Month: August 2024

To gain statistical evidence of miRNA differential expression driven by the environment and/or genotype, we made pairwise comparisons, keeping constant the developmental stage, and evaluating the miRNA modulation among vineyards or between cultivars . The analyses reveal that some miRNAs are differentially expressed between the two genotypes grown in the same environment, but also that a number of miRNAs are modulated by the environment. In particular the number of differentially expressed miRNAs is higher in ripened berries , while no miRNAs are differentially expressed at bunch closure stage . In details, 14 reads are differentially expressed at pea size stage, in at least one comparison, corresponding to 6 distinct miRNA families; 27 reads are modulated at 19 ◦Brix stage, corresponding to 12 miRNA families and 35 reads are differentially expressed in berries at harvest, corresponding to 12 miRNA families. It is worth noting that 4 of the 6 families modulated in the berries at pea size, are still present among the miRNAs differentially expressed in the berries sampled at 19 ◦Brix and at harvest , even though not always in the same comparisons. Some of the modulated miRNAs, both novel and known are intriguingly connected to berry development and secondary metabolism, even though most of the modulated families are still uncharacterized, or with targets not clearly involved in berry ripening and development, and deserve further studies to fully understand their biological roles. Using high throughput sequencing coupled with robust bioinformatics pipelines we analyzed small RNAs derived from the berries of Cabernet Sauvignon and Sangiovese, grown sideby-side in three vineyards, representative of different grapevine cultivation areas in Italy . The size distribution profiles of our libraries were in general consistent with previous reports in berry grapevine, black plastic pots for plants where the 21-nt class was more abundant than the 24-nt class .

Our analysis revealed dynamic features of the regulatory network mediated by miRNAs and other small RNAs, at the basis of genotype-environment interactions. Plants evolved a series of pathways that generate small RNAs of different sizes with dedicated functions . Although the various small RNA classes have been intensively studied, we are still far from understanding how many small RNA pathways exist, and how they are connected . Additionally, new classes of small non-coding RNAs continue to be discovered and many studies demonstrate a substantial redundancy and cross-talk between known small RNA pathways . Estimating the exact percentage of the plant genome covered by small RNA-generating loci still remains a challenge. By applying static cluster analysis, we investigated small RNA abundances across the genome, identifying 4408 small RNAs producing hotspots. We analyzed their expression in different cultivars, environments and developmental stages, highlighting that the majority of the considered small RNA producing regions was modulated in different conditions. This suggests a strong influence of small RNAs in the response to environment in grapevine berries. Only 462 small RNA-generating loci, corresponding to about 10% of the total, were expressed in all the analyzed libraries, possibly involved in essential biological pathways. Comparing the two cultivars, we observed, with few exceptions, that Cabernet Sauvignon berries have a higher number of expressed sRNA-generating loci than Sangiovese berries when collected in the same conditions . Considering the fact that small RNAs are implicated in the regulation of gene expression in several processes , the higher number of small RNAs expressed in Cabernet Sauvignon compared to Sangiovese berries may reflect a buffering effect of small RNAs influencing grapevine response to diverse growing environments. We believe that these characteristics may have contributed to the wide diffusion of Cabernet Sauvignon, allowing its wide cultivation in almost all wine producing countries. This is not the case for Sangiovese whose cultivation is more restricted.

It is worth noting that Sangiovese is considered a very unsettled grapevine cultivar , showing a wide range of variability in response to year, clone and bunch exposure . Differently, Cabernet Sauvignon is a cultivars showing less inter-annual differences in terms, for example, of concentration of secondary metabolites . To better evaluate varietal differences in response to the environment, we calculated the CS/SG ratio for the small RNA producing hotspots in the three vineyards. An interesting example is found in green berries sampled in Riccione. A region on chromosome 4 showed a 390-fold change in the small RNA abundance, when comparing Cabernet vs. Sangiovese . Most of the reads produced in this region are 21 nt long and are also phased in intervals of 21 nt from both strands, typical of a phased locus . The gene in this locus, also known as VvRD22g, encodes a BURP domaincontaining protein, involved in an ABA-mediated abiotic stress response, which persists still after long periods of stress . The small RNAs profile suggests that the locus is regulated by phased siRNAs similarly to the mechanisms already described for PPR, NB-LRR, and MYB gene families . This is a clear example of GxE interactions since the BURP domain gene modulates phased siRNAs production in the two cultivars only when grown in Riccione. When removing the threshold of minimum cluster abundance set to 5 HNA, in the CS/SG ratio, a high number of clusters with fold change greater than 50 was found, where one of the libraries has 0 HNA and the other any number greater than 30 HNA. This fact suggests a very strong modulation of the expression of small RNAs between the two cultivars, which is more or less pronounced depending on the vineyard where the berries were cultivated. A similar situation was observed comparing the expression level of small RNAs between reciprocal hybrids of Solanum lycopersicum and S. pimpinellifolium . The ripening process of grapevine berries is highly affected by the environment and we observed the impact of the environment on the ripening process in the expression of small RNAs.

The most relevant observation is that Riccione is very peculiar in relation to the activation of sRNA hotspots, as indicated by the high number of Riccionespecific clusters and by the extreme modification it induces in the CS/SG ratio : in Riccione in fact this ratio decreases in green berries and increases in ripened berries, and this is not observed in any other vineyard; in addition to this the already discussed example of BURP domain gene, is observed in Riccione, as well. Riccione is the most diverse environment when compared to Montalcino and Bolgheri. Riccione is located at the Adriatic coast and has a temperate sub-littoral climate, while Montalcino and Bolgheri are both located in Tuscany with typically Mediterranean climate. Moreover, both cultivars show a peculiar profile of small RNA loci during berries ripening, in Riccione. The expression of small RNA loci in Cabernet Sauvignon berries drastically changed during development, especially when collected in Riccione , not only in the number of active loci but also in the different genic or intergenic disposition: ripened berries have a 2.6-fold increase in small RNA loci active in genic regions. Differently, when Sangiovese is grown in Riccione, there is a very high number of small RNA loci active in green berries, mainly associated to transposable elements that remains almost stable during development although the proportion of intergenic loci is reduced. Sangiovese berries collected in Montalcino show a 2.5- fold increase of small RNA producing loci during development. Differences during berry development between the cultivars may explain their different behavior in different environments, and the characteristics of each vineyard may favor one or other variety according to their demands. For example, Sangiovese needs a long growing season with sufficient warmth to fully ripen . Consequently, drainage pot cooler environments will require a reprograming of Sangiovese gene expression in order to achieve ripening. Other factors such as composition of soil, level of humidity, photoperiod and density of cultivation may be exerting the same influence on the ripening of the berries triggering the activation of different small RNA loci. Applying a conservative pipeline to the analysis of our 48 small RNA libraries, we recognized 89 known and annotated grapevine miRNAs. In addition, when compared to previous reports in grapevine we identified 7 completely novel miRNAs plus 26 homologous to other plant species, but novel to grapevine. This is a remarkable number considering the stringency of our pipeline and that our study is based only on four developmental stages of berries. The outline of miRNA accumulation across samples is different from that of sRNA-producing loci. While the expression of sRNA-generating regions allows distinguishing very well between ripened and green berries and also between cultivars , the accumulation of miRNAs shows a clear distinction only between ripened and green berries, and when the berries were green, we observe a further dichotomy separating the two cultivars and the two green developmental stages. The same pattern of miRNA accumulation among green and ripened berries of grapevine was observed when we described the miRNA expression atlas of Vitis vinifera . Comparing the distribution of miRNAs expressed throughout our samples, we found a set of 39 miRNAs ubiquitous or nearly ubiquitous to all the libraries, and very few miRNAs specific of a cultivar, vineyard or developmental stage.

All these 39 miRNAs belong to known vvi-miRNA families. With few exceptions, the same set of miRNAs was also found expressed in all the small RNA libraries constructed with different tissues of the grapevine cv. Corvina , where the population of expressed miRNAs appears highly variable apart from a well-defined group of miRNAs, probably related to the basal metabolism. These findings are also consistent with previous report in grapevine where a small number of known tissue-specific miRNAs was described . Considering the ripening process as shown in the heat maps , and the correlation dendrogram, it is clear that most miRNAs are modulated during the developmental process. For some miRNA families, we observed the same peculiar patterns of miRNA accumulation, previously described in the grapevine miRNA atlas , e.g., an increase of accumulation toward ripening for miR156 f/g/i, and a decrease for miR166c/e, miR172d, miR319, and miR396a/b, but this is not the main focus of our paper. To establish genotype and environmental influence on miRNA modulation, we performed a statistical analysis that revealed a number of miRNAs differentially expressed. Being aware of the fact that we had only two biological replicates, we applied the exact test as implemented in the EdgeR package. This test has been recently judged a very robust tool that can be used in experiments similar to our, because of its low false positive rate and relative high true positive ratein the presence of a fold change higher than 4 . Considering berries at the same developmental stages, we compared Sangiovese vs. Cabernet Sauvignon in a given vineyard and Montalcino vs. Bolgheri, Montalcino vs. Riccione, and Bolgheri vs. Riccione keeping the cultivar fixed. In total we performed 9 pairwise comparisons for each developmental stage. In general, we observed that berries at 19 ◦Brix and at harvest show a higher number of differentially expressed miRNAs. The most interesting examples are represented by two novel miRNAs, whose predicted targets are related to the biosynthesis and accumulation of secondary metabolites, which are of crucial importance in grapevine berries, since its quality depends mainly on its metabolites . The candidate grapem1191 is differentially expressed in Sangiovese between Riccione and Bolgheri and was predicted to target the transparent-testa 12 gene that encodes a multidrug secondary transporter-like protein involved in the vacuolar accumulation of the flavonoid proanthocyanidin in different species including grapevine . Also, in grapevine some studies provide evidences that the intracellular transport of acylated anthocyanins is catalyzed by a MATE transporter . The grape-m1355 seems to be involved in four different pathways, all related to secondary metabolites. It is differentially expressed in Montalcino between the two varieties and was predicted to target a cinnamoyl reductase-like protein , which is part of the of the polyphenol biosynthetic pathway ; a cinnamyl alcohol dehydrogenase involved in the lignin biosynthesis ; a phenylacetaldehyde reductase , which catalyzes, in tomato, the last step in the synthesis of the volatile 2-phenylethanol, important for the aroma and flavor of many foods ; and different bifunctional dihydroflavonol 4-reductases . DFR catalyzes the first step in the conversion of dihydroflavonols to anthocyanins and are responsible for the production of colored anthocyanins .

Water use efficiency predictions are further complicated by the results of combination studies of elevated temperature, reduced soil water availability, and elevated CO2, which reveal synergistic effects. In an open top chamber study, combining temperature and CO2 did not result in gs being significantly reduced, contrary to results of elevated CO2 alone . When latent heat is trapped, overheating subsequently decreases the activity of RUBISCO activase, for most plants at temperatures higher than 37°C , and in grapevine between 35-40°C, varying by species . The elevated CO2 and temperature treatments showed an increase in transpiration , and the effects of drought were only temporarily delayed . Temperature and elevated CO2 had an additive effect on plant leaf area for multiple grapevine clones , highlighting that overall higher leaf area without increased WUE could be detrimental for heat stressed vines. Measurements of predawn water potential were more negative in vines at elevated CO2, indicating the demand for soil water availability of vines with increased productivity . Notedly, the production of fine roots was positively impacted by an elevated CO2 treatment, which would theoretically increase water absorption of water available . There remain inconsistent predictions of the effects of elevated CO2 on grapevine whole plant water use efficiency, which seem to be contingent upon other factors such as soil water availability, temperature, and variety of grapevine. With the evidence from these studies of elevated CO2 and combination studies of soil water availability and temperature, grapevines most likely will not benefit from a long-term increase in photosynthesis under elevated CO2. The lack of soil water available and biological temperature thresholds for RUBISCO will limit the gains in photosynthesis, pot with drainage holes and more likely the vines will struggle to release latent heat as temperatures rise.

Grapevine phenology is categorized into four life cycle stages of periodic development: budburst, flowering, veraison, and maturation. The grapevine phenological cycle is a two-year process; bud formation occurs in the first year which develop into shoots in the second year. Therefore, clusters are significantly impacted by the previous year’s climate . For grapevine grown at elevated CO2, advances in phenology compound significantly over seasons . This is likely the result of stored carbon photosynthate from the productive previous year. As a result, it can take several years to observe the effects of elevated CO2 on grapevine phenology , which leads to the question of: “To what extent does elevated CO2 impact the timing of phenological stages over the long-term?” Studies of Arabidopsis, another C3 flowering plant, provide insight to the mechanisms of phenological changes observed in grapevine. Excess carbohydrates may act similarly to phytohormones to delay the upregulation of genes involved in flowering time, as well as cell wall invertases in the meristem that down regulate photosynthesis under treatments of elevated CO2, which leads to earlier flowering . For grapevine, it is possible that excess photosynthate could trigger early flowering through the transfer of carbohydrates from leaves. One of the most robust findings to support this hypothesis is that growth under elevated CO2 results in increased carbohydrate reserves in plants . The sugars produced by photosynthesis contribute only a fraction of the source of carbon needed for rapid growth and development from budbreak to flowering and sugar accumulation in berries at veraison, the remaining needed for these growth spurts is mobilized from long-term storage of total nonstructural carbohydrates in trunks and roots . Over several growing seasons, storage of carbohydrates in the trunk will be impacted by elevated CO2 and could therefore contribute to shifts in phenology.

