Horticulture

The identification of annotated and novel miRNAs was carried out applying a conservative and robust pipeline as described by Jeong et al. and Zhai et al. , and successfully deployed in various published studies . Shortly, in order to recognize the conserved miRNAs, all small RNAs sequenced in the libraries were initially compared against all annotated vvi-miRNAs deposited in miRBase . Subsequently, the whole set of small RNAs passed through the five filters designed according to the properties of validated plant miRNAs and their precursors , keeping track of known miRNAs throughout the filtering. The filters included, but were not limited to, minimum abundance threshold , size range , maximum hits to the grapevine genome , strand bias , and abundance bias [/total ≥ 0.7]. For each possible precursor found, the most abundant read was retained as the biologically active miRNA and in cases where both the 3′ -end and the 5′ -end reads were highly abundant , the two tags were kept. All the known vvi-miRNAs identified by the pipeline were manually inspected, to ensure that the tags identified as known miRNAs were assigned correctly to their actual precursor, and to retrieve the most abundant isoform within the tags mapping in each precursor.

Regarding the novel miRNA candidates identified using this pipeline, nft channel only those for which the most abundant tag was 20, 21, or 22 nt were retained. They were compared with all the known mature plant miRNAs in miRBase to identify homologs. Finally, novel candidates passed through a manual evaluation of precursor secondary structures, using the plant version of the UEA sRNA hairpin folding and annotation tool and the Mfold web server , with default settings.A miRNA was considered as “expressed” only when the values of both biological replicates were greater than or equal to the threshold set at 10 TP4M. We defined a miRNA as “vineyard-, cultivar-, or stage-specific” when it was expressed only in a given vineyard, cultivar or one specific developmental stage. Differentially expressed miRNAs were identified using the CLCbio Genomics Workbench using multiple comparison analysis. We loaded the total raw redundant reads from our 48 libraries in the CLCbio package and trimmed the adaptors, considering only reads between 18 and 34 nt. We annotated miRNAs against the user defined database, comprehending our set of 122 MIRNA loci and their corresponding mature sequences. For each library, the total counts of read perfectly mapping to the miRNA precursors was considered as the input of the expression analysis. Given the main focus of our work, we aimed at identifying miRNAs differentially expressed between the two cultivars in the same environment and developmental stage , or between the three vineyards in the same cultivar and in the same developmental stage . For this reason, we considered each developmental stage and we performed the Empirical Analysis of digital gene expression , an implementation of the “Exact Test” present in the EdgeR Bioconductor package, as implemented in CLCbio software and estimating tag wise dispersion with pairwise comparisons and setting the significance threshold to FDR-adjusted p ≤ 0.05.

The normalized reads of all miRNAs identified in this study and also the cluster abundances obtained from the static clustering analysis were submitted to another adhoc normalization [log10 or log10 ] for correlation analysis. This normalization was chosen because of the enormous range of abundance values that produced a logunimodal distribution and may cause significant biases in the correlation analysis when performed using TP4M or HNA values. After this addition, a value of zero still corresponds to zero of the log10 function, thus making consistent the comparisons among profiles. The dendrogram was generated using the function hclust and the Pearson correlation was calculated using the function cor in R, based on the log10 or log10 values for miRNAs and sRNA-generating loci respectively. Pearson’s correlation coefficients were converted into distance coefficients to define the height of the dendrogram. Heat maps were produced using MeV based on TP4M values of miRNAs abundance. The Venn diagrams were produced using the function vennDiagram in R, based on the miRNA list for each cultivar, environment and developmental stage.Small RNA libraries were constructed and sequenced for 48 samples of grapevine berries . We obtained a total of 752,020,195 raw redundant reads . After adaptors trimming, 415,910,891 raw clean reads were recovered, ranging from 18 to 34 nt in length . Eliminating the reads mapping to rRNA, tRNA, snRNA, and snoRNA sequences, 199,952,950 reads represented by 20,318,708 distinct sequences, i.e., non-redundant sequences found in the 48 libraries , were perfectly mapped to the V. vinifera PN40024 reference genome . The libraries were analyzed to assess the size distributions of mapped reads. Distinct peaks at 21- and 24-nt were observed in all the libraries. Consistent with previous reports in grapevine and other plant species , the 21- nt peak was the highest, comprising a higher proportion of redundant reads, whereas the 24-nt peak was less abundant.

A few exceptions regarding the highest peak in the small RNA size profile were observed: Ric_SG_ps had the highest peak at 24- nt whereas Mont_CS_ps and Mont_SG_bc did not show a clear difference between the 21- and the 24-nt peak. Using the Pearson coefficients we observed a strong association between the replicates as indicated by the high coefficients . To facilitate access and utilization of these data, we have incorporated the small RNAs into a website . This website provides a summary of the library information, including samples metadata, mapped reads, and GEO accession numbers. It also includes pages for data analysis, such as quick summary of the abundances of annotated microRNAs from grapevine or other species. Small RNA-related tools are available, for example target prediction for user-specified small RNA sequences and matching criteria. Finally, and perhaps most importantly, a customized browser allows users to examine specific loci for the position, abundance, length, and genomic context of matched small RNAs; with this information, coupled with the target prediction output, users can develop and assess hypotheses about whether there is evidence for small RNA-mediated regulation of grapevine loci of interest.In order to investigate whether the overall distribution and accumulation of small RNA is affected by the interaction between different V. vinifera genotypes [Cabernet Sauvignon and Sangiovese ] and environments [Bolgheri , Montalcino and Riccione ], we investigated the regions in the grapevine genome from where a high number of small RNAs were being produced , by applying a proximity-based pipeline to group and quantify clusters of small RNAs as described by Lee et al. . The nuclear grapevine genome was divided in 972,413 adjacent, non-overlapping, hydroponic nft fixed-size windows or clusters. To determine the small RNA cluster abundance, we summed the hits-normalized-abundance values of all the small RNAs mapping to each of the 500 bp clusters, for each library . To reduce the number of false positives, we considered a cluster as expressed when the cluster abundance was greater than the threshold for a given library, eliminating regions where few small RNAs were generated, possibly by chance. Libraries from bunch closure, representing green berries, and 19 ◦Brix representing ripened berries, where used in this analysis. From the 972,413 clusters covering the whole grapevine genome, 4408 were identified as expressed in at least one sample. As showed in Figure 1, CS-derived libraries have a higher number of expressed clusters when compared to SG-derived libraries of the same developmental stage and from the same vineyard. The exceptions were the Sangiovese green berries collected in Riccione and Sangiovese ripened berries collected in Montalcino, which have a higher number of expressed clusters than the respective CS ones. The two cultivars show a completely different small RNA profile across environments. When Cabernet berries were green, a higher number of sRNA-generating regions were found active in Bolgheri than in Montalcino and Riccione. Differently, ripened berries had the highest number of sRNA producing regions expressed in Riccione, while Bolgheri and Montalcino show a similar level of expressed clusters . Sangiovese green berries instead show the highest number of active sRNA-generating regions in Riccione, and this number is twice the number found in Bolgheri and Montalcino that is similar. Ripened berries collected in Montalcino and Riccione show almost the same high level of sRNA-generating clusters, whereas those collected in Bogheri present a lower number . We also noted that when cultivated in Bolgheri, neither Cabernet Sauvignon or Sangiovese change dramatically the number of expressed clusters during ripening, while in Riccione Cabernet Sauvignon shows a 2-fold increase of sRNAproducing clusters, which is not observed in Sangiovese. Next, the small RNA-generating clusters were characterized on the basis of the genomic regions where they map, i.e., genic, intergenic and transposable elements. In general, when the berries were green, the numbers of sRNA-generating loci located in genic and intergenic regions were roughly equal in all environments and for both cultivars, except for Sangiovese berries collected in Riccione, which show a slight intergenic disposition of sRNA-producing regions . Differently, in ripened berries on average 65% of the sRNA-generating loci were in genic regions, indicating a strong genic disposition of the sRNA-producing clusters . The shift of sRNA-producing clusters from intergenic to mostly genic is more pronounced in Cabernet Sauvignon berries collected in Riccione, with an increase of approximately 20% of expressed clusters in genic regions when berries pass from the green to the ripened stage. When comparing the clusters abundance among libraries, we found that 462 clusters were expressed in all libraries. The remaining 3946 expressed clusters were either shared among groups of libraries or specific to unique libraries. Interestingly, 1335 of the 4408 expressed clusters were specific to Riccione-derived libraries . The other two environments showed a much lower percentage of specific clusters, 263 and 140 in Bolgheri and Montalcino respectively . Comparing the expressed clusters between cultivars or developmental stages, we did not observe a similar discrepancy of specific clusters toward one cultivar or developmental stage; roughly the same proportion of specific clusters was found for each cultivar and for each developmental stage . Among the 1335 specific clusters of Riccione, 605 were specific to Cabernet Sauvignon ripened berries of and 499 to Sangiovese green berries. Other smaller groups of expressed clusters were identified as specific to one cultivar, one developmental stage or also one cultivar in a specific developmental stage. When comparing the expressed clusters with the presence of transposable elements annotated in the grapevine genome , we noticed that approximately 23% of the sRNA-generating regions were TE-associated. Sangiovese green berries from Riccione have the highest proportion of TE-associated expressed clusters, while Cabernet Sauvignon ripened berries also from Riccione show the lowest proportion of TE associated expressed clusters. Sangiovese berries have the highest percentage of expressed clusters located in TE when cultivated in Riccione, compared to the other two vineyards. Interestingly, Cabernet Sauvignon berries show the lowest proportion of TE-associated clusters when growing in Riccione , independently from the berry stage. In all the libraries, Long Terminal Repeat retrotransposons were the most represented TE. More specifically, the gypsy family was the LTR class associated with the highest number of sRNA hotspots. The other classes of TE associated with the sRNA-generating regions can be visualized in Figure 3B.To determine the global relationship of small RNA-producing loci in the different environments, cultivars and developmental stages, we performed a hierarchical clustering analysis. As showed in Figure 4, the libraries clearly clustered according to the developmental stage and cultivar and not according to the environments. Ripened and green berries had their profile of sRNA-generating loci clearly distinguished from each other. Inside each branch of green and ripened samples, Cabernet Sauvignon and Sangiovese were also well separated, indicating that, the cultivar and the stage of development in which the berries were sampled modulate the outline of sRNA-producing loci more than the environment. Notwithstanding the evidence that developmental stage and variety have the strongest effect in terms of distinguishing samples clustering, we were interested to verify the environmental influence on small RNA loci expression in the two cultivars. Thus, for each sRNA-generating cluster we calculated the ratio between cluster abundance in Cabernet Sauvignon and Sangiovese in each environment and developmental stage, thereby revealing the genomic regions with regulated clusters, considering a 2-fold change threshold, a minimum abundance of 5 HNA in each library and a minimum sum of abundance of 30 HNA . Figure 5 shows how different environments affect the production of small RNAs.

At the turn of the century, Colorado emerged as a purple state in presidential politics and showed evidence that state level office holders were also trending in a more liberal direction . Some argue that the migration of liberals to Colorado from places like California and elsewhere essentially created “a new Western community – a community of creative classes, childless households and urban professionals who are more likely to vote Democrat than the rural conservatives they are increasingly outnumbering” . However, even as the state shifted to prefer Democratic presidential candidates beginning in 2008, the number of registered Republicans exceeded the number of registered Democrats until 2016. At the time of the 2014 midterm elections, unaffiliated voters constituted the largest voting bloc at 34.8% of the electorate, followed by 32.9% Republican, and 30.9% Democratic. Reforms to the state’s presidential and congressional primary rules passed in 2016 adopted a presidential primary and permitted unaffiliated voters with the opportunity to participate in either the Democratic or Republican primary.

