Month: April 2023

This result demonstrated that SlARF6A targets the promoters of CAB, RbcS, and SlGLK1 genes and positively regulates chlorophyll accumulation, chloroplast development and photosynthesis.Motif analysis showed that the SAMS1 promoter contains the conserved ARF binding site, the TGTCTC box. The transient expression assays showed that the LUC/REN ratios were significantly decreased compared with that of the control, suggesting that SlARF6A negatively regulates the expression of SAMS1 genes . ChIP-qPCR was carried out to confirm the binding of SlARF6A with the SAMS1 promoter in vivo, and the results showed that the promoter sequences containing the TGTCTC of SAMS1 were significantly enriched compared with those with the negative control anti-IgG . The direct binding of SlARF6A protein to the SAMS1 promoter was further verified by EMSA. The results indicated that the SlARF6A protein directly bound to the TGTCTC motif in the SAMS1 promoter . Taken together, SlARF6A can target the SAMS1 promoter and negatively regulate the expression of SAMS1 genes. The data demonstrate that SlARF6A plays an important role in ethylene production and fruit ripening.In this study, we functionally characterized the transcription factor SlARF6A in tomato. However, there are two very similar SlARF6 genes in the tomato genome, namely, SlARF6A and SlARF6B. We also examined the function of SlARF6B using genetic approaches and found no obvious phenotypes in the transgenic RNAi and over expression tomato plants . This may be related to the fact that SlARF6B lacks the AUX/IAA domain in the C-terminus of the protein .Previous studies reported that chlorophyll accumulation increased in Arabidopsis roots when they were detached from shoots,lettuce vertical farming which was repressed by auxin treatment. Mutant analyses showed that auxin inhibits the accumulation of chlorophyll through the function of IAA14, ARF7, and ARF19 in Arabidopsis.

In tomato, SlARF4 plays an important role as an inhibitor in chlorophyll biosynthesis and sugar accumulation via transcriptional inhibition of SlGLK1 expression in tomato. In this study, over expression of SlARF6A resulted in enhanced chlorophyll accumulation and chloroplast development, whereas down regulation of SlARF6A decreased chlorophyll accumulation and chloroplast number in tomato . These results demonstrate that SlARF6A positively regulates chlorophyll accumulation and chloroplast number in tomato. Our study also showed that SlARF6A directly targeted the SlGLK1 promoter and positively regulated SlGLK1 expression . Nguyen et al. reported that over expression of SlGLK1 and SlGLK2 produced dark-green fruits and increased chlorophyll accumulation and chloroplast development. The fact that the phenotypes of SlGLK1 over expression plants resembled those described in the OE-SlARF6A plants further suggests that SlARF6A positively regulates SlGLK1 to improve chlorophyll accumulation and chloroplast development in tomato leaves and fruits. Although SlGLK1 and SlGLK2 have similar functions, SlGLK1 functions largely in leaves, while SlGLK2 functions in fruits. However, SlGLK2 does not account for the chlorophyll phenotypes in OE and RNAi-SlARF6A plants because the ‘Micro-Tom’ variety possesses two null alleles of SlGLK2. In our study, down regulation of SlARF6A reduced SlGLK1 expression and chlorophyll accumulation, whereas over expression of SlARF6A increased SlGLK1 expression and chlorophyll accumulation in leaves and fruits of tomato plants . The data demonstrate that SlGLK1 may be involved in chlorophyll accumulation not only in tomato leaves but also in fruits. Further study is needed to elucidate the important role of SlGLK1 in tomato fruit using CRISPR/Cas9 technologies. The chlorophyll a/b-binding proteins are the apoproteins of the light-harvesting complex of photosystem II . CABs are normally complexed with xanthophylls and chlorophyll, functioning as the antennacomplex, and are involved in photosynthetic electron transport. Meng et al. reported that SlBEL11 directly acted on the promoter of CABs to suppress their transcription. Silencing of SlBEL11 increased the expression of CAB genes, resulting in enhanced chlorophyll accumulation and stability in thylakoid membranes of chloroplasts in green tomato fruit.

In our study, SlARF6A targeted the promoter of CABs, which positively regulated chlorophyll accumulation, chloroplast development and photosynthesis in tomato . Our data further demonstrate important roles of CABs in chloroplast activity and photosynthesis in tomato. Rubisco, a key enzyme in the fixation of CO2, is the ratelimiting factor in the photosynthesis pathway under conditions of saturating light and atmospheric CO2. The RbcL and RbcS genes encode two subunits that form the Rubisco enzyme. The RbcL and RbcS genes are localized to the chloroplasts and to the nucleus, respectively. Our study showed that over expression of SlARF6A increased the expression of the RbcS gene. Moreover, SlARF6A directly targeted the RbcS promoter and positively regulated RbcS expression . In addition, SlARF6A positively affected photosynthesis in the fruits and leaves of tomato plants . Our study demonstrates that SlARF6A has important roles in photosynthesis via the direct regulation of the RbcS gene in tomato. Interestingly, RNA-Seq data showed that the expression levels of SlARF4 and SlARF10 genes were not altered in RNAi-SlARF6A and OE-SlARF6A plants, suggesting that SlARF6A may act on chlorophyll accumulation independently of SlARF4 and SlARF10. However, studies indicate that ARFs must form dimers on palindromic TGTCTC AuxREs to form a stable complex, leading to the possibility that SlARF6A, SlARF4 and SlARF10 could form dimers with each other to regulate chlorophyll metabolism. Further study could focus on the interactions among SlARF6A, SlARF4, and SlARF10 to comprehensively elucidate the effects of the transcriptional regulation of ARFs on chlorophyll accumulation in tomato.Down regulation of SlARF4 increased the photosynthesis rate and enhanced the accumulation of starch, glucose and fructose in tomato fruits. In this study, the increased chlorophyll accumulation and photosynthesis rate in OESlARF6A plants resulted in the increased contents of starch and soluble sugars in fruits . Starch is a dominant factor in the nutrients and flavor of fruits. AGPase catalyzes the first regulatory step in starch synthesis, converting glucose-1-phosphate and ATP into ADP-glucose. This critical catalytic reaction is also a limiting step during starch biosynthesis in potato tubers. Knockdown of SlARF4 increases the expression of AGPase genes and starch content. In this study, SlARF6A was positively correlated with the expression of AGPase genes , suggesting the important role of AGPase genes in starch biosynthesis in tomato. However, the EMSA failed to detect any binding between SlARF6A and the promoters of AGPase genes, even though auxin-responsive motifs were detected in the promoters of AGPase S1 and AGPase S2 genes.

Evidence suggests that sucrose induces the expression of AGPase genes in leaves and fruits in tomato. In this study, over expression of SlARF6A led to increased sucrose content in tomato fruits, while the RNAi-SlARF6A fruits displayed decreased sucrose accumulation . The altered accumulation of starch in OE-SlARF6A and RNAi-SlARF6A lines may be explained by the altered expression of AGPase genes influenced by sucrose in tomato. Over expression of SlARF6A also resulted in increased glucose and fructose content, vertical grow shelf which was likely due to the increased starch content degraded into increased contents of soluble sugars in tomato fruits. Our results are consistent with the notion that incipient starch content determines soluble sugars in the process of fruit development. Our study also provides a valuable method to improve the nutritional value of tomato fruits via regulation of SlARF6A expression.The tomato ARF2A gene was reported to positively regulate fruit ripening. Over expression of ARF2A in tomato resulted in blotchy ripening, and silencing of ARF2A led to retarded fruit ripening. Overexpression of ARF2A in tomato promoted early production of ethylene and expression of ethylene biosynthesis and receptor genes. In this study, SlARF6A negatively regulated fruit ripening and ethylene biosynthesis in tomato fruit . S-adenosyl-L-methionine , synthesized by SAM synthetase from ATP and methionine, is a substrate for ethylene biosynthesis . SAM is converted to ACC by the ACS enzyme, and ACC is then converted to ethylene by ACO. The level of SAM is tightly controlled to integrate developmental signals into the hormonal control of plant development. In Arabidopsis, over expression of SAMS1 increases the SAM and ethylene levels, whereas sam1/2 mutants show the opposite phenotype in seedlings. Similarly, in tomato plants, over expression of SAMS1 results in higher concentrations of ACC and ethylene compared with those in WT plants. These data indicate the important role of the SAMS1 gene in ethylene biosynthesis in plants. In this study, SlARF6A directly targeted the SAMS1 promoter and negatively regulated SAMS1 expression . The regulatory mechanism by which SlARF6A affects fruit ripening and ethylene production in tomato fruits can be explained by the interaction between SlARF6A and the SAMS1 promoter. It is interesting that ethylene and auxin interact with each other to control some plant developmental processes. For example, ethylene controls root growth through regulation of auxin biosynthesis, transport and signaling, while the formation of hypocotyl apical hooks is also regulated in a similar fashion in Arabidopsis. In tomato, knockdown of IAA3 results in both auxin and ethylene phenotypes, suggesting that IAA3 might be the molecular connection between ethylene and auxin. Liu et al. reported that the ethylene response factor SlERFB3 integrated ethylene and auxin signaling through direct regulation of the Aux/IAA27 gene in tomato. Our results indicate that SlARF6A negatively regulates ethylene biosynthesis and that the interaction of SlARF6A and SAMS1 represents an important integrative hub mediating ethylene-auxin cross-talk in tomato. In summary, our results demonstrate that SlARF6A regulates chlorophyll level and chloroplast development by directly binding to the promoters of the SlGLK1, CAB1, and CAB2 genes. SlARF6A also directly targets the RbcS gene promoter, activating RbcS expression and increasing the photosynthetic rate. The increased chlorophyll accumulation and chloroplast activity improve photosynthesis, resulting in the increased accumulation of starch and soluble sugars in tomato. In addition, SlARF6A can act directly on the promoter of SAMS1 and negatively regulate its expression, thereby influencing ethylene production and fruit ripening.

The present study provides new insight into the link between auxin signaling, chloroplast activity, and ethylene biosynthesis during tomato fruit development. Our data also provide an effective way to improve fruit nutrition of horticulture crops via regulation of chlorophyll accumulation and photosynthetic activity.For chlorophyll content determination, the fruits at different developmental stages and leaf tissues were collected and examined based on the methods described by Powell et al.. To determine chlorophyll auto- fluorescence, pericarp was peeled off tomato fruits and observed with a TCS SP2 laser confocal microscope . For transmission electron microscopy, pericarp tissues were examined with an FEI Tecnai T12 twin transmission electron microscope according to the method described by Nguyen et al.. For measurements of photosynthesis rates, the green mature fruits and leaves were measured via a PAM-2500 pulse-amplitude modulation fluorometer . The chlorophyll fluorescence parameter was measured based on the method described by Maury et al..For sugar extraction, 1 g of fruit tissue was collected and ground under liquid nitrogen. Subsequently, 10 mL of 80% ethanol was used for extraction three times at 80℃ for 30 min. After centrifugation, samples were completely evaporated under vacuum and then dissolved in 4 mL of distilled water. Using the dissolved samples, HPLC was carried out to determine the content of sucrose, fructose and glucose. Starch content determination was performed using fruit pellets. Four milliliters of 0.2 M KOH was used to dissolve the pellet by incubating the sample in a boiling water bath for 30 min. Then, 1.48 mL of 1 M acetic acid with 7 units of amyloglucosidase was employed to hydrolyze each sample for 45 min. Finally, 10 mL of distilled water was adopted to dissolve the sample, and then the dissolved sample was used for starch content measurement. For metabolite measurement, HPLC analysis was conducted using an Agilent 1260 Series liquid chromatograph system with a vacuum degasser, an autosampler, a binary pump, and a diode array detector controlled by Agilent ChemStation software. A precolumn and a Waters XBridge Amide column were used for analysis. The separation was performed via an isocratic solvent system with solvent A and solvent B , while the mobile phase was maintained at 75% B for 15 min for elution. The column temperature was maintained at 38 °C, and the solvent flow rate was 0.6 mL/ min. Meanwhile, the injection volume was 10 μL for each sample. With a drift tube temperature at 80 °C, the detection system for HPLC was an ELSD 2000, and air was used as the carrier gas with a flow rate of 2.2 L/min. Finally, the contents of glucose, fructose, sucrose and starch in tomato fruits were determined based on the methods described by Geigenberger et al..

Our results show that the greatest variation in stem water d18O and plant water sources occurred during the frontal season and initiation of the dry season in February, whenG. floribundum was shedding old leaves and growing new leaves, but P. piscipula maintained its leaves from the previous wet season . Contrary to what was expected, P. piscipula took water primarily from shallow sources regardless of the month, although some contribution from deeper sources has the potential to occur. Rain also appeared to be an important source for this species. This implies that P. piscipula could have a very well developed shallow root system that allows rapid water uptake after a precipitation event. On the other hand, G. floribundum took water from topsoil and bedrock, the latter being a more important source in the dry season. This suggests a deeper root system than G. floribundum. Overall, our results indicate that the contrasting early and late dry season leaf loss phenology of these two species is not simply determined by rooting depth, but rather a more complicated suite of species-based characteristics based on opportunistic use of dynamic water sources, the balance between carbon gain and water loss, and maintenance of water potential at the end of the dry season. These results are consistent with other studies demonstrating a broad array of coordinated strategies for dealing with seasonal drought in tropical forests . A primary factor determining differences in leaf loss phenology between the two studied species appears to be the maintenance of water potential. G. floribundum consistently exhibited more positive water potential values than P. piscipula, suggesting that G. floribundum has a limited capacity to tolerate negative water potential and moderates water use in a manner that maintains bulk leaf water potential at relatively more positive values compared to P. piscipula . This could provide an advantage of maximizing carbon gain during the dry season when light availability is high.

