The outcome of this R&D effort will be the identification of hardware design approaches and efficient software algorithms that can be implemented as part of an ITS Upgrade and similar detector systems in other collider detectors. We have established a number of benchmark observables of heavy-flavor production at high and low-energy scales that became a strategic part for the future heavy-ion program at the LHC. Moreover, these observables connecting the precision of the single particle tracking and full jet reconstruction put stringent requirements on the performance of the new Si-detector developed for experiments at high collision rates. A major achievement was to establish a working environment for massive computing simulations using the High-Performance Computing clusters . The lightweight framework combines the conventional computing strategy with the possibility to leverage high capacity and parallelism within the HPC for the future simulation tasks. These developments, utilizing the multi-node and multi-core architecture will provide us with the necessary framework for the studies of the time-stamp based digitalization software for the purpose of future detectors. We have started the first tests of the novel setup. On the hardware side of the project, the main performance benchmarks of the principal elements of the readout system have been identified. The processing chip architecture will be defined within the first quarter of 2015 we will be able to devise the necessary hardware design approaches during the second year of the LDRD funding. Enabling more efficient flow of electrons across the boundary between living and human-made systems is critical for bio-energy technologies, including harvesting energy from wastewater and efficient synthesis of fuels from sunlight and CO2. Over the last 20 years, proteins that mediate electron transfer across this abiotic-biotic interface have been identified, purified,mobile vertical grow tables and structurally characterized in isolation. Yet, there remains a dearth of information about the in operando structure of the protein-material interface.

Our overall goal is to address these critical structure-function knowledge gaps so that we can redesign proteins for more efficient electron transfer to materials. The Mtr pathway of Shewanella oneidensis MR-1 is currently the best understood extracellular electron transfer pathway. It consists of a periplasmic decaheme cytochrome c , an outer membrane non-cyt c porin-like protein and an outer membrane decaheme cyt c . The outer membrane decaheme cyts c are the most unique and important components of this pathway since these are the proteins that transfer electrons to abiotic surfaces such as minerals or electrodes. However, there is little information about the protein-material interaction and there is no information on which amino acid residues of cyt c are recognized or associated with the material. This project seeks to uncover mechanisms of interaction between these cyt c and materials . Obtaining adequate amounts of outer membrane decaheme cyts c for structural studies is challenging because of intrinsic difficulty of expressing decaheme cyts c and their low solubility. This year we overcame these challenges to express and purify two variants of the decaheme cyt c, MtrF. First, we expressed a lipidated MtrF with a histidine-tag at its C-terminal. This protein was then purified in the presence of mild detergent using both affinity chromatography and ion exchange chromatography to greater than 90% purity with a net yield of ~1 mg protein/L culture. To eliminate the need for detergent, which will in turn facilitate structural characterization, we also expressed an MtrF variant lacking any lipid modification. To do so, we created a DNA construct containing the MtrB signal sequence, followed by the MtrF coding sequence and a Cterminal histidine-tag. This protein was secreted directly into the culture media, and we successfully purified it to greater than 90% purity at a yield of ~0.5 mg/L culture using a single affinity chromatography step.

We also characterized both the lipidated and non-lipidated MtrF to assess whether they are fully matured decaheme cyts c. UV-Vis spectroscopy of these proteins shows the characteristic peaks of c-type cytochromes at 408, 535, and 552 nm. Additionally, Electrospray Ionization Mass Spectrometry shows that the mass of these proteins is equal to the expected mass of decaheme MtrF. Thus, we conclude that these proteins are indeed fully matured MtrF. Lastly, we have established assays to measure the binding affinity of MtrF to inorganic materials. Specifically, we have shown that the fluorescence of MtrF is quenched upon exposure to certain materials. This measurement will allow us to determine the partition coefficient of MtrF for different materials, and thus greatly add to our understanding of the thermodynamics of proteinmaterial binding. Our most significant accomplishment has been the development of a flexible and inexpensive method for assaying the phenotypes of thousands of genes in parallel using transposon mutagenesis and DNA barcode sequencing. The key to the approach is the introduction of random DNA barcodes into the transposon. A mutant library for a given microbe is characterized a single time using the time-consuming and expensive TnSeq protocol. All subsequent assays to measure mutant fitness for thousands of genes in parallel only require the quantification of the DNA barcodes, a simple and inexpensive assay termed BarSeq. To date, we have applied RB-TnSeq to 21 diverse bacteria and generated over 3,000 whole genome mutant fitness profiles, representing ~9 million gene fitness measurements. We have identified phenotypes for over 20,000 bacterial genes including thousands of genes with no previous known function. We are using these data to predict gene function in diverse species using correlations in mutant fitness across hundreds of diverse growth conditions. In the current year of LDRD funding, we are working to extend the RB-TnSeq to additional, diverse microorganisms relevant to bio-fuel production, bioremediation, and nutrient cycling including archaea. In addition, we are working to increase the efficiency of transposon mutagenesis in diverse microorganisms by engineering new vector variants.

Lastly, we are developing computational tools to enable the microbiology community at large to mine our functional genomic datasets and to globally infer gene function across all sequenced microbial genomes using homology-based methods. There is a strategic imperative for investment in bio-manufacturing infrastructure at Berkeley Lab. In the 2013 State of the Union address, Barack Obama said: “I’m announcing the launch of three more of these manufacturing hubs, where businesses will partner with the Departments of Defense and Energy …. And I ask this Congress to help create a network of fifteen of these hubs and guarantee that the next revolution in manufacturing is Made in America.” While the complete specifications for these fifteen national manufacturing hubs have yet to be announced, given the Administration’s 2012 National Bio-economy Blueprint, it is very likely that there will be at least one biological manufacturing hub. This FY14 lab-wide strategic LDRD aims to place Berkeley Lab in a dominant position from which to lead a competitive effort that brings a bio-manufacturing hub to Northern California. While Berkeley Lab is uniquely positioned to leverage DOE investments in the JGI, NERSC, KBase, JBEI, and the ABPDU for bio-manufacturing competitive advantage,mobile vertical farm the capabilities and expertise at these facilities have yet to be integrated and successfully demonstrated as a ‘one stop-shop’ from target molecule identification to industry-ready microbial production strains. Operationally, this requires bio-manufacturing pipeline component standardization and interoperability; the ability to generate, QC, and track large numbers of DNA constructs; seamless integration of microbial strain construction with high-throughput functional assessment; and machine intelligence to learn from previous successes and failures to drive forward the next design iteration. This strategic LDRD aims integrate existing and develop new Berkeley Lab capabilities and expertise to create revolutionary bio-manufacturing infrastructure. This infrastructure will enable the rapid design, implementation, and assessment of target molecule production by iteratively uncovering and resolving critical biosynthesis bottlenecks. This LDRD aims to tackle a stress-test and a challenging biosynthesis demonstration project to drive a compelling success story narrative, while in parallel addressing key infrastructure gaps. Demonstrating the ability to go from target molecule to functional construct within a year for important and valuable targets will cement Berkeley Lab’s leadership in bio-manufacturing. Our major accomplishment in FY14 was to design and very nearly complete the DNA construction of a refactored actinorhodin antibiotic pathway . This is an impressive feat, as it constitutes a record-breaking refactored secondary metabolite pathway at 22 genes and 30 kbp in length. [In early FY15, we completed and validated the construction of the refactored actinorhodin pathway, transformed the construct into a modified Streptomyces coelicolor host lacking its native actinorhodin pathway, and detected the production of actinorhodin.] Sample preparation and analytical mass spectrometry methods were developed for actinorhodin and other secondary metabolites including violacein, our selected biosynthesis stress-test project target. The completion of the DNA construction of a 60,000-variant refactored violacein pathway combinatorial library is anticipated in the first half of FY15. Significant progress was also made in the development of machine learning algorithms for processing the violacein combinatorial library production data, and in the further development of DNA construction process tracking and assistance software.We succeeded in collecting “pseudo-SINBAD” data from our synchrotron beamline after careful re-alignment of the spindle. Collecting back-scattered reflections and extracting anomalous differences form them proved tractable, and indeed easier than expected.

The problems of non-isomorphism and reflecting crystal design proved to be significant, and we have new projects now investigating these two fronts. Non-isomorphism is a problem that must be overcome for any future multi-crystal macromolecular crystallography endeavor to move forward, and this fact is growing in appreciation in the XFEL and synchrotron communities alike. We discovered that ambient humidity can have a dramatic impact on isomorphism, and we are preparing a manuscript on this phenomenon, which suggested apparatus for controlling it. The design of a high-angle soft-X-ray monochromator also requires further development. This technology is critical for self-seeding in the soft and “tender” X-ray FELs currently under development, so we anticipate significant interest in it. What we learned is that the art of polishing and etching these crystals is absolutely key to preventing severe degradation of the beam emittance at each bounce, but existing manufacturing methods are optimized only for silicon and new etching protocols must be worked out for alternative materials. We also found that the current status of dynamical theory of reflection is severely lacking in the soft X-ray region, and we re-formulated this theory from first principles. We believe a highZ and large-cell material is optimal if crystals of sufficient quality can be prepared. Bismuth Telluride is a promising candidate. Nitrogen is an essential component of proteins and consequently a key element for life and cell development. Mineral N is often limited for plants, which consequently reduces plant growth and biomass yield. While this practice has been partly responsible for the ‘green revolution,’ it has come at high environmental and economic costs. In natural ecosystems, plants have developed strong relationships with microbes to cope with the low availability of essential nutrients such as N. For example, plant rhizospheres contain N2-fixing bacteria that are able to fix atmospheric N2 without the requirement of forming symbiotic association with a host-plant, however several of them depend on plant root exudates for carbon supply. Furthermore, endophytic bacteria colonizing root, stem and leaves of plants with N2-fixing function have been identified in several plants. The aim of this project is to optimize benefits from interactions between plants and free N2-fixing bacteria communities to provide adequate amounts of assimilable N to host-crops. This would reduce fertilizer consumption and carbon footprint of feed stock production, and greatly improve the sustainability of biomass production. Several additional N-fixing strains were isolated from plant isolates were phylo-typed based on 16S rDNA analyses. The genome sequences of several diazotrophic strains isolated were obtained through collaboration with JGI. Genomic comparison and extensive phenotypic characterizations were performed with a few of these strains. Most of the endophytes were capable of utilizing a broad range of carbon substrates. We analyzed the ability of the endophytic diazotrophs to fix atmospheric N2 under varying temperature in-vitro.Results indicated that N-fixation occurred optimally under optimal growth conditions. In addition, biochemical and genomic studies revealed that a bacterial strain could produce from a plant exuded compound the plant growth promoting plant hormone. This discovery led us to the engineering a plant to boost bacterial production of the plant hormone . Coupled with the ability to provide fixed N2 to the plants, this is an extremely desirable trait that can be utilized for sustainable agriculture of biofuel crops such as Switchgrass.

Our second significant accomplishment was that we developed methods of addressing and removing image artifacts on a very large number of images, allowing extraction of the porespace for analysis. All FIB-milled samples of heterogeneous samples have artifacts resulting from imperfect beam shape and the nature of sample heterogeneities, and these have previously hindered extraction of porespace. Third, we successfully simulated single-phase flow through the identified and discretized porespace from one of our largest image sets and are processing our largest image set for flow simulation. The overarching aim of the proposed research is to improve understanding of the degree to which ecological and land management processes influence the physical Earth system and feedback to climate change. The specific goals of the current proposal are aimed at evaluating and improving models of land-atmosphere interactions and climate change field experiments intended to reveal ecosystem responses to climate change. Land surface models are used to represent the properties of vegetation and soils that influence weather and climate through their effects on energy, water and greenhouse gas fluxes. Until recently, land management and cropping systems have not been represented well by these models even though crop growth and management practices have potentially large impacts on many aspects of weather and climate. We are using ground based and satellite observations, including from the ARM site and Ameriflux network towers in agricultural regions to validate and improve a coupled atmosphere-land surface model, WRF-CLM. Such model improvements will improve predictions of weather and climate in agricultural regions,hydroponic nft and in regions undergoing changing land management.

Initial efforts focused on biogeophysics have laid the groundwork for evaluation and improvement of modeled biogeochemistry. Climate change experiments are used to quantify vegetation responses to altered climate states, and mechanisms underlying the responses. Infrared heating is increasingly used to manipulate temperatures. This method has rarely been evaluated in terms of its effects on winter and early spring conditions, nor have comparisons been drawn across multiple sites with the same treatment implementation. We are using data from existing experiments in the Rocky Mountains to examine snowpack and soil responses to the climate manipulations. A key accomplishment in FY14 has been submission of papers describing a regional climate-land surface model that includes dynamic crop growth and irrigation, and its application to assess effects of irrigation decline on heat waves in agricultural regions. The first paper is still in review at Climate Dynamics, while the second was rejected by Geophysical Research Letters and is in revision for Environmental Research Letters. Another analysis was completed to evaluate how irrigation influences land-atmosphere coupling strength. This analysis was presented at the AGU Fall Meeting in December 2014 and is in preparation for a journal submission in FY15. Further progress was made in spinning up an offline version of CLM with crops in the Southern Great Plains – a complex agricultural region with intensive observations. With Noah Molotch we revised and submitted a manuscript for publication describing the effects of experimental heating on snow accumulation and melt, and comparing this to a similar site further south and to a snow pack model. The paper was recently accepted for publication pending minor revision at Forest and Agricultural Meteorology. We also conducted initial analysis of gap-filled microclimate data from multiple warming experiments for two separate papers; one focused on the relationship between spatial variance in soil temperature and the mean over the growing season, and another describing heating effects on soil microclimate. The first analysis was presented in a poster by PhD candidate Danielle Christiansen at the MtnClim meeting in Utah in October 2014.

