There were four replicate hydroponic containers for each of the six treatments

For old wood, the new polish slightly reduces the emission factor while for new wood the polish increases the emission factor slightly but the difference is likely more a function of the age of the wood than the polish. For example, given the old wood where the emissions are already low, adding the new polish may provide an apparent sink for the formaldehyde as it accumulates in the coating. In contrast, for the new wood, the initial emission rate is high and the coating may simply add a diffusion layer that increases the time that the emission from the wood paneling takes to drop to a constant level. With or without the coating, the new wood is clearly the major source of formaldehyde emissions among the materials tested. The aging of the formaldehyde emissions and the affect of the polish coating were tested further by removing the backing plate from the new wood with new polish after the initial conditioning and testing period was complete and reversing the material to expose the unfinished face. Our premise was that the formaldehyde diffuses to and accumulates at the surface of the material when the surface is covered so the initial emissions after uncovering the surface are expected to be high then drop with time towards an constant value. The results are illustrated in Figure 5. The initial test of the polished side was repeated 4 times over 15 days.

To test the emissions of aldehydes from the unfinished side of the same new wood material,pots with drainage holes the sample was flipped to expose the unfinished side and the polished side was sealed and the sample was returned to the test chamber to test the unfinished face. The first measurement found formaldehyde emissions from the unfinished face significantly in excess of the polished side but the emissions decreased rapidily over the next week. The material was flipped again exposing the poished side again that had been sealed for a week and the emission factor doubled but resumed dropping over the next day. Overall, the results indicate that the emission factor of formaldehyde from the new wood with new polish is approaching that of the new wood with no applied polish over time. The standard emission factors for formaldehyde from each of the main wall surface materials listed in Table 3 are compared to field measured values for the PBC building that were collected previously using Equation 3 along with the building parameters listed in Table 4. The results are listed in Table 5 for each material and each floor as a range of concentrations estimated with ACH values representing the maximum and 50% of the maximum accounting for the fact that the demand response system will likely run the ventilation at less than the maximum value. These ranges are summed for the total wall area based on loading factors for each material and the range of total concentraions are compared to the measurements in the last row of the table. Assuming no other significant loss pathways for formaldehyde, the three primary wall materials can easily account for the measured concentrations.

According to the National Center for Health Statistics , there have been increasing pharmaceutical prescriptions for the past 30 years, and they have almost tripled in the past 14 years alone . For agriculture, in 2013 over 6.6 million kg of the 9.1 million kg of antibiotics used were to increase production . Common Bcontaminants of emerging concern^  can be excreted by both humans and animals with little or no change in the chemical structures and, not surprisingly, they have been appearing in wastewater, and in some cases tap water, over the past few years .Most wastewater treatment facilities are not capable of removing all pharmaceuticals , resulting in these compounds being found in effluent. In addition, during heavy storms, untreated wastewater overflow can release even higher concentrations of some pharmaceuticals, which then directly contaminate the environment . Many of these compounds can be found at biologically active concentrations in surface waters around the world . In addition, there is also an increased effort to use reclaimed wastewater in drought-affected areas , resulting in increased exposure. In agriculture/livestock operations, pharmaceuticals are found in manure that is used as fertilizer for feed and crops, effectively compounding the pharmaceutical concentrations . Current research shows that these chemicals tend to be both pseudopersistant in soil and detrimental to soil microbes . Our recent studies of the effects of pharmaceuticals on aquatic insects show that at concentrations found in reclaimed water, CECs can alter development of the medically important mosquito Culex quinquefasciatus, its susceptibility to a common larvicide, and its larval microbial communities .

Female Megaselia scalaris, which are ecologically important detritivores, also displayed an increased developmental period, which could jeopardize the population’s survival when exposed to CECs . Also, the common agricultural pest Trichoplusia ni was negatively affected by antibiotics through a plant matrix . A common birth control agent, 17α-ethinylestradiol, and Bisphenol-A, a common plasticizer, have been shown to cause deformities in the midge Chironomus riparius . However, aquatic insects’ constant exposure to these CECs is likely greater than most terrestrial insects. Interestingly, many CECs were not designed to specifically to impact microbes but have been shown to affect microbial communities. For example, the mental stimulant caffeine can alter biofilm respiration, and diphenhydramine, an antihistamine, has been shown to modify the microbial community of lake biofilms . Therefore, accurately predicting the consequences of specific CECs, even in model insects, currently is difficult if not impossible. This problem is exacerbated by a general lack of information regarding effects of pharmaceuticals and other CECs on the microbial communities of terrestrial insects. Arthropods, such as insects and crustaceans, rely on hormones to grow, develop, mate, and produce pigmentation . However, many pharmaceuticals, especially mammalian sex hormones, are structurally similar to chemicals that these organisms rely on for development. These pharmaceuticals bind to receptors and either increase or disable their counterparts’ natural function. Endocrine disruption has been noted in birds, reptiles, and arthropods, primarily in the modification of primary and secondary sexual characteristics, and changes in courtship behaviors . While most arthropod hormones do not closely match those of mammals, their molting hormone is very similar in structure to the mammalian female sex hormone 17β-estradiol. Increased molting events and inhibition of chitobiase, the enzyme responsible for digestion of the cuticle during insect molting, have been noted in crustaceans exposed to mammalian hormones . In addition to these effects, pharmaceuticals have been shown to have delayed cross-generational effects . Aphids are phloem-limited hemimetabolous insects . Myzus persicae is polyphagous, highly cosmopolitan, and an efficient vector of plant viruses . This insect overwinters in the egg stage on Prunus species, and when their host plants are over-populated and/or stressed, they begin producing alates to disperse and colonize new plants . The sexual forms are also alates and are formed in autumn temperatures wherever peaches or suitable host plants are available . Economically, M. persicae is most damaging in the spring, when the insects hatch and feed on new peach leaves, and serve as vectors of over 100 different plant viruses . The aphid microbiome has been extensively studied and is well understood, making aphids excellent models for microbial community and biological research . Previous research has determined that antibiotics can reduce fecundity, reduce population growth, and increase mortality of aphids . Previous findings were usually due to the reduction of Buchnera, a key symbiont that provides required nutrients the aphids cannot make themselves or acquire from their diet . Currently,drainage pot there is no information regarding pharmaceutical effects at the concentrations found in reclaimed water on the growth and development of phloem-limited insects or their microbial community composition. Many herbivores can be exposed to these contaminants after the CECs enter surface waters, soil, and plants from wastewater reuse and unintended discharge.

There is minimal information available regarding effects of CECs when translocated through plants to terrestrial insects, especially those with specialized feeding techniques. Depending on the acquisition and sequestration by their host-plant species, insects with phloem-limited feeding methods, such as aphids, could have either reduced or increased exposure to CECs. Because previous research demonstrated a substantial change in both the biology and microbial communities of other insects when treated with ecologically relevant levels of CECs , and since aphid growth and development rely on symbionts, we hypothesized that aphids could be affected in similar ways. To test this hypothesis, we conducted bio-assays of aphids reared on a key host plant, Capsicum annuum, exposed to CECs at concentrations found in reclaimed water. Any effects would have potentially important implications from agricultural perspectives. Also, as there is currently little information on effects of CECs on terrestrial insects acquired through a plant matrix, our findings would have possible interest for integrated pest management research.Bell peppers were grown from seeds in 10.16-cm2 pots in UC soil mix no. 3 and fertilized with Miracle Gro nutrient solution at labeled rate and watered as needed in the UCR greenhouse. When plants were approximately 10 cm tall, their roots were washed with D.I. water and they were transplanted to 475-mL Mason jars . Mason jars were coated with FolkArt Multi-Surface acrylic paint on the outside to prevent root exposure to light. Jars were filled with hydroponic growth media containing CEC concentrations described in Table 5.1 with average pH of 7.0 ± 0.5 as in Pennington, Rothman, Dudley, et al. . Treatment media were prepared utilizing stock solutions of treatment compounds dissolved in 5:45 methanol:D.I. water with aliquots of < 500 μL being dissolved in 18 L. Growth media were stored at room temperature in blackened 19-L tanks to protect the CECs from photo degradation and to prevent algal growth. Hydroponic growth media were drained, by Erlenmeyer filter flask and vacuum, and replaced every 3 days to hinder bacterial and fungal growth and maintain CEC concentrations. After filtering through a HEPA-CAP air filter, house air was bubbled into jars through black irrigation tubing to aerate the hydroponic growth media. Each container included one of five CEC treatments or an untreated control hydroponic solution, and was used to water four plants. Plants grew 3 weeks before 10 M. persicae were placed evenly on two fully expanded leaves per plant.Data regarding population growth were collected daily and the experiment was ended after 2 weeks. Three life-stage groupings were collected from each plant, with a minimum sample size of 20 individuals per life stage , and stored in 200 proof ethanol at 62 ± 2 °C until DNA extractions were performed. Plants were separated into parts , weighed, and immediately frozen at − 62 ± 2 °C.Our work demonstrates that the selected CECs did not affect population dynamics or microbial communities of M. persicae reared on bell peppers. Many plants will translocate CECS . However, some plants can metabolize and/or sequester xenobiotics in tissues other than phloem, thereby removing the CEC exposure to aphids . As aphid species rely heavily on the endosymbiont Buchnera species to grow and develop, many aphid populations treated with high concentrations of antibiotics will not survive . However, aphid microbial communities were not affected when treated with antibiotics and other CECs at the low concentrations found in reclaimed water, which is possibly why there were no discernable effects on the aphid population as a whole. While treatments used in our study have previously been demonstrated to have negative effects for at least two other species of insects , this work suggests that aphids are either not exposed to CECs through their host plant, or their bacterial symbionts are not sensitive to them, or depleted enough, to alter their basic biology. Plants treated with antibiotics typically have lower levels of intracellular calcium due to chelation . However, in our study, we did not notice any obvious signs of calcium stress , possibly due to the use of a hydroponic solution which contains more than enough metal ions to provide adequate nutrients to the plants, even with some chelating. We did notice an overall decrease in mass for plants treated with antibiotics likely due to a slowed growth rate from direct action of the antibiotics on plant growth .

Root exudates alter pH and the chemical composition around roots

However, there was a significant difference in the glycosyltransferase activity in radish seedlings treated with diazepam for 7 d and 28 d, although a distinct pattern in the changes of the enzyme activity was absent . For the 7 d cultivation experiment, a significant decrease in glycosyltransferase activity was observed in the shoots of radish seedlings when compared to the control . In contrast, no significant change in glycosyltransferase activity was observed in the shoots of cucumber seedlings when exposed to diazepam . In the 28 d cultivation experiment, only the cucumber seedlings exhibited significant differences in the enzyme activity, with an increase in activity detected in the shoots and a decrease in the cucumber buds . Even though we did not detect oxazepam-glucuronide in the exposed plants, changes in the glycosyltransferase activity indicated that conjugation might have occurred with the parent and its metabolites, including those not examined in this study, or at levels below our detection capability. In addition,vertical farming it may be postulated that rapid phase III metabolism may have limited the accumulation of such conjugates in the plant tissues, making the conjugates transient metabolites. In previous studies, glycosyltransferase was observed to catalyze the detoxification of ibuprofen in Phragmite australis during a 21 d exposure .

Further, the formation of a glucose conjugate has been considered to be a major detoxification pathway for several environmental contaminants . These studies together suggest the importance of phase II metabolism in the metabolic fate of pharmaceuticals in higher plants.Plant roots shape their environment in various ways and are in turn shaped by physiochemical properties of the surrounding soil. Roots affect soil by dislocating particles, by polymer production, and by the release of a wide variety of small molecules . Overall, these processes result in the formation of larger soil aggregates which increase water-holding capacity and influence the stability of soil organic carbon . Plant induced changes in chemistry can lead to weathering of minerals and alter the composition of microbial communities . Further, root exudates can enhance root penetration of soils . Although the effects of plant presence on soils and microbial communities have been a major research questions for over a century , relatively few studies have sought to understand the physiochemical effects of plant growth substrates on plant physiology and exudation. Plant morphology and exudations can be influenced by both physical and chemical properties of soil particles. Typically, particles range from small to large and determine physical parameters such as water-holding capacity of soils . It has been shown that 1-mm beads reduce root and shoot growth, elevate root:shoot ratios, and alter root morphology of maize when compared to hydroponic growth .

Root morphology can be altered by adsorption of root exudate metabolites to substrate such as activated carbon . Natural environments, such as soils, can also affect both root exudate profiles and morphology ; however, determination of causal factors may be confounded by variables such as granule size, chemistry, and microbial community composition. Soil minerals differ in structure and surface charge, thereby governing their interaction with dissolved organic compounds . Results suggest that substrate chemistry can alter exudation. For example, aluminum ions present in stone wool are thought to increase exudation of organic acids in maize . In contrast to particle chemistry, the effect of particle size on exudation is less clear. Particle size could alter exudation in multiple ways. Particle size reduces root growth rates, which limits exudate dispersal. In addition, diffusion depends on substrate size and can thus be a limiting factor for dispersal of exuded compounds . Exudates are mainly produced by root tips : when root morphology is altered by particle size, the number and structure of root tips can be altered, possibly changing the quality and quantity of exudates. Particle size also influences the composition of microbial communities . Microbes differentially metabolize exudates and secrete secondary metabolites , further changing the presence of exuded compounds. d) Particle size can modulate plant chemistry . Here, we investigate the effect of particle size and chemistry on root morphology and exudation in Brachypodium distachyon.

