Author: naturehydro

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Maximize Growth Potential: Unleashing the Power of Hydroponic Grow Systems

Given that Huanglongbing is related to ROS-overproduction, ROS-modulating NMs may have significant impact on the disease course. In the nanozyme field, novel ROS-triggering nanozymes are constantly being synthesized. NADPH oxidase, also referred to as RBOH, is a transmembrane enzyme complex that controls the generation of superoxide, which plays an important role in immune signalling pathways. A recent study synthesized a Fe–N-doped graphene nanomaterial that could mimic the activity of NADPH oxidase by efficiently catalysing the conversion of NADPH into NADP+ , subsequently triggering the generation of oxygen radicals. Given these demonstrated ROS-generating properties, FeNGR nanozymes may be applied to cultivate stress-resistant crops. To date, no studies have employed this nanozyme with NADPH oxidase-like activity for stress tolerance enhancement. Thus, the linkage between plant science and nanozyme fields of study could significantly move this field forward. Last, applications of NMs in agriculture need to consider the potential environmental and human health risks. For example, the impacts of NMs on non-target biota needs to be evaluated. The impacts of NMs on soil microbial and fungal communities that are critical to nutrient uptake of plants, as well as carbon and nitrogen cycling, must be evaluated. In addition, the bio-accumulation of NMs, particularly in edible tissues, needs to be investigated to avoid potential transfer in food chains that could negatively impact human or ecosystem health. However, it is clear that sustainable nano-enabled strategies to promote crop species’ tolerance to abiotic and biotic stresses Several studies report the uptake of emerging contaminants from hydroponic solutions, spiked soils and soil irrigated with TWW or amended with bio-solids or animal manure . However,plastic plant containers these studies often report conflicting or contradictory results concerning the rate of uptake and the extent of translocation.

Hydroponic studies often exhibit higher rates of uptake than those observed in spiked or amended soils studies. For example, in a study conducted by Boonsaner and Hawker the maximum concentration of antibiotics in plant tissues was reached within 2 d in spiked water but took 6-8 d when plants were grown in contaminated soils. In hydroponic systems, plant uptake and translocation are largely driven by the contaminants water solubility, log Kowand/or the pH of the hydroponic solution and the potential for ionization, log Dow, . Whereas in soils, soil-specific processes such as soil-pore water partitioning, and transformations in soil, also contribute to contaminant uptake and accumulation in plants. Thus, the uptake rate and translocation of a contaminant in plants can vary widely depending upon soil and environmental conditions. For example, a soil with higher organic matter content can limit plant uptake of organic contaminants, due to stronger contaminant adsorption than a soil with lower organic matter content . Also, shifts in soil pH can result in ionization of ionizable organic contaminants, affecting the rate of plant uptake . Antibiotics constitute one of the most extensively used pharmaceuticals classes for both human and livestock and as such are nearly ubiquitously detected in wastewater effluent, bio-solids and livestock manure . Relatively more studies have been reported on terrestrial plant uptake and translocation of antibiotics than other pharmaceuticals in the agro-environment, including studies conducted in hydroponic growth solutions, greenhouses, and under field conditions .In hydroponic growth solution, the antibiotic sulfamethoxazole was taken up in the roots and translocated to leaves of four vegetable plants, including lettuce , spinach , cucumber , and pepper plants , with the concentration report to be significantly greater in the roots .

In a 55 day hydroponic study, three antibiotics, i.e., tetracycline, cephalexin, and sulfamethoxazole, were found to be taken up and translocated into edible tissues of pakchoi , with concentrations ranging from 6.9 – 11.8, 26.4 – 48.1, and 18.1 – 35.3 µg kg-1 for tetracycline, cephalexin and sulfamethoxazole, respectively . Several studies have also explored plant uptake of antibiotics from spiked soils . For example, Boxall et al., exposed carrot and lettuce plants to soils spiked with 1 mg kg-1 of 7 antibiotics, i.e., sulfadiazine, trimethoprim, tylosin, amoxicillin, enrofloxacin, florfenicol, and oxytetracycline. After 103 d and 152 d cultivation, antibiotics were quantified in both crops. However, the concentrations varied considerably among different antibiotics and between plant species. For example, amoxicillin was detected at < 1 µg kg-1 in lettuce tissues but was 24 µg kg-1 in carrot tissues . Three sulfonamides, i.e., sulfadiazine, sulfamethazine, and sulfamethoxazole, were also reported to be taken up by pakchoi cultivated in spiked-soils, with sulfamethoxazole having the highest concentration among the three antibiotics throughout the 49 d cultivation . To better predict environmentally relevant risks from antibiotic uptake to human consumption, several studies have been carried out on crops grown in soils irrigated with spiked TWW and/or amended with livestock manure . These studies showed that food crops were capable of taking up and accumulating antibiotics from wastewater and/or manure-amended soils; however, the levels were often very low. For example, chlortetracycline was taken up by corn , green onion , and cabbage that were grown in soils amended with antibioticspiked manure . However, the concentrations were low . Sulfamethoxazole and lincomycin were found to accumulate in lettuce tissues at concentrations up to 125 µg kg-1 and 822 µg kg-1 , respectively, after irrigation with antibiotic-spiked synthetic wastewater at 1 mg L-1 , . Similarly, in field studies, crops irrigated with TWW were found to take up antibiotics, including but not limited to, roxithromycin, clindamycin, ciprofloxacin, sulfamerazine, and sulfamethoxazole . However, in nearly every case the concentration of antibiotics in plant tissues was negligibly low. Nonsteroidal anti-inflammatories are the most commonly consumed class of pharmaceuticals in the world .

As such they are ubiquitously found in TWW, bio solids, and surfaces water . They have been reported to accumulate in soils that receive TWW or bio solids . Several studies have explored the potential for uptake and translocation of NSAIDs in plants, including in hydroponic systems, amended soils, and field studies . NSAIDs have a wide range of physicochemical range properties and, as such, have displayed vastly different uptake and translocation rates . For example, in a hydroponic study the NSAID diclofenac was observed to accumulate only in the roots of four vegetables while relatively high levels of acetaminophen were detected in the leaves . Similarly, a study exploring plant uptake of 14C labeled naproxen and diclofenac from hydroponic solutions showed that two vegetables, i.e., lettuce and collard greens , were capable of accumulating both compounds, and both plants accumulated significantly more diclofenac than naproxen . Radish and ryegrass were shown to absorb and accumulate diclofenac from soils spiked with the chemical at an initial concentration of 1 mg kg-1 . However, after 40 d cultivation, the concentration of diclofenac in the plants was very low < 1 µg kg-1 . Greenhouse studies using soils amended with bio solids and field studies using TWW irrigation considered the uptake of NSAIDs under environmentally relevant conditions. For example, Cortés et al. conducted a greenhouse study in which soybeans and wheat were cultivated in bio solids-amended soils for 60 and 110 d. However, none of the four NSAIDs was detected in the plant shoots. On the other hand, in a long-term field study , diclofenac was found relatively high levels in the fruits of tomato plants after prolonged irrigation with TWW, as compared to sulfamethoxazole and trimethoprim . Further, in another field study, naproxen was detected in the edible tissues of various vegetables irrigated with TWW or TWW fortified with the chemical at 250 ng L-1 and grown until maturity . Several NSAIDs have also been considered in the investigation of potential metabolism of pharmaceuticals in plant cell cultures and whole plants . The metabolism of diclofenac was investigated in four different plant systems, including a horseradish hairy root culture , barley , Arabidopsis thaliana cell culture,blueberry container and Arabidopsis thaliana whole plants . However, the formation of diclofenac metabolites differed significantly by plant systems. For instance, while phase I hydroxylation was observed in all the systems, the horseradish hairy root cultures and barley formed a glucopyranoside as the major Phase II metabolite . Arabidopsis thaliana, on the other hand, produced acyl-glutamatyl-diclofenac as the major Phase II metabolite via direct conjugation . Direct conjugation of naproxen and ibuprofen with glutamic acid and glutamine was also observed in Arabidopsis thaliana plants . The metabolism of acetaminophen has also been studied in multiple plant systems, including horseradish hairy root cultures and Indian mustard . In these studies, direct glucuronisation, glucosidation, and sulfation were observed along with the formation of a reactive metabolite N-acetyl-pbenzoquinoneimine . Taken together these studies have highlighted the ability of plants to uptake and transform NSAIDs. Several classes of psychiatric pharmaceuticals have been detected in TWW and bio-solids including antidepressants, mood stabilizers, and antianxiety agents . Of these compounds, carbamazepine has been likely considered in probably the most in the agroenvironment due to its stability during wastewater treatment and in the environment .

Carbamazepine has been often reported to be taken up by plants in both field and laboratory settings . In hydroponic systems, carbamazepine has been shown to accumulate in both roots and shoots of multiple plant species, including lettuce, spinach, cucumber, and peppers . Cucumber was found to readily translocate carbamazepine when cultivated in hydroponic systems . However, a high rate of translocation was not observed in cabbage plants cultivated in hydroponic systems . In greenhouse studies, carbamazepine was reported to be taken up by cucumbers and ryegrass grown in soils irrigated with TWW and urine . In addition, Shenker et al., reported that uptake into cucumbers was negatively correlated with soil organic matter content. In fields irrigated with TWW, trace levels of carbamazepine was found to accumulate in different parts of various vegetables . Carbamazepine was also reported to transfer to humans after consumption of contaminated vegetables . The metabolism of carbamazepine in plants has also been investigated . In carrot cell cultures five phase I metabolites of carbamazepine were observed to form over 22 d . Further, 10,11-epoxycarbamazepine and 10,11-dihydroxycarbamazepine have been reported in carrots and sweet potatoes grown in fields irrigated with CEC-spiked TWW . Fluoxetine is an antidepressant prescribed for both human and animal consumption , resulting in fluoxetine being commonly detected in environmental samples . In hydroponic cultivations fluoxetine was taken up by cauliflower and accumulated in the stems and leaves . In a greenhouse study exploring plant uptake of fluoxetine from soils irrigated with TWW and amended with bio solids fluoxetine accumulated in the roots , but, it was not translocated to the leaves . In addition, fluoxetine displayed an opposite uptake pattern to that for carbamazepine, and showed a greater accumulation in plants grown in bio-solid-amended soils as opposed to soil irrigated with TWW . Benzodiazepines, are one of the most prescribed classes of pharmaceuticals . Of these, diazepam is among the most commonly detected pharmaceuticals in TWW, with concentration ranging from ng L-1 to low µg L-1 . Benzodiazepines have been shown to be taken up and accumulate in tissues of plants grown in treated hydroponic solutions or soils . In hydroponic solutions, diazepam has been observed to accumulate in both the leaves and roots of lettuce, spinach, cucumber, and pepper with BCF of 10-100 ]. Further, in a greenhouse study exploring the uptake of seven benzodiazepines , both silver beets and radish crops took up and accumulated all seven benzodiazepines from the treated-soil . Oxazepam was found to have the highest accumulation in both plants, with concentrations up to 14.2 µg g-1 in silver beets and 5 µg g-1 in radishes . However, the fate of these pharmaceuticals in the agro-environment is still relatively unexplored, even though their physicochemical properties indicate a high potential for uptake by plants .A multitude of antimicrobials and preservatives are used in health and grooming products, collectively known as personal care products . Personal care products have garnered increased scientific attention due to their presence in surface waters and concerns that some of these antimicrobials and preservatives may be endocrine disruptors . Of these, triclocarban and triclosan have been amongst the best studied compounds in the terrestrial environment due to their ubiquitous occurrence in bio-solids and relative stability in soils after bio-solid application .

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Growing Up Without Ground: The Basics of Hydroponic Crop Cultivation

There may be some regulation controlled by a threshold value for Cd plant status, in both species. Long-term contamination with 0.1 µM would be below this threshold, thanks to the PC sequestration for example. On the other hand, long-term contamination with 10 µM would exceed this limit and result in down-regulation of the transport proteins of the HATS, possibly because of excess free Cd in the cytosol or because of some signal from the shoots. For both plants, cell wall sorption efficiency appears to be improved by the high Cd concentration in the growth solution, whereas the low concentration had generally no significant impact. The increase in the cell wall binding efficiency after high internal Cd accumulation may be related to the down-regulation of intracellular uptake. However, the reduction in symplastic influx is very low and cannot account for the increase observed at the apoplastic level. On the contrary, the up-regulation of the adsorption rate may well account for the decrease in the symplastic uptake. The apparent up-regulation of Cd binding properties may be due to modifications of the root cell-wall adsorption characteristics, particularly the root CEC. Cadmium stress is known to affect cell wall composition. First, Cd increases the proportion of acidic pectins . Secondly, the cell wall CEC may be increased through regulation of enzymes. For instance, pectinmethylesterase has been suggested to be stimulated in the outer cell wall domains of Cd-stressed plants, resulting in a strong decrease in the methylesterification of the acidic pectins. Thus, Cd strongly increases the acid pectins/esterified pectins ratio, hence the higher CEC, particularly in the middle lamellae . This low degree of esterification enhances the adsorption of all metallic trace elements , improving the plant tolerance of the metal. As dry mass did not vary with the level of contamination,40 litre pot there may be no significant difference in the proportion of young roots and then no decrease in the root CEC due to the age of roots.

