Tag: vertical farming

Instead, a single insertion event is approved for commercialization and then must be transferred via standard back crossing to other varieties. This is highly inefficient and often makes it difficult to regain the unique properties of all the diverse varieties.The IR-4 program could also assist with chemical residue testing and with other aspects of meeting the regulatory requirements for release of transgenic horticulture.Leaves growing in sunny locations have comparatively high photosynthetic capacities, Rubisco activity, rates of electron transport, and rates of dark respiration . Some species are restricted to sunny or shady locations, and the leaves of these plants are often genetically adapted to their characteristic light environment. The leaves of other species, including those that are naturally exposed to particularly variable light environments, acclimate to local conditions . Acclimation to extended changes in light enhances net assimilation and nitrogen use efficiency while decreasing vulnerability to high light stress .The local light environment influences the morphological development of leaves in many species, resulting in comparatively thick leaves in bright locations . Fully expanded leaves have a limited capacity for morphological change , and acclimation by these leaves requires biochemical changes in carboxylation, electron transport, and light harvesting, as well as modifications to chloroplast structure and orientation . Monocotyledons with basal meristems, long leaves, and dense canopies may represent a case where photosynthetic acclimation by biochemical change is particularly advantageous. The grass Lolium multi-florum exhibits a strong capacity for local photosynthetic acclimation along the length of a leaf . The leaves of plants like Lolium are produced in dark or dim conditions at the base of plants, and, over time,dutch bucket hydroponic are pushed to the upper part of the canopy. Typha latifolia , at all monocot that forms dense and highly productive mono specific stands in wetlands , may provide an even more extreme example. T. latifolia ramets originate from rhizomes that are buried in sediment, submerged under water, and often shaded by a dense layer of litter and existing plants.

Initial leaf growth is supported by carbohydrates that are either mobilized from rhizomes or translocated from older leaves. Depending on sediment thickness and water depth, and the density of the litter layer and existing canopy, the lower 50–100 cm of a Typha leaf may experience almost total darkness . These characteristics make Typha a useful experimental system for investigating the acclimation capacity of morphologically mature leaves. Basal growth in Typha allows the separation of leaf age from light environment; the oldest segments of Typha leaves are exposed to the brightest light, as opposed to plants with apical meristems, where the youngest leaves are in bright conditions. We investigated the photosynthetic capacity of T. latifolia leaves over time following step changes in shading at different locations along leaves. We hypothesized that morphologically mature Typha leaves have a strong ability for local acclimation, and that individual leaf segments acclimate to the local light level autonomously from the rest of the leaf.Two-month-old sun and shade grown plants with several fully expanded leaves were placed on a bench under high light, and a pair of fully expanded leaves from each plant were selected for experimentation. Individual leaf segments between 20 and 45 cm from the tip were exposed to either sun or shade during the 15-day transfer experiment using cylinders of 80% neutral shade cloth, creating the full combination of segments exposed to constant low light , constant high light , low to high light , or high to low light . Additionally, a set of segments on the same leaves were exposed to either constant high light or low to high light . All treatment combinations and locations were replicated six times. The photosynthesis rate under bright light , stomatal conductance and dark respiration rate were measured every two or three days for two weeks in the middle of the sun and shade segments , on six replicate plants using a portable gas exchange system . Afull sun was measured at a PPFD of 2000 mol m−2 s−1 and Rd was measured in darkness after allowing 3–5 min for equilibration. Leaf temperature was controlled at 25 ◦C and reference CO2 concentration at 370 mol mol−1. The leaf to air vapor pressure deficit ranged from 0.6 to 1.5 kPa. Photosynthetic light response curves were measured after leaves had fully acclimated to a change in light .

A full sun was calculated as the photosynthetic rate at 2000 mol m−2 s−1; Amax was calculated by extrapolating the regression to infinite light; Rd was calculated as the y-intercept; the apparent quantum yield was calculated as the slope extrapolated to darkness. The light response curves were started at high light , and assimilation was measured in response to stepwise PPFD decreases until full darkness. Stomatal conductance decreased gradually in response to light decreases, and increased gradually in response to light increases. This sluggish stomatal response either led to lower rates of photosynthesis for light curves run from dim to bright conditions relative to curves run from bright to dim conditions, or forced unreasonably long equilibration times. Moreover, midday field and greenhouse observations showed that leaves exposed to a continuous PPFD of 2000 mol m−2 s−1 for ∼15 min exhibited a steady CO2 assimilation. We therefore opted to carry out light curves from bright to dark conditions, but acknowledge that lags in stomatal adjustment may have resulted in somewhat higher Ci for the light curves than would have been observed for fully equilibrated leaves. Nonetheless, we emphasize that our study is comparative, and the key is consistency across treatments; we executed the light curves the same way for all treatments and leaf segments. Nitrogen concentration , and leaf mass per area , were measured on the leaf segments used for gas exchange. Nitrogen was determined using the micro Kjeldahl technique; samples were oven dried, ground in a Wiley mill, weighed, digested, and nitrogen concentration was determined with an auto analyzer .We characterized the vertical gradients of light and photosynthetic characteristics during midday sunny conditions in August 2004. The PPFD profile was measured through the canopy at 48 different locations in the SJFM using a horizontal quantum sensor mounted on a 2 m handheld pole. Each profile consisted of ten individual measurements recorded with a datalogger at 0.0, 0.6, 1.2, and 3.0 m above the sediment surface. The 3.0 m measurement was above the canopy. LAI was measured at the base of the canopy with a LI-COR LAI-2000, assuming non-clumped leaves and without distinguishing between live leaves and litter. Photosynthetic light response curves were measured on three segments of fully expanded leaves from 5 different plants. The cross section of leaves changed from flat at the tip to triangular at the base, and it was not possible to seal the chamber on leaf segments further than 100 cm from the tip.The parameters derived from the light response curves, the nitrogen content, and the leaf mass per area,dutch buckets system were compared between treatments using Univariate ANOVA or t tests.

The effect of light treatment was analyzed by Student’s t-test. Univariate ANOVAs and Tukey tests were used to compare Afull sun, Amax, gs and Rd between the light treatments within each sampling period. The effects and interactions of treatment and time following transfer were analyzed with multivariate analysis of variance ; this analysis corrected F values due to temporal auto-correlation. MANOVA does not require the response variables to be equally correlated, assuming an unstructured variance–covariance matrix . The effect of leaf position on the photosynthetic parameters of leaves growing in natural conditions was analyzed with three paired t-tests, because of the high variation among leaves. Statistical analyses were performed with JMP software version 7.0 and Minitab statistical software version 15.Our results confirm previous reports that species from highly variable light environments have a strong capacity for photosynthetic acclimation. In the case of T. latifolia, light heterogeneity is created by the combination of a basal meristem and a dense canopy of live leaves and litter . Typha leaves are exposed to markedly different light environments as they grow and individual segments are pushed upward . The upper segments of leaves in the field, which occurred in a brighter environment, had higher rates of CO2 uptake . Previous field studies on T. latifolia have also reported large CO2 assimilation and gs gradients along leaves . We hypothesize that the patterns of leaf photosynthesis and conductance in Typha reflect four properties. Mature Typha leaf segments are morphologically preformed to function in high light and allow high rates of Afull sun, regardless of the current or growth environment. Mature Typha leaf segments contain sufficient amounts of nitrogen to support high rates of Afull sun, regardless of the current or growth environment. Mature Typha leaf segments rapidly reallocate nitrogen between active and inactive pools in response to local light availability; acclimation occurs at a local level and does not require nitrogen translocation into or out of a leaf segment. The controls on stomatal conductance remain constant over time; the patterns of conductance can be explained based on simple, short-term adjustments that act to maintain a nearly constant Ci concentration despite the changes in Afull sun and the physical environment. We interpret these patterns as a highly plastic strategy that maximizes carbon gain by a monocot growing in a vertically heterogeneous light environment. The construction of leaves that are morphologically capable of high rates of Afull sun is a simple consequence of the spatial decoupling of the growth environment from Fig. 5. Midday photosynthetic photon flux density at 0, 0.6, 1.2, and 3.0 m above the soil surface at the San Joaquin Freshwater Marsh . The lower three locations were within the canopy; the 3.0-m observation was above the canopy. Typha latifolia light response curves measured at the SJFM as a function of distance from the leaf tip . Each curve is the mean ± 1 standard deviation of 5 curves on different plants. The continuous line is the best-fit non-rectangular hyperbola. that experienced later in life. The strategy of investing in leaves that have a morphological capability for high rates of CO2 uptake appears advantageous given a situation where it is difficult to predict which leaves will ultimately experience high light conditions, and where fully expanded leaves are unable to morphologically adjust to a change in light. High rates of Afull sun come at the cost of high Rd. A leaf with a low Afull sun in a shady site has a more favorable carbon balance than a leaf with a high Afull sun in the same environment; the carbon savings associated with reduced Rd more than offset the loss of potential photosynthesis during occasional sunflecks. The rapid down regulation of Afull sun following transfer to shade would be expected to improve the C balance of leaf segments by decreasing Rd. The initial changes in Rd following light change were probably tied to the changes in leaf photosynthetic activity, and the energy requirements to process and export carbohydrates, as well as changes in protein turnover . Subsequent changes in Rd may have been associated with changes in the biosynthesis and/ordegradation of cellular components, such as Rubisco, cytochrome f, and chloroplast ATPase . The amount of nitrogen in leaf segments remained nearly constant over time, leading us to hypothesize a fraction of the nitrogen in shaded segments is stored in inactive pools and is rapidly activated following transfer to high light. These changes may include adjustments in partitioning among carboxylation, electron transport and light harvesting, chloroplast ultra structure, volume, and orientation . The high N content of shaded segments should not be viewed as wasteful. These nutrients can be reabsorbed and reallocated to the rhizome during senescence; a high reabsorption efficiency of P and N has been reported for Typha dominguensis . Moreover, this strategy allows a leaf segment to rapidly and autonomously respond to a change in light availability, without importing or exporting nitrogen to or from other leaf segments or organs.Plants are the primary producers on earth, assimilating carbon dioxide by daytime photosynthesis for the biogenesis of all essential structures. This carbon assimilate is partitioned primarily into sugars and starch in the autotrophic ‘sources’ with a portion of the sugars allocated to the heterotrophic ‘sinks’ to support growth of the latter.

A cross-species comparison of environmental associations suggests some similarities in the genetic mechanisms involved in climatic tolerances across conifer genera. For each of four European conifer species in the Italian Alps, 6–18 SNPs were associated with precipitation/temperature PC axes . There was some overlap between species in the genes represented, including heat shock proteins, and cell wall construction and carbohydrate metabolism genes .Gene expression studies have identified a range of genes that may be involved in drought responses, but these results are not easily connected to the results of physiological or provenance response studies. First, RNA transcripts reflect the genes being expressed at a particular instant, whereas morphological or physiological traits are the result of processes acting over a longer time. Second, most gene expression studies do not examine differences between populations. Although some evidence suggests that stronger gene expression changes during stress are associated with greater growth or survival, different genotypes and demographic stages can show significant differences in gene expression changes . A few studies have begun to address this. Provenances of P. pinaster differed in the expression response of two dehydrin genes, as well as in physiology and mortality rates . Similarly, three genotypes of P. taeda differed in their gene expression responses to drought and re-watering . More such studies are needed, but care must be taken to distinguish between drivers of expression differences. For instance, a more drought-sensitive tree might express higher levels of dehydrins at a given drought stage because the leaf water potential has dropped faster than in a drought-resistant tree, whereas the resistant tree might express higher levels of dehydrins than the sensitive tree at a given leaf water potential.

Genome scan and G2E association studies can be useful tools in the search for genes responsible for local adaptation. Although such studies can identify loci at which allele frequencies differ between environments,nft system it is not always clear how these differences are connected to phenotypic differences, and thus what traits are under selection in a given environment. This is where QTL and G2P association studies are useful.Most conifer QTL studies have focused on wood traits, growth or yield. Of the two that have examined drought tolerance, the first identified four significant and four suggestive QTLs for d13C in P. pinaster, none of which co-located with QTLs for ring width . The second examined a wider range of traits – photosynthesis , chlorophyll fluorescence, gs, d13C, intrinsic WUE and specific leaf area – in F1 cross seedlings of P. pinaster when well watered or after 1 or 2 wk without water, and identified 28 significant and 27 suggestive QTLs . Locations of the QTLs for each trait varied by time point. Candidate genes within the QTLs were identified : those for gs and WUEi included stomatal regulation, ABA signaling and cell wall construction genes; those for d13C included an aquaporin; and those for chlorophyll fluorescence included transcription factors and a histone chaperone.However, only a few studies have investigated drought tolerance in conifers , with less success. All such studies used d13C as the focal trait. As we argue in Section VI, other traits would probably yield results that are more helpful for the understanding of drought responses. Gonzalez-Martinez et al. examined 41 candidate stress response genes of P. taeda, using 61 tree families planted at two sites. However, drought stress was probably mild, and they only identified one strongly associated gene and one weakly associated gene at each site. A later study on the same species examining 3938 SNPs identified seven new associations with d13C . Four of the associations were with unknown proteins, with only a transcription factor probably involved in the ABA-mediated stress response having an obvious connection to drought responses. G2P and G2E association studies complement one another, with the first identifying loci linked to targeted traits, but not whether these loci are under selection in nature, and the second doing the opposite.

