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.

At a minimum, they are examples of environmental technologies that predate the rise of sustainability and widespread environmental awareness in landscape architecture. They also suggest a rich legacy of technological innovation born from the same environmental pragmatism and innovation that define contemporary landscape architecture. It is humbling to think that 30-plus years before the word “sustainability” found its current usage in environmental fields and the language more broadly, a tinkerer in New Jersey invented a permeable turf paver in 1940 through which the “roots of seedlings may take root, and thereby provide an interlocking connection between adjoining blocks.” Or that an inventor in Mahopac, New York, had the prescience in 1932 to devise a technique for anchoring trees to buildings and structures 80 years before they adorned the Bosco Verticale in Milan . Or that an early innovator in the geotechnical arts integrated the process of decomposition into a slope stabilization system in 1933, many years before ideas of weathering became common to landscape practice. These inventions provide us with important antecedents to the word “sustainability” and challenge landscape architects to reevaluate the relationship among innovation, early adoption of technology, professional practice, academic research, and implementation in the built environment. One of the most important figures in the history of innovation in landscape-related technologies is Stanley Hart White, a professor of landscape architecture at the University of Illinois from 1922 to 1959. White patented the first known vertical garden in 1938, roll bench yet the idea emerged in his writings and sketches as far back as 1931 .

His technology integrated a steel structural framework with hydroponic substrate, internal irrigation, and vegetation to provide fine sheets of greenery as a background and camouflage for the modern garden. White prototyped the “Vegetation-Bearing Architectonic Structure and System” in his Urbana garden shed more than 60 years before the emergence of the vertical garden in the contemporary built environment.The merits of this invention may ultimately pale in comparison to the precedent he established for a landscape architect-cum-inventor and technological innovator. White translated modern landscape theory, advances in building sciences, and emergent hydroponics into patent legalese, formulating the origins of vegetation-bearing architecture and pioneering green modernism. At that moment, landscape theory and technology converged at a 1:1 scale. It is hard to recall an instance in the annals of history when the vanguard of landscape thinking intersected so narrowly with the reification of technology, making White’s simultaneous commitment to theory and technology important beyond the invention of the vertical garden. What is the relationship between landscape architecture and technology? Are we simply purveyors and consumers legally chartered to select and specify the next best ready-made in construction documents? Is our use of computers for drawing, mapping, or rendering of future scenarios akin to true invention? Do we have a legacy of inventors and tinkerers who reify, curate, and improve the technologies that materialize our future sustainable and ecological cities? These questions are existential, as progress toward sustainable, ecological, and equitable future landscapes is in part technological determinism at work . Whoever develops the technology that leads us forward in these realms determines, in part, the future configuration of our cities and broader society. Currently, innovation in landscape-related technology is primarily the task of disciplines tangentially oriented to landscape architecture such as material sciences, industrial design, computer science, geotechnical and civil engineering, horticulture, furniture manufacturing, fabrication, and others concerned with the technical, material, and tectonic dimensions of landscape systems. Isn’t this the scope of landscape architecture? I believe it is! The patent archive now boasts thousands of landscape-related technologies, from Subsurface Upflow Wetland Systems to Reinforced Slope Planting Structures and Segmental Bio-retention Basin Systems .

Innovation in this sector will continue to thrive; the question is whether or not landscape architects will lead the way. P. vulgaris is characterized by a particular evolutionary history. Recent analyses based on sequence data presented clear evidence of the Mesoamerican origin of common bean, which was most likely located in México . The expansion of this species to South America resulted in the development of two ecogeographic distinct genetic pools with partial reproductive isolation . After the formation of these genetic pools -between 500,000 and 100,000 years ago – domestication took place, independently in the Mesoamerican and the southern Andean regions of the American continent . Genome analysis of BAT93 and G19833 , P. vulgaris sequenced model genotypes, has initially revealed interesting differences, for example between their genome size and number of annotated genes . The common bean is the most important legume for human consumption. In less favored countries from Latin America and Africa, common bean are staple crops serving as the primary source of protein in the diet. Soil acidity in these tropical regions is a major constraint for crop productivity, usually resulting in a combination of nutrient deficiency and metal toxicity . In acidic soils, aluminum toxicity is the primary factor of growth restriction, resulting in the inhibition of root growth and function, as well as in the increased risk of plants to perish of drought and mineral deficiencies, thus decreasing crop production.In Arabidopsis, the regulation of root growth is modulated by an ABC transporter‐like protein, annotated as ALUMINUM SENSITIVE PROTEIN 3 , which is localized in the tonoplast, suggesting a role in Al vacuolar sequestration . The LOW PHOSPHATE ROOT 1 ferroxidase, an ALS3– downstream protein of the phosphate-deficiency signaling pathway, is involved in root growth inhibition, by modulating iron homeostasis and ROS accumulation in root apical meristem and elongation zone . In root cells, AlT can affect multiple areas, as the plasma membrane, the cell wall and symplastic components . Common bean is known to be highly sensitive to AlT but this sensitivity is genotype-dependent .

