and supplemented with 0.5mM GSH accumulated Cd, but no other metals, to wild-type levels. These results suggest that GSH is required for Cd retention in leaves. On the other hand, GSH supplemented to the roots reduced Cd in the leaves of both opt3-2 and wild-type plants likely because GSH trapped Cd in roots of both opt3-2 and wild-type plants . Glutathione has recently been shown to play a critical role in Fe signaling in yeast by stabilizing FeS clusters in the cytosol . In Arabidopsis, GSH is also important to maintain proper homeostasis and crosstalk between Zn and Fe metabolism . To test whether long-distance transport of GSH is important for proper shoot-to-root signaling and homeostasis of trace metals in roots, we measured the constitutive high activity of the root ferric reductase in opt3-2 after foliar application of GSH . In all cases, including the application of foliar GSH, GSH applied to roots, or foliar application of Fe, the activity of the root ferric reductase remained constitutively high in opt3-2. We also tested whether iron applied directly to roots or complexed with GSH, citrate, or nicotianamine is sufficient to repress the high activity of the Fe chelate reductase in opt3-2 roots. Supplemental Figure 6B shows that iron alone , or in complex with GSH, nicotianamine, or citrate, cannot down-regulate the constitutive iron-deficiency response in opt3-2 back to wild-type levels.We have identified an Arabidopsis mutant, opt3-2, that over-accumulates Cd in seeds and roots but, unexpectedly, under-accumulates Cd in leaves . Cadmium distribution throughout the plant is an orchestrated process dictated by root uptake, root-to-shoot translocation through the xylem, and redistribution of Cd from leaves to sink tissues via the phloem. opt3-2 displays constitutive up-regulation of IRT1, a root transporter with broad specificity for heavy metals including Cd . Over-accumulation of Cd, Zn, Fe, and Mn in roots may be explained by the constitutively high expression of IRT1. However, under-accumulation of Cd in leaves and over-accumulation of Cd in seeds, which is different from essential metals , is inconsistent with the high expression of IRT1 . Nutrients, water, and heavy metals are mobilized from leaves into seeds through the phloem . Accumulation of metabolites in sink tissues and under-accumulation in source tissues is best described as an increased redistribution process, likely through the phloem . Notably, of the analyzed metals, only Cd under-accumulates in leaves . These results suggest that, in contrast to the broad specificity of heavy metal uptake at the root level, metal-specific mechanisms mediate the remobilization of heavy metals from leaves to sink tissues. In addition to the altered distribution of heavy metals within the plant leading to over-accumulation of Cd in seeds, opt3-2 also shows hypersensitivity to Cd at the seedling stage . Both the increased accumulation of Cd in seeds and the Cd hypersensitivity of seedling growth are restored to wild-type levels by ectopically expressing OPT3, demonstrating that the altered redistribution of Cd through the plant is the result of the reduced expression of OPT3 in opt3-2 .The opt3-2 mutant displays a constitutive iron-deficiency response in roots, while the leaves properly respond to iron levels as indicated by wild-type levels of ferritin expression , suggesting that the iron status response is mainly disrupted in roots. In plants, the root iron-deficiency response is regulated by local signals within the root and also by unknown systemic signals originating from aerial tissues .

Interand intraspecific variations in traits that could affect C dynamics are considered

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 .

Extending through both reservoirs was a tube system consisting of a water lift and an air-line

ITNI was originally utilized to assess nitrogen deposition in agriculturally influenced ecosystems . Agricultural plants and cultivated crop species such as corn, rye, and wheat among others, have traditionally been utilized as ITNI study species. Additionally, the ecosystems under investigation were heavily influenced by applied nitrogen fertilizer. The latest investigation into ITNI, as of 2014, has been in peat bogs of Germany . This investigation is considered semi-natural, the peat bogs in question have been drained due to surrounding agricultural and livestock operations and therefore represent an ecosystem that is highly influenced by agricultural processes and is altered significantly by human intervention . To date, no investigators have used the ITNI method to measure Ndeposition in arid or semi-arid regions. The application of the critical load concept in semi-arid ecosystems is hampered by uncertainties in estimating dry and gaseous nitrogen deposition . Coastal sage scrub habitat is especially prone to underestimations of N loading because of arid conditions and the difficulty involved in measuring dry deposition and gas exchange. Since rainfall is usually a minor component of nitrogen loading in coastal sage scrub habitats,containers size for raspberries new methods of measuring nitrogen deposition must be employed. Underestimation of critical loads can misinform environmental policy and regulation with potentially negative effects on ecosystem restoration efforts.

Therefore, my overall goal is to use the ITNI method to better assess and account for all nitrogen uptake pathways by coastal sage scrub and provide a more accurate measurement of atmospheric nitrogen deposition in arid and semiarid ecosystems. The main objective of this chapter was to test the concept of isotope dilution and nitrogen deposition measurement using the ITNI system. It was intended that the ITNI treatments identify the movement and level of equilibrium of 15N in the module system. A specific objective was to test the suitability of the ITNI method in a non-agricultural setting, in which we investigated Coastal Sage Scrub habitats and utilized non-agricultural species such as common California native and Mediterranean invasive plants. By employing coastal sage scrub species in the ITNI method, we can demonstrate the potential to determine species-specific nitrogen deposition rates which would be useful in plant community landscape deposition mosaics of non-agriculturally influenced systems. Our two ITNI study sites were selected based on previous nitrogen deposition data produced by traditional measurement techniques . Both ITNI sites lie along a well-described nitrogen deposition gradient in southern California . My main study site, denoted as “Riverside”, is located at the United States Department of Agriculture Forest Service Pacific Southwest Research Station situated 0.6 miles south of UC Riverside. This location is representative of an urban area receiving elevated levels of nitrogen deposition.

ITNI modules were installed in a fenced workyard containing native and invasive plant species, including California buckwheat,California sagebrush, summer mustard , red brome, and ripgut brome among others. Using conventional methods for deposition monitoring, previous studies have estimated annual N deposition at Riverside to be 14 kg N ha -1 yr -1 . Motte Rimrock Reserve, denoted as “Motte,” is located in the city of Perris, California. Perris is a thirty two square mile city of approximately 70,000 residents. Motte Reserve is managed by the University of California Natural Reserve System which offers protected habitat for California native animal and plant species. Motte contains Riversidian coastal sage scrub and riparian woodlands in addition to native and invasive grasslands. Representative Riversidian species include California buckwheat, California sagebrush, brittle bush, white sage, and black sage. Summer mustard, red brome and ripgut brome are representative Mediterranean invasive plants at the site. Motte, with its buffer of protected habitat surrounded by rural and suburban style developments, represents a moderate nitrogen deposition site . ITNI modules were placed in the middle of the reserve, in an open field adjacent to the reserve manager’s office and dormitory. This site was selected for its lack of obstructing structures or trees and its visibility by Reserve Staff to protect valuable ITNI components. Using conventional methods for deposition monitoring, previous studies have estimated annual N deposition at Motte Reserve to be 12.1 kg N ha -1 yr -1 .Seeds of invasive plants were harvested from Sycamore Canyon Wilderness Park, an open-area preserve in the city of Riverside.

