Tag: vertical agriculture

Allocations to root and grain usually were greatest at ambient CO2, and those to chaff and shoots at either sub-ambient or elevated CO2. Grain typically contained the largest proportion of total N, P, Zn, and Cu, although the organ with the largest percentage of Cu varied with CO2 treatment among NO− 3 -supplied plants. Plants at sub-ambient and elevated CO2 allocated more Cu to the grain, while those at ambient CO2 allocated more to the roots. In general shoots received the majority of K, S, B,Ca, and Mg for all N and CO2 treatments. Ammonium-supplied plants allocated slightly more Mn to the roots at sub-ambient CO2, but allocated increasing amounts to the shoots at the expense of the roots as CO2 concentration increased. In contrast, NO− 3 -supplied plants allocated most of the Mn to the shoots. Ammonium-supplied plants typically allocated more resources to the chaff while NO− 3 -supplied plants allocated a greater percentage of elements to the roots.No other study to our knowledge has examined the influence of N form on plant nutrient relations at three different atmospheric CO2 concentrations. Overall, N form affected growth, total plant nutrient contents, and nutrient distribution in senescing wheat shoots, grain, and roots. The influence of NH + 4 and NO− 3 on growth and nutrient status were so distinct that they should be treated as separate nutrients and not bundled into a general category of N nutrition. Wheat size and nutrition at senescence responded to CO2 concentration in a non-linear manner. As was previously shown , we found that plants supplied with NH4 + were more responsive to CO2 concentration than those supplied with NO− 3 . Although not explicitly addressed here because of the heterogeneity of variances,vertical grow racks interactions between CO2 and N treatments likely existed for a number of the biomass and nutrient measures.

Most nutrient concentrations were generally higher in NH4 + – supplied plants, with the exceptions of NO− 3 − N, Mg, B, and Mn, which were generally higher in NO− 3 -supplied plants. Phytate, which hinders human absorption of Zn and Fe , showed little variation at ambient and elevated CO2 between NH4 + and NO− 3 -supplied plants, which, in conjunction with the observed greater bio-available of Zn in NH + 4 -supplied plants, may have consequences for human nutrition. Distribution of nutrients to the shoots, roots, chaff, and grain in response to CO2 concentration and N form was also non-linear and varied by nutrient. The data support our hypothesis that NO− 3 -supplied plants would show a more limited biomass and yield enhancement with CO2 enrichment than NH4 + -supplied plants. Nevertheless, mean biomass and yield decreased from ambient to elevated CO2 in both NO− 3 – and NH4 + -supplied plants in contrast to biomass increases in prior work on wheat seedlings . NO− 3 – supplied plants allocated more biomass to roots and had larger root:shoot ratios than NH4 + -supplied plants regardless of CO2 concentrations as has been reported previously , but increased root mass at elevated CO2 concentration for NO− 3 -supplied plants reported previously were not observed here. The shoot biomass data suggest that growth differences measured early in the lifespan of wheat supplied with NH4 + or NO− 3 or NH4 + do not necessarily carry through to senescence. This may be due in part to a shift in NO− 3 assimilation to the root , allowing NO− 3 -supplied plants to compensate for the decrease in shoot NO− 3 assimilation that occurs at elevated atmospheric CO2 concentrations . The decrease in yield and biomass measures at elevated CO2 concentrations does not agree with field observations where wheat yields as well as overall biomass increased with elevated CO2 . Similarly, our results that the greatest values for other yield measures occurred at ambient CO2 concentrations varies from the literature. Conflicting results, however, have also been reported .

Many of the field and open top chamber studies were grown under natural light and thus received substantially greater photosynthetic flux density than our chamber-grown plants. These higher light conditions would be more favorable to biomass accumulation. Also, these studies typically applied high amounts of mixed N fertilizer , and yields and biomass have been found to be greater under mixed N nutrition than under either NH4 + or NO− 3 alone . Finally, the wheat cultivar we used is a short-statured variety that has rarely been used in other studies and may have accounted for some of the differences between our study and other published data. Our results that NH4 + -supplied plants had greater yield and yield components than NO− 3 -supplied plants at ambient CO2 have been observed previously . Wang and Below observed greater numbers of kernels head−1 and KN in plants supplied NO− 3 that was not observed here. Their study, however, supplied NH4 + at relatively high levels . Several studies have found that incipient NH4 + toxicity can start appearing at N levels as low as 0.08–0.2 mM NH4 + , although the onset of NH4 + toxicity depends on light level and solution pH . The poorer performance of the NH4 + treatment in Wang and Below , therefore, might derive from NH4 + toxicity. We have previously determined that the 0.2 mM NH4 + -supplied to our plants to be sufficiently high for normal growth, but low enough to avoid toxicity problems under our experimental conditions .Our second hypothesis, that nutrient concentrations are differentially affected by the inorganic N form supplied to the plants and CO2 enrichment, was supported by our data. CO2 concentration and N form interactions may alter tissue demands for nutrients. For many nutrients, ratios between different elements are typically maintained within a narrow range . CO2 concentration and N form may disturb the balance between different nutrients, leading to a cascade of changes in demand, accumulation, and allocation among the different plant tissues .Some portion of the greater response of NH4 + -supplied plants to CO2 derived from a dilution effect from the greater biomass at ambient CO2 concentrations .

Total amounts of nutrients tended to decline with CO2 enrichment for NH4 + -supplied plants, which had the greatest amounts of macro/micro-nutrients at sub-ambient CO2 . These results have not been observed in other published studies . Growth chamber studies, however, tend to have more exaggerated differences among treatments than field and greenhouse experiments , and N source cannot be well-controlled in field and greenhouse experiments. The observed increase in NO− 3 −N concentration with CO2 concentration in NO− 3 -supplied plants has been reported previously , and adds further support to the hypothesis that elevated CO2 concentrations and the resulting decrease in photorespiration inhibit shoot NO− 3 photoassimilation. Nevertheless, tissue NO− 3 − N concentrations observed here were substantially lower than those in the earlier study . Again, this may derive from difference in life stages in the two studies. Most of the N available to the plant for grain filling comes from N translocation rather than uptake from the substrate . Probably, the plants continued to assimilate plant NO− 3 using a non-photorespiratory dependent process such as root assimilation after root N uptake slowed or stopped. Loss of NO− 3 through root efflux to the nutrient solution also may have contributed to the lower concentration of NO− 3 − N. The partitioning and accumulation of all mineral elements was affected in some manner by the CO2 treatment and N form supplied to the plants. Observations that cation concentrations decrease under NH4 + supply relative to NO− 3 supply were not apparent in this study. Again, this could be partly due to the relatively low concentration of NH4 + -supplied in our study, the age of the plants at harvest, and differences among wheat cultivars. Allocation of nutrients within the plant followed similar trends for both N forms,vertical hydroponics with the exceptions of Mn and Cu . Interestingly, in NO− 3 -supplied plants, shoot Mn concentrations increased slightly with CO2, and these plants allocated far more Mn to the shoots than NH4 + -supplied plants at all CO2 concentrations. Manganese has been found to activate Rubisco in place of Mg2+ and the Rubisco-Mn complex has been observed to decrease Rubisco carboxylase activity while minimally affecting or even enhancing oxygenase activity . The slight increase in shoot Mn with CO2 corresponded to a large 23% decrease in Mg concentration. Manganese, which can act as a cofactor for glutamine synthetase , was also the only nutrient that NH4 + -supplied plants allocated agreater percentage to the roots at the expense of the shoots. NO− 3 – supplied plants typically allocated a higher percentage of most nutrients to the roots, as has been reported previously .

Phytate, which forms complexes with divalent cations, has been found to hinder human Zn and Fe absorption during digestion and thus has been labeled an “anti-nutrient.” It may serve a number of valuable functions, however, including roles as an anti-oxidant and anti-cancer agent . Phytate is also the major repository of grain P, and variation in P supply to the developing seed is the major determinant of net seed phytate accumulation . To our knowledge, no published studies have explicitly looked at how phytate is affected by CO2 concentration. Elevated CO2 has been found to have a much larger negative impact on Zn and Fe concentrations than on P in wheat . Several studies have observed that P increases slightly with CO2 concentration, and because the majority of P is tied up in phytate, this may cause increases in grain phytate concentrations as atmospheric CO2 rises. As a result, bio-available Zn and Fe–Zn and Fe not bound to phytate – is expected to decrease even further . Nonetheless, we did not observe such trends in macro- and micro-nutrient concentrations in this study. The mechanism behind these contrasting results is not clear, although the environmental conditions and nutrient solution in which the plants were grown likely had some role. The modeled data demonstrated only a small negative impact of CO2 concentration on bio-available Zn concentrations , which was unexpected. Indeed, the grain from NO− 3 -supplied plants actually showed a slight increase in bio-available Zn between ambient and elevated CO2. These results combined with the differences in grain bio-available Zn between NH4 + and NO− 3 -supplied plants demonstrates that N form may differentially affect the nutritional status of this important nutrient, especially in less developed countries that might be more dependent on phytate-rich grains for their Zn nutrition . The milling process removes some, if not most, of the phytate and grain mineral content with the bran fraction of the grain . Regardless, with over 50% of the human population suffering from Zn deficiencies, even small increases in bio-available Zn would be beneficial . This modeling exercise, however, is not a prediction of how increasing CO2 will affect wheat nutrition so much as illustrates that N source may mediate, to some extent, the effects of CO2 on phytate and bio-available Zn, and that N source will become an even more important agricultural consideration in the future. In summary, both CO2 concentration and N form strongly affect biomass and yield in hydroponically grown wheat, as well as nutrient concentrations in above- and below ground tissues. Interactions among plant nutrient concentrations,CO2 concentrations, and N form are complex and non-linear. The impact of N form and CO2 concentration on the mechanisms affecting nutrient accumulation and distribution requires further research and extension to more realistic and agriculturally relevant growing conditions found in greenhouse and field studies. Of course, in greenhouse and field studies, control of N source is limited and control of atmospheric CO2 concentration is expensive. The effects of CO2 and N form on agriculture and human nutrition observed here are interesting and suggest a new area of research on mitigating the effects of climate change on agriculture. The supply of fertilizers or addition of nitrification inhibitors that increase the amount of available NH4 + may have beneficial effects for human nutrition, particularly in regards to micro-nutrient deficiencies such as Zn and Fe that currently affect billions of people worldwide.

Weighed samples were digested using H2SO4-H2O2 and the samples were measured using the machine Auto Analyzer 3 .Recently, cadmium accumulation in soybean seed is one of the serious agricultural problems. However, characteristics of Cd transport and accumulation in soybean plants are unclear so far. In this study, it was characterized that the mechanism of Cd transport in soybean plants using mixed tracer, which included 107Cd and 109Cd . We described the dynamics of 107Cd transport in the test plants using the positron-emitting tracer imaging system ; which presents a movie of changing distribution of a positron-emitting radioactive tracer within an intact test plant . The 109Cd was used in the experiments with autoradiography and well-type counter to study Cd distribution within the same plant individuals. Soybean plants were grown with hydroponic culture in a growth chamber. Plants at beginning seed stages were transplanted to plastic tubes containing 0.5 mM CaCl2 solution and subjected to the experiments.Cd tracer was added to the CaCl2 solution with 0.1 µM non-radioactive Cd. 107Cd is positron-emitting tracer and detectable by PETIS, but 109Cd is not. The imaging was performed for 36 hours. After the PETIS experiments,equipment for vertical farming the test plants were transferred to a fresh culture solution excluding Cd and grown. The plants were sampled at about 2, 3 and 5 days after the point of Cd feeding. The sampled plants were pressed with heated iron. 109Cd distribution in the test plants was analyzed using autoradiography and well-type counter. In the field of view of PETIS, Cd appeared in the shoot base about a few hours after Cd feeding and moved upward through the stems.

In most cases, Cd reached the upper most node within 36 hours. With the autoradiography, weak signal was detected in the seeds and pods at 2 days after Cd feeding, while no signal was detected in the leaves. It indicates that Cd is transferred to the pods and seeds without passing through leaves by this moment. Results with well-type counter revealed that approximately 90% of Cd absorbed by the test plants was distributed into the roots at 2 days after Cd feeding. The distribution of Cd in the roots decreased at 3 and 5 days after Cd feeding. On the other hand, the distribution of Cd in the seeds, pods, leaves and petioles with time. These results suggest that a large part of Cd accumulated in the roots first, and then moves into the shoot parts on a time scale of day. Approximately 750 million tons of wheat are produced worldwide every year , but further increases are required to feed a growing human population. One understudied area that can contribute to these yield increases is the role of different root architectures on wheat adaptation to different soils. Although some progress has been made in the understanding of root development and architecture in Arabidopsis , this knowledge is lacking in grass species . There have been some examples of phenotypic selection of root architecture in breeding programs , but those methods are laborious and can be accelerated by a better understanding of the genes controlling wheat root architecture. Rye , a close relative of wheat, is more tolerant to water shortages than wheat, and has been reported to have a more robust root system. The translocation of the short arm of rye chromosome one to wheat chromosome 1B contributes to above ground biomass and better performance under drought stress . To address bread making quality problems associated with the 1RS.1BL translocation , a recombinant 1RS chromosome including two wheat 1BS chromosome segment introgressions was developed to eliminate the two rye regions associated with the bread-making quality problems . We introgressed the newly engineered chromosome into the spring wheat variety ‘Hahn’ and generated 1RS/1RSww near isogenic lines .

