Category Archives: Agriculture

Hydroponic Agriculture: Nurturing Crops in the Absence of Soil

The capabilities of SR FTIR spectromi-croscopy for the direct detection of intracellular biochemical responses to exposures to dilute concentrations of OCs and PAHs will have significant impacts in future research methodology of environmental toxicology.Bio-assays used in aquatic toxicology have taken a prominent position among analytical tests for identifying and measuring environmental hazards. Such bio-assays have been developed for testing a variety of organic and inorganic chemicals, as well as effluents, surface waters and sediment samples for acute and chronic toxicity. Many bio-assays using higher organ-isms such as fish, protozoa and algae have been executed, but are labor- and equipment-intensive, costly and complex. More importantly, these aquatic bio-assays do not provide quantitative information on the impact of pollutants on biological treatment systems. In the view of national and international regulations, regulatory agencies are also supporting the development of new toxicity screening procedures that are sensitive, inexpensive and easier to perform. The use of bacterial in vitro assays such as the Microtox Assay has become an attractive alternative to traditional fish and invertebrate methods for toxicological screening. These new assays have been developed to assess the toxicity of various environmental agents, validated and recognized by several standards organizations. The purpose of this study was to apply selected microbial test protocols to assessing the toxicity of hazardous metals such as cadmium and lead. These metals have been reported to pose a high level of hazard to ecological and human health.A Microtox assay was carried out to measure the relative acute toxicity of metal producing data for the calculation of lead concentration effecting 50% reduction in light output . For each test run, two controls without lead,pot blueberries eight sample/lead dilutions and two replicates were done. Tests were carried out on various percentages of the original lead concentration . The sensitivity of the strain of bio-luminescent bacteria was tested for quality control purposes. Growth and oxygen uptake experiments were performed following previously described protocols.

Descriptive statistics were applied to calculate the means+SD of all data sets associated with specific metal concentrations. Specific growth and oxygen depletion rates were computed as slopes of graphical representations of raw data versus times. The toxic end-points expressed as 50% growth inhibition concentration or as 50% oxygen depletion concentration were next derived from graphical presentations of these specific rates versus metal concentrations. Activity quotients were calculated to determine the degree of toxicity associated with lead exposure. Linear regression analysis was performed to determine the relationship between lead concentrations and the times required for 50% reduction in oxygen uptake .Bioluminescence was used as an endpoint for measuring the effect of Cd and Pb to Vibrio fischeri. For both Cd and Pb, a strong dose-response relationship was determined. The concentrations of Cd and Pb effecting 50% reduction in bioluminescence were computed to be 0.79+0.12 mg/L and 0.34+0.03 mg/L, respectively; indicating that Pb was more toxic than Cd. A strong dose-response relationship was also found in the tests with the mixed population of microorganisms. Figure 1 shows the growth patterns obtained from exposure to lead of the mixed population of microorganisms. Data presented in this figure show an overall increase in bacterial growth with the increase in holding/incubation time. These data also show significant reductions in maximum growths with increasing concentrations of lead. EC50 values were computed to be 4.50+0.04 mg/L, and 3.50+0.02 mg/L for Cd and Pb, respectively. Figure 2 presents the dissolved oxygen uptake rates by the mixed population of microorganisms exposed to various concentrations of lead. In general, curves presented in this figure indicated that individual rates of oxygen uptake decreased as lead concentrations increased. The mean values of EC50 were 5.00+0.42 mg/L for Cd, and 3.80+0.04 mg/L for Pb. These data indicated that the mixed population of microorganisms was about 10 times less sensitive to lead toxicity than the marine bacterium, Vibrio fischeri. Data also showed a strong correlation between TD50s and lead concentrations, indicating a time-response relationship with regard to lead toxicity. A similar result was obtained in experiments with cadmium. Data obtained from this research clearly point out the significance of using microbiological systems for acute toxicity testing in aquatic toxicology. Bio-assays employed in the present investigation fulfilled the requirement criteria of fast toxicity screening based on their simplicity, speed, cost effectiveness and the fact that bacteria grow rapidly, represent a low trophic level, and thus provide sensitive early warning data of environmental impacts at higher trophic levels.

Of the three bio-systems evaluated, the Microtox was the most sensitive; yielding an EC50s that was only about one tenth of values recorded in batch cultures . Although batch systems were time-consuming , and relatively less sensitive than the Microtox, they provided valuable information on the toxic effects of lead on microbial growth and respiration. Also, they were easier to perform, and required less expensive equipment compared to the high cost of the Microtox analyzer. Trinitrotoluene is a munitions chemical that was produced and used on an enormous scale during World Wars I and II in shells, bombs, grenades, demo-lition explosives and propellant compositions. 2,4-Dinitrotoluene , and 2,6-Dinitrotoluene , on the other hand, are used in the manufacture of dyes, in munitions as smokeless propellant powders, and as gelatinizing and plasticizing agents in both commercial and military explosive compositions. Both 2,4-DNT, and 2,6-DNT are produced through denitration of toluene with nitric acid in the presence of concentrated sulfuric acid. Small amounts of DNT isomers also occur as byproducts in the production of TNT. Significant amounts of TNT- and DNT-contain-ing waste waters arising from their preparation and production at Army ammunition plants have been identified in soils, surface water and ground water after leaching from disposal sites. Exposure to TNT and DNTs has been associated with numerous health effects. However, limited scientific information is available regarding the environmental fate, ecotoxicity and health effects of these nitroaromatic compounds. We have performed the Microtox, Mutatox and CAT-Tox assays to determine the acute toxicity, genotoxicity and molecular mechanisms by which these munitions chemicals exert their toxicity. Acute and genotoxicity tests were carried out, using a Microtox/Mutatox Model 500 Toxicity Analyzer System. The Microtox procedure measured the relative acute toxicity of lead, producing data for the calcula-tion of lead concentration effecting 50% reduction in light output . For each test run, two controls without lead, eight samples/chemical dilutions and two replicates were done. The Mutatox Assay was conducted according to the standard test protocol. Nonglowing or dark mutant strains of luminescent bacteria were exposed to the test substance , and the amount of light emitted was measured with the Mutatox Analyzer. The sample-induced reversion from nonglowing to lumi-nescent phenotype was used to indicate the genotoxicity of the sample.

Prepared samples were mixed and preincubated in a water bath at 35 ± 0.5°C for 45 minutes. After preincubation, samples were incubated at 27 ± 5°C for 16, 20 and 24 hours, and the potential genotoxic response of the luminescent bacte-ria was determined at each time period by measuring the light intensity of each cuvette using the Mutatox Model 500 Analyzer. The positive response was defined as the light output of at least two times the light intensity of the reagent control blank. The mammalian Gene Profile Assay was performed for measuring differential gene expression in the human liver hepatoma cell line, HepG2. Thirteen recombinant cell lines and the parental HepG2 Cell line were plated over two 96-well microplates. The cell lines were dosed at five TNT concentrations and incubated at 37°C, 5% CO2, for 48 hours. After the incubation period,square plastic plant pots the total protein was measured by the Bradford method, at 600 nm using a microplate reader. A standard sandwich ELISA was performed and in the final step horseradish peroxidase catalyzed a color change reaction that was measured at 405 nm. The parental HepG2 cell line was dosed in the same manner as the recombinant cell lines, and was used to perform a MTT-based cellular viability assay at 550 nm.Polycyclic aromatic hydrocarbons are a family of compounds that includes some potent car-cinogens that are ubiquitous in the environment. The major metabolic pathway for ingested or inhaled PAHs to water-soluble derivatives is oxidative activation by cytochrome P4501A1 followed by detoxification by phase II enzymes like glutathione S-transferases, especially GSTM1. Interindividual variation in PAH metabolism exists due to genetic polymorphisms in the genes coding for these enzymes. The GSTM1 gene is frequently deleted in individuals, resulting in reduced detoxification. Several single-base changes have been identified in the CYP1A1 gene that appear to result in increased susceptibility to various cancers in these individuals. Because PAHs present a threat to human health, human exposure to PAHs has to be monitored in occu-pational settings. While PAHs consist of hundreds of different aromatic compounds, pyrene is typically present in all of these mixtures. Pyrene is metabolized primarily to 1-hydroxypyrene and detoxified as 1OHP sulfate or glucuronide conjugate and excreted via the urine. Through simple enzymatic methods, these conjugating molecules can be cleaved. Therefore urinary 1OHP is the most commonly used biomarker of exposure to PAHs. Recently, a number of investigators have reported differences in the quantity of urinary PAH metabolites in individuals with poly-morphisms or variations in key enzymes involved in the metabolism of xenobiotics. These findings suggest the need for clarification of the effects of polymor-phisms on the metabolism of pyrene. To investigate the role of these polymorphisms, we have undertaken a study to measure 1OHP levels.Genetic polymorphisms: Peripheral blood lympho-cytes are isolated from the blood samples using Histoprep density separation media . DNA is extracted from the PBLs using standard phenol chloro-form extraction methods. Polymorphisms are analyzed by published procedures: For CYP1A1, the procedure described by Cascorbi et al. is followed.

For identification of CYP1A1 M1, a 899 bp fragment is amplified, then digested with MspI which cuts the variant fragment into a 693 and 206 bp fragment. For the identification of the CYP1A1 M2 polymorphism, a 204 bp DNA fragment is amplified, then subjected to digestion with BsrDI, which cuts the wildtype into a 149 and 55 bp fragment. Restriction enzyme digested PCR products are separated by agarose gel elec-trophoresis. GST analysis is performed using a multiplex PCR that co-amplifies the GSTM1 and GSTT1 genes . An actin DNA frag-ment is co-amplified as an internal control. The absence of a GSTM1 or GSTT1 band in the presence of the actin band indicates a GST gene deletion. Analysis of 1OHP: Methods for analysis of 1OHP follow the protocol by Whiton et al. , which involves overnight enzymatic digestion of all conju-gated forms of pyrene in a 25 ml urine sample, organic extraction of 1OHP, and reverse phase HPLC analysis and quantitation of the 1OHP peak.Inorganic pyrophosphate is an intermediate compound generated by a wide range of metabolic processes, including biosynthesis of various macromolecules 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. In Arabidopsis, a tonoplast-localized proton-pumping pyrophosphatase AVP1 was shown to be the key enzyme for cytosolic PPi metabolism in different cell types of various plants. This enzyme activity has been correlated with the important function that AVP1 plays in many physiological processes. Arabidopsis fugu5 mutants lacking functional AVP1 show elevated levels of cytosolic PPi and display heterotrophic growth defects resulting from the inhibition of gluconeogenesis. This important role in controlling PPi level in plant cells is reinforced by a recent study showing that higher-order mutants defective in both tonoplast and cytosolic pyrophosphatases display much severe phenotypes including plant dwarfism, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Moreover, the tonoplast-localized H+ -PPase AVP1 appears to be a predominant contributor to the regulation of cellular PPi levels because the quadruple knockout mutant lacking cytosolic PPase isoforms ppa1 ppa2 ppa4 ppa5 showed no obvious phenotypes.

Nutrient-Rich Innovation: The Benefits of Hydroponic Crop Production

Nanoceria was found to be non toxic for Danio rerio embryos exposed up to 200 mg l−1 nanoceria during 72 h.Table S1† illustrates the diversity in the measured effect concentration of nanoceria. Even for a given species, the results varied widely between studies. For example, Lee et al. showed significant mortality of D. magna after 96 h of expo-sure to 1 mg l−1 of 15 and 30 nm nanoceria103 while no toxicity was measure in D. magma after the same duration at 10 mg l−1 or a 48 h exposure at 1000 mg l−1 nanoceria.Van Hoecke et al. exposed D. magna to higher concentrations of 14, 20, and 29 nm nanoceria for 21 days, and found an LC50 of approximately 40 mg l−1 for the two smaller particles and 71 mg l−1 for the 29 nm particles.When combining all aquatic toxicity data, including C. elegans , no trends were observed between the nanoparticle size and the toxicity. We observed one extreme value, which is a report of reduction in life span of C. elegans at a concentration of 0.172 μg L−1 . 92 Some have suggested that the toxicity at low concentration can be explained by differences in the aggregation state as a function of concentration. NPs may be less aggregated at lower concentration.105 However, the nanoceria used in this study were positively charged, coated with hexa-methyleneteramine . It is possible that this coating rendered nanoceria much more toxic. Another Fig. 1 depicts the median of the lowest observed effect concentration and the EC10 or LC10 toward different species. This figure illustrates the high variability of the observed LOEC/EC10 between studies for a same organism . Based on the LOEC/EC10, the more sensitive species is the cyanobacte-rium Anabaena, while the least sensitive is Daphnia magna. No toxicity was observed up to 5000 mg Ce/L for the zebrafish Danio rerio and Thamnocephalus platyurus Fig. 1. It is noteworthy that exposure models predict concentrations significantly lower than those for which ecotoxicity investigations have encountered toxic effects. Therefore, nanoceria might not have any impact at environmental concentrations,growing pot despite the fact that some results are more worrying. More-over, most of the nano-ecotoxicology performed on aquatic organisms used a single species or a short trophic links and do not take into account important parameters such as the colloidal destabilization of the nanoceria, their interactions with organic molecules/ particles naturally occurring or bio-excreted, or the flux between compartments of the ecosystems .

