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.

A support vector machine algorithm was used to define 200 genes whose expression in diagnostic leaves allowed the most accurate discrimination between P-replete and P-starved plants grown hydroponically in the glasshouse. Table 1 lists the twenty genes whose expression in diagnostic leaves discriminates with the greatest power between P-replete and P-deficient potato plants growing hydroponically in the glasshouse. As an independent test of the diagnostic transcriptome, diagnostic leaves were sampled from plants growing in the field at HRI-Wellesbourne. An analysis of gene expression in these leaves correctly identified that this potato crop was P-deficient. Thus, the diagnostic transcriptome can identify P-deficiency in field-grown potato crops and, therefore, has potential as a viable management tool for precision agriculture. It will allow farmers to monitor the immediate physiological P status of their crops and, thereby, optimise Pi-fertiliser applications and reduce P losses to the environment and commercial losses due to P-deficiency. Plant roots shape their environment in various ways and are in turn shaped by physiochemical properties of the surrounding soil. Roots affect soil by dislocating particles, by polymer production,vertical growing and by the release of a wide variety of small molecules.Overall, these processes result in the formation of larger soil aggregates which increase water-holding capacity and influence the stability of soil organic carbon . Plant induced changes in chemistry can lead to weathering of minerals and alter the composition of microbial communities . Further, root exudates can enhance root penetration of soils .

Although the effects of plant presence on soils and microbial communities have been a major research questions for over a century , relatively few studies have sought to understand the physiochemical effects of plant growth substrates on plant physiology and exudation. Plant morphology and exudations can be influenced by both physical and chemical properties of soil particles. Typically, particles range from small to large and determine physical parameters such as water-holding capacity of soils . It has been shown that 1-mm beads reduce root and shoot growth, elevate root:shoot ratios, and alter root morphology of maize when compared to hydroponic growth . Root morphology can be altered by adsorption of root exudate metabolites to substrate such as activated carbon . Natural environments, such as soils, can also affect both root exudate profiles and morphology ; however, determination of causal factors may be confounded by variables such as granule size, chemistry, and microbial community composition. Soil minerals differ in structure and surface charge, thereby governing their interaction with dissolved organic compounds . Results suggest that substrate chemistry can alter exudation. For example, aluminum ions present in stone wool are thought to increase exudation of organic acids in maize . In contrast to particle chemistry, the effect of particle size on exudation is less clear. Particle size could alter exudation in multiple ways. Particle size reduces root growth rates, which limits exudate dispersal. In addition, diffusion depends on substrate size and can thus be a limiting factor for dispersal of exuded compounds . Exudates are mainly produced by root tips : when root morphology is altered by particle size, the number and structure of root tips can be altered, possibly changing the quality and quantity of exudates. Particle size also influences the composition of microbial communities .

Microbes differentially metabolize exudates and secrete secondary metabolites , further changing the presence of exuded compounds. d) Particle size can modulate plant chemistry . Here, we investigate the effect of particle size and chemistry on root morphology and exudation in Brachypodium distachyon. Specifically, we asked three questions: a) whether root morphology of a model grass is altered in physically restricted conditions as observed in other species, b) if and how the exudate profile changes with particle size, and c) how root morphology and exudation are influenced by substrate chemistry. To facilitate these studies, we used a sterile environment enabling us to focus on plant metabolism without the additional layer of microbial metabolism present in a natural environment. B. distachyon growth and exudation profiles were comparted on various inert substrates in a range of defined particle sizes relative to hydroponic solution. We found that particle size had a significant effect on root weight and root length; however, particle size did not influence composition of root exudates. A defined mixture of soil metabolites was used to evaluate sorption to particles. We further demonstrated that clay sorbed a large degree of exudates, altering the amount of exudates freely available around root. These clay-sorbed exudates could further support growth of a rhizobacterium. Our results highlight the importance of considering soil structure and chemistry when studying plant–soil interactions.Different particle sizes and surface chemistries were chosen to investigate how root morphology and exudation is affected in various plant growth substrates. The particle sizes chosen corresponded to large, intermediary, and small particles . Glass beads were chosen as an inert system with defined sphere diameter, sand as an inert and natural system, and clay as a natural system with a reactive surface. The mineral composition of the sand substrates was determined as more than 98% quartz, whereas the clay was a mixture of 51% opalCT, 37% mica-illite, 10% quartz, and trace amounts of K-feldspar and calcite . The chemical properties of the substrates were assessed by determining the sorption of a mixture of more than forty metabolites belonging to various chemical classes that have also been found in root exudates .

The recovery rate of the various metabolites from the glass beads and the 4-mm sand was comparable to the defined medium control without substrate, whereas the recovery rate from the250-µm sand and from clay were lower by approximately 30% and 70%, respectively . Consistently, differences between clay and other substrates explained 84% of the variance in a principal component analysis, and only 8% of the variance accounts for differences between the control, glass beads, and sand . The metabolites depleted by clay belonged to a variety of chemical classes, among them charged compounds, such as organic acids and ammonium salts , and other nitrogenous compounds , and of comparatively polar compounds such as sugars . We confirm that as expected, clay particles sorb a variety of metabolites from the defined medium.Exudation could be limited by diffusion in our experimental systems with small particle sizes. A diffusion test with a dye resulted in fastest diffusion in controls without substrate added . The diffusion rate of the dye decreased with lower diameters in glass beads and followed a logarithmic trend. In 4-mm sand, the diffusion rate was initially similar to 1-mm glass beads, but then resembled more 0.5-mm beads. For clay, diffusion similar to 1-mm or 0.5-mm beads was observed initially, but subsequently, the dye front ceased moving, likely due to sorption of the negatively charged dye. Based on this analysis, in our experimental setup, exudates would require a minimum diffusion rate of 1.25 cm/h to reach the edge of the glass jar in which the plants were grown . Thus, diffusion was not limiting in glass beads with a diameter equal to or >1 mm, but might be limited in substrates with smaller diameters . This confirms, that as expected, sand and glass beads are inert substrates, whereas clay strongly sorbs a variety of metabolites. In addition, exudation may be limited by diffusion in substrates with particle sizes smaller than 1 mm.The aforementioned substrates were used to investigate how Brachypodium distachyon root morphology and exudation was affected in these experimental systems compared to a hydroponic control. Plants were grouped according to their behavior in the different substrates: plants with weights and root morphology similar to hydroponic controls were termed “big” , and plants with distinct weight and root morphology were termed “small” . The root fresh weight of plants grown in 3-mm glass beads, 4-mm sand, 250-µm sand, and clay was comparable to the hydroponic control, whereas roots grown in 2-mm, 1-mm, and 0.5-mm glass beads and 5-µm sand were significantly smaller . The shoot fresh weight of plants grown in 5-µm sand were significantly smaller compared with plants grown in hydroponics, and all other conditions . The altered root and shoot weights resulted in decreased root/shoot ratios for clay,vertical farm system and 2-mm and 1-mm glass beads-grown plants, and an increased ratio of 5-µm sand-grown plants . Root length and number were assessed for first-order roots , second-order roots , and higher order roots. The total root length correlated with particle size, with maximal length for hydroponically and clay grown roots, approximately 30% shorter root systems for 3-mm beads-, 4-mm sand- and 250-µm sand-grown roots, and 50% or shorter root systems for 1-mm beads-, 2-mm beads-, 0.5-mm beads-, and 5-µm sand-grown roots . First-order root lengths were significantly decreased by more than half for all substrates except for clay and 4-mm sand , whereas the second-order root length was decreased by 40%–70% in 2-mm beads-, 1-mm beads-, and 0.5-mm bead-grown roots, and by ~85% in 5 µm sand-grown roots . Higher order root lengths varied more within one experimental treatment, with a trend for higher total lengths in hydroponics and clay compared with glass beads and sand, and significantly lower lengths in 5-µm sand . Interestingly, root length had a higher Pearson correlation coefficient when correlated with particle size than root numbers. Only roots grown in 1-mm beads and 5-µm sand showed a statistically significant reduction in root number compared with hydroponic controls, which is a result of the large variability in total root number of hydroponically grown plants . The observed reduction in root number originated from a reduced number of secondary and higher order roots.

