Tag: hydroponic

DAB polymerizes in the presence of H2O2 and endogenous peroxidase to form a brownish-red precipitate that can be easily visualized using bright-field microscopy. After staining, trimmed sheath segments were mounted in 50% glycerol. Images were acquired digitally and further processed with the Olympus analySIS cell^F software.To assess the ISR-triggering capacity of S. plymuthica IC1270, susceptible rice plants were grown in soil containing IC1270 bacteria, and subsequently challenged with several fungal pathogens exhibiting different modes of infection. In these ISR bio-assays, the resistance-inducing potential of IC1270 was compared to that of P. aeruginosa 7NSK2, a well-studied PGPR strain which we previously uncovered as a potent activator of induced resistance responses in rice. We first tested whether root colonization by S. plymuthica IC1270 exerts a protective effect against infection by the hemibiotrophic ascomycete M. oryzae, causal agent of the devastating rice blast disease and a major threat to food security worldwide. By 4 days post-inoculation , leaves of control, non-induced plants displayed typical water-soaked, diamond-shaped lesions, developing conidia at the center of each lesion by 6 dpi. In contrast, IC1270-colonized plants exhibited a marked reduction in the number of these susceptible-type lesions, producing a resistance phenotype mimicking that of quantitative trait loci-governed intermediate resistance . This resistance type is characterized by the abundance of small necrotic non-sporulating lesions,flood and drain table less than 2 mm in diameter, 60 to 72 h post-inoculation . Consistent with our previous findings, treatment with P. aeruginosa 7NSK2 resulted in a substantial reduction of disease as well.

No significant differences in the number of susceptible-type lesions could be observed between IC1270- and 7NSK2-treated plants, indicating that IC1270 and 7NSK2 are equally effective in suppressing M. oryzae. Because IC1270 clearly inhibited the growth of M. oryzae in dual culture experiments , possible systemic plant colonization by the rhizobacteria was checked. However, in all bio-assays performed, IC1270 bacteria were absent from sheaths or leaves of root induced plants, indicating that bacterial colonization remained confined to the root zone . Although such spatial separation does not rule out the possibility that IC1270-conferred protection might result from long-distance translocation of bacteria-produced allelochemicals to systemic leaves, the latter is rather unlikely as pilot experiments aimed at elucidating the bacterial traits underpinning IC1270-ISR revealed that mutants defective in the global response regulator protein GacA, which controls the synthesis of various anti-fungal metabolites , were as effective as wild-type IC1270 in reducing rice blast disease severity . The cumulative data therefore strongly suggest that the beneficial protective activity exerted by S. plymuthica IC1270 is based on activation of the plant’s defensive repertoire, rather then being caused by microbial antagonism. To test the spectrum of effectiveness of this IC1270-mediated ISR, we next assayed for induction of resistance against the sheath blight pathogen, Rhizoctonia solani, and the brown spot pathogen, Cochliobolus miyabeanus, both of which are considered necrotrophic fungi. In contrast to M. oryzae, which sequentially invades living cells, R. solani and B. oryzae kill host cells at very early stages in the infection, leading to extensive tissue damage. As shown in Fig 1B, both IC1270 and 7NSK2 failed to reduce disease caused by R. solani.

This impaired ISR response was not due to insufficient root colonization as bacterial counts in the rhizosphere of treated rice seedlings were comparable to those obtained in the M. oryzae bio-assays . Interestingly, in all four independent experiments, IC1270 pretreatment favored subsequent infection by R. solani, causing an average 39.6% increase in disease severity relative to non-induced controls. A similar trend was observed when challenging with C. miyabeanus, with IC1270 consistently promoting vulnerability to the latter pathogen . Root colonization by 7NSK2, however, yielded variable results. No significant differences between control and 7NSK2- treated plants could be observed in three bio-assays, whereas in the two remaining assays, root treatment with7NSK2 rendered rice seedlings substantially more susceptible to brown spot. In all experiments, mock-inoculated control plants remained healthy, and no apparent differences in appearance, size, or weight of control, 7NSK2 or IC1270-treated plants were observed prior to challenge infection . Thus, under the experimental conditions used in this study, root treatment with the ISR-inducing bacteria did not lead to detectable effects on plant growth that could have affected the growth or development of the respective pathogens. Collectively, these findings demonstrate that S. plymuthica IC1270 plays an ambivalent role in the rice induced resistance network, acting as a potent elicitor of resistance to the hemibiotroph M. oryzae while promoting susceptibility to the necrotrophs C. miyabeanus and R. solani.To begin to unravel the defense mechanism underpinning IC1270-mediated ISR, we analyzed the cytological alterations associated with restriction of M. oryzae in IC1270-induced plants using the intact leaf sheath method designed by Koga and associates. In this system, intact leaf sheaths of control, non-induced and IC1270-treated plants of the highly susceptible rice variety CO39 were routinely inoculated by injecting a conidial suspension of the virulent blast isolate VT7.

