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.

The institutional framework advanced in Rausser and Johnson are satisfied by a constitutional smart contract . When structuring the constitutional design, any prescription must essentially define: the degree of centralization; the balance of power; identifying interest groups; the space of issues over which those interests can negotiate; the degree of consensus that is sufficient to conclude negotiations; and the appropriate course if negotiations break down .Second, smart contracts can provide legal and regulatory infrastructure that allow for the strict reinforcement of the constitution. In particular, the security or private property, enforcement of contracts, and assignment of liability for wrongful damage must be established . This is consistent with the Institutional Analysis and Development framework’s scope and payoff rules . This allows DAO partnerships to have greater transparency and “prescriptive force,” or the knowledge and acceptance of a rule leads individuals to recognize that if they break the rule, other individuals may hold them accountable .Third, DAOs, by design, admit that the collective interest of the PPRDP is able, for crucial matters, to rise above immediate self-interest of any particular participants . This is accomplished by allocating the majority of tokens to public sector agents, who have internal incentives and mechanisms to preserve the advancements of fundamental knowledge. Fourth, provisions that discourage collusive activities coupled with policies that provide opportunities to partners who have a comparative advantage are key in achieving sustainable economic growth . The transparency and accurate logging of data using block chain technology allows for these provisions to be fulfilled.

While DAOs have a systemic risk of shadow centralization, in which a cabal of rent-seeking interest groups will collude to gain majority decision-making power, a governance provision of the constitutional smart contract will provide the public sector partner, or universities,vertical grow rack system with 51% of all voting rights to order to assure the partnership will ultimately be to conduct fundamental research with positive spillovers in the space industry. One possible approach to structure the partnership is summarized here. After the PPRDP fee is paid, partners will receive “soul bound” governance tokens , or irrevocable tokens that cannot be sold or transferred to another wallet, to join the DAO. Because of its unique attributes, SBTs can accurately represent and store an entity’s credentials, history with the PPRDP , as well as implement reputation-based voting, which reduces the occurrence of Sybil attacks and can incentivize active and meaningful participation. Because SBTs can create “novel markets with decomposable, shared rights, and permissions” , control rights for IP are less susceptible to IP theft and administrative transaction costs. With these tokens, PPRDP partners have an active say into the governance structure and process. The amount of tokens unlocked will decrease over time; a company who joins the PPRDP in the first year will be allocated more tokens in comparison to a company who joins in the fourth year. The university partner would allocate its governance tokens to researchers, professors, and staff members in order to satisfy the decentralization requirement. Other than governance power, a key utility in holding SBTs is the eligibility to buy security tokens for research projects that are in progress within the PPRDP. Security tokens reflect the potential market value of research discoveries that may well lead to patents and/or commercial applications; such tokens are akin to equity shares of startup companies. During the inception phase, an initial coin offering will be conducted with a valuation that estimates what an investor would be willing to spend on an IP of a similar type. Based on the market share percentage and choice, a PPRDP partner would own either: a proportionate percentage of the patent’s income streams ; right of first refusals; exclusive licensing rights or proportionate payout from another PPRDP partner.

Ultimately, a PPRDP in which the sharing and structure for sharing any value of discoveries are determined by the PPRDP.PPRDP participants can sell security tokens with one another at market prices or sell it back to the university at a 25% discounted price should they lose faith or have high opportunity costs for other research projects. In other words, liquidity is provided for all PPRDP partners with mechanisms to prevent premature investment retractions, mitigating liquidity concerns industry partners are burdened with while hedging financial risk for public partners. In fact, PPRDP partners are incentivized to provide research personnel or in-kind services in exchange for additional security tokens and increased probability of research discoveries. No more than 51% of total security tokens offering should be sold before the maturity of IPs; the public sector would collectively maintain a majority stake in all holdings to protect the public sector interest and research agenda by prohibiting collusive action from participating private firms.Engineering resistance for agricultural improvement presently incorporates diverse strategies to mitigate the crop losses imposed by pathogens. For instance, traditional breeding has been strengthened and streamlined with the advent of new molecular markers for rapid selection of desired traits. Transgenic technology provides a complement to some of the weaknesses inherent in traditional breeding. This includes altering the expression of endogenous components from specific and broad-based pathogen resistance signaling pathways as well as utilizing genes from other species. Further analyses are providing insight into other means of inducing inherent plant defense responses through refined chemical and hormone treatments. The control of viruses and a bacterial pathogen using transgene expression in planta to initiate RNA silencing has also shown great promise.Shortly after the re-discovery of Mendel’s laws of heritability, qualitative traits were identified in wheat that conferred resistance to the rust pathogen Puccinia striiformis . Subsequently, numerous qualitative loci have been identified in diverse plant species that confer resistance .