Therefore, the insignificant effect of low Cd contamination on the apoplastic adsorption rate could be accounted for by the existence of some Cd-stress threshold below which there is no regulation mechanism. A long-lasting tolerance to aluminum ion is an essential phenotype for perennial plants growing on strong acid soils for longer periods. There is increasing evidence that plants with superior Al tolerances are relatively easily found in woody plants, such as tropical plantation trees Melaleuca cajuputi and Paraserianthes falcataria . Some woody plants, including tea, hydrangea, and Melastoma malabathricum, are also known as Al-accumulators that retain large amounts of Al in their above ground organs . However, the mechanisms responsible for high Al tolerance or high Al accumulation in woody plants remain to be elucidated. Our preliminary screening has successfully identified that root elongation in seedlings of Cinnamomum camphora, an evergreen tree widely distributed or planted in China and its neighboring regions, is much less inhibited even at high Al concentrations in a simple ionic solution at least for several days. Our finding is consistent with a study that reported no growth reduction in C. camphora seedlings against Al in a nutrient solution for 5 weeks . As a first step in understanding long-term Al tolerance mechanisms in seedlings of C. camphora, we employed a pulse Al exposure every two days for 60 days in measurements of root elongation and Al accumulation in each organ. To understand Al transport mechanisms in shoots, we also examined the Al accumulation patterns in branch cuttings of C. camphora. Root architecture influences nutrient and water uptake, anchorage, and mechanical support, interactions with microbes, and responses to various abiotic stress factors . Since water and mineral supply are often limited in the soil, a plant with a more extensive root system exhibits higher performance with regard to the tolerance of drought and poor nutrient conditions . Several factors, including root angle, root growth rate, and root types, influence root architecture . Root growth requires the successive formation of new cells from stem cells in the root apical meristem , and the progeny of such stem cells divide rapidly and enter the elongation/differentiation zone . To maintain root meristem activity, the rates of cell division and differentiation have to be coordinated .

Plant hormones greatly influence the balance between cell division and cell differentiation . In addition, the interaction between cytokinin and auxin determines the size of the RAM through the regulation of the genes involved in auxin signaling and/or transport to ensure an appropriate auxin gradient . The rice root system consists of one seminal root, numerous adventitious roots, and lateral roots that emerge from the other two types . Lateral roots are the major components involved in the absorption of nutrients and in interactions with the surrounding soil environment . Lateral root formation represents a complex developmental process modulated by several hormones, including auxin and ethylene . Well defined and closely coordinated cell division activities give rise to lateral root primordia . While lateral roots originate from pericycle cells adjacent to xylem poles in Arabidopsis , pericycle and endodermal cells located near phloem poles are the origins of lateral roots in rice and maize . Their development is initiated by the asymmetric division of the pericycle cells, and subsequent divisions result in the formation of dome-shaped, multilayered, lateral root primordia . After the initiation of asymmetric division, the primordia emerge, form active meristems, and break through the epidermal cells to become new lateral roots. Auxin is essential for various steps in the course of root development—from cell fate acquisition to meristem initiation, emergence, and elongation . In Arabidopsis, auxin is mainly synthesized in young apical tissues of the shoots and roots . Indole-3-acetic acid is considered the major form of auxin, with tryptophan being its precursor . Among the four pathways of IAA biosynthesis from Trp, the indole-3-pyruvic acid pathway is the major pathway in Arabidopsis . In the IPyA pathway, tryptophan aminotransferases convert Trp into IPyA, and YUCCAs synthesize IAA from IPyA, a rate-limiting step for the pathway .

In rice, FISH BONE encodes a Trp aminotransferase; loss of function results in pleiotropic abnormal phenotypes, which include small leaves with large lamina joint angles, unusual vascular development, and defects in root development, which are all consistent with a decrease in internal IAA levels . Mutations in CONSTUTIVELY WILTED1result in narrow and rolled leaves, in addition to the decreased growth of lateral and crown roots . Conversely, the over expression of OsYUC1 causes an increase in IAA accumulation, and auxin-overproducing phenotypes are observed . Such phenotypes are subject to the presence of the transcription factor WUSCHEL-RELATED HOMEOBOX 11 , a key regulator of root development . In rice, auxin induces WOX11 transcription,collection drainage which establishes the YUCCA–auxin–WOX11 module for root development . Ethylene also controls root development. Treatment with low concentrations of an ethylene precursor, 1-aminocyclopropane- 1-carboxylic acid , promotes the initiation of lateral root primordia. In contrast, exposure to higher ACC concentrations inhibits such initiation considerably, while also promoting the growth of already existing lateral root primordia . The regulation is linked tightly with auxin . For example, ethylene application results in the accumulation of auxin at the tip of Arabidopsis primary roots through the promotion of auxin synthesis mediated by WEAK ETHYLENE INSENSIVE2/ANTHRANILATE SYNTHASE α1 and WEI7/INSENSIVE2/ ANTHRANILATE SYNTHASE β1 . WEI2 and WEI7 encode the α and β subunits, respectively, of anthranilate synthase , a rate-limiting enzyme in the biosynthesis of the auxin precursor Trp . In rice, ethylene also increases endogenous IAA concentrations in the roots; however, the effect is minimized in mutants defective in YUC8/REIN7, which participates in auxin biosynthesis . The homeobox genes are critical for growth and development because they regulate cell fate and plant specificity . A family of zinc-finger homeodomain proteins has an N-terminal conserved domain containing several cysteine and histidine residues for potential zinc binding, in addition to a C-terminal domain containing a homeodomain . Most ZF-HD proteins do not have an intrinsic activation domain, which suggests that interactions with other factors are necessary for transcriptional activation . In addition, all 14 members of the ZF-HD gene family in Arabidopsis are predominantly expressed in floral tissues and play key roles in their development . One member, AtHB33, which is negatively regulated by ARF2, is required for seed germination and primary root growth . Among the 11 ZF-HD genes in rice, the over expression of OsZHD1 and OsZHD2 induces leaf curling by controlling the number and arrangement of bulliform cells . Here, we report that the over expression of OsZHD2 in rice improves root growth by enhancing meristem activity. We demonstrated that the homeobox protein elevated ethylene concentrations by increasing the transcript levels of ethylene biosynthesis genes. We further obtained ChIP assay data that revealed an interaction between OsZHD2 and the chromatin of ACS5. Analyses of transgenic rice plants carrying DR5::GUS and DR5::VENUS revealed that the expression of the DR5 reporter gene was induced following treatment with ACC, an ethylene precursor. The results suggest that OsZHD2 increases the biosynthesis of ethylene and subsequently auxin, which stimulates root growth.We isolated a rice mutant plant with an extensive root system from a population of activation tagging lines, in which the expression of a gene is enhanced by multiple copies of the 35S enhancer introduced using T-DNA .

In Line 3A-13017, the root biomass increased significantly . At 8 DAG the seminal roots were 27% longer in the activation plants than in the WT . Their lateral roots were also much longer than in the WT at a similar stage. At the upper parts of the seminal roots, the mutant lateral roots were 144% longer than those of the WT . This activation line also had more lateral roots—230 per seminal root for Line 3A-13017 versus 179 laterals per seminal root for the WT . However, the density of lateral roots did not differ significantly between the genotypes , which indicated that the increase in the number of lateral roots was largely due to the mutant plants having longer primary roots. We located T-DNA 5 kb downstream from the stop codon of OsZHD2 in the transgenic line . Its expression was significantly higher than that of the control, potentially because of the 35S enhancer elements in the T-DNA border region . We designated this activation line as OsZHD2-D.qRT-PCR analysis revealed that the expression level of OsZHD2 was significantly higher in the root tips when compared with levels in the total root . In addition, the expression level of OsZHD2 was significantly higher in the basal parts of shoots including the SAM compared with upper parts of the shoots that contain leaf blades and sheathes . RNA in situ hybridization experiments revealed that OsZHD2 transcripts were abundant in the root tip regions . Several homeobox genes have been identified as key regulators of cell proliferation and specification at the early stages of embryogenesis in plants. Among 107 homeobox genes identified in the rice genome, the expression profiles from 93 members in different tissues during various developmental stages have been analyzed . The results of the analyses revealed that OsZHD2 is highly expressed in the SAM . To evaluate whether OsZHD2 induces meristem activity, we treated seedling plants with 10 µM EdU, a thymidine analog, for 2 h to visualize the S-phase cells that actively incorporate EdU into DNA . The assay results revealed that OsZHD2-D had a higher number of S-phase cells in the RAM compared with the number of cells in the WT . The RAM region is defined based on the number of cells in a file that extend from the quiescent center to the first elongated cell . Quantifying such epidermis cells in the meristem region of lateral roots revealed that the number increased significantly in the activation line—25 versus 15 for the WT —which suggested that enhanced OsZHD2 expression led to the elongation of the RAM region.To confirm that the phenotypes observed from OsZHD2-D were due to the elevated expression levels of OsZHD2, we generated transgenic plants that expressed full-length OsZHD2 cDNA under the control of the maize Ubi promoter . From six independently transformed plants, we selected two lines, OX2 and OX4, which expressed OsZHD2 at high levels . Both had more extensive root systems compared with those of the out segregated WT . Their seminal roots and lateral roots were also significantly longer , and the plants had more lateral roots than the WT . However, the density of lateral roots did not vary among genotypes .

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Hydroponics Unveiled: A Deep Dive into Modern Agriculture Techniques

Interestingly, in companion cells of the phloem, AVP1 was also shown to be localized to the plasma membrane and function as a PPi synthase that contribute to phloem loading, photosynthate partitioning, and energy metabolism. On the other hand, AVP1 is also believed to contribute to the establishment of electrochemical potential across the vacuole membrane, which is important for subsequent vacuolar secondary transport and ion sequestration. Constitutive over expression of AVP1 improves the growth and yield of diverse transgenic plants under various abiotic stress conditions—including drought, salinity, as well as phosphorus and nitrogen deficiency—although the mechanism remains to be fully understood. Taken together, AVP1 serves as a multi-functional protein involved a variety of physiological processes in plants, some of which await to be fully understood. Magnesium is an essential macro-nutrient for plant growth and development, functioning in numerous biological processes and cellular functions, including chlorophyll biosynthesis and carbon fixation. Either deficiency or excess of Mg in the soil could be detrimental to plant growth and therefore plants have evolved multiple adaptive mechanisms to maintain cellular Mg concentration within an optimal range. In higher plants, the most well-documented Mg2+ transporters belong to homologues of bacterial CorA super family and are also called “MRS2” based on their similarity to yeast Mitocondrial RNA splicing 2 protein. Several members of the MGT family mediate Mg2+ transport in bacteria or yeast as indicated by functional complementation as well as 63Ni tracer assay . In plants, they have been shown to play vital roles in Mg2+ uptake, translocation,square planter pots and homeostasis associated with their different subcellular localizations and diverse tissue-specific expression patterns. For instance, MGT2 and MGT3 are tonoplast localized and possibly involved in

Mg2+ partitioning into mesophyll vacuoles; MGT4, MGT5, and MGT9 are strongly expressed in mature anthers and play a crucial role in pollen development and male fertility. MGT6 and MGT7 are shown to be most directly involved in Mg homeostasis because knocking-down or knocking-out either of the genes leads to hypersensitivity to low Mg conditions. MGT6 encodes a plasma membrane-localized high-affinity Mg2+ transporter and mediates Mg2+ uptake in root hairs, particularly under Mg-limited conditions . MGT7 is also preferentially expressed in roots and loss-of-function of MGT7 caused poor seed germination and severe growth retardation under low-Mg conditions. Double mutant of mgt6 and mgt7 displayed a stronger phenotype than single mutants, suggesting that MGT6 and MGT7 may be synergistic in controlling Mg homeostasis in low-Mg environment conditions. In contrast to considerable research on Mg transport and homeostasis under Mg deficient conditions, the regulatory mechanisms required for adaptation to excessive external Mg remain poorly understood. Recent studies suggested that MGT6 and MGT7 are essential for plants to adapt to both normal and high Mg conditions. The mgt6 mutant displayed dramatic growth defects with a decrease in cellular Mg content in the shoot, when grown under high Mg2+. Grafting experiments further suggested a shoot-based mechanism for Mg2+ detoxification although the exact role of MGT6 in this process is still not clear. More importantly, a core regulatory pathway consisting of two calcineurin B-like Ca sensors partnering with four CBL-interacting protein kinases has been established that allows plant cells to sequester Mg2+ into plant vacuoles, thereby protecting plant cells from high Mg2+ toxicity. In this study, we identified the tonoplast pyrophosphatase, AVP1, as an important component in high Mg2+ tolerance in Arabidopsis. Furthermore, by analyzing the avp1-4 mgt6 double mutant and avp1-4 cbl2 cbl3 triple mutant, we showed that the role of AVP1 in high-Mg tolerance was independent of previously reported MGT6 or CBL/CIPK-mediated pathway. Instead, our results suggested a novel link between high Mg2+ stress and PPi homeostasis in plants. Reducing the PPi concentration in the cytoplasm and increasing the acidification of vacuoles represent the two main biochemical functions of AVP1.

