Month: March 2023

Spot s presented homology with the At4g27270 protein whose molecular function is to interact selectively with FMN, and also presents oxidoreductase activity. From the 6 spots not detected in root tip extracts from Fe-deficient plants as compared to the controls , 3 were identified by MALDI-MS . Proteins matched were oxalate oxidase , peroxidase and caffeoyl CoA Ometyltransferase .Changes induced by Fe-deficiency and Fe-resupply in the root tip metabolome were evaluated by non-biased gas chromatography mass spectrometry metabolite profiling. A total of 326 metabolites were present in at least 80% of the samples of at least one treatment, and 77 of them were identified. Partial least square analysis shows a good separation between +Fe and -Fe root tips . Iron-deficient samples were closer to the 24 h and 72 h YZ samples than to the 72 h WZ ones. On the other hand, the 72 h WZ samples were closer to the +Fe samples than to the -Fe ones.Iron deficiency and/or resupply caused significant changes in the levels of 62 out of the 77 identified metabolites. Metabolite level data were normalized to the mean response of the +Fe treatment; response ratios, defined as the level in a given treatment divided by the level in the +Fe control, are indicated in Table 2.Large increases were found for some organic acids , some sugars , nicotianamine and 2-aminoadipic acid. The response ratio of oxalic acid decreased markedly in -Fe conditions, whereas those of other aminoacids, N compounds, lipid metabolites and others did not show large changes when compared to the Fe-sufficient controls.

Twenty-four hours after Fe-resupply, there was a dramatic coordinated increase in the root tip response ratios of galactinol, raffinose, lactobionic acid, cellobiose and nicotianamine when compared to those found in Fe-deficient roots,vertical aeroponic tower garden whereas the response ratios of sucrose, myoinositol, citrate and malate decreased. Seventy-two hours after Fe resupply, the response ratios of galactinol, raffinose, cellobiose, nicotianamine and many other compounds had decreased in the YZ areas, whereas in the WZ the response ratios were very low. The response ratio of many of the lipids increased moderately in all Fe resupplied samples. Metabolites in the coenzyme, glycolysis, oxidative stress, pentose phosphate pathway and signalling categories did not show large response ratio changes with Fe resupply.The changes induced by Fe deficiency in the root tip proteome and metabolome from sugar beet plants grown in hydroponics have been studied. More than 140 proteins and 300 metabolites were resolved in sugar beet root tip extracts. Iron deficiency resulted in significant and higher than 2-fold changes in the relative amounts of 61 polypeptides, and 22 of them were identified. Out of 77 identified metabolites, 26 changed significantly with Fe deficiency. In general, our results are in agreement with previous transcriptomic, proteomic and enzymatic studies on Fe-deficient roots. Our data confirm the increases previously found in proteins and metabolites related to carbohydrate metabolism and TCA cycle pathways. Two major changes induced by Fe deficiency in roots are described in this study for the first time: the increase in DMRL synthase protein concentration and gene expression, and the increase in RFO sugars. The largest change found in the proteome map of root tip extracts from sugar beet plants grown in Fe deficiency conditions corresponded to DMRL synthase, which was detected de novo in Fe-deficient root tips, and is the protein with the highest concentration in these gels . This enzyme catalyses the fourth step of Rbfl biosynthesis, and Rbfl is the precursor of Rbfl sulphates, FMN and FAD, the last one being a cofactor for the root plasma membrane Fe reductase.

The expression of BvDMRL decreased drastically 24 h after the addition of Fe to Fe-deficient plants, whereas DMRL synthase protein abundance and Rbfl and Rbfl sulphate concentrations did not change significantly with Fe-resupply in the YZ of root tips , suggesting that the turnover of this protein is slow. Accumulation in Fe-deficient roots of flavin compounds, including Rbfl and Rbfl 3′ – and 5′-sulphate is a characteristic response of sugar beet and other plant species. The exact role of flavins in Fe deficiency is unknown, and it has been hypothesized, based on the similar location of flavin accumulation and Fe reduction and on the fact that the Fe reductase is a flavin-containing protein, that free flavin accumulation may be an integral part of the Fe-reducing system in roots from Strategy I plants. On the other hand, these compounds are secreted to the growth media at low pH and, assuming high concentrations at the secretion site, they could mediate extracellular electron transfer between soil Fe deposits and root Fe reductase as it has been described for flavin phosphates secreted by some bacteria. Moreover, excreted flavins could also act as a plant-generated signal that could influence rhizosphere microbial populations, indirectly affecting Fe availability. A major change in carbohydrate metabolism was the large increase in RFO compounds that occurs in roots with Fe deficiency. This increase was higher than that found for sucrose . The total concentrations of raffinose and galactinol were also determined by HPLC-MS, and concentrations of both compounds in the 35-80 nmol g FW-1 range were found in Fe-deficient and Fe-resupplied root tips , whereas concentrations in the +Fe treatment were one order of magnitude lower. The sum of the raffinose and galactinol concentrations in the -Fe, 24h, 72hWZ, 72hYZ and +Fe tissues was 13.9, 7.4, 2.2, 5.1 and 0.6% of the total sucrose, respectively, supporting the relevance of the RFOs changes with Fe status. RFOs have diverse roles in plants, including transport and storage of C and acting as compatible solutes for protection in abiotic stresses .

Other explanationfor the large increase in the relative amounts of RFOs could be the ability to function as antioxidants; galactinol and raffinose have hydroxyl radical scavenging activities similar to other soluble antioxidants such as glutathione and ascorbic acid. Since ROS damage and ROS detoxification strategies have been observed in Fe-deficient roots, the increase in RFO concentration could help to alleviate ROS damage produced under Fe deficiency. Moderate increases in sugars commonly found in cell walls such as cellobiose, xylonic acid and arabinose, which may indicate cell wall modifications, were measured in sugar beet Fe-deficient root tips. Changes in cell wall metabolism have been also described in Fe-deficient tomato roots. On the other hand, it has been described that cell wall damage generates oligosaccharides that can act as signalling molecules in stresses such as wounding. The increase in RFOs could also act as a long distance Fe-deficiency signal via phloem sap transport. This is the first description of RFOs accumulation in plants under Fe deficiency, and the physiological implications of this increase deserve further consideration. Most of the proteins found to be up-accumulated in sugar beet root tips by Fe deficiency were identified as carbohydrate catabolism enzymes, including 5 of the 10 glycolytic pathway enzymes ,vertical gardening in greenhouse one of the citric acid cycle and fructokinase. Increases in the activities and concentrations of several glycolytic enzymes in root extracts with Fe deficiency have been previously found, including fructose 1,6-bisphosphate aldolase, enolase, triosephosphate isomerase and GADPH. Also, increases in the activities and concentrations of several enzymes of the citric acid cycle with Fe deficiency have been previously reported in root extracts, including MDH. Results are also in agreement with micro-array gene analysis in Fe-deficient A. thaliana roots. Increases in the amount of PEPC have been found at the protein level, but this enzyme, with a molecular mass of 110 kDa, was not in the range used in our 2-D gels. Up-regulation of carbohydrate catabolism in roots of plants grown in Fe deficient conditions is probably a result of an increased demand of energy and reducing power in roots needed to sustain the increased activity of H+-ATPase and Fe reductase. Also, two spots corresponding to different subunits of F1 ATP synthase increased in 2-D gels from Fe deficient root tips, further supporting the higher energy requirement in these roots. Moreover, our results show an increase in the amount of formate dehydrogenase, an enzyme related to the anaerobic respiration, in Fe-deficient roots, confirming the results of enzyme and transcriptional analysis. Anaerobic respiration is an alternative pathway for energy production when oxidative phosphorylation is impaired. Metabolite studies revealed large increases in organic acids, including a 20-fold citric acid increase. These increases in TCA cycle organic acids with Fe deficiency are coupled with increases in glycolysis and root C fixation by PEPC, and provide an anaplerotic, non-autotrophic C source for leaves which have otherwise reduced photosynthetic rates.

Malate and citrate could also be pumped from the cytosol to the mitochondria via a di-tricarboxylate carrier where they would allow a higher turnover of reducing equivalents. A significant decrease in oxalic acid concentration was observed in Fe deficient root tips, and similar decreases have been reported in Fe-deficient tomato roots. The implications of oxalate concentration decreases with Fe deficiency are still not known, since the role of oxalic acid in plants is quite different from that of the other organic acids, and for a long time it has been considered as a toxin or a metabolic end product . Regarding N and amino-acid compounds, a large increase was measured for nicotianamine, which has been described to play a role in cytosolic Fe availability. A comprehensive representation of the metabolomic and proteomic changes taking place in root tips under Fe deficiency and resupply is shown in Figure 4. Red and yellow symbols indicate major and moderate increases in metabolites and proteins compared to the Fe-sufficient controls. Blue and green symbols indicate major and moderate decreases in metabolites and proteins compared to the controls. Besides the major increases in RFOs and DMRL, Fe deficiency induced significant changes in root tip metabolism, mainly associated to increases in carbohydrate catabolism, glycolysis and TCA cycle and to a lesser extent in aminoacid and nitrogen metabolism . Similar changes were observed in the 24 and 72h YZ Fere supplied roots, whereas the WZ of 72 h Fe-resupplied plants did not show major changes when compared to +Fe plants . On the other hand, the relative amount of lipid metabolism compounds did not change markedly in Fe-deficient roots, whereas Fe resupply caused a moderate increase in this type of metabolites .Sugar beet was grown as described elsewhere. “Monohil” was always used, with the exception of raffinose and galactinol analysis, which was carried out with “Orbis”. After seed germination in vermiculite and 2 weeks in half-strength Hoagland’s nutrient solution with 45 μM Fe-EDTA, plants were transferred into 20 L plastic buckets containing half strength Hoagland’s nutrient solution with either 0 or 45 μM Fe-EDTA. The pH of the Fe-free nutrient solution was buffered at approximately 7.7 by adding 1 mM NaOH and 1 g L-1 of CaCO3. In the Fe resupply experiments, plants grown for 10 d in the absence of Fe were transferred to 20 L plastic buckets containing half strength Hoagland’s nutrient solution, pH 5.5, with 45 μM Fe-EDTA. The root sub-apical region from Fe-sufficient plants , Fe-deficient plants , Fe-deficient plants resupplied with Fe for 24 h and Fe-deficient plants resupplied with Fe for 72 h was collected with a razor blade and immediately frozen in liquid N2. The specific regions of root sampled were: in the case of +Fe, -Fe and 24 h plants, the first 10 mm from the root apex ; in the case of 72 h Fe resupplied roots two zones were sampled separately, the first 5 mm from the root apex, where a new white zone had developed , and the next 5 mm, comprising the still swollen and yellow root zone . Samples were taken at approximately 4 h after light onset in the growth chamber.Protein extracts were obtained as described elsewhere and protein concentration was measured with RC DC Protein Assay . A first dimension isoelectric focusing separation was carried out on ReadyStrip IPG Strips , using a linear pI gradient 5-8. Strips were loaded in a PROTEAN IEF Cell and focused at 20°C, for a total of 14000 V.h. For the second dimension polyacrylamide gel electrophoresis , IPG strips were placed onto 12% SDS-PAGE gels to separate proteins between 10 and 100 kDa. Proteins were stained with Coomassie-Blue R- 250 and results analyzed with the PDQuest 8.0 software.

During this rule making, the US EPA will also reexamine the NSR requirements applicable to existing non attainment areas, in order to address issues of fairness among existing and new non attainment areas. The transitional classification will be available for any area attaining the one-hour standard but not attaining the eight-hour standard at the time the US EPA promulgates the new rules.To encourage early planning and attainment for the eight-hour standard, the US EPA will make the transitional classification available to areas not attaining the eight-hour standard that will need additional local measures beyond the regional transport strategy, as well as to areas that are not affected by the regional transport strategy, provided they meet certain criteria. To receive the transitional classification, these areas must submit an attainment SIP prior to the designation and classification process in the year 2000. The SIP must demonstrate attainment of the eight-hour standard and provide for the implementation of the necessary emission reductions on the same time schedule as the regional transport reductions.By submitting these attainment plans earlier than would have otherwise been required, these areas would be eligible for the transitional classification and would achieve cleaner air much sooner than otherwise required.The majority of areas not attaining the one-hour standard have made substantial progress in evaluating their air quality problems and developing plans to reduce emissions of ozone-causing pollutants. These areas will be eligible for the transitional classification provided that they attain the one-hour standard by the year 2000, and comply with the appropriate provisions of section above depending upon which conditions they meet.For areas not eligible for transitional classification, their work on planning and control programs to meet the one-hour standard by their current attainment date should advance toward meeting the eight-hour standard.