Most of these loci do not confer broad-spectrum resistance; rather, the resistance is limited to subgroups within pathogen species. Likewise, diverse isolates of a particular pathogen species have been shown to contain loci that prevent the pathogen from successfully causing disease on the host . Flor developed a model based upon classical genetics using flax, Linum ultissimom, and the fungal pathogen Melamspora lini. His ‘gene-for-gene’ theory states that the plant resistance locus [R] and the pathogen avirulence determinant [avr] must both be present toobserve phenotypic resistance in the host . For any given plant and potential pathogen pair, an incompatible interaction is the product of these two loci. If the plant lacks the R locus or the pathogen lacks the avr determinant, then a compatible interaction is the outcome. The R gene products are hypothesized to act as receptors for the products of the avirulence locus. Thus Flor’s findings demonstrate that for many host–pathogen relationships,vertical farming companies resistance [R] and avirulence [avr] loci dictate the outcome of varying combinations of host and pathogen genotypes. R gene-mediated resistance is an economical method to control losses in the field and breeders have mobilized R genes into virtually all improved lines. Often this genetic mechanism of resistance lacks long-lasting durability in the field. In terms of Flor’s ‘gene-for-gene’ theory, the pathogen responds to selection pressure by altering or eliminating the avirulence determinant. When the pathogen eliminates the cognate avr gene product, the R gene can no longer perceive the product of the avirulence locus. Plants have responded to these dynamic genetic changes of the pathogen by generating their own clusters of diverse resistance loci . Over the past decade,vertical farming compa a number of R loci and avr loci have been cloned from diverse species of plants and pathogens.As noted above, monogenic resistance frequently is not durable in the field when exposed to high levels of pathogen pressure. One strategy to control pathogen outbreaks in the field is to cultivate plants with diverse genetic backgrounds within a single field. This agricultural practice has been shown to dramatically reduce yield losses caused by the rice fungal pathogen Magnaporthe grisea in repeated field studies . For many crops, utilization of this approach may provide a mechanism for conferring long-term, durable resistance. For some crops, planting and harvesting a field planted with diverse germplasm may not always be practical. Thus it would be useful to develop disease control strategies that would benefit farming practices that still rely on monculture. The use of gene pyramiding offers an attractive mechanism by which multiple resistance loci, each recognizing a unique range of isolates of a pathogen species, may be incorporated into a single cultivar . The combined presence of these R loci ensures that only pathogens devoid of all cognate avirulence loci perceived by the R loci combination can cause disease and. With the advent of molecular markers tightly linked to resistance loci, simultaneous selection of multiple resistance loci has been facilitated. This strategy of marker assisted selection also allows for the selection against undesirable chromosomal segments known to carry unfavorable agronomic traits as well as the successful incorporation of recessive resistance genes.

The interaction of cultivated rice, Oryza sativa, and a bacterial pathogen, Xanthomonas oryzae pv. oryzae offers a clear example of the benefits of gene pyramiding. One set of gene pyramiding experiments was performed using two completely dominant resistance genes, Xa21 and Xa4, and two recessive genes, xa5 and xa13 . Each of these resistance genes confers resistance to distinct isolates of Xoo and individually these R genes have been overcome by known field isolates . The efficacy of the disease resistance conferred by combinations of these four different resistance loci was evaluated in controlled environments as well as in field studies.These results demonstrate the utility and efficacy of gene pyramiding for generating broad-spectrum resistance to Xoo, although this finding does not suggest that combinations of four genes will lead to durable resistance per se. One caveat in gene pyramiding is that any given resistance locus may have variable penetrance. It should not be assumed that each R gene introgressed into any genetic background will display full phenotypic resistance. Therefore each set of R genes incorporated into a cultivar must be evaluated systematically requiring a significant investment of time. However, it is clear that gene pyramiding offers an attractive mechanism for combining the individual specificities of R genes as well as taking advantage of their synergistic effects to generate broad-spectrum resistance.Occasionally, the loss or mutation of an avirulence locus is associated with reduced pathogen fitness. In pepper, bacterial spot disease has been controlled effectively using the Bs2 resistance locus in breeding programs. The durability of Bs2 is due to the widespread conservation of the avrBs2 locus in diverse pathogen isolates of Xanthomonas campestris pv. vesicatoria. Mutation of avrBs2 prevents wild type levels of bacterial growth on susceptible pepper cultivars lacking Bs2 . Another example is found in rice where a functional avrXa7 avirulence locus is required for full virulence of Xoo . Isolates carrying an avrXa7 mutation can cause disease in the presence of the Xa7 R gene although greenhouse tests demonstrated that the severity of disease was reduced . Tests have further demonstrated that spontaneous mutants of avrXa7 could be recovered from Xa7 plants in the field; however these virulent isolates did not persist. Field tests over six years demonstrated that the presence of Xa7 was sufficient to prevent any Xoo epidemics even when avrXa7 mutants had appeared . In contrast, these field tests demonstrated that epidemics were common in fields planted with rice carrying the Xa10 R gene. Xoo with mutations in avrXa10 displayed no loss of fitness in controlled studies and disease in the field was common on Xa10 plants over the same 6-year analysis . These results indicate that some R genes can confer durable resistance if loss of their cognate avirulence locus confers some fitness penalty for the pathogen. Using such R/avr combinations may be a rational strategy for generating durable resistance.An alternate strategy to breeding is to directly introduce a cloned resistance gene into a plant via transgenic technology. Introduction of a gene by transgenic means can overcome the limitations of traditional breeding, namely interspecies sterility. Additionally, transgenic technologies allows multiple genes to be inserted simultaneously. However, validation of the function of the transgene and its stability and heritability after transformation requires a significant investment of time and resources. Further, the transgenic lines must also undergo subsequent analysis for agronomic traits before release.