In a greenhouse study of fruiting cuttings where sugar accumulation in berries was measured, elevated CO2 increased the rate of ripening correlated with the photosynthetic rate . The effect of elevated CO2 on phenology was greater than the treatment of temperature elevated by 4°C . Therefore, an increase in total nonstructural carbohydrates could be a driver of advances in phenology long term, on its own, as well as with concomitant increases in growing season temperatures. Carbohydrate reserves regulate the growth and differentiation of flowers, which only occurs after the grapevine shoot is resource independent from the rest of the vine . These findings suggest that with an increase in carbon reserves stored as starch in roots, trunks and canes, second season shoots may grow faster and achieve independence earlier in the growing season. This could contribute to early flowering as a result of lifted competition for resources between vegetative and reproductive growth. In contrast, long-term studies in grapevine decreasing the leaf to fruit ratios decreased essential reserves of the TNC in the roots . The well-known viticultural technique of strategic leaf removal has been shown to delay maturation, highlighting the importance of carbon availability for phenological development . While the mechanism for phenological shifts in grapevine grown under elevated CO2 is under-studied, these shifts have been quantified using FACE experiments. The combination of elevated CO2 and temperature in OTC caused an advance in flowering time by three days and veraison by two weeks . The impact of elevated CO2 on phenological timing is greatest during the period between fruit set to veraison and this impact increases when combined with a temperature treatment . During fruit set, elevated CO2 treatments with and without temperature treatments increased total soluble solids , as well as decreased anthocyanins and malic acid concentration, which would contribute to an earlier veraison and harvest . However, the impact of high temperature may have a greater impact on this phenological period .

Grapevines vulnerable to frost damage will suffer from early budburst, with subsequent losses in yield . One consequence of increased shoot vigor at elevated CO2 is the expected increase in bud fertility, which will likely increase the number of flowers per vine . Changes in cluster density and phenological timing impact the carefully articulated annual harvest. Unbalanced sugar/acid ratios resulting from early harvest decrease the quality of grapes and wine produced, discussed further in the “Berry and Wine Chemistry” section below . Shifting the lifecycle of grapevine will have a global impact on winegrape production.Fruit composition is a major area of concern for growers and winemakers alike, especially aromatic compounds. The changes in pest interactions, physiology, and timing of veraison in response to elevated CO2 will collectively impact the resulting grape and wine quality . For successful wines, in the grape berry there is a balance of acid and sugar at harvest. Increasing atmospheric carbon available impacts the balance as ripeningadvances and sugar accumulation is accelerated . Flavonoids and anthocyanins are important for the flavor, color, and mouthfeel of wine. The molecular analysis from the original Italian FACE experiments showed increases in total flavonoids, total anthocyanins, and total non-anthocyanin flavonoids in the wine produced with carbon enriched grapes grown at 700 mg/L , which typically would affect the color and mouthfeel of wine. Interestingly, a subsequent experiment using 500 mg/L CO2 open top chambers determined there were significant increases in ethyl 2-methylbutyrate , isoamyl acetate , ethyl hexanoate , ethyl octanoate , butyric acid , and isovaleric acid concentrations and a significant decrease in ethyl acetate concentration in wines produced from enriched CO2 grapes after one year , which contribute to the balance of floral and fruity characteristics in wines . In the second year they found lower methionol , 1-octanol , and 4- ethylguaiacol , and they found higher ethyl lactate and linalool concentrations, large pot with drainage although these changes in berry chemistry did not appear to significantly affect the quality of wine produced . These results agree with early studies led by Bindi et al. that did not find significant effects on the quality of wine produced from grapes grown at elevated CO2 . Although the changes observed in compounds contributing to flavor have been noted as so far insignificant for quality, a major concern for winemakers is the increase in alcohol content resulting from an increase in sugar concentrations in berries, as a result of higher CO2 concentrations . In the past, winemakers have added sugar to the fermentation to increase the final alcohol percentage , depending on legal regulations for winemaking.However, in recent years winemakers have begun removing sugar through processes like reverse osmosis in order to prevent alcohol levels from rising . Overall, elevated CO2 is altering the balance of sugar accumulation, the levels tartaric and malic acids in berries and wine, and the impact on wine quality continues to be investigated . The most recent FACE studies on grapes continue to evaluate the berry chemistry and quality developing over years of exposure to elevated CO2. The VineyardFACE in Germany analyzed must from grapes after pressing and did not find a significant increase in sugar content from conditions of carbon enrichment . The Gonçalves et al. study also concluded that changes in water availability and heat stress could change their predictions in wine quality.

We should expect that with the shifts in phenology and physiological changes to berries, early harvest will impact the quality of grapes in terms of reaching maturation too quickly . Viticulturists could also anticipate altered physiological demands to have long-term impacts on berry quality . In contrast to the ecological pressures discussed above, the rates of some fungal infections may be reduced in elevated CO2 scenarios. With higher carbon allocation to roots, grapevine mycorrhizal colonization may be promoted by elevated CO2 , which has been shown to protect grapevine against the nematode Xiphinema index by stimulating defense gene response . A study of elevated CO2 on several varieties of grapevine seedlings showed a reduced severity of the infection of Xanthomonas campestris pvviticola, a vector of bacterial canker in immature grapevine . This may be the result of lower stomatal conductance ; with stomatal aperture reduced, there is less opportunity for bacteria to invade the leaf pores . Also, researchers recorded a reduced instance and severity of powdery mildew infection in cv Barbera, at elevated CO2 . The Geisenheim VineyardFACE site recorded changes in the bunch architecture but did not see an increase in the frequency of B. cinera, botrytis bunch rot, a necrotrophic fungus, occurrence . Changes in leaf chemistry phenotype, specifically carbon content, , will increase the pressure of grapevine pests in future climates. Increasing available carbon dioxide, without a concomitant increase in nutrient levels in the soil, leads to an increase in C:N ratios in leaves . Insects consume at higher rates when nitrogen has been diluted to meet their nitrogen intake needs and chewing insect pests will generally eat more leaf tissue in elevated carbon dioxide scenarios . Elevated CO2 increased individual survival rates and increased the fecundity of female mealybugs, which eat phloem of grapevine damaging the temporal and perennial plant tissue . The European grapevine moth, Lobesia botrana, is a major problem for European vineyards, affecting both the berries and flowers of grapevines; and has already invaded North and South American vineyards . L. botrana is also responsible for spreading Ochratoxin A-producing Aspergillus fungi, which typically spikes in occurrence during hotter and drier years . At higher temperatures simulating future climate conditions, L. botrana female growth rate and pupal mass increased , while researchers found a down regulation of expression of ethyleneresponsive factors, which suggests grapevines can become more vulnerable to herbivory or abiotic stress under future climate change as these are the major stress and defense response factors . A comprehensive study of soil and elevated CO2 showed the decomposition pathway is altered by the carbon-, nitrogen-, and phosphorus-acquiring enzymes in the soil with a significant increase in nematode density . More than 4,000 plant-parasitic nematodes exist, posing a well-known global issue for grapevine, reducing total crop production by 8.8- 14.6%, and one of the worst threats from the nematode Xiphinema index is GLRV . Under elevated CO2 conditions, if ethylene is suppressed and salicylic acid is increased, it is likely that grapevine will struggle with an increase in pest and disease vectors such as nematodes and fungi . Grapevines largely rely on human intervention for defense against pests and diseases , and this reliance could increase in future climates. Consider the grapevine “immune system” as weakened in terms of chemical defense, but some altered carbon dynamics under elevated CO2 may be beneficial for reducing severity of pest pressure.

The very concept is embedded in the colonial relationship itself, implying as it does that there is some land that is not sacred. Moreover, the federal government did not allow “sacred land” as a legal land-claim defense until the 1970s, and its documentation must meet standards defined by the federal government, a bizarre requirement since the federal courts seldom demonstrate much wisdom in cosmological or religious matters. Thus Indian spiritual connections to the environment are frequently examined and negotiated in light of contemporary residential development, land management activities, and outdoor recreation as well as legislative activities and the outcomes of litigation. Spiritual connections are seldom straightforward however. Stephen Jett suggested that there may be varying degrees of sanctity for Navajo, although he is unclear about whether such a continuum emanates from his own analysis of the situation or from Navajo interpretations. In addition, James Griffith explained in detail the multi-valent possibilities of places where ideas of sacredness held by the Tohono O’Odham, Yaqui, Mexican, and Anglo American peoples intersect. Also, Linea Sundstrom attempted to set the record straight for the Black Hills using archival documents in an ethnohistorical reconstruction identifying what is held sacred in the area, since when, and by whom, including at least seven tribes. The secular popularity of the subject of sacred lands has not come without repercussions. People are dehumanized and cultural complexity trivialized when non-Indian environmentalists furate on indigenous ecological spirituality and activity while ignoring other significant aspects of community and culture. Bruce Willems-Braun makes this point about marginalization of the Nuuchah-nulth of British Columbia, square pot who often have been denied an active role in debates over wilderness preservation because a perspective on the environment has frequently been imposed on them rather than asserted by them.

Three decades ago Brian Goodey called on geographers to work as researcheradvocates in support of Indian tribes and their economic development. Sincethat time, land use, economic development, and tourism, especially related to gambling, have undergone rapid change in Indian Country. When coordinated with tribal interests, research into these areas generally has been viewed positively by tribal governments. There are a number of geographers and their colleagues who are working for tribes directly or as consultants, or are pursuing research on these topics independently. Data problems are a major concern among these researchers, especially difficulties encountered in using census and Bureau of Indian Affairs or Department of Indian Affairs and Northern Development , the Canadian equivalent of the BIA, data for analysis of population and economic and community development. A critical issue in tribal planning is the need to establish a framework for government-to-government relations between tribal and city, county, and state governments when work must be accomplished in a regional context. A major applied research effort on this topic was carried out and summarized by Shirley Solomon, a geographer working with the Northwest Renewable Resource Center in Seattle. This collaboration produced extensive coordination of tribal groups and local governments. One study found that access to health services was severely restricted on the Round Valley Reservation in northern California not only because of its extreme distance from comprehensive medical facilities, but also because trained medical and dental professionals could not be consistently retained. Another found that Montana Indian women were twice as likely as non-Indians to have to travel outside the county to a birthing facility and even more likely to have poor obstetric care anywhere near where they live.

None of this will come as a surprise to readers familiar with rural life, especially life in Indian Country. But geographers are novices to health care planning and are only just beginning to realize their expertise may be of use in examining aspects of health care such as location, access, travel distance, and sense of place. The new multicultural conceptions of health and well-being now beginning to replace standard medical-geography models undoubtedly will encourage more interest in Indian health issues. Federal legislation in the 1980s and 1990s made gambling a potentially significant component of reservation economic development, the impact of which was examined in two edited books and several articles by geographers. The case studies indicate rapid growth in gambling accompanied by some economic benefit accruing to most participating tribes. Long-term stability, increasing competition from other gambling facilities, subsequent social polarization, and the politics of wealth redistribution both inside and outside reservation boundaries are recognized as problems. Regional studies for Connecticut, the Dakotas, New Mexico, and Oklahoma also are included in these publications. The sale of American Indian arts and crafts and the associated appeal of cultural tourism have long played a role in Indian economic life, although the benefits have been distributed unevenly from region to region. In the past, such activities were generally directed and controlled by non-Indians, and the bulk of revenues and proceeds went to non-Indians. While there are some exceptions, this pattern often continues today. Several geographers have studied the consequences of tourism for Native populations and the increased effort to gain local control of tourism facilities and activities. Geographers also have begun applying postcolonial or other social theory to American Indian studies. Generally anglophonic postcolonial studies have emerged from and focus on the British colonial experience; accordingly, postcolonial American Indian research is well represented within Canadian universities, most notably the University of British Columbia. These researchers seek to deconstruct and assess the imprint of a European worldview on the lands, minds, and bodies of Indians. They draw inspiration from postcolonial literature and French intellectuals Henri Lefebvre, Jacques Lacan, Jacques Derrida, and Michel Foucault.

