Horticulture

These toxic elements can be bio-available to terrestrial and aquatic organisms, including crop plants, and could be further bio-accumulated via the food chain causing damage to humans.Since these metals cannot be degraded, current remediation approaches include excavation or capping, with a very high cost and damage to ecosystems.In many cases, these options are not economically feasible, when the contamination is very wide-spread as is the case of many contaminated farmlands and river beds.Compared to ex-situ remediation technologies, in-situ decontamination does not require excavation and transport of contaminated sediment and soil to off-site treatment or disposal facilities, thus it is generally a more practical and economical approach.Conventional insitu soil remediation technologies used for industrial sites contaminated with heavy metals include soil washing/flushing,nft hydroponic chemical immobilization, electro kinetic extraction, and phytoremediation.While these technologies may be appropriate for small scale remediation, they quickly become cost-prohibitive at larger scales.The cost of phytoremediation does not increase much with scale, but the accumulation of metals in the plants presents ecological risks and an eventual disposal cost.

Capping sediments essentially destroys habitat, and the capping may be removed during a large storm event, reexposing the contaminated media.Thus, there is an urgent need to find better methods to sequester heavy metals to reduce human and ecological risk and ensure better food security.Chelating agents, for instance, ethylene diaminetetraacetic acid , are widely used as extractive agents for heavy metals decontamination.Due to its strong metal chelating ability and low cost, EDTA has been used as a metal extraction agent in soil washing.However, soil washing can result in unintended mobilization of metals and other pollutants that can be more easily transported by groundwater, and EDTA itself can pose issues as secondary pollution.Thus, a suitable supporting material for EDTA and other chelating agents would minimize the potential unintended environmental implications.Previously we developed super-paramagnetic EDTA-functionalized nanoparticle adsorbents for water treatment, which were shown to remove a wide range of metal ions with high sorption capacity.To date, most nanoscale adsorbents have been applied to the decontamination of aquatic systems, while very few studies have investigated sediment and soil remediation.We have also demonstrated that nanoparticles can readily transport vertically into deeper soil, driven by gravity.Thus, we set out to develop a new type of high density nanoscale adsorbent, which can remove heavy metal ions during its downward transport, significantly reducing their bioavailability.For this study, we selected tungsten oxidenanoparticlesas the dense core, which is a relatively low-cost material with high density and low ecotoxicity, to develop the dense nanocomposites that can transport vertically through the porous medium.

We first report on the synthesis of EDTA-based Ligand DNPs.We then demonstrate the sorption capacity of Ligand DNPs for Cd2+ and Pb2+.Next, we evaluate the removal efficiency of Ligand DNPs for Cd2+ and Pb2+ in two different natural porous matrices.Finally, we report on the insitu remediation performance of Ligand DNPs for Cd2+ and Pb2+ during gravity-driven vertical transport in these media.The results demonstrate that Ligand DNPs can be applied for effective in-situ metal decontamination from soils and sediments.Pyridine and toluene were purchased from Alfa Aesar.triethoxysilanewas purchased from Sigma-Aldrich.Cadmium chloride anhydrous, lead chloride, ethylenediaminetetraacetic acid , and tris aminomethane were purchased from Fisher Scientific.Diethyl ether and sodium dihydrogen phosphate were purchased from Acros Organics.Standard Suwannee River natural organic matter was obtained from the International Humic Substances Society.A NOM stock solution was prepared by mixing a known amount of NOM with DIwater for 24 h.The pH of the stock solutions was adjusted to 8 with 0.1 M and 0.01 M NaOH and HCl.All chemicals were used as received, without further purification.All solutions were prepared with deionized water from a Barnstead NANOpure Diamond water purification system.Similar to our previous synthesis strategies, the core-shell Ligand DNPs were prepared in two steps.The WO3 nanoparticles were coated with APTES to form a silane polymer layer via hydrolysis reaction.Then, the surface was modified with EDTA by forming the amide bonds between the EDTA’s carboxylic acid groups and APTES coating’s amino groups.

WO3 nanoparticles were dispersed into 40 mL toluene in a flask.After mixing well, 0.4 mL APTES was added to attach an amino group to the WO3 particles.Then the flask was connected to a reflux system , which was then rotated at 30 rpmin a water bath at 90˚C, and refluxed for 2 h.After the solution cooled to room temperature , 2 mM EDTA and 60 mL pyridine were added.The mixture was again rotated at 30 rpm in a water bath at 90˚C in the reflux system for 2 h.After the solution cooled down to room temperature, 100 mL sodium bicarbonate was added to adjust pH to 8.0.Deionized water was used to rinse the particles twice and then decanted.The same rinsing procedure was performed twice with ethanol and then diethyl ether.The particles were dried at room temperature for 24 h, and stored in a capped bottle prior to use.Transmission electron microscopy images were obtained using a JEOL 1230 Transmission Electron Microscope operated at 80 kV.Scanning electron microscopy studies were performed on a FEI XL40 Sirion FEG Digital Scanning Microscope.The surface area and pore volume of Ligand DNPs were determined using a Micromeritics 3Flex Porosimeter.The functional groups of the Ligand DNPs were detected using a Fourier transform infrared spectrometer on a Nicolet iS 10 FT-IR Spectrometer.Two representative soils were used in this study, as examples of the application of Ligand DNPs to treat contaminated porous media.A grassland soil was collected from a flat, well drained grassy area at the Sedgwick Reserve in Santa Ynez, CA , and farmland soil was collected from a fallow field at an organic farm in Carpinteria, CA.The permit for collecting soil samples was authorized by Brenda Juarez.Soil properties can be found in the Supporting Information , in S1 Table in S1 File.Soils were air dried, sieved through a 2 mm mesh, and stored at 4˚C until use.The physicochemical properties of the sieved soil samples, including pH, texture, saturation percent, soluble salts, cation exchange capacity , conductivity, organic content, bulk density, and exchangeable NH4, NO3, K, and PO4, were characterized in our previous study, and available in the SI, shown as S1 Table in S1 File.Total W, Cd, and Pb concentrations of each soil were measured by digesting ~0.3 g soil samples in 10 mL 1:3 HNO3: HCl at 200˚C for 1.5 h in a microwave digestion system , followed by analysis via inductively coupled plasma mass spectroscopy.In order to simulate Cd or Pb contamination, 20 g of each type of soil were placed in 50 mL conical test tubes, mixed with 40 mL of 10 mg/L Cd2+ or Pb2+ solution on an end-over-end shaker with a speed of 70 rpm at room temperature for 7 days to ensure sufficient equilibration time.Then, the tubes were centrifuged at 10,000 rpm for 20 min to separate soil and the residual Cd2+ or Pb2+ solution, and the supernatant was collected for residual Cd2+ or Pb2+ concentration determination by ICP-MS.Soil saturated with Cd2+ or Pb2+ was preserved at 4˚C for the sorption studies.Air dried soil saturated with Cd2+ or Pb2+ was digested with 1:3 HNO3:HCl at 200˚C for 1.5 h in a microwave digestion system,nft system then analyzed via ICP-MS to determine the total Cd or Pb content.For batch sorption experiments, 20.0 mg of Ligand DNPs were first dispersed in 5 mL DI water, then mixed with 10 g of Cd2+ or Pb2+ contaminated soil , in 50 mL conical tubes at pH = 7.Then, these tubes were mixed on the end-over-end system with a speed of 70 rpm at room temperature for 7 days, to ensure sufficient equilibration time.Adsorption kinetics studies were carried out at the previously stated conditions but for a set amount of time, varying from 6-h, to 12-h, 24-h, 2-day, 3-day, and 7-day.The dosage of Ligand DNPs ranged from 3, to 5, 10, 15 and 20 mg to study the adsorption isotherms at pH 7.To evaluate the potential effect of NOM on the remediation performance of Ligand DNPs, the adsorption isotherms were conducted by first dispersing 3, 5, 10, 15 or 20 mg of Ligand DNPs in 5 mL NOM solution , then mixing with 10 g of each type of contaminated saturated soil for 7 days.

After mixing the Ligand DNPs with contaminated saturated soil for the specified time, the supernatant and soil were separated by centrifugation.Due to the high density, the immobilized heavy metals adsorbed by Ligand DNPs would be spun down.The treated soil was collected from the top layer to avoid the possible heavy metal binding Ligand DNPs, and then dried in an oven at 60˚C for 72 h, then digested for total Cd or Pb content analysis via ICP-MS.All experiments were conducted at ambient temperature.To investigate the decontamination capability of Ligand DNPs during gravity-driven transport through soil saturated with Cd2+ or Pb2+, first the contaminated soil was packed into 15 mL conical tubes.An opening with a diameter of 2 cm was made at the bottom of the tubes as the outlet of the system.Suspensions of 20, 40, 60, 80 and 100 mg of Ligand DNPs were dispersed in 5 mL DI water, respectively, and then evenly applied onto the top of each conical tube.After applying the Ligand DNP suspension and allowing the suspension to drip out, the soil columns were placed in horizontal position and air dried overnight then oven dried at 60˚C for 72 h.The dried soil was carefully removed from the conical tube in 3 cm segments, labeled top, middle, and bottom section.Sub-samples were weighed, then digested for Cd or Pb content analysis.The Ligand DNPs were mixed with the two Cd or Pb contaminated soils at pH 7 for 7 days to evaluate their isothermal sorption performance.As shown in Fig 2, the removal efficiency gradually increased as the dosage of Ligand DNPs increased, since this increases the number of active sites.The Ligand DNPs exhibited higher Cd or Pb removal efficiency when applied to farmland soil compared to grassland soil.As shown in S1 Table in S1 File, grassland and farmland soil exhibited significantly different physicochemical characteristics, particularly the organic and ionic concentrations.The CECof grassland soilis considerably higher than the CEC of farmland soil , which results in higher retention of cations, including Cd2+ and Pb2+, leading to much lower desorption from the contaminated soil to the soil-water interface.In addition, as shown in S1 Table in S1 File, the electrical conductivity was 142.1 μm/cm for farmland soil and 18.9 μm/cm for grassland soil, indicating a higher concentration of ionsin the leachate of farmland soil compared to grassland soil.Thus, there can be a higher soil-water interface concentration of Cd2+ or Pb2+ in farmland soil compared to grassland soil, which increases the accessibility and interaction between the active sites of Ligand DNPs and Cd2+ or Pb2+.In both Cd2+ and Pb2+ contaminated soil remediation scenarios, Ligand DNPs achieved higher removal efficiencies on contaminated farmland soil than grassland soil.Ligand DNPs exhibited higher removal efficiencies of Pb2+ from both farmland and grassland soils compared to Cd2+, which agrees with the sequence of their EDTA complex formation constants : 18.04 for Pb2+ and 16.46 for Cd2+.It suggests that the complexation between Pb2+ or Cd2+ and the EDTA-functionalized surface is the dominant removal mechanism.The time-dependent removal of Pb2+ or Cd2+ by Ligand DNPs in contaminated soil was evaluated in batch studies, as shown in Fig 3.Ligand DNPs showed quick removal of Pb2+ in contaminated farmland soils, with over 75% of maximum removal efficiency achieved in the first 6 hours, and a minor increase from 1 to 7 days, when Pb2+ in contaminated grassland soils were treated with Ligand DNPs.Thus, the sorption equilibrium of bio-available Pb2+ with Ligand DNPs can be rapidly reached within 1–2 days, with mixing, in both farmland and grassland soils.Similar removal performance was observed when applying Ligand DNPs for Cd2+ soil remediation, as over 70% of the maximum removal efficiency could be achieved in the first 6 hours for both soils.However, it took up to 3 days of mixing to achieve Cd2+ sorption equilibrium , suggesting Ligand DNPs exhibit a faster removal rate for Pb2+ than Cd2+, which is due to the stronger binding constant with EDTA.NOM concentration in the soil typically ranges from 0.5% to 5%.In the current study, the original grassland soil had a higher organic content than the farmland soil , showing a relatively wide range of organic content.In addition, soluble NOM can interfere with, or compete for, metal cation sorption.In order to evaluate the effect of soluble NOM on the removal of Pb2+ or Cd2+ using Ligand DNPs, an extra 1% NOM was spiked into the Pb2+ or Cd2+ contaminated soils.

