November 2023 – Page 2 – Nature Ecrogrow

Even cultivated vegetables contain less ω3 fatty acids than do plants in the wild

Sweet potato leaves with N contents at CNC showed the same maximum photosynthesis rate as was observed when their N content was significantly below their CNC, and the leaf biomass was still increasing . For rhizomatous plants, the rhizomes have always been considered a sink for excess carbohydrates. The results of our P. australis study to-date suggests that the roots, as well as the rhizomes are tissues in which ‘excess’ carbohydrates can be incorporated. Experiments on storage root crops, strongly suggest that potassium, which causes phloem vessels to be wider, increases the storage root biomass . Wider phloem vessels would allow for more effective transport of photosynthates away from the leaves where they are produced, where they cannot be incorporated anymore. Thus avoiding a build-up of these photosynthesis products in their production vessel. A study by Arp showed that photosynthetic acclimation originally attributed to the high CO2 treatment, occurs if the plants are completely root and rhizome bound. The time until this drop in photosynthesis rate occurred was directly related to the diameter of the pots in which the experimental plants were grown . This photosynthesis rate reduction was not observed in field grown plants without limitations on root and rhizome growth . If the plants physically do not have more room to grow roots and rhizomes, no new roots or rhizomes can be available to receive the translocated photosynthates,plant raspberry in container the photosynthesis products will indeed accumulate in the production vessel , and production will be lowered to a minimum maintenance level.

With a N/C ratio of 0.00869 + 0.00037 rhizome CNC is 4.37x smaller than that of the leaves , the rhizomes can store 4.37 times as much C per molecule or gram of N in their tissues than the leaves can. At this time, the root N/C ratios have not been analyzed yet. Based on the growth of the roots of the no-nitrogen plants, especially when compared to that of the nitrogen supplied plants, we expect the N/C ratio of the roots to be low as well. The picture, based on our results to-date, of growth allocation in P. australis and the role of leaf N content suggest that rhizome and root growth continues after leaf N content has significantly decreased and the amount of senesced leaf tissue is almost as much as the amount of green leaves. Since the carbohydrates that ‘fueled’ this biomass increase were produced by photosynthesis and translocated from the leaves, the active ingredient of systemic herbicides applied at this time would still be transported to these roots and rhizomes, that are the target of the herbicide treatment.Essential fatty acids, both ω6 and ω3 , have been part of the diet since the beginning of human life. It appears that human beings evolved consuming a diet that was much lower in saturated fatty acids than is today’s diet . However, over the past 150 years the balance between ω6 and ω3 in our diet has been upset. The current Western diet is very high in ω6 fatty acids . Intake of ω3 fatty acids is much lower today because of the decrease in fish consumption and the industrial production of animal feeds rich in grains containing ω6 fatty acids, leading to production of meat rich in ω6 and poor in ω3 fatty acids . The same is true for cultured fish and eggs . Thus, modern agriculture, with its emphasis on production, has decreased the ω3 fatty acid content in many foods. Alternative sources of PUFA are therefore desirable, and the concept of obtaining them from higher plants in commercial and sustainable quantities is particularly attractive.

Purslane is the richest source of ω3 PUFAs of any terrestrial green leafy vegetable yet examined , with an extremely good ratio of ω6 to ω3 fatty acids as well as antioxidants, such as α-tocopherol, ascorbic acid, β-carotene and gluthathione, minerals, vitamins and proteins . Purslane is a heat- and drought-tolerant plant, and is an important vegetable crop in southern Europe and Asia. It is eaten fresh, cooked or dried and interest in cultivating it as a food crop has increased all over the world in recent years since its identification as a rich source of ω3 PUFAs and antioxidants . Moreover, purslane is promising for providing both, novel biologically active substances and essential compounds for human nutrition. The increased interest in the potential health benefits associated with the consumption of long-chain ω-3 fatty acids has led to the sale of supplements and fortified foods containing these fatty acids. In these contexts the identification of functional foods, like purslane, that could be suitable for human consumption it is very important. Boron is one of the essential elements for plant growth and development. Deficiency of B has been identified as a serious agricultural issue in more than 100 crops in 80 countries . Limitation of B impairs growth of young tissues and seed set, which results in depressed quality and quantity of agricultural products. In rice, B content is up to 10-time lower than those of dicot plants . And thus rice young seedling is relatively resistant to B limited condition compared to dicot plants such as Arabidopsis . However, the effect of B limitation until the reproductive phase is little known in rice. In this study, we evaluate the growth and yield of rice subjected to B deficient condition by hydroponic experiments. We previously reported that a boron efflux transporter OsBOR1 plays a crucial role in efficient root-to-shoot translocation in rice . Over-expression of AtBOR1 improved growth and seed fertility in Arabidopsis plants under B deficient condition .

But this strategy, up-regulation of native B transporter to achieve the tolerance to B deficiency, has not been applied for the crop so far. We herein generated several independent lines of transgenic rice plants over expressing rice BOR1 and characterised the phenotypes of these transgenic rice plants under B deficient condition. Nitrogen, an important and abundant element, is required for life on earth. As a component of DNA, proteins, and other living building blocks, it is also a limiting nutrient for primary production in ecosystems. The majority of the Earth’s atmosphere is comprised of nitrogen, as N2, however this inert gaseous form is not readily utilized by biological processes and must be transformed into usable, or reactive, nitrogen compounds. Humans contribute to nitrogen emissions and consequent deposition through fossil fuel burning, industrial activities and through agricultural practices . Natural sources of reactive atmospheric nitrogen include biological emissions from microorganisms and lighting fixation Fossil fuel burning and the application of fertilizers for modern agriculture have greatly increased the availability of reactive,plastic seedling pots gaseous nitrogen in the atmosphere. Fossil fuel burning, resulting from industrial and transportation sector pollution emissions, occurs when nitrous oxides are emitted from combustion engines. Additionally, the use of fossil fuels in industrial practices such as coal energy production and the creation of synthetic fertilizers contribute reactive nitrogen species to the atmosphere, mostly in the forms of N2O and NH3, respectively . However, the greatest contributor of anthropogenically released nitrogen emissions is from agricultural practices . The addition of commercial fertilizers to farmed soils releases nitrogen emissions to the atmosphere. This pathway includes microbial metabolism of the fertilized nitrogen through nitrification and resulting denitrification, resulting in mainly N2O reactive gas emissions and in the volatilization of ammonia from decaying plant and animal wastes and manures Biological nitrogen fixation is accomplished by bacteria and archaea microorganisms, independently, or symbiotically, with plants. This process converts N2 to ammonia and into nitrogen containing proteins. These usable forms of nitrogen are readily metabolized forms for plant growth and subsequent trophic cycling. Decay of plant and animal matter also results in the production of ammonia which is further transformed to nitrate by nitrifying bacteria, and then to NO, HONO, and N2O gaseous emissions during intermediate denitrification processes . These emissions account for gaseous release of nitrogen that then can be taken up by plant stomata, deposited onto plant surfaces via dry deposition, dissolved in rain and consequently rained out as part of storm events . Additionally, storm events and cloud activity can further add to atmospheric contributions of reactive nitrogen by lightning. This occurs through high energy splitting of N2 into elemental N and subsequent combination with oxygen resulting in gaseous nitrogen oxides, such as NO3 . Excess nitrogen, in the form of nitrogen deposition, can have profoundly negative effects on ecosystems. Nitrogen fertilization is detrimental in that this provides large amounts of a macro nutrient that is otherwise limited in terrestrial ecosystems. The addition of this limited nutrient can cause critical load responses from microbes, algae, and plants and cause nitrogen saturation in ecosystems . Critical loads, or the highest level of pollution, which will not harm sensitive elements of ecosystems are identified by ecosystem responses to excess pollution.

Exceeding critical loads in ecosystems can cause vegetation-type conversions, invasive species proliferation, pollution runoff in watersheds and subsequent eutrophication in aquatic systems, among other detrimental effects . In Southern California, California coastal sage scrub plant assemblages have declined due to urbanization, pollution, and the invasion of exotic species . Riversidian California coastal sage scrub includes species such as California Buckwheat , California sagebrush , brittle bush , and other assorted sage species . Endangered species such as the California gnatcatcher, Stephen’s kangaroo rat, and quino checker spot butterfly are dependent on dense, highly diverse coastal sage scrub stands . One factor in the decline of coastal sage scrub is the significant increase of nitrogen emissions due to anthropogenic activities. Though NOx emissions have decreased in Southern California since 2005, nitrogen pollution emissions continue to artificially fertilize CSS from fossil fuel combustion and industrial processes . As nitrogen enters CSS habitat through dry deposition, it artificially fertilizes the otherwise nitrogen limited ecosystem . This can cause CSS decline through stimulation of invasive, annual Mediterranean plants which readily consume the excess available nitrogen to out-compete slower growing native plants. The result is fragmented CSS habitats and significant losses of CSS diversity Due to the ecological importance of coastal sage scrub and similar semi-arid plant communities, critical loads have been calculated for coastal sage scrub and other ecosystems of the western United States . However, these loads are based upon imperfect measurements of nitrogen exposure. New approaches, such as the Integrated Total Nitrogen Input Method , are needed to better assess the risks to these ecosystems including the role excess nitrogen deposition plays in continued and sustained invasion from Mediterranean exotics. The principal underlying the ITNI method is relatively simple. A closed plant liquid-sand system is created in a greenhouse and the system is isotopically enriched with 15N-labelled nitrogen. Next the PLS system is placed into the ambient environment where it isotopically equilibrates with atmospheric N. To determine the total amount of nitrogen input from wet, dry and gaseous deposition, the ITNI method utilizes the concept of isotope dilution . Since the 15N content of atmospheric N is significantly less than the 15N content of the PLS system, the concentration of 15N gradually declines in the plants, sand and liquid. At the time of harvest, the sand, plant parts, and nutrient liquid are all sampled for nitrogen quantity and isotope abundances; these measurements are used to determine how much the 15N tracer had been diluted and how much nitrogen has been gained which are used to calculate the amount of incoming nitrogen deposition from the natural field conditions. Previously, multiple methods were required to quantify all pathways of N deposition to a system , but the ITNI method can theoretically resolve this problem with isotope ratio measurements. Moreover, the ITNI method also accounts for nitrogen directly taken up through leaf stomata, an important process that is neglected by traditional deposition collectors . The PLS system contains a plant growing in a nitrogen-free soil composed of silica sand and watered by a liquid reservoir containing plant nutrients and 15N-labelled NO3 – or NH4 + . In most studies, a hydroponic system is used to transport the nutrient liquid from the vessel to the plants, and allow drainage back into the liquid reservoir. Once grown and labeled with 15N, the entire PLS system can be transported to the field and exposed for periods of weeks to months. At the end of field exposure, the entire plant and all of the sand and liquid system parts are harvested for isotope ratio and elemental analysis.

HPLC grade acetonitrile and methanol were used for extraction along with ultra pure water

Since its introduction in 1961 sulfamethoxazole has been widely prescribed due to its potency against both gram-positive and gram-negative bacteria . Currently, sulfamethoxazole has been detected from ng L-1 to µg L-1 in surface and effluent waters and µg kg-1 to mg kg-1 in soils and manure .Recent long-term studies of waste-water application under realistic field conditions have highlighted the potential for sulfamethoxazole to be taken up and translocated in crop plants, including to the fruit . The structures of sulfamethoxazole metabolites, including conjugates from Phase II metabolism, were identified using high-performance liquid chromatography coupled with time-of-flight high-resolution mass spectrometry and further quantified using ultra-high performance liquid chromatography in tandem with a triple quadrupole mass spectrometry . Furthermore, Phase III terminal products in the form of bound residues were quantified using 14C labeling. Arabidopsis thaliana cells were selected as the experimental organism due to their extensive use in the literature, commercial availability, and their membership in the commonly consumed Brassica family . Further, Arabidopsis thaliana plants are found worldwide under several common names and are consumed by a wide variety of animals as well as humans . Cucumber was selected in the hydroponic experiment due to the fact that it is often consumed raw, rapid growth,planting blueberries in containers and amiability to soilless culture .Non-labeled sulfamethoxazole was purchased from MP Biomedicals . Sulfamethoxazole-d4 was purchased from C/D/N Isotopes and 14C-labeled sulfamethoxazole was obtained from American Radiolabeled Chemicals .

