Category Archives: Agriculture

Cultivo de bayas en macetas: Consejos para cultivar arándanos y frambuesas con éxito

La jardinería en recipientes abre interesantes posibilidades para cultivar deliciosas bayas, ofreciendo una solución para aquellos con espacio limitado o condiciones de suelo menos que ideales. Los arándanos y las frambuesas, conocidos por su sabor dulce y ácido, pueden cultivarse con éxito en recipientes con los cuidados y la atención adecuados. En este artículo, exploraremos el potencial del cultivo de arándanos y frambuesas en macetas,maceta 25l junto con consejos esenciales para una cosecha próspera.

Arándanos en contenedores:

Selección de contenedores:

Elija recipientes grandes con un tamaño mínimo de 5 galones para cada planta de arándanos. Opta por recipientes de materiales duraderos, como plástico o cerámica, y asegúrate de que tengan agujeros de drenaje para evitar que se encharquen.
Mezcla de tierra:

Utilice una mezcla de tierra ácida y con buen drenaje diseñada específicamente para los arándanos. Puedes encontrar fórmulas premezcladas o crear la tuya propia combinando musgo de turba, corteza de pino y perlita. Mantener el pH adecuado (entre 4,0 y 5,5) es crucial para el éxito de los arándanos.
Requisitos de luz solar:

Los arándanos crecen bien a pleno sol. Coloque las macetas en un lugar donde reciban al menos 6-8 horas diarias de luz solar directa. Considere la posibilidad de rotar los recipientes de vez en cuando para garantizar una exposición uniforme.
Riego:

Los arándanos prefieren un suelo constantemente húmedo pero no encharcado. Riegue en profundidad cuando la capa superior del suelo esté seca. Cubra la superficie con paja de pino o virutas de madera para retener la humedad y eliminar las malas hierbas.
Abonado:

Utilice un fertilizante de liberación lenta y formación ácida formulado específicamente para los arándanos. Aplíquelo siguiendo las instrucciones del envase, normalmente en primavera y a principios de verano. Evite fertilizar en exceso, ya que esto puede provocar desequilibrios de nutrientes.
Poda:

Pode las plantas de arándanos para mantener una forma compacta y fomentar el desarrollo de nuevos brotes. Elimine las ramas muertas o débiles y aclare las zonas abarrotadas para mejorar la circulación del aire.
Frambuesas en contenedor

Tamaño del contenedor:

Las frambuesas se adaptan mejor a la jardinería en recipientes que los arándanos. Seleccione recipientes con un tamaño mínimo de 15-20 galones para cada planta de frambueso. Los recipientes más grandes ofrecen más espacio para el sistema radicular de la planta.
Mezcla de tierra:

Utilice una mezcla para macetas con buen drenaje y rica en materia orgánica. Una mezcla de compost, tierra de jardín y perlita o vermiculita funciona bien. Asegúrese de que el pH de la tierra esté entre 5,5 y 6,5 para un crecimiento óptimo de la frambuesa.
Luz solar y temperatura:

Los frambuesos crecen bien a pleno sol, pero pueden tolerar la sombra parcial. Asegúrese de que sus macetas reciban al menos 6 horas diarias de luz solar. Considere la posibilidad de colocar las macetas estratégicamente para proteger las plantas del intenso calor de la tarde en climas más cálidos.
Riego:

Las frambuesas prefieren una humedad constante, especialmente durante la época de fructificación. Riegue cuando la capa superior del suelo esté seca y cubra la superficie con mantillo para retener la humedad. Tenga cuidado de no regar en exceso,cultivo de frambuesas ya que las frambuesas son susceptibles a la pudrición de la raíz en condiciones de encharcamiento.
Estructuras de soporte:

Instala un enrejado o un sistema de soporte para las frambuesas con el fin de evitar que las plantas se vuelvan pesadas a medida que crecen. Esto es crucial para soportar el peso de los tallos cargados de fruta.
Poda:

La poda regular es esencial para los frambuesos. Elimine los tallos gastados después de la fructificación y corte las ramas laterales para estimular el crecimiento. Esto ayuda a mantener la salud de la planta y promueve una mejor producción de fruta.
Consejos generales para el cultivo de frambuesas en contenedores:

Polinización:

Las bayas cultivadas en contenedor pueden beneficiarse de la polinización manual, especialmente si cultiva variedades que no se autopolinizan. Agite suavemente las plantas o utilice un cepillo suave para transferir el polen entre las flores.
Protección invernal:

En los climas más fríos, aísle los recipientes para proteger las raíces de las temperaturas bajo cero. Traslade las macetas a un lugar protegido o envuélvalas con arpillera para aislarlas.
Controle los niveles de pH:

Compruebe con regularidad los niveles de pH de la tierra de sus contenedores para asegurarse de que se mantienen dentro de los límites recomendados para cada tipo de baya. Ajústelo si es necesario utilizando enmiendas como azufre o cal.
Rote los recipientes:

Gire los recipientes de vez en cuando para favorecer una exposición uniforme a la luz solar en todos los lados de las plantas. Esto ayuda a evitar un crecimiento desigual y garantiza que todas las partes de la planta reciban la luz solar adecuada.
Inspecciones periódicas:

Vigile de cerca sus bayas cultivadas en contenedor para detectar signos de plagas o enfermedades. La detección precoz permite intervenir con rapidez y mantener la salud de las plantas.
Conclusión:

Cultivar arándanos y frambuesas en recipientes no sólo es factible, sino también gratificante. Con la elección adecuada de recipientes, mezclas de tierra y un cuidado diligente, podrá disfrutar de una abundante cosecha de bayas frescas cultivadas en casa. Tanto si dispone de un pequeño patio como de un balcón, la jardinería en recipientes es una solución versátil para llevar el delicioso sabor de los arándanos y las frambuesas hasta la puerta de su casa.

Mastering Hydroponics: A Guide to Successful Indoor Growing

The homogenate was centrifuged at 13,000 g and 4 °C for 20 min. The activity of glycosyltransferase was measured immediately by mixing 100 µL of supernatant with 0.95 mL of the reaction mixture containing 50 mM PBS , 2 mM MgCl2, 2 mM uridine 5′-diphosphoglucose, 3.125 mM 4- nitrophenyl β-D-glucuronide, 3.125 salicin and 0.95 mL of 1 mM 2,4,5-trichlorophenol . The assay mixture was incubated at 30 °C for 30 min, and then stopped by adding 10 µL of phosphoric acid. After centrifugation at 13,000 g for 5 min, the supernatant was collected and diluted with HPLC grade acetonitrile and 0.1% trifluoroacetic acid . The enzyme activity was determined using an Agilent 1200 series HPLC paired with UV detector and a Thermo Scientific Acclaim™ 120 C18 5-µm column . An isocratic flow was set with 1 mL min-1 70:30 mobile phase A and mobile phase B for 10 min. The TCP-glucoside was detected at 205 nm. A six-point TCP standard calibration curve was used to determine activity. All treatments in the A. thaliana cell incubation experiment were conducted in triplicate, and all hydroponic cultivations were conducted using four replicate jars containing individual plants to account for potential loss of plants. Calibration curves with standards of diazepam, diazepam-d5, nordiazepam, temazepam, oxazepam and oxazepam-glucuronide were used for quantification with the r 2 values of at least 0.99 for all analytes. A limit of detection of 1 ng mL-1 and a limit of quantification of 3 ng mL-1 for diazepam and its metabolites were determined through preliminary experiments. For oxazepam-glucurnonide the LOD was 3 ng mL-1 and the LOQ was 5 ng mL-1 . LODs and LOQs were calculated based on a signal to noise ratio of 3 and 10, respectively. Individual peaks were detected and integrated using TargetLynx XS software from MassLynx platform . Data were analyzed with StatPlus and graphed using Prism 6 GraphPad software . Results were calculated as the mean ± standard deviation . The Student’s t-test was used to test significant differences in the extractable and non-extractable radioactivity and glycosyltransferase activity at α = 0.05. Systematic differences in the concentration of diazepam in plant tissues were assessed using one-way ANOVA with Fisher’s Least Significant Difference post-hoc .Active plant metabolism of diazepam was validated using a range of controls.

No diazepam was detected in the non-treated media or the cell blanks,hydroponic grow kit and there was no significant degradation of diazepam in the cell-free media, suggesting no contamination or significant abiotic transformation. Moreover, no significant difference was seen in cell mass between the chemical-free control and the treatments, indicating that diazepam did not inhibit the growth of A. thaliana. Furthermore, no significant amount of diazepam was adsorbed to the cell matter in the non-viable cell control. In contrast, diazepam dissipated appreciably from the media containing viable cells, with the average concentration decreasing from 698 ± 41.5 to 563 ± 8.93 ng mL-1 after 120 h of incubation, a decrease of nearly 20% . Parallel with the dissipation in the medium, diazepam was detected in the A. thaliana cells, with the highest level appearing after 48 h and a substantial decrease thereafter . The decrease in diazepam level in the cell fit a first-order decay model and yielded a half-life of about 68 h . This half-life was in comparison to a biological half-life of 48 h in humans , indicating a moderate persistence in plant cells. Out of the four known diazepam metabolites only nordiazepam and temazepam were detected in the A. thaliana cells over the 120 h incubation. Temazepam was detected first, with the highest concentration being observed at 12 h, which was followed by a decrease to 58.6 ± 17.0 ng g-1 at the end of the 120 h cultivation. Nordiazepam gradually increased over the 120 h incubation time from 128 ± 61.0 ng g-1 at 6 h to 535 ± 92.0 ng g-1 at the end of incubation . These results correlated with their behavior in the human body, as nordiazepam displayed one of the most prolonged biological half-lives of the benzodiazepine family , while temazepam had a significantly shorter half-life . The parallels observed between human and plant metabolism in this study and others is intriguing, as it indicates that we may be able to use the knowledge of biologically active metabolites formed during human metabolism as a guide to study their formation and longevity in environmental compartments such as higher plants. The complementary use of 14C labeled diazepam facilitated the determination of the fraction of diazepam and its metabolites that were incorporated into the cell matter , which could not be determined using traditional extraction and analytical methods. We observed that the radioactivity in the media decreased while the extractable and bound residue fractions increased over the 120 h incubation .

The extractable radioactivity in the viable cells increased to 113 ± 31 dpm g-1 at 120 h . The bound residues increased steadily to a final level of 1120 ± 224 dpm g-1 , indicating that A. thaliana cells were capable of metabolizing and then sequestering diazepam and its metabolites, likely invacuoles and cell walls. The formation of these bound residues is commonly regarded as a detoxification pathway of xenobiotics in higher plants. Diazepam was found in the cucumber and radish seedlings following a 7 d cultivation at the higher concentration and a 28 d of cultivation following treatment at the lower concentration . After treatment with 1 mg L-1 with diazepam for 7 d, a significantly higher concentration of diazepam was observed in the roots as compared to the shoots in radish seedlings whereas diazepam was more evenly distributed throughout the entire plant of cucumber seedlings . However, after the 28 d cultivation following the lower concentration treatment, this pattern appeared to be different for both plant species. In the radish plants, diazepam was more evenly distributed in the roots, but was significantly lower in the shoots . In the cucumber plants there was a significantly higher concentration in roots and a significantly lower concentration in the shoots . These differences may be due to variations in metabolism between the two species, as well as dynamic changes as a function of contact time in both plant growth and its ability to metabolize and translocate diazepam. Similar metabolites to those in A. thaliana cells were found in seedlings grown in the nutrient solution spiked with diazepam, with nordiazepam being predominant . In the 7 d and 28 d cultivation experiments, temazepam was found to be the second major metabolite in the leaves of the cucumber seedlings, and the level was higher in the 7 d cucumber seedlings than the 28 d plants . Oxazepam was detected in the leaves of both plant species after the 7 d cultivation . The higher accumulation of diazepam and the biologically active metabolites in the leaves may have ecotoxicological ramifications; for example, many insects consume leaves, even if they are not edible tissues for humans . Our results were in agreement with recent findings in Carter et al. , in which they observed the formation of nordiazepam,hydroponic indoor growing system temazepam and oxazepam in radish and silver beet plants exposed to diazepam and chlordiazepoxide. They similarly showed nordiazepam to be the major metabolite with oxazepam and temazepam constituting a much smaller fraction at the end of 28 d cultivation in soil. However, in that study, the authors did not track the formation of these metabolites over time or influence of treatment concentrations.

