Month: January 2023

A distribution ratio value of one indicates that the rate of prey available in the capture zone is equal to that in the water above the sea anemone . A ratio greater than one indicates that more prey were swimming in the water above the sea anemone than were swimming in the water that passed through the sea anemone’s capture zone.Capture efficiency was defined as the proportion of encountered prey that was captured.Trapping efficiency was calculated rather than feeding efficiency because the duration of experiments was short relative to the average ingestion times for sea anemones , thus most captured and retained prey were not ingested during the videos. The duration of the experiments was chosen to minimize the chances that prey would show a decrease in swimming and escape behaviors . Using prey encountered as the denominator when calculating rates for both capture and trapping efficiencies enabled these efficiencies to be compared with published feeding efficiency data for a zooplanktivorous fish . In addition, these efficiencies could be directly related to ecological models that estimate suspension feeding rates from encounter rates. We found that increasing the “strength” of ambient water flow enhanced rates of some steps in the feeding process and decreased others for a passive suspension-feeding predator eating zooplanktonic prey that have strong escape responses. Encounter rates normalized to capture volume depend on the speed of the water moving through the capture zone,dutch bucket for tomatoes and on the turbulence of the flow that stirs the water carrying new prey into prey-depleted water in the capture zone. Therefore, we expected that higher peak wave velocities and turbulence would enhance encounter rates for sea anemones, and this effect was observed for dead copepods.

We also found that encounter rates were greater in strong waves than in weak waves for living copepods, but there was high variation in encounter rates for these swimming prey, thus the difference was not statistically significant. If the tentacles of a predator are deformed by hydrodynamic forces, the volume of the capture zone can be reduced as the velocity of ambient water increases , thereby reducing the rate of encounters in strong waves. However, such deformation of the capture zone did not occur for A. elegantissima in the flow regimes used in our experiments, and all rates were normalized to capture volume. In weak waves more copepods swam above rather than in the capture zone of the sea anemone, whereas in strong waves the copepods were evenly distributed vertically within the water column near the sea anemone. We expected that this difference in vertical distribution would further enhance encounter rates in strong waves compared with weak waves. However our results suggest the variability in the behavior of living copepods plays an important role in shaping encounter rates. We expected that capture rates would be higher for sea anemones feeding in strong waves than in weak waves. We observed that fewer of the copepods passing through the capture zone executed escape maneuvers that avoided the predators’ tentacles in strong waves than in weak waves. Similarly, Heidelberg et al. found that zooplankton could avoid or escape benthic suspension-feeding corals under conditions of slow flow. Likewise, Robinson et al. showed that in weak waves , copepods executed escape maneuvers that enabled them to avoid being captured by a siphon that simulated suction feeding by a predatory fish, whereas in strong waves , the copepods were unable to detect hydrodynamic cues of the siphon and did not swim to avoid capture. Thus, Robinson et al. found higher capture rates for their siphon in strong waves than in weak ones. In our study of A. elegantissima we also found that capture rates were 75% greater in strong waves than in weak waves, but this difference was not significant due to the high variability of encounter rates and the low capture rates of A. elegantissima .

Retention rates were the same for both conditions of flow , and represented less than 0.5% of the prey encountered by a sea anemone. In turbulent and wavy flow, hydrodynamic forces can sweep captured prey off the tentacles of the predator . In slow flow with low turbulence, fewer prey are encountered and captured per time, but a greater proportion of them are retained by the predator than in faster, more turbulent flow. The net result is that the rate of retention of prey of a passive suspension-feeding benthic predator did not change as wave peak velocities and turbulence increased. Nematocysts on the tentacles of sea anemones adhere to prey that contact the tentacles. The adhesive strength of the nematocysts of A. elegantissima was found to be independent of habitat, availability of food, exposure to light, and species of symbiont . This suggests that the lower retention of prey by A. elegantissima in strong waves was not due to physiological differences in the ability of tentacles to hold onto prey, but rather was due to higher hydrodynamic forces dislodging prey in the more rapid flow. Stronger waves increased feeding efficiency for benthic zooplanktivorous fish but had no effect on feeding efficiency for a passive suspension-feeding sea anemone . In both cases, the escape behavior of zooplanktonic prey in slow flow resulted in lower capture rates than in faster flow. As peak velocities and turbulence increased, fewer of the prey moving through the capture zone were stimulated to execute escape maneuvers in response to either type of predator. Although the fish reduced feeding effort and the time spent feeding during a wave cycle, their foraging efficiency improved in stronger waves. These active predators were able to modify their behavior in a way that minimized expenditure of energy for foraging in faster, more turbulent flow, yet their feeding rates increased because fewer of their prey tried to escape capture. In contrast, passive suspension feeders do not swim after their prey and thus probably expend less energy per prey captured than do darting fish. Passive A. elegantissima maintained the same trapping efficiency in both weak and strong waves because, although they capture more prey per unit time in stronger waves, they also lose more of the prey that they catch in the faster, more turbulent flow.

This study reveals the importance of both the behavior of the prey and the flow of ambient water in determining the predation rates of benthic predators. In slow water feeding rates on non-swimming or weakly swimming zooplanktonic prey might be higher than on prey with strong escape responses. Furthermore, studies of feeding by benthic predators on passive particles that have no swimming behavior might overestimate feeding rates. Likewise, feeding studies of shallow-water benthic predators carried out in flumes with steady-state water flow that does not mimic the waves and turbulence to which such predators are exposed in nature could yield unrealistic feeding rates because actively-swimming prey might be able to avoid predators more readily in steady flow with less turbulence and no back-and-forth flow of waves, and the ability of predators to hold on to captured prey exposed to steady drag forces might be different from their retention abilities when prey are exposed to the pulsatile hydrodynamic forces in turbulent waves. Predators capture prey in complex and variable environments. In the ocean, bottomed welling organisms are subjected to water currents, waves, and turbulent eddies. For benthic predators that feed on small animals carried in the water , flow not only delivers prey but can also shape predator-prey interactions.Turbulent flow can stir the fluid environment, enhance prey delivery ,blueberry grow pot reduce the ability of the prey to detect and avoid predation , or wash prey off capture surfaces . How does flow impact predator-prey interactions between a benthic suspension feeder and zooplanktonic prey? Studies of passive suspension feeding have been done experimentally in unidirectional flow for corals , bryozoans , sea pens , and sea anemones , but few studies have examined the effects of waves and turbulence on suspension feeding . Experiments have generally focused on the consumption of non-motile prey, yet actively swimming zooplankton can contribute significantly to the diet of passive suspension feeders . Brine shrimp neutrally-buoyant cysts, or hatched nauplii have been used as live prey . A small number of studies of benthic suspension-feeding predators that used zooplankton prey suggest prey swimming and escape responses might impact capture rates. Although Artemia spp. nauplii can swim, they do not exhibit escape behavior. Previous research on this ubiquitous feeding strategy has been useful in estimating how much suspension feeding can contribute to ecological links between pelagic and benthic communities. A common measure of interactions between predator and prey are encounter rates . For sedentary predators that rely on ambient water to deliver food, encounter rates are dependent on the ambient flow and prey behavior. Humphries suggested the efficiency of particle capture might be higher than estimated for filter feeders in low flow. Chapter 2 suggests that retention is not 100% as is often assumed, and can be a small fraction of the initial encounter rate . The presence of neighbors affects flow around benthic suspension feeders. Okamura found that the feeding rate of an encrusting bryozoan colony was enhanced in the presence of a neighboring colony. The feeding current from the upstream colony drew currents closer to the substratum so that downstream zooids captured more prey.

Passive suspension feeders do not generate their own feeding current. However, ambient flow over a bumpy surface of organisms can generate eddies from which extended tentacles can capture prey from turbulent wakes . The objective of this study was to measure how ambient water flow, prey swimming behavior, and the presence of neighbors affect predation by benthic passive suspension feeders. I addressed this question using sea anemones, Anthopleura elegantissima , which live in wave-dominated flow habitats , feed on a variety of zooplankton that exhibit different swimming behaviors, and live in dense colonies surrounded by conspecifics . In this study we used prey with different swimming behaviors: 1) the calanoid copepod Acartia spp., which has a well-characterized escape response , 2) heat-killed Acartia spp., which are non-swimming prey with the same size, shape, and drag as living Acartia spp., and 3) nauplius larvae of Artemia spp., which are swimming prey with no escape behavior. We examined the effects on feeding of the peak water velocities and turbulent kinetic energy of the wavy ambient water flow, prey swimming and escape maneuvers, and upstream and downstream neighbors. Understanding how zooplankton swimming and the effect of neighboring suspension feeders in realistic flow conditions can contribute to predictions about the link between pelagic and benthic communities based upon flow and prey type. All sea anemones, Anthopleura elegantissima, were collected in October 2012 and May 2013 from Horseshoe Cove, in the Bodega Marine Reserve along the Sonoma Coast in California . The clone from which sea anemones were selected was the same bed over which flow measurements were collected . Sea anemones that were next to one another and positioned away from the edges of the clone were selected. Since A. elegantissima forms genetically identical polyps by binary fission, adjacent sea anemones were likely from the same clone though genetic testing was not performed. Sea anemones were gently peeled from the rock, and each individual was placed in an air-filled plastic bag. The bags were transported to the University of California Berkeley in a cooler kept at 10-15°C. The anemones were housed in a 19-L aquarium filled with recirculating filteredsea water with a salinity of 35‰. The aquarium was kept in a temperature-controlled cold room at 10-15 °C and exposed to a photoregime with 12 hours dark and 12 hours light provided by full-spectrum fluorescent bulbs . The sea anemones were placed on a suspended plastic mesh substratum to prevent attachment to the aquarium walls, and were fed hatched Artemia spp. nauplii once a day, but were not fed 24 hours before use in flume experiments. For flume experiments, sea anemones were transported to the University of North Carolina Wilmington via overnight delivery. Individual sea anemones were placed in plastic bags that were filled with oxygen. The bags were packed into a Styrofoam cooler over a base of ice packs and a middle cushioning layer of newsprint. Upon arrival sea anemones were removed from the plastic bags and housed under aquarium conditions identical to those previously described.

The relation between islands as non spaces and designed places is a convoluted one, but it is a relation that must be kept in mind in the discussion that follows. I will take this section of the introduction to explain my use of the concept of design as it relates to the scientific and social construction of islands and the themes of visitation, vulnerability, and biocomplexity. As mentioned above, the phrase itself, “islands by design,” is the name of a Bahamian environmental assessment and design consultation firm whose projects have included the design plans and use concepts for large marinas and planned developments on several islands. Much like “living laboratory,” I have borrowed this idea and redefined it to suit my purposes. I also borrow here from the History of Science including the history of Anthropology, and I pick examples from Island Archeology, Island Ecology, Tourism Studies and Urban Studies to talk about the power of design and the way it can engender various forms of experience.A columnist in one of The Bahamas’ national papers stated in print in 2007 that, “planning is the key to sustainability,” and notions of design and planning were conspicuous throughout my work with field researchers, conservation managers, and government officials. Conservation and management organizations are currently going through the process of designing and developing science based management plans for everything from individual protected areas to national protected area master plans with guidance and design models from international conservation organizations and consultants; The design of scientific field research projects is often as important a product as the research results when it comes to forms of knowledge created by research scientists and students; Design for the curricula and scope of a new degree program in “Small Island Sustainability” at the College of The Bahamas is currently underway; The newly created Ministry of Environment is redesigning the roles of its own governmental institutions,u planting gutter becoming a central player in the planning processes for projected social and economic development in the country; the Department of Sustainable Tourism is redesigning the ways in which the islands of The Bahamas should be marketed to foreign visitors and reconceptualizing the form tourism infrastructure should take in various locations; The Ministry of Tourism is considering how to redesign its “tourism product” to not only mitigate and adapt to threats of climate change but to discover ways in which to profit from a newly climate aware target traveler.

