Tag: hydroponic

In separate studies NPR1 over-expression and enhanced resistance are correlated either with elevated or earlier expression of PR gene transcripts, thus supporting this theory . Along with stimulating PR gene expression and priming plants to respond to infection, high NPR1 expression levels enhance the sensitivity of plants to chemicals and fungicides including BTH, fosetyl, and Cu2 . NPR1 is also required for a BTH-induced defense priming indicating a great potential for coupling both chemical and transgenic disease strategies through plants expressing NPR1 . For instance, plants can be engineered to over-express NPR1 so that a lower chemical dose is required to confer efficient disease resistance. Genes with high sequence similarity to NPR1 can be found in Arabidopsis, tobacco, tomato, rice and maize suggesting that this regulator will be conserved among many plant species. Over expression of NPR1 in rice has been shown to enhance resistance to the rice bacterial blight pathogen Xoo . Studies with a putative rice homolog of NPR1 indicate that over-expression of the endogenous rice gene can also provide protection against Xoo . However, unlike in Arabidopsis, rice over-expressing NPR1 grown under sub-optimal conditions display a detrimental growth phenotype. These types of observations may predict an overall phenotype that will need to be further investigated when strongly over-enhancing SAR pathway components in plants . Together these data suggest that, in general,growing vegetables in vertical pvc pipe crop plants contain defense signaling components similar to those found in Arabidopsis.

Potentially, over-expression of other endogenous signaling components other than NPR1 may also be able to provide enhanced plant protection.Biotic infections that stimulate localized host cell death can stimulate SAR in a wide variety of plants, as indicated above. Similarly, root colonization by non-pathogenic Rhizobacteria, can stimulate induced systemic resistance . This resistance is distinct from SAR, but interestingly shares one of the same components, NPR1and can work additively with SAR to mount a heightened defense response . Induction of the ISR response requires that plants are able to properly respond to signals triggered by JA and by the plant hormone ethylene. ISR is not functional in Arabidopsis mutants that are nonresponsive to ethylene, although the SAR response remains intact . Presently, most work utilizing ISR with field grown crops focuses on bio-control. For example, when tomato plants or seeds are treated with dried Rhizobacteria spores, the severity of infection by the tomato mottle virus is reduced . It is notable that the ISR pathway shares components with other defense pathways. Thus, altering the amount of a single component involved in multiple pathways, such as NPR1, may have unintended pleiotropic effects, both favorable and unfavorable that will need to be addressed before application in the field. Plants induce defense responses not only against bacterial, fungal and viral pathogens but also against pests that can cause wounding. Thus, other biotic inducers of resistance include herbivorous insects. The chemical JA is important for triggering resistance to these pests . The defense pathways controlling insect defense and other induced responses are partially antagonistic.

Treatment of plants with SA and BTH can inhibit the induction of JA induced genes and conversely, application of JA reduces the defenses triggered by ISR inducers . However, it appears that the SA and JA pathways can also, in some situations, act in concert to promote defenses against at least a subset of pathogens. In Arabidopsis, in studies where both SA and JA are applied to a plant these chemicals can work additively to protect plants against Pseudomonas syringae pv. tomato . Potentially, upregulation of one induced resistance pathway may impart costs to a number of other pathways. With such complexity inherent in defense responses, it becomes clear that thorough field tests performed under multiple environmental, developmental and pathogen stressors will be essential for any plants engineered for enhanced resistance.Basic research is providing an ever-expanding arsenal of genes with which to engineer disease resistance. Several of these genes have already proven useful and more will undoubtedly be discovered. However, the limitations and costs to using this technology are just starting to be explored. A thorough understanding of these areas will be increasingly important as the tools identified by basic research in plant defense mechanisms are applied more frequently to commercial crops. Previously, only a few studies have attempted to look at the costs, for example, in fitness to plants induced for one of these resistance responses and even fewer still of these studies have been with the economically important cereal crops .Most of the genes involved in broad-spectrum resistance have yet to be inserted as transgenes into crops. Therefore, investigations into the costs of induced resistance have started by assaying the effects of using chemical inducers. Heil et al. have studied the fitness of wheat plants treated with BTH in the absence of pathogens.

