Horticulture

One possible explanation was that the experimental conditions varied between the assays for basal defense and DCA-mediated immunity. In contrast to the analysis of DCA-mediated immunity , there was no pre-treatment with compound or mock solution before being sprayed with pathogen in the basal defense assays . It was observed in other experiments that this type of pretreatment affected the experimental outcome and indicated that pre-treatment of Arabidopsis plants with mock solution reduced the extent of susceptibility to Hpa. To examine if any of these synthetic elicitors induced phytotoxicity, trypan blue staining of seedlings after saturation treatment , where seedlings were grown and treated in liquid medium, was used. Dark blue staining, indicating cell death, was prevalent in 100% of the seedlings treated for 24 h with 300 µM of DCA, CMP144, CMP994, CMP202, CMP199, and CMP384 . Cell death was evident starting at treatments of 100 µM for CMP199. Therefore, the absence of GUS expression at 100 µM CMP199 was likely due to compound-induced phytotoxicity. For all synthetic elicitors cell death was not observed at concentrations showing effective GUS reporter activation ,blueberry grow pot indicating that cell death was not responsible for the GUS reporter activation.

Based on these preliminary assays, these synthetic elicitors were clearly potent inducers of GUS expression in the pCaBP22::GUS lines. Additional study was warranted to scrutinize their unique properties and ascertain their potential for future research use. To accurately quantify the defense-inducing activity of these eight novel synthetic elicitors, a dose-response analysis measuring the inhibition of HpaNoco2 spore development in three-week-old Col-0 plants was performed . For this experiment Col-0 seedlings were sprayed with the respective compound or mock solution and challenged with HpaNoco2 24 h later. Hpa spore formation was evaluated 7 dpi. Each synthetic elicitor behaved similarly during either Hpa defense or the reporter gene assays. CMP199 showed nearly 70% inhibition of spore development at 1 µM and 100% at 10 µM. CMP199 which showed a high activity in GUS saturation treatments, proved to be a more efficient defenseinducer compared to DCA at every concentration tested . This finding was important since this was the first time that a synthetic elicitor more potent than DCA had been discovered by our program. CMP199 showed great potential for future use. In the defense assays CMP199 and DCA provided most efficient protection against HpaNoco2, in contrast CMP384 was only able to induce significant levels of immunity at the highest tested concentration . The weakest of the examined new synthetic elicitors, CMP384 and CMP608 mediated only a 30% inhibition of spore development at 100 µM. The remaining five compounds exhibited intermediate levels of defense-inducing activity, with 55-75% inhibition of spore development at 100 µM. Different inducers of the same biological response may act antagonistically, additively, or synergistically. Synergistic activities produce a greater effect than the sum of their independent effects on the system.

A suite of synthetic elicitors with distinct modes of action, acting synergistically, may have great potential as environmentally safe pesticides, as they may trigger strong defense responses at extremely low concentrations when applied in combination. In addition, synergistically acting synthetic elicitors may be useful for plant defense network studies by facilitating the discovery of cooperative crosstalk mechanisms linking distinct compound-triggered pathways. Two redundantly acting compounds should not induce pCaBP22-333::GUS expression when each is applied at concentrations below their minimally active concentration. Additively acting compounds should activate the reporter when combined at concentrations equaling the sum of their minimally active ones. However, these compounds should not activate the reporter when applied at lower combined concentrations. If two compounds activate reporter expression at combined concentrations lower than the sum of their minimally active concentrations, their effects are synergistic. Finally, if two compounds act antagonistically the reporter gene response triggered by one compound should be reduced by application of the second compound. Antagonistically acting compounds would be tested at concentrations guaranteed to induce pCaBP22- 333::GUS expression. Here, the synergy of DCA, CMP442 , or CMP199 under saturation treatment conditions was tested. Consistent with previous observations, CMP199 proved to be the most potent of these three synthetic elicitors . CaBP22-333::GUS expression was scored visually based upon intensity of color. The minimal concentration of CMP199 for inducing the pCaBP22-333::GUS reporter was 100 nM, whereas that of DCA was 250 nM, and that of CMP442 was 1 µM. When any two of the synthetic elicitors DCA, CMP442 or CMP199 were combined the resulting responses were neither synergistic nor antagonistic .

GUS expression was weakly evident after the mixture of 50 nM each of DCA and CMP199 . This combination does not indicate synergism because if it was, GUS expression should have been stronger resulting in a more intense blue color. Instead, it appeared that this interaction was additive given that they activated the reporter at concentrations equaling the sum of their minimally active ones. Further experimentation will be necessary to determine more precisely the minimally active concentration of CMP199 to confirm that the tested compounds do not act synergistically. A concentration below 250 nM of DCA must be used in conjunction with CMP442 to distinguish between these two options. However, it is certain that they did not act strongly synergistic, as 50 nM of each, which is below their minimally active concentration, did not induce GUS expression. Finally, CMP199 and CMP442 appeared to act additively when used together. If they acted synergistically, a mix of 50 nM of each compound would have induced GUS expression. Instead 50 nM CMP199 and 250 nM CMP442 induced GUS in an additive manner. Saturation treatments were revealing about the role each synthetic elicitor plays in the activation of defense. Nonetheless, a more comprehensive defense assay may be necessary to confirm these observations while testing a wider range of concentrations. The complex responses associated with the activation of the plant immune system can be dissected and studied with the aid of synthetic elicitors activating the system at distinct points. These synthetic elicitors may serve as powerful tools to complement traditional genetics used to study plant defense networks. The versatility and reversibility of many synthetic elicitors makes the identification of a wider range and spectrum of these compounds a priority. Building on the success experienced by Knoth et al. in their identification of DCA, eight more synthetic elicitors were reported on here. Experiments demonstrated that most of these elicitors, excluding CMP608 and CMP384, have the ability to induce pCaBP22-333::GUS expression at concentrations of at least 10 µM. These low concentrations increase the probability of high target specificity,hydroponic bucket decreasing the likelihood of off-target or unwanted side effects . At synthetic elicitor concentrations that induced defense, no herbicidal effects were observed, while all compounds proved to be phytotoxic at higher concentrations. Each new synthetic elicitor discussed here adheres to Lipinski’s rule of five suggesting they may all be readily absorbed by plant cells. These rules state that properties that favor bioactive compounds include a molecular weight of less than 500 g/mol, a lipophilicity value of more than five, less than five hydrogen-bond donors, and less than 10 hydrogen-bond acceptors . CMP60, CMP994, and CMP202 share a phenyl-1H-pyrazol-1-ylmethanone skeleton as a common substructure.However, several closely related analogs act as potent inhibitors of human neutrophil elastase, antiinflammatory, analgesic, anticonvulsant, anticholinergic, antihistaminic, antibacterial, antiepileptic, and antifungal agents . CMP384 analogs, but not CMP384 itself, were reported to have antimicrobial activities . Of the remaining compounds, the exact structures or closely related analogs of CMP144, CMP174, and CMP199 are not referenced to in any biological studies. The wide range of structures and their apparent uniqueness offers many promising candidates for potential synthetic elicitors. In HpaNoco2 defense assays, the novel synthetic elicitor CMP199 outperformed DCA with a lower active concentration. A lower active concentration may reflect a higher degree of target specificity. Also, a major goal of this project was the identification of compounds with different modes-of-action. A suite of functionally distinct synthetic elicitors is likely to allow for more control and flexibility when studying the plant defense network and is likely to facilitate the creation of novel environmentally friendly pesticides. CMP199 will be further examined by other lab members to determine if its mode of action is distinct from that of DCA.

To determine if the available synthetic elicitors acted synergistically, dual combinations of DCA, CMP199, and CMP442 were tested at a range of different concentrations for each compound. This experiment demonstrated that these synthetic elicitors are likely to act in an additive, but not synergistic, manner. As a confirmation of this data, additional assays should be performed testing disease resistance inducibility with these combinations of synthetic elicitors. These experiments are necessary since it is possible that the GUS assay is not of sufficient sensitivity to display synergistic effects since. Pathogen assays would provide quantitative data instead of the qualitative data provided by the visually read GUS assays. The plant immune system is a highly complex web of interactions that allow plants to modulate their responses to the type of infecting pathogen. Currently, only a small number of defense network components have been identified and characterized. The identification of additional elements that compose this web will lead to a better understanding of plant defense and new strategies to combat diseases. In microarray experiments performed by Knoth et al. , the ACID cluster was identified as a set of genes upregulated at time points corresponding to synthetic elicitor-mediated disease resistance. These 137 ACID genes were hypothesized to be important for plant defense. More than one quarter of the 137 ACID members encode kinases . Of the 16 ACID genes examined in this study, 12 encoded protein kinases. Protein kinases often act as regulatory elements controlling plant defense responses. To increase the probability of success in the functional analysis of ACID members, genes encoding protein kinases were preferentially chosen for study. ACID3 encoded a TIR-NBS-LRR class disease resistance protein and ACID8 a LRR-receptor like protein kinase. ACID9 and ACID13 encoded LRR transmembrane protein kinases and ACID12 was a receptor like protein kinase. Additionally, ACID1, ACID7, ACID11, ACID10, ACID14, ACID15, and ACID16 were genes that encoded for proteins with potential kinase activity. The remaining four ACID genes did not encode protein kinases. Of the remaining genes: ACID2 is an armadillo/beta-catenin repeat family protein, ACID4 a remorin family protein, ACID6 a member of EXO70 gene family, and ACID5 is an ankyrin repeat family protein. These four genes were included based on the availability of two independent homozygous T-DNA insertion lines. For ACID1, ACID4, ACID6, ACID7, ACID8, ACID9, ACID10, ACID12, ACID13, and ACID15 both tested T-DNA mutant lines exhibited enhanced Hpa susceptibility. This indicated that the mutations in/near the genes themselves were likely responsible for the respective defense phenotypes, as opposed to unknown mutations. Only one of the two tested T-DNA insertion lines for ACID5, ACID11, and ACID16 exhibited reduced basal defense to Hpa. It is likely that in each case the second line contained its T-DNA insertion in a position not capable of reducing gene function. For example, the insertion in acid11-1 is located in an exon and caused enhanced susceptibility to HpaNoco2. The insertion in acid11-2 did not affect HpaNoco2 resistance; this T-DNA located in the promoter of ACID11 did not to significantly reduce ACID11 expression. Neither the acid16-2 nor acid16-3 line displayed a defense phenotype. The insertions in these two mutants were intronic and might be spliced out of the ACID16 pre-mRNA . In contrast, the insertion in acid16-1, which is located in an exon, caused enhanced susceptibility to HpaNoco2. The insertion in the promoter in acid5-2, but not the exonic insertion in acid5-1, resulted in a defense-related phenotype. One possible explanation for this is that the insertion in the promoter abolished proper expression of this gene, while the exonic insertion results in a truncated, but functional, protein. Alternately, the insertion loci predicted by TAIR may not be accurately annotated . Among the acid mutants lines that exhibited reduced basal defense phenotype, only acid4-1, acid4-2, acid6-1, acid6-2, acid7-1, acid7-2, acid8-1, acid13-1, acid13-2, acid15-1, and acid15-2 displayed reduced levels of the respective transcripts. The observation that some T-DNA insertions did not affect endogenous ACID levels was not entirely unexpected.