The second was included in invited talks at Utah State and Penn State Universities in 2014. The purpose of this project is to observe—in situ, and over a range of length- and timescales—how changes in chemical conditions affect the conformation and reactivity of natural organic matter relevant to both soil and shale systems. We believe an ambitious but attainable goal will be to explore such chemistry at the smallest relevant scales, from NOM aggregates and ultimately to single macromolecules. Natural organic matter is a complex mixture of organic molecules and associated metals and is ubiquitous in the near earth surface environment. In the form of soil organic matter and dissolved organic matter, NOM chemistry impacts practically every meaningful ecological process in soils, sediments, ground waters, surface waters, and marine systems. This reduced carbon is important on a mass basis for global carbon cycling, and its’ long-term fate is a major outstanding question for predicting the feed backs associated with global climate change. As kerogens in sedimentary rocks, NOM chemistry plays a key role in energy production capacity, efficiency and associated environmental impacts. A core approach in this project is the application of recent technical developments from the Schuck group in surface-enhanced and tip-enhanced Raman scattering for nanoscale analysis.Raman scattering was chosen as the central analytical technique because it has good sensitivity to organic matter composition and compatibility with aqueous environments. We will also plan to take advantage of the technique of nano-FTIR spectroscopy and X-ray ptychography to explore the nano-scale structure of organic matter in natural shales. The results are highly complementary to information obtained by electron and X-ray imaging and spectroscopy.

Our most significant accomplishment to date has been to show that the naturally occurring iron oxide, magnetite, can be used as a SERS substrate to probe the interaction of organic molecules with natural mineral surfaces. We believe this development is the first demonstration of SERS behavior in a natural metal oxide and will be broadly useful to the study of NOM dynamics. A manuscript on these results has been prepared by Namhey Lee and is currently under review at Journal of Physical Chemistry Letters. We have also collected preliminary ptychography data on a natural shale sample. While data processing is still underway, we believe that this too is a world unique measurement and will have important implications for our understanding of the nano-scale structure of shale materials. Urban farm systems are a dynamic assembly of interdependent systems , but there is a fundamental lack of an understanding of how these systems work independently and as a group. Consequently there is only a vague understanding of how commercially and environmentally sustainable urban farming systems can be designed and operated. While there are many innovations around the country in each of the areas described above, for urban farming efforts to be replicable, scalable and cost effective, rigorous scientific knowledge needs to inform their design and implementation. To have impact at scale, technology needs to be developed and tested in partnership with those who would be the on-theground urban farmers. We propose a partnership with three cities: Oakland, CA; Toledo, OH; Chicago, IL. The goal would be to develop a rich relationship with existing urban agricultural efforts already going on in these cities and use these relationships to inform ongoing development decisions; test and fine tune new technologies as they are developed, and understand the implementation framework that will support adoption of proposed technologies. Specific tasks that would be conducted in partnership with these places would include: Initial scan of existing urban agriculture efforts and assets: Each of the proposed cities already has existing urban agricultural efforts. Understanding the scope and scale of these efforts, the innovations that are already being put into play,hydroponic channel and the challenges and barriers being experienced will be an important grounding for this work. Creation and regular engagement of a partner’s group in each city. We propose in each city to create a partner’s group which would include between 8 to 10 groups involved in urban agricultural efforts. Quarterly meetings with these partner groups would inform larger project decision-making. Testing of new urban agriculture methods, strategies or tools: As the work evolved these partner groups would serve as the on-the-ground testers of new technologies. Although CO2 has been used as an injection fluid for enhanced oil recovery for decades, CO2-EOR remains inefficient. The primary limitation of CO2-EOR effectiveness has been the low viscosity and high mobility of CO2 that results in injected CO2 bypassing oil resulting limited displacement efficiency. After decades of extensive research and practice on ways to reduce CO2 mobility, the CO2-bearing foam approach seems more promising. A variety of surfactants have been designed for generating foams, however, their commercial applications are still lacking. The major barriers limiting industrial applications of CO2 foam-EOR include the high costs of surfactants, compare to the price of oil. Concerns over environment impacts also exist for some of these surfactants. If a CO2-compatible surfactant that overcomes these limitations can be identified, such a discovery will benefit oil recovery, while at the same time increase the incentive to implement carbon capture and increase the capacity of geologic carbon sequestration in depleted oil reservoirs. The overall objective of this project is to develop a new and unconventional material, which is less expensive, non-toxic and highly effective as a substitute of conventional synthetic surfactants, for increasing CO2 viscosity and control its mobility in EOR. The Earth’s near surface sediments contain deposits of natural organic matter produced primarily from degradation of plants.

We hypothesize that these organic deposits contain a large fraction of surfactant-like material. This material can be extracted, and dispersed in the form of nanoparticulates when re-dissolved in water. These nano-particulates can stabilize supercritical CO2- brine foams, and are more cost-effective and environmentally friendly than synthetic surfactants currently used in CO2 foam-EOR. Our most significant accomplishment has been to identify the sources of NSS , and all has been successfully tested as source materials for NSS extraction . We have developed a method to produce the NBS. The NBS is capable of generating and stabilizing dense scCO2-in-water foams containing CO2 at up to 80 volume% with controllable effective viscosities up to 40 cP, three orders of magnitude higher than that of pure CO2 at the same pressure and temperature. We measured interfacial tension of NSS liquid solutions paired with N2, and paired with scCO2 as function of NSS solution concentrations. The NBS performs as an effective surfactant, significantly reducing interfacial tensions between the N2-water and the supercritical CO2- water interfaces. We have also just built a modified high pressure and high temerature foam generator and successfully tested foam-stability vs. temperature. The NSS is a N2- and CO2- compatible and high quality surfactant. We are in the process of optimizing NSS extraction method, estimating extraction costs, and optimizing laboratory CO2-EOR tests in cores. The purpose of this project is to develop a small suite of highly energy-efficient and low-cost medical devices, in order to enable affordable healthcare in rural Sub-Saharan Africa and South Asia. The specific devices developed as a part of this—identified in LIGTT’s “50 Breakthroughs” study—include an infant warmer , and continuing development of a portable solar-powered vaccine refrigerator . For the infant warmer, the key technical questions that needed to be overcome were the choice of a safe, low-cost phase change material which could maintain the appropriate temperature for more about 6 hours; a low-cost, comfortable , safe and sturdy external material; and an overall system design. For the vaccine refrigerator, the key technical question was the appropriateness of thermoelectric materials to effectively and efficiently transfer heat away from the vaccine chambers. Early-stage, conceptual architectural design has suffered radical changes in recent years, due to development on powerful modeling tools that allow fast, parametric modeling of complex building geometries, rapid-prototyping of 3D models, and links to diverse performance simulation tools. As parametric design progressively becomes the preferred method for early-stage conceptual design, the use of Generative Design Methods represents the next step in this process, by introducing performance-based optimization into architectural design, namely in terms of low-energy design. As proofs of concept, this project explored the implementation of Generative Design Methods for optimization at multiple design scales, namely: 1) Design optimization and prototyping of a light redirecting system; main research questions addressed were: a) Currently available methods/tools to use in generative design optimization; b) Reliability and error margins introduced by these methods; c) obstacles for more accurate methods; 2) Whole-building early stage design, for multi-criteria lighting, heating and cooling energy optimization. Light Redirecting System: The system simulated was built in light-weight aluminum, with a 3M high reflectance film applied on top of louvers , and a lower face coated in 70% light reflectance white mate paint, to reduce glare to occupants.

Experimental data from an aeroponic system were selected to evaluate the described approach to model the influence of temperature on the vertical penetration of the root system. The experimental setup in an aeroponic system with no soil and no water movement has the advantage of evaluating the effects of temperature on root growth while limiting the effects of other factors. The experiment was conducted in a greenhouse with bell pepper at the Jacob Blaustein Institutes for Desert Research, in Midreshet Ben-Gurion, Israel . The objective was to evaluate the effect of three different root zone temperatures on root and plant growth. Six bell pepper plants were cultivated from 7 Jan. to 20 Feb. 2015 in aeroponic pots mounted on top of the aeroponic systems. The aeroponic apparatus comprised circular pots made from plastic material with a diameter of 50 cm and a depth of 14 cm. Within each thermally isolated aeroponic pot, misters were fixed to produce the desired fine mist sprayed directly onto the plant roots. The computer-controlled spraying varied from 8-s sprayings at 1-min intervals, depending on the growth stage of the plants and the temperature of the greenhouse. Three different water temperatures were applied to each treatment, namely 7, 17, and 27°C. The air and root zone temperatures were measured daily. The air temperatures in the greenhouse were 25°C during the day and 18°C at night. The treatments were replicated twice, leading to six tanks in total. The maximum rooting depth of each plant was observed four times during the 44 d of plant growth. In the aeroponic systems,rolling bench all other factors that affect the growth and development of roots were rendered insignificant during the treatment.

The implemented modeling approach to simulate the temperature dependent vertical root penetration was evaluated by comparing modeling results with the measured maximum rooting depths in the experimental aeroponic system with bell pepper. Because two approaches were implemented in HYDRUS to describe both time-dependent potential root growth and the temperature stress factor, four combinations are thus available to describe the temperature-dependent root growth. All four models were tested against measured maximum rooting depths to validate their ability to describe the temperature- and time-dependent vertical root penetration. The four combinations are summarized in Table 3. Were the these root growth models able to properly simulate the influence of temperature on vertical root penetration, a single combination of model parameters for each model that could reproduce the measured maximum rooting depths for all temperature treatments would have to exist. An overview of the model parameters that have to be specified for each model is given in Table 4. Table 4 shows that, depending on the model, four or six parameters have to be specified to model temperature-dependent root growth. In a complex soil water flow model such as HYDRUS, four to six additional parameters can significantly increase the calibration effort and parameter uncertainties. The temperature-dependent modeling approach was tested outside of the HYDRUS implementation and within a MATLAB environment. The goal of the evaluation was to determine whether the combination of the time-dependent root growth functions and the temperature stress functions were able to reproduce the measured rooting depths under the given boundary conditions.

A global sensitivity analysis was conducted using the Sobol¢ method to reveal the key parameters of each model and to determine the contribution of the uncertainty of each parameter to the uncertainty of the model output. The Sobol¢ method is based on variance decomposition and provides the impact of each parameter and its interactions with other parameters on the model output . This type of global sensitivity analysis can be applied to nonlinear and non-monotonic models and is a widely used tool for sensitivity analysis studies. Its ability to account for interactions between model parameters is an important advantage of the Sobol¢ method .An overview of studies using the Sobol¢ method for sensitivity analysis in hydrological modeling was provided by Song et al. . The method has already been applied with the HYDRUS software package by Li et al. , Brunetti et al. , and Wang et al. . Sobol¢ proposed that the total variance of the model output can be decomposed into component variances of individual parameters and their interactions. The first-order sensitivity index quantifies the main effect of the ith parameter, Xi . This sensitivity index denotes the part of the total variance due to Xi without considering the interactions with other parameters. The total-order sensitivity index additionally includes the proportion of the variance due to the interactions of Xi with the other parameters. The values of the indices vary from 0 to 1, where 0 stands for no influence and 1 for a high influence on the variance.The number of parameter sets in the sensitivity analysis of the four models was set to 10,000. This number was initially set higher than in studies of Brunetti et al. and Zhang et al. to avoid a time-consuming convergence analysis of the sensitivity analysis and to achieve a higher accuracy of the sensitivity analysis, which increases with an increasing number of model runs.