This is particularly restrictive for horticultural crops, in which many varieties are required to meet different seasonal production requirements and diverse consumer preferences, and any single variety has a relatively small market share. For example, dozens of different types and varieties of lettuce are grown throughout the year as production shifts between summer and winter locations in California, Arizona and Florida. Some agronomic seed companies budget $50 million for the full commercialization of a new biotech crop, in addition to the standard costs for developing and marketing a traditional variety. Given the small acreage of horticultural crops and their much lower overall value, it is difficult to justify the investment in transgenic horticultural crops. For example, the total U.S. market for iceberg lettuce seed is about $27 million. A typical single variety is worth about $150,000 to $250,000 during its 5-year market lifetime, which suggests that garnering a large market share of lettuce varieties with significant added value would be necessary in order to pay for the additional costs imposed on biotech varieties.Despite this gloomy picture, regulatory strategies may be possible that would protect public and environmental safety while decreasing the cost of introducing biotech specialty crops . Plant breeding companies employing biotechnology can manage and reduce regulatory costs by carefully and deliberately determining the necessary testing requirements. Costs can be reduced by focusing development on biotech genes that have already been commercialized in agronomic crops, since expensive toxicity studies done on a new protein produced in a biotech agronomic crop can be used for the same protein produced in a biotech horticultural crop.

The USDA IR-4 program conducts and pays for the collection of efficacy and safety data on pest-control chemicals for “minor” or “specialty” crops, which include most horticultural crops . A new,vertical farm expanded “biotech IR-4” program focused on full crop registration, including EPA, USDA and FDA requirements, could benefit horticultural crops being developed in universities, government laboratories and small companies. This is particularly critical for the next generation of transgenic products, which will be more consumer-oriented and specific to horticultural crops. Because horticultural products in the pipeline are likely to have altered nutritional or quality traits, specific safety tests will be required that cannot rely on data generated for agronomic crops. Without a program like IR-4, testing requirements could preclude such products from ever being developed and reaching the market. As demonstrated by Calgene and Zeneca with their early tomato products, consumers are receptive to labeled products that have clear quality or price benefits. However, focusing entirely on consumer-oriented traits would forgo valuable benefits for crop production, such as virus resistance, which could have enormous advantages for producers that would not be readily recognized by consumers. As further experience is gained with biotech methods, regulatory requirements should be relaxed for categories of products posing little health or environmental risk. In addition, generic crop and gene approvals , rather than the current “event-specific” approach , would do much to encourage further development of such products. Around the world, farmers desire and in some cases demand the benefits that can come from the improved varieties. In India, for example, extensive precommercialization field trials of insect-resistant cotton found average yield increases of 80% along with a 68% reduction in insecticide use . Farmers saw the value of the varieties and grew 25,000 acres of insect-resistant cotton in 2001, prior to government approval .

Similarly, a significant percentage of soybeans in Brazil was grown from herbicide-resistant seeds smuggled into the country from Argentina and propagated by farmers, as Brazilian courts held up their release despite governmental approval. While planting of insect-resistant corn has not been approved in Mexico, Mexican workers returning from the United States have brought back seed corn for planting, and biotech food grain sold in Mexico has also been planted. At the 2002 Institute of Food Technologists’ annual meeting, E.C.D. Todd of Michigan State University reported that Thai farmers are smuggling and planting biotech seeds from China. While the distribution of biotech varieties outside of legal channels cannot be condoned, these examples illustrate that farmers are aware of the advantages these varieties can deliver. As research continues at many companies, universities and government laboratories, biotech horticultural products having similar attractions for growers and consumers may overcome the current financial and logistical hurdles facing their commercial development.Despite vocal opposition, agricultural biotechnology continues to advance. China has made significant strides in commercializing GE horticultural crops over the past 10 years and may well become the world’s leader during the next 10 years . China was the first country to commercialize biotech plants, beginning with field production of thousands of acres of virus-resistant tobacco in 1988, followed by virus-resistant tomatoes and sweet pepper in 1994 . In the mid-1990s, China was criticized by an American delegation for having only a provincial and not a national product-approval system. For several years afterward, it was difficult to determine whether further commercial plantings of biotech crops occurred in China . Interestingly, China established 1997 as the “official” commercialization date for biotech cotton, tomato, sweet pepper and petunia, which is when the crops were authorized by the agricultural-biotechnology safety office of the Chinese Ministry of Agriculture .