Vegetation at Sycamore Canyon consists mainly of Mediterranean invasive plants such as, ripgut, red brome, summer mustard, Russian thistle, among others, with small pockets of native species such as California buckwheat and California sagebrush. Sycamore Canyon was chosen as a seed collection site because of its proximity to both the ITNI study sites. Additionally, ecotypes present this Sycamore Canyon were representative of species adapted to both experimental sites. Invasive species used in the ITNI experiments included Bromus rubensin Deployment 1 and Hirschfeldia incanain Deployments 2 and 3. Bromus rubens and Hirschfeldia incana seeds were harvested from mature plants within Sycamore Canyon during September 2013. Approximately 600 seeds from Bromus rubens were carefully removed with nitrile gloves and placed into a labeled paper bag. Hirschfeldia incana seeds were harvested by selecting senescing mature plants and removing twigs that contained seed pods. The twigs were placed into a labeled paper bag, where the seed pods were broken open by gloved hand to expose the seeds. Seeds of California buckwheat , the native plant used in the ITNI experiment, were acquired from S&S Seeds . The California Buckwheat seeds were shipped in one-ounce sealed plastic bags and, upon arrival, were transferred into a paper bag for storage. Since California buckwheat is a winter germinating species, seeds were stored in a refrigerator for two weeks to increase germination success by breaking any dormancy induced by warm weather conditions. After refrigeration, the seeds were transferred into a petri dish for sepal removal. California buckwheat seeds, or kerns,big plastic pots are actually covered by sepals, or leftover petals from the flowering body that senesce around the seed. The function of the sepal is to protect the seed until germination conditions are correct for growth. To further induce germination, the sepals were removed from the seeds using tweezers. Seeds were propped up on the petri dish, blunt side down, and pushed with the tweezers until the seed was extracted from the sepal material. Successfully extracted kerns were checked for viability by examining for breaks in the seed coat and using a light source to verify that the endosperm inside the seed was still viable. Seeds with breaks in the seed coat and those that did not have a plump endosperm were discarded. Those that passed the viability inspection were placed in another petri dish for germination. A 1 mg/ml solution of gibberellic acid was used to increase the germination rate of California buckwheat, which typically exhibits less than a 65% success rate . The seeds were placed in a petri dish with a single paper towel folded into the bottom of the dish. Approximately 100 seeds were placed on top of the towel and then the gibberellic acid solution was pipetted on top of the seeds and left to soak in the solution overnight. After twenty four hours, the seeds were rinsed with DIW to remove any remaining gibberellic acid and then transferred onto water-dampened paper towel. Approximately 50 seeds were transferred onto each petri dish, using tweezers to place seeds equidistant apart along the entire bottom petri dish. After seeds were evenly distributed, a second paper towel was folded to the shape of the petri and gently laid on top of the seeds. This towel was also damped with Nanopure water without leaving standing water in the petri dish. Once this was complete, the lids were loosely placed on the dishes to help maintain moisture and placed on an east-facing windowsill to provide sunlight. Water was added to the dishes every morning to ensure proper moisture in the dish for germination. On cloudy days a 60W soft-light desk lamp was used to provide additional lighting.

California buckwheat seeds typically took about 3 weeks to fully germinate into small seedlings while summer mustard germinated in about 3 days. Therefore, native seedlings were always started a month prior to an ITNI deployment to improve seedling survival and viability. For Deployment 1, red brome was sown directly into the modules and therefore there was no pre-ITNI germination period for these seedlings. Red brome was sown directly into modules because the time to maturity, and subsequent senescence was much shorter than the other species used in the ITNI modules. ITNI modules were constructed from General Hydroponics Water Farm Modules , obtained from Discount Hydroponics in Riverside, California. Each module utilized a two reservoir system in which a solid growth medium, in this case sand, was placed in a small reservoir with a perforated bottom. To prevent loss of sand, the bottom of the sand reservoir was lined with 80 µm Nitex fabric. The sand reservoir drained into a lower liquid reservoir that contained the liquid growth media and isotope tracer .These tubes joined in the liquid reservoir via a junction piece at the bottom of the liquid reservoir. At the top of the tube system, a watering ring delivered liquid throughout the upper sand reservoir. Water lift was generated when air was pumped into the bottom of the water lift tube . A single DC-powered DC-20 model 12 Volt air compressor, controlled by a Hydrofarm 7 Day Dual Outlet Digital Timer, provided compressed air to the ITNI modules. The pump was powered by AC power at Riverside, and at Motte we used a deep-cycle lead-acid battery that was kept charged by a 30 Watt, 12 Volt, Unlimited Solar Off-Grid Solar Panel. To discourage microbial growth, the ITNI module components were rinsed with a dilute bleach solution, scrubbed with a bottle brush and then rinsed copiously with deionized water. The liquid reservoirs were labeled with volume markings, which were used in conjunction with a clear level-tube connected to the front of the liquid reservoirs to maintain a constant liquid level in the modules. To protect this tubing, and prevent nitrogen deposition into this extra orifice, a plastic sheath was used to cover the tube when not in use for adjusting and checking water levels. All ITNI modules were assembled in a greenhouse to prevent contamination from prolonged outdoor exposure before deployment. The liquid reservoir was filled with nine liters of deionized water. The air lift, water lift tubing, and watering ring were assembled with all junctions connected. Nitex was laid in the sand reservoir and was marked where the opening for the air and water lift was present. At this mark a small X was cut into the Nitex to allow the air and water lift to perforate, but not allow extra space for sand to escape into the liquid reservoir. The sand reservoir was stacked into the liquid reservoir container. The assembled air and water lift was slid through the hole in the Nitex, about half way up the shaft of the assembly. This assembled piece was then installed, with the air and water lift junction piece passing through the opening in the sand reservoir, and the assembly resting with the Nitex on the perforated sand reservoir bottom. This action left the watering ring just below the height of the sand reservoir when assembled. Once the ITNI hardware components were in place, #16 Silver Sand was added to the sand reservoirs. Each module was filled to two inches below the top of the sand reservoir to reduce sand losses to wind and by transport. The watering ring was adjusted to sit just a millimeter or so above the sand; this was done to prevent any “splattering,” of nutrient solution from bubbles in the water lift and to prevent interference by birds drinking water from under the ring . The sand used in the ITNI modules was composed of sub-angular quartz grains with an effective size of 0.5 to 0.7 mm and is a type commonly used in sand-blasting operations. The #16 Silver Sand was baked at 150 to 200 °C before commercial packaging, resulting in a moisture content of < 0.1% by weight and an essentially sterile media.