Taken together, these data indicate that MsLEC1 and MsLEC2 genes, as well as the orthologous gene MaLEC, are expressed in the youngest cells of indeterminate nodules of both alfalfa and white sweetclover. Moreover, the genes that encode these soluble lectins are also expressed in root tips. MsENOD40 gene expression. We hypothesized that expressing the lectin transgenes would not only have an effect on overall nodule phenotype, but also on downstream nodulin gene expression. We analyzed the expression of MsENOD40, which is expressed within a few hours after rhizobial inoculation . Similar to MsLEC1, MsENOD40 is expressed in the youngest cells of indeterminate nodules and also in root meristems . Accumulation of MsENOD40 RNA was significantly higher in RNA isolated from LEC1AS and LEC2AS nodules than in RNA derived from vector controls, in spite of high variability . The highest mean MsENOD40 RNA accumulation was found for LEC1AS plants, which exhibited the severest symbiotic abnormalities.Lectin concentration is very low in the roots of Phaesolus vulgaris in the absence of Rhizobium phaseoli, but infection causes an increase in the quantity of lectin in the roots . However, lectin levels remain relatively constant in pea roots upon inoculation with R. leguminosarum bv. viciae . Thus,flood and drain table the significance of changes or lack thereof in the regulation of lectin expression levels during nodulation of legume roots is unclear.

Earlier, we found that MsLEC2 mRNA accumulated in uninoculated alfalfa roots , and here, we report that its accumulation increased in the roots of nodulated plants as well, possibly due to the initiation of nodule primordia or, in the case of sweet clover, more lateral roots . Nevertheless, it is difficult to assess the importance of MsLEC2 function in the alfalfa-S. meliloti symbiosis. Most LEC2AS plants did not differ from vector controls in the symbiotic parameters examined, nor did they show any obvious alterations in nodule development or morphology. However, symbiotic parameters measured in some of the LEC2AS plants were at a level intermediate between that of the vector controls and LEC1AS plants. These data collectively suggest that MsLEC2 may have a subtle role in the alfalfa-S. meliloti symbiosis. In contrast, all of the symbiotic parameters that were examined in LEC1AS plants were clearly abnormal. In addition, MsLEC2 mRNA accumulation was up-regulated in many of the LEC1AS nodules and roots of nodulated plants. Moreover, the most abnormal LEC1AS nodule used for Northern blot analysis had the highest level of MsLEC2 mRNA accumulation. These results suggest that the MsLEC1 gene product may repress MsLEC2 expression during symbiosis and that there may be a relationship between the level of MsLEC2 expression and normal symbiotic development. The Mtlec2 gene, which is 93% homologous to MsLEC2, may be inessential for nodulation in M. truncatula because it apparently is a pseudogene.

Although the Mtlec2 promoter was active in mature Medicago varia nodules, it was not active in uninoculated or nodulated M. varia roots . In alfalfa, MsLEC3 is a pseudogene whereas MsLEC2 is not, a finding that is consistent with our results showing MsLEC2 mRNA accumulation in nodules, as well as in uninoculated and nodulated alfalfa roots. . This lectin has been proposed to function as a storage protein. We did not detect MsLEC1 mRNA accumulation in nodules using Northern blots containing total RNA, but we were able to detect it in both root and nodule meristems using in situ hybridization methods. These data correlate with those whereby a promoter-GUS fusion of the Mtlec1 gene was found to be active in mature nodules of transgenic M. varia plants but not with its localization in the nodule peripheral tissue instead of the nodule meristem . However, blue color indicating GUS expression was observed in developing nodule primordia of the M. varia Mtlec1–gusA transgenic plants . Our in situ data also demonstrated MsLEC1 mRNA accumulation in root tips, but the Mtlec1–gusA fusion was not expressed in the root meristems of transgenic M. varia. To help resolve these differences, we examined the expression of MaLEC, which codes for a putative soluble lectin, in white sweet clover. We detected MaLEC mRNA in white sweet clover nodule and root meristematic regions, suggesting that these are the main sites of expression for soluble lectin genes in these organs. The symbiotic abnormalities of LEC1AS plants are consistent with the in situ hybridization data, demonstrating that MsLEC1 is expressed in alfalfa nodules. It appears that MsLEC1 expression at the correct level at nodule initiation and in cells in zones I and II of the nodule may be important for regulation of nodule number , as well as for the regulation of nodule size and persistence . Interestingly, the antisense-MsLEC1 mRNA also accumulated in nodules at almost undetectable levels , in spite of its transcription being driven by the strong cauliflower mosaic virus 35S promoter.

These results suggest that the accumulation of the antisense-MsLEC1 mRNA is regulated in some unknown manner. The abnormally large number and size of nodules seen on LEC1AS plants were unexpected. Based on studies in which lectins promoted nodulation and nodulation-related responses , we predicted that smaller, uninfected nodules would have developed. Indeed, the majority of nodules produced by LEC1AS plants were small, undeveloped, and senesced prematurely . However, although infection thread formation appeared normal, at least based on the organized arrangement of rhizobia in the curled root hairs , it is not known whether or not the premature senescence exhibited by the LEC1AS nodules is due to a lack of MsLEC1 transcript accumulation in nodule meristematic tissues or to a defect in persistence of the infection threads. The latter seems less likely because the rhizobia were not affected by their course through the nodules; large numbers of bacteria were recovered from both LEC1AS and LEC1ST nodules . Based on our previous studies, when the MsLEC1 gene is disturbed, a disorganized proliferation of embryonic and vegetative tissues results . In this report, we have shown that following inoculation with Rm1021, the LEC1AS transgenic root nodules that result are also highly aberrant. In contrast, no abnormal vegetative or reproductive development was detected in the LEC1ST plants , although some abnormal nodulation was observed. This result is compatible with the finding that sensesuppression-induced symbiotic abnormalities are usually milder than those from antisense suppression and further suggests that symbiotic processes may be more sensitive to alterations in MsLEC1 expression. In addition to finding that MsLEC2 gene expression was upregulated in the LEC1AS transgene-containing tissues, we also found that MsENOD40, an early nodulin gene, was expressed at relatively high levels in LEC1AS nodules. The MsENOD40 gene has been shown to be up-regulated in response to cytokinin application, and it has been proposed that nodule development may be influenced by changes in the endogenous cytokinin to auxin ratio . LEC1AS plants exhibit excessive nodule formation, a result that is consistent with an increase in the level or responsiveness to cytokinin. Moreover, LEC1AS plantlets frequently formed severe teratomas with minimal root development , and mature LEC1AS roots were poorly developed , further suggesting an excessive cytokinin response. A mechanism whereby lectin could mediate phytohormone levels and interactions is hypothetical at this time, but hydrophobic ligands, including auxins and cytokinins,rolling bench are known to bind to some soluble legume lectins, albeit to sites independent of the sugar-binding site . Taken together, our findings indicate that the expression of the MsLEC1 and MsLEC2 genes, especially the MsLEC1 gene, is important in the compatible symbiotic interaction between alfalfa and S. meliloti. This hypothesis is consistent with both lectin gain-of-function and loss-of-function experiments. How lectins promote compatible symbiotic interactions is unclear, particularly because lectins with similarity to legume lectins have been found in plant families in addition to the Fabaceae . In Arabidopsis moreover, numerous genes encoding receptor kinases with legume lectin domains have been uncovered , and similar proteins have now been identified in Medicago truncatula . The finding that other plant families have genes that encode proteins with legume lectin domains implies that legume lectins are derived from a lectin gene that was already present in an ancestral flowering plant . Indeed, many proteins that appear to be specific to the legumeRhizobium interaction seem to be recruited from proteins that are common to both legumes and nonlegumes, e.g., NORKand HAR1/ NARK . NORK extracellular sequencelike genes are found not only in nonlegumes, including several grasses and Arabidopsis, but also in a gymnosperm . Similarly, HAR1/NARK genes are very similar to CLAVATA1, a serinethreonine kinase that is important for restricting the floral meristem in Arabidopsis.