Leaf organic d13C and d18O values support this observation, because P. piscipula showed consistently higher d13C values than G. floribundum ,vertical farm coupled with lower d18O indicating that the decrease in photosynthetic carbon isotope discrimination was associated with greater stomatal conductance and greater photosynthesis . Greater photosynthesis in P. piscipula is consistent with maintaining a canopy of leaves later into the dry season. Thus, our results are most consistent with maintenance plant water potential to maximize carbon gain during the onset of the dry season. The observation that P. piscipula appeared to use shallower water sources and maintained its canopy of leaveslater into the dry season was not expected. Part of this pattern is driven by the capability of P. piscipula to utilize dynamic sources of water, such as the cold front precipitation during the frontal season . This makes sense, because the Laja bedrock layer was a poor source of water at all times, and soil pockets, which are available, but heterogeneous in distribution, were always better sources of water than rock layers . Water content of soil/ bedrock sources changed along the year suggesting a different seasonal contribution to plant water uptake. The hg of topsoil in the wet and frontal seasons were very similar and three times greater than values measured in the dry season. The Sascab bedrock layer could be a significant source of water in the wet and frontal seasons, but not in the dry season. Soil pockets had two times more water than topsoil in the dry season suggesting that they could be an important source of water for trees during the dry season. In the dry season, the rock profile had hg between 1 and 5 % but nearly all were less than 1 %. These values were slightly lower than those reported by Querejeta et al. and Hasselquist et al. in nearby areas, which suggest that the bedrock was subjected to a greater evaporation during this study. The d18O values of water in this study integrated processes ranging from evaporation of soil and bedrock water sources, transpiration of tree species, and precipitation events. In the wet season, enriched values of d18O of water in topsoil 10–15 cm and trees revealed the occurrence of a depleted precipitation event that occurred on October 21, 1 day before sampling, bringing 19.7 mm of water . Furthermore, a frontal system including cold front #3, the tropical wave #37, as well as the remnants of tropical storm Kiko that formed in the pacific, converged on the study area days before the wet season sampling in October 2007 .

Hurricanes, tropical storms and cold fronts generally have lower stable isotope ratios than convective precipitation events . For example, Perry et al. recorded d18O values of -9.91 % for precipitation during tropical storm Mitch in 1998, and precipitation events ranging from -6 to -10 % for d18O have been recorded in the vicinities of the study area . Consequently, depleted oxygen values in soil 15–30 cm and P. piscipula and G. floribundum trees could be accredited to precipitation originated from these events. Soil pockets also showed more negative values than rock, suggesting that depleted rain water reached this layer. During dry season measurements in February 2008, the d18O of topsoil 0–15 cm was more positive than groundwater suggesting another depleted source of water. Cold front #29, which occurred 4 days before sampling and brought 33.9 mm , could be the main source of water. The strength of the dry season promoted the enrichment of all water sources respect to earlier samplings. However, the more negative value of topsoil from 0 to 5 cm could be affected by dew water since this soil sampling was done early in the morning. More negative d18O values in topsoil than ground water have also been observed by Saha et al. in similar environmental conditions in Miami, associated with water condensation occurring at night in the upper soil layers. Condensation has been shown to deplete d18O soil water 10–15 cm depth by 5 % . Condensation can also account for up to 47 % of total transpiration . Surface dew is easily generated when temperatures go below the dew point at night or in early morning . Under tropical conditions in Tahiti, Clus et al. reported average dew yields of 0.102 mm of dew during the dry season. Therefore, condensed water could be an important source for P. piscipula. Overall, our results indicate that variation in phenology between these two deciduous tropical dry-forest tree species, which vary in the timing of their deciduousness, is not akin to the relatively large variation in rooting depth that can occur between tropical evergreen and deciduous species , but rather reflects the diversity of plant physiological strategies that occur in tropical forest .Insertional mutagenesis is a powerful strategy for gene identification and functional genomics in plants . While the T-DNA approach is applicable to the model plants Arabidopsis and rice, where effective transformation methods are available,nft vertical farming it may not be feasible in many other plant species whose transformation is inefficient.

Transposon can be alternatively used for insertional mutagenesis in those plants, since the generation of new insertions occurs through crossing or propagation rather than through transformation. Supported by the United States National Science Foundation-Plant Genome Program . S.Q. was supported by the Research Start-up Grants of Zhejiang Academy of Agricultural Sciences , China. P.B.F.O. was supported by EU FP5 and FP6 projects CerealGene Tags and CEDROME . J.-S. J. was supported by the World Class University and the Crop Functional Genomics Center projects, Korean Ministry of Education, Science and Technology, Korea.The maize Ac-Ds transposable element has been shown to be active in the plant kingdom widely .Ds insertion libraries have been generated in Arabidopsisand rice . However, the current strategies of transposon tagging are usually slow and labor intensive and have several drawbacks. For example, in the presence of Ac transposase , transposed Ds elements may continue secondary transpositions. Unstable Ds insertions and serial transposition events may cause untagged mutations because imprecise excision or a transposition footprint can result in a mutation that is no longer associated with the transposon . Another problem is that the Ac-Ds transposable elements are highly active in rice and can transpose early in newly transformed callus cells , which results in many sibling plants carrying the same Ds insertions and consequently decreasing gene tagging efficiency. In the present study, we constructed 12 Ac-Ds transposon tagging vectors based on three approaches: AcTPase controlled by glucocorticoid binding domain/VP16 acidic activation domain/Gal4 DNA-binding domain chemical inducible expression system; deletion of AcTPase via Crelox site-specific recombination that was initially triggered by Ds excision; and suppression of early transposition events in transformed rice callus through a dual-functional hygromycin resistance gene in a novel Ds element. We have tested these vectors in transgenic rice and characterized the transposition events. Our results showed that these vectors are useful in functional genomics of rice and they will be useful for other crop plants as well.We constructed Ac-Ds transposon tagging vectors using a GVG-inducible expression system . The vectors pJJ86 and pDs-Ac-GVG carry an in cis two-element system that consists of Ds, 35S:GVG that expresses the chimeric GVG transcription activator, and AcTPase controlled by a GVG-inducible promoter. The inducible promoter is transactivated through interaction between GVG and the 4xGAL4-upstream activating sequence .

The transactivating activity of GVG is regulated by treatment with the steroid chemical dexamethasone . The Ds element in pJJ86 contains the 4x CaMV 35S enhancers for activation tagging , while the Ds in pDsAc-GVG does not. Excision of Ds from pJJ86 can be detected because in the resulting T-DNA fragment, the β-glucuronidase gene is driven by a CaMV 35S promoter. We also constructed a two-vector tagging system in which GVG-inducible AcTPase and Ds are in separate vectors . The strategy of the two-vector system is that transgenic plants carrying the GVG-inducible AcTPase and Ds are generated, respectively, and the AcTPase and Ds are combined in F1 by genetic crosses. In this case, Ds is mobilized in the presence of AcTPase in F1 plants, but stabilized after it is uncoupled from AcTPase in the subsequent generation. To test whether the inducible Ac-Ds system is functional in rice, we transformed rice cultivar Nipponbare with pJJ86. Independently transformed rice calli were cultured for 5 d on media with DEX to induce expression of AcTPase. Because Ds transposition can be detected by GUS activity, the DEX-treated calli and untreated controls were stained for GUS activity. DEX treatment of pJJ86-transformed calli exhibited stronger GUS staining than controls , indicating that the DEXinducible system in this vector is functional in rice. At the same time, there was low background of GUS activity in the untreated rice calli , suggesting that some background transposition occurred in the pJJ86 transformants.Because the Ac-Ds transposable elements are active in newly transformed callus cells and early transposition events lead to the same Ds insertions in sibling plants, we constructed a novel Ds element, designated HPT-Ds, and used the hygromycin resistance gene to suppress transposition. The pHPT-Ds1 vector carrying HPT-Ds and GVGinducible AcTPase in cis is shown in Figure 1E. The HPT gene in HPT-Ds has the same intron and triple splice acceptors as in the gene-trap Ds . Because HPT-Ds is immediately downstream of maize ubiquitin 1 promoter in T-DNA, the Ubi:HPT-Ds fusion confers hygromycin resistance, and transformed rice cells are thereby selected on hygromycin media. In case of transposition, HPT-Ds in the rice genome may not have a promoter nearby for transcription and the rice cells lose hygromycin resistance and can be counter-selected by hygromycin. To examine the efficacy of the HPT-Ds element, we made a test construct containing Ubi:HPT-Ds and confirmed the function of the Ubi-driving HPT gene in a rice transformation experiment. A total of 250 rice calli were transformed using a particle bombardment method and hygromycin-resistant cells were selected from 30 callus explants after 50 d of selection on hgromycin media. In constructing the pHPT-Ds1 vector, HPT-Ds was cloned between Ubi and GUS so that transposant cells canbe detected by GUS assay. The pHPT-Ds2 vector is similar to pHPT-Ds1 except that pHPT-Ds2 carries a Bar gene and transposition can be selected by herbicide resistance . pHPT-Ds1 was introduced into rice cultivar Nipponbare.

In addition, the scale of the application required to have an impact on the atmospheric C level is unclear at this point. More long-term and standardized studies, under different environmental conditions, of below ground carbon fluxes, integrating models and measurements are needed. C sequestration through plant-microbe interaction is still in its exploratory phase. As more worldwide attention is drawn towards mitigating elevated atmospheric C level, hopefully more global collaborative interdisciplinary research efforts will be directed towards assessing the conditions required for successful application of plant-microbe C sequestration. Nitrogen is an essential macro-nutrient for plant growth and development and most terrestrial plants absorb nitrate as their main nitrogen source. In agricultural systems, nitrate supply directly affects plant growth and crop productivity. In many developed and developing countries, excessive nitrogen fertilizer is applied in agriculture,commercial greenhouse supplies while the nitrogen use efficiency of crops is low. Therefore, a large fraction of the applied nitrogen cannot be taken up by plants and is lost into the environment, resulting in serious problems such as eutrophication and nitrate pollution of underground water. These problems must be addressed. One approach is to improve the NUE of crops, which could reduce the load of nitrogen fertilizers on farm land and natural ecosystems. Elucidating the mechanisms and the underlying network of nitrate regulation would provide a theoretical basis and guiding framework for improving NUE.A part of the nitrate imported into cells is reduced and assimilated into amino acid through a series of enzymes including nitrate reductase , nitrite reductase , glutamine synthase , and glutamate synthase .

Nitrate acts as a nutrient and as an important signal to regulate gene expression, plant growth, and development.The short-term effect is referred to as the primary nitrate response, in view of the fact that many genes can be regulated after a short period of exposure to nitrate inputs. Indeed, some genes involved in nitrate transport and reduction are induced in a matter of minutes. The long-term effects include the impact of nitrate on plant growth and development after a longer period of time, including effects on the morphogenesis of roots, plant flowering, seed dormancy, stomatal closure independent of abscisic acid, the circadian rhythm, and the transport of auxin. Among these aspects, the effects of nitrate on root development are well studied and several essential genes involved in this process have been identified. Here we review the genes involved in the primary nitrate response and describe their functions in nitrate signaling . Then we summarize the relationship between nitrate availability and root system architecture and the roles of the characterized genes that control root growth and development in response to local and systemic nitrate signals .In the late 1990s, molecular components involved in nitrate signaling were identified in bacteria and fungi. In Escherichia coli, both NARX and NARQ containing a P-box domain were found to be responsible for nitrate binding and could activate the nitrate-regulating proteins NarL and NarP, which are essential for nitrate sensing. Therefore, these two genes are nitrate regulators in E. coli. In fungi, two transcription factors NirA and Nit4 have been identified as important nitrate regulators. NirA is needed for the expression of nitrate reductase and Nit4 may interact with nitrate reductase directly. Both proteins were demonstrated to activate their target genes that can respond to nitrate. In plants, some genes encoding proteins required for nitrate assimilation, transport, and energy and carbon metabolism are rapidly induced after nitrate treatment.

These are regarded as primary nitrate-responsive genes. Scientists have characterized a few of the regulators playing important roles in primary nitrate responses, mainly by employing methodologies in forward and reverse genetics as well as systems biology. NRT1.1, also called CHL1 and NPF6.3, belongs to the NRT1/PTR family. Previously, NRT1.1 was identified as a dual-affinity nitrate transporter working in both low and high nitrate concentrations. Subsequently, it was shown that NRT1.1 controlled root architecture by acting as a potential nitrate sensor. Then in 2009 it was found that NRT1.1 is involved in the primary nitrate response. Using a forward genetic screen, the Crawford lab identified a mutant with a defective response to nitrate, and the mutation was localized to NRT1.1. Characterization of the mutant revealed that expression of the primary nitrate-responsive genes NIA1, NiR, and NRT2.1 was significantly inhibited when plants were grown in the presence of ammonium. Interestingly, the regulatory role of NRT1.1 was lost when ammonium was absent because the expression of these nitrate-responsive genes was restored in the mutant without ammonium, indicating that other nitrate sensor were present and dominated in the absence of ammonium. The Tsay lab also showed that a null mutant of NRT1.1, chl1-5, lost both nitrate uptake and primary nitrate response functions. They then described an allele of NRT1.1that was defective in nitrate uptake but not nitrate regulation. These results indicate that the primary nitrate response was defective in the mutant chl1-5 but not in chl1-9, and the function of NRT1.1 in nitrate signaling is independent of its uptake activity, thereby identifying NRT1.1 as a nitrate sensor. This research also found that when NRT1.1 was phosphorylated at a low nitrate concentration, it was involved in maintaining the low-level primary response; when it was dephosphorylated under a high nitrate concentration, it led to a high-level primary response. More recent work has shown that NRT1.1-mediated regulation is quite complex in that it activates distinct signaling mechanisms.