Several transcription factors were differentially expressed in this study. GO enrichment showed the molecular function GO term ‘DNA-binding transcription factor activity’ was significantly enriched. In addition to the MYB77 transcription factor gene described earlier, a GRAS transcription factor gene, HAM3, was DE in this study. GRAS transcription factors were previously found to play a role in berry development and ripening in grapes, tomato, and citrus. This transcription factor showed increased expression later in the season when fruit were grown on trifoliate root stock, suggesting the root stock influences its role in improved citrus fruit quality. The largest phenotypic differences seen in mature fruit grown on trifoliate compared to rough lemon root stock were in the levels of total soluble sugar and titratable acid in ripe fruit. The levels of sugars and acids and their ratio in fleshy fruits is one of the most important determinants of sensory traits such as taste and flavor. Two genes were identified as differentially expressed that could play a role in the accumulation of these compounds. Firstly, a P-type ATPase was DE in fruit growing on trees grafted onto trifoliate versus rough lemon.

This gene was down-regulated at time two,hydroponic nft system but upregulated at time three . Studies have proposed a number of ATPases as proton pumps that are responsible for organic acid accumulation in citrus fruit. The reduced expression of this ATPase gene later in the season in fruit grown on rough lemon root stocks could contribute to the lower accumulation of titratable acid levels in these fruits. This ATPase gene identified in this study was not identified in the previous citrus studies, but the regulation of acid accumulation is a complex, as can be seen in other fruits, such as papaya and apple. It is possible this is a graftinduced effect observed with these specific root stocks, which were not examined in the previous studies. Secondly, a homolog of Arabidopsis BETAFRUCT4 was down-regulated in fruit of trees grown on trifoliate root stock compared to rough lemon at time three . This gene encodes a vacuolar invertase. Decreased expression of vacuolar invertases has been associated with increased sucrose content and accelerated ripening. Interestingly, by using an antisense acid invertase gene in transgenictomato to reduce acid invertase activity, fruit displayed higher levels of sucrose, as well as smaller fruit. We see similar trends in sugar accumulation and alterations in fruit size in this study. Klann et al. suggested that the water influx that drives fruit expansion is closely related to the concentration of osmotically active soluble sugars and therefore, all genotypes accumulate water until they reach a similar threshold of soluble sugar concentration. This could also contribute to the increased size of fruit grown on rough lemon fruit compared to trifoliate root stocks.

This study did not identify any statistically significant differentially expressed miRNAs from our fruit small RNA seq data. Therefore, potential miRNAs that target DEGs were predicted. An in-house R-script was used to select for miRNA-mRNA interaction pairs with an expected negative correlation in gene expression. These pairs were identified for the ten genes described above. All ten miRNA genes and their target mRNAs were detected by qRT-PCR. Pearson correlation coefficient value between the relative expression level detected by qRT-PCR and by RNA-sequencing was highly significant with r = 0.94. Of the ten interaction pairs, eight followed expected fold changes between time points . Therefore, it is likely that these eight target mRNAs are being regulated to some extent by their respective miRNA. Only two pairs do not follow the expected inverse relationship between time points, suggesting those mRNAs are not being regulated post transcriptionally by their miRNAs. This has been observed in previous integrated miRNA-mRNA studies. There have also been reports of target genes having a negative or positive feedback regulation on their respective miRNA, which could be another explanation for the inconsistent correlations seen in this study.Citrus is now grown in more than 140 countries in tropical, subtropical and Mediterranean regions. It is one of the most economically important crops in the world.This reduces the juvenile phase, allowing for the trees to produce fruit many years earlier than would trees grown from seed. Due to the large variation in growing conditions and climate in the regions where citrus is grown, different citrus root stocks are required to improve yield and fruit quality in numerous diverse climates, as well as resist various pests and diseases.

Root stocks impart certain traits to the scion and the effects of root stocks can be large. The most significant impacts are on growth, vigor and yield, tree nutrition, stress resistance, and fruit quality. The root stock effects on various aspects of tree growth and fruit development are well documented, but the molecular mechanisms underlying most of these differences are unknown. Previous studies have shown changes in the transcriptome of various root stock genotypes, especially in response to biotic and abiotic stressors. These types of changes have been seen in Arabidopsis, corn, mulberry, tomato, and poplar. In citrus, gene expression profiling has been used to understand root stock effects and responses to biotic and abiotic factors. In another study, expression studies of leaves from mandarin grafted onto various root stocks were analyzed in order to explain root stock effects on the growth of scions. There is extremely limited tissue-specific transcriptome knowledge in citrus, especially for root tissue. A small number of studies have evaluated trifoliate, trifoliate hybrid, and mandarin root transcriptomes in response to citrus diseases, but these studies each assessed only one genotype. Only recently has an RNA-seq based approach been used to establish a reference transcriptome for citrus and of the 28 samples used in the study,nft channel only two were obtained from roots. The root samples collected for this study were sour orange and trifoliate genotypes, but samples were grouped by organ to perform differential expression and subsequent analyses. To our knowledge, there are no comparative studies of citrus root transcriptomes between genotypes. In plants, the root system is critical for plant growth and development. It serves the functions of anchorage, nutrient and water uptake, and is the main boundary between the plant and its soil environment. Root growth relies on a specific set of signals that involves hormone signaling, availability of nutrients and carbon supply. There is a large degree of genotypic variation in crop plant root systems that can influence the plants growth and production including root length, root density, root angle, lateral root number, and root:shoot ratio. These parameters can impact the plant’s size, tolerance to biotic and abiotic stressors, and ability to uptake water and nutrients. For this reason, grafting, a process which connects the roots of one plant to the scion of another, has been widely using in plant breeding programs in order to improve vigor, alter plant architecture, enhance tolerance to disease and abiotic stress, and contribute to the quality of crops. In citrus, root stocks are bred for a variety of traits that are imparted to scions, such as tree size, yield, tolerance to salt, cold, and drought, tolerance to various pests and diseases, and improved fruit quality. Many studies in citrus have been conducted to assess the impacts of genetically differing root stocks on these traits. However, the study of molecular mechanisms behind root attributes lags far behind above ground tissues in plants, especially in perennial crops. Understanding the genetics of how root systems develop, and the regulatory controls of these processes will help optimize the improvement of yield and quality in citrus. Root system length, growth, and architecture control the ability of plants to respond to various stress conditions. The development of the root system and its architecture is determined by genetic factors interacting with numerous environmental factors. Plants must adapt to their environment by controlling their physiological reactions and morphogenesis. This can create complex root system architectures. For example, different root types can produce lateral roots that significantly extend the elaborate root system and allow the plant to search the soil for water and nutrient-rich areas. The increase in lateral root formation allows plants to more easily uptake these essential molecules in order to survive in unfavorable conditions.

Root stock genotypes exhibiting higher abilities to adapt to stress and create more extensive root systems improve nutritional status and water uptake, which can increase marketable yield. A clear example of this was seen in grafted mini-watermelon and tomato plants. Studies in Arabidopsis, rice, and corn have identified several genes that influence root development and root system architecture. In citrus, transcriptomic studies have been performed to understand the effect of root stocks on growth and in response to cold, nutrient deficiency, and fungal inoculation. Besides these studies, little effort has been invested into studying the effects of citrus root stocks at the molecular level, especially genotype-specific effects and their relation to fruit quality. Plants have evolved to cope with a constantly changing environment, modifying the root system architecture in response to nutrient availability and soil microorganisms. This flexibility requires fine tuning of gene expression. Among the molecules that control root development, small RNAs play a vital role in regulating genes at the post transcriptional level in plants. The most well-studied class of sRNAs are microRNAs , which are approximately 21 nucleotides in length and are produced from noncoding transcripts. Mature miRNAs have been shown to negatively regulate gene expression at the post-transcriptional level by specific binding and subsequent cleavage of their target mRNAs, or by the repression of target translation. Increasing evidence demonstrates that plant miRNAs play critical roles in almost all biological and metabolic processes. A review of miRNAs in roots indicated that they participate in root development, the modulation of root architecture, and root biotic interactions. This review focuses on numerous studies using Arabidopsis and legume plants as models. In citrus, miRNAs profiling has revealed their involvement in adaptation to nutrient deficiency, drought and salinity stress, and pathogen infection. However, no information, to date, is available about the role of miRNAs underlying differences in fruit quality observed between citrus root stocks. In the present study, trees grafted onto four root stocks were chosen from a root stock trial at the University of California, Riverside to assess for various fruit quality traits; Argentina sweet orange, Schaub rough lemon, Carrizo citrange, and Rich 16-6 trifoliate orange. Generally speaking, rough lemon root stocks produce the highest yield and fruit size, but this fruit is often of lower quality . Trifoliate orange root stock, when well adapted, produces high quality fruit, with high yield on smaller trees. Carrizo citrange root stocks produce intermediate yield with good fruit quality. Sweet orange root stocks produce good quality fruit but are very susceptible to various citrus diseases, and therefore are rarely used as root stocks for commercial trees. An RNA-seq approach was used to assess differences in gene expression between root stocks that produce fruit with varying quality traits with the aim of identifying genes that could potentially play a role in improvement of fruit quality. Moreover, miRNA expression profiles were obtained for each of the root stocks to identify potential regulatory mechanisms associated with their target genes. The grafted trees were part of a root stock trial conducted at the Citrus Research Center and Agricultural Experiment Station at the University of California, Riverside that included 28 root stocks. Trees were planted in 2011 in a randomized block design with ten replications. Trees were planted on berms, irrigated with mini sprinklers according to soil moisture sensors, and treated with fertilizers and pesticides according to standard commercial practices. Plastic mulch was used to cover the berms to suppress weeds and retain soil moisture. Trees were grown in a fine sandy loam and surface soil pH was 7.3 at the time of planting. It is important to note that no trees with Huanglongbing have been identified at UCR. For sequencing, roots from two biological replicate trees were harvested at four time points throughout the 2014-2015 growing season . Young, newly growing fibrous root tissue was collected from the first 6 inches of soil along the outer edge of the canopy.

In their findings, as root biomass increased beyond a certain threshold it negatively impacted grain yields, perhaps due to increased costs of maintaining a large root system. This point appears worthy of detailed study as it may point to which root system traits will be beneficial in a given environment and how they might impact yields if conditions become favorable. Perhaps a large root system may be beneficial when water is limited; however, will a large root system remain an advantage if water becomes sufficient? These types of questions need to be answered before efforts are made toward modifying crop root system traits. Understanding the genetics of root system traits does not have to wait until we have all the answers. Since root traits are highly plastic and regulated by a number of small-effect loci it will likely take some time to unravel these complex traits. Yaseen and Malhi reported that wheat genotypes varied significantly in their allocation of dry matter in roots and shoots. Frageria also demonstrated that root dry weight was genetically controlled as well as being influenced by the environment. Currently,grow table the neglect of selecting for root system traits is mainly due to the difficulty of measuring root system traits of field-grown plants .

As our understanding of root system genetics is improved new tools such as genetic markers associated with desired traits can be utilized by breeders for marker-assisted breeding efforts. Two systems were used in the experiments described below. One consisted of tubes fitted with a plastic sleeve filled with sand. A filter paper covering two holes punctured at the bottom of the sleeve allowed for drainage. In the standard system water is applied to the top of the tube . In a modified version an additional plastic sleeve was fitted to allow for water to be delivered from the bottom up.The second system consisted of pots lined with a plastic sleeve, filled with sand and four holes punctured in the plastic for drainage. Pots were brought above water holding capacity and allowed to drain for 24 hours before being planted. Peters Excel fertilizer was injected to the irrigation water at a 1:100 ratio. Every irrigation event in the experiments included this ratio of fertilizer.Three doubled haploid bread wheat populations were used to measure total root biomass at various stages of growth. Parents were selected for their root biomass among 16 spring wheat land races and modern cultivars tested by Waines et al. . Cv. Sonora ranked among the highest, Foisy was intermediate, and CBdeM had a low total root biomass. These parents have other contrasting phenotypes for traits such as drought tolerance, plant height, days to heading, awn type, and seminal root angle. Detailed information about genotyping, linkage mapping and general descriptions of each population can be found in the first chapter of this dissertation. Populations Sonora x CBdeM , Sonora x Foisy , and CBdeM x Foisy have 146, 141, and 128 lines respectively. In 2013 the three parental lines were evaluated for root biomass in 80cm tubes using the standard method.