The combination of these approaches is useful for the identification of genes and traits under selection in natural settings, but so far few studies have taken this approach. Eckert et al. tested the association of SNPs with five phenotypic traits and 11 environmental variables across 10 P. lambertiana populations around Lake Tahoe. This study identified six genes associated with phenotypic traits , and 31 associated with environmental PCs. Two genes were associated with both a trait and an environmental axis, including a glucose transport protein associated with d13C and environmental variables linked to water availability. A study focusing on multiple drought response traits and a larger number of SNPs might be able to identify more genes that have variants associated with both environmental gradients and drought tolerance traits. Some traits and processes involved in drought response have been better studied at the genetic level than others . Provenance studies have indicated that differences in stomatal control and shoot growth are often involved in local adaptation to drought, and all other study types have identified the genes likely to be involved . However, although root growth has also been identified as important by provenance studies, root-growth-related genes have not been identified. Conversely, although genes related to resistance traits, such as changes in carbohydrate metabolism, and protective and pathogen defense molecule production, have been identified in expression or association studies, these traits have been largely ignored in provenance studies. Finally, xylem traits, including refilling ability, have not been the focus of any genetic study type.Tree improvement programs that aim to increase growth potential and stress resistance face the challenges of long generation times, the need for large-scale field experiments and the late expression of traits such as wood density . Genomic selection, already routinely used in livestock breeding, has been proposed as a method of speeding up this process by using marker-predicted breeding values for phenotypes of interest . This approach is suitable for species with low LD and for traits with complex genetic architectures as it uses thousands of markers with effects that are estimated simultaneously . As with traditional phenotypic selection, accuracy is likely to be greatest when tests are carried out in environments similar to the target environment, because of the high likelihood of geno type 9 environment interactions .

Several recent studies have demonstrated the potential of genomic selection approaches for traits of interest to forestry. Resende et al. carried out an early evaluation of genomic selection in P. taeda, making use of clonally replicated individuals grown on four sites and genotyped at 4825 SNPs. They found that the accuracy of prediction models within sites ranged from 0.63 to 0.75 for diameter and height,hydroponic gutter and estimated that the breeding cycle could be speeded up by 50% with this method. Gamal El-Dien et al. used GBS to genotype over 1000 interior spruce trees over three sites that had been pheno typed for yield and wood attributes, and found that the incorpo ration of genomic information produced more accurate heritability estimates. Genomic estimated breeding values were most accurate when data from multiple sites were used to fit the model. Of even more relevance to selection for drought tolerance, Jaramillo-Correa et al. identified 18 SNPs associated with climatic PC axes in P. pinaster, and found that the frequency of locally advantageous alleles at these loci correlated with population level survival rates in a common garden at the hot/dry end of the species range. Together with the growth trait analyses, these results suggest that association techniques could be applied to predict breeding values for overall drought tolerance or particular drought tolerance traits even though only some of the loci involved have been identified. There is evidence of significant potential for selection approaches to improve drought responses in conifers. Provenance studies have shown evidence of genetic differentiation between populations in drought responses, and genome scan and G2E associations are finding evidence of natural selection on within-species genetic variation. Second, heritabilities for drought tolerance traits, when these have been examined, tend to be moderate to high. The calculation of heritability requires pedigree information: parent– offspring or sibling and half-sib comparisons. Narrow-sense heritability is the fraction of the variance in a trait attributable to additive genetic variation, as opposed to environmental and non additive genetic variation. Because heritability depends on both genetic variation in the population assessed and the degree of variation caused by the environment, estimates are not transferable between situations. In P. pinaster, estimates of d13C narrow-sense heritability ranged from 0.17 to 0.41, depending on how many individuals of what populations were assessed in what sites; and ring width and height growth rates were also moderately heritable . In the same species, heritability of P50 was 0.44, but this was driven more strongly by low levels of other sources of variation rather than high additive genetic variation . Across species, measured heritabilities for d13C range from the very high 0.7 for Araucaria cunninghamii to < 0.1 for P. taeda . Managers of wild forests are often focused on ensuring the resilience and function of the ecosystem rather than productivity. G2E and G2P association studies may help to identify seed sources that could be ‘preadapted’ to projected conditions for replanting in wild lands. However, wild trees face a range of challenges, including disease and competition, as well as drought . Stand structure and soil properties may also directly affect how trees experience drought stress. Studies that integrate stand level processes with genetic testing can further bridge gaps between genetic experiments and forest-scale management. Restoration projects could be used as experiments to test genomic predictions of survival and growth in a given environment, as well as the effects of genetic composition and diversity of the planted population on restoration success.

Common garden, gene expression and genetic association studies all have different strengths and weaknesses, and none alone will answer the question of how genetic differences affect drought tolerance . As described previously, a combination of different types of association study may help to identify loci that are under selection in the wild and the traits they influence. Similarly, gene expression studies could easily be combined with common garden studies of adults or seedlings to address whether differences in drought responses between populations or genotypes are a result of differences in gene sequences, gene expression patterns or both.Many studies to date have focused on WUE, often using d13C as a proxy. As discussed above, however, WUE is a ratio of changes in photosynthesis and transpiration, which can both vary, and higher WUE may or may not be associated with greater survival or growth in dry conditions. Moreover, different measures of WUE are not entirely consistent. We therefore recommend that future studies use survival and/or growth during and following drought as the metric of overall ‘drought tolerance’, and measure photosynthesis and water loss separately if these are processes of interest. The time involved in the measurement of traits for hundreds or thousands of individuals has encouraged the focus on easily measured d13C, but much progress has been made in high-throughput phenotyping techniques . For instance, thermal and long wave infrared sensors can measure leaf temperature or stomatal conductance, near and short-wave infrared sensors can measure leaf water content, and fluorescence sensors can measure chlorophyll content and photosystem efficiency .There are several traits and processes that have been suggested to be important for drought response by physiological studies, but about which there is little genetic information . Genetic studies frequently identify genes related to carbohydrate metabolism and transport as having altered expression or allele frequencies depending on water availability. It is difficult to make sense of these patterns because the link between these metabolic changes and tree function and survival during drought is still unclear. We also know relatively little about which species can refill cavitated xylem, under what circumstances and by what mechanisms. Thus, it is difficult to determine whether any genes identified by expression or G2E studies are involved in this process. Similarly, how roots and root growth respond to changes in water availability, and what genes are involved in these responses, remain poorly understood. Although the measurement of root architecture can be complex, high-throughput methods are being developed for this as well .Most experimental studies, including those looking at gene expression, have focused on seedlings.

The first target analyzed is human butyrylcholinesterase , an enzyme that can act as a bio-scavenger to counteract the effects of cholinesterase inhibitors such as sarin and that is a candidate for bio-defense countermeasures in several countries. While this product would encounter market dynamics that are different from other commercial products, it is nevertheless designed to satisfy an important component of public safety and merits review. Currently, BuChE is extracted from outdated human blood supplies, but it can also be made recombinantly in cell culture, transgenic animals, and plant systems. The second case study focuses on the cellulase complex, a mixture of 4–6 enzymes used to saccharify cellulosic feed stocks for the production of ethanol as a fuel extender. This target was selected for study because, for more than 30 years, the cost of cellulases has been a major impediment to the economic viability of cellulosic ethanol programs. Cellulases were also selected because they represent an extremely cost sensitive product class on which to conduct case studies. We reasoned that if plant-based manufacturing showed economic promise for this class, then the economically advantageous production of less cost-sensitive biotherapeutics and other products might also be anticipated. In contrast to BuChE, which consists of a purified molecule, the cellulase complex would be expressed in plants that are cultivated near the cellulosic feedstock and the bio-ethanol refinery and stored as silage without purification; the semidried catalyst biomass is mixed on demand with the cellulosic feed stock to initiate saccharification followed by fermentation. This approach varies significantly from previous approaches in which cellulase enzymes are produced via fermentation processes using native or engineered microorganisms.

For the cellulase case study, the plant-based cellulase production process is compared with a recent technoeconomic analysis of cellulase enzymes produced from Trichoderma reesei fer mentation using steam-exploded poplar as a nutrient source.The technoeconomic modeling for both case studies was performed using SuperPro Designer, Version 9.0 , a software tool for process simulation and flow sheet development that performs mass and energy balances,rolling bench equipment sizing, batch scheduling/debottlenecking, capital investment and operating cost analysis, and profitability analysis. This software has been used to estimate cost of goods in a variety of process industries including pharmaceuticals produced by fermentation and plant made pharmaceuticals. It is particularly useful at the early, conceptual plant design stage where detailed engineering designs are not available or warranted. Super Pro Designer was chosen because it has built-in process models and an equipment cost database for typical unit operations used in the biotechnology industry, such as bioreactors, tangential flow ultrafiltration and diafiltration, chromatog raphy, grinding/homogenization, and centrifugation. There are some unit operations and processes used in the case studies that are currently not included in SuperPro Designer, such as indoor or field plant cultivation, plant harvesting, vacuum agroinfiltration, and screw press/disintegrator. For the butyrylcholinesterase case study, SuperPro Designer’s “Generic Box” unit procedure was used to model these unit operations. For the cellulase case study, the indoor unit operations were modeled with the same software while the field production calculation and costs were tracked in Microsoft Excel spreadsheets. Unless otherwise noted, the costs of major equipment, unit operation-specific labor requirements and costs , pure components, stock mixtures, heat transfer agents, power and consumables used in the analyses were determined using the SuperPro Designer built-in equipment cost model and default databanks. For the cellulase case study, the program’s parameters such as water costs and total capital investment distributed cost factors were set to be the same as those used in the model described in Klein-Marcuschamer et al.; this SuperPro Designer model is also available at the Joint Bio-energy Institute technoeconomic analysis wiki site .

Additional case study specific design parameters were selected based on experimental data from journal articles, patent literature, the authors’ laboratory, interviews with scientists and technologists conducting the work cited, technical specification sheets or correlations, heuristics, or assumptions commonly used in the biotechnology and/or agricultural industry. The case study models were based on a new “greenfield” facility, operating in batch mode, although annual production costs neglecting the facility dependent costs were also determined to predict annual production costs using an existing facility. For the butyrylcholinesterase case study, annual operating time of 7920 hours for the facility was used with indoor grown Nicotiana benthamiana plants.For the cellulase case study, since the tobacco plants are grown in the field, it is assumed that plant growth occurs for 215 days of the year and the indoor facility is in operation for 127 days per year . For comparative purposes in the cellulase case study, the laboratory/QA/QC costs were neglected since they were neglected in the JBEI model and such costs are likely to be a minor component for the industrial enzyme case study.For the butyrylcholinesterase case study, the process flow sheet was split into separate modules to better understand the contributions of various process segments.Process flow and unit operations were derived from published methods and results from a number of sources as indicated in each case study, and from interviews with leading gene expression, agronomy, and manufacturing scientists and engineers who have participated in the development and scale-up of the processes described. On the basis of this information, the SuperPro Designer software was applied to calculate material inputs and outputs, bulk, and per-dose or per-unit costs.The two AI classes evaluated in these studies are produced in Nicotiana host plants. Nicotiana species, notably N. tabacum, N. excelciana, and N. benthamiana, are preferred hosts for PMB manufacture due to their metabolic versatility, permissive ness to the propagation of various viral replicons, and high expression yields achievable with a wide range of targets, as reviewed by Pogue et al., De Muynck et al., Thomas et al., Gleba et al., and others. Use of these hosts for production of clinical trial materials is also familiar to FDA and other regulatory agencies, thus facilitating Nicotiana’s acceptance in regulation-compliant manufacturing.

