Author: ecogrow

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.

Naturally occurring methyl iodide can also cause the abiotic methylation of phenolic contaminants

Demethylation is a common phase I metabolism typically catalyzed by cytochrome P450 enzymes that are ubiquitous in humans, terrestrial organisms and aquatic organisms. Sometimes demethylation can also occur via the catalysis of esterase or non-enzymatic hydrolysis for -COOCH3 and result in the formation of carboxyl groups.Methylation of CECs, on the other hand, is a phase II metabolism typically catalyzed by methyltransferases.Various substrates are susceptible to the activity of methyltransferases, such as nucleic acids, lipids and many xenobiotics.Unlike other phase II metabolism, methylation usually leads to increased hydrophobicity, but it is considered a detoxification pathway in most cases.64 Methylation and demethylation are among the most common transformations for CECs in the environment. For example, triclosan was methylated during the WWTP treatment, and the derived methyl triclosan was frequently detected in TWW along with triclosan, sometimes at even higher concentrations.Acetaminophen was reported to methylate during microbial degradation in soil. TBBPA monomethyl ether and dimethyl ether were frequently detected in the environment along with TBBPA, sometimes at higher concentrations. TBBPA MME and TBBPA DME were also formed through abiotic methylation in the natural presence of methyl iodide in aquatic environments.Biotic methylation of TBBPA was also observed to occur through biologically mediated transformations in sediments,earthworms ,vertical tower planter and plants. Methylation of BPA was promoted by Mycobacterium strains like PYR-1 and PCP1.Methylation of diclofenac was observed in aquatic invertebrates.

Demethylation of common CECs has been previously reported as well, such as the O-demethylation of naproxen in humans, terrestrial plants, microbes and soils, and the N-demethylation of diazepam in humans, terrestrial plants and aquatic organisms.Several studies have also shown the demethylation of methylated CECs back to the parent compound. For example, methyl triclosan was back converted to triclosan in A. thaliana and fish.Demethylation of TBBPA DME and TBBPA MME back to TBBPA was observed in pumpkin plants. The back conversion of diclofenac methyl ether in aquatic invertebrates was also reported.As an important type of TPs, methylated CECs are usually more hydrophobic than their corresponding parent compounds, and therefore may pose increased ecological risks. For example, consistently higher concentrations and BCFs of methyl triclosan, as compared with triclosan, were observed in fish, snails and algae collected from TWW-impacted streams.BPA mono- and dimethyl ether were more toxic to the development of zebrafish embryos than BPA itself.Diclofenac methyl ether showed greater bioaccumulation and further higher acute toxicity to H. azteca and G. pulex than diclofenac.However, exceptions exist to this general rule. For instance, the methyl ethers of TBBPA were less toxic to zebrafish development and earthworms in terms of acute exposure.Lower bio-accumulation factors of methyl triclosan than triclosan was reported in algae .These studies suggested that changes in environmental behaviors of CECs induced by methylation were molecule-specific. The investigation of back-and-forth conversion between methylation and demethylation of CECs is important to obtain a more complete understanding of the environmental cycling of CECs. This previously neglected conversion circle implies prolonged persistence of such contaminants in the environment. This transformation circle needs to be further investigated for more comprehensive and accurate risk assessment for CECs that are susceptible to such reactions.

In addition, research is needed to quantitatively evaluate differences in non-target toxicity between CECs and their methylated or demethylated derivatives. Although methylation and demethylation of several CECs in agroecosystems and aquatic invertebrates were studied previously,our overall knowledge of the transformation potential, fate and ecological risks of these methylated or demethylated TPs is limited. Many CECs contain active functional groups such as hydroxyl, methoxyl, carboxyl, ester and amide groups in their chemical structures, making them susceptible to methylation or demethylation under biotic and abiotic conditions. However, the specific molecular properties that promote methylation or demethylation need to be better understood. For example, methyltransferases in plants are involved in many important metabolic activities;therefore, the similarity in chemical structures to the endogenous biomolecular substrates may influence the potential for methylation of xenobiotics in plants. The bond strength of the chemical bond connecting the methyl group and the major fragment may also affect the potential for demethylation of CECs. Furthermore, methylated or demethylated TPs may be demethylated or methylated back to the parent CECs, respectively.This previously ignored metabolic circle may effectively prolong the persistence of CECs in the environment and lead to unrecognized environmental risks. Changes in environmental behaviors induced by methylation or demethylation are poorly understood with few experimental observations. Methylation of CECs can lead to increases in bio-accumulation, and acute and developmental toxicity in organisms,while exceptions also exist.Demethylation of CECs also does not necessarily lead to lower bio-accumulation and toxicity.Methylation and demethylation may also affect the transport, translocation and persistence of CECs by inducing changes in their hydrophobicity, solubility and pKa. The fate of demethylated and methylated TPs of CECs in agroecosystems and aquatic environments warrants a more systematic evaluation.

Given the large and ever-increasing number of CECs in the environment, it is unrealistic to experimentally investigate all CECs, let alone their methylated and/or demethylated TPs.The incorporation of modeling tools, such as QSARs and models based on the use of molecular descriptors, can help predict environmental behaviors and provide an alternative way to assessing the risks of CECs and their TPs. Such modeling approaches need to be validated and refined using experimental data. In conclusion, changes in the environmental behaviors of CECs induced by simple transformation reactions such as methylation and demethylation need to be systematically explored through rigorously designed experiments. The experimental data should be further incorporated into existing models to help validate the utility of models and also allow the prediction for a wide range of CECs for which experimental data may never be available. Contaminants of emerging concern refer to contaminants that are recently discovered in the environment and may pose potential adverse effects, such as developmental toxicity and endocrine disruption, to non-target organisms and human health at environment-relevant concentrations. Because of their widespread use, CECs are ubiquitously present at trace levels in treated wastewater and bio-solids. Many CECs contain reactive functional groups such as hydroxyl, carboxyl, and amide, making them susceptible to biotic and abiotic transformations during treatment at wastewater treatment plants. Therefore, in addition to the parent form of CECs, transformation products are also often present in treated wastewater and bio-solids, sometimes at even higher concentrations . Treated wastewater and bio-solids have been increasingly applied to agricultural lands in recent years in beneficial reuse practices, which serves as a conduit for plants to be contaminated with CECs and their TPs, posing potential human health and ecological risks . Methylation and demethylation are among the most common transformation reactions for many CECs. Biotic demethylation is a phase I metabolism process facilitated mainly by cytochrome P450 enzymes that are ubiquitous in organisms. For example, as a common pharmaceutical itself, nordiazepam is also a demethylated metabolite of diazepam excreted after oraladministration in humans . Likewise, demethylation can convert naproxen to O-desmethyl naproxen , and methylparaben to 4-hydroxybenzoic acid through microbially mediated phase I metabolism. Abiotic demethylation of herbicides and some CECs was also observed after advanced oxidation processes during wastewater treatment. Therefore,lettuce vertical farming demethylated counterparts are among the most commonly observed TPs of CECs. Biotic methylation is a phase II metabolism mediated by methyltransferases. Methylated acetaminophen, i.e., pacetanisidide , is a major metabolite of acetaminophen in soil. Methyl triclosan is the primary TP of the antimicrobial triclosan after WWTP treatment. Tetrabromobisphenol A , a brominated flame retardant, was found to be O-methylated by microbes, as well as in pumpkin plants and earthworms. The addition or loss of a methyl group during transformations alters a compound’s physicochemical properties 31, which may subsequently affect its fate and risk in the environment. As uptake of CECs into plants is known to depend closely on a chemical’s physicochemical properties, such as lipophilicity, it may be hypothesized that methylation or demethylation changes a chemical’s behavior and fate in the soil plant continuum.

