Tag: hydroponic

Most of the variation for in situ exudates was explained by differences between exudates collected in clay, compared with other conditions, which is evident from a principal component analysis . Similarly, in pairwise comparisons, clay-collected exudates showed the most distinct metabolites , followed by 250 µm sand-collected exudates with 5%–18% distinct metabolites . In contrast to in situ exudates, in vitro exudates exhibited similar metabolite profiles when analyzed with a principal component analysis , and fewer metabolites had statistically significant abundances in pairwise comparisons . Notably, in vitro exudates of clay-grown plants showed a comparable number of distinct metabolites in pairwise comparisons with plants grown in other substrates, suggesting that the in situ differences observed between exudate profiles of clay grown plants and plants grown in other conditions resulted from the presence of the clay, and not from an altered plant metabolism.To further investigate differences in in situ-collected exudates, exudate profiles of the groups by their particle sizes and “big beads,” “small beads,” “big sand,” and “small sand” were compared . In a principal component analysis, exudate profiles of hydroponic and “big beads” exudates overlapped, whereas “big beads” versus “small beads” and “big sand” versus “small sand” separated . Pairwise comparisons showed thirteen distinct metabolites between “big beads” and “small beads,”vertical indoor farming and three distinct metabolites between “big sand” and “small sand.” Two thirds of metabolites were more abundant in “big beads” than “small beads,” among them nucleobases and derivatives, as well as organic acids. Four phenolic acids were more abundant in “small beads” versus “big beads.” In “small sand,” two nitrogenous compounds were higher abundance than in “big sand,” a nucleobase, , and an amino acid derivative .

To further examine the differences between clay-grown and hydroponically grown in situ exudates, a multi-variant test was used to compare metabolite abundances between the two conditions. Most of these metabolites were nitrogenous, with more than half containing a heterocyclic nitrogen group. Among these metabolites were nucleic bases, nucleosides and derivatives with acidic groups, amino acids with acidic and/or basic groups, and linear as well as phenolic organic acids. Two nitrogenous metabolites, an organic acid and choline-O-sulfate with an acidic and a basic group, were more abundant in clay-collected exudates . These compounds were not detected in exudates of hydroponically grown plants, in in vitro-collected exudates of clay-grown plants, or in clay control samples without plants , which suggests that these compounds were released from clay only in the presence of plants.Since clay particles were found to strongly sorb exudate metabolites, we wondered whether the sorbed metabolites were accessible to a plant-associated bacterium, supporting microbial growth. Thus, we first determined the desorption rate of metabolites from the various substrates by determining the metabolite recovery rate from glass beads, sand, and clay incubated with defined medium. As shown previously , the metabolite recovery rate was comparable between the no substrate control and glass beads , lower in sand , and the lowest for clay . The metabolite recovery from washes was 4%–14% for all substrates, indicating all substrates had similar desorption rates. Growth of the rhizobacterium Pseudomonas fluorescens on sand or glass beads pre-incubated with defined medium resulted in the same optical density change as growth on particles pre-incubated with water , indicating that these substrates did not retain metabolites supporting growth. Incubation of the bacterium with clay pre-incubated with defined medium however did result in bacterial growth. As the control incubation of clay with defined medium also showed a small increase in OD, presumably as a result of fine particles, the data presented in Figure 6 are normalized by no-bacterial clay control samples .

These data show an increase in OD of bacteria grown on clay pre-incubated with defined medium, indicating that the bacteria are able to utilize the sorbed metabolites for growth. As an additional control experiment, the pre-incubated clay was incubated with water for three days under sterile conditions , allowing for desorption of metabolites from clay particles. The supernatant was subsequently pipetted into a new well, and bacteria were added and allowed to grow for another three days. This experiment resulted in no-bacterial growth , suggesting that bacterial presence is needed to desorb metabolites from clay particles. We conclude that this particular rhizobacterium is capable of desorbing exudate metabolites from clay to support growth.Growth of B. distachyon in particles with different sizes resulted in various morphological changes. A decrease in particle size resulted in decreased root weight, total root length, and in total root number, although the last parameter correlated less strongly . Root weight correlated positively with shoot weight, total root length, and total root number, indicating a dependency of the different parameters. Notably, the morphology of B. distachyon grown in glass beads or sand was not directly comparable: Plants grown in 5-µm sand had higher root weight and total root length than plants grown in 0.5-mm glass beads. The three-dimensional particle arrangement and other differences between the substrates, such as texture, might account for root morphological differences observed between glass bead and sand-grown plants. For glass bead-grown plants, the reduction in total root length was caused by a reduction in second-order root length, whereas the primary order root length was reduced in all sizes smaller than 3 mm. These trends for reduced root weight and root length but not root number are in line with observations made for maize grown in 1 mm versus hydroponic conditions . However, these previous studies noted an even larger decrease in shoot than in root weight, whereas B. distachyon shoot weight did not change significantly in our experimental conditions. Similar results were found for lettuce grown in three different soils, where root fresh weight and morphology changed, but shoot weight was not affected . The constant B. distachyon shoot weight might indicate sufficient nutrient uptake even by smaller root systems in the environments investigated. Thus, in future studies, it might be interesting to evaluate how the different root systems as generated herewith different substrate sizes further respond to altered nutrient levels.

One could expect an additional change in root morphology with different nutrient starvation conditions, for example, root systems optimized for phosphate scavenging form numerous, short lateral roots, whereas roots optimized for nitrogen uptake exhibit fewer, but long lateral roots . Phosphate movement is hindered by particles with a charged substrate, whereas nitrate movement is less affected by soil chemistry . Thus, changes in root morphology of plants grown in phosphate-limited clay might be distinct from plants grown in phosphate-limited sand or glass beads. Plants grown in soil may exhibit additional changes in root morphology and metabolism, as shown for B. distachyon grown in a sterile soil extract, which showed reduced root length, and elongated root hairs and which depleted a variety of metabolites from soil extract . Root systems further respond to local alterations in soil structure, such as to the presence of micro- or macropores, or to air pockets . Investigations of local root morphology responses in heterogeneous settings with multiple, defined substrate sizes and chemistries will thus shed more light onto how plants respond to soil physiochemistry on a spatial and time scale. Multiple systems exist in which such experiments could be attempted, ranging from EcoFAB model systems to rhizotron designs .B. distachyon exhibited significantly altered root morphology when grown in particles with various sizes, with root weight, and root lengths differing between conditions. The exudate profile however was very similar for these plants when collected in vitro , and exudate extraction volumes were normalized by root fresh weight before measurement. Thus indicating that exudation per root fresh weight is constant. As root weight correlated with both, total root length and with total root number, an additional method was needed to determine whether the number of roots or the root length was important for exudation. In the literature, root tips are often mentioned as predominant sites of exudation for several reasons: a) cell wall suberization of this young tissue is still low , b) exudates have been imaged around root tips , and c) more microbes associate with tips compared with other root sections . Few studies exist investigating spatial patterning of exudation, but some examples suggest that other tissues besides root tips might be involved in exudation. For example, vertical growing towers the localization of the malate transporter ALMT1 in Arabidopsis is confined to the root tip in untreated roots, but expands to the entire root system when treated with an activator, aluminum . This suggests differential malate exudation from different parts of the root, depending on the environment. Similarly, strigolactone exudation is environment-dependent, with its transporter PDR1 expressed in single cells along most of the roots . In addition, microbes do not only colonize root tips, but also prominently sites of lateral root emergence, and are found throughout the root system of plants . Distinct microbial populations, associated with B. distachyon seminal and nodal roots, as well as for nodal root tips versus nodal root bases , could be influenced by differential exudation by these organs. We used mass spectrometry imaging to investigate exudation across roots. These data cannot directly be compared to the root morphology and LC/MS data for technical reasons and the fact that the exudates were collected from three-week-old plants, whereas the imaging experiment was performed with seedlings due to technical limitations. Some ions were observed to be most abundant around the root tip, whereas others were also found in the root elongation and maturation zone, or all along the root axis. In addition, some ions were detected on the root itself, which could mean that they are part of the cell wall, or that they have a low diffusion speed.

Despite these limitations, our data suggest that root exudation is a spatially complex process. Exudation might take place in different ways: Root tip-exuded metabolites might diffuse, due to the absence of Casparian strips or secondary cell walls, or might be actively transported. Metabolites exuded from older root tissues are more likely to be transported, either by channels facilitating diffusion, or by active transport proteins. Future studies are needed to investigate the role of various root zones in exudation to determine which tissues are involved in exudation of various compounds and if exudation differs between root types.Root exudate metabolite profiles were unaltered when plants were grown in different particle sizes. As the root weight, root number, and root length correlated and the exudation of compounds was spatially complex, we conclude that exudation profiles are similar across different root morphologies. However, these investigations were limited and may be better informed by comparison of exudation profiles in plants with more radically altered root morphologies, in plants without secondary roots or root hairs. Exudation profiles were also comparable between plants grown in clay, sand, or glass beads, when collected in vitro. This suggests that the physiochemical environment does not alter plant metabolism, as long as other factors such as nutrient levels, light intensity, and humidity, remain unchanged. However, the exudate metabolite profile of clay- versus sand- or glass bead-grown plants was clearly different for in situ exudates . A recent study found differences in sorghum exudates of plants grown in clay, sand, and soil . In this study, exudates were collected from roots with rhizosphere substrate still attached. The largest difference in this dataset was observed between soilgrown and sand- or clay-grown plants, which might be explained by soil-derived metabolites co-extracted with root exudates . The authors showed some ions to be specifically up- or down-regulated in exudates of clay- versus sand grown plants, but their effect was not strong enough to separate the two conditions in a principal component analysis . This may be explained by their exudate collection method, which was a mixture between the in situ and in vitro conditions utilized here. Recently, it was suggested that root tips might detect the concentration of rhizosphere metabolites, altering root morphology and exudation accordingly . Thus, clay-grown plants should exhibit an altered root morphology compared to hydroponically grown plants, as clay sorbs a significant amount of exudates, changing the metabolite concentration around the root tip. However, the root morphology of clay-grown plants is statistically not different from hydroponically grown plants .

Wheat can utilize either form alone , but mixed N nutrition typically produces the best grain yields and quality in hydroponically grown and field-grown plants .Ammonium requires less energy to assimilate into organic compounds , but can prove toxic if it accumulates to high concentrations within plant tissues . Nitrate is generally the predominant form available in aerated, temperate agricultural soils , and may accumulate within plant tissues to high concentrations without toxicity . In wheat, the N form supplied has been found to influence many physiological parameters profoundly including biomass , leaf area , tillering , seed mass , protein content , and mineral nutrient acquisition and distribution , although such differences can vary among cultivars . The presence of NH4 + , as either a sole N source or in mixed N nutrition, increased organic N concentration in shoots, roots, and grain and decreased partitioning of dry matter to the roots in wheat . Decreased cation uptake has been found in wheat under NH4 + nutrition , although results varied among cultivars . For example, NH4 + nutrition decreased whole plant and shoot accumulations of K, Cu, Ca, Mg, Fe, Mn, and Zn . Nutrient allocation to plant tissues also varied between N forms. NH4 + -fed plants distributed a smaller percentage of total P, K, Cu, and B to roots relative to NO3 + -fed plants . Also, a greater percentage of reduced N was allocated to the shoots in NH4 + -fed plants . Elevated atmospheric concentrations of CO2 alter growth and N dynamics of wheat and other C3 plants. Under elevated CO2, wheat has lower protein and N concentrations ,vertical grow racks and lower macro- and micro-nutrients concentrations . Grain phytate concentrations are also thought to increase or remain the same under elevated CO2,and in conjunction with decreased concentrations of micro-nutrients, bio-available Zn and Fe are expected to decrease even further under elevated CO2 , as these micro-nutrients form indigestible complexes with phytate.

By contrast, wheat yields , harvest index , whole plant biomass , shoot biomass , and root biomass typically increase under CO2 enrichment. In addition, elevated CO2 concentration can increase tillering , nitrogen use efficiency , and micro/macro-nutrient use efficiencies. The influence of elevated CO2 on many of these characteristics may vary among cultivars and research protocols . Wheat grown under CO2 enrichment behaves differently under NO− 3 and NH4 + nutrition. Exposure to elevated CO2 inhibits NO− 3 photoassimilation in wheat as well as in all other C3 and C3– C4 intermediate plants tested . At elevated CO2, NH4 + -fed plants showed greater increases in leaf area and smaller decreases in shoot protein concentration than NO− 3 -fed plants , which could have consequences for human nutrition. Vegetative plants receiving NH4 + had greater shoot, stem, and root biomass at elevated CO2 . Wheat receiving NO− 3 grew slower at elevated CO2 than at ambient CO2 . Shoot NO− 3 concentrations in NH4 + -fed plants were undetectable while those in NO− 3 -fed plants increased by 62% with CO2 enrichment . This increase was associated with an inhibition in NO− 3 and NO− 2 reductase activities under elevated CO2 . The interaction between atmospheric CO2 concentration and inorganic N form and how it influences plant growth and nutrient concentrations has not been examined in wheat or any other crop species grown to senescence. Here, we grew wheat hydroponically in controlled environment chambers and measured mineral nutrition, biomass, and nutrient allocation in response to three concentrations of atmospheric CO2 and two forms of N nutrition . We tested the following hypotheses: plant nutrient concentrations and allocation patterns will respond differently to CO2 enrichment under the two N forms, and NO− 3 -fed plants will show a smaller biomass and yield enhancement in response to CO2 enrichment than NH4 + -fed plants as a result of CO2 inhibition of shoot NO− 3 assimilation. Also, we observed both differences in the Zn concentration between plants grown on NH4 + and NO− 3 and a strong dependence of Zn absorption on Zn and phytate concentration, indicating that phytate and bio-available Zn are affected by N form and CO2. Therefore, we used the well supported Miller equation to estimate how N and CO2 might impact a hypothetical human population. Iron, another important micro-nutrient that forms complexes with phytate, was not analyzed because we observed no significant differences in iron concentrations between the N forms and because how best to estimate Fe absorption in humans is still uncertain . Wheat seeds were surface sterilized for one minute in 2.6% sodium hypochlorite solution and thoroughly rinsed with DDI water.