Furthermore, a rice homolog of AtNRT1.1has been identified, and variations in this gene in the rice sub-species indica have been identified as boosting the absorption of nitrate and the transport of nitrate from roots to shoots, and potentially enhance NUE in rice.Another important nitrate regulator is the transcription factor NLP7, which belongs to the NIN -like protein family in Arabidopsis. The NIN protein family was originally found to function in the initiation of nodule development in legume species and these family members are conserved in higher plants and algae . The NIT2 protein is a homologue of the NIN family in Chlamydomonas and can bind to the promoter of the nitrate reductase gene. In Arabidopsis,vertical grow NLP7 has been demonstrated to be an important positive regulator of primary nitrate response as the induction of the nitrate-responsive genes NIA1, NIA2, NRT2.1, and NRT2.2 is inhibited and nitrate assimilation is also impaired in nlp7 mutants. The function of NLP7 in nitrate signaling was further confirmed by the identification of the nlp7 mutant in an effort to explore novel nitrate regulators by using a forward genetics approach. ChIP-chip analysis revealed that NLP7 could bind 851 genes including genes involved in N metabolism and nitrate signaling, such as NRT1.1, CIPK8, LBD37/38, and NRT2.1. A recent study found that NLP7 could regulate the expression of NRT1.1 in the presence of ammonium and bind directly to the promoter of NRT1.1. These findings illustrate that NLP7 works upstream of NRT1.1 directly when ammonium is present. NLP7 can also activate or repress nitrate-responsive genes. The Arabidopsis thaliana genome encodes nine NLPs, all of which contain the conserved RWP-RK domain and the PB1 domain. Members of this family can be divided into four subgroups: NLP1 and 2, NLP4 and 5, NLP6 and 7, and NLP8 and 9 . Yeast one-hybrid screening using four copies of the nitrate response cis-element illustrated that all NLPs could bind to the NRE element. In response to nitrate, the transcript levels of NLP genes are not regulated, but examination of an NLP7-green fluorescent protein fusion revealed that localization of NLP7 was modulated by nitrate via a nuclear retention mechanism. Recently, this localization of NLP7 was identified to occur when Ser205 in NLP7 was phosphorylated in vivo in the presence of nitrate. Suppression of the NLP6 function resulted in the down regulation of nitrate-responsive genes, indicating that NLP6 is also a master nitrate regulatory gene involved in primary response. Further characterization has shown that the N-terminal region of NLP6 is necessary for its activation in response to nitrate signaling. Furthermore, using over expression lines, NLP7 was revealed to significantly improve plant growth under nitrogen-poor and -rich conditions. Moreover, ZmNLP4 and ZmNLP8, maize homologs of AtNLP7, play essential roles in nitrate signaling and assimilation and promote plant growth and yield under low nitrate conditions, implying that they may be potential candidates for improving the NUE of maize. In addition to NLPs, reverse genetics has identified LBD37/38/39 to be important nitrate regulators. LBD37/38/39 belong to a gene family encoding zinc-finger DNA binding transcription factors and are strongly induced by nitrate. Further characterization revealed that over expression of LBD37/38/39 can repress the expression of nitrate-responsive genes including NRT2.1, NRT2.2, NIA1, and NIA2, indicating that the three LBD members function as negative regulators in nitrate signaling. Recently, following advances in bio-informatics and global sequencing analysis, systems biology approaches have been developed and successfully applied to plant nitrogen research. The transcription factors SPL9, TGA1, and TGA4 have been sequentially identified by systems approaches.Research has demonstrated that miR156 can target SPL9 and a mutation in the miR156 caused over expression of SPL9.

Accordingly, miR156-resistant SPL9 transgenic plants were investigated and it was revealed that the transcript levels of NRT1.1, NIA2, and NIR significantly increased in response to nitrate, demonstrating that SPL9 plays a negative role in the primary nitrate response. TGA1 and TGA4 belong to the bZIP transcription factor family and are induced by nitrate in roots. Interestingly, induction of TGA1 and TGA4 is inhibited in chl1-5 and chl1-9 mutants after nitrate treatment, implying that the regulation of TGA1 and TGA4 by nitrate is affected by nitrate transport, but not the signaling function of NRT1.1. Transcriptome analysis of the roots of tga1 tga4 double mutant plants revealed that most of the genes differentially expressed in the double mutant were regulated by nitrate. Among these target genes of TGA1 and TGA4, induction of NRT2.1 and NRT2.2 was substantially reduced in the double mutants. Further analysis demonstrated that TGA1 could bind to NRT2.1 and NRT2.2 promoters to positively regulate their expression. These results all serve to suggest that TGA1 and TGA4 play important roles in the primary nitrate response.Recently, Shuichi’s lab found that nitrate-inducible GARP-type transcriptional repressor1 proteins act as central regulators in nitrate signaling. Co-transfection assays revealed that NIGT1-clade genes including NIGT1.1/HHO3, NIGT1.2/HHO2, NIGT1.3/HHO1, and NIGT1.4/HRS1 were all induced by nitrate and were redundant in repressing the nitrate-dependent activation of NRT2.1. EMSA and chromatin immunoprecipitation–quantitative PCR analysis further showed that NIGT1.1 could directly bind to the promoter of NRT2.1. Transcriptome and co-transfection analysis also illustrated that the expression of NIGT1s was auto regulated and controlled by NLPs. In addition, NIGT1.1 can suppress the activation of NRT2.1 by NLP7. Further investigation suggested that the regulation of NRT2.1 by NIGT1.1 and NLP7 is independent due to their distinct binding sites. A genome-wide survey discovered the potential target genes that might be regulated by both NLP-mediated activation and NLP–NIGT1 transcriptional cascade-mediated repression or the NLP–NIGT1 cascade alone. Furthermore, phosphate starvation response 1 , the master regulator of P-starvation response, also directly enhanced the expression of NIGT1-clade genes, serving to reduce nitrate uptake. CIPK8 and CIPK23 are calcineurin B-like -interacting protein kinases. CIPK8 is induced rapidly by nitrate and down regulated in the chl1-5 mutant. Analysis of two independent T-DNA insertion lines showed that induction of NRT1.1, NRT2.1, NIA1, NIA2, and NiR by nitrate was reduced in cipk8 mutants indicating that CIPK8 works as a positive regulator in the primary nitrate response. Further investigation revealed that CIPK8 regulated the nitrate-induced expression of NRT1.1 and NRT2.1 under higher but not lower nitrate conditions , suggesting that CIPK8 functions as a positive regulator when nitrate is replete.CIPK23 can be induced by nitrate and down regulated in the chl1-5 mutant like CIPK8 . Expression of the nitrate responsive gene NRT2.1 was upregulated in the cipk23 mutants after nitrate treatment, indicating that CIPK23 plays a negative role in primary nitrate response. This gene is essential for the affinity switch of NRT1.1: it interacts with NRT1.1 and phosphorylates NRT1.1 at T101 under low nitrate concentrations to enable NRT1.1 to operate as a high affinity nitrate transporter, while it dephosphorylates NRT1.1 when nitrate is replete so that NRT1.1 functions as a low-affinity nitrate transporter.

As cities work to fulfill their role in providing basic services to citizens, farmers are pointing out an important opportunity to provide refrigerated transportation, storage, and organizational infrastructure to transfer all possible produce grown on urban farms to the best distribution sites. Communication platforms, transport systems, and streamlined procurement in this arena following from other regional “food hub” models could improve the landscape for urban food distribution dramatically . All urban farm respondents are also engaged in closed-loop waste cycles: through composting all farm waste onsite and collecting food scraps from local businesses, farms are involved in a process of regeneration, from food debris to soil.Through extending the UAE framework from farms to urban policy and planning conversations, more efficient pathways for addressing food insecurity in part through strategic centers of urban production and distribution can emerge in cities of the East Bay and elsewhere in the United States. Finally, agroecology relies on the co-creation and sharing of knowledge. Top-down models of food system transformation have had little success. Urban planners have an opportunity to address food insecurity and other urban food system challenges including production, consumption, waste management and recycling by co-creating solutions with urban farmers through participatory processes and investing in community-led solutions. In our systematic review of the literature on whether urban agriculture improves urban food security, we found three key factors mediating the effect of UA on food security: the economic realities of achieving an economically viable urban farm, the role of city policy and planning, and the importance of civic engagement in the urban food system . A radical transformation toward a more equitable, sustainable and just urban food system will require more responsible governance and investment in UA as a public good,livestock fodder system that is driven by active community engagement and advocacy.

Coastal salt marshes are a vital interface between terrestrial and marine ecosystems, providing erosion protection, secure nurseries, runoff filtration, and critical habitat for threatened species . Despite the value of these ecological services, most salt marshes have been lost or degraded by human activities . In fact, 91% of the wetlands in California have been drained and reclaimed for other uses, and the few that remain exist in altered states . To restore ecosystem services lost to these changes requires re-establishment of healthy salt marsh vegetation . Tidal inundation regimes create salinity and moisture gradients that covary with elevation, driving variations in abiotic conditions that can restrict plants to specific zones within the ecotone . An ecotone is a transition between two ecological systems with a steep environmental gradient, such as salinity and moisture levels . At lower elevation in the ecotone, frequent inundation ensures regular soil saturation and salinity values close to that of seawater, and species intolerant of these conditions are restricted to higher elevations . In the upper ecotone tides are infrequent, and in the absence of rainfall, salts are concentrated in the soil via evaporation. In Atlantic coastal salt marsh, year-round rainfall prevents buildup of salts, creating relatively benign growing conditions in the upper ecotone . In contrast, the dry summers of Mediterranean climates drive high evaporation rates that concentrate salts in the soil, making conditions at higher elevations more stringent during warmer parts of the year . Due to the elevation gradient, tidal regimes, and variable rainfall, salt marsh ecotones develop salinity and moisture zones that can intersect in ways that impact species differently. Change within these zones can be driven by environmental factors as well as human influence, and impose short or long-term effects; for example, heavy rain can temporarily dilute the salinity gradient, while the breaching of a dike can restore a salinity gradient where it has long been absent .Removal of non-native species creates large bare soil patches, where higher evaporation rates concentrate salts in the soil .

Restoration activities can thereby intensify naturally occurring moisture and salinity gradients present, affecting success of salt marsh revegetation efforts. If the relative influence of these stressors varies across species, planting strategies that account for these differences could improve restoration outcomes. This knowledge would facilitate planting species into zones where stress levels are tolerable. For instance, Distichlis spicata can tolerate a wide range in salinity, but little is known about its sensitivity to drought . Intelligent placement of D. spicata might therefore depend on its water requirement rather than salinity limitation. Salinity affects water uptake, transport, and transpiration, requiring plants to have adaptations to survive in saline soils . Salt secretion via salt glands is the most common method of salt removal for non-succulent plants; however, salt can also be removed via salt hairs. Both methods require the plant to take up salt and eliminate it through specialized organelles . Another common strategy is succulence, which dilutes ions to non-lethal levels, allowing plants to survive in high salinity environments Salinity exclusion in the roots is a third method, though it is much less common . Soil moisture also affects plant performance, because water uptake, photosynthesis, and turgor pressure can be reduced under dry or high salinity conditions . Both low soil moisture and high soil salinity can decrease plant water potential, which is measured as the negative pressure required to move water through the plant. The lower the value, the more difficult it is to take up and move water through the plant , possibly affecting growth and survival . For this reason, water potential is often used as an indicator of stress. Here, we applied watering treatments varying in salinity and volume to determine the relative influence of each on halophyte plant performance. We expected to observe a more negative water potential for plants in drought or high salinity treatments compared to plants in saturation or freshwater treatments. In addition, salinity and drought stress should exhibit interactive effects, such that combinations of moderate salinity and drought also reduce performance.