In 2010, the evaluation of the root morphological traits related to AlT of 36 P. vulgaris genotypes revealed that Andean genotypes were more resistant to Al than Mesoamerican ones . Mendoza-Soto et al. reported that Mesoamerican common-bean plants subjected to high Al levels for short periods showed decreased root length as well as characteristic symptoms of AlT, such as ROS accumulation, callose deposition, lipoperoxidation and cell death in roots. Along other regulators, plant response to metal toxicity involves also microRNAs as part of the regulatory mechanisms. These molecules are a class of non-coding small RNAs of about 21 nucleotides in length, regulating gene expression at post-transcriptional level, guided by sequence complementarity, inducing cleavage or translational inhibition of the corresponding target transcript . The relevance of miRNA regulation in heavy metal tolerance is well documented; it has been demonstrated that heavy metal-responsive miRNAs show differential expression according to the toxicity level. Target genes of these miRNAs generally encode transcription factors that transcriptionally regulate networks relevant for the response to heavy metals. Additionally these encode transcripts for proteins that participate in metal absorption and transport, protein folding, antioxidant system, phytohormone signaling, or miRNA biogenesis and feedback regulation . High-throughput small RNA sequencing analyses have identified miRNAs that respond to AlT in roots of different plants species, however their function in response to AlT is largely unknown. Some of the target genes cleaved by AlT-responsive miRNAs encode disease resistance proteins, transcription factors or auxin signaling proteins . Our previous research indicated that P. vulgaris is no exception to this phenomenon. We identified common-bean miRNAs that respond to Al,commercial greenhouse supplies these include conserved miRNAs that are Al-responsive in other plant species -i.e. miR319, miR390, miR393- and also miR1511 . miRNAs from the miR1511 family have been identified in non-legume plants like strawberry  and poplar tree , although in the latter its nature as a miRNA has been discussed as it has been considered as part of a retrotransposon . Regarding legumes, miR1511 has been identified in Medicago truncatula and soybean . Also, miR1511 was identified in Mesoamerican common-bean cultivars, being more abundant in flowers and roots . However, this miRNA was not identified when analyzing the Andean G19833 reference genome . Genetic variation in MIR1511 has been reported in a comparative genotyping analysis of different Asian accession of domesticated soybean as well as its wild type progenitor Glycine soja. While sequences of mature miR1511 and miR1511* were found in G. max accessions, the sequences of annual wild G. soja showed insertion/deletion in the stem-loop region of MIR1511 that included complete or partial deletions of mature miR1511 sequence . Updated research indicates that the miR1511 target gene is not conserved in the different plants where it has been identified. In strawberry, the miR1511 targets an LTR retrotransposon gene . Inconsistencies about the nature of miR1511 target gene also hold for legume species. For instance, different targets have been proposed for soybean ranging from genes coding for proteins involved in the regulation of nitrogen metabolism to proteins relevant in plant cell development .

While in other species such as M. truncatula target genes have been searched but have not been identified. The SP1L1 transcript has been proposed as the common-bean miR1511 target , however despite several efforts from our and other groups this prediction could not be experimentally validated. These results suggested a species-specific selection of the corresponding target thus it was essential to experimentally validate the nature and possible function of the miR1511 target gene in common bean. Recent analyses led us to predict an ABC-2-type transporter-related gene, annotated as Aluminum Sensitive Protein 3 , as the target for miR1511. In this work we present its experimental validation. In addition, we genotyped MIR1511 in ecogeographically different common-bean cultivars and investigated the role of miR1511 and its corresponding target in the regulation of plant response to AlT. The comparison of MIR1511 sequence from BAT93 vs. G19833 P. vulgaris reference sequences showed a 58-bp deletion in the G19833 genotype. Such deletion comprised around 57% of pre-miR1511 sequence and included 7-bp and 10-bp of mature and star miR1511, respectively . To explore this phenomenon at a larger scale within the Phaseolus genus, we analyzed Genotyping-By-Sequencing data from 87 genotypes originated from a single genetic population , called non-admixed genotypes. These included genotypes from three Phaseolus species and different populations of wild P. vulgaris: three populations from the Mesoamerican , one from the Andean , and one from the Northern Peru–Ecuador gene pools . All the genotypes belonging to the Andean gene pool and part of the Mesoamerican genotypes displayed a truncated MIR1511, in contrast to the Northern Peru– Ecuador genotypes and the other Phaseolus species that presented a complete version of the MIR1511 in their genome. A population clustering of P. vulgaris genotypes confirmed these results and showed that in the three Mesoamerican populations only a part of the MW1 cluster presented the MIR1511 deletion . Predicted target genes for P. vulgaris miR1511 include SP1L1-like  and isopentyl-diphosphate delta-isomerase , previously reported , and a protein with unknown function and the Aluminum Sensitive Protein 3 , from our recent bio-informatic analysis. From these predicted targets, ALS3 is the only one possibly related to AlT, as reported for Arabidopsis , and showing an adequate binding-site penalty score , thus the 5’RLM-RACE assay was used to experimentally validate the ALS3 mRNA cleavage site. As shown in Figure 3a, a significant number independently cloned transcripts mapped to the predicted site of cleavage, between the nucleotides at positions 457 and 458 of the transcript, which corresponds to position 9 and 10 of the predicted miR1511 binding site, thus confirming a miR1511-induced degradation. The other two degradation events mapped to 7 nucleotides upstream and 17 nucleotides downstream of the miRNA-associated degradation site, suggesting random degradation. An additional action of miR1511 to induce translation inhibition of ALS3 mRNA in common bean, cannot be excluded. miR1511 target genes differ among plant species . In order to evaluate the specificity of the miR1511/ALS3 regulatory node in common bean, we analyzed the miR1511/ALS3 binding site sequence alignment from eight model plant species, including five legumes, which contain a precursor gene of miR1511 in their genome . Because of the deletion in MIR1511 from the G19833 genotype, we used the mature miR1511 and the corresponding ALS3 binding site sequences from the BAT93 Mesoamerican genotype, as representative of P. vulgaris.

An agro-economic approach examines the effect on crop productivity of experimentally controlled changes in water, temperature and other possible influences on yield, such as soil characteristics. This approach is, however, subject to the criticism that it overlooks adaptations or adjustments that farmers might make to changes in climate variable – for example, changing the crop mix to something more suited to the new conditions. Recent work in this area has tried to allow for this sort of adjustment. It is possible to use existing variation, across regions down to the county level in the U.S., in temperature, precipitation, soil quality and so on, to estimate a relationship between agricultural land value, on the one hand, and these input variables on the other. Presumably farmers have adjusted their crop choices to reflect long run climate and other differences in different areas. The estimated relationship can then be used to predict the impact on farmland value of projected future changes in the climate variables, holding constant other influences on value such as soil quality, and even socioeconomic variables such as population and income level in a county. When this is done, researchers have found that the likely impact of the temperature and precipitation changes associated with a doubling of atmospheric concentrations of greenhouse gases, principally carbon dioxide from the combustion of fossil fuels, on the value of farmland in the U.S. is quite modest, and may even be positive. One recent study summarizing and reviewing work in this area concludes that the warming associated with a doubling of atmospheric concentrations of greenhouse gases will result in a net gain of $8.4 billion annually for the U.S. economy, with the largest component a benefit to agriculture of $11.3 billion . I am currently engaged in work with my colleague, Michael Hanemann, and graduate student, Wolfram Schlenker,round plastic pots to adopt the econometric or statistical approach of relating farmland value to climate and other influences. We come to strikingly different conclusions.