These were grown for 30 days and 60 days as preliminary evaluations at two growth stages. Plants were grown in a factorial design with four replications. Doubled haploid progeny were grown in one gallon pots with four replications in a randomized complete block design for 21 and 28 days in 2014 and 2015 respectively. The 2016 experiments included 100 randomly selected progeny from each population with three replications due to constraints on greenhouse space. Plants were grown until heading . Plants were watered as needed to keep the sand at water holding capacity for the duration of the experiments. To gain a broad perspective about the relationship between shoot and root biomass, data from multiple experiments were combined. Data sets from experiments running for a similar duration were combined to fit into a similar scale. Following these criteria three sets of data were created. The first set was created from the data of the trade off experiments described below and data kindly provided by Dr. Harun Bektas . The second set is from the data collected during the allelic variation experiments and the mapping population data collected in 2016 mentioned bellow. The third set is from the 2014 and 2015 mapping population experiments described below. Raw values for individual were used to create scatter plots fitted with a Loess smoothing curve with an alpha of 0.75 with a quadratic degree using statistical analysis software, Statistix.Plants were grown to maturity in the modified tube system and the pot system. Seeds of Pavon and Yecora Rojo were imbibed for 24 hours before planting. For each cultivar two treatments plus a control were run in a factorial design with two replications in fall 2015 and winter 2016. The control was given 500mL of water daily, from the top down.

The fist treatment received water from the bottom only starting at two weeks of growth. Water from the bottom was kept at the furthest point at which the roots reached within the sand as visible through the clear plastic sleeve of two check tubes. As the roots grew into the water profile the water level was continuously dropped until the roots reached the bottom of the tube. The second treatment received water from both the top and the bottom daily. Water from the bottom was maintained at 50cm to prevent roots from growing deeper and 500mL of water was added each day from the top. The pot experiments had three replications in fall 2015 and winter 2016 setup in a factorial design having three treatments and a control. Each pot, for both cultivars,ebb flow table was kept at water holding capacity until three different phenological stages: booting, heading and anthesis at which point water was cut entirely. Treatments are termed as drought-at booting, drought-at-heading, and drought-at-anthesis. After that point, any plant showing severe water stress was given water to prevent death. This point was determined when leaves began to wilt and curl beyond mild symptoms. The control was given ample water to maintain the sand at water holding capacity throughout the experiment. During the experiments days to booting, days to heading, days to anthesis, and days to maturity were recorded. Days to booting was recorded at the point when the flag leaf emerged. Days to heading was recorded at the point when the head split the boot. Days to anthesis was recorded at the point when the first anthers became dehiscent. Days to maturity was recorded at the point when the grain was ready to harvest. At maturity the total number of tillers and total number of fertile tillers were counted. Heads were harvested and the shoots were cut at the soil surface to separate them from the roots. Shoot biomass is reported without grain yield included in the total biomass. Heads were threshed to record grain yield for each plant. For the tube experiments, root length was measured and root biomass above 30 cm was separated from the root biomass bellow 30 cm and weighed separately. The analyses of variance for traits in each experiment were based on mean values of the experimental units and considered significant where p ≤ 0.05. Genotype means were used for LSD all-pairwise comparisons where α = 0.05. In experiments involving the mapping populations, broad sense heritability and Pearson‟s correlation coefficients for shoot and root biomass were calculated on a mean basis across four replications. Genomic regions associated with root and shoot biomass were detected by the software package IciMapping using linkage maps for the mapping populations as described previously and the mean value of four seedlings of each genotype from four replicates in 2014 and 2015; in 2016 the mean value of three seedlings from three replications was used. The composite interval mapping method with a step of 1cM was used and the threshold for QTL detection was determined using 1000 permutations where α = 0.05. Markers in the linkage maps were renamed using the index number provided by Wang et al. preceded by the chromosome designation. In total 6,353 data points were included to create the scatter plots for the combined data analysis of the relationship between root and shoot biomass . The first set included 1,243 data points . The second set had 1,342 points . The third set had a total of 3,768 data points .

In the first set of data, root mass versus shoot mass and grain yield were plotted as those experiments went until maturity. All other experiments were concluded earlier having only shoot and root biomass collected. Data for the fall and winter 2016 trade off experiments were not significantly different so the data were combined. In the pot experiments Pavon 76 did not show significant differences between drought treatments and the control for days to booting, days to heading, or days to anthesis with means of 49.1, 54.3, and 59.1 days respectively. However, days to maturity showed significant differences between the treatments and the control, with means of 92.8 and 137.5 days respectively. The total number of tillers was also significantly different between the treatments and the control with means of 9.3 and 34.3, respectively. The total number of fertile tillers showed significant differences within treatments as well as between treatments and the control. Plants experiencing drought-at booting had means of 5.5 fertile tillers, those at-heading and at-anthesis 8.3 fertile tillers, and the control had means of 32.2 fertile tillers per plant. For shoot biomass, the treatments showed significantly less biomass than the control with a mean of 11.9 grams versus 36.0 grams for the control. Root biomass was significantly different between plants receiving drought-at-anthesis versus those at-booting and plants receiving drought-at-heading had a similar mean when compared to either of the other treatments. Mean root biomass for plants receiving drought at booting, heading, and anthesis were 3.5, 4.9, and 5.1 grams respectively. All drought treatments were significantly less than the control which had a mean total root biomass of 7.1 grams. Grain yield for the drought treatments were significantly less than the control which had a mean yield of 51.3 grams. Plants receiving drought-at-heading yielded the second highest with a mean of 12.0 grams and both the drought-at-booting and drought-at-anthesis treatments yielded the lowest with a mean of 6.3 grams. Similar to Pavon 76 in the pot experiments, Yecora Rojo did not show significant differences between drought treatments and the control for days to booting, days to heading, or days to anthesis with means of 29.6, 36.7, and 41.6 days, respectively. Days to maturity showed a significant difference between the treatments and the control with means of 77.9 and 106.5 days, respectively. The total number of tillers for the drought treatments was 6.6 which was significantly lower than the control with 9.5 tillers per plant. Treatments showed significant differences with the drought-at-booting and drought-at-anthesis treatments having means of 5.0 and 7.0 fertile tillers respectively. Plants receiving drought-at-heading were intermediate between the other two treatments with a mean of 6.0 tillers per plant which was not significantly different the other treatments. All treatments had lower means than the control which had 8.8 fertile tillers per plant. For shoot biomass all treatments were similar with a mean of 3.1 grams which was significantly different from the control of 4.7 grams. The control had the highest root biomass with 2.6 grams per plant and the drought-at-anthesis treatment had the next largest root biomass with 1.9 grams per plant. Both the booting and heading treatments had a mean of 1.3 grams per plant. Grain yield also showed significant difference within the treatments and between the treatments and the control. The control yielded 11.3 grams per plant, the drought-at-anthesis yielded 5.7 grams per plant, and the drought-at heading treatment yielded 4.7 grams per plant which was not significantly different from the drought-at-anthesis treatment or the drought-at-booting treatment, and the drought-at booting treatment yielded the lowest with 3.6 grams per plant. In the tube experiments Pavon did not show significant differences between the treatments and control for days to booting, days to heading, days to anthesis, or days to maturity with means of 55.13, 60.6, 64.8, and 125.2 respectively.

Cultivars Sonora, Foisy, and Chiddam Blanc de Mars were selected. The primary criterion was the total root biomass with Sonora ranked among the highest, Foisy was intermediate, and CBdeM had a low total root biomass. Additionally, the three also have other contrasting phenotypes for traits such as drought tolerance, plant height, days to heading, awn type, and seminal root angle. These parents are what could be considered as “traditional cultivars” in that they were all selected from land races. Sonora was selected from a land race in Durango, Mexico and is known for good drought tolerance but its height makes it susceptible to lodging. Cv. Foisy was selected by Mr. Foisy in Oregon in 1865 and typically yields more than CBdeM and Sonora. CBdeM originates from Ville de Paris, France, and was selected from an English land race. None of these cultivars have a place in commercial agriculture today but still are grown by traditional or artisan farmers as so called heirloom varieties of wheat for bread making. More information about the parents can be found on the UC Davis small grains web page in the 2011 California cultivar descriptions publication . Crosses were made in a triangular manner to form a set of “nested” mapping populations with any given two populations having a single parent in common so that we get the populations Sonora x CBdeM , Sonora x Foisy , and CBdeM x Foisy.

This design provides a built in system for verification of QTLs across populations and genetic backgrounds. For each of the three populations ca. 200 lines were planted on July 11th, 2013 in an air-conditioned greenhouse on the UC campus in Riverside, California,growing lettuce hydroponically in one gallon pots with two plants per pot. These were used for seed increase, leaf tissue for DNA extraction, and for phenotyping of simple traits. Doubled haploid plants in each pair were compared and expected to be identical, however, not all were and any lines with clear differences between the two plants were discarded. A second seed increase was planted on April 4th, 2014 in a similar manner and was also used to collect phenotype data. During this increase plants were grown under 18 hour days with supplemental lighting. In 2015 a two-location field trial was established. Experiments were planted in October 2015 and harvested by May 2015. The two locations were the University of California, Riverside Agricultural Experiment Station in Riverside, California, and at the Coachella Valley Agricultural Research Station . Experiments were set up in randomized augmented designs with three check varieties replicated in each block. The check varieties were Blanca Grande 515, Summit 515, and Cal Rojo.

There were 32 blocks per treatment per location with 16 plots per block. Additional “blank” plots of Summit 515 were planted to make blocks square but were not included in the analysis. Each plot consisted of six rows spaced 20cm apart and 122cm long planted at a density of 560 seeds per plot. Each location had two treatments, one well-irrigated and the other which received limited irrigation after 60% of the genotypes were booting. The well-irrigated treatment received water as needed based upon soil moisture and plant indications. For the limited irrigation treatment water was withheld until plots showed moderate to severe wilting at which point they were irrigated to prevent death.The R statistical package “lmerTest” was used to obtain the predicted mean values for all traits evaluated which included; days to heading, plant height, yield/m2 , and 1000 grain weight . SNP calls from Genome Studio were converted into “A” and “B” genotypes by comparison against parental scores for each population. Markers that were polymorphic between parents for each population were imported to JoinMap 4.1 and used to construct linkage maps. Chromosome and marker index number were used to name markers previously mapped by Wang et al. , for example 5A_6716, and markers that were not previously mapped were named using an underscore and the marker index number with no chromosome indication. Identical individuals were excluded from the genotypes used to construct linkage maps and likely arose as artifacts from the DH procedure or were a result of labeling errors.

