To produce 18 m3 of three-dimensional printed habitat structure on Mars encompassing a volume of 120 m3 for six crew, a current estimate of shipped mass cost is 24 T, of which 11.4 T is dry salts to mix with the MgO in the Martian soil for printer feed stock.Substituting this mixture with PHB, which has a density of 1250 kg m23 , the required 18 m3 of habitat has a mass of 22 500 kg.Consider the following unoptimized bio-manufacture scenario: two 2000 l bioreactors, each at a working volume of 1500 l, resulting in the production of 111.6 kg d21 of PHB.In this scenario, 202 days are needed to synthesize 22 500 kg of PHB, an identical duration as that required for methane bio-manufacture.The total empty mass of the bioreactors is 1.6 T , an 86% savings over the 11.4 T of shipped dry salts.Drawn power can be calculated to be 7.221 W using, and the volumetric size of the bioreactors works out to be 9.896 m3.It should be possible to improve the PHB accumulation percentage of A.platensis with synthetic biology approaches, because such techniques have allowed the cyanobacterium Synechococcus sp.PCC7002 to stably accumulate PHA at up to 52% of the cell dry mass when using the complementation of a cyanobacterial recA null mutation with the Escherichia coli recA gene on a plasmid expression system.Synthetic biology efforts to combine the benefits of C.necator with the cyanobacterium SynechocysThis sp.PCC 6803 of §3.3 have also been documented.Such efforts are important because they lend credence to the idea of one organism that is capable of producing multiple commodities: fuel, biopolymers and as we shall see,dutch bucket hydroponic possibly pharmaceuticals as well.At present, workarounds for accelerated pharmaceutical expiry involve the launch of one or more small unmanned spacecraft to restock crew supplies as needed.

However, the practicality of these missions hinges on the location of a supply depot, conducive launch opportunities, travel time, etc., with corresponding implications on astronaut health in the event of a medical emergency.Thus, the cost of pharmaceutical shelf-life includes the cost of crew safety in addition to the cost of a resupply mission.The former is not easy to quantify but is regarded as paramount to reduce.The advantageous role of biotechnology in pharmaceutical manufacture is known, and examples include antibiotics, antibiotic alternatives such as bacteriophages , and even cancer chemotherapy.Biotechnology can also facilitate the synthesis of antibiotics such as doxycycline and tetracycline from an acetate substrate, albeit with involved steps.To tackle the typically complicated substrate sequence and pathway identification problem to desirable chemical targets, a computerized suite of tools has been recently developed.This platform also suggests the amount of synthetic biological engineering that is necessary to accomplish the manufacture of the target.For instance, Anderson et al.explain how acetaminophen can be produced by modifying the chorismate pathway , wherein p-aminobenzoic acid that is made from chorismate is transformed into 4-aminophenol using the 4ABH gene taken from a mushroom, and this 4-aminophenol can then be used as a substrate to produce acetaminophen with the E.coli nhoA gene.Unfortunately, this strain is not directly suitable for space application, on account of the reduced availability of the resources that the strain uses.However, it should be possible to implement the above path to acetaminophen by harnessing a similar chorismate pathway in an autotroph of electronic supplementary material, table S1 that uses readily available inputs on space missions.The cyanobacterium SynechocysThis sp.PCC 6803, for example, has the genes for chorismate synthase and the trpG and trpE genes that are homologues of the respective E.coli pabA and pabB genes, allowing it to generate an analogue of p-aminobenzoic acid called anthranilate.Moreover, anthranilate can also be acted upon by the 4ABH gene and 4-aminobenzoate hydroxylase.It is therefore plausible that acetaminophen can be synthesized by SynechocysThis sp.PCC 6803 after the insertion of the 4ABH and nhoA genes , and testing of this hypothesis in the near-future is anticipated.

As Ungerer et al.indicate, unnatural gene insertions into SynechocysThis sp.PCC 6803 and subsequent organism optimization can allow for a substantial rate of production of a desired target.It is possible to estimate the volumetric yield of acetaminophen bio-manufacture in SynechocysThis sp.PCC 6803 with the data in: an equivalent carbon output for the reported productivity rate of 171 mg l21 d21of ethylene occurs when 1.5 mmol l21 d21 of acetaminophen is produced, i.e.230 mg l21 d21.This yield is sufficient, because pharmaceuticals require low concentrations of an active ingredient, for example, a single tablet dose of acetaminophen is 325 mg.Hence, even accounting for losses, only a few days of acetaminophen manufacture starting from protected, inactive bacteria will be required to replenish stocks of the pharmaceutical that have expired early because of space radiation.This renders crews independent from resupply spacecraft that could take at least 210 days to arrive after Earth launch.Moreover, the anticipated productivity rate should render the pharmaceutical production apparatus eminently portable and suitable for use on long voyages, because the required bioreactor working volumes need only be around 2 l or less.According to Stoker, this volume in a 3 l bioreactor would draw about 43.7 mW.A calcareous soil with low organic matter content was collected, air-dried and sieved through a 5-mm screen.Sewage sludge was collected, air dried and grounded to about 5 mm granules, and applied as such to each soil according to the treatments.The soil and SS were analyzed for general characteristics, nutrients and heavy metals according to the standard procedures.The major soil and SS characteristics are showed in Table 1.Greenhouse pot experiments with the following treatments were conducted in a randomized complete block design with four replications: 1) zero SS application ; 2) 20 ton SS ha-1; 3) 40 ton SS ha-1; 4) 80 ton SS ha-1; 5) 160 ton SS ha-1; 6) 80 ton Cd-enriched SS ha-1; 7) 160 ton Cd-enriched SS ha-1; and 8) 80 kg DAPha-1 that represented the recommended fertilizer rate.The SS was enriched with Cd by addition of CdCl2 to a Cd concentration of 50 mg kg-1.Each pot was filled with 7 kg air dry soil, air dry soil-SS mixture or air dry soil fertilized with DAP per treatments.Three radish seedlings per pot were planted in seven-liter pots.Plants were watered on alternate days to maintain water content at approximately field capacity.At the end of the growing period , the whole plants were harvested from each pot.

The fresh weights of both shoots and tubers were recorded.Plant parts were oven-dried at 700C for 48 hours, then dry weights recorded.Plant parts were ground to a fine powder using a laboratory mill with 0.5 mm sieve.The milled plant samples were analyzed for nutrients and heavy metals by standard procedures.The soil at the end of the experiment was analyzed for the same parameters mentioned above.Addition of original and Cd-enriched SS increased shoot N content compared to both control and DAP fertilizer treatments.All rates from 40 to 160 ton SS ha-1 of both original and Cd-enriched SS gave the highest shoot and tuber N contents with no significant differences among each treatment.The tuber N content was the lowest for the DAP fertilizer treatment and was even lower than that of the non fertilized control treatment; possibly this decline could be attributed to the dilution effect caused by the higher growth with DAP fertilizer application.Shoot P content increased with increasing rates of SS and was the lowest for the control,dutch buckets system and then the DAP fertilizer treatments.Tuber P content was higher with SS application being the highest with higher rates of SS.The lowest tuber P content was obtained with the control treatment and the DAP fertilizer treatment gave a value higher than that obtained by the control and the SS rate of 20 ton ha-1.Both shoot and tuber K contents were not affected by SS application except by the highest rate of the Cd-enriched SS which might attributed to the increased concentration caused by the stunted growth.Increased plant concentrations of N, P and K with SS application is mainly attributed to the increased levels of the available forms of these nutrients in the soil following SS application.Moreover, Oudeh et al.reported that sludge contains large amounts of N and P, and found an increase in their levels in SS-treated soil when compared to a control or even to the chemical fertilizer treatments.It should be mentioned however, that excessive application of N and P by high rates of sludge application can be detrimental to crop production and the environment.On the other hand, other researchers reported that little differences were found between Thissue concentrations of N, P, and K in corn grown on SS amended soil versus the control.However, it has been reported by Rubin et al.that applying SS at a rate sufficient to meet the plant’s N requirement would provide an excess of P but insufficient K.Therefore, they stated that balancing the amount of nitrogen applied in the SS with the plant’s requirement is an important management factor to avoid nutrient imbalance.Shoot Fe concentration increased with application of SS at rates equal to or higher than 40 ton ha-1.The highest two application rates resulted in lower shoot Fe when the SS was enriched with Cd.Similar trends, but with lower values, was obtained for tuber Fe concentration.Concentrations of Mn in the shoot increased with SS addition, with the highest values obtained by the highest two rates.The treatments effect on tuber Mn concentration was not significant.Application of SS at rates equal to or higher than 40 ton ha-1, similarly or equivalently increased both shoot and tuber Zn concentrations over other treatments.Addition of 80 and 160 ton ha-1 of SS, with or without Cd enrichment, resulted in the highest Cu concentrations in both the shoot and the tuber of radish.The highest shoot Cd concentration was obtained with the application of the highest rate of the Cd-enriched SS followed by the next highest rate.Application of original SS increased shoot Cd only at the highest rate of 160 ton SS ha-1.A similar trend was observed with tuber Cd concentration.SS may facilitate higher mobility for heavy metals through the effect of dissolved organic compounds from the applied SS that form soluble complexes with Cd and Zn and to a lesser extent with Cu and Pb.It is well documented that most of the heavy metal accumulation occurs in the roots of vegetable crops.Differences between root and leaf content was larger for Cd than for Pb, indicating that the translocation of Pb from roots to leaves is more limited than for Cd.

Frost and Ketchum found higher Cd in leaves with SS application.On the other hand, Oudeh et al.found that Zn and Cu but not Cd concentrations were higher in roots than in shoots.In this study, such a differentiation in Pb and Cd distribution were not observed.DTPA-extractable Fe, Mn, Zn and Cu increased with increasing SS application rates.Addition of 160 ton of both SS and Cd-enriched SS ha-1 resulted in the highest values for DTPA-extractable micro-nutrients.On the other hand, DTPA-extractable Pb increased similarly by all rates of SS application rates.Both the control and the DAP fertilizer treatments resulted in the lowest values of the DTPA-extractable Pb.Addition of the Cden riched SS resulted in the highest DTPA-extractable Cd being higher at the higher application rate.Comparatively, much lower DTPA-extractable Cd levels were obtained for the application of the original SS.However, the highest two rates of the original SS yielded higher values of DTPA-extractable Cd compared to the lower rates, but the highest levels of Cd were found with the addition of the Cd-enriched SS.The relationships between the DTPA-extractable Cd and the Cd concentration in radish shoot and tuber were significant and followed quadratic equations.The Cd concentration tended to slowly increase with increasing DTPA-extractable Cd within the range from 0.05 to 0.15 mg kg-1, after that a steep increase was observed.It can be summarized that application of SS to calcareous soil improved soil organic matter, available P, and micro-nutrients as well as the plant growth.However, application of higher rates was unnecessary for plant growth and yet has the potential to cause nutrient imbalance in the soil.Cd-enriched SS resulted in stunted growth at the highest rate and tend to accumulate Cd in the soil and plant parts.Therefore, it can be concluded that SS application to calcareous soils improves plant growth and soil fertility but at high rates of SS and SS rich in Cd should be avoided.

Differences in root impedance have been demonstrated to have a major impact on penetration resistance.Fifteen blocks measuring 33 x 33m were marked out in an even grid with five blocks in each of three north-south columns representing the three treatment replicates.Blocks were separated from each other by 3m wide strips which were sown with grass seed after the first trial year was sown.Within each block, trial plots measured 1.55m wide by 2.1m long and were sown at 250 seed m-2 in early November 2007 or at 360 seed m-2 in early April 2008 for winter and spring varieties, respectively.Fifty-six lines of winter barley and 64 lines of spring barley, identified as a population useful for association genetics , were sown to each cultivation treatment.Prior to the year of sampling, the trial was sown in three successive seasons.The data presented in this paper were derived from samples taken from only the plough treatment, which represents the typical agricultural practice for the region,strawberry gutter system and minimum tillage, which is gaining popularity because of fuel costs and perceived benefits to soil conservation.Flag leaf samples were taken at growth stage 21 from both barley plants grown in pots and each genotype grown in the field and samples were kept on ice until they could be frozen.Samples were freeze dried before milling to a flour.One hundred mg of shoot material was digested in 5 mL of 18 M H2SO4 at 360 °C for 20 mins, after which an excess of 30% hydrogen peroxide was added until digests cleared.