The enzyme is a globular, tetrameric serine esterase with a molecular mass of approximately 340 kDa and a plasma half-life of about 12 days; the plasma 1/2 is largely a function of correct sialylation. BuChE has several activities, including the ability to inactivate organophosphorus nerve agents before they can cause harm. With the recent use of chemical nerve agents such as sarin, there is continued interest on the part of many governments in stockpiling BuChE as a countermeasure. Currently BuChE is purified from outdated blood supplies; however, the high cost of this route and its low supply limit its utility. It has been estimated that extraction of BuChE from plasma to produce 1 kg of enzyme, which would yield small stockpile of 2,500 400- mg doses, might require extraction of the entire US blood supply.Large amounts of the enzyme are required for effective prophylaxis because of the 1 : 1 enzyme/substrate stoichiometry needed for protection against OP agents. Not surprisingly, recombinant routes have been explored and the enzyme can in fact be produced by microbial fermentation,grow table hydroponic animal cell culture, and transgenic goats and stably or transiently expressed in Nicotiana, albeit at modest levels of 20–200 mg/kg fresh weight biomass, with yield improvements being the target of ongoing research. The bacterial product is nonfunctional and the mammalian cell culture products do not have the plasma 1/2 needed for prophylaxis and may be difficult and expensive to scale, as discussed by Huang et al.. Goat milk produced BuChE can be obtained at 1–5 g/L milk, but consists mostly of dimers, is undersialylated and has short plasma 1/2. While expression yields are impressive, transgenic animal sources face challenges of herd expansion to satisfy emergency demand, as well as potential adventitious agent issues, and these challenges need further definition. Furthermore, of these options, only plant-based bio-synthesisyields an enzyme that is sialylated and appears to reproduce the correct tetrameric structure of the native human form in sufficient yield to be commercially attractive; hence, the plant-based route became the basis for our modeling exercise. Not surprisingly, the plant route for BuChE manufacture is also the subject of continued DARPA interest and support .BuChE can be produced sta bly in recombinant plants or transiently in nonrecombinant plants by viral replicons delivered by agrobacterial vectors introduced into the plants via vacuum-assisted infiltration. Relative to stable transgenic plants, the advantages of speed of prototyping, manufacturing flexibility, and ease of indoor scale-up are clearly differentiating features of transient systems and explain why this approach has been widely adopted in the manufacture of many PMP . In our analysis of BuChE, we used expression yields from several sources that evaluated various Agrobacterium mediated expression systems, including Icon Genetics’ mag nICON expression technology. Magnifection should be familiar to most readers of this volume as it has been applied in R&D programs throughout the world and its features have been the topic of multiple original studies and reviews ; therefore, the method is not described here in further detail. Likewise, the process of vacuum assisted infiltration has been described in detail by Klimyuk et al., Gleba et al., and others and is not further explained here.For BuChE, we mod eled the use of an N. benthamiana transgenic line modified to express the mammalian glycosylation pathway, beginning with a mutant host lacking the ability to posttranslationally add plant-specific pentoses but with the ability to add galactosyl and sialic acid residues to polypeptides, based on work recently reported by Schneider et al. .

Use of this host obviates the need to enzymatically modify the plant-made polypeptide in vitro after recovery to ensure the presence of correct mammalian glycan, a procedure that could substantially increase the cost of the AI. A glycan engineered host can be produced in two ways, by stable transformation or via use of multi-gene agrobacterial vectors. The feasibility of sialylation via the latter approach was shown recently by Schneider et al. for BuChE. Although there is an extra element of time required to develop a stable transgenic host compared to the transient modification of a pathway, the availability of a transgenic plant obviates the need to manufacture several Agrobacterium vectors carrying the genes for the product and two binary vectors carrying genes for the sialylation pathway; a procedure that would require additional capital and operational investments to generate multiple inocula in large scale. Therefore, for modeling upstream processes, we assumed that transgenic seed was available and that the resultant BuChE would have mammalian glycans and form tetrameric structures, and hence its biological activity and plasma half-life would be comparable to the native human enzyme.To model downstream purification of BuChE, we assumed harvest and extraction at 7 days after inoculation. Biomass disruption was by homogenization, followed by filtration and clarification, as generally described, but with modifications required for scale-up as indicated in Results and Discussion. Purification of the enzyme was by procainamide affinity chromatography. In the overall process, plant growth, inoculation, and product accumulation steps occur indoors in controlled environments, and extraction, clarification, and final purification of BuChE take place in classified suites, so that manufacturing and release of the enzyme can be compliant with FDA cGMP guidance for human therapeutics. Design premises for this process, specific assumptions used in modeling, and resultant cost calculations are presented .Cellulases currently under evaluation in bio-ethanol programs are all produced by microbial fermentation. Despite decades of research on lowering cellulase manufacturing costs, these enzymes still account for 20–40% of cellulosic ethanol production costs. Hence, lowering the cost of the biocatalyst is critical to the eventual adoption of bio-fuel processes that utilize renewable plant biomass feed stocks without competing with food or feed supplies. An alternative to fermentation produced cellulases is the production of these enzymes in crop plants, with the ultimate goal of producing cellulases at commodity agricultural prices. This process concept was modeled to estimate enzyme and ethanol costs produced by this approach. Should such a process for cellulases prove economically viable, it might encourage the production of other cost-sensitive PMB as well as bio-materials, food additives, and industrial reagents.Scale requirements and cost limitations of cellulases for bio-fuel applications constrained us to model production to open fields, with minimal indoor operations. We initially surveyed two scenarios for inducing production of cellulases in field-grown plants. The first was adaptation of the typical agroinfiltration method.

Further, most farmers strongly suggested the need for crop insurance that compensates in value terms, but they expressed no strong preference among compensations based on gross sales, profits, or production costs.Financial variables examined were off-farm incomes, gross sales, debts, and assets. Clearly, the portion of house hold income risk attributable to variation in farm income decreased as the share of off-farm income rose. For our sample, an average of 63 percent of income came from off-farm sources. A sizable segment of farmers, as many as 25 percent, derived less than 1 percent of their in come from farming in the year sampled. This is consistent with the observation that many of the farms were quite small, many farms operated at a loss in any given year, and there was a relatively large number of so-called “hobby” farms in California. Gross agricultural sales averaged about $0.4 million per farm for the entire sample. Vegetable farms averaged $1.1 million in sales, followed by ornamental crop farms with $0.8 million, and orchard farms with $0.3 million. About 6 percent of fruit/nut farms had sales of more than $1 million, compared to 29 percent for vegetable farms and 13 percent for ornamental farms. Agricultural sales were negatively correlated with off farm income share and positively correlated with acreage. Revenue per acre decreased as acreage increased. Given that specialty crops vary widely in unit value and in value per acre, this indicated that farms with fewer acres tended to grow crops with a high value per acre. Farms in our sample had an average of $1.4 million in assets and $0.6 million in debts. The average debt-to asset ratio was close to 0.5. This ratio is much higher than the 0.16 debt-to-asset ratio reported by the United States Department of Agriculture for all American agriculture in 2003. When viewing assets and debts as financial inputs necessary to generate revenue, the ratio of financial input to gross sales was highest for vegetables and lowest for orchard crops.This study provides a detailed statistical profile of an important segment of California agriculture, the horticultural crop industry.

The information provided is based on a unique survey of growers of horticultural crops, also known as specialty crops,vertical grow table that was conducted during the spring of 2002 at the request of the Risk Management Agency of the United States Department of Agriculture . This report presents data about horticultural industries in California and about the risk management attitudes, approaches, and needs of farmers producing these commodities. Specialty crops are diverse. These crops can best be defined by exclusion—as all agricultural crops excluding grain crops , oilseeds , cotton, peanuts, and tobacco. The bulk of specialty crops consist of fruits and nuts, vegetables, and ornamental crops . The industries featured in this study accounted for more than $16 billion of gross farm revenue in 2001. This value was more than 90 percent of the state’s total crop value and 60 percent of total agricultural value produced in California at the farm level. These industries are also important nationally. California accounts for 37 percent of the total value of horticultural crop production in the United States. In the past, these industries have expanded steadily in California, adding more than 300,000 acres between 1992 and 1997 . In the future, California’s horticultural industries are expected to continue to expand in size and importance. For the most part, horticultural growers have not been major recipients of farm program subsidies and have had relatively little government support compared to growers of commodities such as grains, oilseeds, cotton, sugar, and dairy products. Some horticultural crops have been eligible for USDA crop insurance programs and ad hoc disaster assistance, promotion assistance, and miscellaneous support, but the degree of subsidy has been small—typically around 5 percent of total value, compared to 30 to 50 percent and higher for grains, oilseeds, and cotton . Horticultural crops differ from other kinds of crops in their product characteristics, production processes, and market environments and thus in their risk characteris tics. The design of public policy for these crops must reflect management of their unique risks. Knowledge of market variables and grower risk behavior is essential to developing effective risk management tools for horticultural crops.

Unfortunately, while studies on traditional crops abound, little research has been done on horticultural crops. The objective of this survey was to generate wide-ranging statistical information that can be used broadly to better understand the horticultural crop industry, its sources of risk, and typical responses to those risks. The statistical profile of California’s horticultural producers presented here is the most exhaustive ever undertaken for this group. It draws on survey data collected from approximately one-third of all horticultural crop producers in the state. This report presents a large volume of information concisely. To do so, we summarize the methodology used to collect and tabulate the data; provide an over view of the seven topics addressed; and discuss the primary results. The discussion is organized by issue and includes a narrative describing the main findings for each topic. Selected figures and tables are included. The narrative is supplemented with a data section in the Appendix, which is organized into three parts. The first provides the response rate for each question in the survey. The second contains data tables organized by commodity category. The tables supplement the information presented in the narrative section with further disaggregated analysis. The last part of the Appendix provides the actual survey instrument.The first stage of the study, the survey of specialty crop growers, involved developing a questionnaire. The questionnaire was developed specifically for specialty crop growers based on the format of a survey instrument used previously , with input from RMA and from researchers who conducted an identical study in Florida, Pennsylvania, and New York. The California Agricultural Statistical Service assisted in formatting the questionnaire to facilitate its implementation. The final version of the survey instrument is presented in Appendix 3. We established the sample frame by defining a mini mum number of acres required for a farm to qualify for the study using information from CASS’s database. To be included in the study, a farm had to have at least five acres of perennial crops or at least two acres of annual specialty crops . This limit was designed to exclude very small farms that were unlikely to be commercial operations.

The acreage criterion was applied to CASS’s database, which contains information on more than 60,000 farms in California . A total of 31,864 farms met the acreage limit with the crops selected for the survey. CASS conducted two rounds of mailings and one round of telephone interviews to collect completed surveys. In total, the two survey mailings garnered 7,391 responses. Those mailings were followed by telephone interviews of growers who had not responded by mail, which collected an additional 7,746 responses. In total, 15,137 responses were received . Relatively few farmers answered all 25 survey questions, which required responses in 192 cells. Under some “usability” criteria on the completeness of the DATA COLLECTION AND AGGREGATION answers, some responses were discarded.1 In total, 10,410 observations were entered into an electronic database file that was then transferred to the authors. Our primary analysis used only the horticultural-crop based sample, mobile vertical grow tables which consisted of 10,200 observations.Among non crop categories, aquaculture producers provided the largest number of observations, allowing some statistical analysis of that industry. We provide data tables for aquaculture in Appendix 2 but omitted aquaculture from the narrative analysis. Note that sample size used in our analysis varies depending on the question being analyzed. Survey responses varied in degree of completeness, and valuable information could have been lost if only fully completed responses were used. Thus, to maintain the maxi mum sample size, different sub-samples were used, depending on the usability and appropriateness of the data provided, in analyzing particular issues. Information on sample size is included in most of the table presentations.Several mountain ranges in California create the dominant Central Valley and smaller coastal valleys where much of the state’s agricultural production is concentrated. The large Central Valley consists of the Sacramento Valley, which lies north of the San Francisco Bay Delta, and the San Joaquin Valley, which lies south of the delta. The Central Valley is encircled by the Cascade ranges and Klamath Mountains to the north, the Sierra Nevada Mountains to the east, the coastal ranges to the west, and the Tehachapi Mountains to the south. The coastal ranges also create a long strip of valleys, including, for example, Napa Valley and Salinas Valley. Climates in the region are affected by the cool cur rents of the Pacific Ocean and various mountain ranges. Temperatures in coastal regions are relatively mild while inland areas are hotter. Almost all of the state’s rain and snowfall occurs during late fall and winter . The majority of California’s water sup ply originates in the northern mountain regions of the state. Land for specialty crops is nearly all irrigated via ground water and various district, state, and federal water storage and distribution systems . California has 58 counties. In our analysis, we aggregated the counties into 11 regions with similar geographic and climatic characteristics as shown in Figure 1. The Sacramento Valley and San Joaquin Valley are together referred to as the Central Valley.California’s specialty crops include more than 200 individual crops.