Precipitation during the growing season is virtually nonexistent. Yet California agriculture is profitable, and the value of farmland here is quite high relative to other areas. The explanation, of course, is that crop yields are not related to precipitation during the growing season. Instead, they depend on irrigation, from stored ground or surface water. For surface water especially, what matters is how much snow falls in the Sierras, and when it melts and runs off into streams that feed the large surface reservoirs that in turn supply local irrigation districts. A statistical analysis that simply relates local precipitation to local crop yields, or the value of local cropland, may – and has – come to the misleading conclusion that the relationship between precipitation and value is negative, since in California and the arid west generally some very high-valued farmland receives little or no rainfall during the growing season.Consider a scenario in which over the next several decades the atmospheric concentration of greenhouse gases doubles, and average temperature in the U.S. rises by about five degrees Fahrenheit . To compensate for the higher temperatures, farmers in areas without irrigation would invest in irrigation facilities, as was done earlier in California and elsewhere. Proponents of the econometric approach might argue – indeed, have argued – that this is taken into account in the estimated relationships. Currently irrigated farmland has a high value because the investment in irrigation was profitable, and was undertaken for that reason, as were other adjustments to climate. This is true, but is misleading as a guide to the impact of future warming. The difficulty is that existing irrigation facilities have been heavily subsidized. For example, in California it has been estimated that even after decades of operation, farmers have paid just 18% of the capital, operations and maintenance cost of the federal Central Valley Project. It is clear that, at a minimum, subsidies of this magnitude to agricultural water users – which have in the past been capitalized into the value of the land – are unlikely to be forthcoming in the future, due to changes in what we might call the fiscal climate.

Apart from the issue of subsidies, it appears that irrigation water will be more expensive in the future than it has been in the past. Again drawing on the California experience, the State Water Project delivers water from a storage and conveyance system constructed in the 1960s to irrigation districts in the Tulare Lake Basin at a wholesale cost of about $80 per acre-foot. However, the State Water Project has only about 60% of the supply capacity originally planned in 1960. If the system were now completed, current estimates are that the new water would cost on the order of $300-$450 per acre-foot. For both reasons, cost increases and reduced subsidies, the net benefit, as reflected in the value of agricultural land, from the construction of new irrigation facilities, is likely to be much less than what can be inferred from a statistical study that reflects historic costs and subsidies.One way to proceed, in these circumstances, is to do the statistical analysis on just areas of rainfed, as opposed to irrigated, agriculture. For the U.S., this involves nearly 80% of the counties , so there is no shortage of observations. When we do this, we find that the estimated relationship between precipitation during the growing season and farmland value is no longer negative. With the costless, or very low cost, option of irrigation out of the picture, the effect on farmland value is unambiguously negative. Under various different weighting schemes for the individual county observations, undertaken for technical reasons, the distribution of damages associated with a doubling of the atmospheric concentration of greenhouse gases converges around a median figure of $215 billion. This is the estimated loss in value of agricultural land. Assuming a real interest rate of 5%, this translates into an annual loss of just under $11 billion, as compared to the previous estimate, noted above, of an annual net gain of just over $11 billion. The $11 billion loss estimate needs to be qualified, or at least further interpreted. To derive an estimate for the U.S. as a whole, the impact of warming on irrigated areas, prominently including California, must be added back in. If this is positive, the loss to U.S. agriculture as a whole would be reduced, perhaps even transformed into a net benefit, as in the earlier estimate.

Research we are undertaking for California suggests that this will not be the case. Warming is expected to lead to changes in the pattern of precipitation that will have a negative impact on agriculture in the state, apart from any impact due directly to temperature. The mix of rain and snow, during the winter rainy season, will shift to somewhat more rain, and less snow, than under present conditions. The snow that does fall in the Sierras will melt and run off somewhat earlier in the year. Thus less water will be flowing into the reservoirs, and available for agriculture – and other uses – when demand is highest, in the late spring and summer. On the other hand, winter rain and early spring snow pack runoff into the reservoirs can be expected to exacerbate flooding, much as in the winter of 1997 when an unusually warm storm system moving through the state dumped heavy rains, rather than snow, in the mountains and resulted in major flooding up and down the Central Valley. Adding in an estimate of the impact of warming on existing irrigated areas in the U.S. is thus unlikely to reduce the $12 billion in losses estimated for non-irrigated areas, much less to convert the losses to gains. Another qualification to the results does however suggest that they may overstate the magnitude of potential losses from warming, and also has implications for policy. By excluding irrigation as an option in areas currently without it, we do not allow for the possibility that the cost of construction and operation of new irrigation infra- structure may be less than the losses otherwise suffered. Clearly it is not appropriate to assume that new irrigation will be forthcoming at historic costs, and under historic subsidies, but it is certainly possible that in some areas at least the full cost will be less than the losses without it. This is a question to be investigated on a region specific,hydroponic bucket indeed a project-specific, basis. What is indicated is a benefit/cost analysis of new water projects, where the benefit is the loss in value to agriculture and other sectors predicted to result without the project.Trade Commission jointly administer AD and CVD law . The DOC first determines whether a commodity is being dumped or subsidized and then the USITC decides whether or not the U.S. industry has been injured as a result. The DOC procedure is much less transparent than the USITC procedure. Although it seems too amazing to be true, the DOC rules in favor of the U.S. industry in 95% of the cases. The safeguard law is jointly administered by the USITC and by the President in that the USITC determines whether injury has resulted to the domestic industry and then issues a recommendation to the President for no relief or for a specific method of relief. The President then decides whether or not to heed the recommendation of the USITC or to choose an alternative method or no method for relief. Many other countries have trade remedy laws that are very similar to those in the United States. Traditionally, the United States, EU, Australia and Canada have filed the most AD and CVD cases against foreign suppliers, but more recently, developing countries have filed a growing number of cases.

