¿Se ofrecen opciones ecológicas de macetas plásticas al por mayor?

Sí, en la actualidad hay opciones ecológicas de macetas plásticas disponibles al por mayor. Estas macetas suelen estar fabricadas con materiales plásticos biodegradables, compostables o reciclados, lo que ayuda a reducir el impacto ambiental en comparación con las macetas plásticas convencionales. Algunas características comunes de estas opciones ecológicas incluyen:

  1. Materiales biodegradables: Las macetas están hechas de plásticos biodegradables, lo que significa que se descomponen naturalmente con el tiempo, reduciendo así la acumulación de residuos plásticos.
  2. Materiales compostables: Algunas macetas plásticas ecológicas están hechas de materiales compostables, lo que significa que pueden descomponerse en condiciones de compostaje, convirtiéndose en nutrientes para el suelo.
  3. Plástico reciclado: Otras opciones utilizan plástico reciclado en su fabricación, contribuyendo a reducir la cantidad de plástico nuevo producido y fomentando la economía circular.
  4. Diseños sostenibles: Además de los materiales utilizados,cultivo de la frambuesa algunas empresas se esfuerzan por adoptar prácticas sostenibles en la fabricación y diseño de las macetas, minimizando el uso de recursos y energía.

Al buscar opciones ecológicas al por mayor, es recomendable contactar a proveedores especializados en productos sostenibles, empresas de suministros para jardinería e incluso explorar opciones en ferias comerciales relacionadas con la horticultura y la jardinería sostenible. La demanda de productos respetuosos con el medio ambiente está en aumento, lo que ha llevado a un aumento en la disponibilidad de opciones ecológicas en varios sectores, incluido el de las macetas plásticas.

¿Cuáles son las opciones más económicas de macetas plásticas al por mayor?

Las opciones más económicas de macetas plásticas al por mayor pueden variar según varios factores, como el proveedor, la calidad del material y el tamaño de la orden. Aquí hay algunas sugerencias para encontrar opciones económicas:

  1. Comparar Precios: Investiga y compara precios de diferentes proveedores. Puedes hacer esto visitando sus sitios web,macetas por mayor contactándolos directamente o utilizando plataformas en línea especializadas en ventas al por mayor.
  2. Negociar Descuentos por Volumen: Muchos proveedores ofrecen descuentos por volumen o compras al por mayor. Negocia con los proveedores para obtener precios más bajos al realizar pedidos de mayor cantidad.
  3. Proveedores en Línea: Explora plataformas en línea que conectan a compradores con proveedores al por mayor. Estos sitios a menudo tienen una amplia variedad de opciones y competencia, lo que puede llevar a mejores precios.
  4. Buscar Ofertas y Promociones: Estate atento a ofertas especiales, promociones o liquidaciones. Algunos proveedores ofrecen descuentos temporales o promociones para compras al por mayor en determinados momentos del año.
  5. Proveedores Directos: Considera contactar directamente a fabricantes de macetas plásticas. Al eliminar intermediarios, es posible que encuentres precios más competitivos.
  6. Comprar en Temporada Baja: En algunos casos, los precios pueden ser más bajos durante la temporada baja de jardinería. Aprovecha estos momentos para obtener mejores ofertas.
  7. Explorar Proveedores Locales: Busca proveedores locales que puedan ofrecer precios competitivos debido a menores costos de envío.
  8. Tiendas de Outlet o Liquidación: Algunas tiendas de outlet o liquidación pueden tener existencias de macetas plásticas a precios reducidos. Sin embargo, esto puede variar según la ubicación y la disponibilidad.

Recuerda siempre equilibrar el precio con la calidad del producto. Asegúrate de que las macetas plásticas cumplan con tus estándares de durabilidad y funcionalidad, incluso al buscar opciones más económicas.

Formas Creativas de Reutilizar Macetas de Plástico: Más Allá del Jardín

Las macetas de plástico desempeñan un papel vital en el cuidado de nuestras plantas, pero ¿qué sucede cuando ya no son necesarias para su propósito original? En lugar de relegarlas al contenedor de reciclaje, considera las numerosas formas creativas de reutilizar las macetas de plástico. Desde la organización doméstica hasta soluciones innovadoras de jardinería,cultivar frambuesas estos versátiles contenedores pueden encontrar una nueva vida en varios aspectos de tu hogar y jardín.

Almacenamiento en Interiores y Exteriores:

Las macetas de plástico, especialmente las más grandes, se pueden reutilizar como contenedores de almacenamiento. Úsalas para organizar herramientas pequeñas, guantes de jardinería o incluso juguetes infantiles. La durabilidad de las macetas las hace adecuadas para soluciones de almacenamiento tanto en interiores como en exteriores. Lienzos de Pintura para la Creatividad:

Convierte tus macetas de plástico en obras maestras artísticas utilizándolas como lienzos. Deja que fluya tu creatividad con pinturas, marcadores o decoupage para transformar macetas simples en piezas decorativas únicas. Estas creaciones personalizadas pueden agregar un toque de estilo a tu jardín o espacio interior. Comederos o Baños para Aves DIY:

Convierte las macetas de plástico más pequeñas en comederos o baños para aves. Cuélgalas de las ramas de los árboles o colócalas estratégicamente en tu jardín. Agrega perchas o crea recipientes de agua poco profundos para atraer a tus amigos emplumados, convirtiendo tu jardín en un refugio para la vida silvestre local. Innovaciones en Jardinería Vertical:

Apila macetas de plástico más pequeñas verticalmente para crear un jardín de hierbas único o una exhibición floral. Este enfoque de jardinería vertical no solo maximiza el espacio, sino que también añade un elemento estéticamente agradable a tu jardín o balcón. Organizadores de Hierbas y Especias:

Coloca macetas de plástico en una bandeja o estante para crear una estación compacta y organizada de hierbas y especias. Etiqueta cada maceta con el nombre de la hierba o especia, facilitando el acceso y el mantenimiento de un jardín de cocina bien ordenado. Bandejitas para Germinación de Semillas:

Las macetas de plástico son excelentes para iniciar plántulas. Reutilízalas como bandejitas para la germinación de semillas colocando varias macetas pequeñas dentro de un contenedor más grande. Este vivero improvisado de plántulas se puede mover fácilmente para optimizar la exposición al sol. Portavelas para Ambiente:

Corta la parte superior de las macetas de plástico para crear elegantes portavelas. Coloca macetas de diferentes tamaños para añadir un toque único a tu espacio exterior. La naturaleza translúcida de algunos plásticos puede crear hermosos patrones cuando se iluminan con la luz de las velas. Marcadores de Plantas DIY:

Corta macetas de plástico más grandes en tiras y úsalas como marcadores de plantas DIY. Etiqueta cada tira con el nombre de la planta e insértala en el suelo. Esta solución simple pero efectiva te ayuda a realizar un seguimiento de la diversa vida vegetal de tu jardín. Proyectos de Manualidades para Niños:

Las macetas de plástico son un recurso fantástico para proyectos de manualidades infantiles. Desde crear mini jardines hasta construir esculturas imaginativas,maceta 30 litros la naturaleza resistente y liviana de estas macetas las hace ideales para emprendimientos creativos y prácticos. Separadores Organizativos:

Las macetas de plástico más grandes se pueden cortar en secciones y reutilizar como separadores en cajones o estantes. Úsalas para mantener artículos como calcetines, accesorios o suministros de oficina separados y organizados. Conclusión:

Reutilizar las macetas de plástico no solo reduce los residuos, sino que también abre la puerta a un mundo de posibilidades creativas. Ya sea que busques mejorar tu experiencia en jardinería o agregar un toque de innovación a tu hogar, estas ideas muestran la versatilidad de las macetas de plástico más allá de su propósito inicial. Aprovecha tu creatividad y dale a estos modestos contenedores una segunda vida en tu hogar y jardín.

Efficient Indoor Gardening: Discover the Benefits of Hydroponic Grow Systems

Triclocarban and triclosan have been reported to be taken up by several crop species from hydroponic solutions. For example, after exposure to an aqueous solution mixture of triclocarban and triclosan 11 different food crops, cucumber, tomato, cabbage, okra , pepper , potato , beet, onion , broccoli, celery , and asparagus , were capable of taking up both compounds. However, translocation from roots to the aerial tissue was ≤1.9% for triclocarban and ≤ 3.7% for triclosan after 1 month of exposure . Similarly, Wu et al. found triclocarban and triclosan to have a translocation factor < 0.01 in four vegetables cultivated in a hydroponic solution with two initial exposure concentrations . In a greenhouse study, triclocarban and triclosan were taken up in radish, carrot, and soybeans from bio solid-amended soils and, the greatest concentration was observed in the carrot root after 45 d of treatment and decreased thereafter . However, in a three-year field study in which soils were amended with bio solids in accordance with Ontario providence agricultural practices, the concentration of triclosan and triclocarban in the plant tissues was relatively steady and low . Plants have also been shown to metabolize triclosan, forming 33 metabolites in horseradish cell cultures with the majority being phase II conjugates . Further, one transformation product of the triclosan, methyl-triclosan, has been widely detected in environmental samples and is known to have greater toxicity than the parent compound . Parabens are common preservatives used in cosmetics, and among the most commonly detected CECs in TWW and bio-solid. Parabens are of concern due to their endocrine disrupting potential . Parabens have been widely detected in surface waters and sediments . However,raspberry container knowledge of their behavior, uptake, and transformation in terrestrial systems is comparatively limited.

Methyl paraben was unstable in soil after application of bio-solids, with the maximum concentration of 14.1 µg kg-1 reached after 5 h and decreasing to < 1 µg kg-1 after 35 d . In a bio solid amended field, methyl paraben was the lone paraben detected in the bio solids but was not quantifiable in tomatoes, sweet corn, carrot and potatoes . The above studies highlight the potential for CECs to enter the terrestrial environment, accumulate in plant tissues, and undergo transformations in plants. However, the wide variations in plant uptake and translocation rates under different soil and environmental conditions are currently not well understood and warrant further investigation. Further, it must be noted that the majority of currently published studies have focused on many of the same 20 or so CECs and explored their uptake in mostly the same plant species . There are over 1500 pharmaceutical compounds, alone, currently in circulation . Further, many of the current models have been shown to overestimate the concentration of CECs in plant tissues . In addition, no models have been able to take into account plant metabolism when determining the concentration and risk of CECs in terrestrial plants. More research is needed on a wider swath of CECs with different physicochemical properties in a wider range of plants to improve risk assessment. Transformation of CECs in the environment, including through plant metabolism also needs to be further investigated to better understand their fate and risks in the terrestrial environment. Antibiotic exposure in plants has been widely studied due to previously observed phenotypic toxicity. Several studies showed decreases in root length and changes in shoot development of various plants exposed to several different classes of antibiotics including sulfamides, fluoroquinolones, and penicillins . Most of these studies were conducted at antibiotic concentrations greater than those of environmental relevance and/or utilized artificial or hydroponic growth media. For instance, shoot and root growth of pinto beans grown in a nutrient solution spiked with two antibiotics, chlortetracycline and oxytetracycline, significantly decreased in a dose-dependent manner . Enrofloxacin, a fluoroquinolone, induced hormetic and toxic effects on post-germination growth in lettuce, cucumber, radish and barley plants at concentrations ranging from 0.005 to 50 mg L-1 in laboratory conditions . Seed germination has also been studied as a potential biological end-point to assess toxicity to antibiotic exposure . The exposure effects on seed germination vary considerably by plant species and exposure chemical. In filter paper tests, sweet oat , rice and cucumber seeds were negatively impacted when the seeds were exposed to aqueous solutions of increasing concentrations of six antibiotics, i.e., chlortetracycline, tetracycline, tylosin, sulfamethoxazole, sulfamethazine, and trimethoprim .