Despite their frequent occurrence, the environmental significance of simple transformation reactions such as methylation and demethylation is often overlooked. In this study, we compared plant accumulation and translocation of four pairs of compounds differing only in a methyl group in their structures using two plant models, Arabidopsis thaliana cells and wheat seedlings. Four CECs and their respective methylated or demethylated counterparts were chosen as the test compounds because of their widespread use and occurrence in the environment . Of these compounds, DM-diazepam is not only a TP of diazepam, but also a pharmaceutical itself, and DM-methylparaben is not just a TP of methylparaben, but also an industrial raw material. Results from this study contribute to a better understanding of the implications of simple transformation reactions such as methylation and demethylation on the environmental behavior and potential risks of CECs.All analytical standards were purchased with reported purities ≥ 98%. Acetaminophen, diazepam, DM-diazepam, d5-diazepam , naproxen, DM-methylparaben and methylparaben were purchased from Sigma-Aldrich . M-Acetaminophen was purchased from Santa Cruz . DM-naproxen and d4-methylparaben were purchased from Toronto Research Chemicals . d4-Acetaminophen and d3-naproxen were purchased from C/D/N Isotopes . HPLC-grade methanol, acetonitrile, methyl tert-butyl ether and acetone were purchased from Fisher Scientific . Ultrapure water was generated by an in-house Milli-Q water purification system . Radioisotope labeled compounds were not used in this study, and therefore the uptake efficiency or mass balance of the target compounds in plants could not be derived. The A. thaliana cell suspension was obtained from the Arabidopsis Biological Resource Center at Ohio State University and was maintained in the laboratory at 24 °C and 130 rpm in NT-1 media with constant light . An aliquot of the A. thaliana cell culture was added to fresh, autoclaved NT-1 media , and incubated for 3 d, after which each cell suspension was spiked with individual compounds to arrive at an initial concentration of 1 mg/L. Control treatments included positive and negative control groups containing CECs spiked in nonviable cells , CECs in blank culture solution, or viable cell culture solution without CECs. Each treatment was prepared in triplicate, and was sampled at 1, 3, 6, 11, 24, 48, and 96 h. Entire samples were transferred to polypropylene centrifuge tubes and were immediately centrifugated at 3500 rpm for 30 min. The cell matter was stored at -80 ˚C until further analysis, and the supernatant was transferred into a 40 mL glass bottle and stored at -20 ˚C until further analysis.Wheat seedlings used in this study were germinated from seeds to avoid potential background contamination. Sterilized seeds were germinated on a moist filter paper on a tray in the dark at room temperature. The tray was then transferred into a growth chamber after 2 d for seedlings to grow. When the seedlings grew to about 5 cm in height, they were transplanted into a 50-mL polypropylene centrifuge tube wrapped in aluminum foil and then cultivated in the growth chamber. Initially filled with water, the solution in the tubes was replaced to 1/4 strength and then 1/2 strength Hoagland® nutrient solution at 2-day intervals to allow wheat seedlings gradually acclimating to the nutrient media. Once acclimated, the media were replaced with 30 mL fresh 1/2 strength Hoagland® nutrient solution spiked with a single compound of interest from individual stocks to reach a nominal chemical concentration of 1 mg/L. Water was added to each tube every other day to make up the lost water throughout the incubation experiment. Triplicate containers were sacrificed at 0, 3, 6, 12, 24, 48, 96, 168 and 240 h after the treatment. Plants were rinsed with deionized water, dried with paper towels, and separated into roots and shoots. The nutrient solutions remained in the centrifuge tubes and separated plant tissues were stored at -80 ℃ until further analysis. Transpiration stream concentration factors of target compounds were not measured in wheat seedlings in this study, due to challenges in collecting adequate amount of xylem sap for analysis.Deuterated compounds were used as surrogates during extraction for QA/QC. Extraction of nutrient solutions from A. thaliana cells and wheat seedlings was carried out using a similar method to a previous study 39, with minor modifications. Briefly, 50 μL of the surrogate solution was added to a 5 mL aliquot of nutrient solution. The nutrient solution samples were then extracted by HLB cartridges .

A large fraction of the QTL were identified for differential traits

A similar pattern was observed in Feldman et al. 2017, in which the authors identified QTL on chromosome 2 at 96cM, 5 at 109cM, 7 at 99cM, and 9 at 127 cM which were associated with water use efficiency using the same A10 x B100 RIL population . Though the processes of water and ion uptake are independent, there is a strong relationship between the two; ion homeostasis depends upon transpiration rate, active transport, and membrane permeability, all of which are affected by the water status of the plant. As drought and density of planting are both variables that impact the available water supply, it appears that alteration in the water status of Setaria substantially perturbs the ionome.This additionally suggests that, while the ionome can be interrogated with individual ions, a multi-elemental approach is more likely to identify regions of the genome with weak signal, or those that evince pleiotropy.Rb is a striking example of this, with 13 of its 15 QTL identified via differential analysis. Combined with this trait’s high heritability within each experiment and low repeatability across experiments, these data suggest that there is a strong genotype by treatment component in Rb content in this RIL population. This genotype by treatment effect was apparent in many elements that were assayed, with an average of 74% of the ion specific QTL identified in the differential traits. The preponderance of differential QTL was not universal,vertical farm as within treatment mapping allowed for the identification of 50% or more of the QTL found for Mo and Sr, suggesting that the homeostasis of these elements experiences a smaller degree of environmental perturbation. Several QTL identified in this study overlie genes known to be associated with the control of elemental concentration. One example is Sevir.

G251200, the ortholog of MOT2 in A. thaliana. MOT1, a MOT2 paralog, is responsible for a large fraction of the variation in Mo content in A. thaliana . Although MOT1 has an ortholog in S. viridis on chromosome 9, it was not identified by any of the QTL in this study. This finding suggests that either 1) the RIL population contains allelic diversity in MOT2 that is not present at the chromosome 9 locus or 2) the MOT2 locus in Setaria is responsible for more of the variation in Mo content in this species. Additionally Sevir.5G106900, imputed from the A. thaliana gene ESB1, underlay 10 QTL, the majority of which were identified in the PC QTL mapping. ESB1 is involved in the production of the casparian strip, with mutants in A. thaliana developing increased suberin levels and disordered casparian strips, as well as altered levels of many ions. The identification of QTL in this region in both the DN13 and DR14 experiments and for several different treatments, as well as in the first principal component for both DN13 and DR14 suggests that the Setaria ESB1 ortholog plays a role in a variety of conditions related to water status. The B100 haplotype for this region produces a decrease in Mo as compared to the A10 allele, which is consistent with the relationship between the WT and the esb1 allele seen in A. thaliana. Given the central role played by the casparian strip in water homeostasis, this gene is a good candidate for explaining the coincidence of WUE and ionomic QTL. The translocation of the short arm of rye chromosome 1 from the cultivar Petkus into the long arm of wheat chromosome 1B confers improved tolerance to several abiotic and biotic stresses. Although several genes for resistance to biotic stresses are no longer effective, the1RS.1BL translocation is still widely used because of its beneficial effects on grain yield and improved abiotic stress tolerance . We have previously shown that the presence of a short segment of wheat 1BS chromosome from cultivar Pavon in the distal region of the 1RS translocation was associated with reduced grain yield, biomass, and canopy water status relative to near-isogenic lines carrying the complete 1RS chromosome arm . Carbon isotope discrimination data showed that the lines with the complete 1RS chromosome arm achieve higher yields and better water status through increased access to water throughout the season, rather than through water conservation . A subsequent field study showed that the improved water status of the isogenic lines with the 1RS chromosome was associated with enhanced root density below 20 cm relative to the lines with the 1RSRW chromosome .

Changes in root architecture in the field were correlated with drastic changes in root development in hydroponic growth systems, where the 1RSRW line showed a regulated arrest of the seminal root apical meristem ∼2 wk after germination. By the same time, the 1RSRW plants displayed altered gradients of reactive oxygen species in the root tips and emergence of lateral roots close to the RAM . In this study, we performed exome captures for 1RS, 1RSRW, and its parental lines T-9 and 1B+40 . We show that, as a result of a distal inversion between 1RS and 1BS chromosome arms, T-9 and 1B+40 have duplicated 1BS and 1RS orthologous regions in opposite orientations and that a crossover between these chromosomes resulted in a duplicated 1RS region colinear to the inserted 1BS segment in 1RSRW. Using these genetic stocks, we demonstrate that the dosage of the genes in the duplicated region plays an important role in the regulation of the seminal root growth. We also describe a radiation mutant with a deletion in the inserted 1BS segment and the adjacent 1RS region that restored long roots, confirming the importance of the dosage of the genes in this region on root development. Finally, we identified 38 genes within this deletion and used published RNA-sequencing data and annotation to discuss their potential as candidates for the genes regulating seminal root elongation in wheat.Previous field studies demonstrated that cultivar Hahn carrying the standard 1RS.1BL translocation had longer roots, better access to water, and significantly higher grain yields than isogenic Hahn lines carrying the 1RSRW chromosome . Hydroponic studies confirmed that 2 wk after germination, the roots in Hahn-1RSRW showed a significant reduction in the elongation rate, altered gradients of reactive oxygen species, and the emergence of lateral roots close to the RAM . This earlier reduction in root growth rates in 1RSRW relative to 1RS was also observed in this study, even in experiments that showed variable overall root growth responses . We initially assumed that the 4.8 Mb 1BS segment in the 1RSRW chromosome arm was the result of a homologous recombination event between the overlapping 1BS segments of lines T-9 and 1B+40  and that, therefore, the 1BS wheat genes have replaced the orthologous 1RS rye genes.Given the known positive effect of the 1RS translocation on drought tolerance in wheat, we hypothesized that the lost 1RS genes were the cause of shorter roots in 1RSRW. However, the exome capture sequencing of 1RS and 1RSRW demonstrated that both the 1BS and its orthologous 1RS segment were still present in 1RSRW, disproving our original hypothesis.