These reforms resulted in an immediate increase in the share of unaffiliated voters, nft system which continues to grow. The most recent figures from the Secretary of State’s office indicate that nearly half of the state’s active voters are registered as unaffiliated. Registered Democrats and Republicans constitute an additional 26% and 23.3% of the electorate, respectively . This near parity in major-party voter registration masks the share of the electorate inclined to support Democratic candidates and liberal policy reforms. A recent survey of Coloradoans suggests that unaffiliated voters trust the Democratic party by large margins over its Republican counterparts. On the issues of abortion and climate change, unaffiliated voters trusted the Democratic party by margins of about 45 percentage points. Voters also trusted Democrats by wide margins on the issues of health care , protecting public lands , improving public education , and supporting the working- and middle-class . Respondents also slightly preferred Democrats on the issues of inflation/cost of living and improving the economy . The single issue where unaffiliated voters expressed more trust in Republicans was reducing crime and promoting public safety . As Democrats continue to expand their political power, Republicans have fewer options available to obstruct liberal policy reforms. Democrats successfully repealed the death penalty amidst filibuster threats and near unanimous Republican opposition in 2020, for example.

According to an analysis of ideological polarization in state legislatures over time, Shor and McCarty conclude that Colorado surpassed California as the most ideologically polarized legislature in the nation in 2017 . Figure 1 shows estimates of party ideology in select states using responses to the National Political Awareness Test . The chasm between parties is wide in many Western states including Arizona, California, New Mexico, and Washington, but the rapid polarization of the Colorado legislature across a relatively short period of time is particularly notable. A steady march to the right has positioned the Colorado Republican party as the most conservative in the West, and by 2020, the California Democrats were the only Western party ranked as more liberal than Colorado Democrats . Consistent gains averaging one seat in the Senate and 2.4 seats in the House across recent election cycles has positioned the Democratic party on the cusp of super majority status in both chambers. Democrats have viewed these large majorities as a mandate from the electorate to advance a progressive agenda alongside like minded Democratic governors. The parties remain deeply divided on contentious issues such as abortion, gun control, health care, and election administration.While a unified majority has the votes to impose its will on a numerically disadvantaged minority party, the Colorado General Assembly has a history of protecting minority rights. In recent terms, Republicans have used dilatory tactics to slow progressive policy change including requests to read bills in their entirety. This trend began over objections to Democratic efforts to impose greater regulations on the extraction industry.

In response to more frequent Republican motions to read the full text of bills, sometimes thousands of pages long, the Democrats attempted negotiation, but ultimately decided to have multiple computers read the text aloud at an intelligible speed of about 650 words per minute. In Markwell v. Cooke the Colorado Supreme Court held by a 4-3 vote that the “cacophony” method to read bills in a manner that produced “unintelligible sounds” for hours at a time failed to comply with article V, section 22 of the state’s constitution .3 This ruling emboldened Republicans to continue using stalling tactics to obstruct the Democratic majority. After gaining additional seats in the 2022 elections, Democrats sought to advance several controversial measures over considerable Republican opposition. Although the legislature has a norm of unlimited debate, House Rule 14 permits a majority vote as sufficient to limit further debate on a bill to a minimum of one hour following a successful vote. In the past neither party seemed inclined to invoke Rule 14 to end debate, in part because majority party leaders recognized that they may find themselves in the minority at some point in the not so distant future. However, with substantial Republican opposition to gun control reform and greater confidence that Democrats can maintain their majorities, Democrats employed the rarely used cloture-type mechanism to advance progressive gun control reforms. These included increasing the minimum age to purchase firearms to 21-years-old , imposing a 3-day waiting period for gun purchases , expanding the criteria for making reports under the state’s “red-flag” law , and imposing greater liability for firearms manufacturers . Justifying the decision to invoke Rule 14 for the first time in at least 10 years after enduring legislative debates that at times extended overnight into the early morning, House Speaker Julie McCluskie said, “Filibustering and delay tactics, by having bills read at length, is not why we were voted into office. We were voted into office to consider and debate policy. At the time we invoked House Rule 14, we wanted to drive more productive conversations” . Health care reforms regarding reproductive rights and gender-affirming health care coverage likewise involved bitter partisan conflict and further Republican delays. Since the existence of term limits has restricted the time legislators may serve, few current Democrats have institutional memories of serving in the minority party. The next session of the General Assembly will have zero Democratic lawmakers who previously served in the minority party. This collective inexperience serving as a minority party likely contributed to a greater willingness among Democrats to impose their will on numerically disadvantaged conservatives. With less fear of a tit-for-tat retribution in the near-term future, Democrats proceeded to invoke Rule 14 more than a dozen times in 2023 to advance their policy goals on a range of issues deemed worthy of such norm breaking.Governor Polis submitted a 2024–2025 budget proposal of $15.9 billion in General Fund spending to the General Assembly on November 1, 2023. This General Fund spending balance represents a 6.0% increase from the prior year’s enacted budget. The budget letter emphasizes Polis’ spending priorities pertaining to “five bold goals for the second term,” which includes affordable housing, hydroponic gutter public safety, health care, property taxes, and renewable energy . Public polling data suggest many Coloradoans have economic concerns related to inflation, cost of living, affordable housing, and property taxes . Housing affordability is an issue that resonates across the state but especially across the Front Range and in mountain communities. The presentation of the governor’s budget to the legislature highlights many areas of investment in this area, led by an effort to develop “More Housing Now.”

Seeking to promote greater housing affordability, the governor’s budget proposes several key initiatives to this end including a $136.7 million housing package to promote development and produce a greater supply of housing units. Governor Polis also proposes $65 million in tax credits and funds for local governments to develop new housing near mass transit nodes. The budget further proposes an additional $18 million and $16 million to support local development of accessory dwelling units and further tax credits for new housing units, respectively. As it was when he first took office, education remains among Polis’ top priorities. The FY 24–25 budget proposes new investments of $561 million in primary and secondary education, which would increase per pupil funding by about $700. Another notable investment in education this year is $141.3 million to fully eliminate the budget stabilization factor to fully fund K-12 education. This milestone represents the elimination of the budget stabilization factor for the first time in nearly 15 years. Polis’ budget also seeks to further advance a key campaign pledge from his first run for governor—providing universal pre-kindergarten for every child in the state. Polis signed a bill in 2022 providing all 4-year-olds 10 hours per week of tuition-free preschool in public schools, churches, or childcare centers. The current budget proposes an additional investment of $4.3 million to support the universal pre-K program as well as $8 million to further support science education programming in public schools. Regarding higher education, the budget proposes $33.4 million to colleges across the state to prevent tuition increases that exceed half of the inflation rate. Additional investments of about $9 million are devoted to student scholarships, financial aid, and various student support programs. Another priority in the governor’s budget is public safety. According to data from the National Insurance Crime Bureau, Colorado had the highest rate of automobile thefts in the nation in 2020 and 2021 . The state is also ignominiously among the national leaders in catalytic converter theft, which spurred police departments to offer complementary catalytic converter theft prevention kits to the public. The budget allocates $14.4 million for further auto-theft prevention efforts, and $7.5 million for additional crime prevention programs at the community level. Other spending proposals to promote public safety include nearly $40 million for evidence based crime prevention to “reduce auto theft, protect communities against targeted violence, support community-led crime prevention, leverage new technology to prevent crime, helpvictims recover, and prevent gun violence” . The budget also proposes $1 million to promote Department of Corrections efforts to reduce recidivism. Regarding climate change and environmental protection, the Governor’s Office reaffirmed its commitment to reach 100% renewable energy by 2040 with its recent unveiling of a “Roadmap to 100% Renewable Energy by 2040.” The policy goals in the administration’s roadmap included modernizing the public utilities commission, supporting local government innovation, developing green jobs, promoting energy efficiency, saving consumers money, encouraging public transportation use, incentivizing zero-emission vehicles, and moving toward zero emission buildings. The budget proposal includes increased funding for climate and environmental projects in the Denver metro area as well as rural parts of the state including $14 million to improve air quality, $10 million to support sustainable agricultural practices, and $2 million to replace gas-powered landscaping equipment owned by the state with electric alternatives.Given the wide ideological gulf between the Democratic and Republican parties in Colorado, it may come as a surprise that large majorities of enacted legislation in recent years passed with bipartisan support. Nearly 95% of bills passed in 2021 and 2022 had bipartisan support. However, partisan divisions became more pronounced recently as Democratic majorities sought to advance a number of controversial progressive reforms. The percentage of bipartisan enactments decreased to 88% in 2023 and coincided with a greater proportion of measures enacted by a party line vote. The proportion of party line votes doubled from the prior year to 6.8% in 2023, while the percentage of bills with zero Republican support similarly increased to reach nearly 12% . Greater ideological polarization and divergent partisan preferences on hot-button issues produced greater acrimony between the parties, which was likewise evident during this year’s budgetary debate. An analysis of voting behavior during the last term concluded that Democrats passed considerably more bills with zero Republican support in 2023. However, intra-party divisions occasionally manifested whereby “moderate Democrats sided with Republicans in voting ‘no’ on some more liberal measures, while progressive Democrats sometimes voted against their more moderate colleagues, especially on criminal justice bills” . At least four bills with sufficient Democratic deffections required at least some Republican support . This was not the case with the state budget as Democrats demonstrated more party unity and few Republicans voted in favor. According to Senator Rachel Zenzinger who serves on the Joint Budget Committee one unique aspect to begin the 2024 budget negotiations was a discrepancy in the state’s population estimate from the U.S.

As presented and described in Fig. 6.7A-C, AFM imaging reveals topographic defects precisely aligned with the regions in the Chern magnet in which magnetism has been destroyed. There are two clearly distinct distributions of defects, with thicknesses that differ by about an order of magnitude. It is possible that these correspond to bubbles between two distinct pairs of layers of the stack. Another possibility is that partial oxidation or deliquescence of the MoTe2 crystal has occured. This crystal is indeed air and moisture sensitive, and degradation can happen even inside a glovebox, as illustrated for a CrI3 crystal in Fig. 6.7D-F. Whatever issue is generating this disorder, it will likely be necessary to resolve it in order to fabricate more sophisticated devices based on this Chern magnet.As in three dimensional crystals, many two dimensional crystals have multiple allotropes that are stable under different conditions. Trilayer graphene is such a material. We label multilayer grapheneallotropes using letters that refer to the relative positions of atoms of different layers, projected onto the two dimensional plane. We have already encountered ABA trilayer graphene in the introduction, and this material has atoms in the third layer aligned to atoms in the first layer. At room temperature and pressure the ABA stacking order is preferred, hydroponic dutch buckets but trilayer graphene has a metastable allotrope, ABC trilayer graphene, that can either be prepared or found naturally occurring.

In ABC trilayer graphene atoms in the third layer are aligned neither with the first nor with the second layer. ABC trilayer graphene has band structure that differs significantly from ABA trilayer graphene, and these differences have important consequences for its properties.The band structure of ABC trilayer graphene at two different displacement fields is illustrated in Fig. 7.1. In the absence of a displacement field, the system is metallic at all electron densities. When a large displacement field is applied to the system, it becomes a band insulator when the Fermi level is tuned between the two resulting bands. This is the regime of displacement field that we will be discussing. ABC trilayer graphene has extremely weak spin-orbit coupling, so the spin degree of freedom is present and more or less completely orthogonal to electronic degrees of freedom, contributing only a twofold degeneracy to the band structure. Just like most other allotropes of graphene, ABC trilayer graphene has valley degeneracy, and this produces an overall fourfold energetic degeneracy of its band structure. This is illustrated in Fig. 7.2. As is abundantly clear from these plots, the bands present in ABC trilayer graphene are not flat; they have extremely large bandwidths. However, the bands do satisfy the flat-bottomed band condition, and as a result we can expect these systems to be able to spin- and valley-polarize without paying significant kinetic energy costs.

A schematic of the device we will discuss is presented in Fig. 7.3A. This device allows us to perform several different experiments: we can tune the electron density and displacement field in the ABC trilayer graphene layer, we can measure in-plane electronic transport , and we can measure the out-of-plane capacitive conductivity as a function of electron density and displacement field. Data extracted from this contrast mechanism is presented in Fig. 7.3B. This dataset is restricted to the hole band; i.e., the bottom band in all of the plots we have so far encountered. Sharp features in this dataset correspond to spontaneous symmetry breaking; these features are marked with the numbers and . The right side of this plot, labelled with an electron density of zero, corresponds to charge neutrality in this system and lies in the gap of the band insulator. Therefore and both correspond to situations in which the hole band is very slightly filled. The valley and spin subbands of ABC trilayer graphene are presented in schematic form in Fig. 7.4A in the absence of electronic interactions. When we tune the Fermi level into these bands and activate interactions, we cannot produce a gap- the bandwidths of these bands are far too high- but we can produce full spin or valley polarization, as illustrated in Fig. 7.4B. The precise situations in which we find this system at and are presented in Fig. 7.4C and D; these situations correspond repsectively to full spin polarization but no valley polarization in and full spin and valley polarization in . Valley polarization couples strongly to transport, generating a large anomalous Hall effect and ferromagnetic hysteresis, as presented in Fig. 7.4E.