This is to name only a few examples of the rootedness of design schemes in the Bahamian milieu. I note that design processes come prior to planning, that planning is an aspect of the enactment of thought and design, and the newspaper quote could more accurately say, “design is the key to sustainability.”“Design,” as a term, implies the creation of plan, an intention, a pattern, a contrivance, and productive work to “create the form or structure of something.” This last implication is one of the most important for me, in that I would like to make the creative and formative aspects of field science, sustainable development, and environmental management explicit. Islands by design, then, references the multiple ways in which nature and natures, life and lives, matter and materials, places, spaces, and objects, etc. become conceptually and aesthetically formed and reformed as part of the work of science-based environmental, touristic, and sustainable planning in The Bahamas. The point is not only that natural science and scientific expertise is touted as the position of authority from whence ecological problems can be diagnosed and framed in certain circles- the point is also that natural science and scientific expertise is in the position to influence and set the terms for how solutions will be studied, formed, and enacted. Experts, albeit a somewhat motley crew with disparate backgrounds when it comes to nationality, discipline, realm of experience and outlook, can be considered the designers of the living laboratory. I see the living laboratory as a site of great productivity in the development of the conditions of possibility. With the notion of islands by design, I am in essence proposing another direction for political ecology or environmental anthropology to take, or perhaps another set of tools to add to the rich set which this unruly sub-discipline already possesses. I think that focusing on and conceptualizing design is one way to attempt to describe the work, The study of scientific laboratories as special spaces for social production became a characteristic of the sociology of science beginning in the 1970’s. Ethnographers of science sought to decentralize the analytic focus on human agency through the study of lab practices with an attention to the ways non human substances have a “material agency” in the production of scientific facts.Latour and Woolgar’s Laboratory Life introduced readers to the daily lives of scientists involved in the construction of such facts. Famously using the trope of an anthropologist in the field attending to “science in the making” they noted that all new laboratory activity hinges on previously constructed and accepted facts embodied in lab instruments and that laboratories undergo continual “micro-processes” of negotiation in their operation as a system of fact construction.

The vast amount of literature, documents, and “material dictionaries” produced by labs become important as part of the fact making process and the material arrangements of the lab produce a reality that would not exist without it.They write, “It is not simply that phenomena depend on certain material instrumentation; rather, the phenomena are thoroughly constituted by the material setting of the laboratory.” The laboratory is therefore a space which embodies the work of other fields, reified in its arrangement and equipment, to legitimate the reality it produces; reality and nature are the byproducts of the scientific stabilization of facts in the lab, rather than the cause. In an important observation, Mol and Law note that laboratory experiments are “simplificatory devices: they seek to tame the many erratically changing variables that exist in the world, keeping some stable and excluding others from the argument.”Their point is that simplifications are used to justify action, yet they qualify this by stating that simplification should not be denounced off-hand as it is a productive force, especially when it comes to knowledge practices. As mentioned above, the notion of The Bahamas as a living laboratory for certain sciences and disciplines is a framing concept for this dissertation. The phrase itself, “living laboratory,” originated from an interview with a Bahamian government official who hoped that the international research community might increasingly come to see The Bahamas as a living laboratory for environmental research,planting gutter and it is a concept I have borrowed and stretched to fit my own interests. This can be differentiated from Harre’s use of the term “living laboratory” to describe how living things become crucial aspects of scientific laboratory experiments.For Harre, living things are transformed, in the space of the lab, into instruments and apparatus for measurement, detection, simulation, and experimentation. An important part of the lab “has always been organic, apparatus and instruments constructed from living materials or materials that were once alive.” He provides the example of the fly, Drosophilia, and genetics research: the fly became a piece of lab equipment in that it was a means of producing specific kinds of knowledge as a standardized organism in a system of production; the fly was a designed artifact. Harre writes, “glass must be skillfully blown to become a flask.So too the living material must beshaped and transformed into devices in the living instrumarium.”

It is this experimentation with living things that he calls the living laboratory and he thus uses the phrase in a slightly different valence and for different purposes. My use of the term is also about living things, but as I will show below, it is about the production and manipulation of ideas framing life and living as much as it is about the scientific use of living things in The Bahamas today. His focus is on animal bodies and individuated organisms, while mine is on the way in which processes and systems are identified and designed as sites for experiments or environmental management and the way these “natural” processes are conceptually linked to other enterprises. Following Latour and Woolgar, but taking them into the living laboratory of The Bahamas, this dissertation starts from the point that nature and its scientific forms, i.e. habitats, ecosystems, biodiversity, etc. are not at all given. These are the reified form of past scientific theories and practices made in other laboratory situations at other times. My research in The Bahamas has been centered around contemporary laboratory situations which can be very generally referred to as social science, ecological and environmental research, and sustainable economic development- processes of human life and living, non-human life and living, and economies of visitation and investment. A focus on current events in The Bahamas could very well entail investigating any one of these arenas, and these realms are discursively separate in that they are often discussed as separate categories of inquiry which can be related post facto. But an investigation of The Bahamas as a living laboratory, as a site for active experimentation and exemplification, requires the recognition that these categories are interrelated and coconstitutional. Therefore, I should say at this point that I am working within my own laboratory situation with this dissertation in that I am, as mentioned above, conducting a sort of experiment by bringing a host of scholarly angles to bear on the situations and events I was and continue to be part of and witness to.For my purposes here, I will define a laboratory as a physical, conceptual, and designed space in which ideas can be tested and processes evaluated through experimentation. A laboratory implies scientific research. A laboratory space also implies that matter and materials are manipulated as part of the process of experimentation to achieve a desired outcome or to discover what comes out of a given design process. A living laboratory extends the notion of the physical and conceptual space for scientific research into “the field”- into “real” time and “real” life processes in situ, though this is importantly no less designed. Manipulation can then become the act of influencing pre-existing components and factors and even creating the possibility for the existence of those very factors. The living laboratory is thus a frame in which I call to attention the shifting relationship between fieldwork and the lab, which “traditionally” are separate physical and conceptual spaces- data in various forms is discovered, collected, and removed from the field, in which scientists are explorers and adventurers, and taken to the lab to be analyzed. A living lab merges the processes of exploration, discovery, collection, and analysis and alters what forms are acceptable and accessible as data. Field ecologists, biologists, and social scientists develop contingent relationships to fields based on specific forms of presence and practice. I have developed this observation in contrast to recent observations about the changing science of genomics in which the active presence of science in the field is said to have lessened and where scientists and students can “get in and out” with their data and samples without caring about the particular relationships and socialities of their field site.I argue that today, the field and the lab are synonymous and “the island is a laboratory” takes on a holistic and socialized meaning for the field life sciences- the what counts as experimental material or the subject of field research is increasingly designed as an amalgam of natural and social systemic processes. As I describe below, this has implications for defining what manner and matter of “life” the life sciences produce knowledge about.The panelists prescribed a form of research for a biocomplexity paradigm, stating that biocomplexity questions needed to be approached in an interdisciplinary manner and that they were different from typical research questions. The complex interactions that occur in the “real world” on multiple scales can only be understood through “combined efforts of scientists” from many disciplines who are allowed to work at the relevant “temporal and spatial scales.”

The modification of JA by jasmonate carboxyl methyltransferase converts it into the volatile compound methyl JA . This reaction is presumed to take place in the cytoplasm . MeJA mediates both developmental processes and defense responses against biotic and abiotic stresses . Additionaly, MeJA is a gaseous compound, and can thus act as airborne signals to mediate inter-plant communication. This means that neighboring plants can be signaled if stressed are present . Research has indicated that a positive feedback loop exists for JA biosynthesis . In Arabidopsis, VSP expression is selectively induced by JA and not OPDA, while both molecules induce defense responses. This demonstrates that multiple mechanisms are responsible for the transduction of JA-related signals, which are selectively activated in response to differing stimuli . The bacterial plant pathogen Pseudomonas syringae produces a JA-Ile-like compound termed coronatine , which suppresses some SA-immune responses by activating the JA signaling pathway . The Arabidopsis mutant coronatine-insensitive 1 was identified due to its insensitivity to plant growth inhibition by COR and OPDA . In addition, this mutant is male sterile and more susceptible to some pathogens and pests . COI1 encodes a 66-kD protein that contains an N-terminal F-box motif and a leucine-rich repeat domain . F-box proteins recruit regulatory proteins as substrates for ubiquitin-mediated degradation. This requires them to associate with Skp1 and Cullin to form an E3 ubiquitin ligase termed SCF complex which is important for JA responses .

JA–Ile facilitates binding of SCFCOI1 with the jasmonate ZIM-domain 1 protein . JAZ proteins are substrates for SCFCOI1 and negatively regulate JAresponses . In Arabidopsis these proteins belong to a 12 member family and contain a conserved motif at the C-terminus . Many JAZ mutants displayed no discernible phenotypes,vertical rack system possibly due to functional redundancy . Mutants of jaz10 are hypersensitive toJA, but the lack of obvious phenotypes in other jaz mutants suggests functional redundancy among other family members . The jaz1 mutant on ther other hand displayed male sterility, JA insensitivity, and increased resistance to infection by the bacterial pathogen P. syringae pv. tomato . The JAZ proteins homo- and hetero-dimerize by way of a conserved TIFY domain and also bind AtMYC2 and interact with COI1 through their C-terminal JAS domains . AtMYC2 is a helix-loop-helix transcription factor that acts as a key regulator of JA responses in plant-microbe interactions . When JA-Ile or COR bind to SCFCOI1 complexes, this promotes the ubiquitination of JAZ proteins leading to their degradation. Once degraded the JAZ-dependent repression on AtMYC2 is relieved and JA-responsive genes are activated . The JAZ proteins then recruit a co-repressor TOPLESS 8 and TPL related proteins through a adaptor protein called Novel Interactor of JAZ . NINJA and TPL proteins act as negative regulators of jasmonate responses . Resistance to specific pathogens conferred through JA signaling show little overlap in transcriptional changes. Molecular recognition of necrotrophic pathogens can trigger increased JA and ET synthesis as well as expression of defense genes, such as PDF1.2. Defensins, one group of ubiquitous peptides involved in innate immune response, are found in organisms ranging from invertebrates to plants. They are small , basic, cysteine-rich proteins encoded by multigene families, similar in complexity to those encoding other defense-related proteins . Defensins were first discovered in rabbits in 1984 and described in wheat and barley in 1990 .

PDF genes were later discovered in Arabidopsis and encode small peptides originally labeled as γ-thionins . γ-thionins were later renamed plant defensins to emphasize their structural similarity to mammalian defensins. The ancestry of defensins is thought to pre-date the divergence of plants and animals . Defensins are described in two separate kingdoms and further divided into five classes. Class 1, Class 2 and Class 3 are found in mammals and birds, while Classes 4 and are found in insects and Class 5 in plants . The primary structures of defensins vary between organisms. PDF proteins contain eight conserved cysteines . They also include a common fold, which is formed by a β-sheet and an α-helix steadied by disulfide bridges and capable of stabilizing the entire protein . Arabidopsis PDF genes can be separated into three families, each encoding closely related peptides. Family 1 consists of PDF1.1-1.5, family 2 consists of PDF2.1–2.6, and family 3 consists of At4g30070 and At5g38330 . The predicted mature peptide sequences encoded by PDF1.2a/b/c are identical. PDF1.2aand PDF1.2c form a tandem repeat on chromosome 5 and PDF1.2b is found on chromosome 2 directly adjacent to PDF1.3 . The high sequence similarity between PDF1.2a/b/c and PDF1.3indicates that little evolutionary time has passed since their duplication. More amino acid variability is evident within the second family but this family also has genes that occur in a tandem array . PDF2.1 is expressed specifically in roots, siliques, and seeds . PDF2.2 is expressed in all organs of healthy plants except stems and seeds . PDF2.3 is expressed in all organs excluding roots and is not upregulated in response to pathogen infection . PDF1.2a is induced in leaves upon infection of pathogens such as Botrytis cinerea . PDF1.1 expression is largely seed specific and may protect seedlings against pathogens . PDF1.1, 1.2, 2.1, 2.2 and 2.3 display distinct organ-specific expression patterns . PDF1.1, PDF2.1, PDF2.2 and PDF2.3 are expressed constitutively which means that most plant tissues constitutively express two or more defensin genes at any given time . Therefore, it is likely that specific peptides may be expressed during specific situations and sites . PDF1.4 and PDF2.4 appear not to contain predicted signal peptide sequences, suggesting they stay in the cytoplasm while the others PDF proteins are secreted.