Phase III metabolism appeared to increase from the 7 d to 28 d cultivation for both radish and cucumber seedlings . Between the plant species, the cucumber seedlings had a greater fraction of non-extractable radioactivity in comparison to the radish seedlings . In the 7 d cultivation experiment, the mass balances came to 99.3% for the cucumber plants but only 58.1% for the radish seedlings . Due to the multiple water changes , a complete mass balance was not attainable for the 28 d cultivation experiment. However, when a proxy mass balance was calculated for both species, a similar pattern was observed. A total of 83.0% of the added 14C radioactivity was calculated for the cucumber treatments while the fraction was 61.3% for the radish plants. This could be due to increased mineralization in the growth media and respiration of 14CO2 through plant in the radish cultures. As mineralization is viewed as the final stage of detoxification , it is likely that the radish plant was more efficient in their ability to detoxify diazepam than cucumber plants. The Brassicaceae family, which includes the common radish, has been shown to be effective for phytoremediation due to their possession of genes that increase tolerance to stressors and activation of enzymes capable of extensive bio-transformations .No detectable level of oxazepam-glucuronide was observed in radish or cucumber seedlings for either the 7 d or 28 d cultivation. However, there was a significant difference in the glycosyltransferase activity in radish seedlings treated with diazepam for 7 d and 28 d, although a distinct pattern in the changes of the enzyme activity was absent . For the 7 d cultivation experiment, a significant decrease in glycosyltransferase activity was observed in the shoots of radish seedlings when compared to the control . In contrast, no significant change in glycosyltransferase activity was observed in the shoots of cucumber seedlings when exposed to diazepam . In the 28 d cultivation experiment, only the cucumber seedlings exhibited significant differences in the enzyme activity, with an increase in activity detected in the shoots and a decrease in the cucumber buds . Even though we did not detect oxazepam-glucuronide in the exposed plants, changes in the glycosyltransferase activity indicated that conjugation might have occurred with the parent and its metabolites, including those not examined in this study, or at levels below our detection capability. In addition, it may be postulated that rapid phase III metabolism may have limited the accumulation of such conjugates in the plant tissues, making the conjugates transient metabolites. In previous studies, glycosyltransferase was observed to catalyze the detoxification of ibuprofen in Phragmite australis during a 21 d exposure . Further, the formation of a glucose conjugate has been considered to be a major detoxification pathway for several environmental contaminants . These studies together suggest the importance of phase II metabolism in the metabolic fate of pharmaceuticals in higher plants. Water scarcity has led to continuously increasing use of municipally treated wastewater in agro-environments, especially in arid and semi-arid regions . Similarly, the use of municipal bio solids to improve soil health is increasing . The land application of TWW and bio solids can introduce contaminants of emerging concern into terrestrial environments . Consequently, a range of CECs have been detected in agricultural soils . Literature pertaining to the effects of CECs in terrestrial ecosystems is, however, limited . The majority of previous research has been concerned with the fate and effects of CECs in plants , and only a few studies have considered CECs in terrestrial invertebrates . One of the most important invertebrates in agricultural fields is earthworm. Earthworms ameliorate agricultural soil structure through the formation of new aggregates and macropores; improving soil tilth, aeration, infiltration, and drainage . Furthermore, earthworms consume plant litter, recycle organic matter and aid in nutrient cycling . Earthworms dominate soil fauna with an average biomass of 10 – 200 g m-2 . Due to their ecological importance and abundance, earthworms are a good candidate for ecotoxicity testing . Herein we sought to understand some of the potential consequences of CECs exposure in earthworms. For this study, we selected theearthworm species Eisenia fetida as the test organism due to their widespread use in the scientific literature and extensive habitat range. For CECs, we considered three pharmaceuticals, i.e., naproxen, diazepam, and sulfamethoxazole, and one cosmetic preservative, i.e., methyl paraben. These four compounds were selected due to their range of physicochemical properties and frequent detections in the environment . Naproxen is a commonly consumed nonsteroidal anti-inflammatory drug that has been often found in TWW and bio solids . Diazepam is a psychoactive compound from one of the most commonly prescribed classes of pharmaceuticals of wastewater treatment plants. Diazepam has also been frequently detected in TWW due to poor removal efficiency . Sulfamethoxazole is an antibiotic and has garnered significant scientific interest due to the growing concern over antibiotic resistance .

From Roots to Results: Understanding the Principles of Hydroponic Cultivation

Mn3O4 NPs also possess excellent ROS-scavenging capacities, exerting multiple enzyme mimicking activities, for example, SOD- and CAT-like, as well as hydroxyl radical scavenging activities. A recent study reported that foliar application of Mn3O4 NPs at 1 mg per plant significantly alleviated salinity stress of cucumber plants. The authors found that Mn3O4 NPs increased endogenous low-molecular-weight antioxidants in the leaves, including resveratrol, chlorogenic acid, dihydroxycinnamic acid, benzenetriol, hydroxybenzoic acid, trihydroxybenzene, quinic acid and catechin. The multifunctional catalytic behaviour of Mn3O4 NPs arise from the coexistence of Mn and Mn oxidation states, and the switch between the II and III valence resembles the mechanism of redox enzymes, which is very similar to CeO2 NPs. In addition to directly acting as ROS-scavenger, NMs can act as carriers to deliver ROS-eliminating compounds to enhance plant stress tolerance. The authors of a recent study designed an ROS-responsive star polymer that successfully alleviated plant stress by simultaneous ROS-quenching and nutrient release. Specifically, RSP was foliar-applied to stressed tomato leaves. The RSP penetrated the leaf epidermis and entered into the chloroplasts where it efficiently eliminated H2O2, which subsequently triggered the release of the nutrient from the polymer. This study highlights the potential of using RSP as an ROS-responsive NM to manage short-term plant stress. Apart from foliar application, ROS-scavenging CeO2 NPs have been applied to roots. It has been reported that CeO2 NPs at 100 and 500 mg l−1 applied to hydroponic cultivated rice can significantly increase the nitrogen levels in roots and shoots by 6–12% and 22–30%, respectively, compared with controls. Similarly, ref. added graphene to the growth media of alfalfa ,macetas redondas and found that the amendment alleviated salinity and alkalinity stresses by modulating antioxidant defence systems and genes related to antioxidant defence and photosynthesis.

The treatment of graphene significantly increased dry biomass of alfalfa by 29.4% and 24.3%, respectively, in salinity and alkalinity conditions. In another study, root delivery of ROS-eliminating CeO2 NPs to hydroponically cultivated rice plants was shown to improve rice tolerance under salinity stress, increasing chlorophyll content and yield. ROS-scavenging NMs have also been reported as a seed treatment agent to improve stress resistance. For example, ref. found that cotton seeds primed with poly-coated CeO2 NPs at 500 mg l−1 for 24 h exhibited significantly increased root vitality by 114% under salt stress. Similarly, ref. found that salt-sensitive rapeseeds seeds primed with poly-coated CeO2 NPs exhibited enhanced salt tolerance by modulating ROS homeostasis. These studies suggest that using ROS-scavenging NMs to treat seeds can alleviate stress at germination stage , although the duration of this protective benefit is currently unknown. Figure 3 summarizes the current known or hypothesized mechanisms by which ROS-scavenging NMs alleviate plant stress.ROS are important signalling molecules that mediate redox signalling pathways and contribute to acclimatization against a range of stresses. One study demonstrated that an ROS wave is required to activate systemic acquired acclimation of plants to heat or high light stresses, highlighting the important biological function of this signalling molecule to the acclimation against abiotic stresses. In addition, an ROS-generating-associated gene respiratory burst oxidase homologue has been shown to be critical to plant stress responses. Given the known response of plants to select lower-level ROS that elicit redox signalling pathways, the concept of pretreating or priming plants with ROS-triggering NMs to stimulate defence systems and acquire systemic acquired acclimation may be an effective strategy to increase stress tolerance. In this strategy, plant stress resistance will be acquired via the initiated adaptive responses by ROS-triggering NMs. ROS-triggering NMs could be used to prime plants through a ‘stress memory’, which provides a mechanism for acclimation and adaptation, thereby improving the tolerance/avoidance abilities. Whereas ROS-scavenging NMs serve as a ‘curative’ strategy, ROS-triggering NMs are more like a ‘preventive’ strategy. Currently, only a limited number of studies have employed ROS-triggering nanozymes to increase plant stress resilience, and the researchers are primarily focusing on silver nanoparticles . AgNPs are known to catalyse ROS generation in cells.

A previous study reported that seed priming with AgNPs enhanced the tolerance of pearl millet to salinity stress by activating the antioxidant enzymes. AgNP seed priming significantly increased the fresh and dry weights of plants by 58% and 34%, respectively, compared with plants grown in 150 mM salt. The underlying mechanisms may be that AgNPs activated defence pathways during seed priming, forming the ‘stress memory’ and subsequently enhancing resilience to stress. The mechanisms for AgNPs generating ROS have been reported in recent studies. By using electron spin resonance, ref. demonstrated that AgNPs can directly produce • OH in the presence of H2O2, and Ag is generated during this process; importantly, Ag ions did not catalyse the production of • OH. Similar results were obtained by another study. A more recent study demonstrated that AgNPs possess peroxidase-mimicking activities, which catalyse oxidation of substrate TMB in the presence of H2O2. The formation of • OH by AgNPs is similar to a Fenton reaction in which AgNPs act as a Fenton-like reagent. Under a changing climate, the frequency of seed exposure to abiotic stresses will increase, which could result in reduced germination and loss of vigour, threatening crop yield. As such, accelerating the germination speed and enhancing the seed vigour are critical. One study reported that AgNP seed priming accelerated the germination speed and yield of Chinese cabbage . Another study showed that AgNP priming promoted the germination, growth and yield of watermelons . Nanoscale zero valent iron , also known to be a Fenton-like reagent, can catalyse the generation of ROS. Another study used nZVI as an ROS-modulator to pretreat rice seeds. This study found that priming generated an optimum level of endogenous ROS via Fenton’s reaction, resulting in higher seed germination rate and greater seed vigour. Unfortunately, the study did not further evaluate whether nZVI priming can increase the stress tolerance of seeds or seedlings, but given the observed hormone biosynthesis upregulation and increased antioxidant enzyme activity, nZVI seed priming should be explored as a potential strategy to promote the stress tolerance of rice and other plant species. Collectively, ROS-modulating NM-based seed treatments may be a promising strategy to mitigate climate-change-associated stress. Under stress conditions, although ROS over-accumulation is common, ROS can still act as signalling molecules, which interplay with other signalling molecules such salicylic acid to activate defence-related genes.

These metabolic processes help plants to establish systemic acquired acclimation or systemic acquired resistance, enhancing the resistance to abiotic or biotic stresses. Therefore, NMs that can trigger the upregulation of defence-related hormones/ signalling molecules or genes may also enhance stress resistance. One study reported that silica nanoparticles at 100 and 1,000 mg l−1 enhanced disease resistance of Arabidopsis plants by up-regulating the production of salicylic acid, a defence hormone and an important signalling molecule. Similarly, another study demonstrated that copper-based NMs successfully alleviated damage of soybeans to sudden death syndrome by triggering the upregulation of a broad array of defence-related genes . One study reported that foliar spray of commercial Cu2 nanoparticles significantly increased antioxidant defence-related genes, for example, SOD, GPX, MDAR and WRKY transcription factor, in cucumber plants, although the potential benefits to stress tolerance were not evaluated. Taken together, these studies demonstrate that NMs that can trigger ROS production or stimulate defence pathways can activate systemic acquired resistance of plants, enhancing the protection against disease or stresses through a classic adaptation response .For both ROS-scavenging NMs and ROS-triggering NMs, efficiently penetrating barriers and entering into plant cells is critical for modulating ROS levels and participating in metabolic activities. As such, an in-depth understanding of the uptake pathways of NMs is fundamental for the efficient application of nanobiotechnology in agriculture. Here, we briefly discuss the possible uptake pathways for NMs into plants, including foliar,maceta de 10 litros root and seed application . Foliar application has been the most extensively used delivery pathway. There are several pathways for the entry of nanoscale particles into leaves, including the cuticle and stomata. One study reported that foliar-applied gold nanoparticles in wheat can reach the mesophyll by either the cuticle or stomata, and move through the plant vasculature. Recent studies have noted that the stomatal uptake pathway is more efficient than the cuticle for Cu-based NPs. In addition, nanoparticle properties and leaf biointerface are important factors influencing the uptake and translocation of NPs. More mechanisms for foliar-uptake pathways can be referenced to excellent reviews. Compared with foliar application, delivering ROS-modulating NMs to plants via the roots has been less investigated. It has been reported that the application of ROS-modulating NMs to plant roots is more effective under hydroponic cultivation than under soil-grown conditions. In the soil system, NMs will undergo aggregation, adsorption and dissolution, confounding interaction with plant roots. A recent study compared the leaf and root application of ROS-scavenging CeO2 NPs on alleviating salt stress of cucumber, and found that foliar-sprayed CeO2 NPs enabled better cucumber salt tolerance than root application. The pathways for NMs entering into the root include apoplastic and symplastic pathways. Using ROS-modulating NMs to prime seeds could be a more cost-effective and environmentally friendly strategy than foliar and root application. This type of approach would not only reduce the release of engineered NMs in the environment but would also result in decreased worker exposure to these materials. Using ROS-modulating NMs to treat seeds might be a promising strategy to increase seed quality, promote growth and increase the yield. Additionally, nano-enabled seed treatment is an efficient way to load mineral nutrients into seeds.