Previous field trials showed that the Hahn 1RS lines had significantly higher yield and better canopy water status than the 1RSWW NILs in both well-watered and water-stressed environments, although the differences were larger in the latter . From a cross between Hahn-1RSWW and Hahn-1RS, we generated two additional NILs, one carrying the distal and the other the proximal wheat segment . The two NILs carrying the distal rye region showed significant improvements in grain yield and canopy water status compared to NILs carrying the distal wheat segment . The 1RSxR NILs also showed higher carbon isotope discrimination and increased stomatal conductance, suggesting improved access to soil moisture relative to the 1RSxW NILs . In the winter of 2013, heavy rains waterlogged a UC Davis experimental field that affected the four 1RS NILs at the early tillering stage. Although the affected areas were irregular, the 1RSxR were less affected than the 1RSxW NILs. Based on this observation and previous results, we hypothesized that the 1RSxR lines might have a more extensive root system than the 1RSxW lines, which helped them tolerate both water logging in this experiment and water shortages in the previously published experiments . The first objective of this study was to characterize the effect of the wheat-rye polymorphism in the distal region of the 1RS.1BL translocation on root architecture in the field, and on plant biomass and grain yield under normal, excessive or reduced irrigation. After we observed that the lines with the distal wheat segment had shorter seminal roots than the lines with the distal rye segment in hydroponic conditions, we also decided to study the effect of these genotypes on seminal root growth rates, distribution of reactive oxygen species, and distribution of lateral roots. The implications of the observed differences in root development and architecture are discussed.In this study, we used four near isogenic lines that showed differences in grain yield in previous work .

The recurrent common wheat parent of these NILs is the spring wheat cultivar ‘Hahn’ developed by the International Maize and Wheat Improvement Center . The Hahn cultivar carries the complete 1RS translocation from rye, and the three NILs differed from Hahn either in the presence of a distal interstitial segment of wheat chromatin , a proximal interstitial segment of wheat chromatin , or both . The interstitial wheat segments were introgressed from the common wheat cultivar ‘Pavon 76’ to eliminate the Sec-1 locus from 1RS and to incorporate the Glu-B3/Gli-B1 locus from 1BS into the 1RS chromosome to improve bread-making quality . The source of this 1RS arm was the rye cultivar ‘Petkus’, and the resulting 1RS.1BL translocation became widely distributed in wheat breeding programs around the world . Controlled water logging experiments were conducted during the 2013-2014 and 2015-2016 growing seasons. An additional experiment was performed in 2014-2015 but it was not analyzed due to severe weed problems. The experiments were planted in November and harvested in June . The two water logging experiments were organized in a split-plot randomized complete block design with four blocks in 2014 and three blocks in 2016. Within each block, the main factor was irrigation treatment, and within each irrigation treatment – block combination, the Hahn 1RS, 1RSWW, 1RSRW, and 1RSWR genotypes were used as sub-plots. The average trait values of the 1RSxR and 1RSxW NILs were compared to determine the effect of the distal rye and wheat chromosome segments. In the 2014 field experiment, each block included two different irrigation regimes as main plots. The first treatment was based on plant needs and normal practices in California’s Sacramento Valley and is designated hereafter as normal irrigation. The second treatment, referred hereafter as water logging, consisted of artificial flooding twice a week starting in late January and ending in late March during the tillering stage, followed by normal irrigation. Water was applied via flood irrigation, and the soil profile remained saturated. While plants were not kept fully or partially submerged, there were persistent pools of water on the soil surface indicating a waterlogged environment. Each genotype was planted in three adjacent 1 m rows with 30.5 cm spacing between rows at a rate of 30 grains per row. Genotypes were separated by an empty row ,vertical farming systems and treatments were separated by a minimum of a border row, an irrigation levee, and another border row, leaving in excess of three meters between experimental units of different treatments. Experimental units were replicated six times within each of the four blocks in an RCBD pattern and were used as sub-samples. At the end of the season, each set of three rows was harvested and grain yield was recorded. The average of the six sub-samples was used as a single data point in the statistical analysis. Canopy Spectral Reflectance measurements were taken for all sub-samples on two days . Sub-samples were averaged within days, and day averages were used as repeated measures. Canopy spectral reflectance measurements were taken with the “ASD HandHeld 2 Pro” spectrometer from Malvern Panalytical. Measurements were taken using a “scanning” method in which 50 measurements were taken on a single plot and averaged to give a single reflectance spectrum. From these measurements, differences in biomass between genotypes were estimated using the Normalized Difference Vegetation Index , which was calculated using the formula /, where R = reflectance at the specified wavelength. In the 2016 field experiment, each block included three irrigation treatments. The first treatment was grown under normal irrigation as described above. The water logging treatment included flood irrigations three times a week, from the beginning of February to the end of February, followed by normal irrigation. The terminal drought treatment was grown under normal irrigation conditions until late March , and no additional irrigations after that point. Within each block–treatment combination, each genotype was machine sown in 2.23 m2 plots , which were combine-harvested at maturity. In 2016, CSR measurements were taken as described above on March 24th , April 6th , April 13th and April 28th .

Days were used as repeated measurements and were analyzed as sub-sub-plots in an RCBD split-split-plot design using conservative degrees of freedom for days and all their interactions . After the CSR measurements were completed, an irrigation pipe ruptured flooding several sections of the experiment on April 29th, resulting in increased variability in the final yield measurements. Flooding was irregular and inconsistent across blocks, with major effects on replications two and three of the drought treatment and replication two of the water logging treatment. The field experiment to estimate root length was conducted after a maize crop harvested in the summer of 2016. The field was organized in an RCBD with six blocks and four genotypes per block. Plots were machine sown in 4.5 m2 plots in November 2016 and were grown under normal irrigation conditions. To obtain soil core samples at specific depths and avoid differential soil compaction, we excavated ~2 m deep trenches cutting perpendicular across the middle of plots including complete blocks one , three and six to expose the root system. We took horizontal soil core samples from the center of each block at 20 cm intervals using a thin-walled copper pipe . Core samples were taken from 20 to 140 cm in the first block and from 20 to 180 cm in blocks three and six after we discovered the presence of roots at 140 cm in block 1. Plants were at the tillering stage at the time of the root sampling.Soil core samples were washed using a hydro-pneumatic elutriation system from Gillison’s Variety Fabrications, Inc. . After washing and sorting white turgid roots from other organic matter and decayed roots of the previous maize crop , we suspended the roots in water and scanned them using an EPSON Expression 11000XL flatbed scanner. Scanned root images were analyzed using the WinRhizo software package. Measurements of dry root biomass are not reported because they were too variable due to small biomass, stray soil contaminants, and changes in ambient moisture. The 20 cm sampling point was not used because the large amount of root biomass and organic matter present in these samples made them difficult to clean and measure. Since all root measurements were performed using soil cores of identical volume we refer to these measures as densities .

At the stage of commercial production, techno-economic analyses can provide essential insights into areas such as scheduling, vendor contracts, continuous improvement, and process intensification.Analysis of these individual factor sensitivities provide a preliminary framework for understanding expected bounds of manufacturing costs. It can also serve as a prioritization tool for vendor selection when considering larger, multi-material contracts, as well as with research and development efforts. This analysis could be strengthened to include a forecasting capacity in future work by integrating market analyses to weight each level of factor variation with a likelihood based on predictive market data. From this information, one could establish an anticipated range of COGS based on key cost factors to holistically define uncertainty and risk.Within the given parameter range for expression level and yearly production volume, COGS is more strongly impacted by the expression level. This behavior is specific to the defined parameter ranges, which were selected based on anticipated needs and expectations. In this study, we assumed that raw material and consumable resource purchase costs per unit are independent of yearly amount purchased. As yearly production increases, economies of scale dictate that the material unit price will decrease. This becomes a more important consideration when evaluating COGS over a wide yearly production range. Figure 5 shows similar behaviors for changes in total COGS with expression level and yearly production. However,lettuce vertical farming there is a dissimilar behavior in the upstream versus downstream contributions to COGS over the parameter range. Varying expression level largely influences the upstream processing COGS, while varying yearly production largely influences the downstream processing COGS.

The main reason is that the costly downstream operations are economically dependent on AMP quantity rather than on stream composition. Additionally, we chose to conservatively fix AMP recovery in the downstream, regardless of expression level. The low upstream COGS sensitivity to yearly production is because of the approximately linear scalability of the production platform. This is a main advantage of plant-based production that makes the scale-up from lab to commercial scale considerably simpler and faster than traditional bioreactor-based production platforms.As yearly production changes, the upstream processing scales in an approximately linear fashion for a given processing strategy. However, one could anticipate that scaling to even higher yearly production could enable higher efficiency upstream processing strategies and thus improve the scaling dynamics of upstream economic contributions.The nicotine-free S. oleracea scenario provides insight into the manufacturing costs associated with nicotine clearance. There are minor differences in plant growth and harvest operations, but the majority of upstream COGS reduction is because of higher product recovery and thus lower biomass requirements for a given yearly production level. Higher product recovery is attributed to the removal of the nicotine clearance chromatography step present in the N. benthamiana base case scenario, as illustrated in Figure 2. The smaller batch size and simpler downstream processing as compared to the N. benthamiana base case scenario result in a 26% reduction in the downstream cycle time and 37% reduction in downstream labor costs, yielding a COGS of $4.92/g AMP.

The field-grown N. tabacum scenario results in the lowest COGS of $3.00/g AMP, providing reasonable justification to pursue this manufacturing process. However, our assumptions do not account for potential upstream difficulties associated with product expression consistency, greenhouse growth, and transplantation of seedlings or crop loss because of adverse weather events throughout the growing season, nor do they account for the downstream difficulties associated with removal of the more viscous N. tabacum host leaf impurities. Future work to experimentally support key assumptions of field growth could add higher confidence and value to this alternative scenario. Additionally, the current growth strategy is based on tobacco production as a commodity good; there may be a different growth strategy that is optimal for recombinant protein production . It is worth noting that this manufacturing process is expected to scale especially well. In our model, we assume that dedicated personnel and upstream equipment are required for transgenic handling. At an annual production level of 500 kg AMP, this results in 17% upstream equipment utilization. This means that as the yearly production demand increases, we expect marginal increases to upstream CAPEX and OPEX. As such, we expect upstream-related COGS to reduce dramatically with increases in yearly production demand.Biotic food sanitizers can be used in a variety of applications to augment traditional food sanitizing treatments against specific high-risk pathogens. Given the differences in food safety practices among food products, it can be difficult to measure the cost of use as a single value. Instead, we focused our discussion on cost of use calculations with application rates representative of AMP use—colicins for control of E. coli on red meats. We chose to investigate this example at several points along beef processing: animal washing, post-slaughter carcass cleaning, and meat product protection. We anticipate an application rate of 2–10 ppm AMP in water for animal and carcass wash or 2–10 mg AMP per kg meat product. It should be pointed out that, according to the recently published paper of Hahn-Löbmann et al. in 2019, the application rates of salmocins, Salmonella -derived bacteriocins, could be up to 10 times lower because of the higher potency of salmocins.Figure 7 shows the cost of use estimates for select technoeconomic scenarios modeled in this study compared to relevant standard sanitizing treatments.

Cost of use assumptions and a sample calculation of those performed to generate the cost of use estimates can be viewed in Table S7 and Calculation S1, respectively. In all three points of intervention, AMP application cost ranges are below or overlapping those of standard treatments. Additional information is needed on application rates and spray volume used in animal washing to reduce AMP cost of use range and increase confidence in cost comparison to standard treatments. On the other hand, AMP cost of use ranges for treatment of meat product overlap significantly with standard interventions, indicating comparable costs. Finally, the AMP cost of use ranges for post-slaughter carcass cleaning suggest that the use of AMP at this beef processing juncture has the potential to be substantially lower in cost than standard treatments.Current food safety practices, although largely effective, result in food borne illnesses that impose a $14 billion annual burden on the US healthcare system. As the looming prevalence of antibiotic resistance grows, so will the impact of food borne illnesses. The need for protection against food borne pathogens is only increasing. Reports as far back as 20 years ago acknowledge that areas of the food industry like the meat sector will need to absorb additional costs to improve food safety levels.We investigated bacteriophagederived lysins and bacteria-derived AMPs to explore the capacity of this class of biotic sanitizers to improve food safety levels in the costsensitive food industry. Although previous studies illustrate the efficacy of AMPs, in this study, we performed a techno-economic analysis of plant-based production of AMPs to better understand the commercialization potential of products produced using this platform. Our analysis predicts a $6.88/g AMP COGS for the base case scenario, $4.92/g for the nicotine-free S. oleracea scenario, and $3.00/g for the field-grown N. tabacum scenario. We also evaluated the sensitivity of the base case COGS to changes in purchase price, expression level, and yearly production. In doing so, we identified economic “hot spots,” which include the large contribution of the soilless plant substrate and downstream labor-dependent costs . The cost of use analysis indicates that AMPs are projected to de-risk food borne disease in beef processing as supplemental sanitizing treatments at only minor economic perturbation across several key processing junctures. It is expected that other food processing operations would yield similar benefits. This techno-economic analysis of plant-based production of AMPs is focused on manufacturing costs and the implications for application costs. In developing this model and analysis, we have identified several areas of importance for future analysis, for example,vertical grow shelf consideration of avoided costs associated with the prevention of food disease and illness. An example of a major avoided cost is that associated with food recall, which includes impact to brand image and loss of sales. A cost–benefit model that includes these avoided costs may provide more complete insights into AMPs as a food sanitizing treatment. In addition, there are social, cultural, and behavioral factors that can impact food safety that are not considered in this economic analysis. In our analysis, we describe plant-based production of AMPs as a food processing aid.