The p parameter depends on the considered model and is either four or six . To calculate the sensitivity indices for all i = 1, …, p parameters, Matrix AB i has to be evaluated p times. The total number of model runs required to calculate the sensitivity indices for all parameters of each model were M = N. Archer et al. suggested using bootstrap confidence intervals to evaluate a suitable accuracy of the sensitivity estimates. For this reason, each estimation of the sensitivity indices was repeated 500 times to evaluate the 25th and 75th percentiles of the sensitivity indices. The small number of repetitions is due the fact that the sensitivity analysis of four models, each including four or six parameters, requires a high level of computational effort. Evaluated percentiles were used only as an additional parameter to assess the sensitivity indices .Rather than using experimental data, which may be subject to various errors and effects of various factors, the sensitivity analysis was performed using a hypothetical data set in which observed rooting depths LObs were generated by running all four models with a predefined parameterization. The cardinal temperatures Tmin, Topt, and Tc were set to 8, 23, and 27°C, respectively. A complete list of parameter values for the model runs is provided in Table 5. The boundary conditions were similar to those of the aeroponic experiments. The temperature was set to 22°C during the first 14 d and to either 7, 17, or 27°C during the remaining time period of 150 d. Additionally,grow table hydroponic a fourth scenario was considered with a temperature of 37°C after the first 2 wk to make sure that the specified cardinal temperatures would lie within the applied boundary conditions to determine their influence on the model output. Each model was thus executed four times with four different temperature boundary conditions to generate data for Lsim in Eq..The Differential Evolution Adaptive Metropolis algorithm was used for optimizing the model parameters and for model calibration. The DREAM algorithm is based on Bayesian statistics; it runs multiple different Markov chains to generate a random walk through the search space. Based on a proposal distribution, the sampler evolves to the posterior distribution by iteratively finding solutions with stable frequencies stemming from the fixed probability distribution . The Gaussian likelihood function was used to summarize the distance between model simulations and corresponding observations. The residuals were assumed to be independent and normally distributed while the measurement error was neglected.Eight Markov chains were run with a set of 5000 generations. The initial state of each chain was sampled from a Latin hypercube. The parameter space of each parameter was defined by using the same boundaries as were used for the sensitivity analysis . These parameter limits also define the search domain for the predominantly physically based parameters . The calculation time for the DREAM optimization of a single model was approximately 10 min, with no parallelization needed on a machine with the following specifications: Intel Core i7–4710HQ CPU with 2.50 GHz of RAM and 12 GB of storage.We evaluating the effects of different factors on root growth with two examples using the new root growth module in HYDRUS-1D and HYDRUS-2D.

Then we analyzed the collected experimental data and the evaluation of the temperature-dependent root growth modeling approach. Therefore, we first collected data on the experimental outcomes. Second, we carried out the sensitivity analysis to evaluate the sensitivity of the modeling results to various input parameters and identified which parameters need to be fitted and which can be set to values from the literature. Third, we used the DREAM optimization approach to analyze the collected experimental data while considering the results of the sensitivity analysis.We used two hypothetical examples that demonstrate the implemented root growth model and the impact of various environmental factors on root growth. In the first example, we used HYDRUS-1D and simulated optimal root growth as well as root growth restricted due to low water availability, temperature, texture, and bulk density. In the second example, we used HYDRUS-2D to again simulate optimal root growth and then root growth affected by a nonuniform distribution of water contents due to asymmetrical irrigation.Figure 2 shows examples of the development of simulated root systems under the influence of various environmental factors compared with the potential development of the root system, independent of environmental factors . The soil profile was considered to be homogenous, consisting of 10% sand, 50% silt, and 40% clay and having the bulk density of 1530 kg m−3. The parameters for the soil hydraulic functions of van Genuchten and Mualem were estimated from the textural information using the Rosetta module of HYDRUS-1D. Time-dependent root growth was simulated using the function of Borg and Grimes . Model 2 from Table 2 was selected to describe the potential root length density distribution. The maximum potential rooting depth was set to 120 cm, and root growth was considered for 90 d . The upper and lower boundary conditions were set to atmospheric boundary conditions with surface runoff and free drainage, respectively. In Scenario 1, the development of the root length densities was simulated for drought conditions, which may cause aeration stress and root senescence . Potential evaporation and transpiration rates were set to increase stepwise at the beginning and remain constant at the end of the simulation. Irrigation took place at irregular intervals and covered only 25% of the potential evapotranspiration. Due to low water availability, the root system was underdeveloped compared with the reference simulation when no restrictions on root growth were considered . The root length density in this scenario is reduced compared with Scenario 0, mainly close to the soil surface because the drought stress is highest in soil horizons with the highest root water uptake. Scenario 2 considered the effects of texture and bulk density on the development of the root length densities and vertical root penetration. The results show that due to the increased soil strength , the potential maximum rooting depth of 120 cm was not reached. The additional stress due to the soil strength negatively influenced the vertical penetration of roots, in addition to the effects of root senescence due to the drought stress in Scenario 1. Scenario 3 considered the influence of different root zone temperatures on the root length density development. In this case, only the option to consider the influence of temperature on root growth was enabled. The development of the root system was simulated using the root growth function of Borg and Grimes in combination with the temperature stress function of Jones et al. . The parameters Tmin, Topt, and t m were set to 5°C, 35°C, and 90 d, respectively. The temperature throughout a soil depth of 150 cm was set to a constant value of 10°C. The temperature at the soil surface was set to 25°C during the first 14 d and to 30 and 7°C in Scenarios 3a and 3b, respectively, until the end of the simulation. The results show that the root system in Scenario 3b, which was exposed to temperatures close to Tmin, was underdeveloped compared with the root system in Scenario 3a, which was exposed to temperatures close to Topt.

Additionally, we wanted to test the contribution by each Phytophthora species to leaf decomposition and to determine if there was any difference depending on leaf senescence based on their differing trophic adaptations. While the inclusion of natural stream water in these experiments is an imperfect approximation of natural conditions, namely in excluding both shredder organisms and other microorganisms eliminated in the holding period, it had the potential to reflect the interaction of the inoculated Phytophthora species with bacteria, protozoa, fungal communities, and possibly micro-invertebrates that persisted in stream water. Though the effect of stream water treatment was not statistically significant in the models, a noticeably higher occurrence of P. ramorum on brown leaves in sterile stream water treatments compared with non-sterilized stream water additions and also higher detection of P. ramorum spores by baiting in sterile compared to non-sterile stream water treatments both indicated that there was some difference between the two treatments. Though a much greater diversity of organisms likely influences this system under natural conditions, our previous research exposing leaves in natural streams demonstrated both P. ramorum and clade 6 Phytophthora species effectively colonize California bay leaves under natural conditions. As expected, based on previous work, both Phytophthora species rapidly colonized more than 60% of the leaf area of green leaves in both experiments. That P. ramorum also persisted on green leaves at high levels for the entire 16 weeks despite the loss of approximately 40% of leaf biomass stands in contrast to our previous findings where its colonization of leaves peaked within a few weeks after exposure in natural streams,ebb and flow table but then rapidly dropped to very low levels as colonization by clade 6 Phytophthora species rose and persisted at high levels.

This is evidence that the reduced recovery of P. ramorum from green leaves in natural streams as decomposition progressed was due to displacement from saprotrophic organisms like clade 6 Phytophthora species. Unfortunately, P. ramorum was completely suppressed from colonizing leaves in combined inoculations with P. gonapodyides and it could not be determined if the pattern observed in field experiments would occur under these simulations when both species were present. The suppression of P. ramorum colonization of green leaves in combined Phytophthora inoculations—consistent across all three experiments—was surprising because both species were effective at colonizing leaves when inoculated alone. One explanation could be that sporulation of P. gonapodyides from mycelial mats occurred earlier than that of P. ramorum and that the latter was therefore precluded from leaves because in all experiments, full colonization of green leaves by P. gonapodyides occurred very rapidly. Indeed, in the first experiment, colonization of P. gonapodyides occurred more rapidly on green leaves than that of P. ramorum . However, baiting two days after inoculation in the second experiment showed that P. ramorum spores wereactive in the microcosms where it was inoculated alone, but almost absent in the combined inoculation microcosms. This suggests that the presence of P. gonapodyides itself may have suppressed sporulation by P. ramorum. The rapid leaf colonization by P. gonapodyides in these microcosms also contrasts with the slower colonization that was observed in natural streams and may be an artifact of high inoculum loads and the relative abundance of substrate. The aim of these experiments was to characterize the capacity of each organism for growing and reproducing from each type of leaf rather than estimating typical colonization and decomposition in streams. Though logistically more difficult to prepare and standardize for an experiment of this magnitude, using sporangia or zoospore inoculum rather than mycelial mats may overcome the problem of uneven inoculum activation, the success of which we have experienced in smaller scale experiments.

Alternatively, the use of colonized plant tissue instead of mycelial mats as a source of inoculum may also produce a different outcome from the suppression of P. ramorum that we found with this approach in mixed inoculations. Interestingly, the kind of succession observed in field experiments did occur in a few control microcosms into which both Phytophthora species were accidentally contaminated . However, the limited occurrence and unknown relative quantity of original inoculum precluded more substantial evaluation. In any case, the suppression of P. ramorum sporulation in treatments where P. gonapodyides was present raises the question of what mechanism was responsible for the effect. It also furthers the impression that P. gonapodyides and other clade 6 Phytophthora species may have a moderating effect on the presence of P. ramorum in streams. The green leaves that we used were of mature cuticle and collected in midwinter and late summer for the first and second experiments, respectively. While some seasonal variation in susceptibility to P. ramorum infection has been reported in California bay leaves, the physical and chemical properties of mature leaves have also been reported to be relatively consistent throughout the year. Our results were similar for both experiments, and therefore, any variation in the leaves was overcome by experiment factors. The extensive colonization of brown leaves by P. gonapodyides and their limited colonization by P. ramorum is consistent with previous work where we showed that the former is a competent saprotroph while the latter is relatively ineffective at colonizing dead tissue. A significant discovery in this work was that P. ramorum colonized yellow, senescent leaves that were still fresh and had an intact cuticle to nearly the same degree as it did green leaves. At this stage, though chloroplasts and most of the protein content are gone from leaves, the cells are expected to be still alive, while in brown leaves that have dried the cells are no longer biologically active.

In fact, colonization of the yellow leaves by P. ramorum was not quite as extensive as its colonization of green leaves in the second experiment, which ran more or less concurrently and in which green and brown leaves were maintained in separate microcosms , though the difference between the separate experiments was not analyzed statistically. Though green leaves are shed into streams as a relatively low proportion of total litter, yellow leaves, often shed directly into streams from trees, constitute a much greater proportion of leaf litter in streams . This indicates that a great proportion of leaf litter in the streams is suitable for colonization by P. ramorum, and conforms to the regular recovery of this pathogen from natural leaf litter. Furthermore, the degree of colonization of yellow leaves by both Phytophthora species remained persistent throughout the 16 weeks, as with green leaves in the other experiments,growing lettuce hydroponically suggesting that the same kind of succession may be expected in these leaves as seen with green leaves in natural streams. Also consistent with previous findings with leaves colonized in naturally infested streams, leaves colonized by both Phytophthora species were generally conducive to sporulation as detected by baiting from the microcosms. Phytophthora gonapodyides was consistently recovered from P. gonapodyides-only and combined Phytophthora species inoculation treatments where it had colonized all green and brown leaves at all sampling points. The results from baiting of P. ramorum spores from microcosms were less regular, but nonetheless, mostly successful from microcosms containing colonized green or yellow leaves and occurred minimally from microcosms containing brown leaves which were colonized at only very low levels. The relatively less frequent recovery of P. ramorum by baiting from microcosms with non-sterilized stream water, not observed for P. gonapodyides, may be the consequence of P. ramorum not being well adapted to sporulation in biologically active aquatic environments or relying on different environmental signals. Nevertheless, these results confirm that both of these Phytophthora species can sporulate from colonized, decomposing leaves, whether green, yellow or brown leaves. Furthermore, at least under these conditions, their spores persisted for weeks and even months after any visible substrate was available, though the effect occurred more definitively and for longer with P. gonapodyides. As P. gonapodyides is not known to produce long-term survival structures, the question arises of how P. gonapodyides persisted so long in the microcosms in the absence of leaves. This observation also stands in contrast to our successful elimination of Phytophthora spores from original stream water collections simply by holding the water at cool temperatures for approximately three weeks. The observed persistence of spores of both Phytophthora species may be the result of an abundance of zoospore cysts due to the compact nature of the microcosms, or perhaps because the spores originated from propagules that would not have been suspended in the water column of the flowing streams. While oomycetes have been acknowledged as decomposers in aquatic environments until recently they have primarily been regarded as acting on non-cellulosic detritus such as insect and animal tissue. As most Phytophthora species are known as plant pathogens, the recent evidence that they may also degrade plant tissue in detritus is not surprising. Parasitism is considered an early characteristic in the evolution of oomycetes, but the possible evolution of a saprotrophic lifestyle from parasitic precursors has been considered for fungi and oomycetes.