Although these magnets occur in an atomic crystal, they are composed entirely of electrons we have forced into the system with an electrostatic gate, and as a result we can expect their magnetizations to be considerably smaller than fully spin-polarized atomic crystals. We will use the nanoSQUID microscope to image these magnetic phases. An optical image of the ABC trilayer graphene device used to produce data for the publications is presented in Fig. 7.5A. A black dashed lineoutlines the region we will be imaging using the nanoSQUID microscope. A nanoSQUID image of this region using AC bottom gate contrast is presented in Fig. 7.5B. This magnetic image was taken in the same phase in which we observe magnetic hysteresis, as presented in Fig. 7.4E. Clearly the system is quite magnetized; we also see evidence of internal disorder, likely corresponding to bubbles between layers of the heterostructure. We can park the SQUID over a corner of the device and extract a density- and displacement field-tuned phase diagram of the magnetic field generated by the magnetization of the device; this is presented in Fig. 7.5C. Electronic transport data of the same region is presented in Fig. 7.5D. The spin magnet has only a weak impact on electronic transport, but the valley ferromagnet couples extremely strongly to electrical resistance. The system also supports a pair of superconductors, including a spin-polarized one; these phases are subjects of continued study. Capacitance data over the same region of phase space is presented in Fig. 7.5E.The first systems with nonzero Chern numbers to be discovered were systems with quantum Hall effects. Quantum Hall insulators behave a lot like Chern magnets but are generally realized at much higher magnetic fields, and Berry curvature in these systems comes from the applied magnetic field, not from band structure. The fact that resistance in these materials is an intrinsic property and not an extrinsic one had implications for metrology that were immediately obvious to the earliest researchers that encountered the phenomenon. All of these devices have resistances that depend only on fundamental physical constants, so a resistance standard composed of these materials need not obey any particular geometric constraints, and can thus be easily replicated. The case for quantum Hall resistance standards was strong enough for the the National Institute for Standards and Technology to rapidly adopt them, and today the Ohm is defined by a graphene quantum Hall resistance standard at NIST.There are some downsides to the quantum Hall resistance standard. The modern voltage standard is a superconducting integrated circuit known as the Josephson voltage standard; it uses Shapiro steps to relate the absolute size of a set of voltage steps to a frequency standard. Because the voltage standard and resistance standard are independently fixed to physical phenomena, bato bucket current standards are necessarily defined by the relationship between these two different standards. Unfortunately, the superconducting integrated circuits used as Josephson voltage standards must be operated in very low ambient magnetic field, because large magnetic fields destroy superconductivity.

This makes them incompatible with the graphene quantum Hall resistance standard, which must operate in large magnetic fields, generally B > 5T. This is a surmountable problem- in practice it is handled by storing the two standards in different cryostats, or with significant magnetic shielding between them- but the significant distance separating the standards reduces the precision with which the current standard can be defined with respect to our current resistance and voltage standards. One possible way to resolve this conflict is to replace the quantum Hall resistance standard with a Chern magnet resistance standard. Chern magnets show quantized anomalous Hall effects at low or zero magnetic field, meaning they can be installed in very close proximity to Josephson voltage standards in calibration cryostats. Unfortunately, doped topological insulators have such small band gaps that even at the base temperatures of dilution fridges, there is enough thermal activation of electrons into the bulk to limit the precision of quantization of the quantized anomalous Hall effect in these systems. This made the class of Chern magnets discovered in 2013 unsuitable as replacements for the graphene quantum Hall resistance standard. Since intrinsic Chern magnets have now been discovered, and are observed to have band gaps considerably exceeding those of doped topological insulators, it might make sense to replace the graphene quantum Hall resistance standard with an intrinsic Chern magnet resistance standard. The ease of replication of the fabrication process of MoTe2/WSe2 makes that material particularly intriguing as a candidate material for a new resistance standard, but over the past few years new intrinsic Chern magnets have been discovered almost every year, so we may soon be discussing much better materials for this application. In any case, it seems possible and perhaps even likely that Chern magnets will supplant quantum Hall systems as resistance standards in the near future.For decades, magnetic memories dominated information storage technology. Magnetic storage media are robust, do not require continuous access to power, survive high temperatures and extreme radiation environments, and are relatively cheap to manufacture. Hard drives, cassette tapes, floppy disks, and other legacy technologies leveraged the many advantages of magnetic information storage to fuel an explosion in affordable information storage, facilitating mass market access to movies, music, and personal computing. Since the heyday of these technologies, however, magnetic information storage has fallen out of favor, for one simple reason: magnetic bits cannot be easily written electronically. Legacy mag-netic storage media address magnetic bits mechanically, which limits their maximum speed; modern flash memories can access data much faster precisely because each bit can be written and read electronically.Of course, that fact didn’t take away the many advantages of magnetic memories, and magnetic memories still persist in a variety of niche applications that depend particularly strongly on one of these advantages. Many computers destined to spend their lives in space still use hard drives, and sensors designed to operate over a wide range of temperatures and with intermittent access to power often use non-volatile magnetic memories as well. This has led researchers to search for phenomena and device architectures that allow magnetic order to be switched either with electrical currents or electrostatic gates. Until recently, the best technology available capable of electronic switching of magnetism used spin-orbit torques. In a spin-orbit torque device, current through a system with a strong spin Hall effect pumps spin into a separate magnet, which is eventually inverted by the torque exerted by those spins. This technology has matured considerably over the past few years, producing a cascade of new records for low current density magnetic switching and even a few consumer products in the memory market. The discovery of the first intrinsic Chern magnets produced a fascinating surprise for this field. The exotic orbital magnet in twisted bilayer graphene was found to be switchable with extremely small pulses of current, and the resulting current-switchable magnetic bits displaced previously realized spin-orbit torque devices as the ultimate limit in low-current control of magnetism. A flurry of theoretical investigation of these systems followed, dedicated primarily to identifying and generalizing the mechanism underlying current control of magnetism in these systems. A few years later, AB-MoTe2/WSe2 joined twisted bilayer graphene, with a similarly small magnetic switching current. In the intervening time, a new phenomenon had been observed- switching of a Chern magnet with an electrostatic gate, in twisted monolayer/bilayer graphene. All of these phenomena represent newly discovered and now more or less well understood mechanisms for controlling magnetic bits electronically, and by the performance metrics used in the literature they reign supreme. Several electronic switching phenomena known in intrinsic Chern magnets are summarized in Fig. 8.3. Chern magnets differ from the magnetic materials used in more traditional magnetic memories in a wide variety of intriguing ways other than their electronic switchability.

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, growing strawberries hydroponically 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. A variety of techniques have been developed for preparing atomically thin flakes from van der Waals materials, but by far the most successful has been scotch tape exfoliation. In this process, a chunk of a van der Waals crystal is placed on a piece of scotch tape. This separates the chunk of van der Waals crystal along its cleavage planes into two pieces of comparable size on opposite sides of the piece of tape. This process is repeated several times, further dividing the number of atomic layers in each chunk with each successive repetition. If we assume we are dividing the number of atomic layers in each piece roughly in half with each round, after N repetitions the resulting crystals should have thicknesses reduced by a factor of 2 1 N . This is enough to reduce each flake to atomic dimensions after a small number of repetitions of the process. This process is, of course, self-limiting; once the flakes reach atomic dimensions, they cannot be further subdivided. Graphite can be exfoliated through this process into flakes one or a few atoms thick and dozens of microns in width, a width-to-thickness aspect ratio of 105 .

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. It also depends on very strong in-plane bonds within the material, which must support the large stresses associated with reaching such high aspect ratios; materials with weaker in-plane bonds will rip or crumble. In practice these materials are almost always processed further after they have been mechanically exfoliated, and the preparation process typically begins when they are pressed onto a silicon wafer to facilitate easy handling. Samples prepared in this way are called ‘exfoliated heterostructures.’ It is of course interesting that this process allows us to prepare atomically thin crystals, but another important advantage it provides is a way to produce monocrystalline samples without investing much effort in cleanly crystallizing the material; mechanical separation functions in these materials as a way to separate the domains of polycrystalline materials. Graphene was the first material to be more or less mastered in the context of mechanical exfoliation, but a variety of other van der Waals materials followed, adding substantial diversity to the kinds of material properties that can be integrated into devices composed of exfoliated heterostructures. Monolayer graphene is metallic at all available electron densities and displacement fields, but hexagonal boron nitride, or hBN, is a large bandgap insulator, making it useful as a dielectric in electronic devices.

Exfoliatable semiconductors exist as well, in the form of a large class of materials known as transition metal dichalcogenides, or TMDs, including WSe2, WS2, WTe2, MoSe2, MoS2, and MoTe2. Exfoliatable superconductors, magnets, and other exotic phases are all now known, and the preparation and mechanical exfoliation of new classes of van der Waals materials remains an area of active research. Once two dimensional crystals have been placed onto a silicon substrate, they can be picked up and manipulated by soft, sticky plastic stamps under an optical microscope. This allows researchers to prepare entire electronic devices composed only of two dimensional crystals; these are known as ‘stacks.’ These structures have projections onto the silicon surface that are reasonably large, but remain atomically thin- capacitors have been demonstrated with gates a single atom thick, and dielectrics a few atoms thick. Researchers have developed fabrication recipes for executing many of the operations with which an electrical engineer working with silicon integrated circuits would be familiar, including photolithography, etching, and metallization. I think it is important to be clear about what the process of exfoliation is and what it isn’t. It is true that mechanical exfoliation makes it possible to fabricate devices that are smaller than the current state of the art of silicon lithography in the out-of-plane direction. However, these techniques hold few advantages for reducing the planar footprint of electronic devices, so there is no meaningful sense in which they themselves represent an important technological breakthrough in the process of miniaturization of commercial electronic devices. Furthermore, and perhaps more importantly, it has not yet been demonstrated that these techniques can be scaled to produce large numbers of devices, and there are plenty of reasons to believe that this will be uniquely challenging. What they do provide is a convenient way for us to produce two dimensional monocrystalline devices with exceptionally low disorder for which electron density and band structure can be conveniently accessed as independent variables. That is valuable for furthering our understanding of condensed matter phenomena, independent of whether the fabrication procedures for making these material systems can ever be scaled up enough to be viable for use in technologies.The properties of crystals differ from the properties of atoms floating in free space because the atomic orbitals of the atoms in a crystal are close enough to those of adjacent atoms for electrons to hop between atoms. The resulting hybridization of atomic orbitals produces quantum states delocalized over the entire crystal with the capacity to carry momentum. This situation is shown in schematic form in Fig. 1.9A. For quantum states delocalized over the entire crystal, growing tomatoes hydroponically position ceases to be a useful basis. Instead, under these conditions we label electronic wave functions by their momenta, kx and ky. The atomic orbitals that prior to hybridization had discrete energy spectra now have energy spectra given by discrete functions of momentum, f. We call these functions electronic bands.Electrons loaded into the electronic bands of a two dimensional crystal will occupy the quantum states with the lowest available energies, so we can specify a maximum energy at which we expect to find electrons for any given electron density. We call that energy EF , the Fermi level. We can raise the Fermi level by adding additional electrons to the crystal, as shown in Fig. 1.9B. We have already discussed how two dimensional crystals naturally allow for manipulation of the electron density, and thus the Fermi level. We have also already discussed how the application of an out-of-plane electric field to a two dimensional crystal will change the structure of the atomic orbitals supported by that crystal.