While the additional local reductions that they will need to achieve the eight-hour standard must occur prior to their eight-hour attainment date , for virtually all areas the additional reductions needed to achieve the eight-hour standard can occur after the one-hour attainment date. This approach allows them to make continued progress toward attaining the eight-hour standard throughout the entire period,low round pots without requiring new additional local controls for attaining the eight hour standard until the one-hour standard is attained. These areas, however, will need to submit an implementation plan within three years of designation as non attainment for the new standard for achieving the eight-hour standard. Such a plan can rely in large part on measures needed to attain the one-hour standard. For virtually all of these areas, no additional local control measures beyond those needed to meet the requirements of Subpart 2, Part D of Title I, would be required to be implemented prior to their applicable attainment date for the one-hour standard. Non attainment areas that do not attain the one-hour standard by their attainment date would continue to make progress in accordance with the requirements of Subpart 2, and the control measures needed to meet progress requirements under Subpart 2 should generally be sufficient for meeting the control measure and progress requirements of Subpart 1, as well .After the 1973 OPEC oil embargo, Congress and the public became concerned about the increasing dependence of the U.S. on foreign oil. Since the price of petroleum products was controlled well below market levels, many individuals thought that conservation should be encouraged through the use of non-price mechanisms. In 1975, Congress enacted the Energy Policy and Conservation Act, which placed a particular emphasis on auto fuel economy since the greatest share of petroleum consumption was used by the automobile sector . “This legislation required that the corporate average fuel economy for new cars be raised gradually from 14.2 miles per gallon in model year 1974 to 27.5 miles per gallon by model year 1985” . From the early 1970s to the mid-80s, the average fuel economy of new domestic automobiles increased more than 100 percent .

Gains in CAFE were achieved by: 1) reducing the weight of automobiles, 2) improving engine and drive train efficiency, 3) reducing tire rolling resistance, and 4) improving the aerodynamics of design. Nevertheless, overall gasoline consumption by light-duty vehicles did not decline sharply and is now higher than ever before. There are several reasons why CAFE has not been a more effective instrument for reducing gasoline consumption. The four major factors influencing fuel consumption include: 1) more vehicles in the fleet, 2) more miles driven per vehicle. From 1985 to 1994, there was a 7.6 percentage point increase in the number of trucks and a 0.2 percentage point decrease in the number of passenger cars. The fuel economy of trucks was notably lower than passenger cars , p4-S, Table 4.4). This growth appears to have been driven mainly by demographics, vehicle prices, and consumer incomes. The second factor, average vehicle miles driven, has also risen, particularly for trucks. In addition, the shift in consumer preferences toward light trucks has had an important impact on gasoline consumption. The total number of vehicles miles traveled has been influenced by low gasoline prices. Not surprisingly, the price of gasoline also appears to affect the average miles per gallon through its influence on consumer preferences for more fuel-efficient vehicles and on the decisions of two car families to drive more fuel-efficient automobiles. Nevertheless, it is important to note that CAFE only directly improves miles per gallon of new vehicles. The overall impacts on the fuel economy of the entire fleet occurs very slowly, as older vehicles are retired. At present, the average fuel economy for the entire fleet is approximately 24 miles per gallon, which is about the same as in 1980. In an evaluation of the effects of CAFE on the nation’s fuel consumption, it is important to recognize two counterproductive effects of these standards. First, CAFE encourages increased driving because it lowers the cost of travel. Second, CAFE can encourage the retention of older, low-mileage vehicles because it adds to the costs of manufacturing new vehicles. Hence, these factors have the potential to inadvertently increase pollution because emissions increase proportionately with miles driven and more than proportionately with the age of the vehicles.

The National Environmental Policy Act is one of the most significant pieces of environmental legislation in U.S. history. Passed by Congress in 1969 and signed into law in 1970, NEPA requires federal agencies to consider the environmental consequences of their actions before executing them. In preparing and passing NEPA, Congress recognized “the profound impact of man’s activity on the interrelations of all components of the natural environment, particularly the profound influences of population growth, high-density urbanization,plastic pots 30 liters industrial expansion, resource exploitation, and new and expanding technological advances”. The language of NEPA recognizes the importance of several things: 1) preserving the environment for future generations; 2) maintaining the safety, health, productivity, and well being of the American people; 3) using the products and materials of the natural environment of the country without diminishing them to the point of destruction; and 4) maintaining a balance between the growing population of the U.S. and the country’s natural resources . NEPA requires all agencies of the federal government to assess the possible adverse environmental impacts of proposed actions and legislation. NEPA applies to actions where FHWA, FTA, or agencies delegated the authority for such decisions have control over project approval. Consequently, NEPA applies to many of the projects to which conformity applies . If a federally proposed project has the potential to yield a significant environmental impact, compliance with the NEPA mandates is accomplished through the preparation of an environmental impact Statement . Under NEPA, all EISs must include: 1) a detailed Statement on the environmental impact of the proposed action; 2) a description of any adverse environmental effects that cannot be avoided should the proposal be implemented; 3) a discussion of alternatives to the proposed action; 4) a treatment of the relationship between local short-term uses of the environment and long-term productivity of the area; and 5) a discussion of any irreversible commitments of resources to be involved in a proposed action ) . In Title II of NEPA, Congress established the Council on Environmental Quality as the administering agency of the Act. NEPA required that CEQ develop a set of regulations for implementing the NEPA mandates. These Regulations are contained at 40 CFR Parts 1500 to 1508. Under the CEQ regulations, federal agencies are required to adopt procedures to ensure that applicable project-related decisions are made in accordance with the policies and purposes of the Act. The US DOT’s FHWA and FTA NEPA regulations are contained at 23 CFR Part 771 .In response to many air pollution problems, California adopted the California Clean Air Act in 1988 . California enacted the legislation in recognition of the fact that most urban areas of the State had not attained federal ambient air quality standards by the federal deadline of August 31, 1988. The CCAA directed the development and implementation of California’s own program to attain the ambient air quality standards at the earliest practicable date. Although a significant portion of the CCAA focuses on attainment of ambient standards in air pollution control districts, the statute directs the CARB to reduce emissions of motor vehicles .

The CCAA added a new section to the Health and Safety Code, Section 43000.5, which States: “the State board should take immediate action to implement both short- and long-range programs of across-the-board reductions in vehicular emissions which can be relied upon by the districts in the preparation of their attainment plans or plan revisions”. The CCAA also amended Section 43013, which added a subsection authorizing standards for specific types of motor vehicles and related equipment. “The State Board may adopt and implement motor vehicle emissions standards, in-use performance standards, and motor vehicle fuel specifications for the control of air contaminants and sources of air pollution which the State board has found to be necessary, cost-effective, and technologically feasible to carry out the purposes of this division” . Finally, the CCAA enacted Section 43018. Section 43018 States that the State Board shall try to achieve the maximum degree of emission reduction in mobile and vehicle emissions to meet the State standards. Section 43018 States that the Board shall take whatever actions are necessary, no later than January 1, 1992, to attain a reduction in the emissions of HC and NOx by December 31, 2000. The Board must also achieve maximum feasible reductions in particulates, CO, and toxic air contaminants. Section 43018 establishes that the Board must adopt standards that result in cost-effective control measures on all motor vehicles and motor vehicle fuels. Finally, Section 43018 “…establishes a specific timetable for the Board to conduct workshops and rule making hearings for specific regulations regarding motor vehicles and motor vehicle fuels.”In summary, the California legislators enacted the CCAA as a result of the State’s recognition of its air pollution problems and its inability to meet the federal ambient air quality standards in many urban areas by August 1988. The Act is ambitious and far-reaching in its goals and objectives. For the first time, a vehicle and its fuel would be treated as a system that would have to meet exhaust emission standards. This integrated approach, based on performance of the vehicle/fuel system, provides flexibility and encourages the vehicle and fuel industries to work together to develop the least polluting and most cost-effective vehicle and fuel technologies. Hence, California was the first State to adopt the most stringent vehicle emissions legislation. It is important to note that California legislators had established these goals and standards prior to enactment of the federal CAA 1990. Although the California regulations were already in place, the CAA of 1990 require the introduction of clean-fuel cars in California beginning in 1996. The CAA of 1990 also provides a voluntary “opt-in” provision that allows other States to adopt the California standards . California is the only State that can set higher emission standards than the federal government; after California has established higher standards other States can then adopt them.

Non-sequential selective dissolution in Na-pyrophosphate and ammonium-oxalate was used to characterize Fe, Al and Si in various pedogenic pools. Total C and N concentrations were determined on ground samples by dry combustion using a Costech C/N analyzer . Soil microbial biomass C and N were measured using chloroform fumigation and direct extraction with 0.5 M K2SO4 . Briefly, 10 g oven-dry equivalent samples were fumigated for 48 h in the dark, and then C and N were extracted with 0.5 M K2SO4. Similar extraction was applied for non-fumigated samples. Total dissolved organic C and total extractable N were measured using a C/N analyzer . The non-fumigated control values were subtracted from fumigated values as an estimate of microbial C and N. A Kec/Ken factor of 0.35 was applied for both C and N . Carbon mineralization was measured in the topsoil and subsoil by incubating duplicate soil samples in the dark under laboratory conditions over a 119-day period. Soil moisture was adjusted to ∼ 80% of field capacity and pre-incubated for one week prior to starting the long-term incubation. Soils were incubated in sealed Mason jars fitted with septa. Carbon dioxide in the head space of each soil sample and blanks with no soil was measured each week using an Infrared Gas Analyzer. The CO2 emission was normalized to initial total C content of each soil and expressed as CO2-C mg kg−1 soil C. In addition, net N mineralization was measured on these same samples at the end of the 119-day incubation by determining concentrations of mineral N in 1 M KCl extracts at time zero and at 119 days. Quantification of NO3 – used the vanadium chloride method and NH4 + the Berthelot reaction with a salicylate analog of indophenol blue.

A correlation analysis was performed to assess soil properties most strongly affected by land-use changes,microgreen fodder system using IBM SPSS Statistics 22. 2013.All soils were well drained with an A horizon overlying Bw horizons that extended to the depth of investigation . Soil particle-size distribution was similar among the four sites with the majority of the horizons having a loam texture . Some distinct changes in particle-size distribution within various pedons are attributable to more recent tephra deposition that resulted in burial of the former soil profile. Bulk density in subsoil horizons was very low , characteristic of soils formed in volcanic ash . Db was also low in the A horizon of the pine forest , but was higher under agricultural management due to traffic compaction resulting in a reduced pore volume. The agricultural soils displayed a distinct increase in Db and a reduction in total porosity in the topsoil horizons compared to the pine forest soil. Given the low bulk densities, total porosity was correspondingly high, ranging between 60 and 77%, with values decreasing in surface horizons with agricultural management. Plant-available soil water was generally in a narrow range with the exception of the surface horizons of the pine forest soil . The water retention capacity varied from 37 to 53% in topsoil horizons and from 45 to 51% in subsoil horizons with the lowest values in the pine forest.Soil pH-H2O increased from very strongly acid in the pine forest and tea plantation to moderately acid in the horticultural crops with fallow and intensive cultivation . Regardless of land use, all soils in this study had low CEC characteristic of acidic Andisols dominated by allophanic materials.The pHKCl-pHH2O values ranging between −0.1 and −0.5 were indicative of a soil colloidal fraction dominated by variable charge materials . Especially notable is the very low base saturation and concentrations of exchangeable Ca and Mg for the PF and TP soils . Exchangeable base cations are a common limiting factor for horticultural production in the studied Andisols since these nutrient cations are extremely low under pine forest.

While the horticultural management practice of applying horse manure and lime did not appreciably increase the measured CEC, it was remarkably effective in increasing exchangeable base cations . For example, exchangeable Ca, Mg and K increased from 1.5, 0.3 and 0.2 cmolc kg−1 in the pine forest to 26.3, 3.5 and 1.0 cmolc kg−1 in the intensive horticultural crops, respectively . The high base saturation of over 100% under horticultural land uses compared to < 23% for the pine forest and tea plantation .Organic C concentration in A horizons was highest in PF and 1.0 to 2.0% lower under agricultural management . In contrast, organic C was lower in the PF subsoil while the agricultural sites had elevated organic C concentrations in several subsoil horizons. Organic C stocks in the upper 100 cm of the soil profile were calculated by summing the organic carbon stocks in each individual horizon were present). Organic carbon stocks followed : TP ≈ IH > FH > PF . The agricultural soils contained more organic carbon than the pine forest soil. While horse manure was added to the IH soil for the past 7 years, the TP and FH soils received no organic matter amendments and still had similar pedon organic matter stocks. As a direct comparison, the IH soil receiving horse manure contained only slightly more organic C than the FH soil located 4 m away that received no horse manure and was fallowed over the past 7 years. Dissolved organic carbon concentrations were appreciably higher in the PF topsoil and throughout subsoil horizons of the TP profile . The horticultural soils tended to have lower overall DOC concentrations than PF and TP land uses. Total N concentrations followed a similar distribution to organic C concentrations among sites with total N stocks in the upper one meter of soil following : IH > FH ≈ TP > PF . The C:N ratio was lowest in the upper 50 cm of the IH and FH soil profiles , while values for PF, TP and lower soil horizons at all sites were generally in the range 16 to 19.