Clade 6 Phytophthora are known to be opportunistic pathogens. Stradling saprotrophic and parasitic lifestyles, stream-resident Phytophthora may play an important role in the early breakdown of leaves and vegetative matter that still contain living cells. As facultative pathogens,clade 6 Phytophthora species can enter living cells and open intact tissues to further colonization by other saprotrophic organisms with less ability to penetrate living tissue. This is analogous to the paradigm of ‘conditioning’ of vegetative litter by pioneer microbial species, though in this case with respect to secondary saprotrophic microorganisms that could not on their own overcome physical and chemical protections still present in senescent but still alive leaf tissue. Our results were consistent with this hypothesis, as green leaves decayed more slowly in the absence of Phytophthora. It is uncertain why in the first experiment green leaves in the treatments with no Phytophthora inoculation decomposed very little over the entire 16 weeks of the experiment. In this experiment, both green and brown leaves were maintained together in microcosms, and it is possible that leachate from the leaves, particularly the brown leaves, may have had an inhibitory effect on some microorganisms. In the second experiment, leaves were leached prior to being deployed in the experiment, and also green and brown leaves were kept in separate microcosms. Green leaves in non-inoculated controls in the second experiment lost biomass to a degree ultimately similar to that of inoculated treatments, albeit at a slower rate. This indicates that other organisms were present that could initiate the decomposition of green leaves through the presence of Phytophthora accelerated it. We attempted additional isolations from some samples of leaves on acidified potato dextrose agar medium and found that the leaves in both controls and inoculated treatments were generally well colonized by a multitude of fungi . The fact that similar fungi occurred on leaves from microcosms prepared with both sterile and non-sterilized stream water suggests that many of these fungi were present on the leaves before entering streams as leaf litter .Additionally, overall there were no differences in decomposition rates between treatments with sterile or non-sterilized stream water added. Decomposition was also similar for leaves colonized by either Phytophthora species, indicating that, though P. gonapodyides is a better adapted saprotroph, both species had a similar effect on the decomposition of live, green and yellow leaves. This would be consistent with Phytophthora having the effect of opening integral tissue to colonization by other saprotrophs that then push decomposition forward. Finally, it is interesting that the presence of fungi in these leaves did not affect the persistence of P. ramorum throughout the experiments, suggesting that they are using different resources and that the successive displacement of P. ramorum in previous work may be specific to competition with other Phytophthora species or similar organisms. Under natural conditions, leaves would be exposed to a greater diversity of organisms, including other oomycetes such as Phytopythium species. As P. gonapodyides can colonize dead leaf tissue, it could be expected that it would contribute to leaf decay in brown leaves as well. This was not observed, as loss of biomass in brown leaves was the same in all treatments unaffected by Phytophthora colonization. The fact that P. gonapodyides substantially colonized brown, senesced leaves, but did not increase the rate of biomass loss raises the question as to what resources the organism uses in this substrate.

Marijuana cultivars are known to have THC levels exceeding 2–24% of inflorescence dry weight whereas hemp cultivars produce substantially less THC but rather high levels of CBD . THCA and CBDA share the same bio-synthetic pathway except for the last step in which THCA synthase and CBDA synthase produce THCA or CBDA, respectively . Recent evidence suggests that the genes encoding the two synthases are allelic . CBD and THC are enatiomers, but only THC elicits psychotropic effects, whereas CBD may mediate anti-psychotropic effects , a difference highlighting the stereo-selectivity of receptors in the human body that bind these compounds. Although classified as a drug without therapeutic value in the United States, ingestion of THC is widely regarded as having effects including pain relief and appetite stimulation, that may, among other things, increase the tolerance of cancer patients to chemotherapy . Dronabinol, a synthetic analogue of THC, is approved for use as an appetite stimulant in the United States as a Schedule III drug . Cesamet , another synthetic analogue, is used as an anti-emetic for patients undergoing cancer therapy. The natural product Sativex is approved for use in the UK and is derived from Cannabis cultivars containing both THC and CBD, and is used to treat pain symptoms associated with multiple sclerosis. Compounds from Cannabis sativa are of undeniable medical interest, and subtle differences in the chemical nature of these compounds can greatly influence their pharmacological properties. For these reasons,berry pots a better understanding of the secondary metabolic pathways that lead to the synthesis of bio-active natural products in Cannabis is needed . Knowledge of genetics underlying cannabinoid biosynthesis is also needed to engineer drug-free and distinctive Cannabis varieties capable of supplying hemp fibre and oil seed.

In this report, RNA from mature glands isolated from the bracts of female inflorescences was converted into cDNA and cloned to produce a cDNA library. DNA from over 2000 clones has been sequenced and characterized. Candidate genes for almost all of the enzymes required to convert primary metabolites into THCA have been identified. Expression levels of many of the candidate genes for the pathways were compared between isolated glands and intact inflorescence leaves.Seeds from the marijuana cultivar Skunk no. 1 were provided by HortaPharm BV and imported under a US Drug Enforcement Administration permit to a registered controlled substance research facility. Plants were grown under hydroponic conditions in a secure growth chamber yielding cannabinoid levels in mature plants as reported in Datwyler and Weiblen . Approximately 5 g of tissue was harvested from mature female inflorescences 8 weeks after the onset of flowering. Tissue was equally distributed into four 50 ml tubes containing 20 ml phosphate buffered saline as described by Sambrook et al. , but made with all potassium salts and mixed at maximum speed with a Vortex 2 Genie for four repetitions of 30 s mixing followed by 30 s rest on ice, for a total of 2 min of mixing. Material was sieved through four layers of 131 mm plastic mesh and the flow-through was split into two 50 ml tubes and spun in a centrifuge for 30 s at 500 rpm. Supernatants were decanted and pellets were resuspended in PBS. The suspensions were combined into one tube and pelleted as before. The resulting pellet was diluted into 100 ll of PBS. Five ll were used for cell counting with a haemocytometer, and the total suspension was estimated to contain 70 000 intact glands. Plant residue was incinerated by a DEA-registered reverse distributor .Quantitative reactions were performed as described previously using primers listed in Supplementary Table 4B at JXB online. Equivalent quantities of RNA isolated from glands and inflorescence associated leaves were used to generate the respective single stranded cDNAs. qPCR reactions containing equal quantities of gland or leaf cDNA were run in duplicate along with reactions containing standards consisting of 100-fold sequential dilutions of isolated target fragments, on a Lightcyler qPCR machine .

Lightcycler software was used to generate standard curves covering a range of 106 to which gland and leaf data were compared. Two biological replicates were used to generate the means and standard deviations shown in Supplementary Table 4A at JXB online. These values were used to compute the gland over leaf ratios and P-values shown in Supplementary Table 4A at JXB online. Raw relative expression data, means, standard deviations, P-values from gland versus leaf t tests, qPCR primer sequences, and representative real-time qPCR tracings are shown in Supplementary Table 4A at JXB online.Anatomical study revealed that glands located on mature floral bracts of female plants are the site of enhanced secondary metabolism leading to the production of THCA and other compounds in Cannabis sativa . These glands are located on multicellular stalks and typically are composed of eight cells . The outer gland surface is composed of a smooth capsule covered by a membrane. The capsule contains exudates derived from the gland cells . The weakly attached glands can easily be separated from the bracts and purified as shown in Fig. 1E and F. An EST library was constructed using RNA isolated from purified glands. Over 100 000 ESTs were cloned. Plasmid DNA was isolated and sequenced from over 2000 clones. Because of the directed orientation of cDNA insertion, sequences are expected to represent the coding strand. After the removal of vectoronly, poor quality sequences, and sequences obviously originating from organelles or ribosomal RNA, the remaining sequences were clustered into 1075 unigenes . Overall, 111 of the unigenes were contigs containing two or more closely related ESTs . Only 14 contigs lacked a similar sequence in the NCBI database. Nine hundred and sixty four of the ESTs were only found once and of these 710 were similar to sequences in the NCBI database . The top three unigenes representing the greatest number of ESTs encoded proteins related to metallothionein, RD22-like BURP domain-containing proteins, and chitin binding hevein-like proteins . All three of these proteins have functions related to biotic or abiotic stress responses . Gene Ontology analysis was performed on the sequence dataset . An analysis of biological function indicates that 27% of the unigenes encode proteins with metabolic activity. Unigenes with NCBI matches encoding proteins with unknown function comprise 14% of the total and another 28% are predicted to be involved in various cellular processes such as protein synthesis and protein degradation.

The specific biochemical steps leading to THCA are proposed to begin with a reaction involving a type III PKS enzyme that catalyses the synthesis of olivetolic acid from hexanoyl-CoA and three molecules of malonyl-CoA . Malonyl-CoA is derived from the carboxylation of acetyl-CoA. ESTs encoding acetyl-CoA carboxylase were identified. Hexanoyl-CoA could be produced by more than one pathway in the trichomes. One route to produce hexanoyl-CoA would involve the early termination of the fatty acid biosynthetic pathway, yielding hexanoyl-ACP . The hexanoyl moiety would then be transferred to CoA by the action of an ACP-CoA transacylase or it would be cleaved by the action of a thioesterase, yielding n-hexanol, which would then be converted into n-hexanoyl-CoA by the action of acyl-CoA synthase. Most of the enzymes needed for this route are represented in the EST database, except for thetransacylase and 2,3-trans-enoyl-ACP reductase . A second route to hexanoyl-CoA would involve the production of hexanol from the breakdown of the fatty acid linoleic acid via the lipoxygenase pathway . A survey of the sequenced ESTs revealed candidate genes encoding the enzymes needed to synthesize linoleic acid from acetyl-CoA by the typical fatty acid biosynthetic pathway in plastids followed by the production of hexanol from linoleic acid via the LOX pathway. An third pathway related to the biosynthesis of branched chain amino acids has been proposed to be involved in the production of short-chain and medium-chain fatty acids . However,hydroponic grow system the enzymes in this pathway [2-isopropylmalate synthase, 3-isopropylmalate dehydratase, 3-isopropylmalate dehydrogenase, and 2- oxoisovalerate dehydrogenase ] were not represented in the Cannabis trichome EST library. After the formation of olivetolic acid, a prenyltransferase is proposed to add a prenyl group derived from geranyl diphosphate to create cannabigerolic acid. GPP is derived from the fusion of two isoprene units . Two different biochemical pathways support the synthesis of isoprenoids in plants . Within the list of unigenes all but one of the enzymatic activities needed to convert pyruvate and glyceraldehyde-3-phosphate into isopentenyl and dimethylallyl diphosphate via the methylerythritol 4-phosphate pathway were represented . This finding is consistent with isotopic studies showing that the GPP cannabinoid precursors are synthesized via this pathway . The formation of GPP is mediated by GPP synthase. Several unigenes related to GPP synthase were identified , however, they were more closely related to other terpene synthases. In particular, CAN36 and CAN55, which possibly were derived from the same gene, and the closely related CAN37, are most similar to hop sesquiterpene synthases HISTS1 and HISTS2 , with an average identity of 56% over the first 160 amino acid residues . CAN41 is most similar to hop monoterpene synthase HIMTS2 .The nature of the prenyltransferase is unknown. However, previous studies identified a soluble aromatic geranylpyrophosphate:olivetolate geranyltransferase in the extract of young leaves with the appropriate activity .

The only EST encoding a predicted prenyltransferase was CAN121. However, the encoded protein is more closely related to members of the membrane-bound chloroplast-localized family of prenyltransferases than to soluble prenyltransferases . The final step in the pathway is mediated by THCA synthase, which mediates the conversion of cannabigerolic acid to THCA . Two ESTs with sequences identical to the previous reportedly THCA synthase were identified .Whereas the nature of the prenyltransferase responsible for the synthesis of cannabigerolic acid is unknown, three unigenes, CAN24, CAN383, and CAN1069, comprising eight, one, and two ESTs, respectively, could encode the PKS activity needed to synthesize olivetolic acid. These were therefore characterized in more detail. All three unigenes were represented by individual ESTs encoding complete PKS polypetides. These were sequenced and compared to related PKS sequences . CAN1069 was identical to a previously identified Cannabis gene encoding a chalcone synthase, and is the most closely related of the PKS sequences to other known chalcone synthases from hop and Arabidopsis . The relationships of hop phlorisovalerophenone synthase , which mediates the conversion of malonyl-CoA and isovaleryl-CoA to phlorisovalerophenone, to CAN24 and CAN383 is less clear . CAN24 and CAN383 show 64.6% identity and are nearly equally similar to hop VPS at 71.2% and 72.0%, respectively. The enzymatic activities encoded by CAN24 and CAN1069 were explored in detail.The tagged proteins were purified on a nickel-containing magnetic bead matrix and were assayed for chalcone and olivetol/olivetolic acid synthase activities . Recombinant protein from CAN1069, but not CAN24, produced reaction products when incubated with coumaroyl-CoA and malonyl-CoA . The reaction products were analysed by LC-MS and peak 2 was found to have a molecular mass and absorption spectrum consistent with naringenin , the major product of chalcone synthases. Both CAN24 and CAN1069 were capable of using malonyl-CoA and hexanoyl-CoA as reaction substrates and LC-MS indicated that products of these enzymes were the same, but neither molecular mass nor the absorption spectrum of this product matched olivetol or olivetolic acid . Results similar to CAN24 were obtained using protein purified from CAN383 .Genes required for THCA production are probably more highly expressed in glands of pistillate inflorescences because this is where THCA is most highly concentrated. To test this hypothesis, the relative expression levels in isolated glands versus young inflorescence-associated leaves of selected unigenes were compared using real-time qPCR. The identity of the genes assayed and the differences in relative expression levels are listed in Table 2 and in Supplementary Table 4A at JXB online. Consistent with this hypothesis, THCA synthase expression was 437 times higher in isolated glands than in leaves. CAN24 was expressed 1600 times higher in glands of the inflorescence than in associated leaves. CAN1069 encoding CHS was also more highly expressed in glands than leaves . The expression of a third PKS, CAN383, was expressed at similar levels in glands and leaves . These results are not explained by poorRNA isolation from leaves as unigene CAN219 encoding chlorophyll A/B binding protein showed elevated leaf expression levels .