A correlation analysis between root and shoot weight, total root length, and total root number of all samples showed a significant correlation of all parameters investigated . Root weight and length, and to a lesser degree root number, correlated with particle size. Overall, clay-grown plants were most similar to hydroponically grown plants regarding tissue weight and root morphology. Plants grown in 3-mm glass beads or 4-mm sand had comparable fresh weight compared to the aforementioned plants, but slightly reduced total root length driven by a reduction in first-order root length. Plants grown in 1-mm and 0.5-mm glass beads exhibited reduced root weight and root length, caused by a reduction in first and second-order root length. Plants grown in 5-µm sand exhibited the largest reduction in tissue weight, root length, and number.To investigate whether changes in root morphology might affect exudation profiles, spatial patterns of exudation were investigated using matrix-assisted laser desorption/ionization mass spectrometry . A total of 24 ions were detected in the vicinity of roots . It was not possible to confidently identify these ions given that the MALDI used is not suited for fragmentation of low m/z ions and the fact that MALDI often results in different ions versus the electrospray ionization used in our liquid chromatography–mass spectrometry analyses. However, despite this lack of identifications, our results suggest differences in spatial patterns of chemical components. Some ions showed higher abundances around root tip and elongation zone, supporting a role of these young root tissues in exudation. Other ions were detected along most of the root axis, suggesting exudation also from older root parts, whereas the location of other ions overlapped with the location of the root, which could either indicate short diffusion distances, or association with the cell wall. Overall, these data suggest that multiple tissues are involved in exudation.To investigate whether altered root morphology and various spatial exudation patterns altered overall exudation profiles, exudates were collected in situ and in vitro . The first collection approach generated exudation profiles shaped by plant metabolism and particle chemistry, whereas the second collection approach generated exudate profiles shaped only by plant metabolism. With both collection steps, approximately 100 metabolites were identified based on comparison of retention times, exact mass, and MS/MS fragmentation patterns compared with authentic standards. The metabolites included organic acids, amino acids and derivatives, sugars and other carbohydrates, nucleic bases, nucleosides, and derivatives. Multivariate statistical analysis revealed that 46% of in situ exuded metabolites were significantly different in pairwise comparisons, compared with 30% of in vitro exuded metabolites . A similar result is evident in hierarchical clustering analysis, with in situ-collected exudates clustering according to biological replicates, and in vitro-collected exudates clustering in less distinct patterns .

Formaldehyde emissions for the old wood paneling with new and old polish, and the drywall were all similar ranging from 10 µg/m2 /h to 22 µg/m2 /h . For the new wood, the formaldehyde emissions were approximately an order of magnitude higher than the other materials for both the polished and unfinished surfaces. The emission results for formaldehyde are illustrated in Figure 4 showing that the polish coating does not seem to significantly change the measured emission factors when the age of the wood paneling is taken into consideration. For old wood, the new polish slightly reduces the emission factor while for new wood the polish increases the emission factor slightly but the difference is likely more a function of the age of the wood than the polish. For example, given the old wood where the emissions are already low, adding the new polish may provide an apparent sink for the formaldehyde as it accumulates in the coating. In contrast, for the new wood, the initial emission rate is high and the coating may simply add a diffusion layer that increases the time that the emission from the wood paneling takes to drop to a constant level. With or without the coating,vertical greenhouse the new wood is clearly the major source of formaldehyde emissions among the materials tested. The aging of the formaldehyde emissions and the affect of the polish coating were tested further by removing the backing plate from the new wood with new polish after the initial conditioning and testing period was complete and reversing the material to expose the unfinished face.

Our premise was that the formaldehyde diffuses to and accumulates at the surface of the material when the surface is covered so the initial emissions after uncovering the surface are expected to be high then drop with time towards an constant value. The results are illustrated in Figure 5. The initial test of the polished side was repeated 4 times over 15 days. To test the emissions of aldehydes from the unfinished side of the same new wood material, the sample was flipped to expose the unfinished side and the polished side was sealed and the sample was returned to the test chamber to test the unfinished face. The first measurement found formaldehyde emissions from the unfinished face significantly in excess of the polished side but the emissions decreased rapidily over the next week. The material was flipped again exposing the poished side again that had been sealed for a week and the emission factor doubled but resumed dropping over the next day. Overall, the results indicate that the emission factor of formaldehyde from the new wood with new polish is approaching that of the new wood with no applied polish over time. The standard emission factors for formaldehyde from each of the main wall surface materials listed in Table 3 are compared to field measured values for the PBC building that were collected previously using Equation 3 along with the building parameters listed in Table 4. The results are listed in Table 5 for each material and each floor as a range of concentrations estimated with ACH values representing the maximum and 50% of the maximum accounting for the fact that the demand response system will likely run the ventilation at less than the maximum value. These ranges are summed for the total wall area based on loading factors for each material and the range of total concentraions are compared to the measurements in the last row of the table.

