However, the concentration of Liberibacter DNA has to be high enough to allow for confirmation by conventional PCR followed by cloning and sequencing. By the time titers are high enough in plants for this, the infection has already spread to adjacent trees—especially if Asian Citrus Psyllid are present. This scenario is completely inadequate if the California citrus industry is to keep HLB out of groves. Several early detection techniques are being evaluated by the California Citrus Research Board. These include both direct measurement of elicitor proteins produced by the bacterium and the detection of small interfering and messenger RNAs, proteins, metabolites, and volatile organic compounds produced by the citrus tree in response to infection. Many of these approaches measure systemic responses. This holds a huge advantage over any PCR technique which is significantly limited by sampling since Liberibacter are not uniformly distributed in an infected tree. Nevertheless, a PCR protocol is urgently needed to validate these early detection methods that would provide acceptable evidence of their efficacy to federal and state regulatory agencies. Droplet digital polymerase chain reaction amplification is such a protocol. The technique utilizes the same qPCR primers and probes and amplified products can be isolated, cloned, and sequenced from ddPCR as with conventional PCR. We have evaluated 87 transect/risk-based survey site samples from around the Hacienda Heights positive with ddPCR that have been evaluated by several of the early detection protocols. Correlation of ddPCR results with those of these methodologies will determine which protocol show the most promise for detecting HLB early enough in the infection process to make tree removal, and therefore elimination of inoculum, feasible.

Huanglongbing in Florida is caused by Candidatus Liberibacter asiaticus , a phloem-limited fastidious α-proteobacterium,10 liter drainage pot which is transmitted by Asian citrus psyllids . HLB is causing an unprecedented crisis for citrus industry in Florida and poses a severe threat to citrus production in California, Texas, and Arizona. Currently, no effective HLB management is available. We aim to control HLB by targeting critical traits of Las to break down its infection cycle. Interestingly, Las contains the General Secretory Pathway , which is important for the viability and secretion of putative Sec dependent effectors of Las. SecA, an ATPase, is vital for the function of the Sec pathway and a good target to develop antimicrobials. We have identified multiple SecA inhibitors with high antibacterial activity to Liberibacters and their relatives. We will represent our recent progress in controlling HLB using SecA inhibitors and other antimicrobial compounds. In addition, our study indicates Las contains a functional salicylic acid hydroxylase which breaks down SA and its derivatives. SA and its derivatives play a central role in plant defenses, e.g., systemic acquired resistance , and are exogenously applied on plants as SAR inducers to control plant diseases. Here, we will present our recent progress in controlling HLB by nullifying SA hydroxylase of Las. Finally, breaking down the interactions of virulence factors and their targets in planta has been suggested to be one strategy to generate disease resistant plants. We will present our recent progress in identifying SDEs and their putative targets. We aim to generate HLB resistant or tolerant plants by disrupting the interaction between SDEs and their targets in citrus. Conventional PCR and real-time PCR methods had been developed and systematically validated for detection of citrus Huanglongbing before the disease was detected and confirmed in Miami, Florida, in 2005. Since then, the two rapid and sensitive multiplex qPCR assays have been used to screen field samples for HLB as part of an on-going survey conducted by state, industry, and/or USDA-APHIS regional laboratories. The two screening qPCR assays can detect all three known species of HLB bacteria: ‘Ca. L. asiaticus’ , ‘Ca. L. africanus’, and ‘Ca. L. americanus’. Since 2005, over 200,000 samples of HLB host plants and Asian citrus psyllids collected mainly in Florida, Texas, South Carolina, Louisiana, Georgia, and California have been tested using the screening qPCR assays. Confirmatory tests which include three multiplex qPCR , three cPCR assays , and sequence analysis of the cPCR amplicons are done exclusively at USDA CPHST Beltsville laboratory on samples that test suspect positive in the screening qPCR assays.

The first regulatory detections of HLB-Las by the states using the screening assays were confirmed by the federal confirmatory assays in new quarantine areas. We combined the two screening qPCR assays and validated them as a single assay . The performance of the new combined assay remained unchanged; however, the cost was reduced by half. We have supplemented our confirmatory assays by developing and validating two additional qPCR assays that target two different conserved genes of Las. Although the current qPCR assay is at least 100-fold more sensitive than cPCR, which in 1996 proved useful for pre-symptomatic detection of the disease, in the future we will evaluate several emerging technologies and/or platforms such as digital PCR and digital sequencing for advanced HLB diagnosis. Huanglongbing , associated with Candidatus Liberibacter asiaticus , is the most devastating disease of citrus and threating the citrus industry in Florida. Early root infection has been suggested to play a central role in HLB disease development and of significance to tree health. Therefore, introduction of beneficial bacteria to roots to promote root health might be an alternative approach to management of HLB. Our recent investigations showed that three beneficial Bacillus and closely relative isolates were able to promote citrus plant development with stronger root systems and delay the development of HLB symptoms and Las populations in greenhouse assays. Here, we characterized the rhizosphere competence of these three isolates in both greenhouse and natural environment. Using culture-dependent and – independent approaches, bacterial populations of these isolates on roots of citrus and in rhizospheric soil were determined following soil inoculation. The bacterial populations on the roots of citrus and in soil one month after inoculation were approximately 5.0 × 104 CFU/g and 5.0 × 103 CFU/g , respectively, for the three isolates. The assays revealed a similarity in rhizosphere competence with survival rates ranging from approximately 0.05% to 0.5% for the three isolates. Further analysis revealed that the rhizosphere competence of these isolates may be associated with various phenotypic traits, including substrate utilization, nitrogen dissimilation, siderophore mediated iron acquisition, stress tolerance, copper resistance, and the production of antimicrobial substances.Globalization has increased long-distance human-mediated pathways for invasive disease introduction. Detection of initial introductions of exotic pathogens/pests is challenging because they occur in very low incidence. Optimal probability of eradication/mitigation depends on early detection prior to spread. The earlier the detection, the more likely the pathogen can be eliminated or the epidemic slowed,plant pot with drainage lessening impact over multiple years. To find point introductions across a broad geographic landscape of mixed agricultural/residential areas requires substantial manpower and fiscal resources. Point introductions often go undetected for prolonged periods until incidence exceeds the lower threshold of sampling sensitivity. The Census/Travel model utilizes probable pathways, parses regions into smaller areas , and predicts the most likely locations in a given geographic area for introduction. The model’s geospatial method uses US census and international travel data combined with a pathosystem’s epidemiological characteristics, i.e., latency; detection sensitivity, reliability of confirmation, reproductive rate, environmental suitability, dispersal rate, ease of control, etc. Combining existing foreign population habitat and international pathway data, the model generates a risk index map to identify locations with the highest introduction potential. The risk map is linked to a survey optimizer that calculates the number of samples to be taken in a given area based on risk, and estimates manpower and fiscal requirements. It also ranks foreign countries by their relative contribution to risk of disease introduction. Risk maps were generated for HLB and other pathogens/pests. For prior disease introductions in Florida with identified distributions, the model performed well and validated known points of introduction. The census/travel model is being integrated into existing risk-based model platforms to optimize early detection surveys in California, Texas, Arizona, in Florida to optimize disease intervention/control.

The model is independent of pathosystem, and can be extend to all States to predict introductions of human, animal, or plant diseases/pests. Citrus Health Management Areas facilitate the coordinated control of ACP populations, the clean-up of abandoned groves, and the removal of infected trees. Growers cooperating within a CHMA have been effective in suppressing ACP populations and slowing the spread of HLB in FL, indicating CHMAs are a viable management strategy. It has been confirmed that ACP populations are decreasing where coordinated spray efforts have been implemented in commercial groves in FL. However, the boundaries of CHMAs in FL are not optimized in size, but constructed primarily on arbitrary boundaries with an attempt to combine resources and garner neighbor participation. There are other various relevant factors, such as urban population size, abandoned groves’ acreage, ratio between commercial citrus and residential area, will have a significant contribution to CHMA performance. Improving from CHMA design deficiency in FL, we intend to construct CHMA boundaries for Central Valley based on estimated HLB/ACP risk level under 1-mile2 grid resolution. Where mixed landscapes exist, an optimal mix of residential and commercial landscape is considered so that regional disease management decisions can be implemented more effectively. Through cluster analysis and spatial statistics, we have developed maps that organize plantings based on the spatial pattern and dynamics of ACP populations and HLB risk. K-means clustering method is preferred to construct CHMA as homogenous as possible for risk. The CHMA size and locations are also optimized for cost-effective management. Through thousands of simulations and optimization, 28 CHMAs are currently proposed for California Central Valley, which explains more than 55% of the total risk variance. For super large CHMAs, sub-CHMAs can be further constructed with approximate balanced between acreage and ownership. A similar methodology can be applied for CHMA construction in other citrus producing areas or states .Citrus Huanglongbing , spread by the psyllid vector, is a devastating disease threatening nearly every citrus producing area worldwide with the exception of Australia and Mediterranean countries. The recent finds of ACP in California central valley emphasize the urgency for regulatory intervention and disease control, as this would pose a major threat to the viability of the citrus industry. Increasing ACP incidence and risk are considered inevitable for the Central Valley. A spatial explicit ACP simulation model is developed with attempt to understand the dynamic of ACP population changes, in particular, how ACP spread in a mixture of residential and commercial citrus landscapes in the Central Valley. A 16 mile2 area of Porterville, CA is used as the baseline for ACP spread simulation. The mathematical model considers the following parameters for ACP progression: psyllid life span and mortality, net reproduction rate in natural environment, psyllid dispersal distance, citrus host type and density, new flush production, and effect of different spray schemes.We then modeled factors influencing ACP population variation and interaction with new flush availability in relation to temperature for a period of 3 years using a daily time step. ACP spread occurs more frequently and faster within commercial citrus clusters, but comparatively slower for low density or well separated residential areas. This study also evaluates the behavior and distribution of ACP by subjected to different spray strategy scenarios. A comparison between simulation outputs confirms that the synchronize rate for coordinated spray plays an important role in slowing ACP epidemic development. Besides justifying the benefit of large-scale disease management, the outcome of this simulation model can also quantify the influence of input epidemiological parameters on ACP development, and can assist management decision-making by running specified scenario-based analyses. Powerful computational tools have enabled us to learn/extract patterns of residential host plant distribution. To a large extent, residential citrus biodiversity/choice is influenced by both the physical environment and preferences of household for specific host types. An understanding of social preferences for dooryard citrus tree is critical to residential citrus host density mapping and modeling. Dooryard citrus preferences are heterogeneous and far from random, where certain factors may be able to explain part of the variation in preference. In addition to local climatic and environmental factors, we postulate that a range of demographic and socioeconomic characteristics can also affect the residential preferences for citrus types.

L-PEACH has been implemented using the general-purpose plant modeling software. This makes it possible to keep the model code compact by delegating generic issues to the general functionality of. As a result, L-PEACH is easy to maintain and conducive to simulated experimentation. Furthermore, it may serve as a template for constructing other functional–structural models that involve solving complex systems of equations in growing plant structures. In a comprehensive review of carbon-based tree growth models, Le Roux et al. pointed out that three critical issues have not been adequately addressed by most models: adequate representation of the dynamic and feedback aspects of carbon allocation on tree structure and carbon acquisition; explicit treatment of carbon storage reserves and remobilization over multiple years; and integration of below ground processes and tree water and nutrient economies into whole-plant function. Although the work is far from complete, the L-PEACH model provides a platform for addressing all these critical issues. By combining the sink-driven carbon partitioning concepts of the original PEACH model into a distributed network of architecturally explicit sources and sinks, factors such as the proximity of individual sinks to other sinks and sources, as well as the transport resistances between these entities, can be accounted for and become involved in growth and carbon allocation outcomes. However, although there are some experimental data to indicate the functional nature of these relationships , more data will be required before the model is fully calibrated. Considerable conceptual experimental research will be necessary to provide quantitative data required for this calibration. This model also explicitly addresses carbohydrate storage in stems and roots during the growing season,growing blueberries in pots and remobilization of stored carbohydrates during the spring growth flush.

In the process of developing this model, we became increasingly aware of the lack of information about the quantitative dynamics of carbohydrate reserves in trees. As pointed out by Le Roux et al. , the lack of knowledge of the mechanisms driving reserve deposition and remobilization is a major obstacle for evaluating the carbon available at any given time, or for relating reserve dynamics with internal and external variables in tree growth models. Based on preliminary data on root starch concentrations in peach trees we have chosen to treat the starch reserve sinks in stems and root segments as compartments that have sink capacities proportional to their annual growth increment. These reserves then become carbon sources during the spring flush . Current model functions related to carbohydrate reserves are based on preliminary data, and it is our intention to test more fully and quantify these aspects of the model in the near future. The inclusion of the water stress/interaction component in the model is an attempt to demonstrate how root function can be incorporated into a dynamic L-system model of this type. As with carbon storage, the relationships between developing water stress and physiology are based more on published conceptual relationships than on precise quantitative data collected for the purpose of calibrating the model. Nevertheless, the potential of this model to simulate functional interactions between root and shoot processes is readily apparent. Similarly, there is clearly the potential to incorporate additional root processes such as nutrient uptake into the model to more fully capture the functional dynamics of root– shoot interactions. Future developments could also involve the integration of existing architecturally based models of carbon transport and partitioning in roots , in order to model root function more explicitly.The Ranunculaceae is a large and complex plant family, including approximately 59 genera and 2,500 species . Pulsatilla Miller, first described in 1753, consists of about 40 species that are restricted to temperate subarctic and mountainous areas in the Northern Hemisphere .