These diverse theoretical currents are brought together in studies of colonial surveillance and control of Indian spaces, the appropriation of such spaces through ethnocentric cartographic or other textual representation, and the various Indian responses to such practices. Nicholas Blomley has employed similar themes, investigating Indian blockades in British Columbia as a mechanism for tribal reappropriation of space, and Robert Galois has attempted a revised empirical overview of historical Kwakiutl settlement free from earlier colonial biases. Explaining the mechanisms of group representation and identity is fundamental in several studies: Berry’s comparative work on waterscapes and representation for Paiute and non-Paiute communities in northern Nevada; Dennis Crow’s inquiry about reservations as “low places”; Deur on the Makah whale hunt revival; Galois on the period of colonial consolidation in British Columbia; Peters’ examination of the apparent conflict between “city person” and “Indian” identities in Canadian cities; and Silvern’s study of treaty-rights conflicts in Wisconsin. Also, the spatial aspects of gender are influential in the way places and people function. Karen Morin examined British women’s constructions of Indians as Others in historic “contact zones” like the railroad depots of the American West, Peters explained the role of Indian females in subverting urban spaces in Canada, and Wishart considered the role and status of women in Pawnee society, and the general place of Indian women as subjects of geographical research. In an unusual link to demographic study, Robert Jackson sifted the evidence of population decline in the US Southwest and developed the thesis that deaths were related not only to disease, but also to place/space destruction and reductions in cohesion, identity, and sense of place. In a similar vein, R. Douglas K. Herman provided insight into changes in Hawai‘ian identity in response to colonization and corresponding shifts in environmental knowledge and language. The question of whether there exists a pan-Indian sensibility about place and space also has been of some interest. In a 1976 interview, N. Scott Momaday proposed and described what he termed a pan-Indian ecological sensibility bound into Indian identity, an ethos distinguished from those of non-Indian North Americans by the presence of what he termed a sense of “reciprocal appropriation.” Then geographers David Stea and Ben Wisner wrote of a panIndian ecological worldview projected outward in solidarity with other indigenous or Fourth World peoples. Since then a few geographers have examined pan-Indianness as an evolving identity crucial to understanding the link between place and action, drainage collection pot and one has written about the men’s movement’s appropriation and wild distortion of elements of this identity.56 Others have shown how Indian identities are materialized in places as disparate as battlefield and massacre sites, cultivated gardens, migration corridors, and interior landscapes of the mind. The Inuit have been of particular interest, as shown in studies attempting to explain their mental landscape representations or “mental maps,” their navigational skills, and their secondary position to environmentalists’ identification with stranded whales and stripped seals. Place names or toponyms are also of longstanding interest to geographers, and another intersection where the work of anthropologists has been influential. Most recently the intimate links among social structure, individual and group identity, and place were illustrated by geographers studying Chinook Jargon, Inupiat, Hawai‘ian, Navajo, and Inuktitut place names. The study of Indian and Inuit maps accelerated during the past ten years and helped revitalize cartography as a discipline with an historically and culturally situated subject matter, and not merely a narrow technical one. A group of writers have examined the interpretations made of Indian or Inuit maps by nineteenth- and twentieth-century explorers and government agents, and the discourses into which these documents entered, including those of contemporary scholarship.60 Map exchanges between Indians or Inuit and Europeans or Euro-North Americans occurred everywhere in North America, but extant examples and thus much of the research effort is concentrated in the continent’s interior plains from Texas to Saskatchewan, and in the Arctic.

These maps are seen as emanating from different assumptions and discourses about the world as a home for humans and non-human others when compared to what Europeans and Euro-North Americans were producing. Indian maps actively disrupted European discourse on geography, history, and identity, and represent one side of what was and still is to some extent an unbridgeable gap in worldviews. The other side, the European and Euro-North American mapping of Indian Country, has also come under scrutiny. This cartography spans the last five hundred years, but geographers have tended to study comparatively recent examples. They have questioned these maps, often finding them ideological weapons serving non-Indian interests rather than the simple and innocent mimetic representations that the general public and their makers imagined them to be. Several others deserve attention because they emanate from Indians or Inuit themselves and represent a new trend of “mapping back” or “counter-mapping” the colonizers. A Zuni Atlas was published because Zuni elders, in their effort to press a land claim, decided it was time to make certain protected geographical information available to the public for the first time. In part this atlas is notable because it reveals some Zuni sacred sites in order to demonstrate land occupancy beyond present reservation boundaries. But it does so using maps drawn at a scale too small for the reader to navigate. Thus the sites were revealed for litigation purposes, but still kept safe from unwanted visitors. The Inuit have been especially active in counter-mapping. First, they created a map series containing their own place names inscribed on Canada’s official topographic maps, then they assembled the Nunavut Atlas, an extensive compilation of wildlife patterns and other environmental data gathered from Inuit elders. These may be viewed both as attempts to overcome disruption of the traditional oral transfer of geographic information from one generation to the next and also as nationalistic outpourings in anticipation of the creation of the new Territory of Nunavut in 1999. Robert Rundstrom has taken note of these developments and their implications in an article and map review. GIS are a completely new technological development within the past two decades but are understood as a natural extension into the digital era of gathering and mapping geographical data. To put it most broadly, GIS are computer systems for the gathering, storage, and manipulation of geographical coordinates and associated statistical data about anything on the earth’s surface, expressions of which can be made in either paper or digital form. They are more than computerized mapping systems because they can transform information in ways uncommon or unknown in traditional map making and they easily link with orbiting satellites and the remotely sensed digital images that satellites transmit. In the past five years, a national American Indian GIS association has developed and is closely linked to the leading corporate manufacturer of GJS software; tribes, therefore, have been very active participants in GIS applications and research. Continued efforts to link the US Census Bureau’s Topologically Integrated Geographic Encoding and Referencing file maps with tribal information has also helped some tribes initiate detailed spatial data analysis of the lands and people administered by tribal government.

The measurement was done using AC current excitations of 0.1 – 20 nA at 0.5 – 5.55 Hz using a DL 1211 current preamplifier, SR560 voltage preamplifier, and SR830 and SR860 lock-in amplifiers. Gate voltages and DC currents are applied, and amplified voltages recorded, with a home built data acquisition system based on AD5760 and AD7734 chips.In crystalline solids, orbital magnetization arises from the Berry curvature of the bands and intrinsic angular momentum of the Bloch electron wave packet. Although the orbital magnetization often contributes—at times substantially—to the net magnetization of ferromagnets, all known ferromagnetism involves partial or full polarization of the electron spin. Theoretically, however, ferromagnetism can also arise through the spontaneous polarization of orbital magnetization without involvement of the electron spin. Recently, hysteretic transport consistent with ferromagnetic order has been observed in heterostructures composed of graphene and hexagonal boron nitride, neither of which are intrinsically magnetic materials. Notably, spin-orbit coupling is thought to be vanishingly small in these systems, effectively precluding a spin-based mechanism. To host purely orbital ferromagnetic order, a system must have a time reversal symmetric electronic degree of freedom separate from the electron spin as well as strong electron-electron interactions. Both are present in graphene heterostructures, where the valley degree of freedom provides degenerate electron species related by time reversal symmetry and a moir´e superlattice can be used to engineer strong interactions. In these materials, a long wavelength moir´e pattern, arising from interlayer coupling between mismatched lattices, vertical towers for strawberries modulates the underlying electronic structure and leads to the emergence of superlattice minibands within a reduced Brillouin zone.

The small Brillouin zone means that low electron densities are sufficient to dope the 2D system to full filling or depletion of the superlattice bands, which can be achieved using experimentally realizable electric fields. For appropriately chosen constituent materials and interlayer rotational alignment, the lowest energy bands can have bandwidths considerably smaller than the native scale of electronelectron interactions, EC ≈ e 2/λM, where λM is the moir´e period and e is the electron’s charge. The dominance of interactions typically manifests experimentally through the appearance of ‘correlated insulators’ at integer electron or hole filling of the moir´e unit cell[13, 19], consistent with interaction-induced breaking of one or more of the spin, valley, or lattice symmetries. Orbital magnets are thought to constitute a subset of these states, in which exchange interactions favor a particular order that breaks time-reversal symmetry by causing the system to polarize into one or more valley projected bands. Remarkably, the large Berry curvature endows the valley projected bands with a finite Chern number, so that valley polarization naturally leads to a quantized anomalous Hall effect at integer band filling. To date, quantum anomalous Hall effects have been observed at band fillings ν = 1 and ν = 3 in various graphene heterostructures, where ν = An corresponds to the number of electrons per unit cell area A with n the carrier density. Although orbital magnetism is generally expected theoretically in twisted bilayer graphene, no direct experimental probes of magnetism have been reported because of the relative scarcity of magnetic samples, their small size, and the low expected magnetization density they are predicted to have.In the absence of significant magnetic disorder ferromagnetic domain walls minimize surface tension. In two dimensions, domain walls are pinned geometrically in devices of finite size with convex internal geometry. As discussed in Fig. 5.15, we observe pinning of domain walls at positions that do not correspond to minimal length internal chords of our device geometry–suggesting that magnetic order couples to structural disorder directly.

This is corroborated by the fact that the observed domain reversals associated with the Barkhausen jumps are consistent over repeated thermal cycles between cryogenic and room temperature. Together, these findings suggest a close analogy to polycrystalline spin ferromagnets, which host ferromagnetic domain walls that are strongly pinned to crystalline grain boundaries ; indeed, these crystalline grains are responsible for Barkhausen noise as it was originally described. Although crystalline defects on the atomic scale are unlikely in tBLG thanks to the high quality of the constituent graphene and hBN layers, the thermodynamic instability of magic angle twisted bilayer graphene makes it highly susceptible to inhomogeneity at scales larger than the moir´e period, as shown in prior spatially resolved studies. For example, the twist angle between the layers as well as their registry to the underlying hBN substrate may all vary spatially, providing potential pinning sites. Moir´e disorder may thus be analogous to crystalline disorder in conventional ferromagnets, which gives rise to Barkhausen noise as it was originally described. A subtler issue raised by our data is the density dependence of magnetic pinning; as shown in Fig. 5.3, Bc does not simply track 1/m across the entire density range, in particular failing to collapse with the rise in m in the Chern magnet gap. This suggests nontrivial dependence of either the pinning potential or the magnetocrystalline anisotropy energy on the realized many body state. Understanding the pinning dynamics is critical for stabilizing magnetism in tBLG and the growing class of related orbital magnets, which includes both moir´e systems as well as more traditional crystalline systems such as rhombohedral graphite. In order to understand the microscopic mechanism behind magnetic grain boundaries in the Chern magnet phase in tBLG/hBN, we used nanoSQUID magnetometry to map the local moir´e superlattice unit cell area, and thus the local twist angle, in this device, using techniques discussed in the literature. This technique involves applying a large magnetic field to the tBLG/hBN device and then using the chiral edge state magnetization of the Landau levels produced by the gap between the moir´e band and the dispersive bands to extract the electron density at which full filling of the moir´e superlattice band occurs .

The strength of this Landau level’s magnetization can be mapped in real space , and the density at which maximum magnetization occurs can be processed into a local twist angle as a function of position . It was noted in that the moir´e superlattice twist angle distribution in tBLG is characterized by slow long length scale variations interspersed with thin wrinkles, across which the local twist angle changes rapidly. These are also present in the sample imaged here . The magnetic grain boundaries we extracted by observing the domain dynamics of the Chern magnet appear to correspond to a subset of these moir´e superlattice wrinkles. It may thus be the case that these wrinkles serve a function in moir´e superlattice magnetism analogous to that of crystalline grain boundaries in more traditional transition metal magnets, pinning magnetic domain walls to structural disorder and producing Barkhausen noise in measurements of macroscopic properties. In tBLG, a set of moir´e subbands is created through rotational misalignment of a pair of identical graphene monolayers. In twisted monolayer-bilayer graphene a set of moir´e subbands is created through rotational misalignment of a graphene monolayer and a graphene bilayer. These systems both support Chern magnets. Both systems are also members of a class of moir´e superlattices known as homobilayers; in these systems, the 2D crystals forming the moir´e superlattice share the same lattice constant, and the moir´e superlattice appears as a result of rotational misalignment, as illustrated in Fig. 5.17A. Homobilayers have many desirable properties; the most important one is that the twist angle can easily be used as a variational parameter for minimizing the bandwidth of the moir´e subbands, container vertical farming producing the so-called ‘magic angle’ tBLG and tMBG systems. Homobilayers do, however, have some undesirable properties. Although local variations in electron density are negligible in these devices, the local filling factor of the moir´e superlattice varies with the moir´e unit cell area, and thus with the relative twist angle. The tBLG moir´e superlattice is shown for two different twist angles in 5.17B-C across the magic angle regime; it is clear that the unit cell area couples strongly to twist angle in this regime, illustrating the sensitivity of these devices to twist angle disorder. The relative twist angle of the two crystals in moir´e superlattice devices is never uniform. Imaging studies have clearly shown that local twist angle variations provide the dominant source of disorder in tBLG . It is hard to exaggerate the significance of this problem to the study of moir´e superlattices. Phenomena discovered in tBLG devices are notoriously difficult to replicate. Orbital magnetism at B = 0 has only been realized in a handful of tBLG devices, and quantization of the anomalous Hall resistance has only been demonstrated in a single tBLG device, in spite of years of sustained effort by several research groups. A mixture of careful device design limiting the active area of devices and the use of local probes has allowed researchers to make many important discoveries while sidestepping the twist angle disorder issue- indeed, some exotic phases are known in tBLG only from a single device, or even from individual scanning probe experiments- but if the field is ever to realize sophisticated devices incorporating these exotic electronic ground states the problem needs to be addressed.There is another way to make a moir´e superlattice. Two different 2D crystals with different lattice constants will form a moir´e superlattice without a relative twist angle; these systems are known as heterobilayers . These systems do not have ‘magic angles’ in the same sense that tBLG and tMBG do, and as a result there is no meaningful sense in which they are flat band systems, but interactions are so strong that they form interaction-driven phases at commensurate filling of the moir´e superlattice anyway. Indeed, many of the interaction-driven insulators these systems support survive to temperatures well above 100 K.