The EC10 and EC50 for seed germination were, however, significantly different depending on the antibiotic and the plant species. Rice seeds exposed to sulfamethoxazole were the most sensitive with an EC10 of 0.1 mg L-1 but tylosin had an EC10 > 500 mg L.-1 On the other hand, cucumber seeds exposed to sulfamethoxazole had an EC10 > 300 mg L-1 but an EC10 of 0.17 mg L-1 for chlortetracycline . Exposure to antibiotics can also change plant nutrient and chemical compositions. For example, irrigation with water spiked with sulfamethoxazole and trimethoprim increased production in carbohydrate and soluble solid contents in tomatoes as compared to the plants irrigated with untreated water . The mechanisms driving the phytotoxicity of antibiotics have also been explored. Antibiotics can be directly toxic to or indirectly affect plants. Indirect adverse effects can arise from antibiotic exposure that detrimentally affects mycorrhizal fungi, a vital plant-microbe interaction . Direct toxicity can result when antibiotics interfere with plant hormones or chemical synthesis pathways, or damage chloroplasts, etc. For example, sulfamethoxazole was shown to directly disrupt the folate synthesis pathway in plants by blocking the action of dihydropteroate synthase . Tetracyclines was shown to interrupt mitochondrial proteostasis and damage plant chloroplasts . Interactions with plant hormones may also play a role in the observed phenotypic phytotoxicity. Erythromycin and tetracycline can promote the production of abscisic acid in plants . Abscisic acid, a stress hormone, is crucial for plant responses to drought, salinity, heavy metals, among other stressors , but antibiotic-induced production of this hormone can cause premature leaf and fruit detachment and inhibit seed germination. Plants, depending upon species, can also detoxify antibiotics through reactions with phase II metabolic enzymes . However, studies so far have shown significant variations among plant species. For example, the antibiotic chlortetracycline was detoxified by glutathione conjugation via glutathione-Stransferase in maize , but glutathione-S-transferase did not efficiently catalyze the conjugation in pinto beans . These detoxification reactions, likely produce a series of conjugated metabolites that have yet to be characterized. Understanding the extent of such conjugation is crucial for estimating the total antibiotic uptake, accumulation, and translocation of antibiotics in plants as the formation of conjugates may mask the total concentration, even though some of these conjugates may retain biological activity .

Several widely used NSAIDs, such as ibuprofen, acetaminophen, and diclofenac are amongst the most studied pharmaceuticals in the environment. Studies have shown that NSAIDs can induce toxicity to plants . Phytotoxicity, however, is often plant species and NSAID specific. For example, ibuprofen has been shown to inhibited root elongation in Sorghum bicolor at high concentrations, with EC50 of 232.64 mg L-1 . However,plastic plants pots in seed germination tests exposure to a hydroponic solution containing 1 mg L-1 ibuprofen, along with other fenamic acid class NSAIDs, increased the length of the primary root in lettuce but had no effect on radish . In the same study, diclofenac was observed to decrease the root-to-shoot ratio in radish seedlings cultivated in a sand/spiked-nutrient solution , but did not significantly affect the seed germination. However, protein content was not affected in maize cultivated in soils irrigated twice with different concentrations of acetaminophen but grain yields and seed germination were negatively impacted in a dose dependent-manner . Plants can metabolize and detoxify NSAIDs. For example, plants were found to detoxify acetaminophen by conjugation with glutathione followed by conversion to cysteine and acetylcysteine conjugates . Similarly, diclofenac was found to be converted to glucose conjugates in barley and horseradish and glutamic acid conjugates in Arabidopsis thaliana . Arabidopsis thaliana cell cultures can detoxify ibuprofen via conjugation with sugars and amino acids .As mentioned above, pharmaceuticals used to treat psychiatric disorders are another group of frequently detected pharmaceuticals in environmental samples, particularly the anticonvulsant carbamazepine . Carbamazepine exposure has been seen to exhibit mycotoxicity to carrot mycorrhizal endpoints by decreasing the production of fungal spores . Similarly, carbamazepine induced leaf necrosis, altered plant hormones and macronutrient concentrations, and reduced root growth at plant tissue concentrations of 1 to 4 mg kg-1 in zucchini cultivated in soil spiked with chemical at 0.1 – 20 mg kg-1 . Information on the toxicity of benzodiazepines and fluoxetine in terrestrial plants is still limited; however, toxicity has been reported in aquatic plantsfor these compounds, indicating that toxicity may also occur after exposure in terrestrial plants .Antimicrobials and preservatives are often added to personal care products to increase shelf life. They pass from the human body, largely unchanged, and ultimately end up in TWW, bio solids, and sewage sludge. . Antimicrobials and preservatives have been detected in agricultural soils after irrigation with TWW and/or the application of bio solids, and can be taken up by plants . Two antimicrobials, triclosan and triclocarban, have attracted more attention because of their potential for endocrine disruption and phytotoxicity . For example, triclosan significantly inhibited plant growth in cucumber and rice seedlings with EC50 of 108 mg kg-1 and 57 mg kg-1 , respectively . Lettuce shoot mass also decreased in a dose-dependent manner after cultivation in soil amended with triclocarban-spiked bio solids . On the other hand, growth of radish, carrot, soybean, spring wheat, and corn plants grown in soils amended with bio solids containing environmentally relevant concentrations of triclosan and triclocarban, improved compared to un-amended soils; likely due to the positive impacts of bio solids addition . Thus, plant species, concentrations, and growth media can significantly affect phytotoxicity of these CECs. Studies exploring the phytotoxicity of individual pharmaceuticals or classes of pharmaceuticals are useful to highlight high-risk compounds and/or the potential mechanism of toxicity.

CECs are, however, often introduced into the environment in complex mixtures and these mixtures can affect the uptake and translocation of individual compounds . Some studies report positive effects on plants exposed to CEC mixtures under environmentally relevant conditions. For instance, TWW irrigation increased tomato and lettuce yield compared to freshwater irrigation . Exposure of lettuce seedlings to a mixture of 11 CECs significantly altered plant metabolic pathways, including the citric acid cycle and pentose phosphate pathway, and decreased chlorophyll content in a dose-dependent manner . Also, exposure to 18 CECs at concentrations ranging from 5 to 50 µg L,-1 induced oxidative stress in cucumber seedlings and caused up regulation of enzymes associated with detoxification reactions . Literature on the toxicity of a number significant CECs to terrestrial plants is still very limited, and many of the studies have utilized concentrations that are orders of magnitude higher than those seen in the environment. Studies on the toxicity of mixtures in terrestrial plants are also limited, but warrant attention as several studies have indicated that mixtures can induce effects not observed from individual compounds . The ability of plants to detoxify these compounds through metabolism also merit further research. Overall, more research is needed on the toxicity of a wider range of CECs in plants under environmentally relevant conditions to more accurately assess the impacts of CECs in the agro-environment. The potential for exposure to, and toxicity of, CECs has been investigated in several aquatic invertebrate species. Toxicity end-points such as endocrine disruption, changes in growth, time to development, and mortality rates have been considered in these studies . Studies addressing the effects of CECs on terrestrial invertebrates are, however, few. Of the published studies on terrestrial invertebrates, the earthworm Eisenia fetida has been examined mainly due to their increased susceptibility and ecological importance . Literature pertaining to toxicities of various classes of CECs to terrestrial invertebrates is discussed below. Like in terrestrial plants, antibiotics can also induce toxicity in terrestrial invertebrates. Exposure to environmentally relevant concentrations of antibiotics caused mortality to earthworms and/or induced oxidative stress and genotoxicity in E. fetida. For instance, high concentrations of tetracycline and chlortetracycline inhibited antioxidant enzymes superoxide dismutase and catalase while these enzymes were stimulated at lower doses , and DNA damage was induced along a dose-dependent curve . Also, chlortetracycline can reduced juvenile earthworm and cocoon counts in E. fetida .

Hydroponics 101: A Comprehensive Guide to Soilless Agriculture

The observations indicated that the increased root biomass phenotype in the activation lines was due to the elevated OsZHD2 expression levels. In addition to the root phenotype, the OX plants and the T-DNA activation line influenced leaf development, so that abaxially curled leaves were observed . The OsZHD2-OX plants exhibited markedly increased root development at 14 DAG . Fresh and dry weights of roots were higher for the transgenic lines than for the out segregated WT . To examine whether the increase in biomass improved nutrient uptake, we analyzed the rate at which N was absorbed from a liquid growth medium containing KNO3. Based on the amount of residual N in the medium, the N concentration reduced rapidly and at a higher rate in OX plants than in the WT plants . The results suggested that the former had a higher N uptake capacity via the roots, which would also imply that the OX plants had a higher capacity to tolerate low-N conditions. To test the hypothesis, we grew the plants under low-N conditions in a growth chamber . In mature plants at the booting stage, the N concentration was 1.5- fold higher in the flag leaves of OsZHD2-OX compared with the flag leaves of the WT . The Pi accumulation rate was also 1.5-fold higher in the flag leaves of OsZHD2-OX than in the WT plants . Seed fertility was markedly higher in the over expression plants. Although <30% of the WT seeds were fertile, >50% of the grains from the OX plants were fertile . The results indicated that the uptake of nutrients increased in OsZHD2-overexpressing plants.