This database uses the same forms as ROBO for recording, and several entries in BIRLDATA can also be found in the ROBO database. BIRLDATA is not available online; however, copies can be requested through BIRL. The United States Department of the Interior Bureau of Land Management also has numerous biological control release records, which are not standardized and have not been imported into any USDA database. The BLM is in the process of launching their own internal database, the National Invasive Species Information Management System , which will catalog biological control agent releases and other treatment types within the agency . While web-based catalogs certainly would be the most convenient way to access information on origin, numbers released, initial establishment, and recent distributions of biological control agents, the scope of the available databases do not encompass all the existing data. A plethora of printed documentation is available in the form of annual reviews, reports of local or regional agencies, catalogs, books, peer-reviewed or unpublished publications, original release forms, etc. Even though most of the documents are easily accessible through official channels , collating all the available data on a group of organisms can be laborious depending on the details needed. More comprehensive volumes include Clausen’s world review of biological control of arthropod pests and weeds. Julien and Griffiths compiled a world catalog for weed biological control agents, listing all attempts undertaken in biological control of weeds up to 1996. One of the most up-to-date summaries on biological control of weeds contains information on the origin, history, and recent distributions of 94 weed biological control agents and 39 targeted weeds in the USA . An updated database is underway, which will provide information on the status of weed biological control agents for the continental USA . The above-mentioned references, along with the ROBO and BIRLDATA databases,plant pot with drainage can be useful starting points in search of the history of given biological control organisms, but the acquired data should be interpreted carefully.

The catalogs rely mostly on published data, while many biological control agent importations remain unpublished , especially those considered failures or if the program was unfinished . More reliable data acquisition may be ensured by focusing on states that are known to maintain extensive databases and release records and conduct intensive biological control programs . Irregular record keeping is a problem for bio-control records, including files on ROBO. The accuracy and reliability of bio-control records often are determined by the available funding for a given program, especially the extent of monitoring establishment and efficacy after releases . Consequently, as the numbers of institutions and personnel involved in biological control increase, the quality of record keeping decreases.Though the permitting process is uniform across agencies, the permits themselves give little information on the fate of biological control agents. Additionally, the long-term monitoring of biological control agents is most often undertaken by various institutions and agencies that become involved at the third stage of releases. These agencies have independently developed different methods for record keeping; moreover, they are solicited but not required by law to submit their records to a national database . Many agencies simply have not adopted the BCDC forms . Along with the development of ROBO, plans also were proposed to establish the US National Voucher Collection of Introduced Beneficial Arthropods . The need for such a collection has long been recognized, but this program was curtailed due to loss of technical support within the BCDC . As a result, the deposition of voucher specimens has not become centralized or regulated by the USDA or any other federal agency. Annual publications, complementary to the ROBO database, listing all biological control releases within the USA, were discontinued after 1985 due to loss of personnel and the general low priority of biological control documentation within the ARS . The situation has not improved in subsequent years; a staff of only one person is responsible for the maintenance of BCDC .

The need to link data on the release of invertebrates as biological control agents across the nations of Europe is increasing . Several levels of standards and regulations have been given by different authorities, including the International Plant Protection Convention , the Organisation for Economic Co-operation and Development , the European Union , and the European and Mediterranean Plant Protection Organization . A main focus in these standards is the assessment of risk of biological control agents to human health and their effects on local biodiversity. In order to obtain permission to study or release biological control organisms, a substantial amount of information is required. For example, EPPO suggests a dossier that includes a list of biological features as well as ‘1) details of the proposed import ; 2) whether the organism was collected from the wild or reared in the laboratory’ . Specific guidelines on release of bio-control organisms also suggested by EPPO include ‘1) the release program should be fully documented as to identity, origin, numbers/quantity released, dates, localities and any other data relevant to assessing the outcome; 2) evaluation of the releases should be planned in advance, to assess the impact of the organism on the target pest and non-target organisms’ . EPPO lists 91 biological control species on their web page , which are currently used commercially in the 50 EPPO countries. It also includes a list of 43 introduced classical bio-control agents in EPPO countries that have successfully established in at least one country. The information includes documentation of both successful and unsuccessful introductions, based on the BIOCAT database from CABI and some EPPO countries. This information can be used to understand differences between successful and unsuccessful introductions. Of the 43 classical bio-control agents, 35 are documented to have been released as a single introduction within each country where they were introduced, 7 are documented to have multiple introductions into at least one of the countries where they were introduced, and one has no information. Four of the 43 species include reference to a failed establishment in at least one country where they were introduced. Currently, there are limitations to biological control data unity and uniformity in Europe, largely due to the many, independent nations involved. First, implementation and execution of bio-control regulation in Europe are at the national level and dependent on the national legislation. That is, international standards are not binding, although often they have been the basis for rules and standards at the national level. Nevertheless, huge differences among European countries both at the legislative and implementation levels exist .

Additionally, the necessary information outlined in the international standards for bio-control research or release does not contain a mandate to include the information in a database. This results in limited available and unified information across Europe .bio-control agents introduced in Australia must go through a government-regulated process that includes importation of the potential agent into containment, host- specificity testing, and eventual release . In New Zealand, host- specificity testing is not currently formally regulated, but the Environmental Risk Management Authority is advising potential applicants of the importance of appropriate testing because approved applications to date typically included extensive host- specificity testing following a centrifugal phylogenetic approach . Another difference between the two countries is that once New Zealand grants full release of a bio-control agent, no monitoring or data collection is required by law,drainage planter pot though post release monitoring is encouraged. A separate approval category called ‘conditional release’ in New Zealand, however, can put additional regulations on approved releases that mandate monitoring, reporting, and record-keeping . In Australia, monitoring of establishment, efficacy, and any non-target effects must be reported to the Australian Quarantine Inspection Service 1 year after release . Finally, in New Zealand, at least a single voucher specimen of any imported potential bio-control species is required to be deposited into the New Zealand Arthropod Collection . This voucher system ensures the correct taxonomic identity for the imported species.Ecologists and evolutionary biologists need to become aware of appropriate available datasets that can be used for understanding the early stages of invasion. Biological control data may provide important insights into these early stages. In order to record and store data that can be useful for future research, possibly by researchers in a different sub-discipline from classical bio-control, data should be reliable and be as complete as possible. Useful information that can be added to these datasets includes: number and sources of original collections that contributed to the founding laboratory population, the breeding colony protocols of the quarantine growth phase , the number of individuals released, the location of each release, and the long-term establishment and recent distribution of biological control releases . These five pieces of information standardized across all biological control laboratories would be basic information that other researchers could use. For example, if these data were available, invasive species biologists could use these data to compare establishment success with the collection area in the native range to investigate questions relating to plasticity versus adaptation. Long-term establishment data collected by the researchers who release and monitor the bio-control agents would allow other investigators to determine adaptation to novel conditions, particularly if the bio-control agent has spread on its own to non-release areas. The documentation of establishment failures also is a priority so that comparisons of failures can be made with species or locations that successfully established. Once establishment is confirmed in the new environment and the bio-control agents begin to spread, the importance and possible constraints of environmental factors could be evaluated. The numbers of individuals released would be useful for relating establishment success or failure to potential genetic bottlenecks or Allee effects. Ideally, all this information would be stored in national , public databases that are globally accessible on the internet.

Recently, there has been a proposal for and description of a new centralized database for arthropod bio-control in the USA that if implemented may help in the accessibility and utility of recorded information. Voucher specimens are not only necessary for positive identification of bio-control agents, but they also would be useful for evolutionary studies if they were preserved at all stages of the biological control process from original collection to recovered samples after release, including periodical sampling from the laboratory colony . Currently, whenever vouchers are required by regulation, they are only required in association with initial import. For example, New Zealand and Nebraska, USA require a deposited voucher of any potential bio-control organism for which release approval is being sought. In addition, a few agencies keep voucher specimens of all biological control organisms that have passed through their laboratories . Voucher collections made throughout the duration of a bio-control program can be housed on-site at the bio-control facility, or they could be donated to nearby museums to be curated in their collections. These specimens would provide morphological and genetic data over the time period for which little is known of the evolutionary processes involved in biological invasion. Finally, published records, either in peer-reviewed literature or on the biological control databases, should include physiological tolerance data and laboratory-rearing conditions . Data that would be informative in modeling establishment success in the field include such factors as optimum egg-laying temperature, temperature required for flight, and population growth rates at three or more temperatures. These data should be easily obtainable from bio-control laboratory protocols, particularly because bio-control laboratories have to determine appropriate temperatures for rapid rearing.Plants introduced via the horticulture trade share several major characteristics with introduced bio-control organisms. Both groups are deliberately introduced, and importation records should exist in some form for both bio-control organisms and horticultural plants. Thus, there is documentation of introduction, unlike most invasive species that arrive undetected. Both horticultural plants and bio-control organisms are generally selected to be preadapted to the local climate of introduction and may be selected for vigorous growth and reproduction among other potentially invasive attributes . Differences, however, also exist. Horticultural plants are most often generalists in their biotic and abiotic requirements because they must be able to grow and thrive in a variety of soil, moisture, and/or light conditions to be commercially viable. Additionally, horticultural species span a wide range of life-history and life-form characteristics, whereas bio-control organisms necessarily tend to be more specialized .

Our discovery resolves a decades old mystery of the molecular underpinnings of white-petal California poppies, and adds to the cultural legacy of the California golden poppy.Commercial California poppy varieties were purchased as seeds from Eden Brothers , Vermont Wildflowers , and Cornucopia . Seeds were germinated in individual pots, and subsequent developing flower buds collected and frozen on dry ice. In some cases, poppy leaf material was also collected. Mature flowers from the same plants were examined and photographed to verify the advertised varieties. We also collected ostensibly wild California poppy flower samples from fields across three San Francisco Bay Area counties. For RNA isolation, plant material was pulverized in liquid nitrogen using a mortar and pestle, and then RNA prepared using the RNeasy Mini kit . Genomic DNA was isolated from commercial seeds, using the Quick-DNA Plant/Seed Miniprep Kit .The Laboratory of Chemical Biodynamics continues to base its research plan on the importance of its role in the application of sophisticated chemical sciences to problems relevant to the mission of the Department of Energy. Our Laboratory is in the unique position that it is staffed mainly by investigators trained in chemistry with an interest in applying these skills 12 to both biological and energy science problems. Our mission is to carry out research that takes advantage of our unique skills, as well as to train young investigators in the fields we so strongly 11 represent. The research in the Laboratory of Chemical Bioclynamics is almost entirely fundamental research. The biological research component is strongly dominated by a long term interest in two main themes which make up our Structural Biology Program. The first interest has to do with understanding the molecular dynamics of photosynthesis. The Laboratory’s investigators are studying the various components that make up the photosynthetic reaction center complexes in many different organisms.

This work not only involves understanding the kinetics of energy transfer and storage in plants,square plastic planter but also includes studies to work out how photosynthetic cells regulate the expression of genes encoding the photosynthetic apparatus. The second biological theme is a series of investigations into the relationship between structure and function in nucleic acids. Our basic mission in this program is to couple our chemical and biophysical expertise to understand how not only the primary structure of nucleic acids, but also higher levels of structure including interactions with proteins and other nucleic acids regulate the functional activity of genes. In the chemical sciences work in the Laboratory, our investigators are increasing our understanding of the fundamental chemistry of electronically excited molecules, a critical dimension of every photosynthetic energy storage process. We are developing approaches not only toward the utilization of sophisticated chemistry to store photon energy, but also to develop systems that can emulate the photosynthetic apparatus in the trapping and transfer of photosynthetic energy. This research is directed toward fundamental understanding of the special chemistry of electronically excited molecules, which is involved in every photosynthetic photon energy storage process. An electronically excited molecule differs from the ground state in orbital occupancy, charge distribution, molecular structure, and chemical reactivities. These differences are the key to photon energy storage. Infrared spectroscopy coupled with matrix isolation provides a powerful diagnostic technique. Absorption features are sharp and informative about molecular structures. With a tunable laser photolysis source, we are attempting to map electronic hypersurfaces. Both unimolecular and bimolecular reactions are under study. To increase our knowledge of matrixinduced surface crossing, we are investigating fluorescence and phosphorescence as well. The compound dimethylamino benzonitrile has received a great deal of attention because in room temperature solutions it displays two fluorescent radiative relaxation paths. These two paths are strongly solvent—and temperature-dependent.