It naturally follows that atomic orbitals so modified will produce different electronic bands, as shown in Fig. 1.9C. It is relatively straightforward to compute how electronic bands will respond to the application of a displacement field. We will be using the momentum and energy basis for the rest of this document; this basis is known as momentum space. The simplest experiment we can perform to probe the electronic properties of a two dimensional crystal in this geometry is an electronic transport experiment, in which a voltage is applied to a region of the crystal with another region grounded, so that electrical current flows through the crystal. We can check if the crystal supports any electrical transport at all, and if it does we can measure the electrical resistance of the crystal this way, in close analogy to how this is done for three dimensional crystals. Crystals will only accept and thus conduct electrons if there are available quantum states at the Fermi level; we call these crystals metals , and they can be identified in band structure diagrams by the intersection of the Fermi level with an electronic band . Crystals without empty quantum states at the Fermi level will not accept and conduct electrons , and they can be identified in band structure diagrams with crystals for which the Fermi level does not intersect with an electronic band . There exists a variety of other experiments we can perform on two dimensional crystals in order to understand their properties. Two dimensional crystals can support electronic transport in the in-plane direction if they are metals, as shown in Fig. 1.10. Capacitors can also support electronic transport in the out-of-plane direction, as long as that electronic transport occurs at finite frequency. The same structure that we use to modify the electron density and ambient out-of-plane electric field of a two dimensional crystal can also be used as a capacitive AC conductor, as illustrated in Fig. 1.11A. The conductance will depend only on the frequency at which an AC voltage is applied and the geometry of the parallel plate capacitor. However, if a two dimensional crystal is added in series, the capacitance of the top gate to the bottom gate may be substantially modified. If the two dimensional crystal is an insulator, electric fields will penetrate it and the capacitance between the two gates will not change. However, if the two dimensional crystal is a metal it will accept electrons and cancel the applied electric field, dramatically reducing the capacitance between the top and bottom gates and neutralizing the AC current through the capacitor. This technique can be used to measure the electronic properties of specifically the bulk of a two dimensional crystal; it is the property that was both calculated and measured in Fig. 1.2C and D. These two techniques are the bread and butter of the experimental study of two dimensional crystals, because they require only the ability to create stacks of two dimensional crystals and access to tools common to the study of all other microelectronic systems. We will discuss a considerable amount of electronic transport and capacitance data as well. However, the primary focus of this thesis will be on systems for which the nanoSQUID microscope can provide important information that is inaccessible to these techniques, and so we will discuss a few such systems next.Consider the following procedure: we obtain a pair of identical two dimensional atomic crystals. We slightly rotate one relative to the other, and then place the rotated crystal on top of the other . The resulting pattern brings the top layer atoms in alignment with the bottom layer atoms periodically, but with a lattice constant that is different from and in practice often much larger than the lattice constant of the original two atomic lattices. We call the resulting lattice a ‘moir´e superlattice.’ The idea to do this with two dimensional materials is relatively new, but the notion of a moir´e pattern is much older, and it applies to many situations outside of condensed matter physics. Pairs of incommensurate lattices will always produce moir´e patterns, and there are many situations in daily life in which we are exposed to pairs of incommensurate lattices, like when we look out a window through two slightly misaligned screens, or try to take pictures of televisions or computer screens with our camera phones. Of course these ‘crystals’ differ pretty significantly from the vast majority of crystals with which we have practical experience, so we’ll have to tread carefully while working to understand their properties.

Our studies suggest a combination of its transmission dynamics and how they are affected by management issues, such as the quantity of shade and the density of planting, plus a variety of control from above elements represent a source of control, which sometimes fails .Understanding the general structure of ecological communities has long been a central goal of ecology, from Haeckel to us. Empiricists commonly, and probably necessarily, focus on the community of X, which is to say an assemblage of species defined by some set of criteria: the fungal community of Lake Wobegon, the community of gall-forming insects of oak trees, the microbial community of the human gut, the community of four ciliate species, and so on. Theoreticians perhaps feel less constraint. In the present article, we have defined the community as the herbivores of the coffee plant and their associates, in which top-down control is the goal of management . The framing of regulation from above from theoretical ecology translates directly into biological control from agroecology. Indeed, in agroecology regulation from above is elementary, large round plant pots in that the top-down agents are frequently obvious . However, stopping at that level of understanding may obscure more than clarify, much as the simple phrase controlled from above may indeed obscure .

Precisely how that control is affected may involve many complicated interactions and contingencies, making, we argue, the framing of complex systems a necessary one. The fungus that attacks the scale is most efficient when the scale is hyper dense at a local level, something that cannot happen unless it is under the protection of a mutualistic ant, which deters the other predator , which, however, is able to take advantage of a spatial pattern that is self-organized through a Turing-like process, and so forth. Indeed, we argue that the understanding we claim to have of this system so far comes from detailed study, both empirical and theoretical, and, most importantly is dramatically enriched through the application of some of the concepts newly developed in the distinct field of complex systems. Almost 10 years ago, some of us published a summary of this overall system , suggesting that understanding it required more than just an identification of who eats whom. This update emphasizes that point. Our narrative in the present article is perhaps a bit heterodox. We study a very complicated system , and we seek to understand it through theoretical ecology. To some, at least in the recent past, this might imply a large-scale computer model or sophisticated data manipulation. Our approach is distinct, recalling the wisdom of Levins’ paper on the strategy of model building. We seek to understand, at a deep level, how this system works, not necessarily for the purpose of predicting its future state.

We offer theoretical propositions, many of which are stimulated by mathematical arguments, but we do not seek what postmodern thinkers would have called a “totalizing discourse” with a large-scale model. Rather, we seek to use recent advances in complex systems as a way of stimulating thought, with the mathematical models that go along with them as “educating our intuition,” as Levins urged frequently. The models themselves represent approximate metaphors for this complex reality, all fitting into a hierarchy of understanding , which is mainly qualitative even though originally formulated through mathematical reasoning.Furthermore, our claim that this is a complex reality is meant to imply something deeper than the obvious claim that it is complicated. It is a complex system. For example, if the only players in the system were Azteca, C. viridis, and A. orbigera, the system wouldn’t be exceptionally complicated , but it would be a complex system, because it would have a clear emergent property . Even adding the phorid would mean two predators and two prey, but the spatial pattern that emerges and the dependence of one system on a second system operative at a completely distinct time scale is an essential structural component of the system as a whole. The emergence would defy understanding if only the separate component parts were studied, which is to say if it were approached from a purely reductionist perspective. If the only players were the ants and the coffee berry borer, but the ants did not exhibit trait-mediated indirect interactions, the system would be complicated but not necessarily complex. This distinction between complicated and complex is important for our narrative. Because it is a complex system, it requires a more holistic approach to understand and manage, and there’s more potential for surprise .

A merely complicated system would not have these characteristics. That our model system is coffee is significant in several ways. First, traditional coffee management, with its characteristic shade trees, helps to create landscapes that are friendly to biodiversity conservation . It is a classic high-quality matrix for all sorts of animals and plants. Second, it involves a commodity that is of extremely high value, sometimes the main source of wealth for entire countries. Third, it is the basis of livelihood for millions of small farmers the world over. Fourth, when properly cultivated with shade, it joins other agroforestry systems in the worldwide struggle against climate change. Given all that, understanding the details of its operation would seem worthwhile, and marshaling recent insights from complex systems to anchor that narrative brings one of the classical questions of community ecology into focus as a practical issue. Consequently, besides being of potential importance for ecology, it makes ecology important for some practical aspects of this important crop. It is, for example, evident from only a qualitative understanding of the control from above system that a key element is the species of ant that nests in the shade trees and that, if those shade trees are eliminated , the whole control structure will be dramatically interrupted. Questions also arise about generality. Does this model system reflect something more general about the structure of control from above, or does it simply reflect interactions of this one particular system? First, most terrestrial systems have a spatial component involved, and framing the spatial component as one in which a subsystem operates to effectively create a spatial pattern in which other subsystems may operate is likely to apply frequently. Indeed, the idea of a predator–prey system generating a Turing pattern may be increasingly appreciated as more research programs interrogate the idea . Second, population dynamics unfolding on this space are likely to be nonlinear, and this nonlinearity will frequently be of the form that critical transitions lead to an alternative equilibrium within hysteretic zones, which may be multiple and constrain the herbivores above which control is being exerted . Third, the idea that multiple herbivores have their own suite of controlling factors is almost certainly true, but the idea that there will be connections, even if weak, with other subcomponents of the control from above, is likely to be characteristic. These three generalities encompass the complex systems topics of Turing pattern formation, critical transitions, hysteresis, chaos, basin boundary collisions, trait-mediated indirect interactions, and scale-dependent spatial processes, all of which are exemplified in our model system, plant pots round and certainly may be embedded in other systems of control from above. The message is not that these particular topics are essential but, rather, that control from above is not the one-dimensional process frequently imagined of a predator guild preying on a prey guild but, instead, a complex community of predators and parasites and diseases that interact with one another in complicated ways to eventually generate a self-organized system that exerts effective control over the herbivory. Much as one might say that the vertebrate circulatory system is responsible for bringing oxygen to each cell in the body, one might similarly simplify and say that natural enemies in the coffee agroecosystem are responsible for the regulation of potential pests. However, it is the heart, the veins, the arteries, exchanges across membranes, and so forth that tell the real story of how the delivery of oxygento our tissues actually happens. It is a complex system, the details of which are certainly of interest to health and healing.

Similarly, in our agroecosystem example, the subsystem that creates large-scale pattern sets the stage for a subsystem involving a predator and a disease that affect regulation of one pest, whereas the community structure of ants determines the efficiency of their predacious activities on a second pest and the disease that helps regulate the first pest is an antagonist to the third pest. This is all to say that yes, it is control from above, but that control is delivered through the ecological complexity of the community of natural enemies. It is misleading to suggest that listing the natural enemies and merely identifying them as such is sufficient. It is only through the lens of the reality of its state as a complex system that we may gain full appreciation of the ecological principle of top-down control, which then can be fully exploited in attempts to aid the management of this important agroecosystem. There is something of a conundrum in this narrative. Although it is clear that knowledge of all the ecological complexity could inform practical decisions that producers might want to make, is such detailed knowledge really necessary to provide useful advice to the farmer? If ecological knowledge of the particular system is primitive, could well meaning agroecological advisors give advice that will have unintended negative consequences? Post-WWII industrial agriculture enthusiasts embraced DDT and other pesticides creating the well-known pesticide treadmill that haunts us still today. Indeed, that is one of the issues that caused many environmentally conscious analysts to call for the science of ecology to be more actively embraced by agricultural planners. However, ecology is complicated. Secondary consequences cannot necessarily be predicted short of detailed study and the normal rules of thumb extrapolated from a few experiments or extralocal traditions could backfire. Perhaps the famous medical practitioner’s oath primum non nocere makes sense in agriculture as well. As farmers seek solutions to perceived problems on their farms, agroecologists rightly wish to use the science of ecology to help. However, frequently , ecological knowledge of the particular system is not very well understood because it is only recently that agroecological advocates have begun to break into the mainstream, and the basic research required to understand some of the vexing problems the farmers face has yet to be done. It is therefore common to use a few rules of thumb: avoid monocultures, don’t poison your natural enemies, maintain healthy soil, and so on. Such rules of thumb, on the basis of perceived ecological rules, for the most part make sense and probably conform well to the admonition primum non nocere. However, it is worth remembering the dust bowl, pest resurgence following pesticides, ocean dead zones, and other consequences that we live with today because a previous generation of farm advocates, equally sincere in their desires to help farmers, were prematurely confident in the ability of their tools to help the farmer.In recent years, a variety of novel two-dimensional van der Waals magnets have been discovered, founding the active field of 2D magnetism. Among these prospective compounds, binary chromium tellurides Cr1–δTe are attractive owing to their rich magnetic properties, as well as inherent chemical and structural compatibility when forming heterostructures with other topological systems, such as tetradymite-type topological insulators or chalcogenide-based Dirac/ Weyl semimetals. Furthermore, the broken time-reversal symmetry and spin-orbit coupling offer unique opportunities for the interplay between spin configurations and reciprocal-space topology. In this regard, ferromagnetic Cr2Te3 with strong perpendicular magnetic anisotropy is an intriguing platform to host non-trivial topological physics, particularly for the high-quality thin films grown by molecular beam epitaxy . An important consequence of the band topology in Cr2Te3 is the Berry curvature underlying the anomalous Hall effect. The intrinsic AHE is topological in nature and a hallmark of itinerant ferromagnets, which has also been observed in more exotic systems even without a net magnetization, such as spin liquids, antiferromagnets, and Weyl semimetals. When SOC coexists with long-range magnetic order, the Berry curvature can be significantly influenced near avoided band crossings, rendering the system an incredibly rich playground combining topology and magnetism.