Concentrations of P in diluted digests were determined by reaction with malachite green.All data are presented as the mean of three replicates and bars represent standard errors of the mean.Significant differences were established using ANOVA, and treatment means compared by LSD.Relationships between both shoot biomass and P concentration in the glasshouse experiment were related to the P added using regression.From these regression it was possible to determine the critical P concentration for barley to reach 75% growth in this soil.Mean data from the different cultivation treatments within each population of genotypes were compared using regression and r2 and 95% confidence intervals derived.The critical P concentration to achieve 75% growth was also plotted.All data were tested for normality prior to analysis.The shoot dry weight of spring barley cultivar Optic increased significantly to an asymptote with increasing P addition to the soil.This growth response was described by an exponential rise to a maximum of the form y = a + b.Using this equation it was possible to calculate the amount of P that needs to be added to this soil to achieve a critical proportion of maximal growth.For the purposes of this experiment 75% of maximal growth was used to define the critical growth of the barley plants, which was achieved by an addition of 175 mg P kg-1 soil.The shoot P-concentration of these plants also increased significantly with the addition of P to soil,however this did not reach an asymptote with the relationship being described by a linear equation.This allowed the calculation of the critical P concentration in the shoots to achieve 75% growth which, on this soil, was 2.56 µg P g-1 DW.These results demonstrate that soils typical of the arable north east of Scotland are responsive to the addition of P when barley is grown and that reasonably large additions of P are required to achieve growth approaching the physiological maximum for the barley.When grown in the field, winter barley genotypes had significantly greater P concentration in flag leaves than spring barley genotypes.This may be an indication of the greater root growth associated with winter germplasm which would allow greater access to P resources.

Within the spring and winter genotypes there was significant variation in shoot P concentration ranging from 1.7 to 3.0 and 1.5 to 2.5 µg P g-1 DM, respectively.Of particular interest was that some of the winter barley genotypes were able to achieve the critical shoot P concentration necessary to achieve 75% maximal growth in this soil under conventional cultivation.In contrast none of the spring genotypes were able to achieve this.Moreover, none of either winter or spring varieties passed the critical level in the minimum till cultivation treatment.There was also a significant impact of the cultivation treatment on the concentration of P in shoots, however this effect was dependent on which set of genotypes were considered.In the winter germplasm the minimum till cultivation treatment caused a decline in the shoot P concentration while in the spring germplasm there was a slight increase in the average shoot P concentration.The impact of the cultivation treatment on the ability of the various genotypes to acquire P is demonstrated even more strongly when the shoot P concentration of the various genotypes is correlated between the different cultivation treatments.The lack of correlation demonstrates that there is no significant relationship between the genotypes, either winter or spring germplasm, when grown in the different cultivation treatments.In fact, only 32% of the winter genotypes fall within the 95% confidence intervals of the relationship, while even fewer, only 19%, of the spring genotypes fall within the corresponding confidence interval.This suggests that the vast majority of barley genotypes have a differential response in P-nutrition to cultivation treatments, with approximately equal numbers being more suited to minimum tillage and vice versa.Interestingly, of the five winter genotypes which showed P concentrations greater than the critical for 75% maximal growth in conventional cultivation treatments, only one demonstrated a suitability for growth in minimum till cultivation.The availability and cycling of P for the conventionally cultivated and minimum till treatments are likely to be quite different due to differences in the biological and physiocochemical conditions of these treatments.For example microbial community structure and size, rooting depth, and water relations are all likely to be different.These differences will go some way to explaining why there is no relationship between genotypes of the association mapping populations between the two treatments.It is likely that the traits which allow some genotypes to successfully acquire P in the conventional plough cultivation treatment will be quite different to those of which benefit P-nutrition in minimum tillage treatment, therefore compromising the use of the genetic variation to predict useful genotypes for the different treatments.Results here are analogous with results on studies on genotypic variation in P-use efficiency in wheat which demonstrate that the ability of the wheat lines to acquire P was greatly dependent on soil type and only a small proportion of the variability in shoot growth and P content was attributable to genotypic differences.

It is therefore imperative that screening for P-use efficiency in cereals be performed on soils rather than in hydroponics or agarand ideally on a range of different soil types and under different agronomic treatments as the genetic control of this trait on one soil is likely to be different under a range of conditions.The development of SNP based high throughput genome wide assays which use hundreds of SNP’s to identify single markers from association mapping populations are a powerful tool to allow genotypic variation identified in studies like this to identify markers for P-use efficiency in barley.However, while there may be significant genotypic variation in these association mapping populations, the genetic component of this variation is not always robust between treatments.In conclusion, we have shown that genotypic variation in P-use efficiency is present in association mapping populations grown in the field.However, such variation is not related between soil cultivation treatments, where differences in root abiotic stresses will have a large impact on root growth and nutrient acquisition.It is therefore important that when screening for markers for multi-mechanistic traits, such as P-use efficiency, that screening occurs in a number of environments so that the robustness of the genotypic variation can be tested.In recent decades,grow strawberry in containers ubiquitous use of groundwater in California and other parts of the world have led to chronic groundwater overdraft and water quality issues.Worldwide recognition of groundwater depletion and its adverse effects on human and environmental well-being has increasingly led to actions, policy, and legislative change to manage water resources jointly and sustainably.For example, the Sustainable Groundwater Management Act formed in 2014 in California requires groundwater users to achieve long term groundwater sustainability by managing groundwater extraction and intentionally replenishing water in groundwater aquifers.One possible technique for groundwater replenishment is agricultural managed aquifer recharge in which farmland is flooded using excess surface water in order to recharge the underlying aquifer.As most agricultural fields have lower infiltration capacities compared with dedicated recharge basins, Ag-MAR is designed to capture high-volume excess surface water by flooding large areas of farmland at relatively low recharge rates of less than one meter per Ag-MAR event.Ideally, flooding for Ag-MAR is preferably done on fallow fields or during crop dormancy periods, when agricultural fields have the potential to serve as percolation basins for groundwater recharge.O’Geen et al.recommended potential areas in California for Ag-MAR using an index that combines five soil characteristics: deep percolation, root zone residence time, chemical properties, topography, and surface conditions.An ideal Ag-MAR site will comprise an effective deep percolation , adequate crop tolerance for flooding, low soil salinity, leveled soil surface, and lack of compaction and erosion.O’Geen et al.identified an area of 22,500 km2 of agricultural land , mostly in the Central Valley, as having excellent to moderately good potential for Ag-MAR.Root zone residence time is defined as the duration of saturated conditions in the soil root zone after water is applied.It is a key factor in Ag-MAR, as prolonged saturated conditions in the rhizosphere can damage perennial crops due to O2 deficiency— hypoxia—a well-known situation in agricultural soils under intensive irrigation.

Root functioning, nutrient and water uptake, vegetative growth, and crop yield are all affected by low O2 concentration.Moreover, in flooded fields, complete depletion of O2—anoxia— may occur, which affects crops severely and can lead to plant mortality.Therefore, quantifying the soil aeration status is of great importance for implementation of Ag-MAR on fields with perennial cropsD.A.Webb], grapes [ViThis vinifera L.], alfalfa [Medicago sativa L.]) with potentially no yield lost.Soil aeration is essential to support aerobic soil respiration which includes O2 consumption by plant roots and microbial population.The O2 level in unsaturated soils depends highly on the gas phase since the O2 concentration in atmospheric air is ∼250 mg L−1 while water in equilibrium with the atmosphere contains dissolved O2of only ∼8 mg L−1.The two transport mechanisms for meeting soil oxygen demand are diffusion of O2 in both the liquid and gas phases and convection of O2 in flowing water or air.However, low diffusion rates in water and the low solubility of O2 in water make the liquid phase contribution for O2 replenishment negligible.Therefore, gas transport is considered the main mechanism for supplying soil O2, and gas diffusion, driven by the O2 concentration gradient between the atmosphere and the soil, is considered the dominant transport process.The composition and magnitude of the gas phase in soils determines the soil aeration status, which controls O2 availability to soil respiration.Soil aeration status can be evaluated by the following quantifiers: volumetric air content , O2 concentration in the gas or liquid phase , O2 diffusion rate and soil redox potential.Under flooded conditions, as expected during Ag-MAR, O2 supply by gas transport is suppressed by soil saturation, as water occupies most of the air-filled soil pores.If ponding occurs, the ponded water layer at the soil surface will act as a barrier that effectively blocks soil gas exchange with the atmosphere, because the diffusivity of O2 in water is 10,000 times lower than in air.Under these conditions, hypoxia is expected to develop rapidly as the result of root respiration, microbial activity, displacement of air by water and impeded soil gas exchange.The depletion rate of O2 from the soil solution and entrapped air pockets will depend on temperature and respiration activity, so depletion will be slow at low temperatures and low organic-matter content.Upon water logging, the rate of decline in soil O2 from ∼21% to 0% can vary, ranging from one to several days.To minimize crop damage during Ag-MAR due to poor soil aeration, an adequate supply of O2 to the root zone must be provided.This can be achieved by natural- or forced-aeration of the root zone.

Nitrogen was applied as NH4N03 by continuous injection with the irrigation water at constant concentrations of 25, 50, and 75 mg Nil. The two irrigation treatments allowed the soil water suction at a 25 em depth to drop to approximately 10 or 30 cb at time of irrigation. Pragmatically the two irrigation treatments resulted in irrigation of approximately every day compared to every other day applications. There was no effect of the irrigation treatment on the yields. During the period of peak production, the lowest yields occurred under the lowest N application treatments. However, this yield deficit appears to have been partially offset by greater production of these low N treatments during the earlier part of the season. The total yields of the lowest N treatment were 8~ to 90 per cent of the maximum and yields were not consistently improved by applying N at higher rates. As the rate of N application increased, an increasingly greater proportion of the N taken up by the crop was partitioned into the vines and foliage. In contrast,flood tray an increase in N application rate from 120 to 585 kg/ha increased the amount of N removed in the fruit by only 40kg/ha. Thus, the total amount of N actually removed from the field depended very little on the rate of N fertilizer application. It is apparent from the fruit yield and N uptake data that although total N removal continued to increase with increasing N application, the additional N assimilated in the two higher treatments did little to improve fruit yield but served primarily to elaborate vegetative material and increase the storage of N in the vines.

Therefore, N fertilizer efficiency expressed in terms of the per cent recovery of applied N, the amount of N used by the plant to increase yield, or the economic return for unit of N applied, decreased substantially at the higher N rates. It should be noted, however, that since almost all of the excess N taken up was partitioned into the vines, a large amount of N could be returned to the soil and should serve as an important N source for subsequent crops. The results of the tomato experiment as well as some of the sprinkler-celery experiments illustrate that relatively high N concentration during the initial stages of growth is important. Even though the plant removal is small in total quantity, the plant has a very small root system and must, therefore, be provided with nitrogen in relatively high concentration to prevent nitrogen deficiency at the early stages of growth. On the other hand, as the plant grows larger and the root system becomes more extensive, supplying nitrogen in the amount necessary for uptake appears to be adequate. Nitrogen applied in excess of this amount may potentially be lost through denitrification or leaching depending upon water-soil interactions.Furrow-irrigated sweet corn was grown on the UCR Experiment Station to test the effects of a nitrification inhibitor and three rates of irrigation water application . On a sandy soil, the nitrification inhibitor significantly increased the average weight of the corn stalks. On a sandy loam soil, the nitrification inhibitor increased average N concentrations per stalk at low rates of water application but had little effect at higher application rates. Generally soil N levels were maintained higher with the nitrification inhibitor, and the effect was more pronounced at higher rates of water application .As an example , researchers at Berkeley Lab use 3D printing to create molds for casting the biocompatible polymer polydimethylsiloxane into the upper portion of a fluidics chamber.

This is subsequently attached to a microscope slide, completing the chamber. This is then placed into a sterile container, providing a gnotobiotic system for studying microbial interactions. This chamber design includes a port for growing the model grass Brachypodium distachyon, tubing that allows sterile introduction and sampling of microbes and metabolites, and a lighting system. EcoFABs have been tested with different growth media , different microbes , and plants . Data collected in duplicated systems in different labs had excellent reproducibility .An EcoPOD is a larger-scale fabricated ecosystem that allows direct and intensive monitoring and manipulation of replicated plant-soil-microbe-atmosphere interactions over the complete plant life cycle. The EcoPODs will be equipped with environmental controls to carefully manipulate and control temperature, humidity, and other important climatic parameters both above and below ground. The EcoPODs will also be outfitted with sensors capable of monitoring soil moisture, oxygen, and specific nutrients, and the output from these sensors will be integrated using computer models to gain a coherent understanding of the environment inside the EcoPOD. Multiple EcoPODs will allow scientists to examine the impact of differences in types of soils, microbes and plants on ecosystem interactions. The first prototype EcoPOD will be installed at Berkeley Lab in November 2020, based on a model developed at the German Centre for Integrative Biodiversity Research and manufactured by the German company Umwelt Geräte Technik . This prototype will be used as a base-case for the development of further sensors and imaging capabilities. A vision for 16 EcoPODs, to be placed in the BioEPIC building at Berkeley Lab, is being developed; with a full complement of EcoPODS up to 4 binary variables can be investigated at one time was composed of a brief introduction, followed by breakout into three groups . By holding it at the Genomic Sciences PI meeting between sessions, we aimed to capture broad perspectives from across the Biological and Environmental Science Biological Systems Science /Bioenergy Research Center portfolio. Each group discussed three different topics, guided by facilitators, and recorded by note-takers: Science that can be enabled by fabricated mesocosms; Defining the key technological needs for these systems How to use fabricated mesocosms to bridge the scale gap between lab and field.