Our second hypothesis was that wheat genes present in the 4.8-Mb 1BS segment inserted in 1RSRW could be responsible for the shorter roots. However, the characterization of the Hahn-T-18 line,nft vertical farming which carries a 17-Mb distal 1BS segment and has no identifiable duplications, provided evidence against this hypothesis. The roots of T-18 were slightly longer than those in 1RS at the initiation of the measurements but showed no significant differences in their root growth rates after that day . When the 1BS segment was combined with the 1RS segment in the Hahn-T-21 and Hahn-1B+40, the roots were significantly longer than the roots of 1RSRW and slightly, but not significantly, shorter than the roots in the control 1RS line . Taken together, these results provided conclusive evidence that the presence of the wheat genes in the 1BS segment alone was not responsible for the short roots 1RSRW and disproved our second hypothesis. Our third, and still current, hypothesis, is that the change in gene dosage generated by the duplications of the 1BS and 1RS colinear regions was responsible for the arrest in the seminal root growth. The lack of differences in root growth rate between T-18 and 1RS between 9 and 28 d suggest that the genes in the 1BS segment are not responsible for the reduced growth rate in 1RSRW during the same period . The 1BS-1RS duplication in T-21 and 1B+40 resulted only in a minor decrease in growth rate relative to 1RS and their final root lengths were significantly longer than in 1RSRW . As T-21 tended to be shorter than 1B+40 in both experiments, we cannot rule out the possibility that their different proximal regions may contribute to modulate the effect of the 2R+2B duplication on root length. These results suggest that adding duplicated 1BS genes has a smaller effect on seminal root growth than adding more copies of the 1RS orthologues. The stronger effect of the 1RS segment was evident in plants heterozygous for the 1RSRW chromosome , which showed seminal root length intermediate to that of 1RS and 1RSRW . Based on this result, we hypothesize that the duplication of the 1RS region in 1RSRW is the main driver for shorter roots in this line, but we do not entirely discard the idea that the genes in the 1BS segment may also contribute to the reduced root growth when combined with additional 1RS orthologues. The dosage hypothesis was reinforced by the hydroponic experiments with the radiation-mutants 1RSWW-del-8 and 1RSWW-del-10 back crossed independently to both to Hahn- 1RSRW and Hahn-1RS. In the hydroponic experiments using the back cross lines segregating for the deletions and 1RSRW, the roots of the deletion lines were significantly longer than those of the sister lines carrying at least one 1RSRW chromosome .

By contrast, in the lines segregating for the deletions and the 1RS chromosome, we observed no significant differences in root length between the homozygous deletions and their sister lines carrying at least one 1RS chromosome . The four consecutive back crosses of 1RSWW-del-8 and 1RSWW-del-10 into 1RSRW and 1RS minimized the chances of a possibly confounding effect of independent deletionsin other chromosomes of the radiation mutants. However, they did not rule out the possibility of a confounding effect of a linked deletion in 1RS. Using the exome capture, we did find a linked missing 1RS region corresponding to the orthologous rye region replaced by the proximal wheat segment in homozygotes for the 1RSWW chromosome. We have previously shown that the proximal wheat segment has no effect on root length and confirmed this result in the hydroponic experiments presented in this study . The exome capture data also allowed us to determine the length of the 1RS deleted segment in the deletion lines and to establish that the 1BS and 1RS deletions include mostly orthologous genes . Therefore, the homozygous 1RSWW-del-8 and 1RSWW-del-10 lines are expected to lose two gene copies in 1BS and two in 1RS, changing the gene dosage from 4R+2B to 2R. This hypothesis explains the identical seminal root size observed in the 1RS and the homozygous deletion lines . One limitation of the exome capture assays is that they are closed systems and some genes are not included, which resulted in annotated genes with no reads. We eliminated those genes for the analysis used to delimit the borders of the 1RS–1BS recombination events or of the duplicated 1RS region . This likely resulted in a slight overestimate of the size of the candidate gene regions and the number of potential candidate genes.Once we established conservative borders of the 1BS and 1RS deleted regions in 1RSRW-del-8/10, we considered all the annotated genes in these regions as candidates regardless of their presence in the exome capture. The 1RSAK58 genome is very close to the 1RS present in our lines, so it probably provides a good representation of the rye candidate gene region. However, the CS RefSeq 1.1 used as 1BS reference is not identical to the 1BS Pavon segment, and therefore, we cannot rule out the possibility of genes present in Pavon that are not present in the wheat reference.

A large amount of NPs were found aggregated at left bottom of the root

The poor translocation of the selected PPCP/EDCs from roots to leaves may be attributed to several factors. The compounds considered in this study have moderately high hydrophobicity with log Kow from 3.35 to 4.48 . Translocation of organic compounds within plants generally decreases with increasing hydrophobicity . Also, roots have higher lipid content than most other plant tissues, and neutral compounds have been shown to be preferentially distributed in tissues with high lipid content . In addition, the rapid conversion of 14C residue to the non-extractable form, as discussed above, may be another important factor for the negligible transfer from roots to other plant tissues.The use of 14C labeling, while giving unique information such as the total chemical accumulation in plant tissues, did not provide insights on the chemical composition of the accumulated residue. It is likely that some of the PPCP/EDCs were transformed in the nutrient solution before they were taken up by plants. The used nutrient solution from hydroponic cultivation was subjected to fractionation on HPLC to characterize the portions of 14C existing as parent compound and transformation products . It is evident that different PPCP/EDCs were transformed to different degrees in the nutrient solution and the presence of plants generally enhanced the transformation. In the no-plant control of DCL and NPX, the majority of 14C was in the form of the parent compound , while the percentage of 14C in the SPE aqueous filtrate or eluted on HPLC prior to the parent compound was very small . The presence of lettuce or collards did not increase the transformation of DCL or NPX, with the exception of the DCL-collards treatment,stackable flower pots where 93.8 ± 6.2% of the recovered activity was detected in the SPE aqueous filtrate.

In contrast, BPA and NP were extensively transformed, even in the absence of plants, and transformation was accelerated in the presence of a plant. For example, 50.3 ± 24.3% of the recovered 14C was identified as the parent in the BPA no-plant control, but collards and lettuce treatments had no detectable BPA. In the presence of a plant, 14C was detected in the aqueous filtrate and in HPLC eluent prior to the retention time for BPA . Extensive transformation of NP was also observed; all of the 14C from lettuce or collards cultivation was found in the aqueous phase of the extraction . The fraction of activity in aqueous phases may be attributed to transformation products that were not retained by the HLB cartridge or solvent phase during solvent extraction . Preliminary experiments showed that an average of 93.6% of 14C-BPA, 84.5% of 14C-DCL, and 92.0% of 14C-NPX were recovered from the HLB cartridges and 97.8% of the spiked 14C-NP was recovered in the solvent phase, while the activity in aqueous phases were below detection. Therefore, 14C in the SPE aqueous filtrate for BPA, DCL, and NPX, or in the aqueous phase for NP, was likely from polar transformation products containing the 14C label. The detection of transformation products in used solution suggests that some of the 14C found in plant tissues may be from transformation products formed in the nutrient solution prior to plant uptake. The demonstrated accumulation of PPCP/EDCs into leafy vegetables suggests a potential risk to humans through dietary uptake. To assess whether the concentrations detected in plant tissues in this study may present a potential human health risk, an individual’s annual exposure was estimated using values from the U.S. Environmental Protection Agency for average daily consumption of leafy vegetables . The annual exposure values ranged from 0.32 × 10−3 mg for BPA-lettuce to 2.14 × 10−2 mg for DCL-collards for an average, 70 kg individual residing in the United States.

To place these amounts in context, the values were then converted to either medical dose or 17β-estradiol equivalents. Both DCL and NPX are commonly available non-steroidal anti-inflammatory pharmaceuticals. Based on typical doses and the observed plant concentrations, an average individual would consume the equivalent of much less than one dose of these medicines in a year due to consumption of leafy vegetables, representing a very minor exposure to these PPCPs. However, it should be noted that DCL has proven ecotoxicity and NPX has shown toxicity in mixture with other pharmaceuticals , so a simple estimation may not encompass all possible human health effects. Both BPA and NP are industrial products known to have endocrine disrupting activity. Bonefeld-Jørgensen et al. calculated the Relative Potency of these compounds as compared to 17β-estradiol , an endogenous estrogen hormone, at activating estrogenic receptors. In Table 4, the exposure values of BPA and NP were estimated as E2 equivalents by dividing by their Relative Potency . When the calculated E2 equivalents of BPA and NP are compared with the Lowest Observable Effect Concentration for E2 , it is obvious that the even the highest expected annual exposure to these compounds by consuming leafy vegetables would not reach the LOEC. This rough calculation suggests that consumption of vegetables would be unlikely to influence an individual’s overall endocrine activity, though caution should be used when considering risk to susceptible population groups. Moreover, it must be noted that the use of hydroponic cultivation likely resulted in greater plant accumulation of these PPCP/EDCs, in relation to soil cultivation, due to the absence of chemical sorption to soil organic matter and minerals.