China currently claims to be second only to the United States in agricultural biotech research, development and cultivation, and China is taking full advantage of uncertainty caused by the European Union’s stance on biotech approvals. Beijing University vice president Chen stated, “I expect that in 10 years between 30% and 80% of the rice, wheat, maize, soya, cotton and oil seed crops in China will be transgenic crops. We can take advantage of this 4-year halt [E.U. moratorium] to turn China into a world power in genetically modified organisms.” China is in an excellent position to develop and create internal markets for biotech horticultural crops and clearly has the opportunity to surpass the United States in biotech crop development. Recently, China erected barriers to the importation of biotech grains, creating confusion for U.S. and world exporters, while backing away from some of its earliest commercial biotech products . It is not known whether this is due to internal concern over biotech products or fear of jeopardizing its own export markets to Europe, or is a trade barrier to allow for additional internal development of biotech products. Greater clarity will occur should this issue come before the World Trade Organization . Regulatory issues and costs are reducing commercial opportunities for new biotech crops in the United States. Of course, China will need to meet the requirements of any country receiving their exports,nft vertical farming but currently it is unclear whether any of China’s biotech products are being exported. Korea and Japan are not likely to press this as a trade issue. Other internal political issues are currently complicating commercialization efforts within China, but these are likely to be only short-term barriers . While the United States falters over biotech fruits and vegetables, China is positioning itself to be the world leader in coming years. For the American horticultural industry, the results could be devastating if the United States loses its current competitive edge and more agricultural production moves overseas.Most plants obtain the majority of their nitrogen through root absorption of NH4 + and NO3 – ions from the soil solution. Roots are thought to assimilate most of the NH4 + near the site of absorption to avoid accumulating the high amounts of free NH4 + that would dissipate the transmembrane proton gradients required for respiratory electron transport and for sequestering metabolites in the vacuole . In contrast, plants can store high concentrations of NO3 – in their tissues without toxic effect and may translocate a large portion of this NO3 – to the shoots . The chemical form of the nitrogen source, NH4 + or NO3 – , influences a myriad of plant processes including responses to CO2 enrichment , and each source elicits distinct patterns of gene expression . Nonetheless, characteristics of the root apex under exposure to physiological levels of NH4 + or NO3 – have received relatively little attention. A previously published study profiled net fluxes of NH4 + , NO3 – , and H+ along the axis of the seminal root in a maize seedling . Local influx was measured by depletion of nitrogen from the bathing solution surrounding the root. When nitrogen was supplied as NH4NO3, net influx of NH4 + was rather uniform through the root cap and root growth zone, When nitrogen was supplied as Ca2, the pattern of nitrogen influx was significantly different: net NO3 – influx increased from 1.5 nmol mm 1 h 1 in the root cap to much higher values, 5.4 and 7.6 nmol mm 1 h 1 at 3.5 and 11 mm, respectively, from the root apex.

The presence of NH4 + inhibited net NO3 – influx. This inhibition together with the observed pattern of H+ fluxes indicated that the entire maizeroot apex absorbed more exogenous NH4 + than NO3 – when both forms were present in the medium. The approach in this study is to compare endogenous ‘deposition rates’ of NH4 + and NO3 – with the values for net influx of the exogenously supplied nitrogen forms reported in the earlier study. This leads to an understanding of the source and sink relationships for nitrogen in the growing root. Deposition rates are defined to be the net rate at which a nutrient is added to or removed from the local tissue element. These rates can be calculated from data on tissue concentration and growth velocity . This kinematic approach has illuminated physiology in a number of studies of plant–environment interactions . Here, the spatial profiles of the nitrogen concentrations provide information about metabolic function. Moreover, comparison of net deposition to exogenous influx shows whether the net import or export is occurring at a particular location. Finally, this study extends the approach to show the total uptake of nitrogen into a tissue element during its expansion and displacement. In the following experiments, maize roots were exposed to a nutrient solution that contained NH4 + or NO3 – as sole N sources, both forms together, or no N source. The concentration of NH4 + or NO3 – in the solutions containing them was 0.1 eq m 3 N, a level consistent with those found in soil solutions . Net deposition rates of NH4 + and NO3 – were calculated and root contents of NH4 + and NO3 – and other solutes were measured along the root axis from the apex to 60 mm and in the xylem sap to assess the relative contributions of NH4 + and NO3 – both to the N budget and to the osmolarity in the apical 60 mm, including the 10 mm that comprised the elongation zone.Individual seedling roots were gently blotted dry before they were rapidly frozen on a thermoelectric cold-plate mounted under a dissecting microscope. Axial sections of 1-mm length were made with a fine razor at 1-mm increments from 1 to 10 mm and at 20, 40, and 60 mm from the apex along each of 10 roots. Root sections from each location were pooled. Root sections pooled from 10 roots were weighed to estimate fresh mass and then oven dried at 50  C and weighed to estimate dry mass. Root sections from another 10 roots were placed directly into the sample chamber of a Wescor 5100 Thermocouple Psychrometer to assess osmolarity. Potassium was extracted from sections from a different 10 roots with a solution of 2% acetic acid and analysed using atomic emission spectrometry . Soluble carbohydrates were extracted with boiling water and analysed via HPLC with mass selective detection . Organic acids were extracted with 80% ethanol solution, evaporated under nitrogen at 50  C, redissolved in 0.1 M H2SO4 plus 0.05% EDTA, and analysed via HPLC with UV detection at 195 and 245 nm . Lastly, sections from 10 other roots were collected in Eppendorf tubes containing 1.5 ml of 1 mol m 3 CaSO4, which was adjusted to pH 3 with H2SO4, sonicated for 30 min, and centrifuged.For each N-treatment, there were three or four replicates of sections pooled from 10 roots each for osmolarity, two replicates for potassium, two for soluble carbohydrates, one for organic acids, and at least three for NH4 + and NO3 – .