The most important way in which heterobilayers differ from homobilayers, however, is in their insensitivity to twist angle disorder. In the small angle regime, the moir´e unit cell area of a heterobilayer is almost completely independent of twist angle, as illustrated in 5.17E-F. A new intrinsic Chern magnet was discovered in one of these systems, a heterobilayer moir´e superlattice formed through alignment of MoTe2 and WSe2 monolayers. The researchers who discovered this phase measured a well-quantized QAH effect in electronic transport in several devices, demonstrating much better repeatability than was observed in tBLG. The unit cell area as a function of twist angle is plotted for three moir´e superlattices that support Chern insulators in 5.17G, with the magic angle regime highlighted for the homobilayers, demonstrating greatly diminished sensitivity of unit cell area to local twist angle in the heterobilayer AB-MoTe2/WSe2. MoTe2/WSe2 does have its own sources of disorder, but it is now clear that the insensitivity of this system to twist angle disorder has solved the replication issue for Chern magnets in moir´e superlattices. Dozens of MoTe2/WSe2 devices showing well-quantized QAH effects have now been fabricated, and these devices are all considerably larger and more uniform than the singular tBLG device that was shown to support a QAH effect, and was discussed in the previous chapters. The existence of reliable, high-yield fabrication processes for repeatably realizing uniform intrinsic Chern magnets is an important development, and this has opened the door to a wide variety of devices and measurements that would not have been feasible in tBLG/hBN. The basic physics of this electronic phase differs markedly from the systems we have so far discussed, and we will start our discussion of MoTe2/WSe2 by comparing and contrasting it with graphene moir´e superlattices. In tBLG/hBN and its cousins, valley and spin degeneracy and the absence of significant spin-orbit coupling combine to make the moir´e subbands fourfold degenerate. When inversion symmetry is broken the resulting valley subbands can have finite Chern numbers, so that when the system forms a valley ferromagnet a Chern magnet naturally appears. Spin order may be present but is not necessary to realize the Chern magnet; it need not have any meaningful relationship with the valley order, since spin-orbit coupling is absent. MoTe2/WSe2 has strong spin-orbit coupling, and as a result, the spin order is locked to the valley degree of freedom. This manifests most obviously as a reduction of the degeneracy of the moir´e subbands; these are twofold degenerate in MoTe2/WSe2 and all other TMD-based moir´e superlattices.

The techniques described above still have some limitations, and chief among them is a limited range of electronic densities that they can reach. Of course, the gold standard of electron density modulation is the ability to completely fill or deplete an electronic band, which requires about one electron per unit cell in the lattice. Chemical doping can achieve enormous offsets in charge density, sometimes as high as one electron per unit cell. This limitation isn’t fundamental and there are some ideas in the community for ways to improve it, but for now it remains true that electrostatic gates can modify electron densities only slightly relative to the total electron densities of real two dimensional crystals. As it stands, electrostatic gating can only substantially modify the properties of a crystal if the crystal happens to have large variations in the number and nature of available quantum states near charge neutrality. For many crystals this is not the case; thankfully it is for graphene, and for a wide variety of synthetic crystals we will discuss shortly. Electrostatic gating of two dimensional crystals was rapidly becoming a mature technology by the time I started my PhD. So where does nanoSQUID magnetometry fit into all of this? A variety of other techniques exist for microscopic imaging of magnetic fields; the most capable of these other technologies recently developed the sensitivity and spatial resolution necessary to image stray magnetic fields from a fully polarized two dimensional magnet, plastic pots 30 liters with a magnetization of about one electron spin per crystalline unit cell, and this was widely viewed within the community as a remarkable achievement.

We will shortly be discussing several ferromagnets composed entirely of electrons we have added to a two dimensional crystal using electrostatic gates. Because of the afore- mentioned limitations of electrostatic gating as a technology, this necessarily means that these will be extremely low density magnets with vanishingly small magnetizations, at least 100 times smaller than those produced by a fully polarized two dimensional magnet like the one in the reference above. It is difficult to summarize performance metrics for magnetometers, especially those used for microscopy. Many magnetometers are sensitive to magnetic flux, not field, so very high magnetic field sensitivities are achievable by simply sampling a large region, but of course that is not a useful option when imaging microscopic magnetic systems. Suffice to say that nanoSQUID sensors, which had been invented in 2010 and integrated into a scanning probe microscope by their inventors by 2012, combine high spatial resolution with very high magnetic field sensitivity. This combination of performance metrics was and remains unique in its ability to probe the minute magnetic fields associated with gate-tunable electronic phenomena at the length scales demanded by the size of the devices. Gate-tunable phenomena in exfoliated heterostructures and nanoSQUID microscopy were uniquely well-matched to each other, and although at the time I started my graduate research only a small handful of gate-tunable magnetic phenomena had so far been discovered in exfoliated two dimensional crystals, nanoSQUID microscopy seemed like the perfect tool for investigating them. This makes SQUIDs excellent magnetic field sensors, with the caveat that they do not sample the magnetic field at a point, but averaged over a region A. Making a SQUID is as easy as depositing a superconducting material onto a surface in the correct shape, and it can be done using many of the same techniques used to produce other microscopic electronic devices, like photolithography and thermal evaporation.

This is sufficient for many applications, but it presents some issues for producing sensors for scanning probe microscopy. Scanning probe microscopy is a technique through which any sensor can be used to generate images; we simply move the sensor to every point in a grid, perform a measurement, and use those measurements to populate the pixels of a two dimensional array . This can of course be done with a SQUID, and many researchers have used SQUIDs fabricated this way to great effect. But the spatial resolution of a scanning SQUID magnetometry microscope is set by the size of the SQUID, and there are limits to how small SQUIDs can be fabricated using photolithography. It is also challenging to fashion these SQUIDs into probes that can be safely brought close to a surface for scanning; photolithography produces SQUIDs on large, flat silicon substrates, and these must subsequently be cut out and ground down into a sharp cantilever with the SQUID on the apex in order to get the SQUID close enough to a surface for microscopy. In summary, the ideal SQUID sensor for microscopy would be one that was smaller than could be achieved using traditional photolithography and located precisely on the apex of a sharp needle to facilitate scanning. As is so often the case when developing new technologies, we have to make the best of the tools other clever people have already developed. In the case of nanoSQUID microscopy, the inventors of the technique took advantage of a lot of legwork done by biologists. Long ago, glass blowers found that hollow glass tubes could be heated close to their melting point and drawn out into long cones without crushing their hollow interiors. Chemists used this fact to make pipettes for manipulating small volumes of liquid, and biologists later used the techniques they developed to fashion microscopic hypodermic needles that could be used to inject chemicals into and monitor the chemical environment inside individual cells in a process called patch-clamping. A rich array of tools exist for producing these structures, called micropipettes, for chemists and biologists. Eli Zeldov noticed that these structures already had the perfect geometry to serve as substrates for tiny SQUIDs. By depositing superconducting materials onto these substrates from a few different directions, one can produce superconducting contacts and a tiny torus of superconductor on the apex of the micropipette. The same group of researchers successfully integrated these sensors into a scanning probe microscope at cryogenic temperatures.

The sizes of these SQUIDs are limited only by how small a micropipette can be made, and since the invention of the technique SQUIDs as small as 30 nm have been realized. We call these sensors nanoSQUIDs, or nanoSQUID-on-tip sensors. A few representative examples of nanoSQUID sensors are shown in Fig. 1.4. A characterization of the electronic transport properties of such a sensor, and in particular the sensor’s response to an applied magnetic field, is shown in Fig. 1.5. NanoSQUID microscopes share many of the core competencies of more traditional, planar scan-ning SQUID microscopes. They dissipate little power, and the measurements they generate are quantitative and can be easily calibrated by measuring the period of the SQUID’s electronic response to an applied magnetic field. Measuring a magnetic field with a SQUID does not require optical access; many other magnetic field measurement techniques do. Together, these facts mean that scanning SQUIDs are often the best tools available for probing extremely low temperature phenomena. NanoSQUID sensors also have many advantages over planar SQUIDs. The most obvious, of course, has already been discussed, and that is their higher spatial resolution. A less obvious advantage- indeed, an advantage that became clear only after the first nanoSQUID sensors were fabricated and tested- is the geometry of the thin superconducting contacts, which under normal circumstances are aligned with the axis of the applied magnetic field. Large magnetic fields tend to destroy superconducting phases, so superconducting devices are all limited by the maximum magnetic fields at which they can operate. This so-called critical field HC is not an intensive property; there is a large-size limit that can be measured and tabulated for different materials, but the critical field of an individual piece of superconductor is a strong function of geometry. A thin superconducting film in the plane of an applied magnetic field can accommodate much higher magnetic field magnitudes than can be accomodated by a large piece of the same superconductor. The bulk limit for lead at low temperature is about 80 mT; we routinely make lead nanoSQUIDs that can survive magnetic fields of 1 T, round plastic pots and we have on occasion made nanoSQUIDs that can survive magnetic fields above 2 T. It turns out that many of the most useful magnetic imaging techniques are limited to low field operation. This thesis will focus primarily on low field phenomena, but there are also many magnetic phenomena that require high magnetic fields to appear, including the quantum Hall effect and a variety of magnetic phase transitions. The nanoSQUID technique is useful for studying these as well. NanoSQUID sensors have some unique disadvantages as well. Like planar SQUIDs, nanoSQUIDs require superconductivity to function, which limits them to fairly low operating temperatures. In planar SQUIDs it is often possible to keep the SQUID itself cold while scanning over a much hotter sample, but nanoSQUID sensors are extremely poorly thermalized to their scan heads, which means that they generally are thermalized either to the surface over which they are scanning or to the black body spectrum of the vessel in which they are contained . This gives nanoSQUID sensors some interesting capabilities, namely that under the right conditions they can function as extremely sensitive scanning probe thermometers, but it also comes with some drawbacks. NanoSQUIDs composed of superconductors with critical temperatures below 4.2 K, the boiling point of helium-4 at atmospheric pressure, must thus have actively cooled thermal radiation shields to operate in very high vacuum, and of course imaging of hot samples is completely out of the question for these sensors.

A variety of exciting opportunities exist for the application of sensitive magnetic imaging techniques to biological systems, and this is not a realistic option for nanoSQUID sensors. NanoSQUIDs are quite fragile and can be easily destroyed by vibrations, necessitating vibration isolation systems, and the superconducting film on the apex of the micropipette is quite thin, typically between 15 and 20 nm, so superconducting materials that oxidize in air will be quickly degraded. Thankfully indium and lead do not oxidize rapidly, but they do oxidize at a finite rate, so nanoSQUIDs composed of these materials only last for a few days when left in air. Storage in high vacuum can improve their lifespan, but generally not indefinitely. In summary, scanning probe microscopes fitted with nanoSQUID sensors can function as magnetometry microscopes with 30-250 nm resolution. They are capable of operating at very low temperatures and magnetic fields of up to several Tesla. Their high sensitivities allow them to detect the minute magnetic fields emitted by electronic phases composed entirely of electrons forced into a two dimensional heterostructure with an electrostatic gate. We will discuss some of the properties of two dimensional heterostructures next. Many crystalline compounds have cleavage planes; that is, planes along which cracks propagate most readily. When such compounds are stressed beyond their yield strength, they tend to break up into pieces with characteristic shapes that inherit the anisotropy of the chemical bonds forming the crystal out of which they are composed. Indeed, this observation was a compelling piece of early evidence for the existence of crystallinity, and even atoms themselves. There exists a class of materials withcovalent bonds between unit cells in a two dimensional plane and much weaker van der Waals bonds in the out-of-plane direction, producing extraordinarily strong chemical bond anisotropy. In these materials, known as ‘van der Waals’ or ‘two dimensional’ materials, this anisotropy produces cleavage planes that tend to break bulk crystals up into two dimensional planar pieces. Exfoliation is the process of preparing a thin piece of such a crystal through mechanical means. In some of these materials, the chemical bond anistropy is so strong that it is possible to prepare large flakes that are atomically thin . These two dimensional crystals have properties quite different from their bulk counterparts. They do have a set of discrete translation symmetries, which makes them crystals, but they only have these symmetries along two axes- there is no sense in which a one-atom-thick crystal has any out-of-plane translation symmetries. For this reason they have band structures that differ markedly from their three dimensional counterparts. As previously discussed, this process cannot be executed on every material. It depends critically on scotch tape bonding more strongly to a layer of the crystal than that layer bonds to other layers within the crystal.

