ecogrow – Page 27 – Nature Ecrogrow

Rooted in Water: The Science and Art of Hydroponic Farming

Parathion also contains a thioester linkage that is analogous to the chemical structure of several chemical warfare agents, including sarin. Consequently, parathion is an excellent model for studying enhanced biodegrada–tion of environmental contaminants. Mineralization of parathion requires three unique catabolic properties: hydrolysis of parathion, mineralization of p-nitrophenol, and mineralization of diethyl thiophos–phate . The objective of this research was to develop a coculture capable of hydrolyzing parathion and degrading its metabolite p-nitrophenol; evaluate the kinetics of the reaction; and test the suitability of the coculture for use in a flow-through biofilm reactor for parathion bio–degradation.Escherichia coli strain SD2 was constructed by introducing plasmid pWM513, harboring the genes for parathion hydrolysis and ampicillin resistance, and plasmid pMAG1, carrying the green fluorescent pro–tein gene and tetracycline resistance, into Escherichia coli DH10B. The plasmids were inserted by electropo–ration and strain SD2 was selected using media containing both ampicillin and tetracycline. Strain SD2 was used together with Pseudomonas putida KT2440 carrying plasmid pPNP, harboring the genes for p–nitrophenol degradation and also tetracycline resistance. Strain KT2440 is naturally resistant to ampicillin, so the two strains could be cultured in media containing both antibiotics, and consequently maintained the genes required to degrade parathion. The ability of the coculture to degrade 0.5 mM parathion was evaluated during growth of the strains in a minimal medium containing glucose as the carbon source. P-nitrophenol accumulated in the medium during the growth of strain SD2 alone,drainage gutter resulting from parathion hydrolysis .

In contrast, the accumulation of p-nitrophenol was only transient in the coculture, as a result of the bio–degradation activity of strain KT2440. Kinetic analysis indicated that 2 mM p–nitrophenol was fully inhibitory to the growth of the coculture; consequently, this concentration represented the upper limit for parathion bio–degradation . The coculture was used to cultivate a biofilm in a par–allel plate flow cell for imaging by confocal microscopy. After 72 hours of growth in continuous mode, the biofilm was stained with a red fluores–cent nucleic acid dye and imaged using a confocal microscope. The dye caused the Pseudomonas strain to appear red, while the Escherichia coli strain appeared green and yellow as a result of the colocalization of green color fluorescing from its green fluorescent protein. The images indicated that the biofilm was dominated by Pseudomonas, although E. coli was stably maintained. The results suggest that the two strains could be used as part of a flow-through biofilm reactor for detoxification of parathion. Several industries in the United States and Puerto Rico are dedicated to the manufacture of filters of different materials, including nitrocellulose . These filters are very important in ensuring the purity of ingredients in food and pharmaceutical industries. However, the nitro–cellulose used for manufacturing these filters is considered a hazardous waste for its ignitability , high flammability and oxidizing properties. The Department of Defense also has sites contaminated with NC since it is a major component in explosives. Nitrocellulose exhibits good chemical stability, which for years made incineration the preferred method for dis–posing of the NC wastes. Alkaline hydrolysis appears to be a rapid process for nitrocellulose degradation, but research is limited. This investigation involves the eluci–dation of the mechanism of alkaline hydrolysis and its combination with biodegradation.Table 1 shows the research methods and tools employed in this study. Every step in Table 1 seems to play an important role for either hydrolysis or bio–degradation itself. Interpretation of the species left after degradation time facilitates analysis. Comparison between liquid and agar media is important to really determine if physical attachment is another factor for better fungus growth. Figure 1 shows a possible precipitation of products after acidification on the 9 mL NaOH treatment.

Figure 2 shows fungus growth from 10 mcL of spores suspen–sion placed on a petri dish containing NC as sole carbon source. HPLC assays have been made on a growth curve in a culture tube . A total of 0.1 g of NC hydrolyzed in 6 mL of SSC buffer was used. The strongest peaks are shown. The sample represents one-third of the total area. A total of 20 mcL were injected in a 50:50 acetonitrile:water mixture. Assays are being done using wavelengths of 210 nm and 214 nm, due to the abundance of probably dicarboxilic acids in the hydrolyzed NC.Polycyclic aromatic hydrocarbons are a con–cern in the environment because they are toxic and carcinogenic. Polycyclic aromatics are more recalci–trant in the soil than other hydrocarbons because they are hydrophobic and tend to migrate into the soil . Most PAHs occur as a result of fossil fuel combustion; thus, high concentra–tions of PAHs are found at the sites of active and inactive oil refineries . This study focuses on oil and petroleum contami–nated soil samples taken from Alameda Naval Air Station at Alameda Point, Calif. Pacific Coast Oil Works refinery used the site between 1879 and 1903. After the refinery closed in the 1930s, the U.S. Army and then the U.S. Navy owned the property. In 1991, jet fuel spilled from the jet engine test facility on the site. Heavy rains resulted in jet fuel in the overflow of storm drains. Damage to the storm drains during the Loma Prieta earthquake in 1989 may have caused ground water contamination. Recent studies showed total petroleum hydrocarbon amounts rang–ing from 100 to 10,000 mg/kg soil. Due to the long term and high concentration of oil in the soil it is likely that there are microorganisms capable of degrading the oil products. This study will examine the use of Inipol EAP 22, the same fertilizer used in the Exxon Valdez oil spill clean-up, to obtain optimal growth conditions of the naturally occurring bacteria using CO2 output to monitor the degradation of PAHs and hydrocarbons. Inipol EAP 22 is particularly attractive for this site since it is oleophilic and should make the strongly sorbed PAH components more bio–available, thereby stimulating bio–degradation.More than 8,000 Department of Defense sites need clean-up efforts .

TNT was the most commonly occurring compound within the contaminated samples from these sites. Fortunately, TNT is biodegradable, making in situ bio–remediation a cost–effective and rapid alternative for site cleanup. The toxicity of nitroarenes and their metabolites have been studied in a variety of biological systems but we have been unable to find any studies related to chemotaxis of nitroaromatic compounds in the scientific literature. Chemotaxis allows bacteria to respond to chemical gradients, seeking higher levels of potential nutrients and lower levels of inhibitors. Organisms that have developed mechanisms with which to beneficially orient themselves with respect to these gradients of different types may have a competitive advantage over other organisms. Also, the value of motility as a survival factor for bacteria in environments where nutrients or harmful agents are discontinuously dis–tributed, e.g., contaminated sites, seems obvious. The present study will target how bacteria can use nitroaromatic compounds as a source of nutrients and as a dispersal mechanism in soil,large square pots especially as it may apply to in situ bio–remediation.Chemotaxis provides a means for bacteria to respond to environmental gradients of potential nutrients and toxins, resulting in direct motility towards or away from these substances . The P. fluorecens bacterial strain used responded impressively according to our predictions . This is the first time that bacteria have been demonstrated to be attracted to explosive compounds. The strong attraction of soil bacteria to DNT was also verified using soil perfusion column leachate from field samples taken at Ft. Ord, Calif. In these leachate tests, the chemotactic indices observed for DNT are some of the highest rates ever observed for any type of chemotaxis, including the ones observed for P. fluorecens for concentrations of 2,4 and 2,6-dinitrotoluene that ranged from 0.12 M to 1.20×10-3 M. The results of this work have important implications for the ecology of TNT-degrading bacteria; it also may suggest ways that micro–environments containing explosives might be controlled to increase bio–degradation rates in situ.More than 8,000 Department of Defense sites need clean-up efforts . In a compilation of soil samples collected from 44 Army ammunition plants, arsenals and depots, 28% contained detectable levels of explosives. TNT was the most commonly occurring compound within the contaminated samples, and was seen in 66% of those samples. Facilities that may be contaminated with explosives include manufacturing plants, ordnance works, Army ammunition plants, ammunition depots, Army and Naval proving grounds, burning grounds, artillery impact ranges, explosives disposal sites, bombing ranges, firing ranges, and ordnance test and evaluation facilities.

Due to its toxicity and recalcitrance, the U.S. Environmental Protection Agency has listed TNT as a priority pollutant . Bio-remediation of organic contaminated soils has proven to be one of the fastest and cheapest remediation technologies available. TNT and its daughter products are highly recalcitrant, especially in highly weathered soils, i.e., soils that have been exposed to the environment for a number of years under sub–optimal microbial activity conditions. The present study examines weathered and unweathered soils and looks at the ability of a number of bio–stimulants to increase total microbial respiration.Two-gallon buckets were converted to soil bioreactors . These reactors use the same CO2 trapping principle as the bio–meter flasks, but allow for a much greater soil sample. The KOH trap was a funnel plugged at the bottom with a rubber stopper, which was held in place with epoxy. These traps rested on top of the soil, and could hold up to 80 ml of solution. Larger ascarite-top filters were made using 50 ml Corning vials. A hole was drilled into the bottom of each vial, through which a piece of plastic tubing would fit. This tube was connected to a stopcock, which let the filtered air into the bucket during the times when the KOH was changed. The attached diagram shows the bio–meter bucket design. The soil in each bucket was mixed well prior to the respiration measurements. After the bucket was sealed, the KOH was added to its trap, and the buckets sat for four days. In this four-day period, the KOH was changed and titrated as necessary, with intervals from an hour to a day, depending on the rate of respiration in the soil samples. These intervals change because a faster respiration rate can create enough CO2 to saturate the KOH solution. Therefore, the KOH monitoring a more active soil sample must be titrated much more often than in a slowly respiring sample. For each bucket, three experiments were run: an unamended control, where the soil was taken directly from the buckets in which it was collected, and lasting 3.57 days, an inorganic nutrient amendment, where 1000 ml of MSM was added to each bucket, and lasting 2.93 days, and an organic nutrient amendment, where an aqueous molasses solution was added to each bucket, and lasting 4.80 days. Respiration was measured daily and samples for HPLC analyses of contaminants were taken initially and at the end of the treatment.The respiration analysis indicates that unweathered soil responds faster and in greater magnitudes to nutrient amendments . The results suggest that the weathered soils may have stressed populations and cannot react as quickly to the amendments, especially because they actually reduce their respiration rate in response to MSM addition. The molasses-amended samples all respired at a greater rate than with the other amendments. This result implies that molasses may be used as an effective nutrient source to increase bacterial activity. Three of the four molasses-amended samples are among the lowest four in concentrations of the suspected contaminant located by the HPLC. The HPLC results also suggest a correlation between bacterial activity and bio–degradation. Glycerol trinitrate , also known as nitroglyc–erin, is extensively utilized for the production of explosives and pharmaceuticals. GTN is a hazardous waste product of increasing environmental concern. Current disposal techniques, such as open-air burning and incineration, are expensive and can produce hazardous waste byproducts. Bio–remediation systems could remediate explosive contaminants at approximately a tenfold lower treatment cost and with increased public acceptance. An ecological investigation of GTN-contaminated sites at the Naval Surface Warfare Station in Maryland resulted in the isolation of a Bacillus thuringiensis/cereus strain able to degrade GTN. HPLC and TLC analysis by other researchers of GTN metabolism in cell-free systems suggested that there was a sequential denitration to dinitrate isomers, mononitrate isomers and ultimately to glycerol.

From Water to Harvest: Exploring the Wonders of Hydroponic Agriculture

ABA is therefore necessary for the stomatal closure we observe in esb1-1. Te elevated ABA concentration we observe in leaves of esb1-1 compared to wild-type supports this conclusion. We also used the esb1-1sgn3-3 double mutant to test if SGN3 is involved in initiating this leaf ABA response. In leaves of the esb1-1sgn3-3 double mutant the elevated expression of a set of ABA signalling and response genes observed in esb1-1 is reduced to below that of wild-type . Further, the reduced stomatal aperture of esb1-1 is also recovered to wild-type levels in this double mutant . SGN3is therefore necessary for the ABA-dependent stomatal closure in response to the defective endodermal diffusion barrier in esb1-1. This raises the question of what links detection of a break in the endodermal diffusion barrier with ABA-driven closure of stomates in the leaf? Removal of endodermal suberin in esb1-1 expressing CDEF1 revealed a significant reduction in ABA-regulated gene expression, and a tendency to increasing stomatal aperture towards wild-type . Thus, increased suberin deposition in the endodermis of the esb1-1 root appears to play a partial role in the ABA controlled reduction in leaf transpiration. We have ruled out a role of local ABA signalling in controlling enhanced suberin deposition at the endodermis in esb1-1 . Using a similar strategy of expressing abi1 in the endodermis, in this case using the SCARECROW promoter , primarily active in the endodermis, we also show that in esb1-1 ABA signalling at the endodermis is not promoting stomatal closure or enhanced ABA signalling in leaves . We note that pSCR is also active in bundle sheath cell, and so ABA-signalling in these cells is also not involved in promoting stomatal closure in esb1-1. Furthermore,blueberry packaging containers enhanced ABA signalling in the endodermis is also not responsible for the initiation of the long-distance response of stomatal closure in leaves, and again it is more likely that suppression of ABA signalling is playing a role.