We encouraged broad discussion including criticism. The charge questions which were provided to all participants are included in Appendix 3.Discussions at the workshop included identifying which scientific questions would benefit from these technologies, challenges in implementing the experiments, benefits of de-risking experiments at smaller scales prior to testing in more complex experimental systems, missing capabilities, as well as the experimental limitations of fabricated ecosystems. Additionally, there was extensive discussion about data standards and sharing. Finally, we discussed approaches for steering and community access.EcoPODs present a larger challenge to understand reproducibility, due to the complexity of the system. EcoPODs are lower-throughput/lower-replication and it would help future users design and plan their experiments if data on reproducibility was available. In addition, some concerns about the reproducibility or relevance of past Ecotron-like experiments were raised. Therefore, it was suggested that a set of EcoPOD experiments are performed which show the extent of reproducibility of data collected. This would include experiments which test reproducibility within one unit between lysimeters . These would test single variables and would be paired with field work. One such project is currently underway funded by Berkeley Lab internal research funding , but further investment in this type of project was considered highly beneficial. These experiments could also provide valuable bench marking datasets which can be used as references for field work. For example, iron-deficient experiments in the EcoPOD would generate sets of signature plant and microbial transcripts and metabolites that can be used to survey for iron deficiency in the field. Because of issues of bio-availability, soil micro-nutrient concentrations are poor indicators of nutrient sufficiency,ebb and flow tray but these signatures would provide insights into what each organism is experiencing. Finally, these experiments would act as a pilot for developing data standards and collaborating with system modelers and field researchers, to ensure that data sets that are collected both in fabricated ecosystems and at field sites are One of the key drivers behind developing the fabricated ecosystem infrastructure at Berkeley Lab was to provide a platform for computational modelers to work with experimental biologists to improve our understanding of these complex ecologies. There is a strong scientific need to try to reconcile models driven by lab data and those driven by field data. By identifying which variables are driving the discrepancies between the field and lab data, it will help to prioritize research to understand why we are not able to accurately describe these processes. As fabricated ecosystems move to more complex microbial communities, the combinatorial possibilities will preclude investigating all possible communities within EcoFABs. Therefore it is critical to make effective use of simulations to predict the most informative community structures. KBase capabilities in flux balance modeling will be central to construction of community models to predict microbial interactions and activities. In particular, it was clear that fabricated ecosystems would be valuable for improving our understanding of the role that heterogeneity has on models. Effects seen in the lab are often stronger than those observed in the field e.g., nutrient responses due to masking from heterogeneity. For example, how “strong” does an effect in the lab have to be to be important in the field. Can we predict this? In addition, the flow of lab to EcoFAB to EcoPOD and field give some unique opportunities to consider the challenges of modeling across scales using data from the different systems. Discussions also emphasized that modelers with experience working with experimental data should be included in experimental teams from the beginning, so that the modeling work is fully integrated with the proposed work. In addition, teams should also include those with experience of collecting and working with field data.

This will enable the scientists to learn to speak the same language from the start, as well as to design experiments that inform different aspects of plantmicrobial-soil science. The Berkeley Lab team are encouraged to help facilitate these interactions, and funding to support these interdisciplinary teams is considered a priority.Experiments using the EcoPODs will control a number of environmental parameters, including light intensity, relative humidity, above and below ground temperature, soil moisture and, potentially, gas concentrations. Sensors used to control these parameters, as well as measures of plant and microbial response to these conditions have the potential to generate terabytes of information for each replication of each experiment. Many of the comments centered on what to do with this flood of data and how to ensure that it was stored in a way to make it fully accessible and understandable to future researchers. There was significant discussion on the best way to proceed for standardization of data. There are already a number of ecology community data and metadata standardization groups out there that are working in this area. Two of the leaders in this area are the National Microbiome Data Collaborative and micro BEnet for metadata standards. It is important to communicate between different groups, such as experimentalists and modelers, so that the collected data will be useful to the larger scientific community. Additionally, some coordination between different experimental disciplines will be necessary.Some data-intensive applications have the potential to create computational bottlenecks. Examples given included multi-spectral imaging of root architecture and next-gen sequencing outputs. Can we leverage efforts by other groups that are working in this area, such as ARPA-E, to manage data on this scale? We will also need to develop more efficient ways of sharing these datasets with other members of the scientific community.Below ground sensing in the field is a major challenge due to access. The EcoFABs have the advantage of allowing visual access to the root system including use of optodes for monitoring pH, CO2, O2, and etc throughout the experimental time course. This allows the user to gather both spatial and temporal information of both root,microbe, and chemistry, as well as any introduced perturbation such as nutrient source. However, the spatial scale is necessarily limited. This is particularly challenging when looking at interactions that take weeks to develop, such as between roots and mycorrhizal fungi, or using larger plant species, such as sorghum. Additionally, there are concerns about the effect of light on the below-group ecosystem which is being addressed by using plastics for the root chamber that don’t transmit light in the range sensed by roots. Future challenges include developing miniaturized sensors, e.g., for soil matrix moisture, that are compatible with the EcoFAB. The EcoPODs have the advantage of being much larger, but with necessarily less visual access to the below ground processes. The capture of the growing root architecture in 3D continues to be a challenge.

Electrical Resistivity Tomography and ElectroMagnetic Induction approaches have been used to image and monitor soil resistivity changes associated with the Root Water Uptake.Recent studies explored the use of multi-frequency Electrical Impedance Tomography to take advantage of the root polarizable nature.Despite these advantages, geophysical methods to date share common limitations regarding root characterization. Geophysical methods developed to investigate geological media: in the case of roots they measure the root response as part of the soil response, see Fig. 1a for the ERT acquisition. Because of the natural soil heterogeneity and variability the resolution and signal characteristics of geophysical methods strongly depend on soil type and conditions. As such, interpretation of the root soil system response is non-unique, hindering the differentiation between roots of close plants and the extraction of specific information about root physiology from the electrical signals. Unlike geophysical methods, the BIA for root investigation developed to specifically target the impedance of plant tissues, limiting the influence of the growing medium. A practical consequence is that BIA involves the application of sensors into the plant to enhance the method sensitivity.The measured BIA responses have been used to estimate root characteristics, such as root absorbing area and root mass . Estimation of these root traits is based on assumptions on the electrical properties of roots . A key assumption is that current travels and distributes throughout the root system before exiting to the soil,mobile grow rack with no leakage of current into the soil in the proximal root position . It is only in the former case, that the BIA signal would be sensitive to root physiology.

Despite the physiological relevance of the BIA assumptions and the number of BIA studies, a suitable solution for the characterization of the current pathways in roots is missing. Thus far, only indirect information obtained from invasive and time-consuming experiments have been available to address this issue . Mary et al. , and Mary et al. 2020 tested the combined use of ERT and Mise A La Masse methods for imaging grapevine and citrus roots in the field. An approach hereafter called inversion of Current Source Density was used to invert the acquired data. The objective of this inversion approach is to image the density and position of current passing from the plant to the soil. The current source introduced via the stem distributes into “excited” roots that act as a distributed network of current sources . Consequently, a spatial numerical inversion of these distributed electric sources provides direct information about the root current pathways and the position of the roots involved in the uptake of water and solutes. The numerical approach used to invert for the current source density is a key component required for such an approach. Mary et al. used a nonlinear minimization algorithm for the inversion of the current source density. The algorithm consisted of gradient-based sequential quadratic programming iterative minimization of the objective functions described in Mary et al. .Because no information about the investigated roots was available, the authors based these inversion assumptions and the interpretation of their results on the available literature data on grapevine root architecture. Consequently, Mary et al. highlighted the need for further iCSD advances and more controlled studies on the actual relationships between current flow and root architecture. In this study, we present the methodological formulation and evaluation of the iCSD method, and discussits applications for in-situ characterization of current pathways in roots. We perform our studies using laboratory rhizotron experiments on crop roots. The main goals of this study were: 1) develop and test an iCSD inversion code that does not rely on prior assumptions on root architecture and function; 2) design and conduct rhizotron experiments that enable an optimal combination of root visualization and iCSD investigation of the current pathways in roots to provide direct insight on the root electrical behavior and validate the iCSD approach; and 3) perform experiments to evaluate the application of the iCSD method on different plant species and growing medium that are common to BIA and other plant studies.

The relationship between hydraulic and electrical pathways has been the object of scientific debate because of its physiological relevance and methodological implications for BIA methods . A key and open question concerns the distribution of the current leakage . The distribution of the current leakage is controlled by 1) the electrical radial and longitudinal conductivities , and 2) by the resistivity contrast between root and soil. With regard to σcr and σcl, when σcl is significantly higher than σcr, the current will predominantly travel through the xylems to the distal “active” roots, which are mostly root hairs. Based on the link between hydraulic and electrical pathways, this is consistent with a root water uptake process where root hairs play a dominant role while the more insulated and suberized roots primarily function as conduits for both water and electric current . On the contrary, if the σcr is similar to σcl, the electrical current does not tend to travel through the entire root system but rather starts leaking into the surrounding medium from root proximal portions. The coexistence of proximal and distal current leakage is in line with studies that suggest the presence of a more diffused zone of RWU, and a more complex and partial insulation effect of the suberization, possibly resulting from the contribution of the cell-to-cell pathways . Soil resistivity can affect the distribution of the current leakage by influencing the minimum resistance pathways, i.e., whether roots or soil provide the minimum resistance to the current flow. In addition, soil resistivity strongly relates to the soil water content, which, as discussed, affects the root physiology. Therefore, information on the soil resistivity, such as the ERT resistivity imaging, has the potential for supporting the interpretation of both BIA and iCSD results. Dalton proposed a model for the interpretation of the plant root capacitance results in which the current equally distributes over the root system. Because of the elongated root geometry this model is coherent with the hypothesis of a low resistance xylem pathway . Numerous studies have applied Dalton’s model documenting the predicted correlation between root capacitance and mass . In fact, recent studies with wheat, soy, and maize roots continue to support the capacitance method . Despite accumulating studies supporting the capacitance method, hydroponic laboratory results of Dietrich et al. and other studies have begun to uncover potential inconsistencies with Dalton’s assumptions. In their work, Dietrich et al. explored the effect of trimming submerged roots on the BIA response and found negligible variation of the root capacitance. Cao et al. reached similar conclusions regarding the measured electrical root resistance .

Urban et al. discussed the BIA hypotheses and found that the current left the roots in their proximal portion in several of their experiments. Conclusions from the latter study are consistent with the assumption that distal roots have a negligible contribution on root capacitance and resistance. Because of the complexity of the hydraulic and electrical pathways, their link has long been the object of scientific research and debate. For recent reviews see Aroca and Mancuso ; for previous detailed discussions on pathways in plant cells and tissues see Fensom , Knipfer and Fricke , and Findlay and Hope ; see Johnson and Maherali et al. in regard to xylem pathways. See Jackson et al. and Hacke and Sperry for water pathways in roots. Thus, above discrepancies in the link between electrical and hydraulic root properties can be, at least to some degree,ebb and flow table attributed to differences among plant species investigated and growing conditions. Among herbaceous plants, maize has been commonly used to investigate root electrical properties . For instance, Ginsburg investigated the longitudinal and radial current conductivities of excited root segments and concluded that the maize roots behave as leaking conductors. Similarly, Anderson and Higinbotham found that σcr of maize cortical sleeves was comparable to the stele σcl. Recently, Rao et al. found that maize root conductivity decreases as the root cross-sectional area increases, and that primary roots were more conductive than brace roots. By contrast, BIA studies on woody plants have supported the hypothesis of a radial isolation effect of bark and/or suberized tissues . Plant growing conditions have been shown to affect both water uptake and solute absorption due to induced differences in root maturation and suberization . Redjala et al. observed that the cadmium uptake of maize roots grown in hydroponic conditions was higher than in those grown aeroponically. Tavakkoli et al. demonstrated that the salt tolerance of barley grown in hydroponic conditions differed from that of soil-grown barley. Zimmermann and Steudle documented how the development of Casparian bands significantly reduced the water flow in maize roots grown in mist conditions compared to those grown hydroponically. During their investigation on the effect of hypoxia on maize, Enstone and Peterson reported differences in oxygen flow between plants grown hydroponically and plants grown in vermiculite. The results reported above and in other investigations are conducive to the hypothesis that root current pathways are affected by the growing conditions, as suggested in Urban et al. . For example, the observations by Zimmermann and Steudle and Enstone and Peterson may explain the negligible contributions to the BIA signals from distal roots under hydroponic conditions . At the same time, the more extensive suberization in natural soil and weather conditions could explain the good agreement between the rooting depth reported by Mary et al. based on the iCSD and the available literature data for grapevines in the field. 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, 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.