In order to dissect if both activities are required in this specific high Mg2+-associated process, we resorted to the transgenic line expressing yeast IPP1 gene under the control of the AVP1 promoter in the fugu5-1 mutant background. IPP1 is a cytosolic soluble protein which is not capable of translocating H+ , thus decoupling the hydrolysis and proton pump activities. Interestingly, our results showed that the severely retarded growth of fugu5-1 mutant plants under high-Mg conditions was completely recovered by expression of the IPP1 gene . The quantitative analysis of seedling fresh weight confirmed the complementation . To extend the phenotypic analysis of the avp1 mutants in mature plants, we examined the phenotype of avp1 mutants using hydroponic culture system. Consistent with the patterns of plant growth on agar plates, the mutant plants exhibited a pronounced growth defect than wild-type plants in the hydroponic solutions supplemented with 15 mM external Mg2+, as revealed by much lower fresh weight and lower chlorophyll content . The IPP1 transgenic line also behaved like wild-type plants but not avp1 mutant under this condition, suggesting that PPi hydrolysis is the key function that AVP1 plays in high-Mg adaptation. Although Mg is an essential macro-nutrient required for plant growth, high concentrations of environmental Mg2+ could be detrimental, and the targets underlying toxic effect of high-Mg are not well understood. In the present study, we characterized multiple avp1 mutant alleles and found they were hypersensitive to high external Mg2+. This finding has not only improved our understanding of the mechanism underlying Mg2+ tolerance but also uncovered a novel physiological function of AVP1 in plants. When the plants were confronted with high Mg stress, sequestration of excessive Mg2+ into the vacuole plays a vital role in detoxification of Mg excess from the cytoplasm . The AVP1 protein predominantly localized in the vacuolar membrane and was a highly abundant component of the tonoplast proteome. Encoded by AVP1, vacuolar H+ -PPase, together with vacuolar H+ -ATPase, plays a critical part in establishing the electrochemical potential by pumping H+ across the vacuolar membrane. This proton gradient, in turn, facilitates secondary fluxes of ions and molecules across the tonoplast. Based on this well-established idea, we hypothesized that avp1 mutants may be impaired in cellular ionic homeostasis and should thus exhibit hypersensitivity to a broad range of ions.

However, unexpectedly, we found that avp1 was hypersensitive only to high external Mg2+ but not to other cations . It was shown that over expression of AVP1 improved plant salt tolerance in quite a few species, which was interpreted as the result of increased sequestration of Na+ into the vacuole. It is thus reasonable to speculate that the tonoplast electrochemical potential generated by AVP1 would likewise favor Mg2+ transport into vacuoles via secondary Mg2+/H+ antiporter. Surprisingly, our subsequent experiments did not support this hypothesis and several lines of evidence suggested that the hypersensitivity of avp1 to high Mg2+ was not due to the compromised Mg2+ homeostasis in the mutant. First, unlike other high Mg2+-sensitive mutants such as mgt6 and the vacuolar cbl/cipk mutants, the Mg and Ca content in the avp1 mutant was not altered as compared with wild type, suggesting that AVP1 may not be directly involved in Mg2+ transport in plant cells. Second, higher order mutants of the avp1-4 mgt6 double mutant and avp1-4 cbl2 cbl3 triple mutant displayed a dramatic enhancement in Mg2+ sensitivity as compared to single mutants. These genetic data strongly suggest that AVP1 does not function in the same pathway mediated by MGT6 and does not serve as a target for vacuolar CBL-CIPK. Moreover,blueberry grow it was previously shown that either vacuolar H+ -ATPase double mutant vha-a2 vha-a3 or the mhx1 mutant defective in the proposed Mg2+/H+ antiporter was not hypersensitive to high Mg2+. These results implicate the vacuolar Mg2+ compartmentalization should be fulfilled by an unknown Mg2+ transporter/channel, whose activity is largely not dependent on the tonoplast ∆pH. Identification of this novel Mg2+ transport system across the tonoplast, which is probably targeted by vacuolar CBL-CIPK complexes, would be the key to understand the mechanism. Third, expression of the cytosolic soluble pyrophosphatase isoform IPP1 could fully rescue the Mg-hypersensitivity caused by AVP1 mutation. These lines of evidence pinpoint PPi hydrolysis, rather than ∆pH-assisted secondary ion transport and sequestration, as the major function of AVP1 in high Mg2+ adaptation. Under high Mg stress conditions, a number of adaptive responses are supposed to take place in plants, including the remodeling of plant morphogenesis as well as reprogramming of the gene expression and metabolite profile. However, very little is known so far and therefore, the molecular components targeted by excessive Mg2+ in plant cells remain obscure. Here, we suggest that the concentration of cellular PPi could be responsive to external Mg supply. Our results showed that extremely high levels of Mg2+ led to inhibition of the PPase activity in Arabidopsis, which in turn, resulted in the elevation of PPi content in the cytosol. Because high level of PPi is very toxic, the efficient removal of PPi by AVP1 under high Mg2+ conditions might become one of the limiting factors for optimal plant growth. This idea is supported by the observation that avp1 mutants accumulated significantly higher PPi content under high Mg2+ conditions compared with normal conditions . Most importantly, heterologous expression of the soluble PPase IPP1 gene rescued high Mg-sensitive phenotype of fugu5-1 , which strongly suggested that high Mg2+ hypersensitivity phenotype in avp1 mutants could primarily be attributed to impaired PPi homeostasis.It would be interesting to investigate how PPi concentrations vary in different Mg2+ conditions and during different plant growth stages.

Recently, cytosolic soluble pyrophosphatases were identified in Arabidopsis, and were shown to physiologically cooperate with the vacuolar H+ -PPase in regulating cytosolic PPi levels. Future studies should clarify if this type of soluble isoenzymes is also involved in the same high-Mg adaptation process. Collectively, our findings provide genetic and physiological evidence that AVP1 is a new component required for plant growth under high external Mg2+ concentrations and functions in regulating Mg2+ tolerance via PPi hydrolysis. For on-plate growth assays, seeds of different genotypes were sterilized with 75% ethanol for 10 min, washed in sterilized water for three times, and sown on Murashige and Skoog medium containing 2% sucrose and solidified with 0.8% phytoblend . The plates were incubated at 4 ◦C in darkness for two days and then were positioned vertically at 22 ◦C in growth chamber with a 14 h light/10 h dark photoperiod. After germination, five-day-old seedlings were transferred onto agarose-solidified media containing various ions as indicated in the figure legends and were grown under 14 h light/10 h dark photoperiod. For phenotypic assay in the hydroponics, 10-day-old seedlings geminated on MS plate were transferred to 1/6 strength MS solution and were grown under the 14 h light/10 h dark condition in the plant growth chamber. Fresh liquid solutions were replaced once a week. After two-week culture, the plants were treated with 1/6 MS solutions supplemented with 15 mM MgCl2. 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 ENM-contaminated bio-solid 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. Gravity-driven vertical transport of TiO2, CeO2, and Cu2 engineered nanomaterials and their effects on soil pH and nutrient release were measured in three unsaturated soils. ENM transport was found to be highly limited in natural soils collected from farmland and grasslands, with the majority of particles being retained in the upper 0-3 cm of the soil profile, while greater transport depth was seen in a commercial potting soil. Physical straining appeared to be the primary mechanism of retention in natural soils as ENMs immediately formed micron-scale aggregates, which was exacerbated by coating particles with Suwannee River natural organic matter . Changes in soil pH were observed in natural soils contaminated with ENMs that were largely independent of ENM type and concentration. These changes may have been due to enhanced release of naturally present pH-altering ions in the soil, likely via substitution processes. This suggests ENMs will likely be highly retained near source zones in soil and may impact local communities sensitive to changes in pH or nutrient availability.

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Harvesting the Future: Hydroponic Agriculture and Global Food Security

Although this coating may not persist on the particles in the environment, what is clear is that the effects of chronic dosing and the effects of coating are critical data gaps that should be evaluated. Also completely lacking are more environmentally realistic exposure scenarios, such as ones using natural waters and soils and also multi-species microcosm or mesocosm studies, although such studies are underway. These studies will bring the importance of environmental transformations and indirect ecological impacts into light. It is possible that community or ecosystem level impacts may be more sensitive than individual level effects. Also more chronic and food chain transfer studies should be encouraged to deal with the possible long term effects from, or accumulations of, the likely persistent nanoceria entities. The current available data do not suggest an immediate risk from acute exposures to nanoceria from use as a fuel additive or mechanical/chemical polishing or planarization. However, the data gaps we have discussed should be addressed before a comprehensive ecological risk assessment can be performed for ceria for chronic exposures or for other exposure pathways. This review lays the foundation for such assessments and clearly identifies the areas where research is most critically needed.The Bioremediation, Education, Science and Technology partnership provides a sustainable and contemporary approach to developing new bio-remedial technologies for U. S. Department of Defense priority contaminants while increasing the representation of underrepresented minorities and women in an exciting new bio-technical field. This comprehensive and innovative bio-remediation education program provides under-represented groups with a cross-disciplinary bio-remediation curriculum and financial support, coupled with relevant training experiences at advanced research laboratories and field sites. These programs are designed to provide a stream of highly trained minority and women professionals to meet national environmental needs. The BEST partnership of institutions and participants benefit from a unique central strategy— shared resources across institutional boundaries.

By integrating diffuse resources, BEST forms a specialized “learning institution without walls,” large plastic pots for plants where participants can receive advanced training at any BEST site, and where research capabilities flow freely among the participating institutions. Ongoing faculty and student exchange programs, video taped lectures, the Rotating Scholars program, and the BEST web-site ensure that all participants are empowered with opportunities to excel. The BEST partnership consists of Lawrence Berkeley National Laboratory in Berkeley, Calif., Jackson State University in Jackson, Miss., Ana G. Méndez University System in Puerto Rico, University of Texas at El Paso , University of Southern Mississippi Gulf Coast Research Lab, and University of California at Berkeley . The BEST program contract to the partnership is man-aged by LBNL for the Army Corps of Engineers, Waterways Experiment Station in Vicksburg, Miss. WES manages the contract for the Army Corps of Engineers and is the contracting entity for DoD. The partnership formed by these participating institutions leverages existing institutional resources by strengthening intramural bio-remediation education and research capabilities, and through outreach pro-grams, to disseminate training and scientific enhancement to other Historically Black Colleges and Universities and Minority Institutions . The BEST institutions are focal points for the development and dissemination of cutting-edge research and technology for the bio-remediation of nitro-aro-matic compounds, polycyclic aromatic hydrocarbons and toxic metals. The multidisciplinary BEST partnership strategy creates a flask-to-field solution that develops laboratory research into technology, and technology into field-scale environmental applications required for the cost-effective restoration of damaged environments. This year saw the addition of the University of Southern Mississippi’s Gulf Coast Research Lab and the University of Texas at El Paso as partners in the BEST program. Both institutions provide significant new personnel and training opportunities for the BEST program. The USM Gulf Coast Research Lab investigators’ focus on PAH and heavy metal phytoremediation along shorelines provides an exciting new focus with increased field opportunities for students. The UTEP investigators are focusing on exciting new metal phy-toremediation techniques using desert plants and exciting new techniques to determine risk assessment with PAHs. This year also saw the passage of the program director-ship at LBNL from Dr. Jenny Hunter-Cevera to Dr. Terry C. Hazen in October 1999. Dr. Hunter-Cevera, who has managed the BEST program at LBNL since its inception, will be sorely missed, but her new position as president of the Maryland Biotechnology Institutes may provide increased opportunities for collaboration for the entire BEST program. Dr. Hazen, who specializes in bio-remediation field applications, has demonstrated or deployed bio-remediation technologies at more than 50 sites around the United States and in Europe. He has five patents in bio-remediation technologies that are licensed by more than 40 companies in the U.S. and Europe.

During the past year, the BEST program has provided minority research training for five high school students, 74 undergraduates, 32 graduate students, three post-doctoral fellows and 10 faculty. Students and fac-ulty investigators have given 43 presentations on BEST research at scientific meetings and have published 17 scientific papers. The program produced a full color brochure and flyers in 1999 for use in recruiting more students, and also sponsored 32 lecture/seminars on bio-remediation. Fourteen videotapes of BEST seminars at LBNL/UCB were distributed to the partner institutions. The BEST program also sponsored a phytoremediation workshop for BEST investigators and students that was attended by more than 60 participants. Additional workshops are planned for the coming year. In this report, the research is organized by subject area, and two-page briefs are presented for each of 28 BEST projects. The projects presented provide a good representation of the state-of-the-science research being done with students in the BEST program – the best of BEST.Over the next 75 years, the U.S. government will undertake what has been called the largest civil works project in world history to restore the environment damaged by previous activities at federal sites, e.g., Department of Defense military bases and Department of Energy nuclear facilities. Legislative action, resulting from concern over the accumulating hazards, has mandated pollution control measures and environmental restoration of hazardous waste at all sites. Estimates of total cleanup costs range from $230 billion to more than half a trillion dollars. Given the trend of diminishing budgets throughout the federal government, future generations could inherit both an environmental and budgetary disaster. The imprecision of the cost estimates results from the lack of knowledge of how “clean” the contaminated sites will need to be. Some of the environmental damage is permanent—cleanup technologies either do not exist or are incapable of remediating the contamination. For DoD bases being closed by the Base Realignment and Closure Program, all toxic sites must be remediated before the site is returned to public use. The projected costs of site restoration using existing technologies are staggering: the estimated cleanup cost is at least $24.5 billion for the 7,313 identified U.S. sites . The pollutants at these sites include chlorinated hydrocarbons, metals, petroleum products, explosives, mixed waste and other organics. DOE also has substantial remediation costs—estimated to be from $90 billion to $200 billion . The domestic private sector presents yet another huge set of remediation problems, dwarfed only by the international problems in Eastern Europe and Russia . There is clearly a need for new cost-effective treatment technologies. Bio-remediation, the use of microor-ganisms to detoxify hazardous waste, promises to provide economical and ecologically sound clean-up strategies. An Office of Technology Assessment analysis concluded that the U.S. does not possess a sufficient pool of qualified environmental professionals, i.e.,blueberry pot the trained scientific personnel required to support this rapidly developing multi-disciplinary field. In response to these national environmental needs, the Bio-remediation Education, Science and Technology Program, funded by DoD, was established in 1996. In a few short years, BEST has pioneered a new and successful model for environmental science and education. This partnership has a highly integrated programmatic focus on the scientific and workforce needs of DoD. Since the inception of the BEST program, a significant number of major milestones and deliverables have been achieved. They are described below. The BEST program has made these dramatic accomplishments by using an approach that combines a training-education element with an integrated research project, described later in this introduction.DoD sites throughout the United States contain highly contaminated soils, groundwater and sediments. These properties pose direct and indirect exposure hazards to humans and wildlife.