Specifically, we asked three questions: a) whether root morphology of a model grass is altered in physically restricted conditions as observed in other species, b) if and how the exudate profile changes with particle size, and c) how root morphology and exudation are influenced by substrate chemistry. To facilitate these studies, we used a sterile environment enabling us to focus on plant metabolism without the additional layer of microbial metabolism present in a natural environment. B. distachyon growth and exudation profiles were comparted on various inert substrates in a range of defined particle sizes relative to hydroponic solution. We found that particle size had a significant effect on root weight and root length; however, particle size did not influence composition of root exudates. A defined mixture of soil metabolites was used to evaluate sorption to particles. We further demonstrated that clay sorbed a large degree of exudates, altering the amount of exudates freely available around root. These clay-sorbed exudates could further support growth of a rhizobacterium. Our results highlight the importance of considering soil structure and chemistry when studying plant–soil interactions.Different particle sizes and surface chemistries were chosen to investigate how root morphology and exudation is affected in various plant growth substrates. The particle sizes chosen corresponded to large, intermediary, and small particles . Glass beads were chosen as an inert system with defined sphere diameter, sand as an inert and natural system, and clay as a natural system with a reactive surface. The mineral composition of the sand substrates was determined as more than 98% quartz, whereas the clay was a mixture of 51% opalCT, 37% mica-illite, 10% quartz, and trace amounts of K-feldspar and calcite . The chemical properties of the substrates were assessed by determining the sorption of a mixture of more than forty metabolites belonging to various chemical classes that have also been found in root exudates . The recovery rate of the various metabolites from the glass beads and the 4-mm sand was comparable to the defined medium control without substrate,vertical garden hydroponic whereas the recovery rate from the250-µm sand and from clay were lower by approximately 30% and 70%, respectively . Consistently, differences between clay and other substrates explained 84% of the variance in a principal component analysis, and only 8% of the variance accounts for differences between the control, glass beads, and sand . The metabolites depleted by clay belonged to a variety of chemical classes, among them charged compounds, such as organic acids and ammonium salts , and other nitrogenous compounds , and of comparatively polar compounds such as sugars . We confirm that as expected, clay particles sorb a variety of metabolites from the defined medium.Exudation could be limited by diffusion in our experimental systems with small particle sizes. A diffusion test with a dye resulted in fastest diffusion in controls without substrate added . The diffusion rate of the dye decreased with lower diameters in glass beads and followed a logarithmic trend. In 4-mm sand, the diffusion rate was initially similar to 1-mm glass beads, but then resembled more 0.5-mm beads. For clay, diffusion similar to 1-mm or 0.5-mm beads was observed initially, but subsequently, the dye front ceased moving, likely due to sorption of the negatively charged dye.

Based on this analysis, in our experimental setup, exudates would require a minimum diffusion rate of 1.25 cm/h to reach the edge of the glass jar in which the plants were grown . Thus, diffusion was not limiting in glass beads with a diameter equal to or >1 mm, but might be limited in substrates with smaller diameters . This confirms, that as expected, sand and glass beads are inert substrates, whereas clay strongly sorbs a variety of metabolites. In addition, exudation may be limited by diffusion in substrates with particle sizes smaller than 1 mm.The aforementioned substrates were used to investigate how Brachypodium distachyon root morphology and exudation was affected in these experimental systems compared to a hydroponic control. Plants were grouped according to their behavior in the different substrates: plants with weights and root morphology similar to hydroponic controls were termed “big” , and plants with distinct weight and root morphology were termed “small” . The root fresh weight of plants grown in 3-mm glass beads, 4-mm sand, 250-µm sand, and clay was comparable to the hydroponic control, whereas roots grown in 2-mm, 1-mm, and 0.5-mm glass beads and 5-µm sand were significantly smaller . The shoot fresh weight of plants grown in 5-µm sand were significantly smaller compared with plants grown in hydroponics, and all other conditions . The altered root and shoot weights resulted in decreased root/shoot ratios for clay, and 2-mm and 1-mm glass beads-grown plants, and an increased ratio of 5-µm sand-grown plants . Root length and number were assessed for first-order roots , second-order roots , and higher order roots. The total root length correlated with particle size, with maximal length for hydroponically and clay grown roots, approximately 30% shorter root systems for 3-mm beads-, 4-mm sand- and 250-µm sand-grown roots, and 50% or shorter root systems for 1-mm beads-, 2-mm beads-, 0.5-mm beads-, and 5-µm sand-grown roots . First-order root lengths were significantly decreased by more than half for all substrates exceptfor clay and 4-mm sand , whereas the second-order root length was decreased by 40%–70% in 2-mm beads-, 1-mm beads-, and 0.5-mm bead-grown roots, and by ~85% in 5 µm sand-grown roots . Higher order root lengths varied more within one experimental treatment, with a trend for higher total lengths in hydroponics and clay compared with glass beads and sand, and significantly lower lengths in 5-µm sand . Interestingly, root length had a higher Pearson correlation coefficient when correlated with particle size than root numbers. Only roots grown in 1-mm beads and 5-µm sand showed a statistically significant reduction in root number compared with hydroponic controls, which is a result of the large variability in total root number of hydroponically grown plants . The observed reduction in root number originated from a reduced number of secondary and higher order roots . A correlation analysis between root and shoot weight, total root length, and total root number of all samples showed a significant correlation of all parameters investigated . Root weight and length, and to a lesser degree root number, correlated with particle size. Overall, clay-grown plants were most similar to hydroponically grown plants regarding tissue weight and root morphology. Plants grown in 3-mm glass beads or 4-mm sand had comparable fresh weight compared to the aforementioned plants, but slightly reduced total root length driven by a reduction in first-order root length. Plants grown in 1-mm and 0.5-mm glass beads exhibited reduced root weight and root length, caused by a reduction in first and second-order root length. Plants grown in 5-µm sand exhibited the largest reduction in tissue weight, root length, and number.To investigate whether changes in root morphology might affect exudation profiles, spatial patterns of exudation were investigated using matrix-assisted laser desorption/ionization mass spectrometry . A total of 24 ions were detected in the vicinity of roots . It was not possible to confidently identify these ions given that the MALDI used is not suited for fragmentation of low m/z ions and the fact that MALDI often results in different ions versus the electrospray ionization used in our liquid chromatography–mass spectrometry analyses. However, despite this lack of identifications, our results suggest differences in spatial patterns of chemical components. Some ions showed higher abundances around root tip and elongation zone, supporting a role of these young root tissues in exudation. Other ions were detected along most of the root axis, suggesting exudation also from older root parts, whereas the location of other ions overlapped with the location of the root, which could either indicate short diffusion distances, or association with the cell wall. Overall, these data suggest that multiple tissues are involved in exudation.To investigate whether altered root morphology and various spatial exudation patterns altered overall exudation profiles, exudates were collected in situ and in vitro . The first collection approach generated exudation profiles shaped by plant metabolism and particle chemistry, whereas the second collection approach generated exudate profiles shaped only by plant metabolism.

The mineralization of PPCP/EDCs was previously examined only in a few studies

Topp and Starratt showed that about 10% of 14C-NP was extractable at 40 d, which was in general agreement with the current study . The formation of bound residue is considered a decontamination process, as the chemical has become an integral part of the soil matrix. In this study, bound residue was quantified by combustion of extracted soil samples. Figure 3.2 shows the fractions of bound residue in the incubated soils at 112 d. The levels of bound residue were significantly different among the PPCP/EDCs and followed the overall order BPA > NP > DCL ≥ NPX. For example, at 112 d of incubation in Maricopa soil, bound residue accounted for 66.2, 36.3, 29.6, and 14.9% of the spiked 14C-labeled BPA, NP, DCL, and NPX, respectively . However, no significant difference was noted among the different soils, except for the sterilized Irvine soil, which had significantly reduced levels of bound residue for most compounds. For example, at 112, bound residue for NP was 45.4 ± 16.3% in Irvine soil, 43.9 ± 8.2% in the compost amended Irvine soil, 17.1 ± 6.5% in the sterilized Irvine soil, 36.3 ± 1.2% in Maricopa soil,vertical gardening systems and 34.8 ± 4.6% in Ventura soil . Few studies have examined bound residues of PPCP/EDCs, as such analysis requires the use of 14C-labeling and combustion of solvent-extracted samples.

In Fent et al. , 79% was determined to be in the form of bound residue following incubation of 14C-BPA for 120 d, which was slightly higher than that found in this study . Kreuzig et al. measured the bound residue at 44 – 78% of the spiked 14C-DCL after 102 d of incubation in two soils, which was greater than in the current study for 14C-DCL . Such differences may be attributed to the different soil properties, such as organic carbon content, and to the specific 14C-labeling positions among the studies. The tendency of an organic compound to become bound is strongly affected by the hydrophobic partitioning to soil organic matter . Since the soils used in this study had similar low organic content , this may explain why the levels of bound residue for a compound were similar among soils. Due to this partitioning process, compounds with higher hydrophobicity typically become more bound than compounds with lower hydrophobicity . DCL and NPX are both ionizable compounds, with pKa values of 4.0 and 4.19, respectively, suggesting that they were partly ionized under the experimental conditions used . Ionic compounds are much more polar than neutral compounds like BPA and NP, which may explain why NP and BPA had more bound residue and less extractable residue than DCL and NPX. Microbially-mediated mineralization represents the complete breakdown of an organic compound and is therefore regarded as an environmentally beneficial decontamination process. Throughout the soil incubation in this study, the mineralized 14CO2 was continuously sequestered in NaOH solution and periodically measured. Figure 3.3 shows the cumulative 14C mineralization for each compound in the different soil treatments.

The final mineralized fractions were significantly different among the different PPCP/EDCs in the same soil and followed the order NPX > DCL > BPA > NP. For example, in Irvine soil the cumulative fraction mineralized at the end of 112 d of incubation reached 74.8 ± 2.4% of the initially spiked amount for NPX, 65.7 ± 3.6% for DCL, 22.9 ± 0.1% for BPA, and only 9.2 ± 3.7% for NP. These differences were likely related to the extractable fraction of each compound that may be bio-available for microbial metabolism . This relationship was supported by the fact that mineralization followed the same order as the abundance of the extractable fraction. In addition, it must be noted that the location of the 14C-label may have also contributed to the different mineralization rates. As shown in Figure 3.1, 14C was present on a substituent group in DCL and NPX, while the aromatic ring was labeled for BPA and NP. The mineralization rate differed among the soil treatments. For BPA and DCL, mineralization in Maricopa soil was more limited in comparison to Irvine or Ventura soil . For example, at the end of the 112 d incubation, the cumulative mineralized fraction for 14C-DCL in Maricopa soil was 49.8 ± 3.9%, lower than that in Irvine or Ventura soil . On the other hand, mineralization for NPX and NP was generally similar among the three soils. For example, the fractions of 14C-NP mineralized after 112 d were 9.2 ± 3.7%, 10.0 ± 0.6%, and 10.7 ± 0.8% for Maricopa, Irvine, and Ventura soils, respectively.

Since the extractable fraction was not different among the soils for a compound, the differential mineralization rates suggested that the variation was likely due to differences in microbial population and activity in these soils. Both Irvine and Ventura soils were from agricultural fields not previously exposed to treated wastewater, while Maricopa soil was from an uncultivated area used as a groundwater recharge basin for over 10 years. It is likely that agricultural cultivation and exposure to treated wastewater, respectively, may have led to the establishment of specific microbial communities in these soils, resulting in preferential transformations of some compounds in a given soil. Sterilization of Irvine soil significantly decreased mineralization of PPCP/EDCs , confirming the role of soil microorganisms in the transformation of these PPCP/EDCs. In addition, amendment of compost to the Irvine soil generally resulted in decreased mineralization, with the exception of NPX which was quickly mineralized in all non-sterilized soils. For example, mineralization of BPA decreased from 22.9 ± 0.1% in Irvine soil to 17.5 ± 0.3% after compost addition .In Fent et al. , 13.1 – 19.3% of the spiked 14C-BPA was mineralized after 120 d in four soils, which was in good agreement with the 14.2 – 22.9% range observed for unmodified soils in this study. Mineralization of 14C-DCL was monitored for 102 d in Kreuzig et al. and was found to be 13% of the spiked amount, which was substantially smaller than that in the current study . This reduced mineralization may be attributed to the difference in the 14C labeling position between the two studies. Topp et al. reported that about 50% of 14C-NPX was mineralized after 27 d of incubation, and the rapid mineralization was in agreement with the current study . In a separate study, Topp and Starratt observed that 40% of the initially spiked 14C-NP was mineralized after 40 d of incubation at 30 °C in a sandy soil ,vertical tower for strawberries which was greater than that measured in this study . On the other hand, Shan et al. reported that only 5% of spiked 14C-NP was mineralized after 58 d of incubation at room temperature. Since 14C-NP was labeled on the aromatic ring in all these studies, the higher mineralization in Topp and Starratt may be partly attributed to the use of a higher incubation temperature in that study. Mineralization was the major loss pathway for DCL and NPX, which amounted to 49.8 – 68.3% and 69.2 – 78.3% of the initially spiked 14C, respectively. In comparison, formation of bound residue appeared to be the predominant dissipation pathway for BPA and NP in the soils considered in this study, accounting for 53.0 – 66.2% and 34.8 – 45.4% of the initially spiked 14C, respectively. At the end of 112 d of incubation, the extractable fraction for each compound was consistently smaller than the mineralized or bound residue fraction, suggesting that these PPCP/EDCs were mostly removed in three months through mineralization or formation of bound residue.