This likelihood, when coupled with the fact that most of the 14C in plant tissues was in the non-extractable form, implies that the actual plant accumulation of these PPCP/EDCs by leafy vegetables grown in uncontaminated fields irrigated with reclaimed water may be negligibly small. On the other hand, bio-solids have been shown to contain some PPCP/EDCs at much higher concentrations than treated wastewater and plant uptake from soil amended with may pose an enhanced human exposure risk. Also, given that many PPCP/EDCs may be preferentially distributed in plant roots as compared to above-ground tissues , the potential risk may be significantly greater for root vegetables such as carrots, radishes, and onions. The occurrence of these and other PPCP/EDCs in leafy and root vegetables should be evaluated in the field under typical cultivation and management conditions. Engineered nanoparticles have attracted great interests in commercial applications due to their unique physical and chemical properties. Increased usage of ENPs has raised concerns in the probability of nanoparticles exposure to environment and entry to food chain. The potential health and environmental impact of ENPs need to be understood. Plants are essential components of ecosystems and they not only provide organic molecules for energy but they can also filter air and water, removing certain contaminants. Definitively, plants play a very important role in uptake and transport of ENPs in the environment. Once ENPs are uptaken by plants and translocated to the food chains,tower garden they could accumulate in organisms and even cause toxicity and bio magnification. Nanoparticles are known to interact with plants and some of those interaction have been studied to understand their potential health and environmental impact, including quantum dots, zinc oxide, cerium oxide, iron oxide, carbon nanotubes, among others. The uptake of various ENPs by different plants was summarized in Table 1. Nanoparticles are known to stimulate morphological and physiological changes in several edible plants. Hawthorne et al. noted that the massof Zucchini’s male flowers were reduced by exposed to CeO2 NPs. Quah et al. observed the browner roots and less healthy leaves of soybean treated by AgNPs, but less effects on wheat treated under same condition. Qi et al. reported that the photosynthesis in tomato leaves could be improved by treated with TiO2 NPs at appropriate concentration. Yttrium oxide ENPs have been broadly used in optics, electrics and biological applications due to their favorable thermal stability and mechanical and chemical durability. One of the most common commercial applications is employed as phosphors imparting red color in TV picture tubes. The environmental effects of yttria ENPs have not been reported. Even though the effects of certain NPs have been studied on several plants, the uptake, translocation and bio-accumulation of yttria NPs in edible cabbage have not been addressed until this study.

This plant species was chosen and tested as part of a closed hydroponic system designed to study nanoparticles movement and distribution in a substrateplant-pest system as a model of a simple and controlled environment. The final test “substrate” used was plain distilled water , in which the tested NPs were mixed. In order to observe the translocation and distribution of ENPs in plants, transmission electron microscopy has been one of the most commonly used techniques to identify the localization at cellular scale in two-dimensions , because it can be used to observe all kinds of ENPs. On the other hand, ENPs with special properties, such as upconversion NPs and quantum dots with a particular band gap can be studied with a confocal microscope with alternative excitation wavelengths to trace the ENPs. Several synchrotron radiation imaging techniques exploiting high energy X-ray have become widely used in plant science, which can measure both spatial and chemical information simultaneously, like micro X-ray fluorescence and computed tomography. In this research, we use synchrotron X-ray microtomography with K-edge subtraction to investigate the interaction of yttria NPs with edible cabbage. By using the KES technique, the µ-XCT can not only detect the chemical and spatial information in 3D, but also analyze the concentration of target NPs. The uptake, accumulation, and distribution mapping of yttria NPs in both micro scale and relatively full view of cabbage roots and stem were investigated. We found that yttria NPs were absorbed and accumulated in the root but not readily transferred to the cabbage stem. Compared with yttria NPs, other minerals were observed along the xylem in both cabbage roots and stem. To the best of our knowledge, few reports have studied the impact of yttria NPs on cabbage plants. In addition, by using µ-XCT with KES technique, the distribution and concentration mapping of nanoparticles in full view of plant root have not been previously reported.The µ-XCT was carried out at Beamline 8.3.2 at the advanced light source, Lawrence Berkley National Laboratory. From scanning energies of 16.5 to 17.2 keV, below and above yttrium K-edge, the X-ray attenuation coefficient sharply increases by a factor of 5. Other elements decrease slightly in their attenuation coefficients over this energy range. The localization of yttria NPs can be identified by the subtraction between two reconstructed image datasets , shown in Fig. 2. The slices collected above and below the K-edge were set with same brightness and contrast settings to fairly compare with each other. The grayscale values of reconstructed slices represent the absorption coefficient; therefore, thebright regions in subtracted slice denote the localization of yttria NPs . Other elements appear dark in subtracted slice marked with a red “▲” . These are inorganic elements which support the growth of cabbage. Some biological structures suffered radiation damage during scanning, resulting in a small amount of shrinkage. The bright regions circled in Fig. 2c were caused by such shrinkage, resulting in a registration mismatch between the images above and below the edge. To identify and map the distribution of yttria NPs, an image segmentation protocol was employed that could highlight regions with yttria without finding these regions corresponding to sample shrinkage. The detailed segmentation process is given in the “Method” section.By using K-edge subtracted image technique with Monochromatic X-ray tomography, the translocation and distribution of NPs in the cabbage root is clear . Figure 3a and b were constructed by 17.2 keV and 16.5 keV reconstructed slice datasets, respectively. Their color maps were based on the transverse slice pixel values/absorption coefficients over the range from 0.2 to 17.8 cm−1 . An obvious difference between 17.2 and 16.5 keV visualization in absorption coefficient of yttria NPs was observed. The distribution of yttria NPs in root was segmented and colored in red . Since yttria NPs were not water-soluble, the water that contained them was kept in constant movement with an air pump working 24/7. However, it seems that the dense roots formed a web-like structure that made the suspended NPs to accumulate and aggregate among the roots.

The model’s applicability was demonstrated with a simple case study

The 2C model does not capture other observed dynamics, such as the down regulation of the Agr system and its role in biofilm formation.These hypotheses predict very different carrier and response probabilities. This is because the former assumes that the adapted state is unaffected by resident microflora while the latter assumes that the adapted and un-adapted states are equally affected. The truth is likely in the middle, i.e., SA in the adapted state are affected by resident microflora to a lesser extent than SA in the unadapted state. Such an approach was not pursued in the spirit of avoiding over-fitting given the limited data. We note that more data collected within the first day of inoculation will help judge the quality of the hypotheses presented in this study, which need to be evaluated on an absolute scale with a goodness of fit test. Confidence in the predictions of the model will improve as more data is gathered, either supporting or refuting the hypotheses. The mechanistic nature of the model enabled direct simulation of repeated exposures from the environment, without having to assume independence between exposure events. This paves the way for more involved modeling efforts such as accounting for healthcare workers and other hospital surfaces that contain MRSA. Such efforts can be challenging for two reasons: the availability of high quality data to model behaviors and the computational effort in simulating stochastic systems. However,aeroponic tower garden system they can supplement our understanding of the environment as a source of MRSA and help devise the most effective control measures in hospitals and the community.

New root phenotyping technological developments are needed to overcome the limitations of traditional destructive root investigation methods, such as soil coring or “shovelomics” . Mancuso and Atkinson et al. provide extensive reviews on the methodological advances on non-destructive root phenotyping, including Bioelectrical Impedance Analysis , planar optodes, geophysical methods, and vibrating probe techniques. These techniques aim to mitigate key limitations of traditional root phenotyping, especially addressing the need for a better and more convenient characterization of the finer roots and root functioning. Advances in non-invasive and in-situ approaches for monitoring of root growth and function over time are needed to gain insight into the mechanisms underlying root development and response to environmental stressors. Geophysical methods have been tested to non-destructively image roots in the field. Ground Penetrating Radar approaches have been used to detect coarse roots . Electrical Resistivity Tomography and Electro Magnetic Induction approaches have been used to image and monitor soil resistivity changes associated with the Root Water Uptake . Recent studies explored the use of multi-frequency Electrical Impedance Tomography to take advantage of the root polarizable nature . Despite these advantages, geophysical methods to date share common limitations regarding root characterization. Geophysical methods developed to investigate geological media: in the case of roots they measure the root response as part of the soil response, see Fig. 1a for the ERT acquisition. Because of the natural soil heterogeneity and variability the resolution and signal characteristics of geophysical methods strongly depend on soil type and conditions. As such, interpretation of the root soil system response is non-unique, hindering the differentiation between roots of close plants and the extraction of specific information about root physiology from the electrical signals. Unlike geophysical methods, the BIA for root investigation developed to specifically target the impedance of plant tissues, limiting the influence of the growing medium.