The strong solvent dependence is attributed to a very large charge separation in the electronically excited state. This excited state is, of course, stabilized in polar solvents which affects the dual fluorescence. To add new information and elucidate further this interesting behavior, we have investigated the fluorescence and phosphorescence of DMABN suspended in various matrices at 10° K. Both inert gas and polar matrices have been investigated. With the three inert gas matrices Ar, Kr, and Xe, both fluorescence and phosphorescence were observed. As the spin-orbit coupling constant of the inert gas increased , the amount of flulrescence decreased and the amount of phosphorescence increased. At the same time, the sum of fluorescence plus phosphorescence increased. This shows that the most important effect of the matrix environment is to increase singlet-triplet surface crossing. This is the first clear-cut demonstration of phosphorescence from DMABN and our spectra display interpretable vibrational fine structure that gives information about the ground state. When a polar matrix, such as ammonia or hydrogen bromide is used, the vibrational fine structure is lost but, surprisingly, the zero-zero frequencies of both the singlet and the triplet transitions are unaffected. The significance of this striking difference from the polar solvent affects observed in solutions is under study. The purpose of this program is to search for and evaluate chemical systems which permit use and storage of near infrared photons, and allow to accomplish their efficient conversion into useful energy. The importance for exploring chemistry with these long wavelength quanta derives from the fact that photochemical reactions which can be initiated by these photons are very sparce despite the fact that more than half of the solar irradiance at ground level lies in the near infrared . Our work is aimed at contributing to this key problem in solar photochemistry in two ways. First, on a fundamental level, we are searching for low energy paths of bimolecular reactions that would allow us to initiate the chemistry with near infrared photons. Secondly, on a level directly aiming at photon storage and conversion, we are studing chemical systems which permit synthesis with near infrared photons, accomplish their storage, and offer a way for efficient conversion of the stored chemical energy into useful energy.

We are examining manganese porphyrin species as potential multi-electron oxidation catalyst for oxygen evolution from water and for other oxidation reaction. In photosynthesis some type of manganese complex is involved in the oxygen evolution process. Manganese porphyrin complexes exhibit a rich variety of oxidation states in which the porphyrin macrocycle is resistant to irreversible redox reactions. These properties make them promising oxidation catalysts, and, in addition, it has recently been shown that manganese porphyrin complexes catalyze the oxidation of olefinic hydrocarbons. Our research is directed at characterizing various highly oxidized manganese porphyrin species and studying their chemistry with the view of judging their potential usefulness in the oxidation cycle of an artificial photosynthesis assembly. The work has proceeded along two parallel pathways. The first is directed at water soluble manganese porphyrins and involves chemical, electrochemical and photochemical studies. However,square pot isolation of intermediate species is frequently easier in organic solvents, and we are also investigating the redox chemistry of manganese tetraphenylporphyrin complexes in organic media. Comparison of similarities and differences in the properties of oxidized manganese porphyrins in aqueous and nonaqueous systems has led to helpful itisights and has suggested new experiments. An effort is underway to prepare a polynuclear manganese complex with some cyclic amines, i.e. cyclams. There is reason to suspect that the multiple manganese sites on such a molecule could provide the capability of bringing two oxygen atoms together to form.For gardeners, California’s climate both charms and challenges. Its charms include rainless summers with warm, sunny days and mild nights, and brief, mild winters. But most of these charms are also challenges. The long, hot summers with no precipitation require frequent irrigation, and the low humidity can further increase the water demand and pest susceptibility of humidity-loving plants. The brief, mild winters can render plants that require a long seasonal chill unsatisfactory in either fall color or fruit production, and allow many pests that would be killed elsewhere by winter freezing to survive and multiply from one year to the next. Because so many commonly used landscape plants are ill-adapted to these climatic conditions, large inputs of water, pesticides and fertilizers are needed to keep them looking their best. With constantly increasing population pressures in the state, there is an increasing demand for water . Due to over watering and the frequent use of pesticides and artificial fertilizers, an increase in undesirable chemicals in urban runoff is a growing and serious problem . In addition to all this, whereas other large states such as Texas have only four U.S. Department of Agriculture plant hardiness zones , California is home to at least seven USDA zones and 24 climate zones as described in Sunset Western Garden Book . Nonetheless, large chain nurseries in particular often sell the same plants from one end of the state to the other, ensuring that many customers who bought something that was lovely in the garden center will eventually be disappointed with a plant unsuitable to their part of the state. So how does one create a lovely landscape with such difficult challenges? The obvious answer is simply to garden with plants that have greater drought-tolerance, fewer pest problems and an adaptation to milder winters.

In fact, in recent years there has been a trend in both public landscapes and home gardens to use more plants with these characteristics. These plants, usually native to California or other areas of the world with Mediterranean-type climates, are sometimes referred to as “low-input” because they require little supplemental water and no chemicals to look their best. Their proper maintenance leaves no negative impact on the environment. The horticulture industry, however, thrives on a constant input of new and beautiful plants to tantalize its customers year to year, and despite the growing demand, plants in the “low-input” category have been relatively few and slow in coming to the mainstream nursery market.Those retail nurseries that do offer or specialize in native plants are often known only to a small, motivated market of knowledgeable gardeners who seek them out. Most are located in coastal areas, away from the large tracts of developing Central Valley urbanization, where polluted runoff into watersheds is an issue. Some are inaccessible to much of the public either by location or limited hours, and have limited distribution to the landscape trade. Many California native plants would be beautiful in urban landscapes, but they have been underused in mainstream retail nurseries and the landscape industry because relatively few species have been available in the numbers needed for large-scale retail distribution. Most of the work on native-plant propagation protocols has been used to produce species for reforestation and revegetation by conservation agencies and affiliates, where the market is driven more by governmental than consumer forces. Little attention, however, has been paid to developing commercially viable propagation protocols for the ongoing addition of new, low-input species to the nursery market, partly because of misconceptions among nurserymen and landscapers that all natives are difficult to propagate, and that few are attractive enough to be appealing to consumers. Nothing could be further from the truth. There are many native species that would be year-round assets to any garden, and any difficulty in propagation is simply a protocol waiting to be discovered. A workable answer to all these concerns is a statewide, coordinated, cooperative, low-input plant introduction program. Many other states and regions of the country have long-established, successful, plant introduction programs that benefit all stakeholders by combining the talents, knowledge and energy of university researchers, extension specialists, arboretum and botanical garden personnel, and members of the wholesale and retail horticulture industry. Just such a program is under way at UC Davis. UC Cooperative Extension researchers, UC Davis Arboretum staff and the California Center for Urban Horticulture are partnering with members of the commercial horticulture industry to provide a channel for the ongoing introduction of beautiful new low-input plants to a wide landscape horticulture market. Although this introduction program is in its infancy, it will entail four basic stages: initial selection, a low water tolerance field trial, zone garden trials and commercial introduction. The overriding goal of the project is to provide consumers with a source of beautiful landscape materials that will thrive in a wide variety of California climate zones with little input of water or chemicals.

The pinyon pine is stih regarded as a distinctively Indian resource to be conserved and respected . At present the main threat to continued plant-food collecting derives from land-development activities such as large-scale open-pit mining operations at prime habitat locations. Conflicts over hunting and fishing rights have been more confrontational, leading, in some instances, to open defiance of state laws requiring hcenses and fishing rights on the Truckee River . Though some special concessions have been made in the state laws, it appears that these problems whl continue to be a source of aggravation into the future. Heavy metal pollution, especially cadmium , is a major environmental issue in China and many other parts of the world . The Guangzhou Food and Drug Administration recently reported that the content of Cd in 44% of sampled rice and rice products exceeded national standards of 0.2 and 0.1 mg/kg respectively , which underlined the severity of Cd pollution in China’s main grain producing areas. Some of these grain producing areas have large mines . Poor management of wastes from mining activities have resulted in severe water contamination at levels of up to 3000e5000 mg Cd/L. Due to water shortages and lack of treatment facilities, water contaminated with nitrate and metals is used for irrigating vegetables and grains , which could lead to high nitrate and heavy metal concentrations in vegetables and grains . While Cd damages lungs, kidneys, liver and reproductive organs , nitrate can cause methemoglobinemia in infants . Hence, the importance of treating this kind of contamination cannot be overemphasized. Nanoscale zerovalent iron , with Fe core and iron oxide shell, has been proposed for the treatment of Cd contamination . However, there is a large discrepancy in Cd removal capacity of nZVI reported , 66.9 mg/g , 769.2 mg/g.

The discrepancies may arise from different initial Cd concentrations, temperature, and water chemistry such as pH ,drainage pot and concentrations of dissolved oxygen , phosphate , and nitrate . According to previous studies , X-ray photoelectron spectroscopy analysis indicated that Cd immobilization by nZVI was mainly through adsorption. However, reduction of Cd to Cd may also occur due to its slightly more positive standard electrode potential than Fe . As demonstrated in this study, X-ray diffraction may help to better understand Cd reduction on the nZVI particles, since it can show characteristic peaks of Cd if present. With regard to nitrate pollution, it has been widely observed that ammonium is the main reduction product in the presence of nZVI, with only a small fraction of nitrite detected; nitrite is regarded as an intermediate . Although ammonium is a toxic pollutant to some organisms , it serves as major nitrogen source for plants . Furthermore, nZVI is mainly transformed into magnetite after reaction with nitrate , which avoids significant increase of Fe2þ or exchangeable Fe concentration. This vastly reduces the potential environmental impact of nZVI . Hence, it is possible to employ nZVI to treat contaminated groundwater after it has been pumped out of the ground. Given the prevalence of nitrate and metal contamination in many regions, it is important to understand the influence of one pollutant on the removal of the other using nZVI. In this study we focused on the interplay between nitrate and Cd. Nitratemay affect Cd removal in two ways: in terms of Cd adsorption, while nitrate may not affect adsorption significantly through changing ionic strength , it may enhance Cd removal by driving solution pH above 9 ; and in terms of Cd reduction, nitrate reduction will consume a large part of Fe and produce iron oxided reducing electron supply and restricting electron flow for Cd reduction. Likewise, the presence ofCd may also have two important implications on nitrate reduction: First, similar to Cu islands , Cd islands compounds or Cd) may be formed on nZVI, which may enhance electron transport to nitrate.

Enhanced electron transport may occur given the lower electrical resistivity of Cd than Fe . Second, if Cd is reduced to Cd, it can reduce nitrate to nitrite , which may lead to an increased nitrite yield ratio.However, nitrite accumulation is undesirable in natural environment as nitrite is highly toxic to several organisms, including humans . Several previous studies have shown that catalysts, such as Cu , Ag , or Au , can facilitate nitrite reduction. In this study, Cd removal performance of nZVI in the presence or absence of nitrate was investigated. In addition, the effect ofCd on nitrate reduction was systematically examined.We also evaluated the potential of nZVI with 1 wt.% Cu, Ag, or Au to treat Cd and nitrate co-pollution with minimal nitrite yield. XRD was employed to detect Cd and characterize the transformation of the nanoparticles under different conditions.Batch Cd adsorption experiments with and without nitrate addition were carried out in a 300 ml 3-neck flask. A 200 mg/L Cd2 stock solution was prepared and used for all experiments. Final Cdconcentration was 10e40 mg/L and the load of iron nanoparticles was 500 mg/L. The flask was agitated by an electromagnetic stirrer at 25  C under Ar atmosphere. In the series of tests with nitrate, the concentration of nitrate was 15 mg-N/L. After reacting for 90 min, solution pH was measured, and aqueous samples were collected for Cd and total Fe analyses. Batch tests for determining Cd removal capacity under different nitrate loads were carried out in a series of 100 ml conical flasks sealed with screw caps. The concentrations of Cd were 50, 100, and 150 mg/L while the concentrations of nitrate were 8, 12, 16 mg-N/L. The load of iron nanoparticles was 500 mg/L. The suspensions were mixed at 200 rpm for 90 min. At the end of the experiment pH was measured , and aqueous samples were removed to determine concentrations of nitrogen compounds and metals , total Fe).