When plants were grown either hydroponically or in the field, water-treated control plants were able to achieve greater biomass than their BTH-treated cohorts. In field experiments, however, significant growth differences were not seen until approximately 6 weeks after treatment. The authors suggest that many of the potential fitness costs associated with induced resistance responses may be masked in laboratory experiments where growth conditions are kept optimal, and support this hypothesis with experiments performed growing plants under differing nitrogen concentrations. In addition, when the age of the plants induced for SAR was considered it was found that the growth-costs of BTH treatment could be reduced if the BTH was applied after the lateral shoot formation was complete . These data also underscore the importance of factoring plant developmental programs into any efficient strategy to enhance plant resistance by chemical treatment or genetic engineering.Another unwanted effect that may arise from transgenic manipulation of genes involved in defense signaling pathways is spontaneous cell death. Spontaneous cell death has been uncovered in many genetic screens for enhanced disease resistance and recently, has been seen in transgenic plants. These mutants and transgenic plants are often collectively referred to as lesion-mimic mutants since they display lesions similar to those observed in a defense response even in the absence of pathogens. This form of cell death in plants is sometimes influenced by alterations in environmental conditions such as light, temperature and humidity . Therefore, both in basic research and in applied experiments, it will be important to understand the parameters controlling cell death. This research is critical not only for optimizing the situations where transgenes and chemicals will be most useful to generate disease resistance, but also to minimize negative effects on important agronomic factors such as development,vertical greenhouse fertility and yield. Several dicot lesion-mimic mutants that lead to enhanced cell death have been well characterized including the Arabidopsis acdand lsdmutants . A recessive mutation in the LSD1 gene leads to a lesion-mimic phenotype that is triggered under long-day light conditions and by treatment with SA and INA .

The lsd1 mutation appears to confer hypersensitivity to these compounds . It is hypothesized that reactive oxygen species accumulate in leaf tissues preceding formation of lesions and that LSD1 normally functions to define the extent of lesion spread by suppressing cell death . ROS accumulation is observed in the initial stages of plant defense responses including during the hypersensitive response . In wild-type plants, the HR precedes the formation of micro-lesions that are correlated with induced resistance and is associated with subsequent resistance to pathogen infection . Altered accumulation patterns of ROS in plants with heightened cell death or an HR suggest that ROS play a central role in regulating plant programmed cell death . Lesion-mimic mutants have also been identified in cereals including rice, maize and barley . In rice, one well-characterized class of lesion-mimics contains the slmutants . Many of the sl mutants display heightened resistance to M. grisea and increased expression of PR-1 and peroxidase genes . Other rice lesion mimic mutants displaying enhanced resistance are the cdrmutants . The cdr mutants also have elevated PR gene expression and cell cultures of the cdr mutants under certain conditions can accumulate H2O2. Thus, a subset of rice lesion mimics may have misregulated levels of ROS . Misregulation of ROS accumulation also occurs in transgenic rice engineered to express the OsRac1gene. Over expression of OsRac1 in a wild type stimulated H2O2 accumulation in leaf tissue and over-expression in an sl background stimulated cell death . While mutant rice genes leading to lesion-mimic phenotypes have only been hypothesized to play a role in ROS regulation, one lesion-mimic-inducing gene from maize, Les22, has been cloned. Les22 encodes a uroporphyrinogen decarboxylase , an enzyme required for chlorophyll and heme biosynthesis . Mutations in the homologous human enzyme lead to the light-induced skin toxicity condition of porphyria. People with mutations in the UROD are predicted to accumulate high levels of uroporphyrin III that upon light excitation can become highly reactive resulting in toxic levels of ROS. While the Les22 mutant phenotype does not appear to be associated with enhanced resistance to pathogens, a recessive Les mutant, les9, displays enhanced resistance to the pathogen, Bipolaris maydis . Another maize lesion-mimic mutant, lls1 , a recessive mimic mutation is associated with enhanced resistance to the maize rust fungus, Puccinia sorghi . The LLS1 gene was cloned and found to encode a novel protein containing two binding motifs resembling aromatic ring-hydroxylating dioxygenase regions suggesting that this gene may also be involved in detoxification, perhaps of a phenolic compound important in mediating cell death . Finally, pathogen resistance is associated with lesion-mimic phenotypes in not only rice and maize, but also barley.The mlo mutation confers a spontaneous cell death phenotype upon pathogen challenge and noticeable formation of structural appositions under epidermal cells. Thus, the mutant phenotype confers a rapid death phenotype to the cells, halting fungal ingress at the point of challenge and preventing a compatible interaction. The wild-type allele MLO prevents cell death when challenged by E. graminus . Many mutants showing lesion-mimic or enhanced cell death phenotypes are associated with enhanced disease resistance. This does not necessarily suggest that cell death is a requirement for defense, or that defense always de-represses cell death pathways. Simply, many defense components will likely have multiple roles in basic metabolism and stress responses throughout the plant that need to be characterized before utilizing these genes for resistance engineering.Many of the examples listed above, may appear as substantial challenges to engineering disease resistance, however, these challenges provide opportunities to create plants that are even more resistant than plants engineered based on our current knowledge. For instance if the already identified components of a signaling pathway are not the best candidates for durable resistance in the field, technologies such as micro-arrays will help to pinpoint novel targets of interest . When mutations involved in disease resistance have already been identified, but are recessive in nature such as the mlo, edr1 and mpk4 mutants, classical breeding strategies can be employed. These mutants cannot be placed into heterologous systems using transgenic technology but, as with gene-pyramiding, they are still useful in breeding. Or, as technology continues to improve, gene knockouts and silencing of homologs may be employed to generate mutants in diverse species. If research continues to suggest crosstalk between ISR, SAR and insect defense signaling pathways, there may be great potential for additive defense effects by manipulating overlapping components. So, while limitations and cost of engineering broad-spectrum defenses warrant much attention, it is useful to look at such challenges as means for streamlining and improving upon current engineering strategies.Another promising strategy for enhancing resistance in plants is the use of RNA homology-dependent silencing to combat viral and bacterial disease . The nature of this silencing has been evaluated in a number of systems where similar phenomena are called by different names; RNAi in animals and quelling in fungi . One conserved step leading to RNA homology dependent silencing is the formation of a double stranded RNA intermediate. This dsRNA intermediate is recognized by an enzymatic complex which targets degradation of all corresponding homologous RNA transcripts . Several cases detailed below illustrate the possibilities for generating disease resistant plants by taking advantage of this inherent biological process.