Although we acknowledge this system as complex, it makes some sense to try and simplify it a little to perhaps gain some deeper insight into its operation. A glance at figure 12 suggests that there are two generalized groups of ant predators on the berry borer: the big ants and the small ants . So we can think of it as a two-predator, one-prey system. But there is an obvious indirect effect because the bigger ants negatively affect the ability of the smaller ants to be predators, as was discussed above. Depending on nest densities, it would seem that because the smaller ants prey on all three life stages of the borer and throughout the year for those that forage in infected berries on the ground, they might, in the end, be more efficient predators than the larger ants. Furthermore, the larger ants have an indirect trait-mediated effect on the smaller ants, reducing their effectiveness. One might argue that it is the existence of the smaller ants that potentially regulates the borers over the long run. Unpublished evidence even supports the idea that the berry borer actually seeks areas that are under protection from the ants, supporting the speculation of Gonthier and colleagues that the berry borer gains protection from smaller predators by preferring to attack berries under the protection of larger ants. This basic speculation was put to an unintended test in 2012. Because of a major outbreak of the coffee rust disease , the coffee landscape where we work was heavily sprayed with a combination of calcium carbonate and copper sulfate, hydroponic bucket a permitted activity for organic agriculture. In figure 13, we show the distribution of P. synanthropica as a heat map based on how many tuna fish baits placed in coffee bushes had swarms of P. synanthropica after about 30 minutes.

It is clear that in a single year, a population of thousands of nests of P. synanthropica simply disappeared. Surveys in subsequent years indicated that the smaller ants in the system began to recuperate from the reduced state they had been in, apparently because of the indirect effects of P. synanthropica . Those small ants that attack the borer within the seed, both on the tree and on the ground, especially increased over the next few years. In figure 14, we show the distribution of several of the species in a 50 × 50 meter subplot within the 45-hectare plot. Note how, during the years 2009–2012, the distribution of P. synanthropica remained relatively constant, perhaps slowly increasing in its area of dominance, at the expense of P. protensa on the ground and S. picea arboreally. Then, after the collapse of P. synanthropica between 2012 and 2013, both of those smaller species began to move into the area previously dominated by P. synanthropica. If the above speculations about how the ant community affects the borer are true, we might expect that the elimination of one of the borer’s predators would result in better overall control of the berry borer. In surveys of the berry borer in 2005 and then repeated in approximately the same area in 2018, the attack rate of the borer went from an average of about 15% of berries infected with borers to less than 1%. Insect populations are notoriously variable and respond to many cues in the environment by increasing and decreasing population numbers, frequently in unpredictable ways. Therefore, although this dramatic decline in borer numbers cannot be directly linked to the change in the ant community structure, it is nevertheless worth noting that the underlying narrative of how that community functions as a system of biological control concords perfectly with the changes observed.In the early 1980s, a specter haunted the coffee growing regions of Central America.

The infamous coffee rust disease had arrived in Brazil, and its eventual spread all the way to Mexico was expected, causing extreme worry among farmers and technical advisors. This worry was certainly justified on the basis of the history of the coffee rust disease in Asia . Great Britain’s expansion in what was then called Ceylon was qualitatively distinct from many of its previous imperial adventures. Planting what was effectively a monoculture of coffee, along with a great deal of infrastructure for the time, it was a remarkable centrally planned agricultural development plan. However, the plan effectively created ideal conditions for any disease that could get a foothold, with its virtually shadeless monoculture and networks of roads and railroads that could help distribute the fungal spores widely. When the disease arrived, it took hold and spread throughout the entire island, eventually causing a complete loss of coffee production . However, the rust scare of the 1980s Mesoamerica turned out to be a bit of a false alarm, at least until 2012. Before that year, the rust was always an irksome constraint on production, but the complete devastation that had been feared when it was discovered in the early 1980s never came to pass; it was a problem, to be sure, but not one to get overly agitated about. But then, without much warning, there was an explosion of coffee rust in the 2012–2013 cycle. Countries in the zone declared emergencies as one of their main sources of income seemed to be threatened with severe disruption. Local governments throughout the affected area provided emergency support to coffee producers and both the United Kingdom and the United States came up with significant international aid, specifically for what rapidly came to be called the most devastating emergency in the history of coffee production throughout the region . There are two ecological questions associated with this episode. First, why did the disease not become rampant for approximately 30 years after its introduction, and, second, what caused the very sudden explosion?

Although the answers to either of these questions remain enigmatic, using tools from complexity science provides us with some ideas. The relevant biology of the rust disease is well known. A windblown spore adheres to the under surface of a leaf and encounters a small amount of moisture, causing germination directly into a stoma. The mycelia grow intercellularly and produce haustoria, which penetrate into the plant cell and absorb nutrients, effectively killing the cell. As the fungus grows within the leaf tissue it eventually forms uridia that contain new spores, exiting the leaf from other stomata, causing the characteristic yellow spots on the under surface of the leaves . The transmission dynamics of the disease are dual , stackable planters with some close plant-to-plant dispersion of spores , especially when plants are close enough to touch one another, along with propagule rain from the general spore load that exists in the atmosphere, especially in areas of high concentration of coffee production, when that coffee is attacked by the rust. From the perspective of an individual coffee plant, there are two sources of rust spores: its local neighbors and the general accumulation of spores in its region—that is, from the overall spore load in the atmosphere. But it is also the case that this coffee plant and all others over a very large region contribute to the spores in the overall spore rain from the atmosphere. Given this narrative, it is easy to imagine a situation in which a generally traditional shade coffee landscape would receive a particular rate of spore rain each year and would contribute a bit to the general pool, but because the shade trees act as windbreaks, much of the spore load is never delivered to the coffee plants. One could imagine an equilibrium in which the rust disease would be endemic but not severe, partly because the wind-borne spores have limited access to the coffee trees, meaning that the increment of spore load in the general atmosphere would be limited. Focusing on the large landscape level, if the abundance of spores in the atmosphere is low, it is likely that the incidence of the disease will also be low. But each epidemic will increase the spore density in the atmosphere. The probability that a given farm will become epidemic is a function of both the spore density in the atmosphere and the dispersion rate from the atmosphere to the farm. Changing focus to the local level, the rate of spread of spores from coffee bush to coffee bush on an average individual farm will partially determine whether the rust within that farm will become epidemic. From the point of view of an individual coffee bush, the danger of being infected by a spore comes from two sources: the atmosphere and neighboring infected plants—a regional source and a local source. Imagine that a forested ecosystem is gradually deforested of both shade trees in the coffee farms and the trees in the natural forest around them, and ask what proportion of the farms could be susceptible to an epidemic of coffee rust? According to a simple model that incorporates both regional and local dispersal , the initial deforestation will generate an increase in the number of farms experiencing an epidemic. That increase is likely to be slow and steady at first, but there will be a specific point at which a critical transition will occur and a large number of farms will suddenly become highly infected. This will happen in the complete absence of any other environmental driver, such as climate change or a new more virulent strain of the disease. Indeed, one study in Costa Rica showed that the incidence of rust disease was correlated with the amount of sun coffee and pasture in the surrounding landscape. It could very well be that the sudden outbreak of coffee rust in 2012 is an example of the inevitability of surprise arising from the formality of a critical transition that we have come to associate with highly nonlinear complex systems .

A cartoon version of this theoretical process is presented in figure 16. An important component of the rust disease system, not yet completely understood, is the existence of several natural enemies of the rust . Providing an example of the sorts of ecological complexity of popular literature, the fungal disease of the first pest we discussed, the green coffee scale , is caused by the same species of fungus that, when given the chance, attacks the coffee rust fungus. That same white halo fungus that attacks the green coffee scale, now acts as a mycoparasite . Because this natural enemy is also a natural enemy of the green coffee scale, the connection to the Azteca ant became obvious early on ; Azteca creates conditions under which the scale insect becomes highly concentrated locally, which attracts the infestation of the white halo fungus and creates local hot spots of spores that disperse locally and attack the rust. Correlative evidence for this hypothesis, prior to the 2012 epidemic of rust, comes from multiple sources . Indeed, there has been considerable discussion at international conferences on the potential of L. lecanii as a spray for the rust disease. Our work suggests that partial control of the rust may naturally occur through this and other agents , although the epidemic throughout Mesoamerica in the 2012–2013 growing season shows the potential for the disease to escape such control, if, in fact, it did exist before that. It is quite a remarkable qualitative impression one gets when examining the rust and its control comparatively. It is endemic but rarely epidemic in Puerto Rico but has maintained a relatively severe status in much of Mesoamerica since 2012 . Examining coffee leaves in Mexico easily reveals the presence of L. lecanii but only after considerable searching effort, whereas in Puerto Rico, it is almost inevitable that, if one encounters the rust on a leaf, it is almost certain that one encounters L. lecanii also. What seems epidemic in Puerto Rico is the L. lecanii that seems to keep the rust under control. In addition to the white halo fungus, the larval form of a small fly, Mycodiplosis hamaelae, preys on the spores directly on the coffee leaf but probably also acts as a dispersal agent, at least locally . The coffee leaf rust continues to plague Latin American coffee farmers.

The emergence of the #MeToo movement also appears to have brought the parties together in an effort to change the prevailing culture in the legislature. The most publicized controversy of the legislative session was the emotionally charged debate surrounding the expulsion of a Democratic legislator following multiple sexual harassment allegations. Representative Steve Lebsock was the subject of multiple allegations of sexual harassment. Three formal complaints were made against him, including one from fellow Representative Faith Winter . Following an independent investigation, which found credible evidence that he had engaged in harassment and retaliation, and a full day of emotional speeches on the House floor, the chamber voted 52 to 9 to expel Lebsock from the legislature. The outcome was far from certain prior to the historic vote, as the expulsion resolution required a two-thirds majority vote in order to remove the incumbent representative. Many who supported removing Lebsock cited a need to change the culture in the state legislature. As a result of the vote, Lebsock became the first state representative to be expelled in more than 100 years. In a strange twist, Lebsock submitted the paperwork to change his party affiliation from Democrat to Republican minutes before the expulsion vote. According to state law, this handed the House vacancy to the state’s Republican Party to fill instead of the Democrats who previously held the seat. This act was clearly intended to spite fellow Democrats who abandoned their support of Lebsock once the extent of his harassing behavior became apparent. A spokesperson for the Colorado GOP initially floated the idea of not filling the seat within 30 days, hydroponic nft system which would allow the Democratic governor the opportunity to appoint a successor.

However, the party ultimately decided to appoint a Republican to temporarily hold the seat, which many expected to be regained by the Democrats in the next election cycle. This did not alter the majority party in the chamber since Democrats hold an eight-seat majority after Lebsock’s expulsion. Lebsock was not the only state legislator rebuked for inappropriate behavior, as an independent investigation found credible evidence that Senator Randy Baumgardner sexually harassed a legislative aide in 2016. Senator Baumgardner resigned a key committee chair position while maintaining his innocence in advance of an expulsion vote in the Senate. While the vote to expel Lebsock was supported by representatives of both parties, the parties appeared more divided in the Senate with regard to the allegations against Baumgardner. Republican leadership ordered Baumgardner to undergo sensitivity training, while Democrats have called for more severe sanctions including expulsion. In early April, Baumgardner survived an expulsion vote with 17 voting in favor and 17 voting opposed. While this fell short of the two thirds majority necessary to remove a lawmaker, potential expulsions from the Senate threatened larger reverberations on policy and the budget as the Republicans held a narrow one-seat majority until Senator Cheri Jahn changed her party affiliation from Democrat to unaffiliated in December 2017. On the legislative front, the second session of the 71st General Assembly convened on January 10, 2018 after a special session called by Governor John Hickenlooper in October 2017 failed to produce any accomplishments. Hickenlooper previously called a special session in 2012 to debate a civil unions bill, which ultimately failed to pass. The 2017 special session similarly adjourned amid partisan gridlock. The governor called this special session to address what was described as a bill drafting error contained in a major reform that passed at the end of the General Assembly’s regular session in 2017. Senate Bill 17-267 imposed many substantial reforms .

Among the most notable of these was the repeal of the existing hospital provider fee structure and the creation of the Colorado Healthcare Affordability and Sustainability Enterprise as a government enterprise fund to collect healthcare sustainability and affordability fees. Importantly, this change made it so hospital provider fees do not count as state revenue for the purposes of the Taxpayer’s Bill of Rights . The bill drafting error concerned a modification to the taxes levied on recreational marijuana sales. When Colorado voters approved Amendment 64 to legalize recreational marijuana use in 2012, the amendment included the following language governing the General Assembly’s authority to levy taxes on retail marijuana sales, “The General Assembly shall enact an excise tax to be levied upon marijuana sold or otherwise transferred by a marijuana cultivation facility to a marijuana product manufacturing facility or to a retail marijuana store at a rate not to exceed fifteen percent prior to January 1, 2017 and at a rate to be determined by the General Assembly thereafter, and shall direct the department to establish procedures for the collection of all taxes levied.” Senate Bill 17-267 collapsed the existing 2.9 percent sales tax on retail marijuana sales and the 10 percent special tax into a single 15 percent state tax. The drafting error occurred because legislators intended to only remove the statewide sales tax, while an exemption allowed municipal governments to impose their own sales taxes on marijuana sales where permitted. The bill, however, failed to include a similar exemption for special districts such as the Denver metro area’s Regional Transportation District and the Scientific and Cultural Facilities District , among several others. This omission meant that these districts could no longer continue collecting tax revenue on marijuana sales as they had before Senate Bill 17-267 went into effect. While the SCFD supports scores of organizations and programs including the Denver Zoo, Denver Art Museum, and Denver Museum of Nature & Science, the funding losses incurred by RTD were arguably more likely to affect Colorado residents.

