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Harvesting the Future: Hydroponic Agriculture and Global Food Security

Although this coating may not persist on the particles in the environment, what is clear is that the effects of chronic dosing and the effects of coating are critical data gaps that should be evaluated. Also completely lacking are more environmentally realistic exposure scenarios, such as ones using natural waters and soils and also multi-species microcosm or mesocosm studies, although such studies are underway. These studies will bring the importance of environmental transformations and indirect ecological impacts into light. It is possible that community or ecosystem level impacts may be more sensitive than individual level effects. Also more chronic and food chain transfer studies should be encouraged to deal with the possible long term effects from, or accumulations of, the likely persistent nanoceria entities. The current available data do not suggest an immediate risk from acute exposures to nanoceria from use as a fuel additive or mechanical/chemical polishing or planarization. However, the data gaps we have discussed should be addressed before a comprehensive ecological risk assessment can be performed for ceria for chronic exposures or for other exposure pathways. This review lays the foundation for such assessments and clearly identifies the areas where research is most critically needed.The Bioremediation, Education, Science and Technology partnership provides a sustainable and contemporary approach to developing new bio-remedial technologies for U. S. Department of Defense priority contaminants while increasing the representation of underrepresented minorities and women in an exciting new bio-technical field. This comprehensive and innovative bio-remediation education program provides under-represented groups with a cross-disciplinary bio-remediation curriculum and financial support, coupled with relevant training experiences at advanced research laboratories and field sites. These programs are designed to provide a stream of highly trained minority and women professionals to meet national environmental needs. The BEST partnership of institutions and participants benefit from a unique central strategy— shared resources across institutional boundaries.

By integrating diffuse resources, BEST forms a specialized “learning institution without walls,” large plastic pots for plants where participants can receive advanced training at any BEST site, and where research capabilities flow freely among the participating institutions. Ongoing faculty and student exchange programs, video taped lectures, the Rotating Scholars program, and the BEST web-site ensure that all participants are empowered with opportunities to excel. The BEST partnership consists of Lawrence Berkeley National Laboratory in Berkeley, Calif., Jackson State University in Jackson, Miss., Ana G. Méndez University System in Puerto Rico, University of Texas at El Paso , University of Southern Mississippi Gulf Coast Research Lab, and University of California at Berkeley . The BEST program contract to the partnership is man-aged by LBNL for the Army Corps of Engineers, Waterways Experiment Station in Vicksburg, Miss. WES manages the contract for the Army Corps of Engineers and is the contracting entity for DoD. The partnership formed by these participating institutions leverages existing institutional resources by strengthening intramural bio-remediation education and research capabilities, and through outreach pro-grams, to disseminate training and scientific enhancement to other Historically Black Colleges and Universities and Minority Institutions . The BEST institutions are focal points for the development and dissemination of cutting-edge research and technology for the bio-remediation of nitro-aro-matic compounds, polycyclic aromatic hydrocarbons and toxic metals. The multidisciplinary BEST partnership strategy creates a flask-to-field solution that develops laboratory research into technology, and technology into field-scale environmental applications required for the cost-effective restoration of damaged environments. This year saw the addition of the University of Southern Mississippi’s Gulf Coast Research Lab and the University of Texas at El Paso as partners in the BEST program. Both institutions provide significant new personnel and training opportunities for the BEST program. The USM Gulf Coast Research Lab investigators’ focus on PAH and heavy metal phytoremediation along shorelines provides an exciting new focus with increased field opportunities for students. The UTEP investigators are focusing on exciting new metal phy-toremediation techniques using desert plants and exciting new techniques to determine risk assessment with PAHs. This year also saw the passage of the program director-ship at LBNL from Dr. Jenny Hunter-Cevera to Dr. Terry C. Hazen in October 1999. Dr. Hunter-Cevera, who has managed the BEST program at LBNL since its inception, will be sorely missed, but her new position as president of the Maryland Biotechnology Institutes may provide increased opportunities for collaboration for the entire BEST program. Dr. Hazen, who specializes in bio-remediation field applications, has demonstrated or deployed bio-remediation technologies at more than 50 sites around the United States and in Europe. He has five patents in bio-remediation technologies that are licensed by more than 40 companies in the U.S. and Europe.