To work under more realistic scenario of exposure, few nano-ecotoxicological studies are now performed in aquatic mesocosms with low doses of nanoceria, chronic and long-term exposure. Such studies will allow obtaining reliable exposure and impact data and their integration into an environmental risk assessment model that is currently missing.Although the data on environmental effects are far from complete, it is useful to consider case studies in order to gain knowledge about key data gaps and to give a first impression of relative risks based on current knowledge. While this case study is useful to point out areas where research is most needed, it is critical to point out the limitations of this case study. First, nanoceria have not yet been detected or measured in environmental media, and the actual environmental concentrations are not known. Second, very little is known about the fate and transport of nanoceria in the environment. Third, the toxicity data base is still very limited. Only a select few ecological receptor species have been tested to date and few if any sub-chronic or chronic exposures have been performed in longer lived organisms or in environmentally realistic exposure scenarios. The following case study characterizes the likely exposure concentrations and compares them to toxicity values for soil and water based on emissions due to combustion of fuels containing nanoceria additives and for discharge of chemical mechanical planarization media into sanitary sewers.Based on Table S1,† with the exception of HMT coated nanoceria, which do not apply to this case study and for which coating controls are lacking, the lowest EC10 value measured so far is 8000 ng l−1 for luminescence inhibition in cyano-bacteria.Previous estimates have been made for nanoceria used as a fuel catalyst and arriving in soil and water following atmospheric discharge106 in the UK based on known market size for this product. Clearly there is a wide disparity between concentrations likely to occur due to fuel catalyst combustion106 and the lowest toxicity values observed so far . However, there remains concern that nanoceria may enter water courses through its uses in specialized industrial polishing or chemical/mechanical planarization.Without specialized local knowledge on where these industrial concerns are located, the quantities of nanoceria used, that are disposed of from the premises, and the capacity of the associated sewage treatment plant, the local receiving water concentrations cannot be predicted.

Unfortunately, knowing global or national consumption of nanoceria in the polishing industry would not allow us to predict water concentrations. This is because the use of the product would not be evenly geographically spaced, or linked directly to human population density. However, it is possible to ask: what discharge would be needed to exceed the 8000 ng L−1 toxicity threshold for aquatic exposures? The dilution factor for sewage effluent recommended by EU risk assessment is 10. So effluent would need to contain 80 μg L−1 nanoceria. However, it is estimated that on entering an WWTP 95% of the nanoceria would enter sludge and only 5% pass through into the effluent.In that case the influent concentration would need to be 1.6 mg l−1 nanoceria. WWTPs are designed around population equivalents which tend to be around 160–200 L per PE per day in the UK so a PE unit would need to discharge 256–320 mg Ce per day to receiving waters. Given the current uses of nanoceria, this only seems likely to occur if a large industrial facility is directly discharging wastewater containing high concentrations of nanoceria directly into a sanitary sewer. Note that a population equivalent is a unit describing a given biodegradable load as measured by its biological oxygen demand.We have comprehensively reviewed what is known for nano-ceria about the environmental releases, methods for detection and characterization, fate and transport, toxicity and likelihood of toxicity in soil and water from acute exposures. Initial estimates of releases suggest that the majority of nanoceria will ultimately end up in landfills, with lesser amounts emitted to air, soil and water in that order. Once nanoceria enters the environment, it has been shown that NOM will have a major impact on their fate, transport and toxicity. As with other nanomaterials, aggregation is a key consideration and this has been shown to be influenced by water chemistry and interactions with natural coatings such as NOM. An important feature of nanoceria with respect to its behavior and toxicity is its valence state. There are several techniques that can characterize this property in environmental and bio-logical media, such as XAS, but most require relatively high concentrations. While we didn’t identify studies that detected nanoceria in natural environments or environmental media, a suite of techniques have been used to detect and character-ize them in complex toxicity testing media and in controlled laboratory studies. Thus, a major data gap and area for future research is the prediction and measurement of actual nano-ceria concentrations in the environment, either from point sources or non-point sources.

As a whole nanoceria appears to exhibit similar aquatic toxicity values other commonly studied manufactured nano-materials. For example,square pot a recent review found that species average LC50 values for Ag nanoparticles ranged from 0.01 mg L−1 to 40 mg L−1 while species mean LC50 values for ZnO ranged from 0.1–500 mg L−1 . 116 The range of EC50 values reported for Ce are similar to those for ZnO. Although reported toxicity data here uses LC10 and LOEC values, the range of species means 0.05–25.9 mg L−1 and many of the reported LC50 values are within the range of 0.1–100 mg L−1 , suggesting similar acute toxicity to ZnO NPs in aquatic expo-sures. This is of course based on the available data, which are predominantly on the toxicity of nanoceria to aquatic organisms, with sediment and terrestrial organism data severely lacking. For example, few if any studies have investi-gated toxicity in sediment dwelling organisms, which are likely to be exposed to nanoceria in the aquatic environment due to aggregation, settling and accumulation of nanoceria in sediment. Given the persistence of nanoceria, chronic studies are lacking as we are aware of only the C. elegans study.Equally important, very few species from few taxonomic groups have been tested. Large taxonomic groups such as insects and gastropods have not been tested and only one non-mammalian vertebrate spe-cies has been tested . Another difficulty is that most of the studies were performed with different nano-particles, doses, duration, organisms, exposure media, and their results are not directly comparable. Perhaps due to these differences, there are no apparent patterns to suggest that, as a whole, particle size has a major impact on toxicity. A problem in conducting realistic toxicity studies is the likely transformation of the free particles into homo or hetero-aggregates or even organic complexes in the real environment. There have been few studies that investigated the impact of size across a wide range of systematically varied particle sizes within a single study. Such studies are needed to definitively establish weather size is important. On the other hand coating may be an important variable given the extreme sensitivity seen with HMT coated particles in C. elegans. Coating was demonstrated to be a major determinant of toxicity in a more well controlled study that systematically varied coating properties and used coating controls.2 Of all of the taxonomic groups, toxicity is most well studied in vascular terrestrial plants. Overt phytoxicity of nano-ceria seems minimal and, while root to shoot translocation of these particles is often measurable it is generally quite low. In summary, although the literature on nanoceria impacts on terrestrial plants is not extensive, it is clear that overt phytotoxicity is minimal, even at excessive exposure concentrations. The data do suggest accumulation of nano-ceria within plant tissues, although the precise form of the element that crosses into the plant and the mechanism driving that process remains unknown. The potential trans-generational effects noted in the literature,as well as the complete lack of information on trophic transfer, are areas of concern. In addition, studies investigating environmentally relevant concentrations, potentially secondary effects from nanoceria exposure, including impacts on symbiotic micro-organisms or on edible tissue nutritional quality, certainly warrant further investigation. As a whole, the aquatic and terrestrial toxicity testing data for animals and microorganisms spans multiple orders of magnitude for acute toxicity values . This large variation can be exhibited within a single species exposed to similar nanoceria. For example, toxicity values for D. magna range from around 1–100 mg l−1 for fairly similar particles. Based on the overall toxicity database, it appears that C. elegans is the most sensitive animal and Anabaena is the most sensitive microorganism tested to date, although an important caveat is that the same endpoints were not com-pared across all species and that exposure systems varied. Interestingly no toxicity was observed in the fish species that has been tested even at extremely high exposure concentrations . Unfortunately, only two fish studies have been reported in the literature. There is a complete lack of toxicity testing data for sediment dwelling organisms, and extremely limited data for soil invertebrates. As a whole the data suggest that acute toxicity is possible at low μg L−1 concentrations in the water column. Data are lacking on soils and sediments, but toxicity values are likely to be far lower. One study indicated toxicity at lower concentrations than these values when 8 nm nanoceria were coated with HMT. Since no coating controls were used, it is critical that the influence of this coating and other similar positively charged coatings be studied using a similar end-point and suitable controls. The use and disposal of any nanoceria containing products with this coating should also be evaluated. It is not clear whether the chronic nature of this exposure or the influence of the coating on uptake and toxicity explain why this toxicity threshold is so low.

Using transient models such as the HYDRUS model has been suggested as an alternative

The differential response of roots to nutritional patchiness is probably a consequence of complex nutrient-specific signal transduction pathways .To investigate the effects of heterogeneous root salinity and nutrient conditions, several split-root tomato experiments were conducted . Water uptake from the saline root-zone dramatically decreased within 8 h of treatment in contrast to the non-saline root-zone, with a more pronounced effect when nutrients were provided only to the non-salinized root-zone . This reduction in water uptake did not correlate with decreased root growth , with the saline root-zone only showing significantly less root growth towards the end of the experiment . The rapidity and consistency of decreased water uptake by roots in the saline zone, from treatment imposition through to Day 9, suggests that a primary physiological response was fol-lowed by a morphological response. To further explore the role of heterogeneous nutrient provision on root activity, complete nutrient solutions were selectively depleted of either N or K+ in the non-saline root half while the other root half received a saline, complete nutrient solution . These treatments provoked a ‘two-phase-response’. Immediately upon treatment application, the saline conditions given to one side of the roots dominated, immediately decreasing water uptake of those roots. Subsequently, water uptake from the saline-treated, nutrient-supplied roots proportionally increased, probably in response to the nutrient deficiency induced by the omission of the nutrient on the non-saline side. This effect was marked when K+ was only present in the saline root half and slight in the case of N. The presence of K+ in the nutrient solution was the most important determinant of root activity even when coinciding with salinity,blueberry box resulting in a notably higher shoot tissue Na+ and Cl− concentration when the sole source of K+ was to the saline root volume .One valuable tool in categorizing and quantifying genetic variation in salt tolerance has been to define crop relative yield responses in terms of threshold salinities up to which yields are unaffected and linear decreases in relative yield with increasing salinity thereafter .

However, it is critical to recognize that these relation-ships have generally always been presented in terms of variation in parameters such as ECe or more occasionally in terms of variation in EC1:5 that relate to the salinity of the soil. However, it is not the salinity of the soil that affects plant growth but the salinity of the soil solution, and thus the ratio of salt to water in the soil. This means that the salinity stress on a plant can be doubled by doubling the salt concentration in a soil or by halving the water concentration of the soil. Furthermore, as soils become drier, plant growth becomes affected by the increasingly negative matrix potentials that develop in soils because of the adhesion of water by soil pores. This view profoundly affects the whole idea of the heterogeneity of salinity stress in soils, because heterogeneity arises because of variable: leaching effects of irrigation or rain-fall on salt concentrations in soil, hydrating effects of irrigation or rainfall on soil water contents, effects of surface soil evaporation increasing salt concentrations by capillarity and decreasing water contents in the soil, and/or water extraction rates of roots and the ion uptake/exclusion capacity, which over time also influence ion and water abundances near the roots.All irrigation water introduces salts to the system and in regions with high evapotranspir-ation and low rainfall, traditional salinity management emphasizes deliberate leaching of salts away from the root-zone while avoiding elevation of the water table to prevent damage to crops . Leaching is usually achieved by applying irrigation water in excess of crop evapotranspirational demands. The fraction of applied water that drains below the root-zone is referred to as the ‘leaching fraction’ and this value is used to coarsely gauge the extent of leaching . Larger leaching fractions generally result in larger zones with a low soil water salinity but may necessitate disposal of large volumes of saline drainage water and may cause additional salinization through capillary rise of saline water by raising the water table , as well as environmental impacts of drainage water disposal. Designing the appropriate leaching fractions needed to avoid yield loss is context-specific and will depend on the crop, soil texture, climate, irrigation system and irrigation schedule, and the salinity of irrigation water being used . Ayers and Westcot developed a simple approach to calculate the leaching requirement based on salt mass balance calculations.

This approach estimates the leaching fraction required to keep the average root-zone salinity below the salinity threshold of the crop, assuming a specific root distribution and a strictly vertical, continual water flow. Approaches like this neglect the spatial non-uniformity of irrigation water application as well as the temporal dynamics of irrigation and water uptake during the season and assume that the average root-zone salinity determines the impact of salinity on the crop . While the physical principles underlying salinity management have not changed since Ayers and Westcott developed these leaching guidelines, management goals have shifted over time to better recognize environmental impacts of nutrient and salinity losses and develop more advanced micro-irrigation and fertigation systems. This has given rise to both new challenges and new opportunities in managing salinity. Challenge 1: Managing salinity under micro-irrigation systems. Spatial patterns of salt accumulation are diverse and differ by irrigation system , with each irrigation system having specific challenges to salinity management. In the simplest case, flood irrigation applies water uniformly across the whole surface . In this case, salinity distribution is approximately uniform in the horizontal direction, but a salinity gradient exists vertically . Assuming sufficient leaching, salinity increases with depth in these systems and uniform leaching of salts below the root-zone causes the salinity within it to be relatively homogeneous. In contrast, applying water to only part of the surface causes strong horizontal salinity heterogeneity, as in furrow irrigation and more advanced micro-irrigation systems. Micro-irrigation aims to target water application to the root-zone, thereby improving water use efficiency by applying less water to regions with low root density and providing an opportunity to deliver water at a rate which matches crop demand. Flood and overhead sprinkler irrigation manage soil moisture and salt content at the field scale, while micro-irrigation approaches management at the root-zone scale. Targeted water application results in targeted leaching, with micro-irrigation leaching salts in zones which are rich with plant roots, while flood irrigation requires additional water to also leach salts from field zones between plants with low root density, making micro-irrigation more efficient than furrow/sprinkler irrigation for managing salinity . When drip and furrow irrigation were compared, drip irrigation sustained higher yields of salt-sensitive crops compared to furrow irrigation when saline groundwater is shallow, while using less water than furrow irrigation .