To facilitate a manageable analysis, crop aggregation was needed. Crop codes were developed using three levels of classification. First, all the commodities were assigned to one of five basic categories: field crops, fruits and nuts, vegetables, ornamental crops, and non crop commodities. The last category included a small number of apiary and aquaculture farmers, but for category-specific analyses, we considered only aquaculture farmers because there were too few apiary farmers for any statistical analysis. Fruits/nuts, vegetables, and ornamentals, which were our focus, were then further divided into subcategories of similar types of crops . The third level of classification identified specific crops. Our data analysis used mostly the first two levels of classification. See Table 1 for a detailed description of the classifications. While classification of fruits and nuts into the second level is self-evident, such classification of vegetables needs discussion. A wide variety of vegetables appears in the data and choosing transparent and intuitive yet manageable groups was difficult. Following USDA guidelines, nine botanical classifications of vegetables were aggregated into six groups, guided by climatic growing conditions and by the number of observations available.Farm Size and Regional Profile discusses regional distributions of production for commodity categories and subcategories. It also provides mean acreage and acreage distributions. Mean acreages have relatively large standard deviations. To supplement this information, the distribution of farmers by acreage class has been included. Information provided on this topic pertains to Questions 1 through 6 . Crop Diversification provides information on patterns of crop diversification across crop categories and subcategories. For example, do farmers of perennial crops diversify into annual crops in the same way that annual crop farmers diversify into perennial crops, or do they tend to diversify within the same crop category? This section also includes information on organic farming. In formation provided in this section was obtained primarily from Questions 4 and 5 . Marketing issues include whether a crop is designated for processing or fresh use, the types of marketing channels used, and whether a farmer’s operation involves both growing and shipping or growing only. Marketing channels typically differ according to end use . Whether an operation grows and ships or only grows concerns crops intended for fresh use only; shipping and packaging are not issues for crops destined for processing, which are typically delivered to the plants in bulk. This section also explores the issue of whether price is predetermined through a contract before the time of sale. This section pertains to Questions 6, 7, and 8 in the survey. Yield, Price, and Profit Fluctuations for the preceding five-year period were explored next. Respondents were asked to provide actual yields for those five years; identify the highest fluctuation in yield, price, and profits during the same period; and indicate the main cause for their lowest profits.

When a garden can be surveyed in its entirety, visitors were more likely to consume it from afar than to indulge in its experiential qualities.Also implicit in the collective negotiated design process and the dynamic edge between the centre and external periphery of the garden was that gardens operate somehow as test-beds for operations at the landscape scale—in the same way as the pavilion is typically revered within architecture as an incubator for more expansive architectural praxis. However, the relationship of the garden to the landscape is far more dialectical than its architectural equivalent, and what goes in the garden is not necessarily an experiment for subsequent deployment in the landscape. The garden is more of a counterbalance than a small fragment of landscape; the two interact of course, but from a garden, a landscape does not necessarily grow. There are certainly exceptions to this rule—such as ‘seed dispersal’ concepts that were popular in the 1980’s where the garden was engineered to disseminate its genetic produce on the wind—but the point is that in these examples the garden is sacrificed to its expansion or duplication into the landscape Nevertheless, while not prefiguring landscape-scale operations, gardens have a more encompassing role as potent cultural litmus papers; as Bernard Lassus notes, ‘gardens have almost always foretold in advance the relationships between … society and nature’.In this regard, gardens are more persuasive as reflectors— either of self or society—than empirical experiments that generate results applicable to the world at large. This efficacy of the garden differentiates it from the landscape on the whole, although when we start to consider the consciously designed landscape as opposed to the general cultural landscape,plastic pots for seedlings the issue becomes more obfuscated.

My interpretation of James Corner’s characterization of the real limits to landscape architectural practice in the world illuminates this convergence. Given that landscape architecture influences only a very small percentage of outdoor construction projects, with other aesthetically unconscious operations undertaking the lion’s share, Corner positions landscape design as a primarily ‘metaphorical and ideological’ rather than solely demonstrative or performative praxis; one that uses its cultural currency to edify and illuminate an ecological message—to provide a foundation on which to reflect, rather than attempt to physically cure the world within its own diminutive footprint.This is, I would argue, is also descriptive of the role of the garden. Therefore, while a garden doesn’t necessarily equate to the landscape, the two genres increasingly converge and overlap in contemporary theory and praxis. At a conceptual level, the university gardens pertinently navigated the convergent muddy territory between gardens as reflectors and gardens as demonstrative landscapes. The move to de-frame is the key mechanism in engaging this terrain, although the one threshold that the design teams had no control over restrains its effectiveness: the fence around the Expo site itself. In this regard, the perimeter boundary is physical but also social; while the frame may enable representation by physically separating nature from the continuum of the world, division is also imposed through less tangible but equally powerful social forces. Indeed, to conflate the picturesque as an example, the ultimate frame was formed less from ha-ha’s or the limits of representation, than along lines of society and class. Beyond entrance gates and perimeter fences, garden shows are historically typically also be framed within these societal terms.Whereas William Kent may have ‘leaped the fence, and saw that all nature was a garden’, 66 to jump or destroy the wall of a horticultural expo is to typically find the periphery of a city, complete with its own implied social delineations.

It is in this context that the dissolution of the physical and psychological frame of the institutionalized expo itself— rather than the frames of the individual gardens within—that is the more potent force in contemporary landscape and urbanism.To provide the highest quantification accuracy when comparing samples one needs to minimize differences introduced in the processing of samples and acquiring the data. This can be best achieved through the introduction of stable isotopes into samples that allow samples to be mixed and then analyzed in the mass spectrometer. The application of metabolic labeling, which uses stable-isotope labeled amino acids in cell culture or 15N nitrogen-containing salts into the whole cell or organism in vivo, enables relative quantifications of proteins on a global scale. In such a quantitative experiment, one sample is labeled with the natural abundance , and the other with a stable isotope of low natural abundance during growth. The samples are mixed, processed, and analyzed by the mass spectrometer. Chemically identical peptides from these light- and heavy-labeled mixed samples co-elute by chromatography into the mass spectrometer, which can distinguish between the light and heavy peptides based on their mass difference, and thereby quantify the difference in peptide, and hence protein abundance between the samples. An alternative stable isotope-based strategy is to chemically tag peptides after enzymatic digestion; the most popular reagents for this strategy are isobaric tagging reagents Tandem Mass Tags . The TMT isobaric tagging reagents allow comparison of a larger number of samples, but the labeling is done at the peptide level after sample digestion and then samples are mixed. In contrast, the metabolic labeling is introduced into the proteins during growth, thus samples can be combined at the beginning, minimizing variations introduced by sample processing that can compromise quantification accuracy .

Although SILAC has been widely used in animal cell lines and has been the gold standard for MS-based proteomics quantification , 15N-labeling based quantitative applications are still quite limited in plants despite it being cheaper . This could be due to the complexity of the data analysis. SILAC pairs are easily identifiable because they have well-defined mass differences as typically only lysine and arginine are labeled. In contrast, in 15N labeling, each amino acid in the expressed proteins is labeled, and therefore, the mass difference in 15N pairs varies depending on the number of nitrogen atoms in their composition. Also, as more amino acids are being labeled, the effect of incomplete incorporation of the heavy isotope can be more pronounced under some conditions, such that isotope clusters of heavy labeled peptides in the survey scan MS1 spectra are generally broader, making it harder to identify the monoisotopic peak.There are very few freely available software tools with work flows that can analyze large-scale 15N labeled samples. Such tools include MSQuant , pFIND , and Protein Prospector . The workflow using MSQuant normally requires manual inspection of the pairs of the light and heavy forms that both fit with expected isotope envelope distribution; those that don’t fit the criteria will be omitted from further analysis . This makes it very time-consuming for a large dataset because of the manual inspection prerequisite. In addition,dutch buckets if both forms need to be present for quantification, then there will be a high false-negative rate for some of those highly biologically interesting proteins which only express in one of the two conditions, or from immuno precipitated samples where those proteins will be only in the bait-IP but may be completely absent in the control IP. Here, we present the 15N quantification workflow based on the free web-based software Protein Prospector . After data search with respective 14N and 15N search parameters, quantification between the light and heavy peptide pairs is done based on the identification of either the light or heavy peptide, or both.Additional features in Protein Prospector include a Cosine Similarity score which can be utilized to reduce manual checking of spectra and a cache function that enables efficient result retrieval through cached result storage. This workflow allows us to report quantifications of thousands of proteins and is applicable to the quantification of the total proteomes, sub-proteomes, and immunoprecipitated samples.This workflow can quantify thousands of proteins simultaneously. We demonstrate its performance using three in different proteins is relatively constant . With less complete labeling, the identification rate of heavy labeled peptides is significantly lower than light due to errors in monoisotopic peak assignment . If the labeling efficiency is achieved 98.5% or above, the identification rate between 14N and 15N search is similar in our experience. High-level labeling depends on three factors: 15N containing salt needs to be over 99% purity; we find 15N chemicals from Cambridge Isotope Laboratories are generally high-purity. The labeling time. We recommend growing Arabidopsis for 14 days to achieve high labeling efficiency. If plants can only be labeled for a shorter time before harvesting, then it is recommended to label the plants for one generation using a hydroponic system and start the experiment using the labeled seeds.

If the Arabidopsis plants are small after 14 days of growth, then the labeling efficiency will be lower, for instance, our acinus-2 pinin mutants are smaller than wild-type plants, therefore the labeling efficiency is lower than wild-type with the same duration of labeling. The availability of the 15N salt. Seeds should not be sown too many on solid-medium plates or in the liquid medium. We recommend the Arabidopsis plants are labeled 14 days or more to achieve high labeling efficiency and high identification rate, but users should be cautious not to stress plants by leaving them on medium for too long. Almost all proteins except seed storage proteins are labeled. These are not synthesized during the seedling stage and therefore they don’t incorporate the 15N labeling during growth and will remain unlabeled.Co-eluting peptides are common problems, especially in highly complex samples, and interfere with quantification. High resolution scans in MS1 reduce peak overlap, improving the accuracy of quantification , so we typically acquire our data at 120K resolution. High mass accuracy in MS2 helps to reduce the false discovery rate . Higher FDR was reported in the 15N sequence assignments due to more isobaric amino acid forms present in 15N labeling when the MS2 fragmentation was done using a low-resolution and mass accuracy QTOF2. To check this possibility in MS2 data acquired at high resolution,we compared the FDR in our seven labeled experiments listed in Table 2. After we combined peptides for 14N and 15N searches together with 1% FDR, we parsed the target and decoy 14N and 15N matches and calculated the FDR separately. We found the 15N data results had a lower FDR than that of 14N data search when MS2 scans were done in the high resolution and high-mass accuracy Orbitrap mass spectrometer . This trend is more pronounced in the higher labeling efficiency datasets. If FDR was calculated based only on first three datasets , there was no significant difference between 14N and 15N FDR, despite the average of FDR is slightly lower in 15N search. When four more Col/sec-5 datasets were included for comparison, the 15N FDR is significantly lower than 14N one, indicating using high-resolution and high-accuracy measurement, the unique mass of 15N modification to the amino acid may empower less random matches in data searches.After each peptide is quantified, they are compiled into protein groups in “Search Compare”. The spread of ratios for peptides from the same proteins are measured using the interquartile range, and Q1 , median, and Q3 are reported, as illustrated in Figures 5A,B and quantification of the pairs can be visualized as Figures 5C,D). Here we include two biological experiments as a demonstration. To calculate the median and Q1, Q3, the log base 10 of all the ratios of the peptides from the same protein are first calculated in Protein Prospector to generate log base 10 of median and Q1, Q3, followed by converting these log values to normal values by raising 10 to the power. To plot these quantification results, an R script is written as in Supplementary Data 1. In the R script, the log base 2 of all the ratios of the peptides are calculated and converted back to normal values by ratio 2 to the power. The plot is the same as displayed in Protein Prospector, no matter base 10 or 2 is used.A median value is preferentially reported instead of a mean value, as outliers, which are not unusual, can significantly skew the mean ratio, whereas median values are more tolerant. In general, the more peptides quantified from a single protein, the more accurate the median number is to the actual ratio.