During the past year, the BEST program has provided minority research training for five high school students, 74 undergraduates, 32 graduate students, three post-doctoral fellows and 10 faculty. Students and fac-ulty investigators have given 43 presentations on BEST research at scientific meetings and have published 17 scientific papers. The program produced a full color brochure and flyers in 1999 for use in recruiting more students, and also sponsored 32 lecture/seminars on bio-remediation. Fourteen videotapes of BEST seminars at LBNL/UCB were distributed to the partner institutions. The BEST program also sponsored a phytoremediation workshop for BEST investigators and students that was attended by more than 60 participants. Additional workshops are planned for the coming year. In this report, the research is organized by subject area, and two-page briefs are presented for each of 28 BEST projects. The projects presented provide a good representation of the state-of-the-science research being done with students in the BEST program – the best of BEST.Over the next 75 years, the U.S. government will undertake what has been called the largest civil works project in world history to restore the environment damaged by previous activities at federal sites, e.g., Department of Defense military bases and Department of Energy nuclear facilities. Legislative action, resulting from concern over the accumulating hazards, has mandated pollution control measures and environmental restoration of hazardous waste at all sites. Estimates of total cleanup costs range from $230 billion to more than half a trillion dollars. Given the trend of diminishing budgets throughout the federal government, future generations could inherit both an environmental and budgetary disaster. The imprecision of the cost estimates results from the lack of knowledge of how “clean” the contaminated sites will need to be. Some of the environmental damage is permanent—cleanup technologies either do not exist or are incapable of remediating the contamination. For DoD bases being closed by the Base Realignment and Closure Program, all toxic sites must be remediated before the site is returned to public use. The projected costs of site restoration using existing technologies are staggering: the estimated cleanup cost is at least $24.5 billion for the 7,313 identified U.S. sites . The pollutants at these sites include chlorinated hydrocarbons, metals, petroleum products, explosives, mixed waste and other organics. DOE also has substantial remediation costs—estimated to be from $90 billion to $200 billion . The domestic private sector presents yet another huge set of remediation problems, dwarfed only by the international problems in Eastern Europe and Russia . There is clearly a need for new cost-effective treatment technologies. Bio-remediation, the use of microor-ganisms to detoxify hazardous waste, promises to provide economical and ecologically sound clean-up strategies. An Office of Technology Assessment analysis concluded that the U.S. does not possess a sufficient pool of qualified environmental professionals, i.e.,blueberry pot the trained scientific personnel required to support this rapidly developing multi-disciplinary field. In response to these national environmental needs, the Bio-remediation Education, Science and Technology Program, funded by DoD, was established in 1996. In a few short years, BEST has pioneered a new and successful model for environmental science and education. This partnership has a highly integrated programmatic focus on the scientific and workforce needs of DoD. Since the inception of the BEST program, a significant number of major milestones and deliverables have been achieved. They are described below. The BEST program has made these dramatic accomplishments by using an approach that combines a training-education element with an integrated research project, described later in this introduction.DoD sites throughout the United States contain highly contaminated soils, groundwater and sediments. These properties pose direct and indirect exposure hazards to humans and wildlife.