Plants of Pulsatilla species are often covered with long, soft hairs. Their flowers are solitary and bisexual, with three bracts forming a bell-shaped involucre. The tepal number is always six, and stamens are generally numerous, with the outermost ones resembling degenerated petals . Most authors have treated Pulsatilla as a subgenus or section of the genus Anemone s.l. . However, Miller , Adanson , and Wang et al. have supported a model that separates Pulsatilla from Anemone as an independent genus. Recent phylogenetic studies have shown that all species within Pulsatilla are clustered in a monophyletic group, which is nested within Anemone . Morphologically, Pulsatilla can easily be distinguished from Anemone s.s., since species of the former have a long, plumose beak on the achenes formed by the persistent style and stamens whereas species of the latter do not. Because the primary goal of the present study is to test the use of DNA barcodes for species in the Pulsatilla clade, we here follow the treatment of Wang et al. and Grey-Wilson , regarding Pulsatilla as a distinct genus. There are eleven species of Pulsatilla found in China, most of which are found primarily in the northern part of the country . Some species of Pulsatilla have been used in traditional Chinese medicine for many years for “blood-cooling” or “detoxification” . In particular, the root of Pulsatilla chinensis Regel is a well-known ingredient included in the Chinese Pharmacopoeia . Many species used in folk medicine have been found to contain pharmacologically useful chemical components, including those with anti-cancer and anti-inflammatory activities . The contents of these components differ in various species, resulting in different clinical pharmacological effects. Thus, in cases where target species can be easily confused with their close relatives, undesired species can be inadvertently collected, resulting in negative effects on drug efficacy and patient safety, as has been shown in other plant groups of medicinal importance in China . Pulsatilla is an especially challenging, complex group. In all treatments published to date, the genus has been treated as comprising two to four subgenera: subgenus Miyakea, which contains only one species, P. integrifolia; subgenus Kostyczewianae, which has only one species, located in Central Asia and northwestern China; subgenus Preonanthus, which includes six species; and the largest subgenus Pulsatilla, which comprises 29 species.

However, Pulsatilla shows a frustratingly complicated pattern of intrageneric morphological variability . The recognition and identification of wild Pulsatilla species based on traditional approaches is difficult due to transitional intraspecific morphological characteristics in many Pulsatilla species. For instance, P. turczaninovii and P. tenuiloba were considered to be two separate species that could be told apart by the number of pairs of lateral leaflets . After carefully checking specimens and population investigation, we found that the leaflet numbers of P. turczaninovii and P. tenuiloba are overlapping,drainage gutter and some individuals have both 4 and 5 pairs of lateral leaflets. Flowers nodding before anthesis is recorded as a diagnostic character of P. campanella, but this character was also found in P. ambigua, P. cernua and P. dahurica, and their flower colors show continuous transitional shades of blue . Thus, these characters are not reliable and make Pulsatilla difficult to identify. DNA barcoding aims to achieve rapid and accurate species recognition by sequencing short DNA sequences or a few small DNA regions . This technology was first developed to identify animal species; for example, Hebert et al. argued that “the mitochondrial gene cytochrome c oxidase I , can serve as the core of a global bio-identification system for animals”. Studies have continued to demonstrate that the COI gene fragment efficiently discriminates among animal species, including amphibians , birds , fish , and insects . In plants, however, frequent recombination and low mutation rates restrict the utility of mitochondrial barcode markers . The search for suitable candidates has therefore focused on chloroplast and nuclear DNA markers , although such markers are not always easy to amplify and sequence in all plant taxa using universal primers. Numerous studies have suggested that four standard barcodes — three from the chloroplast genome [the ribulose-bisphosphate/ carboxylase Large-subunit gene , the maturase-K gene , and the trnH-psbA intergenic spacer and the nuclear ribosomal internal transcribed spacers ] — should be used as core barcode markers for the molecular identification of plants . Significant progress has been made in DNA barcoding in plants . However, the discrimination of closely related species using only molecular data is still a major challenge in some genera . Morphological characters, including the shape of nutritive and reproductive organs, remain highly valuable for plant identification and studies of plant evolution . Micromorphological characters have been shown to have great value for species identification and systematics , and these have rarely been considered by previous barcode studies. However, the combination of morphological data and DNA barcodes may be essential for species discrimination, especially in closely related species . Previous molecular phylogenetic studies have included few species from the genus Pulsatilla . In a recent phylogenetic study of Pulsatilla, few species were from Asia and few individuals were collected for one species . Obtaining DNA barcode data from a dataset created by comprehensive sampling of a taxonomically difficult genus such as Pulsatilla should contribute to understanding the discriminatory potential of barcodes in morphologically complex clades. The establishment of an available barcoding system for Pulsatilla may also facilitate further utilization of these taxa, as well as further research into their taxonomy. In this study, four DNA barcode regions were assessed in 19 species of Pulsatilla. Approximately 50% of the accepted species of Pulsatilla found in Europe and the Americas were included, as were 90% of the species found in China.

Our objectives were to: test the effectiveness of common core DNA barcodes in Pulsatilla, evaluate the resolution of these four barcodes, and use 2- to 4-region combinations to correctly identify individuals. We also aimed to develop a protocol that could effectively discriminate among closely related species, primarily for species discrimination of medicinal plants. In addition, we added micro-morphological analyses of leaf tissue obtained using scanning electronic microscopy to reveal the taxonomic relationships among Pulsatilla.In total, 52 accessions representing 19 Pulsatilla species were involved in this study . This sample covered each of the three subgenera from Asia, Europe, and America. Nine samples were sourced from herbarium specimens, while 43 samples were newly collected. All samples were taxonomically identified using published floras, monographs, and references. In total, one to five individuals per species were sampled from different populations in the wild. Fresh leaves were dried in silica gel upon collection and the longitude, latitude, and altitude of each collection site were recorded using a GPS unit . Voucher specimens were stored in the Herbarium of Northwest A&F University and the US National Herbarium . Singleton species were only used as potential causes of failed discrimination, and were not included in the calculation of the identification success rate. Three members of Anemone, two of Clematis, one of Anemoclema, and one of Hepatica were selected as outgroups for tree-based analyses.In this study, the short DNA sequences ITS and trnH–psbA had the best performance in PCR amplification and sequencing among the four barcode markers . Moreover, successful sequencing rates for sequences ITS and trnH–psbA were over 90% for silica-dried samples but lower for herbarium specimens. These findings are consistent with many previous studies . In addition, the varying lengths of insertions/deletions found at the trnH–psbA loci for different species provide important phylogenetic information and species discrimination power . Thus, sequence alignments of this region must be performed with great care to avoid overestimating substitution events. The rbcL and matK genes are approximately 1,428 bp and 1,570 bp in length, respectively . The greatest problem with rbcL and matK was that it was difficult to amplify them from the degraded DNA isolated from old herbarium specimens, since the short lengths of remaining fragments hampered the extension phase of the PCR for these longer genes. Although some problems may be alleviated by using additional pairs of primers, the amplification and sequencing success rate of the old herbarium samples remained poor. Thus, we were not able to obtain all sequences for all herbarium samples.An ideal DNA barcode should be universal, reliable, cost effective, and show considerable discriminatory power. Because none of the proposed single-locus barcodes perfectly meets all these criteria.Multilocus barcodes can often improve the resolution rate of species identification . In the present study, when evaluated alone, the species resolutions based on tree-building for the three chloroplast regions rbcL, matK, and trnH-psbA were 48.78, 14.63, and 9.30%, respectively.

In fleshy fruits, soluble sugars, including sucrose, fructose, and glucose, are not only essential for fruit growth and development but also central to fruit quality. Fruit taste and flavor is closely related to the composition and concentration of sugars and their balance with acids. As the composition and concentration of sugars at fruit maturity is determined by metabolic and transport processes during fruit development, understanding these processes and their regulation is important for fruit quality improvement. At the center of sugar metabolism in sink cells is the Sucrose cycle, previously named the Sucrose–Sucrose cycle or the futile Sucrose recycle, which consists of the breakdown of sucrose by invertase and sucrose synthase, the phosphorylation of the resulting hexoses and the interconversion between hexose phosphates and UDP-glucose, and the re-synthesis of sucrose via sucrose-6-phosphate synthase and sucrose-6- phosphate phosphatase. This metabolic system connects sugar metabolism with many other metabolic pathways such as glycolysis and tricarboxylic acid cycle, starch synthesis, and cellulose synthesis, and its coordination with the sugar transport system on the tonoplast is expected to determine the partitioning of sugars between metabolism in the cytosol and accumulation in the vacuole. In fleshy fruits, the concentration and distribution of sugars in parenchyma cells are affected via this cycle by developmental processes and environmental factors. However,plastic pots for planting the biochemical regulation of the cycle and the associated transport system is not fully understood.

In apple and many other tree fruit species of the Rosaceae family, sorbitol is a primary end product of photosynthesis and a major phloem-translocated carbohydrate, accounting for 60–80% of the photosynthates produced in apple leaves and transported in the phloem. In source leaves, sorbitol is synthesized from glucose-6-phosphate in a two-step process: G6P is first converted to sorbitol-6-phosphate via aldose-6-phosphate reductase , then followed by dephosphorylation of S6P to sorbitol via S6P phosphatase. The loading of both sorbitol and sucrose into the companion cell-sieve element complex in the phloem is passive and symplastic in apple, but their phloem unloading in fruit involves an apoplastic step.Once released from the SE-CC complex of the phloem in apple fruit, sorbitol is taken up into the cytosol of parenchyma cells by plasma membrane-bound sorbitol transporters and then converted to fructose by sorbitol dehydrogenase ; sucrose is either directly taken up into parenchyma cells by sucrose transporters , or first converted to glucose and fructose by cell wall invertase and then transported into the parenchyma cells via hexose transporters. Compared with plants that transport and utilize only sucrose, such as Arabidopsis, tomato , and poplar , apple is unique in that both sorbitol and sucrose are transported in the phloem and are metabolized in sink organs. It is estimated that >80% of the total carbon flux goes through fructose in apple. Once taken up into parenchyma cells of fruit, both sorbitol and sucrose feed into the Sucrose cycle to meet the carbon requirement for fruit growth and development while excess carbon is converted to starch for storage in plastids or transported into vacuole by sugar transporters for accumulation. Although we have characterized the genes and proteins involved in sugar metabolism and accumulation in apple, it remains unclear how apple trees adjust the Sucrose cycle and the transport system in response to altered supply of sorbitol and sucrose from source leaves.

In transgenic apple trees with antisense suppression of A6PR, leaf sorbitol concentration is dramatically decreased, whereas sucrose concentration is significantly elevated in the source leaves, but neither leaf CO2 assimilation nor plant vegetative growth is altered. The decreased sorbitol synthesis leads to significant changes in the expression profile of key genes in leaf starch metabolism and many stress response genes. In addition to being a key metabolite in carbohydrate metabolism, sorbitol also acts as a signal regulating stamen development and pollen tube growth and resistance to Alternaria alternata in apple. In the shoot tips of the A6PR transgenic plants, both the activity and transcript level of SDH are downregulated, whereas those of sucrose synthase are upregulated in response to a lower sorbitol but higher sucrose supply. Teo et al.reported that fruit of the transgenic apple trees accumulated a higher level of glucose and lower levels of fructose and starch at maturity, but no significant difference was detected in the activity of key enzymes in sugar metabolism, CWINV, neutral invertase , fructokinase , hexokinase , or SPS between the transgenic lines and the untransformed control . Considering that antisense suppression of A6PR has drastically decreased leaf sorbitol level and increased sucrose level, leading to less sorbitol but more sucrose being transported in the phloem; and both transcript levels and activities of SDH and SUSY responded to the altered sorbitol and sucrose supply in the shoot tips of the transgenic plants, we predicted that the decreased supply of sorbitol and increased supply of sucrose would lead to down regulation of sorbitol metabolism and upregulation of sucrose metabolism in the transgenic fruit as well. The discrepancy between the data obtained by Teo at al. and our predicted responses on the activities of sucrosemetabolizing enzymes in the transgenic fruit has prompted us to re-evaluate sugar metabolism and accumulation in the fruit of these transgenic plants to better understand how the Sucrose cycle and the sugar transport system respond to an altered supply of sorbitol and sucrose.