For culture on agar plates, seeds of different genotypes were sterilized with 75% ethanol for 10 min, washed in sterilized water three times, and sown on the Murashige and Skoog medium containing 2% sucrose and 0.8% phytoblend . The plates were incubated at 4 ◦C in darkness for two days and then were positioned vertically in the growth chamber at 22 ◦C under 12 h light/12 h dark photoperiod. After germination, five-day-old seedlings were transferred onto agar-solidified MS media supplemented with Na+ at the indicated concentrations and were grown at 22 ◦C under 12 h light/12 h dark photoperiod. For hydroponic culture, after germination and being grown on the MS plate for ten days, the seedlings were transferred to 1/6-strength MS liquid solutions and were grown under the 12 h light/12 h dark photoperiod in the plant growth chamber. Fresh liquid solutions were replaced once a week. After two-week culture, the plants were treated with 1/6 MS solutions supplemented with a range of NaCl concentrations and were grown under 12 h light/12 h dark photoperiod. We show a representative A. mangium root under hydroponic condition in Figure 1a. A cap-like structure, which was apparently consisted of the plant tissues, covered the entire root apex up to the 5 mm region behind the tip. After the structure was readily sloughed off , the root was covered again with a new one of similar size in a week after the detachment . To evaluate roles of the structure on root growth,stacking pots supplier the whole structure and resulting tissues on root surface were carefully removed with forceps, and then the roots were exposed to 0.5 mM calcium solution containing 0, 100, or 500 μM Al for 48 h.

Al induced a root bending in both roots immediately after the exposure, and the proportion in the occurrence of root bending was higher in Al-treated roots without the cap-like structure . The addition of Al slightly reduced the elongation of roots both with and without a cap-like structure, and the degree in the reduction was larger in structure-less roots . In the absence of Al, the removal of a cap-like structure hardly decreased the root elongation, although it slightly increased the occurrence of root bending . In roots without cap-like structures, this facilitation of root bending may result in a sight decrease in the Al-resistant root elongation. The present study revealed that the presence of a cap-like structure around root apices in A. mangium ameliorates an Al-facilitated bending of root growth direction. Root caps play major roles in root gravitropism through the localization of amyloplast granules and/or the basipetal flow of auxin . Our further analysis of the cap-like structure identified that a major attached position of a structure to the root was confined to columella root cap region, and the rigidity between this connection was enhanced during prolonged Al exposure periods . The undetached state of a cap-like structure may have a role in protecting inner root cap cell function from instant Al-caused damages. An apparent root bending induced by Al seems to require high concentrations, since no similar root bending in Al-sensitive plants has been reported. It is likely that a much lower concentration of Al may be sufficient for the complete arrest of root elongation until root cap cell dysfunction become recognizable as a root bending.

The present study did not find any detrimental role of a floating, cap-like structure around the root in Al-resistant root elongation, similar to previous findings on root caps . In the present study, however, we did not evaluate potential roles of undetached tissues on Al tolerance mechanisms. A cycle of detachment and formation of caplike structures imply that non-detached original tissues in the root surface area of root apex may play roles in the Al-resistant root elongation in A. mangium. Another study of A. mangium revealed that more than half of fibrillary tissue initials separated from the root surface were present beyond the entire root elongation region . In summary, we have found a novel cap-like structure of tissues for the protection of root cap cells at the root apex of A. mangium. Further characterization of the detachment pattern of the tissues from the root surface should provide better understanding of their roles in high Al tolerance mechanisms. Potassium and calcium are key elements for plant physiology, as evidenced by being the highest concentration cations in plants and, together with N and P, among the most crucial elements for plant growth and productivity. K is centrally involved in a wide range of crucial biophysical and biochemical processes in plants, including cell osmotic pressure regulation, growth, regulation of photosynthesis, ion homeostasis, control of cell membrane polarization, enzyme conformation/activation, water conservation and salt resilience. Ca has its own crucial roles including as a significant component in cellular walls, the maintenance of membrane integrity against the passive transfer of H+ , K+ , Na+ , and other monovalent cations, and biochemical signaling. Due to these various essential roles, much research has focused on the mechanisms of K and Ca uptake by and transport within plants. For Ca and Mg, another key nutrient element, isotopic fractionation by plants has been investigated both experimentally in the lab to elucidate processes of acquisition and transport, but also in the field ) as monitors of the local and global biogeochemical cycling of these elements.

The isotopic fractionation by plants of micro-nutrients such as Fe, Cu, Zn, and Mo have also been measured. In the case of K, it has been suggested that terrestrial plants represent a significant reservoir for K, with as much as 40– 70% or more of the dissolved K in the world’s rivers coming from the decay of plant matter, resulting from its crucial role as a plant nutrient. Therefore, potassium isotopic systematics potentially provides a new tool for tracking and quantifying nutrient cycling in ecological systems ; a proxy for global geochemical cycling; and a research avenue for understanding the optimal fertilization for efficient agricultural food production. To fulfill that potential, the degree of K isotopic fractionation by plants needs to be established. Two published studies do suggest that plants may fractionate K isotopes. Deviations in the 41K/39K ratio of ∼1‰ from an in-house reference material favoring the light isotope have been measured by Li et al. in several plant materials , but with no measurement of the K sources. Likewise, in their broad survey of natural geologic and organic materials Morgan et al. found that a banana and a potato purchased in a Scottish grocery had 41K/39K ∼ 0.4‰ lighter and ∼0.1 ‰ heavier respectively than seawater K, but again with no measurements of the K sources. Here we use hydroponically grown plants, with isotopically characterized K and Ca nutrient sources, to quantify and compare the isotopic fractionation of K and Ca by three species of vascular plants: soybean , rice , and wheat . These three plants were chosen to cover a selection of important food crops; and each are C3 plants. We choose to compare K and Ca isotopic fractionation for several reasons. First, the atomic weights of K and Ca are relatively close so that mass fractionations can be easily compared. Second, K and Ca have contrasting roles in plant physiology as described above, and have contrasting valencies. Third, there is a body of experimental data on Ca isotopic fractionation by plants that we can compare our Ca isotopic results to . Methods Soybean, rice, and wheat were grown from seeds and cultivated in a large hydroponic system at the U.C. Berkeley Oxford Facility greenhouse. The hydroponic solution, a modified Hoagland’s solution replete with Ca nitrate and K nitrate was replenished periodically to provide an isotopically constant source of Ca and K during plant growth.Harvested plants were divided broadly for soybeans into root, stem,grow lights supplier and leaf samples, and for rice and wheat plants into samples of roots and leaves. The plant samples were rinsed, dried, and weighed, and then ashed before complete dissolution in high-purity nitric acid. Aliquots were taken of the sample solutions for K and Ca isotopic analyses. K was separated from the sample aliquots using AG50×8 cation resin and 1 M HNO3. The K isotopic analyses were conducted on an IsoProbe MC-ICPMS at Lawrence Berkeley National Lab using a sample-standard bracketing technique where Ar-based mass interferences were removed by the introduction of Ne + H2 gas to the hexapole collision cell. Results are reported as per mil deviations of the 41K/39K ratio relative to an inhouse K standard using a spectroscopic concentration standard . We estimate that our in-house standard on the Bulk Silicate Earth scale of Wang and Jacobsen has an approximate δ41KBSE of +0.5‰. For Ca isotopic analysis, the sample aliquots were spiked with a 42Ca–48Ca double spike before chemical separation using Eichrom DGA resin eluted with 3 N HNO3 and separated Ca collected with DI H2O. The spiked Ca separates were then analyzed for isotopic composition using thermal ionization mass spectrometry with a Triton multicollector instrument using a multi-dynamic Faraday cup routine. The Ca isotopic results are reported as per mil deviations of the 44Ca/40Ca ratio from the Bulk Silicate Earth 44Ca/40Ca of 0.0212035. On this scale our long-term average δ44Ca of SRM915A is −1.0 ± 0.1 2s. Further details of the K and Ca separation and isotopic analyses are presented in the Supporting Information.

Another possible effect to consider with transport across cellular membranes is that ions would need to be desolvated before their passage. Hoffman et al. suggest through molecular dynamics simulations, that water exchange rates for solvated ions has a rate dependence on the mass of the solvated ion such that the lighter isotope is favored in desolvation, resulting in a predicted ∼2‰ fractionation in 41K/39K between a precipitating solid and the solution. However, a further feature of K+ specific channels lies in the process of desolvation/solvation of K+ during transmembrane transport. Solvated K + enters the channel where the K+ is handed off to eight protein bonded oxygen atoms replacing the role of the solvating H2O molecules, minimizing the energy required for desolvation, with resolvation occurring with the exit of K+ from the channel.Therefore, we suggest that isotopic fractionation due to dehydration of K+ accompanying K+channel transport may be minimal. Instead we hypothesize that their size selectivity as argued above is the main source of fractionation of K isotopes. We propose that the HATS, operative under low external K+ , would fractionate K isotopes less that the LATS, which in large part depends on highly selective K+ ion channels. This suggestion is supported by experiments involving cells of a marine diatom where under low external Zn concentration , the observed isotopic fractionation of Zn is four times less than under high Zn external concentration . A difference between HA vs LA transport has also been called upon by Deng et al. to explain differences in Zn isotopic fractionation between Zn nonaccumulating and Zn hyperacumulating plants. For isotopic fractionation of K in plants, a test of this would be examining K isotopic fractionation as a function of external solution K+ concentration. Another possible test would be experiments involving genetically modified plants that are capable of only either HATS or LATS K+ transport to look for contrasts in K isotopic fractionation. Roots are not only vital for anchorage and for acquisition of water and nutrients from the soil, but are also engaged in complex physical and chemical interactions with the soil. Plant roots release approximately 11–40% of their photosynthetically fixed carbon, commonly known as root exudates, into the soil . Root exudates and mucilage act as nutrient sources and as signaling molecules for soil microorganisms, thus shaping the microbial community in the immediate vicinity of the root system . In turn, microbial processes promote plant growth by aiding in nutrient acquisition, plant growth hormone production and bio-control of plant pathogens . The physicochemical characteristics of the surrounding soil are also affected by interactions between roots and the microbial community. This interplay between the different rhizosphere components is affected by spatio-temporal processes, which culminates in dynamic feedback loops that maintain the complex rhizosphere environment with physical, chemical and biological gradients that are distinct from the bulk soil .

The stage height and the width of the weir were used to compute flow using a calibrated stage-flow relationship. Pressure measuring devices including Design Analysis H355 Smartgas bubbler systems or HOBO water level loggers were used to measure stage. Data were collected and recorded every fifteen minutes at all flow stations. The fifteen minute flow data were averaged by day to calculate the daily average flow. Water samples were collected at the inlet and outlet of the wetlands on the same day at approximately two week intervals during the study period. Field sampling consisted of collecting depth-integrated water samples and recording field conditions at sites within the study area using an YSI 6600 sonde . Sondes were calibrated before sampling and calibration was confirmed within twenty-four hours after the sampling event. Water samples were collected in glass 1000 mL bottles , 1000 mL HDPE Trace-Clean narrow mouth plastic bottles , 250 mL HDPE Trace Clean wide mouth plastic bottles , 16×100 mm pretreated chlorine free glass tubes , and 40 mL trace clean vials with PTFE septa in accordance with requirements for different lab analysis and volume requirements. All bottles were rinsed with sample water prior to collection of a depth-integrated sample. Samples were immediately stored at 4°C after sampling and transported to the lab on the day of sampling.Samples were received by the laboratory the same day they were sampled,hydroponic nft logged in and inspected for damage, and stored at 4°C until filtering and analysis.

