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These combinations may be capable of activating specific pathways to differing degrees to achieve pathogen appropriate defense responses. Such stimulators of the plant’s inherent immune responses are likely to be superior to conventional biocidal pesticides that are harmful to both environment and consumers . Knoth et al., previously identified two different synthetic elicitors, DCA and CMP442 – thiazolidine-4-carboxylic acid. Both elicitors induce defense in Arabidopsis against Hpa and DCA also against P. syringae. The modes of action of these compounds are distinct; although both act on the SA-dependent branch of the defense network. CMP442 appears to interfere with defense-signaling processes upstream from SA perception, while DCA activates signaling steps downstream from SA . This screening system focused on compounds targeting components of the SA-dependent sector of the defense network and yielded many candidate synthetic elicitors . To widen the spectrum of functionally distinct synthetic elicitors a screening system was needed that allows for the identification of synthetic elicitors interfering with the JA- and ET-dependent parts of the defense signaling network. A set of five genes was identified in microarray experiments that exhibit SA-independent upregulation in response to infection with Hpa . Four of these genes were members of the PDF family including JA-pathway marker gene PDF1.2b. Because PDF genes are known to response to JA and ET, but not SA,ebb and flow table this set of co-expressed defense-related genes was named JEDI and include PDF1.2a , PDF1.2b , PDF1.2c , PDF1.3 , and JEDI1 .

Here the development of transgenic lines in which select JEDI genes were fused to reporter genes to develop a high-throughput chemical screening system is described. In addition, the creation of transgenic plants with an RNAi transgene silencing transcripts of the closely related PDF members within the JEDI set is reported. Furthermore, JEDI transcripts were hyper induced at 48 hpi in nahG compared to Col-0 . The Hpa-inducibility of the JEDI transcripts was confirmed via reverse transcription -PCRs at 0, 6, and 12 hpi with HpaEmoy2 in two-week-old Col-0 and nahG plants. Compared to their levels at 0 hpi, transcript levels of all JEDI genes were enhanced at 6 hpi and 12 hpi in both Arabidopsis lines. Thus, the trends observed in these RT-PCRs were similar to those in the original microarray data, suggesting that JEDI transcript accumulation in response to HpaEmoy2 recognition is at least partly SA independent. Additional time points were examined after Hpa in Col-0 and nahG.PDF genes are known to be positively controlled by the JA-dependent branch of the plant defense network. However, JA-controlled defense responses have been traditionally associated with immunity against necrotrophic or hemibiotrophic pathogens, such as the fungus B. cinerea or the bacterial pathogen P. syringae . Immunity against strict biotrophs, such as Hpa is believed to be controlled by SA dependent immune responses . However, our observation that JEDI–PDF genes are upregulated during incompatible Hpa interactions suggested that JA-dependent signaling processes can also contribute to immunity to this strictly biotrophic pathogen. Thus, this set of Hpa-responsive genes provided us with an opportunity to study aspects of JA-dependent processes in immunity against Hpa. In order to design a high-throughput screening protocol for the identification of synthetic elicitors targeting the JA-dependent branch of the plant defense network, a previously characterized pPDF1.2b::GUS line was obtained and tested.

Seven-day-old seedlings were grown in liquid growth medium in 96-well plates and incubated for 24 h with 100 µM , 45 µM , or mock solution . A concentration of 45 µM MeJA induces expression of pPDF1.2b::GUS . SA should not induce this reporter . At 24 h after the respective treatments, seedlings were histochemically stained to visualize GUS expression .Even the mock-treatment resulted in GUS expression . Due to the apparent lack of specificity in expression responses a screen with this pPDF1.2b::GUS line, this screen was deemed unfeasible. Therefore, a set of new transgenic lines with fusions of JEDI promoters to reporter genes was developed and tested for their suitability for high-throughput chemical screens. Reporter lines ideal for the planned chemical screens should show no or extremely low background expression and exhibit clear pathogen or JA-inducibility. In addition, the variability of reporter gene expression levels in response to the same treatment should be minimal. A reduction in transcript levels of genes important for plant defense can affect R-gene-mediated resistance and/or basal defense responses. A reduction in basal defense may result in enhanced susceptibility to virulent pathogens . To identify effects on basal defense, the homozygous PDF-RNAi lines were infected with the Col-0 virulent Hpa isolateNoco2 . The interaction of HpaNoco2 with Col-0 is compatible, as this Hpa isolate is not recognized by any Col-0 R gene . Three-week-old plants were spray-infected with 2 ml/pot and 7 days post inoculation the extent of spore formation was quantified. Homozygous PDF-RNAi lines 38, 41, and 48 were in the wrky70-3 background.

Lines were created in the wrky70-3 background to see if knocking down gene expression in this mutant line might display different results than in the Col-0 wild type line. PDF-RNAi lines transformed into the wrky70-3 background displayed similar levels of infection as wrky70-3. In addition, wrky70- 3 and its transgenic lines consistently supported similar levels of spores as Col-0. This is contrary to previously reported data, which demonstrated that wrky70-3 supported more pathogen growth than Col-0 . Experiments performed with Arabidopsis plants at a range of ages indicated that WRKY70 transcript levels are upregulated in an age-dependent manner . In two-week-old Arabidopsis plants WRKY70 transcript levels were lower than in three-week-old plants. While basal defense up to an age of two weeks seems to be dependent on WRKY70, this transcription factor seems not to be required for this immune response in older plants. No morphological abnormalities were apparent in any of the PDF-RNAi lines. Pathogen recognition triggers a highly intricate set of defense responses which are coordinated by a complex regulatory network . Two branches of this network, which are dependent on the signaling molecules SA or JA, respectively, have been previously characterized. In Arabidopsis, SA-dependent signaling can processes induce antimicrobial proteins . Alternately, induction of PDF genes such as PDF1.2 is dependent on JA-dependent regulatory processes. While SA-dependent plant immune response have been primarily associated with resistance against biotrophic pathogens, defense reactions promoted by JA seem mainly to be effective against necrotrophs . Extensive crosstalk between JA- and SA-signaling has been reported as both types of defense signaling processes can be additive, antagonistic,flood table or synergistic dependent on the extent of their induction . Furthermore, successful defense against certain pathogens or pests often requires the coordinated induction of both SA- and JA-dependent defense reactions . High-throughput chemical screens previously identified synthetic elicitors activating SA-dependent plant immune response . A complementary screen to identify synthetic elicitors stimulating JA-dependent defense signaling processes would have great potential for the further decipher plant defense networks and provide molecular probes to uncover crosstalk mechanisms coordinating SA- and JA-dependent defense reactions. Interestingly, in nahG Arabidopsis plants JEDI transcripts appeared to be hyper induced in response to infection by Hpa suggesting they are upregulated by signaling mechanisms normally antagonized or suppressed by SA. As the JEDI set includes several PDF genes which are known to be positively regulated by JA , the hyperinduction of JEDI transcripts in the nahG background may point to a JA-dependent gene induction mechanisms that is counteracted by SA. To decipher roles that the JEDI genes may play in defense and stress responses, publically available microarray data were examined. The expression profiles of PDF1.2a, PDF1.2b, and JEDI1 were available through the Botany Array Resource website . These genes were highly upregulated in response to the oomycete Phytophthora infestans, the fungi Erysiphe orontii, Botrytis cinerea as well the abiotic stimuli wounding, osmotic stress, ultra-violet light B treatment, oxidative stress, and drought.

Additional treatments reported on in BAR, such as: SA, the ET precursor 1-aminocyclopropane-1-carboxylic-acid, the cytokinin zeatin, methyl jasmonate, indole-3-acetic acid , abscisic acid , gibberellic acid , and heat caused no significant changes in the expression profiles of PDF1.2a, PDF1.2b, and JEDI1. Microarray data in the BAR database therefore indicated that a wide range of pathogens and stressors may induce JEDI expression. Wounding triggered the accumulation of JEDI transcripts . However, JEDI transcripts did not accumulate in response to treatment with MeJA . A likely explanation for this unexpected observation is that responses to MeJA were measured at early times after MeJA treatment. , while previous studies showed PDF transcripts to accumulate around 48 h after treatment with jasmonates . Microarray data sets in BAR include responses of Arabidopsis to different types of pathogens, such as the hemibiotrophic oomycete P. infestans, the biotrophic fungus E. orontii, and the necrotrophic fungus B. cinerea . Thus, transcriptome responses of Arabidopsis to a complete set of pathogenic lifestyles are represented in BAR. Of these three different types of pathogens, the hemibiotroph P. infestans induced the strongest accumulation of JEDI transcripts while the other two pathogens induced expression to a lesser degree.Besides canonical JA-response elements, additional putative cis-elements are commonly present in all PDF promoters. These include binding sites of the AP2-domain transcription factor RAV1, which binds to bipartite recognition sequence, one of which is a AP2 domain . GCC boxes are known to mediate responses to ET or JA . In addition, W-box motifs are present in JEDI promoters. W boxes have been mainly associated with SA-dependent regulatory mechanisms, but may have wider roles in defense gene regulation . Furthermore, GATA promoter, and I-box motifs are present JEDI1 and PDF1.2a suggesting that these genes have particularly diverse roles. GATA and I-boxes are light-responsive promoter elements. The JEDI genes also respond to light and many non-defense-related stimuli, which again suggest a diversity of roles for these genes. It has been previously reported that the GCC box is important, but not essential, for the defense-related up-regulation of PDF1.2b . Thus, additional promoter elements must also be involved in the defense-related up-regulation of PDF1.2b . While the created transgenic lines with pPDF::LUC or pPDF::GUS fusions seemed not suitable for new synthetic elicitor screens, as they exhibit high background expression levels, these lines may still be useful for other types of experiments. They can be used to study spatial and temporal patterns of pathogen-induced JEDI gene expression. They may also allow the rough localization of promoter elements mediating responses to various defense related stimuli, such as infection with virulent or avirulent Hpa isolates. The high degree of structural relatedness and similarities of their defense related expression among PDF genes suggested functional redundancy. Studies using single mutants for individual PDF genes have not been reported on in the literature for the defense related functions of these genes. Thus, only a quadruple mutant with combined mutations in each of the four PDF genes is likely to exhibit clear defense-related phenotypes. The construction if such a quadruple mutant, however, is not feasible due to the extremely close linkage of PDF1.3 and PDF1.2b as well as PDF1.2c and PDF1.2a . As the nucleotide sequences of these four PDF-JEDI genes share a high degree of identity, RNAi-based silencing using the pANDA-RNAi vector seemed a feasible strategy. Several Arabidopsis lines containing the PDF-RNAi vector exhibited reduced basal defense against Hpa. However, in these lines transcript levels of only PDF1.2c and PDF1.3 appeared to be reduced. Thus, co-silencing of these two PDF members may be sufficient to overcome functional redundancy within this group of defense genes. Unfortunately, the RT-PCR analyses measuring PDF transcript levels were not consistent. Measurements of the respective transcripts need to be repeated by a more reliable and robust method, such as real-time quantitative RT-PCR. Assays to examine the effect of RNAi-transgene on resistance to other pathogens should be also performed. In any case, this study, which demonstrates a potential role of some PDF genes in basal defense against Hpa indicates that members of this gene family can contribute to immunity against a strict biotroph. While this finding is novel, it is not unexpected, as PDF transcripts were found to accumulate during infections of Arabidopsis with pathogens of different lifestyles, including biotrophs .

Following the activation of initial and local defense responses are a set of delayed and systemic responses that include systemic acquired resistance . SAR is an induced form of defense that is activated remotely from the point of pathogen infection conferring a broad spectrum disease resistance against a variety of pathogens . Like many local defense responses, activation of SAR requires the accumulation of the signaling molecule salicylic acid . A complex regulatory network has been shown to be required for proper regulation of these plant immune responses . Many components of this network are commonly utilized by PTI, basal defense, ETI, and SAR. Major regulators of plant defense responses are protein kinases, which act at various hierarchical levels within the plant defense network . There are more than 1000 protein kinases in Arabidopsis . In particular, receptor protein kinases , Ca2+-dependent protein kinases and mitogen-activated protein kinases have been implicated in the regulation of plant immune responses . RPKs are comprised of a transmembrane domain with amino-terminal extracellular domains implicated in ligand recognition and protein–protein interactions, in addition to a carboxyl-terminal intracellular kinase domain involved in signal transduction . The three major sub-classes of RPKs are differentiated based upon their kinase domain substrate specificities. The sub-classes include: receptor-tyrosine kinases,grow bucket receptor-serine/threonine kinases, and receptor-histidine kinases .