Conventional remedial solutions for contaminated soils and sediments or groundwater are slow and expensive, increase inputs to hazardous waste disposal sites, and can increase human exposure to contaminants. Bio-remediation — the use of microo ganisms to destroy hazardous contaminants or to con-vert them to harmless forms — is an emerging treatment technology that can in many instances restore contaminated environments more quickly, at lower cost and at lower human risk than alternative remediation technologies. Bio-remediation can operate in either an in situ mode where contaminants are treated in place, or in an ex situ mode where contaminants are removed from a contaminated zone for treatment . In situ bio-remediation can be used when excavation is impractical — under buildings, highways, runways, etc. In situ bio-remediation can simultaneously treat soil and groundwater in one step, without the generation of hazardous waste products. In situ contaminant degradation can be achieved by either intrinsic or enhanced bio-remediation. Intrinsic bio-remediation exploits the innate capabilities of indigenous micro-bial communities to degrade pollutants. Enhanced bio-remediation seeks to accelerate in situ microbial activity by isolating and controlling the contaminated site so that the microbial environment can be purposely manipulated to correct nutritional or gas phase limitations. Ex situ treatment seeks to further control the remedial environment by placing the contaminants in an engineered treatment system. Phytore mediation, a process in which plants and asso-ciated microbial communities are used for contaminant bio-degradation or bio-immobilization, is an important and rapidly developing mode of bio-remediation. To realize the full potential benefits of plant and microbial treatment systems at DoD sites, these bio-technologies must be developed and optimized for remediation of DoD priority contaminants by an expanded pool of qualified professionals. It was in response to these DoD environmental needs that the BEST partnership of institutions was established.In order to determine whether plants can stimulate the degradation of PAHs in soil, plant species found in literature on phytoremediation of metal-contaminated sites were selected to measure the removal of PAHs in artificially contaminated soil over a period of 62 days. The plant species used for this experiment were alfalfa , barley , tall fescue and orchard grass . The PAHs were phenanthrene and anthracene, in a mixture of 600 ppm each. As shown in Figures 1 and 2, phenanthrene and anthracene were removed from the soils with plants after 62 days. More than 98% of the phenanthrene was removed during that period while the anthracene removal was found to be between 70 and 90%. The results suggest that the rate of disappearance of phenanthrene in soil was greater than anthracene under the same conditions. From the results, it is also indicated that the disappearance of PAHs in soil depends on the bio-availability of the compounds. Because phenanthrene is approximately 10 times more soluble in water than anthracene, it was expected to be more readily available to microbial degradation than anthracene. Plant-assisted degradation of PAHs is thought to be more effective on PAHs with a higher number of rings and higher molecular weights, such as benzopyrene. Anthracene removal in the soil planted with alfalfa was greater than in the soil without plants, while all the other plants have minimal to no effect on anthracene removal compared to the control soil. Phenanthrene was removed to a greater extent in the soil with alfalfa and tall fescue compared to the control without plants . However, both barley and orchard grass showed no effects of the removal of phenanthrene during that period when compared to the soil without plants. Overall, plants had minimum effect on phenanthrene degradation while anthracene degradation was more dependent on plant species. In order to determine the effect of PAH degradation by plants on bacterial numbers in soil, bacteria were counted in soil during the course of the experiment.Parathion is a widely used organophosphate insecticide which can cause adverse neurological effects if ingested or after dermal exposure. No single microor-ganism has been isolated that is capable of completely mineralizing parathion and its metabolites. Hydrolysis of parathion significantly lowers the toxicity of the parent compound, but results in the formation of a toxic intermediate, the nitroaromatic compound p-nitrophenol.

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Hydroponic Agriculture: Cultivating the Future of Sustainable Farming

Interestingly, suppression of endodermal ABA signalling seems to contribute to the inactivation of aquaporin-mediated Lpr in a wild-type Scheduling low but frequent NO3 − applications, at-tuned to crop demand, allows the crop to take up most of the NO3 − before it passes through the low-salinity zone into the saline fringes. Figure 7 simulates continuous NO3 − application and a scenario which applies NO3 − only every 10 d, while the total amount of NO3 − applied is the same for both simulations. High-frequency applications of NO3 − using drip irrigation in-creased N uptake efficiency in some cases .Both Casparian strips and suberin lamellae, two extracellular hydrophobic barriers located in the wall of endodermal cells of the root, are thought to play important roles in restricting the free diffusion of solutes and water . Casparian strips act as apoplastic barriers not only to block solutes moving into the xylem through the free space between cells, but also to prevent their backfow from the stele to the apoplast of the cor-tex. Suberin lamellae, due to their deposition between the endodermal plasma membrane and secondary cell wall, do not block aploplastic transport but rather limit transcellular transport of nutrients and possibly water at the endodermis. Cross talk between the Casparian strip and suberin lamellae exists, with suberin being deposited in response to disruption of Casparian strips . Tese extracellular barriers are therefore at a cross-road between control of mineral nutrient and water uptake. However, the mechanisms that allow plants to integrate both these barrier functions to enable the simultaneous uptake of sufcient water and mineral nutrients remain under explored. Te dirigent-like protein Enhanced Suberin1 functions in the correct formation of Casparian strips by allowing the lignin, deposited at the Casparian Strip Domain through the action of Peroxidase64 and the Respiratory Burst Oxidase Homolog F ,blueberry packaging to form into a continuous ring. In the absence of this dirigent-like protein defective Casparian strips are formed along with enhanced and early deposition of suberin in the endodermis.

A similar pattern of Casparian strip disruption and response is also observed when the Casparian Strip Domain is disrupted through the loss of Casparian Strip Domain Proteins. Tese changes lead to systematic alterations in the profile of mineral nutrients and trace elements accumulating in leaves, and this phenotype provided the first tool for identification of genes involved in Casparian strip development. Detection of the diffusible vasculature-derived peptides CASPARIAN STRIP INTEGRITY FACTORS 1 & 2 through interaction with the SCHENGEN3 receptor-like kinase is what drives this endodermal response to loss of Casparian strip integrity. Here, we report that detection of a loss of Casparian strip integrity at the root endodermis by the CIFs/SGN3 pathway leads to an integrated local and long-distance response. This response rebalances water and mineral nutrient uptake, compensating for breakage of the Casparian strip apoplastic seal between the stele and the cor-tex. This rebalancing involves both a reduction in root hydraulic conductivity driven by deactivation of aquapor-ins, and limitation of ion leakage through deposition of suberin in endodermal cell walls. This local root-based response is also coupled to a reduction in water demand in the shoot driven by ABA-mediated stomatal closure.Te dirigent-like protein Enhanced Suberin1 functions in the formation of Casparian strips by allowing the correct deposition of lignin at the Casparian strip domain. Te enhanced deposition of suberin in the esb1-1 mutant with disrupted Casparian strips can clearly be observed using the lipophilic stain Fluorol Yellow 088 close to the root tip , and this can be quantified by counting the number of endodermal cells afer the onset of cell expansion to the first appearance of yellow fuorescence . This early deposition of suberin is also verifed by the clear correspondence of FY 088 staining with enhanced promoter activity of known suberin biosynthetic genes, including GPAT5 monitored through both GUS staining and GFP fuorescence , and also others through GUS staining . This is further reinforced by the enhanced expression of known suberin biosynthetic genes in esb1-1 relative to wild-type . To better understand the causal link between Casparian strip integrity and enhanced deposition of suberin, we performed a reciprocal grafing experiment that revealed that the esb1-1 mutation is only required in the root to drive enhanced deposition of suberin at the endodermis, placing the function of ESB1 and the driver for increased suberin in the same tissue .

To determine the cause and effect rela-tionship between damaged Casparian strips and enhanced suberin we carefully monitored the first appearance of both Casparian strips and enhanced suberin in esb1-1. Using lignin staining in the Casparian strip marker line pCASP1::CASP1::GFP, we are able to observe that damaged Casparian strips are visible 2.5 days afer sowing . This is at least 12hr before the first indication of enhanced suberin biosynthesis, which we monitor using promoter activity of suberin biosynthetic genes GPAT5, FAR4, FAR1 and FAR5 . This was also verified by the direct observation of suberin deposition with FY 088 . Te observation that treatment with the CIF2 peptide, normally leaked from the stele through loss of Casparian strip integrity, can enhance suberin deposition in wild-type plants supports our interpretation that enhanced suberin deposition is a response to loss of integrity of the Casparian strip-based apoplastic diffusion barrier. Furthermore, loss-of-function of the receptor-like kinase SGN3, required for sensing of CIFs, blocks the enhanced deposition of suberin in esb1-1 and casp1-1casp3-1 based on a chemical analysis of suberin in esb1-1 , and also on FY 088 staining. We conclude that Casparian strip defects sensed by the CIFs/SGN3 surveillance system lead to enhanced deposition of suberin in the endodermis.Te observation that enhanced suberin is deposited as a response to loss of integrity of the endodermal-based diffusion barrier between stele and cortex, raises the question, what is the function of this increased suberin deposition? Previously, the extent of endodermal suberin has been shown to be part of the response to nutrient status . We therefore tested the selectivity to solutes σNaCl, in roots varying in the extent of suberin deposition and the functionality of Casparian strips. For this, we measured solute leakage into xylem sap of pressurized roots at increasing sodium chloride concentrations in the solution bathing the roots. Taken individually, σNaCl of roots of esb1-1, sgn3-3 and wild-type were not significantly different from one another , which is surprising given the disruption of the Casparian strip-based apoplastic diffusion barrier in both mutants.

However, removal of suberin in esb1-1, by endodermal-specific ectopic expression of a cutinase ,blueberry packaging box caused a significant decrease in σNaCl compared to wild-type plants , and a similar tendency when compared to esb1-1 . This supports the notion that enhanced suberin deposition at the endodermis helps prevent passive solute leakage caused by defects in the Casparian strips of the esb1-1 mutant. We also observed a significant decrease in σNaCl in the double mutant esb1-1sgn3-3 compared to both wild-type and sgn3-3 . It is known that SGN3 is required for the enhanced deposition of suberin that occurs at the endodermis in esb1-1 . Our observation that removal of this enhanced suberin in esb1-1sgn3-3 decreases σNaCl further supports our conclusion that the role of this increased suberin deposition is to limit solute leakage where Casparian strip barriers are disrupted.It has also been suggested that endodermal suberin may impact water permeability, though how is still unclear. To further address the role of enhanced endodermal suberin, we investigated root hydraulic conductivity of esb1-1 and observed a significant reduction by 62% with respect to wild-type . Importantly, this difference in esb1-1 Lpr originates mainly from a reduction in an aquaporin-mediated water transport pathway . We also observed that the azide-resistant water transport pathway was lower in esb1-1 than in wild-type , yet to a lesser extent than the aquaporin mediated pathway. Te dra-matic reduction in aquaporin-mediated Lpr in esb1-1 we observe is an intriguing fnding, which led us to consider if this lack of aquaporin activity in esb1-1 roots is due to a direct output from the CIFs/SGN3 signalling pathway, or if it represents an efect downstream of enhanced suberin deposition. We found that removal of endodermal suberin in esb1-1 through expression of CDEF1 in the endodermis had no further efect on Lpr . This rules out a role for suberin in the reduced aquaporin-mediated Lpr of esb1-1. However, in the esb1-1sgn3-3 dou-ble mutant, as compared to esb1-1, we observed a full recovery of Lpr back to wild-type levels . Loss of Casparian strip integrity in esb1-1 therefore appears to be sensed by the CIFs/SGN3 signalling pathway, which leads to the inactivation of aquaporins, thereby reducing Lpr . To support this conclusion, we show that exogenous application of CIF2 to wild-type plants for 3h induces a reduction in Lpr, and only in the presence of a functional SGN3 . We have established the existence of two critical outputs of the CIFs/SGN3 diffusion-barrier surveillance system. Tese are enhanced deposition of endodermal suberin limiting solute leakage, and the inactivation of root aquaporin activity reducing Lpr. Do these two independent outputs of the CIFs/SGN3 diffusion barrier surveillance system work in parallel, or in series with one response leading to the other? Te fact that removal of endodermal suberin in esb1-1 does not compensate for its reduced Lpr suggests that enhanced suberin deposition is not the cause of the reduced aquaporin-mediated Lpr. However, reduced activity of aquaporins through loss-of-function of the two major aquaporins PIP2;1 and PIP2;2 in the pip2;1pip2;2 double mutant, does cause significant increases in endodermal suberin deposition . A similar increase in suberin is also observed afer treatment with the aquaporin inhibitor sodium azide through observation of the activity of the transcriptional reporter pGPAT5::mCITRINE-SYP122 for suberin biosynthesis. GPAT5 expression is observed to expand toward the root tip after 6 hours only of sodium azide treatment .