Antisense suppression of A6PR significantly decreased sorbitol concentration but increased sucrose concentration while largely maintaining fructose and glucose concentrations in source leaves throughout fruit development in the two transgenic lines relative to the untransformed CK . Sorbitol concentration in the source leaves of antisense line A27 was decreased to ~70% initially and 13% at harvest of that detected in CK. For antisense line A04, sorbitol concentration was decreased to 32% initially and 10% at harvest of the CK level. By contrast, sucrose concentration in the source leaves of A27 and A04 was much higher than in CK throughout fruit development, with larger differences detected at later developmental stages . Concentrations of sorbitol and sucrose were also measured for source leaves, leaf petioles, and fruit pedicels at 75 days after bloom . Compared with CK, antisense lines A27 and A04 had lower concentration of sorbitol, higher concentration of sucrose, and lower molar ratio of sorbitol to sucrose in the source leaves,strawberries in a pot leaf petioles, and fruit pedicels. The abundance of sorbitol followed the order of source leaves > leaf petioles > fruit pedicels .Average fruit fresh weight did not differ significantly between the two antisense lines and CK during fruit development except for about a 10% lower value detected for A27 and A04 at 108 DAB and at harvest . Average fruit dry weight did not show any significant difference throughout fruit development . Dark respiration was ~1.5–1.9-fold higher in A27 and A04 fruits than in CK fruits between 40 and 108 DAB during fruit development, but no significant difference was detected at harvest . Fruit yield per tree was significantly lower in the two antisense lines than in CK, largely due to lower average fruit weight at harvest as fruit number per tree was not significantly different between the two antisense lines and CK .Suppression of sorbitol synthesis in source leaves led to a significant decrease in sorbitol concentration in the fruit of two antisense lines A27 and A04 throughout fruit development, particularly in A04 . However, sucrose concentration was similar in the fruits of the two antisense lines and CK during fruit development with a higher level detected in the transgenic fruit only at 74 DAB. Fructose concentration showed no difference between the transgenic fruit and CK except being slightly lower at 108 DAB in the transgenic fruit. Compared with CK, concentrations of glucose and galactose were much higher throughout fruit development, with larger differences detected at later developmental stages. Concentrations of G6P and fructose-6- phosphate decreased during fruit development and were significantly lower in A27 and A04 than in CK from40 to 108 DAB . At fruit maturity , total soluble solids concentration was significantly higher in A27 and A04 than in CK . Fruit starch concentration did not show obvious difference between the transgenic lines and CK before 74 DAB but was slightly lower in A27 and A04 than in CK after 108 DAB .SDH activity decreased during fruit development and was significantly lower in both A27 and A04 than in CK at each developmental stage . CWINV activity dropped dramatically from 40 to 74 DAB and then remained fairly constant to maturity, but no significant difference was detected between the two antisense lines and CK.

NINV activity decreased throughout fruit development and was ~1.5–2.0-fold higher in A27 and A04 than in CK from 74 DAB to fruit maturity . Vacuolar acid invertase activity showed no significant difference between the two antisense lines and CK except a slightly higher activity detected in A27 and A04 than in CK at 108 DAB. SUSY activity declined during fruit development and was significantly higher in A27 and A04 than in CK from 40 to 108 DAB. FK activity decreased during fruit development and was significantly lower in A27 and A04 than in CK at 40 and 74 DAB. HK activity decreased during fruit development and was significantly higher in both A27 and A04 than in CK from 74 to 134 DAB. SPS activity increased slightly from 40 to 108 DAB and then dramatically to fruit maturity, with a significantly lower activity detected in both A27 and A04 from 40 to 108 DAB.Our data clearly showed that sorbitol concentration was significantly lower, whereas sucrose concentration was significantly higher in the source leaves of 5-year-old transgenic “Greensleeves” apple trees with antisense suppression of A6PR compared with the untransformed CK throughout fruit development. These results are consistent with those reported for the 1-year-old transgenic trees. The higher sucrose concentration in the source leaves is an indication that a larger proportion of the photosynthetically fixed carbon ends up in sucrose over a 24-h period because most of the starch accumulated during the day breaks down for sucrose synthesis at night in the transgenic plants although no difference in the carbon flux to sucrose during the day was detected. As both sorbitol and sucrose diffuse into SE-CC complex from mesophyll cells via plasmodesmata,accumulation of a higher level of sucrose in leaves is expected to facilitate the transport of sucrose in the phloem when less sorbitol is translocated in the transgenic plants. The lower concentration of sorbitol and higher concentration of sucrose in both leaf petiole and fruit pedicel and a smaller ratio of sorbitol to sucrose indicate that significantly less sorbitol but much more sucrose is translocated from leaves to fruit in the transgenic trees, which is consistent with a lower sorbitol but a higher sucrose concentration in the phloem exudates collected from fruit pedicels of these plants. The total amount of carbon translocated to fruit is expected to be very similar between the transgenic lines and the CK because all the trees had a very similar cropload and no significant difference was detected in average fruit dry weight between the transgenic lines and the CK at fruit maturity . These data clearly demonstrate that, when sorbitol synthesis is decreased in the source leaves, more sucrose is synthesized in the leaves and translocated to the fruit, thereby largely maintaining fruit growth and development. This is also consistent with the homeostasis of vegetative growth observed in the transgenic lines. The transgenic trees with decreased sorbitol synthesis grown under our experimental conditions were only slightly smaller after 5 years of growth than those of the untransformed CK . This is consistent with comparable photosynthetic rates measured in the transgenic lines and the untransformed CK throughout the growing season , with the lower rates detected only at fruit harvest being largely related to the leaf brown spots caused by Alternaria alternata in the transgenic lines. However, Teo et al.found that the transgenic trees were much smaller than the CK trees. This discrepancy is likely due to differences in growing conditions between the two locations. As sorbitol is implicated in drought-stress tolerance in apple, these trees might have experienced more drought stress under warm and dry conditions in California than under cool and humid conditions in upstate New York.

Similar to the findings for BrpHMA2, our results suggest that these TF genes may respond to Cd. The coding sequences of the three NAC TFs and three AREB TFs listed above were cloned and submitted to the NCBI database. The last three or four numbers of each gene’s full name was used as the gene name. MEGA5 was used to create a phylogenetic tree of these NAC TF or AREB TF genes and Arabidopsis NAC or AREB genes using the neighbor-joining method. The results revealed that the BrpNAC4584 and BrpNAC895 sequences were closer to those of Arabidopsis ANAC046 and ANAC087, respectively ; in addition, the BrpABI227 and BrpABI678 sequences were closer to that of AtABF4, and the BrpABI449 sequence was more comparable to that of AtABF3 .Our results reveal that BrpHMA2 could be activated by Cd2+ , which is similar to the results found for HMA2 in Arabidopsis. Results suggest that BrpHMA2 is involved in the Cd response of plants. BrpHMA2 was also found to be expressed explicitly in the vascular tissues of roots, stems, leaves, flowers, siliques, and carpopodia, and its protein was localized in the plasma membrane . These results are consistent with previous findings for HMA2 in Arabidopsis, OsHMA2 in rice, and TaHMA2 in wheat. The protein plasma membrane localization and the vascular-specific expression pattern of the genes revealed that HMA2 might function as a membrane transporter in long-distance transport in plants. In recent years,best indoor vertical garden system some studies have investigated the function of HMA2. Most of these studies demonstrated that HMA2 is involved in Zn2+ and Cd2+ transmembrane transport and influences root-to-shoot Zn/Cd translocation.

For example, HMA2 in Arabidopsis is thought to be involved in the outward transport of Zn2+ and Cd2+ from the cell cytoplasm, and HMA2 mutants are more sensitive to Cd stress and exhibit higher Zn or Cd accumulation than wild-type plants in the presence of high levels of Zn2+ or Cd2+ 14,15. The over expression of OsHMA2 in wheat, rice, and Arabidopsis improves root-to-shoot Zn/Cd translocation. In addition, the transformation of TaHMA2 in yeast enhances the resistance of cells to Zn/Cd. In rice, the suppression of OsHMA2 decreases the Zn and Cd concentrations in leaves, increases the retention of Zn in roots and reduces the translocation of Cd and Zn from roots to shoots compared with the results obtained with wild type plants. According to the literature, HMA2 is responsible for Zn2+/Cd2+ efflux from cells, plays roles in Zn and Cd loading to the xylem, and participates in the root-to-shoot translocation of Zn/Cd. However, Yamaji et al. found that OsHMA2 is localized at the pericycle of the roots and in the phloem of enlarged and diffuse vascular bundles in the nodes. Their insertion lines of rice showed decreased concentrations of Zn and Cd in the upper nodes and reproductive organs. The study revealed that the heterologous expression of OsHMA2 in yeast is associated with the influx transport of Zn and Cd. These researchers suggested that OsHMA2in the nodes plays an important role in the preferential distribution of Zn and Cd through the phloem to the developing tissues. Our results also revealed that, in the presence of Cd2+, transgenic Arabidopsis seedlings and yeast over expressing BrpHMA2 showed higher concentrations of Cd and enhanced Cd2+ sensitivity compared with the controls . Thus, we propose that BrpHMA2 functions in Cd2+ transport in the phloem tissue of vascular systems through influx into cells, and the efflux from phloem cells during long-distance transport may be performed by other transporters. The differential function of HMA2 from various plants might come from the tiny difference in amino acids in their function domains; this puzzle requires further investigation.

In this study, we identified the NAC TF gene BrpNAC895, a homolog of Arabidopsis ANAC087 , which could be induced by Cd2+ stress . We confirmed that BrpNAC895 has a role in the response of B. parachinensis to Cd2+ stress by upregulating BrpHMA2 expression through direct binding to the BrpHMA2 promoter using EMSA, ChIP–qPCR, and the transient transformation method with B. parachinensis protoplasts . Previous studies have demonstrated that Arabidopsis ANAC087 is associated with plant programmed cell death . It functions along with the TF ANAC046 to show partial redundancy in coregulating the expression of some PCD genes in the root columella, including ZEN1, BFN1, and RNS3. Whether ANAC087 could participate in regulating Cd transporters in plants has not been reported. Our findings on BrpNAC895 show that this NAC TF has a novel role in upregulating BrpHMA2 expression in response to Cd2+ stress. We also identified the Cd-responsive AREB TF BrpABI 449 , which is a homolog of Arabidopsis ABF3 and can bind to the promoter of BrpHMA2 . ABF3 modulates the response to drought, salt, and other osmotic stresses as a master component in ABA signaling. This TF can also regulate the expression of multiple genes, such as the AGAMOUSlike MADS-box TF family gene SOC1, which is a floralintegrator regulating flowering in response to drought, and the AREB TF ABI5, which is a core component in the ABA signaling pathway in the regulation of seed germination and early seedling growth during exposure to ABA and abiotic stresses. In general, ABF3 can form protein complexes with other TFs. For example, ABF3 forms homodimers or heterodimers with AREB1/AREB2 and acts cooperatively to regulate ABRE dependent gene expression. ABF3 forms a complex with NF-YC3 to promote the expression of the SOC1 gene and thus accelerate flowering and drought-escape responses; ABF3 interacts with NAC072 to regulate RD29A and RD29B expression in response to ABA. Thus, complex formation might be the important functional mechanism by which ABF3 regulates gene transcription.

Using EMSAs and ChIP–qPCR assays, we found that BrpABI449 could directly bind to regions of the BrpHMA2 promoter . The interaction of BrpABI449 and BrpNAC895 was further confirmed by pull-down and BiFC assays . The inhibition of BrpABI449 on the transcriptional regulatory role of BrpNAC895 was detected in the B. parachinensis protoplast transient system . The inhibition by BrpABI449 of the transcriptional regulatory role of BrpNAC895 complex, likely interferes with BrpNAC895’s activity in the transcriptional activation of BrpHMA2 in response to Cd stress. It has also been reported that Cd stress can induce a stress response via ABA signaling. Our results showing that BrpNAC895 and BrpABI449 are upregulated by Cd stress also support this point. The uptake or homeostatic regulation of heavy metals needs proper modulation to ensure plant health. Previous studies have shown that Cd stress induces the MYB TF gene MYB49 in Arabidopsis. This TF may further positively regulate the downstream TF gene bHLH38 and bHLH101 by directly binding to their promoters, and activate iron-regulated transporter 1 to enhance Cduptake. In contrast,growing strawberries vertically Cd stress upregulates the expression of ABI5. ABI5 interacts with MYB49, prevents its binding to the promoters of downstream genes, and functions as a negative regulator to control Cd uptake and accumulation. Our present results also demonstrate a mechanism for controlling the expression of the heavy metal transporter gene BrpHMA2 under Cd stress. We propose that Cd2+ induces the expression of BrpNAC895 and BrpABI449, which might be mediated by ABA signaling. The activation of BrpHMA2 enhances Cd2+ uptake and may induce cell damage. Negative regulation of BrpHMA2 is then achieved by the upregulation of another AREB TF, BrpABI449, which interacts with BrpNAC895 and forms BrpNAC895-BrpABI449 protein complexes to inhibit the BrpHMA2 transcription activated by BrpNAC895 . BrpABI449 could also bind to the promoter of BrpHMA2 directly to compete with BrpNAC895 in binding to the BrpHMA2 promoter. This negative regulation may play a supplementary role in the uptake and transport of Cd.Many plant species of Brassicaceae, including Arabidopsis, turnip, and oil seed rape, can be genetically modified, but the creation of transgenic B. parachinensis remains difficult. Therefore, we over expressed BrpHMA2 in Arabidopsis to investigate the function of BrpHMA2 and established a transient transformation system in B. parachinensis protoplasts to perform gene regulatory network analysis. Protoplasts have been widely used for subcellular protein localization and gene regulation analyses. In this study, the transient transformation of B. parachinensis protoplasts was demonstrated to be a powerful system for ChIP–qPCR analysis. Previous studies have applied a similar approach to Populus trichocarpa and Brassica napus. Although the transient transformation system of B. parachinensis protoplasts was successfully used in this study of molecular mechanisms, the system cannot be easily used for phenotype and physiological analyses. The lack of BrpNAC895 and BrpABI449 transgenic B. parachinensis is a problem that severely limits research on this plant.