The economic incentive to install micro-irrigation systems is context-dependent, with the advantage of micro-irrigation over conventional irrigation becoming less clear when growing salt-tolerant crops or when irrigation water is abundant. Despite its potential to accumulate salts in the root-zone, even subsurface drip can have advantages over salinity management with traditional irrigation. While higher tomato yields justified the expense of installing a subsurface drip irrigation system in California, the same was not true of cotton, which remained lucrative with furrow irrigation ,blueberry package as such salt-tolerant crops tend to tolerate flood irrigation without yield loss provided that irrigation is applied pre-planting to avoid stand establishment losses . In drip irrigation systems with strongly localized water application, salt is not only leached downwards, but significant lateral water movement away from the drip emitter also leaches salt horizontally resulting in salt accumulation in the fringes of the wetted volume . This leads to a strongly heterogeneous small-scale salt distribution where soil salinity levels in the top 20 cm can vary by a factor of more than five within only 40 cm of horizontal distance . Although the extent of horizontal salt movement depends on the soil texture and can be partially controlled by emitter spacing, under micro-irrigation, salts concentrated between emitters near the surface generally have little opportunity to intrude into the root-zone without precipitation, due to surface evaporation and irrigation . It is therefore recommended that crops be arranged close to emitters where salinity is low and that new lines be installed as close as possible to where old lines existed to avoid the need for preseason reclamation leaching . Subsurface drip irrigation results in a different pattern of water flow and salinity accumulation. While water application at the soil surface causes salts to leach downward and outward from the water source, subsurface irrigation causes resident and irrigated salts to flow upward through advection and accumulate above the dripline where plants are present . This accumulation pattern antagonizes the establishment of many row crops be-cause germination is relatively sensitive to salt stress . Such production systems rely on pre-season rain, sprinkler or surface irrigation to leach salts below the drip line where they may be leached downward by subsurface irrigation . Shallow installation of subsurface drip lines is advantageous where sufficient pre-season rains are present as irrigating the soil surface may be avoided altogether . This issue can be mechanically managed in processing tomato by adding soil to planting beds , followed by irrigation to accumulate salts into the uppermost zone of the bed, which is subsequently removed and placed in the furrow between rows, where very little horizontal salt movement occurs . The strong localization of water application in drip irrigation questions the applicability of historical steady-state leaching models to micro-irrigation systems . These models insufficiently account for the highly local nature of micro-irrigation and underestimate both the local leaching fraction experienced by plants and the tolerable EC of irrigation water .

Adequate management of heterogeneous salinity patterns and localized leaching under drip or micro-sprinkler may allow sustainable crop production in soils that would otherwise be deemed too saline for that species.These models account for localized application of water and changes in flow rates over time by explicitly simulating two-dimensional water and solute transport in the root-zone by numerically solving mechanistic models. However, although these models are very strong in depicting physical transport processes, they often oversimplify the description of plant physiological processes governing water and solute uptake. For example, the HYDRUS model neglects that the distribution of water uptake is also affected by nutrient concentrations. Moreover, even if it was possible to perfectly simulate the water, nutrient and salinity dynamics for a given scenario, it would still be unclear how the calculated heterogeneous salinity distribution would translate into plant performance. Incorporating current knowledge of plant responses to heterogeneous conditions might make these models more suitable for evaluating salinity management practices. Challenge 2: How to simultaneously optimize N efficiency and minimize the impact of salinity. The necessity of a leaching fraction for long-term salinity management is coupled with the issue of nutrient loss, especially for nitrate , which exhibits similar leaching potential as Cl−. Any practice designed to remove Na+ or Cl− from the root-zone probably also leaches NO3 − . Although a common problem, few studies have addressed the integrated nature of salinity and nutrient management . While NO3 − and Cl− are subject to very similar transport mechanisms and rates in the soil, their distribution in the soil can nevertheless be quite different, and high Na+ and Cl− concentrations do not necessarily coincide with high NO3 − concentrations. This is because: in contrast to Na+ and Cl−, NO3 − is preferentially taken up by plant roots; and nitrogen fertilizer is deliberately added to the irrigation water during fertigation and is to some degree independent of water application. Understanding crop nitrogen demands and responses to spatially localized nutrients and salinity may help manage fertigation systems to achieve the simultaneous goal of salinity leaching and minimal nitrate loss. By providing nutrients through fertigation in a manner that retains nutrients in the low-salinity zone adjacent to the drip-emitter, roots can avoid exploring the saline fringes of the wetted zones,thus reducing salt exposure. HYDRUS-based modelling suggests that high-frequency applications of small amounts of nitrate, timed toward the end of a fertigation event, can help retain NO3 − in the root-zone adjacent to the irrigation source while allowing salt to be leached to the peripheral root-zone.

All plant growth was modeled to occur indoors using a vertical rack system with hydroponic irrigation

Griffithsin also effectively inhibits transmission of HSV-2 , HCV , SARS-CoV , Ebola , and possibly other viruses yet to be studied. Importantly, Griffithsin appears devoid of cellular toxicity that is associated with other lectins. O’Keefe et al. conducted studies with explants of macaque and rabbit vaginal tissues ex vivo and showed that Griffithsin did not induce changes in the levels of cytokines or chemokines, nor did it alter lymphocyte levels in human cervical tissue nor elicit inflammatory responses in rabbit tissue . The combination of extremely wide viral target range and demonstrated preclinical safety makes Griffithsin potentially useful as a prophylactic and/or therapeutic in multiple and diverse antiviral indications. The potential indications for Griffithsin as a human prophylactic or therapeutic include its use as an active pharmaceutical ingredient in vaginal and rectal microbicides. In spite of the value shown by pre-exposure prophylaxis drugs to prevent HIV transmission, issues of cost, side effects, the potential for development of viral resistance through chronic use of antiretrovirals as prevention modalities, and access to PrEP drugs by under resourced populations remain. These unmet needs could be met by the availability of affordable, safe and effective “on demand” antivirals, especially with Griffithsin as the API and its potential to control co-transmitted viruses such as HIV-1, HSV-2 and HCV during intercourse. Adoption of Griffithsin as a new biologic drug, especially in cost-constrained products such as microbicides, is predicated on the feasibility of a scalable manufacturing process that can supply market-relevant volumes of the API at an acceptable cost of goods sold . Previously, we showed that recombinant Griffithsin can be expressed and isolated with high efficiency using transient gene expression in green plants . Although the process described can be further optimized,growing bags the achieved pilot-scale expression yields of >0.5 g Griffithsin per kg of fresh green biomass , recovery efficiencies of 60–90% overall, and Griffithsin purity of >99% of total soluble protein are already impressive.

In this study, we developed a technoeconomic model for Griffithsin manufacturing using a plant-based system with the goal of estimating API manufacturing cost and determined the factors that have the greatest impact on COGS. The output of our study should serve as a basis for additional process improvements, selection of a commercial-scale manufacturer, and should assist in the identification of future product targets for cost-sensitive markets such as prophylactic microbicides as well as those for less cost-constrained therapeutic indications. Technoeconomic modeling was performed with the widely used SuperPro Designer modeling software . The main analysis in this study was conducted using data available from pilot-scale manufacturing of Griffithsin in Nicotiana benthamiana plants using tobacco mosaic virus -induced transient gene expression, and assuming that manufacturing would take place in an existing and fully equipped state-of-the-art plant-based bio-manufacturing facility. Modeling costs based on existing resources of a contract manufacturing organization instead of a “greenfield” build of a new facility was seen as the most likely scenario for launch of a new product. Our reasoning was that dedicated infrastructure could be built subsequently depending on market demand for the drug. As a result, we did not estimate capital equipment or total capital investment costs, and neglected depreciation, insurance, local taxes and factory expenses in the manufacturing operating cost analysis as these investments would have been made by the CMO. Our analysis assumed a 20% net profit margin/fee assessed by the CMO and this figure was added to the production cost of the product to arrive at the final total product cost. In addition to the techno economic analysis, an Environmental Health and Safety Assessment of the designed process was conducted using the method described by Biwer and Heinzle to evaluate the environmental, health and safety impact of Griffithsin manufacturing using the plant-based system, with the goal of assessing the sustainability of the process. The techno economic modeling for this study was performed using SuperPro Designer , Version 9.5 , a software tool for process simulation and flow sheet development that performs mass and energy balances, equipment sizing, batch scheduling/debottle necking, capital investment and operating cost analysis, and profitability analysis. This software has been used to estimate cost of goods in a variety of process industries including pharmaceuticals produced by fermentation and plant-made pharmaceuticals .

It is particularly useful at the early, conceptual plant design stage where detailed engineering designs are not available or warranted. SuperPro was chosen because it has built-in process models and an equipment cost database for typical unit operations used in the biotechnology industry, such as bioreactors, tangential flow ultrafiltration and diafiltration, chromatography, grinding or homogenization, and centrifugation. There are some specific unit operations and processes used in this study that are currently not included in SuperPro, such as indoor plant cultivation, transplantation, plant harvesting and screw press/disintegrator. Such unit operations were addressed through the “Generic Box” feature of the application. Unless otherwise noted, the maintenance costs of major equipment, unit operation-specific labor requirements and costs , pure components, stock mixtures, heat transfer agents, power and consumables used in the analysis were determined using the SuperPro built-in equipment cost model and default data banks. Additional case study specific design parameters were selected based on experimental data from journal articles, patent literature, the authors’ laboratories, interviews with scientists and technologists conducting the work cited, technical specification sheets or correlations, heuristics, or assumptions commonly used in the biotechnology and/or agricultural industry.Process flow and unit operations were derived from published methods and unpublished results obtained by the authors and collaborators who have participated in the development and scale-up of the process described and in the development of Griffithsin products. On the basis of this information, the SuperPro software was used to select and size equipment for each of the unit operations to achieve the desired production target , simulate the operations by performing material and energy balances, and specify and schedule all operations taking place within each piece of equipment to calculate material inputs and outputs and process times. Costs for raw materials, utilities, consumables, labor, laboratory QA/QC, waste disposal and equipment maintenance were then used to determine annual operating costs, and per-unit mass or per-dose costs . The main case study model was based on an existing plant based manufacturing facility, operating in batch mode, and excluded new capital investments and other facility dependent costs, except for equipment maintenance costs, which were included.

For the downstream portion of the Griffithsin manufacturing process, an annual available operating time of 7,920 h for the facility was used with indoor-grown Nicotiana benthamiana plants. Operating time was based on Holtz et al. for a similar facility, which was designed with overlapping utility capacity and in which the largest single utility unit can be down for maintenance and/or repairs and the utility loads can be maintained with redundant equipment. Likewise, per Nandi et al. it was assumed that the plants would be grown continuously throughout the year . Land costs, upfront R&D, upfront royalties, and regulatory/certification costs were neglected in the model as these costs can vary widely. Griffithsin protein can be produced in plants in a number of ways. These include stable expression in recombinant plants; inducible expression in transgenic plants; transient expression induced directly by tobacco mosaic virus replicons; or via agrobacterial vectors introduced into the plants via vacuum assisted, or surfactant-assisted, infiltration . Relative to stable transgenic plants, the advantages of speed of prototyping, manufacturing flexibility,nursery grow bag and ease of indoor scale-up are clearly differentiating features of transient systems and explain why this approach has been widely adopted in the manufacture of many plant-made pharmaceuticals . In our base-case analysis, we modeled expression of Griffithsin using TMV induction described in Fuqua et al. and results from 3 pilot-scale manufacturing runs because these batches provided the most extensive and complete data set; however, this process has been corroborated in 6 additional manufacturing runs at pilot-scale or larger.icotiana benthamiana host plants are generated from seed and propagated indoors under controlled environmental conditions until sufficient biomass is obtained for inoculation with the TMV vector carrying the Griffithsin gene. The process is summarized as follows. An N. benthamiana Master Seed Bank is generated from seeds obtained from the U.S. Department of Agriculture Repository. For bio-manufacturing, seeds from the TW- 16 line are obtained in bulk and stored securely. The Master Seed Bank is qualified for germination rate , freedom from disease, and genetic uniformity, and stored in sealed containers under temperature-controlled conditions . If the seed batch passes release tests, it becomes the Production Seed Batch and is used in the designated production run . Seedlings are allowed to grow for 21 days under controlled environmental conditions . At this stage, the seedlings are transplanted to accommodate their larger size and moved to another growth room to await inoculation, as described in the following sections.