Concurrently, the potentially bio-available extractable residue greatly diminished. Residues extracted from Irvine, Maricopa, and Ventura soils were further analyzed to characterize the composition of extractable 14C after 14 d and 112 d of incubation. Extracted 14C was identified as the parent compound, transformation products appearing during the HPLC run, and transformation products appearing in the SPE filtrate. The results for Irvine soil are shown in Figure 3.4. Extensive transformation of parent PPCP/EDCs was evident in all soils for most compounds. For example, after 112 d of incubation, parent compounds accounted for only ND – 13.8% of the extractable 14C for BPA and 2.4 – 8.4% for NP. The remaining extractable 14C was in the form of transformation products recovered early in the HPLC run or in the SPE filtrate, suggesting that transformation led to the formation of intermediates more polar than the parent. Differences among soil types were also evident. For example, while no parent compound was detected at the end of the incubation for DCL and NPX in Maricopa soil, the majority of the extractable residue was found as the parent for DCL and NPX in Ventura soil. A first-order decay model was used to fit the dissipation of parent compounds in the different treatments. The calculated half-lives ranged from 1.4 to 5.4 d for all PPCP/EDCs in the unmodified soils . The test compounds were relatively more persistent in Ventura soil, and less persistent in Maricopa soil, likely reflecting differences in the native microbial communities. The half-lives of BPA and NP in this study were generally similar to those previously reported . However, the half-lives calculated for DCL and NPX were somewhat shorter than those reported by Xu et al.  or Lin and Gan . This difference may be caused by the different soils and experimental conditions used. Overall, none of the PPCP/EDCs considered in this study exhibited significant persistence in soil as the parent compound. When compared to chromatograms of the parent compound, many transformation products were evident in the soil extracts, and the relative presence of transformation products in the extractable 14C generally increased over time . For example, in Maricopa soil treated with 14C-NPX, 62.1% of the extractable 14C was associated with transformation products at 14 d and the fraction increased to about 100% at 112 d. For NP treatments, 14C was also detected in the SPE filtrate. Since preliminary experiments showed that 14C-NP was quantitatively retained by the SPE cartridge, the 14C in the SPE filtrate may be assumed to be polar compounds not adsorbed by the cartridge sorbent. The extensive transformation of spiked PPCP/EDCs in soil extracts suggests the importance of considering degradation intermediates in addition to the parent compound . Samples from BPA and DCL treatments were further analyzed on UPLC/MS/MS to tentatively identify degradation intermediates. Authentic standards were used to verify the identity by matching retention time and mass transitions of the isolated peaks . In solvent extracts from soil treated with BPA, 4-hydroxyacetophenone , 4- hydroxybenzaldehyde , and 4-hydroxybenzoic acid were detected as transformation products . While information on BPA degradation in soil is very limited, it was suggested by Spivack et al. that BPA may undergo oxidative rearrangement to form 1,2-bis-2-propanol, which is then dehydrated to 4,4′-dihydroxy-a-methylstilbene. Oxidative cleavage may then result in HBA and HA, and further oxidation of HBA forms HBacid. At 14 d, extracts of Maricopa and Ventura soils treated with DCL showed the presence of 5-hydroxydiclofenac , as well as 2,6-dichlorobenzoic acid in Ventura soilonly. A small amount of 2,4-dichlorobenzoic acid was detected in Irvine soil. At the end of 112 d of incubation, 5HD was detected in all soils, while 24DCB and 26DCB were found in Irvine soil, 24DCB and 3,5-dichlorobenzoic acid in Maricopa soil, and 24DCB in Ventura soil. It is likely that oxidation of DCL led to the formation of 5HD, and both DCL and 5HD may serve as precursors to DCB through Ndealkylation of the biphenyl compounds followed by carboxylation . 4′-Hydroxydiclofenac was analyzed for, but not detected in any sample, in contrast to other observations made using microbial culture or human metabolic enzymes . Only a few previous studies examined the transformation products of PPCP/EDCs in soil, sediment, or sewage. In a soil incubated with gram negative bacteria, Spivack et al. identified some of the same intermediates of BPA as in this study. However, even though degradation of DCL was evaluated in sewage , sediment , and in fungal cultures , no effort was made to identify the specific dichlorobenzoic acid isomers. Little information is available about the toxicity of these transformation products as compared to their parent forms.

Potential detoxification mechanisms have been less reported

Cucumber plants are generally more sensitive to contaminants and their bio-accumulation is higher than many other plants due to their high transpiration rate. Our previous study showed both CeO2 and ZnO NPs triggered more physiological changes in cucumber plants compared to corn plants, which have lower transpiration rates.Here we hypothesized that nano-Cu would induce physiological responses in cucumber plants. To evaluate this, we selected metabolomics studies as a novel approach to understand plant−nanoparticle interactions. We selected a 1 H nuclear magnetic resonance -based environmental metabolomics platform to detect the induced alteration, because NMR can simultaneously detect a variety of metabolites with simple sample preparation.In addition, compared with other “omics”, metabolomics reveals effects downstream of DNA and simultaneously provides a nonspecific assessment of the end result of multiple biological responses. Therefore, 1 H NMR has been employed to evaluate the toxicity of a large variety of environmental contaminants on different organisms.The toxicity and toxicity mechanism of titanium dioxide NPs to earthworms and rats was evaluated via an environmental metabolomics platform using 1 H NMR. Furthermore, most previous studies of the effect of various NPs to plants have concentrated on toxicity.

It is well known that approximately 30−40% of all photosynthetically fixed carbon will be transferred to the rhizosphere as root exudates,outdoor vertical plant stands including organic acids, amino acids, sugars, proteins, phenolic compounds, and CO2.These compounds play an important role in plant stress tolerance and external exclusion of pollutants.Considerable evidence exists that plants upregulate some organic acids, including amino acids, to chelate or complex toxic metals , to hinder their translocation to above ground plant tissues.Murphy et al.reported copper ions induce citrate production in root secretions. Our hypothesis is that root exudates may play an important role in NP mobility and bioavailability, as well as upor down-regulation of metabolite levels due to toxic effects and to induce detoxification. Therefore, the aims of the present study were to investigate the uptake, translocation, bio-accumulation, and toxicity of nano-Cu in cucumber tissues. We used nontargeted 1 H NMR and GC-MS based metabolomics to evaluate the physiological changes induced by nano-Cu in cucumber plants. Mineral nutrient metabolism was also evaluated by determining the elemental content in different tissues.After 7 days exposure to 10 and 20 mg/L of nano-Cu, cucumber plants exhibited significant decrease in root length compared to the control. Exposure to 10 and 20 mg/L of nano-Cu also resulted in root biomass reduction by 11.7% and 30.2%, respectively , but only the effect at 20 mg/L was statistically significant . Similar results have been reported in zucchini, squash, wheat, lettuce, and alfalfa.Stampoulis et al.observed reduced root elongation of zucchini seeds after exposure to 1000 mg/L Cu NPs.

Musante and White reported that exposure to Cu NPs at 100 and 500 mg/L resulted in significant biomass reduction of squash.Lee et al. also found Cu NPs at 200 mg/L affected wheat root elongation.More recently, Hong et al. reported that Cu NPs at a concentration of 20 mg/L significantly reduced the root length of alfalfa and lettuce. It has been reported that copper toxicity results from inhibition or activation of some enzymes in the root zone.For instance, Kennedy and Gonsalves reported Cu inhibited ATPase activity in the plasma membrane of Zea mays roots.Previous studies showed copper toxicity triggered oxidative damage and increased the antioxidative enzymes.Compared to the roots, nano-Cu had no statistically significant impact on stem and leaf biomass. This is consistent with previous reports that roots are the most vulnerable organ under nano-Cu stress. Lee et al. reported that nano-Cu at 200 mg/L affected the root length of wheat, while the threshold to induce shoot length reduction was 800 mg/L. CuSO4 was observed to inhibit root growth but not shoot growth in rice seedlings.Fernandes and Henriques attributed the differential effect of Cu on root and shoot growth to the fact that Cu is mainly bio-accumulated in roots compared to shoots. However, Hong et al. demonstrated that the Cu toxicity is a species-specific response.They showed that although the Cu concentration was high in alfalfa shoots, compared to lettuce, there was no shoot length reduction.In all tissues including root, stem, and leaves, Cu concentration in plants treated with 10 and 20 mg/L of nano-Cu were significantly higher than that in the control . This indicates that copper was taken up by and transported from root to stem and leaves within 7 days.

The distribution patterns of Cu indifferent tissues in both control and nano-Cu treated plants are similar: Cu was predominant in roots , followed by stems , and leaves . This indicates that Cu was sequestered primarily in the root compartment, and only a small percentage was transported to upper tissues. This is consistent with previous reports showing that Cu is located primarily in roots of alfalfa and lettuce.It is interesting to note that the stem/root translocation factors have the tendency to increase with increasing nano-Cu concentration, while the leaf/ stem translocation factors decrease . This indicates cucumber plants tend to retain/ sequester more Cu in their stems. The mechanism underlying this phenomenon is complex, and still unclear. We hypothesize that this is a detoxification or active protective process for the plants to avoid damage to the more vulnerable tissue of roots and leaves. It has been shown that copper in xylem sap is almost 100% bound to amino acids.The stem may secret more ligands, e.g., carboxyl and amino groups, organic acids, glutathione, cysteine, to complex or chelate with Cu when it is in excess.Liao et al.showed evidence that more than 99.6% of total Cu in the xylem sap of tomato plants was in a complexed form, with the xylem sap producing amino acids to chelate Cu and hinder its translocation to leaves. These findings with Cu are consistent with previous reports that plants are able to minimize the adverse effects of excess metals by regulating their distribution and translocation within their organs or cells.As shown in Table 1, in the presence of nano-Cu, nutrient elements were markedly affected. The concentrations of Na, P, S, K, Mo, and Zn decreased in all cucumber tissues in exposed plants. Except K in roots and leaves , and Na in leaves , all the decreases were statistically significant . In addition, nano-Cu decreased Fe and Mg uptake in roots, but not in stems and leaves. Reduced uptake of P, S, and Fe in lettuce and alfalfa by nano-Cu was also observed in hydroponic systems.Because the applied nanoCu concentrations in the two studies are similar, cucumber seems to be more sensitive to nano-Cu than lettuce or alfalfa, probably because of its higher transpiration rate that carries more Cu to plant cells. Our previous study showed that nCeO2 and nZnO NPs decreased Cu and Mo content in cucumber fruits; however, neither nCeO2 nor nZnO affected mineral element accumulation in corn cobs.The decrease of K concentration in all tissues may be the result of leakage mediated by ion channels. Murphy demonstrated that Cu promotes K+ efflux rather than inhibiting K+ uptake in Arabidopsis seedlings.Several studies have shown that iron uptake is decreased by excess Cu.Hong et al. also found nano-Cu significantly decreased Fe uptake in lettuce. Waters and Armbrust found that high Cu supply inhibited the activity of ferric reductase, which is an indicator of Fe demand. This led to decreased demand for Fe by the plant, which subsequently led to less Fe accumulation. Decreased P is due to the formation of Cu−phosphate complexes at the root surface.However, some studies showed the up-regulation of S in the presence of nano-Cu or other stressors,vertical plant rack which is quite different from our finding. It is possible that nano-Cu damages the root cell membrane and leads to leakage of many ions. These elements participate in respiration and photosynthesis and in some enzyme system functions. The decrease of these minerals may lead to othermetabolic changes. For example, Cu-induced Fe deficiency can contribute to decreased leaf chlorophyll content and reduced photosynthesis.Reichman reported that the chlorotic symptoms on young leaves of plant experiencing Cu toxicity could be an induced Fe-deficiency.Principal component analysis was performed as a first step to provide a general overview of trends, grouping, and outliers in the 1 H NMR data.PCA of the metabolomics data set extracted from 12 cucumber leaf samples produced three principal components which explained more than 66.9% of the total variance .