Also, individuals with greater than 10% missing data for marker calls were excluded. Initially linkage groups were generated based upon markers mapped by Wang et al. using eight doubled-haploid mapping populations. This was done on a chromosome by chromosome basis including only markers mapped to a given chromosome. Identical markers for the given chromosome were removed prior to mapping. Groupings were made using the default calculation settings for independence LOD and linkage groups were mapped using the default settings for the maximum likelihood algorithm. For linkage groups that failed to generate maps or lacked a sufficient number of markers, additional markers were added from the unmapped pool of SNPs. To do this, all unmapped markers were selected along with the mapped markers for the given chromosome and then the steps listed above were repeated to give new linkage groups with a more suitable number of markers. These newly added markers were then BLASTed against the wheat arm survey sequence to verify their correct linkage group assignment. Phenotypic data for awn type, days to heading, and plant height collected during 2013, 2014 greenhouse evaluations and 2015 field evaluations were used to map QTLs by the software package ICImapping . For greenhouse data, the linkage maps and mean value for two plants of each doubled haploid line were used to map QTLs and for field data the predicted mean values for each genotype were used. The composite interval mapping method with a step of 1 cM was used and the threshold for QTLs detection was determined using 1000 permutations at α = 0.05. Originally, populations SC, SF, and CF consisted of 257, 244, and 214 lines, respectively. During the 2013 seed increase populations were assessed for vernalization requirement, hybrid necrosis,ebb and flow table and uniformity. Of the 200 lines planted for SC, SF, and CF population about 1, 2, and 7%, respectively, showed what appeared to be segregation but could have arisen from multiple unknown reasons. Winter growth habit appeared in 18.5, 7.5, and 1.5% of the SF, CF and SC populations, respectively. This was despite the fact that all three parents are spring wheats and require no vernalization. The appearance of winter growth habit may reflect some combination of recessive alleles of vernalization genes . Hybrid necrosis was rated on a scale of 1 to 10 with 1 being minor and 10 being lethal. Hybrid necrosis was fairly prevalent in SF with 31% of the genotypes showing some level of the phenotype; the CF population had 25% of the genotypes showing some level of hybrid necrosis and the SC population only 6.5%. Any genotype with a score greater than 3 was excluded from genotyping. Because of possible contamination , sterility and with some lines showing winter growth habit and/or hybrid necrosis, populations were reduced in size. Additionally, population sizes had to be limited to genotyping of 150 lines for several reasons, from the cost/practicality issue to future experiment manageability. However, all non-genotyped lines are preserved and can be accessed, if needed. During the 2014 greenhouse evaluations the remaining 150 lines for each population were characterized for vernalization requirement, days to heading, plant height, and awn type . For field trials populations were reduced to 133, 121, and 115 lines for SC, SF, and CF respectively due to winter habit or late flowering of some lines and lack of seed for others.

During the 2015 field evaluations populations were characterized for days to heading and plant height, awn type and 1000 grain weight . However, not all field data could be analyzed and/or were unreliable, thus distributions of trait values in the field for days to heading, plant height at Coachella Valley are shown in Figure 1.3 for SC, SF, and CF.Using phenotypic data collected during the greenhouse evaluations and the field evaluations QTLs were mapped to verify the quality of the genetic maps and provide some basic genetic information about the three populations. Table 1.1 summarizes regions that were mapped in the SC, SF, and CF populations for awn type, days to heading , plant height , and hybrid necrosis . Only the regions consistent through multiple years are discussed. Table 1.2 demonstrates how QTLs can be verified within and between populations by using awn type QTLs as an example. For simplicity of the exercise lines were classified into two groups: awned and awnless. Two genome regions were consistently identified that explained 23.76 – 92.67% of the phenotypic variation within the populations. The first locus was consistently identified in the SF and CF populations on chromosome arm 5AL. The QTL in the SF population covered a 2.2 cM region between the markers 5A_9620 and 5A_6716 with the peak around 266 cM explaining 36-39% of the phenotypic variation across all years and environments. In the CF population, the QTL covers a 3.2 cM region between 5A_9620 and 5A_6716 with the peak around 169 cM. The QTL explains 31-93% of the phenotypic variation observed in the population across all years and environments. This QTL shares the same two markers in common with the QTL identified in the SF population. Additionally, Mackay et al. mapped the same QTL using a wheat MAGIC population and verified it using an association mapping population. They identified the marker BobWhite_c8266_227 as being the closest linked to the QTL which in these populations mapped to the same genetic location as 5A_6716 identified here. The second QTL was consistently identified in the SC and SF populations as being on the chromosome arm 6BL. In the SC population the QTL covers a 4.2 cM region between markers 6B_606 and 6B_1614 with the peak around 103 cM. It explains 62-73% of the phenotypic variation for this population across all three years and two environments. In the SF population the QTL covered a 0.71 cM region in 2013 and 2014, and a 2.1cM region in 2015 with the peak being around 79 and 80 cM respectively. This QTL explains 23-29% of the phenotypic variation in the population. In 2013 and 2014 the QTL was between 6B_45514 and 6B_606, however, in 2015 it shifted by a couplemarkers to 6B_68633 and 6B_84 covered a larger region. However, its peak was still near the same point and the two markers associated with the phenotype in 2013 and 2014 were present in the 2015 region. The QTL shares the 6B_606 marker in common with that identified in the SC population . These results indicate that Sonora carries the dominant allele for B2 on 6BL and that Foisy has the dominant allele for B1 on chromosome arm 5AL. Since CBdeM is fully awned it must have the hd b1 b2 genotype. The trait „heading date‟ or „days to heading” in wheat is determined by several factors, including vernalization requirement controlled by the Vrn genes , the photoperiod genes play a role in determining the sensitivity to photoperiodism and the Earliness per segenes are responsible for controlling flowering time regardless of photoperiod. In the three populations studied here, five major QTLs were found responsible for the heading date character, located on chromosomes 2D, 5A, 5B, and 5D. Two consistent QTLs on chromosome 2D were identified in the SC and CF populations in 2015. In the SC population the QTL covers a 0.67 cM region with its peak around 112 cM between markers 2Dx_32130 and 2Dx_79444. This QTL explains 18.43% of the phenotypic variation seen in the population and has an average additive effect of 7.69 days. In the CF population the QTL covered a 5.8 cM region with a peak around 47 cM between markers 2Dx_7001 and 2Dx_13208. This QTL explains 70.06% of the phenotypic variation in this population and has an average additive effect of -14.24 days. These QTLs are most likely the Ppd-D1 gene described by Beales et al. . Sonora contributed the day length sensitivity allele in the SC population and Foisy contributed the allele in the CF population. This explains why no segregation for the locus was seen in the SF population.

Food is a powerful frame through which to make the climate change problem more concrete and “close to home,” as it implies both a social and essential daily activity. The garden, meanwhile, provides a useful metaphor for the complex global climate system. The curriculum directly connects climate science to community and local action in the garden, thus linking food and climate systems. This systems-thinking lens aligns with Next Generation Science Standards , something that motivates teacher participation if their schools have adopted NGSS. Through local examples, garden activities and guest speakers, the curriculum connects students to other change makers and empowers them with agency to help build a more sustainable food system in their community. Students learn to think of climate change as more than “just” a science problem: it is a social problem requiring action and responsibility from all levels of society—individual to international. Each of the six lessons involves students in activities that translate regenerative agriculture theory into practice . The curriculum provides opportunities for students to learn scientific facts ,hydroponic grow systems share personal narratives , and enact hands-on solutions to climate change via school gardens .

They learn about the negative effects of elevated CO2 in the atmosphere globally and then help lower CO2 locally through increasing plant photosynthetic activity. The pedagogical framework for the curriculum is inspired by Paolo Freire’s critical pedagogy and other more current framings of a signature pedagogy for sustainable food systems education . Educators facilitate collective learning experiences that are often subversive in nature and seek to disrupt inequitable outcomes, both environmental and social. Curriculum implementation followed a co-teaching model. The researcher-teacher partnership draws on complementary domains of expertise: content expertise from the researcher, and classroom management/student dynamic expertise from the teacher. Two symbiotic goals are addressed using co-teaching as an implementation method: 1) students learn climate change from a content expert, and 2) teachers increase knowledge and competence in climate change instruction, allowing future students to benefit from a better-trained instructor and serving as a form of professional development. Studies have shown repeatedly that the best way to improve student performance across a range of subjects is to boost teacher knowledge and competency . This type of participatory, co-teaching implementation inherently limits ability to statistically analyze a large, representative, or randomly generated dataset of students. It is grounded in social science theory of the qualitative, in-depth case study.

Each school required slightly different implementation of the curriculum. In one case snow days canceled several coteaching sessions, which then had to take place via Skype. Taken as a whole, these four cases shed light on important adjustments that can be made to tailor climate change education interventions to site-specific school needs. Pragmatically, meeting unique school needs is a prerequisite for implementing any non-mandatory education intervention in partnership with schools. The study simultaneously investigates student responses to an experiential climate curriculum, and teacher responses to co-teaching as a form of professional development. The methods used for evaluating curriculum efficacy include 1) semi-structured teacher interviews, 2) student surveys , and 3) participant-site observation. Triangulation of these methods improves the validation of results. Deeper understanding can be gained from a small set of cases on CCE, and best practices can then be applied to a larger universe of schools. More specific to each method, teacher interviews followed a six-question interview guide and were semi-structured in nature. Preliminary student surveys provide a baseline for student knowledge and engagement. Compared with post-intervention surveys, this allows basic statistical analysis to define the effect size in the sample population and whether it is significant. The survey assessment includes 10 knowledge-based questions on climate science and food systems applications, as well as 19 engagement questions asking opinion statements measured on Likert-type scales.

This multi-faceted assessment of climate literacy recognizes that “knowledge about climate change can be divided into several general and overlapping categories: knowledge about how the climate system works; specific knowledge about the causes, consequences, and potential solutions to global warming; contextual knowledge placing human-caused global warming in historical and geographic perspective; and practical knowledge that enables individual and collective action” . The engagement questions adapt the Six Americas survey questions to capture students’ change in engagement towards climate change following the curriculum intervention. Participant and site observation over a six week period captures important features of the school climate, both environmental and social, that help contextualize interpretation of results. The quality of the school garden, behavioral norms, and student informal interactions are all variables of interest for understanding other forms of data collection. In climate literacy evaluations, it is important to understand student intention to take action and follow up to document concrete examples of students taking action, which goes beyond simple survey and interview protocols. Certainly, questions can be posed to students asking whether they feel more empowered to seek out their own additional knowledge and participate in climate actions, but ideally these questions can be followed up with evaluation tools documenting actual action outcomes. This was not possible in the contexts of study reported on below but should be a focus for future student climate literacy evaluations. Results presented and discussed below are broadly relevant to climate change education interventions, with some insights as well into the value of food as an engaging entry point or frame for the climate education conversation. Attitude and engagement questions revealed higher levels of concern along the Six Americas spectrum than the national average. The first 10 questions were adapted almost directly from the Six Americas survey, with some modifications for student-friendly language. An additional nine questions were added dealing specifically with food systems, behavior and climate change. Based on the first 10 questions, students were categorized into the six segments from alarmed to dismissive, with almost all students falling in the top three categories . Students demonstrated an overall increase in engagement although this was difficult to measure with precision due to inconsistencies within individual student response patterns. A preliminary analysis is valid for determining directional effect arrows and assessing whether pilot programs show promise, and thus were adequate for this evaluation. Precision could be added in future iterations by simplifying answer scales so they are consistent, and then quantifying student attitudes on a numerical basis. The survey was a bit long to hold student attention, and survey fatigue was a confounding variable in some cases. Work is underway by the Yale Project on Climate Change Communications to create a four-question survey version for teens,vertical grow table which will be a valuable improvement for future studies. Informal observations and conversations reveal a notable curiosity and interest among youth in learning more about climate change. A commonly expressed sentiment, especially at the outset of the curriculum intervention, is that climate change is an important issue that students feel they should know more about. This is mirrored in national statistics reporting that American teens recognize their limited understanding of climate change, and 70% say they would like to know more about the subject . Post-intervention teacher interview themes revealed a widespread appreciation of coteaching as a mechanism for delivering climate change instruction. All teachers interviewed expressed enthusiasm for having a content expert present to deliver instruction on climate, complementing the garden teachers’ expertise in food-related topics, classroom management and student behavior. The positive response from teachers is important to contextualizing student results, as the more enthusiastic and knowledgeable teachers became about climate change connections in the school garden, the more engaging lessons became for students.Teachers were able to learn from the experience and expressed desire to replicate elements of the curriculum on their own in the future, thus meeting one of the process-specific goals of the research. Interviewees also revealed a common theme of searching for hope and action amidst the daunting reality of climate change; the garden and classroom were often identified as key arenas where hope and solution steps exist. Key quotes from interviews are highlighted in Table 15 below.These results, in particular the challenges highlighted by teachers, closely match national findings on climate change education.