Stock solutions of 14C-sulfamethoxazole and non-labeled sulfamethoxazole were prepared in methanol to reach a specific radioactivity of 1.2 × 103 dpm µL-1 and a chemical concentration of 1.0 mg mL-1 , respectively. Mobile phases were prepared using Optima™ LC/MS grade methanol and deionized water. Standards were prepared in HPLC grade methanol and stored in the dark at -20 °C. All solvents used in this study were purchased from Fisher .PSB-D A. thaliana cell line was purchased from the Arabidopsis Biological Resource Center at the Ohio State University . The cells were maintained in liquid suspension culture at 25 °C and rotated at 130 rpm in the dark according to the ARBC protocol . To explore metabolism of sulfamethoxazole in A. thaliana cells, 7 mL of cell culture was inoculated in 43 mL fresh culture and cultivated for 96 h at 25 °C and 130 rpm in the dark to produce the seed culture. A 30 µL aliquot of the non-labeled stock solution and 10 µL aliquot of 14C-sulfamethoxazole were spiked into 30 mL of A. thaliana cell culture, resulting in a nominal initial concentration of sulfamethoxazole of 1 µg mL-1 and a specific radioactivity of 1.2 × 103 dpm mL-1 . Simultaneously, control treatments were prepared by autoclaving cell suspensions before chemical spiking , flasks containing sulfamethoxazole without cells , and flasks containing live cells but no sulfamethoxazole . These control treatments were used to determine adsorption, abiotic degradation, and potential toxicity to cells. The incubation lasted for 96 h, and triplicate containers were sacrificed at 0, 3, 6, 12, 24, 48 and 96 h. At each sampling interval, the entire culture was transferred to a 50 mL polypropylene centrifuge tube and centrifuged at 10,000 rpm for 15 min. The supernatant was collected and stored at -20 °C until further analysis and the plant cells were placed at -80 °C before freeze-drying for 72 h. After drying, cells were fortified with 50 µL of 10 mg L-1 sulfamethoxazole-d4 as a recovery surrogate. Cells were extracted using a modified method previously established in Wu et al. . Briefly, cells are sonicated in a Fisher Scientific FS110H sonication bath for 20 min with 30 mL acidified DI water followed by centrifugation at 10,000 rpm for 15 min.

The supernatant was decanted into a new 50 mL centrifuge tubes. The cell matter was further extracted using 20 mL methyl tert-butyl ether , followed by 20 mL acetonitrile. The MTBE and acetonitrile supernatants were combined, dried under nitrogen at 35 °C, and re-constituted in 1.0 mL methanol. The extract was then combined with the above water extract. The combined sample extract was loaded onto a preconditioned 150-mg Oasis© HLB solid phase extraction cartridge and eluted with 20 mL methanol. The cleaned extract was dried under nitrogen and further recovered in 1.5 mL 50:50 MeOH:H2O . The growth media was acidified to pH 3 and similarly extracted and cleaned as described above. Extraction recovery for the sample preparation protocol of the cell extract was 46% ± 13 and for the growth media was 57% ± 10. Prior to instrument analysis, both cell and media extracts were transferred to micro-centrifuge tubes and centrifuged at 120,000 rpm in a bench-top SciLogex d2012 centrifuge and further filtered through a 0.22-µm polytetrafluoroethylene membrane into 2 mL glass vials. All final extracts in 2 mL glass vials were stored at -20 °C if not immediately analyzed. At each time interval, 100 µL of the cell material extract or concentrated growth media was added to 6 mL Ultima Gold™ liquid scintillation cocktail to measure the extractable 14C-radioactivity on a Beckman LS 5000TD Liquid Scintillation Counter . Additionally, the extracted cell matter was air dried, and a 10 mg aliquot was combusted on an OX-500 Biological Oxidizer . The evolved 14CO2 was captured in 15 mL Harvey Carbon-14 cocktail II and the 14C activity was measured to derive the fraction of bound residues from Phase III metabolism. Accurate mass data were obtained using an Agilent 1200 series HPLC coupled to an Agilent 6210 time-of-flight high-resolution mass spectrometer with ESI/APCI mixed ion source. The separation was achieved on a Thermo Scientific Hypersil Gold C18 column . Mobile phase A consisted of water containing 0.1% formic acid. Mobile phase B was composed of acetonitrile 0.1% formic acid. The solvent gradient ran from 5% to 100% B in 18 minutes at 0.3mL/min flow rate. Samples were analyzed at the High Resolution Mass Spectrometry Facility in the Chemistry Department at the University of California, Riverside.

Raw data files were obtained and converted to mzXMLfiles using ProteoWizard MSConvert and analyzed using MZmine 2 open software . Candidate metabolites were proposed based on the presence of unique peaks in the treatment that were absent in the controls . Identification uncertainty was determined using the Warwick mass accuracy calculator by comparing theoretical m/z to the observed m/z. To structurally identify sulfamethoxazole metabolites, the uncertainties in confirmation were evaluated against the metabolite identification criteria as outlined in Schymanski et al. . Using the criterion, Level 1 structures are those with direct confirmation against authentic standards. Level 2 structures are probable structures based on library spectrum data, literature data, and experimental information and Level 3 structures are tentative structures derived from strong MS/MS information for the proposed structures, but the position of substitutions could not be determined with certainty . Structural identification for sulfamethoxazole metabolites was determined from accurate mass information and fragmentation patterns received using the QTOF mass analyzer . The information was compared to mass spectra libraries and/or the literature on known human metabolites of sulfamethoxazole. Further, identified metabolites were scanned in the selective ion reaction,container growing raspberries multiple reaction monitoring and MS scan modes using Targetlynx™ software with comparison against authentic standards when available . Results were further compared with literature reporting common enzymes in the metabolism of other xenobiotics in plants to determine the most likely pathways and metabolite structures of sulfamethoxazole. The relative fractions of individual metabolites were determined on the Waters UPLC-TQD MS/MS in the selective ion reaction scan mode. Authentic standards of sulfamethoxazole and N4-acetylsulfamethoxazole were used as an example to verify the identity of proposed structures as well as to quantify these compounds. Retention times, accurate mass, and fragmentation patterns were used for validation. The metabolism of sulfamethoxazole in Arabidopsis thaliana cells was validated using a range of controls. No sulfamethoxazole was detected in the media or cell blanks, and there was no detectable disappearance of sulfamethoxazole in the cellfree media, suggesting the absence of contamination or abiotic transformation. Moreover, no significant difference was seen in the cell mass between the chemicalfree control and the treatments indicating that sulfamethoxazole did not affect the growth of A. thaliana under the experimental conditions. In the non-viable cell control, it was found that sulfamethoxazole was adsorbed to the cell matter, but the fraction did not contribute significantly to the dissipation of sulfamethoxazole from the media . In contrast, in the live cell treatments, sulfamethoxazole dissipated appreciably from the media, with the average concentration decreasing from 246 ± 1.74 ng mL-1 initially to 176 ± 5.23 ng mL-1 after 96 h of incubation .

Concentrations were therefore not adjusted for recovery or loss to adsorption to cell matter or the surfaces of the flask. As there was no significant difference between measured initial concentrations of the cell-free flasks, and viable and non-viable flasks, we assumed that adsorption to the container was insignificant. Concurrent to the dissipation in the medium, sulfamethoxazole was detected in the A. thaliana cells, and the level was the highest at 3 h sampling point decreasing thereafter. The presence of sulfamethoxazole in the live cells provided direct evidence of its uptake into A. thaliana cells. The level of sulfamethoxazole in the cell matter decreased after reaching the maxima at 3 h, Fitting a decrease in the sulfamethoxazole level in the cell to a first-order decay model yielded a half-life of 19.4 h . This was in comparison to a biological half-life of 10 h in humans for sulfamethoxazole . The decrease of sulfamethoxazole in the live A. thaliana cells suggested active metabolism. The use of 14C labeled sulfamethoxazole enabled determination of the fractions of sulfamethoxazole and its metabolites that were incorporated into the cell matter, which could not be characterized using traditional extraction and analytical methods. A rapid increase in the bound residue fraction was observed during the 96 h cell cultivation, while the increase in the extractable residue form in the cells was more gradual . During the incubation, the fraction in bound residues increased steadily to 53 ± 10% at 96 h, clearly suggesting that A. thaliana cells were capable of effectively metabolizing and then sequestering sulfamethoxazole and its metabolites in the cell system. In contrast, the fraction of the extractable residues was relatively low, ranging from 0% to 22 ± 1.6%. Extractable residues in plant metabolism are thought to contain Phase I and Phase II metabolites, including conjugates, while Phase III metabolism results in the incorporation or sequestration of metabolites into the cell wall . Therefore, formation of bound residues may be regarded as detoxification of a xenobiotic in plants. Several previous studies also demonstrated that plant cells and whole plants were capable of metabolizing PPCPs, transforming them into more polar intermediates and sequestering them in their cell walls or vacuoles .In A. thaliana cells, tentative metabolism pathways of sulfamethoxazole were derived by combining spectra data, knowledge of human metabolism of sulfamethoxazole and its degradation in water systems . In the proposed metabolism pathways, sulfamethoxazole underwent Phase I metabolism including oxidation and hydroxylation reactions, which was followed by Phase II metabolism through acetylation and rapid conjugation with glucuronic acid, amino acids, and glutathione . The end products of Phase II metabolism were then further sequestered likely through incorporation into cell walls and other cell components, resulting in the formation of non-extractable bound residues. 4-Nitroso-sulfamethoxazole is a known metabolite in human metabolism. The knowledge of its formation in mammalian livers, structure,and properties was used to detect its presence in the HPLC-TOF MS scans. Subsequent UPLC-MS/MS scans suggested the rapid formation of this intermediate. Individuals with low production of N-acetyltransferase were previously reported to exhibit a high production of hydroxylamine sulfamethoxazole and nitrososulfamethoxazole. In humans, these metabolites were found to be responsible for the adverse side effects associated with the consumption of sulfonamides, such as skin rashes or hives . N4-Acetyl-5-OH-sulfamethoxazole was also previously shown to form by cytochrome-P450 oxidation in mammalian livers . In higher plants, it was likely formed through oxidation reactions mediated by cytochrome-P450 enzymes, a superfamily of enzymes in both plants and mammals . N4-Acetylsulfamethoxazole , sulfamethoxazole-glucuronide and N4-sulfamethoxazole-glutathione conjugate metabolites were all previously found in human metabolism of sulfamethoxazole .

Synergism of root phenotypes should also be considered

The need for aquatic model crops is only exacerbated by increasing market value in indoor hydroponic cultivation systems.Root structural architecture defines the spatial configuration of the root system and variation in RSA can reflect efficient phosphate uptake by plants.Much of the current literature focuses on RSA traits in maize , common bean and Arabidopsis thaliana with RSA shown to be a highly plastic trait, changing in response to the availability of water, nutrients and hormone signalling. For example, ethylene is involved in the promotion of lateral root growth, root hair growth and inhibition of primary root growth under low P conditions. Reduced root growth angle : is one of the most important RSA traits for improving P acquisition in many soil-grown species. In maize and common bean, lower RGA is associated with increased P accumulation and improved growth in P deficient soil where P is concentrated in the topsoil. However, in aquatic systems when P is likely homogenously distributed, a lower RGA is unlikely to be advantageous. Root growth angle therefore is not considered important as a trait for enhance PUE in aquatic plants. Increased lateral root density:also enhances P acquisition by allowing plants to explore outside P-depleted zones. Using maize recombinant inbred lines with contrasting lateral rooting phenotypes, significant differences in phosphorus acquisition, biomass accumulation and relative growth rate were observed under low phosphorus availability. Increased investment in the production of lateral roots was shown to be cost-effective under low P. For aquatic crops,nursery pot enhanced lateral root density is an important trait for enhanced PUE since it increases root surface area for P uptake. Adventitious roots: from above ground structures can enhance topsoil foraging by up to 10% in stratified soil.