The API extraction procedure modeled is per Holtz et al. except that a 1:1 ratio of biomass:buffer is used. Briefly, the aerial parts of the plants containing accumulated Griffithsin are mechanically inverted and cut with a mechanical cutter. The harvested biomass is collected in baskets for transport to the extraction suite, to initiate downstream processing. The harvested biomass fresh weight is determined to calculate the volume of extraction buffer to be added, typically at a rate of 1 kg biomass FW:1 L buffer mix . The pH is adjusted to 4.0 and the mixture is heated to 55◦C for 15 min to help precipitate major host plant proteins. The heated mixture is passively cooled and filtered to yield a crude extract. The crude extract is stirred overnight at 4◦C in the presence of bentonite and MgCl2. This procedure helps remove TMV coat protein , which at this step represents the largest protein impurity in the extract. The suspension is filtered to remove aggregated TMV CP, yielding a clarified and partially purified API-containing solution and is then sterile- filtered . In-process controls are applied throughout downstream processing unit operations to determine reagent volumes and assess yield and quality at key steps. To adequately meet the projected initial annual market demand for a rectal micro-bicidal formulation in the United States, approximately 6.67 million doses of Griffithsin API at 3 mg/dose would be needed. This translates into a production rate of 20 kg of purified Griffithsin API per year. The manufacturing facility to produce the required 20 kg of API per year was assumed to segregate production operations into two broad categories; namely, upstream production and downstream recovery and purification. To accommodate a large number of plants, the facility uses a vertical cultivation design with integrated irrigation and runoff collection system. Each rack is compatible with an integrated transportation infrastructure to move each tray to the next phase of the growth cycle. The upstream portion of the facility houses unit operations for N. benthamiana propagation, inoculation with TMV vector, and Griffithsin protein expression and accumulation. These processes begin with seeding and end when the biomass is taken to harvest. The downstream portion of the facility begins at harvest and continues through purification of the Griffithsin DS. Upstream processing is assumed compliant with good agricultural practices , whereas downstream processing is subject to FDA current good manufacturing practice . The general layout of the upstream growth rooms was adapted from Holtz et al. , and includes one germination chamber for seeds, one pre-inoculation room for biomass growth, and an isolated post-inoculation chamber where N. benthamiana inoculated with TMV expresses and accumulates Griffithsin. Plants are arrayed in equally sized trays under light-emitting diode light systems tuned to the optimized photosynthetic absorbance spectrum of N. benthamianaand are continuously illuminated. The plants are rooted in rock wool cubes held in the trays by polystyrene foam floats and perfused with a nutrient solution . Hydroponic irrigation is on a 12-h cycle and is accomplished via nutrient film technique . We modeled a hydroponic system because the nutrient solution is recycled; hence, water is conserved, and fertilizer runoff is reduced although not eliminated. The mass of nutrient solution taken up by the plants, the cost of the nutrient solution per liter, and the mass of residual nutrient solution that goes to the wastewater treatment system are shown in Supplementary Table 1 in Supplementary Material.

MMC is an interstrand cross-linking agent , and CDDP is an intrastrand cross-linking agent

At the subcellular level, SOG1 is present in the nucleus, consistent with previous findings; however, unlike any previous data in response to other genotoxins, SOG1 does show a change following Al exposure that is ATR-dependent and may be the result of relocalization, morphological changes to the cell identity, as well as possible diminished visualization due to protein degradation. Post-translational modification of SOG1 was determined to be crucial to the regulation of its function. There is no significant transcriptional change to SOG1 expression in the presence of Al, despite protein accumulation subsiding following Al exposure. This suggests that there may be an undetermined mechanism for protein turnover following SOG1 activation. As the p53 functional homologue in Arabidopsis, SOG1 turnover could prove to be a conserved regulatory mechanism as p53 is ubiquitinated by the E3 ligase, MDM2, in mammals . Perhaps this is even a speculative role for ALT2, as WD40 proteins have been shown to associate with E3 ubiquitin ligases. It is unlikely that ALT2 would be responsible for SOG1 turnover as part of an ubiquitin-proteasome pathway as loss of ALT2 would result in inappropriate persistence of SOG1 and lead to hypersensitivity rather than tolerance as is observed. As of yet, evidence only supports that the activation mechanism in response to Al is dependent on ATR, likely through phosphorylation of SOG1 by ATR. Future studies are needed to determine how SOG1 is degraded following ATR-dependent activation in response to Al as well as to other stresses. As shown in Chapter 5, the fourth Al tolerance gene isolated from the als3-1 suppressor screen was SUV2, nursery pots which encodes the Arabidopsis homologue of the mammalian ATR-interacting protein, ATRIP. The suv2-3 mutation represents a premature stop codon in the eighth exon of SUV2 at amino acid 359 of 646.

After establishing the als3-1 suppressor as an Al tolerant suv2 mutation, it was characterized as being part of the same ATR-mediated pathway, further supporting SUV2 as an interacting partner of ATR in Arabidopsis. Additionally, both cell cycle arrest and differentiation of the QC were established to be dependent on SUV2 following Al exposure, and that these responses ultimately lead to endore duplication as a means to terminal differentiation of the root meristem. Thissue- and cell-specific localization of SUV2 was shown to be present within the cytoplasm and nucleus of actively dividing cells of the root tip. There is accumulation of SUV2 throughout the meristematic regions of the root tip in the absence of Al. When grown in an Al toxic environment, SUV2 persists in the meristematic region of the root tip, but this zone has significantly been reduced in size and therefore there is a concomitant reduction of SUV2 in the root organ. At the sub-cellular level, SUV2 is present in the cytoplasm and the nucleus of cells at the root tip in both the absence and presence of Al. It is likely that as Al causes differentiation of the root meristem up to the point of complete stem cell consumption, the zone of cell division becomes reduced while the zone of elongation encroaches closer to the apex i.e. a morphological reduction in zones of root development. This reduction in the meristematic zone may account for the insignificant but observable progressive reduction in SUV2 transcript levels following treatment with increasing amounts of Al.As with SOG1, SUV2 is phosphorylated by ATR in vitro. While in vivo studies are needed in both cases to confirm this post-translational modification, this would be considered a conserved relationship between ATR and SUV2, as this phosphorylation is known to occur in the homologous proteins in yeast and humans . This begs the question: what are the bona fide substrates of ATR in the Al response pathway? While SOG1 has been demonstrated to have 5 ATM/ATR phosphorylation sites, defined as TQ or SQ motifs, and SUV2 is speculated to contain two sites, in a recent phosphoproteomics study, SOG1 was not even identified as a substrate of ATR or ATM in response to ionizing radiation, let alone SUV2 . Some DNA repair factors such as LIG4, UVH3 and MRE11 as well other DNA maintenance and metabolism factors like CHR4, HTA7/HTA10, PCNA1, MCM4 and H2AXA were proven to be ATR and ATM targets in this kinase target study .

While the authors acknowledge that their experimental technique using adverse tryptic cleavage likely was responsible for not identifying SOG1 in their large-scale identification of kinase targets, this large-scale proteomic endeavor only tested IR stress and did not distinguish between ATR versus ATM phosphorylated targets . Despite the substantial contribution these findings offer to the field of plant DNA maintenance and repair, more in depth studies are needed not just for Al responses, but also for the myriad types of damage to plant DNA that must be repaired. Al treatment leads to root growth inhibition due to terminal differentiation by means of endore duplication as visualized by increases in cell and nucleus size of cells of the root tip. Al treatment results in substantial increases in both cell and nuclear size for als3-1 roots, which is consistent with terminal differentiation in conjunction with endore duplication. This increase in size was shown to be dependent on the Al tolerance factors: ATR, ALT2, SOG1 and SUV2. This shows that all four genetic factors control terminal differentiation and endore duplication in response to Al. Previous studies of a sog1 loss-of-function mutant defined a set of SOG1- mediated genes that were inducible by γ-radiation . In response to Al toxicity, SOG1 and ATR have now been established to be acting within the same response pathway, so it was of great interest to determine if SOG1, ATR, ALT2, and SUV2 were responsible for the induction of this established gene set after treatment with Al. The proper conditions to determine whether Al causes changes in known SOG1-mediates genes was determined by a time course study of the persistence of SOG1 accumulation without completed QC differentiation in response to high Al concentrations. After 4 days of growth on highly inhibitory concentrations of Al, the pool of stem cells in the root meristem differentiate into cells no longer capable of dividing; while SOG1 is completely absent from the root tip after 5 to 6 days of growth on high concentrations of Al. Taken together, this established that 3 days of growth in Al was the optimal point at which expression changes would be analyzed, satisfying the need for SOG1 actively inducing transcriptional changes that would lead to root growth inhibition in response to Al. Genes that were selected for expression analysis were found in a previous study as being highly induced following γ-radiation in a SOG1-dependent manner .

From the γ-radiation study, a number of DNA repair genes like BRCA1, the ortholog for the human breast cancer susceptibility gene; PARP2, a key component of microhomology-mediated DNA repair; GMI1, involved in homologous recombination and chromosome maintenance; and members of the RAD family of genes, RAD17 and RAD51. CYCLINB1;1accumulates in the G2 phase of cell cycle progression and is regulated by transcriptional activation. During normal cell cycle progression, in a population of cells like the root rip, a few would be in the G2 phase at a given time expressing CYCB1;1, but an increase in expression would suggest more cells within the cell population were halted in the G2 phase, indicative of a G2 cell cycle arrest. Other transcription factors were induced in this γ-radiation study,plastic planters like TRFL3 and TRFL10, MYB family transcription factors known to have roles in developmental processes, defense responses and DNA maintenance . In total, 16 genes were assayed . Treatment with Al resulted in a measurable increase in expression of the subset of assayed genes in Col-0 wild type compared with no Al, and for als3-1 there was an even larger increase of gene expression. In contrast, an increase in expression of these genes was not observed for any of the Al tolerant single mutants or double mutants in comparison to the respective controls. This indicates that Al triggers an ATR-, ALT2, SUV2- and SOG1- dependent transcriptional program that is similar to that observed following treatment with γ-radiation, providing an important link between the cell cycle checkpoints involved in DNA damage detection and transcription in response to Al. Clearly, cell cycle checkpoints are emerging as key regulators of Al response, indicating that Al-dependent activation of these factors is central to terminal differentiation following chronic exposure to Al. Unlike γ-radiation, this stoppage of root growth is ATM-independent, as demonstrated by real-time PCR analysis of an atm loss-of-function mutant, indicating that at least in respect to Al stress, SOG1 functions downstream of ATR rather than ATM. This is of particular importance since it is indicative of the type of damage that ATR, ALT2, SOG1 and SUV2 are detecting in the context of Al. There are clear transcriptional differences between Al treatment and γ-radiation. Examples of these differences would be the lack of induction of AtRAD21 in the absence or presence of Al, as well as the ATM independent manner of SOG1-dependent transcript induction in Al treated seedlings while they are ATM-dependent upon exposure to γ-radiation. The difference in the role of ATM in response to these two stresses is interesting, especially considering that ATM is largely uninvolved in the Al response despite the requirement of functional ATR, ALT2, SOG1 and SUV2. This indicates that ATR, ALT2, SOG1 and SUV2 comprise an Al-response pathway that does not primarily require ATM. This suggests that each DNA stress results in a unique transcriptional profile that may be revealing in relation to their respective impacts on genomic integrity. With the overwhelming evidence that the DNA damage response factors ATR, ALT2, SOG1 and SUV2 all play a role in detecting Al dependent damage, it was of interest to examine their roles in response to known genotoxins. HU is an inhibitor of ribonucleotide reductase by scavenging free radicals that are used for the reduction of ribonucleotides . This stalls the replication fork due to depletion of deoxyribonucelotides. ATR functions to detect replication fork blocks and single stranded DNA breaks and atr mutants are sensitive to HU .

MMC is an aziridine containing antibiotic isolated from Streptomyces caepitosus . MMC itself does not react with DNA, but once it becomes reduced by quinone, the aziridine opens and allows MMC to attack the DNA . This reaction forms crosslinks across the complementary strands of the DNA double helix, or interstrand cross links . DNA cross linking can also occur between adjacent bases, called intrastrand DNA cross linking. The chemotherapy drug CDDP is a platinum-containing drug that is a neutral molecule until it is activated through a series of spontaneous aquation reactions, which involve the sequential replacement of the cis-chloro ligands of CDDP with water molecules . When the aquation event occurs, this allows the platinum atom to bind to DNA, preferentially guanine bases, which forms DNA–DNA intrastrand crosslinks . DNA cross links block replication because when they go unrepaired, collapse of the replication fork occurs blocking replication and leading to cell death . ALT2 is the Arabidopsis homologue of the human protein, CSA, which monitors conformational changes in DNA as assessed by blockage ofDNA replication and RNA transcription, a known effect caused by DNA cross links. alt2-1 is hypersensitive to MMC and CDDP . SOG1, the plant functional homologue of the human transcription factor p53, is a transcription factor in Arabidopsis responsible for the initiation of DNA damage responses including DNA repair as well as the initiation of endore duplication in plants. sog1 loss-of-function mutants are sensitive to both the replication fork poison, HU, and the DNA cross linking agents, MMC and CDDP. SUV2 is the Arabidopsis functional homologue to the human protein, ATRIP, which is required for recruiting ATR to sites of DNA damage, presumably to regions coated in Replication Protein A . RPA is a heterotrimeritc protein complex, comprised of sub-units RPA1, RPA2, and RPA3, which binds to single stranded DNA to protect it from nuceolytic degradation and hairpin formation, similar to the prokaryotic Single Stranded Binding protein .