All of the metabolites proposed for A. thaliana cells were detected in the cucumber seedlings

N4- acetylsulfamethoxazole was detected in A. thaliana cells and confirmed using its authentic standard. The glucose and glutathione conjugates were detected by comparing the exact mass and fragmentation patterns to proposed spectra libraries for each compound . However, observed difference in the fragmentation patterns indicated that conjugation location differed from in those observed for human metabolism. A similar pattern was observed in previous studies concerning the plant metabolism of pharmaceuticals . The proposed amino acid conjugate in A. thaliana cells is, to the best of our knowledge, the first evidence for their occurrence in higher plants. Conjugation with amino acids has been considered a detoxification pathway for other pharmaceuticals . The structure proposed here for leucylsulfamethoxazole was, in part, based on a m/z of 132.0765, 223.1135 and 255.1677 showing distinct fragments of C6H13NO2 C12H18N2 and C10H12N3O3S . The position of the amino acid on the benzene ring was selected based on optimum stable formation . In the tentative metabolism pathways in A. thaliana cells, sulfamethoxazole underwent Phase I oxidation,raspberries for containers forming 4-nitroso-sulfamethoxazole followed by phase II conjugation with leucine or glutathione. Based on the signal strength, a relatively high level of the N5-leucyl-sulfamethoxazole conjugate was detected at the 3 h sampling point but remained at trace levels for most of the incubation duration, with the exception of the 48h sampling point.

The glutathione conjugate appeared quickly , spiked at 48h, and decreased to a non-detectable level by the end of the cultivation . Conjugation with glucuronic acid was also observed to form quickly , and direct glycosylation of sulfamethoxazole has also been observed in mammals . Another pathway appeared to be acetylation of the sulfamethoxazole amine followed by rapid oxidation to form the S296 and S313 metabolites , with N4-acetylsulfamethoxazole being the predominant metabolite at the end of incubation. It has been shown that this acetylation pathway predominates in human metabolism for detoxification . Future research should be conducted to determine if enzymes similar to those seen in human metabolism of sulfamethoxazole actively participate in its metabolism in plants, such as CYP2C9 and N-acetyltransferases 1 and 2. Similar metabolites were found in cucumber seedlings grown in the nutrient solution containing sulfamethoxazole. Metabolites from Phase I and Phase II metabolism were similarly detected and extensive Phase III sequestration was further observed . When cucumber plants were exposed to 1.0 µg L-1 sulfamethoxazole in nutrient solution, sulfamethoxazole was taken up into the plant, with accumulation primarily in the roots, old leaves, and stems, and absent in the new leaves . When 14C activity was used for calculation, a translocation factor , i.e., the ratio of the concentration in leaves/stems over that in the roots, was estimated to be 0.32. While this value was higher than that reported by Dodgen et al.  it suggested that sulfamethoxazole is not readily translocated in the plant after entering the root. This was consistent with models used to predict the behavior, e.g., diffusion through cell membranes, of similar pharmaceutical compounds .

These models suggested that polar compounds , such as sulfamethoxazole, would have translocation factors from the solution to the xylem ranging from 0.25 to 0.5. Bio-concentration factors were calculated for the roots and the shoots as the ratio of specific radioactivity in the tissue over that in the growth media. The mean BCFR and BCFS were determined to be 1.59 and 0.53, respectively. The calculated BCFR and BCFs were similar to those found for other vegetable species in Dodgen et al. . These studies together suggested that while plants are capable of taking up sulfamethoxazole, it mainly remains in the root with limited potential for translocation to the other organs of the plant . After the 7 d cultivation, sulfamethoxazole parent and metabolites identified in the A. thaliana cell incubation were similarly scanned in cucumber samples from the hydroponic cultivation experiment, including plants tissues and blank control without plants. The proposed metabolites were not detected in the control treatment or the cucumber hydroponic solution, indicating that the transformation occurred within the cucumber seedlings following uptake.However, due to the lack of authentic standards for most of the metabolites or low signal , we did not attempt to quantify individual metabolites in the cucumber plants. In a previous study, Chen et al. investigated the uptake, metabolism, and elimination of sulfamethoxazole in Brassica rapa chinensis and Ipomoea aquatica. In that study, no metabolites of sulfamethoxazole were detected in plant tissue. This could be attributed to a number of factors, such as, differences in extraction protocols, instrument analysis or extensive phase III metabolism that decreased the level of metabolites below the limit of detection. Our findings were in line with previous research conducted with the relatedsulfa-drug sulfamethazine, in which N-acetyl-sulfamethoxazole and hydroxysulfamethoxazole were detected in Zea mays L. plants .

The total amount of extractable and non-extractable residues in the cucumber plants ranged from 94% to 80% indicating that some mineralization occurred. This rate of mineralization in the plant cultivation system was higher than that of sulfamethoxazole in soils. Because plant respiration may contribute to the loss of 14C after mineralization, this finding further highlights the detoxification prowess of higher plants .Global climate change has resulted in shifts in precipitation patterns, causing stress on freshwater resources, especially in arid and semi-arid regions . In many of these areas, demand for water has led to increasing use of municipally treated wastewater . Agriculture has been one of the primary targets for TWW reuse with water districts and governments promoting the adoption of recycled water for irrigation . However, the use of TWW for irrigation may come with potential risks, as TWW is known to contain a wide variety of human pharmaceuticals . The use of pharmaceutical compounds has increased with population growth and economic development, resulting in over 1500 compounds currently in circulation . Their widespread consumption has led to their occurrence in TWW as well as in TWW-impacted surface water . For many of these pharmaceuticals, there is limited knowledge about their potential chronic effects in the environment . Further, many of these compounds can transform in the environment, resulting in the formation of transient or recalcitrant transformation products, many with unknown fates and effects in environmental compartments . Diazepam belongs to the class of psychoactive compounds known as benzodiazepines, one of the most prescribed classes of pharmaceuticals . Diazepam is one of the most commonly detected pharmaceuticals in TWW, with concentration ranging from ng L-1 to low µg L-1 . This is likely due to its extensive use and low removal efficiency during secondary wastewater treatment . In humans, diazepam is primarily metabolized via phase I oxidative metabolism by demethylation to nordiazepam , or hydroxylation to temazepam , and then further oxidized to oxazepam . Oxazepam undergoes phase II metabolism via rapid glucuronidation and then excretion via urine . The three primary metabolites of diazepam are psychoactive compounds,blueberries in pots and each is a prescribed pharmaceutical for treating psychological conditions and alcohol withdrawal symptoms . Both oxazepam and nordiazepam have been commonly detected in TWW, often at µg L-1 levels . However, there is little knowledge about the occurrence, formation, and fate of such metabolites outside the wastewater treatment systems . Several studies have focused on the uptake and accumulation of pharmaceuticals in agricultural plants as a result of TWW irrigation . These studies have demonstrated the capacity of higher plants to take up these compounds; however, until recently, relatively little consideration has been given to their metabolism in plants . Recent studies have shown that higher plants can metabolize xenobiotics similarly to humans with phase I modification reactions followed by phase II conjugation reactions using detoxification enzymes that function as a ‘green liver’ .