In a recent national review of science teachers, the first nationally representative study of science educators to focus on climate change, fewer than half of all teachers reported any formal coursework on climate change, yet over two thirds would like targeted professional development opportunities to allow them to dive in deeper to this complex and emotionally sensitive topic . It is well established that teachers are in need of professional development in order to teach an unfamiliar subject with confidence and competence, and several national leaders in climate education are addressing this . Having a climate science “expert” in the classroom to co-teach a climate change curriculum for the first time is another promising form of PD explored here. Partnerships emerged as a key feature enabling success of food and climate education in schools, mirroring the findings in example 1 above. Partner organizations and individuals are able to provide infrastructure support, outdoor learning environments, guest speakers to reinforce climate education units, and program evaluation assistance. Questions of how to scale impact via partnerships at the district or state level and education policy implications are discussed below. Examining results by school context offers strategies for scaling this type of intervention in rural vs. urban school districts. Students at the Lopez school, with abundant local farm and forest resources to devote to furthering climate curricula endeavors, selected a bio-char experiment as a class climate action project, and will be applying locally produced bio-char to test plots in the school garden to compare with non-treated plots , in partnership with the community. This community-school partnership adds to the body of successful climate change engagement strategies meriting replication, particularly other rural communities where local farmers might be interested in participating in farm to school programming at the school or district level. Experiential food and climate change education is an emerging branch of CCE with great potential, where the school garden provides one context for experiential climate learning while many others are possible . By emphasizing and teaching local forms of food production and consumption, this CCE example seeks to localize climate stewardship and in doing so reduce the carbon footprint of food system products and processes. The food-climate nexus diagram presented in Chapter 1 offers both an impetus for scaling this form of integrated food-climate education, and an example of how to do so while visualizing food-climate interactions. This chapter reports on initial positive results from integrating CCE into both the humanities and school garden classrooms. In the case of humanities-focused CCE, students not only demonstrated gains in climate literacy, but also improved their reading comprehension. Sixth grade students performed at a level equivalent to their eighth-grade peers in terms of listing numerous climate mitigation strategies, and reported both looking up new information and speaking with friends/family about climate change more frequently than all other middle school grades. The examples from school garden classrooms more explicitly adopt and test the hypothesis that experiential CCE is more effective than didactic or lecture-based climate instruction. Results show improvements in student learning and strong student interest in the topic. However, further evaluation methodology development is needed to best capture the impacts on student action and behavior. In order to understand the efficacy of experiential CCE relative to CCE that is not experiential, a controlled experiment would be required that uses the same evaluation methodology for students with and without experiential CCE. This methodology would ideally comprise and observational element where teachers report on student “climate actions” over the course of a defined time period. In future studies, a list of core “climate actions” could be developed as a baseline for evaluators to assess whether students are carrying out these activities . Both food-focused and humanities-focused CCE point at an underlying characteristic of CCE. Rather than being treated as its own subject, or topic to be covered in science classrooms, climate change is an overarching frame that infuses all sorts of school activities, processes, and classrooms, from the transportation that bring students to school, to the food that is served in the cafeteria, to the content students are covering with their mathematics, physics, government, or garden teachers.

Civic engagement advances the idea of creating “public commons” through urban agriculture, an idea related to ecological economics and explored in David Bollier’s book Think Like a Commoner: A Short Introduction to the Life of the Commons. A commons “integrates economic production, social cooperation, personal participation, and ethical idealism into a single package;” it is a paradigm of “self-help and collective gain” and an “alternate self-governance structure for resource management and ‘living well’” . The commons paradigm espouses a political philosophy grounded in grassroots civic activism and proposes different “foundational premises for a new political economy” based on social connections and rediscovering “people’s knowledge” of natural systems in their local contexts . However, we must consider who is able to participate in creating such a space . Who participates, in both policy and urban farming as an activity, is a crucial factor in determining whether outcomes will subvert or reinforce existing power, privilege and structural inequities. As Ramírez states, “While recreating neglected urban spaces into ‘productive’ spaces to grow food is inspiring and beneficial on one level, the prevalence of white bodies inhabiting garden spaces reifies uneven geographies and catalyzes gentrifying forces” . It is the role of inclusive policy processes and watchdog citizen activists to counteract this retrogressive tendency of UA projects. One example of grassroots political action,vertical grow towers working around rather than through institutional channels of policy making, is the Catatumbo Collective’s people-to-people reparations project.

Developed by three immigrant women spearheading an urban agriculture organization in Chicago, the people-to-people reparations map locates minority-run farming projects on a map of the United States, providing a brief description of the project and their specific needs, and then a link or contact info so that visitors can donate directly to the project. They are motivated to publicize agricultural history from a minority person’s perspective and provide a means of public accountability through their mapping project, supporting “those who have borne the brunt of labor exploitation, land theft, and discriminatory agricultural policy” . This project has already led to funding for several farmers’ projects, as well as land gifts to create several minority-owned farms. While the founders recognize the need to continue litigation and action through formal policy channels, they honor the urgency of needing to “start right away” by facilitating “transfers of wealth.” They are also contributing to a more updated database of farmers of color, often underrepresented in USDA farm censuses. There is room for more participatory action research linking researchers to citizens and civic engagement projects . This will allow for data to be shared and transferred more easily, and for the network of UA and food justice participants to strengthen through ties to research institutions and each other. Researchers have an important role to play in addressing data gaps and strengthening the network of urban farmers who have clearly identified needs and are ready to work towards appropriate, measurable solutions.According to the literature, access to urban-produced foods is directly tied to the economic realities of urban farming operations.

Daftary-Steel, Herrera, and Porter make a compelling case for building coalitions to provide the necessary political and financial support to fund UA, as well as tackling the “root causes” of food insecurity through social services. From what limited studies exist, it seems clear that economic viability of urban farms is dependent on income far beyond sales capacity of the urban farm.It is important to communicate to policy makers that urban farms are producing a lot more than pounds of food; they are also “distributing” social goods, creating a “commons”, and providing a connection to nature, community, and education , and these in turn are part of improving community food security. The primary benefits of UA organizations are often education , social integration, economic opportunity, and local environmental quality improvements. Producing enough food to transition a community from “food insecure” to “food secure” is not necessarily going to happen through urban farming alone; however, supplementing food intake with locally produced, healthy fruits and vegetables is an important step in building food security and community health. As such, researchers and UA practitioners may consider generating more robust data on the health, environmental and social benefits of UA to promote among policy makers the idea of UA as a public good, worthy of public investment in the same vein as schools, transportation and education. This is especially true in U.S. cities without strong policies supporting the existence of UA, and with high land values and development pressures.

It is important to acknowledge that urban agriculture is not the only solution to food insecurity and food access and should not be the only forum of support/intervention from policymakers. In fact, in some cases “the emphasis on ‘grow your own’ reinforces self-help and government austerity arguments, absolving government of the responsibility to address the structural and institutional causes of food insecurity” . UA is part of the solutions portfolio to improve food justice and food access, but must be complemented and reinforced by other governance efforts to provide affordable, healthy food through neighborhood groceries, food hubs, cooperative markets, culinary and nutrition education programs, farm to school programs or other means of addressing structural causes of food insecurity . Civic engagement, critical scholarship from multidisciplinary perspectives, and alliances between housing, transportation, and food policy are all necessary components of a UA landscape that improves access and meets the needs of both producers and consumers. In conducting this literature review, using a combination of academic and gray literature, we recognize a significant gap between scholarship and practice. Urban agriculture is not a panacea that will automatically produce all the social, environmental, and economic “goods” attributed in the literature at large without proper structuring or policy frameworks in place. A more realistic, and holistic picture of urban agriculture can be advanced by further rigorous evaluation of what particular organizations are choosing to focus on, how much food they are producing currently , how they are distributing their food, and where they need support. It is not just about whether urban farms have the potential to feed food insecure people, but whether they actually do, depending on locally specific modes of distribution, channels of access, and policy climates. Key ideas from the literature about how to enable socially just, economically viable urban agriculture include a focus on food sovereignty, public investment and/or land rights, “agrihood” developments, and attention to disparate neighborhood race and class dynamics when siting UA operations. Researchers can address key data gaps including the actual tracking and consumption of urban-produced food. We can answer lingering questions including: where does the food go, how much is accessed vs. wasted, what are consumer preferences around accessing urban produced foods, and where do institutions need to fill in gaps in access and/or distribution channels? Results of this literature investigation are next applied to our ongoing study of urban agriculture in the East Bay region of the San Francisco Bay Area,container vertical farming characterized by a high amount of urban agriculture activities , yet undergoing rapid gentrification with persistent high levels of food insecurity and income inequality. Despite growing evidence of the diverse health, education, and environmental benefits of urban agriculture, these vibrant spaces of civic engagement remain undervalued by city policy makers and planners in the United States. Because urban farming takes on many different forms and functions, with intended outcomes that may or may not include yield and profits , thriving urban farms and gardens are under constant threat of conversion to housing or other competing, higher-value land uses due to rising land values, and other city priorities.