For the coarse-textured soil, arsenic concentrations in sampled pinnae ranged up to 1890 mg/kg after 11 weeks and increased 2–3-fold after 21 weeks . Pinnae arsenic concentrations were con siderably lower for the medium-textured soil, never reaching the hyper accumulation threshold . The interaction of soil by time indicated that pinnae arsenic concentrations were lower at 21 weeks in ferns growing in the medium-textured soil com pared to coarse-textured soil. The mass of arsenic accumulated in sampled pinnae increased over time, increasing 4–5 times up to 1.1 mg at 21 weeks in the coarse-textured soil and 2–3 times up to 0.67 mg at 21 weeks in the medium-textured soil . At final harvest, soil type similarly affected fern frond arsenic concentrations and mass of accumulated arsenic per fern yet had the reverse effect on whole plant biomass . Fern arsenic concentrations in coarse-textured soil ferns ranged between 2666 and 3570 mg/kg for the whole plant, up to 10 times higher than the values in medium-textured soil ferns. The total mass of accumulated arsenic in coarse-textured soil ferns ranged from 15.2 to 20.2 mg/fern, about two times higher than in the medium textured soil . However, the fern dry biomass was 3–4 times higher in the medium-textured soil than in the coarse-textured soil, with values between 20.1 and 23.5 g for the whole plant . Soil treatment did not affect whole plant arsenic concentrations , mass of accumulated arsenic , or biomass . Arsenic concentrations were greater by up to 2 orders of magnitude in fern above ground biomass compared to the rhizome and roots . Mass of accumulated arsenic was greater by an order of magnitude in above ground biomass compared to rhizomes . Interactions of plant part biomass by soil and by treatment showed that senescent fronds were larger in the medium textured soil ferns compared to coarse-textured soil ferns, yet were smaller in phosphorus-treated ferns across both soils compared to in other treatments.

Within above ground biomass alone, arsenic concentrations were up to 8 times lower in mature and senescent fronds,hydroponic gutter compared to young fronds. However, total mass of accumulated arsenic was greater in senesced fronds , compared to young fronds across both soils. Here, the interaction of frond age and treatment indicated phosphorus-treated senesced fronds accumulated less total arsenic than young fronds. Whole plant phosphorus concentrations were 20% lower in ferns grown in the medium-textured soil , compared to in the coarse-textured soil . In contrast, mean iron concentrations were two to six times higher in ferns grown in the medium textured soil , compared to in the coarse textured soil . Phosphorus concentrations were higher in phosphorus-treated ferns .Across soils, soil , time , and depth affected pore water total arsenic concentrations . In the coarse textured soil, arsenic concentrations decreased after 3 to 7 weeks, with con centrations higher in the 27 cm depth than surface depths for the remainder of the experiments. In the medium-textured soil, pore water could be extracted from the phosphorus-treated columns till 17 weeks, longer than the control and F. mosseae-inoculated columns, where pore water extraction was not possible as early as 5 weeks into the study. Interactions of soil and time , soil and depth , and soil and treatment showed that in the medium-textured soil, pore water arsenic concentrations slightly increased with time, decreased with depth, and in creased with phosphorus treatment. Arsenic concentrations were very low in the coarse-textured soil pore water, less than 4.4 μg/L, but were higher in the medium-textured soil, with a mean of 13.6 μg/L increasing up to a peak of 109 μg/L in weeks 5 to 9 in the phosphorus treated columns . Concentrations of DOC were less than 32 mg/L in the coarse-textured soil pore water but higher in the medium-textured soil pore water, where they reached 165 mg/L . Depth affected pore water DOC concentrations. Interactions of soil by week and soil by depth showed that DOC concentrations increased with time and depth in the medium-textured soil but not coarse textured soil. Pore water iron concentrations were less than 66 μg/L in the coarse textured soil but higher in the medium-textured soil, up to 164 μg/L, and decreased with time .

A significant soil by depth interaction showed that iron concentrations decreased with depth in the medium-textured soil pore water. Phosphorus concentrations were less than 0.60 mg/L in the coarse textured soil pore water but were higher , up to 3 times those values, in the medium-textured soil pore water, and decreased with time in both soils . Phosphorus and total arsenic concentrations were moderately correlated . Soil , time , depth , and treatment affected pore water pH, which ranged from 6.0 to 8.9 in both soils and increased significantly with depth across both soils . Interactions of soil by time , by depth , and by treatment showed in the medium-textured soil that pH increased over time, decreased with depth, and increased with phosphorus treatment.Effluent elemental concentrations, volume, and cumulative leaching A soil by presence/absence of ferns interaction indicated that effluent arsenic concentrations were higher in the presence of ferns in the medium-textured soil , but that presence of ferns did not affect effluent arsenic concentrations in the coarse-textured soil . Across both soils, effluent arsenic concentrations increased with time . Effluent arsenic concentrations were lower in the medium-textured soil than in the coarse-textured soil . Phosphorus treatment lowered arsenic concentrations in effluent of both soils , regard less of whether ferns were present. Effluent volume and cumulative arsenic loss were greater in the absence of ferns, with up to 67 mL/day effluent leading to cumulative arsenic loss of up to 12.6 μg/day by leaching . Regardless of whether ferns were present, effluent volumes were greater in the coarse-textured soil where effluent flow lasted 22 weeks in the presence of ferns , leading to cumulative arsenic loss of up to 5.9 μg/day by leaching. This cumulative arsenic loss was more than from the medium-textured soil in the presence of ferns, where effluent flow ceased at 7 weeks with 50% less loss by leaching. Although in the presence of ferns effluent volumes were greater in phosphorus-treated columns , this did not lead to greater cumulative loss of arsenic from phosphorus-treated soil .

In the absence of ferns, cumulative arsenic lost from soil was lower in the phosphorus-treated soil .Bulk arsenic K-edge XANES spectra of the coarse-textured soil samples indicated greater abundance of arsenic in rhizosphere soil, compared to whole roots or bulk soil . In contrast,raspberry plant container in medium-textured soils, bulk spectroscopy showed rhizosphere soil arsenic fractions lesser than or equal to those in roots, with even lower abundance in bulk soil . In both soils, a similar or higher fraction of arsenic was found in phosphorus-treated soils compared to control samples . Micro-focused arsenic K-edge XANES spectra from coarse- and medium-textured soil showed that across all sample types, a higher fraction of arsenic was present in medium- than coarse-textured soil . In both textures we found very little evidence of arsenic reduction in aggregates from control soil . Compared to control aggregates, a higher fraction of arsenic was found in aggregates of phosphorus-treated soil in coarse- , Fig. 6C, Table SI-4 and medium- , Fig. SI-6C, Table SI-5 textured soil. Similarly, in medium-textured rhizo sphere soil, μXANES spectra showed a lower fraction of arsenic in control soils, but a higher fraction in phosphorus-treated soil . In coarse-textured soil, the fraction of arsenic on/within whole roots ranged up to 28.5% on control roots, 80.9% on F. mosseae-inoculated roots, and 20.0% on phosphorus-treated roots . In the medium-textured soil, the fraction of arsenic on/within whole roots ranged up to 99.8% on control roots, 106.0% on F. mosseae-inoculated roots, and at least 13.8% on phosphorus-treated roots , but good fits could not be obtained. In contrast, on particles adjacent to roots in medium textured soil the fraction of arsenic was only 18.2% in control soil and 3.5% in F. mosseae-inoculated soil . Bulk iron K-edge XANES spectra indicated iron oxyhydroxides were the most abundant species in coarse textured bulk soil, rhizo sphere soil, and roots . In medium-textured bulk soil, rhizosphere soil, and roots, iron oxyhydroxides were less abundant and were not present at all in phosphorus-fertilized roots . Other mineral groups identified through bulk and iron K-edge μXANES spectra in both coarse- and medium-textured soil included iron silicates, iron silicates, and iron silicates .The large increase in frond arsenic concentrations we observed with increasing soil particle size suggests changes in soil texture have a strong effect on arsenic phytoextraction rates, directly through arsenic phyto availability and/or indirectly through nutrient content and availability. Because arsenic strongly associates with the clay particle size fraction including iron oxides , arsenic phyto availability is lower in soils with higher clay contents . Our findings build on previous work showing P. vittata frond arsenic concentrations decrease as clay content in creases in medium to fine-textured soils and across wider clay content intervals . Even in the presence of apparently highly plant-available arsenic, P. vittata did not use the rhizome as a secondary storage organ, in contrast to previous observations . We showed that under high phytoavailability conditions, arsenic tolerance and hyper accumulation are simultaneous functional traits in P. vittata, if genetically independent . However, effective hyper accumulation—and/or tolerance— appears to exact a metabolic cost. We found lower biomass coupled with higher arsenic concentrations in ferns growing in the coarse textured soil, which suggests that at higher levels of phytoavailable arsenic , biomass decreases as resources are allocated to tolerance and hyper accumulation mechanisms.

In arsenic hyper accumulation, en ergy is used for active transport of arsenic via phosphate transporters, glutathione production, arsenic reduction, transport within xylem, and sequestration . The lower fern biomass could also be a response to the lower nutrient content in the coarse textured soil. Like arsenic, nutrient retention can be greater in soils with higher clay and organic matter contents . Extensive nutrient scavenging in the lower-nutrient coarse textured soil could expend metabolic energy, release arsenic from soil, and increase plant uptake of arsenic, requiring more resource allocation away from bio mass production toward sequestration.The greater fern arsenic accumulation coupled to greater loss of arsenic by leaching observed in the coarse- compared to the medium-textured soil suggests that plant-available arsenic is also available to leach. In the coarse textured soil, characterized by a lower iron content and adsorption capacity, arsenic appeared to leach from soil at all depths, not resorb to soil, and accumulate in pore water, leading to higher effluent arsenic concentrations. The peak in effluent arsenic concentrations in the coarse-textured soil suggests rapid, linear leaching of the most available arsenic fraction, similarly to what was observed in soils with 8% clay , followed by a decrease in concentrations as the most available arsenic fractions are depleted. Moreover, we attribute the greater effluent flow rates and duration in the coarse-textured soil to lower transpiration from the smaller above ground fern biomass. Lower biomass leads to lower transpiration, greater infiltration, and greater leaching of available arsenic, compared to the medium-textured soil which better supported plant growth. Yet even though the most leachable fraction was depleted early on from the coarse-textured soil, the highest fern arsenic concentrations were found in young fronds produced later in the study. This could suggest that arsenic continued to be plant available even after leaching decreased. Even in a soil with low adsorption capacity, the pools of arsenic available for leaching and plant uptake are overlapping but not identical.The lower arsenic leaching observed in the medium-textured soil is consistent with the greater clay and iron content and adsorption capacity, lower leachate volume, and more diffusive transport in this soil. Arsenic sorption and desorption processes appeared to occur at all depths, leading to constant pore water arsenic concentrations with depth, stable effluent arsenic concentrations lower than in the coarse-textured soil, and soil arsenic concentrations that increased with depth, indicating retention of arsenic re leased from the surface depth . Nonetheless, pore water and effluent concentrations in our study were still 4 to 40 times higher than in soils with similar or higher clay content , likely due to influent water pH and application rate simulating maximum daily rainfall conditions for an extended period.

The letter alleges that the new variety contains a piece of technology that in fringes upon a client’s IP claims. Furthermore, the patent owner appears not even to be interested in negotiating a license. And to this day, the legendary variety sits in storage somewhere in a greenhouse or a freezer, unused and sadly neglected. Of course, it is difficult to establish the definitive reasons why a project does not come to fruition, especially when there are numerous factors simultaneously affecting the outcome. Prior patents may be just a convenient excuse — and the patent owners a scapegoat — for tough decisions made to terminate unpromising or economically unattractive projects. Still, while patents do provide convincing incentives for private firms to invest in agricultural research and development , taking the necessary steps to respect the rights of patent ownership does add an additional layer of costs for developing new crop varieties. Economists call these additional costs “transaction costs”; they include legal fees for searching and filing patents and expenses for negotiating and drafting licenses. Royalties paid for using another’s technology are not IP transac tion costs. Rather, they are “rent” paid to use the technology and to compen sate for the R&D expenditures spent to create it. Commercial developers of agricul tural biotechnologies often take mea sures to avoid incurring these IP transaction costs. They may shift their R&D strategies or even acquire other companies to avoid dependence on outside technologies, thereby limiting expenses and preventing the complications and uncertainties inherent in “renting” them . These measures, however, can be costly too. Either way,square nursery pots costs faced under an IP system can, in theory, cancel out the private incentives created by IP to pursue innovation. More troubling, IP can even prevent publicly funded innovation from having its in tended social impact.