The seeds were then rolled up in germination paper saturated with 10 mM CaSO4. The germination paper was placed in a 400 mL beaker with approximately 75 mL of 10 mM CaSO4 solution, covered with a plastic bag and placed in an incubator for four days. Seedlings were transplanted into 20 L tubs filled with an aerated nutrient solution that contained 1 mM CaSO4, 1 mM K2HPO4, 1 mM KH2PO4, 2 mM MgSO4, and 0.2 g L−1 Fe-NaEDTA and micro-nutrients 2HPO4 as the N source, Epstein and Bloom, 2005. The nutrient solution was replaced weekly and an additional 0.2 mM of NO− 3 – or NH4 + − N was added midweek until harvest. The solution volume was maintained by daily addition of deionized water. Solution pH varied between 6.8 and 7.0 for both of the N forms, and the NH4 + and the NO− 3 solutions did not differ by more than 0.1 pH units. The plants were grown in controlled environment chambers set at 23/20˚C day/night at 60–70% relative humidity with a photoperiod of 15 h. The photosynthetic flux density was 375µmol m−2 s −1 at plant height. Plants were subjected to one of three CO2 concentrations: “sub-ambient” , “ambient” , and “elevated” . Sub-ambient CO2 concentrations were maintained by passing air that entered the growth chamber through wet soda lime, a mixture of KOH, NaOH, and Ca2 that was replaced as needed. The elevated CO2 conditions were maintained in an environmental chamber equipped with non-dispersive infrared analyzers for CO2 and valves that added pure CO2 to the incoming air stream to hold the chamber concentration at 720 ppm. The wheat was grown until all above ground parts turned completely yellow. Plant matter was sorted into grain, chaff, shoots, and roots and dried for 48 h at 55˚C. Data on kernel number , kernel mass, number of heads, kernels head−1 , and HI were collected prior to sample preparation for nutrient analysis. A portion of the grain was analyzed for phytate using a modification of the method as described by Haug and Lantzsch . The remainder of the grain as well as the shoots and chaff was bulked into five repetitions per treatment and sent to the UC Davis Analytical Laboratory for nutrient analysis.

The roots of plants for each CO2 × N treatment became entangled within the same tub; therefore, we were unable to separate the roots of the individual plants for analysis. Root data are thus presented as means for each treatment with no standard errors or confidence intervals. Data were analyzed using PROC MIXED . Nitrogen form and CO2 factors were treated as fixed independent variables. We used the Tukey–Kramer Honestly Significant Difference test for mean separation. Probabilities less than 0.05 were considered significant. Because some of the transformed variables did not meet the assumption of homogeneity of variances,vertical farming in shipping containers but one-way ANOVAs met the ANOVA assumptions, we analyzed the results via one-way ANOVAs to gain some information on the interactions between CO2 and N form.We used a database derived from the United Nation’s Food and Agriculture Organization ’s national food balance sheets to estimate the average daily per capita dietary intake of zinc and phytate from 95 different food commodities in each of 176 countries. This database combines FAO data on per capita intake of food commodities with USDA data on the nutrient or phytate content of each of these commodities. More detailed discussion of the creation of this database for the International Zinc Collaborative Group may be found in Wuehler et al. . Using this database, we produced two data sheets: one containing per capita daily dietary intake of zinc from each food commodity for each country and another containing per capita phytate intake from each food commodity for each country. To calculate total dietary zinc and total dietary phytate per country, we summed across the rows of all food commodities for each respective country. To determine the proportion of a population at risk for zinc deficiency from a hypothetical least developed country , we first calculated TDP and TDZ values for a set of 44 countries defined by the United Nations as being least developed. We took the mean TDP and TDZ values for these countries to represent a hypothetical “less developed country.” To calculate the bio-available zinc portion we used the Miller equation . Mean TDZ and TDP values were converted to mg mmol−1 and put into the Miller equation to compute the average per capita TAZ in our hypothetical LDC. The variables TDZ, TDP, and TAZ are described above, and Amax, KP, and KR are constants as described in Miller et al. . We made an assumption that our hypothetical LDC receives half of its phytate and half of its zinc from wheat, which is roughly consistent with many of the LDCs in the FAO database. We analyzed the effect of elevated carbon dioxide levels on TDP, TDZ, and TAZ concentrations in a hypothetical LDC population for both NH4 + and NO− 3 -supplied wheat. To calculate a new TAZ for wheat grown under elevated CO2 conditions, we first calculated the percent change in TAZ from ambient to elevated levels for wheat receiving NH4 + or NO− 3 .

This computed percent change was then applied to half of the hypothetical TDZ and TDP; meanwhile, the other half of the hypothetical TDZ and TDP remained unmodified. Thus, the total new TDP and TDZ is the sum of the unmodified and modified portions. These new TDP and TDZ values for both NH4 + and NO− 3 -supplied wheat were then put into the Miller equation to compute new hypothetical TAZ values for an LDC. Differences and corresponding percent changes between the new TAZ values and the original TAZ value for a LDC were computed to determine the overall affect of elevated CO2 on TAZ in NH4 + and NO− 3 -supplied wheat for an average developing world population. TAZ, TDP, and TDZ concentrations can only be compared within a single N form across the CO2 concentrations due to methodological constraints of the model. Plants supplied NH4 + vs. NO− 3 nutrition reacted differently to CO2 enrichment . Plants supplied NH4 + differed across CO2 treatments for most of the yield and biomass measurements. The greatest values typically were found at ambient CO2 concentrations. Shoot, chaff, grain yield, number of heads, and KN were greatest at ambient CO2 levels. Individual kernel mass was greatest under both ambient and elevated CO2 treatments. HI and kernels head−1 showed no change across CO2 treatments. In contrast, biomass and yield measures of NO− 3 -supplied plants did not differ among the three CO2 concentrations. At sub-ambient CO2, differences between the NH4 + and NO− 3 treatments occurred in shoot biomass and three of the yield components: kernel mass, head number, and kernels head−1 . Ammonium-supplied plants had a larger number of heads while NO− 3 -supplied plants had greater shoot biomass, kernel mass, and kernels head−1 . At ambient CO2, NH4 + -supplied plants had a greater number of heads and greater chaff biomass. Plants supplied NO− 3 had a larger number of kernels head−1 . At elevated CO2, biomass and yield measures did not differ with N treatment. The distribution of nutrients and micro-nutrients among plant parts followed similar patterns in both the NH4 + and NO− 3 – supplied plants, although the NH4 + -supplied plant distributions were slightly more variable .

The Darroch and Frost analysis was conducted nearly 20 years ago, and the interviews were limited to women practicing vaginal intercourse. To our knowledge, a more recent study linking likelihood of product use and price sensitivity has not been conducted, or at least not reported, to include other populations of potential microbicide users such as heterosexual couples practicing anal sex or gay men practicing unprotected rectal intercourse. Nevertheless, the 1999 study established an initial price point and price sensitivity for potential users of microbicides in the USA. Griffithsin has a broader spectrum of antiviral activity than HIV-specific PrEP agents, including activity against HSV-2 and HCV, which are co-transmitted with HIV-1 . Hence, Griffithsin might command a higher price due to its broader antiviral activity and its potential to obviate prevention and treatment costs for co-transmitted viruses. In the USA, the cost of the oral PrEP drug Truvada ranges from $1,300 to over $1,700 per month for the uninsured, but treatment is typically covered by insurance with user co-payments of $80–$150 per month. So even if a Griffithsin-containing microbicide sold for $5 per application , a user of 2 packs per month would pay $100 for the microbicide, which is in the range of PrEP, with the potential added benefit of controlling co-transmitted viruses. Consumers in wealthier economies might be receptive to microbicides costing $1–2 or even more per dose; however, consumers in lesser-developed economies might find $1–2/dose to be prohibitive. Hence, absent subsidies, there exists a continuing need to lower COGS for APIs such as Griffithsin. We can conclude that a COGS of <$0.40/dose of Griffithsin DS as determined in this study, and an estimated user cost of $1– 2/dose, mobile vertical grow racks might enable at least some simpler formulations of the drug to be economically marketed.

For more complex formulations and delivery systems, or for higher doses of the drug, lower COGS for bulk Griffithsin would be desirable.The environmental assessment of the plant-based production of Griffithsin indicates low impact, particularly if the plant nutrient solutions are recycled in a hydroponic system and if waste streams containing TMV are treated in a bio-waste heat or chemical treatment process. The assessment method used, although semi-quantitative, utilizes mass input and output stream data generated by SuperPro, along with independent assessment of compound toxicity and/or environmental impact , and allows comparison between alternative production strategies, process configurations or chemical components used in the manufacturing process. Our low environmental impact assessment for plant-based manufacturing should compare favorably with fermentation based approaches to producing Griffithsin. In the latter, the complexities of purification suggest less efficient utilization of materials and higher disposal volumes, although a side-by-side environmental analysis between the two platforms was not conducted in this study.Upstream, Griffithsin expression rates were based on empirical findings using TMV whole virion as the expression vector, which can achieve typically 0.5–1.0 g Griffithsin/kg plant biomass . An average pilot-scale expression rate of0.52 g/kg was used in our model . Although this expression level is quite good for TMV, higher Griffithsin expression levels can be achieved with different technology. For example, Nomad Bioscience GmbH has achieved Griffithsin expression in N. benthamiana exceeding 2.5 g Griffithsin/kg FW biomass using NomadicTM agrobacterial vectors applied to plants either through vacuum infiltration or agrospray , albeit these results were obtained in small-scale studies. For example, even with the same recovery efficiency of 70% assumed in the current model, the output of Griffithsin at the higher expression level would be 1.75 g API/kg plant material, instead of the current 0.37 g/kg; this represents more than 4.7- times the modeled output of protein per kg biomass.

Under such conditions, the costliest parts of the current process, namely biomass production and upstream procedures, would be lowered by the reduced biomass needs to produce the required 20 kg/year of API. Although a full analysis of the cost of agrobacterial inoculation for Griffithsin production needs to be conducted, we know from similar analyses that economics can be favorably impacted by higher expression efficiencies. We can therefore envision that by using a more efficient induction process the per-dose production cost could be less than the current $0.32. Still other gene expression methods can be considered, including using transgenic plants expressing Griffithsin either in constitutive or inducible systems , which could also lead to higher API accumulation in host plant biomass and potentially lower COGS . Increasing expression yield upstream might shift costs to downstream operations to handle process streams with higher concentrations of API. Definition of the comparative cost benefits of these improvements relative to the current process modeled awaits a subsequent evaluation. From a process standpoint, improvements in the efficiency of lighting technologies and/or incorporating solar panels would reduce upstream utilities costs, one of the major contributors to the upstream operating costs. Improving hydroponic nutrient utilization through recycling and minimizing runoff in the simulation model will reduce raw material costs as well as aqueous waste disposal costs, thereby reducing the COGS. In the downstream portion of the process consumables play a major role, particularly dead-end filters and plate-and frame filters; if these could be replaced with tangential flow filtration systems that utilize reusable, cleanable ceramic filters, downstream operating costs could be further reduced. At the time of this writing, such systems were being considered and their impact on Griffithsin COGS will be the subject of a future analysis. Nitrogen is a vital macro-nutrient for plant growth and development. Plants have evolved a range of mechanisms to adapt to imbalanced nitrogen conditions. In agricultural systems, high-yield of crops relies on application of nitrogen fertilizers. But a large part of nitrogen deposited in the soil can’t be absorbed by plants and is lost to the environment, resulting in severe environmental and ecological pollution. Improving the nitrogen use effciency of crops is the key to solve these problems. Studying the genes and mechanisms involved in regulating nitrogen uptake and assimilation can be a prerequisite for improving NUE of crops, therefore it is of great importance for sustaining agriculture.