Assuming no other significant loss pathways for formaldehyde, the three primary wall materials can easily account for the measured concentrations. To probe the effects of SPIONs on the soybean chlorophyll, highly uniform SPIONs with core size of 9 nm, and either plain, or dextran based negative or positive surface charges were synthesized.Dynamic light scattering and ζ potential measurements show that the electrokinetic potential and average sizes of the SPIONs in different solutions are highly dependent on the surface charges of the SPIONs. The average DLS sizes of the SPIONs with different coatings are 18.9 and 20.3 nm in DI water and phosphate buffered saline , respectively. The DLS results are in good agreement with TEM data. It is essential to plant research had monodisperse NPs, with same physicochemical properties for evaluation real interaction effects with plants. In this study, magnetite NPs was synthesized monodisperse and suitable magnetic properties.There are no specific testing manual for nanomaterials phytotoxicity. The U.S. EPA and OECD are guidelines for testing of chemicals, frequently applied to nanotoxicity assay for plant recommended by these guidelines.19 The plant species recommended have different germination time so exposure to nanomaterials at different stage of growing in the same time. In this study, it was used the TZ method because measurement of seed germination percent in this method is not time-dependent. The plants were treated with NPs and the effects on germination index and root elongation were probed. According to the results, various concentrations of SPIONs, with different charges, do not have significant effects on the germination index . Interestingly, positive and negative SPIONs show significant positive influence on root elongation, where as plain SPIONs have no significant effect; more specifically, plain SPIONs slightly inhibit . This may happened due to the lower protective effect of the polymeric shell in plain SPIONs, compared to the negative and positive SPIONs, resulting in release of more Fe2+ ions in the treatment.

To evaluate this hypothesis, the measured Fe2+concentrations in the growth medium containing SPIONs with and without soybean cultured condition were measured by the Snell 2007 method at 510 nm. Results confirm that the concentration of Fe2+ in the growth medium is increased by the cultivating soybean. The NPs accumulate on the root and seed surface and occlude some water and ion channels. Nanomaterial phytotoxicity is related to its dimensions, chemical composition, surface properties, and ionization of the surface.42,43 To remove the slight observed toxicity of SPIONs, lower concentrations are employed for the further stages.Selected VSM measurement results of various soybean tissues treated with 0.06 mg/mL of SPIONs, with various charges, are shown in Figure 3. It is important to mention that there is no trace of the magnetization signal in the control plant tissue. Because of the accumulation of SPIONs in the root, stem, crown, and leaf, regardless of surface charge of the SPIONs, in all sub apical leaf of treatment plants were detected magnetization signal. The weakest magnetic signal was detected from stem tissue in all treatment. The highest magnetic signal in aerial tissue is detected in the crown, where the root vascular systems change to stem and where we observed the maximum accumulation of NPs. The strongest magnetizations are observed in the roots for soybeans cultured in nutrient solution with plain SPIONs; this may happen because the majority of SPIONs may be absorbed/trapped on the root tissue. It is now well recognized that the surface of NPs are covered by various macromolecules, upon their entrance into the fluids containing bio-molecules.Thus, the abundance of magnetic signal in the root tissue clearly indicates that the absorbance of root exudates macromolecules at the surface of NPs caused SPIONs accumulation on the root surface; this result was completely in agreement with the previous reports on the surface of gold NPs could varies their physicochemical properties and affect their uptakes and traslocations into rizhospher and xylem sap25. It is now well-recognized that NPs surfaces are affected by various chemical and biological elements in biological media.The biological identity of NPs changes in biological milieu with absorption of bio-molecules.According to the magnetization results , it is confirmed that positive and negative SPIONs can penetrate well from growth medium into various soybean tissues, rather than plain NPs; furthermore, positive and negative SPIONs can traverse to stem and leaf and less aggregated in aerial plant tissue, compared to the plain NPs. Zhu et al. investigated uptake and translocation of Fe3O4 NPs by pumpkin in hydroponic conditions by using a vibrating sample magnetometer. The study showed similar result that magnetic signals were detected in root, stem, and leave of plant grown in medium with magnetite NPs.On the other hand, Wang et al.did not observe any uptake of 25 nm Fe3O4 NPs by pumpkin plants. It is hypothesized that it is difficult for the larger size NPs to penetrate through the cell walls and transport across the plasma membranes.Figure 4 shows how SPIONs penetrate into the root, traverse to the xylem,vertical grow towers and translocate into the shoot. Figure 4A indicates that accumulation of SPIONs inside the root tissue is much broader than the aerial part of the plant. The largest amount of agglomerated SPIONs cannot be uptaken by the plant cells and a number of them is incorporated into the cell wall. Figure 4B confirms that SPIONs are diffused toward the interior of the stem parenchyma. Since SPIONs dimension have significant smaller size compared to the cell wall pore and the plasmodesmata width, the SPIONs traverse through the biomembranes and other plant pathways. Figure 4C displays SPIONs infiltrated into the mesotome and parenchyma cell from the leaf veins. SPIONs diffuse from xylem’s sap to aerial tissue with apoplastic flow and symplastic transport.

Transpiration and evaporation stream of water from stomata leaf are responsible for accumulated of SPIONs in the margin of leaves. However, the mechanisms underlying these processes are not understood. Nowack and Bucheli50 speculated NPs may enter plant roots through osmotic pressure, capillary forces, through pores in cell walls and intercellular plasmodesmata, or via the highly regulated symplastic route. Plants have selective uptake and translocation of NPs. The NPs could also diffuse into the intercellular space, the apoplast, and then be adsorbed or incorporated into chelates.The properties of NPs, such as composition, size, shape, and surface charge may affect the uptake and translocation inside plant.Enhancement of chlorophyll content in subapical leaves of soybean is depended on the concentration SPIONs in the growth medium and surface charge of NPs. The mean chlorophyll concentration in the soybean fresh weight exposed to SPIONs is significantly lower than those treated with Fe-EDTA at the same concentration . There is no significant difference in the ratio of chlorophyll a/b in all treatments. For these reasons we suggest that the biosynthesis of chlorophyll a is  influenced differently in comparison to that of chlorophyll b. A suitable linear correlation between chlorophyll a and b, with correlation coefficient over 0.9, suggests that the biosynthesis of main photosynthetic pigments is affected by SPIONs. In this experiment, the SPIONs are sole source of iron in the treatment. The soybean rhizosphere is acidified by protons for releassing of Fe ions from SPIONs and then the iron ions are used inside the plant. But iron ions concentrations are not adequate for soybean growth . The SPIONs could provide iron ions with redox reactions involved in the chloroplast. The biochemical reactions in chloroplast stroma, siderophore in the tylakoidal membranes,and photocatalytic reaction are suggested as factors for iron availability from SPIONs. Other results of previous investigations show low ferrofluid concentrations increase chlorophyll level in bean plantlets.The SPIONs effects on the soybean photosynthesis performance may have not only a biochemical influence but also a magnetic field of the particle  influence on the enzymatic structures in the different stages of the photosynthesis reactions.The effect of magnetic NPs coated with tetra methyl ammonium hydroxide with super paramagnetic properties could  influence the ion flows by changing ion channels properties.Chlorophyll a and b concentrations at subapical leaves of soybean are diminished at more than 45 mg·L−1 of plain SPIONs in the growth medium . The ratio of chlorophyll a to b in all treatments indicates that there is no significant difference on the photosynthesis efficiency between Fe-EDTA and SPIONs as sources of iron. We notice toxicity symptoms lead to brown spots covering the leaf surface in the plants for a culture medium with 60 mg·L−1 Fe-EDTA and plain SPIONs. Iron excess in this treatment could be generating oxidative stress in the leaf cells.Nitrogen is the mineral element that most often limits plant growth and primary productivity in natural and agricultural systems. Plants usually acquire N from the soil in the forms of ammonium and nitrate , and management of these forms is vital to agriculture.