The RTD special district encompasses 2,342 square miles across eight counties. The district includes nearly three million residents in its service area. Ridership statistics from the past three years show that annual ridership on RTD commuter rail, light rail, and bus services exceeded 100 million trips . Analysts projected that the bill drafting error would cost RTD nearly $600,000 per month. SCFD monthly losses were estimated at about $56,000. The special session to correct the bill drafting error was opposed by many Republicans. Some argued that modifying the existing tax structure would require a vote of state residents consistent with TABOR’s requirements. The session ended after two measures to correct the error were rejected in the Senate after passage in the House with mostly Democratic support. Following an unsuccessful special session, Governor John Hickenlooper enters the final year of his second term as governor. Term limits prevent him from running again, although there are several indications that he is preparing to launch a bid for the presidency in 2020. A November 1 letter to the General Assembly’s Joint Budget Committee provided details on the key aspects of the governor’s budget request. The budget proposal for the 2018–2019 fiscal year proposed a total funds budget of $30.5 billion, which is an increase of 3.7 percent from the prior year. The General Fund spending budget of $11.5 billion represents a 2.6 percent increase. After legislators considered nearly 200 amendments to the long bill, the state budget totals enacted into law were close to the numbers proposed by the governor. The state’s final General Fund budget was $11.42 billion with $30.63 billion in total fund spending . The letter to the JBC stated that the governor’s budget request “reflects the dynamic factors that are occurring in Colorado’s economy, nft channel constitutional and statutory requirements, and demand for services from State government” . It further noted that the “passage of S.B. 17-267 has materially and positively changed the State’s financial outlook compared with one year ago, when the request had to close a $500 million funding gap in the General Fund. We believe the request reflects the priorities that Colorado citizens expect and accordingly the request addresses important needs in K-12 and higher education as well as certain essential criminal justice system needs” . In percentage terms, spending on K-12 education represents the largest share of the general fund request at 36 percent, while the spending allotment for higher education budget is 9 percent. Nearly every state department was slated to receive increased funding relative to the prior fiscal year with just two exceptions. The budget approved by the General Assembly ultimately increased funding for every state department except for Labor and Employment. The three sections that follow discuss the state’s demographics, revenue, and spending. Colorado’s population growth continues to present both opportunities and challenges for policymakers. While a growing population provides a larger tax base, affordable housing and transportation problems have also accompanied this rapid population growth.

The 2017 update places the number of Colorado residents at slightly greater than 5.6 million . This figure represents an increase of 11.5 percent since 2010 and a 1.2 percent increase since 2016. Of the 223,260 individuals who moved to Colorado in 2016, the Census estimates that 14 percent immigrated from abroad. The size of this group is larger than the number of new residents who moved from any state in the union. Among current U.S. residents who moved to Colorado, the largest shares arrived from California , Texas , Florida , Arizona , Illinois , New York , Virginia , Missouri , Georgia , and North Carolina . The most popular destinations for Coloradoans moving out of state were California, Texas, Washington, Arizona, and Florida. Because most of these population gains have occurred along the Front Range and in the Denver metro area, the housing market in the state’s largest city remains one of the least affordable in the nation. Among U.S. metropolitan areas, the Denver housing market is the most expensive of any city not located in a coastal state. Median home prices in Denver exceeded the national average by more than $100,000 in 2017, and the average price of a single-family homes sold in Denver exceeded $500,000 for the first time in February 2018 . Many residents priced out of the home-buying market have also encountered affordability challenges in the rental market. According to one real estate research firm, apartment rental rates in Denver have increased by nearly 50 percent since 2010. This is the largest increase in rent for any city outside of the San Francisco Bay area. Increased demand for housing units has spurred development in the Denver metro area as evidenced by the construction of 23,000 new apartments between 2016 and 2017 . A growth in the number of available housing units has increased the vacancy rate in the metro area to 6.4 percent in 2018. This figure is the highest seen in Denver since the 2009 recession. It has also resulted in a modest reversal in the long-term trend of rent increases, as the median monthly rental price in Denver decreased to $1,353 from $1,370 at the end of the third quarter in 2017. Table 1 reports selected data from the Census Bureau and Bureau of Labor Statistics  for Denver County, Colorado, and the United States. As seen in Table 1, per capita and household income in Denver and Colorado remain above the national average. The OSBP projects personal income in Colorado to outpace the national average of 3.1 percent in 2017 with a 5.4 percent growth rate . The 2017 population estimate for Colorado is slightly greater than 5.6 million people. Colorado remains one of the fastest growing states in the nation, although population growth has slowed during the past year. After reaching nearly 2 percent in 2015, population growth fell to 1.4 percent in 2017. This figure remains twice the national growth rate of 0.7 percent. The state projects the number of residents to increase to 5.8 million by the 2020 Census. For most racial minority groups, the state is less diverse than the nation as a whole. Native Americans are one exception to this, as the share of those with American Indian heritage is slightly greater in Denver County and Colorado compared to their percentage nationally . The proportion of residents who identify as Hispanic or Latino is also greater in Colorado and Denver County than in the entire country . The Census estimates that 13.2 percent of current U.S. residents were born abroad. Among all Coloradoans, the percentage of foreign-born residents is about 3 percentage points lower, while the percent foreign born residing in Denver County is nearly 3 percentage points greater.

The antioxidant properties of CGA have been associated with preventing various chronic diseases.To better understand the biosynthesis of antioxidants in blueberry fruit, we identified homologs of previously characterized genes in other species involved in ascorbate, flavonols, chlorogenic acid, and anthocyanin biosynthesis. The key biosynthetic genes for these compounds exhibited a distinct developmental-specific pattern of expression . For example, genes involved in the conversion of leucoanthocyanidins to proanthocyanidins are highly expressed in the earliest and middle developmental fruit stages but not in ripening fruit . Conversely, genes involved in the conversion of leucoanthocyanidins to anthocyanins were highly expressed in mature and ripe fruit but not during early fruit developmental stages . Additionally, paralogs encoding the same anthocyanin pathway enzymes and genes involved in vacuolar localization of proanthcyanidins exhibited similar developmental stage-specific expression patterns. For example, the transcription factor complex MYB-bHLH-WD regulates expression of anthocyanin biosynthetic genes in eudicots. Using the Plant Transcription Factor Database v.4.0 , we identified homologs of transcription factors belonging to 55 gene families, and members of some of these gene families were predicted to be involved in the developmental regulation of flavonoid biosynthesis during blueberry fruit growth , including R2- R3-MYBs, R3-MYBs, bHLHs, and WDRs . These transcription factors also exhibit fruit development-specific expression patterns. In addition, we performed a gene co-expression network analysis to identify meta modules of genes that appear coregulated during fruit development, specifically genes that are associated with phytonutrient biosynthesis. Our analysis identified 1,988 meta modules of co-expressed genes, nft hydroponic of which 428 metamodules contained at least one of the 57 Pfam domains that have been previously categorized as associated with specialized metabolic pathways in plants.

Our analysis revealed that 142 of 428 meta modules were more highly expressed in developing fruit compared to other plant Thissues. Some meta modules showed clear trends of being highly expressed during either early or late fruit development. For example, METAMOD00377 is expressed early in fruit development and contains homologs to known anthocyanin genes OMT, HCT, PAL, and HQT as well as 31 homologs to known transcription factors. In contrast, METAMOD01221 is expressed late in fruit development and contains homologs of HCT, TT19, UFGT, and OMT and contains 10 homologs to known transcription factors. Moreover, we also examined meta modules for genes associated with other biosynthetic pathways that impart unique blueberry fruit characteristics. We identified two meta modules where genes appear to be co-regulated. Metamodule METAMOD00377, which contains Pfam domains associated with terpene, saccharide, and alkaloid specialized metabolism, and METAMOD01221, which contains terpene and saccharide metabolism. These meta modules contained genes that are differentially expressed during fruit development. Overall, the developmental-specific expression patterns of key bio-synthetic genes and their putative transcriptional regulators emphasize the tight regulation of production, conversion, and transport of precursor compounds that lead to the accumulation of antioxidant-related metabolites in blue Berry.The coregulation of genes involved in the biosynthesis of terpenes and saccharides during early and late fruit development described above reflects a coordinated interplay between these metabolites during fruit growth. Both terpenes and sugars contribute to the characteristic flavor of ripened fruit . In blueberry, two components play a central role in flavor perception: taste, which is a balance of sweetness and acidity, and aroma. Blueberry aroma is a complex blend of volatiles that include aldehydes, esters, terpenes, ketones, and alcohols. Previous reports in blueberry showed that the aroma profile varies greatly across different blueberry ecotypes and cultivars . For example, the aroma of high bush blueberry is primarily driven by terpene hydrocarbons and aldehydes -2-hexenal, -2-hexenol, -3-hexenol. Both linalool and geraniol are associated with sweet floral flavor. However, linalool was reported to largely impart the characteristic blueberry flavor when combined with certain aldehydes . Here, we also identified and examined the expression of genes involved in the biosynthesis of linalool. Four of the linalool synthase homologs in tetraploid blueberry are highly expressed during late fruit development .

This pattern of expression coincides with previous reports of linalool accumulation in ripened blueberry fruit . On the other hand, one homolog of linalool synthase, although it was expressed during fruit growth, did not show a clear fruit development-specific pattern. Investigating the underlying factors regulating these enzymes will facilitate genetic manipulations that may lead to further improving blueberry flavor in the future.Superior fruit quality is also associated with sugar levels. During fruit ripening, sugar levels of the endocarp increase by importing hexose symplastically and/or apoplastically. Sugar transporters , sucrose transporter, and tonoplast sugar transporter have been demonstrated to regulate intercellular sugar transport in phloem and fruit. In A. thaliana, all clade III SWEET play a role in sucrose transport, with AtSWEET9 primarily functioning in nectary secretion, while AtSWEET15 is required for seed filling by acting with SWEET11 and SWEET12. In blueberry, the clade III SWEET transporters 9 and 10 were highly expressed during early fruit growth, while clade III SWEET transporter 15 was mainly expressed in ripe fruit . interestingly, one of the blueberry SWEET15 homologs showed a distinct pattern of expression compared to the other three homologs. To the best of our knowledge, we are the first to report on the potential role of these genes during blueberry fruit development. In addition, homologs of A. thaliana TST1 and watermelon ClTST1 and ClTST3 were expressed during fruit ripening in blueberry. Elevated expression of a ClTST1 homolog was observed throughout fruit development, but the ClTST3 homolog showed very low expression. Another gene that is highly expressed during fruit maturation is vacuolar invertase. As described in other systems, its upregulation during fruit ripening coincided with the breakdown of starch to sucrose or a mixture of glucose and fructose, suggesting that it may be involved in the regulation of sugar accumulation in blueberry fruit. It was previously reported that vacuolar invertase modulates the hexose to sucrose ratio in ripening frui. In addition, there are also two sugar transport protein homologs that exhibited developmental specific expression. However, their function remains largely unknown, thus, their potential role in sugar accumulation in the developing berry requires further investigation.