By using transmission electron microscopy, one study observed that FeNPs were absorbed on the watermelon seed coat during the priming process and slowly translocated into the seed endosperm. By using transmission electron microscopy with energy-dispersive X-ray spectroscopy, another study also demonstrated the presence of FeNPs inside the seeds after the priming. These results demonstrate that NPs can effectively penetrate the seed coat and enter into seeds, although the mechanisms of action remain unclear and must be evaluated.Using NMs to modulate ROS homeostasis is a promising strategy to enhance stress tolerance of crop species. It is a rapidly developing area of research, with the vast majority of publications dating from only 2017 to 2022. We propose that additional mechanistic studies are needed to explore the potential of this approach. First, compared with the ROS-scavenging NMs strategy, the ROS-triggering NMs approach remains largely unexplored. Using ROS-triggering NMs to stimulate defence pathways at early growth stage, for example, seed or seedling, could enhance the immunity or resistance of plants to abiotic and biotic stresses. This ‘preventive’ strategy combined with an ROS-scavenging NM-based ‘recovery’ strategy may provide a versatile and effective solution for stress tolerance. Notably, ROS-scavenging nanozymes can sometimes also induce early ROS stimulation to enhance plant stress tolerance. As such, ROS-scavenging nanozymes could be applied as a ‘preventive’ strategy as well. In addition, ROS-triggering NMs may temporarily shift energy and resources to defence pathways, potentially impacting carbon and nitrogen metabolism in ways that could compromise biomass accumulation. As such, the regimen of application will be highly important. For example, one could apply ROS-triggering NMs simultaneously with nutrients in order to more broadly support the plant’s defence system. Abiotic stresses often occur in combination or in succession, and as such, future studies need to evaluate the performance of ROS-modulating NMs under multiple stressor scenarios. Furthermore, a comprehensive understanding of the mechanisms for modulating ROS via NMs to enhance plant stress tolerance is needed prior to deployment of these strategies in the field. The relevant mechanisms include the following: the pathway for NM entry into leaves,roots and especially seeds; the catalytic mechanisms of nanozymes, and how the size, surface charge, pH and environmental conditions impact the catalytic activities of NMs; the intracellular kinetics of ROS-scavenging or ROS-triggering mechanisms of NMs; the precise metabolic pathways by which ROS-triggering NMs enhance plant stress resistance ; and the cellular, biochemical and molecular level response of plants to NMs under different treatment regimens, especially transcriptome and metabolic reprogramming induced by ROS-triggering NMs. The orthogonal approach of transcriptomics, proteomics, metabolomics and epigenetics will be a powerful tool to address these questions. Nano-enabled stress tolerance strategies represent a rapidly developing interdisciplinary field of research. We need to pay attention to new findings from both plant and NMs fields, for instance, new knowledge regarding the response mechanism of plants to abiotic and biotic stresses, and state-of-the-art ROS-regulating nanozymes, which will push forward the application of nanozymes in crop stress tolerance. For example, a study recently reported that Huanglongbing, a devastating disease of citrus, is an immune-mediated disease that stimulates the production of ROS as well as the upregulation of genes encoding ROS-producing NADPH oxidases.

Innovative Growth: Hydroponic Agriculture and the Future of Farming

The Cu grains have about the same size as the electron-dense Cu granules in cells of E. splendens placed in a CuSO4 solution for 30 days .While biological activity clearly modified the original distribution of Cu in the rhizospheres, the Cu species could not be identified from the µ-XRF maps but instead were elucidated using EXAFS spectroscopy. All eight µ-EXAFS spectra from areas in the original soil containing the particle morphologies and chemical compositions observed with µ-XRF can be superimposed on the soil’s bulk EXAFS spectrum , indicating that the initial Cu speciation occurred uniformly. If the initial soil contained various assemblages of Cu species that were distributed unevenly, then we might expect that the proportions of species also would have varied among analyzed areas and been detectable by µ-EXAFS spectroscopy ; however, this was not observed. These spectra match those for Cu2+ binding to carboxyl ligands in natural organic matter, as commonly observed . Elemental Cu. In contrast, only the reference spectrum of elemental copper matches the µ-EXAFS spectra of the 12 hot-spot Cu grains, which are statistically invariant . Photo reduction of Cu2+ in the X-ray beam cannot explain the formation of Cu0 because no elemental Cu was detected in the initial soil by powder and µ-EXAFS, and Cu0 was detected in the two phytoremediated soils at 10 K by bulk EXAFS, and all individual spectral scans from the same sample could be superimposed. At 10 K, radiation damage is delayed , and if Cu had been reduced in the beam, the proportion of Cu0 would have increased from scan to scan which was not observed.The rhizospheres were oxidizing as indicated by the presence of iron oxyhydroxide , absence of sulfide minerals, and the fact that P. australis and I. pseudoacorus are typical wetlands plants with aerenchyma that facilitate oxygen flow from leaves to roots . Thermodynamic calculations using compositions of soil solutions collected below the rhizosphere indicate that Cu+ and Cu2+ species should have been dominant . These points along with the occurrences of nanocrystalline Cu0 in plant cortical cells and as stringer morphologies outside the roots together suggest that copper was reduced biotically. Ecosystem ecology of the rhizosphere indicates synergistic or multiple reactions by three types of organisms: plants, endomycorrhizal fungi, and bacteria.

Normally, organisms maintain copper homeostasis through cation binding to bio-active molecules such as proteins and peptides. When bound,plastic pots for planting the Cu2+/Cu1+ redox couple has elevated half-cell potentials that facilitate reactions in the electron-transport chain. Even though average healthy cell environments are sufficiently reducing , there are enough binding sites to maintain copper in its two oxidized states. Copper is also important in controlling cell-damaging free radicals produced at the end of the electron-transport chain, for example in the superoxide dismutase enzyme Cu-Zn-SOD, which accelerates the disproportionation of superoxide to O2 and hydrogen peroxide. However, unbound copper ions can catalyze the decomposition of hydrogen peroxide to water and more free radical species. To combat toxic copper and free radicals, many organisms overproduce enzymes such as catalase, chelates such as glutathione, and antioxidants . Mineralizationcould also be a defense against toxic copper, but reports of Cu+ and Cu2+ biominerals are rare; only copper sulfide in yeast and copper oxalate in lichens and fungi are known. Atacamite 3Cl in worms does not appear to result from a biochemical defense. Biomineralization of copper metal may have occurred by a mechanism analogous to processes for metallic nanoparticle synthesis that exploit ligand properties of organic molecules. In these processes, organic molecules are used as templates to control the shape and size of metallic nanoparticles formed by adding strong reductants to bound cations. For copper nanoparticles and nanowires, a milder reductants ascorbic acids has been used. Ascorbic acid, a well-known antioxidant, reduces Cu cations to Cu0 only when the cations are bound to organic substrates such as DNA in the presence of oxygen in the dark or via autocatalysis on Cu metal seeds in the absence of stabilizing organic ligands . As an example of synthetic control, pH dependent conformation of histidine-rich peptides has led to larger nanocrystals of Cu0 at pH 7–10 than at pH 4–6 . Plants produce ascorbic acid for many functions and rhizospheres often contain the breakdown products of ascorbic acid, which facilitates electron transfer during mineral weathering . Plants produce more ascorbic acid when grown in soils contaminated with heavy metals including copper . Fungi, which proliferate over plants and bacteria in metal-contaminated soils, can stabilize excess copper by extracellular cation binding or oxalate precipitation , but mechanisms probably also require enzymes, thiol-rich proteins and peptides, and antioxidants .

The formation of electron-dense Cu granules within hyphae of arbuscular mycorrhizal fungi isolated from Cu- and As-contaminated soil suggests that fungi also can produce nanoparticulate copper. Some copper reduction possibly occurred in response to the European heat wave of the summer of 2003 . Elevated expression of heat shock protein HSP90 and metallothionein genes has been observed in hyphae of an arbuscular mycorrhizal fungus in the presence of 2 × 10–5 M CuSO4 in the laboratory . This suggests that a single driving force can trigger a biological defense mechanism that has multiple purposes. Thus, reduction of toxic cations to native elements may increase as rhizosphere biota fight metal stress and stresses imposed by elevated temperatures expected from global warming.Laboratory evidence has shown that plants , fungi , bacteria , and algae can transform other more easily reducible metals, including Au, Ag, Se , Hg, and Te, to their elemental states both intra and extracellularly. When mechanisms have been proposed, they typically have involved enzymes; however, ascorbic acid was implicated when Hg2+ was transformed to Hg0 in barley leaves . Theoretically all of these metal cations could be transformed by a reducing agent weaker than ascorbic acid . However, binding appears to stabilize cationic forms in the absence of a sufficiently strong reductant such as ascorbic acid. Processes used in materials synthesis that were developed with biochemical knowledge might yield clues to other possible, but presently unknown, biologically mediated reactions in different organisms.The discovery of nanoparticulate copper metal in phytoremediated soil may shed light on the occurrence of copper in peats. Native copper likely forms abiotically in the reducing acidic environments of Cu-rich peat bogs . However, swamps by definition are more oxidizing with neutral to alkaline pHs, and they may be ideal sites for biotic formation of metallic Cu nanoparticles. For example, in swamp peats near Sackville, New Brunswick, Canada, copper species unidentifiable by XRD were dissolved only with corrosive perchloric acid , suggesting they may have been nanoparticulate metal formed by active root systems of swamp plants. If swamp peats evolve to bog peats the Cu reduction mechanism could convert to autocatalysis on the initial nanocrystals . The addition of peats that either act as templating substrates or contain nanoparticulate copper could enhance the effectiveness of using wetlands plants for phytoremediation. In contrast to harvesting hyperaccumulators, the oxygenated rhizosphere would become an economic source of biorecycled copper, and rhizosphere containment would prevent copper from entering the food chain via herbivores,plant pot drainage limiting potential risks to humans.Predicting Cd concentrations in plants is essential for controlling Cd entry into the food chain.

Cadmium uptake by plants is the result of root adsorption to cell walls and of absorption through root cell membranes. Concentration-dependent kinetics performed on seedlings of maize and alpine pennycress allowed short-term Cd uptake by both root uptake pathways to be parameterized . However, the uptake parameters were obtained on plants which were previously cultivated without exposure to Cd. The objective of the present work was to assess how chronic exposure of plants to different Cd levels would affect the root adsorption and absorption rates. Indeed, pre-exposure to metals may substantially modify the kinetics of metal uptake. Stimulation of Cd uptake has been reported to occur in Fe deficient conditions , showing some up-regulation of membrane proteins able to transport Cd. Moreover, Larsson et al. studied the effect of prior Cd2+ exposure on Cd uptake by roots of Arabidopsis thaliana, and found some up-regulation of total Cd uptake in the wild type, and down-regulation in the PC-deficient mutant. Furthermore, very few works have investigated the regulation of root cation exchange capacity by prior exposure to metal. In the present study, we investigated the impact of plant Cd content on root Cd uptake characteristics, at both the cell wall and membrane levels. The experiment was performed on two species with contrasting demand for Cd: a hyper accumulating ecotype of alpine pennycress , well-known for its ability to accumulate high concentrations of Zn and Cd in its shoots, and maize , which retains Cd in its roots. The remaining plants were exposed to a one-hour uptake in a radio-labelled solution in order to assess the absorption rate of Cd according to the level of Cd accumulated during the cultivation. Three Cd concentrations in the radio-labelled solution were used: 0.1 µM, 10 µM and 50 µM. Before immersion of roots in the radio-labelled solution, they were rinsed and exposed to a desorption treatment in order both to minimize contamination of the radio-labelled solution with Cd leakage from the cell walls, and to liberate all exchange sites able to adsorb Cd. For that, each root system was immersed for two hours in 80 ml of buffered solution containing 5 mM of Ca2 and 2 mM MES buffer, then for two further hours in 80 ml of buffered solution containing 0.5 mM of Ca2 and 2 mM MES buffer. After quick rinsing in distilled water, roots were immersed in 650 ml of 109Cd radio-labelled solution containing 0.5 mM CaCl2, 2 mM MES buffer and CdCl2 in the three different concentrations. Root exposure lasted one hour, without significant variation of Cd external concentration. Each root system was then separated from shoots before immersion in successive ice-cold MES-buffered baths containing 2 mM CdCl2 and 5 mM CaCl2. This desorption procedure was interrupted by 2 minutes freezing in liquid nitrogen; roots were then thawed by agitation in a warm desorbing bath, and desorption kinetics went on in ice-cold desorbing solutions for the resulting disrupted root cells . Cadmium collected in the desorbing solutions was quantified through 20 ml samples by gamma-counting .