The extract solution is filter-pressed at 25–30 psig to remove the aggregated protein impurities. Filtering has a process time of 1 h and requires a filter area of 3 m2 to handle the 590 kg/batch of the process stream. At this stage, the process loses a further 8% of the Griffithsin but removes all the RuBisCO and 87% of the TMV coat protein impurities. The filtrate from this step is transferred to a second mixing and storage tank, mixed with bentonite clay and magnesium chloride, and stored at 4◦C for a 12-h period. This stage is the bottleneck operation for the downstream process. After the 12-h incubation, the solution is filtered through a second 0.3µm filter press and a 0.2µm inline sterilizing filter. These operations remove the remaining protein impurities leaving a Griffithsin extract with greater than 99% purity but at the cost of losing 6% of the Griffithsin. The second plate-and-frame filter has a filter area of about 3 m2 and will process all of the extract in 1 h. There is approximately 222 g of Griffithsin per batch at the end of the filtration phase. Following the filtrations steps, the Griffithsin extract solution is collected in a storage tank and further purifified using an AxiChrom column with Capto MMC resin to remove residual color and potential non-proteinaceous impurities. To accommodate the 222 g of Griffithsin in solution, 4.9 L of MMC bed resin is needed at a 45 mg/mL binding capacity . The order of the operations for this chromatography step are: Equilibrate, load, wash, elute, and regenerate. In total, chromatography requires 10 h with the load step taking the longest, at 8 h, because approximately 600 L of solution are processed. Chromatography is necessary to decolorize the extract at the expense of losing 4% of the Griffithsin,vertical garden indoor giving a remaining Griffithsin mass of 210 g per batch. The 10 L of eluant process fluid is sent through a viral clearance filter and transferred into a pool/storage tank.

Subsequently, the extract is sent through anultrafiltration/diafiltration cycle to remove salts introduced in the chromatography column. After ultrafiltration, the product is transferred into a storage tank to be mixed with the final formulation components. The concentrated Griffithsin is diluted to give a concentration of 10 g/L Griffithsin in 10 mM Na2HPO4, 2.0 mM KH2PO4, 2.7 mM KCl and 137 mM NaCl at pH 7.4. The final volume of the DS is 21 L per batch. As shown by Figure 4, each batch in the downstream requires 39 h of process time which includes all SIP and CIP operations. As batches move from the upstream portion of the facility every 3.44 days, the remaining time left over in the downstream is set as slack time in the model that may be dedicated toward repair, maintenance, etc. The assumptions and results developed in SuperPro were used to calculate the economics of the process described. Table 2 shows the total operating costs segregated individually for upstream and downstream components. Figure 5 displays process category cost contributions graphically, including percentages of total costs. In upstream operations, the largest cost components are utilities and labor , representing 61% and 32% of total upstream costs, respectively. In downstream operations, labor-dependent costs are the highest contributors at 30% of total downstream costs, followed by consumables at 27% of total downstream costs. Overall, the upstream component represents nearly 57% of the total Griffithsin production cost, which is calculated as just over $106/g protein. For a microbicide dose of 3 mg, the per-dose manufacturing cost is $0.32, excluding any CMO fee. An environmental health and safety assessment was also conducted for this case study following the method of Biwer and Heinzle and the results are found in Supplementary Tables 2–4 in Supplementary Materials. Overall, the process uses chemicals that are not harmful to people or the environment, as can be seen by the low magnitude of input and output Environmental Factor values in Supplementary Table 4. The biggest causes for concern are TMV in the residual biomass, and sodium hydroxide and phosphoric acid used in clean-in-place operations, if released to the environment; however we included costs for a thermal or chemical deactivation step for the TMV-contaminated biomass and pH neutralization for the acid and base cleaning agents which would eliminate the environmental impact of these components.

It should also be noted that the upstream nutrient compounds can be more efficiently recycled to increase nutrient utilization by the plants and reduce water/soil impact. Waste compounds in the downstream process are disposed of through wastewater and bio-waste treatment. An aggregate disposal cost of $0.01 per liter of non-TMV-contaminated aqueous streams and $0.1 per kg of bio-waste is assigned in SuperPro for expenses related to wastewater disposal and thermal/chemical deactivation of bio-waste streams. Compounds introduced during or after the post-inoculation step in the upstream facility are considered as bio-waste since they may contain TMV. This includes spent nutrient solution in the post-inoculation step and retentate streams from plate-and frame and dead-end sterilizing filtration skids. Disposal of TMV-contaminated materials poses low environmental risk. There is extensive industrial experience in disposing of TMV contaminated materials, which can be rendered non-infective by treatment with bleach, heat or detergents, diluted and disposed of as municipal waste . The facility modeled can annually produce 20 kg of the potent antiviral Griffithsin for use in microbicide products. The host used in our modeling was Nicotiana benthamiana. This species was selected because of its aforementioned productivity, but also because our previous report on technoeconomic modeling of Nicotiana-produced therapeutic and industrial products prefaces the work reported herein. In addition, the use of Nicotiana for production of clinical trial materials is also familiar to FDA and other regulatory agencies, thus facilitating Nicotiana’s acceptance in regulation-compliant manufacturing . The API is manufactured in the host Nicotiana benthamiana using tobacco mosaic virus as the expression vector. The upstream plant growth and Griffithsin production operations are adapted from the facility layout detailed byHoltz et al. . Over 158,000 plants are housed in vertically stacked hydroponic grow racks, fitted with high-efficiency LED lights.Each batch of 14,450 plants grows over the course of 38 days and yields a total of 578 kilograms of biomass. Ninety-five batches are seeded and grown annually, with one batch reaching harvest every 3.44 days. The downstream Griffithsin extraction and purification process is scaled up from the pilot industrial scale process presented by Fuqua et al. .