This land use challenge and threat to urban farm land tenure is especially characteristic of U.S. cities like San Francisco, one of the most expensive land and housing markets in the country. Under the current urban agriculture paradigm in the U.S., food justice scholars and advocates either try to quantify and highlight the multiple benefits of UA  or pursue a critical theoretical approach, arguing that urban agriculture can yield unfavorable results if pursued without an equity lens, especially in cities with intense development pressures and gentrification concerns . A productivist focus is problematic, because, while urban agriculture can be an important component of community food security, its other social and ecological benefits are just as, and sometimes more, significant . In this article, we suggest that the current debates around “urban agriculture” in the U.S. often lead to an unhelpful comparison with rural farms regarding yield, productivity, economic viability, and ability to feed urban populations, most notably in the policy arena. Defined in these ways, the radical, transformative potential of urban food production spaces and their preservation often gets lost or pushed to the side in city planning decisions in metropolitan regions such as the San Francisco Bay Area, where the threat of displacement is ubiquitous given high levels of economic inequality and extreme lack of affordable land. In order to facilitate what scholars such as Anderson et al. 2018a refers to as the “agroecological transition,” already underway in many urban food ecosystems around the globe , we argue that applying an agroecological approach to inquiry and research into the diversity of sites, goals, and ways in which food is produced in cities can help enumerate the synergistic effects of urban food producers. This in turn encourages the realization of the transformative potential of urban farming, and an articulation of its value meriting protected space in urban regions. Urban agroecology is an evolving concept that includes the social-ecological and political dimensions as well as the science of ecologically sustainable food production . UAE provides a more holistic framework than urban agriculture to assess how well urban food initiatives produce food and promote environmental literacy, community engagement, and ecosystem services. This paper presents a case study of 35 urban farms in San Francisco’s East Bay in which we investigated key questions related to mission, production , labor, financing, land tenure, and educational programming. Our results reveal a rich and diverse East Bay agroecosystem engaged in varying capacities to fundamentally transform the use of urban space and the regional food system by engaging the public in efforts to stabilize, improve, and sustainably scale urban food production and distribution. Yet, as in other cities across the country, they face numerous threats to their existence, including land tenure, labor costs, development pressure, and other factors that threaten wider adoption of agroecological principles. We begin by comparing the concepts of UA and UAE in scholarship and practice, bringing in relevant literature and intellectual histories of each term and clarifying how we apply the term “agroecology” to our analysis. We pay particular attention to the important nonecological factors that the literature has identified as vital to agroecology, but seldomly documents . We then present findings from a survey of 35 diverse urban farm operations in the East Bay. We discuss the results, showing how an agroecological method of inquiry amplifies important aspects of urban food production spaces and identifies gaps in national urban agriculture policy circles. We conclude by positing unique characteristics of urban agroecology in need of further studies and action to create equitable, resilient and protected urban food systems.Agricultural policy in the United States is primarily concerned with yield, markets, monetary exchange, and rural development. The United States Department of Agriculture defines agricultural activities as those taking place on farms. Farms are defined as “any place from which $1,000 or more of agricultural products were produced and sold, or normally would have been sold, during the year” . Urban agriculture has been proliferating across the country in the last decade on both public and private lands, as both for-profit and nonprofit entities, with diverse goals, missions and practices largely centered on food justice priorities and re-localizing the food system. Yet U.S. agriculture policy has been struggling to keep up. In 2016, the USDA published an Urban Agriculture Toolkit, which aims to provide aspiring farmers with the resources to start an urban farm including an overview of the startup costs, strategies for accessing land and capital, assessing soil quality and water availability, production and marketing, and safety and security .

The program uses an “integrated systems approach” to delivering hands-on education in nutrition, ecology, sustainability, and land stewardship . In practice, this consists of educating students in a garden classroom elective for elementary and middle school, hosting a high school farm elective course where students visit local farms, and preparing and preserving food from the school farm in the cafeteria, where local scratch-cooked meals are served year round. Part of the growing national movement around improving the quality of school meals through locally sourced produce, the LIFE program takes advantage of the exceptional quality of both locally produced foods and farm-based educational opportunities on the island. The LIFE program has been funded by a combination of a large private foundation and smaller donations, fundraisers, grants, and in-kind contributions. It is currently working towards a more sustainable, diversified finance model that will expand those invested in the program’s success as well as allow the educational activities associated with LIFE to grow. LCLT coordinates interns to support the LIFE program in the summer , and staff at the Family Resource Center run a volunteer-based gleaning operation on island orchards that yields up to 5,000 lbs. of fruit for the school cafeteria.

Production has grown steadily at the ½ acre school farm, from 1,400 to over 6,000 lbs. between 2009 and 2016 . The program is currently fundraising to purchase two beef cows raised by island teens for the cafeteria meat supply.The results of this research investigation led to the conclusion that students were not yet connecting their experiential learning activities in the school garden to bigger picture environmental themes and challenges, such as climate change,vertical farming companies greenhouse gas emissions, and the environmental impact of conventional food systems. The gaps in environmental knowledge, which forms an important but not complete piece of the environmental literacy equation became clear to teachers at the school, who sought to fill those gaps with new curricula. In partnership with Lopez school and LIFE staff, I developed a food systems and climate change 6-lesson curriculum, designed to engage students in questions at the climate and food nexus. The curriculum walks through the causes, effects, impact monitoring, and solutions to climate change, through the perspective of the food and agriculture sector. The culmination is a class or school-wide climate action project in partnership with the community, sparking informed hope through taking action. This curricular outcome of the LIFE program is evaluated and discussed further as a pathway to farm-based climate education and applied to other school contexts in Chapter 4. In addition to assessing environmental literacy outcomes, environmental impacts of the LIFE program were also investigated in 2015-2016. Using the Cool Climate Carbon Footprint Calculator for food-related emissions, eliminating the need to transport all meat, fruits and vegetables to the school could save 1.4 tons CO2 annually per person.

Adjusting this metric to account for the 9-month school year and multiplying by approximately 120 students who eat at the school cafeteria per day, this yields approximately 120 tons of CO2 savings annually. K-12 education is inherently social. The director of the Organic Farm School finds hope in the very nature of children working together in school gardens, that they might come to see farming as more social and collaborative, and thus more readily forge pathways into cooperative farming.Midnight’s Farm, a 100-acre property located near the center of Lopez Island, heralds the diversity of its operations from the initial entry point down a gravel driveway. A signpost indicates the direction of the compost operations, yoga studio, farm stand selling beef, pork and vegetables, and wood-fired bakery . In the words of the farm owners, “we farm to steward this wonderfully beautiful piece of earth and for the tangible, hands-dirty love of connecting people to the soil and storing a little bit of carbon there, too.” The land was purchased with savings from a previous career as an Alaskan salmon fishing captain, and the past 20 years have seen a progressive investment in land restoration and diversified agriculture operations. From the establishment of hundreds of trees at the property border to rotational grazing plans for cows on pasture and marshland, to fruit trees and ¼ acre home garden with greenhouse, to a blueberry patch being prepared for planting in 2019, biodiversity continues to grow. Revenue streams are accordingly diverse, with the compost and wood chips bringing in the most revenue annually, followed by Field House vacation rentals, beef and pork products, and vegetable sales.

The Field House, available for short term farm stays, hosts visitors year round and is booked throughout busy summer tourism season, capitalizing on the growing market for agritourism opportunities. The farm has typically provided housing for another couple in a barn apartment, in exchange for regular workdays or some combination of paid labor and housing work-trade. Sustainable Agriculture Interns coordinated by LCLT help out during summer months, and the farm is a popular destination for “WWOOFers” as well . Other Lopez Island youth work on the farm several days a week during the summer. At maximum capacity, the farm hosted nine farm employees during the summer 2019 season. Farming practices are the product of decades of experience, and soil fertility is the product of countless yards of compost and mulch application. In the vegetable garden, dozens of crops feed the farm families and neighbors each year. From spring seeding to bed preparation and transplanting, to weeding, irrigation, harvesting, cover cropping and winter greens cultivation in the greenhouse, every activity has its seasonal rhythm. Several planting strips are gradually converting to no-till farming, with compost, mulch, and broad-forking substituting for the mechanical mixing of the soil. Tilling is associated with carbon release and disturbance of the soil biota, so reducing or eliminating tillage is an effort several farmers are working towards, in balance with weed management. Irrigation ponds, dug on most farm properties, fill up with rain in the winter, and provide water to crops through the dry summer months.It is amended with lime and seeded with beneficial plants to boost nutrient quality of forage materials. The cows contribute to the regeneration of pasture soils, providing aeration from their hooves, growth stimulation from grass consumption, and fertilizer from their manure. David and Faith, the owners of Midnight’s Farm,equipment for vertical farming are passionate about researching and implementing agricultural solutions to climate change on their farm.

Their bookshelves are filled with books such as Grass, Soil, Hope; Dirt to Soil; and Growing a Revolution: Bringing our Soil Back to Life, and their social calendar is filled with attending climate talks and hosting climate researchers from University of Washington , WSU, and other institutions. Most recently they are engaged in a carbon footprint analysis of their compost operation, land use, and cattle herd, in order to understand highest impact opportunities for emissions reduction and carbon removal. The results show that currently the farm is contributing to the sequestration of approximately 250 mtCO2e, via forest cover, marshland, managed pastures, compost production and application, which together more than offset emissions from farm machinery, diesel use, and cattle as shown in Figure 9. David and Faith advocate for a “big tent” approach to food systems transition where many different people and groups can see themselves in a process of growing food with a lighter climate impact, and better human health impact. Their vision rests on a premise of developing strong interpersonal relationships, infusing the work with joy, humor, social connection, and opportunities for personal growth. An onsite yoga studio offers space for interns and farming friends to stretch and reinvigorate bodies feeling the effects of hard physical work. David and Faith continue to articulate better and brighter ideas for the future, such as finding long-term land partners and helping launch a climate farm school on the island, pushing forward the vision of a truly regenerative agroecosystem on Lopez. Orderly rows of greens and vegetables lend a sense of efficiency and purpose to the fields of Lopez Harvest. Successional plantings of diverse lettuce varieties march westward across the field, with the largest plants cut for weekly harvests while each neighboring row showcases one fewer week in the field. 500 lettuce plants go in the ground on Wednesdays, and plants are harvested on Tuesdays and Fridays for twice a week deliveries. The humming schedule of running a successful greens production farm serving the two island grocery stores as well as 5-6 island restaurants and food businesses creates a strong weekly rhythm for farm owner and farm workers. Dig, transplant, bed down, repeat.

Six inch spacing, four rows per planting bed. Finish the row, water it in, keep moving. Lopez Harvest sells lettuce mix, a specialty blend of “Island Greens,” chard, microgreens, arugula, herbs, and various seasonal vegetables and specialty crops to most of the for-profit food retail and business operations on the island. Christine, the farm owner, sends out a “pick list” to all customers a week in advance, takes orders by a certain day, and harvests and delivers all orders herself. This is her answer to the question “what does it take to be a successful small-scale farmer on a small island?” She sells her surplus produce directly to retail and restaurant, finding this to be more profitable than selling at the seasonal weekly Farmer’s Market or direct to consumers. She raises additional vegetables for personal consumption, reducing her own need to purchase store-bought foods, and facilitates a meat-share program where costs and benefits from raising meat chickens are shared among participating households. These non-monetary and cooperative forms of exchange are important to the economic viability of her operations. Christine now receives additional revenue from her participation in a beginning farmer mentoring program, where she earns up to $1,000 annually for mentoring younger farmers in their first year of operation . Her farm is on shared land purchased by three couples, and was acquired with family support, a common method for overcoming high barriers to entry for farmland access . While some rows of her field are planted to commercial crops, others are in rye-vetch cover crop mix gaining fertility for next year, or mustards to deter wire worms. The cover crop is mowed down and incorporated into the beds, with some beds serving as experiments for no-till practices where she has also tried occultation techniques to germinate and kill weeds prior to transplanting. This is difficult to enact on her land due to heavy clay soils that need some disturbance to be made ready for tender transplants and is a work in progress. Commercial crops are rotated onto previously cover cropped beds, a dance between production of plants and soil. In Christine’s mind, “good farming is good for the climate;” she adopts practices when they prove beneficial for her land, crops, soil, and business model, and it just so happens that many of these practices are anointed in academic research as climate mitigating strategies. Christine exemplifies a successful independent, woman-owned business model. She receives seasonal labor support through the LCLT intern program and through informal worktrade agreements with friends and neighbors. Christine is a vocal contributor at the monthly farmer coffees, sharing what she’s learned about effective weed control strategies , and a gifted farmer educator. She collaborates with WSU San Extension on a research project to reduce wire worm pest pressure in lettuce crops and is also a collaborator on the Western SARE bio-char cocompost grant, participating in the field trial and soil/crop data collection processes. Christine recognizes the attractiveness of entering into farming cooperatively or with farm partners but struggles with the difficult proposition of supporting multiple households with limited farm revenue streams and land use restrictions. When it comes to sharing land in her current situation, she would love to be able to build and provide more farm worker housing, but is restricted from doing so by county zoning policies that prevent more than two houses from being built on a parcel designated as “farmland7.” The county zoning codes are ripe for reform, but notoriously difficult to get right in terms of regulatory verbiage that protects farmland from becoming housing developments yet allows for ample and affordable farm worker housing.