Cadmium bound to the apoplast at the end of desorption was also quantified through gamma-counting in dry matter. We previously showed that the sudden unloading of metal in the desorption solution caused by the freezing/thawing procedure represents symplastic Cd, while the gradual desorption before and afterwards, corresponds to leakage from the cell walls, regardless of the external concentration . The HATS of maize and alpine pennycress is not affected at all by the low internal accumulation of Cd during growth. The withdrawal of free Cd ion from the cytosol by the complexation with phytochelatins and eventual transport to the vacuole or to the shoots may depress any mechanism regulating the cytosolic Cd concentrations . On the other hand, the plants do not show the same behaviour for the high short-term exposure concentrations: the 0.1 µM Cd contamination of the growth solution would down-regulate the LATS of maize but not that of alpine penny cress, for which some stimulating effect seems to happen. After high Cd accumulation during growth, the Cd symplastic influx is significantly reduced for both plants. Such a diminution of the intracellular uptake of Cd had already been observed on wheat root protoplasts . It may come from down-regulation of the short-time Cd2+ uptake on both high- and low-affinity transport systems. In our study, this reduction of the Cd short-term uptake is higher for the lower exposure concentrations. This supposes that the down-regulation affects the HATS rather than the LATS. The decrease in Cd intracellular influx can also result from up-regulation of the Cd2+ extrusion from intracellular to extracellular space, through PC-Cd efflux , Cd2+/H+ antiport or vesicle excretion . Another alternative explanation to the lower Cd uptake could be the changed appearance of the root system in the Cd-treated plants. Thus, although approximately the same root masses were achieved in all plants tested, the uptake surface might have been different, favoring Cd uptake in the control plants. Finally, some better sequestration of Cd by the cell walls might decrease the entry rate of Cd through cell membranes.

Hydroponic Agriculture: Nurturing Crops in the Absence of Soil

The capabilities of SR FTIR spectromi-croscopy for the direct detection of intracellular biochemical responses to exposures to dilute concentrations of OCs and PAHs will have significant impacts in future research methodology of environmental toxicology.Bio-assays used in aquatic toxicology have taken a prominent position among analytical tests for identifying and measuring environmental hazards. Such bio-assays have been developed for testing a variety of organic and inorganic chemicals, as well as effluents, surface waters and sediment samples for acute and chronic toxicity. Many bio-assays using higher organ-isms such as fish, protozoa and algae have been executed, but are labor- and equipment-intensive, costly and complex. More importantly, these aquatic bio-assays do not provide quantitative information on the impact of pollutants on biological treatment systems. In the view of national and international regulations, regulatory agencies are also supporting the development of new toxicity screening procedures that are sensitive, inexpensive and easier to perform. The use of bacterial in vitro assays such as the Microtox Assay has become an attractive alternative to traditional fish and invertebrate methods for toxicological screening. These new assays have been developed to assess the toxicity of various environmental agents, validated and recognized by several standards organizations. The purpose of this study was to apply selected microbial test protocols to assessing the toxicity of hazardous metals such as cadmium and lead. These metals have been reported to pose a high level of hazard to ecological and human health.A Microtox assay was carried out to measure the relative acute toxicity of metal producing data for the calculation of lead concentration effecting 50% reduction in light output . For each test run, two controls without lead,pot blueberries eight sample/lead dilutions and two replicates were done. Tests were carried out on various percentages of the original lead concentration . The sensitivity of the strain of bio-luminescent bacteria was tested for quality control purposes. Growth and oxygen uptake experiments were performed following previously described protocols.

Descriptive statistics were applied to calculate the means+SD of all data sets associated with specific metal concentrations. Specific growth and oxygen depletion rates were computed as slopes of graphical representations of raw data versus times. The toxic end-points expressed as 50% growth inhibition concentration or as 50% oxygen depletion concentration were next derived from graphical presentations of these specific rates versus metal concentrations. Activity quotients were calculated to determine the degree of toxicity associated with lead exposure. Linear regression analysis was performed to determine the relationship between lead concentrations and the times required for 50% reduction in oxygen uptake .Bioluminescence was used as an endpoint for measuring the effect of Cd and Pb to Vibrio fischeri. For both Cd and Pb, a strong dose-response relationship was determined. The concentrations of Cd and Pb effecting 50% reduction in bioluminescence were computed to be 0.79+0.12 mg/L and 0.34+0.03 mg/L, respectively; indicating that Pb was more toxic than Cd. A strong dose-response relationship was also found in the tests with the mixed population of microorganisms. Figure 1 shows the growth patterns obtained from exposure to lead of the mixed population of microorganisms. Data presented in this figure show an overall increase in bacterial growth with the increase in holding/incubation time. These data also show significant reductions in maximum growths with increasing concentrations of lead. EC50 values were computed to be 4.50+0.04 mg/L, and 3.50+0.02 mg/L for Cd and Pb, respectively. Figure 2 presents the dissolved oxygen uptake rates by the mixed population of microorganisms exposed to various concentrations of lead. In general, curves presented in this figure indicated that individual rates of oxygen uptake decreased as lead concentrations increased. The mean values of EC50 were 5.00+0.42 mg/L for Cd, and 3.80+0.04 mg/L for Pb. These data indicated that the mixed population of microorganisms was about 10 times less sensitive to lead toxicity than the marine bacterium, Vibrio fischeri. Data also showed a strong correlation between TD50s and lead concentrations, indicating a time-response relationship with regard to lead toxicity. A similar result was obtained in experiments with cadmium. Data obtained from this research clearly point out the significance of using microbiological systems for acute toxicity testing in aquatic toxicology. Bio-assays employed in the present investigation fulfilled the requirement criteria of fast toxicity screening based on their simplicity, speed, cost effectiveness and the fact that bacteria grow rapidly, represent a low trophic level, and thus provide sensitive early warning data of environmental impacts at higher trophic levels.

Of the three bio-systems evaluated, the Microtox was the most sensitive; yielding an EC50s that was only about one tenth of values recorded in batch cultures . Although batch systems were time-consuming , and relatively less sensitive than the Microtox, they provided valuable information on the toxic effects of lead on microbial growth and respiration. Also, they were easier to perform, and required less expensive equipment compared to the high cost of the Microtox analyzer. Trinitrotoluene is a munitions chemical that was produced and used on an enormous scale during World Wars I and II in shells, bombs, grenades, demo-lition explosives and propellant compositions. 2,4-Dinitrotoluene , and 2,6-Dinitrotoluene , on the other hand, are used in the manufacture of dyes, in munitions as smokeless propellant powders, and as gelatinizing and plasticizing agents in both commercial and military explosive compositions. Both 2,4-DNT, and 2,6-DNT are produced through denitration of toluene with nitric acid in the presence of concentrated sulfuric acid. Small amounts of DNT isomers also occur as byproducts in the production of TNT. Significant amounts of TNT- and DNT-contain-ing waste waters arising from their preparation and production at Army ammunition plants have been identified in soils, surface water and ground water after leaching from disposal sites. Exposure to TNT and DNTs has been associated with numerous health effects. However, limited scientific information is available regarding the environmental fate, ecotoxicity and health effects of these nitroaromatic compounds. We have performed the Microtox, Mutatox and CAT-Tox assays to determine the acute toxicity, genotoxicity and molecular mechanisms by which these munitions chemicals exert their toxicity. Acute and genotoxicity tests were carried out, using a Microtox/Mutatox Model 500 Toxicity Analyzer System. The Microtox procedure measured the relative acute toxicity of lead, producing data for the calcula-tion of lead concentration effecting 50% reduction in light output . For each test run, two controls without lead, eight samples/chemical dilutions and two replicates were done. The Mutatox Assay was conducted according to the standard test protocol. Nonglowing or dark mutant strains of luminescent bacteria were exposed to the test substance , and the amount of light emitted was measured with the Mutatox Analyzer. The sample-induced reversion from nonglowing to lumi-nescent phenotype was used to indicate the genotoxicity of the sample.

Prepared samples were mixed and preincubated in a water bath at 35 ± 0.5°C for 45 minutes. After preincubation, samples were incubated at 27 ± 5°C for 16, 20 and 24 hours, and the potential genotoxic response of the luminescent bacte-ria was determined at each time period by measuring the light intensity of each cuvette using the Mutatox Model 500 Analyzer. The positive response was defined as the light output of at least two times the light intensity of the reagent control blank. The mammalian Gene Profile Assay was performed for measuring differential gene expression in the human liver hepatoma cell line, HepG2. Thirteen recombinant cell lines and the parental HepG2 Cell line were plated over two 96-well microplates. The cell lines were dosed at five TNT concentrations and incubated at 37°C, 5% CO2, for 48 hours. After the incubation period,square plastic plant pots the total protein was measured by the Bradford method, at 600 nm using a microplate reader. A standard sandwich ELISA was performed and in the final step horseradish peroxidase catalyzed a color change reaction that was measured at 405 nm. The parental HepG2 cell line was dosed in the same manner as the recombinant cell lines, and was used to perform a MTT-based cellular viability assay at 550 nm.Polycyclic aromatic hydrocarbons are a family of compounds that includes some potent car-cinogens that are ubiquitous in the environment. The major metabolic pathway for ingested or inhaled PAHs to water-soluble derivatives is oxidative activation by cytochrome P4501A1 followed by detoxification by phase II enzymes like glutathione S-transferases, especially GSTM1. Interindividual variation in PAH metabolism exists due to genetic polymorphisms in the genes coding for these enzymes. The GSTM1 gene is frequently deleted in individuals, resulting in reduced detoxification. Several single-base changes have been identified in the CYP1A1 gene that appear to result in increased susceptibility to various cancers in these individuals. Because PAHs present a threat to human health, human exposure to PAHs has to be monitored in occu-pational settings. While PAHs consist of hundreds of different aromatic compounds, pyrene is typically present in all of these mixtures. Pyrene is metabolized primarily to 1-hydroxypyrene and detoxified as 1OHP sulfate or glucuronide conjugate and excreted via the urine. Through simple enzymatic methods, these conjugating molecules can be cleaved. Therefore urinary 1OHP is the most commonly used biomarker of exposure to PAHs. Recently, a number of investigators have reported differences in the quantity of urinary PAH metabolites in individuals with poly-morphisms or variations in key enzymes involved in the metabolism of xenobiotics. These findings suggest the need for clarification of the effects of polymor-phisms on the metabolism of pyrene. To investigate the role of these polymorphisms, we have undertaken a study to measure 1OHP levels.Genetic polymorphisms: Peripheral blood lympho-cytes are isolated from the blood samples using Histoprep density separation media . DNA is extracted from the PBLs using standard phenol chloro-form extraction methods. Polymorphisms are analyzed by published procedures: For CYP1A1, the procedure described by Cascorbi et al. is followed.

For identification of CYP1A1 M1, a 899 bp fragment is amplified, then digested with MspI which cuts the variant fragment into a 693 and 206 bp fragment. For the identification of the CYP1A1 M2 polymorphism, a 204 bp DNA fragment is amplified, then subjected to digestion with BsrDI, which cuts the wildtype into a 149 and 55 bp fragment. Restriction enzyme digested PCR products are separated by agarose gel elec-trophoresis. GST analysis is performed using a multiplex PCR that co-amplifies the GSTM1 and GSTT1 genes . An actin DNA frag-ment is co-amplified as an internal control. The absence of a GSTM1 or GSTT1 band in the presence of the actin band indicates a GST gene deletion. Analysis of 1OHP: Methods for analysis of 1OHP follow the protocol by Whiton et al. , which involves overnight enzymatic digestion of all conju-gated forms of pyrene in a 25 ml urine sample, organic extraction of 1OHP, and reverse phase HPLC analysis and quantitation of the 1OHP peak.Inorganic pyrophosphate is an intermediate compound generated by a wide range of metabolic processes, including biosynthesis of various macromolecules such as proteins, DNA, RNA, and polysaccharides. Being a high-energy phosphate compound, PPi can serve as a phosphate donor and energy source, but it can, at high levels, become inhibitory to cellular metabolism. To maintain an optimal PPi level in the cytoplasm, timely degradation of excessive PPi is carried out by two major types of enzymes: soluble inorganic pyrophosphatases and proton-translocating membrane-bound pyrophosphatases. The importance of maintaining an optimal cellular PPi level has been demonstrated in several different organisms. Genetic mutations that lead to the absence of sPPase activity affects cell proliferation in Escherichia coli. In yeast, inorganic pyrophosphatase is indispensable for cell viability because loss of its function results in cell cycle arrest and autophagic cell death associated with impaired NAD+ depletion. 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.