Conventional remedial solutions for contaminated soils and sediments or groundwater are slow and expensive, increase inputs to hazardous waste disposal sites, and can increase human exposure to contaminants. Bio-remediation — the use of microo ganisms to destroy hazardous contaminants or to con-vert them to harmless forms — is an emerging treatment technology that can in many instances restore contaminated environments more quickly, at lower cost and at lower human risk than alternative remediation technologies. Bio-remediation can operate in either an in situ mode where contaminants are treated in place, or in an ex situ mode where contaminants are removed from a contaminated zone for treatment . In situ bio-remediation can be used when excavation is impractical — under buildings, highways, runways, etc. In situ bio-remediation can simultaneously treat soil and groundwater in one step, without the generation of hazardous waste products. In situ contaminant degradation can be achieved by either intrinsic or enhanced bio-remediation. Intrinsic bio-remediation exploits the innate capabilities of indigenous micro-bial communities to degrade pollutants. Enhanced bio-remediation seeks to accelerate in situ microbial activity by isolating and controlling the contaminated site so that the microbial environment can be purposely manipulated to correct nutritional or gas phase limitations. Ex situ treatment seeks to further control the remedial environment by placing the contaminants in an engineered treatment system. Phytore mediation, a process in which plants and asso-ciated microbial communities are used for contaminant bio-degradation or bio-immobilization, is an important and rapidly developing mode of bio-remediation. To realize the full potential benefits of plant and microbial treatment systems at DoD sites, these bio-technologies must be developed and optimized for remediation of DoD priority contaminants by an expanded pool of qualified professionals. It was in response to these DoD environmental needs that the BEST partnership of institutions was established.In order to determine whether plants can stimulate the degradation of PAHs in soil, plant species found in literature on phytoremediation of metal-contaminated sites were selected to measure the removal of PAHs in artificially contaminated soil over a period of 62 days. The plant species used for this experiment were alfalfa , barley , tall fescue and orchard grass . The PAHs were phenanthrene and anthracene, in a mixture of 600 ppm each. As shown in Figures 1 and 2, phenanthrene and anthracene were removed from the soils with plants after 62 days. More than 98% of the phenanthrene was removed during that period while the anthracene removal was found to be between 70 and 90%. The results suggest that the rate of disappearance of phenanthrene in soil was greater than anthracene under the same conditions. From the results, it is also indicated that the disappearance of PAHs in soil depends on the bio-availability of the compounds. Because phenanthrene is approximately 10 times more soluble in water than anthracene, it was expected to be more readily available to microbial degradation than anthracene. Plant-assisted degradation of PAHs is thought to be more effective on PAHs with a higher number of rings and higher molecular weights, such as benzopyrene. Anthracene removal in the soil planted with alfalfa was greater than in the soil without plants, while all the other plants have minimal to no effect on anthracene removal compared to the control soil. Phenanthrene was removed to a greater extent in the soil with alfalfa and tall fescue compared to the control without plants . However, both barley and orchard grass showed no effects of the removal of phenanthrene during that period when compared to the soil without plants. Overall, plants had minimum effect on phenanthrene degradation while anthracene degradation was more dependent on plant species. In order to determine the effect of PAH degradation by plants on bacterial numbers in soil, bacteria were counted in soil during the course of the experiment.Parathion is a widely used organophosphate insecticide which can cause adverse neurological effects if ingested or after dermal exposure. No single microor-ganism has been isolated that is capable of completely mineralizing parathion and its metabolites. Hydrolysis of parathion significantly lowers the toxicity of the parent compound, but results in the formation of a toxic intermediate, the nitroaromatic compound p-nitrophenol.

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It is our hope that this discussion serves as a figurative road map for drawing your own conclusions

Non-human actants such as soil, water, and organic certification, are not included in this diagram. Further, it only accounts for actors that were encountered during fieldwork and is not meant to be exhaustive. Instead, it hopes to provide a snapshot of the ever-changing networks of actors and actants that were involved during the research period . The practice of mapping out the flows allowed us to identify key nodes of power among the urban agriculture networks in San Diego County, which are indicated on the diagram by use of a darker hue of the parent shade used for each network. For example, Leichtag Foundation is a key node of power in the Coastal Roots Farm actor-network. These actors marshall considerable power in comparison to the other actors enrolled in the networks, whether through the possession of crucial resources such as land and capital, political power, and/or consensus-building. In what follows, we discuss the discoveries we made through examining the vignettes and the network relationships. This discussion provides the results we drew from analyzing the vignettes and the network diagram.The microgeographies of these local commodity circuits had considerable influence on the discursive and material relations present at these sites. Narratives around place drove and legitimatized sites’ growing practices and their approaches to justice, whether based on donations or democratic participation. Further, the characteristics of place drove production, distribution, and consumption practices, which had important implications for justice. Every place in this research had different needs and populations, which drove their place-specific emphases and practices. For example, a mission focused on food sovereignty might be inappropriate in an affluent,plastic plants pots primarily white community like Encinitas . However, this mission is apt in a low-income, minority neighborhood like Southeastern San Diego, which has experienced considerable disinvestment and structural oppression.