An expression rate of 0.52 grams of Griffithsin per kilogram of biomass and a downstream recovery of 70% were used in the base case and give a combined yield of 0.370 grams of Griffithsin per kilogram of harvested biomass. Sterile filtration and CIP/SIP systems facilitate compliance with cGMP guidelines. Downstream processing commences upon the completion of an upstream batch and takes 39.3 h. The stable final formulation is >99% Griffithsin as the API with negligible endotoxin levels. In the model,vertical garden indoor system the upstream costs account for nearly 57% of the total cost of Griffithsin production. Containing both upstream and downstream losses of the protein could significantly reduce COGS. Approximately 12% of the protein API is non-liberated from the homogenized biomass and 18% is lost during downstream polishing steps. Based on the data and assumptions employed in the current analysis, the unit production cost of Griffithsin is estimated to be $0.32 per dose . The model was based on published designs for a commercial scale facility and pilot-scale data on Griffithsin production adapted to the facility described. This type of modeling is useful for determining ranges of API selling price, production capacity and expression level requirements for commercial supply and profitability. In this study we modeled the manufacturing of Griffithsin through a contract manufacturing organization instead of a greenfield build of a new facility because we assumed that that would be the most prudent approach to launching a new product. If the product manufactured using the process modeled is used directly as a vaginal rinse or rectal enema, the additional costs post manufacturing would include transportation, storage, insurance, distribution, marketing, etc., none of which were modeled in this manufacturer-level analysis. If the Drug Substance produced via the process analyzed is further formulated , or used as a component of another device , those costs and other product-specific costs would be additive and were also excluded from our manufacturer-level analysis. The cost of goods calculated by the current model reflflects the manufacturer’s cost of production. We are less certain about the wholesale price of the drug because there is no standard “offff- the-shelf ” profifit margin that can be added to toll manufacturing cost to arrive at a standardized answer. Often scale up to commercial launch volumes of a product requires additional process development and optimization, validation batches, etc., which lead to negotiated transfer prices depending on volume, duration of engagement, license fees, export duties, and other factors, all of which would impact the cost of bulk Griffithsin. Nevertheless, for this discussion we assumed a manufacturer’s fee of 20% of COGS for a total production cost of bulk Griffithsin Drug Substance of $0.38/dose. Additive formulation, storage, distribution, insurance, marketing, sales margins and other costs could lead to a consumer-level use cost of $1-2/dose . This technoeconomic analysis emphasized Griffithsin’s use in microbicides because such products arguably represent the most price-constrained applications of this new drug. We cannot define the target retail price of a Griffithsin microbicide; there is no market reference price for microbicides since no commercial microbicides yet exist. For perspective, the user cost of a Griffithsin microbicide can be benchmarked against pre-exposure prophylaxis with traditional male condoms and PrEP with microbicides containing antiretroviral drugs as a newer alternative. Analyses have been conducted on the cost of prevention modalities and the cost savings to the healthcare system enabled by preventing HIV transmission, with prevention being far more cost effective than treatment in most scenarios.

Walensky et al. conducted an analysis of the cost-effectiveness of a Tenofovirbased PrEP microbicide in South African women. In their cost modeling of a vaginal gel, they multiplied the product cost of $0.32/dose times 2 and by 7.2 to arrive at a product use cost of approximately $5/woman month. However, the price of the microbicide gel used in the study was assumed and region-adjusted and hence pricing in other countries may be different. Terris-Prestholt et al. estimated Tenofovir gel prices of $0.25–0.33 per dose, provided that the gel was used in combination with a condom , from which an additive cost of use of $7–$12/person-month can be derived. Assuming the same average use rate of a Griffithsin containing microbicide applied singly without a condom and priced at $1.00–$2.00 per dose, the cost of use would be $7– <$15/person-month. Whether a higher cost of use discourages adoption of Griffithsin-based microbicides by men and women remains to be shown. A market study by Darroch and Frost of the Alan Guttmacher Institute consisted of detailed interviews of a cross-section of 1,000 sexually active women aged 18–44 in the continental United States. Their statistically rigorous survey identified levels and predictors of women’s concerns about STDs and interest in microbicides, as well as their preferences regarding method characteristics and likelihood of usage versus price of product, with survey sample results extrapolated to the national level. The results showed that of the estimated 12.6 million women aged 18–44 interested in microbicides and concerned about STDs, including HIV, 11.5 million would still be interested in the method even if it were not 100% effective, and 11.0 million would remain interested even if the microbicide did not protect against STDs other than HIV. The same study found that women’s predicted use of a microbicide was affected by price, but interest was still high at $2 per application, or roughly up to 5-times the average price of a male condom. The survey concluded that more than seven million sexually active women in the USA would be interested in a vaginal microbicide even if the product only protected against HIV, was only 70–80% effective and cost them $2 per application . That conclusion was arrived at in 1999; the $2 per application cost back then would be $3.05 in 2018. One can conclude from these results that there is interest in effective yet inexpensive, self-administered HIV and STD prevention modalities even if such products might cost more than conventional prevention methods.

In the cost-constrained markets of food additives and processing aids, these new biotic approaches to food sanitation will need to be accessible at the low selling prices that the food industry is accustomed to, or gain market entrance as a luxury good on the basis of their differentiating features, including worker safety in the preparation and handling of the products, environmentally friendly disposal, non-impact on the organoleptic properties of food, and no or minimal food matrix alteration.Strategies to meet low cost of use can be broadly classified as either pertaining to molecular engineering of the treatment agent or manufacturing science and technology. Substantial research has been done to employ genetic engineering to alter the action of native antimicrobial proteins.For example, the modular structure of the bacteriophage class of enzymes known as endolysins provides a perfect “Lego® block”-like molecular engineering platform to swap the N-terminal catalytic domain or the C-terminal binding domain to create novel hybrid moieties.Although molecular engineering approaches possess substantial potential for human therapeutics, changes to the native structure of antimicrobial proteins for food safety applications bar them from taking advantage of the expedited GRAS marketing allowance pathway. For antimicrobials that are novel, or altered, and hence not “generally recognized” as safe, the alternative marketing approval route requires a full preclinical safety data package, which is a costly and time-consuming process that creates a significant barrier to entry for new food safety interventions, given the above mentioned current pricing structures, regulations, and public perception. Consequently,how to make a vertical garden biotic food safety approaches are more amenable to cost containment through manufacturing science and technology.