It is clear that duplication of NSL, CLAVATA1, and other genes found in nonlegumes has taken place, along with a specialization of their respective proteins for the Rhizobium-legume symbiosis. Gene duplication events often result in the development of new functions for the new proteins. Thus, what makes the rhizobialegume interaction specific may rely more on the details of the interactions between various legume proteins including lectinsand their ligands. Finding a lectinless mutant in an indeterminate nodule-forming legume such as white sweetclover, which appears to have only one gene coding for a soluble lectin, might be one strategy for testing this hypothesis. Alternatively, introducing a gene for a legume lectin, e.g., SBL or PSL into a nonlegume such as Arabidopsis, may also help elucidate whether or not legume lectins can promote colonization of a nonlegume root by rhizobia. Construction of transgenic plants was described previously . Briefly, the MsLEC1 transgene contained 420 bp of DNA encompassing the 3 portion of the open reading frame plus 76 bp of predicted 3 untranslated region. The MsLEC2 transgene contained 400 bp from the 5 portion of the open reading frame beginning 74 bp downstream of the predicted initiator codon . Sense or antisense orientations of transgenes were confirmed using DNA sequence analysis. The CaMV 35S promoter drove transcription of all the transgenes. One plant line of alfalfa cv. Regen SY was used for transformation and regeneration of dozens of independent transformant lines of LEC1AS, LEC1ST, LEC2AS and LEC2ST plants, some of which were grown to maturity for use in nodulation assays. Further control lines containing only the vector used for lectintransgene plants but lacking inserted genes following the promoter were also constructed and were used in nodulation assays. Stable transgene integration and activity, as well as transgene-specific phenotypic effects, have been clearly demonstrated . For nodulation tests, stem cuttings of the transgenic alfalfa plants were placed in sterile 11-liter pans that contained 6 liters of a 1:1 mix of perlite/vermiculite saturated with 2.5 liters of complete Hoagland’s 1 /4-strength nutrient solution and were allowed to root. Stem cuttings were from independent, primary transformant lines because they demonstrated developmental abnormalities that were very similar to progeny resulting from selfing . However, because alfalfa is an outcrossing tetraploid and shows inbreeding depression, it was difficult to obtain progeny plants that survived to maturity. The cuttings were transferred to sterilized Magenta jars containing a similar mix of perlite and vermiculite watered with Hoagland’s 1 /4-strength nutrient solution minus nitrogen. A 5-ml suspension of Sinorhizobium meliloti wild-type strain 1021 cells at an optical density of 600 nm equal to approximately 0.1 to 0.2, labeled either with GUS or with GFP , was added to the Magenta jars after the bacteria were rinsed and diluted in sterile water. One and two weeks after inoculation, the roots were carefully removed from the Magenta jars, were rinsed, and were prepared either for GUS-staining or for viewing under a Zeiss Axiophot fluorescent microscope. Stem cuttings of the transgenic alfalfa plants were made as described above and allowed to root. Cuttings were placed in pots with approximately 400 cm3 of potting soil in a greenhouse. One week before inoculation, nitrogen nutrition was withdrawn from the plants, but other macronutrients were supplied. The potting soil was leached with large quantities of tap water four and one days before inoculation. Rm1021 cells were grown in RDM medium , containing 100 mg of streptomycin per liter to an OD600 of 0.11 or 0.13, depending on the experiment. Rhizobia were pelleted in a clinical centrifuge and were suspended in sterile milli-Q water to an OD600 of 0.1 . Rm1021 suspension was placed on the surface of the potting soil of each plant.

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 micro-channel to enable introduction of microorganisms and solutes to the roots in a spatially and temporally defined manner . A recent microfluidic 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.Thus,vertical grow rack interrogating the micro-scale interactions in bigger, more developed plants is not possible with current micro-fluidic channel configurations.

In addition, technical challenges such as operating the micro-valves and micro-fabrication 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 flowth rough systems are designed to provide clear visual access to the rhizosphere with flexibility 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 platforms such as Github for ease of standardization across different labs and experiments . In fact, a recent multi-lab 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. To date, many rhizosphere microbiome studies and growth chambers systems focus on the impact of plant developmental stage, genotype and soil type on microbial composition and function . On the other hand, predation as a driver in the rhizosphere microbiome remains understudied. For instance, protists are abundant in the soil and are active consumers of bacteria and fungi and play a role in nutrient cycling yet remain an overlooked part of the rhizosphere . Viruses are also pivotal in modulating host communities thereby affecting bio-geochemical cycles but their  influence in the rhizosphere is poorly studied . These predatorprey interactions in the rhizosphere deserve in-depth studies which can be facilitated by these specialized growth chambers. Another area worth investigating in the rhizosphere is in anaerobic microbial ecology. At microbially relevant scales, soils primarily exist as aggregates . Aggregation creates conditions different from bulk soil, particularly in terms of oxygen diffusion and water flow resulting in anoxic spaces within aggregates and  influences the microbial community.The rhizosphere is also rich in a wide range of compounds which can serve as alternative electron acceptors such as nitrate, iron, sulfate and humic substances in the absence of oxygen . However, most anaerobic studies in the rhizosphere focus only on aqueous environments such as water-logged paddy soils despite biochemical and metatranscriptomic evidence pointing to the possibility of anaerobic respiration in the rhizosphere . To fully understand biogeochemical cycles in the rhizosphere, it is imperative to investigate rhizosphere processes in the microscale and to include localized redox conditions as one of the influencing parameters. Microfluidic platforms with its fast prototyping capabilities can be helpful in creating growth chambers designed to stimulate these redox changes. In the study of the rhizosphere microbiome, genetic manipulation strategies are foundational in deep characterization of microbial mechanisms and current manipulation techniques require axenic isolates. However, the uncultivability of a significant portion of soil microorganisms continues to hamper efforts in gaining mechanistic knowledge. Even for culturable isolates,vertical grow table the process of isolation introduces selective pressure and disturbance to the community with inevitable loss of information on spatial interactions. A recent innovation in gene editing technologies using CRISPR-cas systems demonstrated in situ editing of genetically tractable bacteria within a complex community . Coupled with the use of transparent soil-like substrates , the application of such a technique for the editing of in situ rhizosphere microbiome while preserving spatial and temporal associations would indeed bring invaluable insights. Specialized growth chambers using 3D fabrication and microfluidic technologies are primed to facilitate such innovations. Finally, this review revealed that while similarities exist among the different growth chamber systems, many of these systems are bespoke. This makes it difficult to replicate experiments and determine reproducibility which are important cornerstones of scientific advancement. The complexity of rhizosphere interactions also warrant that computational models are essential to gain a better understanding of system level processes . However, predictive modeling requires data from standardized approaches to be comparable between experiments. Thus, future growth chamber systems and designs are encouraged to follow the open science framework to enable standardization to an extent, such as in the case of EcoFABs .

In the 1960s, the state of Punjab led in the adoption of new high-yielding varieties of wheat and rice. Production of these new varieties required innovations in the use of fertilizer and water, which occurred in a complementary manner to the innovation in seed choices. Mechanization of several aspects of farming also became a supporting innovation. Agricultural extension services based in Punjab’s public universities guided farmers in their transition to the new modes of production. Furthermore, an infrastructure of local roads and market towns had been developed by the state government: these, along with central government procurement guarantees, gave farmers access and security in earning income from their produce. In the private sector, new providers of seeds and fertilizer, as well as farm equipment and equipment maintenance services also arose. All of these conditions together created what has been known as the Green Revolution economy. With the Green Revolution, Punjab quickly became the state with the highest per capita income. This ranking persisted into the 1990s, but underlying conditions became less favorable well before then. Gains in agricultural yields and productivity slowed, due to diminishing returns. While India began to grow faster after trade and industrial policy liberalization of 1991 and subsequent creeping reforms in other sectors, agriculture remained locked into the old policies, and Punjab mostly into the old equilibrium. The relative failure of Punjab to transition from agriculture to industry or to modern services means that the state still faces a major challenge in effecting this classical structural transformation needed for growth. This failure has been a major reason in the state’s decline toward the middle of the per capita income rankings of India’s major states. However, agriculture also desperately needs attention, even if it cannot be the only sector that must change to address Punjab’s economic problems. The reasons for not neglecting agriculture are several. First, there is the immediate problem of economic distress in the sector, concentrated among small farmers and agricultural laborers. Second, the current pattern of cropping and water use is leading to a rapid decline in the groundwater table, threatening complete ecological collapse of much of the state’s agriculture. Third, the Green Revolution economy has little or no room for further innovation that would enhance productivity and rural incomes. Any one of these reasons is significant, but put together, they imply a compelling case for considering how innovation in Punjab agriculture can be spurred. This paper considers five challenges to effecting meaningful innovation in Punjab’s agricultural economy. It does not present solutions, but it is hoped that an analysis of obstacles to change can provide fundamental inputs into the process of seeking positive change. The first challenge to innovation is that, in contrast to the 1960s Green Revolution, a post-innovation agricultural economy will be much more complex, with a wider range of crops, requiring more sophisticated production technologies, as well as greater complexity in the entire supply chain. The second challenge is that this more complex agriculture will need more sophisticated infrastructure, since fruits and vegetables are much more perishable than grains such as wheat and rice . Other complementary inputs, such as water, fertilizer, farm equipment and management, will also need to be provided in innovative ways. A third challenge flows from the first two characteristics of complexity and complementarity: the costs of switching to new products and modes of production will entail significant one-time switching costs, as well as new and ongoing risks. Future risks, even if partly covered by insurance, represent a kind of switching cost, albeit less direct than explicit expenditures on shifting farm operations from one set of routines and activities to another. The fourth challenge considered here is more subtle, in that it concerns questions of appropriate balance, rather than movement to a well-defined post-innovation future. Indeed, the challenge is to assess what kinds of innovations can best be implemented in which contexts or situations: in some cases, incremental innovations or adaptation of existing frontier techniques from elsewhere may work, while in other cases, frontier innovations spurred by fundamental research may be required.

The first group includes four genes annotated as defense genes, a function that is likely not closely related with the observed phenotypes. This group includes TraesCS1B02G017500 and TraesCS1B02G0017600 , which encode proteins with NB-ARC and LRR domains characteristic of plant disease-resistance proteins involved in pathogen recognition and activation of immune responses. It also includes TraesCS1B02G017700and TraesCS1B02G0018100 , which are both annotated as defensins, a family of small plant antimicrobial peptides that serve to defend plants against pathogens. A second group includes three genes annotated as having enzymatic or housekeeping functions, which may not be compatible with the developmental nature of the observed changes in the roots of 1RSRW. The first gene in this group, TraesCS1B02G017800, encodes a methionine Smethyltransferase that has been implicated in the volatilization of selenium and in the biosynthesis of S-methylmethionine, a compound that is important in the transport of sulfur . The last two genes in this group encode proteins with chaperon functions. TraesCS1B02G019200 is a tubulin-folding cofactor E involved in the second step of the tubulin folding pathway. TraesCS1B02G019300 encodes a chaperone protein DnaJ,hydroponic gutter which stimulates the heat-shock protein Hsp70’s ATPase activity, stabilizing its interaction with client proteins.