Nutrient-Rich Innovation: The Benefits of Hydroponic Crop Production

Nanoceria was found to be non toxic for Danio rerio embryos exposed up to 200 mg l−1 nanoceria during 72 h.Table S1† illustrates the diversity in the measured effect concentration of nanoceria. Even for a given species, the results varied widely between studies. For example, Lee et al. showed significant mortality of D. magna after 96 h of expo-sure to 1 mg l−1 of 15 and 30 nm nanoceria103 while no toxicity was measure in D. magma after the same duration at 10 mg l−1 or a 48 h exposure at 1000 mg l−1 nanoceria.Van Hoecke et al. exposed D. magna to higher concentrations of 14, 20, and 29 nm nanoceria for 21 days, and found an LC50 of approximately 40 mg l−1 for the two smaller particles and 71 mg l−1 for the 29 nm particles.When combining all aquatic toxicity data, including C. elegans , no trends were observed between the nanoparticle size and the toxicity. We observed one extreme value, which is a report of reduction in life span of C. elegans at a concentration of 0.172 μg L−1 . 92 Some have suggested that the toxicity at low concentration can be explained by differences in the aggregation state as a function of concentration. NPs may be less aggregated at lower concentration.105 However, the nanoceria used in this study were positively charged, coated with hexa-methyleneteramine . It is possible that this coating rendered nanoceria much more toxic. Another Fig. 1 depicts the median of the lowest observed effect concentration and the EC10 or LC10 toward different species. This figure illustrates the high variability of the observed LOEC/EC10 between studies for a same organism . Based on the LOEC/EC10, the more sensitive species is the cyanobacte-rium Anabaena, while the least sensitive is Daphnia magna. No toxicity was observed up to 5000 mg Ce/L for the zebrafish Danio rerio and Thamnocephalus platyurus Fig. 1. It is noteworthy that exposure models predict concentrations significantly lower than those for which ecotoxicity investigations have encountered toxic effects. Therefore, nanoceria might not have any impact at environmental concentrations,growing pot despite the fact that some results are more worrying. More-over, most of the nano-ecotoxicology performed on aquatic organisms used a single species or a short trophic links and do not take into account important parameters such as the colloidal destabilization of the nanoceria, their interactions with organic molecules/ particles naturally occurring or bio-excreted, or the flux between compartments of the ecosystems .

To work under more realistic scenario of exposure, few nano-ecotoxicological studies are now performed in aquatic mesocosms with low doses of nanoceria, chronic and long-term exposure. Such studies will allow obtaining reliable exposure and impact data and their integration into an environmental risk assessment model that is currently missing.Although the data on environmental effects are far from complete, it is useful to consider case studies in order to gain knowledge about key data gaps and to give a first impression of relative risks based on current knowledge. While this case study is useful to point out areas where research is most needed, it is critical to point out the limitations of this case study. First, nanoceria have not yet been detected or measured in environmental media, and the actual environmental concentrations are not known. Second, very little is known about the fate and transport of nanoceria in the environment. Third, the toxicity data base is still very limited. Only a select few ecological receptor species have been tested to date and few if any sub-chronic or chronic exposures have been performed in longer lived organisms or in environmentally realistic exposure scenarios. The following case study characterizes the likely exposure concentrations and compares them to toxicity values for soil and water based on emissions due to combustion of fuels containing nanoceria additives and for discharge of chemical mechanical planarization media into sanitary sewers.Based on Table S1,† with the exception of HMT coated nanoceria, which do not apply to this case study and for which coating controls are lacking, the lowest EC10 value measured so far is 8000 ng l−1 for luminescence inhibition in cyano-bacteria.Previous estimates have been made for nanoceria used as a fuel catalyst and arriving in soil and water following atmospheric discharge106 in the UK based on known market size for this product. Clearly there is a wide disparity between concentrations likely to occur due to fuel catalyst combustion106 and the lowest toxicity values observed so far . However, there remains concern that nanoceria may enter water courses through its uses in specialized industrial polishing or chemical/mechanical planarization.Without specialized local knowledge on where these industrial concerns are located, the quantities of nanoceria used, that are disposed of from the premises, and the capacity of the associated sewage treatment plant, the local receiving water concentrations cannot be predicted.

Unfortunately, knowing global or national consumption of nanoceria in the polishing industry would not allow us to predict water concentrations. This is because the use of the product would not be evenly geographically spaced, or linked directly to human population density. However, it is possible to ask: what discharge would be needed to exceed the 8000 ng L−1 toxicity threshold for aquatic exposures? The dilution factor for sewage effluent recommended by EU risk assessment is 10. So effluent would need to contain 80 μg L−1 nanoceria. However, it is estimated that on entering an WWTP 95% of the nanoceria would enter sludge and only 5% pass through into the effluent.In that case the influent concentration would need to be 1.6 mg l−1 nanoceria. WWTPs are designed around population equivalents which tend to be around 160–200 L per PE per day in the UK so a PE unit would need to discharge 256–320 mg Ce per day to receiving waters. Given the current uses of nanoceria, this only seems likely to occur if a large industrial facility is directly discharging wastewater containing high concentrations of nanoceria directly into a sanitary sewer. Note that a population equivalent is a unit describing a given biodegradable load as measured by its biological oxygen demand.We have comprehensively reviewed what is known for nano-ceria about the environmental releases, methods for detection and characterization, fate and transport, toxicity and likelihood of toxicity in soil and water from acute exposures. Initial estimates of releases suggest that the majority of nanoceria will ultimately end up in landfills, with lesser amounts emitted to air, soil and water in that order. Once nanoceria enters the environment, it has been shown that NOM will have a major impact on their fate, transport and toxicity. As with other nanomaterials, aggregation is a key consideration and this has been shown to be influenced by water chemistry and interactions with natural coatings such as NOM. An important feature of nanoceria with respect to its behavior and toxicity is its valence state. There are several techniques that can characterize this property in environmental and bio-logical media, such as XAS, but most require relatively high concentrations. While we didn’t identify studies that detected nanoceria in natural environments or environmental media, a suite of techniques have been used to detect and character-ize them in complex toxicity testing media and in controlled laboratory studies. Thus, a major data gap and area for future research is the prediction and measurement of actual nano-ceria concentrations in the environment, either from point sources or non-point sources.

As a whole nanoceria appears to exhibit similar aquatic toxicity values other commonly studied manufactured nano-materials. For example,square pot a recent review found that species average LC50 values for Ag nanoparticles ranged from 0.01 mg L−1 to 40 mg L−1 while species mean LC50 values for ZnO ranged from 0.1–500 mg L−1 . 116 The range of EC50 values reported for Ce are similar to those for ZnO. Although reported toxicity data here uses LC10 and LOEC values, the range of species means 0.05–25.9 mg L−1 and many of the reported LC50 values are within the range of 0.1–100 mg L−1 , suggesting similar acute toxicity to ZnO NPs in aquatic expo-sures. This is of course based on the available data, which are predominantly on the toxicity of nanoceria to aquatic organisms, with sediment and terrestrial organism data severely lacking. For example, few if any studies have investi-gated toxicity in sediment dwelling organisms, which are likely to be exposed to nanoceria in the aquatic environment due to aggregation, settling and accumulation of nanoceria in sediment. Given the persistence of nanoceria, chronic studies are lacking as we are aware of only the C. elegans study.Equally important, very few species from few taxonomic groups have been tested. Large taxonomic groups such as insects and gastropods have not been tested and only one non-mammalian vertebrate spe-cies has been tested . Another difficulty is that most of the studies were performed with different nano-particles, doses, duration, organisms, exposure media, and their results are not directly comparable. Perhaps due to these differences, there are no apparent patterns to suggest that, as a whole, particle size has a major impact on toxicity. A problem in conducting realistic toxicity studies is the likely transformation of the free particles into homo or hetero-aggregates or even organic complexes in the real environment. There have been few studies that investigated the impact of size across a wide range of systematically varied particle sizes within a single study. Such studies are needed to definitively establish weather size is important. On the other hand coating may be an important variable given the extreme sensitivity seen with HMT coated particles in C. elegans. Coating was demonstrated to be a major determinant of toxicity in a more well controlled study that systematically varied coating properties and used coating controls.2 Of all of the taxonomic groups, toxicity is most well studied in vascular terrestrial plants. Overt phytoxicity of nano-ceria seems minimal and, while root to shoot translocation of these particles is often measurable it is generally quite low. In summary, although the literature on nanoceria impacts on terrestrial plants is not extensive, it is clear that overt phytotoxicity is minimal, even at excessive exposure concentrations. The data do suggest accumulation of nano-ceria within plant tissues, although the precise form of the element that crosses into the plant and the mechanism driving that process remains unknown. The potential trans-generational effects noted in the literature,as well as the complete lack of information on trophic transfer, are areas of concern. In addition, studies investigating environmentally relevant concentrations, potentially secondary effects from nanoceria exposure, including impacts on symbiotic micro-organisms or on edible tissue nutritional quality, certainly warrant further investigation. As a whole, the aquatic and terrestrial toxicity testing data for animals and microorganisms spans multiple orders of magnitude for acute toxicity values . This large variation can be exhibited within a single species exposed to similar nanoceria. For example, toxicity values for D. magna range from around 1–100 mg l−1 for fairly similar particles. Based on the overall toxicity database, it appears that C. elegans is the most sensitive animal and Anabaena is the most sensitive microorganism tested to date, although an important caveat is that the same endpoints were not com-pared across all species and that exposure systems varied. Interestingly no toxicity was observed in the fish species that has been tested even at extremely high exposure concentrations . Unfortunately, only two fish studies have been reported in the literature. There is a complete lack of toxicity testing data for sediment dwelling organisms, and extremely limited data for soil invertebrates. As a whole the data suggest that acute toxicity is possible at low μg L−1 concentrations in the water column. Data are lacking on soils and sediments, but toxicity values are likely to be far lower. One study indicated toxicity at lower concentrations than these values when 8 nm nanoceria were coated with HMT. Since no coating controls were used, it is critical that the influence of this coating and other similar positively charged coatings be studied using a similar end-point and suitable controls. The use and disposal of any nanoceria containing products with this coating should also be evaluated. It is not clear whether the chronic nature of this exposure or the influence of the coating on uptake and toxicity explain why this toxicity threshold is so low.

Using transient models such as the HYDRUS model has been suggested as an alternative

The differential response of roots to nutritional patchiness is probably a consequence of complex nutrient-specific signal transduction pathways .To investigate the effects of heterogeneous root salinity and nutrient conditions, several split-root tomato experiments were conducted . Water uptake from the saline root-zone dramatically decreased within 8 h of treatment in contrast to the non-saline root-zone, with a more pronounced effect when nutrients were provided only to the non-salinized root-zone . This reduction in water uptake did not correlate with decreased root growth , with the saline root-zone only showing significantly less root growth towards the end of the experiment . The rapidity and consistency of decreased water uptake by roots in the saline zone, from treatment imposition through to Day 9, suggests that a primary physiological response was fol-lowed by a morphological response. To further explore the role of heterogeneous nutrient provision on root activity, complete nutrient solutions were selectively depleted of either N or K+ in the non-saline root half while the other root half received a saline, complete nutrient solution . These treatments provoked a ‘two-phase-response’. Immediately upon treatment application, the saline conditions given to one side of the roots dominated, immediately decreasing water uptake of those roots. Subsequently, water uptake from the saline-treated, nutrient-supplied roots proportionally increased, probably in response to the nutrient deficiency induced by the omission of the nutrient on the non-saline side. This effect was marked when K+ was only present in the saline root half and slight in the case of N. The presence of K+ in the nutrient solution was the most important determinant of root activity even when coinciding with salinity,blueberry box resulting in a notably higher shoot tissue Na+ and Cl− concentration when the sole source of K+ was to the saline root volume .One valuable tool in categorizing and quantifying genetic variation in salt tolerance has been to define crop relative yield responses in terms of threshold salinities up to which yields are unaffected and linear decreases in relative yield with increasing salinity thereafter .

However, it is critical to recognize that these relation-ships have generally always been presented in terms of variation in parameters such as ECe or more occasionally in terms of variation in EC1:5 that relate to the salinity of the soil. However, it is not the salinity of the soil that affects plant growth but the salinity of the soil solution, and thus the ratio of salt to water in the soil. This means that the salinity stress on a plant can be doubled by doubling the salt concentration in a soil or by halving the water concentration of the soil. Furthermore, as soils become drier, plant growth becomes affected by the increasingly negative matrix potentials that develop in soils because of the adhesion of water by soil pores. This view profoundly affects the whole idea of the heterogeneity of salinity stress in soils, because heterogeneity arises because of variable: leaching effects of irrigation or rain-fall on salt concentrations in soil, hydrating effects of irrigation or rainfall on soil water contents, effects of surface soil evaporation increasing salt concentrations by capillarity and decreasing water contents in the soil, and/or water extraction rates of roots and the ion uptake/exclusion capacity, which over time also influence ion and water abundances near the roots.All irrigation water introduces salts to the system and in regions with high evapotranspir-ation and low rainfall, traditional salinity management emphasizes deliberate leaching of salts away from the root-zone while avoiding elevation of the water table to prevent damage to crops . Leaching is usually achieved by applying irrigation water in excess of crop evapotranspirational demands. The fraction of applied water that drains below the root-zone is referred to as the ‘leaching fraction’ and this value is used to coarsely gauge the extent of leaching . Larger leaching fractions generally result in larger zones with a low soil water salinity but may necessitate disposal of large volumes of saline drainage water and may cause additional salinization through capillary rise of saline water by raising the water table , as well as environmental impacts of drainage water disposal. Designing the appropriate leaching fractions needed to avoid yield loss is context-specific and will depend on the crop, soil texture, climate, irrigation system and irrigation schedule, and the salinity of irrigation water being used . Ayers and Westcot developed a simple approach to calculate the leaching requirement based on salt mass balance calculations.