The highest concentrations of inorganic N were found in the IH pedon and were dominated by NO3 – . In contrast to the IH soil dominated by NO3 – , inorganic N concentrations were dominated by NH4 + in the TP, FH and PF soils with the highest value in the TP soil and lowest under FH land use. High P fixation , characteristic of Andisols, was exhibited for all land-use types. Under forest vegetation , the soil P retention was consistent at 97% throughout the entire pedon . Change of land use to TP and FH did not appreciably affect P fixation. However,barley fodder system the IH land use receiving application of horse manure for the past 7 years showed appreciably lower P fixation in the upper 40 cm. Reflecting the high P fixation, available P content was below the detection limit for all horizons of all land-use types, except for the upper horizons of the IH land use .Extractable SO4-S content was considerably higher in the PF and TP pedons as compared to the horticultural pedons . Change of land use from pine forest to agriculture decreased extractable S content. The exception is the TP pedon that contains high S due to application of kieserite as an integral part of tea plantation fertilizer management. Extractable micronutrient concentrations showed the following general order of abundance: Fe ≫ Mn > Cu > Zn . In terms of land-use, micronutrient levels followed the general pattern of IH > FH > TP > PF.There were several significant correlations among soil properties . Oxalate-extractable Sio showed a positive correlation with the clay fraction, while Feo had a strong negative correlation with pH and exchangeable Ca and Mg. In contrast, Alo showed no significant correlations with other soil properties. For organo-metal complexes , Alp had highly negative and positive correlations with the clay fraction and organic C, respectively. However, Fep showed no significant correlations with other soil properties. Soil pH showed a highly negative correlation with P retention and Feo, along with a positive correlation with exchangeable cations , total N and Db. Soil bulk density showed a positive correlation with exchangeable cations and negative correlation with P retention. P retention had a negative correlation with exchangeable cations .Andisols are characterized by low Db and high porosity due to the abundance of amorphous and poorly crystalline materials and organic matter that contribute to highly stable and very well structured soils under natural conditions. However, the low natural Db may change due to anthropogenic activities. The evidence was revealed by soil tillage under intensive horticultural crops contributing to increased Db from compaction by potential destruction of soil aggregates due to physical mixing/abrasion by tillage operations. Tillage was reported to destroy macropore pathways of Andisols in Mexico resulting in a lower infiltration and permeability of topsoil horizons .Chemically, the exchangeable cations have positive significant correlation with Db, indicating the increase in soil exchangeable cations gave rise to the increased soil bulk density . This is probably due to the role of Ca and Mg ions derived from lime and manure in binding soil particles, resulting in the change of soil friable structure under forest to more compact aggregate formation under intensive horticultural cultivation.

The water retention capacity varied from 37 to 53% in topsoil horizons and from 45 to 51% in subsoil horizons with the lowest values in the pine forest . These data indicate that the number of soil pores storing plant-available water is lower in the forest Andisols than those converted for agriculture. In other words, the water retention capacity has increased about 50% following conversion from pine forest to agriculture. This implies that the compaction associated with tillage is responsible for increasing the water retention capacity through conversion of macropores to meso/micropores. The water retention capacity in this study was higher than for cultivated Mexican Andisols reported by Prado et al. . The high water retention in Andisols is caused primarily by their large volume of meso/micropores . Formation of these meso/micropores is greatly enhanced by poorly crystalline materials and soil organic matter . Buytaert et al. studied toposequece of Andisols in south Ecuador and reported the large water storage capacity as revealed by water content ranges from 2.64 g g−1 at saturation, down to 1.24 g g−1 at wilting point. The long-term cultivation of agricultural soils in this study has not caused appreciable degradation to the overall Db, porosity or water retention characteristics of these Andisols. While macroporosity was decreased by tillage, the macropore content of topsoil horizons remained > 15% providing adequate infiltration and soil aeration. The loss of macropores is compensated for by the increase in meso/micropores that contribute to increased plant-available water holding capacity. In spite of the increase of bulk density and loss of macropore capacity, field observations confirmed that the agricultural soils in this study retained their high infiltration capacity with no evidence of surface runoff. In Italy, well developed Andisols on flow-like landslides over 70 years experienced low run off and minimal soil erosion owing to a good infiltration in spite of the high slope steepness and the anthropic pressure associated with land management .The pine forest soil was very strongly acidic owing to the strong leaching regime associated with the isothermic/perudic climatic regime. Applications of lime and more recently horse manure to the IH soil were effective in raising the pH of the horticultural soils . In spite of the low soil pH values in the tea plantation, the potential for Al3+ toxicity was not evident as ascribed to the low exchangeable Al3+ concentrations . Threshold values for Al toxicity are generally considered about 2 cmolc kg−1 for common agricultural crops and 1 cmolc kg−1 for Al-sensitive crops . Andisols dominated by allophanic materials generally contain low KCl-extractable Al concentrations; however, these values may be underestimated due to “induced hydrolysis” of displaced Al and subsequent adsorption of polymeric Al to allophanic materials . The elevated pH associated with the horticultural soils reduced the exchangeable Al3+ concentrations to non-detectable levels , further reducing the potential for Al3+ toxicity. A notable findings in this study was the increase in soil pH and base saturation following land use changes as revealed by the strongly positive correlation between soil pH and exchangeable cations .

In the absence of photo assimilation, the starch stored in the source is degraded to replenish cellular sugars in order to avoid carbon starvation. Therefore, carbon assimilation and utilization is carefully balanced for optimal plant development. Adverse environmental conditions can disrupt the normal starch and sugars levels with repercussions for the ability of the plant to sustain growth. Drought is associated with reduced starch or sugar levels in source tissues. Salinity stress can induce higher starch accumulation in the source or sink of some species, but trigger starch reduction in others. Similarly, chilling stress is associated with accelerated source-starch accumulation or degradation. These observed increases in starch or sugars may be adaptive responses for stress-survival, or may be ‘injury’ responses resulting from the under-utilization of carbon because of growth cessation, regardless, documenting these changes is necessary for a deeper understanding of plant stress response. Feeding plants with 14CO2 is useful for tracking carbon movement, and can inform on changes in carbon allocation due to stress. Available data suggests that stress generally accelerates allocation to the sinks as an adaptive response. Salinity increased flux from source to developing fruits in tomato and to the roots in transgenic rice seedlings. Water-stress elicited a similar distribution pattern in Arabidopsis, with higher 14C allocated to the roots, in beans, where 14C flux to the pods increased, and in rice, where it stimulated 14C mobilization from the stem and allocation to the grain. Additional 14C-allocation studies under varied stress conditions could help to clarify whether or not higher source-sink flux is a universal stress response.

The observed changes in local and distant carbon fluxes in plant tissues during stress result from multiple activities – epigenetic, transcriptional, post-transcriptional and post translational changes,dutch buckets occurring across different spatial and temporal scales, which must be integrated to deliver a cohesive response to stress. Te trehalose-6-phosphate/Sucrose non-Fermented Related Kinase 1 signaling cascade may function in this way. It is critical for plant survival under low carbon and energy conditions, in part through changes in starch metabolism. Te T6P/SnRK1 can also modulate source-sink interactions; therefore, key elements of this regulatory network could potentially be activated for a ‘rewiring’ of whole plant carbohydrate use under stress. Because of the many issues with respect to plant carbon use under stress that remain unresolved, our aim in this work was to investigate changes in carbon partitioning and allocation in response to short-term drought, salinity, and cold stresses. 14CO2-labeling of a single source leaf was used to map whole-plant and intra-tissue changes in carbon use, as it can provide partitioning and allocation data in the same system. Single-leaf labeling permits more accurate tracking of 14C-movement than can be obtained by exposing the entire rosette to the label.By comparing plants exposed to different stresses it may be possible to identify convergent and divergent adaptive responses associated with each unfavorable condition. Starch content was also assayed in the source leaf and the roots of the stressed plants and the data were compared to 14C-starch fluxes to identify how starch metabolism may be regulated to alter sugar distribution. Finally, the transcriptional activity of key genes in the T6P/ SnRK1 pathway was assessed to identify genes associated with changes in carbohydrate levels under abiotic stress. By integrating these data, we present one of the first comprehensive pictures of how Arabidopsis changes carbon flux under short-term environmental stress. This information could be combined with that generated from the wealth of -omics data to broaden our understanding of plant stress response.Our first aim was to investigate how plant source and sink tissues use carbon over the diurnal cycle under normal conditions. One hour before the middle of the day , a single mature, but still developing source leaf was fed with 14CO2 for 5 min. Te labeled source leaf, unlabeled sink leaves, and the roots were harvested separately at MD, at the end of the day , and at the end of the night . MD, ED and EN correspond to 6h, 12h and 24h after dawn. Te percentage of 14C distributed among the source and the sinks was determined.

Within each tissue, the incorporation of 14C into the main metabolites pools: sugars, amino acids, organic acids, starch, protein, and ‘remaining insoluble compounds’ , was established. First, we calculated the percentage of 14C distributed from the source to the sinks. During the day, ~60% of the 14C was retained in the source leaf, but by EN, the percentage of total 14C was evenly distributed among all tissues . Nighttime export of 14C from the source, and its subsequent allocation into the sinks, accounted for the re-distribution. Second, we examined the 14C partitioning between the source and sinks to create a full picture of how allocation and subsequent partitioning were altered. Partitioning in the roots was more dynamic than in the sink leaves, and this difference was amplifed most at ED . In the roots, there was increased incorporation of 14C into metabolites used for growth — i.e. sugars, amino acids, and RICs — and less into those used for storage —i.e. protein and starch — compared to the source. Te pattern of 14C-partitioning in source leaf vs. roots therefore reflected the prioritization of biological processes in each tissue type. Te other change of note occurred at EN, when both sinks incorporated less 14C into organic acids but more into starch compared to the source. This may indicate that the sinks had greater sufficiency with respect to carbon with a relatively reduced need for organic acids as sources of energy compared to the source. Finally, we examined changes in 14C-partitioning over the diurnal cycle . Data at ED and EN were compared to that generated at MD to fully assess how the day-night cycle affected carbon partitioning in different tissues. Te metabolic pools in the source leaf were variable, while those in the sinks were relatively stable. Relative to MD, there was less 14C in the sugar and starch fraction, but an almost 2-fold greater flux into organic acids at EN in the source. Organic acids may serve as the primary substrate for respiration after reductions in the sugar pool. In the roots, at EN, the 14C percentage in sugars decreased, but increased in starch. This indicates that the starch in the roots was accumulated constantly during the diurnal cycle, with more accretion during the night than the day. In contrast, in the sink leaves, the carbon flow into sugars and starch were stable at EN, but there was a 4-fold increase in the 14C partitioned into the RICs, suggestive of nighttime growth processes.How stress altered Arabidopsis carbon use over the diurnal cycle at the cellular and whole plant level was examined.