Most plant RPKs are proteins containing an: extracellular signal sequence, extracellular leucine-rich repeats , a transmembrane helix, and cytoplasmic kinase domain with the serine/threonine consensus sequence . One variant in the RPK group is the receptor-like kinases , which belong to a large family known as the RLK/Pelle family. The Arabidopsis RLK family is divided into 45 subfamilies with over 600 members that comprise more than 2% of the Arabidopsis genome . One of the main criteria that distinguishes these subfamilies is the existence and type of extracellular domain . There are 15 classifications for RLK extracellular domains, which include: CRINKLY4-like, C-type lectin-like, CrRLK1-like, DUF26-like, extensin-like, legume -lectin-like, LRK10-like, LRR-like, LysM-like, PERK-like, RKF3-like, Sdomain-like, thaumatin-like, URK1- like, and WAK-like . The LRR domain is the most common and represents the largest RLK group with 216 members subdivided into 13 subfamilies . Most RLKs have a conserved arginine and an aspartate in the activation loop of subdomain VI, which acts as a kinase activator by enhancing phosphotransferase efficiency . Often kinases with arginine and aspartate are important for developmental regulation, while those without these conserved residues are important in innate immunity . Accordingly, plant RLKs can be further subdivided into two major categories based upon their functions: one is involved in cell growth and development and the other in plant–pathogen interactions and defense responses . Examples of this second group are PRRs: Xa21 from rice and FLS2 from Arabidopsis , which interact with certain MAMP-type epitopes. Xa21 is membrane bound serine/threonine protein that is activated by AxYS22, a 17- amino acid peptide conserved in strains of Xanthomonas.

FLS2 is a transmembrane protein that recognizes a number of bacterial MAMPs including peptides derived from the flagellin such as flg22 . Another RLK, CERK1, belongs to a distinct subfamily and is required for immune signaling triggered by fungal chitin. In addition, CERK1 binds and recognizes bacterial peptidoglycans contributing to immunity against bacteria . Another group of kinases important for defense are CDPKs, which are encoded by a 34-member gene family in Arabidopsis and make up one of the largest family of Ca2+ sensors in plants . Host proteins must be able to sense alterations in Ca2+ levels and respond accordingly . CDPKs have N-terminal protein serine/threonine kinase domains attached through an autoinhibitory junction domain to a C-terminal Ca2+-binding calmodulin-like domain . CDPKs bind Ca2+ at their C-terminal domain, which activates their protein kinase activity and facilitates their function as transducers of Ca2+signals. A possible role of Ca2+ in plant defense was proposed when CDPK transcripts were found to be elevated in tobacco, maize, tomato or pepper in response to pathogens or their elicitors . Upon pathogen recognition, cytosolic Ca2+ levels increase . The duration and amplitude of these increases are specific for the respective defense-related stimulus, resulting in the differential activation of downstream components . Two proteins have been suggested as potential substrates for CDPKs in plant defense: PAL and plasma membrane associated NADPH oxidase . PAL appears to be phosphorylated in bean cells challenged with a general elicitor but the significance of this observation remains to be demonstrated. In addition, a CDPK was shown to enhance NADPH oxidase activity stimulating an oxidative burst in tomato protoplasts although the significance of this interaction is also not clear . Romeis et al., demonstrated defense-associated activation of CDPKs in tobacco cell cultures transformed with the Cf-9 gene from tomato. Cf-9 is responsible for providing resistance to Cladosporium fulvum in the presence of its corresponding avirulence gene Avr9.

They established that the presence of Avr9 and Cf-9 a kinase was phosphorylated, causing an increase in kinase activity. They further demonstrated that this kinase is of the CDPK-type, because it required Ca2+ . This was the first direct demonstration of CDPK enzyme activity in plant defense. Meanwhile it has become clear that CDPKs are important not only in plant defense signaling but also serve as key points of convergence of various regulatory pathways due to their ability to respond to different hormonal or environmental cues . To better understand CDPK function in plant defense, additional pathogen-induced CDPK-phosphorylated substrates need to be identified. Mitogen-activated protein kinase cascades transmit and magnify signals through a phosphorelay mechanism involving: MAPK-kinase-kinases , MAPK-kinases , and MAPKs. They link upstream recognition events to downstream targets and their sequential phosphorylation targets substrate proteins in the cytoplasm or nucleus. MAPK activation is one of the earliest conserved signaling events after pathogen recognition . Many signaling cascades are shared between different activating stimuli . Cross-inhibition, feedback control, and the use of defined scaffolding proteins connecting distinct signaling components are utilized to enforce specific relationships between activating stimuli and the respective biological responses . Cross inhibition is manifested in the mutual inhibition between two pathways . Feedback control can be represented by negative feedback loops, where the activation of one component down-regulates the function of another. Scaffold proteins bring components together, which enhances specificity within signaling chains . Little is known about specific scaffolding proteins within plants. Two putative plant scaffolding proteins include alfalfa OMTK1, which interacts in protoplasts with the MAPK MMK3 in response to H2O2 and Arabidopsis MEKK1, which binds to MKK2 and MPK4 . MAPKs pathways are known to be involved in plant development, programmed cell death, responses to some abiotic stressors, and defense signaling. The Arabidopsis genome codes for 110 MAPK cascade components, which includes 20 MAPKs, ten MAPKKs and 80 MAPKKKs . Few MAPKs have been studied due to their lethal mutant phenotypes in plants . The most well understood MAPKs are MPK4, MPK3, and MPK6,dutch bucket for tomatoes with the latter two acting as positive regulators for defense responses and the former being a negative regulator of SAR. The Flg22 peptide is recognized by the receptor FLS2 which complexes with BRI1-ASSOCIATED KINASE and triggers MAPK signaling cascades. This cascade includes the activation of MPK3, MPK4 and MPK6 . MPK4 and MPK6 are also activated by hrp proteins from some bacteria and their activation results in the induction of PR genes which sometimes encode proteins with antimicrobial activities . Further studies must identify and elucidate MAPK cascades and find ways around the widespread mutant lethal phenotypes which inhibit kinase pathway studies. Traditional mutational analyses have been unable to circumvent functional redundancy and lethal mutant phenotypes . Thus, additional types of experimental approaches are necessary for the continued elucidation of the intricate and elaborate circuits within plant immune networks. One novel approach, chemical genetics, offers distinct advantages over traditional techniques.

Chemical genetics allows bioactive small molecules to be used in a reversible manner, since frequently their effects on organisms are not permanent. In addition, it provides more temporal control over experiments, since chemicals typically interfere with their targets immediately after application. In contrast, the timing of pathogen infections is often poorly reproducible, as the germination of spores or pathogen growth and spread in plants is asynchronous and often highly sensitive to subtle changes in environmental conditions. Chemicals also have the ability to simultaneously affect multiple members of highly-related protein families, permitting the study of biological functions of functionally redundant proteins. Using traditional genetics to knock out the function of an entire gene family often proves difficult or infeasible due to technical challenges and lethal phenotypes. Yet another advantage over traditional genetics is that bioactive chemicals allow for the study of essential gene functions at any stage in development because transiently active compounds can be added at any time or any concentration. In contrast, genetic mutations are of permanent nature. If they confer lethal phenotypes, no studies can be performed. Finally, the function of multiple structurally unrelated genes can be knocked out concurrently by using combinations of chemicals while also varying the concentration of each chemical allowing the study of quantitative relationships between defined stimuli and phenotypes . Chemical genomics requires tens of thousands or even hundreds of thousands of chemicals to be screened for their ability to stimulate a particular phenotype of interest . The increase in demand for chemicals that can manipulate a diverse set of biological processes resulted in the need for inexpensive large and structurally diverse chemical libraries for screening. As a result, the concept of combinatorial chemistry was developed . This high-throughput approach is based on simultaneously occurring synthesis steps. During each step a set of distinct chemical building blocks is used, yielding a vast number of structural combinations, referred to as “a combinatorial libraries”. The ease of this novel form of synthesis made these libraries widely available and cost-effective to many fields of academic research . Thus, the large sample size of available structurally distinct chemicals maximizes the probability that compounds will be identified that induces the desired biological effect. The Eulgem lab uses chemical genomics to identify and characterize synthetic elicitors, which are small drug-like molecules that induce plant defense responses . Their ability to induce defense responses provides us with a highly attractive alternative to conventional pesticides, if proven to be less toxic.This toxicity often leads to off-target effects against other organisms and the environment. As a result, the dangers of pesticide poisoning become more of a concern, making the identification of compounds that are not toxic, but instead stimulate plant’s inherent defenses very appealing. In addition to their potential use as pesticide replacements, synthetic elicitors can also be utilized as highly specific stimuli to perform more refined functional analyses of the plant defense network by interference with distinct network nodes. Their use should allow for the selective activation of certain regulatory circuits within this network. The identification of cellular targets of synthetic elicitors can uncover novel components of the plant immune system. Taken together the use of synthetic elicitors is likely to enable us to gain a deeper and more comprehensive understanding of the structure and function of the plant defense network. This report highlights the functional characterization of some members of the ACID cluster, a group of genes identified by micro-array experiments after treatment with two synthetic elicitors, DCA and INA . These 137 genes were found to be enriched for protein kinases, which may play key roles in plant defense signaling. Of the 16 ACID genes examined, ten were required for full basal defense of Arabidopsis against Hpa. Seven of the ten ACID genes have not been implicated as components of the plant immune system yet. While important for basal defense, these genes were not essential for immunity mediated by two distinct R-genes. Although they are transcriptionally activated by DCA, DCA mediated immunity was not compromised in their mutants. In addition, eight novel synthetic elicitors identified in the screen performed by Knoth et al., were further characterized. Notably, a synthetic elicitor was identified with a substantially lower active concentration than DCA. It was noted that DCA-mediated immunity in the acid mutants did not display the same hypersusceptibility seen in basal defense assays .

miRNAs are post transcriptional regulators that cause down regulation of target genes. Therefore, if a target gene is down-regulated by a miRNA, a negative correlation between miRNA expression and the target mRNA expression is expected. No statistically significant differentially expressed miRNAs were observed in our fruit small RNA seq data, so we instead predicted miRNAs that are potentially targeting DEGs found in fruit tissues. This approach was taken due to the complex regulatory networks that are known to exist in plants and other higher organisms. One miRNA may regulate many genes as its targets, while one gene may be targeted by many miRNAs. Both of these scenarios were observed in citrus roots in response to dehydration and salt stress. To evaluate these potential relationships in this study, the psRNA Target program was utilized, which accepts a list of known plant miRNAs in citrus and the coding sequence of the DEGs reported here to predict miRNAs according to the criteria described by Meyers et al.. Over 15,000 miRNA-mRNA interactions were predicted using psRNA Target. The RNAseq data was then utilized with an in-house R-script in order to select potential interacting pairs with an expected negative correlation in gene expression. After removal of genes that did not have any functional annotation, there were 366 combinations of miRNA-mRNA pairs that showed reciprocal expression patterns. Comparing these genes with the enriched GO terms and KEGG pathways led to several candidate miRNA-mRNA interactions that could be causing changes in fruit traits when differentially expressed between root stocks.

These genes included transcriptional regulators, hormone signal transduction genes, transporters,nft growing system and sugar metabolism genes. Based on the interacting pairs predicted and their relevance to fruit quality, 10 pairs of miRNAs and target mRNAs were selected for validation via qRT-PCR analysis . Samples collected at time points two and three were chosen for validation due to the larger differences in expression levels of genes in fruit grown on trifoliate orange compared to rough lemon root stocks at those times. For qRT-PCR, two biological replicates and three technical replicates were analyzed to quantify expression of each gene. Three miRNAs were up-regulated at both time points, while their target genes were down-regulated, two miRNAs were down-regulated at both time points, while their target genes were up-regulated, and the remaining five miRNAs validated were down-regulated at one time point and up-regulated at the other . The correlation between the relative expression level detected by qRT-PCR and by RNA-sequencing was calculated. Pearson correlation values were highly significant with r = 0.94, which strongly supported the sequencing data . However, certain miRNA-mRNA pairs did not have the expected fold changes from one time point to the next. For example, Csi-miR171a shows an increased fold change from September to November. This should correlate with a decreased fold change from September to November in the target gene , but instead, we see an increase in the target mRNA expression from September to November. Only this pair and Csi-miR1863 – ATEXP1 show this inconsistency. The results for the remaining eight pairs were consistent with their expected expression levels.