Yet are there any good indicators of this stalling beyond just stories and rumors? And if so, can we establish links with IP?Recent U.S. Department of Agriculture registrations for field trials of transgenic crops show that R&D in horticultural crops is lagging when compared with the major row crops. Even leading transgenic horticultural crops such as melon, lettuce, straw berry, grape, apple and sunflower are hardly represented in field trials . Horticultural crops are completely dwarfed by corn, the single most commonly tested transgenic crop, which by itself is the subject of almost half of all transgenic field trials. Of course, U.S. production of any single horticultural crop is far less valuable than U.S. production of corn. Less field-testing is to be expected for less valuable crops. But, even when applying a rough calculation to account for the differences in size and value of individual crops — dividing by the annual value of each crop’s U.S. production — horticultural crops tend to show a greater farm-gate value per field trial. In other words, horticultural crops are subject to fewer genetic field trials, and presumably receive less biotech R&D, for every dollar of crop production. Furthermore, the proportion of transgenic field trials conducted by public-sector research organizations, such as state universities or the USDA, versus the proportion conducted by commercial firms, varies widely by crop type . Public-sector involvement in the field-testing of the 10 leading transgenic crops — mostly major row crops — averages just 15%. Yet, in the next 20 mostly horticultural transgenic crops, public-sector involvement averages much higher, around 40%. These numbers should be interpreted cautiously, as the samples representing many of the horticultural crops are small and the ratios are taken over just a few field trials. For example, 16 field trials have been done on transgenic papaya and only 11 on transgenic walnut . Despite this variability, there appears to be less investment in biotech for horticultural crops than for major row crops, both in absolute terms and relative to overall crop values, while a greater proportion of that smaller R&D investment in horticultural crops comes from the public sector. Involvement by commercial firms in horticultural crops seems to be missing. While this data is too sketchy to conclude outright that commercial firms are under investing in horticultural biotechnology, it al lows us to ask whether they might be, and if so, why.

After a few early excursions into horticultural crops — most notably by Monsanto, As grow and Calgene as well as by Syngenta’s predecessors at Zeneca — major agricultural biotechnology firms have virtually shut down their product development in horticultural crops. Long-shelf-life tomatoes, virus-resistant squash and insect-resistant potatoes have not taken off as did Bt corn and herbicide-tolerant soybeans. Some of the specialized vegetable seed firms, such as PetoSeed , and some of the smaller agricultural biotechnology firms that specialized in vegetable crops, such as DNA Plant Technologies , continued their bio technology efforts a bit longer. Yet those efforts appear to have all but dried up in recent years. Instead, fruit and vegetable seed companies with active research and production activities, such as Seminis, Danson, Golden Valley, Harris Moran and others, continue to pursue their product development goals through conventional breeding techniques. One exception is the Scotts Company, which is currently seeking regulatory approval for a biotech product for golf courses, a glyphosate-resistant bentgrass. Indeed, most of the biotech work in horticultural varieties is conducted in university laboratories doing basic plant science. Occasionally, those projects spin out a commercially interesting trait or technology, but university technology-transfer offices have a hard time finding commercial partners among the seed firms, nurseries or growers’ associations.As with any investment, there is a degree of risk involved in putting re sources into the development of a new transgenic horticultural variety. Future returns are uncertain, and expected re turns are weighed against costs incurred to enter the marketplace. Such considerations also apply, more generally, to public-sector investments in re search. Although the measures of success may be more in terms of scientific advancements than earned profits, the practical importance of a new discovery is still important. .The size and strength of demand for a new transgenic variety will determine the size of returns on the investment. Market uncertainties for agricultural products are nothing new, due to such factors as disruptive competition in supply, cyclical price fluctuations and changes in consumer demand. How ever,ebb and flow tray some food consumers, such as in Europe, are skeptical of foods produced using biotechnology.

While a majority of U.S. consumers seem relatively unfazed by the genetic contents of processed bulk commodities such as soybeans and feed corn, consumers could react more strongly to obvious modifications of products in the produce aisle. Yet specific market uncertainties surrounding the use of transgenics could be addressed by the selection of technologies and traits that deliver real tangible benefits to consumers in ways that are perceived as unambiguously safe.The process of regulatory approvals for GM crops is essential to assure the safety of the technology. The R&D costs associated with gaining approval are considered up-front or “sunk” investments, and they must be spent to gain access to the market. These costs can be greater if the transgenic crop contains novel proteins or pest-control components, as additional assessments are required. In major row crops, investments to obtain regulatory approval can be recouped from the small technology fees charged on each bag of transgenic seed, which are multiplied out over millions of acres planted; however, with horticultural crops the distribution of regulatory costs is often concentrated onto much smaller markets. In many horticultural crops, several different varieties are commercially important. If introgression of the new trait via back crossing is not an option, such as may be the case for clonally propagated varieties that do not breed true, each variety must be separately transformed in the lab, and each must be separately tested and approved. Regulatory costs would add up, but they could not be spread out over nearly as large a market as they could for row crops. Still, returns per acre from horticultural varieties tend to be much higher, and the costs of specialized pesticides replaced by transgenic traits may also be higher. In addition, regulatory costs can be expected to decline as more risk assessments are completed, government agencies become more adept at judging the merits of different biotechnologies, and the policies and procedures become streamlined and finely tuned. In addition, the extension of an approach similar to the IR-4 program, which provides regulatory assistance for pesticides targeted to the needs of specialty crops , could reduce the regulatory burden on transgenic specialty crops.Transaction costs for gaining freedom to operate in the relevant IP protected technologies can be consider able. As with regulatory costs, the total IP transaction costs are independent of market size, and a larger number of transgenic varieties means more costly negotiations and more deals to cut. One industry estimate put the costs of negotiating a single crop genetics deal as high as $100,000.

When multiple patented genetic technologies are stacked in a cultivar, as is increasingly the case, the problem is compounded. Uncertainty over the total amount of IP transaction costs scares off investment in R&D projects, unless the expected returns are particularly attractive. This will continue as long as there is uncertainty in the IP landscape for plant bio-technologies and genetic materials. With the number of patents in this area growing at an exponential rate, IP access could be a deterrent to biotech R&D in horticultural varieties for years to come.IP access is a general problem for all of crop biotechnology. The reasons lie in the cumulative nature of the genetics and bio-technologies embodied in transgenic varieties. Plants are complex systems, and a healthy, productive crop plant has numerous genetic and metabolic pathways functioning together. Those genetics are inherited from breeding stock or can be added using biotechnology. A genetically engineered seed or plant cultivar may contain three different kinds of technological components that can be protected as IP, including the germplasm of the plant variety, the specific genes that confer a new trait and the fundamental tools of biotechnology such as genetic markers, promoters and transformation methods. The IP situation is complicated by a number of additional factors that add to the transaction costs.Different technological components of a transgenic crop variety are covered in the United States under different forms of IP law. If a variety is clonally propagated, the germplasm — the plant variety itself — can be claimed as IP at the U.S. Patent and Trademark Office under a Plant Patent, established in 1930 by the Plant Patent Act to protect against cuttings being taken, repropagated and directly resold under another name. Seed-propagated varieties can be claimed as a form of IP under the USDA system of Plant Variety Protection certification, established by the Plant Variety Protection Act in 1970. And, since 1980 — following a landmark decision by the Supreme Court in Diamond v. Chakrabarty over the patenting of a genetically engineered microorganism — all kinds of “invented organisms,” including novel plant germplasm, have come to be claimed as IP under standard U.S. utility patents. Subsequent technological and legal developments following Diamond v. Chakrabarty now allow utility patents to protect invented genes, proteins and other gene products, as well as biotechnology tools such as transformation of genetic contents, selection using genetic markers, and regulation of expression using genetic promoters. Finally, a significant part of the value of an agricultural variety often lies not in its technological or biological characteris tics perse but rather in its recognition and reputation among consumers in the marketplace. That “brand” name can be protected as IP by registering it as a trademark with the USPTO. The challenges posed by multiple layers of IP law are, if anything, greater for horticultural varieties than for row crops: plant patents, PVPs or utility patents may cover the germplasm; util ity patents typically cover the gene and biotechnology tools used; and trade marks are more often used to protect variety names. In leading row crops such as corn and soybeans, germplasm as well as the genes and bio-technologies are protected more consistently under only utility patents. While trade marks like Roundup Ready or Liberty Link refer to input traits and may be of some value in marketing to farmers, the identities of such agronomic traits command little notice or value from final food consumers.

Once the stress condition was removed, a fraction of the cells recovered the capacity to grow in laboratory media, thereby indicating a potential of the E. coli O1O4:H4 cells to cause human disease. The formation of VBNC bacterial cells on plants also was previously described for Listeria monocytogenes on parsley. The number of viable L. monocytogenes cells was 1 to 2 log higher than the culturable cells recovered from parsley grown in greenhouses at 20uC under low relative humidity ; growth of the VBNC cells was not restored on the plants when the RH was increased to 100%. Although we did not examine for E. coli O157:H7 recovery from the VBNC-like state, future efforts might investigate whether those cells can recover and resume growth either under growth-conducive conditions on lettuce or after removal of the cells from the plants and prior to or after plating for viable cell enumeration. Overall, the toxigenic strain EC4045 survived in similar quantities as ATCC 700728 on lettuce. A recent study showed that certain lineages of E. coli are more commonly associated with plants and presumably have evolved the capacity to tolerate plant associated environments better than E. coli isolated from other sites. Because we only compared two strains, subsequent investigations should examine multiple attenuated and virulent O157:H7 strains isolated from different sites for their capacity to colonize and persist on lettuce under field relevant conditions. Survival of E. coli O157:H7 on lettuce also was measured in two field studies. Culturable amounts of strain ATCC 700728 declined shortly after inoculation onto plants in the field, as we reported previously. Rates of cell decline were similar to E coli O157:H7 on lettuce in the growth chamber directly inoculated in drops with a pipette. Real-time PCR estimates of E. coli O157:H7 ATCC 700728 in lettuce leaf washes showed that this strain was present on the plants immediately after inoculation and 2 h later in quantities equivalent to the inoculum levels. Importantly,flower plastic pots the rapid decline in culturable E. coli during the first hours after application onto plants in the field was not due to an inability to remove the organism from the lettuce or from dispersal and lack of strain attachment.

Rather, it appears that the majority of the E. coli cells in the inoculum either died shortly after application or entered a VBNC state. In contrast to the growth chamber experiment results, the numbers of E. coli O157:H7 cells were below detection by real time PCR within 2 days after inoculation onto field lettuce. These findings suggest that the E. coli O157:H7 cells and genomic DNA were degraded rapidly. Environmental parameters such as solar radiation, heat, and water stress could be responsible for the differences in the stability of E. coli O157:H7 DNA in the field compared with the laboratory. Alternatively, cell maintenance might depend on other microorganisms on the leaves. Also, it is notable that different lettuce cultivars were used in the field and growth chamber studies, which may have impacted survival. The potential for cultivar-dependent effects was shown for E. coli O157:H7 on lettuce cultivars grown under axenic conditions in the laboratory. Because the E. coli O157:H7 ATCC 700728 DNA was degraded on field-grown plants within 2 days after inoculation, it is unlikely that the organism developed a VBNC state, particularly over longer time scales . However, this possibility could not be directly addressed using the PMA real time PCR assay in the field trials. Viable cell amounts measured by culturing and PMA real-time PCR were in agreement immediately after application of ATCC 700728 onto laboratory grown lettuce, but PMA-mediated detection was impaired on plants from the field. For those plants, the viable cell number estimates for strain ATCC 700278 were 10-fold lower as measured by PMA real-time PCR than by colony enumeration and total cell numbers estimated by real-time PCR. These differences might have been due to the higher turbidity or opacity of the lettuce plant washes from the field samples, thereby preventing light from penetrating the suspension during the PMA photo inactivation step. This interference would prevent the inactivation of free PMA, resulting in sufficient quantities of the compound to bind genomic DNA released from viable cells during the subsequent DNA extraction and amplification steps. In addition, the PMA real-time PCR assay was unable to detect low numbers of cells. Attempts to detect the ATCC 700278 strain after concentrating the leaf washes were unsuccessful . Similarly, PMA real-time PCR was found to be more reliable for viable cell detection in diluted wastewater than in pooled and concentrated wastewater samples. Such factors strongly limit the overall usefulness of this approach for field-grown plants. However, this method is informative for examination of E. coli O157:H7 on ‘‘cleaner’’ plants grown in the growth chamber and not exposed to the biotic conditions that are common outdoors.