Tandemly duplicated genes arise as a result of unequal crossing over or template slippage during DNA repair, exhibit high birth-death rates, and typically are in co-regulated clusters in the genome. Smallerscale duplications , which include tandem duplicates, are highly biased toward certain gene families including those involved in specialized metabolism. Furthermore, tandem duplications often results in the increased dosage of gene products and may improve the metabolic flux of rate limiting steps in certain bio-synthetic pathways. Most genes associated with the biosynthesis of antioxidants have at least one tandem duplicate present in the high bush blueberry genome, with tandem array sizes ranging from 2 to 10 gene copies . The largest tandem arrays were found for HQT and HCT genes, which are co-regulated and involved in the CGA pathway . Differences in tandem array sizes were also observed between homoeologous chromosomes for various genes. For example, the C3H gene, which is involved in CGA biosynthesis , was present on all four homoeologous chromosomes but with varying tandem array sizes. One of the homoeologous chromosomes had two copies of C3H, while the other three homoeologous chromosomes had four copies. This suggests that copy number differences of C3H among sub genomes may be due to either selection for gene duplication or loss or, in the case of allopolyploidy, may be due to preexisting gene content differences among the diploid progenitor species. Genes in the anthocyanin pathway with other unique duplication patterns include CHS, CHI, OMT, and UFGT. The gene CHS, involved in the conversion of 4-coumaryl-CoA to naringenin chalcone, has two copies, and both have tandem duplicates in at least three of the homoeologous chromosomes. interestingly, the gene CHI has a single preserved tandem gene duplicate on only one of the homoeologous chromosomes. However, nft system additional copies of CHI were also identified more distantly away from the syntenic ortholog on another homoeologous chromosome, likely involving a transposition event following tandem duplication. The OMT and UFGT genes all have tandem duplicates on all of the homoeologous chromosomes, although with varying array sizes, while the ANR gene involved in the conversion of anthocyanidin to proanthocyanidin is single copy on all homoeologous chromosomes. DFR gene, which is involved inthe conversion of dihydroquercetin/dihyromyricetin to leucoanthocyanidin, has a single tandem duplicate on only one of the homoeologous chromosomes. These findings suggest that there may have been greater selective pressure to retain tandem duplicates for genes encoding enzymes involved in anthocyanin production than conversion to proanthocyanidins. The vast majority of tandem duplicates are eventually lost ; however, in rare instances, some may undergo functional diversification. Gene expression analysis revealed that 83.4% of the tandem duplicates were expressed in at least one transcriptome library with 73.5% expressed in at least one of the fruit developmental stages. This suggests that a subset of these duplicate genes have non-functionalized, subfunctionalized, or neofunctionalized. Future studies are needed to more thoroughly investigate the functions of these genes with more diverse libraries and additional transcriptome analyses.Despite the economic importance of blueberry, molecular breeding approaches to produce superior cultivars have been greatly hampered by inadequate genomic resources and a limited understanding of the underlying genetics encoding important traits. This has resulted in breeders having to solely rely on traditional approaches to generate new cultivars, each with widely varying fruit quality characteristics. For example, our analysis of a diversity panel consisting of 84 cultivars and wild species revealed that ”Draper” has antioxidant levels that are up to 19x higher than other cultivars. Thus, the genome of ”Draper” should serve as a powerful resource to the blueberry community for guiding future breeding efforts aimed at improving antioxidant levels among other important fruit quality traits.

Furthermore, to our knowledge, this is not only the first genome assembly of the cultivated high bush blueberry but is also the first chromosome-scale and haplotype-phased genome for any species in the order Ericales. Ericales includes several other high-value crops and wild species with unique lift history traits . Thus, we anticipate that this reference genome, plus associated datasets, will be useful for a wide variety of evolutionary studies. Here, we also leveraged the genome to identify candidate genes and pathways that encode superior fruit quality in blueberry, including those associated with pigmentation, sugar, and antioxidant levels. Furthermore, we found that genes encoding key bio-synthetic steps in various antioxidant pathways are enriched with tandem gene duplicates. For example, tandem gene duplications have expanded gene families that are involved in the biosynthesis of anthocyanins. This suggests that, in addition to a recent whole genome duplication, tandem duplications may have greatly contributed to the metabolic diversity observed in blueberry . These tandem duplicates may have evolved new functions , possibly involved in the biosynthesis of novel compounds, and/or were selected to improve the metabolic flux of specific bio-synthetic steps that alter the dosage of certain endpoint metabolites. Future studies are needed to further investigate the possible role of tandem duplications in having modified metabolite levels and composition in wild and cultivated blueberry. Our analyses also revealed that high bush blueberry, a tetraploid, likely arose from the hybridization of two distinct parents, possibly allopolyploidy, based on the sequence divergence, unique transposable element insertions, and sub genome expression patterns. Our analyses revealed that the sub genomes in high bush blueberry may be controlling a distinct set of genetic programs . The dominantly expressed sub genome in most surveyed Thissues becomes the lowest expressed during fruit development. This observation is similar to findings in allopolyploid wheat where developmental and adaptive traits were shown to be controlled by different sub genomes. For example, cell type- and stage dependent sub genome expression dominance was observed in the developing wheat grain. We argue that both high bush blueberry and hexaploid wheat, each now with high-quality reference genomes, make excellent systems to further investigate these underlying mechanisms of sub genome dominance. sub genome dominance has far-reaching implications to numerous research areas including breeding efforts. For example, marker-assisted breeding needs to target the correct set of dominant homoeologs given the trait in polyploids that exhibit sub genome dominance. Thus, we anticipate that this genome, combined with improved insights into sub genome dominance, will greatly accelerate molecular breeding efforts in the cultivated high bush blueberry.High-molecular-weight genomic DNA was isolated from young leaf Thissue, following a 72-hour dark treatment, using a modified nuclei preparation method.

Likewise, Nicolás et al. found a better photosynthetic performance after inoculating Crimson grapevines grown in a commercial vineyard. Indeed, a recent meta-analysis demonstrated that AMF exert a positive influence on photosynthetic rates, stomatal conductance, and water use efficiency on both C3 and C4 plants subjected to salt stress . Merlot grapevines did not show changes on their berry primary metabolites as affected by the treatments applied . Similarly, a recent study evaluating the effect of different sustained deficit irrigation and RDI showed no differences in must pH and TSS in Merlot berries in a 4-year field experiment conducted in a hot climate . This lack of effect of the irrigation systems on berry primary metabolism might be due to grapevines were not subjected to a severe water stress . On the other hand, previous studies showed that inoculation with AMF of grapevines vineyards did not affect TSS or TA under field conditions or under controlled conditions and our results corroborated these findings. Regarding secondary metabolism, neither irrigation systems nor AMF inoculation modified flavonol and anthocyanin total content at harvest . Similarly, a 2-year field study conducted in Central valley in Califtornia with Merlot did not report differences on flavonol or anthocyanin skin content due to different irrigation amounts . A previous study conducted on Cabernet Sauvignon subjected to water deficit reported that although flavonol synthesis related genes were up-regulated after the onset of fruit ripening, this did not affect berry flavonol concentration at harvest . Similarly, hydroponic channel previous studies with Tempranillo grown under controlled conditions did not observed differences due to AMF inoculation on the total content of flavonol and anthocyanins in berry skins .

Flavonol composition was affected by treatments. Thus, HII grapevines increased quercetin and decreased syringetin contents in berry skins at harvest in accordance to a previous study . Indeed, it is known that AMF inoculation up-regulated phenyl-propanoid biosynthesis key genes in grapevines in response to pathogens . On the other hand, HI led to decreased contents of quercetins, laricitrins, kaempferols, syringetins, and isorhamnetins. Likewise, Martínez-Lüscher et al. found that in spite of the increase in O-methyl-transferase transcript level, methylated flavonols did not increase under water deficit. These authors suggested that given the higher affinity of OMT for quercetins, the lower concentration of quercetins under water deficit could act as a limiting factor for the synthesis of methylated forms, and our findings corroborated this hypothesis. Regarding anthocyanin composition, berry skins from HI grapevines showed lower contents of di-substituted anthocyanins than the ones of FI grapevines. It is well known that water deficit regulates the expression of key genes of the flavonoid pathway such as the flavonoid 30 -hydroxylase, flavonoid 30 ,50 -hydroxylase, and O-methyltransferase in red cultivars . Therefore, these decreased contents of disubstituted anthocyanins were likely explained by a different regulation of these genes when grapevines are subjected to water deficit. The role of AMF for enhancing phenolic compounds was reported in several studies with potted grapevines. Thus, AMF grapevines showed increased content of resveratrol, viniferins, and pterostilbene , total phenols and quercetin content , and total flavonoids in leaves of different grapevine varieties facing different biotic and/or abiotic stresses. Moreover, increased anthocyanin contents were reported in berries from grapevines grown under water deficit and warming conditions . Similarly, we found a strong relationship between the percentage of mycorrhizal colonization and some flavonoids .

The economic analysis data indicated that AMF inoculation and water management did not affect the cost of labor operations, in spite of irrigating with half amount may lead to decreases in yield. However, this came with reductions of the water footprint that have to be taken into account. It is noteworthy that extreme weather recorded in 2020 could modulate the effects described in this work. Moreover, the mycorrhizal extraradical mycelium coexists with soil microbial communities and the synergistic activity between the AMF, the bacterial communities, and the grapevine modulates the benefits of symbiosis on nitrogen fixation, P solubilization, and production of phytohormones, siderophores, and antibiotics . On the other hand, previous studies demonstrated that the microbiome of vineyards is shaped by cropping management , and little is known about whether these communities stimulate or suppress the extraradicular mycelium activity . Therefore, given the effect of AMF inoculation and different irrigation amounts had on grapevine physiology and berry composition, further studies should consider the potential effects of these management practices on vineyard soil living microbiota. Current research aimed to study how Merlot grapevines responded to AMF inoculation and different water amounts in their first productive year in situ. Our results highlighted the role of AMF inoculation for improving vegetative growth, photosynthetic activity, and water status of grapevines, especially when facing mild water deficits in field grown grapevines. Additionally, a strong relationship between the mycorrhizal colonization of roots and some flavonoids was found, corroboration the effect of AMF for regulating anthocyanin and flavonol metabolisms. Finally, although some berry quality traits and grapevine performance were improved by AMF inoculation under water deficit, AMF inoculation was not sufficient to avoid the yield losses due to water deficit in the first productive year of Merlot when facing a hyper-arid growing season.

It is noteworthy that these results may be affected by edaphoclimatic characteristics and living microbiota in vineyard soils, which should be taken into account before making the decision of inoculating the vineyard. Therefore, this study offer a starting point to assess the effect of AMF inoculation on young vines under real field conditions. However, benefits distinct from simple nutrition, such as phytochemicals have recently become clear. Diets rich in a plethora of phytochemicals can promote a healthy and diverse gut microbiota, reduce intestinal and systemic inflammation, and decrease the risk of colorectal cancer and type 2 diabetes melliThus. Some of these benefits can be observed around the world. Many parts of India have historically low colon cancer incidence rates. The Indian subcontinent has been continuously settled for millennia. Ancient cities in the Indus valley have been dated to the third and fourth millennia BC and some sites are even older. Archaeological evidence of grain cultivation, including several varieties of barley and wheat, has been found in excavations dated to the sixth millennium BC. Wheat is still a staple crop in northern India, and many other grains, including barley, were commonly cultivated and eaten until the 1950s, when wheat and white rice became dominant. Although the country has many diverse cultures, some customs remain common and conventional throughout the nation. One such tradition is the form of main meals where a large round platter, the thali, holds rice or bread and several smaller bowls, or katori, which hold a separate condiment or curry to be eaten with the rice or bread at the diner’s preference. Typical dishes include, but are not limited to, dal , yogurt , and assorted spices and vegetables. The development of agriculture early in its history has allowed India to develop rich traditions around food. These traditions have been deeply influenced by Ayurveda, the ancient Indian system of medicine. In Ayurvedic practice, food is a source of nourishment and medicine, used to both prevent and treat illness. Maintaining a proper balance of Ayurvedic elements through diet is considered an effective way to live a healthy liftestyle. According to the Ayurvedic principles, each meal should contain a balance of the six major flavors. This calls for the many small portions of a thali meal which also easily incorporate variety. A variety of flavors in a meal often indicates the presence of many classes of bio-active compounds . Although these substances may not be macronutrients, vitamins, or minerals, they still impact human health. Polyphenols are perhaps the largest class of bio-active compounds, containing subclasses such as flavonoids, isoflavones, stilbenes, lignans, and tannins. As a flavonoid subgroup, anthocyanins are included in this class. Anthocyanins are of interest in the food industry as nontoxic and water-soluble pigments, as most are colored red, purple, or blue, and many display antioxidant and anti-inflammatory activity. A class of phytochemicals called polyphenols is also found in virtually all plant foods, though their quantity may be reduced by preparation methods. Rich sources of anthocyanins include deeply colored fruits and vegetables, such as blueberries, eggplants, hydroponic dutch buckets and certain carrot and potato cultivars. Given that many phytochemicals exert anti-inflammatory activity via promoting gut bacterial diversity, there is a growing interest in a food-based approach to countering the growing epidemic of inflammation-promoted chronic diseases such as colon cancer.We have learned that no discussion of diet is complete without consideration of the intestinal microbiota. Trillions of bacteria, distributed throughout the gastrointestinal tract from mouth to anus, facilitate digestion and intestinal homeostasis. Structural factors greatly impact the overall makeup of each community. For example, low pH prohibits many pathogenic bacteria from colonizing the stomach and the upper small intestine. The depths of the large intestine, on the other hand, is an ideal habitat for many anaerobes. The gut microbiota is a dynamic community, composed of living organisms that can alter in response to diet, disease, and other environmental pressures. Changes in the intestinal microbiota were first correlated with illness in 1681 when Anton van Leeuwenhoek recorded that the microbial composition of his diarrhea differed from normal fecal samples. Since then, the intestinal microbiome has been closely studied to show how it can be implicated in a variety of conditions ranging from obesity to colon cancer. A great deal of investigation into microbiota has been accomplished in the last decade. Many of these observed changes result in an overall loss of bacterial diversity in the microbiota, indicating that species diversity is associated with health. However, the opposite may be true for cause-consequence relations, but not enough research has been brought to light. High-throughput technologies have driven advances in identifying the trillions of microbes and the metabolic functions that live in the colon.