Transcriptomic and proteomic studies in Fe deficient plants have also reported increases in root transcript and protein abundances, respectively, of enzymes related to the glycolytic and TCA cycle pathways, among others. Iron deficiency also induces an accumulation of organic acids, mainly malate and citrate, in roots. The induction of C metabolism in roots of Fe-deficient plants would not only provide a source of reducing power, protons and ATP for the Fe reductase and H+-ATPase enzymes, but also lead to an anaplerotic root C fixation. Carbon accumulated in roots is exported in the form of organic acids via xylem to leaves, which have otherwise drastically reduced photosynthetic rates when Fe-deficient. The higher energy requirements in Fedeficient root cells are tackled by increasing mitochondrial oxidative processes, and roots from Fe-deficient plants show enhanced respiratory activities and higher O2 consumption rates. On the other hand, the mitochondrial respiratory chain is strongly affected under Fe-deficient conditions, since some of its components are Fe-containing enzymes.Furthermore, an increase in anaerobic metabolism has also been described in Fe-deficient roots,hydroponic vertical farm probably as an strategy to oxidize all the reducing power generated by glycolysis and TCA cycle that can not be easily oxidized in the respiratory chain.

When resupplied with Fe, Fe-deficient plants reorganize its metabolism by readjusting metabolic cycles and changing root morphology towards those typical of Fe-sufficient plants.The most common approach used to study Fe deficiency in roots is to analyze only a small number of genes, proteins and/or metabolites. A more comprehensive knowledge of the processes taking place in Fe-deficient roots has been recently provided by the application of modern techniques allowing for the simultaneous and untargeted analysis of multiple genes or proteins. The aim of this work was to characterize the changes induced in the root tip proteome and metabolome of sugar beet plants in response to Fe deficiency and resupply, in order to provide a holistic view of the metabolic processes occurring in plants under different Fe status.The polypeptide pattern of root tip extracts was obtained by 2-D IEF-SDS PAGE electrophoresis. Real scans of typical 2-D gels are shown in Figure 1; an average number of 141 and 148 polypeptides were detected in Fe-sufficient and Fe-deficient plants, respectively . The total number of spots detected was relatively low when compared to other proteomic studies. Several causes may account for this discrepancy, including i) protein extraction method and amount of protein loaded in the gels, ii) gel size, iii) pI range and iv) sensitivity of the staining method. Averaged 2-D polypeptide maps were obtained using gels of three independent preparations, each from a different batch of plants . To better describe polypeptide changes we built a composite averaged virtual map containing all spots present in both Fe-deficient and control root tip extracts . Iron deficiency caused 2-fold increases in 29 spots and 2-fold decreases in signal intensity in 13 spots . Furthermore, 6 spots were only detected in Fe-sufficient plant samples and 13 spots were only detected in Fe-deficient plants .

All polypeptides in the composite averaged map are depicted again in Figure 1D, to permit annotation of those polypeptides where identification was achieved by matrix assisted laser desorption ionization – time of flight MS . These polypeptides were labeled from a to v as described in Figure 1D, and homologies found are described in detail in Table 1. From the 29 spots that showed increases in signal in root tip extracts of Fe-deficient as compared to Fe-sufficient controls, the 20 more abundant were excised and analyzed by MALDI-MS. Since the sugar beet genome has not been sequenced yet and few sequences are avail-able in the databases, identification was performed by homology searches with proteins from other plant species. From the 20 spots analyzed, 14 proteins were identified . These include proteins related to glycolysis such as fructose 1,6-bisphosphate aldolase , triose-phosphate isomerase , 3-phosphoglycerate kinase and enolase . Three spots gave significant matches to malate dehydrogenase , and two more polypeptides presented homology with α and β subunits from F1 ATP synthase . Other proteins increasing in root tip extracts from Fe-deficient sugar beet plants as compared to the controls were fructokinase and formate dehydrogenase . Also, one spot gave significant matches to a cytosolic peptidase At1g79210/YUP8H12R_1 . Spot n gave significant match to a glycine rich protein, which possibly has a role in RNA transcription or processing during stress conditions. From the 13 spots detected de novo in proteome maps from root tip extracts of Fe-deficient plants , the 6 more abundant were excised and analyzed by MALDI-MS, resulting in only 2 positive matches . These significant matches were found for glyceraldehyde 3-phosphate dehydrogenase and DMRL .Changes in the amount of DMRL as well as DMRL gene expression and flavin analysis were further studied using root tip extracts of Fe-sufficient, Fe-deficient and Feresupplied sugar beet plants .

From the 13 spots showing a decrease in signal intensity in root tip extracts from Fe-deficient plants as compared to controls , 3 were identified by MALDI-MS. Spots q and r gave a significant match to nucleoside diphosphate kinase I and to oxalate oxidase-like germin, respectively. Spot s presented homology with the At4g27270 protein whose molecular function is to interact selectively with FMN, and also presents oxidoreductase activity. From the 6 spots not detected in root tip extracts from Fe-deficient plants as compared to the controls , 3 were identified by MALDI-MS . Proteins matched were oxalate oxidase , peroxidase and caffeoyl CoA Ometyltransferase .Iron deficiency and/or resupply caused significant changes in the levels of 62 out of the 77 identified metabolites. Metabolite level data were normalized to the mean response of the +Fe treatment; response ratios, defined as the level in a given treatment divided by the level in the +Fe control, are indicated in Table 2. Iron deficiency caused significant changes in the response ratios of 26 metabolites. Large increases were found for some organic acids , some sugars , nicotianamine and 2-aminoadipic acid. The response ratio of oxalic acid decreased markedly in -Fe conditions, whereas those of other aminoacids, N compounds, lipid metabolites and others did not show large changes when compared to the Fe-sufficient controls. Twenty-four hours after Fe-resupply, there was a dramatic coordinated increase in the root tip response ratios of galactinol, raffinose, lactobionic acid, cellobiose and nicotianamine when compared to those found in Fe-deficient roots,vertical farm whereas the response ratios of sucrose, myoinositol, citrate and malate decreased. Seventy-two hours after Fe resupply, the response ratios of galactinol, raffinose, cellobiose, nicotianamine and many other compounds had decreased in the YZ areas, whereas in the WZ the response ratios were very low. The response ratio of many of the lipids increased moderately in all Fe resupplied samples. Metabolites in the coenzyme, glycolysis, oxidative stress, pentose phosphate pathway and signalling categories did not show large response ratio changes with Fe resupply.The changes induced by Fe deficiency in the root tip proteome and metabolome from sugar beet plants grown in hydroponics have been studied. More than 140 proteins and 300 metabolites were resolved in sugar beet root tip extracts. Iron deficiency resulted in significant and higher than 2-fold changes in the relative amounts of 61 polypeptides, and 22 of them were identified. Out of 77 identified metabolites, 26 changed significantly with Fe deficiency. In general, our results are in agreement with previous transcriptomic, proteomic and enzymatic studies on Fe-deficient roots.

Our data confirm the increases previously found in proteins and metabolites related to carbohydrate metabolism and TCA cycle pathways. Two major changes induced by Fe deficiency in roots are described in this study for the first time: the increase in DMRL synthase protein concentration and gene expression, and the increase in RFO sugars. The largest change found in the proteome map of root tip extracts from sugar beet plants grown in Fe deficiency conditions corresponded to DMRL synthase, which was detected de novo in Fe-deficient root tips, and is the protein with the highest concentration in these gels . This enzyme catalyses the fourth step of Rbfl biosynthesis, and Rbfl is the precursor of Rbfl sulphates, FMN and FAD, the last one being a cofactor for the root plasma membrane Fe reductase. The expression of BvDMRL decreased drastically 24 h after the addition of Fe to Fe-deficient plants, whereas DMRL synthase protein abundance and Rbfl and Rbfl sulphate concentrations did not change significantly with Fe-resupply in the YZ of root tips , suggesting that the turnover of this protein is slow. Accumulation in Fe-deficient roots of flavin compounds, including Rbfl and Rbfl 3′ – and 5′-sulphate is a characteristic response of sugar beet and other plant species. The exact role of flavins in Fe deficiency is unknown, and it has been hypothesized, based on the similar location of flavin accumulation and Fe reduction and on the fact that the Fe reductase is a flavin-containing protein, that free flavin accumulation may be an integral part of the Fe-reducing system in roots from Strategy I plants [9,28]. On the other hand, these compounds are secreted to the growth media at low pH [6] and, assuming high concentrations at the secretion site, they could mediate extracellular electron transfer between soil Fe deposits and root Fe reductase as it has been described for flavin phosphates secreted by some bacteria. Moreover, excreted flavins could also act as a plant-generated signal that could influence rhizosphere microbial populations, indirectly affecting Fe availability. A major change in carbohydrate metabolism was the large increase in RFO compounds that occurs in roots with Fe deficiency. This increase was higher than that found for sucrose . The total concentrations of raffinose and galactinol were also determined by HPLC-MS, and concentrations of both compounds in the 35-80 nmol g FW-1 range were found in Fe-deficient and Fe-resupplied root tips , whereas concentrations in the +Fe treatment were one order of magnitude lower. The sum of the raffinose and galactinol concentrations in the -Fe, 24h, 72hWZ, 72hYZ and +Fe tissues was 13.9, 7.4, 2.2, 5.1 and 0.6% of the total sucrose, respectively, supporting the relevance of the RFOs changes with Fe status. RFOs have diverse roles in plants, including transport and storage of C and acting as compatible solutes for protection in abiotic stresses. Other explanationfor the large increase in the relative amounts of RFOs could be the ability to function as antioxidants; galactinol and raffinose have hydroxyl radical scavenging activities similar to other soluble antioxidants such as glutathione and ascorbic acid. Since ROS damage and ROS detoxification strategies have been observed in Fe-deficient roots, the increase in RFO concentration could help to alleviate ROS damage produced under Fe deficiency. Moderate increases in sugars commonly found in cell walls such as cellobiose, xylonic acid and arabinose, which may indicate cell wall modifications, were measured in sugar beet Fe-deficient root tips. Changes in cell wall metabolism have been also described in Fe-deficient tomato roots. On the other hand, it has been described that cell wall damage generates oligosaccharides that can act as signalling molecules in stresses such as wounding. The increase in RFOs could also act as a long distance Fe-deficiency signal via phloem sap transport. This is the first description of RFOs accumulation in plants under Fe deficiency, and the physiological implications of this increase deserve further consideration. Most of the proteins found to be up-accumulated in sugar beet root tips by Fe deficiency were identified as carbohydrate catabolism enzymes, including 5 of the 10 glycolytic pathway enzymes , one of the citric acid cycle and fructokinase. Increases in the activities and concentrations of several glycolytic enzymes in root extracts with Fe deficiency have been previously found, including fructose 1,6-bisphosphate aldolase, enolase, triosephosphate isomerase and GADPH . Also, increases in the activities and concentrations of several enzymes of the citric acid cycle with Fe deficiency have been previously reported in root extracts, including MDH. Results are also in agreement with microarray gene analysis in Fe-deficient A. thaliana roots. Increases in the amount of PEPC have been found at the protein level, but this enzyme, with a molecular mass of 110 kDa, was not in the range used in our 2-D gels.