This can be seen in the fact that expression of abi1 in the endodermis, blocking ABA signalling, mimics the efect of esb1-1 on Lpr and stomatal aperture closure . However, these possibilities remain to be further explored. In contrast to these root-based or long-distance effects, the closure of stomata in leaves in response to a root-based CIFs/SGN3 derived signal is mediated by ABA locally in the leaves. We also note that the long distance signal connecting CIFs/SGN3 in roots with reduced leaf transpiration is currently unknown. Interestingly, a root-derived peptide has been recently identified as involved in long-distance signalling. In response to drought stress, CLE25 move from root to shoot and induces ABA accu-mulation in leaves and stomatal closure. Casparian strips have been suggested to play a critical role in forming a barrier to apoplastic diffusion to limit uncontrolled uptake and back fow of solutes from roots reviewed in . However, most Casparian strip mutants only appear to show fairly subtle phenotypic effects, and this has been a source of continued puzzlement. Here, we show that sensing damage to Casparian strips via leakage of the vasculature-derived CIF peptides from the stele into the cortex triggers a mechanism that inactivates aquaporins, promotes enhanced deposition of suberin limiting solute leakage in roots, and reduces transpiration in leaves, which all contribute to increasing solute concentration in the xylem . The overall outcome of this integrated response is a rebalancing of solute and water uptake and leakage. These physiological compensation mechanisms mitigate the loss of Casparian strip integrity, allowing relatively normal growth and development. A key part of this compensation mechanism is the ability of esb1-1 to limit water loss by the shoot by reducing stomatal aperture, in an ABA-dependent manner. This is clearly established by our observation that the esb1-1aba1 double mutant has severely reduced growth and seed production compared to either of the single mutants, and these growth defects can be partially supressed by the exogenous application of ABA .

The mechanisms we have identified are triggered by the loss of Casparian strips integrity. Such an event can occur during biotic stress including root nematodes infestation, and also during developmental processes such as lateral root emergence where Casparian strips are remodelled, suberin deposition occurs, and aquaporin expression is suppressed. Here, we describe novel outputs of the CIFs/SGN3 surveillance system that couple sensing of the integrity of the Casparian strip-based apoplastic diffusion barrier at the endodermis with pathways that regulate both solute leakage and hydraulic conductivity in the root . Long distance signals then connect these root-based responses with compensatory mechanisms in leaves which are mediated by local ABA signalling . Our dis-coveries provide a new framework which integrates our emerging understanding of the molecular development of the Casparian strip and suberin diffusion barriers with two of the major physiological functions required for plant survival – solute and water uptake.In recent years, California has tightened rules for reporting diversions of water for agriculture and other uses. One key challenge has been establishing workable standards for the collection of reliable data on relatively small and remote diversions — such as those for far-flung farms and ranches. Under new legislation, a certification program run by UC Cooperative Extension is helping to solve that problem. The State Water Resources Control Board views ac-curate diversion reporting as a key element of sound water management. “It’s incredibly important to monitor how much water comes into and goes out of the system,” says Kyle Ochenduszko, chief of water rights enforcement at the water board. Diversion reports are fed into a state database and support the orderly allocation of water resources by, for instance, enabling the board’s Division of Water Rights to inform water users when new requests to appropriate water might affect their own supply. Since 1966, the California Water Code has required diverters of surface water, with certain exceptions, to report their diversions to the water board. But in part because the water board lacked fining authority for many years, compliance was poor. In 2009, Senate Bill 8 gave the water board the authority to fine non-compliant diverters an initial $1,000, plus $500 for each additional day of failing to report.

Even so, SB 8 did not stipulate precisely how diversions were to be monitored. Rather, it required diverters to measure their diversions using the “best available technologies and best professional practices,” unless they could demonstrate that such technologies and practices were not locally cost-effective. That is, the requirement left wide latitude for interpretation. So things remained until 2015 — when Senate Bill 88 became law. This piece of legislation, passed amid a historically severe drought, directed the water board to draw up emergency regulations regarding water diversions. The regulations, once completed, required diverters of at least 100 acre-feet of water per year to hire an engineer or appropriately licensed contractor to install all monitoring devices. Now the requirements were clear. But the provision mandating installation by an engineer or contractor prompted an outcry from many smaller diverters, particularly those in remote areas of the state. For most diverters near sizable towns — Redding, say — complying with the regulations was manage-able, with expenses limited to the cost of a monitoring device and the services of an installer. But diverters in remote parts of Modoc County, for example, were looking at bigger bills, says Kirk Wilbur of the California Cattlemen’s Association. For such diverters, compliance might require importing an engineer or contractor from far away,blueberry packaging boxes which would entail significant travel expenses. If a site lacked electricity, as many do, the costs would pile higher . So how to reconcile the interests of the state’s diverters with those of the state? How best to balance the public and the private good? The answer, it turned out, was to empower diverters to install their own monitoring devices — with UCCE playing the empowering role. The idea originated with the Shasta County Cattlemen’s Association. It gained the support of the statewide Cattlemen’s Association. It took shape as proposed legislation in 2017 and was shepherded through the Legislature by Assemblyman Frank Bigelow . It breezed through both chambers with no votes in opposition — not even in committee. “All parties realized,” says Assemblyman Bigelow, “that Assembly Bill 589 would cut compliance costs and, as a result, increase compliance rates — which benefited both the regulators and the regulated community.” Essentially, AB 589 allows water diverters to in-stall their own monitoring devices if they successfully complete a monitoring workshop offered by UCCE. Further, it directed UCCE to develop the workshop in coordination with the water board. Khaled Bali, an irrigation water management specialist at the Kearney Agricultural Research and Extension Center, took the lead in drafting the coursework. “Then we met with the [water] board and got feedback,” Bali says. “We made changes until they said, ‘This looks good.’” Attendees at the workshops, which last three and a half hours, gain a solid foundation in the basic principles of diversion monitoring.

They learn how to monitor flows passing through a ditch, over a weir or through a pipe — or gathering in a pond. They learn how to build or install measuring devices appropriate for each type of diversion and how to calibrate those devices to comply with the state’s accuracy requirements. They learn how to navigate the water board’s rather detailed reporting system. Equipment for monitoring flows through open ditches might be limited to a tape measure, a timing device and a floating object. Installing a monitoring device for a diversion routed over a weir — a simple dam with an edge or notch that allows overflow — re-quires a bit more equipment. But once the installation is complete, the diverter need only read a staff gauge that shows the height of the water spilling over the weir’s crest . Diversions flowing through pipes must be outfitted with flow meters. Diversions feeding into a pond or reservoir can be monitored by tracking the depth of the water with a staff gauge, float or pressure transducer . So far, UCCE has offered the course in about 15 lo-cations, from Yreka to Bakersfield. According to Shasta County UCCE County Director Larry Forero — who teaches the $25 course along with Bali, Tehama County UCCE Advisor Allan Fulton and UC Davis–based UCCE Specialist Daniele Zaccaria — about 1,000 people had earned certificates of completion by early October. Even farmers and ranchers who divert less than 100 acre-feet per year are attending. “I’ve been floored,” says Wilbur, “by the number of diverters who have attended the course even though they aren’t required to — they want to better understand the regulations and make sure they’re doing the right thing.” It probably helps that the registration fee is a fraction of the cost of importing a faraway engineer. Due to their increasing use in a wide variety of beneficial industrial and consumer applications, ranging from use as a fuel catalyst, to chemical and mechanical planarization media, there have been increasing concerns about the potential environmental health and safety aspects of manufactured ceria nanomaterials.1,2 Ce is among the most abundant of the rare earth elements making up approximately 0.0046% of the Earth’s crust by weight .3 For example, Ce concentration in soils range from 2 to 150 mg kg−1 . 4 In Europe, the median concentrations of Ce were 48.2 mg kg−1 in soils, 66.6 mg kg−1 in sediment and 55 ng l−1 in water . There are many naturally occurring Ce containing minerals include rhabdophane, allanite, cerite, cerianite, samarskite, zircon, monazite and bastnasite.The existence of naturally occur-ring ceria nanoparticles is also likely and may play a key rolein controlling dissolved Ce concentrations,6 but precisely how the properties of naturally occurring ceria nanoparticles com-pare to manufactured ceria nanomaterials is unclear. There is concern that nanoceria, due to its small particle size and enhanced reactivity by design, may present unique hazards to ecological receptor species. Of critical importance are the redox properties of ceria which enables it to transition between CeĲIII and Ce, which are the key to understanding its potential toxicity.While there has been somewhat extensive investigation into the mammalian toxicity of ceria ,based on the present review, there has been considerably less effort invested into investigation of the environmental fate and effects of nanoceria. In this critical review, we discuss the likely points of environmental release along product life-cycles and resulting environmental exposure to nanoceria, methods of detection in the environment, fate and transport, as well as the available toxicity literature for ecological receptor species.

Disposal of TMV-contaminated materials poses low environmental risk

As batches move from the upstream portion of the facility every 3.44 days, the remaining time left over in the downstream is set as slack time in the model that may be dedicated toward repair, maintenance, etc. The assumptions and results developed in SuperPro were used to calculate the economics of the process described. Table 2 shows the total operating costs segregated individually for upstream and downstream components. Figure 5 displays process category cost contributions graphically, including percentages of total costs. In upstream operations, the largest cost components are utilities and labor , representing 61% and 32% of total upstream costs, respectively. In downstream operations, labor-dependent costs are the highest contributors at 30% of total downstream costs, followed by consumables at 27% of total downstream costs. Overall, the upstream component represents nearly 57% of the total Griffithsin production cost, which is calculated as just over $106/g protein. For a micro-bicide dose of 3 mg, the per-dose manufacturing cost is $0.32, excluding any CMO fee. An environmental health and safety assessment was also conducted for this case study following the method of Biwer and Heinzle and the results are found in Supplementary Tables 2–4 in Supplementary Materials. Overall, the process uses chemicals that are not harmful to people or the environment, as can be seen by the low magnitude of input and output Environmental Factor values in Supplementary Table 4. The biggest causes for concern are TMV in the residual biomass,grow bags for gardening and sodium hydroxide and phosphoric acid used in clean-in-place operations, if released to the environment; however we included costs for a thermal or chemical deactivation step for the TMV-contaminated biomass and pH neutralization for the acid and base cleaning agents which would eliminate the environmental impact of these components.

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

Ninety-five batches are seeded and grown annually, with one batch reaching harvest every 3.44 days. The downstream Griffithsin extraction and purification process is scaled up from the pilot industrial scale process presented by Fuqua et al. . An expression rate of 0.52 grams of Griffithsin per kilogram of biomass and a downstream recovery of 70% were used in the base case and give a combined yield of 0.370 grams of Griffithsin per kilogram of harvested biomass. Sterile filtration and CIP/SIP systems facilitate compliance with cGMP guidelines. Downstream processing commences upon the completion of an upstream batch and takes 39.3 h. The stable final formulation is >99% Griffithsin as the API with negligible endotoxin levels. In the model, the upstream costs account for nearly 57% of the total cost of Griffithsin production. Containing both upstream and downstream losses of the protein could significantly reduce COGS. Approximately 12% of the protein API is non-liberated from the homogenized biomass and 18% is lost during downstream polishing steps. Based on the data and assumptions employed in the current analysis, the unit production cost of Griffithsin is estimated to be $0.32 per dose . The model was based on published designs for a commercial scale facility and pilot-scale data on Griffithsin production adapted to the facility described. This type of modeling is useful for determining ranges of API selling price, production capacity and expression level requirements for commercial supply and profitability. In this study we modeled the manufacturing of Griffithsin through a contract manufacturing organization instead of a greenfield build of a new facility because we assumed that that would be the most prudent approach to launching a new product. If the product manufactured using the process modeled is used directly as a vaginal rinse or rectal enema, the additional costs post manufacturing would include transportation, storage, insurance, distribution, marketing, etc., none of which were modeled in this manufacturer-level analysis.