Alternately, PDF proteins may overlap while acting distinctly to cover gaps in each-others’ activity spectrum . From the third family found within Arabidopsis, PDF3.2 encodes a protein of 129 amino acids with a C-terminal domain that has the conserved cysteine pattern shared by all plant defensins . PDF3.1 encodes 122 amino acids with 56% identical residues to the protein encoded by PDF3.2. These proteins could be fusion proteins or precursors but more research is necessary to determine their functions . Transgenic expression of PDF genes leads to fortification of tissues against pathogen attack. Two PDF proteins, one from dahlia and one from radish were found to inhibit the growth of Neurospora crassa. One observed response after infection with N. crassa is a change in fungal cell ion fluxes,mobile grow rack but this is believed to be a secondary effect, not a direct result of the PDF anti-fungal activity . There are two models describing possible modes-of action for the antimicrobial activity of PDF proteins. One model proposes that pores form in microbial cell membranes. The second model proposes that PDF proteins bind onto the anionic lipid head groups disrupting the stability of the phospholipid bilayer of microbial cell membranes . Binding sites for these PDF proteins to sphingolipids found on the cell surface in fungal membranes have been demonstrated . This suggests that the antimicrobial activity of PDF proteins is dependent on their ability to bind to a target in the membrane of an infecting pathogen . No similar activity has been shown against pathogenic fungi though. Aside from the many possible roles of PDF genes it has been established that many PDF proteins isolated to date display antifungal activities against a broad range of fungi. Monocot PDF proteins may also inhibit α-amylase, an enzyme found in insect guts . This possibly hampers the insect’s ability to digest plant material . It appears though while some alternately inhibit α-amylase activity and protein synthesis that PDF proteins do not display both activities concurrently. Additionally, most plant defensins do not display antibacterial activity which has been suggested to be a result of a infection pressure from fungal as opposed to bacterial pathogens on plants . Additional biological activities of PDF proteins include the ability to inhibit protein translation in a cell free system, inhibit proteases, and inhibit the growth of microbes . They may also act as mediators of zinc tolerance and inhibitors of ion channels, or exhibit activity against mammalian cells and enzymatic activity controlling the redox state of ascorbic acid . This diverse set of functions may be attributed to differences in the primary structures of defensins. PDF activities range from antimicrobial and insecticidal to anti-parasitic.

Due to their broad range of target organisms, PDF overexpression may be a suitable strategy for crop protection. While R-genes typically confer a narrow range of resistance, PDF genes may provide broad-spectrum resistance against multiple types of pathogens. If introduced into plants PDF genes may offer defensive advantages in addition to enhancing the plants ability to combat biotic stressors . Arabidopsis also contains over 300 Defensin-like genes and the evolution of DEFL-, PDF-, and R-genes bears remarkable similarity that may be due to their evolution through similar mechanisms . Members of these three groups can be found as single genes or in clusters, which arose through duplication, recombination, or diversifying selection . Cis-elements discovered within the promoter of PDF1.2b include a GCC box important for the ET response, a stress responsive G-box, a drought responsive element , and JA-responsive elements and . GCC boxes are commonly found in the promoters of genes which encode defenserelated proteins and are known binding sites for some AP2/ERF transcription factors . G-boxes regulate genes in response to environmental conditions such as red and UV light, anaerobiosis, and wounding and can be bound by certainbasic leucine zipper transcription factors . Transcription factors that positively affect PDF1.2b mRNA levels are the AP2/ERF-domain transcription factors ERF1, ORA59, AtERF1 and AtERF2, as well as the basic leucine zipper transcription factor TGA5 . Negative effects were observed upon over expression of AtERF4 or WRKY70, as well as in a triple mutant, where function of TGA2, TGA5 and TGA6, three redundantly acting TGA bZIP factors, is abolished . TGA2, TGA5 and TGA6 are also commonly required for the activation of JA- and ET-dependent defense mechanisms that counteract necrotrophic pathogens . TGA2 is the only transcription factor, for which direct interaction with the PDF1.2 promoter has been demonstrated yet . ORA59 and ERF1 are believed to bind directly to the promoter and AtERF1 and AtERF2 indirectly . ORA59 is the primary positive regulator of PDF1.2b expression in response to JA/ET and it binds GCC boxes . Since most PDF genes belong to small families of closely related members, studying their function can be difficult using traditional genetic methods. Hpa is a useful model pathogen to study the genetics of plant-pathogen interactions . However, unlike infections with bacterial pathogens, inoculation with Hpa does not result in synchronous and uniform responses in host plants. This is mainly due to the unsynchronized germination of Hpa spores upon spray inoculation. Furthermore, transcription factors and other regulators controlling defense responses are frequently represented by families of functionally redundant members . Additionally, some components of the plant immune system may be essential for plant survival during early development . Hence, conventional genetics is of limited use for the dissection of the plant defense network in this circumstance. Chemical genetics utilizes small molecules to alter in vivo protein functions in a reversible and highly controllable manner . Using such bioactive compounds as synthetic elicitors, a simultaneous and uniform activation of the defense network can be accomplished . This allows for reproducible measurements of the dynamics of molecular processes and physiological responses. In addition, synthetic elicitors may be used to simultaneously knock out families of functionally redundant proteins, theoretically resulting in clear phenotypes. Moreover, the function of essential genes can be studied efficiently by introducing the respective chemical at any stage of development to manipulate their activity . The identification of a collection of novel synthetic elicitors may permit the selective manipulation of defined branches of the defense network. Such elicitors will serve as powerful new tools for the emerging field of systems biology. They are likely to facilitate the stimulation of the defense network with unprecedented precision allowing the examination of the relation of defined signaling events and physiological outputs in a quantitative manner. In the broader context, the ability to manipulate disease resistance pathways using synthetic elicitors can also be exploited for agricultural purposes by using various combinations of chemicals to develop a new generation of pesticides.

Small molecules have the potential to simultaneously knockout the function of closely-related members of protein families . This may permit the study of biological functions of functionally redundant proteins. Using traditional genetics, this can be difficult or infeasible due to technical challenges and lethal phenotypes . Yet another advantage over traditional genetics is that bioactive small molecules allow for the study of essential gene functions at any stage in development because transiently active molecules can be added at any time point during plant development or applied at sub-lethal concentrations. In contrast, genetic mutations are permanent and the analysis of plant lines homozygous for a lethal mutation is challenging or impossible. Finally, multiple unrelated gene functions can be knocked out concurrently by using combinations of different bioactive molecules . For the primary screen,dutch buckets system both forward chemical and mutational screens must be specific, meaning that the read-out has to be specific and serve as an unambiguous proxy for the biological process of interest. Chemical genetics requires the screening of many thousands of chemicals in search of one with the ability to stimulate a particular response of interest . The need for chemicals that can manipulate a large diversity of biological processes resulted in the development of large structurally diverse chemical libraries . The concept of chemical genetics is based on the theoretical assumption that for every existing protein in the biosphere there are hypothetical organic structures capable of binding to it and interfering with its function .

The identification of bioactive compounds interfering with any given biological process or target protein requires screening of libraries representing a large diversity of chemical structures. Of key importance for the identification of bioactive compounds are their physicochemical properties. To be a biologically active compound the substance has to be “drug-like”, which means they must be capable of crossing biological membranes and to remain in an active state in the biological target tissue for a sufficient period of time . Lipinski’s ‘rule-of-five’ states 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 . A large sample size of structurally distinct chemicals maximizes the probability that compounds will be identified that induce the desired biological effect. The identification of proteins targeted by a given compound is an integral step but also typically the bottleneck for most chemical genetics projects. Several strategies exist for target identification, some of those include: affinity chromatography, yeast three-hybrid , protein arrays, and screens for mutants with altered sensitivity to a compound of interest . In affinity chromatography, a compound is tagged and immobilized so that interacting proteins can be purified and then identified. In Y3H the compound of interest is tagged with dexamethasone or methotrexate and then applied to yeast cells. The compound then binds the DEX or MTX binding protein, which is fused to the DNA-binding domain of a transcription factor. The activation domain of the transcription factor is translationally fused to a cDNA library. When the compound interacts with a plant protein a complex is created and this results in the transcription of a reporter gene . In protein arrays a fluorescent- or isotope-labeled small molecule is used to screen protein chips . Finally, in a screen for mutants with altered sensitivity, mutagenized organisms are treated with compound and plants showing responses to the compound that differ from wild type are selected for further study .

There are several examples of successful applications of chemical genetics in plant systems. Armstrong et al., performed a high-throughput chemical screen using a 10,000 compound library with the intent of identifying inhibitors of auxin transcriptional activation . Their screening strategy involved the use of a line that expressed GUS in the root elongation zone after application of auxin. This screen resulted in the identification of 30 compounds showing strong inhibition of GUS expression. Four structurally distinct compounds were further studied based on low active concentrations . Two of these compounds impart phenotypes indicative of an altered auxin response, including impaired root development. The two strongest of these compounds displayed similar growth phenotypes after treatment. Additionally, microarray studies using the later two compounds indicated that similar transcriptional changes were induced by both inhibitors . Chemical genetics became popular in plant biology in the past 10 years and numerous successful applications of this approach in plants have been published during the past five years . In 2007 a number of successful chemical screens were reported. The compound 7-ethoxy-4-methyl chromen-2-one was discovered in a screen of 20,000-compound library based on its ability to cause a swollen root phenotype in Arabidopsis . Using live cell imaging of fluorescently labeled cellulose synthase and microtubules, DeBolt et al., showed that treatment with morlin interferes with cortical micro-tubules to alter the movement of cellulose synthase. This interference resulted in unique cytoskeletal defects which produced shorter and more bundled micro-tubules. Morlin proved highly useful for the study of mechanisms that regulate micro-tubule cortical array organization and how it interacts with cellulose synthase .

In a small screen of 120 bioactive molecules Arabidopsis seedlings were used to identify compounds that inhibited early immune responses. This screen resulted in four hits. These compounds reduced flg22 -activated gene expression of MAMP-responsive ATL2 gene. Two of these four compounds, triclosan and fluazinam, interfere with the accumulation of ROIs and transport of the FLS2 receptor. Additionally, the compound Triclosan, which blocks early immune responses, was used to determine a potential role for lipid signaling in flg22-triggered immunity . Hypostatin was discovered in screens as a compound that inhibits hypocotyl growth in a Arabidopsis accession dependent manner . 11 other accession-selective hit molecules were also identified alongside hypostatin, which is an inhibitor of cell expansion. Additionally, a screen for compounds able to disturb microfibril-cellulose attachment resulted in the identification of cobtorin . This study demonstrated that different Arabidopsis accessions can be used to study the activity of interesting new compounds. The enhancement of plant immune responses by exogenous application of chemicals can be traced back to the treatment of tobacco with SA . While SA, JA, and ET can induce defense response, their use in the field or greenhouse setting is restricted based on their shortcomings as defense inducers that are broadly effective on many plant species . The use of environmentally safe plant defense-inducing chemicals, which boost a plant’s innate immune responses, offers an attractive alternative to pesticides. Linda wants me to reference the body of literature that explored SAR inducers as methods of control – klessig + sa An alternative procedure to protect plants against disease is to activate their own defense mechanisms by specific biotic or abiotic elicitors . The classical type of induced resistance is often referred to as systemic acquired resistance . Sodium salicylate , 2,6- dichloroisonicotinic acid , and benzothiadiazole-S-methyl ester are well-known elicitors of SAR in various plants against disease . The expression of SAR, triggered by either pathogen infection or treatment with NaSA or its functional analogs INA or BTH, is tightly associated with the transcriptional activation of genes encoding pathogenesisrelated proteins . The nonprotein amino acid DL-3-aminon-butanoic acid also activates an induced resistance response. BABA induced resistance, involves SA-dependent, SA independent,dutch buckets and ABA-dependent defense mechanisms, and the importance of these defenses varies according to the nature of the challenging pathogen . Many molecules exist that cause an induction of defenses in plants when applied.