These require a lower metabolic investment than basal roots, however, in uniform soil they can limit P acquisition by hindering the growth of basal roots. In aquatic and f looding-tolerant plants, adventitious roots are important for P uptake within the water column. There is positive correlation between the size of the adventitious root system and P uptake in bittersweet plants under long-term submergence, and as mentioned, adventitious roots are responsible for a higher proportion of P acquisition in watercress than basal roots. Thus, in an aquatic crop such as watercress, increased adventitious root production is a key trait for enhanced PUE.Modelling of root architecture in maize and bean has shown RCA may increase the growth of plants up to 70% in maize and 14% in bean under low P availability largely due to remobilisation of P from dying cells. However, formation of aerenchyma is also associated with reduced root hydraulic conductivity in maize which may impede transport of water, but is unlikely to be relevant to hydroponically grown crops. Most aquatic plants, including watercress, form aerenchyma constitutively in roots and stems to aid internal gas exchange and maintain strength under water pressure. For aquatic crops such as watercress, formation of root aerenchyma is an important trait for selection for enhanced PUE. Root hair density and root hair length: root hair density increases up to 5 times in low P conditions. Using Arabidopsis mutants, Bates & Lynch found hairless plants had lower biomass and produced less seed than wild-type plants at low phosphorus availability. Root hairs increased root surface area by 2.5 to 3.5-fold in barley and wheat , respectively, and there was an almost perfect correlation between P uptake and root hair surface area. Root hair traits vary substantially between genotypes and the genetic control underlying their formation is well understood, thus making them an excellent target for plant breeding programs . Root hair length and density are likely to be important for PUE in aquatic crops as they significantly increase root surface area for P uptake.A modelling approach in Arabidopsis showed that the combined effects of root hair length, root hair density, tip to first root hair distance and number of trichoblast files on P acquisition was 3.7-fold greater than their additive effects. For aquatic species such as watercress, the root ideotype for determining optimal P acquisition remains unknown.

Although, absorption of P through the shoots is still debated, root uptake is generally regarded as the mode of P uptake in aquatic plants. Watercress beds have a fine gravel substrate which contains negligible amounts of P. The substrate within watercress beds is likely too shallow to allow for significant stratification of P, thus a shallower basal root angle would be unlikely to provide much adaptive benefit. In groundwater sources, P may be distributed more homogenously due to turbulent f lowing water, so RGA will not assist within the water column. Nevertheless, even with homogenous P distribution, plants with shallower root systems have been shown to encounter less inter-root competition with roots on the same plant so RGA could provide an adaptive value in this sense. Cumbus & Robinson studied P absorption by the adventitious and basal roots of watercress and found that the adventitious roots absorbed a higher proportion of P at low P concentrations, despite having a lower biomass compared to the basal root tissue. Thus, adventitious roots are also a key trait for analysing watercress PUE. Increased production of lateral roots, adventitious roots and root hairs all increase root surface area, and thus will increase P acquisition from the water and sediment and are important traits for PUE in watercress. In addition, the root cap can account for 20% of the phosphate absorbed by the roots of Arabidopsis. Therefore, increasing the number of roots increases the number of root tips and the number of these “hot spots” for phosphate acquisition.Plants are reliant on phosphate transporters to acquire P from the environment and transport P between tissues, and this includes for aquatic plants. The PHT1 family is the most widely studied group of P transporters and is primarily responsible for P uptake but also has a role for P transport between tissues. A broad range of expression patterns are associated with different PHT1 genes but generally, higher expression of PHT1 genes is associated with improved shoot biomass accumulation and P tolerance. Watercress with higher PHT1 expression may result in improved biomass accumulation in P deficient water, but this has yet to be tested. Additional traits that are important in other crops are organic acid exudation and phosphatase activity. Since these control release of P from organic forms in the soil, they are less relevant to watercress cultivation where P released from bound sources would be rapidly lost to the watercourse.

However, phosphatases that remobilise P from intracellular sources have been identified in Arabidopsis so similar phosphatases could enhance internal P utilisation in watercress.Alongside phosphate acquisition, PUE also refers to more efficient P utilisation associated with re-translocation and recycling of stored P, that relies on effective P transportation within the plant, P scavenging, and use of alternate biochemical pathways that bypass P use. Re-translocation between plant tissues is governed by transporters such as PHT transporters and PHO transporters. Unlike, PHT transporters which regulate P acquisition too, PHO transporters are solely responsible for P transport into vascular tissues and cells. The genetic control underlying these PUE mechanisms is covered in the subsequent section. Alternative P use strategies includes substituting phospholipids in cell walls with sulfolipids and galactolipids. Several enzymes in the glycolytic pathway depend on P so bypass enzymes such as pyrophosphate dependent phosphofructokinase , large pots plastic phosphoenolpyruvate carboxylase and pyruvate phosphate dikinase can be recruited to use pyrophosphate for a P donor and conserve limited ATP pools. Several studies have reported increased PEPC activity under P deprivation. The mitochrondrial electron transport chain responds by utilising non-phosphorylative pathways. Acid phosphatases in intracellular spaces or present in the apoplast can increase P availability by remobilising P from senescent tissues and the extracellular matrix. Both aspects of PUE rely on accurate sensing of the P state within the plant and external environment to alter global gene expression and ensure appropriate responses to upregulate P uptake and P use pathways.QTL for overall PUE metrics as well as QTL for more specific architectural root traits associated with low P tolerance have been identified in several economically important crops including soybean, soybean , rice , maize and common bean. RSA is extremely plastic, subject to effects of hormone signalling, environmental stimuli and under the control of several genes so elucidating these QTL is challenging. Studies on other Brassicaceae species are likely of most genetic relevance for QTL mapping in watercress, however QTL associated with other species such as soybean, rice, sorghum and wheat are summarised in Table 1. P-starved Arabidopsis exhibit longer root hairs and higher root hair density, decreased primary root length and increased lateral root density. Three QTL, were identified which explained 52% of the variance in primary root length. In rapeseed primary root length decreases, lateral root length and density increases with declining P concentration. Several QTL are associated with these changes and many co-locate with QTL for root traits in Arabidopsis. A more recent study used over 13 000 SNP markers to construct a genetic linkage map in rapeseed, where 131 QTL were identified in total across different growth systems and P availabilities. However, only four QTL were common to all conditions, demonstrating strong environmental effects determining these QTL. To date, there is no published literature on QTL associated with aerenchyma formation under low P in any plant species and no studies exist on QTL mapping for root traits in watercress. Identification of QTL and markers associated with PUE could accelerate breeding for nutrient use and reduce the environmental impact associated with watercress cultivation.

Specific genes involved in root architecture are targets for enhanced PUE. Although RSA traits are highly quantitative, a BLAST to the rapeseed reference genome revealed 19 candidate genes related to root growth and genetic responses to low P in Arabidopsis. These genes included AUXIN-INDUCED IN ROOT CULTURES 12involved in auxin-induced production of lateral roots and PHOSPHATE DEFICICENCY RESPONSE 2which is part of growth changes in the plant apical meristem under P deficiency. PDR2 is a major component of the P starvation response and functions togetherwith LPR1 and its close paralog LPR2 as a P-sensitive checkpoint in root development by monitoring environmental P concentration, altering meristematic activity and adjusting RSA. Genes involved in transcriptional control are multifunctional under P deprivation; some have overlapping roles in RSA development, P signalling and P utilisation. They are discussed together here despite partial involvement in P utilisation. PHR1and PHL1code for transcription factors that play critical roles in the control of P starvation responses. PHR1 mediates expression of the microRNA miR399 which modulates the PHO2 gene, responsible for P allocation between roots and shoots and affects expression of other PSR genes such as PHT transporters. SPX transcription factors are important negative regulators of PSR via repression of PHR. The roles of several other transcription factor genes on RSA and other regulatory elements are summarised in Table 2 and Figure 3. Auxin, sugars and other hormones such as cytokinins, ethylene, abscisic acid , giberellins and strigolactones are implicated in phosphate-induced determination of RSA so genes involved in these pathways may be significant candidates. Under low P, auxin levels increase in root hair zones and root tips. Auxin mutants such as taa1and aux1have impaired root hair growth in low P. Expression of the Arabidopsis auxin receptor gene TIR1 increases under low P availability which results in increased sensitivity to auxin and production of lateral roots.Mutants in auxin-inducible transcription factors also have disrupted root hair responses under low P. ROOT HAIR DEFECTIVE 6- LIKE-2and ROOT HAIR DEFECTIVE 6-LIKE-4are responsive to P deficiency and promote root hair initiation and elongation. ARF19 is a key transcription factor promoting auxin-dependent root hair elongation in response to low P. HPS1is involved in regulating the sucrose transporter SUC2 and hps1 mutants exhibit significant P-starvation responses under P-sufficient conditions. Plants with impaired cytokinin receptors CRE1 and AHK3 show increased sugar sensitivity and increased expression of P-starvation genes. ETHYLENE RESPONSE FACTOR070 is a transcription factor critical for root development under P starvation. Though no studies exist for P-associated gene expression changes in watercress, Müller et al. used RNA sequencing approaches to identify responses to submergence in watercress and found several ABA biosynthesis and catabolism genes associated with stem elongation. This study provides a model for using transcriptomic approaches to explore hormone-induced morphological changes in watercress.

Food justice research is undoubtedly concerned with equity

Further, their discursive distance from soilless forms of urban agriculture reflected the lack of emphasis that regional supporting organizations and planning initiatives put on these types of growing methods, as they continue to privilege soil-based ways of farming the city. This research hints at important connections between the way growing sites and organizations in San Diego County represent themselves, including their growing methods, primary topic of interest, and institutional affiliation. Our analysis suggests that soilless sites, which are largely for-profit, tend to focus their website content on the innovative methods they use to grow food in urban environments. In contrast, soil-based organizations tend to represent themselves as centered on community and food access. These broad patterns provide important insights into urban agriculture trends in the county and partly support common assumptions held about the goals and motivations of urban agriculture. However, closer examination tells a more nuanced story. Our results show that no single characteristic, whether the use of technology, institutional affiliation, or primary topic, predicted the way our growing sites and organizations represented themselves in narratives on their websites. There were some trends, but the relationship between growing method and the narrative presented is tenuous at best. Overall, two broad conclusions and future research paths can be drawn from the results of this research. First, a politics of technology that creates fixed connections between certain growing methods and values and uses this connection to assume the motivations of urban agriculture participants is misleading and lacks analytical rigor. If we pay attention to the various ways in which urban agriculture organizations represent themselves,drainage gutter it is clear that this connection between growing methods and values is tenuous. For instance, soilless urban agriculture is often associated with entrepreneurialism and therefore cast aside as profit-driven.

While the majority of our soilless sites in our population were for-profit, the link between growing method, for-profit status, and narrative topic was weak. Capital is an underlying reality of all of our sites, especially in the context of neoliberal governance in which even nonprofits are increasingly reliant on private sources of funding , including philanthropy and revenue-generating social enterprises. Entrepreneurialism, therefore, transcends the use of advanced technology and is more meaningfully connected to broader processes like neoliberalism . Future research should continue to unravel these simplistic constructions that constrain research findings and ignore potential tools for improving urban food landscapes. Second, it is important to acknowledge that the genuine motivations and agendas of actors may not match their public narratives and website content. It is therefore critical for researchers to examine the practices that underlie the narratives and self-reported motivations that we have explored and categorized in this chapter. This analysis will require researchers to embed themselves in local urban agriculture networks to observe urban agriculture in practice. Ethnography offers useful tools for this detailed analysis including in-depth interviews and participant observation that allows researchers to examine the relationship between discursive representations and practices of urban agriculture. This methodology will capture the nuanced, everyday interactions that may be hidden by the narratives presented on websites or even in survey data. Avoiding a politics of technology that interprets the connection between technology and capital to mean a singular profit-motive is imperative for gaining a better understanding of the urban agriculture movement. Soilless urban agriculture sites and organizations engage a plethora of environmental and social concerns. Simply equating technologically-advanced urban agriculture with entrepreneurialism, ignoring additional narratives, and forgoing additional critical inquiry creates blind spots in sustainable and equitable food movements.

Based on the narratives examined here, the two forms of agriculture often share values like improving food access, fostering sustainability, and empowering marginalized groups through education and training. We expect the lines to continue to blur in the future as soil based urban growing becomes more entrepreneurial and soilless growing becomes more prolific and accessible. Preliminary interviews already suggest that this is the case in San Diego County. For instance, UrbanLife Farms is planning construction of a new rooftop, hydroponic farm and will integrate it into their broader mission of education and providing job-training for youth in marginalized communities. Project New Village has also expressed an interest in pursuing these growing methods to further their mission of building community wealth and social capital in Southeastern San Diego. This research sought to ‘untangle’ the connections between growing method and narratives. This is an important step in trying to understand some of the common biases against soilless urban agriculture, many of which are rooted in ideological beliefs that are produced and reproduced through popular narratives. However, we recognize that the narratives advertised by urban agriculture sites and organizations on their websites do not accurately reflect the many values that are embedded in these sites or their practices and advocated by their members. This content analysis can only tell us how urban agriculture sites and organization represent themselves in public forums. Still, this analysis begins the task of unraveling a priori assumptions and examining the narratives that accompany urban agriculture practices. These narratives are important actants in urban agriculture actor-networks and are used by actors to strengthen support and attract funding. Deconstructing these narratives is an important step to unveiling co-optation and hollow branding strategies .