If the Drug Substance produced via the process analyzed is further formulated , or used as a component of another device , those costs and other product-specific costs would be additive and were also excluded from our manufacturer-level analysis. The cost of goods calculated by the current model reflects the manufacturer’s cost of production. We are less certain about the wholesale price of the drug because there is no standard “off- the-shelf ” profit margin that can be added to toll manufacturing cost to arrive at a standardized answer. Often scale up to commercial launch volumes of a product requires additional process development and optimization, validation batches, etc., which lead to negotiated transfer prices depending on volume, duration of engagement, license fees, export duties, and other factors, all of which would impact the cost of bulk Griffithsin. Nevertheless, for this discussion we assumed a manufacturer’s fee of 20% of COGS for a total production cost of bulk Griffithsin Drug Substance of $0.38/dose. Additive formulation, storage, distribution, insurance, marketing, sales margins and other costs could lead to a consumer-level use cost of $1-2/dose . This techno economic analysis emphasized Griffithsin’s use in microbicides because such products arguably represent the most price-constrained applications of this new drug. We cannot define the target retail price of a Griffithsin microbicide; there is no market reference price for micro-bicides since no commercial microbicides yet exist. For perspective,garden grow bags the user cost of a Griffithsin microbicide can be benchmarked against pre-exposure prophylaxis with traditional male condoms and PrEP with micro-bicides containing antiretroviral drugs as a newer alternative. Analyses have been conducted on the cost of prevention modalities and the cost savings to the healthcare system enabled by preventing HIV transmission, with prevention being far more cost effective than treatment in most scenarios . Walensky et al. conducted an analysis of the cost-effectiveness of a Tenofovir based PrEP micro-bicide in South African women. In their cost modeling of a vaginal gel, they multiplied the product cost of $0.32/dose times 2 and by 7.2 to arrive at a product use cost of approximately $5/woman month. However, the price of the microbicide gel used in the study was assumed and region-adjusted and hence pricing in other countries may be different. Terris-Prestholt et al. estimated Tenofovir gel prices of $0.25–0.33 per dose, provided that the gel was used in combination with a condom , from which an additive cost of use of $7–$12/person-month can be derived. Assuming the same average use rate of a Griffithsin containing microbicide applied singly without a condom and priced at $1.00–$2.00 per dose, the cost of use would be $7– <$15/person-month. Whether a higher cost of use discourages adoption of Griffithsin-based microbicides by men and women remains to be shown. A market study by Darroch and Frost of the Alan Guttmacher Institute consisted of detailed interviews of a cross-section of 1,000 sexually active women aged 18–44 in the continental United States. Their statistically rigorous survey identified levels and predictors of women’s concerns about STDs and interest in microbicides, as well as their preferences regarding method characteristics and likelihood of usage versus price of product, with survey sample results extrapolated to the national level.

The results showed that of the estimated 12.6 million women aged 18–44 interested in microbicides and concerned about STDs, including HIV, 11.5 million would still be interested in the method even if it were not 100% effective, and 11.0 million would remain interested even if the microbicide did not protect against STDs other than HIV. The same study found that women’s predicted use of a microbicide was affected by price, but interest was still high at $2 per application, or roughly up to 5-times the average price of a male condom. The survey concluded that more than seven million sexually active women in the USA would be interested in a vaginal microbicide even if the product only protected against HIV, was only 70–80% effective and cost them $2 per application . That conclusion was arrived at in 1999; the $2 per application cost back then would be $3.05 in 2018. One can conclude from these results that there is interest in effective yet inexpensive, self-administered HIV and STD prevention modalities even if such products might cost more than conventional prevention methods. The Darroch and Frost analysis was conducted nearly 20 years ago, and the interviews were limited to women practicing vaginal intercourse. To our knowledge, a more recent study linking likelihood of product use and price sensitivity has not been conducted, or at least not reported, to include other populations of potential microbicide users such as heterosexual couples practicing anal sex or gay men practicing unprotected rectal intercourse. Nevertheless, the 1999 study established an initial price point and price sensitivity for potential users of microbicides in the USA. Griffithsin has a broader spectrum of antiviral activity than HIV-specific PrEP agents, including activity against HSV-2 and HCV, which are co-transmitted with HIV-1 . Hence, Griffithsin might command a higher price due to its broader antiviral activity and its potential to obviate prevention and treatment costs for co-transmitted viruses. In the USA, the cost of the oral PrEP drug Truvada ranges from $1,300 to over $1,700 per month for the uninsured, but treatment is typically covered by insurance with user co-payments of $80–$150 per month. So even if a Griffithsin-containing microbicide sold for $5 per application , a user of 2 packs per month would pay $100 for the microbicide, which is in the range of PrEP, with the potential added benefit of controlling co-transmitted viruses. Consumers in wealthier economies might be receptive to microbicides costing $1–2 or even more per dose; however, consumers in lesser-developed economies might find $1–2/dose to be prohibitive. Hence, absent subsidies, there exists a continuing need to lower COGS for APIs such as Griffithsin. We can conclude that a COGS of <$0.40/dose of Griffithsin DS as determined in this study, and an estimated user cost of $1– 2/dose, might enable at least some simpler formulations of the drug to be economically marketed. For more complex formulations and delivery systems, or for higher doses of the drug, lower COGS for bulk Griffithsin would be desirable. The environmental assessment of the plant-based production of Griffithsin indicates low impact, particularly if the plant nutrient solutions are recycled in a hydroponic system and if waste streams containing TMV are treated in a biowaste heat or chemical treatment process. The assessment method used, although semi-quantitative, utilizes mass input and output stream data generated by SuperPro, along with independent assessment of compound toxicity and/or environmental impact , and allows comparison between alternative production strategies, process configurations or chemical components used in the manufacturing process.

Seeds were wrapped in foil and imbibed at 4°C for 4 days before planting

Since the negative short-term effects of Al on growth are apparently limited given that inhibition is alleviated without checkpoint function, conceivably these checkpoints have evolved to detect the minor strain on an individual’s genomic stability and serve to prevent transmission of Al-dependent genetic deffects to subsequent generations that ultimately would compromise the viability of the population. These checkpoints sacrifice the individual by halting the cell cycle in the root and forcing endore duplication to prevent an Al-dependent generational penalty with regard to heritable genomic integrity. Cells of the shoot meristem generate the floral reproductive organs of the plant, and thus the heritable genetic material. While the root is the most affected organ of the plant in the Al toxic response, Al is reallocated and sequestered to other regions of the plant body as Al-oxalate complexes can transport Al through the xylem from the roots to the shoots . In addition to a root hypersensitive phenotype, shoots of als3-1 grown in Al containing media display reduced cotyledon and leaf expansion, as well as a second shoot apex . Thus shoots are indeed affected by Al toxicity which could pose a threat to plant reproductive processes; therefore, it is plausible that Al-dependent root growth inhibition could serve as a means to preserve genomic integrity of the species. In all likelihood, it is plausible that Al induces an inappropriate activation of the DNA damage response by detecting Al-dependent pseudo-cross links. This could be the result of topological strain on the DNA and/or functional interference with Mg-dependent replication machinery,round nursery pots activating unnecessary repair of the DNA that may actually be the true detrimental effect of Al-dependent root growth inhibition.

Repair processes like nucleotide excision repair and nonhomologus end joining may be at work as shown by hypersensitivity to Al for loss-of function mutants in ALT2 and PARP1, PARP2 and KU80 respectively. The repair mechanisms could then result in subsequent damage inflicted on DNA, much of which may be related to double strand breaks that are regularly observed following Al treatment. The specific repair pathway activated in response to Al treatment is unknown, as well as whether or not this unknown process inflicts damage on the DNA during the repair. If indeed Al is acting to cause pseudo-cross links, it stands to reason that this effect would also inhibit proper DNA repair processes and could further cause damage to the DNA. As such, failure to activate this response pathway prevents the program-inflicted damage and results in roots that can grow normally in the presence of inhibitory levels of Al.While it is speculative at this time as to what the true nature of the effect of Al is on DNA integrity, an ATR-dependent DNA damage response pathway is clearly activated in the presence of internalized Al. Whatever the direct effect of Al is, based on the response factors, it can be speculated how these factors assemble and respond to Al stress based on their functional homology to related proteins: SUV2 likely aggregates at sections of persistent single stranded DNA coated by the heterotrimer, Replication Protein A, known to aggregate to single stranded DNA . SUV2 may act as a homodimer as it has been shown to bind to itself through its N-terminal coiled-coil domain as demonstrated by a yeast-2- hybrid . The proline-rich repeats at the N-terminus of SUV2 suggest a stiff elbow-hinge that could “wag” as a mechanism of recruiting other response factors to the locus.

It is possible that this homodimerization is controlled by phosphorylation of SUV2 since the dimerization domain has two SQ phosphorylation motifs associated with it. SUV2 would act to recruit ATR to this region of DNA in a manner similar to their homologues in yeast and mammals . The interaction of SUV2 and ATR with persistent single stranded DNA should induce autophosphorylation of ATR, and phosphorylation of SUV2 by ATR. It is possible that ATR will also be phophorylating other substrates, such as RPA2 and H2AX, to orchestrate additional responses. Once ATR has activated these sensors, likely in conjunction with other unidentified proteins, they would then induce the signal transduction pathway by activation of SOG1, again, likely through phosphorylation by ATR. Conserved serine-glutamine motifs are preferential ATM and ATR phosphorylation targets in mammals . SOG1 has five SQ motifs in the C-terminal transcriptional activation domain and SUV2 has two SQ motifs near the N-terminus, one of which is within the dimerization domain. Following activation of SOG1, expression of a group of genes is promoted, specifically genes tested from an established set of SOG1-mediated genes involved in a DNA damage response. This subset of Al-induced SOG1 mediated genes includes genes known to repair DNA e.g. BRCA1, RAD51, RAD17, GMI1, and PARP2 and halt the cell cycle e.g. CYCB1;1 as well as more transcription factors e.g. TRFL10,TRFL3, ANAC103 and WRKY25. Perhaps it is these transcription factors, as well as unidentified genes promoted as part of this response that are responsible in some unknown manner for a mechanism that forces a programmatic change in the root tip and QC, thus triggering this tissue to switch to endore duplication and causing terminal differentiation and permanent stoppage of growth of the primary root. While little is known about the placement of ALT2 within this signal transduction pathway, it likely functions as a scaffold protein, perhaps as part of a ubiquitin ligase signaling mechanism, acting analogously to other WD-40 proteins. WD40 repeat proteins are a class of proteins that are generally involved in mediating interactions between other proteins, associating with a variety of protein complexes, including E3 ubiquitin ligases . In eukaryotes, proteins are targeted for degradation via the ubiquitination-proteasome system, but ubiquitination also plays an important role in post-translational modification of proteins in the activation of signaling pathways.

As part of a crucial step in the DNA damage response pathway in mammals, following phosphorylation by ATM or ATR, one of the core histones of the nucleosome, γ-H2AX, is mono-ubiquitinated. This mono-ubiquitination is required for the recruitment of subsequent repair factors like BRCA1 and 53BP1 to both double and single stranded DNA breaks . In plants, CULLINs , which are part of a family of scaffolding proteins, form the largest family of E3 ligase complexes. Arabidopsis proteins containing WD40 domains, including ALT2, are proposed to be capable of interacting with the DDB1-CUL4-ROC1 complex . The recruitment structures and mechanisms are not well understood for CULLIN based ubiquitination signaling; however,plastic flower pots CUL4 has been shown to form a complex with DW40 proteins in response to UV damage that is ATR dependent . This establishes a potential link between ATR and ALT2 in a DNA damage response where resulting cross links would cause a replication fork stall, as Al likely causes.A model for stoppage of root growth following chronic exposure to Al can be developed in accordance with current evidence. In this model, Al impacts DNA in a currently unknown way, likely from a pseudo-cross linking effect resulting in a replication fork stall. Based on the genetic factors responsible for activating the Al dependent DNA damage response, it is a reasonable prediction that such an interaction would hold DNA in a conformation that inhibits replication fork progression. Regardless of the physical consequences of Al on DNA structure or integrity that have yet to be determined, the predicted genotoxic effects of Al are clearly sufficient to activate an ATR-, ALT2-, SOG1- and SUV2-dependent cell cycle checkpoint mechanism as demonstrated by the increase in Al tolerance seen for each loss-of-function mutant. This mechanism functions to promote transcription of a group of genes related to halting the cell cycle and to repair the perceived damaged DNA. Furthermore, it is likely that additional genes are included in this transcriptional response that are related in some unknown manner to a mechanism that forces a programmatic change in cells of the root tip and especially the QC. These genes would trigger cells to differentiate, losing their meristematic identity by switching from a normal cell cycle progression to endore duplication. Ultimately, it is this terminal differentiation that permanently stops growth of the primary root as the primary cause of Al toxicity. While significant work remains to be done, especially in determining the genotoxic consequences of Al that activate this DNA damage response pathway and developing a transcriptional profile of SOG1 targets that lead to inhibited root growth following Al treatment, it is clear that terminal differentiation of the root tip following chronic exposure to Al is an active event mediated by the DNA damage checkpoint factors ATR, ALT2, SOG1 and SUV2. Our understanding of the genomic consequences caused by Al is still in the beginning stages, and more work is needed.