In the past few years, developing countries have filed about 50% of the total number of AD and CVD cases worldwide. Economists generally view AD and CVD laws as nothing more than disguised protectionism that is used to protect domestic industries from foreign competition. As traditional trade barriers are lowered, the use of AD and CVD cases has risen worldwide. The main reason that developing countries have criticized the use of AD and CVD laws in developed countries, is their growing frustration with the protectionist use of these laws. For instance, Brazil was reluctant to fully engage itself in discussions on the Free Trade Area of the Americas because of the continued application of U.S. AD duties on products such as orange juice. This past summer, the filing of AD cases on their exports of raspberries and spring table grapes to the United States troubled Chile. It was no surprise that the U.S. grape and raspberry industries filed their cases while the negotiations for the Chile FTA were in full swing. More recently, U.S. honey producers have also received AD protection from competition from Argentina and China, as well as CVD protection from Argentina, which has certainly come at an inopportune time for Argentine producers. During the 1980 to 2000 time period, over 1300 AD and CVD cases were filed in the U.S., of which approximately 116 were agricultural cases. This means that agriculture has initiated its fair share of cases, because agriculture’s share of the value of U.S. total imports is only about 4%. Import relief law was used less often, as there were only 30 such total cases filed from 1980 to 2000. However, U.S. agriculture filed 8 of these 30 cases, and thus accounted for a rather large share. During this time period, there was no noticeable trend in either overall usage or agricultural usage of AD and CVD law. The outcome of the AD and CVD agricultural cases since 1980 is reported in Table 2, where we note that 41 of the 116 total cases resulted in an affirmative ruling in favor of the U.S. domestic industry. During the past two decades, Canada has been the largest target of U.S. AD and CVD agricultural cases. Apart from Canada, most cases have been filed against developing countries, such as China, Colombia and Mexico. As traditional forms of agricultural trade protection are reduced through the WTO, there will most likely be a growing number of trade remedy cases filed by U.S. agriculture. This will not only obstruct U.S. imports but will also encourage retaliation and increased protectionism in other countries. This is all the more reason to keep trade remedy laws on the WTO negotiating table.

Our critical review of the range and reliability of methods for estimating plant-soil BCR offers insight for environmental scientists who must interpret and apply plant-uptake estimates obtained from models and experiments. In this assessment we have emphasized the importance of confronting uncertainties at each stage of the model development and application. We see that uncertainty emerges at the conceptual model stage as well as during mathematical model formulation and calibration and in model applications. The results above show that uncertainty is not simply a variance propagation or “Monte Carlo” assessment that is used to propagate parameter variance at the model application stage. Instead it is a process that begins at the earliest stage of model development and accrues through model formulation and specific applications. In the studies reviewed in this paper, we find that important uncertainties arise at the first stage of model development—the concept formulation. For plant uptake models that address competing soil-root-leaf and soil-air-leaf pathways, conceptual uncertainties remain a dominant source of overall uncertainty. An important contributor to this conceptual uncertainty is the lack of a consistent definition of BCR for soil uptake in both experiments and models. This leads to confusion and inconsistency in the use of BCR. Because this type of uncertainty is difficult, if not impossible, to quantify, we must develop qualitative methods and classifications to communicate this important source of uncertainty. In evaluating model formulation, we observe large differences among models in their predictions of BCR,livestock fodder system but we discovered no clear basis for selecting one model as more accurate than another. The residual errors reported for many of the models in fitting their calibration data leads us to believe that we do not yet have sufficient data to formulate accurate models. But our review of experiments for the single chemical RDX reveals that much of the uncertainty is attributable to the lack of precision and variability of experimental data used to obtain the BCR values used to calibrate models.

It appears that there are advantages to using more than one compartment in formulating BCR models. But lack of experimental data and the poor state of conceptual knowledge suggests that model uncertainty cannot be reduced by adding large numbers of compartments to the models. Too many plant components in our models lead to over specification. But a single compartment model can miss the combined effect of root and shoot uptake processes. The interaction among measurements, conceptual models, and models leads to the conclusion, contrary to our initial expectation, that it may not be possible to distinguish the relative contributions of overall uncertainty from conceptual uncertainty, measurement variability, and model uncertainty. For example, the variations in the value of BCR obtained from the 81 experiments we considered for RDX span a rather wide range. But it is not clear how much of this variation is attributable to experimental uncertainty and how much to inadequate conceptual models. Often the conceptual model is used to design experiments so that an incorrect conceptual model leads to measurements that are difficult to interpret when they are inconsistent with the concept. Perhaps the variance in the experimental values would be much lower if we understood better how BCR is affected by variables whose impact is not yet fully understood—for example temperature, soil properties, etc. Similarly if we really had a complete and thorough conceptual understanding of the process of uptake, then choosing a mathematical equation would likely be less uncertain. That is, the mathematical model formulation may only appear uncertain because we are using mostly-empirical mathematical relationships to describe a process that we do not understand well enough at a conceptual level. So it is not clear whether we classify this as uncertainty in the mathematical model formulation, or as uncertainty in the conceptual model. In applying model performance evaluation to plant uptake modeling, the results and discussion above lead us to a number of key findings. These include: The conceptual formulation of the bio-concentration ratio has an important, but at this point difficult to quantify, contribution to overall uncertainty. In particular, the concept of different plant components, the selection of dry- versus fresh-mass concentrations, and the use of dynamic or steady state concentration ratio strongly impact the reliability and uncertainty of the resulting BCR model.

When we consider both the performance of models with respect to their calibration experiments and also compare different models, we find that quantitative results for any randomly selected organic chemical have very large model uncertainties. We estimate that in the absence of specific experimental information, the expected uncertainty of a BCR model can be represented by a log normal distribution with a GSD of 10 . This means that without additional information on plant species or without plant- and site- specific measurements, we can only expect a model to predict a BCR within ±1 log units such that there is a 66% likelihood that the actual BCR value is 10 times higher or lower than the value obtained from a model. Based on consideration of a large number of experiments for a single, well-studied compound, RDX, we find that experimental measurements of BCR have large experimental variability and that this experimental variability can be represented by a log normal distribution with a GSD of 3.5 . This indicates much of our observed model uncertainty most likely derives from experimental variability. This leads to the observation that controlled measurements cannot necessarily remove the large uncertainties that derive from BCR models. Comparison for RDX of the relative contributions of model uncertainty and experimental variability to uncertainty in BCR estimates indicates that a large fraction of model uncertainty can be attributed to experimental variability. The variability and complexity of the uptake and transport of chemicals in vegetation cannot be captured by a point-value for BCF, but requires the use of ranges and confidence intervals to communicate the large uncertainties associated with estimating BCRs. In any plant-uptake model used to estimate a BCR, we must develop a process for communicating both the magnitude of the result and the confidence that can be placed in this number. On the part of the assessor this requires a presentation of both qualitative and quantitative uncertainties. Heavy metals such as iron , zinc , copper , and manganese are essential micro-nutrients for all organisms, acting as co-factors in a variety of biological processes. These heavy metals are extremely reactive and can become toxic at high concentrations; therefore, the intracellular concentration of these essential metals must be tightly regulated . Other heavy metals such as cadmium , lead, mercury, and the metalloid arsenic do not have biological functions in plants and are toxic even in trace amounts, disrupting several biochemical activities by displacing essential metals from their respective binding sites .