These missions at our sites were fitting and reflected what was going on in those places and within their communities. This drove not only production practices, but also distribution and consumption – the lack of substantial need in Encinitas led to distribution in “less fortunate” communities outside of the neighborhood in order to fully realize their mission. This distribution pattern resulted in a more geographically dispersed network that engaged multiple communities with disparate experiences in a single commodity circuit. The characteristics of place and the narratives around production and distribution drove the actors and actants that enrolled in these networks. The most successful and stable networks in our cases, Solutions Farms and Coastal Roots Farms, successfully enrolled actors with substantial capital resources such as Leichtag Foundation and Alliance Healthcare. Indeed, Daftary-Steel, Herrera, and Porter convincingly argue that urban agriculture projects can only truly sustain themselves and produce public goods like nutritious food, education, and job readiness with external investment in the absence of “major shifts in our national wage structure” . Three factors, we argue, contribute to this successful enrolment of funders: proximity, measurable outcomes, and narrative content. Powerful actors, especially those with sustaining capital resources, are often not located in areas of the most need like Southeastern San Diego and therefore may have few, if any, ties to the neighborhood. Measurable outcomes also play a role in enrolling actants with capital resources – as we illustrated in Chapter 3, sites that practice distributive justice, which produces more readily quantifiable outcomes, attract more funding because they can illustrate the efficacy of investment. Although Mt. Hope Community Garden is still successful at enrolling philanthropic foundations into its network, investments are relatively small because of the difficulty of quantifying outcomes like participation and social cohesion. The final aspect is the content of narratives associated with each urban agriculture site, which are part of what makes them unique places. These narratives are both produced by the actor-networks and at the same time powerful actants that shape these networks – an important contribution of Actor-Network Theory. Mainstream neoliberal and reformist narratives that focus on social enterprising and food security may be more successful at attracting funding, as opposed to narratives that focus on food sovereignty. Indeed, funders are often less connected to the histories of structural oppression that drive grassroots urban agriculture projects like Mt. Hope Community Garden. This trend results in a situation in which the most disenfranchised find it difficult to enroll actors with crucial financial resources, giving support to the hypothesis that those with significant resources are more successful at attracting funding .

It also reinforces race- and class-based inequalities because projects run by disenfranchised groups, which more often have progressive or radical agendas , struggle to obtain the support necessary to sustain themselves financially. We saw this in our analysis of Mt. Hope Community Garden. The food justice narratives that surround the garden and its parent organization do successfully enroll actors with knowledge and skills to support its activities. However, the garden received considerably less funding from its network members, leaving it at the helm of the City of San Diego and its decision to sell their property. Actants like soil, water, technology, produce, and the narratives attached to them also drive action and enroll actors into the networks supporting urban agriculture commodity circuits. For instance, the produce grown at the sites determines the extent to which the organizations can generate revenue, feed people, and drive their mission. The use of soil and narratives around its ability to foster community are particularly salient at sites like Coastal Roots Farm. Technology and narratives around innovation similarly enroll actors that value modernization and novelty – technology played an important role in Solutions Farms enrollment of Alliance Healthcare and its $1 million-dollar Innovation grant. These actants, as Bosco describes them, allow our case sites to “become what they are” and explain why some networks and the justice activities embedded within them are more sustainable than others . Tracing the many connections and relations across our commodity circuits illustrates that the story is more complicated than the presence or absence of soil. Watercress is a semi-aquatic plant that grows in f lowing shallow freshwater and is found across Europe, Asia, the Americas, the Caribbean, New Zealand and Australia. Watercress is placed within the Brassicaceae family together with several other important food crops including broccoli, kale, cabbage, and mustard. A significant amount of commercial aquatic watercress cultivation is centred in a few locations including Florida in the USA, southern Spain and Portugal, France and the south of England, with 90% of production occurring in Dorset, Hampshire and Wiltshire. These chalky areas provide nutrient-rich spring water and boreholes that directly supply the watercress beds. Phosphate-rich fertiliser is used to boost crop yield; however,blueberry pot this presents a major challenge in watercress production since it results in direct leakage of phosphate into the waterways which have high conservation value.