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, plant plastic pots 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 BLS estimates that Colorado’s unemployment rate in December 2017 was 3.0 percent, while Denver’s unemployment rate was 2.9 percent . Earlier in 2017, the state unemployment reached a record low of 2.3 percent. The other six metropolitan statistical areas tracked by the BLS averaged an unemployment rate of 3.4 percent. Fort Collins had the lowest unemployment rate at 2.5 percent. Only two cities had an unemployment rate greater than the national unemployment rate of 4.1 percent—Grand Junction and Pueblo . Personal income growth among state residents reached nearly 8 percent in 2014, but growth slowed over the next two years. This trend reversed in 2017 as personal income growth increased to 5.4 percent. This growth rate exceeds the national rate by more than 2 percentage points . According to the OSPB, per capita income and wage growth in Colorado over the past year also outpaced the national figures. With regard to party registration in Colorado, voters were nearly evenly divided among Democratic, Republican, and unaffiliated categories at the time of the 2016 election. According to voter registration data from the Secretary of State’s office, the state had nearly 3.3 million active voters in November 2016. A plurality of these voters registered as unaffiliated , while the share of Democratic and Republican were nearly equivalent. In February 2018, the number of active voters decreased relative to November 2016 by about 1.7 percent.

This may be partially attributable to controversy surrounding President Donald Trump’s Commission on Voter Fraud, which made data privacy a concern after the commission requested, “voluminous information on voters, including names, addresses, dates of birth, political affiliations and the last four digits of Social Security numbers, along with voting history” . Fifteen months after the 2016 election, the proportion of unaffiliated voters in the state increased to 36.3 percent, while the share of Democrats and Republicans decreased to 31.1 percent and 30.8 percent, respectively . This change is likely driven by primary election reforms approved by voters in the 2016 election. Voters overwhelmingly approved Proposition 107, which adopted a presidential primary in lieu of the existing caucus system, and Proposition 108, which allowed unaffiliated voters to participate in the party primary of their choice. Previously, unaffiliated voters were prohibited from participating in any primary elections or caucus meetings. Because this reform allows unaffiliated voters to participate in the primary of their choosing, it appears that a substantial number of Coloradans changed their party registration status to take advantage of this new opportunity.Colorado’s economic trajectory remains generally positive. In late March, the Governor’s Office of State Planning and Budgeting released its revised economic forecast. The report summarized the condition of Colorado’s economy by stating, “Colorado’s economy is on solid footing with strong employment growth and expectations of an ongoing expansion. New business formation continues to grow, while Colorado oil production is at record levels. Although much of the state’s economic growth has occurred along the Front Range, stabilizing farmland values and increases in energy prices and production have recently supported rural areas as well. Looking forward, higher costs of living and tight labor market conditions are expected to constrain further growth through the forecast period” . The OSPB characterized the 3.1 percent increase in General Fund revenue in the 2016–2017 fiscal year as decreases in oil, gas, and other commodity prices as the catalysts for the three-year decline in corporate tax revenue beginning in 2014. After falling 21.9 percent in the third year of this recent decline, corporate tax revenue is estimated to increase by 38.6 percent in the current fiscal year. In addressing how federal tax reform may affect state corporate tax revenue, the OSPB projects continued growth in corporate tax revenue, but cautions that “future increases will be constrained by higher business costs, especially for employee compensation, black plastic pots which will reduce profit margins and result in lower tax liabilities” . Taxes from the state’s legal marijuana market continue to grow. According to data released by the Colorado Department of Revenue, total marijuana sales surpassed $1.5 billion in 2017 . Of this total, $1.09 billion in sales came from the retail market, while medical marijuana sales were approximately $0.42 billion. Table 2 reports annual marijuana sales and tax revenue data. Sales have increased each calendar year since the retail market began operation on January 1, 2014, but the growth rate has gradually declined each year. In 2014, marijuana sales totaled nearly $680 million. This figure increased by 45.7 percent in 2015 to $990 million. Sales increased by 31.3 percent and surpassed the $1 billion mark for the first time in 2016 to reach a total of $1.3 billion. Total sales in 2017 amounted to $1.5 billion, which represents a 15.3 percent increase.

If the trend continues, Colorado could expect to see sales numbers plateau since the growth rate has decreased by an average of 15 percentage points each year. Sales data through September 2018 indicate sales of nearly $1.16 billion. The monthly sales average for 2018 puts the state on track for an annual marijuana sales total of about $1.54 billion, which would be the largest sales amount to date and constitute a slight increase of 2.4 percent from 2017.State revenue from the medical and retail marijuana markets in the form of taxes and fees reached nearly $250 million in 2017. This is a 27.8 percent increase from 2016 following a 48.5 percent increase in tax revenue from 2015 to 2016. During the first 10 months of 2018, the state reported tax and fee revenues of $223 million. A linear projection of tax revenue for the remainder of the year suggests that the annual tax revenue would reach nearly $268 million, an increase of about 8 percent from the prior year. The largest annual increase in marijuana taxGovernor Hickenlooper’s November letter to the JBC highlighted education and the criminal justice system as two areas where additional investments were necessary . The proposed and enacted funding levels for state departments and changes from the prior year’s budget are reported in Table 3. In total, the General Fund budget request is $11.5 billion, which is an increase of $292.1 million from the prior year. This represents a 2.1 percent increase, while total fund spending is proposed to increase by 3.7 percent to $30.5 billion. The budget signed by the governor into law on April 30 authorized a General Fund budget of $11.42 billion and $30.63 billion in total fund spending . As the data reported in Table 3 indicate, the governor’s budget proposes increasing the funding levels of all state department relative to last year, exempting the Department of Local Affairs and Department of Labor and Employment. The final budget ultimately increased the funding levels for all state departments except for Labor and Employment. The median proposed increase is 5.1 percent, while the enacted budget imposes a median departmental spending increase of 5.7 percent. In addition to funding existing government programs, the governor’s budget proposed new spending initiatives for the Department of Health Care Policy and Financing , Higher Education , K-12 Education , Department of Corrections , and Department of Human Services , in addition to an increase in the General Funds reserve .Relative to last year’s budget, Governor Hickenlooper’s budget request for the 2018– 2019 fiscal year is much more optimistic. This positive tone is a product of a good economic climate and the successful passage of a major budgetary reform in the prior session. Transitioning hospital provider fees into a government enterprise fund made these funds TABOR exempt. In the 2016–2017 fiscal year, hospital provider fee revenue was $654.4 million. In future years, these funds do not count toward the TABOR revenue cap. The governor emphasized the importance of this reform by noting 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” . Beyond this important budgetary reform, Governor Hickenlooper also lauded the strong upward trajectory of the state’s economy by claiming, “Colorado’s economy continues to outperform nearly every state and the national economy overall” . Statewide unemployment remains low and job creation numbers are strong—approximately 53,000 new jobs are projected for 2018. As the state’s population continues to grow, Hickenlooper’s budget request reflects spending priorities to address increased demand for certain state services. Increases in education spending are one of the most notable aspects of the budget request. K-12 spending is proposed to increase by $84.6 million, which represents an increase of $343.38 per student. According to state estimates, the 178 school districts in Colorado currently serve the educational needs of more than 865,000 students. The Department of Education projects that enrollments will grow by 0.7 percent in the current fiscal year. The education budget request remarks that “additional funding proposed by the Governor will allow local districts and charter schools to decide how to best improve the education opportunities of their students.” As required by Amendment 23, the inflationary increase to the State Education Fund amounts to $8.9 million.

In addition to the worst public health crisis in a century and a major economic recession, the year 2020 also witnessed a highly charged presidential election unlike any the nation has ever seen.The global COVID pandemic had less of an effect on election administration in Colorado— relative to other states—because Colorado has employed a vote by mail system since 2013. Most of the nearly 3.3 million ballots in the 2020 election were submitted by mail or returned to ballot drop-off sites. Turnout among active voters was 86.9 percent, which corresponds to a voting eligible turnout rate of 76.4 percent. The results of the 2020 elections in Colorado produced few legitimate surprises. The Joe Biden and Kamala Harris ticket carried the state with 55.4 percent of the vote to 41.9 percent for Donald Trump and Mike Pence. The party composition of the state’s House delegation remained unchanged, with four Democrats and three Republicans elected to 2-year terms. Political novice and business owner Lauren Boebert defeated Republican incumbent Scott Tipton in a primary election for the state’s third congressional district.

Boebert’s firebrand political style, characterized by incendiary rhetoric and enthusiastic allegiance for President Trump, large plastic growing pots catapulted her to a surprising 9-point primary win over Tipton, who had served five terms in the House. Boebert’s brash campaign included vociferous support for the second amendment and tacit backing of various QAnon conspiracy theories. Despite numerous campaign controversies, Boebert emerged victorious in the general election where she defeated Democrat Diane Mitsch Bush by 6 percentage points in Colorado’s most competitive congressional election of 2020. Party control of the General Assembly remained with the Democrats, who maintained a 41-24 advantage in the House and increased the size of their majority by an additional seat in the Senate where party division stands at 20 Democrats and 15 Republicans. There was not a governor’s election in the 2020 election cycle following the election of Democratic Governor Jared Polis in 2018. The contentious presidential election took center stage as a result, although neither Trump nor Biden held a campaign event in Colorado, and the Democrats ultimately won the presidential popular vote in the state by a substantial margin. As Colorado solidified its status as a blue state in the Electoral College, many polls suggested a more competitive race for U.S.

Senate between Republican incumbent Cory Gardner and former governor John Hickenlooper. Following a short-lived candidacy for the Democratic presidential nomination, Hickenlooper launched a bid to unseat the one-term incumbent Gardner, viewed by many as the most vulnerable Senate Republican running for reelection. Gardner was widely considered the second most vulnerable incumbent Senator in this cycle after Democratic Senator Doug Jones from Alabama. Hickenlooper emerged victorious as expected on election night, defeating Gardner by nearly 10 percentage points receiving 53.5 percent of the vote. Several economic and budget related ballot measures appeared on the statewide ballot in 2020, including a proposal to reduce the state’s income tax rate from 4.63 percent to 4.55 percent, which passed with robust support. Voters also approved Amendment B, which repealed the Gallagher Amendment that established residential and non-residential property tax rates in the Colorado Constitution. Although tax increases have historically been unpopular in the Centennial State, the passage of proposition EE raised taxes on cigarettes, tobacco, and other nicotine products such as e-cigarettes and vaping cartridges. Stemming from recent controversies surrounding the use of state fees to circumvent constitutional requirements imposed by the Taxpayer Bill of Rights , Proposition 117 will require voter approval of future state enterprise funds with projected or actual fee revenue exceeding $100 million within its first five years. The passage of Proposition 118 created a new family and medical leave program funded by a payroll tax split by employers and employees that will allow workers to take up to 12 weeks of paid leave. Colorado’s historical passage rate is about 35 percent, but in 2020 a total of nine of the eleven statewide measures passed.