A practical consequence is that BIA involves the application of sensors into the plant to enhance the method sensitivity. BIA measures the electrical impedance response of roots at a single frequency or over a range of frequencies . The measured BIA responses have been used to estimate root characteristics, such as root absorbing area and root mass . Estimation of these root traits is based on assumptions on the electrical properties of roots . A key assumption is that current travels and distributes throughout the root system before exiting to the soil , with no leakage of current into the soil in the proximal root position . It is only in the former case, that the BIA signal would be sensitive to root physiology. Despite the physiological relevance of the BIA assumptions and the number of BIA studies, a suitable solution for the characterization of the current pathways in roots is missing. Thus far, only indirect information obtained from invasive and time-consuming experiments have been available to address this issue . Mary et al. , and Mary et al. 2020 tested the combined use of ERT and Mise A La Masse methods for imaging grapevine and citrus roots in the field. An approach hereafter called inversion of Current Source Density was used to invert the acquired data. The objective of this inversion approach is to image the density and position of current passing from the plant to the soil. The current source introduced via the stem distributes into “excited” roots that act as a distributed network of current sources . Consequently, a spatial numerical inversion of these distributed electric sources provides direct information about the root current pathways and the position of the roots involved in the uptake of water and solutes. The numerical approach used to invert for the current source density is a key component required for such an approach. Mary et al. used a nonlinear minimization algorithm for the inversion of the current source density.

The algorithm consisted of gradient-based sequential quadratic programming iterative minimization of the objective functions described in Mary et al. . The algorithm was implemented in MATLAB, R2016b, using the fmincon method. Because no information about the investigated roots was available, the authors based these inversion assumptions and the interpretation of their results on the available literature data on grapevine root architecture. Consequently, Mary et al. highlighted the need for further iCSD advances and more controlled studies on the actual relationships between current flow and root architecture. In this study, we present the methodological formulation and evaluation of the iCSD method,dutch buckets for sale and discussits applications for in-situ characterization of current pathways in roots. We perform our studies using laboratory rhizotron experiments on crop roots. The main goals of this study were: 1) develop and test an iCSD inversion code that does not rely on prior assumptions on root architecture and function; 2) design and conduct rhizotron experiments that enable an optimal combination of root visualization and iCSD investigation of the current pathways in roots to provide direct insight on the root electrical behavior and validate the iCSD approach; and 3) perform experiments to evaluate the application of the iCSD method on different plant species and growing medium that are common to BIA and other plant studies.The relationship between hydraulic and electrical pathways has been the object of scientific debate because of its physiological relevance and methodological implications for BIA methods . A key and open question concerns the distribution of the current leakage . The distribution of the current leakage is controlled by 1) the electrical radial and longitudinal conductivities , and 2) by the resistivity contrast between root and soil. With regard to σcr and σcl, when σcl is significantly higher than σcr, the current will predominantly travel through the xylems to the distal “active” roots, which are mostly root hairs. Based on the link between hydraulic and electrical pathways, this is consistent with a root water uptake process where root hairs play a dominant role while the more insulated and suberized roots primarily function as conduits for both water and electric current . On the contrary, if the σcr is similar to σcl, the electrical current does not tend to travel through the entire root system but rather starts leaking into the surrounding medium from root proximal portions. The coexistence of proximal and distal current leakage is in line with studies that suggest the presence of a more diffused zone of RWU, and a more complex and partial insulation effect of the suberization, possibly resulting from the contribution of the cell-to-cell pathways .

Soil resistivity can affect the distribution of the current leakage by influencing the minimum resistance pathways, i.e., whether roots or soil provide the minimum resistance to the current flow. In addition, soil resistivity strongly relates to the soil water content, which, as discussed, affects the root physiology. Therefore, information on the soil resistivity, such as the ERT resistivity imaging, has the potential for supporting the interpretation of both BIA and iCSD results. Dalton proposed a model for the interpretation of the plant root capacitance results in which the current equally distributes over the root system. Because of the elongated root geometry this model is coherent with the hypothesis of a low resistance xylem pathway . Numerous studies have applied Dalton’s model documenting the predicted correlation between root capacitance and mass . In fact, recent studies with wheat, soy, and maize roots continue to support the capacitance method . Despite accumulating studies supporting the capacitance method, hydroponic laboratory results of Dietrich et al. and other studies have begun to uncover potential inconsistencies with Dalton’s assumptions. In their work, Dietrich et al. explored the effect of trimming submerged roots on the BIA response and found negligible variation of the root capacitance. Cao et al. reached similar conclusions regarding the measured electrical root resistance . Urban et al. discussed the BIA hypotheses and found that the current left the roots in their proximal portion in several of their experiments. Conclusions from the latter study are consistent with the assumption that distal roots have a negligible contribution on root capacitance and resistance. Because of the complexity of the hydraulic and electrical pathways, their link has long been the object of scientific research and debate. For recent reviews see Aroca and Mancuso ; for previous detailed discussions on pathways in plant cells and tissues see Fensom , Knipfer and Fricke , and Findlay and Hope ; see Johnson and Maherali et al. in regard to xylem pathways. See Jackson et al. and Hacke and Sperry for water pathways in roots. Thus, above discrepancies in the link between electrical and hydraulic root properties can be, at least to some degree, attributed to differences among plant species investigated and growing conditions. Among herbaceous plants, maize has been commonly used to investigate root electrical properties . For instance, Ginsburg investigated the longitudinal and radial current conductivities of excited root segments and concluded that the maize roots behave as leaking conductors. Similarly, Anderson and Higinbotham found that σcr of maize cortical sleeves was comparable to the stele σcl. Recently, Rao et al. found that maize root conductivity decreases as the root cross-sectional area increases, and that primary roots were more conductive than brace roots. By contrast, BIA studies on woody plants have supported the hypothesis of a radial isolation effect of bark and/or suberized tissues . Plant growing conditions have been shown to affect both water uptake and solute absorption due to induced differences in root maturation and suberization . Redjala et al. observed that the cadmium uptake of maize roots grown in hydroponic conditions was higher than in those grown aeroponically. Tavakkoli et al. demonstrated that the salt tolerance of barley grown in hydroponic conditions differed from that of soil-grown barley. Zimmermann and Steudle documented how the development of Casparian bands significantly reduced the water flow in maize roots grown in mist conditions compared to those grown hydroponically. During their investigation on the effect of hypoxia on maize, Enstone and Peterson reported differences in oxygen flow between plants grown hydroponically and plants grown in vermiculite. The results reported above and in other investigations are conducive to the hypothesis that root current pathways are affected by the growing conditions, as suggested in Urban et al. . For example, the observations by Zimmermann and Steudle and Enstone and Peterson may explain the negligible contributions to the BIA signals from distal roots under hydroponic conditions . At the same time, the more extensive suberization in natural soil and weather conditions could explain the good agreement between the rooting depth reported by Mary et al. based on the iCSD and the available literature data for grapevines in the field.

A potential cause for concern in the model fit is the wide intervals

To develop an adequate model to predict viral transport in plant tissue, it is necessary to couple mathematical assumptions with an understanding of the underlying bio-geochemical processes governing virus removal, plant growth, growth conditions and virus-plant interactions. For example, although a simple transport model without AD could predict the viral load in the lettuce at harvest, it failed to capture the initial curvature in the viral load in the growth medium . An alternative to the AD hypothesis that could capture this curvature is the existence of two populations of viruses as used in Petterson et al. , one decaying slower than the other. However, a closer examination of the double exponential model revealed that it was not time invariant. This means that the time taken to decay from a concentration C1 to C2 is not unique and depends on the history of the events that occurred . Other viral models, such as the ones used in Peleg and Penchina faced the same issues. The incorporation of AD made the model time invariant and always provided the same time for decay between two given concentrations. This model fitting experience showcases how mathematics can guide the understanding of biological mechanisms. The hypothesis of two different NoV populations is less plausible than that of viral attachment and detachment to the hydroponic tank. While it appears that incorporating the AD mechanism does not significantly improve viral load prediction in lettuce shoot at harvest, this is a consequence of force fitting the model to data under the given conditions. Changing the conditions, for example,hydroponic fodder system by reducing viral attachment rate to the tank wall, will underestimate virus load in the lettuce shoot in the absence of AD .

Through this model fitting exercise, we also acknowledge that the model can be significantly improved with new insights on virus plant interactions and more data on the viral transport through plants.However, there is significant uncertainty in the data as well suggesting that the transport process itself is noise prone. Moreover, from the perspective of risk assessment, the variability between dose-response models is higher than the within dose-response model variability . Since within dose-response model variability stems from uncertainty in viral loads at harvest among other factors, the wide intervals do not exert a bigger effect than the discordance from different dose response models.Some parameters are identifiable to a good degree through model fitting, but there is a large degree of uncertainty in the viral transport efficiencies and the AD kinetic parameters. While this could be a consequence of fitting limited number of data points with several parameters, the viral load at harvest and risk estimates were well constrained. This large variation in parameters and ‘usefully tight quantitative predictions’ is termed the sloppiness of parameter sensitivities, and has been observed in physics and systems biology . Well designed experiments may simultaneously reduce uncertainty in the parameters as well as predictions , and therefore increasing confidence in predictions. One possible experiment to reduce parameter uncertainty is recording the transpiration and growth rate to fit Eq. 6 independently to acquire at and bt.An interesting outcome of our analysis is the strong association of risk with plant growth conditions. The health risks from consuming lettuce irrigated with recycled wastewater are highest in hydroponic grown lettuce, followed by soil grown lettuce under Sc2 and the least in soil grown lettuce under Sc1 . This difference in risk estimates stems to a large degree from the difference in AD kinetic constants . Increasing katt, s will decrease risk as more viruses will get attached to the growth medium, while increasing kdet, s will have the opposite effect , as more detached viruses are available for uptake by the plant.