Based on this evidence, we propose the following sequence of events. Casparian strip defects are detected by the apoplastic leakage of CIFs from the stele, being sensed by SGN3. Once activated, SGN3 signals the inactivation of aqua-porins thereby reducing Lpr which in turn leads to the early and enhanced deposition of endodermal suberin. Insuch a model, SGN3 would inhibit aquaporin function, which may appear at variance with the usual activation of aquaporins through phosphorylation. Yet, such an inhibition was recently described in the case of FERONIA, a protein kinase inactivating PIP2; 1 function through an as yet unknown mechanism.Abscisic acid has been shown to be involved in regulating both aquaporin activity reviewed in and suberin deposition, making ABA an interesting can-didate worth exploring for a role in downstream CIFs/SGN3 signalling. To probe this possibility we expressed the dominant negative allele of the regulator of ABA signalling ABA-INSENSITIVE 1 in the endoder-mis of esb1-1 using pELTP::abi1. This abi1 construct specifically blocks ABA signalling at the endodermis and delays suberisation in a wild-type background as previously shown in . In esb1-1, we observed abi1 to have no effect on either the inactivation of aquaporins or the enhanced deposition suberin . We also observe that aquaporin inhibition with sodium azide in the pELTP::abi1-1 line still induces expression of the suberin biosynthesis gene GPAT5 toward the root tip in the pGPAT::mCITRINE-SYP122 line, as observed in wild-type . Based on this, activation of ABA signalling in the endodermis does not link perception of Casparian strip defects with the downstream responses of reduced aquaporin-mediated Lpr or suberin deposition. Suppbackground .The esb1-1 mutant is known to have reduced stomatal apertures and enhanced wilting resistance. This suggests that the CIFs/SGN3 sensing system not only initiates a local root response to Casparian strip integrity but is also involved in initiating long-distance responses in the shoot. We observe reduced stomatal apertures in esb1-1 , and an analysis of the expression of a set of known ABA signalling and response genes in leaves suggest that this stomatal closure is part of an ABA driven response. The aba1 mutation confers a strong ABA deficiency.By generating an esb1-1aba1 double mutant, we investigated the ABA-dependent component in the leaf response we observe in esb1-1. ABA-defciency in esb1-1aba1 suppressed both the reduced stomatal aperture and the activation of expression of ABA signalling and response genes that we observe in esb1-1 .

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The extract solution contains Griffithsin as well as the host and viral protein impurities

To ensure consistency of the nutrient solution, all water was assumed to be treated by reverse osmosis with solution-monitoring for proper pH and dissolved solids content. The three phases of plant growth require a total batch time of 38 days in the upstream portion of the facility. Due to the protracted and continuous nature of plant cultivation, the upstream portion of the facility contains multiple concurrent batches staggered at different stages of growth. When one batch graduates to the next step of production , the trays containing the batch’s biomass are cycled out and the corresponding rack space is immediately filled with a new rotation of trays. We divided the 38-day growth period into 11 concurrent batch periods, with one batch ready to enter downstream purification every 3.44 days. Table 1 is a summary of the number of plants, trays and batches that comprise the upstream facility at any given moment. For model building, batch schedules were calculated under the initial assumption of 24/7 operation for 330 days per year. Plant uptake of nutrients and growth were assumed to be linear reaching 15 g FW per plant at viral inoculation and then increasing in mass to reach 40 g FW per plant at harvest. A 5% failure rate of TMV inoculation was assumed . The Griffithsin expression rate was fixed at 0.52 g/kg FW of harvested biomass, with a downstream recovery of 70%, based on pilot-scale results. Additionally, nutrient solution demand was assumed to match observed biomass growth rates assuming that for each kilogram of nutrient solutions, 0.5 kilogram goes into biomass and the remainder is considered aqueous waste. The materials used, quantities and source are summarized in Supplementary Table 1 in Supplementary Material, plastic growing bag together with clarifying comments and references that were used to assist in the calculations. Using the inputs shown in Supplementary Table 1, the upstream and downstream processes were modeled in SuperPro.

The results generated by the software for the upstream operations are shown in Figure 1, with scheduling shown in the equipment occupancy chart in Figure 2. The following descriptions elaborate on the schema presented in each figure. Griffithsin recovery and purification was modeled as a batch process in a facility with an available operating time of 330 days a year for 24 h a day and 7 days a week. In each year, there are 95 batches total to produce 20 kg of purified Griffithsin API. Since the recovery and purification process only takes 1.6 days, the downstream facility has a significant down time of 2.78 days between batches. Overall, each batch requires 39.6 days from seed planting to formulating the final product, with 38 days upstream and 1.6 days downstream. In Figure 1, the upstream processes are dictated by 11 concurrent batches with each batch being 3.44 days apart from each other. A batch basis of 3.44 days was chosen to decrease equipment idle time and thereby increase downstream equipment utilization efficiency. Despite the 39.6-day batch period and a 332-day operating year, in the model the batch time upstream was reduced to approximately 38 days and the operating year was increased to 365 days to reach the desired 95 batches per year. This was done because SuperPro reproduces uniform results for each year. The goal of the upstream process operations is to produce sufficient biomass to enable isolation of 20 kg Griffithsin per annum. The modeling results show that each batch would produce 578 kg of biomass containing 300 g of Griffithsin, assuming an expression yield 0.52 g API/kg FW biomass . Because induction was modeled using infection with recombinant TMV vector, the three main phases in upstream are germination, pre-inoculation, and post inoculation. The duration of the phases in the model are 21 days, 3 days, and 14 days, respectively. Each batch of N. benthamiana plants goes through a germination phase of 21 days and the germination room is designed with a capacity to grow the 86,700 plants necessary to reach the production goal.

This step of the process uses 90 germination trays, each holding about 960 plants, distributed among 6 batches in the germination room. After 21 days post germination, the N. benthamiana plants are transplanted to a lower density to enable further growth. Thus, seedlings from one germination tray are transplanted into three grow trays , meaning that there are three times the number of trays in pre- and post-inoculation, individually, than in germination. The plant density is 646 plants per m2 in the germination trays and 215 plants per m2 after transplantation. In practice, during transplantation each plant will spend only a few minutes away from its growth environment to minimize transplant shock and undue stress. In the model, the overall time was overestimated to be 3 h to accommodate other necessary procedures, such as moving the plants back to the tray stacks. The transplanted trays are relocated to pre-inoculation rooms that are designed to accommodate the increased area from transplanting for ∼3 days. The pre-inoculation room contains 1 batch, each containing 45 trays with 320 plants per tray. Recombinant TMV for inoculation is produced in and isolated from N. benthamiana. The plant growth model is the same as the rest of N. benthamiana plants. By using infected plants and the purification model defined by Leberman , 4 mg of pure TMV per gram of infected plant material can be recovered . Each batch is equivalent to 14,450 plants distributed on 45 trays. Less than 1 microgram of TMV virion is needed to inoculate each plant . Thus, approximately 14.5 mg of TMV is needed per batch and the necessary amount of TMV to inoculate a batch can be produced from a single N. benthamiana plant. Multiple batches of TMV solution can be made simultaneously and stored at −20◦C . TMV production can be done at lab scale and equipment, labor and material costs are negligible compared to the overall cost of plant maintenance. The isolated TMV is incorporated in diatomaceous earth buffer solution at a concentration of 10 micrograms per 2.5 mL of diatomaceous earth buffer solution, which contains 1% by volume diatomaceous earth and 2% by volume of sodium/potassium-based buffer .

The selected inoculation volume of 2.5 mL is a safe middle value from the range suggested in the literature . In the model, the estimated mixing and transfer time for the solution is 1 h, which starts at the beginning of post-inoculation, so the plants and solution enter the same stage together. A forklift is used to transport the plants into the inoculation room. The plants are inoculated with the diatomaceous earth buffer solution described above with a high velocity spray. Inoculation machines are often custom made and consist of a conveyor traveling through an enclosed cylinder equipped with high pressure spray nozzles aimed at the plants’ aerial structures. Once the inoculation is complete, the trays are conveyed to the post-inoculation growth room,wholesale grow bags which is similar in design to the pre-inoculation growth room; the main difference being its size. The post-inoculation room contains 4 batches at any given time for a total of 180 trays with 320 plants per tray. The scheduling of 3 batches is summarized in the equipment occupancy chart in Figure 2. As shown, seeding, germination, transplant, pre-inoculation, inoculation, and post-inoculation occur sequentially, and the batches are staggered by 3.44 days. The downstream unit operations developed in SuperPro are shown in Figure 3, with scheduling summarized in the equipment occupancy chart shown in Figure 4. The following descriptions elaborate on the schema presented in each figure. At the end of each 3.44 day growing rotation cycle upstream, one batch of N. benthamiana plants is ready to be transferred to downstream processing. This is done by placing each tray of plants onto a conveyor system which leads them to the first phase of downstream operations. The matured plants are first harvested for the green biomass from which the majority of Griffithsin can be recovered with a single extraction. Additional Griffithsin could be recovered from fibrous material by reprocessing and from roots ; however, reprocessing was not included in this model. The automated harvester processes the 578 kilograms of biomass at a rate of 193 kilograms of biomass per hour. With an operational buffer time of 1 h, this process is thus expected to take 4 h. As the biomass is processed by the harvester, it is directly fed into a shredder which further comminutes the biomass to improve Griffithsin recovery. The shredder operates at a capacity of 193 kg of harvested biomass per hour for 2.8 h. The shredded biomass is then mixed with an extraction buffer in a buffer addition tank. For every kilogram of plant material, 1 L of extraction buffer is added. Thus, for 578 kg of N. benthamiana in a batch, approximately 578 L of extraction buffer are added. The resultant solid-liquid mixture has a total volume of about 1,135 L and is sent through a screw press, which is represented as a generic box in the model. The screw press separates the solidliquid slurry leaving a main process fluid stream of plant extract and a waste stream of biomass. A loss about 12% of the original starting Griffithsin was modeled assuming it to be non-liberated from the homogenized biomass. The removal of the biomass leaves a main process stream that contains about 585 L . To facilitate the aggregation of proteinaceous impurities, the extract solution is transferred into a mixing tank and heated to 55◦C for 15 min. The mixture is passively cooled and simultaneously transferred out of the tank and fed into the first 0.3µm plate-and-frame filter. The extract solution is filter-pressed at 25–30 psig to remove the aggregated protein impurities.

Filtering has a process time of 1 h and requires a filter area of 3 m2 to handle the 590 kg/batch of the process stream. At this stage, the process loses a further 8% of the Griffithsin but removes all the RuBisCO and 87% of the TMV coat protein impurities. The filtrate from this step is transferred to a second mixing and storage tank, mixed with bentonite clay and magnesium chloride, and stored at 4◦C for a 12-h period. This stage is the bottleneck operation for the downstream process. After the 12-h incubation, the solution is filtered through a second 0.3µm filter press and a 0.2µm inline sterilizing filter. These operations remove the remaining protein impurities leaving a Griffithsin extract with greater than 99% purity but at the cost of losing 6% of the Griffithsin. The second plate-and-frame filter has a filter area of about 3 m2 and will process all of the extract in 1 h. There is approximately 222 g of Griffithsin per batch at the end of the filtration phase. Following the filtrations steps, the Griffithsin extract solution is collected in a storage tank and further purified using an AxiChrom column with Capto MMC resin to remove residual color and potential non-proteinaceous impurities. To accommodate the 222 g of Griffithsin in solution, 4.9 L of MMC bed resin is needed at a 45 mg/mL binding capacity . The order of the operations for this chromatography step are: Equilibrate, load, wash, elute, and regenerate. In total, chromatography requires 10 h with the load step taking the longest, at 8 h, because approximately 600 L of solution are processed. Chromatography is necessary to decolorize the extract at the expense of losing 4% of the Griffithsin, giving a remaining Griffithsin mass of 210 g per batch. The 10 L of eluant process fluid is sent through a viral clearance filter and transferred into a pool/storage tank. Subsequently, the extract is sent through an ultrafiltration/diafiltration cycle to remove salts introduced in the chromatography column. After ultrafiltration, the product is transferred into a storage tank to be mixed with the final formulation components. The concentrated Griffithsin is diluted to give a concentration of 10 g/L Griffithsin in 10 mM Na2HPO4, 2.0 mM KH2PO4, 2.7 mM KCl and 137 mM NaCl at pH 7.4. The final volume of the DS is 21 L per batch. As shown by Figure 4, each batch in the downstream requires 39 h of process time which includes all SIP and CIP operations.

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Magnesium is a key regulator of metabolic processes such as glycolysis and the Krebs cycle

Studies in yeasts and animal cells suggest that RPA-coating of single stranded DNA act as a signal to activate ATR dependent downstream phosphorylation, primarily through an associated protein called ATRIP . SUV2 is required for repair of UV induced damage, as is its namesake, and suv2 mutants are also sensitive to HU, MMC and CDDP. In sum, ATR, ALT2, SOG1 and SUV2 are all required for DNA damage response as a result of replication fork stalls. This requirement of ATR, ALT2, SOG1 and SUV2 for a plant’s survival in the response to specific genotoxins as seen with HU, MMC and CDDP heightens the curiosity as to what the true nature of Al’s impact on DNA is. As the sensitivity of atr,alt2, sog1 and suv2 mutant roots to defined genotoxins demonstrates, loss of these factors should lead to sensitivity to Al rather than tolerance if Al directly causes DNA damage. It seems counter intuitive that a plant gains Al tolerance by reducing the function of factors necessary for DNA damage response. If atr, alt2, sog1 and suv2 mutant roots can maintain root growth even in the presence of Al, what actual damage is detected by ATR, ALT2, SOG1 and SUV2 in the presence of Al? Identification of four factors that have clear roles in DNA damage responses suggests that a primary effect of Al toxicity is directly related to compromised genomic integrity, with Al possibly serving as a genotoxic agent, whether real or perceived. It is curious that loss of any one cell cycle checkpoint results in increased tolerance to Al rather than sensitivity as is observed with known genotoxic agents like HU, MMC and CDDP. This may suggest that these checkpoints are either so sensitive that even the limited amount of genomic stress that might directly or indirectly occur with Al could activate these factors yet in reality be relatively inconsequential to growth, or that Al is inappropriately perceived as a genotoxic agent by ATR, ALT2, SOG1 and SUV2. Based on the current findings on SOG1 and SUV2 in conjunction with the previous reports on ATR and ALT2,plastic nursery plant pot a DNA damage response is the primary cause of Al dependent root growth inhibition in Arabidopsis.