The score plots from PC1 and PC2 reflect that leaf tissues from the control and nano-Cu treated plants were clearly separated from each other by PC2 reflecting differences in metabolic profiles. However, no difference was found between 10 and 20 ppm treatments, indicating that nano-Cu at either level changed the pattern of metabolites in cucumber leaves. The PCA loading plot identified the regions of the NMR spectra that contribute to the observed differences in PC scores. Further, bins with high weighting score were selected and subjected to one-way ANOVA to identify metabolites significantly regulated due to nano-Cu stress. A total of 25 bins were found to be significantly altered in cucumber leaves in response to nano-Cu exposure .In total, 22 metabolites were identified as significantly altered using Chenomx . Most of the altered metabolites are secondary metabolites. Among the five up-regulated metabolites, 4-aminobutyrate , acetylglucosamine, and phenyllactate have previously been shown to be related to stress response. GABA is a nonprotein amino acid. In plants there are numerous observations of a rapid accumulation of GABA in response to biotic and abiotic stress.Zulak reported that GABA levels were rapidly increased in elicitor-treated opium poppy cell cultures.Acetylglucosamine, an amino-sugar, plays an important role in cell signaling. It has been reported that inflammation induced by bacterial infection can result in increased release of amino sugars from mammalian host cells.Phenyllactate and p-hydroxyphenyllactate were found to decrease ROS production in both mitochondria and neutrophils.Those metabolites were further analyzed with MetaboAnaylst 2.0 to identify the major perturbed metabolic pathways induced by nanoCu. The pathway impact value threshold was set as 0.1.Results showed that none of the pathways were disturbed by nano-Cu in cucumber leaves.Extracts of root exudate from control and nano-Cu treated plants were analyzed by 1 H NMR followed by PCA analysis. PCA analysis revealed clear separation in the root exudate metabolomics profiles collected from control and nano-Cu treated cucumber plants along PC1 . The PCA loadings identify the regions of NMR spectra that contribute to the differences in PC1 . In the region of organic acids , a number of bin areas increased in the presence of nano-Cu, indicating nano-Cu and released Cu2+ increased the level of a number of amino acids or organic acids . The aromatic region comprises many characteristic signals of secondary metabolites,which play a crucial role in plant defense to environmental stress.As shown in Figure S5 C, nano-Cu/Cu ions altered the pattern of some secondary metabolites. We further identified those bins to corresponding compounds. However, due to the extremely low concentration and high baseline, it was difficult to link those bin areas to specific compounds using the Chenomx NMR Suite. Thus, GC-MS was used for identification and quantification. A total of 156 metabolites in root exudates were identified by GC-MS. To visualize the general differences between control and nano-Cu treated plants, the 156 identified metabolites were normalized and analyzed by Partial Least Squares Discriminant Analysis using online resources, which is a supervised clustering method to maximize the separation between groups. The score plot shows that cucumber root exudate exposed to different concentrations of nano-Cu are clearly separated along the first principal axis , which explained 30.4% of the total variability. This indicates nano-Cu considerably altered the metabolic profiles of cucumber root exudate, which is consistent with the NMR data. Using parameters of variable importance in projection score,a total of 56 metabolites were found to be responsible for this separation . For the metabolites ofinterest, hierarchical clusters analysis was performed by grouping the samples into clusters based on the similarity of their metabolite abundance profiles. Figure 3 presents the resulting heat map for the selected metabolites, which indicates that some metabolites, including lysine, threonine, phenylalanine, glycine, serine, proline, isoleucine, alanine, valine, leucine, beta-alanine, 4-hydroxybenzoate, benzoic acid, 2- hydroxyvaleric acid, pelargonic acid, salicylic acid, lactic acid, were up-regulated by nano-Cu. However, some metabolites were down-regulated by nano-Cu, including parabanic acid, 3- propionic acid, N-acetylmannosamine, erythritol, pimelic acid, dehydroascorbic acid, N-acetyl-Dgalactosamine, fructose-6-phosphate, hexonic acid, vanillic acid, and citric acid.

The level of chemicals in the cell matter decreased quickly thereafter

Methanol, and then water, 7 mL each, were added to each HLB cartridge for precondition, followed by the addition of the sample and then 5 mL of 5% methanol in water for clean-up. For elution and collection of the target analytes, a final pass-through of 15 mL methanol was performed. The resulting methanol eluent was collected in a glass tube, dried using a nitrogen evaporator, reconstituted in 1 mL methanol-water mixture , and filtered through a 2 mm PTFE filter into a 1.5 mL HPLC vial for instrumental analysis. Plant cell matter and wheat tissues were freeze dried at -50 ˚C for at least 72 h to remove moisture and weighed. Before extraction, 50 μL of a depurated compound was added to each sample as the recovery surrogate. Samples were firstly extracted with 10 mL MTBE via sonication for 30 min. The sonication process was then repeated with 10 mL fresh MTBE one additional time and 10 mL acetonitrile twice. Extracts from the extraction were combined and dried by a nitrogen evaporator, followed by reconstitution in 1 mL methanol and dilution with 20 mL water. The resulting liquids were cleaned up with HLB cartridges using a similar protocol as described above. The final extracts were dried under a gentle nitrogen gas flow, reconstituted in 1 mL methanol: water ,vertical grow shelf and filtered through a 2 mm PTFE filter before instrument analysis. No significant difference was found in the biomass between the treated groups and control groups for experiments using A. thaliana cells. No target analytes were detected in the method blanks.

The concentrations of target analytes in control groups with no cells, or with nonviable cells, varied in the range of 93.4-116.0% of the spiked concentration at the end of exposure as compared to the initial concentrations, suggesting stability of these compounds under abiotic conditions. The individual compounds were found to be taken up by live A. thaliana cells. The levels in the plant cell matter reached maxima within 3 h for all compounds except acetaminophen, which exhibited the highest accumulation at 6 h into the incubation .At the end of 96-h cultivation, the level in the cell matter was < 0.3 μg/g for most compounds, suggesting rapid metabolism in viable plant cells and likely excretion into the aqueous medium. Among the different compounds, DM-diazepam and diazepam appeared to be accumulated to higher levels than the other compounds and were also more recalcitrant to metabolism. After 96 h of incubation, 4.78 ± 0.90 μg/g of DM- diazepam or 3.63 ± 1.74 μg/g of diazepam still remained in the A. thaliana cells. In previous studies, CECs including acetaminophen, diazepam and naproxen were found to be readily metabolized in different plant species 40–42 . The CECs and their methylated/demethylated derivatives showed different accumulation potentials in A. thaliana cells. For example, acetaminophen was detected in the A. thaliana cells at significantly higher concentrations than M-acetaminophen at any given sampling time point . After 6 h of incubation, 6.10 ± 1.57 μg/g of acetaminophen was found in the A. thaliana cells, while the level was only 2.52 ± 0.57 μg/g for M-acetaminophen . In comparison, methylparaben was found to accumulate more than DM-methylparaben at all sampling time points.

For example, at 1 h, methylparaben was found at 11.6 ± 2.81 μg/g in the cell matter, while DMmethylparaben at only 0.06 ± 0.00 μg/g. The difference in accumulation by A. thaliana cells between DM-methylparaben and methylparaben may be partly attributed to the fact that DM-methylparaben was present mostly in an ionized form in the nutrient media . Negatively charged chemicals are known to not easily cross the negatively charged cell walls and membranes and are limited in their plant uptake 43,44. Like methylparaben, higher concentrations of naproxen than DM-naproxen were also detected in the cell matter throughout the exposure time . After 1 h of incubation, 12.31 ± 2.46 μg/g of naproxen was found to be in the cell matter, while the level was only 2.10 ± 0.40 μg/g for DM-naproxen. However, no statistically significant difference was observed between diazepam and DM-diazepam in their levels in A. thaliana cells duringthe exposure experiment. This may be attributed to the fact that log Kow of DM-diazepam is similar to that of diazepam . As the test chemicals were taken up by A. thaliana cells, the levels of CECs and their methylated or demethylated derivatives in the culture media concurrently decreased. In the culture media, the concentration of DM-methylparaben and methylparaben, and DM-naproxen and naproxen all decreased rapidly, and their level fell below the detection limit after just a few hours into the incubation . In comparison, the decrease of acetaminophen and M-acetaminophen, and DM-diazepam and diazepam was relatively slower, with 0.12-0.40 mg/L, or 12-40% still remaining in the cell culture media after 48 h of exposure. The dissipation of CECs and their methylated or demethylated derivatives in the culture media was further fitted to the first-order decay model, and the fit was generally good, with R2 > 0.63. The half-life T1/2 was then calculated from the first-order rate constant . The estimated T1/2 values were very small for methylparaben, DM-methylparaben, and naproxen. The dissipation of DM-naproxen was so rapid that T1/2 could not be derived.

Methylation appeared to increase T1/2 for acetaminophen and DM-diazepam, with statistically significant difference . A mass balance approach was not followed in this study, as subsequent transformation products in the A. thaliana cells were not characterized. Given that the compounds considered in this study were stable under abiotic conditions, the rapid dissipation in the culture media and limited accumulation in the A. thaliana cellssuggested that the CECs and their methylated or demethylated counterparts underwent rapid metabolism in the A. thaliana cells. In the case of acetaminophen, methylparaben, and naproxen, demethylation introduced a hydroxyl or carboxyl group into the molecule. As shown in previous studies, compounds with a hydroxyl or carboxyl functional group can undergo rapid conjugation with various biomolecules in plants. The conjugated intermediates are substantially larger in molecular size and may become “immobilized” once formed in the A. thaliana cells. Future research should consider the formation of conjugates for demethylated compounds and understand the fate and risks of such plant-origin conjugates. Uptake and translocation of the paired compounds were further measured in wheat plants grown hydroponically in nutrient solutions. Roots and shoots of wheat seedlings were collected and analyzed separately to understand the in-plant translocation. Target CECs and their methylated or demethylated counterparts showed great stability in hydroponic solution without wheat seedlings, with recoveries ranging from 99.1-125.7% of the initial spiked concentration after 240 h incubation. No compounds of interest were detected in the untreated hydroponic solution or wheat seedlings. In general, the level of chemicals in the plant tissues first increased and then decreased, suggesting uptake into the roots from the hydroponic media, followed by translocation from roots into shoots and/or metabolism in the plant. All CECs and their methylated or demethylated TPs were detected in wheat roots, and the concentrationswere much higher than those in shoots, indicating generally limited translocation . Among the different CECs, acetaminophen and DM-naproxen were not detected in wheat shoots, while DM-methylparaben was only found occasionally at trace levels. The accumulation of acetaminophen was also limited in the roots,vertical hydroponic which may explain its absence in the shoots. From a previous study, after formation from naproxen through demethylation, DM-naproxen was found to metabolize readily through phase II and phase III pathways in A. thaliana cells. The rapid metabolism of DM-naproxen in plants may have contributed to its absence in the shoots. Higher concentrations were consistently detected for M-acetaminophen than acetaminophen in both wheat roots and shoots . In wheat shoots, only Macetaminophen was detected, suggesting that methylation rendered acetaminophen more mobile and a greater potential to translocate from roots to shoots. In general, DMmethylparaben was found to be taken up more rapidly than methylparaben into wheat roots and reached 20.66 ± 2.78 μg/g at 6 h after the treatment . In comparison, the highest level of methylparaben in roots was observed at 12.34 ± 1.33 μg/g after 96 h of exposure. However, methylparaben consistently exhibited much higher concentrations than DM-methylparaben in the shoots, suggesting a greater potential for translocation for methylparaben . Both compounds were found to undergo rapid metabolism, and their levels after 10 d of incubation were considerably lower than at earlier time points in the roots, while essentially no DM-methylparaben was found in the shoots. As the demethylated derivative of naproxen, although DM-naproxen was taken up quickly and reached 33.32 ± 8.41 μg/g in wheat roots after 24 h, it appeared to be rapidly metabolized , as only 0.33 ± 0.02 μg/g DM-naproxen was detected in the roots after 10 d.

In comparison, naproxen was accumulated in both roots and shoots at consistently higher concentrations than DM-naproxen throughout the experiment . In wheat shoots, DM-naproxen was consistently below the detection limit, suggesting limited translocation, and/or rapid transformations in the roots via pathways such as conjugation. Both DM-diazepam and diazepam showed significant accumulation in wheat plant . At the end of 10-d exposure, 32.74 ± 0.64 μg/g and 13.12 ± 2.79 μg/g of diazepam were detected in roots and shoots, respectively, while the corresponding values were 15.36 ± 1.51 μg/g and 11.81 ± 0.40 μg/g for DM-diazepam, suggesting active translocation after entry in the roots. Among the four pairs of compounds considered in this study, diazepam and DM-diazepam have the largest log Kow . Between diazepam and DM-diazepam, the root accumulation of DM-diazepam was greater than diazepam during the first few sampling time points; however, an opposite trend was observed after 48 h of incubation, where the level of diazepam appeared to be significantly greater than DM-diazepam . Levels of both diazepam and DMdiazepam in the shoots increased over time, and there was no statistically significant difference between their concentrations at the same time points. It must be noted that unlike the other compounds considered in this study, demethylation of diazepam does not introduce a hydroxyl group into the structure and therefore, the almost identical accumulation of diazepam and DM-diazepam may be attributed to their similar physicochemical properties . As CECs and their methylated or demethylated derivatives were taken up by wheat seedlings, their levels in the nutrient solution decreased . The rate of dissipation was similar between acetaminophen and M-acetaminophen, and between methylparaben and DM-methylparaben. However, diazepam and naproxen appeared to decline at a slower rate than their demethylated counterparts . Consequently, the estimated T1/2 values were also significantly longer for diazepam and naproxen than their demethylated derivatives . The prolonged availability of diazepam and naproxen in the nutrient solution may have contributed to their relatively high accumulation in wheat seedlings . No significant correlation was found between log TF and log Dow in this study , likely due to the limited number of compounds considered in this study. Wu et al. observed a generally negative correlation for pharmaceuticals and personal care products in lettuce, spinach, cucumber and pepper. Another study conducted by Li et al. did not show any significant correlation between log TF and log Kow for neonicotinoids in Japanese mustard. Different treatments, plant species and compounds were used in those studies, suggesting that the translocation of xenobiotics in plants may be affected by not only the physicochemical properties of the xenobiotics, but also the inherent characteristics of plants. In addition, plants have a cascade of enzymes that are capable of facilitating metabolic transformations, and metabolism affects TF, as rapid metabolism in the root would translate into a diminished TF. Also, weak acidic CECs dissociated in the cytosol could be repelled by the negatively charged cell membranes, and therefore, become “trapped” in root cells, which may also limit their translocation. Active metabolism, such as conjugation with endogenous plant biomolecules, and the possible “ion trap” in root cells, likely contributed to the lack of apparent translocation for acetaminophen, DM-naproxen and DM-methylparaben in this study. Tools like ChemAxon could help predict basic properties of organic compounds, including TPs that do not always have experimentally derived values. It is feasible to incorporate changes in physicochemical properties, using either experimentally derived or estimated values, into well-established empirical relationships to evaluate the potential influence of common transformation reactions such as methylation and demethylation on plant uptake for a large range of CECs in the scenarios of beneficial reuse of treated wastewater effluent and bio-solids.