Arabidopsis seedlings were exposed to salinity stress using 100 and 200mM NaCl, to osmotic stress using 150 and 300mM mannitol, and to cold stress by exposing roots to 0 °C cold at the beginning of photoperiod. Afer 5hours of stress treatment,grow bucket a single mature source leaf was fed with 14CO2 for 5min. Sampling was done as previously described. Osmotic stress. Carbon allocation was negatively affected by osmotic stress, and the inhibition grew in severity as the stress progressed . By EN, mild and severe mannitol stress increased the percentage of 14C in the source, and decreased it in the roots . This could reffect reduced carbon export due to enhanced source activities, inhibited carbon export from the source, reduced sink strength, or a combination thereof under osmotic stress. Carbon partitioning within the source was also modulated to a greater extent than in the sinks . At MD, both mild and severe osmotic stress reduced the 14C-partitioned into starch but increased 14C-partitioning into organic acids in the source, presumably for respiratory use. Six hours later, only severe osmotic stress had this effect leading to greater 14C flux into osmoprotectants — sugars, organic acids, and amino acids — at the expense of the storage compounds . Te 14C-flux into these osmoprotectants also increased in both sinks at the expense of the RICs, with the latter decreasing drastically in the roots. Salinity stress. Te most obvious change was the percentage of 14C allocated from source leaf into roots, which decreased significantly by EN under both mild and severe NaCl stress . Te 14C-use in source leaf was more responsive to salinity compared to the sinks . Severe salinity stress decreased 14C-partitioning into starch but increased partitioning into sugars, amino acids, and organic acids during the day in the source. At MD, more 14C was partitioned into sugars in the sink leaves, but 6h later at ED the 14C in sugars was stable, with reduced flux into starch and proteins. This indicates that 12h after the stress treatment, carbon was diverted from storage and preferentially partitioned into sugars for osmoprotection. In the roots, less 14C was partitioned into the RICs at ED and EN compared to the control, which suggest a shift away from investing 14C into resources normally used for root growth under salinity. This may have led to increased 14C accumulation into sugars at the end of night because they were under-metabolized. Interestingly, proteins were the only metabolite affected by both mild and severe salinity stress in both source and sink leaves, while it was unchanged in the roots.Further, unlike sink leaves, the source had increased 14C label in protein at MD . Te changes in 14C partitioning and allocation in response to different levels of salinity stress are summarized as follows: the source leaf partitioned less 14C into storage compounds but more 14C into osmoprotectants in response to severe salinity stress; sink tissues showed a differential response to salinity stress: similar to the source leaf, the sink leaves showed reduced 14C in storage compounds, however, roots tissue had reduced 14C in structural compounds; and the amount of 14C imported into roots tissue was inhibited by salinity; this might be due to reduced sink activity, inhibited phloem transport, or a combination thereof. Cold stress. Te percentage of 14C in root tissues was significantly reduced by cold stress at the end of night, showing similarity to tissues under osmotic and salinity stress . Carbon allocation was not affected by low temperature during the day , but carbon partitioning was highly regulated in the source leaf , especially at the end of day. Te most notable difference was that the 14C-flux into starch and RICs decreased relative to the control plants. Te decrease in starch was high at MD but lessened during the diurnal cycle, while the opposite was true for the RICs, where inhibition intensified over the day. In the source, there were also higher fluxes into sugars, amino acids, and organic acids from MD to ED. Cold also triggered increased 14C into the protein pool at MD, and decreased it at ED. At EN, the 14C in RICs strongly decreased, with a corresponding strong increase in sugars. Cold stress therefore stimulated more 14C partitioning into sugars over the diurnal cycle in the source leaf. Te sinks were less affected by cold than the source. In sink leaves, there was increased carbon flow into sugars during the day and decreased carbon into starch at night, with no difference in RICs. In contrast, the roots had increased 14C in the sugar pool at night, and reduced partitioning into the RICs . This change of 14C partitioning suggests reprioritization of reserves with a greater flux towards sugars for osmoprotection at the expense of other pathways.Te 14CO2 labeling experiment showed that starch is the most dynamic metabolite pool that changed under all types of abiotic stresses used in this study. 14C-flux into starch was down-regulated by abiotic stress, and the regulation depended on the time of day and tissue type examined. Under control conditions, 14C-partitioning into starch was stable during the day but decreased at night in the source leaf . However, this pattern was disrupted under salinity and cold stress due to reduced carbon flow into starch. In contrast to the source leaf, 14C in starch in sink leaves did not change during the day even under stress. In roots, the percentage of 14C into starch normally increased by EN, and interestingly, this partitioning was maintained under osmotic stress, but not under salinity and cold stress.

For instance, Pinus nigra ssp. laricio adults have been observed to follow an isohydric strategy, whereas seedlings in a glasshouse experiment did not . Although it is more complicated to impose drought treatments on adults, drought experiments have been carried out on adult trees using networks of rain shields/ gutters to intercept precipitation and direct it away from the trees . This water can also be re-directed to other plots to create ‘well watered’ treatments. For the most part, these studies have been carried out on natural populations. However, if they were coupled with provenance study plantings, one could test for population or genotypic differences in adult drought response. Likewise, apart from some long-term provenance studies , most experiments span a few days to a few months. In order to investigate drought resilience and legacy effects, more multi-year studies are needed.The length and intensity of drought can affect which trait combinations result in greater fitness. In Section IV.1, we mentioned the great diversity of methods used to induce or measure drought stress treatments in gene expression studies. The same diversity is found in G2P and provenance studies as well. There is a need to assess: whether environmental treatments roughly match the range of conditions in the environments in which the target species does or might grow; how environmental treatments relate to plant stress measures ; and whether traits, responses or genotypes associated with drought tolerance in the glasshouse or laboratory predict performance in the field. In addition, studies testing longer term drought treatments are lacking, as are those that explicitly test variable combinations of drought length and severity.

Future work should address these gaps.In most of the genetic studies cited above,u planting gutter a relatively high proportion of the genes expressed or linked to phenotypes or environmental gradients of interest either have unknown or poorly defined functions. Behringer et al. , for instance, found that, of the 832 transcripts analyzed for gene ontology, 538 either had no database hits or could not be assigned to a biological process. Although this could be partly addressed with further studies intraditional model organisms, such as Arabidopsis, analysis of loblolly pine and Norway and Sitka spruce genome sequences suggests that there could be thousands of conifer-specific gene families . This shortcoming must be addressed by further development of model systems in conifers.The tomato is a functional genomics model for fleshy-fruited species and is one of the most popular and economically important crops globally . However, storage at temperatures below 12.5°C followed by rewarming to room temperature, compromises fruit quality, hampering the post harvest handling of this commodity . This cold-induced damage to the fruit called post harvest chilling injury may only be detectable as a loss of flavor, or in severe cases, as fruit spoilage, the extent of which depends on the storage temperature, length of exposure, genotype and fruit developmental stage . The progression of PCI in fruit tissues is complex. It is marked by a loss of selective membrane permeability, increased solute leakage, reactive oxygen species accumulation and metabolic dysfunction . After the fruit is transferred to room temperature for rewarming or reconditioning, higher respiration ensues within days , and within a week, secondary symptoms such as uneven color formation, surface pitting, water soaking and decay are visible . Symptoms are more intense in green compared to riper fruit, since maturation processes are disrupted by chilling . Because of the negative effect on tomato quality and shelf-life, our goal is to better understand PCI development and regulation in this species. Some aspects of the disorder or approaches used here may be relevant for other PCI-sensitive species.

First, we investigated the spatial and temporal evolution of PCI in the whole tomato fruit using MRI. Most studies of tomato PCI have focused on the pericarp, ignoring the internal tissues, which can account for 30% and 70% of the fresh mass of round and cherry tomatoes, respectively. Tao et al. , investigated changes in chilled ‘Micro-Tom’ fruit using non-invasive MRI. They showed that the columella and locular region differed from the pericarp in their response to cold, which has implication for understanding the underlying causes of PCI. The fruit in that study were subjected to a severe cold stress , since this genotype is not as sensitive to chilling temperatures as many commercial varieties . Further, only one developmental stage was chosen . It is not known if their findings are applicable to other cultivars, storage conditions or maturation stages. Second, we investigated if 5-azacytidine could alter PCI. This chemical inhibits DNA and RNA methylation , epigenetic modifications that regulate gene expression, in response to developmental and environmental stimuli in a tissue-specific manner . DNA methylation is a key regulatory process for tomato fruit ripening ; injecting AZA in round tomato fruit accelerated ripening . It was shown that chilling-induced reductions in red fruit volatiles correlated with methylation of key ripening genes. Co-regulation of the ripening and cold response regulatory networks in fruit undergoing chilling stress seems likely . Since differential methylation is essential to both processes, we wanted to determine if AZA could influence PCI symptoms in tomato fruit. In this study, two questions were asked: 1) is it possible to detect spatio-temporal differences in chilled tomato fruit differing in maturation stage, and temperature × time of storage by low-resolution MRI?, and 2) would AZA influence PCI response? For the former, we used commercial cherry tomatoes and mild to moderate chilling stress. For the latter, fruit were injected with AZA weekly in order to detect changes in PCI by methylation , specifically on respiratory activity. Fruit from a commercial cherry cultivar and the functional genomics model ‘Micro-Tom’ were used in this study.At this developmental stage in ‘Sweet 100’, the pericarp, columella and locular tissue showed a differentiated pattern in terms of their D-values after 7 days of chilling . Values were highest in the pericarp followed by the locular tissue and columella. Similar patterns were seen in freshly-harvested breaker fruit .

These three tissues have heterogeneous transcriptional and metabolic profiles due to their distinct origin and functionality . This likely contributed to the distinct D-values observed. When D-values for each region were compared as over each chilling period,planting gutter no changes were observed except for the columella in fruit held at 5°C. Unchanged D-values may be due to cold-induced reductions in free water movement within tissues, and pectin solubilization . Fruit exposed to warmer temperatures, i.e., after storage at the control temperature for 7 days, or after transfer from the cold to 20°C, showed more dynamism in D-values. The different tissue fractions, which had distinct D-values during chilling, changed and became more similar when exposed to warmer temperatures . These non-chilling temperatures may have allowed ripening and other physiological events to take place, leading to these changes.Figure 3A shows the D-values of ‘Sunsugar’ ripened fruit. These data, gathered from breaker, pink, and red fruit immediately after harvest, suggest that as ripening progresses, the D-values of the columella and locular region become more similar . Ripening increases the proportion of free water and metabolites within tissues, due to liquefaction of the locules and breakdown of the structural components of the cell . These changes may have underscored the increased D values seen here, and in other studies . A similar occurrence was seen when red fruit was stored at 2.5°C for 5 days . When D-values for each region were compared over time, there was no significant difference. Tissue liquefactionin red fruit was so extended as a consequence of ripening, that cold did not generate any detectable increase by the MRI, or did not increase membrane leakage since it was already fluid. The observations of pink fruit stored in the cold and then rewarmed are less clear. Both chilling-induced damage during low-temperature storage, and ripening-related tissue deconstruction during rewarming would lead to increased membrane permeability and D values , thus making it difficult to attribute higher D values to one or the other biological phenomenon. There are some points to emphasize with respect to the data when analyzed across cultivars and conditions. First, pericarp D-values did not vary as much as those in the columella and locular regions . Second, there was a weak correlation between MRI derived values for the pericarp and the physical changes caused by cold, visible on the pericarp e.g., poor color development, pitting and decay as reported by the CII data . In contrast, there was more synchrony for the columella and CII which is similar to the data published by Tao et al. . Surprisingly, the locular fraction showed a similar r-value to the pericarp when CII was considered. Therefore, other mechanisms besides the increased water mobility we were able to detect under the experimental conditions used, may have a higher contribution to the development of chilling induced external symptomatology. Third, different D-values were recorded in the three tissues as ripening progressed: they decreased in the pericarp, increased in the columella and were unchanged in the locular tissue , exemplifying the unique response of each tissue-type.

Fourth, MRI could only detect changes after transfer of chilled fruit to room temperature. Loss of membrane selective permeability due to a cold-induced membrane phase transition was not sufficiently advanced to produce detectable increases in free water mobility during cold storage. This supports the view that, increased membrane permeability is unlikely to be one of the earliest events in PCI response, but occurs at a significant rate during rewarming .Fruit undergoing PCI normally exhibit a transitory burst of CO2 when transferred from chilling to room temperature, which acts as a reliable marker for the early stages of cold injured tissue . If AZA-treated fruit show differences in respiratory activity after cold stress compared to the water-treated fruit, this could beindicative of an effect of methylation on PCI. Different responses were observed across varying cold stress in ‘Micro-Tom’ and ‘Sun Cherry’ and are described in turn.AZA affected respiration in fruit stored at both cold and control temperatures across the rewarming period. After 21-days at 2.5°C, AZA led to a lower respiratory rate compared to the water-control fruit during reconditioning , suggesting that AZA might moderate chilling injury in ‘Micro-Tom’. In contrast, at 12.5°C, AZA-treated fruit had higher respiratory rates compared to the water-treated fruit after storage . This effect was likely a result of accelerated climacteric respiration caused by AZA-accelerated ripening . AZA may have varying effects in different conditions, which is consistent with the fact that methylation patterns are diverse across developmental stages with various environmental stimuli .This cultivar was more susceptible to PCI than ‘Micro-Tom,’ and may show a different response to AZA-treatment. In all experiments, an increase in respiration was observed after transfer of fruit to 25°C following cold storage . Effect of AZA was evaluated across reconditioning period . To understand the effect of AZA on respiratory rates in the absence of chilling stress, fruit held at 12.5°C were examined over the entire storage period. AZA treatment led to higher respiration after 14 days , likely due to accelerated ripening. In contrast, water injected fruit showed increases in respiration later – after 21 and 28-day storage . This may be due to a ‘delayed’ climacteric response relative to that in the AZA-fruit. AZA affected the respiratory activity of post harvest chilled fruit after reconditioning. As expected, fruit stored at 2.5°C exhibited a higher respiratory burst than those held at 5°C, while it was minor in fruit at the control temperature , indicating severe chilling injury at lower temperatures . Unlike ‘Micro-Tom’, AZA had no effect on ‘Sun Cherry’ fruit exposed at 2.5°C for 21 days or less, nor fruit held at 5°C for 7 days . AZA did influence fruit respiration after storage at 2.5 or 5°C for 28 days . Extreme fungal growth on fruit upon rewarming made it hard to further evaluate effect of AZA on respiration in ‘Sun Cherry’ .Plants have been transported around the world for centuries, as agricultural commodities, ornamental species or inadvertent contaminants of imported materials. Naturalized plants are those that have spread out of cultivated areas, including gardens, into more wild areas, and invasive plants are the subset of naturalized species that cause ecological or economic harm. In general, only a small proportion of plants introduced into a new region have been invasive plants.