New techniques, such as the transient reprogramming of plant traits via the transfection of RNA based viral vectors using Agrobacterium and gene editing combined with fast-treated Agrobacterium coculture, may be useful approaches for comprehending gene function concerning physiology and for the further application of modifications of gene function to effectively control the accumulation of Cd in B. parachinensis.Reduced pod shattering is an important breeding target in many crops, including common bean . In the wild, many legumes benefit from seed dispersal mediated by explosive pod dehiscence, known as pod shattering. During the domestication process, the trait has been strongly reduced across most legume taxa . Despite this, some market classes of common bean have persistently high levels of pod shattering, leading to reduced yields and a constrained harvest window. This issue is particularly problematic in semiarid environments, which cause pods to become brittle and fracture more easily . Common bean is a vital source of protein and micro-nutrition for hundreds of millions of people globally . The crop was independently domesticated in Middle America and the Andes , leading to the species’ two major domesticated gene pools. These are additionally subdivided into several ecogeographic races, each with a long history of adaptation to specific environmental conditions . In particular, members of the Middle American ecogeographic race Durango are adapted to the semiarid highland environments of northern Mexico and the southwestern USA, whereas the Middle American raceMesoamerica inhabits humid lowland regions of Mexico, Central America and lowland South America. Useful alleles from any major gene pool can readily be moved into others, and crosses between races have major untapped potential for breeders . Seven independent domestication events occurred in the Phaseolus genus, including close relatives of common bean such as Lima bean , runner bean year bean and tepary bean . An improved genetic understanding of pod shattering in common bean will be useful for improvement of numerous other domesticated legumes that suffer from pod shattering . Several genes are known to influence resistance to pod shattering in common bean , and the genes involved vary by gene pool. In the Middle American domesticated beans, the locus Phaseolus vulgaris Pod dehiscence 1 on chromosome Pv03 is associated with a major reduction in pod shattering . The shattering resistance allele is found at high frequency in race Durango, but is nearly absent in market classes belonging to race Mesoamerica or the Andean gene pool . This is a major target for improvement in these classes. Orthologs of this Pv03 gene may also regulate pod shattering in other species, such as cowpea , chickpea and soybean , where the orthologous locus plays a role in adaptation to arid climates by modifying the extent of twisting in pod valves . A possible second locus on chromosome Pv08 in Middle American beans has been proposed to reduce pod shattering , but a relatively small sample size of these individuals has hindered the study of this allele. The Pv08 QTL is also believed to have a major effect in Andean beans, so a deeper investigation of this QTL could provide insight on whether it has evolved in parallel between domestication events. A recently discovered QTL on Pv05, in immediate vicinity of PvMYB26, is associated with a loss of dehiscence in the Andean gene pool . This locus was mapped in detail in a biparental recombinant inbred population , which also found significant QTLs on Pv04 and Pv09 in the same population. The role of the Pv05 and Pv09 loci were identified in parallel in a diversity panel of Andean beans , which also identified significant loci on Pv03 and Pv08. PvMYB26 was subsequently found to be differentially expressed between dehiscent and non-dehiscent individuals, leading to major differences in development of cell walls in the suture . Other loci, including St and To , control strong fiber development in pod sutures and pod walls , respectively, and the mutant variants are found only in snap beans grown as a vegetable. St has been mapped to Pv02, and To has been mapped to Pv04 .

Specialized equipment used for harvesting distant food resources was left afield where needed . Equipment depots of this kind are discussed below. Of food caches, ceramic ollas hidden away in rock crevices and small shelters would be the most readily recoverable archaeologically and the most readily recognizable as actual caches. We would expect them to be uncommon occurrences at field camps or other locations far distant from residential sites. The woven platform granaries at residential sites would leave little recognizable evidence in the archaeological record. The term “forager” is applied to hunter-gatherers who moved about the landscape identifying exploitable food resources, mapping onto those resources, and seasonally moving the base camp to the place where the resources occurred. Residence lasted as long as the resources held out or untD other resources became available elsewhere. Foraging peoples probably were more keyed into the location and timing of food resources than any other hunter gatherers in prehistory. They knew where, when, and how to get food; had they not, such groups would have gone extinct, or at least gone hungry, with their first major error in subsistence planning. Ethnoarchaeological studies suggest that caching of foodstuffs by foragers was discouraged because of the constant need to move the base camp; it merely exacerbated the already uneven distribution of resources in time and space. Caching in such contexts ensured that the food would be where the people were not,vertical growing towers and someone would have to go back and get it later.

Caching of food would have been favored, however, if the foraging group intended to loop back and reoccupy the base camp for another purpose later in the season . In such cases, stored foods could augment those obtained on a subsequent visit. Concealment, such as in a rock-lined cache pit in a rock shelter, may have been important if the site was expected to be used by other groups during the interim. Such joint use of sites by two or more groups may have occurred in areas where water sources were few. Nomadic hunter-gatherers following a forager strategy occupied most of the California desert region in ethnographic times. These groups included the Panamint Shoshoni , Kawaiisu , and Southern Paiute-Chemehuevi , and they typified a lifeway common to almost the entire desert area for thousands of years. Little specific information is available on the caching behavior of California desert foragers. What information is available suggests that they adopted a collector strategy when resources were abundant and then regularly cached food. They cached autumn resources such as pinenuts collected to sustain the group over winter. At such times, the forager strategy definitely was abandoned. Steward reported that a single family might harvest at least 450 kg. of pine nuts in a good year. Storage on this scale meant that people often were compelled to spend the winter at or near the place of storage. If a decision was made to remain in the mountains where the nuts were gathered, the field camp became the winter village. Often, however, the winter village was located some distance away, preferably at a lower elevation that offered a more favorable climate and other amenities. In such cases, some of the nuts were transported to the village and the remainder were cached where collected. Descriptions of pine nut caches vary according to region and author, and are sketchy at best. Some accounts say the cones or nuts were buried in the ground and covered with grass and rocks; others suggest they were buried but not in a pit; still others suggest they were cached and covered with brush and earth within a circle of rocks .

AH agree the caching occurred outdoors in the pinyon groves, rather than in rock shelters. A thorough and systematic study of such caches has not been accomplished, but they should leave a conspicuous and decipherable record on the landscape. Field gear, such as pinyon hooks and poles, seemingly would have required little maintenance and special care, and would have been of little value for anything but harvesting pine nuts. Pinyon harvesting gear would have been left, usually in trees, where last used. Pointed or chiselended hardwood poles for extracting agave hearts would have been useful for little else, and likely would have been left near where agave was obtained. We have observed large block milling stones left in plain view as site furniture on the surface in patches of wild bunch grass at various locations in southern and western Nevada and eastern California. These milling stones mark the resource patch and also the field camp or work area from which the resource was harvested. Elsewhere in the same region we have seen similar milling stones around ephemeral ponds or lakes, perhaps left there as site furniture to be used when aquatic resources appeared at irregular intervals. Equipment of this nature likely would have been available for use by anyone in the group. The place it was left thus would not have been secret, and the equipment would have been used on subsequent visits to harvest the same resources. Such equipment would have been too heavy to carry away readily and almost indestructible anyway if someone else chanced upon it and used it. Because site furniture often tended to consist of large objects or facilities that remained at the site, anyone who used the place was likely to bring them into service. The situation can be described no more clearly than thus: “Upon arrival at a known site, one generally searches for the ‘furniture’ and pulls it ‘up’ out of its matrix for reuse. This means that large items of site furniture get continuously translated ‘upward’ if a deposit is forming” . A distinction must be made, however, between visible site furniture that is publicly accessible, and concealed site furnishings not intended for use by others.

Highly specialized and maintained personal equipment such as snare or trap bundles of the kind found at Ord Shelter, San Bernardino County, California , many similar examples reported by Janetski from across the American West, and a cache of dead fall triggers from Fortymile Canyon, Nye County, Nevada , are examples of equipment caches. Hidden equipment of this nature would have been retrieved from the cache and put to use only by the person that owned and cached it. The place of concealment would not have been generally known, and such gear would enter the archaeological record upon the death of its owner. A cache of basketry, ceramic, and metal containers was reported by King from Joshua Tree National Monument. This equipment cache was secreted in a small rock shelter. Whether it represents the activities of foragers, who left it for use on a subsequent trip to the same area, or whether it represents a field camp of collectors, cannot be determined on the basis of available information. Whatever the case,container vertical farming the equipment appears to be of too specialized and personal a nature to be considered site furniture. In few or no cases involving equipment of these kinds do we believe specialized caching or storage pits would have been used. Highly cared-for personal equipment would have been hidden away where it was safe; site furniture would have been given only the necessary care to ensure its presence the next time it was needed. Put another way, personal equipment was cached ; site furniture simply was abandoned . The models of hunter-gatherer organizational strategy discussed with reference to aboriginal life in the California deserts were derived largely from observations on remnant hunter gatherer populations in various parts of the world. In no case was the group being studied as residentially stable as those we have characterized as collectors in southeastern California, nor were they part-time horticulturalists. The models are idealized extremes that are of limited value to characterize the actual range of variation in aboriginal settlement and subsistence behavior in the California deserts. They tend to characterize extremes beyond the actual range of variation displayed by any group. Many ethnographic groups that were primarily foragers employed a combination of the two strategies, or switched from one strategy to the other and back again as circumstances changed, even in the course of a single year. Their organizational strategy therefore changed, and in some cases they may have occupied the same site more than once in a given year, each time with different agendas . The models are heuristic devices that we employ to force us to consider the nature of hunter gatherer organization and how it might help us characterize site function. WTien used in that way, they are useful; they simply cannot be taken too seriously or they lose their intended utility. Construction of better theoretical models is necessary for understanding the role and significance of food caches and the full range of food caching behavior in prehistory, and using such insights for anticipating other aspects of the archaeological record. Improved models will come from reanalysis of ethnographic accounts and from additional ethnoarchaeological observations, but the real contribution must come from carefully collected data from prehistoric contexts. AH hunter-gatherers probably manipulated their environments to one degree or another. Horticulture is merely one form of environmental manipulation, and it is an integral part of prehistoric cultural adaptations on parts of the Colorado Plateau, where during early Anasazi times extensive use was made of rock-lined cache pits that apparently were concealed in rock shelters.

Models that attempt to explain caching behavior should therefore not be limited strictly to non-horticultural hunter-gatherers, or they become even less useful for broad application to understanding prehistoric adaptations in the California deserts, the Southwest, and elsewhere. Indian Hill Rock shelter is a large overhang in Anza-Borrego Desert State Park of southeastern California . This region Ues m ethnographic Kamia territory on the eastern slope of the Peninsular Ranges and west of the Imperial Valley just north of the international border. Elevations in the desert range from below sea level to over 1,000 m. To the west, the Peninsular Range rises to nearly 2,000 m. Extensive excavations disclosed that aboriginal use of Indian Hill Rock shelter probably began as early as 5,000 years ago. Fairly continuous but non-intensive use of the site occurred into the historic period. Although ethnographic records for the region document irrigation of crops by the Kamia at nearby Jacumba, higher in the mountains to the southwest , no evidence of horticulture was found in the excavations. AH available evidence recovered from the shelter suggests a non-horticultural, hunter-gatherer adaptation, although it is recognized that the same group that used the shelter may have been involved in horticultural pursuits elsewhere in their seasonal round, at least in very recent times. The archaeological deposit is nearly two meters deep. The artifact assemblage includes abundant manos and metates, projectile points, hammer stones, and debitage. The assemblage suggests use of the shelter by groups engaged in hunting and plant gathering, but industries such as basketry and skin working are poorly represented. In short, the assemblage suggests the activities of task groups, not the residue of daily living. Excavations were conducted both within the shelter and in the exposed area to the front. Rock-lined cache pits or cists were encountered throughout the lower levels of the deposit, but only in the sheltered area behind the dripline. The upper levels ^ contain little evidence of cache pits, but instead contain ceramic sherds. Although radiocarbon analyses are still in progress, we anticipate that the levels containing abundant ceramics span no more than the last 1,000 years. Eleven rock-lined cache pits were exposed in the sandy deposits at Indian Hill Rock shelter. None of these was covered ; all had been opened and emptied of their contents in antiquity . Thus, what remained of many of the cache pits were the rock-lined or paved floors and some portions of the rock-lined walls. For this reason, no complete measurements for diameter, depth, or volume are meaningful. Several other features also may have been cache pits, but were lacking enough structural detail to be certain; these were recorded as “rock clusters.” No evidence of burning was found, so the features were not hearths. Three general methods of construction were noted during excavation: overlapping slabs; slabs placed in a mosaic fashion; and large, irregular rocks and/or milling stone fragments, sometimes chinked with smaller rocks.