This led to a critical insight that gut plays as dynamic of a role in metabolism as the liver. The proximity of these microbes to the intestinal mucosa and gut lymphoid Thissue explains the critical role they play in health and disease. Indeed, dysbiosis plays a significant role in the development of inflammatory bowel disease, obesity, and colon cancer. Emerging evidence suggests that diet can directly influence the content and composition of gut microbiota. Thus, understanding the complex interactions between diet, gut microbiota, and the host are crucial in prevention and treatment of chronic diseases that plague our society. Studies in murine models have shown rapid changes in the gut bacteria of mice being switched quickly from a standard diet to a high-calorie diet back to a standard diet. In humans, surveys show that diets high in fiber correlate with higher microbial diversity and reduced populations of Enterobacteriaceae, including Escherichia and Shigella species. Marked differences are also seen during consumption of animal- vs. plant-based diets. While nutrients in the diet will affect intestinal microbes, other substances present in food may also have an effect. For example, most anthocyanins are not absorbed into the bloodstream in the small intestine, and so they stay in the gastrointestinal tract until they reach the colon. There, they can affect the colonic microbiota in multiple ways. Firstly, anthocyanins have antioxidant activity that can reduce inflammation-induced oxidative stress on the gut bacteria. Secondly, anthocyanins are a potential carbon source, which bacteria can metabolize, resulting in increased growth of certain microbes. Lastly, bacterial metabolism of anthocyanins produces a variety of metabolite byproducts, some of which have antimicrobial effects on enteric pathogen species including Escherichia coli.Chronic intestinal inflammation is a hallmark of certain bowel disorders, such as ulcerative coliThis and Crohn’s disease, which are two major forms of inflammatory bowel diseases , and IBD is also considered a risk factor for colorectal cancer. In the latter, inflammation is generally low-grade but persists over a long period of time. Diet composition can promote or suppress chronic inflammation. Low-fiber high-calorie diets, which are typical in Western countries, may directly promote inflammation, or as already discussed, indirectly promote this through dysbiosis. Indeed, some dietary patterns associated with chronic inflammation are also linked to the reduction of total microbial diversity and imbalances in intestinal microbial groups. Furthermore, some bacteria, including E. coli, can flourish during low-grade inflammation, where thinning of the intestinal mucus layer occurs and allows for more direct interaction between the host’s cells and the intestinal bacteria.

Commercial standards of epicatechin, malvidin-3-O-glucoside, and quercetin-3-O-glucoside were used for the quantification of flavan-3-ols, anthocyanins, and flavonols, respectively.Cost estimates on labor operations and gross income per hectare were calculated based on yield and net returns per hectare . Water footprint was calculated as described by Zotou and Tsihrintzis . Briefly, for the green component of the WF , precipitation data during the growing season was obtained from the CIMIS Station and estimated as m3 /ha to obtain the total green consumed water volume . Then the value was divided by the yield expressed as ton/ha. The blue component of the WF was calculated with the total irrigation water amount that grapevines received per hectare, and this blue consumed water volume value was divided by the yield . The gray component of the WF was not calculated given that our experimental conditions avoided the use of fertilizers. Then, the total WF was estimated as the sum of green WF and blue WF. statistical analyses were performed in R-Studio version 3.6.1 for Windows. All the monitored parameters were fit in linear mixed-effect models by using the lmer function from lme4 package with AMF inoculation , irrigation treatment , and their combination as fixed factors, and replicate as random factor . Then, pairwise contrasts were conducted with function lsmeans from lsmeans package using the Kenward–Roger method and Tukey adjustment for p-values. Previously, for gas exchange parameters, stem water potential, mycorrhizal colonization, and flavonoid contents a mixed-effect model including sampling date as fixed factor was run . However, as the treatment effect seemed to be independent in the sampling date , sampling date was removed from the analysis to assess the effect of treatments for each sampling date. Finally, blueberry packaging box correlations between the percentage of mycorrhizal inoculation and flavonoid contents were calculated with the Pearson’s test using the same software.

The comparison between the growing season of the experiment and the reference data for the same period within the last 20 years showed that 2019–2020 was warmer and drier . Thus, average daily temperature was 0.5◦C higher, especially in August, which reached 1.8◦C more, and precipitation of 530 mm less compared to the average, hence, the 2020 growing season was an extreme year regarding temperature and rainfall. Native mycorrhizal colonization was determined before treatment application and no differences between them were observed . The mycorrhizal colonization intensity was analyzed after 3 months of treatment application to ensure the establishment of the mycorrhizal symbiosis, which frequently take place after 2–4 months of inoculation. Similar patterns in AMF colonization intensity were observed in both, 3 months after inoculation and at harvest, where roots from inoculated grapevines showed percentages of colonization values threefold higher than non-inoculated ones . In addition, we observed increased AMF colonization rates along the growing season as shows the significant effect of the sampling date and its interaction with the AMF inoculation . Relative mycorrhizal dependency index allows assessing the dependency of a crop on the mycorrhizal symbiosis to achieve its maximum growth at a given environmental condition. Under FI conditions, RMD values were lower than 100% indicating that the mycorrhizal association impairs the vegetative growth of grapevines; however, RMD values for HI conditions highlighted the role of the mycorrhizal symbiosis for improving grapevine growth under water deficit conditions . Grapevine vegetative growth was also monitored during the 2020 growing season by measuring the green pruning weight, trunk diameter, and leaf area . Measurements before treatment showed no differences between the different plants concerning trunk diameter , corroborating the effect of treatments modulating vegetative growth of vines. Irrigation amount was the main factor affecting both vegetative growth and yield, with grapevines subjected to HI decreasing them . However, as RMD reported AMF inoculation impair the grapevine growth estimated as trunk diameter and as green pruning weight when vines were FI, whereas under conditions, inoculated vines improved their growth . Finally, the leaf area to fruit ratio was not affected by treatments applied. The contents of minerals measured in leaf blades were not affected by AMF inoculation or applied water amount in our experiment . Plant water status was determined by monitoring the SWP each 2 weeks at noon during the growing season.

The SWP values ranged between −0.8 and −1.3 MPa at harvest suggesting that the amount of applied water was successful in reaching the SWP target during the growing season. Irrigation amount was the main factor affecting the water status of vines especially at the end of the growing season. However, before veraison AMF inoculation could increase the grapevine water status under HI conditions . The calculation of the seasonal integral of SWP showed the same pattern; hence, siSWP was mainly affected by irrigation system with HI plants being the most stressed vines . Gas exchange parameters monitored during the season are shown in Figure 3. Carbon assimilation rates increased through the growing season, and were affected by the interaction between AMF inoculation and irrigation amounts . Thus, FII plants showed the highest values of AN at fruit set and harvest, while FINI grapevines increased AN after veraison. Leaf evapotranspiration was slightly modified by treatments at the beginning on the season but no effect was observed later in the season. On the other hand, although no differences in instantaneous water use efficiency were recorded at harvest, AMF inoculated plants showed a better WUE during berry development and ripening . Finally, stomatal conductance was highly affected by the interaction between AMF inoculation and irrigation system during the whole season . Thereby, AMF inoculation of HI plants mitigated the reduction of gs . Primary metabolites and berry fresh weight are presented in Table 4. Must pH, TA, and TSS were not affected by treatments. However, BFW was modified by treatments; hence, AMF inoculation increased BFW of FI plants and decreased in HI . Flavonols and anthocyanins were monitored through berry ripening. The effect of AMF inoculation and irrigation systems on berry skin flavonol content and composition was modulated during the growing season as indicated by the significant interaction of treatments with the sampling dates . At mid ripening, the berry skin flavonol content increased in HINI grapevines . Similarly, quercetin-3-O-glucoside and laricitrin- 3-O-glucoside decreased with AMF inoculation under HIconditions . At harvest, myricetin and quercetin derivatives were the most abundant flavonols found in Merlot berry skins, accounting for more than 40% of the total flavonols. Irrigation treatment was the main factor affecting flavonol content and composition as indicated by the decrease in quercetin, laricitrin, kaempferol, isorhanmetin, and syringetin derivative contents in HI grapevines . It is noteworthy to highlight the increased content of quercetin-3-O-galactoside in HII grapevines . At mid ripening the main anthocyanin was cyanidin-3- O-glucoside, which accounted for ca. 20% .

The total anthocyanin content of Merlot berry skins was not affected by treatments but HI treatment decreased the contents of some anthocyanin derivatives . At harvest, the total anthocyanin content in Merlot berry skins was not affected by different treatments . Malvidin was the most abundant anthocyanin detected in Merlot berry skins , with contents ranged between 23.1% for HINI plants and 28.7% from FII but none of the malvidin derivatives were affected by treatments . The main changes in anthocyanin composition were due to irrigation treatments, thus, HI ledto decreased contents of cyanidin and peonidin derivatives . Finally, an analysis of the relationship between the percentage of AMF colonization and the main flavonoid contents was conducted . The intensity of the AMF colonization had a significant positive relationship with total cyanidins , total peonidins , and total quercetins . In the last decades, warming trends in viticulture areas have been described worldwide . Likewise, weather data recorded during 2020 growing season in Oakville, CA, United States , suggested more stressful conditions for grapevines comparing to the average of last 20 years, blueberry packaging containers challenging their production and quality. Indeed, a recent study based on climate indices suggested a reduction of 8,700 km2 for the Califtornia land suitable for grapevine cultivation by mid-21st century . Within this scenario, smart-farming techniques are mandatory for adaptation and mitigation to guarantee the future of the wine making industry and for reducing potential water conservation issues. Colonization analysis of Merlot grapevine roots indicated that AMF inoculated integrated with the native communities colonizing grapevine roots . Thus, we found that the percentage of mycorrhizal colonization was two to three fold higher in mycorrhizal inoculated treatments compared to non-inoculated ones. However, no differences in mycorrhizal colonization due to water amount received by plants were evident in accordance with a previous study conducted on fruit bearing cuttings . In contrast, a study conducted on own-rooted Cabernet Sauvignon field grapevines reported increased frequency of arbuscules and reduced fine root production when an additional water deficit was applied to the regulated deficit irrigation plot, suggesting that plants could compensate the lower density of fine roots in vines facing water deficit by increasing AMF colonization . These discrepancies between studies may be explained by the fact that grapevines responded to the degree of water deficit from the previous growing season. Thus, Schreiner et al. observed increased arbuscular colonization at bloom, before the onset of differences between the treatments they applied whereas under our experimental conditions, water amounts received by Merlot grapevines the previous season did not differ. AMF colonization data also confirmed the seasonality effect on mycorrhizal colonization and the reinforcement that AMF inoculation exerts on native mycorrhizal colonization . Without imposed water stress, AMF inoculation impaired vegetative growth as indicated in the RMD index. However, when grapevines were subjected to HI treatment, AMF-inoculated vines grew better as indicated by the RMD, green pruning, and trunk diameter. Nevertheless, leaf area was not enhanced after AMF inoculation according to previous studies , which would explain that AMF inoculation was not sufficient to avoid the yield loss due to HI treatment. It is well established that AMF inoculation enhances mineral nutrition of grapevines presumably by a greater exploration of soil by the external hyphal network of the AMF resulting in more efficient roots for obtaining nutrients from soils . Moreover, it was recently reported that the inoculation of grapevines with AMF under controlled conditions led to the upregulation of nutrient transport genes . In spite of the consensus about AMF enhancing grapevine nutrient uptake, contradictory results are reported about increased mineral nutrient content due to the symbiosis . Leaf or petiole mineral nutrient content might be useful for the diagnostic of soil mineral deficiencies allowing growers to manage them. However, concentration of mineral nutrients does not provide accurate information on nutrient uptake or allocation of nutrient in various organs . Therefore, although no differences on the mineral nutrient content in leaf blades were observed, mineral uptake was presumably enhanced by AMF inoculation given the growth promotion recorded in mycorrhizal plants under HI conditions. Furthermore, Balestrini et al. recently reported that although mineral nutrient uptake genes were upregulated after inoculation with different inoculants , the degree of upregulation differed between them, suggesting a specific response to a specific inoculum. Similarly, Nogales et al. did not find accumulation of minerals in grapevine leaves after AMF inoculation with the exception of P, which was enhanced and decreased after F. mosseae and R. irregulare inoculations, respectively. Grapevine water status monitored during the growing season showed that irrigation amounts were the main factor affecting the plant water status. Thus, according to previous work FI plants were maintained under well-watered conditions with values of midday SWP higher than −0.9 MPa and/or gs higher than 200 mmol m−2 s −1 . On the other hand, grapevines subjected to HI were not exposed to a severe water stress as they never reached values of SWP and gs lower than −1.5 MPa and 50 mmol m−2 s −1 , respectively, considered detrimental for grapevine development . We did not measure any SWP differences due to the AMF inoculation when plants were subjected to FI. However, within HI plants, AMF inoculation tended to result in higher SWP values in accordance with previous studies . Therefore, a higher AMF occurrence in the root zone has been related to improve water status of vines by increasing water uptake presumably by increasing the mycorrhizal structures, mainly arbuscules . Accordingly, we observed that photosynthetic performance of AMF inoculated Merlot grapevines was improved .