Continued testing of DNA damage response mutant responses to Al can give us the opportunity to further elucidate how genomic maintenance factors are involved in this biological problem. In addition to the value of gaining a better understanding of the role of DNA damage response factors and cell cycle checkpoints in mediating Al-dependent DNA damage, Al toxicity represents a novel and biologically relevant model for studying ATR dependent mechanisms in the DNA damage response in general.For all growth experiments, seedlings were surface sterilized, vernalized, and etiolated before planting. Seeds were immersed in 70% ethanol and then washed 4 times with sterile water. Seeds were then immersed in 50% bleach for 5 minutes, after which seeds were washed 4 times with sterile water. The AlCl3 soaked gel environment was sterilely prepared by pouring a lower gel layer consisting of 80 mL of nutrient medium plus 0.125% gellan gum in Nunc Lab-Tek Extra-Depth Polystyrene Dishes 100 x 25 mm . Nutrient medium consisted of 2 mM KNO3, 0.2 mM KH2PO4, 2 mM MgSO4, 0.25 mM 2SO4, 1 mM Ca2, 1 mM CaSO4, 1 μM MnSO4, 5 μM H3BO3, 0.05 μM CuSO4, 0.2 μM ZnSO4, 0.1 μM CaCl2, 0.02 μM Na2MoO4, 0.001 μM CoSO4, and 1% sucrose. Al was introduced by overlaying the solidified lower layer with 20 mL of “soak solution” containing the proper concentration of AlCl3. Trail soak solution was made consisting of the nutrient solution medium described above, while only brought to 90% of the intended volume. 50 mL trail solutions were made consisting of 45 mL the slightly concentrated nutrient medium, X mL 25mM AlCl3, Y μL 0.1 N KOH and Z mL diH2O . The trail soak solution was made to determine the amount of 0.1 N KOH to use to adjust the pH of the nutrient soak containing AlCl3. The amount of base to add was determined empirically by adjusting the pH on an aliquot of the soak solution containing AlCl3. The amount of base determined from this trial soak solution was added to the actual soak solution prior to adding AlCl3. The sterilized soak solution was allowed to equilibrate with the lower layer for 2 days and was then poured off. This method was used for all concentrations of AlCl3 for plants grown in a gel soaked environment. In hydroponics experiments, Al-screening media was sterilely prepared as above without gellan gum and AlCl3. Seeds were sowed on 250-μm mesh, polypropylene screen in Parter Medical Products Quad Perti Dish 100 X 15 mm . After 6 days of growth unless otherwise specified, screens were transferred to new Al screening media supplemented with either 0 μM, 25 μM AlCl3 or 50 μM AlCl3. For treatment with hydroxyurea , mitomycin C , bleomycin , or cisplatin were added to plant nutrient media plus sucrose . Seeds were sowed and allowed to grow for seven days, after which roots were measured. For experiments on plant nutrient media plus sucrose , the medium consisted of 5 mM KNO3, 2.5 mM KH2PO4, 2 mM MgSO4, 2 mM Ca2, 50 μM FeEDTA, 1 μM MnSO4, 100 nM CaCl2, 100 nM CoSO4, 5 nM H3BO3, 50 nM CuSO4, 20 nM NaMoO4, 0.8 M Sucrose, 0.8% agar. Plants were grown in 24-hour light at 20°Cin I-36LLVL biological incubator . After one week, plants were repotted in Sunshine Special Blend potting soil with controlled release fertilizer, 15-9-12 + minors . Plants were grown in 24- hour continuous light at 22°C in a plant growth room with Sylvania Gro-Lite fluorescent bulbs until maturity.

Determination of the nature of Al genotoxicity is currently at the initial stages of investigation

While defining Al responsive mechanisms is suggestive of the type of damage Al causes, it is crucial to define the genotoxic consequence of Al that triggers this response, whether real or perceived. Additionally, unanswered questions also persist regarding which factors participate in conjunction with ATR and ALT2 to respond to Al-dependent damage since clearly Al responsive stoppage of root growth is a multi-step process progressing through cell cycle arrest to terminal differentiation associated with endore duplication or DNA replication without cytokinesis. Therefore, not only is further identification of Al tolerance factors crucial to our understanding of Al response signaling, it is also of critical importance to determine how these factors function together to promote Aldependent cell cycle arrest causing terminal differentiation and subsequent plant growth inhibition. While the genotoxic consequences of Al in plants have yet to be elucidated, it is clear from our als3-1 suppressor mutagenesis approach that components of the DNA damage response machinery are mediating root growth inhibition in response to the likely negative impact of Al on DNA structure, integrity,flower display buckets or conformation. The identification of ATR and ALT2 as mediators of Al-dependent root growth inhibition presents new strategies and opportunities to engineer crop species capable of growing in Al toxic soils.

Based on the critical roles of ATR and ALT2 in mediating Al-dependent root growth inhibition and the current knowledge gained from the field of plant DNA damage, further studies need to be done to identify mediators and effectors within the ATR- and ALT2-mediated Al response pathway, especially those responsible for controlling cell cycle arrest, damage repair, and subsequent promotion of endocycling at the root apical meristem to stop root growth. Our understanding of the genomic consequences caused by Al is still in the beginning stages, and more work is needed. Continued testing of DNA damage response mutant responses to Al can give us the opportunity to elucidate further how genomic maintenance factors are involved in this biological problem. In addition to the value of gaining a better understanding the role of DNA damage response factors and cell cycle checkpoints in mediating Al-dependent DNA damage, Al toxicity represents a novel and biologically relevant model to study ATR dependent mechanisms in the DNA damage response in general.In previous studies, seeds of als3-1 were mutagenized with ethyl methanesulfonate and M2 seedlings were screened for roots capable of sustained growth in the presence of 0.75 mM AlCl3 in a soaked gel environment. Identified seedlings were rescued and allowed to set seeds, after which progeny were rescreened to identify bona fide als3-1 suppressors. In order to further study Al-dependent terminal differentiation of the Arabidopsis root, an als3-1 suppressor mutant was chosen from this screen that was capable of sustained root growth in comparison to als3-1 in the presence of a range of AlCl3 concentrations for further analysis. Subsequent work showed this mutant to be an allele of SUPPRESSOR OF GAMMA RESPONSE1 .

Since six mutant alleles have been previously published , this suppressor will be referred to as sog1-7.The sog1-7;als3-1 double mutant was studied further to determine if the als3- 1 suppression resulted from increased Al resistance or tolerance. Internalization of Al has been associated with deposition of the β-1,3-glucan, callose, in the plasmodesmata between cells in the root tip . Seedlings of Col-0 wild type, als3-1, and sog1-7;als3-1 were grown hydroponically for 6 days in 0 μM AlCl3 , and then exposed to either 0 or 25 μM AlCl3 . This is a concentration that causes inhibition of wild-type root growth in hydroponic growth conditions for 24 hours. After these treatments, the seedlings were stained with Aniline Blue to detect callose with the use of fluorescent microscopy. Roots of sog1- 7;als3-1 accumulated callose similarly to both Col-0 wild type and als3-1 which is consistent with plants being tolerant to internalized Al . This suggests that while callose deposition is correlated with Al toxicity and has been suggested to be integral to Al dependent stoppage of root growth, it may not primarily responsible for Al inducible growth inhibition . To determine whether sog1-7;als3-1 showed Al-responsive gene expression, it was tested whether sog1-7;als3-1 showed increases in transcripts known to be induced following Al exposure, as would be expected for enhanced Al tolerance rather than increased Al exclusion. For this experiment, seedlings of Col-0 wild type, als3-1, and sog1-7;als3-1 were grown hydroponically for 6 days, after which seedlings were exposed to 0 or 25 μM AlCl3 for 24 hours. Following this, roots were collected and total RNA was isolated for a Northern analysis with the Al-inducible probes ALS3 and ALMT1 . When grown in the presence of Al, Col-0 wild type, als3-1, and sog1-7;als3-1 all resulted in an increase in expression of both Al-responsive genes.

This further supports characterization of sog1-7 as an Al tolerant mutation, as sog1-7 suppresses the hypersensitivity of als3-1 following the internalization of Al . To quantify total Al that accumulated in the root tissue of Col-0 wild type, als3-1, and sog1-7;als3-1, dried and ashed root tissue was subjected to inductively coupled plasma-optical emission spectrometry . For this experiment, seedlings were grown hydroponically for 6 days in the absence of Al, after which roots were exposed to 0 or 50 μM AlCl3 for 24 hours. Root tips were subsequently harvested, washed with nutrient medium, dried, and then ashed in pure HNO3 in preparation for analysis. All Al-treated root samples showed significant and equivalent accumulation of Al, demonstrating that the sog1-7 mutation is not excluding Al internalization to suppress the als3-1 hypersensitive response . Considering the quantitative uptake of Al by root tissue along with callose deposition and Al-inducible gene expression, evidence supports that the sustained root growth for the sog1-7;als3-1 mutant is due to enhanced Al tolerance rather than Al exclusion from the root tip.A map-based cloning approach was used to identify the nature of the als3-1 suppressor mutation. For this, the als3-1 line carrying the suppressor mutant in the Col-0 background was crossed to an als3-1 line that had been introgressed into the La-0 background . Because of the recessive nature of the als3-1 suppressor mutation, F2 progeny from the cross were grown on gel plates soaked with 0.75 mM AlCl3 ,flower bucket and seedlings with roots that were capable of sustained growth were rescued. Following isolation of genomic DNA, PCR-based analyses were conducted and showed that the als3-1 suppressor mutation localized to the top arm of Arabidopsis chromosome 1 . Fine mapping resulted in a genetic window that allowed identification of candidate genes for sequence analysis. The als3-1 suppressor mutation was subsequently found to be in exon 4 of At1g25580, which was previously reported as the ATM-regulated transcription factor SOG1 that is responsible for initiation of endoreduplication following exposure to DNA damage agents . The als3-1 suppressor mutation represents an amino acid substitution in the predicted NAC domain of this NAM , ATAF1/ATAF2, CUC family transcription factor . To confirm the suppression of the als3-1 phenotype is caused by a single base change in At1g25580, functional complementation was subsequently performed using a full-length genomic SOG1 construct that was previously reported . Seedlings of Col-0 wild type, als3-1, sog1-7;als3-1, and sog1-7;als3-1 carrying a wild-type genomic version of SOG1 were grown in the presence of 0.75 mM AlCl3 in a soaked gel environment. After 7 days of growth, roots were assessed for terminal differentiation. Introduction of a wild-type genomic version of SOG1 into sog1-7;als3-1 fully restored Al hypersensitivity to sog1-7;als3-1, as demonstrated by the transgenic root being terminally differentiated in a manner indistinguishable from Al-treated als3-1 . While there have been six previously published alleles of sog1, only one allele has been maintained since publication, sog1-1 .

This sog1 mutant represents a single amino acid mutation from R to G at residue 155 of SOG1. In order to determine if this allele could also suppress the als3-1 phenotype, the double mutant, sog1-1;als3-1 was generated. Subsequently, seedlings of Col-0 wild type, als3-1, sog1-1;als3-1 and sog1-7;als3-1 were grown in the presence of 0.75 mM AlCl3 in a soaked gel environment. After 7 days of growth root tips were assessed for terminal differentiation. The sog1-1 allele was capable of suppressing the extreme Al response of als3-1 in a manner indistinguishable from sog1-7, since both sog1-1;als3-1 and sog1-7;als3-1 failed to exhibit the severe root growth inhibition seen for Al-treated als3-1 . To analyze sog1-7 roots growth without the als3-1 mutation in the genetic background, sog1-7 was backcrossed to Col-0 wild type, and homozygous sog1-7 F2 progeny were identified by PCR analysis. Col-0 wild-type and sog1-7 seedlings were then grown for 7 days in the absence or presence of increasing concentrations of AlCl3 in a soaked gel environment, after which root lengths were measured. In the absence of als3-1, the sog1-7 mutant roots showed greater growth than wildtype roots in the presence of a range of normally highly inhibitory levels of AlCl3 . This indicates that SOG1 has a prominent role in actively halting root growth following Al treatment. To determine if SOG1 expression is regulated by Al, real-time PCR analysis was performed. Col-0 wild-type seedlings were grown in a hydroponic environment for 6 days and subsequently treated with 0, 25, or 100 μM AlCl3 for 24 hours. Root tissue was collected, total RNA was isolated for cDNA synthesis and RT-PCR was performed using SOG1 specific PCR oligonucleotide primers . There was no indication that SOG1 is transcriptionally induced by Al. Similarly, seedlings of sog1-7 were grown in a 0 μM AlCl3 hydroponic environment for 7 days and subsequently analyzed with real-time PCR and the sog1- 7 mutation was not found to affect transcript stability since Col-0 wild type and sog1-7 showed comparable levels of SOG1 transcript . In previous studies, loss-of-function mutations in cell cycle checkpoint factors ATR and ALT2 resulted in increased root growth in the presence of Al . This tolerance was correlated with failure to arrest cell cycle progression in conjunction with forced quiescent center differentiation. In order to determine if this is also true for roots of a sog1 loss of-function mutant, sog1-7 was crossed to either a transgenic Arabidopsis line carrying a reporter for cell cycle progression, CYCB1;1:GUS , or a reporter for QC status, QC46:GUS . Seedlings of Col-0 wild type and sog1-7 carrying the CYCB1;1:GUS reporter were grown in the absence or presence of 0.75 mM AlCl3 in a soaked gel environment for 7 days, after which they were stained for GUS activity. Col-0 wild type carrying the CYCB1;1:GUS reporter results in a substantial increase in GUS activity following exposure to Al . This is consistent with a large number of root cells being incapable of exiting the G2 phase of mitosis and incapable of proceeding into actual cell division. Unlike previous reports for the atr-4 and alt2-1 loss-of-function mutations where GUS reporter levels were eliminated , sog1-7 carrying CYCB1;1:GUS had substantially reduced levels of the CYCB1;1:GUS reporter compared with Col-0 wild type . This may indicate that the role of SOG1 in Al-dependent inhibition of cell cycle progression at the G2 phase, while likely acting in conjunction with ATR and ALT2, may also function through other factors to prevent CYCB1;1 turnover. Consistent with prior results, it was found that Al treatment results in loss of the QC as measured by QC46 dependent GUS activity that is localized to the root stem cells . For this analysis, QC46:GUS transgenic seedlings in either the Col-0 wild type or sog1-7 backgrounds were grown for 7 days in the absence or presence of 1.50 mM AlCl3 in a soaked gel environment, after which seedlings were stained to visualize the QC. Both Col-0 wild-type and sog1-7 roots had QC46:GUS accumulation in the absence of Al . Treatment with high levels of Al in Col-0 wild type resulted in the loss of the QC, but not in sog1-7 . This indicates that SOG1 plays an active role in differentiation of the QC following Al treatment and likely functions as a step in the transition to endore duplication in the root tip.In support of this model, it was found that Al treatment leads to terminal differentiation in conjunction with substantial increases in cell and nucleus size in als3-1 roots. For this analysis, Col-0 wild- type, als3-1, atr-4;als3-1, alt2-1;als3-1, and sog1-7;als3-1 plants were grown in the absence or presence of 0.75 mM AlCl3 in a soaked gel environment for 7 days, after which seedlings were fixed and stained with 4’,6-diamidino-2-phenylindole .