Plant-derived small molecules other than SA, JA, and ET have been identified as important to controlling or preventing disease in plants. Some of these phytohormones include abscisic acid , brassinosteroids, gibberellin, cytokinin, and auxin . Like the interactions between SA, JA and ET, other molecules are important for the tailoring of defense responses . Activation of ABA signaling processes and its biosynthesis have also been shown to promote plant disease . Alternatively, brassinosteroid treatment has been shown to enhance resistance against some biotrophs, mediate abiotic stress responses through NPR1, and induce PR1 expression . Gibberellic acid can induce increases in ROS accumulation and attenuation of JA signaling . Cytokinins, which affect cell division and morphogenesis, can also enhance the SA response and thus promote resistance against biotrophs . Finally, auxin signaling may suppress SA biosynthesis and signaling, while SA attenuates auxin signaling . These plant hormones are integral to the function of plants. While many of these hormones were not originally associated with defense, new research suggests they all have roles in coordinating plant response to pathogen invasion. Every year billions of dollars are spent on pesticides which leave residues on produce that control pests and pathogens but can be harmful to consumers and the environment. Such off-target effects make the study of interactions between plants and pathogens an integral field for the reduction of conventional pesticide use. Using model pathosystems, such as Arabidopsis thalianaand Hyaloperonospora arabidopsidis , many important questions related to plant disease resistance are being addressed. A complex transcriptional network controls plant immune responses. Of key importance for the regulation of this defense network, are protein kinases that act at various stages during defense activation. Chemical genomics can be used to study these different stages. Plant defense-inducing molecules identified and characterized using chemical genomics will be valuable tools for the dissection of the plant defense network and will serve as leads for the development of novel environmentally safe pesticides. Genes from the Arabidopsis ACIDcluster are coordinately inducible by the synthetic elicitors DCA and INA. This cluster is enriched for genes encoding protein kinases. Using a forward genetics approach it was demonstrated that 10 of 16 ACID members tested are required for full immunity of Arabidopsis against Hpa. Seven of these 10 ACID members have not been implicated in plant immunity before. In addition, eight novel synthetic elicitors identified and characterized via chemical genomics were reported on here, one of which, called CMP442, is a more potent defense inducer than DCA or INA. While effective in crop protection pesticides leave residues on produce and have off target effects . This makes the design of novel green pesticides highly attractive. Also, the elucidation of the finer points of interactions between plant and pathogen is integral for the design of new approaches to more efficiently and safely prevent crop diseases. The model plant Arabidopsis thaliana and the oomycete Hyaloperonospora arabidopsidis are a naturally coevolving pathosystem with a high level of intra-species genetic diversity . Use of this and other model interactions has revealed that plants have a complex inducible immune system that protects wild species and crops from pathogen infections. When plants recognize the presence of an infecting pathogen, a multitude of signaling events are triggered that ultimately lead to efficient defense . Some of the early responses after resistance -gene recognition include changes in ion fluxes, synthesis of reactive oxygen species , alterations in gene transcription, which can be followed by a hypersensitive response , where the plant cells surrounding the point of infection die to restrict pathogen growth . An ancient and fundamental form of plant defense involves conserved microbe-associated molecular patterns . MAMPs are recognized by plant pattern-recognition receptors resulting in the activation of a complex defense response. This form of plant immunity is referred to as pattern-triggered immunity . A second form of immunity is based upon the recognition of pathogen-secreted effector molecules, which are proteins that promote pathogen virulence in the plant. Here the plant is capable of recognizing the presence of pathogen effectors, or their cellular effects, by disease Rproteins. R-proteins constitute a second class of plant immune receptors, besides PRRs, and induce a strong defense response, which often includes HR. This form of immunity is called effector-triggered immunity . In the absence of a cognate R-protein, the secretion of effectors enables pathogens to successfully infect their hosts. During such compatible interactions, plants can still mount a weakened immune response, called basal defense. Basal defense typically limits the spread of pathogens but is not capable of fully preventing disease .

Trifoliate orange, known for its high-quality fruit and tolerance to various biotic and abiotic stressors was compared to rough lemon which historically produces high yield and large fruit that are lacking in flavor. In Chapter 1, I report a detailed analysis of the temporal changes and genotypic differences in gene and miRNA expression in root tissue of different root stocks. To the best of my knowledge, this is the first comparison of root transcriptomes performed in citrus. In Chapter 2, expression data from fruit of trees grafted onto the four different root stocks was assessed. Changes in expression throughout development were linked to fruit quality variation. Additionally, the role of miRNAs in regulating the biological and metabolic processes that were affected in each of these chapters was investigated. The results provide a global examination of the molecular mechanisms underlying graft-induced changes in citrus fruit development and ripening. Citrus is grown in more than 140 countries and is one of the most economically important crops in the world. The total production of citrus in the United States in the 2018 growing season was 6.1 million tons on a total of 679 thousand acres. California produced 58 percent of the United States total, producing 3.6 million tons of citrus on 278 thousand acres. Approximately 75 percent of California’s citrus production is sold to the fresh market opposed to being processed into other commodities, such as juice. Oranges accounted for 64 percent of the total citrus produced in the United States and were valued at $1.8 billion, according to the United States Department of Agriculture. Citrus trees are rarely grown from seed and virtually all commercial citrus is propagated by grafting.

This reduces the juvenile phase,hydroponic gutter allowing for the trees to produce fruit many years earlier than those grown from seed. Root stocks impart certain traits onto the scion and the effects of root stocks can be large. The most significant impacts are on growth and vigor, tree nutrition, stress resistance, and fruit quality. In citrus, phenotypic differences in fruit quality have been well documented. However, understanding of the molecular mechanisms underlying these differences is lacking, especially regulatory mechanisms. Previous studies in apple, grape, sweet cherry, and other fruit crops have examined transcriptome changes in various root stock-scion combinations. In citrus, gene expression profiling has been used to understand root stock effects on growth of trees and responses to biotic and abiotic factors. Many transcriptomic studies have also been performed in citrus to elucidate fruit ripening and development in commonly grown citrus cultivars. To date, none of these reports have linked the genetic effects of citrus root stocks to fruit quality. Fruit growth and development and the mechanisms underlying fruit quality are complex. Signal transduction systems regulate many aspects of fruit ripening32. During citrus development, the ABA-signal pathway may act as a central regulator of ripening, combined with other hormones, including auxin and ethylene. A recent study showed that ABA is a positive regulator of citrus ripening and exogenously applied ABA regulates citrus fruit maturation, suggesting that ABA metabolism plays a crucial role in citrus fruit development and ripening35. Previous studies identified Protein phosphatase 2C family proteins as negative regulators of ABA signaling. PP2C dephosphorylates and inactivates a SNF1-related kinases family 2 protein, which is a positive regulator of ABA-response pathways.

Plants with an inactive form of PP2C were hypersensitive to ABA, causing increased activation of ABA-responsive genes. ABA-signaling response has also been linked with drought-stress tolerance. This study suggested that ABA accumulation is associated with a decrease in relative water content and Romero et al. suggest that ABA increases caused by dehydration upregulate levels of PP2C. Auxin, another phytohormone important for fleshy fruit development, regulates many growth and development processes. The auxin-signaling pathway regulates transcription of hundreds of auxin-inducible genes. Promoters of these auxin-responsive genes contain auxin-responsive elements , which bind the auxin-response factor family of transcription factors. ARF activity is regulated in part by Aux/IAA genes, which are transcriptional repressors of the auxin response. In the absence of auxin, Aux/IAA proteins dimerize with ARFs and recruit corepressors of the TOPLESS family, which in turn recruit chromatin-remodeling proteins that stabilize the repressed state. When auxin is present, it acts as a “glue” between Aux/IAAs and F-box proteins that are part of a ubiquitin protein ligase complex. This causes polyubiquitination and subsequent degradation of Aux/IAAs, which releases its repression, leading to the activation of auxin-regulated genes. Together with ABA and other hormones, auxin regulates several aspects of fruit development, including fruit set, fruit size, and ripening related events. Additionally, prior studies have indicated that small RNAs may play a regulatory role in fruit development and ripening. Small RNAs are a type of single-stranded, non-coding RNA that is typically 20-24 nucleotides in length, of which microRNAs are the most extensively researched class and are known to post-transcriptionally down regulate the expression of target mRNAs through mRNA cleavage or translational inhibition.

In strawberry, miR159 was shown to act as a ripening regulator by targeting a MYB transcription factor, which plays a crucial role in the ripening process51. Several examples of miRNA involvement in fruit development and maturation have been described in a variety of crop species, including apple, grape, peach, blueberry, date palm, and tomato. miRNAs that suppress specific transcription factors that are thought to be regulators of citrus fruit development and ripening have also been identified58. However, the expression profiles of miRNAs in various scion-root stock combinations and their subsequent impact on fruit quality have not yet been evaluated. In this study, trees grafted on 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. In general, rough lemon root stocks produce the highest yield and fruit size, but fruit is of lower quality, containing lower acidity and lower levels of total soluble solids, also known as the “dilution effect”. On the other hand, trees on trifoliate orange produce smaller, high quality fruit with high yield on often smaller trees. Carrizo citrange root stocks produce intermediate yield with good fruit quality. Sweet orange root stocks produce good quality fruit, but trees are very susceptible to various citrus diseases. An RNA-seq approach was implemented to investigate differences in gene expression in fruit due to genetically varying root stocks,hydroponic nft channel with the aim of identifying genes that could potentially play a role in improvement of fruit quality.

Furthermore, miRNA expression profiles were obtained for each of the root stocks to identify potential regulatory mechanisms associated with their target genes.Fruit were harvested in January at the end of the 2016 growing season when fruit were ripe. Total yield was recorded. Ten fresh fruit per tree were collected and analyzed for the following traits: weight, height, width, rind color, rind texture, peel thickness, internal texture and taste. The juice was then pooled from all ten fruit and percent juice, total titratable acid , and total soluble solids for each pool were calculated at the UC ANR Lind cove Research and Extension Center. The average for each trait of the ten fruit per tree were considered one biological replicate and ten biological replicate trees were sampled for statistical analyses. Statistical differences in fruit quality between fruit from trees on different root stocks were evaluated using an analysis of variance test and Chi-squared test. The differences among treatment means were evaluated by Fisher’s Least Significant Difference test and Duncan’s Multiple Range Test. Data were considered to be statistically significant when P < 0.05.The juice vesicles of three representative fruit per tree from two biological replicates of each of the four root stock genotypes at the four collection time points were subjected to RNA-seq . Samples were ground in liquid nitrogen and total RNA was extracted from ~200 mg tissue using the ZR Plant RNA MiniPrep™ kit per manufacturer’s instructions. An Agilent Bioanalyzer was used to confirm the integrity of the total RNA. The RNA with a RIN value greater than seven qualified for RNA-seq. For messenger RNA-seq, sequencing libraries were created using TruSeq Stranded mRNA Library Preparation Kit according to the manufacturer’s protocol. For small RNA-seq, sequencing libraries were created using TruSeq Small RNA Library Preparation Kit according to the manufacturer’s protocol. Each library was prepared for multiplexing with a unique indexed primer. Quantification of all libraries was performed with Nanodrop and Qubit fluorometer. The library size distribution and quality were measured with an Agilent Bioanalyzer. Multiplexed libraries were sequenced in a single lane on an Illumina NextSeq 500 instrument at the University of California, Riverside Genomics Core facility. An average of 11 samples were sequenced per lane. The data analysis was carried out using the RNA-seq workflow module of the systemPipeR package available on Bioconductor60.

Quality reports were created with the FastQC function. Citrus clementina v 1.0 genome assembly and annotations were downloaded from JGI’s portal . Sequencing reads were then mapped against the Citrus clementina v 1.0 reference genome using the Bowtie2 alignment suite for small RNAs and HISAT2 alignment suite for messenger RNAs. Messenger RNA raw reads were counted in a strand-specific manner. Known miRNA gene coordinates, required for counting, were acquired by downloading all known plant miRNAs from the plant miRNA database, aligning these sequences to the Citrus clementina v 1.0 reference genome using Bowtie2 with perfect alignment, and extracting the alignment coordinates. Small RNA raw reads were then counted at the known miRNA locations using the summarize Overlaps function. Sample-wise correlation analysis was performed using rlog transformed expression values generated by the DESeq2 package. In this study, RNA-seq generated reads that mapped to 19,359, 19,124, 19,336, and 19,374 citrus genes in samples from fruit of trees grafted onto sweet orange, Carrizo citrange, rough lemon, and trifoliate orange root stocks, respectively. With criterium of at least 2-fold difference and a p-value less than 0.05 , a total of 1,633 differentially expressed genes were identified between genotypes at one or more time points . There were 684 genes found to be DE between rough lemon and sweet orange root stocks, 388 DEGs between Carrizo citrange and sweet orange, 361 DEGs between trifoliate orange and sweet orange, 178 DEGs between rough lemon and Carrizo citrange, 395 DEGs between trifoliate orange and Carrizo citrange, and 855 DEGs between trifoliate and rough lemon. None of these DEGs overlapped in all 6 comparisons . The majority of the DEGs were specific to one pairwise comparison, but the largest overlap of was a group of 122 DEGs that were commonly shared between RL-SO, CZ-SO, TF-RL, and TF-CZ. Due to the large number of DEGs observed between fruit grafted onto trifoliate orange and rough lemon root stocks and the fact that the largest phenotypic differences in fruit quality traits were generally seen when comparing fruit grown on these root stocks, we primarily focused on this contrast for the remainder of this study. DEGs uniquely belonging to this comparison are more likely to play a role in the phenotypic changes seen when fruit are grown on trifoliate orange versus rough lemon root stock. Gene Ontology and pathway enrichment analyses were conducted to explore the functions of genes that were DE in trees on different root stocks. GO categorization showed that the molecular function GO terms ‘DNA-binding transcription factor activity’ and ‘transferase activity’ were significantly enriched . Genes associated with photosynthesis and located in the photosynthetic membrane were also enriched . KEGG pathway analysis revealed that genes for plant-hormone signal transduction, carotenoid biosynthesis, and fructose and mannose metabolism were significantly enriched when comparing fruit grown on trifoliate to rough lemon root stocks . The hormone-signaling-related pathway included DEGs involved in auxin, gibberellin , abscisic acid , ethylene , and jasmonic acid signaling . Visualization of fold changes using MapMan software revealed that several genes in the ABA and GA pathways were down-regulated in fruit grown on rough lemon compared to trifoliate root stocks.Many genes involved in other cellular responses, as well as transporters were also DE . To further understand the genetic influence of root stocks on fruit quality, we focused on the expression changes of miRNAs and their target genes.