The market penetration rates used for the “worlds” and run years to be modeled will be based on the results from the expert interviews and workshops . Although these results will be used as a basis, some variation will be allowed to conduct a sensitivity analysis during the modeling effort.ATSC is the dynamic optimization of signal cycle times and phases to synchronize signals along selected arterial routes. If all the signal controllers along a route are assigned a common cycle time, users can experience a progressive green wave along the route. An ATSC system can also incorporate dynamic actuation of traffic signals to respond to changes in traffic demand. All traffic signals in the SMART Corridor study area are linked to the Los Angeles Department of Transportation ATSAC System. ATSAC is an interconnected and coordinated signal system which monitors and manages surface street traffic. Traffic is monitored by detectors and traffic surveillance cameras and various timing programs are automatically implemented in response to fluctuating traffic demands.A set of signal input files will be created to represent the alternative scenarios being modeled, as discussed in the section “Proposed Model Runs.” The UC Berkeley team did not intend to simulate ramp metering; consequently, they did not code any of the ramp meters in the Santa Monica Freeway Corridor into the link file. In addition, the INTEGRATION model has not been previously tested with regard to its ability to model ramp metering. As a result,ebb and flow ramp metering is not likely to be directly included in the scope of the modeling effort for the current research project.The ETC technology makes use of a pocket-sized tag containing a radio transponder that is placed inside a vehicle’s windshield.

The transponder transmits radio frequencies to an electronic antenna as the vehicle passes through the toll collection lane, and the appropriate fare is deducted automatically. Such systems have the capacity to move five times as many vehicles as conventional toll lanes, reducing congestion, fuel consumption, and air pollution at toll plazas. As well as saving travel time for motorists, ETC systems are viewed as very convenient . No ETC facilities currently exist along the SMART Corridor. Since the Corridor is only 11 miles long, it would only be feasible to deploy one ETC facility. Recent discussion regarding the implementation of ETC and the modeling of this technology for this project have centered around the issue of HOT lanes . It has been argued that this is the most reasonable application of ETC within the Santa Monica Freeway Corridor. It is not yet clear whether the INTEGRATION model is capable of simulating the deployment of HOT lanes. This will be determined shortly through continuing discussions with the model developers. An ETC facility can be modeled within INTEGRATION by changing the characteristics of a link or links on the network at the location of the ETC facility. Prohibitions can also be assigned to certain vehicle types for use of certain links or lanes .En-route driver information consists of real-time information about traffic conditions, incidents, construction, transit schedules, and weather conditions, which are provided to drivers once travel begins. Real-time traffic conditions can be obtained from surveillance stations or from probe vehicles that drive through the network and transmit data back to the control center. The primary travel behavior impact of such a system would be caused by drivers choosing better routes to their destination or changing their destination or both. Mode choice and trip chaining formation may also be influenced. Information can be provided via in-vehicle displays or voice messages or both, via Highway Advisory Radio , or externally via CMS. This study will attempt to model the impact of in-vehicle information and also CMS. HAR may also be contrasted to CMS.

The INTEGRATION model allows the user to define five different vehicle types in terms of the frequency of information that they receive and a measure of the error likely to be inherent in the information received. The parameters for these vehicle types are defined in the master control file, as described in an earlier section. Nodes within the network will be coded to act as information nodes that transmit traveler information to vehicle types that are coded to receive it.Route guidance systems provide simple instructions to a user to follow a suggested route in order to reach a specified destination. Such information can be provided at the start of a trip or throughout the trip or both with regular updates to an in-vehicle device. Real-time route guidance systems update directions based on real-time traffic conditions, status of transit systems, and road construction/closures. The directions consist of simple instructions for turns or other upcoming maneuvers. The effectiveness of a route guidance system is a function of the market penetration levels of in-vehicle or hand-held route guidance devices, the quality of the information provided, and the response of users to the information provided . This study will model en-route route guidance systems within the context of the scenarios that have been developed.According to Professor Hempel, approaches to environmental policy are becoming more “integrated.” This increased integration takes the form of mandating “ecosystem management” and “sustainable communities:” i.e., air, water, and land use issues are considered together rather than separately. In addition, as environmental policy is increasingly subsumed under “sustainable development,” policies protecting the “environment” may make greater efforts to balance economic, social, and environmental concerns. Balancing multiple goals is extremely complex, according to Professor Hempel, and policies must be informed by large amounts of technical information and data. New technologies, such as Geographic Information Systems and ITS, can help generate this information and data to assist policymakers in taking such an integrated approach to environmental protection.