Nitrate and ammonium are the main nitrogen forms used by plants and most crops, like maize and wheat, take up nitrate as the major nitrogen source. In addition to its nutrient role, nitrate acts also as a signaling molecule for plants. It regulates the expression levels of many genes, including genes directly involved in nitrate assimilation, namely NIAs, NiR, and some NRTs. It is also involved in many adaptive responses of plants, such as root development and architecture, seed dormancy, fowering time, circadian system, leaf development, stomatal movements, and auxin transportation. Nitrate is taken up into plants by nitrate transporters and high afinity and low afinity nitrate uptake systems have been identified. Four gene families have been identified that encode nitrate transporters in Arabidopsis: NRT1/PTR , NRT2 , CLC , and SLAC1/SLAH. Among these families, NRT1/PTR belongs to the low afinity transport system, and NRT2 belongs to the high afinity transport system. NRT1.1 , which belongs to NRT/PTR family, functions in nitrate uptake as both high afinity and low afinity transporter.In addition to the nitrate transport systems, genes involved in nitrate signaling have also been identified. Most of these genes were found to function in root architecture or primary nitrate responses. Te genes functioning in root architecture include the ANR1, the first molecular component isolated by classic molecular genetics approach, is a MADS box transcription factor and positively regulates lateral root branching under sufficient nitrate condition. miR393/AFB3 and NAC4 have been demonstrated to regulate the root system architecture in nitrate signaling using systems approach. Te split-root assays indicated that TCP20 was involved in systemic nitrate signaling for root foraging. Recently, TCP20 was found to regulate root meristem growth under nitrogen starvation and to interact with NLP6&7. HHO1 and HRS1 are two nitrate-responsive transcription factors isolated by genome-wide analyses. They function in the repression of primary root growth under both phosphate starvation and nitrate supply conditions. During last several years,vertical garden growing the nitrate regulatory factors involved in the primary nitrate response have been identified. NRT1.1, in addition to its transport function, was identified to work as a nitrate sensor. Te study on the crystal structure of NRT1.1 has demonstrated that Tr101 phosphorylation is essential for nitrate transport rate and provides further insights into its transport mechanisms. CIPK8 and CIPK23 which belong to CBL-interacting protein kinase family are important players in responding to primary nitrate. CIPK8 works positively while CIPK23 functions negatively in nitrate regulation. Te expression of both CIPK8 and CIPK23 is regulated by NRT1.1. Recently, NRG2 which is an essential nitrate regulatory gene was isolated by forward genetics screen. NRG2 acts as a positive nitrate regulatory factor and modulates NRT1.1 expression and can interact with NLP7. Additionally, several transcription factors were identified to be involved in primary nitrate response, for example, NLP6, NLP7, LBD37/38/39, TGA1, TGA4, and SPL9. NLP7 is NIN-like protein and acts as an important nitrate positive regulator. NLP7 was isolated by reverse genetics strategy and the nlp7 mutants exhibit a nitrogen-starved phenotype. Te nitrate condition can affect the NLP7′s nuclear retention. Previous studies have demonstrated that the nitrate response cis-element NRE can be bound by NLPs and contain a DNA-binding domain RWP-PK and protein-protein interaction domains typeI/II Phox and Ben1p. ChIP-chip assays showed that NLP7 could bind 851 genes containing NRT1.1, NRT2.1, LBD37/38. In addition, over expression of NLP7 can increase plant biomass, nitrogen uptake, total nitrogen content, and expression levels of genes involved in nitrogen assimilation and signaling.

Moreover, NLP7 can control plant root growth under both N-limited and N-rich conditions. NLP6 also functions positively in nitrate regulation, is retained in the nucleus in nitrate-treated plants and can activate the expression of nitrate-responsive genes. LBD37/38/39 are negative regulators in nitrate signaling. Tey are involved in primary nitrate response and can affect nitrogen status, growth, and nitrogen-dependent shoot branching. TGA1, TGA4, and SPL9 were isolated by systems approach. TGA1 and TGA4 belong to bZIP transcription factor family and TGA1 can bind to the promoters of NRT2.1 and NRT2.2. SPL9 is demonstrated to be a nitrate regulatory hub. Although these nitrate regulatory genes have been identified, our understanding of the nitrate regulatory gene network is still incomplete. For example, both NLP7 and NRT1.1 play essential roles in regulating nitrate signaling and ChIP-chip assay showed that NLP7 might bind NRT1.1, however, their relationship and underlining mechanism remain unclear. In this paper, we investigated the relationship between NRT1.1 and NLP7 in nitrate regulation. Our analyses reveal that NLP7 acts as a positive regulatory factor upstream of NRT1.1 when NH4 + is present and modulates the nitrate signaling function of NRT1.1. NLP7 might function in another pathway to regulate nitrate signaling independent of NRT1.1. In addition, transcriptome data showed that four GO terms related to nitrogen were regulated by NRT1.1 as well as NLP7 in nitrate signaling, providing more evidence to support our above conclusion. Furthermore, the ChIP and EMSA assays indicated that NLP7 could bind to specific regions of the NRT1.1 promoter. Our findings not only further elucidate the relationship between NRT1.1 and NLP7, but also provide insights into the network of the nitrate regulatory genes.To study the relationship between NLP7 and NRT1.1, the expression levels of NRT1.1 was detected firstly under potassium nitrate and ammonium nitrate conditions. Figure 1a showed that the transcript levels of NRT1.1 in the nlp7 mutants were not notably changed under potassium nitrate condition, but was significantly decreased in mutant plants under ammonium nitrate condition . This indicates that the expression levels of NRT1.1 can be modulated by NLP7 in the presence of NH4 +. In order to test if NLP7 is regulated by NRT1.1, we tested NLP7 expression in chl1-5 and chl1-13 mutants in potassium nitrate and ammonium nitrate mediums. Te expression of NLP7 was not changed in the nrt1.1 mutants . This result indicates that NRT1.1 may not regulate the expression of NLP7. We also tested the NRT1.1 expression response to nitrate in WT and the nlp7 mutants. qPCR results showed that the induction of NRT1.1 by nitrate was notably decreased in the nlp7 mutants, indicating that NLP7 affects the response of NRT1.1 to nitrate .To elucidate the relationship between NLP7 and NRT1.1, the single mutants: nlp7-4 and chl1-13 which contain the nitrate-responsive NRP-YFP transgene, both of which were isolated by our mutant screens described previously were crossed to obtain the double mutant chl1-13 nlp7-4.

Large Scale Biology Corporation previously investigated N. tabacum outdoor field-grown production of recombinant proteins and personnel involved in that work recommended pursuit of this agronomic approach, with special consideration of field condition variability on product consistency.40 N. tabacum is used instead of N. benthamiana for its increased resilience to agricultural pathogens and weather fluctuation.The upstream processing model is adapted from a techno-economic analysis of plant-made cellulase produced in the field.The upstream and downstream processing model flow sheets are graphically depicted in Supplementary Information, Figure S1 and Figure S2. A complete list of changes to the base case scenario assumptions can be viewed in Table S6.The base case manufacturing facility scenario produces 500 kg of AMP per year at 92% purity including a 42% loss in extraction, downstream processing, and formulation. This yearly production is achieved in 91 manufacturing batches, each with a 42.3-day duration, which process 1.22 million plants per batch with an expression level of 1 g AMP per kg plant FW for a yearly total of 111 million plants processed. The facility plant inventory is 14.7 million plants, which is divided into 12 concurrent batches of plant growth. Initialization of batches is staggered by 3.42 days. The AMP is produced and recovered through a series of manufacturing steps: plant growth, ethanol induction, incubation, harvest, extraction, clarification, concentration, chromatographic purification, vertical farming technology buffer exchange, and formulation. The upstream processing recipe cycle time is 41.4 days and has been designed as the production bottleneck; the downstream processing recipe cycle time is 0.91 days and is thus executed well within the allowable stagger time between plant harvest cycles.

To meet the yearly production demand of 500 kg AMP, upstream processing must produce 867 kg AMP to offset the 42% downstream processing loss. Each upstream processing batch yields 9,520 kg N. benthamiana plant FW containing 9.52 kg AMP, which represents 10% of the total soluble protein .This results in 866,000 kg N. benthamiana plant FW processed over the course of the 91 annual batches, grown in 111,000 units of soilless plant substrate with 1.30 million liters of plant nutrient. Of the annual plant nutrient volume, 436,000 L are sent to waste while the remainder is utilized during plant growth. A total of 7,410 L of 4% ethanol are consumed annually for induction.Manufacturing batches continue directly from upstream to downstream processing; batches are not pooled, and thus 91 downstream processing batches are executed annually. Each batch begins with the upstream production of 9,520 kg N. benthamiana plant FW and 9.52 kg AMP. Screw press extraction results in a stream mass flow of 11,200 kg per batch . After microfiltration and ultrafiltration the stream is considerably reduced to 476 kg per batch . The product stream is eluted from the cation exchange chromatography at 236 kg per batch . The product stream is then diafiltered for a buffer exchanged product stream of 230 kg per batch . The final spray dry formulation results in 9.06 kg formulated product per batch .The base case manufacturing facility requires $50.1 million CAPEX and $3.44 million/year OPEX. The AMPs’ cost of goods sold is calculated to be $6.88/g. Figure 3 shows an economic assessment of upstream and downstream processing. Upstream processing represents 58% of overall operating expenditures , and downstream processing makes up the remaining 42% of operating costs. Of the $2.01 million/year upstream OPEX, the seeding operation represents the majority of the cost. Chromatography and ultrafiltration/diafiltration operations represent the majority of downstream processing OPEX of $1.43 million/year. The downstream CAPEX accounts for 62% of the overall CAPEX with the clarification and UF/DF filtration units representing the largest portion of the downstream capital investment costs.To evaluate the impact of AMP expression level and facility AMP production level, we developed models for a 500 kg AMP/year production level with different AMP expression levels ranging from 0.5 to 5 g AMP/kg FW , and for an expression level of 1 g AMP/kg FW over a range of AMP production levels from 100 kg AMP/year to 1,000 kg AMP/year .

Note that in all cases, the unit operations were resized to meet the design requirements. COGS decreases with diminishing returns as a function of expression level, as can be seen in Figure 5. To illustrate this point, consider that an increase of expression level from 0.5 to 1 g/kg FW results in $4.43/g decrease in COGS, while an increase from 4 to 5 g/kg FW results in $0.22/g decrease in COGS. These changes are equivalent to 39% and 6% reductions, respectively. Also note that at low expression levels the upstream operating costs contribute more to the COGS, whereas at high expression levels downstream operating costs contribute more to the COGS. This is reasonable because the number of plants per batch will increase as expression level decreases, thus requiring more soilless growth media, seeds, and nutrients. CAPEX follows a similar trend with expression level; however, the downstream process is the main contributor to CAPEX, except for very low expression levels . The majority of COGS and CAPEX variation with expression level is attributable to upstream processing, with downstream process costs remaining fairly consistent over the range of expression levels considered. COGS also decreases with diminishing returns as a function of yearly production capacity. Downstream processing is the main contributor to COGS at low production levels while upstream processing is the main contributor at high production levels; at 100 kg/year, downstream processing represents 64% of the COGS, while at 1,000 kg/year the contribution is reduced to 35% of the COGS. Within the given parameter range for expression level and production capacity, COGS shows a higher sensitivity to expression level. Figure 6 shows N. benthamiana FW per batch as a function of expression level and yearly production demand. As expected, biomass requirements are reduced at higher expression levels and lower yearly production demand. At all yearly production levels, significant diminishing returns for increases to expression level are evident within the selected range expression level.

The nicotine-free S. oleracea scenario produces 500 kg AMP/year at 1 g AMP/kg FW with 66% product recovery and 63% purity formulation . Manufacturing batches require ~10% fewer plants than the base case at 1.08 million SS. oleracea plants/batch, and a correspondingly lower plant inventory of 11.1 million plants. The upstream processing duration remains consistent with the base case, while the downstream processing time is reduced to 0.67 days after removal of the nicotine clearance chromatography step of the base case scenario. The S. oleracea manufacturing facility requires $46.5 million CAPEX and $2.50 million/year OPEX. In this scenario, AMPs are manufactured at a COGS of $4.92/g. The field-grown N. tabacum scenario produces 500 kg AMP/year at 1 g AMP/kg FW with 58% product recovery and 92% purity formulation . There are 63 manufacturing batches yearly of 13,900 N. tabacum plants per batch within the late March to late October growing season of the US Midwest/South. The lower number of plants is because of the much larger size of field grown N. tabacum plants compared with indoor grown N. benthamiana plants. The total inventory during steady-state operation is 619,000 plants. The upstream processing duration is 88.4 days, and the larger batches increase the downstream processing time to 1.08 days per batch. The N. tabacum manufacturing facility, including dedicated outdoor field equipment for transgenic handling, requires $27.5 million CAPEX and $1.51 million/- year OPEX. We have neglected labor costs associated with overseeing environmental release of transgenic material, the United States Department of Agriculture Biotechnology Regulatory Services regulatory application, and routine USDA Animal and Plant Health Inspection Service inspections. In this scenario, AMPs are manufactured at a COGS of $3.00/g. A comparison of the capital investment, production costs, and AMP COGS for the N. benthamiana base case, nicotine-free S. oleracea, and field-grown N. tabacum scenarios is shown in Table 2.A greenfield single-product bio-manufacturing facility was chosen to reflect the current whole plant protein bio-manufacturing environment in the United States. There is significant, yet limited, existing manufacturing capacity, most of which is positioned for pharmaceutical-grade production. For smaller annual production demands vertical tower planter, a single- or multi-product contract manufacturing organization model would also be viable. These trends are also reflected globally. The yearly production was determined to meet the demand of a projected market share anticipated for a product of this nature . The number of yearly batches was determined to fully utilize upstream plant growth capacity while leaving idle time for downstream equipment, which is likely to require more maintenance. Future work could include optimization of the plant inventory size, and thus batch size, to maximize the discounted cash flow rate of return over the project lifetime. The optimization will need to identify a balance in the fluctuation of equipment-associated CAPEX and labor- and utility-associated OPEX for both the upstream and downstream. The low-purity requirement of the AMP at 92% is associated with the selection of plant-based production and is a distinct advantage over traditional production platforms for food safety applications. Leafy plant extracts are safe for consumption and routinely consumed as a staple of human diet; when the impurities of the host organism are Generally Recognized as Safe for consumption, there is considerably lower burden on downstream processing. The major focus is redirected from product application safety to product stability and functionality in the presence of the host impurities. Depending on the application rate and consumer consumption, we expect that formulations of 50–95% purity could be employed. Therefore, this analysis represents an upper bound for the anticipated production costs.