Populations Sonora x CBdeM , Sonora x Foisy , and CBdeM x Foisy have 146, 141, and 128 lines respectively. The DH lines were phenotyped using a modified cigar roll method of Zhu et al. . The system is similar to the Cyg germination growth pouches and the gel based system of Bengough et al. . It consists of two plexi-glass plates 20 cm x 30 cm fitted with spacers, germination paper, racks holding the plates upright and tubs used to hold water. One hundred seeds were counted and weighed to estimate average seed weight. Seeds of similar size and weight for each genotype were imbibed in water for 24 hr prior to planting. Germination paper wetted with deionized water was placed on one of the two plates, and two seeds of the same genotype were placed with embryos down 5 cm below the top edge of the paper and 8 cm apart. This set up was covered by a second sheet of wet germination paper and a second sheet of plexiglass. The entire set-up was clipped together and placed upright into tubs of water about 8cm deep; this water level was maintained constant throughout the experiment. Seedlings were grown for 7 days at room temperature without supplemental lighting in a head house at the University of California, Riverside between February 2014 to May 2014 and November 2014 to February 2015. Experiments were setup in a randomized complete block design with four replications where replications were treated as blocks. Each replication had two plants from every genotype. After 7 days plates were removed from the tubs, disassembled, 4x8ft rolling benches and seminal roots were imaged using a hand held digital scanner set to 300 DPI.

To acquire images, the top sheet of germination paper was carefully removed and the top plexi-glass plate was laid over the seedlings so the scanner could be passed over from above. Seminal root angles were measured using the angle tool in ImageJ . For each plant, the angle between the first pair of seminal roots was measured at approximately 3cm below the embryo of the seed , as shown in Figure 1. The analysis of variance for seminal root angle and number was based on mean values of the experimental units. Broad sense heritability was calculated on a mean basis across four replications. Genotype means were used to calculate Pearson‟s correlation coefficients for seminal root number, seed weight, and seminal root angle. Genomic regions associated with traits of interest were detected by the software package IciMapping using linkage maps for these populations as described previously and the mean value of 8 seedlings of each genotype from four replicates. 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. QTL consistent between years within populations and/or consistent between populations were considered as verified QTL and named according to McIntosh Catalogue of gene Symbols for Wheat . Following the format of previous publications an uppercase “Q” in the name signifies strong verification of the QTL and lowercase “q” was used to name QTL that were consistent but warrant further investigation. In most cases associations of root system characteristics with specific genome region varied between populations and within populations from one year to the next. Over two years of the experiment and with all three populations taken together, a total of 31 genomic regions showed statistically significant associations with the seminal root angle and number . Seminal root angle was associated with 12 chromosome regions in the SC population, located on chromosomes 2D, 3B, 4A, 5A, 6A, 6B, 6D, and 7B. In the SF population, five regions on 2D, 5B, 6B, and 7B were identified and another five regions were identified in the CF population, on chromosomes 5B, 6A, and 7A.

The chromosome region with the single largest effect for the seminal root angle was located on chromosome 2D in the SC population. Its estimated effect was equivalent to 7.33° of the total root angle, and it was responsible for 21.42% of the population variation. The region with the lowest, but statistically significant effect for root angle was identified in the CF population, accounting for an estimated 2.90° of the root angle and explaining 9.40% of the variation observed in this population. For the seminal root number, nine genomic regions were identified in the three populations. Of these, four were identified in the SC population, on chromosomes 4A, 5B and 7A. The SF population had only one region, on chromosome 4B. The remaining three regions were identified in the CF population on chromosomes 1B, 6B, and 7D. The region with the largest effect was on chromosome 4A in the SC population, with an estimated effect of -0.25 roots per seedling explaining 17.32% of the total variation. The region with the lowest but statistically significant effect was identified on chromosome 6B in CF, with an estimated effect of 0.15 roots per seedling, explaining 8.41% of the population‟s variation. For the purpose of this study, only those genome regions that showed consistent associations with specific traits within a given population over both years were considered as verified QTLs . In the SC population three such regions were identified, located on chromosomes 2D, 6A, and 7B . The region on chromosome 2D was 4.17 cM region with a peak at 113 cM between markers 2Dx_79444 and 2Dx_77420 in 2014. It accounted for 25.99% of the phenotypic variation seen in the population that year. In 2015, the region was located between markers 2Dx_32130 and 2Dx_79444 covering a 0.67 cM with a peak at 112 cM. That year it explained 21.42% of the phenotypic variation seen. The allele for wider seminal root angle was contributed by Sonora. The second QTL was located on chromosome 6A. In 2014 it was between markers 6A_72189 and 6A_55084 covering a 4.90 cM region with a peak at 151 cM. It explained 7.04% of the phenotypic variation that year. In 2015, this QTL formed a peak at 155 cM between markers 6A_55084 and 6A_21174, it coved 1.35 cM and explained 7.21% of the variation for the trait. The allele for wider seminal root angle was contributed by Sonora.Each of the three tested population showed large phenotypic variation for both seminal root traits measured in this study.

The largest range in seminal root angle was between Sonora and CBdeM with average seminal root angles of 108.73° and 63.31° respectively . The least difference, but still statistically significant,flood and drain table was between CBdeM and Foisy which have more similar seminal root angles of 63.31° and76.95° respectively. The distribution patterns among progenies imply considerable trait complexity.All three parents typically had five seminal roots with few variations between replication giving averages of 4.36, 4.38, and 4.49 seminal roots for SC, SF, and CF respectively. The occurrence of less than five seminal roots is likely explained by environmental interaction and associations with seed weight. Since all parents typically develop five seminal roots it is not surprising that the three populations have similar means and ranges. As will be discussed later, the lack of consistent QTLs for seminal root number may suggest that this trait is heavily influenced by the environment and seed weight. However, one consistent QTL was identified which also suggests that there is a genetic component as well. Additionally, heritability values were relatively high for both traits in all populations but it does not seem to promise any ease of selection for breeding efforts. As will be discussed it certainly doesn’t hint at simplicity for the genetics of these traits.The 90K SNP array was used on eight mapping populations of doubled haploids to order SNPs along individual chromosomes and 44,345 of those were mapped to one or more of 46,977 loci . Due to differences in polymorphism among different sets of parents, only a fraction of all mapped markers can be expected to be useful in any given pairwise combination. Moreover, as distribution of crossover can vary substantially between different pairs of parents the actual genetic map position of any given marker may also differ . To facilitate utilization of the maps generated using the 90K SNP chip, Wang et al. 2014 created a consensus SNP map of wheat, based on the tested eight populations. In essence, this map provides average marker positions for all polymorphic markers of their study and may be used to coordinate maps generated for different populations. As it was explained in an earlier chapter, total lengths of maps for each of the three populations here varied but more importantly, at times very few common markers were present in specific chromosome regions. For verified QTLs, that is for consistent associations between specific DNA markers and genome regions consistently showing up in replications, the consensus map was used to allocate those to specific regions and used DNA sequence data of the closest associated marker to blast against the wheat sequence survey on the URGI database and determine its actual location. In this fashion, relative locations of QTLs identified in this study can be compared to all previous results and can be verified in the future. This approach makes it possible to use even those DNA markers that were not polymorphic between two parents of a given population increasing the resolution of a mapping exercise. This study identified 31 genomic regions associated with seminal root angle and seminal root number in three populations. Most of these regions were unique to specific populations and varied from year to year. This implies that these traits are far from simple, as proposed by Oyanagi and do not appear to be controlled by single loci. It must be pointed out that compared to other studies on seminal root traits, the results presented here appear to be better supported by experimental data. Using a single population of 103 doubled haploids Hamada et al. were unable to identify a QTL for seminal root angle; two QTLs for deep root ratio appeared on chromosomes 1B and 5D. Another QTL, for seminal root, was found on chromosome 5A. None of the regions consistently identified in this study appear to be located on chromosomes of Hamada et al . In another study, Christopher et al. identified 12 QTLs for seminal root angle and number in a single mapping population of bread wheat consisting of 184 individuals. The QTLs for seminal root angle were located on chromosomes 2A, 3D, 5D, 6A, and 6B; those for seminal root number on chromosomes 1B, 3A, 3B, 4A, and 6A. While some chromosomes are the same as those identified in this study, none are on chromosomes verified as valid QTL in this study: 2DS, 6AL and 7BS for seminal root angle and 4BL for seminal root number. In another study Liu et al. again identified a total of 12 QTLs for seminal root angle and number. Seven of those, for seminal root angle were on chromosomes 1A, 2B, 3A, 3B, and 7D and five for seminal root number on 2B, 3B, 3D, 5A, and 7A. Again, there are some genome regions in common with this study but none appear to be are similar to our verified QTL. Most studies employ a single mapping population. Beavis demonstrated that in populations numbering 100 progeny, the QTL effects were greatly overestimated, in populations with 500 progeny the QTL effects were slightly overestimated while populations with 1000 individuals produced estimates close to the actual magnitude of QTL effects. That study highlighted the necessity for larger populations and the need for verification of QTL across populations. Beavis did not address the issue of mapping in parallel populations sharing common parents. To the best of our knowledge only a couple of studies made use of two or more populations in studying root system traits: Zhang et al. used three related recombinant inbred line populations with a single common parent and Kabir et al. used two unrelated populations. Zhang et al. identified QTLs for seminal root number on chromosomes 1D, 2A, 2B, 2D, 3A, 3B, 4A, 4D, 5A, 5D, 6A, 6B, and 7B.

This discrepancy could be due to misannotations in the genome or to the existence of alternative biosynthetic pathways. In any case, the addition of phenylalanine and histidine greatly improved the growth and phosphite oxidation rate of FiPS-3 cultures. The growth benefit conferred by these two amino acids was similar to that of yeast extract or casamino acids and was probably due to their ability to serve as organic carbon and nitrogen sources. FiPS-3 has previously been characterized as a strict anaerobe, and the presence of cytochrome c oxidase genes in its genome has been attributed to an uncharacterized mechanism of nitrite resistance, although this hypothesis was never tested . Here I show that FiPS-3 is not only capable of growing under aerobic conditions, but indeed grows better in the presence of oxygen than in controls containing no electron acceptor. This observation, coupled with the presence of cox genes in its genome, suggests that FiPS-3 is capable of oxygen respiration. This is an unexpected result given that oxygen tolerance is rare among sulfate reducing bacteria and that the capacity for aerobic respiration is rarer still, with only a handful of oxygen-respiring sulfate reducers having been documented so far . However,hydroponic indoor growing system the fact that FiPS-3 grew at a similar rate in the presence of oxygen as it did under sulfate-reducing conditions is puzzling, since oxygen is a substantially more favorable electron acceptor from a thermodynamic point of view and should, therefore, allow for faster growth. This may be indicative of a failure on the part of FiPS-3 to adequately cope with oxidative stress, which imposes a growth cost under aerobic conditions.