These chaperon proteins play important roles under plant stress but are unlikely to play an important role in the phenotypic differences we observed under optimal hydroponic conditions. The third group includes genes involved in regulatory processes or in cell growth or division, processes more likely to be involved in the observed developmental changes in root growth . TraesCS1B02G017900 encodes an E3 ubiquitin-protein ligase CHIP-like protein that ubiquinate heat shock misfolded client proteins, targeting them for proteasomal degradation. Since E3 ubiquit inprotein ligases can ubiquitinate and regulate multiple targets, we could not rule it out as a potential candidate gene. We also included in this group the genes TraesCS1B02G018900 and TraesCS1B02G0019100, which encode 64% similar small GTP-binding proteins from the RAB family. These conserved proteins serve as molecular switches in signal transduction and play important roles in intracellular membrane trafficking, cross-talk with plant hormones and regulation of organogenesis, polar growth, and cell division , all functions that seem relevant to the observed differences in root development. TraesCS1B02G018700, TraesCS1B02G019700, and TraesCS1B02G019800 encode 12-oxophytodienoate reductase-like proteins involved in the biosynthesis of jasmonic acid. Since hormones can affect multiple developmental traits, these are also strong candidate genes. Finally, TraesCS1B02G020200 encodes a wall associated receptor kinase . These serine–threonine kinases are involved in signaling and cell expansion, making it an interesting candidate for the differences in root length observed in 1RSRW.

For the Cd-sensitive cultivar , addition of Cd significantly decreased SOD activities in roots compared with the control, which was intensified with increasing Cd concentrations . The activity of SOD was increased by 47.3%, 12.0% and 9.6% in the plants treated with Cd plus Si compared with the corresponding Cd treatments without Si, respectively . For the Cd-tolerant cultivar , very similar changes were noted in SOD activity in the Cd treatments with or without Si added, with an exception that no significant differences in SOD were found between the Cd1 treatment alone and the control . For the sensitive cultivar , CAT activity in the Cd treatment significantly decreased with increasing Cd concentrations compared with the control. Addition of Si significantly increased CAT activity in Cd-stressed pakchoi roots compared with Cd treatment alone throughout the whole experiment . For example, addition of Si increased CAT activities by 3.7%, 28.4% and 25.7%, respectively, at 0, 0.5 and 5.0 mg L-1 Cd, compared with the corresponding Cd treatments alone. For the Cd-tolerant cultivar , very similar results were obtained of CAT activities in the Cd treatments with or without Si, with an exception that addition of Si did not result in significant differences in CAT activities between the lower and the higher Cd treatments . For the Cd-sensitive cultivar, addition of Si significantly increased APX activities in roots by 55.1% compared with the control. The activity of APX was 16.7% higher in the Cd1 plus Si treatment than in the Cd1 treatment alone, compared to 11.4% at the Cd2 level . For the Cd-tolerant cultivar, very similar changes were observed in APX activities in the Cd treatments with or without Si, with an exception that significant increases in APX activity were found between the Cd plus Si treatment and the Cd treatment alone .Engineered nanoparticles have attracted great interests in commercial applications due to their unique physical and chemical properties. Increased usage of ENPs has raised concerns in the probability of nanoparticles exposure to environment and entry to food chain.

Plants are essential components of ecosystems and they not only provide organic molecules for energy but they can also filter air and water, removing certain contaminants. Definitively, plants play a very important role in uptake and transport of ENPs in the environment. Once ENPs are uptaken by plants and translocated to the food chains, they could accumulate in organisms and even cause toxicity and bio magnification. Nanoparticles are known to interact with plants and some of those interaction have been studied to understand their potential health and environmental impact, including quantum dots, zinc oxide, cerium oxide, iron oxide, carbon nanotubes , among others. The uptake of various ENPs by different plants was summarized in Table 1. Nanoparticles are known to stimulate morphological and physiological changes in several edible plants. Hawthorne et al. noted that the mass of Zucchini’s male flowers were reduced by exposed to CeO2 NPs. Quah et al. observed the browner roots and less healthy leaves of soybean treated by AgNPs, but less effects on wheat treated under same condition. Qi et al. reported that the photosynthesis in tomato leaves could be improved by treated with TiO2 NPs at appropriate concentration. Yttrium oxide ENPs have been broadly used in optics, electrics and biological applications due to their favorable thermal stability and mechanical and chemical durability.One of the most common commercial applications is employed as phosphors imparting red color in TV picture tubes. The environmental effects of yttria ENPs have not been reported. Even though the effects of certain NPs have been studied on several plants, the uptake, translocation and bio-accumulation of yttria NPs in edible cabbage have not been addressed until this study. This plant species was chosen and tested as part of a closed hydroponic system designed to study nanoparticles movement and distribution in a sub-strateplant-pest system as a model of a simple and controlled environment. The final test “substrate” used was plain distilled water , in which the tested NPs were mixed.

In order to observe the translocation and distribution of ENPs in plants, transmission electron microscopy has been one of the most commonly used techniques to identify the localization at cellular scale in two-dimensions , because it can be used to observe all kinds of ENPs. On the other hand, ENPs with special properties, such as up conversion NPs and quantum dots with a particular band gap can be studied with a confocal microscope with alternative excitation wavelengths to trace the ENPs. Several synchrotron radiation imaging techniques exploiting high energy X-ray have become widely used in plant science, which can measure both spatial and chemical information simultaneously, like micro X-ray fluorescence and computed tomography. In this research, we use synchrotron X-ray microtomography with K-edge subtraction to investigate the interaction of yttria NPs with edible cabbage. By using the KES technique, the µ-XCT can not only detect the chemical and spatial information in 3D, but also analyze the concentration of target NPs. The uptake,hydroponic nft channel accumulation, and distribution mapping of yttria NPs in both micro scale and relatively full view of cabbage roots and stem were investigated. We found that yttria NPs were absorbed and accumulated in the root but not readily transferred to the cabbage stem. Compared with yttria NPs, other minerals were observed along the xylem in both cabbage roots and stem. To the best of our knowledge, few reports have studied the impact of yttria NPs on cabbage plants. In addition, by using µ-XCT with KES technique, the distribution and concentration mapping of nanoparticles in full view of plant root have not been previously reported.The µ-XCT was carried out at Beamline 8.3.2 at the advanced light source, Lawrence Berkley National Laboratory. From scanning energies of 16.5 to 17.2 keV, below and above yttrium K-edge, the X-ray attenuation coefficient sharply increases by a factor of 5. Other elements decrease slightly in their attenuation coefficients over this energy range. The localization of yttria NPs can be identified by the subtraction between two reconstructed image datasets , shown in Fig. 2. The slices collected above and below the K-edge were set with same brightness and contrast settings to fairly compare with each other. These are inorganic elements which support the growth of cabbage. Some biological structures suffered radiation damage during scanning, resulting in a small amount of shrinkage. The bright regions circled in Fig. 2c were caused by such shrinkage, resulting in a registration mismatch between the images above and below the edge. To identify and map the distribution of yttria NPs, an image segmentation protocol was employed that could highlight regions with yttria without finding these regions corresponding to sample shrinkage. The detailed segmentation process is given in the “Method” section.By using K-edge subtracted image technique with Monochromatic X-ray tomography, the translocation and distribution of NPs in the cabbage root is clear . Figure 3a and b were constructed by 17.2 keV and 16.5 keV reconstructed slice datasets, respectively. Their color maps were based on the transverse slice pixel values/absorption coefficients over the range from 0.2 to 17.8 cm−1 . An obvious difference between 17.2 and 16.5 keV visualization in absorption coefficient of yttria NPs was observed. The distribution of yttria NPs in root was segmented and colored in red . A large amount of NPs were found aggregated at left bottom of the root. Since yttria NPs were not water-soluble, the water that contained them was kept in constant movement with an air pump working 24/7. However, it seems that the dense roots formed a web-like structure that made the suspended NPs to accumulate and aggregate among the roots.

Uptake of NPs by the root has been observed at primary and lateral root junction as well according to the transverse slice. Figure 2a is one transverse slice localized at the arrow in Fig. 3c showing the junction between primary root and lateral root. We found that the yttria NPs were absorbed by the lateral roots, and particulates began to accumulate along the outer epidermis of primary roots with limited entrance into the vascular tissue of the primary root. It might happen that endodermal cell walls were blocking the entrance of aggregated yttria NPs into vascular tissue. This is shown in the upper section of the 3D visualization where no yttria NPs were observed above the root system. Besides the full view of the translocation in the cabbage root system, the distribution of yttria NPs at the micro-scale within a lateral root was detected and investigated . Figure 4a shows the localization of the micro-scale lateral root visualization. The 3D visualization of micro-scale was built by the segmented transverse reconstructed slices, and the red regions were localized yttria NPs . It is clear that roots are able to uptake the yttria NPs in ground tissue , which appear to accumulate in the root with limited entrance of yttria NPs into vascular tissue being transported through the xylem. Xylem vessels are small with diameters usually smaller than 1 μm in vegetables like cabbage plants to over 100 μm in vessels found in trunks of large trees. Vessels allow nutrients contained in water to be distributed throughout the plant. For NPs, however, if they aggregate, the blockage is expected, that is what we have observed in this study. Long term studies might show that yttria NPs might provide more negative than positive effects on plant growth and development as found with other NPs. Using K-edge subtraction image technique with dualenergy X-ray scanning, the concentration of target NPs can be calculated. This method has been discussed elsewhere. For the cabbage shoot, no yttria NPs were observed , which means that no yttria NPs transported from roots to shoots. As we found no yttria NPs entering vascular tissues of primary root, the yttria NPs accumulated making it difficult to be transported by xylem from the root to the rest of the plant.

Similar to what has been observed in other plant species, supplying rice leaves with G/GO resulted in the sustained production of H2O2within the apoplast , whereas a mixture of xanthine and xanthine oxidase was found to generate both superoxide and H2O2, the latter by dismutation. Treatment with either compoundor with the enzymes alone had no significant effect on disease development compared to buffer-treated control leaves . However, infiltration of G/GO or X/XO dramatically reduced the size of the necrotic lesions incited by M. oryzae infection . By contrast, pre-treatment with G/GO or X/XO mixtures strongly stimulated necrosis induced by R. solani . By 60 hours after infection, the majority of ROS-treated and Rhizoctonia-inoculated leaves showed extensive necrosis and were almost completely deteriorated . Enhanced ROS generation also greatly enhanced lesion formation by C. miyabeanus, suggesting a common pathogenicity mechanism for both these necrotrophs .Although exogenous catalase did not significantly alter lesion development, infiltration of rice leaves with a specific catalase inhibitor, 3-aminotriazole,grow table hydroponic prior to inoculation, was indistinguishable from the G/GO- or X/XO-treated leaves. No lesions were detected in leaves infiltrated with ROS-producing mixtures, catalase or 3-AT alone, as previously reported.