Figure 1.12 shows that seven of the miRNAs had increased expression levels in November compared with September, while three miRNAs decreased in expression from the during fruit development. The objective of this study was to correlate changes in gene expression of grafted citrus trees to effects in fruit quality due to varying root stocks. In this study, four root stocks were chosen from a root stock trial with Washington navel orange scion in Riverside, CA to assess for various fruit quality traits; Argentina sweet orange, Schaub rough lemon, Carrizo citrange, and Rich 16-6 trifoliate orange. Fruit quality data was collected from fruit grown on each of the four root stocks at the end of the growing season when fruit were ripe. In the present study, weight, height, width, rind color, rind texture, peel thickness, internal texture, juice weight, percent juice, total soluble solid and titratable acid levels were measured. The total yield and average fruit weights were markedly higher in navel orange fruit from trees grafted onto rough lemon compared to sweet orange, Carrizo citrange, or trifoliate orange root stocks. The rind thickness was also greatest on rough lemon root stocks compared with the other root stock-scion combinations. The most substantial differences could be seen in total soluble solids and acid levels. The highest levels of sugars and acids were found in fruit grown on Carrizo citrange. Trifoliate orange and sweet orange root stocks produced fruit with only slightly lower sugar and acid levels, while rough lemon produced fruit with significantly lower levels . This is consistent with the previously mentioned reports of root stock effects on fruit quality3,7-14. Presently, there is very little understanding of how root stocks influence citrus fruit quality, especially at the level of gene regulation. In this study, an integrated mRNA and miRNA high throughput sequencing analysis in fruit grafted onto genetically diverse root stocks was performed to help resolve potential mechanisms of root stock-scion effects on fruit quality. In the present study, RNA-seq was used to investigate transcriptome differences in the fruit of ‘Washington’ navel sweet orange grafted onto different root stocks and explore genes that may influence fruit quality traits. Juice vesicles from fruit grafted onto four genetically diverse root stocks at four different fruit development periods were sequenced. The RNA-seq approach detected a similar number of genes in all samples .

A large number of these genes were identified as differentially expressed over the course of fruit development, which is consistent with previous studies of transcriptome changes during fruit ripening in sweet orange . However, most genes showed similar temporal expression patterns among all root stock genotypes. Furthermore, only ~15% of the genes were genotype-specific . Therefore, the remainder of this study focused on DEGs identified between these root stock genotypes during fruit development.The majority of the differentially expressed genes are observed in comparisons involving rough lemon root stocks, especially compared to trifoliate orange. This is consistent with the observed differences in fruit quality traits, as fruit of trees grafted on rough lemon root stock showed consistent significant differences from fruit of trees grafted on the other three root stocks in many of the traits measured . These results suggest that rough lemon and trifoliate root stocks show the greatest effects on the scion and are good candidates to identify graft-related genes playing a role in fruit quality. The largest and most significant changes in gene expression between root stocks were observed at time points two and three . Among the DEGs were several genes with functions involved in fruit quality traits, such as those relating to starch and sucrose metabolism, fructose metabolism,nft hydroponic system and hormone signaling related genes. KEGG pathway analysis displayed plant hormone signal transduction, carotenoid biosynthesis, and fructose and mannose metabolism pathways to be significantly enriched. Several genes involved in various hormone-signaling pathways were DE, mainly genes in the abscisic acid and auxin-response pathways. Several genes involved in these pathways were chosen to validate the RNA-seq data by qRT-PCR due to their potential biological significance regarding root stock effects on fruit quality. ABA has been known to be a regulator of fruit ripening and response to abiotic stress in non-climacteric fruit. AHG1, a homolog of Arabidopsis PP2C family protein, was DE in this study. This gene was slightly up-regulated when comparing fruit of trees grafted on trifoliate to fruit of trees grafted on rough lemon root stock at time two and significantly down-regulated at time three . Upregulation of AHG1 is in accordance with previous studies showing this gene being induced by water stress, which may have occurred in September.

The down regulation of this gene later in the season could be correlated with increased fruit maturation in fruit grown on trifoliate root stocks. This is in agreement with a study in tomato where suppression of PP2C expression led to increased ABA accumulation and higher levels of ABA-signaling genes that increase the expression of ABA-mediated ripening-related genes. Auxin signal transduction is mediated by Aux/IAA and ARF genes. Aux/IAA proteins are negative regulators of the auxin signal transduction pathway. In this study, a gene encoding an Aux/IAA protein, IAA16, was up-regulated in fruit grown on trifoliate compared to rough lemon root stocks at time two and three . A previous study revealed that a gain-of-function mutation in IAA16 displayed reduced response to auxin and ABA, which led to reduced plant growth89. Silencing of related Aux/IAA genes increased fruit size in tomato due to auxin control of cell expansion and elongation. In addition to Aux/IAA, another early auxin-response gene, SAUR78, was DE in this study. This gene was down-regulated in fruit grown on trees grafted onto trifoliate compared to rough lemon root stocks at time two and three . Small Auxin Up RNA genes are a group of auxin-inducible proteins. SAUR78 over expression lines in Arabidopsis increased plant growth through interaction with ethylene receptor. Other SAUR genes have also been shown to promote cell expansion. Furthermore, a MYB77 gene encoding a transcription factor was DE in this study, displaying a slight increase in expression in fruit grown on trifoliate root stock at time two, but a large decrease in expression at time three . This gene was previously described as a regulator of the auxin signal transduction pathway. This protein was shown to interact with ARFs to promote plant growth. Interestingly, the effects of MYB77 in Arabidopsis were found to be increased by endogenous exposure to ABA and further promote plant growth. While these two studies were performed in roots, this transcription factor was shown to be involved in citrus fruit ripening, where it was highly correlated with ABA and suggested to have a similar function in response to the hormone28 . Although there were not statistically significantly differences seen in other genes in the auxin- and ABA-signaling pathways, trends could be observed during hierarchical clustering of these genes. Many of the genes within a family shared common expression levels and generally follow the predicted regulatory patterns in their respective pathways . Taken together, the changes in ABA- and auxin responsive genes suggest a potential mechanism for induced ripening by trifoliate root stock and larger fruit produced when rough lemon is used as a root stock. The expansion phase of citrus fruit development involves cell enlargement and water accumulation. Given the changes in hormone-signaling pathways that likely lead to changes in fruit size, other genes related to fruit growth, such as transporters and genes related to cell wall metabolism were investigated. This led to the identification of two DEGs that could be influencing fruit size. The first, a Plasma membrane Intrinsic Protein 2gene encoding an aquaporin was down-regulated in fruit grown on trifoliate root stock . Water import in plants is mediated by aquaporins and essential for cell expansion. These genes were highly expressed in expanding green grapes and one was identified as a candidate gene under the QTL for berry weight. PIP genes were also associated with an increase in volume of fruit in apple and strawberry. The second DEG, an expansin , was also down-regulated in fruit grown on trifoliate root stock . Expansins play various roles in fruit development, including cell elongation and cell wall softening. A homolog of EXP1 in tomato was expressed during green fruit cell division and expansion with maximum accumulation of EXP1 during the late phase of green fruit expansion and early maturation. The increase in expression of these two genes in fruit grown on rough lemon root stock could contribute to the larger fruit size observed. In addition to cell division and cell expansion, during fruit development, fruit softening is also an important feature that relies on cell wall metabolism. The Trichome Birefringence-Likegene, which encodes a protein required for cellulose biosynthesis, was identified in our study as DE. Mutations in this gene caused a reduction in the amount of pectins and an increase in pectin methylesterase activity. PME catalyses the demethylesterification of pectin, which may undergo depolymerisation by glycosidases. TBL23 was up-regulated in fruit grown on trifoliate root stock compared to rough lemon , suggesting a potential role in fruit softening during citrus ripening. Transcription factors also play an important role in plant development and fruit ripening.

Several of these chromosomes also showed significant associations with this study, but the verified region on 4B was not among them. Similar to our results, individual QTL were almost always exclusive to each population. Kabir et al. identified QTLs for root number on 1B, 2A, 3A, 4A, 4D, and 7A and no QTL was consistent across the two populations. These observations suggest two explanations: either seminal root number is sensitive to environmental effects and many statistically significant associations detected in all studies are spurious, or this trait is controlled by a large number of genes, in different combinations in each parental line. No single locus appears to have a large dominant effect, perhaps with the exceptions of the loci on chromosomes 2DS and 4BL in our study. Unfortunately the two studies of Zhang et al and Kabir et al did not investigate seminal root angle so we have no insight into how that trait behaved in both cases. Within our populations, the expression of seminal root angle QTLs were also highly dependent upon year and population, however, five QTLs were consistent across both years . One of those QTL, QRA.ucr-2D, rolling bench was also verified within two of the three populations . This QTL accounted for the largest proportion of the phenotypic variation of all verified QTLs. This could potentially be due to the greater phenotypic difference between the two parents in the SC and SF populations which allow for greater detection of QTL .

Using relative genetic map distances this QTL appears to be the same QTL as identified by Bektas with a large effect upon other root traits such as deep root weight. Bai et al. also reported a QTL on chromosome 2D for seminal root biomass. In other cases such as QRA.ucr-6A and QRA.ucr-7B the QTLs consistently appeared in the SC population in both years; however, they do not appear in other populations. Given the crossing pattern used in the development of the populations, and even with an assumption that the QTL donor carries the same allele as Foisy, segregation should have been observed in the CF population, but it was not. Perhaps this is because this QTL explains a small percentage of the total phenotypic variation and its effect is overshadowed, hence undetectable, by segregation of different allelic combinations within the SF population. In the CF population qRA.ucr-5B and qRA.ucr-7A varied more from one year to the next than any other QTL, and no association with common markers were detected, even though on the consensus map of Wang et al. all these associated markers fall within 10-20cM of one another. Because the effect of this specific genome region was reproducible it is deserving of further study. These shifts of QTL positions are often associated with changes in the total amount of variation explained by the QTL between years. For example, qRA.ucr-5B in the CF population explained 9.40 % of the phenotypic variation in 2014 but 19.50% in 2015. These QTL appear verified as they produce significant effects in both years, however, their effects were not detected in the other two populations.

This may be an effect of considerable plasticity of the characters measured, illustrating technical difficulties in precise phenotyping. On the other hand, this may hint at the existence of closely linked loci within the same family, each with a minor effect on the total expression of the character, and minor variation within the environment from one year to the next may cause shifts in the locus/loci responsible, thus changing marker associations in the region. These examples could potentially be shedding light on the plasticity of QTL for seminal root angle in light of environmental cues. New techniques such as the clear pot method proposed by Richard et al. may provide less variability by reducing the experimental error. Unraveling the genetics of seminal root angle in wheat may prove to be a longer road than in other crops like rice. Uga et al. identified DEEPER ROOTING 1as a gene controlling the gravitropic response of roots and thus the angle of root growth. Higher expression of DRO1 caused roots to grow more downward and when introduced into a shallow rooted cultivar it improved grain yield under drought by enabling access to water deeper in the soil profile. It is likely simpler to study quantitative traits like seminal root angle within the smaller diploid genome of rice. Although there is synteny between rice and wheat within the region where DRO1 was identified, QTL in that region were not identified. Until recently rice was the closest relative of wheat that had information about seminal root angle genetics. However, researchers interested in barley have now begun to study seminal root traits as well . Using the clear pot method demonstrated by Richard et al. they were able to identify seven QTLs for seminal root angle and number . Using cross species analysis they were able to identify 10 common genes underlying root trait QTLs in barley, wheat, and sorghum.

Perhaps as seminal root angle is unraveled in barley, a closer relative to wheat than rice, it will provide insights which may aid in our understanding of wheat seminal root trait genetics. Seminal root angle and number appear to be interrelated and both appear to be related to seed weight . In the SC population root number and angle are negatively correlated so that seeds with more seminal roots have narrower angles and vice versa. This correlation explained 22% and 30% of the variation seen in 2014 and 2015 respectively. In the SF population seminal root number and seed weight were positively correlated so that heavier seeds tended towards a higher number of seminal roots. That correlation explained 36% and 45% of the variation in 2014 and 2015 respectively. In the CF population all these characters are correlated where seminal root number is positively correlated with seed weight and seminal root angle is negatively correlated with number and weight. The correlation between root number and seed weight explained 46% of the variation in 2014 and 2015, seminal root number and angle explained 24% and 33% in 2014 and 2015 respectively,grow table hydroponic and the correlation of seminal root angle and seed weight explained 23% and 35% of the variation. These results show that in the CF population a significant amount of the variation can be explained by these interactions. This is interesting in that those two parents have more similar seminal root angles . Since seed weight explained a significant amount of the variation for root number and angle it could mean loci for seminal root angle or number in CF are actually seed weight QTLs. The only way to ascertain which character is actually monitored is to map QTLs for seed weight and test their associations with those found for seminal root angle and number. In the SC and SF population no QTL for seed weight was similar to that mapped for root angle and number. However, in the CF population two QTLs for seed weight were in similar positions to QTLs for root angle and number . The QTL for seed weight on chromosome 1B clearly overlaps with the QTL for root number on 1B, each sharing common markers in both years. Of the QTLs mapped for root number in CF this QTL on 1B was the only one observed in both years. For the QTL on 5B there are not any overlapping markers for the root angle QTL and seed weight QTL, however, the QTLs for root angle on 5B shift from one year to the next making this region suspect and deserving of further inquiry. Of the QTLs for root angle in the CF population the QTL on 5B explained the greater portion of variation seen in the population over two years. Since so much variation is explained by the interaction of seed weight with angle and number it is not a major leap to assume this region could be associated with seed weight and inadvertently associated with root angle. Given those results, coupled with the correlation analysis, it seems that seed weight is a major factor, if not the major factor, in the CF population giving rise to most differences in seminal root traits. These interactions between seed weight, seminal root angle and seminal root number indicate the high complexity of root traits.