In humans, Cd exposure has been linked to cancer in the kidneys, lungs, and prostate, and severe Cd poisonings can result in neurological disorders and pulmonary and renal failure . While occupational exposure and tobacco products are associated with a high risk of Cd poisoning, consumption of contaminated plant-based foods represents the major source of Cd exposure in the general public . Many cases of widespread cadmium poisonings have been attributed to consumption of contaminated seeds in Thailand, China, Japan, and Australia . However, the molecular mechanisms and genes mediating the loading of both essential and nonessential heavy metals into seeds remain largely unknown. Metal accumulation and distribution in plants consist of several mechanisms, including: metal uptake into roots, xylem-loading and transport to the shoot, and phloem-mediated redistribution of metals from mature leaves to sink tissues, including younger leaves, roots, and seeds . Cadmium enters the root through the Fe transporter IRT1, which shows broad substrate specificity towards divalent metals including Fe2+, Zn2+, Mn2+, and Cd2+ . Once inside the cell, metals bind to different ligands, according to specific affinities, and these metal–ligand complexes can be stored in different cellular compartments or distributed to other tissues through the vasculature . Because of the broad substrate specificity of IRT1 for divalent metals, transcriptional regulation of the Fe-deficiency response,fodder system trays including up-regulation of IRT1, will also have an impact on the uptake of non-essential heavy metals such as Cd. In plants, the root iron-deficiency response is regulated by local signals within the root and also by systemic signals originating from leaves . Two major transcriptional networks have been identified to mediate the Fe-deficiency response at the root level in Arabidopsis: the FIT network and the POPEYE network .The identification of mutants showing a constitutive Fe-deficiency response even when Fe is supplied in sufficient amounts plus experiments where the constitutive root response is restored by foliar application of Fe suggest that mobile Fe is required for proper shoot-to-root signaling . However, the transporters, ligands, and the chemical speciation of the putative phloem-mobile molecule mediating the systemic Fe signaling have not yet been clearly identified. Here, we report that opt3-2, an Arabidopsis mutant carrying an insertion in the 5’ UTR of the oligopeptide transporter gene OPT3 , over-accumulates significant levels of Cd in seeds. We present evidence suggesting that this Cd over-accumulation may be the result of an enhanced transport of Cd through the plant, making opt3-2 a suitable background for studying long-distance transport of non-essential heavy metals. We further show that OPT3 is targeted to the plasma membrane and is preferentially expressed in the phloem.

The Fe/Zn/Mn uptake transporter IRT1 and other ironstarvation-induced genes are constitutively up-regulated in opt3-2. Interestingly, shoot-specific expression of OPT3 restores metal homeostasis and IRT1 up-regulation in roots showing that OPT3 is the first identified molecular component of the network transferring information on the iron status from leaves to roots. Moreover, Fe mobilization between leaves is impaired in opt3-2, suggesting that OPT3 mediates the movement of Fe out of the leaves, and this transport is required for proper communication between leaves and roots and maintenance of the trace-metal homeostasis in Arabidopsis. Understanding phloem-mediated signaling, transport, and seed-loading mechanisms of both essential and non-essential heavy metals will help to develop strategies for excluding toxic metals from seeds and enhance the nutritional value of grains and plant-based products.Members of the Arabidopsis oligopeptide transporter family have been shown to mediate the transport of a broad spectrum of peptides . Glutathione and phytochelatins are peptides that mediate tolerance and long-distance transport of heavy metals ; therefore, we screened mutants in the Arabidopsis OPT family for differential accumulation of Cd in seeds. A mutant of the Arabidopsis OPT3 gene, opt3-2, showed the strongest over-accumulation of Cd in seeds . To test whether this Cd over-accumulation had an effect on seedling growth, assays were performed on plates in the presence and absence of Cd. Figure 1B shows that opt3-2 is hypersensitive to Cd when grown on medium containing 50 μM CdCl2. To determine whether the increased Cd concentration in opt3-2 seeds was due to a systemic over accumulation of Cd throughout the plant, opt3-2 seedlings were grown hydroponically for 6 weeks, exposed to 20 μM CdCl2 for 72h and the metal concentration of roots and leaves was measured by ICP–OES . The roots of opt3-2 over-accumulated Cd compared to wild-type; however, unexpectedly, Cd concentrations in leaves were almost five-fold less than those of wild-type plants . Conversely, seeds of opt3-2 plants show a large increase in Cd levels compared to wild-type seeds .To determine whether the altered distribution of Cd in opt3-2 correlated with the distribution of essential metals in plant tissues, the levels of Zn, Fe, and Mn in opt3- 2 were also measured and compared to wild-type plants . No dramatic differences in the concentration of Zn and Mn in seeds were found between wild-type and opt3-2 . However, in contrast to Cd accumulation, opt3-2 over-accumulated significant levels of Zn and Fe in leaves compared to wild-type . In roots, the concentration of Fe, Zn, and Mn was increased in opt3-2 compared to wild-type . The different distribution of Cd in aerial parts of the plants suggests that the mechanisms mediating accumulation of metals in opt3-2 leaves is different for Cd compared to the essential metals Fe, Zn, and Mn.

However, due to the short lifetimes of PET radioisotopes, only short biological processes, such as photosynthesis, may be imaged. In contrast, single gamma-ray emitting radio tracers used in single photon emission computed tomography are typically metals and do not easily label organic molecules. However, many trace element metals are essential to a plant’s survival . An active area of plant research is studying hyper accumulation of metals in plants using a radioisotope of that metal; commonly studied metals include: Cd, Zn, Mn, Co, and Ni . Other potential applications for SPECT imaging include: studying plant ion transport in xylem , studying metabolic processes such as tracers for phloem transport , and studying signaling by labeled exogenous peptides or proteins . One further advantage of imaging systems based on gamma-ray detection is the possibility of detecting the interactions of multiple radioisotopes simultaneously as the gamma-rays that they emit have distinct energies that can be distinguished from each other by the detector. For example, simultaneous imaging of 65Zn and 109Cd would enable teasing apart the competition dynamics in their uptake. However, there is an inherent trade off in increased sensitivity of the UCD-SPI system with spatial resolution. Spatial resolution at the mm-scale could be obtained using a collimator to better define the spatial origin of detected gamma rays, but this would lead to a greatly reduced event rate in the system. For the high-energy gamma rays of 65Zn, collimation is a particular challenge. Given the hours-long time scale of the transport studied here, it is possible that the choice of a collimator could have provided improved event positioning while preserving a usable event rate. A possible hybrid approach could have included using an insertable/removable collimator to acquire an alternating combination of two types of images: high sensitivity-low spatial resolution without the collimator; and low sensitivity-higher spatial resolution with a collimator. However, led grow lights the use of a collimator for this system is unexplored thus far.Zn uptake into the symplast in the outer root layers and loading into the apoplastic xylem stream are well understood on molecular level.