The combined effect of the AD parameters depends on their magnitudes and is portrayed in Supplementary Fig. S5. This result indicates that a better understanding of the virus interaction with the growth environment can lead to an improved understanding of risk. More importantly, this outcome indicates that soil plays an important role in the removal of viruses from irrigation water through adsorption of viral particles. An investigation focused on understanding the influence of soil composition on viral attachment will help refine the transport model. The risk predicted by this dynamic transport model is greater than the EPA annual infection risk as well as the WHO annual disease burden benchmark. The reasons for this outcome are many-fold. First, there is a significant variability in the reported internalization of viruses in plants. In research of data for modeling NoV transport in plant, we filtered the existing data using the following criteria: 1) human NoV used as the seed agent, 2) quantitative viral results in growth medium and different locations of the plant. Based on these criteria, the data from DiCaprio et al. represent the best available data on viral internalization and transport in lettuce. However, it is also important to note that a similar study by Urbanucci et al. did not observe human NoV internalization in lettuce. This discrepancy could be due to the specific subspecies of the plant and growth conditions used in the studies. In addition, minor changes such as damages in roots or decrease in humidity of the growing environment can promote pathogen internalization . Alternatively, tracking viral transport through the growth medium and plant is challenging, which may yield false results due to reaction inhibitions in genome amplification and poor detection limit. The risk outcome of this study is conservative because it assumes an individual consumes the wastewater irrigated lettuce daily for an entire year. This assumption and the corresponding higher risk estimates are only applicable to a small portion of consumers, while most consumers in the U.S. are likely to have a more diverse diet.

While the model outcomes presented here represent the best attempt given the available data, it is also possible that the internalization observed by DiCaprio et al. is an extreme case and typical internalization is lesser. As previously discussed by others , risk estimates by different NoV dose-response models differed by orders of magnitude. This study primarily aims to introduce a viral transport model without advocating any one dose-response model. We hope the future refinement of pathogen dose-response models will reduce variability in risk estimates. The risk of consuming lettuce grown in soil as predicted by SalesOrtells et al. is higher than our predictions, although the results of DiCaprio et al. were used in both studies. This is a consequence of considering the greater adsorption capability of soil, which is not reflected when assuming a simple input:output ratio. Using different inoculating concentrations of NoV, body weight and consumption rate distributions also contributed to difference in the outcomes but to a lesser extent. Parameters for crisp head lettuce were obtained from several different sources, each possibly using a different sub-variety of crips head. Yet, global sensitivity analysis showed insensitivity of risk estimates to several assumed and fitted parameters , lending confidence to the approaches taken to parametrize the model. The importance of taking the dynamics of viral transport is underscored by the sensitivity to tli, h in hydroponic and tht, s in soil cases. This suggests that given no change in lettuce consumption, changes in irrigation schedule can affect risk outcome. Such arguments were not possible with the approach of Sales-Ortells et al. . In soil grown lettuce,fodder system the high sensitivity to kp indicate the role of plant specific processes in mediating risk outcome.In addition to a transport model predicting the NoV load in lettuce, we explore the strategies to reduce the risk of NoV gastroenteritis by increasing holding time of the produce after harvesting or using bigger hydroponic culture volumes. Although neither strategy could significantly alleviate the risks, the process highlights two strengths of modeling: 1) It provides mathematical support for arguments that would otherwise be less convincing; 2) It predicts outcomes of experiments without the physical resources required to perform them. For instance, the model can be used to explore alternate irrigation schedules to reduce the NoV internalization risk. Modeling also helps encapsulate our understanding of the system and generate hypotheses. For example, simple first order decay did not produce the trend observed in the water, which suggests that additional mechanisms are at play. We postulate the attachment of virus particles on the walls of the hydroponic system as one possible mechanism and examined the fit of the model. Although viral attachment to glass or other materials has been observed before , here it stands as a hypothesis that can be tested. In addition to generating and testing hypotheses, some of our model assumptions raise broader questions for future research. For example, it was assumed that viruses are transported at the rate of transpiration from the growth medium to the roots, yet not much is known regarding the role of roots in the internalization of viruses. Investigating the defense mechanisms of plants’ roots to passive viral transport, i.e. through rhizosphere microbiome interactions, may shed light on the broad understanding of plant and microbe interactions. The question of extending this model to other pathogen and plant systems draws attention to the dearth of data in enabling such efforts. While modeling another virus may not require changes to the model, understanding transport in other plants can be challenging.

Data required includes models for growth rate and transpiration, plant growth characteristic including density, water content, as well as internalization studies to determine transport efficiencies. However, from the perspective of risk management, lettuce may be used as the worst-case scenario estimate of risk in water reuse owing to its high consumption with minimal pathogen inactivation by cooking. This worst-case scenario can be used to set water quality standards for irrigation water for production of fresh produce eaten raw. The models can also be extended to include pathogen transport to the plant tissue from manure/ biosolids that are used as organic fertilizer.It is impossible to separate the management of nitrogen fertilizer from that of irrigation water in irrigated agriculture. Methods of application, timing, and amounts applied are key concerns both for fertilization and for irrigation water application. While many experiments have characterized crop and soil responses to one variable, relatively few have endeavored to study the interaction of fertilizer management sytems with irrigation management systems. The approach taken in this project was to examine interactions between these two centrally important components of agricultural production, with the ultimate objective of improving recommendations for the use of water and fertilizers in irrigated agriculture.Both greenhouse and field trials were established, at the Agricultural Field Stations at University of California, Riverside and at South Coast Field Station, Santa Ana. Greenhouse trials were undertaken to assess basic relationships between water and nitrogen supply and uptake, while field trials emphasized use of current agricultural production technology to test relationships in the fielk. Most of the research has been published, either as graduate theses or 1n scientific journals. These publications will be referenced throughout this report, and complete details of the research can be found within them.Greenhouse trials were first conducted to determine the relationship between minimum N03 -N concentration and N and water accumulation by tomatoes and lettuce . Over a wide range of solution N03-N concentrations, the ratio of N uptake to water uptake was constant, at approximately 100 fig N/L. This suggested that a constant, continuous supply of N in the irrigation water could supply the necessary nutrient without providing an excess. Experiments in soil columns with Romaine lettuce and Swiss Chard demonstrated that Chard could very efficiently decrease the solution N concentrations to near zero before water passed out the bottom of the column; lettuce was much less efficient. Tomatoes were grown in soil columns with sealed head spaces through which acid-scrubbed air was passed. Columns were irrigated frequently with water containing 0, 50, 100, or 200 fig NIL. Half of the treatments received irrigation water dripped on the surface at the base of the plant, and the other half received it 2.5 em below the soil surface. Urea-ammonium-nitrate was the source of all N. Less than 0.1% of the applied N was trapped as volatilized NH3 , even in the “most likelytt treatment .

The implementation of an ecosystem approach would not be perfect

This is a standards based approach, similar to that used by CARB to determine additionality for its current offset protocols, because it sets a single, uniform threshold that must be met for an offset credit to be issued. As long as the average rate of sequestration was accurate, variability between different projects may not matter because the effects of carbon dioxide are largely not localized and so a reduction in one location is just as good as a reduction in another location. Therefore, it may be administratively favorable and just as environmentally effective to calculate the average acreage required to sequester one metric ton of carbon dioxide, then calculate the offset credits to be issued to each offset project based on the acreage being offered for the offset project.Different averages could be calculated for different regions, as soil type varies greatly by region.However, this approach would prove misleading if only plots with lower-than-average sequestration ability engaged in the program. This may occur if soil with higher sequestration ability can also support higher value uses than offset credit generation. If this is the case, the actual soil sequestered under the program would fall below the program’s projected average. This may be indeterminable until the value of the offset credits is established in a market, although prices within a market can always fluctuate and thus not provide the desired stability that alternative uses may provide. Many of these same problems exist in other types of offset projects which have been approved and are currently in use despite their potential accounting inconsistencies.Additionally, by using this standards-based approach, it is very likely that some projects’ carbon sequestration abilities would be lower than the offset program’s average and would be issued more offset credits than would be issued if the projects’ actual carbon sequestration had been measured. This will always be an issue with a standards-based approach, as seen in the Citizens Climate Lobby litigation. Another option is to require a variable amount of acreage per offset credit under an offset project based on how much carbon that particular land is actually estimated to sequester,ebb flow cannabis based on factors that affect a particular land’s sequestration abilities.