Nomad Bioscience has reported successful substitution of the agroinfiltration step with “agrospray,” a technique in which a suspension containing the Agrobacterium inoculant is admixed with a small amount of surfactant and sprayed onto the leaves of host plants. This approach eliminates the necessity to grow plants in containers , a requirement imposed by the mechanics of the vacuum infiltration treatment in current procedures. Concomitantly, it also eliminates the cost of setting up and operating commercial-scale vacuum chambers, robotic tray manipulators, biomass conveyer systems, and so forth. Thus, this new approach should enable large-scale field inoculation of plants with agrobacteria and the production of bio-logics with more favorable economics. While we modeled the costs of producing cellulases via the agrospray approach, the sheer volume of enzymes needed for commercial-scale cellulosic ethanol processes necessitated a large investment in inoculum production infrastructure, including multiple fermentation trains and associated processing equipment. Further, the most efficient method of inoculating large areas was by aerial spraying, a procedure that not only entailed higher cost but that would also face regulatory uncertainties over spraying GM bacteria. We opted instead for an alternative model using trans genic N. tabacum plants, each line of which carries an ethanol-inducible gene for one component enzyme of the cellulase complex. Synthesis of the cellulase is triggered by application of a dilute solution of ethanol onto the leaves,grow table a process that has been demonstrated in small scale using a double-inducible viral vector.

We assumed that the dilute ethanol solution would be applied via ground irrigation systems that are currently used in agricultural practices, instead of aerial tankers. It was also assumed that the ethanol would be taken off as a side stream from the associated ethanol production facility that uses the cellulase enzymes. In so doing, we obviated the need to produce multiple inocula of GM bacteria and deliver them via aerial spraying. We were also able to model higher biomass density as well as higher expression yields of the enzymes in planta. These changes resulted in multiple economic benefits and were therefore adopted in our calculations.Issues that are important in PMP, such as mammalian-like glycosylation or other post translational modifications, high purity, or specific formulation, are not relevant in the manufacture of cellulases and hence we modeled the use of conventional Nicotiana species in the production of the several enzymes necessary for complete saccharification of feedstock.In this case study, we modeled the use of stable transgenic N. tabacum varieties, each modified to express one cellulase protein upon induction with dilute ethanol. The process is based on inducible release of viral RNA replicons from stably integrated DNA proreplicons. A simple treatment with ethanol releases the replicon leading to RNA amplification and high-level protein production. To achieve tight control of replicon activation and spread in the non-induced state, the viral vector has been deconstructed, and its two components, the replicon and the cell-to-cell movement protein, have each been placed separately under the control of an inducible promoter.

In greenhouse studies, recombinant proteins have been expressed at up to 4.3 g/kg FW leaf biomass in the ethanol-inducible hosts, but seed lines for field application have yet to be developed. In our modeling, we assumed that each transgenic line would have been already field tested and available for implementation. We also assumed that large-scale stocks of each transgenic seed would need to be produced and have included this unit operation in our cost calculations. Because cellulases are needed in different ratios to effect saccharification of different feed stocks, we assumed that seeds would be mixed at the appropriate ratios and that the seed mixtures would be planted directly in the field. At maturity, what one would expect is a field of plants representing all the needed cellulase classes in the appropriate ratio for the intended feed stock. The current method of hydroponic cultivation of seedlings for transplantation to open fields, a common commercial tobacco cultivation practice to ensure germination and plants with good leaf size and quality, was substituted by direct seeding for more favorable economics. For example, traditionally tobacco may be grown at 12,000–16,000 plants/ha depending on variety. Higher-density seedling production for nontraditional uses of tobacco has been reported, targeting planting densities of over 86,000 plants/ha. While transplanting ensures germination and quality, there is an economic limit to the scale at which it can be deployed with highly cost-sensitive AI, leading to interest in direct seeding practices. Experimental high density cultivation studies via direct seeding have reported 400,000 to over 2 million plants/ha and biomass yields exceeding 150 mt/ha. Our modeling included these higher-density practices to determine economic impact.In contrast to typical PMP products, the cellulases would not be extracted after accumulation; rather, the plants would be mechanically harvested and transported to a centralized facility for silaging and storage. Since the cellulase enzymes need to be continuously supplied to the saccharification process in the bioethanol plant and the harvested tobacco is only available for a limited period during the year, the silage inventory would increase during the tobacco-harvesting period and would decrease during the fall/winter.

Cellulase activity in the ensilaged biomass is expected to be stable during the off-season storage. For feedstock conversion, cellulase-containing biomass would be mixed with pretreated lignocellulosic feedstock under controlled conditions to effect saccharification. Although not considered in this economic analysis, this feedstock replacement could also reduce corn stover feedstock requirements and associated costs. After separation of solids, the sugar solution would be fermented conventionally into ethanol, followed by distillation. The overall process we modeled is based on the US National Renewable Energy Laboratory process described by Humbird et al, with substitution of fungal cellulase production in the NREL model by the cellulases stored as silage described herein. Design premises for this process, specific assumptions used in modeling,ebb flow table and the resultant cost calculations are presented .Table 3 shows the total capital investment and annual operating costs for the plant-made rBuChE facility at an expression level of 500 mg/kg FW plant biomass . The annual operating costs are shown with and without facility dependent costs to simulate a new facility and use of an existing facility, respectively. Table 4 shows the resulting rBuChE cost per dose for both cases. Table 3 shows the breakdown of the capital investment and operating costs for the plant-made rBuChE and indicates that the unit production costs are estimated to be about $234/dose if facility dependent costs are not included in the annual operating costs or about $474/dose if these costs are included. Most of the capital cost and a significant portion of the operating costs are associated with the recovery and purification of rBuChE. Our base case assumed rBuChE expression of 500 mg/kg FW because that is a target expression level in ongoing research at several institutions. If a currently achievable level of 100 mg/kg FW is used instead , the costs increase to $1,210/dose and $430/dose when including and excluding facility dependent costs, respectively. In any scenario examined, the production costs in plants are significantly lower than the estimated production costs for blood-derived BuChE . We recognize that additional modification or formulation of the plant-produced enzyme might be necessary or desirable prior to adoption for human use and that such additional modifications would increase the cost of the AI. For example, Geyer et al. reported improved pharmacokinetics of PEGylated plant-produced BuChE relative to the nonmodified enzyme. However, because consensus on the preferred options for modification has not yet been reached, we omitted these additional steps from our calculations.The following premises and assumptions were used for evaluation of cellulase bio-manufacturing in open fields. Due to the fact that this process is specialized and due to the scale and input requirements of a modern bio-fuels operation, our analysis included the construction of a new, dedicated manufacturing facility to provide the required cellulase enzymes for a large scale cellulosic ethanol facility . Figure 4 shows the process operations required for cellu lase enzyme production on a per-batch basis. The flow sheet on the top shows the blending tank needed for preparation of the ethanol induction solution to be applied in the field, and the flow sheet on the bottom shows the transport and storage operations following harvest of the transgenic tobacco.Table 6 shows the total capital investment and annual operating costs for the production of 2.87 million kg of cellulase enzymes per year at an expression level of 4 g cellulase/kg FW tobacco biomass and a plant density of 130 metric tons of biomass per hectare per year.

The table also indicates the corresponding costs obtained from the JBEI model for fungal fermentation-based production of approximately the same amount of cellulase enzymes per year . For the base case study, the plant-based system results in a >30% reduction in unit production costs for the cellulases as well as an 85% reduction in the required capital investment. For the plant-based cellulase production system, the major contributors to the unit production cost were the costs associated with tobacco cultivation , the costs associated with ethanol spraying , followed by the costs associated with ethanol dilution, transporting and storage , and seed costs . The differences in total capital investment and annual operating costs for the two cellulase production platforms are not surprising, since the fungal fermentation area alone requires twelve 288,000-L fermenters along with the seed train necessary to provide the inoculum for the production fermenters. The differences between the two systems would be expected to be even larger if the total capital investment included additional factors for associated piping, instrumentation, insulation, electrical facilities, buildings, yard improvements, and auxiliary facilities because these would be reflected in the facility dependent component of the annual production costs. Figure 5 shows the effect of biomass density on the unit production costs for cellulase enzyme using the ethanol induced tobacco system and indicates, as expected, that the cost of goods decreases as tobacco biomass density increases. In agronomic studies with field-seeded tobacco cultivated at high density, biomass yields exceeding 150 mt/ha have been achieved; higher field densities may be possible with selected varieties and specialized agronomic practices.At the eastern boundary of the Sahel lies the Greater Horn of Africa , a region of northeastern Africa with highly food-insecure countries that are particularly vulnerable to interannual and decadal swings in precipitation totals . Both the GHA and Sahel receive the bulk of their precipitation during the June–September boreal summer season. These regions experienced a significant decline in JJAS precipitation during the 1970s and 1980s due to anomalous warming of the South Atlantic and Indian Oceans and subsequent shifts in moisture transports and upper-air flow . Following the 1980s, warming in the North Atlantic appears to have caused a precipitation recovery throughout much of the Sahel . In the GHA, however, the extent of post-drought recovery is unclear. Recent studies suggest drying over GHA may have continued into the 2000s , but the cause of this potential drying and decoupling of precipitation trends in the GHA from those of the Sahel remains unexplored. Previous studies have identified a variety of drivers of JJAS precipitation and drought in the GHA. While JJAS wind trajectories toward the GHA are quite variable from year to year, the rainforest region of the Congo Basin appears to be the main moisture source to Sudan and much of the Ethiopian Highlands, and the Indian Ocean is the main moisture source for eastern portions of Ethiopia and Kenya . South of the center of the North African surface low, deep convection and enhanced moisture transport from the south and west fuel monsoonal precipitation during JJAS in the Sahel and GHA. Historically, for both the Sahel and GHA, the primary driver of interannual and longer-term variability of summer precipitation is the north-south displacement of jets, zone of maximum convection, and southern boundary of the thermal low over northern Africa , all of which appear to be influenced by tropical sea-surface temperature anomalies . Some research also suggests that increased tropospheric aerosol concentration works to suppress the northward migration of the JJAS rainbelt into northern Africa, potentially reducing precipitation in the GHA . Further, relationships exist between the strength of the Indian monsoon and GHA precipitation . Overall, drought in the GHA tends to occur when there is a reduction in the amount of moisture reaching the GHA from the tropical Atlantic Ocean and Congo Basin region.

From this information, we examined fluctuation patterns that could exist specific to a region or crop category and linked the information with the main source of the lowest profit. Information presented for this topic was obtained from Questions 9, 10, and 11 . Risk Management examined farmers’ perceptions of risk and, in particular, the extent to which risk management tools are available and used. Respondents were asked to rank ten risk sources in order of importance and eight risk management tools in the order of preference. For each risk management tool, the survey also asked about its availability and whether it had been used by the farmer. Also included was information on their receipt of government disaster payments or loans. This section used data from Questions 12, 13, and 14 . Crop Insurance was one of the risk management tools covered in the previous section, but it was then given more extensive coverage. This section summarized in formation on respondents’ history of crop insurance purchases, reasons why they did or did not purchase crop insurance, and suggestions for improving the role of crop insurance. Information presented includes the mean rank ing and distribution of ranks. The relevant survey section for this data was Questions 15 through 22 . Financial Characteristics deals with off-farm income, gross agricultural sales, assets, and debts to provide the distributions of these variables and examine the existence of any systematic distribution patterns. Questions 23, 24, and 25 in the survey were relevant to this section.

Crop diversification is well recognized as a risk management tool . However, little information is available concerning the extent of diversification or the mix of crops used in diversification by horticultural producers. As a risk-reducing tool,mobile vertical farm crop diversification plays a role in pricing crop insurance and is likely to be incorporated as a discount factor in future crop insurance premiums. To implement degree of diversification into the crop insurance premiums structure, decision makers need to know the extent to which crops have been diversified. This section sheds some light on the issue. Figure B1 shows the share of fruit/nut and vegetable farmers who grew a single crop. Seventy percent of fruit/ nut farmers were single-crop growers as opposed to 26percent of vegetable farmers. This implied that crop di versification was more common for vegetable growers than for fruit/nut growers, which was consistent with our expectation that diversifying into multiple crops is more manageable for annual crops than for perennial crops. The tendency toward single-crop production, however, varied by crop. For example, for fruits/nuts the share of single-crop farmers ranged between 35 and 83 percent, depending on the crop. As shown in Figure B2, grapes were most commonly a single crop , while stone fruits were least frequently so . Table B1 presents the diversification patterns and mean acreages. The patterns and extents of diversification for fruit/nut and vegetable farms were very different. Of the 30 percent of fruit/nut farms that were diversified, most were diversified with other fruit/nut crops. However, of the 74 percent of diversified vegetable farms, only 26 percent were diversified using other vegetable crops; 48 percent were diversified with crops in other categories. This indicated that fruit/nut farmers rarely diversify into other crop categories and that diversification across crop categories is more common for vegetable farms, particularly with field crops.