In addition to more integrated approaches, another trend in environmental policy is the increasing use of market-based approaches, with the latest example in California being the energy deregulation scheme. Professor Hempel believes that excessive reliance on market approaches raises environmental concerns. If price is the final arbiter of what is deemed “environmentally acceptable,” policymakers must be sure that those prices reflect full social and environmental costs. Fully reflecting these costs is not always possible,however. For example, when asking “what is the price of a tree” , this price will not reflect the full value of the tree because the real value is in its “ecological inter-connection” . Nature, according to Professor Hempel, is not just a resource component and cannot always be reduced to a separable level.The Sacramento Metropolitan region is expected to grow in population and employment opportunities over the next ten years. This region is expected to absorb another 700,000 people,greenhouse benches and employment is projected to increase between 40 and 50 percent . A majority of this growth will be in Sacramento County. In fact, downtown Sacramento can expect a 38 percent increase in employment and a 37 percent growth in population . Job growth is projected to be strong in the: 1) I-80 corridor between Roseville and Rocklin, 2) I-5 corridor around North Natomas, 3) Rancho Cordova, and 4) West Sacramento. Strong residential growth is expected in Franklin/Laguna, the Vineyard Area I-5/Route 99 corridor, Roseville/Rocklin corridor, and the North Natomas . Many transportation issues arise with an increase in demographics. One issue that will affect many people is congestion. With the growth in population and employment, vehicle trips will increase by approximately one third . Daily VMT will increase from 36,212,000 miles in 1996 to 22,202,000 miles in 2005 . With the increased demand on the freeway network, it is predicted that congestion will increase four fold . Along with the increased travel due to residents living in the metropolitan area, many long distance travelers, passing through the area, will also contribute to congestion in the region . In addition, suburb to suburb travel is rising quickly. In fact, suburb to suburb trips are increasing at a faster rate than suburb to downtown trips . Many of these trips were not anticipated by transportation planners as the area’s transportation system is not designed to carry the large volume of suburb to suburb trips . As a result, many of the corridors are congested. At present, other travel mode choices are very limited, and public transit most often requires that individuals make transfers . Currently, this region supports a light rail and bus system. Many critics have Stated that there is a lack of public transit in this region. Many citizens would like to expand the light rail system and provide more express buses . The goal of increased transit is to reduce congestion and increase the mobility of those who cannot drive by making public transportation more convenient. In a survey distributed in Sacramento and Yolo counties, the respondents preferred improvements to public transit over street widening . Air pollution is directly related to vehicle emissions. Currently, the Sacramento Metropolitan Area is classified as severe non-attainment area for ozone under the federal standards . Other measurements of air quality include PM-10 and CO. In Sacramento, both of these pollutant also are classified as non-attainment .

The baseline figures for the pollutants are given in Table B-6 below. Most of the pollutants decrease over time, with the exception of NOx and PM-10. Specifically, PM- 10 increases slightly, and NOx remains constant .The experience of Bacon et al. highlighted the difficulties associated with coding and calibrating the various aspects of a transportation network. Since the focus of this current research project is on evaluating the energy and environmental impacts of various ITS deployment scenarios, and not on calibrating a network or testing simulation models, it is highly desirable to make use of an existing calibrated network. As the selected simulation model is INTEGRATION, it is also desirable to have a network already coded for this model. In this light, the choices include: 1) the Urbansville and Thruville networks ; 2) the Smart Corridor ); and 3) the TravTek Orlando network .As mentioned earlier, we have selected the I-10 smart corridor for our simulation modeling exercise in this project. The main advantages of the Smart Corridor network are that it is partly calibrated, and it is the only California-based network of these options. There are advantages to using the Urbansville and Thruville networks, not the least of which is that they are already available to the project team. However, the work by Mitretek has been somewhat conceptual in nature and has not consisted of the calibration of an actual traffic network. The Urbansville network is a representation of Detroit, Michigan and its suburbs, consequently, it cannot be correctly calibrated with real-world measured traffic flows. Is it essential to have an accurately calibrated network to simulate the relative impacts of various ITS strategies involving a range of technologies/user services? It can be argued that if the same network is used for each scenario, the degree of calibration may not be critical, but the use of a calibrated network is likely to make the results of the study more credible. In addition to the requirement for a carefully coded network for simulation efforts, there is a need for information about the specific operational characteristics of the technologies to be modeled. The literature reviews and collection of FOT data that has occurred over the last year has provided the project team with much of this necessary information. One of the useful products of this research may be the specification of data needs from FOTs in order to be able to conduct detailed assessments of environmental benefits. This was addressed as an important issue by the ‘Expert Panel’ meeting held by Apogee in Washington, DC. .Presently, streambed restorations in California are actively pursued by a number of agencies, government jurisdictions, water districts, public and private land trusts, and research scientists. In general, most of these programs are governed by legal requirements related to the Clean Water Act and are implemented under the dual jurisdiction of both state and federal agencies. Nearly all programs implemented under this regulatory framework are properly classified as horticultural restoration programs.