Copper nanoparticles and copper oxide nanoparticles are widely used in different fields

Since the deletion mutants showed similar root length as the 1RS line, we decided to focus on the 14 genes expressed in roots that were deleted in both the 1RS duplication and the adjacent and orthologous 1BS insertion . Although the annotated functions of these genes based on conserved domains and homology will require further experimental validation, the list is useful to summarize their inferred functions and to provide a preliminary idea of potential candidate genes. The first group includes four genes annotated as defense genes, a function that is likely not closely related with the observed phenotypes. This group includes TraesCS1B02G017500 and TraesCS1B02G0017600 , which encode proteins with NB-ARC and LRR domains characteristic of plant disease-resistance proteins involved in pathogen recognition and activation of immune responses. It also includes TraesCS1B02G017700and TraesCS1B02G0018100 , which are both annotated as defensins, a family of small plant antimicrobial peptides that serve to defend plants against pathogens. A second group includes three genes annotated as having enzymatic or housekeeping functions, which may not be compatible with the developmental nature of the observed changes in the roots of 1RSRW. The first gene in this group, TraesCS1B02G017800, encodes a methionine Smethyltransferase that has been implicated in the volatilization of selenium and in the biosynthesis of S-methylmethionine,indoor vertical farming a compound that is important in the transport of sulfur .

The last two genes in this group encode proteins with chaperon functions. TraesCS1B02G019200 is a tubulin-folding cofactor E involved in the second step of the tubulin folding pathway. TraesCS1B02G019300 encodes a chaperone protein DnaJ, which stimulates the heat-shock protein Hsp70’s ATPase activity, stabilizing its interaction with client proteins. These chaperon proteins play important roles under plant stress but are unlikely to play an important role in the phenotypic differences we observed under optimal hydroponic conditions. The third group includes genes involved in regulatory processes or in cell growth or division, processes more likely to be involved in the observed developmental changes in root growth . TraesCS1B02G017900 encodes an E3 ubiquitin-protein ligase CHIP-like protein that ubiquinate heat shock misfolded client proteins, targeting them for proteasomal degradation. Since E3 ubiquitinprotein ligases can ubiquitinate and regulate multiple targets, we could not rule it out as a potential candidate gene. We also included in this group the genes TraesCS1B02G018900 and TraesCS1B02G0019100, which encode 64% similar small GTP-binding proteins from the RAB family. These conserved proteins serve as molecular switches in signal transduction and play important roles in intracellular membrane trafficking, cross-talk with plant hormones and regulation of organogenesis, polar growth, and cell division , all functions that seem relevant to the observed differences in root development.

TraesCS1B02G018700, TraesCS1B02G019700, and TraesCS1B02G019800 encode 12-oxophytodienoate reductase-like proteins involved in the biosynthesis of jasmonic acid. Since hormones can affect multiple developmental traits, these are also strong candidate genes. Finally, TraesCS1B02G020200 encodes a wall associated receptor kinase . These serine–threonine kinases are involved in signaling and cell expansion, making it an interesting candidate for the differences in root length observed in 1RSRW.Nanoparticles are materials with at least two dimensions between 1 and 100 nm. The small size and large surface area provide NPs with different physical strength, chemical reactivity, electrical conductivity, magnetism, and optical effects, compared to bulk materials. These special properties allow NP utilization in electronics, engineering, energy production, catalysis, pharmaceutics, cosmetics, textiles, food industry, and agricultural products, among others.Recent statistics indicate that 1317 products containing NPs were on the market in 2010, and it has been estimated that there was a global release of 22 000–80 400, 1100–29 200, and 590–4800 metric tons of engineered nanomaterials into soil, water and atmosphere, respectively. The increase in NP utilization has raised concerns about their release into the environment and possible impacts on living organisms.Very few studies have reported the effects of these two Cu-based NPs to terrestrial plants.Shah and Belozerova15 found that the shoot/ root ratio in lettuce plants treated for 15 days with 0.013% nCu in soil was 2.7, while in control plants the ratio was 1.4. This suggests nCu affects lettuce growth. Another report indicates that, compared to control, 1000 mg nCu/L reduced by 77% the root length and by 90% the biomass of Zucchini grown in hydroponics.

Similarly, Musante and White16 observed that nCu not only affected root length and biomass of zucchini, but also reduced the transpiration volume by 51% in plants exposed to 100 mg nCu/L and 61% in plants treated with500 mg nCu/L. Lee et al. reported that the seedlings’ length of Mung bean and wheat were reduced by 60% and 75%, respectively, when exposed to 1000 mg nCu/L. They also observed less reduction in shoot growth compared to root, which could be associated with low translocation of NPs from roots to shoots.nCuO has also shown to affect plants in different ways. Reports indicate that nCuO did not affect seed germination in zucchini10 and maize ;however, other reports mentioned root length reduction in wheat and duckweed ,and DNA damage in radish , perennial ryegrass , and annual ryegrass .The activity of antioxidant enzymes such as catalase and ascorbate peroxidase can be affected in Cu exposed plants. These enzymes, which are overproduced under abiotic or biotic stress, protect plants from reactive oxygen species damage. Excess ROS can damage proteins, lipids, and DNA.Hou et al.reported that Cu2+, up to 10 mg L1 , increased CAT activity in duckweed in a concentration-dependent manner; however, a reduction in CAT activity was observed when Cu2+ was higher than 10 mg L 1 . A similar trend was observed in APX activity on duckweed treated with copper sulfate.25 nCuO were also found to increase CAT activity in cucumber and wheat.Besides the effects of nCuO on seedlings growth and CAT and APX activity, no reports were found about the effects of Cu NPs/compounds on nutrient uptake by crop plants. Recent literature has shown that nanoparticulate forms of Cu are more effective against pathogenic fungi than the corresponding bulk forms.Thus, very likely, in the near future, there will be an intensive use of nCu and nCuO in agricultural practices. In addition, CuPRO 2005 and kocide 3000 2- based materials have been cataloged as nanoparticulate Cu species. These compounds are intensely used in agricultural production due to their fungicidal properties.Thus,hydroponic vertical farming the possibility of Cu-based NPs contamination of food supply and entrance into the food chain is increasing. However, it is not well understood yet if their effects are different from those of Cu bulk materials. Thus, three Cu-based NPs and other Cu compounds were evaluated in lettuce and alfalfa . The plants were selected because they are intensely produced and exposed to pesticides. Alfalfa is one of the most important forage crops worldwide and lettuce is cultivated worldwide and eaten in a raw form by many people. In this study, root and shoot elongations were measured with a ruler, macro and micro-nutrients’ uptake by using inductively couple plasma-optical emission spectroscopy , and enzyme activity with UV-Vis.The Cu particles used in this study include nanoparticulate CuO , micron-sized Cu and CuO , nanoparticulate Cu , Kocide 3000 , and CuPRO 2005 . Reagent grade CuCl2 salt was obtained from Sigma Aldrich. The size and surface charge of particles at pH 7 were determined by measuring hydrodynamic diameter and zeta potential using a Zetasizer Nano-ZS90 . A previous study showed that phosphate buffer, at the concentration used, had only minimal effects on zeta potential measurements. Primary particle size and morphology were determined via scanning electron microscopy equipped with an Oxford INCA energy-dispersive X-ray spectroscopy probe. Copper content of each particle was determined via ICP-AES .The main copper phase and crystal structure of particles were determined via X-ray diffraction . Cu NPs/compounds’ suspensions/solutions were prepared at 0, 5, 10, and 20 mg L 1 in modified Hoagland’s nutrient solution36 and homogenized by sonication in a water bath at 25 C for 30 min.

There were four replicates per treatment.Seeds of alfalfa Mesa variety and black seeded Simpson lettuce were stirred in 4% ClONa solution for 30 min, rinsed with deionized water three times and kept in DI for 24 h. Subsequently, seeds were rolled in auto claved wet germination paper towels, as described by Carrillo-Casta˜ neda et al.Ten drops of antimycotic/antibiotic solution were added to the seeds before the paper was rolled. The rolls were put into Mason jars containing approximately 10 ml of DI, incubated in the dark for four days and exposed to light for one day. After that, the young plants were transferred into magenta boxes containing 300 mL of modified Hoagland’s nutrient solution. All the boxes and lids were covered with aluminum foil to prevent algae growth. Aquarium pumps were used to aerate boxes, which were put in an Environmental Growth Chamber with light intensity of 300 mmol m2 s 1 , 25/ 20C day/night temperature, and 65% relative humidity. After 10 days of growth in the nutrient solution, seedlings were transferred to magenta boxes containing the Cu NPs/ compounds suspended in nutrient solution and were cultivated for 15 days. Subsequently, plants were removed from the growth medium, washed with tap water and rinsed with DI. The length of the primary root and shoot for each seedling was measured with a ruler and the samples were saved for further analyses. There were 20 young plants per replicate.Major physicochemical properties of the particles are presented in Table S1. nCuO has particles between 10–100 nm, but much larger particles were found in nCu . A reasonable fraction of bulk CuO particles were less than 1 mm but bulk Cu particles were much larger and diverse in morphology. Bulk Cu contains dendritic, plate-like, and irregularly-shaped particles . Kocide 3000 and CuPRO 2005 mainly consist of micron-sized spherical particles, and the main copper phase in them are orthorhombic Cu2. While bulk Cu only contained Cu as the main phase, the presence of Cu2O was observed in nCu, possibly due to higher reactivity of the nanoparticles which may lead to slight oxidation during synthesis and/or storage. All the particles were negatively charged at pH 7 as confirmed by zeta potential measurement. The presence of a small amount of carbon was observed in the nanoparticles, possibly from a surfactant used by the manufacturer to stabilize the NPs. In addition to carbon, Kocide 3000 and CuPRO 2005 also contain oxygen, sodium, aluminum, and silicon .The size of roots of alfalfa and lettuce plants treated for 15 days with Cu NPs/compounds is shown in Fig. S1. After 15 days of treatment, the roots of plants treated with Cu NPs/compounds, showed brown color compared to controls. As seen in Fig. S1,at all concentrations, all Cu NPs/compounds significantly reduced root length in both plant species. The shortest root in lettuce and alfalfa occurred in plants treated with 20 mg L 1 nCuO and nCu, respectively. The reduction of root length was 49.5% 7.8% in lettuce and 47.6% 1.0% in alfalfa. Similar results have been reported for Mung bean, zucchini, and wheat.10,16,17 Lee et al.17 reported that nCu at 200 mg L 1 affected the roots of wheat, while only at 800 mg L1 reduced wheat shoot length. Another report indicates that 10 mg nCuO/L reduced the length of root and shoot in radish seedlings by 46% and 4%, respectively.21 Moreover, 1000 mg nCuO/L, decreased the root and shoot length of radish by 97% and 79%, respectively. In our study, although the Cu concentration was high in alfalfa shoots, compared to lettuce, there was no shoot length reduction; however, bulk Cu, CuPRO 2005, Kocide 3000, and CuCl2 at 20 mg L 1 , significantly reduced lettuce shoot length . This suggests a species specific response to Cu toxicity. A previous report indicated that in Vigna unguiculata, Cu caused a reduction in shoot growth because it also caused a reduction in Fe.In our study, almost all the Cu NPs/compounds reduced Fe in tissues of lettuce and alfalfa . We hypothesize that differences on Cu accumulation were responsible for the different effects of Cu NPs/compounds on plant growth in both plant species. Copper in tissues triggers direct production of ROS via Fenton and induces root growth inhibition.All Cu compounds, at all concentrations, reduced P uptake in roots of both plant species .

CEC is defined as the total sum of exchangeable cations that are adsorbable by the soil

Uptake of NPs by the root has been observed at primary and lateral root junction as well according to the transverse slice. Figure 2a is one transverse slice localized at the arrow in Fig. 3c showing the junction between primary root and lateral root. We found that the yttria NPs were absorbed by the lateral roots, and particulates began to accumulate along the outer epidermis of primary roots with limited entrance into the vascular tissue of the primary root. It might happen that endodermal cell walls were blocking the entrance of aggregated yttria NPs into vascular tissue. This is shown in the upper section of the 3D visualization where no yttria NPs were observed above the root system. Besides the full view of the translocation in the cabbage root system, the distribution of yttria NPs at the micro-scale within a lateral root was detected and investigated . Figure 4a shows the localization of the micro-scale lateral root visualization. The 3D visualization of micro-scale was built by the segmented transverse reconstructed slices, and the red regions were localized yttria NPs . It is clear that roots are able to uptake the yttria NPs in ground tissue ,stacking flower pot tower which appear to accumulate in the root with limited entrance of yttria NPs into vascular tissue being transported through the xylem.