For comparison with R gene-mediated ETI, we also included the VT7- resistant variety C101LAC, the latter being a near-isogenic line of CO39 carrying the blast resistance genes Pi-1 and Pi-33.Similarly, quantitative recording of attempted blast infections revealed no significant differences in the number of unsuccessful penetration events, indicating that both IC1270-mediated ISR and R-gene-conditioned ETI are unlikely to impede pre-penetration development by M. oryzae . On the other hand, epidermal cells were found to respond to fungal ingress through various cellular reaction types depicted at 48 hpi in Fig 2A. A susceptible reaction was manifested as a type 1 phenotype in which extensively branched invasive hyphae vigorously invaded living epidermal cells with little or no visible host response. Interaction phenotype 2, on the other hand, was characterized by prompt arrest of fungal growth in the first-invaded epidermal cell, a phenomenon associated with enhanced vesicular activity and browning of the anti-clinal cell walls, while a type 3 reaction represented infection sites in which fungal invasion was curtailed shortly after penetration due to development of HR-like cell death, as indicated by the characteristic aggregation of the cytoplasm and a bright autofluorescence of the anticlinal cell walls. As expected, sheath cells of noninduced, susceptible CO39 plants inoculated with virulent VT7 predominantly mounted a type 1 reaction, whereas HR was the prevailing plant response in the incompatible interaction between VT7 and C101LAC. Most conspicuously, IC1270-induced CO39 sheath cells displayed an interaction profile resembling that observed in VT7-invaded sheaths of genetically resistant C101LAC, with type 3 reactions accounting for approximately 60% of all interactions by 48 hpi . At later stages of infection, M. oryzae had massively colonized the epidermis and mesophyll of CO39 sheaths causing extensive host damage as evidenced by the ubiquitous presence of cellular debris and fragmented remnants of host cell walls around invasive hyphae in the mesophyll . By contrast, in resistant C101LAC, as well as in IC1270-induced CO39, invading hyphae were largely trapped within hypersensitively dying cells in the epidermal layer, preventing fungal passage to the underlying tissue. Because rapid accumulation of phenolic compounds is a hallmark of rice defense against M. oryzae, we also examined the effect of IC1270 pre-treatment on the level of autofluorescence. Autofluorescence was detectable as early as 18 hpi, irrespective of IC1270 treatment or the level of resistance of the cultivars used . However, similar to what was observed in resistant C101LAC,rolling bench root treatment of CO39 with IC1270 caused the frequency of autofluorescent appressorial sites to increase rapidly from 18 hpi onward, reaching a level of 60 and 100% of all interactions by 24 and 36 hpi, respectively . By contrast, in non-induced CO39 cells, less than 6% of the appressorial sites showed autofluorescence 24 hpi, indicating that root colonization by IC1270 primes rice sheath cells for accelerated deposition of autofluorescent phenolic compounds at sites of attempted pathogen invasion. Along with the high frequency of hypersensitively reacting cells, these observations suggest that IC1270- mediated ISR and R-gene-conditioned ETI act, at least in part, through a similar set of defense reactions.There is ample evidence demonstrating the active involvement of reactive oxygen species , and H2O2 in particular, in the induction, signaling and execution of blast resistance in rice.

Furthermore, in the course of previous studies, we demonstrated that pyocyanininduced H2O2 micro-bursts are primordial for the onset of P. aeruginosa 7NSK2-mediated ISR against M. oryzae. Taking these facts into account, we sought to extend ourcytological analysis of ISR elicited by IC1270 by monitoring the spatiotemporal patterns of pathogenesis-related H2O2 production. In planta accumulation of H2O2 was visualized using an endogenous peroxidase-dependent staining procedure with 3,3′-diaminobenzidine . In these DAB assays, reddish-brown precipitates are deposited at the sites of H2O2 accumulation. No DAB accumulation was observed in mock-inoculated controls, regardless of IC1270 treatment or the inherent level of resistance of the cultivars used. However, comparative analysis of H2O2 production in pathogen-inoculated seedlings revealed the occurrence of a wide range of distinct DAB staining patterns that could be grouped into five categories . The first type comprised interaction sites in which DAB accumulation was not detectable despite massive fungal colonization of both penetrated and neighboring epidermal cells. Conversely, interaction sites displaying H2O2 accumulation in the primary invaded epidermal cell following spread of the invasive hyphae into neighboring cells were classified as a type II reaction. Type III interaction sites were characterized by the ubiquitous occurrence of DAB-positive vesicle-like bodies targeted to the invading hyphae. A type IV reaction referred to intracellular DAB staining tightly associated with the characteristic cytoplasmic aggregates of HR-expressing cells , while interaction sites displaying wholecell DAB accumulation were scored as a type V reaction. Importantly, when the DAB solution was supplemented with ascorbate, staining was abolished, indicating that the staining was due to H2O2 . Leaf sheath cells of susceptible CO39 were characterized by the high ratio of H2O2-negative type I reactions, accounting for 78% and 67% of all interaction sites by 36 and 48 hpi, respectively . In some incidences , H2O2accumulated in the initially penetrated epidermal cell following the formation of an extensively branched mycelium in the neighboring cells. Yet, this type II reaction seemingly occurred too late to effectively stall the pathogen. IC1270-induced CO39 cells, on the other hand, exhibited a strikingly different set of responses in that type I reactions, reaching a level of 33% at 36 hpi, were no longer discernible by 48 hpi. The rapid decline in the frequency of type I reactions from 36 hpi onward corresponded to an approximately 15% increase in the frequency of both type III and type V reactions. HR-like cell death of attacked epidermal cells, seen at approximately 52% of all interaction sites, was always associated with H2O2 accumulation in the cytoplasmic aggregates, beginning 32 hpi. Although not identical, by 48 hpi the H2O2 signature of IC1270-treated CO39 plants showed substantial similarity to that observed in the incompatible interaction between C101LAC and VT7, thereby further emphasizing the possible mechanistic similarities between IC1270-mediated ISR and R-protein-dictated ETI. Starting 50 hpi, a strong accumulation of H2O2 was found in CO39 mesophyll cells that appeared to collapse, whereas in samples from IC1270-induced CO39 or C101LAC sheaths, DAB staining in the mesophyll layer was seldom observed . However, at these late infection stages, massive H2O2 accumulation is most likely a consequence of progressive cellular destruction and overtaxed anti-oxidative capacities, and hence, a chaotic reaction associated with susceptibility, rather than a controlled defense response restricting cellular accessibility for M. oryzae. Together these results clearly demonstrate the potential of IC1270 to prime rice for augmented generation of epidermis-localized H2O2.In light of the well-documented ability of ROS to serve multiple defense-related signaling functions, sometimes with opposite effects in different contexts, we asked whether the ability of IC1270 to boost pathogenesis-related H2O2 generation might account for the differential effectiveness of IC1270-mediated ISR against M.oryzae, R. solani and C. miyabeanus. To address this question, we examined the effect of manipulating the oxidative stress in pathogen-inoculated leaves on subsequent disease development. To artificially raise the level of ROS in inoculated leaves, detached leaves were pressure-infiltrated with mixtures of glucose plus glucose oxidaseand xanthine plus xanthine oxidase .