The effort required to simulate a network is directly proportional to the size and complexity of the network.” One of the limitations encountered with the INTEGRATION model was the inability to establish a run with the network loaded entirely with unguided vehicles. Hence, the base run for the ATIS investigations was carried out with a network loaded entirely with guided vehicles. The research by Bacon et al. also highlighted the difficulty that detailed simulation models have in determining origin-destination demand patterns. Hence, it was recommended that a travel demand model be applied to generate origin-destination matrices for future simulation exercises. These investigations revealed that for a minor incident, a 100 percent IVIS system could offset the adverse traffic effects. However the effect of the major incident was only partially offset by all vehicles in the system being guided vehicles. Bacon et al. did not comment on how the results might change if not all vehicles were guided. There is a generally accepted theory that a threshold exists for increasing benefits from the penetration of IVIS technologies. Glassco et al. State that “the value of information to the ITS user decreases as the proportion of the population receiving that information increases. The value of information to the overall system, however, generally increases as the proportion of the population receiving that information increases.” The exception to this is when a very large proportion of the population reacts to the information, in which case the system performance may decrease. Gardes and May found that more than half of the system benefits resulting from the presence of guided vehicles on a portion of the Santa Monica Freeway corridor was possible with the first five percent of guided vehicles.

An average travel time savings of 3.8 percent resulted from the presence of five percent guided vehicles,blueberry grow pot compared to 5.6 percent for 25 percent guided vehicles, and 6.2 percent for 100 percent guided vehicles. Additionally, Gardes and May found that the travel time benefits of route guidance were similar under incident and non-incident scenarios. Another interesting finding of the study by Bacon et al. study was the conclusion that the effect of “distorted” information on total trip time was almost as adverse as a major incident upon which motorists receive perfect information. Despite the difficulties encountered with applying the INTEGRATION model, Bacon et al. still considered it to be the most suitable model for such studies. Unfortunately, this same study made no attempt to assess the environmental impacts. Nevertheless, this project team intends to consider extending their work to evaluate energy and environmental impacts.Glassco et al. found that adaptive signal control strategies and traveler information services have positive impacts on user and system efficiency for both recurrent congestion and non-recurrent congestion scenarios. However, the modeling studies revealed greater benefits resulting from these ITS user services, under non-recurrent congestion conditions . One specific finding was that vehicles that bypass incidents in an inter-city corridor network due to a route guidance system can reduce travel delays by 50 percent or more. The potential benefits of the ITS user services modeled were found to be greater when the network experiences a greater deviation from expected conditions. Hence, these ITS technologies may be most beneficial for a highly variable transportation system. In a highly predictable system, traffic control systems and travelers tend to adapt their plans according to the anticipated congestion. This process can take place without the influence of ITS technologies such as traveler information services. One of the interesting key findings of the study by Glassco et al. is that “the value of traveler information is proportional to the value of the alternative opportunities available. When the time required to follow an alternate route or to shift mode is high, then the timeliness and accuracy of travel time prediction is more critical.”

This would implies that pre-trip information has greater potential for providing benefits than en-route information, as once a traveler has started a trip he is not likely to have the opportunity to change mode, and by definition the number of route choices decreases for any trip as the traveler gets closer to the destination. In addition, once the trip has begun, the choice has already been made to make the trip at that time. If information was provided prior to the trip, warning of high levels of congestion, travelers have the option of postponing or even canceling the trip. Glassco et al. concluded that perhaps one of the most positive results of their studies of ATIS user services was that a relatively small number of probe vehicles are needed to support adequate network surveillance. They found that an unguided probe population as low as one percent can provide over 50 percent of the benefit of full surveillance, and that the value of route guidance with low probe percentages can be increased by updating travel times more frequently than every ten minutes. It is more effective to use unguided vehicles as probes than guided vehicles, since if all guided vehicles are being routed around an incident, the route guidance system does not learn that congestion has dissipated when the incident is cleared. Van Aerde and Rakha present the methodology and results of a modeling study of the Orlando TravTek operational field test. This test involved the deployment of 100 vehicles equipped with in-vehicle information systems for a one-year period. The modeling study attempted to extrapolate from the available field data the expected performance of a TravTek type system for levels of market penetration ranging from one percent to 100 percent, in terms of measures of effectiveness such as vehicle stops, fuel consumption, vehicle emissions, and accident risk. The Orlando TravTek network consisted of 2,700 links and 90 origin-destination zones, with a traffic loading of approximately 65,000 vehicles. The INTEGRATION model was used to simulate recurring congestion scenarios and freeway incidents for the afternoon peak period, with a small number of simulations of increased and reduced traffic demand to represent future and off-peak conditions, respectively. Consistent with findings by Glassco et al. , travel time benefits were found to be greater for the higher levels of congestion. An interesting finding relating to the much debated issue of latent demand, was that the LMP can be increased to provide travel time savings that have the potential to offset much of the induced demand that may be generated.