The cost sensitivity of the food industry is the most significant barrier to the adoption of new food sanitizing treatments, such as antimicrobial protein preparations. Plant-based platforms have the potential for producing market-relevant volumes of AMPs at competitive costs, because they do not require expensive bioreactors and culture media. In recent studies, we have shown that plants such as Nicotiana benthamiana, spinach, and leafy beets are an attractive and scalable production platform for production of AMPs, including antibacterial colicins, salmocins, and bacteriophage endolysins. We have previously reported expression levels as high as 3 g/kg plant fresh weight .In this study, we address cost sensitivity with a comprehensive techno-economic analysis of plant-based production of AMP for food safety applications. We used laboratory scale results and working process knowledge from pilot and commercial processes to develop a process simulation model using SuperPro Designer® to assess the commercial viability of the production platform and to identify economic “hotspots” to help guide future research and development. A selection of recently published studies on the techno-economics of N. benthamiana plant-based production of a variety of recombinant proteins is summarized in Table 1.To our knowledge, this study is the first techno-economic analysis of a plant-based production platform for AMPs as food safety additives.Nicotiana benthamiana is used as the plant host organism in the base case scenario. Nicotiana benthamiana is used extensively for indoor plant molecular farming applications based on its rapid growth, genetic tractability, susceptibility to agrobacterium transformation, and high expression levels of recombinant proteins.The species is used in the commercial scale production of therapeutics and vaccines by companies such as Kentucky BioProcessing Inc. ,Medicago Inc. ,and iBio CMO.The modeled facility is designed to accommodate a previously reported process using transgenic N. benthamiana featuring a doubleinducible viral vector, developed by Icon Genetics GmbH.

Published results demonstrate minimal background expression of recombinant protein until the induction of deconstructed viral RNA replicons from stable DNA proreplicons is triggered by 1–20% ethanol applied as a spray on the leaves and/or a drenching of the roots, to achieve expression levels as high as 4.3 g/kg plant FW.Although the more common Agrobacterium-mediated transient expression production platform enables rapid production of recombinant target molecules,this transgenic system obviates the need for additional expenses associated with Agrobacterium tumefaciens preparation, vacuum infiltration, and agrobacterium-introduced endotoxin removal.The simulated manufacturing facility is composed of two separate process models/flow sheets: the upstream processing models the plant growth, ethanol induction, and product generation, which feeds into the downstream processing model for purification of the product from the process and product impurities to meet food processing aid specification. Quality assurance , quality control , and laboratory costs associated with good agricultural and collection practices for upstream processing and FDA food industry current good manufacturing practice for downstream processing are included in the design. Equipment, materials of construction, and prices are also modeled on food cGMP standards.The location of commercial-scale plant molecular farming operations of Kentucky Bio-Processing Inc. was selected as the basis for location-dependent costs. Location-dependent costs are based on values obtained from publicly available Owensboro, Kentucky municipal pricing charts . The simulated manufacturing facility is assumed to be a greenfield single-product bio-manufacturing facility that is operational 24 hr per day and 7 days per week with an annual operating time of 90% or 329 days per year. Independent market analyses project a reasonable base case facility production level of 500 kg AMP per year for food safety applications of interest . To meet this demand, the proposed facility employs three-layer vertically stacked indoor plant cultivation stages designed for hydroponic host plant growth in a soilless substrate to support the plant and its roots. The cultivation stages are equipped with a light-emitting diode lighting system and a recirculating ebb and flow hydroponic water supply. The cultivation stage plant growth is divided into a series of trays that advance unidirectionally across the plant cultivation room toward automated plant harvesters and further downstream processing.

Automated belts convey harvested plant tissue to the double-stack disintegrator and further downstream processing.The upstream processing model flow sheet is graphically depicted in Figure 1. Transgenic N. benthamiana seeds consumed in upstream processing are generated in-house from validated Working Seed Banks, which were in turn generated from validated Master Seed Banks. The seed bank release testing includes germination efficiency >95%, confirmation of growth kinetics, and viral testing. CAPEX related to seed generation are excluded, but associated seed production costs are included in the estimate of $9.50/g seed . The seeds are set in soilless plant substrate at a density of 94 N. benthamiana seeds per 30 × 50 cm tray. The seedlings are cultivated hydroponically during the plant growth phase to reach manufacturing maturity by 35 days. Nutrient solution for plant growth is recirculated with minimal waste and routinely monitored and adjusted for consistent quality based on pH and conductivity. At manufacturing maturity, the plants are transferred to an induction space, complete with a separate hydroponic reservoir, curtains for temporary enclosure,vertical planters for vegetables and double rail spray booms. Recombinant expression of AMP is induced over the course of 1 hr via root drenching and aerial tissue spraying with a combined 0.01 L of 4% ethanol per kg FW plant tissue. The plants are then moved to the incubation phase. Post-induction plants are expressing recombinant AMP, and so the nutrient solution is circulated via a separate feed tank and hydroponic reservoir. The nutrient solution in the incubation phase may contain trace levels of ethanol, which may prematurely initiate AMP production and impair plant growth kinetics. AMP accumulates in the N. benthamiana tissue over the course of 6 days. The nutrient solution in the incubation phase is not recirculated between batches, but sent to bio-waste instead, amounting to an overall 23% plant uptake of the nutrient solution. The spent nutrient solution in the incubation phase is treated as bio-waste to address trace amounts of the viral expression vector that may be present in solution.The downstream processing model flow sheet is graphically depicted in Figure 2. Downstream processing begins with plant harvest. This starts with automated harvester collection of aerial N. benthamiana plant tissue. The spent soilless plant substrate is sent to waste along with the remaining N. benthamiana root matrix. The disposal costs for this step are considered negligible and are not explicitly calculated in the model. There are several routes possible for disposal of plant growth substrate such as composting on site, using it for mulch on facility landscape, collection by farmers for spreading on agricultural land, and, as a last resort, sending it to a landfill. It may be possible, and more cost effective, to sterilize and reuse the growth media but this was not considered in the model. The harvested trays are cleaned in an automated washer with 0.1 L of water per tray. The harvested plant tissue is conveyed via automated belts to extraction, which starts with a double-stack disintegrator to reduce plant biomass particle size.