The nature of these interactions has not been tested but it appears plausible that when five seminal roots are initiated they occupy greater space at the developing point of the embryo than when only three roots are initiated. This may force the inner pair of roots more downward, thus reducing the angle between them and explain why more seminal roots is correlated with narrower angles of growth and why those with less seminal roots have a tendency toward wider angles. Additionally, heavier seeds are correlated with higher seminal root numbers which then may influence the association of seed weight with seminal root angle. Perhaps this argument is overly simplistic, and it does not begin to explain why these three characters are correlated only in some populations, and why the levels of interaction change from one population to the next. Another explanation could be linkage of loci for individual traits which could make them difficult to tease apart. In any case, these correlations underscore the complexity of these traits and call for further dissection of each trait and their interactions, so that actual genetic effects are studied. Those interactions could lend new dimensions of complexity when considering the inheritance of seminal root angle and number. These findings also provide new information for considerations when designing future projects centered on these traits. Another point to be made is that QTLs in other studies should be further verified and looked at again through this perspective. As far as we know, other studies did not map QTLs for seed weight when an interaction was observed with seminal root traits. Since the green revolution, semi-dwarf high yielding wheat cultivars have become a standard in commercial production. The semi-dwarf character of wheat lead to a threefold increase in grain yield and provided food security for developing countries . These green revolution wheats were selected for under high-input farming practices which led to a decrease in root biomass . A greater understanding of root traits and how those traits relate to whole plant strategies may enable breeders to increase yields under drought conditions . This understanding can only come by actively studying the root system in a controlled environment and until the relationship of root and shoot traits is better understood we cannot determine how to improve a plants ability to be productive in a fluctuating environment. Roots absorb water and nutrients while also anchoring the plant to the soil. The shoots utilize those resources for photosynthesis and are the site of sexual reproduction. All these functions must work together in coordination for the plant to thrive within its environment. In general plants maintain a fairly strict harmony between shoot and root biomass partitioning . However, during different growth and developmental stages the partitioning of biomass does fluctuate. In the early stages of growth resource allocation and biomass accumulation is focused towards the roots but that shifts considerably as the plant reaches flowering with the major part of photosynthates directed to the shoots . These general principles were supported by Frageria who demonstrated that the root-to-shoot ratio in wheat, as well as other crops, decreased as plants advanced in age. For these reasons it is essential to understand what effect any changes to these general principals may have upon yield within wheat and other crops as well. Increased root biomass increases grain yield under limited or rain-fed environments . This is likely due to the ability of a larger root system to absorb water and nitrogen from the soil; an added benefit is reduced leaching and agricultural run-off . What remains unclear is if increasing root biomass will continue to increase grain yields. This issue has been touched upon in wheat by Maheepala et al. leaving plenty of room for further inquiry and testing.

Other related taxa include uncultured clones from a variety of engineered and natural environments, including: wastewater digesters in France and the United States, estuarine sediments in Taiwan, peat wetlands in Japan, sinkholes in Mexico, and hydrothermal vents in the Pacific Ocean . Whether or not the capacity for DPO is a common feature of this clade remains to be seen, as there is evidence of lateral acquisition of phosphite oxidation genes in FiPS-3 as well as in several APO-capable bacteria . Indeed, whether Phox-21 itself is capable of growth by DPO coupled to CO2 reduction in pure culture has yet to be confirmed, since efforts to isolate this organism have so far proven unsuccessful.The work presented in Chapter 3 revealed the presence in my wastewater enrichments of a novel bacterium from an uncultured clade within the Deltaproteobacteria whose abundance was strongly correlated with DPO activity. Based on this evidence, I hypothesized that this bacterium, designated as strain Phox-21, was the organism responsible for phosphite oxidation in my cultures. However, as my attempts to isolate Phox-21 were unsuccessful, I was unable to confirm that it was capable of carrying out this metabolism in pure culture. Therefore, I decided to perform a metagenomic analysis of some of the DNA samples I had previously collected for 16S rRNA gene analysis in order to look for possible functional markers of DPO, such as the ptx-ptd genes,grow hydroponic in the genome of Phox-21. In addition to establishing a more conclusive link between Phox-21 and DPO, I also hoped that genomic profiling of this organism would reveal its broader metabolic characteristics and provide insights that could aid in its isolation and culturing.

Furthermore, I expected the metagenomic dataset to shed light on the metabolic capabilities of other enrichment community members, thus providing a wider ecological context for the role of DPO in this system.Coates Genomics Sequencing Laboratory at UC Berkeley for sequencing on an Ilumina HiSeq 2000 . Ilumina sequencing reads were trimmed for quality and filtered using Sickle v1.33 with a quality threshold value of 28 and good-quality paired-end reads were then merged using IDBA-UD v1.0 . Merged reads from all samples were combined and assembled using MEGAHIT v1.0.2 with default parameters . MEGAHIT is an assembler developed specifically for metagenomic reads that uses succinct de Bruijn graphs and an iterative multiple k-mer size strategy. Merged reads from each sample where then mapped backed to the combined assembly using BWA-MEM v0.7.10 with default parameters in order to assess sequencing coverage . Contigs from the combined assembly were binned into individual genomes using the Anvi’o v1.1.0 platform . Anvi’o generates hierarchical clusters of related contigs using both tetranucleotide frequency and coverage across samples as the clustering parameters. The platform also provides a visualization interface that allows the user to further refine the contig clusters into genome bins based on coverage, GC content, and phylogenetic marker genes. Furthermore, Anvi’o assigns taxonomic lineages to the genome bins based on the presence of phylogenetic marker genes. Genome bins generated with Anvi’o were subsequently assessed for completeness and contamination based on the presence of lineagespecific, conserved, single-copy marker genes using the automated bin evaluation tool CheckM v1.0.1 . CheckM calculates ‘completeness’ based on the number of expected marker genes that are present in a given bin and ‘contamination’ based on the number of marker genes that are present in multiple copies and have less than 90% amino acid identity to each other.

High-quality genomes were submitted to the Integrated Microbial Genomes database for gene calling and annotation . IMG utilizes Prodigal v2.50 for identification of protein-coding genes, which are then functionally annotated using a custom, manually-curated pipeline based on BLAST and HMMER searches against multiple protein databases .The presence of a ptx-ptd gene cluster in the genome of Phox-21, as well as its higher abundance during phosphite-oxidizing conditions, clearly indicates that this is the organism responsible for DPO in our enrichments. In addition, the observed CO2 dependence of DPO in our enrichments coupled with the fact that no other terminal electron acceptors were added to our media implies that Phox-21 is capable of growing by coupling phosphite oxidation to CO2 reduction. The presence of a formate dehydrogenase complex FdhAB similar to that of M. thermoacetica in Phox-21 provides a putative means by which CO2 reduction could occur. However, the absence of key WLP genes suggests that this organism is unable to generate acetyl-CoA from CO2 alone and therefore is not a true autotroph. Furthermore, it lacks an electron transport chain and thus appears to be incapable of energy conservation through oxidative phosphorylation. Instead, we propose that Phox-21 couples phosphite oxidation to CO2 reduction to formate by means of FdhAB and uses the energy generated by this reaction to assimilate organic carbon sources such as acetate . Based on thermodynamic calculations and physiological evidence, Schink et al. have previously proposed that FiPS-3 is able to conserve energy during DPO by directly generating ATP as well as NADH from the oxidation of phosphite. This putative substrate-level phosphorylation step during DPO is likely mediated by PtdFHI and would allow for energy conservation in the absence of membrane-associated electron transport .

ATP produced in this manner could be used by Phox-21 to incorporate acetate into biomass via AcsM and the partial TCA cycle as well as to run the proton and sodium translocating ATP synthases in reverse in order to establish an ion motive force across the cell membrane . The resulting sodium ion gradient could drive the RNF complex to reduce ferredoxin, which could then serve as an electron donor for pyruvate synthesis by the PFOR enzyme as well as for NADPH production by the NfnAB complex . The FocA transporter could serve both to import acetate for assimilation and to export formate from the cell as a metabolic waste product . However,mobile grow system in the presence of nitrite, formate could be re-oxidized to CO2 by FdoGHI coupled to the reduction of nitrite by NrfAH . This reaction would contribute to the maintenance of a proton motive force and would also yield ammonia, which could be imported into the cell via the AmtB transporter to serve as a nitrogen source. Our metabolic model predicts that Phox-21 should require an organic carbon substrate such as acetate for growth and should also excrete formate into the medium as a product of CO2 reduction. A requirement for organic carbon may at least partly explain the stimulatory effect of rumen fluid on DPO, since rumen fluid has been shown to contain as much as 60 mM acetate in addition to various carbohydrates, organic acids, amino acids, and fatty acids . Additionally, Phox-21 is predicted to be incapable of synthesizing alanine, histidine, threonine, or THF, all of which were absent from our original growth media but may be present in rumen fluid. However, attempts to grow our enrichments in media supplemented with acetate, THF, and amino acids but lacking rumen fluid have so far resulted in substantially lower phosphite oxidation rates, indicating that there may be other components in the rumen fluid that promote the growth of Phox-21. Acetate present in rumen fluid amended cultures could also have served as a growth substrate for the Tepidanaerobacter strains and methanogens present in the communities.

Both Tepidanaerobacter genomes have all the genes for the carbonyl branch of the WLP but lack a formate dehydrogenase and an uptake hydrogenase, which is consistent with previous genomic analysis of Tepidanaerobacter acetatoxydans and indicates that these organisms are capable of syntrophic acetate oxidation but not of autotrophic growth . Likewise, both Methanoculleus sp. EBM-46 and Methanococcoides sp. EBM-47 appear to be capable of acetoclastic methanogenesis, although only EBM-46 has the genes necessary for growth on H2/CO2 and formate as well. The remaining enrichment community members had no WLP genes and mostly lacked genes involved in respiratory processes and were therefore likely involved in the fermentation of organic acids, amino acids, and carbohydrates present in the rumen fluid. DPO was discovered over a decade ago in the marine sediment isolate D. phosphitoxidans FiPS-3, but there had so far been no additional reports of this metabolism in other environments. This study, therefore, represents only the second ever observation of DPO and the first ever description of a community structure and metagenome related to this metabolism. Furthermore, the organism responsible for phosphite oxidation in our system, Phox-21, is a novel bacterium belonging to a candidate order within the Deltaproteobacteria that currently has no cultured representatives. Although my attempts to isolate Phox-21 were unsuccessful, metagenomic analysis revealed the presence of a ptx-ptd cluster in its genome, similar to the one found in FiPS-3. Previous work has shown that ptxD and ptdC are necessary for phosphite oxidation in FiPS-3 . Additionally, as part of this study, I found that ptdFGHI are significantly upregulated in FiPS-3 in the presence of phosphite. That ptdCFHI are also found in Phox-21, but not in any other genome currently available in the IMG database, is further evidence that these genes play an important role in DPO. Interestingly, ptdG is present in FiPS-3 but not in Phox-21, which suggests that this gene may not be essential for DPO in every organism, although it may still be required by FiPS-3. However, much work is still needed in order to elucidate how energy for growth is conserved during DPO. The involvement of PtdFHI in this process has yet to be experimentally confirmed and the mechanism of action of these enzymes has not been determined. Whether ATP is indeed produced from substrate-level phosphorylation during DPO is another outstanding question that requires additional experiments, both in vivo and in vitro, in order to conclusively address. It is assumed to function as a phosphite/phosphate antiporter based on its homology to known antiporters, but this assertion still needs to be tested. The presence of an incomplete Wood-Ljungdahl Pathway in Phox-21 was unexpected, since we had previously assumed that it was capable of growing autotrophically. Nonetheless, this predicted requirement for organic carbon could at least partially explain why the growth of Phox-21 improved so markedly when rumen fluid was added to the enrichments. Although there have been previous reports of organisms with incomplete WLPs , to our knowledge, this study provides the first genomic evidence of an organism capable of using CO2 as a terminal electron acceptor but not as a carbon source. Due to its extremely low redox potential, phosphite is the only known biological electron donor that could drive the reduction of CO2 to formate while generating enough energy to produce ATP for the incorporation of acetate into biomass. As such, this unprecedented metabolism may be unique to DPO-capable organisms. However, as discussed above, the feasibility of this proposed metabolism hinges on whether or not DPO can generate ATP through substrate-level phosphorylation, which is still an open question. Still, the apparent lack of any electron transport chain components in Phox-21 suggests that under phosphite-oxidizing, CO2-reducing conditions, energy conservation in this organism would have to proceed exclusively by means of substrate-level phosphorylation. Ultimately, though, this metabolic model still needs to be experimentally confirmed. I have so far been unable to detect significant formate production or acetate consumption under DPO conditions in my enrichments. However, it may not be possible to detect these processes in enrichment cultures due to the presence of other community members capable of using formate and producing acetate. Obtaining a pure culture of Phox-21 would therefore greatly facilitate any future investigations of its physiology. The environmental prevalence and phylogenetic diversity of DPO-capable organisms remains unclear. Like FiPS-3, Phox-21 belongs to the Deltaproteobacteria, but the sample size of known DPO-capable organisms is still far too small to allow us to determine whether this metabolism is restricted to a specific phylogenetic group. There is evidence that the ptx-ptd genes in FiPS-3 were acquired by lateral gene transfer, but we do not know how common it is for these genes to be horizontally propagated in the environment or what the phylogenetic range of these events might be.