Similar results were observed in LUX ARRHYTHMO mutants in diploid wheat, another component of the evening complex . These results suggest that in the temperate cereals, the evening complex of the circadian clock acts as a transcriptional repressor of PPD1 . Interestingly, two LUX-binding sites are present in the PPD1 promoter, including one in the region deleted in the Ppd-A1a allele. In barley, changes in photoperiod have been shown to have rapid effects on the expression of circadian clock genes . However, we did not observe significant changes in the expression profiles ofany of the core circadian clock genes after 21 NBs, suggesting that changes in the clock played a limited role in the induction of flowering by NBs . Moreover, the strong delay in heading time observed in the Kronos and Paragon ppd1-null mutants under NBmax demonstrated that PPD1 is the major driver of the acceleration of heading time by NBs. This does not rule out the possibility that the circadian clock may play an important role in the regulation of the intermediate steps between PPD1 and FT1 induction or in the PPD1-independent photoperiod pathway in the temperate grasses.In this study, we show that while a single NB as short as 15 min in duration is sufficient to induce PPD1, the peak of expression is not observed until 3 h after the NB . This timeline of events suggests that additional molecular steps may be involved in the transcriptional activation of PPD1 following the initial short exposure to white light. NB responses have previously been shown to be rapid, hydroponic equipment and red-light NBs of 2 min were shown to be sufficient to accelerate flowering . The short length of the light pulse required to trigger the NB response is consistent with a role of the phytochromes in the initial steps of the NB response. In Arabidopsis, conversion of phytochromes from Pfr to Pr forms occurs within 5 min of exposure to high radiance R light, and 2 min of R light treatment is sufficient to initiate the phosphorylation of PIFs, which are direct targets of activated phytochromes .

Phosphorylated PIFs are targeted for degradation by the 26S proteasome, triggering downstream transcriptional responses within 15 min of the light signals . The time lag between the light application and the up-regulation of PPD1 transcript levels suggests the existence of intermediate molecular steps. Based on the involvement of wheat PHYB and PHYC in the light activation of PPD1 transcription and the known interactions between phytochromes and PIFs in Arabidopsis, we hypothesize that the degradation of one or more PIFs acting as PPD1 transcriptional repressors may be involved in the light activation of this gene. A putative PIF binding site is present within the region of the PPD1 promoter that is deleted in the Ppd-A1a allele . According to this hypothesis, the application of FR after NB reduces Pfr levels and limits the degradation of this putative PIF, thereby maintaining some transcriptional repression of PPD1 . Although NBs do not perfectly mimic the LD response, there are several similarities between the two processes, particularly in the PPD1-dependent photoperiodic response. Both processes are dependent on the PHYB/PHYC-mediated light activation of PPD1, both processes require multiple inductive cycles to accelerate flowering, and in both NBs and in plants carrying the Ppd-A1a allele, expression of PPD1 during the night is associated with accelerated flowering. Based on these similarities and on previous studies, we propose a tentative working model for the PPD1-dependent photoperiodic regulation of flowering in wheat . According to this model, flowering is accelerated only when the light-induced expression of PPD1 coincides with the expression and/or activity of one or more circadian-regulated factor required for the induction of FT1. Under LD, but not under SD, PPD1 expression coincides with the putative additional factor, inducing FT1 expression .

When NBs are applied in the middle of the night, light-induced PPD1 expression coincides with a peak of the putative additional factor, resulting in maximal activation of FT1 and early flowering . Although NBs applied earlier or later than this point still result in the induction of PPD1, these NBs no longer coincide with a peak of the putative circadian-regulated factor required for the activation of FT1. In Arabidopsis, the sensitivity of the flowering response to the induction of FT expression is most effective when FT is artificially induced during the evening and early night , suggesting that the timing of FT induction can also carry information relevant to the acceleration of flowering. Other studies support the hypothesis that the timing of PPD1 induction is critical for flowering. In wheat plants carrying the Ppd-A1a allele conferring reduced sensitivity to photoperiod, PPD1 is expressed during darkness . Therefore, even in non-inductive SD photoperiods, PPD1 expression coincides with the peak activity of the putative circadian-regulated factor required for the activation of FT1 and the induction of flowering . This last result suggests that no light stimuli are required to induce FT1 and flowering when PPD1 is mis-expressed during the night. However, in both the phyB-null and phyC-null mutants, the relatively high transcript levels of PPD-A1a were insufficient to induce FT1. A possible explanation for this observation is that PHYB and PHYC are important for some of the intermediate molecular steps required for the FT1 up-regulation by PPD1.The putative additional factor required for FT1 induction is likely to be regulated by the circadian clock, with its expression or activity peaking between 6 and 10 h after dusk under a SD photoperiod of 16 h of darkness. This putative factor could function to stabilize or activate the PPD1 protein or be an additional factor that acts either in a complex with PPD1 or downstream of PPD1 to activate FT1. Alternatively, PPD1 may activate a protein that degrades a repressor of FT1 or induce epigenetic changes in FT1 or other intermediate genes.