The transitory increase in the cellular gallic acid concentration may regulate the expression/activity of UGT that convert gallic acid to the glucoside derivatives . Indeed, gallic acid 3-Oand 4-O-glucosides accumulated in the ugt84a23 ugt84a24 hairy roots . In addition, transcriptome and real-time qPCR analyses identified 11 UGTs with increased expression in ugt84a23 ugt84a24 and one of the candidate UGTs, PgUGT72BD1, exhibited regioselective glucosylation of gallic acid at the 4-OH position . However, none of the candidate UGTs produced gallic acid 3-O-glucoside, suggesting that the gallic acid 3-O-glucosylation activity may be regulated at a level other than transcription.Therefore, even though PgUGT72BD1 is expressed in the wild-type pomegranate roots and hairy roots, gallic acid is mainly used for the biosynthesis of β-glucogallin by PgUGT84A23 and PgUGT84A24 due to their much higher catalytic efficiencies than PgUGT72BD1. Indeed, our previous metabolite profiling analysis did not identify gallic acid 4-Oglucoside in any pomegranate tissues. These results also suggest that the primary role of PgUGT72BD1 in pomegranate roots could be glycosylating aglycones other than gallic acid. Intriguingly, HT production was not completely abolished in ugt84a23 ugt84a24 , suggesting that there could be additional UGT contributing to β-glucogallin formation in pomegranate. Of the 17 so far defined UGT phylogenetic groups in plants,vertical farming in shipping containers PgUGT72BD1 belongs to group E that contains UGT71, UGT72, and UGT88 gene families .

Regioselective glycosylation of hydroxybenzoic acids was previously reported for members of group E UGTs, including AtUGT71B1 that only glycosylates the 4-OH position and AtUGT71C1, AtUGT71C4, and AtUGT72B1 that specifically glycosylate the 3-OH position. Six amino acids are conserved in the hydroxybenzoic acid/ gallic acid 3-O or 4-O UGTs but distinct between the two groups of regioselective UGTs . The function of these amino acids in determining the regioselectivity of the corresponding UGTs can be explored by site-directed mutagenesis and enzyme assays. In addition, once the gallic acid 3-O UGT is cloned in pomegranate, the protein sequences and structural features of the gallic acid 3-O and 4-O UGTs can be compared to identify the key amino acid for regioselectivity. Furthermore, it was proposed that the regioselectivity for hydroxycoumarins was switched among the UGT71, UGT72, and UGT88 families during the evolution of group E UGTs. It will be interesting to understand whether regioselectivity switching event for gallic acid also occurred among these UGT gene families.Haplotype phasing and navigating between allelic and nonallelic variation are the major challenges in assembling genomes of out crossing species with high levels of heterozygosity such as found in members of the genus Juglans. Genome sequencing targeting out crossing plants employed inbred lines, haploids, and megagametophytes to avoid heterozygosity. Interspecific hybrids offer another strategy to avoid heterozygosity. Since the genome of an interspecific hybrid is usually comprised of haploid genomes of the parental species, interspecific hybrids have the same advantages for genome sequencing as haploids, but are usually easy to produce. Technical difficulties with allocating scaffolds to parental genomes have precluded the deployment of hybrids in genome sequencing.

Using an interspecific hybrid between cultivated walnut and its wild relative J. microcarpa, we describe here a novel approach to sequencing hybrid genomes which results ina cost-effective high-quality genome assembly for both parents. The cultivated Persian/English walnut, Juglans regia, is native to Asia whereas J. microcarpa is native to North America, where it occurs in riparian areas in the southwestern USA. Both species are wind-pollinated, highly heterozygous, and intolerant of inbreeding. Their hybrids are infertile. Both have a genome size of about 600 MB with n = 16. English walnut is an important nut crop with 3.8 million tons harvested worldwide in 2017 . Walnut production has been steadily increasing in part due to health benefits derived from including walnuts in the human diet. In the USA, English walnut trees are grown commercially using rootstocks chosen for their ability to tolerate such soilborne pathogens as Phytophthora spp., lesion nematodes, and Agrobacterium tumefaciens, which are all serious pathogens of walnut. The commercial hybrid rootstock J. microcarpa × J. regia possessing tolerance to soil borne diseases is extensively used in walnut production in California. The development of genomic resources for walnut and its wild relatives, including reference-quality genome sequences, will accelerate genetic improvement of walnut scions and rootstocks. Juglans and its relatives in the family Juglandaceae are members of order Fagales, which includes many important forest trees. Reference-quality genome sequences will facilitate comparative genomics and will advance biology of this important group of woody perennials. Recent attempts to sequence the heterozygous genome of English walnut using traditional approaches resulted in assemblies with 4402 scaffolds with N50 = 640 kb and 25,670 scaffolds with N50 = 310 kb.

An attempt to sequence the heterozygous genome of J. microcarpa resulted in an even more fragmented assembly with 329,873 scaffolds with N50 = 136 kb.The novel sequencing approach described here exploits the synergy between long-read sequencing and optical genome mapping. The average length of reads produced with long read platforms exceeds the lengths of a vast majority of plant long-terminal-repeat retrotransposons , which results in a dramatically improved sequence assembly. Contigs or scaffolds assembled from long reads are sufficiently long to be aligned on genome-wide optical maps, which can be assembled with very high accuracy, even for large or polyploid plant genomes. Alignments of the optical maps of a hybrid onto the optical maps of its parents will assign contigs to parental genomes, while also serving as an assembly quality control. We used the assembled J. regia and J. microcarpa genome sequences in conjunction with the Juglandoid wholegenome-duplication, and the Juglandaceae fossil record to calibrate the molecular clock rate for woody perennials. We used the calibrated molecular clock to estimate the time of divergence of Juglans species and other woody perennials. Based on synteny within Juglans genomes, we allocated the 16 Juglans chromosomes produced by the Juglandoid WGD into eight homoeologous chromosome pairs and analyzed their evolution. Finally, we exploited the contiguity of the assemblies in the analyses of the structure and evolution of Juglans telomeres and centromeres and the distribution of disease resistance genes in the J. regia and J. microcarpa genomes.Next, we constructed two optical maps for the hybrid and one for each of its parents . The N50 of the optical contigs ranged from 1.31 to 2.90 Mb. The parental maps consisted of ‘haploid’ regions, in which the haplotypes were similar enough to collapse into a single contig, and ‘diploid’ regions, in which the haplotypes were dissimilar enough to be assembled into separate contigs . The haploid and diploid regions were identified by a map self-alignment . The mean length across the phased regions was 108 Mb in Serr and 33 Mb in 31.01 . Due to the inclusion of phased haplotypes in a map, the sum of the total lengths of the Serr and 31.01 optical maps was 13.4% longer than the length of the MS1-56 optical map . To ascertain whether the genome of our hybrid was complete, we compared the total length of its optical map to the sum of the lengths of the optical maps of the parents,vertical grow racks which we edited to disregard the redundant haplotypes from the phased regions . The total length of the edited maps of Serr and 31.01 differed from the length of the map of MS1-56 by only 1 Mb . We therefore concluded that the hybrid genome was a complete representation of the parental genomes. We aligned the sequence contigs on the optical map of the hybrid, stitched them into scaffolds , and allocated 40 of them comprising 99.85% of the hybrid genome assembly into the parental genomes with the aid of the optical maps of the parents . The remaining 224 scaffolds representing 0.15% of the hybrid genome sequence were too short to be aligned on an optical map and were aligned on Illuminasequence of the J. regia cv Chandler for assigning to parental genomes. Finally, we ordered and oriented scaffolds on high density genetic maps producing 16 pseudomolecules for each of the two genomes. They had only five gaps of unknown lengths ; the remaining gaps were estimated based on the optical map alignments. We then reduced gap lengths or closed them entirely with unassigned contigs or contigs produced with the 10X Genomics technology . We mapped 89X Illumina reads of the hybrid to the genome assemblies and detected 48,717 and 51,789 indels and 748 and 1,205 base substitutions in the Serr and 31.01 assemblies, respectively . We subsequently corrected these errors and produced the fifinal assemblies JrSerr_v1.0 and Jm31.01_v1.0 .Of the genes annotated on the Serr pseudomolecules, 26,403 were collinear with genes on the J. microcarpa pseudomolecules . The two genomes differed by 28 inversions involving > 3 collinear genes, 21 segmental duplications, 3 intra- and 14 inter-chromosomal interstitial translocations, but no terminal translocation . Only 90 of the JrSerr_v1.0 genes were not detected in the Jm31.01_v1.0 assembly.

We computed Ks divergence among the J. regia and J. microcarpa genes to analyze gene duplication and divergence. The Ks plot showed three peaks . The first peak mostly consisted of Ks values between orthologous genes in the two genomes and reflected their divergence. The second peak coincided with the major Ks peak in self-searches within the Serr and 31.01 genomes and reflected the divergence of paralogous genes, which originated by the Juglandoid WGD. The third peak coincided with the major peak in self-searches within the grape genome and reflected divergence between genes duplicated by the whole genome triplication first described in the grape genome. These inferences were confirmed by self-alignment plots , a plot of the JrSerr_v1.0 pseudomolecules against the grape pseudomolecules , and a plot against the Amborella trichopoda scaffolds.We analyzed gene collinearity along homoeologous chromosomes within the Serr genome and detected 38 paracentric inversions involving >3 collinear genes and 20 intra- and 16 inter-chromosomal interstitial translocations. The homoeologues did not differ by any terminal translocations and retained a 1:1 relationship. Using the grape genome as an outgroup, we assigned rearrangements to phylogenetic branches and computed the rates of their accumulation. The rates ranged from 0.4 to 1.4 major rearrangements per MY . The collinearity analysis showed that the 16 J. regia chromosomes can be built from 142 major synteny blocks making up the 19 grape chromosomes. Of them, 122 were shared by the J. regia homoeologous chromosomes, and were in the same order along the them . These rearrangements must have taken place prior to the Juglandoid WGD. We found 43.7% of the J. regia genes collinear with genes on the grape pseudomolecules .In each pair of homoeologous chromosomes within a Juglans genome, one chromosome contained more genes than the other . We denoted the homoeologues with more genes as “dominant” and those with fewer genes as “subdominant,” allocated the 16 Juglans chromosomes into eight homoeologous chromosome pairs, which we arranged in descending order based on the number of genes in the dominant chromosome , and renamed the chromosomes . There were 18,179 and 17,093 genes in the dominant subgenome and 13,107 and 12,304 genes in the subdominant subgenome in JrSerr_v1.0 and Jm31.01_v1.0, respectively . Both dominant and subdominant subgenomes had fewer genes than are in the A. trichopoda genome, which was not subject to a recent WGD . This comparison indicates that both dominant and subdominant Juglans subgenomes have lost genes since the WGD. The difference in gene loss between dominant and subdominant homoeologues was nearly constant among the homoeologous pairs , suggesting that the rate of gene loss was intrinsic to a subgenome. We subdivided homoeologous chromosomes into sections delineated by successive pairs of collinear genes and counted the numbers of singleton genes in the intervening intervals . In the JrSerr_v1.0 pseudomolecules, 13,791 singletons were on the dominant chromosomes but only 8,261 were on the subdominant chromosomes . The numbers of singletons per interval varied little along each chromosome, except for the proximal regions, indicating that gene loss was uniform along the chromosomes and occurred by many deletions involving one or few genes along a chromosome . In each chromosome, deletions were larger and more frequent in the proximal region than the rest of the chromosome, as the numbers of singletons per interval were greatly elevated in proximal regions. We also expressed the numbers of singletons per 2-Mb non-overlapping windows. Most of the windows in subdominant chromosomes contained fewer singletons than those in the dominant homoeologues . Since the divergence of the J. regia and J. microcarpa lineages 8 MYA, 28.7% of singletons were lost from the subdominant chromosomes in J. microcarpa but only 14.9% from the corresponding intervals in the dominant homoeologues . Thus, the factor that caused the asymmetry in fractionation has persisted in the subgenomes since their origin to the recent past. In 22 RNAseq datasets , genes on the dominant chromosomes were on average transcribed significantly more than their paralogues on the subdominant chromosomes .