Mutational approaches using the model plant species Arabidopsis have become an important complement to these studies

Outside of organic acid release as a form of Al tolerance or resistance, a logical but unproven mechanism of Al tolerance is root-mediated alkalinization of the rhizosphere, since Al toxicity is dependent on the pH of the growth environment. Despite the appeal of this process as an Al tolerance mechanism, there is only one report that clearly demonstrates this role in Al tolerance. An Al tolerant mutant in Arabidopsis was shown to release similar amounts of organic acids to wild-type seedlings, indicating that this mutant has a different mechanism of Al tolerance. It was then found that its mechanism of Al tolerance was correlated with an Alactivated root apical H+ influx. This H+ influx resulted in an increase in rhizosphere pH at the surface of the root tip, which was significant enough to decrease Al activity around the root tip . In addition to the various resistance mechanisms described, there has been limited evidence that modification of extra- and intracellular anionic sites can have a positive impact on Al resistance. Polyamines are small aliphatic polycationic molecules that could compete with Al ions for binding sites at the cell wall and membrane to prevent Al from entering the cell . Plant polyamines are detected in actively growing tissues and under stress conditions. They also have been connected to the control of cell division, embryogenesis, root formation, fruit development and ripening, and responses to biotic and abiotic stresses .There have been two reports that describe the relationship between Al and polyamines; one report has demonstrated the positive effects of polyamines on Al tolerance, while another has discussed the effects of Al on cellular polyamines. In saffron, 1mM polyamines were able to reduce Al toxicity. Polyamines were also able to decrease H2O2 content in the presence of Al, as well as decrease Al accumulation at the roots . In cell cultures of a woody plant Catharanthus roseus, it was observed that polyamine levels increase upon Al exposure. Spermine levels increased by two- to three-fold after 24 and 48 hours of exposure to Al, and putrescine levels slightly increased after four hours of exposure,black flower buckets but was surprisingly followed by a sharp decrease . This suggests that even non-chelating molecules can have a significant impact on Al tolerance in plants. It has been argued based on these studies that Al exclusion must be a rapid response to minimize Al uptake and subsequent Al-dependent stress.

Interestingly, much of the findings on Al exclusion mechanisms have arisen from studies that move roots from a no Al environment to one that has highly toxic levels with the research focused on the immediate responses to Al. It is hard to imagine a real world situation in which roots go from an environment with little to no Al to one that has highly inhibitory concentrations. Therefore, it is arguable whether the approach of studying immediate responses to Al is necessarily relevant to Al toxicity in soils since stoppage of root growth in such an environment is likely due to chronic long-term exposure to Al. Consequently, it is of critical importance to determine the toxic effects of Al as it accumulates within plant tissue.It has been very difficult to determine other mechanisms of Al tolerance and resistance in agriculturally relevant plants due to issues such as genome size, availability of knockout lines, generation time, and difficulty in creating transgenic lines. The use of Arabidopsis thaliana has evaded this issue by utilizing the model plant that has a smaller diploid genome, an extensive library of knockout lines, and short generation time . Additionally Arabidopsis has similar sensitivity to Al in comparison to other crop plants and shows classic signs of Al toxicity, making Arabidopsis a suitable plant model for Al toxicity for crop species . The mechanisms of Al resistance have been intensively studied on crop species using natural genetic variation within and across species, such as wheat and maize. While clearly an insightful approach that has given extensive knowledge on Al exclusion mechanisms, this work is limited based on currently existing variability with regard to growth in the presence of Al. Beyond the obvious advantages of using a model species, Arabidopsis has a similar Al toxic threshold to many agriculturally relevant crop species making it a valuable system for investigating how plants sense and respond to Al through the identification of mutants with altered growth capabilities in the presence of Al . This has been particularly true with regard to identification ofAl sensitiveArabidopsis mutants, which have reduced root growth in the presence of Al likely due to deffects in mechanisms required for Al exclusion, Al detoxification, or response to Al-dependent damage.

By screening for Al sensitive Arabidopsis mutants, eight complementation groups were identified indicating that Al toxicity is complex, which is to be expected considering the likely number of factors involved in mechanisms of Al resistance and tolerance . Most importantly, as will be discussed later, identification of these als mutants has allowed for use of a suppressor mutagenesis approach that has resulted in identification of factors that are important for Al-dependent stoppage of root growth. In order to find factors that are required for Al stress response, a genetic approach was taken to identify Arabidopsis mutants that exhibit increased Al sensitivity. Mutant lines were generated through ethyl methanesulfonate mutagenesis of Arabidopsis thaliana ecotype Col-0 wt. M2 seedlings were subsequently screened for their response to Al by identifying seedlings with normal growth in the absence of Al, but restricted growth on low levels of Al. The seedlings were grown on a two-layer gel system, with the upper layer consisting of nutrient medium with no added Al and the lower layer consisting of the same nutrient medium equilibrated with a subtoxic level of AlCl3. Any seedlings that could grow normally through the upper layer but could not penetrate the lower layer were isolated. Each of these Al sensitivemutants likely represents deffects in genes that are required for mechanisms for Al resistance or tolerance. Two of the identified mutants, als1-1 and als3-1 have been studied in depth. Both of these mutations represent a recessive loss of function mutations resulting in greater than wild type root growth inhibition in the presence of sub-threshold levels of AlCl3. The first mutant characterized was als3-1 due to its extreme sensitivity to Al at low levels. When grown on Al asl3-1 shows complete arrest of growth of the primary root and shoot meristems. The roots of als3-1 are stunted and have a swollen club shaped root apex, with root hairs at or near the root tip when grown on Al media. Also, als3-1 roots do not initiate any lateral roots, but can produce secondary roots from the base of the hypocotyl. Growth of lateral roots can reinitiate if als3-1 plants are removed from Al media, but the primary root is irreversibly inhibited by the Al treatment. This response differs from the roots of wild type plants, which are able to fully recover from the Al treatment. Following Al treatment the shoot phenotype of als3-1 shows reduced cotyledon expansion. als3-1 shoots show a delayed growth recovery in comparison to the root, which does not recover at all. Leaf expansion is blocked for several days after the Al recovery.

In addition, the first true leaves of als3-1 develop abnormally following Al treatment. They are severely stunted with very few trichomes, poor leaf expansion,french flower bucket and irregular shaped epidermal cells with a rough leaf surface. The leaves that developed after Al treatment also do not expand but became a disorganized cluster of leaf pegs that eventually expand without petiole development. Eight days after removal from Al, a second shoot apex forms that develops relatively normally except for a greater number of rosette leaves and some fused inflorescences. This shoot phenotype is found to only in als3-1 plants that were challenged with Al and has not been described for als mutants in general. Since this mutation is completely Al-dependent, it was hypothesized that als3-1 represents a factor required for Al-tolerance or resistance. This factor was found to be specific for Al tolerance since als3-1 did not show increased sensitivity to other metals such as copper, nickel, cadmium or lanthanum and does not display any other growth deffects in low pH without Al. Staining of als3-1 roots with hematoxylin and morin, two stains that bind to Al, resulted in similar intensity of staining to wild type, suggesting that als3-1 mutation does not alter the amount of Al uptake . This was later confirmed using ICP-OES . Although there was no difference in the amount of Al uptake, when wild type and als3-1 roots were stained with hematoxylin, wild type plants showed a diffuse pattern of surface bound Al extending from the root apex to the mature region of the root, while als3-1 roots displayed intense staining just proximal to the root tip . Map-based cloning of als3-1 showed that it represents a defect in a gene that encodes an ABC-like transporter homologous to bacterial ybbM, which is a metal resistance protein from Escherichia coli. Based on this similarity and the localization pattern of ALS3, which shows it predominantly at the plasma membrane of root cortical cells and cells of the vasculature, it was proposed that it redistributes Al away from the most sensitive plant tissues in order to maintain cell division . Loss of ALS3 as in the als3-1 mutant would result in inappropriate accumulation of Al in vulnerable areas such as the root tip and would consequently cause growth arrest at levels of Al that have no measurable effect on wild type. GUS staining of plants harboring the ALS3 promoter fused with GUS indicates that ALS3 expression is localized primarily to the sieve tube elements of the phloem in all plant organs, and trichoblast cell files and immature root hairs. GUS activity was also found in the epithem tissue of the hydathodes but not in the actual water pore of the hydathode. Since Northern analysis determined that ALS3 is Al inducible, GUS analysis of Al treated lines resulted in a shift of expression from the root epidermis to the root cortex.

From these results, ALS3 was hypothesized to mediate Al transport within the plant, transporting Al from sensitive tissues from the plant such as the root apical meristem in order to sequester it in less sensitive tissues or also to the hydathodes for excretion by guttation. It is hypothesized that by disrupting this partial ABC transporter, there is inappropriate accumulation of Al in the root, leading to severe symptoms of Al toxicity . Consistent with the importance of ALS3 to Al tolerance, an ALS3 homolog was identified in rice, called STAR2. Although both ALS3 and STAR2 are required for plant Al tolerance, the expression patterns and cellular localization differ. STAR2 is only expressed in roots upon Al treatment and is located in all cell types except for the epidermal cells in the mature root zone . In contrast, ALS3 is expressed at a basal level in the vasculature throughout the plant and its expression is dramatically increased in the Arabidopsis root tip following exposure to Al . Similar to ALS3, STAR2 contains several transmembrane domains that likely form a pore or channel that is involved in substrate movement. Both ALS3 and STAR2 lack an ATPase domain, making them unusual with regard to ABC transporters that often have the transmembrane domains and ATPase domain all as part of one protein. While a separate ATPase domain containing protein partner has not been found for ALS3 , rice STAR2 was shown to interact with another protein, STAR1, which contains an ATPase domain . The STAR1/STAR2 complex functions together as a bacterial-type ABC transporter that is speculated to transport UDP-glucose, although it is currently unclear as to how the transport of UDP-glucose by STAR1/2 is responsible for rice Al tolerance . Other Arabidopsis mutants that have been identified with altered responses to Al include als1 and als7. Both als1 and als7 were identified in the original screen for Arabidopsis mutants with Al hypersensitivity. als1-1 was also identified by mapped based cloning and subsequently characterized. Similarly to als3- 1, als1-1 has an extreme increase in Al sensitivity.

There are few published studies on the genetics of tolerance to chilling temperatures in tomato

Our results reveal that BrpHMA2 could be activated by Cd2+ , which is similar to the results found for HMA2 in Arabidopsis. Results suggest that BrpHMA2 is involved in the Cd response of plants. BrpHMA2 was also found to be expressed explicitly in the vascular tissues of roots, stems, leaves, flowers, siliques, and carpopodia, and its protein was localized in the plasma membrane . These results are consistent with previous findings for HMA2 in Arabidopsis, OsHMA2 in rice, and TaHMA2 in wheat. The protein plasma membrane localization and the vascular-specific expression pattern of the genes revealed that HMA2 might function as a membrane transporter in long-distance transport in plants. In recent years, some studies have investigated the function of HMA2. Most of these studies demonstrated that HMA2 is involved in Zn2+ and Cd2+ transmembrane transport and influences root-to-shoot Zn/Cd translocation. For example, HMA2 in Arabidopsis is thought to be involved in the outward transport of Zn2+ and Cd2+ from the cell cytoplasm, and HMA2 mutants are more sensitive to Cd stress and exhibit higher Zn or Cd accumulation than wild-type plants in the presence of high levels of Zn2+ or Cd2+ 14,15. The over expression of OsHMA2 in wheat, rice, and Arabidopsis improves root-to-shoot Zn/Cd translocation. In addition, the transformation of TaHMA2 in yeast enhances the resistance of cells to Zn/Cd. In rice, the suppression of OsHMA2 decreases the Zn and Cd concentrations in leaves, increases the retention of Zn in roots and reduces the translocation of Cd and Zn from roots to shoots compared with the results obtained with wild type plants. According to the literature, HMA2 is responsible for Zn2+/Cd2+ efflux from cells, plays roles in Zn and Cd loading to the xylem,procona buckets and participates in the root-to-shoot translocation of Zn/Cd. However, Yamaji et al. found that OsHMA2 is localized at the pericycle of the roots and in the phloem of enlarged and diffuse vascular bundles in the nodes. Their insertion lines of rice showed decreased concentrations of Zn and Cd in the upper nodes and reproductive organs.

The study revealed that the heterologous expression of OsHMA2 in yeast is associated with the influx transport of Zn and Cd. These researchers suggested that OsHMA2in the nodes plays an important role in the preferential distribution of Zn and Cd through the phloem to the developing tissues. Our results also revealed that, in the presence of Cd2+, transgenic Arabidopsis seedlings and yeast over expressing BrpHMA2 showed higher concentrations of Cd and enhanced Cd2+ sensitivity compared with the controls . Thus, we propose that BrpHMA2 functions in Cd2+ transport in the phloem tissue of vascular systems through influx into cells, and the efflux from phloem cells during long-distance transport may be performed by other transporters. The differential function of HMA2 from various plants might come from the tiny difference in amino acids in their function domains; this puzzle requires further investigation.In this study, we identified the NAC TF gene BrpNAC895, a homolog of Arabidopsis ANAC087 , which could be induced by Cd2+ stress . We confirmed that BrpNAC895 has a role in the response of B. parachinensis to Cd2+ stress by upregulating BrpHMA2 expression through direct binding to the BrpHMA2 promoter using EMSA, ChIP–qPCR, and the transient transformation method with B. parachinensis protoplasts . Previous studies have demonstrated that Arabidopsis ANAC087 is associated with plant programmed cell death . It functions along with the TF ANAC046 to show partial redundancy in coregulating the expression of some PCD genes in the root columella, including ZEN1, BFN1, and RNS3. Whether ANAC087 could participate in regulating Cd transporters in plants has not been reported. Our findings on BrpNAC895 show that this NAC TF has a novel role in upregulating BrpHMA2 expression in response to Cd2+ stress. We also identified the Cd-responsive AREB TF BrpABI 449 , which is a homolog of Arabidopsis ABF3 and can bind to the promoter of BrpHMA2 . ABF3 modulates the response to drought, salt, and other osmotic stresses as a master component in ABA signaling. This TF can also regulate the expression of multiple genes, such as the AGAMOUS like MADS-box TF family gene SOC1, which is a floralintegrator regulating flowering in response to drought, and the AREB TF ABI5, which is a core component in the ABA signaling pathway in the regulation of seed germination and early seedling growth during exposure to ABA and abiotic stresses .