The identification of this clock-regulated putative factor involved in the PPD1 activation of FT1 is an outstanding question of the PPD1-mediated photoperiodic response in wheat.Plant-based pharmaceutical production is appealing given its inexpensive facility and production cost, linear scale-up, the absence of animal pathogens, and capability to produce complex proteins and perform post-translational modifications, which overcomes one or more drawbacks of traditional recombinant protein expression systems such as animal cell culture and bacterial fermentation.Much work has been carried out using stably transformed plants, but the significantly reduced development and production timeline makes transient expression of proteins in whole plants a particularly attractive option, cutting the time to bring critical medications to the market during a pandemic. Vacuum agroinfiltration is the most widely used method for uniformly introducing agrobacterium harboring an expression cassette containing a gene of interest into plant tissue given its natural ability to transfer T-DNA into plant cells, which is ideal for transient protein production in plants. Although a plant-based recombinant protein production system provides distinct advantages over traditional systems, the differences between N-glycosylation of proteins produced in plants and humans could limit the use of plant systems for the production of glycoprotein-based pharmaceuticals. In higher eukaryotes, the initial steps of N-glycosylation processing are well conserved between plants and human, resulting in oligomannose-type N-glycosylation. However, late N-glycosylation maturation in the Golgi apparatus is kingdom-specific, and thus results in different N-glycosylation on proteins produced in plants compared with human. These plant-specific glycans may lead to potential safety issues such as hypersensitivity or allergy, as plant-specific α-fucose and β-xylose are known to be important IgE binding determinants of plant allergens. Thus, if these plant-specific glycans are present in an injected pharmaceutical product, the glycoprotein could trigger immunological response, or at least, result in a short circulation half-life. N-glycans of proteins produced from mammals are often terminated in β-galactose and sialic acid; sialic acid is particularly important as it typically increases the circulation half-life of proteins. There are a number of ways to avoid incorporating plant-specific glycans in the product such as adding a signal sequence at the C-terminus of the target protein to retain it within ER, or RNAi-mediated knock-down of α-fucose and β-xylose. These methods require modification to either the protein sequence or to the expression system, which can potentially affect protein structure and require a long developmental time. As an alternative, the use of small molecule inhibitors of intracellular glycosidases is a highly flexible bio-processing approach for controlling protein N-glycosylation patterns in transient agroinfiltration processes, and it is the approach investigated in this study. Here, we report an easy and fast way to modify N-glycosylation of recombinant proteins produced transiently in N. benthamiana through the addition of kifunensine in the agrobacterium suspension prior to vacuum agroinfiltration,vertical grow table which avoids modification to protein sequence or expression system while producing recombinant protein with oligomannose-type N-glycans that are similar between plant and human. Oligomannose-type N-glycan is preferred for the HIV-1 viral vaccine development as a vast majority of broad and potent neutralizing antibody responses during HIV-1 infection target mannose-glycan-dependent epitopes. In addition, monoclonal antibodies with oligomannose N-glycans show increased ADCC activity and affinity for FcγRIIIA. Protein N-glycosylation starts in the endoplasmic reticulum , where N-glycan precursors Glc3Man9GlcNAc2 are first synthesized, followed by the removal of terminal Glc residues, resulting in Man9GlcNAc2 structures. Then, a single α1,2 linked mannose is removed by ER class I α-mannosidase, producing Man8GlcNAc2 structures. The trimming of α1-2 mannose residues continues with the action of Golgi class I α-mannosidases in cis-Golgi to give Man5 structures. Kifunensine is a highly selective inhibitor of class I α-mannosidases in both plants and animals, and it has been used in cell cultures to produce recombinant proteins with oligomannose-type N-glycans. Although the general effects of kifunensine and other alkaloid-like processing glycosidases inhibitors are well understood, for the most part this information comes from cell culture system studies. Meanwhile, the study of kifunensine on whole-plant transient protein expression through agroinfiltration is new.

There are only two published papers on whole-plant kifunensine treatment, where kifunensine was supplied hydroponically throughout the whole incubation period, which requires larger quantities of kifunensine, a more expensive hydroponic system and constant monitoring especially at large scale as compared to our method. In addition, it was also shown that hydroponic kifunensine treatment resulted in dramatic decrease of protein expression level which was not observed with our method. In this study, Fc-fused capillary morphogenesis gene-2 , an anthrax decoy protein, served as a model protein, which contains single N-glycosylation site within its Fc domain . CMG2-Fc is a potent anthrax decoy protein as shown previously, where the CMG2 domain binds to anthrax protective antigen and prevents the anthrax toxin from entering the cell. Meanwhile, the presence of Fc domain significantly increases the serum half-life, which prolongs therapeutic activity owing the slower renal clearance for larger sized molecules and interaction with the salvage neonatal Fc-receptor. CMG2-Fc thus can be used as potent anthrax therapeutic and prophylactic without frequent redosing. The expression levels of CMG2-Fc produced transiently in wild type N. benthamiana under kifunensine treated and untreated conditions were measured with a sandwich ELISA, and protein N-glycosylation profiles were evaluated with mass spectrometry for kifunensine treated and untreated conditions. The findings in this study can be applied for N-glycosylation modification of other plant recombinant proteins when oligomannose-type N-glycans lacking core fucose are preferred, without the need to modify protein sequence and/or subcellular targeting.The CMG2-Fc expression levels in crude leaf extract were quantified through a sandwich ELISA to confirm the expression of CMG2-Fc, and to evaluate the effect of kifunensine on protein expression. The ELISA relies on binding of CMG2-Fc through the Fc region to protein A coated on a 96-well plate. A secondary anti-Fc polyclonal antibody linked to a horseradish peroxidase enzyme binds to the CMG2-Fc allowing colorimetric detection. The potential interference of plant host cell proteins and nonspecific binding were determined to be negligible. Twenty wild-type 5–6-week old N. benthamiana plants were divided equally into experimental and control groups, agro-infiltrated and incubated for 6 days, then whole leaves were extracted under identical conditions to determine protein expression. Kifunensine at a concentration of 5.4 µM was included in the agrobacterium suspension in the Kifunensine group. This kifunensine concentration was chosen as a starting point by taking the average of concentrations used in a previous CHO cell culture study, as no reference concentration is available for vacuum infiltration of kifunensine.