Of the traditional field crops, cotton and groundnuts are the most labour intensive, but still demand considerably less labour than flue-cured tobacco. From the social perspective, an important advantage of these crops is that cotton and groundnuts both require casual workers to help at harvest time and so provide employment opportunities for workers beyond each farmer’s permanent labour force. Maize, soybeans and wheat, on the other hand, use considerably less labour with few jobs created for casual workers. For LSC farmers, this can be an important advantage since these crops avoid the trouble and expense of hiring outside workers. A comparison of total variable costs for tobacco and other traditional smallholder crops grown in Natural Region II is given in Table 10. As with the LSC sector, these data show that traditional field crops all cost much less to grow than tobacco on a per hectare basis. This is especially important to understanding the decisions smallholders make since only very few farmers have reliable access to seasonal credit or other sources of crop finance. Of the traditional field crops, cotton is generally the most expensive, but special input schemes help alleviate this pressure. It should also be kept in mind that smallholder farmers mostly cultivate less than a full hectare of most crops so that the actual costs and returns from each enterprise will less than the per hectare results shown below. At the national level,mobile vertical grow racks most maize in Zimbabwe is grown by communal and resettlement farmers for whom this is the principal food crop.

Although other food crops are of greater importance outside Natural Region II, virtually every smallholder farmer cultivates maize to some extent or another to meet at least some of their subsistence needs. Compared with LSC farmers, sales to the GMB are more common and most smallholder farmers also plan to earn at least some cash income from maize each year. Smallholder farmers normally account for 55% to 70% of total sales to the GMB. The financial indicators for smallholder maize are summarised in Table 15 for farmers who sell all of their output for cash. Management practices vary considerably in the smallholder sector and the low input level for communal and resettlement farmers is based on growers using recycled seed without fertiliser; all other production levels are for hybrid maize with more intensive use of fertiliser at each successive management level. Compared with LSC farmers, the results for smallholder maize are generally more favourable. Although crop profits are still very low compared with other smallholder enterprises, maize does at least return a positive gross and net profit at each management level. It should be noted, however, that for all models for hybrid maize the costs of production are greater than the estimated gross returns indicating that farmers must have some other source of cash income to sustain this activity. To the extent that tobacco is often grown in rotation with maize on smallholder farms, it is clearly important to include a similar high-value crop as part of the strategy for household and even national food security. Even for smallholders not growing tobacco, the crop can still help finance improved maize production through remittance income sent by family members or from casual employment on LSC tobacco farms. The imputed returns for small holder maize by marketing arrangement are summarised below.

In interpreting these data, it should be kept in mind that the optimal technological choice and marketing arrangement depends on each household’s own consumption requirements and availability of storage space. In these terms, production at the high and medium levels is much more profitable compared with low-input maize because of the additional grain harvested. Nevertheless, because the estimated profits are positive in all cases, the data show that some cash sales can still be justified at each production level to raise money for minor household expenses. Cotton is Zimbabwe’s third most valuable agricultural export after tobacco and horticulture and is a key economic sector in terms of contribution to GDP, employment and export earnings. More than 200 000 smallholder households derive their livelihood directly from cotton, which is well suited to production in more marginal agricultural areas. About 80% of the lint produced each year is exported with the balance used by a small but vibrant textile industry with numerous downstream jobs. Cottonseed is used for crushing and supplies a large portion of Zimbabwe’s vegetable oil and cake used in stock feed. Over the past five years, communal and resettlement farmers have accounted for 80% to 90% of total plantings by area and typically produce 60% to 80% of Zimbabwe’s total crop. The total annual export value for cotton normally ranges from USD 61 to 75 million depending on price and yield. The cotton industry was deregulated in 1995 with the commercialisation of the Cotton Marketing Board.Two other companies have since entered the industry and the resulting competition has benefited all cotton growers in the form of better market related prices, improved efficiencies and better service. Each buyer sets their own prices based on world market conditions and distinguish between four grades of smallholder cotton to encourage good crop management and post-harvest handling.

This system also contributes to Zimbabwe’s very good reputation as a supplier of top quality lint. Compared with most neighbouring countries, Zimbabwe enjoys a very good ginning out turn of about 41% lint per unit of seed cotton. This is mostly because of a highly developed seed breeding and multiplication system that ensures all farmers start with good gene stock. Because lint is the most valuable product of cotton, this allows more favourable prices to be paid to farmers than in countries with a lower GOT. Compared with Zambia for example , producer prices in Zimbabwe are normally 20% higher in USD terms depending on world market conditions. About 98% of all cotton in Zimbabwe is handpicked which further contributes to the good quality and high prices on offer. Per hectare yields are highly dependent on adequate pest control and, like tobacco,vertical garden growing cotton makes intensive use of agro-chemicals and other purchased inputs. To help smallholder farmers afford these costs, each ginning company operates their own input scheme. Group lending, for example, has been used to good effect by The Cotton Company, which only extends credit through associations. Under this scheme, each association member must sign an agreement stating that the entire group will be ineligible for credit next year if just one individual defaults by selling outside the system. One of the new private companies, on the other hand, operates a voucher system in which farmers can choose to accept part of their payment in the form of a voucher exchangeable for inputs later in the year. Although this means that farmers are effectively put in the position of extending credit to the scheme operator, most growers have welcomed this system, both as a form of protection from inflation and as a way to avoid the problem of saving cash until later in the year to buy inputs. Compared with other traditional field crops, the results for LSC cotton are reasonably attractive. Of the traditional field crops, only wheat offers a potential for greater farmer profits. Although far less profitable than tobacco and other high-value crops, cotton is normally grown on large plots of 100 hectares or more and so can represent an important source of income when high yields are achieved. This is not necessarily true with low-input management and irrigated production is more profitable in every case despite higher production costs. As a handpicked crop, cotton is very labour intensive and requires an estimated 100 to 233 days of casual labour per hectare . This is slightly more than the estimated casual labour requirements for LSC tobacco.Compared with all other smallholder crops, the results for cotton are quite favourable and help explain why so many growers give high priority to this enterprise. Although somewhat expensive compared with other alternatives, the input schemes described above help cover many of these costs and cotton is not normally difficult to afford. On the other hand, input support packages do not always cover the costs of producing at the more intensive management levels and farmer profits are considerably greater with high and medium input use than with more typical low input management . To the extent that communal farmers are able to afford more intensive production as a result of remittances sent by family members working on LSC tobacco farms, a shift away from tobacco could impact the cotton sector.

The sensitivity indicators for cotton are among the best compared with all other smallholder crops and show that the good results for this enterprise are extremely robust. Cotton demands a large amount of labour, especially for good weed control and picking. Spraying is normally done with handheld knapsack sprayers. Smallholder farmers produce roughly 95% of Zimbabwe’s total groundnut crop, mainly on very small plots for home consumption. Groundnuts are one of the most important sources of protein in rural diets and are grown by nearly every smallholder farmer to some extent or another. Recently, total plantings by communal and resettlement farmers has varied between 100 000 to 140 000 hectares per annum. Only about 5% of these groundnuts are grown as a cash crop however and total deliveries to the GMB have been less 3 000 metric tons per year for the past three years. Smallholder groundnuts are generally not suitable for export due to problems with mixed and outdated varieties, and also because of problems with aflatoxin. To the extent that groundnuts can be promoted as a cash enterprise, the most urgent challenge is to develop a seed multiplication program to produce the varieties demanded by international buyers. Most LSC farmers grow long-season flamingo nuts as a dryland crop; only about one third of total production is irrigated. These pink skin confectionery nuts have many the characteristics sought by international buyers, but are not normally grown in sufficient quantities to attract European interest. Most trade therefore is with South Africa and Australia and any LSC groundnuts not exported are normally used domestically for the manufacture of peanut butter. An important advantage of groundnuts is that the crop performs well on the same sandy soils suited to tobacco and so makes an ideal rotation crop. Nevertheless, groundnuts are normally grown on small plots on most farms with only about one third of the area used for tobacco given to this activity. The total area planted to LSC groundnuts had fallen to just 1 000 hectares in recent years compared with more than 8 000 hectares annually in the early 1990s. The financial indicators LSC groundnuts are summarised in Table 19. Except with low input management, these data show that irrigated groundnuts are roughly twice as profitable compared with dryland management at each corresponding production level. Compared with other traditional LSC crops, on the other hand, cotton and wheat both offer higher producer profits. Although only about one third of total production is under irrigation, when high yields are achieved, the estimated gross profits from groundnuts equal about 22% of the pre -harvest cash costs for medium input dryland tobacco. In other words, the income from every 4.6 hectares of irrigated groundnuts, has the potential to finance roughly one hectare of flue-cured tobacco. In terms of labour requirements, groundnuts typically use some casual workers to help with harvesting and are more labour intensive compared with all other traditional field crops except cotton. The quantitative results for smallholder groundnuts are summarised in Table 20. These data show that groundnuts are relatively inexpensive for smallholders to grow, but that farmer profits are also very low. Because most households produce groundnuts mainly as a food crop, however, the imputed food security value should also be taken into account and returns would be greater than shown if the measured against the cost of buying groundnuts throughout the year. On the other hand, most farmers only cultivate a very small area of groundnuts and rarely grow the crop over an entire hectare.Importantly, these data show that groundnuts can be an attractive cash enterprise with the potential to generate similar net earnings to cotton. In order for this potential to be realised, however, farmers must have access to improved seed so as to produce a uniform crop with the characteristics international buyers demand.

The life cycle analysis could look at a future steady state after full application, but a more sophisticated analysis would consider expected rates of hardscape replacement and market penetration of new practices over the analysis period. The analysis could include use stage interactions with other systems, vehicles in particular. Initially, a greatly simplifying assumption would be that all hardscape would need to meet the same functional requirements with regard to vehicle interaction. Effects of hardscape on vehicles in cities include vehicle degradation and increased fuel use due to roughness, and increased fuel use due to energy consumption from deformation of the pavement itself . Very few cities manage their pavements based on roughness, and many do not want to manage roughness because of difficulties in handling roughness caused by manholes and other accommodations for under-pavement utilities. Deformation related energy consumption is primarily an issue for asphalt pavement carrying heavy, slow-moving trucks on hot days. Processes associated with those hardscape material flows, both inside and outside the system boundary would be modeled, as well as transportation of the materials, following LCA principles. The flows are the scaling factors for the processes at the urban area scale. Alternative scenarios for changes in the amount and types of materials, structures, changes in the amount of hardscape in different functional types , and changes in the amount of recycling within the urban boundary to reduce material transportation,vertical planters for vegetables and changes in the processes both within and outside the urban boundary to supply urban hardscape could be compared with current practices.

First-level analysis would look at the life cycle material flows, such as materials by type, massdistances of transportation and where applicable, land area needed within the urban area for stockpiling and processing. Second-level analysis would include calculation of important midpoint LCA indicators providing quantification of impacts of changes in flows. Hydrologic modeling of the most intense storm events would be performed to consider the hydrological impacts of changes in urban hardscape to make it more permeable, including partially and fully permeable pavement where they can both be used to also meet other functions. The effects of the changes in material flows and impacts from changing those flows caused by use of this type of hardscape would also be analyzed.In general, the quantification framework developed in this study could help identify where on the curve shown in Figure 5 we stand as of today. Implementation of better practice can cut down imports and flows resulting in lower costs and other environmental impacts.Hardscape covers large portions of urban surface areas, and has potentially large influence on air emissions, truck traffic and its associated problems, and the potential for flooding. Modeling the inflows of hardscape materials and the outflows of demolished hardscape and other rockbased products from buildings and other civil infrastructure is expected to provide a means to find solutions for reducing these flows and their impacts. Modeling of urban hydrology with respect to the effects of hardscape on surface and groundwater flows from precipitation is expected to provide a means to find solutions that will reduce the risk of flooding and improve groundwater recharge. The sustainability of urban areas can potentially be improved through changes in the built infrastructure of urban hardscapes. A conceptual framework does not exist, but is needed, that considers the entire urban area in order to evaluate high-level effects of changes in the design, construction and renovation of urban hardscape.

The original idea for combining UM and LCA to investigate urban hardscapes came from a plenary presentation at the Pavement Life Cycle Assessment Symposium in Davis, CA by Stephanie Pincetl from UCLA . Using Pincetl and her colleagues’ work as a starting point, a system’s approach that considers rock products and hydrological flows was selected for the modeling. An urban area can be considered as a system, with the city or developed urban area limits defining the system boundary as shown in Figure 6. From the standpoint of material and resource flows, this system can hypothetically be optimized if it maximizes reuse of existing resources within the system boundary; i.e., energy and material flows are generated, consumed, and restored within the system. Hardscape materials are primarily sand, gravel and crushed stone, collectively called aggregate, by mass, with small amounts of binding agents such as asphalt binder and Portland Cement, and even smaller amounts of other materials such as recycled tire rubber, other polymers, and chemical additives. A substantial part of most pavement structures is comprised of base and subbase layers consisting of compacted aggregates. For pavement surface materials, aggregates comprise 80 to 85 percent by volume of typical asphalt concrete and 62 to 68 percent by volume of concrete . On a mass basis, aggregate is about 95 percent of the mass of asphalt concrete and 85 percent of the mass of hydraulic cement concrete because the aggregate is much denser than the binding agents. The facts that aggregate makes up the large portion of hardscape materials, compacted aggregate is very dense , and aggregate must typically be hauled to cities from quarries has strong effects on the fuel use for its transportation and on the damage to the roads over which it is hauled.