Plants were grown in a paddy field under normal N supply. There were no obvious phenotypic differences between the over expression plants and the WT up to maturity. Their architectures were almost identical,growing blueberries including plant height, panicle length, total spikelet number, and fertile seed number . However, the 100-grain weight was higher in the OsZHD2 OX lines . The increase in seed weight was potentially due to increased N uptake.Lateral roots began to emerge from both the WT and the activation lines at 3 DAG. By 4 DAG, the WT laterals were ~0.5 cm long, while those of the activation line were slightly longer . The difference in lengths became more pronounced as the plants grew . We performed transcriptome analyses using mRNA prepared from the total root samples of WT and OsZHD2-D plants at 4 DAG and at 6 DAG . At 4 DAG, 68 genes were up-regulated and 384 genes were down-regulated at least 2-fold in OsZHD2-D . At 6 DAG, 513 genes were up-regulated and 524 were down-regulated at least 2-fold in OsZHD2-D plants . At both stages, 22 transcripts were commonly up-regulated while 54 transcripts were down-regulated at least 2-fold . To verify the RNA sequencing data, we selected four genes that were up-regulated at both stages, in addition to CYCD4;1 and ERF3, which increased only at 6 DAG, and ABCC7 and PUB64, which were down-regulated at both stages . qRT-PCR analyses revealed that their expression patterns were similar to the patterns observed in the results of our RNA sequencing analyses . The findings suggested that the sequence data were reliable.The 22 genes that were up-regulated at both 4 and 6 DAG included two associated with ethylene biosynthesis, S-adenosylmethionine synthetase 2 and ACC oxidase 2 , which suggested that ethylene influenced the root phenotypes .

Ethylene biosynthesis begins with the conversion of methionine to S-adenosylmethione by S-adenosylmethione synthetase, with ATP as a co-substrate . In the following step, ACC is formed from S-adenosylmethione by ACC synthase . The final step is the synthesis of ethylene from ACC by ACC oxidase . Our qRT-PCR assay confirmed that the expression of OsSAM2 and OsACO2 indeed increased in OsZHD2-D lateral roots at both stages . Genes encoding ACS were not placed on the list of induced genes because the differences in transcript levels between WT plants and transgenic plants were <2-fold. However, qRT-PCR analyses revealed that ACS5 transcript levels increased in OsZHD2-D at both stages . Ethylene production measurements from 8 DAG plants showed that OsZHD2-D samples accumulated more ethylene in their roots , shoots , and the whole plant when compared with the WT plants. To examine whether OsZHD2 binds directly to ethylene biosynthesis genes, we performed ChIP assays using transgenic plants over expressing OsZHD2-Myc. Promoter regions P3, P4, and P5 of ACS5 chromatin were enriched by Myc antibodies .To investigate whether the accumulation of ethylene was the major factor responsible for the OsZHD2-D seedling root phenotypes, we investigated the effects of an ethylene biosynthesis inhibitor AVG which reduces ethylene production by blocking ACS activity . The addition of 3 µM AVG reduced lateral root growth in WT plants and rescued the enhanced lateral root growth phenotypes of OsZHD2-D . However, low concentrations of AVG did not affect the lateral growth of OsZHD2-D as well as that of the WT . To examine whether the restoration was due to decreased meristem activity, we performed EdU labeling. The results of the experiment demonstrated that the application of AVG reduced the root meristem activity of the WT and OsZHD2-D significantly .

The results suggest that OsZHD2 enhances meristem activity in the apical region of roots by inducing ethylene accumulation.To determine whether exogenous ethylene treatment promotes root development, 3 DAG seedlings were transferred to MS medium containing various concentrations of ACC. Lateral root length increased significantly when plants were supplied with 10 nM ACC . Previously reported results have suggested that ethylene induces auxin biosynthesis by stimulating the expression of Rice Anthranilate Synthase Alpha-subunit, which encodes an enzyme producing anthranilate, a precursor of Trp . To investigate whether ethylene increases auxin concentrations in rice, we generated transgenic DR5::GUS plants expressing the GUS gene under the synthetic auxin-responsive promoter and their lateral roots exhibited weak GUS expression at the tips and in the basal regions . When plants were exposed to 10 nM ACC, GUS activity was higher in the treated roots than in the control plants grown in the absence of ACC . Staining was also observed in the area between the tips and basal regions where GUS activity had not been observed prior to treatment with ACC. Consistent with the GUS assay results, the GUS transcript levels increased in ACC-treated roots . We also used a DR5::VENUS plant that expressed the yellow fluorescent protein under the influence of the DR5 promoter . The treatment of the plants with ACC increased VENUS signal in the tips and the central stele of the lateral roots . The results of such experiments suggest that ethylene induced auxin biosynthesis in the RAM. The results of qRT-PCR analyses revealed that 10 nM ACC induced the expression of OASA2 as well as auxin biosynthesis genes, TAR2 and YUCCA7, with peaks observed 6 h after treatment . According to the observations, a low concentration of ethylene could induce auxin biosynthesis in rice lateral roots. In Arabidopsis,square plant pot ethylene enhances auxin biosynthesis by increasing the expression of WEI2/ASA1 and WEI7/ASB1, two genes encoding AS sub-units . In rice, OASA1 and OASA2 encode the AS α-subunit . According to the RNA-Seq assay results, OASA2 expression was higher in OsZHD2-D roots , which was validated using qRT-PCR analyses . The major IAA biosynthesis route is the IPyA pathway, which is mediated by TAA/TARs and YUCCA in Arabidopsis . Our RT-PCR results showed that TAR2 was induced in OsZHD2-D roots . We also observed that YUCCA7 expression was higher in the activation line . An analysis of the DR5::GUS plants showed that the expression levels of the GUS reporter were significantly higher in OsZHD2-D roots . All the findings above suggested that OsZHD2 induced IAA biosynthesis. Notably, strong staining was observed in the proximal area of the root tips of OsZHD2-D, which also indicated that OsZHD2 promoted auxin accumulation in the growing region.

To investigate whether AVG treatment affects DR5::GUS expression in OsZHD2-D, we applied 3 µM AVG to the DR5::GUS plants in the WT and OsZHD2-D background. Visualization of GUS expression showed that the reporter expression was decreased by AVG in both plants . This observation supports that the increased auxin biosynthesis in OsZHD2-D was due to elevated ethylene levels. To examine whether OsZHD2 binds directly to auxin biosynthesis genes, we performed ChIP assays using transgenic plants over expressing OsZHD2-Myc. However, we were unable to observe any significant binding of OsZHD2 to the promoter regions of TAR2 and YUCCA7 . To confirm OsZHD2-D phenotypes, we analyzed expression patterns of ethylene and auxin biosynthesis genes in OsZHD2-overexpressing plants. The results of qRT-PCR analyses revealed that expression levels of ethylene and auxin biosynthesis genes are increased in OsZHD2-overexpressing plants .To further study the functional role of OsZHD2, we generated oszhd2 null mutants using the CRISPR/Cas9 [clustered regularly inter spaced short palindromic repeats / CRISPR-associated protein 9] system . Analyses of two independently obtained bi-allelic oszhd2 mutants revealed that the lengths of their seminal roots and lateral roots did not vary considerably from those of the WT and heterozygous plants. The lack of obvious phenotypic changes was potentially due to genetic redundancy. OsZHD2 encodes ZF-HDs, a protein group that includes 11 members in rice . Among them, OsZHD2 is the most homologous to OsZHD1, with 80% identity and 84% similarity at the amino acid sequence level. Plants that over express OsZHD1 exhibit an abaxially curled and drooping leaf phenotype similar to that observed in OsZHD2-OX plants. We isolated a T-DNA tagging line in which T-DNA was inserted 136 bp upstream of the start ATG codon . The expression of OsZHD1 was reduced significantly in the tagging line . For the mutant, no obvious alteration was observed in the phenotype .Since oszhd1 and oszhd2 single mutants exhibited normal root growth, we generated oszhd1 oszhd2 double mutants using the CRISPR/Cas9 system to target the conserved sequence . In the double mutants, lateral root development diminished significantly , indicating that OsZHD1 and OsZHD2 redundantly play roles in the regulation of such development. The transcript levels of SAM2, ACS5, ACO2, OASA2, TAR2, and YUCCA7 also decreased in the oszhd1 oszhd2 double mutants , supporting our hypothesis that the OsZHD genes are involved in the control of the biosynthesis of ethylene and auxin. To observe whether exogenous ethylene treatment would stimulate lateral root development in oszhd1 oszhd2 double mutants, seedlings were grown on N6 medium with or without 1 µM ACC . In the ACC-treated plants, the lengths of the lateral roots of oszhd1 oszhd2 double mutants increased more than the lengths of the lateral roots of the WT plants . These results indicate that the changes in the root architecture observed in oszhd1 oszhd2 double mutants are at least in part due to the defective ethylene biosynthesis.The over expression of OsZHD2 increased ethylene levels and enhanced the expression of genes linked to its biosynthesis. The OsZHD2 transcript is preferentially present in the meristem regions where ACS5 is expressed . Therefore, the primary role of OsZHD2 in root development appears to be the induction of ethylene production by inducing ACS5 expression. Although ethylene generally functions as a growth inhibitor, it occasionally promotes growth, particularly in semi-aquatic plants . Leaf, stem, and root development can be positively regulated by ethylene at relatively low concentrations . In addition, ethylene induces lateral root initiation near the growing root tip and promotes the emergence of lateral root primordia . The overproduction of ethylene through the application of exogenous ACC inhibits lateral root initiation but induces outgrowth of already existing primordia . These observations reported in previous studies support our hypothesis that OsZHD2 enhances root growth by increasing ethylene production in the root tips.Using plants expressing the GUS or VENUS markers under the influence of the DR5 promoter, we showed that a low concentration of ACC induced auxin accumulation in the growing region of lateral roots. We also demonstrated that ethylene increases the expression of auxin biosynthesis genes, including OASA2, TAR2, and YUCCA7 . Expression of the marker genes was promoted strongly in the region near the root tips of OsZHD2-D plants . The above expression trend was similar to that for ACC-induced GUS activity .

Cultivating the Future: The Rise and Benefits of Hydroponic Farming

Few studies have investigated the influence of environmental conditions on ENM uptake and toxicity, particularly throughout the entire plant life cycle. Here, soil-grown plants were exposed until maturity to TiO2, CeO2, or Cu2 ENMs under different illumination intensities, in different soils, and with different nutrient levels. Fluorescence and gas exchange measurements were recorded throughout growth and tissue samples from mature plants were analyzed for metal content. ENM uptake was observed in all plant species, but was seen to vary significantly with ENM type, light intensity, nutrient levels, and soil type. Light intensity in particular was found to be important in controlling uptake, likely as a result of plants increasing or decreasing transpiration in response to light. Significant impacts on plant transpiration, photosynthetic rate, CO2 assimilation efficiency, water use efficiency, and other parameters related to physiological fitness were seen. The impacts were highly dependent on environmental conditions as well as ENM and soil type. Notably, many of these effects were found to be mitigated in soils with limited ENM mobility due to decreased uptake. These results show that abiotic conditions play an important role in mediating the uptake and physiological impacts of ENMs in terrestrial plants.Nanotechnology has the potential to enhance or revolutionize many fields of study, including medicine, transportation, energy storage, personal care, construction, environmental remediation, military applications, manufacturing, and scientific research. Reflecting this broad applicability, nanotechnology has become a multi-billion dollar industry in spite of being in its infancy, and is expected to reach a global market value of over half a trillion U.S. dollars by the end of the decade.With this in mind, and with nanomaterials currently used in nearly 2000 consumer products and many industrial applications,plastic planting pots concerns have naturally arisen about the health and environmental impacts of the manufacture, use, and disposal of this new and extremely varied class of materials.