Excess Cd uptake in plants normally induces the accumulation of reactive oxygen species in plants and has severe consequences, such as chromosome aberrations, protein inactivation, membrane damage, and and further leading to leaf chlorosis and root growth inhibition. Furthermore, accumulation of Cd in crops enhances the risk of Cd poisoning in humans and animals. Brassica species have been identified as Cd hyperaccumulators. Brassica parachinensis L.H. Bailey is a leafy vegetable widely consumed in China, Europe, and other regions of the world. Thus, elucidating the molecular mechanisms of Cd accumulation in this plant is essential for developing effective strategies to control Cd accumulation in the plant’s edible parts. Cd accumulation in plant tissues generally involves a three-step process: absorption and accumulation of Cd in roots from the soil, translocation of Cd to the shoot via vascular tissue, and Cd storage in leaves. The HMA , ZIP , and Nramp families are among the transporter families that have been identified as being involved in these processes. Our previous transcriptome analyses of B. parachinensis also showed that differentially expressed genes enriched in the gene ontogeny terms ‘transmembrane transport’ and ‘metal ion transport’ may be involved in response to Cd, including genes encoding members of some transporter families, such as the subfamily C of ATP-binding cassette proteins and HMAs. HMAs,growing strawberries vertically which belong to the P1B subfamily of the P-type ATPase super family, have been extensively investigated in the model plant Arabidopsis as well as in some crop plants, and the main focus of these studies has been on their functions.

For example, eight members of HMAs have been identified in Arabidopsis thaliana, and among these, AtHMA1–AtHMA4 are thought to specifically transport divalent cations, such as Zn2+, Cd2+, Co2+, and Pb2+ [10]. AtHMA2 is generally regarded as a Zn2+- ATPase. It contains a conserved short metal binding domain in the N-terminus and a long metal binding domain in the C-terminal end; Zn2+-binding affinity was detected in both domains, and Cd2+- and Cu+-binding affinity was detected in the Nterminal domain. Some studies showed that AtHMA2 functioned as an efflux to drive the outward transport of metals from the cell cytoplasm and responsible for cytoplasmic Zn2+ homeostasis and Cd detoxification. Some researchers proposed that AtHMA2 together with AtHMA4 played key roles in the long-distance root to-shoot transport of Zn2+ and Cd2+ by loading these ions into the xylem. Similar results were also reported in wheat TaHMA2. However, it seems that OsHMA2 in rice has a different role. The enhanced sensitivity to Cd and tolerance to zinc deprivation afforded by heterologous expression of OsHMA2 in yeast cells suggest that OsHMA2 functions as a Cd influx transporter. These studies showed that HMA2 and its subfamily members in different plants may function differently. There is a lack of thorough knowledge of the role of BrpHMA2 in Cd hyper accumulation in the leafy vegetable B. parachinensis. The function of BrpHMA2 and the mechanisms that regulate its expression must be elucidated. Previous studies have indicated that plants employ a universal and conserved approach to regulate the transcription of heavy metal uptake and tolerance genes. For example, in a bean , PvMTF-1 , which could be induced by PvERF15 , may regulate the expression of the stress-related gene PvSR2 and confer Cd tolerance to the plant.

In Arabidopsis, two basic helix–loop–helix transcription factors , FIT and PYE , modulate iron deficiency responses by regulating the expression of IRT1 and FRO2, whereas the bHLH TFs IAA-leucine resistant 3 and bHLH104 can form heterodimers and bind to specific elements in the promoter of PYE to regulate PYE. NAC TFs are members of the most prominent TF families in plants. These TFs play essential roles in diverse biological processes, such as growth, development, senescence, and morphogenesis, and are widely involved in various signaling pathways in response to different phytohormones and multiple abiotic and biotic stresses. For example, NAC019, NAC055, and NAC072 negatively regulate drought stress-responsive signaling. NAC096 is associated with drought stress. It could exert its function via a mechanism like that of basic leucine zipper protein -type TFs to bind specifically to abscisic acid – responsive elements in the promoters of several drought stress-responsive genes. This finding implies that NAC096 and bZIP-type TFs can sometimes regulate the same target genes. Studies have also shown that the core DNA-binding sequences of NACRE and ABRE are PyCACG and PyACGTGG/TC , respectively. In a previous study, we identified a few NAC and AREB TFs triggered by Cd stress in B. parachinensis. However, their functions remain unclear. To clarify the molecular mechanisms of Cd accumulation in B. parachinensis, the function of a Cd-responsive metal ion transporter gene BrpHMA2 and the coregulation of BrpHMA2 transcription by two TFs were examined in this study. The findings reveal a precise regulatory mechanism in B. parachinensis in response to Cd stress.We previously analyzed the Cd-induced mRNA transcriptome of B. parachinensis and found that several HMA homologs were substantially expressed under Cd stress. We cloned one of the HMA2 homologs and constructed the phylogenetic tree of this HMA2 homolog with other HMAs in A. thaliana, Oryza sativa, Zea mays, and Alfred stone crop by the neighbor-joining method using MEGA5. The results revealed that the sequence of this HMA2 homolog is closer to that of the AtHMA2 gene , and thus it was named BrpHMA2.