In higher plants, phase I and phase II reactions are followed by a phase III sequestration, resulting in the formation of bound residues . Many of these studies have also highlighted a chemical-specific and species-specific nature of plant metabolism of pharmaceuticals. In this study, we examined the uptake and biotransformation of diazepam in higher plants. Arabidopsis thaliana cells were used for an initial kinetic evaluation and metabolic profiling . Cucumber and radish seedlings were then used under hydroponic conditions to understand metabolism of diazepam and its effect on selected metabolic enzymes in whole plants. PSB-D A. thaliana cell line was purchased from the Arabidopsis Biological Resource Center at Ohio State University and cultured in a liquid culture suspension at 25 °C and 130 rpm in the dark. Cell cultures were maintained in accordance with the ARBC maintenance protocol . The A. thaliana seed culture was produced by inoculating 7 mL of cell culture into 43 mL fresh growth media, followed by 96 h cultivation at 25 °C on a rotary shaker in the dark. After 96 h, 3 mL of the seed culture was inoculated into 27 mL fresh growth media to create an approximate initial cell density of 3.3 g . Flasks were spiked with 30 µL of a stock solution of diazepam and 10 µL of a 14C-diazepam stock solution to yield an initial concentration of 1 µg mL-1 and a specific radioactivity of 7.4 × 103 dpm mL-1 with an initial methanol content of 0.13% . Simultaneously, autoclaving cell suspension flasks prepared control treatments before chemical spiking , flasks containing diazepam without cells , and flasks containing living cells without diazepam . Control treatments were used to determine adsorption, abiotic degradation, and potential toxicity to cells. Flasks were incubated for 120 h in triplicate and sacrificed at 0, 6, 12, 24, 48 and 96 h for sampling and analysis. At each sampling time point, samples were collected and centrifuged at 13,000 g for 15 min in 50 mL polypropylene tubes. The supernatant was collected and stored at -20 °C until further analysis. Cells were immediately stored at -80 °C and then freezedried for 72 h. After drying, each sample was spiked with 50 µL of 10 mg L-1 diazepam-d5 as a surrogate for extraction-recovery calibration and extracted using a method from Wu et al. , with minor modifications. Briefly, cells were sonicated for 20 min with 20 mL methyl tert-butyl ether and then 20 mL of acetonitrile and centrifuged at 13,000 g for 15 min. The supernatants were combined and concentrated to near dryness under nitrogen at 35 °C and then reconstituted in 1 mL of methanol. The cells were then extracted with 20 mL acidified deionized water and the supernatant was combined with the methanol extract for clean-up. Prior to clean-up, 100 µL of cell material extract and growth media were combined with 5 mL liquid scintillation cocktail I to measure the radioactivity in the extractable form on a Beckman LS500TD Liquid Scintillation Counter . Clean-up was carried out using solid phase extraction with 150 mg Waters Oasis© HLB cartridges that were preconditioned with 7 mL methanol and 14 mL deionized water. Samples were loaded onto cartridges and then eluted with 20 mL methanol under gravity. The eluate was dried under nitrogen and further recovered in 1.5 mL methanol:water . After re-suspension extracts were transferred to microcentrifuge tubes and centrifuged at 12,000 g in a tabletop d2012 Micro-Centrifuge . Samples were further filtered through a 0.22-µm polytetrafluoroethylene membrane into 2 mL glass vials and stored at -20 °C before analysis. Extraction of growth media was done after adjusting the solution to pH 3 using HCl, and followed by SPE with Waters HLB cartridges, as described above. The extraction recoveries for the tissues and media were 88 ± 7% and 80 ± 14%, respectively.Hydroponic cultivations were carried out using cucumber and radish seedlings. Seeds were purchased from Lowes and germinated in a commercially labeled organic potting soil in a growth chamber . After the appearance of the first true leaf, uniform seedlings were selected, rinsed with distilled water, and individually placed in amber jars containing 900 mL hydroponic solution . After 3 d of adaption, plants were exposed to diazepam by spiking with 100 µL of the above stock solutions to reach a nominal concentration of 1 mg L-1 and an initial specific radioactivity of 2.5 × 103 dpm L-1 . The cultivation lasted for 7 d.

Plants also respond to P starvation by utilising alternative metabolic pathways

For P acquisition, the PHT gene family controlling P transport provides several candidate genes. In Arabidopsis, the PHTgenes that encode phosphate transporters responsible for transport of P anions are well characterised and are grouped into four families . PHT proteins other than PHT1 are involved in the uptake, distribution and remobilisation of P within the plant, however, PHT1 in the plasma membrane is the most important . Phosphate stress induces expression of these genes. However, the use of PHT transporters in plant breeding has been limited by P toxicity and other side effects of unbalanced P regulation associated with the over expression of some transporter genes. For example, OsPHT1;9 and OsPHT1;10 over expressing rice plants have reduced biomass under high phosphate compared to wild-type plants. Accessory proteins, encoded for by genes like PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR , are also important for proper functioning of P transporter genes. Homologs of PHT1 transporter genes, transcriptional factors including the SPX gene family, and genes involved in RSA determination such as PDR2 and LPR1/2 could be candidate genes for improving phosphate acquisition in watercress, but these genes have yet to be identified in aquatic crops.The transcriptional regulation of PUE is complex: there is some overlap with genes involved in both phosphate acquisition and phosphate utilisation, such as the global transcriptional regulation by PHR1 and PHL1. Here we target genes primarily involved in utilisation,square planter pots including those responsible for P transport within the plant, alternate metabolic pathways, and internal Pscavenging.

Using an Arabidopsis Affymetrix gene chip, changes in global gene expression have been analysed in response to P deprivation. The expression of 612 genes was induced and 254 genes suppressed including upregulation of phosphate transporters such as PHT1 genes and PHO1;H1. Genes involved in protein biosynthesis were down regulated during deficiency, likely representing P recycling strategies. PHT transporters also play a role in P utilisation through re-translocation of P within the plant. Five of the 13 maize PHT1 genes are induced in other tissues such as leaves, anthers, pollen and seeds, suggesting PHT1 involvement in diverse processes such as root to-shoot distribution. PHO1 is another central element responsible for P homeostasis and transport. Pho1 mutants exhibit P deficiency in the shoots due to lack of P loading into the xylem vessels. Phosphatases are important for remobilisation of fixed P. Eleven genes encoding different purple acid phosphatases were reported to be upregulated under P starvation in Arabidopsi. The Arabidopsis genome encodes 29 purple acid phosphatases, some of which are excreted into the soil, such as PAP12 and PAP26. Only phosphatase activity within the plant is relevant to watercress breeding: in a f lowing water system, any P made available around the roots by secreted phosphatases would rapidly wash away. As well as being a major secreted phosphatase, PAP26 is regarded as the predominant intracellular acid phosphatase in Arabidopsis and is upregulated two-fold under Pdeficiency. PAP26 functions in PUE by scavenging P from intracellular and extracellular P-ester pools, to increase P availability in the plant. Homologs of PAP26 should be investigated in watercress.