The disintegrated tissue is then sent to a screw press with an extraction ratio of 0.5 extraction buffer:plant FW for acidic extraction. The extraction buffer and conditions for efficient N. benthamiana extraction have been reported.39 All buffer compositions can be viewed in Table S5. A plant-made AMP purification protocol uses similar acidic extraction to remove N. benthamiana host proteins.The plant extract is clarified using tangential flow microfiltration. The clarified stream is then ultra filtered with additional tangential flow filtration using a 10 kDa molecular weight cutoff to a concentration factor of 20. The AMP in the retentate stream is then purified with cation exchange column chromatography in a bind-and-elute mode of operation. The AMP is eluted isocratically in elution buffer . The purified stream is subjected to one final tangential flow filtration procedure for buffer exchange into phosphate-buffered saline with a diafiltration factor of 3. The purified formulation is spray dried and filled in 1-L plastic bags to obtain the final bulk AMP. All downstream processing water in direct contact with the product stream is reverse osmosis water. All equipment from extraction to formulation are sanitized post processing with a clean-in-place procedure consisting of a prerinse with municipal water, caustic wash with 0.5 M NaOH, postrinse with municipal water, acid wash with 0.5% HNO3, and a final rinse with RO water.Base case scenario outputs were used to identify parameters with significant impact on process economics. We focused the scenario analysis on two different classes of parameters: facility performance parameters and resource purchase costs. Facility performance parameters are defined as inputs that directly impact the physical outputs of the model. Typical biotechnology facility performance parameters include host organism expression level, unit operation recovery, and yearly production level. We chose to investigate expression level and yearly production level. To analyze the impact of facility performance parameters, we set a parameter range based on working process knowledge and then developed a model derived from the base case scenario for each parameter increment within the range. Facility performance parameter changes result in a cascade of changes to the model inputs and outputs; each model is adapted to the resulting stream composition and throughput of the given parameter value while maintaining the constraints of the fixed base case scenario process inputs. Resource purchase costs are defined as inputs that directly control the economic impact of resource utilization for outputs of the model. For the purpose of this analysis, purchase price parameters are contained to cost items within OPEX.Alternative facility design scenarios were developed as comparative models to more broadly explore the context of the base case scenario process economics. The alternative scenario models were designed in alignment with base case scenario inputs unless otherwise noted; each alternative scenario was chosen to isolate the impact of a key facility design assumption. The first scenario investigates an alternative transgenic leafy plant host organism, spinach cultivar Industra, for the base case scenario indoor growth and ethanol-inducible expression. Some colicins have been successfully expressed in S. oleracea plants; however, their expression levels were approximately 10 times lower than that in N. benthamiana so additional research is needed to increase production levels.Several salmocins and lysins can be expressed at high levels in spinach, which is comparable to expression levels in N. benthamiana.The primary distinction in this alternative plant host organism is the lack of nicotine, the major alkaloid in Nicotiana species. In the base case scenario, significant downstream processing emphasis is placed upon nicotine removal. The upstream and downstream processing model flow sheets are graphically depicted in Figure 1 and Figure 2. A complete list of changes to the base case scenario inputs can be observed in Table S4. The second scenario investigates outdoor field-grown transgenic ethanol-inducible Nicotiana tabacum as an alternative to an indoor plant growth facility.

Previous research in our laboratory demonstrated that tomato seedling roots and crowns became highly susceptible to P. capsici following a brief exposure of the roots to salt stress . These plants generally regained turgor during the course of the stress treatment, but remained in a predisposed state in the absence of visible stress FIGURE 4 | ABA-related gene expression in inoculated ‘New Yorker’ tomato seedling roots following an episode of salt stress. NCED1 and TAS14 expression in non-stressed roots and in roots after 18 h exposure to 0.2 M NaCl/0.02 CaCl2, with and without inoculum . NCED1 and TAS14 expression was normalized against Cyp and UK. Values are the means ± SE from two experiments, with roots from fifive plants pooled per sample and six samples analyzed for each treatment mean per time point. Note that seedlings were removed from the salt stress and returned to 0.5X Hoaglands during the course of analysis. symptoms for up to 24 h following removal from the salt. The salt stress effect on disease appears to operate through an ABAdependent mechanism, as evidenced by the loss of predisposition in ABA-deficient mutants and partial complementation with exogenous ABA to restore the predisposition phenotype . Salinity stress also has been shown to make roots more attractive to Phytophthora zoospores . In the present study, chemoattraction of P. capsici zoospores to exudates from salt-stressed roots was significantly greater than to exudates from non-stressed roots. However,hydroponic vertical farm exudates from salt-stressed roots of wild-type tomato plants and ABA-defificient mutants were equally attractive . Thus, differences in root attraction to zoospores cannot explain the differences in disease severity between wild-type and ABA-deficient plants.

These results reinforce our view that the determinative effects of stress-induced ABA in predisposition occur during infection, invasion and colonization, rather than during pre-infection events related to root exudation, zoospore attraction and initial contact with the root . Our results also affirm an earlier study on salinity-induced susceptibility to Phytophthora root rot that pointed to a strong effect of the stress on host defenses . P. capsici is a hemibiotroph, establishing haustoria in host cells during the early stages of infection, and then necrotizing host tissue as the infection progresses . Confocal imaging revealed the presence of haustoria in infected tomato roots that appeared as simple protrusions into root cells , closely resembling those described in the literature for Phytophthora haustoria . After reviewing dozens of P. capsici infections in non-stressed and salt-stressed roots, we concluded that haustoria are present in both treatments. Therefore, it does not appear that P. capsici alters its fundamental infection strategy in salt-stressed tomato roots. The only clear distinction apparent between treatments was the increased rate of colonization, as reflected in greater abundance of hyphae in the salt-stressed roots relative to the controls. While the pathogen’s infection strategy does not appear to change, based on microscopic FIGURE 6 | P. capsici colonization 48 hpi on ‘New Yorker’ and NahG tomato seedlings non-stressed or salt stressed with 0.2 M NaCl/0.02 M CaCl2 for 18 h. Colonization estimated by qPCR of pathogen DNA. Letters indicate significant differences at P = 0.05 . Values are the means ± SE from three experiments, with five samples, each from a separate seedling, for each treatment within each experiment . examination, it is possible that P. capsici alters its strategy in other ways, such as the timing or pattern of display of effectors. We attempted to measure expression of putative and known P. capsici effector genes believed to correspond to the switch from biotrophy to necrotrophy.Pathogen RNA proved difficult to recover during early infection and later as plant tissues died, and so we were unable to detect alterations in effector expression as a function of treatment.

Transcriptome analyses using deep sequencing as reported in a study of P. capsici on tomato leaves may prove to be better able to address this question . Endogenous ABA levels are tightly regulated in the plant by balancing biosynthesis, catabolism and conjugation . NCED1 expression in roots during the 18 h salt stress treatment generally corresponded with salt-induced ABA accumulation that we reported in our previous study . Similar findings in Phaseolus vulgaris showed stress-induced expression of NCED, with accumulation of NCED protein and ABA occurring within a 2 h window . While stimuli have been described that upregulate NCED1 gene expression, relatively little information is available regarding mechanisms for its down regulation. In drought-stressed Arabidopsis, ABA production and expression of NCED3is correlated with the level of available carotenoid substrates . NCED1 expression in tomato roots may diminish as ABA levels decline or as external stresses are removed. Possible post-transcriptional and/or post-translational regulation of NCED1/NCED cannot be ruled out, as suggested for regulation of AAO , the terminal step in ABA synthesis . Following an episode of salt stress and inoculation with P. capsici, NCED1 transcript levels returned topre-stress levels in tomato roots and remained at basal levels in all treatments throughout the 48 h infection time course . However, we saw no evidence for NCED1 induction or ABA accumulation during infection with P. capsici. This is in contrast to Arabidopsis infected by Pst, which induces AtNCED3 and ABA accumulation in leaves . Expression of TAS14, which encodes a tomato dehydrin, is triggered by osmotic stress and ABA . When over expressed in tomato, TAS14 confers partial drought and salinity tolerance . In our study, TAS14 increased rapidly after salt stress onset and remained elevated throughout the course of the stress treatment. Similar to NCED1, TAS14 did not show altered expression following P. capsici infection, and in the case of salt treatment, TAS14 expression returned to basal levels within 24 hpi . The possibility of P. capsici-derived ABA was of interest because some plant pathogenic fungi produce ABA , and some stramenopiles such as the malarial pathogen, Plasmodium falciparum, are capable of ABA synthesis . However, we did not detect ABA in P. capsici culture filtrates or mycelium by immunoassay , and genes encoding the necessary biosynthetic enzymes are not evident in oomycete genomes . Furthermore, we found no evidence that P. capsici infection further engages the pathway as part of its infection strategy, either in non-stressed or salt-stressed tomato plants.