In general, ABF3 can form protein complexes with other TFs. For example, ABF3 forms homodimers or heterodimers with AREB1/AREB2 and acts cooperatively to regulate ABRE dependent gene expression. ABF3 forms a complex with NF-YC3 to promote the expression of the SOC1 gene and thus accelerate flowering and drought-escape responses; ABF3 interacts with NAC072 to regulate RD29A and RD29B expression in response to ABA. Thus, complex formation might be the important functional mechanism by which ABF3 regulates gene transcription. Using EMSAs and ChIP–qPCR assays, we found that BrpABI449 could directly bind to regions of the BrpHMA2 promoter . The interaction of BrpABI449 and BrpNAC895 was further confirmed by pull-down and BiFC assays . The inhibition of BrpABI449 on the transcriptional regulatory role of BrpNAC895 was detected in the B. parachinensis protoplast transient system . The inhibition by BrpABI449 of the transcriptional regulatory role of BrpNAC895 complex, likely interferes with BrpNAC895’s activity in the transcriptional activation of BrpHMA2 in response to Cd stress. It has also been reported that Cd stress can induce a stress response via ABA signaling. Our results showing that BrpNAC895 and BrpABI449 are upregulated by Cd stress also support this point. The uptake or homeostatic regulation of heavy metals needs proper modulation to ensure plant health. Previous studies have shown that Cd stress induces the MYB TF gene MYB49 in Arabidopsis. This TF may further positively regulate the downstream TF gene bHLH38 and bHLH101 by directly binding to their promoters, and activate iron-regulated transporter 1 to enhance Cduptake. In contrast, Cd stress upregulates the expression of ABI5. ABI5 interacts with MYB49, prevents its binding to the promoters of downstream genes, and functions as a negative regulator to control Cd uptake and accumulation. Our present results also demonstrate a mechanism for controlling the expression of the heavy metal transporter gene BrpHMA2 under Cd stress. We propose that Cd2+ induces the expression of BrpNAC895 and BrpABI449, which might be mediated by ABA signaling. BrpNAC895 then promotes the transcription of BrpHMA2 by binding directly to its promoter . The activation of BrpHMA2 enhances Cd2+ uptake and may induce cell damage. Negative regulation of BrpHMA2 is then achieved by the upregulation of another AREB TF, BrpABI449, which interacts with BrpNAC895 and forms BrpNAC895-BrpABI449 protein complexes to inhibit the BrpHMA2 transcription activated by BrpNAC895 .

BrpABI449 could also bind to the promoter of BrpHMA2 directly to compete with BrpNAC895 in binding to the BrpHMA2 promoter. This negative regulation may play a supplementary role in the uptake and transport of Cd.Many plant species of Brassicaceae, including Arabidopsis, turnip, and oil seed rape, can be genetically modified, but the creation of transgenic B. parachinensis remains difficult. Therefore, we over expressed BrpHMA2 in Arabidopsis to investigate the function of BrpHMA2 and established a transient transformation system in B. parachinensis protoplasts to perform gene regulatory network analysis. Protoplasts have been widely used for sub-cellular protein localization and gene regulation analyses. In this study,procona florida container the transient transformation of B. parachinensis protoplasts was demonstrated to be a powerful system for ChIP–qPCR analysis. Previous studies have applied a similar approach to Populus trichocarpa and Brassica napus. Although the transient transformation system of B. parachinensis protoplasts was successfully used in this study of molecular mechanisms, the system cannot be easily used for phenotype and physiological analyses. The lack of BrpNAC895 and BrpABI449 transgenic B. parachinensis is a problem that severely limits research on this plant. New techniques, such as the transient reprogramming of plant traits via the transfection of RNA based viral vectors using Agrobacterium and gene editing combined with fast-treated Agrobacterium coculture, may be useful approaches for comprehending gene function concerning physiology and for the further application of modifications of gene function to effectively control the accumulation of Cd in B. parachinensis.Abiotic stresses, especially those which affect the water relations of the plant such as low temperatures, may decrease plant growth and yield. The majority of plants will suffer damage when exposed to freezing temperatures , but plants of tropical or sub-tropical origin also suffer damage when exposed to chilling temperatures . Exposure of roots to chilling temperatures decreases root hydraulic conductance , and can result in water stress and chilling injury within a few hours of exposure . The susceptibility to water stress induced by root chilling in species of tropical and sub-tropical origin is a concern for agricultural production in Mediterranean climates such as California, where exposure to cold soils in the spring can affect seedling establishment because soil temperatures under an open canopy may be colder than air temperatures . Cultivated tomato is a classic example of a chilling-sensitive crop . It was domesticated from the wild cherry tomato, which is native to mesic, tropical environments . A related wild tomato species, S. habrochaites, grows in the Peruvian Andes at altitudes up to 3300 m and thrives in xeric habitats and at chilling temperatures detrimental to S. lycopersicum . Upon exposure to root chilling conditions, the root hydraulic conductance of both tomato species decreases, but S. habrochaites closes its stomata rapidly in response to chilling stress, thereby maintaining water potential and shoot turgor, whereas the stomata of S. lycopersicum remains open and the shoots wilt . Other agronomically important crops of tropical or sub-tropical origin such as maize and rice respond to root chilling in a manner consistent with that of cultivated tomato . An improved understanding of the underlying mechanisms of root chilling tolerance in wild S. habrochaites would contribute to a better general understanding of chilling sensitivity in crops of tropical and sub-tropical origins.A review by Venema et al. focused on physiological effects of chilling and noted that wild tomato species were promising sources of genetic tolerance to chilling.

Oyanedel et al. evaluated a back cross inbred line population derived from S. habrochaites acc. LA1777 for growth traits under chilling temperatures and reported QTL for higher biomass accumulation on chromosomes 2, 3, and 9. Elizondo and Oyanedel evaluated tomato introgression lines containing S. habrochaites acc. LA1777 introgressions on chromosomes 2 and 3 in the field under low temperatures . The ILs had higher growth rates but lower fruit set than the parental lines in response to an increase in the number of hours of chilling temperatures. To investigate the genetic basis of shoot turgor maintenance under root chilling, Truco et al. used an interspecific BC1 population derived from chilling-susceptible S. lycopersicum cv. T5 and chilling-tolerant wild S. habrochaites acc. LA1778 to map QTL for this trait. Three QTL for shoot turgor maintenance under root chilling were identified on chromosomes 5, 6, and 9. The largest effect QTL located on chromosome 9 accounted for 33 % of the trait phenotypic variance . We designated this QTL stm9 for shoot turgor maintenance, chromosome 9. Subsequently, QTL stm9 was fine-mapped to a 2.7-cM region on the short arm of chromosome 9 between markers T1670 and T1673 . Easlon et al. determined that tomato ILs containing the short arm of chromosome 9 from chilling-tolerant S. lycopersicoides and S. habrochaites maintained shoot turgor under root chilling. Here we high-resolution mapped QTL stm9 using recombinant sub-near-isogenic lines and compared high resolution mapped QTL stm9 to the S. lycopersicum reference genome for initial identification of potential candidate genes and regulatory sequences . Our longer term goal is to identify and functionally test candidate genes and regulatory sequences from S. habrochaites and determine the causal gene or polymorphisms for QTL stm9.A population of near-isogenic lines containing the chromosome 9 region from S. habrochaites acc. LA1778 in an otherwise completely S. lycopersicum cv. T5 background was marker-selected and used for fine-mapping, as described in Goodstal et al. . For high-resolution mapping of stm9, we created and marker selected recombinant sub-near-isogenic lines as follows.

Reliance on biotechnology can increase the risk of forward biological contamination

Trace elements and small-usage compounds can be transported from Earth, or in some cases extracted from the Martian regolith. In the case where power is provided from photocollection or photovoltaics, light energy will vary with location and season, and may be critical to power our bioreactors. Although photosynthetic organisms are attractive for FPS, a higher demand for carbon-rich feedstocks and other chemicals necessitates a more rapid and efficient CO2 fixation strategy. Physicochemical conversion is inefficient due to high temperature and pressure requirements. Microbial electrosynthesis , whereby reducing power is passed from abiotic electrodes to microbes to power CO2 reduction, can offer rapid and efficient CO2 fixation at ambient temperature and pressure . MES can produce a variety of chemicals including acetate , isobutanol , PHB , and sucrose , and therefore represents a filexible and highly promising ISRU platform technology . Biological N2-fixation offers power- and resource-efficient ammonium production. Although photoautotrophic N2 fixation with, for example, purple non-sulfur bacteria, is possible, slow growth rates due to the high energetic demand of nitrogenase limit throughput . Therefore, heterotrophic production with similar bacteria using acetate or sucrose as a feed stock sourced from electromicrobial CO2-fixation represents the most promising production scheme, and additionally benefits from a high degree of process redundancy with heterotrophic bioplastic production. Regolith provides a significant inventory for trace elements and, when mixed with the substantial cellulosic biomass waste from FPS processes, can facilitate recycling organic matter into fertilizer to support crop growth. However, regolith use is hampered by widespread perchlorate , indicating that decontamination is necessary prior to enrichment or use. Dechlorination can be achieved via biological perchlorate reduction using one of many dissimilatory perchlorate reducing organisms . Efforts to reduce perchlorate biologically have been explored independently and in combination with a more wholistic biological platform . Such efforts to integrate synthetic biology into human exploration missions suggest that a number of approaches should be considered within a surface bio-manufactory.

A biomanufactory must be able to produce and utilize feed stocks along three axes as depicted in Figure 5: CO2-fixation to supply a carbon and energy source for downstream heterotrophic organisms or to generate commodity chemicals directly, N2-fixation to provide ammonium and nitrate for plants and non-diazotrophic microbes,macetas para viveros and regolith decontamination and enrichment for soil-based agriculture and trace nutrient provision. ISRU inputs are sub-module and organism dependent, with all sub-modules requiring water and power. For the carbon fixation sub-module , CO2 is supplied as the carbon source, and electrons are supplied as H2 or directly via a cathode. Our proposed bio-catalysts are the lithoautotrophic Cupriavidus necator for longer-chain carbon production [e.g., sucrose ] and the acetogen Sporomusa ovata for acetate production. C. necator is a promising chassis for metabolic engineering and scale-up , with S. ovata having one of the highest current consumptions for acetogens characterized to date . The fixed-carbon outputs of this sub-module are then used as inputs for the other ISRU sub-modules in addition to the ISM module . The inputs to the nitrogen fixation sub-module include fixed carbon feed stocks, N2, and light. The diazotrophic purple-non sulfur bacterium Rhodopseudomonas palustris is the proposed bio-catalyst, as this bacterium is capable of anaerobic, light-driven N2 fixation utilizing acetate as the carbon source, and has a robust genetic system allowing for rapid manipulation . The output product is fixed nitrogen inthe form of ammonium, which is used as a feed stock for the carbon-fixation sub-module of ISRU along with the FPS and ISM modules. The inputs for the regolith enrichment sub-module include regolith, fixed carbon feedstocks, and N2. Azospira suillum is a possible bio-catalyst of choice due to its dual use in perchlorate reduction and nitrogen fixation . Regolith enrichment outputs include soil for the FPS module , H2 that can be fed back into the carbon fixation sub-module and the ISM module, chlorine gas from perchlorate reduction, and waste products. Replicate ISRU bioreactors operating continuously in parallel with back-up operations lines can ensure a constant supply of the chemical feed stocks, commodity chemicals, and biomass for downstream processing in ISM and FPS operations. Integration of ISRU technologies with other biomanufactory elements, especially anaerobic digestion reactors, may enable complete recyclability of raw materials, minimizing resource consumption and impact on the Martian environment .