Nicotiana benthamiana has been developed as an efficient recombinant protein expression platform. Except for nicotine and traces of anabasine, the N. benthamiana leaf constituents are considered safe for human consumption. Therefore, the processing and quality control are centered on host alkaloid reduction. Processing with a single cation exchange column can provide log reduction in the nicotine level in the product stream to <10 ng nicotine/mg TSP in the formulated product. Based on this reduction, the maximum daily intake of nicotine from the use of colicin as a food safety AMP would be much lower than is encountered in everyday consumption of Solanaceae plants such as peppers, tomatoes, or potatoes.15Vertical farming is just beginning to receive commercial interest as an agricultural solution for year-round, locally grown produce free of pesticides. As the vertical farming industry continues to gain traction, technological advances and process intensification will arise that substantially reduce manufacturing costs for both vertical farming of agricultural crops and plant molecular farming. For example, efficient capture and recirculation of water lost to transpiration will greatly reduce water requirements. Continued development of light emitting diode systems is expected to further improve growth rates, which should help reduce CAPEX, utility costs, and plant growth cycle time. Based on this current techno-economic analysis, advances in plant substrate processing strategies have particularly high potential for economic gain. Soilless plant substrate represents 41% of the overall OPEX in the N. benthamiana base case scenario. A single reuse of the soilless plant substrate prior to disposal would lower the overall OPEX by ~21% in the reduction of the cost of consumables. Reuse of the plant substrate can be achieved by either regrowth of harvested plants or a second round of seeding to generate new plants. In the former situation, manufacturing cost reductions would also include those associated with seeding and tray cleaning operations.Techno-economic analysis provides critical information at all stages of a project’s lifetime. Efficiency of internal research and development in biotechnology companies has suffered in recent years.Technoeconomic analysis is a useful tool for improving this efficiency through identification of key economic-influencing parameters and insights into the commercialization potential of the proposed technology. This preliminary analysis provides early indicators of success potential and reduces the risk of investment for key stakeholders. Furthermore, scenario analysis can guide research and development prioritization to maximize return on investment. In the base case model of this study, a change in the expression level from 0.5 to 1 g AMP/kg FW resulted in 20-fold greater COGS savings than from 4 to 5 g AMP/kg FW. This knowledge makes it clear that there is a significant economic incentive to improve expression levels, but only up to a point. Refinement of the analysis with pilot-scale data further strengthens the analysis and provides perspective to inform future scale-up work.

In Arabidopsis, a tonoplast-localized proton-pumping pyrophosphatase AVP1 was shown to be the key enzyme for cytosolic PPi metabolism in different cell types of various plants. This enzyme activity has been correlated with the important function that AVP1 plays in many physiological processes. Arabidopsis fugu5 mutants lacking functional AVP1 show elevated levels of cytosolic PPi and display heterotrophic growth defects resulting from the inhibition of gluconeogenesis. This important role in controlling PPi level in plant cells is reinforced by a recent study showing that higher-order mutants defective in both tonoplast and cytosolic pyrophosphatases display much severe phenotypes including plant dwarfism, ectopic starch accumulation, decreased cellulose and callose levels, and structural cell wall defects. Moreover, the tonoplast-localized H+ -PPase AVP1 appears to be a predominant contributor to the regulation of cellular PPi levels because the quadruple knockout mutant lacking cytosolic PPase isoforms ppa1 ppa2 ppa4 ppa5 showed no obvious phenotypes. Interestingly, in companion cells of the phloem, AVP1 was also shown to be localized to the plasma membrane and function as a PPi synthase that contribute to phloem loading, photosynthate partitioning, and energy metabolism. On the other hand, AVP1 is also believed to contribute to the establishment of electrochemical potential across the vacuole membrane, which is important for subsequent vacuolar secondary transport and ion sequestration. Constitutive over expression of AVP1 improves the growth and yield of diverse transgenic plants under various abiotic stress conditions—including drought, salinity, as well as phosphorus and nitrogen deficiency—although the mechanism remains to be fully understood. Taken together, AVP1 serves as a multi-functional protein involved a variety of physiological processes in plants, some of which await to be fully understood. Magnesium is an essential macro-nutrient for plant growth and development, functioning in numerous biological processes and cellular functions,nft vertical farming including chlorophyll biosynthesis and carbon fixation. Either deficiency or excess of Mg in the soil could be detrimental to plant growth and therefore plants have evolved multiple adaptive mechanisms to maintain cellular Mg concentration within an optimal range.

In higher plants, the most well-documented Mg2+ transporters belong to homologues of bacterial CorA super family and are also called “MRS2” based on their similarity to yeast Mitocondrial RNA splicing 2 protein. Several members of the MGT family mediate Mg2+ transport in bacteria or yeast as indicated by functional complementation as well as 63Ni tracer assay. In plants, they have been shown to play vital roles in Mg2+ uptake, translocation, and homeostasis associated with their different sub-cellular localizations and diverse tissue-specific expression patterns. For instance, MGT2 and MGT3 are tonoplast localized and possibly involved in Mg2+ partitioning into mesophyll vacuoles; MGT4, MGT5, and MGT9 are strongly expressed in mature anthers and play a crucial role in pollen development and male fertility. MGT6 and MGT7 are shown to be most directly involved in Mg homeostasis because knocking-down or knocking-out either of the genes leads to hypersensitivity to low Mg conditions. MGT6 encodes a plasma membrane-localized high-affinity Mg2+ transporter and mediates Mg2+ uptake in root hairs, particularly under Mg-limited conditions. MGT7 is also preferentially expressed in roots and loss-of-function of MGT7 caused poor seed germination and severe growth retardation under low-Mg conditions. Double mutant of mgt6 and mgt7 displayed a stronger phenotype than single mutants, suggesting that MGT6 and MGT7 may be synergistic in controlling Mg homeostasis in low-Mg environment conditions. In contrast to considerable research on Mg transport and homeostasis under Mg deficient conditions, the regulatory mechanisms required for adaptation to excessive external Mg remain poorly understood. Recent studies suggested that MGT6 and MGT7 are essential for plants to adapt to both normal and high Mg conditions. The mgt6 mutant displayed dramatic growth defects with a decrease in cellular Mg content in the shoot, when grown under high Mg2+. Grafting experiments further suggested a shoot-based mechanism for Mg2+ detoxification although the exact role of MGT6 in this process is still not clear. More importantly, a core regulatory pathway consisting of two calcineurin B-like Ca sensors partnering with four CBL-interacting protein kinases has been established that allows plant cells to sequester Mg2+ into plant vacuoles, thereby protecting plant cells from high Mg2+ toxicity. In this study, we identified the tonoplast pyrophosphatase, AVP1, as an important component in high Mg2+ tolerance in Arabidopsis.

Furthermore, by analyzing the avp1-4 mgt6 double mutant and avp1-4 cbl2 cbl3 triple mutant, we showed that the role of AVP1 in high-Mg tolerance was independent of previously reported MGT6 or CBL/CIPK-mediated pathway. Instead, our results suggested a novel link between high Mg2+ stress and PPi homeostasis in plants. The originally reported T-DNA insertional mutant avp1-1 contains an additional T-DNA insertion causing phenotypes unrelated to AVP1 mutation. We thus characterized another T-DNA insertion line avp1-4for this study. The avp1-4 mutant carried a T-DNA insertion in the third exon of AVP1 as further confirmed by PCR analysis and DNA sequencing . The avp1-4 homozygous mutants lacked detectable AVP1 transcripts , and its tonoplast PPi hydrolysis activity was considerably diminished, to only 10% of wild type . Compared with wild-type plants , avp1-4 mutants exhibited no obvious phenotypic changes during the life cycle including vegetative and reproductive periods , which is quite different from avp1-1, because pleiotropic phenotypes observed in avp1-1 are caused by mutation in the GNOMgene. We examined the phenotype of avp1-4 plants under multiple ionic stress conditions and found that avp1-4 mutant and wild-type seedlings grew similarly on the MS medium and did not show hypersensitive response to most of the ionic stresses such as 60 mM Na+ , 60 mM K+ , 40 mM Ca2+, 100 µM Zn2+, 40 µM Cu2+, or 100 µM Fe3+ . However, the growth of avp1-4 seedlings were severely impaired when 20 mM MgCl2 was supplemented . To validate the hypersensitivity of avp1-4 to MgCl2, we grew the seedlings of the mutant together with the wild-type plants on the 1/6 MS medium containing various levels of Mg2+, the avp1-4 mutant plants were clearly stunted as compared with Col-0 , although the primary root length of avp1 was comparable to that of Col-0 . In addition, we also studied one more mutant allele of AVP1 gene in the Wassilewskija background, designated as avp1-3, and another three mutant alleles of AVP1, fugu5-1, fugu5-2, and fugu5-3 in the Col-0 background. Measurements of seedling fresh weight confirmed a severe growth inhibition by 8 mM MgCl2 in both avp1-4 and avp1-3 mutants, as compared with their respective wild-type counterparts . Consistently, we also found that high-Mg sensitivity phenotypes in the three fugu5 mutants were comparable to those in avp1-4 . Together, these results suggested that AVP1 is required for Mg2+ tolerance in Arabidopsis. To verify that the observed phenotypes in the avp1 mutants are caused by a defect in AVP1, we conducted a complementation test in avp1-4 background. A coding sequence fragment of AVP1 was introduced into the avp1-4 mutant, and several homozygous transgenic lines were obtained . Phenotypic analysis of two representative lines showed that oblong-shaped cotyledons of avp1-4 when germinated on MS media containing low sucrose or in soil were fully restored to normal shape .

In addition, seedling growth defects of avp1-4 under high-Mg conditions were also completely rescued . Root length and shoot fresh weight of the transgenic lines under high Mg conditions were similar to those of the wild type . These data further confirmed that loss-of-function in AVP1 was indeed the causal mutation for the high-Mg hypersensitive phenotype of avp1-4.Reducing the PPi concentration in the cytoplasm and increasing the acidification of vacuoles represent the two main biochemical functions of AVP1. In order to dissect if both activities are required in this specific high Mg2+-associated process, we resorted to the transgenic line expressing yeast IPP1 gene under the control of the AVP1 promoter in the fugu5-1 mutant background. IPP1 is a cytosolic soluble protein which is not capable of translocating H+ , thus decoupling the hydrolysis and proton pump activities. Interestingly, our results showed that the severely retarded growth of fugu5-1 mutant plants under high-Mg conditions was completely recovered by expression of the IPP1 gene .To extend the phenotypic analysis of the avp1 mutants in mature plants, we examined the phenotype of avp1 mutants using hydroponic culture system. Consistent with the patterns of plant growth on agar plates,indoor vertical farming the mutant plants exhibited a pronounced growth defect than wild-type plants in the hydroponic solutions supplemented with 15 mM external Mg2+, as revealed bymuch lower fresh weight and lower chlorophyll content . The IPP1 transgenic line also behaved like wild-type plants but not avp1 mutant under this condition, suggesting that PPi hydrolysis is the key function that AVP1 plays in high-Mg adaptation. To address the contribution of PPi hydrolysis activity to high-Mg tolerance, we directly measured V-PPase activity and PPi content under normal and high-Mg conditions. Under normal conditions, PPi hydrolysis activity of two avp1 mutant alleles was reduced by ∼85%, whereas activity from two complementary lines was comparable to the wild-type control . Consistently, the amount of PPi from both mutants was increased by ∼50% . After grown for three days on 15 mM Mg2+, all the plants displayed reduced PPi hydrolysis activity and higher PPi content. However, thePPi elevation of mutant plants during high Mg2+ stress was significantly higher than that of wild type . Altogether, these results strongly indicate that the dampened hydrolysis of cytosolic PPi is the major reason for the increased Mg sensitivity in the avp1 mutants.To assess whether increased Mg2+ sensitivity in the avp1 mutant is associated with Mg2+ homeostasis, we measured the Mg content in wild-type and mutant plants using ICP-MS. When 8 mM Mg2+ was added to the growth medium, Mg content in either shoot or root in all the plants was strikingly elevated, but no significant difference between wild-type and mutant plants in Mg content was observed. . Considering Ca and Mg often affect each other in their uptake and transport, we also measured the Ca content in the same plants.