What kind of Al dependent DNA damage these factors are detecting is still unknown; however, in concurrence with research from the fields of Al toxicity and DNA damage responses, there could be a multitude of sources of this damage, including but not limited to: Reactive Oxygen Species, phosphate deficiency leading to dNTP depletion, competition with Mg2+ causing ATP depletion and enzymatic dysfunction, and topological strain affecting replication fork stalls or reduced transcriptional capabilities.Chronic high levels of Al exposure have been shown to result in peroxidation of lipids within the membranes of cells . Lipid peroxidation is likely a downstream result of Al damage, perhaps caused by Reactive Oxygen Species known to be generated by Al . ROS can cause DNA damage, where damage to individual bases may also be implicated, and could be tested for in a comet assay. In peripheral blood lymphocytes treated with Al, a high incidence of oxidized bases, particularly purines and apurinic/apyramidinic sites, were attributed to Al-generated ROS . In the human genome such base lesions are indeed repaired by ATR-mediated nucleotide excision repair . In Arabidopsis, over expression of a variety of factors in the antioxidant pathway have resulted in increased Al tolerance in Arabidopsis such as glutathione S-transferase and peroxidase . However, a loss-of-function mutant of At4g10500, an uncharacterized member of the 2-oxoglutarate and Fe-dependent oxygenase super family, tested for possible scavenging of ROS in response to Al exposure did not show any phenotypic changes in response to Al . Additionally, if Al-generated ROS were indeed primarily responsible for root growth inhibition, theloss-of-function mutants for DNA damage response factors would result in heightened sensitivity rather than tolerance to Al. Unrepaired damage caused by ROS leads to oxidative damage of lipids, amino acids, and DNA which can lead to cell death. Although ROS is likely a detrimental symptom of Al exposure, as Al toxicity is a complex and widely destructive biological assault, it is unlikely that ATR, ALT2, SOG1, and SUV2 are detecting damage caused by ROS.Al toxicity and phosphate deficiency typically coexist due to acid soil conditions that promote Al bio-availability while simultaneously reduce Pi uptake by the roots , and ALS3 has been identified as a required factor in a Pi starvation response in a sucrose-dependent manner . While Pi deficiency has many varied symptoms as phosphorous is required for photophosphorylation, genetic metabolism, transportation of nutrients, and phospholipid composition of cell membranes , it has dire effects on DNA replication as massive quantities of Pi are needed in the form of dNTP’s that are polymerized in order to form DNA as well as in the ATP consumed in the polymerization reaction. dNTP depletion caused by Pi deficiency would cause replication fork stalls, and lead to replication catastrophe, much like HU treatment.

A Pi starvation growth assay where seedlings of Col-0 wild type, als3-1, and atr- 4;als3-1 could be tested for Pi starvation responses. If atr-4 is capable of suppressing the als3-1 phosphate sensitivity, this could resolve whether or not Pi deficiency plays a predominant role in the Al-dependent damage response. However, damage caused by Pi deficiency would logically result in heightened Al sensitivity rather than tolerance as is observed for the loss-of-function mutants for ATR, ALT2, SOG1 and SUV2. Alternatively, with sucrose being a required factor for Pi sensitivity in als3-1, an Al growth assay where seedlings of Col-0 wild type and als3-1 are grown in the absence of sucrose with increasing amounts of AlCl3 could assess the ALS3-dependent PI starvation response. If Al toxicity is actually the result of Pi starvation, als3-1 should grow similarly to Col-0 wild type in the absence of sucrose, rather than a hypersensitive response to Al. It seems highly improbable given that Al exposure forces terminal differentiation through means of endore duplication, requiring rounds of DNA replication, as possible mechanism to inhibit root growth caused by Pi deficiency. Al3+ ions complete with magnesium ions for binding sites on the plasma membrane and decrease the uptake of magnesium into the root. Increasing concentrations of available magnesium in soil or nutrient media or over expressing magnesium transport genes enhance Al resistance as increased magnesium released into the rhizosphere competes with Al. Magnesium is the predominant ionic regulator of metabolism, largely through its role as a cofactor for all phosphoryl transfers in the cell.It also acts as a second messenger for growth factors in regulation of protein synthesis and is required to maintain genomic stability. Besides its stabilizing effect on DNA and chromatin structure, magnesium is an essential cofactor in almost all enzymatic systems involved in DNA processing. Most obvious in DNA replication, its function is not only charge-related, but very specific with respect to the high fidelity of DNA synthesis . Furthermore, as an essential cofactor in nucleotide excision repair, base excision repair and mismatch repair, magnesium is required for the removal of DNA damage generated by environmental mutagens, endogenous processes, and DNA replication . More studies are warranted to study how Al interferes with the function of magnesium in plants under Al toxic conditions. Al growth responses in the presence of excess magnesium could be tested for atr-4;als3-1, alt2;als3-1, sog1-7;als3-1, and suv2;als3-1 in comparison with Col-0 wild type and als3-1 to determine if magnesium supplementation can alleviate the effects of Al toxicity.

This test would likely be indicative of Al resistance, showing magnesium outcompeting Al for entry into the cells of the root. If Al interferes with DNA replication machinery,seedling starter pot in vitro investigations such as PCR assays could be tested for amplicon lengths in the presence of Al to measure processivity, as such assays has been demonstrated in yeast . Such assays could be performed with commercially available DNA ploymerases, as Arabidopsis polymerase enzymes are not readily available.The effects of Al at the nuclear level are poorly understood. Al rapidly accumulates at high levels in root meristem nuclei and is especially concentrated around interphase chromatin as well as mitotic figures . Al does not appear to have a base composition preference and it is likely that this lack of base discrimination is due Al binding to the phosphate backbone of DNA . This could result from an electrostatic attraction of Al3+ to the negative charges of the phosphodiester bonds. Such an association could increase the rigidity of euchromatin and relax supercoiled heterochromatin destabilizing genome topology through an ever-fluxing torsional tug-of-war. DNA gyrases and topoisomerases regulate topological strains such as supercoiled and relaxed DNA, especially caused by replisome progression, and are required to prevent replication fork stalls caused by supercoiled DNA in front of unwinding . Loss-of-function mutants of topI or topII could be tested for growth responses to Al. Perhaps in reality, overexpression mutants of a topoisomerase would be capable of counteracting the strain Al exerts on DNA and should be generated and tested. However, despite the effectiveness of topoisomerases to alleviate replication induced topological strains, their functions would not rid the nucleus of Al3+ and would likely be unable to ameliorate the affect of Al on the whole genome. Such a topological strain caused by binding of Al to DNA or chromatin could condense DNA molecules and inhibit cell division by reducing its capacity to provide proper DNA separation as is necessary for DNA replication and mitotically relevant transcriptional events . Others have shown that Al causes DNA compaction, as well as compaction of chromatin, potentially through inhibition of unwinding of genomic DNA since Al3+ will raise the Tm of the double helix . Al has been shown to precipitate DNA out of solution in vitro, especially templates normally found in transcriptionally active euchromatin, extending the implication that Al compaction could lead to transcriptional repression. Importantly, many of these gene-silencing effects can be explained due to the extraordinary charge density of the Al ion, and perhaps this alone is reason enough to disrupt DNA processes that activate checkpoint responses. While gene silencing may indeed result from internalized Al, this is likely not a significant consequence of the effect of Al on DNA, as Al-inducible gene expression is a confirmed response to the internalization of Al . However, it is not unreasonable to predict that such conformational changes to the DNA could be perceived by ATR as being deleterious to replication fork progression. Such a topological strain on DNA caused by Al may cause a conformational change reminiscent of covalent crosslinkers, such as MMC and CDDP. Since Al3+ is expected to have high affinity for the negatively charged phosphodiester backbone of DNA and would presumably interact with this backbone differently than divalent cations and could cause an electrostatic interaction where Al acts as a non-covalent pseudo-crosslink. If Al acted as a pseudo-crosslinker that disrupts or restricts unwinding of DNA during processes such as DNA replication and RNA transcription, this may trigger ATR-dependent checkpoint activation, similar to a replication fork stall caused by a true DNA crosslink. As discussed, ATR, ALT2, SOG1 and SUV2 are all required to respond to DNA cross linking agents and all are linked to Al-dependent stoppage of root growth .Many uncertainties persist in regards to the nature of the unknown damage detected by ATR, ALT2, SOG1 and SUV2. It seems counter intuitive that a plant gains tolerance to an agent that causes DNA damage by reducing the function of a factor necessary for DNA-damage detection. Peculiarly, the genomic stability of Arabidopsis roots is secure enough to endore duplicate following Al exposure, indicating that unlike true DNA cross links, the effect of Al on the DNA is not severe enough to inhibit multiple rounds of DNA synthesis phases that would be required for the endore duplication cycle. Yet, visualization of micro-nuclei formation in cells of the root tip indicates that real breakage of chromosomes occurs following long term exposure to Al. Once more, it is perplexing that ATM is largely uninvolved with the Al response despite the consequent DNA breakage in the formation of micro-nuclei. Perhaps these factors force root growth inhibition in the presence of Al in order to prevent passage of minor damage to subsequent generations.

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Histochemical staining of Arabidopsis plants homozygous for full length ALS1 fused to GUS was performed

ALS1 encodes a half type ABC transporter that was localized to the vacuolar membrane of root tip cells, suggesting that it may be important for compartmentalization of internalized Al . Map based cloning revealed that the als1-1 mutation is a single amino acid substitution in this previously uncharacterized half type ABC transporter. It contains two distinct domains: the N-terminal domain contains at least four predicted transmembrane regions and appears to have a structure typical of the membrane spanning regions of ABC transporters and the C-terminal domain has all the required motifs associated with functional ATPases of ABC transporters . ALS1 also has significant homology to two distinct ABC transporters with different functions. ALS1 is homologous to mammalian TAP-type transporters associated with the endoplasmic reticulum to move short polypeptides for antigen processing. ALS1 is also homologous to yeast mitochondrial-localized subfamily B ABC transporter, Atm1p, and vacuolar localized Ycf1p. Atm1p is an essential component in the mitochondrial export of Fe/S clusters to the cytoplasm, indicating that it is essential for iron homeostasis in yeast. Ycf1p is required for movement of cadmium-glutathione into the yeast vacuole and loss of Ycf1p results in cadmium hypersensitivity. It is possible that ALS1 functions similarly to Atm1p and Ycf1p, thus transporting a metal complex in Arabidopsis . It was determined that ALS1 is primarily limited to vascular tissue in the roots, leaves, stems and flowers,bucket flower with high activity throughout the distal portion of the root tip. High GUS activity was also detected in the hydathodes and the area surrounding the water pore. ALS1 was found to be exclusively localized to the vacuolar membrane using ALS1 fused to GFP .

It was proposed that ALS1 is potentially transporting a metal complex from the cytosol to the vacuole of the cell, similar to two yeast homologs Atm1p and Ycf1p. This would allow sequestration of Al into the vacuole and remove interaction of the Al cation within the cytosol. Due to this activity, it is possible that ALS3 loads and unloads Al from the phloem for movement of Al to less sensitive cells, while ALS1 transports intracellular Al from the cytosol to the vacuole for sequestration. Mutation of either of these two factors can lead to inappropriate accumulation of Al in sensitive tissues within the plant . ALS7/SLOWWALKER encodes a transcription factor that among other things is required for regulation of expression of genes whose products participated in the production of polyamines such as spermine . Since als7-1 has Al hypersensitive roots, it is expected that reduced production of polyamines lowers the protective effect of these multi-charged cations because of reduced capacity to compete with Al3+ for binding to anionic sites within the root tip. Anionic targets of Al3+ are expected to include negative charges in the plant cell wall as well as symplastic targets such as genomic DNA, which directly binds to polyamines such as spermine and spermidine . Aluminum resistantmutations have been identified by using EMS mutagenized Arabidopsis seedlings that had enhanced root growth on a phytotoxic level of AlCl3. From this screen, five lines were explored further in depth. Four of these lines were mapped to a similar location and all had increased organic acid release of malate and/or citrate. The fifth line did not have enhanced organic acidrelease, suggesting the resistance is conferred in a manner other than organic acid release, which has already been described previously. Unfortunately, fine scale mapping could not be accomplished due to the difficultly of identifying a population that had a clear Al resistant phenotype .