Excess phosphate results in eutrophication of aquatic ecosystems, a process where nutrient enrichment of water sources results in excessive algal and plant growth, and subsequent disruption of ecosystem community dynamic. Approximately 90% of watercress farms in the UK are on, or upstream of, a Site of Special Scientific Interest , increasing the pressure to minimise phosphate release.Phosphate is vital for plant survival; it forms the phosphodiester bonds that link nucleotides in nucleic acids and is critical for the structure of proteins and carbohydrate polymers, for powering cells through the release of phosphate from ATP and for regulating several metabolic pathways Symptoms of P deficiency are retarded growth, increased root:shoot biomass, decreased leaf area and often dark green or purplered colouration in severely deficient plants due to anthocyanin production. Ninety percent of the global demand for phosphorus is used for food production, however, rock phosphate is a limited resource with estimates that reserves could be exhausted in the next 50–100 years . In addition, most of the remaining rock phosphate reserves areunder the control of only a few countries, with Morocco and the Western Sahara holding over 70% of the total reserves, making it sensitive to political instability. This, combined with increasing costs of extraction and issues of eutrophication,make reducing fertiliser use an important global driver. The high reactivity and low solubility of phosphates make them commonly the growth-limiting nutrient for plants. Accounting for fertiliser application, approximately 30% of global cropland area exhibits soil P deficits although global P imbalances in water sources have not been investigated to any significant extent.Like soil, P in aquatic systems is also divided into different fractions based on solubility and reactivity in aquatic systems, with dissolved orthophosphate the most bio-available. P in water adsorbs to oxides and tightly binds with carbonates in the same manner as when in soil. However, the P inputs to natural water systems and the interaction with P in bed sediments is altered. This creates a dynamic source of phosphorus that transfers between particulate and dissolved forms, between bed sediments and the water column, and between dead and living material . In a watercress bed, the sediment is shallow gravel and thus P uptake from water likely represents the major P source. This is ref lected in a study by Cumbus and Robinson who found that a greater proportion of P was absorbed by the adventitious roots of watercress , compared to basal roots. However, some organic detritus held within the sediment should still be considered. Phosphate dynamics in hydroponic agricultural systems such as watercress beds have not been studied, representing a knowledge gap, but P is likely uniformly distributed due to f lowing water and regular maintenance of P concentrations. Since P retention in sediments is high, P delivery into freshwater systems is largely governed by release from point sources such as sewage treatment works , leaking septic tanks, and from excess fertiliser application. Globally, domestic sources contribute 54% to total P inputs into freshwater systems, 38% from agriculture and 8% from industry. Although substantial steps towards P reduction in fresh waters have been made over the last 50 years there is still much to be done, with only 40% of European surface waters currently in good ecological status.