Consistent with Mg-Ca antagonism, the Ca content in both wild-type and avp1 mutant plants was evidently lower when plants were grown under high external Mg2+ conditions, but Ca content in the shoots and roots in avp1 mutants was similar to that in wild-type plants . These data suggest that both Mg and Ca homeostasis are not altered in the avp1 mutants, which are consistent with the earlier conclusion that PPi hydrolysis rather than vacuolar acidification is responsible for AVP1 function under high-Mg stress.In Arabidopsis, the magnesium transporter MGT6 is important for controlling plant Mg2+ homeostasis and adaptation to both low- and high-Mg conditions. To investigate the functional interaction between AVP1 and MGT6, we created a double mutant that lacks both AVP1 and MGT6transcripts . We next tested the sensitivity of avp1-4 mgt6 double mutant to high external Mg conditions. When grown on the 1/6 MS medium containing 0.25 mM Mg2+, the mgt6 and avp1-4 mgt6 plants showed obvious growth retardation compared with Col-0 and avp1-4 seedlings, resulting from mgt6 mutation that renders plants hypersensitive to low Mg2+ . When the medium Mg2+ levels reached 1 mM, the growth of mgt6 and avp1-4 mgt6 mutants appeared comparable to that of wild-type . Notably, in the presence of high Mg levels such as 4 mM and 6 mM Mg2+ , avp1-4 mgt6 double mutant exhibited more severe inhibition of shoot growth with significantly lower fresh weight and more reduced chlorophyll content as compared to either mgt6 or avp1 single mutant. The enhanced sensitivity of the avp1-4 mgt6 double mutant suggest that AVP1 and MGT6 may represent two independent functions that are required for plant tolerance to high Mg2+ stresses. Int. J. Mol. Sci. 2018, 19, x FOR PEER REVIEW 7 of 15 resulting from mgt6 mutation that renders plants hypersensitive to low Mg2+ . When the medium Mg2+ levels reached 1 mM, the growth of mgt6 and avp1-4 mgt6 mutants appeared comparable to that of wild-type . Notably, in the presence of high Mg levels such as 4 mM and 6 mM Mg2+ , avp1-4 mgt6 double mutant exhibited more severe inhibition of shoot growth with significantly lower fresh weight and more reduced chlorophyll content as compared to either mgt6 or avp1 single mutant. Although Mg is an essential macro-nutrient required for plant growth, high concentrations of environmental Mg2+ could be detrimental, and the targets underlying toxic effect of high-Mg are not well understood.

Among plant species analyzed, P. vulgaris was the only one that showed a binding-site penalty score lower than 5, corresponding to a score recommended to consider a small RNA-target binding as probably functional. For other species, the high penalty scores, ranging from 7.5 to 9, indicate a very low probability for the existence of a functional miR1511/ALS3 regulatory node .A previous study about the response of different Andean and Mesoamerican common-bean cultivars to AlT showed that Andean genotypes are more tolerant to this abiotic stress, as compared to Mesoamerican genotypes . Our phylogenetic analysis revealed that all the Andean genotypes present a deleted version of the MIR1511 that would result in the absence of functional mature miR1511 . Previous work from our group showed that common-bean miR1511 expression responds to AlT stress . Here we analyzed the regulation of miR1511 and ALS3, as well as the early effects of AlT in roots of common-bean plants from the Mesoamerican BAT93 genotype vs. Andean G19833 genotype, with a deleted MIR1511 . Common-bean plantlets from BAT93 and G19833 genotypes were grown in hydroponic conditions either in control or AlT treatments, for up to 48 hrs. First, we performed the expression analysis of miR1511 and ALS3 target gene, using qRT-PCR . In AlT-stressed BAT93 plants, the transcript accumulation level of mature miR1511 gradually decreased, reaching more than half at 24 hours post-treatment ,vertical grow while at 48 hpt it returned to values close to those of time 0 . As expected, G19833 plants did not express mature miR1511 . The transcript level of ALS3 target gene increased in AlT treatment. The ALS3 transcript accumulation was significantly higher in G19833 roots, which lack miR1511, compared to BAT93 roots .

At 6 hpt, ALS3 expression in G19833 roots almost doubled and remained unchanged up to 48 hpt, when transcript accumulation in BAT93 and G1988 roots reached similar levels . To further study the role of miR1511/ALS3 in the physiological reaction of common-bean plants to high Al levels, we aimed to over express the miR1511 precursor in transgenic roots. As long as stable transformation of Phaseolus vulgaris plants is, to date, nearly impossible, we chose to use BAT93 and G19833 composite plants -with untransformed aerial organs and transgenic roots . As long as common bean is recalcitrant to stable transformation, this method is an alternative to demonstrate miRNA functionality . The miR1511-overexpressing composite plants as well as control plants, transformed with empty vector , were grown in AlT and control treatments. The expression level of miR1511 and ALS3 were determined by qRT-PCR in roots from composite plants harvested at 48 hpt . A two-fold accumulation of miR1511 transcript was observed in BAT93 OE1511 roots from plants grown in either treatment, compared to EV . In G19833 EV roots, the absence of miR1511 was confirmed, however a significant accumulation of miR1511 mature transcript was observed in OE1511 roots, albeit at lower levels than the level from BAT93 OEmiR1511 roots . In control treatment, both genotypes showed lower expression level of ALS3 in OEmiR1511 vs. EV roots. In addition, increased ALS3 transcript levels were observed in AlT stressed roots from both genotypes, as compared to control treatment . The primary and earliest symptoms of plants subjected to AlT stress is an inhibition of lateral root formation and root growth due to the alteration of root cell expansion and elongation, inhibiting cell division . On this basis, we analyzed the root architecture phenotype of BAT93 and G19833 OEmiR1511 and EV plants, grown under AlT or control treatments for 48 h . The growth rate of root length, width and area as well as the number of lateral roots, was calculated from the difference of each value at 48 hpt vs. time 0. The BAT93 EV plants under AlT showed decreased rates of each root parameter , thus indicating the drastic effect of high Al on root development. In contrast, G19833 EV plants showed higher tolerance to AlT evidenced by similar rate of the root length, area, width and lateral root number in stress vs control treatments . These results are in agreement with those previously reported indicating a higher tolerance to AlT of Andean common-bean genotypes compared to Mesoamerican genotypes . Surprisingly, in G19833 plants genetically engineered for the expression of mature miR1511, the effect of root phenotype was evident.

The rate of root length, area, width and lateral root number of G19833 OEmiR1511 AlT-stressed plants significantly decreased as compared to EV plants, showing reduced levels similar to those from BAT93 stressed plants . In A. thaliana, primary root growth inhibition under phosphate limitation or AlT is mediated by ALS3 and LPR1, a ferroxidase . LPR1 acts downstream of ALS3 and its expression is epistatic to ALS3 expression . To determine if LPR1 could be involved in the different response to AlT of BAT93 vs. G19833 plants, we measured the accumulation of LPR1 transcripts in similar AlT conditions as those from Figure 4. The transcript level of LPR1 gene decreased in AlT treatment. In AlT BAT93 roots, the transcript level of LPR1 gradually decreased reaching half of the initial expression at 48 hpt. In AlT G19833 roots, the LPR1 expression was significantly lower compared to BAT93 roots from 6 hpt to 24 hpt . At 48 hpt, LPR1 transcript reached similar levels in roots from both genotypes . The LPR1 expression pattern was opposite to the ALS3 expression profile in AlT-stressed roots , indicating an epistatic relation between these two genes and the possible participation of LPR1 together with ALS3 in the control of common-bean root growth under AlT. In order to determine if miR1511 indirectly controls LPR1 expression via the regulation of ALS3 transcript, we evaluated the LPR1 transcripts accumulation in transgenic roots from OEmiR1511 and EV composite plants, growing in Alt vs control conditions. In both BAT93 and G19833 roots, a significant increase of LPR1 transcript accumulation was observed in OEmiR1511 roots from plants grown in either treatment, compared to EV roots . In AlT treatment, roots from both genotypes showed significant lower LPR1 transcript level compared to roots from control condition. Again, LPR1 expression pattern was the opposite compared to that of ALS3 in the same transgenic root samples , thus indicating the probable epistatic relationship between these two genes and the indirect regulation of miR1511 on LPR1 expression.In plants, microRNA genes have a higher birth and death rates than protein-coding genes . For various authors, the miRNAs’ evolution rate generates a reservoir of adaptability for gene regulation . Due to this high evolutionary turnover rate, new miRNA families and members emerge, while others lose their regulatory role and disappear from genomes of phenotypically close species or genotypes. In soybean, MIR1511 is subjected to this mechanism. Htwe et al.,reported two altered versions of MIR1511 alleles in some annual wild soybean genotypes, whereas no deletion was found in G. max and perennial wild soybean MIR1511. Here, we report a similar phenomenon for P. vulgaris MIR1511 genotypic variations. Only part of the MW1 subgroup of P. vulgaris Mesoamerican genotypes and all the Andean genotypes analyzed displayed a 58 bp-deletion in the miR1511 precursor gene compared to the corresponding sequence of P. dumosus, P. coccineus, the PhI gene pool and the rest of P. vulgaris Mesoamerican genotypes .

As MIR1511 is present in non-legume species, the most parsimonious hypothesis to explain the evolution process associated with this event is to consider a deletion of part of miR1511 precursor sequence. In contrast to soybean, where probably two deletion events were required for the generation of two alternative MIR1511 alleles, our results suggest a single deletion event in the common ancestor of a part of MW1 Mesoamerican subgroup and the Andean genotypes for the generation of a different allele of miR1511 precursor gene. This single MIR1511 deletion event hypothesis supports the Mesoamerican model proposed by Ariani and colleagues where the Mesoamerican gene pool is the ancestral population from which the other gene pools have derived. The fact that the PhI gene pool contains an untruncated version, as do the other closely-related Phaseolus species included in this analysis, further confirms the sister-species status of the PhI gene pool, now known as P. debouckii . P. debouckii also contains ancestral, i.e., non-derived, sequences for phaseolin seed protein and chloroplast DNA . Based on the MIR1511 phylogenetic history presented here , we propose an addendum to this model where AW gene pool genotypes derived from one, or more,indoor growers member of the MW1 Mesoamerican subgroup. A clear distinct geographical distribution pattern was observed among the P. vulgaris genotypes featuring the MIR1511 deletion and the ones with an unaltered allele . MIR1511 deletion occurred in genotypes originating from the northern and southern extreme limits of the common-bean distribution in Latin American area. Such distribution pattern correlates with the annual precipitation pattern reported for the American continent , indicating that genotypes with MIR1511 deletion originated from areas with significantly less precipitation as compared to areas where genotypes with unaltered MIR1511 originated . Drought makes soil not suitable for agriculture; it tends to increase soil concentration of different compounds that would result in plant toxicity, including aluminum toxicity, which is an important plant growth-limiting factor . The harsh soil conditions of areas where P. vulgaris genotypes lacking of MIR1511 originated probably forced these common-bean populations to adapt and favored selection of genotypes with higher AlT tolerance. In this work, we showed the experimental validation of a target gene for P. vulgaris miR1511. We validated the miR1511-induce cleavage of ALS3 transcript, an ABC transporter participating in Al detoxification in plants . However, additional action of miR1511 by translation repression of ALS3 cannot be excluded. Other proposed target genes for P. vulgaris miR1511 are not related to plants AlT response and show high binding-site penalty score, thus improbable to be considered as functional in the AlT response.We interpret that the MIR1511 deletion resulting in lack of mature miR1511 allowed common-bean adaptation to high Al in the soils by eliminating the negative regulation of ALS3 transcript and the accumulation of LPR1, in the first 48 hpt, thus increasing its tolerance to AlT and favoring plant growth. Interestingly, similar characteristics hold for the soybean MIR1511-deletion case where the origin of soybean genotypes featuring a MIR1511-altered allele is geographically correlated with areas susceptible to high Al concentration in soil due to presence of drought in these regions .

High aluminum levels in soil mainly affect plant roots; aluminum can be allocated to different sub-cellular structures thus altering the growth of the principal root and the number of lateral roots . In this sense, it has been observed that AlT-stressed plants favor the transport of chelated Al to vacuoles and from roots, through the vasculature, to aerial tissues that are less sensitive to Al accumulation . In Arabidopsis and other plants, ALS1 and ALS3, from the ATP-binding cassette transporter family, are involved in Al detoxification and enhance tolerance to AlT. ALS3 is located in the tonoplast, the plasma membrane of root cortex epidermal cells, and in phloem cells throughout the plant . It has been shown that Arabidopsis als3 mutants are more sensitive to AlT exhibiting extreme root growth inhibition, compared to wild type plants . Recent studies on the role of Arabidopsis ALS3 in root growth inhibition revealed its regulation via the inhibition of the STOP1-ALMT1 and LPR1 pathways, which indirectly control ROS accumulation in roots via the modulation of Fe accumulation . Furthermore, Arabidopsis ALS3 expression is induced by excess Al , a phenomenon we observed in common-bean plants as well . Common-bean ALS3 expression doubled after 6 hours under AlT in roots from G19833 plants, while in stressed roots from BAT93 plants a similar level was reached only after 48 h of treatment . The opposite expression profile was found for the ALS3-epistatic gene LPR1, in the same samples . Our data on the different ALS3 and LPR1 expression level from both genotypes indicate that the absence of the negative regulator miR1511 in G19833 plants allows a faster response to AlT. Although the level of mature miR1511 decreased in BAT93 roots up to 24 h of after exposure to high Al, this level seems sufficient to induce degradation of ALS3 transcript, which showed reduced levels compared to G19833 roots, and an increase of LPR1 expression . Our analysis of root architecture in common-bean plants showed that G19833 Andean genotype plants are more tolerant to AlT as compared to Mesoamerican BAT93 plants .