She outlines three specific opportunities for incorporating climate education into social studies classrooms through tweaks to what is already happening, rather than major curriculum overhauls: 1) in elementary school states and regions studies, where studying the climate of the state or region is already an explicit objective, 2) middle school global geography classes, and 3) in high school current events classes. She sees these as opportunities to “lean into the climate change challenges and how people are addressing them in different contexts” , while minimizing instructional trade offs. The Director concurs, adding, “for independent schools this change is very easy. But for public schools, there’s so much you can do with this curriculum too. If you have to teach about government, geography, or history you can use pieces of this [integrated into pre-existing units and curriculum mandates]” . Preparing teachers to be effective conduits of climate education is a crucial step in humanity’s response to climate change, as “teachers occupy a social role as cultural authority in traditional classroom contexts and are therefore differentially powerful actors in educational spaces” . Teachers are important influencers of what “comes to matter” in the classroom based on their ability to mobilize and communicate their own knowledge effectively . A recent National Academies Board on Science Education report concludes that “ultimately, the ability of the elementary and secondary school systems to provide comprehensive climate literacy education will depend on the systematic availability of quality curriculum resources,berry pots impact of curriculum mandates such as state standards and assessment, and, importantly, the preparation of teachers” .

The Lowell curriculum coordinator is keen to put these recommendations into practice, through co-facilitating a Summer Institute for Climate Education with Climate Generation at Lowell School in Summer 2019. The 3-day institute will be geared towards Humanities educators. In order to scale up and improve existing CCE PD offerings, policy support and funding will be necessary, as well as additional CCE research focused on evaluating PD opportunities and how well they address the needs of both science educators and those from other disciplines. The opportunity to provide integrated Social Studies + STEM PD trainings around climate change is a key area for growth as more states consider adopting climate literacy and education policies. Based on our findings that humanities-focused climate education shows promise in engaging students through narrative, storytelling, and local community projects, and building upon climate communications research that similarly emphasizes a storytelling approach, humanities CCE should be further implemented and investigated through comparative analyses and case studies in other locations that employ a similar climate literacy assessment methodology. This study will benefit greatly from replication particularly in public schools, which are more challenging environments in which to innovate on curriculum content, but more reflective of the U.S. student experience. The time for further study is ripe, with national polls indicating public opinion is strongly in favor of climate education, at 79% supporting the teaching of climate change in public schools . Students receiving the humanities-focused climate curriculum exhibit academic gains in reading comprehension and enthusiasm for the curriculum content. Nonetheless, as seen above, the action/solutions focus can be strengthened by better incorporating authentic and meaningful student climate action projects in the local community. This emphasis on experiential learning may lead to even more effective outcomes as students become empowered to act on their climate concerns.

This calls for additional partnerships and planning in order to implement, and may include field trips, hands-on schoolyard greening projects, contacting local elected officials, and developing community-school collaborations that allow for service-learning projects to have meaningful impacts. Future studies should focus on the student action elements of CCE, and how to most effectively build the education into action pathway, as the “action” piece remains elusive and difficult to measure in many CCE curriculum studies and climate literacy assessments. To investigate this question, I next look at bringing climate education to school gardens and educational farms.This second example of climate education assesses the efficacy of implementing an interdisciplinary, experiential food and climate change curriculum in school garden classrooms. Outcomes of interest include student climate literacy and teacher professional development. Questions of how to teach and research climate education are explored via a participatory teaching + research project with school gardens in Oakland, CA and Lopez Island, WA. Initial results show improvement in student learning and engagement as well as teacher preparation. Both qualitative and quantitative data are analyzed through student surveys, teacher interviews, and site observation; however, further qualitative methodologies to study process of climate and food literacy development are needed. Specifically, this example makes a case for new forms of assessment to capture the multiple dimensions of food and climate literacy including knowledge, engagement, and action/behavior change. It builds on existing climate, environmental, and food literacy research and provides insights for how food systems and climate research can better integrate with K-12 education development.

Building off of expressed desires for implementing climate education in school garden classrooms , this study examines problems of method in two dimensions: methods of delivering climate instruction, and methods of evaluating climate literacy. It is a case of experiential curriculum development and piloting in San Juan County, WA and Oakland, CA. Climate change communications, CCE, and food literacy literatures offer useful strategies for developing students into informed decision makers capable of addressing climate change in their communities . Drawing on the existing bodies of scholarship, the curriculum features climate education activities in the school garden in order to evaluate whether this represents an effective experiential climate education strategy. This study addresses the research questions: 1) What are best practices or effective strategies for delivering climate change curriculum that leads to increases in student climate literacy? 2) How do we currently measure and study climate literacy? And 3) What is the impact on teacher competency and student climate literacy of a 6-week experiential climate curriculum taught in school garden classrooms? Experiential climate change education engages students in hands-on activities and projects that are solution-oriented alongside the presentation of climate science. This approach builds on the best practices of both experiential learning theory and climate change communications by incorporating personal action accompanied by reflection and fostering hope and positive engagement around a complex global issue. By making climate education experiential, it is more salient and actionable for students rather than paralyzing. A school garden is commonly identified by school and district leaders as an effective platform for experiential learning opportunities that also can boost academic performance, attendance, behavior, and student health . Garden educators often recognize the climate benefits of local food production, but this connection is seldom passed along to student gardeners. Thus, gardens were chosen as the context of study for implementing an experiential climate curriculum. The pilot schools represent “early adopters” of climate-friendly schoolyards and climate change curriculum,hydroponic grow system which is not yet widespread in the U.S. . I use a small sample size to generate further hypotheses on what strategies work for bringing student beliefs, attitudes, and behaviors in line with well-defined actions/solutions for climate mitigation. The case studies are therefore an opportunity to learn from schools where climate education shifts are already underway, one in a rural agricultural setting and the other school district in a progressive urban environment known for food justice activism. Both contexts are examples of high community awareness of climate change : thus, if particular CCE strategies cannot succeed in these contexts, the chances for their widespread success are low.They are the top-performing district in Washington state for farm to school activities according to the USDA annual Farm to School Census .

The school garden at the Lopez Island School is a thriving agro-ecological example of a ½ acre food production center that meets most of the fruit and vegetable needs of the school cafeteria, producing over 6,000 pounds of produce annually. However, in-school efforts on integrated energy and climate change education are a self-identified area for improvement. The Lopez school site became a pilot case for the food and climate curriculum based on results from a prior research study on the district’s Farm to School program . Through surveys and interviews with school leaders in spring 2016, I identified climate change curriculum intervention goals in collaboration with teachers, administrators, and students. Follow up focus groups provided the impetus for coteaching a food and climate curriculum in collaboration with the garden teacher. In spring 2017, I implemented a curriculum pilot in the Lopez Island Sustainable Practices classroom for students in grades 8-10. The curriculum pilot was accompanied by pre- and post-surveys for student climate literacy, as well as a teacher interview to debrief the co-teaching model of instruction .Oakland Unified School District is a national leader in the school lunch reform movement. The district is working towards establishing school gardens at all schools, as well as a Central Kitchen and urban farm to provide centralized distribution of locally grown produce to school cafeterias. However, like the San Juan Islands, leadership in school food systems and local sourcing is not yet accompanied by corresponding leadership in climate change education. There is interest at the district and school level to incorporate climate change into school garden classrooms and more thoroughly across multiple school subjects, but preliminary action steps are just beginning. Partnerships with community groups and local universities are seen as a desirable and realistic way to incorporate climate change into both science and garden classrooms. In consultation with the OUSD School Gardens Coordinator and the University of California Cooperative Extension Bay Area Urban Agriculture Advisor, I identified 3 schools for a climate change curriculum pilot in spring and fall 2017. The pilots were assessed with the same student survey and teacher interview guide used at the Lopez school. Pre-implementation interviews with educators and students in the San Juan Islands and Oakland Unified School District show that there is a strong interest in incorporating climate change into school garden educational curriculum, accompanied by a need for training in order to do so effectively. Adults often recognize the climate change connection to their farm to school activities but acknowledge that students are not yet taught about these connections. Adding to the education-action gap, teachers often do not feel qualified to teach students about climate change without being content experts themselves. Preliminary program evaluations and discussion themes at National Farm to School conferences indicate that the education core element is lagging behind the other core Farm to School program elements: presence of school gardens and local procurement . The lack of comprehensive or strategic integration of garden based education into broader environmental and climate educational contexts leaves many garden educators searching for curriculum independently and not utilizing garden classroom time to its full potential; climate change is a glaring omission in all garden curricula and evaluative studies examined for this research project. Furthermore, integration of program evaluation methodologies and consistent tracking of farm to school activities remains a challenge for researchers and practitioners .Figure 20 summarizes the chain of activities relevant for this study, which addresses method in two dimensions: pedagogical method and evaluation method . The methods for curriculum development followed processes common to teacher training programs, and incorporate best practices from both critical pedagogy and experiential learning theory. Critical and experiential pedagogy places the teacher as designers of the educational experience, as coaches or facilitators, and students as leaders of their own learning. Both teachers and students have agency to ask and answer questions, and outcomes are necessarily more fluid and less predictable than pedagogy that lends itself to standardized test-taking. Importantly, critical pedagogy implies an embedded project of unearthing and subverting oppression . The curriculum content is the product of collaboration and feedback with education nonprofits and partner teachers, following participatory action research principles. Initial ideas and activity outlines emerged following interviews and focus groups with school garden educators in the San Juan Islands in spring 2016, as well as conversations with Oakland garden educators in fall 2016. A key partner in the curriculum development process is the nationally recognized nonprofit Climate Generation: A Will Steger Legacy, the source of the climate curriculum that was modified to provide a food/ag frame around the six-lesson structure.

The concept of a food shed in the distribution literature, “like its analogue the watershed, can serve as a conceptual and methodological unit of analysis that provides a frame for action” . Food shed analysis “provides a way to assess the capacity of regions to feed themselves” through proximate location of food production, distribution and consumption . Applying this concept, Peters et al. found that 34% of New York State’s total food needs could be met within an average distance of 49 miles. The food shed, embedded in the local food systems and short food supply chain concepts, is a useful organizing principle for city planners to consider when designing effective food distribution networks, such as the example highlighted in integrating a farm into a housing development project in the South under the title of a “civic agriculture community,” facilitating proximate, affordable distribution channels. This exemplifies planning with a food shed lens by specifying areas at the neighborhood scale for semi-commercial agriculture, neighborhood CSA, residential kitchen gardens, and residential development in order to build food access and ease of distribution into the neighborhood fabric. If urban farmers aren’t able to easily distribute their produce to consumers, either through sales or other forms of distribution, questions of improving food access are jeopardized as well, revealing the inter connectedness of the food systems framework from production to distribution to consumption. Planning for improved urban food distribution includes ideas such as food hubs, agri-hood developments,vertical farm system public storage and transportation options, food aggregating facilities or organizations, mobile food distribution, or state investment in public markets .

Mobile food distribution options are modeled and shown to increase access in Buffalo, NY, in Widener et al.’s theoretical analysis . Agri-hoods have gained increasing mention in local news outlets as a real estate trend in “Development Supported Agriculture , and as many as 200 currently exist or are under construction across the country” . They facilitate distribution by colocating food producers and consumers on strategically planned sites, providing shared infrastructure resources, and making land access affordable for farmers by cross-subsidizing with real estate development. Cooper’s report on food hubs in the south, a form of aggregating supply to enable expanded market access, highlights grassroots solutions developed by and for farmers of color, yet “major challenges [remain] associated with developing and maintaining food hubs within a racial equity framework” . Here again, the Google Alerts provide useful insights from gray literature and local news outlets into recent and effective strategies for city planners, be it food hubs, mobile food distribution options, online platforms for gleaning, second harvest, crop swaps, or distributing excess produce from backyard gardens. These are also areas that stand to benefit from additional scholarly research in terms of quantifying impact on consumption, food insecurity, and nutrition, expanding evaluations of urban food systems to include non-monetary and informal distribution mechanisms. Integrating the access and distribution literature from above, we identified three themes that speak to the efficacy of urban agriculture in meeting food access goals: economic viability, policy and planning models, and civic engagement.In this section, we consider the economics of urban agriculture and the “economic marginalization” that prevents many operations from meeting all the social and environmental benefits of urban agriculture within a for-profit or capitalist-oriented production scheme.