A nanomaterial is defined as a material with at least one dimension in the size range of approximately 1 to 100 nm.3 Here, the term “engineered nanomaterial”is used to differentiate intentionally designed and manufactured nanomaterials from those produced incidentally by natural or anthropogenic processes. The extreme size of ENMs, and the high surface area to volume ratio that comes along with it, typically results in unique properties not found in larger scale or dissolved materials of the same composition. For example, quantum dots, nanoscale particles composed of semiconducting materials , can utilize quantum band gap phenomena to fluoresce in a narrow range of wavelengths, which are highly dependent on their diameter.4 Additionally, ENMs can be extremely reactive due to their high surface area relative to their volume . These novel properties are simultaneously the source of global interest in ENMs and the main issue of concern in terms of the impacts to human health and environmental safety, as regulations for a bulk or dissolved material may not be appropriate for ENMs of the same composition. Additionally, since ENMs can have radically different behavior depending on their composition, size, shape, doping agents, coatings, and/or the characteristics of the media they are present in, a predictive framework for the fate, transport, and toxicity of ENMs in a variety of environments and organisms is needed to effectively regulate ENMs throughout their life cycles. The majority of the current production, use, and disposal of engineered nanomaterials occur in terrestrial environments, and consequently terrestrial ecosystems are and will increasingly be some of the largest receptors of ENMs at all stages of their life cycles.

In particular, soil is predicted to be one of the major receptors of ENMs due to ENMcontaminated biosolid fertilizer and nanopesticide application to agricultural fields, runoff from landfills or ENM-bearing paints, or atmospheric deposition.Both agricultural and natural systems are at risk to ENM contamination via these release scenarios, which makes it necessary to understand the interactions between ENMs, soils, and soil organisms such as plants in order to predict their impacts in real-world scenarios.The goal of the research presented in this thesis was to uncover some of the underlying mechanisms controlling the following processes under environmentally relevant conditions: how ENMs move through unsaturated soils, the effects ENMs have on key soil properties, the uptake and distribution of ENMs in plants, and how ENMs influence plant growth and physiology. These topics were addressed using methods approximating real-world scenarios as closely as possible while maintaining reproducibility and analytical power. The holistic approach utilized here differs fundamentally from that of many studies currently published on these subjects, which use reductionist experimental design to attempt to break down the complex ENM-soil-plant system into simplified components. Reductionist methods can be powerful in providing detailed information about well-understood systems, but when addressing systems as complex and poorly-understood as these designing experiments to closely mimic real-world scenarios can give insight into key controlling mechanisms that can then be targeted for further study. An example of this can be found in Chapter 2, which shows that the main mechanism impeding ENM transport through unsaturated natural soils is physical straining of large ENM aggregates formed via interaction with ions in the soil solution, not through electrostatic attraction or repulsion as was predicted by several studies using well-dispersed ENMs in typical saturated columns of washed quartz sand.As mentioned, Chapter 2 discusses tracking the movement and characteristics of three metal oxide ENMs through three soils, with ENMs being either coated with natural organic matter or uncoated.

In contrast to studies such as those cited above, which use active pumping to push ENMs through water-saturated media, ENM transport in this study was in unsaturated soils and was driven solely by gravity. This was done in order to more closely simulate conditions likely to occur in the real world, as some of the major predicted exposure scenarios involve ENMs entering from the top layers of soil, which are typically unsaturated. Chapter 3 looks at the effects ENM contamination has on several soil properties, which is a subject that is poorly represented in the literature. Metal oxide ENMs like those used in this study have characteristics that make them likely to influence soil properties in some way, such as being similar in composition to naturally-occurring clay minerals that are important in controlling nutrient retention, soil porosity, and organic content.Additionally, they are amphoteric, that is, capable of producing both H+ and OHions depending on their crystal structure and the composition of the media they are in and thus potentially altering soil pH. Soil pH and nutrient availability are both critically important to plants and other soil organisms and were therefore targeted in this chapter. Chapters 4 and 5 explore two aspects of the same system: how plants grown to maturity in ENM-contaminated soils uptake and distribute ENMs throughout their tissues,plastic grow pots and how their growth and physiological processes are affected by the presence of ENMs. Keeping with the theme of designing experiments to predict ENM behavior in real-world scenarios, aspects of the environmental conditions the plants were grown under, specifically illumination intensity and soil nutrient levels, were varied in order to mimic some of the range of conditions plants growing under real conditions would experience.

This was done in a series of three experiments. First, the model plant Clarkia unguiculata was grown to maturity under two illumination intensities in a potting soil with and without receiving additional fertilizer in order to determine the effects of nutrient and light stress. Second, C. unguiculata was again grown to maturity under two illumination intensities, but this time in two natural soils, a grassland soil and an agricultural soil. This was done to see how these plants respond to ENM exposure in soils with different properties beyond nutrient levels. Finally, two crop plants, wheat and radishes, were grown to maturity under two illumination intensities in the grassland and agricultural soils, respectively. This was done in order to see the effects of ENM exposure on plants from different taxonomic groups that are also economically important. By varying one condition throughout this set of experiments, information can be passed from one to the next that could provide additional insight into the key factors at play. ENM mobility in the subsurface is governed by several processes of varying influence, including dissolved ion and pH-induced aggregation, coating by organic and inorganic molecules, sorption to organisms and other media components, and physical straining through soil pore spaces. In particular, chemical and electrostatic interactions with soil clay particles have been implicated as key factors in the subsurface movement of raw or coated ENMs. This has been demonstrated for TiO2 1 and uncoated, citrate-coated, and phosphatecoated CeO2 ENMs2 in soil and implied as the method of retention in other studies.Sorption can occur via electrostatic attraction between charged clay surfaces and oppositely charged ENMs5 or chemically through a dehydration reaction similar to the binding of phosphate or iron oxides to clays. Sorption to organic matter and organisms in soil may also take place through similar mechanisms. The specific organic compounds present in subsurface waters will also differ over geographic area with soil and vegetation type due to the presence of plant root exudates and bacterial communities, which will result in different coatings being available to ENMs in different areas. There is also the possibility of physical straining and collection at airwater-soil interfaces when flowing through porous media like soil. Physical straining of high aspect ratio ENMs in soil has been demonstrated with single-walled carbon nanotubes and implicated as a primary retention mechanism for nanoscale Fe0 in a sandy loam soil.

As aggregation caused by high ionic strength, pHs near the PZC, or coatings increases, physical straining becomes more likely, particularly in soils like Vertisols or Ultisols that are characterized by small pore sizes. Two hypotheses were addressed in these series of experiments. The first hypothesis was that ENM transport would be limited to the upper layers of soil, but particles coated with NOM would penetrate further into the soil due to increased electrostatic repulsive forces as a result of their more negative surface charge.The second hypothesis was that particles would be transported further through potting soil than agricultural or grassland soils due to the greater density and clay contents of the two natural soils causing increased physical straining and electrostatic/chemical sorption. Stock suspensions of CeO2, Cu2, and TiO2 ENMs were prepared by suspending dry ENM powders in 18.2 MΩ cm Nanopure water and sonicating for 30 min in a bath sonicator . Stock suspensions were sonicated for 10 min after dilution to the desired concentration and used within 24 hr. Suwannee River NOM stock solutions were prepared as described in Zhou and Keller 18 . Hydrodynamic diameter and ζ-potential of TiO2, CeO2, and Cu2 ENMs with and without NOM were measured via dynamic light scattering at 20oC by preparing 10 mg L-1 ENM suspensions with and without the addition of 1 mg L-1 NOM in Nanopure water and in soil solution extracts through dilution of a 100 mg L-1 stock, probe sonicating for 2 sec at 20% amplitude with a Misonix Sonicator S- 4000 .ENM transport through the three soils was tested by loosely packing 2.5 cm diameter x 16.34 cm long cylindrical plastic columns with air-dried soil. Due to their different densities, 17.5 ± 0.1 g potting soil, 136 ± 1 g grass soil, or 167 ± 1 g farm soil were needed to completely fill the columns. To simulate gravity-driven transport of ENMs in suspension, 50 mL of 100 mg L-1 TiO2, CeO2, or Cu2 ENM suspensions with or without the addition of 10 mg L-1 NOM were slowly applied to the top of the column. The resulting soil ENM concentrations were on the high end of those currently predicted for metal oxides in soil,but were well within the concentrations predicted for biosolids.Hence, the soil ENM concentrations used in this experiment may be indicative of those found in soils repeatedly amended with biosolids. After ENM application, columns were allowed to drain overnight, oven dried at 60°C for 72 hours, and split into 3 cm segments, ~0.3 g subsamples of which were weighed, digested, in 10 mL 1:3 HNO3:HCl at 200°C for 1.5 hours in a microwave digestion system followed by analysis via inductively coupled plasma atomic emission spectroscopy . This technique was sufficient to dissolve the soil and ≥90% of TiO2, CeO2, and Cu2. Detection limits for all elements tested were approximately 5 μg L-1 . Standard solutions and blanks were measured every 15-20 samples for quality assurance.

Rooted in Water: The Science and Art of Hydroponic Farming

Parathion also contains a thioester linkage that is analogous to the chemical structure of several chemical warfare agents, including sarin. Consequently, parathion is an excellent model for studying enhanced biodegradation of environmental contaminants. Mineralization of parathion requires three unique catabolic properties: hydrolysis of parathion, mineralization of p-nitrophenol, and mineralization of diethyl thiophosphate . The objective of this research was to develop a coculture capable of hydrolyzing parathion and degrading its metabolite p-nitrophenol; evaluate the kinetics of the reaction; and test the suitability of the coculture for use in a flow-through biofilm reactor for parathion biodegradation.Escherichia coli strain SD2 was constructed by introducing plasmid pWM513, harboring the genes for parathion hydrolysis and ampicillin resistance, and plasmid pMAG1, carrying the green fluorescent protein gene and tetracycline resistance, into Escherichia coli DH10B. The plasmids were inserted by electroporation and strain SD2 was selected using media containing both ampicillin and tetracycline. Strain SD2 was used together with Pseudomonas putida KT2440 carrying plasmid pPNP, harboring the genes for pnitrophenol degradation and also tetracycline resistance. Strain KT2440 is naturally resistant to ampicillin, so the two strains could be cultured in media containing both antibiotics, and consequently maintained the genes required to degrade parathion. The ability of the coculture to degrade 0.5 mM parathion was evaluated during growth of the strains in a minimal medium containing glucose as the carbon source. P-nitrophenol accumulated in the medium during the growth of strain SD2 alone,drainage gutter resulting from parathion hydrolysis .