Future research should continue to examine the narratives that growing sites and organizations use to promote themselves and the agendas of their diverse actors involved in growing sites and organizations. Indeed, a whole network of people with different backgrounds, personal experiences, decision-making power, and motivations create and reinforce narratives around urban agriculture, not just the directors who likely inspire the content emphasized in mission statements and websites. Further, researchers should engage more detailed methods like ethnography to examine the practices and hidden power dynamics that underlie these narratives. Although many scholars are already embedding themselves in their local urban agriculture networks, participating and observing, to better understand motivations and power relations , few have critically explored the role of technology and considered the breadth of networks shaping urban agriculture. These networks extend beyond garden gates and warehouse walls into composting facilities, federal buildings, local media offices, ethnic markets, Whole Foods supermarkets, farm to table restaurants,plastic gutter and consumers’ kitchens. Future work should examine these networks in full, accounting for the multitude of actors, narratives, and practices driving the discursive and material realities of urban agriculture in the Global North. Tensions surrounding the use of advanced technology in urban agriculture are often rooted in competing understandings of social justice grounded in assumptions regarding the role of land, labor, and capital . These different conceptualizations of justice are particularly evident in debates around the benefits of soil-based and soilless urban agriculture. Such debates have recently pitted food scholars and advocates against each other at a variety of professional meetings including the recent Food Tank™ Summit in San Diego, California. In these contexts, where organizers typically seek to present a ‘balanced’ perspective by including multiple interest groups on panels, discussions of the future of urban agriculture often act as carriers for different yet simplified narratives of food justice, in which the urban food movement is envisioned at a metaphorical fork in the road with the choice of either a high-tech, entrepreneurial or a nature-based, grassroots future. Social justice, specifically food justice, plays an important role in these dichotomous and divisive arguments. Arguably, all forms of urban agriculture, regardless of their relationship to the soil, have the potential to promote or prevent social justice.

Therefore, it is necessary to examine how urban agriculture initiatives, with various degrees of technological intensity, define and do justice. This research seeks to evaluate the justice narratives and practices that shape three urban agriculture spaces with social missions in San Diego County. Urban agriculture thrives in this county and is increasingly diverse including soil-based and soilless growers – both of which are represented in our study sites. I compare these three spaces by focusing on land, labor, and capital and their relationship to distribution, participation, and recognition – three key aspects of justice. Specifically, I assess the outcomes and opportunities generated at each site that produce benefits for marginalized groups such as increased food access, improved self-sufficiency, job training, community engagement, participation in local food system planning and decision-making, and ownership of resources. At the same time, I examine the sociospatial contexts– geography, regional economies, demographics, and institutional environments – that contribute to sites’ ability to produce benefits for marginalized communities. Justice is a central concept in urban agriculture with ‘social justice’ often cited as a goal of urban food projects in the United States. In general, food justice is concerned with addressing exploitation, racism, and oppression within the food system. It is expressed to varying degrees under monikers such as food security, food justice, and food sovereignty – all of which rely on particular understandings of justice . Food security is undoubtedly the least radical of the three. It is a reformist strategy that focuses on market-based interventions – like increasing access to food retailers – and regulatory reform to ensure that individuals have access to food . Programs such as SNAP , food banking, and initiatives to increase access to supermarkets all fall under the purview of food security. The food movement, which seeks more transformational approaches to food systems, is often concerned with strategies like food justice and food sovereignty that address inequities beyond access to food and tend to focus on communities rather than individuals . Food justice is broadly defined as the idea that every person has the right to access affordable, healthful, and culturally appropriate food produced in an ethical and environmentally sound way . It is a progressive strategy that focuses on removing the disparities, especially those based on race, class, and gender, that underlie food system inequities . As such, food justice looks beyond food itself and begins to address the multiple ways in which cultural, social, economic and political inequality shapes our food system, including the production, distribution, and consumption of food. The localization of food production, which allows for greater connections and accountability, has been a common approach to reduce these disparities. Food sovereignty, arguably the most radical of the three , is defined as “the right of peoples and governments to choose the way food is produced and consumed in order to respect our livelihoods, as well as the policies that support this choice” . Here, the distribution of power, particularly power in planning and managing food systems, is key . This perspective, which has been embraced in the Global South, typically implies a rejection of capitalism and neoliberalism that are viewed as causing inequality and preventing communities from being in control of their own food ways. Often, this perspective translates into building alternative and self-sufficient food systems, including supporting community oriented projects and indigenous practices. Geographer David Harvey argues that “different socio-ecological circumstances imply quite different approaches to the question of what is just or not” . In the United States, the dominant perspective is distributive justice – the idea that outcomes such as jobs, health, and income must be fairly distributed among citizens . This approach to justice underlies concepts like food security, as well as food justice , although the two differ in their approach to fairness – the prior typically stressing equality and the latter emphasizing equity . Equality is a prolific theme in food access research where the argument is made that all people should have equal access or the right to food. However, focusing on equality of outcomes has been widely critiqued for its failure to account for the broader social contexts that produce injustice such as patterns of suburbanization , racial and economic segregation , white privilege , and individual mobility . Equity-based distributive justice is still concerned with outcomes; however, it provides more insights into the social context of injustice and considers the “historical antecedents of inequality” including “slavery, exploitation, and dispossession of the land, labor, and products of women, the poor, and people of color” . Opportunities such as access to resources like land and capital also become important in equity-based distribution.

Total N was quantified in selected tissues and time points for the control and RNAi line

The current study uses multiple time point sampling of an expanded profile of mineral concentrations and contents of all shoot organs in NAM knockdown and control lines. This sampling allows the quantification of N and mineral remobilization as contributors to final grain protein and mineral content, and provides a better understanding of the physiological effects of the NAM genes. In addition, experimental treatments have been included to test the remobilization capacity of Fe and Zn in these lines, and radiolabelled Zn was used to quantify the short-term translocation to grain. The information presented here will inform future genomic and systems level studies designed to understand genes and processes that can be targeted to increase grain mineral concentrations for the bio-fortification of foods.The lines used in this study were the NAM RNAi lines designed to reduce expression of all NAM family members and its non-transgenic control . The transformed line is Bobwhite, a semi-dwarf, hard, white, spring, common wheat variety. Wheat seeds were imbibed at 4 C for 3 d and allowed to germinate in darkness at room temperature for 4 d. Seedlings were planted in commercial potting mix and vermiculite at a 2:1 ratio in 17 cm diameter, 17 cm tall pots, three plants per pot, and placed in a growth chamber . Pots were placed in trays with two pots of each line per tray. Plants were watered as needed by sub-irrigation with a nutrient solution of the following composition: 1.2 mM KNO3, 0.8 mM Ca2, 0.8 mM NH4NO3, 0.3 mM KH2PO4, and 0.2 mM MgSO4. Plants were sampled at anthesis of the first emerged head ,25 liter round pot and at 14, 28, 35, 42, and 56 d after anthesis , with an additional harvest at 70 DAA for the RNAi line.

At each sampling, the number of tillers was noted, and plants were cut with a scalpel into the following parts: heads, peduncles, stems, lower leaves, and flag leaves. For each plant, organs from all tillers were pooled and a total of 4–6 plants from two separate pots were analysed per time point. Tissues were dried for 48 h in a drying oven at 60 C, and dry weights were obtained. After drying, heads were separated into grain, rachis, and florets, grains were counted, and these parts were weighed.Wheat seeds were imbibed as described above. Seedlings were planted in plastic cups with plastic beads for support, then placed in lids over 4.5 l containers, 10 plants per pot, in a growth chamber with the settings as described above. Plants were sampled at anthesis of the first emerged head , and at 42 DAA for the Fe deficiency and control treatments . Since 0 Zn plants matured more rapidly than control or 0 Fe plants, plants were sampled at 35 DAA for Zn deficiency and control treatments. At each sampling, the number of tillers was noted, and plants were cut with a scalpel into the following parts: heads, peduncles, stems, lower leaves, and flag leaves. Usually, all tillers from a plant were collected, although the occasional late-emerging tiller was discarded. Thus, 2–5 tillers from a minimum of two plants per time point were collected. Organs from each tiller were analysed separately, then average values were calculated. Tissues were dried for 48 h in a drying oven at 60 C, and dry weights were obtained. After drying, grain was removed from heads, and weighed separately.For mineral analysis of potting mix-grown plants, above ground organs were dissected and organs from all tillers of each plant were pooled as described above. All tissues except florets were ground in a stainless-steel coffee mill.

Duplicate sub-samples of approximately 250 mg were weighed into glass tubes, and digested in nitric:perchloric acid for 1 h at 100 C, then gradually to 200 C until the sample was taken to dryness. Samples were then resuspended in 15 ml 2% nitric acid. All acids were trace metal grade and water was filtered through a MilliQ system to 18 MX resistivity. Mineral concentrations were determined by ICP-OES . The mineral content of the tissues was calculated by multiplying tissue DW by each mineral concentration. For mineral analysis of hydroponically grown plants, a different digestion procedure was used . Whole organs were weighed into glass tubes, and digested in 2.0 ml nitric acid overnight, then at 125 C for 1.5 h. 1.5 ml 30% H2O2 was added and samples were digested for 1 h. A second 1.5 ml volume of H2O2 was added and samples were digested for 1 h. The temperature was then increased to 200 C and samples were evaporated to dryness. Residues were dissolved in 15 ml 2% nitric acid. Mineral concentrations were determined by ICP-OES. The mineral content of the tissues was calculated by multiplying tissue DW by each mineral concentration. For N analysis, tissue samples were dried to constant weight and ground to a fine powder using a ball mill. The samples were analysed for N concentration by a continuous-flow mass spectrometer at the University of California, Davis Stable Isotope Facility.Wheat plants were grown in hydroponics as described above. The dates of anthesis were noted, and at mid-grain fill , plants were moved from a complete nutrient solution to a complete solution spiked with 65Zn at 1 lCi l 1 for continuous labelling experiments, and at 4 lCi l 1 for pulse labelling experiments. All labellings were initiated between 3–4 h into the photoperiod. Plants were not removed from the growth chamber during the labelling period. For continuous labelling, the plants remained in the labelling solution for up to 24 h. For pulse-labelling, the plants were removed from the labelling solution after 3 h and rinsed for 10 min in complete, unlabelled nutrient solution, then placed in fresh complete, unlabelled nutrient solution. At 12 h or 24 h after the commencement of labelling, shoots were excised and cut into lower leaves, stems, flag leaf, peduncle, and heads.

Heads were ovendried for 4–12 h, then the grains were removed. All tissues were quantified for 65Zn by gamma counting.Control and RNAi plants grew similarly in terms of appearance and total plant size . This similarity was also true for individual plant organs, although some tissues differed at some time points. Total grain weight, on a per head basis, was similar between the two lines. Weight of individual kernels was nearly identical and reached maximum values by 35–42 DAA, although total grain dry weight continued to increase as a result of increased seed numbers at later time points. Across sampling points, RNAi lines had a higher number of grains per head, although these differences were significant only at 14 DAA and 35 DAA . It was observed that, as described previously by Uauy et al. ,greenhouse ABS snap clamp the most notable difference between the two lines was delayed leaf yellowing of the RNAi line. The complete data set of mineral contents for the potting mix-grown plants is presented in Supplementary Table S1 at JXB online. At anthesis, Fe and the contents of most other minerals were similar in the vegetative organs of both lines , suggesting that NAM genes had no effect on the content of these minerals to this point in development. Total Zn content was slightly lower in the RNAi line at anthesis . At grain maturity , the total shoot contents of Fe, Zn, and N were similar in both lines , indicating that total uptake and accumulation of each mineral was not significantly affected by the NAM genes. Total vegetative Fe content decreased between anthesis and maturity in the control line . Although the decrease in content between anthesis and maturity was not significant, the RNAi line total vegetative Fe content did not decrease, but rather increased significantly . Total vegetative Zn decreased only in the control line . Comparing the quantity of mineral remobilized from all vegetative tissues to the quantity of mineral in the grain pool at maturity , the net remobilized Fe and Zn could account for between 13.0% of grain Fe and 42.6% of total grain Zn content in the control line, assuming that all of each mineral demonstrating net remobilization was translocated to the grain . In the control line, Fe content decreased over time in lower and flag leaves, stems, peduncle, and rachis, indicating net remobilization. Contrary to this, Fe remained constant or accumulated over time in all tissues of the RNAi line . This was especially marked in the peduncle of RNAi plants, which accumulated 286% of the initial Fe content. Zinc content decreased significantly in all vegetative tissues of the control line In the RNAi line, Zn was remobilized from both the flag leaf and lower leaves, but the percentage change was lower than the control . Zinc decreased in the RNAi line until 35 DAA in stem, peduncle, and head tissues, after which the content increased .