This approach estimates the leaching fraction required to keep the average root-zone salinity below the salinity threshold of the crop, assuming a specific root distribution and a strictly vertical, continual water flow. Approaches like this neglect the spatial non-uniformity of irrigation water application as well as the temporal dynamics of irrigation and water uptake during the season and assume that the average root-zone salinity determines the impact of salinity on the crop . While the physical principles underlying salinity management have not changed since Ayers and Westcott developed these leaching guidelines, management goals have shifted over time to better recognize environmental impacts of nutrient and salinity losses and develop more advanced micro-irrigation and fertigation systems. This has given rise to both new challenges and new opportunities in managing salinity. Challenge 1: Managing salinity under micro-irrigation systems. Spatial patterns of salt accumulation are diverse and differ by irrigation system , with each irrigation system having specific challenges to salinity management. In the simplest case, flood irrigation applies water uniformly across the whole surface . In this case, salinity distribution is approximately uniform in the horizontal direction, but a salinity gradient exists vertically . Assuming sufficient leaching, salinity increases with depth in these systems and uniform leaching of salts below the root-zone causes the salinity within it to be relatively homogeneous. In contrast, applying water to only part of the surface causes strong horizontal salinity heterogeneity, as in furrow irrigation and more advanced micro-irrigation systems. Micro-irrigation aims to target water application to the root-zone, thereby improving water use efficiency by applying less water to regions with low root density and providing an opportunity to deliver water at a rate which matches crop demand. Flood and overhead sprinkler irrigation manage soil moisture and salt content at the field scale, while micro-irrigation approaches management at the root-zone scale. Targeted water application results in targeted leaching, with micro-irrigation leaching salts in zones which are rich with plant roots, while flood irrigation requires additional water to also leach salts from field zones between plants with low root density, making micro-irrigation more efficient than furrow/sprinkler irrigation for managing salinity . When drip and furrow irrigation were compared, drip irrigation sustained higher yields of salt-sensitive crops compared to furrow irrigation when saline groundwater is shallow, while using less water than furrow irrigation .

The economic incentive to install micro-irrigation systems is context-dependent, with the advantage of micro-irrigation over conventional irrigation becoming less clear when growing salt-tolerant crops or when irrigation water is abundant. Despite its potential to accumulate salts in the root-zone, even subsurface drip can have advantages over salinity management with traditional irrigation. While higher tomato yields justified the expense of installing a subsurface drip irrigation system in California, the same was not true of cotton, which remained lucrative with furrow irrigation ,blueberry package as such salt-tolerant crops tend to tolerate flood irrigation without yield loss provided that irrigation is applied pre-planting to avoid stand establishment losses . In drip irrigation systems with strongly localized water application, salt is not only leached downwards, but significant lateral water movement away from the drip emitter also leaches salt horizontally resulting in salt accumulation in the fringes of the wetted volume . This leads to a strongly heterogeneous small-scale salt distribution where soil salinity levels in the top 20 cm can vary by a factor of more than five within only 40 cm of horizontal distance . Although the extent of horizontal salt movement depends on the soil texture and can be partially controlled by emitter spacing, under micro-irrigation, salts concentrated between emitters near the surface generally have little opportunity to intrude into the root-zone without precipitation, due to surface evaporation and irrigation . It is therefore recommended that crops be arranged close to emitters where salinity is low and that new lines be installed as close as possible to where old lines existed to avoid the need for preseason reclamation leaching . Subsurface drip irrigation results in a different pattern of water flow and salinity accumulation. While water application at the soil surface causes salts to leach downward and outward from the water source, subsurface irrigation causes resident and irrigated salts to flow upward through advection and accumulate above the dripline where plants are present . This accumulation pattern antagonizes the establishment of many row crops be-cause germination is relatively sensitive to salt stress . Such production systems rely on pre-season rain, sprinkler or surface irrigation to leach salts below the drip line where they may be leached downward by subsurface irrigation . Shallow installation of subsurface drip lines is advantageous where sufficient pre-season rains are present as irrigating the soil surface may be avoided altogether . This issue can be mechanically managed in processing tomato by adding soil to planting beds , followed by irrigation to accumulate salts into the uppermost zone of the bed, which is subsequently removed and placed in the furrow between rows, where very little horizontal salt movement occurs . The strong localization of water application in drip irrigation questions the applicability of historical steady-state leaching models to micro-irrigation systems . These models insufficiently account for the highly local nature of micro-irrigation and underestimate both the local leaching fraction experienced by plants and the tolerable EC of irrigation water .

Adequate management of heterogeneous salinity patterns and localized leaching under drip or micro-sprinkler may allow sustainable crop production in soils that would otherwise be deemed too saline for that species.These models account for localized application of water and changes in flow rates over time by explicitly simulating two-dimensional water and solute transport in the root-zone by numerically solving mechanistic models. However, although these models are very strong in depicting physical transport processes, they often oversimplify the description of plant physiological processes governing water and solute uptake. For example, the HYDRUS model neglects that the distribution of water uptake is also affected by nutrient concentrations. Moreover, even if it was possible to perfectly simulate the water, nutrient and salinity dynamics for a given scenario, it would still be unclear how the calculated heterogeneous salinity distribution would translate into plant performance. Incorporating current knowledge of plant responses to heterogeneous conditions might make these models more suitable for evaluating salinity management practices. Challenge 2: How to simultaneously optimize N efficiency and minimize the impact of salinity. The necessity of a leaching fraction for long-term salinity management is coupled with the issue of nutrient loss, especially for nitrate , which exhibits similar leaching potential as Cl−. Any practice designed to remove Na+ or Cl− from the root-zone probably also leaches NO3 − . Although a common problem, few studies have addressed the integrated nature of salinity and nutrient management . While NO3 − and Cl− are subject to very similar transport mechanisms and rates in the soil, their distribution in the soil can nevertheless be quite different, and high Na+ and Cl− concentrations do not necessarily coincide with high NO3 − concentrations. This is because: in contrast to Na+ and Cl−, NO3 − is preferentially taken up by plant roots; and nitrogen fertilizer is deliberately added to the irrigation water during fertigation and is to some degree independent of water application. Understanding crop nitrogen demands and responses to spatially localized nutrients and salinity may help manage fertigation systems to achieve the simultaneous goal of salinity leaching and minimal nitrate loss. By providing nutrients through fertigation in a manner that retains nutrients in the low-salinity zone adjacent to the drip-emitter, roots can avoid exploring the saline fringes of the wetted zones,thus reducing salt exposure. HYDRUS-based modelling suggests that high-frequency applications of small amounts of nitrate, timed toward the end of a fertigation event, can help retain NO3 − in the root-zone adjacent to the irrigation source while allowing salt to be leached to the peripheral root-zone.

All plant growth was modeled to occur indoors using a vertical rack system with hydroponic irrigation

Griffithsin also effectively inhibits transmission of HSV-2 , HCV , SARS-CoV , Ebola , and possibly other viruses yet to be studied. Importantly, Griffithsin appears devoid of cellular toxicity that is associated with other lectins. O’Keefe et al. conducted studies with explants of macaque and rabbit vaginal tissues ex vivo and showed that Griffithsin did not induce changes in the levels of cytokines or chemokines, nor did it alter lymphocyte levels in human cervical tissue nor elicit inflammatory responses in rabbit tissue . The combination of extremely wide viral target range and demonstrated preclinical safety makes Griffithsin potentially useful as a prophylactic and/or therapeutic in multiple and diverse antiviral indications. The potential indications for Griffithsin as a human prophylactic or therapeutic include its use as an active pharmaceutical ingredient in vaginal and rectal microbicides. In spite of the value shown by pre-exposure prophylaxis drugs to prevent HIV transmission, issues of cost, side effects, the potential for development of viral resistance through chronic use of antiretrovirals as prevention modalities, and access to PrEP drugs by under resourced populations remain. These unmet needs could be met by the availability of affordable, safe and effective “on demand” antivirals, especially with Griffithsin as the API and its potential to control co-transmitted viruses such as HIV-1, HSV-2 and HCV during intercourse. Adoption of Griffithsin as a new biologic drug, especially in cost-constrained products such as microbicides, is predicated on the feasibility of a scalable manufacturing process that can supply market-relevant volumes of the API at an acceptable cost of goods sold . Previously, we showed that recombinant Griffithsin can be expressed and isolated with high efficiency using transient gene expression in green plants . Although the process described can be further optimized,growing bags the achieved pilot-scale expression yields of >0.5 g Griffithsin per kg of fresh green biomass , recovery efficiencies of 60–90% overall, and Griffithsin purity of >99% of total soluble protein are already impressive.

In this study, we developed a technoeconomic model for Griffithsin manufacturing using a plant-based system with the goal of estimating API manufacturing cost and determined the factors that have the greatest impact on COGS. The output of our study should serve as a basis for additional process improvements, selection of a commercial-scale manufacturer, and should assist in the identification of future product targets for cost-sensitive markets such as prophylactic microbicides as well as those for less cost-constrained therapeutic indications. Technoeconomic modeling was performed with the widely used SuperPro Designer modeling software . The main analysis in this study was conducted using data available from pilot-scale manufacturing of Griffithsin in Nicotiana benthamiana plants using tobacco mosaic virus -induced transient gene expression, and assuming that manufacturing would take place in an existing and fully equipped state-of-the-art plant-based bio-manufacturing facility. Modeling costs based on existing resources of a contract manufacturing organization instead of a “greenfield” build of a new facility was seen as the most likely scenario for launch of a new product. Our reasoning was that dedicated infrastructure could be built subsequently depending on market demand for the drug. As a result, we did not estimate capital equipment or total capital investment costs, and neglected depreciation, insurance, local taxes and factory expenses in the manufacturing operating cost analysis as these investments would have been made by the CMO. Our analysis assumed a 20% net profit margin/fee assessed by the CMO and this figure was added to the production cost of the product to arrive at the final total product cost. In addition to the techno economic analysis, an Environmental Health and Safety Assessment of the designed process was conducted using the method described by Biwer and Heinzle to evaluate the environmental, health and safety impact of Griffithsin manufacturing using the plant-based system, with the goal of assessing the sustainability of the process. The techno economic modeling for this study was performed using SuperPro Designer , Version 9.5 , a software tool for process simulation and flow sheet development that performs mass and energy balances, equipment sizing, batch scheduling/debottle necking, capital investment and operating cost analysis, and profitability analysis. This software has been used to estimate cost of goods in a variety of process industries including pharmaceuticals produced by fermentation and plant-made pharmaceuticals .

It is particularly useful at the early, conceptual plant design stage where detailed engineering designs are not available or warranted. SuperPro was chosen because it has built-in process models and an equipment cost database for typical unit operations used in the biotechnology industry, such as bioreactors, tangential flow ultrafiltration and diafiltration, chromatography, grinding or homogenization, and centrifugation. There are some specific unit operations and processes used in this study that are currently not included in SuperPro, such as indoor plant cultivation, transplantation, plant harvesting and screw press/disintegrator. Such unit operations were addressed through the “Generic Box” feature of the application. Unless otherwise noted, the maintenance costs of major equipment, unit operation-specific labor requirements and costs , pure components, stock mixtures, heat transfer agents, power and consumables used in the analysis were determined using the SuperPro built-in equipment cost model and default data banks. Additional case study specific design parameters were selected based on experimental data from journal articles, patent literature, the authors’ laboratories, interviews with scientists and technologists conducting the work cited, technical specification sheets or correlations, heuristics, or assumptions commonly used in the biotechnology and/or agricultural industry.Process flow and unit operations were derived from published methods and unpublished results obtained by the authors and collaborators who have participated in the development and scale-up of the process described and in the development of Griffithsin products. On the basis of this information, the SuperPro software was used to select and size equipment for each of the unit operations to achieve the desired production target , simulate the operations by performing material and energy balances, and specify and schedule all operations taking place within each piece of equipment to calculate material inputs and outputs and process times. Costs for raw materials, utilities, consumables, labor, laboratory QA/QC, waste disposal and equipment maintenance were then used to determine annual operating costs, and per-unit mass or per-dose costs . The main case study model was based on an existing plant based manufacturing facility, operating in batch mode, and excluded new capital investments and other facility dependent costs, except for equipment maintenance costs, which were included.