The environmental impacts of imports of new materials and exports of waste materials into and out of the urban area can be assessed using LCA-type calculations. If material use is reduced through recycling and reuse within urban boundaries, these environmental benefits or impacts of such changes can be quantified by comparison to current conditions. Potential unintended negative consequences of materials reuse include increased land use and environmental problems from storage and processing of reused materials in the urban area, which can also be quantified by UM-LCA. Innovative approaches to hardscape systems that move towards a balance in demand and supply from reuse of material resources will require less material from outside the system boundaries and may require less transportation. Furthermore, the system may potentially produce less waste and pollution in the urban area and will export less waste and/or pollution out of the system. Opportunities to move closer to such a system can be analyzed based on the flows of import and export of resources, waste and emissions within and outside the system for environmental impacts in a life cycle perspective. Changes in hardscape extent and technology that improve permeability so as to capture, detain and infiltrate storm water can be analyzed with respect to flooding, storm water quality and groundwater recharge. Some of the means for increasing the permeability of hardscape include the use of permeable pavement, bio-swales, and catchments,vertical farming technology all of which increase the infiltration of storm water through the surface. All of these approaches also slow the rate of runoff, and where infiltration occurs, reduce total runoff. There are various types of permeable pavement available, all of which include a sub-surface porous layer made with gravel to retain or detain the water.The needs of different urban areas for improvements in flood control, water quality and recharge will differ greatly depending on current and predicted climate change effects on precipitation patterns, their topography, size, drainage systems and land use patterns. Together, the potential changes in hardscape systems to be more permeable and re-usable within an urban system can be analyzed together with the proposed framework.The UM model accounts for the energy and material flows within urban areas and between the urban area and its surroundings, helping researchers to study the interactions of nature and human systems, affecting the environmental impact of a city and effects on human and natural ecosystems. Kennedy et al. defined urban metabolism as “the sum total of the technical and socioeconomic processes that occur in cities resulting in growth, production of energy and elimination of waste” .

Although others have studied the relationship of cities to their surroundings, Wolman in 1965 presented metabolism concept to address air and water quality in the U.S. and determined that increased urban population and industry growth created problems of water and air pollution. Furthermore, Wolman postulates that it is not the depletion of the water resource, but rather its mismanagement, that creates water problems. In subsequent years numerous cities worldwide including Brussels, Cape Town, Hamburg, Hong Kong, Lisbon, York, London, Singapore, Stockholm, Sydney, Tokyo, Toronto, and Vienna have been examined using the UM approach . Kennedy et al. studied data from urban metabolism studies since 1965 for eight metropolitan regions around the world and analyzed four fundamental cycles of energy, materials, water and nutrients. The authors found that water inputs per capita for six studies since the 1990s were higher than the four studies from the early 1970s. Due to the expansion of the cities, water table levels were found to be lower because of increased water demand for some cities such as Beijing and Mexico City while several other cities in the world water tables were found to be rising and water quality being highly affected mainly due to the discharge of wastewater into the ground. An increase in commercial and industrial waste was seen, however, the cities that had implemented recycling strategies at a large scale were producing less waste. The data also air pollution emissions over the same period. The study concluded that the urban metabolism results were different from city to city. Li et al. developed a general framework of an urban ecological infrastructure . Blue land , green land , grey land , exits and arteries build up the UEI system. All the sub-systems are tightly integrated and distributed amongst each other. Researchers have investigated transportation and street networks within the ‘grey land’ category have not been looked at in detail and almost no literature exists to guide roadway and other hardscape design in urban areas in light of responding to climate change effects on precipitation . Different methods such as the emergy or material flow analysis can be used in the UM approach. MFA is the most common and internationally accepted method that can be used to study the flows of resources into a city , followed by processing and consumption of resources, and flows of resources out of the city and emissions production . MFA approaches are typically focused entirely on the flows of materials or substances. However, researchers have also applied the UM approach under the umbrella of the three pillars of sustainability  and developed tools such as the integrated UM analysis tool . Others have suggested studying systems in a life cycle perspective and understanding urban sustainability by coupling UM and LCA, as is done in this white paper . Goldstein et al. applied the UM-LCA framework to five global cities and assessed the environmental impacts directly and indirectly caused by the mass and energy flows through the cities . The author concluded that the results of the study are first gross estimates due to inadequate data. Chester et al. presented evidence that better understanding of sustainable urban systems could be achieved by integrating UM and LCA frameworks, which would help in understanding physical flows and urban infrastructure system problems . A predecessor to this application of the UM-LCA framework to urban hardscape, Chester et al.used LCA to estimate the environmental impacts of building and operating parking spaces. Fraser and Chester used UM-LCA type analyses to estimate the materials and emissions investment in streets and roads in the Los Angeles area over time and the transition from heavy impacts from initial construction to increasing impacts from vehicle use and increasing demands for financial resources for maintenance and rehabilitation . This latter work indicates the importance of looking at urban impacts over a long enough analysis period, per LCA methods, to capture changes in impacts as the hardscape infrastructure both grows, or potentially shrinks if hardscape is converted to buildings or back to green space, and ages. Reyna and Chester evaluated the age and rate of demolition of building stock in Los Angeles . Recycling of concrete from building demotion recycling of all types of existing hardscape are the sources of raw aggregate based materials for new hardscape, the use of which is one of the cases proposed for exploration later in this document. LCA data and analysis approaches for use in an UM-LCA framework have greatly improved over the last ten years.

While the utility of these scores could be improved with faunal densities, they provide preliminary insight about what types of microhabitats and which environmental variables may be important to specific services at specific sites. Methane seeps have been recognized as essential fish habitat and this trait-based approach could provide additional evidence for spatial protections. The focus on ecosystem services provides a targeted effort that can help guide research and management priorities. In the case of the Del Mar seep, Grupe et al. found higher densities of commercially-valuable species at the active seep than in adjacent, background areas. Our results somewhat contrast because we found no significant differences in ecosystem services scores among the active seep and background area during the Del Mar dive. This suggests that the Del Mar area, in general, contributes more to fisheries and carbon services than our other study sites. However, background areas could potentially be influenced by the seep through movement of chemicals and animals from the seep to adjacent areas . One drawback to using deep-sea imagery for trait-based ecosystem services assessment is the need for visual evidence. The traits in Table 4.3 are not exhaustive of characteristics that can contribute to fisheries or carbon services, but they were ascertainable from our dive videos. While deep-sea imagery may not be able to confirm regulating services,vertical farming tower for sale like metatranscriptomics could , it does provide insight on animal behavior that can support ecosystem services. For example, mid-water fish would often be seen near the benthos and swimming into it , which has been previously observed .

This could potentially represent an important benthic-pelagic interaction that contributes to carbon export.Expensive and limited ship time can make it difficult to collect ideal video data. The need to juggle multiple scientific goals during each ROV dive makes it impractical to conduct quantitative visual transects, and to generate sufficient number of replicate transects. This can create downstream constraints for data analysis such as lack of quantitative measures of densities and difficulty standardizing observations. Best practices for collecting pictures and videos from deep-sea sampling instruments could be useful . Random and independent sampling could facilitate data analysis. Replicates could also be useful, in which cabled observatories may be appropriate . ROV and AUV transects should be conducted with consistent altitude, zoom, and speed, as well as with a scale for size . The resulting data can then be used to calculate faunal densities and other diversity metrics . A quantitative transect would also allow for comparison among locations and time periods . Accurate maps of the seafloor before dive operations can help ensure best use of time , but perhaps most important are the designation of detailed scientific goals and objectives prior to surveying.Environmental measurements should be made in association with imagery being taken. Physical and chemical properties, such as temperature, oxygen, and hydrogen sulfide at seeps, are important parameters that help shape the biological communities . Porewater chemistry influences the sediment community , which can contribute to fisheries services and carbon services . Scientific tools exist to assess water chemistry such as in situ mass spectrometers that can be mounted on ROVs and niskin bottles that can be used to sample water at discrete depths. These environmental properties can help explain differences in diversity and distribution, and provide insight on how communities may change with human impact such as climate change .

As imaging technology continues to advance, the resolution of pictures and videos becomes increasingly helpful for post-analysis . Imagery should be analyzed consistently, which may mean cross-referencing protocols and morphotypes if more than one person is conducting the analysis. Human bias is inherent to current image analysis but can be minimized with training . As more deep sea imagery is analyzed and libraries are produced, there are possibilities to incorporate machine learning algorithms in collaboration with computer science and programming . Machine learning techniques could facilitate mining of existing deep-sea imagery data. They often sit in labs untouched, representing an underutilized source of knowledge. These pictures and videos provide an opportunity to generate knowledge for habitats that are rarely visualized and are data-limited. Imagery is also routinely collected for non-biological purposes, such as instrument deployment, which could be an additional source of data.The approach developed in this study can serve as an environmental decision-making tool, such as in the designation of spatial protections, consideration of ecosystem service trade offs, and understanding of context-dependent roles of methane seeps. This analysis can identify areas of high ecosystem services provision, such as the Del Mar seep that had relatively high fisheries and carbon scores, which may be important for designating essential fish habitat or marine-protected areas . An ecosystem-services approach can also investigate trade offs that may need to be considered during the environmental decision-making process . For example, if methane seeps provide differential ecosystem services, one prioritization metric for spatial protections could be weighted ecosystem services scores . Lastly, results from this paper advance our understanding of ecosystem services associated with methane seeps. They highlight the context-dependent role of methane seeps in providing fisheries and carbon services along environmental gradients. For example, while the combination of seepage and low oxygen seemed to suppress ecosystem services scores at Point Dume, the Palos Verdes and Del Mar ecosystem services seemed to benefit from at least some seep activity.

Development and urbanization transform coastal landscapes by replacing vegetation with impermeable surfaces. Subsequent precipitation events can lead to decreased water infiltration, modified water flows, and introduction of contaminants into storm water runoff . As a result, flooding and property damage, increased safety and health risks, and environmental damage can occur . Urban planners and developers have traditionally addressed storm water runoff issues by building drainage systems that connect directly to large bodies of water or treatment plants. The former have been found to further alter hydrology and degrade water quality , while the latter can be energetically and chemically intensive . Natural storm water treatment systems are emerging as an alternative storm water management strategy . NTS are human-made systems that use natural processes to capture and treat storm water runoff. They come in different forms: bio-retention systems , infiltration basins and trenches, permeable pavements, dry wells and ponds, treatment wetlands, and combinations. Table 5.1 provides a summary,hydroponic vertical farm although there are differing opinions on what constitutes an NTS . Additionally, some of the listed systems can also treat wastewater, functioning in similar ways but obtaining water from different sources. This paper focuses primarily on bio-retention systems because they host biological communities as part of their design. Because NTS function as built ecosystems, they can support diverse ecosystem services, defined as direct and indirect benefits humans obtain from ecosystems . Ecosystem services associated with NTS have long been acknowledged , but they have largely been ignored by monitoring programs and economic valuation efforts, which have been limited to targeted water functions. Co-benefits have been described for other green spaces such as offsetting carbon emissions , cooling local temperatures , cultural services , and benefits to human health and well-being . The incorporation of ecosystem services and other co-benefits into environmental decision-making can present urban planners and developers with additional benefits, costs, and trade offs to consider in order to make optimized decisions . We use Los Angeles County as a case study because it experiences periodic water crises, hosts a dense human population near the coast, and has policy in place to encourage the use of NTS. There are spatial and temporal mismatches in the supply and demand for water in California, most precipitation occurring remotely from agricultural and metropolitan hubs that need consistent sources of water irrigated and imported . Additionally, California droughts and water shortages are predicted to increase in frequency and magnitude due to anthropogenic climate change .