Its inability to grow in a well-aerated culture lends support to this hypothesis and highlights the fact that the actual dissolved oxygen concentrations it was exposed to in unmixed cultures were likely well below the concentrations present in the head space. As such, it may be more accurate to regard FiPS-3 as a microaerophile rather than a true facultative aerobe. Furthermore, it is also possible that FiPS-3 was not actually growing by aerobic respiration in oxygen-containing cultures but rather growing by sulfate reduction with the oxygen serving to re-oxidize any sulfide produced back to sulfate. Even though sulfate was not added to the aerobic cultures, even a small amount carried over from the inoculum could have supported the observed growth if the sulfide produced was continuously reacting with oxygen to replenish the sulfate pool available to the cells. Further experiments, such as quantification of cox gene expression levels during aerobic growth or assessment of aerobic growth in the presence of complex IV inhibitors, are needed in order to distinguish between these two scenarios. In my FiPS-3 cultures, growth by DPO led to the precipitation of struvite or hydroxyapatite crystals when magnesium or calcium were added to the media. Schink et al. have previously observed the formation of struvite crystals during phosphite oxidation by FiPS-3 cultures. They attributed this phenomenon to the accumulation of inorganic phosphate to high concentrations in the extracellular milieu and the subsequent reaction of this phosphate with magnesium ions present in the medium to form crystalline mineral precipitates. This process of DPO-dependent biomineralization exploits the large difference in solubility between phosphite and phosphate and could potentially be applied to the development of bio-concrete. Bio-concrete refers to ‘self-healing’ concrete that incorporates bio-mineralizing microorganisms in order to help seal cracks that develop over time and thus improve durability and strength .

Most of the research conducted in this area has so far focused on the precipitation of calcium carbonates by heterotrophic bacteria . However, the precipitation of calcium and magnesium phosphate minerals by means of a chemolithoautotrophic DPO-capable bacterium may present an alternate approach for the advancement of this technology. Although upregulation of the PtdF protein in the presence of phosphite has been previously reported , this is the first evidence of increased expression of the entire ptx-ptd gene cluster under phosphite-oxidizing conditions. This finding affirms the connection between the ptd genes and DPO and lends further support to the hypothesis that these genes are necessary for growth by means of phosphite oxidation. It is important to note that 1 mM phosphate was present in both growth conditions, so that the increased expression of these genes was not due to phosphate starvation, which is known to induce expression of ptx genes in APOcapable organisms . However, the substantial difference in expression levels seen between the ptx and ptd genes was unexpected and implies that ptxDE and ptdFCGHI may represent two distinct functional modules that are differentially regulated. The fact that ptxDE are also present in APO-capable organisms in combination with an alternate phosphite transporter also supports the notion that the ptx and ptd genes represent separate modules. As discussed in Chapter 1, PtxD is known to be the enzyme responsible for phosphite oxidation both in APO organisms and in FiPS-3, while the function of the predicted transcriptional regulator ptxE remains a mystery. It is possible that the role of ptxE is actually to promote transcription of ptdFCGHI in the presence of phosphite, which could explain the higher levels of expression seen for these genes. Given that PtxD is highly efficient at turning over phosphite , the transport and energy conservation steps may in fact represent the bottlenecks for growth by DPO. Increased expression of the ptd genes could, therefore, be a way to compensate for the relative inefficiency of these processes compared to the phosphite oxidation step itself. Furthermore, the exceptionally high level of ptdF expression compared to the other ptd genes suggests that this enzyme may catalyze the ratelimiting step in the pathway.

Overall, my RNAseq results highlight the importance of the ptd genes during DPO-dependent growth of FiPS-3, but exactly what their functional roles are and how they are regulated remains to be elucidated. It is essential, going forward, to determine the minimal set of genes that are necessary and sufficient for growth by DPO,vertical rack system either through targeted knockouts in FiPS-3 or through expression of candidate genes in heterologous hosts. DPO enzymes could then be purified and characterized in vitro in order to determine their mechanisms of action. Unfortunately, my attempts to carry out targeted gene deletions in FiPS-3 as well as to heterologously express the full ptx-ptd gene cluster in D. balticum SaxT and in E. coli have sofar proven unsuccessful. Nonetheless, I hope that my efforts to improve the growth of FiPS-3 under both anaerobic and aerobic conditions will aid in the development of genetic tools in this organism that will allow future researchers to address some of these unanswered questions. Furthermore, the ability of FiPS-3 to precipitate phosphate minerals as a byproduct of DPO holds great promise as a potential mechanism for bioconcrete production. However, before this process can be applied on an industrial scale, further research must be undertaken in order to establish the optimal conditions for biomineralization by FiPS-3 as well as to assess the material properties of the different mineral products.Killed controls showed no phosphite oxidation even after 29 weeks and the rate of phosphite oxidation increased over time. The persistence of phosphite under abiotic conditions is congruent with its kinetic stability resulting from the high activation energy needed to break the P-H bond in the phosphite molecule . Moreover, the extent of phosphite oxidation observed in our enrichments is evidence of a dissimilatory process involved in energy metabolism as opposed to a purely assimilatory process. Phosphate accumulated in the media over time at a rate that corresponded to that of phosphite depletion and reached a final concentration that matched the amount of phosphite consumed . Assuming a phosphorus content of ~30 fg/cell , a bacterial culture would have to assimilate only about 300 µM phosphate in order to reach a density of ~1×109 cells/mL. This means that the phosphite consumed in our enrichments far exceeded what would have been required for use as a phosphorus source. In fact, there was more than enough phosphate initially present in the cultures to fulfill the phosphorus needs of growing cells in the absence of phosphite supplementation. The fact that microbial growth rates were significantly higher in phosphite-oxidizing cultures compared to no-phosphite controls provides further evidence that phosphite was acting as an electron donor and energy source for cellular metabolism. Phosphite oxidation and growth only occurred in media amended with CO2 and/or HCO3 – indicating that DPO was coupled to CO2 reduction, as has been shown for FiPS-3 . The rates of phosphite oxidation seen in our rumen fluid amended enrichments are comparable to those seen in cultures of FiPS-3 . However, our enrichments were incubated at 37o C and FiPS-3 can only grow at temperatures between 15 and 30o C . Indeed, our microbial community analysis confirmed the absence of any 16S rDNA reads belonging to the Desulfotignum genus in our enrichments.

There was, however, a clear shift in community composition in the presence of phosphite, with OTU 33 , OTU 21 , and OTU 28 being the most enriched taxa. Addition of rumen fluid to the enrichments greatly enhanced the rate of phosphite oxidation and also substantially altered the microbial community composition. Interestingly, OTUs 33 and 28 were no longer observed in the community. Instead, OTU 21 was further enriched to about 40% of the total community. The tight correlation seen between phosphite oxidation and Phox-21 abundance strongly supports its functional role in DPO. It remains unclear why OTUs 33 and 28 were enriched in our original cultures but not in the presence of rumen fluid. Given that rumen fluid was added to the cultures in an attempt to enhance the growth of OTU 33, its decline was particularly surprising. It is possible that these organisms benefited from the presence of phosphite under low-nutrient conditions, but were then outcompeted when nutrient-rich rumen fluid was added. Also, the fact that these taxa had very low initial abundances means that even moderate amounts of growth would have led to large increases in fold change, which would have magnified the effect of any ancillary growth benefits that these organisms may have received. In any case, the addition of rumen fluid did enhance the growth of Phox-21, which was an unexpected outcome, but perhaps not too surprising given the rich variety of nutrients and cofactors present in rumen fluid .The strong inhibitory effect of molybdate on phosphite oxidation and Phox-21 abundance in a culture not dependent on sulfate reduction was likewise unexpected, since molybdate is known to be a potent and selective inhibitor of sulfate reduction. It acts as a futile substrate for the enzyme ATP sulfurylase, resulting in depletion of the cell’s ATP pool . However, the addition of sulfate did not enhance phosphite oxidation, stimulate sulfidogenesis, or enhance the growth of Phox-21. It is possible that molybdate is targeting the phosphite oxidation pathway directly or affecting some other unrelated pathway, but further work is needed in order to confirm the mechanism of inhibition. Sulfite also appears to inhibit phosphite oxidation, which is further evidence of a potential incompatibility of the sulfate reduction pathway and phosphite oxidation in our enrichment. It is not clear whether the inhibition by sulfite was due to a direct interaction with the phosphite oxidation pathway or to a broader toxicity. PtxD, the phosphite oxidase found in FIPS-3 as well as in most APO-capable bacteria, is known to be inhibited by sulfite in vitro . This enzyme, if present in Phox-21, would be a likely target of sulfite inhibition. However, sulfite is also known to have a broad antimicrobial activity at concentrations as low as 600 µM , which may have also played a role in its inhibition. Phox-21 belongs to the GW-28 candidate order, a poorly studied clade within the Deltaproteobacteria with no known cultured representatives. Its closest relatives are all uncultured clones from different anaerobic waste treatment sites. Clone GW-28 was found in a household biogas digester in China , clone POMEbac42 was found in a food waste digester in Singapore , clone De3155 was found in alkaline landfill leachate sediment in China , and clone QEEB2BG06 was found in a mesophilic wastewater digester in Germany .

The survey was distributed to the 5th, 6th, 7th, and 8th grades, representing 116 students. The 6th grade results represent the “experimental” group in this semi-experimental design, while the 5th and 7th grade students represent “control” groups who did not receive the climate change curriculum focus in their humanities classes. The 8th grade students are used for additional comparative analysis as they study climate change for 12 weeks in science class, and are thus a form of comparison between science and social studies-based climate curriculum.Interview subjects represented school staff , teachers and the partner non-profit Climate Generation , for a total of 5 in-depth interviews of approximately 1 hour in length. By the nature of this study as a participatory research partnership, one of the interview subjects is also a co-author of this paper. Rather than muddying the waters of analysis or representing a conflict of interest, it is our belief and intention that co-authorship provides necessary clarity and depth of perspective on the curriculum pilot by partnering a climate education researcher with an educator interested in critically analyzing, improving, and disseminating a climate curriculum innovation. Interviews were semi-structured, and focused on the following topics: a) impacts of teaching climate change through humanities, b) student and parent responses to curriculum, c) process of developing curriculum and/or delivering instruction , and d) advice and recommendations for other schools. The classrooms observations took place towards the end of the school year, in April, and notes were recorded for analysis by one co-author . The other co-author contributed insights from her observations and experience working in the school over the course of the year in her interview . Observational research methods bring critical insights and context to inform interpretation of results,vertical grow tables revealing personal motivations and helping to uncover process dynamics leading to end results. Survey results were compared across grades and analyzed using Google Forms data analysis as well as basic statistical analyses.

Google Forms is a commonly used educational technology, and thus using this method has the advantage of making the process easily replicable to teachers, schools, and education organizations seeking to gather their own data on similar interventions. Disentangling confounding variables and adjusting for student baseline academic performance and demographics are additional avenues for future research but were not the focus of this study. Interviews were manually coded for themes and analyzed by type to understand differences in reaction between teachers vs. other adults involved in curriculum development and piloting. Interview themes inform next steps for Lowell as well as other schools seeking to implement climate change curriculum through a humanities focus, and guide researchers studying effective strategies and evolving trends in climate education. They reveal strategic improvements possible for the curriculum, as well as hypotheses for testing in larger CCE research explorations .The climate literacy survey was administered to 30 5th graders, 36 6th graders, 27 7th graders, and 23 8th graders, for a total of 116 students. The 6th graders averaged 74.4% correct response on the nine multiple choice questions, while the 5th graders averaged 60.4% correct, 7th graders 69.5% correct, and 8th graders 74.8% correct . The 6th graders had higher correct response rates for five out of the nine MC questions; in the remaining four, the 7th grade had the highest response rate on two questions, and the 8th grade had the highest response rate in the other two. Asked about the global temperature rise limit specified at the United Nations Conference of the Parties talks in Paris, the 6th graders outperformed their peers by the greatest margin, perhaps due to the focus on UN climate conferences leading up to a mock UN climate negotiations activity included in the curriculum . In the open response questions regarding solutions for lowering CO2 levels and mitigating climate change, the 6th grade students matched the 8th graders in terms of total number of responses across various categories , and were the only grade in which a student mentioned the single most impactful individual action to mitigate climate change: have fewer children .