Since als3-1 has such a profound phenotype that is specific to Al stress, it presented a unique opportunity to use this mutation to help identify factors that confer increased Al tolerance and/or resistance using a suppressor mutagenesis approach. EMS chemical mutagenesis on als3-1 was performed and plants that masked als3-1 hypersensitivity on low levels of Al were isolated. The als3-1 suppressor lines represent mutations in factors that confer increased Al resistance and/or tolerance. By using this method, 12 strong suppressor mutants were identified, and 3 were initially chosen for further analysis. All three gave similar phenotypes and all mapped to the same region. Therefore only one of those lines was studied further, alt1-1. When the alt1- 1;als3-1 suppressor mutant was grown in the presence of increasing concentrations of AlCl3, its growth not only reversed AlCl3 hypersensitivity of als3-1 but it also had increased root growth compared to Col-0 wt in higher levels of AlCl3. It was found that alt1-1 is a mutation that does not exhibit any enhancement in Al exclusion, due to callose accumulation and Al content of alt1-1 being similar to wild type . Through map-based clonging, alt1-1was discovered to be a loss of function mutation in ATAXIA TELANGEICTASIA MUTATED AND RAD3-RELATED. In higher eukaryotes, ATR encodes a cell cyclecheckpoint that senses DNA damage as part of a signal transduction pathway capable of halting the cell cycle in order to repair damaged foci . In Arabidopsis, ATR is known to signal repair of persistence of single-stranded DNA, single-stranded DNA breaks, replication fork stalls, and cross links . Alt1-1 confers a single amino acid substitution of G1098E in the highly conserved yet uncharacterized UME domain, which is speculated to function in protein-protein interactions. A second alt allele, alt1-2 was also the results of a point mutation in ATR a L2553F substitution in the conserved phosphatidylinositol 3- and 4-kinase domain of ATR . Both of these dominant-negative mutant alleles were originally found because of their capability to suppress the hypersensitivity phenotype of als3-1 .

Further work has shown that a T- DNA insertion allele, atr-2, is also capable of fully suppressing als3- 1, thus supporting the argument that alt1-1and alt1-2 are mutations that reduce the function of ATR even though they are dominant . This is particularly evident when one considers the response of atr-2 and atr- 4 to other DNA-damage agents, which only adds to the conundrum of why Al activates this ATR-dependent pathway. While these mutants are Al tolerant, as demonstrated by their capacity to maintain root growth due to failure to arrest cell cycle progression and force QC differentiation, atr-2 and atr-4 roots exposed to different DNA-damage agents such as the replication fork poison hydroxyurea and the DNA cross linkers cisplatin and Mitomycin C exhibit extreme sensitivity . From these results, ATR isabsolutely necessary to repair stalled replication forks as well as DNA crosslinks yet loss of this repair factor confers Al tolerance. It is difficult to reconcile increased Al tolerance with loss of such a key DNA-damage response factor, although it could be argued that Al may bind to DNA in a manner similar to covalent crosslinkers but without the extreme detrimental effects of these cross linkers. This argument is based on the likelihood that Al would interact with DNA electrostatically in a reversible manner, with binding likely holding DNA in an unfavorable conformation that subsequently is perceived as a replication fork stall by ATR. Interestingly, even though treatment with Al has been shown to result in DSBs and micronuclei , a loss-of-function atm mutant was incapable of suppressing the Al hypersensitivity phenotype of als3-1suggesting that DSBs are not important to activating the DNA-damage checkpoint following Al treatment. The second als3-1 suppressor mutant identified is alt2-1, which is a loss-of function mutation in the cell cycle checkpoint TANMEI/ALT2. ALT2 encodes a WD- 40 motif containing protein homologous to an integral component of the mechanism required for response to DNA damage, AtCSA,cut flower bucket which is part of the DNA- damage binding machinery . WD-40 proteins are commonly found in many different biochemical pathways, serving as scaffolds for protein complexes. In some cases, such proteins participate in SCF ubiquitin ligase complexes in order to tag target proteins for degradation. Such a role for ALT2 in response to Al is consistent with the observation that CULLIN4, which is a key component of SCF ubiquitin ligases, interacts directly with DWD motif containing proteins . Such a function is distinctly different from what was previously described for human CSA, which functions in TCNER to monitor for conformational changes in DNA as assessed by blockage of transcription . Cooperation of a WD-40 motif containing protein with CULLIN4 is characteristic of Global Genome Nucleotide Excision repair and is independent of RNA polymerase II . At this point, it is unclear in which if either NER response pathway ALT2 may participate although the potential linkage of ALT2 to either is consistent with Al acting as a genotoxin. Like atr-2 and atr-4, alt2-1 falls into the same conundrum since it fails to halt the cell cycle and force differentiation of QC in the presence of a normally inhibitory concentration of Al yet it is highly sensitive to DNA cross linkers .

A double mutant representing the loss-of-function of both ATR and ALT2 showed no additive Al tolerance compared with the single mutants, suggesting that ATR and ALT2 act together to detect Al-dependent damage and actively halt root growth. Interestingly, alt2-1 does not exhibit hypersensitivity to the replication fork poison HU while it does show extreme sensitivity to CDDP and MMC, which is consistent with it being a key regulator of cell cycle progression following exposure to DNA-damage agents . Therefore, at this point the genotoxic nature of Al has yet to be defined. However, it is clear from the unbiased als3-1 suppressor screens, in which DNA damage response factors were identified as Al tolerance mutations, an ATR- and ALT2-dependent cell cycle checkpoint pathway is key to stoppage of root growth and promotion of terminal differentiation following Al treatment. Based on the results with ATR and ALT2, one could argue that Al-dependent DNA damage is a critical determinant of root growth inhibition, yet the effects of Al on the nucleus are just beginning to be elucidated. Al rapidly accumulates to high levels in root meristem nuclei and is especially concentrated around interphase chromatin as well as mitotic figures . It is hypothesized that Al binds to the phosphate backbone of DNA , which would be expected to result from an electrostatic attraction of Al to the negative charges of the phosphodiester bonds. Such an association could increase the rigidity of euchromatin and relax supercoiled heterochromatin destabilizing genome topology through an ever-fluxing torsional tug-of-war. Binding of Al to DNA or chromatin could condense DNA molecules and inhibit cell division by reducing its capacity to provide a viable template for replication, mitotically relevant transcriptional events, and even proper DNA separation . It is not unreasonable to predict that such conformational changes could be perceived by ATR as being deleterious to replication fork progression, thus activating this cell cycle checkpoint. It should be noted that chromosomal aberrations resulting in DNA breakage and intra-strand cross linking are a reported consequence of chronic exposure to Al that lead to micro-nuclei formation although it is not clear how relevant these are to stoppage of root growth since ATM does not appear to have a role in this process . Clearly, further studies are necessary to define if and how Al interacts with genomic DNA, especially since it is the Al3+ species that is predicted to bind to the negatively charged DNA backbone, yet intracellular pH is not expected to favor the formation of this species. In the end, utilizing Al toxicity as a real world model to study DNA damage in plant systems presents a novel system to study DNA damage response without the use of rare chemotherapy drugs or types of radiation not found in earthly environments. Ultimately, it seems counter intuitive that a plant gains tolerance to an agent that causes DNA damage by reducing the function of a factor necessary for DNA damage detection. This certainly begs the question—if atr and alt2 mutant roots can maintain root growth even in the presence of Al, what actual Al-dependent damage is detected by these cell cycle checkpoints? It cannot be ruled out that inappropriate activation of the cell cycle checkpoint machinery and the repair mechanisms that they regulate may actually be the cause of the damage such as DSBs and micro-nuclei. Consequently, one explanation could be that failure to activate this pathway prevents the program-dependent accumulation of the damage and results in roots that can grow in normally inhibitory levels of Al.

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Several QTL for chilling tolerance in rice have been high-resolution mapped and candidate genes identified

The heterozygous BC4 line 04GH0030 from Goodstal et al. containing a single copy of the S. habrochaites allele for fine-mapped QTL stm9 was used to generate a BC5 segregating for only the chromosome 9 region of interest from S. habrochaites. The BC5S1 generation was marker genotyped to identify recombinant individuals within the fine-mapped stm9 region flanked by markers T1670 and T0532 . Self-pollinated seed was collected from individuals chosen for their chromosome 9 introgressions, and these BC5S1 individuals were marker screened for further recombination events within the chromosome interval containing stm9. Self-seed from two heterozygous BC5S1 individuals and two heterozygous BC5S2 individuals were screened for recombinants via marker-assisted selection . Individuals that contained recombination events within the chromosome 9 fine-mapped stm9 region were selected, grown to maturity, and allowed to self-pollinate to produce seed of fully homozygous individual sub-NILs in the BC5S2 or BC5S3 generation . Recombinant homozygous individual sub-NILs were allowed to self-pollinate to generate ample seeds for replicated experiments. Phenotyping experiments were performed with one representative line from each recombinant class. All plant materials were grown in greenhouses at UC Davis. Seeds were planted in 73-cell flats containing soil media. Flats were watered daily, and plants were fertilized with a 10:30:20 NPK solution once a week. Greenhouses containing plants in flats, pots, and hydroponic tanks were maintained at ambient conditions of 25–37 °C with 55–80 % relative humidity during the day, and 18–25 °C with 20–55 % relative humidity at night. Plants from which seed was to be collected were transferred at the 4th to 5th true leaf stage to individual 8-L pots filled with soil media, and grown to maturity to obtain seed.After 2 weeks of growth in flats containing soil media ,procona London container the roots of two seedlings of each sub-NIL or control were carefully washed free of soil media in deionized water and transferred to a hydroponic growth tank set at 20 °C containing a modified Hoagland solution at 20 % of full strength .

Efforts were made to use vigorous seedlings of similar size. Plants were grown in the hydroponic tank for 1 week under ambient illumination in the greenhouse, with constant aeration and circulation of the nutrient solution. Subsequently, the plants were randomized and transferred to a separate refrigerated hydroponic tank containing fresh modified Hoagland solution at 20 % strength. Plants were acclimated overnight at a solution temperature of 20 °C. The following morning supplementary lighting was provided by one 1000 W metal halide lamp starting at 7:00 am and used throughout the experiment to maintain a light level above 1000 µmol m−2 s −2 PAR. The tank solution was maintained at 20 °C for 1 h after the supplementary lighting was turned on, and then the tank temperature was decreased to 6 °C. The tank was held at 6 °C for 2 h prior to phenotyping. Each experiment was conducted as a Randomized Complete Block Design and repeated in two seasons , with days as blocks and two replicate plants of each genotype per block. In addition to the two individual plants per recombinant sub-NIL, each replication of the experiment included controls: four plants of chilling-sensitive S. lycopersicum cv. T5, and two plants of a chilling-tolerant NIL, 03GH1322 that was also used as a tolerant control by Goodstal et al. . Six repetitions of the experiment that were conducted from May 9th to June 13th comprise the Spring data set, and four repetitions of the experiment that were carried out from October 7th to October 20th comprise the Fall data set. Plants were individually phenotyped for shoot turgor maintenance under root chilling according to the rating scale described in Goodstal et al. . Briefly, shoot turgor was scored for each plant on a scale of 0–3, with a stmscore of 0 denoting maintenance of shoot turgor, and a stmscore of 3 denoting severe loss of shoot turgor .The chromosomal location of stm9 in our study agrees with Goodstal et al. who fine-mapped stm9 to marker interval T1670–T1673 . We refined the location of stm9 to marker interval H358–T1673, a genetic distance of 0.32 cM. Our data suggests that the gene or polymorphisms controlling the tolerance phenotype are located close to marker H348 and within the marker interval H358–T1673. The chromosomal location of QTL stm9 detected in both data sets was coincident despite the significant Genotype × Season interaction in the ANOVA.

To examine the cause of the significant Genotype × Season interaction in more detail, we plotted recombinant sub-NIL stmscore means across the two seasons to create interaction plots . Inspection of the plots suggests that the changes in sub-NIL mean values across seasons primarily derived from greater chilling susceptibility of susceptible sub-NILs in the Spring than in the Fall . Magnitude differences would cause the size of the LOD peaks to differ among seasons, but not change the peak location, which is in agreement with our results . In addition to the increase in the magnitude of means for stmscore of the susceptible sub-NILs, two sub-NILs were classified as tolerant in the Spring dataset but as susceptible in the Fall dataset . Sub-NIL C4 had a mean of slightly over 1.0 in the Spring data set, and clearly grouped as susceptible in the Fall dataset . None of these lines contain the S. habrochaites introgression for high-resolution mapped stm9, but the introgressions do all flank stm9. These results suggest the possibility that there are environmentally sensitive genetic modifiers of the stmscore phenotype in this region of chromosome 9, and that the interaction of these modifiers with the environment could be causing the significant Genotype × Season interaction. Phenotypic plasticity in the presence of abiotic stress has been noted and reviewed previously . The rank changes seen within the tolerant group may be due to differences in the genomic content of S. habrochaites sequence in the flanking regions of QTL stm9, and not a direct effect of the environment on the gene or polymorphisms controlling the tolerant stm9 phenotype. Previous work in tomato has shown that the stomatal response of a plant when subjected to root chilling conditions differs between susceptible and tolerant phenotypes . Stomatal control is regulated by multiple environmental factors including light, temperature, day length, humidity, and CO2 levels . The Spring experiments were conducted under longer day lengths, higher air temperatures, and lower humidity than the Fall experiments . These seasonal differences affect the conditions in the greenhouse and may have contributed to the significant Genotype × Season interaction, as well as differences in relative response among the sub-NILs in the Spring versus Fall data sets. In the context of phenotypic plasticity, seasonal effects on sub-NIL performance would account for the more gradual separation of means in the Spring dataset compared to the Fall .Low marker density and small population sizes in initial genome-wide QTL mapping studies may bias upwards the estimation of QTL effects due to the inability to resolve closely linked, smaller effect QTL . Consequently,cut flower transport bucket single large effect QTL may resolve or fractionate into multiple, smaller effect QTL after fine- and high-resolution mapping . The original interspecific BC1 population used by Truco et al. to map QTL for shoot turgor maintenance under root chilling consisted of 196 individuals genotyped with 112 markers. Truco et al. mapped a major effect QTL to a 28-cM region on the short arm of chromosome 9 which accounted for 33 % of the phenotypic variation for shoot turgor maintenance under root chilling .