In conclusion, this study illustrates the similarities and differences between controlled studies of human pathogens on plants in a growth chamber and experiments examining the population dynamics of pathogens on plants under production-like conditions in the field. By applying relevant environmental conditions and droplet inoculation in the growth chamber, we were able to more closely mimic the rapid decline in E. coli O157:H7 culturability that was observed after inoculation of this organism onto lettuce plants in the Salinas Valley. Culture-independent assessments con firmed that the pathogen remains on the plant long after application. However, field studies showed that at least for the majority of E. coli O157:H7 cell inoculants, the loss in culturability was most likely due to cell death rather than an inability to form colonies on standard laboratory media. Hence, this work confirmed our observations that low numbers of E. coli O157:H7 persist on lettuce grown in the Salinas Valley and variations in pathogen survival among individual plants are dependent on other unknown factors .Ethnically diverse populations are disproportionately exposed to hazardous environmental materials by virtue of living in close proximity to toxic waste materials. One-half of the uranium in the US is found on American Indian lands, where mining, milling, processing,grow table and waste storage has commonly occurred. From the 1940s to the 1980s, northwestern New Mexico alone contributed 40% of the U.S. U production. The study setting was a prime target of U mining for military purposes from the 1940s to the 1980s. Diné lands were one of the prime targets for mining, contributing thirteen million tons of U ore for military use from 1945 to 1988 and leaving more than 1100 abandoned and partially unreclaimed U mines, mills, and waste piles. The extent of the health threats to the Diné community exposed to these sites is anticipated to be high. Uranium enters the body primarily by inhalation or ingestion , and then it enters the bloodstream and is deposited in tissues, primarily the kidneys and bones. Human and animal studies of those exposed to U have shown kidney toxicity, as well as damage to the liver, muscles, cardiovascular, and nervous systems. Arsenic is a teratogen. Cadmium can accumulate in organs and impair renal function; Lead is associated with adverse effects on the nervous, developmental, renal and reproductive systems. Selenium toxicosis can cause neurological and gastrointestinal problems and endocrine function disruption and is a teratogen in several species of animals; Molybdenum has been shown to be a male reproductive toxicant in animals and humans.

The Dine Network for Environmental Health study worked closely with 20 Navajo chapters or communities to address the concerns of the community and leaders regarding the health effects of environmental exposures to unreclaimed U mines and mill sites. The DiNEH cohort found that 40% of participants lived within 3.2 km of an abandoned U mine, 16% lived near a U mill, and 12.6% of children played on tailing piles or waste dumps. In self-reported data of past exposure, 15.4% utilized materials from abandoned U mine sites to build homes or other structures, 1.8% sheltered livestock in abandoned mines, 12.7% herded their livestock near contaminated sites, and 12.8% said their livestock came into contact with contaminated water. In this population, surface and groundwater utilization is important for human and livestock consumption as well as agriculture. In affected areas, greater than half of the Diné people continue to drink from unregulated water sources. DiNEH data indicate that more than 80% of Diné people haul water for all uses, including irrigation and livestock watering, despite having regulated water in the home. Mutton or lamb meat and organs are primary food staples in this community, and all aspects of the animal are used; there are important cultural uses for the animal. The purpose of this study was to determine if sheep, a harvested primary dietary food staple on Diné lands in northwestern NM, were contaminated with U and other associated heavy metals. Past studies of these areas in NM demonstrated that the consumption of U contaminated food may occur through the ingestion of locally raised livestock and by way of their forage. Food chain contamination in locally harvested food in the Diné community in NM was reported as a plausible exposure pathway . The current study was undertaken to reexamine and contribute more recent data and introduce data not previously reported . This was a descriptive, comparative study examining contamination levels in locally harvested O. aries, their forage, and associated soil and water from reservation areas within a 3.2 km radius of previously mined areas. Data obtained from the DiNEH study cohort served as one of the sources for identifying subjects and samples of food, herbs, water, forage, and soil. Additional participants were recruited by word-of-mouth, home visits, and advertising at public tribal community events. Of the DiNEH cohort respondents, those individuals who reported harvesting sheep were recruited for potential participation in the present study. Sheep chosen represented a range of ages , their proximity to mining structures, and a variety of water sources. Three ewes were included in this study. The individual sheep data are compared and reported to reflect an accurate measure of heavy metal uptake in O. aries tissue with respect to the associated forage, water consumption, and their environment.The study area is a semi-arid to arid region of the American Southwest in northwestern NM on Diné reservation lands . The average precipitation was less than 25 cm per year according to meteorological data for NM for the study period. Despite several decades of longstanding drought in the area, community members still participated in subsistence activities. Two “Chapters” provided sheep and associated samples. The Mariano Lake Chapter is 272 km2 of land mass and the Churchrock Chapter is 233 km2 . Recruitment was initiated on May 2012 and enrollment began in July 2012. All samples were collected from 10 November to 13 December 2012. This study focused on sheep as a food staple and was part of a larger “parent” research project that examined subsistence farming on the reservation, including the metal contamination of herbs.Three sheep came from two different chapters. From 10 November to 13 December 2012, three ewes were contributed to the study. The O. aries tissue samples were collected in the field immediately after slaughter and included muscle, bone, intestine, lung, liver, kidney, and wool. Upon collection, all samples were placed on dry ice and shipped to the University of New Mexico Analytical Chemistry Laboratory Earth and Planetary Sciences Department for storage and analysis. The 13th cortical rib bone samples were sheared from the proximal, middle, and distal portions and composited together after the removal of excess tissue. The proximal, medial, and distal portions of the small intestine were collected and composited. For lung tissue, the samples were derived from each anatomical lobe and composited. Both kidneys were sampled, and the cortex and medulla were composited separately. Composited muscle samples were from the proximal, medial, and distal portions of the gastrocnemius. Of the wool fiber samples, the area over the neck, middle section, and posterior portions of the animal were sampled and composited. All tissues were representative of 1 g of dried tissue. For coupled organs, the tissues collected from the right side of the sheep were labeled as the sample, and one duplicate was obtained for each tissue type from the left side of the animal. A composited duplicate or replicate was obtained for non-dual type organs .

The government was therefore able to go ahead with its eradication policy with few internal restrictions. Even so, the result was not very positive; the rise of the poppy emporium in Colombia amply demonstrated the limits of the government’s public anti-narcotics policy and the dramatic consequences of unremitting prohibition on the part of the United States. The Colombian government did not attack drug trafficking or narco-terrorism on the financial front. In accordance with the logic of the so-called economic liberalization fomented by the government in the early nineties, it made no sense to place restrictions and greater controls on the free movement of capital. In 1993, a report by the Vienna-based United Nations International Board on Fiscal Control of Narcotics recommended that “Colombian legislation consider the laundering of capital resources to be a crime and that banking laws should become stricter in order to allow for multilateral cooperation….”The alleged financing of the presidential election campaign with drug money formed the backdrop to the anti-narcotics policies of President Ernesto Samper’s administration . As months went by, the coercive diplomacy which the United States had hitherto been exerting on Colombia became transformed into “blackmail diplomacy.”The president’s capacity for political survival led him to “North Americanize” the fight against drug trafficking in Colombia; that is to say, the president accepted and implemented a strategy virtually imposed by the United States. The Samper government undertook an all-out chemical eradication campaign far beyond anything seen in the two preceding decades,macetas de plastico por mayor with massive use of glyphosate. Fumigators also employed imazapyr, a more powerful granulated herbicide, and were planning to use tebuthiouron, an even more devastating killer than the others.

Ernesto Samper also became the president who most helped criminalize the drug trade, while in Colombia it became almost impossible to discuss the subject of legalizing drugs, something Samper himself had suggested in the late seventies, given the failure of repressive measures taken at that time by the Turbay administration and fomented by the United States.In 1995, months before the infamous “Frechette Memorandum” began to circulate — a document which suggested that Colombia should adopt legislation and take drastic measures in the anti-drug war — President Samper had launched his “integral plan” announcing, amongst other things, the creation of Operación Resplandor designed to put a definite end to all illegal crops which existed in Colombia in the space of two years.”An all-out eradication policy had been set in place. In 1994 , 4.094 hectares of coca were eradicated. In 1995, the Samper administration eradicated 25,402 hectares; and in 1996, 9,711 hectares. In 1994, the Gaviria and Samper administrations had eradicated 5,314 hectares of poppies. In 1995, the Samper government eradicated 5,074 hectares; and in 1996, 6,044 hectares.Between the years 1995 and 1996, glyphosate was used on a massive scale to destroy illegal crops.Even so, the idea of putting an end once and for all to illegal crops proved again to be illusory. In 1996, the US government estimated that the number of hectares dedicated to the planting of coca in Colombia had reached 53,800 hectares, while independent estimates placed the figure at around 80,000 hectares.This meant that Colombia had surpassed Bolivia, a country which traditionally was second only to Peru as a coca producer in South America. The same official US source estimated that Colombia had 4.133 hectares of marijuana and that the country had produced 63 tons of heroin in 1996. However, Colombians had their greatest surprise of all in 1996 when small farmers from the south, especially from the Caquetá region, suddenly made their presence felt in mass demonstrations and protest marches. Nobody had expected this. It was as if the whole population had discovered overnight, and a little belatedly, that the country had ceased to be the processor of these stimulants and had transformed itself now into something else: a huge grower of illegal crops.

People also came to realize that the state simply did not operate at all in a large and strategic portion of the country’s territory, and that power, at the local level, was in the hands of insurgent groups, especially in those of the FARC . Colombians came to realize as well that violent measures alone were not going to solve the profound and intricate social, political and economic problems which had been incubating for decades in the nation’s geographic wilderness.In sum, fumigating with herbicides in southern Colombia in 1996 turned out to be as useless for dismantling the illegal business of drug dealing as had similar efforts in previous years. The difference was that, in 1996, paramilitary detachments were multiplying at a frightening rate in the south. The political blindness of people in government, police officers and the military, together with the administration’s obsequious submission to United States policies, led to a repeat, in 1997, of the indiscriminate fumigation with herbicides — on a huge scale with glyphosate, to a lesser extent with imazapyr. In 1997, Colombia sprayed 41,847 hectares of coca and 6,962 hectares of marijuana. Twenty-two hectares of coca were eradicated manually, as well as twenty-five hectares of poppies and 261 hectares of marijuana. In just over three years, the government had fumigated more than 100,000 hectares of illegal crops. But paradoxically that only went to prove, as never before, just how mistaken, harmful and counter-productive the chemical destruction of such crops could be; in 1998, almost 110,000 hectares of the national territory were dedicated to plantations of coca, marijuana and poppies. In that year, the Samper administration , and that of Andrés Pastrana , fumigated 66,083 hectares of coca and 2,931 of poppies, and manually destroyed 3,126 hectares of coca, 181 of poppies and 18 of marijuana.Nonetheless, according to the Central Intelligence Agency , the total area planted in coca in 1999 amounted to 120,000 hectares,and the US State Department declared that this had increased to 136,200 hectares in the year 2000. This means that in just four years, from 1996 to 2000, the surface planted in coca in Colombia has doubled; the total number of hectares went from 8,280 to 13,200. An increase in the fumigation of illegal crops has not resulted in a decrease in the area planted with illegal crops,rolling benches nor to a decrease in the production of illegal drugs.

To this evident failure one must add the fact that, on the US market, cocaine and heroin have become both cheaper and purer. It is worth noting, also, that something similar has occurred in Western Europe where, in 1999, a gram of cocaine was worth US$90, and a gram of heroin was fetching US$98. So, the rationale which attempts to justify a strong eradication policy in the centers of supply has proved to be way off the mark. It had been presumed that the massive destruction of illegal drugs where production and processing were taking place was going to lead to less availability of narcotics in the centers of demand, an increase in price for the ultimate consumer and a lowering of standards of purity in the stimulants themselves. Quite the opposite has happened; in the year 2000 one could procure in the United States more drugs of better quality than ever before, and at lower prices. Besides, in terms of illegal drug consumption and of drug-related crime, the United States record has not shown substantial improvement. In 1988 the number of occasional consumers of heroin was reckoned at 167,000; in 1995 it had reached 322,000; while the total number of heroin consumers worldwide went from 692,000 in 1992 to 810,000 in 1995. The overall demand for heroin was 1,800,000 grams per year in 1988, but by 1996 it had soared to 2,400,000.Despite certain laudable achievements in reducing drug consumption in the United States, it is evident that a strong demand still exists. In this context it is worth quoting Bruce Bagley: “Some 13 million US drug users spent approximately US$67 billion on illicit drugs in 1999, making the US market the most lucrative one in the world for Colombian traffickers.”Concomitantly, in 1990 the total number of arrests in the area of drug-related law infringements was 1,089,500, whereas in 1996 the figure had risen to 1,128,647. In 1990, 53 percent of prisoners in federal jails were serving sentences for narcotic-related crimes; in 1995 the statistic had risen to 59.9 percent.Finally, the environmental cost to Colombia of chemical eradication has not been sufficiently studied and quantified. However, it is estimated that “for every hectare of poppies sown, an average of 2.5 hectares of woodlands are destroyed; in the case of coca plantations the ratio is 1 to 4, and for marijuana it is 1 to 1.5.”However the negative effects of herbicide fumigation have not been assessed in this process of forest destruction. What we do know is that the mere fact of fumigation forces the growers to move elsewhere in order to plant their illegal crops, and that entails necessarily a further environmental disaster.Despite the fact that organizations such as Greenpeace, the Worldwide Fund for Nature and Dow Agrosciences are opposed to the use of this herbicide, the United States authorities have insisted that it is quite harmless.