All filtration and preservation of samples were completed within 24 hours. Samples were collected, preserved, stored, and analyzed by methods outlined in Standard Methods for the Analysis of Water and Wastewater , unless otherwise indicated. Total organic carbon and dissolved organic carbon were analyzed on a Teledyne-Tekmar Apollo 9000 by high temperature combustion according to SM 5310 B. Nitrate-nitrogen was quantified using the TL- 2800 ammonia analyzer made by Timberline Instruments . Soluble reactive phosphate-phosphorus was quantified in filtered samples by the ascorbic acid method adapted from SM 4500-P-E. Based on the results of this study, we can begin to envision possible scenarios for the implementation of treatment wetlands in the San Joaquin River Basin. The result of the combined wetland and microcosm studies suggest that 10 cm d-1 is a reasonable estimate of k for wetland soils in the study area. During this study in 2008, flows at Ramona Lake averaged 20,300 m3 d-1 entering a marsh of approximately 81,000 m3 and had an average measured inlet nitrate-nitrogen of 3.4 mg L-1 . There are currently no regulatory limits for nitrate in agricultural drainage. Previous studies suggested that 0.5 mg L-1 can be used as the critical concentration above which nuisance algae problem can occur . Based on this criterion, the wetland areas required for achieving target nitrate effluent concentrations of 0.5 mg L-1 was calculated using Eq. . The relationship between effluent nitrate-nitrogen concentration and wetland size for the Ramona watershed is shown in Figure 2-6. To reach an effluent nitrate concentration of 0.5 mg L-1 , the Ramona watershed would need an area of 51 ha , which is equivalent to increasing the current marsh size approximately 6 times. The marsh size would need to be increased 15 times to reach the same effluent quality with a single pond system. Growers are reluctant to relinquish land from crop production, so accommodation between riparian wetland expansion and economic vitality must be reached.

Ramona Lake receives drainage from over 2,000 hectares of farmland and an expanded pond-in-series treatment wetland would represent approximately 2.5 % of that area. Additionally, these wetlands do not necessarily need to be located on productive farmland, since there are lower-value riparian areas within the levees defining the San Joaquin floodplain that are down-gradient of the agricultural lands . Diversion of irrigation drainage through constructed wetlands before discharge to the San Joaquin River is being further evaluated as a water quality management option by irrigation and drainage districts in the San Joaquin Valley.The functionality of the study sites as riparian buffers was assessed using the California Rapid Assessment Method for Wetlands . CRAM is a scientifically defensible rapid assessment tool that was developed by an association of local, state, and federal groups and allows a field team to assess the overall health of a wetland system, ranging from the best to worst possible conditions for that type of wetland. Using CRAM, a suitable Assessment Area is determined for the type of wetland being assessed. Four main attributes, including buffer and landscape context, hydrology, physical structure, and biotic structure, are broken down into fourteen metrics which are then assigned a score based on four alternate conditions, from best to worst , depending on the wetland type. Scores are changed to numeric values added up and converted to a percentage score for each main attribute and averaged across attributes to determine the wetland’s overall score. Scores can range from 25% to 100%, with 100% representing a fully functional wetland. The buffer and landscape context score measures the ability of the wetland to handle stress from the surrounding area and consisted of the metrics: landscape connectivity, percent of Assessment Areas with buffer, average buffer width, and buffer condition. Hydrology score is a measure of hydroperiod, channel stability, and how connected the wetland is to the adjacent floodplain.

The study area is characterized by a Mediterranean climate, with hot dry summers and cool wet winters.All of the sites were dominated by crop irrigation runoff during the dry season. The water source score was low for all of the sites that were studied, as all of the sites had an impacted hydrology regime. However, the sites still receive natural inputs, such as storm runoff and groundwater seepage. Physical structure assessed the physical diversity of the environment, with a higher diversity promoting a greater number of habitat niches. Structural patch richness measured the number of physical features, which identified the potential habitat and complexity of the wetland. The list of patch types was dependent on the wetland type and was found in the CRAM manual . Biotic structure included living flora in a wetland and was a measure of species richness and abundance and the physical structure of the plant community. The plant community score included three sub-metrics: number of plant layers, number of co-dominant species, and percent invasion. Each plant layer consisted of plants of similar height that made up at least 5% of the possible growing area.The four sites showed temporal and spatial variation in observed bio-degradation rates. Under aerobic conditions, average half-lives of chlorpyrifos were 5 , 16 , 18 and 21 days for Hospital Creek, Ingram Creek, Ramona Lake and San Joaquin River National Wildlife Refuge respectively . For most of the sampling events, degradation rate of chlorpyrifos was highest in Hospital Creek sediments followed by Ingram Creek sediments . Typical chlorpyrifos degradation curves along with formation of its transformation product TCP are shown in Fig. 3-6 to Fig 3-9 for field sediments. At the end of 21 days, chlorpyrifos concentration decreased to less than 1% of its initial concentration in Hospital Creek sediments and about 20% of the initial chlorpyrifos was measurable as TCP. This value is in agreement with previous chlorpyrifos degradation studies conducted on soils,hydroponic channel where TCP accounted for 10% to 30% of the initial chlorpyrifos concentration . TCP concentration reached its peak value at the end of 7 days and did not further accumulate. This may be due to the fact that TCP is subject to degradation . It has been suggested that most of the chlorpyrifos degrading microorganisms are either capable of degrading TCP or can tolerate high TCP concentrations because TCP has antimicrobial properties and can suppress microbial activity if it accumulates . The rate of abiotic chlorpyrifos hydrolysis was 0.02 d-1 in deionized water at pH 7.2 and 30o C , which corresponds to a half-life of 36.5 days, and is consistent with previously reported data . The initial chlorpyrifos concentration decreased by about 25% in killed control flasks over 21 days. The rate of chlorpyrifos degradation in killed control flasks was 0.02 d-1 , not significantly different from the control flasks without sediment, suggesting that abiotic hydrolysis was the dominant mechanism for the observed chlorpyrifos loss in the killed controls. In addition to the analysis of first-order decay curves, first-order degradation kinetics was evaluated by plotting log-mean chlorpyrifos concentrations versus degradation rates . Experiments from 2010 were combined and it was determined that the chlorpyrifos degradation was first-order with respect to the log-mean chlorpyrifos concentration . The slope of the regression line gives an estimate of the average firstorder degradation constant kdeg for field sediments. The first-order degradation rate constants obtained from this analysis was in reasonable agreement with kdeg values obtained from the exponential decay curves . Chlorpyrifos degradation rates can vary widely in different soils with half-lives ranging from 10 to 120 days. Only a few studies have been conducted in sediments, and half-lives ranged from 20 to 24 days in urban stream sediments , 27 to 77 days in nursery recycling pond sediments , and 58 to 144 days in sediments from a constructed wetland . Half-lives obtained in this study were within the range reported in the literature except Hospital Creek and Ingram Creek sites, which had significantly faster degradation rates for most of the sampling events.

The large variation in half-lives observed in previous studies has been attributed to different environmental factors, such as soil type, soil pH, moisture content, temperature, and organic carbon content . Watersheds included in this study have similar cropping practices and sediment properties . Therefore, physical characteristics do not explain the wide range of observed degradation rates. The difference observed in degradation rates in this study may be related to the wet-dry cycle of the typical agricultural drains , which would allow aerobic conditions to prevail, whereas the wetland sites are permanently flooded systems with anaerobic sediments. Hospital and Ingram Creek sediments were typically light brown in color indicative of aerobic conditions whereas the wetland sediments were dark gray indicating reduced conditions. Agricultural drains typically have low flow rates and occasionally dry out during non-irrigation season in late fall and winter; however, high flows and flooded conditions were observed for Hospital Creek during sampling events in 2011 which may account for a consistent decrease in observed removal rates at this site compared to the previous year . To investigate the effect of redox potential on chlorpyrifos degradation, sediments with highest chlorpyrifos degradation capacity from Hospital Creek were incubated under anaerobic conditions. Chlorpyrifos degradation rate was much lower under anaerobic conditions . Half-life of chlorpyrifos in Hospital Creek sediments under anaerobic conditions increased to 92 days . The inhibitory effect of anaerobic conditions on chlorpyrifos degradation has been previously documented for soils and sediments . These results suggest that allowing a wet-dry cycle can enhance the degradation rates of an organophosphate insecticide in these systems by providing aerobic conditions in sediments. The application of a wet-dry cycle is a useful management tool for the rehabilitation of permanently flooded wetlands . The persistence of aquatic wetland plants during wet-dry cycle is ensured by regular reseeding of the population by seed germination . During dry phase, emergent plant species germinate on exposed mudflats and when water returns, they expand by vegetative propagation . Depth and duration of the flooding period will affect how these wetland communities develop. A long period under permanently flooded conditions is not desired both from the ecological point of view, as it would likely result in decreased plant species richness, and from a pesticide management point of view, as sufficient aeration is desired between consecutive irrigation seasons. The proposed wet-dry cycle period would be similar to agricultural drains in the region. The dry phase would encompass the non-irrigation season in late fall and winter, and the wetland would be flooded again in spring and summer when the irrigation season begins. In the dry phase, given the moderate volatility of chlorpyrifos, volatilization would not be a significant dissipation pathway unless spray applications occur. On the other hand, photolysis may contribute to chlorpyrifos degradation in dry sediments if it is exposed to direct sun light.

Supporting information for the assumptions and calculations can be found in the Supplementary Information . In addition to supplying nutrients, this single serving will assist with other aspects of life support. It also serves to revitalize water as 9.7and 22.8liters per day of clean water are released in gaseous form via transpiration, most of which can be recycled for crop cultivation unless needed in other operations, such as for pharmaceutical formulations. These simple calculations highlight the auxiliary value of plants for bio-regenerative life support. The contributions to all aspects of life support depend highly on the crop species and cultivation environment. For example, a previous study using a closed human/plant system has shown experimentally that 11.2 m2 of wheat grown at high light intensity supplies sufficient oxygen for one person. Wheat is one of the most productive crops for oxygen production, which is amplified by the high light intensity used and its tolerance of a 24-h light cycle.In addition to traditional life support metrics like oxygen productivity, crop selection for molecular pharming must also take into account factors such as the efficiency of transformation and characteristics of the host cell protein compared to the product target.Table 2 summarizes the key assumptions that were built into the two test cases. The logic for selection of the production method is shown in Figure 4 and further described in the Supplementary Information . From the perspective of molecular pharming,ebb and flow trays lettuce serves as a fast-growing crop with a small cultivation footprint in which the edible biomass is also the expressible biomass capable of producing pharmaceuticals. Potato represents a slow-growing crop that has the advantage of distinct edible biomass and expressible biomass ; molecular pharming would not significantly impact the total available food resource.

Leaves detached from the intact plant are capable of providing comparable pharmaceutical yields to those from the intact plant. Production of pharmaceuticals in inedible biomass is one way to create physical separation of the food and pharmaceutical streams while maintaining resource flexibility. However, there are situations in which it may be advantageous for merged food and pharmaceutical streams; there are reports in literature on oral delivery of pharmaceuticals in both lettuce and potato tubers. While promising, this technology is still in the early stages of development. As shown in Figure 5, only 10.4 g FWis needed for the Test Case 1 acute disease state countermeasure in potato, while 36.9 g FWis needed for the Test Case 2 chronic disease state countermeasure in lettuce. While these test cases are driven by conservative assumptions of performance well-established in literature, it is important to note that biomass requirements are highly dependent on the rate of pharmaceutical accumulation , medication dose size, and drug delivery modality. Figure 6 illustrates how the total crop biomass demand differs between the two test cases based on the medication demand and over a range of conservatively estimated molecular pharming expression levels, while Figure 6 shows how the biomass requirements depend on drug delivery modality.Since the founding of modern biotechnology with Cohen and Boyer’s discovery of recombinant DNA technology in 1973, biological organisms have risen to prominence as the primary means for producing high value pharmaceutical proteins and other products, most of which are too complex to be economically and sustainably produced using current chemical synthesis approaches. In the half-century since inception of recombinant DNA technology, a plethora of biological platforms have been engineered as factories of recombinant products – microbial culture, eukaryoticcell culture, live animals, cell lysates, and whole plants.