Despite the large initial genetic size of the QTL stm9 region detected by Truco et al. , subsequent fine-mapping by Goodstal et al. and high-resolution mapping in our present study do not provide any evidence of multiple QTL or QTL fractionation. The relatively small genetic size of high resolution mapped stm9 and the lack of QTL fractionation indicates that this level of resolution is suitable for the identification of candidate genes for stm9. There are numerous examples in the literature of environmentally stable, high resolution mapped QTL that have led to candidate gene identification and in some cases subsequent causal gene/ polymorphism determination.These QTL include qCTS12 , qCtss11 , and qCTB7 . Tomato-specific QTL examples include fw2.2, a fruit weight QTL, and se2.1, a stigma exsertion QTL, both identified in progeny derived from S. pennellii, another wild tomato relative . The causal gene underlying QTL fw2.2 was identified by Frary et al. , who proposed that changes in the regulation of ORFX , not changes in the sequence or structure of the expressed protein, are responsible for changes in fruit size. Chen and Tanksley determined the casual mutation underlying se2.1 is a mutation in the Style2.1 promoter that results in a down-regulation of Style2.1 expression during flower development. Collectively, the results from these studies suggest that candidate gene identification and functional testing for QTL stm9 should focus on mutations in regulatory and promoter regions of candidate genes in addition to mutations that may affect the sequence or structure of expressed proteins.Many genes have been identified as being involved directly or indirectly in plant tolerance or resistance to abiotic stresses , including chilling/cold tolerance . Plant responses to abiotic stresses can include multiple pathways that involve a variety of gene products such as receptors, signaling molecules, transporters, transcription regulators, and transcription factors . Many of the identified stress response pathways have been associated with tolerance to a range of abiotic stresses . The plant’s response to abiotic stress may result in both reversible and irreversible activation of stress response pathways . Because of the complex nature of the pathways involved, the specific genotype of the plant also has a large influence on abiotic stress response . Plant responses to abiotic stressors are dependent on the interplay of abiotic stress, environment, and genotype . Therefore, a particular abiotic stress applied in different environmental contexts may result in overlapping, but distinct responses from a single genotype . We analyzed the physical region in the cultivated tomato reference genome that is syntenic to the S. habrochaites QTL stm9 region because an assembled S. habrochaites whole genome sequence is not available. All of the protein products of the S. lycopersicum annotated genes located within 30 kb of the QTL stm9 peak marker have features that are shared with genes involved in responses to water stress and other abiotic stresses. In addition, the majority of the S. lycopersicum genes located within the syntenic high-resolution mapped stm9 region have been implicated in abiotic stress response pathways . It is possible that plant responses to root chilling stress may induce a more complex transcriptional response than other types of water stress such as those caused by salt or polyethylene-glycol , although overlap has been seen in the response to all three stresses . For example, in grape, under root chilling stress only transcripts for protein synthesis and the cell cycle were up-regulated to a lesser extent than under salt or PEG stress. The regulation of plant metabolism, protein metabolism, signal transduction, calcium signaling, stress hormone pathways, and transcription factors were all increased to a greater extent under root chilling in grape . These categories of genes account for the majority of genes located within the syntenic S. lycopersicum QTL stm9 region. While the total number of annotated genes within the S. lycopersicum reference genome region containing QTL stm9 is relatively small, there are no estimates available for S. habrochaites due to the unavailability of assembled whole genome sequence for this wild species. A comparison of the genetic and S. lycopersicum physical maps of the chromosome 9 region containing stm9 shows a variable rate of recombination across this region . The average kbp/cM for marker interval T1670–T1673 is 952 kbp/ cM, whereas for marker interval T1673–T0532, it is 385 kbp/cM. Recombination occurs more frequently in generich euchromatic regions, but can be suppressed due to lack of homology, heterochromatic regions, and/or the presence of repetitive elements . It is possible that this variable rate of recombination is due to the presence of repetitive elements or other local structural polymorphisms affecting the synteny and colinearity of the S. lycopersicum and S. habrochaites genome sequences in this region. In addition, our flow cytometry results indicated that the genome size of S. habrochaites is 1.5 × that of S. lycopersicum .

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Eight growers spanning the major rose-producing areas of California participated in the program

With the new bent-shoot method, plants are grown in raised containers in a modified hydroponics system. Most of the shoots are bent downward at the crown to intercept more light, creating a perennial lower canopy that exists for the 5 to 8 years of crop production. The upper canopy contains only stems that produce flowers, which take 45 to 52 days to develop. The bent-shoot method creates a spatial separation between the harvested flowers and perennial foliage that does not exist in standard roses. Pesticides to control western flower thrips and powdery mildew that are more compatible with mite predators have also recently become available. These developments, coupled with the difficulty that rose growers were facing in controlling spider mites, made us confident that we could develop a successful IPM program that rose growers would adopt. This project was initiated in 2000 with major funding from the Pest Management Alliance Program of the California Department of Pesticide Regulation and was later supplemented with additional funding. The goal of the Alliance project was to foster a team approach to the development and implementation of IPM programs in a given commodity and to document a reduction in traditional pesticide use. Our Alliance team included researchers, county-based advisors, growers, chemical and biological-control industry representatives,30 litre plant pots commodity associations and government officials. Our objective was to develop a cost-effective IPM program for the key pests of cut roses that included sampling, thresholds, biological control and directed sprays of reduced-risk pesticides.Each grower contributed an IPM and a conventional-practice greenhouse; all greenhouses were between 5,000 and 10,000 square feet in size.

All pest management decisions in the IPM greenhouses were based on the IPM program that we developed, while the grower made all pest management decisions in the conventional greenhouses. Data was collected and compared on a weekly basis by trained scouts using a comprehensive sampling plan that provided information about the density of insects, mites and diseases. The project included growers with several different rose varieties and both the bent-cane and hedgerow training techniques, but we kept these two variables standardized within a location. Implementation began in March 2000 and continued until January 2001. Fixed precision sampling plans that had been previously developed for two spotted spider mites and western flower thrips were used in our scouting program. This type of sampling plan was developed through intensive surveys of a crop to determine a pest’s spatial distribution. The degree of acceptable error was decided upon in advance, and the number of samples needed to obtain that precision was calculated using knowledge of the pest’s spatial distribution in the crop. We used a precision of 0.25, which is acceptable for pest management sampling . Generally, as spatial distribution becomes more aggregated , more samples are required to determine pest density with the desired precision. Although they take some effort to develop, these types of sampling plans are often more accurate and efficient than other sampling approaches. This study represents the first use of such plans in a floriculture IPM program. Sampling for all other pests was done during sampling and inspection for two spotted spider mites. Data was collated and summarized by the scouts and then discussed by members of the Alliance team. The scouts then met with the growers to discuss control strategies. Based on thresholds developed for each of the pests, no action was taken; cultural controls were used; biological control agents were released; or a pesticide application was made. Each greenhouse was a replicate, and ANOVA was used to determine whether there were differences between the conventional and IPM treatments.The first leaf above the bend on 38 randomly selected plants was sampled per 10,000 square feet of greenhouse area to estimate mite density at the desired precision.

Plants were classified as infested if the scout found more than five mobile mites on the sampled leaf, or not infested if there were five or fewer. These samples were also used to determine co-occurrence of two spotted spider mites with the predatory mite Phytoseiulus persimilis, and they were inspected for secondary pests and diseases. In addition to the fixed samples, the scouts took directed samples as they walked down each row and noticed damage by insects, mites or pathogens. These plants were flagged for potential spot treatments. In the IPM greenhouses, mite treatments were initiated according to the percentage of infested plants . Chemical controls included azadirachtin , bifenazate and insecticidal soap , all of which provide some level of compatibility with P. persimilis. Releases of predatory mites were based on the cooccurrence of two spotted spider mites and predators on the sampled leaf. Cooccurrence is the percentage of plants with two spotted spider mites on which P. persimilis also occurs. This idea has been discussed in the literature as a theoretical basis for natural enemy releases, but has never been tested in practice . We chose to include this method in our program because our natural enemy supplier recommended it to growers. Additional predatory mites were released when co-occurrence was less than 10%. All predator releases were made to leaves just below those on which two spotted spider mites were present. Predators were kept refrigerated and were released as soon as possible after arrival at the greenhouse, as per the supplier’s instructions.A fixed precision sampling plan for western flower thrips was also developed . This sampling plan used yellow sticky traps and a general threshold of 25 to 50 thrips per trap per week . Three 4-by-6-inch yellow sticky traps with both sides exposed were placed per 10,000 square feet . The traps were placed at flower level and were evenly distributed in the greenhouse . The lower threshold of 25 thrips per trap per week was used in more-susceptible varieties and in areas of heavy thrips pressure. The higher threshold of 50 thrips per trap per week was used in less-susceptible varieties . There is currently no cost-effective biological control agent for western flower thrips in cut roses, so control of this pest in the IPM greenhouses included both cultural and chemical methods.

Although the female thrips lays eggs in the flower or in foliage directly below the flower, the development time for eggs and larvae is longer than the 5 to 6 days between sepal split and flower harvest . Routine flower harvest removes immature thrips from the greenhouse and subsequently there is little thrips reproduction in the rose greenhouse unless open flowers are left on the rose plant. Teerling has measured significantly higher thrips populations in Canadian rose greenhouses when these flowers are not removed. Cultural control was the removal of open flowers, and chemical control was applications of spinosad or azadirachtin directed to the flowers when the thrips-per-trap-perweek threshold was reached. Research on the distribution of thrips in the rose range has revealed that most thrips are found near the developing flower . Based on these findings, we then conducted a trial to determine whether sprays directed toward the flowers would provide control equivalent to full-volume wet sprays. Such a study is critical to the implementation of IPM in the rose range, because a typical full-volume spray in roses may reach hundreds of gallons of water per acre. Such high volume thoroughly wets the foliage, but creates problems with runoff and affects biological control agents regardless of where they are on the plant. In separate rose greenhouses,25 liter pot plastic we initiated a replicated study where rose beds were divided into 20-foot sections and applications of registered pesticides were made using full-volume wet sprays at 275 gallons per acre versus the same material applied just to the upper canopy at 70 gallons per acre . Registered materials — acephate , methiocarb and spinosad — at label-recommended rates were used in the study. There were four replicated, 20-foot rows for each material per volume combination, and applications were made for 4 weeks. At the end of this time, 10 flowers were removed from each section and examined for the presence of thrips. Our effort to introduce IPM principles in the management of powdery mildew centered on an attempt to use a predictive model for powdery mildew of grapevines . The UC Davis powdery mildew risk-assessment model for grapevines is based on the effect of temperature on the reproductive rate of the pathogen following initial plant infection. As temperatures are recorded in vineyards, risk points are accumulated if temperatures are favorable or subtracted if temperatures are not favorable. When risk points reach a predetermined threshold, fungicide application is recommended. This model has been effective in determining if and when fungicide treatments need to be applied to grapevines, and has resulted in effective disease management with significantly reduced fungicide usage in California. In commercial rose greenhouses, growers spray regularly weekly during mildew season in Central California and all year long in Southern California. It is not unusual for half of all yearly pesticide sprays in a rose crop to be for mildew control, presenting a strong argument for matching applications to actual risks. Although the powdery mildew fungus attacking roses is a different species, its response to environmental conditions is similar to that of the species attacking grapevines. For this reason, we sought to determine whether the grapevine mildew model could be easily adapted to greenhouse grown roses. The greenhouses used in this effort were instrumented so that temperature, relative humidity and leaf wetness were measured at 30-minute intervals throughout the day and night. Temperature data was fed into the GMM to add or subtract risk points. In order to correlate actual disease development with the GMM risk points, a trained scout evaluated plants in the greenhouses weekly.

This was accomplished by walking through the greenhouses in a predetermined pattern, stopping at regular intervals and evaluating one plant at each stop-point to assess disease incidence and severity. Disease incidence was determined by the presence or absence of mildew lesions on the leaves of harvestable stems. Disease severity was determined by counting the actual number of lesions on leaves attached to the harvestable stems. This data was used to calculate an overall disease rating for the crop that could be compared to risk predictions based on the GMM. Along with the disease incidence and severity data, we recorded the timing of all chemical fungicide and insecticide applications made by the growers in the IPM houses so that we could evaluate these effects on disease ratings. As a resistance management practice, growers typically varied the fungicide materials used throughout the season. A few fungicides were common across all locations, but growers did differ in some of the materials applied. For example, if powdery mildew became severe in a greenhouse, growers at all locations would typically apply piperalin because of its eradicative properties. Other materials used at the various locations included myclobutanil , chlorothalonil , benzeneacetic acid , azoxystrobin , insecticidal soap and potassium bicarbonate .Plants in both the IPM and conventional greenhouses were inspected for whiteflies, aphids, mealybugs, Botrytis, downy mildew and rust as part of the inspections for twospotted spider mites. The same traps that were used to monitor western flower thrips were also used to monitor white flies and winged aphids. We emphasized the use of materials that were compatible with the P. persimilis predator for control of these pests when necessary.Two spotted spider mites. Predatory mites were successfully used in all of the IPM greenhouses and almost eliminated the need for miticide applications in those houses. A comparison of two spotted spider mite levels under IPM and conventional control across all nurseries revealed that there were significantly more plants with no mites and significantly fewer plants with mites at the two levels measured in the IPM greenhouses . Similar results were observed at the individual nurseries. The cost of IPM during the first 8 weeks was higher than the cost of conventional control . Higher release rates were needed during this startup period for several reasons, including increased predator mortality as growers learned proper release techniques and the desire of some growers to begin biological control when twospotted spider mite densities were greater than the 25% infested threshold. After several releases had been made and predators became established, the release rate dropped and costs for the two control programs were comparable. Western flower thrips.