In contrast, the accumulation of p-nitrophenol was only transient in the coculture, as a result of the biodegradation activity of strain KT2440. Kinetic analysis indicated that 2 mM pnitrophenol was fully inhibitory to the growth of the coculture; consequently, this concentration represented the upper limit for parathion biodegradation . The coculture was used to cultivate a biofilm in a parallel plate flow cell for imaging by confocal microscopy. After 72 hours of growth in continuous mode, the biofilm was stained with a red fluorescent nucleic acid dye and imaged using a confocal microscope. The dye caused the Pseudomonas strain to appear red, while the Escherichia coli strain appeared green and yellow as a result of the colocalization of green color fluorescing from its green fluorescent protein. The images indicated that the biofilm was dominated by Pseudomonas, although E. coli was stably maintained. The results suggest that the two strains could be used as part of a flow-through biofilm reactor for detoxification of parathion. Several industries in the United States and Puerto Rico are dedicated to the manufacture of filters of different materials, including nitrocellulose . These filters are very important in ensuring the purity of ingredients in food and pharmaceutical industries. However, the nitrocellulose used for manufacturing these filters is considered a hazardous waste for its ignitability , high flammability and oxidizing properties. The Department of Defense also has sites contaminated with NC since it is a major component in explosives. Nitrocellulose exhibits good chemical stability, which for years made incineration the preferred method for disposing of the NC wastes. Alkaline hydrolysis appears to be a rapid process for nitrocellulose degradation, but research is limited. This investigation involves the elucidation of the mechanism of alkaline hydrolysis and its combination with biodegradation.Table 1 shows the research methods and tools employed in this study. Every step in Table 1 seems to play an important role for either hydrolysis or biodegradation itself. Interpretation of the species left after degradation time facilitates analysis. Comparison between liquid and agar media is important to really determine if physical attachment is another factor for better fungus growth. Figure 1 shows a possible precipitation of products after acidification on the 9 mL NaOH treatment.

Figure 2 shows fungus growth from 10 mcL of spores suspension placed on a petri dish containing NC as sole carbon source. HPLC assays have been made on a growth curve in a culture tube . A total of 0.1 g of NC hydrolyzed in 6 mL of SSC buffer was used. The strongest peaks are shown. The sample represents one-third of the total area. A total of 20 mcL were injected in a 50:50 acetonitrile:water mixture. Assays are being done using wavelengths of 210 nm and 214 nm, due to the abundance of probably dicarboxilic acids in the hydrolyzed NC.Polycyclic aromatic hydrocarbons are a concern in the environment because they are toxic and carcinogenic. Polycyclic aromatics are more recalcitrant in the soil than other hydrocarbons because they are hydrophobic and tend to migrate into the soil . Most PAHs occur as a result of fossil fuel combustion; thus, high concentrations of PAHs are found at the sites of active and inactive oil refineries . This study focuses on oil and petroleum contaminated soil samples taken from Alameda Naval Air Station at Alameda Point, Calif. Pacific Coast Oil Works refinery used the site between 1879 and 1903. After the refinery closed in the 1930s, the U.S. Army and then the U.S. Navy owned the property. In 1991, jet fuel spilled from the jet engine test facility on the site. Heavy rains resulted in jet fuel in the overflow of storm drains. Damage to the storm drains during the Loma Prieta earthquake in 1989 may have caused ground water contamination. Recent studies showed total petroleum hydrocarbon amounts ranging from 100 to 10,000 mg/kg soil. Due to the long term and high concentration of oil in the soil it is likely that there are microorganisms capable of degrading the oil products. This study will examine the use of Inipol EAP 22, the same fertilizer used in the Exxon Valdez oil spill clean-up, to obtain optimal growth conditions of the naturally occurring bacteria using CO2 output to monitor the degradation of PAHs and hydrocarbons. Inipol EAP 22 is particularly attractive for this site since it is oleophilic and should make the strongly sorbed PAH components more bioavailable, thereby stimulating biodegradation.More than 8,000 Department of Defense sites need clean-up efforts .

TNT was the most commonly occurring compound within the contaminated samples from these sites. Fortunately, TNT is biodegradable, making in situ bioremediation a costeffective and rapid alternative for site cleanup. The toxicity of nitroarenes and their metabolites have been studied in a variety of biological systems but we have been unable to find any studies related to chemotaxis of nitroaromatic compounds in the scientific literature. Chemotaxis allows bacteria to respond to chemical gradients, seeking higher levels of potential nutrients and lower levels of inhibitors. Organisms that have developed mechanisms with which to beneficially orient themselves with respect to these gradients of different types may have a competitive advantage over other organisms. Also, the value of motility as a survival factor for bacteria in environments where nutrients or harmful agents are discontinuously distributed, e.g., contaminated sites, seems obvious. The present study will target how bacteria can use nitroaromatic compounds as a source of nutrients and as a dispersal mechanism in soil,large square pots especially as it may apply to in situ bioremediation.Chemotaxis provides a means for bacteria to respond to environmental gradients of potential nutrients and toxins, resulting in direct motility towards or away from these substances . The P. fluorecens bacterial strain used responded impressively according to our predictions . This is the first time that bacteria have been demonstrated to be attracted to explosive compounds. The strong attraction of soil bacteria to DNT was also verified using soil perfusion column leachate from field samples taken at Ft. Ord, Calif. In these leachate tests, the chemotactic indices observed for DNT are some of the highest rates ever observed for any type of chemotaxis, including the ones observed for P. fluorecens for concentrations of 2,4 and 2,6-dinitrotoluene that ranged from 0.12 M to 1.20×10-3 M. The results of this work have important implications for the ecology of TNT-degrading bacteria; it also may suggest ways that microenvironments containing explosives might be controlled to increase biodegradation rates in situ.More than 8,000 Department of Defense sites need clean-up efforts . In a compilation of soil samples collected from 44 Army ammunition plants, arsenals and depots, 28% contained detectable levels of explosives. TNT was the most commonly occurring compound within the contaminated samples, and was seen in 66% of those samples. Facilities that may be contaminated with explosives include manufacturing plants, ordnance works, Army ammunition plants, ammunition depots, Army and Naval proving grounds, burning grounds, artillery impact ranges, explosives disposal sites, bombing ranges, firing ranges, and ordnance test and evaluation facilities.

Due to its toxicity and recalcitrance, the U.S. Environmental Protection Agency has listed TNT as a priority pollutant . Bio-remediation of organic contaminated soils has proven to be one of the fastest and cheapest remediation technologies available. TNT and its daughter products are highly recalcitrant, especially in highly weathered soils, i.e., soils that have been exposed to the environment for a number of years under suboptimal microbial activity conditions. The present study examines weathered and unweathered soils and looks at the ability of a number of biostimulants to increase total microbial respiration.Two-gallon buckets were converted to soil bioreactors . These reactors use the same CO2 trapping principle as the biometer flasks, but allow for a much greater soil sample. The KOH trap was a funnel plugged at the bottom with a rubber stopper, which was held in place with epoxy. These traps rested on top of the soil, and could hold up to 80 ml of solution. Larger ascarite-top filters were made using 50 ml Corning vials. A hole was drilled into the bottom of each vial, through which a piece of plastic tubing would fit. This tube was connected to a stopcock, which let the filtered air into the bucket during the times when the KOH was changed. The attached diagram shows the biometer bucket design. The soil in each bucket was mixed well prior to the respiration measurements. After the bucket was sealed, the KOH was added to its trap, and the buckets sat for four days. In this four-day period, the KOH was changed and titrated as necessary, with intervals from an hour to a day, depending on the rate of respiration in the soil samples. These intervals change because a faster respiration rate can create enough CO2 to saturate the KOH solution. Therefore, the KOH monitoring a more active soil sample must be titrated much more often than in a slowly respiring sample. For each bucket, three experiments were run: an unamended control, where the soil was taken directly from the buckets in which it was collected, and lasting 3.57 days, an inorganic nutrient amendment, where 1000 ml of MSM was added to each bucket, and lasting 2.93 days, and an organic nutrient amendment, where an aqueous molasses solution was added to each bucket, and lasting 4.80 days. Respiration was measured daily and samples for HPLC analyses of contaminants were taken initially and at the end of the treatment.The respiration analysis indicates that unweathered soil responds faster and in greater magnitudes to nutrient amendments . The results suggest that the weathered soils may have stressed populations and cannot react as quickly to the amendments, especially because they actually reduce their respiration rate in response to MSM addition. The molasses-amended samples all respired at a greater rate than with the other amendments. This result implies that molasses may be used as an effective nutrient source to increase bacterial activity. Three of the four molasses-amended samples are among the lowest four in concentrations of the suspected contaminant located by the HPLC. The HPLC results also suggest a correlation between bacterial activity and biodegradation. Glycerol trinitrate , also known as nitroglycerin, is extensively utilized for the production of explosives and pharmaceuticals. GTN is a hazardous waste product of increasing environmental concern. Current disposal techniques, such as open-air burning and incineration, are expensive and can produce hazardous waste byproducts. Bioremediation systems could remediate explosive contaminants at approximately a tenfold lower treatment cost and with increased public acceptance. An ecological investigation of GTN-contaminated sites at the Naval Surface Warfare Station in Maryland resulted in the isolation of a Bacillus thuringiensis/cereus strain able to degrade GTN. HPLC and TLC analysis by other researchers of GTN metabolism in cell-free systems suggested that there was a sequential denitration to dinitrate isomers, mononitrate isomers and ultimately to glycerol.