We predicted that plant tissue water potential would reflect stress caused by drought and/or salinity, and that more negative values would correlate with reduced survival and growth. Because the natural distribution of salt marsh species differs within the ecotone, where moisture and salinity covary with elevation, we expect treatment effects to vary across species. To test these hypotheses, we subjected five native perennials to eight different watering treatments in the greenhouse. Plant tissue and soil water potential were measured to assess physiological and abiotic effects of treatments, and growth and survival were tracked to assess treatment effects on plant performance. Because the tissue measuring process was time-intensive, we harvested two species at a time to minimize drying of samples. We harvested F. salina and D. spicata tissue at eleven weeks for water potential testing; these species were processed first due to elevated mortality . At thirteen weeks, J. carnosa and E. californica were harvested. S. macrotheca and soil blanks were harvested at the beginning of the fourteenth week. Although we see no evidence of systematic bias resulting from staggered harvest, we cannot rule out the possibility of an effect. Following tissue harvest for each species, we cut green stems and leaves into 0.5 – 1.0 cm lengths before placing them into 15 mL sample cups . We immediately placed lids on cups and wrapped stacks of four cups with Parafilm “M” to prevent moisture loss. We stored tissue samples in a cool,nft channel dry place for a maximum of three days before processing, and we randomized processing order among treatments to avoid biases related to length of storage time. We emptied soil blanks into 1-quart Ziploc bags and sealed them inside a second bag. Soil was homogenized inside bags before dispensing into sample cups. Soil samples were stored in Ziploc bags for approximately one month before processing, due to technical issues with our instrument. Soil water potential was affected by treatment, leading to significantly more negative water potentials in the drought treatment, and in treatments of increasing salinity . There was also a significant interaction between drought and salinity , with the effects of salinity intensifying in the drought treatment . Patterns for tissue water potential were similar with water potential generally declining as salinity increased across all five species . Although plants in the drought treatment received less than half of the water than the saturation treatment, tissue water potential remained similar across watering volume for most species. Nonetheless, drought had a significant effect on tissue water potential for all five species . The effect of salinity and its interaction with drought differed across species; E. californica showed a significant response to salinity and the interaction between drought and salinity. F. salina and J. carnosa showed a significant response to salinity, but not the interaction between drought and salinity. Finally, D. spicata and S. macrotheca did not respond to either salinity or the interaction between drought and salinity. The range of measured tissue water potential varied greatly among species, with D. spicata reaching as low as -12 MPa. In contrast, J. carnosa and S. macrotheca stayed within -1.5 to -3 MPa, and E. californica and F. salina had intermediate values. Our experiment simulated two stressors – drought and salinity – that are important determinants of plant distribution in California coastal wetlands. The treatments resulted in distinct water potential patterns for both soil and plant tissue across species. Soil water potential in particular showed a striking response to treatment, with measurements ranging from ~ -0.5 MPa to -6 MPa – low enough to expect impacts on plant performance.

The soil water potential at which plants are unable to take up sufficient water to compensate stomatal water loss is known as the permanent wilting point; it is often the soil water potential where the plant irreversibly wilts and dies . -1.5 MPa is commonly accepted as the permanent wilting point for glycophytes ; however, Warrick notes that due to the substantial variation in plant species tolerance, some can survive well past the -1.5 MPa permanent wilting point threshold. At the moment, very little research has been done to identify soil water potential thresholds affecting halophyte performance or permanent wilting points. Treatments here clearly affected plant tissue water potential, with readings ranging from -1.5 MPa to -12 MPa; most species remained above -7.5 MPa. For glycophytes, plant tissue water potential of -1.5 MPa generally reduces cell expansion, cell wall synthesis, and protein synthesis. As water potential continues to drop, photosynthesis and stomatal conductance dramatically decrease, while solute and abscisic acid accumulation increases . Hsiao and Acevedo briefly discuss halophytes, but there is insufficient research to draw firm conclusions on halophyte physiological response to decreasing plant tissue water potential. Although the patterns were similar for plant tissue and soil, the magnitude of change was different, so soil water potential cannot be used as a direct indicator of plant tissue water potential. There was a general pattern that suggested increasing salinity led to decreasing tissue water potential. However, only E. californica, F. salina, and J. carnosa showed a significant change in water potential as salinity increased. The observed change was likely due to increased solute concentration in the tissue to compensate for higher solute concentration in the soil . As soil solute concentrations increase, it becomes more difficult for plants to take up water. In response, plants can concentrate solutes in their tissue, creating a hypertonic state that allows continued passive uptake of water . Lack of response to increasing salinity for some species may have resulted from insufficiently stringent treatments. Our highest salinity treatment was 60% seawater, whereas plants in the low ecotone can experience inundation with full-strength seawater. The average low marsh soil has a salinity concentration of 43.9 ppt , while seawater averages about 34.9 ppt, indicating that some species can survive 125.79% seawater. Because our plants only received 60% seawater, or roughly half the concentration plants can experience in the field, it would be useful to repeat this greenhouse experiment with higher salinity treatments. Drought effects can be similar to salinity effects, causing plants to become hypertonic to increase water uptake . Drought significantly affected all species, causing a decrease in water potential when compared to the saturation treatment. It should be noted that although our drought treatment had a significant effect, it is unlikely to replicate true field conditions.

Issues regarding environmental detection, uncertain concentrations, and unknown toxicity are not unique to ENMs; they are also raised for other emerging contaminants.However, because the ENM industry is rapidly evolving and scientists seek to assist in advancing environmentally safe nanotechnology, environmental relevance in ENM hazard assessment should be prioritized. To accomplish this, representatives from academia, industry, and government regulation working in ecotoxicology, exposure modeling, and social science Table S1 addressed four questions within this critical review: What exposure conditions are used in assessing ENM ecotoxicity potential for model organisms? What exposure and design considerations drive mesocosm experiments for assessments of ENM environmental hazards? What is the state of knowledge regarding ENM environmental exposure conditions, via measurements or modeling simulations? How should concepts such as exposure conditions, ENM transformation, dose, and body burden be used in interpreting biological and computational findings for assessing risks? The main objective was to provide context and guidance to the meaning of environmental relevance in ENM environmental hazard assessment. This critical review addresses the four motivating questions and expands on detailed topics that emerged during the project . For each question, there are findings and recommendations. These serve to crystallize what is meant by environmental relevance in ENM ecotoxicology, and further coalesce ENM environmental exposure and hazard assessment endeavors.Many systems, approaches, and conditions have been used to assess ENM ecotoxicity.The applicability and challenges of standardized testing protocols under aquatic conditions have been reviewed and vetted in workshops.Also, the OECD reviewed and vetted its guidelines for testing ENMs in an expert meeting.Readers are referred to those reports for deliberations of ENM standardized testing. Research studies include laboratory-specific low- and high-throughput dose−response evaluations using select media with ENM compositional and receptor variants for assessing uptake and effect mechanism.Investigations include mechanistic gene transcriptional,DNA damage,dutch bucket for tomatoes metabolomics profiling,and transgenerational experiments.

Bottle-scale microcosms partially simulate limited levels of environmental complexity,for example, in using natural soils or sediments with or without plants and associated rhizosphere influences,or using seawater or marine sediments and associated receptors based on expected ENM compartmentalization.Nonstandard microcosms assess ecological end points, for example, microbial community composition and function related to C and N cycling.Environmental factors are examined, including how ENMs interactively affect soil water availability and soil bacterial communities.Single-species experiments that assess ENM bio-association or bio-accumulation precede and motivate using microcosms for assessing dual species trophic transfer and potential bio-magnification.Pristine ENMs, including those with surface functionalization, capping agents, or adsorbed species or coatings,are the most frequently assessed, although released and transformed versions are increasingly studied.Results for textiles, paints, and nanocomposites suggest that released particles significantly transform and age, and exhibit different environmental behavior and effects compared to pristine ENMs.Assessing changes in form and associated behavior or activity across the material’s life cycle are uniquely challenging. Nominal test exposure concentrations vary widely and are sometimes related to scenarios such as repeated applications,accidental spills,or ranges over a spatial gradient.High exposure concentrations may be used:for assessing bio-availability in soil in comparison to simpler media, or to accommodate analytical instrument detection limits. High concentrations are also used to establish no-effect limits, using limit tests; if an effect is not observed, the ENM is assumed to be nontoxic at lower concentrations,although effects could occur at longer exposure times. However, this approach is problematic if agglomeration and sedimentation of ENMs are concentration dependent, or if effect mechanisms do not scale with concentration, which might occur if organisms adaptively respond to toxicants. Some challenges to ENM ecotoxicology are familiar to conventional chemical ecotoxicology while others are unique. With conventional chemicals, toxicity is related to the effective dose of a toxicant molecule crossing the cell membrane and disrupting essential processes. However, ENMs can exert effects as particulates and in a molecular or ionic form depending on the dissolution extent in the medium.

The effective ENM dose which impacts the assay results may be unknown or changing because it varies with media conditions or with dynamic physicochemical interactions of receptors and toxicants.One effective dose metric may be insufficient to characterize an observed effect that could be related to both a physical interaction of the ENM particulate and the dissolved ionic form. Also, bio-availability and effective ENM doses change because ENMs can transform abiotically and biotically during assessment. While such influences on stability exist in conventional ecotoxicology, stability in a particulate exposure must also consider the ENM size distribution, and potential physical changes to the ENM such as dissolution and agglomeration. Therefore, effective and nominal doses may or may not be related. Although multiple ENMs may co-occur in product formulations, different ENMs are infrequently studied together or with cocontaminants.ENMs can conditionally sorb and modulate the toxicity and uptake of other contaminants and vice versa.ENMs can acquire coatings such as natural organic matter ,and age with varying pH and sunlight.Assessment outcomes are affected by media chemistry, physical characteristics, and additives . The exposure concentration at the receptor and consequent effects depend on ENM dissolution perhaps assisted by biotic ligands,and speciation,shed ions,surface associations, and heteroaggregation with colloids and particulate matter,r agglomeration and settling.All of these vary with ENM types and their varying properties, and ambient conditions.Changes in ENM properties may change bioavailability and toxicity.Various test durations and end points have been studied, from acute responses measured by standard test protocols to short-term microbial biodiversity and community composition effects  or plant genotoxicity and nutrient composition changes.Multiple end points, toxicant characterization methods, and experimental controls increase assessment comprehensiveness and reduce artifacts and misinterpretations.Experimental controls based on coatings and dispersants enable determining if the apparent toxicity is attributable to the ENM itself or to ligand or surface groups. Yet whether and how experimental controls are used varies widely, for example metal salts that allow interpreting biological responses relative to ENM dissolution products versus intact ENMs.The appropriate analytical method for quantifying, locating and characterizing organism-associated ENMs depends on ENM chemistry and amounts or concentrations.

For ENMs exhibiting fluorescence or for ENMs containing heavy elements, high resolution microscopy can assess biological uptake and compartmentalization.X-ray synchrotron methods can sensitively locate bio-accumulated metal oxide ENMs and their transformation products in biota.No single analytical tool is suitable for all ENMs; however, the general lack of methodologies that can be routinely implemented to quantify ENM exposure in complex matrices continues to be a major challenge to the fundamental understanding of ecological effects. Alternative testing strategies can simultaneously assess many material types, controls, and concentrations,which is useful for ENMs not available insufficient quantities for microcosms. However, the potential for interferences in screening assay performance, depending on the specific ENM properties and toxicity test conditions,are increasingly recognized, and therefore should be controlled or accounted for in study designs.To date, ENM ecological hazard assessments have not adequately explored numerous well-conceived and plausible exposure scenarios that are founded in theory, hypothesis,blueberry grow pot mechanism and occurrence probability; yet scenarios increase certainty and predictability when addressing nanotechnology related material safety. For instance, hazard assessments have been mainly skewed toward as-produced ENMs without full consideration of potential aging, since aging cannot be fully standardized in a realistic context. However, such studies can and should further be conducted. Biological uptake, and the compartmentalization and speciation of ENMs, are still infrequently studied, limiting possibilities for attributing ENM exposure to effects at biological receptors.Varying degrees of rigor have been applied in designing and incorporating controls that are relevant to experimental questions or hypotheses. Varying degrees of attention are paid to experimental artifacts. While great advancements have been made in ENM ecotoxicology, improvements are needed to increase the environmental relevance of future research.To understand hazards for plausible exposure initiation scenarios, assessment conditions will need to depart from standardized testing protocols.It may be helpful to link or compare data obtained for ENMs under plausible scenarios to those obtained with standard methods using standardized media to facilitate interpreting complex multivariate experiments or comparing results among multiple laboratories. Herein, several recommendations regard exposure conditions that should be used in assessing ENM ecotoxicity .Soil ecotoxicity studies should specify the soil taxonomy and characteristics such as pH, clay content, and organic matter content, using standard methods.Similarly, characterization of sediments should be provided. Natural soils and sediments are preferred, because artificial media do not harbor natural soil communities, and do not reflect chemical and physical characteristics that influence ENM effects and bio-availability. Water characteristics determine ENM agglomeration, dissolution, and other behaviors affecting aquatic system compartmentalization and hence need definition. When adequately documented, media characteristics can be used retrospectively to interpret conditional ENM bio-availability or effect mechanisms.ENMs in testing should represent the particulate material form relevant to the environmental scenario . ENMs should be fully characterized and their history should be adequately described to allow comparing between studies. For toxic coatings,coating identity and degree of coverage should be related to observed effects. Impurities in ENMs introduced during product synthesis and handling should be characterized, since they can sometimes account for apparent ENM ecotoxicity.Nominal concentrations should scale according to exposure scenarios, or to specific objectives such as mechanistic research or quantifying biotic uptake.