Genes involved in lipid metabolism and biosynthesis represent the largest group of core PSI genes in Arabidopsis, demonstrating their importance in PUE under P starvation. Three genes encode “plant-type” PEPC enzymes in Arabidopsis . PPC1 is expressed in roots and f lowers, PPC2 in all organs and PPC3 in only roots. PEPC activity is affected by phosphorylation by PPCK , thus PPCK1 and PPCK2 genes are additional important components for P-bypassing. Membrane phospholipids constitute approximately 20% of the total P in the leaves of P-sufficient plants. This represents a large pool of P that can be remobilised. 7% of the P-responsive genes found by Misson et al.were involved in lipid biosynthesis pathways in Arabidopsis. This includes the genes MGD2 and MGD3 whose expression changed 11-fold and 48- fold under P deficiency, respectively. These genes encode major enzymes for galactolipid biosynthesis and are involved in replacing phospholipids in cell membranes with non-phosphorus lipids such as galactolipid digalactosyldiacylglycerol. PECP1 is involved in the liberation of P from phospholipids and is upregulated under P deprivation, with up to 1785-fold increases in expression reported in roots. PSR2 encodes a phosphatase involved in galactolipid biosynthesis and whose expression increases 174-fold in P deprived seedlings. Both PECP1 and PSR2 have similar roles in the dephosphorylation of phosphocholine in the galactolipid synthesis pathway. However, despite their massive upregulation, it has been observed that inactivation of PECP1 and PSR2 does not alter plant growth or plant P content under P-deprivation so PCho is not likely a major source of P under limiting conditions.

PLDζ1 and PLDζ2 encode phospholipases D zeta 1 and 2 that hydrolyse major phospholipids such as phosphatidylcholine which yields phosphatidic acid and PA phosphatase and releases DAG and P. Phospholipids can also be replaced by sulfolipids. SQD2 is the primary gene in this pathway and encodes an enzyme that catalyses the final step in the sulfolipid biosynthesis. GDPD1 is involved in the formation of glycerol-3- phosphate from phospholipid products , that can be dephosphorylated to release P. Homologs of PHT1 genes responsible for P redistribution, within the plant, genes involved in P scavenging , genes implemented in metabolic pathways that bypass P use including galactolipid biosynthetic pathways and those involved in sulfolipid biosynthesis could be candidate genes for improving phosphate utilisation in watercress.Watercress root research is virtually completely absent in the literature, with no studies on root responses to phosphate availability. Nevertheless, the finite nature of rock phosphate and the fact that watercress cultivation methods have the potential to result in environmental damage , are clear drivers, as with soil-grown crops, to breed for watercress with improved PUE. Uncovering phenotypic traits and the molecular basis for PUE are important early steps in breeding for phosphate use efficiency. No commercial breeding programs exist for watercress worldwide, but germplasm collections are emerging. Development of new watercress varieties is also no doubt limited by the lack of genomic information for this crop. Payne screened a germplasm collection under indoor and outdoor conditions and identified significant variation in several traits including stem length, stem diameter and antioxidant concentrations. Voutsina used RNA-seq to analyse the first watercress transcriptome and identified differences in antioxidant capacity and glucosinolate biosynthesis across a germplasm collection of watercress, with 71% of the watercress transcripts annotated based on orthology to Arabidopsis. Jeon et al. independently used RNA-seq approaches to assemble the watercress transcriptome de novo. They identified 33 candidate genes related to glucosinolate biosynthetic pathways using Arabidopsis glucosinolate genes to search for homologous sequences in the watercress transcriptome. Additionally, a watercress mapping population comprising 259 F2 individuals was established by Voutsina using parents with contrasting nutrient and growth phenotypes. Genotype-by-sequencing of this mapping population enabled the construction of first genetic linkage map for watercress and identified 17 QTL for morphological traits of interest, growing blackberries in containers antioxidant capacity and cytotoxicity against human cancer cells. However, no root traits were assessed in this work. Screening this mapping population and wider germplasm for root traits may reveal individuals with extreme PUE phenotypes, which could allow the development of markers and QTL associated with this complex trait. RNA-seq approaches could ultimately lead to the identification of candidate genes. Together these findings will assist in developing commercial cultivars with a reduced need for phosphate, and a reduced negative environmental impact.

To assess whether homologs of the candidate PUE genes identified in this study exist in watercress, available watercress transcriptome data was mined for 13 key PUE genes selected from Table 2. This included genes whose expression was induced more than tenfold in at least 2 independent studies , plus PHR1, the global regulator of P starvation responses. Annotated transcripts were obtained from transcriptomic studies of watercress by Voutsina et al., 2016; Jeon et al., 2017; Müller et al., 2021 and matches for these candidate genes were assessed by searching for genes using AGI . Across all studies, strong matches were found for PHT1;4, SPX1, PHR1, MDG3, PEPC1, PLDζ1/2, PSR2 and SQD2, with all corresponding e-values ranging from 0 to 3.00E-32. Additionally, Müller et al. identified homologous transcripts for MDG2. Voutsina et al. had transcripts corresponding to PHT1;3, and MDG2, and Jeon et al. had hits for PHT1;2 and PHT1;3. No matching transcripts were found for PHT1;1 in any of the three studies. Where FDR values were < 0.05, changes to expression patterns were noted. Interestingly, Müller et al. also observed varying levels of upregulation of PHT1;4 and PHR1 following submergence, which may suggest a link between phosphate starvation and submergence responses.Being a high-energy phosphate compound, PPi can serve as a phosphate donor and energy source, but it can, at high levels, become inhibitory to cellular metabolism. To maintain an optimal PPi level in the cytoplasm, timely degradation of excessive PPi is carried out by two major types of enzymes: soluble inorganic pyrophosphatases and proton-translocating membrane-bound pyrophosphatases. The importance of maintaining an optimal cellular PPi level has been demonstrated in several different organisms. Genetic mutations that lead to the absence of sPPase activity affects cell proliferation in Escherichia coli. In yeast, inorganic pyrophosphatase is indispensable for cell viability because loss of its function results in cell cycle arrest and autophagic cell death associated with impaired NAD+ depletion. In Arabidopsis, a tonoplast-localized proton-pumping pyrophosphatase AVP1 was shown to be the key enzyme for cytosolic PPi metabolism in different cell types of various plants. This enzyme activity has been correlated with the important function that AVP1 plays in many physiological processes. Arabidopsis fugu5 mutants lacking functional AVP1 show elevated levels of cytosolic PPi and display heterotrophic growth defects resulting from the inhibition of gluconeogenesis. This important role in controlling PPi level in plant cells is reinforced by a recent study showing that higher-order mutants defective in both tonoplast and cytosolic pyrophosphatases display much severe phenotypes including plant dwarfism, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Moreover, the tonoplast-localized H+ -PPase AVP1 appears to be a predominant contributor to the regulation of cellular PPi levels because the quadruple knockout mutant lacking cytosolic PPase isoforms ppa1 ppa2 ppa4 ppa5 showed no obvious phenotypes. Interestingly, in companion cells of the phloem, AVP1 was also shown to be localized to the plasma membrane and function as a PPi synthase that contribute to phloem loading, photosynthate partitioning, and energy metabolism. On the other hand, AVP1 is also believed to contribute to the establishment of electrochemical potential across the vacuole membrane, which is important for subsequent vacuolar secondary transport and ion sequestration. Constitutive over expression of AVP1 improves the growth and yield of diverse transgenic plants under various abiotic stress conditions—including drought, salinity, as well as phosphorus and nitrogen deficiency—although the mechanism remains to be fully understood. Taken together, AVP1 serves as a multi-functional protein involved a variety of physiological processes in plants, some of which await to be fully understood. Magnesium is an essential macro-nutrient for plant growth and development, functioning in numerous biological processes and cellular functions, including chlorophyll biosynthesis and carbon fixation. Either deficiency or excess of Mg in the soil could be detrimental to plant growth and therefore plants have evolved multiple adaptive mechanisms to maintain cellular Mg concentration within an optimal range. In higher plants, the most well-documented Mg2+ transporters belong to homologues of bacterial CorA super family and are also called “MRS2” based on their similarity to yeast Mitocondrial RNA splicing 2 protein. Several members of the MGT family mediate Mg2+ transport in bacteria or yeast as indicated by functional complementation as well as 63Ni tracer assay. In plants, they have been shown to play vital roles in Mg2+ uptake, translocation, and homeostasis associated with their different sub-cellular localizations and diverse tissue-specific expression patterns. For instance, MGT2 and MGT3 are tonoplast localized and possibly involved in Mg2+ partitioning into mesophyll vacuoles; MGT4, MGT5, and MGT9 are strongly expressed in mature anthers and play a crucial role in pollen development and male fertility. MGT6 and MGT7 are shown to be most directly involved in Mg homeostasis because knocking-down or knocking-out either of the genes leads to hypersensitivity to low Mg conditions.