Waste stream processing to recycle essential elements will reduce material requirements in the biomanufactory. Typical feed stocks include inedible crop mass, human excreta, and other mission wastes. Space mission waste management traditionally focuses on water recovery and efficient waste storage through warm air drying and lyophilization . Mission trash can be incinerated to produce CO2, CO, and H2O . Pyrolysis, another abiotic technique, yields CO and H2 alongside CH4 . The Sabatier process converts CO2 and CO to CH4 by reacting with H2. An alternate thermal degradation reactor , operating under varying conditions that promote pyrolysis, gasification, or incineration, yields various liquid and gaseous products. The fact remains however, that abiotic carbon recycling is inefficient with respect to desired product CH4, and is highly energy-intensive. Microbes that recover resources from mission wastes are a viable option to facilitate loop closure. Aerobic composting produces CO2 and a nutrient-rich extract for plant and microbial growth . However, this process requires O2, which will likely be a limited resource. Hence, anaerobic digestion, a multi-step microbial process that can produce a suite of endproducts at lower temperature than abiotic techniques , is the most promising approach for a Mars biomanufactory to recycle streams for the ISM and FPS processes. Digestion products CH4 and volatile fatty acids can be substrates for polymer-producing microbes . Digestate, with nutrients of N, P, and K, can be ideal for plant and microbial growth , as shown in Figure 6. Additionally, a CH4 and CO2 mixture serves as a biogas energy source, and byproduct H2 is also an energy source . Because additional infrastructure and utilities are necessary for waste processing, the extent of loop closure that is obtainable from a treatment route must be analyzed to balance yield with its infrastructure and logistic costs. Anaerobic digestion performance is a function of the composition and pretreatment of input waste streams , as well as reaction strategies like batch or continuous, number of stages, and operation conditions such as organic loading rate, solids retention time, operating temperature, pH, toxic levels of inhibitors and trace metal requirements . Many of these process parameters exhibit trade-offs between product yield and necessary resources. For example, a higher waste loading reduces water demand, albeit at the cost of process efficiency. There is also a potential for multiple co-benefits of anaerobic digestion within the biomanufactory. Anaerobic biodegradation of nitrogen-rich protein feed stocks, for example, releases free NH3 by ammonification. While NH3 is toxic to anaerobic digestion and must thus be managed , it reacts with carbonic acid to produce bicarbonate buffer and ammonium, decreasing CO2 levels in the biogas and buffering against low pH.

The resulting digestate ammonium can serve as a fertilizer for crops and nutrient for microbial cultures.FPS and ISM waste as well as human waste are inputs for an anaerobic digester, with output recycled products supplementing the ISRU unit. Depending on the configuration of the waste streams from the biomanufactory and other mission elements, the operating conditions of the process can be varied to alter the efficiency and output profile. Open problems include the design and optimization of waste processing configurations and operations, and the identification of optimal end-product distributions based on a loop closure metric against mission production profiles, mission horizon, biomanufacturing feedstock needs, and the possible use of leftover products by other mission elements beyond the bio-manufactory. A comparison with abiotic waste treatment strategies is also needed, checking power demand, risk, autonomy, and modularity benefits.Biomanufactory development must be done in concert with planned NASA missions that can provide critical opportunities to test subsystems and models necessary to evaluate efficacy and technology readiness levels . Figure 7 is our attempt to place critical elements of a biomanufactory road map into this context. We label critical mission stages using Reference Mission Architecture -S and RMA-L,macetas por mayor which refer to Mars surface missions with short and long durations, respectively.Beyond contamination, there are ethical issues that concern both the act of colonizing a new land and justifying the cost and benefits of a mission given needs of the many here on earth. Our road map begins with the call for an extensive and ongoing discussion of ethics . Planetary protection policies can provide answers or frameworks to address extant ethical questions surrounding deep-space exploration, especially on Mars . Critically, scientists and engineers developing these technologies cannot be separate or immune to such policy development.We have outlined the design and future deployment of a biomanufactory to support human surface operations during a 500 days manned Mars mission. We extended previous stand-alone biological elements with space use potential into an integrated biomanufacturing system by bringing together the important systems of ISRU, synthesis, and recycling, to yield food, pharmaceuticals, and bio-materials. We also provided an envelope of future design, testing, and biomanufactory element deployment in a road map that spans Earth-based system development, testing on the ISS, integration with lunar missions, and initial construction during shorter-term initial human forays on Mars. The innovations necessary to meet the challenges of low-cost, energy and mass efficient, closed-loop, and regenerable bio-manufacturing for space will undoubtedly yield important contributions to forwarding sustainable bio-manufacturing on Earth. We anticipate that the path towards instantiating a biomanufactory will be replete with science, engineering, and ethical challenges. But that is the excitement—part-and-parcel—of the journey to Mars.Rose production is currently the largest component of California’s $300 million cut-flower industry. In 2001, California growers produced 66% of the U.S. rose crop, with a wholesale value of $45 million . The key pests of cut roses are two spotted spider mites , western flower thrips and rose powdery mildew . The two spotted spider mite is a foliage feeder that extracts the cell contents from leaves. This feeding causes foliar stippling and can disrupt the plant’s photosynthetic and water balance mechanisms . The western flower thrips is both a foliage and flower feeder, although it feeds primarily on flowers in the cut-rose system . Powdery mildew is probably the most widespread and best-known disease of roses. The fungus produces a white, powdery-appearing growth of mycelium and conidia on leaves, which can cause distortion, discoloration and premature senescence. Although it causes some disruption of photosynthesis and transpiration control, the key impact of powdery mildew is reduced aesthetic value caused by the white, powdery spots and leaf distortion. Fresh cut roses are often harvested twice daily, so revised reentry intervals imposed by the U.S. Environmental Protection Agency after pesticide application limit the number of pesticides that are useful in this production system . In addition, the typical number of pesticide sprays applied to roses grown for cut flowers has impeded the implementation of integrated pest management procedures, particularly the use of biological controls. The IPM approach to pest management incorporates all cost-effective control tactics appropriate for the crop, including biological, cultural and chemical controls. Pesticides that target hard-to-kill floriculture pests frequently kill natural enemies as well, which favors continued reliance on conventional pesticides while discouraging the adoption of biological control. Heavy pesticide use against key pests in the greenhouse has resulted in the widespread development of pesticide resistance in western flower thrips , mites , white flies , aphids and leaf miners . The heavy use of pesticides in cut roses is also a worker safety concern in global and local production. California rose growers reached a crisis point about 8 years ago, when pesticide resistance, costs and limited pesticide availability threatened the growers’ ability to effectively manage two spotted spider mites. At the same time, a new cut-rose production system that favors the success of IPM was gaining widespread acceptance. Roses were traditionally grown in soil with a hedgerow training system, where flowers are cut in a manner that gradually creates a 7-foot or taller hedge. The hedges are pruned back annually to about a 3-foot height and the process is begun again.

The far side is occupied by community and government facilities

Most of the search results involved tourist agencies in Nanjing and Shanghai who were advertising the theme park by means of a detailed tourist itinerary. Second, I found a limited number of advertisements for the sale of resettlement houses in the Jinhu New Village. Third, I found accounts from urbanites who had traveled to Jinhu and were commenting on their experiences at the theme park. Most of them seem to have enjoyed themselves and left only very short comments. Last but not least, I encountered one informed and detailed online report by a local resident—entitled “Jinhu New Countryside is a Gambling Game and a Fraud”. Although I have used the internet as an extra source to understand what is going on at the Jinhu site, it is worth noting that the internet has also served as an important arena used by actors involved with Jinhu to gain legitimacy, to voice complaints, to vent anger, and to advertise for economic profit. Jinhu New Village is located in southern Anhui Province about 30 kilometers south of Wuhu City. Altogether it occupies an area of 189 hectares , on a stretch of land running along the east side of the national road G205. G205 is a two-lane paved road built before China’s highway construction boom of the last ten years. The site is situated on a broad plain traversed by numerous canals and lakes. These lakes are the reason why this area would also be chosen as the site for a theme park. Given the relatively high average temperatures and annual precipitation, two harvests are possible each year . The rich agricultural fields in the Jinhu new countryside construction zone once supported twelve traditional villages . Residents of these villages have now all been relocated to Jinhu New Village on the southwest corner of the development zone. Both the Jinhu New Village and the associated theme park are separated from the national G205 road by a stretch of landscaping composed of a long canal, green lawns, and willow trees. As with many New Countryside projects now under construction in China—projects often readily visible to drivers rushing along the gleaming new freeways crisscrossing the country—the Jinhu site is a veritable monument to a particular notion of rural modernization and development.

When driving north along the national highway,maceta 5 litros one encounters first a large billboard explaining that one is approaching China’s “first low-carbon national tourist site.” Behind the billboard, one sees the new houses arranged in a perfect grid, quite distinct from the more haphazard arrangement of farmhouses and fields in the traditional villages . Less than a kilometer beyond the New Village, one reaches the entrance to Jinhu Rural World theme park, situated on the east side of the highway. Here two large billboards announce: “National AAAA tourist site—National modern technology agricultural experimental site—National agricultural tourist demonstration site—Welcome to Jinhu experimental site!” According to the master plan—which appears on one of the billboards— the rural world theme park will eventually have 41 attractions. However, as of June 2012, only 16 projects were finished and open to tourists. Below I analyze the built environment of Jinhu New Village and Jinhu Rural World theme park, paying particular attention to how rural space is undergoing transformation and to how the “New Countryside” contrasts with the old countryside and traditional rural society.One’s first impression of Jinhu New Village is of a modern suburb imported from somewhere in the United States. It is a large housing complex, with houses that are remarkably uniform in structure and physical appearance laid out on a grid consisting of alleyways running either perpendicular or parallel to the national highway. The residences nearest to the highway are two- or three-story townhouses; further from the highway are several rows of new apartment buildings. The aesthetics of the new residences stand in stark contrast to the vernacular architectural style of the old red brick peasant houses, a few of which still stood undemolished just southeast of the New Village. This aesthetic of the New Village is a curious mimicry of American “streetcar” and “sitcom” suburbs, those suburbs that Dolores Hayden describes as refilecting the American “idealized life in single-family houses with generous yards”. More specifically, the dwellings in the New Village, with their white stucco walls, their grey-tile roofs, and their uniformity of appearance, resemble in remarkable ways tract housing in American suburbia—such as in the Los Angeles Basin . As in any American suburb, each house also has a garage, with additional parking available in clearly marked parking spaces along the alleyways. Many aspects of this new Chinese suburbia seem out of place in the Chinese context.

Grass lawns may have particular symbolic significance in American urban and suburban environments , but from a Chinese peasant’s perspective, grass is a weed that infects one’s fields, the last thing one would think of deliberately planting beside one’s home. The garages and parking lots are also curiosities given that very few peasant households in Jinhu own a car. Many peasants currently use their garages to store their agricultural tools. The New Village does not merely emulate American suburbia; it also contains elements of a China-specific vision of the urban modern, as represented by an orderly but dense arrangement of tall buildings. In American suburbia, the ideal consists of single detached family houses placed in a landscaping that tries to imitate the natural environment. However, in Jinhu New Village, one experiences a much greater feeling of density, highlighted by the concentration of residences, including both rows of two- to three-story stucco houses—called bieshu in Chinese—and blocks of six- to seven-story apartment buildings. The term bieshu ordinarily refers to detached houses. At Jinhu, however, they are not free-standing; they are much more like townhouses or row houses than detached single-family houses. One way to explain this preference for a densely built environment is to consider the perceived ecological limitations of Chinese agricultural land. The central government has in recent years expressed concern for China’s “national food security,” leading to various strategies to maximize available arable land, a point I will address later in detail. However, there is another equally plausible explanation. For the Chinese urban elites, tall buildings and uniform orderliness symbolize modern life. For example, many Chinese government officials and nouveaux riches traveling to the U.S. are quite disappointed by their experience—if the U.S. is so modern, why do the vast dense expanses of awe-inspiring skyscrapers exist only in New York City and Chicago, and not in other American cities and towns?10 The orderliness and density of Jinhu New Village, then, is a public advertisement of how fortunate the displaced peasants are to be living in a new modern environment. Besides the modern architectural styles and the orderly arrangement of buildings,cultivo de la frambuesa the modern environment of Jinhu New Village is also refilected inside the homes. Here, one finds a variety of conveniences unavailable in the old villages, including flush toilets, solar powered water heaters, running water, reliable plumbing, built-in gas stoves, garbage collection, and even high-speed internet.

Although my host Mrs. Tang had only a second grade education, she was adamant about the importance of using computers and accessing the internet, by means of which she learned a great deal chatting with other netizens. Indeed, computers and the internet seem generally to be welcome by most peasants in rural China. Other particularly appreciated modern conveniences are showers and hot water, made possible by the solar-powered water heaters installed on the roofs of the houses. I have observed these new water heaters in New Countryside housing projects . Much like the entire Jinhu development site, the space of the New Village is clearly compartmentalized with well-delineated commercial, communal, governmental, and private residential zones. The core of the New Village is organized around a T-shaped axis . Immediately after entering the New Village by the main gate, one faces a two-lane street with commercial shops on both sides . At the far end of this street is a perpendicular street that forms the top of the T. One side of this second street is also occupied by commercial space. A multi-story building houses the police station, the community administrative office , and a “petition office” that handles grievances.Beside the government building is a three-story pre-school and kindergarten, a basketball court, and an indoor market place . Further from the T-shaped axis and on all sides are the private residences. The two- or three-story townhouses stand closer to the national highway; the apartment blocks lie just beyond the community and administration buildings. Throughout the New Village, iron fences separate public spaces from private residential yards, and curbs demarcate the boundaries between pedestrian and vehicular zones of circulation. None of these various ways of compartmentalizing space—derived from Western urban models—were present in the old natural villages. Despite the orderliness and the conveniences of the modern built environment, all is not as it seems, since the new houses come with a variety of extra costs that peasants were not burdened with in their old villages.