Xylem vessels are small with diameters usually smaller than 1 μm in vegetables like cabbage plants to over 100 μm in vessels found in trunks of large trees . Vessels allow nutrients contained in water to be distributed throughout the plant. For NPs, however, if they aggregate, the blockage is expected, that is what we have observed in this study. Long term studies might show that yttria NPs might provide more negative than positive effects on plant growth and development as found with other NPs. Using K-edge subtraction image technique with dualenergy X-ray scanning, the concentration of target NPs can be calculated. This method has been discussed elsewhere. Cadmium is not essential but highly toxic for higher plants even at a trace level, with the clear exception of certain hyper accumulator plant species . There is an increasing body of evidence showing that Si has many direct and indirect beneficial effects on the growth of plants subjected to various forms of abiotic stress including Cd stress . So far, numerous studies have demonstrated that Si can enhance resistance or tolerance to Al , Mn and salt toxicity in plants . By contrast, less work has been done about possible roles of Si in Cd tolerance, although Chen et al. reported that application of silicon-containing steel sludge and furnace slag could decrease Cd uptake by wetland rice. More recently, we have shown that Si-mediated alleviation of Cd toxicity in pakchoi could be attributed to Si-suppressed Cd uptake and root-to-shoot transport . However, the underlying mechanisms are still poorly understood.

More importantly, studies have been focused mainly on the roles of Si in alleviating heavy metal toxicity in Si-accumulating graminaceous plant species such as rice and maize , while less work has been done on the possible roles of Si in dicots such as pakchoi, bean and strawberry that do not accumulate much amount of Si in their tissues . In this study, we show that Si was able to mitigate Cd toxicity in roots of pakchoi, with respect to antioxidant enzymes and non-enzymatic antioxidants, and histochemical characterization using two contrasting pakchoi cultivars that differ greatly in response to Cd exposure. To confirm Si-mediated roles in antioxidative defense system, we performed histochemical staining experiments with Evans blue and Schiff’s reagent . The Evans blue was applied to determine the loss of plasma membrane integrity and the Schiff’s reagent to determine the degree of peroxidation of membrane lipids . For the Cd sensitive cultivar , the roots treated with both concentrations of Cd alone were stained to different extents, and under the higher Cd treatment, the roots were stained extensively. And the roots became lighter straining in the treatment with Cd plus Si compared with the Cd treatment alone. For example, root tips were more heavily stained in the Cd1 treatment than in the Cd1Si treatment. For the Cd-tolerant HYD, very similar changes were also observed in the roots . Furthermore, the Si beneficial effects on the protection of cell membrane against Cd-induced oxidative damage were more significant in the Cd-tolerant plant roots than in the Cd-sensitive plant roots. For the Cd-sensitive cultivar , addition of Cd significantly decreased SOD activities in roots compared with the control, which was intensified with increasing Cd concentrations . The activity of SOD was increased by 47.3%, 12.0% and 9.6% in the plants treated with Cd plus Si compared with the corresponding Cd treatments without Si, respectively .

For the Cd-tolerant cultivar , very similar changes were noted in SOD activity in the Cd treatments with or without Si added, with an exception that no significant differences in SOD were found between the Cd1 treatment alone and the control . For the sensitive cultivar , CAT activity in the Cd treatment significantly decreased with increasing Cd concentrations compared with the control. Addition of Si significantly increased CAT activity in Cd-stressed pakchoi roots compared with Cd treatment alone throughout the whole experiment . For example, addition of Si increased CAT activities by 3.7%, 28.4% and 25.7%, respectively, at 0, 0.5 and 5.0 mg L-1 Cd, compared with the corresponding Cd treatments alone. For the Cd-tolerant cultivar , very similar results were obtained of CAT activities in the Cd treatments with or without Si, with an exception that addition of Si did not result in significant differences in CAT activities between the lower and the higher Cd treatments . For the Cd-sensitive cultivar, addition of Si significantly increased APX activities in roots by 55.1% compared with the control. The activity of APX was 16.7% higher in the Cd1 plus Si treatment than in the Cd1 treatment alone, compared to 11.4% at the Cd2 level . For the Cd-tolerant cultivar, very similar changes were observed in APX activities in the Cd treatments with or without Si, with an exception that significant increases in APX activity were found between the Cd plus Si treatment and the Cd treatment alone .While agriculture in wealthy nations has entered the domain of mass market and industrialization, subsistence farming is still practiced in vast swaths of the developing world. In this model of agriculture, the output of a family’s farm is primarily used to feed that family. This agricultural context can have profound consequences for human health, as such limited diets contribute to micro-nutrient malnutrition. Iron deficiency is the most common deficiency, affecting over 2 billion people including many women of childbearing age and children . The associated costs are immense in terms of both human life and economic output . Because communities are often constrained to specific crop species by environmental and cultural factors, it is necessary to pursue local crop improvement in addition to larger scale efforts. Natural variation within closely related species provides the means to manipulate agronomically important traits, iron uptake among these,danish trolley reducing the severity of iron deficiency in subsistence cultures. Staple cereals including rice and wheat have a wide range of variation in iron uptake and spatial allocation among their cultivars; this has been leveraged previously to enhance their iron uptake properties . Foxtail millet is a critical subsistence crop in Northern China, India, and parts of Africa . Additionally, S. italica and its wild ancestor, S. viridis, are emerging model organisms. S. italica is a relatively recent domesticate, and can be considered a subspecies of Setaria viridis in spite of their phenotypic differences .

Indeed, the two species are capable of cross pollination and exhibit a continuous spectrum of morphological traits. In light of this, they are often denoted Setaria italica subsp. viridis and Setaria italica subsp. italica. Throughout this thesis, groups of individuals containing both subspecies will be referred to as either Setaria or the Setaria species complex. Due to its close relationship with economically important crops like maize and wheat , small stature, relatively short life span, status as a C4 plant, and sequenced genome, S. italica subsp. viridis is becoming a preferred subject for genetic studies . Iron acquisition does not occur in a vacuum; ionic homeostasis is intricately connected . The concentration of iron plays a role in influencing the concentration of other ions through its control over plant height. Ions such as zinc, copper, and manganese share enough properties with iron to be chelated by PS and taken up by the same transporters , though the transporters do seem to be exclusively regulated by iron status. In rice, iron deficiency associated upregulation of the iron transporters OsIRT1 and OsIRT2 contributes to cadmium uptake . Ion interactions with the soil solution also impact the bio-availability of other, complementary ionic species. If there are multiple ions held very tightly to a given colloid within the soil, they may work to ‘shield’ nearby ions from plant roots, thereby decreasing the availability of an ion that is typically fairly accessible . The interconnection of ions in the grasses is not entirely dependent upon iron; magnesium and cadmium compete for translocation in barley , calcium impacts concentrations of manganese, potassium, strontium, rubidium, and zinc . It is clear, then, that the relative concentration of one ion in the soil can impact the ultimate concentration of different ions in the plant, and that any attempt to understand the genetics underlying the homeostasis of one ion must necessarily address the complex dynamics of the complete ionic milieu. The interconnection of the concentration of various ions has led researchers to the concept of the ionome of an organism; that is, its mineral nutrient and trace element composition . The ionome is essentially the inorganic component of an organic system. The study of the ionome is referred to as ionomics. This process relies on the quantitative measurement of the ionic composition of a living organism as it varies with genetic background, developmental time points, and physiological conditions . Ionomics relies on the simultaneous sampling of the entire ionome; this sampling is often conducted with the use of Inductively Coupled Plasma-Mass Spectroscopy . Because iron is central to the growth, development, and ultimate reproductive capacity of plants, the different availabilities of this nutrient in different soils provides a strongly variable selective pressure. Though S. italica appears to have been domesticated from S. viridis in Gansu Province, Northwestern China in around 5900 BP , it has since been cultivated in many diverse regions, including ‘Southeast Europe, South and Central China, the Far East, the Americas, Australia’, and parts of Africa . Given this wide pattern of cultivation, it is likely that S. italica has been cultivated in many different soil types. Indeed, the USDA’s Germplasm Resources Information Network contains 772 S. italica accessions at time of writing. The documentation for these accessions contains information such as the exact coordinates of collection, which indicate the diversity of soils in which S. italica has been cultivated during the last century. The diversity of soil characteristics is commonly defined by the USDA’s 12 order system . These orders differ in a wide variety of properties, many of which are important to ionic homeostasis in the plants that live upon the soil. Among these characteristics are the percentage of organic matter, mean annual soil temperature, base saturation, pH and cation exchange capacity .CEC is largely dependent upon pH: the number of exchangeable cations increases in the soil solution as pH increases . Phytosiderophore uptake in the grasses also depends upon pH, and therefore on soil type, with lower pH contributing to increased PS uptake . There is evidence that soil type influences PS exudation as well: the Tamaro wheat cultivar appears to exude different levels of PS in different soils . This is consistent with the differential induction of PS in media with different iron availabilities . Plants working to obtain mineral nutrients not only face the obstacle of extracting insoluble iron from the soil, they also face significant competition from organisms in the rhizosphere for these resources.

Data were acquired with a MTP DAS-1 resistivity meter with 8 potential channels

To minimize these ambiguities and to develop a more robust approach for non-invasive in-situ root imaging, we aim to develop iCSD inversion code that does not rely on prior assumptions on root architecture and function and use rhizotron experiments to validate the iCSD approach.The phrase “inversion of Current Source Density” was introduced by Łęski et al. to describe the 2D imaging of current sources associated with the brain neural activation. Similar inversion methodologies have been developed for the interpretation of the self potential data, where the distribution of naturally occurring currents is investigated . With regard to active methodologies, Binley et al. developed an analogous approach for detecting pollutant leakage from environmental confinement barriers. Although there are physical and numerical intrinsic differences between application of the iCSD to detect brain neuronal activity and current pathways in roots, we decided to adopt the term iCSD as the general physical imaging of current source density remains valid. With iCSD, we indicate the coupling of ERT and MALM through the proposed numerical inversion procedure for the imaging of the current source density,hydroponic net pots and its correlation with root architecture. We introduce the necessary aspects regarding the ERT and MALM methods in this section. However, we direct the interested readers to more in-depth discussion about the ERT method , and to Schlumberger and Parasnis with regard to the MALM method.

In the following discussion we use ρmed to represent the 2D or 3D distribution of the electrical resistivity in the growing medium . CSD represents the 2D, or 3D, distribution of the Current Source Density within the same medium. In the case of roots, the CSD is controlled by the current conduction behavior of the roots, specifically by the leakage pattern of the root system . Both ERT and MALM are active methods. In these methods the current is forced through the medium by applying a potential difference between two current electrodes. In ERT, both current electrodes are positioned in the investigated medium, while for MALM the positive current pole is installed in the plant stem,similar to BIA . The potential field resulting from the current injection depends on CSD, resistivity of the medium , and boundary conditions. The boundary conditions are known a priori and their impact on the potential field can be properly modeled. In ERT, the current sources correspond to the electrodes used to inject current, allowing us to invert for ρmed. Then, the iCSD accounts for the obtained ρmed and explicitly inverts the MALM data to obtain current source distribution.The rhizotrons used in this study were designed to enable the concurrent direct visualization of the roots and electrical measurements. Rhizotron dimensions were 52 cm × 53 cm × 2 cm , see Fig. 2. Figure 2a shows the rhizotron setup with 64 silver/silver chloride electrodes located on the back viewing surface.

The viewing surfaces were covered with opaque material to stop the light from affecting the development of the roots. The back viewing surface was removable, allowing homogeneous soil packing for the plant experiments and convenient access to the electrodes. Besides the top opening, the rhizotrons were waterproof to enable hydroponic experiments and controlled evapotranspiration conditions during the soil experiments and plant growth. All the experiments were performed in a growth chamber equipped with automatic growth lights and controlled temperature and humidity. The temperature varied with a day/night temperature regime of 25/20 °C. The humidity ranged from 45 to 60%. For both ERT and MALM methods, the electrical potential field is characterized by a set of potential differences measured between pairs of electrodes. It is important to properly arrange the electrodes on the rhizotron viewing surface and design a suitable acquisition sequence to obtain a good sensitivity coverage of the investigated system . This is particularly true for the iCSD, as both ERT and MALM acquisitions affect its result. The 64 electrodes were arranged in a 8 by 8 grid on the back viewing surface of the rhizotron, leaving the front surface clear for the observation . For the ERT, the designed arrangement of the electrodes offers a good compromise between a high coverage on the central part of the rhizotron, which encompasses the root zone, and a sufficient coverage on the rhizotron sides to avoid an excessive ERT inversion smoothness. For the MALM, the arrangement of the electrodes is highly sensitive to the position of the investigated current sources. Because of their central positions, the electrodes are closer to the expected sources of current and thus in the region of maximum potential gradient. Hence, this electrode configuration maximizes the changes in both magnitude and sign of the measured ΔV associated with a change in the CSD distribution.