Synthesis of malate, however, may unduly tax a carbohydrate-limited root apex. Indeed, Ca2 treatment, which accumulated more NO3 – than the other treatments , contained negligible amounts of malate . In conclusion, NH4 + and NO3 – differentially affect the finescale spatial patterns of uptake, export, assimilation, and carbohydrate content along root apices. Moreover, although NO3 – levels are maintained low in the meristem and the apical part of the growth zone, NO3 – clearly needs to be considered as a significant component of the osmotic pool supporting expansion at the base of the growth zone and sustaining the functions of young, mature root tissues.Unlike most commonly occurring coconuts, the edible solid endosperm often called the ‘meat’ or ‘kernel’ is thicker, with a soft jelly-like texture at maturation. This can be attributed to some unusual physiological and bio-chemicaltraits. Makapuno contains high levels of cytokinins which promotes cell proliferation and expansion that results in greater endosperm thickness compared with normal coconuts . The unlimited growth of the solid endosperm makes Makapuno a model system for tumorigenesis studies in higher plants . Makapuno also lacks – galactosidase activity which leads to the accumulation of higher levels of the water-soluble galactomannan rather than the water insoluble mannan found in normal coconuts . This likely alters cell wall structure and adhesion and produces a highly viscous endosperm . Finally, Makapuno has a higher content of moisture and protein in the endosperm but lower crude fat; the latter trait should reduce rancidity which is important to those food processing industries that use coconut in their products .