Solutions Farm’s current co-manager is a graduate of the program

Considering the social factors that shape access to opportunities brings up notions of spatial justice and the idea that rights and opportunities are not evenly distributed. Spatial justice would require space to be reorganized and reconceptualized “to promote equal access to opportunities, foster participation in decision-making, and encourage different ways of being in space” . To paraphrase a popular expression, the growing field is not levelled. Geographers are particularly well-positioned to examine the spatial barriers that prevent people from accessing and growing food. As several observers have noted, distributive ‘food justice’ activities like community gardens and farmer’s markets often take a top-down approach in which decision-making power is relegated to a few leaders such as nonprofit directors and/or managers that may reinforce existing power inequities . Procedural justice, a more radical approach to justice, rejects top-down strategies, instead advocating for participatory, grassroots action. In place of fairness in outcomes, procedural justice promotes fairness in procedures such as participation in regulatory and organizational processes and decision-making . This form of justice is less common and primarily associated with food sovereignty . It is also informing progressive visions of the food movement and many food justice initiatives . Herman and Goodman urge us to cultivate a participatory understanding of food justice to “move beyond the local,blueberry container distributive issues in which it often becomes mired” and focus our efforts instead on building a movement that is itself inclusive and just.

Procedural and distributive forms of justice are not mutually exclusive – in many ways, they inform one another. For example, the distribution of land and capital undoubtedly influences the power people possess to participate in and make decisions around food provisioning. In fact, researchers argue that the most effective concepts of justice are actually ‘trivalent’ – they combine distribution and participation and include recognition of disenfranchised groups . This approach, they argue, is critical, pluralist, unified, and accounts for the particular and everyday experiences of injustice that vary with social context . Some, however, argue that this perspective of justice that is contingent upon time and space, fails to produce a universal notion of justice that might combat global forms of injustice . Indeed, Cadieux and Slocum warn, “If food justice means anything, it may stand for nothing—or, worse, serve to undermine the credibility and rigor of substantive food justice practices,” which, in turn, may make it vulnerable to co-optation . Indeed, researchers must examine not only how actors define food justice, but also how they do food justice . Yet, singular, standardized, and universalist ideals may reinforce insider/outsider mentalities that devalue particular justice practices that do not fit this view . In the face of the dynamic nature of justice, Goodman, Dupuis, and Goodman recommend a reflexive theory of justice that moves away from “the perfect and privileged politics of standard setting” .

This approach sees justice as “a process by which people pursue goals while acknowledging the imperfection in their actions” . This concept of justice is situated , admits conflicts, responds to changing circumstances, and recognizes the nuance of everyday. San Diego County is a growing, diverse metropolitan region of over 3 million people. Its 4,526 square miles are comprised of distinct landscapes including densely populated urban neighborhoods, sprawling suburbs, and open spaces. Urban agriculture thrives in this county with the help of a year-round growing climate and generally supportive regulatory environment. Traditional soil-based models have flourished in this atmosphere and over 90 urban farms and community gardens currently operate in the county. The city’s approval of the Urban Agriculture Incentive Zones Act – a 2014 California bill that provides tax incentives to private landowners who allow urban agriculture on their vacant parcels in urban areas –suggests the trend will continue. Soilless models, specifically hydroponic and aquaponic greenhouses, are also on the rise in the county, particularly in the North County area. The availability of natural resources, specifically water and affordable land, drive urban agriculture locations and practices in the county. Water is increasingly scarce and expensive in southern California and land prices continue to rise throughout the county.

Further, less than 1% of the county’s inhabitants are employed in agriculture. However, interest in urban agriculture is growing – a list of 40 urban growers seeking land for urban agriculture through AB551 in San Diego is available on a website created in support of the bill. The availability of environmental resources is complicated by disproportional access to the economic and social resources required to support these activities including funding, skills, and institutional capacity. Indeed, research indicates that urban agriculture is more often realized as a cultural amenity among white, educated and affluent consumers, who tend to have greater access to these resources . People of color, who disproportionately suffer from hunger, are less likely to have similar access to environmental, economic, and social resources. The region’s socio-spatial landscape supports the thesis of disproportional access to resources. The county itself is highly segregated and race and socio-economic status differences can be read in the landscape. For example, poverty is concentrated in certain areas, specifically in older urban neighborhoods around Downtown San Diego , the South Bay, Oceanside, Vista, and Escondido, as well as a few sparsely populated rural areas. Racial segregation is even more concentrated, following a similar pattern, with most non-white and non-white Latino residents living around downtown and in the South Bay. These spatial patterns are not accidental, but instead reflect racially-biased political and economic decisions, such as mortgage policies, zoning regulations, municipal funding, transportation planning, and real estate practices, as well as histories of suburbanization, and more recently, gentrification. In cities, neoliberal economic development shifts state responsibilities onto the private and nonprofit sector and creates competition among neighborhoods for resources while also supporting do-it-yourself approaches to urban issues such as urban agriculture . US Census data also unveils gross inequities in median incomes . For example, according to American Community Survey data for 2013-2017, the median household income in the county for that period was $70,588. The top 20 percent of households earned 50 percent of the total income; the bottom 20 percent earned just 3 percent. Income varied considerably among racial groups.