At anthesis and grain maturity, total N contents were similar between both lines , suggesting that reduced NAM expression did not alter total N uptake and accumulation. Throughout grain development, total and individual grain N content were significantly lower in the RNAi line than in the control line . As with Zn, RNAi plants remobilized N from flag leaves and lower leaves over the time-course, but to a lesser extent than in the control plants . The greatest differences in flag leaf N remobilization occurred between 35 DAA and 56 DAA . Comparing N content of all shoot vegetative organs between these time points , total shoot N increased by similar quantities between the RNAi and control lines . However, grain of the control line gained 14 mg of N, while grain of the RNAi line increased only by 2.7 mg. Vegetative tissues of control plants showed a net remobilization of 6.6 mg of N, while those of the RNAi line did not show a net remobilization of N, but rather N increased by 5.3 mg. From anthesis to maturity, N was remobilized from the control stem and peduncle , while N content increased in these tissues of the RNAi line . This resulted in significant differences between control and RNAi lines for both grain N and vegetative N at maturity .To characterize further the mineral remobilization in the RNAi lines under different levels of Fe and Zn, plants were grown using hydroponic conditions, either in a complete nutrient solution for the duration of the experiment, or in an Fe- or Zn-deficient solution from anthesis onwards. Withholding Fe and Zn forced plants to rely solely on stored Fe and Zn to supply the grain. This treatment was used to assess potential net remobilization from the vegetative tissue to the grain while preventing uptake and xylem translocation of these minerals during grain fill, although some root-associated Fe and Zn may have supplied a finite quantity of residual mineral content. If the RNAi line was capable of remobilizing minerals, a net loss of Fe or Zn content would be detected in vegetative tissues between anthesis and maturity. The full data set is presented as Supplementary Table S2 available at JXB online. Leaf yellowing was delayed in the RNAi line in hydroponics, similar to potting mix-grown plants. The control and RNAi lines had similar Fe and Zn vegetative contents at anthesis . For total shoots , neither line exhibited significant net remobilization of Fe or Zn when grown on complete nutrient solution, but both remobilized significant quantities of these minerals when grown on deficient nutrient solutions . Iron-deprived RNAi plants had significantly less net remobilization, with a decrease of 40.2% of vegetative Fe content as compared to 65.6% for the control line . Zinc-deprived RNAi plants showed a net remobilization of 69.6% of Zn content compared to 74.8% for the control line, but the difference between lines was not significant . The quantities of Fe and Zn remobilized were more than enough to account for grain mineral content , although total Fe or Zn content in the grain for each line was significantly lower than when these minerals were supplied continuously . Some shoot Fe and Zn may have been translocated to roots to maintain root growth. Roots of both lines grown in complete nutrient solution did not decrease in Fe or Zn content during grain fill .

The type of plant has also been shown to influence the rate of bacterial pathogen internalization

In previous studies in hydroponically growing Romaine lettuce, it was shown that the levels of internalized TV RNA was higher than the levels of infectious virus that could be recovered from the same samples . In addition it was found that employing an RNAse pre-treatment to degrade viral RNA that was not found in an intact viral capsid reduced the level of TV RNA detection . Similarly, in studies investigating murine norovirus and hepatitis A virus internalization in green onions and spinach more samples were found to be positive for viral RNA compared to infectious virus . Two types of growth substrates, soil and hydroponic solution are commonly used in pathogen internalization studies conducted with plants. Pathogen internalization has been detected more often in hydroponically grown plants compared to plants grown in soil . In this study, both hydroponic and soil growing green onions were contaminated with TV at a level of 1 × 106 PFU/ml. Similar to previous studies, we found that internalization of TV via the root of hydroponically growing green onion occurred while no TV internalization occurred in soil grown green onions. The same phenomena was also observed with HAV and MNV, which were not detected in green onion plants grown in soil for up to 20 days . Compared to soil systems,black plastic plant pots a drastic increase in virus internalization was observed in hydroponic systems with both HAV and MNV internalized up to 4 log RT-qPCR units . Most investigators have suggested that the motility in hydroponic solution may provide more opportunity for uptake and internalization into leafy green plants compared to soil systems .

The virus capsid has an ionic charge and it may remain bound to charged particles in the soil matrix reducing viral uptake by the roots . It is also possible that root damage induced by transplanting of seedlings into the hydroponic system also increased internalization in this study and others . In this study, no virus internalization occurred in radishes while there was viral internalization detected in green onions and Romaine lettuce. Internalization of Salmonella Typhimurium was also observed in lettuce and radish but not cress or spinach seedlings . In the same study, radish seedlings were susceptible to Salmonella internalization while mature radishes were resistant, indicating the importance of plant age on pathogen internalization and dissemination . The radishes used in this study were mature . In our study we found no significant decrease in the level of virus detected in the feed water of hydroponically growing radishes. This indicates that no viral uptake occurred via radish root and it is possible that these mature radishes were not permissive to pathogen internalization. Previously it was shown TV was internalized and disseminated in growing strawberry plants while the virus was not detected in peppers of growing green pepper plants . It was found that the green peppers had a significant anti-viral effect on TV and that this may play a role in the lack of internalization in bell pepper plants . In this study, we found that the levels of TV detected in Romaine lettuce increased over the study period, which is indicative of internalization and dissemination with no inhibition. However, the internalization and dissemination kinetics was different in green onions with high levels of internalized virus detected on day 1 post inoculation and decreasing over the study period. This may indicate that an inhibitory compound in the green onion may be inactivating the virus.

Green onions contain a wide array of phenolic compounds which have the potential to act as antimicrobials . We found that sap from the green onion root lead to a 1-log reduction in infectious TV only after 14 days of exposure. Previously, it was found that a component of radishes, trans-4–methylthio-3-butenyl isothiocyanate , possessed antimicrobial activity . In addition, the pigment found in the skin of radishes, anthocyanins, have also been shown to have antiviral activity. Different cyanidin glycosides present in radishes have antiviral activity against influenza A and B viruses and herpes-1 virus . Our data indicates that radish roots are not permissive to virus penetration and internalization; therefore, we investigated whether radishes had antiviral properties. A maximum 1-log reduction in infectious TV titer was achieved after 14 days of exposure of TV to radish sap, indicating only minimal antiviral effects. In conclusion, we have shown that i) human NoV and TV can be internalized in hydroponically grown green onions but not in soil grown green onions, ii) the magnitude of TV internalization is influenced by the level of virus present, and iii) different types of plants have different susceptibility for viral internalization. Salinity stress is a major abiotic stress that has significant adverse effects on crop productivity and yield. These negative effects include interference of root function in absorbing water, as well as the prevention of physiological and biochemical processes such as nutrient uptake and assimilation . Unfortunately, many regions around the world are facing a rapid increase in soil salinity and sodicity. It is estimated that at least 0.3 million hectares of farmland is becoming unusable annually, and another 20–46 million ha are suffering decreases in production potential each year . Nevertheless, even with lower yield potential, these salt-affected farmlands must continue to produce crops so the increasing demand for food can be met and food security concerns mitigated.

The lack of new productive land threatens food security, thus the productivity of existing marginal lands must improve.There are numerous potential solutions for mitigating salt stress, including genetic engineering of plants with salt tolerance and application of exogenous compounds such as hormones, growth regulators, or nanoparticles . Among the potential solutions, selecting plant varieties with high tolerance to salt stress appears to be one of the most promising approaches in utilizing salt-affected soil for crop production . Although some progress has been made using measurement of photosynthetic parameters as a more sensitive method to screen for salt tolerance , the standard process of selecting either conventionally–bred or transgenic salt-tolerant crop lines relies on laborious phenotyping to assess tolerance. Despite the emergence of innovative platforms, precise instrumentation, sophisticated sensors, and rapid development of advanced machine learning and deep learning algorithms, phenotyping is still a barrier to variety development. While DNA sequencing and plant genotyping has rapidly evolved, phenotyping still depends on conventional methods which are not as accurate or efficient. In general, these techniques can be time-consuming, destructive, subjective, and costly. In recent years, non-contact sensing technology, in particular imaging, has been extensively deployed as a potential substitute for conventional methods for high-throughput phenotyping of plants. Thanks to the advances in developing sensors with high spatial and spectral resolution, different imaging sensors including visible, fluorescence, thermal, and spectral imaging are available, each tailored for specific applications. Each of these sensing technologies can vary in their application, as well as limitations, in the context of plant phenotyping . Among these techniques, hyperspectral imaging is uniquely suited to provide insights into the internal activities of plants, leaf tissue structure, leaf pigments, and water content . HSI also provides the ability to investigate physiological dynamics of plants caused by environmental variables , and consequently has drawn substantial attention for plant phenotyping . Few research studies have attempted to identify salt stress in plants using hyperspectral reflectance. In a previous study, three potential indicators including blue, yellow, and red edge positions of vegetation reflectance spectrum were calculated to detect four levels of salt stress imposed on Chinese castor bean . The authors claimed that blue and red edge positions shift to the shorter wavelength in response to salt stress and therefore could be used to detect salt stress. However, the pattern of shifting to the shorter wavelength was not consistent across all treatments and hence further research is required. In another paper,black plastic planting pots the application of HSI to identify plant tolerance to salt stress in a high throughput phenotyping system was reviewed . They concluded more efficient and fully automated methods are required to analyze complex hyperspectral images. To leverage the full potential of HSI, a large high-quality hyperspectral dataset and several preprocessing tasks are necessary .

However, there are two major challenges that hamper the application of HSI. The first major challenge is accounting for the variance caused by the complex interaction between incident light and leaf surfaces due to non-Lambertian reflectance properties. The direction of reflected light is a function of leaf geometry, including leaf angle, and curvature. Several researchers have focused on pre-processing techniques to address the problems related to leaf angle and curvature . One method to resolve this problem is to generate a high-resolution 3D representation of plants by upfront geometric calibration of the hyperspectral camera . However, this proposed method depends on highly intensive processing and is only suitable for close-range imaging. The second major challenge is analyzing the complex and high-dimensional hyperspectral images in order to extract meaningful features and recognize latent patterns associated with the desired phenotyping trait in a more interpretable manner. To address this issue, machine learning and deep learning algorithms can be leveraged. Recent reviews of various ML algorithms emphasize the potential of these methods in the context of agriculture and provide guidelines for plant scientists to deploy them . Singh et al. reported that ML algorithms are a promising approach to analyze large datasets generated by sophisticated imaging sensors mounted to platforms that can cover large areas. Despite several studies that focus on the application of HSI for plant phenotyping, research is limited in the context of handling, processing, and analyzing hyperspectral images. This research was motivated by the need to identify salt tolerant wheat lines to mitigate yield losses due to salinity, and to ultimately maintain or improve production on saline soils. The objectives of this study were to rank wheat lines based on their tolerance to salt stress, assess the difference between the salt tolerance of lines to attain a quantitative ranking rather than a qualitative ranking, and evaluate the feasibility of precise ranking of wheat lines as early as one day after applying salt treatment. We hypothesized that the spectral response of wheat leaves experiencing salt stress would deviate from the control leaves even one day after adding the stress, and this deviation would be larger for a susceptible line compared to a salt tolerant line. To the best of our knowledge, no previous study has investigated early detection of salt tolerant plant lines using advanced phenotyping tools and approaches. This research proposes a machine learning approach to analyze hyperspectral images of wheat lines to rank their salt stress tolerance in a quantitative, interpretable, and non-destructive manner while reducing cost, time, and labor input.To develop analytical methods for analysis of hyperspectral images, four bread wheat lines were selected with varying levels of salt tolerance. The cultivar Kharchia was included as it is historically known to maintain a stable harvest index and yield well in high salt conditions , and the salt-sensitive cultivar Chinese Spring was selected as well . Two additional “unknown” lines were selected for screening from a set of wheat alloplasmic lines developed in Japan . Alloplasmic lines are created by substitution backcrossing to replace the cytoplasmic genomes of one species with those of another while maintaining the original nuclear genome background, and have shown promise for improving stress tolerance and other developmental traits . The two alloplasmic lines selected were Aegilops columnaris KU11-2 [abbreviated co hereafter] and Ae. speltoides aucheri KU2201B [abbreviated sp hereafter] with the cytoplasmic genome type preceding the nuclear genome background, which in this case is Chinese Spring . Screening was performed in a hydroponic system in a Conviron growth chamber to ensure uniform conditions. Hydroponic systems are commonly used to screen plants for salt tolerance, including wheat. In all experiments, growth conditions in the Conviron were set at 22◦C during light conditions and 18◦C during the dark, 16 h photoperiod, 375 µmol m−2 s −1 light intensity, and 50% relative humidity. Three hydroponic tanks were used per treatment . Each hydroponic tank contained a grid of 16 Cone-tainers filled with perlite. Within each tank, there were four genotypes each with four individual replicates . For each treatment , there were three replicate tanks; hence, there were a total of 48 Cone-tainers for each treatment.