They have gone even further; during the Pastrana administration especially, they have been putting pressure on Bogotá to apply a dangerous fungus, fusarium oxysporum, in the process of obligatory eradication. Nonetheless, after almost four years in government, the Pastrana administration has not taken the risk of rethinking the procedure of chemical eradication. On the contrary, since coming to power in August 1998, Pastrana has persisted in an unquestioning policy of intensive fumigation. He has gone even further than his predecessors, in that he accepted the setting up of an Anti-narcotics Battalion within Colombia’s armed forces, in accordance with the wishes of the United States as expressed over the past few years. In 1999, this special unit of 1,200 men under the command of the Colombian army but monitored by “Washington’s magnifying glass,” replaced the anti-narcotics unit of the police force in the most critical of tasks, namely those to do with illegal crops.In 2001, as the so-called “Plan Colombia” went into operation — insofar as it touched on aspects of security and the anti narcotic policy of the United States — three battalions of the Colombian army were charged with combating illegal drugs. In short, there has been nothing new as far as eradication is concerned. Rather things have gone on as usual, in the hope that Colombia’s armed forces, by playing a definitive role in the fight against drugs, will somehow turn things around and produce a fundamental change in favor of the government and of the United States. The risk that Colombia is taking by continuing to obsessively and obsequiously spray crops is an enormous one. By insisting on this unfortunate and counter-productive measure, the government is committing a serious political error and is leading the country to the brink of a catastrophe which will affect both the population and the country’s ecology, but will not effectively help to overcome the drug problem. Chemical eradication has already produced multiple negative effects: for a start, it has contributed to greater devastation of the environment; it has led, also, to an even closer marriage between drug traffickers and paramilitaries and, at the same time, has encouraged guerrilla fronts to depend more than ever on income from the drug trade; it has served to increase corruption at different levels of society; without achieving any positive results, it has involved the government unnecessarily, in some of the most violent aspects of the drug war; it has exposed some of the weakest and most vulnerable members of Colombia’s society — peasants, Indians, poor farmers, and others — to greater threats, often forcing them to migrate and leaving them totally unprotected; and finally it has helped to further stigmatize Colombia in the eyes of the world, despite the fact that no other country has sprayed crops with herbicides to nearly the same extent. Nonetheless, it would seem that nothing is going to change in this regard; the year 2002 will probably see more futile fumigations. To sum up: notwithstanding the intense war being waged to combat it, the drug trade will continue to prosper.

Many insects rely on microbial communities and endosymbionts to grow and develop; however, it has been shown that Lepidoptera species do not have a vertically transmitted microbial community . In addition, because the effects of microbial communities on T. ni survival and development have not been documented, we present these data only to show that microbial communities change when exposed to CECs, and not as a proven factor influencing survival. We found significant shifts in the microbial community in the various life stages examined within the control treatments notably from third instar to subsequent life stages.However, there is one family, Lactobacillaceae, which ap pears in all treatments and life stages in high proportions, except for adults. They are fairly common in insects and can be responsible for at least 70% of the bacterial community . Lactobacillaceae is responsible for ∼42% of the bacteria in all life stages, followed by Pseudomonadaceae, Alcaligenaceae, and Enterobacteriaceae. Lactobacillaceae have been shown to act as beneficial bacteria in Drosophila ; however, its function in T. ni is still unknown. Alcaligenaceae has been shown to be present in other moths , but Lepidopterans are not thought to have a functional microbiome . There are clear patterns regarding the changes in microbial community proportionality according to the heat map . In controls, third-instar microbial communities are relatively evenly spaced by family.Once the insects reach the adult stage, their most predominant family is Pseudomonadaceae. This pattern holds in the acetaminophenand caffeine treatment groups as well. Interestingly, the other treatment groups do not share this pattern. For antibiotic- and hormone-treated T. ni, Lactobacillaceae is the predominant microbial family in the immature stages, but at the adult stage microbial community reverts to predominantly Pseudomonada ceae. We suspect that this is because, once the larvae undergo metamorphosis and shed their gut contents in preparation forpupation,macetas de 9 litros they are no longer exposed to the pressures exerted by the CECs on the microbial community. Fig. 3 provides a visual indication of the changes in the bacterial communities over time.

The increase in β diversity after eclosion could be due to the larvae no longer being exposed to CECs or diet-borne bacteria after being moved to sterile containers. Also, when bacteria are lost as larvae digest their gut contents during pupation, the microbial β diversity could change. Interestingly, the hormone-treated T. ni follow a similar pattern to those exposed to antibiotics, but their ellipses are always much smaller, suggesting the entire insect population is showing a uniform response within their microbial communities. However, in the mixture-treated insects, larvae displayed a greater average diversity in their microbial community structure than either pupae or adults. This finding has not been shown in any single category of treatment, and we suspect the microbes exposed to mixtures could be experiencing potential interactive effects among chemicals . Such interactions should be the focus of future studies along with investigations of plant rhizosphere bacteria, particularly since we found a difference in the Bradyrhizobiaceae family for all treatments. These results show that a terrestrial insect pest of commercial crops can be affected by CECs found in reclaimed wastewater for agricultural use. Our results suggest that CECs found in waste water can impact T. ni growth and development, survivor ship, and alter their microbial communities. Because T. ni is a common agricultural pest found around the world, feeds on a wide variety of plants, and has a history of developing pesticide resistance, its ability to deal with toxins is likely higher than many other insects. In addition, the responses we observed to CECs could have interesting implications for IPM practices on plants such as lowering the amount of pesticides needed or increasing susceptibility to insect pathogens, as has been shown in mosquitoes . These potential effects may be understated because some insects cannot detect the presence of the pharmaceuticals . However, we do not recommend purposefully exposing crops to CECs specifically for the control of insects because our study documented that these pharmaceuticals are translocated into crops and we do not yet know their possible effects on humans if consumed . We specifically want to note that ingestion of these compounds through uptake and translocation by a plant is not the only way T. ni or any other insect would be exposed to these compounds.

Overhead sprinkler irrigation could cause contact absorption by the plants or insects, and simply drinking water on leaves at contaminated sites could ex pose insects to higher concentrations than were found in plant tissues. In fact, the ciprofloxacin concentration used was less than one-third of the highest rate . We urge caution in ex trapolating to plants growing in soil, because variation in soil type and potential soil bacterial degradation could affect persistence [although soil bacteria are often negatively impacted by CECs ]. However, CEC exposures are considered pseudo persistent because they are reapplied with each irrigation. Thus, the effects reported here are likely to be conservative. Additional studies with other insects, particularly those with other feeding strategies, will be necessary before any patterns can be discerned.The correction of metal micro-nutrient deficiencies is a problem still not fully solved in Agriculture. The low solubility of the iron, manganese and zinc oxides in the pH range of calcareous soils contributes, among other factors, to the low availability of these nutrients to plants. The Fe3+ chelate of o,oEDDHA acid, a polyphenolic polyaminocarboxylic acid, and its analogues are the most efficient solution to correct the Fe deficiency with good results in hydroponic and soil conditions. The Mn deficiency is often treated with salts of Mn as Mn sulfate or as the Mn chelate of EDTA or analogous. Zinc sulphate has traditionally been the ‘‘reliable’’ source of Zn fertilizer but other sources of Zn are also available . New chelating agents such as o,pEDDHA -N´acid, a polyphenolic chelating agent with only five available donor groups or EDDS ,mobile vertical farm a biodegradable ligand with similar structure than EDTA, are been considered but there are not studies about the efficiency of the use of metal fertilizer mixes containing these chelating agents. The aim of this work was study the efficacy of the combined application of Fe, Mn, Zn and Cu chelates to correct the deficiencies in soybean in hydroponic solution in the presence of CaCO3. Then the stems of two individual seedlings were wrapped together with polyurethane foam and placed in 500 mL vessels preserved from the light by means of a black cover and with continuous aeration. At this point, treatments were applied in the presence of the MS solution. The Fe was applied as o,oEDDHA/Fe3+ in all the cases since it is known to be one of the best sources for the Fe nutrition .

The Mn, Zn and Cu were applied as o,pEDDHA, EDDS, EDTA, HEDTA or DTPA chelates, with the same chelating agent for the three metals in the same treatment and two additional treatments with Mn and Cu chelated by EDTA and Zn chelated by o,pEDDHA or EDDS. Three controls with no Mn, no Zn and no Mn and Zn with the remaining metal micro-nutrients chelated by EDTA were tested too. Moreover, 0,1g/l of CaCO3 was added to the pots, in order to achieve calcareous soils conditions. The concentrations in the hydroponic solution were 1.00 Fe, 0.375 Mn, 0.250 Zn and 0.150 Cu. The chelate solutions were prepared as described by Alvarez-Fernandez et al. . Three replicate pots per treatment were considered. The plants stayed for 7 days in these conditions.Samples were taken after 7 days of the treatment application. Plants were washed following the procedure described by Álvarez Fernflndez et al. , and fresh and dry weight of leaves, stems and roots determined separately. Then, micro-nutrient concentrations were determined in the plant organs after dry mineralization by atomic absorption spectrophotometry. Plant dry weight at the end of the experiment showed that in the three treatments with Zn-o,pEDDHA, plants had higher values than with the other treatments. In addition, controls with no Mn were more affected than the control with no Zn. It seems that the Mn nutrition have a more relevant effect in the plant weight than the Zn nutrition. Plants treated with DTPA had the lowest values. It is important to indicate that in general the best treatments are those with the chelates of lower stability, while the high stable chelates gives the worst results. This is the consequence of the competition between the plant and the chelating agent for the Zn2+ and Mn2+ as already studied by Halvorson and Lindsay . Then, the results here presented are only valid for hydroponic like cultures. In conclusion, the best treatment for the whole application of Mn and Zn was for the o,pEDDHA ligand that presents the higher levels for Mn and Zn in leaf especially for Zn and in the plant dry weight. It seems that the less stable polyphenolic chelates, like o,pEDDHA, are adequate for the nutrition of Mn and Zn in hydroponics due the low competence of this chelating agent with the plant for the metals. Horticultural crops have high economic, and enrich our lives through their aesthetic and nutritional value. Many horticultural species originate from tropical regions and are sensitive to cold at every stage of their life cycle. Cold stress leads to lower productivity and post-harvest losses in these species, with poor economic and environmental outcomes. Better understanding of the protective mechanisms mediated by hormonal and other signaling pathways may offer solutions to reduce cold-stress induced losses. The papers included in this collection illustrate this concept, examining natural cold-tolerance mechanisms and practical ways for growers to alleviate chilling stress and to reduce crop losses. The studies were remarkably diverse in terms of the species studied , plant organs examined , and approaches used . The papers encompassed the use of basic science, aimed at identifying key genes and their roles in cold signal transduction and protective pathways in fruit and photosynthetic tissues; reverse genetics for proof-of-concept on the hypothesized role of a cold-tolerance transcription factor cloned from an understudied species; and emerging technologies, by using exogenous hormones and signaling compounds to mitigate the harmful effects of chilling. These studies are described below. C-repeat binding factor proteins constitute a transcription factor subfamily known to play a key role in plants against different types of abiotic stress including cold, heat, salinity or dehydration, and thus have been extensively studied. Over expression of CBFs has been used for the development of genetically modified plants with enhanced stress tolerance and for the investigation of the molecular mechanisms underlying plant stress responses. Using this approach, Yang et al. found that over expression of three newly identified longan CBF genes enhanced cold tolerance in Arabidopsis by increasing the content of the osmoprotectant proline, reducing the accumulation of reactive oxygen species , and stimulating the expression of cold-responsive genes. The fact that longan, a cold-sensitive species, showed low expression levels for these three genes, suggests a possible strategy for genetic improvement of cold tolerance in this crop. Cold storage of apples is often used to extend post-harvest storage; however, it leads to superficial scald development, which is a major physiological disorder characterized by necrosis of the hypodermal cortical tissue. Karagiannis et al. applied a multiomics systems approach and created regulatory module networks to compare scald-affected and healthy apple phenotypes. Individual and combinatorial treatments with ozone , which induced scald symptoms, and 1-methylcyclopropene , which reversed O3-stimulated scald effect, were used to identify pathways and gene-to-protein-to-metabolite networks involved in scald prevention and sensitivity. Importantly, 1-MCP-induced scald tolerance correlated with the expression of genes involved in photosynthesis, stress responses, flavonoid biosynthesis, and ethylene signaling in apple peel and key TFs that may control some of these processes. This study represents an important contribution for future functional studies to develop improved apple cultivars to superficial scald. The acquisition of cold tolerance under conditions of varying light quality is essential for plants growing in regions with seasonal variation in both temperature and light . Photo inhibition, i.e., the down regulation of the electron transport chain, reduces plant productivity, but safeguards the photosynthetic apparatus during cold and light stress .