Dose verification, including size distribution and ENM concentration, is also desirable, although heretofore challenging in soil and sediment exposures. ENM physicochemical changes during release and in the environment should be studied to uncover properties of ENMs that reach biological receptors. All potential forms of ENMs, including transformation products and residual reagents used in synthesis, should be accounted for, such that all toxicants can be related to biological responses.Study designs should anticipate dispersing agent effects and the nature of transformed ENMs plus cocontaminants, by including controls to account for effects, fate, and kinetics of ENMs in the test medium.The ENM physicochemical states should be understood before conducting hazard assessments.This is important because some ENMs agglomerate or dissolve or otherwise change in laboratory media, resulting in nonuniform exposures and uncertain bio-availability. Such unevenness precludes relating measured effects to the applied dose. Spatial bio-association, bio-accumulation,and intraorganism compartmentalization should be assessed to locate ENMs and their components.Important advances have been made toward characterizing the physicochemical factors influencing ENM behavior in environmental and test media, and toward utilizing that information to develop standardized methods for conducting ENM ecotoxicity testing.However, aquatic or terrestrial species and even different species belonging to the same order respond differently to ENMs using the same tests. Thus, test species and end points should be carefully chosen to enhance the relevance of ENM ecotoxicity testing. Some complex interrelationships and dependencies between species comprising ecosystems have been described.However, focused research could rationally identify species for routine evaluations; likewise, the scientific rationale behind test species should be reported. Ecosystems are more complex than conditions of routine ENM ecotoxicity evaluations. Thus, research should define an optimal suite of test species and end points to determine the ecosystem response to a given ENM. In general, biological receptors should be chosen for expected exposures stemming from realistic exposure scenarios. For example, relatively insoluble ENMs may, depending on their density, size and agglomeration state, rapidly settle out of suspension and associate with aquatic sediments. In that case, initial hazard assessment could focus on benthic, rather than pelagic, receptor organisms.Conversely, for ENMs that rapidly dissolve under environmental exposure conditions, conventional ecotoxicological exposure scenarios may be applied and receptors chosen to assess dissolution product toxicity. However, ENM dissolution rates vary, and pelagic organisms can be more sensitive than benthic organisms.Thus, both ENM compartmentalization and form must be accounted for when choosing receptors.Multiple effects measurements should be applied to answer research questions.Mechanistically understanding overt toxicity is needed, which may require measuring more omics end points and choosing variables for developing mathematical models to predict toxicity at untested concentrations or conditions.Omics technologies can also identify potential modes of action that are conserved among different species. However, different scientific communities will have varying preferences in defining needs for omics-level investigations.Effects interpretation requires understanding the effective toxicant dose or other basis of impacts.For ENMs, the mass concentration basis of dosing may relate only partially to the effective applied dose, since biological effects often originate from surface interactions with receptors.Furthermore, ENMs are more complex than conventional chemicals because ENM shape, aggregation state and surface area may in- fluence toxicity.Thus, surface area applied has been suggested as a supplemental dosing metric.However, ENM surface area in suspension/solid media is not a straightforward assessment given that ENMs may aggregate with a size distribution that is affected by the medium in which they are dispersed.

These studies demonstrated that this approach can be successfully used in environments under intensive irrigation and fertigation management. Additionally, Ramos et al. reported that similar salinity distributions were obtained when this simple approach of EC modelling using HYDRUS was compared with much more complex predictions involving consideration of precipitation/dissolution and ion exchange as done with UNSATCHEM, particularly when the soil solution is under-saturated with calcite and gypsum. Nitrogen transport was simulated by means of a sequential first-order decay chain, implemented in HYDRUS-2D. Hence, N reaction or transformation processes, other than nitrification, were not considered. Similar assumptions have also been made in previous studies involving modelling of the nitrate transport is soil . We also assumed that inherent soil organic N was mineralised directly into NO3 –N, consistent with other studies . Nitrate was assumed to be present only in the dissolved phase . Ammonium was assumed to adsorb to the solid phase with a Kd value of 3.5 cm3 g 1 . The nitrification of NH4 + –N to NO3 –N thus acts as a sink for NH4 + –N and as a source for NO3 –N. First-order rate constants for solutes in the liquid and solid phases were set to be 0.2 d 1 . These were taken from a review of published data presented by Hanson et al. , and represent the centre of the range of reported values. The longitudinal dispersivity was considered to be 20 cm and the transverse dispersivity was taken as one-tenth of eL. These values have been optimised in similar studies involving solute transport in field soils .In this approach, the drip tubing can be considered as a line source ,vertical hydroponic nft system because in a twin line drip irrigation system with closely spaced drippers the wetted pattern from adjacent drippers merges to form a continuous wetted strip along the drip lines . Water movement was therefore treated as a two-dimensional process .

Our field observations of the wetting pattern on the soil surface during experiments also supported this approach. The transport domain was set as a rectangle with a width of 250 cm and a depth of 150 cm. The transport domain was discretised into 2172 fifinite element nodes, which corresponded to 4191 triangular elements . Observation nodes corresponded to the locations where EnviroSCAN probes and SoluSAMPLERs were installed, at a distance of 10 cm from the emitter source .The nitrogen balance for the mandarin crop was evaluated for two fertigation strategies. First, the fertigation pulse was applied at the beginning of each irrigation event . Second, the fertigation pulse was applied near the end of each irrigation event . It is a common practice that irrigation water is initially and at the end free of fertilizer, to ensure a uniform fertiliser application and flushing of the drip lines . Therefore, fertigation applications were simulated to either start one hour after irrigation started or to end one hour before irrigation stopped. Nitrate management strategies also include a judicious manipulation of irrigation and N fertilizer applications, and increasing or decreasing the frequency of applications. These interventions should improve N uptake by plants and reduce N leaching out of the plant root zone . The evaluated scenarios are described in Table 2. Scenario, S1, illustrates the impact of applying the same volume of water in small irrigation events . Scenarios S2 and S3 then represents the reduction of the irrigation volume application by 10% and 20%, respectively. Scenarios S4 and S5 are based on decreasing the nitrogen application by 10–20%, respectively, while scenarios S6 and S7 represent a combined reduction in irrigation and fertigation by 10–20%, respectively. Five scenarios were executed, in which irrigation was reduced during the second half of the crop season, i.e., between January and August, by 10%, 20%, 30%, 40%, and 50%, respectively.The water contents measured weekly by EnviroSCAN at different depths at a horizontal distance of 10 cm from the dripper, and corresponding values simulated by HYDRUS-2D during the entire growing season are illustrated in Fig. 5. The measured water contents remained similar at 10 and 80 cm cm, fluctuated between 0.1 and 0.2 cm3 cm 3 at 25 and 50 cm, and stayed higher than 0.2 cm3 cm 3 at 110 cm soil depths throughout the growing season, indicating a favourable moisture regime in the crop root zone.

However, the simulated water contents were lower than the measured values during the initial period at a depth of 10 cm and during the mid period at a depth of 110 cm. The simulated values matched the measured values more closely at soil depths of 25 and 50 cm, which is the most active root zone for water and nutrient uptake for citrus . However, the profile average water distribution matched well. The MAE between weekly measured and simulated moisture content values across all locations varied from 0.01 to 0.04 cm3 cm 3 , indicating a good agreement between the two sets of values . Slightly higher temporal MAE values during the mid-season agreed well with the variation shown in Fig. 5. Similarly, the MAE values at 10, 25, 50, 80, and 110 cm soil depths at a 10 cm lateral distance from the dripper also revealed that the variation between measured and simulated water contents remained between 0.02 and 0.04 cm3 cm 3 . However, the differences were slightly higher at 10 cm depth as compared to greater depths . Higher variations at the surface depth are to be expected because this part of the soil profile is influenced by soil evaporation, which peaks in day time and is low at night time, while the assumption of a constant atmospheric boundary flux for daily time steps in the model deviated from the actual transient conditions existing at the surface boundary. Other studies also showed a similar magnitude of variations between measured and predicted water contents.Comparison of simulated electrical conductivities of soil solution with weekly measured values at different depths are shown in Fig. 6. Despite of low irrigation water salinity and low initial soil salinity , the measured ECsw increased in the soil with the onset of irrigation at all depths, except at 150 cm where the increase in salinity occurred only after December 2006. Subsequently, a decreasing trend was observed in ECsw later in the season. The higher amount of irrigation compared to ETC. and an significant amount of precipitation during this period resulted in a reduction in soil solution salinity. On the other hand, the model over-predicted ECsw at a depth of 25 cm from October to December 2006 and under-predicted it at a depth of 100 cm during the same period. However, at a depth of 150 cm, simulated values remained constant till January 2007, indicating a delayed response.

The increase in simulated ECsw values was delayed at 100 and 150 cm depths as compared to measured values. Both set of values matched well at a depth of 50 cm and the profile average of ECsw also showed a close match. It is significant to note that irrigation with good quality water in our study led to the development of significant levels of measured ECsw . However, the ECsw values remained below the threshold of salinity tolerance of orange throughout the season . The MAEs between weekly measured and simulated ECsw in the soil ranged from 0.08 to 0.76 dS m 1 , which are acceptable for a complex and highly dynamic soil system,nft hydroponic system with the exception of a few divergent values obtained between mid October and December . The disagreement in ECsw values during this period was correlated with corresponding fluctuations and low values of water contents, especially at soil depths of 10 and 25 cm and this variability was transferred to the ECsw values. Differences between measured and simulated ECsw values at 50 cm depth were relatively higher than at other depths . The mean MAE at 25, 100, and 150 cm depths ranged from 0.19 to 0.36 dS m 1 , showing a good agreement with the measured values at these depths. The spatial distribution of ECsw in the soil profile at various dates is depicted in Fig. 7. It can be seen that salts remained restricted to roughly the upper 50 cm of the soil profile until December . The salts mass was later pushed deeper due to high rainfall . The downward movement of salts continued in February and March , because in March the amount of irrigation was higher than ETC. . It is pertinent to note here that the ECsw distribution under the dripper remained lower as compared to the adjoining soil at all times, because a continuous water application in this region pushes the salts towards the outer boundary of the wetting front. The drainage flux during and after March transported salts vertically downwards, thereby making the soil directly beneath the dripper relatively salt free by the end of the season. Applying additional water at the end of the season could be a strategy to create a salt free root zone which may encourage vigorous root development, and assist the plant growth in the ensuing season.Comparison of weekly measured and daily simulated nitrate– nitrogen concentrations at different depths in the soil profile is illustrated in Fig. 8. Over-prediction was observed at a depth of 25 cm from October to November 2006, which coincided with similar over-prediction for salinity. Similarly, both measured and simulated values matched well at a depth of 50 cm, while a delayed response in predicted nitrate contents was observed at lower depths. However, a fairly good correspondence was observed between profile averaged NO3 –N contents. The temporal MAE values for NO3 –N ranged from 0.1 to 1.97 mmol L 1 . Similar differences between measured and HYDRUS-2D simulated values were also reported in another study involving simulations of nitrogen under field cropped conditions. Additionally, MAE at a 25 cm depth had a higher value L 1 ) than at greater depths L 1 ). A similar match of nitrate distributions has been reported in other studies as well . The reason for differences in ECsw and NO3 –N values may be partially due to the fact that model reports point values, whereas the Solu SAMPLER draws in solution from a sampling area of a certain volume, the size of which depends on the soil hydraulic properties, the soil water content, and the applied suction within the ceramic cup .

Hence the measured parameters considered in modelling may not represent the inherent spatial variability of the soil. In addition, while a homogeneous soil environment is assumed by the model, the field site could be far more heterogeneous and anisotropic. Also, the model simulations considered only a 2D movement of nitrogen and the nitrification process, while more complex nitrate processes were not taken into account. Ramos et al. documented numerous factors influencing the correspondence between measurements and simulations of water contents and solute concentrations in the soil under drip irrigation conditions and these factors are relevant also for the present investigation. These factors, including those mentioned above, may modify the error in the simulated NO3 –N values. The simulated movement of nitrate–nitrogen in the soil under a mandarin tree at various dates is shown in Fig. 9. Nitrate fertigation increased the nitrogen content in the soil with time, as is evident from an increasing size of the concentration plume below the dripper as the season progressed. This indicates that the plant was not able to take up all nitrogen added through fertigation, and thus nitrogen built up in the soil over time, leading to a maximum concentration values in January . Ultimately, nitrogen started moving downwards after late January, when there was high rainfall and total water additions exceeded ETC. Alva et al. also detected greater variations in NO3 –N concentrations in the 0–15 cm depth horizon, as compared to greater depths in a field experiment involving citrus. The seasonal NO3 –N concentrations in the domain varied from 0.01–7.03 mmol L 1 . Hutton et al. reported higher mobilization of nitrate at a shallower depth under drip irrigation of grapevine, and seasonal root zone nitrate concentrations ranging between 0 and 11.07 mmol L 1 in the Murrumbidgee Irrigation Areas in Australia.

To test whether this difference is due to an artifact of our extraction procedure, we have tried to denature both extracts with a number of techniques; however, leaf polypeptides never broke down to such small components. This result, although preliminary, is the first evidence of a difference in DNA associated proteins from two plant organs and as such, is worth further investigation. Our technical approach is to isolate proteins which bind specifically with the T-DNA regulatory sequences of the root-specific and the leaf-specific T-DNA genes. First, proteins are isolated on the basis of their general DNA-binding capacity. To this effect, protein extracts from each plant organ are run on DNA-cellulose affinity columns to fractionate proteins which show general DNA-binding capacities. The test we use for visualizing DNA-binding proteins is a modified version of the technique published by Bowen et al., 1980: proteins are transferred to nitrocellulose filters and probed with nick-translated labelled DNA. The DNA sequences we are using for these experiments are sub-clones containing the leaf-specific and the root- specific promoters of the pRiA4 T -DNA. The goal is to show that there is indeed a specificity in the recognition of each of these sequences by the two types of extracts and to identify what proteins are’ involved. The difficulty that will be encountered in this part of the project is in defining conditions that will enhance the sequence specificity of the DNA-binding proteins. This has been a major difficulty in the identification of sequence-specific recognition factors. Retarded electrophoretic migration of DNA complexed to proteins has proven to be a useful method in detecting specific over non-specific binding of proteins to isolated sequences: it has recently been used to identify specific transcription factors in SV 40 infected cells, and in O2 induced cytochrome expression in yeast. We are now testing several approaches in order to determine the general and sequence specific DNA-binding properties of the Tobacco root and leaf proteins.