The only two that failed were Proposition 115, which sought to ban abortion 22 weeks following gestation, and amendment C, which would have reformed lottery regulations for charitable organizations. In sum, however, statewide election outcomes resulted in an uncharacteristic number of policy changes, many with important economic consequences. Unified Democratic government remained in place in Denver, which set the stage for the 2021–2022 budget cycle. The three sections that follow discuss the state’s demographics, revenue, and spending.The Census Bureau estimates the state’s population in 2020 to be 5.7 million residents, which is an increase of 14.8 percent from 2010. This population increase has outpaced growth in new housing units over the past decade. Only Utah and Texas have experienced larger growth rates since 2010. Colorado joined Oregon, Montana, Texas, Florida, and North Carolina as the six states that gained at least one seat in the House of Representatives following reapportionment. Because of the redistricting reforms approved by voters in 2018, boundaries for all eight congressional districts will be drawn by an independent redistricting commission. Table 1 presents further demographic and economic data from the Census Bureau and Bureau of Labor Statistics  for Denver County, Colorado, and the United States. The pre-pandemic unemployment rate in Colorado commonly fluctuated between 2 and 3 percent. At the beginning of 2020, the state’s unemployment rate stood at just 2.7 percent. Job losses from the COVID recession produced double-digit unemployment figures for three consecutive months in the spring, with a maximum of 12.1 percent unemployment in April . Industries disproportionately affected included retail trade, accommodation, hospitality, food service, arts, entertainment, and recreation. Similar to patterns in other states, job losses in Colorado were sharper and longer lasting among lower-income earners. State economists reported a change in employment among lower-income individuals of about 35 percent from January to May 2020. After reaching an apex in April, unemployment figures have improved or remained steady in each month thereafter. BLS data indicate that Colorado unemployment returned to single digits in July 2020 and fell below 7 percent in October. Unemployment levels in the state steadily decreased by a monthly average of 0.14 percent, reaching a pandemic low of 5.1 percent in November. According to one estimate, the state had recovered about 85 percent of the COVID job losses, which is better than the national average, but only one metropolitan area—Colorado Springs—had so far exceeded pre-pandemic employment levels . The civilian labor force participation rates of 72.2 percent in Denver County and 67.6 percent in Colorado are both better than the national figure of 63 percent.Per capita and household income levels in Colorado and Denver County remained greater than the national average. Median home prices in the state and county were likewise substantially above the median home price in the United States . The influx of new residents coupled with high real estate prices has kept affordable housing a persistent problem, particularly in the Denver metro area and many mountain communities across the state. For example, large plastic pots the Crested Butte city council recently declared a housing emergency while debating policy reforms to address the affordable housing crisis, including a one-year moratorium on short-term rental licenses. The city was also considering other measures, including easing restrictions on camping and imposing greater taxes on rental or vacation properties. Other innovative efforts to increase the availability of affordable housing in places such as Ouray, Ridgway, and Telluride included revising zoning ordinances to permit greater resident density. Efforts also included designating housing as only available to the local workforce by attaching income and deed restrictions to certain property developments. The rapid growth of the state’s population has also had effects on the composition of the Colorado electorate. According to the state’s active voter rolls, a plurality of voters in Colorado are registered as unaffiliated . Thirty percent of the electorate is registered Democratic, and Republican registration places third at 27.3 percent .

The proportion of unaffiliated voters increased by nearly 5 percentage points since the 2016 election. Contributing factors to this increase are election reforms passed in 2016, which replaced the existing caucus system with a presidential primary, and a second reform that allowed unaffiliated voters to participate in the party primary of their choice. Unaffiliated voters were previously prohibited from participating in primary elections or caucus meetings. Voters in Colorado who self-identified as unaffiliated or independent voted 55-40 in favor of the Democratic ticket in the 2020 presidential election, while the split among ideological moderates was 65-30 in favor of Biden/Harris . Regarding the racial and gender composition of the electorate, exit poll data from 2020 estimate white voters to constitute 79 percent of the electorate. Latino and Latina voters were the largest non-white voting bloc at 12 percent of voters, while the proportion of Black voters was about 3 percent . These numbers, however, do not reflect the actual size of racial minority groups in the state. According to Census data, Colorado’s Hispanic population is nearly 22 percent, the Black population is 4.6 percent, and the Native American population is 1.6 percent. A plurality of the state’s voters in 2020 were white women . An additional 35 percent of ballots were cast by white men. White women voted Democratic in the presidential election by a 2-1 margin, while Biden received 49 percent of the state’s white male vote to Trump’s 48 percent .Decreased economic activity and substantial job losses beginning in March 2020 resulted in a projected decline in state revenue for the first time since 2009. The Office of State Planning & Budgeting revenue forecast projected an overall decrease in state revenue of 4.7 percent to $12.3 billion in the 2020–21 fiscal year . This was a substantial decrease in state revenue but an improvement upon earlier projections, which previously estimated a percentage drop in revenues exceeding double digits. Revenues for the 2021–22 fiscal year, however, are predicted to return to pre-pandemic levels. Prior to the COVID pandemic, individual income tax revenues grew by nearly 5 percent. The projected decline in income tax revenue during the first year of the pandemic was 8.1 percent totaling $8.2 billion . The OPSB projects income tax revenue to rebound in 2022 and beyond—however, not reaching pre-pandemic growth levels, claiming that the “reduction is expected to be short-lived and mitigated by the fact that low income earners were disproportionately impacted by the pandemic” . Such a turnaround would be critical to Colorado’s economic recovery as income taxes represent about two-thirds of General Fund revenues. Corporate income tax collections were also deeply affected by the pandemic and fell by 20.8 percent to $728.3 million in the 2019–20 fiscal year. Economists predict that corporate taxes will continue in a downward trajectory by an additional 10 percent in 2021. Unlike income and corporate taxes, sales tax revenues increased during the pandemic on account of increased online shopping and a gradual rebound in retail sales. Overall, sales tax revenue increased 4.7 percent in 2020, with state projections for an additional 6.5 percent this year. Beginning this year, Colorado will also generate a greater amount of cigarette tax revenue, which equaled $32.5 million in 2020, following the passage of proposition EE. Cigarette taxes increased from 84 cents to $1.94 per pack in 2021 and will reach $2.64 in 2027. Tax rates on other tobacco products, including vaping products containing nicotine, will similarly increase to 62 percent in 2027.Governor Polis’ budget proposal for the 2021–2022 fiscal year proposed a total funds budget of $35.4 billion, which is a 9 percent increase from the prior year. The General Fund spending budget of $13.6 billion is a 20 percent increase of $2.25 billion. His letter to the Joint Budget Committee began by noting the extraordinary challenges and opportunities brought about by the global pandemic, an economic recession, the Black Lives Matter movement for racial justice, and historic wildfires and other natural disasters. Introducing his second budget since the pandemic began, Polis stated, “Each of these crises has exposed weaknesses in our systems of public health and health care, commerce, and education, and highlighted the urgency to take bold climate action in order to protect our Colorado way of life. To meet the moment and to build resiliency for the future, we must make critical investments to position Colorado for a strong, inclusive, and rapid recovery. Colorado will not only recover from these crises, but together we will build back stronger than before” .

The slight difference in mapping rate between the two references can be explained by either the absence of some low-expression transcripts in the ISNT or the residual error rate in isoform sequences. Gene expression analysis using the ISNT as reference showed similar results compared to the Cabernet Sauvignon genome assembly, with a very high correlation of expression level and differential gene expression, and with similar global transcriptomic changes. However, we observed differences in the number of expressed and differentially expressed features that depend on the reference used. Those differences could be explained by the diploid phasing of the Cabernet Sauvignon genome assembly and that multiple ISNT transcripts might correspond to a single gene locus. Nonetheless, similar relative amounts of Biological Process GO terms were found among the differentially expressed genes, confirming that the transcriptome obtained using Iso-Seq captured the transcriptional reprogramming underlying the main physiological and biochemical changes during grape berry development. In addition, gene expression analysis revealed that some private isoforms are significantly modulated during berry development, square pots for plants indicating that in addition to identifying the private gene space, the ISNT reference makesit possible to observe its expression.

In conclusion, this study demonstrates that Iso-Seq data can be used to create and refine a comprehensive reference transcriptome that represents most genes expressed in a tissue undergoing extensive transcriptional reprogramming during development. In grapes, this approach can aid developing transcriptome references and is particularly valuable given diverse cultivars with private transcripts and accessions that are genetically distant from available genome references, like the non-vinifera Vitis species used as rootstocks or for breeding. The pipeline described here can be useful in efforts to reconstruct the gene space in plant species with large and complex genomes still unresolved.Grapevines are susceptible to the highest number of pathogens to infect a single crop, with over 70 viruses detected. In 2008, a new virus was first observed in Napa County, California, which economically threatened grapevines: grapevine red blotch virus. This virus is the causative agent of grapevine red blotch disease, which has been identified in vineyards across the United States, Canada, Argentina, Mexico, South Korea, and India . Reports indicate GRBV primarily spreads through propagation material and secondarily through an insect vector. Spissistilus festinus was shown to successfully transmit GRBV in greenhouse settings, yet this has not been replicated in vineyards to date.

GRBV has been identified as a virus from the Geminiviridae family containing a circular single-stranded DNA genome similar to other geminiviruses. GRBD expresses symptoms of reddening of leaf blades and margins, with reddening of the primary, secondary, and tertiary veins in red grape cultivars. GRBV affects grapevines in various ways. For example, leaves on infected vines show increased levels of sugar, phenolics, particular amino acids, and enzymatic activity related to plant defense, as well as a reduction in carbon fixation. However, the most damaging are the effects on grape composition which has been shown to be detrimental to final wine quality. GRBV delays ripening by decreasing the accumulation ofsugar and anthocyanin in berries, potentially due to the impairment of translocation mechanisms. The virus has variable impacts on primary and secondary metabolites, specifically phenolic and aroma compounds. In summary, detrimental economic impacts to vineyards in the United States could reach $68,548/ha with vine removal being the only current method of alleviation. Consequently, recent research has strived to understand the effects and functioning of GRBV to establish mitigation strategies to alleviate the impact on grape composition and wine quality. The first phase is characterized by cell division and production of seeds, as well as synthesis of tannins and organic acids. The second phase is characterized with the onset of veraison, which is when the grape berry begins to soften and change color. The final and third phase is berry engustment/ripening, where berries increase in size, sugar accumulates, acidity declines, and secondary metabolites such as anthocyanins and aromatic compounds are synthesized inside the berry.

Studies have shown that volatile compounds such as terpenoids and C6 compounds, begin to accumulate in berries after veraison, and are controlled by numerous factors. The synthesis of these compounds in berries is also altered by external factors such as light exposure or pathogens. In addition, these secondary metabolites are crucial to grape growers and winemakers due to their importance in the quality of a final wine. Grape maturity has shown to be a key driver in the composition of a final wine, where later harvested fruit produces wines with lower concentrations of C6 alcohols and higher in concentration of esters. However, the impacts of GRBV on volatile compound abundance in harvested grapes has not been investigated. A plant’s genetic material may influence susceptibility to viral infections. Additionally, rootstocks can impact grapevine physiology and impact the overall composition of a grape berry. For instance, rootstock 110R causes high vigor and high drought tolerance in grapevines; whereas 420A is a rootstock of low to moderate vigor and low drought resistance. Vigor, resulting in greater shoot length and hence leaf area, may impact net carbon assimilation and the translocation of metabolites into the berry, consequently affecting the final wine composition. These hydric differences affecting carbon metabolism in rootstocks can also impact the plant-pathogen interactions. Therefore, it is plausible that severity of GRBD symptoms will be dependent on the interaction between scion cultivar-rootstock. However, this has not been fully investigated. Macro and micro climate fluctuations may also be a factor in pathogen-plant interactions, and should be considered. This study investigated the impact of GRBV on the biosynthesis and accumulation of primary and secondary metabolites in grape berries throughout ripening and at harvest. Additionally, the influence of seasonal and genotypic factors on disease expression within grapevines were studied.Figures 1–3 depict sugar accumulation, anthocyanin levels, TA, and pH through ripening. Sugar accumulation was determined by converting ◦Brix to mg of sugar per berry. Anthocyanin content was lower in RB. grapes when compared to RB grapes for both years and rootstocks during ripening . However, the degree of impact varied depending on season and rootstock. In 2016, both rootstocks were equally impacted throughout ripening regarding sugar accumulation and anthocyanins levels. However, in 2017 sugar accumulation was generally not significantly impacted by disease status. In 2017, grape anthocyanin levels were more significantly impacted for infected vines on 110R rootstock than 420A rootstock, whereas rootstock impact was less apparent in 2016. At harvest in 2016 , CS 110R and 420A rootstocks, respectively, had a 2% and 11% decrease in anthocyanin content and a 12% and 18% decrease in sugar content in RB grapes when compared to RB grapes. In 2017, atharvest , anthocyanin content was 35% and 11% lower in RB when compared to RB , and sugar content was 9% and 7% lower, for 110R and 420A, respectively. By plotting ◦Brix over ripening and fitting a linear trendline, it is possible to compare the rate of ripening for RB and RB grapevines .