The transcript level of BrpHMA2 in seedlings grown hydroponically was examined using reverse transcription–quantitative PCR to investigate the expression pattern of BrpHMA2 in B. parachinensis. According to the results, BrpHMA2 was expressed at higher levels in leaves than in roots. Cd stress may increase BrpHMA2 expression in leaves and roots, although BrpHMA2 expression in leaves fluctuates owing to developmental regulation . The GUS gene was transformed and expressed in Arabidopsis using the promoter of BrpHMA2 to corroborate the expression pattern, and histochemical assays were performed. Instant β-glucuronidase staining for 0.5 hours showed that the GUS signal was visible in the vascular bundles of the leaves and roots of the plants treated with 50 μM Cd 2 for 2 days, but not in vascular bundles ofseedlings that were not treated with Cd . Results from an examination of transcripts of the GUS gene in the reporter line were also consistent with these findings . This showed that BrpHMA2 could be induced by Cd stress. However, when the pBrpHMA2::GUS transgenic seedlings were subjected to GUS staining for 3 hours, a strong GUS signal could be observed in the vascular bundles of the cotyledons, true leaves, stems, petals, filaments, and the carpopodium of the seeds in young siliques. The blue GUS signal was particularly strong in the tissue junction regions where the vascular bundles were clustered . These results indicate that BrpHMA2 may function primarily in transport in vascular tissues. The fluorescent signal of BrpHMA-GFP was detected at the plasma membrane by transient expression analysis in protoplasts of B. parachinensis leaf cells , indicating that BrpHMA2 is localized at the plasma membrane.To further analyze the function of BrpHMA2, BrpHMA2 fused with the galactose-inducible promoter was transformed into a Cd-hypersensitive yeast mutant, ycf. In the presence of the transcriptional inducer galactose, Cd2+ considerably inhibited the growth of yeast cells with heterologous expression of BrpHMA2 compared with that of cells transformed with the empty vector . However, when gene expression was suppressed by the presence of glucose, no growth differences were detected between the cells transformed with BrpHMA2 and those transformed with the empty vector. The Cd content in the heterologous transgenic cells grown in liquid medium was higher than that in the control cells . These results indicate that BrpHMA2 functions as an affluxtype Cd transporter.To determine the TFs responsible for BrpHMA2 expression in B. parachinensis, a cis-element analysis of 2000 bp of the BrpHMA2 promoter was performed. In the promoter region, three ABRE cis elements were identified,vertical farming equipment all of which contain the G-box family core sequence ACGT . The NAC recognition site CGTG is likewise present in these ABREs. In the promoter of BrpHMA2, two additional NAC recognition motifs, CDBS and CACG, were found. Three ABREs , four NACRESs, and four CDBS cis elements were found in the promoter of BrpHMA2 . These findings suggest that certain transcription factors, such as NACs or AREBs, may control BrpHMA2 in B. parachinensis via these cis elements. To confirm this deduction and identify the regulatory pathways involved in the response to Cd stress, the transcriptome of B. parachinensis as mentioned above was used to collect data for the NAC and AREB genes that showed differential expression following Cd stress. Eighteen NAC genes and 11 AREB genes were selected to create a heat map, and three NAC TFs and three AREB TFs were identified as Cd-induced TFs . Their transcription levels were further analyzed by RT–qPCR. The results showed that the NAC TF genes BraA03000895, BraA010004584, and BraA10002796 were upregulated in the roots of the plants exposed to Cd for 1 day . After 4 days of Cd exposure, the AREB TF gene BraA01000449 was induced in roots, and BraA05001227, BraA01000449, and BraA01003678 were induced in leaves . Similar to the findings for BrpHMA2, our results suggest that these TF genes may respond to Cd. The coding sequences of the three NAC TFs and three AREB TFs listed above were cloned and submitted to the NCBI database. The last three or four numbers of each gene’s full name was used as the gene name. MEGA5 was used to create a phylogenetic tree of these NAC TF or AREB TF genes and Arabidopsis NAC or AREB genes using the neighbor-joining method.

The results revealed that the BrpNAC4584 and BrpNAC895 sequences were closer to those of Arabidopsis ANAC046 and ANAC087, respectively ; in addition, the BrpABI227 and BrpABI678 sequences were closer to that of AtABF4, and the BrpABI449 sequence was more comparable to that of AtABF3 .Electrophoretic mobility shift assays were conducted to investigate whether the BrpNAC895 protein directly binds to the promoter of BrpHMA2. Three probes containing NACRES and CBDS motifs on the BrpHMA2 promoter were designed and used for the EMSA. The results revealed that the BrpNAC895-MBP fusion protein could bind to the three probes in vitro . A chromatin immuno precipitation assay was performed using an anti-GFP antibody to precipitate BrpNAC895-GFP fusion proteins expressed in B. parachinensis protoplasts, and three fragments covering the NACRES and CBDS motifs on the BrpHMA2 promoter were designed and used for PCR. Moreover, there is only one base interval between the last two NACRES cis elements, so they were considered as one fragment . Approximately 1.5- to 2-fold enrichment of fragments pF1, pF2, and pF3 was detected compared with those found in the control . The results demonstrate that BrpNAC895 can promote the expression of BrpHMA2 by binding directly to the NACRES and CBDS motifs of its promoter.To investigate the mechanism of BrpHMA2 coregulation by BrpNAC895 and BrpABI449, a ChIP assay was performed by expressing BrpABI449-GFP in B. parachinensis protoplasts to analyze the binding affinity of BrpABI449 with the promoter of BrpHMA2. A qPCR analysis revealed that the BrpABI449 protein was enriched with fragments containing pF2 and pF3 of the BrpHMA2 promoter . We further performed an EMSA to confirm the binding of BrpABI449 to ABRE motifs in the promoter of BrpHMA2. The results proved that BrpABI449 could bind directly to the probes containing ABRE cis elements in the pF2 and pF3 regions of the BrpHMA2 promoter .The roles of BrpNAC895-binding loci in BrpHAM2 transcriptional regulation were investigated by constructing a series of BrpHMA2 promoter mutants by changing CACG/CGTG in the NACRES or CBDS to AAAA . A dual LUC assay was performed using the effector p35S::BrpNAC895 vector and the reporter vector was cotransformed into B. parachinensis protoplasts. Compared with pBrpHMA2::LUC, the cotransformation of p35S::BrpNAC895 with pMUT1::LUC or pMUT3::LUC resulted in much reduced LUC activity, but the cotransformation of the p35S::BrpNAC895 effector with pMUT2::LUC resulted in considerably higher LUC activity . Among the cotransformations of the promoter of BrpHMA2 with two or more mutations, substantially weaker LUC activity could only be seen in the transformations with promoters mutated at both locus 1 and locus 3 . These findings indicate that the mutation in the first and third NACRES motifs reduced the BrpNAC895-activated transcription of BrpHMA2, and these two binding loci may play central roles in the BrpNAC895-activated transcription of BrpHMA2.To elucidate the relationship between NAC and AREB TFs, a bimolecular fluorescence complementation approach was used.