A direct evaluation of traditional manufacturing platforms, such as mammalian cell suspension culture and bacterial fermentation, as alternative scenarios would be a valuable future contribution. To our knowledge, there are no existing direct comparisons of whole plant, microbial fermentation, and mammalian cell culture platforms in the literature. Future work to compare AMP manufacturing in different locations would also add insight into the geographical and national sensitivity of AMP manufacturing process costs. We compare three host plant batch production models in our analysis, all with different manufacturing processes. A valuable future analysis would be to additionally compare alternative operational modes for a single host plant. Continuous manufacturing is a nascent biotechnology process intensification trend that describes processing of a target molecule from raw materials to final product without any hold steps in a continuous flow process. This contrasts with the more traditional batch manufacturing investigated in this analysis, in which discrete batches are processed at time intervals. It is generally accepted that continuous manufacturing reduces facility footprint, buffer usage, and equipment sizing as compared to batch manufacturing. To date, there are no publications of continuous manufacturing using plant-based production. We anticipate that plant-based production is a favorable platform for continuous manufacturing, which can reduce CAPEX costs through the replacement of large steel vessels with small disposable containers; whole plant production does not require disposable containers, as the plant itself functions as the bioreactor. A techno-economic analysis comparing these two manufacturing modes will provide additional insight into the economics of plant-based production.Potassium is a small, univalent cation that is not toxic in plant cells even at rather high concentrations. Healthy plant cells contain high concentrations of K, but the question is why? What does K do inside the plant? The reason we have these questions about plant K may be because K is small and very mobile in plant cells, making it difficult to pin point its exact roles. The cytoplasm in most healthy plant cells contains over 100 mM K. This is precisely the K concentration needed to promote protein synthesis . Every step of protein synthesis requires over 100 mM K for all of the structures involved to form the correct conformations necessary in the process. Correct structures are extremely important for the interactions of m-RNA, t-RNA, small ribosomes, large ribosomes, and the elongating protein. These structures must have the proper conformations in order to come together and then break apart at the right time. The conformations of these structures and their appropriate activities are only correct when they are bathed in high K concentrations. Similar concentrations of other monovalent cations do not produce the proper conformations. There are over 60 important enzymes that require K-activation in order to reach their maximum catalytic activity, and major processes like protein synthesis and starch synthesis involve some of these enzymes . Interestingly, certain plant species are known for their high K requirements. For example, alfalfa crops has a high K requirement for maximum productivity, removing about 42 lbs K in each ton of hay harvested. In trying to determine the reason for this high K requirement, I considered sugar, starch, protein, and oil production of different crops and compared these factors with K removed by the crop . The only factor highly correlated with K removal was protein removal, supporting the connection between K and protein synthesis discussed above. In fact, this correlation was also high when K and protein contents of grain crops were compared . Therefore, if one is growing a crop that produces large quantities of protein/acre, the K requirement will be high. In addition, the cation, K+ , is required to balance the negative charges of the acidic amino acids, aspartate and glutamate, that extend out from the amino acid polymers. Therefore, there are two important reasons why high protein crops require large quantities of K. Boron is an important micro-nutrient element required for all plant species.

In the present study, we characterized multiple avp1 mutant alleles and found they were hypersensitive to high external Mg2+. This finding has not only improved our understanding of the mechanism underlying Mg2+ tolerance but also uncovered a novel physiological function of AVP1 in plants. When the plants were confronted with high Mg stress, sequestration of excessive Mg2+ into the vacuole plays a vital role in detoxification of Mg excess from the cytoplasm. The AVP1 protein predominantly localized in the vacuolar membrane and was a highly abundant component of the tonoplast proteome. Encoded by AVP1, vacuolar H+ -PPase, together with vacuolar H+ -ATPase, plays a critical part in establishing the electrochemical potential by pumping H+ across the vacuolar membrane. This proton gradient, in turn, facilitates secondary fluxes of ions and molecules across the tonoplast. Based on this well-established idea, we hypothesized that avp1 mutants may be impaired in cellular ionic homeostasis and should thus exhibit hypersensitivity to a broad range of ions. However, unexpectedly, we found that avp1 was hypersensitive only to high external Mg2+ but not to other cations . It was shown that overexpression of AVP1 improved plant salt tolerance in quite a few species, which was interpreted as the result of increased sequestration of Na+ into the vacuole. It is thus reasonable to speculate that the tonoplast electro chemical potential generated by AVP1 would likewise favor Mg2+ transport into vacuoles via secondary Mg2+/H+ antiporter. Surprisingly, our subsequent experiments did not support this hypothesis and several lines of evidence suggested that the hypersensitivity of avp1 to high Mg2+ was not due to the compromised Mg2+ homeostasis in the mutant. First, unlike other high Mg2+-sensitive mutants such as mgt6 and the vacuolar cbl/cipk mutants, the Mg and Ca content in the avp1 mutant was not altered as compared with wild type,hydroponic vertical farming suggesting that AVP1 may not be directly involved in Mg2+ transport in plant cells. Second, higher order mutants of the avp1-4 mgt6 double mutant and avp1-4 cbl2 cbl3 triple mutant displayed a dramatic enhancement in Mg2+ sensitivity as compared to single mutants.

These genetic data strongly suggest that AVP1 does not function in the same pathway mediated by MGT6 and does not serve as a target for vacuolar CBL-CIPK. Moreover, it was previously shown that either vacuolar H+ -ATPase double mutant vha-a2 vha-a3 or the mhx1 mutant defective in the proposed Mg2+/H+ antiporter was not hypersensitive to high Mg2+. These results implicate the vacuolar Mg2+ compartmentalization should be fulfilled by an unknown Mg2+ transporter/channel, whose activity is largely not dependent on the tonoplast ∆pH. Identification of this novel Mg2+ transport system across the tonoplast, which is probably targeted by vacuolar CBL-CIPK complexes, would be the key to understand the mechanism. Third, expression of the cytosolic soluble pyrophosphatase isoform IPP1 could fully rescue the Mg-hypersensitivity caused by AVP1 mutation. These lines of evidence pinpoint PPi hydrolysis, rather than ∆pH-assisted secondary ion transport and sequestration, as the major function of AVP1 in high Mg2+ adaptation. Under high Mg stress conditions, a number of adaptive responses are supposed to take place in plants, including the remodeling of plant morphogenesis as well as reprogramming of the gene expression and metabolite profile. However, very little is known so far and therefore, the molecular components targeted by excessive Mg2+ in plant cells remain obscure. Here, we suggest that the concentration of cellular PPi could be responsive to external Mg supply. Our results showed that extremely high levels of Mg2+ led to inhibition of the PPase activity in Arabidopsis, which in turn, resulted in the elevation of PPi content in the cytosol. Because high level of PPi is very toxic, the efficient removal of PPi by AVP1 under high Mg2+ conditions might become one of the limiting factors for optimal plant growth. This idea is supported by the observation that avp1 mutants accumulated significantly higher PPi content under high Mg2+ conditions compared with normal conditions .

Most importantly, heterologous expression of the soluble PPase IPP1 gene rescued high Mg-sensitive phenotype of fugu5-1 , which strongly suggested that high Mg2+ hypersensitivity phenotype in avp1 mutants could primarily be attributed to impaired PPi homeostasis.It would be interesting to investigate how PPi concentrations vary in different Mg2+ conditions and during different plant growth stages. Recently, cytosolic soluble pyrophosphatases were identified in Arabidopsis, and were shown to physiologically cooperate with the vacuolar H+ -PPase in regulating cytosolic PPi levels. Future studies should clarify if this type of soluble isoenzymes is also involved in the same high-Mg adaptation process. Collectively, our findings provide genetic and physiological evidence that AVP1 is a new component required for plant growth under high external Mg2+ concentrations and functions in regulating Mg2+ tolerance via PPi hydrolysis.Arabidopsis thaliana ecotype Columbia and Wassilewskija were used as wild type in this study. The mutants fugu5-1, fugu5-2, fugu5-3, and transgenic plants fugu5-1+IPP1 were offered and characterize by Ferjani. The cbl2 cbl3 double mutant was described in previous studies. The T-DNA insertion mutants avp1-4and mgt6were obtained from the European Arabidopsis Stock Centre and the Arabidopsis Biological Resource Center. The mutant avp1-3was a T-DNA insertion mutant in the Wassilewskija background and obtained from INRA Arabidopsis T-DNA mutant library. Mutants with multiple gene-knockout events were generated by genetic crosses, and homozygous mutant plants were screened from F2 generation and identifified by genomic PCR using primers listed in Supplementary Table S1.For on-plate growth assays, seeds of different genotypes were sterilized with 75% ethanol for 10 min, washed in sterilized water for three times, and sown on Murashige and Skoog medium containing 2% sucrose and solidified with 0.8% phytoblend . The plates were incubated at 4 ◦C in darkness for two days and then were positioned vertically at 22 ◦C in growth chamber with a 14 h light/10 h dark photoperiod. After germination, five-day-old seedlings were transferred onto agarose-solidified media containing various ions as indicated in the figure legends and were grown under 14 h light/10 h dark photoperiod. For phenotypic assay in the hydroponics, 10-day-old seedlings geminated on MS plate were transferred to 1/6 strength MS solution and were grown under the 14 h light/10 h dark condition in the plant growth chamber. Fresh liquid solutions were replaced once a week. After two-week culture, the plants were treated with 1/6 MS solutions supplemented with 15 mM MgCl2. Two-week-old hydroponically grown plants were treated with 1/6 MS solutions containing 0 or 15 mM MgCl2. After two-day treatment, leaves of all the plants were collected to prepare crude membrane as described previously.Plant materials were ground at 4 ◦C with cold homogenization buffer containing 350 mM sucrose, 70 mM Tris-HCl , 3 mM Na2EDTA, 0.2% BSA, 1.5% PVP-40, 5 mM DTT, 10% glycerol, 1 mM PMSF and 1×protease inhibitor mixture . The homogenate was filtered through four layers of cheesecloth and centrifuged at 4000× g for 20 min at 4 ◦C.

The supernatant was then centrifuged at 100,000× g for 1 h. The obtained pellet was suspended in 350 mM sucrose, 10 mM Tris-Mes , 2 mM DTT and 1× protease inhibitor mixture. Pyrophosphate hydrolysis was measured as described in previous studies. The assay solution for PPi hydrolysis activity contained 25 mM Tris-Mes , 2mM MgSO4, 100 µM Na2MoO4, 0.1% Brij 58, and 200 µM Na4P2O7. PPase activity was expressed as the difference of phosphate release measured in the absence and the presence of 50 mM KCl. After incubation at 28 ◦C for 40 min, 40 mM citric acid was added to terminate reactions. For the measurement of inorganic Pi amount, freshly prepared AAM solution acetone, 2.5 mM ammoniummolybdate, 1.25 M H2SO4 was added to the reaction solution, vortexed and colorimetrically examined at 355 nm.Two-week-old hydroponically grown plants were transferred to 1/6 MS solutions containing 0 or 15 mM MgCl2. After two-day treatment, leaves of all the plants were collected and PPi was extracted from leaf tissue as described previously. Leaf samples were ground to powder in liquid nitrogen, suspended with three volumes of pure water, heated at 85 ◦C for 15 min,vertical hydroponic garden and then centrifuged at 15,000 rpm for 10 min. The supernatants were collected and then centrifuged at 40,000 rpm for 10 min. The obtained supernatants were diluted with pure water and subjected to PPi assay using a PPi Assay Kit according to the manufacturer’s instructions. Fluorescence was monitored with a Safire 2 plate reader set at 316 nm for excitation and 456 nm for emission .The Bio-remediation, Education, Science and Technology partnership provides a sustainable and contemporary approach to developing new bio-remedial technologies for U. S. Department of Defense priority contaminants while increasing the representation of underrepresented minorities and women in an exciting new bio-technical field. This comprehensive and innovative bio-remediation education program provides underrepresented groups with a cross-disciplinary bio-remediation curriculum and financial support, coupled with relevant training experiences at advanced research laboratories and field sites. These programs are designed to provide a stream of highly trained minority and women professionals to meet national environmental needs. The BEST partnership of institutions and participants benefit from a unique central strategy— shared resources across institutional boundaries. By integrating diffuse resources, BEST forms a specialized “learning institution without walls,” where participants can receive advanced training at any BEST site, and where research capabilities flow freely among the participating institutions. Ongoing faculty and student exchange programs, video taped lectures, the Rotating Scholars program, and the BEST web-site ensure that all participants are empowered with opportunities to excel. The BEST partnership consists of Lawrence Berkeley National Laboratory in Berkeley, Calif., Jackson State University in Jackson, Miss., Ana G. Méndez University System in Puerto Rico, University of Texas at El Paso , University of Southern Mississippi Gulf Coast Research Lab, and University of California at Berkeley . The BEST program contract to the partnership is managed by LBNL for the Army Corps of Engineers, Waterways Experiment Station in Vicksburg, Miss.