Travel time reductions were found for all levels of market penetration analyzed; however, the most significant marginal benefits were achieved at lower market penetration levels. The maximum reduction in travel time was found to occur for a LMP of 100 percent, where the network travel time saving was 15 percent. In addition to travel time savings, the study considered travel distance savings and found that a 7 percent reduction was possible at an LMP of 100 percent. En-route traveler information can have two conflicting effects on travel distance: distance traveled can be reduced by route guidance information which minimizes navigational errors, and distance traveled may be increased when users receive real-time traffic information and subsequently divert around an incident. The fuel consumption benefits of the TravTek system were predicted to be proportional to the travel time savings, with respect to the various LMPs. The magnitude of potential fuel consumption benefits is related to the topology of the network. That is, if when an incident occurs there are alternative routes available with little or no increase in travel distance, the potential for energy savings is greater. This is similar for emission benefits; however, the relationship between vehicle emissions,square plastic pot and traffic conditions and distance traveled is much more complex than for fuel consumption. HC emissions were found to decrease for all LMPs, with a maximum of 16 percent being achieved at a LMP of 100 percent. CO emission increased up to three percent for LMP’s less than 10 percent, and then decreased up to seven percent for LMP’s up to 100 percent. Van Aerde and Rakha claimed that ATIS may tend to always cause an increase in NOx emissions—they predicted increases in NOx of up to five percent, with the only reduction being one percent at an LMP of 100 percent.FOTs have provided little direct evidence of emission impacts of ITS in general, but they do provide some insights to ETC and ATSC. The US DOT’s comprehensive review of data available from FOTs does not even classify emissions or other environmental impacts in their summary table. Other FOT reviews do extract some limited results specific to ETC and ATSC and other traffic flow control systems, but echo the DOT’s general conclusion that FOTs have provided limited insight into environmental impacts. Little and Wooster reported that the focus of most of the FOTs had been on “technical feasibility and, to a lesser extent, user response.” Further, they note that most of these tests address more conventional traffic management user services, such as dynamic signal coordination. ETC facilities are one of the types of ITS technologies for which FOTs have produced emission assessments. Large emission reductions can be achieved at individual electronic toll facilities; however, the overall benefit is dependent on the frequency of toll plazas. The reductions, although large, are highly localized and may be insignificant at the network level. ATSC has shown significant potential for providing energy and environmental benefits in more than one FOT. Modeling results support the FOT conclusions regarding ETC and ATSC and provide additional insight for traveler information and vehicle guidance. Results reported in US DOT show that the application of the ATMS user services provided a general increase in speed and a reduction in total delay, although they do not analyze emissions specifically. Maximum benefits were achieved with the simultaneous implementation of signal coordination on arterial streets and ramp metering on freeway facilities.

Glassco et al. found that ATSC strategies and traveler information services have positive impacts on user and system efficiency for both recurrent congestion and non-recurrent congestion scenarios. However, the modeling studies showed greater benefits resulting from these ITS user services under non-recurrent congestion conditions . Modeling results reveal mixed results for navigation and traveler information systems. Analysis of Orlando’s TravTek network indicated varying levels of travel time, travel distance, and emissions effects depending on levels of market penetration. HC emissions were found to decrease for all LMPs, with a maximum of 16 percent being achieved at a LMP of 100 percent. CO emission increased up to three percent for LMPs less than 10 percent, and then decreased up to seven percent for LMPs up to 100 percent. Van Aerde and Rakha claimed that ATIS may always tend to cause an increase in NOx emissions. They predicted increases in NOx of up to five percent, with the only reduction being one percent at an LMP of 100 percent.Many researchers are trying to estimate the market for various ITS technologies. Three methods that are popular for estimating the market for ITS technologies include: focus groups with potential market segment members, interviews, focus groups and surveys of ITS experts, and evaluations of the traveler response to ITS demonstration programs . Without actual data and experience, it becomes virtual impossible to accurately predict the market for single and multiple ITS products and services. In market and traveler behavior research, the more innovative the solution or the farther into the future an innovation will be introduced, the more researchers must rely upon survey research methods and participant responses to experimental situations to understand the types of impacts that are probable or even possible. Turrentine and Kurani conclude that an experimental situation will often elicit and engage the decision processes of its participants and reveal participant lifestyle goals.Based on their new technology experience, Turrentine and Kurani conclude that current Stated preference survey methods often produce inaccurate estimates of the demand for unknown and new technologies. Other methods such as interactive Stated response when presented in the context of a household’s actual activity space and modal split data can produce more accurate demand estimates. In market evaluations of electric vehicles , many Stated preference studies often estimate huge price penalties for limited-range vehicles . In general, these studies rely on data from hypothetical-choice experiments in which participants are presented with choice sets of the vehicle. Then, participants are asked to identify the one vehicle, from each of the choice sets, they would be willing to purchase.