Table 3 shows a comparison of current pharmaceutical production platforms based on attributes relevant to their deployment for human health in space. Details of the category definition and system rankings are included in Supplementary Information. There are also new platforms on the horizon for production and drug delivery .Commercial biopharmaceutical manufacturing on Earth is dominated by microbial fermentation and mammalian cell culture. Spread across over 1,700 production facilities globally, there is a commercial production capacity of 4.8 million liters for microbial fermentation and 15.0 million liters mammalian cell culture . Regulatory pathways have been well established, decades of intensive research have seen orders of magnitude increase in productivity, and billions of dollars have been invested into developing a culture-based system infrastructure. However, this established dominance of culture based systems does not easily translate into the implementation of human health in space for several reasons. The most glaring difficulty is with cell culture behavior, both with the cell biology and fluid dynamics, in altered gravity; operation will need to be compatible with micro-gravity for in-flight production and reduced gravity for a Moon or Mars mission. There is a growing body of literature on the development of bioreactors with alternative containment and mixing for micro-gravity. The main existing technical difficulties of culture-based systems in limited resource environments are the expensive and complex equipment requirements and the need for the aseptic operation for growing production host cells. Microbial fermenters and cell culture bioreactors are made of glass and/or a special grade stainless steel for durability and corrosion resistance. Bioreactors are generally designed with a suite of capabilities, including: culture agitation, aeration, sampling, in-line sensing, feedback control systems , cleaning, and sterilization. This complex process equipment lowers general accessibility and increases the workforce specialization of operators, which in turn forms another barrier to application in limited resource environments. The equipment burden of culture-based systems is largely a result of the need to maintain a sterile cultivation environment during operation.

Without adequate environmental protection, cultures are susceptible to contamination by undesired organisms.In addition to complexity, stainless steel bioreactors impose significant mass and volume penalties that might prohibit adoption in a space mission. For example, a typical glass and stainless steel stirred tank reactor for 1 L working volume of culture weighs 3.7 kg, not including liquid culture mass and auxiliary reactor components . A growing trend in culture-based systems is to employ single-use technology for cost-savings in cleaning validation, capital costs, and time. Single-use technology for culture-based systems typically consists of a multi-layered plastic bag used in lieu of, or with support of, a stainless steel vessel. Of specific importance to space missions, these savings could also translate into significant reductions in mass and volume requirements. However, as the name “single-use” states, these plastic bioreactor housings are only used once, introducing significant consumable and waste streams to the pharmaceutical foundry. Therefore, single-use technology may introduce reliance on a stable supply chain for consumables that could strain feasibility in a limited resource environment. The use of recyclable materials for single-use technology has not been commercially implemented but would serve to alleviate these concerns. The hindrance of consumable waste is offset by reduced cleaning requirements and should be evaluated within a mission architecture. For example, if pharmaceutical production is projected to be below a threshold capacity, then the extra consumables required to be flown may be acceptable. Exceptional to the typical culture-based system vulnerabilities, microbial, oxygenic photoautotrophic cultures represent a promising subset of culture-based systems that may be better equipped for supporting human life in space. They share many of the same benefits of molecular pharming; these organisms are able to use available in situ resources as feed stocks, and some have been shown to be quite tolerant to a range of water qualities. Additionally, some of these species have unique advantageous characteristics. They can serve as a food resource, grow under conditions that minimize the probability of contamination, and even be used as bio-fertilizer to improve soil quality and crop productivity. A subset of these organisms,4×8 flood tray including the microalgal species Chlamydomonas reinhardtii and Chlorella vulgaris, and the cyanobacterial species Arthrospira platensis , is categorized by the U.S. Food and Drug Administration as being Generally Recognized as Safe , whereby these organisms are considered edible and are sold commercially as food and nutritional supplements. The edible nature of these organisms presents a potential advantage to pharmaceutical foundries in space in that if the target production molecules are bio-available through simply eating the wet or dry biomass of the production host, no downstream purification is needed. The microbial nature of these organisms provides potential advantages to plant systems. First, microalgae and cyanobacteria have genetic tools that are typically more advanced than those of plants . Although tools for engineering A. platensis have been reported, engineering this organism has remained a challenge in the field. To this end, we have recently developed a genetic toolkit for creating stable mutants of A. platensis that will help unlock this organism for metabolic engineering goals. Second, these organisms have faster growth rates than plants, which enables shorter times to reach the biomass necessary for molecular harvesting. Third, the larger metabolic diversity of microalgae and cyanobacteria compared to plants could help to metabolically engineer target molecules that are difficult or impossible to produce in plants using current technologies. Therefore, these organisms may be well suited for pharmaceutical production, or for enhancing the nutritional load through vitamin supplementation. Thus, GRAS-status microbial oxygenic photoautotrophs are poised to become edible molecular pharming hosts for space missions. As these technologies continue to mature, a detailed techno-economic comparison between plants, microalgae, and cyanobacteria will be needed. It may be that a robust pharmaceutical foundry for space ends up being less about selecting one system and more about selecting a network of systems. It is important that interconnectivity and synergy of different platforms be considered for biological-based production of pharmaceuticals and other high-value products to support human life .

A main distinguishing feature of whole plants as a pharmaceutical production platform is the freedom from complex equipment housing during operation; the supracellular structure of a plant serves as its own natural “bioreactor” for operational controland protection against contamination. This effectively means that molecular pharming can be employed with lower complexity process control systems and equipment. Figure 7 illustrates the simplicity and linear scalability of producing pharmaceuticals in whole plants as compared to culture-based systems. However, an equivalent system masscomparison of molecular pharming and culture-based systems for spaceflight is needed to rigorously evaluate the perceived advantage of molecular pharming simplicity. This self-regulating behavior also suggests that plants may serve as a more robust production platform with higher tolerance to input quality variation for given output product quality. In the literature, the strength of molecular pharming production tolerance as compared to culture-based systems is as yet unproven, but would be a valuable avenue of research to directly investigate.For decades, plants have been identified as important life support objects for human health in space. Here we have presented the need for an Earth-independent pharmaceutical life support system and identified molecular pharming as a strategy to tap into the power of plants to serve as a pharmaceutical foundry to meet that need. Molecular pharming in space has the potential to provide manufacturing capacity to respond to both acute and chronic disease states in space with a relatively small amount of plant biomass. Selecting the set of the most appropriate molecular pharming-based production strategies should be carried out within a reference mission architecture, which considers key attributes that we have laid out here. There are many ways to envision pharmaceutical foundries for interplanetary use. Chemical synthesis is limited in production targets and in reagent supply but it may be necessary when biology is not sufficient or capable . Translating culture based systems from Earth to space utility faces the challenges of cell biology, fluid dynamics, feed stock sustainability, mass/volume penalties, and crew training. Their relatively high productivity may position them as an effective platform for settlement missions to sustain larger populations. Autotrophic cultures are exceptional solutions to several challenges of traditional culture based systems and have more potential as a near term platform. More thorough investigation is needed to select an appropriate set of pharmaceutical foundries. Process mass intensityis a metric recently adopted by the bio-pharmaceutical industry to measure the environmental footprint of production. PMI is defined as the total mass in kg of raw material and consumable inputs to produce 1 kg of active pharmaceutical ingredient. PMI can serve as a useful reference point when performing ESM analyses of pharmaceutical foundries in space.

Copper can also participate in a number of inorganic complexes with compounds found in natural waters, such as sulfate, sulfide, phosphate, chloride, and carbonate. The behavior of different Cu species in the environment is not well understood, and the formation of these various complexes may cause precipitation of ionic copper and alter the surface charge and therefore aggregation and dissolution kinetics of nanoparticulate copper. Solubility for the copper-based ENMs tested in this study have been seen to be enhanced at low pH and by the presence of organic coatings in previous research.Additionally, several copper nanomaterials including Cu2O and CuO have been shown to possess photocatalytic properties,which may pose greater hazard to organisms if suspended in photic surface waters than if sedimented into aphotic sediments. Size, coating, solubility,and photoactivity have all been implicated as playing roles in ENM toxicity and are all affected by water chemistry. Aggregate size is influenced by ionic strength and pH via charge regulation,whereby the effective repulsive surface charge of the ENMs is decreased through ionic shielding and surface de/protonation. Depending on their composition, organic surface coatings can stabilize or destabilize8 particles in suspension and through the same mechanisms alter interactions between organisms and ENMs.Previous research has shown that copper-based ENMs are toxic to a wide range of organisms, including fungi, aquatic and terrestrial plants,estuarine amphipods, daphnids and protozoa,marine worms and clams,and mussels.It is therefore necessary to develop our understanding of how these materials behave once released into the environment in order to predict at-risk populations and properly regulate their manufacture, use, and disposal.In this study,flood table the physiochemical behaviors of three different species of Cu-based ENMs were quantified in eight natural and artificial waters covering a range of IS, pH, and organic content to gain insight into how these particles may behave in the environment.

Additionally, equilibrium speciation modeling was performed to predict transformations of the Cu ENMs. Based on previous work, we hypothesized that aggregation would largely be controlled by the IS of the water, with more saline waters having greater aggregation due to surface charge shielding, and by the presence of dissolved organic matter that will increase electrostatic and steric repulsion between particles. Due to the propensity for larger, heavier aggregates to settle more rapidly, we hypothesized that sedimentation would be directly related to aggregation kinetics and hence controlled by IS and total organic content . We hypothesized that pH would be the key factor in dissolution with more dissolution occurring at lower pH and that the presence of TOC would also cause a small amount of dissolution. Additionally, we hypothesized that nano-Cu would have the greatest dissolution in oxic waters as it oxidized to Cu2+.Briefly, the nano-Cu and nano-CuO ENMs were in the form of a high purity powder with primary particle sizes of 100−1000 and 20−100 nm, respectively. Kocide 3000 is composed of spherical composites on the order of 50 μm made up of irregular nano- to micro-scale Cu2 particles embedded in a primarily carbon-based matrix that rapidly breaks down in water to release the Cu2 particles. These can be seen in the inset of Figure S1. Kocide 3000 was chosen as a representative of commercially available nanocopper containing bio-cides, which often contain other ingredients like dispersants. Properties of the five natural and three artificial waters used here can be found in Table 2, and details on collection and preparation can be found in the Supporting Information. Total organic carbon content of the eight waters was determined using a Shimadzu TOC 5000 analyzer. Conductivity was measured with a traceable bench conductivity meter , and pH was measured using a HACH HQ 40d portable meter.

Aggregation kinetics of Cu-based ENMs were measured by preparing 10 mg L−1 ENM suspensions in each water through dilution of a 100 mg L−1 stock, probe sonicating for 2 s at 20% amplitude with a Misonix Sonicator S-4000 , and then measuring size trends over time at 20 °C via dynamic light scattering . Measurements were taken every 30 s for 1 h. To measure sedimentation over time, the optical absorbency of suspensions identical to those described above were determined in triplicate every 6 min for 6 h at 320 nm with the exception of nano-Cu in lagoon water, seawater, and diluted seawater, which were measured at 520 nm at a concentration of 20 mg L−1 . Nano-Cu is the only of the three particles where copper is primarily in the zerovalent state, and as such it is able to participate in unique chemical reactions prior to oxidation to the +1 and +2 states. One of these is the temporary formation of copper chloride compounds in saline waters , which absorbs strongly at 320 nm, the spectral wavelength that was used to detect solid copper. To test the effects of phosphate on nano-CuO, the sedimentation rates, ζ-potential , and pH of 10 mg L−1 nano-CuO in Nanopure water with the addition of 0, 0.1, 0.2, 0.5, 1, and 2 mg PO4 3− L−1 were measured in triplicate. To measure dissolution, ENM suspensions were prepared and stored at room temperature for 0, 1, 7, 14, 21, 30, 60, or 90 days, at which point they were transferred to Amicon Ultra-4 10 kDa centrifugal filter tubes and centrifuged at 4000g for 40 min with a swinging bucket rotor. Filter retention was insignificant.The filtrate was analyzed using a copper ion selective electrode under consistent lighting conditions to minimize light-induced interference. The filtrate was then oxidized with 1.2 vol % HNO3 and 0.9 vol % H2O2 and analyzed for total copper content via inductively coupled plasma atomic emission spectroscopy , with a detection limit of 50 μg L−1 . Standard solutions were measured every 15 samples for quality assurance. Two parameters related to dissolution were quantified: dissolved copper , the total copper content of the ENMs present as free ions , and aqueous phasecopper , the total copper content of the ENMs in the filtrate, which includes dissolved copper, complexed copper 2, etc., and copper bound by ligands under 10 kDa. The ISE that was used to detect free ionic copper was capable of detecting both Cu1+ and Cu2+, both of which may have been shed by the nano-Cu ENMs, but since Cu1+ undergoes rapid disproportionation26 into Cu and Cu2+ and is readily oxidized to Cu2+ in oxic water , it is unlikely to be present as a free ion in any significant amount.