Peptide treatments were performed in the morning and collected at the indicated times

The larger genome size of S. habrochaites suggests the possibility that the putative loss of function of genes and/or genetic elements in S. lycopersicum may be due to deletions or non-functional null mutations. Matsuba et al. sequenced a functional gene cluster for terpene biosynthesis on chromosome 8 of S. habrochaites acc. 1778 and identified several rearrangements, deletions, and a novel gene when compared to the same gene cluster on chromosome 8 of the S. lycopersicum reference genome. Our prior research suggests that the inability of cultivated tomato to maintain shoot turgor under root chilling is the result of a loss of function in S. lycopersicum . Taken together, the current evidence suggests that the S. habrochaites allele for high-resolution mapped QTL stm9 may not be completely syntenic to S. lycopersicum, and that it may not contain the same genic compliment as the S. lycopersicum allele for stm9. Therefore, although the S. lycopersicum genome sequence is helpful in identifying potential candidate genes for shoot turgor maintenance under root chilling, the genomic sequence of the stm9 region of S. habrochaites is necessary for accurate, well-informed candidate gene identification.Stability of QTL expression for tolerance to abiotic stresses is important for successful deployment of stress tolerance QTL in breeding crop plants. Although a significant Genotype × Season interaction was identified for QTL stm9,procona flower transport containers the potential causes of the interaction suggest that this region would likely be useful as a stable source of root chilling tolerance for breeding. A number of other QTL have been identified as targets for breeding despite a significant Genotype × Season interaction in several species, including barley, rice, and maize .

The phenotypic plasticity likely contributed by the stm9 flanking regions suggest that any future breeding strategies should be undertaken with the smallest introgression possible that still contains the entire high-resolution mapped QTL stm9. The S. habrochaites introgression in sub-NIL C7 contains only the high resolution QTL stm9 region . This sub-NIL was grouped as tolerant in both the Spring and Fall datasets, and gave a consistently low stmscore in both seasons , suggesting it may serve as a suitable potential donor parent source of tolerance to root chilling in breeding programs. Due to the complexity of the abiotic stress response pathway, it is unlikely that the S. habrochaites QTL stm9 allele contains only a single gene conferring shoot turgor maintenance under root chilling. Single causal genes have been identified for a number of major QTL , but other major QTL have been shown to be controlled by two or more causal genes or polymorphisms . Identification and testing of the causal gene or polymorphisms underlying QTL stm9 for tolerance to root chilling will be an important step in the identification of genetic targets for improving stress tolerance of plants exposed to root chilling and other types of water stress through marker assisted breeding. Determination of the gene/polymorphisms responsible for a quantitative trait phenotype is facilitated by genomic approaches . Once a target region is identified via high-resolution mapping, a combination of genomic sequencing, structural genomic analysis, and transcriptome profiling can be used to assist in the identification of candidate genes. Therefore a biologically informed ranking of candidate genes located within the QTL stm9 region will require a combination of S. habrochaites genome sequence for this region as well as transcription profiles for susceptible and tolerant subNILs exposed to root chilling.

It is hoped that a better understanding of the underlying mechanism for tolerance to rapid-onset water stress in wild tomato S. habrochaites may aid in the identification of chilling tolerance genes in other species of tropical and sub-tropical origin.Plant immunity is mediated, in part, by cell surface immune receptors that recognize molecules produced by microbes. For example, the Arabidopsis FLS2 and EFR receptors recognize the flg22 peptide derived from bacterial flagellin and the elf18 peptide derived from elongation factor thermounstable protein, respectively . The rice XA21 receptor recognizes the sulfated RaxX peptide derived from the RaxX protein produced by Xanthomonas oryzae pv. oryzae. XA21, EFR, and FLS2 all contain extracellular leucine rich repeat , transmembrane, and intracellular non-RD kinase domains. These receptor domains are partially interchangeable. For example, the LRR domain from EFR can be fused to the transmembrane and intracellular domain of FLS2 to form a chimeric receptor that responds to elf18 treatments when transiently expressed in Nicotiana benthamiana and Arabidopsis thaliana . The EFR LRR can be fused to the transmembrane and intracellular domain of XA21 to form achimeric receptor that responds to elf18 treatment and confers partial resistance to Xoo in transgenic rice lines . The availability of rapid and reliable assays that measure markers characteristic of immune response activation can help facilitate investigations of innate immune signaling. For example, immune signaling studies of FLS2 and EFR in Arabidopsis have been aided by the availability of rapid and reliable assays . In contrast, studies of the XA21-mediated immune response have been limited by the lack of rapid assays and well-characterized genetic markers. Typically, disease assessments are carried out by measuring lesions on rice leaves or by assessing bacterial populations from infected leaves . In this study we aimed to establish a rapid and efficient assay to monitor the XA21- mediated immune response after bacterial infection. For this purpose, we employed the EFR:XA21:GFP chimera composed of the EFR extracellular domain and the XA21 transmembrane and intracellular kinase domains, tagged with green fluorescent protein .

EFR:XA21:GFP transgenic rice plants are partially resistant to Xoo and detached EFR:XA21:GFP leaves respond to elf18 with stress related gene induction, mitogen-activated protein kinase cascade activation, and reactive oxygen species production . These results indicate that plants expressing the EFR:XA21:GFP chimeric protein are appropriate for studies to identify markers of resistance. We used RNA sequencing to identify genes differentially regulated in elf18 treated EFR:XA21:GFP rice. We then assessed if differentially regulated genes in elf18 treated EFR:XA21:GFP rice leaves were up-regulated in Xoo infected rice leaves expressing full-length XA21, which are resistant to Xoo. We developed a rapid and reliable assay to analyze gene expression in detached rice leaves inoculated with Xoo. We identified 8 DRGs from elf18 treated EFR:XA21:GFP rice that are also specifically up-regulated in detached XA21 rice leaves infected with Xoo.For peptide treatments, wild type Kitaake and progeny from line EFR:XA21:GFP-3-8 Kitaake rice leaves were harvested from plants grown in the greenhouse for 4.5 weeks . 1.5–2 cm leaf sections were collected from expanded adult leaves using surgical grade scissors. Thissue from the leaf base and leaf tip was discarded. Detached leaves were equilibrated overnight in 6-well Costar cell culture plates under constitutive light . For bacterial inoculations, we used detached rice leaves harvested from 4-week old plants grown using a hydroponic growth system as described previously under a light intensity of 280 µmol/. Freshly harvested leaves from Kitaake and Ubi-Myc:XA21 Kitaake rice were cut into 1.5–2 cm pieces and immediately floated on 10 mM MgCl2 solution for mock treatments or 10 mM MgCl2 containing fresh Xoo cell suspensions at O.D.600 of 0.1 . The samples were left overnight under constitutive light and collected 24 h post-inoculation . Leaves were floated on approximately 1.5 mL Xoo cell suspension media in 6-well Corning Costar cell culture plates . The detached leaf infection assay allows a more uniform distribution,procona valencia compared to the scissor inoculation method , of Xoo inoculum by floating leaves on bacterial suspensions. Detached leaves were frozen in liquid nitrogen and powdered using a Qiagen tissue lyser. For tissue from greenhouse grown plants, RNA was extracted from powdered tissue using TRI Reagent and precipitated with isopropanol. For tissue from hydroponically grown plants, RNA was extracted using the Spectrum Plant Total RNA Kit from Sigma-Aldrich. RNA was DNase treated using the TURBO DNase kit from Life Technologies. RNA concentrations were normalized to the lowest sample concentration in each experiment. cDNA was synthesized from 2 µg of total RNA using the High Capacity cDNA Reverse Transcription Kit by Life Technologies. Gene expression changes were determined by 11 Ct method normalized to Actinand compared to mock treated samples. Plant growth, leaf tissue isolation, and treatments were performed as described above. RNA was isolated from untreated Kitaake as well as untreated and elf18 treated EFR:XA21:GFP leaf tissue using the Spectrum Plant Total RNA Kit from Sigma-Aldrich and on-column DNase treated to remove genomic DNA contamination following the manufacturer’s instructions. RNA was quantified using the Quant-IT Ribogreen RNA Assay Kit. Sequences were deposited to the NCBI Sequence Read Archive . RNAseq libraries, sequencing, and reference alignment were performed as described previously . Sample correlation between Kitaake and EFR:XA21:GFP replicates and differential gene expression analysis was performed using the Bioconductor ‘edgeR’ package for R .We generated a PXO99A1 hrpA1 mutant in Philippine race 6 strain PXO99Az, a derivative of strain PXO99 . Xoo was grown in 10 g PSB , 10 g Sucrose, 1 g sodium glutamate , final volume 1L, pH 7.0 or on PSA plates at 28 ◦C.

PXO99A1 hrpA1 was generated by single crossover mutagenesis using the suicide vector pJP5603 . An approximately 500 base pair sequences within hrpA1 was amplified using forward primer 50 -CGGGGTACCGTGCTGCGTGATTTGTCCG-30 and reverse primer 50 – CGCGGATCCTGACTTGGTCGATGCAGTCC-30 and cloned into the multiple cloning site of pJP5603 using the restriction enzyme sites KpnI and BamHI. PXO99A-competent cells were transformed with the suicide plasmids by electroporation and plated to PSA with kanamycin . PXO99A1 hrpA1 colonies with kanamycin resistance were screened by PCR for colonies with single crossover events, which contain the vector disrupting the target gene. PXO99A1 raxST and PXO99A1 raxST complemented strains used in this study were described previously . PXO99A1 raxST evades XA21- mediated immunity while the complemented PXO99A1 raxST strain does not.We analyzed the transcriptomic profile of EFR:XA21:GFP rice lines treated with elf18 to identify genes differentially regulated during this response. We sequenced cDNA from EFR:XA21:GFP leaves treated with 500 nM elf18 for 0.5, 1, 3, 6, and 12 h. We also included untreated EFR:XA21:GFP and Kitaake as controls . Multidimensional scaling of pairwise biological coefficient of variance comparisons for each sample revealed that replicate samples group together . This grouping of biological replicates demonstrates the overall transcriptional similarity between each sample . We identified 2,212 genes that were differentially regulated in EFR:XA21:GFP rice treated with elf18 compared with untreated samples. Using a false discovery rate cutoff of 0.05 and absolute expression log fold change of 2 or greater, we previously reported that the transcriptomic profile of untreated Kitaake compared to untreated EFR:XA21:GFP did not differ significantly . Over the treatment time course, we identified 2,212 DRGs using untreated EFR:XA21:GFP at 0 h as a reference. The number of DRGs that overlap between the elf18 treatment time points are summarized in Fig. 1B and File S1. Of the 2,212 differentially regulated genes, there were 1,420 up-regulated and 792 down-regulated genes. The highest number of DRGs was observed 6 h post elf18 treatment. These results show that elf18 treated EFR:XA21:GFP rice express a substantially different set of genes over time compared to untreated samples. To examine the types of biological processes affected in elf18 treated EFR:XA21:GFP rice, we analyzed GO term enrichment of DRGs using the AgriGo analysis tool . A total of 1,204 out of 1,420 of the up-regulated DRGs and 682 of the 806 down-regulated DRGs had GO annotations. An FDR of 0.05 or less was used to define significantly enriched terms compared to the Michigan State University annotation reference as calculated by the AgriGo tool . Fig. 1C and File S2 summarize the most enriched GO terms in each of the three major DRG clades. Clade 1 contains 1,333 genes that are mostly up-regulated over time. Genes from clade 1 are enriched for metabolic process , response to stimulus and response to stress GO terms . Clade 2 genes are up-regulated across all time points and are enriched for secondary metabolic process , metabolic process and response to stress GO terms . Clade 3 consists of 757 genes that are mostly down-regulated in all time points. Photosynthesis and response to abiotic stimulus are the most enriched GO terms associated with clade 3 genes . We chose 23 DRGs from the elf18 treated EFR:XA21:GFP rice RNAseq dataset with relatively high logFC and low FDR values after 3, 6, and 12 h for detailed analysis.