Building on our earlier work with respect to 7NSK2-mediated ISR, we sought to extend our analysis of the proposed dual role of ROS in rice defense by feeding the pro-oxidative pigment pyocyanin to hydroponically grown rice plants and observe any effects on plant resistance. Opposite to the enhanced resistance observed against M. oryzae, pyocyanin feeding favored subsequent infection by both C. miyabeanus and R. solani . Amending the pyocyanin solution with ascorbate, which has long been recognized as a major antioxidant buffer and free-radical scavenger, severely attenuated the pyocyanin-provoked resistance or susceptibility, corroborating our previous findings. Taken together, these results clearly demonstrate that enhanced ROS levels in inoculated leaves positively influence resistance to M. oryzae, while exerting a negative effect on rice defense to C. miyabeanus and R. solani.Despite the emergence of rice as a pivotal model for molecular genetic studies of disease resistance in cereal crops, molecular information regarding chemically and biologically induced defenses is still largely missing. In an effort to broaden our understanding of the rice induced resistance machinery, we analyzed the host defense responses underpinning ISR triggered by the biocontrol agent S. plymuthica IC1270. The results presented in this study demonstrate that root colonization by IC1270 predisposes rice to undergo a massive oxidative burst and related HR-like cell death at sites of attempted pathogen invasion, a process culminating in heightened resistance to the hemibiotrophic blast pathogen, M. oryzae.

The same treatment, however, rendered plants more susceptible to attack by the necrotrophic pathogens R. solani and C. miyabeanus. Besides tagging ROS and HR-like cell death as two-faced players in the rice defense response, these findings strengthen the argument that rice requires distinct mechanisms for defense against M. oryzae and the necrotrophs R. solani and C. miyabeanus. Mounting evidence indicates that generation of systemic resistance does not necessarily require direct activation of defense mechanisms, but can also result from a faster and stronger activation of basal defenses in response to pathogen attack. For instance, unlike pathogen-induced SAR, classic rhizobacteria-mediated ISR in Arabidopsis is not associated with a direct induction of defense mechanisms, but with priming for augmented defense activation upon challenge inoculation. Other ISR-inducing PGPRs also have been found to enhance the plant’s defensive capacity by hyper-activating pathogen-activated defenses, suggesting that priming for enhanced defense is a common mechanism in PGPR-mediated ISR. The results presented in this study add further support to this concept as root colonization by IC1270 did not cause a strong constitutive resistance phenotype, but ratherprimed plants to hyper-respond to subsequently inoculated pathogens, resulting in excessive defense activation and enhanced resistance to M. oryzae.

This priming effect of IC1270 was borne out by the observation that challenge inoculation of IC1270-colonized plants with M. oryzae entailed a rapid accumulation of autofluorogenic phenolic compounds in and around epidermal cells displaying dense cytoplasmic granulation , two features that are considered as hallmarks of an ETIassociated HR. Comparative profiling of pathogenesis-related H2O2 accumulation in blast susceptible, yet ISR-expressing, and genetically resistant leaf sheath cells, further strengthened the parallels between R protein-mediated ETI and IC1270- triggered ISR priming . Hence, IC1270 appears to protect rice from M. oryzae by reprogramming pathogen attacked epidermal cells to undergo a rapid HR-like response, thereby providing a possible functional interface between rhizobacteria-mediated ISR and avirulent pathogen-induced ETI. Such mechanistic similarities between ISR and ETI are compatible with the idea that defense signals from multiple ‘entry points’ can converge and target overlapping sets of defense effectors. Particularly relevant in this regard is the substantial overlap between gene expression changes and alterations in SA content induced during an avirulent pathogen-triggered ETI response, and those induced by treatment with flg22, an 22-amino-acid epitope of the archetypal MAMP elicitor flagellin. Although unequivocal evidence is still lacking, the striking homologies with the sensitive perception mechanisms for pathogen-derived MAMPs that function in PTI suggest that ISR-triggering rhizobacteria are recognized in a similar manner. In this perspective, it is not inconceivable that the mechanistic parallels between IC1270-mediated ISR and ETI can be traced to converging MAMP- and R-protein-induced defense responses. Furthermore, consistent with the view of ETI as an accelerated and amplified PTI response, such MAMP-orchestrated ISR elicitation may also explain the partial nature of the IC1270-induced resistance against M. oryzae. Apart from S. plymuthica IC1270, several other biological and chemical agents have been shown to be capable of inducing resistance to M. oryzae, among which the SA analog BTH and the redox-active pigment pyocyanin, key determinant of ISR induced by P. aeruginosa 7NSK2. Interestingly, both these resistance inducers appear to mimic IC1270 in that they produce a similar resistance phenotype, characterized by hypersensitively dying cells in the vicinity of fungal hyphae. Although it does not follow that the signaling conduit governing IC1270-mediated ISR is necessarily the same as that leading to pyocyanin- or BTH-inducible blast resistance, such commonalities apparent at the level of defense mobilization suggest that these elicitors may feed into related, if not identical, resistance pathways. Further supporting this hypothesis is the overlap manifest at the level of resistance to attackers, with IC1270, BTH and pyocyanin all being ineffective or even increasing vulnerability to C. miyabeanus and R. solani. Intriguingly, induction of ISR by the PGPR strain P. fluorescens WCS374r appears to rely on a different resistance strategy and was found to be associated with priming for a diverse set of HR-independent cellular defenses, the prompt elaboration of invading hyphaeembedding tubules being a prominent component. Considering this apparent plasticity in the molecular processes leading to induced resistance against M. oryzae, it is tempting to speculate that rice is endowed with multiple blast-effective induced resistance pathways.

The rapid production of ROS during the so-called oxidative burst is a hallmark of the plant’s defense response. Although ROS are generally viewed as initiating agents in the disease resistance network ,grow table accumulating evidence indicates that ROS formation can cascade either to the detriment or benefit of the plant depending on the lifestyle and parasitic habits of the invading pathogen. Hence, ROS can play a dual role in pathogen defense, acting as key players in resistance to biotrophic pathogens on the one hand, while weakening necrotroph resistance by assisting pathogen-induced host cell death on the other. Taking these facts into account, we propose that priming for enhanced ROS generation may likewise function in IC1270-mediated ISR, thereby accounting for the differential effectiveness of this resistance against hemibiotrophic and necrotrophic pathogen assault. Critical to the formation of a hypothesis of primed ROS generation as a key event in ISR by IC1270 was the observation that artificially increased H2O2 levels, either resulting from infiltration of ROS-generating mixtures, inhibition of endogenous catalase activity or hydroponic feeding of pro-oxidative pyocyanin, faithfully mimicked IC1270 in conditioning resistance to M. oryzae but susceptibility to C. miyabeanus and R. solani. Although we are aware that final proof for primed ROS generation as the causal resistance mechanism underpinning IC1270-mediated ISR requires the use of inhibitor compounds able to abrogate the oxidative burst , such scavenger experiments could not be performed since detached leaves, needed for effective infiltration of chemicals in rice, somehow failed to develop ISR. Therefore, we can not rule out the possibility that the altered pathogen response of IC1270-induced plants may result in part from ROS-independent processes. Nonetheless, the involvement of boosted ROS generation in the establishment of IC1270-mediated ISR is apparent. In accordance with previous studies, continuous generation of H2O2 in situ by infiltration of G/GO or 3-AT did not induce any detectable cell death per se, indicating that additional pathogen-induced signals are needed for expression of HR-like cell death. Indeed, current concepts suggest that death of host cells during the HR requires the poised production of nitric oxideand ROS, coupled to simultaneous suppression of the plant’s antioxidant machinery. In view of these data, it could be reasoned that IC1270-mediated priming for potentiated ROS generation might lower the threshold for activation of programmed cell death, thereby blocking the hemibiotroph M. oryzae in its initial biotrophic phase. In line with this concept, there is ample evidence demonstrating that early-produced H2O2 is a central signal leading to the elicitation of a wide range of blast-effective defenses, among which programmed cell death. Most tellingly, Kachroo and associates [84] reported a fungal glucose oxidase gene to sequentially induce H2O2 generation, rapid HR-like cell death and enhanced resistance against M. oryzae when ectopically expressed in young rice plants. On the other hand, it is not inconceivable that IC1270-mediated priming for H2O2 may tilt the ROS-controlled cellular life-or death balance toward death, thereby facilitating subsequent tissue colonization by the necrotrophs R. solani and C. miyabeanus.

This notion is corroborated by recent observations demonstrating that IC1270 pretreatment has no marked impact on the early infection events in C. miyabeanus– or R. solani-challenged plants except for a substantial increase in the number of dying cells preceding the fungal growth front . However, given the myriad defense related plant responses modulated by ROS, other yet unidentified mechanisms also may play a role.Parasitic plants directly invade and rob nutrients from host plants. The consequences can be devastating to the host plant and some of the world’s most pernicious agricultural pests are parasitic weeds. The number of parasitic angiosperms is surprisingly large with over four thousand parasitic species identified in nineteen different plant families. Parasitic plants have a wide diversity of growth habits ranging from the tiny flowered mistletoes that live in the tops of trees to the enormously flowered and rootless Rafflesia whose entire vegetative body is endophytic. The degree to which parasites rely on hostresources also varies. Some obligate parasites, like Rafflesia, have lost photosynthetic capabilities and are fully heterotrophic. Others, like Triphysaria, are facultative parasites that can mature without a host plant but will parasitize neighboring plants when available. The single feature shared by all parasitic plants is the ability to invade host tissues via a haustorium. Haustoria of parasitic plants fulfill multiple functions including host attachment, penetration, and translocation of resources from host to parasite. Interestingly, the competence to develop haustoria has originated in autotrophic ancestors multiple times during the evolution of angiosperms. There are two general hypotheses for the evolutionary origins of haustoria. One hypothesis suggests that the genes encoding haustorium development are derived from nonplant organisms, such as bacteria or fungi, that are endophytic or which have transferred a set of genes required for haustorium formation into the parasite genome. The second is that genes encoding haustorium developmentare derived from those present in autotrophic angiosperms where they fulfill functions unrelated to parasitism. The identification of genes associated with haustorium development will provide insights into the evolutionary origins of plant parasitism. These genes will also elucidate the degree to which haustoria in different parasitic families are encoded by convergent or homologous genetic pathways. Parasitic plants in the Orobanchaceae develop haustoria on their roots in response to contact with host roots. Several molecules, typically products of the phenylpropanoid pathway, have been identified that induce haustorium development when applied to Orobanchaceae roots in vitro.