Furthermore, even among the growers who diversified within their own crop category, the scope of diversification was smaller for fruit/nut farming, as indicated by the average number of crops, 2.56 for fruits/nuts and 3.59 for vegetables . Table B1 also presents mean acreages. Note that the acreage figures in the table are for land that was planted in fruits/nuts or vegetables only. We did this to exclude often extensive field-crop areas and to examine the scale of farmers’ operations for their primary crops relative to various patterns of crop diversification. A cursory observation of the acreage figures indicated that primary crop acreage increased with crop diversification for both fruits/ nuts and vegetables . Also, farms that diversified within a crop category were relatively large. We revisit this issue with more detailed vegetable data later in this report. Table B2 shows the pattern of crop mix for fruit/nut farms, which are diversified predominantly with other fruit/nut crops. The table lists the two types of crops most commonly used for diversification in each subcategory. Judging by the percent of farmers, growers of berries, citrus, stone fruits, and tree nuts have made substantial use of same-category crop diversification. For tree nuts and stone fruits, the diversification patterns were symmetric with substantial cross-diversification between the two groups. The diversification trends for citrus and tropical crops were interesting. While 66 percent of sampled tropical crop growers diversified with citrus, only 28 per cent of citrus farmers diversified with tropical crops . We now turn to vegetables. Table B3 summarizes the pattern of diversification for farmers who grew only vegetables and shows the distributions of those farmers by the number of vegetables grown. While half of the vegetable-only farmers produced a single crop, 9 percent produced more than six different vegetable crops.

When we shifted from all vegetables to the subcategories, diversification patterns varied considerably. This was illustrated with Groups V2 and V5, which showed the highest and lowest levels of diversification. Table B3 also provides mean vegetable acreages for vegetable-only farmers. There was a tendency for farmers with more acres of vegetables to grow a larger variety of vegetable crops, suggesting that large-scale commercial farms engaged in more diversified vegetable production. In other words, the “scope” of diversification was positively related to the scale of the operation. This report does not include a discussion of crop diversification for ornamental crops because of a lack of information. The finest level of diversification we could investigate with the data for ornamental crops was diversification patterns across the three subgroups in the category: floriculture, nursery products, and Christmas trees. Our data indicated that ornamental growers rarely diversified across these groups. Organic farming information is summarized in Table B4. The table combines acres of “organic” and “transitional-organic” plantings and presents the combined area as “organic acreage” . Table B4 shows that 14 percent of vegetable growers practiced organic farming, compared to 6 percent of fruit/nut growers, although organic fruit/nut farms were more numerous. Most organic farmers also grew conventional crops and, on average, they devoted more land to conventional production than to organic production.This section summarizes the survey results on types of output use , marketing channels, and types of operations . Figure C1 shows the distribution of farmers by type of use for their fruits/nuts and vegetables . The two types, “mainly fresh” and “mainly processing,” were defined to include cases in which more than 80 percent of output volume was designated to the listed use. For fruits/nuts,vertical farming racks 71 percent of farmers were characterized as mainly processing and 23 percent as mainly fresh. These figures were almost re versed for vegetables—67 percent of vegetable farmers specialized in fresh-use crops and 26 percent in processing-use crops. For both fruits/nuts and vegetables, only 7 percent of farms supplied both fresh and processing uses . This implied that production of fruits/nuts and of vegetables in California tends to be specialized for either processing or fresh use.4 Also, these figures were consistent with the common observation that, for both vegetables and fruits/nuts, specific uses dictate the varieties grown. For example, Cling peaches are typically destined for canning and the Roma variety of tomatoes is usually made into paste. Relevant marketing channels are determined by whether the crop goes to the fresh market or for processing since the two uses require different post harvest handling techniques. Once harvested, processing crops are shipped directly to a processing plant. Fresh-use crops are usually sorted, packed, and refrigerated before being shipped to wholesale or retail buyers. This implies that specific marketing channels emerge to accommodate the post harvest handling required for each use. Figure C2 lists the marketing channels available for processing crops and the share of farms that used those channels. For fruits/nuts, marketing cooperatives and contracts with a processor were the most widely used marketing channels, accounting for 90 percent of the farms. How ever, for processed vegetables, marketing cooperatives played a relatively small role. Instead, contracts with a processor arranged at a predetermined price predominated.

While contracts with processors were an important marketing avenue for both the fruit/nut and the vegetable categories, the patterns of pricing arrangements with processors were distinctly different. For fruits/nuts, contracts with and without predetermined prices were almost equally important , whereas for processed vegetables, contracts with processors were mostly arranged under predetermined prices .Given the importance of processing use for fruits/nuts, we further investigated their marketing channels by disaggregating the category and looking at subgroups of the crop, as shown in Table C1.Cooperatives were especially important for citrus crops and tree nuts , and predetermined price contracts were particularly prevalent for grapes . The bulk of the grape growers produced wine grapes and, according to a recent survey, 90 percent of wine grape growers in California have either written or oral contracts with wineries . Overall, the data in Table C1 underscored the prevalent role of contracts in the processed fruit/nut industry. For vegetables, crop-specific marketing channels did not deviate much from the overall marketing pattern reported in Figure C2 and disaggregated information is not presented here. Post harvest handling is a crucially important component in marketing fresh-use crops. Thus, large commercial growers sometimes integrate field production with post harvest packing and shipping activities under the same owner. These growers are often referred to as grower/shippers . Table C2 indicates that 9 percent of the fresh-use growers who responded to the survey were grower/shippers. The vegetable industry had the largest proportion of grower/ shippers ; next was the ornamental industry , followed by fruit/nut operations . There is no parallel notion of post harvest handling for ornamentals and, thus, the remainder of the grower/ shipper discussion mostly relates only to fruits/nuts and vegetables. Grower/shippers operate on large scales and usually supply large-scale buyers such as grocery chains and mass-merchandisers , often at a pre-negotiated price. Negotiating the price before market conditions are known has important implications for price risk. Even though the net effect of prefixing the price depends on the structure of market power, a contract with a fixed price tends to reduce price risk. Our survey indicated that 51 of 75 fruit/nut grower/shippers sold, on average, 85 percent of their products at a predetermined price. However, for vegetables, the data indicated that only one grower/shipper sold product at a predetermined price.While grower/shippers typically supply their crops directly to large retailers or wholesalers, the grower-only group tends to market its crops through contracts with shippers or other means. As shown in Table C3, the two major outlets for fruits/nuts are marketing cooperatives and independent shipper/brokers. On the other hand, for vegetables, cooperatives have a minor role, and major roles are played by three marketing channels: direct marketing to consumers , independent shipper/brokers, and direct marketing to commercial buyers. Comparing marketing channels between processed and fresh-use crops, two observations stand out. With no single dominant marketing channel, fresh-use crops are generally marketed through various channels. Nevertheless, for fruits/nuts, the importance of cooperatives is significant—cooperatives are widely used in marketing both fresh and processed fruits/nuts.Production risk is closely linked to yield risk . As a way to measure yield risk, fluctuations in yields were investigated. The survey asked for information on actual annual yields from 1997 to 2001, and complete five-year yield data were obtained from about 45 percent of the respondents . Using the five-year yield data, average yield deviations in percentage were calculated and are reported in Table D1. To arrive at average yield deviations, for each observation we first calculated the simple average using the five-year yields. The percentage deviation from the average yield was then computed for each year . The all-year average deviation was the average of the five-year yield deviations. Table D1 presents the sample mean of all-year deviations by crop category and by crop-specific group. 

If the Q1 and Q3 are quite tight, then the quantification results are quite reliable. If the reciprocal labeling gives similar results, such as SR45, then the quantification should be reliable, even if the total peptides from this protein are only a few. We recommend at least three to four biological experiments be done for quantification, including at least one reciprocal labeling experiment . After the quantification is done, the users can evaluate the quality of the quantification of each protein and peptide of interest. Protein Prospector provides interactive feedback during the quantification process to allow for manual validation of the quantification results or visual assessment of what went wrong in case the ratio is incorrect. For protein quantification, a tight range between Q1 and Q3 often indicates the quantification is reliable. In cases where the range is big and the protein itself is of interest, then users can use the Cosine Similarity score to determine the quality of the matches or manually check them.Once the peptide sequence is identified, the elemental composition of the peptide is generated based on the peptide sequence. The CS score, similar to the Isotope Dot Product “idotp” product used in Skyline , automatically measures the similarity between the experimentally measured isotope pattern and the calculated pattern using the M, M+1, M+2 peaks, thus reducing manual checking time by auto-flagging the contaminated peaks .

The CS score ranges between 1.0 and 0.0 and can be determined by measured peak intensity or area. Figure 6A shows the pair have both good CS scores and the L/H ratio of this peptide is close to the median number of the protein. Figure 6B shows one peptide in the pair of another peptide from the same protein gives a lower CS score ,grow bucket and thus the L/H ratio of this peptide will produce an outlier ratio. Importantly, Protein Prospector takes account of labeling efficiency when calculating the CS score, as low labeling efficiency changes the isotope pattern quite dramatically. It should be noted that the CS score will be less accurate when the peak intensity of the peptide is very low. Users can create a cache file when submitting the quantification in Search Compare. This stores the data required to regenerate the Search Compare report in a JSON file. The cache function is quite useful for various reasons: when the user needs to retrieve the data or manually check the data, there is no need to re-calculate the quantification, which can take many hours for a large dataset. With the cache file, the reports come up quickly for a few seconds rather than hours; Often it is hard to display an HTML peptide report when many proteins or peptides are quantified. The cache function can allow visualization of such reports easily. This workflow allows users to report quantification of thousands of proteins and is applicable to the quantification of the total proteomes, sub-proteomes, and immuno precipitated samples . During the extraction of elution profile of every peptide identified, Protein Prospector averages together scans over a time window but doesn’t fit the peak shape to a Gaussian function. Therefore, each identified peptide/protein will be quantified and none gets discarded due to failing the scoring threshold for fitting the Gaussian function. To get high quality data, we recommend to get 97% or above labeling efficiency to achieve higher ID rates in 15N samples, so more proteins will be reproducibly identified and quantified between different replicates. Data acquired on high resolution and high accuracy instruments will also improve the quality of the dataset. A systematic normalization is normally required before comparing results between different experiments , as the samples are rarely mixed at exactly 1:1.

One choice is to use the median number of all the quantified proteins, or median number of top one hundred abundant proteins . Alternatively, users can use housekeeping proteins that are assumed to not change for normalization. Statistical analysis of quantification data on three or more replicates is advised. Users need to determine how to apply statistical analysis on the data using a separate tool. Benjamini-Hochberg multiple hypothesis test has been used to determine significant regulated PTM peptide groups in 15N metabolic labeled samples . A standardized statistic pipeline for protein quantification is still lacking, particularly a pipeline that can leverage quantification ratios of each peptide from a protein. Our current workflow uses a median value, which takes advantage of the quantification ratios of each peptide but is less affected by outliers than using the mean. However, the statistical power utilizing quantifications from these multiple peptides from single protein has not been explored and awaits development in the future. A targeted quantification strategy is recommended for further analysis of proteins of interest because this provides more accurate quantification and is less likely to have missing values, particularly in the 15N labeled samples . In addition to targeted analysis, data-independent acquisition can also be utilized, which can be done in label free samples or combined with 15N metabolic labeling in the future. DIA benefits from having few missing values, but more efforts will be needed to deconvolve the mixed MS2 spectra in DIA datasets. 15N metabolic labeling has been utilized in studies of analyzing protein synthesis and degradation . These studies are based on incomplete and often low incorporation rates which result in very broad satellite peak distributions and cause 15N labeled peptide isotope clusters to overlap with 14N labeled peptide clusters. As Protein Prospector doesn’t deconvolve the 15N distribution from the 14N distributions , the presented workflow will not provide accurate quantification in this type of study. On the other hand, for chase studies that analyze the assembly kinetics in vitro , the presented workflow can be applied because the proteins involved have a very high labeling efficiency.