From Water to Harvest: Exploring the Wonders of Hydroponic Agriculture

ABA is therefore necessary for the stomatal closure we observe in esb1-1. Te elevated ABA concentration we observe in leaves of esb1-1 compared to wild-type supports this conclusion. We also used the esb1-1sgn3-3 double mutant to test if SGN3 is involved in initiating this leaf ABA response. In leaves of the esb1-1sgn3-3 double mutant the elevated expression of a set of ABA signalling and response genes observed in esb1-1 is reduced to below that of wild-type . Further, the reduced stomatal aperture of esb1-1 is also recovered to wild-type levels in this double mutant . SGN3is therefore necessary for the ABA-dependent stomatal closure in response to the defective endodermal diffusion barrier in esb1-1. This raises the question of what links detection of a break in the endodermal diffusion barrier with ABA-driven closure of stomates in the leaf? Removal of endodermal suberin in esb1-1 expressing CDEF1 revealed a significant reduction in ABA-regulated gene expression, and a tendency to increasing stomatal aperture towards wild-type . Thus, increased suberin deposition in the endodermis of the esb1-1 root appears to play a partial role in the ABA controlled reduction in leaf transpiration. We have ruled out a role of local ABA signalling in controlling enhanced suberin deposition at the endodermis in esb1-1 . Using a similar strategy of expressing abi1 in the endodermis, in this case using the SCARECROW promoter , primarily active in the endodermis, we also show that in esb1-1 ABA signalling at the endodermis is not promoting stomatal closure or enhanced ABA signalling in leaves . We note that pSCR is also active in bundle sheath cell, and so ABA-signalling in these cells is also not involved in promoting stomatal closure in esb1-1. Furthermore,blueberry packaging containers enhanced ABA signalling in the endodermis is also not responsible for the initiation of the long-distance response of stomatal closure in leaves, and again it is more likely that suppression of ABA signalling is playing a role.

This can be seen in the fact that expression of abi1 in the endodermis, blocking ABA signalling, mimics the efect of esb1-1 on Lpr and stomatal aperture closure . However, these possibilities remain to be further explored. In contrast to these root-based or long-distance effects, the closure of stomata in leaves in response to a root-based CIFs/SGN3 derived signal is mediated by ABA locally in the leaves. We also note that the long distance signal connecting CIFs/SGN3 in roots with reduced leaf transpiration is currently unknown. Interestingly, a root-derived peptide has been recently identified as involved in long-distance signalling. In response to drought stress, CLE25 move from root to shoot and induces ABA accu-mulation in leaves and stomatal closure. Casparian strips have been suggested to play a critical role in forming a barrier to apoplastic diffusion to limit uncontrolled uptake and back fow of solutes from roots reviewed in . However, most Casparian strip mutants only appear to show fairly subtle phenotypic effects, and this has been a source of continued puzzlement. Here, we show that sensing damage to Casparian strips via leakage of the vasculature-derived CIF peptides from the stele into the cortex triggers a mechanism that inactivates aquaporins, promotes enhanced deposition of suberin limiting solute leakage in roots, and reduces transpiration in leaves, which all contribute to increasing solute concentration in the xylem . The overall outcome of this integrated response is a rebalancing of solute and water uptake and leakage. These physiological compensation mechanisms mitigate the loss of Casparian strip integrity, allowing relatively normal growth and development. A key part of this compensation mechanism is the ability of esb1-1 to limit water loss by the shoot by reducing stomatal aperture, in an ABA-dependent manner. This is clearly established by our observation that the esb1-1aba1 double mutant has severely reduced growth and seed production compared to either of the single mutants, and these growth defects can be partially supressed by the exogenous application of ABA .

The mechanisms we have identified are triggered by the loss of Casparian strips integrity. Such an event can occur during biotic stress including root nematodes infestation, and also during developmental processes such as lateral root emergence where Casparian strips are remodelled, suberin deposition occurs, and aquaporin expression is suppressed. Here, we describe novel outputs of the CIFs/SGN3 surveillance system that couple sensing of the integrity of the Casparian strip-based apoplastic diffusion barrier at the endodermis with pathways that regulate both solute leakage and hydraulic conductivity in the root . Long distance signals then connect these root-based responses with compensatory mechanisms in leaves which are mediated by local ABA signalling . Our dis-coveries provide a new framework which integrates our emerging understanding of the molecular development of the Casparian strip and suberin diffusion barriers with two of the major physiological functions required for plant survival – solute and water uptake.In recent years, California has tightened rules for reporting diversions of water for agriculture and other uses. One key challenge has been establishing workable standards for the collection of reliable data on relatively small and remote diversions — such as those for far-flung farms and ranches. Under new legislation, a certification program run by UC Cooperative Extension is helping to solve that problem. The State Water Resources Control Board views ac-curate diversion reporting as a key element of sound water management. “It’s incredibly important to monitor how much water comes into and goes out of the system,” says Kyle Ochenduszko, chief of water rights enforcement at the water board. Diversion reports are fed into a state database and support the orderly allocation of water resources by, for instance, enabling the board’s Division of Water Rights to inform water users when new requests to appropriate water might affect their own supply. Since 1966, the California Water Code has required diverters of surface water, with certain exceptions, to report their diversions to the water board. But in part because the water board lacked fining authority for many years, compliance was poor. In 2009, Senate Bill 8 gave the water board the authority to fine non-compliant diverters an initial $1,000, plus $500 for each additional day of failing to report.

Even so, SB 8 did not stipulate precisely how diversions were to be monitored. Rather, it required diverters to measure their diversions using the “best available technologies and best professional practices,” unless they could demonstrate that such technologies and practices were not locally cost-effective. That is, the requirement left wide latitude for interpretation. So things remained until 2015 — when Senate Bill 88 became law. This piece of legislation, passed amid a historically severe drought, directed the water board to draw up emergency regulations regarding water diversions. The regulations, once completed, required diverters of at least 100 acre-feet of water per year to hire an engineer or appropriately licensed contractor to install all monitoring devices. Now the requirements were clear. But the provision mandating installation by an engineer or contractor prompted an outcry from many smaller diverters, particularly those in remote areas of the state. For most diverters near sizable towns — Redding, say — complying with the regulations was manage-able, with expenses limited to the cost of a monitoring device and the services of an installer. But diverters in remote parts of Modoc County, for example, were looking at bigger bills, says Kirk Wilbur of the California Cattlemen’s Association. For such diverters, compliance might require importing an engineer or contractor from far away,blueberry packaging boxes which would entail significant travel expenses. If a site lacked electricity, as many do, the costs would pile higher . So how to reconcile the interests of the state’s diverters with those of the state? How best to balance the public and the private good? The answer, it turned out, was to empower diverters to install their own monitoring devices — with UCCE playing the empowering role. The idea originated with the Shasta County Cattlemen’s Association. It gained the support of the statewide Cattlemen’s Association. It took shape as proposed legislation in 2017 and was shepherded through the Legislature by Assemblyman Frank Bigelow . It breezed through both chambers with no votes in opposition — not even in committee. “All parties realized,” says Assemblyman Bigelow, “that Assembly Bill 589 would cut compliance costs and, as a result, increase compliance rates — which benefited both the regulators and the regulated community.” Essentially, AB 589 allows water diverters to in-stall their own monitoring devices if they successfully complete a monitoring workshop offered by UCCE. Further, it directed UCCE to develop the workshop in coordination with the water board. Khaled Bali, an irrigation water management specialist at the Kearney Agricultural Research and Extension Center, took the lead in drafting the coursework. “Then we met with the [water] board and got feedback,” Bali says. “We made changes until they said, ‘This looks good.’” Attendees at the workshops, which last three and a half hours, gain a solid foundation in the basic principles of diversion monitoring.

They learn how to monitor flows passing through a ditch, over a weir or through a pipe — or gathering in a pond. They learn how to build or install measuring devices appropriate for each type of diversion and how to calibrate those devices to comply with the state’s accuracy requirements. They learn how to navigate the water board’s rather detailed reporting system. Equipment for monitoring flows through open ditches might be limited to a tape measure, a timing device and a floating object. Installing a monitoring device for a diversion routed over a weir — a simple dam with an edge or notch that allows overflow — re-quires a bit more equipment. But once the installation is complete, the diverter need only read a staff gauge that shows the height of the water spilling over the weir’s crest . Diversions flowing through pipes must be outfitted with flow meters. Diversions feeding into a pond or reservoir can be monitored by tracking the depth of the water with a staff gauge, float or pressure transducer . So far, UCCE has offered the course in about 15 lo-cations, from Yreka to Bakersfield. According to Shasta County UCCE County Director Larry Forero — who teaches the $25 course along with Bali, Tehama County UCCE Advisor Allan Fulton and UC Davis–based UCCE Specialist Daniele Zaccaria — about 1,000 people had earned certificates of completion by early October. Even farmers and ranchers who divert less than 100 acre-feet per year are attending. “I’ve been floored,” says Wilbur, “by the number of diverters who have attended the course even though they aren’t required to — they want to better understand the regulations and make sure they’re doing the right thing.” It probably helps that the registration fee is a fraction of the cost of importing a faraway engineer. Due to their increasing use in a wide variety of beneficial industrial and consumer applications, ranging from use as a fuel catalyst, to chemical and mechanical planarization media, there have been increasing concerns about the potential environmental health and safety aspects of manufactured ceria nanomaterials.1,2 Ce is among the most abundant of the rare earth elements making up approximately 0.0046% of the Earth’s crust by weight .3 For example, Ce concentration in soils range from 2 to 150 mg kg−1 . 4 In Europe, the median concentrations of Ce were 48.2 mg kg−1 in soils, 66.6 mg kg−1 in sediment and 55 ng l−1 in water . There are many naturally occurring Ce containing minerals include rhabdophane, allanite, cerite, cerianite, samarskite, zircon, monazite and bastnasite.The existence of naturally occur-ring ceria nanoparticles is also likely and may play a key rolein controlling dissolved Ce concentrations,6 but precisely how the properties of naturally occurring ceria nanoparticles com-pare to manufactured ceria nanomaterials is unclear. There is concern that nanoceria, due to its small particle size and enhanced reactivity by design, may present unique hazards to ecological receptor species. Of critical importance are the redox properties of ceria which enables it to transition between CeIJIII and Ce, which are the key to understanding its potential toxicity.While there has been somewhat extensive investigation into the mammalian toxicity of ceria ,based on the present review, there has been considerably less effort invested into investigation of the environmental fate and effects of nanoceria. In this critical review, we discuss the likely points of environmental release along product life-cycles and resulting environmental exposure to nanoceria, methods of detection in the environment, fate and transport, as well as the available toxicity literature for ecological receptor species.