In the absence of major decomposition pathways, PAHs are removed from the atmosphere by dry and wet deposition, which is considered the major source of PAHs in soil . Although PAHs are considered amongst the most widespread organic pollutants in numerous environmental matrices such as soils, sediments, water, and wastewater, the ultimate sink of atmospheric PAHs is soil . Wild and Jones conducted an investigation to quantify the production, cycling, and storage of PAHs in the United Kingdom and determined that more than 90% of the total PAH burden resided in the surface soil. In addition to atmospheric deposition, PAHs can also enter soil systems through disposal of waste materials, creosote use, road runoff, and fossil fuel spills . The fate of PAHs in soil systems is primarily influenced by PAH hydrophobicity and the physicochemical properties of the soil. Due to their strong hydrophobicity and environmental recalcitrance, PAHs are typically associated with nonaqueous phases in soil where they associate into four main compartments: 1) organic matter; 2) the mineral compartment, which includes exposed soil surfaces and surfaces within pore spaces; 3) nonaqueous-phase liquids; and 4) combustion residues such as soot . The degree to which PAHs are retained within the soil system is controlled primarily by soil properties such as organic matter and soil texture as well as the PAH physicochemical properties.

Because of the nonpolar, hydrophobic nature of PAHs, soil organic matter is considered the most important sorbent of PAHs . The organic matter or total organic carbon can also act as a carrier for the vertical migration of PAHs from the soil surface .Polycyclic aromatic hydrocarbons have a very strong affinity for soil organic matter via π-π interactions between the aromatic structure of PAHs and aromatic moieties of organic matter . In soils with low amounts of organic matter or total organic carbon content,fodder systems for cattle the soil texture plays a critical role in the environmental fate of PAHs. For example, Karickhoff et al. reported an increase in pyrene adsorption coefficients with an increasing clay content . In addition, decreasing particle size is typically associated with concomitant increases in the proportion of HMW and decrease in the proportion of LMW PAHs . The greatest PAH soil concentrations in numerous studies have been observed in the silt-sized soil fraction, which was potentially due to the silt fraction containing the greatest concentration of soil organic matter and its associated aromatic structures for binding . In addition, clay fractions are characterized by a very high specific surface area, abundant surface charge, and a high organic matter density, all of which provide a large number of sorption sites for PAHs . Sorption of PAHs to soils generally entails an initially rapid and reversible phase followed by a period of slow sorption occurring over a period ranging from weeks to years, and this slow sorption leads to a chemical fraction that resists desorption and biodegradation . Increasing contact times between PAHs and soil organic matter or fine soil fractions can also result in the “aging” effect or sequestration of PAHs . This process involves the continuous diffusion and retention of PAHs within the solid phase of organic matter and also in nanopores or voids in the organic matrix, thus blocking PAHs from abiotic and biotic loss processes.

As a result of the strong association of PAHs with the nonpolar soil organic fractions, PAH bioavailability, or PAH concentrations in the aqueous phase that are directly available to soil microbes for degradation, is generally low . The bioavailability of PAHs is determined by two main factors, which are the rate of transfer of PAHs from the soil to the living cell and the rate of uptake and metabolism . Bio-availability is an important concept with regards to PAH-contaminated soil remediation and risk assessment as microbial degradation constitutes the major dissipation pathway for PAHs compared to other processes such as evaporation, photolysis, and plant uptake . The extent and rate of microbial degradation of PAHs in the terrestrial environment is influenced by a variety of abiotic and biotic factors which include temperature, pH, aeration, accessibility ofactors, which are the rate of transfer of PAHs from the soil to the living cell and the rate of uptake and metabolism . Bio-availability is an important concept with regards to PAH-contaminated soil remediation and risk assessment as microbial degradation constitutes the major dissipation pathway for PAHs compared to other processes such as evaporation, photolysis, and plant uptake .f nutrients, microbial population, contaminant bio-availability, and physicochemical properties of the PAH . Typically, the rate of PAH biodegradation is inversely proportional to the number of aromatic rings or molecular weight of the PAH . For example, half-lives of phenanthrene in soil may range from 16 to 126 days, while half-lives of HMW PAHs such as benzo[a]pyrene may Mansour, 2016. Some of the major PAH-degrading genera in soils include Mycobacterium, Sphingomonas, Bacillus, Pseudomonas, and Rhodococcus . The ability of soil microbes to degrade PAHs is determined by 1) the ability of bacteria to transport the PAH into the cell, 2) the physicochemical properties of the PAH as a substrate for available microbial enzymes, and 3) the suitability of the PAH as an inducer for the appropriate transport or degradative enzymes .

As shown in Figure 1.3, there are two primary mechanisms involved in the aerobic metabolism of PAHs by soil bacteria . The principal mechanism for aerobic PAH degradation by soil bacteria involves the dioxygenase/monooxygenase enzymes, which incorporates a hydroxyl group derived from molecular oxygen into the aromatic nucleus, resulting in the oxidation of the aromatic ring to form cis-dihydrodiols . This initial ring oxidation is considered to be the rate limiting step of the PAH bio-degradation process in soil systems . The cis-dihydrodiols are stereoselectively dehydrogenated by cisdihydrodiol dehydrogenases to form dihydroxylated intermediates, called catechols . Subsequently, the catechol may then be cleaved by intradiol or extradiol ring-cleaving dioxygenases through the ortho or meta-cleavage pathway to tricarboxylic acid intermediates such as succinic, fumaric, pyruvic, and acetic acids and acetaldehyde. These TCA intermediates are utilized for cell-protein synthesis and energy by microorganisms with the final production of carbon dioxide and water . Soil bacteria can also degrade PAHs via the cytochrome P450-mediated pathway to form transdihydrodiols . Polycyclic aromatic hydrocarbons are biodegraded by soil microbes in one of two ways, either as the sole carbon and energy source or by cometabolism . Because PAHs occur in the environment as complex mixtures of LMW and HMW PAHs, cometabolism is an important interaction that transforms non-growth substrate PAHs, particularly HMW PAHs, in the presence of growth substrates to enhance PAH degradation . Numerous soil microbes have been isolated that utilizeLMW PAHs such as naphthalene and phenanthrene as their sole carbon source and throughout the past decade, multiple soil bacteria have been discovered and isolated that are capable of utilizing HMW PAHs as sole carbon sources . For example, Mycobacterium vanbaalenii PYR-1, an isolate from an oil-contaminated estuary near the Gulf of Mexico,fodder sprouting system has been utilized to determine the complete pyrene degradation pathway using various metabolic, genomic, and proteomic approaches.Pyrene is often used as a model compound for HMW PAH biodegradation because it is structurally similar to several carcinogenic HMW PAHs.The primary pathway for pyrene degradation by M. vanbaalenii PYR-1 is deoxygenation by dioxygenase and monooxygenase at the C-4 and C-5 positions to produce both cis– and trans-4,5-pyrenedihydrodiol, respectively. The metabolite undergoes further metabolization involving more than 20 enzymatic steps utilizing rearomatization, decarboxylation, and oxygenation to produce phthalate that is further transformed to the TCA cycle via the β-ketoadipate pathway . Another pyrene degradation pathway exists for M. vanbaalenii PYR-1 that involves the oxidation of pyrene at the C-1 and C-2 positions to form O-methylated derivatives of pyrene-1,2-diol as a detoxification step . Mycobacterium vanbaalenii PYR-1 is also capable of degrading or transforming biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, benzo[a]anthracene, and benzo[a]pyrene, thus making this bacterium an effective candidate for the  bio-remediation of PAH-contaminated soils. Because PAHs are toxic, ubiquitous pollutants that are highly resistant to degradation in contaminated soils, remediation of PAH-contaminated sites is critical for protecting human health and the environment. Several physical and chemical PAH remediation technologies such as incineration, excavation and land filling, UV oxidation, and solvent extraction have been used to clean up PAH-contaminated soils.

However, these remediation methods have several negative aspects including cost, regulatory burden, and that some of these conventional methods do not result in PAH dissipation, but rather transfer from one environmental compartment or form to another . These limitations of conventional treatment methods have led to the increased use of bio-remediation techniques at PAH-contaminated sites as they are considered to be safe, environmentally-friendly, and cost-effective . Bio-remediation involves the utilization of biological processes or activity of microorganisms to remove pollutants from contaminated matrices to achieve concentrations that are acceptable according to health and regulatory standards. Bio-remediation technologies can be classified into two main categories, in situ or ex situ. In situ bio-remediation technologies target contaminant removal or attenuation under natural environmental conditions without the need for excavation, whereas ex situ bio-remediation processes involve the physical removal of the contaminated material for remediation off-site . Therefore, in situ remediation practices are particularly effective for widely dispersed contaminants and are typically less expensive than ex situ approaches. Additionally, exposure to site workers to hazardous pollutants is minimal and in situ treatments also allow for remediation in inaccessible environments . Because of these advantages, in situ bio-remediation constitutes approximately 25% of all remediation projects for contaminated sites . However, because in situ bio-degradation does not disturb the contaminated soil, remediation has been found to be more variable due to the natural environmental conditions . Although ex situ bio-remediation is less economical compared to in situ treatments, ex situ bio-remediation methods are less limited by environmental factors that could adversely affect the remediation efficacy and the physical and chemical conditions can be manipulated before and during degradation . Ex situ bio-remediation generally requires less time to achieve efficient contaminant remediation since optimal remediation conditions can be monitored and modified as needed . The successful implementation of PAH-contaminated soil  bio-remediation treatments depends on a multitude of factors that can be categorized into three main domains encompassing PAHs, environmental conditions, and soil microbial communities . Factors involving PAHs include the physicochemical properties of PAHs, concentration and toxicity, and the length of time the PAHs have been in contact with the soil and their associated bio-availability. Because in situ bio-degradation treatments are currently a common type of remediation practice, environmental conditions in soil that must be evaluated for effective PAH bio-degradation include soil type, organic matter content, nutrient availability, moisture, temperature, pH, and presence of oxygen or alternative electron acceptors. Soil microbial transformations are the major process governing degradation of PAHs in contaminated soil, and therefore critical factors related to soil microbes include the presence of a soil microbial community capable of degrading PAHs, which encompasses microbial type, abundance, distribution, acclimation or previous exposure, and metabolic rate. Natural or bio-attenuation is an in situ  bio-remediation technique that involves passive remediation of a PAH-contaminated site without any external alterations . Because there are no site modifications, natural attenuation is considerably cheaper than other  bio-remediation methods. However, one of the major limitations of bio-attenuation is that the process can take extended periods of time to achieve appreciable levels of PAH dissipation. Bio-degradation is especially slow in PAH contaminated sites that have been in contact with non-polar soil domains for a prolonged duration, resulting in decreased PAH bio-availability.Due to the environmental stability of PAHs, the majority of PAH-contaminated sites undergo engineered or enhanced bio-remediation, which involves site modifications to enhance the extent and rate of PAH degradation . Typically, enhanced bio-remediation techniques for PAH-contaminated soils involve bio-augmentation, bio-stimulation, surfactant amendment, phytoremediation, or an integrated combination of bio-remediation techniques.Bio-augmentation is the process in which contaminant-degrading microorganisms are introduced into the soil as single strains or bacterial consortia to increase the rate of PAH bio-degradation. The main advantage of bio-augmentation is the relatively low cost of inoculating soil microorganisms into the soil system.Bio-augmentation is an effective method to increase bio-remediation efficacy for PAH-contaminated soils that possess low numbers of native PAH-degrading soil microbes or when the native soil microbial population does not exhibit sufficient metabolic activity to result in PAH dissipation.