The urban food justice and food sovereignty movements in the U.S. are limited in practice in achieving their more radical or transformative goals due to the fact that they are operating within “a broader framework of [capitalist] market neoliberalism” . The challenge has not been growing enough food per se, but rather “producing and distributing food in ways accessible and affordable for the growing urban poor” while sustaining UA operations in a capitalist, production- and profit-oriented society. Daftary-Steel, Herrera and Porter declare that an urban farm cannot simultaneously provide jobs to vulnerable individuals, provide healthy food to low-income households and generate sustainable income and/or profits from sales. Therefore, what forms of urban agriculture are economically viable in today’s political economy? Operations that provide jobs, job training and professional development but sell mostly to high-end consumers , operations that are volunteer-driven or publicly funded and operations that cross-subsidize healthy food donations with revenues generated from other services besides food production or from crowd-sourced funding . When it comes to economic viability, many urban farming operations openly acknowledge that they are dependent on grants and donations to sustain their operations, which is a double edged sword. On the one hand, as long as an organization can prove itself worthy in receiving grants and donations, it may represent economic viability and long term sustainability. On the other, if the organization is wrapped up in a charismatic individual leader or fails to receive ongoing grant injections beyond one or two initial successes, it will not achieve long-term economic viability.Examples include redistributive business models, barter and exchange networks, food aggregators, food recovery organizations, cooperatives, food hubs, and “agrihoods” . Food hubs are reframed as both tools for provision of market access and self-determination for black farm cooperatives in the South in Cooper’s report with potential to subvert historic racism and economic marginalization of black farmers.

Key to this and other food policy reports in the gray literature is elevating voices and fostering dialogue led by communities of color.While food, and urban agriculture, used to be “strangers to the planning field” or “puzzling omissions” from American Planning Association resources prior to the early 2000s , there has been an increase in academic work in the past 10 years dealing with urban food systems planning. In this section we consider the policy landscape of various city and state efforts to incentivize and create space for urban agriculture. Policy is needed to lower costs for low income consumers and urban farmers seeking land, provide strategic location of distribution sites, and encourage year-round produce supply, often enabled by greenhouse systems in urban farms. Are current policy incentives enough to create expanded food access and community food security from urban farms? Horst et al. would argue no; rather, an explicit commitment to food justice and an “equity lens” is needed for policymakers and planners to create UA spaces that benefit low income and minority communities equally if not more than already advantaged groups. Due to the current landscape of “disparities in representation,vertical indoor farming leadership and funding, and insecure land tenure,” unless these problems are explicitly addressed, “even the most well intentioned initiatives will perpetuate or even reinforce the injustices that practitioners and supporters aim to address” . This sentiment is echoed in Morales’ chapter in Cultivating Food Justice, which calls for “applied research to discover and advance policy objectives related to the antiracist and economic objectives espoused by the Growing Food and Justice Initiative” . This suggests that only by foregrounding issues of race and economic inequality can cities create UA spaces that address food insecurity. In asking the question “Can cities become self-reliant in food?” Grewal and Grewal find that, in a best-case scenario, the City of Cleveland can achieve almost 100% self-reliance in fresh produce needs, poultry and eggs, and honey, but only with huge amounts of planning support . Blum-evitts puts forth a foodshed assessment tool to allow planners to assess local farm capacity in relation to local food needs . Theoretical work such as this is important to advance ideas of what is possible and motivate efforts to make change, although it must constantly stay in dialogue with what is happening in practice and expand beyond a productivist focus on local food systems. Urban farms are, after all, producing a lot more than food, and “increasing food production in cities does not guarantee that people experiencing food insecurity will access that food” . UA is re-valued along a broader spectrum of “products” or outputs in Figure 12 below.Creating urban agriculture incentive zones is one possible approach to policy and planning, likely to benefit the propertied class via tax breaks . Policies such as California’s AB 551, the Urban Agriculture Incentive Zone act, have come under criticism for not going far enough to build a just food system, relying on private rather than public spaces to support UA. It is unclear whether incentive zones will be widely adopted by cities and counties in California, and whether they will meaningfully address food access or food sovereignty, especially when the length of time required to devote a piece of land to urban agriculture is only 5 years.

In cases where tax incentives are used to promote urban agriculture, primary beneficiaries of the policy are often the privileged class of property owners rather than low-income households or non-property-owning urban farmers. Cities with some sort of food policy regulating, allowing for or promoting urban agriculture include the City of Baltimore , City of Somerville, Detroit, Portland, Madison, Seattle, San Francisco, San Jose, Los Angeles, San Diego, Austin, Minneapolis, New Orleans, Milwaukee, Boston, and Chicago12. Many policies allow for commercial sales of urban-produced food within the city as “approved sources” ; allow for value-added processing and sale of urban produced foods in people’s home kitchens ; create tax incentives for property owners to convert land into urban farms ; amend zoning regulations ; or set up urban beekeeping pilot projects . The Baltimore example is especially noteworthy for its long-term planning approach to structuring urban agriculture into the landscape of the city, with considerations for equity in place. However, legalizing the ability to grow food in cities is not enough to promote equity and justice, nor resolve all the legal conundrums related to compliance with the terms of legislation . Creating incentive zones for certain types of UA practices is not the same as creating supportive policies to allow and encourage the existence of the diverse array of practices and practitioners that constitute UA. Especially in cities with growing population and housing pressures , particular attention must be paid in policy making to avoid advancing gentrification and displacement. This is less of a concern in cities without such housing pressures , but development is always a threat that must be considered when siting urban farms on private land. A promising policy direction pioneered by the City of Seattle is to dedicate public lands in low income neighborhoods to UA, which Seattle does through its P-Patch program. Other policy recommendations gleaned from the literature include: creation of a citywide UA task force with citizen representatives; efforts to tie in local “good food” policies with city Climate Action Plans to promote UA and alternative food waste management alongside climate benefits13; devote public lands to urban farms and gardens in perpetuity; “retrofit” affordable housing developments with community gardens ; provide public storage, transport, and aggregation options for urban farmers; and convert corner stores into neighborhood groceries offering fresh produce from local farms. Many of these efforts have potential to address many city priorities at once, for example: food access, nutrition and fitness, transportation, community development and crime reduction . Providing land access for low-income and minority farmers is an important step towards ensuring a food supply that is culturally appropriate, desirable, and marketable to food insecure urban communities. By publicly confronting land insecurity and tenure arrangements, policymakers can directly respond to research on UA’s uneven development .Smaller numbers of citizens are becoming involved in advocating for UA policies and improved zoning regulations that support food access goals, holding cities accountable to UA projects. Through direct participation, citizens are already voting with their feet in favor of UA initiatives . Existing literature states, “‘participants in a community garden continually express a heightened sense of self-esteem gained from sharing knowledge and skills with each other.’ Such community connections can, in some cases, lead towards participation at the larger [policy] level” . By expanding civic engagement into the local policy realm, it is more likely that sites designated, set aside, or incentivized for urban agriculture development will be strategically located, address food insecurity and food justice concerns, and provide long-term access for UA . Civic engagement can take many forms, including participating in neighborhood organizations, contacting elected officials and city councilmembers to communicate multiple values of UA, aligning UA with existing city plans/ordinances, or participating in food policy councils. Citizen volunteers are participating in building community economies, often non-capitalist and non-exploitative in nature .

Distributing the risk, responsibilities, and knowledge-intensive labor among partners is a yet-to-be-thoroughly-tested strategy for overcoming some of the land access challenges facing farmers in the San Juan Island region.Agroecology rests upon an essential foundation of building healthy soil, through ecological cultivation of plants, insects, and food webs governed by the “Law of Return” creating a rich network of life on the farm. On Lopez, land clearing for farming, homesteading, and haying posed a threat to the island’s biological and pedologic resource base starting in the late 19th century with the arrival of European-Americans. Today, there is growing attention around restoring and revitalizing soils, forestland, and ecosystem services. Farms such as Midnight’s Farm are managing land for three purposes: healthy food production, economic viability, and soil carbon storage . Other farms are following suit, seeking to build soil and revitalize land that has been degraded especially from repeated haying. The soils on the island vary across short distances, from sandy and well-drained hilltops to heavy clay and moisture-retaining wetlands. The island geology is mostly rock, with a thin soil layer, not considered ideal for farming activities. In the words of one farmer, “we don’t have much rich farmland for row crops on Lopez, so most of us are in a constant dance to balance income-producing crops with inputs to improve the soil and, therefore, the harvest” . Farmers and ranchers are involved in a suite of soil-building practices out of necessity for maintaining productive small-scale operations year after year. These practices include compost production and application, cover cropping,vertical grow rack system bio-char production and co-composting, crop rotations, intercropping , managed rotational grazing, minimal- or no-till cultivation, and combinations of perennial and annual plantings with animals to create a diverse ecological farming system that takes less than it gives back to the ultimate life-source: the soil.

Farmers receive support, training, and information from researchers at WSU SJC Extension, SJICD, and through annual farmer to farmer workshops. Several farmers collaborated in 2015 to host a visit from the Soil Carbon Coalition’s Peter Donovan in order to sample local soils as a baseline and collect additional samples in later years to measure carbon storage, an important component of soil health. WSU researchers offer regular guidance and workshops around crop rotations and pasture management to improve island soils. Recently, WSU partnered with local farmers and the local bakery to host a Field Day on small scale grain production, part of a soil-building rotation that can enhance fertility in concert with legumes and other crops. Other WSU researchers collaborated on a successfully funded Western Sustainable Agriculture Research and Education grant proposal with five local producers to explore the use of bio-char co-composted with cattle bedding and other woody biomass materials as a soil amendment, which will be applied in experimental trial plots beginning in summer 2020 . The SJICD received a WA State Department of Ecology grant to purchase a no-till seed drill that is shared among islands, and recently used in a sequence of liming and seeding Lopez pastures with diverse seed mixes to restore grassland soil health. While the support and education provided by local agriculture and conservation organizations is essential, there is a constant need for further financial resources to extend and improve educational initiatives and technology pilots.Related to efforts of building healthy soil through crop rotations and appropriate farming decisions, farmers on Lopez are taking steps to provide their own inputs for crop production that do not need to be imported or purchased from off island.At a Department of Ecology-approved facility, the farm produces compost from forest and agricultural debris dropped off from across the island, grinding, composting, and screening materials in an aerated static pile system to create a finished product that is widely applied to local croplands.

Manure and bedding material from the farm’s cattle, pigs, and chickens are valuable feed stocks to the composting process as well. Midnight’s produces over 600 yards of compost annually, which is all applied to Lopez agricultural lands and gardens. Farms also self-compost, recycling waste products in smaller decentralized systems and supplementing with purchased composts. Animals also play a role: “Our pigs really close the loop for us on the farm,” one farmer stated, referring to food and plant scraps she was feeding to her American Guinea hogs who were in the midst of transforming it into high quality meat . More recently, due to wildfire risk mitigation efforts, the island has begun to selectively remove and burn some trees in a controlled, limited oxygen environment to create local bio-char, a potentially valuable soil amendment with implications for increased soil carbon sequestration. Current production is happening at a very small scale, but regional interest in larger-scale bio-char production abounds. Midnight’s Farm initiated a research collaboration between WSU extension, U.C. Berkeley, and five local producers from across Western Washington to address the question: can bio-char be a multi-use farm product that improves farm-based co-composted products and vegetable production, and promotes soil C sequestration? Two regionally sourced bio-chars will be applied to cattle bedding at Midnight’s Farm, and then the bio-char-bedding will be co-composted with other on-farm feed stocks to produce a bio-char-enhanced compost product. Through absorbing Nitrogen and other nutrients from the cattle bedding, the “charged” bio-char is intended to provide valuable fertilizer-like qualities to the compost, reducing the need for other amendments to cropping fields. The research hypotheses are: 1) blending bio-char into cow bedding will result in greater N retention, reducing the potential for environmental loss, 2) adding the bio-char bedding blend to compost will increase nutrient content, thereby adding value to the compost product, and that 3) compost with bio-char as a feed stock will lead to increased soil carbon, cation exchange capacity, and pH when applied to soil . The research team will measure impacts on manure handling, composting, soil quality and crop yields, following field application trials on two local farms . Data will be collected in Spring 2020 on soil profiles before amendment, and again in Fall 2020 on soils and crop yields.

The research underway is based on prior work from local bio-char researcher Kai Hoffman-Krull and others, who have worked with universities in Washington and Montana over the past five years investigating on-farm bio-char soil amendments. They have found through field trials on nearby Waldron Island, WA, that in addition to improving soil C storage, locally produced bio-chars have potential to “significantly improve soil fertility and crop productivity in organic farming systems on sandy soils” . However, there remains controversy around the impacts of bio-char in disparate contexts, evidenced by several meta-analyses pointing out varied outcomes based on pyrolysis and feed stock conditions,mobile vertical grow racks and differential effects of in temperate vs. tropical soils .Pending outcomes of the local study on Lopez and across Western Washington, best practices for creating a locally sourced “complete” soil amendment could be scaled regionally, minimizing “external inputs” on a growing number of small-scale organic farms. The goal of minimizing external inputs extends from farmers to others in the food supply chain, including island bakers of Barn Owl Bakery. Rather than purchase bulk inputs like sugar and wheat for their baked goods, Sage and Nathan are actively pursuing the local cultivation of grains and sugar beets to create their own 100% organic island grown products– sprouted Lopez wheat locally milled into flour for wild leavened breads, fruit scones, flat breads, and weekly specialties incorporating other island grown ingredients. Their work is also supported by local researchers from WSU Extension and a Western SARE grant to understand the impact of seeding rate and fertilizer application on the agricultural performance and baking quality of land race wheat. The goal of local input sourcing is also local waste management and reuse of waste as inputs into other ecological processes. Outputs from some farms become inputs for others, in a cost-minimizing closed-loop cycle for those involved.Transitioning food systems to agroecological practices will not be possible without investing in the “re-skilling” of an agroecological workforce.