This workflow can be also applied to the quantification of post-translational modification with a slight modification. The users will include related PTM search parameters into data search. Instead of reporting median number at protein level, ratios from each peptide are reported and then compared across different replicates. Trait analysis, especially genome-wide trait analysis, is centered on how genetic variation gives rise to phenotypic variation. This type of analysis relies on statistical methods and tools to perform association mapping between causal genetic variants and resulting phenotypes, which can determine the heritability of a trait at a subset of genetic variants and delineate regions of the genome that control the trait, thereby providing markers that can be utilized to accelerate breeding by marker-assisted selection. Because of the great success of genome-wide association studies , hundreds of SNPs conferring genetic variation of complex traits have been identified and reported. However, the genetic structures of most traits remain unexplained, as associated SNPs detected from GWAS explain only a small fraction of heritability and a much smaller percentage of the total phenotypic variance. This is mainly because a number of these studies employed only additive models that fail to account for epistasis,blueberries container or the interaction between multiple loci and the environment. Xu et al. proposed a new linear mixed model for mapping quantitative loci by incorporating multiple polygenic covariance structures. Based on this model, a pipeline for estimating epistatic effects was developed to com prehensively estimate additive effects, dominance effects, and interaction effects between multiple genetic loci. PEPIS allows analysis of genome-wide genetic architectures, including genotype interaction effects , and can thereby explain more than 80% of phenotypic variance. Compared with standard GWAS tools that consider only additive effects, the PEPIS pipeline is equipped with a more complex polygenic linear model that can explain more phenotypic variance. However, neither of these methods can explain nearly 100% of phenotypic variance, as neither considers the interaction between genotypes and environments . Today, the predominant thinking in biology is that the orchestrated expression of many genes in different environmental conditions affects the transcriptome, proteome, and metabolome to produce a final observable phenotype. Recent work in Saccharomyces cerevisiae suggests that GxE can occur at the individual locus level and the group level for multiple loci, leading to environment dependent epistatic interactions. Although Muir et al.conceptualized the partitioning of GxE into two possible inter action types, our mathematical understanding of the genetic and molecular mechanisms by which GxE collectively gives rise to phenotypes is still incomplete. The central dogma of biology is that the genome, tran scriptome, proteome, and metabolome are cascading and connected to the end phenome. The development of life science technologies enables transcriptomic, proteomic, and metabolomic events to be analyzed in detail within the same biological system, allowing the systematic study of a complete biological system.

Out of all the omic data from the same biological system, genomic data generally remain constant across environments, although the same genotype subjected to different environments can produce a wide range of phenotypes by triggering the expressions of different genes, downstream enzymes, and metabolites. Most current association methods and analysis tools perform associa tion mapping based on fundamental relationships between DNA sequence variation and phenotypic variation without addressing environmental variation. GxE can be understood by observing and measuring the expression of genes or metabolites. Harper et al.developed an associative transcriptomic approach to study complex traits in the polyploidy crop species Brassica napus by correlating trait variation with the quantitative expression of genes and sequence variation of transcripts, with the consistent physical positions of the two kinds of associative markers allowing the identification of high-confidence transcription factor candidates. However, their method is based on a pure additive model only, and they make no mention of interaction effects between biomarkers or their contribution to phenotypic variation. To overcome the limitation of standard GWAS that fails to consider the GxG and GxE effects, we extend associative geno mics and transcriptomics into a broader associative omics by systematically integrating all available omic data into one analytical model. Here we propose a new LMM and describe the development of a pipeline for analyzing traits through ome-wide association studies to implement the model. The proposed LMM considers not only the additive effects of each biological marker but also the interaction effect of each marker pair. The marker pairs’ interaction effect introduced here corresponds to two-dimensional association mapping, which is complementary to one-dimensional association mapping in regular GWAS. Consequently, the proposed model and PATO WAS pipeline are not limited to GWAS for genotype-to phenotype mapping ; instead, they are capable of per forming multiple types of ome-wide association studies, such as transcriptome-wide association studies for transcript-to phenotype mapping and metabolome-wide association studies for metabolite-to-phenotype mapping . We submit a rice recombinant inbred line dataset with three omics markers and two agronomic traits to PATOWAS for comprehensive analyses of associative omics. The results demonstrate that our proposed LMM and the pipeline PATO WAS can effectively address the GxG effect and the GxE effect, perform multiple-level associative omics in one platform, and innovatively provide a systems biology view into the traits analyzed.We aimed to systematically integrate multiple associative omic results to provide more biological insights into the phenotypic traits to be analyzed. We first collected a dataset of 210 rice RILs geno typed with 1619 marker bins, profiled with 22,584 transcripts and 1000 metabolites, and phenotyped with two agronomic traits . The phenotypic traits were yield and thousand grain weight , and the omic quantitative markers were bin based genotype data, Affymetrix RNA microarray-based gene expression data, and mass spectrometry-based profiling of metabolite abundance data. We presumed that expressed tran scripts, proteins, and metabolites are prone to vary when sub jected to the environments, while the genetic variants are considerably stable. Therefore, compared with genome-wide genotypic data, we further presumed that measured gene expression and metabolite abundance contain both gene and environment information and expect that associative transcriptomics or metabolomics could explain more phenotypic variance . Motivated by our consideration of genetic epistasis and our desire to explain more phenotypic variance, we next proposed astatistical LMM that considers not only the additive effects of the two components, the lower the residual component and the each marker variant but also the interaction effects of each more phenotypic variance can be explained by the model.

Crucially, fast initial fern growth in the medium-textured soil, likely due to higher nutrient content and/or lower arsenic phytoavailability, led to a decrease in effluent flow and therefore arsenic leaching, as transpiration exceeded water application. In this soil, the fern required less energy to acquire nutrients, released less arsenic from soil via nutrient scavenging, and produced greater biomass such that arsenic concentrations in biomass were lower. The increase in mass of arsenic accumulated from 11 to 21 weeks, coupled with lower final biomass of mature and young compared to senescent fronds, shows that arsenic accumulation continued even as growth slowed. This arsenic accumulation could be due to increased nutrient scavenging associated with drought stress.The increase in arsenic concentrations in leachate in the presence of ferns growing in the medium-textured soil reveals the importance of rhizosphere processes to arsenic release for uptake and leaching. We found that arsenic depletion from the medium-textured soil was the greatest in surface soil where P. vittata roots are primarily located . Moreover, in both soils we calculated that arsenic concentrations in rhizosphere pore water must be greater than those in bulk soil pore water,square planter pot because assuming arsenic concentrations to be the same in rhizosphere and bulk pore water indicated a discrepancy between arsenic intake through transpiration flux, and fern arse nic content. Processes other than mass flow of soluble arsenic from bulk soil to roots must be important for arsenic uptake .

If nutrients availability in soil is lower than P. vittata demand, P. vittata could employ nutrient-scavenging processes that release iron, phosphorus, and therefore arsenic from soil into pore water in the rhizosphere , increasing pore water arsenic concentrations locally to potentially very high concentrations. We suggest that the majority of the arsenic taken up into P. vittata was mobilized directly in the rhizosphere, similarly to others who found greater desorption of cadmium in cadmium hyper accumulator rhizospheric compared to bulk soils . We hypothesize that higher diffusivity due to greater connected pore space in the medium-textured soil could lead to transport of the arsenic re leased in the rhizosphere to the bulk soil, where it is then available for leaching. Similarly, rhizosphere DOC could be transported to the bulk soil and promote release of arsenic. However, in the coarse-textured soil characterized by lower porosity, larger pores, lower saturated fraction, and pre dominantly advective flow, arsenic and DOC released in the rhizosphere did not contribute to bulk leachate arsenic concentrations and, conversely, arsenic in the bulk pore water was not as accessible to the plants.We suggest rhizosphere arsenic mobilization is a byproduct of nutrient scavenging processes, particularly iron-scavenging in the medium-textured soil, where we found higher iron concentrations in ferns and in root zone pore water. Specifically, arsenic release from soil could be coupled to phosphorus and iron release from soil iron oxide minerals . Release processes could include ion exchange, ligand-enhanced dissolution, and reductive dissolution , likely tied to release of root exudates from P. vittata roots . We found primarily oxidized arsenic in our well-drained rhizosphere soil, suggesting processes including ion exchange and ligand-enhanced dissolution, likely coupled to rhizosphere DOC, are more important than reductive dissolution, similarly to in the Pine rhizosphere . Alternately, the predominance of oxidized species could indicate P. vittata preferentially took up reduced species, leaving oxidized species behind. We found evidence of reductive processes in the rhizosphere, with up to 41% of the arsenic present as arsenic in rhizosphere soil, up to 100% of the arsenic present as arsenic on and within roots, and iron phases in rhizosphere soil, suggesting reduced arsenic and iron could play a secondary role in arsenic release and uptake.

A high fraction of surficial arsenic could indicate transport of arsenic toward the root and accumulation in the rhizoplane, with slower uptake of arsenic enriching arsenic relative to arsenic on the root surface. The presence of arsenic on the root surface could also indicate efflux of arsenic from roots, which has been proposed to be a secondary tolerance mechanism in P. vittata and other plants under arsenic stress . In bulk pore water, bulk soils, and soil aggregates, the predominance of arsenic indicates arsenic can leach under oxic conditions. Arsenic availability for leaching, whether due to soil characteristics or influence of plant growth, is not dependent on reducing conditions. Indeed, arsenic mobility in soil increases at the circumneutral to alkaline pore water pH we observed . Arsenic mobilized as arsenic could be oxidized, perhaps coupled to reduction of the moderately-available soil manganese. Leaching of root derived dissolved organic carbon could also increase arsenic release from bulk soil for leaching.Rhizosphere nutrient acquisition processes have a specific significance in the case of hyper accumulators. Infertile soils could characterize the hyper accumulator ecological niche , such that P. vittata employs scavenging techniques and associates with indigenous AMF to acquire necessary phosphorus and other nutrients. We found Glomus spp. including F. mosseae were present across all treatments whether due to colonization by indigenous mycorrhiza or due to inoculation. In the very low nutrient coarse-textured soil, we hypothesize that extensive use of these scavenging processes cost metabolic energy, locally in creased already high arsenic availability, led to high uptake of arsenic and consequently even more energy expenditure to sequester this arsenic, and ultimately resulted in low biomass containing arsenic at high concentrations. The lack of effect of supplemental phosphorus in the coarse textured soil suggests it is a balance of phosphorus and other nutrients which are required to meet P. vittata nutritional needs. In contrast, in the medium-textured soil, we hypothesize the ferns used less energy to acquire nutrients.

Iron scavenging here was successful, apparently meeting fern nutrient needs and therefore limiting “byproduct” arse nicreleased from soil. Hence, P. vittata growing in the medium-textured soil experienced lower metabolic costs and consequently higher biomass until drought stress limited biomass production. In keeping with evolution under phosphorus starvation conditions , our results suggest P. vittata is less tolerant to extractable phosphorus concentrations greater than that of the medium-textured soil . Fronds of P. vittata growing in its native habitat in China were only 0.08% phosphorus, and ferns including P. vittata had the lowest phosphorus content of any flora group in China . We found phosphorus application delayed fern growth in both medium- and coarse-textured soils,hydroponic nft channel as has been shown for tropical forest ferns , leading to smaller senescent fronds containing lower amounts of arsenic.Our findings suggest that P. vittata is a good choice for remediation at the mesoscale, because arsenic uptake in ferns exceeded cumulative loss by leaching by an order of magnitude, and transpiration limited leaching compared to the absence of ferns. Decreased effluent volumes and cumulative arsenic leaching in both soils in the presence of ferns confirms the critical role transpiration plays in limiting water percolation and leaching of soluble, plant available constituents . The leaching to uptake ratio measured in this mesocosm system is not directly scalable to field conditions. We demonstrate that arsenic leaching during phytoextraction depends on soil characteristics, fern growth, and water input/evapotranspiration ratios, and therefore must be measured at the field scale. The constant water application required in our column study design could have increased leaching of arsenic relative to field applications. On the other hand, our experimental design could have limited plant growth and therefore nutrient scavenging activities, which we showed can increase arsenic release from soil. Larger biomass under field conditions could increase the influence of the nutrient scavenging geo chemical processes observed here and lead to increases in arsenic mobilization for both uptake and potential leaching, explaining the excess loss of arsenic from soil observed under field conditions . Counter intuitively, because we showed that P. vittata continued to phytoextract arsenic under drought conditions from the medium-textured soil to effectively limit arsenic leaching, phytoextraction could be best suited for dry soils with lower arsenic availability. Here, even though frond arsenic concentrations were an order of magnitude greater in coarse-textured soil ferns, mass of accumulated arsenic in coarse-textured soil ferns was only 1.2 to 2.4 times that of medium-textured soil ferns, while leached arsenic was also greater in coarse-textured soil, due to the lower biomass and lower transpiration. Alternatively, phytostabilization with species with high transpiration rates but lower iron demand could limit biotic and abiotic arsenic leaching.Such substrates typically have an inorganic and organic component . The organic component provides high porosity, low bulk density, and nutrient retention , which makes Sphagnum peat moss a strongly suitable option with widespread use .