To further investigate the influence of nitrate on Cd removal, two different series of tests were performed. In Series 1, nitrate was not present in the reaction system. 125 mg nZVI were added into 250 ml of a 40 mg-Cd/L solution, the solution pH was adjusted and maintained at 9.0, stirring for 30 min. After stirring, an aqueous sample was collected and analyzed for total Fe and Cd. The remaining mixture was then divided into 5 parts, which were adjusted to and maintained at pH 8.5, 8.0, 7.5, 7.0, or 6.5, stirring for another 30 min. Aqueous samples were then collected from the 5 sub-samples for Cd and Fe analyses. In Series 2, 125 mg nZVI was added into 250 ml of 40 mg/L Cd and 15 mg-N/L nitrate. After 20 min, solution pH increased to 9, and remained stable for ~10 min, so it was not adjusted as in Series 1. As before, an aqueous sample was collected and analyzed. The suspension was divided into 5 parts whose pH was adjusted and kept at 8.5, 8.0, 7.5, 7.0, or 6.5. After stirring for another 30 min, aqueous samples were collected and analyzed. Additionally, we performed a control test to see the effect of pH on Cd removal, by adjusting the pH of 15 mg Cd/L solutions to 7, 7.5, 8.0, 8.3, 8.5, or 9.0, in separate vials. After 5 min mixing and another 5 min standing, 1 ml aqueous media was collected for Cd measurement. Sodium hydroxide and hydrochloric acid were used to adjust and maintain solution pH in all cases. To examine the effect of different Cd loads on nitrate reduction,frambueso maceta nZVI was added into a series of 15 mgN/L nitrate solutions with 10, 20, 30, 40, 50 or 100 mg/L Cd in 100 ml conical flasks sealed with screw caps. After 120 min shaking , samples were collected and nitrogen compounds in solution were analyzed. To investigate the effect of Cd addition on nZVI oxidation, 250 ml of a 500 mg/L nZVI suspension with or without 10 mg/L Cd load was agitated vigorously by an electromagnetic stirrer for 45 min in a 500 ml beaker without seal or Ar protection. Throughout the experiment, oxidation-reduction potential was carefully monitored. In batch tests for determining the effect of pH on nitrate reduction in the presence of Cd, the pH of five 250 ml nitrate solutions with 500 mg/L nZVI and 20 mg/L Cd was maintained for 60 min at pH 7.0, 7.5, 8.0, 8.5, or 9.0, using sodium hydroxide and hydrochloric acid to adjust pH. Aqueous samples were then collected for nitrate, nitrite, and ammonium analyses.

Tests for effect of Me catalysts on nitrate reduction and nitrite yield were carried out in a series of 100 ml conical flasks sealed with screw caps. Freshly-made nZVIeCu, nZVIeAg, or nZVIeAu was added into a 45 mg-N/L nitrate solution with or without 30 mg/L Cd load. The nZVI load was 1500 mg/L. After 90 min, aqueous samples were collected for nitrate, nitrite, and ammonium analyses. Based on the effect of Me catalysts on nitrate reduction, further investigation was done to see the effect of Au on nitrate reduction under different Cd load conditions. nZVIeAu was introduced to a set of 15 mg-N/L nitrate solutions with 10, 20, 30, 40, 50, and 100 mg/L Cd. nZVIeAu concentration was 500 mg/L. Aqueous samples were isolated and analyzed , total Fe after 90 min of shaking at 200 rpm. To simulate Cd and nitrate contaminated groundwater, sodium nitrate and cadmium acetate were added into real groundwater to achieve 15 mg-N/L nitrate and 20 mg/L Cd load. nZVIeAu concentration was still 500 mg/L. After 90 min shaking, aqueous samples were collected and analyzed. Characteristics of the groundwater are shown in Table S1. Nitrite reduction by nZVI or nZVIeAu in the presence of Cd at different pH was performed to better understand nitrite accumulation during nitrate reduction. Aqueous samples were collected for 2 h every 30 min and NO2 , NH4 þ were analyzed. All tests were performed in triplicate.ORP and pH were monitored throughout using a HACH Sc200 . Cadmium and total iron in the collected samples were determined by inductively coupled plasma after filtering with 0.22 mm filter and acidifying with 4% ultrahigh purity HNO3. All nitrogen containing compounds in filtered samples were assessed spectrophotometrically based on a previous study that used a colorimetry technique . A Zetasizer Nano-ZS90 was used to determine the zeta potential of particles. XRD was carried out on a Bruker D8 Advance X-ray diffraction instrument , and the diffraction angle from 10 to 90 was scanned. For preparing samples, the collected solid samples were transferred into a vacuum freeze dryer immediately. After 24 h, the dried samples were analyzed via XRD.As shown in Fig. 1, in the absence of nitrate, 500 mg/L nZVI only completely removed Cd when the initial concentration was 10 mg Cd/L or less. However, with 15 mg-N/L nitrate in solution, complete Cd removal was observed even when the initial Cd concentration was increased to 40 mg/L. Cd removal capacity of nZVI was 40 mg/g in the absence of nitrate, while it reached 80 mg/g when nitrate was present at 15 mg/L. Several studies showed that nZVI reduces nitrate to ammonium, accompanied by increased solution pH ) , which could enhance Cd removal by nZVI through precipitation 2) . Under these conditions we observed that solution pH exceeded 9; while without nitrate the pH was below 8 . Increased pH may therefore be responsible for increased Cd removal capacity of nZVI, due to the presence of nitrate. Additionally, final total Fe concentrations detected in these two reaction systems were also very different. As seen in Fig. 1, total Fe in the supernatant at the end of the experiments exceeded 9 mg/L in all the conditions without nitrate while Fe was not detected in the supernatant of reaction systems with nitrate. The absence of Fe in suspension was probably due to the oxidation to Fe and precipitation stimulated by nitrate.In the series of tests with pH control, Cd removal efficiency decreased as pH decreased both in the presence and absence of nitrate . At pH 9, Cd removal efficiency of both reaction systems were very close.

Alternatively, the presence of microbes and microbe‐derived metabolites that alter plant hormone homeostasis could also cause the phenotype observed in soil extract . Compounds such as tryptophan and salicylate detected in soil extract are reported to alter root morphology , and thus are candidates for causing elongated root hairs. We suggest that the long root hair phenotype observed could be a result of soil extract nutrient levels and specific concentrations of signaling compounds. The determination of the causal factor resulting in the long root hair phenotype represents an important future direction. Root hair length was shown to have a significant impact on how plants grow in natural soils, and how plants interact with their environment. Root hairs alter physical properties of the soil, such as the extension of the rhizosphere and the pore size development in soils . Root hairs also affect biotic interactions by defining the rhizosphere and the amount of C exuded from roots . The complex morphological and metabolic alterations of B. distachyon when grown in soil extract stress the importance of not only considering standard laboratory growth media, but also more natural substrates when studying plant–environment interactions. It would be interesting to investigate how root hair length changes when solid particles, microbial communities, or both are added back to the soil extract used in this study, to investigate morphology changes in a more natural environment. In addition, the observation that increased root hair length was restricted to primary roots but not observed on lateral roots highlights the need for high spatial resolution when measuring root traits, even in a simplified system like the EcoFAB. In conclusion, EcoFABs are reproducible tools to study a variety of topics, and this reproducibility enables interlaboratory studies of plant–environment interactions. Their low cost, flexibility,vertical farming and compatibility with metabolomics studies enables investigations of increasingly complex conditions simulating specific natural environments.

We found that B. distachyon growth in EcoFABs was reproducible across four laboratories for a number of morphological and metabolic traits, including tissue FW and phosphate content, total root length, and metabolic profiles of root tissue and root exudates. In addition, plants grown in soil extract exhibited an altered root : shoot ratio and elongated root hairs, and depleted half of the investigated metabolites from soil extract. An important next step in the development of more field‐relevant EcoFABs will be the ability to include solid materials and microbial communities that reflect additional important aspects of soils. Bagged greens in the market are often labeled “pre-washed,” “triple-washed,” or “ready-to-eat,” and look shiny and clean. But are they really “clean” of harmful microbes? We cannot be so sure. Food safety has been threatened by contamination with human pathogens including bacteria, viruses, and parasites. Between 2000 and 2008, norovirus and Salmonella spp. contributed to 58 and 11% of forborne illnesses, respectively in the United States . In those same years, non-typhoidal Salmonella alone was ranked as the topmost bacterial pathogen contributing to hospitalizations and deaths . In 2007, 235 outbreaks were associated with a single food commodity; out of which 17% was associated with poultry, 16% with beef, and 14% with leafy vegetables that also accounted for the most episodes of illnesses . Apart from the direct effects on human health, enormous economic losses are incurred by contaminated food products recalls. The 8-day recall of spinach in 2006 cost $350 million to the US economy . It should be realized that this is not the loss of one individual, but several growers, workers, and distributors. This is a common scenario for any multi-state food borne outbreak. Additionally, the skepticism of the general public toward consumption of a particular food product can lead to deficiencies of an important food source from the diet. Less demand would in turn lead to losses for the food industry.

Economic analysis shows that money spent on prevention of food borne outbreak by producers is much less than the cost incurred after the outbreak . Contamination of plants can occur at any step of food chain while the food travels from farm to table. Both pre-harvest and post-harvest steps are prone to contamination. Contaminated irrigation water, farm workers with limited means of proper sanitation, and fecal contamination in the farm by animals can expose plants to human pathogens before harvest of the edible parts . After harvest, contamination can occur during unclean modes of transportation, processing, and bagging . Mechanical damage during transport can dramatically increase the population of human pathogens surviving on the surface of edible plants . Control measures to decrease pathogen load on plant surfaces have been defined by the Food Safety Modernization Act and Hazard Analysis and Critical Control Point system . Using chlorine for post-harvest crop handling has been approved by US Department of Agriculture under the National Organic Program. However, some studies indicated that internalized human pathogens escape sanitization . Thus, understanding the biology of human pathogen-plant interactions is now crucial to prevent pathogen colonization of and survival in/on plants, and to incorporate additional, complementing measures to control food borne outbreaks. We reasoned that as plants are recognized vectors for human pathogens, enhancing the plant immune system against them creates a unique opportunity to disrupt the pathogen cycle. In this cross-kingdom interaction, the physiology of both partners contribute to the outcome of the interactions . Bacterial factors important for interaction with plants have been discussed in recent, comprehensive reviews . Plant factors contributing to bacterial contamination is much less studied and discussed. In this review, we highlight current knowledge on plants as vectors for human pathogens, the molecular mechanisms of plant responses to human bacterial pathogens, and discuss common themes of plant defenses induced by phytopathogens and human pathogens.

We have focused on human bacterial pathogens that are not recognized plant pathogens such as Salmonella enterica and Escherichia coli , but yet are major threats to food safety and human health.The leaf environment has long been considered to be a hostile environment for bacteria. The leaf surface is exposed to rapidly fluctuating temperature and relative humidity, UV radiation, fluctuating availability of moisture in the form of rain or dew, lack of nutrients, and hydrophobicity . Such extreme fluctuations, for example within a single day, are certainly not experienced by pathogens in animal and human gut. Thus, it is tempting to speculate that animal pathogens may not even be able to survive and grow in an environment as dynamic as the leaf surface. However, the high incidence of human pathogens such as S. enterica and E. coli O157:H7 on fresh produce, sprouts, vegetables, leading to food borne illness outbreaks indicate a certain level of human pathogen fitness in/on the leaf. The plant surface presents a barrier to bacterial invaders by the presence of wax, cuticle, cell wall, trichomes, and stomata. All except stomata, present a passive defense system to prevent internalization of bacteria. Nonetheless,macetas cuadradas several bacteria are able to survive on and penetrate within the plant interior. The surface of just one leaf is a very large habitat for any bacteria. The architecture of the leaf by itself is not uniform and provides areas of different environmental conditions. There are bulges and troughs formed by veins, leaf hair or trichomes, stomata, and hydathodes that form microsites for bacterial survival with increased water and nutrient availability, as well as temperature and UV radiation protection . Indeed, distinct microcolonies or aggregates of S. enterica were found on cilantro leaf surfaces in the vein region In addition, preference to the abaxial side of lettuce leaf by S. enterica may be is an important strategy for UV avoidance . Conversion of cells to viable but non-culturable state in E. coli O157:H7 on lettuce leaves may also be a strategy to escape harsh environmental conditions . Hence, localization to favorable microsites, avoidance of harsh environments, and survival by aggregation or conversion to non-culturable state may allow these human pathogens to survive and at times multiply to great extent on the leaf surface. As stomata are abundant natural pores in the plant epidermis which serve as entrance points for bacteria to colonize the leaf interior , several studies addressed the question as to whether human bacterial pathogens could internalize leaves through stomata. Populations of E. coli O157:H7 and S. enterica SL1344 in the Arabidopsis leaf apoplast can be as large as four logs per cm2 of leaf after surface-inoculation under 60% relative humidity suggesting that these bacteria can and access the apoplast of intact leaves. Several microscopy studies indicated association of pathogens on or near guard cells. For instance, S. enterica serovar Typhimurium SL1344 was shown to internalize arugula and iceberg lettuce through stomata and bacterial cells were located in the sub-stomatal space . However, no internalization of SL1344 was observed into parsley where most cells were found on the leaf surface even though stomata were partially open . Cells of S. enterica serovar Typhimurium MAE110 , enteroaggregative E. coli , and E. coli O157:H7 were found to be associated with stomata in tomato, arugula leaves, and baby spinach leaves, respectively. In the stem E. coli O157:H7 and Salmonella serovar Typhimurium were found to be associated with the hypocotyl and the stem tissues including epidermis, cortex, vascular bundles, and pith when seedlings were germinated from contaminated seeds.