The cost of high-speed internet access is roughly USD $25 per month, a fee that also includes unlimited local phone calls. Peasants accept this fee more readily than some of the other charges they face, as it provides a service they did not formerly enjoy. By contrast, from the peasant’s perspective, the cost of water and natural gas are more difficult to accept. In the old villages, water was freely drawn from wells; and they used gathered firewood rather than gas for cooking. Most significant of all, however, their cost of living is now much higher because food has to be purchased instead of being produced on their land, a concept utterly alien to the peasants. While staying in the village, the most common complaint I heard was that residents no longer had land for cultivating vegetables and raising domestic animals. Now they had to buy food from supermarkets. Inevitably, village residents have found ways to cut living expenditures by a variety of means. To avoid using running water, women wash vegetables and laundry in the canal built next to the main road. For drinking water, many households invest in a well, which they dig in their small backyards. Not only is well water free, peasants also consider it to be cleaner than tap water. In a similar fashion, to minimize natural gas usage, many households have purchased portable pre-made cylinder-shaped aluminum stoves. Around 5 am every morning, in order to boil water, Mrs. Tang’s father-in-law got up to start a fire in the aluminum stove, using wood collected from demolished houses. This was a common practice: with the stove set right outside the garage door, he would chat—sometimes standing and sometimes sitting on a stool—with two other neighbors who were also doing the same thing. Another way to save on cooking expense is to build an old-style firewood stove in one’s backyard. One couple running a majiang parlor out of their home did just this, despite warnings from the New Village management, who has ordered them to demolish it, on the grounds that it was ugly and damaged the orderly and neat image of the New Village. There are other ways in which, in order to save money, residents resist the management’s efforts to maintain a neat appearance. To mitigate the daily cost of food, residents in Jinhu New Village utilize all sorts of marginal land around the edge of the complex. In some cases, they have gotten rid of the lawn near their houses by spreading herbicides left over from their farming days. Another less destructive way is to dig a hole just large enough to plant pumpkins or other vegetables that grow vertically, plants that can more discretely blend into the lawn and tree landscaping. Chickens are also allowed to roam freely on and around the landscaping . All of these various survival strategies constitute continuities with common practices in the old villages; all are very much part of the peasants’ familiar habitus.But the “landless” peasants residing in the New Village have also turned to other more novel strategies to help them make ends meet. Numerous residents have converted their townhouses for commercial purposes, something they would not have done in their old villages. One common small business is a restaurant.

A representative quality control sample run was used as the reference file to align peaks

After the fusion of Tic20-proteoliposomes with a lipid bilayer, ion channel activity was observed . The total conductance under symmetrical buffer conditions , 250 mM KCl was dependent on the direction of the applied potential: 1260 pS and 1010 pS under negative and positive voltage values, respectively. The channel was mostly in the completely open state, however, individual single gating events were also frequently observed, varying in a broad range between 25 pS to 600 pS . All detected gating events were depicted in two histograms . Two conductance classes were defined both at negative and positive voltage values with thresholds of 220 pS and 180 pS, respectively . Note that gating events belonging to the smaller conductance classes occurred more frequently. The observed pore seems to be asymmetric, since higher conductance classes notably differ under positive and negative voltages. This is probably due to interactions of the permeating ions with the channel, which presumably exhibits an asymmetric potential profile along the pore. Since small and large opening events were simultaneously observed in all experiments, it is very unlikely that they belong to two different pores. The selectivity of Tic20 was investigated under asymmetric salt conditions , 250/20 mM KCl. Similarly to the conductance values, the channel is intrinsically rectifying ,supporting asymmetric channel properties. The obtained reverse potential is 37.0 ± 1.4 mV . According to the Goldman-Hodgkin-Katz approach, this corresponds to a selectivity of 6.5:1 for K+ :Cl- -ions, thus indicating cation selectivity similar to Tic110. To determine the channel’s orientation within the bilayer, two side-specific characteristics were taken into account: the highest total conductance under symmetrical buffer conditions was measured under negative voltage values, and the channel rectifies in the same direction under asymmetrical buffer conditions . Therefore, it seems that the protein is randomly inserted into the bilayer. The pore size was roughly estimated according to Hille et al.. Considering the highest conductance class , a channel length of 1-5 nm and a resistivity of 247.5 Ω cm for a solution containing 250 mM KCl,frambueso maceta taking into account that the conductivity of the electrolyte solution within the pore is ~5 times lower than in the bulk solution, the pore size was estimated to vary between 7.8-14.1 Å.

This is in good agreement with the size of protein translocation channels such as Toc75 in the outer envelope membrane and Tic110 in the IE. Thus, the size of the Tic20 pore would be sufficient for the translocation of precursor proteins through the membrane. NtTic110, as a negative control, did not show any channel activity during electrophysiological measurements, indicating that the measured channel is not the result of a possible bacterial contamination . Considering our data presented here and those published in previous studies, we can conclude that the Tic translocon consists of distinct translocation channels: On the one hand, Tic110 forms the main translocation pore and therefore facilitates import of most of the chloroplast-targeted preproteins; on the other hand, Tic20 might facilitate the translocation of a subset of proteins. This scenario would match the one found in the inner mitochondrial membrane, where specific translocases exist for defined groups of precursor proteins: the import pathway of mitochondrial carrier proteins being clearly separated from that of matrix targeted preproteins. The situation in chloroplasts does not seem as clear-cut, but an analogous separation determined by the final destination and/or intrinsic properties of translocated proteins is feasible. The severe phenotype of attic20-I mutants prompts us to hypothesize that Tic20 might be specifically required for the translocation of some essential proteins. According to cross-linking results, Tic20 is connected to Toc translocon components. Therefore, after entering the intermembrane space via the Toc complex, some preproteins might be transported through the IE via Tic20. On the contrary, Kikuchi et al.presented that Tic20 migrates on BN-PAGE at the same molecular weight as the imported precursor of the small subunit of Rubisco and that tic20-I mutants display a reduced rate of the artificial precursor protein RbcS-nt: GFP. The authors interpreted these results in a way that Tic20 might function at an intermediate step between the Toc translocon and the channel of Tic110. However, being a substantial part of the general import pathway seems unlikely due to the very low abundance of Tic20. It is feasible to speculate that such abundant proteins as pSSU, which are imported at a very high rate, may interact incidentally with nearby proteins or indifferently use all available import channels.

To clarify this question, substrate proteins and interaction partners of Tic20 should be a matter of further investigation. Additionally, a very recent study suggested AtTic20-IV as an import channel working side by side with AtTic20-I. However, detailed characterization of the protein and experimental evidence for channel activity are still missing.Plants have pre-formed and inducible structural and biochemical mechanisms to prevent or arrest pathogen ingress and colonization. These defenses include barriers such as papillae and ligno-suberized layers to fortify cell walls, and low-molecular weight inhibitory chemicals . Plants undergo transcriptional changes upon perception of microbe associated molecular patterns or effectors to induce local and systemic resistance. The oomycete MAMPs, arachidonic acid and eicosapentaenoic acid , are potent elicitors of defense. These eicosapolyenoic acids were first identified as active components in Phytophthora infestans spore and mycelial extracts capable of eliciting a hypersensitive-like response, phytoalexin accumulation, lignin deposition, and protection against subsequent infection in potato tuber discs . Further work demonstrated root treatment with AA protects tomato and pepper seedlings from root and crown rot caused by Phytophthora capsici, with associated lignification at sites of attempted infection . AA has been shown to induce resistance, elicit production of reactive oxygen species, and trigger programmed cell death in members of the Solanaceae and other families . Phaeophyta and Rhodophyta members contain numerous bioactive chemicals that can elicit defense responses in plants . The brown alga, Ascophyllum nodosum, is a rich source of polyunsaturated fatty acids, including AA and EPA, which comprise nearly 25% of its total fatty acid composition . A. nodosum and oomycetes belong to the major eukaryotic lineage, the Stramenopila, and share other biochemical features . Commercial extracts of A. nodosum, used in organic and conventional agriculture as plant bio-stimulants, may also help plants cope with biotic and abiotic stresses. A proprietary A. nodosum extract, Acadian , has been shown to provide protection against bacterial and fungal pathogens . Studies in A. thaliana showed ANE induced systemic resistance to Pseudomonas syringae pv. tomato and Sclerotinia sclerotiorum . Investigation into ANE-induced resistance in A. thaliana and tomato suggest the role of ROS production, jasmonic acid signaling, and upregulation of defense-related genes and metabolites .

As a predominant polyunsaturated fatty acid in ANE, AA may contribute to ANE’s biological activity. In a parallel study we demonstrated AA’s ability to systemically induce resistance and ANE’s capacity to locally and systemically induce resistance in tomato to different pathogens . Further, we showed that AA and ANE altered the phytohormone profile of tomato by modulating the accumulation of defense-related phytohormones . Through RNA sequencing,cultivar frambuesas this same study revealed a striking level of transcriptional overlap in the gene expression profiles of AA- or ANEroot-treated tomato across tested time points . Gene ontology functional analysis of transcriptomic data revealed AA and ANE enriched similar categories of genes with nearly perfect overlap also observed in categories of under-represented genes. Both AA and ANE treatment protected seedings from challenge with pathogens with different parasitic strategies while eliciting expression of genes involved in immunity and secondary metabolism. The shared induced resistance phenotype and extensive transcriptional overlap of AA and ANE treatments suggested similar metabolic changes may be occurring in treated plants. In the current study, untargeted metabolomic analyses were conducted to assess global effects of root treatment with AA and the AA-containing complex extract, ANE, on the metabolome of tomato plants. Fatty acid sodium salts were prepared and stored as previously described . AA stock solution was prepared by dissolving 100 mg of fatty acid salt in 1 mL of 75% ethanol. AA stock solution was subsequently stored in a glass vial at −20°C flushed with N2 to minimize oxidation. A proprietary formulation of A. nodosum extract was diluted with deionized water to a 10% working concentration, which was used to prepare treatment dilutions. All chemicals were diluted to their treatment concentrations with sterile diH2O. Hydroponically reared, 3-weekold tomato seedlings with two fully expanded true leaves were transferred to 1 L darkened treatment containers with their respective root treatment solutions. Following 24, 48, 72, and 96 hours of root treatment, tomato seedlings were removed from treatment containers, and leaves and roots were excised from shoots and flash frozen in liquid nitrogen. Each sample was the pool of roots or leaves of two seedlings with four replications per tissue, treatment, and time point. Samples were transported on dry ice and stored at −70 °C until metabolite extraction. The issue samples were ground in liquid nitrogen using a mortar and pestle and 100 mg was weighed and transferred to a 2-ml bead-beating tube containing four 2.8-mm ceramic beads. All tools and consumables were pre-chilled in liquid nitrogen. After weighing, each sample was removed from liquid nitrogen and kept at −20 °C until addition of extraction solution.One ml of extraction solution was added to each sample which was then vortexed, followed by bead-beating in a bead mill at a speed of 2.9 m/s for one 3-min cycle. After bead-beating, samples were centrifuged at 12k × g for 10 min at 4 °C . Samples were diluted 5-fold using extraction solution and filtered into LC-MS-grade HPLC vials using 0.22-μm PTFE syringe filters. HPLC vials were kept at 4 °C until LC-MS analysis. A blank was prepared by adding 1 ml extraction solution to a bead-beating tube containing beads that was processed equivalently to the samples. In addition, a quality control sample was prepared by combining 20 μl of each of the extracted samples and processed equivalently.Samples were analyzed via high performance liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry controlled by MassHunter software in centroid data mode. Mobile phase A was ultrapure water with 0.05 % formic acid and mobile phase B was acetonitrile with 0.05 % formic acid. Before starting the run, the column , equipped with a guard column , was conditioned for 20 minutes with 95 % mobile phase A and 5 % B. Column temperature was maintained at 40 °C.

The sample injection order was randomized, with individual samples being run consecutively in positive and negative mode. The quality control sample was injected at the beginning and end of the run, as well as after every 12 samples throughout the run to check signal and elution stability. Source parameters were as follows: drying gas temperature of 325 °C and 350 °C , drying gas flow 12 l/min, nebulizer pressure 35 psi, sheath gas temp 375 °C and 400 °C , sheath gas flow 11 l/min, capillary voltage 3500 V and 3000 V , nozzle voltage 0 V and 1500 V , fragmentor 125 V, skimmer 65 V, and octopole 750 V. Acquisition was performed over a mass range of 50 to 1700 m/z using the all-ions MS/MS technique, cycling three different collision energies at an acquisition rate of 3 spectra/s. Simultaneous infusion of a solution of purine and hexakisphosphazine using the reference nebulizer was used throughout the runs for mass calibration. Positive and negative mode raw data files from MassHunter were analysed separately in MS-DIAL before downstream analysis. Tolerances for MS1 and MS2 were set to 0.025 and 0.075 Da respectively . For peak detection, the mass slice width was set to 0.1 DA and the minimum peak height was set to 15,000 which was approximately 3 times the noise level observed in the total ion chromatogram. A linear weighted moving average method was used for peak smoothing, with a smoothing level of 3 scans and a minimum peak width of 5 scans. Deconvolution was performed with a sigma window value of 0.5 and an MS/MS abundance cutoff of 10. The adducts permitted were [M+H]+, [M+NH4]+, [M+Na] +, [M+K]+, [M+H−H2O]+, and [2M+H]+ in positive mode, and [M−H]−, [M−H2O−H]−, [M+Cl]−, [M+Na−2H]−, and [M+K−2H]− in negative mode.MS-DIAL data was cleaned in MS-CleanR in RStudio using the following parameters: minimum blank ratio of 0.8, maximum relative standard deviation of 30, minimum relative mass deffect of 50, maximum RMD of 3000, maximum mass difference of 0.05 and maximum retention time difference of 0.15.

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