For the downstream portion of the Griffithsin manufacturing process, an annual available operating time of 7,920 h for the facility was used with indoor-grown Nicotiana benthamiana plants. Operating time was based on Holtz et al. for a similar facility, which was designed with overlapping utility capacity and in which the largest single utility unit can be down for maintenance and/or repairs and the utility loads can be maintained with redundant equipment. Likewise, per Nandi et al. it was assumed that the plants would be grown continuously throughout the year . Land costs, upfront R&D, upfront royalties, and regulatory/certification costs were neglected in the model as these costs can vary widely. Griffithsin protein can be produced in plants in a number of ways. These include stable expression in recombinant plants; inducible expression in transgenic plants; transient expression induced directly by tobacco mosaic virus replicons; or via agrobacterial vectors introduced into the plants via vacuum assisted, or surfactant-assisted, infiltration . Relative to stable transgenic plants, the advantages of speed of prototyping, manufacturing flexibility,nursery grow bag and ease of indoor scale-up are clearly differentiating features of transient systems and explain why this approach has been widely adopted in the manufacture of many plant-made pharmaceuticals . In our base-case analysis, we modeled expression of Griffithsin using TMV induction described in Fuqua et al. and results from 3 pilot-scale manufacturing runs because these batches provided the most extensive and complete data set; however, this process has been corroborated in 6 additional manufacturing runs at pilot-scale or larger.icotiana benthamiana host plants are generated from seed and propagated indoors under controlled environmental conditions until sufficient biomass is obtained for inoculation with the TMV vector carrying the Griffithsin gene. The process is summarized as follows. An N. benthamiana Master Seed Bank is generated from seeds obtained from the U.S. Department of Agriculture Repository. For bio-manufacturing, seeds from the TW- 16 line are obtained in bulk and stored securely. The Master Seed Bank is qualified for germination rate , freedom from disease, and genetic uniformity, and stored in sealed containers under temperature-controlled conditions . If the seed batch passes release tests, it becomes the Production Seed Batch and is used in the designated production run . Seedlings are allowed to grow for 21 days under controlled environmental conditions . At this stage, the seedlings are transplanted to accommodate their larger size and moved to another growth room to await inoculation, as described in the following sections.

The API extraction procedure modeled is per Holtz et al. except that a 1:1 ratio of biomass:buffer is used. Briefly, the aerial parts of the plants containing accumulated Griffithsin are mechanically inverted and cut with a mechanical cutter. The harvested biomass is collected in baskets for transport to the extraction suite, to initiate downstream processing. The harvested biomass fresh weight is determined to calculate the volume of extraction buffer to be added, typically at a rate of 1 kg biomass FW:1 L buffer mix . The pH is adjusted to 4.0 and the mixture is heated to 55◦C for 15 min to help precipitate major host plant proteins. The heated mixture is passively cooled and filtered to yield a crude extract. The crude extract is stirred overnight at 4◦C in the presence of bentonite and MgCl2. This procedure helps remove TMV coat protein , which at this step represents the largest protein impurity in the extract. The suspension is filtered to remove aggregated TMV CP, yielding a clarified and partially purified API-containing solution and is then sterile- filtered . In-process controls are applied throughout downstream processing unit operations to determine reagent volumes and assess yield and quality at key steps. To adequately meet the projected initial annual market demand for a rectal micro-bicidal formulation in the United States, approximately 6.67 million doses of Griffithsin API at 3 mg/dose would be needed. This translates into a production rate of 20 kg of purified Griffithsin API per year. The manufacturing facility to produce the required 20 kg of API per year was assumed to segregate production operations into two broad categories; namely, upstream production and downstream recovery and purification. To accommodate a large number of plants, the facility uses a vertical cultivation design with integrated irrigation and runoff collection system. Each rack is compatible with an integrated transportation infrastructure to move each tray to the next phase of the growth cycle. The upstream portion of the facility houses unit operations for N. benthamiana propagation, inoculation with TMV vector, and Griffithsin protein expression and accumulation. These processes begin with seeding and end when the biomass is taken to harvest. The downstream portion of the facility begins at harvest and continues through purification of the Griffithsin DS. Upstream processing is assumed compliant with good agricultural practices , whereas downstream processing is subject to FDA current good manufacturing practice . The general layout of the upstream growth rooms was adapted from Holtz et al. , and includes one germination chamber for seeds, one pre-inoculation room for biomass growth, and an isolated post-inoculation chamber where N. benthamiana inoculated with TMV expresses and accumulates Griffithsin. Plants are arrayed in equally sized trays under light-emitting diode light systems tuned to the optimized photosynthetic absorbance spectrum of N. benthamianaand are continuously illuminated. The plants are rooted in rock wool cubes held in the trays by polystyrene foam floats and perfused with a nutrient solution . Hydroponic irrigation is on a 12-h cycle and is accomplished via nutrient film technique . We modeled a hydroponic system because the nutrient solution is recycled; hence, water is conserved, and fertilizer runoff is reduced although not eliminated. The mass of nutrient solution taken up by the plants, the cost of the nutrient solution per liter, and the mass of residual nutrient solution that goes to the wastewater treatment system are shown in Supplementary Table 1 in Supplementary Material.

MMC is an interstrand cross-linking agent , and CDDP is an intrastrand cross-linking agent

At the subcellular level, SOG1 is present in the nucleus, consistent with previous findings; however, unlike any previous data in response to other genotoxins, SOG1 does show a change following Al exposure that is ATR-dependent and may be the result of relocalization, morphological changes to the cell identity, as well as possible diminished visualization due to protein degradation. Post-translational modification of SOG1 was determined to be crucial to the regulation of its function. There is no significant transcriptional change to SOG1 expression in the presence of Al, despite protein accumulation subsiding following Al exposure. This suggests that there may be an undetermined mechanism for protein turnover following SOG1 activation. As the p53 functional homologue in Arabidopsis, SOG1 turnover could prove to be a conserved regulatory mechanism as p53 is ubiquitinated by the E3 ligase, MDM2, in mammals . Perhaps this is even a speculative role for ALT2, as WD40 proteins have been shown to associate with E3 ubiquitin ligases. It is unlikely that ALT2 would be responsible for SOG1 turnover as part of an ubiquitin-proteasome pathway as loss of ALT2 would result in inappropriate persistence of SOG1 and lead to hypersensitivity rather than tolerance as is observed. As of yet, evidence only supports that the activation mechanism in response to Al is dependent on ATR, likely through phosphorylation of SOG1 by ATR. Future studies are needed to determine how SOG1 is degraded following ATR-dependent activation in response to Al as well as to other stresses. As shown in Chapter 5, the fourth Al tolerance gene isolated from the als3-1 suppressor screen was SUV2, nursery pots which encodes the Arabidopsis homologue of the mammalian ATR-interacting protein, ATRIP. The suv2-3 mutation represents a premature stop codon in the eighth exon of SUV2 at amino acid 359 of 646.

After establishing the als3-1 suppressor as an Al tolerant suv2 mutation, it was characterized as being part of the same ATR-mediated pathway, further supporting SUV2 as an interacting partner of ATR in Arabidopsis. Additionally, both cell cycle arrest and differentiation of the QC were established to be dependent on SUV2 following Al exposure, and that these responses ultimately lead to endore duplication as a means to terminal differentiation of the root meristem. Thissue- and cell-specific localization of SUV2 was shown to be present within the cytoplasm and nucleus of actively dividing cells of the root tip. There is accumulation of SUV2 throughout the meristematic regions of the root tip in the absence of Al. When grown in an Al toxic environment, SUV2 persists in the meristematic region of the root tip, but this zone has significantly been reduced in size and therefore there is a concomitant reduction of SUV2 in the root organ. At the sub-cellular level, SUV2 is present in the cytoplasm and the nucleus of cells at the root tip in both the absence and presence of Al. It is likely that as Al causes differentiation of the root meristem up to the point of complete stem cell consumption, the zone of cell division becomes reduced while the zone of elongation encroaches closer to the apex i.e. a morphological reduction in zones of root development. This reduction in the meristematic zone may account for the insignificant but observable progressive reduction in SUV2 transcript levels following treatment with increasing amounts of Al.As with SOG1, SUV2 is phosphorylated by ATR in vitro. While in vivo studies are needed in both cases to confirm this post-translational modification, this would be considered a conserved relationship between ATR and SUV2, as this phosphorylation is known to occur in the homologous proteins in yeast and humans . This begs the question: what are the bona fide substrates of ATR in the Al response pathway? While SOG1 has been demonstrated to have 5 ATM/ATR phosphorylation sites, defined as TQ or SQ motifs, and SUV2 is speculated to contain two sites, in a recent phosphoproteomics study, SOG1 was not even identified as a substrate of ATR or ATM in response to ionizing radiation, let alone SUV2 . Some DNA repair factors such as LIG4, UVH3 and MRE11 as well other DNA maintenance and metabolism factors like CHR4, HTA7/HTA10, PCNA1, MCM4 and H2AXA were proven to be ATR and ATM targets in this kinase target study .

While the authors acknowledge that their experimental technique using adverse tryptic cleavage likely was responsible for not identifying SOG1 in their large-scale identification of kinase targets, this large-scale proteomic endeavor only tested IR stress and did not distinguish between ATR versus ATM phosphorylated targets . Despite the substantial contribution these findings offer to the field of plant DNA maintenance and repair, more in depth studies are needed not just for Al responses, but also for the myriad types of damage to plant DNA that must be repaired. Al treatment leads to root growth inhibition due to terminal differentiation by means of endore duplication as visualized by increases in cell and nucleus size of cells of the root tip. Al treatment results in substantial increases in both cell and nuclear size for als3-1 roots, which is consistent with terminal differentiation in conjunction with endore duplication. This increase in size was shown to be dependent on the Al tolerance factors: ATR, ALT2, SOG1 and SUV2. This shows that all four genetic factors control terminal differentiation and endore duplication in response to Al. Previous studies of a sog1 loss-of-function mutant defined a set of SOG1- mediated genes that were inducible by γ-radiation . In response to Al toxicity, SOG1 and ATR have now been established to be acting within the same response pathway, so it was of great interest to determine if SOG1, ATR, ALT2, and SUV2 were responsible for the induction of this established gene set after treatment with Al. The proper conditions to determine whether Al causes changes in known SOG1-mediates genes was determined by a time course study of the persistence of SOG1 accumulation without completed QC differentiation in response to high Al concentrations. After 4 days of growth on highly inhibitory concentrations of Al, the pool of stem cells in the root meristem differentiate into cells no longer capable of dividing; while SOG1 is completely absent from the root tip after 5 to 6 days of growth on high concentrations of Al. Taken together, this established that 3 days of growth in Al was the optimal point at which expression changes would be analyzed, satisfying the need for SOG1 actively inducing transcriptional changes that would lead to root growth inhibition in response to Al. Genes that were selected for expression analysis were found in a previous study as being highly induced following γ-radiation in a SOG1-dependent manner .

From the γ-radiation study, a number of DNA repair genes like BRCA1, the ortholog for the human breast cancer susceptibility gene; PARP2, a key component of microhomology-mediated DNA repair; GMI1, involved in homologous recombination and chromosome maintenance; and members of the RAD family of genes, RAD17 and RAD51. CYCLINB1;1accumulates in the G2 phase of cell cycle progression and is regulated by transcriptional activation. During normal cell cycle progression, in a population of cells like the root rip, a few would be in the G2 phase at a given time expressing CYCB1;1, but an increase in expression would suggest more cells within the cell population were halted in the G2 phase, indicative of a G2 cell cycle arrest. Other transcription factors were induced in this γ-radiation study,plastic planters like TRFL3 and TRFL10, MYB family transcription factors known to have roles in developmental processes, defense responses and DNA maintenance . In total, 16 genes were assayed . Treatment with Al resulted in a measurable increase in expression of the subset of assayed genes in Col-0 wild type compared with no Al, and for als3-1 there was an even larger increase of gene expression. In contrast, an increase in expression of these genes was not observed for any of the Al tolerant single mutants or double mutants in comparison to the respective controls. This indicates that Al triggers an ATR-, ALT2, SUV2- and SOG1- dependent transcriptional program that is similar to that observed following treatment with γ-radiation, providing an important link between the cell cycle checkpoints involved in DNA damage detection and transcription in response to Al. Clearly, cell cycle checkpoints are emerging as key regulators of Al response, indicating that Al-dependent activation of these factors is central to terminal differentiation following chronic exposure to Al. Unlike γ-radiation, this stoppage of root growth is ATM-independent, as demonstrated by real-time PCR analysis of an atm loss-of-function mutant, indicating that at least in respect to Al stress, SOG1 functions downstream of ATR rather than ATM. This is of particular importance since it is indicative of the type of damage that ATR, ALT2, SOG1 and SUV2 are detecting in the context of Al. There are clear transcriptional differences between Al treatment and γ-radiation. Examples of these differences would be the lack of induction of AtRAD21 in the absence or presence of Al, as well as the ATM independent manner of SOG1-dependent transcript induction in Al treated seedlings while they are ATM-dependent upon exposure to γ-radiation. The difference in the role of ATM in response to these two stresses is interesting, especially considering that ATM is largely uninvolved in the Al response despite the requirement of functional ATR, ALT2, SOG1 and SUV2. This indicates that ATR, ALT2, SOG1 and SUV2 comprise an Al-response pathway that does not primarily require ATM. This suggests that each DNA stress results in a unique transcriptional profile that may be revealing in relation to their respective impacts on genomic integrity. With the overwhelming evidence that the DNA damage response factors ATR, ALT2, SOG1 and SUV2 all play a role in detecting Al dependent damage, it was of interest to examine their roles in response to known genotoxins. HU is an inhibitor of ribonucleotide reductase by scavenging free radicals that are used for the reduction of ribonucleotides . This stalls the replication fork due to depletion of deoxyribonucelotides. ATR functions to detect replication fork blocks and single stranded DNA breaks and atr mutants are sensitive to HU .