Eutrophication of watercourses is also prevalent across the UK: in the most recent analysis, 55% of river water bodies in England failed to meet the revised P standards for good ecological status. Eutrophication is both an economic as well as environmental issue. In the US, the economic damage of eutrophication equates to $2.2 billion annually, due to losses in recreational water use, waterfront real estate, recovery of endangered species and drinking water. Naturally, phosphate levels in chalk aquifers are less than 20 μg/l, however, inputs of phosphate rapidly increase these concentrations above P targets downstream of watercress farms. In the river Itchen where several watercress farms are located, total SRP load comes predominantly from sewage treatment works but watercress beds can be responsible for up to 62% of the total reactive phosphate in some chalk streams, suggesting room for improvement in P management. Casey and Smith found watercress beds increased mean P concentrations which may cause undesirable growth of algae and disruption of community dynamics. One important strategy to tackle this problem of eutrophication is through plant breeding. By breeding watercress varieties with improved phosphorus use efficiency , the impact of watercress farming on eutrophication could be minimised. To date, no breeding for nutrient use has been conducted in watercress even though P release represents a clear issue in watercress production.Phosphate use efficiency is defined as the capacity for biomass production using the P absorbed . Here PUE is used as a broader term that also encompasses phosphate acquisition efficiency, defined as the ability to take up P, as has been used in several studies. Plant traits underpinning PUE can be observed at the macroscopic, microscopic, and molecular levels and we consider their relevance to future breeding for enhanced PUE. To date, knowledge on P acquisition by aquatic plants only covers the effectiveness of plants for phytoremediation , rather than breeding for PUE in aquatic crops such as water chestnut , water spinach , lotus and watercress. Present information does not cover morphological or genetic components to improve PUE in aquatic species, and with new plant species emerging as suggested model organisms, watercress is offered as a model crop for aquatic systems.

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This interpretation correlates well with the position of three TGAT core motifs

Transcription factor binding sites were identified by consulting multiple online prediction tools which quickly found over two hundred predicted cis-motifs, many of which had low probability scores. The odds of identifying functional cis-motifs were increased in a few select cases by adding 5bp sequences on either side of the core motif, based on previously identified target sites for WUS , ARF1, and ARR1. The enlarged biding sites were then mapped to the CLV3 genomic sequence, tolerating up to 2 mis-matches in the flanking regions. In order to account for the presence of transcription factors whose cis-motifs are not currently known, MEME analysis were employed to identify motifs shared between genes that are co-expressed with CLV3. Overall, 231 potential cis-motifs and transcription factor binding sites were identified. Most were randomly distributed over the entire CLV3 genomic sequence, but irregular clusters could be recognized near the coding region. The largest cluster occurred in the upstream 500bp of the 5’ promoter, while up to three smaller clusters occurred in the 3’ enhancer region . The list of potential factors was then filtered to include those found inside the previously identified CLV3 regulatory regions, which left just 157 predictions . Many of the remaining predictions were found to have overlapping sequences, though it is unclear how well this might predict their actual function in-vivo. One notable example of this phenomenon is a predicted MYB-like binding site located at -155bp, which was predicted by four different databases. In other cases, two structurally different transcription factors were predicted to have overlapping cis-motifs, such as the bZIP/homeodomain pair Opaque-2/ALFIN-1 in the 3’ enhancer region. Interestingly,large plastic pots for plants the data also revealed four partial miR414 targets, three of which overlapped with the DNA/Mariner family transposable element At2gTE50670 in the 3’ enhancer , and the fourth occurred in the 3rd exon.

In an alternative approach to identify unknown cis-motifs, phylogenetic footprinting was used to compare CLV3 orthologous sequences from different species. In this method, functional regulatory structures can be identified by their conservation over evolutionary time, which often requires little more than performing a sequence alignment. The method is also quite robust, as previous studies found that the identified footprints matched 80 and 85% of known transcription factor binding sites. To begin this analysis, three CLV3 orthologs were identified by their syntenic relationships within the Brassicaceae using the tools in the Brassica Genome.org database. Their cDNA sequences were aligned with 27 CLE family paralogs identified in A. thaliana in order to identify features that were unique to CLV3 orthologs, before expanding the search to additional species. This analysis revealed three potentially unique traits that might be used to distinguish orthologs from the multitudes of closely related CLE genes. These included three consecutive histidines at the C terminal end of the CLE motif, a C-terminal oligo extension, and a 3-exon gene structure, all of which had been previously identified in the CLV3 sub-group. Additional orthologs were then identified using tBlastn searches against the AtCLV3 protein, for which nine species which met the criteria described above: Brachypodium distachyon, Oryza sativa, Ricinus communis, Glycine max, A. thaliana, Arabidopsis lyrata and Brassica rapa, Capsella grandiflora, and Camelina sativa. No AtCLV3 orthologs were identified in the gymnosperms, basal angiosperms, or the Asteriids using these search parameters. The Euphorbiaceae and Fabaceae each contributed one species in the closely related Eurosiids I, while the monocots are represented by two species in the Poaceae. As a result, this sampling is heavily biased towards the Brassicaceae family , which provide more than half of the total number of species. In order to footprint the promoter regions, initial sequence alignments were performed using 8kb genomic fragments, containing up to 5kb of upstream and downstream sequences on either side of the coding region. However, little or no homology was found when all nine orthologs were aligned simultaneously. This was not improved by removing monocot clade, as the two grass orthologs failed to align with each other.