The average mass of CMG2-Fc per kg leaf fresh weight was 717 and 874 mg/kg leaf FW for the Kifunensine and Kifunensine samples, respectively . The control group expression level was consistent with previous results. These data suggest that the addition of kifunensine in the agro-infiltration process was not detrimental to transient protein production, and in this case, it resulted in a 22% increase in CMG2-Fc yield. This allows the use of kifunensine for modification of glycosylation profiles without compromising protein expression. Total soluble protein content of whole leaf extract of Kifunensine and Kifunensine groups were similar as shown in Figure 1, which indicates that kifunensine does not have significant impact on plant protein synthesis in general.In this study, the influences of one-time kifunensine vacuum infiltration on the expression level, N-glycan profile of a recombinant protein, mobile vertical grow tables namely CMG2-Fc, produced transiently in N. benthamiana plants were evaluated in both whole-leaf extract and AWF. We found that kifunensine had a positive impact on protein production when supplied in the agroinfiltration solution; specifically, we observed a 22% increase of protein expression with kifunensine treatment condition, presumably owing to its suppressing effects on ER-associated degradation pathway.

This finding is consistent with previous observations in multiple mammalian cell culture systems, and there is no reason to suspect that this will not be the case for other eukaryotic systems, including plant systems. Plants were monitored visually throughout the incubation period, and there were no significant phenotypical differences between kifunensine-treated and control groups.In the case of a whole-plant study, Roychowdhury et al. have shown that the yield of recombinant cholera toxin B sub-unit dropped by 30% and 75% when kifunensine was supplied at 5 µM hydroponically for 3 days and 5 days post agroinfiltration, respectively. In contrast, we observed slight increase in protein yield when kifunensine was infiltrated in leaf tissue instead of being supplied hydroponically. Thus, the lower protein yield they observed may have resulted from continued application in the hydroponic solution, and it is eliminated when kifunensine was supplied directly to leaf tissue through vacuum infiltration. In addition, in the hydroponic study, the target protein was retained in ER, while the model protein in our study and other cell culture studies were targeted for secretion. This difference in protein targeting may also play a role in protein yield changes upon kifunensine treatment. Kifunensine is known to inhibit enzymatic activity of class I α-mannosidases, and thus should stop mannose trimming in the first place to yield single Man9 N-glycan structures. However, we observed multiple oligomannose-type N-glycans with mannose residues ranging from 3 to 9, although the most abundant structure was Man9.

This observation is consistent with cell culture kifunensine studies, where multiple oligomannose-type N-glycans were detected under kifunensine treatment. This could potentially due to the difference in inhibition efficacy of kifunensine towards class I α-mannosidases isoforms, which results in an incomplete inhibition of mannose trimming from Man9 structure. Also taking enzyme kinetics into consideration, depending on the ER concentrations of Man9 glycoprotein substrate, class I α-mannosidases, and kifunensine, enzymatic mannose trimming from Man9 could take place even if the amount of active ER α-mannosidase I is low. Although mannose trimming was not completely inhibited at Man9 structure, this method still showed the ability to significantly modify glycosylation using a simple bio-processing approach. It is likely that Man9 abundance can be further increased if treated with higher concentration of kifunensine, but it is not necessary if the goal is to eliminate the production of plant-specific complex N-glycans. Although in this case, CMG2-Fc can be purified easily from whole-leaf extracts through a one-step purification with Protein A chromatography, in many cases, multiple steps of chromatography are required to purify a target protein from a large pool of host native proteins when a highly selective affinity tag is not present. This could result in low protein yield and difficulties to achieve high purity, which is typically required for therapeutic recombinant protein products. Targeting proteins to the apoplast allows the collection of target protein in AWF, which contains much lower levels of plant native proteins than whole-leaf extract since only secreted proteins are collected, thus lowers the downstream process complexity. In this case, CMG2-Fc purity and concentration increased by 3.9-folds and 4.4-folds, respectively, when collected in AWF versus in whole-leaf extract.

A similar trend was observed in kifunensine-treated samples, which confirms that kifunensine does not affect protein secretion, allowing secretion of CMG2-Fc with oligomannose-type glycoforms. The increase in purity and concentration was consistent with a previous study on harvesting a target protein from plant AWF. Hence, AWF collection is a feasible method for recombinant protein harvesting, which avoids contamination with intracellular host cell proteins, and is particularly valuable when target protein is hard to purify. Together with kifunensine treatment, apoplast-targeted recombinant protein without any plant-specific glycoforms can be transiently produced in N. benthamiana, and likely in other plants as well. Products can be collected at high concentration and purity from AWF, containing predominantly oligomannose-type N-glycans. Further studies should focus on determining how long the inhibition effect of kifunensine lasts after the one-time vacuum infiltration by monitoring the protein glycoform profile at multiple time points after vacuum infiltration, and the threshold concentration of kifunensine that results in a complete N-glycan shift from plant complex-type to oligomannose-type for other glycoproteins, particularly those with more N-linked glycosylation sites. In addition, the protein expression kinetics should be compared between kifunensine-treated and untreated groups to maximize target protein yield. Depending on the desired glycoform, this method can also be applied to other N-glycan processing inhibitors such as castanospermine, deoxynojirimycin, and swainsonine.The transient expression of CMG2-Fc is achieved by whole-plant vacuum infiltration of N. benthamiana with Agrobacterium tumefaciens EHA 105 containing binary vector for CMG2-Fc expression and Agrobacterium tumefaciens containing the pBIN binary vector for expression of the RNA silencing suppressor P19 from Tomato bushy stunt virus. Each A. tumefaciens strain was cultured separately in 20 mL of Luria-Bertani media with selection antibiotics for 18 h in the dark at 28 ◦C, on an orbital shaker at 250 rpm. Then, 8 mL of each culture is transferred to 250 mL LB media and incubated for 18 h in the dark at 28 ◦C at 250 rpm shaking to further amplify the cell population. The A. tumefaciens cells were collected by centrifugation at 1800× g for 30 min at 15 ◦C, and the cell density was quantified through absorbance measurement at 600 nm. Both A. tumefaciens strains were resuspended into infiltration buffer with a final cell density of 0.4 for each strain . Six-week old N. benthamiana plants were turned upside down and submerged into the agrobacteria suspension, followed with vacuum infiltration for 1 min after vacuum pressure reached 20 inches Hg. Infiltrated plants were then incubated in a growth chamber at 20 ◦C and 90% humidity for 6 days. All leaves were cut from the petioles were used for AWF collection or stored at −80 ◦C for whole leaf protein extraction.Microplate wells were coated with unlabeled Protein A at a concentration of 0.05 mg/mL in phosphate-buffered saline at 37 ◦C for 1 h, mobile vertical farm and then blocked with 5% nonfat dry milk in PBS buffer. Crude plant extract and purified CMG2-Fc standards were added to wells and incubated at 37 ◦C for 1 h. CMG2-Fc bound to Protein A on the plate was detected by adding 50 µL of horseradish peroxidase -labeled goat anti-human IgG at concentration of 0.4 µg/mL and incubated for 1 h at 37 ◦C.

Between each of these steps, microplates were washed with PBS with 0.05% v/v of Tween-20. Finally, the protein concentration was quantified by adding 100 µL of TMB substrate . Plates were incubated at room temperature for 10 min, followed by addition of 100 µL 1N HCl to stop the reaction. The TMB substrate reacts with HRP, allowing colorimetric detection of CMG2-Fc levels.Site-specific N-linked glycosylation analysis including measurement of the glycopeptide relative abundance of the protein was analyzed with mass spectrometry. Samples were first reduced with 2 µL of 550 mM dithiothreitol at 65 ◦C for 50 min to break the disulfide bond between cysteine amino acids. Then 4 µL of 450 mM iodoacetamide was added as an alkylation reagent for 25 min. Samples were placed in the dark environment to prevent the loss of IAA efficacy. After denaturing, proteins were digested with 1 µg of trypsin for 18 h in a 37 ◦C water bath. The digestion process was stopped by placing samples at −20 ◦C for around one hour. An Agilent 1290 infinity ultra-high-pressure liquid chromatography system coupled to an Agilent 6495 triple quadrupole mass spectrometer was used for N-glycosylation analysis. For sample separation, the analysis column used on the UHPLC system was an Agilent Eclipse plus C18 column . To protect the analysis column, an Agilent Eclipse plus C18 trap column was used to trap samples first. The mass spectrometer was operated using the dynamic multiple reaction monitoring mode which is a targeted tandem MS mode and Agilent MassHunter Quantitative Analysis B.05.02 software was used for data analysis. In the MRM method, glycopeptides were quantified with the glycopeptide mass used as the precursor ion and the common oxonium fragments with m/z 204.08 and 366.14 used as product ion. The concentration of glycopeptide in ion counts was normalized to the total ion counts of glycopeptides in the sample for the relative abundance calculation. The rhizosphere is the area around the plant root in soil where microorganisms are densely populated and dynamic interactions between the plants and microorganisms occur. These complex interactions and the assemblage of microorganisms are established, shaped, and maintained by the plant root. Plants exude photosynthetically fixed carbons as nutrient sources, secondary metabolites, and signaling molecules to populate root ecological niches with beneficial microorganisms. This “rhizosphere effect” has important implications for plant growth and protections against biotic and abiotic stressors, and for geochemical carbon cycling in soil environments. With the expected rise of severe, climate-change-related weather conditions such as drought and the growing need to increase plant productivity with sustainable agricultural practices, it is vital to gain a mechanistic understanding of the rhizosphere effect and engineer the rhizosphere to improve plant growth and resilience. To understand the mechanisms of the highly dynamic processes occurring in the rhizosphere, in situ interrogation of the system with high spatiotemporal resolution is necessary. However, due to the underground nature of the root system and the sheer complexity of the microbiome in the highly heterogeneous soil environment, studying root and microbial interactions has been challenging.

For example, in a typical rhizosphere analysis experiment, the plant is uprooted from soil in the field or a pot at a defined time point, and the microbiome and other relevant chemicals are sampled in a destructive manner. This practice of uprooting the plant is limiting because the spatial information on microbial community members is lost and the same plant generally cannot be sampled over time. Further, there are many confounding variables, such as chemical reactions with minerals in soil that complicate the analysis and make the studies less reproducible and relevant across different locations and environments. To overcome these experimental hurdles, researchers have designed various types of specialized devices such as rhizotrons and microfluidics devices to improve specific aspects of sampling, analytics, and manipulation of the rhizosphere system, albeit often deviating substantially from the natural system. Fluorescent microscopy is a promising tool for noninvasive in situ imaging of the microbial and root interactions with high spatiotemporal resolution. Developing in situ rhizosphere imaging methods is made especially more relevant as the microbial community colonization of the root and the persistence and succession patterns are likely dynamic and dependent on the developmental stage of the plant. Better knowledge in this can help guide synthetic microbial community inoculation protocols to optimize plant productivity. A notable development of in situ imaging devices is a microfluidic root chip by Massalha et al., in which real-time imaging of the microbial colonization of Arabidopsis thaliana seedlings was captured by high-resolution fluorescent confocal microscopy.