WES manages the contract for the Army Corps of Engineers and is the contracting entity for DoD. The partnership formed by these participating institutions leverages existing institutional resources by strengthening intramural bio-remediation education and research capabilities, and through outreach programs, to disseminate training and scientific enhancement to other Historically Black Colleges and Universities and Minority Institutions . The BEST institutions are focal points for the development and dissemination of cutting-edge research and technology for the bio-remediation of nitro-aromatic compounds, polycyclic aromatic hydrocarbons and toxic metals. The multidisciplinary BEST partnership strategy creates a flask-to-field solution that develops laboratory research into technology, and technology into field-scale environmental applications required for the cost-effective restoration of damaged environments. This year saw the addition of the University of Southern Mississippi’s Gulf Coast Research Lab and the University of Texas at El Paso as partners in the BEST program.The USM Gulf Coast Research Lab investigators’ focus on PAH and heavy metal phytoremediation along shorelines provides an exciting new focus with increased field opportunities for students. The UTEP investigators are focusing on exciting new metal phytoremediation techniques using desert plants and exciting new techniques to determine risk assessment with PAHs. This year also saw the passage of the program directorship at LBNL from Dr. Jenny Hunter-Cevera to Dr. Terry C. Hazen in October 1999. Dr. Hunter-Cevera, who has managed the BEST program at LBNL since its inception, will be sorely missed, but her new position as president of the Maryland Biotechnology Institutes may provide increased opportunities for collaboration for the entire BEST program. Dr. Hazen, who specializes in bio-remediation field applications, has demonstrated or deployed bio-remediation technologies at more than 50 sites around the United States and in Europe. He has five patents in bio-remediation technologies that are licensed by more than 40 companies in the U.S. and Europe. During the past year, the BEST program has provided minority research training for five high school students, 74 undergraduates, 32 graduate students, three post-doctoral fellows and 10 faculty. Students and faculty investigators have given 43 presentations on BEST research at scientific meetings and have published 17 scientific papers. The program produced a full color brochure and flyers in 1999 for use in recruiting more students, and also sponsored 32 lecture/seminars on bio-remediation. Fourteen videotapes of BEST seminars at LBNL/UCB were distributed to the partner institutions.

These data agree with those reported by Blair et al. . The miR1511 over expression in transgenic BAT93 roots increased the root growth sensitivity to Al and, moreover, an increased sensitivity to AlT was observed in G19833 composite plants engineered for miR1511 expression . These data support the hypothesis that miR1511 induces degradation of ALS3 transcripts thus delaying the adequate root response to AlT stress. Therefore, absence of miR1511 resulting in diminished ALS3 transcript degradation appears to be an evolutionary advantage to Al contamination in soils, leading to an inhibition of the LPR1 pathways, a faster relocation of chelated Al to vacuole and Al-tolerant aerial tissues and a lesser effect on root growth, a phenomenon that partially explains why P. vulgaris Andean genotypes are more resistant to AlT than Mesoamerican ones . Overall, the current results about AlT in arid environments, combined with previous results by other authors, illustrates the complexity of adaptation to drought conditions. Tolerance of these conditions encompass mechanisms of growth and development like root depth and reshaping of the root profile, persistent growth despite drought conditions , continued translocation of photosynthesis from pod walls into seeds resulting in a high pod harvest index , and Al detoxification under unfavorable edaphic conditions . In turn, this knowledge helps designing and interpreting experiments in the introgression of genetic diversity for drought tolerance from wild to domesticated common-bean and breeding drought tolerant common bean, in general . In conclusion, our study reports an original case of the gene evolution of a single MIRNA the MIR1511, within Phaseolus vulgaris and close relatives, allowing adaptation to the aluminum toxicity abiotic stress.Phaseolus vulgaris G19833 and BAT93 MIR1511 sequences were obtained from the Phaseolus vulgaris release v2.1, from Phytozome 12 database, and from NCBI Whole Genome Shotgun database ,danish trolley respectively. Sequences for the phylogenetic tree display in Figure 1 were obtained from the SNPhylo analysis performed by Ariani et al. , based on the collection of representative SNPs located in sequences at least 5 kb from an annotated feature .

Only sequences from non-admixed genotypes were selected for the analysis , constituting a total of 87 sequences from three Phaseolus species , including three populations for the Mesoamerican , one for the Andean , and one for the Northern Peru–Ecuador P. vulgaris gene pool. Sequence alignment was performed thanks to MUSCLE algorithm .Accession PI430191 was used as out group for rooting the phylogenetic tree. Evolutionary analyses were conducted in MEGA X . GBS sequence reads from the 87 selected genotypes were pre-processed as previously described and mapped to the BAT93 MIR1511 sequence using the BWA-MEM algorithm as described by Ariani et al. . MIR1511 was considered complete if at least one read was mapped to the miR1511 mature sequence and at least another read mapped to another part of the miR1511 precursor sequence. When at least one read was mapped to another part of the miR1511 precursor sequence and none on the miR1511 mature sequence, MIR1511 was considered deleted. For further supporting this analysis we performed an additional validation for the samples with a complete BAT93 MIR1511 sequence. In brief, the reads aligning to the BAT93 MIR1511 sequence were re-mapped to the G19833 reference genome. Alignment results showed a partial alignment to these reads in the homologous region containing the truncated MIR1511 in the G19833 reference, up to the deletion visible in Figure 1a . The architecture of transgenic roots from composite plants under control or AlT treatments was analyzed by determining the growth rate of root length, width, and area, as well as the number of lateral roots formed, using the ImageJ software. For both treatments root measurements were done at the beginning of the experiment and after 48 hrs of growth. As mentioned before, AlT treatment plants were first adapted to hydroponic culture in control treatment . They were then taken out from this culture to be quickly photographed -for subsequent root architecture analysis- and were introduced into a hydroponic culture under AlT treatment for 48 hpt to be harvested and photographed again. Data of growth rate of each parameter represent the difference of the values at 48 and 0 hpt. Each root architecture parameter was determined in transgenic roots from 10 to 15 composite plants for each treatment. Statistical analyses were performed using the Mann-Whitney null hypothesis statistical test. One-third of the fields on earth contain calcareous soil. Plants grown in calcareous soils that are low in iron availability demonstrate decreased growth and yield. Under conditions of low Fe availability, rice plants induce transcriptional responses that promote the uptake of Fe from the soil as ferric Fe–mugineic acid phytosiderophore chelates and ferrous Fe ions.

Thus, an understanding of the mechanisms by which plants such as rice respond to Fe deficiency is required to maintain plant yields and prevent food shortages. In our previous studies, Kobayashi et al. identified two Fe-deficiency-responsive cisacting elements , which confer Fe deficiency-induced expression in rice roots and leaves . We also found two rice transcription factors, IDE-binding factors 1 and 2 , that bind to IDE1 and IDE2, respectively . IDEF1 and IDEF2 belong to uncharacterized branches of the plant-specific transcription factor families ABI3/VP1 and NAC, and they exhibit novel sequence recognition properties. IDEF1 and IDEF2 transcripts are constitutively expressed in both roots and leaves. Transgenic rice plants that express IDEF1 under the control of the IDS2 promoter were found to be tolerant to early Fe deficiency in hydroponic culture and calcareous soil. IDEF1 regulates the ferrous ion transporter gene OsIRT1, the Fe-deficiency-induced transcription-factor gene OsIRO2, and other genes related to Fe deficiency. IDEF2 regulates the metal nicotianamine transporter gene OsYSL2 as well as other Fe-deficiency-related genes. Nevertheless, the specific mechanisms and tangential pathways affected by these key transcription factors have not been elucidated fully. Therefore, delineation of the expression patterns and characteristics of IDEF1 and IDEF2 could help elucidate the response of rice plants to Fe deficiency. To understand the mechanisms by which plants respond to Fe deficiency, we examined the expression patterns of IDEF1 and IDEF2 by promoter–GUS analysis under Fe-sufficient and Fe-deficient conditions. This information could be critical for the creation of rice varieties that grow in problem soils. We analyzed the spatial expression patterns of IDEF1 and IDEF2 during the germination, vegetative, and seed-maturation stages by histochemical localization of GUS staining as described by Inoue et al. and Nozoye et al. . Two kb of the 5’ region upstream of the translation start site was used as the promoter sequence for IDEF1, whereas 2 kb of the 5’ region upstream of the transcription start site was used as the promoter sequence for IDEF2. Rice was transformed with the IDEF1 promoter–GUS or the IDEF2 promoter– GUS by an Agrobacterium-mediated method, and T1 or T2 seeds were obtained for use in the analysis. To induce Fe deficiency, 28-day-old plants were cultivated hydroponically without Fe– EDTA 1-12 days before harvest. For analysis during the flowering and maturing periods, IDEF1 and IDEF2 seeds were cultured in Fe-sufficient artificial soil with fertilizer, and developing seeds were progressively sampled for GUS expression analysis. IDEF1 and IDEF2 expression was observed in both the endosperm and embryo during the early seed germination period. IDEF2 expression was induced in the leaf primordium during germination. IDEF1 and IDEF2 regulate genes related to Fe deficiency as well as other unknown genes. The expression patterns of some Fe-deficiency-induced genes have been investigated during the early germination period . The expression patterns of IDEF1, IDEF2, and OsNAS1 were similar; all were expressed in the embryo and endosperm. Conversely, other Fedeficiency-induced genes such as OsNAS2, OsNAS3, OsNAAT1, OsDMAS1, OsYSL2, and OsIRT1 were not expressed in endosperm tissues. It is speculated that genes involved in phytosiderophore biosynthesis and Fe transport are differentially regulated in the germination and vegetative stages under low Fe conditions. IDEF1 or IDEF2 promoter–GUS plants were grown in hydroponic culture under Fedeficient or Fe-sufficient conditions. The expression patterns of IDEF1 and IDEF2 were found to be similar despite Fe availability. In leaf blades of IDEF1 lines, strong expression was observed in mesophyll cells and in small vascular bundles. Interestingly,vertical aeroponic tower garden the main vascular bundle was not stained in either Fe-sufficient or Fe-deficient leaf samples. It is assumed that the principle function of the main vascular bundle is to transport water and nutrients and that Fe is needed in mesophyll cells and small vascular bundles for photosynthesis. In contrast to IDEF1, IDEF2 was highly expressed in vascular bundles but not in mesophyll cells. In the inner layers of the stem/leaf sheath of IDEF1 lines, mesophyll and small vascular cells demonstrated dense staining, indicating high IDEF1 expression. In root sections, IDEF1 and IDEF2 lines showed strong GUS staining in the secondary roots, which emerge under conditions of Fe deficiency.

This finding suggests that IDEF1 and IDEF2 induce Fe-deficiency-responsive genes such as OsIRT1 and OsIRO2 in secondary roots and that this may play an important role in the uptake and utilization of Fe in low Fe conditions. GUS staining was also found inside the vascular bundles of root sections in IDEF1 and IDEF2 lines. IDEF1 and IDEF2 lines were cultured in soil to investigate spatial expression patterns during the flowering and maturation periods. Prior to anthesis, IDEF1 line pollen showed high expression. After fertilization, the ovary was found to be heavily stained. Expression was also observed in the vascular bundles of the husk during the flowering and early seed development stages. There was strong staining of the embryo and the aleurone layer in the late progress maturation stages . In embryos, the scutellum and leaf primordium were densely stained. Similar to IDEF1, IDEF2 was expressed in most pollen in the flowering stage. IDEF2 was also expressed in immature seeds just after flowering and in the dorsal vascular sections in the late maturation stage. In flowering plants, a primary role for boron is to form a diester cross-link between two monomers of rhamnogalacturonan-II , a pectic polysaccharide present in the cell walls of all vascular plants . Rhamnogalacturonan-II is a structurally complex domain of pectin , which comprises 12 different monosaccharides that are linked together by at least 20 different glycosidic linkages . Nevertheless, its structure is largely conserved in vascular plants . The majority of RG-II exists in the wall as a dimer that is generated by forming a borate diester between the D-apiose of side chain A of two RG-II molecules. The inability of RG-II to properly assemble into a dimer results in the formation of cell walls with abnormal biochemical and bio-mechanical properties and has a severe impact on plant productivity.Nevertheless, the mechanisms that drive the interactions between borate and RG-II are poorly understood . There is increasing evidence that alteration of RG-II structure and cross-linking have severe impacts on plant growth, development and viability. To date, the only characterized RG-II biosynthetic enzymes are the rhamnogalacturonan xylosyl transferases , which catalyze the transfer of xylose from UDP-xylose to fucose to form ɑ-xylose–fucose in vitro . Inactivation of RGXT1 and -2 has no discernible effect on plant growth or RG-II structure , implying redundancy of function, whereas mutations affecting RGXT4 lead to defects of root and pollen tube growth that are lethal . Mutations that prevent the synthesis of UDP-Api and CMP-Kdo are also lethal and provide further evidence for the essential role of RG-II in plant growth . In the dwarf Arabidopsis mutant murus 1 L-galactose replaces L-fucose in several cell wall polysaccharides, including RGII, because the plant is unable to produce GDP-fucose in its shoots as it lacks GDP-D-mannose-4,6-dehydratase GMD1 . This has been shown to result in the incomplete formation of the A side-chain of RG-II, which in turn reduces the stability of the borate cross-linked dimer . Thus, the structural integrity of RG-II is probably important for its biological functions. Pectic and hemicellulosic polysaccharides are synthesized in the Golgi apparatus using activated donor substrates, typically in the form of nucleotide diphosphate-linked sugars . However, most NDP sugars are synthesized in the cytosol . Thus, NDP-sugar transporters are required to provide substrates for glycan synthesis . The Golgi-localized NST sub-family, which forms part of clade IIIa of the NST/triose phosphate transporter super family , comprises four members related to GONST1 , the first nucleotide sugar transporter described in Arabidopsis . The members of this family are the only Arabidopsis NSTs that contain a predicted GDP-binding motif .