These include investigating factors influencing the formation of DNA-protein complexes in solution,ebb flow tray such as salt conditions, length of reaction and subsequent washes, and competition experiments with random sequence DNA such as calf-thymus DNA. If necessary, the DNA-cellulose column fractionation will be more elaborate to yield protein species in significantly purified form so as to reduce the background of non-specific binding. DNA-protein complex formation will also. be tested using smaller probes i.e purified promoter sequences from the plasmid that bear the leaf and root pRi T-DNA regulated genes. We hope thus to discriminate between sequence specificity and general binding affinities of the proteins now being isolated. This approach will focus on the other aspect of tissue-specificity i.e a study of the DNA sequences that interact with cellular factors to control the activity of specialized genes. It is also the most straightforward part of the project proposed. We would like to define what the DNA sequences are that interact with the proteins that are being isolated-in 1-. This we will determine by DNA foot-printing experiments: DNA-protein complexes will be formed -using purified protein fractions from the root or leaf extracts and purified restriction fragments containing sequences of the pRiA4 T-DNA for the root and leaf-specific promoters. It will thus be possible to identify the precise positions of the binding-sites of the tissue-specific proteins in the 5’upstream regions of the two genes under consideration. . Foot printing experiments are difficult if the proteins required to complex with the DNA are in low abundance. Hence, this experiment depends on previous work to attain a semi-purification of the proteins required. The initial aim of this work will be to introduce the plasmids we have from the Ri TDNA which contain genes expressed preferentially in different tissues, into protoplasts from these tissues. We will study the expression of the genes on these plasmids at the RNA and protein level.· The DNA will be introduced into the protoplasts either by transfection or by electroporation, both techniques are presently being used in our lab. The assays in this case will be short term expression assays in which the RNA from the protoplasts which carry the plasmids ,. will be isolated within 48 hr of introduction of the DNA.

The RNA encoded by the gene driven from the plasmid promoter will be quantified. To study expression at the level of protein the promoters will be isolated and linked to in vitro to the gene coding for the chloramphenicol acetyltransferase enzyme . CAT activity will be measured after introducing the DNA into protoplasts. A large part of this work will be directed at maintaining the tissue specific expression of the rotoplast by adjusting the growth conditions during the course of the experiment. We will modulate the culture media by adjusting the ratio of auxin to cytokinin and determine the effect on tissue specific expression. Also, we will investigate the involvement of oligosaccharins in determining the tissue specific expression of these plasmids. After the transient expression experiments are underway, plants will be regenerated containing these modified Ri T-DNAs. Protoplasts will be isolated from root and leaf tissues of these plants, and nuclei will immediately be tested for transcriptional activity, using the technique of Ackerman et al., CAT activity will be measured in parallel, as it is easily detectable in small amounts of material. By addition to root-nuclei of extracts from leaf tissues and harvesting of the subequent transcription complexes, it is hoped that it will be possible to activate the silent root-specific promoter . This assay will be used to test the factors isolated in the DNA binding work described above for tissue-specific activators. The overall goal of this program is to develop an understanding of the processes involved in hydrocarbon production in plants. Specifically, we are interested in the mechanisms and control of isoprenoid biosynthesis. Acquiring this basic information will be necessary before we can genetically manipulate plants to increase hydrocarbon yields. We use the latex isolated from laticifer cells of the Euphorbia lathyris plant for our biosynthetic studies. These cells are the site of both the biosynthesis and the storage of large quantities of sterols . We are currently examining the final steps of sterol synthesis, the epoxidation and cyclization of squalene, to determine if more than one squalene cyclase is ,.- involved in sterol synthesis. We are also attempting to identify the organelle involved in the conversion of mevalonic acid to sterols. A secondary interest is the’ synthesis of sesquiterpenes which are a better candidate for direct use as fuel than are the triterpenoids.

The manipulation of a plant towards increased sesquiterpene production at the expense of sterol product is thus a long-term goal of our research. This requires an understanding of the processes involved, specifically the initial cyclization of farnesyl pyrophosphate ,flood and drain tray the branch point at which sesquiterpene synthesis diverts from the general isoprenoid biosynthetic pathway. It may be possible to increase the hydrocarbon content of plants by changing the environment around these plants. We have found that by increasing the day length from about 12 hours to 16. hours and by maintaining a constant day/night temperature region, we observed about a 9-fold increase in the activity of 3-hydrox-3-methyglutaryl-Coenzyme A Reductase . Since this enzyme plays a key role in the synthesis of isoprenoids, it is possible that hydrocarbon production is sensitive to environmental controls. Controlled changes in environmental conditions could restrict growth while having only small effects on the photosynthetic rate . If applied when the plant approached maturity, this could result in an increase in assimilates available for partitioning into isoprenoid biosynthesis. We also hope to identify the internal controls of carbon allocation, since they also could be manipulated to increase plant hydrocarbon production. To investigate these posiblities we are observing the effects of three different environmental variables on hydrocarbon production: salinity stress, water stress .and nitrogen deficiency. After establishing the growth conditions necessary to grow E. lathyris hydroponically, we completed the preliminary study of salinity stress. Extractions of the hydrocarbons and sugars will be performed on the water-stressed plants. However, experiments utilizing nutrient stress have not been successful to this point as low levels of nitrogen cause rapid senescence and death. The effects of salinity on growth were determined from changes in shoot length, total fresh weight and root and shoot dry weights. Changes in the photosynthetic apparatus were determined from chlorophyll content, thylakoid proton gradient formation~ and in vivo fluorescnece patterns. Changes in the levels of energy-rich compounds were determined by heptane and methanol extractions of the dried plant. Only the shoot portion of the plant was used for this extraction as this is what would be available for harvest. The preliminary results indicate that salinities of 50 mM NaCI and greater affect both growth and photosynthesis, though the reduction in growth is sharper than the reduction in photosynthesis. Results of extraction experiments with plant components also indicate that increased salinity causes an increase in carbon allocation to the heptane and methanol fractions, with most of the increase occurring in the carbohydrate-rich methanol fraction. Salinization also caused a 3-fold increase in HMGR activity. To obtain a more comprehensive understanding of carbon flow through E. [athyris, we performed some preliminary carbon-labeling experiments. Now that we have established that it is possible to use isotope labeling to monitor latex production and follow carbon translocation in E. [athyris, this technique will be used further in stress experiments. trlterpenols, and their fatty acid esters.

To understand the bio-synthetic processes involved in the production of sterols. it is necessary to elucidate the structures of these compounds. We have previously identified four of the six major triterpenols as cyloartenol, 24-methylenecyc1oartenol, lanosterol and 24-methylenelanosterol. With the results obtained from 3H-NMR, chemical shift reagents, 13C-NMR and optical rotation we have identified the fifth compound as eupha-7,24- dienol. Wehav~ made a preliminary identification of the sixth compound as euphol on the basis of its behavior in gas chromatography. In most plant systems the initial product of squalene cyclization is cycloartenol and not lanosterol as found in animals and fungi.’ However, since lanosterol is a major component of E. [athyris latex, we are attempting to determine if lanosterol is a product of cyc1oartenol, produced via separate, parallel pathway, or is a precursor of cyc1oartenol. To determine if one of the first two mechanisms is responsible for squalene cyclization, we have synthesized deuterium-labeled MVA to be used as a susbtrate to follow its latex-catalyzed conversion to lanosterol. . One of the major difficulties in this analysis is the high level of endogenous sterols .present in the latex. These make the small amount of newly synthesized deuterated compounds difficult to detect by gas chromatography-mass spectrometry. Attempts at delipidation by extraction with organic solvents such as butanol and diisopI:opyl ether greatly reduced the level of triterpenols but also. destroyed all bioysnthetic activity. Purification by rate sedimentation and isopycnic centrifugation has reduced the lipid level to some extent, but we are still trying to find the proper analytical conditions that will allow us to employ the GC-MS to observe the deuterium label. By choosing special sequences for synthetic DNA oligomers we have been able to form stable “sticky end” dimers in solution from DNA hairpin with dangling ends. The sequences are chosen with a central section which is not complementary to any other in the molecule, which is flanked by a self-complementary sequence. Such sequences have been shown previously to lead to hairpin formation. We have modified these sequences to include a self complementary dangling tail, which allows two such molecules to associate in solution. We have observed the imino proton spectra from these molecules to confirm that such a dimer is indeed the stable form in solution. The imino protons have been assigned using nuclear Overhauser effect difference spectra, and confirm that the dimer is essentially a single continuous double helix in solution. On each of the backbone strands, however, there is a break and a missing phosphate. We have now collected complete two dimensional NOE data sets of two different sequences. In these spectra, when collected at sufficiently short mixing times, the cross peak intensities reflect the inverse sixth power of distances between protons.

The desire to increase detector count by up to two orders of magnitude requires a fresh approach. We identified components in current detector systems that are currently not scalable, and proposed to improve their scalability. The five areas that we have chosen to focus on are: Monolithic fabrication of inductors and capacitors for frequency-multiplexed readout, monolithic fabrication of a silicon lenslet array for optical coupling of the detectors, spray application of anti-reflection coatings for silicon lenslet arrays, fully automated wire bonding to the a detector array, and automated electrical inspection of detector array. Current CMB experiments using frequency-domain multiplexing use printed circuit boards with thousands of hand-soldered capacitors in the cryogenic readout electronics. We have developed a monolithic technology at LBNL Micro Systems Lab to fabricate superconducting inductors and capacitors on silicon wafers. We achieved resonances with high quality factor, predictable resonance peak location, and high yield. Using lithographic fabrication greatly reduces cost and gives a scalable technology with an order of magnitude increase in multiplexing factor. We have successfully machined monolithic silicon lenslet arrays from single crystal silicon., which can replace hand-assembed arrays. We have also developed a plasma sprayed ceramic anti-reflection coatings that can replace our current hand-molded coatings. We have also succeeded in wire bonding to our detector wafers with an automatic wire bonder at LBNL. We are bonding at a 100 micron pitch wire bond, which gives higher connection density and lower cost than the manual wire bonding in current use. Finally, we have developed automated electrical inspection that can characterize thousands of detectors per wafer automatically and rapidly. Previously each detector was hand tested for electrical continuity. New telescope techniques that can explore objects in deep space with high resolution and sensitivity in near-IR region are highly demanded for the study of properties of dark energy and dark matter over time. However,rolling benches near-IR observations from the Earth’s surface are extremely challenging because of the bright background emitted from the atmosphere.

Fortunately, these 300+ OH emission lines are intrinsically very narrow, covering only a small fraction of the total spectrum, and the continuum between the lines is typically as dark as zodiacal light, the background level directly observed from space. Therefore, selectively filtering the OH emission light will enable high sensitive deep space near-IR measurements from the ground. We are developing an innovative optical filter system for highly sensitive near-IR observation by effectively removing the OH emission lines from the Earth’s atmosphere. Different from natural materials, the physical properties of a metamaterial is not primarily dependent on its chemical constituents, but rather upon the structures of the building blocks which are much smaller than light wavelengths. The IR filter design is investigated through numerical simulations and we are exploring effective thin film deposition strategies for large number of layers with controlled roughness and stress. The metamaterial near-IR filter system will be fully compatible with existing telescopes and other instruments because of the large acceptance angle, moderate diameter, and thin filter thickness. By tailoring effective optical properties of the nanoscale layered structures, our metamaterial filter system can provide sharp spectral filtering line-width and high transmission of residual signals, which will be otherwise impossible for traditional techniques based on natural materials. Through the design of the nanoscale structures of the filter system and the development of related nanofabrication techniques, we have developed a multilayer algorithm to realize the desired performance. We have also investigated the fabrication process. Our most significant accomplishment is the design of an integral multilayer-filter system. The system can eliminate a large number of OH emission lines in the spectrum from 1.5~1.8 µm where most of skyline noise comes from. Our approach combines the Needle Optimization and the Tunneling method to search for the global minimum of the merit function. We adopt the scheme of distributed IR filters, and different sub-units are assembled to form an integral filter system with a figure of merit around three. Our method can be generally applicable to a broader bandwidth. We have also explored proper fabrication process of growing multilayer thin films with controlled thicknesses and surface roughness. Although we have achieved more than 30 layers of Silica and TiO2 thin films with the thickness of each layer monitored in situ, we realized that it is very challenging to fabricate the full structure with current state-of-art thin film technologies, which requires the deposition of over 1000 layers with total thickness in the order of millimeters and the roughness of each layer less than 1% of its thickness.