The need to control thrips with pesticides often limits the use of biological control in floriculture crops

The monitoring program and the use of reduced risk pesticides to control western flower thrips worked very effectively in the IPM greenhouses. This was a critical component of the entire program, because thrips are considered the key pest of roses. Significantly fewer western flower thrips were caught in the IPM houses than in the conventional houses across all nurseries. The largest differences in thrips levels between the two treatments occurred during the summer months, when western flower thrips pressure is generally highest . There were also greater fluctuations in the overall densities of western flower thrips in the conventional houses, as well as more variation between individual conventional houses during the time of peak thrips pressure, compared to the IPM greenhouses. We attribute both of these observations to the regular removal of open flowers in the lower canopy that occurred under IPM but not in the conventional houses. Powdery mildew. Our attempt to use the grape mildew model without modification to predict powdery mildew infection in greenhouse-grown roses was not satisfactory. The GMM is based on a sustained temperature threshold of 70°F to 85°F, which is a little higher than optimum for my celial growth of the rose mildew pathogen . For this reason, we attempted to improve the performance of the model by running it with a temperature range of either 65°F to 85°F or 65°F to 80°F. Generally, we found the model to be of limited value in Southern California; it showed a high level of disease risk most times of the year, and disease was a chronic problem. There was no clear start to a mildew season, and there was little success in identifying environmental changes associated with changes in disease pressure. On the other hand, Central California greenhouses did appear to have a seasonal component to disease,25 litre plant pot with powdery mildew on greenhouse roses starting in early spring and tapering off by early fall.

However, even under these conditions, the model was not successful in identifying triggering events. For example, there was a poor relationship between the powdery mildew index in a greenhouse near Monterey when the model was run with a temperature range of 65°F to 85°F . This relationship was improved somewhat by running the model for the same data using a temperature range of 65°F to 80°F . However, there were many times in the spring and early summer when the PMI indicated high disease risk but no disease was evident on the crop . We have no explanation for these persistent failures. Perhaps there was no inoculum in the greenhouse; perhaps we were not fully aware of all fungicide treatments; or perhaps greenhouse humidity is interacting in a way that confounds the model. Clearly a model that could predict the most opportune times for applying fungicide treatments to control powdery mildew on roses would be beneficial. We were encouraged by the fact that the model never indicated low risk when there was in fact significant disease , and that we sometimes saw a rise in mildew incidence after a rise in the index with an appropriate latent period lag . However, our research showed that the UC Davis powdery mildew risk assessment model for grapevines is not easily adapted to the challenge of powdery mildew on greenhouse roses. Additional research is needed to develop a more suitable modeling platform before it will be possible to effectively advise growers regarding risk periods. Secondary pests. Effective IPM implementation was hindered at two sites by the citrus mealybug . This pest is generally not a problem for rose growers until IPM is implemented, when the cessation of broad-spectrum pesticide applications can allow this pest to develop. It is generally a problem only at sites where roses are or were grown adjacent to other flower crops such as Stephanotis, an important citrus mealybug host plant. Unfortunately, natural enemies of the citrus mealybug are not regularly available at the commercial level, and the most effective mealybug pesticides are detrimental to spider mite predators.

We are working with the natural enemy suppliers to try to change this situation, and we continue to evaluate reduced-risk pesticides for efficacy against the citrus mealybug.Overall, we believe that the rose IPM program was successful. For example, most of the growers participating in the study wanted to abandon their conventional treatments in favor of using a biological control, predatory mites, to control two spotted spider mites; we allowed them to do so after we felt that enough data had been collected for a good comparison of the IPM and conventional treatments. Future work should concentrate on reducing the sampling effort while still collecting sufficient information to support good pest management decisions. In addition, more work is needed on refining the predictive powdery mildew model as well as on developing effective IPM techniques for secondary pests. This program represents the first and largest effort to demonstrate and implement an IPM strategy on floriculture crops in the United States. Drawing on the partnerships that are central to the Pest Management Alliance concept, we have shown that high-quality roses can be produced with substantially fewer pesticides and with the incorporation of biological control into mainstream floriculture. Effective partnering with the biological control industry has also been a hallmark of this program. This has led to the widespread use of predatory mites in commercial rose production in California, representing the largest use of biological control by the floriculture industry in the United States.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,30 litre plant pots bulk 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, 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.Previous transcriptomic work revealed a high level of congruency in differentially expressed genes in AA- and ANE-treated tomato seedlings compared to H2O-treated controls . To further this line of investigation, the metabolomic profiles of AA- and ANEroot-treated plants were compared to H2O after 24-, 48-, 72-, and 96-hours exposure to their respective treatments. Locally-treated roots and distal leaves were harvested, flash frozen, extracted for metabolites, and subsequently analyzed via LC-MS, which with underivatized samples primarily captures the nonvolatile metabolome . Partial least squares score plots of tomato root tissue revealed distinct clustering by treatment irrespective of time point .

No overlap was observed in the 95% confidence ellipses for any treatment group. Likewise, heat map visualization of the log10 signal of metabolites showed clear clustering of metabolomic profiles by treatment . Features displayed in the heat map were filtered from the total dataset with a p-value < 10−6 and absolute fold change > 5 in roots. Less defined clustering was observed in PLS score plots of distal leaf tissue across sampled time points . Ellipses representing the 95% confidence interval for both H2O and AA treatments both partially overlap with the ANE treatment group. Similarly, a heat map depicting metabolite log10 signal showed more diffuse clustering by treatment . Visualized metabolites from leaves displayed in the heat map used a p-value < 0.001 and an absolute fold-change > 2. These findings are reflective of distal tissue not directly treated with either elicitor. Changes in the distal leaves were not as robust as in the directly treated roots, likely due to diminution of systemic signals that effect metabolic changes throughout the plant. An assessment of the total annotated features across the metabolomic analysis revealed shared and unique annotated features between roots and leaves . Roots and leaves share 44 features with leaves displaying the largest number of unique metabolic features . There were 330 unique identified features in leaves compared to 223 features unique to root tissue . A comparison of differential metabolic features for AA- and ANE-treated plants compared to the H2O control revealed robust overlap for both roots and leaves . AA- and ANE-treated roots shared 68 differential metabolic features, with AA and ANE treatments possessing 37 and 29 differential features unique to each, respectively . Less overlap was observed in leaves with 39 shared differential metabolites, with AA- and ANE-root treated plants displaying 34 and 19 uniquely differential metabolites, respectively . Chemical enrichment analyses were conducted to identify classes of metabolites whose accumulation was locally or systemically altered in AA- and ANE-root-treated tomato seedlings. Enrichment analyses of metabolites whose mean signal was significantly changed in AA- or ANE-treated plants compared to H2O identified numerous affected chemical classes . These changes were most robust in directly treated roots compared to distal leaves. Treatment of tomato seedlings with AA showed strong modulation of metabolomic features classified as triter penoids and linoleic acid and derivatives in roots. AA-treated roots also showed increases in hydroxycinnamic acids and derivatives and fatty acyl glycosides of mono- and disaccharides. ANE-treated roots showed modulation in the accumulation of triterpenoids, steroidal glycosides, and hydroxycinnamic acids and derivatives. Similar to AA-treated plants, the roots of plants treated with ANE also showed increases in metabolites classified as fatty acyl glycosides of mono- and di-saccharides. Although less striking than the chemical enrichment analysis of roots, leaf tissue of root treated plants did reveal an altered metabolome . These changes in metabolite accumulation occurred most prominently at 96 hours, the latest tested time point. Increases in sesquiter penoids and steroidal saponins were seen in leaves of AA-treated plants at 96 hours.

Nothing remains in the theme park of traditional agrarian social relations

The cemetery is fenced with iron bars, thus compartmentalizing the space much like other parts of the Jinhu site. Inside the cemetery, tombs are densely arranged based on a grid plan, quite similar to the arrangement of houses in the New Village. Instead of being in the form of tumuli—as were all tombs in traditional China and as are tombs commonly encountered in rural China today—tombs in the Jinhu cemetery are flat with standing steles, similar to those found in Western cemeteries. By use of this format of cemetery, the village dead were now confined to a neat,organized Western version of modernization, much like, as described above, the living villagers residing in Jinhu New Village. This Western-style format also served to make more efficient use of land, an important element in the logic of rural development of particular concern to central government planners.In sum, what do we make of the built environment at Jinhu New Village and Jinhu Rural World theme park? It is quite clear that New Countryside planners were not interested in a nostalgic presentation of the elements of old rural society. They were after something revolutionary and new, producing a built environment that highlighted scientific agricultural technologies, hygienic agriculture, and a compartmentalized space labeled for outsiders. Perhaps the most curious element of this vision of the rural modern evident at Jinhu involves the mimicry of American suburbia. Neither the lawns nor the stucco architecture have any precedence in traditional China. In peasant society,macetas 30 litros where grass is deemed a weed, lawns are particularly out of place.

There is little doubt, then, that this aesthetic represents a deliberate attempt to modernize by imitating the American model. American suburbia today, however, is the culmination of an almost two hundred year history, partly the outcome of developments in transportation technologies since the1820s, including the railroad, streetcars, and automobiles . In the U.S., suburbanization was partly accompanied by the decline of cities that accompanied a nation-wide deindustrialization. China has experienced none of these conditions. Unlike U.S. suburbia, Jinhu New Village has not developed organically over time; it involves an imagined modernity based on an imported model from the West now being imposed on rural Chinese society. As Lisa Rofel points out, modernity in China does not neatly replicate the hypothetical transnational European model, but entails “successive imaginaries of modernity” as an alternative to the Eurocentric “anthropology of modernity” as typically conceived. Karl Polanyi’s analysis of the process of “disembeddedness” described in The Great Transformation is relevant here . In traditional rural society, villagers are “embedded” into a social economy where economic transactions are combined with social interactions. In Jinhu Rural World, agrarian society is entirely commodified following the rules of the market economy for the sale of its urban consumers. The Jinhu New Village residents are disembedded from their traditional social economy when they lost their farmland and were moved out of their traditional villages. Though technically classified by the government as “peasants who have lost their land” , villagers are more accurately described as precarious wage laborers, now dependent on capitalists. What is happening in today’s China resembles the story Polanyi told about the early stages of the Industrial Revolution and the rise of capitalism in eighteenth-century Europe.

It is compelling to observe a stark contrast between China’s highly developed industry in urban areas and its “precapitalist” condition in the hinterland and the countryside. Though the Chinese economy has emerged as the second largest in the global arena, the significance of the Jinhu case suggests that China’s “great transformation” has merely started. Rural modernization and the “disembeddedness” of rural society underscore the drastically enhanced tension between two coexisting worlds—a “modern” New Countryside and an older traditional rural society, the world created by businessmen and government planners and the world of the longtime residents of Jinhu. One might construe this tension as leading to the triumph of modern scientific techniques, of machines, and of computer-based management over a dying traditional world. The new rural world, as refilected in the theme park, is devoid of the “backward” peasantry. The New Village is largely empty of young able-bodied men and women, who have gone elsewhere to find work. And those peasants who insist on staying behind in their village have neither land to farm nor much chance to find employment. They are left to eke out an existence, often by trying to maintain their old ways in an alien environment, for example by trying surreptitiously to grow a few crops on the New Village landscaping. Another way of viewing this tension, however, is to see it as a continuation of a longstanding Chinese economic phenomenon. In her book China’s Motor, Hill Gates has argued that, going back to imperial times, China’s economy was driven by two motors: a top-down state-driven “tributary” economy, and a grass roots small petty bourgeois-driven economy satisfying the needs of the people. In so far as it is the product of central planning, many New Countryside projects including Jinhu Rural World theme park, can be thought of as examples of the former “motor.”