Only one particle size of clay was used here, and diffusion rates were lower in this environment. In systems with low diffusion rates, exudate concentration is likely higher around the roots, which might lead to higher exudate re-uptake than in systems with larger particle sizes . Clays with different particle sizes might provoke a root morphology and exudation profile distinct from glass bead-grown plants and is worth further investigation. Further, substrate particle size might be a factor defining the amount of exudates present in soils.The largest difference in exudate profiles observed was between in situ clay-grown plants and other in situ conditions. Notably, the distinct exudation of clay-grown plants disappeared when exudates were collected in vitro, indicating that the differences observed resulted from the presence of clay, and not from an altered exudation of compounds by B. distachyon. About 20% of compounds were distinct between hydroponic and clay exudates, and most of these compounds were reduced in abundance in the presence of clay. Among these compounds were organic acids, amino acids, and nucleosides. When clay was incubated with a defined medium, 75% of compounds were reduced in abundance,container vertical farming among them negatively and positively charged compounds, as well as neutral compounds. The higher metabolite retention by clay in the defined medium experiment compared with the plant experiment might be due to several factors: The clay was incubated for two hours with the defined medium, but for three weeks with plants producing exudates.

Although exudates were also collected for two hours in the plant experiment, the clay was likely already saturated to some degree with exudates. The quantification of exudate amounts at different plant developmental stages in future studies would enable a better estimation of the total amount of compounds exuded and would correct for the difference in the two experimental setups. The reduction of metabolite abundance in the presence of clay is most likely due to its high ion exchange capacity, compared with quartz-based particles such as sand or glass beads . Previous studies investigating sorption of bacterial lysates to ferrihydrite found a depletion of more than half of the metabolites . Similarly, incubation of bacterial lysates with a soil consisting of 51% sand, 28% silt, and 21% clay resulted in low metabolite recovery rates . These findings are consistent with our data. Interestingly, two nitrogenous metabolites were higher in abundance in exudates of in situ clay-grown plants, . These compounds were not detected in clay negative controls, or in in vitro exudates of clay-grown plants, making it likely that the presence of plants leads to the release of these compounds from clay. Multiple examples exist in literature that describe a release of compounds from minerals by specific exudates. For example, plant-derived organic acids such as malate and citrate solubilize mineral-bound phosphate , and plant-derived oxalate releases organic compounds bound to minerals, making them available to microbial metabolism . Altered exudation depending on the growth substrate was also described for tomato, cucumber, and sweet pepper growing in stone wool, with higher exuded levels of organic acids and sugars compared with glass bead-grown plants .

The authors suggest that the presence of aluminum ions in stone wool might be responsible for the altered exudation observed. As the authors did not investigate in vitro-collected exudates of stone wool grown plants, it is unclear to which degree the observed effect was due to changes in plant metabolism or due to the presence of stone wool. In soils, metabolite sorption to minerals can lower decomposition rates . Also, the amount of clay in soil is correlated with retention of labeled carbon in soils . In clay-dominated soils, the size of clay particles shapes how much carbon can be retained: large clay aggregates were found to adsorb more carbon than smaller aggregates . Here, we only investigated one size of clay particles. Thus, it would be prudent to investigate the sorption behavior of clays with different particle sizes, and the ability of microbes to subsequently desorb these compounds. In natural systems, the presence of large amounts of clay with a specific particle size likely results in the sorption of plant-derived compounds to particles, changing the direct availability of these compounds to heterotroph organisms and, thus, altering soil processes.Microbes can release sorbed compounds from minerals, and they likely preferentially colonize minerals that are associated with compounds missing from the environment . The rhizobacterium Pseudomonas fluorescens utilized in this study was indeed able to desorb metabolites from clay, utilizing them as a carbon source for growth . In soils, root exudation creates zones with high metabolite concentrations.Although P. fluorescens was able to grow on particles conditioned with exudates, it did not grow on the effluent of the washed particles.