We believe this difficulty is resulted from the mathematical limitations of Needle Optimization approach we employed, and a much better mathematical tool must be developed. We are exploring a better design in collaboration with Prof. James A. Sethian, a mathematician to optimize the astrofilter structure with less number of layers and less sensitive to the fabrication errors of surface morphology. Compost is one of the main organic fertilizers. For optimum growth and balanced nutrition, plants need to be fertilized, because Turkish soils are very low in plant nutrients, especially in phosphorus and zinc. With rising environmental concerns about heavy fertilization polluting the soil and water, it is very important to produce mycorrhizal inoculation in order to reduce the amount of chemical fertilizers. Also very recently there has been a great demand for organic production. Since plants are strongly mycorrhiza-dependent, it is meaningful to produce mycorrhiza-inoculated seedlings. Mycorrhizal fungi are the most common symbiotic organisms, occurring on nearly 90% of plant species. Most horticultural plants are colonized by arbuscular mycorrhizal fungi , whose presence can enhance the growth of the host plant . Mycorrhizal fungi can expand effectiveness of root surface area for better nutrient and water uptake. Also it has been reported by Douds et al. that mycorrhizal fungi seem to be stimulated in cropping systems which incorporate crop rotations, green manures, reduced tillage and minimize pesticides and chemical fertilizers while utilizing organic amendments. Since the last century human population has risen. Accordingly, there is a huge demand for food. In order to have sufficient food, more chemical input has been used in agriculture in the last 40 years. Consequently, soil quality and plant health decreased. Soil biological fertility and ecological balance have been damaged. Once soil fertility is damaged, it is rather difficult to recover soil sustainability. Therefore, there is a great demand for rehabilitation of soil by adding organic amendments. It is possible to protect the soil environment by using organic sources, such as compost and mycorrhizal applications. Perner et al. reported that the addition of compost in combination with mycorrhizal inoculation can improve nutrient status and flower development of plants grown on peat-based substrates. Labidi et al. studied the influence of added compost and arbuscular mycorrhiza on production of extra-radical mycelia in Acacia cyanophylla, finding that compost addition enhanced the production of AM mycelia in all treatments in Saharan ecosystems. Since there is compost material in natural ecosystem,ebb and flow bench and in the same environment mycorrhiza exists, it was hypothesized that different compost materials have different effects on mycorrhizal inoculation. The role of mycorrhiza in ecological systems still needs to be studied. In forest ecosystems, there is a large amount of compost, and plant roots get benefit from mycorrhiza.

It is important to do research on the role of several composting materials on horticultural plant seedling quality and growth. The experiment was carried out in a greenhouse at Çukurova University, Faculty of Agriculture, Department of Soil Science, Adana, Turkey. 14 different compost materials were produced. Tomato and pepper plants were inoculated with G. caledonium. A level of 1000- spores per pot was placed 3 cm below the seeds. Non-mycorrhizal plants also received the same amount of mycorrhizal spore free medium. The experiment was arranged in a randomized complete block design in three replications. Plants were fertilized with a mixture of 70% compost, 18% ground basaltic tuff, 10% andesitic tuff and 2% rock phosphate. Seedlings were then produced in two growth media, consisting of organic fertilizer, soil and sand and andesitic tuff, soil and organic fertilizer . Plants were then inoculated with G. caledonium, while control plants received the same amount of growth medium free from mycorrhizal spores. Roots were stained and analyzed for mycorrhizal fungal root colonization based on the gridline intersection method. At harvest time, plant length, stem diameter, shoot and root dry matter, nutrient content and root colonization were determined. The results showed that the mycorrhizal inoculation significantly increased tomato and pepper plant length and that plant diameter depended on compost materials. The efficiency of mycorrhiza, growth media and compost applications were different. The results showed that the mycorrhizal inoculation significantly increased pepper plant length and diameter . In both growth media, animal manure , chicken manure, plant material, straw, and different plant material were determined as the best compost material for tomato growth . 6:3:1 mixed growth media was better than 1:1:1 . In the two growth media, domestic waste, animal fertilizers , plant material and sewage sludge were determined to be the best compost material for pepper plant growth . 6:3:1 mixed growth media was better than 1:1:1 . Pepper and tomato plants grown in 1:1:1 growth medium showed a high response to mycorrhizal inoculum. Mycorrhizal colonization was investigated and it was found that mycorrhizal colonization levels differed by compost materials. As shown previously, mycorrhizal fungi can improve the performance of seedling quality by stimulating nutrient uptake . In the present experiment, effects of different compost materials on plant parameters were more pronounced than the effects of mycorrhizal inoculation. This may be directly related to the nutrient content of compost materials. Soil biological properties, such as useful microorganism, are important for soil quality. Also the interactions between micro organisms, such as mycorrhiza, soil-borne fungi and nematodes are important for sustainable agriculture.In exchange for plant carbohydrates they increase the uptake of immobile nutrients, such as P, Zn, and Cu, and also NH4 + -N, K and Mn . Horticultural cultivation is becoming widespread in the Mediterranean coasts of Turkey. Soils in this region have high levels of clay and lime, which cause P, Zn, Fe and Mn deficiency; consequently, the major problem in the region is nutrient deficiency in several plant species . Cakmak et al. reported that zinc deficiency is a critical nutritional problem for plants and humans in Turkey. Since the Zn is an essential element for several enzymes in plants, Zn deficiency reduces the plant growth dramatically. Zn deficiency can be alleviated by fertilization. However the recent rise in the use of fertilizers has affected both human health and ecosystems. In the last few decades, it has been observed that a reduction of fertilizer input resulted in the improvement of soil quality. We tested whether mycorrhizal inoculation of seedlings could completely or partially substitute fertilizer application. In general, horticultural plants are mycorrhizal dependent. Our earlier results showed that mycorrhizal seedlings are more resistant to environmental stress factors, such as water deficiency and hot temperature. Under field conditions, the effect of mycorrhizal inoculation on the mortality of seedling was tested and it was found that inoculated seedlings had a greater survival rate than non inoculated plants . It is essential to screen efficient AM fungi in order to get the maximum benefit from mycorrhiza for a particular host. Lee and George have shown that G. mosseae inoculated cucumber plants had increased P, Zn, and Cu concentrations, and mycorrhizal hyphae transported those nutrients to the plants. Since most horticultural crops are grown under controlled nursery conditions before being transplanted to the greenhouse or open field, it is possible to inoculate the seedlings in the nursery. The effect of inoculation with several mycorrhizal species on seedling survival and plant growth nutrient uptake and root infection of cucumbers, melons, watermelons and marrows were studied. Several field experiments were carried out on an Arik clay-loam soil, which was classified as an Entic Chromoxerert in the Agricultural Experimental Station of Çukurova University, Adana, in southern Turkey, whose prevailing climate is Mediterranean. Soil is calcareous and pH is 7.7 and organic matter content is 1.46 %. Honey melon , watermelon , cucumber , and marrow seeds were sown in a sand: soil: organic matter growth medium.

The double-curvature geometry of the system was optimized using a Genetic Algorithm, with experiments being performed in three stages: Stage 1 – Radiance only ; Stage 2 – Radiance + BSDF material ; Stage 3 – BSDF + Radiance 3-Phased Method. The second round of experiments focused on Stage 2 and Stage 3 only. Main conclusions drawn were: under direct beam radiation, the difference between the two methods is significant, with the 3-Phase Method performing up to 34% better in simulating the direct solar component. Without a strong direct solar component, the two methods have a closer performance, simulating diffuse light within a closer range, but Radiance+BSDF still has a significant error rate of up to 10%, thus proving unacceptable for this end. Rapid Prototyping was done initially done using 3D printing, with white plastic coated with 97% reflectance film, and its light transmission measured with the Integrating Sphere at LBNL. A final, industry-grade prototype was designed and budgeted, with an extra thin aluminum profile, 0.020 inches thick, built with 6063 Alloy, T6 temper, and coated with 3M D50A Specular Film Protected, with 98.5% visible light reflectance. A prototype sized for Flexlab, including casting dies, was budgeted at only $1200 by SAPA, but construction was not authorized. Final validation of simulation methods developed against physical measurements was thus not possible. Conclusions regarding product development: for optically complex systems, typical Rapid Prototyping methods do not display the necessary optical accuracy; however, measurements with the goniophotometer require uniform samples, and no larger than one square inch. Thus,hydroponic bucket for optically complex systems with non-uniform geometry, the only current option is building a full-scale prototype for physical testing.

If product development is to be made a priority, new alternatives should be developed to bridge between computer simulations and full scale, expensive and difficult to build prototypes. Whole Building Analysis with 3D Pareto Multi-criteria optimization: building variables under study were: floor stacking patterns, glazing and shading systems characteristics, and shading panes offset, for each orientation, and skylight dimensions. The multi-Objective Metrics and Goals applied were: illuminance levels , Cooling Energy Use Intensity and Heating Energy Use Intensity . Results suggested that Whole Building Multi-criteria Analysis is a fast and effective way of testing complex building alternatives at early design stages, providing useful, operative information for decision making in very short amounts of time, in comparison to standard parametric simulation. The large penetration of renewable energy sources requires flexible resources to manage the variability in generation and demand. Increased observability, improved modeling, and more detailed simulation algorithms are necessary to quantify the impact of intermittent generation to the power network, and – at the same time – efficiently plan the operation of the flexible resources. In order to study the interactions of such complex systems, co-simulation platforms are deemed necessary. The Virtual Grid Integration Laboratory is a modular co-simulation platform designed to study interactions between demand response strategies, building comfort, communication networks, and power system operation. It combines three different simulation tools and incorporates functions for the optimal management of both the grid and the flexible resources. First, research was conducted to determine the appropriate simulation tools for VirGIL and the interfaces to couple them. The Functional Mockup Interface has been selected for coupling the simulation tools. FMI provides a standardized interface, which allows for a very modular co-simulation architecture, where several different modules can be added, exchanged, and tested. FMI enables battery modeling, Electric Vehicle simulation, and advanced optimization functions to be coupled in the future.

DIgSILENT Power factory has been selected as the power system simulator. Using a widely used commercial power system simulation platform will help reduce the barriers to the industry for adopting such platforms, investigate and subsequently deploy demand response strategies in their daily operation. Modelica has been selected as the building modeling language. Modelica is an acausal modeling language, which allows for efficient simulation. Given the complexity of the building models and controls, simulation speed and accuracy are important factors. OMNET++, an established open-source network simulator, has been selected as the communication networks simulation tool. An FMI wrapper was developed for Power factory and OMNET++. VirGIL is the first tool worldwide that will connect Power factory, and OMNET in a co-simulation environment over FMI. Detailed Energy Plus models for existing buildings in LBNL campus were converted to RC models through the BRCM toolbox, and then converted to the Modelica language. Model reduction algorithms were used to reduce the model complexity but maintain the accuracy as much as possible. Studies to compare the accuracy have been carried out. All simulation tools are coupled through FMI to a master algorithm, which is implemented in Ptolemy II. To increase VirGIL’s simulation speed, novel simulation algorithms, notably the Quantized State Simulation algorithm, have been developed in Ptolemy. The first case study of VirGIL was on the LBNL distribution grid. Real data have been used for the power network and the modeling of Building 71. In the second year, we plan to simulate more complex systems, and extend VirGIL by adding advanced optimization algorithms for power system planning and building operation. Research shows that electricity sub-metering can lead to a 10%-30% reduction in electricity use in commercial buildings, and it is likely these savings are available in residential and industrial facilities too. The cost to install available electricity metering technology is very high resulting in virtually no market penetration and an inability to achieve the available savings. We aim to demonstrate the core technologies needed for electricity metering technology that has one-tenth the installed cost of today’s solutions. This new solution will provide sufficient accuracy and time resolution to enabling the retro- and continuous commissioning as well as distributed resource grid integration activities needed for a low carbon society. The core technologies behind power meter are voltage and current measurement, and we will demonstrate non-contact measurement of both quantities.

A suite of sensors will be installed on the surface of circuit breakers in electrical panels, and this installation can be done with minimal training and without an electrician. The sensors will measure the magnetic and electric fields passing through the face of the breaker thousands of times per second, and a set of inverse electromagnetic algorithms will estimate voltage and current in real-time. Each sensor unit will accurately estimate power under a variety of conditions and also compensate for external error sources automatically and without user driven calibration. Our most significant accomplishment is the successful demonstration of accurate voltage, current, and power measurement using a combination of commercially available sensors, custom electronics, and custom electromagnetic and statistical auto-calibration algorithms. We built a proof-of-concept sensor, installed it on various circuit breakers, and demonstrated measurement of voltage, current, and power under laboratory and real-world conditions. We also demonstrated the ability to reliably mitigate the impact of interfering fields from nearby currents. We have also designed an advanced sensor capable of measuring magnetic field in multiple locations and in multiple directions. This technology is critical for reducing the complexity of the installation, improving interference mitigation,stackable planters and enabling improved auto-calibration algorithms. The vector fields that are now measurable are useful for key aspects of advanced auto-calibration algorithms that require no reference information. We are currently applying these results to the design of new sensors and auto-calibration algorithms. The new sensors use a combination of commercially available components and CMOS multi-axis magnetic field sensors. The new algorithms utilize newly identified statistical properties of the primary and interfering signals as well as new inverse electromagnetic analysis techniques. The goal of the first year of this 2-year LDRD was to construct a mass spectrometer that will allow quantitative analysis of gas consumption and gas evolution from electrochemical or chemical systems. This spectrometer is envisioned to become a critical component of a comprehensive and systematic approach designed to understand fundamental reactions occurring in metal-air batteries, Li-ion batteries, CO2 reduction catalysis, oxygen reduction catalysis, and other systems where gas evolution occurs. The differential electrochemical mass spectrometer was constructed over the first 9 months of FY2014 and is now fully operational. The DEMS was custom-built to provide many unique features that allow in-situ, real-time quantitative analysis of gas consumption/evolution in electrochemical cells, which can then be related to Coulometry to more clearly understand electrochemical processes. This technique is extraordinarily powerful and sensitive and provides the capability to assign electrochemical currents to specific reactions. By combining this information with that provided by other spectroscopic and classic electrochemical characterization techniques, complex electrochemical processes can be clearly understood and more readily controlled. Construction and calibration of the DEMS was completed in June 2014, allowing it to have all of the useful features necessary to quantify gas consumption and evolution from electrochemical systems. We are currently exploring three avenues of research with this capability: new electrolyte systems for Li-air batteries , outgassing of Li-ion battery materials at high voltages, and magnesium corrosion in aqueous electrolyte systems to understand limitations of aqueous Mg-air batteries. Proposals have been submitted to the NSF and JCAP II in an attempt to secure funding for aqueous electrocatalysis research . Among the interesting scientific outcomes of our endeavors, we have identified that oxygen out gassing of high voltage non-stoichiometric, Li Li-ion battery cathode materials occurs above 4.6 V and continuously occurs if the electrode is held above that potential, implying that O2 evolution is a parasitic process that should be avoided. Furthermore, CO2 evolution at potentials much lower than 4.6V occurs in this system, implying that the electrode catalyzes an unwanted side reaction.