However, following Gates’ rationale, the place of informal businesses, though often less visible, deserves closer analysis as a two-track economy that incorporates both bottom-up survival strategies and a top-down state generated social system. In Jinhu New Village, the majiang parlor, computer repair shop, clothing factory, restaurants, and general stores are all informal businesses of this sort, unregistered with the state, and hence subject neither to state regulations nor to state taxation. Although the Chinese regime often portrays itself as the mastermind behind post- 1978 development, in many urbanizing centers, including Jinhu New Village, the most vigorous sector of the local economy frequently seems to involve the informal economy, consisting of businesses operating out of private homes. Indeed, in Jinhu, the officially designated commercial spaces along the T-shaped axis remain largely unoccupied.China is commonly portrayed as a classic example of top-down authoritarian rule, albeit an authoritarian rule that is “fragmented” in nature. James Scott portrayed Maoist China as the epitome of the authoritarian regime when discussing the disastrous potential consequences of top-down decision making during the Great Leap Forward of the 1950s. Other more recent events are frequently presented as evidence of China’s authoritarianism, including the repression of the Tiananmen Square student protests of 1989 and of the Falun Gong sect. On the surface,macetas 5 litros the “New Countryside” campaign appears to involve top-down centralized decision-making. Not only was the campaign first announced as part of a five year plan, but the government also maintains complete control of the major sources of funding. However, although top-down authoritarian decision-making is evident in many state-led projects in contemporary China, the New Countryside affords the possibility of complicating our understanding of how central government campaigns are put into practice at the local level by revealing the complex relations between multiple participating actors, namely the central government, local state officials, business entrepreneurs, peasants, and an emerging middle class of urban consumers of the products of the countryside. The first actor to consider is the central government. The central government’s justification for rural development is focused on the macro-level. More specifically, its concerns can be reduced to three tangible issues: sannong, “national food security” , and ecological sustainability. As previously explained, sannong refers to the accumulated problems arising from uneven economic development between east coast regions and hinterland regions, between urban and rural societies, and between industrial and agricultural sectors. These problems have increasingly attracted the Chinese government’s attention, partly due to a growing concern for maintaining social stability. The New Countryside campaign was initially designed to be a national level policy for resolving the sannong problem, partly through “agricultural modernization” and a process of “national agricultural comprehensive development” . Yet, the central government has also increasingly worried about maintaining an adequate food supply as China’s agricultural sector is displaced by its industrial sector. The government has recently proclaimed a “red line of 120 million hectares of cultivated land” , needed to guarantee adequate food production.

It is largely for reasons of “national food security” that the government has asked local authorities to “save and consolidate” fragmented farmland. The call to save arable land has, thus, become a source of legitimacy for many rural projects, including the Jinhu case. A third issue concerns “ecological” matters. The government has become aware that rapid economic growth has come at the expense of ecological degradation. Realizing the risk of long-term unsustainability, the government has recently recognized the importance of improvements to the environment. It is worth noting that these current concerns of the Chinese central government derive from a globalized discourse. As David Harvey points out, in the past decade, food insecurity, energy, and environmental crises have become a new subject of development discourse all over the world. Whereas the central government is preoccupied primarily with macro-level issues, it falls on local actors—especially county, township, and village level officials—to implement these policies across China’s vast rural society. The logic of rural development as viewed by local state officials can be reduced to two main goals. The first is to implement central policies to the satisfaction of the central government. By turning their efforts into a success story—usually as measured by local economic growth—local officials can guarantee their career advancement in the bureaucracy. Equally important, however, is the second goal of making money on the side by profiting personally from local economic growth. While implementing central policies, partly by consolidating land, such officials are able to converge their own interests and concerns with those of the central government. In fact, land development for residential housing is the single most lucrative business and the engine of economic growth in today’s China, including the hinterlands . At the same time, dedicating land to the production of food, especially grain staples like rice and wheat, is far less profitable, partly due to government fixing of grain prices. Thus, while expropriating and consolidating peasant land is done in the name of the central government’s agenda of protecting national food security, the reality is more complex. The houses of peasants are torn down after all in order to make them the designated consumers of newly built rural residential housings. Moreover, after consolidation, the land may be used for purposes other than grain production, in this case, for a theme park nominally dedicated to the theme of agricultural production. The third group of actors involved in New Countryside rural development are the business entrepreneurs. As is universally true of all capitalists, the entrepreneurs’ primary objective is to make a profit. In order to do this, they need to attract consumers, while simultaneously adhering to local and central government regulations and ensuring that local peasants do not resist violently. In some sense, then, the local capitalists serve as the glue that joins together the various agendas of the other actors in the local arena. In China, real estate developers—including those involved in Jinhu Rural World—adhere to a logic of land development that involves placing some unused land in reserve for future expansion. In urban centers, land is held in reserve because it is always more profitable to sell additional housing projects after a site has attracted the attention of consumers during an initial phase of development.13 In the countryside, the rationale for holding land in reserve is somewhat different. Given the difficulty of having the peasants removed and resettled in New Villages, a process described in some detail below, a major priority is to keep them from returning to their farmlands while awaiting the financial capacity to develop the land in its entirety. For example, in Jinhu Rural World, as of summer 2012, only 16 out of 41 attractions had been opened to the public. Rather than renting out the land to farmers to cultivate until it was ready for development, the former farmland has been left fallow for the past few years and fenced off to keep local residents from accessing it. The fourth group of actors are, of course, the peasants themselves. In contrast to the profit motive driving rural development by entrepreneurs, the peasants generally adhere to a “subsistence ethic” that demands that all village resources be fully exploited for the production of food.

Additional information on model parameters can be found in the Supporting Information

The findings of our study coincide with the well-characterized role of lignin and its intermediates in plant defense. This work characterizes local and systemic metabolic profiles of AA- and ANE-treated tomato with the oomycete-derived MAMP, AA, and the AA-containing biostimulant, ANE. AA and ANE profoundly alter the tomato metabolome toward defense-associated secondary metabolites with notable overlap in enriched metabolite classes compared to H2O control. Further investigation is required to elucidate the functional contribution of these metabolic features in AA- and ANE induced resistance and, more broadly, plant immunity. Our study adds to the understanding of MAMP-induced metabolomes with implications for further development of seaweed-derived bio-stimulants for crop improvement. Although copper-based engineered nanomaterials currently comprise a relatively small fraction of global ENM production ,their toxicity and life cycle characteristics raise concerns regarding their environmental risk. For example, a common use for Cu-based ENMs is as the active ingredient in marine antifouling paints or agricultural biocides,where they are directly introduced into the environment as intentionally toxic substances. Copper based ENMs are somewhat unique among the most widely used ENMs in that they can participate in redox reactions to form three oxidation states: Cu0 , Cu1+, maceta 25 litros and Cu2+. Copper can also participate in a number of inorganic complexes with compounds found in natural waters, such as sulfate, sulfide, phosphate, chloride, and carbonate.

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

Additionally, equilibrium speciation modeling was performed to predict transformations of the Cu ENMs. Based on previous work, we hypothesized that aggregation would largely be controlled by the IS of the water, with more saline waters having greater aggregation due to surface charge shielding, and by the presence of dissolved organic matter that will increase electrostatic and steric repulsion between particles. Due to the propensity for larger, heavier aggregates to settle more rapidly, we hypothesized that sedimentation would be directly related to aggregation kinetics and hence controlled by IS and total organic content . We hypothesized that pH would be the key factor in dissolution with more dissolution occurring at lower pH and that the presence of TOC would also cause a small amount of dissolution. Additionally, we hypothesized that nano-Cu would have the greatest dissolution in oxic waters as it oxidized to Cu2+.Aggregation kinetics of Cu-based ENMs were measured by preparing 10 mg L−1 ENM suspensions in each water through dilution of a 100 mg L−1 stock, probe sonicating for 2 s at 20% amplitude with a Misonix Sonicator S-4000 , and then measuring size trends over time at 20 °C via dynamic light scattering . Measurements were taken every 30 s for 1 h. To measure sedimentation over time, the optical absorbency of suspensions identical to those described above were determined in triplicate every 6 min for 6 h at 320 nm with the exception of nano-Cu in lagoon water, seawater, and diluted seawater, which were measured at 520 nm at a concentration of 20 mg L−1 . Nano-Cu is the only of the three particles where copper is primarily in the zerovalent state, and as such it is able to participate in unique chemical reactions prior to oxidation to the +1 and +2 states.

One of these is the temporary formation of copper chloride compounds in saline waters , which absorbs strongly at 320 nm, the spectral wavelength that was used to detect solid copper. To test the effects of phosphate on nano-CuO, the sedimentation rates, ζ-potential , and pH of 10 mg L−1 nano-CuO in Nanopure water with the addition of 0, 0.1, 0.2, 0.5, 1, and 2 mg PO4 3− L−1 were measured in triplicate. To measure dissolution, ENM suspensions were prepared and stored at room temperature for 0, 1, 7, 14, 21, 30, 60, or 90 days, at which point they were transferred to Amicon Ultra-4 10 kDa centrifugal filter tubes and centrifuged at 4000g for 40 min with a swinging bucket rotor. Filter retention was insignificant.The filtrate was analyzed using a copper ion selective electrode under consistent lighting conditions to minimize light-induced interference. The filtrate was then oxidized with 1.2 vol % HNO3 and 0.9 vol % H2O2 and analyzed for total copper content via inductively coupled plasma atomic emission spectroscopy , with a detection limit of 50 μg L−1 . Standard solutions were measured every 15 samples for quality assurance. Two parameters related to dissolution were quantified: dissolved copper , the total copper content of the ENMs present as free ions , and aqueous phasecopper , the total copper content of the ENMs in the filtrate, which includes dissolved copper, complexed copper 2, etc., and copper bound by ligands under 10 kDa. The ISE that was used to detect free ionic copper was capable of detecting both Cu1+ and Cu2+, both of which may have been shed by the nano-Cu ENMs, but since Cu1+ undergoes rapid disproportionation26 into Cu and Cu2+ and is readily oxidized to Cu2+ in oxic water ,maceta redonda it is unlikely to be present as a free ion in any significant amount. Visual MINTEQ was used to predict speciation and complex formation in the natural waters based on the parameters given in Table 2. Aggregation of nano-Cu and Cu2 particles was characterized by three phases in the 1 h time period measured: immediate aggregation to roughly 5−10 μm in the first few seconds post sonication, a downward trend in aggregate size from 0 to 10 min that was likely due to sedimentation of the largest aggregates out of the water column, and a stable phase in which aggregate diameters averaged 700−2000 nm. Aggregation of nano-Cu and nano-CuO followed the trends outlined in our hypothesis with a few instructive exceptions discussed below, but Cu2 had similar aggregation behaviors in all waters. The polydispersity indices reported from the DLS analysis for Cu2 and nano-Cu were near the arbitrary cutoff value of 1 at all time points in all waters, indicating very broad size distributions. Average aggregate size and statistical groupings for all three ENMs can be found in Table 3. AverageCu2 aggregate size in the third phase did not vary significantly with water type , which may be due to the large proportion of dispersants and other non-Cu ingredients in Kocide 3000. However, despite its high polydispersity, nano-Cu aggregate size correlated significantly with water type . Nano-Cu aggregate size correlated well with IS except in wastewater and storm runoff, which had the highest organic contents of the waters tested by a wide margin. In wastewater, nano-Cu aggregates were smaller than would be predicted by its moderate ionic strength, but aggregates in storm runoff were comparable to those found in the most saline waters. This counter intuitive behavior may be explained by the very low rate of sedimentation of nano-Cu in storm runoff resulting in larger aggregates being retained in the zone measured by DLS.