The electrode diameter was 1.5 mm. The penetration of the electrodes into the rhizotron was 4 mm ± 1 mm. To evaluate the possible distorting effects of the densely populated electrodes on the potential field distribution, a test was performed with low conductivity water . The test showed no resistivity anomalies, which may be caused by the presence of the electrodes . Therefore, while rhizotron setups with electrodes only on the sides were successfully adopted , we found that the current setup represents a better solution for iCSD experiments .For the ERT acquisition over the 2D grid of electrodes, we chose a dipole-dipole skip 2 configuration. For each skip 2-couple of injection electrodes the remaining skip-2 couples of electrodes were used as potential dipoles . The associated complete set of reciprocals was also acquired, the resulting acquisition sequence contained 3904 data points . Following the ERT data acquisition, the MALM data acquisition required little setup adjustments and time. As the two current electrodes are fixed, the use of a multichannel resistivity meter significantly reduced the acquisition time and, consequently, supported the acquisition of more robust data sets. Electrode 1 was used to inject the current into the plant stem, while electrode 64 was used as a return electrode in the growing medium . The remaining 62 electrodes were used to map the resulting potential field. A sequence with 204 ΔVs was used. Considering the grid in Fig. 2a, the sequence included the vertical, horizontal, and diagonal ΔVs between adjacent electrodes. While 61 ΔVs would provide all the independent differences,blueberry grow pot the 204 ΔV sequence was preferred because of its redundancy and consequent lower sensitivity to acquisition errors. The acquisition time remained relatively short as the multichannel instrument was optimized with fixed current electrodes that allowed 8 ΔVs to be measured at once.

The iCSD inversion that we developed was based on the physical principles of a bounded system in which linearity and charge conservation were applied to decompose the investigated CSD distribution into the sum of point current sources. This provided a discrete representation of the root system portions where the current leaks from the roots into the surrounding medium. Because of the linearity of the problem, the collective potential field from multiple current sources is the linear combination of their individual potential fields. As such, the measured ΔV can be viewed as and decomposed into the sum of multiple ΔVs from a set of possible current sources. These possible current sources are namedViRTual electrodes . As purely numerical electrodes, they are simulated by mesh nodes representing possible current sources, but with no direct correlation with the real electrodes used during data acquisition. Basically, the VRTe were distributed to represent a grid over which the true CSD distribution is discretized. In order to account for any possible CSD, a 2D grid of 306 VRTe was arranged to cover the entire rhizotron . The charge conservation law implies that the sum of the current fractions associated with the VRTe has to be equal to the overall injected current, which is provided by the resistivity meter. If we normalize the injected current to be equal to 1, the sum of the VRTe weights has to be 1 as well. Briefly, for Ohm’s law, normalizing the current to 1 is equivalent to calculating the resistance, R, from ΔV. Then, the use of R simplifies the presentation of the numerical problem. Once the VRTe nodes are added to the ERT-based ρmed structure, the potential field associated with each of the VRTes is simulated with BERT. From these simulated potential fields, the same sequence of 204 R is extracted, each corresponding to a single VRTe. Each extracted sequence contains the resistances that would be measured in the laboratory if all the current sources were concentrated at the VRTe point .Synthetic numerical and laboratory experimental tests were performed in order to evaluate the capabilities of the setup and inversion routine to couple the ERT and MALM approaches for the iCSD. In the numerical tests both the true source response and VRTe responses were calculated with BERT. Figure 3 shows an explanatory numerical test with inversion of a point source, and the associated Pareto front that was used to select the optimum regularization strength. As this first experiment was performed to specifically test the inversion routine, a homogeneous ρmed was used in order to avoid influence from the baseline resistivity distribution complexity. For the second experiment, the laboratory tests were conducted. Because of the ρmed heterogeneity of any experimental system, these laboratory tests need to include the ERT inversion, and the use of the obtained ρmed as input in the iCSD.

The true current sources were obtained using insulated metallic wires inserted into the rhizotron . The insulating plastic cover was removed at the tips of the metallic wires to obtain the desired current sources. Six experimental tests were performed using different numbers and positions of these current sources. The rhizotron was filled with tap water and left to equilibrate to achieve steady state conditions of water temperature and salinity, thus minimizing ρmed heterogeneity and changes during the experiment. Changes in ρmed during the ERT and MALM acquisition periods would make the ERT-based ρmed less accurate and compromise the iCSD. To make sure ρmed was stable, a second ERT was performed after the MALM acquisition and compared with the initial measurement. The conductivity of the solution was also measured in several locations of the rhizotron with a conductivity meter to validate theρmed obtained from the ERT inversion. This setup allowed the acquisition of good quality data sets since less than 5% of the data were discharged during the data processing. Because of the controlled laboratory conditions, the ρmed obtained with the ERT was stable and consistent with the direct conductivity measurements. The quality of the ERT inversion was also confirmed by comparing the model responses with the acquired data . Similarly, the acquired iCSD data were plotted against the resistances calculated with the CSD distribution obtained from the iCSD. The tests also allowed a more informed definition of the VRTe grid. For our setup, a spacing of 3 cm provided a good compromise between resolution, stability, and duration of the iCSD routine. The 3-cm spacing also agrees with the ERT resolution, which would not support a higher iCSD resolution. Successive numerical tests were based on the 8- source laboratory tests shown in Fig. 4. These tests aimed to 1) link laboratory and numerical tests to evaluate the influence of the numerical iCSD routine and laboratory setup on the overall iCSD stability and resolution; 2) account for a more complex CSD, given by the 8 wire-tip sources that were used to simulate distal current pathways; and 3) account for possible ρmed heterogeneity. To address goals 1 and 2, the position of the 8 sources was replicated in the numerical tests and a test with homogeneous ρmed was included to simulate the water resistivity of the laboratory tests. To address goal 3, heterogeneous ρmed were tested.In order to account for the heterogeneous ρmed the following modeling steps were carried out. First, a true ρmed was assigned to the mesh cells of the rhizotron ERT model. We included homogeneous, linear, and quadratic resistivity profiles in the y direction, see Fig. 5. Second, the ERT acquisition was simulated with the ERT laboratory sequence and 3% of Gaussian error, in line with reciprocal and stacking errors observed in the laboratory data sets. Third, the forwarded ERT data sets were inverted following the exact laboratory procedure.

Nitrogen hastened economic maturity of the celery

Ammoni losses does not appear to be a very significant pathway for loss. Soil pH decreased markedly a few centimeters from the emitter .An experiment was conducted to evaluate celery response, N uptake and N leaching as related to form, amount, and method of N-fertilizer application under drip irrigation . Fertilizer forms included ammonium sulfate and a slow release fertilizer applied to the soil before planting and urea ammonium nitrate applied with the irrigation water during the growing season. AS and SR were applied at 314 kg Nlha and UAN was applied at 168-, 336-, and 504 kg N/ha. Two irrigation variables were 400 and 500 rom of water application. The experiment was conducted on San Ernigdio sandy loam soil at the University of California South Coast Field Station near Santa Ana. Celery seedlings were-obtained from a commercial nursery and planted in two rows per bed. The distance between centers of each bed was 1 m. Three row spacings were used for each treatment. Bi-wall drip irrigation tubing was placed on the soil surface down the center of each bed equidistant from each celery row. The soil-applied fertilizer was placed approximately 5 em below the soil surface,fodder system for sale immediately below the drip line. All of the AS and SR fertilizer was applied prior to transplanting. Ten per cent of the N to be applied with the drip irrigation water was applied through the drip lines prior to planting.

Thereafter, UAN was applied during each irrigation to the appropriate rows until approximately one month before harvest after which fertilizer injection was stopped. Irrigation was usually done three times a week and occasionally a fourth irrigation was applied during hot, dry, windy periods. There was no marketable yield on the plot which did not receive N fertilizer. Treatments receiving UAN with the irrigation water had higher yields with 500 as compared to 400 rom water application. On the other hand, higher yields were obtained under the 400 as compared to the 500 rom irrigation treatment for the AS and SR fertilizer treatments. Furthermore, at comparable N application rates, the soil applied N resulted in significantly lower yields than application of N with the irrigation water. With the higher water application, more N leached below the root system than at lower irrigation rates, based on the pattern of N distribution in the profile at the end of the experiment. Application of N.with the irrigation water is superior to application of the N to the soil in a preplant operation. Split applications to the soil are mechanically impractical with drip irrigation. Celery requires continuously high soil water content which results in which leaching 105ses of soil-applied fertilizer. Applying the N with the irrigation water made it available to the plant continuously over the growing season and apparently much of the N was extracted by the root system as the water flowed past the roots; there was very little difference in the amount of N leached beyond the root zone for the different water application treatments. Nevertheless, there was some deep N movement with the drip system even under low N application.

The SR fertilizer produced a higher yield than AS. Nitrogen was retained within the granules and slowly released during the growing period, and was thus partially protected from leaching. In fact,observation at the end of the experiment indicated that not all of the N had been released from the capsules by harvest. The N left in the soil after cropping is a potential source for groundwater pollution. Whether the N does in fact get carried to the groundwater depends upon a number of subsequent factors such as precipitation and/or irrigation which will carry the N downward. A barley crop was grown on the experimental area after the celery crop was removed without further N application to measure residual N in the profile which could be removed by barley. For the plots receiving UAN, there was an increase in N uptake from plots receiving increasing amounts of UAN during the celery crop. For comparable amounts of N application, much more N was taken up in the barley on the plots receiving the SR as compared to the AS and UAN treatments. This is partially a result of the N retained in the capsules and also the N in the profile which had moved beyond the celery roots but remained accessible to the deeper rooted barley crop. For both AS and SR there was more N uptake from the 400 mm irrigation treatment as compared to the 500 rom irrigation treatment, suggesting that the higher water treatment moved some N beyond the depth of recovery by the barley plant. Two experiments were conducted at the University of California South Coast Field Station on celery using a sprinkler irrigation system . This system was chosen to experimentally apply a continuous water amount variable across the plot to determine the interaction between fertilizer N and water application on celery gro~h. This was accomplished by using “line source” irrigation where water application is highest next to the sprinkler line and decreases at increasing distances from the sprinkler line.

A standard sprinkler system applied water uniformly over the entire plot with fertilizer. Then irrigation was applied through a single line which provided high water application near the line which decreased with distance away from the line. In one experiment, different fertilizer N treatments were applied to the soil in rows perpendicular to the line source. This provided an opportunity to determine the growth under the different fertilizer treatments at different rates of water application. In the second experiment, some of the N was applied with the irrigation water under the standard irrigation and again these plots were perpendicular to the line source which allowed a comparison of celery growth in response to differential water application. In experiment 1, a comparison is made between preplant and no preplant fertilizer treatment and different amounts of N applied with the irrigation water. The preplant fertilizer was AS whereas the injected fertilizer was DAN. In experiment 2, all of the N was applied to the soil in bands with none being applied with the irrigation water.There are apparently counterbalancing factors in the effect of quantity of water application to celery. The celery prefers high soil water availability to maintain high turgor, rapid growth, and succulence. On the other hand, large applications of water could leach N from the root zone and decrease its availability to the plant. Since N was applied with the irrigation water throughout the growing season,fodder growing system it could partially compensate for N that might be leached. This was observed to be the case under the drip irrigation system. However, on the AS150 UAN150 treatment, the added leaching of preplant N under the highest water application may not have been sufficiently compensated for by the N applied in the irrigation water. For the soil-applied fertilizer treatments the results from the control and AS300 treatments clearly illustrate the interaction between water and N availability as dual determinants of crop yield. The highest yields under these two treatments were observed at the intermediate water application and decreased yields were found under the lower and higher water application. Presumably the reduced yields at lower water application was because of less than optimum water content and the decreased yield at high water application was due to low N availability, much of the N having leached from the root zone. Since all N in both of these treatments was in the soil at the beginning, this N was exposed to the greatest potential for leaching loss. A small experiment was conducted on celery using furrow irrigation , In this experiment, four rates of N fertilizer were applied. All treatments received 100 kg N/hapreplant as ammonium phosphate. The remainder of the N was applied as side dressed N split in two equal increments of urea applied 29 and 49 days after celery transplanting. At harvest time, there was no significant difference in yield for the three highest N application rates but the lowest had significantly lower yield. Even though the final yield was not significantly different for the three higher application rates, there was increasing rate of growth with increasing N application during the early stages, with the growth rate reaching a peak and then diminishing thereafter.