Disposal of TMV-contaminated materials poses low environmental risk

As batches move from the upstream portion of the facility every 3.44 days, the remaining time left over in the downstream is set as slack time in the model that may be dedicated toward repair, maintenance, etc. The assumptions and results developed in SuperPro were used to calculate the economics of the process described. Table 2 shows the total operating costs segregated individually for upstream and downstream components. Figure 5 displays process category cost contributions graphically, including percentages of total costs. In upstream operations, the largest cost components are utilities and labor , representing 61% and 32% of total upstream costs, respectively. In downstream operations, labor-dependent costs are the highest contributors at 30% of total downstream costs, followed by consumables at 27% of total downstream costs. Overall, the upstream component represents nearly 57% of the total Griffithsin production cost, which is calculated as just over $106/g protein. For a micro-bicide dose of 3 mg, the per-dose manufacturing cost is $0.32, excluding any CMO fee. An environmental health and safety assessment was also conducted for this case study following the method of Biwer and Heinzle and the results are found in Supplementary Tables 2–4 in Supplementary Materials. Overall, the process uses chemicals that are not harmful to people or the environment, as can be seen by the low magnitude of input and output Environmental Factor values in Supplementary Table 4. The biggest causes for concern are TMV in the residual biomass,grow bags for gardening and sodium hydroxide and phosphoric acid used in clean-in-place operations, if released to the environment; however we included costs for a thermal or chemical deactivation step for the TMV-contaminated biomass and pH neutralization for the acid and base cleaning agents which would eliminate the environmental impact of these components.

It should also be noted that the upstream nutrient compounds can be more efficiently recycled to increase nutrient utilization by the plants and reduce water/soil impact. Waste compounds in the downstream process are disposed of through wastewater and bio-waste treatment. An aggregate disposal cost of $0.01 per liter of non-TMV-contaminated aqueous streams and $0.1 per kg of bio-waste is assigned in SuperPro for expenses related to wastewater disposal and thermal/chemical deactivation of bio-waste streams. Compounds introduced during or after the post-inoculation step in the upstream facility are considered as bio-waste since they may contain TMV. This includes spent nutrient solution in the post-inoculation step and retentate streams from plate-and frame and dead-end sterilizing filtration skids. There is extensive industrial experience in disposing of TMV contaminated materials, which can be rendered non-infective by treatment with bleach, heat or detergents, diluted and disposed of as municipal waste .The facility modeled can annually produce 20 kg of the potent antiviral Griffithsin for use in microbicide products. The host used in our modeling was Nicotiana benthamiana. This species was selected because of its aforementioned productivity, but also because our previous report on technoeconomic modeling of Nicotiana-produced therapeutic and industrial products prefaces the work reported herein. In addition, the use of Nicotiana for production of clinical trial materials is also familiar to FDA and other regulatory agencies, thus facilitating Nicotiana’s acceptance in regulation-compliant manufacturing . The API is manufactured in the host Nicotiana benthamiana using tobacco mosaic virus as the expression vector. The upstream plant growth and Griffithsin production operations are adapted from the facility layout detailed byHoltz et al. . Over 158,000 plants are housed in vertically stacked hydroponic grow racks, fitted with high-efficiency LED lights. The environment is controlled and monitored for compliance with good agricultural practices . Each batch of 14,450 plants grows over the course of 38 days and yields a total of 578 kilograms of biomass.

Ninety-five batches are seeded and grown annually, with one batch reaching harvest every 3.44 days. The downstream Griffithsin extraction and purification process is scaled up from the pilot industrial scale process presented by Fuqua et al. . An expression rate of 0.52 grams of Griffithsin per kilogram of biomass and a downstream recovery of 70% were used in the base case and give a combined yield of 0.370 grams of Griffithsin per kilogram of harvested biomass. Sterile filtration and CIP/SIP systems facilitate compliance with cGMP guidelines. Downstream processing commences upon the completion of an upstream batch and takes 39.3 h. The stable final formulation is >99% Griffithsin as the API with negligible endotoxin levels. In the model, the upstream costs account for nearly 57% of the total cost of Griffithsin production. Containing both upstream and downstream losses of the protein could significantly reduce COGS. Approximately 12% of the protein API is non-liberated from the homogenized biomass and 18% is lost during downstream polishing steps. Based on the data and assumptions employed in the current analysis, the unit production cost of Griffithsin is estimated to be $0.32 per dose . The model was based on published designs for a commercial scale facility and pilot-scale data on Griffithsin production adapted to the facility described. This type of modeling is useful for determining ranges of API selling price, production capacity and expression level requirements for commercial supply and profitability. In this study we modeled the manufacturing of Griffithsin through a contract manufacturing organization instead of a greenfield build of a new facility because we assumed that that would be the most prudent approach to launching a new product. If the product manufactured using the process modeled is used directly as a vaginal rinse or rectal enema, the additional costs post manufacturing would include transportation, storage, insurance, distribution, marketing, etc., none of which were modeled in this manufacturer-level analysis.

If the Drug Substance produced via the process analyzed is further formulated , or used as a component of another device , those costs and other product-specific costs would be additive and were also excluded from our manufacturer-level analysis. The cost of goods calculated by the current model reflects the manufacturer’s cost of production. We are less certain about the wholesale price of the drug because there is no standard “off- the-shelf ” profit margin that can be added to toll manufacturing cost to arrive at a standardized answer. Often scale up to commercial launch volumes of a product requires additional process development and optimization, validation batches, etc., which lead to negotiated transfer prices depending on volume, duration of engagement, license fees, export duties, and other factors, all of which would impact the cost of bulk Griffithsin. Nevertheless, for this discussion we assumed a manufacturer’s fee of 20% of COGS for a total production cost of bulk Griffithsin Drug Substance of $0.38/dose. Additive formulation, storage, distribution, insurance, marketing, sales margins and other costs could lead to a consumer-level use cost of $1-2/dose . This techno economic analysis emphasized Griffithsin’s use in microbicides because such products arguably represent the most price-constrained applications of this new drug. We cannot define the target retail price of a Griffithsin microbicide; there is no market reference price for micro-bicides since no commercial microbicides yet exist. For perspective,garden grow bags the user cost of a Griffithsin microbicide can be benchmarked against pre-exposure prophylaxis with traditional male condoms and PrEP with micro-bicides containing antiretroviral drugs as a newer alternative. Analyses have been conducted on the cost of prevention modalities and the cost savings to the healthcare system enabled by preventing HIV transmission, with prevention being far more cost effective than treatment in most scenarios . Walensky et al. conducted an analysis of the cost-effectiveness of a Tenofovir based PrEP micro-bicide in South African women. In their cost modeling of a vaginal gel, they multiplied the product cost of $0.32/dose times 2 and by 7.2 to arrive at a product use cost of approximately $5/woman month. However, the price of the microbicide gel used in the study was assumed and region-adjusted and hence pricing in other countries may be different. Terris-Prestholt et al. estimated Tenofovir gel prices of $0.25–0.33 per dose, provided that the gel was used in combination with a condom , from which an additive cost of use of $7–$12/person-month can be derived. Assuming the same average use rate of a Griffithsin containing microbicide applied singly without a condom and priced at $1.00–$2.00 per dose, the cost of use would be $7– <$15/person-month. Whether a higher cost of use discourages adoption of Griffithsin-based microbicides by men and women remains to be shown. A market study by Darroch and Frost of the Alan Guttmacher Institute consisted of detailed interviews of a cross-section of 1,000 sexually active women aged 18–44 in the continental United States. Their statistically rigorous survey identified levels and predictors of women’s concerns about STDs and interest in microbicides, as well as their preferences regarding method characteristics and likelihood of usage versus price of product, with survey sample results extrapolated to the national level.

The results showed that of the estimated 12.6 million women aged 18–44 interested in microbicides and concerned about STDs, including HIV, 11.5 million would still be interested in the method even if it were not 100% effective, and 11.0 million would remain interested even if the microbicide did not protect against STDs other than HIV. The same study found that women’s predicted use of a microbicide was affected by price, but interest was still high at $2 per application, or roughly up to 5-times the average price of a male condom. The survey concluded that more than seven million sexually active women in the USA would be interested in a vaginal microbicide even if the product only protected against HIV, was only 70–80% effective and cost them $2 per application . That conclusion was arrived at in 1999; the $2 per application cost back then would be $3.05 in 2018. One can conclude from these results that there is interest in effective yet inexpensive, self-administered HIV and STD prevention modalities even if such products might cost more than conventional prevention methods. The Darroch and Frost analysis was conducted nearly 20 years ago, and the interviews were limited to women practicing vaginal intercourse. To our knowledge, a more recent study linking likelihood of product use and price sensitivity has not been conducted, or at least not reported, to include other populations of potential microbicide users such as heterosexual couples practicing anal sex or gay men practicing unprotected rectal intercourse. Nevertheless, the 1999 study established an initial price point and price sensitivity for potential users of microbicides in the USA. Griffithsin has a broader spectrum of antiviral activity than HIV-specific PrEP agents, including activity against HSV-2 and HCV, which are co-transmitted with HIV-1 . Hence, Griffithsin might command a higher price due to its broader antiviral activity and its potential to obviate prevention and treatment costs for co-transmitted viruses. In the USA, the cost of the oral PrEP drug Truvada ranges from $1,300 to over $1,700 per month for the uninsured, but treatment is typically covered by insurance with user co-payments of $80–$150 per month. So even if a Griffithsin-containing microbicide sold for $5 per application , a user of 2 packs per month would pay $100 for the microbicide, which is in the range of PrEP, with the potential added benefit of controlling co-transmitted viruses. Consumers in wealthier economies might be receptive to microbicides costing $1–2 or even more per dose; however, consumers in lesser-developed economies might find $1–2/dose to be prohibitive. Hence, absent subsidies, there exists a continuing need to lower COGS for APIs such as Griffithsin. We can conclude that a COGS of <$0.40/dose of Griffithsin DS as determined in this study, and an estimated user cost of $1– 2/dose, might enable at least some simpler formulations of the drug to be economically marketed. For more complex formulations and delivery systems, or for higher doses of the drug, lower COGS for bulk Griffithsin would be desirable. The environmental assessment of the plant-based production of Griffithsin indicates low impact, particularly if the plant nutrient solutions are recycled in a hydroponic system and if waste streams containing TMV are treated in a biowaste heat or chemical treatment process. The assessment method used, although semi-quantitative, utilizes mass input and output stream data generated by SuperPro, along with independent assessment of compound toxicity and/or environmental impact , and allows comparison between alternative production strategies, process configurations or chemical components used in the manufacturing process.