As indicated by the slope of the best fit line, the rate of ripening was always higher for RB data vines when compared to RB data vines, with the exception for CS 420A in 2017. In addition, large square plant pots the rate was also lower in 2016 than 2017 across virus status and rootstocks. Interestingly, the difference in the rate of ripening between RB and RB data vines was larger in 2016 than in 2017 which correlates to the larger differences in accumulated sugar at harvest. In 2017, the rate was lower for CS 420A than CS 110R across virus status.The volatile compound profiles of RB and RB grapes were determined in both 2016 and 2017 seasons . PCA was performed to plot the variability between RB and RB grape samples . Between 80.6- 94.5% of the variance is explained by the PCA in Figures 4 and 5. For CS 110R, only the significantly different volatile compounds between RB and RB are plotted. There were, respectively ten and nine significant volatile compounds that explained the difference between treatments for CS 110R in 2016 and 2017. For CS 420A, the volatile compounds that contributed most to the variance of the PCA were plotted, due to few volatile compounds being significantly different. This selection was based on the squared cosine which shows the importance of the volatile compounds to explain the variance in the data. For CS 420A, in 2016, only cis-3-hexen-1-ol was significantly different, and in 2017, only β-linalool and β-citronellol were significantly different with a α level of 0.95.After veraison, volatile compound accumulation begins in grapes and changes through ripening. However, the impacts of GRBV on grape volatile compounds have not been investigated. We found 35 different aromatic compounds in grapes from the two rootstocks over two seasons, of which 24 were similar between the two years studied . In 2016, across rootstock, RB grapes were generally lower in volatile compound levels than RB , except for C6 compounds such as 2-hexenal, hexanal, cis-3-hexen-1-ol, trans-2-hexen-1-ol, and hexanol . These C6 volatile compounds are synthesized in the grape skin through the lipoxygenase pathway, are generally responsible for green or grassy aromas and accumulate in CS grapes up to 18 ◦Brix . With the exception of hexanol, the levels of these compounds begin to significantly decrease thereafter, with a 67% decrease in grapes at 25 ◦Brix when compared to grapes at 18 ◦Brix. These observations correlate with the common finding that GRBV causes a delay in ripening, with green aromas being present and correlated with the lower sugar accumulation. On the other hand, RB grapes were highly correlated with monoterpenes such as limonene, ß-myrcene, α-terpinene, geranial and p-cymene , which are responsible for floral and fruity aromas. These compounds have been associated with CS grapes at harvest and are known to increase through grape ripening and may decrease at over ripeness. In addition, RB grapes were also highly correlated with esters such as ethyl acetate, ethyl hexanoate, hexyl acetate, and ethyl butyrate. Although ester formation is mainly related to yeast or bacteria metabolism during winemaking , grapes are also known to synthesize esters. Anthraniloyl-coenzyme A :methanol acyltransferase is known to be responsible for the formation of methyl anthranilate in grapes and it is also classified as an ester-forming acyltransferase, which could be responsible for the formation of esters in grapes. The esters found in the current work that in general related mostly to RB grapes, are known to produce red and black fruit aromas. Collectively, these results confirm that RB grapes underwent normal ripening processes and produced more fruity aromas, while RB grapes at harvest have aroma characteristics more related to early ripening stages.Results indicated that in 2016 GRBD had a larger impact regarding sugar accumulation, pH, TA, and final sugar content than in 2017. In addition, the harvest dates were two to three weeks later in 2017 than in 2016. These observations can potentially be explained by the difference in temperature between the two seasons. In 2017, Napa County experienced a heat wave from 26 August–11 September, where nine days were over 35 ◦C, and four days were over 40 ◦C. The cumulative growing degree days for both years can be seen in Figure 1e. Extreme heat conditions during grape maturation have been shown to inhibit enzymatic activity and halt the biosynthesis of metabolites inside the grape berry. Inhibition of these processes due to heat leads to decreases in sugar accumulation and increases in acidity in healthy fruit. This is thought to be caused from a decrease in rate of translocation of sugars from leaves to fruit, through the reduction in photosynthesis at temperatures greater than 30 ◦C. The rate of ripening in 2017 was faster than 2016prior to the heat spike . However, during the heat spike in late August to harvest, sugar accumulation plateaued resulting in extended harvest times in 2017. In addition, research has shown that temperature can alter virus-induced gene silencing which is triggered with the infection of a virus as a plant-derived defense mechanism to downregulate the genes of interest. Previous work on other plant species infected with a geminivirus has shown that the extent of gene silencing is related to temperature. Specifically, Chellappan et al. showed that temperatures over 30 ◦C induced gene silencing, which interfere with gene expression, resulting in decreases in viral DNA accumulation and decreases in symptoms.

It could be hypothesized that in the 1st year bearing 100% of the clusters, while not showing a reduction in soluble solids, may have taken a toll on plant reserves. However, neither root mass nor starch content were impacted by the crop level in our work. Palliotti and Cartechini performed cluster thinning on three varieties over three seasons and found that cluster thinning did not affect must soluble solids. In years where rainfall was more abundant , results of cluster thinning were compensated with larger berries , with compensation of berry size similar to our results. Precisely, these kinds of results are those that disturbed the correlation between leaf area to fruit mass and berry total soluble solids . This suggested that larger berries may offer higher resistance to increases in berry total soluble solids regardless of leaf area to fruit mass . This hypothesis was supported by the response of grapevines submitted to water deficits that had smaller berries with higher soluble solids despite having much lower carbon assimilation rates .Development, blueberry packaging which encompassed the timing of all physiological events recorded , was delayed clearly by defoliation when treatments were in place, which excluded bud break and flowering.

The initiation of each of these pheno-phases is quite complex as it may require more than one preexisting condition. For instance, the release from dormancy is often associated to the fulfillment of a chilling/thermal time accumulation requirement , which supported the observation that all grapevines in the same site as this experiment would have a similar date of bud break. However, entering and exiting dormancy is also concomitant with major events of mobilization of soluble carbohydrates that condition the response of the latent bud . Similarly, veraison may be modeled with thermal time but ultimately requires a sucrose stimulus . Leaf senescence of deciduous plants is largely induced by shorter days and cooler temperatures, but as evidenced in our work, defoliation treatments delayed it. Other studies have suggested that leaves are able to sense source strength and delay leaf senescence accordingly . However, in our work, source strength was achieved through more leaves rather than better leaf net carbon assimilation performance. In both experimental years we witnessed the 33%L treatments assimilate more carbon compared to 100%L to compensate. However, it remains to be seen in future works if this is in fact a carbon starvation effect or an artifact of plant water status.

In fact, high sugar levels are one of the signals inducing natural leaf senescence , and this can be modulated. Interestingly, this response was not conditioned by sink strength or differences in leaf area in the final year, only by the practice of defoliation in our study.In the third year of study, no treatments were applied and therefore, all effects observed are attributable to cumulative effect of previous years’ conditions. The so-called carryover effects have been discussed in relation to indirect observations, where the treatments were applied for several years or when historical series were analyzed . In the case of defoliation, much direct evidence of carryover effects exists. For instance, Jermini et al. showed how defoliation caused by downy mildew induced severe reductions in the successive year’s yield. Bennett et al. also reported severe reductions in yield, and these were attributed to reductions in clusters per vine, to reductions in berries per cluster, but not to changes in berry mass. In that study, there were changes in inflorescence per vine and flowers per inflorescence. Therefore, promoting root and canopy growth over the years has a strong cumulative effect on yields. Alternate bearing is an issue in some tree fruit crops such as mango, avocado, olive, pistachio, citrus, etc. [reviewed by Monselise and Goldschmidt ] and fruit removal in those crops is not only performed aiming for in-season effects, but also to maintain consistent yield over the seasons. The carryover effects of crop level in grapevine have been reported less frequently than defoliation.

Our results suggested that in fact grapevine is a perennial crop not very sensitive to alternate bearing. Yield was associated with dormant season precipitation or root and shoot starch content at budburst . In our results, starch content of roots was only affected by defoliation in July and September samplings, which are coetaneous with sucrose stimulus to berry ripening. As root starch content fully recovered in all treatments, root mass was the only factor that would explain changes in yield in the successive season . In fact, in 2019, grapevines that were defoliated during the two previous seasons had lower root mass and fruit load as a carryover effect, which led to a faster recovery of starch reserves. Likewise, the carry over effects of defoliation were evident in leaf area, berries per cluster, and yield in the final year.After many efforts directed at balancing grapevine canopy by focusing on fruit removal, a renewed focus on maintaining an active leaf area with proper solar radiation exposure to clusters is needed. Our data indicated that carbon balance and translocation were more influenced by leaf area rather than crop level . The canopy leaf area and architecture determined the photosynthetic capacity which in turned initiated the sugar induced growth. Once a large enough plant reduced carbon pool was available, berry enlargement and sugar allocation determined berry size. Canopy size also dictated how fast ripening progressed as well as storage of non-structural carbohydrates. Finally, we did not measure a direct physiological benefit of fruit removal in the primary metabolism of grapevine that could suggest one unit of leaf area would equate to one unit of fruit in the grapevine.The HPA axis maintains a diurnal rhythm marked by a daily peak after waking, a subsequent decline over the course of the day, and a nadir shortly after onset of continuous sleep . The diurnal pattern of HPA activity plays important roles in a variety of metabolic, immunological, and psychological processes that support our day-to-day functioning . In studies of children, the preferred assessment method of HPA axis activity is the collection of saliva and the measurement of cortisol . Cortisol is the “end-product” hormone released into the bloodstream from the adrenal glands—the final step in a biological cascade initiated by the hypothalamus and perpetuated by the pituitary gland. In addition to supporting the orchestration of several other processes , moderate cortisol levels are thought to support effective neural transmission and optimal learning and high-order cognition . In times of acute physiological or psychological stress, the HPA axis mounts a particularly pronounced response, culminating in high levels of cortisol that reach glucocorticoid receptors throughout the body and brain. Working with the ANS, these acute HPA stress responses coordinate the physiological and psychological resources needed to overcome the stressor . Yet, given negative feedback processes, high cortisol levels also play important regulatory roles in down-regulating HPA axis activation, allowing it and other systems to return to baseline . Collectively, blueberry packaging box these complex within- and cross-system dynamics support an organism’s ability to both respond to and recover from the effects of environmental stressors . Newborn infants can mount an HPA axis response to environmental stimuli , and normative circadian rhythms tend to stabilize as infants begin to forego their afternoon naps . However, the span from infancy through early childhood is also a time of meaningful developmental change. Indeed, a growing theoretical and empirical literature indicates that children’s early experiences play a critical role in the organization of their emerging adrenocortical systems .Low-income ecologies present a confluence of distal and proximal risk factors thought to influence children’s developing physiological stress systems and undermine optimal cognitive and social development . For example, children growing up in low-income contexts are more likely to face distal stressors, such as inhospitable and dangerous neighborhoods and inadequate access to services and social capital . Such distal risks are known to have trickle-down effects that undermine parents’ abilities to effectively read, interpret, and respond to their children’s needs . In turn, a convergent literature comprising experimental work with animals as well as observational studies of young children indicates that sensitive and responsive caregiving can support adaptive HPA axis functioning .