Nano-CuO displayed markedly different aggregation trends than the other two particles, aggregates being on average smaller and more monodisperse with PDIs ranging from 0.24−0.36. Additionally, aggregate size significantly increased with time in all waters except freshwater and storm runoff, where aggregate size decreased . Given that there was very little sedimentation or dissolution in these two waters over the measurement period , it appears that the low IS of the storm runoff and freshwater media caused disaggregation to occur. Further evidence for this can be found in previous work,which showed that nano-CuO aggregate size decreased over time in Nanopure water with up to 10 mM NaCl but that at higher ionic strength aggregation occurred. Table 3 shows nano-CuO aggregation has a strong positive correlation with IS for all waters but hydroponic media. The large average aggregate size in hydroponic media is likely a result of the decrease in electrostatic repulsion between particles caused by the pH of the media being near the isoelectric point for nano-CuO .Sedimentation kinetics for nano-Cu, Cu2, and nano-CuO over 6 h are shown in Figure 2. In general, sedimentation follows our hypothesis and shows a positive relationship with ionic strength and an inverse relationship with organic content. However, all three particles show different trends depending on their specific composition, and nano-CuO exhibited an unpredicted stabilizing effect due to the presence of phosphate. Cu2 remained relatively well suspended in all waters but groundwater likely due to the proprietary organic dispersants included in its formulation, which give it a high surface charge3 and a low bulk density .Nano-Cu was stable in high TOC waters, namely wastewater and storm runoff, and unstable in the rest. The instability of nano-Cu in hydroponic media may have been due to the low pH of the media causing increased dissolution and subsequent formation of insoluble Cu32 precipitate . Interestingly, aggregate size does not seem to correlate with sedimentation rate in any of the three ENMs tested here. This suggests that aggregate density , stabilizing coatings, and dissolution/ precipitation may be more important predictors of sedimentation rate. Regardless of dispersants or oxidation state, all three particles were unstable in groundwater. This was likely due to the high bicarbonate and low chloride concentrations found in groundwater, resulting in the formation of insoluble copper carbonates. Speciation modeling predicts that in groundwater all three particles will precipitate as malachite 2) at equilibrium . Lagoon water and seawater also had relatively high amounts of HCO3 −, but due to their high Cl− content, atacamite 3) is predicted to be the dominant form at equilibrium. This suggests that these particles are unstable in saline waters. The trends in nano-CuO sedimentation rates can largely be explained as functions of water ionic strength and phosphate content, with waters being grouped into those with and without detectable PO4 3− and IS accounting for order within those groups . For example, waters with undetectable levels of PO4 3− had the highest sedimentation rates by a wide margin and showed increasing sedimentation with increasing IS. To further investigate these trends, the ζ-potential, pH, and sedimentation rates of nano-CuO in Nanopure water with increasing PO4 were measured. Nano-CuO sedimentation rates across a range of seawater/freshwater mixtures were also measured. Figure 3 shows that sedimentation rate increases linearly with IS and slows over time. This has implications for estuarine environments and other areas where waters of varying salinity mix, as it suggests nano-CuO and similar ENMs may sediment from the water column when moving from areas of low salinityto areas of high salinity. Figure 4 shows that PO4 3− has a variable effect on the sedimentation rate of nano-CuO in Nanopure water, causing increased sedimentation at the lowest concentration , decreased sedimentation from 0.2 to 0.5 mg L−1 , and having no effect at 1.0 or 2.0 mg L−1 PO4 3−.

The dimension of the channel depends on the type and age of the plant

Split-root systems are widely studied and have been adapted to rhizoboxes as well as to pots and tubes . In the rhizosphere, plants host a wide diversity of bacteria on the surface of the root as well as within roots in the vascular tissue . Due to its abundance and importance, the bacterial community in the rhizosphere is perhaps the most widely studied among other microbial members in the rhizosphere ecosystem. While the study of endophytic bacteria requires inevitable destructive sampling due to its localization, several non-destructive approaches have been developed to study microbes inhabiting the rhizoplane. One of the most widely studied plant-microbe interactions in the rhizosphere is that of the symbiotic relationship between legumes and rhizobia . Once a potential nodule forming bacteria is isolated, it is often required to authenticate its nodule forming phenotype by inoculating on host plants. However, conventional methods such as the use of soil pouches do not allow long term incubation, while “Leonard jars,” consisting of two stacked glass jars forming the top soil layer and the bottom nutrient solution layer, can be expensive and time consuming . A recent study challenges this by describing the use of clear plastic CD cases as minirhizotrons with potential for use in phenotyping root traits such as legume formation, and demonstrated innovation that democratizes research opportunities in rhizosphere research . Other microbial interactions in the rhizosphere, however, may not result in visible changes to the root system and often rely on next-generation omics technologies. As such,macetas plastico physical separation of the rhizosphere from the bulk soil becomes paramount in elucidating changes to microbial community and interactions.

One approach to this end is the use of nylon bags with differing pore sizes . The nylon bag restricts the movement of roots and the soil inside the bag is then regarded as the rhizosphere soil to compare against the surrounding root free bulk soil . Developing further on this concept, Wei et al. designed a specialized rhizobox that allowed repeated non-destructive sampling by adding individual nylon bags of root-free soil surrounding the root compartment which are then used as a proxy for the rhizosphere . These methods allowed easy distinction of the rhizosphere and the bulk soil but, we now know that the rhizosphere community is not only distinct from the bulk soil but also varies with type, part and age of the root, largely as a consequence of varying root exudation patterns . Studying this phenomenon in situ in the soil requires separation of desired roots from others without disturbance to plant growth or soil. To address this, researchers have used a modified rhizobox design with a side compartment to regulate root growth and quarantine specific roots from the main plant chamber . This additionally creates easy distinction between old and new roots and allows testing on specific quarantined roots despite plant age. A study using this set up showed specific microbial chemotaxis toward different exudates on an individual root whereas another showed spatial and temporal regulation of niche differentiation in microbial rhizosphere guilds . Similar physical perturbations to regulate root growth in response to microbial stimuli have also been applied in the micro-scale and are explored in the next section. Our assessment of the major growth chambers showed that most of the systems applied share similarities in basic structural components such as in the use of two parallel sheets in rhizoboxbased devices. While these growth chambers brought many of the rhizosphere processes to light, limitations do exist. One limitation is with the scale of applicability.

Most of these growth systems are mesoscale and can easily reproduce pot scale studies but may not be easily translatable to interactions occurring at the micro-scale nor recapitulate processes occurring at field-relevant scale. The next section describes advances in technology resulting in a new wave of unique devices making use of microfluidic processes and fabricated ecosystems which are specifically made to investigate specific rhizosphere processes. A complex web of biochemical processes and interactions occur in micro-scale dimensions in the rhizosphere. Having the ability to interrogate and manipulate these micro-scale processes and environmental conditions with high spatiotemporal resolution will elucidate mechanistic understanding of the processes. Microfluidics has proven to be a powerful approach to minimize reagent usage and to automate the often-repetitive steps. The micro-scale of the channels also allows precise control of reproducible conditions utilizing the laminar flow and automated fluidic operations . In addition, the microfluidic devices are well integrated with conventional imaging techniques by using a glass slide or coverslip as a substrate bonded with polydimethylsiloxane . These characteristics, as well as the ability to rapidly prototype and reproducibly manufacture using soft lithography technique, have enabled new ways of interrogating and studying the rhizosphere environment in a reproducible manner. Many of the microfluidic devices used for studying the rhizosphere share a similar design concept . They have an opening port, sometimes with pipette tips inserted into the PDMS body where the seed of the seedling rests and a micro-channel where the primary root grows into.

For example, an Arabidopsis thaliana’s seedling is typically grown in a microfluidic device up to 10 days, with chamber dimension around 150 to 200 µm in height, whereas the Brachypodium distachyon seedling chamber is 1 mm in height due to its thicker roots . Media and/or inoculation of the microbiome is achieved through additional channels to the main chamber. The PDMS body with the channels is typically bonded on a 50 mm by 75 mm microscope slide, and is made to accommodate multiple plants to increase throughput. Automated control offers the ability for continuous imaging and manipulation of media conditions with high temporal resolution. One notable example of a microfluidic device for rhizosphere studies is the RootChip, which uses the micro-valves in a PDMS device to control the fluidics . The first study using the RootChip grew 8 Arabidopsis plants on a single device with micro-valves but by the second iteration, the throughput has been doubled indicating rapid technological advances in the field. In addition, all these studies demonstrated spatiotemporal imaging at single-cell resolution and dynamic control of the abiotic environments in the rhizosphere. Another microfluidics-specific application to rhizosphere study is to use the laminar flow to generate the spatially precise and distinct micro-environment to a section of the root as demonstrated by Meier et al. . A young Arabidopsis’ seedling was sandwiched and clamped between two layers of PDMS slabs with microchannel features to tightly control synthetic plant hormone flow with 10 to 800 µm resolution to the root tip area,cultivar arandanos enabling observations of root tissues’ response to the hormones. As many root bacteria produce auxin to stimulate the interactions with the root, this study showed the possible mechanism of microbiome inducing the interaction by stimulating root hair growth. Another application of laminar flow utilized the RootChip architecture by adding the two flanking input channels to generate two co-laminar flows in the root chamber, subjecting a root to two different environmental conditions along the axial direction to study root cells adaptation to the micro-environment at a local level . These studies revealed locally asymmetrical growth and gene pattern regulations in Arabidopsis root in response to different environmental stimuli. Microfluidic platforms have also been successfully employed to study the interactions between the root, microbiome and nematodes in real time . In the systems, additional vertical side channels are connected perpendicularly to the main microchannel to enable introduction of microorganisms and solutes to the roots in a spatially and temporally defined manner . A recent micro-fluidic design incorporated a nano-porous interface which confines the root in place while enabling metabolite sampling from different parts of the root . These studies demonstrated the potential of microfluidics in achieving spatiotemporal insights into the complex interaction networks in the rhizosphere. Despite several advantages of microfluidics in rhizosphere research as described above, some challenges remain. All the microfluidic applications grow plants in hydroponic systems where clear media is necessary for the imaging applications and packing solid substrates in the micro-channels is not trivial.

The micro-scale of the channels limits the applications of these devices to young seedlings. Thus, interrogating the micro-scale interactions in bigger, more developed plants is not possible with current microfluidic channel configurations. In addition, technical challenges such as operating the micro-valves and microfabrication present a barrier to device design and construction for non-specialists. Fabricated ecosystems aim to capture critical aspects of ecosystem dynamics within highly controlled laboratory environments . They hold promise in accelerating the translation of lab-based studies to field applications and advance science from correlative and observational insights to mechanistic understanding. Pilot scale enclosed ecosystem chambers such as EcoPODs, EcoTrons and EcoCELLs have been developed for such a purpose . These state-of-the-art systems offer the ability to manipulate many parameters such as temperature, humidity, gas composition, etc., to mimic field conditions and are equipped with multiple analytical instruments to link below ground rhizosphere processes to above ground observations and vice versa . Currently, however, accessibility to such systems is low as there are only several places in the world which can host such multifaceted facilities due to the requirement of significant financial investments. Switching back to lab-scale systems, a recent perspective paper calls for the need to standardize devices, microbiomes and laboratory techniques to create model ecosystems to enable elucidation of molecular mechanisms mediating observed plant-microbe interactions e.g., exudate driven bacterial recruitment . Toward this goal, open source 3D printable chambers, termed Ecosystem Fabrication devices, have been released with detailed protocols to provide controlled laboratory habitats aimed at promoting mechanistic studies of plant-microbe interactions . Similar to a rhizotron setup, these flow through systems are designed to provide clear visual access to the rhizosphere with filexibility of use with either soil or liquid substrates . Certainly, there are many limitations to these devices in that they are limited to relatively small plants and limit the 3D architecture of the root system. Still, an advantage with the EcoFAB is that its 3D printable nature allows for adaptations and modifications to be made and shared on public data plat forms such as Github for ease of standardization across different labs and experiments . In fact, a recent multilab effort showed high reproducibility of root physiological and morphological traits in EcoFAB-grown Brachypodium distachyon plants . The development of comparable datasets through the use of standardized systems is crucial to advancing our understanding of complex rhizosphere interactions. Open science programs such as the EcoFAB foster a transparent and collaborative network in an increasingly multidisciplinary scientific community. Specialized plant chamber systems are necessary for nondestructive visualization of rhizosphere processes and interactions as all destructive sampling approaches tend to overestimate the rhizosphere extent by 3–5 times compared to those based on visualization techniques . Nonetheless, plants in such chambers are still grown in defined boundaries and suffer from inherent container impacts. For instance, studies have pointed out that container design significantly influences root growth during early developmental stages and leaves lasting impacts on plant health and phenotype . The majority of the lab-based chambers are also centimeter scale and are unlikely to replicate exact field conditions in terms of soil structure, water distribution, redox potential or root zone temperatures . While comparisons between chamber-grown and pot-grown plants show similar outputs , studies comparing plants grown in confined spaces to those directly grown in the field are missing. A recent review mapped the gradient boundaries for different rhizosphere aspects and found that despite the dynamic nature of each trait, the rhizosphere size and shape exist in a quasi-stationary state due to the opposing directions of their formation processes . The generalized rhizosphere boundaries were deducted to be within 0.5–4 mm for most rhizosphere processes except for gases which exceeds > 4 mm and interestingly, they are independent of plant type, root type, age or soil . Bearing this in mind, our assessment of the different growth chambers revealed possible overestimation of rhizosphere ranges in some chamber set ups. For instance, the use of root-free soil pouches representing rhizosphere soil despite being cm-distance away from the rhizoplane. This prompts the need for careful evaluation of new growth chamber designs to ensure accurate simulation of natural rhizosphere conditions.