Spanning approximately 3,460 ft, this levee will be made of compacted clay fill with a unit weight of 110 pcf, and cohesion of 1000 psf. The levee will have a slope of approximately 3H to 1V, a crown width of 16’, and a height of 1.5’ above the 100-year flood free boad elevation. While backside erosion may be a concern, it will be too costly to provide the entire internal perimeter with installed rip rap. Rather, to account for backside erosion, rip rap will be stocked piled on site so that it is readily available for the emergency armoring of the internal portion of the levee. Also, to allow for visual monitoring and ensure adequate maintenance vegetation must be continually removed along the toes of the levees. The Hydroponics component of the proposed system is a synthesis of Nutrient Film Technique and Deep Water Raft Hydroponics to address the needs of the Aquaponics Water Management System. Component design ratios are based on the reliable and robust University of Virgin Island’s 1/8 acre system which has been in operation since the 1980’s and is a model system for commercial aquaponics around globe. Hydroponics includes three main sub-components: Nutrient Film Tubes, Buoyant Rafts, and Anchors. Nutrient Film Tubes are hollow core pipes that transport nutrients to the plant roots. They are constructed from polyvinyl chloride and have holes eight inches apart containing net pots with growing media that supports plant roots. With 36 100-ft tubes per subsystem, each will support 5400 planters for an expected annual production of 11,000 lbs of vegetation. Aquaponics Water Management System will be designed with 600 subsystems,flower pots for sale so there are 3,240,000 planters in total for an approximate annual yield of six million pounds of produce. Buoyant rafts are constructed from PVC encapsulated Styrofoam, which may be recycled from Styrofoam used in packaging or other prior uses.

The buoyant force per volume of Styrofoam raft is approximately 55 lbs per cubic foot. The buoyancy required to keep each subsystem afloat is approximately 35,000 to 40,000 lbs. Therefore Aquaponics Water Management System requires approximately 400,000 cubic ft of Styrofoam rafts to produce approximately 21 to 24 million pounds of buoyancy to keep the system afloat. Anchors are based on a pole and slide method designed to prevent lateral movement of the rafts by wind and wave action while allowing for vertical movement of the rafts when there are changes in water height. The pole is a pier encapsulated with PVC and anchored in the ground by a concrete foundation or helix foundation anchor depending on the engineering load requirements. The loop is designed to slide vertically along the pier and is attached to the rafts as shown in Figure 4. The advantage of hydroponics over terrestrial agriculture is that it allows for more diverse crop production. While corn, asparagus, and sugar beets were abundantly grown on Sherman Island during most of the twentieth century, the majority of crops currently grown are field crop, such as wheat and barley. A variety of leafy vegetables, herbs, and other crops proven compatible with hydroponics can be potentially grown on Sherman Island with this system: artichokes, arugula, asparagus, basil, beets, broccoli, brussels sprouts, cabbage, carrots, cauliflower, celery, cilantro, collard, eggplant, endive, garlic, lavender, leek, lettuce leaf, okra, onions, parsley, parsnips, peas, bell peppers, radishes, raspberries, spinach, strawberries, and tomatoes. Floriculture is also compatible with hydroponics. Combinations of these candidates would be used in Figure 4. Schematic of anchoring system . order to take advantage of seasonal cycles, though some of these crops grow continuously throughout the year. Because the area does not experience extreme temperatures and frost, biennials and perennials could survive in the outdoor environment of the system. This system provides a competitive opportunity to produce crops that are unique to the area, specific to a niche market, and possibly organic. However, additional research needs to be done to test which crops would be successful at growing on site, especially with regards to the effects of seasonal weather, water salinity, bird migration, insect plagues, and other factors that may cause crops to fail.

While the equipment and resources for Aquaculture greatly depends on the fish species, a general design will be described. Incubation jars and tanks of varying sizes are needed to raise the fish. Stocking density depends on the fish size and stage of life and affects the number of tanks needed. Tanks should have water control values. Aerators and pumps are needed to provide oxygen and circulation. For fish health, filters are needed to regulate particulate concentrations in the water to the fish tanks, although not many would be needed by having snails in the tanks for cleaning. Feed may be live or dry, with the latter able to be delivered by automation. Depending on the temperature control needed, heaters or chillers may be necessary. A barn-like structure is necessary to house incubation and larvae tanks while adult fish tanks can be kept outdoors. Adequate piping and electrical wiring is needed among tanks, filters, and controllers. Parameters of concern to monitor are water pH, total ammonia nitrogen concentration, salinity, water temperature, and dissolved oxygen concentration. These parameters can be monitored by sensors and controlled by filters, heaters or chillers, and aerators. Each of the 600 aquaculture subsystems is comprised of 8,240 gallons of rearing tanks, 400 gallons of filtering and degassing tanks, 2,000 gallons of clarifiers, 50 gallons of base addition tanks, and 200 gallons of sump. The Aquaculture system has the potential to produce fish species for several purposes including conservation, fishing or live market sale. At first, conservation of the Delta Smelt or other endangered species was considered, but this prospect was discouraged after speaking with Dr. Joan Lindberg, director of the Fish Conservation and Culture Lab, who explained that Delta Smelt aquaculture is greatly resource-intensive and not economically viable for the proposed location because the California Department of Water Resources is planning other fish conservation efforts in Rio Vista. Therefore, fish rearing of species popular for fishers or sale on the live market would be more feasible for Aquaponics Water Management System.

Expert opinions agree that sturgeon or catfish are good candidates for this system to produce net profit on the live market. In addition, these species are currently fished in the Delta. Numerous literatures on the sturgeon and catfish aquaculture allows for detailed design and protocol to be easily made. Because catfish aquaculture is predominantly in the Southeast of the United States and sturgeon aquaculture research has been completed at U.C. Davis, this report will focus on sturgeon. Sturgeon aquaculture is especially lucrative for its caviar production. While it takes almost nine years for female sturgeon to be mature with eggs, younger sturgeon is valuable for its meat. At about 18 months of age, sturgeon is profitable to sell on the live market. Raising some fish from this age to 36 months allows for sex determination, and females may be further raised for caviar production while males sold for meat. With the 600 subsystems, there is enough capacity for fifteen brood stock and their offspring. This is expected to yield 215,000 fish for sale at 18 months. Since sturgeon is already present in the Delta, unintentional release into the San Joaquin River would not jeopardize the Delta’s fragile ecosystems.The internal cutoff levee encloses the flood storage zone and will serve as a secondary defense should river levees fail. In doing so, it must be able to withstand the principal causes of levee failure: over topping, surface erosion, internal erosion , and slides within the levee embankment or the foundation soil. Any levee constructed on Sherman Island inherits a number of issues that require intensive design, first and foremost being the quality of the foundation soils. The generalized subsurface soil profile and proposed internal levee when the internal area is back flooded to a level of 3ft is shown below in Figure 5. Most concerning is the approximately 40 foot peat layer that immediately underlies the levee. As a result, the foundation soils are extremely weak and compressible, variable, and feature severe underseepage vulnerability, which if left unaddressed can lead to piping and levee failure. The most common method of stabilizing is excavation and replacement of the peat layer however this is not economically feasible because the peat layer is too large. In addition to the problems with the peat layer there is a deep sand layer that has the potential to liquefy during a seismic event. US Army Corp of Engineers standards dictate that levees should be designed to a factor of safety of 1.3 to 1.4, dependent on the time scale for which a levee will be holding back elevated levels of water. Table 1 shows the minimum factors of safety for levee slope stability as designated by the USACE. While the internal levee will not be kept at a high flood level indefinitely,tower garden the levee must maintain at least a factor of safety of 1.4 as it is the critical piece of infrastructure protecting the aquaculture system and the rest of Sherman Island. Modeling of our proposed clay fill levee will determine the optimum back flooding to maintain this factor of safety while equalizing the pore pressure as much as possible. During design and construction of this levee, the consolidation of the peat soil needs to be considered.

Until the peat layer has been compressed, there will be minimal structural stability to the levee. Since, as described by Dr. Seed , for approximately every foot of fill placed the peat will settle approximately a foot. As such, the levee will need to be constructed in stages with a first layer being installed and then allowed to settle. To allow for sufficient consolidation, this layer will remain for approximately a year and a half. At this stage the second layer can be added up to the specifications of the levee design. The levee will be constructed with compacted clay fill with a unit weight of 110 pcf and cohesion of 1000 psf, to best limit flow through the levee.The existing levees that surround Sherman Island feature clearly delineated layers of soil similar to that in the internal portion of the island. Under seepage is a significant issue for these levees with the most common failure concern being piping. Additionally, the levees are constructed of sand rather than low hydraulic conductivity clays. Settlement through the years has led to constant repairs, and as such an irregular levee shape. Figure 6 displays the soil and levee profile that will be used for the geotechnical analysis. This levee is extremely susceptible to failure in high flood water events due to the high head differential between the rivers and subsided land inside Sherman Island. Therefore back flooding the flood storage zone is a favorable approach because it reduces the difference in hydraulic gradient.To determine the effects of back flooding, on the existing levees and the constructed levee, seepage analyses and slope stability analyses parented from the seepage analyses were performed. For the proposed internal levee, the profile was generated from soil borings and CPT tests provided by Caltrans for the Antioch Bridge. The profile for the existing levee was created for the RESIN project and given to us for use by Anna Harvey. This profile is appropriate for the project site as this profile was taken from an area near a previous failure, and has an extremely large peat layer. If the flood management can benefit this failure prone levee, then it can aid in the protection of levees at the southwest site. It should be noted that the actual 100 year and 50 year flood levels are +7.5 ft MSL and +5.5 ft MSL respectively. However, for simplicity of the model and to account for variability such as sea level rise, +8 and +6 ft were chosen, to provide a lower end estimate of factors of safety. The slope stability analyses were run with the Spencer method, as it is most applicable to this situation and is most accurate. . Additionally, the simulations were run assuming no tension crack. The data given in Table 2 from simulations performed demonstrate that back flooding the internal cutoff levee to a level of MSL + 6 ft maintains a factor of safety of 1.4 while MSL + 7 ft maintains a factor of safety of 1.3.

OPT3 is a member of the oligopeptide transporter family and members of this family have been shown to mediate the transport of a broad range of peptides . Arabidopsis OPT3 has also been reported to rescue the ability of yeast mutants defective in Cu and Mn transport to grow on low concentrations of these transition metals . However, so far there is no direct evidence to suggest that OPT3 mediates the transport of transition metals, in the ionic form or complexed with a ligand, or whether OPT3 mediates the transport of a ligand that facilitates the uptake and accumulation of transition metals into the cell. In fact, our OPT3 localization experiments in yeast show that OPT3–YFP fusions are unable to transit out of the ER to the plasma membrane . This intracellular localization of OPT3 makes it difficult to interpret the ability of yeast strains defective in transition metal transport to grow on minimal media when expressing OPT3. Glutathione is a small peptide that has also gained recent attention in metal-status signaling via GSH coordinated intermediaries of the iron–sulfur cluster assembly machinery.Our radiotracer experiments using 35S-GSH and the ferric reductase assay in opt3-2 roots show that leaf-to-leaf movement of GSH was unaffected and that foliar application of GSH does not suppress the constitutive Fe-deficiency response in opt3-2. These results suggest that shoot-to-root transport of GSH alone has little effect on the long-distance signaling of the Fe status in Arabidopsis.Phloem transport plays a key role in delivering nutrients, including metals, hydroponic net pots to developing seeds . However, the mechanisms of toxic heavy metal loading into seeds are largely unknown. Nicotianamine, GSH, and PCs are the main metal-chelating molecules found in phloem sap . Nicotianamine has been shown to form complexes with Fe, Cu, Zn, and Mn, while GSH and PCs preferentially bind to Cd .