These features of the Makapuno coconut are similar to ‘Kopyor’ in Indonesia, ‘Dikiri Pol’ in Sri Lanka, ‘Thairu thengai’ in India , Maphrao Kathi’ in Thailand and Dua Dac Ruot in Vietnam ,vertical planters for vegetables and are all known for their combination of good taste and unique ‘meat’ texture .The unique endosperm of Makapuno may be controlled by a single Mendelian recessive mutation . The described traits are only found in the triploid endosperm and all alleles need to be recessive. Makapuno therefore need to be physically segregated from normal coconut trees to prevent cross pollination. In addition, the highly viscous nature of the inner endosperm makes germination difficult. The need for a triploidhomozygous recessive state and the observed low germination frequency makes Makapuno coconuts a rarity in nature. The result is a sale price that is 3–5 times higher than normal coconuts in South East Asia and that can be 50 times higher than that of normal mature coconuts in Thailand. Although Makapuno is a value-added commodity,to our knowledge, the post harvest behavior of its fruit has not yet been studied. This is important because of the increased demand for fresh coconut fruit in distant markets in Europe and the United States . Previous work showed that normal mature coconut can be stored for 3–5 months post harvest under ambient atmosphere, after which, the liquid endosperm evaporates and the embryo germinates . De-husked mature coconut has a shorter storage life: up to 2 months at 0–1.5 ◦C, or 3 weeks at 12–15 ◦C . Although removal of the husk reduces shelf-life, the cost-benefit analysis is better. These fruit weigh less which lowers long-distance shipping costs and the price can be marked-up as the product is more convenient for the consumer. As described, Makapuno fruit has distinct properties compared to normal coconuts and this would be expected to influence the storage performance and conditions used to maintain its quality.

The overall goal of this study therefore, was to provide basic information on the storage conditions needed to prolong Makapuno coconut storage-life.Fully mature Makapuno coconuts were partially de-husked, leaving a layer of fiber 1–2 cm thick and were transported from Kanchanaburi province to the laboratory at Kasetsart University within 2 days of harvest. Six uniform and damage-free coconuts were packed in strong well ventilated fiberboard cartons with dividers to separate individual fruit and were then stored at 30 ± 2 ◦C, 66 ± 5% relative humidity . Individual fruit was examined for respiration rate, ethylene production rate, weight loss, color, total soluble solid contents , titratable acidity and lipid oxidation of coconut meat expressed as malondialdehyde values and decay at the initial day and 3 days after storage.The overall aim of this work was to determine the conditions necessary to extend the storage life of partially de-husked Makapuno coconuts after harvest. Our observation of Makapuno coconut maturation agreed with the findings of Islam et al. . Makapuno coconut reached early maturation ∼9 months after flowering and became fully mature ∼10–11 MAF. The endosperm is thicker and softer than that of normal coconut and the liquid endosperm filled the rest of cavity. Our data showed no significant difference in respiration and ethylene production of Makapuno coconut harvested between 9 and 11 MAF .Since there are no published data on the post harvest biology of Makapuno that we are aware of, this work is the first evaluation of the compositional, physiological and biochemical changes in Makapuno coconut fruit after harvest. During 3 days of storage at 30 ◦C, the rates of respiration and ethylene production for Makapuno were similar to those reported for normal mature coconut genotypes . However, the respiration rate from disks of Makapuno coconut was found to be higher than that of intact whole fruit.

This may be due to differences between whole fruit and disks in term of the amount of surface area exposed to the atmosphere or the gas transmission and gas solubility through the tissues. Post harvest fruit quality can be partially assessed by evaluating SS, TA and weight loss. The SS of de-husked normal coconut declines but TA increases as the post harvest storage period increases . Normal harvested mature coconuts lose weight gradually over the storage period due to water evaporation from the fruit cavity or due to absorption by the kernel . In contrast, Makapuno coconut in this study exhibited less weight loss probably due to the viscosity of the endosperm, which restricted water loss through evaporation . Consequently,the color, SS and TA of Makapuno meat did not significantly change after 3 days of 30 ◦C storage. One of the major problems with coconut storage is the increasing rancidity of the endosperm that results from lipid oxidation i.e. the oxidative deterioration of lipids. This leads to an undesirable taste and smell during storage. Malondialdehyde is used as an indicator of lipid peroxidation , and generally, higher MDA levels correlate with higher rancidity . Although insignificant , the MDA of the meat increased 2-fold after 3 days at 30 ◦C storage. Compared to other coconuts, lipid oxidation is less problematic for Makapuno during storage,vertical farming technology probably due to the lower crude fat relative to non-Makapuno types . Another major problem of harvested coconut is decay. Deterioration of Makapuno fruit was evident as mold infestation on the husk surface, which also penetrated the fruit ‘eye’ after they were transferred to 30 ◦C for 3 days . The most prevalent fungal molds found on Makapuno husks were Aspergillus spp. and Penicillium spp. but Fusarium spp. and Curvulria spp. were also detected . Treating fruits with 3–5% sodium metabisulfite or linear low-density polyethylene—file wrap could not prevent surface mold or extend Makapuno coconut storage life when stored at 30 or 5 ◦C .As expected, fruit storage-life increased with decreasing temperature. At 5 ◦C the rate of respiration and ethylene production was reduced 3–6 fold and the storage life was increased to 42 days when stored at 5 ◦C rather than 3 days at 30 ◦C. Moreover, following transfer from 5 to 30 ◦C, respiration rates were similar to fruit kept continuously at 30 ◦C and ethylene production did not change , indicating that there was little or no chilling injury using these temperatures and storage times . During the 6-week storage period, fruit weight loss was significant but endosperm color, SS, TA and decay incidence were similar to those that were stored at 30 ◦C for 3 days. This may indicate that most of weight loss caused by loss of moisture from husk but not from the endosperm. Makapuno fruit developed symptoms consistent with post harvest chilling injury when stored at 2 ◦C for 4 weeks. Cold-stored fruits transferred to 30 ◦C for an additional 3 days exhibited signs of deterioration: the meat developed a woolly texture, and a rancid smell and taste coincided with higher MDA and decay scores. Thus, 2 ◦C was more unsuitable for Makapuno coconut storage than 5 ◦C.Generally, enclosing fruit in films, bags or coatings reduces water loss, prevents the spread of disease among batches of stored fruit and establishes an atmospheric composition that slows down deterioration . It was previously reported that high-OTR bags can enhance the shelf life of mature coconuts . To determine the effect of high-OTR on Makapuno coconuts quality,fruits were stored individually in high OTR bags and kept at 5 ◦C and compared to unbagged fruits held at 5 ◦C.