The non-Latino white median household income reached $86,790; however, it was much lower for Latinos and African Americans . This disparity explains the high rate of poverty among these groups – 16% for Latinos and 21% for African Americans compared to 8.3% for non-Latino whites. The county’s social landscape suggests disparate access to the resources needed to support urban agriculture activities. Land is expensive and scare and capital is unevenly distributed since it likely follows income distribution. Nonetheless, soil-based and soilless urban agriculture are popping up throughout the county,growing blueberries in containers and often with a mission to promote social justice. However, what these organizations mean by ‘justice’ and how they practice it, as well as the shared and unique contexts that support and constrain it, remain unknown. Tounpack the complexities of justice in the county, I examine three urban growing spaces, two soil-based and one soilless, that prioritize a social mission, but whose contexts produce differential access to the resources to support it. I analyze these growing spaces using a qualitative research design described in the next section. The neighborhood settings of these urban agriculture enterprises are quite different . Encinitas and Southeastern San Diego represent two ends of the spectrum; their socio-economic landscapes tell two quite different stories. In Encinitas, the median household income is high and poverty and unemployment rates are low. Residents tend to be non-Latino white and many have college degrees. Housing is predominately owner-occupied and the median property values are high . In Southeastern San Diego, the population is denser and more diverse – mainly Latino, but with relatively sizable non-Latino Black and Asian inhabitants. In fact, in the 1950s and 60s, the black population was much larger because Southeastern San Diego was one of the few places in San Diego where African Americans were not restricted by legal covenants to own or rent properties . Today, despite demographic changes, this remains an important element of the neighborhood’s identity. The median household income is significantly lower than in Encinitas. Unsurprisingly, poverty and unemployment rates are higher here, as well. There are fewer people with college degrees and the home-ownership rate and median property value are considerably lower than in the rest of the county. Vista lies somewhere in the middle on economic characteristics such as median income, poverty and unemployment. The city is less dominated by a single race or ethnicity, with almost the same proportion of non-Latino White and Latino residents. However, it has one of the highest percentages of homelessness in the county , which drives local nonprofit organizing.

These neighborhood characteristics contribute to the way the local organizations define and do justice and provide the socio-spatial setting that creates barriers and opportunities for their work.Coastal Roots Farm was established in 2014 as a Jewish community farm to “provide dignified access to fresh food for those who need it most” . The 20-acre, organic farm names food justice, as well as sustainable agriculture and ancient Jewish wisdom, as its founding principles. “What we mean by food justice is that everyone deserves a right to this wonderful food,” says Sharon Goodson, the farm’s Director of Philanthropy in 2016. The farm uses soil-based methods to grow produce that it sells through its Community Supported Agriculture programs; however, the majority of the harvest is donated to local organizations aimed at hunger relief in Encinitas, as well as local Holocaust survivors and their families. The farm hosts monthly ‘pop-up’ farm stands in Vista and at Camp Pendleton – areas with high poverty rates. It also hosts educational events and Jewish farming festivals for the local community. Community members can participate in volunteering activities; however, there are no personal plots for resident use. The farm’s staff tend to have considerable experience in farming and are recruited nationally– for example, the current Farm Manager has over 17 years of experience in farming throughout the Pacific Northwest – as well as nonprofit management. Ellie Honan, the Farm Production Assistant Manager, was drawn to the farm from Minnesota by an apprenticeship. Leadership and management positions are also staffed by applicants with previous nonprofit experience. The farm’s funding comes from Leichtag Foundation, a private foundation dedicated to Jewish life and social entrepreneurship in San Diego County and Jerusalem . Solutions Farms opened in 2012 to serve as a social enterprise for the Solutions for Change family homelessness program. It is a 2-acre, organic, soilless farm that uses commercial aquaponic production to provide workforce training to previously homeless adults who have overcome addiction and may have few of the appropriate skills to build a career to support their families. The farm operates as an LLC and participants are formal employees that receive income, in addition to transformational housing, to support career development and self-sufficiency. Participants on average triple their annual income after their first year in the program to upwards of $20,000, which in turn decreases their dependence on cash aid and food stamps . The program pays specific attention to financial literacy – for instance, program graduates are required to have $3,000 in a personal savings account when they graduate. The structure of the program is top-down, although there are some opportunities for graduates to join the organization as staff if positions are available. The farm primarily grows lettuces that are sold to local restaurants, juice bars, and farmers’ markets in North County. Previously, the farm had a contract with Vista Unified School District; however, the bid was not renewed. The enterprise does not mention ‘justice’ in its promotional material, but the website notes that the farm’s workforce development program has a “worthwhile social purpose” . Funding for the Solutions for Change program comes from governmental support such as affordable housing funds, private foundation grants, and donations. Mt. Hope Community Garden, overseen by the local nonprofit Project New Village, broke ground in 2011. The garden is 1/3 of an acre and has 40 garden beds specifically for local residents to grow food and flowers for personal use and/or sale.

The average grain Zn concentration of potting mix-grown control plants was similar to that of field-grown wheat

In Fe- and Zn-withholding treatments, root mineral content and concentration decreased substantially in both lines. Roots of all lines and treatments continued to grow during grain fill, and were larger at grain maturity than at anthesis, making calculation of remobilization by subtraction impractical. Despite the lack of significant net Fe and Zn remobilization from the total vegetative tissues of plants grown incomplete nutrient solution, the control line exhibited substantial Fe and Zn net remobilization from some vegetative organs . The control line exhibited a 54% and 48.3% decrease in flag leaf Fe and Zn content, respectively, between anthesis and maturity . For both minerals, the RNAi line remobilized significantly less Fe and Zn from the flag leaves. This indicates that the NAM genes influence net remobilization from flag leaves even in the complete nutrient solution. The other minerals tended to accumulate or remain constant in flag leaves between anthesis and 35 DAA, and the effect of the NAM genes was not readily apparent . An exception was Cu, which was also remobilized in both RNAi and control plants . These data suggest that under these experimental conditions,snap clamps ABS pvc pipe clip the effect of NAM genes on mineral remobilization primarily affects remobilizable metal micro-nutrients, i.e. Fe, Zn, and Cu.Over the time-course of the potting mix experiment, grain Ca and Mg concentrations were similar between lines at most time points, while K was higher in the RNAi line .

Copper, Fe, Mn, P, S, and Zn were at lower concentrations in the RNAi line at most time points. In the hydroponics experiments, the RNAi line produced more, although slightly smaller grains per head than the control line, resulting in significantly higher total grain mass per tiller . Across treatments, the total grain mineral content per tiller was higher in the RNAi line than in the control line for Ca and K, and similar for Mg, Mn, P, and S . This similar total grain mineral accumulation spread across a higher total grain weight in the RNAi line resulted in lower concentrations of Mg, Mn, P, and S in this line, relative to the control . Copper, Fe and Zn concentrations were also lower, but by a greater percentage than the other minerals. In terms of total grain accumulation , Fe was lower in the RNAi line relative to the control in complete solution at 35 DAA and 42 DAA , and in the Fe withheld experiment . Grain Zn content in the RNAi line was also significantly lower than the control in complete solution at 35 DAA and 42 DAA , but not in the Zn deficiency treatment .Much of the N imported into wheat grain is derived from protein in vegetative tissues that is degraded to amino acids and recycled by translocation to the grain, i.e. remobilization . It is often assumed that certain minerals supplied to seeds also come from remobilized sources, but, unlike N, specific sources of stored or recycled minerals are unknown, and the few studies that have quantified the contributions of minerals remobilized from vegetative tissues to seeds have reached differing conclusions .