Repeating this pattern, both R. communis and G. max also failed to alignment with each other, or with any of the remaining orthologs. In contrast, conserved regions became clearly visible when the five Brassicaceae species were aligned separately . This result appears to reflect the optimum degree of sequence divergence for this gene, as previous studies have found that orthologs outside of the Brassicaceae were less informative due excessive divergence, whereas sequences obtained entirely within the Brassicaceae have been found to have too little divergence . Three of the remaining species had complete genomic sequences, while the other two consisted of two contigs separated by a gap of unknown size. In the B. rapa ortholog, the gap was located in the 3’ region, and was flanked by 256 and 452 base pair sequences that did not align with any of the other Brasssicaceae orthologs, despite strong sequence conservation in the surrounding regions. This indicates the recent insertion of a large DNA fragment, potentially >700bp in size. Attempts to locate the source of the two end-fragment sequences in the B. rapa genome with BLAST searches, unexpectedly found that each was present in multiple copies, and were distributed across several different chromosomes. No evidence of transposable element sequences were found, so the flanking regions were here interpreted to be contaminating scaffold sequences from the original genome assembly. A similar gap of unknown size occurred in C. grandiflora, where one contig aligned with the CDS and 3’ UTR, while the entire 5’ upstream contig failed to align with any other ortholog. In both cases, the non-aligning sequences were removed from the analysis, providing a final alignment consisting of four orthologs in the 5’ promoter region, and five orthologs spanning the CDS and 3’ UTR. Overall, the five orthologs shared between 27% and 65% sequence similarity, and grouped into two closely related pairs. One pair contained C. grandiflora and C. sativa, and the other contained A. thaliana and A. lyrata. In contrast, B. rapa was found to be distinct from all other Brassicaceae orthologs, which accurately recapitulates its predicted evolutionary relationship with the rest of the family. Upon closer inspection, the coding regions were found to be 79-93% similar, which dropped to just 14-34% in regions with no significant alignments. The initial alignment was considerably fragmented, with many insertions, deletions, and isolated nucleotides. In many cases, the position of these features varied with the settings in the alignment software, and were here interpreted to be artifacts of the alignment procedure.