The differential partitioning of Cd among roots, leaves, and seeds in opt3-2 relative to the essential metals Fe, Zn, and Mn suggests that independent mechanisms mediate the partitioning of essential and non-essential metals, likely as specific metal–chelate complexes. Understanding phloem-mediated transport and seed loading mechanisms of individual metals and metal–ligand complexes will be important to restrict accumulation of toxic metals in seeds while ensuring the accumulation of essential metals. In summary, we show that Arabidopsis OPT3 is expressed in the phloem and functions in the long-distance shoot-to-root signaling of Fe/Zn/Mn status. When OPT3 expression is compromised, there is a mis-regulation of genes mediating uptake and mobilization of trace metals leading to an over-accumulation of cadmium, but not other metals, in seeds. We further show that mobilization of Fe2+ between leaves is impaired in opt3-2 and that targeted OPT3 expression in leaves is sufficient to restore Fe/Zn/Mn status signaling to roots providing molecular information on shoot-to-root Fe-status signaling. Sensing and regulation of trace-metal homeostasis in plants have been long-standing questions in plant biology and the results presented here offer new insights and avenues to advance our understanding of how essential and nonessential metals are accumulated and distributed within plant tissues.Wild-type and opt3-2 seeds were surface-sterilized, stratified at 4°C for 48h in the dark, and germinated under a 16-h light/8-h dark photoperiod. For Cd sensitivity experiments, ¼ MS plates were supplemented with 50 μM CdCl2 and allowed to grow vertically for 14 d. For metal determination in seeds, 2-week-old seedlings were transferred to Sunshine Basic Mix 2 soil supplemented with heavy metals as described . For metal determination in roots and leaves, plants were grown hydroponically as described previously . Elemental analyses were performed by ICP–OES at the UCSD/Scripps Institution of Oceanography analytical facility using dried rosette leaves, roots, or seeds digested overnight in trace-metal grade 70% HNO3 as described previously .In field experiments performed in co-operation with CIMMYT, 16 contrasting maize cultivars were tested for N efficiency and the underlying mechanisms for grain yield formation .

N-efficient cultivars were found to have a high N uptake and dry matter accumulation after anthesis, while N uptake and dry matter production until anthesis were not decisive for N efficiency. Further characteristics of N-efficient cultivars were delayed leaf senescence , a high harvest index and high kernel numbers . N uptake and dry matter production after anthesis have frequently been found to be decisive for grain yield in cultivar comparisons both under low and high N supply . In almost all these cases, cultivars with an improved performance during reproductive growth were also characterized by delayed leaf senescence. The causal relationships between the different traits are not yet clear. Genotypic differences in delayed leaf senescence might improve dry matter production after anthesis and thus increase harvest index and yield. It may also affect N uptake, due to an enhanced C supply to the roots. This view implies a key role for leaf senescence. However, delayed leaf senescence may also be merely a symptom of increased N uptake. To unravel the relationships between leaf senescence and the other traits decisive for N efficiency, correlation coefficients between the traits were calculated . Leaf senescence score 28 days after anthesis, with a high score representing a high ratio of senescent leaves on the plants, was negatively related to N efficiency in all investigated environments. Close relationships between leaf senescence score and dry matter accumulation and N uptake after anthesis, however, were found only in one of the experiments . This finding only partially support the above described assumption that delayed leaf senescence causes improved reproductive growth and N uptake. Surprisingly, leaf senescence score correlated with kernel numbers and harvest index suggesting that leaf senescence changes the pattern of N remobilization to the kernels. Thus, although delayed leaf senescence appears to be a decisive part of N efficiency and is suited as a selection trait for N efficiency, its physiological action remains to be elucidated. To test if leaf senescence might be a suitable selection trait for N efficiency also in short-term experiments, the same 16 tropical maize cultivars that were used for the field studies were grown in hydroponics . Leaf senescence was induced by subjecting the plants to N deficiency.

The progression of leaf senescence was monitored by photosynthesis and leaf chlorophyll measurements that were estimated by SPAD values. Cultivars differed both in SPAD values and photosynthesis rates of old leaves during N deprivation. Photosynthesis rate during leaf senescence proved to be a better indicator for N efficiency in this study than leaf chlorophyll content. Significant negative correlations were found between SPAD values, photosynthesis rates in the nutrient-solution experiment and leaf senescence scores in the field experiments, and positive correlations were found between photosynthesis rates and grain yield under low-N conditions in the field. The data suggests that the assessment of the capacity of a genotype to maintain a higher photosynthetic capacity of old leaves during N deficiency-induced senescence at the seedling stage may be suited as a selection parameter for N efficiency. However, photosynthesis rate during leaf senescence could explain only up to 20 % of the cultivar differences in N efficiency, while leaf senescence in the field experiments could explain 47 % . Enzymes within the chloroplast stroma are degraded early during leaf senescence which could be responsible for the decline in photosynthesis rate . Plant and leafN status at the beginning of the N deficiency period might influence the onset of leaf senescence. Plant-N status is determined by N uptake during early vegetative growth and depends on N supply during that period. An efficient root-N uptake rate during the N depletion period will prolong the N supply to the leaves. Apart from improving leaf-N status, this also increases cytokinin production of the roots , which will also delay leaf senescence . The leaf-senescence rate might also be influenced by the rate of N export from the leaf. The amount of N exported depends upon the breakdown of N compounds within the leaf and thus protease activity, but might also be influenced by sink strength. These findings raise the question if cultivar differences in N deficiency-induced leaf senescence might depend on the initial leaf-N content,blueberry grow pot which may be influenced by the N supply during leaf growth, the N uptake into the leaf or the total plant, or the amount of N which is exported from the leaf. Clarification of these aspects may help simplifying and/or improving the experimental procedure for an evaluation of N deficiency-induced leaf senescence in short-term experiments as a marker for N efficiency. Therefore, photosynthesis rates and leaf-N contents were investigated before and during N deficiency-induced leaf senescence for maize cultivars grown in hydroponics . The plants were pre-cultured at two different N rates thus creating differences in N status. Photosynthesis rates decreased considerably during leaf senescence; however, this was not always related to a decrease in leaf-N content of plants pre-cultured at low-N supply. In leaves of plants pre-cultured at high N supply, photosynthesis rates and leaf-N contents declined more in parallel. The decrease in photosynthesis rate must, therefore, have been governed by other factors than leaf-N status. This suggests that N remobilization was not the initial cause for N deficiency-induced leaf senescence, but may rather reflect leaf-inherent differences in leaf senescence. A dissection of N import and N export from the senescing leaf during the N-deficiency period was performed by 15N labelling.

Although there were only small net changes in leaf-N content during the N-deficiency period at N1, considerable N amounts were exported from and imported into the leaf during this time span . Leaf-N contents before the onset of leaf senescence were more than two times higher at high compared to low-N supply. The amount of N exported during N deprivation was nearly four times higher at high N compared to low N. These results suggest that N export was mainly governed by N availability in the leaf. Cultivar differences in leaf-N content prior to leaf senescence had no impact on leaf-N content during leaf senescence . Unexpectedly, N import represented a quantitatively not negligible part of total leaf-N even during leaf senescence, and cultivar differences in N import were also important for differences in total leaf-N during leaf senescence. Since N import was not related to total plant N uptake , it was probably governed by leaf-inherent factors. Some observations made by quantifying leaf and plant-N flows during N deficiency-induced leaf senescence were unexpected. First, photosynthesis rate decreased earlier and stronger than leaf-N content . This could be due to the degradation of N-containing enzymes within the chloroplast stroma . Alternatively, the declining photosynthesis rate induced leaf senescence and consequently N remobilisation from the leaf . Our results suggest that the decrease in photosynthesis rate might have been caused by a negative feedback regulation due to an accumulation of C assimilates in the leaves, since specific leaf weight increased during N deprivation . Leaf-area growth and thus shoot growth is strongly decreased by N deficiency mediated via cytokinins produced in the roots . A poor leaf growth will lead to a low carbohydrate demand and a low phloem-sap flow from matured leaves to growing leaves. This will also affect N retranslocation, since it could be shown that a low phloem-sap flow also decreases amino acid translocation . Thus, N retranslocation from senescing leaves under N deficiency might be delayed due to a low sink-N demand. N import might have played a decisive role for the induction of leaf senescence, since nitrate influx regulates the induction of leaf senescence . In this study, N import was probably governed by leaf-inherent factors instead of reflecting total plant-N uptake. Nitrate-N enters the leaf by the transpiration stream. Therefore, a decrease in stomatal conductance affects N import. This might be due to the decreased photosynthesis rate or mediated by abcisic acid , which is known to induce stomatal closure. Indeed, differences in ABA contents have been found in senescing leaves of an early-senescing and a stay-green phenotype of maize . The possible carbon and nitrogen flows in the plants which might influence leaf senescence of vegetative plants are summarized in Figure 2. Photosynthesis rates and leaf-N contents of plants pre-cultured at low or high N supply were significantly related to leaf senescence scores at anthesis of the same cultivars grown at low-N stress in the field .

Participants whose data in any run exceeded motion thresholds were discarded from the analysis. First-level analysis was performed by creating a standard regression model with estimated HRF for each condition while the six motion parameters and outlying volumes as determined by ART added to the design matrix as regressors of no interest. For the standard GLM analyses , three sets of contrasts for each participant were created. First, we compared total activation for the Passages against the Baseline condition as well as the Central and Peripheral conditions against the Baseline condition. Then, Central – Baseline and Peripheral – Baseline contrasts were directly compared. Finally, to further understand the potential overlap and specificity of passages as related to scrambled words, we examined the Boolean conjunction of Passages – Baseline and Words – Baseline . To investigate the dynamic processes involved in building a coherent text representation, brain regions were examined that demonstrated increased or decreased activation as a function of time as the participant progressed through the Passages, relative to the Baseline. These temporal analyses determined whether the dynamic process of building a text representation was associated with increased or decreased activation in specific areas. To accomplish these analyses, for each run we modeled each phrase onset as a stick function with a height equal to the difference between phrase onset and the initial phrase of Paragraph 1. For instance, to examine increased activation over time for the Central phrases, if Central phrases were presented at the 3rd, 7th, and 9th TR, the resulting vector would be. The “1” in the vector represents the onset in which the first event of interest occurs. Any proceeding event is weighted in proportion to the time passed between the new event and original event of interest. The resulting vectors were then convolved using the HRF to create conditions of interest and were inserted into the first-level GLM. This formulation allowed us to model a linear relationship,fodder system for sale thus representing temporal/dynamic changes associated with each condition.

To temporally model the Passages against the baseline, we collapsed the Central and Peripheral onset vectors and built the condition of interest using the same formulation above.The neural correlates of expository text comprehension have not previously been examined in fMRI. This study not only sought to identify a network of regions specifically activated for discourse level processing of expository text but, due to the fluctuating cognitive demands within the comprehension of a single text, also examined the neural systems that underlie comprehending the text over time. Finally, to further identify core processes of expository text comprehension, this study aimed to define the functional underpinnings of comprehending textual centrality, which is one key indicator that a reader has formed a coherent mental representation .In expository text comprehension, we see co-activation of left-lateralized language regions and two heteromodal association areas—left AG and PCC/PCU—commonly associated with higher-order cognitive processes . Specifically, the observed language regions have been identified as part of an executive semantic control network , with left IFG and left posterior MTG thought to direct semantic connections to fit the current context, while regions in the anterior temporal lobe are thought to store and integrate specific semantic associations . These regions have been observed to activate for different levels of comprehension , and an examination of the scrambled words condition compared to expository comprehension in our own study shows that regions in this executive semantic control network overlapped for both conditions . From the perspective of hierarchical comprehension, in which reading is comprised of discourse comprehension built on top of single word comprehension, these shared regions could be interpreted as contributing to word level processes only. However, given previous findings indicating these regions are active when processing semantic associations for words and sentences, hierarchical assumptions of functionality may overlook these regions’ complex contributions to reading . This complexity is supported by the temporal analyses discussed below, which show activations of the semantic control network over time that are unique to expository text. The heteromodal regions that we see co-activated with the semantic control network were activated for expository text, not words .

The distinction of these regions as discourse-specific is unsurprising. Both regions have been previously identified as multi-function, cognitive “hubs,” which perform higher-order cognitive processes . In the context of language, left AG is primarily associated with semantic memory, incorporation of semantic information into a coherent whole, and making top-down semantic predictions , while PCC has been noted for its activation at updates in readers’ mental representation of narrative texts . This co-activation of left AG and PCC, along with language regions suggests that expository text comprehension involves a core semantic-processing network which integrates semantic information both at the word- and sentence-level, along with activation of heteromodal regions that more globally update the situation model into a coherent whole.Our findings of posterior midline and left AG in expository text when compared to single word reading is similar to what is reported for narrative, and further supports the possibility that these regions are involved in global comprehension processes which aren’t necessarily dependent on discourse type . Unlike previous findings on narrative comprehension, however, it’s important to note that apart from these left-lateralized activations, our findings suggest that expository text comprehension does not rely on additional regions within the theory of mind network— a network associated with social inference processes and contextualization of narrative text within world knowledge . The absence of other primary hubs of the theory of mind network, particularly the medial prefrontal cortex, emphasizes that narrative and expository texts may have critically different cognitive requirements, stressing the need to examine both text types in order to isolate specific comprehension processes susceptible to dysfunction. A direct study of narrative and expository texts is needed to further explore these comparisons. Contrary to our hypothesis, expository text did not show activation of the dorsal attention network. This could be a result of the fact that our participants were skilled adult readers, and our passages were written at a fourth grade reading level. We created the passages to be highly cohesive, easily decodable, and thus easy to comprehend. However, it is likely that these relatively undemanding passages decreased the overall EF load.