Coconuts in high-OTR bags had a high O2 transmission rate which allowed O2 to escape from the bag resulting in low O2 accumulation inside. Ethylene also accumulated, but the concentration was lower than 0.15 ppm. While the unbagged Makapuno coconut could be stored only for 6 weeks at 5 ◦C, Makapuno coconut kept in high-OTR bag and stored at 5 ◦C lasted for 10 weeks without any sign of deterioration. In addition, high OTR bag and 5 ◦C storage minimized evaporation very effectively as there was almost no weight loss after 10 weeks and there was no change even after transfer to 30 ◦C for 3 additional days . This reduction in weight loss is likely due to the high relative humidity maintained inside the bag. The combination of high-OTR bag and 5 ◦C effectively suppressed the growth of microbes probably via protecting the coconut from direct contact with fungi and preventing moisture condensation on husk surface. The CO2 and O2 concentration were not high or low enough to kill the fungi therefore it is unlikely that the modified atmosphere would affect fungi pathogen metabolism. The MA provided by the high-OTR bag also maintained SS, TA and MDA levels even after transfer to 30 ◦C. After 10 weeks storage in high OTR bags, fruit quality was maintained and coincided with a reduction in water loss. Thus it is hypothesized that the reduction in water loss might be the most important factor prolonging Makapuno storage life when stored in high OTR bags. However, conclusive proof of this hypothesis requires further experiment.What was once limited to the realm of science fiction and theoretical astrophysics models is now within an operational vision of exploring the “final frontier,” otherwise known as outer space. With the advent of cost-effective launch technologies and the gradual deregulation of space launches and flights, the global economic activity in the space industry has begun to surge. While satellite services likely comprise most viable and profitable venture in space, the adoption of novel space-based goods and services, from space tourism to organ bio-printing, show tremendous potential to disrupt even incumbent industries on Earth. The global space industry has increased in value from $162 billion in 2005 to $469 billion in 2021. While government spending increased 19% to add $107 billion to the space industry, much of the value growth can be attributed to the private sector: commercial enterprises provided an estimated $224 billion in goods and services and $138 billion from constructing infrastructure and support . The most recent space industry reports by Citi and Morgan Stanley project a $1 trillion valuation and $100 billion in annual revenues by 2040 . Space-related research and development has expanded dramatically based on the 22% annual increase for the past five years attributable to the private sector, in contrast to the 10% increase for U.S. governmental expenditures in nominal dollars . While NASA and public sector expenditures have stagnated $12 billion2 in the past few decades, “NewSpace” companies,3 or new companies emerging in the private space industry, have invested an estimated $5-6 billion in 2020, up from less than $1 billion in 2010 .

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.