Deficiency of B has been identified as a serious agricultural issue in more than 100 crops in 80 countries . Limitation of B impairs growth of young tissues and seed set, which results in depressed quality and quantity of agricultural products. In rice, B content is up to 10-time lower than those of dicot plants . And thus rice young seedling is relatively resistant to B limited condition compared to dicot plants such as Arabidopsis . However, the effect of B limitation until the reproductive phase is little known in rice. In this study, we evaluate the growth and yield of rice subjected to B deficient condition by hydroponic experiments. We previously reported that a boron efflux transporter OsBOR1 plays a crucial role in efficient root-to-shoot translocation in rice . Over-expression of AtBOR1 improved growth and seed fertility in Arabidopsis plants under B deficient condition . But this strategy, up-regulation of native B transporter to achieve the tolerance to B deficiency, has not been applied for the crop so far. We herein generated several independent lines of transgenic rice plants over expressing rice BOR1 and characterised the phenotypes of these transgenic rice plants under B deficient condition. Rice cultivar Nipponbare was used. Growth experiments were carried out in a green house under natural light condition . After germination on the agar medium containing 0.5 mM CaCl2, seedlings were transferred to a Kimura B hydroponic solution containing 2 mM MES with different B levels: Concentration of B in the solution was 18 µM for B sufficient condition, and 18, 0.18, 0.03 µM for B deficient treatments. Seedlings were grown until grain ripening stage and plant height was measured every other week. At harvest, stackable planters yield components were measured. Concentration of B in flag leaf and husked grain were determined by ICP-MS after digestion using nitric acid and hydrogen peroxide.

Difference in plant height among the B treatments was not clear until 4 weeks after treatments . However, when subjected to lower than 0.18 µM B, reduced plant height was clearly observed according to the growth periods . Visible symptom on leaf blades such as chlorosis was not observed in B deficient-treated plants. On the other hand, the grain yield was greatly reduced by B deficient treatments lower than 0.18 µM B, which was mainly because of decreased numbers of spikelets . Under 0.03 µM B, seedlings showed dwarf phenotype and panicle formation was severely inhibited . Concentration of B in flag leaf and brown rice was also decreased according to the level of B supply . These results suggest that in rice, young seedlings were relatively tolerant to B limited condition, however, continuous low B supply clearly impairs the vegetative and reproductive growth, leading to decreased yields. RT-PCR analysis of 11 independent transgenic lines demonstrated the expression and its variation of introduced gene in transgenic plants . We selected 9 lines with sufficient numbers of seeds and tested the growth under moderate B deficient condition for 2 weeks. The growth of shoots and roots did not differ among 8 transgenic lines and NT . However, among transgenic lines over-expressing introduced OsBOR1, B concentrations in xylem sap of several lines were higher compared to those of NT and transformants with weak expression of introduced gene . These results suggest that increased expression of the rice boron transporter BOR1 enhanced root-to-shoot translocation of boron, which might be resulted in improved B acquisition and further growth under boron deficient condition. Deltas are the pinnacles of life: they provide resources for a diverse array species and it is therefore critical that we protect them. After describing the current issues facing the Sacramento San Joaquin River Delta, CA, US, this report will describe a case study of a water management and agricultural diversification system at Sherman Island which can serve as a demonstration project for future application to deltas around the globe. The magnitude and diversity of California’s agricultural, environmental, industrial, recreational, and urban interests in the Sacramento-San Joaquin Delta emphasize the importance of protecting the Delta infrastructure. Protected by 1,100 miles of levees are over 538,000 acres of farming, 64,000 acres of cities and towns, and 75,000 acres of undeveloped land from flooding and saltwater intrusion the Delta is home to nearly 515,000 people living in seven counties, 500 different plant and animal species, including 20 that are endangered and major transportation and utility infrastructure. 

The Sacramento, San Joaquin, Mokelumne, Cosumnes, Calaveras Rivers and their tributaries flow into the Delta and provide water to over 22 million Californians – over two-thirds of the population. Sherman Island sits on the western edge of the Sacramento-San Joaquin River Delta and is one of the key geographic features protecting the Delta as a water resource. The island is located northeast of the city of Antioch, California, and lies within the jurisdiction of Sacramento County. The Sacramento and San Joaquin rivers meet at its western boundary, which is bordered to the northeast by Three-Mile Slough. Levee instability caused by continued subsidence in the region is a severe risk to cause catastrophic failure. If Sherman Island’s levees were to fail water quality for the entire Delta and most of California, would be compromised, most likely on a timescale of years to decades. Because residents rely on the Delta for their drinking water numerous health issues would result from the Delta being compromised. In addition, the livelihoods of those who utilize the freshwater, such as farmers and industry works, could deteriorate. Delta Bearier Engineering estimates the cost to recover lost water supply and to repair levees, infrastructure, and damaged homes in the event of levee failures to be as high as $2.2 trillion . The increased salinity due to saltwater intrusion will also likely destroy populations of wildlife species. The loss of species has the ladder effect on the diet and shelter of other species; thus, whole ecosystems are susceptible to deterioration. Global climate change is a daunting but undeniable reality, the impacts of which must be considered. Engineered systems must be designed with these effects in mind if they are to remain resilient over the entire life span of the project. In considering just how climate change will affect the Sacramento-San Joaquin Delta, it is important to evaluate the following: Sea-Level Rise Numerous scenarios and global climate models have been developed to predict the effect of climate change on global mean sea level . The Intergovernmental Panel on Climate Change gives an estimated increase in GMSL of 0.1 m – 0.9 m between the year 1990 and 2100 . Similarly, a more recent study suggests sea level will rise by as much as 0.5 m to 1.4 m over the course of the next century . There are several implications for Sherman Island and the Delta given these predictions. Governed by significant tidal inflows, the Delta is very susceptible to increased salinity in inland waterways due to rising sea level . This poses a great threat to the viability of Sherman Island and the Delta as a water resource for the State of California. Saltwater intrusion into the lower Delta could compromise water quality, resulting in reduced agricultural yields and greater stresses on alternative water sources. Additionally, a higher mean sea level also means increased pressure on the already fragile levee system.

Levee heights would need to be drastically increased, or else the combination of increased sea level and major storm events would pose an even greater risk to the system. While sea level rise has greater impact on long term water level variations, it is changes in river flow that have the biggest effect on a short-timescale for the Delta . Most predictions indicate that there will be increased flows during the winter months and reduced flows in spring and summer. From a water resource perspective, this implies that less water will be available as the state approaches times of warmer temperature and increasing agricultural demand. Furthermore, reduced spring flows will invite saltwater intrusion further inland into the Delta at a time when even less water will be available to flush the system and maintain the integrity of water supplies. While it is difficult to apply global climate change models to smaller scale regions such as the Delta, it can be inferred from the available studies that wind velocity and intensity will increase in the area. The serious implications of this matter are that wind velocities in the Delta region determine wind and wave action, two factors which have a significant impact on levee erosion . Consequently, levees weakened by wind and waves are subject to greater risk of failure in the event of high water or severe storm. Changes in average temperature and the amount and type of precipitation are also expected as part of global climate change. The amount and timing of annual runoff is one of the biggest impacts, stackable flower pots as precipitation normally falling in the form of snow will turn to rain, reducing the amount of water available for spring flows from snow melt . Increased temperature will have significant effect on the temperature gradient between the San Francisco Bay Area and the Central Valley, further increasing the intensity of wind velocities . Warmer temperatures will also lead to earlier melting of snow, resulting in reduced water availability for an agriculture-dependent state already plagued by drought. Delta system must consider the needs of global climate change including: flood control, agricultural development, water quality, and environmental sustainability. Consequently, efforts must be made to reverse subsidence, stabilize the fragile levee system, increase economic productivity, and protect vital water and environmental resources. The Aquaponics Water Management System combines hydroponics , aquaculture , and restored wetlands enclosed in flood storage zone. The aquaponics system is a bio-integrated system in which waste byproducts from aquaculture are used as nutrients for plant growth in the hydroponics components. Each aquaponics system consists of fish rearing tanks, solids settling and removal tanks, a bio-filter, the hydroponics rafts and a sump. The first step of the aquaponics cycle is fish eat food and excrete ammonia rich effluent. The effluent is sent through the settling tanks to reduce the amount of suspended organic matter. Next the ammonia is removed and bacteria convert ammonia and nitrites to nitrates in the bio-filter. The nitrate rich water is then pumped to the hydroponics component where plants’ roots hang into the pipes and absorb the nutrients from the water. Once water has reached the end of the hydroponics component, it is collected in a sump and then returned back to the rearing tanks. 

The water flow through the aquaculture and hydroponics systems is shown in Figure 1. The aquaculture components sit outside of the flood management zone while the hydroponics and wetland systems dwell within. The flood storage zone spans 800 acres and is able to store 12,000 to 16,000 acre-feet of water. Figure 2 displays the approximate layout of the system which consists of floating hydroponics rafts that can move up and down with the variations of the water levels but are anchored to prevent lateral movement. In addition, the flood storage zone provides wetland acreage for wildlife habitat restoration, recreation, carbon sequestration, and subsidence reversal. Levees and Siphons function to enclose the flood storage zone, transport and store water during high river water levels as shown in Figure 3.Levees construction and upgrades are the primary infrastructure needed for the flood storage zone. Levees currently bound the northern, southern, and western edges of the Aquaponics Water Management System. The western and southern edges are bounded by Army Corp Levees protecting the Island from Sacramento and San Joaquin rivers, respectively. The northern edge is bounded by Mayberry Slough levees which are not engineered project levees. Therefore, there is little boring data and high uncertainty of the construction materials and stability. Therefore, a sandy berm will be constructed to filter, and buttress the levees to provide support. The largest infrastructure component to construct is the internal cutoff levee to enclose the flood storage zone by bounding the eastern side. This levee will be constructed 1,160 ft west of the Antioch Bridge, encompassing Scour Lake while maintaining a buffer zone between the system and the maintenance setbacks for the Antioch bridge.

To evaluate whether the observed metal accumulation phenotypes were due to impaired expression of the OPT3 gene, the coding sequence of OPT3 was expressed in opt3- 2 under the control of the Cauliflower mosaic virus 35S promoter. Over expression of OPT3 in four independent lines was confirmed by qPCR . opt3-2 contains a T-DNA insertion in the 5’ UTR of OPT3 . Therefore, the residual OPT3 transcript observed in opt3-2 is expected in this knockdown line. OPT3 complementation lines were grown on heavy metal-containing soil, and the metal concentration of their seeds was determined by ICP–OES. Cd accumulation in seeds of the four complemented lines was reduced to wild-type levels . Over expression also rescued the seedling sensitivity of opt3-2 to Cd , indicating that ectopic expression of OPT3 is sufficient to complement the sensitivity and metal accumulation phenotypes of opt3-2.Previous GUS staining experiments have shown that OPT3 is expressed throughout the vasculature; however, localization at a higher resolution has not been evaluated . To identify where in the vasculature OPT3 is preferentially expressed, β-glucuronidase was expressed under the control of the native OPT3 promoter. Under standard growth conditions, GUS staining was negligible; however, under Fe-limiting conditions , staining was clearly observed in the phloem, but not in the pith or endodermis . Consistent with our findings, cell-type-specific microarray data sets show the highest intensity values of OPT3 in the phloem, comparable to the phloem sucrose transporter SUC2 . Thus, two independent approaches show preferential expression of OPT3 in the phloem. To gain insight into the subcellular localization of OPT3, an N-terminal YFP–OPT3 translational fusion was infiltrated into Nicotiana benthamiana leaves. Fluorescence was detected along the cell periphery, indicative of plasma membrane localization . A weaker perinuclear fluorescence and transvacuolar strands were also observed in some cells , indicating that a fraction of the YFP–OPT3 localizes to the endoplasmic reticulum . The ER fluorescence pattern, however, was not present in all cells. Furthermore, Hechtian strands were clearly present connecting the cell wall to the plasma membrane of plasmolyzed leaf cells .These results suggest that OPT3 is a plasma membrane transporter preferentially expressed in the phloem.The Arabidopsis mutant opt3-2 shows a constitutive Fe-deficiency response in roots including the up-regulation of the Fe/Zn/Mn transporter IRT1 . Despite this Fe-deficiency response, Fe sensing in shoots remains intact . The molecular mechanisms mediating shoot-to-root signaling of iron status in plants remain largely unknown.

The impaired iron sensing in roots but not shoots of opt3-2, in conjunction with phloem localization, suggests a possible role of OPT3 in shoot-to-root transport of a signal reporting metal status. To test this hypothesis, the OPT3 coding sequence was expressed in opt3-2 under the control of the shoot-specific chlorophyll a/b binding protein promoter . Shoot specificity of the CAB2 promoter was determined by GUS staining . RT–PCR analyses confirmed that OPT3 is preferentially expressed in the shoots of three independent transgenic lines . The residual OPT3 transcript in opt3-2 roots expressing CAB2pro:OPT3 plants is consistent with the knockdown nature of the opt3-2 allele. Thus, the low level of OPT3 transcript in roots is not sufficient to properly regulate metal homeostasis in roots . Two of the major phenotypes described in opt3-2 are the constitutive iron-deficiency response in roots, as illustrated by high IRT1 expression , and the over-accumulation of Cd in seeds . Thus, we tested whether shoot-specific expression of OPT3 was able to complement both phenotypes. As shown by RT– PCR, IRT1 transcript levels were greatly reduced in the roots of CAB2pro:OPT3-expressing plants compared to the opt3-2 mutant . These results show that shoot-specific expression of OPT3 is sufficient for proper regulation of metal homeostasis, including communication between leaves and roots. Furthermore, the Cd accumulation in CAB2pro:OPT3 seeds was reduced to wild-type levels . Seedling hypersensitivity to Cd was also rescued in the three independent CAB2pro:OPT3– expressing lines . Collectively, these results demonstrate that shoot-specific OPT3 expression is sufficient to complement opt3-2 root phenotypes, suggesting that OPT3 may mediate the long-distance transport of a signaling molecule from leaves to relay information about metal status, thus contributing to whole-plant metal homeostasis.To test whether OPT3 functions in the mobilization of Fe or other molecules, we first assessed the capacity of wild-type and opt3-2 to remobilize Fe from one leaf to other leaves using the radiotracer 59Fe. In these experiments, 59Fe was loaded into a mature leaf as Fe2+ at a slightly acidic pH to resemble the apoplastic pH. The addition of ascorbic acid was used to reduce Fe3+ to Fe2+ and maintain it in the reduced form. Figure 7A shows that Fe can be re-mobilized from one leaf to adjacent leaves in wild-type. In contrast,aeroponic tower garden system opt3-2 shows negligible movement of 59Fe between leaves. Figure 7B shows the 59Fe activity in the four leaves adjacent to the leaf where the 59Fe was originally applied.