Interestingly, regions in the executive semantic control network progressively activate over time in passages alone, despite being activated in both passages and words in the mean analysis . This shows that these semantic regions have a unique activation pattern in expository text comprehension, further supporting findings that they play multifunctional roles interacting with different comprehension levels . Observations of the BOLD signal in these regions and its correlation to the HRF for central or peripheral events suggest that these increases are specifically due to language processes . During discourse comprehension, in order to maintain the reader’s situation model, these semantic networks would necessarily be increasingly relied on over the course of the passage. As the amount of required semantic connections increases, both a greater store of semantic associations and increased executive direction of those associations are required to ensure that new information aligns with and is integrated into the current situation model . It has also been suggested that left IFG and left posterior MTG play a role in integrating modality-specific knowledge into the reader’s situation model, which could also contribute to its increasing activation through comprehension . When looking at regions that decrease over the course of comprehension, we see decreased activation of right IPS in both word and passage conditions . However, compared to words and baseline, the BOLD signal in right IPS shows a marked decrease in activation at central and peripheral events, suggesting that IPS could have a unique relationship with discourse-specific processes . Interestingly, this region has been previously implicated in discourse-level narrative comprehension. Ferstl et al. suggested that right IPS is involved in attentional shifts from local to global aspects of the mental representation of the text. In narrative comprehension compared to scrambled sentences, Yarkoni et al. saw an initial spike of activation in the same region, followed by a linear decrease over the time course of comprehension, attributing the activation pattern to visuospatial updates involved in initial situation model construction. Similarly, when contrasting the first paragraph of expository text to the second paragraph , fodder growing system we see activation in the same region, suggesting that right IPS is more prevalent in the beginning of comprehension than the end. Consequently, decreased activation of right posterior parietal cortex could be indicative of the region’s role in construction of the situation model. The overlapping temporal decrease in scrambled words could reflect readers’ initial attempts to build a situation model despite incoherence, particularly since task types were not identified to readers ahead of the stimuli. However, higher-order interpretations of IPS activations in texts should be treated carefully, as activations could reflect subtle, visual attention differences between tasks. These findings closely reflect Yarkoni et al.’s narrative findings, and support a cross-genre reading model in which visuospatial updating and attention regions are involved in the initial construction of a reader’s mental representation of a text, and executive semantic control areas are increasingly necessary for its maintenance. The similarities between studies suggest not only that there are distinct cognitive stages during text comprehension, but that some of the neural structures underlying these stages may be shared across text genre.Our second aim was to examine the neural correlates associated with the ability to distinguish between a text’s central and peripheral ideas, or readers’ sensitivity to centrality . Skilled readers demonstrate sensitivity to centrality by recognizing and recalling a greater proportion of central than peripheral ideas ; however, identifying central information is a skill known to be particularly vulnerable to disruption among individuals who experience comprehension difficulties . Because sensitivity to centrality is both a critical component of comprehension and one that is vulnerable to disruption, we aimed to explore the neural underpinnings of this process. A direct comparison of mean group-level activation indicates that central text ideas are cognitively distinct from peripheral ideas, eliciting greater activation in textual integration regions when compared to peripheral.

Specifically, reading central relative to peripheral ideas was associated with posterior midline structures, namely PCC and PCU , as well as anterior temporal regions. These findings relate to previous studies of discourse processing that have found PCC and PCU to be associated with forming connections among text ideas , updating story representations , and connecting text-based information to prior knowledge . Additionally, Speer et al. found greater PCC activation when readers processed the points in the text that required the greatest degree of mental model updating. Activation of STG/MTG has also been associated with linking semantic ideas to form a connected narrative . These findings confirm that in addition to readers’ ability to behaviorally distinguish between central and peripheral information, the degree of textual relevancy is associated with a distinct neural network of textual/extra-textual integration and mental representation regions in the comprehender.Comparing central and peripheral activations over time shows that as the text progresses, central ideas recruit different parts of the language network than peripheral ideas. specifically, regions within the executive semantic control network differentiate central and peripheral processing over time, with central ideas increasingly relying on the left IFG, and peripheral ideas activating left anterior MTG independently from and posterior MTG to a greater extent than central ideas. This centrality-driven division between frontal and temporal semantic processing regions can be seen in the BOLD signal, with left IFG and left anterior MTG initially responding generally to the switch from non-word to word stimulus, before demonstrating clear correlation with central and peripheral HRF prediction peaks, respectively . While both temporal and frontal regions are implicated in semantic cognition, it has been suggested that left posterior MTG acts as a general interface between lexical and conceptual knowledge, anterior MTG is involved in specific semantic associations, while left IFG is more context specific, activating for conceptual knowledge that is cued by the preceding text . Consequently, for central textual ideas, which are more semantically-dependent on previous ideas, the IFG is increasingly involved in making appropriate semantic connections to the established context. On the other hand, processing peripheral ideas, or ideas which have Comparing central and peripheral activations over time shows that as the text progresses, central ideas recruit different parts of the language network than peripheral ideas. specifically, regions within the executive semantic control network differentiate central and peripheral processing over time, with central ideas increasingly relying on the left IFG, and peripheral ideas activating left anterior MTG independently from and posterior MTG to a greater extent than central ideas.

The patterns of the IAA levels that developed in less than 10 days in the new shoots on the hanging and upright stem fragments may have resulted from at least two different factors. First, we hypothesize that the overall seasonal pattern resulted from the in situ temperature conditions at the time we sampled the main stem fragment. Secondly, the difference between IAA levels in the new shoots on the hanging and the upright stem fragments may have resulted from the effect of stem orientation on the inter- and intracellular distribution of plant growth regulators in the plant tissues. The near horizontal positioning of that part of the hanging stem where the side shoots were growing from the stem caused these side shoots to grow vertically, perpendicular to the direction of the original stem. This gravitropic response of the side shoots is the result of the different inter- and intracellular distribution of plant growth regulators that resulted from the horizontal orientation of the main stem. Rooting occurred for the intact meristems on both the intact stem fragments and those split lengthwise, with no significant difference in rooting percentages . Zero rooting or shoot growth occurred from meristems that had been cut. Therefore, only stem fragments without or with a damaged meristem will be totally harmless. Even stem fragments as small as a meristem can result in the establishment of a new plant, and through that, the establishment of a new stand/clone. Management personnel active in the removal of Arundo state that based on their observations,grow bucket regeneration from stem fragments is rare, compared to regeneration from rhizome fragments. However, new Arundo plants that had regenerated from stem fragments were observed in situ during this study .

Regeneration from Arundo stem fragments under a series of environmental conditions was also reported by . There may be two reasons for the limited observations of Arundo regeneration from stem fragments in the field. If most eradication efforts that introduce stem fragments into the environment occur in the months of October through February, regeneration from these stem fragments will be inherently low , and the ambient temperature will be low as well. There are limitations in place that prevent eradication activities during the breeding season of the endangered bird species that nest in the river basins that have been invaded by Arundo. The other reason is that after a growing season, it will be difficult to distinguish between Arundo plants that have grown from a stem fragment or from a rhizome fragment , because the new plant will have grown a substantial rhizome . When the a plant has regenerated from a rhizome fragment, not much of the original large rhizome fragment remains after it has supported the regeneration of a new Arundo plant. .Growth of the tissues with the highest CNC, the leaves, stopped earliest , but the leaves remained green. Their continued photosynthesis supplied the carbohydrates for the continued growth of the rhizomes , stems , and to a lesser extent that of the roots . Both below ground tissues, the rhizome and the roots, have a lower CNC level than the leaves. For those parts of the Arundo that is primarily responsible for growth, the leaves and the roots, their growth patterns mirrors their internal N:C ratio. As these tissues near their CNC, their sink strength for photosynthates, and therefore their growth is reduced. Both the leaves and roots have reached their CNC approximately since day 132, and their growth started tapering off since that time. If the experiment would have been continued longer, this would have shown better for the roots. After 60 days, the root masses of the individual A. donax plants could not be separated, and each plant was assigned a quarter of the root biomass, to show that overall root growth was tapering off near the end of the experiment.

The tissue with the lowest CNC, was not, as expected, the rhizome which had a CNC of 0.030 g N/g C, but the stem, for which the N:C ratio went as low as 0.013 . Unlike the leaves, roots, and rhizome, which reached their CNC after approximately 130 days of growth, the N:C ratio did not reach its lowest levels until day 245. The rhizome of Arundo donax act as a storage tissue for reserves. The reserves stored will support stem regrowth from meristems on the rhizome in the spring . In addition to the rhizomes, spring regrowth is also supported by the stem tissues. Unlike the common reed, Phragmites australis, new side shoots grow from the upper section of Arundo stems in the spring . Both tissues are originally stem tissues, and both play a role in the spring regrowth of the Arundo plant. Both the Arundo tissues that support spring regrowth, stems and rhizomes, have CNC levels below that of the leaves of Arundo.This resulted in significantly more stem growth than rhizome growth, with a final stem biomass of 1190 ± 95 g, and a final rhizome biomass of 171 ± 79 g after 334 days of development. For Ipomoea batatas these patterns were the opposite, because the CNC of the reserve storage tissue, the storage roots, was significantly lower than that of the stems . The CNC of both tissues was lower than that of the Ipomoea leaves. The N:C ratio of the leaves was 0.045 ± 0.0014, of the stem 0.017 ± 0.000006, and that of the storage roots was 0.013 ± 0.0011. For this species, as for Arundo, the biomass of the tissue with the lowest CNC, the storage roots, was significantly higher at 181.8 ± 23.8 g DW, than that of the tissue with a higher CNC, the stems, which reached 28.9 ± 7.5 g DW, when the plants had matured.The results of this experiment indicate that the leaf N:C ratio and their CNC can be used as an indicator for good timing of systemic herbicide application, such as that of the glyphosate based Rodeo©. When the N:C ratio of the leaves has been reduced to their CNC, the only major tissues that initially continue growth are the roots, the rhizomes and the stem. The applied glyphosate will be transported to these tissues, with the flow of photosynthates in the phloem supplied by the photosynthetically active leaves. If the glyphosate accumulates in and kills the roots, this will kill the Arundo stand, because the uptake of soil water and nutrients will not be possible any more.

This is not the most likely scenario, because root growth being reduced as they reach their own CNC. After root growth has tapered off, the roots can still function in the uptake of water and nutrients. The glyphosate will also be transported to those tissues that support spring regrowth, thestem and the rhizomes, because their growth continues after the leaves have reached their CNC. Based on physiological studies into the amount of reserves stored in the rhizomes of Arundo throughout the growing season, transport of photosynthates to and storage of reserves in the rhizomes, the major underground plant structure that will support regrowth if the above ground parts of the plant are killed or removed, starts near the end of July and continues through October/November . If this tissue accumulates enough glyphosate due to the use the correct herbicide concentrations and good timing of application, recovery should be reduced to a minimum.Soybean is an important biotech food, vegetable, and field crop that provides oil , protein , and carbohydrate to millions of people worldwide. Furthermore, soybean is a promising sustainable source of biofuels in North America, South America, and Europe . Zinc deficiency has been recognized globally as a major micro-nutrient stress that lowers crop yield and productivity around the world . Zn deficient soils occur in nearly 30% of the world’s arable lands. Selection and breeding of plant genotypes for Zn efficiency , defined as the ability of plants to maintain reasonable yield under Zn deficiency, is considered a sustainable approach to increase plant production on low Zn soils . Considerable differences in response to low Zn stress are known to exist among genotypes of bread wheat , rye, triticale , rice,dutch bucket for tomatoes , and common bean . Variations in shoot or leaf based parameters together with higher internal Zn utilization can be the principal factors in differential ZE in crop plants . Preliminary studies in common bean indicated that leaf physiological parameters such as leaf area are a useful criteria for ZE screening . Currently, there is little information regarding response of stomatal conductance to low Zn stress. Many earlier studies of low Zn stress focused on economically important cereal species. Few studies have been conducted in soybean, and fewer have tested hydroponics as a growing media. It has been shown that critical Zn deficiency level for soybean leaves was 15 µg g-1 . In a field study in Central Turkey, Zn deficient calcareous soils were shown to reduce yield and cause the development of visual symptoms on young leaves of soybean plants . Many soybean genotypes are being developed in the U.S. but little is known about their reaction to low Zn stress. Therefore, the objectives of this study were to: develop a suitable hydroponics-based method for ZE screening of soybean plants to identify more Zn efficient and less Zn efficient genotypes; and detect genotypic ZE variation in soybean using physiological parameters such as leaf area, chlorophyll contents, stomatal conductance, nutrient concentration, and plant biomass. Available Zn concentrations around 1 to 2 pM has already been shown to induce Zn deficiency in bread wheat and common beans .