As a state, California has passed several propositions to protect water supply and quality . In 2004, Los Angeles passed Proposition O which allowed the city to issue up to $500 million to fund projects that increase local water quality . In 2012, Los Angeles adopted its Low Impact Development Ordinance requiring development and redevelopment projects that alter impervious area to mitigate runoff by capturing precipitation and utilizing natural resources where possible. As a result of these environmental conditions and political momentum, green infrastructure and NTS have been broadly distributed throughout Los Angeles County and continue to be implemented. NTS are designed to capture storm water runoff for infiltration or reuse , and have been shown to be effective . Most systems are oriented vertically, using gravity to direct water flow through several layers that generally consist of a ponding area with vegetation, porous filter media, and a drainage zone . Infiltration rates of a system can vary widely depending on variables such as size , age , filter media , and other design factors . Vegetation also plays a role by intercepting precipitation and water flows , and preventing clogging of filter media to maintain infiltration capacity . Additionally, evapotranspiration by plants can account for 15- 20% of inflow . Urban areas with low permeable surface area can be prone to flooding and changes in groundwater recharge . Los Angeles County lacks infrastructure to handle large volumes of storm water , and flooding can occur when unexpectedly high precipitation occurs. This issue occurs in a state that experiences episodic drought , highlighting the need for proper storm water management and use. Targeted storm water infiltration by NTS can provide ecosystem services that help address these challenges.By altering landscapes and hydrology, urbanization can lead to increased flood risk caused by heavy precipitation and storm surge . NTS have been shown to significantly reduce runoff volume and magnitude of high-flow events by capturing and storing storm water runoff. Hatt et al. found reductions in runoff volume of 33% on average, as well as peak flow reductions of at least 80% in three bio-retention systems in Australia. In another field study, Davis reported mean peak flow reductions of 49% and 58% in two test bio-retention systems, as well as delays in flow peaks which can provide urban managers with time to put mitigation measures in place. Most Los Angeles NTS showed visual indicators of flood control services: permeable surfaces that allow for water infiltration, graded landscaping to help direct runoff, and ponding areas designed to temporarily store water. For example, Elmer Avenue Green Street was constructed specifically to address street flooding during precipitation events by incorporating bioswales, biofilters, permeable pavements, and rain barrels . However, additional information is needed in order to quantify these services. Measurements for flood hazard may be relevant , as well as those for flood vulnerability . Spatial models have also been developed, mapping urban surfaces to estimate infiltration capacity . The value of flood control has been extensively studied in the context of wetlands , generally by assessing differences in property damage along a spectrum of wetland area . Brander et al. conducted a meta-analysis to estimate the value of flood control by wetlands which resulted in a median of $20-30 1995 USD per hectare annually. Watson et al. estimated a net present value of less than $100 USD per hectare of wetland annually. NTS usually operate at smaller spatial scales than major wetland restoration projects so the main unknown here is whether NTS of different designs provide more localized flood control.Groundwater is used by more than half of the population in the U.S. and its recharge is an essential component of the water cycle . Major sources of recharge in urban environments include runoff infiltration, and leakages from the water supply and sewage systems . Urbanization can decrease groundwater recharge due to the installation of impermeable surfaces . However, the importation of large volumes of water to meet demand in highly-populated urban areas also leads to significant leakages and recharge . These changes can also degrade water quality, e.g. leakage from sewage systems, saltwater intrusion due to lowering of the water table . NTS can contribute to groundwater recharge by providing permeable surfaces and pore space in their filter media that allow storm water runoff to pass into the soil subsurface.

The historical record there and elsewhere thus suggests that during summer and early fall starches were consumed in far greater number than during the winter, when protein and fat consumption increased proportionally.By 1600, as another example, Western Apache communities had developed a “seasonal cycle” that veered between food gathering, horticulture, and winter hunts.In July they harvested saguaro fruit in the Gila Valley of modern day Arizona. In July they also began a month-long harvest of acorns. Fall and winter were dominated by hunting for animal meats. Similarly, on the Texas plains prior to European contact, Apache communities spent spring and summer in agricultural villages, moving towards hunting dominated nutrition in the winter.While surveying indigenous communities who had avoided the level of European contact suffered by their southern neighbors, Weston A. Price’s Nutrition and Physical Degeneration suggested that similar practices were maintained during the early twentieth century among communities living inside the Rocky Mountain Range in far northern Canada. Moving further towards speculation, or at least a working hypothesis, it is worth considering whether health declined as Native Americans were forced to consume maize and other starches during the winter months, having lost access to ancestral winter hunting grounds. After all, seasonal consumption of carbohydrates could have improved health by limiting the overall production of insulin in the bloodstream . In assessing such a hypothesis, students would be able to consult much recent research on the evolutionary dimension of obesity and metabolic syndrome. Firstly,stacking flower pot tower it has been suggested that the move towards all-year round abundance of food in many populations may have caused over-consumption at times when the human metabolism may otherwise have benefited from calorie reduction and/or greater insulin sensitivity.

The latter has been linked to reduced carbohydrate and protein access in comparison to fat, often during winter. Secondly, a number of studies have shown a correlation between blood vitamin D levels and insulin sensitivity. Thus, from an evolutionary perspective, it may be postulated that some human populations may be adapted to consume more starch during those months when insulin sensitivity is higher due to raised blood vitamin D levels from available sunshine.What, then, might students make of the statements made by the Spanish colonizer Cabeza de Vaca, who lived with Coahuiltecan communities in what is now part of Texas, for eight years from 1538? During winter they hunted buffalo, deer, and javelin, while during the summer and fall they were sustained by fish, plants, and starches such as the mesquite bean. In what has been described as a “feast or famine economy”, animal-based diets in winter would thus give way to gorging thanks to “the ripening of fruit, or tuna, of the prickly pears [which] typically meant days of feasting until the fruit ran out.” During the winter period of hunting for animal proteins and fats, de Vaca noted the ability of Coahuiltecans to maintain aerobic activity for long periods of time: “The men could run after a deer for an entire day without resting and without apparent fatigue. . . one man near seven feet in stature. . . runs down a buffalo on foot and slays it with his knife or lance, as he runs by its side.”Students might even consider the above, and other similar original source testimonies, in light of modern scientific research on fat-adapted aerobic activity, including the use of ketones as an energy source. The metabolic state of ketosis is defined as the elevation of the ketone bodies D-beta-hydroxybutyrate and acetoacetate in the body in response to the consumption of a diet low in glucose and high in fats, or following long periods of fasting.

A number of recent studies have suggested that the metabolic use of ketones may benefit long periods of medium-intensity movement by reducing the need for regular consumption of carbohydrates. They have focused on the ability of endurance athletes to maintain or perhaps even increase performance whilst consuming a high fat diet .Students and researchers might consider the possibility that some indigenous communities prior to European contact would at least have cycled between periods of ketoadaptation and period of glucose-burning, depending on the season. It has certainly been suggested that communities in the northernmost parts of Canada and Alaska have historically utilized ketogenic or keto-adapted diets, burning fat obtained from meat and fish rather than glucose as a primary fuel for many months of the year .Though much more research is needed to determine the nature of fat burning during physical exercise, a useful hypothesis can be gleaned by noting that many Native Americans undertook long hunts over several hours at just the point in the season when they may have benefited from increased endurance due to their fat-adapted metabolic states and high fat consumption. Any European disruption to winter hunts, according to such a proposition, would also have disrupted ancestral metabolic patterns that incorporated seasonal fat-adaptation, or even seasonal ketosis. Fat-adapted metabolic diets have been found to be therapeutic for a number of medical disorders, particularly but not only neuropathies. Evidence of their potential health-promoting benefits, at least in periodic cycles, might also support the hypothesis that disruption to ancestral metabolic cycles could have prevented other benefits that are as yet unknown, pending further scientific research – thus potentially exacerbating Native American susceptibility to infectious diseases following European contact.

Of course, students would be encouraged to examine confounding evidence to such a hypothesis. In this case, for example, they might consider the historical record for Tarahumara communities in Northwestern Mexico, who ran great distances through the colonial era while eating comparatively few animal meat products. A similar association could be found with historic Apaches and Hopis, both of whom ran for long distances with a diet dominated by maize, squash, and beans. To be sure, it is ambiguous whether, historically, Tarahumaras relied solely on plant carbohydrates and proteins from beans, or whether such an account represents a teleological extrapolation from their diets which were examined during the second half of the twentieth century.But students would certainly be able to note the association between their high starch diet and their high-distance aerobic activities . Yet even here, other analytical indicators might become relevant. Several studies have shown low plasma cholesterol levels for modern Tarahumaras, at least in regard to HDL. But other studies have also shown relatively high levels of cardio-respiratory problems in rural populations who have maintained a high starch diet while decreasing their physical movement. That is to say, the problematic effects of high blood glucose might have been mitigated by intense exercise burning the substance for fuel rather than raising insulin to a sub-optimal level.The Tarahumara case study might even lead students to ask a further set of questions, which speak to recent research by O’Keefe and others on burning glucose during periods of aerobic endurance activity. Compared to long-distance aerobic activity in a fat-adapted state, students might ask, what are the long term consequences of burning glucose for fuel in a high state of oxidative stress, such as long distance running and hunting? Oxidative stress can be defined as the increased production of oxidizing species or a significant decrease in the effectiveness of antioxidant defenses, such as glutathione, in association with intense consumption of oxygen during periods of activity . Even if exercise may have prevented glucose from being stored as fat among Tarahumaras, and lowered inflammatory markers,ebb and flow oxidative damage due to their metabolic state during exercise may have portended problematic cardiovascular health outcomes.Ongoing scientific discussions about optimal nutritional health – as defined in peer-reviewed studies – should help to inform our historical understanding of the contact period between Europeans and Native-Americans, from the sixteenth century through to the nineteenth century. Students should be in a position to question the perception of nomadic hunting as the only indigenous Native American nutritional and ecological practice in the three centuries following European contact. The regular or seasonal consumption of hunted wild animal products was not antithetical to horticultural cultivation. Both were often complementary during the pre-contact era, providing micro-nutrients and macro-nutrients that varied according to the season. Distrust of European agriculture – including its perceived association with the spread of diseases – did not require Native-Americans to conceive of themselves as a people who eschewed ecological cultivation altogether. If any generalizations are to be made, rather, students would be better off examining the ways in which Native American populations perceived colonial farming as a threat to their own land management of crops as well as to their continued hunter-gatherer activities outside indigenous cultivated settlements. We ought to examine the existence of both ecological systems in order to consider their distinction from those that followed the European encounter. During the era of contact, horticulture declined. But although hunting and gathering increased in response to colonial pressure on cultivated plants, its character and regional context changed in order to accompany new European technologies, often deployed in new lands. Those communities who migrated to the Great Plains – whether from the East, the West, or the Northern Great Lakes – eventually suffered from diminishing access to hunted animals, without the potential to mitigate that loss through renewed horticulture sustenance.

The same phenomenon also often occurred among Native American communities who remained at the site of first European contact. As is evident in the tragic history of Native American health and ecology in the three centuries after first contact, then, external interventions in the indigenous food system may well have contributed to heightened susceptibility to infectious diseases and near demographic collapse. The modern scientific literature on nutrition and health should inform our understanding of the negative health outcomes that were associated with diminished access to indigenously cultivated crops and/or hunter-gathered animals and plants. In evaluating declining health after European contact, students and researchers should assess the effects of curtailed hunting and gathering, the threat to pre-contact forms of horticulture, and the colonial misunderstanding of their symbiosis. Such an assessment could be informed by our understanding of the nutritional and ecological changes that accompanied the introduction of infectious diseases: a greater threat from zoonotically spread pathogens; diminished access to fats, fat soluble vitamins, proteins and essential minerals from animal and plant sources; an increasing inability to gather potentially beneficial indigenous starches and Resistant Starch sources; a growing threat to seasonal oscillations between winter higher fat diets and summer starches; and even a decline in cyclical ketosis in some regions. In light of current research on the relationship between nutritional density and immunity, and metabolic syndromes, diminished access to indigenous food sources can be related to the greater vulnerability to infectious diseases, above and beyond the differing historical immunities that distinguished Native Americans from Europeans. Questioning or modifying the Biological Exchange thesis should help students and researchers evaluate an important and ongoing question in public policy: historically, since European colonization efforts, why have top-down political interventions in nutrition so often accompanied a decline in ancestral health principles, health, and fertility? Indeed, such a correlation is suggested by events following the three centuries of European colonization, from the mid-nineteenth century to the present day. They might offer important insights as a concluding section or even a postscript to the proposed course, connecting the colonial period to the contemporary era. In 1867, for example, the Treaty of Medicine Lodge required Southern Plains Native Americans to give up land in return for government annuities. The federal government then began supplying them with food handouts, using the industrial and transportation systems developed to supply troops with grains during the Civil War. The containment of Native Americans in reserves severely limited the physical activity to which their communities had grown accustomed over previous centuries. It has been suggested that their decreased energy expenditure would have made them less likely to burn increasing volumes of glucose in their diet, contributing even further to the development of diabetes and other immune disorders through the twentieth century. Students and researchers would do well to examine the correlation between the Treaty of Medicine Lodge, other similar documents, and declining nutritional health among Native Americans during the post-colonial era. Aleš Hrdlička, a medic and an anthropologist, reported in his 1902 Physiological and Medical Observations among the Indians of Southwestern United States and Northern Mexico that obesity and associated “grave disease[s] of the liver” were “exclusively” found among Pima Indians on new reservations, rather than among those who relied on a more traditional system of hunting meats and gathering or cultivating fibrous seeds, chenopods, plants, and starchy tubers.