Compared to wild-type, opt3-2 shows a severe reduction in the quantity of 59Fe mobilized from one leaf to the adjacent leaves , suggesting that OPT3 is required for the reallocation of Fe between plant tissues. In fact, opt3-2 plants over-accumulate Fe in mature leaves compared to wild-type, as visualized by Perls’ staining . Interestingly, over-accumulation of Fe in opt3-2 occurs only in mature leaves but not in young leaves . Moreover, accumulation of Fe in opt3-2 is more evident at the base of the trichomes and near the vasculature in the minor veins but not in the main vasculature, suggesting that, in opt3-2, the reallocation of Fe between leaves is impaired, particularly at advanced stages of leaf development . To test whether OPT3 functions as a Fe2+ transporter similarly to IRT1, we expressed OPT3 in a yeast strain deficient in Fe2+ uptake . As previously shown, IRT1 expression in yeast allows the fet3fet4 strain to grow on minimal media without the addition of extra Fe . OPT3 was unable to rescue the fet3fet4 strain , suggesting that, in yeast, OPT3 does not mediate the uptake of Fe2+ like IRT1. Subcellular localization studies, however, show that OPT3–YFP protein fusions do not localize to the plasma membrane in yeast in contrast to in planta . This mislocalization of OPT3 in yeast precluded further characterization of OPT3 using yeast as a heterologous system. Note that, if fet3fet4 yeast cells were not sufficiently pre-starved of iron, growth of the fet3fet4 mutant was observed, and therefore long-term starvation of yeast was required for these complementation tests. We attempted complementation with different yeast promoters, starting with the strong GAL promoter . Using the phosphoglycerate kinase yeast promoter, OPT3 also did not complement the pre-iron-starved fet3fet4 yeast mutant, consistently with previous studies showing that OPT3 does not complement this yeast mutant . Nevertheless, 59Fe re-mobilization studies suggest that OPT3 is essential for the remobilization of Fe within plant tissues; whether this transport occurs as Fe2+ or as a Fe-ligand complex remains to be determined. OPT3 is a member of the oligopeptide transporter family and some members of this family have been found to have broad substrate specificity for peptides of different length and amino acid composition . To test whether OPT3 mediates the long-distance transport of GSH in planta, we pursued radiotracer experiments to assess the movement of 35S-GSH from one leaf to adjacent leaves . No differences were found between wild-type and opt3-2, suggesting that OPT3 does not participate in the mobilization of GSH between plant tissues. Interestingly, opt3- 2 rosette leaves from plants exposed to 20 μM CdCl2 and supplemented with 0.5mM GSH accumulated Cd, but no other metals, to wild-type levels. These results suggest that GSH is required for Cd retention in leaves. On the other hand, GSH supplemented to the roots reduced Cd in the leaves of both opt3-2 and wild-type plants likely because GSH trapped Cd in roots of both opt3-2 and wild-type plants .

Glutathione has recently been shown to play a critical role in Fe signaling in yeast by stabilizing FeS clusters in the cytosol . In Arabidopsis, GSH is also important to maintain proper homeostasis and crosstalk between Zn and Fe metabolism . To test whether long-distance transport of GSH is important for proper shoot-to-root signaling and homeostasis of trace metals in roots, we measured the constitutive high activity of the root ferric reductase in opt3-2 after foliar application of GSH . In all cases, including the application of foliar GSH, GSH applied to roots, or foliar application of Fe, the activity of the root ferric reductase remained constitutively high in opt3-2. We also tested whether iron applied directly to roots or complexed with GSH, citrate, or nicotianamine is sufficient to repress the high activity of the Fe chelate reductase in opt3-2 roots. Supplemental Figure 6B shows that iron alone , or in complex with GSH, nicotianamine, or citrate, cannot down-regulate the constitutive iron-deficiency response in opt3-2 back to wild-type levels.We have identified an Arabidopsis mutant, opt3-2, that over-accumulates Cd in seeds and roots but, unexpectedly, under-accumulates Cd in leaves . Cadmium distribution throughout the plant is an orchestrated process dictated by root uptake, root-to-shoot translocation through the xylem, dutch buckets for sale and redistribution of Cd from leaves to sink tissues via the phloem. opt3-2 displays constitutive up-regulation of IRT1, a root transporter with broad specificity for heavy metals including Cd . Over-accumulation of Cd, Zn, Fe, and Mn in roots may be explained by the constitutively high expression of IRT1. However, under-accumulation of Cd in leaves and over-accumulation of Cd in seeds, which is different from essential metals , is inconsistent with the high expression of IRT1 . Nutrients, water, and heavy metals are mobilized from leaves into seeds through the phloem . Accumulation of metabolites in sink tissues and under-accumulation in source tissues is best described as an increased redistribution process, likely through the phloem . Notably, of the analyzed metals, only Cd under-accumulates in leaves . These results suggest that, in contrast to the broad specificity of heavy metal uptake at the root level, metal-specific mechanisms mediate the remobilization of heavy metals from leaves to sink tissues. In addition to the altered distribution of heavy metals within the plant leading to over-accumulation of Cd in seeds, opt3-2 also shows hypersensitivity to Cd at the seedling stage . Both the increased accumulation of Cd in seeds and the Cd hypersensitivity of seedling growth are restored to wild-type levels by ectopically expressing OPT3, demonstrating that the altered redistribution of Cd through the plant is the result of the reduced expression of OPT3 in opt3-2 .The opt3-2 mutant displays a constitutive iron-deficiency response in roots, while the leaves properly respond to iron levels as indicated by wild-type levels of ferritin expression , suggesting that the iron status response is mainly disrupted in roots. In plants, the root iron-deficiency response is regulated by local signals within the root and also by unknown systemic signals originating from aerial tissues . OPT3 is a plasma membrane transporter preferentially expressed in phloem cells during iron starvation . Cell-specific micro-arrays and OPT3pro:GUS analysis under Fe-limiting conditions show preferential expression of OPT3 in phloem cells, suggesting a role of OPT3 in long-distance transport processes. Notably, shoot specific expression of OPT3 in the opt3-2 background rescued the constitutively high expression of IRT1 in roots, the seed Cd over-accumulation phenotype, and the seedling sensitivity to Cd . These results suggest that the impaired metal homeostasis in opt3-2 roots is caused by a disruption of the shoot-to-root signaling of the leaf metal status. Thus, OPT3 is the first shoot-expressed gene required for proper communication from leaves to roots to maintain metal homeostasis at the whole-plant level. Several Arabidopsis and tomato mutants displaying an Fe-deficiency response in roots can be rescued by foliar application of Fe ; these experiments suggest that shoot-to-root Fe signaling plays an important role in Fe homeostasis which in turn could also impact the uptake and accumulation of other transition metals such as Zn, Mn, and Cd, as seen in opt3-2 . Foliar application of Fe does not repress the Fe-deficiency response in opt3-2 roots to wild-type levels , suggesting that source-to-sink transport of Fe, or a molecule mediating Fe signaling, is impaired in opt3-2. Radiotracer experiments using 59Fe demonstrate that the movement of Fe between leaves is impaired in opt3-2 ; whether this leaf-to-leaf transport occurs as Fe2+ or as an Fe–ligand complex remains to be determined.

They found that reading the final sentence of expository texts relative to narrative texts elicited a greater increase in N400 amplitude, and they concluded that expository texts required more demanding semantic processing. Eason et al. also reported differences between genres, showing that expository texts placed higher demands on executive function than narrative texts, particularly inferencing and planning/organizing information. EF is thought to be essential to the process of building a coherent text representation because it enables readers to store previously read text ideas as they simultaneously read new ideas and integrate them into their mental representation . While behavioral data certainly support the theoretical significance of EF to reading comprehension in general , Eason et al.’s findings of higher demands on EF for expository text suggest that for this particular text genre, which is critical for acquiring new information, EF is particularly salient.One hallmark of successful reading comprehension is that the reader can distinguish between ideas that are important, or central, to the overall meaning of the text, and those that are less important, or peripheral.The ideas and their connections form a network in the reader’s mind . Some ideas are causally or logically connected to a great number of other ideas and as a result emerge as being important, or central, to the overall meaning of the text, while others have relatively few connections and fall out as being peripheral, or unimportant . A robust finding in the comprehension literature is that skilled readers are more likely to recognize and recall an idea the more central it is to the overall meaning of the text . This finding holds for both narrative and expository texts . van den Broek et al. propose that a reader’s ability to distinguish a text’s central and peripheral ideas, or their sensitivity to structural centrality,hydroponic fodder system is an important indicator of their comprehension ability.

For example, adults show greater sensitivity to structural centrality than do children ; typically developing children show greater sensitivity to centrality than do children with reading disability as well as those with Attention Deficit Hyperactivity Disorder ; and readers show greater sensitivity to centrality when reading in their native compared to foreign language . Importantly, studies suggest that centrality tends to emerge as a feature of the developing text representation. van den Broek used eye-tracking equipment to show that skilled readers fixate more frequently and spend more time reading central ideas than they do peripheral ones. This suggests that centrality is a dynamic construct that emerges as the reader processes a text, consistent with the idea that readers form connections among semantically related text ideas as they read. In theory, the ideas that are most important stand out because they are the ones that have the most connections and are consequently the ones most likely to be recalled. To date, although sensitivity to centrality has been investigated behaviorally, the neural mechanisms remain unknown. Understanding the neural mechanisms underlying sensitivity to centrality may allow for a more specific understanding of normal and disrupted comprehension processes.The current study sought first to identify the neural correlates specific to expository text comprehension, looking both at regions which overlap with single word processing and those which are specific to discourse-level processing. Once the systems for expository text comprehension were identified, we employed temporal analysis techniques to examine how these systems change over the course of building and maintaining a coherent mental representation of the text. We hypothesized that when isolating discourse-level comprehension from word-level comprehension, we would see regions that have previously been implicated by sentence and narrative comprehension, particularly those associated with discourse-level language processing separate from social cognition [bilateral TP, angular gyrus , and PCC] . We predicted that the other traditional language regions, such as left-lateralized inferior frontal gyrus , middle temporal gyrus , and anterior superior temporal sulcus , would most likely be shared by both word and passage tasks, but that these multi-functional regions would behave differently over the temporal course of passage comprehension compared to single-word comprehension .

Additionally, due to prevalence of information organization in expository comprehension, we expected to see activations in the dorsal attention network [dorsolateral prefrontal cortex , intraparietal sulcus , inferior parietal lobule ], which has been associated with the kind of updating, integrating, and immediate planning of information that has been behaviorally described in previous studies on expository comprehension . We consequently hypothesized that over the course of passages alone, semantic control areas shared by words and passages at the mean-level would become increasingly responsive over time in passage comprehension alone due to the increased semantic demands associated with integrating and maintaining new information in a global text representation. Given Yarkoni et al.’s study showing that the parietal visuospatial attention regions are involved in the construction of text , while classic language areas are reflective of the maintenance of readers’ mental models, we hypothesized that along with the emergence of a greater reliance on perisylvian regions over time, in passages we would see a decrease over time of posterior parietal regions. The second goal of the current study was to examine the patterns of neural activation that are uniquely associated with processing central vs. peripheral ideas. While behavioral measures clearly indicate that readers distinguish central from peripheral ideas, both online and when recalling the text, the neurobiological processes that support this fundamental aspect of text comprehension have yet to be explored. Gaining insight into processes that promote a reader’s sensitivity to centrality advances current comprehension models. More focally, it allows for isolating the underlying neural mechanisms supporting processes that may be disrupted in individuals with poor sensitivity to centrality. Such knowledge may eventually inform ways to individualize intervention for problematic reading comprehension. Given previous behavioral findings, we expected there to be unique semantic and integrative regions that differentiate central ideas from peripheral ideas. Finally, we predicted that with the temporal progression of the text, there are changes in the cognitive demands required in differentiating central and peripheral information and integrating those units into the mental model, resulting in temporally dynamic neural systems for different types of textual information.