MMC is an aziridine containing antibiotic isolated from Streptomyces caepitosus . MMC itself does not react with DNA, but once it becomes reduced by quinone, the aziridine opens and allows MMC to attack the DNA . This reaction forms crosslinks across the complementary strands of the DNA double helix, or interstrand cross links . DNA cross linking can also occur between adjacent bases, called intrastrand DNA cross linking. The chemotherapy drug CDDP is a platinum-containing drug that is a neutral molecule until it is activated through a series of spontaneous aquation reactions, which involve the sequential replacement of the cis-chloro ligands of CDDP with water molecules . When the aquation event occurs, this allows the platinum atom to bind to DNA, preferentially guanine bases, which forms DNA–DNA intrastrand crosslinks . DNA cross links block replication because when they go unrepaired, collapse of the replication fork occurs blocking replication and leading to cell death . ALT2 is the Arabidopsis homologue of the human protein, CSA, which monitors conformational changes in DNA as assessed by blockage ofDNA replication and RNA transcription, a known effect caused by DNA cross links. alt2-1 is hypersensitive to MMC and CDDP . SOG1, the plant functional homologue of the human transcription factor p53, is a transcription factor in Arabidopsis responsible for the initiation of DNA damage responses including DNA repair as well as the initiation of endore duplication in plants. sog1 loss-of-function mutants are sensitive to both the replication fork poison, HU, and the DNA cross linking agents, MMC and CDDP. SUV2 is the Arabidopsis functional homologue to the human protein, ATRIP, which is required for recruiting ATR to sites of DNA damage, presumably to regions coated in Replication Protein A . RPA is a heterotrimeritc protein complex, comprised of sub-units RPA1, RPA2, and RPA3, which binds to single stranded DNA to protect it from nuceolytic degradation and hairpin formation, similar to the prokaryotic Single Stranded Binding protein .

Mutational approaches using the model plant species Arabidopsis have become an important complement to these studies

Outside of organic acid release as a form of Al tolerance or resistance, a logical but unproven mechanism of Al tolerance is root-mediated alkalinization of the rhizosphere, since Al toxicity is dependent on the pH of the growth environment. Despite the appeal of this process as an Al tolerance mechanism, there is only one report that clearly demonstrates this role in Al tolerance. An Al tolerant mutant in Arabidopsis was shown to release similar amounts of organic acids to wild-type seedlings, indicating that this mutant has a different mechanism of Al tolerance. It was then found that its mechanism of Al tolerance was correlated with an Alactivated root apical H+ influx. This H+ influx resulted in an increase in rhizosphere pH at the surface of the root tip, which was significant enough to decrease Al activity around the root tip . In addition to the various resistance mechanisms described, there has been limited evidence that modification of extra- and intracellular anionic sites can have a positive impact on Al resistance. Polyamines are small aliphatic polycationic molecules that could compete with Al ions for binding sites at the cell wall and membrane to prevent Al from entering the cell . Plant polyamines are detected in actively growing tissues and under stress conditions. They also have been connected to the control of cell division, embryogenesis, root formation, fruit development and ripening, and responses to biotic and abiotic stresses .There have been two reports that describe the relationship between Al and polyamines; one report has demonstrated the positive effects of polyamines on Al tolerance, while another has discussed the effects of Al on cellular polyamines. In saffron, 1mM polyamines were able to reduce Al toxicity. Polyamines were also able to decrease H2O2 content in the presence of Al, as well as decrease Al accumulation at the roots . In cell cultures of a woody plant Catharanthus roseus, it was observed that polyamine levels increase upon Al exposure. Spermine levels increased by two- to three-fold after 24 and 48 hours of exposure to Al, and putrescine levels slightly increased after four hours of exposure,black flower buckets but was surprisingly followed by a sharp decrease . This suggests that even non-chelating molecules can have a significant impact on Al tolerance in plants. It has been argued based on these studies that Al exclusion must be a rapid response to minimize Al uptake and subsequent Al-dependent stress.

Interestingly, much of the findings on Al exclusion mechanisms have arisen from studies that move roots from a no Al environment to one that has highly toxic levels with the research focused on the immediate responses to Al. It is hard to imagine a real world situation in which roots go from an environment with little to no Al to one that has highly inhibitory concentrations. Therefore, it is arguable whether the approach of studying immediate responses to Al is necessarily relevant to Al toxicity in soils since stoppage of root growth in such an environment is likely due to chronic long-term exposure to Al. Consequently, it is of critical importance to determine the toxic effects of Al as it accumulates within plant tissue.It has been very difficult to determine other mechanisms of Al tolerance and resistance in agriculturally relevant plants due to issues such as genome size, availability of knockout lines, generation time, and difficulty in creating transgenic lines. The use of Arabidopsis thaliana has evaded this issue by utilizing the model plant that has a smaller diploid genome, an extensive library of knockout lines, and short generation time . Additionally Arabidopsis has similar sensitivity to Al in comparison to other crop plants and shows classic signs of Al toxicity, making Arabidopsis a suitable plant model for Al toxicity for crop species . The mechanisms of Al resistance have been intensively studied on crop species using natural genetic variation within and across species, such as wheat and maize. While clearly an insightful approach that has given extensive knowledge on Al exclusion mechanisms, this work is limited based on currently existing variability with regard to growth in the presence of Al. Beyond the obvious advantages of using a model species, Arabidopsis has a similar Al toxic threshold to many agriculturally relevant crop species making it a valuable system for investigating how plants sense and respond to Al through the identification of mutants with altered growth capabilities in the presence of Al . This has been particularly true with regard to identification ofAl sensitiveArabidopsis mutants, which have reduced root growth in the presence of Al likely due to deffects in mechanisms required for Al exclusion, Al detoxification, or response to Al-dependent damage.

By screening for Al sensitive Arabidopsis mutants, eight complementation groups were identified indicating that Al toxicity is complex, which is to be expected considering the likely number of factors involved in mechanisms of Al resistance and tolerance . Most importantly, as will be discussed later, identification of these als mutants has allowed for use of a suppressor mutagenesis approach that has resulted in identification of factors that are important for Al-dependent stoppage of root growth. In order to find factors that are required for Al stress response, a genetic approach was taken to identify Arabidopsis mutants that exhibit increased Al sensitivity. Mutant lines were generated through ethyl methanesulfonate mutagenesis of Arabidopsis thaliana ecotype Col-0 wt. M2 seedlings were subsequently screened for their response to Al by identifying seedlings with normal growth in the absence of Al, but restricted growth on low levels of Al. The seedlings were grown on a two-layer gel system, with the upper layer consisting of nutrient medium with no added Al and the lower layer consisting of the same nutrient medium equilibrated with a subtoxic level of AlCl3. Any seedlings that could grow normally through the upper layer but could not penetrate the lower layer were isolated. Each of these Al sensitivemutants likely represents deffects in genes that are required for mechanisms for Al resistance or tolerance. Two of the identified mutants, als1-1 and als3-1 have been studied in depth. Both of these mutations represent a recessive loss of function mutations resulting in greater than wild type root growth inhibition in the presence of sub-threshold levels of AlCl3. The first mutant characterized was als3-1 due to its extreme sensitivity to Al at low levels. When grown on Al asl3-1 shows complete arrest of growth of the primary root and shoot meristems. The roots of als3-1 are stunted and have a swollen club shaped root apex, with root hairs at or near the root tip when grown on Al media. Also, als3-1 roots do not initiate any lateral roots, but can produce secondary roots from the base of the hypocotyl. Growth of lateral roots can reinitiate if als3-1 plants are removed from Al media, but the primary root is irreversibly inhibited by the Al treatment. This response differs from the roots of wild type plants, which are able to fully recover from the Al treatment. Following Al treatment the shoot phenotype of als3-1 shows reduced cotyledon expansion. als3-1 shoots show a delayed growth recovery in comparison to the root, which does not recover at all. Leaf expansion is blocked for several days after the Al recovery.

In addition, the first true leaves of als3-1 develop abnormally following Al treatment. They are severely stunted with very few trichomes, poor leaf expansion,french flower bucket and irregular shaped epidermal cells with a rough leaf surface. The leaves that developed after Al treatment also do not expand but became a disorganized cluster of leaf pegs that eventually expand without petiole development. Eight days after removal from Al, a second shoot apex forms that develops relatively normally except for a greater number of rosette leaves and some fused inflorescences. This shoot phenotype is found to only in als3-1 plants that were challenged with Al and has not been described for als mutants in general. Since this mutation is completely Al-dependent, it was hypothesized that als3-1 represents a factor required for Al-tolerance or resistance. This factor was found to be specific for Al tolerance since als3-1 did not show increased sensitivity to other metals such as copper, nickel, cadmium or lanthanum and does not display any other growth deffects in low pH without Al. Staining of als3-1 roots with hematoxylin and morin, two stains that bind to Al, resulted in similar intensity of staining to wild type, suggesting that als3-1 mutation does not alter the amount of Al uptake . This was later confirmed using ICP-OES . Although there was no difference in the amount of Al uptake, when wild type and als3-1 roots were stained with hematoxylin, wild type plants showed a diffuse pattern of surface bound Al extending from the root apex to the mature region of the root, while als3-1 roots displayed intense staining just proximal to the root tip . Map-based cloning of als3-1 showed that it represents a defect in a gene that encodes an ABC-like transporter homologous to bacterial ybbM, which is a metal resistance protein from Escherichia coli. Based on this similarity and the localization pattern of ALS3, which shows it predominantly at the plasma membrane of root cortical cells and cells of the vasculature, it was proposed that it redistributes Al away from the most sensitive plant tissues in order to maintain cell division . Loss of ALS3 as in the als3-1 mutant would result in inappropriate accumulation of Al in vulnerable areas such as the root tip and would consequently cause growth arrest at levels of Al that have no measurable effect on wild type. GUS staining of plants harboring the ALS3 promoter fused with GUS indicates that ALS3 expression is localized primarily to the sieve tube elements of the phloem in all plant organs, and trichoblast cell files and immature root hairs. GUS activity was also found in the epithem tissue of the hydathodes but not in the actual water pore of the hydathode. Since Northern analysis determined that ALS3 is Al inducible, GUS analysis of Al treated lines resulted in a shift of expression from the root epidermis to the root cortex.

From these results, ALS3 was hypothesized to mediate Al transport within the plant, transporting Al from sensitive tissues from the plant such as the root apical meristem in order to sequester it in less sensitive tissues or also to the hydathodes for excretion by guttation. It is hypothesized that by disrupting this partial ABC transporter, there is inappropriate accumulation of Al in the root, leading to severe symptoms of Al toxicity . Consistent with the importance of ALS3 to Al tolerance, an ALS3 homolog was identified in rice, called STAR2. Although both ALS3 and STAR2 are required for plant Al tolerance, the expression patterns and cellular localization differ. STAR2 is only expressed in roots upon Al treatment and is located in all cell types except for the epidermal cells in the mature root zone . In contrast, ALS3 is expressed at a basal level in the vasculature throughout the plant and its expression is dramatically increased in the Arabidopsis root tip following exposure to Al . Similar to ALS3, STAR2 contains several transmembrane domains that likely form a pore or channel that is involved in substrate movement. Both ALS3 and STAR2 lack an ATPase domain, making them unusual with regard to ABC transporters that often have the transmembrane domains and ATPase domain all as part of one protein. While a separate ATPase domain containing protein partner has not been found for ALS3 , rice STAR2 was shown to interact with another protein, STAR1, which contains an ATPase domain . The STAR1/STAR2 complex functions together as a bacterial-type ABC transporter that is speculated to transport UDP-glucose, although it is currently unclear as to how the transport of UDP-glucose by STAR1/2 is responsible for rice Al tolerance . Other Arabidopsis mutants that have been identified with altered responses to Al include als1 and als7. Both als1 and als7 were identified in the original screen for Arabidopsis mutants with Al hypersensitivity. als1-1 was also identified by mapped based cloning and subsequently characterized. Similarly to als3- 1, als1-1 has an extreme increase in Al sensitivity.

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