We have also quantified H2 evolution from Mg electrodes immersed in various aqueous electrolytes as a function of anodic and cathodic currents, providing useful insight into the currently poorly understood Mg-corrosion reaction. Vehicle-grid integration can simultaneously transform the electricity market and the automotive market. For the automotive market, VGI can: 1) allow vehicles to meet all corporate average fuel economy requirements and increasingly stringent emissions regulations, 2) move harmful vehicle emissions away from densely populated areas, and 3) provide revenue to offset the capital cost of vehicle electrification. For the electricity market, VGI can: 4) provide a distributed and growing source of grid energy storage, 5) provide better renewables integration, 6) provide a rapidly ramping resource for many electricity markets, and 7) encourage consumers to more closely scrutinize their home electricity bills just like with gasoline or diesel fuel prices. Despite these benefits, the widespread deployment of VGI faces many uncertainties and barriers within both the electricity market and the automotive market. This LDRD project has created the Vehicle-to-Grid Simulator to provide systematic quantitative methods to develop solutions to the electricity market and automotive market barriers to VGI. This LDRD is developing two versions of V2G-Sim, 1) V2G-Sim Analysis, and 2) V2G-Sim Operations. The V2G-Sim Analysis model couples sub-models for: a) driver behavior, b) automated generation of trip-specific drive cycles , c) vehicle power train models of energy usage during a trip, d) vehicle charging, and e) vehicle response to managed charging or V2G algorithms. V2G-Sim Analysis predicts the behavior for individual vehicles/drivers, and then aggregates individual vehicle profiles to generate grid impacts predictions for large numbers of plug-in vehicles . With these coupled sub-models, V2GSim Analysis provides a platform for scenario analysis of PEV deployment for transmission and distribution infrastructure planning, impacts analysis from various PEV managed charging or V2G algorithms, design of market and pricing structures, etc. V2G-Sim Operations builds upon V2G-Sim Analysis to provide temporally- and spatially-resolved forecasting of PEV charging demands and V2G opportunities allowing an aggregator or integrator to bid PEV services onto an electricity market and operate an electricity grid having many PEVs as a resource within the grid while ensuring each vehicle is sufficiently charged when it needs to be. An underappreciated aspect of climate change is how uncertainty about expected changes affects climate mitigation and adaptation measures. In the past, energy and water planners counted on a relatively stable assessment of climate, infrastructure and policy baselines. With climate change, planners face new uncertainties and forecasts of greater variability. Infrastructure plans must be revised which might include a new peripheral canal or a more decentralized electricity grid, mitigation measures might be considered, such as incorporating bio-fuels and improved batteries, and the valuation of water, as a product or as an energy commodity, must be reconsidered.

We have shown that when values from the literature are used for the model-insensitive parameters, the number of calibrated parameters can be reduced for Model A from four to two, for Model C from four to one, for Model B from six to four, and for Model D from six to three. The reduced number of calibrated parameters leads to a reduced complexity of the calibration task and a reduced uncertainty in optimized parameters, especially in connection with the HYDRUS programs. The small number of additional calibration parameters makes the modeling approach of temperature-dependent root growth a convenient add-on to the HYDRUS models. However, the implementation of the influence of other stress factors on the vertical root penetration, as well as on the root length density distribution, into the HYDRUS models still needs to be validated against experimental data.The purpose of this project is to develop the technology necessary for a fiber based laser plasma accelerator to be used for electronic brachytherapy treatment of prostate cancer. Such a device would deliver MeV electrons suitable for medical applications in a small form factor machine. The laser pulse is transported through a hollow core fiber optic that terminates at an embedded gas target where ionization and acceleration occurs. LPAs operate with energy gradients 1000 times larger than conventional accelerators allowing for the energy gain to occur within a millimeter of the treatment area, increasing the therapeutic dose to the tumor site while minimizing collateral irradiance of adjacent healthy tissue. These milestones include, design of a gas jet capable of providing 1-10 MeV electrons, development of a hollow core fiber suitable for transporting the necessary laser energy,ebb and flow trays and establishing a program to evaluate the efficacy of LPA produced electrons as a radiation source. The gas jet target uses a gas profile featuring a sharp density transition to reduce the laser energy needed for electron injection.

Laser plasma simulations will be conducted to establish the gas parameters and physical dimensions necessary to produce MeV electrons. This will be followed by design and fabrication of a gas jet able to deliver the requisite profile. Accomplishments Simulations have successfully shown 1-10 MeV electron beam generation with about 10 mJ of laser energy. This is achieved by relying on breaking of a laser excited plasma wave at a sharp density transition. To realize this density profile with step, a special gas jet has been developed with two distinct gas regions. The second gas region has half the electron density of the first region, and the density transition occurs in a few 10’s of µm that, according to the simulations, is expected to allow electron injection with small amounts of laser energy. The total length of the gas cell is 350 µm, sufficiently small to act as an internal target during treatment. Guided by the simulation results, a design was developed to physically achieve such a density profile. This jet design uses two converging diverging nozzles that are pressure matched at the nozzles’ exit. The resulting flow is known as a slip line where two regions of disparate density can exist in steady state. In order to demonstrate the initial design, the gas jet was constructed at a scale that is fourteen times larger than the original design. This enables easier manufacturing and a rather straightforward measurement of the density drop. Furthermore, the enlarged device would act as an initial laser target for comparison to simulation. A provisional patent was submitted for this novel two-stream gas jet technique. Currently, we are in the process of integrating the target into an existing target chamber that is powered by an existing10 TW laser system. This includes modifying several components of the system to accommodate the new target. In initial experiments, high power laser beams have been focused on the gas target with the longitudinal profile of plasma recombination light showing evidence of a density drop. Quantifying the longitudinal gas density and studies to establish the minimum amount of laser energy that is needed to generate e-beams are scheduled for FY15. Additionally, a second round of simulations that includes ionization effects for the scaled up target is being performed to benchmark experimental results.This proposal is laying the groundwork for the development of computer codes that will be ultimately capable of virtual prototyping and virtual computer experiments on the next generation of supercomputers. The advanced design of future particle accelerators requires consideration of a variety of physical processes that are not appropriately modeled with current techniques and computational power.

We are pursuing the development of a new, highly accurate and scalable solver combining the accuracy of spectral methods with the scalability of finite-difference methods. We propose a crucial change to the scalable algorithm, namely changing the solver portion from FDTD to PSATD solver, to mitigate significant unphysical effects from discretization errors. Traditionally, such a move would compromise the scalability of the PIC method and render the calculation intractable for the required accuracy and spatial resolution. Key to our approach is the design and high-performance implementation of a new solver to take advantage of multilevel parallelism in emerging systems by naturally subdividing the computational domain and workload in a way that is optimally assigned to the heterogeneous computational resources. Our algorithmic research will provide high numerical accuracy and stability. One of our most significant accomplishments is the development of a novel method for highfidelity modeling of the propagation of electromagnetic waves on a supercomputer. The unprecedented level of flexibility in the tuning of accuracy and locality of our method enables very high accuracy while preserving scalability to a very large number of computer cores. Our new solver also offers larger time steps than conventional solvers by removing the standard limitations due to the well-known Courant-Friedrich-Levy condition. Another significant accomplishment is the analysis of the so-called “numerical Cherenkov” instability for our solver in various modes that has lead to the successful development of novel methods that mitigate the instability. The mitigation method has been automated in our ParticleIn-Cell code Warp and is now successfully used by collaborators at other institutions. Strong scaling of the new method was demonstrated on NERSC’s supercomputers Hopper and Edison, yielding near-linear scaling up-to 50,000 cores. During the scalability studies, we also worked to analyze the performance variability of Edison at larger core counts and developed methods to ascertain scalability in the time-sharing environment with the newer Cray Aries interconnect with Dragonfly topology. The scalability of our solver was also contrasted to the scaling of standard global FFTs. The results were presented at the international SC’14 conference and will be documented in a journal publication. We are beginning to develop and study the scalability of the solver on test-bed next generation computers through portable implementations of the solvers, via heterogeneous and emerging programing models. The solver is critical to the further development of the Warp code, which has been selected in a highly competitive competition as a first tier NERSC NESAP application. We are also in the process of extending our methods to three dimensions and applying them to large-scale simulations of laser plasma accelerators.

The goal of this proposal is to develop the scientific user community for the next generation soft x-ray spectro-microscopy and tomography based on x-ray ptychography. X-ray ptychography is a robust diffraction imaging method that can take full advantage of the high brightness of the ALS and which is not limited in resolution by the x-ray optics. The technique we have developed enhances the already high resolution scanning transmission x-ray microscope with the addition of diffraction data and is able to probe chemical species, molecular orientation, magnetization,4×8 flood tray and structural morphology at <10 nm resolution. We will apply this method to difficult problems in the material sciences with a focus on materials for energy and carbon cycle research. In particular, we will study chemical phase transformations in nano-crystals of active battery electrodes, hydration reactions in calcium silicate hydrate, pore evolution in sintered zirconia, and magnetic domain structure in thin magnetic films. Finally, we will further develop in situ methods for high resolution microscopy of electrochemical and hydration reactions in these materials. These studies require the ability to visualize 10 nm scale features embedded in a micron-scale matrix with sensitivity to the chemical species however there currently is no imaging technique with such capabilities. We will develop soft x-ray scanning diffraction microscopy with the study of these systems and provide a microscopy program that will directly and efficiently benefit from the very high brightness of the new COSMIC beamline at the ALS and future beamlines at the ALS-U. This program will readily be extended to other problems in the materials sciences such as the study of porous zeolites for carbon capture and catalysis and the characterization of materials by design like self-assembled nanoparticle aggregates for replacement of non-renewable resources in batteries and solid state devices. Finally, this project will leverage the computational resources at NERSC for the reconstruction of Giga-element datasets and potentially provide experimental feedback to material computation efforts. Using the unique spatial resolution and chemical sensitivity of our imaging method we have directly visualized a two phase chemical transformation and the complicated chemical domain pattern present in the smallest available nano-crystals of LiFePO4. Our measurements were also correlated with high resolution transmission electron microscope images of many such particles to show that these small particles do not suffer from material fracture but still display a complicated domain pattern which does not correlate with the material crystallographic axes as was expected. These results seem to indicate that the superior electrochemical performance of the small particles is due primarily to mechanical issues rather than the fundamental character of the phase transformation itself and provides critical insight into how to engineer higher performing batteries. We have also used our high resolution imaging technique to measure the magnetic domain wall width distribution in a thin film of CoPd, the pore size distribution in sintered Yttria stabilized zirconia, and the x-ray magnetic circular dichroism spectrum in magnetotactic bacteria all with spatial resolutions which are not achievable at any other x-ray facility worldwide.

Storage ring based light sources have been extremely successful over the years, enabling forefront science in many areas. Recent developments in accelerator technology and lattice design open the door for very large further increases in brightness – more than 100 times, particularly by reducing the horizontal emittance. This can greatly enhance the capability of light sources for imaging and spectroscopy. While there are no showstoppers, work was necessary to reduce risk and cost, enable a timely execution, and optimize the properties of the source to the needs of the science case and prospective user community. The goal of this project was to investigate, demonstrate and improve critical technologies necessary for diffraction limited storage ring light sources at the subsystem level. At a later stage, tests will be carried out with some of those new systems using beams in existing light sources. roject Description There is a great need in advanced diffraction gratings at the ALS and other synchrotron facilities. To provide high spectral resolution and high diffraction efficiency the gratings are required to have a variable line spacing , perfect blazed grooves, and smooth groove surface. For Reflective Zone Plates elliptical lines are required to provide two dimensional focusing. Due to limitations of conventional techniques one can only design gratings within a very limited set of variables. New approaches for fabrication of high quality grating in a cost effective manner should be found to make advanced diffraction gratings available. This work aims development of a new grating fabrication process based on Direct Write Lithography technique which is used in semiconductor industry for making high resolution MEMS structures, nanostructures, and consumer microelectronics. A grating pattern can be written by scanning a focused laser or electron beam over a substrate surface coated with a photoresist. The DWL technology will be used as a pattering technique for making diffraction gratings of arbitrary groove density variation and groove shape complexity. The best available DWL systems will be used for making grating prototypes which will be characterized in terms of groove position accuracy, imaging performances, and diffraction efficiency. DWL process will be optimized and eventually combined with wet anisotropic etch technique for making highly efficient blazed gratings.