To correct such artifacts, isolated nucleotides were manually adjusted left or right to maximize local sequence alignments within ±5bp. Where variation in the length of tandem repeats was apparent, gaps were introduced into one or more ortholog sequences to accommodate the largest number of repeats present. Conserved regions were then identified by using a 5bp sliding window to identify regions with more than 60% identity. This window is unusually small compared to previous studies that have used 15-50bp sliding windows, but was chosen here to more accurately reflect the minimum size of known transcription factor binding sites. Where large contiguous conserved regions were found, the presence of small 1-3bp indels within their sequences were used to break them into smaller fragments, as disruption of these sites indicates that they do not contain functional cis-motifs. scattered in the 3’ UTR. Several predicted transcription factor sites were found within the coding regions, but these were interpreted to be non-functional, as previous GUS-reporter systems did not reveal any significant regulatory elements within this region. Among other notable features was a predicted signal peptide in the first exon, identified with signal P 4.0,blueberry pot which was almost entirely conserved and is consistent with the secretion of the mature CLV3 oligopeptide. In addition, the second exon was found to be completely conserved with no In all, 42 conserved regions were identified, ranging in size from 5 to over 111bp long. Fourteen footprints were found in the coding sequence, of which nine of were clustered around the three exons. Only one footprint was found entirely within in the 5’ UTR, and the remaining four were intervening gaps. The second exon also completely overlapped with several predicted transcription factors, including HOX2a, aswell as cytokinin and gibberellic acid responsive motifs. This suggests as-yet unrecognized functional role for the second exon, which might explain why it has been retained in a family that consists largely of single exon genes. The 3rd exon was also highly conserved, although curiously the most conserved region only partially overlapped with the CLE motif and instead included part of the C-terminal extension. In the 3’ UTR, the footprints were found to overlap with potential zinc-finger and MYB binding sites, as well as a cytokinin responsive ARR10 site. In the upstream regulatory region, the 5’ promoter contained ten conserved footprints, eight of which formed a large and nearly contiguous block near the TSS. The two isolated footprints were located at -204bp and -167bp upstream, corresponding to the palindromic Motif#2 and the redundantly predicted MYB binding site, respectively. In the remaining footprints, additional predictions were found for an overlapping AGL15/CBF site, an auxin response element, overlapping GT1 and AGAMOUS sites, and one prediction for a TATA-less promoter. The latter may be related to the position of the only recognizable TATA box-like sequence, which at – 68bp upstream, which is more than double the usual 25-35bp described for other TATA-based promoters. In contrast, the 3’ enhancer region contained seventeen footprints arranged in roughly three clusters, spanning a region nearly 600 bp long. Two of these clusters closely corresponded with the previously noted clusters of predicted transcription factor sites, while the third was distinctly isolated and had no predicted transcription factors. Together, the footprints contained one of the three known WUS binding sites , two predicted AtHB1 binding sites, a cytokinin responsive element , several bZIP motifs, a KNOX-like site, and a predicted cis-motif forNPR1. Strikingly, the majority of the footprints also overlapped with a DNA transposable element in A. thaliana, At2TE50665 . It has previously been implied that WUS controls CLV3 expression in a concentration dependent manner, which is consistent with the close proximity of two demonstrated WUS binding sites .

The region around these two sites also contains several other TAAT cores within a single stretch about 100bp long, much of which is represented by four conserved footprints, which together might form a WUS binding site cluster. However, only the +970 WUS binding site was found to be perfectly conserved, while the other TAAT cores displayed mutations or were interrupted by indel sequences in one or more orthologs. Instead, when the region around the known WUS binding sites was examined in more detail with a 5bp sliding window, a strikingly periodic pattern was observed, where four different conserved motifs were found to be regularly spaced about 15 bp apart. In order from 5’-3’, these motifs were identified as CCGTTGGG, AGTAC, TTGTCAA, and TAATTAATGG , the latter two of which correspond to a predicted W-box motif, and the +970 WUS binding site. In addition, a perfectly conserved sequence was found just 25-36 bp downstream in all orthologs, which consisted almost entirely of tandem repeats containing ATG. The ATG repeats also overlapped with a predicted ALFIN-1 homeodomain/Opaque-2 binding site, suggesting that this sequence may actually represent a modified bZIP motif, or perhaps an atypical homeodomain binding site containing a TGAT core motif. It is not clear how many potential binding sites are present in these ATG repeats, but in consideration of the size of the conserved region, it seems likely that they could accommodate up to three transcription factor proteins simultaneously.The potential functional role of the TGAT motifs is further supported by the observation that they are 4x over-represented in the surrounding 124 bp conserved region, while the TAAT cores actually are 5x under-represented. In addition, pair-wise distance measurements between the two cores revealed a skewed distribution, where few sites were found closer together than the median value of 5bp. When several median-length pairs were aligned, this corresponded to the 13bp motif TAATnnWnnTGAT. When this motif was subjected to Patmatch searches of the A. thaliana genome, it was found to be 26x over represented among the genes directly targeted by WUS. Multiple copies of the 13bp motif were also found in several target genes, including two in the 3’ enhancer of AtCLV3.