Visual MINTEQ was used to predict speciation and complex formation in the natural waters based on the parameters given in Table 2.Aggregation of nano-Cu and Cu2 particles was characterized by three phases in the 1 h time period measured: immediate aggregation to roughly 5−10 μm in the first few seconds postsonication, a downward trend in aggregate size from 0 to 10 min that was likely due to sedimentation of the largest aggregates out of the water column, and a stable phase in which aggregate diameters averaged 700−2000 nm. Aggregation of nano-Cu and nano-CuO followed the trends outlined in our hypothesis with a few instructive exceptions discussed below, but Cu2 had similar aggregation behaviors in all waters. The polydispersity indices reported from the DLS analysis for Cu2 and nano-Cu were near the arbitrary cutoff value of 1 at all time points in all waters, indicating very broad size distributions. Average aggregate size and statistical groupings for all three ENMs can be found in Table 3. AverageCu2 aggregate size in the third phase did not vary significantly with water type , which may be due to the large proportion of dispersants and other non-Cu ingredients in Kocide 3000. However, despite its high polydispersity, nano-Cu aggregate size correlated significantly with water type . Nano-Cu aggregate size correlated well with IS except in wastewater and storm runoff, which had the highest organic contents of the waters tested by a wide margin. In wastewater, nano-Cu aggregates were smaller than would be predicted by its moderate ionic strength,rolling benches but aggregates in storm runoff were comparable to those found in the most saline waters. This counter intuitive behavior may be explained by the very low rate of sedimentation of nano-Cu in storm runoff resulting in larger aggregates being retained in the zone measured by DLS. Nano-CuO displayed markedly different aggregation trends than the other two particles, aggregates being on average smaller and more monodisperse with PDIs ranging from 0.24−0.36. Additionally, aggregate size significantly increased with time in all waters except freshwater and storm runoff, where aggregate size decreased . Given that there was very little sedimentation or dissolution in these two waters over the measurement period, it appears that the low IS of the storm runoff and freshwater media caused disaggregation to occur. Further evidence for this can be found in previous work,3 which showed that nano-CuO aggregate size decreased over time in Nanopure water with up to 10 mM NaCl but that at higher ionic strength aggregation occurred. Table 3 shows nano-CuO aggregation has a strong positive correlation with IS for all waters but hydroponic media. The large average aggregate size in hydroponic media is likely a result of the decrease in electrostatic repulsion between particles caused by the pH of the media being near the isoelectric point for nano-CuO .Sedimentation kinetics for nano-Cu, Cu2, and nano-CuO over 6 h are shown in Figure 2. In general, sedimentation follows our hypothesis and shows a positive relationship with ionic strength and an inverse relationship with organic content. However, all three particles show different trends depending on their specific composition, and nano-CuO exhibited an unpredicted stabilizing effect due to the presence of phosphate. Cu2 remained relatively well suspended in all waters but groundwater likely due to the proprietary organic dispersants included in its formulation, which give it a high surface charge3 and a low bulk density.Nano-Cu was stable in high TOC waters, namely wastewater and storm runoff, and unstable in the rest. The instability of nano-Cu in hydroponic media may have been due to the low pH of the media causing increased dissolution and subsequent formation of insoluble Cu32 precipitate . Interestingly, aggregate size does not seem to correlate with sedimentation rate in any of the three ENMs tested here. This suggests that aggregate density , stabilizing coatings, and dissolution/ precipitation may be more important predictors of sedimentation rate.

Regardless of dispersants or oxidation state, all three particles were unstable in groundwater. This was likely due to the high bicarbonate and low chloride concentrations found in groundwater, resulting in the formation of insoluble copper carbonates. Speciation modeling predicts that in groundwater all three particles will precipitate as malachite 2) at equilibrium . Lagoon water and seawater also had relatively high amounts of HCO3 −, but due to their high Cl− content, atacamite 3) is predicted to be the dominant form at equilibrium. This suggests that these particles are unstable in saline waters. The trends in nano-CuO sedimentation rates can largely be explained as functions of water ionic strength and phosphate content, with waters being grouped into those with and without detectable PO4 3− and IS accounting for order within those groups . For example, waters with undetectable levels of PO4 3− had the highest sedimentation rates by a wide margin and showed increasing sedimentation with increasing IS. To further investigate these trends, the ζ-potential, pH, and sedimentation rates of nano-CuO in Nanopure water with increasing PO4 were measured. Nano-CuO sedimentation rates across a range of seawater/freshwater mixtures were also measured. Figure 3 shows that sedimentation rate increases linearly with IS and slows over time. This has implications for estuarine environments and other areas where waters of varying salinity mix, as it suggests nano-CuO and similar ENMs may sediment from the water column when moving from areas of low salinity to areas of high salinity. Figure 4 shows that PO4 3− has a variable effect on the sedimentation rate of nano-CuO in Nanopure water, causing increased sedimentation at the lowest concentration , decreased sedimentation from 0.2 to 0.5 mg L−1 , and having no effect at 1.0 or 2.0 mg L−1 PO4 3−. Phosphate is well-known for its ability to covalently bond to metal oxides,27,28 giving the potential to significantly alter their surface properties. Figure 5 shows that even at relatively low concentrations of PO4 3− , the ζ-potential of nano-CuO switches from positive to negative and becomes increasingly negative with increasing PO4 3− concentrations. This change in ζ-potential is likely due to the formation of a negatively charged layer of copper phosphate on the surface of the ENMs resulting in enhanced electrostatic repulsion, leading to the effects shown in Figure 4.

There were no help files to guide those who were new to the program. The program was written in FORTRAN to be run in a DOS environment, so naming files was limited to eight characters or less. If there were any input errors, then the program did not indicate what they were, just that there were errors. Data entry involved parsing the salts added to the solution into the individual metals and ligands, calculating their respective concentrations, and then entering these concentrations as the –log into a DAT input file. Some calculations may have taken several iterations, which involved having to save the file, run the calculation, examine the output file, then make appropriate corrections, input the data again, save the file, and make additional calculations. In discussing these issues with the authors of GEOCHEM-PC we were encouraged to build upon and improve the existing program, so that it would work in a Windows XP or Vista environment and would have increased power and function. Included in GEOCHEM-EZ are improvements which would be expected by modern users , while maintaining complete backward compatibility to the GEOCHEM-PC format. A customizable database of common salts has been included, which eliminates the need to parse and to calculate the concentration of each metal or ligand. In addition, berry pots the user is no longer limited to enter concentration as nM, µM, or mM, but can now enter the concentrations as g/L or mg/ L, provided the salts of interest are part of the salts database. These last two features will make data input more rapid and help in eliminating the most common user errors. The program does automatically check for errors in data entry, convergence, and case similarity.

The user can instantly preview input and output files and make necessary corrections , something that formerly involved having to save these files and run the calculations a second or third time. Output files may be customized by filtering the output tables prior to saving the file. Within the Help menu we have included a Unit Converter which can convert any salt in the database from g/L or mg/L to molar concentrations or vice versa. Shown below is the GEOCHEM-EZ interface. This example is for a basal Murashige –Skoog medium, with the salt, metal, and ligand concentrations entered in mg/L. Note that the entries are mostly salts that are contained within the salts database and are accessed via the drop down list on the left side. However, the user may still add individual metals or ligands, if that is preferred. This entry for the M-S medium represents a simple case . Note that there are two tabs open , representing two separate cases that are being run simultaneously. Many cases can be run at the same time, another feature that makes solution analyses more rapid.Why should one use this program to design experimental solutions? Many scientists have modified standard nutrient solutions for hydroponics-based research or for specific experimental treatments without having analyzed these solutions to see whether any precipitation or solids may form because of the changes that they made to the solution composition. Geochem-EZ can help predict potential problems in experimental media. This program can also be used to design sensible chelate buffer systems or to calculate the concentration of a particular ion needed to provide a constant ionic activity. It is also a good way to know whether there is sufficient free activity of important nutrients in the solution of interest.

Often there is the assumption that if the nutrient is part of the solution, then it is readily available to the plant. This is not necessarily true. Interaction with other ions, pH effects, complexation, and precipitation may occur, reducing the free activity of the ion of interest. CO plays a role in the photoperiodic response in several grass species, such as rice , sorghum , and barley . However, these species also possess an additional photoperiod pathway that is not present in Arabidopsis, in which the PHOTOPERIOD1 gene plays a central role. PPD1 encodes a member of the PSEUDO RESPONSE REGULATOR protein family and is homologous to the Arabidopsis circadian clock genes PRR3 and PRR7 . The duplication that originated PRR3 and PRR7 in Arabidopsis and PRR37 and PRR73 in the grasses are independent, and therefore their sub-functionalization is independent . In Arabidopsis, PRR3 and PRR7 encode components of the circadian clock, and their disruption alters the expression of other clock genes . By contrast, variation in PPD1/ PRR37 in the grasses has no impact on the periodicity of the circadian response . These results suggest that after its duplication in the grass lineage, PPD1 evolved as a photoperiod gene that functions as an output of the circadian clock. Most natural variants in the photoperiodic response in wheat are associated with deletions in the promoters of PPD-A1 or PPD-D1 or with differences in PPD-B1 copy number . The promoter deletions in the PpdA1a or Ppd-D1a alleles are associated with the misexpression of PPD1 during the night, the induction of FT1, and the acceleration of flowering under SD . Plants carrying these alleles still flower earlier under LD than under SD and, therefore, will be referred to as “reduced photoperiodic response” alleles . The acceleration of flowering by PPD1 requires its transcriptional activation by light, which is mediated by two members of the phytochrome family, PHYB and PHYC .

The phytochromes absorb light maximally in the red and far-red spectrum and exist as two interchangeable isoforms, the inactive R light absorbing Pr form synthesized in the cytoplasm and the active FR light absorbing Pfr form that is translocated to the nucleus . Upon arriving in the nucleus, Pfr phytochromes interact with bHLH proteins known as PHYTOCHROME-INTERACTING FACTORS , which initiates a cascade of light regulated signaling pathways . During darkness and upon exposure to FR light, Pfr phytochromes revert to the inactive Pr form. Despite the molecular characterization of some of the components of the PPD1-dependent flowering pathway in wheat, there are still large gaps in our knowledge of the mechanisms involved in the light regulation of PPD1 and FT1 and in the perception of photoperiodic differences. In this study, we characterized the response of wheat when exposed to short pulses of light during the long nights of SD photoperiods, which are referred to as night-breaks or NBs hereafter. NB experiments have the advantage of modifying photoperiods without changing the total hours of light received by the plant. NBs cause significant delays in flowering when applied to SD plants grown under SD.These observations demonstrate that the duration of the night is critical to regulate flowering time in many SD plants and that the NB response can be characterized as a transient period of sensitivity to light that inhibits flowering. In rice, a single NB was sufficient to inhibit flowering in SD via the PHYB-mediated transcriptional repression of Hd3a . These observations are consistent with the external coincidence model of flowering, according to which flowering is induced when external light and internal oscillating circadian signals coincide . In this study, we show that NBs accelerate flowering in wheat plants grown under SD and that the response is strongest in the middle of the night. Using ppd1-null mutants, we demonstrate that this response is mediated by PPD1. We also show that although PPD1 transcription is rapidly induced within 1 h of exposure to a single NB, multiple NBs are required for induction of FT1 to high levels and for early flowering. Finally, we show that the magnitude of PPD1 induction in response to NBs increases in accordance with the length of darkness preceding the light signal and that this induction is dependent on active protein synthesis during darkness.When the long nights of SD were interrupted by 1 h pulses of white light at different points of the night , flowering of the Kronos-PS plants was accelerated . The timing of the NB had a strong effect on heading date, with a maximum acceleration when the NB was applied in the middle of the night . Under these conditions, plants headed just 7 d later than those grown in a LD photoperiod . NBs applied either earlier or later than this point had a weaker effect on heading date, although among plants exposed to NBs after 6, 8, or 10 h of darkness,hydroponic grow system heading date was not significantly different . NBs of 15 min given after 8 h of darkness were equally effective in accelerating flowering as 1 h NBs applied at the same time . To characterize the transcriptional responses associated with accelerated flowering in NB, we compared the expression levels of selected flowering time genes in 6-week-old plants grown since germination under NBmax conditions with those maintained in a SD photoperiod. Because allelic variation at the PPD1 loci can affect the expression of each homeolog separately, we measured PPD-A1 and PPD-B1 transcript levels using homeolog specific assays. For all other targets, quantitative reverse transcription -PCR assays that amplify both A and B homeologs were used . In SD-grown Kronos-PS plants, PPD-A1 and PPD-B1 expression levels remained low throughout the night, and FT1 transcripts were not detected at any of the analyzed time points.