This is evident with respect to children’s acute stress responses. For example, young children with secure attachment relationships and more sensitive caregivers tend to show better regulated HPA axis responses when faced with acute psychological stressors . Changes in the quality of children’s caregiving environments have also been linked with their baseline, or resting levels of HPA axis activity. For example, at the more extreme end, children who are moved from very high-risk households into foster care have been found to evince comparatively lower resting cortisol levels than their peers who remain in high-risk homes . It is important to note that similar relations are evident with respect to parenting within the normal range. For instance, in prior work with the same sample as used in the present study, our group showed that higher levels of maternal sensitivity in infancy are predictive lower levels of resting cortisol, after adjusting for income and a number of potential confounds . Beyond psychosocial risks, children growing in the context of economic adversity are more apt be exposed to households that are more densely populated, noisy, disorganized, and unpredictable—aspects typically discussed under the umbrella term chaos . A growing literature suggests that chaotic environments may alter children’s ANS and HPA axis functioning in early and middle childhood. Recent work by researchers in our laboratory suggests similar effects with respect to infants and toddlers , with within-child increases in chaos predictive of contemporaneous increases in resting salivary cortisol in later infancy and toddlerhood.Notably, young children growing up in low-income contexts spend substantial amounts of time in settings outside of their homes—such as non-parental child care. Indeed, in the United States approximately 43% of children in poverty attend regular non-parental care by 9 months of age . A well-developed literature indicates that young children’s early child care experiences also play a meaningful role in their HPA axis functioning. Meta-analytic findings indicate that—compared to their normal diurnal patterns experienced at home—children tend to show cortisol increases across the day on days when they attend child care . Some work suggests these patterns are particularly strong in toddlerhood and the beginning of the early childhood years and for children who attend lower quality child care . There is also some, albeit limited, evidence of long-term effects; for example, Roisman and colleagues found that spending greater proportions of time in center-based care in infancy and early childhood was predictive of children’s subsequent cortisol awakening response in adolescence. Although such effects with regard to child care type have been rather mixed, there is some reason to suspect that heightened exposure to peers—such as often found in center-based child care—may be a salient stressor for young children . For instance, at abehavioral level, the replicated link between greater exposure to center-based child care and subsequently heightened levels of aggression in childhood has been shown to be at least partially explained by the typically higher levels of peer exposure faced by these children . That said, the potential effects of peer exposure on HPA axis functioning in infancy and toddlerhood are largely unknown. Indeed, some work has suggested that heightened peer exposure may be associated with lower levels of HPA activity. For instance, in their generative study of infants and toddlers, Watamura and colleagues found that toddlers who spent more time playing with their peers tended to show lower cortisol levels than their less social classmates. Of course, the direction of this relation remains unclear; for example, perhaps less physiologically stressed children choose to play more with peers . However, it nonetheless raises the possibility that peer exposure may provide valuable opportunities for young children to learn to negotiate such complex social contexts.With rare exception, the extant literature concerning child care and children’s early adrenocortical functioning has been based on rather small, homogeneous samples of children from middle- to upper income families. Informed by findings from studies of children’s behavioral outcomes, there is increasing evidence to suggest that child care effects for children growing up in poverty may be quite different those for children from more affluent home families .

Prior work has typically concerned diurnal cortisol rhythms over the course of the day, within-child child care variation across a more limited span , and rather blunt indices of child care exposure . In contrast, we considered children’s resting cortisol levels at a single point in time, within-child variation over the course of many months, and variation in the actual number of hours children spent in child care per week. Despite these differences , the commonalities between this prior work and the present findings are noteworthy. Taken collectively, the findings suggest that within-child increases in child care exposure—on both short- and long-term developmental time scales—tend to be associated with within-child increases in salivary cortisol, for children from low-risk contexts. In particular, in the present study we found that—holding all other time-invariant confounds constant—fairly long-term shifts in child care exposure across spans of several months may underlie changes in HPA axis function, at least at 24 months of age. This finding with respect to developmental timing is consistent with meta-analytic data suggesting that the link between child care and children’s cortisol increases over the course of the child care day tends to be quite small in infancy yet pronounced toward early childhood .

The cause of these developmental differences remains largely unclear. On the one hand, plastic growing bag it is reasonable to conjecture that the types of experiences that occur in child care, such as exposure to peers, noise, and the divided attention of caregivers, might be particularly salient stressors during infancy. Developmentally, infants lack the internal self-regulatory control that is presumably needed to down-regulate their stress responses . On the other hand, children’s experiences of the same child care context may also be quite different at different points in development . For instance, whereas an infant and early-childhood classroom may maintain the same number of peers, children tend to be more actively engaged with their peers in early childhood . Such normative social differences may begin to explain the emergence of the childcare effect in toddlerhood. This is an important question for future work. Critically, our findings indicated an inversed within-child relation between child care hours and cortisol at 24 month of age, for children from high-risk contexts. For instance, for toddlers with cumulative risk levels that were 1 SD above the mean, increases in weekly child care hours were associated with contemporaneous decreases in children’s cortisol levels. Although this conditional association was somewhat modest—corresponding to a standardized regression coefficient of approximately .14—it is approximately one quarter of the size of the effect of time of day, a known biological mechanism underlying within person cortisol variation. Furthermore, the estimate is robust as a within-person effect, which adjusts for all possible time-invariant confounds.

These findings align with our prior work with respect to testing between-child interaction effect as predictive of children’s resting cortisol levels at 48 months. In that work we found similarly that the direction of the association between child care hours and children’s cortisol levels differed for children from low- versus high-risk contexts . For children experiencing low levels of cumulative risk, attending a greater number of child care hours—on average, between 7 and 35 months—was predictive of comparatively higher cortisol levels, whereas for high-risk children a negative relation was evident. Indeed, the consistency between our prior and present findings is rather remarkable, given the different substantive interpretations of between- versus within-person effects. The former represents one’s average child care exposure compared to other children’s average exposures; the latter represents time-specific increases in child care hours at 24 months, compared to one’s own prior child care hours at 7 and 15 months—irrespective of one’s average level. It is important to note that our prior interactive findings at 48 months and the present interactive findings between 7 and 24 months almost certainly tap distinct effects. As above, the interpretations differ. In addition, 24-month and 48-month cortisol levels are virtually orthogonal . The present findings also align with prior studies that have considered interactive effects between environmental risk and child care exposure in the context of children’s behavioral outcomes. For example, using the same sample as the present study, in prior work we have found that, for children experiencing high levels of household chaos across early childhood, greater weekly hours in child care were predictive comparatively fewer behavior problems. In particular, consistent with the idea of a buffering effect, greater child care exposure ameliorated the detrimental relation between household chaos and children’s social problems.

Similar interactive effect have been noted by others , and work conducted with predominantly lower income samples has increasingly shown that the “beneficial” relations between child care and children’s social and academic outcomes may be stronger and more consistent than those from studies of children from more affluent families . Despite some commonalities emerging across this literature, the mechanisms underlying these interactions remain largely unclear. Our results provide some indication that center based care may play a role. We found that—for children from low-risk contexts—attending greater proportions of one’s time in child care in center-based care was predictive of higher cortisol levels than spending more time on other types of non-parental care. In contrast, no such relation was evident for children from high-risk contexts. To some degree, this is may be inconsistent with prior work. For instance, on the basis of data from their heterogeneous sample, Roisman and colleagues found that greater exposure to center-based care in early childhood was predictive of a lower awakening cortisol levels in adolescence. Of note, though, is that the cortisol awakening response is typically considered to be a process that is distinct from typical HPA axis functioning throughout the day . There is also, however, some reason to suspect that our findings align with some behavioral findings. For instance, there is a good indication that greater exposure to center-based care may be linked to heightened levels of aggression in early childhood —perhaps particularly so for children from more affluent families . Meta-analytic findings indicate a positive correlation between cortisol and aggression levels in early childhood . Indeed, considering the potential secondary effects of cortisol on behaviors and cognition is an important next step we intend to take with these data. However, there is also reason to suspect that the translation of children’s cortisol levels into behavioral effects will not be a simple one. For example, in the present study we also found that children who were exposed to greater numbers of peers, on average, wholesale grow bags between 7 and 24 months, tended to have lower cortisol levels. This is consistent with the extant data , yet it is somewhat inconsistent with the idea that the link between center-based care and heightened levels of aggression is explained by its effects on heightened HPA axis activity: Peer problems may well underlie the effects of center-based care on aggression . Suffice it to say, there is much left to clarify. Interestingly, prior work has been suggested that the negative relation between greater peer play and lower daily cortisol levels may reflect the fact children who are less physically aroused may “select into” greater amounts of peer play . Of course, this may well be the case, yet it is noteworthy that our finding with regard to the number of children in child care—presumably reflecting peer exposure that is considerably less child driven—shows similar negative relation. Although it remains a conjecture to be tested directly, ongoing practice negotiating the social world of one’s peers may, in fact, play a “steeling” or “inoculating” role in the way that children respond physiologically to subsequent peer interactions. Evidence from nonhuman primates suggests that such steeling effects may be common and play an important role in resiliency .As such, our present findings suggest that child care type and peer exposure—at least when considered cumulatively between ages 7 and 24 months—are linked with HPA axis functioning across this period.

However, it is important to note that they do not appear to explain the effects of childcare quantity. Child care quantity, type, and peer exposure were additively and independently predictive of children’s cortisol levels, and no high-order interactions were evident. Like our prior work with children’s 48-month cortisol levels, caregiver responsivity was not related with children’s cortisol levels, between or within persons. Thus, as noted by others in prior work the mechanism underlying the child care quantity effect remains largely unclear. One possibility is that, for children facing the confluence of environmental challenges of poverty, child care may be comparatively less physiologically stressing than their experiences outside of child care. Indeed, the limited available evidence suggests that— unlike their more affluent peers—children from higher risk contexts tend to show a normative diurnal cortisol decline in their cortisol levels across the child care day . If this is the case, then the “beneficial” relation between greater child care exposure and cortisol observed for children from high-risk families may partially reflect a dose–response relation, whereby greater child care exposure leads to less exposure to the pronounced environmental stressors outside of child care. This, of course, remains a question to be addressed directly. Indeed, there are several potentially important mechanisms that were not addressed in the present study—such as the quality of children’s peer relations , teacher–child relationship quality , and degree of classroom chaos —that remain critical areas inquiry, with regard to clarifying the relation between child care exposure and HPA axis functioning.Several of our findings are robust to within-child analyses that accounted for all time invariant confound or between-child analyses that adjusted for several observed control covariates. In fact, with regard to within-child effects, the conservative nature of these estimates may underlie the fact that there were several null within-person relations . On the one hand, these specifications may strengthen the internal validity of our inferences. On the other hand, to the extent that the causal relation between these variables is contingent on cumulative exposure they would be missed by our within-person specification. They allow us to pick up cumulative, traitlike variability in these child care measures—variability that is likely quite meaningful. However, our inferences are only causally unbiased to the extent to which we have accounted for all sources of potential endogeneity . This assumption is typically dubious. As such, we make no causal claims. Nontrivially, though, these potential endogeneity problems would have to explain both selection into cumulative risk and selection into child care, as well as why the direction of the child care effects vary as a function of cumulative risk.As is common with large, comprehensive epidemiological deigns, trade-offs must often be made between breadth and depth. For instance, we were unable to collect multiple saliva samples over the course of a day or across several consecutive days. This would have strengthened the reliability of our cortisol measures and made our findings more directly informative to the extant literature concerning children’s diurnal cortisol patterns . Our saliva samples were collected modally around 10:00 a.m., when the child was at rest, after he or she had had time acclimate to the RAs in the home. There was little evidence that children were particularly stressed by the RAs’ visits. The vast majority of mothers rated their children’s behavior during the visit as being typical. Of the minority who indicated that their children were behaving differently, it was typically due to external reasons . For the few who ascribed differences as being due to the RAs’ visit, they were evenly split between those showing emotions that were comparatively more negative and comparatively more positive than usual. This is consistent with the extant laboratory-based literature that suggests that exposure to novel adults typically fails to evoke an acute stress response—even when it is intended to do so . That being said, we cannot rule out the possibility that our cortisol measures may partially tap reactivity to the RAs’ presence. Finally, by using terms like beneficial to describe, for instance, the negative relation between child care hours and cortisol, we imply that lower cortisol levels may be substantively “better” than higher cortisol levels. Other interpretations are possible. For instance, high levels of cumulative risk could make these children especially vulnerable to child care effects that cause the HPA axis to be underactive . Underactive HPA axis functioning is also a known risk factor for less optimal cognitive functioning and behavior problems . We cannot rule this interpretation out; however, we find it less plausible.