The extract was analyzed by high-performance liquid chromatography

Following the short-term testing, the building managers identified several potential sources of indoor contaminants including composite wood paneling, painted gypsum board walls and high density plastic barrels used as hydroponic containers for the bio-filtration-based air cleaning technology. Both new and aged plywood wall paneling was present in the building and the paneling was coated with a clear polish. The goal of this study was to measure material specific emission factors for VOCs and carbonyls and characterize the potential influence of the polish coating on the wood panel material. All building materials that were tested for emissions were harvested from the PBC building, double wrapped in foil and shipped directly to Lawrence Berkeley National Laboratory for testing. A description of each of the material samples is provided in Table 1. All samples except for the hydroponic drum were cut to 0.023 m 2 . In the laboratory, the sides and backs of the material were sealed with aluminum tape and stainless steel backing plates, respectively, to leave only the front face of the material exposed for testing. Each sample was placed individually in 6-liter stainless steel conditioning chamber as illustrated in Figure 1. The conditioning chambers were closed with Teflon lined lids and held at approximately 22 ˚C and 50% relative humidity to precondition the materials prior to sampling. For new materials, samples are typically preconditioned to allow the emissions to drop to a more relevant value for estimating long-term emission rates. For materials that are allowed to age in the environment, the conditioning period is important to allow chemicals that have partitioned into the material from the environment, e.g.,drainage planter pot chemicals that are not indigenous to the material, to off gas so that the measured emission rates are relevant to the material being tested.

The emission testing generally followed California Specification 01350 and ASTM Standard Guide D-6007-02 using small emission chambers. The approach has been used for a wide range of materials measuring both VOCs and carbonyls as described previously and as summarized below. Four emission chambers installed in a controlled environment oven provide an isolated environment with constant temperature and humidity. The constant humidity was maintained by splitting the flow of dry carbon/HEPA filter air with a portion of the air bubbling through a water bath then re-mixed to achieve the desired humidity for air flowing through each chamber. The chambers, shown in Figure 2, are made of stainless steel and all interior surfaces are coated with Sulfinert® coating to minimize chemical interaction with chamber walls. The chambers are 10.75 L and are operated with an approximate ventilation rate of 1 liter per minute equivalent to 5.6 air changes per hour , or 2.6 m3 [air]/m2 [exposed surface area]/hour. The standard tests are operated at 25 °C and 50% RH. After being pre-conditioned, each test material was transferred to the test chamber and placed on Sulfinert® treated screens resting slightly below the midpoint of the chamber. Each material was allowed to equilibrate in the test chamber for at least 30 minutes after being transferred from the conditioning chamber before testing. Once equilibrated, the air samples were collected directly from the test chamber and analyzed for VOC and Aldehydes as described below. The materials were first tested after 24 hours of conditioning, and then again after at least seven days of conditioning. The first sampling period was used to get information on upper bound emission rates and allow for the identification of the mix of chemicals in the emission stream.

The second sampling period, after seven days conditioning, provides the emission factors that are more relevant to the long-term emission pattern. Additional measurements were collected for the new wood with new polish to further understand how the polish affects the emissions from the material. VOC samples were collected and analyzed following the U.S. Environmental Protection Agency Method TO-17. VOC air samples were collected directly from the chambers by drawing chamber air through multi-sorbent tubes with a primary bed of TenaxTA® sorbent backed with a section of Carbosieve®. A peristaltic pump was used to pull the air through the sample tubes at a rate of approximately 100 mL/min for 1 hour. The flow was measured using a DryCal gas flow meter and was recorded at the beginning and the end of the sampling period. Before subjected to chemical analysis, each sample was spiked with 120ng of gas-phase 1-Bromo-3 Fluoro-Benzene , which was used as the internal standard in the quantification method. Analytes were thermally desorbed from the sampling tubes using a thermodesorption auto-sampler , a thermo-desorption oven , and a cooled injection system . Desorption was performed in splitless mode where the desorbed analytes were refocused on the cooled injection system prior to injection. Desorption temperature for the TDS started at 30 ˚C with a 0.5 minute delay followed by a 60 ˚C ramp to 250 ˚C and a 4 minute hold time. The cooled injection system was fitted with a Tenaxpacked glass liner that was held at -10 ˚C throughout desorption and then heated within 0.2 minutes to 270 ˚C followed by a 3-minute hold time. Compounds were resolved on a GC equipped with a 30 meter HP- 1701 14% Cyanopropyl Phenyl Methyl capillary column with helium flow of 1.2 mL/min.

The initial temperature of the oven was -10 ˚C held for 0.5 minutes then ramped at 5 ˚C/min to 40 ˚C then 3 ˚C/min to 140 ˚C and finally at 10 ˚C/min to 250 ˚C and held for 10 minutes. The resolved analytes were quantified using electron impact mass spectrometry, , with mass to charge ratio limits of 44.0 m/z and 450.0 m/z. The MS was operated in full scan mode with a solvent delay of 3.00 minutes. Compounds were initially identified using NIST mass spectral search program for the NIST/EPA/NIH mass spectral library with identity confirmed and quantified using pure standards. When pure standards were not available, the analyte was reported in terms of toluene equivalence by comparing the instrument response for the total ion chromatogram of the chemical to a multi-point calibration of TIC response for toluene.The volatile carbonyls including formaldehyde, acetaldehyde and acetone are quantified using USEPA Method TO-15. As with the VOC samples, the air was drawn directly from the chambers during sampling. The sampling rate was maintained at less than 80% of the total air flow through the chamber to prevent back flow of unfiltered air into the chamber during testing. The sample air passed through silica gel cartridges coated with 2,4-dinitrophenyl-hydrazine, which quantitatively reacts with the carbonyl functional group effectively trapping the aldehydes and other low molecular weight carbonyl compounds. A peristaltic pump was used to pull the air through the cartridge at a rate of approximately at 800 mL/min for 1 hour. The flow was measured using a DryCal gas flow meter and recorded at the beginning and the end of the sampling period. Prior to analysis, sample cartridges were eluted with 2ml of high purity acetonitrile and the effluent was brought to a final volume of 2 ml.The HPLC was fitted with a C18 reverse phase column and run with 65:35 H2O: Acetonitrile mobile phase at 0.35 mL/minute and UV detection at 360 nm. Multi-point calibrations were prepared for the target analytes using commercially available hydrazone derivatives of formaldehyde,plant pot with drainage acetaldehyde and acetone.All materials were initially tested after only 24-hours of conditioning time. Prior to conditioning, the materials had been tightly wrapped in foil and packaged individually in resealable plastic bags during shipping so the initial emissions were expected to be elevated. The purpose of this in initial testing was to identify the chemicals in the emission stream. A total of forty chemicals were identified in the emission stream from the six materials tested. All chemicals are listed in Table 2 along with steady state concentrations measured after 24-hours of conditioning. All values are listed in Table 2 for comparision but values above the typical method limit of quantification of 0.5 µg/m3 are listed in bold text. The plastic material from the hydroponic drum was tested as received without sealing the back and sides so the exposed area was approximately double that of the other materials. The initial measurements found that except for hexadecane and tetradecane, most of the VOCs from the plastic material, including the aldehdyes, were near or below the minimum detection limit. Therfore, the plastic is not likely a source of indoor contaminants in the indoor environment of the PBC.

The drywall material produced a number of elevated VOCs with two that exceeded the linear range of the analytical method. Drywall is typically a low VOC material although fresh coatings such as paint or plaster can emitt VOCs during curing. The drywall samples tested in this study appear to be freshly painted because the edges were sealed with paint. This might explain the elevated propylene glycol and benzyl alcohol. The new wood paneling had very high levels of formaldehyde both with and without polish although the polished panel had the high test levels of formaldehyde overall. However, the unpolished new wood panel produced a wider variety and higher levels of VOC in the emissions. The old wood paneling produced much lower levels of formaldehyde and the levels of VOCs in general were similar both with new and old polish. After the initial tests were completed to identify the target chemicals in the emission stream, the materials were returned to the conditioning chambers for approximately six more days before measuring the emission factors. Concentrations for the plastic material from the hydroponic drum remained low in the second test so emission factors are not reported for the plastic hydroponic drum material. The standard emission factors determined for the wood paneling and drywall materials are reported in Table 3. The formaldehyde emissions from new wood with new polish were still significantly elevated after 7 days but we suspected that the combination of polish and storage may have increased the time needed for the emission factor to drop to a relativily constant level. To address this, we continued to condition the new wood with new polish for an additional week and re-tested. The additional time needed to condition the new wood with new polish may have been due to a higher capacity of the polish coating for accumulating formaldehyde during storage. This possibility was tested and is discussed further below. The standard emission factors for the materials from the PBC are summarized in Table 3. Several of the chemicals that were initially detected in the materials were no longer detectable in the emission stream after a week of conditioning and are therfore not listed in Table 3. The painted drywall continued to have extremely high levels of benzyl alcohol and propylene glycol as well as quantifiable levels of several other aldehydes , alcohols and esters that may be related to the coating material and/or sorbed into the drywall matrix from the environment. The wood paneling material presented a mix of VOCs depending on if the polish and/or wood were new or old as illustrated in Figure 3. Figure 3 lists the sum of all emission factors for VOC presented as stacked colums with the largest overall emission factors listed in decreasing order from bottom to top on the figure legend. Emission factors listed in Table 3 that are below the approximate limit of quantification of 1.65 µg/m2 /h are not included in Figure 3. Overall the drywall material had the highest sum of individual emission factors with the paneling material emitting 134, 129, 33 and 7 for the new wood no applied polish, old wood new polish, new wood new polish and old wood old polish, respectivily. Formaldehyde emissions for the old wood paneling with new and old polish, and the drywall were all similar ranging from 10 µg/m2 /h to 22 µg/m2 /h . For the new wood, the formaldehyde emissions were approximately an order of magnitude higher than the other materials for both the polished and unfinished surfaces. The emission results for formaldehyde are illustrated in Figure 4 showing that the polish coating does not seem to significantly change the measured emission factors when the age of the wood paneling is taken into consideration.

Arabidopsis thaliana cells were used for an initial kinetic evaluation and metabolic profiling

The mechanisms for plant accumulation of neutral organic compounds have been well studied for pesticides and herbicides, but relatively little work has been reported for PPCP/EDCs. Neutral compounds are thought to be taken up by passive diffusion through the root cell membrane, which is hampered by strong polarity or hydrophobicity . For neutral PPCP/EDCs in this study, a positive linear correlation with log Dow was observed for BCF leaf or BCF root . The effect of hydrophobicity Translocation of compounds from root to aerial tissues may lead to their accumulation in edible leaves or fruits. A translocation factor , the concentration in leaf tissue divided by that in root tissue, was calculated for PPCP/EDCs in each treatment . In this study, atorvastatin, ibuprofen, and sulfamethoxazole were the least translocated , while carbamazepine, meprobamate, and dilantin were the most translocated . The mean TF value was the highest for tomato at 2.90, with a range of 0 – 18.40, followed by carrot at 1.47, with a range of 0 – 13.58, while lettuce showed the least translocation with an average TF of 0.84 and a range of 0 – 5.50. The warm-dry treatment, which induced higher transpiration , also showed greater TF values than the cool-humid treatment. This observation suggested that increased mass flow due to transpiration enhanced the movement of PPPC/EDCs from roots to leaves in this study. To assess the effect of transpiration on TFs of anionic, cationic, and neutral PPCP/EDCs,vertical farms the TF values in each treatment were compared to the mass of nutrient solution transpired by each treatment .