In plants grown in NB conditions from germination, PPD-A1 transcript levels doubled in response to NB, but this homeolog was expressed at very low levels in all assayed time points . By contrast, PPD-B1 transcript levels were approximately 20-fold higher than PPD-A1 before NB and 26-fold higher after NB , suggesting that the PPD-B1 homeolog contributes the majority of PPD1 transcripts in photoperiod-sensitive tetraploid wheat. This result is consistent with a previous study in the hexaploid wheat variety Paragon, where PPD-B1 accounted for 90% of all PPD1 transcripts . PPD-B1 expression was significantly higher in NB than in SD conditions at all time points and was rapidly upregulated by NB, peaking between 1 h and 3 h after the start of the NB . FT1 transcript levels were significantly higher in NB conditions than in SD and showed increased expression 5 h after the start of the NB . Even before exposure to NB, FT1 transcript levels were significantly higher in plants grown under NB since germination than in those grown under SD . FLOWERING LOCUS T2 and VERNALIZATION1 expression levels were also elevated in plants grown in NB, while FLOWERING LOCUS T3 expression was reduced in comparison to SD-grown plants . These results show that the transcriptional regulation of these flowering time genes in NB is similar to their regulation in LD photoperiods .Phytochromes are activated and inactivated following exposure to R and FR light, respectively, so we tested the effects of FR light treatment on the NB response. Kronos-PS plants were grown under two different conditions from germination. In one chamber, plants were exposed to a 1 h NB after 8 h of darkness, and in the other chamber, plants were exposed to the same conditions except that the 1 h NB was followed by a 30 min pulse of FR light. Plants exposed to FR light exhibited a delay of 8.9 d in heading date when compared to control plants, but the difference was not significant . One possible reason for the mild effect of this FR treatment on heading date could be that the exposure to 1 h of white light was sufficient for the irreversible activation of downstream genes or proteins in the flowering induction pathway before the FR light inactivation of the phytochromes. To test this possibility, we applied the NBs as 15 1-min pulses of white light intercalated either with 15 1-min periods of darkness or 15 1-min pulses of FR light . Application of the NB using this protocol was less effective in accelerating heading than when the NB was given as a 1 h block of white light, but the FR treatment had a proportionally larger effect and significantly delayed heading date . At the transcriptional level, PPD-B1 expression was significantly reduced only by the pulsed FR treatment . These results suggest that despite the absolute requirement of PHYB and PHYC function for the NB response, the FR light conditions used in these experiments were not sufficient to abolish the NB response completely.

The chromosomal location of QTL stm9 detected in both data sets was coincident despite the significant Genotype × Season interaction in the ANOVA. To examine the cause of the significant Genotype × Season interaction in more detail, we plotted recombinant sub-NIL stmscore means across the two seasons to create interaction plots . Inspection of the plots suggests that the changes in sub-NIL mean values across seasons primarily derived from greater chilling susceptibility of susceptible sub-NILs in the Spring than in the Fall . Magnitude differences would cause the size of the LOD peaks to differ among seasons, but not change the peak location, which is in agreement with our results . In addition to the increase in the magnitude of means for stmscore of the susceptible sub-NILs, two sub-NILs were classified as tolerant in the Spring dataset but as susceptible in the Fall dataset . Sub-NIL C4 had a mean of slightly over 1.0 in the Spring data set, and clearly grouped as susceptible in the Fall dataset . None of these lines contain the S. habrochaites introgression for high-resolution mapped stm9, but the introgressions do all flank stm9. These results suggest the possibility that there are environmentally sensitive genetic modifiers of the stmscore phenotype in this region of chromosome 9,vertical farm tower and that the interaction of these modifiers with the environment could be causing the significant Genotype × Season interaction.

The rank changes seen within the tolerant group may be due to differences in the genomic content of S. habrochaites sequence in the flanking regions of QTL stm9, and not a direct effect of the environment on the gene or polymorphisms controlling the tolerant stm9 phenotype. Previous work in tomato has shown that the stomatal response of a plant when subjected to root chilling conditions differs between susceptible and tolerant phenotypes . Stomatal control is regulated by multiple environmental factors including light, temperature, day length, humidity, and CO2 levels . The Spring experiments were conducted under longer day lengths, higher air temperatures, and lower humidity than the Fall experiments . These seasonal differences affect the conditions in the greenhouse and may have contributed to the significant Genotype × Season interaction, as well as differences in relative response among the sub-NILs in the Spring versus Fall data sets. In the context of phenotypic plasticity, seasonal effects on sub-NIL performance would account for the more gradual separation of means in the Spring dataset compared to the Fall .Low marker density and small population sizes in initial genome-wide QTL mapping studies may bias upwards the estimation of QTL effects due to the inability to resolve closely linked, smaller effect QTL . Consequently, single large effect QTL may resolve or fractionate into multiple, smaller effect QTL after fine- and high-resolution mapping . The original interspecific BC1 population used by Truco et al. to map QTL for shoot turgor maintenance under root chilling consisted of 196 individuals genotyped with 112 markers. Truco et al. mapped a major effect QTL to a 28-cM region on the short arm of chromosome 9 which accounted for 33 % of the phenotypic variation for shoot turgor maintenance under root chilling . Despite the large initial genetic size of the QTL stm9 region detected by Truco et al. , subsequent fine-mapping by Goodstal et al. and high-resolution mapping in our present study do not provide any evidence of multiple QTL or QTL fractionation.

The relatively small genetic size of high resolution mapped stm9 and the lack of QTL fractionation indicates that this level of resolution is suitable for the identification of candidate genes for stm9. There are numerous examples in the literature of environmentally stable, high resolution mapped QTL that have led to candidate gene identification and in some cases subsequent causal gene/ polymorphism determination. Several QTL for chilling tolerance in rice have been high-resolution mapped and candidate genes identified. These QTL include qCTS12 , qCtss11 , and qCTB7 . Tomato-specific QTL examples include fw2.2, a fruit weight QTL, and se2.1, a stigma exsertion QTL, both identified in progeny derived from S. pennellii, another wild tomato relative . The causal gene underlying QTL fw2.2 was identified by Frary et al. , who proposed that changes in the regulation of ORFX , not changes in the sequence or structure of the expressed protein, are responsible for changes in fruit size. Chen and Tanksley determined the casual mutation underlying se2.1 is a mutation in the Style2.1 promoter that results in a down-regulation of Style2.1 expression during flower development. Collectively, the results from these studies suggest that candidate gene identification and functional testing for QTL stm9 should focus on mutations in regulatory and promoter regions of candidate genes in addition to mutations that may affect the sequence or structure of expressed proteins.Many genes have been identified as being involved directly or indirectly in plant tolerance or resistance to abiotic stresses , including chilling/cold tolerance . Plant responses to abiotic stresses can include multiple pathways that involve a variety of gene products such as receptors, signaling molecules, transporters, transcription regulators, and transcription factors . Many of the identified stress response pathways have been associated with tolerance to a range of abiotic stresses . The plant’s response to abiotic stress may result in both reversible and irreversible activation of stress response pathways . Because of the complex nature of the pathways involved, the specific genotype of the plant also has a large influence on abiotic stress response . Plant responses to abiotic stressors are dependent on the interplay of abiotic stress, environment, and genotype .

Therefore, a particular abiotic stress applied in different environmental contexts may result in overlapping, but distinct responses from a single genotype . We analyzed the physical region in the cultivated tomato reference genome that is syntenic to the S. habrochaites QTL stm9 region because an assembled S. habrochaites whole genome sequence is not available. All of the protein products of the S. lycopersicum annotated genes located within 30 kb of the QTL stm9 peak marker have features that are shared with genes involved in responses to water stress and other abiotic stresses. In addition, the majority of the S. lycopersicum genes located within the syntenic high-resolution mapped stm9 region have been implicated in abiotic stress response pathways . It is possible that plant responses to root chilling stress may induce a more complex transcriptional response than other types of water stress such as those caused by salt or polyethylene-glycol , although overlap has been seen in the response to all three stresses . For example, in grape, under root chilling stress only transcripts for protein synthesis and the cell cycle were up-regulated to a lesser extent than under salt or PEG stress. The regulation of plant metabolism, protein metabolism, signal transduction, calcium signaling, stress hormone pathways, and transcription factors were all increased to a greater extent under root chilling in grape . These categories of genes account for the majority of genes located within the syntenic S. lycopersicum QTL stm9 region. While the total number of annotated genes within the S. lycopersicum reference genome region containing QTL stm9 is relatively small, there are no estimates available for S. habrochaites due to the unavailability of assembled whole genome sequence for this wild species. A comparison of the genetic and S. lycopersicum physical maps of the chromosome 9 region containing stm9 shows a variable rate of recombination across this region .It is possible that this variable rate of recombination is due to the presence of repetitive elements or other local structural polymorphisms affecting the synteny and colinearity of the S. lycopersicum and S. habrochaites genome sequences in this region. In addition, our flow cytometry results indicated that the genome size of S. habrochaites is 1.5 × that of S. lycopersicum . The larger genome size of S. habrochaites suggests the possibility that the putative loss of function of genes and/or genetic elements in S. lycopersicum may be due to deletions or non-functional null mutations. Matsuba et al. sequenced a functional gene cluster for terpene biosynthesis on chromosome 8 of S. habrochaites acc. 1778 and identified several rearrangements, deletions, and a novel gene when compared to the same gene cluster on chromosome 8 of the S. lycopersicum reference genome. Our prior research suggests that the inability of cultivated tomato to maintain shoot turgor under root chilling is the result of a loss of function in S. lycopersicum . Taken together,vertical plant tower the current evidence suggests that the S. habrochaites allele for high-resolution mapped QTL stm9 may not be completely syntenic to S. lycopersicum, and that it may not contain the same genic compliment as the S. lycopersicum allele for stm9. Therefore, although the S. lycopersicum genome sequence is helpful in identifying potential candidate genes for shoot turgor maintenance under root chilling, the genomic sequence of the stm9 region of S. habrochaites is necessary for accurate, well-informed candidate gene identification.Stability of QTL expression for tolerance to abiotic stresses is important for successful deployment of stress tolerance QTL in breeding crop plants. Although a significant Genotype × Season interaction was identified for QTL stm9, the potential causes of the interaction suggest that this region would likely be useful as a stable source of root chilling tolerance for breeding. A number of other QTL have been identified as targets for breeding despite a significant Genotype × Season interaction in several species, including barley, rice, and maize .

The phenotypic plasticity likely contributed by the stm9 flanking regions suggest that any future breeding strategies should be undertaken with the smallest introgression possible that still contains the entire high-resolution mapped QTL stm9. The S. habrochaites introgression in sub-NIL C7 contains only the high resolution QTL stm9 region . This sub-NIL was grouped as tolerant in both the Spring and Fall datasets, and gave a consistently low stmscore in both seasons , suggesting it may serve as a suitable potential donor parent source of tolerance to root chilling in breeding programs. Due to the complexity of the abiotic stress response pathway, it is unlikely that the S. habrochaites QTL stm9 allele contains only a single gene conferring shoot turgor maintenance under root chilling. Single causal genes have been identified for a number of major QTL,but other major QTL have been shown to be controlled by two or more causal genes or polymorphisms.Identification and testing of the causal gene or polymorphisms underlying QTL stm9 for tolerance to root chilling will be an important step in the identification of genetic targets for improving stress tolerance of plants exposed to root chilling and other types of water stress through markerassisted breeding. Determination of the gene/polymorphisms responsible for a quantitative trait phenotype is facilitated by genomic approaches . Once a target region is identified via high-resolution mapping, a combination of genomic sequencing, structural genomic analysis, and transcriptome profiling can be used to assist in the identification of candidate genes. Therefore a biologically informed ranking of candidate genes located within the QTL stm9 region will require a combination of S. habrochaites genome sequence for this region as well as transcription profiles for susceptible and tolerant subNILs exposed to root chilling. It is hoped that a better understanding of the underlying mechanism for tolerance to rapid-onset water stress in wild tomato S. habrochaites may aid in the identification of chilling tolerance genes in other species of tropical and sub-tropical origin.WRC project W-769 focused on obtaining data on the seasonal dynamics of growth, resource allocation, and pollutant allocation in selected wetland plant species grown under conditions of elevated pollution as compared to non-polluted habitats. Recent studies have indicated that pollution from urban runoff, especially storm water discharge, has caused major water quality problems in streams, lakes, and reservoirs, including nutrient enrichment, introduction of toxic materials, turbidity and heavy sediment deposition . Wetlands have the capacity to intercept storm runoff and store storm waters, simultaneously removing suspended solids, and some dissolved pollutants prior to discharge into waterways. Aquatic and semi-aquatic plants play an important role in promoting both nutrient transformation and nutrient removal in aquatic treatment systems.