In this work, net remobilization is defined as the loss of stored mineral content over time from one organ, and subsequent accumulation of that mineral content into another organ. Because net change in mineral content over time is a function of influx and efflux of nutrients, net remobilization will be detected only when efflux exceeds influx. Thus, substantial quantities of a given mineral could pass through an organ without a detectable change in content, resulting in no detectable net remobilization. Likewise, minerals could be remobilized from one subcellular compartment while accumulating in another compartment or in the apoplastic spaces without a change in total content or net remobilization. Since all shoot tissues have been collected and analysed in these experiments, it is possible to assess mineral partitioning to various tissues over time. If the grain mineral pool were to increase while the shoot mineral pool remained constant, then the quantity of mineral translocated to the grain must have passed through the shoot tissues, and would be equal to the quantity entering the shoot during that time period. When comparing lines, if translocation of mineral to the grain is inhibited, the decreased flux will be detected as a relative increase in vegetative mineral content and less of an increase in grain mineral content. Although this discussion will focus on Fe, Zn, and N, other minerals were quantified in order to determine whether the effects of the NAM genes were general in nature, or if certain minerals were disproportionately affected. Quantifying tissue DW and multiple minerals also demonstrated that remobilization did not occur for all minerals, and that observed changes in organ content were not secondary effects of changes in growth or organ mass.

The NAM genes are members of the NAC transcription factor family and were previously shown to affect grain Fe, Zn, and N content in a dosage-dependent manner. Construction of the RNAi line used here and the resultant alterations in NAM transcript levels have been described previously . As transcription factors, NAM proteins are predicted to regulate genes that encode for proteins that carry out physiological processes for nutrient remobilization and/or translocation to grain. The RNAi and control lines used in our work only differ in their relative NAM gene expression, and are otherwise isogenic. Therefore, differences in Fe, Zn, and N dynamics between the control and transgenic lines can be assigned to direct or downstream effects of these genes. Our results indicate that the extent of net remobilization is dependent on availability of mineral inputs and thus will probably be highly dependent on environmental conditions in field-grown plants. In complete hydroponic nutrient solution growth conditions, no significant net remobilization of Fe or Zn was observed in either line. Despite this, grain Fe and Zn contents and concentrations were substantially higher than those from plants grown in potting mix, where remobilization of both Fe and Zn was observed in control lines. When hydroponic plants were deprived of Fe or Zn inputs post-anthesis, net remobilization occurred in both the control and RNAi lines, from shoot tissues and also from roots . Both lines remobilized more than enough of these minerals to account for the entire grain content, although the Fe and Zn quantities accumulated in the grain were substantially lower than in plants on the complete solution treatment. These results suggest that while remobilization and partitioning of Fe and Zn to grain is impaired in the NAM knockdown line, this is not due to a complete inhibition of remobilization,greenhouse snap clamps as the RNAi line is capable of remobilizing minerals under nutrient-limiting conditions. They also suggest that when Fe and Zn are readily available to the roots, and are adequately absorbed into the plant, this source supersedes the need for remobilization from the leaves. In the absence of sufficient Fe and Zn from the soil, the plant can obtain these minerals from the storage forms present in both shoot and root vegetative tissues. These results are consistent with those obtained when P was withheld from wheat plants during grain development .In potting mix growth conditions, net remobilization of Fe and Zn from the control line was observed, but diminished or no net remobilization in the RNAi line. Total accumulation of plant Fe and Zn was similar, but partitioning of these minerals to grain was substantially lower in the RNAi line. In this same experiment, vegetative N content decreased in the control line over time, indicating net remobilization, while there was an increase in N in the RNAi line. Between 35 DAA and 56 DAA, net remobilized N could account for 46% of the increase in grain N in the control line, but accounted for none of the N in the grain of the RNAi line. These results, in combination with the 65Zn experiment that demonstrated decreased short-term translocation of Zn to grain, indicate that the translocation of certain minerals and N to grain is impaired in the NAM knockdown line.

A combination of decreased efflux and sustained influx of minerals into vegetative tissues could account for the lower net remobilization exhibited in the RNAi lines, and could also account for the lower percentage of total Fe, Zn, and N partitioned to grain. Target genes of the wheat NAM transcription factors have not been identified, and the molecular mechanism by which the NAM proteins affect translocation to grain is currently unknown. A micro-array study of senescence in Arabidopsis leaves revealed a large number of up-regulated transporter proteins, including OPTs, YSLs, and ZIPs . It is possible that NAM proteins regulate similar transporter genes in wheat and that these genes are needed for effective Fe and Zn remobilization. Other possible explanations include indirect effects on phloem loading for efflux of Fe, Zn and N from leaves; or an effect on the rate or timing of disassembly of the internal sources of these elements. The latter hypothesis is supported by the observation that lines with a functional copy of the NAM-B1 gene had higher soluble protein and amino acids concentration in the flag leaves than near isogenic lines with a non-functional NAM-B1 gene . The reduced expression of NAM genes and the accompanying delay of normal vegetative development, i.e. senescence, may result in a disruption of the normal source and sink tissue relationship. Delayed senescence and the accompanying degradation of proteins may result in a situation where the substrates for transporters are decreased or not present, or are only present later in the grain-filling period and thus are less efficiently translocated out of source tissues. Hundreds to thousands of proteins are estimated to interact with Zn ions as structural or catalytic components or as substrates , thus substantial quantities of Zn could be released during protein degradation. Similarly, Fe from the degradation of chloroplast proteins could be released during leaf senescence. Delayed degradation of chloroplasts containing these proteins, as suggested by the delay in leaf yellowing in the RNAi line, may explain why remobilization of Fe was inhibited proportionally more than the remobilization of Zn. Since the grain of the RNAi plants grew normally , the movement of water and photo assimilates did not seem to be impaired. This suggests an inhibition of translocation processes more specific to Fe, Zn, and N rather than a general inhibition of phloem transport.However, grain of control line wheat grown in complete hydroponic culture had a Zn concentration approximately five times higher , which parallels the improvements in Zn grain concentrations made via Zn fertilization . The grain Fe concentration of the hydroponic control line was approximately twice that of potting mix-grown plants . In the RNAi line also, Fe and Zn concentrations in grain from plants grown on complete hydroponic solution were significantly higher than in grain from potting mix-grown plants . These results suggest that wheat grain is already capable of accumulating several-fold higher Fe and Zn concentrations than are usually obtained in field situations. Because the RNAi line had lower partitioning of Fe and Zn to grain under both high and low availability, grain concentrations of these nutrients can possibly be increased by improvements in the efficiency of translocation. Indeed, over expression of an Arabidopsis Zn transporter in barley resulted in increased seed Zn concentration . However, constitutive over expression of a Zn transporter in rice resulted in the aberrant distribution of Zn within the plant . Over expression of transporters may need to be targeted spatially and temporally to result in the desired increases of nutrients in the target tissue. The transgenic line also showed reduced translocation of N. It is estimated that grain protein in the control line was 19.7%, while protein in the RNAi line was one-third lower, at 13.0%. These values are higher than normally observed in field situations, possibly as a result of the continuous supply of N to the plants. While decreased expression of the NAM genes negatively affects the accumulation of Fe, Zn and protein in the grains, increasing the transcript levels of the NAM genes above the levels normally found in current commercial varieties can result in increased protein.