To accomplish the goals of the study, an fMRI passage comprehension task was designed in which participants viewed three types of stimuli: coherent expository passages and scrambled words and nonalphanumeric symbols . Within the Passages condition, we delineated the text’s central and peripheral ideas. To examine differences between central vs. peripheral ideas, as well as overall patterns of activation associated with text, we employed a typical general linear model . To examine the emergence of a mental representation of the text, or dynamic changes taking place over time, an approach sensitive to temporal features was taken , whereby examination of neural activation that emerged or diminished over time for various conditions was revealed.Seventeen adults participated in the study. All participants met the following inclusion criteria: native English speakers; normal hearing and vision; no history of major psychiatric illness; no traumatic brain injury/epilepsy; no history of a developmental disability; and no contraindication to MRI. Each participant gave written consent at the beginning of the study, with procedures carried out in accordance with Vanderbilt University’s Institutional Review Board. All participants had a standard score within the average range on a composite of standardized reading tests or had no history of difficulty with reading. Participants received $25 as compensation for a 2-h testing session.Coh-Metrix 2.0 was used to create 8 passages that were equivalent across measures of word concreteness, syntactic simplicity, referential cohesion, causal cohesion, and narrativity. Passages were matched on descriptive factors, including: number of words, average sentence length, and all passages measured a Flesch-Kincaid grade-level between 4.0 and 4.9. To insure that passages were equivalent in difficulty, each of the 8 passages was isolated and compared to the mean of the remaining 7 passages. Passages were considered equivalent when measures were within a 90% confidence interval of the mean of the group of remaining passages. At the end of this process, fodder system the passages were equal across 23 measures of descriptive statistics, vocabulary frequency, word concreteness, syntactic simplicity, referential cohesion, causal cohesion, and narrativity . Four of these passages were used for the Passages condition and four were used for the Words condition , which included words from the passages in randomized order. All passages were 150 words in length. Each sentence was no longer than 13 words. The passages were all expository and included the following topics: Hang Gliding, Wrasses, Velvet Worms, and Hydroponics. Each passage consisted of two paragraphs, the first of which served to introduce the topic while the second elaborated on a particular detail of the subject matter.Using imaging technology to explore the neural correlates of reading comprehension is a challenging task due to the temporal nature of discourse processing. Previous studies have presented the entire paragraph on one screen , but this procedure prohibits comparing how readers process specific aspects of the passage, such as central vs. peripheral ideas, because the block contains both types of information.The most temporally precise presentation method is to present the story one word at a time, and several studies have employed this procedure . When piloting passages using this approach, participants reported that it created an uncomfortable, artificial reading experience, likely in part because readers typically process words up to 14–15 letters to the right of their fixation , and using a single word-by-word presentation prevents this.

The moving window procedure is an alternative method that allows examination of the processing associated with single words. The advantage of this procedure is that the word immediately preceding and following the word under fixation are also visible. Although this allows for a more naturalistic reading experience, the approach was undesirable for this study because it requires a self-paced design, and temporal consistency in the presentation of stimuli is required for group comparisons. To avoid both the above confounds, we presented our passages one meaningful phrase at a time. This procedure enabled us to compare activation related to processing central and peripheral ideas, yet decreased the artificial demands imposed by a word-byword presentation. Each phrase was presented on a separate trial. The phrases included noun phrases, verb phrases, and prepositional phrases, and they ranged from 1 to 6 words in length. The number and type of words presented together determined the phrases’ presentation duration. We allowed 550 ms for each content word and 275 ms for each function word. For timing purposes, we presented no more than three content words per slide and randomized the time between phrases to allow comparison across phrases. The Words condition followed the same presentation format as the Passages condition. The baseline condition was presented between paragraph 1 and paragraph 2 of both the Passages and Words conditions. The purpose of this design was to allow participants’ activation to return to baseline after reading each block . The presentation sequence was: Passage condition, Paragraph 1; Baseline condition; Passage condition, Paragraph 2; Baseline condition; Words condition; Baseline condition. The mean time for the passages block was 78.54 ; Baseline mean = 47.69 ; and Words mean = 82.45 . In all three conditions, 8% of the stimuli were repeated on two consecutive screens. To monitor whether participants attended to the stimuli, participants pressed a button with their right thumb when they detected a phrase repetition or a symbol configuration repetition. Mean percentage correct response was very high .Imaging was performed on a research-dedicated Philips Achieva 3T MR scanner with a 32-channel head coil. Functional images were acquired using a gradient echo planar imaging sequence with 40 slices with no gap and consisted of 4 runs, each 7 min . Other relevant imaging parameters for the functional images are TE = 30 ms , FOV 240 × 240 mm, slice thickness = 3 mm with 0 mm gaps, 75◦ flip angle, TR = 2200 ms, and a matrix size 80 × 80 , implying 3 mm3 isotropic voxels. All functional data were analyzed using MATLAB and SPM8 . The functional data for each participant were slice-timing corrected, aligned to the mean functional image, normalized to MNI space, and spatially smoothed with a 8 mm FWHM Gaussian filter.

The sprouted stem fragments rested on a floating plastic mesh supported by a ring of plastic pipe, on the surface of each trash can’s nutrient solution. A sheet of opaque white plastic was wrapped around and over each trash can to block out sunlight preventing algae growth and high temperatures in the nutrient solution. The nutrient solutions in the containers were monitored two times per week during a 48-week growth period. Each check consisted of the following: addition of enough deionized water to bring the can’s nutrient solution level up to a 100-L mark, determination of the can’s electrical conductivity in full volume. A concentrated Hoagland solution was added to re-establish the conductivity of the nutrient solution to its original value. The pH was adjusted to 5.7. The harvest dates were partially determined by the growth of the plants as the experiment progressed. Harvested plants were separated into apical meristems, green leaf blades, brown leaf blades , green leaf sheaths, brown leaf sheaths , stems, rhizomes, and roots. Plant parts were dried to a constant weight at 60 C. Biomass of the tissue was determined and sub-samples were ground in a Wiley mill to pass a 0.5 mm mesh screen . The nitrogen and carbon contents of the tissues were determined using an organic elemental analyzer . Stem fragments with meristems can root and regenerate new Arundo plants , as has been reported earlier by . There were significant patterns in rooting success of meristems on Arundo stems throughout the growing season. In the winter months of November through January, rooting is low, and success percentage lies below 20%, with the exception of 28 ± 12% rooting for meristems from hanging stems in January . Nearly all meristems rooted from March through September. The speed with which meristems rooted showed a related pattern through the growing season . In the period with the lowest rooting success, t50 had the highest values,dutch buckets system indicating the slowest rooting.

Rooting was most rapid in the months of May through July, a time window that was more narrow that the period in which rooting is most successful.There are no significant differences between the results in plain water and half-Hoagland nutrient solution . The rooting success and speed pattern is similar in soil, but the single replicate meristems do not allow for inclusion in the two-way ANOVA. When compared within each sampling, the rooting success of meristems from hanging stems was significantly higher than that of meristems from upright stems . When split for rooting medium, there was no significant different in rooting success between hanging and upright meristems over time in plain water , but the differences remain significant in the nutrient solution and in soil . Like with rooting success, rooting speed of hanging meristems was significantly faster when compared to that of the upright meristems of the same sampling date . When separated among rooting media, the difference in the speed with which rooting occurs was most pronounced in plain water , but still exists in the nutrient solution and soil . Though these differences in rooting success and the speed of rooting may be statistically significant, they generally are too small to be ecologically significant. Stem diameter at the node where the meristem is placed is an indicator of relative height on the stem, and the age of the meristem. Within stems, there was no relation of rooting success or speed with the diameter of the stem at the point of the meristem, so the older meristems on an Arundo stem do not root better or faster than the younger meristems on Arundo stems.When the temperature of the rooting environment was controlled at 28/15 °C for 14/10 h during the entire growing season the seasonal patterns of rooting success and speed remained, and differences between the seasonal rooting patterns of the fragments from hanging and upright stems emerged . The overall patterns differed slightly from those in the greenhouse experiment, with the lowest rooting by both stem type fragments in February through March. The rooting percentages increased in April, and the highest rooting rates occurred from July through September at 80 – 92% for both stem types.

In October the rooting of the upright stem fragments decreased more than that of those from hanging stems. The lowest rooting rates of the stem fragments from upright stems were 0 – 10% in February and March, while the rooting rates of the hanging stem fragments only decreased to 30% . The positive influence of the seasonal effect in the months of July – September on the rooting rates of both stem types masked the difference that emerged in the Winter and Spring months. The rooting by meristems from upright stems benefits more from this seasonal effect that of meristems from hanging stems. The seasonal effect on the rooting rates of the stem types could be related to a number of environmental factors that change during the growing season, such as temperature, light intensity, and daylength. determined that stem fragments sampled at the same time, but stored at different temperatures, displayed different sprouting percentages when potted and regenerated at the same temperature in a single greenhouse. We hypothesize that the different ambient temperatures prior to sampling in our experiment was an ecophysiological equivalent of the experimental factor “storage temperature” in the Boose and Holt study, and one of the factors involved in the seasonal pattern of A. donax stem fragment rooting.The seasonal differences in the rooting percentages and speed between meristems from upright and hanging stems that was striking under controlled temperature conditions had been much less pronounced in the greenhouse rooting experiment. The results of the greenhouse rooting experiment show that the temperature at the time of rooting influence the effects of the seasonal factor. Environmental effects, such as temperature and inundation, are known to affect the success of invasive plants with either negatively or positively In the greenhouse experiment, the temperatures of the rooting medium varied with the ambient temperatures and solar irradiation, while the temperature of the rooting environment in the growth chamber experiment was the same throughout the growing season. The greenhouse, the temperature of the rooting media in the winter ranged from 0.5 – 2 °C at night to 19 –21 °C during the day. In the spring and summer, solar irradiation increased these temperatures to 16 -18 and 28 – 34 °C, respectively. To test the effect of the temperature at rooting, we tested the rooting of fragments of hanging A. donax stems at different temperatures in April and May, a period that in the year-round temperature controlled experiment the success rates were 45 ± 10% in 1998, and 45 ± 21% in 1999. In this test using constant temperatures, no rooting occurred at 10 °C during the 40 days of the experiment .

At 15 °C, rooting was better than at 10 °C , but significantly less than at 17.5, 20, and 22.5 °C . In the greenhouse experiment, the seasonal pattern of rooting success was present, but the inherent advantage of fragments of the hanging stems in the winter months was masked by the negative effect of the lower temperatures of the rooting media. The temperatures chosen for the year-round temperature controlled experiment were selected to reflect the temperature conditions in the habitats invaded by A. donax in Southern California in the months of April and May. From the results of this April constant temperature experiment, it appears that the lower night temperature in the 28/15 °C for 14/10 h experiment led to a reduction in rooting success from the maximum possible in that month. This reemphasizes the effect of in situ temperature on the success of stem fragment meristem rooting, and the ecological danger of the floating stem fragments in shallow waters. The inherent seasonal pattern observed in the year round temperature controlled experiment may have resulted from cycles in the concentrations of the plant growth regulators that play a role in the growth of the side shoots, and the apical dominance of the top of the main stems. One of the growth regulators that plays a major role in the regulation of apical dominance is indole-3-acetic acid. The effect of IAA on the rooting of axillary bud on A. donax stem fragments throughout the growing season was tested through the use of exogenous IAA in the rooting medium,dutch buckets and the determination of endogenous IAA levels in the shoots that grew from the axillary buds. When the stem fragments and their axillary buds were exposed to 5 and 10 µM IAA in the rooting medium, the difference between the hanging and the upright stems disappeared. The main effect of the exogenous IAA was a significant improvement of the rooting percentage and speed of the upright stem fragments in the winter and spring periods, so that the difference between the two stem types was minimized. The exogenous IAA had little effect on the rooting success and speed of the hanging stem fragments . At 20 µM exogenous IAA, the highest concentration applied, the success rate and the speed of upright stem fragment rooting decreased from the optimum observed at 5 and 10 µM, almost down to the percentages and t50 observed in the absence of the hormone . The IAA in the rooting medium may have reached the axillary bud through the vascular bundles of the main stem piece, directly through the cuticle of the bud itself, which was positioned immediately below the rooting medium surface, or both. In early studies into the effect of IAA onplant growth, the growth regulator was sometimes applied to the leaf tissues, and the position of the axillary bud in the rooting medium could have resulted in a similar situation. There is a striking similarity between the seasonal pattern of the endogenous IAA levels in the shoots that grow from the hanging and upright stem fragments and their seasonal rooting patterns. The lowest IAA levels for both stem types occurred in the spring, and the highest levels in late summer . At the time that the endogenous IAA levels are low, levels in the shoots from the upright stem fragments are significantly lower than in those from the hanging stems. As the IAA levels in the shoots increase with the progression of the growing season, the levels in the shoots from the upright stem fragments increase more than in those from the hanging stems, and the difference between the shoots of the two stem types disappears . The role of this IAA is unclear. Although studies have also shown more complicated mechanisms , IAA produced in the main stem apex plays an important role in the apical dominance, and the growth of side shoots. A difference between the rooting and the endogenous IAA patterns is that the seasonal pattern of endogenous IAA concentrations in the new shoots runs approximately one month behind that of the rooting success. This makes it less likely that the IAA concentrations in these new shoots, of which the growth is initiated prior to root growth, is the direct cause of the rooting of the stem fragments. It is possible that both the rooting pattern and the endogenous IAA pattern result from seasonal variation in another factor, possibly the concentrations of another plant growth regulator. Preliminary enzyme kinetics results have shown higher NADP-dependent indole- 3-acetatealdehyde oxidase activity in the new shoots that developed from the hanging stems in April than in those from the upright stems . This indicates that the IAA measured in the new shoots may be the product of de novo synthesis in these shoots, rather than a trace of the IAA that may have been stored in the main stem fragment that was used in the experiment. IAA plays an important role in tracheary cell differentiation and xylem regeneration . The support for the formation of the vascular system by IAA and its promotion of the formation of adventitious roots may be related, but no causal relationship was determined in this study.