Metabolites were identified following the Metabolomics Standards Initiative conventions, using the highest confidence level , which is identified as at least two orthogonal measures versus authentic chemical standards . Three orthogonal measures were used to compare samples with authentic chemical reference standards: retention time , fragmentation spectra , and accurate mass . Peak height and retention time consistency for the LC/MS run were ascertained by analyzing quality control samples that were included at the beginning, during, and at the end of the run. Internal standards were used to assess sample-to sample consistency for peak area and retention times. Metabolite peak heights were scaled relative to the maximum peak height in any sample within an experiment to allow for relative comparison of peak heights between samples , but not for absolute metabolite level quantification.Chemical classes were assigned to metabolites with the ClassyFire compound classification system.To explore the variation between experimental conditions,vertical vegetable tower the metabolite profiles were PCA-ordinated, and the 95% confidence level was displayed as ellipses for each treatment. Hierarchical clustering analysis with a Bray–Curtis Dissimilarity Matrix was performed with the python 2.7 Seaborn package. Metabolite significance levels were analyzed with the Python SciPy ANOVA test coupled to a python Tukey’s honestly significant difference test with alpha = .05 corresponding to a 95% confidence level.

To test whether metabolites sorbed to clay were accessible for a plant-associated bacterium, the desorption rates of metabolites from different substrates were tested in a first experiment, and the growth rate of Pseudomonas fluorescens WCS415 on various substrates pre-incubated with metabolites was tested in a second experiment. The desorption rate of metabolites from substrates was determined for glass beads , sand , and clay . The substrates were incubated with 50 times concentrated defined medium or with 0× DM for 6 hr at 23°C. The substrates were subsequently washed three times with water, to remove soluble metabolites. The recovered metabolites of all three steps were analyzed by LC/MS, as described above. Substrates were added to a 12-well plate , and 2 ml of 0× DM was added to each well. A Pseudomonas fluorescens WCS415 preculture was grown in 5 ml 20× DM for 16 hr at 30°C, 200 rpm. The culture was pelleted at 4,000 g, 23°C for 5 min, and resuspended in 0× DM. The wells were inoculated with an initial optical density of 0.05 in triplicates. The plates were incubated at 30°C for 3 d , 1 ml of the supernatant was removed to determine OD at 600 nm. Positive growth controls were P. fluorescens grown in the same experimental setup in 50×, 20×, 10×, and 0× DM, but without substrate. A set of negative controls was prepared to account for different variables in the experiment: Substrates incubated with 0× DM with bacteria were set up as a growth control, accounting for metabolites already adsorbed to clay. Substrates incubated with 50× and 0× DM but without bacteria were used to control for changes in optical density of clay caused by DM.

The metabolite desorption experiment was performed by adding 2 cm3 of clay pre-incubated with 50× or 0× DM to a 12-well plate in triplicates, followed by the addition of 2 ml of 0× DM. The plate was incubated for 3 d at 30°C. Subsequently, 1.5 ml of the supernatant was removed by pipetting and placed in a new 12-well plate. Half of the wells were inoculated with P. fluorescens, the other half served as negative controls. OD at 600 nm was determined after 3 d of growth at 30°C.Global climate change has resulted in shifts in precipitation patterns, causing stress on freshwater resources, especially in arid and semi-arid regions . In many of these areas, demand for water has led to increasing use of municipally treated wastewater . Agriculture has been one of the primary targets for TWW reuse with water districts and governments promoting the adoption of recycled water for irrigation . However, the use of TWW for irrigation may come with potential risks, as TWW is known to contain a wide variety of human pharmaceuticals . The use of pharmaceutical compounds has increased with population growth and economic development, resulting in over 1500 compounds currently in circulation . Their widespread consumption has led to their occurrence in TWW as well as in TWW impacted surface water . For many of these pharmaceuticals, there is limited knowledge about their potential chronic effects in the environment . Further, many of these compounds can transform in the environment, resulting in the formation of transient or recalcitrant transformation products, many with unknown fates and effects in environmental compartments .

Diazepam belongs to the class of psychoactive compounds known as benzodiazepines, one of the most prescribed classes of pharmaceuticals . Diazepam is one of the most commonly detected pharmaceuticals in TWW, with concentration ranging from ng L−1 to low μg L−1 . This is likely due to its extensive use and low removal efficiency during secondary wastewater treatment . In humans, diazepam is primarily metabolized via phase I oxidative metabolism by demethylation to nordiazepam , or hydroxylation to temazepam , and then further oxidized to oxazepam . Oxazepam undergoes phase II metabolism via rapid glucuronidation and then excretion via urine . The three primary metabolites of diazepam are psychoactive compounds, and each is a prescribed pharmaceutical for treating psychological conditions and alcohol withdrawal symptoms . Both oxazepam and nordiazepam have been commonly detected in TWW, often at μg L−1 levels . However, there is little knowledge about the occurrence, formation, and fate of such metabolites outside the wastewater treatment systems . Several studies have focused on the uptake and accumulation of pharmaceuticals in agricultural plants as a result of TWW irrigation . These studies have demonstrated the capacity of higher plants to take up these compounds; however, until recently, relatively little consideration has been given to their metabolism in plants . Recent studies have shown that higher plants can metabolize xenobiotics similarly to humans with phase I modification reactions followed by phase II conjugation reactions using detoxification enzymes that function as a ‘green liver’ . In higher plants, phase I and phase II reactions are followed by a phase III sequestration, resulting in the formation of bound residues . Many of these studies have also highlighted a chemical-specific and species-specific nature of plant metabolism of pharmaceuticals. In this study, we examined the uptake and bio-transformation of diazepam in higher plants.Cucumber and radish seedlings were then used under hydroponic conditions to understand metabolism of diazepam and its effect on selected metabolic enzymes in whole plants.PSB-D A. thaliana cell line was purchased from the Arabidopsis Biological Resource Center at Ohio State University and cultured in a liquid culture suspension at 25 °C and 130 rpm in the dark.

Cell cultures were maintained in accordance with the ARBC maintenance protocol . The A. thaliana seed culture was produced by inoculating 7 mL of cell culture into 43 mL fresh growth media, followed by 96 h cultivation at 25 °C on a rotary shaker in the dark. After 96 h, 3 mL of the seed culture was inoculated into 27 mL fresh growth media to create an approximate initial cell density of 3.3 g . Flasks were spiked with 30 μL of a stock solution of diazepam and 10 μL of a 14Cdiazepam stock solution to yield an initial concentration of 1 μg mL−1 and a specific radioactivity of 7.4 × 103 dpm mL−1 with an initial methanol content of 0.13% . Simultaneously, control treatments were prepared by auto-claving cell suspension flasks before chemical spiking , flasks containing diazepam without cells , and flasks containing living cells without diazepam . Control treatments were used to determine adsorption,vertical farming equipments abiotic degradation, and potential toxicity to cells. Flasks were incubated for 120 h in triplicate and sacrificed at 0, 6, 12, 24, 48 and 96 h for sampling and analysis. At each sampling time point, samples were collected and centrifuged at 13,000g for 15 min in 50 mL polypropylene tubes. The supernatant was collected and stored at −20 °C until further analysis. Cells were immediately stored at −80 °C and then freeze-dried for 72 h. After drying, each sample was spiked with 50 μL of 10 mg L−1 diazepam-d5 as a surrogate for extraction-recovery calibration and extracted using a method from Wu et al. , with minor modifications. Briefly, cells were sonicated for 20 min with 20 mL methyl tert-butyl ether and then 20 mL of acetonitrile and centrifuged at 13,000g for 15 min. The supernatants were combined and concentrated to near dryness under nitrogen at 35 °C and then reconstituted in 1 mL of methanol. The cells were then extracted with 20 mL acidified deionized water and the supernatant was combined with the methanol extract for cleanup. Prior to clean-up, 100 μL of cell material extract and growth media were combined with 5 mL liquid scintillation cocktail I to measure the radioactivity in the extractable form on a Beckman LS500TD Liquid Scintillation Counter . Clean-up was carried out using solid phase extraction with 150 mg Waters Oasis© HLB cartridges that were preconditioned with 7 mL methanol and 14 mL deionized water. Samples were loaded onto cartridges and then eluted with 20 mL methanol under gravity. The eluate was dried under nitrogen and further recovered in 1.5 mL methanol:water . After re-suspension extracts were transferred to micro-centrifuge tubes and centrifuged at 12,000g in a tabletop d2012 Micro-Centrifuge . Samples were further filtered through a 0.22-μm polytetrafluoroethylene membrane into 2 mL glass vials and stored at −20 °Cbefore analysis. Extraction of growth media was done after adjusting the solution to pH 3 using HCl, and followed by SPE with Waters HLB cartridges, as described above. The extraction recoveries for the tissues and media were 88 ± 7% and 80 ± 14%, respectively. After extraction, the cell matter was air dried, and a 10-mg sub-sample was removed and combusted on a Biological Oxidizer OX-500 to determine the radioactivity in the non-extractable form. The evolved 14CO2 was captured in 15 mL Harvey Carbon-14 Cocktail II and analyzed on a LSC.Hydroponic cultivations were carried out using cucumber and radish seedlings.

Seeds were purchased from Lowes and germinated in a commercially labeled organic potting soil in a growth chamber . After the appearance of the first true leaf, uniform seedlings were selected, rinsed with distilled water, and individually placed in amber jars containing 900 mL hydroponic solution . After 3 d of adaption, plants were exposed to diazepam by spiking with 100 μL of the above stock solutions to reach a nominal concentration of 1 mg L−1 and an initial specific radioactivity of 2.5 × 103 dpm L−1 . The cultivation lasted for 7 d. A parallel treatment with an initial diazepam concentration at 1 μg L−1 and a specific radioactivity of 2.5 × 102 dpm was included to simulate more realistic exposure levels and to validate the high level treatments. The cultivation lasted for 28 d with the culture solution renewed every 3 d. Plant blanks and treatment blanks were placed alongside the treatment jars. At the end of 7 d or 28 d cultivation, the seedlings were removed from the jars. Before sample preparation, roots were rinsed thoroughly with distilled water. Harvested plants were separated into below ground biomass and above ground biomass . Flowering buds from cucumbers were also separated to observe any potential for accumulation in fruits. Tissues were freeze-dried and then stored at −80 °C until analysis. A 0.2-g aliquot of the dried plant tissue was ground to a fine powder using a mortar and pestle. Samples were extracted and prepared as described above. A six-point TCP standard calibration curve was used to determine activity.Active plant metabolism of diazepam was validated using a range of controls. No diazepam was detected in the non-treated media or the cell blanks, and there was no significant degradation of diazepam in the cell free media, suggesting no contamination or significant abiotic transformation. Moreover, no significant difference was seen in cell mass between the chemical-free control and the treatments, indicating that diazepam did not inhibit the growth of A. thaliana. Furthermore, no significant amount of diazepam was adsorbed to the cell matter in the non-viable cell control. In contrast, diazepam dissipated appreciably from the media containing viable cells, with the average concentration decreasing from 698 ± 41.5 to 563 ± 8.93 ng mL−1 after 120 h of incubation, a decrease of nearly 20% .