However, the dynamics of symplastic movement and patterning of the radial transport have thus far only been modeled to elucidate the timescales of these events . After xylem loading, the mass flow-mediated movement of Zn into the shoot inside the xylem is expected to occur within 30 min in Arabidopsis, as previously shown for water and Cd in xylem sap . From previous SPECT imaging, we have shown that a pulse of radiolabelled pertechnetate moving in the xylem stream reaches the shoot apical meristem of a 2 week old sunflower already in 5 min . The rate-limiting step for root-to-shoot translocation of Zn was proposed to be xylem loading involving HMA4 transporters in both A. halleri and A. thaliana . The dynamics of root-to-shoot Zn flux, however, have so far remained unclear in different species and transgenic lines. Estimates of Zn translocation rates from root to shoot were first obtained by spectroscopy methods of ashed shoot tissues. Early work with metal hyper accumulator Noccaea caerulescens suggested that the speed of root-to-shoot Zn transport was between 20 and 60 hr . Recently, positron imaging of Zn uptake estimated the time for Zn root-to-panicle transport in dwarfed mature rice to be 5.3 hr . Here, we have produced the first Zn root-to-shoot imaging data for A. halleri using UCD-SPI. Zn accumulates within the shoot of A. halleri, consistent with its ability to hyperaccumulate Zn, different from the HMA4 RNAi line. The speed of Zn transport into the shoot in our data as observed with the smoothed standard error show clear shoot accumulation within 5–7 hr, respectively . These results are in line with previous reports for rice . This contrasts strongly with the faster speed of the other xylem-transported compounds, such as water in A. thaliana , Cd in A. halleriand pertechnetate in sunflower , all measured to reach the shoot in 30 min. It should be noted, however, that the experiments demonstrating water transport and Cd transport were carried out using decapitated stems and are thus destructive in nature, but also far more sensitive to small quantities than the method used here. The slower speed of Zn transport indicates that Zn loading into the xylem by HMA4 is slow and under tight control even in the metal hyper accumulator A. halleri. Modelling the radial transport of Zn uptake has indeed indicated that HMA concentration is one of the key determinants of the uptake dynamics . The HMA4 transporter pumps Zn2+ from the root symplasm into the apoplastic xylem sap of A. thaliana . Strongly elevated expression of A. halleri HMA4 was suggested to be responsible for the increased in root-to-shoot translocation of Zn in A. halleri relative to A. thaliana . This conclusion was drawn based on the quantification of shoot Zn concentrations after long-term growth in HMA4-RNAi lines and wild-type A. halleri and in A. thaliana Col-0 . In the same experiment, root Zn concentration was elevated in some A. halleri HMA4 RNAi lines relative to A. halleri wild-type plants and even relative to A. thaliana . HMA4 is critical to the ability of A. halleri to hyper accumulate Zn.

We tested the functional role of HMA4 for A. halleri Zn translocation from root to shoot by imaging the Zn uptake dynamics of A. halleri HMA4-RNAi line relative to A. halleri. We found that the Zn signal in the shoot of HMA4-RNAi line did not increase over our 40-hr imaging period, but conversely, we saw a continuous decrease in shoot Zn signal with significant differences observable at 3 hr . The lack of an increase in shoot Zn confirms that Zn loading into the xylem is abolished in the HMA4-RNAi plants . The continuous decrease in the Zn signal in the shoot ROI seems to reflect bleeding of the strong early Zn signal from the root ROI into the shoot ROI. The dissipating signal through the A. halleri HMA4- RNAi time course could be due to apoplastic 65Zn adsorbed to the cell walls of outer root layers during the 65Zn pulse and not removed by the triple rinsing with Hoagland solution. This cell wall-adsorbed 65Zn would be desorbed into the growth medium during the imaging period by diffusion. The influx of Zn into the root symplasm is very tightly and rapidly regulated in Zn-concentration dependent fashion . Without the loading of Zn into the xylem, Zn builds up in the root symplasm. In the case of A. halleri HMA4-RNAi, the symplasm could be saturated with Zn at 3 hr after the resupply, leading to prevention of further uptake of the cell wall-adsorbed 65Zn and thus higher Zn desorption than Zn uptake into the symplasm. Finally, we compared the dynamics of Zn movement in the Zn hyper accumulator A. halleri with those in the related species A. thaliana, a non-metal hyper accumulator. Based on previous studies comparing Zndeficient to Zn-sufficient plants of A. thaliana and/or A. halleri, the Zn concentrations in our hydroponic solutions can be estimated to result in moderate Zn deficiency . The net concentration of 65Zn in the resupply media over the 24 hr period showed a net decrease, suggesting that Zn was taken up into the shoot, although these levels are variable. We found that Zn resupply after Zn deprivation in A. thaliana did not lead to detectable uptake or change of Zn in the shoot or the root ROI. It is possible that the small size and flat rosette growth habit of A. thaliana affected our ability to detect Zn dynamics. Also, low abundance of HMA4 transporters in A. thaliana roots may lead to much slower dynamics that we were unable to capture. In the absence of quantification of 65Zn levels in the shoot, it is possible, although unlikely, that Zn was not translocated in A. thaliana.Although most plants prevent the accumulation of heavy metals so as to avert toxicity, metal hyper accumulators selectively extract high concentrations of metals from the soil into their shoots without incurring symptoms of toxicity . By using the heavy metal radio label 65Zn and the UCD-SPI imaging system, we gained a more detailed spatiotemporal understanding of the dynamics of metal movement into plants, which may be a path toward the use and understanding of metal hyper accumulating plants for such advantageous applications.Reading comprehension is a complex process that requires the coordination and integration of a number of component cognitive skills. The ability to read single words in isolation is widely accepted as one skill critical to comprehension, but successful reading comprehension does not always directly stem from adequate word identification skills. Some individuals who are skilled word readers are not skilled passage comprehenders , supporting the idea that reading comprehension requires processes above and beyond single word reading. Theoretical models of reading comprehension propose that successful comprehension requires a reader to draw on both text-based information and prior knowledge in order to build a coherent and meaningful mental representation of the text . This mental representation is the reader’s understanding of the text’s deeper meaning; it consists of ideas from the text,strawberry gutter system relevant background knowledge, and inferences the reader makes about things not explicitly stated in the text . Building this mental representation is a dynamic process because cognitive demands change over time. For example, readers are known to spend more time processing words and sentences at the beginning of a text relative to later points.