The plant rhizosphere is also a complex habitat for microorganisms with different life styles including plant beneficial symbionts and human pathogens. Nutritionally rich root exudate has been documented to attract S. enterica to lettuce roots . Although bacteria cannot directly penetrate through root cells, sites at the lateral root emergence and root cracks provide ports of entry for S. enterica and E. coli O157:H7 into root tissues , and in some instances between the epidermal cells . High colonization of S. enterica has been observed in the root-shoot transition area . Once internalized both bacterial pathogens have been found in the intercellular space of the root outer cortex of Medicago truncatula . Salmonella enterica was found in the parenchyma, endodermis, pericycle, and vascular system of lettuce roots and in the inner root cortex of barley . A detailed study on the localization of E. coli O157:H7 in live root tissue demonstrated that this bacterium can colonize the plant cell wall, apoplast, and cytoplasm . Intracellular localization of E. coli O157:H7 seems to be a rare event as most of the microscopy-based studies show bacterial cells in the intercellular space only. Bacterial translocation from roots to the phyllosphere may be by migration on the plant surface in a flagellum-dependent manner or presumably through the vasculature . The mechanism for internal movement of enteric bacterial cells from the root cortex to the root vasculature through the endodermis and casparian strips and movement from the roots to the phyllosphere through the vascular system is yet to be demonstrated. Several outbreaks of S. enterica have also been associated with fruits, especially tomatoes. Salmonella enterica is unlikely to survive on surface of intact fruits raising the question: what are the routes for human pathogenic bacteria penetration into fruits? It has been suggested that S. enterica can move from inoculated leaves , stems, and flowers to tomato fruits. However, the rate of internal contamination of fruits was low when leaves were surface-infected with S. enterica . The phloem has been suggested as the route of movement of bacteria to non-inoculated parts of the plant as bacterial cells were detected in this tissue by microscopy . Figure 1 depicts the observed phyllosphere and rhizosphere niches colonized by bacteria in/on intact plants and probable sources of contamination.Plants are able to mount a generalized step-one response that is triggered by modified/degraded plant products or conserved pathogen molecules. These molecules are known as damage or pathogen associated molecular patterns . In many cases, conserved PAMPs are components of cell walls and surface structures such as flagellin, lipopolys accharides, and chitin . Examples of intracellular PAMPs exist such as the elongation factor EF-Tu . PAMPs are recognized by a diverse set of plant extracellular receptors called pattern-recognition receptors that pass intracellular signals launching an army of defense molecules to stop the invasion of the pathogens.

Each increment was sectioned longitudinally into two halves. One half of the soil was homogenized, large roots and green mosses were removed, and a sub-sample of soil was extracted with 2 M KCl to determine the initial soil depth profile of plant available NH4-N and NO3-N . Nutrient concentrations were standardized per unit dry soil mass using gravimetric water conversion from samples oven dried for more than 48 h at 70°C. The distribution of total carbon and N throughout the soil profile was determined by elemental analysis on similarly collected and processed soil cores collected in 2012 from the nearby NGEE Arctic Intensive site [Iversen et al., 2015b]. Species- specific below ground biomass was determined from the remaining half of the soil from each depth increment. Living roots and rhizomes associated with the target species in each plot were removed from each depth increment using forceps, oven dried at 70°C for more than 48 h, and weighed to determine species specific living below ground biomass, which was converted to unit ground area using the bulk density of the core depth increments. After the initial harvest, we injected a solution of 15NH4Cl in the soil beneath newly located 9 cm ff 9 cm plots in homogenous species patches. We separately targeted three soil horizons for each species: organic, shallow mineral, or the permafrost boundary . Injections were made in a grid pattern of 16 points per plot to ensure homogenous delivery of the tracer solution at a given soil depth. The organic horizon injections were targeted at 3 cm depth, the mineral horizon injections were targeted at 10 cm depth,vertical grow rack and the permafrost boundary injections were targeted at 1 cm above the permafrost boundary .

One week later, the vegetation in the labeled plots was clipped to the moss surface, and soil cores were taken in the center of each labeled plot. Above ground and below ground vegetation was processed and quantified as above, and oven-dried, ground plant tissues were analyzed for 15N using continuous-flow isotope ratio mass spectrometry . Duplicate samples and standards of known 15N concentration were used to ensure the precision and accuracy of the data. The field experiment provides edaphic and vegetation data collected prior to 15N addition to initialize and drive the N-COM model. Then, the total amount of 15N acquired by the plants after the experimental tracer addition was used to test the predictions from the three nutrient competition concepts . The major difference among the three competition models is that only ECA explicitly considers essential root traits for plant-microbe competition. Therefore, first, a comparison between the ECA model and the other two models will inform how root traits control plant-microbe competition. Second, the 15N tracer experiment quantififies the vertical distribution of plant N uptake, which is an emergent pattern of plant-microbe competition. By comparing model predictions with different plantmicrobe competition hypotheses with the observations, we can evaluate how plant-microbe competition hypotheses affect plant N uptake. Third, since Relative Demand and Microbes Win competition hypothesesare widely used by prevailing ESMs, the discrepancy between these two concepts and observations can inform future modeling efforts.N-COM is a process-based model originally developed to represent coupled ecosystem carbon, nitrogen, and phosphorus cycles [Zhu and Riley, 2015; Zhu et al., 2016] based on Equilibrium Chemistry Approximation kinetics [Tang and Riley, 2013], although its structure is sufficiently generic to include any number of substrates and consumers.

The modeling framework mechanistically represents nutrient competition assuming plants and microbes produce specialized nutrient transporter enzymes to react with soil inorganic nitrogen substrates, enzyme-substrate complexes are then formed, these complexes can be transported into cells, and finally, the transporter enzymes are liberated [Button, 1985; Williams and Miller, 2001]. Thus, the binding of substrates to plant nutrient transporter enzymes inhibits the binding between substrate and microbial nutrient transporter enzymes and vice versa. While nutrient diffusivity limitation may constrain plant uptake by affecting substrate affinity [Tang and Riley, 2013], we did not consider diffusivity limitation in this study because 15N was directly added in the rooting zone and the spatial scale of the plots were just a few centimeters. As applied here, N-COM quantifies tundra C and N fluxes in three model layers: organic layer , mineral layer , and near the permafrost boundary layer for Carex aquatilis, Eriophorum angustifolium, and Salix rotundifolia . We focus here on plant 15NH4 + uptake; other nutrient uptake fluxes are described in Zhu et al. [2016]. Competition for NH4 + occurs among roots, nitrifiers, and microbial decomposers. However, nitrifier activity is typically very small in tundra soils [Giblin et al., 1991; Schimel et al., 1996]. We therefore assumed in this study that competition only occurred between roots and microbial decomposers. The model does not represent microbial community and diversity . Since different microbial functional groups may have different enzymatic kinetics, we also assessed uncertainties stemming from this model simplification in our uncertainty analysis .Most model parameters were directly taken from field data . However, several key model parameters were not measured in the field experiment due to logistical constraints, and our derivation of those parameters from literature may have introduced uncertainties.

We quantified the uncertainty associated with the unobserved parameters using a Monte Carlo approach. For ECA model simulations, VMAX and KM were randomly sampled from their observed ranges 500 times for each plant species and for soil microbes. For the Relative Demand and Microbes Win competition models, which do not use kinetics parameters, uncertainty stemmed from the estimate of total plant N demand calculated by dividing plant net biomass production by its C to N ratio. Similarly, we randomly sampled plant net biomass production 500 times from the literature reported range for each species. Uncertainty ranges associated with the above mentioned parameters are reported as error bars in results and figures.The three dominant tundra plant species observed here were dramatically different in terms of their maximum rooting depth, vertical rooting profile , and their prescribed root NH4 + uptake kinetics . However, the distribution of soil NH4 + throughout the soil profile was relatively similar across the three plant monocultures , being higher in the organic layer and near the permafrost boundary , and lower in mineral soil layers . Carex aquatilis is a relatively deep-rooting species . Moreover, it had the highest total root density of the species, most of which was in the organic and surface mineral layers . Eriophorum angustifolium is also a deep-rooting species . However, its root density was much lower compared with Carex aquatilis. Salix rotundifolia also has a relatively high density of roots, of which the vast majority are in the upper 10 cm of soil and ~90% are in the organic layer . This pattern indicates that Salix rotundifolia lacks the ability to access deep soil nutrients and must directly compete with microbial decomposers in surface soils. The 15N tracer data show that Carex aquatilis took up most of its NH4 + from mineral soil layers,vertical planting tower which agrees with its root density profile . Similar to Carex aquatilis, Eriophorum angustifolium took up most of its NH4 + from mineral soil layers . However, the observed uptake profile was in contrast to its root density profile . Although roughly equalamounts of roots existed in the organic layer and mineral layer , mineral layer NH4 + uptake was tenfold higher than in the organic layer. Salix rotundifolia took up 80% of NH4 + from the organic layer . Our modeling of plant-microbe competition using the ECA approach in N-COM generally captured the observed vertical patterns of plant N uptake for all three tundra species . Differences between ECA predictions and observations were small: for Carex aquatilis, ECA overestimated plant N uptake near the permafrost boundary layer and for Eriophorum angustifolium, ECA overestimated plant N uptake at organic layer . Our uncertainty analysis showed that plant-microbe competition was sensitive to the choice of kinetics parameters . However, the plant N uptake patterns were conservative even when considering the full range of uncertainties in derived kinetics parameters. Therefore, we conclude that using literature-derived kinetics parameters introduced modest uncertainty in our model analysis but did not undermine the fidelity of the ECA approach.In addition to the ECA competition hypothesis, two other prevailing hypotheses are employed by ESMs: root nitrogen uptake is based on plant demand and the competition between root and decomposer microbes is scaled with their relative demand and the Microbes Win hypothesis , which assumes roots are completely out competed by microbes and that roots take up nitrogen after microbial demand has been satisfied. Model setup for RD and MIC are described in supporting information Method S3.They each predicted substantial nitrogen uptake near the permafrost boundary layer , in contrast to observed uptake.

These discrepancies occurred because, in these hypotheses, root uptake in shallow soil layers are either completely suppressed or largely suppressed by microbial decomposers . Therefore, they tend to acquire soil nitrogen from deeper in the soil profile, where microbial competition stress is lower. Both ECA and observations indicate that Eriophorum shifts its nitrogen uptake from the organic layer to mineral layer, but not down to the permafrost boundary layer. Overall, by explicitly considering maximum rooting depth, biomass density, and uptake kinetics, ECA is the only hypothesis that captured NH4 + uptake patterns for all three tundra species.For Carex aquatilis and Salix rotundifolia, the observed NH4 + uptake profiles were consistent with the prevailing hypothesis that fine-root biomass density, as functionally absorptive tissues, exerts first-order control on nutrient uptake [De Baets et al., 2007; Vamerali et al., 2003]. For Eriophorum angustifolium, however, the observed uptake profile did not follow the prevailing hypothesis. We showed that this pattern resulted from decreased competition between roots and microbial decomposers in mineral soils. The ECA competition hypothesis as integrated in the N-COM model explicitly represents these competitive interactions and accurately predicted the NH4 + uptake profile . The model results indicate that since Eriophorum angustifolium is a relatively poor competitor for NH4 + , it shifts its uptake profile deeper in the soil, in order to avoid NH4 + competition with microbial decomposers in the organic layer. Root physiology traits suggest that the Eriophorum angustifolium root system is less carbon efficient. In particular, compared with Carex aquatilis, maintenance and growth respiration per gram of root are higher but root longevity is much shorter for Eriophorum angustifolium [Billings et al., 1977; Shaver and Billings, 1975]. Although root morphological traits suggest that Eriophorum angustifolium has higher root length per gram root biomass [Eissenstat et al., 2000], total root density is much lower than Carex aquatilis . Furthermore, the Eriophorum angustifolium NH4 + uptake pattern is also consistent with the idea that microbial activity and N immobilization are highly limited by carbon availability. Compared with mineral soil layers, relatively higher carbon availability in surface organic layer will lead to higher potential of microbial activity and consequently higher microbial N immobilization demand and stronger nitrogen competition between plant and microbe [Booth et al., 2005]. Although both gross nitrogen mineralization and immobilization rates are commonly high in surface soils, net immobilization typically occurs because of strong microbial demand [e.g., Iversen et al., 2011]. Overall, our 15N tracer measurements and modeling analysis at Barrow, Alaska, showed that plant nitrogen uptake patterns emerge from root and soil biotic competition, which could be predicted by essential root traits and appropriate treatment of microbial competitive interaction. Although not studied here, mineral surfaces are also effective competitors for enzymes [Sulman et al., 2014; Tang and Riley, 2015], and further research is required to determine when those processes need to be included in nutrient and carbon cycle models.In this study, we showed that an important complication in predicting arctic tundra vegetation species responses to warming is associated with their different root characteristics, which can affect their ability to compete for elevated nitrogen availability throughout the soil profile. In this sense, explicitly considering key root functional traits is particularly important for studying warming-induced fertilization effects on arctic vegetation. Here we highlight the importance of several essential root traits in controlling nitrogen uptake patterns. First, maximum rooting depth is an important plant functional trait in modeling plant nitrogen uptake and response to arctic warming.