However, increasing expense and competing uses for peat , impacts of its harvest on wetland ecosystems , including loss of peat bogs as a key global C sink , and its perception as unsustainable have spurred recent investigations of substitutes for peat in soil-free substrates, including biomass waste products such as compost and sawdust . Biochar has been recently proposed as a strong candidate to substitute for peat because of its high porosity, low density and high cation-exchange capacity. Biochar is a carbon -rich material produced by pyrolysis of biomass and has been a major subject of study as a soil amendment in the last decade . In addition to providing high nutrient and water retention, replacing peat with BC could offset or reverse the C footprint of soil-free substrates into a net C sink . Evidence to-date suggests neutral or positive effects of BC use in substrates on nutrient availability and plant growth , though many studies examine additions of BC to peat-based substrates, rather than replacing a substrate component such as peat .Evaluating effects of high BC substitution rates on substrate properties and plant growth is necessary to understand the extent to which BC can replace peat. At low amendment or substitution rates, BC has been found to maintain or improve plant growth as a result of increased nutrient availability , reduced nutrient and water loss , and amelioration of peat acidity , though these effects may be BC-specific due to feed stock and pyrolysis influences on BC properties . However, at high substitution rates, substrate properties conducive to plant growth may be compromised. In particular, the high pH of many BCs could result in BC-substituted substrates with pH values unfavorable to plant growth. For example, pelleted wood BC substitution for peat required adjustment of pH due to the liming effect of the BC . The neutral to alkaline pH of BCs and their liming potential means that BC substitution for peat can increase pH beyond optimum for plant growth in potting media . Explicit eva luation of BC effects on substrate pH and plant performance provides a basis to improve design of BC-based substrates and inform trade-offs in this application of BC . The objective of this study was to determine the effects of BC substitution for peat and substrate pH on greenhouse production, using marigold as a model crop. In the United States, the wholesale value of marigolds plants was 30.3 million USD in 2015 . Softwood BC was substituted for peat in a typical 70:30 peat:perlite mixture at 10%v increments. Since many BCs are alkaline and will increase pH of substrates in proportion to the degree of substitution, the effect of adjusting pH of substrates to typical soil free substrate values was also evaluated. Marigold germination and growth were measured over 9 weeks. We hypothesized that under greenhouse conditions , marigold germination and growth would be more sensitive to BC substitution at higher rates and that this would be due to elevated substrate pH. Additionally, we hypothesized that pH adjustment of BC substrates would increase the extent to which this softwood BC could be substituted for peat without compromising plant growth.Marigold var. ‘Crackerjack’ seeds were sown directly in 0.7 L of substrate pre-fertigated to 100% WHC using 0.5% Hoagland solution in 1.2 L polypropylene pots in a greenhouse at the UC Davis Plant Growth Facility. Pots were arranged 18 cm apart in a completely randomized block design with four replicates per substrate-pH treatment . Pots were drip fertigated with 0.5% Hoagland solution at 66 mL d−1 for weeks 1–6 and 99 mL d−1 for weeks 7–9.

For many important horticultural crops, ex ports constitute a large share of output, so FTO under IP must include freedom in foreign markets. Since the various IP rights important for plants are administered nationally, an exporter must check FTO separately in each foreign market. In general, the tools of biotechnology are more likely to be patented in just the major markets — such as the United States, Europe and Japan — and less likely to be patented in countries with smaller markets. Uses of bio-technologies specifically for minor crops are less likely to be widely patented in multiple countries than are uses in important field crops. However, as a result of the International Union for the Protection of New Varieties of Plants agreement first established in 1961, PVP systems are widely available overseas for the protection of clonally propagated varieties, and such varieties do tend to be widely registered in multiple countries. Still, not all types of bio technologies, genes or plant germplasm can be protected in all countries. For example, utility patenting of plants is allowed in only a few countries . Beyond these trends, however, there are no hard-and-fast rules as to which technology will be protected in which country, as each inventor decides where to seek protection . As a result, those seeking FTO are confronted by an often bewildering international patchwork of IP rights, where the negotiations needed for a particular transgenic variety can differ significantly each time it crosses a national border.Unless a new transgenic variety is developed by an integrated effort at a large company backed by a broad IP portfolio, a number of different owners — including companies, individuals,ebb and flow trays universities and even governments — will have valid IP claims over the technologies and genetic contents that end up being included in it.

That means there are numerous owners to track down, negotiations to conduct, billable legal time to hire, and multiple royalty payments to administer. The costs and headaches involved in working out “who owns what” and “who owes what to whom” can balloon into what economists call the “tragedy of the anti commons” and render the development process unfeasible.Given the smaller markets involved, there is less incentive in industry to consolidate IP portfolios around horticultural crops. Also, not one of the public-sector organizations or their typically smaller commercial partners in horticultural crop development has a complete IP portfolio in plant biotechnology.When technologies are patented, it is often not clear who currently owns particular aspects of each technology. This uncertainty is cleared up in the courts through patent interference cases, where attorneys and scientists under take intensive “surveying” of the “property lines” between the patents and technologies in question. Some times these cases drag on for years, keeping key technologies in legal limbo and the R&D community guessing as to who is the rightful owner. Yet, for most registered patents there is no such scrutiny. As a result, the boundaries for a considerable expanse of technological territory are not clearly demarcated, creating considerable uncertainty as to when a new application could be considered to be infringing or “trespassing.” In horticultural crops, the lack of clarity about the scope and validity of patent claims is especially important. Because the markets are smaller, fewer products have been developed and fewer contests have been fought to establish legal precedents. Furthermore, just the anticipation of possible legal costs can shut a project down before it ever gets off the ground.IP covering a crop variety may be sold, licensed or transferred to another organization at any time. The transfer of rights can occur either in part or in whole .

The transfer can happen in just one territory where it is protected or in multiple territories. The transfer of rights for a biotechnology tool or gene could be specified for use in just one crop , in several crops , or in any and all crops. Finally, to make matters worse, the fact that the IP rights have been transferred may be considered commercially sensitive information and not be made public.Any organization managing the release of a new crop variety faces uncertainty about which IP rights actually cover what technologies, who holds those rights in which countries, and to what degree a specific new transgenic variety infringes on those rights. Resolution of such uncertainty is not less costly for crops with smaller market values. Even after reliable information is obtained, uncertainty remains about negotiating the permissions. IP owners are not required to negotiate licenses, and they may feel there is not enough potential revenue in minor crops to make their licensing efforts worthwhile. They may also be concerned about technology stewardship, given the nervousness among consumers about food biotechnology and its status as a hot media topic. They may worry that the mishandling of their technology by a small and relatively inexperienced horticultural player could lead to stronger regulations, potentially eroding that technology’s value in its major crops, or jeopardize public perceptions about biotechnology overall.In response to IP congestion and continuing uncertainties, several leading U.S. public-sector agricultural re search organizations have come together to create the Public Intellectual Property Resource for Agriculture , an organization providing col laborative IP management solutions to public-sector and smaller private-sector players in horticulture . While individual universities and even the USDA have small and uncoordinated IP port folios in plant genetics, together they hold a fairly comprehensive set of technologies that could be useful for developing transgenic varieties .

PIPRA seeks to coordinate the disparate portfolios of its member organizations to support specialty crop applications. With the offices of technology transfer of its member organizations, PIPRA is pursuing several cooperative strategies.First, PIPRA seeks to develop and adopt more precisely focused terms of licensing, with specific distinctions for the “fields of use” to which a technology is licensed. A company that licenses a technology in vented at a university can still get the full benefit of using the technology in those major row crops in their line of business,grow strawberries container even if the license clearly defines and grants exclusive use of the technology in just those crops. Such a license effectively “reserves” the rights to use the technology in any other crops. Horticultural firms could then make separate agreements with the university to use the technology in only their defined specialty crops. An ad vantage of this strategy is that it can also apply to other minor uses, including “alternative” crops and humanitarian applications in staple crops for developing countries . By discriminating be tween big markets and multiple smaller markets — including those with limited commercial value but important social benefits — public-sector scientists could see their inventions earn royalties in the big markets of ma jor row crops while still helping to improve smaller crops or increase food security in world’s poorest regions.A database will, for the first time, list in one place current information about all of the patents of PIPRA’s members and their availability for licensing alongside information about technologies published in the scientific literature , in sufficient detail to identify which technologies can be accessed for which uses. The database will offer a clear, complete and certain “universal listing” of technologies available from PIPRA’s member organizations and the public domain. Commercial patent databases and professional legal staff are available to researchers in large private companies for searching through the “prior art” to make FTO analyses of a new product’s IP position. Such resources are seldom available to academic and government researchers.

The PIPRA database will decrease uncertainty about what cannot be used by showing what can be used.PIPRA is investigating the creation of patent-pooling mechanisms, which would collect IP submitted from its member organizations, package the technologies together and offer unified licenses for the “bundled” IP in a field of use, such as a specific crop, or in a particular state or country. This process mimics, in a virtual way, how large commercial firms have assembled their IP portfolios to provide FTO in major field crops. Its feasibility will depend — at least at the outset — on the extent to which public sector organizations are able and willing to provide access to patents covering key enabling biotechnology tools already licensed to the corporate sector. Even if used to access technologies on just a patent-by-patent basis, coordinated information and streamlined access to academic and government owned IP could help decrease trans action costs and improve efficiency in technology-transfer markets. There is ample room for improvement here, as some have complained that negotiating licenses from universities and government agencies is often less efficient than negotiating licenses from firms. PIPRA can improve public-sector technology transfer for agriculture by providing information, tools and precedents for efficient licensing. Greater opportunities lie in the The costs and headaches involved in working out “who owns what” and “who owes what to whom” can balloon into what economists call the “tragedy of the anti-commons” and render the development process unfeasible. steps being taken to coordinate access, package IP bundles and target uses in lower-value markets such as horticultural crops and traits important for food security in developing countries. These are, generally speaking, areas that commercial firms are not interested or capable of serving. Such collaboration is not surprising, given the history and ethos of cooperation among agricultural experiment stations within the land-grant system. Public-sector institutions also have greater legal flexibility to enter into collective IP management arrangements, given historical antitrust concerns about abuses of patent-coordination efforts in industry. Even more important will be the establishment of ongoing precedents and mechanisms for the treatment of future IP. Academic and government re searchers will go on making important discoveries and inventing new technologies for agriculture. Those future inventions will, from their inception, be handled in ways — such as being listed in the universal database, licensed for targeted “fields of use” and included in IP pools — that will make them accessible in a carefully proscribed manner, not just to top commercial bidders, but to anyone else in the broader agricultural community who can make good use of the technology, including horticultural researchers and growers.Current practices in patenting and intellectual property protection have created barriers to the use of biotechnology and advanced agricultural technologies for the creation and commercialization of new crop varieties. The complex and cumulative nature of biological innovation requires access to multiple technologies that are often exclusively owned or licensed. For example, commercializing a single variety of transgenic tomato could involve obtaining the rights to use a variety of technologies and genes from numerous life-sciences companies, government agencies and universities. Obtaining “freedom to operate” for transgenic crop varieties is difficult. There is considerable uncertainty as to who holds what rights to particular technologies, and negotiating access to those rights is time-consuming and costly. This is a problem for the major international agricultural companies that focus primarily on high-volume crops such as corn, soybeans and cot ton; for research institutions that work on specialty crops grown on much smaller acreages, such as tomatoes, strawberries, apples and cabbage; and for public institutions that work on staple crops for humanitarian use in developing countries. The international agricultural companies have taken steps to solve their FTO problems through mergers and cross-licensing agreements that bring together essential IP components within one company. However, public-sector institutions — such as universities, government agencies, international agricultural research centers and others working on specialty and staple crops — are still struggling to find ways to gain FTO. In addition, donor agencies such as the Rockefeller and McKnight Foundations, which have a long history of investing in agricultural research that benefits subsistence farmers in developing countries, have also found that IP constraints are reducing the flow of technology.Universities and other nonprofit institutions have generated many key patents related to agricultural bio technology and they will most likely remain an important source of innovation. However, no single institution has the complete package of technologies required for commercialization of a biotech variety. Although some institutions are developing ways to deal with these problems, there are still many examples of public-sector inventions that have been licensed exclusively to private-sector partners. In late 2002, representatives of more than a dozen U.S. public-sector agricultural re search institutions joined with the U.S. Department of Agriculture and the Rockefeller and McKnight Foundations to discuss access to patented agricultural technologies for the development and distribution of improved specialty crops and those targeted for the developing world.