While the sample sizes are too small to merit statistical significance, there are other conclusions of significance to be drawn from these results .The curriculum coordinator, who worked most closely on the development process along with Climate Generation staff, recognized the need from the beginning to build a wide platform of support for the idea from stakeholders both within and beyond the school. Several parents as well as staff, teachers, and external partners were present at the very first curriculum planning meetings. According to school staff, the reaction from parents has been overwhelmingly positive, and there is reported evidence of students discussing climate change with their parents. In the Director’s words, “any push back we were expecting has not happened,” .The conversation has spread among parents at the whole school, who are aware of what is going on, and are motivated to keep their students at Lowell because of the new curriculum focus. It is additionally used as a recruitment tool to attract new families– 2018/2019 6th grade recruitment gains are related to the new curriculum, according to staff.The curriculum coordinator summarized the first year implementation as follows: “Students learned how climate change affects a variety of people around the world, and how different people are responding to it. They read fiction and non-fiction texts featuring climate change and studied the interaction of geography, civics, and economics with climate change. Next year we are hoping to add a substantial service-learning aspect to the course, as well” . When asked if middle schoolers are “ready” to learn about climate change, addressing a common concern among educators about an age threshold for talking about a topic as overwhelming as climate change, all respondents answered in the affirmative. In the words of one teacher, students learn about the Holocaust and slavery in middle school, so they’re already dealing with emotionally charged content.

One 6th grade teacher initially felt inclined to avoid the topic due to “gloom and doom” connotations and possible negative emotional responses, preferring to focus instead on developing a love of nature and the outdoors among students. However, he reports having his mind changed by the curriculum pilot experience, and notes that his skepticism about the climate fiction novel in particular proved unwarranted. The students had incredibly positive responses to the cli-fi novel chronicling a girl and her family’s flight from their homeland as climate refugees and struggle to start a new life. Students felt catalyzed to take action: “The Cli-fi novel was groundbreaking for me. The kids really enjoyed it. It’s a weird way for doom and gloom to be exposed to them, but yet their reaction is incredibly surprising, I thought they’d turn off, but it almost seems motivating and inspiring them to learn more” . The Director of the Middle School brought up several noteworthy outcomes from the first year not captured in the student climate surveys. From the school’s internal standardized testing, he noticed a dramatic improvement in the 6th graders’ reading comprehension scores. Students increased by almost two Standard Deviations from their scores last year, an unusually high rate of increase,flower pot wholesale with top students maintaining their performance and the bottom third reaching grade level standards . One key growth point identified in several interviews was the need to expand and improve the solutions focus within the curriculum and provide opportunities for students to take meaningful action locally on climate mitigation options. For example, when students read “The Boy Who Harnessed the Wind,” students build windmills in science class. During the focus on renewable energy, they could also do a community action project around solar incentive programs: Maryland has a program for homeowners to put solar on their roofs, and students could advocate for D.C. to adopt a similar program as the same publicly owned utility covers both jurisdictions. Another idea brought up by staff is to change the end-of-year field trip from New York City to an island in the Chesapeake Bay threatened with disappearance due to sea level rise. The solutions piece is the most complex part to develop, as the content must be laid down first and authentic solutions built on top of that. This is a clear area of focus for future years . During this visit, 6th grade students articulately expressed the difference between climate change and global warming, which they acknowledged they had not known previously.

One student clearly explained the process of hydraulic fracturing in incredible detail, down to the underground wells with cement lining, injecting water at high velocity to release the oil stored in underground pores, producing a lot of wastewater and creating seismic activity . Students were generally excited to share what they had learned, and recounted memorable topics and projects of particular interest to them. This included the “CliMojis” art project, where they created personal Climate Emojis after reading a Washington Post article about a company designing climate change-based emojis, so that texters could communicate their frustrations and anxieties around climate change pictorially . The students read and discussed the article, and then designed their own climojis, synthesizing learning in a visual form. Other significant learning experiences reported were the Fossil Fuel Museum and learning about how bees will be impacted by climate change while reading a novel called The Hour of the Bees. When asked what students were hoping to do about climate change based on their newfound knowledge, students responded with ideas such as spreading awareness, creating more things from plant materials , farming in a way that supports the environment, and putting more plants in the ground. Many students seemed to have clear ideas around how to take action and expressed feelings of hope and empowerment when describing their collective “climate solutions.” As a whole, the Lowell middle school demonstrated much higher levels of knowledge and engagement around climate change than the average American teenager or adult. Based on a 2010 nationally representative survey of American teenagers, knowledge of climate science basic facts was found to be very low . 59% of American adults fall into the “Alarmed” or “Concerned” categories of the YPCCC Six Americas spectrum as of December 2018 compared to 82% of Lowell middle school students. What remains a challenge both nationally and at Lowell is building optimism around our ability to solve climate change: only 8% of youth agreed that we can and will do something to mitigate climate change in a recent study , and a mere 5% of Lowell students indicated they believe their generation will solve climate change. While acknowledging the receptive audience for implementing the curriculum, the results relating to increased student engagement, increased reading scores and favorable response to a humanities-focused climate curriculum are nevertheless significant and worth building on as an approach to middle school climate education. Further hypotheses are generated such as the claim that climate change as an engaging topic can help boost student performance in core academic disciplines , requiring further testing via controlled experiments. The time period between 6th and 8th grade is a significant youth development stage during which students develop capacity in knowledge retention and empathy and gain exposure to many new topics, and yet the 6th graders performed equal to or above the 8th graders on most climate knowledge and engagement questions. They shared information learned with families and friends more often than their 8th grade peers learning about climate change through science only, generating important hypotheses for CCE/IGL scholars . Results and best practices from this case study should be applied intentionally to other classrooms and school contexts. The web of support is a crucial enabling factor as well as the participation of key influencers, which must be identified in other contexts. The Lowell School curriculum coordinator suggests several vehicles for integrating similar curricula into more structured, state-mandated public school subject matter: through choice of reading materials in civics classes, suggested options for student independent research projects, and current events classes at the high school level.

Mack et al. find that 68 urban gardens in Phoenix, AZ are currently serving just 8.4% of “food desert” residents, and through spatial analysis, 53 gardens sited strategically could serve 96.4% of such residents. From these studies, it is clear that UA projects are not necessarily occurring where they are most needed to increase food security. When it comes to spatial analyses, “while a macro-level quantitative study of the potential in terms of land availability shows that it would be feasible to grow the basic daily vegetable needs for the urban poor in the United States, current evidence from urban farms located within lower-income communities shows that such farms are not necessarily feeding the communities in which they are located,” due to a variety of factors including cost of produce and cultural desirability.Barriers to access are not just due to geographic distance, but rather an array of intersecting factors including the high costs of some urban produced foods, especially from commercial or for-profit operations. Fresh, local produce from vertical or rooftop farms such as Gotham Greens , Plenty Higher Ground Farm , Freight Farms or AeroFarms are often sold at a premium to restaurants and grocery stores, and thus unaffordable to low income households . Despite claims that vertical farms can “feed the world in the 21st century” , it remains to be seen if vertical farms can address food access and food justice. Such farms are often following a corporate food system model of profit maximization and resource use efficiency, subscribing to capitalist logics rather than alternative, social-justice-oriented practices. Among for-profit farms, “the few profitable operations tend to be those selling to high-end restaurants and consumers, not to lower-income residents” . The cost of food, especially healthy fresh produce, is often in tension with other high costs of living in urban areas , causing low-income residents to become dependent on emergency food services and food pantries. This intersects with poor nutrition and diet-related diseases- according to the Alameda County Community Food Bank Hunger Study report,vertical towers for strawberries “food is often the most critical factor in our clients’ health”, and 40% of clients are in fair or poor health .

Food banks and food pantries fill important “access gaps” that urban farms could better supplement or address if cost of urban produced food was made more affordable, or through donations to food banks . Low-income households can circumvent the high costs of urban produced food from commercial farms by establishing their own backyard gardens , or adopting plots in community gardens. Through direct participation in UA, in particular food insecure individuals can offset significant percentages of fresh vegetable expenditures , and enhance food security through improved healthy food access . Access via UA participation is certainly enabled when urban farms and gardens are physically proximate to low income neighborhoods, demonstrating the intersection of cost and geography in expanding access. There are abundant examples of non-profit farms that give food away for free or at reduced rates , yet there is little scholarship on the consumption or impact of donations/discounted offerings specifically.High costs of land and development pressures also play a significant role in limiting access to both farming and locally-produced foods, as seen in studies of Chicago, New York City, and the San Francisco Bay Area . High cost of land prevents community gardens from being established in the urban core in Chicago, leads to hundreds of community gardens in NYC slated for redevelopment annually, and drives gentrification and displacement in neighborhoods around urban farms. Land tenure insecurity directly contributes to lack of access as many urban farms formerly serving minority and immigrant populations have been forcibly closed due to development priorities for privately owned lots . A recent article on land security indicators among California urban farmers showed that farms with higher land security also had “more financial and institutional support, and are located in census tracts with higher economic opportunity” . This highlights the necessity of devoting publicly owned lands to urban agriculture in low income and minority neighborhoods, as private lands are highly vulnerable to development pressures, thus jeopardizing any gains realized by social justice oriented urban farms. In contexts where urban farms strive to provide living wage jobs and career or educational opportunities for low-income communities, youth, or formerly incarcerated individuals, it is often challenging to also provide food access to these same communities.

Unless significant grant funding or donations exist, the goals of boosting food security are in tension with capitalist economic realities to pay living wages and sell the product at below-market costs . This speaks to the “unattainable trifecta of urban agriculture,” that is the idea that UA can simultaneously achieve community food security, provide on-the job training and fair living wages, and generate revenue through sales to cover these costs without substantial outside investment , as well as the tension between farm security and food security . In examples such as City Growers and Higher Ground Farms in Boston, organizational efforts to provide jobs and job training lead to marketing of produce to high-end restaurants, retail establishments, farmers markets, and CSAs at prices unaffordable to food insecure households .A fourth important food access barrier cited in the literature relates to cultural acceptability and nutrition education, widely accepted as part of food security definitions . Access to culturally appropriate foods is known to be an important factor , yet little is understood about the effects of urban farms growing culturally relevant foods and its relation to food access. More qualitative research is needed on the cultural acceptability of urban produced foods and how that might correlate with improvements in access. There is increasing evidence of the importance of culturally relevant educational materials around nutrition, food literacy, and culinary skills for improving access and actual consumption of healthy, fresh, urban-produced foods among low income, minority, or immigrant households . Culinary skills and food literacy are becoming focal points of school garden programs , and innovative organizations such as the Green Bronx Machine show how urban agriculture embedded into high-needs schools can directly improve food education,vertical growing which translates into increased access and consumption . Additional research is needed to quantify the impact of educational school gardens on community food security. Recent urban foraging literature is exploring stewardship practices and culturally relevant products gathered by foragers in cities around the world, as well as the sociocultural benefits that result .

From Mien immigrants gathering dandelion bud-shoots in urban parks , to informal urban foragers helping maintain trees and parks in Seattle, WA ranging in age from 23 to 83 , to the value of edible weeds urban foraging is an activity that recognizes certain agroecosystems as “commons” for public access and management. Urban forest justice scholars “recognize the rights of local people to have control over their own culturally appropriate wild food and health systems, including access to natural resources and to the decision-making processes affecting them” . The potential to address food insecurity with foraging and gleaning activities is being explored by organizations such as Ample Harvest and The Urban Farmers in Northern California; Ample Harvest’s online platform supports over 42 million backyard and community gardeners in ending food waste by channeling excess produce to 1 out of every 4 food banks across the country . While some food justice scholars conclude that current shifts toward local, organic, sustainably produced foods are only accessible and affordable to those with higher economic means “or at least the cultural cachet necessary to obtain such foods through barter, trade, or other means of exchange” , the examples above illustrate successful alliances of food justice advocates and local government working to enable sustainable, healthy food access for all urban residents. Through strategic planning and policy design, it may be possible to move beyond ad-hoc successes in linking urban agriculture with food access. The articles reviewed in this section provide a mix of academic studies, theoretical arguments, and policy literature. Additional empirical evidence and longitudinal studies are needed to demonstrate the ability of UA to significantly improve nutrition and food insecurity among urban low-income households over time. Furthermore, consumer preference surveys of urban produced foods are a conspicuous absence in the reviewed access literature. We turn next to food distribution, and the question of how urban produced foods get from the farm to the consumer through various distribution mechanisms.While many articles reviewed mechanisms for channeling rural or peri-urban produced foods into urban areas to increase fresh produce access , very little scholarly data exists on the distribution and accessibility of urban produced foods, and what does exist is largely under-theorized. In fact, very few sources reviewed explicitly name “food distribution” as a key term. Urban agriculture remains a relatively small, yet important percentage of the larger food distribution system in cities: “few, if any, urban agriculture projects, are intended to replace traditional food retail or would claim to lead to food self sufficiency for individuals or for cities” . As such, very little is understood about where and how urban farmers distribute their food including modes of transportation delivery, either individually or in aggregate, and to whom .