In the face of the potentially negative consequences of climate change on agriculture, all avenues of mitigation must be examined, and even small improvements may prove worthwhile.Bio-fuel crops have been developed as an alternative, carbonneutral energy source, among which the perennial C4 grass, Panicum virgatum L. , native to North America, can adapt to a wide range of environments, including those with marginal soils and low water input. However, in order to better manage and optimize this crop for bio-fuel production, it is important to understand the mechanisms that enable its adaptivity, and how nutrient-poor environments impact chemical composition, biomass yield and feed stock quality. A long-standing barrier to this mechanistic understanding lies in the difficulty in characterizing plant chemical composition and quantifying plant-available nutrients at the rhizosphere. Phosphorus is a critical nutrient, and poor P management poses a global risk for environmental sustainability and food security. P limitation severely restricts photosynthesis and reduces CO2 fixation, but upregulates pathways associated with organic acid/ carboxylate exudation. P limitation can also be associated with increasing biosynthesis of defense metabolites, such as increased lignification of cell walls,hydroponic vertical farming systems suggesting that changes in plant carbon allocation in response to P limitation may alter both the yield and the chemical composition of bio-fuel feed stocks, and therefore productivity.

In this light, it may be beneficial to monitor plant available P concentration and plant chemical composition during the growth season, with the goal of improving biomass production and optimizing the chemical composition for improving feed stock quality through active land management, especially when growing in marginal soils. Quantifying soil P available to plants is challenging, especially if attempting to do this dynamically during a growing season. Typical chemical extraction methods quantify only a fraction of the inorganic P pool and are typically measured in top soils prior to planting. Although the P concentration data obtained with these methods have been widely used in the literature to represent total P availability, they are not an accurate measure of P available for plant growth. Perennial grasses such as switch grass produce deep roots that explore and obtain nutrients and water from distinct locations deep into the soil, and these locations vary across the growing season and lifetime of a plant. Further, plants have developed a number of strategies to access P from different types of soil, including the adaptive secretion of compounds such as organic acids, enzymes and side rophores which either mobilize soil P directly, or indirectly through their stimulation of the rhizosphere microbiome and symbiotic fungi. Combined, dynamic growth of roots through a soil profile with distinct concentrations and chemical forms of P, an adaptive allocation of photosynthate below ground, and a micro-biome with typically unknown capacity for P mobilization, makes predicting plant available P a highly complex task.

Meanwhile, there has been renewed interest and some success in predicting plant nutrient levels using spectroscopic methods for remote sensing with the help of machine intelligence. A variety of machine learning tools have been utilized to achieve satisfactory results with independent variables obtained, for example, by visible to near-infrared spectroscopy, to predict nutrient levels in shoots in agricultural crops, total nitrogen content of soils and plant adaptive responses to stress. Most of these tools are linear models such as partial least squares regression, principal component analysis, or support vector machines often with a nonlinear kernel, likely due to their inherent robustness and reduced chance of over fitting.Thus, it is conceivable that a machine learning approach could predict nutrient availability by monitoring the biochemical signatures of plant shoots. However, to our knowledge, this aspect has not been well explored. In this paper, we use a molecular spectroscopic method to determine and quantify the organic P and inorganic P in leaf tissue. This approach also provides important information on overall plant tissue biochemistry that can be used as multi-plex signatures of a plant’s response to environmental conditions. P speciation can be quantified dynamically and feed stock quality for bio-fuel production can be inferred. We then use this tissue biochemical information to infer and evaluate plant-available P using a machine-learning model trained using a dataset from a controlled laboratory experiment. Building off this approach, we used the model to interpret plant spectral data from two field locations where contrasting available P was expected.A series of experiments in sand cultures were performed to evaluate the dose-response of plant tissue chemistry to varying N and P. The chemical signatures in plant leaves varied substantially with P and N availability in the growth media, as shown in Fig. 1. Note that the absorbance data were normalized to the maximum to show the relative concentration changes on the same scale. We observed higher cellulose, lower lignin, lower lipids, and higher organic and inorganic phosphate concentrations in the leaves of plants grown in solution with closer to optimal P concentration, and increased lipid and amide concentrations in solution with closer to optimal N concentration.

The cellulose/ lignin ratio was very sensitive to P concentration, showing a 3-fold increase from <150 μM to 500 μM P, but was not consistently sensitive to N concentration. Note that the P/N concentration likely fluctuated during plant growth and between the fluid replenishment, thus the values here refer to the average concentration through the growth. Since the C/L ratio is an important metric for bio-fuel production, we suggest that higher P concentrations would produce a higher C/L ratio for potentially increased bio-fuel yield. The higher relative amide concentration in plants grown in the lowest concentration of P compared to those in intermediate P concentrations likely reflected severe P-stress in these plants, as soluble nitrogenous compounds including amino-acids and amides accumulate in other species under P-deficiency, and consistent with our other observations. P deficiency was also associated with higher lipid content, as indicated by the increased signal from carbonyl bonds, possibly related to the production of triacylglycerides as storage compounds under P limitation. Note that while lignin concentrations increased gradually with decreasing P , cellulose concentrations showed a threshold effect with a large increase between 30 and 150 μM P, and these opposing responses manifested in a cellulose/lignin ratio that is highly sensitive to P deficiency, but not to N deficiency. The increase in lignin concentration under P deficiency was possibly related to induction of defense genes and defense metabolites and the overall shift to lower cellulose and more lignin may represent a more pathogen-resistant, rigid cell wall.Because of the strong dependence of feed stock chemical composition on soil phosphorus concentration in the controlled growth experiments, we evaluated switch grass growth at two field locations contrasting in soil P availability. The soil texture of these locations differs with the RR site being a sandy loam and 3rd Stbeing a silt loam. Chemical characterization of bulk soil samples indicated a significantly higher Mehlich-III extractable P concentration in RR soils relative to 3rd St , with no significant seasonality observed . In general, plants grew taller in the RR plot than at 3rd St, reaching maximum heights at T4. Our leaf-tissue measurements showed that leaf Pi concentration increased over the course of the growing season in both field experiments. The Pi concentration of leaf tissue collected at RR was higher than that at 3rd St , consistent with the bulk soil characterization,vertical grow rack although the plant Pi as determined by FTIR was more similar than the extractable soil P data might have suggested. This may be expected due to P homeostasis and overall biomass difference. Pi concentration showed an earlier increase in plants at Red River , presumably reflecting greater uptake early on due to higher concentrations of plant-available phosphate in the soil. Further increase in the concentration of Pi, especially in the late season along with the decrease of Po at RR may reflect mobilization of Pi for translocation elsewhere in the plant, including storage tissues that support regrow in the next growing season. The trend of organic phosphates in the lower panel of Fig. 3 shows that, contrary to that of Pi, the concentration of Po plateaued in the later stages of growth at around T4, when the plant reached maximal biomass as indicated by the maximal plant heights . Po concentration decreased significantly during senescence in plants grown on the higher P soils at Red River. This explains the transient sharp increase in Pi described above and the large decrease of Po and total P . Seasonal increase of total P content in the shoots of switch grass has been observed before35, but our spectroscopic method enabled us to dissect P speciation during the growth season.

Meanwhile, we observed a similar trend in concentration of lipid signature in the late growth stage . Since the leaves we collected tended to be younger leaves to be consistent with our sand-based experiments, the maximum P concentration at T4 may reflect a combination of P uptake over the growth period, plus reallocation from old to younger leaves, resulting in higher concentrations of major P-containing molecular classes like phospholipids and/or ribosomal RNA.Because of the critical role of P in the growth of switch grass and its strong correlation with biochemical composition for this bio-fuel species, we believe the seasonal characterization of plant available P in the rhizosphere and P speciation may be beneficial for crop management and improved environmental outcomes. We demonstrate here that a machine-learning model can be used to quantify P availability using the plant leaves themselves as sensors. Since the nutrient concentration in the rhizosphere in a hydroponic substrate is relatively well controlled, this experiment allowed us to develop training data for an ML model. We achieved a principal component regression model with a high R2 of ~1 , which allows us to predict plant-available P concentrations based on the spectral data collected on the leaf tissue from field-grown plants. The predicted P concentrations are shown in Fig. 4a. Note that in a more traditional approach, the model prediction would be further validated by another independent method to evaluate the model’s accuracy. However, such a method for accurate estimation of bio-available P concentration through the soil profile over time does not yet exist in practice. We believe that our model contains an accurate statistical description of the correlation between the P concentrations in the growth media and all the spectral features in younger leaf samples, given the high accuracy achieved with large concentration range and the high affinity of P uptake; thus this model can be used for prediction of the P concentration available to each plant within the rhizosphere. The predicted P concentration available to the plants shows a gradual increase and then a sharp dip in T4 when the plant reached maximal biomass, reflecting an increase in P uptake at T4 and a quick decrease in P uptake at T5, when the shoot senesces. As a perennial plant, switch grass remobilizes and stores P in roots to support the subsequent year’s growth, consistent with previous observations with P remobilization efficiency ranging from 31% to 65% in different ecotypes. The increase in Pi in the tissue at the later stage of growth, the strong correlation of cellulose content with total P concentration, and the large reduction of plant available P in the rhizosphere provided us with a clear picture of the interaction of P availability and tissue composition during the life cycle of switch grass. This may be a consideration in the timing of harvest to achieve optimal bio-fuel yield and reserve P in the root for the optimal growth in the next year. The total P concentration in the roots followed a similar seasonal trend, showing a reduction of total P concentration near the period of maximal growth and at least a partial recovery in T5. The P concentrations measured in roots by ICP-MS at T5, a point at which plants had begun senescing, were similar across the two field locations , with a mean value of 729 ppm at 3rd St and 703 ppm at RR, respectively. The similar root P concentrations at T5 may be reflective of reduced plant P demand during senescence as well as plant P re-allocation and is described further below.