This could be due to the fact that, without context or relevant background knowledge activated to facilitate comprehension, comprehension necessitates more effortful attention to the initial construction of a mental representation . Conversely, later stages of comprehension processes are facilitated by an increasing semantic contextualization . A number of imaging studies have examined the neurobiological correlates of reading comprehension . Patterns of activation emerge when processing discourse that cannot be predicted from models of reading single words, or even single sentences, in isolation . Areas that consistently appear to be unique to processing narrative texts include the dorsal medial prefrontal cortex and bilateral temporal parietal junction, often attributed to social cognition required in story comprehension, bilateral temporal poles , which play a role in generating specific semantic associations in connected text, and posterior medial structures, including posterior cingulate cortex and precuneus , which have been associated with updates in and integration of the reader’s mental model . This demonstrates that reading connected text involves additional processes beyond the phonological, orthographic, semantic, and syntactic processes seen at the word and sentence level. Still, many questions regarding how readers form a coherent text representation remain unanswered. Only a handful of studies have examined how the neural correlates of discourse processing change over the temporal progression of the discourse . Of the few, Xu et al. used fMRI to compare the activation associated with reading the beginning of a story with the activation associated with reading the end of the story . They found that processing the story’s setting and initiating events resulted in strongly left lateralized activation, while processing the story’s outcome resulted in increased activation in right hemisphere perisylvian and extrasylvian regions thought to contribute to inference and contextualization of narrative .These right hemisphere regions have since been related to social cognition processes that may be narrative-specific . This study provides evidence that reading comprehension not only involves processes distinct from those required in single word reading, but also that comprehension demands can vary from point to point within a given text. Similarly, by modifying the cohesiveness of text Yarkoni et al. identified neural regions that showed linear increases in activation as a function of reading time. More specifically, they compared construction processes with maintenance processes . They found that regions in the posterior parietal cortex associated with visuospatial updating and attention are involved in the construction of a reader’s mental model, while perisylvian language areas were more involved in its maintenance. These studies support theoretical models that suggest that building a mental representation of text is a dynamic process in which the cognitive demands shift from one point in the text to the next. Nevertheless, it is important to note that all of the aforementioned fMRI studies on discourse processing have exclusively examined narrative texts; none to date have examined expository texts . However, event-related potential and behavioral studies suggest such genre distinctions are important. For example, Baretta et al. used ERP to distinguish between narrative and expository texts.

The legibility of the names was not as important as the way the surface became progressively dense over the weeks of writing them. Parker realized that the process was “like a record of the soil growing. It’s like when you make your own compost and put it into the soil: it just continues to grow. So this painting may never be finished.”Mechanical eradication of Arundo can be attempted in many different manners. The most frequently used method is the cutting of the above ground material, the plant’s tall stems. Another method of mechanical eradication is digging out the underground biomass, the rhizomes.The large amount of standing above ground biomass, up to 45 kg/m2 impedes the removal of the cut material, because the costs will be too high. The costs associated with the removal of the large biomass of the stems, has led to the use of “chippers” that will cut the stems into pieces of approximately 5 – 10 cm in situ. After these efforts, the chipped fragments are left in place. A small fraction of the fragments left behind after chipping will contain a meristem. The stem pieces of these fragments may have been left intact, or split lengthwise. In the second case the node at which the meristem at located will have been split as well. On many pieces with a meristem, the meristem itself may still be intact. These stem fragments might sprout and regenerate into new Arundo plants . If stems are not cut into small pieces, or removed after cutting, the tall, cut stems can be washed into the watershed during a flood event. This material can accumulate behind bridges and water control structures with possible consequences as described in the introduction.

Meristems on the stems can also sprout, and lead to the establishment of new stands of Arundo at the eradication project site, bato bucket or down river . A. donax stands have a high stem density. The outer stalks of dense stands will start to lean to the outside because the leaves produced during the growing season push the stems in the stand apart. After the initial leaning due to crowding, gravity will pull the tall outside stems almost horizontal . Throughout this report these outside hanging stems will be referred to as “hanging stems”. The horizontal orientation causes hormonal asymmetry in these stems. The main hormones involved are IAA , GA and ethylene . The unusual IAA and GA distributions cause the side shoots developing on these hanging stems, to grow vertically. IAA also plays an important role in plant root development , and may therefore have a stimulative effect on root emergence from the adventious shoot meristem on fragments that originated from hanging stems, that would be absent in stem fragments from upright stems. In a preliminary experiment comparing root emergence between stem fragments from hanging and upright stems, 38% of the hanging stem stem fragments developed roots, while none of the upright stem-stem fragments showed root emergence . These results indicated the need for further study into the possibility that new A. donax plants can regenerate from the stem fragments with shoot meristems that might be dispersed during mechanical Arundo removal efforts. In order to apply herbicides at that time that the rate of downward translocation of photosynthates and herbicide would be greatest, this time period has to be established. Carbohydrate distribution and translocation within indeterminate plants, such as Arundo, results from the balance between the supply of carbon compounds to and the nitrogen concentration in the different plant tissues. Carbon and nitrogen are the most important elements in plant tissues. Due to different diffusion rates of NO3 – and NH4 + in soil water versus that of CO2 in air, and differences in plant N and C uptake rates, plant growth will earlier become nitrogen limited than carbon limited. During plant development tissue nitrogen concentrations are diluted by plant growth , which is mainly based on the addition of carbohydrates to the tissues.