Lopez has a series of educational offerings in place to reach a variety of audiences from K-12 students to beginning and current. At the farmer-to-farmer level, Lopez farmers engage in regular meet ups and events, including the monthly farmer coffee. On the second Wednesday of each month, Lopez farmers gather at the Lopez Grange for an hour of information and resource sharing. Organized in 2019 by Faith Van de Putte, the forum is a meeting of the minds and transactional space for connecting problems with solutions, questions with answers. Where do people get good, affordable organic chicken feed? Who has straw for goat bedding? How do you get rid of persistent weeds like thistle and morning glory? Do deer get into the grain fields through the electric fence? How can we arrange for annual small animal vet clinics to provide appropriate care for our sheep, goats, and pigs? Disease and pest identification and management topics swirl around room, some finding mostly empathy, and others finding a speedier resolution. At a September 2019 coffee, several farmers shared positive results from experimenting with a new Organic Materials Review Institute -approved herbicide called “Weed Slayer,” said to be effective against the pernicious thistles. Underlying these informational exchanges is the challenge of continuing to run the iconic, diversified small farms of Lopez, lauded as beacons of sustainable agriculture and agritourism, yet requiring countless hours of hard work, determination and passion. Lopez farmers recognize that they cannot “go it alone” on their small farms and rely on the support of other farmers as well as researchers from WSU Extension. Two county extension agents were present at a recent coffee gathering to generate a list of future workshop and clinic topics to offer for farmers, as well as to gauge interest in collaborating on planned future research experiments, grants, and educational demonstrations. In addition to educating each other, Lopez farmers educate aspiring farmers primarily through the Lopez Community Land Trust Sustainable Agriculture internship program. Each year on average five interns live and work on one of the islands six main educational farms, learning from the farmer how to seed, transplant, weed, water, and regeneratively farm diverse vegetable varieties and care for animals such as chickens, sheep, pigs, and cows. These interns select several readings and a documentary to discuss with other interns under supervision from Land Trust staff. Interns complement the practical and hands-on skills of farming with bigger picture reflection and dialogue about ideal vs. real food systems, connecting the production element to all the other moving parts of the system. According to the internship program director, the three biggest takeaways for participants are 1) importance of good local food, 2) basic life skills and 3) the experience of living in community. It is an “empowering experience;” however, it is not a formal or comprehensive beginning farmer training program and has thus far not led to the transition of farmland from an aging farmer to a former agricultural intern. There are additional opportunities for young farmer mentor ship through a Beginning Farmer and Rancher Development grant where more experienced farmers receive funds to support and mentor younger farmers as they begin their own operations. This is geared towards new farmers who have already taken steps to start up operations on Lopez or other islands. All farmers, new and old, have a recurring opportunity to learn more about evolving farm practices at the annual San Juan Agriculture Summit , which rotates between Lopez, San Juan, and Orcas Island, and is held in February each year. The Ag Summit began nine years ago at the impetus of the Agricultural Resource Committee and now WSU Extension has taken on the primary organizing role. Topics presented are wide ranging, from soil health to business and marketing to climate change, and feature speakers from all over the Western United States. The Ag Summit is a social as well as educational event, bringing farmers together for dining, dancing, and community building. On Lopez Island, farm to school programming is run through the Lopez Island Farm Education program. It began as a collaboration between LCLT, the Lopez Island School District, Lopez Island Education Foundation, the Family Resource Center, S & S Center for Sustainable Agriculture, the SJI Conservation District, WSU SJC Extension and the Heller Family.

Total soil C and N were strongly associated with EOC and EON, the soil C:N ratio, and POXC. These variables had negative values along axis 2 and thus contrasted with the pattern of soil inorganic N. Weak loading of AMT1.1, NRT2.1, Nii, and GS2 on the first two principal components reflects the lack of association of expression levels of these genes with biogeochemical and plant variables. Non-overlapping confidence ellipses for seven out of 13 fields on the PCA biplot indicated distinct N cycling patterns . Fields 1 and 2, with the highest values along axis 1, had low values of all variables included in the analysis. Field 4 had the highest values along axis 2 corresponding with higher soil NH4 + and NO3 – . Fields 10, 11, 12, and 13 were associated with high values of labile and total soil C and N. Overlapping confidence ellipses of fields 3, 5, 6, 7, 8, and 9 close to the origin indicate similar, moderate values of this suite of variables for these fields. Three groups of fields were identified by k-means cluster analysis of the same 28 variables included in the PCA . Group 1 included fields 1 and 2, which had low mean values for yield , the lowest mean soil C and N and soil inorganic N pools , and the lowest mean value of GS1 relative expression in roots. Groups 2 and 3 had similarly higher mean yield , shoot N, and petiole NO3 – than group 1, but these two groups differed substantially in their soil C and N pools. Group 2 had higher soil NH4 + and NO3 – pools as well as root expression of AMT1.2 while group 3 had higher total and labile soil C pools. Expression of GS1 was similar in both groups. Based on the relative magnitude of F-statistics calculated for each variable,vertical indoor hydroponic system soil C and N, EOC, EON, shoot N, and soil NO3 – at transplant and anthesis were most strongly differentiated across the three groups.

The high F-statistics of AMT1.2 and GS1 relative to other N metabolism genes indicate that root expression of these genes are most responsive to soil N cycling.This study confirms that working organic farms can produce high yields with tightly-coupled N cycling that minimizes the potential for N losses. Such farms had the highest soil C and N and used high C:N organic matter inputs coupled with labile N inputs that resulted in high soil biological activity, low soil inorganic N pools, high expression for a root N assimilation gene, adequate plant N, and high yields. Organic systems trials have previously shown crop N deficiencies that lead to less-than-ideal crop productivity; losses of N when Navail ability is poorly synchronized with crop N demand; or alternatively, that organic production can reduce N losses. But how working organic farms achieve yields competitive with high-input conventional production with low potential for N losses has not been demonstrated. Elevated expression of a key gene involved in root N assimilation, cytosolic glutamine synthetase GS1, in fields with tightly coupled N cycling confirmed that plant N assimilation was high when plant-soil-microbe N cycling was rapid and inorganic N pools were low, thus showing potential as a novel indicator of N availability to plants. Improving biologically-based farming systems will benefit from research that uses novel tools to uncover innovations happening on farms, especially if the research process helps facilitate knowledge exchange among farmers and researchers.To characterize the substantial variation in crop yield, plant-soil N cycling, and root gene expression across 13 fields growing the same crop on similar soil types, we propose three N cycling scenarios: “tightly-coupled N cycling”, “N surplus”, and “N deficient”.

Values of indicator variables suggest differing levels of provisioning, regulating, and supporting ecosystem services in each scenario . Fields in group 3 show evidence of tightly-coupled plant-soil N cycling, a desirable scenario in which crop productivity is supported by adequate N availability but low potential for N loss. Despite consistently low soil NO3 – pools in these fields, well below the critical mid-season level for conventional processing tomatoes in California, total above ground N concentrations were very close to or only slightly below the critical N concentration for processing tomatoes. Tomato yields were also above the county average . This discrepancy between low soil inorganic N pool sizes and adequate tomato N status is due to N pools that were turning over rapidly as a result of efficient N management, high soil microbial activity, and rapid plant N uptake. Composted yard waste inputs with relatively high C:N ratios in concert with limited use of labile organic fertilizers applied during peak plant N demand provided organic matter inputs with a range of N availability. A companion study showed how high potential activities of N-cycling soil enzymes but lower activities of C-cycling enzymes in this set of fields reflect an abundant supply of C but N limitation for the microbial community, thus stimulating production of microbial enzymes to mineralize N. Plant roots can effectively compete with microbes for this mineralized N, especially over time and when plant N demand is high. High root expression of GS1 in these fields indicates that root N assimilation was elevated and thus actual plant N availability and uptake was higher than low inorganic N pools would suggest . Fields from group 2 demonstrated N surplus, showing similar yields to group 3 but with lower total and labile soil C and N and a higher potential for N losses, given much higher soil inorganic N . While actual N losses depend on a host of factors , high soil NO3 – is considered an indicator for N loss potential. Results from a companion study support the idea that soil microbes were C rather than N-limited in these fields, showing higher potential activities of C-cycling soil enzymes but low activities of N-cycling soil enzymes, the inverse of group 3 .

An alternative multivariate clustering approach based on an artificial neural network suggests multiple potential drivers of higher inorganic N pools in these fields, including both management factors and soil characteristics . For instance, field 4 had strong indications of surplus N driven at least in part by a large application of seabird guano , a readily-mineralizable organic N fertilizer, at tomato transplanting when plant N demand is low. In contrast, higher inorganic N in field 8 was likely driven by low plant N demand based on very low soil P availability, which resulted in plant P limitation. These site-specific problems were identifiable due to the focus on variability across similar organic fields and illustrate the need for site-specific approaches to reduce N losses. Finally, the two fields included in group 1 were exemplary of N deficiency, in which low N availability compromises crop productivity but also likely limits N losses within the growing season. While low soil NH4 + and NO3 – concentrations were similar to group 3,vertical farming tower for sale low total and labile soil organic matter and poorly-timed organic matter inputs compromised microbial activity and likely limited N mineralization.Cytosolic glutamine synthetase GS1 encodes for the enzyme that catalyzes the addition of NH4 + to glutamate, the former resulting from either direct uptake of NH4 + from soil or reduction of NO3 – in roots. GS1 is thus the gateway for N assimilation in roots and is upregulated to increase root N assimilation capacity. Similar levels of GS1 expression in groups 2 and 3, in spite of large differences in soil NH4 + and NO3 – concentrations at the anthesis sampling, suggests that plant N availability is indeed higher in group 3 fields than would be expected based on measurement of inorganic N pools alone. The low levels of GS1 expression found in fields with clear N deficiency supports this idea. These results complement recent experimental approaches that showed rapidly increased expression of GS1 in tomato roots in response to a pulse of 15NH4 + -N on an organic farm soil, which was linked to subsequent increases in root and shoot 15N content, even when this pulse did not significantly change soil inorganic N pools. GS1 transcripts and glutamine synthetase enzyme activity also increased with increasing NH4 + and NO3 – availability in sorghum roots, suggesting this response may be widespread among plant species.

Interestingly, inclusion of soil GWC in multiple linear regression models increased the proportion of GS1 expression variability explained to nearly 30% ; soil water content increases microbial activity as well as the mass flow and diffusion of inorganic N to roots. Further research will undoubtedly show how other factors like crop physiological N demand relative to C fixation and P availability increase the interpretability of N uptake and assimilation gene expression in roots.The N cycling scenarios identified on this set of organic fields corresponded at least in part with landscape clusters based on landscape and soil characteristics . Fields that balanced high yields with low potential for N loss and high internal N cycling capacity were part of PAM cluster 1, which had the highest productive capacity rating . Landscape clusters encompassing more marginal soils included both low-yielding fields exhibiting N deficiency or high-yielding fields that used inputs of highly available N like seabird guano to alleviate N deficiency . But these inputs led to the highest soil NO3 – levels and thus came at the cost of higher potential for N loss. Long-term efforts to increase internal soil N cycling capacity would help alleviate both N deficiency and the need for such large inputs of labile N. Whether farmers are willing to invest in management to increase soil N cycling capacity depends in part on how likely they perceive the benefits to be, especially on marginal soils. The discussions that we had with each farmer in this study indicated genuine interest in adaptive management to further tighten plant-soil N cycling, but this may not always be the case. Indeed, the proportion of management vs. inherent soil characteristics responsible for driving differences in N cycling is challenging to untangle. Farmers may allocate more resources to more productive land and likewise fewer resources to more marginal land, or may selectively transition more marginal land to organic management. Documenting the multiple services provided by increases in soil quality and facilitating information exchange among organic growers such as through the landscape approach used here may help build momentum for efforts to improve soil quality and plant-soil-microbe N cycling.The so-called chelation strategy , which is mainly found in graminaceous plants, is based on the excretion of phytosideropores to the rhizosphere. Phytosideropores rapidly chelate Fe, to form Fe-PS chelates that are subsequently transported into the root cells through a specific transporter. The socalled reduction strategy relies on the coordinated action of a membrane bound Fe reductase, that reduces Fe to Fe, an Fe uptake transporter and an H+ -ATPase that lowers the pH of the rhizosphere, is mainly used by non graminaceous plants, including Beta vulgaris. The reduction strategy includes root morphological, physiological and biochemical changes that lead to an increased capacity for Fe uptake. Morphological changes include root tip swelling, development of transfer cells and an increase in the number of lateral roots, leading to an increase in the root surface in contact with the medium. Some plants are able to accumulate and/or release both reducing and chelating substances, such as phenolics and flavins, which may have a role in Fe acquisition. Iron has been shown to down-regulate riboflavin synthesis in flavinogenic yeast strains and some bacteria. In plants, Rbfl and derivatives are accumulated and/ or excreted in Fe-deficient roots and could act as a redox bridge for electron transport to the Fe reductase. Moreover, FRO2 belongs to a super family of flavocytochrome oxidoreductases, and a recent study confirmed that the FRO2 protein contains FAD sequence motifs on the inside of the membrane. Also, a connection between Fe deficiency perception and Rbfl excretion has been described to occur through basic helixloop-helix transcription factors in Arabidopsis thaliana. At the metabolic level, increases in the activity of phosphoenolpyruvate carboxylase and several enzymes of the glycolytic pathway and the tricarboxylic acid cycle have been found in different plant species grown under Fe deficiency.