It is important to focus on the means through which food produced by different types of farm operations travels from farm to consumer, and the processes through which that food is exchanged , as this directly impacts access and consumption. The scholarly literature as well as media stories describe various modes by which fresh produce is distributed in the city to address fresh food access including both formal and informal distribution channels . Applying a distribution lens to the existing literature yields similar results to the food access analysis in that several articles theorize idealized distribution systems, showing the capacity of hypothetical urban and peri-urban farms to supply distribution networks that meet most urban food demands . Others highlight barriers and challenges farmers face in practice around distributing their produce to those in need while maintaining their operations . None, in our search, focus analysis on distribution flows of urban produced foods across a city. Rather, a more common focus is on which distribution channels are best for getting produce, not necessarily urban produced, into the hands of food insecure households or residents of “food deserts” . Is it a corner store, a large supermarket, or small local farm stand within a mile radius that households need to access fresh produce?In the case of corner stores, several studies have built on analyses of the prevalence of corner stores and liquor stores in low-income census tracts and endeavored to study the effects of providing fresh local produce in these stores otherwise carrying largely processed foods and sugary beverages. Results have been mixed, with some cases of pairing urban farms with corner store retailers yielding increases in sales of fresh produce , but others showing no increase and even resistance from corner store operators who feel that this produce will not sell and therefore become a waste disposal issue . Small Farmers markets as distribution sites receive critical assessments in the literature for their ability to serve as distribution channels to low-income consumers. Alison Hope Alkon writes about the closing of a farmers’ market in West Oakland, a historically African American neighborhood, juxtaposed with the white spaces of farmers markets that are thriving in neighboring Berkeley in her book Black, White and Green: Farmers Markets, Race and the Green Economy . She theorizes the promise and limitations of the “green economy” and chronicles the food movement’s anti-capitalist roots yet ultimate manifestation as reproducing capitalist inequalities. Lucan et al.’s study of farmers markets in the Bronx took issue with limited hours of operation, seasonality, affordable common produce, and availability of predominantly healthy foods among farmers markets compared to nearby stores . Accepting Electronic Benefit Transfer payments is a basic prerequisite for farmers markets to be considered accessible to low-income consumers, a concept pioneered by the GrowNYC’s Greenmarket program . While farmers markets in all 50 states now accept food stamps , the price of offerings such as a bunch of kale still exceeds the price of nearby fast food options that may offer a more filling but less nutritious meal option. Some states are moving in the direction of matching EBT funds through various “market match” policies, a step towards improving food distribution and access at farmers markets .

Settlers from the Midwest arrived with cattle, sheep, pigs and chickens, and by 1908 Lopez had a commercial creamery shipping 1,500 lbs of butter each month to the mainland. By the 1920s the islands reach a high in number of farms and farmland, with 566 farmers and over 68,000 acres in production, largely for homesteading and subsistence purposes . Important crops included fruit orchards, strawberries, peas, and beef cattle eventually taking over from dairy herds after the 1948 milk regulations. Homesteading and horticultural production continued through the mid to late 1900s; total farmland acreage fell to its lowest point in the 1970s and increased again up until 2007. Of concern for soil fertility, hay production has been a significant component of farmland increase, contributing to soil depletion over time. Despite its small population size and total agricultural acreage, San Juan County ranks in the top half of Washington counties for value of sales from hogs/pigs and sheep/goats .Development pressures building up in the 1990s and 2000s began to adversely impact farmland and housing access. In 1989, the Lopez Community Land Trust formed in response to a rapidly emerging affordable housing crisis on the island, as home prices rose 190% in one year. The Land Trust immediately focused energies on fundraising and building affordable housing communities, breaking ground on the first set of homes in 1990, and eventually completing the award-winning net zero Common Ground community in 2006, recognized for its innovative integration of straw bale construction, local materials, rainwater catchment system, solar hot water heating, and community solar array. The Land Trust adopted sustainable agriculture as core to its mission from the outset,vertical farming supplies recognizing the need to “provide permanently affordable access to land for such purposes as quality housing, sustainable agriculture and forestry, cottage industries and co-operatives by forever removing the land from the speculative market” . In 1996 LCLT collaborated to bring the nation’s first mobile meat processing unit to the islands to humanely slaughter animals, managed by the newly formed Island Grown Farmers Cooperative .

The mobile processing unit is USDA-inspected and greatly reduces costs from transporting animals off island for slaughter. LCLT helped establish the Lopez Island Farm Education program in 2006, as well as a sustainable agriculture internship program that has funded and placed over 65 interns on island farms to learn regenerative practices for farmer-educators. LCLT initiated the Lopez Island Farm Trust in 2018 to preserve parcels of farmland in perpetuity, starting with the purchase of the historic Stonecrest Farm property for $1,000,000. LCLT’s accomplishments are summarized in Figure 6. Today, farmers specialize in crops such as grass-fed meats, berries, tree fruits, diverse vegetables, grains, fiber, lavender and herbs, as well as value-added products including preserves, cheeses, ciders, and wines . Construction of greenhouses and hoop houses and commercial kitchens has enabled year-round production and preservation of the agricultural bounty. The average size of farms has decreased to 58 acres as the focus is more on small vegetable production than meat operations. Average market value of products sold per farm has decreased as well to just over $13,000, although once farm expenses are factored in, net farm income is -$6,293 . Small scale heritage grain production has re-emerged on several islands, which represents an exciting step towards relocalizing important food supply chains and reclaiming sovereignty that has been taken away from communities through consolidation of food “commodities” . Grains comprise the largest acreage of certified organic crop production in San Juan County at approximately 200 acres in production. Grains also represent new revenue stream for farmers taking advantage of growing interest in sourcing local grains among local bakeries and restaurants. A talk at the San Juan Agricultural summit in 2019 on farming history in the San Juan concluded with the statement that “it is a myth you can’t make a living farming in the islands, but the successful people have been those who have innovated and shown their savvy at investing in new varieties or types of crop and in contacting distant specialty markets” . Looking to the future, continued access to farmland remains a strong concern for the agricultural community, as the island faces heightening pressure for development serving the summer tourism and second home markets, and the ability of the Land Trust to purchase other pieces of island farmland is limited by fundraising capacity.

A 2011 report titled “Growing Our Future: An Agricultural Strategic Action Plan for San Juan County, WA” goes on to state that “appropriate state or local policies and regulations, as well as training, support, and resources are necessary to promote effective transfer, succession, and leasing of farmland for a new generation of farmers. Housing for these new farmers is a major issue as there are no code allowances for adding housing for succession farmers on existing farms, nor is there affordable housing available for new farming efforts on leased land.” Changes to code language are needed to allow for farmland transitions to occur that provide affordable housing options for new farmers. Additional incentives must be put in place to facilitate the active and regenerative farming, rather than degradation and neglect, of farmland parcels. When San Juan County farmland value is estimated at over $13,000 per acre , aging farmers are facing retirement decisions, and farms are operating a net loss financially, the continued economic viability of sustainable agriculture is called into question. Challenges of aging farmers, attracting new farmers with interest in regenerative practices, and affordability of land and farm worker housing are familiar to farming communities nationwide. Lopez has an advantage in facing such challenges through its support network at the island and county levels: LCLT, the County Agricultural Resource Committee, Ag Guild, and WSU San Juan County Extension are active proponents and providers of technical assistance, policy support, financial resources, and outreach geared towards supporting current farmers and attracting new skilled agriculturalists to the island community. Such supports are essential to confronting these and additional challenges related to building a resilient local food system on a chain of islands. When it comes to protecting farmland in the San Juans,vertical lettuce tower the strategic action planning process in 2011 came to the conclusion that “success in protecting farmland will ultimately be defined not only by the amount of farmland conserved, but also by the productive, profitable, and sustainable use of that farmland by local farmers, thereby contributing to a strong, diversified economy that benefits farmers and their community, while also building a viable and resilient local food system” . Supporting “productive, profitable, and sustainable use” of farmland will require action steps outlined as report recommendations, including: 1) Adopt and promote scale-appropriate state and local regulations in order to foster farm businesses and support a thriving local farm economy; 2) promote opportunities for new farmers to establish successful farms; 3) develop adequate access to infrastructure necessary to process and maintain diverse agricultural operations; and 4) expand local and regional marketing opportunities .

Members of the Ag Guild, ARC, and local agricultural stakeholders are working towards many of these goals, discussed further in the section below. Further partnerships are sought with conservation organizations to buy, conserve, and sell farmland parcels to those intending to farm the land regeneratively, meeting both conservation and food production objectives.The literature on agroecology and sustainable food systems highlights critical factors for success in the domain of farming or food production, starting with affordable access to good quality farmland. Other essential “success factors” for sustainable and regenerative agriculture include cultivating soil health, minimizing external inputs, educating farmers to perform knowledge intensive practices, and cultivating human resources to support agroecological transitions . Jules Pretty describes five forms of “capital” that are important to building sustainable agricultural systems: physical, natural, social, human and financial . Following his presentation of agricultural sustainability, each of these forms of capital is considered and analyzed in the case of the Lopez Island food system in the sub-sections below which evaluate land access, soil health, use of inputs, farmer education, and human resources present in the form of farmers practicing regenerative agriculture. The analysis of physical, natural, social and financial capital is based off of informal interviews and conversations with local agriculture organization leaders as well as participation in island agricultural education events. The aging farmer population and farmland transition dilemmas on Lopez are challenges mirrored in agricultural communities nationwide, encompassing both large industrial and smaller scale operations. Several of the island’s most successful farms are led by farmers in their 50s, 60s, and 70s, without a clear plan of who will take over as the current owner-operators seek to retire. The primary mechanisms for farm transfer and new farm establishment are through LCLT, the San Juan Islands Ag Guild, and the real estate market for island farmland. LCLT works towards three goals related to land access: affordable housing, sustainable communities, and farmland conservation. Their most recent initiative, the Lopez Island Farm Trust , was formed in 2018 to spearhead farmland conservation work. LIFT aims to strengthen the local food system and provide affordable access to land through a “comprehensive legal, ethical, and economically viable land lease system.” LIFT seeks to acquire, lease and manage new and historical farms; provide education for beginning farmers; foster business opportunities for regenerative agriculture operations; and encourage multi generational living on the land.

LCLT plans to use the affordable lease template as a model for securing and transitioning other farmland parcels, whether gifts or purchases, to the next generation of regenerative farmers. Ensuring the success of the newly leased Stonecrest Farm operation is essential to the continuation of this work, as facilitating a smooth transition to a new family operation is inherently challenging. It remains to be seen how replicable the Stonecrest Farm purchase is, or the degree to which it can serve as an affordable land access model, due to the difficulty for the land trust to raise large sums of money on a regular basis; “it was a big lift for us,” says LCLT Community Liaison Rhea Miller, of the fundraising effort to purchase Stonecrest. The Ag Guild recently received a three-year Beginning Farmer and Rancher Development grant from the USDA to research and improve access to farmland for new and beginning farmers. The grant collaboration included WSU SJC Extension, the Northwest Ag Business Center, Whidbey Island Conservation District, and the Organic Farm School farmer training program on Whidbey Island. As part of the grant activities, staff at the Ag Guild conduct outreach with beginning farmers interested in accessing farmland and establishing operations in the San Juan Islands, and posts opportunities for farmland access on its website’s “Farmers-to-Farmland” page . The outreach process includes connecting farmers to suitable farmland parcels and providing relevant information about available water sources, markets, local contacts, and housing options. Rather than seeing this as a failure, ensuring opportunity to opt out is an important part of the farmland succession process and ultimately setting up new farmers for success . Recognizing and overcoming challenges of a specific context is an essential part of farmland transitions, with some challenges more easily overcome than others . In most cases, the land tenure for new farmers would be through lease agreements, rather than ownership models, as much of the farmland available in the county is owned by the Land Bank, Preservation Trust, or private individuals open to leasing arrangements with aspiring farmers. There is a divide between the landowning and land leasing populations, with many young people not able to afford to buy into an ownership arrangement. Pathways to cooperative and collective ownership5 of farmland as a land access opportunity are largely absent in the Lopez case study and throughout the Pacific Northwest. Ag Guild staff are very open to the idea of supporting more farmers, both current and new, in establishing cooperative enterprises. Organic Farm School directors are similarly encouraging of this idea, arguing that many new farmers might not be ready to take over an 80-acre parcel of land and put it to productive use immediately, but it might be more appropriate for a group of five to divide up vegetable production, flower production, poultry production, grazing and value added products.