This project-by-project option would theoretically give a more accurate estimate of the amount of carbon sequestered by each project, and would thus give managers a better idea of whether a project has actually offset a whole carbon credit. On the other hand, it would also take much more administrative resources and time to administer due to the variability of different land and the measurements required to calculate that variability, which could hinder the offset program’s implementation.Additionally, project-by-project approaches do not always ensure accuracy. The Kyoto Protocol’s Clean Development Mechanism utilized a project-by-project approach to determine additionality, and the results were reportedly rife with error and under- or over-exaggeration when it was convenient. Experience has shown that not even a project-by-project approach will result in perfect results. However, measures can be taken to try to avoid some of the shortcomings of a project-byproject approach. For example, some have suggested replacing opportunities for subjective determinations by the project proponent or host in the project approval or crediting process with objective criteria.This would decrease the opportunities for the project proponent or host to control the outcome of the project’s approval process. Some of the measurements and logistical work could be contracted out to independent third parties at the project proponent’s expense. This practice is used by certification programs, such as the Forest Stewardship Council and the Sustainable Forestry Initiative, in order to save the certification program the resources that would be required to perform the measurements and verifications themselves. Likewise, establishing similar requirements for an offset program would decrease the resources required from CARB and should be acceptable to the project host and proponent as long as the cost is not prohibitive.Whereas a standards-based approach may make sense for determining an offset project’s additionality for the Livestock Protocol due to the protocol’s relative simplicity, its measurability, and the uncommon use of BCSs without financial incentives, these factors are not as clearly present for a potential agricultural soil carbon sequestration offset program. The amount of methane captured by a BCS digester and subsequently destroyed under the Livestock Protocol is measured by a site-specific meter and thus does not present the same difficulties and variables that exist when measuring soil carbon sequestration.The court in Citizens Climate Lobby indicated that a standards-based approach to determine additionality made sense for the Livestock Protocol because the technology was so infrequently used without the financial incentives from the offset protocol.

This line of thinking may not so clearly comport to a possible agricultural soil carbon sequestration offset program due to the existing prevalence of cropland conservation practices.To determine whether this is true of whatever region would be included in the offset protocol, CARB could commission an outside group to analyze current prevalence as they did when formulating the Livestock Protocol.Even if it was discovered that these conservation practices were generally uncommon, as with BCSs, so that additionality could be satisfied by a standards-based approach, the complications associated with other issues may be so complex and variable that a case-by-case measurement process using an ecosystem approach may still be preferable to determine whether the project’s emission reductions are legitimate and lacking in egregious incidental effects.Increased herbicide use as an incidental effect to agricultural soil carbon sequestration offset projects is unique to this type of offset program, but may also be resolved if approached from an ecosystem approach on a case-by-case basis. First, it would need to be determined whether increased herbicide use is actually a threat for the type of land that is participating in the offset program. If so, the effects of herbicide on the local resources and the increased nitrous oxide emissions should be accounted in the project’s approval process. Actively finding and implementing alternatives to herbicide use that make sense for the particular project host would alleviate the effects of increased herbicide use. One option is to replace increased herbicide use due to not ill and conservation till practices with cover crops in combination with other agricultural practices. At least one study claims that cover crops can greatly reduce the need for herbicide.Unfortunately, it seems that it is difficult to naturally replace the benefits of herbicide, as higher crop yields are reported when using herbicide instead of cover crops.Because cover crops and other agricultural practices do not seem to replicate herbicide, it would be unlikely to see a voluntary decrease in herbicide use. If herbicide use was prohibited or limited under a future offset program and no-till or conservation tillage was a major part of the program, it is likely that farmers would not be interested in participating in the program due to the difficulty or impossibility of balancing these two requirements. A better alternative may be to consider implementing a pesticide management program within the agricultural soil carbon sequestration offset project.

The pesticide management program would differ by project, as different projects would likely have different crops with different surrounding environments and site-specific needs. The pesticide management program utilized by the USDA in the Missouri River Basin study observed a decrease in herbicide use when cropland conservation practices were implemented. These practices included prevention, avoidance, monitoring, and suppression strategies to reduce pesticide use. Prevention includes measures such as using seeds and transplants that are free of pests, preventing weeds from reproducing,ebb and flow tables eliminating hosts for pests and disease organisms, and scheduling irrigation to prevent disease development.Avoidance practices include crop rotation to avoid the pest or disease, planting seeds with genetic resistance to pests, choosing crops that will mature and be harvested before pests or disease develops, and not planting in certain parts of the field that are prone to crop failure from pests and disease.Monitoring includes testing to determine crop rotation selection and when suppression activities are required.Suppression includes cultivating and temperature management for weed control, traps and exclusion devices for pest control, biological control by disrupting mating, and more deliberate and informed use of pesticides as a last resort.A well-functioning ecosystem approach to management requires research and consulting with experts from many different disciplines to construct the program and to evaluate each project on a case-by-case basis. This approach requires resources and time above and beyond what would be required for a standards-based approach, the approach currently favored by CARB in its offset protocols. Even once the experts are secured, scientists may remain too narrowly focused on their specific disciplines to do a full or fair assessment for purposes of an ecosystem approach.An ecosystem approach will identify trade offs, which can create a whole separate discussion of priorities and values that may require an extended time frame for considering any program or project evaluated under the ecosystem approach. Additionally, the court in Citizens Climate Lobby identified problems with offset programs that utilize the project-by-project analyses implicated in an ecosystem approach. Aside from being considered expensive and slow, the case-by case analysis utilized under the Kyoto Protocol’s Clean Development Mechanism is often criticized for being inaccurate due to excessively narrow or broad framing of answers to questions that are supposed to determine whether a project is actually additional to a business-as-usual scenario.Despite these drawbacks, following an ecosystem approach when considering an agricultural carbon sequestration offset program and its subsequent projects would be more meaningful and accurate. It would force decision makers to discuss whether a proposed offset program or project would be causing more overall harm than would be caused without the offset program or project and what sort of trade offs would be made.

Currently, harmful externalities of existing agricultural soil carbon sequestration offset programs seem to be ignored in at least some of the programs that implement the offset projects. For example, monitors for one soil carbon sequestration offset project noted that although herbicides are applied without considering the environmental consequences, “these activities are not part of the project under discussion.”An ecosystem approach would ensure that herbicide use and other possibly harmful externalities would be included in discussions concerning the costs and benefits of offset programs and projects. This is especially important for offset programs and projects, which are in theory neutral—trading one ton of carbon in one location for one ton of carbon or carbon equivalent in another location—and helping to implement the environmental purpose of the carbon market. Certain offset programs would garner less support if it was clear that their overall effect on the environment was a net negative. Thus, the ecosystem approach can help decision makers understand and create an agricultural soil carbon sequestration offset program with acceptable trade offs and incidental effects.Naturally occurred and fertilization-induced soil salinity, and low nutrient use efficiency are significant constraints in modern agriculture production . At the cellular level, the extrusion of Na+ ions at the cell plasma membrane and the compartmentation of Na+ into vacuoles are efficient mechanisms to avoid excessive cytosolic Na+ concentration and maintain an adequate cytosolic K+ /Na+ ratio . The plasma membrane electrical potential difference of root cells is usually maintained around 120 mV, while the tonoplast potential is positive and around 20–40 mV . These potentials allow the root to acquire sufficient K+ via high-affinity transporter systems in K+ – limited soils or via low-affinity transporter systems at normal external K+ supplies. The presence of high external Na+ concentrations suppresses the K+ conductance through LATS and competes with K+ uptake through HATS , causing a decrease in intracellular K+ with a concomitant [K+ ]/[Na+ ] imbalance. The plant vacuolar Na+ /H+ antiporters were shown originally to mediate the electroneutral Na+ /H+ exchange, driving the excess cytosolic Na+ into the vacuole . The NHX proteins belong to the large mono-valent cation/proton transporters family, showing three distinct functional clades . In Arabidopsis, in addition to the plasma membrane-located NHX7 and NHX8, also known as SOS , intracellular NHXs sharing high sequence similarity are further divided into type-I and type-II, based on their subcellular location . Type-I NHXs are vacuolar-located, while type-II NHXs are found at endosome, Trans-Golgi Network /Golgi and prevacuolar compartments .

Climate projections are not included here, but could be included in future analyses

Fragmentation and loss of farmland causes farmers to lose benefits associated with being part of a large farming community, such as sourcing inputs, accessing information, sharing equipment,and supporting processing and shipping operations . This is further exacerbated by loss of agricultural land near the Sacramento River, either due to future flooding or to mitigation of habitat for wild species. Also, by fragmenting the landscape and consuming more land area in the floodplain, urbanization in the A2 scenario could work against the provision of ecosystem services related to water quality, biodiversity conservation, open space and its aesthetic and recreational value . Strengthening the urban community’s interest and support of farmland preservation is a key challenge for mitigation of GHG emissions, and the long‐term viability of agriculture in Yolo County. Historically, urban and suburban development has covered many regions within California that were formerly leading agricultural producers, including the Los Angeles Basin and Orange County, much of the San Francisco Bay Area, and areas of the Central Valley near Fresno, Modesto, Merced, Sacramento, and Stockton. Between now and the year 2050 much additional urbanization is likely near these metropolitan areas, as well as in locations that are at a considerable distance from existing major cities, such as the Salinas Valley and Ventura County. Strategies to preserve agricultural land from urbanization are likely to dovetail with strategies to adapt to climate change and mitigate greenhouse gas emissions, reducing the state’s overall vulnerability to climate change. For example, maintaining a strong greenbelt of agricultural land around existing urban areas and adopting compact urban development policies can greatly reduce GHG emissions , while preserving agricultural production and potentially enhancing ecosystem services. This section considers urbanization implications related to agriculture and climate change,strawberry gutter system based on statewide modeling of 2050 urban growth scenarios, using existing datasets regarding agricultural production, land use, and soils.