For cationic and neutral PPCP/EDCs, significant positive correlation was observed between TF values and the transpired mass , suggesting that translocation of cationic and neutral compounds from root to leaves was influenced by transpiration. The impact of transpiration on TF was similar for both cationic and neutral compounds, as evident from their similar slopes of the regression lines . In contrast, a similar relationship was not found for anionic PPCP/EDCs . Cationic compounds also had significantly greater TF values than neutral compounds or anionic compounds , which suggests that cationic compounds were more likely than the other compounds to translocate from root to leaf tissues. This behavior may be due to the partitioning behavior of the cation molecules; charged molecules of cationic species tend to be sequestered in plant compartments with high pH, such as phloem . On the other hand, TF values for anionic compounds were generally low, which may be due to the ion trap effect in roots that are known for other anionic compounds . The ion trap effect occurs when the neutral fraction moves into root cells and become partly dissociated due to the change in pH inside the cells. The dissociated anions would not be able to quickly diffuse out of the cell into xylem and other plant parts, due to electrical repulsion, causing limited translocation. 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, 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.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 14Cdiazepam 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,vertical plant growing control treatments were prepared by auto claving cell suspension flasks 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 13,000g 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 freeze-dried 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 13,000g 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 cleanup. 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 micro-centrifuge tubes and centrifuged at 12,000g 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 °Cwas greater for root tissues as compared to leaves , likely due to the contribution of adsorption to the accumulation in root tissues. Other studies have suggested that the optimum log Kow value for plant uptake is around 1 – 3.5 . In this study, diazepam, with a log Dow value of 2.82, exhibited the largest BCF values among the neutral compounds considered in this study, which was in agreement with previous observations.Similar metabolites to those in A. thaliana cells were found in seedlings grown in the nutrient solution spiked with diazepam, with nordiazepam being predominant . In the 7 d and 28 d cultivation experiments, temazepam was found to be the second major metabolite in the leaves of the cucumber seedlings, and the level was higher in the7 d cucumber seedlings than the 28 d plants . Oxazepam was detected in the leaves of both plant species after the 7 d cultivation . The higher accumulation of diazepam and the biologically active metabolites in the leaves may have ecotoxicological ramifications; for example, many insects consume leaves, even if they are not edible tissues for humans . Our results were in agreement with recent findings in Carter et al. , in which they observed the formation of nordiazepam, temazepam and oxazepam in radish and silverbeet plants exposed to diazepam and chlordiazepoxide. They similarly showed nordiazepam to be the major metabolite with oxazepam and temazepam constituting a much smaller fraction at the end of 28 d cultivation in soil. However, in that study, the authors did not track the formation of these metabolites over time or influence of treatment concentrations. Phase III metabolism appeared to increase from the 7 d to 28 d cultivation for both radish and cucumber seedlings . Between the plant species, the cucumber seedlings had a greater fraction of nonextractable radioactivity in comparison to the radish seedlings . In the 7 d cultivation experiment, the mass balances came to 99.3% for the cucumber plants but only 58.1% for the radish seedlings . Due to the multiple water changes , a complete mass balance was not attainable for the 28 d cultivation experiment. However, when a proxy mass balance was calculated for both species, a similar pattern was observed. A total of 83.0% of the added 14C radioactivity was calculated for the cucumber treatments while the fraction was 61.3% for the radish plants. This could be due to increased mineralization in the growth media and respiration of 14CO2 through plant in the radish cultures. As mineralization is viewed as the final stage of detoxification , it is likely that the radish plant was more efficient in their ability to detoxify diazepam than cucumber plants. The Brassicaceae family, which includes the common radish, has been shown to be effective for phytoremediation due to their possession of genes that increase tolerance to stressors and activation of enzymes capable of extensive bio-transformations .The activity of glycosyltransferase was measured in the control seedlings as well as seedlings exposed to diazepam for the 7 d and 28 d cultivation experiments . Glycosyltransferase catalyzes the transfer of sugars, such as glucuronic acid, to many types of acceptor molecules, including xenobiotics . The conjugation of glucuronic acid with oxazepam is the major detoxification pathway of diazepam in humans . No detectable level of oxazepam-glucuronide was observed in radish or cucumber seedlings for either the 7 d or 28 d cultivation.

Soil texture and organic carbon content were determined using established methods

Consequently, many PPCPs/EDCs are routinely found in WWTP products . At the same time, land application of treated wastewater and bio-solids is increasing . Although these compounds are usually detected at trace levels in soils and plant tissues , there is continual input of these biologically active compounds. Better knowledge of the extent and composition of PPCP/EDC accumulation in plants is needed to improve our understanding of the current and future risk to human health.As natural resources are stressed by population growth, urbanization, and climate change, previously under-utilized waste materials such as treated wastewater and bio-solids from wastewater treatment plants are increasingly being explored and used. For instance, about 3.6 × 109 cubic meters of treated wastewater is currently reused in the U.S. for purposes including agricultural and landscape irrigation, and water reuse is growing by 15% a year . Similarly, approximately 6 × 106 metric tons of bio-solids are produced each year in the U.S., of which about 60% is applied to land . Regulations governing such reuses are mostly concerned with pathogens, nutrients, and heavy metals . However, studies over the last two decades have shown that numerous anthropogenic chemicals, such as pharmaceutical and personal care products and endocrine disrupting chemicals , are present in treated wastewater and bio-solids . Many of these chemicals are known to have unintended biological effects on non-target organisms at low levels . Therefore, the beneficial reuse of these waste materials for irrigation or soil amendmentintroduces contaminants into the soil environment and may pose risks to terrestrial ecosystems and human beings through dietary exposure . In general, the fate of a xenobiotic in soil includes complete mineralization ,vertical planting tower conversion to transformation products, and formation of bound residue .

Mineralization of a compound is viewed as complete detoxification, while formation of bound residue is also generally considered a decontamination process . In soil, PPCP/EDCs may undergo microbially-mediated transformations, processes that are greatly influenced by both the soil microbial community and the physico-chemical properties of PPCP/EDCs . The formation of transformation products poses unknown risks as the new products may have biological activity . However, to date, most studies on the fate of PPCP/EDCs in soil have only considered removal of the parent compound while ignoring fate pathways. In this study, with the coupled use of 14C-labeling and chromatographic separation, we quantitatively characterized mineralization and formation of bound residue, as well as disappearance of the parent compound and formation of transformation products, of four commonly occurring PPCP/EDCs, i.e., bisphenol A , diclofenac , naproxen, and nonylphenol , under different soil conditions. Several transformation products of BPA and DCL were also identified. These PPCP/EDCs appear frequently in treated wastewater and bio-solids , but little information is available on their complete fate in soil. More knowledge of the complete fate of PPCP/EDCs in soil may be used to improve risk evaluation for land application of treated wastewater and bio-solids. Agricultural soils were collected from the University of California’s South Coast Research and Extension Center in Irvine, CA and from the University of California’s Hansen Agricultural Center in Ventura, CA . A third soil was collected from a treated wastewater recharge basin at the Riparian Preserve at Water Ranch in Maricopa, AZ . Soils were collected from the surface layer . After air-drying, soil was passed through a 2 mm sieve.

To examine the effect of organic matter, a sub-sample of the Irvine soil was amended with sieved redwood compost at 50% to create the Irvine Amended soil treatment. To understand the role of soil microorganisms, another sub-sample of Irvine soil was autoclaved at 121°C for 45 min on two consecutive days to create the Irvine Sterilized treatment.The field capacity of each soil was determined using the pressure chamber method, where -33 J/kg of hydraulic head was applied to saturated soil . Table 3.1 lists selected soil properties. Soil respirometers were constructed by suspending a 2 mL glass vial in a 40 mL amber glass bottle with a screw-cap lined with a septum. During incubation, 1.0 mL of 1M NaOH solution was deployed in the 2 mL vial to trap 14CO2 from mineralization. A syringe needle was inserted through the septum to enable the sampling and refill of the NaOH solution to monitor mineralization kinetics. A working solution was prepared for each 14C-PPCP/EDC in water. Air-dried soil, equivalent to 10 g dry weight, was placed in the amber bottle and spiked with 0.8 mL of a working solution containing about 3 × 105 dpm radioactivity, making an initial concentration in soil of 12.6 µg/kg for BPA, 69.3 µg/kg for DCL, 46.4 µg/kg for NPX, or 52.8 µg/kg for NP. Deionized water was added to reach field capacity in each soil, which equated to 35% of the total water capacity for Irvine soil and Irvine Sterilized soil, 21% for Irvine Amended soil, 47% for Maricopa soil, and 45% for Ventura soil. Each soil sample was manually mixed to achieve homogenization. The sample bottles were closed, and then NaOH solution was injected into each suspended vial. All soil respirometers were incubated at room temperature . Respirometers were opened briefly on a weekly basis for aeration and deionized water was added gravimetrically as needed to maintain the soil water content. On 1, 3, 7, 14, 21, 28, 35, 42, 49, 56, 63, 70, 77, 84, 91, 102, and 112 d after the treatment, the NaOH solution in each respirometer was exchanged with new NaOH solution using a disposable syringe.

The used solution was placed in a 7 mL glass scintillation vial and mixed with 4 mL of Ultima Gold Scintillation Cocktail , followed by measurement of 14C on a Beckman LS 5000TD Liquid Scintillation Counter . On day 0, 3, 14, and 112, three soil samples from each treatment were transferred into a freezer for extraction and analysis of extractable and bound residues. Soil samples were extracted using EPA Method 1694. In brief, soil samples were removed from the freezer and the thawed soil was transferred to a 50 mL polypropylene centrifuge tube. The soil was sequentially extracted with 35 mL of freshly prepared phosphate buffer -methanol twice and 20 mL of methanol once. For each extraction cycle, the centrifuge tubes were mixed at 260 rpm for 1 h on a horizontal shaker and then centrifuged at 2300 rpm for 15 min. The supernatant was decanted into a 100 mL glass flask, from which a 3 mL sub-sample was removed for analysis on LSC todetermine the total extractable 14C residue. The remaining solvent extract was capped and stored at 4 °C until further analysis. After the sequential solvent extraction,vertical hydroponic farming the soil was air-dried in the fume hood and then 1.0 g aliquots were combusted on an OX-500 Biological Oxidizer at 900 °C for 4 min. The evolved 14CO2 was trapped in 15 mL of Harvey Carbon-14 cocktail , followed by measurement on LSC to determine the total bound 14C residue. The recovery of 14C in soil was determined to be 71-110% by combusting spiked soil samples and was used to correct for the actual amount of 14C in soil. The soil extracts were prepared for analysis of parent and transformation compounds by a method modified from Wu et al. . In brief, selected extracts were removed from the refrigerator and mixed with 1200 mL of deionized water, such that methanol was less than 5% of the total solution. The aqueous sample was then passed through a solid phase extraction cartridge at a rate of 5 mL/min. The cartridge was pre-conditioned with 5 mL each of methylene chloride, methanol, and ultra-pure water. A 6 mL sub-sample of the filtrate that passed through the cartridge was collected and analyzed on LSC to determine the presence of any 14C not retained on the solid phase. The cartridges were then dried under nitrogen gas and eluted with 7 mL methanol. The eluent was condensed to 250 µL under a gentle nitrogen flow and transferred to a 2 mL glass vial. The condensing vessel was rinsed with 200 µL of methanol and the rinsate was added to the eluent in the glass vial. A 50 µL aliquot of non-labeled parent standard stock solution was spiked into each vial to make the final sample volume to 500 µL. To characterize the extractable residue, a 50 μL aliquot of the prepared extract was injected into an Agilent 1100 Series high performance liquid chromatography with an ultraviolet detector. A Dionex Acclaim-120 C18 RP column was used for separation at a flow rate of 1.0 mL/min at 35 °C.