These results indicate that salt stress, but not Phytophthora infection, strongly engages the ABA pathway in tomato roots – NCED1 and TAS14 gene expression, and ABA synthesis and accumulation. The SA-induced tomato PR protein, P4, is homologous to PR-1 in tobacco and Arabidopsis. P4 gene expression is induced in tomato leaves by plant activators , pathogens, including Phytophthora infestans, and the oomycete elicitor arachidonic acid . We found that infection of tomato roots by P. capsici strongly induces P4, but exposure of the roots to salt prior to inoculation essentially abolished P4 expression relative to non-stressed, inoculated plants . Similarly, expression of the JA-induced PI-2 was significantly reduced in infected plants that had been previously salt-stressed. Our findings that salt stress prevents pathogen-induced SA- and JA-regulated gene expression are consistent with results in other plant–microbe interactions that demonstrate ABA-mediated suppression of SA and JA defense responses . Tomato plants suppressed in SA accumulation by the nahG transgene are more susceptible to P. capsici than the wild type control plants in both non-stressed and salt-stressed assay formats . This suggests a role for SA-mediated responses in partially limiting P. capsici colonization. However, the proportional increase in pathogen colonization observed in salt-stressed plants relative to non-stressed plants is the same in both WT and NahG backgrounds. Impairment of SA action by salt stress may contribute to increased pathogen colonization; however, we did not see a compounding effect of the SA-deficiency in NahG plants on stress-induced disease severity. Salicylic acid’s role in tomato resistance to P. capsici is complex. In a study using chemical activators that mimic SA action to induce resistance, we found these activators when applied to roots induced systemic protection of tomato leaves against the bacterial speck pathogen , with and without predisposing salt stress . However, these same plant activator treatments afforded no protection against P. capsici, with or without the salt stress treatment. Pst and P. capsici are quite different in their infection strategies and requirements, as well as the organs they attack in the plant, so interpreting differences in disease outcomes following different treatments is a speculative exercise, at best. P. capsici may simply be a more aggressive pathogen relative to Pst,vertical farm and our experimental format is highly conducive to root and crown rot disease. So P. capsici attack overwhelms any chemically induced resistance that is otherwise capable of withstanding Pst challenge. NahG expression may impair a set of SA-mediated defenses that are effective against P. capsici, but differ from a subset, induced by chemical activators, that are insufficient to resist this pathogen. The JA-deficient tomato mutants acx1 and def1 in the ‘Castlemart’ background are compromised in defense against insects and pathogens . Although severity of the predisposition phenotype can vary among tomato cultivars, we were astonished that ‘Castlemart’ and its JA mutants were not predisposed by salt, strongly trending instead toward enhanced resistance . This suggests a stress response in ‘Castlemart’ that is different from other tomato genotypes we have examined in predisposition studies. The reason for this is unclear, and limited resources precluded our further examining predisposition in this cultivar. Unlike the other genotypes used in our study, ‘Castlemart’ is a processing variety with a pedigree that may have incorporated different stress tolerances. It is a determinate variety that was bred for arid climates, and arid zone soils are more commonly associated with salinity . ‘Castlemart’ has been reported to accumulate proteinase inhibitors in response to high salinity . Jasmonic acid and its methyl ester when applied to leaves can induce resistance in tomato to P. infestans . Arabidopsis mutants in JA perception and synthesis are more susceptible to oomycete pathogens. Studies with other oomycete diseases also illustrate JA’s importance in resistance .

We found that exogenous JA enabled tomato roots to respond in a manner that partially offffset the salt stress impairment of PR-protein gene expression . The induction of P4 only during infection of JA-treated plants is reminiscent of the reported sensitization by methyl jasmonate of the plant’s response to eicosapolyenoic acid elicitors released during infection by Phytophthora species and potentiation of JA signaling by the plant activator β-aminobutryic acid . Our results with the tomato genotypes and treatments used in this and previous studies affirms ABA’s dominant effect relative to the salt-induced impacts on SA and JA action during predisposition to Phytophthora root and crown rot. ABA appears to be necessary to predispose tomato seedlings to this disease following acute salt stress. However, results presented here and previously indicate that priming through chemical activation of the SA and JA response networks may partially offset the stress-induced impairment of defense-related gene expression and the increased susceptibility in tomato to certain pathogens. We recognize that the response pathways modulated by ABA, JA and SA during episodic root stress may interact in subtle ways beyond the resolution afforded by the pathosystem and treatments we selected . Comparative transcriptomics, proteomics and metabolomics of plants under predisposing stress should help identify key regulatory features . Studies with additional mutants as well as salt- and drought-tolerant genotypes also may reveal additional variation that could be useful to refine our understanding of the abiotic-biotic stress ‘interactome’ . This information could suggest novel targets to mitigate the impact of root stresses that increase severity of soil borne diseases. Inorganic pyrophosphate is an intermediate compound generated by a wide range of metabolic processes, including biosynthesis of various macro-molecules such as proteins, DNA, RNA, and polysaccharides. Being a high-energy phosphate compound, PPi can serve as a phosphate donor and energy source, but it can, at high levels, become inhibitory to cellular metabolism. To maintain an optimal PPi level in the cytoplasm, timely degradation of excessive PPi is carried out by two major types of enzymes: soluble inorganic pyrophosphatases and proton-translocating membrane-bound pyrophosphatases. The importance of maintaining an optimal cellular PPi level has been demonstrated in several different organisms. Genetic mutations that lead to the absence of sPPase activity affects cell proliferation in Escherichia coli. In yeast, inorganic pyrophosphatase is indispensable for cell viability because loss of its function results in cell cycle arrest and autophagic cell death associated with impaired NAD+ depletion.