Accordingly, our experiments successfully induced Zn deficiency at this concentration level in hydroponics. Based on our results, it appears that hydroponics with chelate buffers is feasible for screening soybean ZE trait. The soybean genotypes tested in this study had considerable variability and physiological responses to low Zn stress in hydroponics. Total leaf area, chlorophyll content, and leaf Zn concentration levels were all high in MZE genotypes. At the same time LZE soybean genotypes had various visible symptoms which indicated unfavorable Zn levels. This is consistent with previous findings that soybean plants showed chlorosis and brown leaf patches in calcareous soils . In terms of overall assessment genotypes “Williams” and “Hampton” were the most Zn efficient and inefficient, respectively . Although chlorosis is the most prominent symptom of low Zn stress, there is limited info on the effect of Zn deficiency on chlorophyll content levels. Leaf chlorophyll content was greater for MZE genotypes such as “Williams” and “Pella86” compared with LZE genotypes. Our results suggest that increased chlorosis was the cause of reduced SPAD levels. This is in agreement with the previous findings on wheat and common beans . The variability in both stomatal conductance and shoot Fe concentration was considerably large . It is interesting to note that Fe concentrations were considerably high for some genotypes such as “Thomas” . The lack of correlation with ZE trait across the genotypes tested may indicate that stomatal conductance could not be used for early detection of Zn stress in soybean. Significant differences between soybean genotypes in shoot Zn and N concentration were observed in low-Zn grown plants in hydroponics. Although there was no significant correlation between shoot nutrient concentration and ZE trait, LZE genotypes were characterized by slightly lower concentration of Zn, Fe, and N . This data are in agreement with previous findings showing that Zn efficient wheat varieties transported more Zn from roots to shoots than Zn inefficient varieties under Zn deficiency in the early field growth stages in bread wheat .As sessile organisms, plants are presented with numerous biotic challenges such as herbivory and pathogen attack. Plants initiate responses to these challenges by harnessing tightly regulated phytohormone networks. Salicylic acid levels increase in plants following pathogen infection and SA is critical for the development of systemic acquired resistance . There are two enzymatic pathways for the generation of SA: one via phenylalanine ammonia lyase and the other via isochorismate synthase . In tomato , Arabidopsis and Nicotiana benthamiana, most pathogen-induced SA appears to be synthesized via the ICS pathway .

Targeted analysis of lipids showed that sphingolipids were significantly upregulated in Ag treatments, indicating disturbance in the vacuolar and plasma membrane of A. thaliana plants . In 4-week old cucumber plants, exposed to 10 and 100 mg/l nAg, three times daily for consecutive 7- days via foliar spray, the responses were similar to ionic Ag exposure . Both AgNPs and Agþ ion exposures altered the accumulation of 76 metabolites involved in activation of carbohydrate metabolism , antioxidant system and defense response . Ag treatments also resulted in decreased accumulation of Gln and Asn, responsible for N fixation, and over-accumulation of salicylic acid, which is a major signaling molecule that activates plant defense and systemic acquired resistance. Exposure to 100 mg/l nAg enhanced the accumulation of linolenic and linoleic acids, the most abundant unsaturated fatty acids in the membrane .They also accumulated polyamines , GABA and its metabolites , which play a predominant role in stress response, nitrogen storage, signal transduction, growth and development. In maize leaves, TCA cycle intermediates, citric acid and a-ketoglutaric acid were increased whereas, succinic acid was decreased, affecting the carbon metabolism. The NPs altered several biological pathways in the leaves including amino acid metabolism , methane metabolism, pantothenate and CoA biosynthesis, carbon metabolism and pyridine metabolism. In addition, nFe3O4 and nTiO2 specifically altered isoquinoline alkaloid biosynthesis and glyoxylate and dicarboxylate metabolism. In roots, all the ENMs affected inositol phosphate metabolism and TCA cycle, however, nFe3O4 and nTiO2 affected ascorbate/aldarate, methane, glycerolipid, glyoxylate, dicarboxylate metabolisms. nTiO2 exposure to 7-day old rice seedlings in hydroponic media for 14-days resulted in elevated levels of metabolites in TCA cycle and pentose phosphate pathway , whereas decreased levels of metabolites involved in the carbohydrate synthesis metabolism including starch and sucrose metabolism, and glyoxylate and dicarboxylate metabolism . Direct exposure to nTiO2 suspension also increased the biosynthesis of fatty acids, amino acids and secondary metabolites in the rice seedlings. In a recent study, Gong et al. reported that a 6- day exposure to 10 and 500 mg/l nY2O3 disturbed carbohydrate metabolism, TCA cycle and amino acid metabolism in maize seeds . An increased accumulation of sugars , nft growing system free amino acids , GABA, and organic acids was attributed to cellular response to nY2O3 stress.

Three weeks-old Phaseolus vulgaris plants were chronically exposed to 250 and 1000 mg/l nCeO2 in the form of suspension every 48 h for 2 weeks via foliar spray or soil application, resulting in a total exposure load of 50 and 200 mg nCeO2 . Foliar application was shown to exhibit stronger negative effects; membrane lipids, lignans , and metabolites involved in pteridin and lignin synthesis were enriched, demonstrating immediate response on structural integrity, whereas most carotenoids, glucosinolates, terpenoids, andphenolic compounds were down-accumulated. In contrast, soil application resulted in overaccumulation of carotenoid and phenolic compounds, whereas polyols and pteridin-related compounds were down-accumulated. A three generational chronic exposure to nCeO2 exhibited higher degree of alterations in the metabolite accumulation in wheat grains compared to two-generational exposure . The altered metabolites were primarily involved in fructose-mannose metabolism, pentose-phosphate, phosphoribosyl pyrophosphate pathways; however, the degree of alteration in the grain metabolites was influenced by soil N availability. Vecerova et al. separated the polar and non-polar fractions of the leaf extracts obtained from barley plants exposed to an aerosol spray of CdO NPs for 21 days . LC-MS was used to determine sugars, phenolics, amino acids and Krebs cycle acids, while fatty acids were analyzed using GC-MS. An increase in the total amino acid content in roots and leaves, indicated protective mechanisms in response to stress. Decrease in levels of sugars in the roots, and phenolic acids in leaves were reported. In a recent study, we performed targeted metabolomics in soybeans exposed to CdS-quantum dots for 14 days in vermiculite soil media, which showed that major metabolic pathways in soybeans including glutathione metabolism, TCA cycle, glycolysis, fatty acid oxidation and biosynthesis were perturbed .In 7-day old A. thaliana plants grown on artificial media spiked with 125 and 1000 mg/l carbon nanodots for 7 days, growth was significantly inhibited compared to control and activated-carbon treatments. The metabolite profiling suggested increased accumulation of carbohydrates in A. thaliana roots and shoots, which indicates impact on the carbohydrate metabolism and cell wall stress . In the Cdot exposed A. thaliana tissues, overaccumulation of various alkaloids, lignan, carotenoids, flavonoids, amino acids, organic acids , and fatty acids were noted, indicating activation of defense response.

A three week chronic exposure of C60 fullerols via foliar spray twice a day for two days to cucumber plants showed degradation of plasma membrane metabolites like linolenic and palmitic acid, whereas increased accumulation of antioxidant metabolites such as 3-hydroxyflavone, 4-vinylphenol dimer, 1,4 benzenetriol, methyl transcinnamate, quinic acid, and dehydroascorbic acid .Plant rhizosphere is rich in metabolites that exude from roots and assist plants to cope with abiotic or biotic stress by building tolerance, defense and resilience . Zhao et al. reported that nCu exposure triggered an increase in the release of amino acid from C. sativus plants to sequester Cu ions at ENM-root interface and decreased citric acid content to control Cu dissolution from nCu, in order to lower Cu bio-availability and subsequent implications . Enhanced levels of ascorbic acid and phenolic compounds in the root exudate confirms increased stress in the plants exposed to nCu . In another study, ENM -treated soil collected after 28 days plant growth showed decreased levels of water-soluble Cu and increased levels of levoglucosan, linolenic acid, 4-hydroxycinnamic acid, allo-inositol, b-mannosylglycerate, gluconic acid, methyl phosphate, and methyl b-D galactopyranoside compared to untreated soil, indicating exudation of compounds involved in defense response . However, since the soil is a concomitant mixture of metabolites from the plant, residual organic matter and the soil microbiome, conclusions on root exudate characterization based on soil metabolomics could be confusing. Thus, sampling is crucial to extract maximum information from root exudate metabolomic studies, which may otherwise lead to inconclusive information. Appropriate control samples must be introduced while comparing ENM effects on root exudates between different plant species or varieties, as secondary metabolite composition vary significantly.Plant genome encodes for 36,795 proteins and the average size of a plant protein is 436 amino acid residues, which is 27% smaller than the average animal protein . Proteins are the key players in plant signaling and stress response as they are directly utilized in processes involved in cellular homeostasis. Characterizing the plant proteome offers functional analysis of the translated regions of the genome; hence, proteomics complements transcriptomic and metabolomic for a comprehensive understanding of underlying cellular mechanism in plants.Proteomic analysis serves as an important tool in crop improvement, protection and phytotoxicity studies as it has the potential to identify the key regulatory proteins, posttranslation modifications and protein-protein interactions in plants in response to an external stimulus.

Probing the ENM-plant interaction at the protein level is necessary to elucidate the cellular processes and identify the candidate proteins involved in ENM response. Using time-resolved experimental designs and by delving at sub-cellular or organelle levels, it is also feasible to identify ENM-responsive proteins involved in specific biological pathways at different growth stages of a plant . A rather unexplored area is the characterization of protein coronas formed around ENMs while interacting with plant tissues under different growth conditions, which also influence the toxicity and mobility of ENMs in plants.Two-dimensional gel electrophoresis separates protein mixtures based on charge in the first dimension and by mass in the second dimension on 2D-gels, which in combination with MS have been used for decades for plant proteome profiling. However, it has its limitations owing to poor sensitivity, low reproducibility, and low throughput. Especially for plant whole lysates that contain an array of secondary metabolites like pigments and phenolics, these compounds can introduce streaking in a gel . With rapid development in MS techniques, quantitative proteomics have evolved from gelbased to gel-free approaches. Like metabolomics,vertical hydroponic nft system proteomics can also be categorized into untargeted and targeted method. Due to the exploratory nature of untargeted proteomics, it is the preferred approach to screen for candidate protein markers to elucidate plant responses to ENM exposure. This approach can facilitate high-throughput analysis of protein abundance across different ENM exposures, tissue types, stages of growth, physiological conditions and stress conditions . In a gel-based approach, proteins are separated by 2DE and the spots showing comparable differences are excised from the gel. The proteins in the gel fractions are digested into peptides, which are then characterized by LC-MS. A typical gel- and label-free quantitative proteomic analysis used for discovery studies employ a bottom-up approach, where the proteins in a sample are cleaved into peptides, which are then separated, identified and quantified using LC-MS/MS . The steps and key challenges in proteomic analysis in ENM-plant interaction study are summarized below.Proteomic analysis demands robust sampling methods aligned with the scope of the study. Combining different organs into one sample must be avoided as the tissue-specific protein information is lost, resulting in confounding interpretation of results. In addition, proteomics result is also prone to variation in response to age, time of harvest, and growth conditions. Hence, control experiments and biological replicates should always be carried out simultaneously and harvested at the same time of the day. Plant tissues must be immediately frozen upon harvest, followed by homogenization into fine powder which can be stored at _x0005_ 80 C. The proteins in the sample are then subjected to precipitation, resuspension in buffer, followed by digestion using protease trypsin, which are then analyzed using LC-MS. The major challenges in sample preparation for proteomics arise from recalcitrant tissues, hydrophobic membrane proteins, presence of lignin, proteases, and metabolites like phenolic compounds, lipids, organic acids, carbohydrates, terpenes, and pigments, and presence of high abundance proteins that may mask variation in low abundance proteins during analysis .Electrospray ionization and Matrix-Assisted Laser Desorption Ionization are the most common platforms used to ionize peptides and proteins. The precise molecular mass of the resulting ions is then analyzed using mass analyzers, such as ion trap, quadrupole, Orbitrap, time-of-flight , and Fourier transform ion cyclotron resonance , which are often used in tandem to achieve higher degrees of ion separation .

In untargeted proteomics using LC-MS/MS, data dependent acquisition is performed, where the highest abundance peptide ions from full MS scans are selected for MS/MS. However, this may generate datasets skewed toward the identification of relatively high abundance proteins, thereby masking and excluding the low abundance proteins from quantification . Several labeling techniques such as isobaric Tags for Relative and Absolute Quantitation and Stable Isotope Labeling by Amino acids in Cell culture are also available which can help to reduce errors introduced during measurement conditions. However, untargeted proteomic analysis requires extensive data processing and is currently challenged by incomplete and limited nature of plant genomic and proteomic databases. In recent times, the use of labelfree shotgun proteomic techniques have become increasingly popular, as they do not restrict the number of proteins identified compared to gel based methods . However, gel-free methods have several drawbacks, such as masking of low abundant peptides and unavailability of protein database for all species.Untargeted proteomics require extensive post acquisition data processing after extracting features using LC-MS. There are several open-access and commercial software tools that can be used for feature extraction, such as MaxQuant, Proteome Discover, OpenMS, etc. The extracted features are subjected to filtering, peak detection, normalization, spectral identification, retention time/peak alignment, statistical analysis, peptide identification, quantitation, protein inference, and data visualization. All the raw and processed proteomics data should be deposited into public data repositories. Targeted proteomics, on the other hand, is hypothesis-driven and provides unparallel specificity and sensitivity for the targeted proteins of biological interest. In targeted proteomics, the mass spectrometer is programed to analyze a pre-selected group of proteins, using SRM, whereby assays are developed on a QqQ to detect fragment-ion signals arising from unique peptides representing each targeted protein.Comparative assessment of plant proteomes in response to ENMs can effectively screen candidate proteins and associated pathways, and potentially elucidate the cross talk between different processes that control the metabolite regulation in the cells. Several studies have employed untargeted proteomics to identify key plant proteins involved in ENMs-associated cellular signaling or stress response.