Mobile phase A was ultra-pure water acidified with 0.2% acetic acid and mobile phase B was acetonitrile. The ratio of mobile phase A to B was 60:40 for BPA, 50:50 for DCL, 60:40 for NPX, and 25:75 for NP, with corresponding UV wavelengths of 280, 284, 278, and 280 nm, respectively, for positioning the parent compounds. The HPLC eluent was fractionated in 1 min increments using an automated fraction collector . Each fraction was mixed with 4 mL of cocktail for analysis of 14C to monitor the distribution of 14C as a function of run time. To identify transformation products, extracts from BPA and DCL treatments were further analyzed on an ACQUITY ultra-performance liquid chromatography system using an ACQUITY UPLC BEH C18 column at 40 °C. Mobile phase A was 0.001% formic acid in water and mobile phase B was methanol. The following mobile phase program was used: 0 – 0.5 min, 5 – 50% B; 0.5 – 12 min, 50 – 100% B; 12 – 13 min, 100% B; 13 – 16 min, 5% B. Analysis was performed with a Waters Micromass triple quadrupole detector equipped with an electrospray ionization source in the negative mode. Parameters of MS/MS were as follows: source temperature, 120 °C; desolvation temperature, 350 °C; capillary voltage, 3.0 kV; cone voltage, 20 V; desolvation gas flow, 600 L/h; cone gas flow, 50 L/h. Standards were run in scan and daughter modes to identify the most robust transition pattern and cone voltage for each compound, and the optimized parameters are listed in Supplemental Table S3.1. Quantitative analysis was performed in the multiple reaction monitoring mode. All data were processed using MassLynx 4.1 software . The extractable fraction of xenobiotics is often used to represent the bio-available fraction that may illicit biological effects . Incubated soil samples were extracted with solvents to determine the extractable residue of spiked 14C-PPCP/EDCs. Figure 3.2 depicts the extractable residue of treatments after 112 d of incubation. For all compounds in all soils, the extractable residue decreased over the incubation period. For example, in Irvine soil spiked with DCL, the extractable 14C decreased to only 6.6 ± 0.2 % at 112 d. The abundance of extractable 14C varied among the PPCP/EDCs, and the general order was NP > BPA > DCL ≥ NPX. For example, in Ventura soil at 112 d, the extractable fraction was 12.9 ± 0.8% for NP, 9.8 ± 0.3% for BPA, 6.8 ± 0.4% for DCL, and 5.6 ± 0.1% for NPX . The level of extractable residue was generally similar among Irvine, Maricopa, and Ventura soils. After sterilization, the level of extractable residue was consistently higher than in the non-sterilized treatment, suggesting that the dissipation of extractable residue was largely due to microbially- mediated transformations. In addition, compost amendment slightly increased the level of extractable residue in Irvine soil. In Fent et al. , no 14C was detectable in the extract of soil treated with 14C-BPA after 120 d, which was in agreement with the present study, where extractable residue in the unmodified soils was low at the end of incubation . In a clayey silt soil and a silty sand soil, Kreuzig et al. reported 5% and 43% extractable 14C after 102 d of incubation following 14CDCL treatment; the difference between soils was attributed to indigenous microbial activity In this study, only 6.6 – 8.1% of 14C-DCL residue was extractable at the end of incubation. Lin and Gan found that after 84 d of incubation, 5% and 40% of the spiked NPX were recovered as the parent compound from a sandy soil and medium loam soil, respectively, while the extractable fraction was only 3.1 – 5.6% in the current study.

Engineered nanomaterials are being used in a wide array of consumer products

Other common transformations may also be of great importance to improve our understanding of environmental risks of CECs. For example, halogenated CECs can be produced during the disinfection process that is commonly used in treating wastewater and drinking water, and such halogenated derivatives may have very different biological activity profiles as well as environmental behaviors from their precursors. Conjugation with endogenous bio-molecules has been widely observed for biologically mediated CEC transformations. For example, conjugates of CECs and/or their metabolites are common in higher plants. Enzymes such as glucuronidases, aminoacylases, and dipeptidases in the human gut and intestine may hydrolyze these conjugates, releasing the parent or metabolites in their free form. Future research is needed for these unique TPs of CECs to obtain a more comprehensive understanding of the environmental fate and risks of CECs.Global climate change leads to more variable and extreme weather conditions that decrease total agricultural production of staple food crops. To cope with less predictable growth conditions under temperate climate conditions during spring and summer, an earlier time point of drilling may be chosen. Cereal plants tiller before winter, and due to an established crown root system the tillers are more resistant against temporary drought stress in spring. After the winter period, however, tillering continues in particular with nitrogen fertilization in spring, even though tiller number might already be sufficient for optimum yield. Thus, there is a need to uncouple tillering from nitrogen supply. To investigate the physiological effect of different N forms on tillering, spring barley was cultured in buffered nutrient solution supplied with stabilized nitrogen forms. Plants were harvested at the end of the vegetative growth phase and analysed for biomass, tiller number,vertical farming systems concentrations of mineral nutrients and cytokinins.

To investigate the effect of nitrogen forms on cytokinin translocation, endogenous cytokinins were determined in xylem exudates or benzyladenine riboside was supplied to the nutrient solution and measured in xylem exudates. Cytokinins were determined by radio immuno assays using specific antibodies. For field trials winter wheat was fertilized with stabilized nitrogen forms in the starter dressing and analysed at the end of the vegetation period for nitrogen levels, phytohormone levels and yield components.The vast majority of ENM studies have examined the acute toxicity of nanoparticles and particle forms to determine if they represent a risk to human health and/or the environment . In studies that examined the effects of metal oxide nanoparticles on plants, most studies have shown low to moderate toxicity, even at relatively high ENM concentrations . However, release of ENMs into the environment may have other subtle effects on plant uptake and use of important nutrients, which could alter growth and development. For example, nitrogen is one of the most important nutrients since it is an essential component of amino acids, proteins and nucleic acids, including the carboxylating enzyme involved in photosynthesis . Although many forms of N occur in soils, not all forms are available to plants. In addition, microbial processing of N affects pools and fluxes of N in soils. Understanding the effects of ENMs on factors such as N uptake and metabolism is important not only to understand plant growth and development, but also for understanding how ENMs may affect ecosystem processes. Previous studies showed the impacts of heavy metal on N metabolism in plants, and it is possible that metal oxide nanoparticles could influence N uptake. Sutter et al. reported that Cd, Pb, and Zn decreased 15N abundance in aquatic moss while Schmidt et al. found that As or As significantly decreased 15N incorporation in Silene vulgaris. These researchers found that metals affected N uptake and protein synthesis which resulted in decreased metabolic activity of plants. We also reported decreases in 15N/14N ratio of wheat treated with cerium oxide nanoparticles , but did not find whether the isotopic changes occurred in the soil, the root rhizosphere, or after N uptake through changes in root or shoot metabolism . In order to help isolate the mechanisms underlying changes in N uptake and/or metabolism in response to ENM exposure, we used hydroponic systems to allow us to control the forms and isotopic ratios of N supplied to the roots, and to minimize the influence of soil interactions external to plant roots.

We selected CeO2-NPs since they are widely used in many technological applications that could reach the environment and interact with terrestrial/agricultural plant species . In this study, the influence of CeO2-NPs on nitrogen metabolism of different forms of N in wheat and barley was explored. The hypotheses were 1) CeO2-NPs do not alter uptake of N or growth in wheat and barley regardless of the form of N supplied, i.e., NO3 – , NH4 + or NH4NO3, 2) shifts in the isotopic ratios of N in leaves and roots in response to the different forms of N supplied are not influenced by CeO2-NPs exposure, and 3) wheat and barley show similar isotopic ratios in response to the different N forms and to CeO2-NP exposure. We chose to study N because CeO2-NPs modified N and 15N abundance in wheat , and we chose wheat and barley because these species vary in response to CeO2-NPs exposure, possibly indicating different modes of action . We tested 500 mg CeO2- NPs/L because this exposure level in soil altered roots, shoots, and grains δ 15N in wheat .The nanomaterial suspensions were prepared as previously described in Rico et al. . The nutrient solution was placed in 150-mL plastic jars . CeO2-NPs were added to the solution then sonicated for 30 mins at 20⁰C with occasional stirring. After sonication, the jars were covered with caps that had three holes where cuttings from 3 mL plastic pipette were fitted to hold two plants and air pumps . Air was constantly supplied using air pumps. All materials used for the hydroponic experiment were sterile and soaked in 10% hypochlorite solution before use. Two nine-day-old wheat or seven-day-old barley seedlings were grown in nutrient solution in growth chamber set at 16-h photoperiod, 20/10˚C, 70% humidity, 300 μmol/m2 -s. At harvest, root and shoot were separated, washed thoroughly with Milli-Q water. After drying in the oven, total biomass was measured. Plant materials were ground and subjected to N and 15N analysis. The table of two-way ANOVA with CE and NS as main factors is presented in Appendix C . For wheat, NS was significant for all parameters measured while CE was significant only for every biomass, N contents, and δ 15N measurements . In barley, NS was also significant for all parameters except root N concentration, whereas CE was significant only for total shoot and plant N contents, root N concentration, and root δ 15N . The global mean of CE were calculated and presented in Table 1. Ce-500 did not affect N concentration but decreased biomass, and all N contents and δ 15N in wheat, but increased global mean shoot and plant N, root N concentration, and root δ 15N in barley.

Our results led to rejection of all three hypotheses tested in this study. N uptake and metabolism plays a central role in all cellular functions in plants, and the shifts observed here in response to CeO2-NP exposure indicate that ENMs have the potential to alter how important nutrients such as N are utilized in plants, even when toxicity is not evident. Our hydroponic experiments removed the chemical and biological complexity of interactions that occur at the root-soil interface in soils in order to better understand possible mechanisms underlying changes in N uptake and metabolism in the two species. Additional studies will be needed to examine CeO2-NP and N interactions in soil-grown plants, and to evaluate the longer-term consequences of changes in N dynamics in plants exposed to CeO2-NPs. CeO2-NPs did not affect the root influx of inorg from NH4 + since total plant and shoot N contents increased without changes in biomass and N concentration . However,vertical growing systems the very high whole-plant and shoot δ 15N discriminations in Ce-500 coupled with its low root δ 15N compared to Ce-0 strongly suggest low influx of 15N into the roots or high efflux of 15Nenriched inorg to outside roots . A previous study showed that discrimination in NH4 + uptake could result from efflux of 15N-enriched NH4 + from inorg . This could happen when NH4 + gets assimilated immediately in the roots which increases the pool of 15N-enriched NH4 +, then the 15N-enriched NH4 + are transported out of the roots. Due to its toxicity, NH4 +, generally is rapidly assimilated or flushed out of the roots . In contrast, data seem to suggest that CeO2-NPs decreased root to shoot translocation of 15N when the wheat seedlings were grown in NO3 – because root δ 15N increased to similar level with shoot δ 15N despite decreased shoot N content and a lack of net change in whole-plant δ 15N discrimination . Wheat was a good discriminator of 15N when the N source was NH4NO3 as shown by notable whole-plant and shoot δ 15N discriminations in Ce-0. Exposure to CeO2-NPs only increased root δ 15N discrimination despite remarkable decreases in biomass production and N content. The decrease in root δ 15N was probably due to the discrimination against 15N similar to what was observed in wheat in NH4 +. It is also probable that shoot δ 15N from Ce-0 and Ce-500 was from δ 15N of the source NO3 – , and the decrease root δ 15N was due to discrimination against 15N from NH4 +. Previous reports also showed that lower N uptake decreased 15N abundance in Cd-treated aquatic moss . In another report, alfalfa plants that exhibited impaired growth features when subjected to high carbon dioxide concentration and water deficiency had negative leaf δ 15N values . In this study, N concentration in both shoot and root did not differ between Ce-500 and Ce-0 indicating that CeO2-NPs did not affect N uptake and that reduced total N content was due to low plant biomass. It is possible that low biomass was due to reduction in macromolecules such as fatty acids and lignins similar to what was observed in rice seedlings exposed to CeO2-NPs . The synchrotron micro-XRF analysis showed that both wheat and barley translocated CeO2- NPs to the shoots regardless of N source in the growth media . We have shown in previous hydroponic studies the uptake of Ce in wheat and barley seedlings , which corroborates the plant uptake of CeO2-NPs recorded in the current study. Our data on speciation is in agreement with data normally reported in the literature regarding the accumulation of CeO2-NPs in plants grown in hydroponic culture solution . Unfortunately, the data does not allow further speciation analysis to determine which part of the plants the reduction occurred. Our findings also revealed that CeO2-NPs were translocated to the shoots suggesting an uptake of CeO2-NPs in barley plants; however, barley seedlings did not exhibit decreases in biomass or 15N uptake . More studies should be performed to understand why CeO2-NPs markedly disturbed N or 15N uptake in wheat than barley. Copper and its compounds have been known to have the ability to inhibit fungi since ancient times and have been used widely in agriculture as fungicides,algaecides,pesticides,and herbicides.There are at least 209 pesticide products registered in California that use copper oxide as an active ingredient.In addition, due to steady increase of drug resistance of bacteria, synthesis and application of novel antibacterial/anti antifungal Cu nanoparticles has increased.Besides antibacterial applications, Cu NPs also have application as additives of livestock and poultry feed.There is increasing concern about the potential for bio-accumulation and toxicity of Cu NPs after their release to the environment. It has been shown in several studies that nano-Cu triggers reactive oxygen species generation and induces oxidative stress in cells, bacteria, and zebrafish.However, very few studies have focused on the toxicity of Cu NPs on terrestrial plants, especially crop plants. Lee et al.,documented that Cu NPs are toxic to mung bean and wheat at concentrations of 335 and 570 mg/L, respectively. Hong et al.reported that even at the level of 5−20 mg/L, Cu NPs significantly reduced the root length of alfalfa and lettuce and altered their nutrient uptake.