When plant growth becomes nitrogen limited, the tissue will maintain the minimum nitrogen content needed for the nucleic acids and proteins that maintain metabolic function. At this low tissue nitrogen content, there is not enough nitrogen in an individual cell to provide the nucleic acids and proteins to support the metabolism of two cells, therefore the cells cannot divide. This means that the tissue cannot grow anymore , until it receives a new supply of nitrogen. When plant tissues cannot grow due to nitrogen limitation, they cannot incorporate or store additional carbohydrates. This lower physiological limit of tissue nitrogen content, at which no more cell division or incorporation of carbon is possible, is called the critical nitrogen content of the tissue . The CNC is expressed on a carbon basis .The CNC can be determined for whole plants , and for the different functional tissues of the plant. Different plant parts perform different functions, and therefore have different minimum nitrogen requirements for metabolism maintenance. In previous research with the dicotyledonous storage root perennial Ipomoea batatas , it was determined that the most photosynthetically active tissues, the leaves, and the fibrous roots, which are involved in nutrient uptake, have the highest CNC of all vegetative plant tissues . The storage roots of I. batatas had a significantly lower CNC than any of the other Ipomoea tissues. The difference between the actual tissue nitrogen content and the CNC determines the capacity of these different plant tissues to incorporate or store carbohydrates. Tissues with nitrogen contents that are above the CNC can still incorporate or store carbohydrates. These tissues have a positive carbon sink strength . Photosynthetically active tissues that have reached their CNC will not incorporate the produced carbohydrates, because that would dilute the nitrogen content of these tissues below the CNC, and metabolism would be impaired. Instead, the photosynthetically active tissues deposit the produced carbohydrates in the phloem, which transports them to those tissues that still have the ability to incorporate or store carbohydrates .

This is how leaves, that because of their high CNC loose the ability to incorporate the photosynthates in their own tissues relatively early during the development of the plant, can still produce photosynthates and translocate them down to the reserve storage organs, such as I. batatas storage roots, which maintain their positive carbon sink strength, and tissue growth , the longest of all plant tissues, due to their low CNC.Regeneration from stem fragments starts with the growth of a new stem from the meristem. Root growth from the meristem always follows shoot growth, and not all meristems with shoots will grow roots. Therefore, rooting of the stem fragment was selected as the criteria of successful regeneration from a meristem on a stem fragment. The stem fragments were checked for rooting three times per week. Rooting success was calculated in percent of all meristems in the container. The speed with which rooting occurred was expressed is t50. That is the number of days needed for 50% of the total number of meristems that eventually would root in a container, to root. When a stem fragment had rooted, the diameter of the stem at the point of the meristem was determined,dutch bucket hydroponic to assess the effect of the relative age of the meristem on the speed with which they rooted. The effect of the temperature at the time of rooting was tested for hanging A. donax stem fragments at two times in the growing season. In April 1998, 32 stem fragments were randomly distributed over 4 containers with aerated nutrient solution each at 10 and 20 °C. The stem fragments were monitored every day for rooting. Rooting success was calculated in percent of all meristems in each individual container, and mean rooting percentages were calculated for the four replicate containers for each fragment type. The speed with which rooting occurred was expressed using t50. This experiment was repeated in April 1999 at 15, 17.5, and 22.5. A repeat 20 °C treatment was included in the 1999 experiment, to allow for comparison with the 1998 experiment. Rooting of Arundo donax stem fragments under controlled temperatures at different exogenous indole acetic acid concentrations. Hanging stems and upright stems of A. donax were collected along the Santa Ana River, in Riverside County, CA, every month from February, 1999 through May, 2000. Eighty meristem containing fragments of each stem type were cut and surface sterilized. Twenty replicate fragments each , were randomly assigned to a control and three different exogenous IAA treatment levels . The fragments were placed in individual culture tubes , that had been auto claved containing 15 ml of plant growth medium with 4.4 g/L MS salts, 2 g/L Gel Gro, and the assigned concentration of IAA .

Each stem fragment was placed with its lower end in the rooting medium and its meristem submerged but near the interface of the rooting medium and the air. The upper end of the stem fragment extended above the rooting medium. The tubes were placed in a climate-controlled chamber with a temperature/light regime of 14 h of 27 °C in the light, and 10 h of 15 °C in the dark. The rooting of the fragments in the tubes was monitored daily for 30 days. Rooting success was calculated as percent of all meristems at the different IAA concentrations. The speed with which rooting occurred was expressed for each month using the t50 for each IAA concentration and stem type combination. The remaining meristem containing fragments from the hanging and upright A. donax stems that were collected monthly to test the effect of exogenous IAA were stored in tap water in separate containers. The containers were placed in the growth chamber under the temperature and light regime described previously, and the water was aerated. After approximately 10 days, the meristems on the fragments had developed into shoots and roots. Three replicate samples of approximately 10 g of the new shoot material were harvested from the stem fragments of both the hanging and the upright stems, and placed on ice. For the extraction of endogenous IAA, the samples were dipped in grinding media , placed in glass tubes, and flash frozen in liquid nitrogen. The tissues were homogenized for 2 minutes in 20 ml grinding medium with an Omnimixer . The homogenates were incubated on a wrist action shaker for 20 minutes and filtered through micro-cloth. The filtered solution was centrifuged at 10,000 rpm in a JA-17 rotor, and the supernatant was saved. The tissue material on the filter and the centrifugation pellet were combined and incubated again in 10 ml grinding media, and the sample was filtered and centrifuged as before. The supernatants were combined and their volume was reduced to 1 ml using a speed vacuum evaporator at 37 C. The concentrated samples were centrifuged at 13,000 rpm for 10 minutes at 4 °C. The supernatants were filtered through a 2 µm syringe filter . The resulting tissue extracts were stored in 1.5 ml centrifuge tubes, and stored at -80 °C. For the HPLC, the extracts were eluted from a C18 reverse phase column with an analytical SB-18 guard column , with a gradient solution of 20-35% acetonitrile with 20 mM sodium acetate at a flow rate of 1.5 ml/min. The samples were monitored using a spectrofluorimeter detection system at an excitation wavelength of 280 ± 10 nm, and an emission wavelength of 350 ± 10 nm. The IAA peaks of the sample extracts were identified and quantified using 0.1 and 0.5 µM standard solutions.The critical nitrogen content of Arundo leaf tissue was determined in a hydroponics experiment. One hundred Arundo stem fragments were collected in June 1998 from the Santa Ana River near River Road in Riverside county. In the greenhouse, the stem fragments were placed in water for 2 weeks to allow for root and shoot growth. After two weeks, 48 young plants that sprouted from the meristems on the stem fragments were randomly selected for use in the experiment. Four stems were placed in each of eleven 120-liter plastic containers, that were filled with 100 L aerated, half strength Hoagland nutrient solution .