This suggests that the organism is able to release mineral-bound metabolites as an additional source for growth—a trait that supports competitiveness and survival in the rhizosphere. Root-associated bacteria have distinct exudate substrate preferences from bulk soil bacteria , which might also define the kind of compounds bacteria are able to release from minerals . Our results are further evidence that minerals play an important role in plant–microbe interactions by sorbing root exudates, which can later be solubilized by microbes for growth. We conclude that alteration in particle size affects root morphology in B. distachyon. Root exudation was constant per root fresh weight, and the exudate metabolite profiles were similar across root morphologies. Mass spectrometry imaging detected ion abundances across various regions of the root system, suggesting involvement of different tissues in exudation. Exudates were strongly sorbed by clay, significantly reducing the availability of free metabolites. Some of the clay-bound metabolites however could be utilized by a rhizobacterium for growth. Soil clay content thus is likely an important factor to consider when investigating root exudates or plant–microbe interactions in natural environments.Substrates with various particle sizes and surface chemistries were chosen as experimental systems to assess changes in root morphology and exudation. The particle sizes used correspond to large soil particles , intermediary particles , and small particles . Glass beads constitute an inert experimental system, for which the diameter of the spheres is defined, and the particles have a defined mineral composition . The sand and clay substrates constitute more natural environments than glass beads . The sand and clay mineral composition was either defined by the manufacturer or determined here. To assess the chemical properties of the substrates, the sorption of metabolites to the substrates was assessed by incubating them with a defined medium . The defined medium consisted of amino acids, organic acids, sugars, nucleobases, nucleosides, and others, see Table S2. The various substrates were sterilized, and the defined medium was prepared as a sterile, 20 µM equimolar solution. The substrates were fully submerged in the defined medium in sterile conditions and incubated at 24°C for 8 hr. The sterility of the system was confirmed by plating an aliquot on LB plates, followed by a 3-day incubation. The defined medium was removed by pipetting. The recovered volume was recorded; for substrates with smaller particle sizes, the entire volume could not be reclaimed. Samples were filtered through a 0.45-µm filter and frozen at −80°C. See “Liquid chromatography–mass spectrometry sample preparation” for sample processing.The frozen samples were lyophilized , resuspended in 3 ml LC/MS grade methanol , vortexed three times for 10 s, sonicated for 20 min in a water bath at 24°C, and incubated at 4°C for 16 hr for salt precipitation. Samples were then centrifuged for 5 min at 5,000 g and 4°C, and supernatants were transferred to new micro-centrifuge tubes and evaporated at 24°C under vacuum until dry. The dried extracts were resuspended in 500 µl LC/MS grade methanol,hydroponic vertical garden and the above procedure centrifugation, drying and resuspension procedure were repeated. Finally, samples were resuspended in 100% LC/MS grade methanol with 15 µM internal standards , with the solvent volume being proportional to the root biomass .In the various experimental systems used here, exudation rates could be limited by diffusion. To determine the diffusion rates of various substrates, sterilized substrates were added to pipettes with a 50 ml volume. The pipettes were sealed at the bottom with parafilm, placed vertically, 50 ml of substrate was added, and approximately 25 ml of sterilized 0.5× MS was added to fully immerse submerse the substrate . The experimental setup was sterile, but the experiment was conducted in non-sterile conditions.

Congo red 4B was solubilized in water at a concentration of 20 mg/ ml, and 250 µl of the dye was added simultaneously to pipettes containing the various substrates. The front of the dye was followed recorded over time up to 4.5 hr. Initially, the movement of the dye front was supported by mass flow .Brachypodium distachyon Bd21-3 seeds were dehusked and sterilized in 70% v/v ethanol for 30 s, and in 6% v/v NaOCl with 0.1% v/v Triton X-100 for 5 min, followed by five wash steps in water. Seedlings were germinated on 0.5× Murashige & Skoog plates in a 150 µmol/ m2 s −1 16-hr light/8-hr dark regime at 24°C for three days. Weck jars were rinsed five times with MilliQ water, sprayed with 70% v/v ethanol, treated with UV light for 1 hr in a laminar flow hood, and dried over night. The jars were filled with 150 ml of the respective substrate, and 50 ml of 0.5× MS basal salts liquid medium. Three seedlings were transferred into each jar, with the roots buried in the substrate. As a control, jars without substrate were prepared: PTFE mesh was cut to fit the size of the jar, and autoclaved. Three openings were cut into the mesh to hold the seedlings. The mesh was transferred to jars with 50 ml 0.5× MS medium. For each condition, an experimental negative control was prepared containing substrate, but no seedlings. The experimental control jars were treated the same as the jars containing plants. To enable gas exchange, two strips of micropore tape were placed across the jar opening, and the lid was set on top and wrapped with micropore tape to ensure sterility. Plants were grown in a 16-hr light/8-hr dark regime at 24°C with 150 µmol/m2 s −1 illumination, and the growth medium was replaced weekly: The old medium was removed by pipetting, and new 0.5× MS was added. Sterility of the jars was tested in week 3 by plating 50 µl of medium on Luria-Bertani plates, following by three days incubation at 24°C. Plants were grown for 21 days before exudate collection and root morphology determination.Mass spectrometry imaging was used to investigate spatial patterning of root exudation across the root system. Brachypodium distachyon seeds were sterilized and germinated on 0.5 MS plates as described above. A stainless steel MALDI plate was cleaned with 100% v/v ethanol, and a 7 × 7 cm square of aluminum foil was affixed to the plate with double-sided scotch tape. The foil was overlayed with 4 ml 0.1% ultrapure agarose to create a thin layer of agarose. Four-day-old seedlings were transferred to the agarose layer, and gentle pressure was applied with a spatula to embed the roots in the agarose. The stainless steel plate was transferred into a petri dish plate to keep humidity constant and incubated for 6 hr in a growth chamber with 150 µmol/m2 s −1 illumination and 24°C. MALDI matrix was prepared as follows: 10 mg/ml a-cyano-4-hydroxycinnamic acid and 10 mg/ml Super-DHB were dissolved in 75% v/v methanol, 24.9% LCMS-grade water, and 0.1% formic acid. The plate with the seedlings was removed from the growth chamber, leaves were cut to ensure flatness of the sample, and the sample was sprayed with MALDI matrix, which simultaneously desiccated the tissue. The plate was incubated for 24 hr in a vacuum desiccator until completely dry. Mass Spectrometry Imaging was performed using a 5,800 MALDI TOF/TOF in positive reflector MS mode with an Nd:YAG laser acquiring spectra over a range of 50−2000 Da and accumulating 20 shots/spot. The 4,800 Imaging Tool software was used to raster across the sample and record spectra in x-y step-sizes of 75 × 75 μm. Data viewing and image reconstruction were performed using OpenMSI .Ion chromatograms corresponding to metabolites represented within our in-house standard library were extracted from LC/MS data with Metabolite Atlas.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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