Through mechanical weathering processes, these clay fragments become incorporated into the soil and may provide a long-term source of PAH contamination in the environment . Polycyclic aromatic hydrocarbons are ubiquitous environmental contaminants and 16 PAHs are considered priority pollutants by the U.S. .There have been a few ecotoxicological evaluations concerning the large PAH concentrations from clay target fragments, but these studies have reported that the PAHs elicit low toxicity in aquatic organisms . This low toxicity was determined to be primarily due to the low bio-availability of PAHs resulting from the process of making clay targets in which the PAHs in the binding agent are bound under heat and pressure with dolomitic limestone . In addition, due to their aromatic nature and hydrophobicity, PAHs typically bind to nonpolar soil domains such as organic matter, further decreasing their bio-availability . However, a recent clay target ecotoxicity study using Eisenia andrei showed that the content of clay fragments in soils was correlated with PAH bio-accumulation in the terrestrial soil organism, suggesting that direct ingestion can be a more important route of exposure and potentially explain the lack of toxicity in exposed aquatic organisms . Clay-target contaminated site evaluations have also concluded that the elevated PAH concentrations in the soil from the clay target fragments pose an unacceptable level of risk to future potential residents and current site workers.

There are several PAH remediation strategies involving physical, chemical, biological,stackable flower pots and thermal technologies; however, conventional PAH removal methods such as incineration, excavation, and land filling are expensive and inefficient . Because of these issues, biological remediation practices such as bio-augmentation and phytoremediation have become preferred in situ treatment technologies as they are considered to be cost-effective and more environmentally friendly for the cleanup of PAH-contaminated soils . However, biological remediation operations can also be ineffective due to the limited PAH soil bio-availability that is a consequence of the clay target manufacturing process and the physicochemical properties of these compounds, which can be further exacerbated by the aging effect in field-contaminated soils . These PAH bio-availability limitations can be overcome through the use of surfactants that increase the desorption of PAHs from the soil to the aqueous phase, thus increasing their bio-availability to the degrading soil microbes . Bio-surfactants such as rhamnolipids or glycolipids offer an environmentally-friendly alternative to synthetic surfactants and are becoming more economically-feasible through the use of low-cost substrates and offer distinct advantages to synthetic surfactants such as reduced toxicity, high biodegradability, and greater stability under different temperature, pH, or salinity conditions . In practical surfactant-enhanced PAH contaminated soil remediation applications, mixtures of surfactants are commonly used to take advantage of the potential synergistic effects that can result in increased solubilization at a reduced effective surfactant concentrations . The bioaugmentation of biosurfactant-producing soil microbes has also been shown to be an effective strategy for the remediation of PAH-contaminated soils. For example, M. vanbaalenii PYR-1, a glycolipid-producing microorganism isolated from an oilcontaminated estuary near the Gulf of Mexico, has been shown to enhance PAHsolubility and degradation in PAH-contaminated soils .

Another in situ biological remediation treatment commonly used to increase PAH bio-availability is phytoremediation, or the use of plants and the associated rhizosphere to restore contaminated sites . Phytoremediation is considered to be an effective, low-cost alternative to cleanup large contaminated sites . The PAH bio-availability is enhanced in the plant rhizosphere, as plant roots secrete root exudates that promote PAH desorption from the soil matrix . In addition, plant roots may release enzymes that play a key role in the degradation of PAHs including oxygenases, dehydrogenases, phosphatases, and lignolytic enzymes . Finally, plant roots also provide easily degradable carbon sources and other nutrients that increases microbial biomass, diversity, and activity, contributing to enhanced PAH degradation through direct metabolism or co-metabolism . Because of the numerous benefits provided by the rhizosphere, grass species are recommended for phytoremediation treatments due to their extensive fibrous root systems and large root surface area, and hence more extensive interactions between PAHs and the rhizosphere microbial community . Typically, the primary contaminants of concern during the remediation of outdoor shooting range soils are heavy metals from ammunition; however, large concentrations of PAHs from the clay target fragments remain in the contaminated soil and could possibly become more bioavailable during the remediation of the metals.Brij-35 nonionic surfactant and sodium dodecyl sulfate anionic surfactant were purchased from Sigma-Aldrich. Rhamnolipid biosurfactant isolated from P. aeruginosa NY3 was purchased from AGAE Technologies .Diatomaceous earth, Ottawa sand, and all GC-MS grade solvents used in this study were purchased from Thermo Fisher Scientific . All substrates utilized for soil enzymatic analyses were purchased from Tokyo Chemical Industry Co., .

Bermudagrass, switch grass, and lettuce [Lactuca sativa] seeds were purchased from Lowe’s.A Vista coarse sandy loam was collected manually using a shovel from the 0-15 cm soil depth of an abandoned shooting range located near Lake Elsinore, California that was littered with clay target fragments with no prior soil remediation or waste removal from the site. The collected soil was air-dried for 5 d at approximately 23 °C and sieved through a 2-mm stainless-steel mesh screen.The soil pH and electrical conductivity were determined potentiometrically in a 1:2 soil-to-water suspension . Total metal analysis was carried out using an Optima 7300 DV inductively coupled, argon-plasma optical emission spectrometer following U.S. EPA Method 3050B after a 6-h digestion in a mixture of nitric acid, hydrogen peroxide, and hydrochloric acid at 95 °C .Mycobacterium vanbaalenii PYR-1 was stored at -80 °C in a 30% glycerol stock and the inoculum was prepared according to a previous method in MBS solution amended with pyrene as a carbon source . The CMC of Brij-35 and rhamnolipid biosurfactant was determined previously . The CMC of the Brij-35/SDS surfactant mixture was determined by measuring the surface tension of surfactant solutions over a concentration range using a Du Noüy ring-tensiometer and using the inflection in the plot of surface tension against surfactant concentration. The CMC was determined to be 0.099 mM at 0.5/0.5 molar fraction, which was similar to a previous study .After the soil was thoroughly mixed, 1 kg soil was placed in a stainless-steel bowl and 150 mL of distilled water was added and mixed to achieve a soil water potential of approximately -33 kPa determined by a soil tensiometer. For the M. vanbaalenii PYR-1 bioaugmented treatments, 15 mL M. vanbaalenii PYR-1-MBS solution was added to yield approximately 106 CFU/g soil and thoroughly mixed . The same procedure using only the MBS solution was added to the non-inoculated, or native, soil as the control. The PYR-1-MBS solution was reapplied every 2 months by adding the inoculum solution into the soil rhizosphere 5 cm below the soil surface . Once the soil treatments were prepared, the soil was added to the phytoremediation sample containers,flower pots for sale which consisted of 800-mL glass jars that were first painted on the outside with black paint, followed by aluminum enamel to prevent exposure to light . The pots contained approximately 50 g of 2-cm diameter gravel at the bottom to allow for accumulation of any excess soil water . Bermudagrass and switch grass seeds were surface-sterilized by three sequential washings in 0.1% sodium hypochlorite, followed by two rinses with sterile distilled water . Bermudagrass and switch grass seeds were planted at a rate of 20 seeds/pot and sealed with plastic wrap for 1 week for optimal seedling emergence conditions. After 2 weeks, plants were thinned to 8 plants/pot and amended with a commercial fertilizer for bermuda grass establishment. Treatments were then fertilized monthly with 100 mg/kg-N as urea, and 12.5 mg/kg-P as monobasic potassium phosphate . Due to the potential toxicity of surfactants to emerging plant seedling , surfactant addition at 50 mg/kg was initiated 1 week after plant thinning and initial fertilizer application. Since rhamnolipid biosurfactants have been previously shown to be degraded by the soil microbial community and are considered more biodegradable than the synthetic surfactants used in this study, surfactants at the initial rate were reapplied to the soil surface every 40 d .

Each pot was placed randomly in one of four blocks, each containing one replication of all treatment combinations in a climate-controlled growth chamber . The PAH phytoremediation experiment was continued for 8 months in the growth chamber under a 12/12 hour day/night period at 23±1/19±1 °C and 40% relative humidity. The average light intensity was obtained through fluorescent and incandescent lighting in the growth camber . Each pot was weighed daily for 8 months and the soil moisture was gravimetrically adjusted to 20% by application of distilled water . The quantity of distilled water added to the soil to achieve proper soil moisture was not adjusted for vegetation biomass produced during the study. Plant shoots were trimmed to a height of 5 cm every 3 months in order to stimulate continuous plant growth . At the end of the 8-month phytoremediation experiment, plant shoots and roots were separated from the soil as described in section 2.7. Once the vegetation was removed, the soil was sieved to pass through a 2-mm sieve and separated into two subsamples. The first soil subsample was air-dried for 7 d at approximately 23 °C in the dark and used for PAH analysis and toxicity assay . The second soil subsample was used for soil enzyme analysis and kept at fieldmoist conditions and analyzed within 1 week after the termination of the experiment. set at 60 °C and then raised at 5°C/min to 280°C . Quantification of PAHs was performed using an internal standard-normalized calibration curve and coefficients of determination for all calibration curves fulfilled the requirement of R2 ≥ 0.99. Soil dehydrogenase soil activity was analyzed by the use of 2–3- -5-phenyl tetrazoliumchloride as a substrate . A 1.0-g soil aliquot was mixed with Tris buffer and INT substrate in a stoppered 100-mL Erlenmeyer flask, and the mixture was incubated for 2 h at 40 °C in the dark. After incubation, the mixture was extracted using 10 mL N,Ndimethylformamide:ethanol mixture for 1 h at 23 °C in the dark and shaken every 20 min. Immediately after filtration, iodonitrotetrazolium formazan formation was measured colorimetrically at 464 nm against the reagent blank using a UV-Visible spectrophotometer . Soil dehydrogenase activity was expressed as µg INTF produced/g dry soil 2h. Soil polyphenol oxidase activity was measured by the utilization of pyrogallic acid as a substrate to form purpurogallin . Ten mL of 1.0% pyrogallol was added to 1.0 g soil sample and incubated at 30 °C for 2 h at 200 rpm. Afterwards, 5 mL of citrate-phosphate buffer was added to the treatment to stop the reaction, followed by the addition of 35 mL ether and shaking for 30 min at 200 rpm. The colored ether with dissolved purple gallic prime was measured colorimetrically at 430 nm on a UV-Visible spectrophotometer.The polyphenol oxidase activity was expressed as mg purpurogallin produced/g dry soil 2h. Control assays for each soil enzyme activity included autoclaved soil treatments, assays without soil, and assays without substrate addition during incubation . Results of soil enzyme activities are reported on an oven-dry-weight basis.At the end of the phytoremediation experiment, plant shoots were cut at the soil surface and rinsed with distilled water to remove any adhering soil. Approximately 4 g shoot subsample was taken and freeze-dried for PAH extraction and the remaining shoots were dried to a constant weight at 55 °C and weighed to determine total shoot biomass. The freeze-dried plant shoots were ground to pass a 2-mm, stainless-steel mesh screen using a Wiley Mill Grinder and 2 g was used to determine PAH shoot concentrations using procedures similar to those for soil PAH extraction . Plant roots were manually collected from the soil using forceps, placed on a 500-µm stainless-steel sieve, and thoroughly rinsed with distilled water to remove any adhering soil particles. Approximately 2 g root subsample was taken and freeze-dried and 1 g was used to determine PAH root concentrations similar to shoot analysis. The remaining plant roots were dried to a constant weight at 55 °C and weighed to determine total root biomass. The lettuce seed toxicity assay was performed to evaluate changes in phytotoxicity before and after remediation treatments by following a method in Cofield et al. . Briefly, 100 g soil at 85% water-holding capacity was placed in a 150 mm ´ 15 mm Petri dish and 40 lettuce seeds were evenly distributed and pressed into the soil.