The lowest N treatment had not yet reached its peak grow~h rate at harvest time. These data illustrate that celery growth can be stimulated by high N but that with sufficient time the effects of N application rate on yield tend to diminish. Two experiments were conducted using furrow irrigation with broccoli as the test crop . The first experiment was conducted on a San Emigdio sandy loam soil very low in inherent fertility. The experimental variables consisted of three fertilizer N application rates , two irrigation treatments , and two N application procedures . One-third of the fertilizer was applied to the soil in a band adjacent to the plants prior to planting on all plots including those which were to receive N with the irrigation water. Thereafter the soi1- applied fertilizer was applied in two side dressings of 16-20-0 fertilizer or injected into the irrigation water in equal proportions during each irrigation. Hereafter the treatments will be referred to “inject” where fertilizer was applied with the irrigation water and “soil-applied” when fertilizer was applied as the two side dressings. Irrigation frequency was the same for both irrigation treatments but 30% more water was applied at each irrigation for the ET +30 treatment than for the ET treatment. Fertilizer was injected into the irrigation water continuously by a proportioning unit except at the end of each irrigation when the lines were flushed with water. The furrows were dammed at the end to avoid runoff. Under the lower irrigation treatment there were no differences in yield due to method of applying the N. Under the higher irrigation there was consistently higher yield when the N was applied with the irrigation water as compared to side dressing. Furthermore, there was lower yield under each fertilizer rate for the higher irrigation treatment. Irrigation to replenish water loss through ET provided for both high yields and higher N use efficiency as compared to excessive irrigation. Potential advantages of injecting N with the irrigation water include savings of labor and energy for the two side dress operations and avoiding plant damage during the side dress operations. In this experiment, efficient energy, N, and water use were consistent with high crop production. A second experiment was conducted with broccoli and furrow irrigation on the Sorrento loam. The furrows were 90 ill long. Yields were higher under the higher N application as compared to the lower application and for a given N application the average yields were always higher for the side dressing as compared to applying the N with the irrigation water. More N was removed in the crop than applied as fertilizer for the lower application rate and a very high percentage of the N applied under the higher rate was removed in the crop. Tensiometers installed indicated there was very little water movement deep into the profile, thus minimizing leaching losses. Under these conditions, N is used efficiently and growth appears to be related to the rate of application. In the second experiment, there was higher average yield when the fertilizer was applied as side dressing rather than injected with the irrigation water whereas there was no difference in the first experiment. The more uniform application throughout the row with continuous injection in the first experiment may have been a factor as compared to injection during part of the irrigation cycle during the second experiment. No harvestable yield resulted from unfertilized plants. There was a trend toward decreased plant growth with increased water application for the treatment which did not receive N, due to leaching of the small amount of N in the profile. The lower fertilizer treatment resulted in fairly uniform growth at different water contents except at the very dry end where there was relatively less growth, while the plots receiving the higher fertilizer treatments had a trend toward higher production with higher water content.

The activity of NHXs plays a significant role in the transport of K+ into the vacuole

In the rice genome, there are at least five NHX members with OsNHX1-OsNHX4 belonging to the type-I and OsNHX5 to the type-II . The operation of the NHXs also affects the pH gradients across the different luminal compartments . Both type-I and type-II NHXs have different subcellular localization; they may have different mechanisms in salt tolerance, and other not yet defined functions. Large efforts have been paid to increasing the plant salt tolerance by over expressing NHX orthologue genes from different species. In rice, over expression of the NHX1 homologue genes from Oryza sativa, Chenopodium glaucum and Atriplex dimorphostegia resulted in the enhanced tolerance to salt stress . However, these NHX proteins actually mediate both Na+ /H+ and K+ /H+ exchange and their functions cannot be solely explained by accumulating Na+ into vacuole . The type-II NHXs have also been shown to be involved in salt tolerance. AtNHX5 and AtNHX6 are the only two type-II NHXs inArabidopsis. Although their function appears to be redundant, the double knockout Atnhx5nhx6 displayed high sensitivity to salt stress . In tomato, the over expression of LeNHX2, a type-II NHX located in prevacuolar compartments and Golgi,industrial drying racks enhanced salt tolerance at high external K+ levels . Plants use a number of strategies to deal with high salinity .

Halophytes rely heavily on the homoeostasis of three major inorganic ions to maintain their osmotic and turgor pressure under saline conditions, while glycophytes predominantly increase the synthesis of compatible solutes . For salt tolerant plants grown in high external saline conditions, the efficient compartmentation of Na+ into vacuole and other cell organs via intracellular NHXs is of importance, together with the extrusion of Na+ at the roots, via plasma membrane-bound antiporters such as SOS . Jerusalem artichoke belonging to the same Asteraceae family as sunflower is an herbaceous perennial plant that have potential as a bio refinery crop . H. tuberosus is highly tolerant to infertile, drought and saline stresses and its variety Nanyu No. 1 could fully emerge in the coastal region containing 1% salt at soil surface layer and grow well in soil containing 0.5%–0.6% salt or under irrigation with 50%–75% sea water . In this study, we isolated two putative NHX genes, HtNHX1 and HtNHX2, from H. tuberosus cv. NY-1 and examined their roles in enhancing the tolerance of salt stress and nutrient deficiency in rice, Arabidopsis and yeast. Our results indicate that HtNHX2 could function in enhancing the plant tolerance to salinity stress and improving plant nutrient efficiency.Considering the essential roles of K+ in balancing uptake and distribution of anions, particularly, nitrate and phosphate and improving plant growth, we characterized the effects of expressing HtNHX1 and HtNHX2 on enhancing the tolerance of rice to nutrient deficiency. The reduction in nutrient supply to 1/4 of its full strength decreased the growth and biomass of WT and HtNHX1-expressing rice equally while did not affect the growth of HtNHX2-expressing rice plants .

The HtNHX2 lines showed about 35%, 25% and 45% increase in total biomass, N and P contents, respectively, in comparison with WT , indicating that HtNHX2 expression could remarkably enhance the root acquisition of N and P, thus improving plant growth under limited nutrient supply conditions. In contrast, N and P contents in the HtNHX1 lines were lower than those in WT. 15N quantification analysis of rice grown in low N and K conditions showed that HtNHX2 expression did not alter root N contents, but significantly increased shoot N contents , suggesting the enhanced N translocation from shoot to root, in addition to the increase in total N uptake. The notion of the role of HtNHX2, but not HtNHX1, in enhancing K+ uptake and mediating N and P accumulation was further supported by the growth of rice plants in infertile paddy fields . Compared to WT, HtNHX1 lines showed 20% lower straw weight and 40% lower grain yield, resulting in significant lower grain harvest index due to a larger portion of unfilled grains . The HtNHX1 lines contained significant higher total N, similar P and lower K+ in the straw at mature stage . Remarkably, the HtNHX2 lines showed 45% increase of total grain yield and 90%, 40% and 13% higher N, P and K+ contents in the straw .Plant NHX-type antiporters play important roles in salt tolerance and the maintenance of cellular K+ and Na+ homoeostasis and the regulation of intracellular pH. In this study, we cloned two highly identical salt-stress up-regulated NHX orthologues genes, HtNHX1 and HtNHX2 from H. tuberosus . HtNHX1 and HtNHX2 share identical 50 -UTR, 30 -UTR and coding sequences, except for a fragment of consecutive 342 bp in HtNHX1 which is absent in HtNHX2. Whether the two genes were alternatively spliced or evolutionary independently generated is not clear. According to their sequences, both genes are predicted to belong to the type-I family and to locate at the vacuole .

The ability of HtNHX1 but not HtNHX2 to confer resistance to hygromycin in yeast and Arabidopsis and different cellular localization in rice would suggest functional differences between the two isoforms. Notably, the role of HtNHX2 in conferring tolerance to nutrient deficiency, in addition to conferring tolerance to salinity, provides a novel insight on NHX functions .We observed that HtNHX1 or HtNHX2 improved rice tolerance to salt stress , but they did not have significant influence on growth and uptake of K+ and Na+ at normal growth conditions . At salinized conditions, HtNHX1 increased rice K+ and Na+ accumulation, that is keeping the same ratio of K+ /Na+ as that in WT . This indicated that HtNHX1 indistinguishably transported K+ and Na+ , which is similar to that of AtNHX1 and AtNHX2 in Arabidopsis . In addition, the K+ and Na+ contents of plant expressing HtNHX1 were higher in the roots and the shoots than in WT plants , supporting the role of HtNHX1 as type-I NHXs in sequestering Na+ into the vacuoles . Interestingly, the accumulation and distribution of K+ and Na+ in plants expressing HtNHX2 differed from that seen in HtNHX1 plants, as HtNHX2 plants accumulated relatively more Na+ and K+ . These results were in agreement with the localization of HtNHX2 to intracellular compartments other than the vacuole . Endosomal trafficking and the vesicle fusion to the vacuole are important components of the response of plant cells to salinity. Transporters playing roles in cation sequestration, such as NHX1 and H+ -pumps ,commercial greenhouse benches depend on vesicular trafficking for their delivery to the tonoplast. Thus, the expression of endosomal NHXs may influence protein trafficking from the Golgi/TGN to the vacuoles , necessary for the response to high salinity. The upregulation of endosomal NHXs, such as AtNHX5 in response to salt stress, but not to osmotic shock, supported the role of endosomal AtNHXs . In addition, salinity induced bulk endocytosis, promoted the rapid increase in vacuolar volume and the accumulation of sodium into the vacuole . These results suggest that the over expression of endosomal NHXs, such as HtNHX2, contributed to salt tolerance through several mechanisms: increasing vesicle fusion to the vacuole, contributing to the accumulation of Na+ and by increasing the trafficking of transporters that could explain the improved nutrient uptake in the transgenic plants. HtNHX1 and HtNHX2 rescued the salt sensitivity of the Arabidopsis Atnhx5 nhx6 double mutant at the same extent , but HtNHX1 was more effective than HtNHX2 in conferring tolerance of Arabidopsis to external hygromycin . It has been shown that AtNHX5 and AtNHX6 located at endosomes, TGN and prevacuolar compartments and nhx5 nhx6 double knockouts displayed abnormal vesicular trafficking and sensitivity to salinity . In rice, HtNHX2 showed similar cellular localization as AtNHX5 ; thus, it is plausible that HtNHX2 may play a similar role as AtNHX5 in the regulation of endosomal ion and pH homoeostasis. Nevertheless, these results should be taken with some caution as both HtNHX1 and HtNHX2 were expressed under the control of a constitutive promoter and abnormal localization due to unregulated expression cannot be ruled out.Potassium is a plant essential nutrient, and vacuolar K+ plays roles in the regulation of cellular volume and tissue expansion. Although plants grown under different K+ supply can change their vacuolar K+ contents, cytosolic K+ is maintained at a relative constant level of around 60–100 mM .

The over expression of AtNHX1 in tomato led to increased vacuolar K+ accumulation and higher tissue K+ contents. In the knockout Atnhx1, Apse et al. reported a reduction in K+ /H+ and Na+ / H+ exchange and reduced cell expansion. Bassil et al. observed lower vacuole pH and K+ concentration in the nhx1 nhx2 mutant, further supporting the role of AtNHX1 and AtNHX2 in driving the uptake of K+ into the vacuole. The expression of HtNHX2 improved plant growth and grain yield at low K+ concentrations , and the tissue K+ contents of the transgenic plants were higher than the WT and HtNHX1-expressing plants. Notably, HtNHX2 localization appeared to be similar to that of AtNHX5 , that is intracellular vesicles, TGN. , thus associated with vesicular trafficking and storage protein sorting . In yeast, nhx1 mutants have been shown to have impaired vacuolar biogenesis and protein sorting . In tomato, LeNHX2 colocalized with prevacuolar and Golgi markers and appeared to be more selective to K+ than Na+ . Interestingly, HtNHX2 lacks a continuous 114 amino acids stretch at predicted six to eight transmembrane domains of HtNHX1 . This stretch also comprises a segment of eight amino acids that is absent in type-II NHXs . Although the occurrence of a shorter HtNHX2isoform retaining its transport function has not been reported before, shorter AtNHX1 isoforms have been reported . Using DNA shuffling mutagenesis, Xu et al. generated a 296 amino acids deleted AtNHX1, AtNHXS1, in which, several transmembrane domains and the C-terminus hydrophilic tail containing the CaM-binding domain were deleted. Interestingly, such large fragment deletion did not alter its vacuolar localization but increased the Na+ /K+ selectivity and Na+ /H+ exchange activity, enhancing the NaCl tolerance of yeast expressing AtNHXS1 . Notably, plants expressing the endosomal HtNHX2 accumulated more K+ and Na+ than the transgenic rice expressing the vacuolar HtNHX1. Although it is possible to speculate that the deletion in HtNHX2 resulted in increased transport activity of the antiporter, a detailed structure/function analysis is needed to assess this point. The increased tolerance to nutrient deficiency displayed by transgenic rice plants expressing HtNHX2 was associated with increased K+ contents and enhanced N assimilation as seen by the increase in 15N translocation from root to shoot and the amounts of N, P and K+ in the straw of plants grown in infertile soil . K+ is the major accompanying ion for the translocation of NO3 and sucrose in plants . About 40%–90% of root acquired K+ could be retranslocated from the shoot via the phloem and recycled through the roots , and K+ recycling in plants can act as an important signal for feedback control of nutrient uptake . It could be speculated that the expression of HtNHX2 improved the synthesis/delivery of the transporters of N, P and K+ to plasma membranes by improving vesicular trafficking and/or protein targeting . The functions of NHX-type transporters have been extensively studied , and the effects of the over expression of NHXs on salt tolerance have been shown in different crop species . Here, we show that although the expression of HtNHX1 conferred salt tolerance, only the expression of HtNHX2, a shorter isoform ot the known type-II NHXs, conferred salt tolerance under nutrient stress conditions.The tubers of H. tuberosus genotype NY-1 after dormancy were germinated on moist sand in an incubator. Uniformly germinated slices of the buds were selected, sown in sands and then transplanted into 1/2 Hoagland nutrient solution in the greenhouse as described previously . After emergence of the fourth leaf, the seedlings were treated with 50, 100, 200 and 300 mM NaCl for 24 h or with 100 mM NaCl for 12 h before the sampling for analysis of HtNHX1 and HtNHX2 expression. Each treatment was replicated three times. Arabidopsis thaliana wild-type and nhx5 nhx6 double knockout mutants were provided by Professor Eduardo Blumwald at University of California .