Seeds were wrapped in foil and imbibed at 4°C for 4 days before planting

Since the negative short-term effects of Al on growth are apparently limited given that inhibition is alleviated without checkpoint function, conceivably these checkpoints have evolved to detect the minor strain on an individual’s genomic stability and serve to prevent transmission of Al-dependent genetic deffects to subsequent generations that ultimately would compromise the viability of the population. These checkpoints sacrifice the individual by halting the cell cycle in the root and forcing endore duplication to prevent an Al-dependent generational penalty with regard to heritable genomic integrity. Cells of the shoot meristem generate the floral reproductive organs of the plant, and thus the heritable genetic material. While the root is the most affected organ of the plant in the Al toxic response, Al is reallocated and sequestered to other regions of the plant body as Al-oxalate complexes can transport Al through the xylem from the roots to the shoots . In addition to a root hypersensitive phenotype, shoots of als3-1 grown in Al containing media display reduced cotyledon and leaf expansion, as well as a second shoot apex . Thus shoots are indeed affected by Al toxicity which could pose a threat to plant reproductive processes; therefore, it is plausible that Al-dependent root growth inhibition could serve as a means to preserve genomic integrity of the species. In all likelihood, it is plausible that Al induces an inappropriate activation of the DNA damage response by detecting Al-dependent pseudo-cross links. This could be the result of topological strain on the DNA and/or functional interference with Mg-dependent replication machinery,round nursery pots activating unnecessary repair of the DNA that may actually be the true detrimental effect of Al-dependent root growth inhibition.

Repair processes like nucleotide excision repair and nonhomologus end joining may be at work as shown by hypersensitivity to Al for loss-of function mutants in ALT2 and PARP1, PARP2 and KU80 respectively. The repair mechanisms could then result in subsequent damage inflicted on DNA, much of which may be related to double strand breaks that are regularly observed following Al treatment. The specific repair pathway activated in response to Al treatment is unknown, as well as whether or not this unknown process inflicts damage on the DNA during the repair. If indeed Al is acting to cause pseudo-cross links, it stands to reason that this effect would also inhibit proper DNA repair processes and could further cause damage to the DNA. As such, failure to activate this response pathway prevents the program-inflicted damage and results in roots that can grow normally in the presence of inhibitory levels of Al.While it is speculative at this time as to what the true nature of the effect of Al is on DNA integrity, an ATR-dependent DNA damage response pathway is clearly activated in the presence of internalized Al. Whatever the direct effect of Al is, based on the response factors, it can be speculated how these factors assemble and respond to Al stress based on their functional homology to related proteins: SUV2 likely aggregates at sections of persistent single stranded DNA coated by the heterotrimer, Replication Protein A, known to aggregate to single stranded DNA . SUV2 may act as a homodimer as it has been shown to bind to itself through its N-terminal coiled-coil domain as demonstrated by a yeast-2- hybrid . The proline-rich repeats at the N-terminus of SUV2 suggest a stiff elbow-hinge that could “wag” as a mechanism of recruiting other response factors to the locus.

It is possible that this homodimerization is controlled by phosphorylation of SUV2 since the dimerization domain has two SQ phosphorylation motifs associated with it. SUV2 would act to recruit ATR to this region of DNA in a manner similar to their homologues in yeast and mammals . The interaction of SUV2 and ATR with persistent single stranded DNA should induce autophosphorylation of ATR, and phosphorylation of SUV2 by ATR. It is possible that ATR will also be phophorylating other substrates, such as RPA2 and H2AX, to orchestrate additional responses. Once ATR has activated these sensors, likely in conjunction with other unidentified proteins, they would then induce the signal transduction pathway by activation of SOG1, again, likely through phosphorylation by ATR. Conserved serine-glutamine motifs are preferential ATM and ATR phosphorylation targets in mammals . SOG1 has five SQ motifs in the C-terminal transcriptional activation domain and SUV2 has two SQ motifs near the N-terminus, one of which is within the dimerization domain. Following activation of SOG1, expression of a group of genes is promoted, specifically genes tested from an established set of SOG1-mediated genes involved in a DNA damage response. This subset of Al-induced SOG1 mediated genes includes genes known to repair DNA e.g. BRCA1, RAD51, RAD17, GMI1, and PARP2 and halt the cell cycle e.g. CYCB1;1 as well as more transcription factors e.g. TRFL10,TRFL3, ANAC103 and WRKY25. Perhaps it is these transcription factors, as well as unidentified genes promoted as part of this response that are responsible in some unknown manner for a mechanism that forces a programmatic change in the root tip and QC, thus triggering this tissue to switch to endore duplication and causing terminal differentiation and permanent stoppage of growth of the primary root. While little is known about the placement of ALT2 within this signal transduction pathway, it likely functions as a scaffold protein, perhaps as part of a ubiquitin ligase signaling mechanism, acting analogously to other WD-40 proteins. WD40 repeat proteins are a class of proteins that are generally involved in mediating interactions between other proteins, associating with a variety of protein complexes, including E3 ubiquitin ligases . In eukaryotes, proteins are targeted for degradation via the ubiquitination-proteasome system, but ubiquitination also plays an important role in post-translational modification of proteins in the activation of signaling pathways.

As part of a crucial step in the DNA damage response pathway in mammals, following phosphorylation by ATM or ATR, one of the core histones of the nucleosome, γ-H2AX, is mono-ubiquitinated. This mono-ubiquitination is required for the recruitment of subsequent repair factors like BRCA1 and 53BP1 to both double and single stranded DNA breaks . In plants, CULLINs , which are part of a family of scaffolding proteins, form the largest family of E3 ligase complexes. Arabidopsis proteins containing WD40 domains, including ALT2, are proposed to be capable of interacting with the DDB1-CUL4-ROC1 complex . The recruitment structures and mechanisms are not well understood for CULLIN based ubiquitination signaling; however,plastic flower pots CUL4 has been shown to form a complex with DW40 proteins in response to UV damage that is ATR dependent . This establishes a potential link between ATR and ALT2 in a DNA damage response where resulting cross links would cause a replication fork stall, as Al likely causes.A model for stoppage of root growth following chronic exposure to Al can be developed in accordance with current evidence. In this model, Al impacts DNA in a currently unknown way, likely from a pseudo-cross linking effect resulting in a replication fork stall. Based on the genetic factors responsible for activating the Al dependent DNA damage response, it is a reasonable prediction that such an interaction would hold DNA in a conformation that inhibits replication fork progression. Regardless of the physical consequences of Al on DNA structure or integrity that have yet to be determined, the predicted genotoxic effects of Al are clearly sufficient to activate an ATR-, ALT2-, SOG1- and SUV2-dependent cell cycle checkpoint mechanism as demonstrated by the increase in Al tolerance seen for each loss-of-function mutant. This mechanism functions to promote transcription of a group of genes related to halting the cell cycle and to repair the perceived damaged DNA. Furthermore, it is likely that additional genes are included in this transcriptional response that are related in some unknown manner to a mechanism that forces a programmatic change in cells of the root tip and especially the QC. These genes would trigger cells to differentiate, losing their meristematic identity by switching from a normal cell cycle progression to endore duplication. Ultimately, it is this terminal differentiation that permanently stops growth of the primary root as the primary cause of Al toxicity. While significant work remains to be done, especially in determining the genotoxic consequences of Al that activate this DNA damage response pathway and developing a transcriptional profile of SOG1 targets that lead to inhibited root growth following Al treatment, it is clear that terminal differentiation of the root tip following chronic exposure to Al is an active event mediated by the DNA damage checkpoint factors ATR, ALT2, SOG1 and SUV2. Our understanding of the genomic consequences caused by Al is still in the beginning stages, and more work is needed.

Continued testing of DNA damage response mutant responses to Al can give us the opportunity to further elucidate how genomic maintenance factors are involved in this biological problem. In addition to the value of gaining a better understanding of the role of DNA damage response factors and cell cycle checkpoints in mediating Al-dependent DNA damage, Al toxicity represents a novel and biologically relevant model for studying ATR dependent mechanisms in the DNA damage response in general.For all growth experiments, seedlings were surface sterilized, vernalized, and etiolated before planting. Seeds were immersed in 70% ethanol and then washed 4 times with sterile water. Seeds were then immersed in 50% bleach for 5 minutes, after which seeds were washed 4 times with sterile water. The AlCl3 soaked gel environment was sterilely prepared by pouring a lower gel layer consisting of 80 mL of nutrient medium plus 0.125% gellan gum in Nunc Lab-Tek Extra-Depth Polystyrene Dishes 100 x 25 mm . Nutrient medium consisted of 2 mM KNO3, 0.2 mM KH2PO4, 2 mM MgSO4, 0.25 mM 2SO4, 1 mM Ca2, 1 mM CaSO4, 1 μM MnSO4, 5 μM H3BO3, 0.05 μM CuSO4, 0.2 μM ZnSO4, 0.1 μM CaCl2, 0.02 μM Na2MoO4, 0.001 μM CoSO4, and 1% sucrose. Al was introduced by overlaying the solidified lower layer with 20 mL of “soak solution” containing the proper concentration of AlCl3. Trail soak solution was made consisting of the nutrient solution medium described above, while only brought to 90% of the intended volume. 50 mL trail solutions were made consisting of 45 mL the slightly concentrated nutrient medium, X mL 25mM AlCl3, Y μL 0.1 N KOH and Z mL diH2O . The trail soak solution was made to determine the amount of 0.1 N KOH to use to adjust the pH of the nutrient soak containing AlCl3. The amount of base to add was determined empirically by adjusting the pH on an aliquot of the soak solution containing AlCl3. The amount of base determined from this trial soak solution was added to the actual soak solution prior to adding AlCl3. The sterilized soak solution was allowed to equilibrate with the lower layer for 2 days and was then poured off. This method was used for all concentrations of AlCl3 for plants grown in a gel soaked environment. In hydroponics experiments, Al-screening media was sterilely prepared as above without gellan gum and AlCl3. Seeds were sowed on 250-μm mesh, polypropylene screen in Parter Medical Products Quad Perti Dish 100 X 15 mm . After 6 days of growth unless otherwise specified, screens were transferred to new Al screening media supplemented with either 0 μM, 25 μM AlCl3 or 50 μM AlCl3. For treatment with hydroxyurea , mitomycin C , bleomycin , or cisplatin were added to plant nutrient media plus sucrose . Seeds were sowed and allowed to grow for seven days, after which roots were measured. For experiments on plant nutrient media plus sucrose , the medium consisted of 5 mM KNO3, 2.5 mM KH2PO4, 2 mM MgSO4, 2 mM Ca2, 50 μM FeEDTA, 1 μM MnSO4, 100 nM CaCl2, 100 nM CoSO4, 5 nM H3BO3, 50 nM CuSO4, 20 nM NaMoO4, 0.8 M Sucrose, 0.8% agar. Plants were grown in 24-hour light at 20°Cin I-36LLVL biological incubator . After one week, plants were repotted in Sunshine Special Blend potting soil with controlled release fertilizer, 15-9-12 + minors . Plants were grown in 24- hour continuous light at 22°C in a plant growth room with Sylvania Gro-Lite fluorescent bulbs until maturity.