The actual complexities of urban‐agriculture interactions require a great deal of monitoring and interdisciplinary synthesis that is beyond our scope, e.g., urban heat island or ozone effects may lead to additional vulnerabilities for agriculture with climate change. Our aim here is instead to present an initial overview of potential agricultural adaptation and vulnerability effects related to urbanization, and to suggest directions for further research. The strong policy framework in California for GHG mitigation under AB 32, the Global Warming Solutions Act of 2006, has drawn attention to the fact that California’s urban planning framework is in a state of uncertainty and potential transition. SB 375, the Sustainable Communities and Climate Protection Act of 2008, requires Metropolitan Planning Organizations within the state to prepare “sustainable communities strategies” that show how each region will meet GHG‐reduction targets through integrated land use, housing, and transportation planning. As of 2011, MPOs are just beginning to develop such plans. SB 375 is widely seen as having the potential to usher in a new era of land use planning in California, in which regional “blueprints” will be adopted to manage and reduce urban and suburban expansion . However, it is by no means clear how the California Air Resources Board or the legislature will react to ensure that such potential is in fact met. In addition, as of 2010 every county and municipality in the state must now consider GHG emissions within their General Plans and associated Environmental Impact Reports . Since 2007, the state Attorney General’s office has frequently threatened legal action against those jurisdictions that do not include planning alternatives to reduce GHG emissions . The California Air Resources Board is also strongly encouraging local governments and large institutions to prepare Climate Action Plans and GHG emissions inventories, and many have already done so.

These actions mean that local governments are now more actively exploring land use planning alternatives to mitigate GHG emissions and adapt to climate change. Although political resistance to growth management will certainly continue, such trends mean that in the future the state’s local governments are more likely to consider growth management scenarios that respond to the twin goals of preserving agricultural land and responding to climate change. This institutional and political environment affects our analysis below, and will be referred to when appropriate.To analyze the impact of future urbanization scenarios on agricultural landscapes within California within the context of climate change, we relied on modeling done by the UC Davis Information Center for the Environment using UPlan software under a separate portion of this Climate Change Vulnerability and Adaptation Study for California. We then performed additional analyses on the UPlan projections for 2050, using statewide data on agriculture, land use, and soils. UPlan is a geographic information system ‐based land use allocation model developed by ICE and used for urban planning purposes by more than 20 counties in California, including a number of rural counties in the San Joaquin Valley . It is particularly useful for large‐scale urban growth scenarios in rural areas, and has been used in a research context to analyze urbanization effects on natural resources , urbanization effects on wildfire risk , and the effect of land use policies on natural land conversion . Using UPlan, researchers first develop a base of GIS information related to geographical features such as roads, rivers and streams, floodplains, parkland, and existing urban areas. They then supply demographic inputs within future urban growth scenarios. Researchers also specify geographical features that are likely to attract urban growth , discourage growth , or prevent growth , and assign weightings to each. For example, freeway interchanges may attract development, since builders desire the locational advantages.

Designation as prime farmland may discourage development, since local governments may take this factor into account within their zoning and growth management policy making, and farmers may participate in the Williamson Act or other programs designed to discourage urbanization. Acquisition of land as public open space will prevent urbanization altogether, thus making a “mask” designation appropriate within UPlan. Relying on the combined weightings for each 50‐meter grid cell, UPlan allocates the future population increase across four residential land use types , and several nonresidential land use types . The result is a spatial projection of future urbanization with designations for each land use type. ICE staff developed two main UPlan scenarios for statewide mapping within this project scenario of urban development. The other is a “smart growth” alternative that clusters development into nodes, specifies somewhat higher densities, and places more development within existing city borders. Such scenarios reflect growth management philosophies within the state during recent decades; many local and regional planning agencies have developed similar alternatives within their own planning processes. The ICE SG scenario is relatively conservative and does not assume any dramatic changes to current planning policies. In reality, over the past two decades,grow strawberry in containers development within the state near large metropolitan areas has become increasingly compact and focused on infill sites rather than greenfield locations. The California agricultural areas most affected by urbanization between now and 2050 will not necessarily be those with the greatest overall amount of new urban and suburban development. Rather, other factors will come into play. These include the amount of agricultural base remaining within the region, the extent to which urban development fragments agricultural landscapes, and the extent to which farmers benefit from increased access to urban markets. If there is relatively little agricultural base left, as is currently the case around some of the state’s large metropolitan areas, then it becomes more difficult for farmers to find suppliers, processors, and other agricultural support functions . This may affect farm operations on a crop‐by‐crop basis. For example, there is only one processor of apples left in Sonoma County, formerly home to extensive apple orchards, and if that facility closes, then production of classic varieties such as Gravensteins will become difficult . If urban development fragments agricultural land into isolated pockets separated by roads, subdivisions, office parks, and other urban facilities, then it becomes more difficult for farmers to move equipment from field to field, and conflicts may arise with new suburban residents over noise, odor, and potential spraydrift associated with farming operations. Fragmentation may also reduce the benefits farmers receive from being part of a large farming community, such as sourcing inputs, accessing information, sharing equipment, and supporting processing and shipping operations . Impacts on agriculture from urbanization will then be disproportionate to the land area covered. On the other hand, urbanization can benefit agriculture if it increases access to markets . This factor is likely to benefit some types of agriculture more than others. Specialty production of fruits, vegetables, meats, and dairy products for use by restaurants, distribution through high‐end grocery stores, and sale at farmers’ markets and through community‐ supported agriculture networks is likely to benefit. Conversely, production of grains and lower‐ value fruits and vegetables is not likely to see a boost from the presence of local markets, since farmers primarily sell these bulk commodities to large‐scale processing facilities for regional, national, or international distribution .Addressing climate change is a priority issue for Californians and involves individuals, businesses, and government.

The Global Warming Solutions Act of 2006 seeks to reduce the emission of greenhouse gases to 1990 levels by 2020. This legislation goes into effect gradually, so that people will have time to implement the necessary actions to come into compliance by the 2020 deadline. Some businesses, however, are proactive on climate change mitigation, and are signing up through mechanisms such as the Climate Action Registry to become leaders and early adopters of GHG emission‐reduction programs. By making progress toward carbon neutrality ahead of deadlines, these companies may qualify for incentive programs and be recognized as environmental leaders. Among such leaders are a number of wine companies that are managing their vineyard lands and adjoining forests that maximize biomass on the landscape and balance the emissions generated in their production processes. This paper is a case study about one such company, Fetzer/Bonterra Vineyards, who has set their objectives to reduce their GHG emissions and use renewable sources to meet much of its energy demands. As an environmentally conscious business, and a major grower and producer of wines, Fetzer/Bonterra attempts to achieve a balance between habitat conservation, ecologically based organic production, production goals, and financial profit. When the company purchased ranches for growing grapes in Mendocino County, a decision was made to maintain a large fraction of that land in natural habitat without livestock grazing. This was based on an environmental ethic to combine wine production with conservation of the landscape’s natural integrity. This approach also included a series of sustainability measures . To learn more about the carbon storage and dynamics on its land, Fetzer/Bonterra collaborated with researchers at the University of California Davis to conduct an assessment of the distribution and magnitude of carbon stored across the vineyard‐woodland landscape. The main goal was to find a way to assess carbon stocks to determine the absolute and relative amounts of carbon stored in different vegetation and land use types. Fetzer/Bonterra’s rationale behind the assessment was to identify the relative value of the different vegetation types on their land in terms of contributing to the positive, or offset, side of their carbon budget. Because the study also collected data on the different woody plant species, information on the diversity of plant communities was obtained. The species and community diversity data make it possible to assess the relationship between carbon stocks and biodiversity, and to show how habitat type affects the magnitude of C stocks. This approach will allow vineyard managers to prioritize non‐vineyard land for carbon storage, biodiversity and habitat conservation, and eventually other types of ecosystem services, such as keeping steep slopes and stream corridors forested to protect against erosion and sediment loading in waterways. Greater carbon stocks in forests is to be expected, but it is significant to recognize that Fetzer/Bonterra uses a management approach for a combination of perennial woody crops and conserved habitat that maximizes the contribution of the heterogeneous landscape to total carbon stocks. Using this Fetzer/Bonterra case study experience as an example, this paper showcases the important role that California agricultural landscapes can play in climate change adaptation and mitigation strategies.